Lin Chang Sheng-Huei, Vermeulen Jenny, Biboy Jacob, Gaynor Erin C, Vollmer Waldemar, Frirdich Emilisa
Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada.
Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom.
Front Microbiol. 2025 Sep 9;16:1641976. doi: 10.3389/fmicb.2025.1641976. eCollection 2025.
The helical morphology of is maintained by its peptidoglycan (PG) layer and influences its success as a pathogen. Periplasmic PG hydrolases that cleave the PG glycan backbone and peptide sidechains (such as carboxypeptidases and endopeptidases) are critical for proper cell function and/or growth and are important in the PG remodeling required for cell shape generation and any morphological alterations. The shape is determined by PG hydrolases Pgp1 (DL-carboxypeptidase), Pgp2 (LD-carboxypeptidase) and Pgp3 (DD-carboxypeptidase/DD-endopeptidase), as well as a group of M23 peptidase domain containing proteins with previously uncharacterized activity: CJJ81176_1105, CJJ81176_1228, and CJJ81176_0166. Using a PG cleavage assay, we showed that 1105 and 1228 have DD-carboxypeptidase/DD-endopeptidase activity, and 0166 is a DD-carboxypeptidase. We renamed 1105, 1228, and 0166 to Pgp4 (peptidoglycan peptidase 4), Pgp5, and Pgp6, respectively. Pgp6 is the first described M23 peptidase with substrate selectivity on monomeric pentapeptides. Sequence comparisons between the DD-carboxypeptidase Pgp6 and the DD-carboxypeptidase/DD-endopeptidase Pgp3 (with an available crystal structure) and their corresponding orthologs revealed that Pgp6 contains insertion sequences in the M23 peptidase domain not present in Pgp3. Modeling of Pgp6 predicted that the insertion sequences would restrict the active site groove, only allowing entrance of a smaller substrate. This provides a possible explanation for the lack of Pgp6 DD-endopeptidase activity. To our knowledge, Pgp6 is the first reported DD-carboxypeptidase in the M23 peptidase superfamily. Deletions in , and resulted in mutants with varying curved rod morphologies and changes in PG muropeptide profiles in comparison to wild type and each other. Using these mutants, we examined the effect of deleting these genes on properties affecting pathogenesis and survival: motility, biofilm formation, autoagglutination, the ability to transition to a coccoid form, growth under varying pH, susceptibility to antimicrobial compounds, and adherence, invasion and intracellular survival in human epithelial cells. Each mutant showed distinct phenotypic changes to each other, indicating they are not functionally redundant. This also further supports the correlation between morphology and morphology-related genes with pathogenic potential.
其螺旋形态由肽聚糖（PG）层维持，并影响其作为病原体的致病性。能够切割PG聚糖主链和肽侧链的周质PG水解酶（如羧肽酶和内肽酶）对于细胞的正常功能和/或生长至关重要，并且在细胞形状生成和任何形态改变所需的PG重塑中发挥重要作用。其形状由PG水解酶Pgp1（DL-羧肽酶）、Pgp2（LD-羧肽酶）和Pgp3（DD-羧肽酶/DD-内肽酶）以及一组含有M23肽酶结构域且活性未知的蛋白质：CJJ81176_1105、CJJ81176_1228和CJJ81176_0166决定。通过PG切割试验，我们发现1105和1228具有DD-羧肽酶/DD-内肽酶活性，0166是一种DD-羧肽酶。我们分别将1105、1228和0166重新命名为Pgp4（肽聚糖肽酶4）、Pgp5和Pgp6。Pgp6是首个被描述的对单体五肽具有底物选择性的M23肽酶。DD-羧肽酶Pgp6与DD-羧肽酶/DD-内肽酶Pgp3（具有可用晶体结构）及其相应的直系同源物之间的序列比较显示，Pgp6在M23肽酶结构域中含有Pgp3中不存在的插入序列。Pgp6的建模预测，插入序列会限制活性位点凹槽，仅允许较小底物进入。这为Pgp6缺乏DD-内肽酶活性提供了一种可能的解释。据我们所知，Pgp6是M23肽酶超家族中首个被报道的DD-羧肽酶。与野生型以及彼此相比，Pgp4、Pgp5和Pgp6的缺失导致突变体具有不同的弯曲杆状形态以及PG muramopeptide谱的变化。利用这些突变体，我们研究了缺失这些基因对影响致病性和生存的特性的影响：运动性、生物膜形成、自凝集、转变为球菌形态的能力、在不同pH下的生长、对抗菌化合物的敏感性以及在人上皮细胞中的黏附、侵袭和细胞内存活。每个突变体彼此之间都表现出明显的表型变化，表明它们在功能上并非冗余。这也进一步支持了其形态与形态相关基因与致病潜力之间的相关性。
