Exploring the pan-surfome of Streptococcus suis: Looking for common protein antigens

Streptococcus suis is a swine and human pathogen for which no commercial vaccine is still available. Conserved and broadly distributed surface proteins have become the chosen targets for the development of efficacious vaccines that could overcome the problems of non-heterologous protection of bacterins or capsule polysaccharide-based vaccines. In this work, we have analyzed by proteomics a collection of 39 strains obtained from infected pigs. The isolates belonged to 19 of the most prevalent serotypes during the last years. We have applied the "shaving" approach to define the "pan-surfome" or the set of both common and unique surface proteins identified in such strains. This set was constituted by 113 proteins. We have categorized them for their potential for further vaccination studies, based on their distribution among strains and their a priori accessibility to antibodies. According to these criteria, the cell-wall protein SsnA appears to be the best candidate from this list, as it was that with the widest distribution among the analyzed pathogen types, it showed to be highly immunogenic and highly accessible to antibodies, as demonstrated by flow cytometry.     

A Pneumococcal Protein Array as a Platform to Discover Serodiagnostic Antigens Against Infection

Pneumonia is one of the most common and severe diseases associated with Streptococcus pneumoniae infections in children and adults. Etiological diagnosis of pneumococcal pneumonia in children is generally challenging because of limitations of diagnostic tests and interference with nasopharyngeal colonizing strains. Serological assays have recently gained interest to overcome some problems found with current diagnostic tests in pediatric pneumococcal pneumonia. To provide insight into this field, we have developed a protein array to screen the antibody response to many antigens simultaneously. Proteins were selected by experimental identification from a collection of 24 highly prevalent pediatric clinical isolates in Spain, using a proteomics approach consisting of “shaving” the cell surface with proteases and further LC/MS/MS analysis. Ninety-five proteins were recombinantly produced and printed on an array. We probed it with a collection of sera from children with pneumococcal pneumonia. From the set of the most seroprevalent antigens, we obtained a clear discriminant response for a group of three proteins (PblB, PulA, and PrtA) in children under 4 years old. We validated the results by ELISA and an immunostrip assay showed the translation to easy-to-use, affordable tests. Thus, the protein array here developed presents a tool for broad use in serodiagnostics.Streptococcus pneumoniae, also known as the pneumococcus, is a Gram-positive pathogen recognized as a major cause of pneumonia worldwide (1). It resides as a commensal in the nasopharynx of healthy carriers, but in susceptible individuals this bacterium can spread to other body locations and cause disease. The main group risks are the elderly, immunocompromised people and infants. In fact, ∼800,000 children die each year because of pneumococcal disease, of which >90% of these deaths occur in developing countries (2, 3). In addition, a high number of pneumococcal infection cases are diagnosed in the developed countries and can be associated with high morbidity in children and are an important factor that influences quality of life and produces significant mortality in adults (4, 5). There are licensed polysaccharide-based vaccines to prevent pneumococcal infections, but their efficacy is limited (3). Therefore, pneumococcal pneumonia remains as an important health problem and once it has occurred, early diagnosis with accurate diagnostic methods is essential in order to provide patients with prompt and appropriate therapy and hence to improve outcome (1).Although the major burden of pneumococcal infections is caused by pneumonia, the ability to identify S. pneumoniae as a causative agent in lung infections in children is quite limited. Blood cultures are often negative (6, 7). The BinaxNOW test, which measures teichoic acid, is less specific in children than in adults, because healthy carriage in infants can produce false positive results (8). The amplification and quantification of pneumococcal genes (namely spn9802, ply or pcpA) by PCR has been also used, but with lower sensitivity than culture in blood samples in adults and inability to discriminate between carriage and disease in nasopharyngeal and sputum samples (9–11).The detection of antibody serological markers by any immunoassay is widely used for early diagnosis, epidemiological surveillance, or evaluation of vaccine immunogenicity against many pathogens, including the pneumococcus (6, 12, 13). Serological diagnosis of pneumococcal disease based on a single antigen is often challenging, because of the interference of natural antibodies elicited by previous colonization events. Therefore, to better discriminate between diseased and healthy people, a combination of antigens would be desirable. To this regard, proteomics offers an excellent platform to develop the necessary more sensitive and specific immunoassays that can be used for the aforementioned purposes.It is well assumed that surface proteins are those with the highest chance to raise an effective immune response against pathogen infection, as they are sufficiently exposed and accessible to both T and B cells (14, 15). Protein arrays are powerful tools to interrogate the pattern of host humoral responses to infections (16), which allows the study of many antigens simultaneously with a small amount of sample (17) to select a set of antigens with optimal sensitivity and specificity (18, 19). In this work, we have selected a set of 95 pneumococcal surface proteins by experimental identification, using the proteomic approach of “shaving” of live cells with proteases and further liquid chromatography-tandem MS (LC/MS/MS)¹ analysis (20). After producing the selected proteins as recombinant fragments, we have developed the first pneumococcal surface protein chip and probed it with a collection of sera from infected and control children, in order to find proteins that differentiate between pneumococcal or nonpneumococcal infection/health status. Three proteins were proven to discriminate with optimal sensitivity, specificity, and accuracy between nonpneumococcal disease and disease status for <4-year-old children. As a proof-of-concept, we have developed an immunostrip assay with such proteins, obtaining the same sensitivity, specificity, and accuracy, thus demonstrating the power of high-throughput technologies for discovering diagnostic biomarkers of infection and its possible translation to an easy-to-use clinical tool.     

A surface protein of Streptococcus suis serotype 2 identified by proteomics protects mice against infection

Streptococcus suis serotype 2 is a major Gram-positive swine pathogen, causing also zoonoses. We describe here the immunoprotective activity in an in vivo animal model of a serotype-2 cell wall protein, designated Sat, which was identified by a previously validated proteomics approach consisting of the protease digestion of live bacteria and the selective recovery of exposed domains, followed by LC/MS/MS analysis. Increased survival rate (80%) and decreased bacterial burden were observed in mice immunized with a recombinant Sat fragment, suggesting that this protein is a potential vaccine candidate against serotype-2 infection.     

Exploring anaerobic environments for cyanide and cyano-derivatives microbial degradation

Applied Microbiology and Biotechnology - Cyanide is one of the most toxic chemicals for living organisms described so far. Its toxicity is mainly based on the high affinity that cyanide presents...     

Identification of Potential New Protein Vaccine Candidates through Pan-Surfomic Analysis of Pneumococcal Clinical Isolates from Adults

Purified polysaccharide and conjugate vaccines are widely used for preventing infections in adults and in children against the Gram-positive bacterium Streptococcus pneumoniae, a pathogen responsible for high morbidity and mortality rates, especially in developing countries. However, these polysaccharide-based vaccines have some important limitations, such as being serotype-dependent, being subjected to losing efficacy because of serotype replacement and high manufacturing complexity and cost. It is expected that protein-based vaccines will overcome these issues by conferring a broad coverage independent of serotype and lowering production costs. In this study, we have applied the “shaving” proteomic approach, consisting of the LC/MS/MS analysis of peptides generated by protease treatment of live cells, to a collection of 16 pneumococcal clinical isolates from adults, representing the most prevalent strains circulating in Spain during the last years. The set of unique proteins identified in all the isolates, called “pan-surfome”, consisted of 254 proteins, which included most of the protective protein antigens reported so far. In search of new candidates with vaccine potential, we identified 32 that were present in at least 50% of the clinical isolates analyzed. We selected four of them (Spr0012, Spr0328, Spr0561 and SP670_2141), whose protection capacity has not yet been tested, for assaying immunogenicity in human sera. All of them induced the production of IgM antibodies in infected patients, thus indicating that they could enter the pipeline for vaccine studies. The pan-surfomic approach shows its utility in the discovery of new proteins that can elicit protection against infectious microorganisms.     

Another turn of the screw in shaving Gram-positive bacteria: Optimization of proteomics surface protein identification in Streptococcus pneumoniae

Bacterial surface proteins are of outmost importance as they play critical roles in the interaction between cells and their environment. In addition, they can be targets of either vaccines or antibodies. Proteomic analysis through "shaving" live cells with proteases has become a successful approach for a fast and reliable identification of surface proteins. However, this protocol has not been able to reach the goal of excluding cytoplasmic contamination, as cell lysis is an inherent process during culture and experimental manipulation. In this work, we carried out the optimization of the "shaving" strategy for the Gram-positive human pathogen Streptococcus pneumoniae, a bacterium highly susceptible to autolysis, and set up the conditions for maximizing the identification of surface proteins containing sorting or exporting signals, and for minimizing cytoplasmic contamination. We also demonstrate that cell lysis is an inherent process during culture and experimental manipulation, and that a low level of lysis is enough to contaminate a "surfome" preparation with peptides derived from cytoplasmic proteins. When the optimized conditions were applied to several clinical isolates, we found the majority of the proteins described to induce protection against pneumococcal infection. In addition, we found other proteins whose protection capacity has not been yet tested. In addition, we show the utility of this approach for providing antigens that can be used in serological tests for the diagnosis of pneumococcal disease.