Isolation, Characterization And Application Of Endogenous Plasmids From Haemophilus Parasuis Field Isolates

Haemophilus parasuis are commensal bacteria in swine upper respiratory tract that invade in body and cause severe systemic infection named Glasser’s disease, which is characterized by polyserositis, arthrithis and meningitis. The disease was reported to have broken out and prevailed in all major pig-breeding countries, and the prevalence is increasing with the change of breeding mode. H. parasuis has become an important bacterial pathogen impacting pig industry world-wide, and it is urgent to analyze and demonstrate its molecular pathogenesis in order to generate a highly effective and comprehensive strategy for the prevention and control of this disease. The high-throughout approaches including complete genome sequencing, comparative genomics, proteomics and transcriptomics have been successively applied in H. parasuis and a number of potential virulence factors were screened out. To validate these assumptions and inferences, conventional genetic manipulation tools are indispensable. Currently, however, the lack of genetic manipulation tools is the bottleneck that hampers more intensive studies on pathogenesis of H. parasuis and hence restricts the development of prevention and control strategies. Therefore, to construct a genetic manipulation system adaptable to the pathogen is a primary task in this field. In this study, native plasmids were identified from field strains of H. parasuis and components in the plasmids were used to construct shuttle vector, expression vector and temperature-sensitive replication vector adapted to the pathogen, and the electroporation method for H. parasuis was optimized, allowing an over-expression of particular genes in H. parasuis, providing necessary technical support for the gene-deleted strains and laying a substantial foundation for basic researches in this filed. The main content are as following:1. The screening of native plasmids in Haemophilus parasuis isolates110 H. parasuis strains isolated from 2005 to 2007 from different pig farms in China were screened and 10 strains were identified to harbor endogenous plasmids. The complete sequencing was conducted in 6 plasmids, namely pYZ18, pYC93, pFZ51, pWH24, pZK15 and pHN61, and the open reading frames were annotated by using ORF Finder and Blast tools in NCBI website. The sequence of plasmid pZK15 was identical to that of pYC93. Three of the 6 sequenced plasmids, namely pYZ18, pYC93 and pZK15, have a replication mode that does not require the plasmid-encoded replication protein. The identity of the later two was probably due to the horizontal transfer of the same plasmid. Plasmid pYZ18 has a replication region close to that of pYC93 and pZK15 but not completely the same, with a similarity of 81% in nucleotide sequence. Thus, we assumed that these 3 plasmids have the same replicate mode but the results showed their copy numbers differed a lot, probably because the sites of sequence difference were associated with the control of plasmid copy numbers. The replication modes of the remaining 3 plasmids require the involvement of plasmid-encoded replication protein. The replication regions of pHN61 and pFZ51, including the sequence encoding replication protein and non-encoding region, were completely identical and differed a lot from that of pWH24. Five of the 6 plasmid harbor resistance genes different from each other, except for cryptic plasmid pWH24. The licosamide resistance gene lnuC was fist identified in Gram-negative bacteria, and tetH and its inhibitory gene tetR were also first discovered in H. parasuis. Moreover, pZK15, with 7 resistance genes, is the first multiple-resistance plasmid identified in H. parasuis. Based on a rough estimation of copy numbers of the plasmids, as well as the stability and range of replicon distribution, we selected elements including the replication origin of pYC93 and ampicillin and chloromycetin resistance gene expression cassettes as the components to construct vectors for genetic manipulation in H. parasuis.2. The construction of shuttle vector pSHK4The shuttle vector between E.coli and the research target is the most fundamental component of the genetic manipulation system, which requires two basic factors:a replicon with stable heredity in two host bacteria, and a screening marker suitable to two host bacteria. According to the previous studies on resistance gene cassettes that function in Pasteurellaceae, a Tn10 derived kanamycin resistance gene frequently used in vector tools was selected as resistance screening marker for the shuttle vector. The replicon of shuttle vector combined the replication origin of H. parasuis endogenous plasmid pYC93 and that of commercialized cloning vector ColEl. The multiple cloning site of commercialized cloning vector was amplified and the four necessary components was integrated and linked via overlapping PCR followed by self-ligation reaction, generating a shuttle vecot pSHK4 with stable heredity in E.coli and H. parasuis. Restriction site Ncol and Ndel were introduced between three individual components, where DNA sequences can be inserted in need of subsequent experiments to construct other vectors with more extensive functions. Considering the high copies and plasmid DNA production in large quantity, we transformed the serovar reference strains and field isolates of H. parasuis with the shuttle vector, and systemically studied the eletroporation method of the bacteria. By optimizing the preparation of competent cells and each step in the procedure of eletroporation, we have generally overcome the transformation barrier which is comprehensive in H. parasuis strains. After transforming pSHK4 into E.coli and H. parasuis, the bacteria were passaged in medium without antibiotics for 25 generations and the vector was stably present in both of host bacteria, indicating its genetic stability as a shuttle vector.3. The construction of expression vector pK3EXOn the basis of shuttle vector, we introduced elements including the transcription promoter of housekeeping gene rpsJ of H. parasuis SH0165, lacO gene, lacI gene, histidine tag sequence and transcription terminator, resulting in expression vector pK3EX, whose expression can be induced by IPTG in H. parasuis. The transcription promoter was amplified from the genome of haemophilus parasuis strain SH0165, lacO gene, lacI gene and histidine tag sequence were amplified from commercialized vector pET28a and transcription terminator was amplified from endogenous plasmid pHN61. The location of regulatory gene lacO is between promoter sequence and multiple cloning site, which is the combing site for lacI gene product. The natural promoter of H. parasuis wza gene was inserted upstream to lacI to ensure expression of lacI in H. parasuis and to reduce the background expression of cloned gene to the minimum level. Upstream to transcription terminator were 6His tag sequence and following stop codon, for the sake of easy purification of expression product by affinity chromatography. We successfully cloned H. parasuis SH0165 gene aidA between SaiI and XbaI sites on pK3EX vector and obtained recombinant plasmid pK3EX-AT1, which was then transformed into strain 174. An obvious over-expression in strain 174 was validated by Western blotting. Using the 6His tag in the fusion expression product, the target protein AidA was purified via Ni affinity column. The construction of expression vector provides a powerful tool for researches on virulence factors of H. parasuis. Over-expression of endogenous genes and investigation on the phenotypic changes of over-expressing strains will help understand the role that target genes play in the survival or pathogenesis for this pathogen. In addition, an expression vector can also be applied in the construction of the complementary strain of delete mutantions. Generally, the successful establishment of H. parasuis expression vector pK3EX is contributive to more profound understanding of the pathogenesis of this important swine bacterial pathogen.4. The construction of vectors with temperature-sensitive replicationIn this study, the pYC93 replication origin was replaced by synthesized temperature-sensitive replication origin in the shuttle vector pSHK4, generating vector pSHK TS with temperature-sensitive replication that can be used to construct knockout mutants of H. parasuis without resistance marker. This mutagenesis strategy allows convenience in deletion mutation for more than one genes in one mutan. Besides, deletion mutants without resistance markers are potential to be directly used as attenuated vaccines. It will make the construction of genetic engineering vaccines possible while studying virulence factors of a bacterial pathogen. In the practice, however, it is quite easy to obtain strains of single exchange in which the plasmid was integrated into H. parasuis genome, but the colonies that grew after resolvation of plasmid from genome by the second exchange were all reverse wild type. Considering it is perhaps because the mixture of mutants cultured in vitro with wild type leads to a lack of competitive advantage and they finally fail to survive, we further reformed vector structure, by placing kanamycin resistance gene in the middle of multiple cloning site, adding another ampicillin or chloromycetin resistance gene onto the plasmid backbone and thus creating other two vectors with temperature-sensitive replication, namely pAK5TS and pCK5TS. Moreover, FRT sequence was introduced into the two flanks of kanamycin resistance gene cassette in pAK5TS and pCK5TS, making it possible to eliminate the resistance gene in the genome by FLP site-specific recombinase in mutants. In this way, it not only avoids the polarity effects caused by the insertion of resistance gene into mutation site, but also contributes to more clear demonstration of molecular basis of phenotypic change of the mutants. It is expectable that multiple genes can be successively knocked-out in one strain and those mutants whose virulence were significantly reduced can be directly used as attenuated vaccines for the prevention of Glasser’s disease in swine.