Listeria monocytogenes and Shigella
Listeria monocytogenes vs Shigella diseases..
Listeriosis usually from ingesting contaminated food, causes mild gastroenteritis but can cause sepsis in immuno-compromised people.
Dysentery (Shigella dysenteriae) from accidental fecal-oral from infected person, causes bloody diarrhea.
Identify Mycobacterium tuberculosis by..
acid-fast stain of sputum, IFN-gamma release assay, qPCR, Tuberculin skin test
Yersinia enterolitica and Yersinia pseudotuberculosis
Cause acute gastroenteritis, spread via contaminated food/water, Inv (invasin) binds directly to B1 integrins.
Rickettsiae rickettsi disease and vector vs Rickettsiae prowazekii
Prowazekii transmitted via flea bite and causes epidemic typhus
Both invade endothelial cells that line blood vessels.
How do obligate intracellular pathogens like Chlamydia and Rickettsia differ from E. coli?
All the pathogens are Gram-negative but Chlamydia and Rickettsia have much smaller genomes compared to E. coli and lack key metabolic pathways, so they have to use host's nutrients instead.
How does the motility of spirochetes compare to the motility of other flagellated bacteria like E. coli?
Flagella are a PAMP that are located extracellularly can be recognized by TLR-5. Spirochetes have periplasmic flagella with screw-like motility that evades the immune system.
Legionella virulent factors to survive inside host cells:
Dot/Icm or Type IV SS: expressed after invasion, recruits ER vesicles and adds layers of membrane around the ingested bacteria so that an ER-like vacuole forms (where the bacteria replicate in). Requires specific effector proteins to do this, though many of the effector proteins function redundantly.
In what ways are fungal pathogens different from mammalian cells? How does this affect what processes can be targeted with anti-fungal drugs?
Fungal pathogens have cell membranes composed of ergosterol which is similar to cholesterol that’s found in humans. Anti-fungal drugs are restricted to carbohydrates found in the cell wall. Fungal pathogens have cell walls with chitin and glucans. Antifungal drugs can target beta-glucan synthesis and ergosterol synthesis, but because fungi are otherwise similar to mammalian cells, this limits the number of processes that can be targeted with antifungal drugs as compared to bacteria.
Western blot:
Gentamycin assay to measure invasion of cultured human cells in the lab by a bacterial pathogen
T3SS function
Gram-negative bacteria, the T3SS needle tip binds to receptors on M cells and secrete effector proteins. Effector proteins stimulate uptake by non-phagocytic epithelial cells by modulating the actin cytoskeleton.
Specialized protein secretion system that injects specific “effector” proteins directly from the bacterial pathogen into the cytoplasm of a host eukaryotic cell.
Shigella vs E. coli Similarities and Differences:
Closely related to E. coli gram-negative rod. Both chromosomes are syntenous (genes in same order). E. coli and Salmonella are motile compared to Shigella who is non-motile outside of cells (lacks flagella).
Shigella has lost some virulence genes compared to E. coli genome due to reductive evolution and has a large viral chromosome and has 2 pathogenicity cells. Shigella can only survive and reproduce intracellularly.
Method for monitoring phagosome maturation..
Using fluorescently marked bacteria add Ab against late endosomal or lysosomal proteins, observe through fluorescent microscopy
Yersinia pestis factors required for survival in fleas:
hms expopolysaccharide (on linear chromosome): biofilm formation in flea midgut, EPS clumps and binds heme which allows for proventriculus colonization, flea that is blocked with biofilm blocks ingestion of blood and causes flea to throw up and regurgitate infected blood into new host.
Compare and contrast Rickettsia and Listeria. What are the similarities and differences between these intracellular pathogens?
Similarities: both Listeria and Rickettsia can escape the phagosome to replicate in the cytoplasm of host cells and use actin-based motility to move inside of cells and spread from cell to cell.
Differences: Listeria infect epithelial cells in GI tract and Rickettsia infects endothelial cells lining blood vessels. Listeria is transmitted by eating contaminated foods while Rickettsia is transmitted by insect bite. Listeria is Gram-positive, Rickettsia is Gram-negative. Listeria only uses actin based motility to spread from cell to cells. Rickettsia also use additional protein Sca4 that disrupts tight junctions between cells for spreading from cell to cell.
What is the infectious form, and the replicative form?
Elementary bodies (Eb): infectious form, have stronger membranes due to cross-linked disulfide bonds and are smaller than reticulate bodies.
Reticulate bodies (Rb): replicative form, weaker membranes due to no cross-links, larger than Eb.
Unique features of Borrelia genome vs E. coli genome
E. coli has one circular chromosome wherease Borrelia species have one linear chromosome with closed ends and multiple circular plasmids. The ends of the linear chromosomal and plasmid DNA have hairpin telomeres, where the 5’ and 3’ ends of the two DNA strands are joined together. This stabilizes the linear DNA, protecting it from degradation by nucleases.
Legionella Dot/Icm effector proteins:
manipulate Rab1 GTPase. SidM (GEF) activates Rab1 GTPase that’s involved in recruiting ER vesicles, LidA binds Rab1 GTPase on the Legionella vacuole membrane to retain Rab1 on the Legionella vacuole.
What are three main classes of anti-fungal drugs and how do they kill or inhibit growth of fungi?
Azoles (fluconazole): targets ergosterol by inhibiting Erg11 in ergosterol synthesis pathway, forms toxic ergosterol.
Polyenes: targets ergosterol by forming a “sponge” around the cell membrane by directly binding to the cell membrane, but it can be toxic because it can also bind cholesterol.
Echinocandins: targets enzyme B-glucan synthase, induces cell wall stress response.
Epidemiology methods: RFLP analysis with pulsed-field gels vs. SNP typing
RFLP analysis:
Immuno-fluorescence microscopy assay to measure invasion of cultured human cells in the lab by a bacterial pathogen
What virulence factors does Shigella require to invade host epithelial cells? What virulence factors does Shigella require to spread from cell to cell?
IPaBCD form the tip of the TSS needle are needed to invade. To spread between cells, Shigella needs IpaBC to escape from the phagosome, and IcsA localizes to one pole of the bacterial cell and polymerizes actin behind the bacteria.
Granulomas are composed of...
Infected macrophages surrounded by T cells that are surrounded by fibroblasts.
plasminogen activator (Pla) located on pPLP1 plasmid: targets mammalian plasminogen which activates plasmin, activates a protease cascade that degrades skin associated proteins via MMP proteins and allows for infection to spread to lymphatic tissues.
F1 fimbriae – encoded on pFra. Act like a capsule to prevent opsonization and phagocytosis at the bite site.
Rickettsia entry and spread
OmpA and other adhesin proteins -> initial adhesion to membrane -> lyses phagolysosome via phospholipases -> RicA induces cell to cell spread and Sca4 cause disruption of tight junctions.
Describe the replication cycle of Chlamydia trachomatis.
Eb invade cell via endocytosis and enter as Eb (infectious form), a vacuole forms in the cell and Eb differentiate into Rb and replicate inside vacuole created by MDMP and sphingomyelin. The cells then exit vacuole and differentiate back into Eb, exit cell via lysis or endocytosis where the inclusion (vacuole) fuses with the PM.
How does spirochetes flagella affect immune evasion?
Flagella are a PAMP that are located extracellularly can be recognized by TLR-5. Spirochetes have periplasmic flagella with screw-like motility that evades the immune system.
Describe one molecular mechanism by which fungal pathogens can evolve to become resistant to anti-fungal drugs.
Duplication of parts of certain chromosome arms (aneuploidy). The chromosomes that are amplified have genes related to resistance to the drug (azoles duplication one arm of chromosome 5 increases copy number of Erg11 and also increases expression of a transcription factor that up-regulates drug-efflux.
Bead agglutination assay:
Methods for determining the function of secreted effector proteins of a T3SS
What virulence factor is responsible for the increased virulence of Shigella dysenteriae compared to other Shigella species? What conditions can induce expression of this virulence factor? What host cells does it target? How can this virulence factor be acquired by other bacteria?
Shiga toxin (STX) encoded on a bacteriophage. Antibiotics induce expression by causing damage to DNA, activates RecA and removes that phage repressor. Targets epithelial cells in intestine to cause bloody diarrhea and endothelial cells in kidney, leading to hemolytic uremic syndrome (blood in urine). STX acquired by other bacteria via infection with lysogenic bacteriophage that carries the toxin gene.
B subunit binds Gb3 glycosphingolipid found on intestinal epithelial cells and kidney endothelial cells. A subunit depurinates (removes an adenine) from 28S rRNA and inhibits protein synthesis leading to cell death.
How does M. tuberculosis prevent phagosome-lysosome maturation fusion in macrophages?
PtpA is a phosphatase that removes a VPS phosphate, prevents recruitment of V-ATPase complex or buildup of V-ATPase on phagosomal membrane. Prevents acidification of phagosome.
SapM is a phosphatase secreted by SecA2 system that prevents the recruitment of late-endosomal protein Rab7, prevents fusion of lysosome and phagosome. Removes phosphates from PI3P on phagosome membrane.
pYV proteins (TS33 Ysc system; Yops effectors):
YopE: inhibit Rho GTPase activity which are involved in regulating vesicle trafficking and cytoskeletal rearrangement by acting as a GAP that stimulates GTP to GDP-bound (inactive) form. Less actin polymerization means that the cell cannot phagocytose Yersinia.
YopJ: a Ser/Thr acyl-transferase that puts acetyl-group on Ser/Thr residues involved in signal transduction pathway from TLR. Acetylated kinases can't be activated so signal transduction is disrupted. No inflammatory cytokines are produced in response to PAMPs binding TLRs.
YopM: localizes to the nucleus, interacts with 2 host kinase proteins and inhibits production of IL-10 and IL-1B. Affects inflammasome activation (inhibits pyrin activation) and pyroptosis (inhibits Caspace-1).
Describe a genetic technique you could use to express a fluorescent protein in Chlamydia.
Clone FP in a shuttle vector that has the complete chlamydial plasmid (w/Chlamydia ori), and an E.coli ori so that the plasmid can be propagated and transformed into E.coli. Introduce the plasmid into Chlamydia via transformation.
What proteins help Borrelia burgdorferi to evade the complement system and how do they function?
CRASP proteins. Some bind to complement factor H, Factor H-like H proteins, and factor H related proteins. Binding to the factor H related proteins inhibits C5-C5a conversion (recruitment of phagocytes) and targeting factor H and factor H-like H proteins accelerate degradation of C3b so that the bacteria are not opsonized as efficiently (prevents MAC formation).
What four main features fungal pathogens must have to cause disease?
Thermotolerance (ability to grow at 37C), uptake of nutrients found within host for growth, ability to breach innate barriers to infection/exposure to the pathogen (often acquired via inhalation), evade immune response.
General methods for producing site-directed insertional mutants in obligate intracellular pathogens like Chlamydia.
Site-directed mutants can be generated using a suicide plasmid with an antibiotic resistance marker to replace the gene of interest with the antibiotic resistance marker.
What Listeria virulence factors are required for invasion of host cells? How do these proteins trigger uptake? How is expression of these proteins regulated?
Master regulator of virulence PrfA is only translated to protein at high temp (body temp). An RNA hairpin structure blocks access to the ribosome to the ribosome binding site on mRNA at lower temps. At higher temps, the hairpin melts, allowing access of the ribosome to the mRNA to begin translation.
Internalins are a family of adhesin/invasins: receptor binding triggers endocytosis to internalize bacteria. InIA is adherence to epithelial cells, binds to E-cadherin (calcium-dependent cell-cell adhesin). InIB has adherence to many cell types, binds the ubiquitously expressed MET receptor for hepatocyte growth factor.
What is one mechanism of isoniazid resistance in Mycobacterium tuberculosis? Why would a PCR assay be useful to determine if the bacteria are resistant?
Mutation of the catalase enzyme KatG which convert INH-prodrug to its active form.
Mutation in InhA which makes mycolic acids for the cell wall, overexpression prevents inhibition by active form of INH.
Why must Yersinia pestis replicate to high numbers in the blood of rodent hosts?
Fleas only take a small blood meal, so there has to be high numbers to ensure colonization of the flea.
How can mutant strains of Chlamydia be generated?
Site-directed mutants can be generated by using a suicide plasmid with an antibiotic resistance marker to replace the gene of interest w/antibiotic resistance marker. Random mutants can be generated by chemical mutagenesis via treatment w/EMS.
Borrelia burgdorferi and Borrelia hermsii both use antigenic variation of surface proteins to evade antibodies. Which antigen variation system is most similar to PilE antigenic variation in Neisseria gonorrhoeae and why?
The Borrelia burdorferi VlsE antigenic variation system is most similar to PilE antigenic variation in Neisseria gonorrhoeae. Both cases have single expression site that has a promoter and start codon for protein translation, and multiple silent cassettes that don’t encode for the full-length protein—only parts of it. These silent cassettes get copied into the expression site; it can be either the entire silent sequence or small segments of the silent sequence that recombine in, resulting in potentially thousands of different sequence variants. The DNA sequence at the silent sites never changes in the recombination process.
Many fungal pathogens are dimorphic, meaning they have two different physical forms. What are the two forms, generally? What causes the switch between the two forms? For Histoplasma, what virulence factors are involved in triggering this switch and what do they do?
Grow as filamentous molds in their natural environment, inside the host they grow as yeast. Temperature change causes a switch between the two forms.
Drk1 histidine kinase and Ryp1 transcriptional regulator sense changes in temperature, Ryp1 effects production of alpha-glucan.
CRISPR-Cas9 screens:
Use a cell line that expresses Cas9 and transduce the cell line with a plasmid containing sgRNA to target a specific host cell gene. Infect cells expressing sgRNA with the pathogen, extract the plasmids out of the whole cells and sequence the sgRNAs.
What Listeria virulence factors are required for escape from the phagosome? For spread to neighboring cells? Why is replication in the cytoplasm beneficial for Listeria?
Listeriolysin O (LLO) is a pore forming toxin that inserts into the phagosome membrane at acidic pH. PLCs are phospholipases that degrade the phagosome membrane phospholipids.
ActA is required for spread which polymerizes actin behind cell to push cell forward.
Replication in the cytoplasm allows bacteria to avoid all harsh conditions associated with fusion of the phagosome with the lysosome like acid and degradative enzymes. Also allows access to all cytoplasmic nutrients and avoids all parts of immune system located at the mucosal surface like antibodies and complement.
What nutrients does M. tuberculosis use for growth in the host? What metabolic pathways/enzymes does it require to grow using these nutrients?
Host lipids, have no ETC or access to CAC.
Uses beta-oxidation and glyoxylate shunt. Beta-oxidation breaks down lipids to acetyl-CoA units so that it can enter CAC. Isocitrate lyase enzyme in glyoxylate shunt allow for bacteria to bypass steps of CAC -> sugar fermentation
How are clonal strains of Chlamydia carrying the mutation of interest isolated?
Individual mutant strains can be isolated from growing the bacteria on a layer of McCoy cells that are overlayed with soft agar, bacteria released by an infected cell will only be able to infect neighboring cells to create a clear zone.
The agar can contain antibiotics that select for the marker from the suicide plasmid. Bacteria can then be isolated from individual plaques via stabbing into the soft agar with a pipette.
How is the antigenic variation system in the other Borrelia species different?
Borrelia hermsii are different because its Vlp or Vsp genes don’t encode full-length protein, whole silent gene gets recombined into the single expression locus. There are limited number of possibilities of Vsp or Vlp genes expressed on the surface of the bacteria.
What is one important receptor of the innate immune system that recognizes fungal pathogens? What fungal molecule does this receptor recognize? How does Histoplasma capsulatum avoid recognition by this receptor?
Dectin-1, recognizes beta-glucan. Histoplasma produces alpha+beta glucan where alpha-glucan is presented on the outside of beta-glucan to avoid Dectin-1 detection and evade inflammatory response.
Gene analysis via RNA sequencing of host genes in infected vs. uninfected cells or tissues:
capture mRNA using poly-A tails, convert to cDNA sequence. If the cDNA generates lots of sequencing reads at a location, it’s a highly expressed gene.
Tuberculosis vaccine
BCG, attenuated live vaccine, made through serial passaging. Deletion of ESX-L secretion system.
How can complementation of mutant Chlamydia strains be done?
Complementation is done by cloning gene into Chlamydia plasmid then introducing that plasmid by transformation.
Differences between PilE and VlsE systems:
Differences from Neisseria include B. burgdorferi VlsE antigenic variation system does involve homologous DNA sequences but does not require RecA. Neisseria PilE antigenic variation does require RecA and does happen in lab culture conditions.
What are the two dimorphic forms of Coccidioides immitis? How does the form that is found during infection of mammals help these fungi to cause disease?
In the soil it grows as hyphae, after inhalation they differentiate into spherules where endospores form inside, upon lysis thousands of endospores are released and can reform into spherules.
Genetic screen: RNAi
silence gene expression (affects mRNA translation to protein), cells are transfected with lentivirus containing different silenced RNA.
How are Chlamydia infections diagnosed?
Overlay sample of chlamydia cells with layer of McCoy cells. Stain with iodine to detect cytoplasmic inclusion bodies.
RT qPCR with use of primers that are specific for Chlamydia glycogen plasmid.
How is capsule used to diagnose meningitis caused by Cryptococcus? How is this similar to or different from the capsules of bacterial pathogens that can cause meningitis? How does capsule increase the virulence of Cryptococcus?
Cryptococcus capsules are composed of two sugars: GXM, GalXM and mannoproteins. Decreases uptake by phagocytes, increases resistance to stressors encounted in the macrophages phagosome, prevents fungi from being degraded, prevents activation oof the complement system, it masks other surface Ag from detection by Ab and masks cell wall components from detection by innate immune sensors (Dectin-1). The capsule polysaccharide is also less immunogenic compared to other surface molecules.
Cryptococcus capsules are composed of two sugars: GXM, GalXM and mannoproteins. Decreases uptake by phagocytes, increases resistance to stressors encounted in the macrophages phagosome, prevents fungi from being degraded, prevents activation oof the complement system, it masks other surface Ag from detection by Ab and masks cell wall components from detection by innate immune sensors (Dectin-1). The capsule polysaccharide is also less immunogenic compared to other surface molecules.
Transgenic animals:
Analysis of pathogen growth and ability to cause disease in transgenic animals that are lacking genes encoding important components of the immune system.
What effector protein causes actin polymerization?
TARP
What are Crypotococcus titan cells? What are 2 ways that they help Cryptococcus to cause disease?
Titan cells are enlarged yeast cells that have high ploidy (16+ copies of genome) and are too large to be taken up by phagocytes. Titan cells walls are composed of a thickened layer of chitin (replaces beta-glucan), they induce a Th2 type immune response that’s not as effective.