The identification and characterization of novel persistence genes in chlamydia trachomatis

Abstract

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that can infect the eyes, genital tract, and disseminate to lymph nodes in humans. Many C. trachomatis infections are clinically asymptomatic and can become chronic if left untreated. When humans are infected with C. trachomatis, a cytokine that is produced is interferon-gamma (IFN-γ). In vitro, IFN-γ stimulates expression of the host enzyme indoleamine 2,3-dioxygenase. This enzyme converts free intracellular tryptophan to N-formylkynurenine. Tryptophan starvation induces C. trachomatis to enter a viable-but-nonculturable state termed persistence, which has been proposed to play a key role in chronic Chlamydial disease. To circumvent host induced tryptophan depletion, urogenital strains of C. trachomatis encode a functional tryptophan synthase (TS). TS synthesizes tryptophan from indole and serine, allowing Chlamydia to reactivate from persistence. Transcriptomic analysis revealed C. trachomatis differentially regulates hundreds of genes in response to tryptophan starvation. However, genes that mediate entry, survival, and reactivation from persistence remain largely unknown. Using a forward genetic screen, we identified six Susceptible to IFN-γ mediated Persistence (Sip) mutants that have diminished capacities to reactivate from persistence with indole. Mapping the deleterious persistence alleles in three of the Sip mutants revealed that only one of the mutants had a mutation in TS. The two other Sip mutants mapped had mutations in CTL0225, a putative integral membrane protein, and CTL0694, a putative oxidoreductase. Neither of these genes plays a known role in tryptophan synthesis. However, amino acid (AA) competitive inhibition assays suggest that CTL0225 may be involved in the transport of leucine, isoleucine, valine, cysteine, alanine, and serine. Additionally, metabolomics analysis indicates that all free amino acids are depleted in response to IFN-γ, making this amino acid transporter essential during persistence. Taken together we have identified two new chlamydial persistence genes that may play a role in chronic chlamydial disease.