A hypothesis explaining why so many pathogen virulence proteins are moonlighting proteins

A hypothesis explaining why so many pathogen virulence proteins are moonlighting proteins Abstract Moonlighting or multitasking proteins refer to those proteins with two or more functions performed by a single polypeptide chain. Proteins that belong to key ancestral functions and metabolic pathways such as primary metabolism typically exhibit moonlighting phenomenon. We have collected 698 moonlighting proteins in MultitaskProtDB-II database. A survey shows that 25% of the proteins of the database correspond to moonlighting functions related to pathogens virulence activity. Why is the canonical function of these virulence proteins mainly from ancestral key biological functions (especially of primary metabolism)? Our hypothesis is that these proteins present a high conservation between the pathogen protein and the host counterparts. Therefore, the host immune system will not elicit protective antibodies against pathogen proteins. The fact of sharing epitopes with host proteins (known as epitope mimicry) might be the cause of autoimmune diseases. Although many pathogen proteins can be antigenic, only a few of them would elicit a protective immune response. This would also explain the lack of successful vaccines based in these conserved moonlighting proteins. moonlighting proteins, vaccines, virulence proteins, host immune response, epitope, conservation INTRODUCTION Moonlighting and multitasking proteins refer to those proteins with two or more functions performed by a single polypeptide chain. Moonlighting proteins present alternative functions (named canonical and moonlighting) that are mostly affected by cellular localization, cell type, oligomeric state, concentration of cellular ligands, substrates, cofactors, products or post-translational modifications (Huberts 2010; Copley 2012; Jeffery 2014). Usually multitasking proteins are experimentally revealed by serendipity. The appearance of a new function within a polypeptide can become an advantage for the microorganism because it permits a lower number of genes and proteins, thus, making its genome more compact. In any case, these proteins complicate the interpretation of knock-outs, DNA arrays, metabolomics, systems biology, drug pharmacokinetics, pharmacodynamics and toxicity assays/analyses. It is remarkable that moonlighting is typically exhibited by proteins that are ubiquitous and belong to an ‘ancestral’ function or pathway, for example primary metabolism. Currently, two updated multitasking/moonlighting protein databases exist: MultitaskProtDB-II (Franco et al. 2018) and MoonProt (Chen et al. 2018). MultitaskProtDB-II contains 694 multitasking proteins. A remarkable fact is that 25% of these known multitasking proteins present a moonlighting function related to the pathogen's virulence activity (Table S1, Supporting Information). Moreover, their canonical function is to be an enzyme of key pathways like glycolysis or Krebs cycle (Henderson and Martin 2011; Amblee and Jeffery 2015). It is remarkable that these proteins are intracellular and, without having a canonical signal peptide or secretion motifs, they go outside the microorganism cell membrane by a still unknown mechanism. These facts rise two major questions: Why is the canonical function of these virulence proteins mainly from ancestral key biological functions (especially of primary metabolism)? And, why are they shared by many pathogen species? The high percentage of moonlighting proteins of our database that have been reported as pathogen virulence factors prompts a question: Are most of them true virulence factors? Since the function of pathogen moonlighting proteins is related to key metabolism, hence essential proteins, it is difficult to generate mutants or full-gene knock-outs in order to perform direct experimental demonstrations on their virulence involvement. However, some newly designed and naturally existing mutants do not lose the essential canonical function but become non-virulent in in vivo challenges. For example, in anaerobic conditions bacteria lack an operative glycolytic pathway. Neisseria meningitidis, which uses fructose-1,6 biphosphate aldolase as an adhesin, loses the adherence capacity to the epithelial and endothelial cells when isogenically mutated, but this capacity returns in the complementation assay (Tunio et al.2010). Another example of modification of a glycolytic enzyme without losing its canonical function is glyceraldehyde phosphate dehydrogenase of Streptococcus pyogenes. Here, a small hydrophobic amino acid sequence has been added reducing the amounts of GAPDH at the surface of the microorganism and this mutant loses its virulence in mice (Jin, Agarwal and Pancholi 2011). Finally, another example of a bacterial moonlighting protein acting as a toxin is GroEL, the archetypal category of virulence factors. This protein is an insect neurotoxin in Enterobacter aerogens, but is inactive in Escherichia coli when mutated in three amino acids (Yoshida et al.2001). We propose a hypothesis based on our previous work (Amela, Cedano and Querol 2007). In that work, we suggested that, given that one of the most important tasks of the immune system is the differentiation between self and non-selfantigens, this system would discard eliciting protective antibodies against pathogen proteins sharing epitopes with host proteins (epitope mimicry), because this could be the cause of autoimmune diseases (Benoist and Mathis 2001). It means that although many pathogen proteins can be antigenic only a few of them would elicit a protective immune response. RESULTS AND DISCUSSION As said before, most of the moonlighting proteins belong to the central metabolism (glycolysis, Krebs cycle…) and their enzyme amino acid sequences are highly conserved throughout evolution (Henderson and Martin 2011; Amblee and Jeffery 2015). Therefore, they probably share epitopes. Obviously, this has a particular importance for the design and development of successful subunit recombinant vaccines, especially with the advent of reverse vaccinology. Figure 1 shows a multialignment of nine representatives of one of the most prevalent virulence moonlighting proteins, enolases, versus the human orthologous protein, alpha-enolase. The predicted B-cell epitopes are highlighted in yellow in all the enolase protein sequences. As can be seen, many human epitopes overlap with highly conserved amino acid sequence stretches present in all enolases. According to our hypothesis the human immune system would not elicit protective antibodies against the pathogen's enolases. As we have previously described, there are examples in which even a lower number of shared epitopes can be responsible of an autoimmune response (Amela, Cedano and Querol 2007). Table S2 (Supporting Information) shows other examples of other pathogen's moonlighting enzymes (GAPDH, PDK, PMD…) aligned with the human orthologous counterpart. As can be seen, they also match stretches of amino acid sequence and overlap predicted epitopes. Figure 1. View largeDownload slide Clustal Omega multialignment of human and pathogen enolases. Highlighted in yellow shows the predicted B-cell epitopes in all the enolase protein sequences. Asterisks depict fully conserved amino acids. Most human predicted epitopes match to highly conserved amino acids stretches. Microorganisms are as follows: AERHY = Aeromonas hydrophila; NEIMB = Neisseria meningitidis; BORBU = Borrelia burgdorferi; BACAN = Bacillus anthracis; STAAU = Staphylococcus aureus; STRPN = Streptococcus pneumoniae; STRPY = Streptococcus pyogenes; STRMU = Streptococcus mutans; STRSU = Streptococcus suis. Figure 1. View largeDownload slide Clustal Omega multialignment of human and pathogen enolases. Highlighted in yellow shows the predicted B-cell epitopes in all the enolase protein sequences. Asterisks depict fully conserved amino acids. Most human predicted epitopes match to highly conserved amino acids stretches. Microorganisms are as follows: AERHY = Aeromonas hydrophila; NEIMB = Neisseria meningitidis; BORBU = Borrelia burgdorferi; BACAN = Bacillus anthracis; STAAU = Staphylococcus aureus; STRPN = Streptococcus pneumoniae; STRPY = Streptococcus pyogenes; STRMU = Streptococcus mutans; STRSU = Streptococcus suis. In fact, it has been reported that streptococcal enolase cross-react with human enolase and may be involved in autoimmune conditions and complications following infection (Fontan et al. 2000; Cole et al. 2005). An exhaustive inspection of Violinet database shows that no moonlighting protein is, as a vaccine, in the market, which is a good indicator of being a true protective antigen. Few (some chaperones such as GroEL and hsp70) are even in the lower Violinet status (research) —some chaperones such as GroEL and hsp70. Moreover, in all the cases involving moonlighting proteins, the assays have been done on mice, guinea pig or rabbit showing quite a low level of protection (≤20%) or the authors merely indicate the presence of ‘some degree’ of immune response. According to Violinet, the recombinant proteins that have reached the market (or are close to doing it, like an ebola vaccine) are shown in Table 1. Table 1. Current recombinant protein vaccines. Pathogen  Host  Recombinant protiens  Neisseria meningitidis  Human  NHBA, NadA and FHbp  Borrelia burgdorferi  Human  OspA  Bordetella pertussis  Human  fhaB  Human papilloma virus  Human  L1  Hepatitis B virus  Human  Capsid protein  Zaire ebola virus  Human  Vp35  Pig circovirus  Pig  Capsid protein  Pasteurella multocida serotype D  Pig  Dermonecrotoxin  Pathogen  Host  Recombinant protiens  Neisseria meningitidis  Human  NHBA, NadA and FHbp  Borrelia burgdorferi  Human  OspA  Bordetella pertussis  Human  fhaB  Human papilloma virus  Human  L1  Hepatitis B virus  Human  Capsid protein  Zaire ebola virus  Human  Vp35  Pig circovirus  Pig  Capsid protein  Pasteurella multocida serotype D  Pig  Dermonecrotoxin  View Large Table 1. Current recombinant protein vaccines. Pathogen  Host  Recombinant protiens  Neisseria meningitidis  Human  NHBA, NadA and FHbp  Borrelia burgdorferi  Human  OspA  Bordetella pertussis  Human  fhaB  Human papilloma virus  Human  L1  Hepatitis B virus  Human  Capsid protein  Zaire ebola virus  Human  Vp35  Pig circovirus  Pig  Capsid protein  Pasteurella multocida serotype D  Pig  Dermonecrotoxin  Pathogen  Host  Recombinant protiens  Neisseria meningitidis  Human  NHBA, NadA and FHbp  Borrelia burgdorferi  Human  OspA  Bordetella pertussis  Human  fhaB  Human papilloma virus  Human  L1  Hepatitis B virus  Human  Capsid protein  Zaire ebola virus  Human  Vp35  Pig circovirus  Pig  Capsid protein  Pasteurella multocida serotype D  Pig  Dermonecrotoxin  View Large A BLASTP pair alignment of these proteins versus the human and mammal proteomes shows that the output states No significant similarity found. These results agree with our hypothesis that the host, in order to avoid an autoimmune response, avoids eliciting protective antibodies against pathogen proteins with which it shares epitopes. Therefore, pathogen evolution would positively select those virulence proteins whose amino acid sequence is conserved to some degree. Our hypothesis would explain the lack of successful subunit vaccines present in the market that are based on these moonlighting proteins. On the other hand, due to the degree of conserved sequence and shared and epitopes, it should exist cross-strain protective immunity using these moonlighting proteins as subunit vaccines, which is not the case. For all these reasons, a strategy based on designing a vaccine using a moonlighting protein as the main antigen might be unsuccessful. MATERIALS AND METHODS Pathogen virulence proteins that are moonlighting were collected from MultitaskProtDB-II (Franco et al. 2018), (see Table S1,Supporting Information). The vaccine candidate proteins were obtained from the database Violinet (He et al. 2014), which contains 800 proteins that have been tested as subunit vaccines (recombinant or isolated) and then purified. These proteins can be real marketed vaccines, licensed as successful ones, or having only a ‘research’ status. Continuous B-cell epitopes of the human orthologous proteins of the previously said pathogen virulence proteins (i.e. enolases) were predicted using the algorithm BepiPred (Larsen, Lund and Nielssen 2006). Protein sequence alignments were performed with BLASTP of the NCBI server (Altschul et al. 1997), and multialignments were done with Clustal–Omega of the EBI Server (Li et al. 2015). Both analyses were performed under the server default parameters. SUPPLEMENTARY DATA Supplementary data are available at FEMSPD online. FUNDING This research was supported by Ministerio de Economía y Competitividad of Spain [BFU2013-50176-EXP, BIO2013-48704-R and BIO2017-84166R], by the Centre de Referència de R+D de Biotecnologia de la Generalitat de Catalunya, and from the Comisión Coordinadora del Interior de Uruguay. Conflict of interest. None declare. REFERENCES Altschul SF, Madden TL, Schäffer AA et al.   Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res  1997; 25: 3389– 402. Google Scholar CrossRef Search ADS PubMed  Amblee V, Jeffery C. Physical features of intracellular proteins that moonlight on the cell surface. PLoS One . 2015; 10: e0130575. doi: 10.1371 /journal.pone.0130575. Google Scholar CrossRef Search ADS PubMed  Amela I, Cedano J, Querol E. Pathogen proteins eliciting antibodies do not share epitopes with host proteins: a bioinformatics approach. PLoS One  2007; 2: e512. Google Scholar CrossRef Search ADS PubMed  Benoist C, Mathis D. Autoimmunity provoked by infection: how good is the case for T cell epitope mimicry? Nat Immunol  2001; 2: 797– 801. Google Scholar CrossRef Search ADS PubMed  Chen C, Zabad S, Liu H et al.   MoonProt 2.0: an expansion and update of the moonlighting proteins database. Nucleic Acids Res  2018; 46: D640– 4. Google Scholar CrossRef Search ADS PubMed  Cole JN, Ramirez RD, Currie BJ et al.   Surface analyses and immune reactivities of major cell wall-associated proteins of group A Streptococcus. Infect Immun  2005; 73: 3137– 46. Google Scholar CrossRef Search ADS PubMed  Copley SD. Moonlighting is mainstream: paradigm adjustment required. Bioessays  2012; 34: 578– 88. Google Scholar CrossRef Search ADS PubMed  Fontan PA, Pancholi V, Nociari MM et al.   Antibodies to streptococcal surface enolase react with human alpha‐enolase: implications in poststreptococcal sequelae. J Infect Dis  2000; 182: 1712– 21. Google Scholar CrossRef Search ADS PubMed  Franco-Serrano L, Hernández S, Calvo A et al.   MultitaskProtDB-II: an update of a database of multitasking/moonlighting proteins /moonlighting proteins. Nucleic Acids Res  2018; 46: D645– 8. Google Scholar CrossRef Search ADS PubMed  Henderson B, Martin A. Bacterial virulence in the moonlight: multitasking bacterial moonlighting proteins are virulence determinants in infectious disease. Infect Immun  2011; 79: 3476– 91. Google Scholar CrossRef Search ADS PubMed  He Y, Racz R, Sayers S et al.   Updates on the web-based VIOLIN vaccine database and analysis system. Nucl Acids Res  2014; 42: D1124– 32. Google Scholar CrossRef Search ADS PubMed  Huberts DH, van der Klei IJ. Moonlighting proteins: an intriguing mode of multitasking. Biochim Biophys Acta  2010; 1803: 520– 5. Google Scholar CrossRef Search ADS PubMed  Jeffery CJ. An introduction to protein moonlighting. Biochem Soc Trans . 2014; 42: 1679– 83. Google Scholar CrossRef Search ADS PubMed  Jin H, Agarwal S, Pancholi V. Surface export of GAPDH/SDH, a glycolytic enzyme, is essential for Streptococcus pyogenes virulence. mBio . 2011; 2: e00068– 11. Google Scholar CrossRef Search ADS PubMed  Larsen JEP, Lund O, Nielssen M. Improved method for predicting linear B-cell epitopes. Immunome Res  2006; 2: 2. Google Scholar CrossRef Search ADS PubMed  Li W, Cowley A, Uludag M et al.   The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Res  2015; 43: W580– 4. Google Scholar CrossRef Search ADS PubMed  Tunio SA, Oldfield NJ, Berry A et al.   The moonlighting protein fructose-1,6 biphosphate aldolase of Neisseria meningitidis adherence to human cells. BMC Microbiol . 2010; 76: 605– 15. Yoshida N, Okeda K, Watanabe E et al.   Protein function. Chaperonin turned insect toxin. Nature . 2001; 411: 44. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Pathogens and Disease Oxford University Press

A hypothesis explaining why so many pathogen virulence proteins are moonlighting proteins

Loading next page...
 
/lp/ou_press/a-hypothesis-explaining-why-so-many-pathogen-virulence-proteins-are-C6uA6JltV9
Publisher
John Wiley & Sons Inc.
Copyright
© FEMS 2018.
ISSN
2049-632X
eISSN
2049-632X
D.O.I.
10.1093/femspd/fty046
Publisher site
See Article on Publisher Site

Abstract

Abstract Moonlighting or multitasking proteins refer to those proteins with two or more functions performed by a single polypeptide chain. Proteins that belong to key ancestral functions and metabolic pathways such as primary metabolism typically exhibit moonlighting phenomenon. We have collected 698 moonlighting proteins in MultitaskProtDB-II database. A survey shows that 25% of the proteins of the database correspond to moonlighting functions related to pathogens virulence activity. Why is the canonical function of these virulence proteins mainly from ancestral key biological functions (especially of primary metabolism)? Our hypothesis is that these proteins present a high conservation between the pathogen protein and the host counterparts. Therefore, the host immune system will not elicit protective antibodies against pathogen proteins. The fact of sharing epitopes with host proteins (known as epitope mimicry) might be the cause of autoimmune diseases. Although many pathogen proteins can be antigenic, only a few of them would elicit a protective immune response. This would also explain the lack of successful vaccines based in these conserved moonlighting proteins. moonlighting proteins, vaccines, virulence proteins, host immune response, epitope, conservation INTRODUCTION Moonlighting and multitasking proteins refer to those proteins with two or more functions performed by a single polypeptide chain. Moonlighting proteins present alternative functions (named canonical and moonlighting) that are mostly affected by cellular localization, cell type, oligomeric state, concentration of cellular ligands, substrates, cofactors, products or post-translational modifications (Huberts 2010; Copley 2012; Jeffery 2014). Usually multitasking proteins are experimentally revealed by serendipity. The appearance of a new function within a polypeptide can become an advantage for the microorganism because it permits a lower number of genes and proteins, thus, making its genome more compact. In any case, these proteins complicate the interpretation of knock-outs, DNA arrays, metabolomics, systems biology, drug pharmacokinetics, pharmacodynamics and toxicity assays/analyses. It is remarkable that moonlighting is typically exhibited by proteins that are ubiquitous and belong to an ‘ancestral’ function or pathway, for example primary metabolism. Currently, two updated multitasking/moonlighting protein databases exist: MultitaskProtDB-II (Franco et al. 2018) and MoonProt (Chen et al. 2018). MultitaskProtDB-II contains 694 multitasking proteins. A remarkable fact is that 25% of these known multitasking proteins present a moonlighting function related to the pathogen's virulence activity (Table S1, Supporting Information). Moreover, their canonical function is to be an enzyme of key pathways like glycolysis or Krebs cycle (Henderson and Martin 2011; Amblee and Jeffery 2015). It is remarkable that these proteins are intracellular and, without having a canonical signal peptide or secretion motifs, they go outside the microorganism cell membrane by a still unknown mechanism. These facts rise two major questions: Why is the canonical function of these virulence proteins mainly from ancestral key biological functions (especially of primary metabolism)? And, why are they shared by many pathogen species? The high percentage of moonlighting proteins of our database that have been reported as pathogen virulence factors prompts a question: Are most of them true virulence factors? Since the function of pathogen moonlighting proteins is related to key metabolism, hence essential proteins, it is difficult to generate mutants or full-gene knock-outs in order to perform direct experimental demonstrations on their virulence involvement. However, some newly designed and naturally existing mutants do not lose the essential canonical function but become non-virulent in in vivo challenges. For example, in anaerobic conditions bacteria lack an operative glycolytic pathway. Neisseria meningitidis, which uses fructose-1,6 biphosphate aldolase as an adhesin, loses the adherence capacity to the epithelial and endothelial cells when isogenically mutated, but this capacity returns in the complementation assay (Tunio et al.2010). Another example of modification of a glycolytic enzyme without losing its canonical function is glyceraldehyde phosphate dehydrogenase of Streptococcus pyogenes. Here, a small hydrophobic amino acid sequence has been added reducing the amounts of GAPDH at the surface of the microorganism and this mutant loses its virulence in mice (Jin, Agarwal and Pancholi 2011). Finally, another example of a bacterial moonlighting protein acting as a toxin is GroEL, the archetypal category of virulence factors. This protein is an insect neurotoxin in Enterobacter aerogens, but is inactive in Escherichia coli when mutated in three amino acids (Yoshida et al.2001). We propose a hypothesis based on our previous work (Amela, Cedano and Querol 2007). In that work, we suggested that, given that one of the most important tasks of the immune system is the differentiation between self and non-selfantigens, this system would discard eliciting protective antibodies against pathogen proteins sharing epitopes with host proteins (epitope mimicry), because this could be the cause of autoimmune diseases (Benoist and Mathis 2001). It means that although many pathogen proteins can be antigenic only a few of them would elicit a protective immune response. RESULTS AND DISCUSSION As said before, most of the moonlighting proteins belong to the central metabolism (glycolysis, Krebs cycle…) and their enzyme amino acid sequences are highly conserved throughout evolution (Henderson and Martin 2011; Amblee and Jeffery 2015). Therefore, they probably share epitopes. Obviously, this has a particular importance for the design and development of successful subunit recombinant vaccines, especially with the advent of reverse vaccinology. Figure 1 shows a multialignment of nine representatives of one of the most prevalent virulence moonlighting proteins, enolases, versus the human orthologous protein, alpha-enolase. The predicted B-cell epitopes are highlighted in yellow in all the enolase protein sequences. As can be seen, many human epitopes overlap with highly conserved amino acid sequence stretches present in all enolases. According to our hypothesis the human immune system would not elicit protective antibodies against the pathogen's enolases. As we have previously described, there are examples in which even a lower number of shared epitopes can be responsible of an autoimmune response (Amela, Cedano and Querol 2007). Table S2 (Supporting Information) shows other examples of other pathogen's moonlighting enzymes (GAPDH, PDK, PMD…) aligned with the human orthologous counterpart. As can be seen, they also match stretches of amino acid sequence and overlap predicted epitopes. Figure 1. View largeDownload slide Clustal Omega multialignment of human and pathogen enolases. Highlighted in yellow shows the predicted B-cell epitopes in all the enolase protein sequences. Asterisks depict fully conserved amino acids. Most human predicted epitopes match to highly conserved amino acids stretches. Microorganisms are as follows: AERHY = Aeromonas hydrophila; NEIMB = Neisseria meningitidis; BORBU = Borrelia burgdorferi; BACAN = Bacillus anthracis; STAAU = Staphylococcus aureus; STRPN = Streptococcus pneumoniae; STRPY = Streptococcus pyogenes; STRMU = Streptococcus mutans; STRSU = Streptococcus suis. Figure 1. View largeDownload slide Clustal Omega multialignment of human and pathogen enolases. Highlighted in yellow shows the predicted B-cell epitopes in all the enolase protein sequences. Asterisks depict fully conserved amino acids. Most human predicted epitopes match to highly conserved amino acids stretches. Microorganisms are as follows: AERHY = Aeromonas hydrophila; NEIMB = Neisseria meningitidis; BORBU = Borrelia burgdorferi; BACAN = Bacillus anthracis; STAAU = Staphylococcus aureus; STRPN = Streptococcus pneumoniae; STRPY = Streptococcus pyogenes; STRMU = Streptococcus mutans; STRSU = Streptococcus suis. In fact, it has been reported that streptococcal enolase cross-react with human enolase and may be involved in autoimmune conditions and complications following infection (Fontan et al. 2000; Cole et al. 2005). An exhaustive inspection of Violinet database shows that no moonlighting protein is, as a vaccine, in the market, which is a good indicator of being a true protective antigen. Few (some chaperones such as GroEL and hsp70) are even in the lower Violinet status (research) —some chaperones such as GroEL and hsp70. Moreover, in all the cases involving moonlighting proteins, the assays have been done on mice, guinea pig or rabbit showing quite a low level of protection (≤20%) or the authors merely indicate the presence of ‘some degree’ of immune response. According to Violinet, the recombinant proteins that have reached the market (or are close to doing it, like an ebola vaccine) are shown in Table 1. Table 1. Current recombinant protein vaccines. Pathogen  Host  Recombinant protiens  Neisseria meningitidis  Human  NHBA, NadA and FHbp  Borrelia burgdorferi  Human  OspA  Bordetella pertussis  Human  fhaB  Human papilloma virus  Human  L1  Hepatitis B virus  Human  Capsid protein  Zaire ebola virus  Human  Vp35  Pig circovirus  Pig  Capsid protein  Pasteurella multocida serotype D  Pig  Dermonecrotoxin  Pathogen  Host  Recombinant protiens  Neisseria meningitidis  Human  NHBA, NadA and FHbp  Borrelia burgdorferi  Human  OspA  Bordetella pertussis  Human  fhaB  Human papilloma virus  Human  L1  Hepatitis B virus  Human  Capsid protein  Zaire ebola virus  Human  Vp35  Pig circovirus  Pig  Capsid protein  Pasteurella multocida serotype D  Pig  Dermonecrotoxin  View Large Table 1. Current recombinant protein vaccines. Pathogen  Host  Recombinant protiens  Neisseria meningitidis  Human  NHBA, NadA and FHbp  Borrelia burgdorferi  Human  OspA  Bordetella pertussis  Human  fhaB  Human papilloma virus  Human  L1  Hepatitis B virus  Human  Capsid protein  Zaire ebola virus  Human  Vp35  Pig circovirus  Pig  Capsid protein  Pasteurella multocida serotype D  Pig  Dermonecrotoxin  Pathogen  Host  Recombinant protiens  Neisseria meningitidis  Human  NHBA, NadA and FHbp  Borrelia burgdorferi  Human  OspA  Bordetella pertussis  Human  fhaB  Human papilloma virus  Human  L1  Hepatitis B virus  Human  Capsid protein  Zaire ebola virus  Human  Vp35  Pig circovirus  Pig  Capsid protein  Pasteurella multocida serotype D  Pig  Dermonecrotoxin  View Large A BLASTP pair alignment of these proteins versus the human and mammal proteomes shows that the output states No significant similarity found. These results agree with our hypothesis that the host, in order to avoid an autoimmune response, avoids eliciting protective antibodies against pathogen proteins with which it shares epitopes. Therefore, pathogen evolution would positively select those virulence proteins whose amino acid sequence is conserved to some degree. Our hypothesis would explain the lack of successful subunit vaccines present in the market that are based on these moonlighting proteins. On the other hand, due to the degree of conserved sequence and shared and epitopes, it should exist cross-strain protective immunity using these moonlighting proteins as subunit vaccines, which is not the case. For all these reasons, a strategy based on designing a vaccine using a moonlighting protein as the main antigen might be unsuccessful. MATERIALS AND METHODS Pathogen virulence proteins that are moonlighting were collected from MultitaskProtDB-II (Franco et al. 2018), (see Table S1,Supporting Information). The vaccine candidate proteins were obtained from the database Violinet (He et al. 2014), which contains 800 proteins that have been tested as subunit vaccines (recombinant or isolated) and then purified. These proteins can be real marketed vaccines, licensed as successful ones, or having only a ‘research’ status. Continuous B-cell epitopes of the human orthologous proteins of the previously said pathogen virulence proteins (i.e. enolases) were predicted using the algorithm BepiPred (Larsen, Lund and Nielssen 2006). Protein sequence alignments were performed with BLASTP of the NCBI server (Altschul et al. 1997), and multialignments were done with Clustal–Omega of the EBI Server (Li et al. 2015). Both analyses were performed under the server default parameters. SUPPLEMENTARY DATA Supplementary data are available at FEMSPD online. FUNDING This research was supported by Ministerio de Economía y Competitividad of Spain [BFU2013-50176-EXP, BIO2013-48704-R and BIO2017-84166R], by the Centre de Referència de R+D de Biotecnologia de la Generalitat de Catalunya, and from the Comisión Coordinadora del Interior de Uruguay. Conflict of interest. None declare. REFERENCES Altschul SF, Madden TL, Schäffer AA et al.   Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res  1997; 25: 3389– 402. Google Scholar CrossRef Search ADS PubMed  Amblee V, Jeffery C. Physical features of intracellular proteins that moonlight on the cell surface. PLoS One . 2015; 10: e0130575. doi: 10.1371 /journal.pone.0130575. Google Scholar CrossRef Search ADS PubMed  Amela I, Cedano J, Querol E. Pathogen proteins eliciting antibodies do not share epitopes with host proteins: a bioinformatics approach. PLoS One  2007; 2: e512. Google Scholar CrossRef Search ADS PubMed  Benoist C, Mathis D. Autoimmunity provoked by infection: how good is the case for T cell epitope mimicry? Nat Immunol  2001; 2: 797– 801. Google Scholar CrossRef Search ADS PubMed  Chen C, Zabad S, Liu H et al.   MoonProt 2.0: an expansion and update of the moonlighting proteins database. Nucleic Acids Res  2018; 46: D640– 4. Google Scholar CrossRef Search ADS PubMed  Cole JN, Ramirez RD, Currie BJ et al.   Surface analyses and immune reactivities of major cell wall-associated proteins of group A Streptococcus. Infect Immun  2005; 73: 3137– 46. Google Scholar CrossRef Search ADS PubMed  Copley SD. Moonlighting is mainstream: paradigm adjustment required. Bioessays  2012; 34: 578– 88. Google Scholar CrossRef Search ADS PubMed  Fontan PA, Pancholi V, Nociari MM et al.   Antibodies to streptococcal surface enolase react with human alpha‐enolase: implications in poststreptococcal sequelae. J Infect Dis  2000; 182: 1712– 21. Google Scholar CrossRef Search ADS PubMed  Franco-Serrano L, Hernández S, Calvo A et al.   MultitaskProtDB-II: an update of a database of multitasking/moonlighting proteins /moonlighting proteins. Nucleic Acids Res  2018; 46: D645– 8. Google Scholar CrossRef Search ADS PubMed  Henderson B, Martin A. Bacterial virulence in the moonlight: multitasking bacterial moonlighting proteins are virulence determinants in infectious disease. Infect Immun  2011; 79: 3476– 91. Google Scholar CrossRef Search ADS PubMed  He Y, Racz R, Sayers S et al.   Updates on the web-based VIOLIN vaccine database and analysis system. Nucl Acids Res  2014; 42: D1124– 32. Google Scholar CrossRef Search ADS PubMed  Huberts DH, van der Klei IJ. Moonlighting proteins: an intriguing mode of multitasking. Biochim Biophys Acta  2010; 1803: 520– 5. Google Scholar CrossRef Search ADS PubMed  Jeffery CJ. An introduction to protein moonlighting. Biochem Soc Trans . 2014; 42: 1679– 83. Google Scholar CrossRef Search ADS PubMed  Jin H, Agarwal S, Pancholi V. Surface export of GAPDH/SDH, a glycolytic enzyme, is essential for Streptococcus pyogenes virulence. mBio . 2011; 2: e00068– 11. Google Scholar CrossRef Search ADS PubMed  Larsen JEP, Lund O, Nielssen M. Improved method for predicting linear B-cell epitopes. Immunome Res  2006; 2: 2. Google Scholar CrossRef Search ADS PubMed  Li W, Cowley A, Uludag M et al.   The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Res  2015; 43: W580– 4. Google Scholar CrossRef Search ADS PubMed  Tunio SA, Oldfield NJ, Berry A et al.   The moonlighting protein fructose-1,6 biphosphate aldolase of Neisseria meningitidis adherence to human cells. BMC Microbiol . 2010; 76: 605– 15. Yoshida N, Okeda K, Watanabe E et al.   Protein function. Chaperonin turned insect toxin. Nature . 2001; 411: 44. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Journal

Pathogens and DiseaseOxford University Press

Published: Apr 30, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off