7 Invasion & Intracellular Growth

• Obligate intracellular pathogens = can only reproduce inside of host cells, cannot live outside host cells

• Facultative intracellular pathogens = can reproduce either inside or outside of host cells

cellular immune response

Humoral immunity is the aspect of immunity that is mediated by macromolecules – including secreted antibodies, complement proteins, and certain antimicrobial peptides – located in extracellular fluids. Humoral immunity is named so because it involves substances found in the humors, or body fluids.

• One mechanism relies on effector proteins secreted by the pathogen → effector proteins trigger entry into host cell

• Second mechanism relies on surface proteins expressed on the pathogen that bind to receptors on the host cell → resulting in entry

(a) bacteria attaching to host cell surface deliver effector proteins into host cell via specialized secretion systems

(b) effectors interact with cellular targets that regulate host cell actin dynamics, causing membrane ruffling beneath the invading bacteria

(c) eventually leads to bacterial engulfment and internalization into a membrane-bound vacuole through a macropinocytosis-like process

(a) bacterial surface proteins bind receptors on host cell membrane, activating signaling cascades that control actin cytoskeleton

(b) actin-driven pseudopods which fuse in a zippering membrane process progressively entrap bacteria

(c) bacteria are progressively engulfed until a phagosome is formed

secrete effector molecules that cause protrusions of membrane ruffles that bring the bacterial cell in

Salmonella and Shigella

Salmonella Pathogenicity Island 1 (SPI1)-encoded T3SS translocates effectors that drive “trigger”-mediated invasion of host cells

• subset of T3SS1 effectors (SipA, SipC, SopB, SopE, SopE2) act in concert to induce massive localised rearrangements of actin and the plasma membrane (and activate signaling pathways)

After digestion of contaminated food, Yersinia pseudotuberculosis traverses through the gastrointestinal tract until the terminal ileum

• Present on their surface is the outer membrane (OM) protein invasion

• plays a crucial role during first phases of infection by facilitating efficient translocation across the intestinal epithelial barrier by binding beta-1 integrins expressed on surface of host cells - ZIPPER

Avoidance of the microbicidal lysosome

• Achieved by either subverting endosomal trafficking and remodeling of the phagosome into a hospitable vacuole

• Promoting phagosomal membrane disruption and escaping to the cytosol

• enhanced protection against immune recognition probably plays a key role

However, inherent challenges to a vacuolar lifestyle

• need for reprogramming endosomal traffic to avoid destruction by lysosomes

• vacuole remodeling to allow the acquisition of nutrients to support pathogen replication

These cells are known to initiate mucosal (at mucosal membranes of the intestines) immunity responses on the apical membrane of the M cells and allow for the transport of microbes and particles across the epithelial cell layer from the gut lumen to the lamina propria where interactions with immune cells can take place

When host cells, such as immune cells and phagocytes, actively endocytose pathogens

• Although phagocytosis allows the pathogen to gain entry to the host cell, in most cases, the host cell kills and degrades the pathogen by using digestive enzymes

• However, some intracellular pathogens have the ability to survive and multiply within phagocytes

• Normally, when a pathogen is ingested by a phagocyte, it is enclosed within a phagosome in the cytoplasm

• phagosome fuses with a lysosome to form a phagolysosome, where digestive enzymes kill the pathogen

They prevent fusion of phagosome with lysosome

• remain alive and dividing within the phagosome

(i) secrete effector proteins into cytosol via the SPI-2 encoded T3SS-2 and prevent fusion of the phagosome with the lysosome

(ii) within the SCV (Salmonella containing vacuole), Salmonella proliferate resulting in cytokine secretion

(iii) macrophage undergoes apoptosis, and Salmonella escapes innate immune system

L. monocytogenes and Shigella produce proteins that lyse the phagosome before it fuses with the lysosome

• Allows bacteria to escape into cytoplasm to multiply

• e.g. Shigella escape from phagosome/endosome by secretion of IpaB protein

→ Once inside phagosome/endosome, IpaB/C complex on tip of T3SS is inserted into membrane

→ Virulence factors are secreted into cytoplasm via T3SS, triggering host cell apoptosis, cytoskeleton rearrangement etc.

→ Virulence factors are also secreted into phagosome/endosome via assembled T3SS

→ Secreted IpaB protein forms a tetramer complex with inserted membrane and ion channels form

→ IpaB ion channels allows inflow/outflow of small molecules

→ Phagosome/endosome disrupted

Pathogens utilize host cell actin cytoskeleton to move in the cytosol of infected cells and project into adjacent cells through formation of membrane protrusions

• Formed protrusions resolve into vacuoles from which pathogen escapes, thereby gaining access to the cytosol of adjacent cells

Listeria monocytogenes

• Eating food that has been contaminated (raw milk products) with L. monocytogenes can result in life-threatening infections (pregnant women!)

• Bacteria first invade small intestinal epithelial cells

(a) Listeria invades host cells via a zipper mechanism

• interaction of surface internals InlA and InlB with host cell surface receptors E-cadherin and Met

(b) Escapes from phagosome before fusion with lysosome occurs

• action of secreted proteins, pore-forming toxin LLO, and phosphatidylinositide phospholipase C (PI-PLC)

(c) Replicates in cell cytosol, and

(d) Spreads by actin polymerization

• propels the bacteria unidirectionally

(e) Promotes cell-to-cell spreading of Listeria

(f) Rupture of two-membrane vacuole is mediated by action of LLO and PC-PLC

Most are unable to carry out energy metabolism and lack many biosynthetic pathways

→ entirely dependent on host cell to supply them with ATP (adenosine triphosphate) and other intermediate molecules

• Chlamydial growth cycle involves transformation between distinct forms

  • elementary body (EB) and reticulate body (RB)

• Highly infectious EB attaches to epithelial cells and induces ingestion by host cell

  • EB are metabolically inactive and represent the extracellular Chlamydia growth form

• Once ingested into a phagosome, fusion of the phagosome with the host lysosome is prevented

  • ensures EB survival

• EB reorganises within the phagosome into a metabolically active RB

  • RBs are noninfectious but can replicate and do so by binary fission

• Several stimuli, including antibiotics and cytokines, can drive chlamydia into a persistent state

  • lasts until removal of exogenous stressor

• If persistence is avoided, or if infection is reactivated from persistence, the RB will ultimately reorganise back into EB

• → released from host cell to infect surrounding epithelial cells

• Coxiella burnetii is an obligate intracellular bacterium that causes acute and chronic infections

  • Agent of Q fever

• After inhalation by a host, C. burnetii invades and replicates within alveolar macrophages without alerting the innate immune system

  • ‘stealth pathogen’

• Inside macrophages, bacterium replicates within a compartment that is very similar to a phagolysosome, termed the Coxiella- containing vacuole (CCV)

• Although an intracellular parasite under natural conditions, C. burnetii is culturable using specially designed axenic media

Group of obligate intracellular vector-borne Gram-negative bacteria that include many organisms of clinical and agricultural importance

• Anaplasma spp.

• Ehrlichia chaffeensis

• Rickettsia spp.

• Orientia tsutsugamushi

Each have a different method of intracellular survival

A degradative pathway by which cytosolic content, organelles and pathogens are delivered to lysosomes as part of cellular homeostasis and innate immunity