College of Life Sciences, Anhui Normal University, Wuhu, China.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Appl Environ Microbiol. 2024 Apr 17;90(4):e0228423. doi: 10.1128/aem.02284-23. Epub 2024 Mar 6.
Halocins are antimicrobial peptides secreted by haloarchaea capable of inhibiting the growth of other haloarchaea or bacteria. Halocin H4 (HalH4) is secreted by the model halophilic archaeon ATCC 33500. Despite attempts to express heterologously in and subsequent careful renaturation procedures commonly employed for haloarchaeal proteins, no active halocin was obtained. However, it was discovered that the antihaloarchaeal activity of this halocin could be activated through cleavage by halolysin R4 (HlyR4), a serine protease also secreted by ATCC 33500. Replacement of the cysteine at the number 115 amino acid with glycine and deletion of the internal trans-membrane region (15 aa) markedly abolished HalH4's antihaloarchaeal activity. Compared to the N-terminus, the C-terminal amino acid sequence was found to be more crucial for HalH4 to exert its antihaloarchaeal activity. Mass spectrometry analysis revealed that the biologically active antihaloarchaeal peptide produced after hydrolytic cleavage by HlyR4 was the C-terminus of HalH4, suggesting a potential mechanism of action involving pore formation within competitor species' cell membranes. Taken together, this study offers novel insights into the interplay between halocins and secreted proteases, as well as their contribution to antagonistic interaction within haloarchaea.
The antihaloarchaeal function of halocin H4 (HalH4) can be activated by extracellular proteases from haloarchaea, as demonstrated in this study. Notably, we report the first instance of halocin activation through proteolytic cleavage, highlighting its significance in the field. The C-terminus of HalH4 (CTH4) has been identified as the antihaloarchaeal peptide present in hydrolysates generated by HlyR4. The CTH4 exhibited inhibitory activity against a range of haloarchaeal species ( spp., spp., spp., spp., and spp.), as well as selected bacterial species ( spp. and spp.), indicating its broad-spectrum inhibitory potential across domains. The encoding gene of halocin HalH4, , from the model halophilic archaeon ATCC 33500 can be expressed in without codon optimization.
卤素有抗菌肽由haloarchaea 分泌能够抑制其他 haloarchaea 或细菌的生长。卤素 H4 (HalH4) 是由模型嗜盐古菌 ATCC 33500 分泌的。尽管试图在和随后的仔细复性程序中表达异源，通常用于 haloarchaeal 蛋白质，但没有获得活性卤素。然而，人们发现这种卤素的抗 haloarchaeal 活性可以通过halolysin R4 (HlyR4) 的切割激活，HlyR4 也是由 ATCC 33500 分泌的一种丝氨酸蛋白酶。用甘氨酸取代第 115 位氨基酸的半胱氨酸，并删除内部跨膜区（15 个氨基酸），明显消除了 HalH4 的抗 haloarchaeal 活性。与 N 端相比，C 端氨基酸序列对 HalH4 发挥抗 haloarchaeal 活性更为重要。质谱分析表明，经 HlyR4 水解切割后产生的具有生物活性的抗 haloarchaeal 肽是 HalH4 的 C 端，这表明其作用机制可能涉及竞争物种细胞膜内形成孔道。总的来说，这项研究为卤素和分泌蛋白酶之间的相互作用以及它们对 haloarchaea 内拮抗相互作用的贡献提供了新的见解。
本研究表明，halocin H4 (HalH4) 的抗 haloarchaeal 功能可被 haloarchaea 细胞外蛋白酶激活。值得注意的是，我们首次报道了通过蛋白水解切割激活卤素的实例，这凸显了它在该领域的重要性。HlyR4 产生的水解产物中的 HalH4 的 C 端（CTH4）已被鉴定为存在的抗 haloarchaeal 肽。CTH4 对一系列 haloarchaea 物种（ spp.， spp.， spp.， spp. 和 spp.）以及选定的细菌物种（ spp. 和 spp.）表现出抑制活性，表明其具有广谱抑制潜力。来自模型嗜盐古菌 ATCC 33500 的卤素 HalH4 的编码基因 ，无需密码子优化即可在 中表达。
