Influence of dTMP on the Phenotypic Appearance and Intracellular Persistence of Staphylococcus aureus

Abstract

Thymidine-dependent small-colony variants (SCVs) of Staphylococcus aureus are frequently associated with persistent and recurrent infections in cystic fibrosis patients. The phenotypic appearance of S. aureus SCVs or normal-colony variants (NCVs) is postulated to be affected by the intracellular amount of dTMP. This hypothesis was proven by metabolic pathway assays revealing altered intracellular dTMP concentrations, followed by investigation of the associated phenotype. Inhibition of the staphylococcal thymidylate synthase, which generated intracellular dTMP from dUMP, using 5-fluorouracil and co-trimoxazole resulted in an SCV phenotype. Inhibition of a nucleoside transporter, which provided the bacterial cell with extracellular thymidine, caused growth inhibition of SCVs. In turn, reversion of SCVs to NCVs was achieved by supplying extracellular dTMP. High-performance liquid chromatography additionally confirmed the intracellular lack of dTMP in SCVs, in contrast to NCVs. Moreover, the dTMP concentration is postulated to influence the intracellular persistence of S. aureus. Cell culture experiments with cystic fibrosis cells revealed that clinical and co-trimoxazole-induced SCVs with a diminished amount of dTMP showed significantly better intracellular persistence than NCVs. In conclusion, these results show that the dTMP concentration plays a key role in both the phenotypic appearance and the intracellular persistence of S. aureus.Staphylococcus aureus is an important human pathogen and causes diseases ranging in severity from minor skin infections to life-threatening conditions, such as endocarditis, pneumonia, and sepsis (15, 16). Small-colony variants (SCVs) are naturally occurring subpopulations of S. aureus with distinctive phenotypic and pathogenic traits (22). In contrast to the normal-colony variant (NCV) of S. aureus, SCVs produce tiny, nonhemolytic colonies that are not pigmented or have minor pigmentation and they have altered expression of virulence genes and auxotrophism for distinct growth factors (12,21,23,28). With regard to pathogenic traits, S. aureus SCVs are associated with persistent, relapsing, and treatment-resistant infections and have been isolated from clinical specimens from patients with osteomyelitis, device-related infections, brain abscesses, soft tissue infections, and cystic fibrosis (CF) lung disease (1,4,14,25,30). The pathogenesis of S. aureus SCV infection is not fully understood; however, it has been shown that clinical SCVs persist longer in eukaryotic cells than corresponding S. aureus strains with the normal phenotype (2, 31). This intracellular survival of S. aureus SCVs is a sophisticated mechanism that probably contributes to pathogenicity insofar as the intracellular location provides a niche for the bacteria, where they are protected against host defenses and antibiotic therapy.With regard to auxotrophism for growth factors, two main groups of S. aureus SCVs are recovered from clinical specimens: (i) SCVs that are dependent on menadione or hemin, indicating defici...

Thymidine-Dependent Staphylococcus aureus Small-Colony Variants: Human Pathogens That Are Relevant Not Only in Cases of Cystic Fibrosis Lung Disease

“…To the best of our knowledge, the observation that a metabolite of thymidine, i.e., dTMP, and not thymidine itself is abundant in human specimens with necrotic cells has not been described so far. This finding is of crucial importance, since proliferating TD-SCVs of S. aureus have been demonstrated to utilize extracellular dTMP as a growth factor in vitro (24).…”

“…While SCVs dependent on hemin or menadione can be isolated from patients with osteomyelitis, persistent soft tissue infection, and device-related infections (15,17,20,21), thymidine-dependent SCVs (TD-SCVs) are well-known in the context of cystic fibrosis (CF) lung disease, especially after prior use of trimethoprimsulfamethoxazole (SXT) (2,7,9). It has been shown recently that random mutations in the thymidylate synthase-encoding thyA gene which lead to an intracellular lack of dTMP are responsible for the formation of the TD-SCV phenotype (1,4,24) and that hypermutability due to a defect DNA mismatch repair system favors the acquisition of these mutations (3). As TD-SCVs are apparently able to use thymidine and/or the metabolite dTMP from the environment, these variants are able to bypass the antibiotic effect of folic acid antagonists and consequently are resistant to SXT (7,13,24).…”

“…It has been shown recently that random mutations in the thymidylate synthase-encoding thyA gene which lead to an intracellular lack of dTMP are responsible for the formation of the TD-SCV phenotype (1,4,24) and that hypermutability due to a defect DNA mismatch repair system favors the acquisition of these mutations (3). As TD-SCVs are apparently able to use thymidine and/or the metabolite dTMP from the environment, these variants are able to bypass the antibiotic effect of folic acid antagonists and consequently are resistant to SXT (7,13,24).…”

Thymidine Auxotrophic Staphylococcus aureus Small-Colony Variant Endocarditis and Left Ventricular Assist Device Infection

“…Thymidine auxotrophs are generally considered resistant to TMP-SMX due to utilization of extracellular deoxythymidine monophosphate, bypassing the pathway targeted by TMP-SMX (7,15,28). The ideal approach to assessing the antimicrobial susceptibility of thymidine-dependent S. aureus SCVs is unclear.…”

“…Best described for S. aureus, the purported mechanisms behind their persistence include antimicrobial resistance (4), enhanced biofilm formation (24,25), and the ability to exist for prolonged periods in the intracellular milieu (2,26,28). Conceivably, purulent secretions containing deteriorating cells, the DNA of which can be degraded by S. aureus DNase, present around the LVAD could result in a supply of thymidine, supporting growth of thymidine-dependent S. aureus (7,15,28). Thymidine auxotrophic S. aureus SCVs have been frequently isolated from the lungs of cystic fibrosis patients and (less frequently) from patients with chronic soft tissue infection, tympanitis, bronchitis, peritonitis, and bacteremia (7) but have not been previously reported in association with endocarditis, pacemaker infection, or LVAD infection.…”