Exposure to Moderate Glycosuria Induces Virulence of Group B Streptococcus.

To explore whether glycosuria induces virulence of uropathogens, in turn facilitating urinary tract infection (UTI), we exposed group B Streptococcus (GBS) strain 10/84 to human urine plain or with 300 mg/dL glucose (mimicking moderate glycosuria). Exposure to moderate glycosuria significantly augmented bacterial growth, kidney bacterial burden in a mouse model of ascending UTI, and virulence characteristics and expression of corresponding genes. Exposure to glycosuria increased GBS adherence to human bladder epithelial cell line and expression of corresponding PI2a fimbrial gene, antimicrobial peptide LL-37 resistance and bacterial surface charge modulating dltA, and GBS hemolytic ability and expression of genes encoding pore-forming toxins.

Human Urine Alters Methicillin-Resistant Staphylococcus aureus Virulence and Transcriptome.

Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of hospital-associated urinary tract infections (UTI), especially in catheterized individuals. Despite being rare, MRSA UTI are prone to potentially life-threatening exacerbations such as bacteremia that can be refractory to routine antibiotic therapy. To delineate the molecular mechanisms governing MRSA urinary pathogenesis, we exposed three S. aureus clinical isolates, including two MRSA strains, to human urine for 2 h and analyzed virulence characteristics and changes in gene expression. The in vitro virulence assays showed that human urine rapidly alters adherence to human bladder epithelial cells and fibronectin, hemolysis of sheep red blood cells (RBCs), and surface hydrophobicity in a staphylococcal strain-specific manner. In addition, transcriptome sequencing (RNA-Seq) analysis of uropathogenic strain MRSA-1369 revealed that 2-h-long exposure to human urine alters MRSA transcriptome by modifying expression of genes encoding enzymes catalyzing metabolic pathways, virulence factors, and transcriptional regulators. In summary, our results provide important insights into how human urine specifically and rapidly alters MRSA physiology and facilitates MRSA survival in the nutrient-limiting and hostile urinary microenvironment. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is an uncommon cause of urinary tract infections (UTI) in the general population. However, it is important to understand MRSA pathophysiology in the urinary tract because isolation of MRSA in urine samples often precedes potentially life-threatening MRSA bacteremia. In this report, we describe how exposure to human urine alters MRSA global gene expression and virulence. We hypothesize that these alterations may aid MRSA in acclimating to the nutrient-limiting, immunologically hostile conditions within the urinary tract leading to MRSA UTI.

Electronic Cigarette (E-Cigarette) Vapor Exposure Alters the Streptococcus pneumoniae Transcriptome in a Nicotine-Dependent Manner without Affecting Pneumococcal Virulence.

The effects of electronic cigarette (e-cigarette) vapor (EV) exposure on the physiology of respiratory microflora are not fully defined. We analyzed the effects of exposure to vapor from nicotine-containing and nicotine-free e-liquid formulations on the virulence and transcriptome of Streptococcus pneumoniae strain TIGR4, a pathogen that asymptomatically colonizes the human nasopharyngeal mucosa. TIGR4 was preexposed for 2 h to nicotine-containing EV extract (EVE+NIC), nicotine-free EV extract (EVE-NIC), cigarette smoke extract (CSE), or nutrient-rich tryptic soy (TS) broth (control). The differences between the treatment and control strains were explored using transcriptome sequencing (RNA sequencing [RNA-Seq]), in vitro virulence assays, and an in vivo mouse model of acute pneumonia. The analysis of RNA-Seq profiles revealed modest changes in the expression of 14 genes involved in sugar transport and metabolism in EVE-NIC-preexposed TIGR4 compared to the control, while EVE+NIC or CSE exposure altered expression of 264 and 982 genes, respectively, most of which were involved in metabolism and stress response. Infection in a mouse model of acute pneumonia with control TIGR4 or with TIGR4 preexposed to EVE+NIC, EVE-NIC, or CSE did not show significant differences in disease parameters, such as bacterial organ burden and respiratory cytokine response. Interestingly, TIGR4 exposed to CSE or EVE+NIC (but not EVE-NIC) exhibited moderate induction of biofilm formation. However, none of the treatment groups showed significant alterations in pneumococcal hydrophobicity or epithelial cell adherence. In summary, our study reports that exposure to EV significantly alters the S. pneumoniae transcriptome in a nicotine-dependent manner without affecting pneumococcal virulence.IMPORTANCE With the increasing popularity of e-cigarettes among cigarette smoking and nonsmoking adults and children and the recent reports of vaping-related lung illness and deaths, further analysis of the adverse health effects of e-cigarette vapor (EV) exposure is warranted. Since pathogenic bacteria such as Streptococcus pneumoniae can colonize the human nasopharynx as commensals, they may be affected by exposure to bioactive chemicals in EV. Hence, in this study we examined the effects of EV exposure on the physiology of S. pneumoniae strain TIGR4. In order to differentiate between the effects of nicotine and nonnicotine components, we specifically compared the RNA-Seq profiles and virulence of TIGR4 exposed to vapor from nicotine-containing and nicotine-free e-liquid formulations. We observed that nicotine-containing EV augmented TIGR4 biofilms and altered expression of TIGR4 genes predominantly involved in metabolism and stress response. However, neither nicotine-containing nor nicotine-free EV affected TIGR4 virulence in a mouse model.

Cigarette Smoke Extract-Exposed Methicillin-Resistant Staphylococcus aureus Regulates Leukocyte Function for Pulmonary Persistence.

Cigarette smoke (CS) predisposes exposed individuals to respiratory infections not only by suppressing immune response but also by enhancing the virulence of pathogenic bacteria. As per our observations, in methicillin-resistant Staphylococcus aureus strain USA300, CS extract (CSE) potentiates biofilm formation via the down-regulation of quorum-sensing regulon accessory gene regulator. Because accessory gene regulator is a global regulator of the staphylococcal virulome, in the present study we sought to identify the effects of CS exposure on staphylococcal gene expression using RNAseq. Comparative analysis of RNAseq profiles revealed the up-regulation of important virulence genes encoding surface adhesins (fibronectin- and fibrinogen-binding proteins A and B and clumping factor B) and proteins involved in immune evasion, such as staphylocoagulase, staphylococcal protein A, and nuclease. In concurrence with the RNAseq data, we observed: (1) significant up-regulation of the ability of CSE-exposed USA300 to evade phagocytosis by macrophages and neutrophils, a known function of staphylococcal protein A; and (2) twofold higher (P < 0.001) number of CSE-exposed USA300 escaping neutrophil extracellular trap-mediated killing by neutrophils as a result of CS-mediated induction of nuclease. Importantly, in three different mouse strains, C57BL6/J, Balb/C, and A/J, we observed significantly higher pulmonary bacterial burden in animals infected with CSE-exposed USA300 as compared with medium-exposed control USA300. Taken together, these observations indicate that bioactive chemicals in CS induce hypervirulence by augmenting the ability of USA300 to evade bactericidal functions of leukocytes, such as phagocytosis and neutrophil extracellular trap-mediated killing.

Investigating the role of nucleotide-binding oligomerization domain-like receptors in bacterial lung infection.

Lower respiratory tract infections (LRTIs) are a persistent and pervasive public health problem worldwide. Pneumonia and other LRTIs will be among the leading causes of death in adults, and pneumonia is the single largest cause of death in children. LRTIs are also an important cause of acute lung injury and acute exacerbations of chronic obstructive pulmonary disease. Because innate immunity is the first line of defense against pathogens, understanding the role of innate immunity in the pulmonary system is of paramount importance. Pattern recognition molecules (PRMs) that recognize microbial-associated molecular patterns are an integral component of the innate immune system and are located in both cell membranes and cytosol. Toll-like receptors and nucleotide-binding oligomerization domain-like receptors (NLRs) are the major sensors at the forefront of pathogen recognition. Although Toll-like receptors have been extensively studied in host immunity, NLRs have diverse and important roles in immune and inflammatory responses, ranging from antimicrobial properties to adaptive immune responses. The lung contains NLR-expressing immune cells such as leukocytes and nonimmune cells such as epithelial cells that are in constant and close contact with invading microbes. This pulmonary perspective addresses our current understanding of the structure and function of NLR family members, highlighting advances and gaps in knowledge, with a specific focus on immune responses in the respiratory tract during bacterial infection. Further advances in exploring cellular and molecular responses to bacterial pathogens are critical to develop improved strategies to treat and prevent devastating infectious diseases of the lung.

The NLRP3 Inflammasome Is Dispensable in Methicillin-Resistant Staphylococcus aureus Urinary Tract Infection.

The NLRP3 inflammasome is a cytoplasmic complex that senses molecular patterns from pathogens or damaged cells to trigger an innate immune defense response marked by the production of proinflammatory cytokines IL-1ß and IL-18 and an inflammatory death called pyroptosis. The NLRP3 inflammasome is activated in the urinary tract by a variety of infectious and non-infectious insults. In this study, we investigated the role of the NLRP3 inflammasome by comparing the pathophysiology of methicillin-resistant Staphylococcus aureus (MRSA) ascending UTI in wild-type (WT) and Nlrp3-/- mice. The difference in the bacterial burden detected in the urinary tracts of MRSA-infected WT and Nlrp3-/- was not statistically significant at 6, 24, and 72 h post-infection (hpi). The levels of pro-inflammatory cytokines and chemokines as well as the numbers of granulocytes recruited to bladder and kidney tissues at 24 hpi were also similar between Nlrp3-/- and WT mice. The histopathological analysis of MRSA-infected bladder and kidney sections from Nlrp3-/- and WT mice showed similar inflammation. Overall, these results suggest that MRSA-induced urinary NLRP3 activity does not play a role in the pathophysiology of the ascending UTI.

Defining the Roles of Pyruvate Oxidation, TCA Cycle, and Mannitol Metabolism in Methicillin-Resistant Staphylococcus aureus Catheter-Associated Urinary Tract Infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of complicated urinary tract infection (UTI) associated with the use of indwelling urinary catheters. Previous reports have revealed host and pathogen effectors critical for MRSA uropathogenesis. Here, we sought to determine the significance of specific metabolic pathways during MRSA UTI. First, we identified four mutants from the Nebraska transposon mutant library in the MRSA JE2 background that grew normally in rich medium but displayed significantly reduced growth in pooled human urine (HU). This prompted us to transduce the uropathogenic MRSA 1369 strain with the transposon mutants in sucD and fumC (tricarboxylic acid [TCA] cycle), mtlD (mannitol metabolism), and lpdA (pyruvate oxidation). Notably, sucD, fumC, and mtlD were also significantly upregulated in the MRSA 1369 strain upon exposure to HU. Compared to the WT, the MRSA 1369 lpdA mutant was significantly defective for (i) growth in HU, and (ii) colonization of the urinary tract and dissemination to the kidneys and the spleen in the mouse model of catheter-associated UTI (CAUTI), which may be attributed to its increased membrane hydrophobicity and higher susceptibility to killing by human blood. In contrast to their counterparts in the JE2 background, the sucD, fumC, and mtlD mutants in the MRSA 1369 background grew normally in HU; however, they displayed significant fitness defects in the CAUTI mouse model. Overall, identification of novel metabolic pathways important for the urinary fitness and survival of MRSA can be used for the development of novel therapeutics. IMPORTANCE While Staphylococcus aureus has historically not been considered a uropathogen, S. aureus urinary tract infection (UTI) is clinically significant in certain patient populations, including those with chronic indwelling urinary catheters. Moreover, most S. aureus strains causing catheter-associated UTI (CAUTI) are methicillin-resistant S. aureus (MRSA). MRSA is difficult to treat due to limited treatment options and the potential to deteriorate into life-threatening bacteremia, urosepsis, and shock. In this study, we found that pathways involved in pyruvate oxidation, TCA cycle, and mannitol metabolism are important for MRSA fitness and survival in the urinary tract. Improved understanding of the metabolic needs of MRSA in the urinary tract may help us develop novel inhibitors of MRSA metabolism that can be used to treat MRSA-CAUTI more effectively.

Cigarette smoke increases Staphylococcus aureus biofilm formation via oxidative stress.

The strong epidemiological association between cigarette smoke (CS) exposure and respiratory tract infections is conventionally attributed to immunosuppressive and irritant effects of CS on human cells. Since pathogenic bacteria such as Staphylococcus aureus are members of the normal microbiota and reside in close proximity to human nasopharyngeal cells, we hypothesized that bioactive components of CS might affect these organisms and potentiate their virulence. Using Staphylococcus aureus as a model organism, we observed that the presence of CS increased both biofilm formation and host cell adherence. Analysis of putative molecular pathways revealed that CS exposure decreased expression of the quorum-sensing agr system, which is involved in biofilm dispersal, and increased transcription of biofilm inducers such as sarA and rbf. CS contains bioactive compounds, including free radicals and reactive oxygen species, and we observed transcriptional induction of bacterial oxidoreductases, including superoxide dismutase, following exposure. Moreover, pretreatment of CS with an antioxidant abrogated CS-mediated enhancement of biofilms. Exposure of bacteria to hydrogen peroxide alone increased biofilm formation. These observations are consistent with the hypothesis that CS induces staphylococcal biofilm formation in an oxidant-dependent manner. CS treatment induced transcription of fnbA (encoding fibronectin binding protein A), leading to increased binding of CS-treated staphylococci to immobilized fibronectin and increased adherence to human cells. These observations indicate that the bioactive effects of CS may extend to the resident microbiota of the nasopharynx, with implications for the pathogenesis of respiratory infection in CS-exposed humans.

Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.

The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal (also known as neutrophil gelatinase associated lipocalin, siderocalin, lipocalin 2) sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH-sensitive mechanism. As catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal-catechol-Fe(III) complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal-siderophore interactions but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.

A Systematic Review of the Literature Examining the Effects of Cigarette Smoke and e-Cigarette Vapor on the Virulence of Human Pathogenic Bacteria.

The bioactive chemicals in cigarette smoke (CS) and e-cigarette vapor (EV) may affect pathogenic bacteria in the nasopharyngeal microflora, which may have implications on the pathophysiology of respiratory infections in cigarette smokers and e-cigarette users. In this systematic review, we seek to synthesize the research evidence supporting this hypothesis. To address the central research question, "what is known from the published, peer-reviewed literature about the effects of cigarette smoke or e-cigarette vapor exposure on the physiology of human pathogenic bacteria?", we screened the PubMed®, Web of ScienceTM, and ScienceDirect databases for reports examining the virulence characteristics and gene expression in human pathogenic bacteria exposed to either CS or EV. The principal conclusion from our analysis is that exposure to either CS or EV induces the virulence of respiratory pathogenic bacteria in a strain-dependent manner, which may in turn facilitate respiratory infections in cigarette smokers and e-cigarette users. In addition, we present evidence that nicotine and reactive oxygen species are the main chemicals responsible for CS/EV-mediated alterations in bacterial physiology. We note limitations that this review does not examine reports describing the alterations in host respiratory physiology or nasopharyngeal dysbiosis caused by CS/EV exposure. Future research to determine whether CS/EV-mediated augmentation of bacterial virulence indeed plays a role in human respiratory tract infections is warranted.

Systematic Review of Literature Examining Bacterial Urinary Tract Infections in Diabetes.

This systematic review addresses the central research question, "what is known from the published, peer-reviewed literature about the impact of diabetes on the risk of bacterial urinary tract infections (UTI)?" We examine the results from laboratory studies where researchers have successfully adapted mouse models of diabetes to study the pathophysiology of ascending UTI. These studies have identified molecular and cellular effectors shaping immune defenses against infection of the diabetic urinary tract. In addition, we present evidence from clinical studies that in addition to diabetes, female gender, increased age, and diabetes-associated hyperglycemia, glycosuria, and immune impairment are important risk factors which further increase the risk of UTI in diabetic individuals. Clinical studies also show that the uropathogenic genera causing UTI are largely similar between diabetic and nondiabetic individuals, although diabetes significantly increases risk of UTI by drug-resistant uropathogenic bacteria.

Glycosuria Alters Uropathogenic Escherichia coli Global Gene Expression and Virulence.

Uropathogenic Escherichia coli (UPEC) is the principal etiology of more than half of urinary tract infections (UTI) in humans with diabetes mellitus. Epidemiological data and studies in mouse model of ascending UTI have elucidated various host factors responsible for increasing the susceptibility of diabetic hosts to UPEC-UTI. In contrast, diabetic urinary microenvironment-mediated alterations in UPEC physiology and its contributions to shaping UPEC-UTI pathogenesis in diabetes have not been examined. To address our central hypothesis that glycosuria directly induces urinary virulence of UPEC, we compared virulence characteristics and gene expression in human UPEC strains UTI89 (cystitis) and CFT073 (pyelonephritis), exposed for 2 h in vitro to urine from either male or female donors that was either plain or supplemented with glucose to mimic glycosuria. Compared to control UPEC exposed to nutrient-rich culture medium, lysogeny broth, glycosuria-exposed UPEC exhibited significant increase in biofilm formation and reduction in the hemagglutination of Guinea pig erythrocytes (a measure of type 1 piliation). In addition, the analysis of UTI89 transcriptome by RNA sequencing revealed that 2-h-long, in vitro exposure to glycosuria also significantly alters expression of virulence and metabolic genes central to urinary virulence of UPEC. Addition of galactose as an alternative carbon source affected biofilm formation and gene expression profile of UPEC to an extent similar to that observed with glucose exposure. In summary, our results provide novel insights into how glycosuria-mediated rapid changes in UPEC fitness may facilitate UTI pathogenesis in the diabetic urinary microenvironment. IMPORTANCE Uropathogenic Escherichia coli (UPEC) is an important causative agent of urinary tract infections in diabetic humans. We examined the effects of in vitro exposure to glycosuria (presence of glucose in urine) on the virulence and gene expression by UPEC. Our results show that glycosuria rapidly (in 2 h) alters UPEC gene expression, induces biofilm formation, and suppresses type 1 piliation. These results offer novel insights into the pathogenesis of UPEC in the urinary tract.

Inerolysin, a cholesterol-dependent cytolysin produced by Lactobacillus iners.

Lactobacillus iners is a common constituent of the human vaginal microbiota. This species was only recently characterized due to its fastidious growth requirements and has been hypothesized to play a role in the pathogenesis of bacterial vaginosis. Here we present the identification and molecular characterization of a protein toxin produced by L. iners. The L. iners genome encodes an open reading frame with significant primary sequence similarity to intermedilysin (ILY; 69.2% similarity) and vaginolysin (VLY; 68.4% similarity), the cholesterol-dependent cytolysins from Streptococcus intermedius and Gardnerella vaginalis, respectively. Clinical isolates of L. iners produce this protein, inerolysin (INY), during growth in vitro, as assessed by Western analysis. INY is a pore-forming toxin that is activated by reducing agents and inhibited by excess cholesterol. It is active across a pH range of 4.5 to 6.0 but is inactive at pH 7.4. At sublytic concentrations, INY activates p38 mitogen-activated protein kinase and allows entry of fluorescent phalloidin into the cytoplasm of epithelial cells. Unlike VLY and ILY, which are human specific, INY is active against cells from a broad range of species. INY represents a new target for studies directed at understanding the role of L. iners in states of health and disease at the vaginal mucosal surface.

Cigarette smoke inhibits airway epithelial cell innate immune responses to bacteria.

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

Rational manipulation of mRNA folding free energy allows rheostat control of pneumolysin production by Streptococcus pneumoniae.

The contribution of specific factors to bacterial virulence is generally investigated through creation of genetic "knockouts" that are then compared to wild-type strains or complemented mutants. This paradigm is useful to understand the effect of presence vs. absence of a specific gene product but cannot account for concentration-dependent effects, such as may occur with some bacterial toxins. In order to assess threshold and dose-response effects of virulence factors, robust systems for tunable expression are required. Recent evidence suggests that the folding free energy (ΔG) of the 5' end of mRNA transcripts can have a significant effect on translation efficiency and overall protein abundance. Here we demonstrate that rational alteration of 5' mRNA folding free energy by introduction of synonymous mutations allows for predictable changes in pneumolysin (PLY) expression by Streptococcus pneumoniae without the need for chemical inducers or heterologous promoters. We created a panel of isogenic S. pneumoniae strains, differing only in synonymous (silent) mutations at the 5' end of the PLY mRNA that are predicted to alter ΔG. Such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. These studies provide proof-of-principle for further investigation of mRNA ΔG manipulation as a tool in studies of bacterial pathogenesis.

α-Intercalated cells defend the urinary system from bacterial infection.

α-Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC-dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.

ß-Hemolysin/cytolysin of Group B Streptococcus enhances host inflammation but is dispensable for establishment of urinary tract infection.

Group B Streptococcus (GBS; Streptococcus agalactiae) is a major human pathogen that disproportionately affects neonates and women in the peripartum period and is an emerging cause of infection in older adults. The primary toxin of GBS, ß-hemolysin/cytolysin (ßH/C), has a well-defined role in the pathogenesis of invasive disease, but its role in urinary tract infection (UTI) is unknown. Using both in vitro and in vivo models, we analyzed the importance of ßH/C in GBS uropathogenesis. There were no significant differences in bacterial density from the bladders or kidneys from mice infected with wild-type or isogenic ßH/C-deficient GBS, and competitive indices from co-infection experiments were near 1. Thus, ßH/C is dispensable for the establishment of GBS-UTI. However, ßH/C-sufficient GBS induced a more robust proinflammatory cytokine response in cultured bladder epithelial cells and in the urinary tracts of infected mice. Given the near ubiquity of ßH/C-expressing strains in epidemiologic studies and the importance of local inflammation in dictating outcomes and sequelae of UTI, we hypothesize that ßH/C-driven inflammatory signaling may be important in the clinical course of GBS-UTI.

Emergence of the epidemic methicillin-resistant Staphylococcus aureus strain USA300 coincides with horizontal transfer of the arginine catabolic mobile element and speG-mediated adaptations for survival on skin.

UNLABELLED: The arginine catabolic mobile element (ACME) is the largest genomic region distinguishing epidemic USA300 strains of methicillin-resistant Staphylococcus aureus (MRSA) from other S. aureus strains. However, the functional relevance of ACME to infection and disease has remained unclear. Using phylogenetic analysis, we have shown that the modular segments of ACME were assembled into a single genetic locus in Staphylococcus epidermidis and then horizontally transferred to the common ancestor of USA300 strains in an extremely recent event. Acquisition of one ACME gene, speG, allowed USA300 strains to withstand levels of polyamines (e.g., spermidine) produced in skin that are toxic to other closely related S. aureus strains. speG-mediated polyamine tolerance also enhanced biofilm formation, adherence to fibrinogen/fibronectin, and resistance to antibiotic and keratinocyte-mediated killing. We suggest that these properties gave USA300 a major selective advantage during skin infection and colonization, contributing to the extraordinary evolutionary success of this clone. IMPORTANCE: Over the past 15 years, methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem. It is likely that adaptations in specific MRSA lineages (e.g., USA300) drove the spread of MRSA across the United States and allowed it to replace other, less-virulent S. aureus strains. We suggest that one major factor in the evolutionary success of MRSA may have been the acquisition of a gene (speG) that allows S. aureus to evade the toxicity of polyamines (e.g., spermidine and spermine) that are produced in human skin. Polyamine tolerance likely gave MRSA multiple fitness advantages, including the formation of more-robust biofilms, increased adherence to host tissues, and resistance to antibiotics and killing by human skin cells.

The Ngal reporter mouse detects the response of the kidney to injury in real time.

Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarker characteristics that link the protein to the injured organ have not yet been described. We generated an Ngal reporter mouse by inserting a double-fusion reporter gene encoding luciferase-2 and mCherry (Luc2-mC) into the Ngal (Lcn2) locus. The Ngal-Luc2-mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real time. In the kidney, Ngal-Luc2-mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ- and cell-specific relationship with tubular stress, which correlated with the level of urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2-mC mice also revealed both the onset and the resolution of the injury, and the actions of NF-κB inhibitors and antibiotics during infection. Thus, imaging of Ngal-Luc2-mC mice and cells identified injurious and reparative agents that affect kidney damage.