Group B streptococci (GBSs) are the leading cause of neonatal meningitis. GBSs enter the CNS by penetrating the blood-brain barrier (BBB), which consists of specialized human brain microvascular endothelial cells (hBMECs). To identify GBS factors required for BBB penetration, we generated random mutant libraries of a virulent strain and screened for loss of hBMEC invasion in vitro. Two independent hypo-invasive mutants possessed disruptions in the same gene, invasion associated gene (iagA), which encodes a glycosyltransferase homolog. Allelic replacement of iagA in the GBS chromosome produced a 4-fold decrease in hBMEC invasiveness. Mice challenged with the GBS ∆iagA mutant developed bacteremia comparably to WT mice, yet mortality was significantly lower (20% vs. 90%), as was the incidence of meningitis. The glycolipid diglucosyldiacylglycerol, a cell membrane anchor for lipoteichoic acid (LTA) and predicted product of the IagA glycosyltransferase, was absent in the ∆iagA mutant, which consequently shed LTA into the media. Attenuation of virulence of the ∆iagA mutant was found to be independent of TLR2-mediated signaling, but bacterial supernatants from the ∆iagA mutant containing released LTA inhibited hBMEC invasion by WT GBS. Our data suggest that LTA expression on the GBS surface plays a role in bacterial interaction with BBB endothelium and the pathogenesis of neonatal meningitis.

Group A streptococcus (GAS) is a leading cause of severe, invasive human infections, including necrotizing fasciitis and toxic shock syndrome. An important element of the mammalian innate defense system against invasive bacterial infections such as GAS is the production of antimicrobial peptides (AMPs) such as cathelicidins. In this study, we identify a specific GAS phenotype that confers resistance to host AMPs. Allelic replacement of the dltA gene encoding D-alanine-D-alanyl carrier protein ligase in an invasive serotype M1 GAS isolate led to loss of teichoic acid D-alanylation and an increase in net negative charge on the bacterial surface. Compared to the wild-type (WT) parent strain, the GAS ⌬dltA mutant exhibited increased susceptibility to AMP and lysozyme killing and to acidic pH. While phagocytic uptake of WT and ⌬dltA mutants by human neutrophils was equivalent, neutrophil-mediated killing of the ⌬dltA strain was greatly accelerated. Furthermore, we observed the ⌬dltA mutant to be diminished in its ability to adhere to and invade cultured human pharyngeal epithelial cells, a likely proximal step in the pathogenesis of invasive infection. Thus, teichoic acid D-alanylation may contribute in multiple ways to the propensity of invasive GAS to bypass mucosal defenses and produce systemic infection.

Streptococcus pneumoniae is one of the few species within the group of low-G ؉C gram-positive bacteria reported to contain no D-alanine in teichoic acids, although the dltABCD operon encoding proteins responsible for D-alanylation is present in the genomes of two S. pneumoniae strains, the laboratory strain R6 and the clinical isolate TIGR4. The annotation of dltA in R6 predicts a protein, D-alanine-D-alanyl carrier protein ligase (Dcl), that is shorter at the amino terminus than all other Dcl proteins. Translation of dltA could also start upstream of the annotated TTG start codon at a GTG, resulting in the premature termination of dltA translation at a stop codon. Applying a novel integrative translation probe plasmid with Escherichia coli lacZ as a reporter, we could demonstrate that dltA translation starts at the upstream GTG. Consequently, S. pneumoniae R6 is a dltA mutant, whereas S. pneumoniae D39, the parental strain of R6, and Rx, another derivative of D39, contained intact dltA genes. Repair of the stop codon in dltA of R6 and insertional inactivation of dltA in D39 and Rx yielded pairs of dltA-deficient and dltA-proficient strains. Subsequent phenotypic analysis showed that dltA inactivation resulted in enhanced sensitivity to the cationic antimicrobial peptides nisin and gallidermin, a phenotype fully consistent with those of dltA mutants of other gram-positive bacteria. In addition, mild alkaline hydrolysis of heat-inactivated whole cells released D-alanine from dltAproficient strains, but not from dltA mutants. The results of our study suggest that, as in many other low-G؉C gram-positive bacteria, teichoic acids of S. pneumoniae contain D-alanine residues in order to protect this human pathogen against the actions of cationic antimicrobial peptides.Teichoic acids (TAs) are polymers with a relatively wide structural diversity that are present at the surfaces of many gram-positive bacteria (18). The most common types of TAs are comprised of either polyglycerol phosphate or polyribitol phosphate chains of variable length that are substituted with glycosyl residues or D-alanyl esters, or both. TAs may be covalently linked to peptidoglycan (wall teichoic acids [WTAs]) or anchored in the cytoplasmic membrane by their glycolipid moiety (lipoteichoic acids [LTAs]). As major constituents of the surfaces of gram-positive bacteria, TAs have an impact on a number of important biological processes, such as autolysis (60), binding of cations (25) and surface-associated proteins (9, 29), adhesion (1, 61), biofilm formation (21), coaggregation (13), resistance to antimicrobial agents (50, 51), protein secretion (46), acid tolerance (7), stimulation of immune response (20, 41), and virulence (1, 14, 52). In most of these processes, the degree of D-alanylation of TAs has been shown to be of outstanding importance. Addition of D-alanine to TAs reduces the negative charge of the cell envelope, thereby influencing the binding and interaction of various compounds. Incorporation of D-alanine in LTAs is accomplished in a t...

SummaryMany Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies.

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In Staphylococcus carnosus, the nreABC (for nitrogen regulation) genes were identified and shown to link the nitrate reductase operon (narGHJI) and the putative nitrate transporter gene narT. An nreABC deletion mutant, m1, was dramatically affected in nitrate and nitrite reduction and growth. Transcription of narT, narGHJI, and the nitrite reductase (nir) operon was severely reduced even when cells were cultivated anaerobically without nitrate or nitrite. nreABC transcripts were detected when cells were grown aerobically or anaerobically with or without nitrate or nitrite. NreA is a GAF domain-containing protein of unknown function. In vivo and in vitro studies showed that NreC is phosphorylated by NreB and that phospho-NreC specifically binds to a GC-rich palindromic sequence to enhance transcription initiation. This binding motif was found at the narGHJI, nir, and narT promoters but not at the moeB promoter. NreB is a cytosolic protein with four N-terminal cysteine residues. The second cysteine residue was shown to be important for NreB function. In vitro autophosphorylation of NreB was not affected by nitrate, nitrite, or molybdate. The nir promoter activity was iron dependent. The data provide evidence for a global regulatory system important for aerobic and anaerobic metabolism, with NreB and NreC forming a classical two-component system and NreB acting as a sensor protein with oxygen as the effector molecule.Staphylococcus carnosus, traditionally used as a starter culture in the production of raw fermented sausages, reduces nitrate to ammonia in two steps: (i) nitrate is taken up and reduced by a dissimilatory nitrate reductase to nitrite, which is subsequently excreted, and (ii) after depletion of nitrate, the accumulated nitrite is imported and reduced by an NADHdependent nitrite reductase to ammonia, which then accumulates in the medium. Nitrate reductase is a membrane-bound enzyme involved in energy conservation, whereas nitrite reductase is a cytosolic enzyme involved in NADH reoxidation. The absence of oxygen and the presence of nitrate and/or nitrite induce nitrate reductase and nitrite reductase activities. Nitrite reduction is inhibited by nitrate and by high concentrations of nitrite (Ն10 mM), whereas nitrate reduction is not influenced by nitrite and ammonia (19).Although the amino acid sequences of the S. carnosus nitrate reductase and nitrite reductase enzymes are similar to those of the corresponding Escherichia coli proteins, we have found evidence that the regulatory proteins and operator sequences differ (21,24).In E. coli, expression from the nir promoter (P nir ) is dependent both on FNR (fumarate and nitrate reductase regulation) and on NarL or NarP (36, 37). Recently, Browning et al. (7) have shown that P nir is repressed by three DNA binding proteins, i.e., Fis (factor for inversion stimulation), integration host factor (IHF), and H-NS (histone-like nucleoid structuring protein), and that NarL and NarP can relieve IHF-and Fismediated repression but are unable to counteract H-NS-mediat...

SummaryThe nreABC ( n itrogen re gulation) operon encodes a new staphylococcal two-component regulatory system that controls dissimilatory nitrate/nitrite reduction in response to oxygen. Unlike other twocomponent sensors NreB is a cytosolic protein with four N-terminal cysteine residues. It was shown that both the NreB-cysteine cluster and Fe ions are required for function. Isolated NreB was converted to the active form by incubation with cysteine desulphurase, ferrous ions and cysteine. This activation is typical for FeS-containing proteins and was reversed by oxygen. During reconstitution an absorption band at 420 nm and a yellow-brownish colour (typical for an FNR-type iron-sulphur cluster formation) developed. After alkylation of thiol groups in NreB and in the cysteine mutant NreB(C62S) almost no ironsulphur cluster was incorporated; both findings corroborated the importance of the cysteine residues. Comparison of the kinase activity of (i) the reconstituted (ii) the unreconstituted, and (iii) the unreconstituted and deferrated NreB-His indicated that NreB kinase activity depended on iron availability and was greatly enhanced by reconstitution. NreB is the first direct oxygen-sensing protein described in staphylococci so far. Reconstituted NreB contains 4-8 acid-labile Fe and sulphide ions per NreB which is in agreement with the presence of 1-2 iron-sulphur [4Fe-4S] 2+ clusters of the FNR-type. Unlike FNR, NreB does not act directly as transcriptional activator, but transfers the phosphoryl group to the response regulator NreC.

Staphylococcus aureus is exposed to multiple antimicrobial compounds, including oxidative burst products and antibiotics. The various mechanisms and regulatory pathways governing susceptibility or resistance are complex and only superficially understood. Bacillus subtilis recently has been shown to control disulfide stress responses by the thioredoxin-related YjbH protein, which binds to the transcriptional regulator Spx and controls its degradation via the proteasome-like ClpXP protease. We show that the S. aureus YjbH homolog has a role in susceptibility to the disulfide stress-inducing agent diamide that is similar to that in B. subtilis, and we demonstrate that the four cysteine residues in YjbH are required for this activity. In addition, the inactivation of YjbH led to moderate resistance to oxacillin and other ␤-lactam antibiotics, and this phenotypic change was associated with higher penicillin-binding protein 4 levels and increased peptidoglycan crosslinking. Of note, the impact of YjbH on ␤-lactam susceptibility still was observed when the four cysteines of YjbH were mutated, indicating that the roles of YjbH in disulfide stress and ␤-lactam resistance rely on different types of interactions. These data suggest that the ClpXP adaptor YjbH has more target proteins than previously thought, and that oxidative burst and ␤-lactam resistance mechanisms of S. aureus are closely linked.Staphylococcus aureus is a major human pathogen causing a wide spectrum of diseases that range from mild skin infections to life-threatening septicemia, pneumonia, and toxic-shock syndrome (27). ␤-Lactam antibiotics such as oxacillin are among to the most effective drugs against S. aureus infections, but their usefulness is continuously decreasing because ␤-lactam-resistant strains, particularly methicillin-resistant S. aureus (MRSA), are spreading in hospitals and, more recently, in the community at large, alarming international health authorities (11). Resistance is based on ␤-lactamases (penicillin-resistant S. aureus) or on an alternative peptidoglycan-biosynthetic enzyme, the penicillin-binding protein 2a (MRSA) (5, 14). In addition, mutations in a variety of proteins affecting cell wall biosynthesis and turnover can affect ␤-lactam susceptibility, leading either to hypersensitivity or to moderate levels of resistance (3,5,9,17,24,36). In many cases it remains unclear how the mutated proteins affect staphylococcal ␤-lactam resistance, and the underlying mechanisms remain only superficially understood.During the process of infection, S. aureus is engulfed by phagocytic cells such as neutrophils and macrophages, and the bacteria are exposed to the microbicidal activity of these cells.One of the most powerful antimicrobial mechanisms is based on the production of reactive oxygen species (ROS) or nitrogen species (RNS) by phagocyte NADPH oxidase and nitric oxide synthase, respectively (19,29). In addition, ROS also are generated during incomplete electron transfer in the bacterial respiratory chain (21). Therefore, even nonpath...