In Haemophilus influenzae Rd KW20, we identified a gene, adhC, which encodes a class III alcohol dehydrogenase (AdhC) and has S-nitrosoglutathione reductase activity. adhC exists on an operon with estD, which encodes an esterase. Divergent to the adhC-estD operon is the Haemophilus influenzae nmlR gene (nmlR HI ), which encodes a MerR family regulator that is homologous to the Neisseria MerR-like regulator (NmlR). Analysis of an nmlR HI mutant indicated that expression of the adhC-estD operon is regulated by NmlR HI in strain Rd KW20. Chromosomal inactivation of either adhC or nmlR HI resulted in sensitivity to S-nitrosoglutathione and decreased S-nitrosoglutathione reductase activity. Examination of the NmlR HI -AdhC system in the genome sequences of nontypeable H. influenzae strains R2846, R2866, and 86-028NP identified significant variations. The adhC gene of 86-028NP was predicted to be nonfunctional due to a premature stop codon. Polymorphisms in the operator/promoter region of R2866 resulted in reduced enzyme activity. This correlated with an increased sensitivity to S-nitrosoglutathione. The adhC-nmlR HI system was examined in thirty-three clinical isolates (both capsular and nontypeable strains). Nucleic acid sequence data showed that only strain 86-028NP contained a premature stop codon. There were some variations in the DNA sequence of the operator/promoter region which altered the nmlR HI promoter. However, the clinical isolates still possessed S-nitrosoglutathione reductase activity and showed at least the equivalent ability to grow in the presence of S-nitrosoglutathione as Rd KW20. These data suggest that the nmlR HI -adhC system has a role in the defense against nitrosative stress in Haemophilus influenzae.
BackgroundadhC from Haemophilus influenzae encodes a glutathione-dependent alcohol dehydrogenase that has previously been shown to be required for protection against killing by S-nitrosoglutathione (GSNO). This group of enzymes is known in other systems to be able to utilize substrates that form adducts with glutathione, such as aldehydes.ResultsHere, we show that expression of adhC is maximally induced under conditions of high oxygen tension as well as specifically with glucose as a carbon source. adhC could also be induced in response to formaldehyde but not GSNO. An adhC mutant was more susceptible than wild-type Haemophilus influenzae Rd KW20 to killing by various short chain aliphatic aldehydes, all of which can be generated endogenously during cell metabolism but are also produced by the host as part of the innate immune response.ConclusionsThese results indicate that AdhC plays a role in defense against endogenously generated reactive carbonyl electrophiles in Haemophilus influenzae and may also play a role in defense against the host innate immune system.
Nickel acts as a co-factor for a small number of enzymes in bacteria. Urease is one of the two nickel-dependent enzymes that have been identified in Haemophilus influenzae; glyoxalase I is the other. However, nickel has been suggested to have roles in H. influenzae that can not attributed to the function of these enzymes. We have previously shown that in the H. influenzae strain Rd KW20 the inability to acquire nickel led to alterations to the cell-type; an increased biofilm formation and changes in cell surface properties. Here we report the differences in the genome wide gene expression between Rd KW20 and a strain incapable of importing nickel (nikQ); revealing a link between intracellular nickel levels and genes involved in metabolic pathways, stress responses and genes associated with surface factors such as type IV pili. We have then taken a strain previously shown to use type IV pili both in biofilm formation and for twitching motility (86-028NP) and have shown its homologous genes (NTHI1417-1422; annotated as cobalt transporter, cbiKLMOQ) did import nickel and mutations in this locus had pleiotropic effects correlating to stress response and motility. Compared to wild type cells, the nickel depleted cells were more electronegativity charged, they aggregated and formed a biofilm. Correct intracellular nickel levels were also important for resistance to oxidative stress; the nickel depleted cells were more sensitive to oxidative stress. The nickel depleted cells were also non-motile, but the addition specifically of nickel returned these cells to a wild type motility state. We have also analysed the role of nickel uptake in a naturally, urease negative strain (the blood isolate R2866) and depleting intracellular nickel (a nikQ mutant) in this strain effected a similar range of cell functions. These data reveal a role for the capacity to acquire nickel from the environment and for the correct intracellular nickel levels as part of H. influenzae stress response and in signalling for a switch to a sessile bacterial lifestyle.
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