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Item Astabiotics: Antimicrobial Signal Transduction Activators(Office of the Vice Chancellor for Research, 2013-04-05) Fortney, K. R.; Krieger, A.; Chen, L.; Zhang, Z.-Y.; Pollok, Karen E.; Wolfe, A. J.; Zhou, D.; Smith, S. N.; Mobley, H. L. T.; Spinola, S. M.The increasing prevalence of multi-drug resistant Gram-negative bacteria is a major public health concern. All available antibiotics are inhibitors of targets essential for virulence or growth. CpxRA is a highly conserved bacterial signal transduction system that responds to extracytoplasmic membrane stress. CpxA is a sensor with kinase and phosphatase activity; upon activation, CpxA donates a phosphate group to CpxR, activating a transcriptional response. Activation of CpxRA reduces the flow of protein traffic through the cytoplasmic membrane, dramatically reducing the expression of virulence determinants. Activation of CpxRA abolishes the virulence of Salmonella Typhimurium in mice. We found that activation of CpxRA crippled the ability of Haemophilus ducreyi to cause disease in experimentally infected human volunteers. Using an Escherichia coli reporter strain, we developed a high throughput screen to detect compounds that activate CpxRA. In a pilot screen of 36,000 compounds, we identified 1 class of compounds that shifts the equilibrium of CpxA to kinase activity, activating CpxR. Based on its potency, the calculated effective dose of the lead compound (a nitroindole) was 10 mg/kg. Female mice tolerated 100 mg/kg of the nitroindole given twice a day for 3 days. A CpxRA activating mutant constructed in uropathogenic E. coli (UPEC) was severely impaired in a murine urinary tract infection model; thus, activation of CpxRA is a valid treatment strategy for UPEC. However, when female mice were challenged with UPEC and treated with 100 mg/kg of the nitroindole using the schedule above, there were no differences in the recovered CFU in the urine, bladder, and kidney of sham and compound-treated mice. Future studies will include medicinal optimization of the nitroindole and identification and optimization other leads that activate CpxRA. Although our pilot test of efficacy was negative, astabiotics have great potential as broad-spectrum, adjunctive therapies to existing antimicrobials for treatment of Gram-negative infections.Item Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism(Wiley, 2017-09) Kashyap, Des R.; Kuzma, Marcin; Kowalczyk, Dominik A.; Gupta, Dipika; Dziarski, Roman; Medicine, School of MedicineMammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram-positive and Gram-negative bacteria through simultaneous induction of oxidative, thiol and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP-induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP-Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidoreductases to oxygen. CpxRA two-component system was a negative regulator of PGRP-induced oxidative stress. By contrast, PGRP-induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn2+ through influx of extracellular Zn2+ ) were mostly independent of oxidative stress. Thus, manipulating pathways that induce oxidative, thiol and metal stress in bacteria could be a useful strategy to design new approaches to antibacterial therapy.Item Both MisR (CpxR) and MisS (CpxA) Are Required for Neisseria gonorrhoeae Infection in a Murine Model of Lower Genital Tract Infection(American Society for Microbiology, 2017-08-18) Gangaiah, Dharanesh; Raterman, Erica L.; Wu, Hong; Fortney, Kate R.; Gao, Hongyu; Liu, Yunlong; Jerse, Ann E.; Spinola, Stanley M.; Microbiology and Immunology, School of MedicineDuring infection, Neisseria gonorrhoeae senses and responds to stress; such responses may be modulated by MisRS (NGO0177 and NGO0176), a two-component system that is a homolog of CpxRA. In Escherichia coli, CpxRA senses and responds to envelope stress; CpxA is a sensor kinase/phosphatase for CpxR, a response regulator. When a cpxA mutant is grown in medium containing glucose, CpxR is phosphorylated by acetyl phosphate but cannot be dephosphorylated, resulting in constitutive activation. Kandler and coworkers (J. L. Kandler, C. L. Holley, J. L. Reimche, V. Dhulipala, J. T. Balthazar, A. Muszyński, R. W. Carlson, and W. M. Shafer, Antimicrob Agents Chemother 60:4690-4700, 2016, https://doi.org/10.1128/AAC.00823-16) showed that MisR (CpxR) is required for the maintenance of membrane integrity and resistance to antimicrobial peptides, suggesting a role in gonococcal survival in vivo Here, we evaluated the contributions of MisR and MisS (CpxA) to gonococcal infection in a murine model of cervicovaginal colonization and identified MisR-regulated genes using RNA sequencing (RNA-Seq). The deletion of misR or misS severely reduced the capacity of N. gonorrhoeae to colonize mice or maintain infection over a 7-day period and reduced microbial fitness after exposure to heat shock. Compared to the wild type (WT), the inactivation of misR identified 157 differentially regulated genes, most of which encoded putative envelope proteins. The inactivation of misS identified 17 differentially regulated genes compared to the WT and 139 differentially regulated genes compared to the misR mutant, 111 of which overlapped those differentially expressed in the comparison of the WT versus the misR mutant. These data indicate that an intact MisRS system is required for gonococcal infection of mice. Provided the MisR is constitutively phosphorylated in the misS mutant, the data suggest that controlled but not constitutive activation is required for gonococcal infection in mice.