PBP4 Emerges as a Target for Resistant Staphylococcal Infections

A 2026 review identifies Staphylococcus aureus penicillin-binding protein 4 as a promising but unproven target for tackling antibiotic resistance and recurrent osteomyelitis. PBP4 is a highly conserved, low-molecular-weight transpeptidase that is not required for growth under conventional laboratory conditions, yet it appears to influence both unusual forms of methicillin-resistant S. aureus and the invasion of tiny channels within infected bone. The authors argue that selective inhibitors could eventually serve as adjuvants to existing antibiotics, but emphasize that the evidence remains largely preclinical and that the protein’s biochemical and clinical roles have not yet been fully resolved.

The interest in PBP4 is unusual because it acts on the same cellular construction system targeted by major classes of antibiotics without itself being an essential target. In S. aureus, peptidoglycan is assembled from repeating N-acetylglucosamine and N-acetylmuramic acid units whose stem peptides are crosslinked to strengthen the bacterial cell wall. High-molecular-weight penicillin-binding proteins, or PBPs, perform much of this work. PBP1 supports cell division, PBP2 participates in septal peptidoglycan synthesis, and PBP3 contributes to peripheral cell-wall construction. By contrast, PBP4 is a class C low-molecular-weight enzyme that can crosslink preassembled peptidoglycan and exchange noncanonical D-amino acids at peptide termini. Deleting pbp4 reduces peptidoglycan crosslinking and makes bacterial cells more elastic, consistent with a role in repairing or modifying the wall after initial construction.

Biochemical findings provide evidence that PBP4 directly transpeptidizes peptidoglycan. Purified protein can incorporate D-amino acids into muropeptides even when abundant water is present, a property expected of a transpeptidase rather than a carboxypeptidase. Its sequence contains the SXXK, SXN and KTG motifs characteristic of PBP active sites. Serine 75 is thought to attack the D-Ala-D-Ala terminus of a stem peptide and form an acyl-enzyme intermediate, after which an adjacent peptide or its pentaglycine bridge can become the acceptor. Wild-type cells incorporate fluorescent noncanonical D-amino acids into their walls, whereas pbp4 deletion sharply reduces this activity. Some findings nevertheless leave open the possibility that PBP4 also recruits other enzymes, particularly PBP2, to support crosslinking in intact cells. The proposed catalytic sequence remains largely inferred from sequence conservation, structural analogy and in vitro results rather than established through a full mechanistic analysis.

PBP4 has also emerged in studies of a resistance phenotype that does not depend on the conventional mecA gene. mecA encodes PBP2A, an extra transpeptidase that lets S. aureus continue crosslinking peptidoglycan when the native PBP2 is inhibited by beta-lactams. In a stepwise selection experiment, researchers exposed a mecA-deficient strain to increasing ceftobiprole concentrations. The resulting mutant resisted ceftobiprole, ceftaroline and other beta-lactams without producing PBP2A. It carried a 36-base-pair duplication upstream of pbp4 and two amino-acid substitutions near the enzyme’s predicted active site. Moving the promoter duplication into the parent strain increased PBP4 production 20- to 40-fold and produced a resistant phenotype, while the PBP4 substitutions alone did not do so. These observations associate elevated PBP4 activity with resistance but do not fully establish the molecular mechanism. Possible explanations include altered transpeptidase activity, changes in peptidoglycan remodeling, or sequestration of beta-lactams.

Clinical-isolate studies support the relevance of this route to resistance. Deleting pbp4 produced 16-fold reductions in oxacillin and nafcillin resistance in community-acquired MRSA strains, although the deletion did not have the same effect in the hospital-acquired strains examined. In another survey of 185 mecA-independent MRSA clinical isolates, 8 carried pbp4 mutations, 22 carried gdpP mutations, and 155 carried mutations in both regulatory or coding regions associated with the two genes. Moving pbp4-related mutations into a susceptible background increased resistance to nafcillin, oxacillin and ceftaroline, and combining them with loss of gdpP produced high-level resistance in the reported experiments. The molecular basis for that synergy remains unresolved. A separate structural study found PBP4-mediated hydrolysis of ampicillin and cefotaxime, but whether PBP4 acts as a beta-lactamase during infection is unknown. Together, these findings suggest that mecA-based testing may underestimate the prevalence of beta-lactam-resistant S. aureus.

A second disease-related function involves osteomyelitis, a bone infection that is difficult to eradicate because bacteria can occupy osteocyte lacuno-canalicular networks, or OLCNs. These interconnected channels are roughly 100 to 600 nanometres wide within mineralized bone, whereas a staphylococcal cell is about one micrometre across, meaning the bacterium must deform to enter. Scanning electron microscopy has shown that S. aureus colonizes OLCNs in chronically infected mouse bone and in samples from diabetic patients. Within these spaces, bacteria are shielded from host defenses and antibiotics, creating a plausible reservoir for reinfection. A transposon screen found that pbp4 disruption prevented OLCN invasion in a laboratory canalicular model and in animals. Atomic-force-microscopy studies indicate that PBP4 contributes to cell-wall stiffness, which may help bacteria withstand confinement and maintain wall integrity, although the molecular mechanism connecting PBP4 activity to invasion has not been established.

That link between wall mechanics and invasion suggests a possible dual function for a PBP4 inhibitor. One application would be to weaken a resistance mechanism and restore the activity of beta-lactams such as ceftobiprole or ceftaroline, which inhibit PBP2A and have been used against MRSA. Another would be to prevent bacteria from entering bone channels before establishing a protected reservoir. The review proposes using such a drug as an adjuvant rather than as a stand-alone antibiotic, including during prophylaxis or treatment for severe open tibial fractures. This setting has a relatively high osteomyelitis incidence: a retrospective study of 235 patients with type III open diaphyseal tibial fractures found 16.6% developed osteomyelitis. By contrast, infection following total joint replacement is reported in only 1% to 2% of patients, creating a much larger obstacle to demonstrating a preventive effect. The proposed strategy remains preclinical and has not been tested as a clinical intervention.

Early chemical studies offer support for the concept without establishing a therapy. A high-throughput screen identified six compound classes that reduced or eliminated beta-lactam resistance in cells overexpressing a PBP4 variant. The compounds did not reduce bacterial viability in laboratory culture, bound PBP4 more selectively than other PBPs, and inhibited fluorescent D-alanine incorporation into peptidoglycan and OLCN invasion in vitro. Phenyl urea and benzothiophene inhibitors received particular attention, and structure-guided analogues showed stronger activity than the parent compounds. Some analogues also reversed PBP2A-mediated resistance, indicating that compounds initially considered PBP4-selective may inhibit additional PBPs. Their selectivity therefore requires careful evaluation before therapeutic effects could be attributed specifically to PBP4. These results concern cell-based assays and in vitro invasion models; they do not establish reduced infection, improved outcomes or safety in patients.

Cefoxitin has served as an informative probe because it has greater affinity for PBP4 than for other S. aureus PBPs. At sub-antimicrobial concentrations, it restored beta-lactam susceptibility in PBP4-associated resistant strains and reversed serum-associated daptomycin tolerance linked to cell-wall thickening. Cefoxitin is unsuitable as an ideal selective PBP4 drug, however: it is not orally available, requires frequent intravenous dosing and carries risks including nephrotoxicity, disruption of gut microbiota and selection of resistance. More selective non-antimicrobial inhibitors might avoid some of these liabilities, but targeting a protein that becomes important during resistance or pathogenesis can itself create evolutionary pressure. Reduced drug binding, efflux, altered permeability and other bypass mutations remain plausible routes of resistance and must be assessed experimentally for each compound. The review also notes that the expectation of less resistance to inhibitors of non-essential targets cannot be assumed and should be tested target by target.

The review concludes that PBP4 deserves continued investigation as an antimicrobial target because it links a noncanonical resistance phenotype with bacterial invasion of protected bone niches, potentially allowing one class of compounds to address two clinical problems. Its 99.07% amino-acid sequence identity across surveyed S. aureus genomes suggests that a shared inhibitor might be possible across lineages, while related PBP4 changes in Enterococcus faecalis and Pseudomonas aeruginosa indicate broader relevance. Nevertheless, no PBP4 inhibitor has yet demonstrated safety or efficacy in humans. The immediate path is to clarify the enzyme’s resistance mechanism, optimize compounds for true PBP4 selectivity, test whether resistance emerges, and evaluate adjuvants in populations with a sufficiently high osteomyelitis event rate. Until those steps are completed, PBP4 remains an emerging target with mechanistic promise rather than an established treatment strategy.

Subject of Research: Preclinical development of PBP4 inhibitors for antibiotic resistance and staphylococcal osteomyelitis

Article Title: Penicillin binding protein 4: an emerging Staphylococcus aureus therapeutic target

Article References: Gondil, V. S., Stefaniak, M. A., Melander, C., & Dunman, P. M. (2026). Penicillin binding protein 4: an emerging Staphylococcus aureus therapeutic target. Frontiers in Cellular and Infection Microbiology, 16, 1940058. https://doi.org/10.3389/fcimb.2026.1940058

Image Credits: AI Generated

DOI: 10.3389/fcimb.2026.1940058

Keywords: Staphylococcus aureus, penicillin-binding protein 4, PBP4 inhibitors, antibiotic resistance, MRSA, beta-lactams, osteomyelitis, peptidoglycan, ceftaroline, ceftobiprole, Penicillin, binding