Immunoproteomics Analysis Of Whole Cell Bacterial Proteins Of Riemerella Anatipestifer And Construction Of The Sip Gene Deletion Mutant

Riemerella anatipestifer infection is one of the most economically important diseases confrontingthe duck industry throughout the world. It accounts for significant economic losses due to high mortality,weight loss, condemnations, downgrading, and salvage. R. anatipestifer belongs to the genus Riemerellain the family Flavobacteriaceae. Currently,at least21serotypes have been identified,but only very poorcross-protection has been observed among different serotypes of R. anatipestifer. Moreover, little isknown about the molecular basis of its pathogenesis and the virulence factors involved.To uncover the pathogenesis of the bacteria, develop novel vaccine candidates and serologicaldiagnosis marker, in this study, an immunoproteomic assay was used to identify immunogenic proteinsamong the whole cell bacterial proteins of R. anatipestifer; on the other hand, sip gene deletion mutantwas constructed, and the roles of siderophore interacting protein (SIP) in the pathogenicity and ironacquisition of R. anatipestifer was also analyzed.1. Immunoproteomics analysis of whole cell bacterial proteins of Riemerella anatipestiferIn this study, an immunoproteomic assay was firstly used to identify immunogenic proteins amongthe whole cell bacterial proteins of R. anatipestifer virulent strain Th4, with Duck antiserum against R.anatipestifer Th4(Serotype2), CH3(Serotype1), HXb2(Serotype10), duck convalescent serum againststrain Yb2(Serotype2) and na ve duck serum.We identified64protein spots by duck antiserum againstR. anatipestifer Th4duck antiserum. Immunogenic proteins on a duplicate gel were excised andidentified by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) and peptide mass fingerprinting (PMF), a total of34immunogenic proteins werefound, With the exception of OmpA and GroEL. In addition, TonB-dependent outer membrane receptorwas found to be a cross immunogenic antigen among serotypes1,2and10of R. anatipestifer. This willhelp us reveal the pathogenic mechanism, develop new vaccines and serological diagnosis methods.2. Construction and characterization of R. anatipestifer CH3△sipIn this study, we constructed R. anatipestifer CH3△sip by allelic exchange through a recombinantsuicide vector,which included the flanking DNA sequences of sip gene of R. anatipestifer ashomologous arms and a spectinomycin resistance cassette.The transformants were screened on TSBagar plates containing spectinomycin and Kanamycin, and on TSB agar. The transconjugants in whichthe sip gene was replaced by a Spc~Rcassette were selected and thus the sip mutant strain,CH3△sip, hadbeen generated.The analysis of biological characteristics showed that the virulence of CH3△sip mutantto ducklings was27times decreased than that of the parent strain CH3. When TSB supplemented with100μM2’-2’-dipyridyl (DIP), an iron chelator, and the growth of the CH3△sip mutant wassignificantly slower than that of CH3(p<0.05), and this could be recovered by complemented CH3△sipmutant.The resulted suggested that SIP played a critical role in the iron acquisition.This study will helpto further study the iron acquisition system and their roles in the pathogenicity of R. anatipestifer.