A role for sigma factor B in the emergence of Staphylococcus aureus small-colony variants and elevated biofilm production resulting from an exposure to aminoglycosides

Abstract

Staphylococcus aureus small-colony variants (SCVs) and biofilms are linked to chronic infections. It is known that the presence of aminoglycoside antibiotics may contribute to the emergence of SCVs and it is thought that molecular mechanisms are involved in the ability of S. aureus to adopt this phenotype. No study has addressed the possible role of the stress- and colonization-related alternative sigma factor B (SigB) in the emergence of SCVs, although a sustained SigB activity was reported in these variants. Here, we demonstrate that SigB is involved in the emergence of SCVs resulting from an exposure to a sub-inhibitory concentration of aminoglycosides. Monitoring of gene expression in an aminoglycoside-treated prototypical strain or in clinical SCVs showed the activation of SigB, whereas the accessory gene regulator (agr) system was not. Furthermore, gentamicin-treated prototypical bacteria and SCVs had an increased ability to form biofilm only in a SigB functional background. The administration of a sub-inhibitory concentration of gentamicin significantly increased the formation of SCVs for a prototypical strain but not for the sigB mutant in a mouse model of S. aureus-induced mastitis. Collectively, our results show that SigB may positively influence the appearance of S. aureus SCVs and the production of biofilm upon aminoglycoside exposure.

Introduction

Staphylococcus aureus is an opportunistic pathogen which can cause multiple types of serious infections with high morbidity rates but also persistent and recurring infections. The ability of S. aureus to cause diverse diseases has been linked to the numerous virulence factors which allow the bacterium to evade host defenses, to adhere to or destruct host tissues and to induce sepsis syndromes [1]. Diverse and complex regulatory networks control the expression of these virulence factors in a time- and environment-dependent manner.

It is thought that the transition from the colonization to a dissemination phase during infection is controlled by the accessory gene regulator (agr) system [2], [3], whereas stress responses and adhesin expression needed for host tissues colonization are positively influenced by the activity of the alternative transcription sigma factor B (SigB) [4], [5], [6], [7]. During the exponential growth of prototypical S. aureus strains in vitro, the transcription of the hld gene which encodes RNAIII, a regulatory RNA, is induced through the agr quorum-sensing dependent two-component system. The agr system impacts on the expression of several exotoxins (e.g., α-hemolysin) and proteolytic enzymes, and represses the expression of cell-surface proteins involved in colonization [2]. On the other hand, SigB plays an important role in regulating genes in response to environmental stresses, as for example, during stationary phase, heat exposure and change in osmotic pressure (for review see [8]). An important consequence of SigB activity is the up-regulation in expression of a multitude of cell-surface proteins such as the fibronectin-binding protein A (FnBPA) and the down-regulation of a variety of exotoxins [4], [9]. The genes regulated by SigB also include another global regulator of virulence, sarA (staphylococcal accessory regulator) [4], [9]. SarA modulates the expression of several virulence factors either by stimulating RNAIII transcription or by pathway(s) independent of the agr system. Overall, the sarA locus affects at least 120 genes in S. aureus, up-regulating the expression of hemolysins as well as the FnBPs [10]. To add further complexity, there is a possible interplay between SigB and agr since mutants in the sigB operon show an altered agr activity [11].

Previous studies have demonstrated that the formation of biofilm by S. aureus can be influenced by agr [12], [13], SigB [12], [13], [14], [15] and SarA [13], [16]. It has been shown that bacterial biofilm may confer protection from host defenses as well as tolerance to at least some antibiotics, biocides and hydrodynamic shear forces [17]. The intrinsic resistance of biofilm-associated bacteria to antibiotics may be explained by a reduced growth rate that alters their susceptibility to antibiotics that target cell wall biosynthesis whereas a reduced oxidative metabolism impedes uptake of aminoglycoside antibiotics. It was estimated that biofilm formation is involved in 65% of nosocomial infections in the United States [18] and most of chronic infections [19], [20]. More particularly, S. aureus intra-mammary infections often lead to chronic and difficult-to-treat bovine mastitis [21], [22], [23], and genomic loci involved in biofilm formation are found in most clinical bovine mastitis strains [24], [25]. Biofilm formation also appears to play a role in lung infections persisting in cystic fibrosis (CF) patients [26].

S. aureus small-colony variants (SCVs) are often described as a sub-population of slow-growing respiratory deficient bacteria that are auxotroph for hemin or menadione with a reduced susceptibility to aminoglycosides [27]. Interestingly, SCVs are frequently isolated from chronic infections [27], [28 ] as in the case of infections of the airways of CF patients [7], [29 ], [30], [31] and infections of the bovine mammary gland during mastitis [30], [32]. Several studies have shown that S. aureus SCVs possess an increased capacity to invade and persist in host cells [7], [29 ], [33], which may help the establishment of chronic infections by offering the bacterium a protection against the immune system and the action of antibiotics [33], [34]. Using SCVs isolated from cystic fibrosis patients, we have previously demonstrated that SigB activity influences the transcription of several virulence factors and is associated with an increased capacity to invade and persist within host cells [7]. In addition, we have also recently shown that CF-isolated SCVs are locked into a colonization state as a result of a sustained SigB activity and expression of genes encoding cell-surface proteins [6], [7]. Interestingly, many biofilm-associated genes are transcribed in SCVs isolated from cystic fibrosis patients [7] and the SCV phenotype was recently associated with an increased in biofilm formation [35].

It has been shown that some environmental factors such as the mammalian intracellular milieu [36] and the presence of aminogycosides such as gentamicin and tobramycin [27], [37], [38], [39], [40] may contribute to the emergence of the SCV phenotype. Noteworthy, aminoglycosides are often used for treatment of infections in CF patients [26] and have also been used to treat bovine mastitis [41], [42]. It is thought that the ability of S. aureus to switch between the SCV and the prototypical wild-type phenotype may involve underlying regulatory and/or genetic mechanisms [38]. Although results from Schaaff et al. [39] suggest that mutations play a role in the development of SCVs, experiments were performed in the S. aureus strain 8325-4, which is a strain characterized by a lower SigB activity resulting from a “natural” deletion in the rsbU locus [43]. Therefore, questions about the possible influence of the alternative sigma factor B on the emergence of the SCV phenotype are still unanswered at this time [27], [39], [44].

The aim of this work was to investigate the possible role played by SigB in the emergence of the SCV phenotype upon an exposure to aminoglycosides. We found that the activity of SigB contributes to the emergence of SCVs resulting from an exposure to aminoglycosides by comparing the quantity of SCVs generated by a prototypical strain and its sigB mutant. While SigB-dependent genes were up-regulated in both SCVs isolated from cystic fibrosis patients and from bovine mastitis, and also in prototypical bacteria exposed in vitro to aminoglycosides, the agr system was not activated. Interestingly, the observed modulations in gene expression were consistent with a SigB-dependent increased in biofilm formation in the clinical SCV isolates as well as in the gentamicin-treated prototypical bacteria. A role for SigB in the emergence of the SCV phenotype in response of prototypical bacteria to an aminoglycoside treatment was also established in vivo in a mouse model of S. aureus-induced mastitis.