Structural and kinetic analyses of penicillin-binding protein 4 (PBP4)-mediated antibiotic resistance in Staphylococcus aureus

Abstract

Edited by Chris Whitfield Methicillin-resistant Staphylococcus aureus (MRSA) causes serious community-acquired and nosocomial infections worldwide. MRSA strains are resistant to a variety of antibiotics, including the classic penicillin and cephalosporin classes of ␤-lactams, making them intractable to treatment. Although ␤-lactam resistance in MRSA has been ascribed to the acquisition and activity of penicillin-binding protein 2a (PBP2a, encoded by mecA), it has recently been observed that resistance can also be mediated by penicillin-binding protein 4 (PBP4). Previously, we have shown that broad-spectrum ␤-lactam resistance can arise following serial passaging of a mecA-negative COL strain of S. aureus, creating the CRB strain. This strain has two missense mutations in pbp4 and a mutation in the pbp4 promoter, both of which play an instrumental role in ␤-lactam resistance. To better understand PBP4's role in resistance, here we have characterized its kinetics and structure with clinically relevant ␤-lactam antibiotics. We present the first crystallographic PBP4 structures of apo and acyl-enzyme intermediate forms complexed with three late-generation ␤-lactam antibiotics: ceftobiprole, ceftaroline, and nafcillin. In parallel, we characterized the structural and kinetic effects of the PBP4 mutations present in the CRB strain. Localized within the transpeptidase active-site cleft, the two substitutions appear to have different effects depending on the drug. With ceftobiprole, the missense mutations impaired the K m value 150-fold, decreasing the proportion of inhibited PBP4. However, ceftaroline resistance appeared to be mediated by other factors, possibly including mutation of the pbp4 promoter. Our findings provide evidence that S. aureus CRB has at least two PBP4-mediated resistance mechanisms.

Identification of Penicillin Binding Protein 4 (PBP4) as a critical factor for Staphylococcus aureus bone invasion during osteomyelitis in mice

“…It can be assumed that pbp4 expression is tightly controlled, as it is dynamically tuned in response to local antibiotic concentrations [49]. Recently, studies have shown that PBP4 may play an important role in S. aureus resistance to β-lactam antibiotics [40,[50][51][52][53]. Therefore, other factors contributing to the regulation of pbp4 activity should be investigated further, including wall teichoic acids, shown to act as spatial and temporal regulations of PBP4 mediated peptidoglycan crosslinking [54], and PBP2, which has been shown to interact with PBP4 during cell wall synthesis [55].…”

Constructing and deconstructing the bacterial cell wall

“…Presumably in order to accommodate the added steric demand of this collaboration, a new, longer amino terminus for acyl‐enzyme acceptance is created by addition of a (Gly) 5 pentapeptide to the ε‐amine of the lysine . Moreover, yet additional crosslinking activity is provided by a second transpeptidase (PBP4) either as a result of resistance mutation and/or increased expression . Hence, S. aureus has two primary resistance mechanisms against β‐lactam antibiotics: the plasmid‐encoded BlaZ β‐lactamase (useful against early‐generation β‐lactams) and PBP2a (useful against all but the most recent generation of β‐lactams, such as ceftaroline and ceftobiprole).…”

“…[149][150][151][152] Moreover, yet additional crosslinking activity is provided by a second transpeptidase (PBP4) either as a result of resistance mutation and/or increased expression. [153][154][155][156][157][158] Hence, S. aureus has two primary resistance mechanisms against β-lactam antibiotics: the plasmid-encoded BlaZ β-lactamase (useful against early-generation β-lactams) and PBP2a (useful against all but the most recent generation of β-lactams, such as ceftaroline and ceftobiprole). As noted previously, both resistance mechanisms are activated only when S. aureus detects the presence of a β-lactam.…”

“…As noted above PBP2a complements a second S. aureus PBP, PBP2. This fully functional pair is abetted by a third PBP (PBP4, by a mechanism not yet fully deciphered) and by “auxiliary enhancing factors,” such as the addition of the (Gly) 5 extension to the amine on the peptide stem customarily used for the crosslinking of the peptidoglycan. The subtle basis for the irrefutable ability of PBP2a to discriminate in favor of its peptidoglycan substrate and against β‐lactam inactivators emerged from analysis of the crystal structure of the soluble enzyme (alone, and following acylation of its serine by various β‐lactams) .…”

Genetic Profiling of Methicillin-resistant Staphylococcus aureus in An African Hospital by Multiplex-PCR

“…The hallmark of Methicillin resistant Staphylococcus aureus (MRSA) is the proliferation of antimicrobial resistance and MRSA nasal carriage often precedes infection [1]. The feat of this bacterium as a pathogen is directly linked to its range of virulence and immune evasion factors that enable the bacterium to dodge host defenses [2].…”