Transcript March 12 lecture presentation

"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
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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
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Sometimes, the bacteria/virus makes mistakes-too virulent.
Influence pandemic-HIV?
Sometimes, the bacteria/virus is not virulent enough.
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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.
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The phenotype or property under investigation should be
associated with pathogenic members of a genus or pathogenic
species.
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Specific inactivation of the gene(s) associated with the suspected
virulence trait should lead to a measurable loss in pathogenicity
or virulence.
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Reversion of the mutated gene should lead to restoration of
pathogenicity: complementation.
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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 +/- toxin
E.coli
Staphylococcus
Streptococcus
E.coli easy
Staph-Strep
more difficult
Facultative Intracellular
Listeria
Salmonella
Shigella
Mycobacterium TB
Easy!
Obligate Intracellular
Chlamyidia
Rickettsia
Mycobacterium leprae
No genetics
Facultative intracellular: can live inside host cells and outside
Obligate intracellular: can live only inside host cells
Extracellular: 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 pH
An 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
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Mark phage with Km
Treat cells with DNA damage agent
mitomycin C
Harvest culture supernatants
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
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IVET: In vivo expression
technology
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Results of a lot of mutant hunts
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Pathogens contain genes not found in their closely related counter parts
Genes specific to the pathogens are organized into islands, islet, atolls.
That is, specific regions that are unique to the pathogens.
Pathogenicity islands encode those functions needed for the pathogen
to causes a successful infection. It still needs the rest of the
chromosome!
Pathogenicity islands have different G+C content than the backbone
chromosome. Islands tend to be A+T rich, especially in Salmonella and
E.coli.
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.
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
Spi-4 Macrophage Survival?
0
Spi-3 Macrophage Survival
Spi-5 Unknown
Salmonella Chromosome
4.9106 Bases
Spi-1 Invasion
Spi-2 Macrophage Survival
50
Virulence Plasmid
120kb
The secretion apparatus may be an old flagella
or phage?
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Salmonella Pathogenesis - INTRODUCTION
• 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
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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.
1829- P.Ch.A. Louis in Paris separated typhoid from other fevers.
1884-Gaffkey Germany isolated Salmonella Typhi from spleens of infected
patients
1896- first heat killed vaccine by Pfeiffer and Kalle.
1920 to 1940-Kaufman and White developed the serotype classification of
Salmonella
1952-Zinder and Lederberg-discovered genetic transduction using phage
P22
1973-Bruce Ames developed the “Ames Test” to determine mutagenic
activity of chemicals
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
Host adapted: humans
Systemic Infection resulting in
Enteric fever with little diarrhea
Vaccine available
Carrier state
Salmonella Typhimurium
Non-host adapted
Rarely extra-intestinal but
usually the cause of diarrhea
No vaccine available
No long-term carrier state
Properties of SlyA
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slyA is found in many Gram-negative bacteria, both pathogenic and
and non-pathogenic
The sequence is conserved
SlyA has been classified in the MarR family of small molecular
weight transcriptional regulatory proteins
SlyA mutants of Salmonella are profoundly attenuated for virulence
SlyA mutants are unable to survive and replicate in host
macrophages
Originally thought to be a toxin, but later discovered not to be.
The PhoP and SlyA Regulons
Overlap
SlyA
PhoP
nanA
Flagella
Spi-1
mgtBC
phoN
phoP
pmrD
sodCI
mig-5
envE
mig-14
pagC
pagD
pagK
pagJ
pgtE
ugtL
virK
ycbY
ydhJ
ydhI
yggM
yhcN
ssrAB
nmpC
STM1249
STM1328
STM1498/1499
marA
PhoP/PhoQ in Salmonella
PhoP/PhoQ two component regulatory system
1. Was identified in the first gene hunt for Salmonella vir genes
2. Is needed for virulence
3. Transmits the signal from the host to the bacteria for survival inside
epithelial cells, phagocytes, and in the lumen of intestine
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
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SlyA
The end