Related Experiment Video
Updated: Jun 21, 2025
Development and Assessment of Intracellular Infection Models for Staphylococcus aureus
Published on: January 17, 2025
Clinical and in vitro models identify distinct adaptations enhancing Staphylococcus aureus pathogenesis in human
Dustin R Long1, Elizabeth A Holmes2, Hsin-Yu Lo2
1Division of Critical Care Medicine, Department of Anesthesiology and Pain Medicine, University of Washington School of Medicine, Seattle, Washington, United States of America.
Abstract:
Staphylococcus aureus is a facultative intracellular pathogen of human macrophages, which facilitates chronic infection. The genotypes, pathways, and mutations influencing that phenotype remain incompletely explored. Here, we used two distinct strategies to ascertain S. aureus gene mutations affecting pathogenesis in macrophages. First, we analyzed isolates collected serially from chronic cystic fibrosis (CF) respiratory infections. We found that S. aureus strains evolved greater macrophage invasion capacity during chronic human infection. Bacterial genome-wide association studies (GWAS) identified 127 candidate genes for which mutation was significantly associated with macrophage pathogenesis in vivo. In parallel, we passaged laboratory S. aureus strains in vitro to select for increased infection of human THP-1 derived macrophages, which identified 15 candidate genes by whole-genome sequencing. Functional validation of candidate genes using isogenic transposon mutant knockouts and CRISPR interference (CRISPRi) knockdowns confirmed virulence contributions from 37 of 39 tested genes (95%) implicated by in vivo studies and 7 of 10 genes (70%) ascertained from in vitro selection, with one gene in common to the two strategies. Validated genes included 17 known virulence factors (39%) and 27 newly identified by our study (61%), some encoding functions not previously associated with macrophage pathogenesis. Most genes (80%) positively impacted macrophage invasion when disrupted, consistent with the phenotype readily arising from loss-of-function mutations in vivo. This work reveals genes and mechanisms that contribute to S. aureus infection of macrophages, highlights differences in mutations underlying convergent phenotypes arising from in vivo and in vitro systems, and supports the relevance of S. aureus macrophage pathogenesis during chronic respiratory infection in CF. Additional studies will be needed to illuminate the exact mechanisms by which implicated mutations affect their phenotypes.
Insights
Staphylococcus aureus evolves enhanced macrophage invasion during chronic infections. This study identified key gene mutations contributing to its pathogenesis in macrophages, crucial for understanding persistent respiratory infections.
Area of Science:
- Microbiology
- Infectious Diseases
- Genetics
Background:
- Staphylococcus aureus is a facultative intracellular pathogen.
- Chronic infections, particularly in cystic fibrosis (CF) patients, are facilitated by S. aureus's ability to infect human macrophages.
- The genetic basis for this macrophage pathogenesis is not fully understood.
Purpose of the Study:
- To identify gene mutations in S. aureus that influence its pathogenesis in macrophages.
- To compare genetic adaptations occurring during in vivo (chronic CF infections) and in vitro (laboratory selection) conditions.
Main Methods:
- Analysis of serially collected S. aureus isolates from chronic CF respiratory infections.
- Genome-wide association studies (GWAS) on in vivo isolates.
- In vitro passaging of laboratory strains with human THP-1 derived macrophages.
- Whole-genome sequencing of adapted strains.
- Functional validation using transposon mutants and CRISPR interference (CRISPRi).
Main Results:
- S. aureus strains evolved increased macrophage invasion capacity during chronic human infection.
- GWAS identified 127 candidate genes associated with in vivo macrophage pathogenesis.
- In vitro selection identified 15 candidate genes.
- Functional validation confirmed 95% of in vivo-associated genes and 70% of in vitro-associated genes contribute to virulence.
- 37 validated genes included 17 known virulence factors and 27 newly identified genes.
- 80% of disrupted genes positively impacted macrophage invasion, suggesting loss-of-function mutations.
Conclusions:
- This study reveals novel genes and mechanisms contributing to S. aureus macrophage pathogenesis.
- It highlights differences in genetic adaptations between in vivo and in vitro systems.
- The findings support the relevance of S. aureus macrophage pathogenesis in chronic respiratory infections in CF patients.
