Editorial
Clinical implications of glycoproteomics for Acinetobacter baumannii Expert Review of Proteomics Downloaded from informahealthcare.com by SUNY Health Sciences Center on 03/31/15 For personal use only.
Expert Rev. Proteomics 12(1), 1–3 (2014)
Rachel L Kinsella University of Alberta, Biological Sciences, Edmonton, AB, Canada
Nichollas E Scott University of British Columbia, Vancouver, BC, Canada
Mario F Feldman Author for correspondence: University of Alberta, Biological Sciences, Edmonton, AB, Canada Tel.: +1 780 492 6105 [email protected]
The opportunistic human pathogen Acinetobacter baumannii persists in the healthcare setting because of its ability to survive exposure to various antimicrobial and sterilization agents. A. baumannii’s ability to cause multiple infection types complicates diagnosis and treatment. Rapid detection of A. baumannii infections would likely improve treatment outcomes. Recently published Acinetobacter glycoproteomic data show the prevalence of O-linked glycoproteins, suggesting the possibility for an O-glycan-based detection technology. O-glycan biosynthesis is required for protein glycosylation and capsular polysaccharide production in A. baumannii. Recent publications demonstrate key roles for protein glycosylation and capsular polysaccharide in the pathogenicity of A. baumannii. Targeted antimicrobial development against O-glycan biosynthesis may produce new effective treatment options for A. baumannii infections. Here, we discuss how the data gathered through Acinetobacter glycoproteomics can be used to develop technologies for rapid diagnosis and reveal potential antimicrobial targets. In addition, we consider the efficacy of glycoconjugate vaccine development against A. baumannii.
Acinetobacter baumannii is a Gramnegative opportunistic pathogen of increasing concern to healthcare systems [1]. A. baumannii has acquired resistance to multiple antibiotics, which, coupled to its ability to survive standard control measures, desiccation, and a diverse range of sterilizing agents, has resulted in its ability to persist in clinical settings and cause serious infections worldwide [1]. Nosocomial A. baumannii infections of high-risk groups, such as immunocompromised individuals and patients whom have undergone invasive surgery, complicate treatment options leading to increased mortality and morbidity. Within these high-risk groups, A. baumannii can cause a variety of infections, but most often manifests as pneumonia, sepsis, soft-tissue infections, urinary tract infections or meningitis [1]. The broad tissue tropism of A. baumannii represents a significant diagnostic challenge as infection by this agent at different sites can present as a range of symptoms. Because immediate intervention is required to
control A. baumannii infections, the lack of rapid detection of this organism is a major obstacle in preventing and controlling A. baumannii-associated conditions. Epidemiological research on A. baumannii may provide antibiotic resistance profiles and tissue tropisms associated with specific strains. The development of a serotyping system for A. baumannii would aid in rapid detection and strainto-strain differentiation. This would be clinically beneficial for the rapid and more effective treatment of A. baumannii. Because of detectable variation in the polysaccharides present at Gram-negative cell surfaces, carbohydrate structures have been the basis for technologies used to detect and differentiate bacterial strains for >40 years. Examples of these are the Escherichia coli O antigen polysaccharide-based serotyping system, the Brucellosis detection technology based on O antigen structure, and the group B Streptococci capsular polysaccharide-based serotyping [2,3]. Unlike many other Gram-negative bacteria, the surface of
KEYWORDS: Acinetobacter baumannii • Acinetobacter diagnosis • clinical relevance • glycoproteomics • mass spectrometry • O-linked glycosylation • proteomics • ZIC-HILIC
informahealthcare.com
10.1586/14789450.2015.987756
Ó 2014 Informa UK Ltd
ISSN 1478-9450
1
Expert Review of Proteomics Downloaded from informahealthcare.com by SUNY Health Sciences Center on 03/31/15 For personal use only.
Editorial
Kinsella, Scott & Feldman
A. baumannii lacks the conventional O antigen attached to lipid A. However, it is covered by other carbohydrate structures, including capsular polysaccharide and O-linked glycoproteins. These structures are ideal to be used as diagnostic markers. Similar to other bacteria [4,5], all pathogenic Acinetobacter strains can post-translationally modify proteins by the addition of diverse glycan structures [6–9]. In Acinetobacter species, O-linked glycosylation is mediated by the conserved O-oligosaccharyltransferase, PglL, which attaches an O-glycan to hydroxylated amino acids (Serine or Threonine) on multiple proteins [8]. The biosynthesis of the O-glycan is encoded by a single genetic locus also responsible for generation of the capsule [7,9]. Consistent with the extreme genetic diversity of this locus [10,11], A. baumannii has the most diverse O-linked glycosylation system to date, with extensive glycan diversity existing both between strains and microheterogeneity within strains [7]. Despite this diversity, O-linked glycans from Acinetobacter species share similar properties, consisting of 3–5 carbohydrate residues, with limited branching and negatively charged sugars [7]. These common features suggest a shared function of the O-linked/capsule monomers across strains. Consistent with this hypothesis, clinical strains of A. baumannii are highly resistant to complement-mediated killing in a capsule-dependent manner [7,9], and the attachment of these glycans to protein substrates is also essential for biofilm formation and virulence [7–9]. Although these findings demonstrate the importance of O-glycan biosynthesis and protein glycosylation for virulence, their precise contribution to these phenotypes is unknown. The use of mass spectrometry and proteomic approaches has been instrumental in identification and characterization of Acinetobacter glycosylation and its glycoproteome [9–11]. The use of selective enrichment of glycopeptides using zwitterionic hydrophilic interaction liquid chromatography [10] coupled to multiple fragmentation approaches provides a robust platform for the analysis of diverse glycopeptides. This approach enabled the identification of diverse and multimeric forms of the O-glycan and the determination of the glycosylation sites using submicrogram amounts of sample [9–11]. With increasing refinement to these proteomic approaches, it is now possible to assess glycosylation in a quantitative manner using techniques, such as dimethyl labeling [12,13]. Furthermore, by using ultraviolet photodissociation information on carbohydrate stereochemistry can be gathered from approximately 1/1000 of the sample amount needed for Nuclear Magnetic Resonance spectroscopy [14]. The application of these developments within the field of bacterial glycosylation analysis paired with continuous refinement of mass spectrometry instrumentation [15] enables higher quality data to be generated in shorter periods of time, facilitating the characterization of glycosylation within this genus. With these tools, it is now possible to start addressing the role of protein glycosylation in Acinetobacter biology and the potential use of glycoconjugates as diagnostic markers.
2
Together with others, we have discovered the carbohydrate biosynthesis locus responsible for the production of the O-glycan and its polymerization into the capsular polysaccharide [7,9–11]. As mentioned earlier, the capsular polysaccharide is required for resistance to complement-mediated killing, and therefore survival in the host. A conserved initiating glycosyltransferase, PglC, is responsible for attaching the first sugar of the O-glycan to the lipid carrier and is required for protein glycosylation and capsular polysaccharide production [9]. Deletion of pglC results in sensitivity to complement-mediated killing and inability to colonize mice [10]. The increasing prevalence of multidrug resistant A. baumannii infections worldwide demands the development of new antimicrobials to fight this pathogen. The conservation of PglC across Acinetobacter species makes this enzyme an excellent target for antimicrobial development against A. baumannii. A. baumannii’ s ability to survive on abiotic and biotic surfaces in the hospital environment provides a potential source of infection to susceptible surgical or immunocompromised patients. Prophylactic vaccination against A. baumannii is a potential solution to this concern. Conjugations of O antigens or capsular polysaccharides to a protein carrier have been successfully used as effective licensed vaccines against Haemophilus influenzae, Neisseria meningitidis, Salmonella and Streptococcus pneumoniae [3]. Within these conjugated vaccines, the carbohydrate portion provides serological specific immunity, whereas the protein component initiates long-lasting immunity [3]. We suggest that a glycoconjugate vaccine, specific for the O-linked glycans, may be an effective means to prevent A. baumannii infections within high-risk groups. We recognize that the intrastrain glycan variability displayed by A. baumannii complicates glycoconjugate vaccine design; however, the prominent surface exposed nature of the O-linked glycans produced by each strain makes these antigens ideal targets. Because of the diversity in the O-glycan structures produced across A. baumannii, further epidemiological studies would be beneficial in determining whether there is a highly prevalent or extreme-disease associated serotype, which would make the best vaccine candidate. In addition, multivalent glycoconjugate vaccinations against highly prevalent A. baumannii serotypes may be effective. Financial & competing interests disclosure
RL Kinsella is funded by NSERC and Alberta Innovates Technology Futures. NE Scott is supported by the National Health and Medical Research Council of Australia (NHMRC) Overseas (Biomedical) Fellowship (APP1037373) and the Medical Smith Foundations for Health Research Trainee Post-Doctoral Fellow (award #5363). MF Feldman is supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant, and is also a CHIR investigator. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Expert Rev. Proteomics 12(1), (2014)
Glycoproteomics for A. baumannii
glycoprotein substrates. PLoS One 2013; 8(5):e62768
References 1.
Expert Review of Proteomics Downloaded from informahealthcare.com by SUNY Health Sciences Center on 03/31/15 For personal use only.
2.
3.
4.
Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008;21(3):538-82 Ciocchini AE, Serantes DA, Melli LJ, et al. Development and validation of a novel diagnostic test for human brucellosis using a glycoengineered antigen coupled to magnetic beads. PLoS Negl Trop Dis 2013; 7(2):e2048 Astronomo RD, Burton DR. Carbohydrate vaccines: developing sweet solutions to sticky situations? Nat Rev Drug Discov 2010;9(4):308-24 Iwashkiw JA, Vozza NF, Kinsella RL, et al. Pour some sugar on it: the expanding world of bacterial protein O-linked glycosylation. Mol Microbiol 2013;89(1):14-28
5.
Nothaft H, Szymanski CM. Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol 2010;8(11):765-78
6.
Schulz BL, Jen FE, Power PM, et al. Identification of bacterial protein O-oligosaccharyltransferases and their
informahealthcare.com
7.
8.
Scott NE, Kinsella RL, Edwards AV, et al. Diversity within the O-linked protein glycosylation systems of Acinetobacter species. Mol Cell Proteomics 2014;13(9): 2354-70 Iwashkiw JA, Seper A, Weber BS, et al. Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog 2012;8(6): e1002758
9.
Lees-Miller RG, Iwashkiw JA, Scott NE, et al. A common pathway for O-linked protein-glycosylation and synthesis of capsule in Acinetobacter baumannii. Mol Microbiol 2013;89(5):816-30
10.
Hu D, Liu B, Dijkshoorn L, et al. Diversity in the major polysaccharide antigen of Acientobacter baumannii assessed by DNA sequencing, and development of a molecular serotyping scheme. PLoS One 2013;8(7):e70329
Editorial
11.
Kenyon JJ, Hall RM. Variation in the complex carbohydrate biosynthesis loci of Acinetobacter baumannii genomes. PLoS One 2013;8(4):e62160
12.
Lithgow KV, Scott NE, Iwashkiw JA, et al. A general protein O-glycosylation system within the Burkholderia cepacia complex is involved in motility and virulence. Mol Microbiol 2014;92(1):116-37
13.
Boersema PJ, Raijmakers R, Lemeer S, et al. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 2009;4(4):484-94
14.
Madsen JA, Ko BJ, Xu H, et al. Concurrent automated sequencing of the glycan and peptide portions of O-linked glycopeptides anions by ultraviolet photodissociation mass spectrometry. Anal Chem 2013;85(19): 9253-61
15.
Herbert AS, Richards AL, Bailey DJ, et al. The one hour yeast proteome. Mol Cell Proteomics 2014;13(1):339-47
3
Clinical implications of glycoproteomics for Acinetobacter baumannii Expert Review of Proteomics Downloaded from informahealthcare.com by SUNY Health Sciences Center on 03/31/15 For personal use only.
Expert Rev. Proteomics 12(1), 1–3 (2014)
Rachel L Kinsella University of Alberta, Biological Sciences, Edmonton, AB, Canada
Nichollas E Scott University of British Columbia, Vancouver, BC, Canada
Mario F Feldman Author for correspondence: University of Alberta, Biological Sciences, Edmonton, AB, Canada Tel.: +1 780 492 6105 [email protected]
The opportunistic human pathogen Acinetobacter baumannii persists in the healthcare setting because of its ability to survive exposure to various antimicrobial and sterilization agents. A. baumannii’s ability to cause multiple infection types complicates diagnosis and treatment. Rapid detection of A. baumannii infections would likely improve treatment outcomes. Recently published Acinetobacter glycoproteomic data show the prevalence of O-linked glycoproteins, suggesting the possibility for an O-glycan-based detection technology. O-glycan biosynthesis is required for protein glycosylation and capsular polysaccharide production in A. baumannii. Recent publications demonstrate key roles for protein glycosylation and capsular polysaccharide in the pathogenicity of A. baumannii. Targeted antimicrobial development against O-glycan biosynthesis may produce new effective treatment options for A. baumannii infections. Here, we discuss how the data gathered through Acinetobacter glycoproteomics can be used to develop technologies for rapid diagnosis and reveal potential antimicrobial targets. In addition, we consider the efficacy of glycoconjugate vaccine development against A. baumannii.
Acinetobacter baumannii is a Gramnegative opportunistic pathogen of increasing concern to healthcare systems [1]. A. baumannii has acquired resistance to multiple antibiotics, which, coupled to its ability to survive standard control measures, desiccation, and a diverse range of sterilizing agents, has resulted in its ability to persist in clinical settings and cause serious infections worldwide [1]. Nosocomial A. baumannii infections of high-risk groups, such as immunocompromised individuals and patients whom have undergone invasive surgery, complicate treatment options leading to increased mortality and morbidity. Within these high-risk groups, A. baumannii can cause a variety of infections, but most often manifests as pneumonia, sepsis, soft-tissue infections, urinary tract infections or meningitis [1]. The broad tissue tropism of A. baumannii represents a significant diagnostic challenge as infection by this agent at different sites can present as a range of symptoms. Because immediate intervention is required to
control A. baumannii infections, the lack of rapid detection of this organism is a major obstacle in preventing and controlling A. baumannii-associated conditions. Epidemiological research on A. baumannii may provide antibiotic resistance profiles and tissue tropisms associated with specific strains. The development of a serotyping system for A. baumannii would aid in rapid detection and strainto-strain differentiation. This would be clinically beneficial for the rapid and more effective treatment of A. baumannii. Because of detectable variation in the polysaccharides present at Gram-negative cell surfaces, carbohydrate structures have been the basis for technologies used to detect and differentiate bacterial strains for >40 years. Examples of these are the Escherichia coli O antigen polysaccharide-based serotyping system, the Brucellosis detection technology based on O antigen structure, and the group B Streptococci capsular polysaccharide-based serotyping [2,3]. Unlike many other Gram-negative bacteria, the surface of
KEYWORDS: Acinetobacter baumannii • Acinetobacter diagnosis • clinical relevance • glycoproteomics • mass spectrometry • O-linked glycosylation • proteomics • ZIC-HILIC
informahealthcare.com
10.1586/14789450.2015.987756
Ó 2014 Informa UK Ltd
ISSN 1478-9450
1
Expert Review of Proteomics Downloaded from informahealthcare.com by SUNY Health Sciences Center on 03/31/15 For personal use only.
Editorial
Kinsella, Scott & Feldman
A. baumannii lacks the conventional O antigen attached to lipid A. However, it is covered by other carbohydrate structures, including capsular polysaccharide and O-linked glycoproteins. These structures are ideal to be used as diagnostic markers. Similar to other bacteria [4,5], all pathogenic Acinetobacter strains can post-translationally modify proteins by the addition of diverse glycan structures [6–9]. In Acinetobacter species, O-linked glycosylation is mediated by the conserved O-oligosaccharyltransferase, PglL, which attaches an O-glycan to hydroxylated amino acids (Serine or Threonine) on multiple proteins [8]. The biosynthesis of the O-glycan is encoded by a single genetic locus also responsible for generation of the capsule [7,9]. Consistent with the extreme genetic diversity of this locus [10,11], A. baumannii has the most diverse O-linked glycosylation system to date, with extensive glycan diversity existing both between strains and microheterogeneity within strains [7]. Despite this diversity, O-linked glycans from Acinetobacter species share similar properties, consisting of 3–5 carbohydrate residues, with limited branching and negatively charged sugars [7]. These common features suggest a shared function of the O-linked/capsule monomers across strains. Consistent with this hypothesis, clinical strains of A. baumannii are highly resistant to complement-mediated killing in a capsule-dependent manner [7,9], and the attachment of these glycans to protein substrates is also essential for biofilm formation and virulence [7–9]. Although these findings demonstrate the importance of O-glycan biosynthesis and protein glycosylation for virulence, their precise contribution to these phenotypes is unknown. The use of mass spectrometry and proteomic approaches has been instrumental in identification and characterization of Acinetobacter glycosylation and its glycoproteome [9–11]. The use of selective enrichment of glycopeptides using zwitterionic hydrophilic interaction liquid chromatography [10] coupled to multiple fragmentation approaches provides a robust platform for the analysis of diverse glycopeptides. This approach enabled the identification of diverse and multimeric forms of the O-glycan and the determination of the glycosylation sites using submicrogram amounts of sample [9–11]. With increasing refinement to these proteomic approaches, it is now possible to assess glycosylation in a quantitative manner using techniques, such as dimethyl labeling [12,13]. Furthermore, by using ultraviolet photodissociation information on carbohydrate stereochemistry can be gathered from approximately 1/1000 of the sample amount needed for Nuclear Magnetic Resonance spectroscopy [14]. The application of these developments within the field of bacterial glycosylation analysis paired with continuous refinement of mass spectrometry instrumentation [15] enables higher quality data to be generated in shorter periods of time, facilitating the characterization of glycosylation within this genus. With these tools, it is now possible to start addressing the role of protein glycosylation in Acinetobacter biology and the potential use of glycoconjugates as diagnostic markers.
2
Together with others, we have discovered the carbohydrate biosynthesis locus responsible for the production of the O-glycan and its polymerization into the capsular polysaccharide [7,9–11]. As mentioned earlier, the capsular polysaccharide is required for resistance to complement-mediated killing, and therefore survival in the host. A conserved initiating glycosyltransferase, PglC, is responsible for attaching the first sugar of the O-glycan to the lipid carrier and is required for protein glycosylation and capsular polysaccharide production [9]. Deletion of pglC results in sensitivity to complement-mediated killing and inability to colonize mice [10]. The increasing prevalence of multidrug resistant A. baumannii infections worldwide demands the development of new antimicrobials to fight this pathogen. The conservation of PglC across Acinetobacter species makes this enzyme an excellent target for antimicrobial development against A. baumannii. A. baumannii’ s ability to survive on abiotic and biotic surfaces in the hospital environment provides a potential source of infection to susceptible surgical or immunocompromised patients. Prophylactic vaccination against A. baumannii is a potential solution to this concern. Conjugations of O antigens or capsular polysaccharides to a protein carrier have been successfully used as effective licensed vaccines against Haemophilus influenzae, Neisseria meningitidis, Salmonella and Streptococcus pneumoniae [3]. Within these conjugated vaccines, the carbohydrate portion provides serological specific immunity, whereas the protein component initiates long-lasting immunity [3]. We suggest that a glycoconjugate vaccine, specific for the O-linked glycans, may be an effective means to prevent A. baumannii infections within high-risk groups. We recognize that the intrastrain glycan variability displayed by A. baumannii complicates glycoconjugate vaccine design; however, the prominent surface exposed nature of the O-linked glycans produced by each strain makes these antigens ideal targets. Because of the diversity in the O-glycan structures produced across A. baumannii, further epidemiological studies would be beneficial in determining whether there is a highly prevalent or extreme-disease associated serotype, which would make the best vaccine candidate. In addition, multivalent glycoconjugate vaccinations against highly prevalent A. baumannii serotypes may be effective. Financial & competing interests disclosure
RL Kinsella is funded by NSERC and Alberta Innovates Technology Futures. NE Scott is supported by the National Health and Medical Research Council of Australia (NHMRC) Overseas (Biomedical) Fellowship (APP1037373) and the Medical Smith Foundations for Health Research Trainee Post-Doctoral Fellow (award #5363). MF Feldman is supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant, and is also a CHIR investigator. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Expert Rev. Proteomics 12(1), (2014)
Glycoproteomics for A. baumannii
glycoprotein substrates. PLoS One 2013; 8(5):e62768
References 1.
Expert Review of Proteomics Downloaded from informahealthcare.com by SUNY Health Sciences Center on 03/31/15 For personal use only.
2.
3.
4.
Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008;21(3):538-82 Ciocchini AE, Serantes DA, Melli LJ, et al. Development and validation of a novel diagnostic test for human brucellosis using a glycoengineered antigen coupled to magnetic beads. PLoS Negl Trop Dis 2013; 7(2):e2048 Astronomo RD, Burton DR. Carbohydrate vaccines: developing sweet solutions to sticky situations? Nat Rev Drug Discov 2010;9(4):308-24 Iwashkiw JA, Vozza NF, Kinsella RL, et al. Pour some sugar on it: the expanding world of bacterial protein O-linked glycosylation. Mol Microbiol 2013;89(1):14-28
5.
Nothaft H, Szymanski CM. Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol 2010;8(11):765-78
6.
Schulz BL, Jen FE, Power PM, et al. Identification of bacterial protein O-oligosaccharyltransferases and their
informahealthcare.com
7.
8.
Scott NE, Kinsella RL, Edwards AV, et al. Diversity within the O-linked protein glycosylation systems of Acinetobacter species. Mol Cell Proteomics 2014;13(9): 2354-70 Iwashkiw JA, Seper A, Weber BS, et al. Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog 2012;8(6): e1002758
9.
Lees-Miller RG, Iwashkiw JA, Scott NE, et al. A common pathway for O-linked protein-glycosylation and synthesis of capsule in Acinetobacter baumannii. Mol Microbiol 2013;89(5):816-30
10.
Hu D, Liu B, Dijkshoorn L, et al. Diversity in the major polysaccharide antigen of Acientobacter baumannii assessed by DNA sequencing, and development of a molecular serotyping scheme. PLoS One 2013;8(7):e70329
Editorial
11.
Kenyon JJ, Hall RM. Variation in the complex carbohydrate biosynthesis loci of Acinetobacter baumannii genomes. PLoS One 2013;8(4):e62160
12.
Lithgow KV, Scott NE, Iwashkiw JA, et al. A general protein O-glycosylation system within the Burkholderia cepacia complex is involved in motility and virulence. Mol Microbiol 2014;92(1):116-37
13.
Boersema PJ, Raijmakers R, Lemeer S, et al. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 2009;4(4):484-94
14.
Madsen JA, Ko BJ, Xu H, et al. Concurrent automated sequencing of the glycan and peptide portions of O-linked glycopeptides anions by ultraviolet photodissociation mass spectrometry. Anal Chem 2013;85(19): 9253-61
15.
Herbert AS, Richards AL, Bailey DJ, et al. The one hour yeast proteome. Mol Cell Proteomics 2014;13(1):339-47
3
