Division of Infection and Immunity, International Institute for Zoonosis Control, Hokkaido University, Sapporo, Japan.
GenEndeavor LLC, 26219 Eden Landing Rd, Hayward, CA, 94545, USA.
BMC Microbiol. 2024 Sep 17;24(1):351. doi: 10.1186/s12866-024-03492-1.
Bacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA's roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished.
We used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar.
Through LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3' end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567.
B. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA.
蜡样芽胞杆菌是一种革兰氏阳性、产芽孢的细菌，它产生一系列与细菌生态位适应和各种感染发展相关的效应子。其中 EsxA 是一种分泌蛋白，其分泌依赖于 VII 型分泌系统（T7SS）的 EssC 成分。尽管推测 EsxA 可能参与芽孢形成，但它在细菌细胞内的作用仍不清楚。然而，蜡样芽胞杆菌的 T7SS 库尚未被报道，其功能也尚未确定。
我们使用模式菌株蜡样芽胞杆菌 ATCC14579 通过同源重组生成ΔessC 突变体，使用归巢内切酶 I-SceI 介导的无标记基因替换。通过液相色谱-串联质谱（LC-MS/MS）比较分析了模式菌株和ΔessC 突变体的培养上清液。我们进一步生成 T7SSb 特异性基因突变，以探索 T7SSb 依赖性效应子的管家作用。通过经典的 Schaeffer-Fulton 染色方法观察蜡样芽胞杆菌 ATCC14579 及其突变体的芽孢形成过程。通过在 LB 琼脂上计数集落形成单位来确定本研究中每个菌株的孢子活力。
通过 LC-MS/MS，我们在野生型菌株的培养上清液中鉴定出一对几乎相同（94%）的效应蛋白 EsxA，属于 WXG100 蛋白超家族的 sagEsxA 样亚家族，而在ΔessC 突变体中则没有。对来自不同蜡样芽胞杆菌菌株的 T7SSb 基因簇进行同源性分析，发现其 3'端的 essC 编码了额外的底物，具有多样性。删除 esxA1 和 esxA2 基因既不会改变细胞形态也不会影响生长速度，但ΔesxA1ΔesxA2 缺失导致可存活的孢子数量明显减少，且整体芽孢形成过程较慢。在 24 小时培养过程中，野生型细胞中有超过 80%形成内生孢子，而在ΔesxA1ΔesxA2 突变体中不到 5%。野生型和ΔesxA1ΔesxA2 的最大孢子比例分别为 0.96 和 0.72。总之，这些结果表明 EsxA1 和 EsxA2 协同作用，是蜡样芽胞杆菌 ATCC14567 芽孢形成所必需的。
蜡样芽胞杆菌 ATCC14579 拥有两种几乎相同的依赖于 T7SSb 的效应子，属于 sagEsxA 样蛋白。同时删除这些效应子的编码基因显著延迟和减少了芽孢形成，这是 EsxA 的一个新发现。
