Direct Delivery

Of Bacterial Toxins

• Some bacteria are able to directly deliver their toxins into the cytoplasm of eukaryotic cells through a contact-dependent secretion system

• direct injection is both efficient and selective

• The potency of intracellularly acting toxins is derived, in part, from their mode of action

– most are enzymes that catalyze the covalent modification of specific molecular targets.

• To be successful intracellularly acting toxins must access their substrates inside target cells.

• To overcome the membrane barrier, intracellularly acting toxins are either injected directly into host cells by pathogenic microbes or, alternatively, enter cells in a manner that is microbe-independent

• The eukaryotic plasma membrane is a formidable gatekeeper that effectively restricts macromolecules such as toxins from passing freely into cells.

• Some gram-negative pathogens inject Some gram-negative pathogens inject their toxins into the cytosol of host cells their toxins into the cytosol of host cells through bacterial transport machines that through bacterial transport machines that function as macromolecular syringesfunction as macromolecular syringes

• The syringes, which resemble either The syringes, which resemble either bacterial flagella or conjugative pili, bacterial flagella or conjugative pili, facilitate the direct passage of toxin facilitate the direct passage of toxin effectors from bacterial cells into effectors from bacterial cells into eukaryotic cells by processes referred to as eukaryotic cells by processes referred to as Type III or Type IV secretion mechanisms Type III or Type IV secretion mechanisms

• Type III and Type IV secretion machines do Type III and Type IV secretion machines do not allow the free exchange of proteins not allow the free exchange of proteins between prokaryotic and eukaryotic cellsbetween prokaryotic and eukaryotic cells– They appear to selectively regulate which toxin They appear to selectively regulate which toxin

effectors pass through the syringe needleeffectors pass through the syringe needle

Injection mechanisms limit the extent to Injection mechanisms limit the extent to which a pathogen can remodel the host, which a pathogen can remodel the host, because a bacterium can directly affect because a bacterium can directly affect only a single target cell at any given time.only a single target cell at any given time.

Pathogenic microbes exert a broader Pathogenic microbes exert a broader sphere of influence by releasing a bolus of sphere of influence by releasing a bolus of toxin that can act upon many cells within a toxin that can act upon many cells within a given tissue and/or diffuse away to given tissue and/or diffuse away to modulate one or more types of cells at modulate one or more types of cells at multiple locations within the host.multiple locations within the host.

How do the toxins affect eukaryotic cells?

Toxins can affect signal transduction such as phosphorylation

Yersinia uses a protein kinase YpkA and a protein tyrosine phosphatase YopH

YpkA and YopH are injected into the cell through Type III secretion system

YpkA and YopH are able to paralyze macrophages before they can kill the bacteria

YopH dephosphorylates cytoskeletal proteins disrupting phagocytosis

EPEC TirEPEC Tir (translocated intimin receptor) is (translocated intimin receptor) is a protien found in enteropathogenic E. colia protien found in enteropathogenic E. coli

EPEC TirEPEC Tir is injected via Type III secretion is injected via Type III secretion systemsystem

EPEC Tir EPEC Tir becomes part of the eukaryotic becomes part of the eukaryotic cell membrane and functions as a receptor cell membrane and functions as a receptor for a bacterial adhsin called intiminfor a bacterial adhsin called intimin

EPEC infection is characterized by EPEC infection is characterized by destruction of host-cell intestinal microvilli, destruction of host-cell intestinal microvilli, act in rearrangement within epithelial act in rearrangement within epithelial cells, and the formation of a raised cells, and the formation of a raised platform or pedestal at the site of bacterial platform or pedestal at the site of bacterial attachmentattachment

Toxins can act on small G proteins and affect the cytoskeleton of the host cell SopE is injected into eukaryotes from

Salmonella via type III secretion system It binds and activates small G proteins (Rac

and Dcd42) Causes membrane ruffling and actin

cytoskeleton reorganization This mediates the uptake of the bacteria into

the eukaryotic cell

P. aeruginosa P. aeruginosa Exoenzyme SExoenzyme S has ADP- has ADP-ribosylating activity that ADP-ribosylates ribosylating activity that ADP-ribosylates the small G protein Rasthe small G protein Ras

The toxin is injected via a type III secretionThe toxin is injected via a type III secretion It causes the cytoskeleton to collapse and It causes the cytoskeleton to collapse and

the cell morphology to changethe cell morphology to change The result is the rounding of the cell The result is the rounding of the cell

►YopEYopE is injected via type III secretion is injected via type III secretion system from system from YersiniaYersinia

► It is able to paralyze macrophage It is able to paralyze macrophage phagocytosis by causing actin phagocytosis by causing actin depolymerization depolymerization Resulting in the disruption of cell Resulting in the disruption of cell

stress fibers; hence rounding of stress fibers; hence rounding of cells/loss of cell shapecells/loss of cell shape

What to take note of

• Bacteria can use structures similar to conjugative pili to insert their toxins directly to the cell

• The bacteria inject their toxins through Type III or Type IV secretion systems

• This process is more efficent, but it can only affect one cell at a time

• The toxins can act on signal transduction pathways, small G proteins and the cytoskeleton of the eukaryotic host cell