Deletion of hypothetical wall teichoic acid ligases in Staphylococcus aureus activates the cell wall stress response.

The Staphylococcus aureus cell wall stress stimulon (CWSS) is activated by cell envelope-targeting antibiotics or depletion of essential cell wall biosynthesis enzymes. The functionally uncharacterized S. aureus LytR-CpsA-Psr (LCP) proteins, MsrR, SA0908 and SA2103, all belong to the CWSS. Although not essential, deletion of all three LCP proteins severely impairs cell division. We show here that VraSR-dependent CWSS expression was up to 250-fold higher in single, double and triple LCP mutants than in wild type S. aureus in the absence of external stress. The LCP triple mutant was virtually depleted of wall teichoic acids (WTA), which could be restored to different degrees by any of the single LCP proteins. Subinhibitory concentrations of tunicamycin, which inhibits the first WTA synthesis enzyme TarO (TagO), could partially complement the severe growth defect of the LCP triple mutant. Both of the latter findings support a role for S. aureus LCP proteins in late WTA synthesis, as in Bacillus subtilis where LCP proteins were recently proposed to transfer WTA from lipid carriers to the cell wall peptidoglycan. Intrinsic activation of the CWSS upon LCP deletion and the fact that LCP proteins were essential for WTA-loading of the cell wall, highlight their important role(s) in S. aureus cell envelope biogenesis.

Induction kinetics of the Staphylococcus aureus cell wall stress stimulon in response to different cell wall active antibiotics.

BACKGROUND: Staphylococcus aureus activates a protective cell wall stress stimulon (CWSS) in response to the inhibition of cell wall synthesis or cell envelope damage caused by several structurally and functionally different antibiotics. CWSS induction is coordinated by the VraSR two-component system, which senses an unknown signal triggered by diverse cell wall active agents. RESULTS: We have constructed a highly sensitive luciferase reporter gene system, using the promoter of sas016 (S. aureus N315), which detects very subtle differences in expression as well as measuring > 4 log-fold changes in CWSS activity, to compare the concentration dependence of CWSS induction kinetics of antibiotics with different cell envelope targets. We compared the effects of subinhibitory up to suprainhibitory concentrations of fosfomycin, D-cycloserine, tunicamycin, bacitracin, flavomycin, vancomycin, teicoplanin, oxacillin, lysostaphin and daptomycin. Induction kinetics were both strongly antibiotic- and concentration-dependent. Most antibiotics triggered an immediate response with induction beginning within 10 min, except for tunicamycin, D-cycloserine and fosfomycin which showed lags of up to one generation before induction commenced. Induction characteristics, such as the rate of CWSS induction once initiated and maximal induction reached, were strongly antibiotic dependent. We observed a clear correlation between the inhibitory effects of specific antibiotic concentrations on growth and corresponding increases in CWSS induction kinetics. Inactivation of VraR increased susceptibility to the antibiotics tested from 2- to 16-fold, with the exceptions of oxacillin and D-cycloserine, where no differences were detected in the methicillin susceptible S. aureus strain background analysed. There was no apparent correlation between the induction capacity of the various antibiotics and the relative importance of the CWSS for the corresponding resistance phenotypes. CONCLUSION: CWSS induction profiles were unique for each antibiotic. Differences observed in optimal induction conditions for specific antibiotics should be determined and taken into account when designing and interpreting CWSS induction studies.

Unveiling electrotransformation of Moraxella catarrhalis as a process of natural transformation.

The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhalis.

Cold shock response of the UspA1 outer membrane adhesin of Moraxella catarrhalis.

Colonization of the human nasopharynx exposes Moraxella catarrhalis, a common cause of otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, to sudden downshifts in temperature, occurring when the host breathes cold air. We investigated whether in vitro cold shock influences the expressions of the outer membrane adhesins UspA1 and hemagglutinin, which are considered virulence factors, and of an M. catarrhalis homolog of recA, a housekeeping gene, which in Escherichia coli is induced by cold shock. Quantitative real-time reverse transcriptase PCR was used for measuring mRNA copy number. A screening experiment revealed that a cold shock at 26 degrees C maximally induced the copy number of uspA1. In comparison with 37 degrees C conditions, a 1-hour cold shock at 26 degrees C increased copy numbers of uspA1 and recA by 2.5-fold (11.2 +/- 1.8 versus 4.5 +/- 0.8 copies/CFU) and 2.7-fold (0.30 +/- 0.10 versus 0.11 +/- 0.06), respectively, but did not induce transcription of hag. Exposure to 26 degrees C increased surface expression of UspA1, as assessed by fluorescence-activated cell sorter analysis, and resulted in a significant increase in adherence of strain O35E to Chang human conjunctival cells (97.1% +/- 2.0% versus 48.3% +/- 9.2% at 37 degrees C; P = 0.01). Cold shock induction of uspA1 and recA was detected in strains belonging to either phylogenetic subpopulation of M. catarrhalis (16S rRNA types 1 and 2/3) and was most pronounced in type 2/3 strains (4- to 25-fold for uspA1), which do not express detectable amounts of UspA1 protein at 37 degrees C. These data indicate that cold shock at a physiologically relevant temperature of 26 degrees C induces the expression of at least one virulence factor (UspA1). To our knowledge, no similar data are available for other nasopharyngeal pathogens.

Moraxella catarrhalis strains with reduced expression of the UspA outer membrane proteins belong to a distinct subpopulation.

The outer membrane proteins UspA1 and UspA2 are candidate antigens for a Moraxella catarrhalis vaccine. We previously reported that 103 of 108 isolates (95%) from young children expressed UspA1 detected by reactivity with the monoclonal antibody mAb24B5. The aim of the present study was to investigate mechanisms controlling UspA1 expression by analysis of five mAb24B5 non-reactive isolates. Four of these strains were characterized by (i) decreased or absent transcription of uspA1 and uspA2 and (ii) clustered mutations and deletions in the promoter region of both uspA1 and uspA2. Antigenic or phase variation were not responsible for reduced levels of UspA1 expression. While mAb24B5-positive isolates expressing normal levels of uspA1 and uspA2 mRNA belonged to the previously described 16S rRNA type 1 phylogenetic group, these four mAb24B5-negative isolates were found to belong to the 16S rRNA gene types 2 or 3. The remaining mAb24B5-negative isolate (#610) belonged to 16S rRNA type 1 and exhibited a posttranscriptional defect of UspA1 expression defined by normal levels of uspA1 mRNA and both recombinant and in vitro expression of mAb24B5-reactive UspA1. In conclusion, M. catarrhalis clinical isolates exhibiting reduced expression of UspA1 and UspA2 belonged to a distinct phylogenetic subpopulation. A UspA-based vaccine is unlikely to be effective against such isolates.

Mucosal immune response to specific outer membrane proteins of Moraxella catarrhalis in young children.

BACKGROUND: Moraxella catarrhalis is an important cause of otitis media. A number of candidate antigens for a future infant otitis media vaccine have been identified, but their mucosal immunogenicity induced by nasopharyngeal M. catarrhalis colonization has not been characterized. The aim of this study was to determine the salivary IgA response to M. catarrhalis outer membrane proteins (OMP) in young children. METHODS: Children ages 1 to 24 months evaluated for acute respiratory tract infection were prospectively enrolled. M. catarrhalis nasopharyngeal colonization was determined by (1) selective culture and (2) detection by reverse transcription-PCR of messenger RNA specific for the OMP UspA1 and UspA2. Salivary IgA responses were detected by immunoblot analysis of M. catarrhalis OMP. Isogenic knockout mutants for UspA1, UspA2, hemagglutinin (Hag), transferrin-binding protein B (TbpB) and CopB were constructed for identification of specific target OMP. RESULTS: Sixty-six patients were studied. The rates of M. catarrhalis colonization by culture, reverse transcription-PCR for messenger RNA and mRNA were 40, 94 and 58%, respectively. Anti-M. catarrhalis salivary IgA was detected in 62 patients (94%). IgA directed against a >250-kDa antigen (assigned to UspA1/UspA2 by mutant analysis) and a 200-kDa antigen (Hag) were detected in 65 and 70% of patients, respectively. Bands at 80 to 85 kDa (82%) consisted of IgA directed against monomeric UspA2, TbpB and CopB. CONCLUSIONS: colonization occurring in early infancy is associated with a consistent mucosal immune response directed against the UspA proteins, Hag and other OMP. The data suggest that several M. catarrhalis OMP are immunogens of the nasopharyngeal mucosal immune system of infants.

Low-level resistance to rifampin in Streptococcus pneumoniae.

Rifampin is recommended for combination therapy of meningitis due to beta-lactam-resistant Streptococcus pneumoniae. High-level rifampin resistance (MIC, > or =4 mg/liter) has been mapped to point mutations in clusters I and III of rpoB of the pneumococcus. The molecular basis of low-level resistance (MICs, > or =0.5 and <4 mg/liter) was analyzed. Spontaneous mutants of clinical pneumococcal isolates were selected on Columbia sheep blood agar plates containing rifampin at 0.5, 4, 10, or 50 mg/liter. Low-level resistance could be assigned to mutations in cluster II (I(545)N, I(545)L). Sensitive (MIC, <0.048 mg/liter) wild-type strains acquired low-level resistance at a rate approximately 10 times higher than that at which they acquired high-level resistance (average mutation frequencies, 2.4 x 10(-7) for low-level resistance versus 2.9 x 10(-8) for high-level resistance [P < 0.0001]). In second-step experiments, the frequencies of mutations from low- to high-level resistance were over 10 times higher than the frequencies of mutations from susceptibility to high-level resistance (average mutation frequencies, 7.2 x 10(-7) versus 5.0 x 10(-8) [P < 0.001]). Mutants with low-level resistance were stable upon passage. Sequencing of a clinical isolate with low-level resistance (MIC, 0.5 mg/liter) revealed a Q(150)R mutation upstream of cluster I. The frequencies of mutations to high-level resistance for this strain were even higher than the rates observed for the in vitro mutants. Therefore, a resistance-mediating mutation located outside clusters I, II, and III has been described for the first time in the pneumococcus. In vitro low-level rifampin resistance in S. pneumoniae could be mapped to cluster II of rpoB. Mutants of pneumococcus with low-level resistance may be selected in vivo during therapy in tissue compartments with low antibiotic concentrations and play a role in the development of resistance.

The outer membrane proteins UspA1 and UspA2 of Moraxella catarrhalis are highly conserved in nasopharyngeal isolates from young children.

UspA1 and UspA2 of Moraxella catarrhalis are vaccine candidates. The aims of this study were to determine: (1) the frequencies of occurrence and (2) the degrees of conservation of two surface-exposed epitopes of the uspA1 and uspA2 genes and their respective gene products in 108 nasopharyngeal isolates from young children. The uspA1 and uspA2 genes were detected in 107 (99%) and 108 (100%) isolates, respectively. Twenty-three of 108 uspA2 genes (21%) were identified as the variant gene uspA2H. One-hundred and five isolates (97%) expressed the mAb17C7-reactive epitope shared by UspA1 and UspA2, and 103 isolates (95%) reacted with the UspA1-specific mAb24B5. The only isolate which lacked a uspA1 gene demonstrated reduced adherence to HEp-2 cells and complement sensitivity. The data indicate that both uspA genes and the expression of at least two surface-exposed epitopes are virtually ubiquitous in isolates from a population at risk for otitis media. A vaccine capable of inducing a bactericidal immune response against the mAb17C7- and/or mAb24B5-reactive epitopes appears promising.