Dual 4‐ and 5‐phosphatase activities regulate SopB‐dependent phosphoinositide dynamics to promote bacterial entry

Salmonella are able to invade non‐phagocytic cells such as intestinal epithelial cells by modulating the host actin cytoskeleton to produce membrane ruffles. Two type III effector proteins SopB and SopE play key roles to this modulation. SopE is a known guanine nucleotide exchange factor (GEF) capable of activating Rac1 and CDC42. SopB is a phosphatidylinositol 4‐phosphatase and 5‐phosphatase promoting membrane ruffles and invasion of Salmonella through undefined mechanisms. Previous studies have demonstrated that the 4‐phosphatase activity of SopB is required for PtdIns‐3‐phosphate (PtdIns(3)P) accumulation and SopB‐mediated invasion. We show here that both the 4‐phosphatase as well as the 5‐phosphatase activities of SopB are essential in ruffle formation and subsequent invasion. We found that the 5‐phosphatase activity of SopB is likely responsible for generating PtdIns‐3,4‐bisphosphate (PtdIns(3,4)P₂) and subsequent recruitment of sorting nexin 9 (SNX9), an actin modulating protein. Intriguingly, the 4‐phosphatase activity is responsible for the dephosphorylation of PtdIns(3,4)P₂ into PtdIns(3)P. Alone, neither activity is sufficient for ruffling but when acting in conjunction with one another, the 4‐phosphatase and 5‐phosphatase activities led to SNX9‐mediated ruffling and Salmonella invasion. This work reveals the unique ability of bacterial effector protein SopB to utilize both its 4‐ and 5‐phosphatase activities to regulate phosphoinositide dynamics to promote bacterial entry.     

SulA-induced filamentation in Salmonella enterica serovar Typhimurium: effects on SPI-1 expression and epithelial infection

Aims: Salmonella enterica serovar Typhimurium is capable of adopting a filamentous phenotype in response to damage. How this adaptive response affects bacterial virulence is unclear. We have examined the hypothesis that filamentation affects the ability of Salmonella to infect host cells. Methods and Results: Expression of the cell division inhibitor SulA in Salm. Typhimurium SL1344 from an arabinose‐inducible plasmid resulted in filamentation. We examined expression of the type 3 secretion system (T3SS) encoded by Salmonella pathogenicity island 1 (SPI‐1) using SL1344 expressing a chromosomal PprgH‐gfp reporter. Single cell analysis of SulA‐induced SL1344 PprgH‐gfp revealed a relationship between increasing cell length and decreasing propensity for prgH expression, but there was no evidence of a significant change in prgH expression evident at the whole population level. Filamentous Salm. Typhimurium were capable of initiating membrane ruffling on MDCK epithelial cells, but only nonfilamentous bacteria (<6 μm) invade. Conclusions: Induction of SulA expression in Salmonella inhibits septation. Increasing filament length is associated with down‐regulation of SPI‐1 gene expression, but a significant proportion of filaments retain the ability to produce SPI‐1 T3SS and induce membrane ruffles on epithelia. Despite an active SPI‐1 T3SS, filamentous Salmonella are unable to invade epithelial cells. Significance and Impact of the Study: Our findings that filamentous Salmonella can express an invasive phenotype but fail to invade cells suggest that their presence in food does not constitute an immediate risk of infection until septation occurs. The described SulA expression model provides a convenient model for studying the impact of filamentation in the absence of additional stresses.     

Regulation of Salmonella-induced membrane ruffling by SipA differs in strains lacking other effectors

The Salmonella pathogenicity island 1 (SPI-1) type three secretion system (TTSS) is essential for Salmonella invasion of host cells through its triggering of actin-dependent membrane ruffles. The SPI-1 effectors SipA, SopE, SopE2 and SopB all have actin regulating activities and contribute to invasion. The precise role of actin regulation by SipA in Salmonella invasion remains controversial since divergent data have been presented regarding the relationship between SipA and membrane ruffling. We hypothesized that the contribution of SipA to membrane ruffling and invasion might vary between Salmonella strains. We compared the effects of SipA deletion on Salmonella enterica serovar Typhimurium ( S.  Typhimurium) strains that possess or lack SopE. Loss of SipA reduced invasion in the early stages of infection by SopE⁺ and SopE^- strains but the number of membrane ruffles elicited was unaffected. Salmonella strains lacking both SipA and SopE induced ruffles with very different morphology from those induced by wild-type strains or ones lacking single effectors, including the presence of highly dynamic finger-like protrusions and numerous filopodia. A similar phenotype was found for sipA ^- sopE ^-, sipA ^- sopE2 ^- and sipA ^- sopB ^- mutants. Thus, SipA plays a more prominent role in induction of invasion-competent membrane ruffles by Salmonella lacking a full complement of SPI-1 effectors.     

Quantitative insights into actin rearrangements and bacterial target site selection from Salmonella Typhimurium infection of micropatterned cells

Reorganization of the host cell actin cytoskeleton is crucial during pathogen invasion. We established micropatterned cells as a standardized infection model for cell invasion to quantitatively study actin rearrangements triggered by Salmonella Typhimurium (S. Tm). Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape avoiding strong cell‐to‐cell variations, a major limitation in classical cell culture conditions. S. Tm induced F‐actin‐rich ruffles and invaded micropatterned cells similar to unconstrained cells. Yet, standardized conditions allowed fast and unbiased comparison of cellular changes triggered by the SipA and SopE bacterial effector proteins. Intensity measurements in defined regions revealed that the content of pre‐existing F‐actin remained unchanged during infection, suggesting that newly polymerized F‐actin in bacteria‐triggered ruffles originates from the G‐actin pool. Analysing bacterial target sites, we found that bacteria did not show any preferences for the local actin cytoskeleton specificities. Rather, invasion was constrained to a specific ‘cell height’, due to flagella‐mediated near‐surface swimming. We found that invasion sites were similar to bacterial binding sites, indicating that S. Tm can induce a permissive invasion site wherever it binds. As micropatterned cells can be infected by many different pathogens they represent a valuable new tool for quantitative analysis of host–pathogen interactions.     

A role for the Salmonella Type III Secretion System 1 in bacterial adaptation to the cytosol of epithelial cells

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that invades the intestinal epithelium. Following invasion of epithelial cells, Salmonella survives and replicates within two distinct intracellular niches. While all of the bacteria are initially taken up into a membrane bound vacuole, the Salmonella‐containing vacuole or SCV, a significant proportion of them promptly escape into the cytosol. Cytosolic Salmonella replicates more rapidly compared to the vacuolar population, although the reasons for this are not well understood. SipA, a multi‐function effector protein, has been shown to affect intracellular replication and is secreted by cytosolic Salmonella via the invasion‐associated Type III Secretion System 1 (T3SS1). Here, we have used a multipronged microscopy approach to show that SipA does not affect bacterial replication rates per se, but rather mediates intra‐cytosolic survival and/or initiation of replication following bacterial egress from the SCV. Altogether, our findings reveal an important role for SipA in the early survival of cytosolic Salmonella.     

Outer membrane protein OmpV mediates Salmonella enterica serovar typhimurium adhesion to intestinal epithelial cells via fibronectin and α1β1 integrin

Salmonella typhimurium is an invasive Gram‐negative enteric bacterium, which causes salmonellosis, a type of gastroenteritis in humans and typhoid‐like symptoms in mice. Upon entering through the contaminated food and water, S. typhimurium adheres, colonises, and invades intestinal epithelial cells (IECs) of the small intestine. In this study, we have shown that upon deletion of the outer membrane protein OmpV, there is a significant decrease in adherence of S. typhimurium to the IECs, indicating that OmpV is an important adhesin of S. typhimurium. Further, our study showed that OmpV binds to the extracellular matrix component fibronectin and signals through α1β1 integrin receptor on the IECs and OmpV‐mediated activation of α1β1, resulting in the activation of focal adhesion kinase and F‐actin modulation. Actin modulation is crucial for bacterial invasion. To the best of our knowledge, this is the first report of an adhesin mediated its effect through integrin in S. typhimurium. Further, we have observed a decrease in pathogenicity in terms of increased LD₅₀ dose, lesser bacterial numbers in stool, and less colonisation of bacteria in different organs of mice infected with Δompv mutant compared with the wild‐type bacteria, thus confirming the crucial role of OmpV in the pathogenesis of S. typhimurium.     

The Annexin A2/p11 complex is required for efficient invasion of Salmonella Typhimurium in epithelial cells

The facultative intracellular pathogen, Salmonella enterica, triggers its own uptake into non‐phagocytic epithelial cells. Invasion is dependent on a type 3 secretion system (T3SS), which delivers a cohort of effector proteins across the plasma membrane where they induce dynamic actin‐driven ruffling of the membrane and ultimately, internalization of the bacteria into a modified phagosome. In eukaryotic cells, the calcium‐ and phospholipid‐binding protein Annexin A2 (AnxA2) functions as a platform for actin remodelling in the vicinity of dynamic cellular membranes. AnxA2 is mostly found in a stable heterotetramer, with p11, which can interact with other proteins such as the giant phosphoprotein AHNAK. We show here that AnxA2, p11 and AHNAK are required for T3SS‐mediated Salmonella invasion of cultured epithelial cells and that the T3SS effector SopB is required for recruitment of AnxA2 and AHNAK to Salmonella invasion sites. Altogether this work shows that, in addition to targeting Rho‐family GTPases, Salmonella can intersect the host cell actin pathway via AnxA2.     

Membrane ruffling requires coordination between type Ialpha phosphatidylinositol phosphate kinase and Rac signaling

Membrane ruffle formation requires remodeling of cortical actin filaments, a process dependent upon the small G-protein Rac. Growth factors stimulate actin remodeling and membrane ruffling by integration of signaling pathways that regulate actin-binding proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of many actin-binding proteins and is produced by the type I phosphatidylinositol phosphate kinases (PIPKIs). Here we show in MG-63 cells that only the PIPKIalpha isoform is localized to platelet-derived growth factor (PDGF)-induced membrane ruffles. Further, expression of kinase dead PIPKIalpha, which acts as a dominant negative mutant, blocked membrane ruffling, suggesting that PIPKIalpha and PIP2 participate in ruffling. To explore this, PIPKIalpha was overexpressed in serum-starved cells and stimulated with PDGF. In serum-starved cells, PIPKIalpha expression did not stimulate actin remodeling, but when these cells were stimulated with PDGF, actin rapidly reorganized into foci but not membrane ruffles. PIPKIalpha-mediated formation of actin foci was independent of both Rac1 and phosphatidylinositol 3-kinase activities. Significantly, coexpression of dominant active Rac1 with PIPKIalpha in PDGF-stimulated cells resulted in membrane ruffling. The PDGF- and Rac1-stimulated ruffling was inhibited by expression of kinase-dead PIPKIalpha. Combined, these data support a model where the localized production of PIP2 by PIPKIalpha is necessary for actin remodeling, whereas formation of membrane ruffles required Rac signaling.     

Role of the ESCRT‐III complex in controlling integrity of the Salmonella‐containing vacuole

Intracellular pathogens need to establish specialised niches for survival and proliferation in host cells. The enteropathogen Salmonella enterica accomplishes this by extensive reorganisation of the host endosomal system deploying the SPI2‐encoded type III secretion system (SPI2‐T3SS). Fusion events of endosomal compartments with the Salmonella‐containing vacuole (SCV) form elaborate membrane networks within host cells enabling intracellular nutrition. However, which host compartments exactly are involved in this process and how the integrity of Salmonella‐modified membranes is accomplished are not fully resolved. An RNA interference knockdown screen of host factors involved in cellular logistics identified the ESCRT (endosomal sorting complex required for transport) system as important for proper formation and integrity of the SCV in infected epithelial cells. We demonstrate that subunits of the ESCRT‐III complex are specifically recruited to the SCV and membrane network. To investigate the role of ESCRT‐III for the intracellular lifestyle of Salmonella, a CHMP3 knockout cell line was generated. Infected CHMP3 knockout cells formed amorphous, bulky SCV. Salmonella within these amorphous SCV were in contact with host cell cytosol, and the attenuation of an SPI2‐T3SS‐deficient mutant strain was partially abrogated. ESCRT‐dependent endolysosomal repair mechanisms have recently been described for other intracellular pathogens, and we hypothesise that minor damages of the SCV during bacterial proliferation are repaired by the action of ESCRT‐III recruitment in Salmonella‐infected host cells.     

Structural and functional characterization of SiiA,an auxiliary protein from the SPI4‐encoded type 1 secretion system from Salmonella enterica

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)‐encoded type one secretion system (T1SS) and the SPI1‐encoded type three secretion system (T3SS‐1). The SPI4‐encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure–function relationships in SiiA, a non‐canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA‐PD). The crystal structure of SiiA‐PD displays homology to that of MotB and other peptidoglycan (PG)‐binding domains. SiiA‐PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA‐MotB homology is paralleled by a predicted SiiB‐MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.     

RNAi screen of Salmonella invasion shows role of COPI in membrane targeting of cholesterol and Cdc42

The pathogen Salmonella Typhimurium is a common cause of diarrhea and invades the gut tissue by injecting a cocktail of virulence factors into epithelial cells, triggering actin rearrangements, membrane ruffling and pathogen entry. One of these factors is SopE, a G‐nucleotide exchange factor for the host cellular Rho GTPases Rac1 and Cdc42. How SopE mediates cellular invasion is incompletely understood. Using genome‐scale RNAi screening we identified 72 known and novel host cell proteins affecting SopE‐mediated entry. Follow‐up assays assigned these ‘hits’ to particular steps of the invasion process; i.e., binding, effector injection, membrane ruffling, membrane closure and maturation of the Salmonella‐containing vacuole. Depletion of the COPI complex revealed a unique effect on virulence factor injection and membrane ruffling. Both effects are attributable to mislocalization of cholesterol, sphingolipids, Rac1 and Cdc42 away from the plasma membrane into a large intracellular compartment. Equivalent results were obtained with the vesicular stomatitis virus. Therefore, COPI‐facilitated maintenance of lipids may represent a novel, unifying mechanism essential for a wide range of pathogens, offering opportunities for designing new drugs.     

IpgB1 is a novel Shigella effector protein involved in bacterial invasion of host cells. Its activity to promote membrane ruffling via Rac1 and Cdc42 activation

Shigella, the causative agent of bacillary dysentery, is capable of inducing the large scale membrane ruffling required for the bacterial invasion of host cells. Shigella secrete a subset of effectors via the type III secretion system (TTSS) into the host cells to induce membrane ruffling. Here, we show that IpgB1 is secreted via the TTSS into epithelial cells and plays a major role in producing membrane ruffles via stimulation of Rac1 and Cdc42 activities, thus promoting bacterial invasion of epithelial cells. The invasiveness of the ipgB1 mutant was decreased to less than 50% of the wild-type level (100%) in a gentamicin protection or plaque forming assay. HeLa cells infected with the wild-type or a IpgB1-hyperproducing strain developed membrane ruffles, with the invasiveness and the scale of membrane ruffles being comparable with the level of IpgB1 production in bacteria. Upon expression of EGFP-IpgB1 in HeLa cells, large membrane ruffles are extended, where the EGFP-IpgB1 was predominantly associated with the cytoplasmic membrane. The IpgB1-mediated formation of ruffles was significantly diminished by expressing Rac1 small interfering RNA and Cdc42 small interfering RNA or by treatment with GGTI-298, an inhibitor of the geranylgeranylation of Rho GTPases. When IpgB1 was expressed in host cells or wild-type Shigella-infected host cells, Rac1 and Cdc42 were activated. The results thus indicate that IpgB1 is a novel Shigella effector involved in bacterial invasion of epithelial cells via the activation of Rho GTPases.     

Shigella deliver an effector protein to trigger host microtubule destabilization,which promotes Rac1 activity and efficient bacterial internalization

Shigella deliver a subset of effectors into the host cell via the type III secretion system, that stimulate host cell signal pathways to modulate the actin dynamics required for invasion of epithelial cells. Here we show that one of the Shigella effectors, called VirA, can interact with tubulin to promote microtubule (MT) destabilization, and elicit protrusions of membrane ruffling. Under in vitro conditions, VirA inhibited polymerization of tubulin and stimulated MT destabilization. Upon microinjection of VirA into HeLa cells, a localized membrane ruffling was induced rapidly. Overexpression of VirA in host cells caused MT destruction and protruding membrane ruffles which were absent when VirA was co-expressed with a dominant-negative Rac1 mutant. Indeed, Shigella but not the virA mutant stimulated Rac1, including the formation of membrane ruffles in infected cells. Importantly, the MT structure beneath the protruding ruffling was destroyed. Furthermore, drug-induced MT growth in HeLa cells greatly enhanced the Shigella entry. These results indicate that VirA is a novel type of bacterial effector capable of inducing membrane ruffling through the stimulation of MT destabilization.     

Presence of SopE and mode of infection result in increased Salmonella‐containing vacuole damage and cytosolic release during host cell infection by Salmonella enterica

Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen that has evolved sophisticated molecular mechanisms to establish an intracellular niche within a specialised vesicular compartment, the Salmonella‐containing vacuole (SCV). The loss of the SCV and release of STM into the cytosol of infected host cells was observed, and a bimodal intracellular lifestyle of STM in the SCV versus life in the cytosol is currently discussed. We set out to investigate the parameters affecting SCV integrity and cytosolic release. A fluorescent protein‐based cytosolic reporter approach was established to quantify, time‐resolved, and on a single cell level, the release of STM into the cytosol of host cells. We observed that the extent of SCV damage and cytosolic release is highly dependent on experimental conditions such as multiplicity of infection, type of host cell line, and STM strain background. Trigger invasion mediated by the Salmonella Pathogenicity Island 1‐encoded type III secretion system (SPI1‐T3SS) and its effector proteins promoted cytosolic release, whereas cytosolic bacteria were rarely observed if entry was mediated by zipper invasion. Presence of SPI1‐T3SS effector SopE was identified as major factor for damage of the SCV in the early phase after STM invasion and sopE‐expressing strains showed higher levels of cytosolic release.     

Inhibition of the PtdIns(5) kinase PIKfyve disrupts intracellular replication of Salmonella

3‐phosphorylated phosphoinositides (3‐PtdIns) orchestrate endocytic trafficking pathways exploited by intracellular pathogens such as Salmonella to gain entry into the cell. To infect the host, Salmonellae subvert its normal macropinocytic activity, manipulating the process to generate an intracellular replicative niche. Disruption of the PtdIns(5) kinase, PIKfyve, be it by interfering mutant, siRNA‐mediated knockdown or pharmacological means, inhibits the intracellular replication of Salmonella enterica serovar typhimurium in epithelial cells. Monitoring the dynamics of macropinocytosis by time‐lapse 3D (4D) videomicroscopy revealed a new and essential role for PI(3,5)P₂ in macropinosome‐late endosome/lysosome fusion, which is distinct from that of the small GTPase Rab7. This PI(3,5)P₂‐dependent step is required for the proper maturation of the Salmonella‐containing vacuole (SCV) through the formation of Salmonella‐induced filaments (SIFs) and for the engagement of the Salmonella pathogenicity island 2‐encoded type 3 secretion system (SPI2‐T3SS). Finally, although inhibition of PIKfyve in macrophages did inhibit Salmonella replication, it also appears to disrupt the macrophage's bactericidal response.     

Involvement of SipA in modulating actin dynamics during Salmonella invasion into cultured epithelial cells

Salmonella entry into epithelial host cells results from the host actin cytoskeleton reorganization that is induced by a group of bacterial proteins delivered to the host cells by the Salmonella type III secretion system. SopE, SopE2 and SopB activate CDC42 and Rac1 to intercept the signal transduction pathways involved in actin cytoskeleton rearrangements. SipA and SipC directly bind actin to modulate the actin dynamics facilitating bacterial entry. Biochemical studies have indicated that SipA decreases the critical concentration for actin polymerization and may be involved in promoting the initial actin polymerization in Salmonella-induced actin reorganization. In this report, we conducted experiments to analyze the in vivo function(s) of SipA during Salmonella invasion. SipA was found to be preferentially associated with peripheral cortical actin filaments but not stress fibres using permeabilized epithelial cells. When polarized Caco-2 cells were infected with Salmonella, actin cytoskeleton rearrangements induced by the wild-type strain had many filopodia structures that were intimately associated with the bacteria. In contrast, ruffles induced by the sipA null mutant were smoother and distant from the bacteria. We also found that the F-actin content in cells infected with the sipA mutant decreased nearly 80% as compared to uninfected cells or those infected with the wild-type Salmonella strain. Furthermore, expression of either the full-length or the SipA(459-684) actin-binding fragment induced prominent punctuate actin assembly in the cortical region of COS-1 cells. These results indicate that SipA is involved in modulating actin dynamics in cultured epithelial cells during Salmonella invasion.