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Urinary tract infection (UTI) is a disease of extremely high incidence in both community and nosocomial settings. UTIs cause significant morbidity and mortality, with approximately 150 million cases globally per year. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTI and is generally treated empirically. However, the rapidly increasing incidence of UTIs caused by multidrug-resistant UPEC strains has led to limited available treatment options and highlights the urgent need to develop alternative treatment and prevention strategies. In this study, we performed a comprehensive analysis to define the regulation, structure, function, and immunogenicity of recently identified UPEC vaccine candidate C1275 (here referred to as IrmA). We showed that the irmA gene is highly prevalent in UPEC, is cotranscribed with the biofilm-associated antigen 43 gene, and is regulated by the global oxidative stress response OxyR protein. Localization studies identified IrmA in the UPEC culture supernatant. We determined the structure of IrmA and showed that it adopts a unique domain-swapped dimer architecture. The dimeric structure of IrmA displays similarity to those of human cytokine receptors, including the interleukin-2 receptor (IL-2R), interleukin-4 receptor (IL-4R), and interleukin-10 receptor (IL-10R) binding domains, and we showed that purified IrmA can bind to their cognate cytokines. Finally, we showed that plasma from convalescent urosepsis patients contains high IrmA antibody titers, demonstrating the strong immunogenicity of IrmA. Taken together, our results indicate that IrmA may play an important role during UPEC infection., IMPORTANCE Uropathogenic E. coli (UPEC) is the primary cause of urinary tract infection (UTI), a disease of major significance to human health. Globally, the incidence of UPEC-mediated UTI is strongly associated with increasing antibiotic resistance, making this extremely common infection a major public health concern. In this report, we describe the regulatory, structural, functional, and immunogenic properties of a candidate UPEC vaccine antigen, IrmA. We demonstrate that IrmA is a small UPEC protein that forms a unique domain-swapped dimer with structural mimicry to several human cytokine receptors. We also show that IrmA binds to IL-2, IL-4, and IL-10, is strongly immunogenic in urosepsis patients, and is coexpressed with factors associated with biofilm formation. Overall, this work suggests a potential novel contribution for IrmA in UPEC infection.

In bacteria, copper (Cu) can support metabolic processes as an enzymatic cofactor but can also cause cell damage if present in excess, leading to intoxication. In group B Streptococcus (GBS) a system for control of Cu efflux based on the canonical cop operon supports survival during Cu stress. In some other bacteria, genetic systems additional to the cop operon are engaged during Cu stress and also contribute to Cu management. Here, we examined genetic systems beyond the cop operon in GBS for regions that contribute to survival of GBS in Cu stress using a forward genetic screen and probe of the entire bacterial genome. A high-density mutant library, generated using pGh9-ISS1, was used to expose GBS to Cu stress and compared to non-exposed controls en masse. Nine genes were identified as essential for GBS survival in Cu stress, whereas five genes constrained GBS growth in Cu stress. The genes encode varied factors including enzymes for metabolism, cell wall synthesis, transporters and global transcriptional regulators. Targeted mutation of the genes validated their roles in GBS resistance to Cu stress. Notably, several genes, including stp1, yceG, plyB and rfaB were also essential for resistance to Zn stress. Excepting copA, the genes identified are new to the area of bacterial metal ion intoxication. We conclude that a discrete and limited suite of genes beyond the cop operon in GBS contribute to a repertoire of mechanisms used to survive Cu stress in vitro and achieve cellular homeostasis.Significance StatementGenetic systems for copper (Cu) homeostasis in bacteria, including Streptococci, are vital to survive metal ion stress. Genetic systems that underpin survival of GBS during Cu stress, beyond for the archetypal cop operon for Cu management, are undefined. We show that Streptococcus resists Cu intoxication by utilizing a discrete and limited suite of genes beyond the cop operon, including several genes that are new to the area of bacterial cell metal ion homeostasis. The Cu resistome of GBS defined here enhances our understanding of metal ion homeostasis in GBS.

Bacteria can utilize Copper (Cu) as a trace element to support cellular processes; however, excess Cu can intoxicate bacteria. Here, we characterize the cop operon in group B streptococcus (GBS), and establish its role in evasion of Cu intoxication and the response to Cu stress on virulence. Growth of GBS mutants deficient in either the copA Cu exporter, or the copY repressor, were severely compromised in Cu-stress conditions. GBS survival of Cu stress reflected a mechanism of CopY de-repression of the CopA efflux system. However, neither mutant was attenuated for intracellular survival in macrophages. Analysis of global transcriptional responses to Cu by RNA-sequencing revealed a stress signature encompassing homeostasis of multiple metals. Genes induced by Cu stress included putative metal transporters for manganese import, whereas a system for iron export was repressed. In addition, copA promoted the ability of GBS to colonize the blood, liver and spleen of mice following disseminated infection. Together, these findings show that GBS copA mediates resistance to Cu intoxication, via regulation by the Cu-sensing transcriptional repressor, copY. Cu stress responses in GBS reflect a transcriptional signature that heightens virulence and represents an important part of the bacteria’s ability to survive in different environments.ImportanceUnderstanding how bacteria manage cellular levels of metal ions, such as copper, helps to explain how microbial cells can survive in different stressful environments. We show how the opportunistic pathogen group B Streptococcus (GBS) achieves homeostasis of intracellular copper through the activities of the genes that comprise the cop operon, and describe how this helps GBS survive in stressful environments, including in the mammalian host during systemic disseminated infection.

In many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD+ ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae, also known as group B streptococcus (GBS). We present structures of Rex bound to NAD+ and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium. The structures reveal the molecular basis of DNA binding and the conformation alterations between the free NAD+ complex and DNA-bound form of Rex. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also expression of the monocistronic rex gene as well as virulence gene expression. Rex enhances GBS virulence after disseminated infection in mice. Mechanistically, NAD+ stabilizes Rex as a repressor in the absence of NADH. However, GBS Rex is unique compared to Rex regulators previously characterized because of its sensing mechanism: we show that it primarily responds to NAD+ levels (or growth rate) rather than to the NADH/NAD+ ratio. These results indicate that Rex plays a key role in GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to harvest nutrients from the host., Author summary The Rex family transcription repressors serve critical roles in control of cellular metabolism. Pathogenic microorganisms often use metabolic cues to regulate their metabolism and virulence functions. Here, we structurally and functionally characterized Rex in the facultative aerobic Gram-positive coccus Streptococcus agalactiae, also known as group B streptococcus (GBS). GBS is a leading cause of meningitis in newborn babies and infants. Rex interacts with both the oxidized and reduced form of the cofactor nicotinamide adenine dinucleotide (NAD). The reduced form (NADH) abrogates DNA binding whereas the oxidized form (NAD+) enhance binding of Rex to DNA. We show that the cellular concentration of NAD+ is the main effector of Rex binding to DNA. A drop in the concentration of NAD+ is triggered when cells do not find a specific sugar such as glucose in the environment. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also its own expression as well as virulence gene expression. Together our results identify GBS Rex as an important transcription regulator critical for GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to efficiently utilize nutrient resources provided by the host.

Zinc (Zn) is an essential trace element for normal bacterial physiology but divergently, can intoxicate bacteria at high concentrations. Here, we define the molecular systems for Zn detoxification in Streptococcus agalactiae, also known as group B Streptococcus, and examine the effects of resistance to Zn stress on virulence. We compared the growth of wild-type bacteria and mutants deleted for the Zn exporter, czcD, and the response regulator, sczA, using Zn-stress conditions in vitro. Macrophage antibiotic protection assays and a mouse model of disseminated infection were used to assess virulence. Global bacterial transcriptional responses to Zn stress were defined by RNA-sequencing and qRTPCR. czcD and sczA enabled S. agalactiae to survive Zn stress, with the putative CzcD efflux system activated by SczA. Additional genes activated in response to Zn stress encompassed divalent cation transporters that contribute to regulation of Mn and Fe homeostasis. In vivo, the czcD-sczA Zn-management axis supported virulence in the blood, heart, liver and bladder. Additionally, several genes not previously linked to Zn stress in any bacterium, including most notably, arcA for arginine deamination also mediated resistance to Zn stress; representing a novel molecular mechanism of bacterial resistance to metal intoxication. Taken together, these findings show that S. agalactiae responds to Zn stress by sczA regulation of czcD, with additional novel mechanisms of resistance supported by arcA, encoding arginine deaminase. Cellular management of Zn stress in S. agalactiae supports virulence by facilitating bacterial survival in the host during systemic infection.Importance StatementStreptococcus agalactiae, also known as group B streptococcus, is an opportunistic pathogen that causes various diseases in humans and animals. This bacterium has genetic systems that enable Zinc (Zn) detoxification in environments of metal stress, but these systems remain largely undefined. Using a combination of genomic, genetic and cellular assays we show that this pathogen controls Zn export through CzcD to manage Zn stress, and utilizes a system of arginine deamination never previously linked to metal stress responses in bacteria to survive metal intoxication. We show that these systems are crucial for survival of S. agalactiae in vitro during Zn stress and also enhance virulence during systemic infection in mice. These discoveries establish new molecular mechanisms of resistance to metal intoxication in bacteria; we suggest these mechanisms are likely to operate in other bacteria as a way to sustain microbial survival in conditions of metal stress, including in host environments.

The bladder is innervated by primary afferent nerve fibres that detect bladder distension and, through projections into the spinal cord, provide sensory input to the central nervous system circuits regulating bladder sensation and function. Uropathogenic E. coli (UPEC) bacteria are the primary cause of urinary tract infection (UTI) in adults, inducing clinical symptoms characterised by exaggerated bladder sensation, including urgency, frequency, and pelvic pain. However, the mechanisms underlying UTI-induced modulation of bladder afferent function are yet to be explored. Here, we isolated supernatants from the bladders of female mice acutely infected with UPEC (strain CFT073), or those sham-treated with phosphate buffered saline. Supernatants were then applied into the bladder lumen of healthy donor mice, and multiunit bladder afferent nerve responses to distension measured ex-vivo. Supernatant constituents from UPEC or sham-treated mice were analysed using a mouse cytokine multiplex assay. Supernatants from UPEC-infected mice significantly enhanced bladder afferent firing to distension in the absence of changes in muscle compliance. Further evaluation revealed that UPEC supernatants exclusively sensitised high-threshold bladder mechanoreceptors to graded bladder distension and also recruited a population of "silent nociceptors" to become mechanosensitive, thereby amplifying bladder afferent responses to physiological stimuli. UPEC supernatants contained significantly elevated concentrations of a range of cytokines released from innate immune cells, including but not limited to TNF-α, IL-1β, IL-6, IL-17, IFN-gamma, and MCP-1. These data provide novel mechanistic insight into how UPEC-mediated UTI induces bladder hypersensitivity and the symptoms of frequency, urgency, and pelvic pain.

Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH), encoded by gapC, is a glycolytic enzyme that is associated with virulence and immune-mediated protection. However, the role of GAPDH in cellular cytokine responses to S. agalactiae, bacterial phagocytosis and colonization of the female reproductive tract, a central host niche, is unknown. We expressed and studied purified recombinant GAPDH (rGAPDH) of S. agalactiae in cytokine elicitation assays with human monocyte-derived macrophage, epithelial cell, and polymorphonuclear leukocyte (PMN) co-culture infection models. We also generated a S. agalactiae mutant that over-expresses GAPDH (oeGAPDH) from gapC using a constitutively active promoter, and analysed the mutant in murine macrophage antibiotic protection assays and in virulence assays in vivo, using a colonization model that is based on experimental infection of the reproductive tract in female mice. Human cell co-cultures produced interleukin (IL)-1β, IL-6, macrophage inflammatory protein (MIP)-1, tumour necrosis factor (TNF)-α and IL-10 within 24 h of exposure to rGAPDH. PMNs were required for several of these cytokine responses. However, over-expression of GAPDH in S. agalactiae did not significantly affect measures of phagocytic uptake compared to an empty vector control. In contrast, oeGAPDH-S. agalactiae showed a small but statistically significant attenuation for persistence in the reproductive tract of female mice during the chronic phase of infection (10-28 days post-inoculation), relative to the vector control. We conclude that S. agalactiae GAPDH elicits production of multiple cytokines from human cells, and over-expression of GAPDH renders the bacterium more susceptible to host clearance in the female reproductive tract. One-sentence summary: This study shows Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase, an enzyme that functions in glycolysis, gluconeogenesis and virulence, modifies phagocytosis outcomes, including cytokine synthesis, and effects bacterial persistence in the female reproductive tract.

In bacteria, guaA encodes guanosine monophosphate synthetase that confers an ability to biosynthesize guanine nucleotides de novo. This enables bacterial colonization in different environments and, while guaA is widely distributed among Bacteroidetes and Firmicutes, its contribution to the inhabitation of the human microbiome by commensal bacteria is unclear. We studied Streptococcus as a commensal urogenital tract bacterium and opportunistic pathogen, and explored the role of guaA in bacterial survival and colonization of urine. Analysis of guaA-deficient Streptococcus revealed guanine utilization is essential for bacterial colonization of this niche. The genomic location of guaA in other commensals of the human urogenital tract revealed substantial cross-phyla diversity and organizational structures of guaA that are divergent across phyla. Essentiality of guaA for Streptococcus colonization in the urinary tract establishes that purine biosynthesis is a critical element of the ability of this bacterium to survive and colonize in the host as part of the resident human microbiome.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an evolutionarily conserved essential enzyme in the glycolytic pathway. GAPDH is also involved in a wide spectrum of non-catalytic cellular 'moonlighting' functions. Bacterial surface-associated GAPDHs engage in many host interactions that aid in colonization, pathogenesis, and virulence. We have structurally and functionally characterized the recombinant GAPDH of the obligate intracellular bacteria Chlamydia trachomatis, the leading cause of sexually transmitted bacterial and ocular infections. Contrary to earlier speculations, recent data confirm the presence of glucose-catabolizing enzymes including GAPDH in both stages of the biphasic life cycle of the bacterium. The high-resolution crystal structure described here provides a close-up view of the enzyme's active site and surface topology and reveals two chemically modified cysteine residues. Moreover, we show for the first time that purified C. trachomatis GAPDH binds to human plasminogen and plasmin. Based on the versatility of GAPDH's functions, data presented here emphasize the need for investigating the Chlamydiae GAPDH's involvement in biological functions beyond energy metabolism.

Urinary tract infections (UTI) frequently progress to chronicity in infected individuals but the mechanisms of pathogenesis underlying chronic UTI are not well understood. We examined the role of interleukin (IL)-17A in UTI because this cytokine promotes innate defense against uropathogenic Escherichia coli (UPEC). Analysis of UPEC persistence and pyelonephritis in mice deficient in IL-17A revealed that UPEC CFT073 caused infection at a rate higher than the multidrug resistant strain EC958. Il17a-/- mice exhibited pyelonephritis with kidney bacterial burdens higher than those of wild-type (WT) mice. Synthesis of IL-17A in the bladder reflected a combination of γδ-T and TH 17 cell responses. Analysis of circulating inflammatory mediators at 24h postinoculation identified predictors of progression to chronicity, including IL-6 and monocyte chemoattractant protein-1 (MCP-1). Histological analysis identified infiltrating populations of neutrophils, NK cells, and γδ T cells in the bladder, whereas neutrophils predominated in the kidney. Analysis of the contribution of flagella to chronicity using hyper-flagellated and fliC-deficient UPEC in WT and Il17a-/- mice revealed that, in a host that is deficient for the production of IL-17A, flagella contribute to bacterial persistence. These findings show a role for IL-17A in defense against chronic UTI and a contribution of flagella to the pathogenesis of infection.

Interleukin-10 is part of the immune response to urinary tract infection (UTI) due to E. coli, and it is important in the early control of infection in the bladder. Defining the mechanism of engagement of the immune system by the bacteria that enables the protective IL-10 response is critical to exploring how we might exploit this mechanism for new infection control strategies. In this study, we reveal part of the bacterial flagellar apparatus (FliC) is an important component that is sensed by and responsible for induction of IL-10 in the response to UPEC. We show this response occurs in a TLR5-dependent manner. Using infection prevention and control trials in mice infected with E. coli, this study also provides evidence that purified FliC might be of value in novel approaches for the treatment of UTI or in preventing infection by exploiting the FliC-triggered bladder transcriptome., Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) engages interleukin-10 (IL-10) as an early innate immune response to regulate inflammation and promote the control of bladder infection. However, the mechanism of engagement of innate immunity by UPEC that leads to elicitation of IL-10 in the bladder is unknown. Here, we identify the major UPEC flagellar filament, FliC, as a key bacterial component sensed by the bladder innate immune system responsible for the induction of IL-10 synthesis. IL-10 responses of human as well as mouse bladder epithelial cell-monocyte cocultures were triggered by flagella of three major UPEC representative strains, CFT073, UTI89, and EC958. FliC purified to homogeneity induced IL-10 in vitro and in vivo as well as other functionally related cytokines, including IL-6. The genome-wide innate immunological context of FliC-induced IL-10 in the bladder was defined using RNA sequencing that revealed a network of transcriptional and antibacterial defenses comprising 1,400 genes that were induced by FliC. Of the FliC-responsive bladder transcriptome, altered expression of il10 and 808 additional genes were dependent on Toll-like receptor 5 (TLR5), according to analysis of TLR5-deficient mice. Examination of the potential of FliC and associated innate immune signature in the bladder to boost host defense, based on prophylactic or therapeutic administration to mice, revealed significant benefits for the control of UPEC. We conclude that detection of FliC through TLR5 triggers rapid IL-10 synthesis in the bladder, and FliC represents a potential immune modulator that might offer benefit for the treatment or prevention of UPEC UTI. IMPORTANCE Interleukin-10 is part of the immune response to urinary tract infection (UTI) due to E. coli, and it is important in the early control of infection in the bladder. Defining the mechanism of engagement of the immune system by the bacteria that enables the protective IL-10 response is critical to exploring how we might exploit this mechanism for new infection control strategies. In this study, we reveal part of the bacterial flagellar apparatus (FliC) is an important component that is sensed by and responsible for induction of IL-10 in the response to UPEC. We show this response occurs in a TLR5-dependent manner. Using infection prevention and control trials in mice infected with E. coli, this study also provides evidence that purified FliC might be of value in novel approaches for the treatment of UTI or in preventing infection by exploiting the FliC-triggered bladder transcriptome.

Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) engages interleukin-10 (IL-10) as an early innate immune response to regulate inflammation and promote the control of bladder infection. However, the mechanism of engagement of innate immunity by UPEC that leads to elicitation of IL-10 in the bladder is unknown. Here, we identify the major UPEC flagellar filament, FliC, as a key bacterial component sensed by the bladder innate immune system responsible for the induction of IL-10 synthesis. IL-10 responses of human as well as mouse bladder epithelial cell-monocyte cocultures were triggered by flagella of three major UPEC representative strains, CFT073, UTI89, and EC958. FliC purified to homogeneity induced IL-10 in vitro and in vivo as well as other functionally related cytokines, including IL-6. The genome-wide innate immunological context of FliC-induced IL-10 in the bladder was defined using RNA sequencing that revealed a network of transcriptional and antibacterial defenses comprising 1,400 genes that were induced by FliC. Of the FliC-responsive bladder transcriptome, altered expression of il10 and 808 additional genes were dependent on Toll-like receptor 5 (TLR5), according to analysis of TLR5-deficient mice. Examination of the potential of FliC and associated innate immune signature in the bladder to boost host defense, based on prophylactic or therapeutic administration to mice, revealed significant benefits for the control of UPEC. We conclude that detection of FliC through TLR5 triggers rapid IL-10 synthesis in the bladder, and FliC represents a potential immune modulator that might offer benefit for the treatment or prevention of UPEC UTI. IMPORTANCE Interleukin-10 is part of the immune response to urinary tract infection (UTI) due to E. coli, and it is important in the early control of infection in the bladder. Defining the mechanism of engagement of the immune system by the bacteria that enables the protective IL-10 response is critical to exploring how we might exploit this mechanism for new infection control strategies. In this study, we reveal part of the bacterial flagellar apparatus (FliC) is an important component that is sensed by and responsible for induction of IL-10 in the response to UPEC. We show this response occurs in a TLR5-dependent manner. Using infection prevention and control trials in mice infected with E. coli, this study also provides evidence that purified FliC might be of value in novel approaches for the treatment of UTI or in preventing infection by exploiting the FliC-triggered bladder transcriptome.

Streptococcus agalactiae, also known as group B Streptococcus, is an aetiological agent of urinary tract infection (UTI) in adults, including cystitis, pyelonephritis and asymptomatic bacteriuria (ABU). Whereas ABU-causing S. agalactiae (ABSA) have been shown to grow and achieve higher culture denstity in human urine compared to uropathogenic S. agalactiae (UPSA) other phenotypic distinctions between S. agalactiae isolated from different forms of UTI are not known. Here, we define the hemolytic activities and biofilm-formation of a collection of clinical isolates of UPSA, ABSA and recurrent S. agalactiae bacteriuria (rSAB) strains to explore these phenotypes in the context of clinical history of isolates. A total of 61 UPSA, 184 ABSA, and 47 rSAB isolates were analyzed for relative hemolytic activity by spot assay on blood agar, which was validated using a erythrocyte lysis suspension assay. Biofilm formation was determined by microtiter plate assay with Lysogeny and Todd-Hewitt broths supplemented with 1% glucose to induce biofilm formation. We also used multiplex PCR to analyze isolates for the presence of genes encoding adhesive pili, which contribute to biofilm formation. Comparing the hemolytic activities of 292 isolates showed, surprisingly, that ABSA strains were significantly more likely to be highly hemolytic compared to other strains. In contrast, there were no differences between the relative abilities of strains from the different clinical history groups to form biofilms. Taken together, these findings demonstrate a propensity of S. agalactiae causing ABU to be highly hemolytic but no link between clinical history of UTI strains and ability to form biofilm.

Uropathogenic E. coli (UPEC) is the major cause of urinary tract infections and a frequent cause of sepsis. Nearly half of all UPEC strains produce the potent cytotoxin hemolysin, and its expression is associated with enhanced virulence. In this study, we explored hemolysin variation within the globally dominant UPEC ST131 clone, finding that strains from the ST131 sublineage with the greatest multidrug resistance also possess the strongest hemolytic activity. We also employed an innovative forward genetic screen to define the set of genes required for hemolysin production. Using this approach, and subsequent targeted mutagenesis and complementation, we identified new hemolysin-controlling elements involved in LPS inner core biosynthesis and cytoplasmic chaperone activity, and we show that mechanistically they are required for hemolysin secretion. These original discoveries substantially enhance our understanding of hemolysin regulation, secretion and function., Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections. Nearly half of all UPEC strains secrete hemolysin, a cytotoxic pore-forming toxin. Here, we show that the prevalence of the hemolysin toxin gene (hlyA) is highly variable among the most common 83 E. coli sequence types (STs) represented on the EnteroBase genome database. To explore this diversity in the context of a defined monophyletic lineage, we contextualized sequence variation of the hlyCABD operon within the genealogy of the globally disseminated multidrug-resistant ST131 clone. We show that sequence changes in hlyCABD and its newly defined 1.616-kb-long leader sequence correspond to phylogenetic designation, and that ST131 strains with the strongest hemolytic activity belong to the most extensive multidrug-resistant sublineage (clade C2). To define the set of genes involved in hemolysin production, the clade C2 strain S65EC was completely sequenced and subjected to a genome-wide screen by combining saturated transposon mutagenesis and transposon-directed insertion site sequencing with the capacity to lyse red blood cells. Using this approach, and subsequent targeted mutagenesis and complementation, 13 genes were confirmed to be specifically required for production of active hemolysin. New hemolysin-controlling elements included discrete sets of genes involved in lipopolysaccharide (LPS) inner core biosynthesis (waaC, waaF, waaG, and rfaE) and cytoplasmic chaperone activity (dnaK and dnaJ), and we show these are required for hemolysin secretion. Overall, this work provides a unique description of hemolysin sequence diversity in a single clonal lineage and describes a complex multilevel system of regulatory control for this important toxin.

INTRODUCTION AND OBJECTIVES:Uropathogenic E. coli (UPEC) is the primary cause of adult urinary tract infection (UTI) inducing the clinical symptoms of urgency, urinary frequency, and acute pelvic p...

Uropathogenic

Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections (UTIs). The multidrug-resistant E. coli sequence type 131 (ST131) clone is a serious threat to human health, yet its effects on immune responses are not well understood. Here we screened a panel of ST131 isolates, finding that only strains expressing the toxin hemolysin A (HlyA) killed primary human macrophages and triggered maturation of the inflammasome-dependent cytokine IL-1β. Using a representative strain, the requirement for the hlyA gene in these responses was confirmed. We also observed considerable heterogeneity in levels of cell death initiated by different HlyA+ve ST131 isolates, and this correlated with secreted HlyA levels. Investigation into the biological significance of this variation revealed that an ST131 strain producing low levels of HlyA initiated cell death that was partly dependent on the nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, with this response being associated with a host-protective role in a mouse UTI model. When the same ST131 strain was engineered to overexpress high HlyA levels, macrophage cell death occurred even when NLRP3 function was abrogated, and bladder colonization was significantly increased. Thus, variation in HlyA expression in UPEC affects mechanisms by which macrophages die, as well as host susceptibility vs. resistance to colonization.-Murthy, A. M. V., Sullivan, M. J., Nhu, N. T. K., Lo, A. W., Phan, M.-D., Peters, K. M., Boucher, D., Schroder, K., Beatson, S. A., Ulett, G. C., Schembri, M. A., Sweet, M. J. Variation in hemolysin A expression between uropathogenic Escherichia coli isolates determines NLRP3-dependent vs. -independent macrophage cell death and host colonization. Refereed/Peer-reviewed

Melioidosis, caused by the bacterium Burkholderia pseudomallei , is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs.

Group B Streptococcus (GBS) is a gram-positive bacterium associated with various diseases in humans and animals. Many studies have examined GBS physiology, virulence and microbe-host interactions using diverse imaging approaches, including fluorescence microscopy. Strategies to label and visualize GBS using fluorescence biomarkers have been limited to antibody-based methods or non-specific stains that bind DNA or protein; an effective plasmid-based system to label GBS with a fluorescence biomarker would represent a useful visualization tool. In this study, we developed and validated a green fluorescence protein (GFP)-variant-expressing plasmid, pGU2664 that can be applied as a marker to visualize GBS in experimental studies. The synthetic, constitutively active CP25 promoter drives strong and stable expression of GFPmut3 in GBS carrying pGU2664. GBS maintains GFPmut3 activity at different phases of growth. Application of fluorescence polarization enables easy discrimination of GBS GFPmut3 activity from the auto-fluorescence of culture media commonly used to grow GBS. Differential interference contrast microscopy, in combination with epifluorescence microscopy to detect GFPmut3 in GBS, enabled visualization of bacterial attachment to live human epithelial cells in real time. Plasmid pGU2664 was also used to visualize phenotypic differences in the adherence of wild-type GBS and an isogenic gene-deficient mutant strain lacking the control of global virulence regulator CovR in adhesion assays. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications. We discuss the advantages and consider the limitations of this fluorescent biomarker system developed for GBS. Importance Section Group B Streptococcus (GBS) is a bacterium associated with various diseases in humans and animals. This study describes the development of a strategy to label and visualize GBS using a fluorescence biomarker, termed green fluorescence protein (GFP)mut3. We show that this biomarker can be successfully applied to track the growth of the bacteria in liquid medium, and enables the detailed visualization of GBS in the context of live human cells in real time microscopic analysis. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications.

The opportunistic pathogen Streptococcus agalactiae is the major cause of meningitis and sepsis in a newborn’s first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme and quinone are available in the environment, and a functional respiratory chain is required for full virulence. Remarkably, it is shown here that the entire respiratory chain of S. agalactiae consists of only two enzymes, a type 2 NADH dehydrogenase (NDH-2) and a cytochrome bd oxygen reductase. There are no respiratory dehydrogenases other than NDH-2 to feed electrons into the respiratory chain, and there is only one respiratory oxygen reductase to reduce oxygen to water. Although S. agalactiae grows well in vitro by fermentative metabolism, it is shown here that the absence of NDH-2 results in attenuated virulence, as observed by reduced colonization in heart and kidney in a mouse model of systemic infection. The lack of NDH-2 in mammalian mitochondria and its important role for virulence suggest this enzyme may be a potential drug target. For this reason, in this study, S. agalactiae NDH-2 was purified and biochemically characterized, and the isolated enzyme was used to screen for inhibitors from libraries of FDA-approved drugs. Zafirlukast was identified to successfully inhibit both NDH-2 activity and aerobic respiration in intact cells. This compound may be useful as a laboratory tool to inhibit respiration in S. agalactiae and, since it has few side effects, it might be considered a lead compound for therapeutics development., IMPORTANCE S. agalactiae is part of the human intestinal microbiota and is present in the vagina of ~30% of healthy women. Although a commensal, it is also the leading cause of septicemia and meningitis in neonates and immunocompromised adults. This organism can aerobically respire, but only using external sources of heme and quinone, required to have a functional electron transport chain. Although bacteria usually have a branched respiratory chain with multiple dehydrogenases and terminal oxygen reductases, here we establish that S. agalactiae utilizes only one type 2 NADH dehydrogenase (NDH-2) and one cytochrome bd oxygen reductase to perform respiration. NADH-dependent respiration plays a critical role in the pathogen in maintaining NADH/NAD+ redox balance in the cell, optimizing ATP production, and tolerating oxygen. In summary, we demonstrate the essential role of NDH-2 in respiration and its contribution to S. agalactiae virulence and propose it as a potential drug target.

Uropathogenic Escherichia coli (UPEC) cause the majority of community-acquired urinary tract infections (UTIs). Quantitation of bacteriuria (the number of bacteria in urine) is important for diagnostic approaches and in diverse research applications. Most UPEC strains express hemolysin, the expression of which has been correlated with the severity of UTI in murine models of infection. In this study, we sought to develop and optimise a quantitative Polymerase Chain Reaction (qPCR) assay for enumeration of hemolysin-positive UPEC in urine. Using recombinant plasmid pJET1.2::hlyD, which we termed pGU2470, the sensitivity range and linearity of amplification of qPCR was determined using primers and a probe targeting hlyD. Whole-cell preparations containing UPEC were quantified for hlyD copy number using CT values and compared to standards prepared with a known amount of pGU2470. We compared the efficiency of the assay for analysis of human and mouse urine because mouse models of human UTI are frequently used for investigating UPEC UTI. Urine samples were collected from healthy adults and mouse infection assays and used to assess any potential inhibitory effects of urine on the qPCR. The linear quantitative range of the qPCR (i.e. sensitivity) in detecting UPEC genomes was 103 copies/ml; qPCR-derived estimates of UPEC bacteriuria (based on number of genomes detected) were, on average, ten-fold higher that culture-based estimates. Finally, the frequencies of positive and negative predictions of UPEC in urine using the qPCR were equivalent to colony count methods. This assay provides an alternative to culture-based approaches for quantitation of UPEC bacteriuria in research studies.

Results from microbiome studies on oral cancer have been inconsistent, probably because they focused on compositional analysis, which does not account for functional redundancy among oral bacteria. Based on functional prediction, a recent study revealed enrichment of inflammatory bacterial attributes in oral squamous cell carcinoma (OSCC). Given the high relevance of this finding to carcinogenesis, we aimed here to corroborate them in a case-control study involving 25 OSCC cases and 27 fibroepithelial polyp (FEP) controls from Sri Lanka. DNA extracted from fresh biopsies was sequenced for the V1 to V3 region with Illumina’s 2 × 300–bp chemistry. High-quality nonchimeric merged reads were classified to the species level with a prioritized BLASTN-based algorithm. Downstream compositional analysis was performed with QIIME (Quantitative Insights into Microbial Ecology) and linear discriminant analysis effect size, while PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was utilized for bacteriome functional prediction. The OSCC tissues tended to have lower species richness and diversity. Genera Capnocytophaga, Pseudomonas, and Atopobium were overrepresented in OSCC, while Lautropia, Staphylococcus, and Propionibacterium were the most abundant in FEP. At the species level, Campylobacter concisus, Prevotella salivae, Prevotella loeschii, and Fusobacterium oral taxon 204 were enriched in OSCC, while Streptococcus mitis, Streptococcus oral taxon 070, Lautropia mirabilis, and Rothia dentocariosa among others were more abundant in FEP. Functionally, proinflammatory bacterial attributes, including lipopolysaccharide biosynthesis and peptidases, were enriched in the OSCC tissues. Thus, while the results in terms of species composition significantly differed from the original study, they were consistent at the functional level, substantiating evidence for the inflammatory nature of the bacteriome associated with OSCC.

Interleukin-17 (IL-17) is a pro-inflammatory cytokine involved in the control of many different disorders, including autoimmune, oncogenic, and diverse infectious diseases. In the context of infectious diseases, IL-17 protects the host against various classes of microorganisms but, intriguingly, can also exacerbate the severity of some infections. The regulation of IL-17 expression stems, in part, from the activity of Interleukin-23 (IL-23), which drives the maturation of different classes of IL-17-producing cells that can alter the course of infection. In this review, we analyze IL-17/IL-23 signalling in bacterial infection, and examine the interconnecting mechanisms that link immune regulation, host genetics, and microbial virulence in the context of bacterial pathogenesis. We consider the roles of IL-17 in both acute and chronic bacterial infections, with a focus on mouse models of human bacterial disease that involve infection of mucosal surfaces in the lungs, urogenital, and gastrointestinal tracts. Polymorphisms in IL-17-encoding genes in humans, which have been associated with heightened host susceptibility to some bacterial pathogens, are discussed. Finally, we examine the implications of IL-17 biology in infectious diseases for the development of novel therapeutic strategies targeted at preventing bacterial infection.

Summary Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic E scherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional programme associated with intramacrophage survival, we performed host–pathogen co‐transcriptome analyses using RNA sequencing. Mouse bone marrow‐derived macrophages (BMMs) were challenged over a 24 h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated and quantified the mammalian and bacterial transcriptomes. Bone marrow‐derived macrophages responded to the two UPEC strains with a broadly similar gene expression programme. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes up‐regulated at 24 h post‐infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co‐cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment.

The purpose of this study was to establish a three-dimensional fluorescent tooth model to investigate bacterial viability against intra-canal medicaments across the thickness and surface of root dentine. Dental microbial biofilms (Enterococcus faecalis and Streptococcus mutans) were established on the external root surface and bacterial kill was monitored over time against intra-canal medicament (Ca(OH)2 ) using fluorescent microscopy in conjunction with BacLight SYTO9 and propidium iodide stains. An Olympus digital camera fitted to SZX16 fluorescent microscope captured images of bacterial cells in biofilms on the external root surface. Viability of biofilm was measured by calculating the total pixel area of green (viable bacteria) and red (non-viable bacteria) for each image using ImageJ® software. All data generated were assessed for normality and then analysed using a Mann-Whitney t-test. The viability of S. mutans biofilm following Ca(OH)2 treatment showed a significant decline compared with the untreated group (P = 0.0418). No significant difference was seen for E. faecalis biofilm between the Ca(OH)2 and untreated groups indicating Ca(OH)2 medicament is ineffective against E. faecalis biofilm. This novel three-dimensional fluorescent biofilm model provides a new clinically relevant tool for testing of medicaments against dental biofilms.

Here we report the complete genome sequence of Streptococcus agalactiae strain 874391. This serotype III isolate is a member of the hypervirulent sequence type 17 (ST-17) lineage that causes a disproportionate number of cases of invasive disease in humans and mammals. A brief historical context of the strain is discussed.

Here we report the complete genome sequence of Streptococcus agalactiae strain 874391. This serotype III isolate is a member of the hypervirulent sequence type 17 (ST-17) lineage that causes a disproportionate number of cases of invasive disease in humans and mammals. A brief historical context of the strain is discussed.

There is increasing interest in the possible role of the oral microbiome in oral squamous cell carcinoma (OSSC). However, studies on the possible association between the oral bacteriome and OSCC remain inconclusive, while the potential role of the “mycobiome” in oral carcinogenesis has never been explored. I hereby present results from our recent studies on the bacteriome and mycobiome associated with OSCC based on analysis of samples from Yemeni and Sri Lankan cohorts. Tissue biopsies were obtained from the cases while deep buccal swabs or fibro-epithelial polyps were collected from the controls. Sequencing of the bacterial V1-V3 region of the 16S rRNA gene and the fungal ITS2 region using Illumina’s 2x300 bp chemistry was employed to study the bacteriome and mycobiome, respectively. Merged reads were classified to species level using a BLASTN-algorithm. Downstream analyses were performed using QIIME, PICRUSt, and LEfSe. Fusobacterium nucleatum subsp. polymorphum, Pseudomonas aeruginosa, Prevotella and Campylobacter spp. were more abundant in OSCC samples while Streptococcus mitis and Rothia spp. were overrepresented in the controls. Functionally, inflammatory bacterial attributes including bacterial mobility, flagellar assembly, bacterial chemotaxis and LPS synthesis were enriched in the tumors. Mycobiome analysis revealed a dysbiotic fungal community dominated by C. albicans in association with OSCC.