Explore the vast range of titles available.

Vibrio parahaemolyticus is a marine bacterium that thrives in warm climates. It is a leading cause of gastroenteritis resulting from consumption of contaminated uncooked shellfish. This bacterium harbors two putative type VI secretion systems (T6SS). T6SSs are widespread protein secretion systems found in many Gram-negative bacteria, and are often tightly regulated. For many T6SSs studied to date, the conditions and cues, as well as the regulatory mechanisms that control T6SS activity are unknown. In this study, we characterized the environmental conditions and cues that activate both V. parahaemolyticus T6SSs, and identified regulatory mechanisms that control T6SS gene expression and activity. We monitored the expression and secretion of the signature T6SS secreted proteins Hcp1 and Hcp2, and found that both T6SSs are differentially regulated by quorum sensing and surface sensing. We also showed that T6SS1 and T6SS2 require different temperature and salinity conditions to be active. Interestingly, T6SS1, which is found predominantly in clinical isolates, was most active under warm marine-like conditions. Moreover, we found that T6SS1 has anti-bacterial activity under these conditions. In addition, we identified two transcription regulators in the T6SS1 gene cluster that regulate Hcp1 expression, but are not required for immunity against self-intoxication. Further examination of environmental isolates revealed a correlation between the presence of T6SS1 and virulence of V. parahaemolyticus against other bacteria, and we also showed that different V. parahaemolyticus isolates can outcompete each other. We propose that T6SS1 and T6SS2 play different roles in the V. parahaemolyticus lifestyles, and suggest a role for T6SS1 in enhancing environmental fitness of V. parahaemolyticus in marine environments when competing for a niche in the presence of other bacterial populations.

The initial binding of bacteria to host cells is crucial to the delivery of virulence factors and thus is a key determinant of the pathogen's success. We report a multivalent adhesion molecule (MAM) that enables a wide range of Gram-negative pathogens to establish high-affinity binding to host cells during the early stages of infection. MAM7 binds to the host by engaging in both protein-protein (with fibronectin) and protein-lipid (with phosphatidic acid) interactions with the host cell membrane. We find that MAM7 expression on the outer membrane of a Gram-negative pathogen is necessary for virulence in a nematode infection model and for efficient killing of cultured mammalian host cells. Expression of MAM7 on nonpathogenic strains produced a tool that can be used to impede infection by Gram-negative bacterial pathogens. Targeting or exploiting MAM7 might prove to be important in combating Gram-negative bacterial infections.

It has been proposed that bacterial membrane proteins may be synthesized and inserted into the membrane by a process known as transertion, which involves membrane association of their encoding genes, followed by coupled transcription, translation and membrane insertion. Here, we provide evidence supporting that the pathogen Vibrio parahaemolyticus uses transertion to assemble its type III secretion system (T3SS2), to inject virulence factors into host cells. We propose a two-step transertion process where the membrane-bound co-component receptor (VtrA/VtrC) is first activated by bile acids, leading to membrane association and expression of its target gene, vtrB , located in the T3SS2 pathogenicity island. VtrB, the transmembrane transcriptional activator of T3SS2, then induces the localized expression and membrane assembly of the T3SS2 structural components and its effectors. We hypothesize that the proposed transertion process may be used by other enteric bacteria for efficient assembly of membrane-bound molecular complexes in response to extracellular signals. It has been proposed that bacterial membrane proteins may be produced via ‘transertion’, or concurrent transcription, translation and membrane insertion from membrane-associated genes. Here, Kaval et al. provide evidence supporting that Vibrio parahaemolyticus uses transertion to assemble a transmembrane complex (type III secretion system) used to inject virulence factors into host cells.

Key Points On infection, bacterial pathogens interact with host membranes to trigger various cellular processes through different mechanisms. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways that are involved in vesicle trafficking. Many bacterial effectors manipulate phosphoinositide (PI) homeostasis at the plasma membrane to destabilize actin dynamics and alter the morphology of the membrane. This facilitates the entry of pathogens or, in other cases, damages the cells by disrupting membrane integrity and eventually leading to rapid cell lysis in the later stage of infection. Some pathogens use bacterial phosphatases or PI adaptor proteins to form intracellular vacuoles that are derived from host membranes in order to establish a replicative niche. Altered PI levels at the surfaces of these vacuoles as a result of the activity of bacterial phosphatases can block phagosomal maturation to avoid lysosomal fusion. The GTPase signalling pathway is often targeted by bacterial pathogens to manipulate the actin cytoskeleton and endosomal trafficking. RAB GTPases , which have an important role in vesicular trafficking pathways, are recruited to bacterium-containing vacuoles, where their active state can be differentially regulated by effectors. Bacterial effectors mimic GTPase-activating protein (GAP) or guanine nucleotide exchange factor (GEF) activity to target RHO-family GTPases that are key regulators of actin dynamics. This results in loss of cell shape, motility and ability to phagocytose pathogens. Autophagy is one of the cellular defence mechanisms against the invasion of pathogenic bacteria. However, some pathogens have evolved strategies to subvert autophagy to their own advantage by establishing autophagic vesicles as their replicative niche. This allows them to survive inside host cells and avoid lysosomal degradation. Some bacterial effectors are speculated to induce autophagy during infection. This may not only protect the bacteria from degradative enzymes and immune responses, but also provide nutrients from cellular debris. For extracellular pathogens, inducing autophagy helps prevent phagocytosis. Bacterial pathogens secrete a range of effector proteins to target the signalling pathways that regulate host cell membranes. Here, Orth and colleagues describe the bacterial effectors that target phosphoinositide signalling, GTPase signalling and autophagy, and discuss how targeting these pathways can alter host membrane dynamics. Bacterial pathogens interact with host membranes to trigger a wide range of cellular processes during the course of infection. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways involved in vesicle trafficking. Such changes facilitate the entry and replication of the pathogen, and prevent its phagocytosis and degradation. In this Review, we describe the manipulation of host membranes by numerous bacterial effectors that target phosphoinositide metabolism, GTPase signalling and autophagy.

The production of antimicrobial reactive oxygen species by the nicotinamide dinucleotide phosphate (NADPH) oxidase complex is an important mechanism for control of invading pathogens. Herein, we show that the gastrointestinal pathogen Vibrio parahaemolyticus counteracts reactive oxygen species (ROS) production using the Type III Secretion System 2 (T3SS2) effector VopL. In the absence of VopL, intracellular V. parahaemolyticus undergoes ROS-dependent filamentation, with concurrent limited growth. During infection, VopL assembles actin into non-functional filaments resulting in a dysfunctional actin cytoskeleton that can no longer mediate the assembly of the NADPH oxidase at the cell membrane, thereby limiting ROS production. This is the first example of how a T3SS2 effector contributes to the intracellular survival of V. parahaemolyticus, supporting the establishment of a protective intracellular replicative niche.

Cancer evolves through a multistep process that occurs by the temporal accumulation of genetic mutations. Tumor-derived exosomes are emerging contributors to tumorigenesis. To understand how exosomes might contribute to cell transformation, we utilized the classic two-step NIH/3T3 cell transformation assay and observed that exosomes isolated from pancreatic cancer cells, but not normal human cells, can initiate malignant cell transformation and these transformed cells formed tumors in vivo. However, cancer cell exosomes are unable to transform cells alone or to act as a promoter of cell transformation. Utilizing proteomics and exome sequencing, we discovered cancer cell exosomes act as an initiator by inducing random mutations in recipient cells. Cells from the pool of randomly mutated cells are driven to transformation by a classic promoter resulting in foci, each of which encode a unique genetic profile. Our studies describe a novel molecular understanding of how cancer cell exosomes contribute to cell transformation. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that major issues remain unresolved (see decision letter ).

Bacteria use diverse mechanisms to kill, manipulate, and compete with other cells. The recently discovered type VI secretion system (T6SS) is widespread in bacterial pathogens and used to deliver virulence effector proteins into target cells. Using comparative proteomics, we identified two previously unidentified T6SS effectors that contained a conserved motif. Bioinformatic analyses revealed that this N-terminal motif, named MIX (marker for type six effectors), is found in numerous polymorphic bacterial proteins that are primarily located in the T6SS genome neighborhood. We demonstrate that several MIX-containing proteins are T6SS effectors and that they are not required for T6SS activity. Thus, we propose that MIX-containing proteins are T6SS effectors. Our findings allow for the identification of numerous uncharacterized T6SS effectors that will undoubtedly lead to the discovery of new biological mechanisms.

Pore-forming proteins (PFPs) represent a functionally important protein family, that are found in organisms from viruses to humans. As a major branch of PFPs, bacteria pore-forming toxins (PFTs) permeabilize membranes and usually cause the death of target cells. E. coli hemolysin ClyA is the first member with the pore complex structure solved among α-PFTs, employing α-helices as transmembrane elements. ClyA is proposed to form pores composed of various numbers of protomers. With high-resolution cryo-EM structures, we observe that ClyA pore complexes can exist as newly confirmed oligomers of a tridecamer and a tetradecamer, at estimated resolutions of 3.2 Å and 4.3 Å, respectively. The 2.8 Å cryo-EM structure of a dodecamer dramatically improves the existing structural model. Structural analysis indicates that protomers from distinct oligomers resemble each other and neighboring protomers adopt a conserved interaction mode. We also show a stabilized intermediate state of ClyA during the transition process from soluble monomers to pore complexes. Unexpectedly, even without the formation of mature pore complexes, ClyA can permeabilize membranes and allow leakage of particles less than ~400 Daltons. In addition, we are the first to show that ClyA forms pore complexes in the presence of cholesterol within artificial liposomes. These findings provide new mechanistic insights into the dynamic process of pore assembly for the prototypical α-PFT ClyA.

Coordinated assembly and disassembly of integrin-mediated focal adhesions (FAs) is essential for cell migration. Many studies have shown that FA disassembly requires Ca2+ influx, however our understanding of this process remains incomplete. Here, we show that Ca2+ influx via STIM1/Orai1 calcium channels, which cluster near FAs, leads to activation of the GTPase Arf5 via the Ca2+-activated GEF IQSec1, and that both IQSec1 and Arf5 activation are essential for adhesion disassembly. We further show that IQSec1 forms a complex with the lipid transfer protein ORP3, and that Ca2+ influx triggers PKC-dependent translocation of this complex to ER/plasma membrane (PM) contact sites adjacent to FAs. In addition to allosterically activating IQSec1, ORP3 also extracts PI4P from the PM, in exchange for phosphatidylcholine. ORP3-mediated lipid exchange is also important for FA turnover. Together, these findings identify a new pathway that links calcium influx to FA turnover during cell migration.

Vibrio parahaemolyticus is a globally disseminated Gram-negative marine bacterium and the leading cause of seafood-borne acute gastroenteritis. Pathogenic bacterial isolates encode two type III secretion systems (T3SS), with the second system (T3SS2) considered the main virulence factor in mammalian hosts. For many decades, V. parahaemolyticus has been studied as an exclusively extracellular bacterium. However, the recent characterization of the T3SS2 effector protein VopC has suggested that this pathogen has the ability to invade, survive, and replicate within epithelial cells. Herein, we characterize this intracellular lifestyle in detail. We show that following internalization, V. parahaemolyticus is contained in vacuoles that develop into early endosomes, which subsequently mature into late endosomes. V. parahaemolyticus then escapes into the cytoplasm prior to vacuolar fusion with lysosomes. Vacuolar acidification is an important trigger for this escape. The cytoplasm serves as the pathogen’s primary intracellular replicative niche; cytosolic replication is rapid and robust, with cells often containing over 150 bacteria by the time of cell lysis. These results show how V. parahaemolyticus successfully establishes an intracellular lifestyle that could contribute to its survival and dissemination during infection. IMPORTANCE The marine bacterium V. parahaemolyticus is the leading cause worldwide of seafood-borne acute gastroenteritis. For decades, the pathogen has been studied exclusively as an extracellular bacterium. However, recent results have revealed the pathogen’s ability to invade and replicate within host cells. The present study is the first characterization of the V. parahaemolyticus ’ intracellular lifestyle. Upon internalization, V. parahaemolyticus is contained in a vacuole that would in the normal course of events ultimately fuse with a lysosome, degrading the vacuole’s contents. The bacterium subverts this pathway, escaping into the cytoplasm prior to lysosomal fusion. Once in the cytoplasm, it replicates prolifically. Our study provides new insights into the strategies used by this globally disseminated pathogen to survive and proliferate within its host. The marine bacterium V. parahaemolyticus is the leading cause worldwide of seafood-borne acute gastroenteritis. For decades, the pathogen has been studied exclusively as an extracellular bacterium. However, recent results have revealed the pathogen’s ability to invade and replicate within host cells. The present study is the first characterization of the V. parahaemolyticus ’ intracellular lifestyle. Upon internalization, V. parahaemolyticus is contained in a vacuole that would in the normal course of events ultimately fuse with a lysosome, degrading the vacuole’s contents. The bacterium subverts this pathway, escaping into the cytoplasm prior to lysosomal fusion. Once in the cytoplasm, it replicates prolifically. Our study provides new insights into the strategies used by this globally disseminated pathogen to survive and proliferate within its host.