Student Reader header
Biology Political Science History Chemistry Physics Workbook Twitter
Parasitology    →    ©

Foodborne illnesses can be caused by microbes two different ways: poisoning and infection. Food poisoning results from ingesting a bacterial exotoxin. Onset of food poisoning symptoms is rapid. Food infection results from ingesting a food contaminated with bacteria. Onset of food infection symptoms is relatively slow (sometimes several days).

Kind Examples
Intracellular Bacteria Mycobacterium species; Listeria monocytogenes.
Intracellular Fungi Pneumocystis carinii; Candida albicans.
Intracellular Parasites Leishmania spp..
Viruses All viruses are intracellular pathogens. For example, herpes.
Salmonella spp.
  • S. thyphimurium is usual cause of human salmonellosis
  • Gram negative, motile rods, non-sporeforming
  • common in the intestinal tract of fowl
  • produce enteroendotoxins
  • Salmonella spp. found in poultry, eggs, cantaloupe, tomatoes, cilantro, alfalfa sprouts, clover sprouts, mung-bean sprouts, orange juice
S. thyphimurium
  • approx. 10,000 cells are sufficient to cause an infection
  • salmonellosis arises after cells have grown in the intestine
  • symptoms include sudden onset of headache, chills, vomiting, and diarrhea, and a subsequent fever
Escherichia coli O157:H7
  • VTEC strain; i.e., verocytotoxic E. coli
  • also EHEC; i.e., enterohemorrhagic E. coli
  • over half of the cases of infection in the U.S. result from tainted meat
  • a dirty cow’s coat contacts the meat during skinning, or fecal matter from the intestines contacts the meat during evisceration
  • also raw fruit juice, unwashed lettuce
  • incubation period of 1-3 days after ingestion
  • cases usually present as diarrhea, possibly with severe abdominal cramps
  • sometimes only mild diarrhea, or no symptoms
  • more serious cases lead to low platelet count and renal failure (HUS – hemolytic uremic syndrome)
  • bacteria adhere to intestinal mucosa and secrete verocytotoxin
  • toxin cleaves the 28S rRNA of the 60S ribosomal subunit (stops protein synthesis)
  • damages cells of lower intestine reduces ability to absorb fluid (if blood vessels are damaged, this also results in bloody diarrhea)
Staphlococcus aureus
  • most common type of food poisoning
  • Gram positive
  • produces six enterotoxins, which are heat stable
  • can cause diarrhea, vomiting, and nausea, generally within six hours of ingestion
  • S. aureus is often the food poisoning culprit in unrefrigerated mayo, meat, poultry, cream-filled baked goods, etc.
Clostridium botulinum
  • Gram positive, anaerobic, spore-forming
  • lives in soil or water
  • spores may contaminate food (or livestock) before harvest (or slaughter)
  • if correctly processed (to kill spores), no problem
  • however, if spores germinate (initiate growth), even a tiny amount of the toxin can be highly poisonous
  • botulism is very severe… often fatal
  • neurotoxin paralyzes breathing
  • heat labile 80°C for 10 min. destroys toxin
  • poisoning usually occurs in foods that are not cooked after processing
  • smoked meats, canned beans or vegetables, sushi
Ergot Produces many chemical compounds 12 are psychoactive (ergotamines)

Virulence factors are cellular properties which promote pathogenesis. Without these virulence factors, pathogenesis is attenuated or eliminated. These virulence factors can be:

Factor Overview
Adherence Colonization and contact..
Invasins Tissue and cell invasion.
Toxins Motility and chemotaxis.
Nutrition Compete for nutrients.
Evasion Evasion and suppression of immune response.

Introduction & History

The outcome of a microbe-host encounter depends on host defense and the strain of microbe. Disease is not the desire outcome: pathogens evolved to cause disease, but are evolving away from virulence. Humans are accidental hosts.

Louis Pasteur (1822-1895), the father of microbiology, developed:

  • The germ theory of disease
  • Rabies, anthrax, chicken cholera
  • Heat based sterilization techniques
  • Pasteurization
  • Vaccines and immunization

Robert Koch (1843-1910), the father of microbial pathogenesis, developed:

  • Methylene blue staining of bacterial cells
  • Solid bacterial cultures
  • Chemical disinfectants
  • Discovered the tuberculosis bacillus (1882)
  • Discovered the cholera bacillus (1883)
  • 1905 Nobel Laureate in Medicine for the discoveries on tuberculosis.
  • Koch’s Postulates

Alexander Fleming discovered penicillin in 1929, thereby beginning the era of antibiotics. He won the 1945 Nobel Prize for this discovery.

  • “Fleming kept his cultures 2-3 weeks before discarding them. When he looked at
    one set he noticed that the staphylococcus bacteria seemed to be dissolving. The
    mold that contaminated the culture was a rare organism called penicillium. He left
    the culture on the lab bench and went on vacation. While he was away the culture
    was subjected to a cold spell followed by a warm one – the only conditions under
    which the discovery could be made.”

Joshua Lederberg discered bacterial conjugation in 1946. Bacterial conjugation and mating has become the basis for genetic mapping, recombination, and gene discovery. He won the 1958 Nobel Prize for this discovery.

Since 1953, a molecular revolution has ensued due to discoveries and developments:

  • Watson and Crick: DNA double helix
  • Genetic code
  • Central dogma
  • Enzymes to manipulate DNA (DNAP, restriction enzymes, ligases)
  • Gene cloning and genetic engineering

See Koch’s Postulates

There are 3 major categories of pathogenic bacteria:

  • Gram-Positive and -Negative
  • Cocci/Rods
  • Spirochetes (helically shaped & neither gram-positive or -negative)

Importance of Bacteriopathology Research

A human body has 1013 eukaryotic cells and 1014 prokaryotic cells. Microbial infections include:

  • Oral microbial infections (cavity and gum disease)
  • Diarrhea and enteric bacteria
  • Tuberculosis and mycobacterium
  • Ulcer and helicobacter infection
  • Urinary tract infection
  • STD

There an 800% chance for a person to contract a microbial infection during their lifetime. $200 billion are spent annually treating microbial infections, with $55 billion spent annually treating oral microbial infections (cavities and gum disease), $19 billion spent annually treating ulceres, and $10 billion annually spent treating non-HIV/AIDS STDs. Microbial infections accounted for 57 million deaths in 2002 according to the World Heath Organization (microbial shown in red and non-microbial in blue):

  • Tuberculosis = 11 million
  • Pertussis/tetanus = 0.6 million
  • Diarrheal diseases = 1.8 million
  • Respiratory infections = 3.8 million
  • Malaria = 1.2 million
  • Injuries (car accidents to war) = 5.2 million
  • Cancer = 7.1 million
  • Cardiovascular disease = 16.6 million

Overview

It is important to approach and study bacterial pathogenesis at both a cellular (diagnosis using Koch’s postulates and treatment by killing pathogen and blocking transmission) and molecular level (using molecular Koch’s postulates and treatment by targeting virulence factors).

Evading and suppressing host immune response
•Protective capsule
•Phase variation
•Type III secretion system
•Immunotoxins
•Repelled by or resistance to nitric oxide
•Surviving phagocytosis
•Degrade complement
•Ig degradation or Ig binding ability
•Bacteria induced apoptosis
•etc

Colonization Bacterium occupies and multiplies in a particular area of the body
Infection Colonization of the body by a bacterium capable of causing disease
Disease Infection that produces symptoms
Symptoms Effects of bacterial infection apparent to infected person or animal
Virulence Ability of a bacterium to cause disease (also pathogenicity)
Virulence Factor Bacterial product or strategy that contributes to virulence or pathogenicity

To study bacterial infection at the cellular level, there are 3 forms of cultures:

Tissue Models Amidst Tissue (and Organ Culture) Models, Human, mouse, rat, bird, and insect tissues are commonly used. Entire organs, such as skins, kidneys, livers, brains, and lungs are commonly used as well. Multiple cell types, such as epithelial, fibroblast, endothelial, and macrophage are also used often.
Animal Models Animals such as mice, rats, rabbits, dogs, monkeys, fish, and worms are used. Humanized animals are animals which have been genetically engineered to contain human genes.
Human Patients Ethical issues arise when using human volunteers. Retrospective studies of outbreaks, statistical analyses of public health data, and human volunteers are all examples of using human patients.

Detecting and monitoring bacterial infections. Classical methods innclude bacterial culture of infected tissues and em analysis of infected tissues. Detecting and monitoring bacterial infection, like modern in vivo imaging, whole body fluorecsent imaigng of E. coli GFP in various organs.

Virulence Factors

Factor Overview
Colonization

Genes associated with bacterial colonization, such as pili and adhesins.

Type 1 fimbriae Fimbriae in Enterobacteriaceae which bind specifically to mannose terminated glycoproteins on eukaryotic cell surfaces.
Sex pilus A specialized pilus that binds mating procaryotes together for the purpose of DNA transfer.
Common pili Same as fimbriae.
Fimbriae Filamentous proteins on the surface of bacterial cells that may behave as adhesins for specific adherence.
Ligand A surface molecule that exhibits specific binding to a receptor molecule on another surface.
Receptor A complementary macromolecular binding site on a (eukaryotic) surface that binds specific adhesins or ligands.
Adhesin A surface structure or macromolecule that binds a bacterium to a specific surface.
Teichoic acids and lipoteichoic acids (LTA) Cell wall components of Gram-positive bacteria that may be involved in nonspecific or specific adherence v
Lipopolysaccharide (LPS) A distinct cell wall component of the outer membrane of Gram-negative bacteria with the potential structural diversity to mediate specific adherence. Probably functions as an adhesin.
Capsule A detectable layer of polysaccharide (rarely polypeptide) on the surface of a bacterial cell which may mediate specific or nonspecific attachmen.
Glycocalyx A layer of exopolysaccharide fibers on the surface of bacterial cells which may be involved in adherence to a surface.
Invasion Aka infection factors, genes that allow a bacterium to invade tissues such as invasins. Invasins are bacterial extracellular substances which act against the host by breaking down primary or secondary defenses of the body. Invasins are proteins (enzymes) that act locally to damage host cells and/or have the immediate effect of facilitating the growth and spread of the pathogen. The damage to the host as a result of this invasive activity may become part of the pathology of an infectious disease.
Disease

genes that are responsible for producing symptoms, such as toxins. Toxigenesis, or the ability to produce toxins, is a mechanism by which many bacterial pathogens produce disease. Toxins can act by protein cleavage (intracellular proteases), direct damage to cell membranes, host protein modification, host signal transduction pathway modification, and as superantigens. At a chemical level, there are two types of bacterial toxins:

Endotoxins Endotoxins (aka Lipopolysaccharides or LPS) are bound to Gram-negative cell walls, and proteins, which are released from bacterial cells and may act at tissue sites elsewhere in the body.
Exotoxins The extracellular diffusible toxins are exotoxins.
Motility/Chemotaxis
Biofilm Formation
Quorum Sensing
Immune Interference Evasion and suppression of host immune response

Virulence Gene Regulation

There are several ways a strain can become pathogenic: by gaining genes specific to the pathogen, absence of a suppressor locus in the pathogen, allelic differences between genes shared by pathogen and nonpathogen, and by differential regulation of the same genes.

Virulence genes can be regulated by:

  • Environmental cues (CO2, temperature, iron, pH, etc)
  • 2-component regulatory systems
  • Quorum sensing
  • Genetic rearrangement: fimA & RecA- dependent pilin antigenic variation
  • Contact-dependent regulation

Secretory Pathways

Gram (+) and Gram (-) cells have different secretory pathways. This is due to morphological difference, with Gram (+) cells having 3 compartments (cytoplasm, membrane, and extracellular) and Gram (-) cells having 5 compartments (cytoplasm, inner membrane, periplasm, outer membrane, and extracellular). Gram (+) cells use the sec-dependent general secretory pathway (types II and V), and Gram (-) cells use the sec-independent pathway (types I, III, and V) as well as the autotransporter (type V).

Competition assay tests virulence factor. Mix mutant and WT strains of bacteria. If mutant has defect in virulence, this will be detected by examining ratio of mutant:WT at assay endpoint.

How Can Resistance Occur?
  1. Underproduction of proteins required for drug activation.
  2. Underproduction of a membrane protein that is necessary for uptake of the drug (methotrexate resistance in Leishmania).
  3. Overproduction of the target enzyme resulting from DNA amplification: Dihydrofolate reductase-thymidylate synthetase (DHFR-TS) overproduction leading to methotrexate resistance.
  4. Mutation in a cellular protein that results in decreased affinity of the drug and its target.
  5. Overproduction of a membrane glycoprotein that pumps drugs out of the cell.

Introduction & History

Alexander Fleming discovered penicillin (the 1st antibiotic) in 1929. As more antibiotics have been developed, selective pressure for antibiotic resistance has increased. As genes encoding resistance are transferred between bacteria, antibiotics gradually become effective against fewer bacteria. As a result, new antibiotics must perpetually be discovered.

Approximately 2 million nosocomial infections/yr occur, resulting in ~80,000 patient deaths. Strains of Staph. aureus and Enterococcus resistant to conventional antibiotics are widespread, and the total cost of additional health care due to resistant strains is ~$5 billion.

Agriculture and aquaculture account for ~40-50% of all antibiotic use in the US. Tetracycline derivatives are used at subtherapeutic doses to augment the size of pigs and chickens, and to reduce farm animal infection rates. As a result, though, groundwater and manure from farms oftentimes carry Tetr (tetracycline resistant) bacteria which contaminate the ecosystem.

Currently, there are three precautions implemented to abate the development of antibiotic resistance:

  • Educating the medical community about infection prevention
  • Conservative use of antibiotics like vancomycin to slow evolution of antibioticr strains.

Spread of Resistance Genes

An example of a drug resistance plasmid is pB10. It was isolated in a waste water treatment plant and confers resistance to antimicrobials (amoxicillin, streptomycin, sulfonamides, and tetracycline) and mercury. It is a mosaic plasmid, meaning it has regions from several plasmids combined via recombination. It has at least 5 distinct mobile genetic elements (most conferring resistance).

Resistance genes can get around by gene transfer (plasmids, transposons, and conjugative transposons) or bacterial intermediaries (non-pathogenic strains are resistance gene reservoirs).

Example: Tetracyclines

Tetracycline antibiotics include doxycycline (clinical use) and tetracycline, chlortetracycline, and oxytetracycline (agricultural use). Tetracycline readily crosses the peptidoglycan layer of Gram-positive and outer membrane of Gram-negative bacteria and inhibits translation. Tetracycline is classified as a “broad spectrum”antibiotic because it is effective against a wide range of organisms. In Gram negative bacteria, tetracycline is transported though the outer membrane as a metal-chelatedcomplex (probably Mg2+) by the OmpFand OmpC porin channels. Accumulation of the cationic tetracycline-Mg complex in the periplasmis facilitated by the Donnanpotential of the membrane. Dissociation of the cationic complex allows the low molecular weight, lipophilictetracyclinesto penetrate the cytoplasmic membrane. In Gram-positive cells the peptidoglycan layer is porous to low molecular weight compounds allowing entry into the cell and bind the ribosome.

Mechanisms of resistance against tetracylines:

  • Effluxers: transporters move tetracyclines across cell membrane (often antiporters, with tetracycline out and H+ in). Effluxer proteins may have broad specificity, meaning they are mutlidrug transporters.
  • Ribosomal Protection: Proteins bind the ribosome to prevent tetracyclines from binding. tetM resembles EF-G and displaces tetracycline, but does not replace EF-G. Mutations to rRNA alter binding pockets where tetracyclines bind (16s rRNA).
  • Enzymatic: enzymes modify and inactivate tetracyclines

There are many Tetr genes. Those marked with asterisks are restricted to Gram-positive cells. Gene families are separated by semicolons.

  • Efflux: tetA, tetB, tetD, tetE, tetG, tetH, tetI, tetJ, tetZ*, tet30, tet31, tet33*, tet38, tet39; tetK*, tetL*; otrB*, tcr3*, otrC*; tetP(A); tetV; tetY; tet35
  • Ribosomal protection: tetM*, tetO*, tetS*, tetW*; tetQ*, tetT*, tet36*; otrA*, tetP(B), tet*; tet32*
  • Enzymatic: tetX, tet34, tet37

There are two ways that toxins are released into the environment. Some cells will secrete toxins while other cells will release toxins upon cell lysis. Exotoxins are secreted by the cell while endotoxins are released upon cell lysis. Endotoxins are usually a lipopolysaccharide or a cell wall protein. There are three ways for exotoxins or endotoxins to have a toxic effect on the host:

  1. Lipolysaccharide (LPS) is a superantigen causing septicemia (toxic shock).
  2. Enterotoxins affect the GI tract.
  3. Cytotoxin destroys cells by inhibiting protein synthesis or damaging membranes.
Name Organism Activity
Anthrax Toxin Bacillus anthracis Edema Factor (EF) is an adenylatecyclase that causes increased levels in intracellular cyclic AMP in phagocytes and formation of ion-permeable pores in membranes (hemolysis).
Adenylate cyclase toxin Bordetella pertussis Acts locally to increase levels of cyclic AMP in phagocytes and formation of ion-permeable pores in membranes (hemolysis).
Cholera enterotoxin (ctx) Vibriocholerae ADP ribosylation of G proteins stimulates adenlyate cyclase and increases cAMP in cells of the GI tract, causing secretion of water and electrolytes
E. coli LT toxin Escherichia coli Similar to cholera toxin
E. coli ST toxin Escherichia coli Stimulates guanylatecyclase and promotes secretion of water and electrolytes from intestinal epithelium
Shiga toxin Shigelladysenteriae Enzymatically cleaves rRNAresulting in inhibition of protein synthesis in susceptible cells
Perfringensentero toxin Clostridium perfringens Stimulates adenylate cyclase leading to increased cAMPin epithelial cells
Botulinum toxin Clostridium botulinum Zn++dependent protease that inhibits neurotransmission at neuromuscular synapses, resulting in flaccid paralysis
Tetanus toxin Clostridium tetani Zn++dependent protease that inhibits neurotransmission at inhibitory synapses resulting in spastic paralysis
Diphtheria toxin (dtx) Corynebacterium diphtheriae ADP ribosylationof elongation factor 2 leads to inhibition of protein synthesis in target cells
Exotoxin A Pseudomonas aeruginosa Inhibits protein synthesis; similar to diphtheria toxin
Anthrax toxin (LF) Bacillus anthracis Lethal Factor (LF) is a Zn++dependent protease that induces cytokine release and is cytotoxicto cells by an unknown mechanism
Pertussistoxin (ptx) Bordetella pertussis ADP ribosylationof G proteins blocks inhibition of adenylatecyclasein susceptible cells
Staphylococcus enterotoxins* Staphylococcus aureus Massive activation of the immune system, including lymphocytes and macrophages, leads to emesis
Toxic Shock Syndrome Toxin
(TSST-1)*
Staphylococcus aureus Acts on the vascular system causing inflammation, fever and shock
Exfoliatintoxin* Staphylococcus aureus Cleavage of epidermal cells (intradermal separation)
african sleeping sickness trypanosoma brucei
T. brucei gambiense T. brucei rhodesiense T. brucei brucei
Distribution West Africa East Africa
Vector Glossina palpalis G. morsitans, palpides. G. palpalis, fuscipes, tachinoides
Disease Chronic (mo’s-y’rs) Acute (w’ks-mo’s)
Parasitemia Low High
CNS Spread Late Early
Host Human Humans & Game Cattle & Game
HDL Rxn? No No Yes

Aspect Overview
Vector The tsetse fly, encompassing all species of the genus Glossina. Only newly hatched tsetse flies are competent to transmit the disease. Tsetse flies are a poor vector since less than 1% of flies are infected.
Transmission Trypanosoma brucei species are transmitted by tsetse bites. The tsetse fly contains trypanosomes in its probiscus and salivary glands; thus, T. brucei species are known as saliva-type or salivarian trypanosomes.
Life Cycle
Stage Overview
Short Stumpy Able to infect the tsetse fly vector.
Procyclic Able to infect a mammalian host.
Metacyclic Pre-adaption occurs. Mitochondrial genes stop expression by losing cristi.
Long Slender No functioning mitochondria — glycolytic lifestyle.
G0
Short-Stumpy Bloodstream Trypomastigote

The non-dividing short-stumpy bloodstream trypomastigote infects the fly upon ingestion of a blood meal. While the meal is retained within the midgut, Trypanasoma brucei differentiates into a procyclic form.

Procyclic Form

The procyclics divide by binary fission within the insect midgut. After about two weeks, some procyclics migrate from the midgut through the hemocoel to reach the salivary glands. At this point the procyclic form differentiates through an epimastigote stage into a non-dividing metacyclic trypomastigote stage.

Non-Dividing Metacyclic Trypomastigote

The non-dividing metacyclic trypomastigote is infectious for the mammalian host. Metacyclic trypomastigotes are found in the salivary glands ~20 days after the bloodmeal; each bite transfers ~40,000 trypomastigotes; only 400 trypomastigotes are needed to initiate infection.

Dividing Metacyclic Trypomastigote

Metacyclic trypomastigotes replicate at the site of infection. There may be an immune response causing a trypanosomal chancre at the site of the bite. From the bite wound, the trypomastigotes move via the lymphatics to the lymph nodes and then to the bloodstream where they differentiate into the long-slender form.

Long-Slender

Long-slender bloodstream trypomastigotes divide by binary fission in the bloodstream. The long-slender trypomastigotes are not infectious for the fly. On occasion, the long-slender protozoa differentiate into the short-stumpy protozoa to continue the cycle in the tsetse fly.

Disease

Causes Nagana in animals and African Sleeping Sickness in humans. Symptoms include Winterbottom’s sign and swollen lymphnodes on neck.

Stage Overview
Primary Stage Metacyclic trypomastigotes are introduced subcutaneously and multiply. In 2-3 days the bite site is itchy, swollen and red. After 6 days a trypanosomal chancre may develop at the bite site. This is oft dismissed as an innocent boil.
Blood Stage Generalized infection manifests as fever, sometimes accompanied by malaise, headache and joint pain. Trypanosomes are found in the bloodstream 5-12 days after infection. Trypanosoma gambiense are scarcer than T. rhodesiense in the bloodstream. Trypanosomes infiltrate the lymphatic system, and the influx of B-cells causes lymphadenopathy. In a Trypanosoma gambiense infection, swollen cervical (neck) lymph nodes are referred to as Winterbottom’s sign. The lysis of trypanosomes releases toxins that TNF-α secretion by macrophages, thus causing cyclic (relapsing) cachexia (fever) with a cycle of 7-10 days.
Late Rhodesian In the Late Stage of Rhodesian Sleeping Sickness, it is just a few weeks before the parasite enters the CNS from the lymphatics. Death is quick upon CNS involvement, if myocarditis was not already fatal.
Late Gambian In the Late Stage of Gambian Sleeping Sickness, the parasite invades the CNS within one or more years and causes personality changes including insomnia and irritability. Inflammatory changes cause a demyelinating meningoencephalitis, causing cerebral edema, hemorrhages, pericarditis, and anemia. The encephalopathy leads to apathy, somnolence and coma. Death is usually due to intercurrent infections such as pneumonia.
Detection
Technique Overview
Epidemiology Domestication of cattle occurs only outside the tsetse area. Losses in meat production, milk yield and tractive power are estimated to cost approximately $500 million annually and, if lost potential in livestock and crop production are also considered, the disease costs Africa an estimated $5 billion per year (1994 prices). Complete control of tstetsewould result in an increase in beef production of 1.5 million tons per annum. However, this would also have a massive impact on the use of land and significantly reduce the possibilities for wild life in Africa. (From http://www.icp.ucl.ac.be/~opperd/parasites/)
Aspect Overview
Agent Trypanosoma cruzi (aka Schizotrypanum cruzi) is a protozoan parasite and the causative agent of Chagas Disease. It is found primarily in Central and South America.
Vector

Reduviids (aka kissing bugs) are the Trypanosoma cruzi vector. Several genera of Reduviidae transmit T. cruzi.

Vector Overview
Triatoma infestans Frequently invades homes (mud and stick huts); domestic transmission cycle.
Rhodnius prolixus Resides in rural settings or forests; silvatic transmission cycle.
Panstrongylus megistus Same as Rhodnius prolixus
Transmission Reduviids transfer Trypanosoma cruzi when they defecate into a wound while taking a blood meal. Oftentimes the mammal inadvertently rubs the feces into the bite itself due to itch. T. cruzi is thus a dung-type or stercorarian trypanosome. Transmission via blood transfusions have made Chagas Disease not just a rural disease but an urban disease as well. Between 1960 and 1989, infection of blood in South American cities’ banks ranged from 1.7% in Sao Paulo, Brazil to 53.0% in Santa Cruz, Bolivia, a percentage far higher than that of hepatitis or HIV infection. Transmission by blood transfusion has spread to Los Angeles due to immigration from Central America. In Los Angeles, 2% of the blood donors in a 1993 study were seropositive. Five cases of Chagas Disease in the US in 1990-1993 came from blood transfusion or organ transplants.
Life Cycle

The protozoan parasite Trypanosoma cruzi is the agent of Chagas Disease. It has an insect cycle and a mammalian cycle. In the mammalian host Trypanosoma cruzi survives inside a host cell (intracellular amastigote) then bursts out (extracellular trypamastigote) then is uptaken into a reduviid consuming a blood meal.

Epimastigote
Metacyclic Trypomastigote
Event Overview
Transmission An infected triatomid defecates onto a bite wound or mucosal surface of the vertebrate host. When the host scratches the bite, metacyclic trypomastigotes in the triatomid feces are pushed into the wound.
Attachment Metacyclic trypomastigotes bind macrophages via receptor-mediated attachment
Lysosome Vacuole Within the macrophage, the trypomastigote recruits lysosomes. The lysosomes fuse together and encapsulate the trypomastigote in a lysosome-derived vacuole.
Vacuole Bursts The trypomastigote secrets TcTOX, an antigen similar to the lytic complex C9 that lyses the vacuole. The trypomastigotes enter the cytoplasm.
Differentiation The metacyclic trypomastigote differentiates into an amastigote and divides.
Amastigote
Event Overview
Replication The amastigotes go through nine cycles of intracellular replication in 4-5 days.
Differentiation Differentiate into trypomastigotes.
Trypomastigotes
Event Overview
Rupture The host cell ruptures and the parasites are released into the bloodstream, where they are disseminated throughout the body.
Disease
Stage Overview
Acute Symptoms include: Romaña’s Sign; fever; hepatosplenomegaly; and trypomastigotes in blood. The acute phase lasts 2-8 weeks and has 10% mortality.
Indeterminate No parasites are evident in blood. Amastigotes nest in muscle tissue, particularly of the heart. A Chagasic Heart has a myriad of amastigote clusters. Anti-Trypanosoma cruzi antibodies are present.
Chronic Chronic Chagas Disease symptoms include: nerve degeneration; megaesophagus (25%); megacolon (30%); and cardiomyopathy (80%) including arrhythmia, blocks, cardiomegaly and apical aneurism. Arrhythmia due to Chagas Disease is characterized by a unique and diagnostically useful pattern.
Detection
Technique Direct? Overview
Xenodiagnosis Direct.
PCR Direct.
Microscopy Direct. Unfortunately the parasite is not abundant in the blood during indeterminate and chronic infections, leading to diagnosis via microscopy in only 1% of these cases. Also morphological similarities between Trypanosoma rangeli and T. cruzi contribute to misdiagnosis.
IFA Indirect.
Epidemiology

Chagas Disease is prevalent in South and Central America, having infected 15-20 million people and with over 100 million people at risk. Up to 60% of the population is serologically positive for Trypanosoma cruzi in some endemic areas. Once considered an exotic rarity, improved diagnostic methods have revealed Chagas Disease as one of the most widespread Latin America infectious diseases. Over 20% of patients had Chagas Disease in one hospital in Goiania. Chronic Chagas Disease causes most cases of sudden death in young adults of Latin America due to its weakening of heart muscle.

Chagas Disease is mainly a disease of third world countries where reduviids infest substandard houses with dirt floors, mud walls and thatched roofs. Chagas Disease is combatted by spraying housing with insecticide and by replacing adobe with modern materials. Hexochlorocyclohexane (BHC) is an ideal insecticide due to its low cost, low non-insect toxicity and high activity in mud walls (which lead to rapid attenuation of most other insecticides). Applications vary in frequency from monthly to twice annually. Another prevention method is the replacement of adobe with modern materials impervious to reduviid infestation. These two techniques form the core of the Southern CONE Initiative.

Spirochetes are helically shaped, either resembling a corkscrew or a flat wave. Spirochetes have hidden flagella and tremendous antigenic variation, thus making them potentially potent pathogens. Spirochetes thrive in blood, saliva and other nutrient-rich environment; they are susceptible to (and avoid via chemotaxis) H2O2 and other free radicals. Interestingly, Borrelia species do not have LPS in their outer membranes and are neither gram-positive nor -negative. Their genome lacks any LPS biosynthesis genes. The eight genera of spirochetes are listed below.

Genera Pathogenicity Associated Diseases
Borrelia only pathogenic Lyme disease; relapsing fever; borreliosis.
Brachyspira parasitic or pathogenic Swine dysentery.
Brevinema parasitic (white-footed mouse)
Cristispira parasitic (gastropods)
Leptonema free-living or parasitic
Leptospira free-living parasite or pathogen Leptospirosis.
Spirochaeta free-living
Treponema parasitic or pathogenic Syphilis; periodontitis; yaws.

The life cycles of B. burgdoreri (Lyme Disease) and Treponema (Syphilis) are shown below.

Organism Life Cycle
B. burgdoreri
  1. Inoculation in the skin area.
  2. Invasion into the bloodstream.
  3. Leaving bloodstream and entering tissues throughout the body.
Treponema
  1. 1° Stage: Initial inoculation at genital areas and the appearance of a single sore (called a chancre).
  2. 2° Stage: The appearance of rash and mucous membrane lesions at different areas of the body, fever, fatigue, headache etc.
  3. Late Stage: Damage to internal organs, including brain, nerves, eyes, heart, blood vessels, liver, bones and joints.

Below that is an overview of the primary virulence factors for in vivo spirochete pathogens. Signal transduction, motility, and chemotaxis mutants are defective in tissue penetration.

Virulence Factor Overview
Motility Spirochetes have periplasmic flagella which rotate around the cytoplasmic cylinder. Movement is rotational (snakelike) and this allows translocation in highly viscuous environments. The spirochete motility swarming assay screens fla- mutants via colony size (big colonies indicate the motile WT strain, while compact colonies indicate immotile fla- mutants). Upon isolation of fla- mutants, biochemical approaches may be used to identify which genes have been altered to induce stagnancy.
Chemotaxis

How is chemotaxisis achieved in spirochetes? They swim in presence of attracts and flex in presence of repellents. There are numerous examples of bacterial chemotaxis (enteric bacteria seek nutrients; rhizobium seek plant root; thermophiles seek heat; agrobacterium seek woundsites). However, it is less clear what spirochetes seek. Possibilities are tick salivary glands, blood, specific tissues, and immune cell evasion.

The capillary chemotaxis assay is designed to determine attracts and repellents for B. burgdorferiand and T. denticola. It is performed as follows:

  • Step 1
  • Step 2
  • Step 3
  • Step 4
  • Attractants: Serum & Saliva
  • Repellents: H2O2, and Ethanol

Flexing and swimming. The chemotactic system has 2 receptor system. The flagellar motors are special because they can go CW and CCW. Depending on the particular flagellar motor there can be coordinated or uncoordinated movement.

CheW is attached to CheA (CheW is an accessory protein acting as a coupling protein). PR methylates MCP, reducing the activity.

Pathogenic Spirochetes Chemotaxis Genes
Borrelia burgdorferi 2 CheA, 3 CheY, 3 CheW, 5 MCP, 1 CheX, 2 CheR
Treponema pallidum 1 CheA, 1 CheY, 2 CheW, 4 MCP, 1 CheX, 1 CheR
Treponema denticola 1 CheA, 1 CheY, 1 CheW, 21MCP, 1 CheX
Immune Evasion
  • Unexposed flagellar filaments.
  • Running away from oxygen radicals.
  • Leaving bloodstream and targeting specific tissues for good hiding places.
  • Very limited surface proteins.
  • Many spirochetes (such as Borrelia) have no LPS.
  • Antigenically variable surface proteins such as Vlsproteins.
  • Multiple circular and linear plasmids with extensive recombination rates.
  • Production of extracellular proteases.
  • others

Borreliasppdo are neither gram-negative nor -positive, and Borrelia genome data reveals no LPS biosynthesis genes (Borellia lack LPS). The major surface proteins of pathogenic spirochetes are:

  • OspA(outer surface protein A)
  • OspB(outer surface protein B)
  • OspC(outer surface protein C)
  • OspD(outer surface protein D
  • Erps(OspErelated proteins)
  • DbpA(decorinbinding protein)
  • VlsE(Antigenicallyvariable proteins)

OspA and Lymevaccine LYMErix. OspA was found in abundance by B. burgdorferi grown in laboratory medium. OspAbased Lyme vaccine was developed. Upregulated in tick larva as it becomes a nymph. Downregulated in nymph as it takes a blood meal. Not expressed on bacteria in mammal until late in infection.

In Vivo Survival
Challenge Solution
Highly viscous body fluids Corkscrew-like movement
Numerous immune cells No LPS, few surface proteins, unexposed flagella, etc.
Low free iron concentration Proteins use manganese in place of iron
Abundant peptides & few carbohydrates Spirochetes degrade polypeptides for nutrition.
No cytochromes nor TCA enzymes.

Helicobacter pylori secretes urease to maintain a basic/neutral environment. Lophotrichous tufted flagella, powered by a proton gradient, give it motility. Helicobacter pylori is extracellular and does not need to be intracellular to replicate. They have a tuft at one end, and this is mediated by that they are encased in a membrane. The structure of VacA is like a flower petal such that it might almost form a pore but it is unclear what it does with pathogenicity.

  • 1881 & 1893: Spiral organisms found in stomach contents of dogs and other mammals
  • 1938: Spiral organisms first described in human gastric mucosa
  • 1975: First attempts to culture gastric bacteria
  • 1982: Helicobacter pylori cultured and shown to cause ulcers

Helicobacter pylori is associated with gastritis, duodenal and gastric ulcers and gastric adenocarcinomas. Gastric adenocarcinomas are the 14th leading cause of death in the world, and are believed to be the 8th leading cause by 2010. This is largely because infection in the lumen of the stomach is not accessible to immunocytes and macrophages.

90% of duodenal ulcers and 70% of gastric ulcers are Helicobacter pylori positive. Presence of gastritis is a risk factor for duodenal ulces and ulcer relapse. Cure of Helicobacter pylori infection leads to a dramatic reduction in ulcer relapse rate. Addition of antibiotics to acid suppressive therapy increases speed of healing of acute ulcers.

Helicobacter pylori and Human Ulcer Diseases

Transmission:
Ancient association with humans. Believed to be present in human stomachs before. Human migration started ~100,000 years ago. Overlap between genetically distinct H. pylori and human populations.

Transmission

Developing Countries
  • 80% population colonized
  • Colonization during childhood
  • Parent-child transmission (genotyping)
  • Strains persist and re-infection rare
  • Very low rate of ulcers!
  Industrialized Nations
  • 20-50% population colonized
  • Colonization later in life
  • Transmission between spouses rare
  • Rare childhood infection rate (<20%)

Colonizaiton → immunomodulation → host remodeling → symptoms

1. Oral ingestion
2. Transit of gastric environment
a) Adult stomach pH ~ 1-2
b) Infection occurs in early childhood, higher stomach pH
3. Penetrating gastric mucus
a) Thick (viscous), and sloughing action
4. Attachment
a) Allows bacteria to persist in stomach
5. No competition for H. pylori once established on the mucosal
surface
Route of infectionRoute of infection

Interesting genes of Interesting genes of Helicobacter pyloriHelicobacter pylori

Listeria monocytogenes is a small gram-positive coccobacillus between .5 and 2µm long. It is widely distributed, as it can infect mammals, birds, fish, ticks, and crustacea. It is microaerophilic (it is an obligate aerobe but is viable in minimally oxygenated environments) and can ferment sugars to produce various acids. It has an optimum tmperature of 37°C but is viable at temperatures as low as 2.°C.

The GI tract is the portal of entry for extrauterine infections. Mucosal colonization and invasion occurs with lowered host defenses. Once in the circulation, Listeria monocytogenes has tropism for the fetus, placenta, and CNS. Listeria from the infected placenta disseminate to the liver, spleen, lungs, & CNS.

Listeria monocytogenes are readily phagocytosed by macrophages following the activation of the alternative pathway of complement. Within resting macrophages, Listeria can survive and multiply. L. monocytogenes survive and multiply within the cytoplasm of mammalian cells and spread to adjacent cells via pili. They are able to spread between cells without even leaving the cytoplasmic environment.

1949, Germany, University of Halle. In 85 newborns or stillborn infants granulomas were detected histopathologically in various organs such as liver, spleen, brain, lung and skin. Granulomatis infantiseptica. J. Potel, then H.P.R. Seeliger

Some quick information on Listeria monocytogenes:

  • L. monocytogenes is 20x more likely to infect pregnant women, and can cause abortion, premature birth, stillbirth and death-after-birth.
  • If an infected baby is born alive, it is at high risk for septicemia, pneumonia, diarrhea, seizures, skin lesions and meningitis.
  • L. monocytogenes can enter the GI tract in immunocompromised individuals (cancer & renal patients).
  • AIDS patients are 100x more likely to contract L. monocytogenes and develop meningitis (the most common form of listeriosis, with 30% mortality).
  • Septic infections result in endocarditis and pneumonia.

Listeriosis, the set of clinical symptoms related to a malignant Listeria monocytogenes infection, is most prevalent in pregnant mothers, fetuses, newborns, cancer patients, and AIDS patients. The elimination of a L. monocytogenes infection depends upon a cell-mediated immunity (CMI) response (meaning antibodies play no role). Interestingly,=, about 5% of the healthy human population carries the bacterium. This beckons the question: What are the virulence factors for L. monocytogenes?

Pathogenesis

Sequential events associated with pathogenesis:

  • Entry into the host GI tract (via contaminated food or water) or transplacentally.
  • Invasion/phagocytosis of various cell types: intestinal epithelial cells, Peyer’s patches, macrophages, liver parenchymal cells, hepatocytes.
  • Intracellular multiplication requires escape from phagocytic vesicle.
  • Cell-to-cell spread via pili.

There are four assays used to investigate Listeria Monocytogenes pathogenesis:

Hemolysis Listeria Monocytogenes are hemolytic due to Listeriolysin O (LLO), a protein encoded by hlyA. To analyze hemolytic activity qualitatively, look for a zone of hemolysis are Listeria colonies grown on blood agar. To analyze hemolytic activitivy quantitatively dilute Listeria culture supernatent and test its ability to cause 50% lysis of a red blood cell suspension.
Egg Yolk Opacity Assay Listeria Monocytogenes produces lecithinase or phopholipase. These enzymes can be detected by growing L. monocytogenes on agar plates that have a 5% egg yolk overlay. Lecithinase and phospholipases will cause zones of opacity.

Assessing intraccelular growth and cell-to-cell spread:

  • Listeria monocytogenes are added to mammalian cells such that 1 out of 10 to 50 cells become infected.
  • They are then incubated (1hr), washed, and fresh medium is added containing gentamicin.
  • Cells are lysed after 1, 2, 3 and 4 hours and the released bacteria are plated.
  • Microscopic examination: After 8 hours, the cultures are analyzed by EM.
  • Macroscopic examination: culture is overlayed with 0.5% agarose, incubated for 48hrs and then stained with neutral red (vital stain).

LD50 Virulence Testin in Mice: Mice are inoculated either orally or intravenously with various doses of Listeria Monocytogenes bacteria, and the dose at which 50% of the animals die is determined.

ListeriolysinO

ListeriolysinO (LLO) is one of 22 members of cholesterol-dependent cytolysins secreted by Gram-positive bacteria. The best characterized of these are perfringolysinO (PFO) and streptolysinO (SLO). ListeriolysinO is unique because it acts in a vacuole, while the others act extracellularly on host cell membranes. Genetically replacing LLO with PFO results in Listeria that escape from the vacuole, but kill the host cell upon growth in the cytosol. Therefore, it can be deduced that LLO is active in the vacuole but inactive in the cytosol.

There are three reasons why ListeriolysinO is less toxic in the cytosol:

Enzymatically ListeriolysinO is enzymatically more active at acid pH’s than at neutral pH’s.
PEST ListeriolysinO amino acid sequence analyiss shows the presence of a PEST-like sequence region (rich in proline, glutamate, serine and threonine) at the N-terminal region of the protein. This PEST sequence may negate LLO toxicity in the cytosol by targeting LLO for proteolytic degradation, or by making it a target of MAP kinase phosphorylation.
NLS Region ListeriolysinO has a nuclear localization sequence (NLS region) which may localize LLO to the nuclear membrane. The nuclear membrane does not possess cholestrol, and LLO is cholestrol-dependent.

Listeria monocytogenes’ Cell-To-Cell Spread

Cossart et al theorized that Lecithinase activity was important for cell-to-spread spread. They tested this theory:

  • Create L. monocytogenes Tn917 insertion mutants
  • Screen for Lecithinase mutants in the pool of Tn917 insertion mutants
  • LUT12 was found to be Lecithinase- but LLO+ (the hlyA gene remained intact)

The properties of Lecithinase were examined by running tests on LUT12:

  • LUT12 intracellular growth rate the same as wild type (but stops after 3hrs).
  • LUT12 LD50 = 108.6 (wild type LD50 = 104) so Lecithinase is avirulent
  • LUT12 unable to form plaques in 3T3 cells.

In addition, EM analysis of LUT12 showed there was:

  • Escape from the phagosome
  • No cell-to-cell spread
  • No actin clouds nor actin tails

To discover which genes were involved in cell-to-cell spread, scientists cloned the locus of Tn917 insertion and performed sequence analysis. They found that Tn917 had inserted into actA, a gene which had been previously cloned and sequenced. Based on this finding, it was very likely that actA played a crucial role in cell-to-cell spread. Scientists confirmed this with the following experieent:

  • Introduce “in-frame” non-polar deletion mutations into hlyA and actA.
  • Compare these constructs with wild type L. monocytogenes.

Scientists found that actin polymerization and cell-to-cell defents of LUT12 were due to an actA mutation by insertion of Tn917, and the Lecithinase phenotype was due to polar effects on plcB. Since actA is responsible actin polymerization (known informally as comet tail formation), scientists set out to discover how actin polymerization is associated with L. monocytogenes. intracellular movement:

  • Potoroo (a kind of rodent) kidney epithelial cells faciliate analysis using fluorescent microscopy.
  • Phalloidin-Rhodamine Conjugate is a molecule composed of:
    • Phalloidin (amanita mushroom toxin), which binds to polymerized actin (F-actin) and
    • Rhodamine, a fluorescent molecule that emits red light after excitation.
  • Potoroo kidney cells were infect with L. monocytogenes and stained with phalloidin-rhodamine.
  • Fluorescence in the comet tails was measured.

Scientists found that actin filament density decreases exponentially as the distance from the pole of the bacterium increases.

How does actinpolymerization result in intracellular & cell-to-cell movement of Listeria Monocytogenes?

  • What are the actin filament dynamics in the comet tail?
  • Potoroo kidney epithelial cells were microinjected with rabbit skeletal muscle “globular” actin (G-actin) covalently coupled to “caged resorufin” (CR).
    • Experimental approach: CR R(red fluorescence)360ηm UV
    • Potorookidney cells then infected with Listeria. CR-actinwill be incorporated into the comet tail.
    • A laser was used to focus UV (360ηm UV) on a single spot on the comet tail.
    • The spot was imaged by fluorescence and the bacterium imaged by phase contrast microscopy.

Examined 22 bacterial cells by 4 parameters:

  • Filament half-life (exponential decay curve: 100% →50%)
  • Speed (distance moved over time, 55 sec)
  • Tail length (measured from back end of bacterium to tail tip)
  • Distance from “spot” to bacterium

How is actinpolymerization restricted to one pole of the bacterial cell?

  • Hypothesized that actinpolymerization is polar because ActAis polar.
  • Used immunoelectronmicroscopy (IEM): anti-ActA antibody that was conjugated to colloidal gold (10ηm particles).

Is polarized localization of ActArequired for unidirectional movement?

  • Observations: LytA of Streptococcus pneumoniae’s autolysis. LytAbinds to the cell wall of S. pneumoniae. LytAwill also bind to DEAE cellulose.C-terminal domain of LytAis sufficient for this binding but insufficient to mediate cell lysis.
  • Experimental approach:Fused the C-terminal domain of LytAto ActA.

Labeled LytA-ActAwith FITC. DEAE: diethylaminoethanol
-To show that if actinfilaments polymerized in a polar manner,
movement would result →S. pneumoniae.-Took advantage of the fact that S. pneumoniaecell division

Transcytosis is mediated by pili which are the first mediators of attachment, then retraction of pili, then attachment of

Term Overview
Anthroponosis Disease with humans as only vertebrate hosts.
Zoonosis Disease transmitted among wild animals (reservoir hosts) and humans.
Reservoir Host Wild animal that maintains infection in nature.
Procyclic Related to the beginning of the parasite’s life cycle.
Metacyclic Related to the infective stage of the parasite’s life cycle.
VSG Variable surface glycoprotein
ESAG Expression site associated glyocoprotein
COG A bluster of orthologous genes (COG).
3-Way COG A 3-Way COG is orthologous between three species.
2-Way COG A 2-way COG is orthologous between two species. .
Synteny A phenomenon where a COG consists of five or more genes.
Aspect Overview
Vector Sandflies are pool feeders — they prick their victim and suck up the resulting pool of blood. They are infected upon ingesting infected macrophages.
Transmission
Life Cycle
Amastigote
Event Overview
Macrophage Amastigotes are within the macrophages.
Ingestion Upon ingestion by the sandfly, amastigotes transform into promastigotes.
Promastigote
Event Overview
Gut The promastigotes in the gut of the female sandfly attach to the midgut-hindgut epithelium where they divide by binary fission for 8-20 days after initial ingestion of blood.
Metacyclic Promastigote
Event Overview
Pharynx A decline in tetrahydrobiopterin triggers metacyclogenesis, whereby the promastigotes move forward to the pharynx.
Blockage Metacyclic promastigotes secrete the promastigote secretory gel (aka PSG) to block the pharynx. The PSG plug forces the stomodeal valve open and extends into the pharynx region. Metacyclic promastigotes are concentrated at the anterior pole of the plug but are found along the foregut in both the cibarium and proboscis.
Feeding When such a sandfly attempts to feed, it must egest the plug by regurgitating a bolus of metacyclic promastigotes — “clearing its throat”. This bolus is injected into the bite wound. There is no infection of the sandfly’s salivary glands.
Reservoir
Life Cycle
Stage Overview
Disease
Leishmaniasis Species Patient Reservoir Vector
Old Localized Cutaneous L. major (moist)
L. tropica (dry)
Human Dog, rodent Phlebotomus spp (Sandfly)
New Cutaneous L. mexicana Human Dog, rodent Phlebotomus spp (Sandfly)
Diffuse Cutaneous L. aethiopica Human Hyrax Phlebotomus spp (Sandfly)
New Dry Cutaneous L. amazonensis Human Dog, rodent. Lutzomyia
Muco-Cutaneous L. braziliensis
L. guyanensis
(both viannia)
Human Dog, rodent. Phlebotomus spp (Sandfly)
Visceral L. donovani Human Human Phlebotomus spp (Sandfly)
Visceral L. infantum None, zoonosis Dogs Phlebotomus spp (Sandfly)
Leishmania/HIV Co-infection is an emerging disease, particularly in southern Europe where 25-70% of adult Visceral Leishmaniasis are related to HIV infection, and 1.5-9.5% of AIDS cases suffer from from newly acquired or reactivated Visceral Leishmaniasis.
Parasite Distribution
L. tropica Urban areas of Mediterranean, Middle East, Pakistan and parts of India.
L. major Rural (esp desert) areas of Central Asia, South Russia, Middle East and Africa.
Parasite Distribution
L. braziliensis
L. mexicana
Central and South America.
L. infantum Africa, Asia and South Europe.
L. chagasi South America
Detection
Technique Overview
Discovery Overview
Kala Azar Kala Azar (aka Black Fever) has existed in India and China for centuries. Kala Azar was considered to be a communicable malaria-like disease that showed relapses, emaciation, as well as enlargement of the liver and spleen, and spread slowly acrossthe continents along the trade routes.
Parasite Discovery In 1900, Scottish Army doctor William Leishman found Leishmania donovani in stained splenic smears from a soldier suffering from a fever contracted at Dum-Dumin, India. Leishman’s observations were published in 1903. At the same time, a Professor of Physiology at Madras University named Charles Donovan described a similar parasite in splenic biopsy smears. The causative agent of Kala Azar was thus found.
Distribution In 1924 the Kala-Azar Commission noted that the distribution of a sandfly (Phlebotomus argentipes) in India tightly overlapped that of Kala Azar.
Sandfly Vector In 1939, Smith, Haldar and Ahmed discovered that if flies were given a blood meal and then disallowed to take additional bloodmeals (though kept alive on raisins), then flagellates grew so numerous that they blocked the pharynx as happens with plague bacilli in fleas. These workers then subjected hamsters to the bite of blocked sandflies and each hamster became infected.
Human Infection, I In 1941, human transmission of leishmaniasis was demonstrated when Adler and Ber successfully infected volunteers with Leishmania major by the bite of Phlebotomus papatasi.
Human Infection, II In 1942, Swaminath, Shortt and Andersen allowed 6 human volunteers to be bitten by infected Phlebotomus argentipes and all developed Kala Azar.
Aspect Overview
Vector Horsefly. However, Trypanosoma evansi has no lifecycle in its vector.
Transmission
Life Cycle
Stage Overview
Disease

Causes Surra in horses and castle.

Stage Overview
Detection
Technique Overview
Discovery Overview
Vinchucas

As early as the Colonial Period, Portugese and Spanish missionaries in Latin America described attacks by vinchucas, biting blood-sucking bugs.

Instead of ordinary bedbugs … these are bugs bigger and more pernicious to the inhabitants … they are as big as the tip of a little finger, long brownish and in the shape of beetles. They live in the ceiling of the houses and get out at night guided by the smell of peopleasleep, and getting down on the beds, bite cruelly, making a big wheal and sucking up to a half a thimble full of blood. While they suck blood they do it with such care and sweetness that it cannot be felt; but when they withdraw full they leave an unbearable pain and itching.
Kissing Bugs Carlos Chagas was sent by Oswaldo Cruz on an antimalarial campaign in Minas Gerais preceding the construction of a rail line there. After a year in Minas Gerais, Chagas was informed by railroad engineer Cantarino Mota of blood-sucking bugs in local huts called barbeiros (kissing bugs) due to their tendency to bite faces of sleeping people. Curious if these bugs transmitted human or animal disease, Chagas studied Panstrongylus megistus, a blood-sucker of the reduviid subfamily triatominae. Chagas found in its hindgut contents numerous flagellates resembling stages of a trypanosome Chagas had described from a marmoset. Chagas sent infected triatomine bugs to Cruz’ medical institute in Rio de Janeiro, where they were allowed to bite monkeys. After 30 days trypanosomes were found in the monkeys’ peripheral blood. Other animals (rabbits, pigs, dogs and other monkeys) were subsequently found susceptible to infection.
Human Blood Chagas was now convinced that Kissing Bugs were also the vector of a human disease. In 1909, two or three weeks after finding Trypanosoma cruzi in triatomines and a cat, Chagas was called to treat a seriously ill 2 year old named Bernice. She suffered from fever, hepatosplenomegaly and lymphadenopathy; her blood teemed with trypanosomes similar to those Chagas had found in the marmoset. He wrote that,

Examination between cover glass and slide revealed the existence of flagellates in good number and fixing and staining of blood films made it possible to characterize the parasite’s morphology and to identify it with Schizotrypanum cruzi.
Correlation By 1911, human disease had been correlated to reduviidae. In that year, Chagas identified dividing Trypanosoma cruzi in heart muscle. In 1912, Chagas found that the armadillo was a reservoir host. The parasite was named after Carlos Chagas’ mentor and employer Oswaldo Cruz. At 29 years of age, Carlos Chagas had described the agent, vectors and animal and human symptoms and existence of a new disease. Unfortunately, Chagas had enemies including Charles Donovan and German microbiologist Krause. Krause denounced Chagas’ findings and his work was forgotten for decades.
Aspect Overview
Agent Trypanosoma rangeli is a protozoan parasite morphologically similar to T. cruzi and with an overlapping geographical distribution. T. rangeli infects humans and other animals but does not have pathogenic activity. However it is devastating to its insect vector R. prolixus.
Vector

Transmitted by the Reduviid (aka kissing bug) genera Rhodnius prolixus which resides in rural settings or forests.

Transmission Rhodnius prolixus discharges Trypanosoma rengali via its saliva whilst taking a blood meal.
Life Cycle
Disease
Detection
Epidemiology
Discovery Overview
Sleeping Sickness Human Sleeping Sickness was observed by Arab doctors as early as 1375. In 1702, English naval surgeon John Atkins described sleepy distemper in Africans living along the Guinea Coast. In 1803, physician Thomas Winterbottom worked in the Sierra Leone colony and published an account of African lethargy. Winterbottom recognized a telltale clinical characteristic: swelling of the cervical lymph nodes.
Human Infection In May 1901, a 42-year-old English laborer on the steamships plying the Gambia River developed a fever. Doctors treated him with quinine to no avail. His blood was examined for malaria parasites but none were found. However, his blood was laced with trypanosomes. In 1902, this trypanosome of humans was named Trypanosoma gambiense by Joseph Everett Dutton of the Liverpool School of Tropical Medicine.
Gambian Epidemic In 1901, a severe epidemic of Sleeping Sickness broke out in Uganda. The Royal Society of London sponsored a commission headed by Bruce to investigate its cause. By 1902 the commission had discovered that the distribution of Winterbottom’s Sign corresponded with the distribution of Sleeping Sickness. Further, Dr. Aldo Castellani examined cerebrospinal fluid from a Sleeping Sickness patient and found trypanosomes. Out of 34 Sleeping Sickness patients, 20 had trypanosomes in their cerebrospinal final; none of 12 control cases had trypanosomes. Bruce suspected that the tse-tse fly transmitted Gambian Sleeping Sickness. When it was found that a tsetse fed on a human patient could transmit the disease to monkeys, it was concluded: Sleeping Sickness is a human tsetse disease.
Discovery Overview
Surra Disease In 1880, Griffith Evans, an English veterinarian in Punjab, India, found trypanosomes in the blood of horses, mules and camels suffering from a fatal wasting disease called surra (aka African Trypanosomiasis today). Inoculating healthy animals with trypanosome-infected blood produced surra; Evans was convinced the trypanosome was a parasite. In 1899, another research identified a biting stable fly as the vector. The trypanosome was later known as Trypanosoma evansi.
Discovery Overview
First Sightings In 1841, G. Valentinin Berne of Switzerland examined trout blood and saw a protozoan propelled by an undulating membrane. This was a trypanosome. In 1843, David Gruby of Paris discovered a similar organism in frog blood and called it Trypanosoma sanguinis. Its etymology is: the Greek word trypano, meaning auger- or screw-like; the Greek word soma, meaning body; and the Latin word sanguinis, meaning blood. Trypanosomes were considered curiosities with no applied importance.
Discovery Overview
Cattle Decimation In the early 1890s, British colonial farmers in Zululand saw their European cattle breed (Nebu) decimated by a wasting disease called Nagana, the Zulu word for depression. The native cattle breed (N’Dama) was unaffected by Nagana.
Identifying Parasite In 1894, Bruce was sent to investigate Nagana. He examined blood from diseased cattle and described a rapidly vibrating protozoan lashing amidst erithrocytes. Bruce successfully used Koch’s Postulates to establish this protozoan parasite as the agent of Nagana.
Epidemiology In 1895, Bruce hypothesized and proved about the Nagana parasite that: wild game were the reservoir; the tsetse fly infesting the same land as the wild game was the vector; and that tsetse fly bites were the mode of transmission. The parasite would later be named Trypanosoma brucei brucei.
Discovery Overview
Rhodesian Trypanosome In 1910, J. W. Stephens of the Liverpool School of Tropical Medicine discovered a new species of trypanosomes: it was from a Sleeping Sickness patient who acquird the disease in 1909 in Rhodesia. Trypanosoma gambiense and its vector Glossina palpalis do not exist in Rhodesia. The Rhodesian Sleeping Sickness was more acute and the parasites had a unique morphology. Stephens called them Trypanosoma rhodesiense.
T. rhodesiense In 1912, Bruce headed a commission near Lake Nyssa to investigate Rhodesian Sleeping Sickness. Bruce found Trypanosoma rhodesiense in the blood of 1/3 of the 180 game animals examined. Bruce compared the morphology of T. rhodesiense with T. brucei from cases of Nagana and found the parasites to be identical. Bruce concluded that T. rhodesiense and T. brucei are responsible for the same disease The tsetse species Glossina morsitans and G. palidipes were found to be the vectors of the East African parasite T. rhodesiense (instead of G. palpalis, G. fuscipes and G. tachinoides for the West African parasite T. brucei brucei).
Aspect Overview
Vector Tsetse fly.
Transmission
Reservoir Wild animals
Life Cycle
Stage Overview
Disease

Causes Nagana in cattle.

Stage Overview
Detection
Technique Overview
Key Points on Trypanosomes’ Flagellum
Gross flagellum structure same in BSF and PCF. There are differences, many of which are not understood.
Flagellum is attached to cell body along its length. This is mediated by well-organized desmosome-like juctions between the membranes of the flagella and parasite.
Direction of motility is toward the flagellum tip. Thisis the result of a tractile flagellar beat that initiates at the tip of the flagellum and migrates to the base.
The flagellum is extracellular at all stages of infection. Therefore, parasite depends on its own motility for dissemination in the host and vector.

What Are the Uses of the Flagellum in Trypanosomes?
Motility This is critical for development and pathogenesis
Host cell attachment
Cell division
Morphogenesis This encompasses cell size, shape and form; organelle positioning and inheritance.
Immune Evasion
Endocytosis and secretion
Sensory organelle
Trypanosomal Flagellum Research
Which genes?
  1. Bioinformatics
  2. Proteomics/mass-spec
  3. Mutant screens
What do they do?
  1. Inducible RNAi
  2. Gene knockouts
  3. Site-directed mutants
How to assemble?
  1. EM: TEM, SEM, CryoET
  2. X-Ray Crystallogray

Trypanosoma brucei Flagellum
Conserved
  1. 9+2 axoneme.
  2. Outer/inner dyneins.
  3. Radial spokes.
  4. Nexin links.
Novel
  1. FAZ (attachment to cell body).
  2. PFR
  3. Stationary central pair.
  4. Expanded gene families.
    1. DRC
    2. Nexins
    3. Tip-to-base repeat

Trypanosoma brucei has a single flagellum 10-20 microns in length that is attached to the subpellicular cytoskeleton along the length of the cell body. The flagellum is always extracellular since the parasite is dependent upon its own motility for migration within the insect and animal host.

Dynein Regulatory Complex The DRC is part of a signal transduction pathway that regulates dynein. Simultaneous dynein activation causes paralysis; dynein activity must be regulated. The only known DRC subunit is trypanin.
Flagellar Attachment Zone FAZ
Dynein Regulatory Complex DRC

Intestinal Protozoan Parasites Giardia lamblia & Entamoeba histolytica

G. lamblia E. histolytica
Excystation Site Triggered by gastric acid. Lower small intestines.
Colonization Site Upper small intestine. Large bowel lumen. Sometimes invades mucosa, liver, lung and brain.
Attachment Adhesive disc. Lectins.
Nutrients Absorbs from host. Phagocytosis of RBCs & tissue bacteria.
Stage Overview
Ingestion Infection is acquired by ingestion of mature cysts
Excystation Cysts pass through the stomach, where gastric acid (low pH) induce them to excystate and emerge in the lower stomach or upper small intestine as a trophozoite.
Colonization The trophozoites colonize the small intestine and are the disease-causing stage.
Adherence The trophozoite adheres to the epithelia via its ventral adhesive disc, and can cause malabsorption from the intestines and diarrhea.
Encystation Trophozoites that migrate to the distal small intestine are triggered by high pH and bile salts to undergo encystation. The resulting cyst is non-dividing and has a rigid cell-wall that allows it to survive outside the host.
Defecation Cysts are passed in the feces of the host and mature and survive externally until they are ingested by another host.
Giardia lamblia Life Cycle

Infection of the host results when cysts are ingested. Trophozoites (growing, motile parasite stage) emerge from the cyst via excystation in the proximal small intestines. Some trophozoites colonize the small intestine, while others travel to distal intestine where they encyst so that they may be defecated. The life cycle is completed when a new host ingests these defecated cysts.

Giardia lamblia Transmission

Person-to-person contact and ingesting contaminated water are the most frequent methods of contracting this fecally transmitted infection. Since Giardia lamblia infects many mammals, mountain and forest streams are often contaminated with cysts defecated by local wildlife. Filtration, boiling or chemical treatment of stream water is critical for preventing Giardia lamblia infection.

Giardia Trophozoite

The Giardia lamblia trophozoite has two nuclei, each with equal DNA content and activate transcription. The trophozoite also has eight flagella in four pairs. Additionally, the Giardia lamblia trophozoite has an axostyle and ventral adhesive disc. It reproduces by binary fission and can undergo encystation to become a cyst.

Excystation

In vivo, occurs in stomach/upper small intestine motile, feeding trophozoite emerges in the small intestines. In vitro, induced by: low pH, as is found in the stomach; or protease treatment at pH 8, mimicking the upper small intestines.

Encystation

In vivo, occurs in lumen of small intestines. Trophozoites undergoes developmental change, synthesizes and exports cyst wall components to its outer surface. In vitro, it is induced by: high pH and high bile salt, mimicking lower small intestines.

Giardia Cyst

The Giardia lamblia cyst has a protective coat that allows the parasite to exist outside the host; this is necessary for transmission. Unlike the trophozoite, it has four nuclei, no visible flagella and stains for cyst-specific proteins. The Giardia lamblia cyst is resistant to cold, chlorine and disinfectants; but is sensitive to heat, freezing and desiccation. The cyst wall is composed of leucine-rich cyst wall protein (CW1-4) and polymers of galactosamine and N-acetylgalactosamine.

Mechanisms of Encystation

Encystation involves synthesis of a cell wall requiring specific secretion of cyst wall components. Encystation-specific secretory vesicles (ESV) are induced at the beginning of encystation. ESVs deliver cyst wall components (CW1 and CW2) to the trophozoite surface. Inhibition of encystation blocks transmission of giardiasis.

VSP & Antigenic Variation
Giardia lamblia’s plasma membrane is covered by Variant Specific Protein (VSP). There are 50-150 different VSP genes encoded in the Giardia lamblia genome. Regulated expression of these genes results in antigenic variation.
Antigenic variation is the ability of a single parasite to change the proteins its presents to the host’s immune system. There is a 30-250 kDA, 25 amino acid membrane spanning region that is conserved; it is cysteine-rich and is laden with CXXC motifs.

Presence and Location of Proteins
Tagging Use recombinant DNA techniques to introduce a tag in the gene of interest. The tag is usually <15 amino acids and inserted into a non-critical domain of the protein. The plasmid with the gene encoding this protein is introduced into the cell and immunofluorescence assays (IFA) using an antibody that specifically recognizes the “tag” are conducted on fixed cells to detect and localize the protein.
Fluorescence Use recombinant DNA to fuse a large naturally fluorescent protein (ie, GFP) to the protein of interest. The plasmid with the gene encoding this fusion protein is introduced into the cell and living cells can be viewed, in real time, to watch movement of the GFP-fusion protein.
Conventional IFA Isolate the protein of interest or its gene and make a recombinant protein. Use the protein to make an antibody that recognizes it and use microscopy on fixed cells to detect the protein.
Immunoblots Cell lysate is fractionated via SDS-PAGE, then electrophoreticaly transferred from gel onto a membrane matrix. The membrane is reacted with an antibody of interest. This verifies protein presence but does not reveal localization.
Applying These Techniques: Antigenic Variation
Antigenic variation was studied via conventional IFA against the VSP TSA417.
Clone A TSA417 plasmid was transfected into E. coli.
Purify Affinity chromatography was used to purify the protein.
Antibody An animal was used to make a TSA417 antibody.
Giardia G. lamblia were chemically fixed onto slides.
Reaction Fixed cells were treated with anti-TSA417 antibody.
Label Fourescent secondary antibody labelled bound cells.
Over 85% of Giardia lamblia are positive for TSA417, indicating the presence of this surface antigen.
Assay For Presence of a Specific RNA
Northern blots and RT-PCR can assay for presence of steady-state RNAs. To examine whether gene expression is being controlled at the transcription level, this procedure is used:
Purify RNA is purified from all stages of in vitro switching.
Separate RNAs are size-fractionated via gel electrophoresis.
Blot RNAs are transferred to a blot and specific RNAs are detected with labelled probes.

Antigen (VSP) Switching Occurs
Induction TSA417+ cells were induced to encyst/excyst.
TSA417? The newly differentiated cells were stained for surface TSA417.
Previously TSA417+ cells become TSA417- upon encystation or excystation. Antigen switching occurred during differentation.
How Is VSP Switching Controlled?

Selective intracellular transport to the cell surface?

  • Does IFA results address this?
  • Do the Immunoblot results address this?

Protein Stability or Production?

  • Rapid, specific degradation of specific VSP proteins?

Selective translation of specific VSP mRNA?

  • RNA Stability or Production?
  • Rapid, specific degradation of specific VSP mRNA?
  • Selective transcription of specific VSP mRNA?

A nature paper published in 2008 that suggests RNAi (post-transcriptional regulation) is used to assure only a single VSP protein is made at any given time 1.

Conclusions on Antigenic Switching, Part I

During encystation/excystation expression of the “old” VSP is turned-off. The newly emerged trophozoite, following encystation/excystation, expresses a “new” VSP on the surface. Antigenic switching occurs during differentiation – upon completion of the life cycle when a parasite is transmitted from one host to another.  Differentiation-associated switching of surface antigens may explain the common occurrence of repeated giardiasis. 

Conclusions on Antigenic Switching, Part II

Previously infected hosts can be re-infected by their own cysts because new variants are not recognized by existing antibodies and can thus recolonize the intestines.  Antigenic Variation allows the parasite to thrive within a community allowing it to reinfect hosts previously infected by other variants.  Enhances the probability of parasites being transmitted to reservoir animals that are partially immune to other VSP variants.

1Prucca et al. (2008) Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature 456: 750-754.
Entamoeba histolytica is the third parasitic killer worldwide. Despite this, it infects many more than it kills. It has a worldwide distribution and lacks a vector, instead transmitting as cysts via fecal-oral route. Poor sanitation leads to contaminated water and a high incidence of this disease. It is diagnosed via microscopy, ELISA and/or PCR. Entamoeba histolytica is a huge single-celled and single-nucleus amitochondriate that colonizes the large intestines and lyses tissues (Giardia lamblia colonizes the small intestines and has two nuclei). It engulfs bacteria and red blood cells for nourishment, and moves using pseudopodia. In place of mitochondria, E. histolytica has intracytoplasmic vacuoles, and also crystalline bodies containing ribonucleoprotein helices.
Entamoeba histolytica secretes proteinases that dissolve host tissues, kills host cells on contact, and engulfs red blood cells. E. histolytica trophozoites also invade the intestinal mucosa, lysing host cells and causing ulcers (amoebic colitis). Amoebas can breach the mucosal barrier and travel through the portal circulation to the liver, where they cause abscesses that are 100% fatal if untreated. Amoebic liver abscesses grow inexorably and, at one time, were almost always fatal, but now even large abscesses can be cured by one dose of antibiotic. A variety of virulence factor are currently being investigated to better define pathogenic mechanisms. Entamoeba histolytica (pathogen) is a distinct species from Entamoeba dispar (a harmless commensal).

Life Cycle of Entamoeba histolytica
Ingestion Cysts are ingested.
Transform Cysts differentiate into trophozoites in stomach.
Division Trophozoite multiplies in small intestine.
Invasion Trophozoites invade the colon.
Some trophozoites encyst in the colon and pass in the feces to continue the cycle. In a small percentage of infections, the trophozoite also invades other organs, such as the liver and brain, and causes abscesses.
Factors Implicated in Pathogenesis
GalNAc lectin Adheres mucin/cells, serum resistance.
Fibronectin/collagen Adheres extracellular matrix receptors.
Cysteine proteinases Invasion through the extracellular matrix.
Amoebapore Lysis of target cells.
Phospholipases Lysis of target cells.
Cytoskeleton Adhesion plates, endocytosis, motility.

GalNAc Lectin: Adherence to Mucin/Cells
What’s the Deal?

Killing of human cells requires adherence of parasite to target cells. Entamoeba histolytica has a lectin protein that binds carbohydrates on the host cell, specifically galactose and N-acetyl-D-galactosamine. Hence, the Entamoeba histolytica lectin is known as GalNAc Lectin.

What Attaches?

Addition of 50mM galactose or N-acetyl-D-galactosamine to host cells in vitro blocked the adherence of the parasite to the host cells. This assay indicated that sugar was involved in adherence. However, it was unclear whether the sugar moieties were on the parasite or host cells.

But Which Has the Sugar?

Host cells were found that had a glycosylation enzyme mutation such that terminal Gal/GalNAc sugars were added to host surface proteins. This strain was resistant to killing by E. histolytica, indicating that the sugars required for parasite-host attachment are on the host.

What’s the Parasite Lectin?
Affinity chromatography identified the protein for adherence:
Starve Cells were starved for methionine.
Label Labelled methionine was added to label all proteins.
Lyse Cells were lysed and run through a Gal/GalNAc column.
Wash Column was washed until no radiolabel came off.
Elute Gal was added in excess to elute bound proteins.
Two proteins were found, which turned out to be the 170 kDa heavy and 35kDa light chains, normally connected as a heterodimer via a disulfide bond. The heavy subunit contains a carbohydrate recognition domain that binds host cell sugars. The C-terminal region is involved in intracellular signaling. Each subunit is encoded by a separate gene, of which there are multiple different copies.
Many Roles of GalNAc Lectin?
From here, mass spectrometry identified the protein sequence and then PCR was used to amplify and clone the gene. With the gene in hand, it can be used to induce overexpression or to knock out the gene in the parasite.
The following functions of GalNAc Lectin were identified:
Adhesion GalNAc Lectin adheres to host cells.
Apoptosis GalNAc Lectin induces apoptosis of host cells.
Virulence Parasite virulence proportional to amount of lectin.
Uptake Mediates uptake of gut bacteria for feeding.
Encystation GalNAc Lectin binding induces encystation in gut.
Antibodies Inducing antibody response → acquired immunity.

Ameobapore: Lysis of Target Cells
Ameobapore proteins are a family of 5kDa (77 amino acid) proteins whose three members have ~50% similarity to each other. Amoebapores form β-barrel pores in membranes that allow the pathogen to lyse the host cells after binding via GalNAc lectins. One amoebapore is specifici to eukaryotic membranes and another to prokaryotic membranes. Multiple types of ameobapores mediate lysis of different cell types (i.e. host cells that are attacked versus bacteria cells that are engulfed for nourishment). Note that the membrane lipids of Entamoeba histolytica are impervious to ameobapore activity.
To study amoebapores, comparisons were made between Entamoeba histolytica and E. dispar. These two species are extremely similar except that Entamoeba dispar is a harmless commensal. These two species may be distinguished via PCR, morphology and lytic activity. RNA silencing can mimic the effect of E. dispar. AmoebaporeA is specific to eukaryotic membranes. It was reduced via RNA silencing to undetectable levels. AmoebaporeA- trophozoites were introduced into SCID mice and caused significantly smaller amebic liver abscesses compared to the parental strain.

Cysteine Proteinases: Invasion Through Extracellular Matrix to Reach Cells
At least six cysteine proteases are expressed in Entamoeba histolytica and none are expressed in E. dispar — this indicates they are likely virulence factors. Some cysteine proteases are secreted and can disrupt a host cell monolayer; CP5 causes host cells to roll up into little balls and detach from the dish.
Comparing the genome sequences of CP5 in E. histolytica and a homologous gene in E. dispar, it seems that E. dispar has accumulated enough mutations in this gene to have a non-functional version.

Metronidazole is marketed as Flagyl and is chemically 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole. It selectively kills anaerobic cells. The only other approved drug for treatment of trichiomoniasis is tinidazole; a strain resistant to one drug is oft resistant to the other. In Trichomonas vaginalis it is activated in the hydrogenosome by ferredoxin; in Entamoeba hystolytica and Giardia lamblia it is activated in the cytosol by other proteins.

Chemotherapy: The Basics

Metronidazole is the main treatment for T. vaginalis. Its derivative tinidazole is so similar that a strain resistant to one drug is oft resistant to the other. In the T. vaginalis hydrogenosome, these drugs are activated when ferredoxin transfers an electron to metronidazole’s NO2 group and a lethal free radical forms. Sensitive parasites lacking hydrogenosomes (Giardia and Entamoeba) activate the drug in the cytosol.

Chemotherapy: Resistance

Drug-resistant parasites were identified by culturing parasites from patients and measuring the mean lethal metronidazole concentration required for killing. Some strains were 10-50x more resistant to metronidazole. What is the basis of resistance?  Are resistant parasites deficient in drug activation? Western blots, northern blots, complements, knockouts, microscopy and centrifugation were all used to answer these two questions.

Western & Northern Blots

Western (protein) and northern (mRNA) blots showed that drug-resistant strains had low levels of the hydrogenosomal protein ferredoxin and the related protein PFO. Other underexpressed proteins included malic enzyme and hydrogenase.

Ferredoxin Knockout

Ferredoxin was knocked out with NeoR plasmids. Restriction enzyme, northern and immunoblot analysis verified ablation of ferredoxin. Other genes were unaffected, except for hydrogenosomal PFO (↓↓) and cytosolic hydrogenase (↑↑).

Minimal Lethal Concentration

The minimal lethal concentration (MLC) of metronidazole was about the same for the parental strain and the ferredoxin knockout. Loss of ferredoxin alone therefore does not cause Trichomonas vaginalis cells to become resistant to metronidazole.

Ferredoxin Complementation

The metronidazole-resistant Trichomonas foetus strain KV1/MR100 was transformed with a ferredoxin plasmid. The minimal lethal concentration of un-transformed and transformed cells was then compared. Tranformation with ferredoxin significantly restored metronidazole sensitivity.

Hydrogenosome Morphology

Sensitive and resistant Trichomonas vaginalis strains underwent microscopy. Hydrogenosomes were equal in quantity across all strains, but were 3-4x smaller in resistant strains. Centrifugation found that resistant strains’ hydrogenosomes were less dense and more homogeneous.

Interpretation

These results indicate that a loss of hydrogenosomal proteins is involved in drug resistance, but it is unclear if this is a cause or effect. Restoring ferredoxin to a resistant strain increases sensitivity; however, knocking out ferredoxin from a sensitive strain does not confer resistance.

Conclusions, Part I

Drug resistant strains lack one or more hydrogenosomal proteins. Highly drug resistant strains lack multiple hydrogenosomal proteins, and their hydogenosomes are smaller, less dense and more homogenous (formed a narrow band upon centrifugation).

Conclusions, Part II

Ferredoxin complementation in a resistant strain confers sensitivity, but knocking out ferredoxin has no effect. Undetected ferredoxin genes may remain; or a ferredoxin- and PFO-independent pathway may exist.

Trichomonas vaginalis Genome

The Trichomonas vaginalis genome is ~160 Mb and haploid. It has over 26,000 genes, with 70% being repetitious. There are seven ferredoxin genes, varying 30-90% in identity to the one knocked out.

MDR Protein: What Is It?

Multi-Drug Resistant Protein (aka MDR or P-glycoprotein) is a ~170 kDa glycoprotein. It is targeted to the plasma membrane, where it inserts twelve transmembrane domains and thereby forms a pore.

MDR Protein: How Does It Work?

MDR can bind and pump out numerous drugs, driving the reaction via ATP hydrolysis using its own ATPase domain. In resistant strains it is overexpresed.

What Is Tetraspanin?

Tetraspanins (aka TSPs) are eukaryotic membrane proteins involved in proliferation, adhesion, migration, fusion and signal transduction. Tetraspanin proteins contain: four transmembrane domains; a short extracellular loop (EC1); a longer extracellular loop (EC2) and short N-terminal and C-terminal tails.

More Information

Extracellular Domain 2 (EC2) contains: a constant region with three α-helices; and a variable region with sites for protein-protein interactions. Making a C-terminal deletion of the cytoplasmic tail produces a dominant-negative mutation. TSP6ΔCt was engineered lacking the 16 last C-terminal amino acids.

What Is Trichomonas vaginalis?

Trichomonas vaginalis is the most common sexually-transmitted protozoan parasite. It is an extracellular parasite, a facultative anaerobe and lacks mitochondria (it is amitochondriate) and peroxisomes. Trichomonas vaginalis has a unique organelle called the hydrogenosome. It resides in the urogenital tract and is transmitted via sexual intercourse. It lacks a cyst stage, meaning it cannot survive outside the host. Its trophozoite has one anterior nucleus, five flagella (four at one end and one along the cell), an axostyle, unique cytoskeletal proteins and a hydrogenosome.

Trichomonas vaginalis Epidemiology

Worldwide there are over 300 million cases of non-viral sexually-transmitted infections reported annually: 170 million by Trichomonas; 89 million by Chlamydia; 62 million by gonorrhea; and 12 million by syphilis. Trichomonas is the most common infectious protozoan in Europe and North America.

Trichomonas vaginalis Symptomology

Trichomonas vaginalis infects both men and women. Symptoms are variable. In men it is often asymptomatic but may cause irritation, prostatitis and urethritis. In women it may cause vaginitis and pregnancy problems (preterm delivery; low birth weight; and increased infant mortality). Long-term untreated infections are associated with infertility in men and women.

Trichomonas vaginalis & HIV

Trichomonas
 vaginalis causes 
punctate
 hemorrhages
. In an HIV-negative person, this gives HIV ready access of white blood cells. A similar mechanism is responsible for increased susceptibility to cervical and prostate cancer. In an HIV-positive person, the hemorrhages leak the virus and causes a higher concentration of HIV in the ifnected area. Epidemiological
 studies
 in 
Africa
 indicate
 a 
2-3 
fold 
increase
 in 
HIV
 transmission 
in
 people 
infected 
with 
Trichomonas vaginalis.



Life Cycle of Trichomonas vaginalis
Diagnostic Stage

Trophozoite is present in vaginal and prostatic secretions and urine.

Division

Multiplies by longitudinal binary fission.

Infective Stage

Trophozoite in vagina or orifice of urethra.


Trophozoite Virulence Factors

Trichomonas vaginalis virulence factors are poorly defined.

  1. Cell-cell and cell-matrix interaction proteins. Trichomonas vaginalis trophozoites are extracellular and must therefore bind host cell surfaces somehow.
  2. Extracellular cysteine proteases and other proteases.
  3. Pore-forming proteins.
  4. Terminal N-acetylglucosamine (GlcNAc) and Galactose (Gal). Trichomonas vaginalis’ surface has a high-density coating of a lipophosphoglycan (LPG) (~2.7×106 per cell).

Is LPG A Virulence Factor? Part I

To determine if LPG is a virulence factor, the adherence and cytotoxicity of Trichomonas vaginalis LPG mutants is assayed.

Mutagenesis Chemical mutagenesis was performed with ethyl methanesulfonate (EMS). Transposons failed.
Selection Incubate mutagenized populations with the lectin ricin. Ricin binds to &beta1,4 galactose residues in LPG. Centrifugation removes agglutinated (LPG+) cells and remaining cells are LPGx
Study LPGx mutants were fluorescently stained with ricin to verify their mutation. Remaining polysaccharides are purified and assayed for adherence and cytotoxicity to vaginal epithelium.
Complement Complementation experiments identify the mutant gene encoding the LPG.

Is LPG A Virulence Factor? Part II

Binding LPG mutants did not bind RCA120 nor wheat germ agglutinin, indicating an inability to bind Gal and GlcNAc, respectively.
Adhesion &
Cytotoxicity

Wild-type and LPG-mutant adhesion/cytotoxicity to vaginal epithelial cells was studied in vitro.

  1. Ectocervical cells grown to ~70-80% confluency as monolayers.
  2. Parasites are labeled with cell-tracker blue and added to ectocervical cells at 37°C and 5% CO2
  3. T. vaginalis infections proceed for indicated time points before analyses.

LPG mutants were dramatically ineffective at adhesion (number of parasites adhered) and cytotoxicity (lactate dehydrogenase release).

OK. LPG Is A Virulence Factor. What Sugars Are Crucial?

Are there host cell receptors that interact with LPG? To answer this question, identify possible host cell lectins that acts as adhesion factors for T. vaginalis, using wild-type and LPG mutant parasites for comparison.

WT LPG In wild-type cells, LPG monosaccharide composition is primarily four sugars: ~30% rhamnose; ~15% xylose; ~25% galactose; ~25% GlcNAc.
LPGx Mut LPG mutants with altered virulence have significantly reduced levels of GlcNAc and galactose, while rhamnose and xylose are largely unaffected.
WT LPG++ Certain wild-type parasites are highly adherent. Their LPG has a high proportion of galactose.

Does Trichomonas vaginalis LPG Bind Galectin-1?

Incubate Incubate galectin-1 with Trichomonas vaginalis in presence of non-competing (sucrose) or competing (lactose) sugars.
Separate Separate and collect unbound galectin-1 by centrifugation (Sample U).
Elute Elute parasite-bound galectin-1 and collect via centrifugation (Sample E).
Parasites Collect remaining parasites (Sample P)
Blot Western blot the three samples and stain with a galectin-1 antibody.

Galectin-1 is present in all three samples incubated with a non-competing sugar. This is because some galectin-1 remained unbound (Sample U), plenty was bound to the parasite (Sample E) and a small amount of bound organisms had remained in the un-eluted portion (Sample P).

When incubated with a competing sugar, galectin-1 is only present in Sample U. This is because some galectin-1 remained unbound (Sample U), and the only binding that did occur was between galectin-1 and lactose; no galectin-1 bound the parasites (Samples E and P).

This experiment was repeated with LPGx mutant Trichomonas vaginalis and these mutants did not bind galectin-1. Furthermore, wild-type LPG can compete parasite binding to LPG (yieleded results as when lactose was used as a competing sugar) but mutant LPG cannot compete parasite binding.

Additional experiments corroborate that host galectin-1 binds to parasite LPG. Increasing galectin-1 levels in host cells increases parasite binding. RNAi knockouts of galectin-1 have hugely reduced parasite binding.


T. vaginalis LPG Conclusions, Part I

LPG is a parasite adherence factor that binds host cell surface galectin-1. LPGx mutants have reduced adherence and cytotoxicity to cervical epithelial cells. LPGx sugar composition has reduced galactose and GlcNAc (glucosamine). Wild-type Trichomonas vaginalis mutants bind host galectin-1 in a sugar-specific manner. LPGwild-type can compete galectin-1 binding to Trichomonas vaginalis, but not LPGx.

T. vaginalis LPG Conclusions, Part II

Binding between the host and the parasite is enhanced when the host cell surface is saturated with galectin-1 capable of dimerizing; this indicates that galectin-1 and LPG cross-link. Binding is reduced when galectin-1 is knocked down via RNAi, but binding is restored when galectin-1 levels are replenished. This demonstrates that parasite LPG adherences to epithelial cells via a carbohydrate-mediated binding to host cell galectin-1


Searching the Genome for Protein Virulence FactorsThe genome is large (~160 Mb) and highly (~65%) repetitive. There are likely ~60,000 genes present. It has 59 repeat families, including retro/transposons and viral-like genes. Of interest are massively expanded gene families. There are 927 protein kinases, 658 BspA-like genes and 328 small GTPases. Moderate expansion is also common, including a family of 24 SAPLIPs.
SAPLIPs & HemolysisSome Trichomonas vaginalis strains are hemolytic while others are not. Trichomonas vaginalis Strain G3 lyses ~60% of RBC in 90 minutes, while Strain SS22 is non-hemolytic. Examining SAPLIP expression was informative: hemolytic Strain G3 upregulated SAPLIP 10B by a magnitude of ~20, while non-hemolytic Strain SS22 did not upregulate SAPLIP 10B (it ↑↑ SAPLIP 12 instead).

Any Surface Proteins? Tetraspanins!
Biotinylation Surface proteins were biotinylated. Biotin can’t permeate the membrane and thus labels surface proteins. Biotin binds terminal amine groups.
Washing Cells were lysed and run over a streptavidin column. Straptavidin binds biotin tightly and specifically even amidst harsh washes to remove unbound contaminants.
Elution Proteins bound to biotin (in turn bound to streptatividin) can be eluted by a change in pH.
MudPIT MudPIT separates populations of proteins.
TSP-1,6,8 were found to be highly abundant on pathogenic strain surfaces. In animals, TSP is involved in proliferation, adhesion, migration, fusion and signal transduction.
IFA TSP-1,6,8 were tagged with IFA to very their surface expression pattern.
TSP-1,8 are found on the plasma membrane in microdomains. TSP-6 is a flagellar protein. IFA tagging was repeated in the presence of ectocervical cells. TSP-1 was upregulated after 4 hours. TSP-8 was upregulated after 30 minutes (dropping off after six hours). TSP-6 showed a bimodal curve with expression heightened at 15-30min and again at 4hrs; furthermore, TSP-6 relocalized to intracellular vacuoles from 0.5-1hrs and migrated again to flagella at 3-6hrs. Since adhesion to host cells usually occurs within 30 minutes, TSP-6 is the only one possibly involved.
A Closer Look at TSP-6
A mutant tetraspanin (TSP6ΔCt) was engineered that did not localize to the flagella, and instead targeted the plasma membrane and vacuoles. Localization did not change in presence of host cells. To test if this impacted parasite migration, a matrigel assay was performed.
Parasites Parasites were placed in wells at one end of a matrigel.
Media An extract rich in ECM proteins was placed at the opposite end.
Incubate The matrigel was incubated at 37°C.
Measuring Migrated cells were counted.

Trichomonas vaginalis migration was reduced by ~40% in cells transfected with TSP6ΔCt relative to those transfected with TSP6.

T. vaginalis TSP-6 Conclusions, Part I

Trichomonas vaginalis surface proteins and secreted proteins, such as SAPLIPs, are likely involved in pathogenesis. Studies identified abundant T. vaginalis membrane tetraspanins: two (TSP-1,8) were transmembrane; a third (TSP-6) was mostly flagellar. In the presence of vaginal epithelial cells, TSP-6 migrates from flagella to intracellular vesicles and back to flagella.

T. vaginalis TSP-6 Conclusions, Part II

Truncation of TSP-6’s C-terminus results in loss of flagella localization and a decrease in Trichomonas vaginalis migration — it is likely involved in parasite migration through host cell extracellular matrix. TSP-6’s role in pathogenesis is unclear, but it appears to be involved in sensing host cells, perhaps leading to outside-in signaling, and also in cellular migration.

Apicoplasts Are Organelles & Have Genomes

The apicoplast (aka plastid) is an organelle found exclusively in the apicomplexan phylum. The apicoplast has a 35kb genome and is surrounded by 4 membranes. Although its genome can be isolated and sequenced, the apicoplast itself cannot be isolated. The apicoplast is also known as a plastid, as drugs against chloroplasts and prokaryotes also kill apicomplexan parasites.

Apicoplasts Encode Localized Proteins

Phylogenetics and GFP tagging reveal that nuclear-encoded proteins of apicoplast origin localize to the apicoplast. Thus, localization of apicoplast proteins is regardless of where they are encoded. Apicoplast proteins have three domains: hydrophobic signal sequence; transit peptide; and mature protein domain. Localization requires the signal sequence and transit peptide.

Apicoplast Protein Function

Identifying protein function via proteomics requires isolated organelles. Unfortunately, the apicoplast cannot be isolated. Thus a common sense approach has been used: all the proteins required for fatty acid synthesis were found in the apicoplast; thus, the apicoplast can probably synthesize fatty acids.

Four Apicoplast Membranes

The apicoplast likely became encased in four membranes via a double endosymbiotic event. The chloroplast arose by engulfment of a cyanobacteria by a plant/algae ancestor. An algae was then engulfed by the ancestor of all apicomplexans. Thus an apicoplast organelle arose with four membranes.

What Is It Related To?

The apicoplast genome was sequenced and found to be plastid-like. It encodes rRNA, tRNAs, ribosomal proteins and 5-6 genes related to chloroplast genes. Furthermore, drugs targeting prokaryotic and chloroplast enzymes also kill apicomplexans. Thus, it seems that the apicoplast arose from a chloroplast. Also, nuclear-encoded plastid-like proteins — ie, acyl carrier protein (ACP) or p59 — localize to the apicoplast. The same goes for proteins of plastid origin. But how?

How Does It Localize?

Deletion and restoration identified an evolutionarily conserved apicoplast targeting signal at the N-terminus. Known as the ACP, this bipartite signal contains: a signal sequence to engage secretion; and a plastid-targeting domain to target the plastid. Fusing GFP to ACP targeted it the plastid. Analyzing numerous ACPs led to a consensus amino acid sequence. All translated proteins were screened for this motif at their N-terminus. This identifies putative apicoplast proteins.

So How Did It Arise?

Nuclear genes, morphology, biochemistry and pharmacology places apicomplexans with ciliates and dinoflagellates. However, presence of plastids suggest placement with plants and algae. This paradox is resolved by two stage lateral genetic transfer: an ancestral plant/algae cell engulfed a cyanobacteria; this evolved into a free-living algae; this algae was engulfed by an ancestral apicomplexan. Plastids and mitochondria arose via engulfment of eubacteria by eukaryotes.

engulfment eubacteria algae eukaryote chloroplast apicoplast apicomplexan

What Is Toxoplasma?

Toxoplasma gondii is an obligate intracellular parasite that grows and divides inside a host cell and has no extracellular lifecycle. Toxoplasma gondii’s importance rests upon its worldwide distribution and ability to infect almost any mammalian or avian cell. Felines (where its sexual stage occurs) are Toxoplasma gondii’s definitive host; it lacks a vector.

Toxoplasma Sexual Stage

The sexual stage allows for the creation of diversity and possibly more virulent strains. Toxo undergoes an asexual stage in humans with 2 forms: 1) tachyzoite (which is similar to but not exactly the same as the trophozoite so don’t mix these up) 2) bradyzoite (which is a tissue cyst similar but again not the same as the cysts formed by Giardia and Entamoeba).

Toxoplasma Genome

Toxoplasma gondii has a haploid genome, making it amenable to loss of function mutations (achievable by chemical mutagenesis). Toxoplasma gondii can also undergo homologous recombination, making possible full knockouts of certain genes. The Toxoplasma gondii genome has been sequenced, making gene identification easier.

Contracting Toxoplasma

Humans can contract this by eating under cooked meat or cat feces. Toxoplasma gondii has a worldwide distribute, with 15-30% of Americans infected and over 70% of Europeans. Many people are already infected but because they are healthy they are asymptomatic. Toxo cause a lifelong chronic infection that is never cleared. Toxoplasma gondii only causes serious problems in immunocompromised individuals and unborn children when their mother gets a primary infection. It is the leading cause of death among AIDS patients. As an apicomplexan parasite, Toxoplasma gondii is often used as a model system because it is much easier to work with than other apicomplexan parasites such as plasmodium.

Toxoplasma Organelles

Apicoplast or plastid which has some features in common with the chloroplast and like the mitochondria and chloroplast it originated from an endosymbiotic event. The important organelles to know are the micronemes, the rhoptries, and the dense granules as these all appear to be secretory organelles that originated from the golgi (not from endosymbiosis) and they are all involved in host cell invasion. Conoid is a microtubule based tip on the parasite body (don’t worry about this one too much). Another structure to keep in mind is the inner membrane complex which is also involved in host cell invasion.


Toxoplasma gondii Life Cycle
Humans and other animals are intermediate hosts. Cats are the definitive host as they harbor the Toxoplasma gondii sexual cycle.
Release Cat releases infective oocysts.
Ingestion Intermediate host ingests infective oocysts.
Differentiation Infective oocysts becomes a tachyzoites.
Invasion Tachyzoites may invade host cells.
Dormancy Tachyzoites may become dormant pseudocysts containing bradyzoites.
Pseudocysts Tazhyzoites may become pseudocysts activated during an immune system breakdown.
Where Does It Divide?
Toxoplasma gondii can invade many tissues, including muscle, brain, eye and intestinal epithelium, and is eve found free in the blood in heavy infections. Each tachyzoite forms an independent parasitophorous vacuole inside the host cell. Inside this vacuole, the parasite divides mitotically (via endodyogeny) with up to 64 parasites within a single vacuole.
When Does It Divide?
Unabated tachyzoite replication bursts the cell and releases tachyzoites that can infect new host cells. An active immune response induces a halt in division and the parasite encases itself in a membrane to become a pseudocyst. Within a pseudocyst are dormant bradyzoites that can survive for decades. They reactive when the immune system is compromised.
When Does Toxoplasmosis Occur?
Toxoplasmosis arises in immunosuppressed patients (cancer, transplants, HIV) and also in fetuses when the mother becomes infected during pregnancy. Congenital toxoplasmosis causes hydrocephalus, chorioretinitis, cerebral calcification, seizures and severe developmental delays.

How Does Toxoplasma gondii Invade the Host Cell?

As an intracellular parasite, Toxoplasma gondii must avoid lysosomal destruction. It accomplishes this by active invasion into a parasite vacuole that filters out host cell transmembrane proteins which prevents the vacuole from being targeted to the lysosome (this filtering is accomplished by rhoptry proteins). Invasion also involves an actin-based gliding motility which requires the secretion of adhesive proteins from the micronemes

Microneme

Micronemes secrete adhesive proteins that anchor in the parasite plasma membrane. The adhesive proteins attach to the host plasma membrane to mediate the initial attachment of the parasite to the host cells. Micronemes secrete additional proteins which bind the parasite’s actin-myosin motor through a parasite protein called aldolase. This connection drives motility/invasion.

Rhoptry

Rhoptry proteins form the moving junction, which is the ring that forms around the parasite cell through which the parasite squeezes into the host cell. Rhoptry proteins also act a molecular sieve to filter out host cell transmembrane proteins from the parasite vacuole to keep the vacuole from being targeted to the lysosome.

Dense Granule

Dense granules are involved in remodeling the parasite vacuole so the parasite can live, grow and divide within it.


A Closer Look: Microneme
Micronemes fuse with the parasite plasma membrane to release microneme proteins onto the parasite's surface. At this point the microneme proteins can interact with the host cell. These are transient surface proteins that only exist on the surface during invasion. The release of microneme proteins is regulated by calcium. This was identified by treatment with a calcium ionophore and a separate treatment with a calcium chelator; supernatant (secreted proteins) was compared to cell lysate (total protein) using SDS-PAGE and immunoblots.
Ionophore Treatment with a calcium ionophore caused an increase in microneme proteins in the supernatant. Thus, an increase in secretion occurred.
Chelator Treatment with a calcium chelator caused a decrease in microneme proteins in the supernatant. Thus, a decrease in secretion occurred.

Microneme proteins attach to the host cell, and also to the parasite’s actin-myosin motor. Myosin is anchored in the inner membrane complex and drives host cell invasion by the parasite. Aldolase mediates the connection between microneme proteins and the actin-myosin motor. As the parasite invades the host cell, a protease cleaves microneme adhesion proteins. This releases them from the parasite plasma membrane. Microneme protein, aldolase and protease activity is collectively the glideosome.

A Closer Look: Rhoptry Body Proteins (ROPs)
Rhopty Body Proteins (ROPs) modulate host cell function. They are found in the bulbous body of the rhoptries. Once secreted into the host cell, they are localized to specific areas (ie, the nucleus) where they exert their function. This is determined via a microarray experiment.
Infected Human cells are cultured with T. gondii.
Uninfected Another batch of human cells are cultured alone.
Isolate mRNA mRNA is isolated from each cultures.
Make cDNA cDNA is prepared from the mRNA by adding fluorescent nucleotides. The infected sample may use red nucleotides and the uninfected sample may use green nucleotides. It is important to use differently colored nucleotides.
Hybridization cDNA from both samples is hybridized to a microarray chip containing every human gene.

A yellow spot means the gene is expressed equally in both infected and uninfected cells. A red spot means the gene is induced in infected cells. If the spot is green then the gene is repressed in infected cells. Typical of a general response, proinflammatory genes are upregulated. Some other changes were induced by Toxoplasma gondii infection but they were unremarkable.


A Closer Look: Rhoptry Neck Proteins (RONs)
Rhoptry Neck Proteins (RONs) are involved in formation of the moving junction, and are a molecular sieve that removes host cell transmembrane proteins from the parasite vacuole. The moving junction is the interface between the host and parasite cells during invasion. This same moving junction and sieve activity occurs in Plasmodium as well. RONs are found in the narrow neck of the rhoptries. When rhoptries were first discovered, they were isolated and then a proteomics approach (much like that used for hydrogenosomal proteins) was used to determine their function.
Separation First the protein contents of the rhoptries were separated out on a gel via SDS-PAGE.
Isolation Each band of protein was cut out of the gel.
Digestion Proteins were digested with trypsin.
Specrometry Mass spectrometry determined each protein’s amino acid sequence.
Analysis The sequences were analyzed to determine the function of each protein.
Micronemes: Perforin-Like Proteins
Toxoplasma gondii contains a perforin-like protein localized to the micronemes. Perforin-like proteins are involved in pore formation. Researchers thought that perhaps this protein is involved in parasite egress (bursting) from the host cell.
Knockout studies revealed that perforin-like protein was not essential for egression in vitro. However, egression was slower in its absence. When induced by calcium to egress, wild-type parasites took ~2 minutes while mutants took ~20 minutes.
However, in vivo this perforin-like protein is essential. The delay in bursting out of the host cell makes the parasite vulnerable to the host immune system, and infection does not take hold.

Can Toxoplasma gondii Proteins Access the Host Cell?
This method reveals the location of T. gondii proteins:
Cyto D Cyto D disrupts actin, stopping invading parasites.
Antibodies A fluorescent antibody for the protein of interest will reveal if its present in the host cell.
Rhoptry proteins access the host cell via evacuoles. One rhoptry protein is targeted to the nucleus. Also, a rhoptry kinase (ROP16) is secreted to the host cell nucleus and modulates host STAT3 signaling and IL12 production. Some ROP proteins secreted into the host cell likely modulate host cell pathways.
So They Do. But How?
It is unclear how parasite proteins get inside the host cell — the current model is called kiss and spit. In this model, the parasite breaches the host cell membrane and injects proteins into the host cell. There is a spike in conductivity before invasion, indicating a possible breaching of the host cell.
Infects Humans
Organism Disease
Plasmodium Malaria
Toxoplasma Toxoplasmosis
Cryptosporidium Cryptosporidiosis
Infects Animals
Organism Disease
Eimeria Coccidiosis
Theileria Theileriosis
Babesia Babesiosis
Neospora Neosporosis
Apicomplexans Have Three Genomes
Genome Overview
Mitochondrial 6 kb tandem repeat; CO1, COII, Cyb; Fragmented rRNA
Nuclear 11 chromosomes; 80 x 106 bp haploid; Map unit 150-300 kb
Apicoplast 35 kb genome; Plastid like rRNA; rpoB, tufA, clpC
Apicomplexans Have Apically Specialized Organelles
Organelle Purpose Overview
Polar Ring Microtubules
Conoid
Organelle Purpose Overview
Micronemes Invasion Cell attachment
Rhoptries Invasion Vacuole formation
Dense Granules Invasion Modify vacuole
Secretory/Exocytic Organelles

The apical complex consists of four organelles: micronemes; rhoptries; dense granules; and conoid. The former three are needed for host cell invasion, whereby each organelle sequentially discharges its contents.

Micronemes Mediate cell attachment (T0-15s).
Rhoptries Involved in vacuole formation (T20-50s).
Dense Granules Modifies intracellular vacuole (T60-1200s).
apicomplexan organelles diagram
What Is Malaria?

Malaria is the world’s most deadly parasitic disease. Malaria’s agent is the obligate intracellular opportunistic apicomplexan Plasmodium. The vector for Plasmodium is the female anopheles mosquito (only females take blood meals). Plasmodium vivax and Plasmodium falciparum cause approximately 95% of malaria cases.

Where Is Malaria?

Malaria is found through-out the subtropics and tropics. Malaria was also once prevalent in the Missippissi delta, but was eradicated in the 1950s by DDT (insecticide) and advanced hygiene. About ½ of the world’s population lives in endemic areas. There are 300 million people infected with malaria.

It’s An Apicomplexan

Plasmodium has the characteristic apicomplexan organelles: an apicoplast; micronemes; rhoptries; dense granules; and the conoid. Occurring via the same pathway as in Toxoplasma, invasion is a coordinated effort of micronemes, rhoptries and dense granules, ending in a vacuole that does not fuse with the lysosome.

Species Pathogens Distribution
Plasmodium vivas Benign tertian Most widespread. Occurs in the tropics, subtropics and warm temperate regions.
Plasmodium ovale Bening tertian The tropics of Asia, West Africa, South Africa.
Plasmodium malariae Benign quartan Rare. Spottily found in subtropics and warm temperate regions.
Plasmodium falciparum Malignant tertian Tropics and subtropics.

Life Cycle, Part I: Infection
Infection Sporozoite enters human host via blood meal.
Travel The sporozoite travels to the liver.
Merozoite Sporozoite transforms into the merozoite form.
Invasion Merozoite invades a red blood cell.
Life Cycle, Part II: Erythrocyte Stage
Ring Stage Merozoite has a ring-like DNA structure.
Trophozoite Merozoite has grown into a large blob.
Schizont Merozoite has divided into dot-like merozoites and gametocytes. Gametocytes are macro or male.
Burst Merozoites and gametocytes burst from the cell.
During this cycle the parasite forms a food vacuole which is essential for its survival as it is required to breakdown host cell proteins and release free amino acids for use by the parasite.
Life Cycle, Part IIIA: Uptake by Mosquito
Uptake Gametocytes are take up by a new mosquito.
Life Cycle, Part II: Erythrocyte Stage

How Is Hemoglobin Digested?

The parasite primarily breaks down hemoglobin, a process which requires falcipan and plasmepsins I and II. These enzymes are specific to and required by the parasite, making them theoretically good drug targets. Sadly, many similar genes are able to compensate for each other’s loss — no drug was general enough to target all these genes whilst remaining specific enough to be harmlesst.

Finding the Enzymes

Biochemical, genome and proteomic approaches were used to characterize the specific proteases required for free amino acid generation (falcipan and the plasmepsins). One approach was to overexpress one of these Plasmodium genes in E. coli to make milligrams of the protein. Highly concentrated protein was induced to crystallize. FInally, X-ray crystallography determined the structure.

Heme → Hemozoin

When hemoglobin is digested, the toxic heme groups released are polymerized into inactive hemozoin. Chloroquine blocks the formation of hemozoin and the free heme that accumulates is toxic to the parasite. Most malaria is now chloroquine-resistant, however. Resistance arose when government of endemic countries fed the drug to the people in quantities insufficient for eradication but sufficient for selection.


Vaccines

Targets:
(1) Sporozoite – designed to prevent infection
(2) Merozoite – designed to reduce severe & complicated manifestations
(3) Gametocyte – designed to arrest the development of parasite in mosquito – transmission blocking

Picture 3.png
Picture 2.png
Picture 1.png

Major problems:
•  highly reproductive organisms
•  high levels of antigenic variability between strains & species
•  high mutation rate of genes encoding surface proteins
•  antigenic variation programmed in parasite

Invasion of Plasmodium is similar to Toxoplasma: Apical organelles are essential;  parasite resides in vacuole that does not fuse with lysosome. Malaria, like Toxoplasma, requires the coordinated discharge of 3 organelles derived from the secretory pathway – micronemes, rhoptries & dense granules to invade heptacytes (sporozoites) and red blood cells (merozoites).

Infected Red Blood Cells – I-RBC

Gametocytes released in blood are transmitted to new mosquito intracellular parasites breakdown host cell proteins to obtain free amino acids

Plasmodium trophozoite (green) inside the red blood cell (gray) forms a food vacuole (also called a digestive vacuole) (red) that is required for survival. RBC proteins digested in food vacuole.

Picture 4
Picture 5

Identify the specific proteases required for free amino acid generation – biochemistry and/or genomics, proteomics
Overexpress this Plasmodium gene in E. coli to make milligram Quantities – problem with T-A richness of malaria genes
Using high concentrations of protein promote it to form crystals
Determine the structure of the crystallized protein using X-ray Crystallograpy.

Food vacuole proteases purified and X-ray crystal structures determined
Ribbon structure of food vacuole protease- Plasmepsin II – drug target?
Good candidate – it’s active site is sufficiently different from similar proteases in humans

Polymerization of heme (ferriprotoporyphin) into insoluble hemozoin “malaria pigment”

Anti-Malarial Drugs

Quinones – Chloroquine, Quinine, Mefloquine
Fansidar (Pyrimethamine + Sulfadoxine)
Atovaquone
Artemisinin – current drug of choice – resistance arising?
BIG Problem: Drug Resistance to Multiple Drugs spread through-out Asia, Africa and South America — and much faster, too. Deaths caused by malaria on the rise due to spread of multi-drug resistant parasites.

Pathological Conditions Associated with Malaria
•  Hemolytic anemia
•  Splenomegaly & hepatomegaly
•  Renal damage
•  Cerebral damage
•  Immunosupression

Susceptibility to Malaria

•  Blacks less susceptible than whites
Duffy blood group antigen – receptor for P. vivax
•  People with sickle-cell trait less susceptible

Plasmodium can hide in cells (liver or RBC) and reactivate later

Tag Cloud