Role of FNR and FNR-regulated, sugar fermentation genes in Neisseria meningitidis infection

Abstract

While it is generally accepted that anaerobic metabolism is required during infection, supporting experimental data have only been described in a limited number of studies. To provide additional evidence on the role of anaerobic metabolism in bacterial pathogens while invading mammalian hosts, we analysed the effect of the inactivation of FNR, the major regulatory protein involved in the adaptation to oxygen restrictive conditions, and of two of the FNR-regulated genes on the survival of Neisseria meningitidis serogroup B (MenB) in vivo. We found that fnr deletion resulted in more than 1 log reduction in the meningococcal capacity to proliferate both in infant rats and in mice. To identify which of the FNR-regulated genes were responsible for this attenuated phenotype, we defined the FNR regulon by combining DNA microarray analysis and FNR-DNA binding studies. Under oxygen-restricted conditions, FNR positively controlled the transcription of nine transcriptional units, the most upregulated of which were the two operons NMB0388-galM and mapA-pgmbeta implicated in sugar metabolism and fermentation. When galM and mapA were knocked out, the mutants were attenuated by 2 and 3 logs respectively. As the operons are controlled by FNR, from these data we conclude that MenB survival in the host anatomical sites where oxygen is limiting is supported by sugar fermentation.

Fig. 1

Transcription profile analysis of FNR-dependent genes and computational analysis of MenB FNR consensus sequence. A. Hierarchical clustering of gene expression profiles of wild-type MC58 and MC-fnrKO strains grown under oxygen restriction compared by DNA microarray analysis. Red and green bars indicate down- and upregulated genes in the wild-type strain respectively. The zoomed panel represents the upregulated cluster of FNR-dependent genes whose expression ratios are shown in the table. Blue and purple identify genes belonging to the same transcriptional units. B. Analysis of the FNR consensus sequence derived from the multiple sequence alignment of the upstream regions of the nine clustered upregulated transcriptional units. The graph reports the representation of each nucleotide of the derived consensus sequence above the MC58 genomic nucleotide frequency. The most conserved region of the derived consensus is highlighted in red. Pair-wise alignment of the derived MenB FNR consensus with the previously proposed E. coli FNR consensus is reported below. C. Frequency of FNR-regulated genes carrying the FNR consensus sequence. The graph indicates the number of genes (in blue) regulated at different time points and the percentage of regulated genes (in white) with an FNR box in their promoter-proximal region.

Fig. 2

EMSA analysis of FNR-regulated genes. PCR-amplified upstream regions were incubated with different concentrations (nM) of purified Ec FNR and Nm FNR proteins and DNA protein binding was followed by gel retardation analysis in the presence of salmon sperm DNA. To assess FNR binding specificity, reactions containing a 10-fold molar excess of each specific unlabelled DNA region were also included (lanes 400+C and 800+C). NMB0808 was used as negative control as its expression was unaffected by FNR deletion and no FNR consensus sequence was identified in its promoter-proximal region.

Fig. 3

Transcription analysis of galM and mapA genes in wild-type and mutant strains. Total RNA from wild-type and mutant strains was reverse transcribed and the derived cDNAs were amplified using gene-specific primer pairs and analysed by agarose gel electrophoresis. All PCR reactions from non-reverse transcribed RNAs were negative (not shown). The genetic organization of the amplified regions is schematized above each corresponding gel. Arrows indicate the position of the primers used for the amplification reactions and bp numbers indicate the size of each PCR product. Molecular weight standards are given on the right side of each gel.