Sialic acid mediated transcriptional modulation of a highly conserved sialometabolism gene cluster in Haemophilus influenzae and its effect on virulence

Abstract

Background: Sialic acid has been shown to be a major virulence determinant in the pathogenesis of otitis media caused by the bacterium Haemophilus influenzae. This study aimed to characterise the expression of genes required for the metabolism of sialic acid and to investigate the role of these genes in virulence.

Results: Using qRT-PCR, we observed decreased transcriptional activity of genes within a cluster that are required for uptake and catabolism of 5-acetyl neuraminic acid (Neu5Ac), when bacteria were cultured in the presence of the sugar. We show that these uptake and catabolic genes, including a sialic acid regulatory gene (siaR), are highly conserved in the H. influenzae natural population. Mutant strains were constructed for seven of the nine genes and their influence upon LPS sialylation and resistance of the bacteria to the killing effect of normal human serum were assessed. Mutations in the Neu5Ac uptake (TRAP transporter) genes decreased virulence in the chinchilla model of otitis media, but the attenuation was strain dependent. In contrast, mutations in catabolism genes and genes regulating sialic acid metabolism (siaR and crp) did not attenuate virulence.

Conclusion: The commensal and pathogenic behaviour of H. influenzae involves LPS sialylation that can be influenced by a complex regulatory interplay of sialometabolism genes.

Figure 1

The sialometabolism gene cluster of H. influenzae. Indicated are the catabolism and transport groups of genes, each gene is represented by an arrow indicating the direction of transcription. The HI numbers corresponding to the reading frame designation in the strain Rd genome sequence are given above the arrows and the gene names below. 0 indicates the position of the CRP binding sequence.

Figure 2

T-SDS-PAGE analyses of LPS isolated from wild type (wt) strains Rd, 375 and 486 and their respective mutants. Panels (a) and (d) show profiles of LPS without (-) and with (+) neuraminidase treatment. The wt or mutant strains are indicated above each lane. Shown are: panels (a) and (b), strain Rd; panel (c), strain 375; panel (d), strain 486.

Figure 3

Resistance (% survival) of H. influenzae strains to the killing effect of normal human serum. 500 organisms of strain Rd (panel a) or NTHi 486 (panel b) or derived mutants were added to different (doubling) dilutions of pooled human serum; percentage survival of inoculum of bacteria (y-axis) is shown for varying serum concentrations (x-axis). Each point is the averaged result of 3 independently performed experiments, error bars (1 standard deviation) are shown.

Figure 4

Effect of mutation of siaP, siaR and crp on bacterial counts of H. influenzae strains from the middle ear of chinchillas when compared to wild type strains. Animals were inoculated with between 60 and 100 organisms directly into the middle ear bullae. Each data point represents the average number of organisms ml^-1 of exudate or washings from the middle ear for typically four animals at different times (days) following inoculation. Shown are wild type and isogenic strains for: panel (a), NTHi 486; panel (b), Rd; panel (c), NTHi 375. The lower detection limit is a bacterial count of 2.00.

Figure 5

PCR amplification for cDNA of sialometabolism genes from strain Rd showing co-transcripts for adjacent gene pairs. cDNA was made after bacteria were grown in BHI in the presence of sialic acid. RT-PCR products shown are in lane 2, nagA/nagB; lane 3, nagB/nanA; lane 4, nanA/siaR; lane 5, siaR/nanK; lane 6, nanK/nanE; lane 7, siaP/siaQM; lane 8, siaQM/HI0148. Lane 1 shows the 1 kb DNA ladder marker with the 1.6 kb band marked by an arrow.

Figure 6

q-PCR data for sialometabolism genes of H. influenzae. In each panel, the y-axis shows the quantity of mRNA, relative to the frdB control gene, for cDNA from wild type or mutant strains following growth in the presence (+) or absence (-) of exogenous Neu5Ac (x-axis). Shown are: panel (a) siaP; panel (b) nanE; panel (c) siaR. Each value shown below the x axis represents the results from 3 separate experiments utilising independent cDNA and mRNA preparations and each q-PCR reaction was run in triplicate. The error bars indicate the standard deviations derived for the respective data.