SspH2 As Anti-inflammatory Candidate Effector And Its Contribution In Salmonella Enteritidis Virulence

Salmonella are Gram-negative bacteria represent one such example of a successful pathogen.Salmonella one of a numerous examples pathogens in the world of host-pathogen interactions.Salmonella pathogenesis is dependent on its ability to enter and replicate within host cells.Bush and pull of war between a pathogen and its host has been one of the most incredible stories in the field of microbial pathogenesis for centuries.The outcome of an infection is the result of a balance between the in vivo environment where the bacteria survive and grow and the regulation of fitness genes at a level sufficient for the bacteria to retain their characteristic rate of growth in a given host.It is of greatest interest to understand the mechanisms behind the successful habitation of a pathogen inside the host body.Salmonella can cause food poisoning,gastrointestinal inflammation,typhoid fever depending on the Salmonella serotypes and the host.After overpass the intestinal mucosa,Salmonella infects macrophages and spreads via the blood to the liver and spleen where it multiplies intracellularly.The ability of Salmonella to survive persistently within the host,while fighting against the complex anti-bacterial immune system,is attributable to a repertoire of virulence-associated genes known as Salmonella Pathogenicity Islands(SPIs).The way of entrance and the strategy to continuously survive inside the target cells varies according to the type of cell and depends on the sequential expression of particular genes by Salmonella.Following invasion the bacteria remain within a modified phagosome known as the Salmonella containing vacuole(SCV),within which they will survive and replicate.Invasion and SCV biogenesis are dependent on two Type ? Secretion Systems(T3SS),T3SS1&T3SS2,which are used to translocate distinct units of bacterial effector proteins into the host cell.It is apparent that SCV biogenesis is a really dynamic process involving extensive cell reprogrammed process such as apoptosis,membrane remodeling,interactions with the endolysosomal pathway,actin rearrangements and microtubule-based movement and tubule extension.Salmonella pathogenesis studies to date have been focused on Salmonella Typhimurium,while the pathogenesis of Salmonella Enteritidis,is poorly understood.The interaction between Salmonella Enteritidis and the host immune system still create a question mark in the field of immunology.This study was designed to study the characteristics of the SspH2 as effector from SPI2 to investigate the effects of the gene in host immune response and it is contribution on SE virulence.To better understand the pathogenesis effects of SspH2,we successfully constructed the recombinant strains C50336-sspH2-and C50336-sspH2+based on Salmonella Enteritidis C50336-WT strain.In the following parts we will outline the findings of each experiment to detect(i)the characterizations of sspH2 gene(ii)role of SspH2 effector in bacteria survival inside the host cells and(iii)eliciting immune responses in host.1.Construction and characteristics analysis of a SspH2 mutant of Salmonella EnteritidisIn this section SspH2 gene(?2.367 kb)deleted mutant and complementary strains of SE C50336 were constructed using ?-red recombinant system and pBR322 mediated vector,respectively.The biological characteristics such as growth rate,LD50 and virulence were evaluated between the constructed strains C50336-SspH2-and C50336-SspH2+ and their parent C50336-WT.The growth rate was consistent with all strains.The animal test showed that the LD50 of C50336-SspH2-was-30 fold higher than that of the other two strains.To better understand the the role of the SspH2 gene in survival and replication,cellular infection model was done by 1×107CFU from each strain used Caco2-BBE cell line.The analysis of data showed the copy number of internalized bacteria was low in Mutant group compared to WT and Complementary groups,8h p.i.record the high peak.While at 24h p.i.,WT and Complementary strains continue to replicate by high rate compared to Mutant group.These results demonstrated that SspH2 effector strongly has a role in invasion and may participate in the late stage of SCV biogenesis process.In vivo study was established as an exploratory experiment to clarify SspH2 effector role in SE C50336 pathogenicity.C57BL/6 mice were infected by 108 CFU and bacteriological counts from tissue homogenates and histological scores were done.The result of colonization in internal organs showed that SspH2 increase the bacteria colonization in internal organs of mice.The rate of virulence in Mutant was low that leads to SspH2 effector increase the virulence of SE C50336.2.The role of SspH2 effector in host immunity elicitingIn this part qRT-PCR technique was used to investigate the role of SspH2 effector in host immune response.in vitro and in vivo RNA extracted was used,the result clearly showed that exposure of villus-like Caco-2 cells and tissues from C57BL/6 mice infected by S.Enteritidis C50336 mutant strain reported low level of IL-8 and increase the level IL-I?,INF-?,IL-12b and iNOS cytokines.That leads to SspH2 is anti-inflamatory effector.Principal FindingsThese study help in developing an understanding the contribution in virulence and immune-response effects of the SspH2 effector.The outputs will shed light and put baseline information on SspH2 effector role in SE life cycle.Under this present data we can conclude(1)the result of LD50 lead to show that SspH2 increase the virulence of SE C50336(30-fold)(2)Looking at different profiles of cytokines expression SspH2 has anti-inflammatory effects in cell response.(3)It is a virulence gene increase the severity of disease.(4)Increase the colonization of SE in internal organs.(5)It is a "Ghost gene" masking the race of Salmonella and presenting the profile of cytokines as anti-inflammatory response by downregulation the pro-inflammatory cytokines expression level.