RESUMEN
Staphylococcus aureus modulates the host immune response directly by interacting with the immune cells or indirectly by secreting molecules (secretome). Relevant differences in virulence mechanisms have been reported for the secretome produced by different S. aureus strains. The present study investigated the S. aureus secretome impact on peripheral bovine mononuclear cells (PBMCs) by comparing two S. aureus strains with opposite epidemiological behavior, the genotype B (GTB)/sequence type (ST) 8, associated with a high within-herd prevalence, and GTS/ST398, associated with a low within-herd prevalence. PBMCs were incubated with different concentrations (0%, 0.5%, 1%, and 2.5%) of GTB/ST8 and GTS/ST398 secretome for 18 and 48 h, and the viability was assessed. The mRNA levels of pro- (IL1-ß and STAT1) and anti-inflammatory (IL-10, STAT6, and TGF-ß) genes, and the amount of pro- (miR-155-5p and miR-125b-5p) and anti-inflammatory (miR-146a and miR-145) miRNAs were quantified by RT-qPCR. Results showed that incubation with 2.5% of GTB/ST8 secretome increased the viability of cells. In contrast, incubation with the GTS/ST398 secretome strongly decreased cell viability, preventing any further assays. The GTB/ST8 secretome promoted PBMC polarization towards the pro-inflammatory phenotype inducing the overexpression of IL1-ß, STAT1 and miR-155-5p, while the expression of genes involved in the anti-inflammatory response was not affected. In conclusion, the challenge of PBMC to the GTS/ST398 secretome strongly impaired cell viability, while exposure to the GTB/ST8 secretome increased cell viability and enhanced a pro-inflammatory response, further highlighting the different effects exerted on host cells by S. aureus strains with epidemiologically divergent behaviors.
Asunto(s)
Enfermedades de los Bovinos , MicroARNs , Infecciones Estafilocócicas , Animales , Bovinos , Staphylococcus aureus/genética , Leucocitos Mononucleares , Secretoma , Antiinflamatorios , Infecciones Estafilocócicas/veterinariaRESUMEN
Staphylococci and streptococci are common causes of intramammary infection in small ruminants, and reliable species identification is crucial for understanding epidemiology and impact on animal health and welfare. We applied MALDI-TOF MS and gap PCR-RFLP to 204 non-aureus staphylococci (NAS) and mammaliicocci (NASM) and to 57 streptococci isolated from the milk of sheep and goats with mastitis. The top identified NAS was Staphylococcus epidermidis (28.9%) followed by Staph. chromogenes (27.9%), haemolyticus (15.7%), caprae, and simulans (6.4% each), according to both methods (agreement rate, AR, 100%). By MALDI-TOF MS, 13.2% were Staph. microti (2.9%), xylosus (2.0%), equorum, petrasii and warneri (1.5% each), Staph. sciuri (now Mammaliicoccus sciuri, 1.0%), arlettae, capitis, cohnii, lentus (now M. lentus), pseudintermedius, succinus (0.5% each), and 3 isolates (1.5%) were not identified. PCR-RFLP showed 100% AR for Staph. equorum, warneri, arlettae, capitis, and pseudintermedius, 50% for Staph. xylosus, and 0% for the remaining NASM. The top identified streptococcus was Streptococcus uberis (89.5%), followed by Strep. dysgalactiae and parauberis (3.5% each) and by Strep. gallolyticus (1.8%) according to both methods (AR 100%). Only one isolate was identified as a different species by MALDI-TOF MS and PCR-RFLP. In conclusion, MALDI-TOF MS and PCR-RFLP showed a high level of agreement in the identification of the most prevalent NAS and streptococci causing small ruminant mastitis. Therefore, gap PCR-RFLP can represent a good identification alternative when MALDI-TOF MS is not available. Nevertheless, some issues remain for Staph. haemolyticus, minor NAS species including Staph. microti, and species of the novel genus Mammaliicoccus.
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Enfermedades de los Bovinos , Enfermedades de las Cabras , Mastitis Bovina , Enfermedades de las Ovejas , Infecciones Estafilocócicas , Animales , Bovinos , Femenino , Cabras , Mastitis Bovina/diagnóstico , Leche , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de Restricción , Ovinos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/veterinaria , Infecciones Estafilocócicas/veterinaria , Staphylococcus , Streptococcus/genéticaRESUMEN
BACKGROUND: In a collaboration between animal and human health care professionals, we assessed the genetic characteristics shared by non-aureus staphylococci (NAS) infecting humans and dairy ewes to investigate their relatedness in a region concentrating half of the total National sheep stock. We examined by PCR 125 ovine and 70 human NAS for biofilm production, pyrogenic toxins, adhesins, autolysins genes, and accessory gene regulator (agr) locus. The microtiter plate assay (MPA) was used for the phenotypic screening of biofilm production. Ovine NAS included S. epidermidis, S. chromogenes, S. haemolyticus, S. simulans, S. caprae, S. warneri, S. saprophyticus, S. intermedius, and S. muscae. Human NAS included S. haemolyticus, S. epidermidis, S. hominis, S. lugdunensis, S. capitis, S. warneri, S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis. RESULTS: Phenotypically, 41 (32.8%) ovine and 24 (34.3%) human isolates were characterized as biofilm producers. Of the ovine isolates, 12 were classified as biofilm-producing while the remaining 29 as weak biofilm-producing. All 24 human isolates were considered weak biofilm-producing. Few S. epidermidis isolates harbored the icaA/D genes coding for the polysaccharide intercellular adhesin (PIA), while the bhp, aap, and embp genes coding biofilm accumulation proteins were present in both non-producing and biofilm-producing isolates. Fifty-nine sheep NAS (all S. epidermidis, 1 S. chromogenes, and 1 S. haemolyticus) and 27 human NAS (all S. epidermidis and 1 S. warneri) were positive for the agr locus: agr-3se (57.8%) followed by agr-1se (36.8%) predominated in sheep, while agr-1se (65.4%), followed by agr-2se (34.6%) predominated in humans. Concerning virulence genes, 40, 39.2, 47.2%, 52.8, 80 and 43.2% of the sheep isolates carried atlE, aae, sdrF, sdrG, eno and epbS respectively, against 37.1, 42.8, 32.8, 60, 100 and 100% of human isolates. Enterotoxins and tsst were not detected. CONCLUSIONS: Considerable variation in biofilm formation ability was observed among NAS isolates from ovine and human samples. S. epidermidis was the best biofilm producer with the highest prevalence of adhesin-encoding genes.
Asunto(s)
Biopelículas , Staphylococcus , Adhesinas Bacterianas/genética , Animales , Enterotoxinas , Femenino , Humanos , Ovinos , Staphylococcus/genética , Virulencia/genéticaRESUMEN
BACKGROUND: Listeria monocytogenes is a ubiquitous Gram-positive bacterium responsible for a severe foodborne disease in humans, and contaminated dairy products can be an important source of infection. Typically, infected dairy ruminants show clinical manifestations including encephalitis, septicemia, abortion, and diarrhea, but may also become asymptomatic carriers and shed L. monocytogenes in the feces acting as an important source of viable bacteria. Isolation from individual goat milk has been documented very rarely, and chronic, asymptomatic intramammary infection by L. monocytogenes with continuous milk shedding of viable bacteria has never been described in this dairy species. CASE PRESENTATION: At the routine controls, cheese and bulk milk were positive for L. monocytogenes in a herd of 200 lactating Alpine goats, but none showed clinical signs of listeriosis. Individual milk was subjected to bacterial culture and a clinically healthy goat was identified as affected by a chronic intramammary infection (IMI) by L. monocytogenes. The goat had never shown clinical signs of mastitis or other diseases. Her right half-udder milk was positive to L. monocytogenes in two consecutive samples collected one week apart, as demonstrated by bacterial culture and molecular analysis. Mammary tissues collected after culling were also positive to L. monocytogenes by culture. Histological examination highlighted a chronic interstitial mastitis with leukocyte infiltration, atrophy of the alveoli and presence of corpora amylacea. Immunohistochemistry (IHC) and immunofluorescence (IF) confirmed the presence of high numbers of bacteria in the lumen of mammary alveoli, with intracellular bacteria mainly located in macrophages, but also present in neutrophils and epithelial cells. After culling of the positive goat, bulk tank milk tested negative to L. monocytogenes at the following controls. CONCLUSION: This study demonstrates that L. monocytogenes can establish a chronic, subclinical IMI in goats with high numbers of bacteria shed in milk, representing a source of contamination for the herd and its dairy products. This underscores the importance of frequently monitoring all dairy herds that sell directly milk and/or fresh cheese and indicates that a chronic L. monocytogenes IMI should also be considered as source of bacteria when bulk tank milk contamination is detected in a dairy goat farm.
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Enfermedades de las Cabras/microbiología , Listeria monocytogenes/aislamiento & purificación , Listeriosis/veterinaria , Mastitis/veterinaria , Animales , Queso/microbiología , Industria Lechera , Femenino , Contaminación de Alimentos/análisis , Enfermedades de las Cabras/diagnóstico , Cabras , Italia , Listeriosis/microbiología , Mastitis/microbiología , Leche/microbiologíaRESUMEN
This research communication reports the evaluation of cathelicidin in dairy goat milk for its relationship with the somatic cell count (SCC) and microbial culture results. Considering the limited performances of SCC for mastitis monitoring in goats, there is interest in evaluating alternative diagnostic tools. Cathelicidin is an antimicrobial protein involved in innate immunity of the mammary gland. In this work, half-udder milk was sampled bimonthly from a herd of 37 Alpine goats along an entire lactation and tested with the cathelicidin ELISA together with SCC and bacterial culture. Cathelicidin and SCC showed a strong correlation (r = 0.72; n = 360 milk samples). This was highest in mid-lactation (r = 0.83) and lowest in late lactation (r = 0.61), and was higher in primiparous (0.80, n = 130) than in multiparous goats (0.71, n = 230). Both markers increased with stage of lactation, but cathelicidin increased significantly less than SCC. In addition, peak level in late lactation was lower for cathelicidin (5.05-fold increase) than for SCC (7.64-fold increase). Twenty-one (5.8%) samples were positive to bacteriological culture, 20 for coagulase-negative staphylococci and one for Streptococcus spp.; 18 of them were positive to the cathelicidin ELISA (85.71% sensitivity). Sensitivity of SCC >500 000 and of SCC >1 000 000 cells/ml was lower (71.43 and 23.81%, respectively). Therefore, the high correlation of cathelicidin with SCC during the entire lactation, along with its lower increase in late lactation and good sensitivity in detecting intramammary infection (IMI), indicate a potential for monitoring subclinical mastitis in dairy goats. However, based on this preliminary assessment, specificity should be improved (40.41% for cathelicidin vs. 54.57 and 67.85% for SCC >500 000 and >1 000 000 cells/ml, respectively). Therefore, the application of cathelicidin for detecting goat IMI will require further investigation and optimization, especially concerning the definition of diagnostic thresholds.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Cabras/fisiología , Lactancia/fisiología , Leche/citología , Animales , Péptidos Catiónicos Antimicrobianos/química , Femenino , CatelicidinasRESUMEN
The first characterization of the sheep fecal microbiota was recently reported, as obtained by using a multi meta-omic approach. Here, the mass spectra generated by single-run LC/high-resolution MS in the context of that study were reanalyzed using a host-specific database, in order to gain insights for the first time into the host fecal proteome of healthy Sarda sheep. On the whole, 5349 non-redundant tryptic peptide sequences were identified, belonging to 1046 different proteins. The "core" fecal proteome (common to all animals) comprised 431 proteins, mainly related to biological processes as immune response and proteolysis. Proteins involved in the immune/inflammatory response and peptidases were specifically investigated. This dataset provides novel insights into the repertoire of proteins secreted in the sheep intestinal lumen, and constitutes the basis for future shotgun and targeted proteomics studies aimed at monitoring changes in the sheep fecal proteome in response to production variables, infectious/inflammatory states, and variations in the gut microbiota. Data are available via ProteomeXchange with identifier PXD006145.
Asunto(s)
Proteínas Bacterianas/metabolismo , Heces/microbiología , Mucosa Intestinal/metabolismo , Proteoma/análisis , Ovinos/microbiología , Animales , Mucosa Intestinal/microbiología , Análisis de Secuencia de ProteínaRESUMEN
In veterinary medicine, assay performance is often affected by the lack of species-specific diagnostic tools. Reliable biomarkers might be identified by investigating biological fluids of the species of interest, but protein sequence databases are often incomplete and human-specific devices for reducing sample complexity might fail when applied to animal plasma. Here, seven commercial methods based on different capturing agents (anti-human antibodies, affinity ligands, mixture of antibodies and ligands, and combinatorial peptide ligand libraries) are applied to cat plasma and evaluated in terms of yield, identified proteins/ peptides, and relative abundance by high-resolution shotgun proteomics and label-free quantitation. As a result, anti-human antibody-based methods are unsatisfactory. Most fail in reducing albumin and immunoglobulins, and some lead to a substantial removal of other highly abundant proteins, probably because of nonspecific interactions. A protein A/dye ligand-based method is efficient in reducing immunoglobulins, fibrinogen, and apolipoprotein A1 and A2, but not albumin, and protein identifications do not increase. Only peptide ligand libraries flatten the dynamic range, and increased protein identification (59.0%). Albumin and immunoglobulins are successfully depleted (60.7% and 35.9%, respectively). Although further studies will be required for reinforcing our observations, this work can provide a useful guide for cat plasma pretreatment in biomarker discovery studies.
Asunto(s)
Biomarcadores/sangre , Proteínas Sanguíneas/aislamiento & purificación , Proteínas Sanguíneas/metabolismo , Proteoma/análisis , Animales , Gatos , Cromatografía de Afinidad , Cromatografía Liquida , Espectrometría de Masas en TándemRESUMEN
Background: Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. Methods: Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the N-terminal and C-terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivities relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay. Conclusions: The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.
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Equinococosis/diagnóstico , Echinococcus granulosus/inmunología , Proteínas del Helminto/química , Proteínas del Helminto/inmunología , Lipoproteínas/química , Lipoproteínas/inmunología , Secuencia de Aminoácidos , Animales , Equinococosis/inmunología , Ensayo de Inmunoadsorción Enzimática , Proteínas del Helminto/síntesis química , Humanos , Lipoproteínas/síntesis química , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
Paratuberculosis (PTB) or Johne's disease is a contagious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Ovine PTB is less understood than bovine PTB, especially concerning paucibacillary infection and its evolution into clinical disease. We combined shotgun proteomics, histopathology and immunohistochemistry for the characterization of ileal tissues collected from seven asymptomatic sheep negative to serum ELISA, positive to feces and tissue MAP IS900 and F57 PCR, histologically classified as paucibacillary, actively infected, together with 3 MAP-free controls (K). Following shotgun proteomics with label-free quantitation and differential analysis, 96 proteins were significantly changed in PTB vs K, and were mostly involved in immune defense processes and in the macrophage-MAP interaction. Principal component analysis (PCA) of protein abundances highlighted two PTB sample clusters, PTB1 and PTB2, indicating a dichotomy in their proteomic profiles. This was in line with the PCA of histopathology data and was related to features of type 2 (PTB1) and type 3a (PTB2) lesions, respectively. PTB2 proteomes differed more than PTB1 proteomes from K: 43 proteins changed significantly only in PTB2 and 11 only in PTB1. The differential proteins cathelicidin, haptoglobin, S100A8 and S100A9 were evaluated by immunohistochemistry. K tissues were negative to cathelicidin and haptoglobin and sparsely positive to S100A8 and S100A9. PTB tissues were positive to all four proteins, with significantly more cells in PTB2 than in PTB1. In conclusion, we described several pathways altered in paucibacillary PTB, highlighted some proteomic differences among paucibacillary PTB cases, and identified potential markers for disease understanding, staging, and detection.
Asunto(s)
Íleon/patología , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Paratuberculosis/patología , Enfermedades de las Ovejas/patología , Animales , Infecciones Asintomáticas , Biomarcadores/análisis , Ensayo de Inmunoadsorción Enzimática/veterinaria , Heces/microbiología , Femenino , Íleon/microbiología , Inmunohistoquímica/veterinaria , Paratuberculosis/microbiología , Reacción en Cadena de la Polimerasa/veterinaria , Proteoma , Proteómica , Ovinos , Enfermedades de las Ovejas/microbiologíaRESUMEN
BACKGROUND: Canine mammary tumors represent the most common neoplasm in female dogs, and the discovery of cancer biomarkers and their translation to clinical relevant assays is a key requirement in the war on cancer. Since the description of the 'Warburg effect', the reprogramming of metabolic pathways is considered a hallmark of pathological changes in cancer cells. In this study, we investigate the expression of two cancer-related metabolic enzymes, transketolase (TKT) and transketolase-like 1 (TKTL1), involved in the pentose phosphate pathway (PPP), an alternative metabolic pathway for glucose breakdown that could promote cancer by providing the precursors and energy required for rapidly growing cells. RESULTS: TKT and TKTL1 protein expression was investigated by immunohistochemistry in canine normal (N = 6) and hyperplastic glands (N = 3), as well as in benign (N = 11) and malignant mammary tumors (N = 17). TKT expression was higher in hyperplastic lesions and in both benign and malignant tumors compared to the normal mammary gland, while TKTL1 levels were remarkably higher in hyperplastic lesions, simple adenomas and simple carcinomas than in the normal mammary glands (P < 0.05). CONCLUSIONS: This study reveals that the expression of a key PPP enzyme varies along the evolution of canine mammary neoplastic lesions, and supports a role of metabolic changes in the development of canine mammary tumors.
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Enfermedades de los Perros/enzimología , Glándulas Mamarias Animales/enzimología , Neoplasias Mamarias Animales/enzimología , Transcetolasa/biosíntesis , Animales , Western Blotting , Perros , Femenino , Hiperplasia/enzimología , Hiperplasia/veterinaria , Técnicas para Inmunoenzimas , Glándulas Mamarias Animales/patologíaRESUMEN
Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Proteínas Fúngicas/metabolismo , Rhodotorula/metabolismo , Biotecnología , Carotenoides/biosíntesis , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional/métodos , Proteínas Fúngicas/genética , Ontología de Genes , Proteómica/métodos , Rhodotorula/genética , Análisis de Secuencia de Proteína , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: We have previously demonstrated that the hydroxylated biphenyl compound D6 (3E,3'E)-4,4'-(5,5',6,6'-tetramethoxy-[1,1'-biphenyl]-3,3'-diyl)bis(but-3-en-2-one), a structural analogue of curcumin, exerts a strong antitumor activity on melanoma cells both in vitro and in vivo. Although the mechanism of action of D6 is yet to be clarified, this compound is thought to inhibit cancer cell growth by arresting the cell cycle in G2/M phase, and to induce apoptosis through the mitochondrial intrinsic pathway. To investigate the changes in protein expression induced by exposure of melanoma cells to D6, a differential proteomic study was carried out on D6-treated and untreated primary melanoma LB24Dagi cells. METHODS: Proteins were fractionated by SDS-PAGE and subjected to in gel digestion. The peptide mixtures were analyzed by liquid chromatography coupled with tandem mass spectrometry. Proteins were identified and quantified using database search and spectral counting. Proteomic data were finally uploaded into the Ingenuity Pathway Analysis software to find significantly modulated networks and pathways. RESULTS: Analysis of the differentially expressed protein profiles revealed the activation of a strong cellular stress response, with overexpression of several HSPs and stimulation of ubiquitin-proteasome pathways. These were accompanied by a decrease of protein synthesis, evidenced by downregulation of proteins involved in mRNA processing and translation. These findings are consistent with our previous results on gene expression profiling in melanoma cells treated with D6. CONCLUSIONS: Our findings confirm that the curcumin analogue D6 triggers a strong stress response in melanoma cells, turning down majority of cell functions and finally driving cells to apoptosis.
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Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/farmacología , Curcumina/análogos & derivados , Redes Reguladoras de Genes/efectos de los fármacos , Melanoma/metabolismo , Proteómica/métodos , Compuestos de Bifenilo/química , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Curcumina/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Melanoma/tratamiento farmacológico , Mitocondrias/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
To date, most metaproteomic studies of the gut microbiota employ stool sample pretreatment methods to enrich for microbial components. However, a specific investigation aimed at assessing if, how, and to what extent this may impact on the final taxonomic and functional results is still lacking. Here, stool replicates were either pretreated by differential centrifugation (DC) or not centrifuged. Protein extracts were then processed by filter-aided sample preparation, single-run LC, and high-resolution MS, and the metaproteomic data were compared by spectral counting. DC led to a higher number of identifications, a significantly richer microbial diversity, as well as to reduced information on the nonmicrobial components (host and food) when compared to not centrifuged. Nevertheless, dramatic differences in the relative abundance of several gut microbial taxa were also observed, including a significant change in the Firmicutes/Bacteroidetes ratio. Furthermore, some important microbial functional categories, including cell surface enzymes, membrane-associated proteins, extracellular proteins, and flagella, were significantly reduced after DC. In conclusion, this work underlines that a critical evaluation is needed when selecting the appropriate stool sample processing protocol in the context of a metaproteomic study, depending on the specific target to which the research is aimed. All MS data have been deposited in the ProteomeXchange with identifier PXD001573 (http://proteomecentral.proteomexchange.org/dataset/PXD001573).
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Heces/microbiología , Microbioma Gastrointestinal/genética , Microbiota/genética , Proteómica , Humanos , Biosíntesis de Proteínas/genética , Proteoma/genéticaRESUMEN
Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and antimicrobial proteins that are released extracellularly by neutrophils and other immune cells as a means for trapping and killing invading pathogens. Here, we describe NET formation in milk and in mammary alveoli of mastitic sheep, and provide a dataset of proteins found in association to these structures. Nucleic acid staining, immunomicroscopy and fluorescent in-situ hybridization of mastitic mammary tissue from sheep infected with Streptococcus uberis demonstrated the presence of extranuclear DNA colocalizing with antimicrobial proteins, histones, and bacteria. Then, proteomic analysis by LTQ-Orbitrap Velos mass spectrometry provided detailed information on protein abundance changes occurring in milk upon infection. As a result, 1095 unique proteins were identified, of which 287 being significantly more abundant in mastitic milk. Upon protein ontology classification, the most represented localization classes for upregulated proteins were the cytoplasmic granule, the nucleus, and the mitochondrion, while function classes were mostly related to immune defence and inflammation pathways. All known NET markers were massively increased, including histones, granule proteases, and antimicrobial proteins. Of note was the detection of protein arginine deiminases (PAD3 and PAD4). These enzymes are responsible for citrullination, the post-translational modification that is known to trigger NET formation by inducing chromatin decondensation and extracellular release of NETs. As a further observation, citrullinated residues were detected by tandem mass spectrometry in histones of samples from mastitic animals. In conclusion, this work provides novel microscopic and proteomic information on NETs formed in vivo in the mammary gland, and reports the most complete database of proteins increased in milk upon bacterial mastitis.
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Trampas Extracelulares/metabolismo , Mastitis/veterinaria , Neutrófilos/metabolismo , Enfermedades de las Ovejas/inmunología , Infecciones Estreptocócicas/veterinaria , Streptococcus/fisiología , Animales , Trampas Extracelulares/microbiología , Femenino , Humanos , Glándulas Mamarias Animales/microbiología , Glándulas Mamarias Animales/patología , Mastitis/inmunología , Mastitis/microbiología , Leche/citología , Leche/microbiología , Neutrófilos/microbiología , Ovinos , Enfermedades de las Ovejas/microbiología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiologíaRESUMEN
BACKGROUND: The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking. EXPERIMENTAL DESIGN: DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity. RESULTS: DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility. CONCLUSIONS: These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.
RESUMEN
BACKGROUND: The zootechnical performance of three different commercial feeds and their impact on liver and serum proteins of gilthead sea bream (Sparus aurata, L.) were assessed in a 12 week feeding trial. The three feeds, named A, B, and C, were subjected to lipid and protein characterization by gas chromatography (GC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. RESULTS: Feed B was higher in fish-derived lipids and proteins, while feeds C and A were higher in vegetable components, although the largest proportion of feed C proteins was represented by pig hemoglobin. According to biometric measurements, the feeds had significantly different impacts on fish growth, producing a higher average weight gain and a lower liver somatic index in feed B over feeds A and C, respectively. 2D DIGE/MS analysis of liver tissue and Ingenuity pathways analysis (IPA) highlighted differential changes in proteins involved in key metabolic pathways of liver, spanning carbohydrate, lipid, protein, and oxidative metabolism. In addition, serum proteomics revealed interesting changes in apolipoproteins, transferrin, warm temperature acclimation-related 65 kDa protein (Wap65), fibrinogen, F-type lectin, and alpha-1-antitrypsin. CONCLUSIONS: This study highlights the contribution of proteomics for understanding and improving the metabolic compatibility of feeds for marine aquaculture, and opens new perspectives for its monitoring with serological tests.
RESUMEN
In this study, the proteome profile of European sea bass (Dicentrarchus labrax) muscle was analyzed using two-dimensional electrophoresis (2-DE) and tandem mass spectrometry with the aim of providing a more detailed characterization of its specific protein expression profile. A highly populated and well-resolved 2-DE map of the sea bass muscle tissue was generated, and the corresponding protein identity was provided for a total of 49 abundant protein spots. Upon Ingenuity Pathway Analysis, the proteins mapped in the sea bass muscle profile were mostly related to glycolysis and to the muscle myofibril structure, together with other biological activities crucial to fish muscle metabolism and contraction, and therefore to fish locomotor performance. The data presented in this work provide important and novel information on the sea bass muscle tissue-specific protein expression, which can be useful for future studies aimed to improve seafood traceability, food safety/risk management and authentication analysis. This work is also important for understanding the proteome map of the sea bass toward establishing the animal as a potential model for muscular studies.
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Lubina/metabolismo , Proteínas de Peces/química , Perfilación de la Expresión Génica , Músculo Esquelético/metabolismo , Proteoma/química , Animales , Electroforesis en Gel Bidimensional , Proteínas de Peces/fisiología , Redes y Vías Metabólicas/fisiología , Contracción Muscular/fisiología , Músculo Esquelético/química , Espectrometría de Masas en TándemRESUMEN
Non-aureus staphylococci and mammaliicocci (NASM) are associated with bovine mastitis and increased milk somatic cell count (SCC) but their relationships with mammary gland health at the species level are not clearly defined. Regional differences have also been reported in their specific prevalence. The implementation of MALDI-TOF MS in milk microbiology is generating large and dependable datasets with the potential of providing useful epidemiological information. We present the retrospective analysis of 17,213 milk samples sent to our laboratory in 2021-2022, including 13,146 quarter samples from cows with subclinical (SCM) or clinical mastitis (CM) from 104 farms, and 4,067 composite herd survey (HS) samples from 21 farms. NASM were isolated from 21.12% of SCM, 11.49% of CM, and 15.59% of HS milk samples. The three most frequently identified NASM in SCM milk were Staphylococcus chromogenes (33.33%), S. haemolyticus (26.07%), and S. epidermidis (10.65%); together with S. microti and S. hyicus, these species were significantly more prevalent in quarters with SCM (p < 0.05). The three most frequently identified NASM in CM milk were S. chromogenes (31.69%), S. haemolyticus (21.42%), and Mammaliicoccus sciuri (18.38%), although no significant associations were found between these NASM species and CM. The three most frequently identified NASM in HS milk were S. chromogenes (44.49%), S. epidermidis (17.84%), and S. haemolyticus (17.23%), with S. chromogenes being isolated in all the farms sending HS milk (100%). In conclusion, this retrospective study provides the first information on the NASM species isolated from cow milk in Italy, expanding our knowledge on the epidemiology of NASM at the species level and providing further insights into their relationships with mammary gland health in modern dairy farms.
Asunto(s)
Enfermedades de los Bovinos , Mastitis Bovina , Infecciones Estafilocócicas , Femenino , Bovinos , Animales , Estudios Retrospectivos , Leche/microbiología , Infecciones Estafilocócicas/veterinaria , Granjas , Mastitis Bovina/epidemiología , Mastitis Bovina/microbiología , Enfermedades de los Bovinos/microbiologíaRESUMEN
The ability of Salmonella species to adhere to surfaces and form biofilms, leading to persistent environmental reservoirs, might represent a direct link between environmental contamination and food processing contamination. The purpose of this study was to investigate the biofilm-forming ability of 80 multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL) producing Salmonella enterica serovar Infantis strains isolated from the broiler food chain production through whole genome sequencing (WGS), PCR, and morphotype association assays. Biofilm formation was quantified by testing the strains at two different temperatures, using 96-well polystyrene plates. The rough and dry colony (rdar) morphotype was assessed visually on Congo red agar (CRA) plates. Based on our results, all tested S. Infantis strains produced biofilm at 22 °C with an rdar morphotype, while at 37 °C, all the isolates tested negative, except one positive. Most isolates (58.75%) exhibited strong biofilm production, while 36.25% showed moderate production. Only 5 out of 80 (6.25%) were weak biofilm producers. WGS analysis showed the presence of the fim cluster (fimADF) and the csg cluster (csgBAC and csgDEFG), also described in S. Typhimurium, which are responsible for fimbriae production. PCR demonstrated the presence of csgD, csgB, and fimA in all 80 S. Infantis strains. To our knowledge, this is the first study comparing the effects of two different temperatures on the biofilm formation capacity of ESBL producing S. Infantis from the broiler production chain. This study highlights that the initial biofilm components, such as curli and cellulose, are specifically expressed at lower temperatures. It is important to emphasize that within the broiler farm, the environmental temperature ranges between 18-22 °C, which is the optimum temperature for in vitro biofilm formation by Salmonella spp. This temperature range facilitates the expression of biofilm-associated genes, contributing to the persistence of S. Infantis in the environment. This complicates biosecurity measures and makes disinfection protocols on the farm and in the production chain more difficult, posing serious public health concerns.
RESUMEN
Non-aureus staphylococci and mammaliicocci (NASM) are the microorganisms most frequently isolated from milk. Given their numerosity and complexity, MALDI-TOF MS is one of the preferred species identification approaches. Nevertheless, reference mass spectra for the novel species Staphylococcus borealis were included only recently in the Bruker Biotyper System (MBT) library, and other species of veterinary interest such as S. rostri are still absent. This work provides an updated picture of the NASM species found in milk, gained by retrospectively analyzing the data relating to 21,864 milk samples, of which 6,278 from clinical mastitis (CM), 4,039 from subclinical mastitis (SCM), and 11,547 from herd survey (HS), with a spectrum library including both species. As a result, S. borealis was the second most frequently isolated NASM (17.07%) after S. chromogenes (39.38%) in all sample types, with a slightly higher percentage in CM (21.84%), followed by SCM (17.65%), and HS (14.38%). S. rostri was also present in all sample types (3.34%), reaching 8.43% of all NASM in SCM and showing a significant association (p < 0.01) with this condition. Based on our findings, the presence of S. borealis and S. rostri in milk and their potential association with mastitis might have been overlooked, possibly due to the difficulties in differentiating these species from other closely related NASM. Our results indicate that S. borealis could be a more frequent contributor to bovine udder infections than previously thought and that S. rostri should also not be underestimated considering its significant association with SCM.