Understanding how bacteria adapt to different environmental conditions is crucial for advancing knowledge regarding pathogenic mechanisms that operate during infection as well as efforts to develop new therapeutic strategies to cure or prevent infections. Here, we investigated the transcriptional response of Neisseria gonorrhoeae, the causative agent of gonorrhea, to L-lactate and glucose, two important carbon sources found in the host environment. Our study revealed extensive transcriptional changes that gonococci make in response to L-lactate, with 37% of the gonococcal transcriptome being regulated, compared to only 9% by glucose. We found that L-lactate induces a transcriptional program that would negatively impact iron transport, potentially limiting the availability of labile iron, which would be important in the face of the multiple hydrogen peroxide attacks encountered by gonococci during its lifecycle. Furthermore, we found that L-lactate-mediated transcriptional response promoted aerobic respiration and dispersal of biofilms, contrasting with an anaerobic condition previously reported to favor biofilm formation. Our findings suggest an intricate interplay between carbon metabolism, iron homeostasis, biofilm formation, and stress response in N. gonorrhoeae, providing insights into its pathogenesis and identifying potential therapeutic targets.IMPORTANCEGonorrhea is a prevalent sexually transmitted infection caused by the human pathogen Neisseria gonorrhoeae, with ca. 82 million cases reported worldwide annually. The rise of antibiotic resistance in N. gonorrhoeae poses a significant public health threat, highlighting the urgent need for alternative treatment strategies. By elucidating how N. gonorrhoeae responds to host-derived carbon sources such as L-lactate and glucose, this study offers insights into the metabolic adaptations crucial for bacterial survival and virulence during infection. Understanding these adaptations provides a foundation for developing novel therapeutic approaches targeting bacterial metabolism, iron homeostasis, and virulence gene expression. Moreover, the findings reported herein regarding biofilm formation and L-lactate transport and metabolism contribute to our understanding of N. gonorrhoeae pathogenesis, offering potential avenues for preventing and treating gonorrhea infections.

Transcriptional regulator MtrR inhibits the expression of the multidrug efflux pump operon mtrCDE in the pathogenic bacterium Neisseria gonorrhoeae. Here, we show that MtrR binds the hormonal steroids progesterone, β-estradiol, and testosterone, which are present at urogenital infection sites, as well as ethinyl estrogen, a component of some hormonal contraceptives. Steroid binding leads to the decreased affinity of MtrR for cognate DNA, increased mtrCDE expression, and enhanced antimicrobial resistance. Furthermore, we solve crystal structures of MtrR bound to each steroid, thus revealing their binding mechanisms and the conformational changes that induce MtrR.

GdhR is a transcriptional repressor of the virulence factor gene lctP, which encodes a unique l-lactate permease that has been linked to pathogenesis of Neisseria gonorrhoeae, and loss of gdhR can confer increased fitness of gonococci in a female mouse model of lower genital tract infection. In this work, we identified a single nucleotide polymorphism (SNP) in gdhR, which is often present in both recent and historical gonococcal clinical strains and results in a proline (P)-to-serine (S) change at amino acid position 6 (P6S) of GdhR. This mutation (gdhR6) was found to reduce GdhR transcriptional repression at lctP in gonococcal strains containing the mutant protein compared to wild-type GdhR. By using purified recombinant proteins and in vitro DNA-binding and cross-linking experiments, we found that gdhR6 impairs the DNA-binding activity of GdhR at lctP without an apparent effect on protein oligomerization. By analyzing a panel of U.S. (from 2017 to 2018) and Danish (1928 to 2013) clinical isolates, we observed a statistical association between gdhR6 and the previously described adenine deletion in the promoter of mtrR (mtrR-P A-del), encoding the repressor (MtrR) of the mtrCDE operon that encodes the MtrCDE multidrug efflux pump that can export antibiotics, host antimicrobials, and biocides. The frequent association of gdhR6 with the mtrR promoter mutation in these clinical isolates suggests that it has persisted in this genetic background to enhance lctP expression, thereby promoting virulence. IMPORTANCE We report the frequent appearance of a novel SNP in the gdhR gene (gdhR6) possessed by Neisseria gonorrhoeae. The resulting amino acid change in the GdhR protein resulted in enhanced expression of a virulence gene (lctP) that has been suggested to promote gonococcal survival during infection. The mutant GdhR protein expressed by gdhR6 had a reduced ability to bind to its target DNA sequence upstream of lctP. Interestingly, gdhR6 was found in clinical gonococcal strains isolated in the United States and Denmark at a high frequency and was frequently associated with a mutation in the promoter of the gene encoding a repressor (MtrR) of both the mtrCDE antimicrobial efflux pump operon and gdhR. Given this frequent association and the known impact of these regulatory mutations, we propose that virulence and antibiotic resistance properties are often phenotypically linked in contemporary gonococcal strains.