MisR/MisS two-component regulon in Neisseria meningitidis

Abstract

Two-component regulatory systems are involved in processes important for bacterial pathogenesis. Inactivation of the misR/misS system in Neisseria meningitidis results in the loss of phosphorylation of the lipooligosaccharide inner core and causes attenuation in a mouse model of meningococcal infection. One hundred seventeen (78 up-regulated and 39 down-regulated) potential regulatory targets of the MisR/MisS (MisR/S) system were identified by transcriptional profiling of the NMBmisR mutant and the parental wild-type meningococcal strain NMB. The regulatory effect was further confirmed in a subset of target genes by quantitative real-time PCR and beta-galactosidase transcriptional fusion reporter assays. The MisR regulon includes genes encoding proteins necessary for protein folding in the bacterial cytoplasm and periplasm, transcriptional regulation, metabolism, iron assimilation, and type I protein transport. Mutation in the MisR/S system caused increased sensitivity to oxidative stress and also resulted in decreased susceptibility to complement-mediated killing by normal human serum. To identify the direct targets of MisR regulation, electrophoretic mobility shift assays were carried out using purified MisR-His(6) protein. Among 22 genes examined, misR directly interacted with 14 promoter regions. Six promoters were further investigated by DNase I protection assays, and a MisR-binding consensus sequence was proposed. Thus, the direct regulatory targets of MisR and the minimal regulon of the meningococcal MisR/S two-component signal transduction system were characterized. These data indicate that the MisR/S system influences a wide range of biological functions in N. meningitidis either directly or via intermediate regulators.

FIG. 1.

β-Galactosidase reporter activities of MisR-regulated genes. The reporter fusion was integrated into a permissive chromosomal locus as a single copy. β-Galactosidase activities of the wild type and misS and misRS mutants grown in GC broth to mid-log phase are expressed as Miller units and correspond to mean values for at least three independent experiments, each with triplicate measurements. Black bars, reporter strains in a wild-type background; gray bars, reporter strains with a misS::aphA-3 mutation; hatched bars, reporter strains with a misRS::aphA-3 double mutation.

FIG. 2.

EMSA results of MisR-promoter interaction. ³²P-labeled promoter fragments (∼5 fmol) were generated by T4 kinase phosphorylation, mixed with increasing amounts of MisR∼P (lanes 2 to 4) and MisR (lanes 5 to 7) for 20 min at 30°C, and then subjected to gel electrophoresis. Free probe is shown in lane 1. MisR∼P was generated by incubation with 50 mM acetyl phosphate for 30 min at 37°C. The amounts of MisR protein are 68 pmol (lanes 2 and 5), 136 pmol (lanes 3 and 6), and 272 pmol (lanes 4 and 7). The last three panels with six samples show the results of representative competition experiments with bfrA, tdfH, and B0556. Lane 1, probe alone; lane 2, probe and MisR; lanes 3 and 5, 2 μg specific and nonspecific competitor DNA, respectively; lanes 4 and 6, 4 μg specific and nonspecific competitor DNA, respectively.

FIG. 3.

Results of DNase I protection assays of the coding (A) and noncoding (B) strands of the fkpA promoter and the noncoding strand of the mtr promoter (C) by MisR∼P. ³²P-end-labeled promoter fragments were incubated with increasing amounts of MisR∼P for 20 min at 30°C and then subjected to DNase I digestion. MisR∼P was prepared freshly by reaction with 150 mM acetyl phosphate at 37°C for 30 min. The amounts of MisR∼P used in panels A and B were 0, 85, 170, 340, and 510 pmol and in panel C were 0, 42.5, 85, 170, 340, 680, and 1,360 pmol. Dideoxy-chain termination sequences corresponding to the probes are shown in the order of G, A, T, and C. The vertical bars indicate protected regions.

FIG. 4.

(A) Consensus alignment of MisR-binding sites. Regions protected from DNase I from misR, hmbR, mtr, hlyB, hlyD, and fkpA promoters are aligned by the BioProspector program (31). The bold letters indicate sequences identical to the 15-bp consensus motif. K, G or T; W, A or T; R, A or G; H, any bases except G; N, any bases. (B) Graphic representation of the consensus MisR-binding motif generated by the WebLogo program (6, 45). The overall height of the stack indicates the sequence conservation at that position, while the height of bases within the stack indicates the relative frequency of each base at that position.

FIG. 5.

(A) Results of serum bactericidal assays. NMB (wild type, filled diamond), M7 mutant (unencapsulated, filled square), NMBmisR mutant (misR::aphA-3, filled triangle), YT0310 mutant (misS::aphA-3, open circle), YT0336 mutant (misRS::aphA-3, asterisk), and C1001 mutant (misR::aphA-3/Ptrc::misR, filled circle with dotted line) strains grown to log phase and resuspended in HEPES-minimal essential medium buffer were exposed to 5, 10, and 25% NHS. The viable cell counts (n = 4) were determined after 30 min of incubation, and the percentages of survival were calculated by comparing these values to the viable counts at time zero. *25% indicates treatment with 25% heat-inactivated serum for 30 min. Averages and standard deviations for at least three independent experiments are shown. Student's t test with a two-tailed hypothesis indicates significant differences between the wild-type strain and three mutants at 10 and 25% serum levels (P ≤ 0.05). (B) Results of capsule-specific whole-cell ELISA. The OD₄₀₅ reading of the serogroup B wild-type strain NMB was used as 100% for normalizing the OD₄₀₅ readings of the YT0310 and the NMBmisR mutants. The nonencapsulated mutant M7 (synA::Tn916) was included as a negative control (n = 3).