Type IV pilus is a dynamic structure

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Type IV pilus is a dynamic structure

• PilT mutant have extralong pili that are TM- and
  fail to take up DNA or phage

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Neisseria pathogenesis

• Two human pathogens
• Neisseria gonorrhea STD
• Neisseria meningiditis meningitis, sepsis
• Humans are the only reservoir
• No animal model

3
Diverse, redundant adhesins

• Type IV pili antigenic variation
• CD46 is a receptor
• Opa proteins phase variation (on/off),
  antigenic variation
• 2 classes of receptor HSPGs, CD66
• Different subclasses of CD66 receptor use
  different internalization pathways
• Required for human infections
• Opc protein phase variation via mutations in
  poly-C tract in promoter region
• LOS
• Porins

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Antigenic variation
PilS genes
Reciprocal recomb Recomb during
transformation Recomb between chromosomes
PilE gene
Phase variation
ATG__________GGGGGGGG____Stop (41
aa) ATG__________GGGGGGGGG_________________stop
(991 aa)
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Adhesion Invasion

• In vitro systems
• Organ culture (FTOC, NPOC)
• Tissue culture T84 cells
• Initial stages attach as microcolonies
• Elongation of microvilli/filopodia towards
  bacteria
• Motility dispersal over epithelial surface
• Later times, disperse from microcolonies, pili
  disappear, intimate association with host cell
  membrane
• Engulfment, invasion, transcytosis

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• Pilus-induced cortical plaques
• Begin within min, lasts for hrs
• PTyr, actin, ezrin, transmembrane glycoproteins
• PilT dependent

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Pilus-mediated host cell signaling

• Piliated GC/MC or semipurified pili trigger
  cysotolic Ca flux.
• Requires PilC1
• Blocked by antibodies to CD46
• Depletion of Ca2i ? ?bacterial adherence
• May stimulate lysosomal exocytosis and release of
  Lamp1 (substrate for IgA protease)
• Recall T. cruzi!

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Type IV pili induces host signaling

• Host micorarrays compared wt to pilT mutant
• Upregulated MAPK and other stress-activated
  pathway and induces genes that protect cells from
  apoptosis
• Can be mimicked by applying artificial force on
  membrane

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Two classes of Opa receptors

• HSPGs bind some Opa variants ? binding/internaliza
  tion
• Localized recruitment of HSPG receptors, F-actin,
  P-tyr
• Stimulation of Lipid hydrolysis enzymes PC-PLC
  and Sphingomyelinase
• CD66
• Multiple modes of entry

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Different CEACAMS specify different entry modes
CEACAM 3 CEACAM 1
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Neisseria exemplify diversity

• Multiple adhesins/receptors
• Antigenic phase variation
• Multiple pathways of entry

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Bacterial entry into cells

• Provides access to nutrient-rich environment
• Protection from immune system
• Access to new or deeper tissues
• Must survive in new hostile environment

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Why bacteria might NOT want to enter into cells

• Harsh environment
• Limited nutrients and iron
• Presentation to immune system
• Limited possibilities for sex or other forms of
  genetic exchange

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Routes of invasion

• Phagocytosis (actin-dependent)
• Entry into professional phagocytes
• IgFc-receptor
• C3b CR1 receptor
• iC3b CR3/CR4 receptor
• Accum of complement components w.o. being lysed
• Delivery to lysosome unless pathogen interferes
  with intracellular trafficking
• Induced endocytosis/phagocytosis (actin-dep)
• Entry into non-professional phagocyte via
  pathogens ligand
• Active induction of internalization

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• Active invasion (actin-independent)
• Active entry without triggering host uptake
  pathways
• Apicomplexan parasites have motile invasive
  stages (zoites)
• Probably does not involve specific adhesins
• Route of entry specifies endosomal fate
• Toxoplasma subcellular localiz depends on route
  of entry

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Zipper Hypothesis
Phagocytosis Ingestion of solid material gt 1m
in diameter
Phagocytosis requires repeated sequential
interactions between the phagocyte membrane and
target particle
Implications Cell changes and signaling events
are very local Exquisite cellular discrimination
among surface receptors
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Multiple stepsMultiple steps at which microbes
can interfere

• Early signaling events
• FcG phosphorylation of ITAM by Src-gtdocking of
  SH2 proteins (Syk)-gtPI3K activation, PIP2-gtPIP3,
  DAG, PKC
• Actin polymerization via Rho GTPases
• Phagosome maturation
• Kiss and run
• Reactive oxygen generation

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Phagocytosis is complexDictyostelium Genetics
Cardelli, Traffic, 2000
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Mammalian phagocytosis complex and dynamic
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Distinct GTPase Effectors in Different Modes of
Phagocytosis
Type III effectors can modulate GTPases and
affect phagocytosis
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Effector mechanisms resulting from phagocytic
receptor engagement
Fc receptor engagement- proinflammatory CR3-
partially inflammatory Proinflammatory effector
mechanisms NADPH Oxidase (CGD) iNOS PLA2
Recruitment of TLRs TNFa, IL-1, etc.
Recruitment of effector mechanisms can be
blocked by pathogens
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Entry into non-prof phagocytes

• Bacterial adhesins that mimic eukaryotic
  signaling molecules
• Yersinia, EPEC, Listeria
• Zipper
• Deliver/inject bacterial molecules that
  manipulate host cell cytoskeleton
• Salmonella, Shigella, Helicobacter
• trigger mechanism (resemble growth-factor-induce
  d membrane ruffling

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2 modes of entry
Zipper Bacterial ligand binds to host cell
receptor
Trigger Bacterial injects molecules into host
cell
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Steps in Zipper uptake

• Contact adherence
• Actin independent
• Receptor clustering
• Phagocytic cup formation
• Actin polymerization
• Membrane extension
• Phagocytic cup closure and retraction
• Actin depolymerization

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Yersinia diseases

• Y. pestis
• Bubonic plague
• Transmitted by flea
• Y. pseudotuberculosis, Y. entercolitica
• Both cause food-borne GI illnesses
• Oral-fecal transmission
• First understanding of bacterial internalization
• Type III secretion discovered inhibits
  internalization
• Mostly extracellular pathogen

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• Isberg Falkow 1985
• Cosmid library of Y. pseudotb introduced into E.
  coli K12
• Selected for ADHESION
• Screened for INVASION using Gentamycin protection
  assay, confirmed by EM
• Identified the inv gene
• Yersinia ?inv mutant defective in binding
  invasion
• Gent protection assay identified another invasin,
  Ail
• WHY MIGHT THIS APPROACH NOT WORK?

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Inv gene product

• 103 kD OMP
• Attachment and entry functions cannot be
  separated
• C-terminal 192 aa is necessary sufficient to
  direct entry

Y
MBP-Inv fusion
Anti-MBP
Y
SA
Protein A
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b1 integrins are the receptor

• Made affinity column of soluble form of inv
• Biotinylated solubilized membrane extracts b1
  integrins
• Abs to b1 integrins block Yersinia invasion
• Suggests that entry into non-phagocytic cells
  related to entry into phagocytic cells (CR are
  comprised of integrins)
• b1 integrins are receptors for Fn

Y
Y
Y
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Why does the same receptor bind Fn but
internalize Inv?

• Inv Fn recognize different portions of b1
  integrins?
• Abs that block Inv binding also block Fn binding
• RGD peptides inhibit both
• Fn is a competitive inhibitor of Inv binding
• Binding and invasion domains of Inv cannot be
  genetically separated
• Is it just affinity?
• Bacteria coated with anti-a5b1 mAbs uptake
  dependent upon affinity of mAbs for receptor

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Inv Fn have very different structures

• Conserved location of Critical Asp residue
• Convergent evolution

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• Physical nature of interaction
• High affinity interaction Kd of Fn-a5b1
  100-fold greater than for Inv-a5b1
• Invasin monomers multimerize multimerization is
  required for uptake
• Increased local concentration of integrin
  receptors

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• Lectin domain of Inv is inflexible may stabilize
  interaction w/receptor

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Proof of zipper model
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Downstream events

• b1 integrins bind talin, a-actinin, FAK,
  paxillin, IPCA-1
• b1 integrin cytoplasmic tail req for bacterial
  uptake
• Some clues provided by type III secreted
  anti-invasins
• YopH tyrosine phosphatase that targets FAK,
  Cas, paxillin, Fyb
• YopE Rho-family GTPase activating protein (GAP)
• Cdc42 independent
• Rac activation of Arp 2/3, independent of N-WASP

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How does Yersinia access b1 integrins?

• M cells are shallow cells specialized in
  transcytosis of antigens
• Overlay Peyers patches
• Many pathogens access sub-intestinal tissues via
  M cells

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How does Yersinia access b1 integrins?

• B1 integrins expressed on the BL surface of
  epithelial cells.

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Other Yersinia adhesins pH6ag, Ail Yad A

• Also required for binding/or and internalization
• Differential expression
• Inv expressed at RT
• Ail expressed at 37
• Ail mutants show cell-type specific invasion
  defect
• YadA binds Fn
• pH6 ag binds glycosphingolipids

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Role of multiple adhesins in vivo
Wt Inv

• InvA mutant is delayed in colonizing PPs (PO)
• LD50 not affected
• YadA mutant dec virulence PO or IP
• InvAil double mutant is avirulent

Intragastric inoculation
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Yersinia inhibits its own invasion using type III
secreted effectors

• YopE GAP for Rho, Rac, Cdc42
• YopH tyrosine phosphatase, targets focal
  adhesion proteins (p130cas, FAK, paxillin, FyB,
  SKAP-HOM)
• YopT cysteine protease that cleaves isoprenoid
  moiety of RhoA at C-terminus
• YopO/YpkA serine threonine kinase activated by
  G actin and binds to Rho GTPases
• Roles in vivo Macrophages? Lymphocytes?
  Suppression of oxidative burst? Ca signaling?

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Inv homologs are found in other GNs

• Intimin (EPEC, EHEC, Citrobacter freudii)
• Located on LEE PAI
• Homology in N-terminal 500 aa (OM localization
  export of C-terminus

Intimin Invasin
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EPEC

• Common cause of diarrhea in children in
  developing countries
• Causes attaching/effacing lesions
• Several virulence factors
• Type IV bundle forming pilus (plasmid-encoded)
• Intimin adhesin
• Type III secretion system
• Type III secreted receptor tir
• Other type III secreted proteins

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EPEC translocates its own receptor (Tir) for
intimin
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EPEC inhibits its own invasion
PI3 Kinase
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EPEC

• EPEC uses intimin as a ligand for binding
• Secretes and translocates its own receptor into
  the host cell (Tir)
• Causes interesting cytoskeletal rearrangements
• Inhibits its own invasion via type III secreted
  proteins
• Tir has similarities to H. pylori CagA
• Translocated into host cell membrane
• Variable tyrosine phosphorylation
• Does EPEC move from pedestal to pedestal along
  surface of cells?
• How does this relate to disease (diarrhea?)

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Listeria

• Causes sepsis and meningitis in immunocompromised
  adults, abortion in pregnant women, neonatal
  infections, rarely GI illness
• Food-borne

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possibly the most famous work of art in
microbial pathogenesis
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Internalin a family of adhesins

• Screened Tn library of L. monocytogenes for
  mutants UNABLE to invade
• Defined the Internalin gene family, a family of
  23 genes
• InlA B are necessary for invasion
• InlA expressed in L. innocua or enterococcus is
  SUFFICENT to confer internalization
• InlA or InlB is SUFFICENT for beads to be
  internalized

GW domains Cell surface Association
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The real thing
InlA-coated beads
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E-cadherin is a receptor for Internalin A

• Affinity chromatography using InlA
• Identified E-cadherin
• Ca-dependent cell-cell adhesion through
  homophilic interactions between extracellular
  domain
• BL localization!!!
• Cytoplasmic domain interacts w/catenins actin
  cytoskeleton
• N-cadherin does not work

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• LRR and IR regions of InlA sufficient
• Ectodomain of E-cadherin is sufficient for
  bacterial adherence
• Cytoplasmic domain of E-cadherin is required for
  InlA-mediated internalization
• Fusion protein of E-cadherin ectodomain fused to
  C-terminus of a-catenin sufficient to promote
  InlA-mediated entry

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How can E-cadherin mediate cell-cell contacts and
bacterial entry?

• In cell-cell contact, adjacent cell counteracts
  force generated by actin-cadherin complex
• For bacteria or InlA-coated beads, no
  counter-acting force-gtbacterial entry
• Also, multiple E-cadherin-InlA contacts-gt
  zippering

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Using receptor hetergeneity to create a better
animal model

• InlA is not sufficient to mediate internalization
  into mouse cells
• Chicken L-cam
• hEcadP16
• mEcadE16 -
• hEcadE16E -
• mEcadE16P
• Explains why no role for InlA
• in mouse model of listeria!

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Transgenic mouse expressing E-cadherinE16P are
permissive for Listeria infection

• Strain Animal LD50 IV LD50 po
• Wt Mouse 105 gt5 x 1010
• ?inlA Mouse 105 gt5 x 1010
• Wt Guinea pig 5 x 107 1011
• ?inlA Guinea pig 5 x 107 gt 1011
• Wt Mouse E16P lt 5 x 1010
• ?inlA Mouse E16P gt5 x 1010

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InlB-mediated internalization 3 receptors?

• gC1q-R by affinity chromatography
• Requires Gly-trp repeats of InlB
• Subcellular localization function of gC1q-R
  controversial
• gC1q-R and HSPG binds GW domains and may
  dissociate InlB from bacterial surface to host
  cell surface
• C-Met (HGF receptor) (educated guess based on
  activation of specific signaling pathways
• PO4 of Shc, Gab1, Cbl
• PI3K and c-Met recruited to site of bacterial
  contact
• Membrane ruffling sensitive to wortmannin,
  genistein, CytD
• InlB interacts through LRR
• GAGs bound to cell surface proteoglycans
• Requires Gly-trp repeats of InlB
• May enhance interaction with C-met

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• InlB necessary sufficient for activation of PI
  3-kinase and tyrosine phosphorylation of Gab1,
  Cbl, Shc

• Vav (GEF)-Rac-PAK- WAVE-Arp2/3, cofilin -LIM
  kinase recruited, then cofilin inactivated by LIM
  kinase
• HGF InlB bind to different portions of the
  C-met receptor

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Unanswered questions

• What is the relative importance of InlA and InlB
  in vivo?
• How does Listeria access E-cadherin, a BL protein
• What is the relationship of gC1qR and c-met?
• How do two closely related ligands trigger
  different pathways
• Are the pathways synergistic?
• E-cadherin and C-met are both at cell junctions
• How does signaling result in internalization?