Protein Secretion System Research Articles

Twin-arginine translocation (Tat) mutants in Salmonella enterica serovar Typhimurium have increased susceptibility to cell wall targeting antibiotics.

ABSTRACTThe twin-arginine translocation (Tat) system is a protein secretion system that is conserved in bacteria, archaea and plants. In Gram-negative bacteria, it is required for the export of folded proteins from the cytoplasm to the periplasm. There are 30 experimentally verified Tat substrates in Salmonella, including hydrogenase subunits, enzymes required for anaerobic respiration and enzymes involved in peptidoglycan remodeling during cell division. Multiple studies have demonstrated the susceptibility of tat mutants to antimicrobial compounds such as SDS and bile; however, in this work, we use growth curves and viable plate counts to demonstrate that cell wall targeting antibiotics (penicillins, carbapenems, cephalosporins and fosfomycin) have increased killing against a Δtat strain. Further, we demonstrate that this increased killing is primarily due to defects in translocation of critical Tat substrates: MepK, AmiA, AmiC and SufI. Finally, we show that a ΔhyaAB ΔhybABC ΔhydBC strain has an altered ΔΨ that impacts proper secretion of critical Tat substrates in aerobic growth conditions.

Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

Extreme genetic diversity in the type VII secretion system of Listeria monocytogenes suggests a role in bacterial antagonism.

The type VII protein secretion system (T7SS) has been characterized in members of the phyla Actinobacteria and Firmicutes. In mycobacteria the T7SS is intimately linked with pathogenesis and intracellular survival, while in Firmicutes there is mounting evidence that the system plays a key role in interbacterial competition. A conserved membrane-bound ATPase protein, termed EssC in Staphylococcus aureus, is a critical component of the T7SS and is the primary receptor for substrate proteins. Genetic diversity in the essC gene of S. aureus has previously been reported, resulting in four protein variants that are linked to specific subsets of substrates. Here we have analysed the genetic diversity of the T7SS-encoding genes and substrate proteins across Listeria monocytogenes genome sequences. We find that there are seven EssC variants across the species that differ in their C-terminal region; each variant is correlated with a distinct subset of genes for likely substrate and accessory proteins. EssC1 is most common and is exclusively linked with polymorphic toxins harbouring a YeeF domain, whereas EssC5, EssC6 and EssC7 variants all code for an LXG domain protein adjacent to essC. Some essC1 variant strains encode an additional, truncated essC at their T7 gene cluster. The truncated EssC, comprising only the C-terminal half of the protein, matches the sequence of either EssC2, EssC3 or EssC4. In each case the truncated gene directly precedes a cluster of substrate/accessory protein genes acquired from the corresponding strain. Across L. monocytogenes strains we identified 40 LXG domain proteins, most of which are encoded at conserved genomic loci. These loci also harbour genes encoding immunity proteins and sometimes additional toxin fragments. Collectively our findings strongly suggest that the T7SS plays an important role in bacterial antagonism in this species.

The Salmonella effector protein SopD targets Rab8 to positively and negatively modulate the inflammatory response.

The food-borne bacterial pathogen Salmonella Typhimurium uses a type III protein secretion system to deliver multiple proteins into host cells. These secreted effectors modulate host cell functions and activate specific signalling cascades that result in the production of pro-inflammatory cytokines and intestinal inflammation. Some of the Salmonella-encoded effectors counter this inflammatory response and help to preserve host homeostasis. We demonstrate that the Salmonella effector protein SopD, which is required for pathogenesis, functions to both activate and inhibit the inflammatory response by targeting the Rab8 GTPase, a negative regulator of inflammation. We show that SopD has GTPase activating protein activity for Rab8, and therefore inhibits this GTPase and stimulates inflammation. We also show that SopD activates Rab8 by displacing it from its cognate guanosine dissociation inhibitor resulting in the stimulation of a signaling cascade that suppresses inflammation. We solved the crystal structure of SopD in association with Rab8 to 2.3 Å resolution, which reveals a unique contact interface underlying these complex interactions. These findings show the remarkable evolution of a bacterial effector protein to exert both agonistic and antagonistic activities toward the same host cellular target to modulate the inflammatory response.

Cytophaga hutchinsonii SprA and SprT Are Essential Components of the Type IX Secretion System Required for Ca2+ Acquisition, Cellulose Degradation, and Cell Motility.

The type IX secretion system (T9SS) is a novel protein secretion system, which is found in and confined to the phylum Bacteroidetes. T9SS is involved in the secretion of virulence factors, cell surface adhesins, and complex biopolymer degrading enzymes to the cell surface or extracellular medium. Cytophaga hutchinsonii is a widely distributed bacterium, which is able to efficiently digest cellulose and rapidly glide along the solid surfaces. C. hutchinsonii has a full set of orthologs of T9SS components. However, the functions of most homologous proteins have not been verified. In C. hutchinsonii, CHU_0029 and CHU_2709 are similar in sequence to Flavobacterium johnsoniae T9SS components SprA and SprT, respectively. In this study, the single deletion mutants of chu_0029 (sprA) and chu_2709 (sprT) were obtained using a complex medium with the addition of Ca2+ and Mg2+. Single deletion of sprA or sprT resulted in defects in cellulose utilization and gliding motility. Moreover, the ΔsprA and ΔsprT mutants showed growth defects in Ca2+- and Mg2+-deficient media. The results of ICP-MS test showed that both the whole cell and intracellular concentrations of Ca2+ were dramatically reduced in the ΔsprA and ΔsprT mutants, indicating that SprA and SprT are both important for the assimilation of trace amount of Ca2+. While the assimilation of Mg2+ was not obviously influenced in the ΔsprA and ΔsprT mutants. Through proteomics analysis of the cell surface proteins of the wild type and mutants, we found that the ΔsprA and ΔsprT mutants were defective in secretion of the majority of T9SS substrates. Together, these results indicate that SprA and SprT are both essential components of C. hutchinsonii T9SS, which is required for protein secretion, Ca2+ acquisition, cellulose degradation, and gliding motility in C. hutchinsonii. Our study shed more light on the functions of SprA and SprT in T9SS, and further proved the link between the T9SS and Ca2+ uptake system.

Functional metagenomic and enrichment metatranscriptomic analysis of marine microbial activities within a marine oil spill area.

Microorganisms can degrade petroleum hydrocarbons, providing the advantages of low cost and few side effects towards ecosystems. Here, we evaluated the mechanisms of microbial degradation of marine petroleum hydrocarbon using metagenomics and metatranscriptomics approaches in order to provide new insight into microbial degradation of petroleum hydrocarbon. Seawater samples were collected at a depth of ∼8m from an area near a drilling platform in the Bohai Bay and metagenomic sequencing was used to evaluate the functional potential of these marine microbial communities. Metatranscriptomic sequencing, fluorescence in-situ hybridization experiments, and flow cytometry were also performed on the microbial communities of samples subjected to 12 different culture conditions. The data were also subjected to Weighted Gene Co-expression Network Analysis (WGCNA) and co-transcription data visualization to evaluate co-transcription of gene functions. Metagenomic sequencing indicated the presence of numerous genes that were related to petroleum hydrocarbon metabolism. Further, the high co-transcription of genes in multiple pathways, indicated that groups of genes were synergistically transcribed to metabolize petroleum hydrocarbons. Metatranscriptomics also showed that microbial metabolism was highly active in the enrichments and that the transcription of a large number of prokaryotic replication and repair genes were significantly up-regulated including those encoding for the type VI secretion system (T6SS) protein, DNA polymerase I, thymidine phosphorylase, mevalonate kinase, and two-component systems. Concomitantly, the transcription of ribosomal genes involved in translation and photosynthetic genes involved in energy metabolism were down-regulated. Overall, oil and oxygen presence can increase the oil-degradation rates and related genes' transcription. Lot different metabolisms are co-regulated to exploit nutrients derived from the metabolism of petroleum hydrocarbons. Our analysis of metagenomic, metatranscriptomic and degradation data in this study show that a widespread gene spectrum involved in oil-degradation and the cooperation among genes is of great importance.

Advances in Understanding Activation and Function of the NLRC4 Inflammasome.

Innate immune receptors initiate a host immune response, or inflammatory response, upon detecting pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Among the innate immune receptors, nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play a pivotal role in detecting cytosolic PAMPs and DAMPs. Some NLRs can form a multiprotein cytosolic complex known as the inflammasome. Inflammasome activation triggers caspase-1–mediated cleavage of the pore-forming protein gasdermin D (GSDMD), which drives a form of inflammatory cell death called pyroptosis. Parallelly, activated caspase-1 cleaves immature cytokines pro–IL-1β and pro–IL-18 into their active forms, which can be released via GSDMD membrane pores. The NLR family apoptosis inhibitory proteins (NAIP)-NLR family caspase-associated recruitment domain-containing protein 4 (NLRC4) inflammasome is important for mounting an immune response against Gram-negative bacteria. NLRC4 is activated through NAIPs sensing type 3 secretion system (T3SS) proteins from Gram-negative bacteria, such as Salmonella Typhimurium. Mutations in NAIPs and NLRC4 are linked to autoinflammatory disorders in humans. In this review, we highlight the role of the NAIP/NLRC4 inflammasome in host defense, autoinflammatory diseases, cancer, and cell death. We also discuss evidence pointing to a role of NLRC4 in PANoptosis, which was recently identified as a unique inflammatory programmed cell death pathway with important physiological relevance in a range of diseases. Improved understanding of the NLRC4 inflammasome and its potential roles in PANoptosis paves the way for identifying new therapeutic strategies to target disease.

Structure and mechanism of the proton-driven motor that powers type 9 secretion and gliding motility.

Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor, and a proton-driven motor that powers gliding motility and the Type 9 protein secretion system (T9SS) in Bacteroidetes bacteria. Here, we present cryo-EM structures of the gliding motility/T9SS motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identifies protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane.

The type VI secretion system of Xanthomonas phaseoli pv. manihotis is involved in virulence and in vitro motility

BackgroundThe type VI protein secretion system (T6SS) is important in diverse cellular processes in Gram-negative bacteria, including interactions with other bacteria and with eukaryotic hosts. In this study we analyze the evolution of the T6SS in the genus Xanthomonas and evaluate its importance of the T6SS for virulence and in vitro motility in Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of bacterial blight in cassava (Manihot esculenta). We delineate the organization of the T6SS gene clusters in Xanthomonas and then characterize proteins of this secretion system in Xpm strain CIO151.ResultsWe describe the presence of three different clusters in the genus Xanthomonas that vary in their organization and degree of synteny between species. Using a gene knockout strategy, we also found that vgrG and hcp are required for maximal aggressiveness of Xpm on cassava plants while clpV is important for both motility and maximal aggressiveness.ConclusionWe characterized the T6SS in 15 different strains in Xanthomonas and our phylogenetic analyses suggest that the T6SS might have been acquired by a very ancient event of horizontal gene transfer and maintained through evolution, hinting at their importance for the adaptation of Xanthomonas to their hosts. Finally, we demonstrated that the T6SS of Xpm is functional, and significantly contributes to virulence and motility. This is the first experimental study that demonstrates the role of the T6SS in the Xpm-cassava interaction and the T6SS organization in the genus Xanthomonas.

Pathogenic bacterial toxins and virulence influences in cultivable fish

Bacterial virulence factors play a major role in infection, which provides devastating effect on the aquaculture industry. Bacterial toxins are the major virulence factors that influence the roles of host cells and take control of living organisms’ vital processes to facilitate microbial infection. Various other bacterial virulent components such as adhesins, capsular polysaccharides, lipopolysaccharides, bacterial flagellum, pili and microbial siderophore provide a diverse effect in the microbial pathogenesis and express a variety of pathogenic molecules including aebABCEG, HutA, cpsD and pPHDD1. Bacterial protein secretion system secretes enzymes and toxins from bacterial cytoplasm to the host membrane targeting the immune cells and promoting biofilm formation. However, as they enter, the host body expresses various immune molecules that interact to utilize the pathogen recognition receptors. Toxins are powerful molecules formed by a broad range of bacterial pathogens attacking host cells and playing key roles in the dialogue between host and pathogen. They are important virulence factors that are often enough to decide the outcome of the infection. Hence, a detailed view on the virulence molecules majorly exotoxins from various bacterial pathogenic strains that are necessary to understand the pathogenesis. This review focuses on bacterial toxins and various other virulence factors, which influence the pathogenesis in fish.

Identification of Nanobodies Blocking Intimate Adherence of Shiga Toxin-Producing Escherichia coli to Epithelial Cells.

Therapeutic antibodies (Abs) inhibiting bacterial adhesion to host epithelia are an attractive option to reduce the load of Shiga toxin-producing E. coli (STEC) in the intestine of the patient and also in the bovine reservoir, thereby minimizing the risk of STEC contamination in the food chain. Of particular interest are recombinant single-domain Ab fragments called nanobodies (Nbs) derived from the variable domain of camelid heavy chain-only antibodies (VHH). The outer membrane adhesin intimin and the translocated intimin receptor (Tir) are essential for the attachment of STEC to host epithelia. In addition, EspA filaments of the bacterial type III protein secretion system are needed for Tir translocation into the host cell. Given their importance for bacterial adhesion and colonization, we developed Nbs against intimin, Tir and EspA proteins of STEC serotype O157:H7. Here, we report the screening methods used to isolate inhibitory Nbs blocking intimin-Tir protein-protein interaction, actin-pedestal formation, and intimate adhesion of STEC to epithelial cells in vitro. First, we describe how VHH gene repertoires can be produced as Nbs secreted by E. coli using the α-hemolysin (HlyA) protein secretion system. Next, we report the methods for identification of inhibitors of intimin-Tir protein-protein interaction and of STEC intimate adhesion to HeLa cells in culture. These methods can be adapted for the screening of Nbs against different adhesin-receptor complexes to block the adhesion of other pathogens to host cells.

Genome Analysis of Antimicrobial Resistance Genes and Virulence Factors in Multidrug-Resistant Campylobacter fetus Subspecies Isolated from Sheath Wash

Campylobacter fetus subspecies are mostly characterized by reproductions problems in cattle and sheep. This study aimed to study the genetic profile and assess the genes mechanism of resistance and their virulence factors using genome sequence analysis. A total of 59 confirmed Campylobacter fetus subspecies based on molecular assays and DNA sequencing were subjected to antimicrobial susceptibility test against 14 antibiotic agents representing the five classes of antibiotics using the disc diffusion method. In addition, sequencing the genome of all strains induced complete resistance against all tested antibiotics. The results of the antimicrobial test indicated that 54.4% had a resistance profile, 26.3% were intermediate, while 19.3% were observed to be susceptible. The Whole Genome Sequencing (WGS) result revealed the presence of different genes, such as Broad-specificity multidrug efflux pump and 16S rRNA (guanine527-N7)-methyltransferase (gidB), efflux pump conferring antibiotic resistance (MacA and MacB), protein-altering cell wall charge conferring antibiotic resistance (PgsA), which have never been reported in Campylobacter fetus subspecies. The WGS also revealed the presence of genes that involved in colonization, adhesion, motility, and invasion, such as type IV secretion system protein (VirD4), S-Layer, cytolethal distending toxin (A, B, and C), Campylobacter invasion antigen (CiaB), and fic domain protein (fic) were among important CDS. The presence of these uncommon genes explains the resistance of Campylobacter fetus subspecies against different tested antibiotics. The results of this study can be used to implement molecular surveillance of Campylobacter fetus subspecies and conduct further studies on the resistance mechanism in these subspecies.

Mechanisms involved in the adaptation of Escherichia coli O157:H7 to the host intestinal microenvironment.

Host adaptation of pathogens may increase intra- and interspecies transmission. We showed previously that the passage of a clinically isolated enterohemorrhagic Escherichia coli (EHEC) O157 strain (125/99) through the gastrointestinal tract of mice increases its pathogenicity in the same host. In this work, we aimed to elucidate the underlying mechanism(s) involved in the patho-adaptation of the stool-recovered (125RR) strain. We assessed the global transcription profile by microarray and found almost 100 differentially expressed genes in 125RR strain compared with 125/99 strain. We detected an overexpression of Type Three Secretion System (TTSS) proteins at the mRNA and protein levels and demonstrated increased adhesion to epithelial cell lines for the 125RR strain. Additional key attributes of the 125RR strain were: increased motility on semisolid agar, which correlated with an increased fliC mRNA level; reduced Stx2 production at the mRNA and protein levels; increased survival at pH 2.5, as determined by acid resistance assays. We tested whether the overexpression of the LEE-encoded regulator (ler) in trans in the 125/99 strain could recreate the increased pathogenicity observed in the 125RR strain. As anticipated ler overexpression led to increased expression of TTSS proteins and bacterial adhesion to epithelial cells in vitro but also increased mortality and intestinal colonization in vivo. We conclude that this host-adaptation process required changes in several mechanisms that improved EHEC O157 fitness in the new host. The research highlights some of the bacterial mechanisms required for horizontal transmission of these zoonotic pathogens between their animal and human populations.

The outer-membrane protein MafA of Neisseria meningitidis constitutes a novel protein secretion pathway specific for the fratricide protein MafB

ABSTRACT MafB proteins are toxins secreted by Neisseria spp. which are involved in interbacterial competition. Their secretion mechanism has so far not been elucidated. Each strain can produce several MafB variants. On the chromosome, the mafB genes are localized on genomic islands also containing mafA genes. MafA proteins have a role in virulence with reported activities in adhesion and transcytosis of pathogenic Neisseria, a priori unrelated to MafB activities. In this study, we investigated the possible involvement of MafA in the transport of MafB across the outer membrane of Neisseria meningitidis. In wild-type strains, proteolytic fragments of MafB proteins were detected in the extracellular medium. In the absence of MafA, secretion was abrogated, and, in the case of MafBI, full-length and truncated polypeptides were detected inside the cells and inside outer-membrane vesicles. MafBI secretion required its cognate MafA, whereas MafBIII could use any MafA. Heterologous expression in Escherichia coli showed that MafBIII is transported to a cell-surface-exposed, i.e. protease-accessible, location in a MafA-dependent way. MafA itself was found to be localized to the outer membrane, forming large oligomeric complexes. As homologs were found in diverse bacteria, the Maf system represents a new protein secretion system in Gram-negative bacteria.

Virulence genotyping of drug resistant Pseudomonas aeruginosa clinical isolates in Egypt using multiplex PCR

Pseudomonas aeruginosa is a serious Gram- negative pathogen capable of causing several nosocomial and community acquired infections. It produces many toxins such as: exotoxin A, pyocyanin, elastase and type III secretion system proteins. P. aeruginosa is genotyped according to the produced type III secretion system protein into invasive and cytotoxic genotypes. This study aimed to determine the prevalent virulence genotypes as well as the prevalence of five virulence genes using multiplex PCR. The antimicrobial resistance of the collected 134 P. aeruginosa isolates was detected using the disc diffusion method. The five exotoxin genes were detected by conventional multiplex PCR. Seventy percent of isolates were multidrug resistant. The phz, lasB, toxA, exoS and exoU genes were detected in 93%, 90%, 88%, 57% and 43% of isolates, respectively. Fifty two percent of isolates represented the invasive genotype and 38.8% represented the cytotoxic genotype. Clinical P. aeruginosa strains exhibit high antimicrobial resistance. The invasive and cytotoxic genotypes are associated with different types of clinical specimens and both genotypes can co-exist in the same isolates. Therefore, following up the antibiotic resistance of P. aeruginosa and its virulence factors and produced toxins is recommended to judge the bacterial virulence in outbreaks and epidemiologic studies.

Comparative proteomic analysis reveals novel insights into the interaction between rice and Xanthomonas oryzae pv. oryzae

BackgroundBacterial blight, which is caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease worldwide. Rice introgression line H471, derived from the recurrent parent Huang-Hua-Zhan (HHZ) and the donor parent PSBRC28, exhibits broad-spectrum resistance to Xoo, including to the highly virulent Xoo strain PXO99A, whereas its parents are susceptible to PXO99A. To characterize the responses to Xoo, we compared the proteome profiles of the host and pathogen in the incompatible interaction (H471 inoculated with PXO99A) and the compatible interaction (HHZ inoculated with PXO99A).ResultsIn this study, a total of 374 rice differentially abundant proteins (DAPs) and 117 Xoo DAPs were detected in the comparison between H471 + PXO99A and HHZ + PXO99A. Most of the Xoo DAPs related to pathogen virulence, including the outer member proteins, type III secretion system proteins, TonB-dependent receptors, and transcription activator-like effectors, were less abundant in the incompatible interaction than in the compatible interaction. The rice DAPs were mainly involved in secondary metabolic processes, including phenylalanine metabolism and the biosynthesis of flavonoids and phenylpropanoids. Additionally, some DAPs involved in the phenolic phytoalexin and salicylic acid (SA) biosynthetic pathways accumulated much more in H471 than in HHZ after the inoculation with PXO99A, suggesting that phytoalexin and SA productions were induced faster in H471 than in HHZ. Further analyses revealed that the SA content increased much more rapidly in H471 than in HHZ after the inoculation, suggesting that the SA signaling pathway was activated faster in the incompatible interaction than in the compatible interaction.ConclusionsOverall, our results indicate that during an incompatible interaction between H471 and PXO99A, rice plants prevent pathogen invasion and also initiate multi-component defense responses that inhibit disease development.

Porcine Enterotoxigenic Escherichia coli Strains Differ in Their Capacity To Secrete Enterotoxins through Varying YghG Levels.

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens for humans and farm animals such as pigs. Porcine ETEC strains induce diarrhea through the production of heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (pSTa/STb). Although LT secretion levels differ between porcine ETEC strains, and this has been linked to virulence, it is unclear whether ST secretion levels also differ between porcine ETEC strains. In addition, the molecular mechanism underlying different LT secretion levels has not been elucidated. In this work, multiple porcine ETEC strains were assessed for their capacity to produce and secrete the enterotoxins LT, pSTa, and STb. The strains differed greatly in their capacity to secrete LT, pSTa, and STb. Remarkably, in some strains, periplasmic production did not correlate with their ability to secrete LT, resulting in high periplasmic production and low LT secretion levels. Furthermore, the results indicated that the type II secretion system (T2SS) protein YghG plays a regulatory role in controlling LT secretion levels. These findings highlight YghG as an important mediator of the secretion of the heat-labile enterotoxin LT by porcine ETEC strains and provide better insights into ETEC enterotoxin secretion.IMPORTANCE Enterotoxigenic E. coli strains are a major health concern. Enterotoxins secreted by enterotoxigenic E. coli are crucial for diarrhea induction. Enterotoxin secretion levels differ between strains; however, it is currently unclear what drives these differences. The discrepancy in the production and secretion capacities of enterotoxins in ETEC is important to clarify their function involved in diarrhea induction. Our results further deepen our understanding of how type II secretion system (T2SS) components of ETEC control enterotoxin secretion levels and may lay the foundation for a better understanding of ETEC molecular pathogenesis.

Role of Recipient Susceptibility Factors During Contact-Dependent Interbacterial Competition.

Bacteria evolved multiple strategies to survive and develop optimal fitness in their ecological niche. They deployed protein secretion systems for robust and efficient delivery of antibacterial toxins into their target cells, therefore inhibiting their growth or killing them. To maximize antagonism, recipient factors on target cells can be recognized or hijacked to enhance the entry or toxicity of these toxins. To date, knowledge regarding recipient susceptibility (RS) factors and their mode of action is mostly originating from studies on the type Vb secretion system that is also known as the contact-dependent inhibition (CDI) system. Yet, recent studies on the type VI secretion system (T6SS), and the CDI by glycine-zipper protein (Cdz) system, also reported the emerging roles of RS factors in interbacterial competition. Here, we review these RS factors and their mechanistic impact in increasing susceptibility of recipient cells in response to CDI, T6SS, and Cdz. Past and future strategies for identifying novel RS factors are also discussed, which will help in understanding the interplay between attacker and prey upon secretion system-dependent competition. Understanding these mechanisms would also provide insights for developing novel antibacterial strategies to antagonize aggressive bacteria-killing pathogens.

Genomic Characterisation of Mushroom Pathogenic Pseudomonads and Their Interaction with Bacteriophages.

Bacterial diseases of the edible white button mushroom Agaricus bisporus caused by Pseudomonas species cause a reduction in crop yield, resulting in considerable economic loss. We examined bacterial pathogens of mushrooms and bacteriophages that target them to understand the disease and opportunities for control. The Pseudomonas tolaasii genome encoded a single type III protein secretion system (T3SS), but contained the largest number of non-ribosomal peptide synthase (NRPS) genes, multimodular enzymes that can play a role in pathogenicity, including a putative tolaasin-producing gene cluster, a toxin causing blotch disease symptom. However, Pseudomonas agarici encoded the lowest number of NRPS and three putative T3SS while non-pathogenic Pseudomonas sp. NS1 had intermediate numbers. Potential bacteriophage resistance mechanisms were identified in all three strains, but only P. agarici NCPPB 2472 was observed to have a single Type I-F CRISPR/Cas system predicted to be involved in phage resistance. Three novel bacteriophages, NV1, ϕNV3, and NV6, were isolated from environmental samples. Bacteriophage NV1 and ϕNV3 had a narrow host range for specific mushroom pathogens, whereas phage NV6 was able to infect both mushroom pathogens. ϕNV3 and NV6 genomes were almost identical and differentiated within their T7-like tail fiber protein, indicating this is likely the major host specificity determinant. Our findings provide the foundations for future comparative analyses to study mushroom disease and phage resistance.

Shigella flexneri Disruption of Cellular Tension Promotes Intercellular Spread

SUMMARYDuring infection, some bacterial pathogens invade the eukaryotic cytosol and spread between cells of an epithelial monolayer. Intercellular spread occurs when these pathogens push against the plasma membrane, forming protrusions that are engulfed by adjacent cells. Here, we show that IpaC, a Shigella flexneri type 3 secretion system protein, binds the host cell-adhesion protein β-catenin and facilitates efficient protrusion formation. S. flexneri producing a point mutant of IpaC that cannot interact with β-catenin is defective in protrusion formation and spread. Spread is restored by chemical reduction of intercellular tension or genetic depletion of β-catenin, and the magnitude of the protrusion defect correlates with membrane tension, indicating that IpaC reduces membrane tension, which facilitates protrusion formation. IpaC stabilizes adherens junctions and does not alter β-catenin localization at the membrane. Thus, Shigella, like other bacterial pathogens, reduces intercellular tension to efficiently spread between cells.