Study Of Haemophilus Parasuis HAPS₂096 Mediated Resistance Agaist Cecropin B

Haemophilus parasuis（HPS） is a common pathogenic bacteria causing Glasser‘s disease in pigs, which is infective in all age stages. With many clinical serotypes, this disease is difficult to control and usually associated with other virus. Antibiotics are the best preference in treatment due to the lack of effective vaccine, but more and more drug resistant HPS has been found due to the abuse of antibiotics. Cecropin B（CB） is an antimicrobial peptide with broad antibacterial spectrum naturally found in Hyalophora cecropia. There is a great potential in investigation and development for CB as a new clinical antibiotic. But previous study has verified a stable resistant strain of HPS against CB.The HAPS₂096 gene encoded a periplasmic substrate binding protein（PSBP）, which is essential for an ABC type amino acid transporter. ABC transporter is a key factor in drug resistance in bacteria. In this study, we verified that gene HAPS₂096 is a key factor in CB resistance in HPS, and tried to explain the role it plays in CB resistance.1. Construction of HAPS₂096 deletion mutant strain and its complemented strainGenetic engineering was used in construction of the mutant strain. A suicidal vector harboring homologous arms of HAPS₂096 has been constructed and naturally transferred into HPS0135 to get a HAPS₂096 deletion mutant strain with kanamycin resistance. On the other hand, HAPS₂096 open read frame with its promotor was cloned into a modified endogenous vector and transferred into the mutant strain to generate a complementary strain. A control strain with the same vacant vector was generated at the same time. All the strains were confirmed by PCR, polarity effect verification, and RT-PCR.2. CB bactericidal assay of each strainA serial of CB concentrations has been set according to the MIC, and the bacterial number was set as 105CFU/m L. After incubated with 0.25μg/ml CB for 1h, the survival of the wild type was 12.02%, which is significantly higher than 5.16% of the mutant strain（p<0.05）. Similar result was generated in 0.125μg/ml CB. The survival of the wild type is 26.62%, significantly higher than 13.09% of the mutant strain（p<0.05）.After incubated in 0.25μg/ml CB for 1h, the survival of the vacant vector control strain was 1.81%, significantly lower than the complementary strain 6.82%（p<0.05）.In the agar diffusion test, after incubated with 1μg/m L for 20 h, a more obvious bacteriostatic circle could be observed in mutant strain, but it was not visible in the wild type.3. Immunoelectron microscropy analysis of the mutant strain and wild type after CB treatmentDifferent strains were incubated with 0.5μg/ml CB for 0.5h, followed by strained with immunogold labled antibody. The CB particles and the morphology of different strains could be observed by immunoelectron microscropy. The cell membrane of the wild type strain was smoother and intact. However, the mutant strain showed blurring cell membrane and cytoplasm. Broken cells presented much more in the mutant strain than the wild type. CB mainly accumulated on the cell menbrane in the mutant strain, but in the wild type, it mainly located in cytoplasm.4. Far Western-blotThe fusion protein GST-PSBP could be obtained by cloning and expressing gene HAPS₂096. Far Western-blot showed that CB combined with the fusion protein but not the GST tag, by implying anti-CB antibody after incubation with CB.In conclusion, the periplasmid substrate binding protein of HPS ABC type amino acid transporter plays an important role in CB resistance. This HAPS₂096 could combain with CB and transfer to cytoplasm to protect the cell membrane from damage.