Effects of Penicillin-Binding Protein 4 Overproduction in<i>Staphylococcus aureus</i>

Henze, Uta; Roos, Martin; Berger‐Bächi, Brigitte

doi:10.1089/mdr.1996.2.193

Cited by 28 publications

(26 citation statements)

References 21 publications

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“…In S. aureus, PBP4 has been shown to catalyze both transpeptidase and carboxypeptidase reactions (19). Consistent with the presence of a transpeptidase activity are studies suggesting that PBP4 is required for the high level of cross-linking within S. aureus peptidoglycan (15,41). Our results are in agreement with this since increased expression of pbpD ( Fig.…”

Section: Discussionsupporting

confidence: 91%

Transcription analysis of the Staphylococcus aureus gene encoding penicillin-binding protein 4

1997

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The high level of cross-linking found in Staphylococcus aureus peptidoglycan is dependent on the low-molecular-weight penicillin-binding protein PBP4. Recently, the PBP4 gene, pbpD, was cloned and shown to be adjacent to and divergently transcribed relative to the putative ABC-type transporter gene, abcA. Disruption of abcA (in strain KB400) was previously shown to result in heightened resistance to several antibiotics known to interact with PBP4, suggesting that the regulation of pbpD is affected by abcA. In this report, this hypothesis was confirmed by use of a Northern (RNA) blot analysis which revealed increased accumulation of pbpD-specific transcripts in KB400 compared to that in the wild-type strain, 8325-4. By using reverse-phase highperformance liquid chromatography to examine the structure of the peptidoglycan, it was demonstrated that the increased expression of pbpD resulted in an increased level of peptidoglycan cross-linking in the staphylococcal cell wall. Promoter fusion studies demonstrated that the abcA mutation caused approximately 7-fold and 100-fold increases in pbpD and abcA promoter activities, respectively. Primer extension experiments revealed that these genes have long, untranslated leader sequences that result in a transcriptional overlap of 80 bp. Interestingly, deletion of a 26-bp region containing an inverted repeat sequence resulted in the loss of expression from both the abcA and the pbpD promoters. These data provide evidence that abcA and pbpD are under the control of a common regulatory mechanism that may involve the transport function of the abcA gene product.Methicillin-sensitive strains of Staphylococcus aureus produce four penicillin-binding proteins (PBPs), 1 to 4, that are involved in the assembly of the cell wall peptidoglycan. Growth studies using cefotaxime and cephalexin, ␤-lactam antibiotics that preferentially interact with PBP2 and PBP3, respectively, have provided insight into the physiological roles of these PBPs (9). Exposure of S. aureus cells to cefotaxime resulted in extrusion of cytoplasm and cell lysis, while exposure to cephalexin resulted in cell enlargement and the cessation of septation (9). Based on these data, it was hypothesized that PBP2 and PBP3 are essential for cell viability and function as the primary peptidoglycan transpeptidase and a septation-associated transpeptidase, respectively (9). PBP4, the only low-molecular-weight PBP produced by S. aureus, has been shown to be involved in secondary cross-linking. Wyke et al. (41) reported that mutants lacking PBP4 contained a hypo-cross-linked cell wall, providing evidence that this protein, although nonessential, plays an important role in the high level of cell wall cross-linking seen in S. aureus. In agreement with this are the studies of Kozarich and Strominger (19) demonstrating that purified PBP4 is unique among the low-molecular-weight PBPs in that it possesses transpeptidase and carboxypeptidase activities.Methicillin-resistant S. aureus produces a fifth PBP, termed PBP2a, that is the prima...

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Section: Discussionsupporting

confidence: 91%

Transcription analysis of the Staphylococcus aureus gene encoding penicillin-binding protein 4

1997

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“…The cephamycin MIC for an MRSA strain was 100 mg liter Ϫ1 , whereas that for its isogenic mutant, which was defective in PBP2, was 3 mg liter Ϫ1 (20). In vitro induction of overexpression of PBP4 in an MSSA strain resulted in methicillin resistance (11). Ceftriaxone, an expanded-spectrum cephalosporin, has also recently been advocated as a presumptive test for MRSA; its performance (95% sensitivity and 97% specificity) (5) was lower than those of cephamycins.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Three Techniques for Detection of Low-Level Methicillin-Resistant Staphylococcus aureus (MRSA): a Disk Diffusion Method with Cefoxitin and Moxalactam, the Vitek 2 System, and the MRSA-Screen Latex Agglutination Test

et al. 2002

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Very-low-level methicillin-resistant Staphylococcus aureus (MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible S. aureus (MSSA). We evaluated the performances of three methods for detection of low-level methicillin resistance: the disk diffusion method using the cephamycin antibiotics cefoxitin and moxalactam, the Vitek 2 system (bioMérieux), and the MRSA-screen test (Denka). Detection of the mecA gene by PCR was considered to be the "gold standard." We also determined the sensitivity of the oxacillin disk diffusion method with 5-and 1-g disks and that of the Oxascreen agar assay with 6 mg of oxacillin liter ؊1 for detection of MRSA. We compared the distributions of MICs of oxacillin and cefoxitin by the E-test (AB Biodisk), and those of moxalactam by dilutions in agar, for MRSA and MSSA isolates. The 152 clinical isolates of S. aureus studied were divided into 69 MSSA (mecA-negative) and 83 MRSA (mecA-positive) isolates, including 63 heterogeneous isolates and 26 class 1 isolates (low-level resistance). The cefoxitin and moxalactam disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm, and moxalactam inhibition zone diameters were <24 mm. The Vitek 2 system and the MRSA-screen test detected 94 and 97.6% of all MRSA isolates, respectively. The sensitivities of the 5-and 1-g oxacillin disks were 95.2 and 96.4%, respectively, whereas that of the Oxascreen agar screen assay was 94%. All of the tests except the 1-g oxacillin disk test were 100% specific. For the class 1 MRSA isolates, the sensitivity of the Vitek 2 test was 92.3%, whereas those of the MRSA-screen test and the disk diffusion method with cefoxitin and moxalactam were 100%. Therefore, the cefoxitin and moxalactam disk diffusion methods were the bestperforming tests for routine detection of all classes of MRSA.

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“…First of all, though B. subtilis encodes a large number of LMW PBPs, the loss of one or more does not adversely affect the cell shape of this organism (25,26). And yet, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae each express only a single DD-carboxypeptidase, and the loss of this LMW PBP does have morphological consequences in these bacteria (12,30). This implies the enzyme also plays a pivotal role in the coccoid bacteria.…”

Section: Discussionmentioning

confidence: 99%

Endopeptidase Penicillin-Binding Proteins 4 and 7 Play Auxiliary Roles in Determining Uniform Morphology ofEscherichia coli

et al. 2004

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The low-molecular-weight (LMW) penicillin-binding protein, PBP 5, plays a dominant role in determining the uniform cell shape of Escherichia coli. However, the physiological functions of six other LMW PBPs are unknown, even though the existence and enzymatic activities of four of these were established three decades ago. By applying fluorescence-activated cell sorting (FACS) to quantify the cellular dimensions of multiple PBP mutants, we found that the endopeptidases PBP 4 and PBP 7 also influence cell shape in concert with PBP 5. This is the first reported biological function for these two proteins. In addition, the combined loss of three DD-carboxypeptidases, PBPs 5 and 6 and DacD, also impaired cell shape. In contrast to previous reports based on visual inspection alone, FACS analysis revealed aberrant morphology in a mutant lacking only PBP 5, a phenotype not shared by any other strain lacking a single LMW PBP. PBP 5 removes the terminal D-alanine from pentapeptide side chains of muropeptide subunits, and pentapeptides act as donors for cross-linking adjacent side chains. As endopeptidases, PBPs 4 and 7 cleave cross-links in the cell wall. Therefore, overall cell shape may be determined by the existence or location of a specific type of peptide cross-link, with PBP 5 activity influencing how many cross-links are made and PBPs 4 and 7 acting as editing enzymes to remove inappropriate cross-links.

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Effects of Penicillin-Binding Protein 4 Overproduction inStaphylococcus aureus

Cited by 28 publications

References 21 publications

Transcription analysis of the Staphylococcus aureus gene encoding penicillin-binding protein 4

Transcription analysis of the Staphylococcus aureus gene encoding penicillin-binding protein 4

Evaluation of Three Techniques for Detection of Low-Level Methicillin-Resistant Staphylococcus aureus (MRSA): a Disk Diffusion Method with Cefoxitin and Moxalactam, the Vitek 2 System, and the MRSA-Screen Latex Agglutination Test

Endopeptidase Penicillin-Binding Proteins 4 and 7 Play Auxiliary Roles in Determining Uniform Morphology ofEscherichia coli

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