Identification of Widespread Adenosine Nucleotide Binding in Mycobacterium tuberculosis

SummaryComputational prediction of protein function is frequently error-prone and incomplete. In Mycobacterium tuberculosis (Mtb), ∼25% of all genes have no predicted function and are annotated as hypothetical proteins, severely limiting our understanding of Mtb pathogenicity. Here, we utilize a high-throughput quantitative activity-based protein profiling (ABPP) platform to probe, annotate, and validate ATP-binding proteins in Mtb. We experimentally validate prior in silico predictions of >240 proteins and identify 72 hypothetical proteins as ATP binders. ATP interacts with proteins with diverse and unrelated sequences, providing an expanded view of adenosine nucleotide binding in Mtb. Several hypothetical ATP binders are essential or taxonomically limited, suggesting specialized functions in mycobacterial physiology and pathogenicity

Phosphoproteomics analysis of a clinical mycobacterium tuberculosis Beijing isolate : expanding the mycobacterial phosphoproteome catalog

CITATION: Fortuin, S., et al. 2015. Phosphoproteomics analysis of a clinical mycobacterium tuberculosis beijing isolate : expanding the mycobacterial phosphoproteome catalog. Frontiers in Microbiology, 6:6, doi:10.3389/fmicb.2015.00006.The original publication is available at www.frontiersin.orgReversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.http://journal.frontiersin.org/article/10.3389/fmicb.2015.00006/fullPublisher's versio

Costing analysis of levofloxacin as antibiotic prophylaxis for pediatric household contacts of multi-drug resistant tuberculosis patients in a South African setting

Background The incidence of TB in children under 15 years, accounts for 8% of the global TB burden. In 2018, the World Health Organisation (WHO) estimated that there were approximately 11 000 multi-drug resistant (MDR) TB cases in South Africa. Despite having very clear guidelines on TB treatment programs and management, availability of inexpensive diagnostic tests, curative and preventive therapies, and the widespread use of the BCG vaccines, South Africa continues to have the highest the number of MDR-TB cases per capita. Levofloxacin is used as part of the group of fluoroquinolones in the drug regimen recommended in the treatment of MDR-TB patients. In addition to investigating the clinical impact of levofloxacin as preventative antibiotic therapy, the expected costs of the intervention will be a critical input to determining feasibility and costs effectiveness, which will inform policy and implementation considerations. Methods We performed a cost analysis on using existing data from the Tuberculosis Child Multi-drug-resistant Preventative Therapy (TB-CHAMP) trial, conducted from a TB control program perspective. We used data from 510 childhood household contacts of MDR-TB patients in South Africa that were treated with levofloxacin for 6 months as a preventative therapy for MDR-TB. In our analysis we evaluated the estimated health system cost associated with provision of levofloxacin to childhood contacts of MDRTB patients in South Africa. Results The mean total cost of treating a child household contact, irrespective of their weight band is ZAR 5,289.79. When the cost were analysed by weight categories we found that the cost increased by weight category; ZAR 2,146.78 (under 5 kg), ZAR 4,714.58 (between 5-15.9 kg) and ZAR 6,606.67 (over 16 kg). We performed a comprehensive sensitivity analysis and found that the scheduled clinic visits were the major cost driver. Aside from the scheduled visits we observed that there was an increase in additional health service utilization for children with a weight more than 5kg. Conclusion We envisage that based on our analysis we will be able to inform policy decisions about the management and prevention of childhood household contacts of MDR-TB patients in developing TB themselves

The evolution of the Mycobacterium tuberculosis proteome in response to the development of drug resistance

Thesis (PhD)--Stellenbosch University, 2013.ENGLISH ABSTRACT: This study is the first of its kind to highlight the importance of using the latest state of the art technology available in the field of proteomics as a complementary tool to characterize the proteome of members of the Mycobacterium tuberculosis Beijing lineage which have been linked to outbreaks and drug resistance of Tuberculosis (TB). Our label-free comparative analysis of two closely related M. tuberculosis strains with different transmission patterns and levels of virulence highlighted numerous factors that may alter metabolic pathways leading to hyper-virulence whereby the strain was able to rapidly replicate in the host and cause extensive disease. This comparative analysis clearly demonstrated that both instrumentation and analysis software impacts on the number of proteins identified and thereby the interpretation of the proteomic data. These proteomes also served as substrates for the discovery of phosphorylation sites, a field of research that reflects a significant knowledge gap in the field of M. tuberculosis. By using differential separation techniques in combination with the state of the art mass spectrometry we described the phosphorylation sites on 286 proteins. This was the first study to document phosphorylation of tyrosine residues in M. tuberculosis. By this means, our data set further extend and complement previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis. Using advanced mass spectrometry methods we further investigated the impact of the in vivo evolution of rifampicin resistance on the proteome of a rifampicin-resistant strain containing a S531L rpoB mutation. We identified the presence of overabundant proteins which could provide novel insight into potential compensatory mechanisms that the bacillus uses to reduce susceptibility to anti-TB drugs. Our findings suggest that proteins involved in a stress response may relate to an altered physiology enabling the pathogen to tolerate and persist when exposed to anti-TB drugs. Together this suggests that structural changes in the RNA polymerase precipitated a cascade of events leading to alterations of metabolic pathways. In addition, we present the first comprehensive analysis of the effect of rifampicin on the proteome of a rifampicin resistant M. tuberculosis isolate suggesting that rifampicin continues to influence the biology of M. tuberculosis despite the presence of an rpoB mutation. Our analysis showed alterations in the cell envelope composition and allowing the bacterium to survive in a metabolically dormant/persistent growth state. The results presented in this study illustrate the full potential of using a proteomic approach as a complementary molecular technique to select promising candidate molecules and genes for further characterization using the tools of molecular biology.AFRIKAANSE OPSOMMING: Die huidige studie is ‘n eerste van sy soort, deur die nuutste gevorderde tegnologie in die proteomika veld te gebruik. Die proteoom van lede van die Mycobacterium tuberculosis Beijing stam, wat die oorsaak is van tuberkulose (TB) uitbrake en ook weerstandige TB, is gekarakteriseer. Ons merkervrye vergelykende analise van twee naby verwante M. tuberculosis stamme met verskillende vlakke van oordraagbaarheid en virulensie, beklemtoon verskeie faktore wat metaboliese paaie mag verander, wat kan ly tot hiper-virulensie, wat die TB-stam in staat stel om vinniger te repliseer in die gasheer en ‘n uitgebreide siektetoestand kan veroorsaak. Die analise het duidelik gewys dat die toerusting wat gebruik word, sowel as die sagteware ‘n invloed kan hê op die hoeveelheid proteïne wat geïdentifiseer kan word en daardeur intrepretasie van proteomika data kan beïnvloed. Hierdie proteome dien as substrate vir die ondekking van fosforilasie setels, ‘n veld van navorsing wat dui op ‘n gaping in ons kennis van M. tuberculosis. Deur gebruik te maak van differensiële skeidingstegnieke en moderne spektrometrie beskryf ons fosforileringsetels in 286 proteine. Hierdie is die eerste studie wat fosforilasie van tirosien residue in M. tuberculosis beskryf. Hierdeur komplimenteer en brei ons data die huidige kennis oor gefosforileerde peptiede en fosforilasie setels in M. tuberculosis uit. Deur gebruik te maak van gevorderde massa spektrometriese tegnieke het ons verder ook die impak van in vivo evolusie van rifampicin weerstandigheid op die proteoom van ‘n rifampicin weerstandige TB-stam met die algemene S531L rpoB mutasie ondersoek. Ons het proteïne geïdentifiseer wat in groot hoeveelhede voorkom en kan nuwe insigte gee tot potensiele kompenserende meganismes wat deur die bacillus gebruik word om vatbaarheid vir anti-TB middels te verminder. Ons bevindings dui daarop dat proteïene betrokke in ‘n stresreaksie mag lei tot ‘n verandering in fisologie wat die patogeen in staat stel om anti-TB middels te verdra en te volhard in die teenwoordigheid van sulke middels. Saam impliseer dit dat ‘n ketting van gebeure wat lei tot veranderinge in metaboliese paaie, word vooraf gegaan deur strukturele veranderinge in die RNS polimerase. Tesame hiermee bied ons ook die eerste omvattende analise aan van die effek wat rifampicin op die proteoom van ‘n rifampicin weerstandige M. tuberculosis isolaat het, en wat aan die hand doen dat rifampicin voordurend die biologie van M. tuberculosis beïnvloed, ten spyte van die teenwoordigheid van ‘n rpoB mutasie. Ons analise dui op veranderinge in die samestelling van die selomhulsel wat die bakterie toelaat om te oorleef in ‘n metabolies dormante staat. Die resultate wat in hierdie studie aangebied word illustreer die volle potensiaal van ‘n proteomiese benadering as komplementêre molekulêre tegniek om belowende kandidaat molekules en gene te kies vir verdere karakterisering, deur gebruik te maak van molekulêre tegnieke.The National Research Foundation (RSA),Norwegian Research Council (Norway)National Institute of Health –Forgarty (USA)Southern Africa Consortium for Research Excellence-Welcome Trust (SACORE) (United Kingdom)Kwazulu-Natal Research Institute for Tuberculosis and HIV (K-RITH) (USA

Data_Sheet_3_Proteomics analysis of the p.G849D variant in neurexin 2 alpha may reveal insight into Parkinson’s disease pathobiology.docx

Parkinson’s disease (PD), the fastest-growing neurological disorder globally, has a complex etiology. A previous study by our group identified the p.G849D variant in neurexin 2 (NRXN2), encoding the synaptic protein, NRXN2α, as a possible causal variant of PD. Therefore, we aimed to perform functional studies using proteomics in an attempt to understand the biological pathways affected by the variant. We hypothesized that this may reveal insight into the pathobiology of PD. Wild-type and mutant NRXN2α plasmids were transfected into SH-SY5Y cells. Thereafter, total protein was extracted and prepared for mass spectrometry using a Thermo Scientific Fusion mass spectrometer equipped with a Nanospray Flex ionization source. The data were then interrogated against the UniProt H. sapiens database and afterward, pathway and enrichment analyses were performed using in silico tools. Overexpression of the wild-type protein led to the enrichment of proteins involved in neurodegenerative diseases, while overexpression of the mutant protein led to the decline of proteins involved in ribosomal functioning. Thus, we concluded that the wild-type NRXN2α may be involved in pathways related to the development of neurodegenerative disorders, and that biological processes related to the ribosome, transcription, and tRNA, specifically at the synapse, could be an important mechanism in PD. Future studies targeting translation at the synapse in PD could therefore provide further information on the pathobiology of the disease.</p

Data_Sheet_2_Proteomics analysis of the p.G849D variant in neurexin 2 alpha may reveal insight into Parkinson’s disease pathobiology.docx

Parkinson’s disease (PD), the fastest-growing neurological disorder globally, has a complex etiology. A previous study by our group identified the p.G849D variant in neurexin 2 (NRXN2), encoding the synaptic protein, NRXN2α, as a possible causal variant of PD. Therefore, we aimed to perform functional studies using proteomics in an attempt to understand the biological pathways affected by the variant. We hypothesized that this may reveal insight into the pathobiology of PD. Wild-type and mutant NRXN2α plasmids were transfected into SH-SY5Y cells. Thereafter, total protein was extracted and prepared for mass spectrometry using a Thermo Scientific Fusion mass spectrometer equipped with a Nanospray Flex ionization source. The data were then interrogated against the UniProt H. sapiens database and afterward, pathway and enrichment analyses were performed using in silico tools. Overexpression of the wild-type protein led to the enrichment of proteins involved in neurodegenerative diseases, while overexpression of the mutant protein led to the decline of proteins involved in ribosomal functioning. Thus, we concluded that the wild-type NRXN2α may be involved in pathways related to the development of neurodegenerative disorders, and that biological processes related to the ribosome, transcription, and tRNA, specifically at the synapse, could be an important mechanism in PD. Future studies targeting translation at the synapse in PD could therefore provide further information on the pathobiology of the disease.</p

Data_Sheet_1_Proteomics analysis of the p.G849D variant in neurexin 2 alpha may reveal insight into Parkinson’s disease pathobiology.docx

Parkinson’s disease (PD), the fastest-growing neurological disorder globally, has a complex etiology. A previous study by our group identified the p.G849D variant in neurexin 2 (NRXN2), encoding the synaptic protein, NRXN2α, as a possible causal variant of PD. Therefore, we aimed to perform functional studies using proteomics in an attempt to understand the biological pathways affected by the variant. We hypothesized that this may reveal insight into the pathobiology of PD. Wild-type and mutant NRXN2α plasmids were transfected into SH-SY5Y cells. Thereafter, total protein was extracted and prepared for mass spectrometry using a Thermo Scientific Fusion mass spectrometer equipped with a Nanospray Flex ionization source. The data were then interrogated against the UniProt H. sapiens database and afterward, pathway and enrichment analyses were performed using in silico tools. Overexpression of the wild-type protein led to the enrichment of proteins involved in neurodegenerative diseases, while overexpression of the mutant protein led to the decline of proteins involved in ribosomal functioning. Thus, we concluded that the wild-type NRXN2α may be involved in pathways related to the development of neurodegenerative disorders, and that biological processes related to the ribosome, transcription, and tRNA, specifically at the synapse, could be an important mechanism in PD. Future studies targeting translation at the synapse in PD could therefore provide further information on the pathobiology of the disease.</p

MetaNovo: An open-source pipeline for probabilistic peptide discovery in complex metaproteomic datasets.

BackgroundMicrobiome research is providing important new insights into the metabolic interactions of complex microbial ecosystems involved in fields as diverse as the pathogenesis of human diseases, agriculture and climate change. Poor correlations typically observed between RNA and protein expression datasets make it hard to accurately infer microbial protein synthesis from metagenomic data. Additionally, mass spectrometry-based metaproteomic analyses typically rely on focused search sequence databases based on prior knowledge for protein identification that may not represent all the proteins present in a set of samples. Metagenomic 16S rRNA sequencing only targets the bacterial component, while whole genome sequencing is at best an indirect measure of expressed proteomes. Here we describe a novel approach, MetaNovo, that combines existing open-source software tools to perform scalable de novo sequence tag matching with a novel algorithm for probabilistic optimization of the entire UniProt knowledgebase to create tailored sequence databases for target-decoy searches directly at the proteome level, enabling metaproteomic analyses without prior expectation of sample composition or metagenomic data generation and compatible with standard downstream analysis pipelines.ResultsWe compared MetaNovo to published results from the MetaPro-IQ pipeline on 8 human mucosal-luminal interface samples, with comparable numbers of peptide and protein identifications, many shared peptide sequences and a similar bacterial taxonomic distribution compared to that found using a matched metagenome sequence database-but simultaneously identified many more non-bacterial peptides than the previous approaches. MetaNovo was also benchmarked on samples of known microbial composition against matched metagenomic and whole genomic sequence database workflows, yielding many more MS/MS identifications for the expected taxa, with improved taxonomic representation, while also highlighting previously described genome sequencing quality concerns for one of the organisms, and identifying an experimental sample contaminant without prior expectation.ConclusionsBy estimating taxonomic and peptide level information directly on microbiome samples from tandem mass spectrometry data, MetaNovo enables the simultaneous identification of peptides from all domains of life in metaproteome samples, bypassing the need for curated sequence databases to search. We show that the MetaNovo approach to mass spectrometry metaproteomics is more accurate than current gold standard approaches of tailored or matched genomic sequence database searches, can identify sample contaminants without prior expectation and yields insights into previously unidentified metaproteomic signals, building on the potential for complex mass spectrometry metaproteomic data to speak for itself

On the impact of the pangenome and annotation discrepancies while building protein sequence databases for bacteria proteogenomics

CITATION: Machado, K. C. T., et al. 2019. On the impact of the pangenome and annotation discrepancies while building protein sequence databases for bacteria proteogenomics. Frontiers in Microbiology, 10:1410, doi:10.3389/fmicb.2019.01410.The original publication is available at https://www.frontiersin.orgENGLISH ABSTRACT: In proteomics, peptide information within mass spectrometry (MS) data from a specific organism sample is routinely matched against a protein sequence database that best represent such organism. However, if the species/strain in the sample is unknown or genetically poorly characterized, it becomes challenging to determine a database which can represent such sample. Building customized protein sequence databases merging multiple strains for a given species has become a strategy to overcome such restrictions. However, as more genetic information is publicly available and interesting genetic features such as the existence of pan- and core genes within a species are revealed, we questioned how efficient such merging strategies are to report relevant information. To test this assumption, we constructed databases containing conserved and unique sequences for 10 different species. Features that are relevant for probabilistic-based protein identification by proteomics were then monitored. As expected, increase in database complexity correlates with pangenomic complexity. However, Mycobacterium tuberculosis and Bordetella pertussis generated very complex databases even having low pangenomic complexity. We further tested database performance by using MS data from eight clinical strains from M. tuberculosis, and from two published datasets from Staphylococcus aureus. We show that by using an approach where database size is controlled by removing repeated identical tryptic sequences across strains/species, computational time can be reduced drastically as database complexity increases.https://www.frontiersin.org/articles/10.3389/fmicb.2019.01410/fullPublisher's versio