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Background: PBP4, a low-molecular-weight PBP in Staphylococcus aureus, is not considered to be a classical mediator of β-lactam resistance. Previous studies carried out by our group with laboratory strains of S. aureus demonstrated the ability of PBP4 to produce β-lactam resistance through mutations associated with the pbp4 promoter and/or gene. Recent studies of β-lactam-resistant clinical isolates of S. aureus have reported similar mutations associated with pbp4., Objectives: To determine if pbp4-associated mutations reported among clinical strains of S. aureus mediate β-lactam resistance., Methods: The pbp4 promoters and genes bearing mutations from clinical isolates were cloned into a heterologous host. Reporter, growth and Bocillin assays were performed to assess their role in β-lactam resistance. X-ray crystallography was used to obtain acyl-enzyme intermediate structures of the WT and mutant PBP4 with nafcillin and cefoxitin., Results: Of the five strains that contained pbp4 promoter mutations, three strains exhibited enhanced expression of PBP4. The R200L mutation in pbp4 resulted in increased survival in the presence of the β-lactams nafcillin and cefoxitin. Further, introduction of either a promoter or a gene mutation into the genome of a WT host increased the ability of the strains to resist the action of β-lactams. The four high-resolution X-ray structures presented demonstrate the binding pose of the β-lactams tested and provide hints for further drug development., Conclusions: Mutations associated with the pbp4 promoter and pbp4 gene altered protein activity and mediated β-lactam resistance among the clinically isolated strains that were studied., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: PBP4 is typically considered unimportant for conferring high-level β-lactam resistance in Staphylococcus aureus. Mutations in PBP4 have been associated with β-lactam non-susceptibility among natural strains of S. aureus. We have previously shown that PBP4 can mediate high-level β-lactam resistance in laboratory-generated strains passaged in β-lactam antibiotics. Mutations in the pbp4 promoter that up-regulate its expression and missense mutations that surround PBP4's active site were detected in high frequencies among passaged strains, suggesting PBP4 plays a key role in resistance. How these mutations participate in PBP4's ability to provide high-level β-lactam resistance is unknown., Objectives: To determine whether enzymatic activity of PBP4 is required for high-level β-lactam resistance and to investigate how the pbp4-associated mutations provide β-lactam resistance., Methods: The catalytic activity of PBP4 was disabled through introduction of a serine to alanine point mutation in its active site (Ser-75→Ala) in a representative and well-studied passaged strain, CRB. pbp4 promoter and missense mutations detected in CRB were reconstituted in a WT strain individually and in combination. β-Lactam resistance of the resultant strains was evaluated by population analysis. Bacterial peptidoglycan composition of the pbp4 mutants was evaluated with and without antibiotic treatment using LC., Results: PBP4 inactivation imparted complete β-lactam susceptibility of CRB. Reconstitution of PBP4 missense mutations alone did not impart β-lactam resistance, but did so in synergism with pbp4 promoter mutation. A similar synergistic interaction of pbp4 mutations was observed in enhanced peptidoglycan cross-linking upon antibiotic treatment., Conclusions: PBP4's activity and overexpression both contribute to high-level β-lactam resistance.

Community-associated (CA) infections with methicillin-resistant Staphylococcus aureus (MRSA) are on a global rise. However, analysis of virulence characteristics has been limited almost exclusively to the US endemic strain USA300. CA-MRSA strains that do not produce Panton-Valentine leukocidin (PVL) have not been investigated on a molecular level. Therefore, we analyzed virulence determinants in a PVL-negative CA-MRSA strain, ST72, from Korea. Genome-wide analysis identified 3 loci that are unique to that strain, but did not affect virulence. In contrast, phenol-soluble modulins (PSMs) and the global virulence regulator Agr strongly affected lysis of neutrophils and erythrocytes, while α-toxin and Agr had a major impact on in vivo virulence. Our findings substantiate the general key roles these factors play in CA-MRSA virulence. However, our analyses also showed noticeable differences to strain USA300, inasmuch as α-toxin emerged as a much more important factor than PSMs in experimental skin infection caused by ST72., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Phenol-soluble modulins (PSMs) are a recently discovered family of amphipathic, alpha-helical peptides that have multiple roles in staphylococcal pathogenesis and contribute to a large extent to the pathogenic success of virulent staphylococci, such as Staphylococcus aureus. PSMs may cause lysis of many human cell types including leukocytes and erythrocytes, stimulate inflammatory responses, and contribute to biofilm development. PSMs appear to have an original role in the commensal lifestyle of staphylococci, where they facilitate growth and spreading on epithelial surfaces. Aggressive, cytolytic PSMs seem to have evolved from that original role and are mainly expressed in highly virulent S. aureus. Here, we will review the biochemistry, genetics, and role of PSMs in the commensal and pathogenic lifestyles of staphylococci, discuss how diversification of PSMs defines the aggressiveness of staphylococcal species, and evaluate potential avenues to target PSMs for drug development against staphylococcal infections., (Published 2014. This article is a US Goverment work and is in the public domain in the USA.)