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There is evidence that in infected cells in vitro the meningococcal HrpA/HrpB two-partner secretion system (TPS) mediates the exit of bacteria from the internalization vacuole and the docking of bacteria to the dynein motor resulting in the induction of pyroptosis. In this study we set out to study the role of the HrpA/HrpB TPS in establishing meningitis and activating pyroptotic pathways in an animal model of meningitis using a reference serogroup C meningococcal strain, 93/4286, and an isogenic hrpB knockout mutant, 93/4286Ω hrpB . Survival experiments confirmed the role of HrpA/HrpB TPS in the invasive meningococcal disease. In fact, the ability of the hrpB mutant to replicate in brain and spread systemically was impaired in mice infected with hrpB mutant. Furthermore, western blot analysis of brain samples during the infection demonstrated that: i. N. meningitidis activated canonical and non-canonical inflammasome pyroptosis pathways in the mouse brain; ii. the activation of caspase-11, caspase-1, and gasdermin-D was markedly reduced in the hrpB mutant; iii. the increase in the amount of IL-1β and IL-18, which are an important end point of pyroptosis, occurs in the brains of mice infected with the wild-type strain 93/4286 and is strongly reduced in those infected with 93/4286Ω hrpB . In particular, the activation of caspase 11, which is triggered by cytosolic lipopolysaccharide, indicates that during meningococcal infection pyroptosis is induced by intracellular infection after the exit of the bacteria from the internalizing vacuole, a process that is hindered in the hrpB mutant. Overall, these results confirm, in an animal model, that the HrpA/HrpB TPS plays a role in the induction of pyroptosis and suggest a pivotal involvement of pyroptosis in invasive meningococcal disease, paving the way for the use of pyroptosis inhibitors in the adjuvant therapy of the disease.

Background Pseudomonas aeruginosa intestinal carriage rates are significantly higher in immunosuppressed individuals and hospitalized patients who therefore have increased risk of infections and antibiotic-associated diarrhea. To combat intestinal dysbiosis and decolonize P. aeruginosa from gastrointestinal tract, we investigated the anti-adherence and gut microbiota modulation properties of marine prebiotic fucoidans. Methods Proteomic analysis of culture supernatant was performed by LC–MS/MS. Using lectin-based enzyme-linked immunosorbent assay, hemagglutinin domain interaction and inhibition with biomolecules were studied. We investigated the role of nutritional grade fucoidans in a mouse model and used 16S ribosomal RNA sequencing to examine fecal microbiota composition. Results Analysis of culture supernatant proteins indicated the secretion of two-partner secretion (TPS) family proteins, including TpsA1/CdiA2 and TpsA2/CdiA1. Lectin like activity at the N-terminal of TpsA due to a conserved hemagglutinin domain (Pfam identifier [ID] PF05860) mediates binding to mucins that carry multiple fucosylated glycans. Fucose-rich sulfated polysaccharides (fucoidans) and sulfated dextrans were found to be potent inhibitors of the recombinant N-terminal hemagglutinin domain of TpsA (TpsA-NT-HAD) binding to mucins. In a mouse model, antibiotic-induced dysbiosis was essential for P. aeruginosa gastrointestinal colonization. After prophylactic oral fucoidans supplementation, a higher proportion (60%) of the mice were decolonized over time and resisted re-colonization, this was associated with remarkable expansion of Bacteroides (post-infection day-3 abundance, 29–50%) and consequential reductions in bloom of Enterobacteriaceae and Enterococcaceae populations. In the non-supplemented group, Parabacteroides mediated recovery from dysbiosis but failed to decolonize P. aeruginosa . Conclusions Supplementing diet with marine prebiotic fucoidans can mediate earlier recovery from dysbiosis and decolonization of P. aeruginosa from gut by inhibiting secreted virulence factor (TpsA/CdiA) interaction with mucins and promoting the growth of beneficial Bacteroides population. We suggest the prophylactic use of nutritional grade fucoidans to decolonize P. aeruginosa from gastrointestinal tract of at-risk individuals to prevent infection and transmission of colonizing P. aeruginosa .

ExlA (also called exolysin) is a recently discovered virulence factor secreted by a subset of Pseudomonas aeruginosa strains in which a type 3 secretion system is lacking. exlA -positive strains were identified worldwide in the clinic, causing several types of infectious diseases, and were detected in various locations in the environment. ExlA possesses pore-forming activity and is cytolytic for most human cell types. It belongs to a class of poorly characterized bacterial toxins, sharing a similar protein domain organization and a common secretion pathway. This review summarizes the recent findings regarding ExlA synthesis, its secretion pathway, and its toxic behavior for host cells.

Belonging to the two-partner secretion family of proteins, contact-dependent growth inhibition (CDI) systems mediate interbacterial antagonism among closely related Gram-negative bacteria. The toxic portion of a large surface protein, BcpA/CdiA, is delivered to the cytoplasm of neighboring cells where it inhibits growth. Translocation of the antibacterial polypeptide out of the producing cell requires an associated outer membrane transporter, BcpB/CdiB. Some bacteria, including many Burkholderia species, encode multiple distinct CDI systems, but whether there is interaction between these systems is largely unknown. Using Burkholderia cepacia complex species as a model, here we show that related BcpB transporters exhibit considerable secretion flexibility and can secrete both cognate and non-cognate BcpA substrates. We also identified an additional unique Burkholderia dolosa CDI system capable of mediating interbacterial competition and demonstrated that its BcpB transporter has similar relaxed substrate specificity. Our results showed that two BcpB transporters (BcpB-2 and BcpB-3) were able to secrete all four of the B. dolosa BcpA toxins, while one transporter (BcpB-1) appeared unable to secrete even its cognate BcpA substrate under the tested conditions. This flexibility provided a competitive advantage, as strains lacking the full repertoire of BcpB proteins had decreased CDI activity. Similar results were obtained in Burkholderia multivorans , suggesting that secretion flexibility may be a conserved feature of Burkholderia CDI systems. Together these findings suggest that the interaction between distinct CDI systems enhances the efficiency of bacterial antagonism. The Burkholderia cepacia complex (Bcc) is a group of related opportunistic bacterial pathogens that occupy a diverse range of ecological niches and exacerbate disease in patients with underlying conditions. Contact-dependent growth inhibition (CDI) system proteins, produced by Gram-negative bacteria, contain antagonistic properties that allow for intoxication of closely related neighboring bacteria via a secreted protein, BcpA. Multiple unique CDI systems can be found in the same bacterial strain, and here we show that these distinct systems interact in several Bcc species. Our findings suggest that the interaction between CDI system proteins is important for interbacterial toxicity. Understanding the mechanism of interplay between CDI systems provides further insight into the complexity of bacterial antagonism. Moreover, since many bacterial species are predicted to encode multiple CDI systems, this study suggests that interactions between these distinct systems likely contribute to the overall competitive fitness of these species.

is a Gram-negative bacterial pathogen that normally resides as a commensal in the human nasopharynx but occasionally causes disease with high mortality and morbidity. To interact with its environment, it transports many proteins across the outer membrane to the bacterial cell surface and into the extracellular medium for which it deploys the common and well-characterized autotransporter, two-partner and type I secretion mechanisms, as well as a recently discovered pathway for the surface exposure of lipoproteins. The surface-exposed and secreted proteins serve roles in host-pathogen interactions, including adhesion to host cells and extracellular matrix proteins, evasion of nutritional immunity imposed by iron-binding proteins of the host, prevention of complement activation, neutralization of antimicrobial peptides, degradation of immunoglobulins, and permeabilization of epithelial layers. Furthermore, they have roles in interbacterial interactions, including the formation and dispersal of biofilms and the suppression of the growth of bacteria competing for the same niche. Here, we will review the protein secretion systems of and focus on the functions of the secreted proteins.

Neisseria meningitidis and Neisseria gonorrhoeae are the only pathogenic species of the genus Neisseria. Although these two species are closely related, they specialized on survival in completely different environments within the human host—the nasopharynx in the case of N. meningitidis versus the urogenital tract in the case of N. gonorrhoeae. The genetic background of these differences has not yet been determined. Here, we present a comparison of all characterized transcriptional regulators in these species, delineating analogous functions and disclosing differential functional developments of these DNA-binding proteins with a special focus on the recently characterized regulator FarR and its contribution to divergent host niche adaptation in the two Neisseria spp. Furthermore, we summarize the present knowledge on two-partner secretion systems in meningococci, highlighting their overall expression among meningococcal strains in contrast to the complete absence in gonococci. Concluding, the decisive role of these two entirely different factors in host niche adaptation of the two human pathogenic Neisseria species is depicted, illuminating another piece of the puzzle to locate the molecular basis of their differences in preferred colonization sites and pathogenicity.

Initially identified in pathogenic Gram-negative bacteria, the two-partner secretion (TPS) pathway, also known as Type Vb secretion, mediates the translocation across the outer membrane of large effector proteins involved in interactions between these pathogens and their hosts. More recently, distinct TPS systems have been shown to secrete toxic effector domains that participate in inter-bacterial competition or cooperation. The effects of these systems are based on kin vs. non-kin molecular recognition mediated by specific immunity proteins. With these new toxin-antitoxin systems, the range of TPS effector functions has thus been extended from cytolysis, adhesion, and iron acquisition, to genome maintenance, inter-bacterial killing and inter-bacterial signaling. Basically, a TPS system is made up of two proteins, the secreted TpsA effector protein and its TpsB partner transporter, with possible additional factors such as immunity proteins for protection against cognate toxic effectors. Structural studies have indicated that TpsA proteins mainly form elongated β helices that may be followed by specific functional domains. TpsB proteins belong to the Omp85 superfamily. Open questions remain on the mechanism of protein secretion in the absence of ATP or an electrochemical gradient across the outer membrane. The remarkable dynamics of the TpsB transporters and the progressive folding of their TpsA partners at the bacterial surface in the course of translocation are thought to be key elements driving the secretion process.

The type 5 secretion system (T5SS) is one of the more widespread secretion systems in Gram-negative bacteria. Proteins secreted by the T5SS are functionally diverse (toxins, adhesins, enzymes) and include numerous virulence factors. Mechanistically, the T5SS has long been considered the simplest of secretion systems, due to the paucity of proteins required for its functioning. Still, despite more than two decades of study, the exact process by which T5SS substrates attain their final destination and correct conformation is not totally deciphered. Moreover, the recent addition of new sub-families to the T5SS raises additional questions about this secretion mechanism. Central to the understanding of type 5 secretion is the question of protein folding, which needs to be carefully controlled in each of the bacterial cell compartments these proteins cross. Here, the biogenesis of proteins secreted by the Type 5 secretion system is discussed, with a focus on the various factors preventing or promoting protein folding during biogenesis.

Background Two-partner secretion systems in Gram-negative bacteria consist of an outer membrane protein TpsB that mediates the secretion of a cognate TpsA protein into the extracellular milieu. TpsA proteins have diverse, often virulence-related functions, and some of them inhibit the growth of related bacteria. In Neisseria meningitidis , several functions have been attributed to the TpsA proteins. Downstream of the tpsB and tpsA genes, several shorter tpsA -related gene cassettes, called tpsC , are located interspersed with intervening open-reading frames (IORFs). It has been suggested that the tpsC cassettes may recombine with the tpsA gene as a mechanism of antigenic variation. Here, we investigated (i) whether TpsA of N. meningitidis also has growth-inhibitory properties, (ii) whether tpsC cassettes recombine with the tpsA gene, and (iii) what the consequences of such recombination events might be. Results We demonstrate that meningococcal TpsA has growth-inhibitory properties and that the IORF located immediately downstream of tpsA confers immunity to the producing strain. Although bioinformatics analysis suggests that recombination between tpsC cassettes and tpsA occurs, detailed analysis of the tpsA gene in a large collection of disease isolates of three clonal complexes revealed that the frequency is very low and cannot be a mechanism of antigenic variation. However, recombination affected growth inhibition. In vitro experiments revealed that recombination can be mediated through acquirement of tpsC cassettes from the environment and it identified the regions involved in the recombination. Conclusions Meningococcal TpsA has growth-inhibitory properties. Recombination between tpsA and tpsC cassettes occurs in vivo but is rare and has consequences for growth inhibition. A recombination model is proposed and we propose that the main goal of recombination is the collection of new IORFs for protection against a variety of TpsA proteins.

Uncovering the secretome of Yersinia pestis is a necessary measure to better understand the virulence of this plague-causing bacterium. Using bioinformatics methods, the components of all the secretion systems known to date in the Y. pestis KIM genome were mapped, including several systems identified by this study. It was found that this organism possesses Sec, twin-arginine translocation, signal recognition particle, Omp85/YaeT, type I, type II, type III, type VI, chaperone/usher, autotransporter, and two-partner secretion (TPS) systems, but lacks a type IV secretion system. The TPS systems caught the authors' attention, for they are used by several bacterial pathogens for the secretion of large virulence determinants. Reverse transcriptase-PCR analysis confirmed the gene expression of three TPS systems in Y. pestis KIM; bioinformatics studies indicated that the exoproteins of these TPS systems are putative hemolysins and adhesins. Results thus suggest that these are functional systems, capable of secreting virulence proteins that might contribute to plague infection. This is the first report on the complete secretome of pathogenic Y. pestis KIM.