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A Proteolytic Transmembrane Signaling Pathway and Resistance to β-Lactams in Staphylococci

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Science  09 Mar 2001:
Vol. 291, Issue 5510, pp. 1962-1965
DOI: 10.1126/science.1055144

Abstract

β-Lactamase and penicillin-binding protein 2a mediate staphylococcal resistance to β-lactam antibiotics, which are otherwise highly clinically effective. Production of these inducible proteins is regulated by a signal-transducing integral membrane protein and a transcriptional repressor. The signal transducer is a fusion protein with penicillin-binding and zinc metalloprotease domains. The signal for protein expression is transmitted by site-specific proteolytic cleavage of both the transducer, which autoactivates, and the repressor, which is inactivated, unblocking gene transcription. Compounds that disrupt this regulatory pathway could restore the activity of β-lactam antibiotics against drug-resistant strains of staphylococci.

β-Lactam antibiotics are the most effective drugs for the treatment of staphylococcal infections, yet they often cannot be used because many strains are resistant. Resistance is due to production of either β-lactamase or an extra penicillin-binding protein, PBP 2a (1, 2). β-Lactamase, encoded by blaZ, inactivates penicillin by hydrolysis of its β-lactam ring. PBP 2a, encoded by the chromosomal genemecA, in methicillin-resistant strains of staphylococci, confers resistance not only to penicillin, but also to all β-lactam antibiotics. PBP 2a, which is probably a transpeptidase (3), can substitute for other PBPs but, because of its low affinity for binding β-lactams, is unbound at clinically relevant concentrations of antibiotic, allowing cell wall synthesis to continue (4). Although β-lactamase and PBP 2a are genetically and biochemically distinct, both are regulated by similar sensor-transducer and repressor proteins. Their regulatory proteins are homologs of each other and of those controlling expression of inducible β-lactamase in Bacillus licheniformis(5–7). In staphylococci, the genes for the sensor-transducer (blaR1 or mecR for β-lactamase or PBP 2a, respectively) and the DNA binding repressor protein (blaI or mecI) are located immediately upstream of the structural gene (blaZ ormecA) and are transcribed in the opposite direction as a polycistronic message. Repressor binds as a homodimer to palindromic sites within the mec and bla intergenic promoter regions, nucleotide sequences of which are 57% identical, blocking transcription of both structural and regulatory genes (8–10). Either bla or mec regulatory genes can control production of PBP 2a and β-lactamase because of the high degree of homology of the two systems (8, 11), although regulation in clinical isolates is principally by blabecause of deletions or mutations in the mec regulatory genes that weaken repressor activity (12, 13).

β-Lactam binding to the extracellular, penicillin-binding domain of the sensor transducer generates a transmembrane signal that results in the removal of repressor from DNA binding sites, allowing for transcription of both structural and regulatory genes (10,14). The details of this signaling mechanism have been a mystery. Induction of β-lactamase is accompanied by proteolysis of BlaI with conversion of 14-kD BlaI into an ∼11-kD fragment (10). To determine its site of cleavage, we tagged BlaI with the 11–amino acid c-Myc sequence (15–17). Under inducing conditions, identically sized 11-kD BlaI fragments were detected in the Staphylococcus aureus transformants expressing wild-type BlaI or BlaI tagged at its COOH-terminus, indicating a COOH-terminal location of the cleavage site (Fig. 1A). Accordingly, BlaI tagged with c-Myc sequence at the NH2-terminus generated a BlaI fragment that migrated more slowly than that of COOH-terminus–tagged BlaI (Fig. 1B).

Figure 1

Western blot analysis of BlaI in whole cells of S. aureus strain RN4220 transformants, grown under noninducing (–) and inducing (+) conditions (29). (A and B) WT is a transformant with pCH2278 (11, 24), which is a wild-type blalocus cloned into pRN5542 (30). C-Myc indicates the transformant with c-Myc sequence EQKLISEEDLN (17, 19) tagged onto the COOH-terminus of BlaI (15, 16), and N-Myc indicates that c-Myc sequence was tagged onto the NH2-terminus of BlaI. (C) Lanes N101F→AA indicate the transformant with these amino acid substitution mutations (N101A, F102A) introduced into BlaI. (D) Lanes H201→A and E202→A are transformants with these point mutations (H201A and E202A, respectively) introduced into the H201EXXH zinc metalloprotease motif of BlaR1. (E) Lanes R293→A are the transformant containing this substitution mutation (R293A) introduced into BlaR1. For induction of β-lactamase, log-phase organisms were grown in tryptic soy broth with and without CBAP (10 μg/ml). Cells were pelleted by centrifugation, resuspended in lysis buffer [lysostaphin (200 μg/ml), 10 mM MgCl2, deoxyribonuclease (15 μg/ml), ribonuclease (15 μg/ml), and 20 mM tris-Cl (pH 7.6)], and incubated at 37°C for 30 min. Samples were electrophoresed through a 12% SDS–polyacrylamide gel electrophoresis (PAGE) gel. Western blotting was performed with the alkaline phosphatase detection method by using the rat antiserum to BlaI as the primary antibody (31). The antiserum was raised in Sprague-Dawley rats injected with purified GST-BlaI fusion protein.

The specific cleavage site within BlaI was located by engineering a construct where the glutathione S-transferase (GST) gene,gst, was fused in-frame to the COOH-terminus ofblaI (18). The NH2-terminal amino acid sequence of cleaved GST-BlaI fusion product was determined to be FAKNEELNN (19), which localized the site of cleavage between residues N101 and F102 residing within the sequence NH2-KSLVL N101F102AKNEELNN. Substitution mutation of A101A102 for N101F102(N101A, F102A) at this site both prevented proteolysis of BlaI (Fig. 1C) and inducible expression of β-lactamase (20). Thus, site-specific proteolytic cleavage of BlaI near its COOH-terminus is required for reversal of transcriptional repression in this signal transduction pathway.

BlaR1 is a prime candidate for the protease that cleaves BlaI. Deletion or mutation of blaR1 prevents β-lactamase induction and proteolysis of BlaI (10, 11). In addition, a H201EXXH zinc metalloprotease signature motif is present within the predicted 186–amino acid cytoplasmic domain (14,21). The histidine residues of metalloproteases with this signature motif are essential for zinc binding, and the glutamic acid residue is a catalytic base (22). Two site-directed mutations, His201 → Ala201 (H201A) and Glu202 → Ala202 (E202A), were introduced into BlaR1. Either mutation prevented the cleavage of BlaI (Fig. 1D) and induction of β-lactamase.

Zinc metalloproteases typically are autocatalytic proenzymes activated by intramolecular cleavage (23). We have previously reported the presence of an inducible ∼35-kD PBP in transformants of the methicillin-resistant strain COL containing intact blaR1 but not in a transformant in which blaR1 had been disrupted (11, 24), suggesting that the 35-kD PBP was a cleavage product of BlaR1. Cleavage of BlaR1 was confirmed by using antiserum against a synthetic peptide identical in sequence to that within the BlaR1 cytoplasmic domain (Fig. 2A). An inducible 33- to 35-kD peptide, as well as a small amount of a larger protein, corresponding to full-length 66-kD BlaR1 was detected in the transformant COL631.

Figure 2

(A) Western blot of BlaR1 in membranes of strain COL631, a transformant of the methicillin-resistant strain COL into which pCH631 has been introduced (24). Each lane indicates the growth of cells under noninducing (–) or inducing (+) conditions. BlaR1 was detected by a rabbit antiserum, which was prepared in New Zealand rabbits by intradermal immunization with a 14–amino acid synthetic peptide identical to the sequence S281HSFNGKKSLLKRR294of the BlaR1 cytoplasmic domain. Post indicates postimmunization serum, and Pre indicates preimmune serum. Numbers at the left indicate the position of migration of molecular weight markers (in kilodaltons). β-Lactamase induction and immunoblotting was performed as described in Fig. 1. (B) Western blot with monoclonal antibody to His6 in whole-cell lysates of RN4220 transformants, uninduced (–) and induced (+). WT indicates the RN4220 (pCH2278) transformant containing wild-type bla. Lanes 6-His are a transformant with His6 tagged onto the COOH-terminus of BlaR1 (15, 16) and wild-type H201EXXH motif. Lanes 6-His + E202→A are a transformant with His6-tagged BlaR1 plus this substitution mutation in its HEXXH motif. BlaR1 was detected by immunoblotting with monoclonal antibody to His6(Invitrogen).

The specific site at which BlaR1 is cleaved and the participation of the HEXXH motif in its proteolysis were investigated with a pair ofS. aureus strain RN4220 transformants into which a COOH-terminus His6-tagged BlaR1 fusion had been introduced. In one transformant, the zinc metalloprotease motif was wild-type, H201EXXH, and in the other, a E202A substitution mutation had been introduced. An inducible, His6-tagged 33-kD protein (but no 66-kD protein) was detected in the transformant containing intact HEXXH sequence but not in the transformant with the E202A mutation (Fig. 2B). Thus, the zinc metalloprotease signature motif of BlaR1 is not only required for the cleavage of BlaI, but also is required for its own cleavage.

The His6-tagged 33-kD polypeptide was then purified from lysates of whole cells (18). Its NH2-terminal amino acid sequence was determined to be RLINIKEAN, localizing the specific cleavage site within the sequence KKSLIKR293RLINIKEA of the BlaR1 cytoplasmic domain. A transformant containing mutant BlaR1 [Arg293 → Ala293 (R293A)] did not degrade BlaI (Fig. 1E), and β-lactamase was not detectable under noninducing or inducing conditions. Thus, the signal initiating proteolysis of BlaI and release of repression also involves site-specific proteolysis of BlaR1.

To determine whether BlaR1 generation could be autocatalytic or required a staphylococcal co-factor, we cloned blaR1 tagged at its 3′ end with a His6 sequence (to monitor for cleaved gene products) under control of an isopropyl-β-d-thiogalactopyranoside (IPTG)–inducible promoter into a tightly regulated Escherichia coliexpression vector. His6-tagged peptides of ∼19, 31 to 36, and 61 kD were expressed in the presence of IPTG (Fig. 3). The amount of the 31-kD peptide was relatively increased in the presence of the β-lactam inducer, 2-(2′-carboxyphenyl)benzoyl-6-aminopenicillanic acid (CBAP). These results are consistent with autocatalysis of BlaR1 triggered by binding of inducer.

Figure 3

Western blot of His6-tagged BlaR1 peptides purified from E. coli strain BL21(DE3) (Invitrogen). blaR1 with His6 tagging sequence and its intact ribosome binding site was amplified by PCR from theS. aureus RN4220 transformant described in Fig. 2 and cloned downstream of the T7 promoter into pBluescript SK(Stratagene). The T7 promoter with tagged blaR1 was amplified by PCR and recloned into a low-copy-number plasmid, pACYC184 (New England BioLabs, Beverly, Massachusetts), which was used to transform E. coli strain DH5α. The resulting plasmid, p184R6H, was used to transform BL21(DE3). The BL21(DE3) (p184R6H) transformant was grown to an optical density at 600 nm of ∼0.5, and BlaR1 expression was induced with IPTG in the presence or absence of CBAP (10 μg/ml) for 1 hour. Cell lysate equivalent to 10 ml of culture was subjected to metal affinity precipitation by using the Talon Metal beads (Clontech). Beads were boiled for 5 min in SDS sample buffer, and samples were loaded on an SDS-PAGE gel for immunoblotting with monoclonal antibody to His6 (Invitrogen). Numbers at the right indicate the position of migration of molecular weight markers (in kilodaltons). Lanes indicate the presence (+) or absence (–) of IPTG and CBAP in the growth medium. Relative amounts of protein were determined by scanning densitometry.

The sequence of events during induction of β-lactamase appears to be as follows. Bacteria detect β-lactam antibiotic by its binding to the penicillin-binding domain of the sensor protein, BlaR1, only a few copies of which are present in the membrane. Binding promotes rapid autocatalytic cleavage of BlaR1, which appears to be a prometalloprotease. The activated metalloprotease either directly cleaves BlaI or promotes or participates with one or more cofactors in BlaI cleavage to generate an 11-kD fragment containing the DNA binding domain and a 3-kD fragment containing the dimerization domain. Repression is reversed by converting the BlaI monomer to a form that is incapable of dimerizing and binding. Loss of BlaI from its intergenic operator sites allows transcription of blaZ. β-Lactamase is then expressed, resulting in resistance. Because BlaR1 once cleaved can no longer transmit signal, intact BlaR1 must be continually generated in order to detect antibiotic in the environment, which explains why its production is also up-regulated. As the antibiotic concentration decreases, BlaR1 is no longer autoactivated, BlaI is no longer cleaved, and equilibrium shifts back to the intact repressor, which can again dimerize, bind DNA, and turn the system off. The simplest model for signal transduction would involve direct cleavage of BlaI by BlaR1. However, Cohen and Sweeney (25) have reported that a putative third chromosomal regulatory element, blaR2, was also involved in β-lactamase induction. Our results do not rule out the participation of such an additional element or elements.

Regulation by a transmembrane signal transmitted by sequential proteolytic events has not been described in bacteria. The β-lactamase regulatory system differs from two bacterial systems in which protease activity is involved in gene regulation or transmembrane signaling (26–28). The E. coli FtsH membrane metalloprotease is a constitutively expressed enzyme that degrades the heat-shock transcriptional factor σ32, which affects transcription of several genes (27). There is, however, no sensor component of this system. The Bacillus subtilis membrane protease SpoIIGA (28) perhaps most closely resembles β-lactamase gene regulation. SpoIIGA protease activity is increased in the presence of an endogenously produced ligand and converts the transcription factor precursor pro-σE into its active form. However, SpoIIGA activation does not involve proteolysis. Thus, regulation of β-lactam resistance in staphylococci occurs through a specific pathway that may offer potential targets for drug development.

  • * To whom correspondence should be addressed. E-mail: chipc{at}itsa.ucsf.edu

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