Global regulation of Staphylococcus aureus genes by Rot.

Staphylococcus aureus produces a wide array of cell surface and extracellular proteins involved in virulence. Expression of these virulence factors is tightly controlled by numerous regulatory loci, including agr, sar, sigB, sae, and arl, as well as by a number of proteins with homology to SarA. Rot (repressor of toxins), a SarA homologue, was previously identified in a library of transposon-induced mutants created in an agr-negative strain by screening for restored protease and alpha-toxin. To date, all of the SarA homologues have been shown to act as global regulators of virulence genes. Therefore, we investigated the extent of transcriptional regulation of staphylococcal genes by Rot. We compared the transcriptional profile of a rot agr double mutant to that of its agr parental strain by using custom-made Affymetrix GeneChips. Our findings indicate that Rot is not only a repressor but a global regulator with both positive and negative effects on the expression of S. aureus genes. Our data also indicate that Rot and agr have opposing effects on select target genes. These results provide further insight into the role of Rot in the regulatory cascade of S. aureus virulence gene expression.

GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes: direct activation of Cdc42.

Members of the genus Brucella are intracellular alpha-Proteobacteria responsible for brucellosis, a chronic disease of humans and animals. Little is known about Brucella virulence mechanisms, but the abilities of these bacteria to invade and to survive within cells are decisive factors for causing disease. Transmission electron and fluorescence microscopy of infected nonprofessional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry. Cell uptake of B. abortus was negatively affected to various degrees by actin, actin-myosin, and microtubule chemical inhibitors. Modulators of MAPKs and protein-tyrosine kinases hampered Brucella cell internalization. Inactivation of Rho small GTPases using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522, and TcdA significantly reduced the uptake of B. abortus by HeLa cells. In contrast, cytotoxic necrotizing factor from Escherichia coli, known to activate Rho, Rac, and Cdc42 small GTPases, increased the internalization of both virulent and non-virulent B. abortus. Expression of dominant-positive Rho, Rac, and Cdc42 forms in HeLa cells promoted the uptake of B. abortus, whereas expression of dominant-negative forms of these GTPases in HeLa cells hampered Brucella uptake. Cdc42 was activated upon cell contact by virulent B. abortus, but not by a noninvasive isogenic strain, as proven by affinity precipitation of active Rho, Rac, and Cdc42. The polyphasic approach used to discern the molecular events leading to Brucella internalization provides new alternatives for exploring the complexity of the signals required by intracellular pathogens for cell invasion.

Decreased amounts of cell wall-associated protein A and fibronectin-binding proteins in Staphylococcus aureus sarA mutants due to up-regulation of extracellular proteases.

Data have been presented indicating that Staphylococcus aureus cell surface protein can be degraded by extracellular proteases produced by the same bacterium. We have found that in sarA mutant cells, which produce high amounts of four major extracellular proteases (staphylococcal serine protease [V8 protease] [SspA], cysteine protease [SspB], aureolysin [metalloprotease] [Aur], and staphopain [Scp]), the levels of cell-bound fibronectin-binding proteins (FnBPs) and protein A were very low compared to those of wild-type cells, in spite of unaltered or increased transcription of the corresponding genes. Cultivation of sarA mutant cells in the presence of the global protease inhibitor alpha(2)-macroglobulin resulted in a 16-fold increase in cell-bound FnBPs, indicating that extracellular proteases were responsible for the decreased amounts of FnBPs in sarA mutant cells. The protease inhibitor E64 had no effect on the level of FnBPs, indicating that cysteine proteases were not involved. Inactivation of either ssp or aur in the prototype S. aureus strain 8325-4 resulted in a threefold increase in the amount of cell-bound FnBPs. Inactivation of the same protease genes in a sarA mutant of 8325-4 resulted in a 10- to 20-fold increase in cell-bound protein A. As the serine protease requires aureolysin to be activated, it can thus be concluded that the serine protease is the most important protease in the release of cell-bound FnBPs and protein A.

Regulation of virulence determinants in Staphylococcus aureus.

The pathogenicity of Staphylococcus aureus depends on the combined action of more than 40 different extracellular toxins, enzymes and cell surface proteins. A global regulator agr controls the production of many of these virulence factors by a regulating RNA molecule, RNAIII. Most of the virulence genes regulated by RNAIII are also regulated by SarA and a family of homologous proteins. The Sar proteins appear to repress transcription of individual virulence genes or sets of genes. As some Sar proteins also repress one or more sar homologous genes an increased production of a single Sar protein can result in decreased expression of some virulence genes, and an increased expression of others. Results are presented suggesting that RNAIII might function as an antirepressor, binding one or more of the Sar proteins.

In vivo proteolytic degradation of the Escherichia coli acyltransferase HlyC.

Escherichia coli hemolysin (HlyA) is the prototype toxin of a major family of exoproteins produced by Gram-negative bacteria known as "repeats in toxins." Only fatty acid-acylated HlyA molecules at residues Lys564 and Lys690 are able to damage the target cell membrane. Fatty acylation of pro-HlyA is dependent on the co-synthesized acyltransferase HlyC and the acylated form of acyl-carrier protein. By using a collection of hlyA and hlyC mutant strains, the processing of HlyC was investigated. HlyC was not detected by Western blot in an E. coli strain encoding hlyC and hlyA, but it was present in a strain encoding only hlyC. The hlyC mRNA pattern, however, was similar in both strains indicating that the turnover of HlyC does not occur at the transcriptional level. HlyC was detected in Western blots of cell lysates from an E. coli strain encoding HlyC and a HlyA derivative where both acylation sites were substituted. Similar results were obtained when HlyC was expressed in a hlyA mutant strain lacking part of a putative HlyC binding domain, indicating that this particular HlyA region affects HlyC stability. We did not detect HlyC in cell lysates from hlyC mutants with different abilities to acylate pro-HlyA, suggesting that the degradation of HlyC is not related to the HlyA acylation process. The protease systems ClpAP, ClpXP, and FtsH were found to be responsible for the HlyA-dependent processing of HlyC.

Identification and characterization of SarH1, a new global regulator of virulence gene expression in Staphylococcus aureus.

The global regulators agr (accessory gene regulator) and sarA (staphylococcal accessory regulator) have been reported to be both activators and repressors of virulence gene expression in Staphylococcus aureus. How the effector of the agr system, RNAIII, interacts with target gene promoters is unknown. SarA, on the other hand, is a DNA-binding protein, which binds to conserved DNA motifs immediately upstream of both positively and negatively regulated promoters. Here, we searched for additional regulators that could explain the differential effects of RNAIII and SarA. Four differently regulated genes (hla, alpha-toxin; hld, RNAIII; spa, protein A; ssp, serine protease) were analysed for binding of potential regulatory proteins to the corresponding promoter DNA fragments, linked to magnetic beads. One protein (29 kDa), with affinity for all four promoters, showed a high degree of similarity to SarA and was named SarH1 (Sar homologue 1). Expression of sarH1 was strongly repressed by sarA and agr. Analysis of hla, hld, ssp and spa mRNAs in sarH1, sarA and agr mutants, and in sarA/sarH1 and agr/sarH1 double mutants, revealed that sarH1 has a strong repressive effect on hla and an activating effect on spa transcription. SDS-PAGE analysis of secreted proteins from the different mutants showed that the production of several other exoproteins was affected by sarH1. In conclusion, we show that both the agr-dependent suppression of protein A production and the sarA-dependent stimulation of alpha-toxin production is mediated via a new regulator, SarH1, which belongs to a family of Sar homologues.

Regulation of agr-dependent virulence genes in Staphylococcus aureus by RNAIII from coagulase-negative staphylococci.

Many of the genes coding for extracellular toxins, enzymes, and cell surface proteins in Staphylococcus aureus are regulated by a 510-nucleotide (nt) RNA molecule, RNAIII. Transcription of genes encoding secreted toxins and enzymes, including hla (alpha-toxin), saeB (enterotoxin B), tst (toxic shock syndrome toxin 1), and ssp (serine protease), is stimulated, while transcription of genes encoding cell surface proteins, like spa (protein A) and fnb (fibronectin binding proteins), is repressed. Besides being a regulator, RNAIII is also an mRNA coding for staphylococcal delta-lysin. We have identified RNAIII homologs in three different coagulase-negative staphylococci (CoNS), i.e., Staphylococcus epidermidis, Staphylococcus simulans, and Staphylococcus warneri. RNAIII from these CoNS turned out to be very similar to that of S. aureus and contained open reading frames encoding delta-lysin homologs. Though a number of big insertions and/or deletions have occurred, mainly in the 5' half of the molecules, the sequences show a high degree of identity, especially in the first 50 and last 150 nt. The CoNS RNAIII had the ability to completely repress transcription of protein A in an RNAIII-deficient S. aureus mutant and the ability to stimulate transcription of the alpha-toxin and serine protease genes. However, the stimulatory effect was impaired compared to that of S. aureus RNAIII, suggesting that these regulatory functions are independent. By creating S. epidermidis-S. aureus RNAIII hybrids, we could also show that both the 5' and 3' halves of the RNAIII molecule are involved in the transcriptional regulation of alpha-toxin and serine protease mRNAs in S. aureus.

RNase E, the major player in mRNA degradation, is down-regulated in Escherichia coli during a transient growth retardation (diauxic lag).

The endoribonuclease RNase E plays a major part in mRNA degradation in Escherichia coli in addition to its role in processing rRNA. RNase E is encoded by an essential gene, rne, also known as ams and hmp, which is autoregulated post-transcriptionally. Here we report a transient decrease in the steady state level of the full-length rne transcript and a corresponding decline in the amount of the protein and enzymatic activity. During this period an mRNA fragment, lacking an intact 5' end, accumulates. This down-regulation of RNase E occurs under aerobic growth conditions in rich medium during a short diauxic lag in mid-exponential phase; it most likely reflects an exhaustion of a not yet identified medium compound which is followed by switching on a new metabolic pathway. During this lag, the levels of bulk protein are maintained. Our results suggest that a transient drop in the intracellular RNase E level is a means of cells to retard mRNA turnover in a period of adjustment to medium utilization. Furthermore, the here described regulation of the rne transcript and its cognate gene product seems to occur by an RNase E-independent mechanism responsive to changes in growth conditions.

Incomplete activation of Escherichia coli hemolysin (HlyA) due to mutations in the 3' region of hlyC.

Mutational analysis of the carboxy-terminal region of Escherichia coli HlyC was performed by site-directed mutagenesis. Replacement of residue Val-127 or Lys-129 reduced the activity of HlyC to about 30 or 60%, respectively, of that of the wild type, while replacement of Gly-128 reduced the activity to less than 1% of the wild-type level. Complete inactivation of HlyC was caused by a double mutation, replacement of Gly-128 with valine and of Lys-129 with isoleucine. Analysis of culture supernatants from mutants with reduced hemolytic activity by two-dimensional gel electrophoresis revealed the production and simultaneous secretion of nonacylated, monoacylated, and fully acylated HlyA forms, demonstrating impairment of the acylation reaction, possibly due to a decreased affinity of HlyC for the individual HlyA acylation sites.

Transcription of Staphylococcus aureus fibronectin binding protein genes is negatively regulated by agr and an agr-independent mechanism.

The production of cell surface proteins in Staphylococcus aureus is generally down-regulated in the postexponential growth phase by the global regulator agr. The effector of this regulation is the RNAIII molecule, which is encoded within the agr locus. RNAIII seems to regulate most target genes at the level of transcription, but it also has an effect on the translation of some genes. To study the role of agr on the expression of fibronectin binding proteins (FnBPs), we investigated the transcription and translation of fnb genes in agr mutant strain WA250 and its parent strain, 8325-4. The results show that fnb genes are negatively regulated by agr and also by an agr-independent mechanism that restricts fnb mRNA synthesis to the early exponential phase of growth. Transcription and Western blot analysis of cell-associated FnBPs demonstrated that synthesis of both FnBPA and FnBPB in the wild-type and agr mutant strains took place preferentially during the first hour of growth and rapidly decreased after the second hour. We also confirmed previous results showing that the agr mutant strain has an increased capacity to bind fibronectin compared to its parent agr+ strain. However, while the concentrations of fnb mRNAs and proteins differed by a factor of 16 between the strains, the difference in fibronectin binding was only twofold, indicating that the binding of fibronectin to the bacteria is not proportional to the amount of FnBPs on their surface.

Variation in the agr-dependent expression of alpha-toxin and protein A among clinical isolates of Staphylococcus aureus from patients with septicaemia.

In Staphylococcus aureus synthesis of many virulence factors is regulated by the agr locus. The regulatory molecule RNAIII, induced by agr, activates transcription of the alpha-toxin gene, hla, while it acts as a repressor of the protein A gene, spa. Forty clinical strains of S. aureus from human blood cultures were analysed for alpha-toxin and protein A production. An inverse correlation between alpha-toxin and protein A production was found in most strains. The levels of alpha-toxin and protein A production varied significantly among strains, which indicates various levels of the regulator, RNAIII. This was confirmed by selecting strains producing different amounts of alpha-toxin, showing that the variations in toxin production are due to the variations of RNAIII transcript. However, in one of the selected strains which produced high levels of alpha-toxin, OR153, although RNAIII is also strongly expressed, the specific hla mRNA was unexpectedly low. One partial explanation for the high alpha-toxin production by this clinical isolate might be its lack of extracellular proteases.

Detection of the response regulator AgrA in the cytosolic fraction of Staphylococcus aureus by monoclonal antibodies.

The expression of many virulence genes in Staphylococcus aureus is controlled by a regulatory RNA molecule, RNAIII, which is encoded by the agr locus. Transcription RNAIII requires the activity of the agrA, B, C and D genes, which code for components of a quorum sensing signal transduction system. In this report we describe the overexpression and purification of the response regulator, AgrA. Monoclonal antibodies were produced and used to detect AgrA in the cytosolic fraction of S. aureus cells. Purified AgrA did not bind to the RNAIII promoter region in a DNA mobility shift experiment. This confirms previous results obtained with protein extracts from agr+ and agr- cells.

Transcriptional control of the agr-dependent virulence gene regulator, RNAIII, in Staphylococcus aureus.

Many of the genes coding for extracellular toxins, enzymes and cell-surface proteins in Staphylococcus aureus are regulated by a 510 nt RNA molecule, RNAIII. Expression of the RNAIII gene is positively controlled by the closely linked agr operon, which encodes a multicomponent signal-transduction system, and by an unlinked operon called sar. We have analysed the 120 bp region that separates the RNAIII promoter, P3, from the divergent agr promoter, P2. By transcription analysis, it was shown that P3 can function in trans of the agr operon. A stretch of 53 bp upstream of P3, containing an interrupted repeat of 7 bp, was found to be required for the agr-dependent expression of RNAIII. A single cytoplasmic protein was shown to bind to at least two sites within this regulatory region. The protein, which was absent in extracts from a sarA mutant, was identified as the sarA product by N-terminal amino acid sequencing. A DNA fragment from the P2 region, encompassing an almost identical repeated DNA motif, competed for the same protein. No interaction between the regulatory DNA sequence and any agr-dependent products could be demonstrated. The results of this study suggest that P3 and P2 are controlled by a mechanism involving the binding of the SarA protein to multiple sites within the regulatory regions immediately upstream of each promoter, and the as yet unknown activity of AgrA.

Activation of alpha-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII.

The synthesis of virulence factors in Staphylococcus aureus is controlled by a regulatory RNA molecule, RNAIII, encoded by the agr locus. Transcription of genes coding for secreted toxins and enzymes is stimulated, while transcription of cell-surface protein genes is repressed by RNAIII. In the case of staphylococcal alpha-toxin, RNAIII also seems to stimulate translation by an independent mechanism. In this report we show that in a mutant lacking RNAIII the rate of alpha-toxin (hla) production relative to the cellular concentration of hla mRNA was reduced 10-fold as compared with the wild-type strain. A 75% complementarity between the 5' end of RNAIII and the 5' untranslated region of the hla transcript suggests a direct interaction between the RNAs. A complex of RNAIII and hla mRNA was demonstrated in extracts of total RNA from the wild-type strain, and also with in vitro synthesized RNAs. Ribonuclease T1 digestion experiments revealed that the ribosome binding site of the hla transcript is blocked by intramolecular base-pairing. Hybridization with RNAIII prevents this intramolecular base-pairing and makes the hla mRNA accessible for translation initiation. This is, to our knowledge, the first example of an 'antisense RNA' that stimulates translation of the target mRNA.

The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine arthritis model.

We have studied the role of the accessory gene regulator (agr) of Staphylococcus aureus as a virulence determinant in the pathogenesis of septic arthritis. At least 15 genes coding for potential virulence factors in Staphylococcus aureus are regulated by a putative multicomponent signal transduction system encoded by the agr/hld locus. agr and hld mutants show a decreased synthesis of extracellular toxins and enzymes, such as alpha-, beta-, and delta-hemolysin, leucocidin, lipase, hyaluronate lyase, and proteases, and at the same time an increased synthesis of coagulase and protein A as compared with the wild-type counterpart. We have used a recently described murine model of S. aureus-induced arthritis to study the virulence of S. aureus 8325-4 and two agr/hld mutants derived from it. Sixty percent of the mice injected with the wild-type strain developed arthritis, whereas agrA and hld mutants displayed joint involvement in only 10 and 30%, respectively. In addition, 40% of the mice inoculated with the wild-type strain displayed an erosive arthropathy; such changes were not detectable at all in mice inoculated with the agrA mutant. Serum levels of interleukin-6, a potent B-cell differentiation factor, were significantly higher (P < 0.001) in the mice inoculated with the wild-type strain than in those inoculated with the agrA mutant counterpart. Overall, our results suggest that the agr system of S. aureus is an important virulence determinant in the induction and progression of septic arthritis in mice.

Retarded RNA turnover in Escherichia coli: a means of maintaining gene expression during anaerobiosis.

In this study, we extend earlier observations on the influence of growth rate on mRNA stability and rRNA processing in Escherichia coli during continuous culture, to the effect of anaerobiosis. During slow anaerobic growth (generation time 700 min) both ompA and bla mRNA had a prolonged half-life compared to that during slow aerobic growth and the processing of 9S RNA was even more profoundly retarded, which indicated a general slowing of mRNA turnover. The latter was confirmed by a nearly fourfold increase in the functional half-life of bulk mRNA. In spite of this difference in stability, steady state levels of RNA, as judged by those of the ompA and 9S transcripts, were the same in aerobic and anaerobic cells at a given growth rate. Furthermore, we found that RNA synthesis during anaerobiosis was a fraction of that observed during slow aerobic growth and it is proposed that this offsets the general increase in mRNA stability. Our data therefore suggest that a constant level of RNA is maintained by matching the rate of decay to the level of RNA synthesis.

Intraocular levels of cefuroxime in inflamed rabbit eyes.

The pharmacokinetics of cefuroxime was studied in the inflamed rabbit eye employing subconjunctival, intravitreal and combined intravitreal-intravenous routes of administration to study the intraocular levels and the duration of minimum inhibitory concentrations (MIC) of the antibiotic in the vitreous and in the aqueous. A standard inoculum of viable S. aureus was injected into the vitreous of 36 pigmented rabbits to establish experimental endophthalmitis. A biological method was used for the antibiotic assay. Penetration of systemically and subconjunctivally administered-cefuroxime into the inflamed vitreous was poor. Intraocular inflammation increased the clearance of the intravitreally injected cefuroxime. A dose of 75 mg subconjunctivally produced levels in the aqueous far exceeding MIC for over six hours. Penetration of intravitreally injected cefuroxime into the aqueous was poor, inconsistent and short lasting. Following a single intravitreal injection of 1000 micrograms cefuroxime, levels exceeding the MIC for common ocular pathogens persisted in the vitreous for at least 24 hours but supplementation with intravenous cefuroxime neither increased the intraocular levels nor delayed the clearance of the intravitreally injected antibiotic.

Decay of ompA mRNA and processing of 9S RNA are immediately affected by shifts in growth rate, but in opposite manners.

By growing Escherichia coli in continuous cultures at various growth rates, we provide definitive evidence that the stability of the ompA mRNA is growth rate dependent. Shifting fast-growing cells into physiological salt buffer led to an immediately increased rate of ompA mRNA decay and to an instantly decreased rate of 9S RNA conversion into 5S rRNA. Shifting slowly growing cells into fresh medium had the opposite effect for each of the two RNA species. The observed regulatory patterns underline the need of cells to adjust the output of ompA and 9S RNAs in response to growth rate changes. At all growth rates and throughout all shift experiments, the half-life of bla mRNA was constant. A stabilization of the ompA transcript was even observed when slowly growing cells were shifted into fresh medium already containing the transcriptional inhibitor rifampicin. A hybrid bla transcript with the 5' untranslated region from the ompA gene behaved similarly to the wild-type ompA messenger in response to a shift in growth rate. In agreement with this result, we found that the same type of 5' cleavages as have been previously shown to initiate the decay of the ompA transcript seem to be involved in stability regulation. In E. coli the degradation of mRNA has been shown to depend on the ams/rne gene. This gene controls the stability-related cleavages in the ompA transcript, catabolic processes, and the cleavages which process the 9S rRNA into 5S RNA, an anabolic process. We discuss these results with respect to the ams/rne gene and the related nuclease activities that control the ompA and 9S RNA cleavages.

The secretory S complex in Bacillus subtilis is identified as pyruvate dehydrogenase.

We have cloned the operon for the Bacillus subtilis S complex, which has been suggested to be a component of the protein secretion machinery. The S-complex operon was found to encode 4 proteins, which were identified as subunits of pyruvate dehydrogenase (PDH). The Staphylococcus aureus membrane-bound ribosome protein (MBRP) complex has been considered to be a counterpart of the B. subtilis S complex. Here, we sequenced a fragment of the MBRP operon encoding the C-terminal part of E1 beta, the entire E2 and the N-terminal part of the E3 subunit of PDH, thus conclusively confirming the PDH identity of the MBRP complex as well. It appeared unlikely that PDH could be a primary component in protein secretion, thus disproving the previous hypothesis of the role of the S complex. However, attachment of the S complex (PDH) to the membrane and ribosomes may produce a biologically significant interaction.

Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase.

We have cloned the operon coding for the Bacillus subtilis S complex, which has been proposed to be a component in protein secretion machinery. A lambda gt10 library of B. subtilis was screened with antiserum directed against the Staphylococcus aureus membrane-bound ribosome protein complex, which is homologous to the B. subtilis S complex. Two positive overlapping lambda clones were sequenced. The S-complex operon, 5 kilobases in size, was shown to contain four open reading frames and three putative promoters, which are located upstream of the first, the third, and the last gene. The four proteins encoded by the operon are 42, 36, 48, and 50 kilodaltons in size. All of these proteins were recognized by antisera separately raised against each protein of the S. aureus membrane-bound ribosome protein and B. subtilis S complexes, thus verifying the S-complex identity of the lambda clones. Sequence analysis revealed that all four proteins of the B. subtilis S complex are homologous to the four subunits of the human pyruvate dehydrogenase (PDH). Also, the N terminus of the 48-kilodalton protein was found to have 70% amino acid identity with the N-terminal 211 amino acids, determined so far, from the E2 subunit of B. stearothermophilus PDH. Furthermore, chromosomal mapping of the S-complex operon gave a linkage to a marker gene located close to the previously mapped B. subtilis PDH genes. Thus, the S complex is evidently identical to the B. subtilis PDH, which has been shown to contain four subunits with molecular weights very similar to those of the S complex. Therefore, we propose that the S complex is not a primary component of protein secretion.