Subversion of membrane transport pathways by vacuolar pathogens

Abstract

Mammalian phagocytes control bacterial infections effectively through phagocytosis, the process by which particles engulfed at the cell surface are transported to lysosomes for destruction. However, intracellular pathogens have evolved mechanisms to avoid this fate. Many bacterial pathogens use specialized secretion systems to deliver proteins into host cells that subvert signaling pathways controlling membrane transport. These bacterial effectors modulate the function of proteins that regulate membrane transport and alter the phospholipid content of membranes. Elucidating the biochemical function of these effectors has provided a greater understanding of how bacteria control membrane transport to create a replicative niche within the host and provided insight into the regulation of membrane transport in eukaryotic cells.

Figure 1.

Mtb and S. Typhimurium manipulate the fate of their vacuole through modification of phosphoinositide metabolism. (A) Normal maturation of a phagosome containing nonpathogenic bacteria. After phagocytosis, bacteria reside in a vacuole showing similarities with early endosomes, notably presenting the small GTPase Rab5. Rab5 recruits the PI3 kinase hVPS34 that produces PI3P at the phagosome surface. The presence of PI3P is required for the maturation of the phagosome to phagolysosome by the recruitment of a subset of proteins, including EEA1. (B) Mtb creates a replicative niche by manipulating PI3P metabolism. Mtb blocks the activation of hVPS34 at its vacuole by ManLAM, thereby preventing PI3P production. The mechanism for this block involves the inhibition of Ca²⁺ rise, which is necessary for hVPS34 activation through a cascade involving calmodulin. Moreover, Mtb secretes SapM, a phosphatase that could be involved in depleting the vacuole from any residual PI3P. Finally, Mtb can expand its vacuole by recruitment of endosome vesicles. This recruitment could be achieved by the Mtb lipid PIM, a phosphoinositide analogue. (C) The S. Typhimurium T4SS effector SopB generates PI3P at the vacuole membrane. A possible mechanism for PI3P enrichment at the early S. Typhimurium vacuole is an indirect modulation of hVPS34 recruitment by SopB. This results in a prolonged and increased presence of PI3P at the vacuole surface. The presence of a high amount of PI3P induces subsequent recruitment of PI3P-binding proteins, including SNX1, SNX3, and PIKfyve, which were shown to be required for the maturation of the S. Typhimurium–containing vacuole (SCV).

Figure 2.

Intracellular bacteria subvert the eukaryotic cytoskeleton to create a replicative niche. Two representative examples are depicted here. S. Typhimurium uses microtubules to form SIFs. SIFs are typical structures of the SCV. The mechanism of their formation, which involves microtubules, is not completely understood. At least two T3SS effectors, SifA and PipB2, play a role in SIF formation. They target the mammalian proteins SKIP and kinesin-1 and altogether induce protrusion and expansion of tubules from the SCV. C. trachomatis actively induces polymerization of actin and intermediate filaments to consolidate its large vacuole. This relies on an unknown bacterial effector and the host GTPase RhoA.

Figure 3.

Pathogens manipulate host GTPases. (A) Positive regulation of host GTPases. The S. Typhimurium effector SopE binds and activates Rho/Rac/Cdc42 GTPases, thereby stimulating assembly of an actin-based network at the plasma membrane. SopE acts as a GEF and catalyzes the exchange of GTP for GDP. Similarly, the L. pneumophila effector DrrA functions as a GEF for the small GTPase Rab1 (a key GTPase involved in ER to Golgi and intra-Golgi vesicular transport) and activates it on the LCV. (B) Negative regulation of host GTPases. Bacteria have evolved several ways to negatively regulate GTPases. First, bacteria encode GAPs, such as SptP and LepB, to catalyze the hydrolysis of GTP to GDP to inactivate GTPases. Second, bacteria encode enzymes, such as VopS, IbpA, and DrrA, that inhibit downstream signaling of GTPases by posttranslationally modifying them with AMP. AMPylated Rac failed to interact with its effector p21-activated kinase (PAK). Similarly, AMPylated Rab1 was unable to bind to its effector MICAL-3 (microtubule-associated monoxygenase, calponin, and LIM domain containing 3). Recently, the L. pneumophila effector SidD has been shown to act on Rab1 to remove this AMP modification. Third, a novel modification on Rab1 and Rab35 was reported whereby the L. pneumophila effector AnkX modified the class II switch region of the Rabs with a phosphocholine moiety. This modification was observed in Rabs bound to both GDP and GTP. Phosphocholination of Rab35-GDP prevented its binding to its GEF connecdenn (CD). Finally, effectors such as YopT function as proteases to cleave Rho GTPases to inhibit their function.