title: Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression.
abstract: Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronic S. aureus infections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS; thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations in thyA were leading to inactivity of TS proteins, and TS inactivity led to tremendous impact on S. aureus physiology and virulence. Whole DNA microarray analysis of the constructed ΔthyA mutant identified severe alterations compared to the wild type. Important virulence regulators (agr, arlRS, sarA) and major virulence determinants (hla, hlb, sspAB, and geh) were downregulated, while genes important for colonization (fnbA, fnbB, spa, clfB, sdrC, and sdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The ΔthyA mutant was strongly attenuated in virulence models, including a Caenorhabditis elegans killing model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed that thyA activity has a major role for S. aureus virulence and physiology. Importance: Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus carry mutations in the thymidylate synthase (TS) gene (thyA) responsible for de novo synthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistant S. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact on S. aureus virulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.
authors: Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany.
authors: Andre
authors: A
authors: Kriegeskorte
authors: Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany.
authors: Desiree
authors: D
authors: Block
authors: Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany.
authors: Mike
authors: M
authors: Drescher
authors: Institute of Hygiene, University Hospital of Münster, Münster, Germany.
authors: Nadine
authors: N
authors: Windmüller
authors: Institute of Hygiene, University Hospital of Münster, Münster, Germany.
authors: Alexander
authors: A
authors: Mellmann
authors: Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany.
authors: Cathrin
authors: C
authors: Baum
authors: Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany.
authors: Claudia
authors: C
authors: Neumann
authors: Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors: Nicola Ivan
authors: NI
authors: Lorè
authors: Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors: Alessandra
authors: A
authors: Bragonzi
authors: Institute for Animal Physiology, Westfalian Wilhelms-University, Münster, Germany.
authors: Eva
authors: E
authors: Liebau
authors: Institute for Animal Physiology, Westfalian Wilhelms-University, Münster, Germany.
authors: Patrick
authors: P
authors: Hertel
authors: Integrated Functional Genomics, Core Unit of the Interdisciplinary Center for Clinical Research, University Hospital of Münster, Münster, Germany.
authors: Jochen
authors: J
authors: Seggewiss
authors: Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany.
authors: Karsten
authors: K
authors: Becker
authors: Department of Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin, USA.
authors: Richard A
authors: RA
authors: Proctor
authors: Georg
authors: G
authors: Peters
authors: kahl@uni-muenster.de.
authors: Barbara C
authors: BC
authors: Kahl
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D011014:Pneumonia / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D013203:Staphylococcal Infections / Q000235:genetics / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins