Rhodomyrtone decreases Staphylococcus aureus SigB activity during exponentially growing phase and inhibits haemolytic activity within membrane vesicles.

Sigma factor B (SigB) controls the expression of Staphylococcus aureus genes including virulence factors and plays a role in the bacterial secretion system through membrane vesicle production. Inhibition of SigB could attenuate SigB dependent virulence and secretion system. The objective of this study was to determine the effects of rhodomyrtone on SigB and virulence factors related to SigB. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of rhodomyrtone against 67 clinical methicillin-resistant S. aureus isolates were 0.25-8 µg/ml, which were similar to those of vancomycin. Using luciferase gene fused to SigB dependent promoters of asp23, five time reduction in SigB activity was observed when the bacteria were treated with rhodomyrtone for 3 h. Rhodomyrtone significantly reduced SigB activity in a concentration dependent manner in exponentially growing cells (P < 0.05). In addition, sigB mutant was more sensitive towards increasing concentrations of rhodomyrtone than the wild type and yabJ-spoVG mutant. Rhodomyrtone at 0.625 µg/ml reduced the growth of sigB mutant by approximately 99%, compared with the yabJ-spoVG mutant and the wild type. Membrane vesicles were significantly reduced in the bacterial cells when treated with 0.5 × MIC rhodomyrtone (P < 0.05). Decreased haemolytic activity was detected within rhodomyrtone-treated membrane vesicles. The results indicated that rhodomyrtone inhibited S. aureus SigB activity during exponentially growing phase and inhibited haemolytic activity within membrane vesicles.

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.

Fourteen years of plant proteomics reflected in Proteomics: moving from model species and 2DE-based approaches to orphan species and gel-free platforms.

In this article, the topic of plant proteomics is reviewed based on related papers published in the journal Proteomics since publication of the first issue in 2001. In total, around 300 original papers and 41 reviews published in Proteomics between 2000 and 2014 have been surveyed. Our main objective for this review is to help bridge the gap between plant biologists and proteomics technologists, two often very separate groups. Over the past years a number of reviews on plant proteomics have been published . To avoid repetition we have focused on more recent literature published after 2010, and have chosen to rather make continuous reference to older publications. The use of the latest proteomics techniques and their integration with other approaches in the "systems biology" direction are discussed more in detail. Finally we comment on the recent history, state of the art, and future directions of plant proteomics, using publications in Proteomics to illustrate the progress in the field. The review is organized into two major blocks, the first devoted to provide an overview of experimental systems (plants, plant organs, biological processes) and the second one to the methodology.

Surface-Exposed Protein Moieties of Burkholderia cenocepacia J2315 in Microaerophilic and Aerobic Conditions.

Burkholderia cepacia complex infections remain life-threatening to cystic fibrosis patients, and due to the limited eradication efficiency of current treatments, novel antimicrobial therapies are urgently needed. Surface proteins are among the best targets to develop new therapeutic strategies since they are exposed to the host's immune system. A surface-shaving approach was performed using Burkholderia cenocepacia J2315 to quantitatively compare the relative abundance of surface-exposed proteins (SEPs) expressed by the bacterium when grown under aerobic and microaerophilic conditions. After trypsin incubation of live bacteria and identification of resulting peptides by liquid chromatography coupled with mass spectrometry, a total of 461 proteins with ≥2 unique peptides were identified. Bioinformatics analyses revealed a total of 53 proteins predicted as localized at the outer membrane (OM) or extracellularly (E). Additionally, 37 proteins were predicted as moonlight proteins with OM or E secondary localization. B-cell linear epitope bioinformatics analysis of the proteins predicted to be OM and E-localized revealed 71 SEP moieties with predicted immunogenic epitopes. The protegenicity higher scores of proteins BCAM2761, BCAS0104, BCAL0151, and BCAL0849 point out these proteins as the best antigens for vaccine development. Additionally, 10 of the OM proteins also presented a high probability of playing important roles in adhesion to host cells, making them potential targets for passive immunotherapeutic approaches. The immunoreactivity of three of the OM proteins identified was experimentally demonstrated using serum samples from cystic fibrosis patients, validating our strategy for identifying immunoreactive moieties from surface-exposed proteins of potential interest for future immunotherapies development.

Identification of Potential Bioactive Peptides in Sheep Milk Kefir through Peptidomic Analysis at Different Fermentation Times.

Sheep farming is an important socioeconomic activity in most Mediterranean countries, particularly Spain, where it contributes added value to rural areas. Sheep milk is used in Spain mainly for making cheese, but it can be used also for making other dairy products, such as the lactic-alcoholic fermentation product known as kefir. Dairy products have health benefits because, among other reasons, they contain molecules with biological activity. In this work, we performed a proteomics strategy to identify the peptidome, i.e., the set of peptides contained in sheep milk kefir fermented for four different periods of time, aiming to understand changes in the pattern of digestion of milk proteins, as well as to identify potential bioactive peptides. In total, we identified 1942 peptides coming from 11 different proteins, and found that the unique peptides differed qualitatively among samples and their numbers increased along the fermentation time. These changes were supported by the increase in ethanol, lactic acid, and D-galactose concentrations, as well as proteolytic activity, as the fermentation progressed. By searching in databases, we found that 78 of the identified peptides, all belonging to caseins, had potential biological activity. Of these, 62 were not previously found in any milk kefir from other animal species. This is the first peptidomic study of sheep milk kefir comprising time-course comparison.

SARS-CoV-2 Seroprevalence Study in Pediatric Patients and Health Care Workers Using Multiplex Antibody Immunoassays.

SARS-CoV-2 infection has become a global health problem specially exacerbated with the continuous appearance of new variants. Healthcare workers (HCW) have been one of the most affected sectors. Children have also been affected, and although infection generally presents as a mild disease, some have developed the Pediatric Inflammatory Multisystem Syndrome Temporally Associated with SARS-CoV-2 (PIMS-TS). We recruited 190 adults (HCW and cohabitants, April to June 2020) and 57 children (April 2020 to September 2021), of whom 12 developed PIMS-TS, in a hospital-based study in Spain. Using an in-house Luminex assay previously validated, antibody levels were measured against different spike and nucleocapsid SARS-CoV-2 proteins, including the receptor-binding domain (RBD) of the Alpha, Beta, Gamma, and Delta variants of concern (VoC). Seropositivity rates obtained from children and adults, respectively, were: 49.1% and 11% for IgG, 45.6% and 5.8% for IgA, and 35.1% and 7.3% for IgM. Higher antibody levels were detected in children who developed PIMS-TS compared to those who did not. Using the COVID-19 IgM/IgA ELISA (Vircell, S.L.) kit, widely implemented in Spanish hospitals, a high number of false positives and lower seroprevalences compared with the Luminex estimates were found, indicating a significantly lower specificity and sensitivity. Comparison of antibody levels against RBD-Wuhan versus RBD-VoCs indicated that the strongest positive correlations for all three isotypes were with RBD-Alpha, while the lowest correlations were with RBD-Delta for IgG, RBD-Gamma for IgM, and RBD-Beta for IgA. This study highlights the differences in antibody levels between groups with different demographic and clinical characteristics, as well as reporting the IgG, IgM, and IgA response to RBD VoC circulating at the study period.

Surfome analysis as a fast track to vaccine discovery: identification of a novel protective antigen for Group B Streptococcus hypervirulent strain COH1.

Safe recombinant vaccines, based on a small number of antigenic proteins, are emerging as the most attractive, cost-effective solution against infectious diseases. In the present work, we confirmed previous data from our laboratory showing that whole viable bacterial cell treatment with proteases followed by the identification of released peptides by mass spectrometry is the method of choice for the rapid and reliable identification of vaccine candidates in Gram-positive bacteria. When applied to the Group B Streptococcus COH1 strain, 43 surface-associated proteins were identified, including all the protective antigens described in the literature as well as a new protective antigen, the cell wall-anchored protein SAN_1485 belonging to the serine-rich repeat protein family. This strategy overcomes the difficulties so far encountered in the identification of novel vaccine candidates and speeds up the entire vaccine discovery process by reducing the number of recombinant proteins to be tested in the animal model.

Glass Slide-Printed Protein Arrays as a Platform to Discover Serodiagnostic Antigens Against Bacterial Infections.

Infectious diseases represent a major cause of morbidity and mortality worldwide. Early detection of infections is capital for managing life-threatening cases. So far, traditional diagnostic methods such as microbiological cultures are slow and, sometimes, inaccurate. In the molecular era, high-throughput techniques are essential for providing tools that are able to diagnose in a fast and reliable way, as well as they can be used for monitoring the humoral response of groups of people in a program of epidemiological surveillance when an outbreak occurs, or when a vaccine is being evaluated. Antigen-based protein microarrays are an ideal means for these purposes, as they can carry up to thousands of protein antigens from pathogenic sources and be probed with sera from different human groups (acute or chronic infected people, convalescent, controls). For the diagnosis of bacterial infections, the best antigens are in principle the surface proteins, as they have the highest chances to raise an effective immune response. Here we describe a general protocol for fabricating a glass slide-based protein microarray using recombinant bacterial surface antigens, according to our own expertise in the study of pneumococcal disease. The probing with human sera aims to evaluate differences between diseased and healthy people, in order to discover discriminating antigens that can be used, after appropriate validation, in further easy-to-use formats such as immunostrips.

Approaching In Vivo Models of Pneumococcus-Host Interaction: Insights into Surface Proteins, Capsule Production, and Extracellular Vesicles.

Infections caused by the Gram-positive bacterium Streptococcus pneumoniae have become a major health problem worldwide because of their high morbidity and mortality rates, especially in developing countries. This microorganism colonizes the human upper respiratory tract and becomes pathogenic under certain circumstances, which are not well known. In the interaction with the host, bacterial surface structures and proteins play major roles. To gain knowledge into gradual changes and adaptive mechanisms that this pathogen undergoes from when it enters the host, we mimicked several in vivo situations representing interaction with epithelial and macrophage cells, as well as a condition of presence in blood. Then, we analyzed, in four pneumococcal strains, two major surface structures, the capsule and extracellular vesicles produced by the pneumococci, as well as surface proteins by proteomics, using the "shaving" approach, followed by LC-MS/MS. We found important differences in both surface ultrastructures and proteins among the culture conditions and strains used. Thus, this work provides insights into physiological adaptations of the pneumococcus when it interacts with the host, which may be useful for the design of strategies to combat infections caused by this pathogen.

Comparative Exoproteome Analysis of Streptococcus suis Human Isolates.

The swine pathogen Streptococcus suis is a Gram-positive bacterium which causes infections in pigs, with an impact in animal health and in the livestock industry, and it is also an important zoonotic agent. During the infection process, surface and secreted proteins are essential in the interaction between microorganisms and their hosts. Here, we report a comparative proteomic analysis of the proteins released to the extracellular milieu in six human clinical isolates belonging to the highly prevalent and virulent serotype 2. The total secreted content was precipitated and analyzed by GeLC-MS/MS. In the six strains, 144 proteins assigned to each of the categories of extracellular or surface proteins were identified, as well as 680 predicted cytoplasmic proteins, many of which are putative moonlighting proteins. Of the nine predicted signal peptide-I secreted proteins, seven had relevant antigenic potential when they were analyzed through bioinformatic analysis. This is the first work comparing the exoproteome fraction of several human isolates of this important pathogen.

Extracellular Vesicles from Different Pneumococcal Serotypes Are Internalized by Macrophages and Induce Host Immune Responses.

Bacterial extracellular vesicles are membranous ultrastructures released from the cell surface. They play important roles in the interaction between the host and the bacteria. In this work, we show how extracellular vesicles produced by four different serotypes of the important human pathogen, Streptococcus pneumoniae, are internalized by murine J774A.1 macrophages via fusion with the membrane of the host cells. We also evaluated the capacity of pneumococcal extracellular vesicles to elicit an immune response by macrophages. Macrophages treated with the vesicles underwent a serotype-dependent transient loss of viability, which was further reverted. The vesicles induced the production of proinflammatory cytokines, which was higher for serotype 1 and serotype 8-derived vesicles. These results demonstrate the biological activity of extracellular vesicles of clinically important pneumococcal serotypes.

Omics and Bioinformatics Approaches to Identify Novel Antigens for Vaccine Investigation and Development.

Despite the outstanding technological advances achieved in the last few decades, infectious diseases remain a major societal challenge. From the variolation carried out in ancient China during the 15th century to the more advanced RNA and DNA vaccines presently available, vaccines have been proven as highly effective therapeutic tools to combat various infectious diseases. Vaccine research and development is now empowered with recent advances in Omics sciences and the developments of powerful bioinformatics tools. This Special Issue has gathered a total of nine original papers, including seven research papers and two reviews, illustrating the use of Omics data and bioinformatics in the research, design and development of vaccines against pathogens and cancer. The integration of knowledge from Omics and Bioinformatics will certainly boost vaccine research and development, leading to novel therapeutic tools against new and old pathogens and cancer in the near future.

Proteomic and Bioinformatic Analysis of Streptococcus suis Human Isolates: Combined Prediction of Potential Vaccine Candidates.

Streptococcus suis is a Gram-positive bacterium responsible for major infections in pigs and economic losses in the livestock industry, but also an emerging zoonotic pathogen causing serious diseases in humans. No vaccine is available so far against this microorganism. Conserved surface proteins are among the most promising candidates for new and effective vaccines. Until now, research on this pathogen has focused on swine isolates, but there is a lack of studies to identify and characterize surface proteins from human clinical isolates. In this work, we performed a comparative proteomic analysis of six clinical isolates from human patients, all belonging to the major serotype 2, by "shaving" the live bacterial cells with trypsin, followed by LC-MS/MS analysis. We identified 131 predicted surface proteins and carried out a label-free semi-quantitative analysis of protein abundances within the six strains. Then, we combined our proteomics results with bioinformatic tools to help improving the selection of novel antigens that can enter the pipeline of vaccine candidate testing. Our work is then a complement to the reverse vaccinology concept.

Characterization of the Burkholderia cenocepacia J2315 Surface-Exposed Immunoproteome.

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.

Search of Potential Vaccine Candidates against Trueperella pyogenes Infections through Proteomic and Bioinformatic Analysis.

Trueperella pyogenes is an opportunistic pathogen, responsible for important infections in pigs and significant economic losses in swine production. To date, there are no available commercial vaccines to control diseases caused by this bacterium. In this work, we performed a comparative proteomic analysis of 15 T. pyogenes clinical isolates, by "shaving" live cells, followed by LC-MS/MS, aiming at the identification of the whole set of surface proteins (i.e., the "pan-surfome") as a source of antigens to be tested in further studies as putative vaccine candidates, or used in diagnostic tools. A total of 140 surface proteins were detected, comprising 25 cell wall proteins, 10 secreted proteins, 23 lipoproteins and 82 membrane proteins. After describing the "pan-surfome", the identified proteins were ranked in three different groups based on the following criteria: to be (i) surface-exposed, (ii) highly conserved and (iii) widely distributed among different isolates. Two cell wall proteins, three lipoproteins, four secreted and seven membrane proteins were identified in more than 70% of the studied strains, were highly expressed and highly conserved. These proteins are potential candidates, alone or in combination, to obtain effective vaccines against T. pyogenes or to be used in the diagnosis of this pathogen.

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome.

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.

Multivariate discriminant analysis distinguishes metal- from non metal-related biomarker responses in the clam Chamaelea gallina.

Molecular biomarkers are among the most sensitive and earliest responses to pollutants. However, lack of detailed knowledge on variability of responses and their possible seasonal variation limit their use. In addition, the seasonality of biological processes modulates the response of organisms to pollutant stressors. Using multivariate statistics, we have studied the influence of environmental and biological factors on the response of a battery of molecular biomarkers in the clam Chamaelea gallina collected along the South-Spanish littoral. Multivariate discriminant analysis clearly distinguished biomarker response between clean and polluted areas, using heavy metals as indicator of pollution. Such differences disappeared when the dataset was normalised for metal content, thus indicating that pollution was the main significant cause of the changes observed between clean and polluted sites. In conclusion, this work shows that, when applying a complete biomarker panel, multivariate statistical tools can be used to discern pollutant- from non pollutant-related responses.

Proteomic analysis of goat milk kefir: Profiling the fermentation-time dependent protein digestion and identification of potential peptides with biological activity.

Kefir is a fermented dairy product, associated to health benefits because of being a probiotic and due to the presence of molecules with biological activity. In this work, we have profiled the peptide composition of goat milk kefir at three different fermentation times using a peptidomics approach, in order to study changes in peptide concentrations and patterns of protein digestion throughout the fermentation time. We identified 2328 unique peptides corresponding to 22 protein annotations, with a maximum of peptides found after 24 h fermentation. We established different digestion patterns according to the nature of the proteins, and quantified the changes in the peptides appearing in all the fermentation times. We also identified 11 peptides that matched exactly to sequences with biological activity in databases, almost all of them belonging to caseins. This is the most comprehensive proteomic analysis of goat milk kefir to date.

Overcoming function annotation errors in the Gram-positive pathogen Streptococcus suis by a proteomics-driven approach.

BACKGROUND: Annotation of protein-coding genes is a key step in sequencing projects. Protein functions are mainly assigned on the basis of the amino acid sequence alone by searching of homologous proteins. However, fully automated annotation processes often lead to wrong prediction of protein functions, and therefore time-intensive manual curation is often essential. Here we describe a fast and reliable way to correct function annotation in sequencing projects, focusing on surface proteomes. We use a proteomics approach, previously proven to be very powerful for identifying new vaccine candidates against Gram-positive pathogens. It consists of shaving the surface of intact cells with two proteases, the specific cleavage-site trypsin and the unspecific proteinase K, followed by LC/MS/MS analysis of the resulting peptides. The identified proteins are contrasted by computational analysis and their sequences are inspected to correct possible errors in function prediction. RESULTS: When applied to the zoonotic pathogen Streptococcus suis, of which two strains have been recently sequenced and annotated, we identified a set of surface proteins without cytoplasmic contamination: all the proteins identified had exporting or retention signals towards the outside and/or the cell surface, and viability of protease-treated cells was not affected. The combination of both experimental evidences and computational methods allowed us to determine that two of these proteins are putative extracellular new adhesins that had been previously attributed a wrong cytoplasmic function. One of them is a putative component of the pilus of this bacterium. CONCLUSION: We illustrate the complementary nature of laboratory-based and computational methods to examine in concert the localization of a set of proteins in the cell, and demonstrate the utility of this proteomics-based strategy to experimentally correct function annotation errors in sequencing projects. This approach also contributes to provide strong experimental evidences that can be used to annotate those proteins for which a Gene Ontology (GO) term has not been assigned so far. Function annotation correction would then improve the identification of surface-associated proteins in bacterial pathogens, thus accelerating the discovery of new vaccines in infectious disease research.

"Shaving" Live Bacterial Cells with Proteases for Proteomic Analysis of Surface Proteins.

Surface proteins are essential molecules for the interplay between cells and the environment. They participate in many biological processes including transport, adhesion, cell-cell recognition, signaling, and other cell interactions. In pathogenic microorganisms, these molecules may act as virulence or cytotoxicity factors. Analyzing the set of surface proteins is critical to understand these processes and to identify possible targets that can be the starting point for other studies or discoveries (e.g., vaccines or diagnostics). Here I describe a proteomic procedure to identify in a fast and reliable way a set of surface-exposed proteins in bacteria, the methodology of which can be adapted to other biological systems (unicellular fungi, parasites). The protocol presented here involves "shaving" the cells cultured in broth with proteases followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and analysis of the generated peptides. This method overcomes some important limitations of the first-generation, gel based proteomics techniques, and the "shaving" approach allows one to identify which domains from identified proteins are more accessible to proteases. These identified proteins have the highest potential to be recognized by antibodies, and thus permits the identification of potential epitopes or antigens.