The impact of Elaeagnus angustifolia root exudates on Parafrankia soli NRRL B-16219 exoproteome.

Root exudates from host plant species are known to play a critical role in the establishment and maintenance of symbiotic relationships with soil bacteria. In this study, we investigated the impact of root exudates from compatible host plant species; Elaeagnus angustifolia on the exoproteome of Parafrankia soli strain NRRL B-16219. A total of 565 proteins were evidenced as differentially abundant, with 32 upregulated and 533 downregulated in presence of the plant exudates. Analysis of the function of these proteins suggests that the bacterial strain is undergoing a complex metabolic reprogramming towards a new developmental phase elicited in presence of host plant root exudates. The upregulation of Type II/IV secretion system proteins among the differentially expressed proteins indicates their possible role in infecting the host plant, as shown for some rhizobia. Additionally, EF-Tu, proteins upregulated in this study, may function as an effector for the T4SSs and trigger plant defense responses. These findings suggest that Parafrankia soli may use EF-Tu to infect the actinorhizal host plant and pave the way for further investigations of the molecular mechanisms underlying the establishment of symbiotic relationships.

Constitutive proteins of lumpy skin disease virion assessed by next-generation proteomics.

IMPORTANCE: Lumpy skin disease virus (LSDV) is the causative agent of an economically important cattle disease which is notifiable to the World Organisation for Animal Health. Over the past decades, the disease has spread at an alarming rate throughout the African continent, the Middle East, Eastern Europe, the Russian Federation, and many Asian countries. While multiple LDSV whole genomes have made further genetic comparative analyses possible, knowledge on the protein composition of the LSDV particle remains lacking. This study provides for the first time a comprehensive proteomic analysis of an infectious LSDV particle, prompting new efforts toward further proteomic LSDV strain characterization. Furthermore, this first incursion within the capripoxvirus proteome represents one of very few proteomic studies beyond the sole Orthopoxvirus genus, for which most of the proteomics studies have been performed. Providing new information about other chordopoxviruses may contribute to shedding new light on protein composition within the Poxviridae family.

Human gut microbiome and metabolite dynamics under simulated microgravity.

The Artificial Gravity Bed Rest - European Space Agency (AGBRESA) study was the first joint bed rest study by ESA, DLR, and NASA that examined the effect of simulated weightlessness on the human body and assessed the potential benefits of artificial gravity as a countermeasure in an analog of long-duration spaceflight. In this study, we investigated the impact of simulated microgravity on the gut microbiome of 12 participants during a 60-day head-down tilt bed rest at the :envihab facilities. Over 60 days of simulated microgravity resulted in a mild change in the gut microbiome, with distinct microbial patterns and pathway expression in the feces of the countermeasure group compared to the microgravity simulation-only group. Additionally, we found that the countermeasure protocols selectively increased the abundance of beneficial short-chain fatty acids in the gut, such as acetate, butyrate, and propionate. Some physiological signatures also included the modulation of taxa reported to be either beneficial or opportunistic, indicating a mild adaptation in the microbiome network balance. Our results suggest that monitoring the gut microbial catalog along with pathway clustering and metabolite profiling is an informative synergistic strategy to determine health disturbances and the outcome of countermeasure protocols for future space missions.

The future of spaceflight will involve missions beyond the International Space Station or the Moon and astronaut's health will be challenged by a harsh space environment for longer periods. In the last decade, the intestine has gained importance in dictating overall physiology and we explore it as an additional indicator of health during our ground-based bed rest study simulating microgravity for 60 days. Through the analysis of fecal proteins, we compile the catalog of microbes colonizing the gut of the 12 participants along with the implicated biological activity of the proteins and another 9 lipid analytes. We found specific microbes associated with recovery or healthy status in our subjects to be increased during spaceflight countermeasure conditions and inverse observations in subjects subjected to perilous spaceflight simulation. Our approach improves the functional characterization of the gut by the use of noninvasive methodology correlating the microbial composition of human stool samples with physiological status.

Discriminating Susceptibility of Xanthine Oxidoreductase Family to Metals.

The xanthine oxidoreductase (XOR) family are metal-containing enzymes that use the molybdenum cofactor (Moco), 2Fe-2S clusters, and flavin adenine dinucleotide (FAD) for their catalytic activity. This large molybdoenzyme family includes xanthine, aldehyde, and CO dehydrogenases. XORs are widely distributed from bacteria to humans due to their key roles in the catabolism of purines, aldehydes, drugs, and xenobiotics, as well as interconversions between CO and CO2. Assessing the effect of excess metals on the Rubrivivax gelatinosus bacterium, we found that exposure to copper (Cu) or cadmium (Cd) caused a dramatic decrease in the activity of a high-molecular-weight soluble complex exhibiting nitroblue tetrazolium reductase activity. Mass spectrometry and genetic analyses showed that the complex corresponds to a putative CO dehydrogenase (pCOD). Using mutants that accumulate either Cu+ or Cd2+ in the cytoplasm, we show that Cu+ or Cd2+ is a potent inhibitor of XORs (pCOD and the xanthine dehydrogenase [XDH]) in vivo. This is the first in vivo demonstration that Cu+ affects Moco-containing enzymes. The specific inhibitory effect of these compounds on the XOR activity is further supported in vitro by direct addition of competing metals to protein extracts. Moreover, emphasis is given on the inhibitory effect of Cu on bovine XOR, showing that the XOR family could be a common target of Cu. Given the conservation of XOR structure and function across the tree of life, we anticipate that our findings could be transferable to other XORs and organisms. IMPORTANCE The high toxicity of Cu, Cd, Pb, As, and other metals arises from their ability to cross membranes and target metalloenzymes in the cytoplasm. Identifying these targets provides insights into the toxicity mechanisms. The vulnerability of metalloenzymes arises from the accessibility of their cofactors to ions. Accordingly, many enzymes whose cofactors are solvent exposed are likely to be targets of competing metals. Here, we describe for the first time, with in vivo and in vitro experiments, a direct effect of excess Cu on the xanthine oxidoreductase family (XOR/XDH/pCOD). We show that toxic metal affects these Moco enzymes, and we suggest that access to the Moco center by Cu ions could explain the Cu inhibition of XORs in living organisms. Human XOR activity is associated with hyperuricemia, xanthinuria, gout arthritis, and other diseases. Our findings in vivo highlight XOR as a Cu target and thus support the potential use of Cu in metal-based therapeutics against these diseases.

Frankia alni Carbonic Anhydrase Regulates Cytoplasmic pH of Nitrogen-Fixing Vesicles.

A phyloprofile of Frankia genomes was carried out to identify those genes present in symbiotic strains of clusters 1, 1c, 2 and 3 and absent in non-infective strains of cluster 4. At a threshold of 50% AA identity, 108 genes were retrieved. Among these were known symbiosis-associated genes such as nif (nitrogenase), and genes which are not know as symbiosis-associated genes such as can (carbonic anhydrase, CAN). The role of CAN, which supplies carbonate ions necessary for carboxylases and acidifies the cytoplasm, was thus analyzed by staining cells with pH-responsive dyes; assaying for CO2 levels in N-fixing propionate-fed cells (that require a propionate-CoA carboxylase to yield succinate-CoA), fumarate-fed cells and N-replete propionate-fed cells; conducting proteomics on N-fixing fumarate and propionate-fed cells and direct measurement of organic acids in nodules and in roots. The interiors of both in vitro and nodular vesicles were found to be at a lower pH than that of hyphae. CO2 levels in N2-fixing propionate-fed cultures were lower than in N-replete ones. Proteomics of propionate-fed cells showed carbamoyl-phosphate synthase (CPS) as the most overabundant enzyme relative to fumarate-fed cells. CPS combines carbonate and ammonium in the first step of the citrulline pathway, something which would help manage acidity and NH4+. Nodules were found to have sizeable amounts of pyruvate and acetate in addition to TCA intermediates. This points to CAN reducing the vesicles' pH to prevent the escape of NH3 and to control ammonium assimilation by GS and GOGAT, two enzymes that work in different ways in vesicles and hyphae. Genes with related functions (carboxylases, biotin operon and citrulline-aspartate ligase) appear to have undergone decay in non-symbiotic lineages.

An ancient coronavirus from individuals in France, circa 16th century.

OBJECTIVES: At the time when the COVID-19 pandemic was responsible for more than six million deaths worldwide, the antiquity of coronaviruses remains undefined. We investigated individuals buried during the 16th century in France for the direct and paleoserological diagnosis of the coronavirus. METHODS: The 2011-2012 excavation of Abbey Saint-Pierre in Baume-Les-Messieurs, France uncovered 12 skeletons of individuals from the 13th to the 18th century. The total proteins extracted from dental pulps were subjected to microbial paleoserology, targeting SARS-CoV-2, human-associated coronavirus (HCoV)-229E, and OC43 antigens and for coronavirus peptide research using metaproteomics, in parallel to negative controls. RESULTS: Three peptide sequences totaling 36 amino acids indicative of a coronavirus were retrieved from the dental pulp remains collected from two individuals buried circa 16th century, in whom paleoserology confirmed a specific immunological response against modern-day SARS-CoV-2 and HCoV-229E. CONCLUSION: We provide serological and proteomic evidence for a betacoronavirus with no modern correspondent, infecting populations in the 16th century, extending the antiquity of coronaviruses by more than three centuries. Historical, archaeozoological, and paleoproteomic data suggested close contacts between these two individuals and domestic swine, cattle, and poultry, suggesting an ancient zoonotic coronavirus. Coronaviruses have been undesirable companions of populations long before the ongoing coronavirus disease 2019 outbreak emerged.

Mass spectrometry detection of monkeypox virus: Comprehensive coverage for ranking the most responsive peptide markers.

The recent and sudden outbreak of monkeypox in numerous non-endemic countries requires expanding its surveillance immediately and understanding its origin and spread. As learned from the COVID-19 pandemic, appropriate detection techniques are crucial to achieving such a goal. Mass spectrometry has the advantages of a rapid response, low analytical interferences, better precision, and easier multiplexing to detect various pathogens and their variants. In this proteomic dataset, we report experimental data on the proteome of the monkeypox virus (MPXV) recorded by state-of-the-art shotgun proteomics, including data-dependent and data-independent acquisition for comprehensive coverage. We highlighted 152 viral proteins, corresponding to an overall proteome coverage of 79.5 %. Among the 1371 viral peptides detected, 35 peptides with the most intense signals in mass spectrometry were selected, representing a subset of 13 viral proteins. Their relevance as potential candidate markers for virus detection by targeted mass spectrometry is discussed. This report should assist the rapid development of mass spectrometry-based tests to detect a pathogen of increasing concern.

Correction: Pujic et al. The Proteogenome of Symbiotic Frankia alni in Alnus glutinosa Nodules. Microorganisms 2022, 10, 651.

The authors wish to make the following corrections to this paper [...].

Taxonomical and functional changes in COVID-19 faecal microbiome could be related to SARS-CoV-2 faecal load.

Since the beginning of the pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) the gastrointestinal (GI) tract has emerged as an important organ influencing the propensity to and potentially the severity of the related COVID-19 disease. However, the contribution of the SARS-CoV-2 intestinal infection on COVID-19 pathogenesis remains to be clarified. In this exploratory study, we highlighted a possible link between alterations in the composition of the gut microbiota and the levels of SARS-CoV-2 RNA in the gastrointestinal tract, which could be more important than the presence of SARS-CoV-2 in the respiratory tract, COVID-19 severity and GI symptoms. As established by metaproteomics, altered molecular functions in the microbiota profiles of high SARS-CoV-2 RNA level faeces highlight mechanisms such as inflammation-induced enterocyte damage, increased intestinal permeability and activation of immune response that may contribute to vicious cycles. Uncovering the role of this gut microbiota dysbiosis could drive the investigation of alternative therapeutic strategies to favour the clearance of the virus and potentially mitigate the effect of the SARS-CoV-2 infection.

Identification and Characterization of Marine Microorganisms by Tandem Mass Spectrometry Proteotyping.

The vast majority of marine microorganisms and their functions are yet to be explored. The considerable diversity they encompass is an endless source of knowledge and wealth that can be valued on an industrial scale, emphasizing the need to develop rapid and efficient identification and characterization techniques. In this study, we identified 26 microbial isolates from coastal water of the NW Mediterranean Sea, using phylopeptidomics, a cutting-edge tandem mass spectrometry proteotyping technique. Taxonomical identification at the species level was successfully conducted for all isolates. The presence of strains belonging to the newly described Balneolaeota phylum, yet uncharacterized at the proteomics scale, was noted. The very first proteomics-based investigation of a representative of the Balneolaeota phylum, Balneola vulgaris, is proposed, demonstrating the use of our proteotyping workflow for the rapid identification and in-depth molecular characterization, in a single MS/MS analytical run. Tandem mass spectrometry proteotyping is a valuable asset for culturomic programs as the methodology is able to quickly classify the most atypical isolates.

The Proteogenome of Symbiotic Frankia alni in Alnus glutinosa Nodules.

Omics are the most promising approaches to investigate microbes for which no genetic tools exist such as the nitrogen-fixing symbiotic Frankia. A proteogenomic analysis of symbiotic Frankia alni was done by comparing those proteins more and less abundant in Alnus glutinosa nodules relative to N2-fixing pure cultures with propionate as the carbon source. There were 250 proteins that were significantly overabundant in nodules at a fold change (FC) ≥ 2 threshold, and 1429 with the same characteristics in in vitro nitrogen-fixing pure culture. Nitrogenase, SuF (Fe-Su biogenesis) and hopanoid lipids synthesis determinants were the most overabundant proteins in symbiosis. Nitrogenase was found to constitute 3% of all Frankia proteins in nodules. Sod (superoxide dismutase) was overabundant, indicating a continued oxidative stress, while Kats (catalase) were not. Several transporters were overabundant including one for dicarboxylates and one for branched amino acids. The present results confirm the centrality of nitrogenase in the actinorhizal symbiosis.

Critical Assessment of MetaProteome Investigation (CAMPI): a multi-laboratory comparison of established workflows.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

Increasing the power of interpretation for soil metaproteomics data.

BACKGROUND: Soil and sediment microorganisms are highly phylogenetically diverse but are currently largely under-represented in public molecular databases. Their functional characterization by means of metaproteomics is usually performed using metagenomic sequences acquired for the same sample. However, such hugely diverse metagenomic datasets are difficult to assemble; in parallel, theoretical proteomes from isolates available in generic databases are of high quality. Both these factors advocate for the use of theoretical proteomes in metaproteomics interpretation pipelines. Here, we examined a number of database construction strategies with a view to increasing the outputs of metaproteomics studies performed on soil samples. RESULTS: The number of peptide-spectrum matches was found to be of comparable magnitude when using public or sample-specific metagenomics-derived databases. However, numbers were significantly increased when a combination of both types of information was used in a two-step cascaded search. Our data also indicate that the functional annotation of the metaproteomics dataset can be maximized by using a combination of both types of databases. CONCLUSIONS: A two-step strategy combining sample-specific metagenome database and public databases such as the non-redundant NCBI database and a massive soil gene catalog allows maximizing the metaproteomic interpretation both in terms of ratio of assigned spectra and retrieval of function-derived information. Video abstract.

Recombinant myelin oligodendrocyte glycoprotein quality modifies evolution of experimental autoimmune encephalitis in macaques.

Experimental autoimmune encephalitis (EAE) is a well-recognized model for the study of human acquired demyelinating diseases (ADD), a group of inflammatory disorders of the central nervous system (CNS) characterized by inflammation, myelin loss, and neurological impairment of variable severity. In rodents, EAE is typically induced by active immunization with a combination of myelin-derived antigen and a strong adjuvant as complete Freund's adjuvant (CFA), containing components of the mycobacterial wall, while myelin antigen alone or associated with other bacterial components, as lipopolysaccharides (LPS), often fails to induce EAE. In contrast to this, EAE can be efficiently induced in non-human primates by immunization with the recombinant human myelin oligodendrocyte glycoprotein (rhMOG), produced in Escherichia coli (E. coli), purified and formulated with incomplete Freund's adjuvant (IFA), which lacks bacterial elements. Here, we provide evidence indicating how trace amounts of bacterial contaminants within rhMOG may influence the course and severity of EAE in the cynomolgus macaque immunized with rhMOG/IFA. The residual amount of E. coli contaminants, as detected with mass spectrometry within rhMOG protein stocks, were found to significantly modulate the severity of clinical, radiological, and histologic hallmarks of EAE in macaques. Indeed, animals receiving the purest rhMOG showed milder disease severity, increased numbers of remissions, and reduced brain damage. Histologically, these animals presented a wider diversity of lesion types, including changes in normal-appearing white matter and prephagocytic lesions. Non-human primates EAE model with milder histologic lesions reflect more accurately ADD and permits to study of the pathogenesis of disease initiation and progression.

Ionizing-radiation-resistant Kocuria rhizophila PT10 isolated from the Tunisian Sahara xerophyte Panicum turgidum: Polyphasic characterization and proteogenomic arsenal.

A new strain belonging to the genus Kocuria, designed PT10, was isolated from irradiated roots of the xerophyte Panicum turgidum. Isolate PT10 is a Gram-positive, coccoid, aerobic and ionizing-radiation (IR)-resistant actinobacterium. PT10 has shown an ability to survive under extreme conditions, such as gamma irradiation, desiccation and high concentration of hydrogen peroxide. Phenotypic, chemotaxonomic and comparative genome analyses support the assignment of strain PT10 (LMG 31102 = DSM 108617) as Kocuria rhizophila. The complete genome sequence of PT10 consists of one chromosome (2,656,287 bps), with a 70.7% G + C content and comprises 2481 protein-coding sequences. A total of 1487 proteins were identified by LC-MS/MS profiling. In silico analyses revealed that the proteome of the oxidation-tolerant PT10 possesses several features explaining its IR-resistant phenotype and many adaptive pathways implicated in response to environmental pressures - desiccation, cold, reactive oxygen species and other stressors.

Shotgun proteomics analysis of SARS-CoV-2-infected cells and how it can optimize whole viral particle antigen production for vaccines.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and is continuing to spread rapidly around the globe. No effective vaccine is currently available to prevent COVID-19, and intense efforts are being invested worldwide into vaccine development. In this context, all technology platforms must overcome several challenges resulting from the use of an incompletely characterized new virus. These include finding the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles. Here, we describe a shotgun tandem mass spectrometry workflow, the data produced can be used to guide optimization of the conditions for viral amplification. In parallel, we analysed the changes occurring in the host cell proteome following SARS-CoV-2 infection to glean information on the biological processes modulated by the virus that could be further explored as potential drug targets to deal with the pandemic.

Proteotyping SARS-CoV-2 Virus from Nasopharyngeal Swabs: A Proof-of-Concept Focused on a 3 Min Mass Spectrometry Window.

Rapid but yet sensitive, specific, and high-throughput detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is key to diagnose infected people and to better control the spread of the virus. Alternative methodologies to PCR and immunodiagnostics that would not require specific reagents are worthy to investigate not only for fighting the COVID-19 pandemic but also to detect other emergent pathogenic threats. Here, we propose the use of tandem mass spectrometry to detect SARS-CoV-2 marker peptides in nasopharyngeal swabs. We documented that the signal from the microbiota present in such samples is low and can be overlooked when interpreting shotgun proteomic data acquired on a restricted window of the peptidome landscape. In this proof-of-concept study, simili nasopharyngeal swabs spiked with different quantities of purified SARS-CoV-2 viral material were used to develop a nanoLC-MS/MS acquisition method, which was then successfully applied on COVID-19 clinical samples. We argue that peptides ADETQALPQR and GFYAQGSR from the nucleocapsid protein are of utmost interest as their signal is intense and their elution can be obtained within a 3 min window in the tested conditions. These results pave the way for the development of time-efficient viral diagnostic tests based on mass spectrometry.

Estimating relative biomasses of organisms in microbiota using "phylopeptidomics".

BACKGROUND: There is an important need for the development of fast and robust methods to quantify the diversity and temporal dynamics of microbial communities in complex environmental samples. Because tandem mass spectrometry allows rapid inspection of protein content, metaproteomics is increasingly used for the phenotypic analysis of microbiota across many fields, including biotechnology, environmental ecology, and medicine. RESULTS: Here, we present a new method for identifying the biomass contribution of any given organism based on a signature describing the number of peptide sequences shared with all other organisms, calculated by mathematical modeling and phylogenetic relationships. This so-called "phylopeptidomics" principle allows for the calculation of the relative ratios of peptide-specified taxa by the linear combination of such signatures applied to an experimental metaproteomic dataset. We illustrate its efficiency using artificial mixtures of two closely related pathogens of clinical interest, and with more complex microbiota models. CONCLUSIONS: This approach paves the way to a new vision of taxonomic changes and accurate label-free quantitative metaproteomics for fine-tuned functional characterization. Video abstract.

Post-production modifications of murine mesenchymal stem cell (mMSC) derived extracellular vesicles (EVs) and impact on their cellular interaction.

In regards to their key role in intercellular communication, extracellular vesicles (EVs) have a strong potential as bio-inspired drug delivery systems (DDS). With the aim of circumventing some of their well-known issues (production yield, drug loading yield, pharmacokinetics), we specifically focused on switching the biological vision of these entities to a more physico-chemical one, and to consider and fine-tune EVs as synthetic vectors. To allow a rational use, we first performed a full physico-chemical (size, concentration, surface charge, cryoTEM), biochemical (western blot, proteomics, lipidomics, transcriptomics) and biological (cell internalisation) characterisation of murine mesenchymal stem cell (mMSC)-derived EVs. A stability study based on evaluating the colloidal behaviour of obtained vesicles was performed in order to identify optimal storage conditions. We evidenced the interest of using EVs instead of liposomes, in regards to target cell internalisation efficiency. EVs were shown to be internalised through a caveolae and cholesterol-dependent pathway, following a different endocytic route than liposomes. Then, we characterised the effect of physical methods scarcely investigated with EVs (extrusion through 50 nm membranes, freeze-drying, sonication) on EV size, concentration, structure and cell internalisation properties. Our extensive characterisation of the effect of these physical processes highlights their promise as loading methods to make EVs efficient delivery vehicles.

Proteases as Secreted Exoproteins in Mycoplasmas from Ruminant Lungs and Their Impact on Surface-Exposed Proteins.

Many mycoplasma species are isolated from the ruminant lungs as either saprophytes or true pathogens. These wall-less bacteria possess a minimal genome and reduced metabolic capabilities. Accordingly, they rely heavily on their hosts for the supply of essential metabolites and, notably, peptides. Seven of 13 ruminant lung-associated Mycoplasma (sub)species were shown to possess caseinolytic activity when grown in rich media and assessed with a quantitative fluorescence test. For some species, this activity was detected in spent medium, an indication that proteases were secreted outside the mycoplasma cells. To identify these proteases, we incubated concentrated washed cell pellets in a defined medium and analyzed the supernatants by tandem mass spectrometry. Secreted-protease activity was detected mostly in the species belonging to the Mycoplasma mycoides cluster (MMC) and, to a lesser extent, in Mycoplasma bovirhinis Analyzing a Mycoplasma mycoides subsp. capri strain, chosen as a model, we identified 35 expressed proteases among 55 predicted coding genes, of which 5 were preferentially found in the supernatant. Serine protease S41, acquired by horizontal gene transfer, was responsible for the caseinolytic activity, as demonstrated by zymography and mutant analysis. In an M. capricolum mutant, inactivation of the S41 protease resulted in marked modification of the expression or secretion of 17 predicted surface-exposed proteins. This is an indication that the S41 protease could have a role in posttranslational cleavage of surface-exposed proteins and ectodomain shedding, whose physiological impacts still need to be explored.IMPORTANCE Few studies pertaining to proteases in ruminant mycoplasmas have been reported. Here, we focus on proteases that are secreted outside the mycoplasma cell using a mass spectrometry approach. The most striking result is the identification, within the Mycoplasma mycoides cluster, of a serine protease that is exclusively detected outside the mycoplasma cells and is responsible for casein digestion. This protease may also be involved in the posttranslational processing of surface proteins, as suggested by analysis of mutants showing a marked reduction in the secretion of extracellular proteins. By analogy, this finding may help increase understanding of the mechanisms underlying this ectodomain shedding in other mycoplasma species. The gene encoding this protease is likely to have been acquired via horizontal gene transfer from Gram-positive bacteria and sortase-associated surface proteases. Whether this protease and the associated ectodomain shedding are related to virulence has yet to be ascertained.