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