Abstract
Staphylococcus aureus small-colony variants (SCVs) are associated with chronic, persistent, and relapsing courses of infection and are characterized by slow growth combined with other phenotypic and molecular traits. Although certain mechanisms have been described, the genetic basis of clinical SCVs remains often unknown. Hence, we adapted an episomal tool for rapid identification and investigation of putative SCV phenotype-associated genes via antisense gene silencing based on previously described Tnl0-encoded tet-regulatory elements. Targeting the SCV phenotype-inducing enoyl-acyl-carrier-protein reductase gene (fabI), plasmid pSN1-AS‘fabI’ was generated leading to antisense silencing, which was proven by pronounced growth retardation in liquid cultures, phenotype switch on solid medium, and 200-fold increase of antisense ‘fabI’ expression. A crucial role of TetR repression in effective regulation of the system was demonstrated. Based on the use of anhydrotetracycline as effector, an easy-to-handle one-plasmid setup was set that may be applicable to different S. aureus backgrounds and cell culture studies. However, selection of the appropriate antisense fragment of the target gene remains a critical factor for effectiveness of silencing. This inducible gene expression system may help to identify SCV phenotype-inducing genes, which is prerequisite for the development of new antistaphylococcal agents and future alternative strategies to improve treatment of therapy-refractory SCV-related infections by iatrogenically induced phenotypic switch. Moreover, it can be used as controllable phenotype switcher to examine important aspects of SCV biology in cell culture as well as in vivo.
Highlights
The small-colony variant (SCV) phenotype represents a slow growing subpopulation of Staphylococcus aureus associated with chronic, persistent, and recurring infections, which are difficult to diagnose and challenging in terms of treatment (Proctor et al, 2006; Vaudaux et al, 2006; Kahl et al, 2016)
This study focused on the adaption of the expression plasmid pRAB11 (Helle et al, 2011) for the identification of genes potentially triggering the phenotype switch from WT to SCV
Since SCV-caused infections are difficult to treat, it is of major interest to identify and characterize those genes, which are involved in the phenotypic switch from the WT to the SCV phenotype
Summary
The small-colony variant (SCV) phenotype represents a slow growing subpopulation of Staphylococcus aureus associated with chronic, persistent, and recurring infections, which are difficult to diagnose and challenging in terms of treatment (Proctor et al, 2006; Vaudaux et al, 2006; Kahl et al, 2016). The identification of the genetic mechanisms leading to the phenotype switch are prerequisite to prevent SCVrelated recurrence and chronic infection and to develop new antimicrobials not selecting for SCVs. Hitherto, only some phenotype switch-related genes and mechanisms were identified by in vitro generation of knockout mutants (including accC, accD, aroD, cspB, hemA, hemB, menA, menB, menD, plsX, sdhCAB, and thyA) and sequencing approaches (including accC, accD, aroB, aroC, aroD, ecfA, ecfT, fabF, fabI, hemA, hemB, hemC, hemD, hemE, hemG, hemH, menA, menB, menC, menE, menF, relA, stp, and thyA) (von Eiff et al, 1997; Bates et al, 2003; Schaaff et al, 2003; Chatterjee et al, 2008; Lannergård et al, 2008; Duval et al, 2010; Gao et al, 2010; Gaupp et al, 2010; Parsons et al, 2011, 2013, 2014; Köser et al, 2012; Wakeman et al, 2012; Hammer et al, 2013; Dean et al, 2014; Painter et al, 2015; Lin et al, 2016; Cao et al, 2017; Zhang et al, 2017; Bazaid et al, 2018; Giulieri et al, 2018; Schleimer et al, 2018; Vestergaard et al, 2018).
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