Department of Biology, College of Science, University of Al-Qadisiyah, Al Diwaniyah, Iraq.
Department of Basic Science, College of Nursing, University of Kufa, Kufa, Iraq.
Braz J Microbiol. 2023 Jun;54(2):609-618. doi: 10.1007/s42770-023-00957-9. Epub 2023 Mar 27.
Shigellosis remains a worldwide health problem due to the lack of vaccines and the emergence of antibiotic-resistant strains. Shigella (S.) dysenteriae has rigid peptidoglycan (PG), and its tight regulation of biosynthesis and remodeling is essential for bacterial integrity. Lytic transglycosylases are highly conserved PG autolysins in bacteria that play essential roles in bacterial growth. However, their precise functions are obscure. We aimed to identify, clone, and express MltC, a unique autolysin in Escherichia (E.) coli C41 strain. The purification of recombinant MltC protein was performed using affinity chromatography and size-exclusion chromatography methods. The PG enzymatic activity of MltC was investigated using Zymogram and Fluorescein isothiocyanate (FITC)-labeled PG assays. Also, we aimed to detect its localization in bacterial fractions (cytoplasm and membrane) by western blot using specific polyclonal anti-MltC antibodies and its probable partners using immunoprecipitation and mass spectrometry applications. Purified MltC showed autolysin activity. Native MltC showed various locations in S. dysenteriae cells during different growth phases. In the Lag and early stationary phases, MltC was not found in cytoplasm and membrane fractions. However, it was detected in cytoplasm and membrane fractions during the exponential phase. In the late stationary phase, MltC was expressed in the membrane fraction only. Different candidate protein partners of MltC were identified that could be essential for bacterial growth and pathogenicity. This is the first study to suggest that MltC is indeed autolysin and could be a new drug target for the treatment of shigellosis by understanding its biological functions.
志贺氏菌病仍然是一个全球性的健康问题，这是由于缺乏疫苗和抗生素耐药菌株的出现。志贺氏菌（S.）痢疾有刚性肽聚糖（PG），其生物合成和重塑的严格调节对于细菌的完整性至关重要。溶菌酶是细菌中高度保守的 PG 自溶酶，在细菌生长中起着重要作用。然而，它们的确切功能尚不清楚。我们的目的是鉴定、克隆和表达大肠杆菌 C41 株中独特的自溶酶 MltC。使用亲和层析和分子筛层析方法纯化重组 MltC 蛋白。使用 Zymogram 和荧光素异硫氰酸酯（FITC）标记的 PG 测定法研究 MltC 的 PG 酶活性。此外，我们还旨在通过使用特异性多克隆抗 MltC 抗体的 Western blot 检测其在细菌部分（细胞质和膜）中的定位，并通过免疫沉淀和质谱应用检测其可能的伴侣。纯化的 MltC 表现出自溶酶活性。天然 MltC 在不同生长阶段的 S. dysenteriae 细胞中显示出不同的位置。在迟滞期和早期稳定期，细胞质和膜部分中未发现 MltC。然而，在指数生长期检测到它在细胞质和膜部分。在晚期稳定期，MltC 仅在膜部分表达。鉴定了 MltC 的不同候选蛋白伴侣，这些伴侣可能对细菌生长和致病性至关重要。这是第一项研究表明，MltC 确实是一种自溶酶，通过了解其生物学功能，可能成为治疗志贺氏菌病的新药物靶点。
