鸭疫里氏杆菌B739_RS00825基因缺失株抗血红素毒性和氧化应激损伤以及定殖能力分析

B739_RS00825-deleted strain of Riemerella anatipestifer CH-1:heme detoxication,oxidative stress resistance,and colonization

【目的】血红素可作为细菌重要的铁离子来源,然而转运过多的血红素也会对细菌造成毒性。细菌通过调节、外排、螯合等多种方式减轻血红素毒性作用。鸭疫里氏杆菌(Riemerella anatipestifer,RA)是一种感染鸭及其他禽类的革兰氏阴性病原菌。前期研究表明,该菌编码血红素转运系统,且能够从血红蛋白获取血红素,然而该菌是否编码血红素解毒蛋白未知。本研究以编码一氧化氮合成酶的基因B739_RS00825为研究对象,分析其在抗血红素毒性和氧化应激损伤以及定殖能力中的功能。【方法】构建B739_RS00825缺失株,并通过测定生长曲线、细菌存活率、毒力及定殖等试验方法鉴定其在抗血红素毒性、抗氧化应激损伤、宿主致病中的功能。【结果】与RA CH-1相比,RA CH-1ΔB739_RS00825在添加过量血红素的培养基中生长不受影响;然而与RA CH-1Δfur相比,RA CH-1ΔfurΔB739_RS00825在含血红素培养基中的生长明显受到抑制且对H2O2的抵抗力降低;B739_RS00825基因在氧化应激条件下及fur缺失株中明显上调;与RA CH-1相比,RA CH-1ΔB739_RS00825在雏鸭的毒力和定殖能力无明显减弱。【结论】B739_RS00825基因参与了该菌抗血红素毒性和抗氧化应激反应的功能,且受铁摄取调节子(ferric uptake regulator,Fur)调控。然而,B739_RS00825基因并未参与该菌在宿主体内的定殖。

[Objective]The heme of host is an important iron source for pathogenic bacteria.However,excessive heme can cause damage to bacteria.Bacteria can reduce the heme toxicity through regulation,efflux,and chelation.Riemerella anatipestifer is a Gram-negative bacterium that infects ducks and other birds.The available studies have demonstrated that R.anatipestifer encodes a heme transport system to obtain heme from host hemoglobin.However,we are not clear whether this bacterium encodes heme detoxification protein or not.In this study,we analyzed the roles of B739_RS00825 encoding nitric oxide synthase in heme detoxification,oxidative stress resistance,and host colonization.[Methods]We constructed the B739_RS00825-deleted strain and studied its roles in heme detoxification,oxidative stress resistance,and host colonization through establishing the growth curve and determining the survival rate under H2O2 stress,the lethality to ducklings,and the colonization ability in ducklings.[Results]Compared with that of R.anatipestifer CH-1,the growth of CH-1ΔB739_RS00825 in the medium supplemented with excessive heme was not affected.However,compared with CH-1Δfur,CH-1ΔfurΔB739_RS00825 showed significantly inhibited growth in the medium containing excess heme and weakened resistance to H2O2.The transcription of B739_RS00825 was significantly upregulated under oxidative stress and in CH-1Δfur.Compared with CH-1,CH-1ΔB739_RS00825 did not show attenuated lethality or colonization in ducklings.[Conclusion]Gene B739_RS00825 was involved in the heme detoxification and oxidative stress resistance of R.anatipestifer and it was regulated by ferric uptake regulator(Fur).However,the gene was not involved in the lethality or colonization of R.anatipestifer in ducklings.