The Energy-Coupling Factor Transporter Module EcfAA’T, a Novel Candidate for the Genetic Basis of Fatty Acid-Auxotrophic Small-Colony Variants of Staphylococcus aureus

Abstract

Staphylococcal small-colony variants (SCVs) are invasive and persistent due to their ability to thrive intracellularly and to evade the host immune response. Thus, the course of infections due to this phenotype is often chronic, relapsing, and therapy-refractory. In order to improve treatment of patients suffering from SCV-associated infections, it is of major interest to understand triggers for the development of this phenotype, in particular for strains naturally occurring in clinical settings. Within this study, we comprehensively characterized two different Staphylococcus aureus triplets each consisting of isogenic strains comprising (i) clinically derived SCV phenotypes with auxotrophy for unsaturated fatty acids, (ii) the corresponding wild-types (WTs), and (iii) spontaneous in vitro revertants displaying the normal phenotype (REVs). Comparison of whole genomes revealed that clinical SCV isolates were closely related to their corresponding WTs and REVs showing only seven to eight alterations per genome triplet. However, both SCVs carried a mutation within the energy-coupling factor (ECF) transporter-encoding ecf module (EcfAA’T) resulting in truncated genes. In both cases, these mutations were shown to be naturally restored in the respective REVs. Since ECF transporters are supposed to be essential for optimal bacterial growth, their dysfunction might constitute another mechanism for the formation of naturally occurring SCVs. Another three triplets analyzed revealed neither mutations in the EcfAA’T nor in other FASII-related genes underlining the high diversity of mechanisms leading to the fatty acid-dependent phenotype. This is the first report on the ECF transporter as genetic basis of fatty acid–auxotrophic staphylococcal SCVs.

Novel Research Models for Staphylococcus aureus Small Colony Variants (SCV) Development: Co-pathogenesis and Growth Rate

“…It is known that they form through auxotrophy in elements of the electron transport chain and ATP production (Kahl et al, 2003a;Proctor et al, 2006). These are either single or a combination of auxotrophy in the biosynthesis of menadione, hemin or thymidine (Kahl et al, 2003a;Kohler et al, 2008;Melter and Radojevič, 2010;Maduka-Ezeh et al, 2012;Dean et al, 2014;Horiuchi et al, 2015) CO 2 (Thomas, 1955) and fatty acids (Schleimer et al, 2018). Many SCV isolates have no defined auxotrophism (Edwards, 2012) and various mutations in the electron transport chain that result in SCV (Proctor, 2019) are not observed in clinical isolates.…”

“…The use of genetically stable SCVs (sSCV) has greatly improved our understanding of SCVs (Kriegeskorte et al, 2014a;. Models of sSCV S. aureus include mutants auxotrophic to menadione (Schaaff et al, 2003;Lannergård et al, 2008;Dean et al, 2014;Pader et al, 2014), hemin (Balwit et al, 1994;Von Eiff et al, 1997;Schaaff et al, 2003), thymidine (Balwit et al, 1994;Von Eiff et al, 1997;Besier et al, 2007;Chatterjee et al, 2008;Kriegeskorte et al, 2014a;Kittinger et al, 2019), fatty acids (Bazaid et al, 2018;Schleimer et al, 2018) CO 2 (Thomas, 1955;Gómez-González et al, 2010), chorismite synthesis (precursor for aromatic amino acids and menaquinone biosynthesis) (Zhang et al, 2017), selection in gentamicin (Balwit et al, 1994) and serial passage in mice models immunized against capsular polysaccharide (Tuchscherr et al, 2008).…”

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

“…The small-colony variant (SCV) phenotype represents a slow growing subpopulation of Staphylococcus aureus associated with chronic, persistent, and recurring infections, which are particularly difficult to diagnose and challenging in terms of treatment (Proctor et al, 2006; Vaudaux et al, 2006; Kahl et al, 2016). The colony morphology and physiological characteristics of SCVs differ widely from the WT, not only due to slower growth, pinpoint colonies, reduced or no pigmentation and hemolytic activity, respectively, but also in several biochemical traits such as altered expression of virulence factors, decreased respiration and coagulase activity as well as frequently by auxotrophism for hemin, menadione, thymidine, or fatty acids (Proctor et al, 2006; Seggewiß et al, 2006; Kriegeskorte et al, 2011, 2014; Proctor et al, 2014; Schleimer et al, 2018).…”

“…The identification of the genetic mechanisms leading to the phenotype switch are prerequisite to prevent SCV-related recurrence and chronic infection and to develop new antimicrobials not selecting for SCVs. Hitherto, however, 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). Besides these SCVs triggered by mutational events restricted to one gene locus, SCVs being the consequence of combined mutations in two or more genes were also rarely described (Hammer et al, 2013; Bui and Kidd, 2015; James et al, 2019).…”

“…Moreover, selective disruption of genes essential for growth is not feasible. Aggravating the analysis of clinical SCVs so far, indubitable identification of genes causative for the phenotype switch is only possible for stable SCVs that already underwent mutational adaption and by comparison with a revertant phenotype that spontaneously emerged from the SCV phenotype (Becker et al, 2006; Schleimer et al, 2018). Besides the rarely isolated stable clinical SCVs, in vitro selection for stable SCVs mandatory for WGS as well as for transcriptomics and proteomics can be performed by cultivation in the presence of sublethal concentrations of certain antibiotics (e.g., aminoglycosides) (Balwit et al, 1994) or by serially passaging bacteria in the presence of HeLa cells using modified gentamicin protection assays (McLean et al, 2019).…”