Construction Of Bacillus Subtilis Spore Mutants And Their Immunomod?lation Effects On Porcine Alveolar Macrophages(M?3D4/2)

Bacillus subtilis is one of the probiotics that have been widely used for enhancing animal intestinal health.Bacillus subtilis can exert immunoreg?latory functions in the form of vegetative cells or spores in vitro.However,spores can better adapt to the gastrointestinal environment than vegetative cells and play an important role in immune-modulation.However,there is limited information about the immune-regulation mechanism of spores.In order to investigate immune-regulatory functions of Bacillus subtilis spore mutants on porcine alveolar macrophages(M?3D4/2),three experiments were performed as follows:(1)Six genes spoIVA?saf A?cotE?cotX?cotZ and cgeA,which are crucial for assembly of the spore coat,were knocked out from chromosomal DNA of Bacillus subtilis 1A747,individually.(2)We tested the growth,spor?lation and germination abilities of mutant strains B.subtilis spoIVA~-?B.subtilis safA~-?B.subtilis cot E~-?B.subtilis cotX~-?B.subtilis cotZ~-and B.subtilis cgeA~-.Transmission electron microscope was used to observe the changes of the spore coat.(3)After mutant strains were co-cultured with M?3D4/2 cells,mRNA expressions of Toll-like receptors and key genes in MAPK?NF-?B signalings in M?3D4/2cells were measured by qRT-PCR.In addition,the expression levels of cytokines were detected by ELISA.The results were summarized as follows:1.The sequencing results showed that the spore coat mutant strains of Bacillus subtilis were successfully constructed.2.The sporulation ability of B.subtilis spoIVA~-was seriously impaired.So,B.subtilis spoIVA~-was not used for subsequent experiments.In contrast,other mutants could sporulate and germinate normally like the wild strain.Transmission electron microscopy of the spores revealed that the spore structure of B.subtilis spoIVA~-was disordered,the spore inner coat of B.subtilis safA~-was fuzzier and thinner than the wild strain,and B.subtilis cotE~-spore didn't show obvious outer coat.3.All of the mutant strains spores significantly increased TLR4 activation at mRNA level(P<0.05).Among the 5 mutant strains,B.subtilis cotX~-had the strongest ability to increase the transcriptional level of MyD88?IRAK1?NF-?B2 and the production of IL-1?and IL-8(P<0.05).B.subtilis cgeA~-promoted the transcription of NF-?B2 and production of all of the pro-inflammatory cytokines(P<0.05).B.subtilis cotE~-activated MyD88 and NF-?B2 followed by enhancement of the expression of IL-8 and TNF-?(P<0.05).With B.subtilis safA~-,the mRNA level of IRAK1 and the production of IL-1??IL-8 and TNF-?were improved(P<0.05).B.subtilis cotZ~-also had the capacity to enhance the expression of inflammatory cytokines(IL-1?,IL-8 and TNF-?)in M?3D4/2 cells without affecting MyD88 signaling pathway(P<0.05).In conclusion,Bacillus subtilis showed immune-regulatory effects on the M?3D4/2 cells.The spore coat proteins played vital roles in the effects.B.subtilis cotX~-and B.subtilis cotE~-transmited signals through the MyD88-dependent pathway.At the same time,there might be other pathways involved and they will be studied in the future.