Olivia Steele-Mortimer

Endoplasmic Reticulum-Mediated Phagocytosis Is a Mechanism of Entry into Macrophages

Phagocytosis is a key aspect of our innate ability to fight infectious diseases. In this study, we have found that fusion of the endoplasmic reticulum (ER) with the macrophage plasmalemma, underneath phagocytic cups, is a source of membrane for phagosome formation in macrophages. Successive waves of ER become associated with maturing phagosomes during phagolysosome biogenesis. Thus, the ER appears to possess unexpectedly pluripotent fusion properties. ER-mediated phagocytosis is regulated in part by phosphatidylinositol 3-kinase and used for the internalization of inert particles and intracellular pathogens, regardless of their final trafficking in the host. In neutrophils, where pathogens are rapidly killed, the ER is not used as a major source of membrane for phagocytosis. We propose that intracellular pathogens have evolved to adapt and exploit ER-mediated phagocytosis to avoid destruction in host cells.

Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia

Salmonella enterica is an intracellular bacterial pathogen that resides and proliferates within a membrane-bound vacuole in epithelial cells of the gut and gallbladder. Although essential to disease, how Salmonella escapes from its intracellular niche and spreads to secondary cells within the same host, or to a new host, is not known. Here, we demonstrate that a subpopulation of Salmonella hyperreplicating in the cytosol of epithelial cells serves as a reservoir for dissemination. These bacteria are transcriptionally distinct from intravacuolar Salmonella. They are induced for the invasion-associated type III secretion system and possess flagella; hence, they are primed for invasion. Epithelial cells laden with these cytosolic bacteria are extruded out of the monolayer, releasing invasion-primed and -competent Salmonella into the lumen. This extrusion mechanism is morphologically similar to the process of cell shedding required for turnover of the intestinal epithelium. In contrast to the homeostatic mechanism, however, bacterial-induced extrusion is accompanied by an inflammatory cell death characterized by caspase-1 activation and the apical release of IL-18, an important cytokine regulator of gut inflammation. Although epithelial extrusion is obviously beneficial to Salmonella for completion of its life cycle, it also provides a mechanistic explanation for the mucosal inflammation that is triggered during Salmonella infection of the gastrointestinal and biliary tracts.

Taking Possession: Biogenesis of the Salmonella‐Containing Vacuole

The Gram‐negative pathogen Salmonella enterica can survive and replicate within a variety of mammalian cells. Regardless of the cell type, internalized bacteria survive and replicate within the Salmonella‐containing vacuole, the biogenesis of which is dependent on bacterially encoded virulence factors. In particular, Type III secretion systems translocate bacterial effector proteins into the eukaryotic cell where they can specifically interact with a variety of targets. Salmonella has two distinct Type III secretion systems that are believed to have completely different functions. The SPI2 system is induced intracellularly and is required for intracellular survival in macrophages; it plays no role in invasion but is categorized as being required for Salmonella‐containing vacuole biogenesis. In contrast, the SPI1 Type III secretion system is induced extracellularly and is essential for invasion of nonphagocytic cells. Its role in post‐invasion processes has not been well studied. Recent studies indicate that Salmonella‐containing vacuole biogenesis may be more dependent on SPI1 than previously believed. Other non‐SPI2 virulence factors and the host cell itself may play critical roles in determining the intracellular environment of this facultative intracellular pathogen. In this review we discuss the recent advances in determining the mechanisms by which Salmonella regulate Salmonella‐containing vacuole biogenesis and the implications of these findings.

Salmonella type III effectors PipB and PipB2 are targeted to detergent-resistant microdomains on internal host cell membranes

The intracellular pathogen, Salmonella enterica, translocates type III effectors across its vacuolar membrane into host cells. Herein we describe a new Salmonella effector, PipB2, which has sequence similarity to another type III effector, PipB. In phagocytic cells, PipB2 localizes to the Salmonella‐containing vacuole (SCV) and tubular extensions from the SCV, Salmonella‐induced filaments (Sifs). We used the specific targeting of PipB2 in macrophages to characterize Sifs in phagocytic cells for the first time. In epithelial cells, PipB2 has a unique localization pattern, localizing to SCVs and Sifs and additionally to vesicles at the periphery of infected cells. We further show that the N‐terminal 225‐amino‐acid residues of PipB2 are sufficient for type III translocation and association with SCVs and Sifs, but not peripheral vesicles. Subcellular fractionation demonstrated that both PipB and PipB2 associate with host cell membranes and resist extraction by high salt, high pH and to a significant extent, non‐ionic detergent. Furthermore, PipB and PipB2 are enriched in detergent‐resistant microdomains (DRMs), also known as lipid rafts, present on membranes of SCVs and Sifs. The enrichment of Salmonella effectors in DRMs on these intracellular membranes probably permits specific interactions with host cell molecules that are concentrated in these signalling platforms.

Salmonella trafficking is defined by continuous dynamic interactions with the endolysosomal system.

Following invasion of non‐phagocytic host cells, Salmonella enterica survives and replicates within a phagosome‐like compartment known as the Salmonella‐containing vacuole (SCV). It is now well established that SCV biogenesis, like phagosome biogenesis, involves sequential interactions with the endocytic pathway. However, Salmonella is believed to limit these interactions and, in particular, to avoid fusion of terminal lysosomes with the SCV. In this study, we reassessed this process using a high‐resolution live‐cell imaging approach and found an unanticipated level of interaction between the SCV and the endocytic pathway. Direct interactions, in which late endosomal/lysosomal content was transferred to SCVs, were detected within 30 min of invasion and continued for several hours. Mechanistically, these interactions were very similar to phagosome–lysosome fusion because they were accompanied by rapid acidification of the SCV, could be blocked by chemical perturbation of microtubules or vacuolar acidification and involved the small GTPase Rab7. In comparison with vacuoles containing internalized Escherichia coli or heat‐killed Salmonella, SCVs did show some delay of fusion and acidification, although, this appeared to be independent of either type III secretion system. These results provide compelling evidence that inhibition of SCV–lysosome fusion is not the major determinant in establishment of the Salmonella replicative niche in epithelial cells.

The Salmonella effector protein PipB2 is a linker for kinesin-1

Understanding the mechanisms of Salmonella virulence is an important challenge. The capacity of this intracellular bacterial pathogen to cause diseases depends on the expression of virulence factors including the second type III secretion system (TTSS-2), which is used to translocate into the eukaryotic cytosol a set of effector proteins that divert the biology of the host cell and shape the bacterial replicative niche. Yet little is known about the eukaryotic functions affected by individual Salmonella effectors. Here we report that the TTSS-2 effector PipB2 interacts with the kinesin light chain, a subunit of the kinesin-1 motor complex that drives anterograde transport along microtubules. Translocation of PipB2 is both necessary and sufficient for the recruitment of kinesin-1 to the membrane of the Salmonella-containing vacuole. In vivo, PipB2 contributes to the attenuation of Salmonella mutant strains in mice. Taken together, our data indicate that the TTSS-2-mediated fine-tuning of kinesin-1 activity associated with the bacterial vacuole is crucial for the virulence of Salmonella.

HilD-mediated transcriptional cross-talk between SPI-1 and SPI-2

The acquisition of new genetic traits by horizontal gene transfer and their incorporation into preexisting regulatory networks have been essential events in the evolution of bacterial pathogens. An example of successful assimilation of virulence traits is Salmonella enterica, which acquired, at distinct evolutionary times, Salmonella pathogenicity island 1 (SPI-1), required for efficient invasion of the intestinal epithelium and intestinal disease, and SPI-2, essential for Salmonella replication and survival within macrophages and the progression of a systemic infection. A positive regulatory cascade mainly composed of HilD, HilA, and InvF, encoded in SPI-1, controls the expression of SPI-1 genes, whereas the two-component regulatory system SsrA/B, encoded in SPI-2, controls expression of SPI-2 genes. In this study, we report a previously undescribed transcriptional cross-talk between SPI-1 and SPI-2, where the SPI-1–encoded regulator HilD is essential for the activation of both the SPI-1 and SPI-2 regulons but at different times during the stationary phase of growth in Luria-Bertani medium. Our data indicate that HilD counteracts the H-NS–mediated repression exerted on the OmpR-dependent activation of the ssrAB operon by specifically interacting with its regulatory region. In contrast, HilD is not required for SPI-2 regulon expression under the in vitro growth conditions that are thought to resemble the intracellular environment. Our results suggest that two independent SPI-2 activation pathways evolved to take advantage of the SPI-2–encoded information at different niches and, in consequence, in response to different growth conditions.

The Mechanism of Salmonella Entry Determines the Vacuolar Environment and Intracellular Gene Expression

Macrophages are an important intracellular niche for Salmonella particularly for systemic infection. The interaction of Salmonella with these cells is mediated by two type III secretion systems (TTSS), encoded on Salmonella pathogenicity islands 1 and 2 (SPI1, SPI2), which mediate distinct phases of the pathogen–host cell interaction. The SPI1 TTSS mediates invasion whereas the SPI2 TTSS is required for intramacrophage survival. Importantly, however, Salmonella can enter macrophages by either SPI1‐dependent invasion or host cell‐mediated phagocytosis. Here, we investigated how the mechanism of internalization affects the intracellular environment and TTSS gene expression. Intracellular bacterial survival depended on the method of entry, because complement‐opsonized and SPI1‐induced Salmonella initiated replication within 8 h whereas immunoglobulin G (IgG)‐opsonized and non‐opsonized Salmonella were initially killed. Analysis of vacuolar pH showed that acidification of the Salmonella‐containing vacuole occurred more rapidly for non‐opsonized or SPI1‐induced Salmonella compared with IgG‐opsonized or complement‐opsonized Salmonella. Finally, quantitative polymerase chain reaction was used to compare the transcriptional profiles of selected SPI1 and SPI2 regulon genes. We found that the magnitude of SPI2 gene induction depended on the mechanism of internalization. Unexpectedly, SPI1 genes, which are rapidly downregulated following SPI1‐mediated invasion, were induced intracellularly following phagocytic uptake. These results reveal another level of complexity in pathogen–macrophage interactions.

The Salmonella SPI1 effector SopB stimulates nitric oxide production long after invasion.

The ability of Salmonella enterica to invade and replicate within host cells depends on two type III secretion systems (TTSSs) encoded on pathogenicity islands 1 and 2 (SPI1 and SPI2). The current paradigm holds that these systems translocate two classes of effectors that operate sequentially and independently. In essence, the SPI1 TTSS mediates early events (i.e. invasion) whereas the SPI2 TTSS mediates post‐invasion processes (i.e. replication, vacuole maturation). Contrary to this model, we have found in infected macrophages that a SPI1 effector, SopB/SigD, increased inducible nitric oxide synthase levels and nitric oxide production, host cell process previously known only to be a target of the SPI2 TTSS. Furthermore, SopB protein and message persist many hours after invasion. Our findings reveal an unanticipated potential for dialogue between the SPI1 and SPI2 TTSS and the host cell response.

Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.

The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella‐containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono‐ubiquitinated. Substitution of these six lysine residues with arginine, SopB‐K6R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB‐dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro‐survival kinase Akt/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB‐K6R mutant. At later times, fewer SCV were decorated with SopB‐K6R compared with SopB. Instead SopB‐K6R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.