Porphyromonas gingivalis, an oral bacterium associated with periodontal disease, requires haemin for growth. Although several multigenic clusters encoding haemin-uptake systems are present on the genome of P. gingivalis, little is known regarding their transcriptional organization and expression. This study identified a 23 kDa iron-regulated haemin-binding protein encoded by a larger than previously reported variant of hmuY. It was shown that the hmu locus is larger than previously reported and is composed of six genes, hmuYRSTUV, encoding a novel hybrid haemin-uptake system. The locus has an operonic organization and the transcriptional start site is located 292 bp upstream of hmuY. The data indicate that the regulation of the operon is iron-dependent. Interestingly, differential regulation within the operon was demonstrated, resulting in excess of the hmuYR message encoding the outer-membrane proteins when compared to the full-length transcript. In addition, the hmuY transcript is more prevalent than the hmuR transcript. Secondary structure analysis of the hmuYRSTUV mRNA predicted the formation of several potential stem-loops in the 59 ends of hmuR-and hmuS-specific mRNAs, consistent with the differential regulation observed. Finally, it was demonstrated that haemin binding and uptake are elevated in iron-depleted conditions and are reduced 45 % and 70 %, respectively, in an hmu-deficient strain when compared to the parental strain, indicating that the hmu locus plays a major role in haemin acquisition in P. gingivalis. Since homologues of the hmu locus were also found in Bacteroides fragilis, Bacteroides thetaiotaomicron and Prevotella intermedia, these findings may have implications for a better understanding of haemin acquisition in those organisms as well.

Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Cadependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF, vraSR contributes to DAP resistance in the present group of clinical strains. Staphylococcus aureus is the most common Gram-positive pathogen among skin and soft tissue infections (2). Methicillin resistance in S. aureus is mediated by the acquisition of a penicillin-binding protein (PBP), PBP 2a, which has decreased affinity for ␤-lactam antibiotics but can continue to cross-link the cell wall once the native PBPs (i.e., PBPs 1 to 4) have been inactivated (23). A distinctive feature for most methicillin-resistant S. aureus (MRSA) strains is the heterogeneous expression of resistance, characterized by a small proportion (Յ0.1%) of the population expressing a high level of homogeneous resistance while most of the other isolates in the population express resistance to 10 g/ml (12, 15, 43). Daptomycin (DAP) is a cyclic anionic lipopeptide antibiotic that is produced by Streptomyces roseosporus (3) and is approved for treatment of skin and skin structure infections as well as treatment of bacteremia and right-side endocarditis caused by MRSA (1). The mechanism of action involves disruption of cytoplasmic membrane function, resulting in depolarization and cell death due to disruption of critical metabolic functions, such as protein, DNA, and RNA synthesis (2).The incidence of DAP resistance in clinical isolates is very low, and resistant strains display small increases in MIC (2). The exact m...

Although it is estimated that 20-30% of the general human population are carriers of Staphylococcus aureus, this bacterium is one of the most important etiological agents responsible for healthcare-associated infections. The appearance of methicillin resistant S. aureus (MRSA) strains has created serious therapeutical problems. Detailed understanding of the mechanisms of S. aureus infections seems necessary to develop new effective therapies against this pathogen. In this article, we present an overview of the biochemical and genetic mechanisms of pathogenicity of S. aureus strains. Virulence factors, organization of the genome and regulation of expression of genes involved in virulence, and mechanisms leading to methicilin resistance are presented and briefly discussed.

Methicillin-resistant

The present results support the notion that SOS response is mechanistically involved in generating mutations that, in addition to mecA induction, allow the selection of a highly oxacillin-resistant population.

No abstract

The SOS response, a conserved regulatory network in bacteria that is induced in response to DNA damage, has been shown to be associated with the emergence of resistance to antibiotics. Previously, we demonstrated that heterogeneous (HeR) MRSA strains, when exposed to sub-inhibitory concentrations of oxacillin, were able to express a homogeneous high level of resistance (HoR). Moreover, we showed that oxacillin appeared to be the triggering factor of a β-lactam-mediated SOS response through lexA/recA regulators, responsible for an increased mutation rate and selection of a HoR derivative. In this work, we demonstrated, by selectively exposing to β-lactam and non-β-lactam cell wall inhibitors, that PBP1 plays a critical role in SOS-mediated recA activation and HeR-HoR selection. Functional analysis of PBP1 using an inducible PBP1-specific antisense construct showed that PBP1 depletion abolished both β-lactam-induced recA expression/activation and increased mutation rates during HeR/HoR selection. Furthermore, based on the observation that HeR/HoR selection is accompanied by compensatory increases in the expression of PBP1,-2, -2a, and -4, our study provides evidence that a combination of agents simultaneously targeting PBP1 and either PBP2 or PBP2a showed both in-vitro and in-vivo efficacy, thereby representing a therapeutic option for the treatment of highly resistant HoR-MRSA strains. The information gathered from these studies contributes to our understanding of β-lactam-mediated HeR/HoR selection and provides new insights, based on β-lactam synergistic combinations, that mitigate drug resistance for the treatment of MRSA infections.

Methicillin resistance in Staphylococcus aureus is primarily mediated by the acquired penicillin-binding protein PBP 2a, which is encoded by mecA. PBP 2a acts together with native PBP 2 to mediate oxacillin resistance by contributing complementary transpeptidase and transglycosylase activities, respectively. In this study, we have investigated a phenotype of ␤-lactam dependence in a clinical methicillin-resistant S. aureus strain (strain 2884D) obtained by in vitro selection with ceftobiprole. 28884D, which grew very poorly in blood agar, required the presence of the ␤-lactam antibiotics to grow. On the basis of this observation, we hypothesized that a gene or genes essential for growth were dependent on oxacillin induction. Identification and analysis of genes regulated by oxacillin were performed by both real-time reverse transcription-PCR and spotted microarray analysis. We found that mecA was constitutively expressed in strain 2884D and that the constitutive expression resulted from perturbations in the two systems involved in its regulation, i.e., MecI/ MecR1 (staphylococcal chromosome cassette mec type I) and BlaI/BlaR1 (nonfunctional penicillinase operon). PBP 2 appeared to be poorly induced by oxacillin in 2884D. Further analysis of the PBP 2 two-component VraSR regulatory system showed that it was nonfunctional, accounting for the lack of response to oxacillin. Together, these results support the notion that limited PBP 2 availability may have led 2884D to become dependent on oxacillin-mediated mecA induction as a required survival mechanism.Methicillin-resistant Staphylococcus aureus (MRSA) is an increasingly common cause of nosocomial infections and now is also appearing in community populations (25). The therapeutic agents available for the treatment of staphylococcal infections have been limited as a result of acquired resistance to the actions of the most active antimicrobials, especially ␤-lactams. Methicillin resistance in S. aureus is mediated by the acquisition of a new drug-resistant target, a penicillin-binding protein (PBP), PBP 2a, which has a decreased affinity for ␤-lactam antibiotics, but it can continue to cross-link the cell wall once the native PBPs (i.e., PBPs 1 to 4) have been inactivated. PBP 2a is encoded by the mecA gene, which is located on a 21-to 67-kb genomic island called staphylococcal chromosome cassette mec (SCCmec) (18). The transcription of mecA can be regulated by two distinct but related sets of regulatory genes. One of them is mecI/mecR1, which is located upstream of mecA and which is divergently transcribed from mecA. The second set is the homologous regulatory element of staphylococcal penicillinase, blaI/blaR1 (23). MecI and BlaI are repressors that can each repress mecA transcription as well as blaZ transcription (10,23,33). In contrast, MecR1 and BlaR1 are the corresponding sensor transducers which, in contrast to MecI and BlaI, are specific for the cognate repressors and cannot substitute for each other (23). Sensing of the signals of these transmembrane repres...