Lectures 20 and 21 - Colonization and Invasion

3 stages of BAC infection

Colonization (upregulation of virulence genes)→Persistence (Upregulation of persistence genes, downregulation of colonization genes)→Spread (Upregulation of genes that induce escape of host into environment, Coughing, vomiting, etc.)

Biofilms and Attachment

Resists physical forces that sweep cells away, defends against phagocytosis and penetration of toxins. Nutrient rich surfaces.

Environmental Biofilm

Act as source of infection, Legionnaire’s Disease

Bacterial Colonization Biofilms

Plastic catheters, dental or knee implants.

Borrelia burgdorferi

Causal agent of Lyme disease. Passes through skin by tick bite.

Penetration of mucus in intestine

M cells do not have a thick layer of mucus, so Bacterial cell may pass through them. Some can also produce sIgA1 proteases which sever the sIgA1 bond that keeps them trapped in mucus.

Pili

commonly shed. The advantage of this is that the pili can be made of different pilus proteins which can help in evading the host’s immune response.

Phase variation of pilin genes in Neisseriria gonorrhoeae

on/off switch.
PilE: pilin structural gene
pilA: Response regulator. Engages DNA to regulate gene expression
pilB: Sensor (unclear what environmental signal it recognizes). In the membrane

Antigenic variation of pilin genes in Neisseriria gonorrhoeae

Changing the amino acid sequence of surface proteins.
Homologous recombination between pilS and pilE. The altered pilE is expressed but c’somal copies of the pilS remain unchanged since the altered form was not integrated into the chromosome.

Adhesins in Gram+

exhibit both pili-like and non-pili adhesins. Streptococcus and Corynebacteria.
S. pyogenes bears structures that resemble pili but are formed of multimers of M protein which helps prevent phagocytosis, not an adhesin. The adhesin it does encode is Protein F1

FAK host protein

Recruited by bacteria during integrin mediated attachment to provide a tighter binding. If absent, the bacterial attachment is not as tight.

Brucella abortus

colonizes the placenta. 4C sugar erythritol increases the regulation of virulence factors when Brucella grows on the compound.

Importance of Iron

Fe is associated with lactoferrin, transferrin, ferritin, and heme. During Bac infection, body sequesters Fe even further. BAC have competition with host for Fe.

Siderophores

lwo molecular weight with high affinity for Fe. 2 classes: Hydroxamates and Catechols.

Hydroxamate

sent out into extracellular environment, interacts with ferric Fe+3 and then binds to cytoplasmic receptor, reduced in the cell to ferrous Fe+2, the free Hydroxamate can be used again.

Using host proteins that contain Fe

Transferrin, lactoferrin, and hemoglobin can be bound by the BAC to use as a disguise and to remove Fe from the compounds.

Other Fe obtaining strategies

Toxin production: host cells are killed to induce the release the Fe stores.
Iron Abstinence: Some BAC have evolved to not need Fe at all or use another element like Manganese (Mn) like Borrelia burgdorferi.

BAC that escape into the cytoplasm of phagocytic cells

Intracellular BAC have to survive host cell defenses. Escape vacuole before phagolysomal fusion.

BAC that invade non-phagocytic cells like epithelial cells

Penetration with invasins. Shigella
Vacuole escape with listeriolysin and phospholipases (pore forming). Listeria
Intra and intercellular movement/cell-to-cell spread with ActA. Listeria
Actin-based motility (use of host proteins for movement) used by Shigella and Listeria.

General Secretion pathway

most common way of protein secretion.
Stage 1: Signal peptides/Leader sequences. 15-26AA.

Sec translocation

Stage 1: signal peptides and leader sequences. The peptides are 15-26AA long and start with a couple positively charges residues followed by some hydrophobic residues.
SecB: tetramer that delivers pre-protein in its unfolded state to the secA-secYEG integral membrane complex. It then dissociates from the pre-protein and is recycled.
The ATPase SecA functions as the ATP-dependent motor that drives the translocation reaction. During the transport process the signal peptide is removed – hydrophobic residues trap it in the cytoplasmic membrane.

Signal Recognition Particle (SRP) pathway

Bacterial system similar to SRP pathway in eukaryotic cells. Similar to Sec secretion pathway, but more hydrophobic. USE GTP

Twin-arginine Translocation (TAT) Pathway

Exports folded substrate proteins through tightly sealed membrane. Energy source comes from Proton Motive Force, so they DO NOT NEED NTP or GTP. This system is not found in all bacterial species. Signal peptide contains twin-arg (-RR-).

Tat appartatus

Comprised of TatA,B, and C. Substrate binds to TatBC complex, this triggers recruitment of TatA. Translocon formed and translocation occurs.

Type I secretion

Sec-independent. Move substrate proteins across both inner and outer membranes in a single step. Signal sequence is at the C terminus. Transport slow floding proteins, energy burned comes from ATP. ATP binding cassette (ABC) protein exporters. Substrates include toxins, proteases, lipases, etc. The secreted proteins fold in the external environment

Pilus assembly and Type II Secretion

Sec-Dependent. Pili assembly and the main terminal branch. ATP, Type 4 pili = Type II secretion….secretin

P pili/Type 1 pili

intrinsic capacity for self-assembly. Periplasmic chaperone prevent assembly in periplasm. Usher protein guides subunits through outer membrane where they will assemble.

Type 4 pili

This pili begins at the periplasmic face of the cytoplasmic membrane. The apparatus extends through a pore in the outer membrane. ~20 accessory proteins. Synthesized when needed and can be retracted.

Type III The secretion needle

Sec-Independent. Inject proteins directly into host cell cytoplasm. Across inner and outer membranes. The secreted molecules never see antibodies or other aspects of the immune system since they bypass any membrane. Triggered by intimate contact with host cell and driven by ATP hydrolysis. Recognition signal appears to involved with N-terminus amino acids or 5’ UTR of mRNA in some cases. Protein folding occurs in host cells. Very similar structure to flagellar basal body.

Conjugation-like secretion: Type IV secretion

Sec-Independent. Involved export of proteins across 3 membranes, inner, outer, and eukaryotic membranes. Assembles an extracellular needle like structure. Includes bacterial conjugation systems. Complex of 12 or more proteins. Tube-like structure that transfer proteins into the host. Can carry DNA. Driven by ATP hydrolysis.

T4S in Legionella pneumophila

Dot/Icm system. Direct injection of proteins into cytoplasm of macrophages. Subvert phagosome:lysosome fusion pathway.

T4S in Helicobacter pylori

CagA. Direct delivery of oncoprotein CagA from bacteria to gastric epithelial cell cytoplasm. Inflammatory response induced. Cag PAI associated with highly virulent strains of H. pylori.

T4S in Bordetella pertussis

Ptl System. Secretes pertussis toxin. Toxin components secreted with Sec pathway and assembled in periplasm; secreted across outer membrane into extracellular space

Type V secretion

SEC-Dependent, ATP, Autotransporter proteins secreted. Largest group of secreted proteins in Gm- bacteria. Most involved in pathogenesis: proteases, toxins, adhesins, invasins. Localized to outer membrane. Large proteins. Long signal sequence found at N-terminus. The OM translocon forms a Beta-Barrel with the outer membrane through which the functionally active domain is threaded out into the extracellular milieu.

Autotransporter proteins: IcsA

Shigella spp. Protein required for actin based motility

Autotransporter proteins: Pertactin

Bordetella spp. Adhesin found in the vaccine (aP)

Autotransporter proteins: VacA

H.pylori. vacuolating protein.

Type 6 Secretion

Sec-Independent. No obvious N terminal signal sequence required. ATP powered. Usually lethal effector proteins are secreted into cells via contact-dependent processes. Can target Eukaryotic or Bacterial cells.

Secretions systems specific to Gm+ bacteria

Proteins need to interact strongly with Bac cell surface to prevent secretion to surrounding media.

1. Sortase enzymes: mediate covalent attachment of secreted proteins to peptidoglycan.

2. Some proteins have transmembrane anchors or can be attached to membrane lipids as lipoproteins on the cell surface.

3. Bind peptidoglycan or Teichoic acid through binding domains like LysM.

4. Form ionic or hydrophobic contacts with specific proteins embedded in the cell membrane of to the membranes themselves.

Cytolysin-Mediated Translocation (CMT)

Sec dependent, Powered by ATP. Gm+ bacterial method of secretion Capable of secreting proteins directly into the cytoplasm of host cells. Very controlled in comparison to Gm- bacteria. SLO and SPN disrupt host cell membrane. Toxins affect host only if S. pyogenes is producing an adhesin that allows adhesion to host cell membrane