Recent cases of infections caused by community-acquired methicillin-resistant Staphylococcus aureus(MRSA) (CA-MRSA) strains in healthy individuals have raised concerns worldwide. CA-MRSA strains differ from hospital-acquired MRSAs by virtue of their genomic background and increased virulence in animal models. Here, we show that in two common CA-MRSA isolates, USA300 and MW2 (USA400), a loss of penicillin binding protein 4 (PBP4) is sufficient to cause a 16-fold reduction in oxacillin and nafcillin resistance, thus demonstrating that mecA, encoding PBP2A, is not the sole determinant of methicillin resistance in CA-MRSA. The loss of PBP4 was also found to severely affect the transcription of PBP2 in cells after challenge with oxacillin, thus leading to a significant decrease in peptidoglycan cross-linking. Autolysis, which is commonly associated with the killing mechanism of penicillin and ␤-lactams, does not play a role in the reduced resistance phenotype associated with the loss of PBP4. We also showed that cefoxitin, a semisynthetic ␤-lactam that binds irreversibly to PBP4, is synergistic with oxacillin in killing CA-MRSA strains, including clinical CA-MRSA isolates. Thus, PBP4 represents a major target for drug rediscovery against CA-MRSA, and a combination of cefoxitin and synthetic penicillins may be an effective therapy for CA-MRSA infections.

Staphylococcus aureus RN4220, a cloning intermediate, is sometimes used in virulence, resistance, and metabolic studies. Using whole-genome sequencing, we showed that RN4220 differs from NCTC8325 and contains a number of genetic polymorphisms that affect both virulence and general fitness, implying a need for caution in using this strain for such studies.Staphylococcus aureus is a versatile pathogen that is responsible for the majority of nosocomial and community-acquired infections. One of the biggest challenges in treating staphylococcal infections is that many S. aureus strains have developed resistance against various antibiotics. In contrast to clinical isolates, RN4220 is a commonly used laboratory strain that is characterized by a mutation in the sau1 hsdR gene, making it restriction deficient and hence an ideal intermediate cloning host. RN4220 was originally derived from NCTC8325-4 using UV and chemical mutagenesis (10). NCTC8325-4, in turn, was derived from an early clinical isolate, NCTC8325 (also known as PS47 or RN1), cured of three prophages (16). Indeed, RN4220 is not a suitable candidate for the study of antibiotic resistance, because newer methicillin-resistant S. aureus (MRSA) lineages have evolved [e.g., ST239 (hospital), ST80, and ST59 (community)], presumably due to recombination events between lineages. RN4220 is also known to harbor a small deletion in rsbU, a gene within the stress-induced sigB operon, which renders it deficient in B expression. Additionally, RN4220 shows a ⌬agr mutant phenotype and does not produce ␣-hemolysin despite producing small amounts of RNAIII in late log phase (24). Recent studies revealed that a deletion of cvfB (encoding conserved virulence factor B) in RN4220 resulted in diminished agr expression, with a reduction in hla expression, protease production, and virulence, in a silkworm model of systemic infection (14), but the loss of hemolytic activity in the cvfB mutant of RN4220 was found to be due to a defective agr locus and not attributable to the cvfB mutation (14). In another study, SrrAB, a two-component regulatory system, was found to repress the transcription of RNAIII of the agr locus in RN4220 (20,27). However, the interpretation of virulence and of the associated regulatory data in these studies with RN4220 is suspect due to an inherent agr defect in this strain (24). Finally, O'Neill showed by comparative sequencing that NCTC8325-4, which was thought to be identical to its parent NCTC8325 except for the deletion of three prophages, possesses previously undescribed polymorphisms that may influence the virulence and pathogenicity of NCTC8325-4 (18). As a result of these issues, it is extremely important to delineate an accurate picture of the mutations in RN4220, given the polymorphisms in this strain which can have an impact upon virulence and resistance phenotype.Using Illumina Solexa-based whole-genome sequencing (paired end) (P. Mayer, L. Farinelli, and E. Kawashima, U.S. patent application WO98/44151), we obtained the whole-genome sequence of st...

The activation of type I IFN signaling is a major component of host defense against viral infection, but it is not typically associated with immune responses to extracellular bacterial pathogens. Using mouse and human airway epithelial cells, we have demonstrated that Staphylococcus aureus activates type I IFN signaling, which contributes to its virulence as a respiratory pathogen. This response was dependent on the expression of protein A and, more specifically, the Xr domain, a short sequence-repeat region encoded by DNA that consists of repeated 24-bp sequences that are the basis of an internationally used epidemiological typing scheme. Protein A was endocytosed by airway epithelial cells and subsequently induced IFN-β expression, JAK-STAT signaling, and IL-6 production. Mice lacking IFN-α/β receptor 1 (IFNAR-deficient mice), which are incapable of responding to type I IFNs, were substantially protected against lethal S. aureus pneumonia compared with wild-type control mice. The profound immunological consequences of IFN-β signaling, particularly in the lung, may help to explain the conservation of multiple copies of the Xr domain of protein A in S. aureus strains and the importance of protein A as a virulence factor in the pathogenesis of staphylococcal pneumonia.

SummaryThe ZmpC zinc metalloproteinase of Streptococcus pneumoniae , annotated in the type 4 genome as SP0071, was found to cleave human matrix metalloproteinase 9 (MMP-9). The previously described IgA protease activity was confirmed to be specifically linked to the IgA1-protease/SP1154 zinc metalloproteinase. MMP-9 is a protease cleaving extracellular matrix gelatin and collagen and is activated by proteolytic cleavage like most proteases. MMP-9 is a human protease and is involved in a variety of physiological and pathological matrix degrading processes, including tissue invasion of metastases and opening of the blood-brain barrier. While TIGR4 (serotype 4) and G54 (serotype 19) pneumococcal genome strains have a highly conserved copy of zmp C, the genome of R6 (a derivative of serotype 2 D39 strain) lacks zmp C. Both the analysis for zmp C presence and MMP-9 cleavage activity in various pneumococcal strains showed correlation of ZmpC with MMP-9 cleavage activity. When assaying clinical isolates of S. pneumoniae , the zmpC gene was not found in any of the nasal and conjunctival swab isolates, but it was present in 1 out of 13 meningitis isolates and in 6 out of 11 pneumonia isolates. In a murine pneumonia model, infection with a zmpC -mutant reduced mortality at 3-4 days post-infection by 75%, when compared with infection with wild-type strains. These data indicate that the ZmpC pneumococcal protease may play a role in pneumococcal virulence and pathogenicity in the lung.

The role of chromosomally encoded toxin-antitoxin (TA) loci in bacterial physiology has been under debate, with the toxin proposed as either an inducer of bacteriostasis or a mediator of programmed cell death (PCD). We report here that ectopic expression of MazF Sa , a toxin of the TA module from Staphylococcus aureus, led to a rapid decrease in CFU counts but most cells remained viable as determined by differential Syto 9 and propidium iodide staining after MazF Sa induction. This finding suggested that the toxin MazF Sa induced cell stasis rather than cell death. We also showed that MazF Sa selectively cleaves cellular mRNAs in vivo, avoiding "important" transcripts such as recA, gyrB, and sarA mRNAs in MazF Sa -induced cells, while these three mRNAs can be cleaved in vitro. The results of Northwestern blotting showed that both sarA and recA mRNAs bind strongly to a putative RNA-binding protein. These data suggest that S. aureus likely undergoes stasis by protecting selective mRNA with RNA-binding proteins upon the expression of MazF Sa in vivo.Many bacteria have chromosomally encoded toxin-antitoxin (TA) loci in which the toxin and antitoxin genes exist in an operon and are coexpressed to form a TA complex. The toxin is stable, while the antitoxin is labile and can be degraded in vivo by host proteases (e.g., ClpP or Lon in Escherichia coli). Under conditions of stress whereby transcription of the TA operon is repressed and which hence preclude the continuous synthesis of the labile antitoxin, the more-stable toxin can unleash its toxic effect to inhibit cell growth. However, metabolic stresses, such as amino acid and carbon source starvation, have been shown to induce transcription of E. coli mazEF and other TA loci in E. coli (7,9,14). Studies with several toxin systems indicate that many toxins are probably sequence-specific endoribonucleases. For instance, MazF of E. coli cleaves mRNA at ACA sites both in vitro and in vivo (30), while the RelE toxin, also from E. coli, cleaves mRNA positioned at the ribosomal A site both in vitro and in vivo, with cleavage occurring between the second and third bases of the A site codon