The Role And Regulation Of Type â…¥ Secretion System 2 Core Component VgrG In Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli (APEC) could cause avian colibacillosis with different clinical symptoms such as airsacculitis, synovitis, salpingitis peritonitis. Avian infected with APEC, accompaning by other pathogens are economically devastating to poultry industries.Microbial pathogenicity is a complex phenomenon encompassing diverse mechanisms. Pathogenic bacteria utilize secretion system to delivery the virulence factors to the enviroment or into host cells, which play essential roles during infection. T6SS has been identified in more than one fourth of all sequenced bacterial genomes. The stucture of T6SS is homologous to the T4 phage. Previous studies showed that the T6SS contributes to pathogenicity in many pathogenic bacteria. Furthermore, the functions of T6SS in nonpathogenic bacteria-host interactions or interbacterial interactions were also discovered. Recent study identified three T6SS loci (T6SS1, T6SS2 and T6SS3) in APEC. However, the roles of these T6SS is remain unclear. Previous findings have demonstrated various classes of regulators sensitive to environmental cues that specifically modulate the activity of the T6SS. The ferric-uptake regulator (Fur) protein is a key modulator of iron-dependent gene expression in bacteria. Expression of T6SS in two opportunistic enteric pathogens, E. tarda and EAEC, is repressed directly at the transcriptional level by Fur. T6SS can be regulated by other systems, such as Two-component regulatory systems (TCS). T6SS secreted proteins were only detected in the late stage of infection by Salmonella and Pseudomonas aeruginosa, which was due to that TCS SsrB negative regulate the T6SS effectively expressed in the early stage of infection. T6SS is a newly discovered bacterial secretion system, its pathogenic mechanism in APEC is not clear. Core component VgrG not only compose the structure of T6SS, but also is the effector of T6SS. Thus, the effects of T6SS core component VgrGon the virulence of APEC were determined. Moreover, we determined the roles of cpxR in virulence and regulation of T6SS2 in APEC, which is important to study the pathogenecity of APEC.1. Cloning and expression of T6SS2 core component VgrG in avian pathogenic Escherichia coliWe disigened the primers based on the sequence of T6SS2 vgrG gene of APEC DE719. The vgrG gene was amplified from DE719 genomic DNA by polymerase chain reaction (PCR). The PCR products were cloned into plasmid pET-30a via restriction endonuclease digestion. The recombinant plasmid was transformed into E. coli BL21 (DE3). The VgrG-His fusion protein were expressed by isopropyl-beta-D-thiogalactopyranoside (IPTG) induction at a final concentration of 1 mM. The 73 kDa VgrG-His fusion protein was purified and the final protein concentration was determined by the Bradford method. The polyclonal anti-VgrG antibodies were produced in New Zealand White rabbits.2. Construction and characterization of T6SS2 vgrG gene mutant strain and complementary strain in avian pathogenic Escherichia coli.In order to determine the effect of T6SS2 core component VgrG on the virulence of APEC, the vgrG gene mutant strain was conducted using Red homologous recombination system. The vgrG gene was complementary into mutant strain by plasmid pSTV28. Then, the growth curve, motility, biofilm formation, adhesion and invasion capacity to DF-1 cells and virulence to duck of wild-type strain, mutant strain and complementary strain were determined. The mutant strain and complementation strain showed well genetic stability, and deletion of vgrG did not affect growth kinetics, motility, nor the biofilm formation of APEC. The inactivation of vgrG in APEC DE719 led to the increased adhesion capacity to DF-1 cells. However, the vgrG gene mutant strain showed significantly decreased survival ability and attenuated virulence in ducks. These data indicated that T6SS2 core component VgrG was involved in the process of APEC infection, which would help us to comprehensive understand the pathogenicity of APEC.3. Construction and characterization of cpxR gene mutant strain and complementary strain in avian pathogenic Escherichia coliTo examine the roles of cpxR in virulence and regulation of T6SS2 in APEC, the cpxR gene mutant strain and its complementary strain were constructed using Red homologous recombination system and plasmid pSTV28. Then, the growth curve, motility, biofilm formation, adhesion and invasion capacity to DF-1 cells and virulence to duck of wild-type strain, mutant strain and complementary strains were determined. Meanwhile, we investigated the effect of cpxR gene on the expression of T6SS2 core components. The mutant strain and complementation strain showed well genetic stability, and deletion of cpxR did not affect growth kinetics, motility and biofilm formation of APEC. The inactivation of cpxR resulted in reduced serum resistance, decreased invasion capacity to DF-1 cells, decreased survival and attenuated virulence in ducks. The deletion of cpxR resulted in the decreased expression of T6SS2genes vgrG、hcp1、hcp2, which might be a reason for the decreased invasion capacity and defective virulence.