Both MisR (CpxR) and MisS (CpxA) Are Required for Neisseria gonorrhoeae Infection in a Murine Model of Lower Genital Tract Infection

Abstract

During 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.

Keywords: CpxRA; MisRS; Neisseria gonorrhoeae; RNA-Seq; envelope stress; genes; infection; mice.

FIG 1

(A and B) Competitive infection of BALB/c mice with the FA1090 and FA1090 misS::kan (A) or the FA1090 and FA1090 misR::erm (B) strains. Solid circles indicate mice from which both WT and mutant bacteria were recovered, and open symbols indicate mice from which only WT bacteria were recovered. The competitive index was calculated as follows: [FA1090 misS::kan or FA1090 misR::erm/FA1090 (output)]/[FA1090 misS::kan or FA1090 misR::erm/FA1090 (input)]. Horizontal bars indicate geometric mean competitive indices. For cultures from which no mutant CFU were recovered, a value of 20 CFU (limit of detection) was used in the calculation. (C and D) Average numbers of mutant CFU and average calculated numbers of WT CFU recovered from coinfected mice over time.

FIG 2

Noncompetitive infection of BALB/c mice with the FA1090, FA1090 misS::kan, and FA1090 misR::erm strains. (A) Percentage of BALB/c mice colonized with the indicated N. gonorrhoeae strains over time. Statistical significance was observed between the capacities of the WT strain and the misS (P = 0.004) and misR (P = 0.002) mutant strains to maintain colonization (as determined by a log rank [Mantel-Cox] test). (B) Colonization load in mice that maintained infection for 8 days, expressed as average log₁₀ CFU per milliliter of vaginal swab suspension. Significantly fewer CFU were recovered over time from mice inoculated with either mutant than from mice inoculated with WT bacteria. (P = 0.005 and 0.003 for misR and misS mutant bacteria, respectively, as determined by 2-way ANOVA with Bonferroni's multiple-comparison test).

FIG 3

Venn diagram showing the numbers of genes differentially expressed in FA1090 versus FA1090 misR::erm, FA1090 misS::kan versus FA1090 misR::erm, and FA1090 misS::kan versus FA1090. The up- and downregulated genes or operons are indicated by up and down arrows, respectively. The total numbers of genes differentially expressed are indicated in boldface type outside the Venn diagram.

FIG 4

qRT-PCR validation of RNA-Seq data. (A) Fold change in expression levels of target genes in the misS mutant relative to the misR mutant. The expression levels of target genes were normalized to that of rps15. The data represent the means ± standard deviations from four independent experiments. (B) Correlation between the fold changes derived from qRT-PCR and those derived from RNA-Seq.

FIG 5

Effect of deletions of misR and misS on gonococcal survival in the presence of normal human serum (A) and heat shock (B). (A) Percent survival of FA1090, FA1090 misS::kan, and FA1090 misR::erm cells after exposure to 50% pooled normal human serum. Percent survival was calculated as follows: (geometric mean CFU after exposure to active NHS/geometric mean CFU before exposure to active NHS) × 100. (B) Percent survival of FA1090, FA1090 misS::kan, and FA1090 misR::erm cells after exposure to heat shock at 42°C. Percent survival was calculated as follows: (geometric mean CFU after heat shock/geometric mean CFU before heat shock) × 100. The data are means ± standard deviations from four independent experiments. *, P < 0.05; **, P < 0.01.