SUMMARY Pore-forming toxins (PFTs) are the most common bacterial cytotoxic proteins and are required for virulence in a large number of important pathogens, including Streptococcus pneumoniae , group A and B streptococci, Staphylococcus aureus , Escherichia coli , and Mycobacterium tuberculosis . PFTs generally disrupt host cell membranes, but they can have additional effects independent of pore formation. Substantial effort has been devoted to understanding the molecular mechanisms underlying the functions of certain model PFTs. Likewise, specific host pathways mediating survival and immune responses in the face of toxin-mediated cellular damage have been delineated. However, less is known about the overall functions of PFTs during infection in vivo . This review focuses on common themes in the area of PFT biology, with an emphasis on studies addressing the roles of PFTs in in vivo and ex vivo models of colonization or infection. Common functions of PFTs include disruption of epithelial barrier function and evasion of host immune responses, which contribute to bacterial growth and spreading. The widespread nature of PFTs make this group of toxins an attractive target for the development of new virulence-targeted therapies that may have broad activity against human pathogens.

Lactobacillus iners is a common constituent of the human vaginal microbiota. This species was only recently characterized due to its fastidious growth requirements and has been hypothesized to play a role in the pathogenesis of bacterial vaginosis. Here we present the identification and molecular characterization of a protein toxin produced by L. iners. The L. iners genome encodes an open reading frame with significant primary sequence similarity to intermedilysin (ILY; 69.2% similarity) and vaginolysin (VLY; 68.4% similarity), the cholesterol-dependent cytolysins from Streptococcus intermedius and Gardnerella vaginalis, respectively. Clinical isolates of L. iners produce this protein, inerolysin (INY), during growth in vitro, as assessed by Western analysis. INY is a pore-forming toxin that is activated by reducing agents and inhibited by excess cholesterol. It is active across a pH range of 4.5 to 6.0 but is inactive at pH 7.4. At sublytic concentrations, INY activates p38 mitogen-activated protein kinase and allows entry of fluorescent phalloidin into the cytoplasm of epithelial cells. Unlike VLY and ILY, which are human specific, INY is active against cells from a broad range of species. INY represents a new target for studies directed at understanding the role of L. iners in states of health and disease at the vaginal mucosal surface.The cholesterol-dependent cytolysins (CDCs) are a family of protein toxins produced by a wide range of Gram-positive bacteria. CDCs share several characteristics, including a fourdomain structure, a requirement for membrane cholesterol for efficient activity, and an ability to form large pores in host cells, exceeding 150 Å in diameter (38). In general, soluble CDC monomers are secreted into the extracellular environment and bind to target cell membranes through direct recognition of cholesterol or, in the cases of the human-specific toxins vaginolysin (VLY) from Gardnerella vaginalis and intermedilysin (ILY) from Streptococcus intermedius, via recognition of human CD59 on the target cell surface (12,14,21). Following membrane association, CDCs oligomerize to form a prepore structure, a process that is dependent upon the availability of cholesterol (22)(23)(24)34). In many cases, CDCs are required for virulence for their cognate organisms, and rather than acting solely as cytolytic toxins, CDCs may have more sophisticated roles in disease pathogenesis (15,18,30). Understanding CDC evolution and host specificity is of considerable interest and has been limited by incomplete knowledge of the diversity of the CDC family. In particular, characterization of cytolysins most closely related to those in which host specificity has evolved may provide additional insights into the mechanism and effects of such restriction.Lactobacillus iners is a relatively recently recognized member of the human vaginal microbiota (9, 33, 42) that was initially overlooked because of its inability to grow on de ManRogosa-Sharpe agar, which is normally used to isolate vaginal lactobacilli. In healthy women...

Preterm birth and fetal demise are likely the direct result of toxin-induced damage and inflammation rather than differences in efficiency of ascension into the upper genital tract. These data demonstrate a distinct contribution of βH/C to GBS chorioamnionitis and subsequent fetal infection in vivo and showcase a model for this most proximal step in GBS pathogenesis.

The p110delta isoform of class I phosphoinositide 3-kinase (PI3K) plays a major role in B cell receptor signaling, while its p110gamma counterpart is thought to predominate in leukocyte chemotaxis. Consequently, emphasis has been placed on developing PI3Kgamma selective inhibitors to treat disease states that result from inappropriate tissue accumulation of leukocytes. We now demonstrate that PI3Kdelta blockade is effective in treating an autoimmune disorder in which neutrophil infiltration is required for tissue injury. Using the K/BxN serum transfer model of arthritis, in which neutrophils and leukotriene B(4) (LTB(4)) participate, we show that genetic deletion or selective inhibition of PI3Kdelta diminishes joint erosion to a level comparable to its PI3Kgamma counterpart. Moreover, the induction and progression of joint destruction was profoundly reduced in the absence of both PI3K isoforms and correlated with a limited ability of neutrophils to migrate into tissue in response to LTB(4). However, the dynamic interplay between these isoforms is not pervasive, as fMLP-induced neutrophil extravasation was primarily reliant on PI3Kgamma. Our results not only demonstrate that blockade of PI3Kdelta has potential therapeutic value in the treatment of chronic inflammatory conditions, but also provide evidence that dual inhibition of these lipid kinases may yield superior clinical results.

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Bacterial vaginosis is a highly prevalent and poorly understood polymicrobial disorder of the vaginal microbiota, with significant adverse sequelae. Gardnerella vaginalis predominates in bacterial vaginosis. Biofilms of G. vaginalis are present in human infections and are implicated in persistent disease, treatment failure, and transmission. Here we demonstrate that G. vaginalis biofilms contain extracellular DNA, which is essential to their structural integrity. Enzymatic disruption of this DNA specifically inhibits biofilms, acting on both newly forming and established biofilms. DNase liberates bacteria from the biofilm to supernatant fractions and potentiates the activity of metronidazole, an antimicrobial agent used in the treatment of bacterial vaginosis. Using a new murine vaginal colonization model for G. vaginalis, we demonstrate >10-fold inhibition of G. vaginalis colonization by DNase. We conclude that DNase merits investigation as a potential nonantibiotic adjunct to existing bacterial vaginosis therapies in order to decrease the risk of chronic infection, recurrence, and associated morbidities.

The human upper respiratory tract, including the nasopharynx, is colonized by a diverse array of microorganisms. While the host generally exists in harmony with the commensal microflora, under certain conditions, these organisms may cause local or systemic disease. Respiratory epithelial cells act as local sentinels of the innate immune system, responding to conserved microbial patterns through activation of signal transduction pathways and cytokine production. In addition to colonizing microbes, these cells may also be influenced by environmental agents, including cigarette smoke (CS). Because of the strong relationship among secondhand smoke exposure, bacterial infection, and sinusitis, we hypothesized that components in CS might alter epithelial cell innate immune responses to pathogenic bacteria. We examined the effect of CS condensate (CSC) or extract (CSE) on signal transduction and cytokine production in primary and immortalized epithelial cells of human or murine origin in response to nontypeable Haemophilus influenzae and Staphylococcus aureus. We observed that epithelial production of interleukin-8 (IL-8) and IL-6 in response to bacterial stimulation was significantly inhibited in the presence of CS (P < 0.001 for inhibition by either CSC or CSE). In contrast, epithelial production of beta interferon (IFN-␤) was not inhibited. CSC decreased NF-B activation (P < 0.05) and altered the kinetics of mitogen-activated protein kinase phosphorylation in cells exposed to bacteria. Treatment of CSC with antioxidants abrogated CSC-mediated reduction of epithelial IL-8 responses to bacteria (P > 0.05 compared to cells without CSC treatment). These results identify a novel oxidant-mediated immunosuppressive role for CS in epithelial cells.

BackgroundNext-generation sequencing of transposon-genome junctions from a saturated bacterial mutant library (Tn-seq) is a powerful tool that permits genome-wide determination of the contribution of genes to fitness of the organism under a wide range of experimental conditions. We report development, testing, and results from a Tn-seq system for use in Streptococcus agalactiae (group B Streptococcus; GBS), an important cause of neonatal sepsis.MethodsOur method uses a Himar1 mini-transposon that inserts at genomic TA dinucleotide sites, delivered to GBS on a temperature-sensitive plasmid that is subsequently cured from the bacterial population. In order to establish the GBS essential genome, we performed Tn-seq on DNA collected from three independent mutant libraries—with at least 135,000 mutants per library—at serial 24 h time points after outgrowth in rich media.ResultsAfter statistical analysis of transposon insertion density and distribution, we identified 13.5 % of genes as essential and 1.2 % as critical, with high levels of reproducibility. Essential and critical genes are enriched for fundamental cellular housekeeping functions, such as acyl-tRNA biosynthesis, nucleotide metabolism, and glycolysis. We further validated our system by comparing fitness assignments of homologous genes in GBS and a close bacterial relative, Streptococcus pyogenes, which demonstrated 93 % concordance. Finally, we used our fitness assignments to identify signal transduction pathway components predicted to be essential or critical in GBS.ConclusionsWe believe that our baseline fitness assignments will be a valuable tool for GBS researchers and that our system has the potential to reveal key pathogenesis gene networks and potential therapeutic/preventative targets.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2741-z) contains supplementary material, which is available to authorized users.