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).

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

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

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

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

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.

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 1^st 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 Tet^r (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 antibiotic^r 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 Tet^r 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.

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.

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

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.

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 14^th leading cause of death in the world, and are believed to be the 8^th 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

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:

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:

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

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.

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

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

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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.

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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