Local Generation of Kynurenines Mediates Inhibition of Neutrophil Chemotaxis by Uropathogenic Escherichia coli.

During epithelial infections, pathogenic bacteria employ an array of strategies to attenuate and evade host immune responses, including the influx of polymorphonuclear leukocytes (PMN; neutrophils). Among the most common bacterial infections in humans are those of the urinary tract, caused chiefly by uropathogenic Escherichia coli (UPEC). During the establishment of bacterial cystitis, UPEC suppresses innate responses via multiple independent strategies. We recently described UPEC attenuation of PMN trafficking to the urinary bladder through pathogen-specific local induction of indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolic enzyme previously shown to have regulatory activity only in adaptive immunity. Here, we investigated the mechanism by which IDO induction attenuates PMN migration. Local tryptophan limitation, by which IDO is known to influence T cell longevity and proliferation, was not involved in its effect on PMN trafficking. Instead, metabolites in the IDO pathway, particularly L-kynurenine, directly suppressed PMN transepithelial migration and induced an attached, spread morphology in PMN both at rest and in the presence of chemotactic stimuli. Finally, kynurenines represent known ligands of the mammalian aryl hydrocarbon receptor (AHR), and UPEC infection of Ahr(-/-)mice recapitulated the derepressed PMN recruitment observed previously in Ido1(-/-)mice. UPEC therefore suppresses neutrophil migration early in bacterial cystitis by eliciting an IDO-mediated increase in local production of kynurenines, which act through the AHR to impair neutrophil chemotaxis.

Induction of indoleamine 2,3-dioxygenase by uropathogenic bacteria attenuates innate responses to epithelial infection.

Uropathogenic Escherichia coli (UPEC) are the chief cause of urinary tract infections. Although neutrophilic inflammation is a hallmark of disease, previous data indicate that UPEC promotes local dampening of host innate immune responses. Here, we show that UPEC attenuates innate responses to epithelial infection by inducing expression of indoleamine 2,3-dioxygenase (IDO), a host enzyme with previously defined roles in adaptive immune regulation. UPEC induced IDO expression in human uroepithelial cells and polymorphonuclear leukocytes (PMN) in vitro and in bladder tissue during murine cystitis via a noncanonical, interferon-independent pathway. In the bladders of UPEC-infected IDO-deficient mice, we observed augmented expression of proinflammatory cytokines and local inflammation, correlated with reduced survival of extracellular bacteria. Pharmacologic inhibition of IDO also increased human PMN transepithelial migration. Stimulation of IDO expression therefore represents a pathogen strategy to create local immune privilege at epithelial surfaces, attenuating innate responses to promote colonization and the establishment of infection.

YbcL of uropathogenic Escherichia coli suppresses transepithelial neutrophil migration.

Uropathogenic Escherichia coli (UPEC) strains suppress the acute inflammatory response in the urinary tract to ensure access to the intracellular uroepithelial niche that supports the propagation of infection. Our understanding of this initial cross talk between host and pathogen is incomplete. Here we report the identification of a previously uncharacterized periplasmic protein, YbcL, encoded by UPEC that contributes to immune modulation in the urinary tract by suppressing acute neutrophil migration. In contrast to wild-type UPEC, an isogenic strain lacking ybcL expression (UTI89 ΔybcL) failed to suppress transepithelial polymorphonuclear leukocyte (PMN) migration in vitro, a defect complemented by expressing ybcL episomally. YbcL homologs are present in many E. coli genomes; expression of the YbcL variant encoded by nonpathogenic E. coli K-12 strain MG1655 (YbcL(MG)) failed to complement the UTI89 ΔybcL defect, whereas expression of the UPEC YbcL variant (YbcL(UTI)) in MG1655 conferred the capacity for suppressing PMN migration. This phenotypic difference was due to a single amino acid difference (V78T) between the two YbcL homologs, and a majority of clinical UPEC strains examined were found to encode the suppressive YbcL variant. Purified YbcL(UTI) protein suppressed PMN migration in response to live or killed MG1655, and YbcL(UTI) was detected in the supernatant during UPEC infection of bladder epithelial cells or PMNs. Lastly, early PMN influx to murine bladder tissue was augmented upon in vivo infection with UTI89 ΔybcL compared with wild-type UPEC. Our findings demonstrate a role for UPEC YbcL in suppression of the innate immune response during urinary tract infection.

Streptolysin S inhibits neutrophil recruitment during the early stages of Streptococcus pyogenes infection.

In contrast to infection of superficial tissues, Streptococcus pyogenes infection of deeper tissue can be associated with a significantly diminished inflammatory response, suggesting that this bacterium has the ability to both promote and suppress inflammation. To examine this, we analyzed the behavior of an S. pyogenes mutant deficient in expression of the cytolytic toxin streptolysin S (SLS-) and evaluated events that occur during the first few hours of infection by using several models including injection of zebrafish (adults, larvae, and embryos), a transepithelial polymorphonuclear leukocyte (PMN) migration assay, and two-photon microscopy of mice in vivo. In contrast to wild-type S. pyogenes, the SLS- mutant was associated with the robust recruitment of neutrophils and significantly reduced lethal myositis in adult zebrafish. Similarly, the mutant was attenuated in embryos in its ability to cause lethality. Infection of larva muscle allowed an analysis of inflammation in real time, which revealed that the mutant had recruited PMNs to the infection site. Analysis of transepithelial migration in vitro suggested that SLS inhibited the host cells' production of signals chemotactic for neutrophils, which contrasted with the proinflammatory effect of an unrelated cytolytic toxin, streptolysin O. Using two-photon microscopy of mice in vivo, we showed that the extravasation of neutrophils during infection with SLS- mutant bacteria was significantly accelerated compared to infection with wild-type S. pyogenes. Taken together, these data support a role for SLS in the inhibition of neutrophil recruitment during the early stages of S. pyogenes infection.

contribution of genetically restricted, methicillin-susceptible strains to the ongoing epidemic of community-acquired Staphylococcus aureus infections.

BACKGROUND: Within the current worldwide epidemic of community-acquired Staphylococcus aureus infections, attention has focused on the role of methicillin-resistant strains. We characterize methicillin-susceptible strains that also contribute to this epidemic. METHODS: We tracked cultures from abscess specimens submitted to the microbiology laboratory at St. Louis Children's Hospital and examined Panton-Valentine leukocidin (PVL) genes in methicillin-susceptible S. aureus (MSSA) isolates. We further characterized some isolates by multilocus sequence typing, pulsed-field gel electrophoresis, antibiotic susceptibility, accessory gene regulator (agr) allele, and presence of the arcA gene of the arginine catabolic mobile element. RESULTS: From 1999 to 2007, we detected a 250-fold increase in cultures of abscesses yielding methicillin-resistant S. aureus (MRSA) and a 5-fold increase in abscess cultures yielding MSSA. MSSA isolates from abscesses and wounds were more likely to encode PVL than isolates from other sources. In contrast to PVL-negative isolates of MSSA, which were genetically diverse, PVL-positive isolates were predominantly multilocus sequence typing type 8 and agr type 1. More than half of PVL-positive MSSA isolates were resistant to erythromycin and susceptible to clindamycin with the absence of inducible resistance, a pattern uncommon in PVL-negative MSSA but frequent in the USA300 clone of MRSA. In addition, pulsed-field gel electrophoresis of PVL-positive MSSA strains revealed the USA300 pattern. CONCLUSIONS: In addition to methicillin-resistant strains, the current epidemic of S. aureus infections includes infections caused by methicillin-susceptible strains that are closely related genetically and share phenotypic characteristics other than susceptibility to methicillin. These findings suggest that factors other than methicillin resistance are driving the epidemic.

Attenuation of human neutrophil migration and function by uropathogenic bacteria.

The establishment of bacterial infections at mucosal epithelial surfaces is determined by the balance of virulence attributes of the pathogen with the activity of innate host defenses. Polymorphonuclear leukocytes (PMN) are key responders in many bacterial infections, but the mechanisms by which pathogens subvert these early responses to establish infection are largely undefined. Here, we model early interactions between human PMN and the primary cause of urinary tract infections, namely uropathogenic Escherichia coli (UPEC). Our objective was to define virulence phenotypes of uropathogens that permit evasion of PMN activity. We show that UPEC strains, as compared with laboratory and commensal E. coli, resist phagocytic killing and dampen the production of antimicrobial reactive oxygen species by PMN. Analysis of the transcriptional responses of PMN to E. coli strains revealed that UPEC exposure downregulates the expression of PMN genes that direct pro-inflammatory signaling and PMN chemotaxis, adhesion, and migration. Consistent with these data, UPEC attenuated transepithelial neutrophil recruitment in an in vitro model of acute infection and in a murine model of bacterial cystitis. We propose that these UPEC strategies are important in the establishment of epithelial infection, and that the findings are germane to bacterial infections at other epithelial surfaces.

Virulence gene expression in human community-acquired Staphylococcus aureus infection.

Isolates of methicillin-resistant Staphylococcus aureus (MRSA) were once linked uniformly with hospital-associated infections; however, community-acquired MRSA (CA-MRSA) now represents an emerging threat worldwide. To examine the association of differential virulence gene expression with outcomes of human infection, we measured transcript levels of target staphylococcal genes directly in clinical samples from children with active known or suspected CA-MRSA infections. Virulence genes encoding secreted toxins, including Panton-Valentine leukocidin, were highly expressed during superficial and invasive CA-MRSA infections. In contrast, increased expression of surface-associated protein A was linked only with invasive disease. Comparisons with laboratory-grown corresponding clinical isolates revealed that tissue-specific expression profiles reflect the activity of the staphylococcal accessory gene regulator during human infection. These results represent the first demonstration of staphylococcal gene expression and regulation directly in human tissue. Such analysis will help to unravel the complex interactions between CA-MRSA and its host environmental niches during disease development.

Contribution of invariant residues to the function of Rgg family transcription regulators.

The Rgg family of transcription regulators is widely distributed among gram-positive bacteria, yet how these proteins control transcription is poorly understood. Using Streptococcus pyogenes RopB as a model, we demonstrated that residues invariant among Rgg-like regulators are critical for function and obtained evidence for a mechanism involving protein complex formation.

Comparative functional analysis of the lac operons in Streptococcus pyogenes.

Having no known environmental reservoir, Streptococcus pyogenes, a bacterium responsible for a wider variety of human diseases than any other bacterial species, must rely on its host for metabolic substrates. Although a streptococcal aldolase, LacD.1, has been adapted to virulence gene regulation, both LacD.1 and a paralogous protein, LacD.2, are predicted to function in the tagatose 6-phosphate pathway for lactose and galactose utilization. In order to gain insight into the mechanism of the LacD.1 regulatory pathway and the role of genome context in the emergence of LacD.1's novel regulatory functions, we compared the function and regulation of the Lac.1 and Lac.2 loci. The Lac.1 operon is not inducible, and regulation by LacD.1 is independent of a functional tagatose 6-phosphate pathway and enhanced by the conserved truncation of upstream Lac.1 genes. In contrast, Lac.2 expression is sensitive to environmental carbohydrates, and LacD.2, not LacD.1, contributes to growth on galactose. Thus, we conclude that the Lac.1 locus has been specialized to participate in regulation, leaving efficient utilization of carbohydrate sources to the Lac.2 locus. The adaptation of LacD for transcription regulation may be an underappreciated strategy among prokaryotes, as homologues of this multifaceted enzyme are present in a broad range of species.

Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a model for in vivo gene expression.

For a pathogen such as Streptococcus pyogenes, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analyses of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we determined that the gene encoding the SpeB cysteine protease is up-regulated over the course of infection in a murine soft-tissue model. Conditions were identified, including growth phase, acidic pH, and an NaCl concentration of <0.1 M, that were required for expression of speB in vitro. Analysis of global expression profiles in response to these conditions in vitro identified a set of coregulated genes whose expression patterns showed a significant correlation with that of speB when examined during infection of murine soft tissues. This analysis revealed that a culture medium that promotes high levels of SpeB expression in vitro produced an expression profile that showed significant correlation to the profile observed in vivo. Taken together, these studies establish culture conditions that mimic in vivo expression patterns; that growth phase, pH, and NaCl may mimic relevant cues sensed by S. pyogenes during infection; and that identification of other environmental cues that alter expression of speB in vitro may provide insight into the signals that direct global patterns of gene expression in vivo.

A novel adaptation of aldolase regulates virulence in Streptococcus pyogenes.

Regulation of virulence factor expression is critical for pathogenic microorganisms that must sense and adapt to a dynamic host environment; yet, the signal transduction pathways that enable this process are generally poorly understood. Here, we identify LacD.1 as a global regulator of virulence factor expression in the versatile human pathogen, Streptococcus pyogenes. LacD.1 is derived from a class I tagatose-1,6-bisphosphate aldolase homologous to those involved in lactose and galactose metabolism in related prokaryotes. However, regulation of transcription by LacD.1 is not dependent on this enzymatic activity or the canonical catabolite repression pathway, but likely does require substrate recognition. Our results suggest that LacD.1 has been adapted as a metabolic sensor, and raise the possibility that regulation of gene expression by metabolic enzymes may be a novel mechanism by which Gram-positive bacteria, including S. pyogenes, coordinate multiple environmental cues, allowing essential transcription programs to be coupled with perceived nutritional status.

Recombinant cytochromes c biogenesis systems I and II and analysis of haem delivery pathways in Escherichia coli.

Genetic analysis has indicated that the system II pathway for c-type cytochrome biogenesis in Bordetella pertussis requires at least four biogenesis proteins (CcsB, CcsA, DsbD and CcsX). In this study, the eight genes (ccmA-H) associated with the system I pathway in Escherichia coli were deleted. Using B. pertussis cytochrome c4 as a reporter for cytochromes c assembly, it is demonstrated that a single fused ccsBA polypeptide can replace the function of the eight system I genes in E. coli. Thus, the CcsB and CcsA membrane complex of system II is likely to possess the haem delivery and periplasmic cytochrome c-haem ligation functions. Using recombinant system II and system I, both under control of IPTG, we have begun to study the capabilities and characteristics of each system in the same organism (E. coli). The ferrochelatase inhibitor N-methylprotoporphyrin was used to modulate haem levels in vivo and it is shown that system I can use endogenous haem at much lower levels than system II. Additionally, while system I encodes a covalently bound haem chaperone (holo-CcmE), no covalent intermediate has been found in system II. It is shown that this allows system I to use holo-CcmE as a haem reservoir, a capability system II does not possess.

Mutations in cytochrome assembly and periplasmic redox pathways in Bordetella pertussis.

Transposon mutagenesis of Bordetella pertussis was used to discover mutations in the cytochrome c biogenesis pathway called system II. Using a tetramethyl-p-phenylenediamine cytochrome c oxidase screen, 27 oxidase-negative mutants were isolated and characterized. Nine mutants were still able to synthesize c-type cytochromes and possessed insertions in the genes for cytochrome c oxidase subunits (ctaC, -D, and -E), heme a biosynthesis (ctaB), assembly of cytochrome c oxidase (sco2), or ferrochelatase (hemZ). Eighteen mutants were unable to synthesize all c-type cytochromes. Seven of these had transposons in dipZ (dsbD), encoding the transmembrane thioreduction protein, and all seven mutants were corrected for cytochrome c assembly by exogenous dithiothreitol, which was consistent with the cytochrome c cysteinyl residues of the CXXCH motif requiring periplasmic reduction. The remaining 11 insertions were located in the ccsBA operon, suggesting that with the appropriate thiol-reducing environment, the CcsB and CcsA proteins comprise the entire system II biosynthetic pathway. Antiserum to CcsB was used to show that CcsB is absent in ccsA mutants, providing evidence for a stable CcsA-CcsB complex. No mutations were found in the genes necessary for disulfide bond formation (dsbA or dsbB). To examine whether the periplasmic disulfide bond pathway is required for cytochrome c biogenesis in B. pertussis, a targeted knockout was made in dsbB. The DsbB- mutant makes holocytochromes c like the wild type does and secretes and assembles the active periplasmic alkaline phosphatase. A dipZ mutant is not corrected by a dsbB mutation. Alternative mechanisms to oxidize disulfides in B. pertussis are analyzed and discussed.