RESUMEN
Clostridioides difficile-associated infection (CDI) is a health-care-associated infection caused, as the name suggests, by obligate anaerobic pathogen C. difficile and thus mainly transmitted via highly resistant endospores from one person to the other. In vivo, the spores need to germinate into cells prior to establishing an infection. Bile acids and glycine, both available in sufficient amounts inside the human host intestinal tract, serve as efficient germinants for the spores. It is therefore, for better understanding of C. difficile virulence, crucial to study both the cell and spore states with respect to their genetic, metabolic, and proteomic composition. In the present study, mass spectrometric relative protein quantification, based on the 14N/15N peptide isotopic ratios, has led to quantification of over 700 proteins from combined spore and cell samples. The analysis has revealed that the proteome turnover between a vegetative cell and a spore for this organism is moderate. Additionally, specific cell and spore surface proteins, vegetative cell proteins CD1228, CD3301 and spore proteins CD2487, CD2434, and CD0684 are identified as potential protein markers for C. difficile infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Esporas Bacterianas/metabolismo , Biomarcadores/metabolismo , Cromatografía Liquida/métodos , Clostridioides difficile/citología , Clostridioides difficile/patogenicidad , Enterocolitis Seudomembranosa/microbiología , Humanos , Espectrometría de Masas en Tándem/métodos , VirulenciaRESUMEN
Spores of Bacillus cereus pose a threat to food safety due to their high resistance to the heat or acid treatments commonly used to make food microbiologically safe. Spores may survive these treatments and later resume growth either on foodstuffs or, after ingestion, upon entering the gut they are capable of producing toxins, which cause either vomiting or diarrhea. The outer layers of the spore, the spore coat and exosporium, consist primarily of proteins that may serve as potential biomarkers for detection. The major morphogenetic protein CotE is important for correct assembly and attachment of the outermost layer, the exosporium, and by extension retention of many proteins. However, characterization of the proteins affected by deletion of CotE has been limited to electrophoretic patterns. Here we report the effect of CotE deletion on the insoluble fraction of the spore proteome through liquid chromatography-Fourier transform tandem mass spectrometry (LC-FTMS/MS) analysis. A total of 560 proteins have been identified in both mutant and wild-type spore coat isolates. A further 163 proteins were identified exclusively in wild-type spore isolates indicating that they are dependent on CotE for their association with the spore. Several of these are newly confirmed as associated with the exosporium, namely BC_2569 (BclF), BC_3345, BC_2427, BC_2878, BC_0666, BC_2984, BC_3481, and BC_2570. A total of 153 proteins were only identified in ΔCotE spore isolates. This was observed for proteins that are known or likely to be interacting with or are encased by CotE. Crucial spore proteins were quantified using a QconCAT reference standard, the first time this was used in a biochemically heterogeneous system. This allowed us to determine the absolute abundance of 21 proteins, which spanned across three orders of magnitude and together covered 5.66% ± 0.51 of the total spore weight. Applying the QconCAT methodology to the ΔCotE mutant allowed us to quantify 4.13% ± 0.14 of the spore total weight and revealed a reduction in abundance for most known exosporium associated proteins upon CotE deletion. In contrast, several proteins, either known or likely to be interacting with or encased by CotE (i.e., GerQ), were more abundant. The results obtained provide deeper insight into the layered spore structure such as which proteins are exposed on the outside of the spore. This information is important for developing detection methods for targeting spores in a food safety setting. Furthermore, protein stoichiometry and determination of the abundance of germination mediating enzymes provides useful information for germination and outgrowth model development.
Asunto(s)
Bacillus cereus/química , Proteínas Bacterianas/genética , Proteoma/genética , Esporas Bacterianas/química , Secuencia de Aminoácidos , Bacillus cereus/genética , Bacillus cereus/metabolismo , Proteínas Bacterianas/metabolismo , Cromatografía Liquida , Microbiología de Alimentos , Eliminación de Gen , Ontología de Genes , Humanos , Anotación de Secuencia Molecular , Proteoma/química , Proteoma/aislamiento & purificación , Proteoma/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Coloración y Etiquetado/métodos , Espectrometría de Masas en TándemRESUMEN
Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZâ»ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilamentâ»ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Proteínas del Citoesqueleto/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , División Celular/genética , Reactivos de Enlaces Cruzados/química , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Lisina/química , Lisina/genética , Lisina/metabolismo , Espectrometría de Masas/métodos , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida/métodos , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Programas InformáticosRESUMEN
Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram-positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique interprotein cross-linked peptides with <1% false discovery rate by mass spectrometry and genome-wide database searching. Nearly 60% of the interprotein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and ß' subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth. Data are available via ProteomeXchange with identifier PXD006287.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Glutaratos/química , Mapeo de Interacción de Proteínas/métodos , Succinimidas/química , Secuencia de Aminoácidos , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Reactivos de Enlaces Cruzados/química , Medios de Cultivo/química , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glutamato Deshidrogenasa/química , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Biogénesis de Organelos , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Especificidad de la Especie , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
The endospore is the dormant form of Bacillus subtilis and many other Firmicutes. By sporulation, these spore formers can survive very harsh physical and chemical conditions. Yet, they need to go through germination to return to their growing form. The spore inner membrane (IM) has been shown to play an essential role in triggering the initiation of germination. In this study, we isolated the IM of bacterial spores, in parallel with the isolation of the membrane of vegetative cells. With the use of GeLC-MS/MS, over 900 proteins were identified from the B. subtilis spore IM preparations. By bioinformatics-based membrane protein predictions, ca. one-third could be predicted to be membrane-localized. A large number of unique proteins as well as proteins common to the two membrane proteomes were identified. In addition to previously known IM proteins, a number of IM proteins were newly identified, at least some of which are likely to provide new insights into IM physiology, unveiling proteins putatively involved in spore germination machinery and hence putative germination inhibition targets.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Esporas Bacterianas/metabolismo , Proteínas Bacterianas/clasificación , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Proteínas de la Membrana/clasificación , Microscopía Electrónica de Transmisión , Proteoma/clasificación , Esporas Bacterianas/ultraestructura , Espectrometría de Masas en TándemRESUMEN
Time-series transcript- and protein-profiles were measured upon initiation of carbon catabolite repression in Escherichia coli, in order to investigate the extent of post-transcriptional control in this prototypical response. A glucose-limited chemostat culture was used as the CCR-free reference condition. Stopping the pump and simultaneously adding a pulse of glucose, that saturated the cells for at least 1h, was used to initiate the glucose response. Samples were collected and subjected to quantitative time-series analysis of both the transcriptome (using microarray analysis) and the proteome (through a combination of 15N-metabolic labeling and mass spectrometry). Changes in the transcriptome and corresponding proteome were analyzed using statistical procedures designed specifically for time-series data. By comparison of the two sets of data, a total of 96 genes were identified that are post-transcriptionally regulated. This gene list provides candidates for future in-depth investigation of the molecular mechanisms involved in post-transcriptional regulation during carbon catabolite repression in E. coli, like the involvement of small RNAs.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Glucosa/deficiencia , Proteoma , Transcriptoma , Reactores Biológicos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Marcaje Isotópico , Análisis por Micromatrices , Anotación de Secuencia Molecular , Isótopos de Nitrógeno , Factores de TiempoRESUMEN
Bacillus weihenstephanensis is a subspecies of the Bacillus cereus sensu lato group of spore-forming bacteria known to cause food spoilage or food poisoning. The key distinguishing phenotype of B. weihenstephanensis is its ability to grow below 7 °C or, from a food safety perspective, to grow and potentially produce toxins in a refrigerated environment. Comparison of the proteome profile of B. weihenstephanensis upon its exposure to different culturing conditions can reveal clues to the mechanistic basis of its psychrotolerant phenotype as well as elucidate relevant aspects of its toxigenic profile. To this end, the genome of the type strain B. weihenstephanensis WSBC 10204 was sequenced and annotated. Subsequently, the proteome profiles of cells grown at either 6 or 30 °C were compared, which revealed considerable differences and indicated several hundred (uncharacterized) proteins as being subproteome- and/or temperature-specific. In this manner, several processes were newly indicated to be dependent on growth temperature, such as varying carbon flux routes and a different role for the urea cycle. Furthermore, a possible post-translational regulatory function for acetylation was suggested. Toxin production was determined to be largely independent of growth temperature.
Asunto(s)
Bacillus cereus/genética , Proteínas Bacterianas/genética , Genoma Bacteriano , Procesamiento Proteico-Postraduccional , Proteoma/genética , Acetilación , Bacillus cereus/metabolismo , Proteínas Bacterianas/metabolismo , Ciclo del Carbono/fisiología , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Enterotoxinas , Microbiología de Alimentos , Isoformas de Proteínas , Proteoma/metabolismo , Análisis de Secuencia de ADN , Temperatura , Urea/metabolismoRESUMEN
Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.
Asunto(s)
Candida glabrata/química , Pared Celular/química , Proteínas Fúngicas/análisis , Proteoma/análisis , Candida glabrata/aislamiento & purificación , Candidiasis/microbiología , Humanos , Espectrometría de Masas , ProteómicaRESUMEN
Bionumbers and bioestimates are valuable tools in biological research. Here we focus on cell wall-related bionumbers and bioestimates of the budding yeast Saccharomyces cerevisiae and the polymorphic, pathogenic fungus Candida albicans. We discuss the linear relationship between cell size and cell ploidy, the correlation between cell size and specific growth rate, the effect of turgor pressure on cell size, and the reason why using fixed cells for measuring cellular dimensions can result in serious underestimation of in vivo values. We further consider the evidence that individual buds and hyphae grow linearly and that exponential growth of the population results from regular formation of new daughter cells and regular hyphal branching. Our calculations show that hyphal growth allows C. albicans to cover much larger distances per unit of time than the yeast mode of growth and that this is accompanied by strongly increased surface expansion rates. We therefore predict that the transcript levels of genes involved in wall formation increase during hyphal growth. Interestingly, wall proteins and polysaccharides seem barely, if at all, subject to turnover and replacement. A general lesson is how strongly most bionumbers and bioestimates depend on environmental conditions and genetic background, thus reemphasizing the importance of well-defined and carefully chosen culture conditions and experimental approaches. Finally, we propose that the numbers and estimates described here offer a solid starting point for similar studies of other cell compartments and other yeast species.
Asunto(s)
Candida albicans/citología , Pared Celular/metabolismo , Saccharomyces cerevisiae/citología , Candida albicans/metabolismo , Candida albicans/fisiología , Procesos de Crecimiento Celular , Pared Celular/química , Proteínas Fúngicas/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologíaRESUMEN
Resistance characteristics of bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of eight days, using metabolically (15)N labeled mature spores as reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Microbiología de Alimentos , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Cromatografía Liquida , Reactivos de Enlaces Cruzados , Calor , Proteómica , Esporas Bacterianas , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
It was demonstrated that the tetracycline resistance plasmid in Escherichia coli resembling K-12 23:06 containing the E. coli plasmid DM0133 could be transferred to tetracycline sensitive E. coli K-12 MG1655 YFP. The sensitive recipient strain has a slight metabolic advantage in continuous fermentation in absence of tetracycline pressure and as a result the numbers of the resistant recipient strain increase during fermentation. In presence of tetracycline pressure the sensitive strain is eliminated, but when it acquires tetracycline resistance the strain has still the same metabolic advantage as its sensitive parent strain in absence of tetracycline. Here a model will be shown that could explain the rate of transformation of a sensitive into a resistant recipient strain and its subsequent growth during continuous fermentation. According to the model the probability of formation of mutants would be much higher at the dilution rate of 0.09 compared to 0.28, whereas the growth of mutants would be much faster at high dilution rate. The growth model shows how the recipient mutants and the donor cells behave in relation to the dilution rate and the number of mutants. Apart from a deterministic model describing the growth rate of both the donor strain and the resistant recipient strain a stochastic model was developed that is particularly useful when low numbers of mutants are formed.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Resistencia a la Tetraciclina , Tetraciclina/farmacología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Biológicos , Transformación BacterianaRESUMEN
The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using (15)N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the ß-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.
Asunto(s)
Candida albicans/fisiología , Pared Celular/metabolismo , Candida albicans/citología , Quitina/metabolismo , Quitinasas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Técnicas de Inactivación de Genes , Glucano Endo-1,3-beta-D-Glucosidasa/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteoma/metabolismo , Estrés FisiológicoRESUMEN
PA (phosphatidic acid) is a lipid second messenger involved in an array of processes occurring during a plant's life cycle. These include development, metabolism, and both biotic and abiotic stress responses. PA levels increase in response to salt, but little is known about its function in the earliest responses to salt stress. In the present study we have combined an approach to isolate peripheral membrane proteins of Arabidopsis thaliana roots with lipid-affinity purification, to identify putative proteins that interact with PA and are recruited to the membrane in response to salt stress. Of the 42 putative PA-binding proteins identified by MS, a set of eight new candidate PA-binding proteins accumulated at the membrane fraction after 7 min of salt stress. Among these were CHC (clathrin heavy chain) isoforms, ANTH (AP180 N-terminal homology) domain clathrin-assembly proteins, a putative regulator of potassium transport, two ribosomal proteins, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and a PI (phosphatidylinositol) 4-kinase. PA binding and salt-induced membrane recruitment of GAPDH and CHC were confirmed by Western blot analysis of the cellular fractions. In conclusion, the approach of the present study is an effective way to isolate biologically relevant lipid-binding proteins and provides new leads in the study of PA-mediated salt-stress responses in roots.
Asunto(s)
Proteínas de Arabidopsis/aislamiento & purificación , Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , Ácidos Fosfatidicos/metabolismo , Raíces de Plantas/química , Tolerancia a la Sal , Algoritmos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Unión Proteica/efectos de los fármacos , Proteoma/análisis , Proteoma/efectos de los fármacos , Proteoma/aislamiento & purificación , Tolerancia a la Sal/genética , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/fisiologíaRESUMEN
Bacillus cereus, responsible for food poisoning, and Clostridium difficile, the causative agent of Clostridium difficile-associated diarrhea (CDAD), are both spore-forming pathogens involved in food spoilage, food intoxication, and other infections in humans and animals. The proteinaceous coat and the exosporium layers from spores are important for their resistance and pathogenicity characteristics. The exosporium additionally provides an ability to adhere to surfaces eventually leading to spore survival in food. Thus, studying these layers and identifying suitable protein targets for rapid detection and removal of spores is of the utmost importance. In this study, we identified 100 proteins from B. cereus spore coat, exosporium and 54 proteins from the C. difficile coat insoluble protein fraction. In an attempt to define a universal set of spore outer layer proteins, we identified 11 superfamily domains common to the identified proteins from two Bacilli and one Clostridium species. The evaluated orthologue relationships of identified proteins across different spore formers resulted in a set of 13 coat proteins conserved across the spore formers and 12 exosporium proteins conserved in the B. cereus group, which could be tested for quick and easy detection or targeted in strategies aimed at removal of spores from surfaces.
Asunto(s)
Bacillus cereus/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Clostridioides difficile/metabolismo , Proteoma/metabolismo , Esporas Bacterianas/metabolismo , Bacillus cereus/fisiología , Adhesión Bacteriana , Clostridioides difficile/fisiología , Farmacorresistencia Bacteriana , Microbiología de Alimentos , Peptidoglicano/metabolismo , Estructura Terciaria de ProteínaRESUMEN
The opportunistic fungal pathogen Candida albicans has developed various ways to overcome iron restriction in a mammalian host. Using different surface proteins, among them membrane- and wall-localized glycosylphosphatidylinositol (GPI) proteins, it can exploit iron from host haemoglobin, ferritin and transferrin. Culturing C. albicans in rich medium supplemented with the ferrous iron chelator bathophenanthroline disulfonic acid or in the minimal medium yeast nitrogen base resulted in a strong decrease of the iron content of the cells. MS analysis of the changes in the wall proteome of C. albicans upon iron restriction showed a strong increase in the levels of the GPI-modified adhesin Als3, which also serves as a ferritin receptor, and of the GPI-modified CFEM (common in fungal extracellular membranes) domain-containing proteins Csa1, Pga7, Pga10, and Rbt5. The wall levels of the GPI-modified proteins Hyr1, the adhesin Als4 and the copper- and zinc-containing superoxide dismutase Sod4 also strongly increased, whereas the levels of Tos1 (a non-GPI protein) and the GPI-modified adhesin Als2 strongly decreased. Strikingly, peptides derived from the CFEM domain of the haem-binding proteins Csa1, Pga10 and Rbt5 were capable of forming iron adduct ions during MS analysis, consistent with a key role of this domain in haem binding.
Asunto(s)
Candida albicans/química , Candida albicans/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Proteínas Fúngicas/análisis , Hierro/metabolismo , Proteoma/análisis , Candida albicans/crecimiento & desarrollo , Medios de Cultivo/química , Perfilación de la Expresión Génica , Espectrometría de Masas , Proteínas de la Membrana/análisisRESUMEN
The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose-grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate-grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose-grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate-grown cells. We identified mating and pheromone-regulated proteins that were exclusive to lactate-grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa-specific and other niche-specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.
Asunto(s)
Candida albicans/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Proteoma/metabolismo , Antifúngicos/farmacología , Biopelículas , Candida albicans/metabolismo , Candida albicans/fisiología , Pared Celular/química , Pared Celular/metabolismo , Pared Celular/ultraestructura , Farmacorresistencia Fúngica , Proteínas Fúngicas/metabolismo , Glucosa/metabolismo , Glucosa/farmacología , Ácido Láctico/metabolismo , Ácido Láctico/farmacología , Pruebas de Sensibilidad Microbiana , Presión Osmótica , Fenotipo , Proteoma/efectos de los fármacos , Estrés FisiológicoRESUMEN
Qualitative phenotypic changes are the integrated result of quantitative changes at multiple regulatory levels. To explain the temperature-induced increase of glycolytic flux in fermenting cultures of Saccharomyces cerevisiae, we quantified the contributions of changes in activity at many regulatory levels. We previously showed that a similar temperature increase in glucose-limited cultivations lead to a qualitative change from respiratory to fermentative metabolism, and this change was mainly regulated at the metabolic level. In contrast, in fermenting cells, a combination of different modes of regulation was observed. Regulation by changes in expression and the effect of temperature on enzyme activities contributed much to the increase in flux. Mass spectrometric quantification of glycolytic enzymes revealed that increased enzyme activity did not correlate with increased protein abundance, suggesting a large contribution of post-translational regulation to activity. Interestingly, the differences in the direct effect of temperature on enzyme kinetics can be explained by changes in the expression of the isoenzymes. Therefore, both the interaction of enzyme with its metabolic environment and the temperature dependence of activity are in turn regulated at the hierarchical level.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Glucólisis , Isoenzimas/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Isoenzimas/química , Espectrometría de Masas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , TemperaturaRESUMEN
Enzyme reprofiling in bacteria during adaptation from one environmental condition to another may be regulated by both transcription and translation. However, little is known about the contribution of translational regulation. Recently, we have developed a pulse labeling method using the methionine analog azidohomoalanine to determine the relative amounts of proteins synthesized by Escherichia coli in a brief time frame upon a change in environmental conditions. Here we present an extension of our analytical strategy, which entails measuring changes in total protein levels on the same time scale as new protein synthesis. This allows identification of stable and labile proteins and demonstrates that altered levels of most newly synthesized proteins are the result of a change in translation rate rather than degradation rate. With this extended strategy, average relative translation rates for 10 min immediately after a switch from aerobiosis to anaerobiosis were determined. The majority of proteins with increased synthesis rates upon an anaerobic switch are involved in glycolysis and pathways aimed at preventing glycolysis grinding to a halt by a cellular redox imbalance. Our method can be used to compare relative translation rates with relative mRNA levels at the same time. Discrepancies between these parameters may reveal genes whose expression is regulated by translation rather than by transcription. This may help unravel molecular mechanism underlying changes in translation rates, e.g. mediated by small regulatory RNAs.
Asunto(s)
Anaerobiosis/genética , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Biosíntesis de Proteínas , Proteoma , Alanina/análogos & derivados , Alanina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteoma/genética , Proteoma/metabolismoRESUMEN
Fluconazole is a commonly used antifungal drug that inhibits Erg11, a protein responsible for 14α-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14α-methylated sterols, which causes increased membrane fluidity and drug permeability. Surface-grown and planktonic cultures of Candida albicans responded similarly to fluconazole at 0.5 mg/liter, showing reduced biomass formation, severely reduced ergosterol levels, and almost complete inhibition of hyphal growth. There was no evidence of cell leakage. Mass spectrometric analysis of the secretome showed that its composition was strongly affected and included 17 fluconazole-specific secretory proteins. Relative quantification of (14)N-labeled query walls relative to a reference standard mixture of (15)N-labeled yeast and hyphal walls in combination with immunological analysis revealed considerable fluconazole-induced changes in the wall proteome as well. They were, however, similar for both surface-grown and planktonic cultures. Two major trends emerged: (i) decreased incorporation of hypha-associated wall proteins (Als3, Hwp1, and Plb5), consistent with inhibition of hyphal growth, and (ii) increased incorporation of putative wall repair-related proteins (Crh11, Pga4, Phr1, Phr2, Pir1, and Sap9). As exposure to the wall-perturbing drug Congo red led to a similar response, these observations suggested that fluconazole affects the wall. In keeping with this, the resistance of fluconazole-treated cells to wall-perturbing compounds decreased. We propose that fluconazole affects the integrity of both the cellular membranes and the fungal wall and discuss its potential consequences for antifungal therapy. We also present candidate proteins from the secretome for clinical marker development.