Strategies for Intracellular Survival of Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality that is prevalent in tropical regions of the world. A key component of the pathogenesis of melioidosis is the ability of B. pseudomallei to enter, survive, and replicate within mammalian host cells. For non-phagocytic cells, bacterial adhesins have been identified both on the bacterial surface and associated with Type 4 pili. Cell invasion involves components of one or more of the three Type 3 Secretion System clusters, which also mediate, at least in part, the escape of bacteria from the endosome into the cytoplasm, where bacteria move by actin-based motility. The mechanism of actin-based motility is not clearly understood, but appears to differ from characterized mechanisms in other bacterial species. A small proportion of intracellular bacteria is targeted by host cell autophagy, involving direct recruitment of LC3 to endosomes rather than through uptake by canonical autophagosomes. However, the majority of bacterial cells are able to circumvent autophagy and other intracellular defense mechanisms such as the induction of inducible nitric oxide synthase, and then replicate in the cytoplasm and spread to adjacent cells via membrane fusion, resulting in the formation of multi-nucleated giant cells. A potential role for host cell ubiquitin in the autophagic response to bacterial infection has recently been proposed.

Keywords: Burkholderia; adhesion; autophagy; intracellular survival; melioidosis; pathogenesis; ubiquitination.

Figure 1

Schematic representation of selected steps in the intracellular lifestyle of B. pseudomallei. B. pseudomallei can be internalized by either phagocytic or non-phagocytic cells. In non-phagocytic cells PilA and the adhesins BoaA and BoaB are critical for uptake. For the internalization step in non-phagocytic cells the Bsa T3SS structural proteins BipD (translocator), BsaQ (structural component), and BopE (effector) are required. The IrlRS two-component signal transduction system is predicted to regulate the expression of other key gene(s) involved in internalization. For internalization in phagocytic cells the Bsa T3SS putative effector BopA and the alternate sigma factor RpoS play minor roles. The Bsa T3SS structural proteins BsaZ, BsaQ, and BsaU, the translocator protein BipD and the putative effector BopA are critical for membrane disruption and escape from the endocytic vesicle. Once in the cytoplasm B. pseudomallei can replicate and move by actin-based motility in a BimA-dependent fashion. B. pseudomallei LPS only weakly stimulates IFN secretion, which results in reduced iNOS expression and NO production. Furthermore, B. pseudomallei induces expression of suppressor of cytokine signaling 3 (SOCS3) and cytokine-inducible Src homology 2-containing (CIS) proteins which inhibit the Janus kinases – signal transducers and activators of transcription (JAK–STAT) signaling pathway and concomitant iNOS activation. Purine (PurM, PurN), histidine (HisF), and para-aminobenzoate (PabB) biosynthetic pathways are important for intracellular replication and survival, while the B. pseudomallei T6SS-1 Hcp protein, the alternate sigma factor RpoS, and the Bsa T3SS protein BipB are involved in stimulating cell-to-cell fusion and the formation of MNGC. NOD2 refers to nucleotide-binding oligomerization domain-containing protein 2. Green arrows are used to indicate progression of time. Red T-bar arrows indicate inhibitory interactions. Secretion system components are color coded with effectors in purple, translocators in black, and structural components in red.

Figure 2

Possible fates of B. pseudomallei in infected macrophages. Following phagocytic uptake by macrophages, bacteria are first located within phagosomes. The majority of wild-type bacteria can escape from the phagosome into the cytosol (pathway B) in a process which is largely uncharacterized but involves the Bsa T3SS. Once free in the cytosol bacteria activate BimA-mediated actin-based motility and replicate (pathway C). Potentially some cytosolic bacteria may be sequestered in canonical autophagosomes (pathway D), but the available evidence suggests that this occurs very infrequently. The autophagy marker protein LC3 can be recruited to bacteria-containing phagosomes, a process designated LC3-associated phagocytosis (LAP) which stimulates further phagosomal maturation via recruitment of other proteins, and the subsequent fusion of phagosomes with lysosomes, leading to bacterial killing (pathway A). It is possible that bacteria may escape from LAP, but definitive evidence that this occurs is presently lacking.