Outer membrane proteomics of Pasteurella multocida isolates to identify putative host-specificity determinants

Abstract

Pasteurella multocida is a Gram-negative bacterium responsible for diseases affecting a broad range of farm-reared animals. Although there is an association between capsular serotype and disease, the molecular basis of host specificity is poorly understood. Outer membrane proteins (OMPs) are at the interface of bacterium and host and are likely to play important roles in host specificity and disease. Two classes are of particular importance—adhesins that are adapted for colonization of specific host niches and iron-acquisition proteins that allow pathogens to acquire iron from host-specific iron complexes. A comparative analysis of the outer membrane (OM) proteome of eight P. multocida isolates associated with disease of avian, bovine, ovine and porcine species was performed to identify putative host-specificity determinants. Isolates were cultured in iron-replete media, and also in iron-limited conditions to mimic the iron-limited host environment, and induce expression of iron-regulated OMPs expressed in vivo. The OMP-rich sarcosyl-insoluble cell fraction was isolated and the OMPs were separated by SDS-PAGE and identified by matrix-assisted light desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The expressed proteome was compared with the in silico predicted proteome from the genome sequence of P. multocida strain Pm70, using PSORTb and Proteome Analyst subcellular localization software. In iron-rich conditions isolates were clustered into three groups based on high molecular weight (HMW) OMP similarity. Isolates responsible for invasive disease were clustered into a single group. Putative colonization OMPs were present in isolates recovered from different host species, but showed molecular weight heterogeneity. Such proteins are good candidates for further study as disease or host-specificity determinants, as variation between these proteins may be a consequence of adaptation to different host niches. HMW OMPs were identified as being involved in iron-uptake. However, isolates associated with different diseases and host species expressed different iron-uptake proteins, or regulated expression differently, suggesting adaptation to specific host niches.