Mycobacterial PE/PPE Proteins at the Host-Pathogen Interface

The mycobacterial PE/PPE proteins have attracted much interest since their formal identification just over a decade ago. It has been widely speculated that these proteins may play a role in evasion of host immune responses, possibly via antigenic variation. Although a cohesive understanding of their function(s) has yet to be established, emerging data increasingly supports a role for the PE/PPE proteins at multiple levels of the infectious process. This paper will delineate salient features of the families revealed by comparative genomics, bioinformatic analyses and genome-wide screening approaches and will summarise existing knowledge of subcellular localization, secretion pathways, and protein structure. These characteristics will be considered in light of findings on innate and adaptive host responses to PE/PPE proteins, and we will review the increasing body of data on B and T cell recognition of these proteins. Finally, we will consider how current knowledge and future explorations may contribute to a more comprehensive understanding of these intriguing proteins and their involvement in host pathogen interactions. Ultimately this information could underpin future intervention strategies, for example, in the area of new and improved diagnostic tools and vaccine candidates

Recent developments in the application of flow cytometry to advance our understanding of Mycobacterium tuberculosis physiology and pathogenesis

CITATION: Parbhoo, T., Sampson, S.L. & Mouton, J.M. 2020. Recent developments in the application of flow cytometry to advance our understanding of Mycobacterium tuberculosis physiology and pathogenesis. Cytometry Part A, 97(7):683-693. doi:10.1002/cyto.a.24030The original publication is available at https://onlinelibrary.wiley.com/journal/15524930The ability of the bacterial pathogen Mycobacterium tuberculosis to adapt and survive within human cells to disseminate to other individuals and cause active disease is poorly understood. Research supports that as M. tuberculosis adapts to stressors encountered in the host, it exhibits variable physiological and metabolic states that are time and niche-dependent. Challenges associated with effective treatment and eradication of tuberculosis (TB) are in part attributed to our lack of understanding of these different mycobacterial phenotypes. This is mainly due to a lack of suitable tools to effectively identify/detect heterogeneous bacterial populations, which may include small, difficult-to-culture subpopulations. Importantly, flow cytometry allows rapid and affordable multiparametric measurements of physical and chemical characteristics of single cells, without the need to preculture cells. Here, we summarize current knowledge of flow cytometry applications that have advanced our understanding of the physiology of M. tuberculosis during TB disease. Specifically, we review how host-associated stressors influence bacterial characteristics such as metabolic activity, membrane potential, redox status and the mycobacterial cell wall. Further, we highlight that flow cytometry offers unprecedented opportunities for insight into bacterial population heterogeneity, which is increasingly appreciated as an important determinant of disease outcome. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.https://onlinelibrary.wiley.com/journal/15524930Publishers versio

Small molecule efflux pump inhibitors in Mycobacterium tuberculosis: a rational drug design perspective

Drug resistance in Mycobacterium tuberculosis (M. tuberculosis) complicates management of tuberculosis. Efflux pumps contribute to low level resistance and acquisition of additional high level resistance mutations through sub-therapeutic concentrations of intracellular antimycobacterials. Various efflux pump inhibitors (EPIs) have been described for M. tuberculosis but little is known regarding the mechanism of efflux inhibition. As knowledge relating to the mechanism of action and drug target is central to the rational drug design of safe and sufficiently selective EPIs, this review aims to examine recent developments in the study of EPIs in M. tuberculosis from a rational drug development perspective and to provide an overview to facilitate systematic development of therapeutically effective EPIs. Review of literature points to a reduction in cellular energy or direct binding to the efflux pump as likely mechanisms for most EPIs described for M. tuberculosis. This review demonstrates that, where a direct interaction with efflux pumps is expected, both molecular structure and general physicochemical properties should be considered to accurately predict efflux pump substrates and inhibitors. Non-competitive EPIs do not necessarily demonstrate the same requirements as competitive inhibitors and it is therefore essential to differentiate between competitive and non-competitive inhibition to accurately determine structure activity relationships for efflux pump inhibition. It is also evident that there are various similarities between inhibitors of prokaryotic and eukaryotic efflux pumps but, depending on the specific chemical scaffolds under investigation, it may be possible to design EPIs that are less prone to inhibition of human P-glycoprotein, thereby reducing side effects and drug-drug interactions

Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions

BACKGROUND: The PE and PPE multigene families of Mycobacterium tuberculosis comprise about 10% of the coding potential of the genome. The function of the proteins encoded by these large gene families remains unknown, although they have been proposed to be involved in antigenic variation and disease pathogenesis. Interestingly, some members of the PE and PPE families are associated with the ESAT-6 (esx) gene cluster regions, which are regions of immunopathogenic importance, and encode a system dedicated to the secretion of members of the potent T-cell antigen ESAT-6 family. This study investigates the duplication characteristics of the PE and PPE gene families and their association with the ESAT-6 gene clusters, using a combination of phylogenetic analyses, DNA hybridization, and comparative genomics, in order to gain insight into their evolutionary history and distribution in the genus Mycobacterium. RESULTS: The results showed that the expansion of the PE and PPE gene families is linked to the duplications of the ESAT-6 gene clusters, and that members situated in and associated with the clusters represent the most ancestral copies of the two gene families. Furthermore, the emergence of the repeat protein PGRS and MPTR subfamilies is a recent evolutionary event, occurring at defined branching points in the evolution of the genus Mycobacterium. These gene subfamilies are thus present in multiple copies only in the members of the M. tuberculosis complex and close relatives. The study provides a complete analysis of all the PE and PPE genes found in the sequenced genomes of members of the genus Mycobacterium such as M. smegmatis, M. avium paratuberculosis, M. leprae, M. ulcerans, and M. tuberculosis. CONCLUSION: This work provides insight into the evolutionary history for the PE and PPE gene families of the mycobacteria, linking the expansion of these families to the duplications of the ESAT-6 (esx) gene cluster regions, and showing that they are composed of subgroups with distinct evolutionary (and possibly functional) differences

Prevalence of pyrazinamide resistance across the spectrum of drug resistant phenotypes of Mycobacterium tuberculosis

Pyrazinamide resistance is largely unknown in the spectrum of drug resistant phenotypes. We summarize data on PZA resistance in clinical isolates from South Africa. PZA DST should be performed when considering its inclusion in treatment of patients with rifampicin-resistant TB or MDR-TB

Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics

Abstract Background Transcriptional responses required to maintain cellular homeostasis or to adapt to environmental stress, is in part mediated by several nucleic-acid associated proteins. In this study, we sought to establish an affinity purification-mass spectrometry (AP-MS) approach that would enable the collective identification of nucleic acid-associated proteins in mycobacteria. We hypothesized that targeting the RNA polymerase complex through affinity purification would allow for the identification of RNA- and DNA-associated proteins that not only maintain the bacterial chromosome but also enable transcription and translation. Results AP-MS analysis of the RNA polymerase β-subunit cross-linked to nucleic acids identified 275 putative nucleic acid-associated proteins in the model organism Mycobacterium smegmatis under standard culturing conditions. The AP-MS approach successfully identified proteins that are known to make up the RNA polymerase complex, as well as several other known RNA polymerase complex-associated proteins such as a DNA polymerase, sigma factors, transcriptional regulators, and helicases. Gene ontology enrichment analysis of the identified proteins revealed that this approach selected for proteins with GO terms associated with nucleic acids and cellular metabolism. Importantly, we identified several proteins of unknown function not previously known to be associated with nucleic acids. Validation of several candidate nucleic acid-associated proteins demonstrated for the first time DNA association of ectopically expressed MSMEG_1060, MSMEG_2695 and MSMEG_4306 through affinity purification. Conclusions Effective identification of nucleic acid-associated proteins, which make up the RNA polymerase complex as well as other DNA- and RNA-associated proteins, was facilitated by affinity purification of the RNA polymerase β-subunit in M. smegmatis. The successful identification of several transcriptional regulators suggest that our approach could be sensitive enough to investigate the nucleic acid-associated proteins that maintain cellular functions and mediate transcriptional and translational change in response to environmental stress

FAD binding, cobinamide binding and active site communication in the corrin reductase (CobR)

Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I)

Antimycobacterial Activity, Synergism, and Mechanism of Action Evaluation of Novel Polycyclic Amines against Mycobacterium tuberculosis

Mycobacterium tuberculosis has developed extensive resistance to numerous antimycobacterial agents used in the treatment of tuberculosis. Insufficient intracellular accumulation of active moieties allows for selective survival of mycobacteria with drug resistance mutations and accordingly promotes the development of microbial drug resistance. Discovery of compounds with new mechanisms of action and physicochemical properties that promote intracellular accumulation, or compounds that act synergistically with other antimycobacterial drugs, has the potential to reduce and prevent further drug resistance. To this end, antimycobacterial activity, mechanism of action, and synergism in combination therapy were investigated for a series of polycyclic amine derivatives. Compound selection was based on the presence of moieties with possible antimycobacterial activity, the inclusion of bulky lipophilic carriers to promote intracellular accumulation, and previously demonstrated bioactivity that potentially support inhibition of efflux pump activity. The most potent antimycobacterial demonstrated a minimum inhibitory concentration (MIC99) of 9.6 μM against Mycobacterium tuberculosis H37Rv. Genotoxicity and inhibition of the cytochrome bc1 respiratory complex were excluded as mechanisms of action for all compounds. Inhibition of cell wall synthesis was identified as a likely mechanism of action for the two most active compounds (14 and 15). Compounds 5 and 6 demonstrated synergistic activity with the known Rv1258c efflux pump substrate, spectinomycin, pointing to possible efflux pump inhibition. For this series, the nature of the side chain, rather than the type of polycyclic carrier, seems to play a determining role in the antimycobacterial activity and cytotoxicity of the compounds. Contrariwise, the nature of the polycyclic carrier, particularly the azapentacycloundecane cage, appears to promote synergistic activity. Results point to the possibility of combining an azapentacycloundecane carrier with a side chain that promotes antimycobacterial activity to develop dual acting molecules for the treatment of Mycobacterium tuberculosis

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

Recombination in pe/ppe genes contributes to genetic variation in Mycobacterium tuberculosis lineages

Background: Approximately 10 % of the Mycobacterium tuberculosis genome is made up of two families of genes that are poorly characterized due to their high GC content and highly repetitive nature. The PE and PPE families are typified by their highly conserved N-terminal domains that incorporate proline-glutamate (PE) and proline-proline-glutamate (PPE) signature motifs. They are hypothesised to be important virulence factors involved with host-pathogen interactions, but their high genetic variability and complexity of analysis means they are typically disregarded in genome studies.// Results: To elucidate the structure of these genes, 518 genomes from a diverse international collection of clinical isolates were de novo assembled. A further 21 reference M. tuberculosis complex genomes and long read sequence data were used to validate the approach. SNP analysis revealed that variation in the majority of the 168 pe/ppe genes studied was consistent with lineage. Several recombination hotspots were identified, notably pe_pgrs3 and pe_pgrs17. Evidence of positive selection was revealed in 65 pe/ppe genes, including epitopes potentially binding to major histocompatibility complex molecules.// Conclusions: This, the first comprehensive study of the pe and ppe genes, provides important insight into M. tuberculosis diversity and has significant implications for vaccine development