LSU BIOL 4215 Salmonella: Invasion and Evasion

Salmonella

gram negative pathogen- outbreakes often caused by contaminated food

InvA

a gene that allows Salmonella cells to invade epithelial cells through an Injectisome. Making them inv+.

FlhA

a gene involved in the assembly of flagella

interesting relationship found between InvA and FlhA

InvA is homologous to FlhA

pathogenicity islands

refers to gene clusters responsible for virulence

SPI-1 and SPI-2

salmonella pathogenicity islands that each encode a separate type III secretion system

Injectisome

mechanism for injecting toxins into host cells

Flagella

whiplike tails found in one-celled organisms to aid in movement

TIIISS

one of the bacterial secretion systems used by bacteria to secrete their effector proteins into the host's cells to promote virulence and colonisation, type of injectisome

cryoelectron tomography

can provide images of multi- protein complexes/structures by EM and computer rendering in 3D

discovered through cryoelectron tomography about TIIIS

TIIIS structural similarities across species

use of TIIISS by pathogens

moves proteins called effectors into host cells

animal pathogens that use type III secretion machinery

salmonella, E.coli, Shigella flexneri, pseudomonas aeruginosa, yersinia pestis, chlamydia trachomatis

though many animal pathogens use type 3 secretion, why do they have different effects on host cells?

the specificity of the pathogens effect on the host cell lies in the nature of the secreted effectors

How many proteins are injected into host cells by salmonella?

over 40

Salmonella effectors mechanisms of host manipulation (9)

modulation of GTPases, Repurposing of host enzymes, phosphorylation, ubiquitylation, Ribosylation, Acylation, Acetylation, Proeolysis, GlcNAcylation

Phosphorylation

The metabolic process of introducing a phosphate group into an organic molecule.

Ubiquitylation

Process by which one or more ubiquitin molecules are attached to a protein substrate molecule, which often results in the degradation of the tagged protein

GlcNAcylation

the post-translational, covalent attachment of a single N-acetylglucosamine (GlcNAc) to Ser or Thr residues of proteins

Ribosylation

The transfer of ADP-ribose from NAD+ onto Arginine, Glutamate, or Aspartate residues.

-catalyzed by ribosyltransferase

Acylation

When an acyl group (-COR) is added to a molecule

Acetylation

addition of acetyl group

Proteolysis

the breakdown of proteins or peptides into amino acids by the action of enzymes

What awas discovered in the 1988 study by Finlay, Gumbiner, and Falkow

The researchers observed that bacterial entry was associated with dramatic reorganization of host cell actin cytoskeleton, leading to membrane ruffling at the site of bacterial contact. The salmonella actively invades epithelial cells

Required for Salmonella invasion

TIIISS

SopE

Salmonella effector required for membrane rufflign and invasion

actin polymerization

Process of actin monomers forming filaments.

Membrane ruffling requires

actin polymerization

Actin

A globular protein that links into chains, two of which twist helically about each other, forming microfilaments in muscle and other contractile elements in cells; component of cytoskeleton

How does SopE promote actin polymerization?

activates members of the Rho family of GTPases, particularly Rac1 and Cdc42, which are key regulators of actin cytoskeleton dynamics.

G proteins

A class of proteins that reside next to the intracellular portion of a receptor and that are activated when the receptor binds an appropriate ligand on the extracellular surface.

First TIIISS effector to have a function assigned to it

SopE

Structural basis for reversible activation of a Rho protein by SopE

SopE has no amino acid similarity with any known GEF, binds Cdc42 differently from any known GEF

Inactive Rho=

less actin polymer

active Rho

more actin polymer

how does salmonella alter host cell shape

by activating and deactivating small GTPases

Explain steps of Salmonella altering cell shape

SopE protin into cytoplasm, binds to Rac

SopE acts as guanin-nucleotide exvhange factor to favour GTP form of Rac

Rac stimulates membrane ruffling

bacterial uptake

SptP protin binds to GTP Rac, restoring GTPase activity

Cell shape is restored

Proteins that mimic active G-protein created by bacterium

Map (EPEC), IpgB (Shigella), SifA (Salmonella)

WxxxE motif

a conserved sequence that function as guanine nucleotide exchange factors (GEFs) for Rho GTPases.

Yersinia pestis

plague, spread by fleabite or inhaled, systemic infection

Yersina pestis virulance

resistant to phagocytosis and macrophages, replicate extracellularly

Plasmid encoded type III secretion system

How many were killed by black death in the 14th century

50 million people

YopE is a GAP

inactivating a G protein and turning off phagocytosis in immune cells

Yersinia Strategy

remain extracellular, avoid phagocytosis

THe MAP kinase pathway is also targeted by

T3SS effectors

How odes Yersinia YopJ inhibit kinase activation

inhibites mitogen-activated protin kinase (MPK) and nuclear tacter kB(NFkB) signaling pathways used in innate immune response by preventing activation of family of MAPK kinases

How does YopJ act as acteyltransferase

uses acetyl-coenzyme A to modify serine and threonine residues in activation loop of MAPKK6, blocking phosphorylation

What type of pathway is a common target for T3SS effectors

MAP kinase pathways

How does phosphothreonine lyase activity (ex. Shigella OspF) prevent re-phosphorylation?

removes phosphates from MAPK threonine residues

A potential target for virulence drugs

TIIISS