Mol' Micro'

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Mol. Micro. Pathogenesis March 06,
2009 Dr. Joseph Vogel jvogel_at_borcim.wustl.
edu 314-747-1029
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• Reading assignment for Wed
• Pizzaro-Cerda Cossart. Bacterial Adhesion and
  Entry into Host Cells. Cell. 2006. 124715.
• Kubori T Galan JE. Temporal regulation of
  Salmonella virulence effector function by
  proteasome-dependent protein degradation. Cell.
  2003. 115333-42.

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Review of type I to V secretion systems
Desvaux et al Trends Microbiol. 2004
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• 4 proteins no longer secreted into supernatant
  of the mutant
• Defective infection could be due to
  hypersenstive response to secreted proteins
  (suggests the imps encode an injection system)

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• A Vibrio cholerae protein (VasK) with homology
  to Legionella IcmF was identified as being
  induced in a rabbit model of infection
• (Das S et al. 2002. Biochem Biophys Res Commun.
  295922-8.)
• Identifed a unique gene cluster containing IcmF
  in Vibrio cholerae and other proteobacteria
  through in silico analysis
• (Das S Chaudhuri K. 2003. In Silico Biol.
  3287-300.)
• Called the gene cluster IAHP (IcmF associated
  homologous proteins)

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Legionella pneumophila Dot/Icm type IV secretion
system

• IcmF is a large inner membrane protein
• Inactivation of icmF destabilizes the Lp T4SS
  (assembly factor)
• (Sexton Vogel. 2004. Infection Immunity)

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Type VI secretion systems
Cascales. 2008. EMBO Reports. 9735-741.
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Type VI secretion systems

• Regulation
• Sigma 54 activator -- transcriptional
• PpkA (kinase) -- post-transcriptional
• PppA (phosphatase) -- post-transcriptional
• Fha (forkhead protein) -- post-transcriptional
• Assembly factors
• IcmF and DotU/IcmH and ClpV ATPase
• Secreted substrates
• Hcp (hemolysin co-regulated protein)

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Another secreted substrate is VgrG
(valine-glycine repeat)

• VgrG - needle injection systems?
• N-terminus shares structural features with the
  phage tail of bacteriophage T4
• C-terminus is probably the effector domain (e.g.
  Vibrio cholerae VgrG1 resembles actin
  cross-linking domain of RtxA toxin

Pukatzki S. et.al. PNAS 200710415508-15513
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Type VI secretion systems
Microbiology. 2008 Jun154(Pt 6)1570-83.
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Cell envelope of Mycobacterium tuberculosis
Abdallah et al. 2007. Nature Reviews Micro.
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• ESX or Type Seven Secretion System (T7SS)
• BCG
• Mycobacterium bovis Bacille Calmette-Guerin
• Isolated by prolonged passage of a virulent
  strain of M. bovis
• Tuberculosis vaccination strain
• Protects animals when challenged with a lethal
  dose of virulent tubercle bacilli
• Efficacy for preventing Tb in human adults is
  questionable

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• ESX or Type Seven Secretion System (T7SS)
• BCG
• Continual passage ended up with 38 ORFs lost
• One region is RD1 (region of difference 1)
• RD1 9.5 kb of DNA 9 ORFs (including ESAT-6
  CFP-10)
• Secreted proteins
• ESAT-6 (early secreted antigen target 6 kDa)
• CFP-10 (culture filtrate protein, 10 kDa)
• Two secreted proteins of unknown function
• Originally identified as immunodominant antigens
  of M. tb.
• Both lack an obvious Sec signal sequence
• Maybe Esx is a novel secretion system?

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ESX-1 secretion system (T7SS)
Abdallah et al. 2007. Nature Reviews Micro.

• Key experiments
• BCG complete RD1 -gt restore secretion of
  ESAT-6 increases virul.
• Deletion of RD1 from M. tuberculosis attenuates
  the strain

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• ESAT-6 and CFP-10 interact and are co-dependent
  for stability and secretion
• Last 7 aa of CFP10 interacts with Rv3871
  (ATPase)
• ESX-1 signal sequence is portable, i.e.
  necessary sufficient
• Rv3871 interacts with Rv3870 in the membrane
• Even more EspA, Rv3615c, ESAT-6, and CFP-10
  are also dependent on each other for export

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Gram positive bacteria encode T7SSs
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Terminology Secretion active protein
transport across the bacterial
membrane(s) Translocation transport of
proteins across the host membrane
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Three methods to show translocation 1.
Immunofluoresence (localization of Yops)
YopH (everywhere)
YopE (perinuclear)
YpkA (inner surface of plasma membrane)
yopB yopD (translocation deficient)
Yops are green HeLa cell membrane is
red Cellular Micro Fig 13.5
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Three methods to show translocation 2. Yop-Cya
reporter strategy (Cornelis 1994)
Cornelis G. et al. 1998. Microbiol Mol Biol Rev.
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Three methods to show translocation 3. Assay by
host phosphorylation
Day JB, Ferracci F, Plano GV. Mol Microbiol.
2003. 47(3)807-23. Translocation of YopE and
YopN into eukaryotic cells by Yersinia pestis
yopN, tyeA, sycN, yscB and lcrG deletion mutants
measured using a phosphorylatable peptide tag and
phosphospecific antibodies.
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Adherence
Chaperone/Usher-mediated pilus biogenesis
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EPEC (enteropathogenic E. coli)

• EPEC induces a characteristic lesion called an
  attaching and effacing phenotype
• After intimate attachment to intestinal
  epithelial cells via adhesins (e.g. type IV
  pili), bacteria induce localized effacement of
  absorptive microvilli
• Results in the formation of pedestal-like
  structures on which the bacteria perch
• Locus of enterocyte effacement (LEE) -- encodes
  a T3SS

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Adherence -- EPEC uses a T3SS
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EPEC

• T3SS exports Tir (translocated
  intimin receptor)
• Tir is injected into host cells and inserts into
  membrane where it functions as the receptor for
  the outer membrane protein intimin
• Tir is phosphorylated by host kinases and
  recruits Nck, N-WASP, Arp2/3 complex
• Leads to nucleation of actin and formation of
  pedestals

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Invasion Intracellular Niche
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Why invade?

• Advantages
• immune avoidance
• to cross a mucosal barrier
• dispersal in the host
• to avoid competitors
• gain access to nutrients

Challenges - must survive host defenses at the
new site - route of entry (how to get in) - must
survive defenses within the cell, i.e. avoid or
survive within the lysosomal environment
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Choice of target cell consequences

• 1. Professional phagocytes
• Examples macrophages and PMNs)
• Have many inherent systems for recognition and
  ingestion of microbes
• phagocytosis
• Ingest particles gt 200 nm in size
• Actin dependent
• Involves various signal transduction events
  including tyrosine phosphorylation and small GTP
  protein activation (Rho, Rac, Cdc42)
• 2. Non-professional phagocytes
• Do not have many inherent systems for
  recognition and ingestion of microbes
• Are capable of endocytosis (receptor mediated
  event in which actin plays a secondary role)

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Choice of target cell consequences (professional
vs. non- professional phagocytes)

• 1. Professional phagocytes
• Advantages for the pathogen
• Host does the work
• Doesnt necessarily require dedicated entry
  mechanism on the part of the pathogen
• Disadvantage for the pathogen
• Professional phagocytes are dedicated killers
• Examples of pathogens that replicate in
  professional phagocytes Mycobacterium
  tuberculosis, Listeria monocytogenes, Salmonella
  typhimurium, Histoplasma capsulatum, Toxoplasma

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Choice of target cell consequences (professional
vs. non- professional phagocytes)

• 2. Non-professional phagocytes
• Advantages for the pathogen
• Pathogen does not have to deal with the potent
  anti-microbial systems present in phagocytes
• Disadvantage for the pathogen
• Pathogen must find a way to directly invade or to
  stimulateuptake by the host cell
• Examples of pathogens that replicate in
  non-professional phagocytes
• Salmonella typhimurium, Listeria monocytogenes,
  Yersinia enterocolitica, Toxoplasma gondii,
  Neisseria meningitidis

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Entry systems

• Endocytosis Viral
• Phagocytosis
• Receptor mediated Leishmania
• Lysosome recruitment Trypanosoma cruzi
• Induced Endocytosis
• Zipper Yersinia/Listeria
• Trigger Salmonella/Shigella
• Cell to Cell Spread Listeria/Shigella
• Active Invasion Toxoplasma/Plasmodium

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Triggered vs Zippered Entry
Tight envelopment of the pathogen by the host
cell membrane - via high-affinity binding of a
pathogen surface protein with a
transmembrane receptor on the host cell
Stimulation of cytoskeletal changes underneath
the site of interaction - causes a ruffling like
process that internalizes the pathogen
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Cossart Sansonetti. Science 2004
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Invasion Assays (gentamicin protection)

• Gentamicin
• Inhibits protein synthesis by binding to the L6
  protein of the 50S ribosomal subunit
• Bacteriocidal (not bacteriostatic)
• Membrane impermeant

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Invasion Assays (gentamicin protection)
1. Grow an overnight culture
2. Plate to detect input numbers
3. Infect monolayer
5. Add gentamicin and incubate
x
x
x
x
x
x
4. Incubate (allow time for internalization of
the bacteria)
6. Lyse host cell and plate to determine
of Intracellular bacteria
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Invasion Assays (double immunofluorescence)
1. Grow an overnight culture
4. Stain the extracellular bacteria (e.g. red)
5. Stain the intracellular bacteria (e.g. green)
2. Infect monolayer
3. Incubate (allow time for internalization of
the bacteria)
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• Several examples of invasion
• Yersiniae
• Salmonella
• Listeria

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Example 1 Enteropathogenic Yersiniae

• Y. enterocolitica Y. pseudotuberculosis are
  food borne pathogens
• Primary sites of multiplication are Peyers
  patches and
• mesenteric lymph nodes (MLNs)
• The target cell is the M-cell (Yersiniae invade
  and transcytose in order to cross the
  intestinal epithelial barrier)

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Example 1 Enteropathogenic Yersiniae
Utilize Yersinia protein called invasin to
invade via a zipper mechanism of entry
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Invasin

• Invasin is a large (986aa) outer membrane
  protein (type V)
• An inv mutant shows 1000-fold reduction in
  invasion of host cells
• Invasin is sufficient to promote entry into host
  cells
• (actually just the D4D5 C-terminal domain is
  sufficient but less efficient)
• Receptor for invasin is a subset of b1integrins
• In intestine, only M cells have b1 integrins on
  the luminal surface

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Invasin structure
Ig-like immunoglobulin like-folds
CTLD C-type-lectin-like-domain
Hamburger ZA, Brown MS, Isberg RR, Bjorkman PJ.
Crystal structure of invasin a bacterial
integrin-binding protein. Science. 1999.
286291-5.
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Integrins

• Expressed on the surface of many mammalian cells
  (involved in a variety of activities including
  cell-cell adhesion, cell-matrix adhesion, cell
  signaling, and inflammation)
• Natural ligands are extracellular matrix
  proteins
• (e.g. collagen, fibronectin, and laminin)
• However, these natural ligands bind but are not
  internalized
• (very odd because invasin and fibronectin bind to
  the exact same site on b1 integrins)
• So what makes binding by invasin trigger
  internalization?

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• Invasin binds integrin with 100-fold higher
  affinity
• High affinity binding allows zippering or
  enveloping of the bacteria because the ligand is
  able to recruit so many receptors
• Invasin can also dimerize via D2 domain
• High affinity binding dimerization leads to
  clustering of receptor
• b1 integrin cytoplasmic domain needs to cluster
  in order to signal to cytoskeleton, thus
  inducing internalization

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Invasin mediated uptake

• Players include
• Invasin b1-integrins
• FAK (focal adhesion kinase - tyrosine kinase)
• c-Src, p130Cas, and Crk
• PI-3 kinase
• ARF6
• Rac1 activation
• N-WASP?
• Arp2,3 complex actin

Cossart Sansonetti. Science 2004
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Example 2 Salmonella typhimurium

• Foodborne pathogen, with
• initial site of multiplication
• in the Peyers patch
• Also targets the M-cells
• gt20 bacterial proteins are
• required for the entry process
• Internalized Salmonella
• remains in a vacuole where
• it replicates

Trigger mechanism characterized by massive
ruffles and clustering of F-actin beneath the
entering bacteria
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• SPI-1 T3SS is required for delivery of effector
  molecules that stimulate the signal
  transduction events resulting in the ruffling
  and triggered endocytosis
• Some SPI-1 effector proteins are encoded at
  SPI-1 locus (not all)
• However, all of these genes have features
  suggestive of horizontal acquisition

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How do the Salmonella effectors get the job done?

• SipB and SipC form the translocon/pore for
  secretion of the other effectors into the host
  cell (similar to YopB/YopD/LcrV from Yersinia)
• SipC also initiates actin nucleation via its
  C-terminal domain

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How do the Salmonella effectors get the job done?

• SopE
• Directly binds and activates small GTP binding
  proteins Cdc42 and Rac acting as a GEF (guanine
  nucleotide exchange factor)
• Activated Cdc42 and Rac gt stimulates
  cytoskeleton rearrangements by interacting with
  WASP family proteins that in turn bind and
  activate Arp2/3 complex involved in actin
  nucleation - thereby inducing membrane ruffling

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How do the Salmonella effectors get the job done?

• SopB gt phosphatidylinositol phosphatase
  stimulates actin rearragements indirectly by
  activating Cdc42
• SipA gt stabilizes the actin filaments by
  inhibiting their depolymerization and forms a
  complex with the actin bundling protein of the
  host, plastin

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Jorge Galans Yin and Yang

• Salmonella is internalized by triggering profuse
  membrane ruffling via the action of various T3SS
  substrates
• However following internalization, Salmonella
  needs to restore the normal cellular
  architecture, i.e. stop ruffling
• It does by injecting, SptP, into the host cell
• How does this work? Assigned reading (Kubori T
  Galan JE. Cell. 2003.)

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Example3 Listeria monocytogenes

• In humans, disease is most common in pregnant
  women, newborns, and the immunocompromised
• Typically food borne but can also be transmitted
  to the fetus by the mother
• Infects a wide range of cell types including
  macrophages

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Listeria monocytogenes
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Listeria virulence genes

• Most of the genes are clustered (10 kb region
  of chromosome)
• Region is absent from nonpathogenic Listeria
  species
• The inlAB operon is located in another region
• However all of these genes are under the control
  of the transcriptional regulator PrfA

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Listeria monocytogenes invasion genes (inlAB)
inlAB were identified by screening a library of
transposon mutants of L. monocytogenes for loss
of invasion of Caco-2 cells
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InlA (internalin)

• Expression of inlA alone in L. innocua confers
  invasion of Caco-2 cells
• Latex beads coated with internalin are
  internalized - thus internalin is sufficient to
  confer uptake
• The receptor for internalin was identified by
  affinity chromatography as E-cadherin (human)
• E-cadherin is a transmembrane glycoprotein that
  plays a role in tissue cohesion and architecture
• The intracellular domain interacts with catenins
  which interact with the actin cytoskeleton

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Internalin A mediated entry by Listeria

• Players include
• InlA
• E-cadherin catenins
• Unconventional myosin (myosin VIIa)
• Vezatin (myosin VIIa ligand)
• Rac1 activation
• Arp2,3 complex actin

Cossart Sansonetti. Science 2004
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InlB

• inlB mutants gt InlB plays a role in invasion of
  some cells (hepatocyte-like cell lines, HeLa,
  Vero, CHO cells fibroblasts, not Caco-2 cells)
• inlB mutants do not replicate as well as WT in
  mouse liver
• InlB
• Found on the bacterial surface and in culture
  sup
• Has LRR repeats (loosely attaches to cell
  surface by C-terminal repeats)
• Latex beads coated with InlB are efficiently
  internalized by Vero cells
• The receptor for InlB was recently shown to be
  the Met receptor tyrosine kinase

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Internalin B mediated entry by Listeria

• Players include
• InlB
• Met (transmembrane receptor tyrosine kinase)
• Cbl, Gab1, Shc
• PI 3-kinase (generate PIP3)
• Rac1 activation
• WAVE, VASP, profilin
• Arp2,3 complex actin

Cossart Sansonetti. Science 2004
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Listeria monocytogenes
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Listeria monocytogenes

• Within 30 minutes of entry, the bacteria lyse
  the vacuole and are found in the cytoplasm
• This is primarily due to the activity of LLO
  (listeriolysin O), but two phospholipases also
  contribute
• LLO is actually a vacuole specific cytolysin, in
  part because it
• has an acidic pH optimum (the vacuole is at pH
  5.9)
• L.m. mutants lacking LLO do not escape from
  phagosome
• Expression of LLO by B. subtilis is sufficient
  to lead to escape from the vacuole
• ActA is expressed at the pole of the bacterium
  and is required to mediate actin nucleation and
  actin based motility

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Actin-based motility (Listeria)
Listeria moves 10-15 mm/min (can sprint at rates
up to 36 mm/min in some cell types)
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Induced uptake (review)

• Zippering mechanism
• Yersinia (invasin)
• Listeria (internalin)
• Triggering mechanism
• Salmonella (TTSS)
• Shigella (TTSS)

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End of Day 3 (go back to your labs and do some
real work)