AUTHOR=Häffner Nicola , Bär Julian , Dengler Haunreiter Vanina , Mairpady Shambat Srikanth , Seidl Kati , Crosby Heidi A. , Horswill Alexander R. , Zinkernagel Annelies S. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.01415

DOI=10.3389/fmicb.2020.01415

ISSN=1664-302X

ABSTRACT=<p><italic>Staphylococcus aureus</italic> 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, <italic>S. aureus</italic> isolated from invasive infections often show a dysfunction in the accessory gene regulator (<italic>agr</italic>), a major virulence regulatory system in <italic>S. aureus</italic>. To assess whether intracellular environment and <italic>agr</italic> 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 <italic>S. aureus</italic> wild type strains and their isogenic <italic>agr</italic> knock out mutants as well as a natural dysfunctional <italic>agr</italic> 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 <italic>S. aureus</italic> strains with an <italic>agr</italic> knock out predominantly resided in a neutral environment, whereas wild type strains and an <italic>agr</italic> complemented strain resided in an acidic environment. <italic>S. aureus agr</italic> 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 <italic>S. aureus</italic> wild type strain 6850, but not in its <italic>agr</italic> mutant indicating a pH dependent formation of SCVs in the wild type strain. The in-depth understanding of the interplay between intracellular persistence, <italic>agr</italic> function and pH should help to identify new therapeutic options facilitating the treatment of chronic <italic>S. aureus</italic> infections in the future.</p>