The glycoprotease CpaA secreted by medically relevantAcinetobacterspecies targets multipleO-linked host glycoproteins

Abstract

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 A. 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.IMPORTANCECpaA 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 of 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 towards additional roles in bacteria-host interactions. We propose that CpaA belongs to an expanding class of functionally-defined glycoproteases that targets multiple O-linked host glycoproteins.

Architecturally complex O -glycopeptidases are customized for mucin recognition and hydrolysis

“…Indeed, some peptidases target only mucins or glycoproteins having mucin-type glycosylation, indicating that they tolerate glycosylation. In the case of some peptidases, they even rely on glycosylation as a key substrate recognition determinant (5)(6)(7)(8)(9)(10).…”

“…Pfam 13402 or the "peptidase_M60 family" was classified through characterization of a founding member combined with bioinformatics analysis (9). This revealed a mucin-active peptidase with the defining feature of relatedness to insect enhancins, the presence of a zinc-metallopeptidase gluzincin motif (HEXXHX(8,28)E), and multimodularity (i.e., the occurrence of noncatalytic carbohydrate-binding modules; CBMs) (11,12).…”

InvL, an invasin-like adhesin, is a type II secretion system substrate required for Acinetobacter baumannii uropathogenesis

“…Second, the effector is extruded through the outer membrane secretin (designated GspD herein) via the assembly of a pseudopilus (18)(19)(20)(21)(22). In Acinetobacter, the T2SS is responsible for secretion of several proteins, including the lipases, LipA, LipH, and LipAN, the protease, CpaA, and the γ-glutamyltransferase, GGT (23)(24)(25)(26)(27)(28). For A. baumannii strain ATCC 17978, a meningitis isolate from 1951, mutation of the T2SS results in attenuation in bacteremia and pneumonia models of infection (23,29).…”

Revealing the human mucinome

“…Toward this goal, enzymes derived from microorganisms known to colonize mucosal environments have shown promise for developing tools specifically suited to characterize mucin-domain glycoproteins [32][33][34][35][36][37][38] . We recently characterized a panel of such enzymes, termed mucinases, and showed that each of them harbor unique peptide-and glycan-based cleavage motifs 39 .…”