"it may be s--- to you, but it is my bread and butter." stanley falkow, ph.d

"It may be s--- to you, but it is my bread and butter." Stanley Falkow, Ph.D.

Genetics of Pathogens

• Life styles of pathogen• Methods to identify genes needed for

virulence• Environmental signals for virulence

gene regulation• Specific Examples

The Main Points of Pathogenesis

• The pathogens are usually one step ahead• The host is one step behind• Why are we not all dead?• We are not all dead nor are all pathogens eliminated?

Bacteria do not have brains and they do not seek you out.

The purpose of a bacterium is to make bacteria.Life does not exist in pure culture.Life does not exist in logarithmic growth-

stationary phase and hungry is the usual state

• Sometimes, the bacteria/virus makes mistakes-too virulent. Influence pandemic-HIV?

• Sometimes, the bacteria/virus is not virulent enough.

Robert Koch (1876)• Koch's postulates

– find microorganism in all cases of the disease, that are absent in healthy animals

– isolate the microorganism from diseased host in pure culture

– infect a healthy animal with the microorganism and get the same disease

– Isolate the same microorganism in pure culture from the infected host

Molecular Koch’s Postulates.

• The phenotype or property under investigation should be associated with pathogenic members of a genus or pathogenic species.

• Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence.

• Reversion of the mutated gene should lead to restoration of pathogenicity: complementation.

• Current molecular pathogenesis research is held to this standard. It is not always possible to satisfy the third rule for technical or physiological reasons.

Pathogen classes: Barriers to genetics?

Extra-cellular +/- toxinE.coliStaphylococcusStreptococcus

Facultative IntracellularListeriaSalmonellaShigellaMycobacterium TB

Obligate IntracellularChlamyidiaRickettsiaMycobacterium leprae

No geneticsEasy!E.coli easyStaph-Strep more difficult

Facultative intracellular: can live inside host cells and outsideObligate intracellular: can live only inside host cellsExtracellular: lives on the surface of the host.

Regulation of cholera toxin production in Vibrio cholerae

-cholera toxin produced at 37°C, physiologic salt 150mM, amino acids, slightly basic pH: basically conditions that would exist in the small intesting -ToxR-ToxS produced at 25-30°C, acid pHAn example of temporal and spatial regulation of regulatory molecules

Cholera toxin is encoded on a phage, called CT-regulation of phage expression is also controlled by the SOS pathway.

Cholerae toxin is encoded on a prophage and is mobile

Cholerae Toxin

Expression of the prophage genes and toxin is controlled by the SOS pathway

Induction of SOS causes induction of the entire phage and cholerae toxin production. LexA is cleaved, the RstR repressor protein comes off, and the phage gene expression is induced.Repression mechanism is similar to lambda phage cII system.

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Shiga toxin is enocode on a lambda-like phage. Production of shiga toxin is controlled by a similar system in lambda phage. Conditions that induce the phage also induce the production of toxin.



Shiga toxin is encoded on a phage, H-19B-E.coli O157:H7 “hamburger E.coli” HUS in humans-production of Shiga toxin regulated, condition that mimic bowel -Antibiotic treatment of people with HUS is lethal!-Antibiotic can induce the phage and also lyse cells filled with toxin 1. Mark phage with Km

2. Treat cells with DNA damage agent mitomycin C

3. Harvest culture supernatants4. Determine titer and transduce Amp

marker

Results: DNA damaging agents induced the phage and toxin production!

Mechanism for horizontal gene transfer

Brute force method



STM: Signature Tagged Mutagenesis





IVET: In vivo expression technology

Results of a lot of mutant hunts

1. Pathogens contain genes not found in their closely related counter parts

2. Genes specific to the pathogens are organized into islands, islet, atolls. That is, specific regions that are unique to the pathogens.

3. Pathogenicity islands encode those functions needed for the pathogen to causes a successful infection. It still needs the rest of the chromosome!

4. Pathogenicity islands have different G+C content than the backbone chromosome. Islands tend to be A+T rich, especially in Salmonella and E.coli.

5. PA’s can encode a specialized secretion apparatus designed to transfer effector proteins into the host. The proteins are specifically designed to alter host cell function. The proteins usually interact with a specific host protein or class of proteins.

6. Some but not all PA’s have inserted in a rare tRNA seltRNA. This insertion event does not interrupt the tRNA, but provides a powerful selection for the PA’s presence.

General structure of a genomic island. -in pathogens, called PA: Pathogenicity Island-usually PAs are inserted into rare tRNA, selenocysteine tRNA-tRNA is sometime duplicated and still functional or a new one is carried on the incoming island-this provides a selection to hold the island in place

Chromosomal Location of Salmonella Pathogenicity Islands

0

Spi-1 Invasion

Spi-5 Unknown

Spi-2 Macrophage Survival

Spi-4 Macrophage Survival?

Spi-3 Macrophage Survival

50

Salmonella Chromosome

Virulence Plasmid120kb

4.9106 Bases

The secretion apparatus may be an old flagella or phage?

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Salmonella Salmonella Pathogenesis - Pathogenesis - INTRODUCTIONINTRODUCTION

• Salmonella is an enteric pathogen capable of infecting a wide variety of vertebrate hosts

• Not all infected hosts exhibit clinical symptoms of Salmonella infection

• Non-typhoidal Salmonella serotypes cause enteritis and occasional systemic infection

• Certain Salmonella serovars exhibit host adaptation but the mechanisms are poorly understood

• Salmonella is a facultative intracellular pathogen adapted to survival in host mononuclear phagocytes

• Genetic tools are available to dissect virtually every aspect of the Salmonella-host interaction

Great Moments in Salmonella History

1. First isolated by Theobald Smith. S. Choleraesuis from porcine intestine around 1885. He worked for Daniel Salmon, who had nothing to do with the work.

2. 1829- P.Ch.A. Louis in Paris separated typhoid from other fevers. 3. 1884-Gaffkey Germany isolated Salmonella Typhi from spleens of

infected patients 4. 1896- first heat killed vaccine by Pfeiffer and Kalle. 5. 1920 to 1940-Kaufman and White developed the serotype

classification of Salmonella 6. 1952-Zinder and Lederberg-discovered genetic transduction using

phage P22 7. 1973-Bruce Ames developed the “Ames Test” to determine

mutagenic activity of chemicals 8. September 1984-Salmonella Typhimurium used to restaurants in

the The Dalles, OR.

“Typhoid Mary” was Mary Mallon

Major Differences between S.Typhi and S.Typhimurium

Salmonella Typhi Salmonella TyphimuriumHost adapted: humans Non-host adaptedSystemic Infection resulting in Rarely extra-intestinal but Enteric fever with little diarrhea usually the cause of diarrheaVaccine available No vaccine availableCarrier state No long-term carrier state

Properties of SlyA1. slyA is found in many Gram-negative bacteria, both

pathogenic and and non-pathogenic2. The sequence is conserved 3. SlyA has been classified in the MarR family of small

molecular weight transcriptional regulatory proteins4. SlyA mutants of Salmonella are profoundly attenuated

for virulence5. SlyA mutants are unable to survive and replicate in host

macrophages6. Originally thought to be a toxin, but later discovered not

to be.

SlyA

mgtBC phoNphoP pmrDsodCImig-5

envEmig-14pagCpagD pagKpagJpgtEugtLvirKycbY

PhoP

ssrABnmpC

STM1249STM1328

STM1498/1499marA

The PhoP and SlyA Regulons Overlap

ydhJ ydhIyggMyhcN

nanAFlagella

Spi-1

PhoP/PhoQ two component regulatory system1. Was identified in the first gene hunt for Salmonella vir

genes2. Is needed for virulence3. Transmits the signal from the host to the bacteria for

survival inside epithelial cells, phagocytes, and in the lumen of intestine

PhoP/PhoQ in Salmonella

Possible mechanisms by which PhoP and SlyA coordinately

control Transcription of target genes:

PhoP regulates the transcription of slyA directly. NO

PhoP and SlyA interact together at target loci. Not formally excluded, but probably not

PhoP controls post translational modification of SlyA or the production of a ligand needed for SlyA activity. Focus of current research

The end

"it may be s--- to you, but it is my bread and butter." stanley falkow, ph.d

Documents

production of toxin

production of shiga

cholerae toxin production

phage expression

shiga toxin reg

entire phage

phage gene expression

vibrio choleraecholera