Chun Fang ZHOU, Xueyang CHEN, Xiongyan LIANG, Xiaowei FANG, Keli GAO, Jing CHEN, Yufang GU, Yuying YANG

The Effect of Single Amino Acid Substitution in SecA2 on Protein Translocation and Pathogenicity of Listeria monocytogenes

Listeria monocytogenes is an important zoonotic pathogen that cause severe listeriosis with high mortality in immunosuppressive humans. Infection and pathogenicity of L. monocytogenes was mediated by several surface proteins that translocated by secretion systems. Our previous genomic study showed the secretion systems of the virulent and low-virulent strains were different in secA2 and two hollin genes. To confirm whether the pathogenicity of the two strains was determined by the difference observed in secretion system. We deleted secA2 and the two hollin genes to compare the pathogenic phenotypes. Our data showed that secA2 but not the two hollin genes affected the pathogenic phenotypes. To further confirm whether the single base mutant in secA2 affected the protein pathogenicity and translocation ability of SecA2, we complemented the secA2 deletion mutant strain with secA2Lm850658 and secA2M7, which encode SecA2 with Asn567 and Lys567, respectively. Our data showed that secA2 mutant complement with secA2Lm850658 instead of secA2M7 significantly improved the adhesion and invasion ability to epithelial cells Caco-2 and bacterial load in mice liver and spleen at both 24 and 48 h post infection. Cell surface protein analysis indicated that only SecA2 with Asn567 could restore the protein translocation ability. Taken together, our study demonstrated that single amino acid mutant in SecA2 affected the protein translocation and pathogenicity of L. monocytogenes for the first time.

SecA2’de Tek Amino Asit Yerdeğiştirmesinin Listeria monocytogenes’te Protein Translokasyonu ve Patojenite Üzerine Etkisi

Listeria monocytogenes önemli bir zoonotik patojen olup immunsupresyone insanlarda yüksek mortaliteye sahip şiddetli listeriozise neden olur. L. monocytogenes enfeksiyonu ve patojenitesi sekresyon sistemi tarafından salınan çeşitli yüzey proteinleri tarafından oluşturulur. Yapılan önceki genomik çalışma virülent ve düşük-virülent suşların sekresyon sistemlerinin secA2 ve iki hollin genlerinde farklı olduğunu gösterdi. İki suşun patojenitelerindeki farkın sekresyon sistemlerinde gözlenen farktan kaynaklanıp kaynaklanmadığı araştırıldı. Çalışmada, patojenik fenotipleri karşılaştırmak amacıyla secA2 ve iki hollin geni çıkarıldı. Elde edilen sonuçlar iki hollin geninin değil de secA2 geninin patojenik fenotipi etkilediğini gösterdi. secA2 geninde tek baz mutantının protein patojenitesini ve SecA2’nin translokasyon kabiliyetini etkileyip etkilemediğini test etmek amacıyla secA2 gen delesyon mutant suşunu sırasıyla Asn567 ve Lys567’i kodlayan secA2Lm850658 ve secA2M7 ile tamamladık. Elde edilen veriler, secA2Lm850658 ile tamamlanan secA2 mutantının anlamlı derecede Caco-2 epitel hücrelerine adezyon ve invazyon kabiliyeti ile fare karaciğer ve dalak dokularında enfeksiyon sonrası 24 ve 48. saatlerde bakteriyel yığılmayı artırdığını gösterdi. Hücre yüzey protein analizi, sadece Asn567’li SecA2’nin protein translokasyon kabiliyetini koruyabildiğini gösterdi. Tüm sonuçlar değerlendirildiğinde, L. monocytogenes SecA2’de tek amino asit mutasyonunun protein translokasyonu ve patojeniteyi etkilediği ilk kez gösterilmiştir.

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