Article Characterization of the Burkholderia cenocepacia J2315 Surface-Exposed Immunoproteome 1, 1, 1, Sílvia A. Sousa * , António M.M. Seixas y , Manoj Mandal y, Manuel J. Rodríguez-Ortega 2 and Jorge H. Leitão 1,* 1 iBB–Institute for Bioengineering and Biosciences, 1049-001 Lisbon, Portugal; [email protected] (A.M.M.S.); [email protected] (M.M.) 2 Departament of Biochemistry and Molecular Biology, Córdoba University, 14071 Córdoba, Spain; [email protected] * Correspondence: [email protected] (S.A.S.); [email protected] (J.H.L.); Tel.: +351-2184-19986 (S.A.S.); +351-2184-17688 (J.H.L.) Both authors contributed equally to the work. y Received: 31 July 2020; Accepted: 3 September 2020; Published: 6 September 2020 Abstract: Infections by the Burkholderia cepacia complex (Bcc) remain seriously life threatening to cystic fibrosis (CF) patients, and no effective eradication is available. A vaccine to protect patients against Bcc infections is a highly attractive therapeutic option, but none is available. A strategy combining the bioinformatics identification of putative surface-exposed proteins with an experimental approach encompassing the “shaving” of surface-exposed proteins with trypsin followed by peptide identification by liquid chromatography and mass spectrometry is here reported. The methodology allowed the bioinformatics identification of 263 potentially surface-exposed proteins, 16 of them also experimentally identified by the “shaving” approach. Of the proteins identified, 143 have a high probability of containing B-cell epitopes that are surface-exposed. The immunogenicity of three of these proteins was demonstrated using serum samples from Bcc-infected CF patients and Western blotting, validating the usefulness of this methodology in identifying potentially immunogenic surface-exposed proteins that might be used for the development of Bcc-protective vaccines. Keywords: Burkholderia cepacia complex; surface-exposed moieties; liquid chromatography–tandem mass spectrometry (LC-MS/MS); immunoreactive proteins; cystic fibrosis; serum samples; immunoreactivity 1. Introduction The Burkholderia cepacia complex (Bcc) presently comprises at least 23 closely related bacterial species [1–3] that emerged in the 1980s as important pathogens for patients suffering from cystic fibrosis (CF). Bcc bacteria can be found in a wide range of environments including water, soil and the rhizosphere of plants, being able to survive in hospital settings and pharmaceutical aqueous solutions [4]. Despite their presence in the environment, Bcc bacteria are opportunistic pathogens capable of causing life-threatening infections in the respiratory tract of immunocompromised patients, patients with chronic granulomatous disease (CGD) and especially patients suffering from CF, the most common lethal inherited genetic disease among Caucasians [5]. The majority of Bcc-infected CF patients experience an increased decline in pulmonary function, which is associated with chronic infection and exacerbation episodes [5]. However, some patients can also develop a rapid and fatal necrotizing pneumonia known as the cepacia syndrome [5,6]. These factors, coupled with the organism’s armory of virulence factors and the intrinsic resistance of Bcc bacteria to several clinically available antimicrobials, render chronic infections virtually untreatable [5,7–9], making infections by Bcc a major concern since Vaccines 2020, 8, 509; doi:10.3390/vaccines8030509 www.mdpi.com/journal/vaccines Vaccines 2020, 8, 509 2 of 22 Vaccines 2020, 8, x 2 of 21 Bccthe a clinical major outcomeconcern since is highly the clinical variable outcome and unpredictable is highly variable [10]. Considering and unpredictable all the above, [10]. Considering a strategy to alleradicate the above, Bcc bacteriaa strategy from to CFeradic patientsate Bcc is bacteria imperative. from Since CF patients there is nois eradicationimperative. therapySince there presently is no eradicationavailable [11 therapy], new strategiespresently available to deal with [11], Bcc new infections strategies are to ofdeal major with interest. Bcc infections Currently, are of no major such interest.strategies Currently, exist, but no a wide such variety strategies of approaches exist, but a andwide antigens variety areof approaches under study and for theantigens development are under of studya possible for the vaccine development against Bccof a [ 12possible–15]. vaccine against Bcc [12–15]. BacteriaBacteria use thethe proteinsproteins expressed expressed on on their their surface surface to to interact interact with with their their environment, environment, being being also alsoexposed exposed to the to immune the immune system system of the of host. the Therefore, host. Therefore, surface-exposed surface-exposed proteins proteins are appealing are appealing antigens antigensfor the development for the development of new of therapeutic new therapeutic strategies strategies for the for eradication the eradication of bacterial of bacterial infections infections [16]. [16].However, However, a wide a wide range range of characteristics of characteristics including including low low abundance, abundance, hydrophobic hydrophobic natures natures and and low lowsolubility solubility make make these these proteins proteins di ffidifficultcult to to study, study, especially especially using using first-generationfirst-generation gel-dependent proteomicsproteomics approaches [17–19]. [17–19]. In In order order to to uncover uncover novel novel surface-exposed surface-exposed antigens antigens to to be be studied studied for for thethe development development of of new new therapeutic therapeutic strategies strategies for for th thee eradication eradication of of Bcc Bcc infections, infections, a a bioinformatics analysisanalysis ofof thethe surface-exposed surface-exposed proteins proteins of B. of cenocepacia B. cenocepaciaJ2315 andJ2315 their and immune their immune profile was profile performed was performedin this work. in The this proteins work. identifiedThe proteins were identified experimentally were validated experimentally using a surfacevalidated “shaving” using a approach surface “shaving”comprising approach the digestion comprising of live intact the digestion cells with of proteases live intact and cells the recoverywith proteases of the cells’ and surface-exposedthe recovery of themoieties, cells’ followedsurface-exposed by their analysismoieties, by followed liquid chromatography–tandem by their analysis by liquid mass chromatography–tandem spectrometry (LC-MS/MS) mass(Figure spectrometry1). In this way,(LC-MS/MS) using a (Figure sensitive 1). andIn this gel-free way, using approach, a sensitive the limitations and gel-free associated approach, with the limitationsmembrane associated protein analysis with membrane can be overcome. protein analysis This approach can be overcome. also grants This the approach acquisition also ofgrants new theinformation, acquisition as of it new allows information, the identification as it allows of the the domains identification of the identifiedof the domains proteins of the that identified are more proteinsaccessible that to are proteases more accessible and therefore to proteases to the and host th immuneerefore to system the host and immune antibodies system [17 and]. The antibodies surface “shaving”[17]. The surface approach “shaving” has been approach applied mainlyhas been to applied Gram-positive mainly bacteria,to Gram-positive owing to theirbacteria, characteristic owing to theirthicker characteristic cell walls that thicker confer cell a walls greater that resistance confer a togreater cell lysis. resistance Nonetheless, to cell lysis. it has Nonetheless, also been applied it has alsoto Gram-negative been applied to bacteria, Gram-negativ unicellulare bacteria, fungi andunicellular also larvae fungi of and parasitic also larvae helminth of parasitic worms [17helminth,20–22]. wormsDifferent [17,20–22]. levels of successDifferent were levels reported of success in each were case, reported depending in each on case, factors depending such as protein on factors structure such asand protein microorganism structure and surface microorganism topology. surface topology. Figure 1. Schematic representation of the steps involved in the identification of surface-exposed Figure 1. Schematic representation of the steps involved in the identification of surface-exposed proteins using the surface-shaving approach for live B. cenocepacia cells. proteins using the surface-shaving approach for live B. cenocepacia cells. Despite the increase in popularity of this technique, it had never been applied to bacteria of the Bcc,Despite and therefore, the increase this pioneering in popularity work of was this envisioned.technique, it With had thisnever purpose, been applied and to to discover bacteria surface of the Bcc,proteins and exposedtherefore, to this the pioneering host during work the infection was envisi process,oned. theWith protocol this purpose, was optimized and to discover for B. cenocepacia surface proteinsJ2315 using exposed growth to conditions the host thatduring mimic the the infe environmentction process, in thethe lungs protocol of CF was patients. optimized The CF for lung B. cenocepaciahas steep oxygen J2315 using gradients, growth usually conditions with low that oxygen mimic concentrations the environment within in the the lungs typically of CF thick patients. mucus The CF lung has steep oxygen gradients, usually with low oxygen