Bacterial physiology: envelopes

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Bacterial physiology envelopes beyond

• Bacterial envelopes GP GN
• Specialized surface structures flagella, pili,
  capsule
• Protein export systems

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Bacteria have complex envelopes and surface
structures

• Essential for viability
• Composed of unique components
• Target for antibiotics
• Protects against environmental stresses
• Bile salts, low pH, low osmotic pressure
• Ligands for adherence
• Resist phagocytosis
• Trigger innate immune response, sepsis
• Antigenic variation between bacteria

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Gram stainHans Christian Gram 1844

• Divides bacteria into 2 (3) classes
• Gram positive, gram negative, poorly staining
  (includes TB ((Acid fast))

Pink
Purple
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Examples
GNC
GPC
GNR
GPR
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Gram negative vs gram positive envelope
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Gram Negative Membranes

• Inner (cytoplasmic) membrane
• Active transport
• Respiratory chain components
• energy transducing systems
• H-ATPase proton pump
• Biosynthetic enzymes for membrane phospholipids,
  PG, LPS, capsule
• Periplasm
• Peptidoglycan
• Degradative enzymes, B-lactams
• Binding proteins, signaling molecules
• Outer membrane
• Porins, tranporters,LPS
• Specific uptake of maltose, B12, nucelosides,
  iron
• Murein lipoprotein tethers OM to PG
• Asymmetric
• inner phospholipid leaflet
• Outer leaflet LPS, Mg

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E. Coli Peptidoglycan

• Alternating GlNAc MurNAc connected by beta 1,4
  glycosidic bond
• Peptide side chains
• L-ala, D-glu, DAP, D-ala
• Interpeptide bonds via transpeptidases
• PCNs block transpeptidation

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Differences between GP GN PG

• L-Lys in place of DAP
• More frequent interpeptide cross-linking
• GPs more sensitive to PCNs because of absence of
  OM
• GNs have single layer, GPs have multiple layers
  of PG

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GN Outer membrane
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LPS has 3 components
Species or serotype- specific
Genus-specific ag
Toxic, Anchors in OM, TRL4 ligand
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Gram positive envelope
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Gram positive cell wall

• Peptidoglycan is major constituent
• Sugar chains (glycan backbone) cross-linked via
  peptides
• target of PCN, cephalosporins, vancomycin
• Ligand for TLR2
• Polar 2 to sugars charged amino acids

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Techoic Lipotechoic acid

• Techoic acid Negatively charged polyglycerol or
  polyribotol phosphate polymers, covalently linked
  to PG in GPs
• Lipotechoic acid Techoic acid w/lipophilic
  glycolipid anchored in cytoplasmic membrane
• TLR2 ligand
• Adhesin for Streptococcus spp.

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Bacterial capsules

• Outermost layer (up to 10 u) of some GP GN
  bacteria
• Usually viscous polysaccharide
• Not essential for viability
• Sometimes is less discrete
• Slime
• Help microbes resist phagocytosis
• Shield from complement

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Flagella

• Generally found on rods (GN GP)
• Rarely on cocci, which are adapted for dry
  environments
• Long (3-12 um) filamentous hollow cylindrical
  structure
• Allows swimming
• Motility and chemotaxis

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Flagellar localization

• Polar or peritrichous location

Vibrio
Bartonella
E. coli
Spirillum
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Flagellar structure

• Filament (flagellin protein)
• Hook connects filament to basal body
• Basal body anchors hook, imparts motion
• Motor
• M S rings Cytoplasmic membrane
• P ring periplasm
• L ring outer membrane
• Highly regulated protein machine

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Swimming/chemotaxis

• Counterclockwise rotation of basal body? whirling
  of helical filament
• Driven by proton motive force, not ATP
• Assays
• Flagellar stains
• Swimming through semisolid medium (0.3 agar)
• Direct microscopic observation of living bacteria

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Flagella and pathogenesis

• Swimming/chemotaxis important for motility and
  virulence
• Co-regulated with other virulence factors (TCP in
  V. cholera)
• Adhesion (Salmonella, Pseudomonas)
• Protein export apparatus (Salmonella)
• Early stages in biofilm formation
• Flagellin protein is immunogenic
• Salmonella flagellin is a presumptive ligand for
  TLR5

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Pili/fimbriae

• Mostly found on GNR, rarely GPR
• Short, hairlike structures
• Not involved in swimming or swarming motility

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Functions of Pili

• Conjugation (E. coli F pilus)
• DNA uptake (Neisseria)
• Phage receptors
• Adhesion
• Early steps in biofilm formation
• Twitching motility (Pseud aeruginosa)
• Resist phagocytosis
• Antigenic variation (Neisseria gonorrhea)

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The protein secretion problem
?
Outer membrane
GN
Inner membrane
?
PG
GP
Inner membrane
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Gram Positives
Sortase
IM
Type I GSP Tat
Sec-dep
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Gram Negatives
Type II
Type IV
Chaperone/ Usher
Type V
OM
IM
Type I GSP Tat
Type III
Sec-dep
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Targeting Translocation Release
Pre-protein translocase
Signal peptidase
ATPase
Multiple SecA homologs
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Gram Positives
Sortase
IM
Type I GSP Tat
Sec-dep
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Cell surface proteins in GPs

• C-terminal LPXTG sorting motif
• Followed by hydrophobic domain tail of charged
  residues
• Found in gt 100 proteins, many important in
  pathogenesis
• New drug target
• Sec-dependent secretion, then recognized and
  cleaved by sortase (membrane associated
  transpeptidase)
• Heroic effort to clone sortase
• Found in all GPs examined to date
• All encode gt 1 sortase ?functions
• S. Aureus SrtA mutants ?virulence in mice
• Structure has been solved

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So what does sortase do?

• Cysteine transpeptidase
• LPXT?G
• Amide link between T carboxyl group and NH2 group
  of pentaglycine crossbridge

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Specialized Protein Export Systems in GNs
?

• Allow transport across OM
• Special issues
• Folded vs unfolded protein
• No ATP or other energy sources at OM
• Self-energized or harness energy from IM
• Terminal branches of the GSP ie sec-dependent
• Sec-independent
• Some protein export machines evolved from
  organelle biogenesis systems (Flagella, pili)

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Gram Negatives
Type II
Type IV
Chaperone/ Usher
Type V
OM
IM
Type I GSP Tat
Type III
Sec-dep
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Tat (twin arginine translocase)

• Found in GP and GN
• Related to ?pH-dependent protein import pathway
  of chlorplasts
• Involves TatA-E protein products
• Longer ss with invariant RR motif, less
  hydrophobic
• Translocates pre-folded proteins across IM
• Substrates usually bound to co-factor
• Respiratory and photosynthetic electron transport
  proteins
• Can transport oligomers
• Can feed into other pathways that allow export
  across the OM (Phospholipase in Pseudomonas)

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Gram Negatives
Type II
Type IV
Chaperone/ Usher
Type V
OM
IM
Type I GSP Tat
Type III
Sec-dep
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Chaperone/usher pathway

• Assembly of adhesins (P type I pili)
• Periplasmic chaperone (PapD) OM usher (PapC)
• Thin flexible tip fibrillum connected to rigid
  helical rod
• Pilus subunits bind to chaperone release of
  subunit in periplasm, proper folding, caps
  interactive surfaces
• Usher forms translocation channel for pilin
  assembly
• PapC forms ring-shaped oligomers
• Similar structures in type IV pili and type III
  secretion
• Donor strand complementation

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Gram Negatives
Type II
Type IV
Chaperone/ Usher
Type V
OM
IM
Type I GSP Tat
Type III
Sec-dep
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Autotransporter Secretion (type V)

• 1st step is Sec-dep secretion across IM
• Diverse substrates
• Proteases, toxins, adhesins, invasins
• Substrate has 3 domains
• N-terminal sig seq
• Internal passenger
• C-terminal B-domain
• B-domain forms B-barrel pore structure, allowing
  passenger domain to pass
• Once secreted, passenger domain is retained or
  clipped off
• Does not req ATP for OM transit
• Some proteins req single accessory factor (B.
  pertussin FHA)

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Examples

• N. gonorrhea IgA protease
• H. pylori VacA
• H. influenza Hla fibrillar protein
• Remains attached to OM and serves as adhesin

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Gram Negatives
Type II
Type IV
Chaperone/ Usher
Type V
OM
IM
Type I GSP Tat
Type III
Sec-dep
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Type II secretion
Secretin
Chaperone
Energizer
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Type II secretion

• Substrates include extracellular enzymes toxins
  from GNs
• Cholera toxin (activates euk AC)
• 1st step is Sec-dependent
• Forms AB5 in periplasm
• Secretion structure is related to type IV pili
  (substrate is pili vs toxins) and to DNA uptake
  systems (B. subtilis)

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Gram Negatives
Type II
Type IV
Chaperone/ Usher
Type V
OM
IM
Type I GSP Tat
Type III
Sec-dep
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Type IV secretion

• Homologous to bacterial conjugation and VirB
  system of Agrobacterium
• translocation of bacterial proteins into euk cell
• Helicobacter CagA inserted into Euk membrane,
  and associates with SHP-2, alters cell shape
• Legionella secretes a GEF into cytoplasm while
  residing in vacuole
• Bordatella pertussis PTX secretion
• VirB export system best understood

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Type IV secretion Legionella, H. pylori, Pertussis
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• Initial transport across IM is sec-dependent
• Probably periplasmic intermediate
• VirB proteins are membrane associated, interact
  w/each other, present in multiple copies
• VirB4 B11 contain NTP binding activity may
  energize secretion
• Protein-protein interactions may drive assembly
  of minimal transport complex NTP binding may be
  required for secretion
• VirB7-10 may form complex that spans periplasm
• No secretin homolog instead, VirB2 is major TFP
  and VirB5 is minor pilus component
• Pilus may serve as secretion tube for
  translocation of proteins or DNA into euk cells

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Type IV secretion in H. Pylori

• Type IV secretion system encoded by the CAG PAI
• CagA is a substrate for type IV secretion
• Inserted into host cell membrane,
• Phosphorylated by Src
• Assoc w/SHP-2 phosphatase
• Disrupts localization of ZO-1/JAM/tight junctions
• Disrupts cell polarity
• Necessary and sufficient for morphological
  changes in host cells

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Legionella

• Can transport DNA
• several effectors now identified
• RalF
• Sec7 homolog
• Modifies vacuolar compartment

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Gram Negatives
Type II
Type IV
Chaperone/ Usher
Type V
OM
IM
Type I GSP Tat
Type III
Sec-dep
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Type I secretion (ABC transporters)

• Secretes toxins, proteases, lipases
• Related export proteins in euks
• Sec-independent
• No periplasmic intermediate
• C-terminal 60 aa secretion signal
• 3 components
• IM ABC transporter
• IM fusion protein (MFP)
• OMP (not req in GPs)
• E. coli a-hemolysin export
• OMP TolC (porin-like B-barrel which extends into
  periplasm)
• MFP forms trimer that contacts ABC and OMP
• MFP may prevent periplasmic intermediate
  formation by forming closed channel between IM
  OM

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Gram Negatives
Type II
Type IV
Chaperone/ Usher
Type V
OM
IM
Type I GSP Tat
Type III
Sec-dep
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Type III secretion

• Injects effector molecules from bacteria
  directly into eukaryotic cell
• Disrupts host signaling pathways?evade host
  defense mechanisms
• Sec-independent
• Can be separated into 2 steps secretion
  translocation
• Secretion involves gt 25 proteins that form
  channel across IM OM
• Evolutionarily related to flagellar apparatus
• Translocation apparatus itself is secreted by the
  TTSS
• Translocation apparatus include YopB YopD,which
  can form a pore in lipid bilayers

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Type III Secretion System
Sec-dependent Secretion
Bacteria
Host cell
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Type III secretion
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Type III secretion

• Contact-dependent secretion pathway gt20
  components
• Can be separated into two steps
• Export out of bacteria (lab conditions)
• Translocation into host cell
• No N-terminal cleavage but secretion signal is in
  5' end
• Secretion apparatus conserved among pathogens,
  the effector molecules differ

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The needle complex
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What is the signal for TTSS

• Bipartite signal sequence
• N-terminus is necessary and sufficient for
  secretion (not translocation)
• Middle portion binds to chaperone
• What does chaperone do?
• Does not protect effector from degration
• Does keep it from aggregating
• Temporal control of effector secretion?
• Not all effectors have identified chaperones?

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Effectors

• Highly variable between different species and
  strains
• Pseudomonas possesses ExoS or ExoU, but rarely
  both
• Horizontally acquired, often reside in PAIs
• ExoU flanked by IS sequences
• Other effectors carried on phage remnants
• Clearly target eukaryotic proteins
• YopH tyrosine phosphatase that targets focal
  adhesion proteins
• YopJ/P targets MAP kinase
• SopE GEF for Cdc42 Rac
• Often have two separate domains with 2 distinct
  activities and targets
• SptP N-terminal GAP domain and C terminal
  tyrosine phosphatase domain

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Bacterial ammunition diverse strategies for
targeting the host cells

• Diverse export systems
• Some are sec-dependent (type II and IV)
• Some require folding in periplasm
• Some directly secreted across IM OM
• Diverse recognition signals
• N (Sec, type III)- or C (type I)-terminus
• Protein vs mRNA
• Related substrates transported by diverse
  pathways
• AB5 toxins by type II and type IV
• Proteases by type I or type II