Biomedical subjects
Publications and source records attributed to Brigitte Berger-Bächi.
Bacteria have adapted a variety of different ways to acquire antibiotic resistance, fostering the rapid development of resistance within a short evolutionary time. The general genetic basis of events leading to and promoting antibiotic resistance formation in bacteria are presented and exemplified by showing the evolution of methicillin, glycopeptide, linezolid, and ketolide resistance in Staphylococcus aureus.
Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue (saCOL1786) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the DeltaccpA mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of S. aureus by down-regulating RNAIII, the effector molecule of the agr locus, and altering the transcription patterns of hla, encoding alpha-hemolysin, and spa, encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant S. aureus strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant S. aureus strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased cap operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.
DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of -131 mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsbA.
Isogenic Staphylococcus aureus strains with different capacities to produce sigma(B) activity were analyzed for their ability to attach to fibrinogen- or fibronectin-coated surfaces or platelet-fibrin clots and to cause endocarditis in rats. In comparison to the sigma(B)-deficient strain, BB255, which harbors an rsbU mutation, both rsbU-complemented and sigma(B)-overproducing derivatives exhibited at least five times greater attachment to fibrinogen- and fibronectin-coated surfaces and showed increased adherence to platelet-fibrin clots. No differences in adherence were seen between BB255 and a DeltarsbUVWsigB isogen. Northern blotting analyses revealed that transcription of clfA, encoding fibrinogen-binding protein clumping factor A, and fnbA, encoding fibronectin-binding protein A, were positively influenced by sigma(B). Sigma(B) overproduction resulted in a statistically significant increase in positive spleen cultures and enhanced bacterial densities in both the aortic vegetations and spleens at 16 h postinoculation. In contrast, at 72 h postinoculation, tissues infected with the sigma(B) overproducer had lower bacterial densities than did those infected with BB255. These results suggest that although sigma(B) appears to increase the adhesion of S. aureus to various host cell-matrix proteins in vitro, it has limited effect on pathogenesis in the rat endocarditis model. Sigma(B) appears to have a transient enhancing effect on bacterial density in the early stages of infection that is lost during progression.
Inactivation of hemB in Staphylococcus aureus strain Newman resulted in a small-colony phenotype and was accompanied by an altered expression pattern of global regulators and control of virulence factor production. Transcription profiles followed over 15 h by Northern blot analyses revealed that transcripts of the global regulators arl, rot, sae, sarR, sarS, srr, svrA, and sigB disappeared after the exponential phase and that both agr transcripts were completely absent in the hemB mutant. Apart from a general concentration of transcriptional activity to the exponential phase, premature gene expression was observed for rot, hla, and spa. Nevertheless, reported sigmaB-dependent transcripts, such as sarC and clfA, were produced throughout the 15-h growth period monitored. The absence of these transcripts in a hemB sigB double mutant demonstrated their dependence on sigmaB and indicated an unexpected, permanent sigmaB activity in the hemB mutant. Variations in the extents of the directly sigmaB-controlled asp23, rsbVW-sigB, and sarC transcripts argue for additional factors modulating sigmaB activity. This study provides the first extended synopsis of the transcriptional patterns of different regulators over the entire growth cycle in the widely used Newman strain.
The alternative sigma factor sigma(B) of Staphylococcus aureus controls the expression of a variety of genes, including virulence determinants and global regulators. Genetic manipulations and transcriptional start point (TSP) analyses showed that the sigB operon is transcribed from at least two differentially controlled promoters: a putative sigma(A)-dependent promoter, termed sigB(p1), giving rise to a 3.6-kb transcript covering sa2059-sa2058-rsbU-rsbV-rsbW-sigB, and a sigma(B)-dependent promoter, sigB(p3), initiating a 1.6-kb transcript covering rsbV-rsbW-sigB. TSP and promoter-reporter gene fusion experiments indicated that a third promoter, tentatively termed sigB(p2) and proposed to lead to a 2.5-kb transcript, including rsbU-rsbV-rsbW-sigB, might govern the expression of the sigB operon. Environmental stresses, such as heat shock and salt stress, induced a rapid response within minutes from promoters sigB(p1) and sigB(p3). In vitro, the sigB(p1) promoter was active in the early growth stages, while the sigB(p2) and sigB(p3) promoters produced transcripts throughout the growth cycle, with sigB(p3) peaking around the transition state between exponential growth and stationary phase. The amount of sigB transcripts, however, did not reflect the concentration of sigma(B) measured in cell extracts, which remained constant over the entire growth cycle. In a guinea pig cage model of infection, sigB transcripts were as abundant 2 and 8 days postinoculation as values found in vitro, demonstrating that sigB is indeed transcribed during the course of infection. Physical interactions between staphylococcal RsbU-RsbV, RsbV-RsbW, and RsbW-sigma(B) were inferred from a yeast (Saccharomyces cerevisiae) two-hybrid approach, indicating the presence of a partner-switching mechanism in the sigma(B) activation cascade similar to that of Bacillus subtilis. The finding that overexpression of RsbU was sufficient to trigger an immediate and strong activation of sigma(B), however, signals a relevant difference in the regulation of sigma(B) activation between B. subtilis and S. aureus in the cascade upstream of RsbU.
The majority of methicillin-resistant Staphylococcus aureus (MRSA) isolates, recovered in 2003 at the Department of Medical Microbiology in Zürich, Switzerland, belonged to major clones that are circulating worldwide. Staphylococcal cassette chromosome mec type IV (SCCmec-IV), harbored by half of the isolates, was found in sequence type 217 (ST 217), which is an allelic variant of epidemic MRSA-15 (designated EMRSA-15), in a new local ST 617 descending from clonal complex CC 8 and in low-level oxacillin-resistant strains of multiple genetic lineages characteristic of community-onset MRSA. SCCmec-I, SCCmec-II, and SCCmec-III were in the minority, and four MRSA isolates had complex, rearranged SCCmec elements. A novel SCCmec-N1 of approximately 30 kb, associated with a dfrA gene and a ccr 4-related recombinase complex, was identified in a large number of low-level oxacillin-resistant isolates, which descended from the successful clonal complex CC 45 and are spreading among intraveneous drug users. In contrast, the SCCmec types of oxacillin-resistant coagulase-negative staphylococci (MRCNS) were of completely different composition. SCCmec type I (SCCmec-I) and SCCmec-II were more frequent than in the MRSA, while fewer contained SCCmec-IV. The other MRCNS displayed 11 different, complex patterns, suggesting frequent recombination between different SCCmec elements. With one ccr-negative exception, these strains amplified between one and three different ccr products, indicating either new varied complexes or multiple ccr loci. This suggests the presence of novel SCCmec types in MRCNS and no extensive interspecies SCCmec transfer between MRSA and MRCNS.
OBJECTIVE: To elucidate factors that contribute to the development of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Forty-nine MRSA isolates were subjected to passage selection with vancomycin to isolate mutants with reduced susceptibility to vancomycin. One mutant was chosen for detailed molecular and biochemical characterization. RESULTS: Five vancomycin-resistant mutants (vancomycin MICs, 6-12 mg/L) were obtained in vitro from five MRSA parent isolates. Upon acquisition of vancomycin resistance, all mutants showed a concomitant decrease in oxacillin resistance. In one particular MRSA strain, selection for vancomycin resistance repeatedly produced deletions and rearrangements, including loss of the mecA gene. Pleiotropic phenotypical changes, such as yellow pigment formation, loss of haemolysis, thickened cell wall, increased resistance to lysostaphin and reduced cell wall turnover were observed in this mutant. CONCLUSION: Acquisition of vancomycin resistance in one MRSA strain triggered mecA deletion suggesting that this deletion, coupled to other rearrangements and/or mutations, may be responsible for the increased vancomycin resistance phenotype.
Staphylococcus aureus peptidoglycan is cross-linked via a characteristic pentaglycine interpeptide bridge. Genetic analysis had identified three peptidyltransferases, FemA, FemB and FemX, to catalyse the formation of the interpeptide bridge, using glycyl t-RNA as Gly donor. To analyse the pentaglycine bridge formation in vitro, we purified the potential substrates for FemA, FemB and FemX, UDP-MurNAc-pentapeptide, lipid I and lipid II and the staphylococcal t-RNA pool, as well as His-tagged Gly-tRNA-synthetase and His-tagged FemA, FemB and FemX. We found that FemX used lipid II exclusively as acceptor for the first Gly residue. Addition of Gly 2,3 and of Gly 4,5 was catalysed by FemA and FemB, respectively, and both enzymes were specific for lipid II-Gly1 and lipid II-Gly3 as acceptors. None of the FemABX enzymes required the presence of one or two of the other Fem proteins for activity; rather, bridge formation was delayed in the in vitro system when all three enzymes were present. The in vitro assembly system described here will enable detailed analysis of late, membrane-associated steps of S. aureus peptidoglycan biosynthesis.
Glucosamine-6-P occupies a central position between cell wall synthesis and glycolysis. In the initial steps leading to peptidoglycan precursor formation glucosamine-6-P is processed sequentially to UDP-N-acetylglucosamine, while to enter the glycolysis pathway, glucosamine-6-P is isomerized by NagB to fructose-6-P. Although we could not demonstrate NagB activity, nagB inactivation significantly reduced growth. Mutational analysis showed that NagA was involved in glucosamine-6-P formation from N-acetylglucosamine-6-P, and GlmS in that from fructose-6-P. Inactivation of glmS prevented growth on glucose as sole carbon source, which resumed after complementation with N-acetylglucosamine. Transcription of glmS as well as the amount of GlmS was reduced in the presence of N-acetylglucosamine. This and the preferential incorporation of N-acetylglucosamine over glucose into cell wall material showed that N-acetylglucosamine was used exclusively for cell wall synthesis, while glucose served both cell wall synthesis and glycolysis. These observations suggest furthermore GlmS to be the key and only enzyme leading from glucose to cell wall synthesis in Staphylococcus aureus, and show that there exists a tight regulation and hierarchy in sugar utilization. Inactivation of nagA, nagB or glmS affected the susceptibility of S. aureus to cell wall synthesis inhibitors, suggesting an interdependence between efficiency of cell wall precursor formation and resistance levels.
The experimental deletion of the tcaRAB region has been shown to increase teicoplanin resistance in Staphylococcus aureus. By sequential genetic complementation of a tcaRAB mutant, we identified tcaA as the key gene within tcaRAB that is responsible for changes in glycopeptide resistance levels. Northern blot analysis of the tcaRAB region showed that the tcaA gene is expressed only weakly over the growth cycle and is strongly inducible by teicoplanin. Among some clinical isolates tested, glycopeptide-intermediate-resistant (GISA) strains Michigan and SA137/93G were found to have truncated tcaA genes. While the former carries a nucleotide insertion that creates a premature stop codon, the latter was found to harbor an IS256 insertion. Complementation of these two GISA strains with a functional tcaA allele reduced their levels of teicoplanin and vancomycin resistance five- to eightfold and twofold, respectively. The data presented here indicate that inactivation of tcaA contributes to and plays a relevant role in glycopeptide resistance in S. aureus clinical isolates.
Transformation of a type I SCCmec element into Staphylococcus aureus yielded highly oxacillin-resistant transformants with a reduced growth rate. Faster-growing variants could again be selected at the cost of reduced resistance levels, demonstrating an inverse correlation between oxacillin resistance levels and growth rate.
TcaR, which shares sequence homology with MarR-like transcriptional regulators, has been identified as a novel Staphylococcus aureus regulator affecting the expression of the global regulatory element SarS (SarH1), as well as that of the cell surface-associated protein SasF (N315-SA2439). Microarray analysis, confirmatory Northern blots, and genetic complementation experiments showed that TcaR upregulates sarS and thus spa transcription. In addition, it attenuates whole-length transcription of sasF, thereby producing a truncated transcript lacking the 3' terminus, which codes for the cell wall anchor motif. Hence, in strains containing an intact tcaR gene, TcaR is likely to decrease the amount of the surface-associated protein SasF and to increase that of the surface-associated protein A. The widely used laboratory strains derived from NCTC8325 were found to be natural, truncated mutants of tcaR, harboring an inactive TcaR and therefore expressing very low levels of sarS. The data presented here identified TcaR as a further activator of sarS, and a modulator of sasF expression that has to be taken into account in studies of virulence gene expression in S. aureus.
Microarray-based analysis of the transcriptional profiles of the genetically distinct Staphylococcus aureus strains COL, GP268, and Newman indicate that a total of 251 open reading frames (ORFs) are influenced by sigmaB activity. While sigmaB was found to positively control 198 genes by a factor of > or =2 in at least two of the three genetic lineages analyzed, 53 ORFs were repressed in the presence of sigmaB. Gene products that were found to be influenced by sigmaB are putatively involved in all manner of cellular processes, including cell envelope biosynthesis and turnover, intermediary metabolism, and signaling pathways. Most of the genes and/or operons identified as upregulated by sigmaB were preceded by a nucleotide sequence that resembled the sigmaB consensus promoter sequence of Bacillus subtilis. A conspicuous number of virulence-associated genes were identified as regulated by sigmaB activity, with many adhesins upregulated and prominently represented in this group, while transcription of various exoproteins and toxins were repressed. The data presented here suggest that the sigmaB of S. aureus controls a large regulon and is an important modulator of virulence gene expression that is likely to act conversely to RNAIII, the effector molecule of the agr locus. We propose that this alternative transcription factor may be of importance for the invading pathogen to fine-tune its virulence factor production in response to changing host environments.
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Protein-protein interactions play an important role in all cellular processes. The development of two-hybrid systems in yeast and bacteria allows for in vivo assessment of such interactions. Using a recently developed bacterial two-hybrid system, the interactions of the Staphylococcus aureus proteins FemA, FemB and FmhB, members of the FemABX protein family, which is involved in peptidoglycan biosynthesis and beta-lactam resistance of numerous Gram-positive bacteria, were analysed. While FmhB is involved in the addition of glycine 1 of the pentaglycine interpeptide of S. aureus peptidoglycan, FemA and FemB are specific for glycines 2/3 and 4/5, respectively. FemA-FemA, FemA-FemB and FemB-FemB interactions were found, while FmhB exists solely as a monomer. Interactions detected by the bacterial two-hybrid system were confirmed using the glutathione S-transferase-pulldown assay and gel filtration.
A novel membrane-associated protein, MsrR, was identified in Staphylococcus aureus which affects resistance to methicillin and teicoplanin, as well as the synthesis of virulence factors. MsrR belongs to the LytR-CpsA-Psr family of cell envelope-related transcriptional attenuators and was shown to be inducible by cell wall-active agents, such as beta-lactams, glycopeptides, and lysostaphin. The expression of msrR peaked in the early exponential growth phase and decreased sharply thereafter. msrR mutants showed increased sarA transcription and an earlier and higher expression of RNAIII, resulting in altered expression of virulence factors such as alpha-toxin and protein A. These observations suggest that MsrR is a new component involved in sarA attenuation and the regulatory network controlling virulence gene expression in S. aureus.
Individual strains of Staphylococcus aureus have different capacities to become internalized by osteoblasts. Here we report that the levels of sigma(B) expressed by S. aureus correlate with the capacity of this bacterium to be internalized by osteoblasts. However, sigma(B) is not essential for internalization and does not necessarily account for the differences in the capacities of strains to be internalized.