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

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.

Haemophilus influenzae biofilms: hypothesis or fact?

Many publications state that nontypeable Haemophilus influenzae (NTHi) produces biofilms. Here, we review many of the publications that have led to acceptance by some that NTHi expresses a biofilm-specific phenotype as a distinct part of its life cycle. Biofilm formation was originally invoked to explain the failure to culture NTHi from middle-ear effusions, recalcitrance to antibiotics and its pathogenic behaviour. We argue that the current evidence for NTHi biofilm formation in vitro and in vivo is inconclusive. We consider that NTHi biofilm is hypothesis not fact, and although it might yet prove to be correct, there has been little or no consideration of alternative interpretations for the in vitro and in vivo observations. Uncritical acceptance of a distinctive NTHi biofilm phenotype has the potential to mislead and could confuse and compromise research efforts aimed at improving management and prevention of NTHi diseases of the human respiratory tract.

Induction of the SOS regulon of Haemophilus influenzae does not affect phase variation rates at tetranucleotide or dinucleotide repeats.

Haemophilus influenzae has microsatellite repeat tracts in 5' coding regions or promoters of several genes that are important for commensal and virulence behaviour. Changes in repeat number lead to switches in expression of these genes, a process referred to as phase variation. Hence, the virulence behaviour of this organism may be influenced by factors that alter the frequency of mutations in these repeat tracts. In Escherichia coli, induction of the SOS response destabilizes dinucleotide repeat tracts. H. influenzae encodes a homologue of the E. coli SOS repressor, LexA. The H. influenzae genome sequence was screened for the presence of the minimal consensus LexA-binding sequence from E. coli, CTG(N)(10)CAG, in order to identify genes with the potential to be SOS regulated. Twenty-five genes were identified that had LexA-binding sequences within 200 bp of the start codon. An H. influenzae non-inducible LexA mutant (lexA(NI)) was generated by site-directed mutagenesis. This mutant showed increased sensitivity, compared with wild-type (WT) cells, to both UV irradiation and mitomycin C (mitC) treatment. Semi-quantitative RT-PCR studies confirmed that H. influenzae mounts a LexA-regulated SOS response following DNA assault. Transcript levels of lexA, recA, recN, recX, ruvA and impA were increased in WT cells following DNA damage but not in lexA(NI) cells. Induction of the H. influenzae SOS response by UV irradiation or mitC treatment did not lead to any observable SOS-dependent changes in phase variation rates at either dinucleotide or tetranucleotide repeat tracts. Treatment with mitC caused a small increase in phase variation rates in both repeat tracts, independently of an SOS response. We suggest that the difference between H. influenzae and E. coli with regard to the effect of the SOS response on dinucleotide phase variation rates is due to the absence of any of the known trans-lesion synthesis DNA polymerases in H. influenzae.

Destabilization of tetranucleotide repeats in Haemophilus influenzae mutants lacking RnaseHI or the Klenow domain of PolI.

A feature of Haemophilus influenzae genomes is the presence of several loci containing tracts of six or more identical tetranucleotide repeat units. These repeat tracts are unstable and mediate high frequency, reversible alterations in the expression of surface antigens. This process, termed phase variation (PV), enables H.influenzae to rapidly adapt to fluctuations in the host environment. Perturbation of lagging strand DNA synthesis is known to destabilize simple sequence repeats in yeast and Escherichia coli. By using a chromosomally located reporter construct, we demonstrated that the mutation of an H.influenzae rnhA (encoding RnaseHI) homologue increases the mutation rates of tetranucleotide repeats approximately 3-fold. Additionally, deletion of the Klenow domain of DNA polymerase I (PolI) resulted in a approximately 35-fold increase in tetranucleotide repeat-mediated PV rates. Deletion of the PolI 5'>3' exonuclease domain appears to be lethal. The phenotypes of these mutants suggest that delayed or mutagenic Okazaki fragment processing destabilizes H.influenzae tetranucleotide repeat tracts.

Mutations in Haemophilus influenzae mismatch repair genes increase mutation rates of dinucleotide repeat tracts but not dinucleotide repeat-driven pilin phase variation rates.

High-frequency, reversible switches in expression of surface antigens, referred to as phase variation (PV), are characteristic of Haemophilus influenzae. PV enables this bacterial species, an obligate commensal and pathogen of the human upper respiratory tract, to adapt to changes in the host environment. Phase-variable hemagglutinating pili are expressed by many H. influenzae isolates. PV involves alterations in the number of 5' TA repeats located between the -10 and -35 promoter elements of the overlapping, divergently orientated promoters of hifA and hifBCDE, whose products mediate biosynthesis and assembly of pili. Dinucleotide repeat tracts are destabilized by mismatch repair (MMR) mutations in Escherichia coli. The influence of mutations in MMR genes of H. influenzae strain Rd on dinucleotide repeat-mediated PV rates was investigated by using reporter constructs containing 20 5' AT repeats. Mutations in mutS, mutL, and mutH elevated rates approximately 30-fold, while rates in dam and uvrD mutants were increased 14- and 3-fold, respectively. PV rates of constructs containing 10 to 12 5' AT repeats were significantly elevated in mutS mutants of H. influenzae strains Rd and Eagan. An intact hif locus was found in 14 and 12% of representative nontypeable H. influenzae isolates associated with either otitis media or carriage, respectively. Nine or more tandem 5' TA repeats were present in the promoter region. Surprisingly, inactivation of mutS in two serotype b H. influenzae strains did not alter pilin PV rates. Thus, although functionally analogous to the E. coli MMR pathway and active on dinucleotide repeat tracts, defects in H. influenzae MMR do not affect 5' TA-mediated pilin PV.