Quantitative Proteomic Analysis Reveals Changes in the Benchmark Corynebacterium pseudotuberculosis Biovar Equi Exoproteome after Passage in a Murine Host

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.Fil: Marques Da Silva, Wanderson. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasil. Institut National de la Recherche Agronomique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carvalho, Rodrigo D. De Oliveira. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Dorella, Fernanda A.. Universidade Federal de Minas Gerais; BrasilFil: Folador, Edson L.. Universidade Federal da Paraíba. Centro de Biotecnologia; BrasilFil: Souza, Gustavo H. M. F.. Waters Corporation; BrasilFil: Pimenta, Adriano M. C.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Figueiredo, Henrique C. P.. Universidade Federal de Minas Gerais; BrasilFil: Le Loir, Yves. Institut National de la Recherche Agronomique; FranciaFil: Silva, Artur. Universidade Federal do Pará; BrasilFil: Azevedo, Vasco. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasi

Extracellular Vesicles and Their Role in <em>Staphylococcus aureus</em> Resistance and Virulence

Staphylococcus aureus is a pathogen of great importance to clinical and veterinary medicine. Recently, there has been a growing interest in S. aureus extracellular vesicles (EVs) in the pathogenesis of this bacterium. Released by living cells into the extracellular milieu, EVs are membranous structures carrying macromolecules such as proteins, nucleic acids, and metabolites. These structures play several physiological roles and are, among others, considered a mechanism of intercellular communication within S. aureus populations but also in trans kingdom interactions. S. aureus EVs were shown to transport important bacterial survival and virulence factors, such as β-lactamases, toxins, and proteins associated with bacterial adherence to host cells, and to trigger the production of cytokines and promote tissue inflammation. In this chapter, we will review the main studies regarding S. aureus EVs, including their composition and roles in host-pathogen interactions, and the possible applications of EVs for vaccines and therapy development against staphylococcal infections

Heterologous expression of Brucella abortus GroEL heat-shock protein in Lactococcus lactis

BACKGROUND: Brucella abortus is a facultative intracellular pathogen that mainly infects cattle and humans. Current vaccines rely on live attenuated strains of B. abortus, which can revert to their pathogenic status and thus are not totally safe for use in humans. Therefore, the development of mucosal live vaccines using the food-grade lactic acid bacterium, Lactococcus lactis, as an antigen delivery vector, is an attractive alternative and a safer vaccination strategy against B. abortus. Here, we report the construction of L. lactis strains genetically modified to produce B. abortus GroEL heat-shock protein, a candidate antigen, in two cellular locations, intracellular or secreted. RESULTS: Only the secreted form of GroEL was stably produced in L. lactis, suggesting a detrimental effect of GroEL protein when intracellularly produced in this bacterium. Only trace amounts of mature GroEL were detected in the supernatant fraction of induced lactococcal cultures, and the GroEL precursor remained stacked in the cell fraction. Attempts to raise the secretion yields were made, but even when GroEL was fused to a synthetic propeptide, secretion of this antigen was not improved. CONCLUSION: We found that L. lactis is able to produce, and to secrete, a stable form of GroEL into the extracellular medium. Despite the low secretion efficiency of GroEL, which suggest that this antigen interacts with the cell envelope of L. lactis, secretion seems to be the best way to achieve both production and protein yields, regardless of cellular location. The L. lactis strain secreting GroEL has potential for in vivo immunization

Molecular characterisation of Staphylococcus aureus strains isolated from small and large ruminants reveals a host rather than tissue specificity.

International audienceStaphylococcus aureus is an important pathogen in domestic ruminants. The main objective of this study was to determine the similarity of epidemiologically unrelated S. aureus isolates from bovine, ovine, and caprine hosts regardless the locus of isolation (nares and udder). By pulsed-field gel electrophoresis, seven major pulsotypes were identified among 153 isolates recovered from 12 different regions of France as well as from Brazil, the USA and Belgium. Typing of the accessory gene regulator (agr) and capsular (cap) serotype was carried out on all the isolates and revealed the predominance of agr I and III and of cap8 regardless the ruminant host species. Screening for methicilin-resistant S. aureus (MRSA) was carried out by disk diffusion and revealed a prevalence of only 3.2% of MRSA among the strains tested. These results suggest the existence of a host rather than tissue specificity among S. aureus isolates colonising the ruminant species and suggest a limited transmission of those isolates between large (bovine) and small (ovine-caprine) ruminants. The agr class and cap types correlated with pulsotype clusters rather than with a specific host species. Antimicrobial resistance appears not to have contributed to the predominance of any given genotypes, and MRSA prevalence appears very low in ruminant isolates

Exfoliative Toxins of Staphylococcus aureus

Virulent strains of Staphylococcus aureus secrete exfoliative toxins (ETs) that cause the loss of cell‐cell adhesion in the superficial epidermis. S. aureus ETs are serine proteases, which exhibit exquisite substrate specificity, and their mechanisms of action are extremely complex. To date, four different serotypes of ETs have been identified and three of them (ETA, ETB and ETD) are associated with toxin‐mediated staphylococcal syndromes related to human infections leading to diseases of medical and veterinary importance

Metagenomic Approaches for Investigating the Role of the Microbiome in Gut Health and Inflammatory Diseases

The human gut microbiota makes fundamental contributions to host metabolism and immune system. Therefore, perturbations in its composition, a process known as dysbiosis, have an important role in the development of several chronicle diseases, mainly intestinal inflammatory disorders. Culture-independent molecular methods are allowing scientific community to uncover substantive findings, thus giving a more detailed description of the human intestinal microbiota. This chapter presents a review on current metagenomic approaches, based on next-generation sequencing technologies, for investigating bacterial taxonomic classification and predictive function associated with the human gut in health and disease. In this context, we describe recent studies that have been trying to elucidate important alterations in microbiome composition across individuals according to delivery mode, aging, diet and medication that might be linked to susceptibility to immune-mediated diseases. A description of the main bacterial taxa and genes acting in dysbiosis during inflammation, focusing on chronic inflammatory bowel diseases and colorectal cancer, is also explored in this chapter

Development of serological proteome analysis of mastitis by Staphylococcus aureus in ewes.

International audienceStaphylococcus 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

Molecular Basis of Virulence in Staphylococcus aureus Mastitis

S. aureus is one of the main pathogens involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable, ranging from subclinical to gangrenous mastitis. This work represents an in-depth characterization of S. aureus mastitis isolates to identify bacterial factors involved in severity of mastitis infection

Milk Microbiota: What Are We Exactly Talking About?

The development of powerful sequencing techniques has allowed, albeit with some biases, the identification and inventory of complex microbial communities that inhabit different body sites or body fluids, some of which were previously considered sterile. Notably, milk is now considered to host a complex microbial community with great diversity. Milk microbiota is now well documented in various hosts. Based on the growing literature on this microbial community, we address here the question of what milk microbiota is. We summarize and compare the microbial composition of milk in humans and in ruminants and address the existence of a putative core milk microbiota. We discuss the factors that contribute to shape the milk microbiota or affect its composition, including host and environmental factors as well as methodological factors, such as the sampling and sequencing techniques, which likely introduce distortion in milk microbiota analysis. The roles that milk microbiota are likely to play in the mother and offspring physiology and health are presented together with recent data on the hypothesis of an enteromammary pathway. At last, this fascinating field raises a series of questions, which are listed and commented here and which open new research avenues