MxiA, MxiC and IpaD Regulate Substrate Selection and Secretion Mode in the T3SS of <i>Shigella flexneri</i>

Type III secretion systems (T3SSs) are central virulence devices for many Gram-negative bacterial pathogens of humans, animals & plants. Upon physical contact with eukaryotic host cells, they translocate virulence-mediating proteins, known as effectors, into them during infection. T3SSs are gated from the outside by host-cell contact and from the inside via two cytoplasmic negative regulators, MxiC and IpaD in Shigella flexneri, which together control the effector secretion hierarchy. Their absence leads to premature and increased secretion of effectors. Here, we investigated where and how these regulators act. We demonstrate that the T3SS inner membrane export apparatus protein MxiA plays a role in substrate selection. Indeed, using a genetic screen, we identified two amino acids located on the surface of MxiA's cytoplasmic region (MxiAC) which, when mutated, upregulate late effector expression and, in the case of MxiAI674V, also secretion. The cytoplasmic region of MxiA, but not MxiAN373D and MxiAI674V, interacts directly with the C-terminus of MxiC in a two-hybrid assay. Efficient T3S requires a cytoplasmic ATPase and the proton motive force (PMF), which is composed of the ΔΨ and the ΔpH. MxiA family proteins and their regulators are implicated in utilization of the PMF for protein export. However, our MxiA point mutants show similar PMF utilisation to wild-type, requiring primarily the ΔΨ. On the other hand, lack of MxiC or IpaD, renders the faster T3S seen increasingly dependent on the ΔpH. Therefore, MxiA, MxiC and IpaD act together to regulate substrate selection and secretion mode in the T3SS of Shigella flexneri

How do the virulence factors of shigella work together to cause disease?

Shigella is the major cause of bacillary dysentery world-wide. It is divided into four species, named S. flexneri, S. sonnei, S. dysenteriae, and S. boydii, which are distinct genomically and in their ability to cause disease. Shigellosis, the clinical presentation of Shigella infection, is characterized by watery diarrhea, abdominal cramps, and fever. Shigella's ability to cause disease has been attributed to virulence factors, which are encoded on chromosomal pathogenicity islands and the virulence plasmid. However, information on these virulence factors is not often brought together to create a detailed picture of infection, and how this translates into shigellosis symptoms. Firstly, Shigella secretes virulence factors that induce severe inflammation and mediate enterotoxic effects on the colon, producing the classic watery diarrhea seen early in infection. Secondly, Shigella injects virulence effectors into epithelial cells via its Type III Secretion System to subvert the host cell structure and function. This allows invasion of epithelial cells, establishing a replicative niche, and causes erratic destruction of the colonic epithelium. Thirdly, Shigella produces effectors to down-regulate inflammation and the innate immune response. This promotes infection and limits the adaptive immune response, causing the host to remain partially susceptible to re-infection. Combinations of these virulence factors may contribute to the different symptoms and infection capabilities of the diverse Shigella species, in addition to distinct transmission patterns. Further investigation of the dominant species causing disease, using whole-genome sequencing and genotyping, will allow comparison and identification of crucial virulence factors and may contribute to the production of a pan-Shigella vaccine

Composition and activity of the non-canonical Gram-positive SecY2 complex

The accessory Sec system in Streptococcus gordonii DL1 is a specialized export system that transports a large serine-rich repeat protein, Hsa, to the bacterial surface. The system is composed of core proteins SecA2 and SecY2 and accessory Sec proteins Asp1–Asp5. Similar to canonical SecYEG, SecY2 forms a channel for translocation of the Hsa adhesin across the cytoplasmic membrane. Accessory Sec proteins Asp4 and Asp5 have been suggested to work alongside SecY2 to form the translocon, similar to the associated SecY, SecE, and SecG of the canonical system (SecYEG). To test this theory, S. gordonii secY2, asp4, and asp5 were co-expressed in Escherichia coli. The resultant complex was subsequently purified, and its composition was confirmed by mass spectrometry to be SecY2-Asp4-Asp5. Like SecYEG, the non-canonical complex activates the ATPase activity of the SecA motor (SecA2). This study also shows that Asp4 and Asp5 are necessary for optimal adhesion of S. gordonii to glycoproteins gp340 and fibronectin, known Hsa binding partners, as well as for early stage biofilm formation. This work opens new avenues for understanding the structure and function of the accessory Sec system

The Needle Component of the Type III Secreton of Shigella Regulates the Activity of the Secretion Apparatus

Gram-negative bacteria commonly interact with eukaryotic host cells by using type III secretion systems (TTSSs or secretons). TTSSs serve to transfer bacterial proteins into host cells. Two translocators, IpaB and IpaC, are first inserted with the aid of IpaD by Shigella into the host cell membrane. Then at least two supplementary effectors of cell invasion, IpaA and IpgD, are transferred into the host cytoplasm. How TTSSs are induced to secrete is unknown, but their activation appears to require direct contact of the external distal tip of the apparatus with the host cell. The extracellular domain of the TTSS is a hollow needle protruding 60 nm beyond the bacterial surface. The monomeric unit of the Shigella flexneri needle, MxiH, forms a superhelical assembly. To probe the role of the needle in the activation of the TTSS for secretion, we examined the structure-function relationship of MxiH by mutagenesis. Most point mutations led to normal needle assembly, but some led to polymerization or possible length control defects. In other mutants, secretion was constitutively turned “on.” In a further set, it was “constitutively on” but experimentally “uninducible.” Finally, upon induction of secretion, some mutants released only the translocators and not the effectors. Most types of mutants were defective in interactions with host cells. Together, these data indicate that the needle directly controls the activity of the TTSS and suggest that it may be used to “sense” host cells

The Architecture of the Cytoplasmic Region of Type III Secretion Systems

Type III secretion systems (T3SSs) are essential devices in the virulence of many Gram-negative bacterial pathogens. They mediate injection of protein effectors of virulence from bacteria into eukaryotic host cells to manipulate them during infection. T3SSs involved in virulence (vT3SSs) are evolutionarily related to bacterial flagellar protein export apparatuses (fT3SSs), which are essential for flagellar assembly and cell motility. The structure of the external and transmembrane parts of both fT3SS and vT3SS is increasingly well-defined. However, the arrangement of their cytoplasmic and inner membrane export apparatuses is much less clear. Here we compare the architecture of the cytoplasmic regions of the vT3SSs of Shigella flexneri and the vT3SS and fT3SS of Salmonella enterica serovar Typhimurium at ~5 and ~4 nm resolution using electron cryotomography and subtomogram averaging. We show that the cytoplasmic regions of vT3SSs display conserved six-fold symmetric features including pods, linkers and an ATPase complex, while fT3SSs probably only display six-fold symmetry in their ATPase region. We also identify other morphological differences between vT3SSs and fT3SSs, such as relative disposition of their inner membrane-attached export platform, C-ring/pods and ATPase complex. Finally, using classification, we find that both types of apparatuses can loose elements of their cytoplasmic region, which may therefore be dynamic

Steps for Shigella Gatekeeper Protein MxiC Function in Hierarchical Type III Secretion Regulation

Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions

The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements