Pathogenesis of Streptococcus urinary tract infection depends on bacterial strain and β-hemolysin/cytolysin that mediates cytotoxicity, cytokine synthesis, inflammation and virulence

Streptococcus agalactiae can cause urinary tract infection (UTI) including cystitis and asymptomatic bacteriuria (ABU). The early host-pathogen interactions that occur during S. agalactiae UTI and subsequent mechanisms of disease pathogenesis are poorly defined. Here, we define the early interactions between human bladder urothelial cells, monocyte-derived macrophages, and mouse bladder using uropathogenic S. agalactiae (UPSA) 807 and ABU-causing S. agalactiae (ABSA) 834 strains. UPSA 807 adhered, invaded and killed bladder urothelial cells more efficiently compared to ABSA 834 via mechanisms including low-level caspase-3 activation, and cytolysis, according to lactate dehydrogenase release measures and cell viability. Severe UPSA 807-induced cytotoxicity was mediated entirely by the bacterial β-hemolysin/cytolysin (β-H/C) because an β-H/C-deficient UPSA 807 isogenic mutant, UPSA 807 "cylE, was not cytotoxic in vitro; the mutant was also significantly attenuated for colonization in the bladder in vivo. Analysis of infection-induced cytokines, including IL-8, IL-1β, IL-6 and TNF-α in vitro and in vivo revealed that cytokine and chemokine responses were dependent on expression of β-H/C that also elicited severe bladder neutrophilia. Thus, virulence of UPSA 807 encompasses adhesion to, invasion of and killing of bladder cells, pro-inflammatory cytokine/chemokine responses that elicit neutrophil infiltration, and β-H/C-mediated subversion of innate immune-mediated bacterial clearance from the bladder

Complete Genome Sequence of Serotype III Streptococcus agalactiae Sequence Type 17 Strain 874391.

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

Type 2 NADH dehydrogenase is the only point of entry for electrons into the Streptococcus agalactiae respiratory chain and is a potential drug target

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

Streptococcus agalactiae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) elicits multiple cytokines from human cells and has a minor effect on bacterial persistence in the murine female reproductive tract

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 analyzed 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, tumor 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 affects bacterial persistence in the female reproductive tract

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract 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

Higher salivary expression of S100A12 in patients with ulcerative colitis and chronic periodontitis

[Extract] Our research group has a special interest in the association of inflammatory bowel disease (IBD) and chronic periodontitis, an inflammation that affects the alveolar bone and can lead to tooth loss [1]. Both diseases are related to an imbalance between the immune-inflammatory response and the dysbiotic microbiota. However, a plausible biological explanation for a local effect of IBD on the periodontal tissue destruction is still missing

A comparative study of extradural anesthesia using 0.75% ropivacaine, 0.75% ropivacaine with fentanyl, and 0.75% ropivacaine with buprenorphine for cesarean section from a rural teaching hospital in India

Context: The study was to compare the analgesic efficacy of three different epidural solutions - ropivacaine, ropivacaine fentanyl mixture, and ropivacaine buprenorphine mixture - for cesarean section. Materials and Methods: This was a prospective, randomized, controlled, double blind study carried out in primi parturients undergoing elective cesarean section with singleton fetus. A total of 102 parturients in the age group of 20-35 years, American Society of Anesthesiologists (ASA) I or II scheduled for elective cesarean under continuous epidural anesthesia were divided into three groups using a computer-generated random number list. The test dose (3 ml 2% lignocaine with 15 μg adrenaline) and 0.75% ropivacaine 12 ml were given to all parturients. In addition, normal saline 1 ml, fentanyl 50 μg, and buprenorphine 300 μg were given to Group I, II, and III respectively. Sensory block, motor block, analgesia, maternal effects, fetal outcome, and surgeons′ and parturients′ satisfaction were evaluated. Results: Onset of sensory block was faster in the fentanyl and buprenorphine groups compared to ropivacaine group (9.94 ± 0.48, 10.72 ± 0.26 versus 14.59 ± 0.34). Duration of sensory block was prolonged in buprenorphine group as compared to fentanyl and ropivacaine groups (120.41 ± 4.31) versus (95.68 ± 3.28, 98.28 ± 3.42). Duration of analgesia was prolonged in buprenorphine group compared to fentanyl and ropivacaine groups (516.38 ± 29.14 versus 327.06 ± 12.41, 285.78 ± 10.10). It proved to be safe for mother and fetus. The surgeon and the parturients were satisfied with the mode of anesthesia. Conclusion: Ropivacaine 0.75%, ropivacaine 0.75% with fentanyl 50 mg, or buprenorphine 300 mg provided safe anesthesia when given extradurally for cesarean section. Addition of both fentanyl and buprenorphine to ropivacaine hastened the onset of sensory block, while addition of buprenorphine provided prolonged excellent postoperative analgesia

Conserved bacterial de novo guanine biosynthesis pathway enables microbial survival and colonization in the environmental niche of the urinary 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

Urinary tract infection of mice to model human disease: Practicalities, implications and limitations

Abstract: 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

The Streptococcus agalactiae virulence regulator CovR affects the pathogenesis of urinary tract infection

<b>Background</b>\ud \ud - <i>Streptococcus agalactiae</i> can cause urinary tract infection (UTI). The role of the <i>S. agalactiae</i> global virulence regulator, CovR, in UTI pathogenesis is unknown\ud \ud <b>Methods</b>\ud \ud - We used murine and human bladder uroepithelial cell models of UTI and <i>S. agalactiae</i> mutants in <i>covR</i> and related factors, including -hemolysin/cytolysin ( -h/c), surface-anchored adhesin HvgA, and capsule to study the role of CovR in UTI.\ud \ud <b>Results</b>\ud \ud - <i>covR</i>-deficient serotype III <i>S. agalactiae</i> 874391 was significantly attenuated for colonization in mice and adhesion to uroepithelial cells. Mice infected with <i>covR</i>-deficient <i>S. agalactiae</i> produced less pro-inflammatory cytokines compared to wild type (WT) 874391. Acute cytotoxicity in uroepithelial cells triggered by <i>covR</i>-deficient but not WT 874391 was associated with significant caspase-3 activation. Mechanistically, <i>covR</i> mutation significantly altered the expression of several genes in <i>S. agalactiae</i> 874391 that encode key virulence factors, including -h/c and HvgA, but not capsule. Subsequent mutational analyses revealed that HvgA and capsule, but not the -h/c, exerted significant effects on colonization of the murine urinary tract <i>in vivo</i>.\ud \ud <b>Conclusion</b>\ud \ud - S. agalactiae CovR promotes bladder infection and inflammation, and adhesion to and viability of uroepithelial cells. The pathogenesis of S. agalactiae UTI is complex, multifactorial and influenced by virulence effects of CovR, HvgA and capsule