RESUMO
The human pathogen Mycobacterium tuberculosis typically causes lung disease but can also disseminate to other tissues. We identified a M. tuberculosis (Mtb) outbreak presenting with unusually high rates of extrapulmonary dissemination and bone disease. We found that the causal strain carried an ancestral full-length version of the type VII-secreted effector EsxM rather than the truncated version present in other modern Mtb lineages. The ancestral EsxM variant exacerbated dissemination through enhancement of macrophage motility, increased egress of macrophages from established granulomas, and alterations in macrophage actin dynamics. Reconstitution of the ancestral version of EsxM in an attenuated modern strain of Mtb altered the migratory mode of infected macrophages, enhancing their motility. In a zebrafish model, full-length EsxM promoted bone disease. The presence of a derived nonsense variant in EsxM throughout the major Mtb lineages 2, 3, and 4 is consistent with a role for EsxM in regulating the extent of dissemination.
Assuntos
Doenças Ósseas , Mycobacterium marinum , Mycobacterium tuberculosis , Tuberculose , Animais , Humanos , Peixe-Zebra , Tuberculose/microbiologia , Macrófagos/microbiologia , Proteínas de Bactérias/genéticaRESUMO
The pathological accumulation of cholesterol is a signature feature of Niemann-Pick type C (NPC) disease, in which excessive lipid levels induce Purkinje cell death in the cerebellum. NPC1 encodes a lysosomal cholesterol-binding protein, and mutations in NPC1 drive cholesterol accumulation in late endosomes and lysosomes (LE/Ls). However, the fundamental role of NPC proteins in LE/L cholesterol transport remains unclear. Here, we demonstrate that NPC1 mutations impair the projection of cholesterol-containing membrane tubules from the surface of LE/Ls. A proteomic survey of purified LE/Ls identified StARD9 as a novel lysosomal kinesin responsible for LE/L tubulation. StARD9 contains an N-terminal kinesin domain, a C-terminal StART domain, and a dileucine signal shared with other lysosome-associated membrane proteins. Depletion of StARD9 disrupts LE/L tubulation, paralyzes bidirectional LE/L motility and induces accumulation of cholesterol in LE/Ls. Finally, a novel StARD9 knock-out mouse recapitulates the progressive loss of Purkinje cells in the cerebellum. Together, these studies identify StARD9 as a microtubule motor protein responsible for LE/L tubulation and provide support for a novel model of LE/L cholesterol transport that becomes impaired in NPC disease.
Assuntos
Cinesinas , Células de Purkinje , Animais , Camundongos , Cinesinas/genética , Proteômica , Transporte Biológico , Lisossomos , Camundongos KnockoutRESUMO
The ESX-1 (ESAT-6-system-1) system and the protein substrates it transports are essential for mycobacterial pathogenesis. The precise ways that ESX-1 substrates contribute to virulence remains unknown. Several known ESX-1 substrates are also required for the secretion of other proteins. We used a proteo-genetic approach to construct high-resolution dependency relationships for the roles of individual ESX-1 substrates in secretion and virulence in Mycobacterium marinum, a pathogen of humans and animals. Characterizing a collection of M. marinum strains with in-frame deletions in each of the known ESX-1 substrate genes and the corresponding complementation strains, we demonstrate that ESX-1 substrates are differentially required for ESX-1 activity and for virulence. Using isobaric-tagged proteomics, we quantified the degree of requirement of each substrate on protein secretion. We conclusively defined distinct contributions of ESX-1 substrates in protein secretion. Our data reveal a hierarchy of ESX-1 substrate secretion, which supports a model for the composition of the extracytoplasmic ESX-1 secretory machinery. Overall, our proteo-genetic analysis demonstrates discrete roles for ESX-1 substrates in ESX-1 function and secretion in M. marinum.
Assuntos
Proteínas de Bactérias , Mycobacterium marinum , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Mycobacterium marinum/patogenicidade , Transporte Proteico , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Amino acid substitutions (AASs) alter proteins from their genome-expected sequences. Accumulation of substitutions in proteins underlies numerous diseases and antibiotic mechanisms. Accurate global detection of AASs and their frequencies is crucial for understanding these mechanisms. Shotgun proteomics provides an untargeted method for measuring AASs but introduces biases when extrapolating from the genome to identify AASs. To characterize these biases, we created a "ground-truth" approach using the similarities betweenEscherichia coli and Salmonella typhimurium to model the complexity of AAS detection. Shotgun proteomics on mixed lysates generated libraries representing â¼100,000 peptide-spectra and 4161 peptide sequences with a single AAS and defined stoichiometry. Identifying S. typhimurium peptide-spectra with only the E. coli genome resulted in 64.1% correctly identified library peptides. Specific AASs exhibit variable identification efficiencies. There was no inherent bias from the stoichiometry of the substitutions. Short peptides and AASs localized near peptide termini had poor identification efficiency. We identify a new class of "scissor substitutions" that gain or lose protease cleavage sites. Scissor substitutions also had poor identification efficiency. This ground-truth AAS library reveals various sources of bias, which will guide the application of shotgun proteomics to validate AAS hypotheses.
Assuntos
Escherichia coli , Proteômica , Proteômica/métodos , Substituição de Aminoácidos , Escherichia coli/genética , Peptídeos/genética , Peptídeos/química , ProteínasRESUMO
Bottom-up proteomics (BUP) produces rich data, but visualization and analysis are time-consuming and often require programming skills. Many tools analyze these data at the proteome-level, but fewer options exist for individual proteins. Sequence coverage maps are common, but do not proportion peptide intensity. Abundance-based visualization of sequence coverage facilitates detection of protein isoforms, domains, potential truncation sites, peptide "hot-spots", and localization of post-translational modifications (PTMs). Redundant stacked-sequence coverage is an important tool in designing hydrogen-deuterium exchange (HDX) experiments. Visualization tools often lack graphical and tabular-export of processed data which complicates publication of results. Quantitative peptide abundance across amino acid sequences is an essential and missing tool in proteomics toolkits. Here we created PrIntMap-R, an online application that only requires peptide files from a database search and FASTA protein sequences. PrIntMap-R produces a variety of plots for quantitative visualization of coverage; annotation of specific sequences, PTM's, and comparisons of one or many samples overlaid with calculated fold-change or several intensity metrics. We show use-cases including protein phosphorylation, identification of glycosylation, and the optimization of digestion conditions for HDX experiments. PrIntMap-R is freely available, open source, and can run online with no installation, or locally by downloading source code from GitHub.
Assuntos
Peptídeos , Proteômica , Proteômica/métodos , Software , Proteoma/metabolismo , Sequência de AminoácidosRESUMO
Mycobacterial pathogens pose a sustained threat to human health. There is a critical need for new diagnostics, therapeutics, and vaccines targeting both tuberculous and nontuberculous mycobacterial species. Understanding the basic mechanisms used by diverse mycobacterial species to cause disease will facilitate efforts to design new approaches toward detection, treatment, and prevention of mycobacterial disease. Molecular, genetic, and biochemical approaches have been widely employed to define fundamental aspects of mycobacterial physiology and virulence. The recent expansion of genetic tools in mycobacteria has further increased the accessibility of forward genetic approaches. Proteomics has also emerged as a powerful approach to further our understanding of diverse mycobacterial species. Detection of large numbers of proteins and their modifications from complex mixtures of mycobacterial proteins is now routine, with efforts of quantification of these datasets becoming more robust. In this review, we discuss the "genetic proteome," how the power of genetics, molecular biology, and biochemistry informs and amplifies the quality of subsequent analytical approaches and maximizes the potential of hypothesis-driven mycobacterial research. Published proteomics datasets can be used for hypothesis generation and effective post hoc supplementation to experimental data. Overall, we highlight how the integration of proteomics, genetic, molecular, and biochemical approaches can be employed successfully to define fundamental aspects of mycobacterial pathobiology.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Genômica , Infecções por Mycobacterium/metabolismo , Mycobacterium/metabolismo , Proteoma/metabolismo , Humanos , Mycobacterium/genética , Mycobacterium/patogenicidade , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/microbiologia , Proteoma/análiseRESUMO
Small proteins of up to â¼50 amino acids are an abundant class of biomolecules across all domains of life. Yet due to the challenges inherent in their size, they are often missed in genome annotations, and are difficult to identify and characterize using standard experimental approaches. Consequently, we still know few small proteins even in well-studied prokaryotic model organisms. Mass spectrometry (MS) has great potential for the discovery, validation, and functional characterization of small proteins. However, standard MS approaches are poorly suited to the identification of both known and novel small proteins due to limitations at each step of a typical proteomics workflow, i.e., sample preparation, protease digestion, liquid chromatography, MS data acquisition, and data analysis. Here, we outline the major MS-based workflows and bioinformatic pipelines used for small protein discovery and validation. Special emphasis is placed on highlighting the adjustments required to improve detection and data quality for small proteins. We discuss both the unbiased detection of small proteins and the targeted analysis of small proteins of interest. Finally, we provide guidelines to prioritize novel small proteins, and an outlook on methods with particular potential to further improve comprehensive discovery and characterization of small proteins.
Assuntos
Archaea/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Espectrometria de Massas/métodos , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bactérias/genética , Proteínas de Bactérias/genética , Biologia Computacional , Regulação da Expressão Gênica em Archaea/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologiaRESUMO
Genome-wide transcriptomic analyses have revealed abundant expressed short open reading frames (ORFs) in bacteria. Whether these short ORFs, or the small proteins they encode, are functional remains an open question. One quarter of mycobacterial mRNAs are leaderless, beginning with a 5'-AUG or GUG initiation codon. Leaderless mRNAs often encode unannotated short ORFs as the first gene of a polycistronic transcript. Here, we show that polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression. Detailed mutational analysis shows that one polycysteine short ORF controls expression of the downstream genes. Our data indicate that ribosomes stalled in the polycysteine tract block mRNA structures that otherwise sequester the ribosome-binding site of the 3'gene. We assessed endogenous proteomic responses to cysteine limitation in Mycobacterium smegmatis using mass spectrometry. Six cysteine metabolic loci having unannotated polycysteine-encoding leaderless short ORF architectures responded to cysteine limitation, revealing widespread cysteine-responsive attenuation in mycobacteria. Individual leaderless short ORFs confer independent operon-level control, while their shared dependence on cysteine ensures a collective response mediated by ribosome pausing. We propose the term ribulon to classify ribosome-directed regulons. Regulon-level coordination by ribosomes on sensory short ORFs illustrates one utility of the many unannotated short ORFs expressed in bacterial genomes.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Mycobacterium smegmatis/genética , Fases de Leitura Aberta/genética , Peptídeos/genética , Elementos de Resposta/genética , Cisteína/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , RNA Mensageiro/genética , Sítio de Iniciação de TranscriçãoRESUMO
Potent immunosuppressive mechanisms within the tumor microenvironment contribute to the resistance of aggressive human cancers to immune checkpoint blockade (ICB) therapy. One of the main mechanisms for myeloid-derived suppressor cells (MDSCs) to induce T cell tolerance is through secretion of reactive nitrogen species (RNS), which nitrates tyrosine residues in proteins involved in T cell function. However, so far very few nitrated proteins have been identified. Here, using a transgenic mouse model of prostate cancer and a syngeneic cell line model of lung cancer, we applied a nitroproteomic approach based on chemical derivation of 3-nitrotyrosine and identified that lymphocyte-specific protein tyrosine kinase (LCK), an initiating tyrosine kinase in the T cell receptor signaling cascade, is nitrated at Tyr394 by MDSCs. LCK nitration inhibits T cell activation, leading to reduced interleukin 2 (IL2) production and proliferation. In human T cells with defective endogenous LCK, wild type, but not nitrated LCK, rescues IL2 production. In the mouse model of castration-resistant prostate cancer (CRPC) by prostate-specific deletion of Pten, p53, and Smad4, CRPC is resistant to an ICB therapy composed of antiprogrammed cell death 1 (PD1) and anticytotoxic-T lymphocyte-associated protein 4 (CTLA4) antibodies. However, we showed that ICB elicits strong anti-CRPC efficacy when combined with an RNS neutralizing agent. Together, these data identify a previously unknown mechanism of T cell inactivation by MDSC-induced protein nitration and illuminate a clinical path hypothesis for combining ICB with RNS-reducing agents in the treatment of CRPC.
Assuntos
Carcinoma Pulmonar de Lewis/imunologia , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Células Supressoras Mieloides/imunologia , Neoplasias de Próstata Resistentes à Castração/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patologia , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Masculino , Camundongos , Células Supressoras Mieloides/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/patologiaRESUMO
Stable heavy-isotope labeling is commonly used in quantitative proteomics. Several common techniques incorporate deuterium (2 H) as the heavy isotopic label using reductive amination with formaldehyde. Compared with alternatives, dimethyl labeling reagents are inexpensive and the labeling chemistry is simple and rapid. However, the substitution of hydrogen by deuterium can introduce subtle changes in peptides' polarities, leading to a shift in chromatographic retention times between deuterated and nondeuterated peptides that can lead to quantification deviations. Capillary zone electrophoresis has emerged as a complementary separation for ESI-MS-based proteomics, including targeted and quantitative approaches. The extent to which the deuterium isotope effect impacts CZE-based proteomics, which separates peptides based on their S/N ratios, has not been investigated. To address this issue, CZE was used to analyze dimethyl labeled E. coli tryptic digests in 100 min single-shot analyses. The median migration time shift was 0.1 s for light versus heavy labeled peptides, which is 2.5% of the peak width. For comparison, nUHPLC-ESI-MS/MS was used to analyze the same sample. In UPLC, deuterated peptides tended to elute earlier than nondeuterated peptides, with a retention shift of 3 s for light versus heavy labeled peptides, which is roughly half the peak width. This shift in separation time did not have a significant effect on quantitation for either method for equal mixing ratios of the light-intermediate-heavy isotope labeled samples.
Assuntos
Deutério/química , Eletroforese Capilar/métodos , Proteoma/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida de Alta Pressão , Formaldeído/química , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Proteoma/química , Espectrometria de Massas em TandemRESUMO
Diabetic foot ulcers are characterized by hypoxia. For many patients, hyperbaric oxygen (HBO) therapy is the last recourse for saving the limb from amputation, for which the molecular basis is not understood. We previously identified the active form of matrix metalloproteinase-9 (MMP-9) as responsible for diabetic foot ulcer's recalcitrance to healing. Transcription of mmp-9 to the inactive zymogen is upregulated during hypoxia. Activation of the zymogen is promoted by proteases and reactive oxygen species (ROS). We hypothesized that the dynamics of these two events might lead to a lowering of active MMP-9 levels in the wounded tissue. We employed the full-thickness excisional db/db mouse model to study wound healing, and treated the mice to 3.0 atm of molecular oxygen for 90 minutes, 5 days per week for 10 days in an HBO research chamber. Treatment with HBO accelerated diabetic wound healing compared to untreated mice, with more completed and extended reepithelialization. We imaged the wounds for ROS in vivo with a luminol-based probe and found that HBO treatment actually decreases ROS levels. The levels of superoxide dismutase, catalase, and glutathione peroxidase-enzymes that turn over ROS-increased after HBO treatment, hence the observation of decreased ROS. Since ROS levels are lowered, we explored the effect that this would have on activation of MMP-9. Quantitative analysis with an affinity resin that binds and pulls down the active MMPs exclusively, coupled with proteomics, revealed that HBO treatment indeed reduces the active MMP-9 levels. This work for the first time demonstrates that diminution of active MMP-9 is a contributing factor and a mechanism for enhancement of diabetic wound healing by HBO therapy.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Pé Diabético/metabolismo , Oxigenoterapia Hiperbárica , Metaloproteinase 9 da Matriz/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cicatrização , Animais , Catalase/metabolismo , Modelos Animais de Doenças , Precursores Enzimáticos/metabolismo , Glutationa Peroxidase/metabolismo , Camundongos , Receptores para Leptina/genética , Superóxido Dismutase/metabolismoRESUMO
ESX (ESAT-6 system) export systems play diverse roles across mycobacterial species. Interestingly, genetic disruption of ESX systems in different species does not result in an accumulation of protein substrates in the mycobacterial cell. However, the mechanisms underlying this observation are elusive. We hypothesized that the levels of ESX substrates were regulated by a feedback-control mechanism, linking the levels of substrates to the secretory status of ESX systems. To test this hypothesis, we used a combination of genetic, transcriptomic, and proteomic approaches to define export-dependent mechanisms regulating the levels of ESX-1 substrates in Mycobacterium marinum WhiB6 is a transcription factor that regulates expression of genes encoding ESX-1 substrates. We found that, in the absence of the genes encoding conserved membrane components of the ESX-1 system, the expression of the whiB6 gene and genes encoding ESX-1 substrates were reduced. Accordingly, the levels of ESX-1 substrates were decreased, and WhiB6 was not detected in M. marinum strains lacking genes encoding ESX-1 components. We demonstrated that, in the absence of EccCb1, a conserved ESX-1 component, substrate gene expression was restored by constitutive, but not native, expression of the whiB6 gene. Finally, we found that the loss of WhiB6 resulted in a virulent M. marinum strain with reduced ESX-1 secretion. Together, our findings demonstrate that the levels of ESX-1 substrates in M. marinum are fine-tuned by negative feedback control, linking the expression of the whiB6 gene to the presence, not the functionality, of the ESX-1 membrane complex.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium marinum/genética , Fatores de Transcrição/metabolismo , Sistemas de Secreção Tipo VII/genética , Retroalimentação Fisiológica , Mycobacterium marinum/metabolismoRESUMO
The ESX-1 (ESAT-6 system 1) secretion system plays a conserved role in the virulence of diverse mycobacterial pathogens, including the human pathogen Mycobacterium tuberculosis and M. marinum, an environmental mycobacterial species. The ESX-1 system promotes the secretion of protein virulence factors to the extracytoplasmic environment. The secretion of these proteins triggers the host response by lysing the phagosome during macrophage infection. Using proteomic analyses of the M. marinum secretome in the presence and absence of a functional ESX-1 system, we and others have hypothesized that MMAR_2894, a PE family protein, is a potential ESX-1 substrate in M. marinum We used genetic and quantitative proteomic approaches to determine if MMAR_2894 is secreted by the ESX-1 system, and we defined the requirement of MMAR_2894 for ESX-1-mediated secretion and virulence. We show that MMAR_2894 is secreted by the ESX-1 system in M. marinum and is itself required for the optimal secretion of the known ESX-1 substrates in M. marinum Moreover, we found that MMAR_2894 was differentially required for hemolysis and cytolysis of macrophages, two lytic activities ascribed to the M. marinum ESX-1 system.IMPORTANCE Both Mycobacterium tuberculosis, the cause of human tuberculosis (TB), and Mycobacterium marinum, a pathogen of ectotherms, use the ESX-1 secretion system to cause disease. There are many established similarities between the ESX-1 systems in M. tuberculosis and in M. marinum Yet the two bacteria infect different hosts, hinting at species-specific functions of the ESX-1 system. Our findings demonstrate that MMAR_2894 is a PE protein secreted by the ESX-1 system of M. marinum We show that MMAR_2894 is required for the optimal secretion of mycobacterial proteins required for disease. Because the MMAR_2894 gene is not conserved in M. tuberculosis, our findings demonstrate that MMAR_2894 may contribute to a species-specific function of the ESX-1 system in M. marinum, providing new insight into how the M. marinum and M. tuberculosis systems differ.
Assuntos
Proteínas de Bactérias/metabolismo , Eritrócitos/microbiologia , Hemólise , Interações Hospedeiro-Patógeno , Mycobacterium marinum/genética , Mycobacterium marinum/patogenicidade , Animais , Proteínas de Bactérias/genética , Macrófagos/microbiologia , Camundongos , Mycobacterium tuberculosis , Proteômica , Células RAW 264.7 , Tuberculose/microbiologia , Virulência , Fatores de Virulência/genéticaRESUMO
Capillary zone electrophoresis (CZE) can produce high-resolution separations of biological samples, including microbial mixtures. The study of complex populations of microorganisms using CZE is limited because most detectors have limited sensitivity, are destructive, and provide limited information for microbial identification. To address these issues, we developed an integrated capillary zone electrophoresis apparatus to fractionate bacteria from complex mixtures. We deposited fractions onto nutrient agar in a Petri dish for microbial culturing, and we subjected the observed colonies to Sanger sequencing of a phylogenetic marker, the 16S rRNA gene, for microbial identification. We separated and cultured both a single bacteria species, the model Gram-negative organism Escherichia coli, and a complex environmental isolate of primary sewage effluent. Sequence analysis of the 16S rRNA genes from this mixture identified 15 ± 5 distinct bacterial species per run. This approach requires minimal manipulation of microbial populations and combines electrophoretic fractionation of bacterial cells with automated collection for accurate identification of species. This approach should be applicable to microorganisms in general and may enable discrimination of physiologically different cells in complex assemblages, such as in microbiome samples.
Assuntos
Eletroforese Capilar/métodos , Escherichia coli/isolamento & purificação , Escherichia coli/classificação , Escherichia coli/genética , Filogenia , RNA Ribossômico 16S/classificação , RNA Ribossômico 16S/genética , Esgotos/microbiologia , Águas Residuárias/microbiologiaRESUMO
N-terminal acetylation (NTA) is a post-transcriptional modification of proteins that is conserved from bacteria to humans. In bacteria, the enzymes that mediate protein NTA also promote antimicrobial resistance. In pathogenic mycobacteria, which cause human tuberculosis and other chronic infections, NTA has been linked to pathogenesis and stress response, yet the fundamental biology underlying NTA of mycobacterial proteins remains unclear. We enriched, defined, and quantified the NT-acetylated populations of both cell-associated and secreted proteins from both the human pathogen, Mycobacterium tuberculosis, and the nontuberculous opportunistic pathogen, Mycobacterium marinum. We used a parallel N-terminal enrichment strategy from proteolytic digests coupled to charge-based selection and stable isotope ratio mass spectrometry. We show that NTA of the mycobacterial proteome is abundant, diverse, and primarily on Thr residues, which is unique compared with other bacteria. We isolated both the acetylated and unacetylated forms of 256 proteins, indicating that NTA of mycobacterial proteins is homeostatic. We identified 16 mycobacterial proteins with differential levels of NTA on the cytoplasmic and secreted forms, linking protein modification and localization. Our findings reveal novel biology underlying the NTA of mycobacterial proteins, which may provide a basis to understand NTA in mycobacterial physiology, pathogenesis, and antimicrobial resistance.
Assuntos
Proteínas de Bactérias/química , Mycobacterium marinum/química , Mycobacterium tuberculosis/química , Peptídeos/análise , Processamento de Proteína Pós-Traducional , Acetilação , Proteínas de Bactérias/classificação , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cromatografia Líquida , Biologia Computacional/métodos , Humanos , Mycobacterium marinum/isolamento & purificação , Mycobacterium marinum/metabolismo , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/metabolismo , Peptídeos/química , Proteólise , Especificidade da Espécie , Coloração e Rotulagem/métodos , Espectrometria de Massas em Tandem , Tuberculose Pulmonar/microbiologiaRESUMO
Mycobacterial pathogens use the ESAT-6 system 1 (Esx-1) exporter to promote virulence. Previously, we used gene disruption and complementation to conclude that the MMAR_0039 gene in Mycobacterium marinum is required to promote Esx-1 export. Here we applied molecular genetics, proteomics, and whole-genome sequencing to demonstrate that the MMAR_0039 gene is not required for Esx-1 secretion or virulence. These findings suggest that we initially observed an indirect mechanism of genetic complementation. We identified a spontaneous nonsense mutation in a known Esx-1-associated gene which causes a loss of Esx-1 activity. We show that the Esx-1 function was restored by nonsense suppression. Moreover, we identified a polar mutation in the ppsC gene which reduced cellular impermeability but did not impact cytotoxicity in macrophages. Our studies reveal insight into Esx-1 export, nonsense suppression, and cell envelope lipid biogenesis.
Assuntos
Proteínas de Bactérias/genética , Códon sem Sentido , Regulação Bacteriana da Expressão Gênica , Mycobacterium marinum/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/metabolismo , Mycobacterium marinum/patogenicidade , Fenótipo , Transporte Proteico , VirulênciaRESUMO
Capillary zone electrophoresis (CZE) is emerging as a useful tool in proteomic analysis. Interest arises from dramatic improvements in performance that result from improvements in the background electrolyte used for the separation, the incorporation of advanced sample injection methods, the development of robust and sensitive electrospray interfaces, and the coupling with Orbitrap mass spectrometers with high resolution and sensitivity. The combination of these technologies produces performance that is rapidly approaching the performance of UPLC-based methods for microgram samples and exceeds the performance of UPLC-based methods for mid- to low nanogram samples. These systems now produce over 10 000 peptide IDs in a single 100-min analysis of the HeLa proteome.
Assuntos
Proteoma/isolamento & purificação , Eletroforese Capilar/normas , Humanos , Proteômica/métodos , Melhoria de Qualidade , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Modern mass spectrometry-based methods provide an exciting opportunity to characterize protein expression in the developing embryo. We have employed an isotopic labeling technology to quantify the expression dynamics of nearly 6000 proteins across six stages of development in Xenopus laevis from the single stage zygote through the mid-blastula transition and the onset of organogenesis. Approximately 40% of the proteins show significant changes in expression across the development stages. The expression changes for these proteins naturally falls into six clusters corresponding to major events that mark early Xenopus development. A subset of experiments in this study have quantified protein expression differences between single embryos at the same stage of development, showing that, within experimental error, embryos at the same developmental stage have identical protein expression levels.
Assuntos
Proteômica , Xenopus laevis/embriologia , Animais , Evolução Molecular , Proteoma , Proteômica/métodos , Xenopus laevis/metabolismoRESUMO
Bacterial pathogens are exposed to toxic molecules inside the host and require efficient systems to form and maintain correct disulfide bonds for protein stability and function. The intracellular pathogen Francisella tularensis encodes a disulfide bond formation protein ortholog, DsbA, which previously was reported to be required for infection of macrophages and mice. However, the molecular mechanisms by which F. tularensisâ DsbA contributes to virulence are unknown. Here, we demonstrate that F. tularensisâ DsbA is a bifunctional protein that oxidizes and, more importantly, isomerizes complex disulfide connectivity in substrates. A single amino acid in the conserved cis-proline loop of the DsbA thioredoxin domain was shown to modulate both isomerase activity and F. tularensis virulence. Trapping experiments in F. tularensis identified over 50 F. tularensisâ DsbA substrates, including outer membrane proteins, virulence factors, and many hypothetical proteins. Six of these hypothetical proteins were randomly selected and deleted, revealing two novel proteins, FTL_1548 and FTL_1709, which are required for F. tularensis virulence. We propose that the extreme virulence of F. tularensis is partially due to the bifunctional nature of DsbA, that many of the newly identified substrates are required for virulence, and that the development of future DsbA inhibitors could have broad anti-bacterial implications.
Assuntos
Dissulfetos/metabolismo , Francisella tularensis/enzimologia , Francisella tularensis/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Deleção de Genes , Isomerases , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Análise de Sobrevida , Tularemia/microbiologia , Tularemia/patologia , Fatores de Virulência/genéticaRESUMO
Top-down proteomics offers the potential for full protein characterization, but many challenges remain for this approach, including efficient protein separations and effective fragmentation of intact proteins. Capillary zone electrophoresis (CZE) has shown great potential for separation of intact proteins, especially for differentially modified proteoforms of the same gene product. To date, however, CZE has been used only with collision-based fragmentation methods. Here we report the first implementation of electron transfer dissociation (ETD) with online CZE separations for top-down proteomics, analyzing a mixture of four standard proteins and a complex protein mixture from the Mycobacterium marinum bacterial secretome. Using a multipurpose dissociation cell on an Orbitrap Elite system, we demonstrate that CZE is fully compatible with ETD as well as higher energy collisional dissociation (HCD), and that the two complementary fragmentation methods can be used in tandem on the electrophoretic time scale for improved protein characterization. Furthermore, we show that activated ion electron transfer dissociation (AI-ETD), a recently introduced method for enhanced ETD fragmentation, provides useful performance with CZE separations to greatly increase protein characterization. When combined with HCD, AI-ETD improved the protein sequence coverage by more than 200% for proteins from both standard and complex mixtures, highlighting the benefits electron-driven dissociation methods can add to CZE separations.