Intramacrophage Proliferation Ability Of Salmonella SpiC Mutants And Screening Of Drugs Against Intracellular Salmonella

Salmonella spp.is one of the important zoonotic pathogens that can cause serious diseases on humans and animals.When Salmonella infects host,it can proliferate in intestinal epithelial cells and survive in macrophages,and enter the bloodstream for systemic infection.The survival of Salmonella in macrophages is an important factor for its spreading outside the intestinal tract,which cause systemic infection.The anti-inflammatory cytokine IL-10 and Salmonella containing vacuole(SCV)can protect intramacrophage Salmonella not to be killed,but the mechanisms on Salmonella inducing IL-10 expression and promoting SCV formation in macrophages are unclear.The proliferation of intramacrophage Salmonella is mainly regulated by T3SS2.SpiC is a major effector protein of T3SS2 and mediates the function of T3SS2,but the correlation of SpiC with Salmonella intramacrophage proliferation and IL-10 expression also remains unclear.SarA is one of T3SS2 effectors,which can induce macrophages to produce high level of IL-10 by activating STAT3 to improve the proliferation ability of intramacrophage Salmonella Typhimurium.SifA is an important T3SS2 effector that maintains the integrity of the SCV membrane,which enables Salmonella to stay in the SCV and escape host immune response.In this study,the spiC mutant of Salmonella Typhimurium was constructed,and the sarA and sifA mutants were used as control strains to analyze the proliferation and IL-10 level in macrophages.In addition,it was further verified whether spiC mutants of other three Salmonella serotypes had same abilities of promoting proliferation and inducing IL-10 in macrophages.Intracellular Salmonella is killed difficultly by common antibacterial drugs because they can not enter cells,and persist in macrophages to cause host long-term infection.At present,the proliferation and persistence of intracellular Salmonella is a major problem in the clearance of salmonellosis in livestock and poultry industry.In order to eradicate salmonellosis,some safe and effective drugs against intracellular Salmonella shall be found by the strategy of "old drugs-new uses",and so one drug against intracellular Salmonella was screened out from 66 drugs by using a model of Salmonella spiC mutant with strong intramacrophage proliferation ability,and its antibacterial effect was further evaluated.1 Intramacrophage proliferation ability of Salmonella spiC mutantsAccording to the principle of homologous recombination,SL1344ΔspiC,SL1344ΔsarA,SL1344ΔspiCΔsarA,SL1344ΔsifA and SL1344ΔspiCΔsifA were constructed by using the suicide plasmid pGMB152,and their biological characterizations were analyzed.The results showed that the biochemical characteristics and growth rate of these mutants were consistent with the wild-type strain,and there was no significant difference in formation of biofilm.Swarm motility of spiC mutant increased,while that of SL1344ΔsarA decreased.The loss of spiC,sarA and sifA genes had no effect on the adhesion rate and invasion rate of Salmonella to RAW264.7 cells.Compared with the wild-type strain,SL1344ΔspiC could induce macrophages to produce high level of IL-10,and its intramacrophage proliferation ability significantly increased;SL1344ΔsarA induced low level of IL-10,and its intracellular proliferation ability decreased.Complete SCV could not be formed after sifA gene being deleted,and the proliferation ability in macrophages decreased.However,SL1344ΔspiC enabled macrophages to form many SCVs.Therefore,the spiC mutant increased the ability of intracellular proliferation by inducing high level of IL-10 and forming SC Vs.Further,the spiC mutants of Salmonella Enteritidis,Salmonella Dublin,and Salmonella Pullorum also had the same abilities of promoting intramacrophage proliferation and inducing high level of IL-10 with the spiC mutant of Salmonella Typhimurium,which confirmed that the phenomenon about the spiC mutants of different Salmonella serotypes in promoting the proliferation of Salmonella in macrophages and inducing high level of IL-10 was universal.2 Screening and evaluation of drugs against intracellular SalmonellaTo screen drugs against intracellular Salmonella needed a model.Although the wild-type strain of Salmonella could survive and proliferate in macrophages,but its high virulence could cause high inflammatory response,wild-type Salmonella was quickly eliminated by the host,which was not suitable for drugs screening.Salmonella spiC mutant had high proliferation ability in macrophages with long-term persistence,which was a good model to screen some drugs against intracellular Salmonella.Nifuratel(NIF)with high intracellular antibacterial activity and low cytotoxicity was selected as a candidate drug,then its antibacterial effect was further evaluated.MICs of NIF against STm SL1344,SD Sdu189,SE C50041 and SP S06004 were 200μM,200 μM,100 μM and 25 μM,respectively,and the MICs against multidrug-resistant Salmonella Typhimurium TZF 10 and TX 2-7 were 400 μM and 200μM.NIF had good intracellular antibacterial activity in murine macrophages RAW264.7 and J774A.1,avian macrophages HD-11 and human epithelial cells HeLa.At the same time,NIF could also reduce intracellular the number of Salmonella Typhimurium,Salmonella Enteritidis,Salmonella Dublin,Salmonella Pullorum,Shigella,Listeria monocytogenes and multidrug-resistant Salmonella Typhimurium.Animal experiments showed that NIF reduced the death rate in Salmonella-infected mice with lethal dose and the bacterial colonization in the liver and spleen of mice.25 μM NIF completely killed SE C50041 in RAW264.7,while its extracellular MIC was 100 μM.NIF might had other way to function.Preliminary exploration of its intracellular antibacterial mechanism showed that NIF might indirectly reduce the level of IL-10 by inhibiting STAT3 and p-STAT3 to decrease the proliferation ability of intracellular Salmonella.In conclusion,compared with the wild-type strain,STm SL1344ΔspiC improved the intracellular proliferation ability in macrophages,induce macrophages to secrete high level of IL-10.The spiC mutants of Salmonella Enteritidis,Salmonella Dublin and Salmonella Pullorum had the same abilities of intramacrophage proliferation,which proved the universality of this phenomenon.By a model that spiC mutant could persist for long-term because of high proliferation ability in macrophages,NIF was screened out to show good the effect against intracellular different Salmonella serotypes in different kinds of cells.It also decreased the proliferation ability of extracellular Salmonella and the death rate in mice infected with wild-type Salmonella.These results not only broadened our understanding on the mechanism of intracellular Salmonella proliferation,but also provided new ways to clear salmonellosis in livestock and poultry industry.