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

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
M. Florencia Haurat ◽  
Nichollas E. Scott ◽  
Gisela Di Venanzio ◽  
Juvenal Lopez ◽  
Benjamin Pluvinage ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Glycosylation Site ◽  
Type Ii Secretion ◽  
Target Sequence ◽  
Acinetobacter Species ◽  
Content Type ◽  
Host Interactions ◽  
Type Ii Secretion System ◽  
Glycosylation Sites ◽  
Bona Fide

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 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. IMPORTANCE 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.

scholarly journals The glycoprotease CpaA secreted by medically relevant Acinetobacter species targets multiple O-linked host glycoproteins

2020 ◽  
Author(s):  
M. Florencia Haurat ◽  
Nichollas E. Scott ◽  
Gisela Di Venanzio ◽  
Juvenal Lopez ◽  
Benajmin Pluvinage ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Glycosylation Site ◽  
Type Ii Secretion ◽  
Target Sequence ◽  
Acinetobacter Species ◽  
Host Interactions ◽  
Type Ii Secretion System ◽  
Glycosylation Sites ◽  
Bona Fide ◽  
Insight Into

ABSTRACTGlycans 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.

scholarly journals Xylose-Inducible Promoter Tools for Pseudomonas Species and Their Use in Implicating a Role for the Type II Secretion System Protein XcpQ in the Inhibition of Corneal Epithelial Wound Closure

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Jake D. Callaghan ◽  
Nicholas A. Stella ◽  
Kara M. Lehner ◽  
Benjamin R. Treat ◽  
Kimberly M. Brothers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wound Closure ◽  
Inducible Promoter ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Content Type ◽  
Corneal Epithelial ◽  
Type Ii Secretion System ◽  
Promoter System

ABSTRACT Tunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and Pseudomonas fluorescens. The Pxut promoter, derived from the P. fluorescens xut operon, was incorporated into a broad-host-range pBBR1-based plasmid and was compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. Green fluorescent protein (GFP) fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate the cloning of genes toxic to E. coli to generate plasmids. The Pxut promoter was activated at a lower inducer concentration than PBAD in P. fluorescens, and higher gfp levels were achieved using Pxut. Flow cytometry analysis indicated that Pxut was leakier than PBAD in the Pseudomonas species tested but was expressed in a higher proportion of cells when induced. d-Xylose as a sole carbon source did not support the growth of P. aeruginosa or P. fluorescens and is less expensive than many other commonly used inducers, which could facilitate large-scale applications. The efficacy of this system was demonstrated by its use to reveal a role for the P. aeruginosa type II secretion system gene xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species. IMPORTANCE Pseudomonas species are enormously important in human infections, in biotechnology, and as model systems for investigating basic science questions. In this study, we have developed a xylose-inducible promoter system, evaluated it in P. aeruginosa and P. fluorescens, and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type II secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

scholarly journals Porcine Enterotoxigenic Escherichia coli Strains Differ in Their Capacity To Secrete Enterotoxins through Varying YghG Levels

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Haixiu Wang ◽  
Raquel Sanz Garcia ◽  
Eric Cox ◽  
Bert Devriendt
Keyword(s):  
Escherichia Coli ◽  
Secretion System ◽  
Farm Animals ◽  
Health Concern ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Content Type ◽  
E Coli ◽  
Type Ii Secretion System ◽  
Heat Labile

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens for humans and farm animals such as pigs. Porcine ETEC strains induce diarrhea through the production of heat-labile enterotoxin (LT) and/or heat-stable enterotoxins (pSTa/STb). Although LT secretion levels differ between porcine ETEC strains, and this has been linked to virulence, it is unclear whether ST secretion levels also differ between porcine ETEC strains. In addition, the molecular mechanism underlying different LT secretion levels has not been elucidated. In this work, multiple porcine ETEC strains were assessed for their capacity to produce and secrete the enterotoxins LT, pSTa, and STb. The strains differed greatly in their capacity to secrete LT, pSTa, and STb. Remarkably, in some strains, periplasmic production did not correlate with their ability to secrete LT, resulting in high periplasmic production and low LT secretion levels. Furthermore, the results indicated that the type II secretion system (T2SS) protein YghG plays a regulatory role in controlling LT secretion levels. These findings highlight YghG as an important mediator of the secretion of the heat-labile enterotoxin LT by porcine ETEC strains and provide better insights into ETEC enterotoxin secretion. IMPORTANCE Enterotoxigenic E. coli strains are a major health concern. Enterotoxins secreted by enterotoxigenic E. coli are crucial for diarrhea induction. Enterotoxin secretion levels differ between strains; however, it is currently unclear what drives these differences. The discrepancy in the production and secretion capacities of enterotoxins in ETEC is important to clarify their function involved in diarrhea induction. Our results further deepen our understanding of how type II secretion system (T2SS) components of ETEC control enterotoxin secretion levels and may lay the foundation for a better understanding of ETEC molecular pathogenesis.

scholarly journals Live Attenuated Salmonella Vaccines Displaying Regulated Delayed Lysis and Delayed Antigen Synthesis To Confer Protection against Mycobacterium tuberculosis

2011 ◽  
Vol 80 (2) ◽  
pp. 815-831 ◽  
Author(s):  
María Dolores Juárez-Rodríguez ◽  
Jiseon Yang ◽  
Rebin Kader ◽  
Praveen Alamuri ◽  
Roy Curtiss ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Signal Sequence ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Content Type ◽  
Degree Of Protection ◽  
Amino Terminal ◽  
Type Ii Secretion System ◽  
Cellular Immune

ABSTRACTLive recombinant attenuatedSalmonellavaccine (RASV) strains have great potential to induce protective immunity againstMycobacterium tuberculosisby deliveringM. tuberculosisantigens. Recently, we reported that, in orally immunized mice, RASV strains delivering theM. tuberculosisearly secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10) antigens via theSalmonellatype III secretion system (SopE amino-terminal region residues 1 to 80 with two copies of ESAT-6 and one copy of CFP-10 [SopENt80-E2C]) afforded protection against aerosol challenge withM. tuberculosis. Here, we constructed and evaluated an improvedSalmonellavaccine againstM. tuberculosis. We constructed translational fusions for the synthesis of two copies of ESAT-6 plus CFP-10 fused to the OmpC signal sequence (OmpCSS-E2C) and amino acids 44 to 338 of antigen 85A (Ag85A294) flanked by the signal sequence (SS) and C-terminal peptide (CT) of β-lactamase (BlaSS-Ag85A294-BlaCT) to enable delivery via theSalmonellatype II secretion system. The genes expressing these proteins were cloned as an operon transcribed from Ptrcinto isogenic Asd+/MurA+pYA3681 lysis vector derivatives with different replication origins (pBR, p15A, pSC101), resulting in pYA4890, pYA4891, and pYA4892 for SopENt80-E2C/Ag85A294synthesis and pYA4893 and pYA4894 for OmpCSS-E2C/Ag85A294synthesis. Mice orally immunized with the RASV χ11021 strain engineered to display regulated delayed lysis and regulated delayed antigen synthesisin vivoand harboring pYA4891, pYA4893, or pYA4894 elicited significantly greater humoral and cellular immune responses, and the RASV χ11021 strain afforded a greater degree of protection againstM. tuberculosisaerosol challenge in mice than RASVs harboring any other Asd+/MurA+lysis plasmid and immunization withM. bovisBCG, demonstrating that RASV strains displaying regulated delayed lysis with delayed antigen synthesis resulted in highly immunogenic delivery vectors for oral vaccination againstM. tuberculosisinfection.

scholarly journals Enterotoxigenic Escherichia coli Secretes a Highly Conserved Mucin-Degrading Metalloprotease To Effectively Engage Intestinal Epithelial Cells

2013 ◽  
Vol 82 (2) ◽  
pp. 509-521 ◽  
Author(s):  
Qingwei Luo ◽  
Pardeep Kumar ◽  
Tim J. Vickers ◽  
Alaullah Sheikh ◽  
Warren G. Lewis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enterotoxigenic Escherichia Coli ◽  
Effective Vaccine ◽  
Type Ii Secretion ◽  
Intestinal Epithelial ◽  
Content Type ◽  
E Coli ◽  
Type Ii Secretion System ◽  
Efficient Access ◽  
Genes Encoding

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) is a leading cause of death due to diarrheal illness among young children in developing countries, and there is currently no effective vaccine. Many elements of ETEC pathogenesis are still poorly defined. Here we demonstrate that YghJ, a secreted ETEC antigen identified in immunoproteomic studies using convalescent patient sera, is required for efficient access to small intestinal enterocytes and for the optimal delivery of heat-labile toxin (LT). Furthermore, YghJ is a highly conserved metalloprotease that influences intestinal colonization of ETEC by degrading the major mucins in the small intestine, MUC2 and MUC3. Genes encoding YghJ and its cognate type II secretion system (T2SS), which also secretes LT, are highly conserved in ETEC and exist in other enteric pathogens, including other diarrheagenicE. coliandVibrio choleraebacteria, suggesting that this mucin-degrading enzyme may represent a shared virulence feature of these important pathogens.

scholarly journals Deciphering the Effect of the Different N-Glycosylation Sites on the Secretion, Activity, and Stability of Cellobiohydrolase I from Trichoderma reesei

2014 ◽  
Vol 80 (13) ◽  
pp. 3962-3971 ◽  
Author(s):  
Feifei Qi ◽  
Weixin Zhang ◽  
Fengjie Zhang ◽  
Guanjun Chen ◽  
Weifeng Liu
Keyword(s):  
Trichoderma Reesei ◽  
Target Genes ◽  
Structural Integrity ◽  
Catalytic Domain ◽  
Glycosylation Site ◽  
Cellobiohydrolase I ◽  
Regular Secondary Structure ◽  
Content Type ◽  
Extracellular Secretion ◽  
Glycosylation Sites

ABSTRACTN-linked glycosylation modulates and diversifies the structures and functions of the eukaryotic proteome through both intrinsic and extrinsic effects on proteins. We investigated the significance of the three N-linked glycans on the catalytic domain of cellobiohydrolase I (CBH1) from the filamentous fungusTrichoderma reeseiin its secretion and activity. While the removal of one or two N-glycosylation sites hardly affected the extracellular secretion of CBH1, eliminating all of the glycosylation sites did induce expression of the unfolded protein response (UPR) target genes, and secretion of this CBH1 variant was severely compromised in a calnexin gene deletion strain. Further characterization of the purified CBH1 variants showed that, compared to Asn270, the thermal reactivity of CBH1 was significantly decreased by removal of either Asn45 or Asn384 glycosylation site during the catalyzed hydrolysis of soluble substrate. Combinatorial loss of these two N-linked glycans further exacerbated the temperature-dependent inactivation. In contrast, this thermal labile property was less severe when hydrolyzing insoluble cellulose. Analysis of the structural integrity of CBH1 variants revealed that removal of N-glycosylation at Asn384 had a more pronounced effect on the integrity of regular secondary structure compared to the loss of Asn45 or Asn270. These data implicate differential roles of N-glycosylation modifications in contributing to the stability of specific functional regions of CBH1 and highlight the potential of improving the thermostability of CBH1 by tuning proper interactions between glycans and functional residues.

scholarly journals Functional Characterization of EpsC, a Component of the Type II Secretion System, in the Pathogenicity of Vibrio vulnificus

2011 ◽  
Vol 79 (10) ◽  
pp. 4068-4080 ◽  
Author(s):  
Won Hwang ◽  
Na Yeon Lee ◽  
Juri Kim ◽  
Mi-Ae Lee ◽  
Kun-Soo Kim ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Outer Membrane Proteins ◽  
Functional Characterization ◽  
Secretion System ◽  
Host Cells ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Content Type ◽  
Type Ii Secretion System ◽  
Secreted Factors

ABSTRACTEpsC, one of the components comprising the type II secretion system (T2SS), was isolated from a human-pathogenic bacterium,Vibrio vulnificus, to evaluate its role in eliciting virulence. AnespC-deleted mutant ofV. vulnificusdisplayed a reduced cytotoxicity to the human cell line HEp-2 and an attenuated virulence in a mouse model. This mutant exhibited dramatic defects in the secretion of diverse extracellular proteins, such as outer membrane proteins, transporters, and the known secreted factors, notably, a hemolysin (VvhA) and an elastase (VvpE). A defect in its secretion of proteins was restored by intranscomplementation of the intactepsCgene. Analyses of cellular fractions revealed that VvhA and VvpE of theΔepsCmutant were not excreted outside the cell but were present mainly in the periplasmic space. Examination of aV. vulnificusmutant deficient in TolC, a component of the T1SS, showed that it is not involved in the secretion of VvhA and VvpE but that it is necessary for the secretion of another major toxin ofV. vulnificus, RtxA. Therefore, the T2SS is required forV. vulnificuspathogenicity, which is mediated by at least two secreted factors, VvhA and VvpE, via facilitating the secretion and exposure of these factors to host cells.

scholarly journals Photobacterium damselae subsp. damselae Major Virulence Factors Dly, Plasmid-Encoded HlyA, and Chromosome-Encoded HlyA Are Secreted via the Type II Secretion System

2015 ◽  
Vol 83 (4) ◽  
pp. 1246-1256 ◽  
Author(s):  
Amable J. Rivas ◽  
Ana Vences ◽  
Matthias Husmann ◽  
Manuel L. Lemos ◽  
Carlos R. Osorio
Keyword(s):  
Secretion System ◽  
Transcriptional Level ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Marine Animals ◽  
Content Type ◽  
Type Ii Secretion System ◽  
Photobacterium Damselae ◽  
Prepilin Peptidase

Photobacterium damselaesubsp.damselaeis a marine bacterium that causes septicemia in marine animals and in humans. Previously, we had determined a major role of pPHDD1 plasmid-encoded Dly (damselysin) and HlyA (HlyApl) and the chromosome-encoded HlyA (HlyAch) hemolysins in virulence. However, the mechanisms by which these toxins are secreted remain unknown. In this study, we found that a mini-Tn10transposon mutant in a plasmidless strain showing an impaired hemolytic phenotype contained an insertion inepsL, a component of a type II secretion system (T2SS). Reconstruction of the mutant by allelic exchange confirmed the specific involvement ofepsLin HlyAchsecretion. In addition, mutation ofepsLin a pPHDD1-harboring strain caused an almost complete abolition of hemolytic activity against sheep erythrocytes, indicating thatepsLplays a major role in secretion of the plasmid-encoded HlyApland Dly. This was further demonstrated by analysis of different combinations of hemolysin gene mutants and by strain-strain complementation assays. We also found that mutation of the putative prepilin peptidase genepilDseverely affected hemolysis, which dropped at levels inferior to those ofepsLmutants. Promoter expression analyses suggested that impairment of hemolysin secretion inepsLandpilDmutants might constitute a signal that affects hemolysin and T2SS gene expression at the transcriptional level. In addition, singleepsLandpilDmutations caused a drastic decrease in virulence for mice, demonstrating a major role of T2SS andpilDinP. damselaesubsp.damselaevirulence.

scholarly journals The Type II Secretion System and Its Ubiquitous Lipoprotein Substrate, SslE, Are Required for Biofilm Formation and Virulence of Enteropathogenic Escherichia coli

2012 ◽  
Vol 80 (6) ◽  
pp. 2042-2052 ◽  
Author(s):  
Deborah L. Baldi ◽  
Ellen E. Higginson ◽  
Dianna M. Hocking ◽  
Judyta Praszkier ◽  
Rosalia Cavaliere ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Content Type ◽  
Type Ii Secretion System ◽  
Heat Labile ◽  
Outer Membrane Lipoprotein ◽  
Biofilm Development

ABSTRACTEnteropathogenicEscherichia coli(EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenicE. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease.

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