| M. haemolytica is a Gram-negative coccobacillus which resides in the upper respiratory tract of healthy calves. In animals immunocompromised by stress, the microorganism gets inhaled into and colonizes the lung, causing bovine pneumonic pasteurellosis. It is hypothesized that M. haemolytica coordinates its gene expression in response to environmental signal(s), 'switching' between the commensal and pathogenic states. This report is the first study of environmental sensing mechanisms in this microorganism. Five putative pairs of two-component signal transduction systems were identified in the M. haemolytica genome sequence through BLAST searches; these are ArcBA, CpxRA, NarQP, PhoRB and TtrSR. The nitrate and nitrite sensor NarQ and response regulator NarP were further investigated. Multiple sequence alignments showed high sequence conservation between M. haemolytica NarQP their homologues. Domain analyses located two N-terminal transmembrane helices in the NarQ and a C-terminal helix-turn-helix DNA binding motif in NarP, both typical for these proteins. A narP knock-out mutant strain was generated by replacing narP with a chloramphenicol resistant cassette, plpcat, through double homologous recombination. This mutant, DeltaNarP7, lost its ability to respond to the addition of NaNO3 in the media, while an altered growth rate and differential protein expression was observed for the parental strain, SH1217, grown with NaNO3 supplementation. Western inmunoblot analysis revealed differential expression of virulence-associated proteins such as leukotoxin (lktA) and adhesin (AhsA) in SH1217 grown with NaNO3 supplementation, but not in DeltaNarP7. A proteomic analysis using ISO-DALT 2D electrophoresis and liquid chromatography-mass spectrometry identified seventeen more proteins for which their expression were altered. Proteins overexpressed in response to NaNO3 supplementation include the iron transporters FbpA and YfeA, a superoxide dismutase and proteins involved in translation. The altered expression of these proteins are mediated by NarP, since the response was absent in DeltaNarP7. Putative NarP binding sequences have been found in the promoter regions of some of the genes, such as fbpA. These proteins shown to be under the control of NarP are thought to aid in the prevalence of M. haemolytica in its host during pathogenesis of BPP, either through enhancing the effectiveness of nitrate respiration or by assisting to evade host defense mechanisms. |
