Defining the interaction of the protease CpaA with its type II secretion chaperone CpaB and its contribution to virulence in Acinetobacter species

Abstract

, , and are a frequent cause of multidrug-resistant, healthcare-associated infections. Our previous work demonstrated that M2 possesses a functional type II secretion system (T2SS) that is required for full virulence. Further, we identified the metallo-endopeptidase CpaA, which has been shown previously to cleave human Factor V and deregulate blood coagulation, as the most abundant type II secreted effector protein. We also demonstrated that its secretion is dependent on CpaB, a membrane-bound chaperone. In this study, we show that CpaA expression and secretion are conserved across several medically relevant species. Additionally, we demonstrate that deletion of results in attenuation of M2 virulence in moth and mouse models. The virulence defects resulting from the deletion of were comparable with those observed upon abrogation of T2SS activity. The virulence defects resulting from the deletion of are comparable with those observed upon abrogation of T2SS activity. We also show that CpaA and CpaB strongly interact, forming a complex in a 1:1 ratio. Interestingly, deletion of the N-terminal transmembrane domain of CpaB results in robust secretion of CpaA and CpaB, indicating that the transmembrane domain is dispensable for CpaA secretion and likely functions to retain CpaB inside the cell. Limited proteolysis of spheroplasts revealed that the C-terminal domain of CpaB is exposed to the periplasm, suggesting that this is the site where CpaA and CpaB interact Last, we show that CpaB does not abolish the proteolytic activity of CpaA against human Factor V. We conclude that CpaA is, to the best of our knowledge, the first characterized, virulence factor secreted by species.

Assessing Acinetobacter baumannii Virulence and Persistence in a Murine Model of Lung Infection

“…At 36 hpi, bacterial burdens were assessed in lungs (A), livers (B), and spleens (C). Due to mortality with 3X10 9 CFU inoculum and low dissemination to the spleen with 3X10 8 CFU, an inoculum of 1X10 9 CFU was chosen [8,9]. …”

Catabolism of 3-hydroxypyridine byEnsifer adhaerensHP1: a novel four-component gene encoding 3-hydroxypyridine dehydrogenase HpdA catalyzes the first step of biodegradation

“…It was reported that SRPBCC domains have a deep hydrophobic ligand-binding pocket that can bind diverse ligands and spans all three kingdoms of life, but its function remains unclear. Moreover, SRPBCC-containing genes play a wide range of roles, including strawberry fruit ripening [22] , regulation of anthocyanin accumulation [23] , encoding type II secretion chaperones [24] , functioning in the stabilization of mRNA [25] , catalyzing the free radical reactions of polyunsaturated fatty acids [26] , and α-pyridone ring formation [27] . Hydroxylases that hydroxylate the C6 of pyridine, such as 3-succinoylpyridine dehydrogenase (SpmABC) [28] and picolinic acid dehydrogenase (PicA) [29] , include SRPBCC-containing components and are fused with [2Fe-2S] clusters, whereas most pyridine hydroxylases, such as NdhLSM and KdhLMS, have no SRPBCC domain (Figure 4A).…”

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

“…Among these T2SS effectors, the zinc-metallo-endopeptidase CpaA is the best-characterized and most abundant effector secreted by several medically relevant Acinetobacter strains (7,8,12). We demonstrated that CpaA stability and secretion depends on the membranebound chaperone CpaB, which is encoded adjacent to CpaA (7,8). The N-terminal transmembrane domain of CpaB anchors the protein to the membrane, whereas its C-terminal domain interacts directly with CpaA in the periplasm (8).…”

“…We demonstrated that CpaA stability and secretion depends on the membranebound chaperone CpaB, which is encoded adjacent to CpaA (7,8). The N-terminal transmembrane domain of CpaB anchors the protein to the membrane, whereas its C-terminal domain interacts directly with CpaA in the periplasm (8). Removal of the CpaB transmembrane domain results in secretion of the CpaA-CpaB complex (8).…”

“…Although the T2SS machinery is conserved across Acinetobacter spp, the effector repertoire is diverse and varies from strain to strain (7,9,10) Approximately 20 to 60 T2SS effectors are secreted by any given Acinetobacter strain, and these proteins are involved in lipid assimilation, serum resistance, colonization of various host tissues, antibiotic resistance, and biofilm formation (3,4,7,11). Among these T2SS effectors, the zinc-metallo-endopeptidase CpaA is the best-characterized and most abundant effector secreted by several medically relevant Acinetobacter strains (7,8,12). We demonstrated that CpaA stability and secretion depends on the membranebound chaperone CpaB, which is encoded adjacent to CpaA (7,8).…”