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Aspergillus flavus and Aspergillus fumigatus are important human pathogens that can infect the lung and cornea. During infection, Aspergillus dormant conidia are the primary morphotype that comes in contact with the host. As the conidial surface-associated proteins (CSPs) and the extracellular proteins during the early stages of growth play a crucial role in establishing infection, we profiled and compared these proteins between a clinical strain of A. flavus and a clinical strain of A. fumigatus. We identified nearly 100 CSPs in both Aspergillus, and these non-covalently associated surface proteins were able to stimulate the neutrophils to secrete interleukin IL-8. Mass spectrometry analysis identified more than 200 proteins in the extracellular space during the early stages of conidial growth and germination (early exoproteome). The conidial surface proteins and the early exoproteome of A. fumigatus were enriched with immunoreactive proteins and those with pathogenicity-related functions while that of the A. flavus were primarily enzymes involved in cell wall reorganization and binding. Comparative proteome analysis of the CSPs and the early exoproteome between A. flavus and A. fumigatus enabled the identification of a common core proteome and potential species-specific signature proteins. Transcript analysis of selected proteins indicate that the transcript-protein level correlation does not exist for all proteins and might depend on factors such as membrane-anchor signals and protein half-life. The probable signature proteins of A. flavus and A. fumigatus identified in this study can serve as potential candidates for developing species-specific diagnostic tests. Key points• CSPs and exoproteins could differentiate A. flavus and A. fumigatus.• A. fumigatus conidial surface harbored more antigenic proteins than A. flavus.• Identified species-specific signature proteins of A. flavus and A. fumigatus.

The outermost constituent of many bacterial cells is represented by an S-layer, i.e., a semiporous lattice-like layer composed of self-assembling protein subunits called S-layer proteins (Slps). These proteins are involved in several processes, such as protecting against environmental stresses, mediating bacterial adhesion to host cells, and modulating gut immune response. Slps may also act as a scaffold for the external display of additional cell surface proteins also named S-layer associated proteins (SLAPs). Levilactobacillus brevis is an S-layer forming lactic acid bacterium present in many different environments, such as sourdough, milk, cheese, and the intestinal tract of humans and animals. This microorganism exhibits probiotic features including the inhibition of bacterial infection and the improvement of human immune function. The potential role of Slps in its probiotic and biotechnological features was documented. A shotgun proteomic approach was applied to identify in a single experiment both the Slps and the SLAPs pattern of five different L. brevis strains isolated from traditional sourdoughs of the Southern Italian region. This study reveals that these closely related strains expressed a specific pattern of surface proteins, possibly affecting their peculiar properties.

The pathogenicity of L. pneumophila , the causative agent of Legionnaires’ disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted “protein sociology” of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

The protozoa Leishmania donovani causes visceral leishmaniasis (kala-azar), the third most common vector-borne disease. The visceral organs, particularly the spleen, liver, and bone marrow, are affected by the disease. The lack of effective treatment regimens makes curing and eradicating the disease difficult. The availability of complete L. donovani genome/proteome data allows for the development of specific and efficient vaccine candidates using the reverse vaccinology method, while utilizing the unique sequential and structural features of potential antigenic proteins to induce protective T cell and B cell responses. Such shortlisted candidates may then be tested quickly for their efficacy in the laboratory and later in clinical settings. These antigens will also be useful for designing antigen-based next-generation sero-diagnostic assays. L. donovani's cell surface-associated proteins and secretory proteins are among the first interacting entities to be exposed to the host immune machinery. As a result, potential antigenic epitope peptides derived from these proteins could serve as competent vaccine components. We used a stepwise filtering-based in silico approach to identify the entire surface-associated and secretory proteome of L. donovani, which may provide rationally selected most exposed antigenic proteins. Our study identified 12 glycosylphosphatidylinositol-anchored proteins, 45 transmembrane helix-containing proteins, and 73 secretory proteins as potent antigens unique to L. donovani. In addition, we used immunoinformatics to identify B and T cell epitopes in them. Out of the shortlisted surface-associated and secretory proteome, 66 protein targets were found to have the most potential overlapping B cell and T cell epitopes (linear and conformational; MHC class I and MHC class II).

The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s) among the cell surface associated proteins (SAPs) of that would be valuable in the development of an alternative diagnostic assay for shigellosis, particularly one that could be run using a stool sample rather than serum. The SAPs of clinical isolates of S. , , and were extracted from an overnight culture grown at 37 °C using acidified-glycine extraction methods. Protein profiles were observed by SDS-PAGE. To determine if antibodies specific to certain SAPs were present in both sera and stool suspensions, Western blot analysis was used to detect the presence of IgA, IgG, and IgM. Immunoblot analysis revealed that sera from patients infected with . recognized 31 proteins. These SAP antigens are recognized by the host humoral response during infection. Specific antibodies against these antigens were also observed in intestinal secretions of shigellosis patients. Of these 31 proteins, the 35 kDa protein specifically reacted against IgA present in patients' stool suspensions. Further study illustrated the immunoreactivity of this protein in , and . This is the first report that demonstrates the presence of immunoreactive SAPs in stool suspensions. The SAPSs could be very useful in developing a simple and rapid serodiagnostic assay for shigellosis directly from stool specimens.

Surface-associated proteins from BCG Moreau RDJ are important components of the live Brazilian vaccine against tuberculosis. They are important targets during initial BCG vaccine stimulation and modulation of the host's immune response, especially in the bacterial-host interaction. These proteins might also be involved in cellular communication, chemical response to the environment, pathogenesis processes through mobility, colonization, and adherence to the host cell, therefore performing multiple functions. In this study, the proteomic profile of the surface-associated proteins from BCG Moreau was compared to the BCG Pasteur reference strain. The methodology used was 2DE gel electrophoresis combined with mass spectrometry techniques (MALDI-TOF/TOF), leading to the identification of 115 proteins. Of these, 24 proteins showed differential expression between the two BCG strains. Furthermore, 27 proteins previously described as displaying moonlighting function were identified, 8 of these proteins showed variation in abundance comparing BCG Moreau to Pasteur and 2 of them presented two different domain hits. Moonlighting proteins are multifunctional proteins in which two or more biological functions are fulfilled by a single polypeptide chain. Therefore, the identification of such proteins with moonlighting predicted functions can contribute to a better understanding of the molecular mechanisms unleashed by live BCG Moreau RDJ vaccine components.

Preeclampsia (PE) is the leading cause of maternal and fetal mortality and morbidity. Furthermore, recent studies have reported that miR-145 within the preeclamptic trophoblast debris may cause the high blood pressure via interacting with the maternal endothelium. The aim of the present study was to investigate the functions of miR-145 in PE. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to assess the expression of miR-145 and mucin (MUC1), respectively. TargetScan, miRBase and miRWalk were used to predict the targets of miR-145. Constructed miR-145 mimic plasmids were transfected into HTR-8/SVneo cells for further experiments, including an MTT assay for cell proliferation, Transwell assay for cell invasion and flow cytometry for cell apoptosis analysis. Additionally, the luciferase reporter gene system was employed for target verification. The results demonstrated that miR-145 is downregulated and MUC1 is upregulated in PE tissues and cells compared with normal placenta tissues and cells. The correlation analysis suggests that the expression of miR-145 is negatively correlated with MUC1. Meanwhile, increased proliferation, enhanced invasion and decreased apoptosis of HTR-8/SVneo cells was observed in miR-145 mimic groups compared with mimic control group. Also, the decreased luciferase activity in the miR-145 mimic group indicates that MUC1 may be a target of miR-145. In summary, the results of the present study suggest that miR-145 may serve key roles in the regulation of trophoblast cell proliferation and invasion by targeting MUC1.

To quantitatively evaluate the correlations between the amount of initial burst release and the surface-associated protein, and between the onset time for the second burst release and the matrix polymer degradation. Human serum albumin (HSA) was microencapsulated in polylactide (PLA) and poly-dl-lactide-poly(ethylene glycol) (PELA) with PEG contents of 5, 10, 20, and 30%, respectively, using the solvent extraction procedure based on formation of double emulsion w/o/w. Microspheres with similar particle size (1.7-2.0 microm), similar protein entrapment (2.1-2.8%) but different surface-associated proteins (9.3-53.6%) were used to evaluate the in vitro matrix degradation and protein release profiles. Degradation was characterized by studying the intrinsic viscosity decrease, medium pH change, and weight loss of the microspheres. The matrix degradation and protein release profiles were highly dependent on the polymer composition of the microspheres. Faster decreases in the intrinsic viscosity of recovered matrix polymer, the microspheres weight, and the pH of degradation medium, and earlier onsets for the break in intrinsic viscosity reduction and the mass loss were detected for PELA microspheres with higher PEG content. The hydration and swelling of microspheres matrix contributed greatly to the degradation of matrix polymer. The HSA release showed triphasic profile and involved two mechanisms for all the microsphere samples. Smaller amount of initial burst release, larger gradual release rate, and earlier onset for the second burst release were observed for HSA from matrix polymer with higher PEG content. The extent of the initial burst release was quantitatively related with the surface-associated protein. The second burst release of HSA was observed to occur within 1 week after the onset for mass loss, which was also the break in the intrinsic viscosity reduction rate. Protein release profiles could be rationalized by optimizing the matrix polymer degradation and microsphere characteristics.

Staphylococcus aureus infection of bone causes severe bone damage but the mechanisms responsible remain to be established. We used the protein-rich fraction released by saline extraction of Staphylococcus aureus (SAM) to test the hypothesis that the surface-associated proteins promote bone breakdown by directly activating osteoclasts. Isolated chick osteoclasts were incubated on dentine slices with or without the surface protein fraction from S. aureus. The resulting osteoclastic excavations were measured using confocal reflection microscopic surface mapping. SAM both stimulated the formation of resorption pits and induced the production of pits with larger volume : area ratios..[PUBLICATION ABSTRACT]

This chapter contains sections titled: Introduction Cell Lysis Methods Sample Preparation for 2D GE Fractionation Strategies Sample Preparation for Liquid Chromatography coupled with Mass Spectrometry (LC‐MS) Conclusion References