Transcriptional regulation of a gonococcal gene encoding a virulence factor (L-lactate permease)

GdhR is a GntR-type regulator of Neisseria gonorrhoeae encoded by a gene (gdhR) belonging to the MtrR regulon, which comprises multiple genes required for antibiotic resistance such as the mtrCDE efflux pump genes. In previous work we showed that loss of gdhR results in enhanced gonococcal fitness in a female mouse model of lower genital tract infection. Here, we used RNA-Seq to perform a transcriptional profiling study to determine the GdhR regulon. GdhR was found to regulate the expression of 2.3% of all the genes in gonococcal strain FA19, of which 39 were activated and 11 were repressed. Within the GdhR regulon we found that lctP, which encodes a unique L-lactate transporter and has been associated with gonococcal pathogenesis, was the highest of GdhR-repressed genes. By using in vitro transcription and DNase I footpriting assays we mapped the lctP transcriptional start site (TSS) and determined that GdhR directly inhibits transcription by binding to an inverted repeat sequence located 9 bases downstream of the lctP TSS. Epistasis analysis revealed that, while loss of lctP increased susceptibility of gonococci to hydrogen peroxide (H2O2) the loss of gdhR enhanced resistance; however, this GdhR-endowed property was reversed in a double gdhR lctP null mutant. We assessed the effect of different carbon sources on lctP expression and found that D-glucose, but not L-lactate or pyruvate, repressed lctP expression within a physiological concentration range but in a GdhR-independent manner. Moreover, we found that adding glucose to the medium enhanced susceptibility of gonococci to hydrogen peroxide. We propose a model for the role of lctP regulation via GdhR and glucose in the pathogenesis of N. gonorrhoeae.

GdhR是淋病奈瑟菌（Neisseria gonorrhoeae）的GntR型转录调节因子（GntR-type regulator），由隶属于MtrR调节子的gdhR基因编码；该调节子包含多种与抗生素耐药相关的基因，例如mtrCDE外排泵基因。既往研究表明，gdhR基因缺失可提升淋病奈瑟菌在雌性小鼠下生殖道感染模型中的定植适合度。本研究采用RNA测序（RNA-Seq）开展转录谱分析研究，以解析GdhR调节子的组成。结果显示，在淋病奈瑟菌菌株FA19的全部基因组基因中，有2.3%的基因受GdhR调控，其中39个基因被激活，11个基因被抑制。在GdhR调节子中，我们发现lctP基因——其编码一种独特的L-乳酸转运蛋白，且已被证实与淋病奈瑟菌致病进程相关——是受GdhR抑制程度最高的基因。通过体外转录实验与DNase I足迹试验，我们定位了lctP的转录起始位点（TSS），并证实GdhR可通过结合位于lctP转录起始位点下游9个碱基处的反向重复序列，直接抑制lctP的转录。上位性分析结果显示：尽管lctP基因缺失会增强淋病奈瑟菌对过氧化氢（H₂O₂）的敏感性，但gdhR基因缺失可提升其耐药性；然而，在gdhR与lctP双基因缺失突变株中，GdhR赋予的这一耐药表型会被完全逆转。我们评估了不同碳源对lctP基因表达的影响，发现在生理浓度范围内，D-葡萄糖（而非L-乳酸或丙酮酸）可通过不依赖GdhR的方式抑制lctP的表达。此外，我们还发现，向培养基中添加葡萄糖会增强淋病奈瑟菌对过氧化氢的敏感性。基于上述实验结果，我们提出了一套模型，用以阐释GdhR与葡萄糖通过调控lctP基因表达，在淋病奈瑟菌致病过程中所发挥的作用。