Staphylococcus aureus is both a commensal and a pathogen of the human host. Survival in the host environment requires resistance to host-derived nitric oxide (NO·). However, S. aureus lacks the NO·-sensing transcriptional regulator NsrR that is used by many bacteria to sense and respond to NO·. In this study, we show that S. aureus is able to sense and respond to both NO· and hypoxia by means of the SrrAB two-component system (TCS). Analysis of the S. aureus transcriptome during nitrosative stress demonstrates the expression of SrrAB-dependent genes required for cytochrome biosynthesis and assembly (qoxABCD, cydAB, hemABCX), anaerobic metabolism (pflAB, adhE, nrdDG), iron-sulfur cluster repair (scdA), and NO· detoxification (hmp). Targeted mutations in SrrAB-regulated loci show that hmp and qoxABCD are required for NO· resistance, whereas nrdDG is specifically required for anaerobic growth. We also show that SrrAB is required for survival in static biofilms, most likely due to oxygen limitation. Activation by hypoxia, NO·, or a qoxABCD quinol oxidase mutation suggests that the SrrAB TCS senses impaired electron flow in the electron transport chain rather than directly interacting with NO· in the manner of NsrR. Nevertheless, like NsrR, SrrAB achieves the physiological goals of selectively expressing hmp in the presence of NO· and minimizing the potential for Fenton chemistry. Activation of the SrrAB regulon allows S. aureus to maintain energy production and essential biosynthetic processes, repair damage, and detoxify NO· in diverse host environments.

CcpA is the global mediator of carbon catabolite repression (CCR) in gram-positive bacteria, and growing evidence from several pathogens, including the group A streptococcus (GAS), suggests that CcpA plays an important role in virulence gene regulation. In this study, a deletion of ccpA in an invasive M1 GAS strain was used to test the contribution of CcpA to pathogenesis in mice. Surprisingly, the ⌬ccpA mutant exhibited a dramatic "hypervirulent" phenotype compared to the parental MGAS5005 strain, reflected as increased lethality in a model of systemic infection (intraperitoneal administration) and larger lesion size in a model of skin infection (subcutaneous administration). Expression of ccpA in trans from its native promoter was able to complement both phenotypes, suggesting that CcpA acts to repress virulence in GAS. To identify the CcpAregulated gene(s) involved, a transcriptome analysis was performed on mid-logarithmic-phase cells grown in rich medium. CcpA was found to primarily repress 6% of the GAS genome (124 genes), including genes involved in sugar metabolism, transcriptional regulation, and virulence. Notably, the entire sag operon necessary for streptolysin S (SLS) production was under CcpA-mediated CCR, as was SLS hemolytic activity. Purified CcpA-His bound specifically to a cre within sagAp, demonstrating direct repression of the operon. Finally, SLS activity is required for the increased virulence of a ⌬ccpA mutant during systemic infection but did not affect virulence in a wild-type background. Thus, CcpA acts to repress SLS activity and virulence during systemic infection in mice, revealing an important link between carbon metabolism and GAS pathogenesis.

Salmonella enterica serovar Typhi, the cause of typhoid fever, is host-adapted to humans and unable to cause disease in mice. Here, we show that S.

Carbon catabolite repression (CCR) allows bacteria to alter metabolism in response to the availability of specific sugar sources, and increasing evidence suggests that CCR is involved in regulating virulence gene expression in many pathogens. A scan of the M1 SF370 group A streptococcus (GAS) genome using a Bacillus subtilis consensus identified a number of potential catabolite-responsive elements (cre) important for binding by the catabolite control protein A (CcpA), a mediator of CCR in gram-positive bacteria. Intriguingly, a putative cre was identified in the promoter region of mga upstream of its distal P1 start of transcription. Electrophoretic mobility shift assays showed that a His-CcpA fusion protein was capable of binding specifically to the cre in Pmga in vitro. Deletion analysis of Pmga using single-copy Pmga-gusA reporter strains found that Pmga P1 and its upstream cre were not required for normal autoregulated mga expression from Pmga P2 as long as Mga was produced from its native locus. In fact, the Pmga P1 region appeared to show a negative influence on Pmga P2 in these studies. However, deletion of the cre at the native Pmga resulted in a reduction of total mga transcripts as determined by real-time reverse transcription-PCR, supporting a role for CcpA in initial expression. Furthermore, normal transcriptional initiation from the Pmga P1 start site alone was dependent on the presence of the cre. Importantly, inactivation of ccpA in the M6 GAS strain JRS4 resulted in a reduction in Pmga expression and Mga protein levels in late-logarithmic-phase cell growth. These data support a role for CcpA in the early activation of the mga promoter and establish a link between CCR and Mga regulation in the GAS.

Nitric oxide (NO·) is a ubiquitous molecular mediator in biology. Many signaling actions of NO· generated by mammalian NO· synthase (NOS) result from targeting of the haem moiety of soluble guanylate cyclase. Some pathogenic and environmental bacteria also produce a NOS that is evolutionary related to the mammalian enzymes, but a bacterial haem-containing receptor for endogenous enzymatically-generated NO· has not been previously identified. Here we show that NOS of the human pathogen Staphylococcus aureus, in concert with an NO·-metabolising flavohaemoprotein, regulates electron transfer by targeting haem-containing cytochrome oxidases under microaerobic conditions to maintain membrane bioenergetics. This process is essential for staphylococcal nasal colonisation and resistance to the membrane-targeting antibiotic daptomycin, and demonstrates the conservation of NOS-derived NO·-haem receptor signaling between bacteria and mammals.

Moraxella catarrhalis is a Gram-negative obligate aerobe that is an important cause of human respiratory tract infections. The M. catarrhalis genome encodes a predicted truncated denitrification pathway that reduces nitrate to nitrous oxide. We have previously shown that expression of both the M. catarrhalis aniA (encoding a nitrite reductase) and norB (encoding a putative nitric oxide reductase) genes is repressed by the transcriptional regulator NsrR under aerobic conditions and that M. catarrhalis O35E nsrR mutants are unable to grow in the presence of low concentrations of nitrite (W. Wang, et al., J. Bacteriol. 190:7762-7772, 2008). In this study, we constructed an M. catarrhalis norB mutant and showed that planktonic growth of this mutant is inhibited by low levels of nitrite, whether or not an nsrR mutation is present. To determine the importance of NorB in this truncated denitrification pathway, we analyzed the metabolism of nitrogen oxides by norB, aniA norB, and nsrR norB mutants. We found that norB mutants are unable to reduce nitric oxide and produce little or no nitrous oxide from nitrite. Furthermore, nitric oxide produced from nitrite by the AniA protein is bactericidal for a Moraxella catarrhalis O35E norB mutant but not for wild-type O35E bacteria under aerobic growth conditions in vitro, suggesting that nitric oxide catabolism in M. catarrhalis is accomplished primarily by the norB gene product. Measurement of bacterial protein S-nitrosylation directly implicates nitrosative stress resulting from AniA-dependent nitric oxide formation as a cause of the growth inhibition of norB and nsrR mutants by nitrite.Moraxella catarrhalis is an obligately aerobic Gram-negative bacterium that colonizes the human upper respiratory tract. For many decades, Moraxella catarrhalis was considered to be a harmless member of the normal flora and was known as Neisseria catarrhalis due to its morphological similarities to commensal Neisseria species (47). Recently, M. catarrhalis has been recognized as an important pathogen in both the upper and lower respiratory tracts (45). M. catarrhalis is the third leading bacterial cause of acute otitis media (32,44,67) in infants and very young children and the second most common bacterial cause of exacerbations of chronic obstructive pulmonary disease (COPD) in adults (43,46,58). It is estimated that 2 to 4 million exacerbations of COPD in the United States are attributable to M. catarrhalis infection each year (46). M. catarrhalis has been implicated in other infections, including community-acquired pneumonia (64), and extremely rarely may cause fatal bacteremia or pneumonia in patients with preexisting health conditions, such as immunodeficiency or impaired airway defenses (57).Studies show that nasopharyngeal colonization with M. catarrhalis is common in infants and young children, and a high rate of colonization is associated with an increased risk of otitis media (16,31). Recent surveys of nasopharyngeal colonization of Streptococcus pneumoniae, nontypeable Haemophilus in...

Coordinate regulation of virulence factors by the group A streptococcus (GAS) Streptococcus pyogenes is important in this pathogen's ability to cause disease. To further elucidate the regulatory network in this human pathogen, the CovR-repressed two-component system (TCS) trxSR was chosen for further analysis based on its homology to a virulence-related TCS in Streptococcus pneumoniae. In a murine skin infection model, an insertion mutation in the response regulator gene, trxR, led to a significant reduction in lesion size, lesion severity, and lethality. Curing the trxR mutation restored virulence comparable to the wild-type strain. The trxSR operon was defined in vivo, and CovR was found to directly repress its promoter in vitro. DNA microarray analysis established that TrxR activates transcription of Mga-regulated virulence genes, which may explain the virulence attenuation of the trxR mutant. This regulation appears to occur by activation of the mga promoter, Pmga, as demonstrated by analysis of a luciferase reporter fusion. Complementation of the trxR mutant with trxR on a plasmid restored expression of Mga regulon genes and restored virulence in the mouse model to wild-type levels. TrxR is the first TCS shown to regulate Mga expression. Because it is CovR repressed, TrxR defines a new pathway by which CovR can influence Mga to affect pathogenesis in the GAS.