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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.

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.

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.

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.

Flagella are expressed on the surface of a wide range of bacteria, conferring motility and contributing to virulence and innate immune stimulation. Host-pathogen interaction studies of the roles of flagella in infection, including due to uropathogenic Escherichia coli (UPEC), have used various methods to purify and examine the biology of the major flagella subunit protein, FliC. These studies have offered insight into the ways in which flagella proteins interact with host cells. However, previous methods used to extract and purify FliC, such as mechanical shearing, ultracentrifugation, heterologous expression in laboratory E. coli strains, and precipitation-inducing chemical treatments have various limitations; as a result, there are few observations based on highly purified, non-denatured FliC in the literature. This is especially relevant to host-pathogen interaction studies such as immune assays that are designed to parallel, as closely as possible, naturally-occurring interactions between host cells and flagella. In this study, we sought to establish a new, carefully optimized method to extract and purify non-denatured, native FliC from the reference UPEC strain CFT073 to be suitable for immune assays. To achieve purification of FliC to homogeneity, we used a mutant CFT073 strain containing deletions in four major chaperone-usher fimbriae operons (type 1, F1C and two P fimbrial gene clusters; CFT073Δ4). A sequential flagella extraction method based on mechanical shearing, ultracentrifugation, size exclusion chromatography, protein concentration and endotoxin removal was applied to CFT073Δ4. Protein purity and integrity was assessed using SDS-PAGE, Western blots with anti-flagellin antisera, and native-PAGE. We also generated a fliC-deficient strain, CFT073Δ4ΔfliC, to enable the concurrent preparation of a suitable carrier control to be applied in downstream assays. Innate immune stimulation was examined by exposing J774A.1 macrophages to 0.05-1 μg of purified FliC for 5 h; the supernatants were analyzed for cytokines known to be induced by flagella, including TNF-α, IL-6, and IL-12; the results were assessed in the context of prior literature. Macrophage responses to purified FliC encompassed significant levels of several cytokines consistent with prior literature reports. The purification method described here establishes a new approach to examine highly purified FliC in the context of host-pathogen interaction model systems.

Uropathogenic

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.

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.

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.

Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract.

Infection with Burkholderia pseudomallei causes melioidosis, a disease with a high mortality rate (20% in Australia and 40% in Southeast Asia). Neurological melioidosis is particularly prevalent in northern Australian patients and involves brain stem infection, which can progress to the spinal cord; however, the route by which the bacteria invade the central nervous system (CNS) is unknown. We have previously demonstrated that B. pseudomallei can infect the olfactory and trigeminal nerves within the nasal cavity following intranasal inoculation. As the trigeminal nerve projects into the brain stem, we investigated whether the bacteria could continue along this nerve to penetrate the CNS. After intranasal inoculation of mice, B. pseudomallei caused low-level localized infection within the nasal cavity epithelium, prior to invasion of the trigeminal nerve in small numbers. B. pseudomallei rapidly invaded the trigeminal nerve and crossed the astrocytic barrier to enter the brain stem within 24 h and then rapidly progressed over 2,000 μm into the spinal cord. To rule out that the bacteria used a hematogenous route, we used a capsule-deficient mutant of B. pseudomallei that does not survive in the blood and found that it also entered the CNS via the trigeminal nerve. This suggests that the primary route of entry is via the nerves that innervate the nasal cavity. We found that actin-mediated motility could facilitate initial infection of the olfactory epithelium. Thus, we have demonstrated that B. pseudomallei can rapidly infect the brain and spinal cord via the trigeminal nerve branches that innervate the nasal cavity.

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1β (IL-1β) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1β secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1β secretion pathway in human macrophages. This has important implications for understanding UTI in humans. Refereed/Peer-reviewed

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Murine models of human UTI are vital experimental tools that have helped to elucidate UTI pathogenesis and advance knowledge of potential treatment and infection prevention strategies. Fundamentally, several variables are inherent in different murine models, and understanding the limitations of these variables provides an opportunity to understand how models may be best applied to research aimed at mimicking human disease. In this review, we discuss variables inherent in murine UTI model studies and how these affect model usage, data analysis and data interpretation. We examine recent studies that have elucidated UTI host–pathogen interactions from the perspective of gene expression, and review new studies of biofilm and UTI preventative approaches. We also consider potential standards for variables inherent in murine UTI models and discuss how these might expand the utility of models for mimicking human disease and uncovering new aspects of pathogenesis.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are a significant health concern, exacerbated by the rapid emergence of multidrug resistant strains refractory to antibiotic treatment. P fimbriae are strongly associated with upper urinary tract colonization due to specific binding to α-D-galactopyranosyl-(1-4)-β-D-galactopyranoside receptors in the kidneys. Thus, inhibiting P-fimbrial adhesion may reduce the incidence of UPEC-mediated UTI. E. coli 83972 is an asymptomatic bacteriuria isolate successfully used as a prophylactic agent to prevent UTI in human studies. We constructed a recombinant E. coli 83972 strain displaying a surface-located oligosaccharide P fimbriae receptor mimic that bound to P-fimbriated E. coli producing any of the 3 PapG adhesin variants. The recombinant strain, E. coli 83972:: lgtCE, impaired P fimbriae–mediated adhesion to human erythrocytes and kidney epithelial cells. Additionally, E. coli 83972::lgtCE impaired urine colonization by UPEC in a mouse UTI model, demonstrating its potential as a prophylactic agent to prevent UTI. Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with an estimated 150 million cases per year [1]. The recurrence rate is high, and infections frequently become chronic after multiple episodes. Bacterial interference using nonvirulent bacteria to prevent infection by pathogens is an alternative therapy for patients with chronic and recurrent UTIs that do not respond to antimicrobial treatment [2]. Escherichia coli 83972 is a clinical asymptomatic bacteriuria (ABU) isolate capable of long-term bladder colonization without causing disease symptoms and has been effectively used as a prophylactic agent for the prevention of UTI in

Uropathogenic Escherichia coli is the primary cause of urinary tract infections, which affects over 60% of women during their lifetime. UPEC exhibits a number of virulence traits that facilitate colonization of the bladder, including inhibition of cytokine production by bladder epithelial cells. The goal of this study was to identify the mechanism of this inhibition. We observed that cytokine suppression was associated with rapid cytotoxicity toward epithelial cells. We found that cytotoxicity, cytokine suppression and alpha-hemolysin production were all tightly linked in clinical isolates. We screened a UPEC fosmid library and identified clones that gained the cytotoxicity and cytokine-suppression phenotypes. Both clones contained fosmids encoding a PAI II(J96)-like domain and expressed the alpha-hemolysin (hlyA) encoded therein. Mutation of the fosmid-encoded hly operon abolished cytotoxicity and cytokine suppression. Similarly, mutation of the chromosomal hlyCABD operon of UPEC isolate F11 also abolished these phenotypes, and they could be restored by introducing the PAI II(J96)-like domain-encoding fosmid. We also examined the role of alpha-hemolysin in cytokine production both in the murine UTI model as well as patient specimens. We conclude that E. coli utilizes alpha-hemolysin to inhibit epithelial cytokine production in vitro. Its contribution to inflammation during infection requires further study.

The molecular mechanisms that define asymptomatic bacteriuria (ABU) Escherichia coli colonization of the human urinary tract remain to be properly elucidated. Here, we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness, and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin, and yersiniabactin and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity and growth in defined iron-deficient medium. No siderophores were detected in an 83972 quadruple mutant deficient in all four siderophore biosynthesis pathways; this mutant did not grow in defined iron-deficient medium but grew in iron-limited pooled human urine due to iron uptake via the FecA ferric citrate receptor. In a mixed 1:1 growth assay with strain 83972, there was no fitness disadvantage of the 83972 quadruple biosynthetic mutant, demonstrating its capacity to act as a “cheater” and utilize siderophores produced by the wild-type strain for iron uptake. An 83972 enterobactin/salmochelin double receptor mutant was outcompeted by 83972 in human urine and the mouse urinary tract, indicating a role for catecholate receptors in urinary tract colonization.

Staphylococcus saprophyticus is an important cause of urinary tract infection (UTI), particularly among young women, and is second only to uropathogenic Escherichia coli as the most frequent cause of UTI. The molecular mechanisms of urinary tract colonization by S. saprophyticus remain poorly understood. We have identified a novel 6.84 kb plasmid-located adhesin-encoding gene in S. saprophyticus strain MS1146 which we have termed uro-adherence factor B (uafB). UafB is a glycosylated serine-rich repeat protein that is expressed on the surface of S. saprophyticus MS1146. UafB also functions as a major cell surface hydrophobicity factor. To characterize the role of UafB we generated an isogenic uafB mutant in S. saprophyticus MS1146 by interruption with a group II intron. The uafB mutant had a significantly reduced ability to bind to fibronectin and fibrinogen. Furthermore, we show that a recombinant protein containing the putative binding domain of UafB binds specifically to fibronectin and fibrinogen. UafB was not involved in adhesion in a mouse model of UTI; however, we observed a striking UafB-mediated adhesion phenotype to human uroepithelial cells. We have also identified genes homologous to uafB in other staphylococci which, like uafB, appear to be located on transposable elements. Thus, our data indicate that UafB is a novel adhesin of S. saprophyticus that contributes to cell surface hydrophobicity, mediates adhesion to fibronectin and fibrinogen, and exhibits tropism for human uroepithelial cells.

Group B Streptococcus (GBS) causes urinary tract infections, but the pathogenic mechanisms underlying GBS urinary tract infections are unknown. We investigated whether uropathogenic GBS can bind to bladder uroepithelium to initiate urinary tract infection. Uropathogenic GBS isolated from a patient with acute cystitis bound to human T24 bladder uroepithelial cells in close association with F-actin in statistically significantly higher numbers compared with nonuropathogenic GBS. In vivo modeling using transurethrally infected mice revealed superior fitness of uropathogenic GBS for bladder colonization and potent uropathogenic GBS-specific up-regulation of interleukin 1alpha during infection. Thus, binding of uropathogenic GBS to uroepithelium and vigorous induction of interleukin 1alpha represents the initial stages of GBS urinary tract infection.

The study established the virulence potential of multidrug-resistant Escherichia coli (MDREC) isolates from nosocomial infections in hospitalised dogs. The isolates were resistant to fluoroquinolones, belonged to two distinct clonal groups (CG1 and CG2) and contained a plasmid-mediated AmpC (CMY-7) beta-lactamase. CG1 isolates (n=14) possessed two of 36 assayed extraintestinal virulence genes (iutA and traT) and belonged to phylogenetic group A, whereas CG2 isolates (n=19) contained four such genes (iutA, ibeA, fimH and kpsMT K5) and belonged to group D. In a mouse gastrointestinal tract colonisation model, colonisation by index CG1 strain C1 was transient, in contrast to the index CG2 strain C2b, which persisted up to 40days post-inoculation. In a mouse subcutaneous challenge model, both strains were less virulent than archetypal group B2 extraintestinal pathogenic E. coli (ExPEC) strain CFT073; strain C1 caused no systemic signs and strain C2b was lethal to only one of six mice. In a mouse urinary tract infection model, strain C2b colonised the mouse bladder over 2 logs higher compared to strain C1. Whilst both groups of canine MDREC appear less virulent than a reference human ExPEC strain, CG2 strains have greater capacity for colonisation and virulence.

The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins.

Apoptosis of murine and human macrophages induced by group B Streptococcus agalactiae (GBS) is likely an important virulence mechanism that is used by the bacteria to suppress the host immune response and to persist at sites of infection. The mechanisms by which GBS induces apoptosis are, however, largely unknown. In this study, we report that in murine macrophages GBS induces unique changes in the regulation and localization of the apoptotic regulators Bad, 14-3-3, and Omi/high-temperature requirement A2 and leads to the release of cytochrome c and the activation of caspase-9 and caspase-3. Furthermore, inhibition of caspase-3 impaired GBS-induced apoptosis of macrophages. The ability to modulate the activity of effector caspases may therefore represent an unexploited avenue for therapeutic intervention in GBS infections.

Burkholderia pseudomallei, the causative agent of melioidosis, is an important pathogen in tropical regions of Australia and Southeast Asia. Antibiotic therapy can be ineffective in patients with acute septicaemic melioidosis. It has been proposed that adjunctive immunotherapy using granulocyte colony stimulating factor (G-CSF) combined with antibiotics may provide an alternative approach to antibiotics alone. We have developed a murine model for melioidosis that allows novel treatment approaches to be investigated. This study looked at the potential for murine G-CSF therapy both alone and as an adjunct in the treatment of acute disseminated B. pseudomallei infection in BALB/c mice. A number of therapeutic variables involving ceftazidime and recombinant murine G-CSF were studied. Surviving mice were sacrificed and splenic bacterial loads were determined. Combining recombinant murine G-CSF with ceftazidime offered no advantage over ceftazidime alone. Pre-treatment with recombinant murine G-CSF did not demonstrate a significant benefit. This would suggest that adjunct immunotherapy using G-CSF is of limited benefit.

Melioidosis is caused by the Gram-negative bacillus Burkholderia pseudomallei. Most clinical reports of disease are from south-east Asia and northern Australia. The organism is intrinsically resistant to most commonly available antibiotics. Standard therapy includes ceftazidime either alone or in combination with co-trimoxazole. The clinical advantage in adding co-trimoxazole has never been determined; nor has the activity of newer, fourth-generation cephalosporins, such as cefepime, been studied in the treatment of this condition. BALB/c mice have been shown to represent an animal model of melioidosis. This animal model was used in this study to compare the efficacy of ceftazidime and cefepime alone or with co-trimoxazole, in the therapy of melioidosis. Antibiotic levels in the mice were determined by HPLC, and dosing was modified to keep plasma antibiotic levels at or above the MIC for the organism-antibiotic combination for a significant part of a 12 h period. Bacterial load, as determined by splenic counts, showed that ceftazidime in combination with co-trimoxazole was the most effective therapeutic option. The animal model described in this study can be used as a preliminary evaluation of therapeutic options for melioidosis.

Cytokine mRNA levels were assessed inBurkholderia pseudomallei-susceptible BALB/c mice andB. pseudomallei-resistant C57BL/6 mice following administration of a sublethal dose of less virulent (LV)B. pseudomallei, a candidate immunogen tested for protection against a highly virulent (HV) challenge. Compared on the basis of the bacterial loads, the cytokine patterns induced by HV and LVB. pseudomalleiwere similar, involving gamma interferon, interleukin-10, and other cytokines. Partial cross-protection betweenB. pseudomalleistrains is shown to be associated with cytokine profiles involving both type 1 and type 2 cytokines.

Melioidosis is a potentially fatal disease that is endemic to tropical northern Australia and Southeast Asia, with a mortality rate of 14 to 50%. The bacterium Burkholderia pseudomallei is the causative agent which infects numerous parts of the human body, including the brain, which results in the neurological manifestation of melioidosis. The olfactory nerve constitutes a direct conduit from the nasal cavity into the brain, and we have previously reported that B. pseudomallei can colonize this nerve in mice. We have now investigated in detail the mechanism by which the bacteria penetrate the olfactory and trigeminal nerves within the nasal cavity and infect the brain. We found that the olfactory epithelium responded to intranasal B. pseudomallei infection by widespread crenellation followed by disintegration of the neuronal layer to expose the underlying basal layer, which the bacteria then colonized. With the loss of the neuronal cell bodies, olfactory axons also degenerated, and the bacteria then migrated through the now-open conduit of the olfactory nerves. Using immunohistochemistry, we demonstrated that B. pseudomallei migrated through the cribriform plate via the olfactory nerves to enter the outer layer of the olfactory bulb in the brain within 24 h. We also found that the bacteria colonized the thin respiratory epithelium in the nasal cavity and then rapidly migrated along the underlying trigeminal nerve to penetrate the cranial cavity. These results demonstrate that B. pseudomallei invasion of the nerves of the nasal cavity leads to direct infection of the brain and bypasses the blood-brain barrier., IMPORTANCE Melioidosis is a potentially fatal tropical disease that is endemic to northern Australia and Southeast Asia. It is caused by the bacterium Burkholderia pseudomallei, which can infect many organs of the body, including the brain, and results in neurological symptoms. The pathway by which the bacteria can penetrate the brain is unknown, and we have investigated the ability of the bacteria to migrate along nerves that innervate the nasal cavity and enter the frontal region of the brain by using a mouse model of infection. By generating a mutant strain of B. pseudomallei which is unable to survive in the blood, we show that the bacteria rapidly penetrate the cranial cavity using the olfactory (smell) nerve and the trigeminal (sensory) nerve that line the nasal cavity.

Genital tract carriage of group B streptococcus (GBS) is prevalent among adult women; however, the dynamics of chronic GBS genital tract carriage, including how GBS persists in this immunologically active host niche long term, are not well defined. To our knowledge, in this study, we report the first animal model of chronic GBS genital tract colonization using female mice synchronized into estrus by delivery of 17β-estradiol prior to intravaginal challenge with wild-type GBS 874391. Cervicovaginal swabs, which were used to measure bacterial persistence, showed that GBS colonized the vaginal mucosa of mice at high numbers (106–107 CFU/swab) for at least 90 d. Cellular and histological analyses showed that chronic GBS colonization of the murine genital tract caused significant lymphocyte and PMN cell infiltrates, which were localized to the vaginal mucosal surface. Long-term colonization was independent of regular hormone cycling. Immunological analyses of 23 soluble proteins related to chemotaxis and inflammation showed that the host response to GBS in the genital tract comprised markers of innate immune activation including cytokines such as GM-CSF and TNF-α. A nonhemolytic isogenic mutant of GBS 874391, Δcyle9, was impaired for colonization and was associated with amplified local PMN responses. Induction of DNA neutrophil extracellular traps, which was observed in GBS-infected human PMNs in vitro in a hemolysin-dependent manner, appeared to be part of this response. Overall, this study defines key infection dynamics in a novel murine model of chronic GBS genital tract colonization and establishes previously unknown cellular and soluble defense responses to GBS in the female genital tract.

Urinary tract infection (UTI) is one of the most common bacterial infections in humans, with uropathogenic Escherichia coli (UPEC) the leading causative organism. UPEC has a number of virulence factors that enable it to overcome host defenses within the urinary tract and establish infection. The O antigen and the capsular polysaccharide are two such factors that provide a survival advantage to UPEC. Here we describe the application of the rpsL counter selection system to construct capsule (kpsD) and O antigen (waaL) mutants and complemented derivatives of three reference UPEC strains: CFT073 (O6:K2:H1), RS218 (O18:K1:H7) and 1177 (O1:K1:H7). We observed that while the O1, O6 and O18 antigens were required for survival in human serum, the role of the capsule was less clear and linked to O antigen type. In contrast, both the K1 and K2 capsular antigens provided a survival advantage to UPEC in whole blood. In the mouse urinary tract, mutation of the O6 antigen significantly attenuated CFT073 bladder colonization. Overall, this study contrasts the role of capsule and O antigen in three common UPEC serotypes using defined mutant and complemented strains. The combined mutagenesis-complementation strategy can be applied to study other virulence factors with complex functions both in vitro and in vivo.

Production of cytokines including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) is an important early-stage host response following infection with intracellular pathogens. Development of immunity to these pathogens is determined to a large extent by the timing and relative level of expression of the cytokines. Numerous studies have shown that early cytokine responses involving interleukin-12 (IL-12) and IFN-γ are important for resistance to intracellular pathogens, whereas responses involving IL-4 and IL-10 increase host susceptibility. These often-indistinct early cytokine responses influence the differentiation of naı̈ve CD4+T helper cells, which later develop into what have commonly been termed Th1- and Th2-type cells. The characterization of CD4+T-helper-cell responses as Th1 or Th2 type is based largely on the cytokine profiles during the specific phase and has been used in recent years to account for the innate resistance and susceptibility of different inbred strains of mice to several intracellular pathogens. Studies investigating cytokine production in terms of CD4+T-helper-cell polarization inBurkholderia pseudomalleiinfection have not been undertaken. In this study, we used semiquantitative reverse transcription-PCR to assess induction of cytokine mRNA in liver and spleen ofB. pseudomallei-susceptible BALB/c and relatively resistant C57BL/6 mice following infection with virulentB. pseudomallei. The levels of mRNA for IFN-γ, TNF-α, IL-1β, IL-6, IL-10, and IL-12 increased in both BALB/c and C57BL/6 mice 24 to 36 h after infection. A comparison of BALB/c and C57BL/6 responses revealed the relative levels of expression of mRNA for several of these cytokines, including IFN-γ, were greater in BALB/c mice, suggesting a role for endotoxic shock and cytokine-mediated immunopathology in the development of acute melioidosis. Early induction of mRNA for the cytokines classically associated with development of Th1- and Th2-type responses was absent or minimal, and induction levels in both strains of mice were similar. During the specific phase, cytokine mRNA profiles occurred as a combination of Th1- and Th2-type patterns. Collectively, these results demonstrate that cytokine mRNA responses in BALB/c and C57BL/6 mice following infection with virulentB. pseudomalleido not develop as polarized Th1- or Th2-type profiles. Considering the role of TNF-α and IFN-γ in the processes of endotoxic shock, these results also indicate that selected cytokines, while important for resistance toB. pseudomalleiinfection, are also potential contributors to immunopathology and the development of acute fulminating disease.

Melioidosis is a potentially fatal disease of both human and animals caused by the bacterium Burkholderia pseudomallei. Disease is endemic in tropical and subtropical regions of Southeast Asia and Northern Australia. The pathogenesis of melioidosis is poorly understood. In particular, the host responses that occur following infection, and the specific host-pathogen interactions that result in the development of either acute or chronic infection are unclear. Using an established murine model, we investigated early proinflammatory cytokine responses believed to be critical in the development of acute and chronic B. pseudomallei infection. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to assess levels of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta) and interleukin 6 (IL-6) in the liver of mice following infection. We demonstrate that the level of mRNA for these cytokines increase moderately in chronic infection in C57BL/6 mice. However, in acute infection in BALB/c mice, mRNA responses for these cytokines were shown to be comparatively greater. These results demonstrate that early proinflammatory cytokine responses are important in the immunopathogenesis of melioidosis.

Streptococcus agalactiae causes urinary tract infection (UTI) in pregnant adults, non-pregnant adults, immune-compromised individuals and the elderly. The pathogenesis of S. agalactiae UTI in distinct patient populations is poorly understood. In this study, we used murine models of UTI incorporating young mice, aged and dam mice to show that uropathogenic S. agalactiae causes bacteriuria at significantly higher levels in aged mice compared to young mice and this occurs coincident with equivalent levels of bladder tissue colonisation at 24 h post-infection (p.i.). In addition, aged mice exhibited significantly higher bacteriuria burdens at 48 h compared to young mice, confirming a divergent pattern of bacterial colonization in the urinary tract of aged and young mice. Multiparous mice, in contrast, exhibited significantly lower urinary titres of S. agalactiae compared to age-matched nulliparous mice suggesting that parity enhances the ability of the host to control S. agalactiae bacteriuria. Additionally, we show that both age and parity alter the expression levels of several key regulatory and pro-inflammatory cytokines, which are known to be important the immune response to UTI, including Interleukin (IL)-1β, IL-12(p40), and Monocyte Chemoattractant Protein-1 (MCP-1). Finally, we demonstrate that other cytokines, including IL-17 are induced significantly in the S. agalactiae-infected bladder regardless of age and parity status. Collectively, these findings show that the host environment plays an important role in influencing the severity of S. agalactiae UTI; infection dynamics, particularly in the context of bacteriuria, depend on age and parity, which also affect the nature of innate immune responses to infection.

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli ; however, Gram-positive organisms, including Streptococcus agalactiae , or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. The genomesequenced prototype UPEC strain CFT073 contains 11 putative AT-encoding genes. In this study, we have performed a detailed molecular characterization of two closely related AT adhesins from CFT073: UpaB (c0426) and UpaC (c0478). PCR screening revealed that the upaB and upaC AT-encoding genes are common in E. coli. The upaB and upaC genes were cloned and characterized in a recombinant E. coli K-12 strain background. This revealed that they encode proteins located at the cell surface but possess different functional properties: UpaB mediates adherence to several ECM proteins, while UpaC expression is associated with increased biofilm formation. In CFT073, upaB is expressed while upaC is transcriptionally repressed by the global regulator H-NS. In competitive colonization experiments employing the mouse UTI model, CFT073 significantly outcompeted its upaB (but not upaC) isogenic mutant strain in the bladder. This attenuated phenotype was also observed in single-challenge experiments, where deletion of the upaB gene in CFT073 significantly reduced early colonization of the bladder.©2012, American Society for Microbiology. All Rights Reserved. © 2012, American Society for Microbiology., This work was supported by grants from the Australian National Health and Medical Research Council (631654), the Australian Research Council (DP1097032), the University of Queensland, the Institut Pasteur, the CNRS URA 2172, the Network of Excellence EuroPathoGenomics, and the European Community (LSHB-CT-2005-512061).

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.

Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1 + vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival. © 2011 Elsevier GmbH.

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.

Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH , present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.

The periplasmic multicopper oxidase (CueO) is involved in copper homeostasis and protection against oxidative stress. Here, we show that the deletion of cueO in uropathogenic Escherichia coli increases its colonization of the urinary tract despite its increased sensitivity to hydrogen peroxide. The cueO deletion mutant accumulated iron with increased efficiency compared to its parent strain; this may account for its advantage in the iron-limited environment of the urinary tract.

Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with the virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter subgroup of proteins. The best characterized of these proteins, antigen 43 (Ag43), is a self-recognizing adhesin that is associated with cell aggregation and biofilm formation in E. coli K-12. The sequenced genome of prototype UPEC strain CFT073 contains two variant Ag43-encoding genes located on pathogenicity islands. The biological significance of both of these genes and their role in UPEC pathogenesis have not been investigated previously. Here we performed a detailed molecular characterization analysis of Ag43a (c3655) and Ag43b (c1273) from UPEC CFT073. Expression of Ag43a and Ag43b in a K-12 background revealed that they possess different functional properties. Ag43a produced a strong aggregation phenotype and promoted significant biofilm growth. Deletion mutants and strains constitutively expressing Ag43a and Ag43b were also constructed using CFT073. When these mutants were analyzed in a mouse model of UTI, Ag43a (but not Ag43b) promoted long-term persistence in the urinary bladder. Our findings demonstrate that Ag43a contributes to UPEC disease pathogenesis and reveal that there are pathogenicity-adapted variants of Ag43 with distinct virulence-related functions.

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract. The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. Deliberate colonization of UTI-susceptible individuals with E. coli 83972 has been used successfully as an alternative approach for the treatment of patients who are refractory to conventional therapy. Colonization with strain 83972 appears to prevent infection with UPEC strains in such patients despite the fact that this strain is unable to express the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we investigated the growth characteristics of E. coli 83972 in human urine and show that it can outcompete a representative spectrum of UPEC strains for growth in urine. The unique ability of ABU E. coli 83972 to outcompete UPEC in urine was also demonstrated in a murine model of human UTI, confirming the selective advantage over UPEC in vivo. Comparison of global gene expression profiles of E. coli 83972 grown in lab medium and human urine revealed significant differences in expression levels in the two media; significant down-regulation of genes encoding virulence factors such as hemolysin, lipid A, and capsular polysaccharides was observed in cells grown in urine. Clearly, divergent abilities of ABU E. coli and UPEC to exploit human urine as a niche for persistence and survival suggest that these key differences may be exploited for preventative and/or therapeutic approaches.

Group B streptococcus (GBS; Streptococcus agalactiae) induces apoptosis of macrophages, and this may be an important mechanism GBS uses to suppress immune responses. The mechanisms whereby GBS induces apoptosis have not been identified. We studied GBS infection in murine macrophage-like J774A.1 cells and analyzed gene expression before apoptosis. Tumor necrosis factor (TNF)-alpha , interleukin (IL)-1, and inducible nitric oxide synthase (iNOS) gene expression coincided with apoptosis. Inhibition of iNOS gene expression by use of N(G)-monomethyl-L-arginine (NMMA) inhibited apoptosis, whereas inhibition of TNF-alpha and IL-1 biological activity did not. Macrophages from congenic iNOS-deficient mice were less susceptible to apoptosis than were macrophages from C57BL/6 mice. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced apoptosis without infection, confirming its proapoptotic effect. NMMA did not impair the microbicidal activity of macrophages, however, and SNAP was not bactericidal against GBS in vitro. In human monocyte-derived macrophages (HMDMs), NO production was minimal, even after costimulation with IFN-gamma and lipopolysaccharide. Dose-dependent apoptosis of HMDMs occurred without a significant NO response. Thus, NO is an important mediator of GBS-induced murine macrophage apoptosis but does not contribute to antimicrobial activity or cytotoxicity in HMDMs. HMDMs and murine macrophages are killed by GBS by alternative, NO-independent mechanisms. Future studies of host-cell machinery commandeered by GBS to bring about apoptosis will be important for understanding the role played by apoptosis in defense against this important human pathogen.

Burkholderia pseudomallei, the etiological agent of melioidosis, causes significant mortality in endemic regions, but little is known regarding the immune mechanisms required for successful protective immunity. To establish a model of immunization that could be used to study this we screened a library of B. pseudomallei strains for immunogenicity in mice. BALB/c mice were immunized with test strains, and 2 weeks later were given a lethal challenge (LC) of virulent B. pseudomallei. Among 49 strains tested, a single strain, CL04, exhibited strong immunoprotective capacity. Interestingly, CL04 had been cultured from a patient with chronic colonization of B. pseudomallei, which is a rare phenomenon. Mice immunized with 0.1 x LD50 (5 x 10(3) CFU) of CL04 had significantly better survival and lower bacterial loads after LC compared to naïve controls. Dose-response analysis demonstrated more robust immunity after higher immunizing doses, and bacterial inactivation by gamma irradiation diminished the protective effect, indicating a requirement for viable organism for immunity. CL04-induced immunity was demonstrated both in B. pseudomallei-susceptible BALB/c and -resistant C57BL/6 mice. We investigated the gene profile of CL04-induced immunity by analyzing responses to immunization using cDNA microarray. Unique responses involving granulocyte macrophage colony stimulating factor (GM-CSF), the proapoptotic regulator Bad and cyclin-dependent kinase (CDK5) were detected in immunized mice, but these responses were absent in naïve-LC mice. Further, responses differed between mouse strains, indicating dependence on host genetic background. This model will be useful in identifying elements of the immune response required for successful adaptive immunity against B. pseudomallei.

Group B streptococcus (GBS) induces apoptosis in macrophages. Growth conditions minimizing beta-hemolysin expression, such as high glucose, reduce apoptosis. We constructed an isogenic mutant strain of GBS 874391 lacking the beta-hemolysin structural gene cylE and investigated the role that beta-hemolysin plays in apoptosis of J774 macrophages. Viability of macrophages infected with wild-type or cylE GBS was similar and significantly less than that of macrophages infected with GBS grown in high-glucose media. Thus, apoptosis in GBS-infected macrophages is dependent not on beta-hemolysin but on a factor coregulated with beta-hemolysin by glucose.

Melioidosis is a disease of the tropics caused by the facultative intracellular bacterium Burkholderia pseudomallei. In human infection, increased levels of IFN-gamma in addition to the chemokines interferon-gamma -inducible protein 10 (IP-10) and monocyte interferon-gamma -inducible protein (Mig) have been demonstrated. However, the role of these and other chemokines in the pathogenesis of melioidosis remains unknown. Using BALB/c and C57BL/6 mice as models of the acute and chronic forms of human melioidosis, the induction of mRNA was assessed for various chemokines and CSF (G-CSF, M-CSF, GM-CSF, IP-10, Mig, RANTES, MCP-1, KC and MIP-2) in spleen and liver following B. pseudomallei infection. Patterns of chemokine and CSF induction were similar in liver and spleen; however, responses were typically greater in spleen, which reflected higher tissue bacterial loads. In BALB/c mice, high-level expression of mRNA for all chemokines and CSF investigated was demonstrated at day 3 postinfection, correlating with peak bacterial load and extensive infiltration of leucocytes. In contrast, increased mRNA expression and bacterial numbers in C57BL/6 mice were greatest between 4 and 14 days following infection. This paralleled increases in the size and number of abscesses in liver and spleen of C57BL/6 mice at days 3 and 14 postinfection. Earlier induction of cytokine-induced neutrophil chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) mRNA was demonstrated in spleen, while MIP-2, MCP-1, IP-10 and Mig were demonstrated in liver of BALB/c mice when compared to spleen and liver of C57BL/6. The magnitude of cellular responses observed in the tissue correlated with increased levels of the chemokines and CSF investigated, as well as bacterial load. Compared with C57BL/6 mice, greater infiltration of neutrophils was observed in liver and spleen of BALB/c mice at day 3. In contrast, early lesions in C57BL/6 mice predominantly comprised macrophages. These results suggest that the inability of BALB/c mice to contain the infection at sites of inflammation may underlie the susceptible phenotype of this mouse strain towards B. pseudomallei infection.

Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei isolates from Australia and Papua New Guinea. Among 42 non-arabinose-assimilating (ara(-)) isolates, LD,(, ranged from 10 to > 10(6) CFU. There were numerous correlations between virulence and disease presentation in patients; however, this was not a consistent observation. Virulence did not correlate with isolate origin (i.e. clinical vs environmental), since numerous ara- environmental isolates were highly virulent. The least virulent isolate was a soil isolate from Papua New Guinea, which was arabinose assimilating (ara(+)). Stability of B. pseudomallei virulence was investigated by in vivo passage of isolates through mice and repetitive in vitro subculture. Virulence increased following in vivo exposure in only one of eight isolates tested. In vitro subculture on ferric citrate-containing medium caused attenuation of virulence, and this correlated with changes in colony morphology. Pulsed-field gel electrophoresis and randomly amplified polymorphic DNA typing demonstrated that selected epidemiologically related isolates that had variable clinical outcomes and different in vivo virulence were clonal strains. No molecular changes were observed in isolates after in vivo or in vitro exposure despite changes in virulence. These results indicate that virulence of selected B. pseudomallei isolates is variable, being dependent on factors such as iron bioavailability. They also support the importance of other variables such as inoculum size and host risk factors in determining the clinical severity of melioidosis. (C) 2001 Editions scientifiques et medicales Elsevier SAS.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in tropical and subtropical regions of South-East Asia and Northern Australia. Antimicrobial therapy regimens for treatment of acute septicemic melioidosis are of variable efficacy. Ceftazidime is the current antibiotic of choice and is commonly administered with other agents such as cotrimoxazole or doxycycline. The emergence of resistant strains of B. pseudomallei and the persistence of high mortality rates prompted the present study. Using an established mouse model of acute disseminated B. pseudomallei infection, we compared the efficacy of ceftazidime versus cefpirome in combination with cotrimoxazole or chloramphenicol therapy in vivo. Control mice that were infected but did not receive antibiotic therapy died within 96 hours of infection. No deaths occurred in treatment groups receiving either cephalosporin or cotrimoxazole, despite the demonstrated resistance of B. pseudomallei to cotrimoxazole in vitro. The mortality rate in treatment groups receiving either cephalosporin and chloramphenicol was 66%. These results demonstrate a comparable level of efficacy between ceftazidime and cefpirome for treatment of acute B. pseudomallei infection in mice.

The mechanisms involved in the pathogenesis of melioidosis, caused by the intracellular bacterium Burkholderia pseudomallei, are unclear. C57BL/6 mice are resistant to infection, while BALB/c mice are highly susceptible. Previous studies have demonstrated that peritoneal exudate cell preparations enriched for macrophages are capable of effectively eliminating intracellular pathogens. In this study we present evidence showing that interaction of macrophages with lymphocytes is necessary for efficient anti-B. pseudomallei activity.

Burkholderia pseudomallei is the aetiological agent of melioidosis, a life-threatening bacterial disease occurring in many species of animals, including man. Infection in humans commonly manifests as one of three clinical presentations: acute, subacute or chronic disease. Investigations were undertaken to assess the suitability of BALB/c and C57Bl/6 mice as animal models for the different forms of human melioidosis. The course of infection in BALB/c mice was similar to that which occurs in acute human infection. By contrast, infection of C57Bl/6 mice appeared to mimic chronic human melioidosis. While BALB/c mice suffered a rapidly-progressive bacteraemia which resulted in host death by 96 h, C57Bl/6 mice were able to prevent this, and typically remained asymptomatic for up to 6 weeks. LD50 values of 4 cells and 2.5 x 10(4) cells for BALB/c and C57Bl/6 mice, respectively, reflect these observations. The heightened level of resistance to B. pseudomallei observed in C57Bl/6 mice was suggested to have a genetic basis, when the susceptibilities of first filial and reciprocal backcross generations were examined. Growth kinetics of B. pseudomallei within BALB/c and C57Bl/6 peritoneal exudate cell (PEC) cultures were examined to investigate PEC microbicidal efficiency as a determinant of host susceptibility. C57Bl/6 PEC cultures exhibited greater microbicidal efficiency towards B. pseudomallei when compared to BALB/c cells, indicating that susceptibility may be determined by non-specific, cellular mechanisms. Collectively, these results suggest that the BALB/c and C57Bl/6 strains of mice may provide excellent models for acute and chronic human melioidosis, respectively.