Effects Of Streptococcus Pneumoniae Autolysin LytA On ISG Responses Of Macrophages

Objective: To investigate whether Streptococcus pneumoniae(S.pn)autolysin LytA influences the ISG15 conjugation system in macrophages through affecting the abnormal accumulation of S.pn-DNA in the cytoplasm of macrophages,and further regulates the phagocytic ability of macrophages.Background: S.pn autolytic enzyme LytA encoded by lyt A gene acts on peptidoglycan of bacterial cell wall to hydrolyze it,causing S.pn autolysis in the stationary growth phase and the release of a variety of cytoplasmic contents,such as Ply and S.pn-DNA.Studies have shown that the abnormal cytosolic S.pn-DNA in cells upregulates the expression of IFN-Ⅰ and ISGs through DAI/STING/IRF3 signaling pathway.ISG15,a kind of Ub L protein,is one of the most robustly upregulated ISGs,which has been proved to play an essential role in host defenses against virus,bacteria and fungus infection.However,whether S.pn autolysin LytA affects ISG15 conjugation during S.pn infection remains elusive.Previous studies suggested that LytA deficiency inhibits the release of S.pn-DNA,and S.pn-DNA can induce ISG15.Therefore,we speculate that S.pn autolysin LytA regulates phagocytic capacity of macrophages by affecting the abnormal accumulation of S.pn-DNA in the cytoplasm of macrophages and influnceing IFN-Ⅰ responses and its downstream ISG15 expression.Methods and Results: 1.RAW264.7 cells,PEMs,A549 cells and MEF cells were divided into NC group,Lipo 2000 treared group,S.pn D39 infected group and S.pn-DNA stimulated group.q PCR and WB were then used to determine the expression of ISG15 and ISGylated proteins.Compared to NC group and Lipo 2000 treated group,S.pn D39 and S.pn-DNA stimulation significantly upregulated the levels of ISG15 and ISGylation;and the expression of ISG15 was dose-dependent on IFN-β.2.RAW264.7 cells were divided into NC group,S.pn D39 infected group and S.pn D39 Δlyt A infected group.The levels of ISG15 and ISGylation were detected by q PCR and WB in order to explore the affect of S.pn autolysin LytA on ISG15 conjugation system.Compared to NC group and S.pn D39 infected group,ISG15 and ISGylated proteins were significantly increased in S.pn D39 Δlyt A infected macrophages.3.RAW264.7 cells were divided into NC group,S.pn D39 infected group,S.pn D39 Δlyt A infected group and r LytA+S.pn D39 Δlyt A stimulated group and were immunofluorescence stained.The abnormal cytosolic DNA in macrophages was observed under a laser scanning confocal microscope and the copy numbers of 16 S r DNA and gyr B,the conserved consequences of S.pn genome,were quantified by q PCR.The amount of cytosolic S.pn-DNA in S.pn D39 Δlyt A infected macrophages was significantly higher than that in S.pn D39 group,while exogenous r LytA supplementation could inhibit this abnormal accumulation.4.RAW264.7 cells were divided into NC group,S.pn D39 infected group,S.pn D39 Δlyt A infected group and r LytA+S.pn D39 Δlyt A stimulated group.Then phagocytosis and killing tests were carried out and the expression of inflammatory cytokines TNF-α and IL-1β was measured by q PCR.The phagocytosis of S.pn D39 Δlyt A infected macrophages was enhanced while killing ability was attenuated when compared to S.pn D39 infected group,which could be reversed by exogenouse r LytA supplementation.Additionaly,the levels of inflammatory cytokines TNF-α and IL-1β were significantly increased in S.pn D39 Δlyt A infected group than in S.pn D39 group.Conclusions: This study was the first to report that the important virulence protein LytA of S.pn inhibited ISG15 conjugation activated by S.pn-DNA by reducing abnormal cytosolic S.pn-DNA accumulation in macrophages and prevented the phagocytosis of macrophages to S.pn by inducing bacterial autolysis.We testified that the loss of S.pn LytA further significantly upregulated ISG15 level induced by S.pn and increased inflammatory cytokines including TNF-α and IL-1β expression in macrophages.We also found that the phagocytosis of macrophages to S.pn D39 Δlyt A was enhanced and the clearance of it was promoted.Our finding will help to further understand the regulatory mechanisms of immune responses mediated by bacterial autolysis and bacterial DNA release,providing new experimental basis for the prevention and treatment of S.pn infectious diseases.