Artificial Selection for Pathogenicity Mutations in Staphylococcus aureus Identifies Novel Factors Relevant to Chronic Infection

Abstract

Adaptation of Staphylococcus aureus to host microenvironments during chronic infection involves spontaneous mutations, yet changes underlying adaptive phenotypes remain incompletely explored. Here, we employed artificial selection and whole-genome sequencing to better characterize spontaneous chromosomal mutations that alter two pathogenicity phenotypes relevant to chronic infection in S. aureus: intracellular invasiveness and intracellular cytotoxicity. We identified 23 genes whose alteration coincided with enhanced virulence, 11 that were previously known and 12 (52%) that had no previously described role in S. aureus pathogenicity. Using precision genome editing, transposon mutants, and gene complementation, we empirically assessed the contributions of individual genes to the two virulence phenotypes. We functionally validated 14 of 21 genes tested as measurably influencing invasion and/or cytotoxicity, including 8 newly implicated by this study. We identified inactivating mutations (murA, ndhC, and a hypothetical membrane protein) and gain-of-function mutations (aroE Thr182Ile, yhcF Thr74Ile, and Asp486Glu in a hypothetical peptidase) in previously unrecognized S. aureus virulence genes that enhance pathogenesis when introduced into a clean genetic background, as well as a novel activating mutation in the known virulence regulator gene saeS (Ala106Thr). Investigation of potentially epistatic interactions identified a tufA mutation (Ala271Val) that enhances virulence only in the context of purine operon repressor gene (purR) inactivation. This project reveals a functionally diverse range of genes affected by gain- or loss-of-function mutations that contribute to S. aureus adaptive virulence phenotypes. More generally, the work establishes artificial selection as a means to determine the genetic mechanisms underlying complex bacterial phenotypes relevant to adaptation during infection.

Comparative Transcriptome Analysis Reveals Differentially Expressed Genes Related to Antimicrobial Properties of Lysostaphin in Staphylococcus aureus

“…These differences between mutant 1801_2010, WT Newman_2010, and Newman_WT strains in Figure 1 indicate these genetic changes are potentially responsible for phenotypic variation. However, a phenotypic trait caused by genetic sources of variation could include additive variance, dominant variance, environmental variance (e.g., organismal adaptation), and epistatic variance [ 30 , 31 ].…”

Variable Release of Lipoteichoic Acid From Staphylococcus aureus Bloodstream Isolates Relates to Distinct Clinical Phenotypes, Strain Background, and Antibiotic Exposure

“…LTA biosynthesis is a highly conserved function in Gram-positive organisms. Point mutations causing amino acid variants can possibly alter protein production, protein-protein interactions, protein structure, and/or protein function ( McLean et al, 2019 ). Previous studies have similarly identified genetic differences in LTA biosynthesis leading to structural changes in Streptococcus suis strains, which may have implications for influencing the host immune response, though this has not been extensively studied in S. aureus clinical strains ( Gisch et al, 2018 ).…”

Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants

“…Aggravating the analysis of clinical SCVs so far, indubitable identification of genes causative for the phenotype switch is only possible for stable SCVs that already underwent mutational adaption and by comparison with a revertant phenotype that spontaneously emerged from the SCV phenotype (Becker et al, 2006; Schleimer et al, 2018). Besides the rarely isolated stable clinical SCVs, in vitro selection for stable SCVs mandatory for WGS as well as for transcriptomics and proteomics can be performed by cultivation in the presence of sublethal concentrations of certain antibiotics (e.g., aminoglycosides) (Balwit et al, 1994) or by serially passaging bacteria in the presence of HeLa cells using modified gentamicin protection assays (McLean et al, 2019). By using these methods, however, the SCV-selective mutations only occur in the corresponding drug target genes or genes essential for antibiotic uptake (Schaaff et al, 2003; McLean et al, 2019).…”

“…Besides the rarely isolated stable clinical SCVs, in vitro selection for stable SCVs mandatory for WGS as well as for transcriptomics and proteomics can be performed by cultivation in the presence of sublethal concentrations of certain antibiotics (e.g., aminoglycosides) (Balwit et al, 1994) or by serially passaging bacteria in the presence of HeLa cells using modified gentamicin protection assays (McLean et al, 2019). By using these methods, however, the SCV-selective mutations only occur in the corresponding drug target genes or genes essential for antibiotic uptake (Schaaff et al, 2003; McLean et al, 2019). Further approaches selecting for a stable SCV phenotype include a chemostat to generate steady-state growth conditions with low nutrients and low growth rate for a prolonged time and with specific chemical stress (Bui and Kidd, 2015; Bui et al, 2015).…”