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Serological proteome analysis of Corynebacterium pseudotuberculosis isolated from different hosts reveals novel candidates for prophylactics to control caseous lymphadenitis
... In a complementary approach, immune-reactive exoproteins of two C. pseudotuberculosis strains were studied in a serological proteome analysis using blood sera from goats and sheep. With 13 immuno-reactive proteins identified from both strains, also this proteomic approach revealed putative targets for vaccine development [72]. Six of the identified proteins from the core immune-proteome were of unknown functions, surface layer protein A, cell-surface hemin receptor HtaA and two trehalose corynomycolyl transferases were related to cell envelope functions and resuscitation-promoting factor plays a role in the stress response and virulence. ...
... Six of the identified proteins from the core immune-proteome were of unknown functions, surface layer protein A, cell-surface hemin receptor HtaA and two trehalose corynomycolyl transferases were related to cell envelope functions and resuscitation-promoting factor plays a role in the stress response and virulence. In addition, neuraminidase (sialidase) and invasion-associated protein p60 are part of the accessory seroproteome and putatively related to the cell surface and virulence [72]. ...
Within the genus Corynebacterium, six species are potential carriers of the tox gene, which encodes the highly potent diphtheria exotoxin: Corynebacterium diphtheriae, Corynebacterium belfantii, Corynebacterium rouxii, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis and Corynebacterium silvaticum. Based on their potential to infect different host species and cause either human infections, zoonotic diseases or infections of economically important animals, these bacteria are of high scientific and economic interest and different research groups have carried out proteome analyses. These showed that especially the combination of MS-based proteomics with bioinformatic tools helped significantly to elucidate the functional aspects of corynebacterial genomes and to handle the genome and proteome complexity. The combination of proteomic and bioinformatic approaches was also used to discover new vaccine and drug targets. In addition, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been established as a fast and precise tool for the identification of these bacteria.
... C. diphtheriae (Hansmeier et al., 2006;Möller et al., 2021b), C. ulcerans (Bittel et al., 2018), (Möller et al., 2020a) and C. pseudotuberculosis Seyffert et al., 2011Seyffert et al., , 2014Silva et al., 2013Rees et al., 2015;Silva et al., 2017b;Raynal et al., 2018;Sá et al., 2021). ...
... MS-based characterization approaches were intensively used to investigate the proteome of C. pseudotuberculosis, in respect to adaption to environmental stress , as well as the exoproteome Silva et al., 2013;Silva et al., 2017a), membrane proteins (Raynal et al., 2018;Rees et al., 2015), the variation in the proteome of biofilm forming and non-biofilm forming strains (Sá et al., 2021) and the identification of novel vaccine targets (Seyffert et al., , 2014. Beside the analysis of bacteria grown under laboratory conditions proteomic studies of C. pseudotuberculosis with direct contact to the host (Rees et al., 2015;Silva et al., 2017a) or animal serum (Raynal et al., 2018) demonstrated changes of the bacterial proteome during infection. ...
Diphtheria toxin (DT) expressing Corynebacterium diphtheriae is the etiological agent of the classical respiratory diphtheria. Since the introduction of diphtheria toxoid vaccines cases of respiratory diphtheria declined significantly over the years. Nevertheless, local outbreaks, e.g. in the former Soviet Union in 1990s, in Venezuela or Yemen are reported and consistently associated with poor access to vaccination. Despite the constant high global vaccination coverage, the number of reported diphtheria cases increased within the last years. However, beside C. diphtheriae, other pathogenic corynebacteria e.g. C. ulcerans, C. pseudotuberculosis and the novel identified C. silvaticum are known to cause severe infections in humans and animals. Regardless of the medical and veterinary importance, only few virulence factors besides diphtheria toxin and phospholipase D are known. In frame of this thesis, the basic molecular and proteomic characterization and characterization of the infection process of pathogenic corynebacteria were carried out. A group of non-toxigenic tox-gene bearing (NTTB) C. ulcerans, isolated from wild animals, were assembled into a wild life cluster. Dangel and co-workers first designated this group as a novel species Corynebacterium silvaticum, in 2020. A member of this novel species is strain W25, who was identified as C. ulcerans, firstly (Busch et al., 2019). Phylogenomic analyzes revealed, that strain W25 belongs to a novel species along with a C. pseudotuberculosis strain PO100/5 and C. ulcerans KL1196 (Möller et al., 2020b). Genome analyses revealed 172 unique genes in C. silvaticum and two missing enzymes, a pullulanase type I and a 1,4-α amylase, which results in a lack of the ability to ferment starch. The main characteristic of this species is the NTTB phenotype. An examination of the tox-gene sequence in W25 showed a two base (GG) insertion which results in a pseudogene due to a frameshift mutation. RNA hybridization experiments and detection methods for the diphtheria toxin approved the NTTB phenotype of C. silvaticum W25. An investigation of the virulence potential of C. silvaticum showed that 17 niche and virulence factors out of 23 known are present in C. silvaticum. A proteomic analysis identified a secreted protein with a predicted trypsin-like serine protease function and homologous to the virulence factors Vsp1 and Vsp2 from C. ulcerans, which contributed to 88.1 % of the secretome (Möller et al., 2020a). Another approach to investigate the virulence potential of C. silvaticum, are cell culture experiments. The studies showed a detrimental effect to human and animal epithelial cell lines. Moreover, C. silvaticum revealed a cell line dependent low adhesion and invasion rates to epithelial cells. Since cell culture do not represent a complete living organism, the invertebrate model organism Galleria mellonella was used to approve the virulence potential of C. silvaticum (Möller et al., 2021a). Microbial proteomics contributed to a better understanding of human and animal pathogens. Based on mass spectrometric (MS) techniques, the proteomic analysis of pathogenic corynebacteria was an additional focus of this thesis. Mass spectrometry was used to confirm the expression of a novel virulence factor from C. diphtheriae and C. ulcerans, the Shiga-like toxin (Weerasekera et al., 2019c). Beside the identification of single proteins, MS was used to characterize whole proteomes of C. diphtheriae strain ISS3319 grown under different culture conditions (Möller et al., 2021b). A label-free approach revealed, up and down regulated proteins as an adaption to changing growth conditions. Interestingly, besides up- and down-regulated proteins and newly expressed proteins a substantial number of virulence factors was present under all three growth conditions tested, which indicates a pre-adaption of C. diphtheriae to host conditions. An adaption of C. diphtheriae due to a vaccine induced immunity was also suspected in the literature. For toxoid vaccine production, the purification of the toxins is based on centrifugation and filtration steps. The inactivation of toxins is achieved by formaldehyde cross-linking. This unspecific inactivation process may result in protein complexes including various proteins besides DT. Those side products are thought to trigger the host immune system and lead to an adaption of C. diphtheriae. A principal investigation of the protein content of common used Diphtheria Tetanus Pertussis (DTP) vaccine formulations, using MS were carried out (Möller et al., 2019a, b, c). Vaccines from different countries and manufacturers as well as a cellular and whole cellular pertussis vaccines were analyzed. The analysis revealed complex protein mixtures in all vaccines with various proteins besides the main components. A bioinformatic analysis identified proteins with antigenic potential which therefore may contribute to an adaption of the respective bacteria. This vaccine-induced immunity was approved for C. diphtheriae. For this purpose, human sera were used as primary antibody for Western Blot analysis. The human sera showed a strain-dependent antibody binding and permits conclusions of probable antibody driven decreased fitness of C. diphtheriae.
... Rodeando esta región se identificaron células CD5 + , así como células CD4 + y CD8 + distribuidas a través del tejido linfático. Generalmente en las lesiones caseosa inmaduras se encuentran linfocitos CD4 + y en las lesiones más desarrolladas la concentración de células CD8 + es predominante, lo que se relaciona con el mecanismo del sistema inmune para combatir la diseminación de los macrófagos infectados (70,71) . ...
... En cabras los cambios histopatológicos observados en el tracto reproductivo y nódulos linfáticos después de la inoculación experimental con C. pseudotuberculosis, revelaron infiltración leucocítica, así como congestión generalizada, degeneración, infiltración de células del estroma y necrosis en los ovarios (71) . El estudio de la respuesta del sistema inmune en un modelo experimental permitió establecer que la respuesta humoral comienza entre el día 6 y 11 post infección. ...
La linfadenitis caseosa es una enfermedad que afecta la producción ovina y caprina a nivel mundial. El agente etiológico es una bacteria Gram positiva, intracelular facultativa denominada Corynebacterium pseudotuberculosis biovar ovis. La enfermedad puede cursar con un desarrollo cutáneo o visceral, provocando deterioro en la condición física del animal, así como pérdidas en la producción de leche y carne, decomiso de las canales, rechazo de las pieles y como consecuencia, grandes pérdidas económicas. El estudio de los factores de virulencia y los mecanismos de patogénesis han permitido comprender esta enfermedad, así como establecer las moléculas diana para el desarrollo de nuevas vacunas. Existen vacunas comerciales disponibles a nivel mundial; sin embargo, la protección conferida por éstas no ha sido eficaz en el control de la enfermedad. Actualmente el uso de nuevas tecnologías ha permitido la obtención y caracterización de proteínas con potencial inmunogénico para el desarrollado de nuevas vacunas, las cuales podrían ser una alternativa para incrementar la protección. En el presente trabajo se exponen los principales factores de virulencia de Corynebacterium pseudotuberculosis, sus implicaciones en la patogénesis y las tendencias actuales en las formulaciones vacunales.
... Phospholipase D (PLD) [7] and Endoglycosidase CP40 [8] are virulence factors of Corynebacterium pseudotuberculosis biovar ovis, a gram-positive rod that cause Caseous lymphadenitis (CLA), a source of economic loss to the sheep and goat industries. CLA characteristic lesions are associated with encapsulated cutaneous and visceral abscesses with pyogranulomas formation, a response of the immune system to contain the infections in the host's tissues, but which makes it difficult to eliminate the bacteria [9,10]. ...
Mapping B and T cell epitopes constitutes an important action for peptide vaccine design. PLD and CP40 virulence factors of Corynebacterium pseudotuberculosis biovar ovis, a causal agent of Caseous Lymphadenitis, have been evaluated in a murine model as good candidates for vaccine development. Therefore, the goal of this work was to in silico analyze B and T cell epitopes of the PLD and CP40 proteins of a Mexican isolate of Corynebacterium pseudotuberculosis ovis. The Immune Epitope Data Base and Resource website was employed to predict the linear and conformational B-cell, T CD4+, and T CD8+ epitopes of PLD and CP40 proteins of Corynebacterium pseudotuberculosis ovis Mexican strain 2J-L. Fifty B cell epitopes for PLD 2J-L and forty-seven for CP40 2J-L were estimated. In addition, T CD4+ and CD8+ cell epitopes were predicted for PLD 2J-L (MHC I:16 epitopes, MHC II:10 epitopes) and CP40 2J-L (MHC I: 15 epitopes, MHC II: 13 epitopes). This study provides epitopes, paying particular attention to sequences selected by different predictor programs and overlap sequences as B and T cell epitopes. PLD 2J-L and CP40 2J-L protein epitopes may aid in the design of a promising peptide-based vaccine against Caseous Lymphadenitis in Mexico.
... Os animais do grupo controle negativo receberam como placebo uma dose de solução salina estéril (PBS). Research, Society and Development, v. 11, n. 12, e440111234549, 2022 (CC BY 4. (Seyffert et al., 2014). Os genes que codificam para as proteínas recombinantes de C. pseudotuberculosis foram selecionados e submetidos ao desenho de versões sintéticas, com otimização de códons para expressão em sistemas heterólogos. ...
Corynebacterium pseudotuberculosis é o agente etiológico da linfadenite caseosa (LC), uma doença infecciosa crônica que afeta ovinos e caprinos. Numerosos testes sorológicos foram desenvolvidos para detectar a doença, sendo o Ensaio Imunoenzimático (ELISA) um dos mais utilizados devido às suas vantagens de sensibilidade e especificidade. O presente estudo teve como objetivo avaliar a reatividade de um painel de antígenos de Corynebacterium pseudotuberculosis em caprinos experimentalmente infectados com linhagens não formadoras e formadoras de biofilme. Foram utilizados 18 caprinos da raça Canindé sendo: grupo 1 - animais controle negativo, grupo 2 - caprinos infectados com bactéria não formadora de biofilme e grupo 3 - caprinos infectados com linhagem formadora de biofilme. Foram testados os antígenos recombinantes CP40 e PLD, antígenos de fração de membrana (F3) e antígenos secretados de C. pseudotuberculosis (BHI). A sensibilidade e especificidade dos testes ELISA indireto para todos os antígenos foram de 100%. No entanto, os valores de densidade óptica (DO) registrados no ELISA BHI foram menores que os encontrados nos outros testes ELISA, sugerindo que antígenos específicos podem apresentar melhor sensibilidade e especificidade no diagnóstico da LC.
... This preliminary study combined the serum of goats infected with C. pseudotuberculosis and extracellular proteins of 1002_ovis. In a second study, Seyffert et al. (2014) utilized a pool of serum from goat and sheep infected by C. pseudotuberculosis and comparative proteomics to identify immune-reactive proteins. The comparative analysis between the exoproteome of 1002 and C231 allowed identifying 13 immunoreactive proteins non-redundant Interestingly, six immune-reactive exoproteins (Cphp1, Cphp2, Cphp3, Cphp4, Cphp6, and Cphp7) with unknown functions identified in the core-exoproteome are exclusive of C. pseudotuberculosis. ...
Background
Corynebacterium pseudotuberculosis is a Gram-positive facultative intracellular pathogen and the etiologic agent of illnesses like caseous lymphadenitis in small ruminants, mastitis in dairy cattle, ulcerative lymphangitis in equines, and oedematous skin disease in buffalos. With the growing advance in high-throughput technologies, genomic studies have been carried out to explore the molecular basis of its virulence and pathogenicity. However, data large-scale functional genomics studies are necessary to complement genomics data and better understating the molecular basis of a given organism. Here we summarize, MS-based proteomics techniques and bioinformatics tools incorporated in genomic functional studies of C. pseudotuberculosis to discover the different patterns of protein modulation under distinct environmental conditions, and antigenic and drugs targets.
Methodology
In this study we performed an extensive search in Web of Science of original and relevant articles related to methods, strategy, technology, approaches, and bioinformatics tools focused on the functional study of the genome of C. pseudotuberculosis at the protein level.
Results
Here, we highlight the use of proteomics for understating several aspects of the physiology and pathogenesis of C. pseudotuberculosis at the protein level. The implementation and use of protocols, strategies, and proteomics approach to characterize the different subcellular fractions of the proteome of this pathogen. In addition, we have discussed the immunoproteomics, immunoinformatics and genetic tools employed to identify targets for immunoassays, drugs, and vaccines against C. pseudotuberculosis infection.
Conclusion
In this review, we showed that the combination of proteomics and bioinformatics studies is a suitable strategy to elucidate the functional aspects of the C. pseudotuberculosis genome. Together, all information generated from these proteomics studies allowed expanding our knowledge about factors related to the pathophysiology of this pathogen.
... pseudoTB virulence factors that may be used as novel vaccine targets (Santana-Jorge et al., 2016;Seyffert et al., 2014;Silva et al., 2018;Silva et al., 2013). ...
Caseous lymphadenitis (CL) is a chronic bacterial infection caused by Corynebacterium pseudotuberculosis (C. pseudoTB) that affects small ruminants. This disease has a worldwide prevalence and results in significant economic losses to the sheep and goat industries. Antibiotics have had limited success in treating CL, due to the difficulty of penetrating the dry, thick-walled abscesses that characterize this disease. Essential oils are complex bioactive compounds that have been increasingly explored as sources of antimicrobial activity. Due to the nature of these oils, tissue and wood penetration may be possible, enabling topical and environmental treatment (e.g.; disinfectants of farm surfaces, such as feeders or shearing equipment). The purpose of this project was to evaluate certain essential oil components (EOCs) with known antibacterial properties for their bactericidal effects on C. pseudoTB using a standard disk diffusion assay. The most effective inhibition of C. pseudoTB growth in vitro was due to β-citronellol, carvacrol, thymol, and trans-cinnamaldehyde. These EOCs were able to inhibit C. pseudoTB growth at concentrations as low as 10 mg/ml. Essential oils and their components can also have toxic effects on eukaryotic cells. For this reason, a cell viability assay on a line of mammalian fibroblast cells (Buffalo rat liver cells) was conducted to evaluate the cytotoxicity of the EOCs that were most effective at inhibiting C. pseudoTB growth. Based on direct microscopic observations, EOCs damaged mammalian cells; this was not reflected in the cell viability assay. Therefore, further research is needed on the toxicity of these components before use in vivo.
... Apesar da importância econômica da LC, C. pseudotuberculosis tem poucos genes de virulência caracterizados, porém vários estudos já foram desenvolvidos e algumas proteínas já foram descobertas como chaperonas, serina-proteases, transportadoras de ferro, permeases de oligopeptídeo, proteínas ligadas à resposta ao estresse oxidativo que estão envolvidas com a patogenicidade da bactéria e estão associados à adesão, invasão celular, sobrevivência e proliferação na célula hospedeira. Há ainda, outras proteínas que são responsáveis pela síntese da parede celular e que participam de processos fisiológicos no crescimento bacteriano (Moraes et al., 2014;Pacheco et al., 2011;Seyffert et al., 2014). Silva et al. (2013) (Hodgson et al., 1992;. ...
A linfadenite caseosa é uma enfermidade que acomete os caprinos e ovinos causando prejuízos econômicos. É uma doença infectocontagiosa de ocorrência mundial, principalmente nos países em desenvolvimento, com ocorrência em todo rebanho brasileiro, especialmente nos Estados do Nordeste, notadamente na Bahia e em Pernambuco. Apesar de ter uma alta prevalência, a prevenção, o diagnóstico e o tratamento são negligenciados, aumentando os riscos de contaminação entre os rebanhos e consequentemente a diminuição da produtividade destes animais e a qualidade do produto ao consumidor. Alguns métodos de prevenção estão sendo desenvolvidos, utilizando bactérias mortas, vivas ou inativadas, podendo usar ou não a tecnologia do DNA recombinante. A virulência de Corynebacterium pseudotuberculosis, está associada com os lipídeos da parede celular e a fosfolipase D, bem como a produção de biofilmes. A produção desses genes de virulência aumenta a sobrevivência e a multiplicação do patógeno. O objetivo do estudo foi abordar nessa revisão os impactos econômicos provocados pela Linfadenite Caseosa, epidemiologia, patogenia e fatores de virulência, bem como sinalizar informações sobre diferentes aspectos entre cepas de C. pseudotuberculosis produtoras e não produtoras de biofilme, buscando fornecer subsídios que possam contribuir para o aprimoramento de vacinas e diagnóstico desta enfermidade dos pequenos ruminantes.
... Caseous Lymphadenitis (CLA) is a chronic infectious disease characterized by purulent lesions and formation of abscesses in organs and superficial and internal lymph nodes [1,2]. The etiological agent of CLA is the bacterium Corynebacterium pseudotuberculosis [3,4], which presents a higher prevalence in sheep and goat herds [5,6]. Currently there is no effective prophylaxis for CLA. ...
... Moreover, putative antigenic proteins were identified through reverse vaccinology (Soares et al., 2013a). Some studies have shown that extracellular proteins are related to the pathogenic process of C. pseudotuberculosis (Wilson et al., 1995;Billington et al., 2002;Pacheco et al., 2012;Seyffert et al., 2014). However, phospholipase D (Pld) exotoxin, which contributes to bacterial spread in the host, is considered the major virulence factor of this pathogen (McKean et al., 2007). ...
Corynebacterium pseudotuberculosis biovar equi is the etiologic agent of ulcerative lymphangitis. To investigate proteins that could be related to the virulence of this pathogen, we combined an experimental passage process using a murine model and high-throughput proteomics with a mass spectrometry, data-independent acquisition (LC-MSE) approach to identify and quantify the proteins released into the supernatants of strain 258_equi. To our knowledge, this approach allowed characterization of the exoproteome of a C. pseudotuberculosis equi strain for the first time. Interestingly, the recovery of this strain from infected mouse spleens induced a change in its virulence potential, and it became more virulent in a second infection challenge. Proteomic screening performed from culture supernatant of the control and recovered conditions revealed 104 proteins that were differentially expressed between the two conditions. In this context, proteomic analysis of the recovered condition detected the induction of proteins involved in bacterial pathogenesis, mainly related to iron uptake. In addition, KEGG enrichment analysis showed that ABC transporters, bacterial secretion systems and protein export pathways were significantly altered in the recovered condition. These findings show that secretion and secreted proteins are key elements in the virulence and adaptation of C. pseudotuberculosis. Collectively, bacterial pathogenesis-related proteins were identified that contribute to the processes of adherence, intracellular growth and evasion of the immune system. Moreover, this study enhances our understanding of the factors that may influence the pathogenesis of C. pseudotuberculosis.
... In C. diphtheriae, it was demonstrated that a protein with trans-sialidase activity promotes cellular invasion [63,64]. In addition, NanH was reported to be immunoreactive in the immunoproteome of 1002_ovis, showing the antigenicity of this protein [65]. Interestingly, genomic difference in relation to gene involved in the adhesion and invasion process, also already were observed between biovar ovis strain and biovar equi strains, mainly in genes related to pilus [10,12]. ...
Background
Corynebacterium pseudotuberculosis is a pathogen classified into two biovars: C. pseudotuberculosis biovar ovis, the etiologic agent of caseous lymphadenitis and C. pseudotuberculosis biovar equi, which causes ulcerative lymphangitis. The available whole genome sequences of different C. pseudotuberculosis strains have enabled identify difference of genes related both virulence and physiology of each biovar. To evaluate be this difference could reflect at proteomic level and to better understand the shared factors and the exclusive ones of biovar ovis and biovar equi strains, we applied the label-free quantitative proteomic to characterize the proteome of the strains: 1002_ovis and 258_equi, isolated from goat (Brazil) and equine (Belgium), respectively.ResultsFrom this analysis, we characterized a total of 1230 proteins in 1002_ovis and 1220 in 258_equi with high confidence. Moreover, the core-proteome between 1002_ovis and 258_equi obtained here is composed of 1122 proteins involved in different cellular processes, which could be necessary for the free living of C. pseudotuberculosis. In addition, 120 proteins from this core-proteome presented change in abundant with statistically significant differences. Considering the exclusive proteome, we detected strain-specific proteins to each strain. When correlated, the exclusive proteome of each strain and proteome with change in abundant, the proteomic differences, between the 1002_ovis and 258_equi, this related to proteins involved in cellular metabolism, information storage and processing, cellular processes and signaling.Conclusions
This study reports the first comparative proteomic study of the biovars ovis and equi of C. pseudotuberculosis. The results generated in this study provide information about factors which can contribute to understanding both the physiology and the virulence of this pathogen.
... Different strategies like the transposon mutagenesis have been adopted to identify C. pseudotuberculosis biovar ovis exported proteins [7]. Additionally, comparative proteomics has been applied to characterize the extracellular proteome of C. pseudotuberculosis biovar ovis, as well as, the extracellular immunoproteome (strains C231_ovis and 1002_ovis) [8][9][10][11]. In these studies, some proteins of the strain 1002_ovis, suspected to be virulence factors, were not detected suggesting this strain presents a low virulence. The surface proteome of C. pseudotuberculosis biovar ovis was also characterized using bacterial strains isolated from the lymph nodes of naturally infected sheep. ...
Background
Corynebacterium pseudotuberculosis biovar ovis, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, C. pseudotuberculosis changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the C. pseudotuberculosis infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage.
Results
The experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of C. pseudotuberculosis. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen.
Conclusions
This study shows that C. pseudotuberculosis modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of C. pseudotuberculosis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-017-0925-6) contains supplementary material, which is available to authorized users.
... Esses estudos moleculares sofreram grande avanço após o sequenciamento do genoma de diversas cepas de C. pseudotuberculosis, e modelos vacinais baseados em bactérias recombinantes têm apresentado resultados promissores . Uma promessa tem sido a análise proteômica de proteínas reconhecidas por soros de pequenos ruminantes naturalmente infectados pela bactéria, a qual tem revelado novos alvos para o desenvolvimento de vacinas (Seyffert et al. 2014). Apesar das pesquisas em vacinas estarem direcionadas, em sua grande parte, para pequenos ruminantes, as mesmas podem ser importantes para fundamentar novos antígenos a serem empregados na imunoprofilaxia da doença em equídeos. ...
Resumo: Corynebacterium pseudotuberculosis é o agente causador da linfadenite caseosa em caprinos e ovinos, sendo responsável por significativas perdas econômicas na ovinocaprinocultura mundialmente. Esta bactéria Gram-positiva também infecta equinos, causando desde quadros assintomáticos até infecções sistêmicas, podendo levar o animal a óbito. Especificamente no Brasil, não foram relatados casos de infecção em equinos, mas acredita-se que, devido à convivência de pequenos ruminantes infectados com equinos em diversas propriedades rurais, seja natural que ocorra a infecção desses animais. A presente revisão tem como objetivo fornecer informações sobre a bactéria C. pseudotuberculosis, sobre os aspectos epidemiológicos e clínicos da infecção em equídeos, bem como sobre técnicas de manejo para sua prevenção.
... Caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis, is a chronic infectious disease, affecting mainly wild and domestic small ruminants in which abscesses develop in the lymph nodes and (more rarely) in the lungs (Muller et al., 2011;Seyfert et al., 2014). Once CLA is present in a herd, control of the disease is difficult, since C. pseudotuberculosis is readily transmitted between animals (O'Reilly et al., 2008). ...
The treatment of postoperative infection caused by multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), has become an intractable clinical challenge owing to its low therapeutic efficacy and high risk of recurrence. Apart from imperfect antibacterial therapies, induction of insufficient immunogenicity required for the successful clearance of a pathogen may also contribute to the problem. Herein, we utilized an ultra-micro photosensitizer, AgB nanodots, using photothermal therapy, photodynamic therapy, and Ag⁺ ion sterilization to efficiently clear invading MRSA both in vitro and in vivo. AgB nanodots were also found to upregulate host immunogenicity in a murine model and establish immunological memory by promoting the upregulated expression of danger signals that are commonly induced by stress-related responses, including sudden temperature spikes or excess reactive oxygen production. These stimulations boost the antibacterial effects of macrophages, dendritic cells, T cells, or even memory B cells, which is usually defined as infection-related immunogenic cell death. Hence, the proposed AgB nanodot strategy may offer a novel platform for the effective treatment of postoperative infection while providing a systematic immunotherapeutic strategy to combat persistent infections, thereby markedly reducing the incidence of recurrence following recovery from primary infections.
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Corynebacterium pseudotuberculosis est une bactérie pathogène intracellulaire facultative, divisée en deux biovars : C. pseudotuberculosis ovis, agent de la lymphadénite caséeuse (petits ruminants), et C. pseudotuberculosis equi, responsable de lymphangites ulcéreuses (chevaux), mammites (bovins) et d’œdèmes cutanés (buffles). La génomique fonctionnelle a pour but d'élucider le rôle que joue chaque gène dans l'organisme, ainsi que l'interaction de ces gènes dans un réseau biologique. Au cours de ce travail de thèse, différentes stratégies protéomiques ont été adoptées pour l’étude fonctionnelle du génome de C. pseudotuberculosis : i) l’analyse du protéome extracellulaire des souches 1002_ovis et C231_ovis a permis la caractérisation totale de 60 protéines différentes de C. pseudotuberculosis dont 18 protéines sont régulées différemment ;ii) le protéome de la souche 1002_ovis été analysé en réponse au stress nitrosant révélant 58 protéines différentiellement régulées et impliquées dans différents processus biologiques susceptibles de favoriser la survie à ce stress ; iii) les souches 1002_ovis et 258_equi ont été utilisées pour induire des infections expérimentales sur modèle souris. L’analyse de leur protéome extracellulaire avant et après passage en série sur souris a permis d’identifier 250 protéines différentiellement régulées touchant diverses fonctions. Pour conclure, ce travail a permis de générer une base de données de protéines et de valider la fonctionnalité de différents gènes jusqu’alors prédits in silico seulement et des informations importantes sur les facteu
Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis, an infectious disease that affects sheep and goats worldwide. Even though the pathogenic process involved in this disease is well known, only a few virulence factors of C. pseudotuberculosis have been described so far, making it difficult to develop vaccines and therapies to control outbreaks. Recently studies have been conducted to better understand this pathogen and its behaviour under harsh conditions that resemble the intrahost environment. The harmful effects in bacteria exposed to adverse situations are neutralized through a refined gene control driven by transcriptional regulators belonging to the family of sigma factors. Among these regulators, the extracytoplasmic function (ECF) sigma factors stand out for their involvement in the interaction between the bacterial cell and its surrounding environment. We discuss herein the advances in the research related to the ECF sigma factors in C. pseudotuberculosis, addressing their potential roles in stress response and pathogenesis.
Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis, a disease that predominantly affects small ruminants, causing significant economic losses worldwide. As a facultative intracellular pathogen, this bacterium is exposed to an environment rich in reactive oxygen species (ROS) within macrophages. To ensure its genetic stability, C. pseudotuberculosis relies on efficient DNA repair pathways for excision of oxidative damage such as 8-oxoguanine, a highly mutagenic lesion. MutY is an adenine glycosylase involved in adenine excision from 8-oxoG:A mismatches avoiding genome mutation incorporation. The purpose of this study was to characterize MutY protein from C. pseudotuberculosis and determine its involvement with DNA repair. In vivo functional complementation assay employing mutY gene deficient Escherichia coli transformed with CpmutY showed a 13.5-fold reduction in the rate of spontaneous mutation, compared to cells transformed with empty vector. Also, under oxidative stress conditions, CpMutY protein favored the growth of mutY deficient E. coli, relative to the same strain in the absence of CpMutY. To demonstrate the involvement of this enzyme in recognition and excision of 8-oxoguanine lesion, an in vitro assay was performed. CpMutY protein was capable of recognizing and excising 8-oxoG:A but not 8-oxoG:C presenting evidences of glycosylase/AP lyase activity in vitro. In silico structural characterization revealed the presence of preserved motifs related to the MutY activity on DNA repair, such as catalytic residues involved in glycosylase/AP lyase activity and structural DNA-binding elements, such as the HhH motif and the [4Fe4S] cluster. The three-dimensional structure of CpMutY, generated by comparative modeling, exhibits a catalytic domain very similar to that of E. coli MutY. Taken together, these results indicate that the CpmutY encodes a functional protein homologous to MutY from E. coli and is involved in the prevention of mutations and the repair of oxidative DNA lesions.
We report the complete genome sequence of
Corynebacterium pseudotuberculosis
262, isolated from a bovine host.
C. pseudotuberculosis
is an etiological agent of diseases with medical and veterinary relevance. The genome contains 2,325,749 bp, 52.8% G+C content, 2,022 coding sequences (CDS), 50 pseudogenes, 48 tRNAs, and 12 rRNAs.
ABSTRACT: Staphylococcus aureus is a major cause of mastitis in ruminants. In ewe mastitis, symptoms range from subclinical to gangrenous mastitis. S. aureus factors or host-factors contributing to the different outcomes are not completely elucidated. In this study, experimental mastitis was induced on primiparous ewes using two S. aureus strains, isolated from gangrenous (strain O11) or subclinical (strain O46) mastitis. Strains induced drastically distinct clinical symptoms when tested in ewe and mice experimental mastitis. Notably, they reproduced mild (O46) or severe (O11) mastitis in ewes. Ewe sera were used to identify staphylococcal immunoreactive proteins commonly or differentially produced during infections of variable severity and to define core and accessory seroproteomes. Such SERological Proteome Analysis (SERPA) allowed the identification of 89 immunoreactive proteins, of which only 52 (58.4%) were previously identified as immunogenic proteins in other staphylococcal infections. Among the 89 proteins identified, 74 appear to constitute the core seroproteome. Among the 15 remaining proteins defining the accessory seroproteome, 12 were specific for strain O11, 3 were specific for O46. Distribution of one protein specific for each mastitis severity was investigated in ten other strains isolated from subclinical or clinical mastitis. We report here for the first time the identification of staphylococcal immunogenic proteins common or specific to S. aureus strains responsible for mild or severe mastitis. These findings open avenues in S. aureus mastitis studies as some of these proteins, expressed in vivo, are likely to account for the success of S. aureus as a pathogen of the ruminant mammary gland
In the current study, the applicability of the quantification of gamma interferon (IFN-γ) levels for the detection of animals infected with Corynebacterium pseudotuberculosis and for determining caseous lymphadenitis (CLA) clinical status was evaluated. Peripheral blood leukocytes were collected from CLA nonendemic areas animals, from CLA seropositive animals without clinical signs of the disease, and from seropositive animals presenting CLA clinical signs. The leukocytes were stimulated with C. pseudotuberculosis-secreted antigens that were concentrated by the three-phase partitioning technique. An ovine IFN-γ enzyme-linked immunosorbent assay was used to quantify IFN-γ production. Goats and sheep with CLA had higher IFN-γ levels than uninfected seronegative animals. Leukocytes from sheep with CLA chronic abscesses produced higher IFN-γ levels when compared with seropositive sheep without CLA clinical signs, but this difference was not significant in goats. The sensitivity of the assay was 55.8% and 56%, whereas the specificity was 100% and 93%, for goats and sheep, respectively. In conclusion, IFN-γ is a potential marker for the determination of CLA infection status in small ruminants; however, further research is needed to improve assay sensitivity.
Background
Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats.
Results
An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins.
Conclusions
Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.
Background: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. Results: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. Conclusions: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.
We conducted a seroepidemiological survey to determine the prevalence of caseous lymphadenitis (CLA) in goat herds in Minas Gerais state, Brazil. Serum samples were collected from goats (n=676) from 108 rural properties in 2001, covering most of the sub-regions of this ca. 586,500 square kilometer state. Antibodies against Corynebacterium pseudotuberculosis secreted proteins were detected by an indirect enzyme-linked immunosorbent assay (ELISA). Most of the animals (78.9%) tested positive for CLA; 98% of flocks presented at least one seropositive animal. Goats managed under an extensive production system had a significantly higher seroprevalence of CLA than those in intensive and semi-intensive operations. The age distribution of the animals in the flocks affected the prevalence of this disease; however, goat breed did not. We found seropositivity against C. pseudotuberculosis to be highly prevalent in these Brazilian goat herds; consequently, appropriate management practices for the control of CLA should be implemented.
The IRMa toolbox provides an interactive application to assist in the validation of Mascot search results. It allows automatic filtering of Mascot identification results as well as manual confirmation or rejection of individual PSM (a match between a fragmentation mass spectrum and a peptide). Dynamic grouping and coherence of information are maintained by the software in real time. Validated results can be exported under various forms, including an identification database (MSIdb). This allows biologists to compile search results from a whole study in a unique repository in order to provide a summarized view of their project. IRMa also features a fully automated version that can be used in a high-throughput pipeline. Given filter parameters, it can delete hits with no significant PSM, regroup hits identified by the same peptide(s) and export the result to the specified format without user intervention.
Availability:
http://biodev.extra.cea.fr/docs/irma (java 1.5 or higher needed).
Corynebacterium pseudotuberculosis continues to cause considerable economic losses in ovine and caprine herds worldwide, causing caseous lymphadenitis. Nevertheless, the immunology of this disease is relatively unknown. Novel antigens may provide vaccines that are more effective and improve diagnostic methods for better control of this disease. The available commercial vaccines are not able to fully protect susceptible animals, cannot be used in all host species and are not licensed for use in many countries. Recent studies on the genomics of C. pseudotuberculosis and on its molecular determinants of virulence should bring us new alternatives for more effective vaccine formulations.
The gene of Listeria monocytogenes that encodes a major extracellular protein (p60) was cloned in Escherichia coli. The gene was designated iap, as p60 was previously shown to represent an invasion-associated protein (M. Kuhn and W. Goebel, Infect. Immun. 57:55-61, 1989). The recombinant E. coli clone expressed p60, as shown by immunoblotting. The complete nucleotide sequence of iap was determined. The deduced amino acid sequence of p60 (484 amino acids) contains a putative N-terminal signal sequence of 27 amino acids and an extended repeat region consisting of 19 threonine-asparagine units. Hybridization with the entire iap gene revealed the presence of homologous sequences in most other Listeria species. In contrast, a 400-base-pair internal iap probe which contained the whole repeat region hybridized only with genomic DNA from L. monocytogenes. Four oligonucleotides previously described as specific probes for the detection of L. monocytogenes (A. R. Datta, B. A. Wentz, D. Shook, and M. W. Trucksess, Appl. Environ. Microbiol. 54:2933-2937, 1988) were shown to be part of the iap gene.
Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment. The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of Corynebacterium diphtheriae subsp. mitis and evaluated their adherence to human group B erythrocytes and HEp-2 cells. A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl(3). The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively. Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells. Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions. Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface. Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in D-mannose and D-galactose residues on the surfaces of both strains. Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of C. diphtheriae surface carbohydrate moieties. The significant changes in the degree of lectin binding specific for D-mannose, D-galactose and sialic acid residues may have an effect on binding of host cells. The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems. For C. diphtheriae, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes.
To estimate absolute protein contents in complex mixtures, we previously defined a protein abundance index (PAI) as the number of observed peptides divided by the number of observable peptides per protein (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome. Res. 12, 1231-1245). Here we report that PAI values obtained at different concentrations of serum albumin show a linear relationship with the logarithm of protein concentration in LC-MS/MS experiments. This was also the case for 46 proteins in a mouse whole cell lysate. For absolute quantitation, PAI was converted to exponentially modified PAI (emPAI), equal to 10PAI minus one, which is proportional to protein content in a protein mixture. For the 46 proteins in the whole lysate, the deviation percentages of the emPAI-based abundances from the actual values were within 63% on average, similar or better than determination of abundance by protein staining. emPAI was applied to comprehensive protein expression analysis and to a comparison study between gene and protein expression in a human cancer cell line, HCT116. The values of emPAI are easily calculated and add important quantitation information to proteomic experiments; therefore we suggest that they should be reported in large scale proteomic identification projects.
Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis (CLA), a common disease in small ruminant populations throughout the world. Once established, this disease is difficult to eradicate because drug therapy is not effective and because the clinical detection of infected animals is of limited efficiency. We reviewed the microbiological, biochemical and taxonomic features of C. pseudotuberculosis, general aspects of infection, the main virulence determinants and currently available commercial vaccines. We also examined the current molecular strategies for the study of virulence in C. pseudotuberculosis, including the latest research on the identification of novel virulence factors and genes, which will help us to better understand the biology of this microorganism. This knowledge may also contribute to the development of improved CLA vaccines, including subunit and DNA-based types, as well as to improve the diagnosis, treatment and control of this disease.
Bacterial pathogens operate by attacking crucial intracellular pathways in their hosts. These pathogens usually target more than one intracellular pathway and often interact at several points in each of these pathways to commandeer them fully. Although different bacterial pathogens tend to exploit similar pathway components in the host, the way in which they 'hijack' host cells usually differs. Knowledge of how pathogens target distinct cytoskeletal components and immune-cell signalling pathways is rapidly advancing, together with the understanding of bacterial virulence at a molecular level. Studying how these bacterial pathogens subvert host-cell pathways is central to understanding infectious disease.
The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein-protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall-targeting beta-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein-protein interaction as one way of regulating its activity.
Staphylococcus aureus is an opportunistic pathogen in dairy ruminants where it is found in healthy carriage and can be a major cause of mastitis. A better knowledge of the host-pathogen interactions is needed to tackle this serious animal health problem. This study aimed at identifying S. aureus proteins differentially expressed by S. aureus in nasal colonization versus mastitis. Serological proteome analysis (SERPA) was used to examine protein samples prepared from culture supernatants of S. aureus strains originally isolated from gangrenous mastitis and nasal carriage (O11) or subclinical mastitis (O46) and to compare patterns of immune-reactive proteins. These staphylococcal proteins were revealed by sera obtained from ewes suffering from S. aureus mastitis and by sera obtained from healthy nulliparous ewes (i.e. no lactation and no mastitis or other symptoms) that were nasally colonized by S. aureus. Altogether 49 staphylococcal immune-reactive proteins were identified in this study. Patterns of proteins revealed by sera from infected- or healthy carrier- animals were comparable and analysis singled out one immune-reactive protein, N-acetylmuramyl-L-alanine amidase, which was recognized by each of the 6 sera from infected animals, when tested individually, and not by the sera of healthy carriers. This is the first study that compares the S. aureus seroproteome in colonization versus mastitis context in ruminants. These results open avenues for studies aiming at a better understanding of the balance between infection and commensal lifestyle in this opportunistic pathogen and at new prevention strategies.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Caseous lymphadenitis (CLA) is a chronic disease affecting small ruminants caused by Corynebacterium pseudotuberculosis and is responsible for significant economic losses. Previous studies have reported that a set of C. pseudotuberculosis secreted proteins react with sera from infected goats. Mapping of the secretome will shed light on the pathogenesis of CLA. We have hereby identified six immunoreactive secreted proteins of C. pseudotuberculosis by 2D Western blotting using sera from goats with CLA and characterized them by mass spectrometry. The information retrieved will support studies leading to the development of efficient vaccines and diagnostic kits.
The flexibility of Commercial-Off-The-Shelf (COTS) SRAM based FPGAs is an attractive option for the design of artificial satellites, however, the functional verification of HDL-based designs is required and is of fundamental importance. Formal verification using model checking represents a system as formal model that are automatically generated by synthesis tools. On the other hand, the properties are represented by temporal logic expressions and are traditionally manually elaborated, which is susceptible to human errors increasing the costs and time of the verification. This work presents a new method for automatic property generation for formal verification of Hardware Description Language (HDL) based systems. The industrial case study is a communication subsystem of an artificial satellite, which was developed in cooperation with the Brazilian Institute of Space Research (INPE).
This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS(E)), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT(®). A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis.
Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis a chronic infectious disease affecting small ruminants. The 2D-DIGE technique was used to compare the exoproteomes of two C. pseudotuberculosis biovar ovis strains isolated from goat (strain 1002) and sheep (strain C231). Seventeen proteins differentially produced were identified here. Nine proteins appeared over-produced in the exoproteome of 1002 goat strain and 8 in that of C231 sheep strain. These proteins were related to various biological functions, such as the cell envelope, respiratory metabolism and proteolysis. This proteomic analysis revealed strain-specific exoproteins although each of the corresponding genes was found in both strain genomes. Such differential expression pattern may reflect inter-strain differences in adaptation to a specific host, in pathogenicity and or in antigenicity of this pathogenic bacterium.
Staphylococcus aureus is an opportunistic pathogen in dairy ruminants where it is found in healthy carriage and can be a major cause of mastitis. A better knowledge of the host-pathogen interactions is needed to tackle this serious animal health problem. This study aimed at identifying S. aureus proteins differentially expressed by S. aureus in nasal colonization versus mastitis. Serological proteome analysis (SERPA) was used to examine protein samples prepared from culture supernatants of S. aureus strains originally isolated from gangrenous mastitis and nasal carriage (O11) or subclinical mastitis (O46) and to compare patterns of immune-reactive proteins. These staphylococcal proteins were revealed by sera obtained from ewes suffering from S. aureus mastitis and by sera obtained from healthy nulliparous ewes (i.e. no lactation and no mastitis or other symptoms) that were nasally colonized by S. aureus. Altogether 49 staphylococcal immune-reactive proteins were identified in this study. Patterns of proteins revealed by sera from infected- or healthy carrier- animals were comparable and analysis singled out one immune-reactive protein, N-acetylmuramyl-L-alanine amidase, which was recognized by each of the 6 sera from infected animals, when tested individually, and not by the sera of healthy carriers. This is the first study that compares the S. aureus seroproteome in colonization versus mastitis context in ruminants. These results open avenues for studies aiming at a better understanding of the balance between infection and commensal lifestyle in this opportunistic pathogen and at new prevention strategies.
Staphylococcus aureus is a major agent of mastitis in ruminants worldwide. So far, efficient measures for its prophylaxis (including vaccination) have proven to be unsuccessful and there is a need for a better understanding of the host response to udder infection by S. aureus. Serological proteome analysis (SERPA) is a promising technique that can be used to identify S. aureus immuno-dominant determinants providing that bacterial culture conditions used to grow S. aureus strains for protein sample preparation mimic the context of mastitis. A S. aureus strain was used in experimental mastitis to generate sheep serum used to determine the best growth conditions for SERPA. Sera collected in the field from different ewes suffering from mastitis by S. aureus were used to confirm experimental observations. Three different culture media (BHI, whey and iron-depleted RPMI) were tested. The influence of aeration and growth phase on protein production was also evaluated by immuno-detection of protein samples prepared from cultures grown in different conditions and obtained from different culture fractions (supernatant, cell wall, and total lysates). Our results showed that culturing in iron-depleted RPMI with (secreted proteins, prepared from stationary phase) or without aeration (cell wall proteins, prepared from early stationary phase, and total proteins, prepared from exponential phase) is the condition that best mimics growth in vivo during mastitis and this in vitro growth condition is to be used henceforth in experiments involving SERPA.
Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, a disease that affects goats and sheep, and can cause severe economic losses. In this study, four different antigenic extracts were obtained from the attenuated strain T1, which was isolated in the state of Bahia (Brazil). Forty-four Canindé breed goats were divided in five groups, each receiving a different antigen solution and saline buffer as a control. The humoral response was monitored through the identification of specific IgG by indirect ELISA and Western Blotting, and the production of IFN-gamma was followed in order to observe the activation of cellular response. After twelve weeks of antigen inoculation, the animals were challenged with 2 x 10(5)CFU of a wild strain, also isolated in Bahia, and necropsy was performed on all animals twelve weeks afterwards. It was observed that the attenuated bacteria gave a protection of 33.3%, in addition to the weak humoral response elicited. Animals inoculated with secreted antigen associated with Freund's incomplete adjuvant and oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) showed a strong humoral response, but this inoculation could not prevent the spread of challenge bacteria in the majority of animals. These results demonstrate the immunogenic potential of the attenuated T1 strain in the development of a vaccine against caseous lymphadenitis in goats.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The permeability properties and the exclusion limits of the crystalline surface layers (S layers) of two selected strains of Bacillus stearothermophilus were investigated. Measurements were performed of passive solute uptake into the intracellular space of native or glutaraldehyde-treated sacculi. Native sacculi were prepared from whole cells by extracting the cytoplasmic membrane with Triton X-100 under conditions which preserved the integrity of the S layer and the peptidoglycan-containing layer. The permeability barrier was found to consist of three adjacent layers, namely, the S layer, the peptidoglycan-containing layer, and an incomplete S layer attached to the inner face of the peptidoglycan-containing layer. In glutaraldehyde-treated sacculi the peptidoglycan was digested after stabilizing the S-layer lattice by chemical cross-linking. The solutes selected for the uptake measurements were mannose, proteins, and dextrans of increasing molecular weights. The S layers of both strains allowed free passage for molecules with a molecular weight of up to 30,000 and showed sharp exclusion limits between molecular weights of 30,000 and 45,000, suggesting a limiting pore diameter of about 4.5 nm.
To evaluate the efficacy of a commercially available bacterin-toxoid vaccine for preventing Corynebacterium pseudotuberculosis-induced abscesses in sheep.
Prospective randomized controlled trial.
31 mixed-breed sheep seronegative for C pseudotuberculosis.
Sheep were randomly assigned to vaccinate (n = 20) or nonvaccinate (11; control) groups. Sheep in the vaccinate group received 2 doses of serial A or serial B bacterin-toxoid vaccine at 4-week intervals. Serologic testing was conducted after vaccination to document an antibody response to vaccination. All sheep were challenge inoculated with virulent C pseudotuberculosis organisms 32 weeks after the second vaccination. Twenty weeks after challenge inoculation, all sheep were examined for external and internal abscesses secondary to C pseudotuberculosis infection.
Vaccinated sheep developed an antibody response to both components of the vaccine, as measured by use of ELISA tests. After challenge inoculation, vaccinated sheep had significantly less external, internal, and total abscesses than control sheep.
Vaccination of sheep with a commercially available bacterin-toxoid against C pseudotuberculosis could substantially decrease the prevalence and number of abscesses that form secondary to C pseudotuberculosis infection.
Corynebacterium diphtheriae and Corynebacterium ulcerans use haemin and haemoglobin as essential sources of iron during growth in iron-depleted medium. C. diphtheriae and C. ulcerans mutants defective in haemin iron utilization were isolated and characterized. Four clones from a C. diphtheriae genomic library complemented several of the Corynebacteria haemin utilization mutants. The complementing plasmids shared an approximately 3 kb region, and the nucleotide sequence of one of the plasmids revealed five open reading frames that appeared to be organized in a single operon. The first three genes, which we have termed hmuT, hmuU and hmuV, shared striking homology with genes that are known to be required for haemin transport in Gram-negative bacteria and are proposed to be part of an ABC (ATP-binding cassette) transport system. The hmuT gene encodes a 37 kDa lipoprotein that is associated with the cytoplasmic membrane when expressed in Escherichi coli and C. diphtheriae. HmuT binds in vitro to haemin- and haemoglobin-agarose, suggesting that it is capable of binding both haemin and haemoglobin and may function as the haemin receptor in C. diphtheriae. This study reports the first genetic characterization of a transport system that is involved in the utilization of haemin and haemoglobin as iron sources by a Gram-positive bacterium.
By data mining in the sequence of the Corynebacterium glutamicum ATCC 13032 genome, six putative mycolyltransferase genes were identified that code for proteins with similarity to the N-terminal domain of the mycolic acid transferase PS1 of the related C. glutamicum strain ATCC 17965. The genes identified were designated cop1, cmt1, cmt2, cmt3, cmt4, and cmt5 ( cmt from corynebacterium mycolyl transferases). cop1 encodes a protein of 657 amino acids, which is larger than the proteins encoded by the cmt genes with 365, 341, 483, 483, and 411 amino acids. Using bioinformatics tools, it was shown that all six gene products are equipped with signal peptides and esterase domains. Proteome analyses of the cell envelope of C. glutamicum ATCC 13032 resulted in identification of the proteins Cop1, Cmt1, Cmt2, and Cmt4. All six mycolyltransferase genes were used for mutational analysis. cmt4 could not be mutated and is considered to be essential. cop1 was found to play an additional role in cell shape formation. A triple mutant carrying mutations in cop1, cmt1, and cmt2 aggregated when cultivated in MM1 liquid medium. This mutant was also no longer able to synthesize trehalose di coryno mycolate (TDCM). Since single and double mutants of the genes cop1, cmt1, and cmt2 could form TDCM, it is concluded that the three genes, cop1, cmt1, and cmt2, are involved in TDCM biosynthesis. The presence of the putative esterase domain makes it highly possible that cop1, cmt1, and cmt2 encode enzymes synthesizing TDCM from trehalose monocorynomycolate.
The corynemycolyltransferase proteins were identified from Corynebacterium glutamicum and Corynebacterium efficiens genomes using computational tools available in the public domain. Three-dimensional models were constructed for corynemycolyltransferases based on the crystal structures of related mycolyltransferases in Mycobacterium tuberculosis using the comparative modeling methods. The corynemycolyltransferases share overall an alpha/beta-fold characteristic of the mycolyltransferases despite low sequence identity (<20%) shared by some of the corynemycolyltransferases. However, a significant difference is observed in the region between amino acid residues Trp82-Trp97 and Ala222-Asn223 corresponding to mycolyltransferases. The specificity pockets defined by interactions with the trehalose substrate observed in the crystal structure complex of Ag85B mycolyltransferase (PDB code: 1F0P) suggests that trehalose may not bind some corynemycolyltransferases. This is due to critical mutations in corynemycolyltransferase binding subsites that lead to loss of equivalent side-chain interactions with trehalose and unfavorable steric interactions, particularly, in the case of cmytC gene and the protein corresponding to the gene identifier CE0356 with the equivalent Ala222-Asn223 "long insertion loop". Further, the fibronectin binding region (Phe58-Val69), in mycolyltransferases associated with mediating host-pathogen interactions in M. tuberculosis comprises amino acid residue mutations in the corresponding region in the soil bacterium--Corynebacterium corynemycolyltransferases, that suggest a different epitope and therefore possible lack of binding to fibronectin. The corynemycolyltransferase cmytA responsible for the cell shape formation and for maintaining the cell surface integrity is associated with a C-terminal domain that we have recently shown to comprise tandem amino acid sequence repeats that is likely to be associated with a regular secondary structural motif.
Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals.
Caseous lymphadenitis (CLA) of sheep, caused by Corynebacterium pseudotuberculosis, has been a significant disease in the majority of sheep-rearing regions for over a century. Because of the chronic and often sub-clinical nature of the infection, it has proved difficult to control and prevalence is high in many parts of the world, which in turn leads to significant economic losses for farmers. This review describes the important characteristics of C. pseudotuberculosis and examines the pathogenesis and epidemiology of the infection in sheep. The review also discusses the immune response to infection and describes the methods that have been developed to control CLA, with particular emphasis on the use of vaccination and serological testing.
Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis Effects of iron limitation on adherence and cell surface carbohydrates of Corynebacterium diphtheriae strains
- Le Maré Chal
- C Jan
- G Even
- S Pulido
- C Guibert
- J M Hernandez
- D Franç
- P Schrenzel
- J Demon
- D Meyer
- E Berkova
- N Thiery
- R Vautor
- Le Loir
- Y Moreira
- L O Andrade
- A F Vale
- M D Souza
- S M Jr
- R Asad
- L M Asad
Le Maré chal, C., Jan, G., Even, S., Pulido, C., Guibert, J.M., Hernandez, D., Franç, P., Schrenzel, J., Demon, D., Meyer, E., Berkova, N., Thiery, R., Vautor, E., Le Loir, Y., 2011. Staphylococcus aureus seroproteomes discriminate ruminant isolates causing mild or severe mastitis. Vet. Res. 42, 35. Moreira, L.O., Andrade, A.F., Vale, M.D., Souza, S.M., Hirata Jr., R., Asad, L.M., Asad, N.R., Monteiro-Leal, L.H., Previato, J.O., Mattos-Guaraldi, A.L., 2003. Effects of iron limitation on adherence and cell surface carbohydrates of Corynebacterium diphtheriae strains. Appl. Environ. Microbiol. 69, 5907–5913.