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In this chapter, detailed procedures for stable isotope labeling with amino acids in cell culture, SILAC labeling of yeast auxotroph, optimization and evaluation of phosphopeptide enrichment, and sample preparation and analysis by high-resolution LC-MS/M, identification of phosphosites, and quantification methods are described.We report methods for the application of double SILAC to yeast using a combination of labeled lysine and labeled arginine.The combination of SILAC-based quantitation with phosphopeptides enrichment by TiOsub2/subin a batch that enables measurement of protein posttranslational modifications is a powerful application to analyze the global phosphoproteome for studies in signaling pathways.

The transition between yeast and hyphal morphologies plays a crucial role in the pathogenicity of Candida albicans. Recent studies have pointed out the great relevance of extracellular vesicles (EVs) secreted by microorganisms in a wide variety of biological processes including interaction with the host. Therefore, the main objective of this work was to compare the EVs secreted by yeast and hyphal forms to shed light on C. albicans-host interaction. EVs were obtained by ultracentrifugation of the culture medium supernatant and analysed by mass spectrometry. They were characterized by transmission electronic microscopy (TEM) and dynamic light scattering (DLS). DLS and TEM analysis showed that yeast EVs were significantly bigger than hyphal EVs, being most of them in the range between 400 to 500nm while hyphal EVs were ranged mostly around 100-200nm. Proteomic analysis showed greater protein diversity in hyphal EVs when compared to yeast EVs (up to 1700 different proteins identified versus 300), although less amount of total protein was obtained. Gene Ontology (GO) analysis showed that yeast EVs were enriched in surface proteins while hyphal EVs, although containing also most of these surface proteins, were also significantly and exclusively enriched in proteins involved in protein metabolism (ribosomal proteins, many aminoacid-pathway enzymes and proteasome) and cellular transport. The differences between YEVs and HEVs also prompted a different immune host response, as tested with macrophage cell cultures and human sera from patients with invasive candidiasis. All these differences point out a possible different biogenesis and roles of EVs secreted by both morphologies.

Fungal infections are a global health problem. Of them, those produced by Candida albicans are the most important, with a reduced arsenal of antifungals and an increasing problem of antifungal resistance. Thus, the discovery of new antifungal targets and drugs remains interesting. Metformin is a biguanide administered as a first-line treatment for Type II Diabetes Mellitus and it has recently been published its anti-Candida action, especially against C. glabrata, and its synergistic effect with other antifungals. Our studies of the effect of metformin on C. albicans have revealed an inhibition of growth, filamentation and other phenotypes important for virulence. Although metformin has been described as an AMPK agonist, its mechanism of action is partly unknown. To deepen into the anti-Candida mechanism of action, we have addressed the differential proteomic study. A set-up of the conditions for the proteomic study has been carried out, fixing a concentration of 50mM of metformin, 6 h of treatment at 37°C in RPMI medium and with 60 rpm of agitation. The proteomic study using the Labelfree technique and 4 biological replicas, allowed the identification and quantification of a total of 1899 proteins, 206 of them presenting differences in abundance due to metformin exposure. Of these, 127 increased and 79 decreased due to the action of the drug. The most relevant functions of these proteins are related to antifungal response, filamentation, biofilm formation and metabolism, being 9 essential proteins for the microorganism that could be new antifungal targets.

Candida albicans is a commensal fungus that causes systemic infections in immunosuppressed patients. In order to deal with the changing environment during commensalism or infection, C. albicans must reprogram its proteome. Characterizing the stress-induced changes in the proteome that C. albicans uses to survive should be very useful in the development of new antifungal drugs. We studied the C. albicans global proteome after exposure to hydrogen peroxide (H2O2) and acetic acid (AA), using a DIA-MS strategy. More than 2000 C. albicans proteins were quantified using an ion library previously constructed using DDA-MS. C. albicans responded to treatment with H2O2 with an increase in the abundance of many proteins involved in the oxidative stress response, protein folding and proteasome-dependent catabolism, which led to an increased proteasome activity. The data revealed a previously unknown key role for Prn1, a protein similar to pirins, in the oxidative stress response. Treatment with AA resulted in a general decrease in the abundance of proteins involved in amino acid biosynthesis, protein folding, and rRNA processing. Almost all proteasome proteins declined, as did proteasome activity. Apoptosis was observed after treatment with H2O2, but not AA. A targeted proteomic study of 32 proteins related to apoptosis in yeast supported the results found by DIA-MS and allowed the creation of an efficient method to quantify relevant proteins after treatment with stressors (H2O2, AA, and amphotericin B). This approach also uncovered a main role for Oye32, an oxidoreductase, suggesting this protein as a possible apoptotic marker common to many stressors.

Human gut microbiota can be studied through the characterization of microorganisms present in feces. Metaproteomics has arisen as a good approach to investigate this vast community. However, the processing of fecal samples in order to obtain the largest number of proteins from gut microbiota to be subsequently analyzed by means of metaproteomics is a challenge. Here we describe a protocol to approach this task. It includes two main steps: the first step of humectation and dispersion of the feces, followed by the separation of microorganisms from other fecal components such as roughage and food debris, and the second step in which microbial cells are broken up and microbiota proteins recovered for MS analysis. Detailed procedures for sample preparation, protein extraction, trypsin digestion, and mass spectrometry analysis for gut microbiota samples are provided.

In fungi, metals are associated with the expression of virulence factors. However, it is unclear whether the uptake of metals affects their pathogenicity. This study aimed to evaluate the effect of iron/copper in modulating pathogenicity and proteomic response in two clinical isolates of C. neoformans with high and low pathogenicity. Methods: In both isolates, the effect of 50 µM iron and 500 µM copper on pathogenicity, capsule induction, and melanin production was evaluated. We then performed a quantitative proteomic analysis of cytoplasmic extracts exposed to that combination. Finally, the effect on pathogenicity by iron and copper was evaluated in eight additional isolates. Results: In both isolates, the combination of iron and copper increased pathogenicity, capsule size, and melanin production. Regarding proteomic data, proteins with increased levels after iron and copper exposure were related to biological processes such as cell stress, vesicular traffic (Ap1, Vps35), cell wall structure (Och1, Ccr4, Gsk3), melanin biosynthesis (Hem15, Mln2), DNA repair (Chk1), protein transport (Mms2), SUMOylation (Uba2), and mitochondrial transport (Atm1). Increased pathogenicity by exposure to metal combination was also confirmed in 90% of the eight isolates. Conclusions: The combination of these metals enhances pathogenicity and increases the abundance of proteins related to the main virulence factors.

Candida albicans is a commensal fungus that causes systemic infections in immunosuppressed patients. In order to deal with the changing environment during commensalism or infection, C. albicans must reprogram its proteome. Characterizing the stress-induced changes in the proteome that C. albicans uses to survive should be very useful in the development of new antifungal drugs. We studied the C. albicans global proteome after exposure to hydrogen peroxide (H2O2) and acetic acid (AA), using a data-independent acquisition mass spectrometry (DIA-MS) strategy. More than 2,000 C. albicans proteins were quantified using an ion library previously constructed using data-dependent acquisition mass spectrometry (DDA-MS). C. albicans responded to treatment with H2O2 with an increase in the abundance of many proteins involved in the oxidative stress response, protein folding, and proteasome-dependent catabolism, which led to increased proteasome activity. The data revealed a previously unknown key role for Prn1, a protein similar to pirins, in the oxidative stress response. Treatment with AA resulted in a general decrease in the abundance of proteins involved in amino acid biosynthesis, protein folding, and rRNA processing. Almost all proteasome proteins declined, as did proteasome activity. Apoptosis was observed after treatment with H2O2 but not AA. A targeted proteomic study of 32 proteins related to apoptosis in yeast supported the results obtained by DIA-MS and allowed the creation of an efficient method to quantify relevant proteins after treatment with stressors (H2O2, AA, and amphotericin B). This approach also uncovered a main role for Oye32, an oxidoreductase, suggesting this protein as a possible apoptotic marker common to many stressors. IMPORTANCE Fungal infections are a worldwide health problem, especially in immunocompromised patients and patients with chronic disorders. Invasive candidiasis, caused mainly by C. albicans, is among the most common fungal diseases. Despite the existence of treatments to combat candidiasis, the spectrum of drugs available is limited. For the discovery of new drug targets, it is essential to know the pathogen response to different stress conditions. Our study provides a global vision of proteomic remodeling in C. albicans after exposure to different agents, such as hydrogen peroxide, acetic acid, and amphotericin B, that can cause apoptotic cell death. These results revealed the significance of many proteins related to oxidative stress response and proteasome activity, among others. Of note, the discovery of Prn1 as a key protein in the defense against oxidative stress as well the increase in the abundance of Oye32 protein when apoptotic process occurred point them out as possible drug targets.

BackgroundEchinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets.ObjectivesWe determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates.MethodsTwenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival.ResultsF659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin–calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity.ConclusionsOur findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis.

The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1Δ cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1Δ mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.

In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective strategy to study the protein cargo from MSC-derived EVs.

In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective strategy to study the protein cargo from MSC-derived EVs.

Pseudomonas aeruginosa is an important opportunistic human pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under different conditions and from diverse cellular compartments have been joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1, the highest coverage among bacterial PeptideAtlas datasets. The proteins included cover 89% of metabolic proteins, 72% of proteins involved in genetic information processing, 83% of proteins responsible for environmental information processing, more than 88% of the ones related to quorum sensing and biofilm formation, and 89% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. The manuscript describes the building of the PeptideAtlas and the contribution of the different proteomic data used. Significance: Pseudomonas aeruginosa is among the most versatile human bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies.

Candida albicansis a commensal fungus that causes systemic infections in immunosuppressed patients. In order to deal with the changing environment during commensalism or infection,C. albicansmust reprogram its proteome. Characterizing the stress-induced changes in the proteome thatC. albicansuses to survive should be very useful in the development of new antifungal drugs. We studied theC. albicansglobal proteome after exposure to hydrogen peroxide (H2O2) and acetic acid (AA), using a DIA-MS strategy. More than 2000C. albicansproteins were quantified using an ion library previously constructed using DDA-MS.C. albicansresponded to treatment with H2O2with an increase in the abundance of many proteins involved in the oxidative stress response, protein folding and proteasome-dependent catabolism, which led to an increased proteasome activity. The data revealed a previously unknown key role for Prn1, a protein similar to pirins, in the oxidative stress response. Treatment with AA resulted in a general decrease in the abundance of proteins involved in amino acid biosynthesis, protein folding, and rRNA processing. Almost all proteasome proteins declined, as did proteasome activity. Apoptosis was observed after treatment with H2O2, but not AA. A targeted proteomic study of 32 proteins related to apoptosis in yeast supported the results found by DIA-MS and allowed the creation of an efficient method to quantify relevant proteins after treatment with stressors (H2O2, AA, and amphotericin B). This approach also uncovered a main role for Oye32, an oxidoreductase, suggesting this protein as a possible apoptotic marker common to many stressors.IMPORTANCEFungal infections are a worldwide health problem especially in immunocompromised patients and patients with chronic disorders. Invasive candidiasis, mainly caused byC. albicans, are among the most common fungal diseases. Despite the existence of treatments to combat candidiasis the spectra of drugs available are limited. For the discovery of new drug targets is essential to know the pathogen response to different stress conditions. Our study provides a global vision of proteomic remodeling inC. albicansafter exposure to different agents such as hydrogen peroxide, acetic acid and amphotericin B that can cause apoptotic cell death. This results revealed the significance of many proteins related to oxidative stress response and proteasome activity among others. Of note, the discovery of Prn1 as a key protein in the defence against oxidative stress as well the increase in the abundance of Oye32 protein when apoptotic process occurred point out them as possible drug targets.

Pseudomonas aeruginosa is an important opportunistic pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under very different conditions and from diverse cellular compartments have been analyzed and joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1. This is the highest published coverage among the eight bacterial PeptideAtlas datasets currently available. The proteins in the Pseudomonas PeptideAtlas cover 84% of metabolic proteins, 71% of proteins involved in genetic information processing, 72% of proteins responsible for environmental information processing, more than 80% of proteins related to quorum sensing and biofilm formation, and 81% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. Here we describe how this resource was built and some of the physiologically important proteins of this pathogen.SignificancePseudomonas aeruginosa is among the most versatile bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies.

Metaproteomics is as a promising technique for studying the human gut microbiota, because it can reveal the taxonomic profile and also shed light on the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing and microbial cell disruption methods for metaproteomic analyses of human gut microbiota. An unsupervised principal component analysis showed that different methods produced similar human gut microbial taxonomic profiles. An unsupervised two-way hierarchical clustering analysis identified the microbial taxonomic signatures associated with each method. Proteobacteria and Bacteroidetes identification was favored by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads. Ascomycota identification was enhanced by using large-sized glass beads during sample processing for stool dispersion. Euryarchaeota identification was improved with a combination of small and medium-sized glass beads for cell disruption. Assessments of the relative abundance of Firmicutes, Actinobacteria and Spirochaetes improved when ultrasonication was performed before cell disruption with glass beads. The latter method also increased the overall number of identified proteins. Taxonomic and protein functional analyses of metaproteomic data derived from stool samples from six healthy individuals showed common taxonomic profiles. We also detected certain proteins involved in microbial functions relevant to the host and related mostly to particular taxa, such as B12 biosynthesis and short chain fatty acid production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. Finally, in this metaproteomic study we identified several human proteins, mostly related to the anti-microbial response, which could contribute to determining the beneficial and detrimental relationships between gut microbiota and human cells in particular human diseases.

Extracellular vesicles (EVs) have been described as mediators of microorganism survival and interaction with the host. InCandida albicans, a relevant commensal fungal pathogen, the dimorphic transition is an important virulence trait in candidiasis. We have analyzed EVs secreted by yeast (YEVs) or hyphal cells (HEVs) fromC. albicans, finding interesting differences in both size distribution and protein loading. In general, HEVs were smaller and carried a much more diverse protein cargo than YEVs, including most of the proteins identified in YEVs, which were mainly cell surface proteins. Virulence factors such as phospholipases, aspartic proteases (Saps), adhesins and invasins, and the precursor protein of candidalysin toxin Ece1p were also detected. HEVs were also enriched in proteasomal and ribosomal proteins, and in enzymes from amino acid biosynthetic pathways, all involved in protein metabolism, as well as proteins related to intracellular protein transport and components of the ESCRT pathway related to exosome biogenesis. Both types of EV presented immune reactivity with human sera from patients suffering invasive candidiasis. In our conditions, only HEVs could elicit the release of TNFα by activated macrophages. This first analysis ofC. albicansHEVs shows their relevance to pathogenesis and possible new diagnostics or treatments.

Antibiotic resistance and hospital acquired infections are on the rise worldwide. Vancomycin-resistant enterococci have been reported in clinical settings in recent decades. In this multiomics study, we provide comprehensive proteomic and transcriptomic analyses of a vancomycin-resistant

Antibiotic resistance and hospital acquired infections are on the rise worldwide. Vancomycin-resistant enterococci have been reported in clinical settings in recent decades. In this multiomics study, we provide comprehensive proteomic and transcriptomic analyses of a vancomycin-resistant Enterococcus faecalis clinical isolate from a patient with a urinary tract infection. The previous genotypic profile of the strain C2620 indicated the presence of antibiotic resistance genes characteristic of the vanB cluster. To further investigate the transcriptome of this pathogenic strain, we used whole genome sequencing and RNA-sequencing to detect and quantify the genes expressed. In parallel, we used two-dimensional gel electrophoresis followed by MALDI-TOF/MS (Matrix-assisted laser desorption/ionization-Time-of-flight/Mass spectrometry) to identify the proteins in the proteome. We studied the membrane and cytoplasm subproteomes separately. From a total of 207 analysis spots, we identified 118 proteins. The protein list was compared to the results obtained from the full transcriptome assay. Several genes and proteins related to stress and cellular response were identified, as well as some linked to antibiotic and drug responses, which is consistent with the known state of multiresistance. Even though the correlation between transcriptome and proteome data is not yet fully understood, the use of multiomics approaches has proven to be increasingly relevant to achieve deeper insights into the survival ability of pathogenic bacteria found in health care facilities. L.P. was granted a PhD fellowship by Fundação para a Ciência e a Tecnologia and European Social Fund (SFRH/BD/81307/2011). This work was supported by the Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from Fundação para a Ciência e a Tecnologia/ MCTES (UID/QUI/50006/2019). This research is also a result of the GenomePT project (POCI-01-0145-FEDER- 022184), supported by COMPETE 2020—Operational Programme for Competitiveness and Internationalisation, Lisboa Portugal Regional Operational Programme (Lisboa, 2020), Algarve Portugal Regional Operational Programme (CRESC Algarve, 2020), under the PORTUGAL 2020 Partnership Agreement, through the EuropeanRegionalDevelopment Fund, andbyFundação para a Ciência e a Tecnologia.Work performed in University of La Rioja was supported by project SAF2016- 76571-R of Agencia Estatal de Investigatio´n and FEDER. info:eu-repo/semantics/publishedVersion

Vultures have evolved adaptive mechanisms to prevent infections associated with their scavenging lifestyle. However, food-borne exposure to antimicrobial pharmaceuticals can promote opportunistic infections with adverse outcomes. Here, we used multivariate and network analyses to increase understanding of the behavior of the yeast communities causing oral mycosis outbreaks recently reported in wild nestling cinereous (Aegypius monachus), griffon (Gyps fulvus) and Egyptian (Neophron percnopterus) vultures (CV, GV and EV, respectively) exposed to antibiotics from livestock farming. Common and unique yeast signatures (of Candida, Debaromyces, Diutina, Meyerozyma, Naganishia, Pichia, Rhodotorula, Trichosporon and Yarrowia species) associated with oral mycoses were identified in the three vulture species. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) highlighted that oral lesions from CV and GV shared similar yeast signatures (of major causative pathogens of opportunistic mycoses, such as Candida albicans, Candida parapsilosis and Candida tropicalis), while EV had a distinct yeast signature (of uncommon pathogenic species, such as Candida dubliniensis, Candida zeylanoides, Pichia fermentans and Rhodotorula spp.). Synergistic interactions between yeast species from distinct fungal phyla were found in lesions from CV and GV, but not in EV. These formed co-occurrence subnetworks with partially or fully connected topology. This study reveals that the composition, assembly and co-occurrence patterns of the yeast communities causing oral mycoses differ between vulture species with distinct feeding habits and scavenging lifestyles. Yeast species widely pathogenic to humans and animals, and yeast co-occurrence relationships, are distinctive hallmarks of oral mycoses in CV and GV. These vulture species are more exposed to antibiotics from intensively medicated livestock carcasses provided in supplementary feeding stations and show higher incidence of thrush-like oral lesions than EV. These findings may be useful for development of new initiatives or changes in the conservation of these avian scavengers affected by anthropogenic activities., This work was supported by the Ministry of Economy, Industry and Competitiveness from Spain (grant numbers CGL2009-12753-C02-01/BOS and CGL2010-15726).

Macrophages are involved in the primary human response to Candida albicans. After pathogen recognition, signaling pathways are activated, leading to the production of cytokines, chemokines, and antimicrobial peptides. ATP binding proteins are crucial for this regulation. Here, a quantitative proteomic and phosphoproteomic approach was carried out for the study of human macrophage ATP-binding proteins after interaction with C. albicans. From a total of 547 nonredundant quantified proteins, 137 were ATP binding proteins and 59 were detected as differentially abundant. From the differentially abundant ATP-binding proteins, 6 were kinases (MAP2K2, SYK, STK3, MAP3K2, NDKA, and SRPK1), most of them involved in signaling pathways. Furthermore, 85 phosphopeptides were quantified. Macrophage proteomic alterations including an increase of protein synthesis with a consistent decrease in proteolysis were observed. Besides, macrophages showed changes in proteins of endosomal trafficking together with mitochondrial proteins, including some involved in the response to oxidative stress. Regarding cell death mechanisms, an increase of antiapoptotic over pro-apoptotic signals is suggested. Furthermore, a high pro-inflammatory response was detected, together with no upregulation of key mi-RNAs involved in the negative feedback of this response. These findings illustrate a strategy to deepen the knowledge of the complex interactions between the host and the clinically important pathogen C. albicans.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strain C999 was isolated of a Spanish patient with urinary tract infection. Previous genotyping indicated that this strain presented a multidrug-resistance phenotype and carried beta-lactamase genes encoding CTX-M-15, TEM-1, and OXA-1 enzymes. The whole-cell proteome, and the membrane, cytoplasmic, periplasmic and extracellular sub-proteomes of C999 were obtained in this work by two-dimensional gel electrophoresis (2DE) followed by fingerprint sequencing through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). A total of 602 proteins were identified in the different cell fractions, several of which are related to stress response systems, cellular responses, and antibiotic and drug responses, consistent with the multidrug-resistance phenotype. In parallel, whole genome sequencing (WGS) and RNA sequencing (RNA-Seq) was done to identify and quantify the genes present and expressing. The in silico prediction following WGS confirmed our strain as being serotype O25:H4 and sequence type ST131. The presence of proteins related to antibiotic resistance and virulence in an O25:H4-ST131 E. coli clone are serious indicators of the continued threat of antibiotic resistance spread amongst healthcare institutions. On a positive note, a multiomics approach can facilitate surveillance and more detailed characterization of virulent bacterial clones from hospital environments.

Bacterial physiology is regulated at different levels, from mRNA synthesis to translational regulation and protein modification. Herein, we propose a parameter, dubbed post-transcriptional variation (PTV), that allows extracting information on post-transcriptional regulation from the combined analysis of transcriptomic and proteomic data. We have applied this parameter for getting a deeper insight in the regulon of the Pseudomonas aeruginosa post-transcriptional regulator Crc. P. aeruginosa is a free-living microorganism, and part of its ecological success relies on its capability of using a large number of carbon sources. The hierarchical assimilation of these sources when present in combination is regulated by Crc that, together with Hfq (the RNA-binding chaperon in the complex), impedes their translation when catabolite repression is triggered. Most studies on Crc regulation are based either in transcriptomics or in proteomics data, which cannot provide information on post-transcriptional regulation when analysed independently. Using the PTV parameter, we present a comprehensive map of the Crc post-transcriptional regulon. In addition of controlling the use of primary and secondary carbon sources, Crc regulates as well cell respiration, c-di-GMP mediated signalling, and iron utilization. Thus, besides controlling the hyerarchical assimilation of carbon sources, Crc is an important element for keeping bacterial homeostasis and, consequently, metabolic robustness.

Candida albicans is a commensal microorganism in the oral cavity and gastrointestinal and urogenital tracts of most individuals that acts as an opportunistic pathogen when the host immune response is reduced. Here, we established different immunocompetent murine models to analyze the antibody responses to the C. albicans proteome during commensalism, commensalism followed by infection, and infection (C, C+I, and I models, respectively). Serum anti-C. albicans IgG antibody levels were higher in colonized mice than in infected mice. The antibody responses during gut commensalism (up to 55 days of colonization) mainly focused on C. albicans proteins involved in stress response and metabolism and differed in both models of commensalism. Different serum IgG antibody-reactivity profiles were also found over time among the three murine models. C. albicans gut colonization protected mice from an intravenous lethal fungal challenge, emphasizing the benefits of fungal gut colonization. This work highlights the importance of fungal gut colonization for future immune prophylactic therapies.

Macrophages may induce fungal apoptosis to fight against C. albicans, as previously hypothesized by our group. To confirm this hypothesis, we analyzed proteins from C. albicans cells after 3 h of interaction with macrophages using two quantitative proteomic approaches. A total of 51 and 97 proteins were identified as differentially expressed by DIGE and iTRAQ, respectively. The proteins identified and quantified were different, with only seven in common, but classified in the same functional categories. The analyses of their functions indicated that an increase in the metabolism of amino acids and purine nucleotides were taking place, while the glycolysis and translation levels dropped after 3 h of interaction. Also, the response to oxidative stress and protein translation were reduced. In addition, seven substrates of metacaspase (Mca1) were identified (Cdc48, Fba1, Gpm1, Pmm1, Rct1, Ssb1, and Tal1) as decreased in abundance, plus 12 proteins previously described as related to apoptosis. Besides, the monitoring of apoptotic markers along 24 h of interaction (caspase-like activity, TUNEL assay, and the measurement of ROS and cell examination by transmission electron microscopy) revealed that apoptotic processes took place for 30% of the fungal cells, thus supporting the proteomic results and the hypothesis of macrophages killing C. albicans by apoptosis.

The characterization of pathogen-specific antigenic proteins at the protein species level is crucial in the development and molecular optimization of novel immunodiagnostics, vaccines or immunotherapeutics for infectious diseases. The major requirements to achieve this molecular level are to obtain 100% sequence coverage and identify all post-translational modifications of each antigenic protein species. In this article, we show nearly complete sequence information for five discrete antigenic species of Candida albicans Tdh3 (glyceraldehyde-3-phosphate dehydrogenase), which have been reported to be differentially recognized both among candidemia patients and between candidemia and control patients. A comprehensive description of the top-down immunoproteomic strategy used for seroprofiling at the C. albicans protein species level in candidemia as well as for the chemical characterization of this immunogenic protein (based on high-resolution 2-DE, Western blotting, peptide mass fingerprinting, tandem mass spectrometry and de novo peptide sequencing) is also provided. The top-down characterization data on the speciation of the C. albicans immunome in candidemia presented here are related to our research article entitled “Seroprofiling at the Candida albicans protein species level unveils an accurate molecular discriminator for candidemia” (Pitarch et al., J. Proteomics, 2015, http://dx.doi.org/10.1016/j.jprot.2015.10.022). Keywords: Diagnosis, Candidemia, Invasive candidiasis, Protein species, Serologic response, Biomarkers, Immunoproteomics, Immunome, Serological proteome analysis, Glyceraldehyde-3-phosphate dehydrogenase

To provide new and expanded proteome documentation of the opportunistically pathogen Candida albicans, we have developed new protein extraction and analysis routines to provide a new, extended and enhanced version of the C. albicans PeptideAtlas. Two new datasets, resulting from experiments consisting of exhaustive subcellular fractionations and different growing conditions, plus two additional datasets from previous experiments on the surface and the secreted proteomes, have been incorporated to increase the coverage of the proteome. High resolution precursor mass spectrometry (MS) and ion trap tandem MS spectra were analyzed with three different search engines using a database containing allele-specific sequences. This approach, novel for a large-scale C. albicans proteomics project, was combined with the post-processing and filtering implemented in the Trans Proteomic Pipeline consistently used in the PeptideAtlas project and resulted in 49,372 additional peptides (3-fold increase) and 1630 more proteins (1.6-fold increase) identified in the new C. albicans PeptideAtlas with respect to the previous build. A total of 71,310 peptides and 4174 canonical (minimal non-redundant set) proteins (4115 if one protein per pair of alleles is considered) were identified representing 66% of the 6218 proteins in the predicted proteome. This makes the new PeptideAtlas build the most comprehensive C. albicans proteomics resource available and the only large-scale one with detections of individual alleles., This work has been financially supported in part by project BIO2012-31767Ministerio de Economía y Competitividad, Spain, PROPMT (S2010/BMD-2414) from the Comunidad Autónoma de Madrid, REIPI, Spanish Network for the Research in Infectious Diseases (RD12/0015/0004), and PRB2 projects PT13/0001/0004 and PT13/0001/0008 from the ISCIII. VV held a research contract associated to project BIO2012-31767. These results are lined up with the Spanish Initiative on the Human Proteome Project (B/D-HPP). This work was funded in part by the American Recovery and Reinvestment Act (ARRA) funds through National Institutes of Health from the NHGRI grant RC2HG005805, the NIGMS grants R01GM087221 and 2P50GM076547 to the Center for Systems Biology, the National Institute of Biomedical Imaging and Bioengineering grant U54EB020406, the National Science Foundation MRI grant 0923536, and the EU FP7 ‘ProteomeXchange’ grant 260558.

Invasive Candidiasis (IC) poses a major public health problem worldwide. Despite the introduction of new antifungal agents and changes in clinical practices, its morbidity and mortality rates and healthcare costs remain persistently high. This is mainly because of the serious underlying conditions of infected patients (critically ill or severely immunocompromised patients) and the difficulties encountered in early diagnosing this opportunistic mycosis and initiating prompt and appropriate antifungal therapy. In the light of this great clinical challenge, the past decades have witnessed the development of diverse early detection and therapeutic intervention strategies aimed at minimizing the clinical impact and economic burden of this healthcare-associated infection caused by Candida species. Here, we review the currently available methods for IC diagnosis. These encompass (i) gold standard methods (fungal culture and tissue histopathology), (ii) pathogen-derived biomarker detection tests (PCR, protein antigens, mannan, ß-D-glucan and D-arabinitol-based assays), (iii) host-derived biomarker detection tests (Candida albicans germ tube antibodies or CAGTA, anti-mannan antibodies, other infection-specific antibodies, procalcitonin, serum amyloid A, interleukin 17, interleukin 23 and transforming growth factor ß-based assays), (iv) clinical prediction algorithms (Candida score, Candida colonization index and other prediction rules), and (v) leading-edge molecular, proteomic and immunomic technologies (such as peptide nucleic acid-fluorescent in situ hybridization or PNA-FISH, T2 magnetic resonance or T2Candida assay, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or MALDI-TOF MS, among others). Their strengths, utility, limitations as well as combined use to assist in the diagnosis of this life-threatening and costly fungal infection (including candidemia and deepseated candidiasis) are also discussed.

Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts. Our data also suggest that mesenchymal stem cells constitute a promising source for the detection of missing proteins.

Gardnerella vaginalis is one of the main etiologic agents of bacterial vaginosis (BV). This infection is responsible for a wide range of public health costs and is associated with several adverse outcomes during pregnancy. Improving our understanding of G. vaginalis protein cell surface will assist in BV diagnosis. This study represents the first proteomic approach that has analyzed the exposed proteins on G. vaginalis cell surface using a shaving approach. The 261 G. vaginalis proteins identified using this approach were analyzed with bioinformatic tools to detect characteristic motifs from surface-exposed proteins, such as signal peptides (36 proteins), lipobox domains (17 proteins), LPXTG motifs (5 proteins) and transmembrane alpha-helices (66 proteins). One third of the identified proteins were found to have at least one typical motif of surface-exposed proteins. Furthermore, the subcellular location was examined using two predictors (PSORT and Gpos-mPLoc). These bioinformatic tools classified 17% of the identified proteins as surface-associated proteins. Interestingly, we identified 13 members of the ATP-binding cassette (ABC) superfamily, which were mainly involved in the translocation of various substrates across membranes. To validate the location of the G. vaginalis surface-exposed proteins, an immunofluorescence assay with antibodies against Escherichia coli GroEL was performed to reveal the extracellular location of the moonlighting GroEL. In addition, monoclonal antibodies (mAb) against G. vaginalis Cna protein were produced and used to validate the location of Cna on the surface of the G. vaginalis. These high affinity anti-Cna mAb represent a useful tool for the study of this pathogenic microorganism and the BV.