Fitness Cost of Rifampin Resistance in Neisseria meningitidis: In Vitro Study of Mechanisms Associated with rpoB H553Y Mutation

Abstract

Rifampin chemoprophylaxis against Neisseria meningitidis infections led to the onset of rifampin resistance in clinical isolates harboring point mutations in the rpoB gene, coding for the RNA polymerase β chain. These resistant strains are rare in medical practice, suggesting their decreased fitness in the human host. In this study, we isolated rifampin-resistant rpoB mutants from hypervirulent serogroup C strain 93/4286 and analyzed their different properties, including the ability to grow/survive in different culture media and in differentiated THP-1 human monocytes and to compete with the wild-type strain in vitro. Our results demonstrate that different rpoB mutations (H553Y, H553R, and S549F) may have different effects, ranging from low- to high-cost effects, on bacterial fitness in vitro. Moreover, we found that the S549F mutation confers temperature sensitivity, possibly explaining why it is observed very rarely in clinical isolates. Comparative high-throughput RNA sequencing analysis of bacteria grown in chemically defined medium demonstrated that the low-cost H553Y substitution resulted in global transcriptional changes that functionally mimic the stringent response. Interestingly, many virulence-associated genes, including those coding for meningococcal type IV pili, porin A, adhesins/invasins, IgA protease, two-partner secretion system HrpA/HrpB, enzymes involved in resistance to oxidative injury, lipooligosaccharide sialylation, and capsular polysaccharide biosynthesis, were downregulated in the H553Y mutant compared to their level of expression in the wild-type strain. These data might account for the reduced capacity of this mutant to grow/survive in differentiated THP-1 cells and explain the rarity of H553Y mutants among clinical isolates.

FIG 1

Map of Rif^r-conferring rpoB mutations, growth, and stationary-phase survival of rifampin-resistant strains in complex medium. (A) Map of the N. meningitidis RNA polymerase (RpoB) subunit with the locations of rif clusters N, I, II, and III (top) and the amino acid sequence alignment of rif cluster I from N. meningitidis, E. coli, S. aureus, B. subtilis, M. tuberculosis, and Streptomyces coelicolor A3 (2) (bottom). Numbering begins at the first amino acid (Aa) of the RpoB sequence. Circles above the N. meningitidis sequence, the amino acid substitutions (H553Y, H553R, and S549F) analyzed in this study; asterisks, evolutionarily conserved amino acid residues. (B and C) Growth (B) and stationary-phase survival (C) curves of the mutants and the wild-type strain in GC broth at 37°C and 32°C. The experiments were performed in triplicate with three independent cultures, and statistical significance was examined by the Student t test. Results are indicated as means ± SDs. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 2

Growth and stationary-phase survival of rifampin-resistant strains in minimal medium. Growth (A to E, left) and stationary-phase survival (A to E, right) curves for mutants and the wild-type strain in MCDA medium with increasing sodium concentrations ranging from 21.6 mM to 121.6 mM. The experiments were performed in triplicate with three independent cultures for each medium, and statistical significance was examined by the Student t test. Results are indicated as means ± SDs. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 3

Survival and growth of wild-type strain 93/4286, the H553Y mutant, and the 93/4286RpoB^H553Y transformant in differentiated human THP-1 monocytes and evaluation of sensitivity to nitrosative and oxidative injury. (A and B) Survival and growth of wild-type strain 93/4286 and derivative rifampin-resistant mutants (A) and transformants (B) in THP-1 cells. Differentiated THP-1 cells (10⁵ cells/well) were infected with meningococci at an MOI of 10, treated with gentamicin, and reincubated in RPMI 1640 medium for the indicated times. After saponin lysis, the numbers of CFU from intracellular bacteria were scored. Values are means from at least four independent experiments. Results are shown as the relative number of CFU per well ± SD. (C) Effect of the H553Y substitution on sensitivity to nitrosative injury. The H553Y mutant, transformant 93/4286RpoB^H553Y, and the wild-type strain were grown to OD₆₀₀ of 1.0 (corresponding to about 1 ×10⁹ CFU for each strain) and then exposed to increasing concentrations of the nitric oxide generator SNP, and the viable bacteria were evaluated by the CFU method. Values represent the means from three independent experiments ± SDs. (D) Effect of the H553Y substitution on sensitivity to oxidative killing. Bacteria were incubated with H₂O₂ over a range of concentrations (0 to 0.2 mM) for 20 min in MCDA-2, and the number of surviving bacteria was determined by the CFU method. Data are shown as the means ± SDs from three independent experiments, each with triplicate samples. (E) Semiquantitative analysis of transcriptional changes in the katA gene by real-time RT-PCR. Transcription was performed with retrotranscribed total RNA isolated from the wild-type strain and H553Y mutant that had been grown in MCDA-2 and exposed to increasing concentrations of H₂O₂. Results are reported as the fold change in the level of expression of the katA transcript, normalized to the 16S mRNA level, compared to the level of expression in the wild-type strain grown with 0 mM H₂O₂. (F) Intracellular glutathione levels. The levels of the intracellular glutathione pool in bacterial strains were determined in MCDA-2. The H553Y mutant had a significantly reduced amount of intracellular glutathione after exposure to 0.2 mM H₂O₂. Values represent the means ± SDs from three independent experiments. The statistical significance of all above-mentioned experiments was determined by Student's t test. Asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG 4

Quantitative determination of meningococcal capsular polysaccharide. (A) Immunoblot assay. Wild-type strain 93/4286 or the H553Y mutant grown in MCDA-2 medium to late logarithmic phase was resuspended in 1× PBS, and growth was normalized to an OD₆₀₀ of 1. Dilutions of the bacterial suspensions (0.09 to 0.01 as shown) were transferred onto PVDF membranes by slot blotting. After blotting, the membranes were air dried, blocked in 5% milk in 1× PBS, incubated for 2 h with a primary antibody against serogroup C meningococci, washed in Tween 0.1% in 1× PBS, and then incubated for 1 h with a secondary antibody conjugated with horseradish peroxidase at a 1:5,000 dilution. (B and C) Resorcinol assay for quantitative determination of sialic acid. Wild-type strain 93/4286 or the H553Y mutant was grown to late logarithmic phase either in MCDA-2 medium or in GC medium, as indicated. Extracts enriched in capsular polysaccharide were then prepared, and the sialic acid content was determined by the chromogenic resorcinol reaction with N-acetylneuraminic acid as a standard. (B) The chromogenic reaction in a representative set of samples is shown. (C) The sialic acid content was normalized to the optical density at 600 nm of the bacterial cultures. Asterisks indicate statistical significance (**, P < 0.05; ***, P < 0.005).