Proteomic changes in the milk of water buffaloes (Bubalus bubalis) with subclinical mastitis due to intramammary infection by Staphylococcus aureus and by non-aureus staphylococci

Subclinical mastitis by Staphylococcus aureus (SAU) and by non-aureus staphylococci (NAS) is a major issue in the water buffalo. To understand its impact on milk, 6 quarter samples with >3,000,000 cells/ mL (3 SAU-positive and 3 NAS-positive) and 6 culture-negative quarter samples with <50,000 cells/ mL were investigated by shotgun proteomics and label-free quantitation. A total of 1530 proteins were identified, of which 152 were significantly changed. SAU was more impacting, with 162 vs 127 differential proteins and higher abundance changes (P < 0.0005). The 119 increased proteins had mostly structural (n = 43, 28.29%) or innate immune defence functions (n = 39, 25.66%) and included vimentin, cathelicidins, histones, S100 and neutrophil granule proteins, haptoglobin, and lysozyme. The 33 decreased proteins were mainly involved in lipid metabolism (n = 13, 59.10%) and included butyrophilin, xanthine dehydrogenase/oxidase, and lipid biosynthetic enzymes. The same biological processes were significantly affected also upon STRING analysis. Cathelicidins were the most increased family, as confirmed by western immunoblotting, with a stronger reactivity in SAU mastitis. S100A8 and haptoglobin were also validated by western immunoblotting. In conclusion, we generated a detailed buffalo milk protein dataset and defined the changes occurring in SAU and NAS mastitis, with potential for improving detection (ProteomeXchange identifier PXD012355)

Proteomics Characterization of Cytoplasmic and Lipid-Associated Membrane Proteins of Human Pathogen Mycoplasma fermentans M64

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen

Polyphenols, colour and antioxidant activity changes in four Italian red wines during storage

The evolution, during one year of storage, of the antioxidant activity, phenolic compounds and colour of four red wines, obtained from two different Sardinian important cultivars (Cannonau and Cagnulari) and kept in stainless steel tanks or in bottle, was evaluated. Two samples were subjected to the microoxygenation process, with one of these derived from a late grape harvest. Wines stored in stainless steel tanks showed a decrease in phenolic content and antioxidant activity, while the bottled wine did not show significant variations. The different degree of microoxygenation caused some significant differences with regard to the decrease in phenolic compounds during the storage, being lower in the sample microoxygenated for a longer time and with more oxygen. Correlation coefficients between anthocyanins and antioxidant activity were high for all the samples analysed. Regarding the colour, percent of blue showed a significant increase in microoxygenated wines. PCA, applied to the data set of phenolic compounds showed that Cannonau wine is different from Cagnulari samples, because of the higher content of cinnamic acids. Cagnulari bottled wine differed from the two Cagnulari stored in steel tanks, in particular for the anthocyanins content, confirming the reducing activity of the bottle

Molecular rationale for the impairment of the MexAB-OprM efflux pump by a single mutation in MexA

Efflux pumps of the Resistance-Nodulation-cell Division (RND) superfamily contribute to intrinsic and acquired resistance in Gram-negative pathogens by expelling chemically unrelated antibiotics with high efficiency. They are tripartite systems constituted by an inner-membrane-anchored transporter, an outer membrane factor protein, and a membrane fusion protein. Multimerization of the membrane fusion protein is an essential prerequisite for full functionality of these efflux pumps. In this work, we employed complementary computational techniques to investigate the stability of a dimeric unit of MexA (the membrane fusion protein of the MexAB-OprM RND efflux pump of Pseudomonas aeruginosa), and to provide a molecular rationale for the effect of the G72S substitution, which affects MexAB-OprM functionality by impairing the assembly of MexA. Our findings indicate that: i) dimers of this protein are stable in multiple µs-long molecular dynamics simulations; ii) the mutation drastically alters the conformational equilibrium of MexA, favouring a collapsed conformation that is unlikely to form dimers or higher order assemblies. Unveiling the mechanistic aspects underlying large conformational distortions induced by minor sequence changes is informative to efforts at interfering with the activity of this elusive bacterial weapon. In this respect, our work further confirms how molecular simulations can give important contribution and useful insights to characterize the mechanism of highly complex biological systems

Immunoproteomic characterisation of Mycoplasma mycoides subspecies capri by mass spectrometry analysis of two-dimensional electrophoresis spots and western blot.

Six proteins were found to have a less than 95% amino acid similarity to a closely related Mycoplasma species showing that they warrant further evaluation in development of diagnostic tests. These proteins were a dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex, phosphoglycerate kinase, pyrimidine-nucleoside phosphorylase, 30S ribosomal protein S6, ribulose-phosphate 3-epimerase and D-lactate dehydrogenase