Shengnan SONG, Yajun YANG, Hai JIANG, Meihua YANG, Yuanzhi WANG

Deletion of lpsA Gene of Brucella melitensis Strain M5-90 Promotes Caspase-11 Induced Non-classical Pathways Pyroptosis in Brucella-infected Mouse Macrophage Cells

Brucella utilizes diverse virulence factors to modulate the infectious cycle and lifestyle associated with eukaryotic hosts. Lipopolysaccharides (LPS) play an important role in the establishment of persistent infections of Brucella, but its mechanism in host cell to interfere with the host’s specific signaling pathway related to the elimination of Brucella is still not clear. LpsA, a glycosyl transferase, involves in the biosynthesis of LPS, and may aff ect the intracellular survival of Brucella. This study aimed to investigate the eff ects of lpsA on pyroptosis in Brucella-infected macrophage cells. We constructed the lpsA mutant strain (M5-90ΔlpsA) and the complementary strain (M5-90ΔlpsA-C) of Brucella melitensis strain M5-90 infected mouse macrophages to detect pyroptosis. We found that the inactivation of the lpsA gene weakened the ability of LPS gains access to cytosol during Brucella mutant infections, and reduced the survival of Brucella. Western blot and quantitative real-time PCR assays showed that the mRNA and protein levels of caspase-11 and NOD-like receptor family pyrin domain-containing 3 in M5-90ΔlpsA group were higher than those observed in M5-90 group. These results indicated M5-90ΔlpsA promoted the pyroptosis of RAW264.7 cells (a mouse macrophage cell line) after Brucella infection. The finding reveals that Brucella lpsA plays a partial role in innate immunity and infl ammatory response by inhibiting the LPS-induced atypical pyroptosis pathway.

Brucella melitensis M5-90 Suşunun lpsA Geninin Silinmesi Brucella İle Enfekte Fare Makrofaj Hücrelerinde Kaspaz-11 ile İndüklenen Klasik Olmayan Piroptozis Yolaklarını Teşvik Eder

Brucella, ökaryotik konakçılarla ilişkili enfeksiyon döngüsünü ve yaşam biçimini düzenlemek için çeşitli virülens faktörlerini kullanır. Lipopolisakkaritler (LPS), Brucella’nın kalıcı enfeksiyonlarının oluşmasında önemli bir rol oynar, ancak konakçı hücrede, Brucella’nın ortadan kaldırılmasıyla ilgili konakçının spesifik sinyalizasyonuna müdahale etme mekanizması halen net değildir. Bir glikozil transferaz olan LpsA, LPS’nin biyosentezinde görev alır ve Brucella’nın hücre içi yaşama yeteneğini etkileyebilir. Bu çalışmada lpsA’nın Brucella ile enfekte makrofaj hücrelerinde piroptoz üzerine etkilerinin araştırılması amaçlanmıştır. Piroptozu saptamak için Brucella melitensis M5-90 suşunun lpsA mutantı (M5-90ΔlpsA) ve komplementer suşu (M5- 90ΔlpsA-C) ile enfekte olmuş fare makrofajları oluşturduk. LpsA geninin inaktivasyonunun, LPS’nin Brucella mutant enfeksiyonları sırasında sitozole giriş yeteneğini zayıfl attığını ve Brucella’nın yaşama yeteneğini azalttığını saptadık. Western Blot ve kantitatif real-time PCR deneyleri, M5-90 grubuna oranla M5-90ΔlpsA grubunda kaspaz-11 ve NOD-benzeri reseptör ailesi pyrin domain-containing 3’ün mRNA ve protein seviyelerinin daha yüksek olduğunu gösterdi. Bu bulgular, M5-90ΔlpsA’nın, Brucella enfeksiyonu sonrası bir fare makrofaj hücre hattı olan RAW264.7 hücrelerinde piroptoza yol açtığını gösterdi. Bu bulgu, Brucella lpsA’sının, LPS ile indüklenen atipik piroptoz yolunu inhibe ederek doğal bağışıklık ve enfl amatuar yanıtta kısmi bir rol oynadığını ortaya koymaktadır..

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