Differential Study Of Distinct Virulence Of Nocardia Cyriacigeorgica Strains In Host Cells

Objectives In this study,we compared the virulence of N.cyriacigeorgica ATCC14759 and N.cyriacigeorgica GUH-2,explored their differences in the activation of the innate immune signaling pathway in host cells and in the expression of cytokines in vitro and in vivo,which will lay a foundation for studying the pathogenic mechanisms of Nocardia strains.Methods In order to investigate the effects of the two strains on hosts,we compared the virulence between N.cyriacigeorgica ATCC14759 and N.cyriacigeorgica GUH-2 by measuring cytotoxicity,the ability of host cell invasion,viability in host cells and the survival of BALB/c mice after infection.Western blotting was used to compare the differences in activation of MAPKs(including p38,ERK,and JNK),the NF-?B signaling pathway,and the PI3K/Akt signaling pathway in A549 and RAW 264.7cells.BALB/c mice were infected by nasal drip,and then the bacterial loads in the lung and serum of mice at different time intervals were counted;ELISA was used to detect the expression of cytokines in vitro and in vivo at different time intervals;SPSS 23.0 software was used for statistical analysis.Results A series of virulence-related tests indicated that N.cyriacigeorgica ATCC14759 exhibits higher cytotoxicity in A549 cells and RAW 264.7 cells,higher lethality in mice,superior invasion ability in Hela cells,A549 cells and RAW 264.7 cells at 1 h post-infection,and superior viability in A549 cells and RAW 264.7 cells.Western Blot showed that both these two strains could activate MAPK signaling pathway.Notably,A gradual increase in JNK and p38 levels was observed at in N.cyriacigeorgica GUH-2-infected cells compared to N.cyriacigeorgica ATCC14759-infected cells,whereas the levels of p-ERK were significantly higher in N.cyriacigeorgica ATCC14759-infected cells than in N.cyriacigeorgica GUH-2-infected cells.In the activation of NF-?B signaling pathway,both strains could activate p65,and no significant difference in the degree of activation was observed.Moreover,in the activation of PI3K/Akt signaling pathway,N.cyriacigeorgica ATCC14759 led to a stronger expression in p-AKT levels than N.cyriacigeorgica GUH-2.In the experiment to detect the concentration of cytokines in RAW 264.7 cells stimulated with N.cyriacigeorgica by ELISA,We consistently found higher concentrations of IL-6 and IL-10 in N.cyriacigeorgica GUH-2-infected cells than in N.cyriacigeorgica ATCC14759-infected cells,both at 12 h and at 24 h.In addition,we observed an increase in TNF-?levels upon infection,and the difference between these two groups was not statistically significant.98.35% of the bacteria were eliminated in BALB/c mice at 48 h post-infection when infected with 2×107 CFU N.cyriacigeorgica GUH-2 by nasal drip.In the early stage of infection,the concentrations of IL-2,IL-4,IL-6,IL-10,IL-12,TNF-? and IFN-? in the lung tissue were significantly higher than those in the control group(P <0.01),then the concentrations of these cytokines showed a trend of decline as time goes by;In serum,the concentrations of IL-6(6 h-24 h),IFN-?(12h-24 h)and IL-10(48 h)were higher than those in the control group(P <0.01),the concentrations of IL-12(12 h-48 h)and IL-4(12 h)were significantly inhibited when compared with the control group(P < 0.01),the secretions of TNF-? and IL-2 were at a low level in both the control group and the experimental group(P > 0.05).In addition,there were positive correlations between the concentrations of cytokines and bacterial loads in the lung tissue.Conclusion N.cyriacigeorgica ATCC14759 is more virulent than N.cyriacigeorgica GUH-2: it is more cytotoxic,more viable in host cells,and more lethal to mice after infection.Furthermore,the signaling pathways that are activated upon infection with either of these strains show similarities and differences,suggesting their pathogenesis processes are different.No significant difference was observed in the activation of the NF-?B pathway,however,the requirement for MAPK activation and the release of cytokines upon infection differ between the two strains.Moreover,N.cyriacigeorgica ATCC14759 infection led to higher p-AKT levels than N.cyriacigeorgica GUH-2.In the process of infection with N.cyriacigeorgica in mice,IL-6,TNF-?,IL-12,IL-2,IFN-?,IL-10 and IL-4were involved in the inflammatory response in mice,especially the high concentrations of IL-6,IL-12,IFN-? and IL-10.There were upregulated signaling pathways and cytokines in the pathogenic mechanisms of Nocardia strains.The regulatory network formed by the interaction of cytokines play a significant role in the process of eliminating Nocardia infection.