Sample/practice exam 2019, questions and answers PDF

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MST 1 Practice Short Answer:

The 2 main mechanisms are trigger and zipper mechanisms and both are analogous to regular host mechanisms. The zipper mechanism is analogous to receptor mediator endocytosis. It is passive for the bacteria, and an example of a bacteria that does this is Listeria. Listeria binds to host receptor Ecadherin using adhesins InlA and InlB. This triggers clustering of the receptors around the bacteria and engulfment in a vesicle and enters the endocytic pathway. The zipper mechanism can be mediated by phagocytic cells, such as macrophages or neutrophils. Alternatively, bacteria might use the trigger mechanism of invasion, which is homologous to macropinocytosis, a regular host uptake mechanism. This requires the bacteria to mediate initial loose attachment to the host cell, before expressing T3SS that translocate key effector proteins. These are responsible for modulate host cytoskeleton and causing membrane ruffling that encloses the bacteria into a vesicle. An example of a bacteria that does this is Shigella, which does this to transcytose through M-cells into the gastrointestinal epithelia to access the basolateral surface of endothelial cells where it can infect, replicate and spread. An example of a bacteria that forms a cytosolic niche is Listeria. As mentioned above, Listeria invades the host cell using the zipper mechanism by binding to E-cadherin using the InlA and InlB adhesins. The receptors cluster around the bacteria and it is engulfed in a phagosome. Listeria has an important transcription factor called prfA, which is responsible for binding to promoter regions of genes that are imperative for virulence and thus encourage RNA polymerase binding and gene expression. Some examples of the genes include Hly, an enzyme responsible for degrading the vacuolar membrane and allow the bacteria to escape into the cytosol. Once in the cytosol, Listeria forms actin tails. This is mediated by prfA ensuring the expression of ActA. This is localised to a pole of the bacteria and is translocated to the membrane using Sec translocase. Once at the membrane, it recruits VASP (a molecule like N-WASP) that recognises a motif on ActA. This recruit Arp2/3 which is responsible for nucleating and polymerising F-actin to produce long, branched actin tails that allow Listeria to move intracellularly and inter-cellular i.e. invade neighbouring cells. Note, listeria also has Listeria lysin O or LLO, which also degrades vacuolar membranes to escape into the cytosol.

Phagocytosis results in the engulfment of a bacteria by a phagocytic cell. It enters the cell in a vacuole called a phagosome, which will mature through the endocytic pathway with the assistance of endosomal compartments that distribute specific membrane markers in a “kiss and run” way. The

early endosome makes transient contact with the phagosome, distributing markers such as Rab5 GTPase and early endosomal antigen 1 (EEA1) to produce an early phagosome. The late endosome donates markers such as lysosomal associate membrane protein (LAMP), Rab7 and cathepsin proteases to produce a late phagosome. This then matures into a phagolysosome. The environment inside the vacuole becomes more and more hostile throughout the endocytic pathway, with reducing pH, increasing ROS and pH-dependent enzymes such as proteases, phospholipases and nucleases. Legionella pneumophila is an intravacuolar bacteria that prevents entering the endocytic pathway by disguising itself as the endoplasmic reticulum. After invasion of the cell, it produces a legionellacontaining vacuole (LCV) and builds a Type 4B Dot/Icm secretion system into the vacuolar membrane. This is responsible for secreting effector proteins that modulate host pathways and prove to be essential for the survival of the bacteria. An example of this is RaiF, which recruits host Arf1. Another key example is Legionella’s modulation of host Rab-1 GTPase. In normal circumstances, Rab-1 is found associated with GDP as Rab1-GDP, and with the assistance of Rab-GEF, the GDP is swapped for a GTP and it forms Rab1-GTP. This has functions such as attracting host ER vesicles. To inactivate, Rab-GAP removes the GTP and returns the GDP. Using its Dot/Icm T4BSS, Legionella translocates effector proteins such as SidM. This is an AMPylase i.e. it adds an AMP group to Rab-1, thus activating it and causing the recruitment of ER vesicles to the surface of the LCV. This contains ER markers such as calnexin and ribosomes, and as a result it will not enter the endosomal pathway. SidD is responsible for removing the AMP and de-activating Rab-1, and LepB removes Rab-1 i.e. has GAP activity. Note, there is often redundancy in Legionella effector proteins. AnkX/Lem3 have the same function as SidM/SidD, however add or remove phosphocholine instead of AMP....