Burnier, J. | Search bioRxiv

bioRxiv2025

Strain-resolved CRISPRi-Seq reveals conserved antibiotic vulnerabilities in Staphylococcus aureus

Staphylococcus aureus remains a major clinical threat due to rising antibiotic resistance and high rates of treatment failure. Deciphering the genetic responses to antibiotic pressure and identifying conserved vulnerabilities are essential steps toward developing broadly effective therapies. Here, we constructed strain-resolved CRISPR interference (CRISPRi) libraries targeting all genes in four clinically relevant S. aureus strains spanning major clonal complexes. CRISPRi-seq screens enabled high-resolution mapping of their fitness landscapes and the definition of a core essentialome representing robust targets for antimicrobial intervention. Exposure of the CRISPRi libraries to four mechanistically distinct antibiotics revealed genome-wide susceptibility profiles, identifying both strain-dependent and conserved susceptibility signatures shaped by the drug mode of action and genetic background. Analysis of these conserved vulnerabilities provided insight into antibiotic-specific stress responses and resistance mechanisms. Among the core determinants of vancomycin vulnerability, we identified several previously uncharacterized genes, including a conserved membrane-associated operon, here designated EsrABC, whose disruption markedly increases vancomycin sensitivity in the four strains. Our study provides a genome-wide atlas of S. aureus fitness and conditional vulnerabilities, fully explorable in the here-developed online AureoBrowse platform (https://aureobrowse.veeninglab.com/), revealing candidates for synergistic therapies and potential therapeutic targets.

microbiology↗

bioRxiv2024

A bacterial cell wall repair and modification system to resist host antibacterial factors

Pathogenic bacteria have acquired the ability to resist antibacterial defense mechanisms of the host. Streptococci are common in animal microbiota and include opportunistic pathogens like Group A Streptococcus (GAS) and Streptococcus pneumoniae (pneumococcus). While the conserved streptococcal S protein has been identified as a key factor in GAS virulence, its exact function is unclear. Here, we show that the pneumococcal S protein is crucial for resisting against host-derived antimicrobials by coordinating cell wall modification and repair. Specifically, we show that S proteins are septally localized through their transmembrane domain and contain an extracellular peptidoglycan (PG) binding LysM domain which is required for its function. Protein-protein and genetic interaction studies demonstrate that the pneumococcal S protein directly interacts with a PG synthase, class A penicillin binding protein PBP1a, and the PG deacetylase PgdA. Single-molecule experiments reveal that the fraction of circumferentially moving PBP1a molecules is reduced in the absence of S protein. Consistent with an impaired PBP1a function, streptococci lacking S protein exhibit increased susceptibility to cell wall targeting antibiotics and altered cell morphologies. PG analysis showed reduced N-deacetylation of glycans in the S. pneumoniae S protein mutant, indicating reduced PgdA activity. We show that pneumococci lacking the S protein cannot persist transient penicillin treatment, are more susceptible to the human antimicrobial peptide LL-37 and to lysozyme, and show decreased virulence in zebrafish and mice. Our data support a model in which S proteins regulate PBP1a activity and play a key role in coordinating PG repair and modification. This cell wall sentinel control system provides defense against host-derived and environmental antimicrobial attack.

microbiology↗