A family of conserved bacterial virulence factors dampens interferon responses by blocking calcium signaling.

Interferons (IFNs) induce an antimicrobial state, protecting tissues from infection. Many viruses inhibit IFN signaling, but whether bacterial pathogens evade IFN responses remains unclear. Here, we demonstrate that the Shigella OspC family of type-III-secreted effectors blocks IFN signaling independently of its cell death inhibitory activity. Rather, IFN inhibition was mediated by the binding of OspC1 and OspC3 to the Ca2+ sensor calmodulin (CaM), blocking CaM kinase II and downstream JAK/STAT signaling. The growth of Shigella lacking OspC1 and OspC3 was attenuated in epithelial cells and in a murine model of infection. This phenotype was rescued in both models by the depletion of IFN receptors. OspC homologs conserved in additional pathogens not only bound CaM but also inhibited IFN, suggesting a widespread virulence strategy. These findings reveal a conserved but previously undescribed molecular mechanism of IFN inhibition and demonstrate the critical role of Ca2+ and IFN targeting in bacterial pathogenesis.

Pathogenic E. coli Extracts Nutrients from Infected Host Cells Utilizing Injectisome Components.

Microbiota and intestinal epithelium restrict pathogen growth by rapid nutrient consumption. We investigated how pathogens circumvent this obstacle to colonize the host. Utilizing enteropathogenic E. coli (EPEC), we show that host-attached bacteria obtain nutrients from infected host cell in a process we termed host nutrient extraction (HNE). We identified an inner-membrane protein complex, henceforth termed CORE, as necessary and sufficient for HNE. The CORE is a key component of the EPEC injectisome, however, here we show that it supports the formation of an alternative structure, composed of membranous nanotubes, protruding from the EPEC surface to directly contact the host. The injectisome and flagellum are evolutionarily related, both containing conserved COREs. Remarkably, CORE complexes of diverse ancestries, including distant flagellar COREs, could rescue HNE capacity of EPEC lacking its native CORE. Our results support the notion that HNE is a widespread virulence strategy, enabling pathogens to thrive in competitive niches.

Structural Insights into Type III Secretion Systems of the Bacterial Flagellum and Injectisome.

Two of the most fascinating bacterial nanomachines-the broadly disseminated rotary flagellum at the heart of cellular motility and the eukaryotic cell-puncturing injectisome essential to specific pathogenic species-utilize at their core a conserved export machinery called the type III secretion system (T3SS). The T3SS not only secretes the components that self-assemble into their extracellular appendages but also, in the case of the injectisome, subsequently directly translocates modulating effector proteins from the bacterial cell into the infected host. The injectisome is thought to have evolved from the flagellum as a minimal secretory system lacking motility, with the subsequent acquisition of additional components tailored to its specialized role in manipulating eukaryotic hosts for pathogenic advantage. Both nanomachines have long been the focus of intense interest, but advances in structural and functional understanding have taken a significant step forward since 2015, facilitated by the revolutionary advances in cryo-electron microscopy technologies. With several seminal structures of each nanomachine now captured, we review here the molecular similarities and differences that underlie their diverse functions.

The Versatile Roles of Type III Secretion Systems in Rhizobium-Legume Symbioses.

To suppress plant immunity and promote the intracellular infection required for fixing nitrogen for the benefit of their legume hosts, many rhizobia use type III secretion systems (T3SSs) that deliver effector proteins (T3Es) inside host cells. As reported for interactions between pathogens and host plants, the immune system of legume hosts and the cocktail of T3Es secreted by rhizobia determine the symbiotic outcome. If they remain undetected, T3Es may reduce plant immunity and thus promote infection of legumes by rhizobia. If one or more of the secreted T3Es are recognized by the cognate plant receptors, defense responses are triggered and rhizobial infection may abort. However, some rhizobial T3Es can also circumvent the need for nodulation (Nod) factors to trigger nodule formation. Here we review the multifaceted roles played by rhizobial T3Es during symbiotic interactions with legumes.

Cooperation and cheating orchestrate Vibrio assemblages and polymicrobial synergy in oysters infected with OsHV-1 virus.

Polymicrobial infections threaten the health of humans and animals but remain understudied in natural systems. We recently described the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease affecting oyster production worldwide. In the French Atlantic coast, the disease involves coinfection with ostreid herpesvirus 1 (OsHV-1) and virulent Vibrio. However, it is unknown whether consistent Vibrio populations are associated with POMS in different regions, how Vibrio contribute to POMS, and how they interact with OsHV-1 during pathogenesis. By connecting field-based approaches in a Mediterranean ecosystem, laboratory infection assays and functional genomics, we uncovered a web of interdependencies that shape the structure and function of the POMS pathobiota. We show that Vibrio harveyi and Vibrio rotiferianus are predominant in OsHV-1-diseased oysters and that OsHV-1 drives the partition of the Vibrio community observed in the field. However only V. harveyi synergizes with OsHV-1 by promoting mutual growth and accelerating oyster death. V. harveyi shows high-virulence potential and dampens oyster cellular defenses through a type 3 secretion system, making oysters a more favorable niche for microbe colonization. In addition, V. harveyi produces a key siderophore called vibrioferrin. This important resource promotes the growth of V. rotiferianus, which cooccurs with V. harveyi in diseased oysters, and behaves as a cheater by benefiting from V. harveyi metabolite sharing. Our data show that cooperative behaviors contribute to synergy between bacterial and viral coinfecting partners. Additional cheating behaviors further shape the polymicrobial consortium. Controlling cooperative behaviors or countering their effects opens avenues for mitigating polymicrobial diseases.

Caught in the act: In situ visualization of bacterial secretion systems by cryo-electron tomography.

Bacterial secretion systems, such as the type 3, 4, and 6 are multiprotein nanomachines expressed at the surface of pathogens with Gram-negative like envelopes. They are known to be crucial for virulence and to translocate bacteria-encoded effector proteins into host cells to manipulate cellular functions. This facilitates either pathogen attachment or invasion of the targeted cell. Effector proteins also promote evasion of host immune recognition. Imaging by cryo-electron microscopy in combination with structure determination has become a powerful approach to understand how these nanomachines work. Still, questions on their assembly, the precise secretion mechanisms, and their direct involvement in pathogenicity remain unsolved. Here, we present an overview of the recent developments in in situ cryo-electron microscopy. We discuss its potential for the investigation of the role of bacterial secretion systems during the host-bacterial crosstalk at the molecular level. These in situ studies open new perspectives for our understanding of secretion system structure and function.

The Yersinia Type III Secretion System as a Tool for Studying Cytosolic Innate Immune Surveillance.

Microbial pathogens have evolved complex mechanisms to interface with host cells in order to evade host defenses and replicate. However, mammalian innate immune receptors detect the presence of molecules unique to the microbial world or sense the activity of virulence factors, activating antimicrobial and inflammatory pathways. We focus on how studies of the major virulence factor of one group of microbial pathogens, the type III secretion system (T3SS) of human pathogenic Yersinia, have shed light on these important innate immune responses. Yersinia are largely extracellular pathogens, yet they insert T3SS cargo into target host cells that modulate the activity of cytosolic innate immune receptors. This review covers both the host pathways that detect the Yersinia T3SS and the effector proteins used by Yersinia to manipulate innate immune signaling.

Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli.

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

Catheter-associated urinary tract infection by Pseudomonas aeruginosa progresses through acute and chronic phases of infection.

Healthcare-associated infections are major causes of complications that lead to extended hospital stays and significant medical costs. The use of medical devices, including catheters, increases the risk of bacterial colonization and infection through the presence of a foreign surface. Two outcomes are observed for catheterized patients: catheter-associated asymptomatic bacteriuria and catheter-associated urinary tract infection (CAUTI). However, the relationship between these two events remains unclear. To understand this relationship, we studied a murine model of Pseudomonas aeruginosa CAUTI. In this model, we also observe two outcomes in infected animals: acute symptoms that is associated with CAUTI and chronic colonization that is associated with asymptomatic bacteriuria. The timing of the acute outcome takes place in the first week of infection, whereas chronic colonization occurs in the second week of infection. We further showed that mutants lacking genes encoding type III secretion system (T3SS), T3SS effector proteins, T3SS injection pore, or T3SS transcriptional activation all fail to cause acute symptoms of CAUTI. Nonetheless, all mutants defective for T3SS colonized the catheter and bladders at levels similar to the parental strain. In contrast, through induction of the T3SS master regulator ExsA, all infected animals showed acute phenotypes with bacteremia. Our results demonstrated that the acute symptoms, which are analogous to CAUTI, and chronic colonization, which is analogous to asymptomatic bacteriuria, are independent events that require distinct bacterial virulence factors. Experimental delineation of asymptomatic bacteriuria and CAUTI informs different strategies for the treatment and intervention of device-associated infections.

HilE represses the activity of the Salmonella virulence regulator HilD via a mechanism distinct from that of intestinal long-chain fatty acids.

The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long-chain fatty acids, which are highly abundant throughout the host intestine, directly bind to and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerization, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. Long-chain fatty acids bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerization interface. Our results highlight two distinct, mutually exclusive mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads.

Overexpression of T3SS translocation signals in Salmonella causes delayed attenuation.

Engineering pathogens is a useful method for discovering new details of microbial pathogenesis and host defense. However, engineering can result in off-target effects. We previously engineered Salmonella enterica serovar Typhimurium to overexpress the secretion signal of the type 3 secretion system effector SspH1 fused with domains of other proteins as cargo. Such engineering had no virulence cost to the bacteria for the first 48 hours post infection in mice. Here, we show that after 48 hours, the engineered bacteria manifest an attenuation that correlates with the quantity of the SspH1 translocation signal expressed. In IFN-γ-deficient mice, this attenuation was weakened. Conversely, the attenuation was accelerated in the context of a pre-existing infection. We speculate that inflammatory signals change aspects of the target cell's physiology, which makes host cells less permissive to S. Typhimurium infection. This increased degree of difficulty requires the bacteria to utilize its T3SS at peak efficiency, which can be disrupted by engineered effectors.

Microbial Primer: The bacterial flagellum - how bacteria swim.

Bacteria swim using membrane-spanning, electrochemical gradient-powered motors that rotate semi-rigid helical filaments. This primer provides a brief overview of the basic synthesis, structure and operation of these nanomachines. Details and variations on the basic system can be found in suggested further reading.

The Genetics of Enteropathogenic Escherichia coli Virulence.

In many parts of the world, enteropathogenic Escherichia coli (EPEC) are a leading cause of death in children with diarrhea. Much of what we know about the pathogenesis of EPEC infections is based on the study of one or two prototypic strains that have provided deep insight into the precise mechanisms by which EPEC colonizes the intestine, evades host immunity, and spreads from person to person. In some cases, defining the biochemical activity of the host-interacting effector proteins from these prototypic strains has led to the discovery of novel post-translational protein modifications and new understandings of biology and host-pathogen interactions. However, genomic analysis of recent EPEC isolates has revealed that the EPEC pathotype is more diverse than previously appreciated. Although by definition all strains carry the locus of enterocyte effacement, the effector repertoires of different clonal groups are quite divergent, suggesting that there is still a great deal to learn about the genetic basis of EPEC virulence.

Novel Single Nucleotide Variations Alter Pathogenicity in Korean Isolates of Erwinia amylovora.

Erwinia amylovora, the causal agent of fire blight disease, has become a serious threat to the pome fruit industry in Korea since 2015. In this study, we showed that two new isolates of E. amylovora, Ea17-2187 and Ea19-7, obtained from pear orchards in Anseong, Korea, exhibited unique pathogenicity compared with other isolates thus far. Both were nonpathogenic to immature apple fruits but occasionally caused disease on immature pear fruits at varying reduced rates. Bioinformatic analyses revealed that their genomes are highly similar to those of the type strains TS3128 and ATCC49946 but have different mutations in essential virulence regulatory genes. Ea17-2187 has a single nucleotide substitution in rcsC, which encodes the core components of the Rcs system that activates the exopolysaccharide amylovoran production. In contrast, Ea19-7 contains a single nucleotide insertion in hrpL, which encodes a master regulator of the type III secretion system. In both cases, the mutation can cause premature termination and production of truncated gene products, disrupting virulence regulation. Introduction of the nonmutated rcsC and hrpL genes into Ea17-2187 and Ea19-7, respectively, fully recovered pathogenicity, comparable with that of TS3128; hence, these mutations were responsible for the altered pathogenicity observed. Interestingly, virulence assays on immature pear fruits showed that the hrpL mutant of Ea19-7 was still pathogenic, although its virulence level was markedly reduced. Taken together, these results suggest that the two new isolates might act as opportunistic pathogens or cheaters and that some Korean isolates might have evolved to acquire alternative pathways for activating pathogenicity factors.

Calcium Induces the Cleavage of NopA and Regulates the Expression of Nodulation Genes and Secretion of T3SS Effectors in Sinorhizobium fredii NGR234.

The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR_b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC, genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia-legume interactions.

PtsN in Pseudomonas aeruginosa Is Phosphorylated by Redundant Upstream Proteins and Impacts Virulence-Related Genes.

The bacterial nitrogen-related phosphotransfer (PTSNtr; here, Nitro-PTS) system bears homology to well-known PTS systems that facilitate saccharide import and phosphorylation. The Nitro-PTS comprises an enzyme I (EI), PtsP; an intermediate phosphate carrier, PtsO; and a terminal acceptor, PtsN, which is thought to exert regulatory effects that depend on its phosphostate. For instance, biofilm formation by Pseudomonas aeruginosa can be impacted by the Nitro-PTS, as deletion of either ptsP or ptsO suppresses Pel exopolysaccharide production and additional deletion of ptsN elevates Pel production. However, the phosphorylation state of PtsN in the presence and absence of its upstream phosphotransferases has not been directly assessed, and other targets of PtsN have not been well defined in P. aeruginosa. We show that PtsN phosphorylation via PtsP requires the GAF domain of PtsP and that PtsN is phosphorylated on histidine 68, as in Pseudomonas putida. We also find that FruB, the fructose EI, can substitute for PtsP in PtsN phosphorylation but only in the absence of PtsO, implicating PtsO as a specificity factor. Unphosphorylatable PtsN had a minimal effect on biofilm formation, suggesting that it is necessary but not sufficient for the reduction of Pel in a ptsP deletion. Finally, we use transcriptomics to show that the phosphostate and the presence of PtsN do not appear to alter the transcription of biofilm-related genes but do influence genes involved in type III secretion, potassium transport, and pyoverdine biosynthesis. Thus, the Nitro-PTS influences several P. aeruginosa behaviors, including the production of its signature virulence factors. IMPORTANCE The PtsN protein impacts the physiology of a number of bacterial species, and its control over downstream targets can be altered by its phosphorylation state. Neither its upstream phosphotransferases nor its downstream targets are well understood in Pseudomonas aeruginosa. Here, we examine PtsN phosphorylation and find that the immediate upstream phosphotransferase acts as a gatekeeper, allowing phosphorylation by only one of two potential upstream proteins. We use transcriptomics to discover that PtsN regulates the expression of gene families that are implicated in virulence. One emerging pattern is a repression hierarchy by different forms of PtsN: its phosphorylated state is more repressive than its unphosphorylated state, but the expression of its targets is even higher in its complete absence.

FabR senses long-chain unsaturated fatty acids to control virulence in pathogen Edwardsiella piscicida.

Long-chain unsaturated fatty acids (UFAs) can serve as nutrient sources or building blocks for bacterial membranes. However, little is known about how UFAs may be incorporated into the virulence programs of pathogens. A previous investigation identified FabR as a positive regulator of virulence gene expression in Edwardsiella piscicida. Here, chromatin immunoprecipitation-sequencing coupled with RNA-seq analyses revealed that 10 genes were under the direct control of FabR, including fabA, fabB, and cfa, which modulate the composition of UFAs. The binding of FabR to its target DNA was facilitated by oleoyl-CoA and inhibited by stearoyl-CoA. In addition, analyses of enzyme mobility shift assay and DNase I footprinting with wild-type and a null mutant (F131A) of FabR demonstrated crucial roles of FabR in binding to the promoters of fabA, fabB, and cfa. Moreover, FabR also binds to the promoter region of the virulence regulator esrB for its activation, facilitating the expression of the type III secretion system (T3SS) in response to UFAs. Furthermore, FabR coordinated with RpoS to modulate the expression of T3SS. Collectively, our results elucidate the molecular machinery of FabR regulating bacterial fatty acid composition and virulence in enteric pathogens, further expanding our knowledge of its crucial role in host-pathogen interactions.

Genetic and Environmental Investigation of a Novel Phenylamino Acetamide Inhibitor of the Pseudomonas aeruginosa Type III Secretion System.

Traditional antibiotics target essential cellular components or metabolic pathways conserved in both pathogenic and nonpathogenic bacteria. Unfortunately, long-term antibiotic use often leads to antibiotic resistance and disruption of the overall microbiota. In this work, we identified a phenylamino acetamide compound, named 187R, that strongly inhibited the expression of the type III secretion system (T3SS) encoding genes and the secretion of the T3SS effector proteins in Pseudomonas aeruginosa. T3SS is an important virulence factor, as T3SS-deficient strains of P. aeruginosa are greatly attenuated in virulence. We further showed that 187R had no effect on bacterial growth, implying a reduced selective pressure for the development of resistance. 187R-mediated repression of T3SS was dependent on ExsA, the master regulator of T3SS in P. aeruginosa. The impact of 187R on the host-associated microbial community was also tested using the Arabidopsis thaliana phyllosphere as a model. Both culture-independent (Illumina sequencing) and culture-dependent (Biolog) methods showed that the application of 187R had little impact on the composition and function of microbial community compared to the antibiotic streptomycin. Together, these results suggested that compounds that target virulence factors could serve as an alternative strategy for disease management caused by bacterial pathogens. IMPORTANCE New antimicrobial therapies are urgently needed, since antibiotic resistance in human pathogens has become one of the world's most urgent public health problems. Antivirulence therapy has been considered a promising alternative for the management of infectious diseases, as antivirulence compounds target only the virulence factors instead of the growth of bacteria, and they are therefore unlikely to affect commensal microorganisms. However, the impacts of antivirulence compounds on the host microbiota are not well understood. We report a potent synthetic inhibitor of the P. aeruginosa T3SS, 187R, and its effect on the host microbiota of Arabidopsis. Both culture-independent (Illumina sequencing) and culture-dependent (Biolog) methods showed that the impacts of the antivirulence compound on the composition and function of host microbiota were limited. These results suggest that antivirulence compounds can be a potential alternative method to antibiotics.

A prophage tail-like protein facilitates the endophytic growth of Burkholderia gladioli and mounting immunity in tomato.

A prophage tail-like protein (Bg_9562) of Burkholderia gladioli strain NGJ1 possesses broad-spectrum antifungal activity, and it is required for the bacterial ability to forage over fungi. Here, we analyzed whether heterologous overexpression of Bg_9562 or exogenous treatment with purified protein can impart disease tolerance in tomato. The physiological relevance of Bg_9562 during endophytic growth of NGJ1 was also investigated. Bg_9562 overexpressing lines demonstrate fungal and bacterial disease tolerance. They exhibit enhanced expression of defense genes and activation of mitogen-activated protein kinases. Treatment with Bg_9562 protein induces defense responses and imparts immunity in wild-type tomato. The defense-inducing ability lies within 18-51 aa region of Bg_9562 and is due to sequence homology with the bacterial flagellin epitope. Interaction studies suggest that Bg_9562 is perceived by FLAGELLIN-SENSING 2 homologs in tomato. The silencing of SlSERK3s (BAK1 homologs) prevents Bg_9562-triggered immunity. Moreover, type III secretion system-dependent translocation of Bg_9562 into host apoplast is important for elicitation of immune responses during colonization of NGJ1. Our study emphasizes that Bg_9562 is important for the endophytic growth of B. gladioli, while the plant perceives it as an indirect indicator of the presence of bacteria to mount immune responses. The findings have practical implications for controlling plant diseases.

Calcium-responsive plasmid copy number regulation is dependent on discrete YopD domains in Yersinia pseudotuberculosis.

Yersinia pathogenicity depends mainly on a Type III Secretion System (T3SS) responsible for translocating effector proteins into the eukaryotic target cell cytosol. The T3SS is encoded on a 70 kb, low copy number virulence plasmid, pYV. A key T3SS regulator, YopD, is a multifunctional protein and consists of discrete modular domains that are essential for pore formation and translocation of Yop effectors. In Y. pseudotuberculosis, the temperature-dependent plasmid copy number increase that is essential for elevated T3SS gene dosage and virulence is also affected by YopD. Here, we found that the presence of intracellular YopD results in increased levels of the CopA-RNA and CopB, two inhibitors of plasmid replication. Secretion of YopD leads to decreased expression of copA and copB, resulting in increased plasmid copy number. Moreover, using a systematic mutagenesis of YopD mutants, we demonstrated that the same discrete modular domains important for YopD translocation are also necessary for both the regulation of plasmid copy number as well as copA and copB expression. Hence, Yersinia has evolved a mechanism coupling active secretion of a plasmid-encoded component of the T3SS, YopD, to the regulation of plasmid replication. Our work provides evidence for the cross-talk between plasmid-encoded functions with the IncFII replicon.