Adaption of an Episomal Antisense Silencing Approach for Investigation of the Phenotype Switch of Staphylococcus aureus Small-Colony Variants

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.

A functional menadione biosynthesis pathway is required for capsule production by Staphylococcus aureus

“…aureus to the small colony variant (SCV) or persister phenotype: fabD and menD . SCVs auxotrophic for fatty acids that are more resistant to FAS-II inhibitors, such as triclosan, are associated with mutations in the fabD and fabI genes [22, 23]. SCVs auxotrophic for menadione are more resistant to aminoglycoside antibiotics, such as gentamicin, and are associated with mutations in the menD gene [15, 16].…”

The two-component regulator WalKR provides an essential link between cell wall homeostasis with DNA replication inStaphylococcus aureus

“…The vector pRAB11(pC194 replicon) 34 was modified by PCR to restore the consensus tetO upstream of the tetR gene (IM1290/IM1291) 97 . As described previously, this reduced the impact of elevated level TetR production and allowed leaky expression of the target gene in the absence of aTc.…”

Status quo of tet regulation in bacteria

“…Schleimer et al . ( 2019 ) reported that a restoration of tetR autoregulation of pRAB11 reduced growth retardation, which was attributed to a decreased abundance of the repressor. The resulting plasmid was employed to induce AS‐RNA directed to the essential gene fabI , a gene investigated in similar studies before (Ji et al ., 2004 ; Stary et al ., 2010 ).…”

“…Six of the promoter variants were analysed and all led to lower expression levels in the induced state and tighter repression in the absence of an inducer. Schleimer et al (2019) reported that a restoration of tetR autoregulation of pRAB11 reduced growth retardation, which was attributed to a decreased abundance of the repressor. The resulting plasmid was employed to induce AS-RNA directed to the essential gene fabI, a gene investigated in similar studies before (Ji et al, 2004;Stary et al, 2010).…”