Elsevier

Journal of Proteomics

Volume 133, 5 February 2016, Pages 113-124
Journal of Proteomics

Identification of vaccine candidate antigens of Staphylococcus pseudintermedius by whole proteome characterization and serological proteomic analyses

https://doi.org/10.1016/j.jprot.2015.12.017Get rights and content

Highlights

  • New therapies are urgently needed for methicillin-resistant S. pseudintermedius.

  • MRSP proteome characterization and SERPA identify potential vaccine antigens.

  • 4 highly immunogenic proteins identified: SpsD, AtpA, FABI and predicted lipoprotein

Abstract

The recent emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) has complicated considerably the treatment of infections caused by these bacteria. Therefore new treatment strategies are urgently needed, namely through the development of vaccines towards the control of bacterial infections. Our study describes an extensive characterization of the proteome of S. pseudintermedius through a 2-DE MALDI-TOF/TOF approach, followed by SERological Proteome Analysis (SERPA) to identify potential vaccine candidate antigens. We were able to identify 361 unique proteins, of which 39 are surface proteins. In order to assess the immunogenic potential of S. pseudintermedius proteins, a Western blot analysis of two-dimensional gels was carried out with serum from healthy dogs, dogs with atopic dermatitis infected and not infected with S. pseudintermedius. Only immunogenic areas detected by ≥ 50% of the dogs with atopic dermatitis infected with S. pseudintermedius sera and by < 50% of the healthy dogs sera were excised and identified from Coomassie-colloidal stained gels. The areas identified by IgE were not considered as vaccine targets, because those proteins could induce hypersensitivity. We were able to identify 13 unique proteins after in-gel digestion of selected protein gel spots, with 4 antigenic proteins showing promising features for vaccine development. No specific antibodies were identified in the dogs with atopic dermatitis not infected with S. pseudintermedius sera that could contribute to prevention of infection. The SERPA approach employed in this study revealed novel candidate therapeutic targets for the control of S. pseudintermedius infections.

Introduction

Staphylococcus pseudintermedius is a Gram-positive coccus, which belongs to the Staphylococcus intermedius group (SIG) [1]. Like Staphylococcus aureus, S. pseudintermedius are normally harmless and colonize the skin and mucous membranes of animals [1]. However, they can also be pathogenic by overcoming the hosts' immune system and of clinical interest because they cause a variety of infections among small animals, especially dogs, causing high morbidity [1]. One of the most common diseases caused by S. pseudintermedius is pyoderma in dogs. Pyoderma is usually secondary to atopic dermatitis, a pruritic allergic skin disease that results in disrupted skin barrier and predisposition for secondary S. pseudintermedius infections [2]. S. pseudintermedius produces a wide range of virulence factors, especially proteins. However, not much is known about the role of these proteins during infection [3]. In S. aureus, homologue proteins are produced in a cell density-dependent manner, through the agr system [4]. To initiate infection, S. aureus synthesizes surface proteins, like adhesins, that bind extracellular-matrix molecules and promote colonization [4]. Once colonization has been established the bacteria multiply, activating a density-sensing mechanism that stimulates the production of exoproteins, like exfoliative toxins and leukocidins, therefore promoting the spread of bacteria to other sites [4]. It has been hypothesized that the agr system of S. pseudintermedius may play a comparable role in the pathogenesis of these bacteria in canine pyoderma [3].

Pyoderma is usually treated with antimicrobials [1]. The selective pressure imposed by the long-term administration of antimicrobials can lead to the development of bacterial resistance that could potentially be transmitted to human pathogens, like S. aureus. The recent emergence of methicillin-resistant S. pseudintermedius (MRSP) has complicated considerably the treatment of infections caused by these bacteria. MRSP have become virtually resistant to all the antimicrobials approved for administration in companion animals, which has led to ethical concerns about the use of antimicrobials classified by the World Health Organization as “critically important” for human medicine [1]. In this way new treatment strategies are urgently needed and one way of controlling bacterial infections is through the development of vaccines.

The “omics” era (genomics, transcriptomics and proteomics) has brought new tools that provide information about cell's genetic background, their potential regulatory mechanisms and biological activity of proteins [5]. Proteomics has the advantage of dealing directly with proteins, which are the main players of life processes [6]. Proteome characterization of bacterial species, especially of their surface-exposed protein fractions, can lead to the identification of vaccine candidates, which can then be tested for their ability to induce protective immunity or as passive immunization targets [5], [7], [8]. This can be accomplished in silico, using bioinformatics tools, and/or by combining proteomics with serological analysis (SERPA — SERological Proteome Analysis) [7], [8], [9], [10], [11], [12], [13], [14]. This approach has enabled the identification of vaccine candidates against S. aureus [12], [13], [14].

The aim of this study was to characterize the proteome of S. pseudintermedius through the 2-DE MALDI-TOF/TOF approach and subsequently use a SERPA approach, using immunoblots followed by MS protein identification, to depict potential vaccine candidate antigens. To design potent and generally applicable subunit vaccines, it is necessary to identify those antigens that are recognizable on a wide patient population during infection [9]. For this reason we used serum from dogs with atopic dermatitis and recurrently infected with S. pseudintermedius (ADI) and discarded those proteins identified by healthy dogs (H), to detect proteins only expressed during infection. We used a third group, dogs with atopic dermatitis and not recurrently infected with S. pseudintermedius (ADH), in an attempt to identify antigenic proteins for which specific antibodies were missing or underrepresented in infected patients that could contribute to prevention of infection.

Section snippets

Bacterial strain and culture conditions

S. pseudintermedius strain FMV5819/10 was isolated from a dog with deep pyoderma presented to the Dermatology Service of the Teaching Hospital FMV-UL. This strain belongs to sequence type 71 and has an SCCmec II–III, representing the European MRSP clone ST71-II–III [16]. An overnight culture was inoculated into brain–heart infusion broth (BHIB, Biokar® Solabia Group, Pantin, France) until the mid-exponential phase was reached, corresponding to an OD600 of 0.4–0.5.

Protein fractionation

The fractionation protocol was

Fractionation of S. pseudintermedius FMV5819/10 proteins

To better characterize the proteins from S. pseudintermedius strain FMV5819/10, we performed a fractionation procedure based on previous protocols used in other Staphylococcus species. Fig. 2 and Supplementary Fig. 1 (with the Spots ID) show the 2-DE patterns of S. pseudintermedius FMV5819/10 protein fractions: cell wall associated, membrane and cytoplasmatic. The first fraction was obtained after lysostaphin treatment. This enzyme cleaves specifically the cross bridges of the staphylococcal

Conflicts of interest

The authors have no conflict of interest to declare.

Acknowledgments

This work was funded by FCT — Fundação para a Ciência e Tecnologia, Project PTDC/CVT-EPI/4345/2012 and PhD grant SFRH/BD/68864/2010 to Natacha Couto from the same institution.

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    These authors contributed equally to this work.

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