Structure-Function Studies on the Neisseria gonorrhoeae Multi-Drug Resistance Regulator, MtrR

Abstract

Gonorrhea, caused by the Gram-negative bacterium, Neisseria gonorrhoeae, has been identified as an urgent public health threat due to the rise of multidrug resistance. One common mechanism by which bacteria gain resistance to antibiotics is the overexpression of efflux systems that can export multiple antibiotics/drugs from the bacterial cell. Before the use of antibiotics, bacteria utilized efflux pumps as a defense mechanism against innate host antimicrobials. Over time, these pumps adapted to recognize clinical antibiotics, leading to the development of multidrug resistance. Transcriptional regulation plays a crucial role in maintaining optimal cell viability, because the synthesis of these efflux pumps incurs a significant energetic cost. Hence, finely-tuned transcriptional regulators are essential to sense the environment accurately. They must upregulate the expression of efflux pumps when needed to combat antimicrobial challenges, while repressing expression during times of low demand to conserve energy resources. Obligate human pathogens like N. gonorrhoeae face not only antimicrobial cytotoxic stress but also oxidative stress from the host immune system. Gonococcal cells must possess robust mechanisms to detect and counteract reactive oxygen species for successful colonization and pathogenesis. Oxidative stress can lead to damage in DNA, lipids, and proteins. To mitigate this, bacteria employ a range of defenses, including transcriptional regulators that sense oxidative stress and subsequently regulate genes involved in protein refolding and repair mechanisms. These regulatory mechanisms are crucial for the bacteria's ability to adapt and survive within the hostile environment of the host. Overexpression of the multidrug efflux pump operon mtrCDE, encoding a critical factor of multidrug-resistance in Neisseria gonorrhoeae is repressed by the transcriptional regulator, MtrR (Multiple transferable resistance Repressor). Moreover, MtrR directly represses rpoH, a gene responsible for producing a sigma factor crucial in defending against reactive oxygen species. MtrR's control extends to over 60 genes, either directly or indirectly, setting it apart from other multidrug-binding transcription regulators and emphasizing its potential as an important focus for expanding our knowledge of bacterial global regulators. An in-depth investigation of MtrR will be invaluable in understanding multidrug resistance in gonorrhea infections. MtrR is a member of the TetR family of transcription regulators, members of which are found ubiquitously, and function to “interpret” environmental stresses and intracellular signals, that are then utilized by the bacterium to mount a defense against these potential cell killers. This dissertation aims to explore multiple facets of MtrR, including its DNA recognition mechanism, identification of innate host ligands, clarification of the induction mechanism, and examination of its ability to sense oxidative species. Here, I report the results from a series of in vitro experiments to identify the DNA binding and recognition mechanism of MtrR. MtrR residues involved in specific DNA site recognition was confirmed by structure driven site directed mutagenesis and biochemical experimentation including fluorescence polarization and circular dichroism. Additionally, I report results to identify innate, human inducers of MtrR and to understand the biochemical and structural mechanisms of the gene regulatory function of MtrR. Isothermal titration calorimetry experiments reveal that MtrR binds the hormonal steroids progesterone, -estradiol, and testosterone, all of which are present at significant concentrations at female and male urogenital infection sites as well as ethinyl estrogen, a component of some birth control pills. Binding of these steroids results in decreased affinity of MtrR for cognate DNA, as demonstrated by fluorescence polarization-based assays, and an in vivo increased antimicrobial resistance and expression of the MtrR-repressed mtrCDE and rpoH genes. The crystal structures of MtrR bound to each steroid provided insight into the flexibility of the binding pocket, elucidated residue-ligand interactions, and revealed the conformational consequences of the induction mechanism of MtrR. Three residues, D171, W136 and R176, are key to the specific binding of these gonadal steroids. These studies provide a molecular understanding of the transcriptional regulation by MtrR that promotes N. gonorrhoeae survival in its human host when challenged by innate steroidal antimicrobials. Finally, the study delved into the involvement of MtrR in sensing and responding to reactive oxygen species. This investigation focused on the impact of four cysteine residues per MtrR subunit. MtrR cysteine involvement in sensitizing MtrR to oxidating environments were confirmed by site-directed mutagenesis and biochemical binding assays. Crystallographic studies on oxidized MtrR unveiled potential oxidation states and induced conformational changes. The aim of my work on MtrR is to broaden our understanding of the induction mechanisms of this protein. This work will translate to the fuller understanding of mechanistic function of all members of the TetR protein family, as some bind single inducers with high specificity whilst others are polyspecific for their inducers. Furthermore, my studies will demonstrate how a single transcription regulator is able to fend off two very different dangers: attack by innate and myriad clinically prescribed antibiotics and damage by ROS.