Surface Protein Extracts Research Articles

Abstract LB008: Integrative surfaceome profiling identifies immunotherapeutic targets in osteosarcomas (OS); Preclinical testing of BT1769, an MT1-MMP-targeted Bicycle Toxin Conjugate, in OS by the Pediatric Preclinical Testing Consortium (PPTC)

Abstract Introduction: Toxin-conjugated agents targeting cell-surface antigens (e.g. antibody-drug conjugates) show promising clinical activity for some solid tumors, but none yet for OS. There is an urgent need to identify high-confidence OS cell-surface antigens and evaluate the antitumor activity of toxin conjugates that can deliver cytotoxic payloads to OS tumors expressing these targets. Methods: To identify cell-surface proteins that are highly expressed in OS but not in normal tissues, we prepared surface protein extracts from 4 standard, 4 patient-derived OS cell lines, and 10 OS PDX tissues. Quantitative mass spectrometry (MS) was used to profile the surface proteins. We included the proteomics data of OS and 39 types of normal human tissues via a public MS database (ProteomicsDB). We compared RNA-sequencing data from 101 OS patient samples via the NCI TARGET project with the RNA-sequencing data of 54 types of normal human tissue (GTEx, 17382 samples). We integrated the MS and RNA sequencing datasets to prioritize candidate surface targets by differential and absolute expression at both protein and mRNA levels. The cell-surface expression of the targets was further validated by IHC and flow cytometry with OS cell lines and tumor tissue microarray. BT1769, which consists of a bicyclic peptide targeting the tumor antigen MT1-MMP, linked to the cytotoxin monomethyl auristatin E (MMAE) via a molecular spacer and cleavable linker was tested in 6 OS PDX models using standard PPTC methods at 3 mg/kg given IV weekly x 4. Ewing sarcoma (EWS) models lacking MT1-MMP expression were also tested. Results: MS identified 1156 surface proteins as highly expressed in OS cell lines/PDX tissues. 129 proteins overexpressed in OS were identified from the public MS database. RNA sequencing data identified 195 overexpressed plasma membrane-associated genes in OS compared to normal tissues. The targets identified by both proteomics and transcriptomic data analysis were cross-referenced with existing toxin-conjugated agents. Three potential targets, MT1-MMP, CD276, and MRC2 were validated and MT1-MMP was prioritized because it had the highest absolute expression level in OS. BT1769 significantly inhibited tumor growth in 6/6 OS PDX models when compared to vehicle, with complete response maintained at 6 weeks in 3/6 models. Minimal activity was observed in 2/2 EWS models. Correlation analysis between the MT1-MMP gene expression level, IHC staining intensity and drug response are underway and will be presented. Conclusion: Integrated proteomic and transcriptomic OS surfaceome profiling identified high-confidence OS cell-surface antigens as therapeutic targets. MT1-MMP was highly expressed in most OS patient samples and PDX models, and BT1769 was highly active against select OS PDX models. Citation Format: Yifei Wang, Wendong Zhang, Zhongting Zhang, Xiangjun Tian, Rossana N. Lazcano Segura, Pooja Hingorani, Michael Roth, Jonathan Gill, Douglas Harrison, Zhaohui Xu, Jing Wang, Eric J. Earley, Stephen W. Erickson, Edward A. Kolb, Malcolm A. Smith, Peter Houghton, Raushan Kurmasheva, Tara Gelb, Philip Huxley, Johanna Lahdenranta, Gemma Mudd, Richard Gorlick. Integrative surfaceome profiling identifies immunotherapeutic targets in osteosarcomas (OS); Preclinical testing of BT1769, an MT1-MMP-targeted Bicycle Toxin Conjugate, in OS by the Pediatric Preclinical Testing Consortium (PPTC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB008.

Cell surface and extracellular proteins of potentially probiotic Lactobacillus reuteri as an effective mediator to regulate intestinal epithelial barrier function.

The present study aimed to evaluate the potential of cell surface and extracellular proteins in regulation of intestinal epithelial barrier (IEB) function. Eight potentially probiotic L. reuteri strains were evaluated for presence of mapA gene and its expression on co-culturing with the Caco-2 cells. The ability of untreated (Viable), heat-inactivated, 5M LiCL treated L. reuteri strains as well as their cell-free supernatant (CFS) to modulate expression of IEB function genes (hBD-2, hBD-3, claudin-1 and occludin) was also evaluated. Caco-2 cells were treated with cell surface and extracellular protein extracts and investigated for change in expression of targeted IEB function genes. The results showed that mapA gene is present in all the tested L. reuteri strains and expression of mapA and its receptors (anxA13 and palm) increase significantly on co-culturing of L. reuteri and Caco-2 cells. Also, up-regulated expression of IEB function genes was observed on co-culturing of L. reuteri (viable, heat-inactivated and CFS) and their protein extracts with Caco-2 cells in contrast to down-regulation observed with the pathogenic strain of Salmonella typhi. Therefore, this study concludes that the cell surface and extracellular protein from L. reuteri act as an effective mediator molecules to regulate IEB function.

Abstract 5405: Integrative surfaceome profiling identifies MMP14, MRC2 and CD276 as candidate immunotherapeutic target in osteosarcomas

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor in pediatric patients. However, the survival rate of patients with osteosarcomas has not improved for the last several decades. Current developments in immunotherapeutic strategies may increase the efficacy of these treatments for solid tumors. However, these treatments, such as antibody-drug conjugates (ADCs) or CAR-T cell therapy have not been sufficiently studied in OS because of a lack of known tumor-specific target. In this study, we developed an integrative pipeline combining the transcriptomic data from OS patients and the cell-surface proteomics data generated from a panel of patient-derived OS cell lines and PDX models and relating these to normal tissue expression. RNA sequencing data from 40 high-risk OS patients treated at MD Anderson Cancer Center was pooled with 111 OS patients available through the public TARGET online database. All the tumor data was subsequently compared with normal tissue RNA-sequencing data from the NIH Genotype-Tissue Expression (GTEx) database. The significantly differentially expressed genes (log fold change tumor versus normal >1.5 for each tissue, p < 0.01) were selected. We further filtered this gene list by cell surface protein prediction based on the Compartments subcellular localization database. To determine protein expression levels, we prepared surface protein extracts from a panel of patient-derived OS cell lines and PDX models. Proteomic mass spectrometry was used to profile these OS surface proteins. We also included the proteomics data of OS tissue and 39 types of normal human organs via the public proteomic database. Integrative analysis of the transcriptomic and proteomic data demonstrated MMP14, MRC2, and CD276 are highly expressed in OS compared to normal tissues across all datasets. The expression of the three cell surface proteins was validated by IHC staining with an OS tissue microarray from 100 patients, and western blot with our PDX tissues and patient-derived cell lines. A previously reported preclinical study has shown the CD276 ADC (mCD276-PBD) has good antitumor activity against xenograft models of OS. Multiple preclinical tests including CD276 CAR-T cells, MRC2 ADC and MMP14 Bicycle Drug Conjugate (BT1718) are planned to further validate the therapeutic potential of these targets. Our current findings have not been previously reported in OS. The results might warrant further development of ADC and CAR-T cell therapy for osteosarcomas against these targets. Citation Format: Yifei Wang, Zhongting Zhang, Wendong Zhang, Zhaohui Xu, Xin Zhou, Michael Roth, Jonathan Gill, Douglas Harrison, Pooja Hingorani, Sankar Kannan, Sylvester Jusu, Xiangjun Tian, Jing Wang, Rossana Lazcano, Alexander Lazar, Richard Gorlick. Integrative surfaceome profiling identifies MMP14, MRC2 and CD276 as candidate immunotherapeutic target in osteosarcomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5405.

Comparison of Staphylococcus aureus surface protein extraction methods and immunogenicity

Staphylococcus aureus is the major contagious bovine mastitis pathogen and has no effective vaccine. Strain variation and limited knowledge of common immunogenic antigen/s are among major constraints for developing effective vaccines. S. aureus cell surface proteins that are exposed to the host immune system constitute good vaccine candidates. The objective of this study was to compare two novel S. aureus surface protein extraction methods with biotinylation method and evaluate immune-reactivity of extracted proteins. Surface proteins were extracted from nine genetically distinct S. aureus strains from cases of bovine mastitis. After extraction, bacterial cell integrity was examined by Gram staining and electron microscopy to determine if extraction methods caused damage to cells that may release non-surface proteins. The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and evaluated for immune-reactivity using western blot. Results showed that all three extraction methods provided multiple protein bands on SDS-PAGE. Western blot result showed several immunoreactive surface proteins, in which some proteins strongly (well-resolved, thick, dark, and intense band) reacted across the nine strains tested. The three methods are valid for the extraction of surface proteins and hexadecane, and cholic acid methods are more feasible than biotinylation since both are easier, cheaper, and have minor effects on the bacterial cell. Strongly immune-reactive surface proteins may serve as potential candidates for a vaccine to control S. aureus mastitis in dairy cows.

Abstract 2866: Surfaceome profiling in osteosarcoma: Identification of the candidate immunotherapeutic target

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor in pediatric patients. Current developments in targeted therapeutic strategies show potential clinical implications in cancer treatments. However, immunotherapies such as antibody-drug conjugates (ADCs) or CAR-T cell therapy have not been sufficiently studied in OS because of the lack of tumor-specific target. In this study, we developed a multi-step RNA-seq based pipeline. First, we did RNA sequencing for our OS PDX models and 17 patient-derived OS cell lines. The results are then pooled with the RNQ-seq data from 111 OS patient via the Therapeutically Applicable Research to Generate Effective Treatments project (TARGET). All these tumor data are subsequently compared with normal tissue RNA-sequencing data from NIH Genotype-Tissue Expression (GTEx) database. The significantly differentially expressed genes (log fold change tumor versus normal >1 for each tissue, p < 0.01) are selected. We further filtered this gene list by cell surface protein prediction based on Gene Ontology, the TransMembrane prediction using hidden Markov models (TMHMM), and glycosylphosphatidylinositol (GPI)-anchored protein annotations. Based on the transcriptomic result, ranks of differentially expressed genes were generated. CD276, LCLAT1, and OR8B12 are the surface markers with high ranks. To validate the transcriptomic results, we prepared surface protein extraction of the PDX models and patient-derived OS cell lines. We are profiling these surface proteins by proteomic mass spectrometry. Further validation for the expression level and location of these markers are underway. Our current finding is not previously reported in OS. The results warrant further development of ADC and car-T cell therapy. Note: This abstract was not presented at the meeting. Citation Format: Yifei Wang, Zhongting Zhang, Sankaranarayanan Kannan, Wendong Zhang, Michael Roth, Jonathan B. Gill, Zhaohui Xu, Xiangjun Tian, Jing Wang, Richard Gorlick. Surfaceome profiling in osteosarcoma: Identification of the candidate immunotherapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2866.

Comparison of Staphylococcus aureus Surface Protein ExtractionMethods

Staphylococcus aureus is the major contagious bovine mastitis pathogen and has no effective vaccine. Strainvariation and limited knowledge of common immunogenic antigen/s are among major constraints for developingeffective vaccines. S. aureus cell surface proteins that are exposed to the host immune system constitute goodvaccine candidates. The objective of this study was to compare S. aureus surface protein extraction methods andevaluate immune-reactivity of extracted proteins. Surface proteins were extracted from nine genetically distinct S.aureus strains from cases of bovine mastitis. After extraction, bacterial cell integrity was examined by Gram stainingand electron microscopy to determine if extraction methods caused damage to cells that may release non-surfaceproteins. The extracted proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and evaluated for immune-reactivity using western blot. Results showed that all three extractionmethods provided multiple protein bands on SDS-PAGE. Western blot result showed several immuno-reactivesurface proteins, in which some proteins strongly (well-resolved, thick, dark and intense band) reacted across thenine strains tested. The three methods are valid for the extraction of surface proteins and hexadecane and cholicacid methods are more feasible than biotinylation since both are easier, cheaper and have minor effects on thebacterial cell. Strongly immune-reactive surface proteins may serve as potential candidates for a vaccine to controlS. aureus mastitis in dairy cows.

Rab11a drives adhesion molecules to the surface of endometrial epithelial cells

Is Rab11a GTPase, a regulator of intracellular trafficking, of significance in endometrial functions? Rab11a is an important component of the cascades involved in equipping the endometrial epithelium (EE) with 'adhesiveness' and 'cohesiveness'. Cell adhesion molecules (CAMs) have been investigated extensively for modulation in their endometrial expression during the peri-implantation phase. However, the mechanisms by which CAMs are transported to the EE surface have not received the same attention. Rab11a facilitates transport of specific proteins to the plasma membrane in endothelial cells, fibroblasts, embryonic ectodermal cells, etc. However, its role in the transport of CAMs in EE remains unexplored. In-vitro investigations were directed towards deciphering the role of Rab11a in trafficking of CAMs (integrins and E-cadherin) to the cell surface of Ishikawa, an EE cell line. Towards this, Rab11a stable knockdown (Rab-kd) and control clones of Ishikawa were generated. JAr (human trophoblastic cell line) cells were used to form multicellular spheroids. Pre-receptive (n = 6) and receptive (n = 6) phase endometrial tissues from women with proven fertility and receptive phase (n = 6) endometrial tissues from women with unexplained infertility were used. Rab-kd and control clones were used for in-vitro assays. Live cells were used for biotinylation, JAr spheroid assays, flow cytometry, trans-epithelial electrical resistance assays and wound-healing assays. Lysosome and Golgi membranes were isolated by ultracentrifugation. Confocal microscopy, immunoblotting, qRT-PCR and immunohistochemistry were employed for assessing the expression of Rab11a, integrins and E-cadherin. shRNA-mediated attenuation of Rab11a expression led to a significant (P < 0.01) decline in the surface localization of αVβ3 integrin. Cell surface protein extracts of Rab-kd clones showed a significant (P < 0.05) reduction in the levels of αV integrin. Further, a significant (P < 0.01) decrease was observed in the percent JAr spheroids attached to Rab-kd clones, compared to control clones. Rab-kd clones also showed a significant (P < 0.001) decline in the total levels of E-cadherin. This was caused neither by reduced transcription nor by increased lysosomal degradation. The role of Rab11a in maintaining the epithelial nature of the cells was evident by a significant increase in the migratory potential, presence of stress-fibres and a decrease in the trans-epithelial resistance in Rab-kd monolayers. Further, the levels of endometrial Rab11a and E-cadherin in the receptive phase were found to be significantly (P < 0.05) lower in women with unexplained infertility compared to that in fertile women. Taken together, these observations hint at a key role of Rab11a in the trafficking of αVβ3 integrin and maintenance of E-cadherin levels at the surface of EE cells. N/A. The in-vitro setting of the study is a limitation. Further immunohistochemical localizations of Rab11a and CAMs were conducted on a limited number of human endometrial samples. Rab11a-mediated trafficking of endometrial CAMs in EE cells can be explored further for its potential as a target for fertility regulation or infertility management. This study was funded by the Indian Council of Medical Research (ICMR), the Department of Science and Technology (DST), the Council of Scientific and Industrial Research (CSIR), Government of India. No competing interests are declared.

Comparison of three methods for cell surface proteome extraction of Listeria monocytogenes biofilms.

The cell surface proteome of the foodborne pathogen Listeria monocytogenes, the etiological agent of listeriosis, is critical for understanding the physiological processes associated with stress resistance and persistence in the environment. In this context, the most widespread mode of growth for bacterial cells in natural and industrial environments is in biofilms. Cell surface proteins are, however, challenging to characterize because of their low abundance and poor solubility. Moreover, cell surface protein extracts are usually contaminated with cytoplasmic proteins that constitute the main signal in proteomic analysis. This study aimed to compare the efficiency of three methods to extract and explore surface proteins of L. monocytogenes growing in a biofilm: trypsin shaving, biotinylation, and cell fractionation. Peptide separation and identification were performed by shotgun proteomics using high-performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). The biotinylation method was the most effective in extracting surface proteins, with the lowest rate of contamination by cytoplasmic proteins. Although presenting a higher contamination rate in cytoplasmic proteins, the other two techniques allowed the identification of additional surface proteins. Seven proteins were commonly retrieved by the three methods. The extracted proteins belong to several functional classes, involved in virulence, transport, or metabolic pathways. Finally, the three extraction methods seemed complementary and their combined use improved the exploration of the bacterial surface proteome. These new findings collectively inform future discovery and translational proteomics for clinical, environmental health, and industrial applications.

Miniaturized Digestion and Extraction of Surface Proteins from Candida albicans following Treatment with Histatin 5 for Mass Spectrometry Analysis.

A common approach to isolate surface proteins from fungal and bacterial cells is to perform a proteolytic cleavage of proteins on the surface of intact cells suspended in solution. This paper describes miniaturization of this technique, in which cells are adhered on glass surfaces, and all sample treatments are conducted at μL volumes. Specifically, Candida albicans cells were attached onto HSA-coated glass slides. By depositing the appropriate reagent solutions on the adhered cells, we successfully performed cell washing, treatment with antifugal peptide, Histatin 5, and a proteolysis on intact cells with trypsin. The resulting peptides were subsequently analysed by mass spectrometry. In general, the data obtained was similar to that collected with suspended cells in much larger sample volumes. However, our miniaturized workflow offers the benefit of greatly reducing the consumption of cells and reagents.

Fungal surface protein mediated one-pot synthesis of stable and hemocompatible gold nanoparticles.

Despite their large secretome and wide applications in bioprocesses, fungi remain underexplored in metal nanoparticles (MNP) biosynthesis. Previous studies have shown that cell surface proteins of Rhizopus oryzae play a crucial role in biomineralization of Au(III) to produce gold nanoparticles (AuNPs). Therefore, it is hypothesized that purified cell surface protein may produce in vitro AuNPs with narrow size distribution for biomedical and biocatalytic applications. However, different protein extraction methods might affect protein stability and the AuNP biosynthesis process. Herein, we have explored the extraction of cell surface proteins from R. oryzae using common detergents and reducing agent (sodium dodecyl sulfate (SDS) Triton X-100, and 1,4-dithiothreitol (DTT)) and their effect on the size and shape of the biosynthetic AuNPs. The surface proteins extracted with reducing agent (DTT) and non-ionic detergent (Triton X-100) produce spherical AuNPs with a mean particle size of 16±7nm, and 19±4nm, respectively, while the AuNPs produced by the surface protein extracted by ionic detergent (SDS) are flower-like AuNPs with broader size distribution of 43±19nm. This synthetic approach does not require use of any harsh chemicals, multistep preparation and separation process, favouring environmental sustainability. The biosynthetic AuNPs thus formed, are stable in different physiological buffers and hemocompatible, making them suitable for biomedical applications.

Comparison of Surface Proteomes of Adherence Variants of Listeria Monocytogenes Using LC-MS/MS for Identification of Potential Surface Adhesins.

The ability of Listeria monocytogenes to adhere and form biofilms leads to persistence in food processing plants and food-associated listeriosis. The role of specific surface proteins as adhesins to attach Listeria cells to various contact surfaces has not been well characterized to date. In prior research comparing different methods for surface protein extraction, the Ghost urea method revealed cleaner protein content as verified by the least cytoplasmic protein detected in surface extracts using LC-MS/MS. The same technique was utilized to extract and detect surface proteins among two surface-adherent phenotypic strains of L. monocytogenes (i.e., strongly and weakly adherent). Of 640 total proteins detected among planktonic and sessile cells, 21 protein members were exclusively detected in the sessile cells. Relative LC-MS/MS detection and quantification of surface-extracted proteins from the planktonic weakly adherent (CW35) and strongly adherent strains (99-38) were examined by protein mass normalization of proteins. We found that L. monocytogenes 99-38 exhibited a total of 22 surface proteins that were over-expressed: 11 proteins were detected in surface extracts of both sessile and planktonic 99-38 that were ≥5-fold over-expressed while another 11 proteins were detected only in planktonic 99-38 cells that were ≥10-fold over-expressed. Our results suggest that these protein members are worthy of further investigation for their involvement as surface adhesins.

Zymographic Techniques for the Analysis of Bacterial Cell Wall in Bacillus.

Zymography of cell wall hydrolases is a simple technique to specifically detect cell wall or peptidoglycan hydrolytic activity. The zymographic method can be used for assessing the hydrolytic activities of purified target proteins, cell surface proteins, and proteins secreted to culture. Here, methods of cell wall and peptidoglycan purification, extraction of cell surface proteins containing cell wall hydrolases, and zymographic analysis are described. The purified or extracted proteins are separated by electrophoresis using an SDS gel containing cell wall or peptidoglycan material and then the proteins are renatured in the gel. The renatured cell wall hydrolases in the gel hydrolyze the material around the proteins. The cell wall or peptidoglycan in the gel is stained by methylene blue and the hydrolyzed material cannot be stained, resulting in the detection of cell wall hydrolytic activities of the enzymes on the gel.

Comparison of five methods for direct extraction of surface proteins from Listeria monocytogenes for proteomic analysis by orbitrap mass spectrometry

Extracts of surface proteins, with minimal artifacts from contaminating cytosolic components, are highly desirable for investigating surface factors involved in the attachment and formation of biofilms by bacteria that are problematic in commercial food processing facilities. In this study, we compared the protein profiles of the food pathogen, Listeria monocytogenes, recovered after applying different surface protein extraction methods compiled from the literature: trypsin-enzymatic shaving with BICAM/sucrose or Tris/sucrose buffers (Tryp B+S, Tryp T+S), Tris-buffered urea (UB), lithium chloride (LiCl) and Tris-buffered urea applied with hypotonic-stressed cells (UB-Ghost), and subjected them to liquid chromatography tandem mass spectrometry and protein identification. The data indicate that the UB-Ghost extraction method provides a cleaner extract of surface proteins including the predicted (this study and the literature) or validated members (literature) from L. monocytogenes. This was determined by an accumulative lower unique peptide number exhibited by mass spectrometry for total cytoplasmic proteins among different surface extracts, with a majority of proteins demonstrating hydrophilic properties. The extracted proteins were from different functional categories and have associations with the cell surface, intermediary metabolism, information pathways, or functionally unknown proteins as suggested by in silico analyses performed by other groups (Leger and ListiList). The utilization of an optimized method for surface protein extraction should greatly facilitate identification by LC–MS/MS that could be useful to anyone working on molecular proteomics of bacterial surfaces.

Health-promoting properties exhibited by Lactobacillus helveticus strains.

Many strains belonging to lactobacilli exert a variety of beneficial health effects in humans and some of the bacteria are regarded as probiotic microorganisms. Adherence and capabilities of colonization by Lactobacillus strains of the intestinal tract is a prerequisite for probiotic strains to exhibit desired functional properties. The analysis conducted here aimed at screening strains of Lactobacillus helveticus possessing a health-promoting potential. The molecular analysis performed, revealed the presence of a slpA gene encoding the surface S-layer protein SlpA (contributing to the immunostimulatory activity of L. helveticus M 92 probiotic strain) in all B734, DSM, T80, and T105 strains. The product of gene amplification was also identified in a Bifidobacterium animalis ssp. lactis BB12 probiotic strain. SDS-PAGE of a surface protein extract demonstrated the presence of a protein with a mass of about 50 kDa in all strains, which refers to the mass of the S-layer proteins. These results are confirmed by observations carried with transmission electron microscopy, where a clearly visible S-layer was registered in all the strains analyzed. The in vitro study results obtained indicate that the strongest adhesion capacity to epithelial cells (HT-29) was demonstrated by L. helveticus B734, while coaggregation with pathogens was highly diverse among the tested strains. The percentage degree of coaggregation was increasing with the incubation time. After 5 h of incubation, the strongest ability to coaggregate with Escherichia coli was expressed by T104. The T80 strain demonstrated a significant ability to co-aggregate with Staphylococcus aureus, while DSM with Bacillus subtilis. For B734, the highest values of co-aggregation coefficient was noted in samples with Salmonella. The capability of autoaggregation, antibiotic susceptibility, resistance to increasing salt concentrations, and strain survival in simulated small intestinal juice were also analyzed.

Data from an integrative approach decipher the surface proteome of Propionibacterium freudenreichii.

The surface proteins of the probiotic Propionibacterium freudenreichii were inventoried by an integrative approach that combines in silico protein localization prediction, surface protein extraction, shaving and fluorescent CyDye labeling. Proteins that were extracted and/or shaved and/or labeled were identified by nano-LC–MS/MS following trypsinolysis. This method’s combination allowed to confirm detection of true surface proteins involved in host/probiotic interactions. The data, supplied in this article, are related to the research article entitled “Surface proteins of P. freudenreichii are involved in its anti-inflammatory properties” (Le Maréchal et al., 2014 [6]).

Adhesion properties of potentially probioticLactobacillus kefirito gastrointestinal mucus

We investigated the mucus-binding properties of aggregating and non-aggregating potentially probiotic strains of kefir-isolated Lactobacillus kefiri, using different substrates. All the strains were able to adhere to commercial gastric mucin (MUCIN) and extracted mucus from small intestine (SIM) and colon (CM). The extraction of surface proteins from bacteria using LiCl or NaOH significantly reduced the adhesion of three selected strains (CIDCA 8348, CIDCA 83115 and JCM 5818); although a significant proportion (up to 50%) of S-layer proteins were not completely eliminated after treatments. The surface (S-layer) protein extracts from all the strains of Lb. kefiri were capable of binding to MUCIN, SIM or CM, and no differences were observed among them. The addition of their own surface protein extract increased adhesion of CIDCA 8348 and 83115 to MUCIN and SIM, meanwhile no changes in adhesion were observed for JCM 5818. None of the seven sugars tested had the ability to inhibit the adhesion of whole bacteria to the three mucus extracts. Noteworthy, the degree of bacterial adhesion reached in the presence of their own surface protein (S-layer) extract decreased to basal levels in the presence of some sugars, suggesting an interaction between the added sugar and the surface proteins. In conclusion, the ability of these food-isolated bacteria to adhere to gastrointestinal mucus becomes an essential issue regarding the biotechnological potentiality of Lb. kefiri for the food industry.

Purification and identification of sperm surface proteins and changes during epididymal maturation

Surface membrane proteins have a key role in the sequential interactions between spermatozoa and oocytes. The aim of this study was to characterize protein changes occurring during post-testicular differentiation using a new overall approach to study surface membrane proteins of spermatozoa. A dedicated protocol based on specific purification of surface membrane proteins labeled with sulfo-NHS-SS-biotin was developed for this purpose. Appropriate gel electrophoresis separation and purification methods combined with standard proteomic methods were then used to identify and quantify surface membrane proteins from immature and mature spermatozoa. Membrane-associated proteins were discriminated from integral membrane proteins by differential solubilization. Protein regionalization on the spermatozoon surface was achieved by comparative analysis of the surface protein extracts from the entire spermatozoa and from periacrosomal sperm plasma membranes. Identification of several known proteins and of new proteins related to the process of epididymal maturation showed the reliability of this protocol for specific purification of a subproteome and identification of new sperm membrane proteins. This approach opens up a new area in the search for male fertility markers.

Mining the surface proteome of tomato (Solanum lycopersicum) fruit for proteins associated with cuticle biogenesis

The aerial organs of plants are covered by the cuticle, a polyester matrix of cutin and organic solvent-soluble waxes that is contiguous with the polysaccharide cell wall of the epidermis. The cuticle is an important surface barrier between a plant and its environment, providing protection against desiccation, disease, and pests. However, many aspects of the mechanisms of cuticle biosynthesis, assembly, and restructuring are entirely unknown. To identify candidate proteins with a role in cuticle biogenesis, a surface protein extract was obtained from tomato (Solanum lycopersicum) fruits by dipping in an organic solvent and the constituent proteins were identified by several complementary fractionation strategies and two mass spectrometry techniques. Of the ∼200 proteins that were identified, a subset is potentially involved in the transport, deposition, or modification of the cuticle, such as those with predicted lipid-associated protein domains. These include several lipid-transfer proteins, GDSL-motif lipase/hydrolase family proteins, and an MD-2-related lipid recognition domain-containing protein. The epidermal-specific transcript accumulation of several of these candidates was confirmed by laser-capture microdissection and quantitative reverse transcription-PCR (qRT-PCR), together with their expression during various stages of fruit development. This indicated a complex pattern of cuticle deposition, and models for cuticle biogenesis and restructuring are discussed.

Conidial surface proteins of Metarhizium anisopliae: Source of activities related with toxic effects, host penetration and pathogenesis

Conidial contact with an arthropod surface is the first step of the fungal penetration and infection process. However, conidia of Metarhizium anisopliae have associated components, like enzymes that could be involved in triggering the penetration process and toxic effects that have not yet been well characterized. Fungi produce many enzymes that also are toxic components found in bacteria and animal venoms and thus may be considered as potential virulence factors. In this work, we report several enzymatic activities from spore surface protein extracts. The major proteolytic activities observed in spore surface proteins (SSP) were Pr1 and Pr2 activities, in that order. According to the zymograms obtained, SSP contain different proteases. SSP contain trehalase, exo- and endo-chitinase activities, and seven different chitinase bands which have been observed in zymograms. Activities involved in protection against reactive oxygen species (ROS) were also detected. Two lipolytic enzymes were also detected in lipase zymograms. Phospholipase C activity, closely related to microbial pathogenesis, was detected for the first time in M. anisopliae conidia. These activities described could be an initial step towards understanding the mechanisms involved in the first stage of M. anisopliae infection process and its toxic effects against arthropod hosts.

Two isoforms of eukaryotic phospholipase C in Paramecium affecting transport and release of GPI-anchored proteins in vivo

Surface proteins anchored by a glycosylphosphatidylinositol (GPI) residue in the cell membrane are widely distributed among eukaryotic cells. The GPI anchor is cleavable by a phospholipase C (PLC) leading to the release of such surface proteins, and this process is postulated to be essential in several systems. For higher eukaryotes, the responsible enzymes have not been characterized in any detail as yet. Here we characterize six PLCs in the ciliated protozoan, Paramecium, which, in terms of catalytic domains and architecture, all show characteristics of PLCs involved in signal transduction in higher eukaryotes. We show that some of these endogenous PLCs can release GPI-anchored surface proteins in vitro: using RNA i to reduce PLC expression results in the same effects as the application of PLC inhibitors. With two enzymes, PLC2 and PLC6, RNA i phenotypes show strong defects in release of GPI-anchored surface proteins in vivo. Moreover, these RNA i lines also show abnormal surface protein distribution, suggesting that GPI cleavage may influence trafficking of anchored proteins. As we find GFP fusion proteins in the cytosol and in the surface protein extracts, these PLCs obviously show unconventional translocation mechanisms. This is the first molecular data on endogenous Paramecium PLCs with the described properties affecting GPI anchors in vitro and in vivo.