Phenotypic heterogeneity and temporal expression of the capsular polysaccharide in Staphylococcus aureus

Abstract

SummaryBacteria respond to ever-changing environments through several adaptive strategies. This includes mechanisms leading to a high degree of phenotypic variability within a genetically homogeneous population. In Staphylococcus aureus, the capsular polysaccharide (CP) protects against phagocytosis, but also impedes adherence to endothelial cells and/or matrix proteins. We analysed the regulation of core biosynthesis genes (capA-P) necessary for CP synthesis using single-cell assays (immunofluorescence and promoter-activity). In persistent human carriers, we found a distinct subpopulation of nasal S. aureus to be CP positive. In vitro, cap expression is also heterogeneous and strongly growth-phase dependent. We asked whether this peculiar expression pattern (earlyOff/lateHeterogen) is orchestrated by the quorum system Agr. We show that the Agr-driven effector molecule RNAIII promotes cap expression largely via inactivation of the repressor Rot. High NaCl, deletion of CodY or Sae also resulted in higher cap expression but did not change the earlyOFF/ lateHeterogen expression pattern. Activity of the quorum system itself is largely homogenous and does not account for the observed heterogeneity of cap expression or the strictly growth phase dependent expression. Our findings are in contrast to the prevailing view that quorum sensing is the main driving force for virulence gene expression when bacterial cell densities increase.

Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster in Staphylococcus aureus

“…and Table ). On exponentially growing bacteria, no CP was detectable, but upon reaching stationary phase approximately 40% of the population became CP‐positive, which is consistent with previous results (George et al , ). With SigB being a regulator of late genes, we first investigated the effect of constitutive sigB expression on CP synthesis during growth.…”

“…Interestingly, CP synthesis was commonly found to be strictly growth phasedependent and detectable only after post-exponential growth phase (Poutrel et al, 1995;Dassy and Fournier, 1996;Pohlmann-Dietze et al, 2000;Cunnion et al, 2001;George et al, 2015). In addition, not all bacteria in a population are CP-positive as revealed by flow cytometry and immunofluorescence (IF) of in vitro-and in vivo-grown bacteria (Poutrel et al, 1997;Pohlmann-Dietze et al, 2000;George et al, 2015;Conlon et al, 2016). As only nonencapsulated cells are able to adhere to endothelial cells (Pohlmann-Dietze et al, 2000), while CP protects bacterial cells from phagocytosis (Karakawa et al, 1988;Thakker et al, 1998;Portoles et al, 2001), it is likely that CP heterogeneity provides better adaptability of the population as a whole.…”

“…In general, P cap activity correlates with CP synthesis, indicating that regulation occurs predominantly at the transcriptional level (Ouyang et al, 1999;Meier et al, 2007;Jansen et al, 2013;Hartmann et al, 2014;George et al, 2015). Yet, the data to explain the molecular mechanisms of cap regulation are puzzling.…”

“…For the quorum sensing system Agr, it was shown that Agr-mediated cap activation occurs via inactivation of the repressor Rot (George et al, 2015). Rot is known to bind to several target genes; however, its binding motif is ill-defined (Killikelly et al, 2015).…”

Stochastic and Differential Activation of σB and PrfA in Listeria monocytogenes at the Single Cell Level under Different Environmental Stress Conditions

“…While stochastic activation following salt stress has been demonstrated previously for σ B (Utratna et al, 2012), our data provide novel insights into PrfA activation. Stochastic regulation of virulence gene expression has been demonstrated in other microbial species, including the cap genes in S. aureus (George et al, 2015), the type three secretion system 1 in Salmonella Typhi (Arnoldini et al, 2014), and flagella synthesis in S. enterica (Stewart and Cookson, 2014). The PrfA operon includes genes that encode secreted factors, e.g., the pore-forming toxin, LLO, which facilitates L. monocytogenes escape from the phagocytic vacuole in host cells, a mechanism that potentially lends itself to labor-sharing.…”