The Glycoprotease CpaA Secreted by Medically Relevant Acinetobacter Species Targets Multiple O-Linked Host Glycoproteins.

CpaA is a glycoprotease expressed by members of the Acinetobacter baumannii-calcoaceticus complex, and it is the first bona fide secreted virulence factor identified in these species. Here, we show that CpaA cleaves multiple targets precisely at O-glycosylation sites preceded by a Pro residue. This feature, together with the observation that sialic acid does not impact CpaA activity, makes this enzyme an attractive tool for the analysis of O-linked human protein for biotechnical and diagnostic purposes. Previous work identified proteins involved in blood coagulation as targets of CpaA. Our work broadens the set of targets of CpaA, pointing toward additional roles in bacterium-host interactions. We propose that CpaA belongs to an expanding class of functionally defined glycoproteases that targets multiple O-linked host glycoproteins. Glycans decorate proteins and affect their biological function, including protection against proteolytic degradation. However, pathogenic, and commensal bacteria have evolved specific glycoproteases that overcome the steric impediment posed by carbohydrates, cleaving glycoproteins precisely at their glycosylation site(s). Medically relevant Acinetobacter strains employ their type II secretion system (T2SS) to secrete the glycoprotease CpaA, which contributes to virulence. Previously, CpaA was shown to cleave two O-linked glycoproteins, factors V and XII, leading to reduced blood coagulation. In this work, we show that CpaA cleaves a broader range of O-linked human glycoproteins, including several glycoproteins involved in complement activation, such as CD55 and CD46. However, only CD55 was removed from the cell surface, while CD46 remained unaltered during the Acinetobacter nosocomialis infection assay. We show that CpaA has a unique consensus target sequence that consists of a glycosylated serine or threonine residue after a proline residue (P-S/T), and its activity is not affected by sialic acids. Molecular modeling and mutagenesis analysis of CpaA suggest that the indole ring of Trp493 and the ring of the Pro residue in the substrate form a key interaction that contributes to CpaA sequence selectivity. Similar bacterial glycoproteases have recently gained attention as tools for proteomic analysis of human glycoproteins, and CpaA appears to be a robust and attractive new component of the glycoproteomics toolbox. Combined, our work provides insight into the function and possible application of CpaA, a member of a widespread class of broad-spectrum bacterial glycoproteases involved in host-pathogen interactions.

CpaA是由鲍曼不动杆菌 - 醋酸钙不动杆菌复合群成员表达的一种糖蛋白酶，它是在这些物种中鉴定出的第一种真正的分泌型毒力因子。在此，我们表明CpaA恰好能在脯氨酸残基之前的O - 糖基化位点切割多个靶标。这一特征，连同唾液酸不影响CpaA活性这一观察结果，使得这种酶成为用于生物技术和诊断目的的分析O - 连接的人类蛋白质的一种有吸引力的工具。先前的研究已确定参与血液凝固的蛋白质是CpaA的靶标。我们的研究拓宽了CpaA的靶标范围，指向其在细菌 - 宿主相互作用中的其他作用。我们提出CpaA属于一类不断扩大的功能明确的糖蛋白酶，其靶向多种O - 连接的宿主糖蛋白。 聚糖修饰蛋白质并影响其生物学功能，包括防止蛋白水解降解。然而，致病菌和共生菌已经进化出特定的糖蛋白酶，克服了碳水化合物造成的空间位阻，在糖基化位点精确地切割糖蛋白。医学相关的不动杆菌菌株利用其II型分泌系统（T2SS）分泌糖蛋白酶CpaA，它有助于致病性。先前，已表明CpaA能切割两种O - 连接的糖蛋白，即因子V和因子XII，导致血液凝固降低。在这项工作中，我们表明CpaA能切割更广泛的O - 连接的人类糖蛋白，包括几种参与补体激活的糖蛋白，如CD55和CD46。然而，在医院感染不动杆菌的检测中，只有CD55从细胞表面被去除，而CD46保持不变。我们表明CpaA具有独特的共有靶序列，由脯氨酸残基之后的糖基化丝氨酸或苏氨酸残基（P - S/T）组成，并且其活性不受唾液酸影响。对CpaA的分子建模和诱变分析表明，色氨酸493的吲哚环和底物中的脯氨酸残基环形成一种关键的相互作用，这有助于CpaA的序列选择性。类似的细菌糖蛋白酶最近作为人类糖蛋白蛋白质组分析的工具受到关注，并且CpaA似乎是糖蛋白质组学工具箱中一个强大且有吸引力的新成员。总之，我们的工作深入了解了CpaA的功能和可能的应用，CpaA是参与宿主 - 病原体相互作用的一类广泛存在的广谱细菌糖蛋白酶的成员。