Intracellular Environment and agr System Affect Colony Size Heterogeneity of Staphylococcus aureus

Abstract

<i>Staphylococcus aureus</i> causes chronic and relapsing infections, which may be difficult to treat. So-called small colony variants (SCVs) have been associated with chronic infections and their occurrence has been shown to increase under antibiotic pressure, low pH and intracellular localization. In clinics, <i>S. aureus</i> isolated from invasive infections often show a dysfunction in the accessory gene regulator (<i>agr</i>), a major virulence regulatory system in <i>S. aureus</i>. To assess whether intracellular environment and <i>agr</i> function influence SCV formation, an infection model was established using lung epithelial cells and skin fibroblasts. This allowed analyzing intracellular survival and localization of a panel of <i>S. aureus</i> wild type strains and their isogenic <i>agr</i> knock out mutants as well as a natural dysfunctional <i>agr</i> strain by confocal laser scanning microscopy (CLSM). Furthermore, bacterial colonies were quantified after 1, 3, and 5 days of intracellular survival by time-lapse analysis to determine kinetics of colony appearance and SCV formation. Here, we show that <i>S. aureus</i> strains with an <i>agr</i> knock out predominantly resided in a neutral environment, whereas wild type strains and an <i>agr</i> complemented strain resided in an acidic environment. <i>S. aureus agr</i> mutants derived from an intracellular environment showed a higher percentage of SCVs as compared to their corresponding wild type strains. Neutralizing acidic phagolysosomes with chloroquine resulted in a significant reduction of SCVs in <i>S. aureus</i> wild type strain 6850, but not in its <i>agr</i> mutant indicating a pH dependent formation of SCVs in the wild type strain. The in-depth understanding of the interplay between intracellular persistence, <i>agr</i> function and pH should help to identify new therapeutic options facilitating the treatment of chronic <i>S. aureus</i> infections in the future.