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

Jenkins GA, Figueira M, Kumar GA, Sweetman WA, Makepeace K, Pelton SI, Moxon R, Hood DW - BMC Microbiol. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Infectious Diseases Group, University of Oxford Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX39DS, UK.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: To investigate in more detail the interdependence of genes involved in sialometabolism, we compared gene expression in wild type and mutant strains following growth in the presence or absence of exogenous Neu5Ac. RT-PCR analysis of total RNA extracted from strain Rd mutated in each of the genes nanA, siaR, nanK, nanE, siaP, siaQM, HI0148 and crp was performed using internal pairs of primers specific for each gene of interest (Table 1) and the levels of expression compared using the RT-PCR amplification product for the housekeeping gene, frdB, as a control between samples. The level of transcript for each sialometabolism gene was generally greater in the siaR mutant background when compared to the wild type strain, although the results proved difficult to quantify (data not shown). This would be consistent with SiaR exerting a regulatory (negative) effect on sialometabolism gene expression, i.e. acting as a repressor [12]. The corresponding change in expression of multiple genes might suggest some co-regulation or co-dependence. Using primer pairs targeted against the 5' and 3' ends of adjacent genes across the region, RT-PCR analysis showed some co-transcripts for most gene pairs across the sialometabolism region (Figure 5).


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

Jenkins GA, Figueira M, Kumar GA, Sweetman WA, Makepeace K, Pelton SI, Moxon R, Hood DW - BMC Microbiol. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2836998&req=5

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.
Mentions: To investigate in more detail the interdependence of genes involved in sialometabolism, we compared gene expression in wild type and mutant strains following growth in the presence or absence of exogenous Neu5Ac. RT-PCR analysis of total RNA extracted from strain Rd mutated in each of the genes nanA, siaR, nanK, nanE, siaP, siaQM, HI0148 and crp was performed using internal pairs of primers specific for each gene of interest (Table 1) and the levels of expression compared using the RT-PCR amplification product for the housekeeping gene, frdB, as a control between samples. The level of transcript for each sialometabolism gene was generally greater in the siaR mutant background when compared to the wild type strain, although the results proved difficult to quantify (data not shown). This would be consistent with SiaR exerting a regulatory (negative) effect on sialometabolism gene expression, i.e. acting as a repressor [12]. The corresponding change in expression of multiple genes might suggest some co-regulation or co-dependence. Using primer pairs targeted against the 5' and 3' ends of adjacent genes across the region, RT-PCR analysis showed some co-transcripts for most gene pairs across the sialometabolism region (Figure 5).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Infectious Diseases Group, University of Oxford Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX39DS, UK.

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.

Show MeSH
Related in: MedlinePlus