Site-directed mutagenesis of Mycobacterium tuberculosis and functional validation to investigate potential bedaquiline resistance-causing mutations.

Molecular detection of bedaquiline resistant tuberculosis is challenging as only a small proportion of mutations in candidate bedaquiline resistance genes have been statistically associated with phenotypic resistance. We introduced two mutations, atpE Ile66Val and Rv0678 Thr33Ala, in the Mycobacterium tuberculosis H37Rv reference strain using homologous recombineering or recombination to investigate the phenotypic effect of these mutations. The genotype of the resulting strains was confirmed by Sanger- and whole genome sequencing, and bedaquiline susceptibility was assessed by minimal inhibitory concentration (MIC) assays. The impact of the mutations on protein stability and interactions was predicted using mutation Cutoff Scanning Matrix (mCSM) tools. The atpE Ile66Val mutation did not elevate the MIC above the critical concentration (MIC 0.25-0.5 µg/ml), while the MIC of the Rv0678 Thr33Ala mutant strains (> 1.0 µg/ml) classifies the strain as resistant, confirming clinical findings. In silico analyses confirmed that the atpE Ile66Val mutation minimally disrupts the bedaquiline-ATP synthase interaction, while the Rv0678 Thr33Ala mutation substantially affects the DNA binding affinity of the MmpR transcriptional repressor. Based on a combination of wet-lab and computational methods, our results suggest that the Rv0678 Thr33Ala mutation confers resistance to BDQ, while the atpE Ile66Val mutation does not, but definite proof can only be provided by complementation studies given the presence of secondary mutations.

The impact of genotype on the phenotype of Mycobacterium tuberculosis ΔsufR mutants.

Iron-sulphur (FeS) cluster biogenesis is a tightly regulated process in vivo. In Mycobacterium tuberculosis (Mtb), SufR functions as a transcriptional repressor of the operon encoding the primary FeS cluster biogenesis system. Previously, three independently isolated mutants (ΔRv1460stop_1.19, ΔRv1460stop _5.19 and ΔRv1460stop _5.20) harbouring the same deletion in sufR, displayed different growth kinetics in OADC supplemented 7H9 media. To investigate this discrepancy, we performed whole genome sequencing of the 3 mutants and the wild-type progenitor. Single nucleotide polymorphisms (SNPs) were identified in 3 genes in the ΔRv1460stop_1.19 mutant and one gene in the ΔRv1460stop_5.20 mutant. Phenotyping of the ΔRv1460stop_5.19 mutant, which had no additional SNPs, revealed increased susceptibility to clofazimine, DMNQ and menadione, while uptake and survival in THP-1 cells were not significantly different from the wild-type strain. Given that these results differ from those reported for other sufR deletion mutants (ΔSufRMTB and MtbΔSufR), they suggest that the position of the sufR deletion and the genotype of the progenitor strain impact the resulting phenotype.

Investigating Mycobacterium tuberculosis sufR (rv1460) in vitro and ex vivo expression and immunogenicity.

Iron is vital metal for Mycobacterium tuberculosis infection, survival, and persistence within its human host. The mobilization of sulphur (SUF) operon encodes the primary iron-sulphur (Fe-S) biogenesis system in M. tuberculosis and is induced during iron limitation and intracellular growth of M. tuberculosis, pointing to its importance during infection. To study sufR expression at single cell level during intracellular growth of M. tuberculosis a fluorescent reporter was generated by cloning a 123 bp sufR promoter region upstream of a promotorless mcherry gene in an integrating vector. Expression analysis and fluorescence measurements during in vitro culture revealed that the reporter was useful for measuring induction of the promoter but was unable to detect subsequent repression due to the stability of mCherry. During intracellular growth in THP-1 macrophages, increased fluorescence was observed in the strain harbouring the reporter relative to the control strain, however this induction was only observed in a small sub-set of the population. Since SufR levels are predicted to be elevated during infection we hypothesize that it is immunogenic and may induce an immune response in M. tuberculosis infected individuals. The immune response elicited by SufR for both whole blood assay (WBA, a short term 12-hr stimulation to characterise the production of cytokines/growth factors suggestive of an effector response) and lymphocyte proliferation assay (LPA, a longer term 7-day stimulation to see if SufR induces a memory type immune response) were low and did not show a strong immune response for the selected Luminex analytes (MCP-1, RANTES, IL-1b, IL-8, MIP-1b, IFN-g, IL-6 and MMP-9) measured in three clinical groups, namely active TB, QuantiFERON positive (QFN pos) and QFN negative (QFN neg) individuals.

The Mycobacterium smegmatis HesB Protein, MSMEG_4272, Is Required for In Vitro Growth and Iron Homeostasis.

A-type carrier (ATC) proteins are proposed to function in the biogenesis of Fe-S clusters, although their exact role remains controversial. The genome of Mycobacterium smegmatis encodes a single ATC protein, MSMEG_4272, which belongs to the HesB/YadR/YfhF family of proteins. Attempts to generate an MSMEG_4272 deletion mutant by two-step allelic exchange were unsuccessful, suggesting that the gene is essential for in vitro growth. CRISPRi-mediated transcriptional knock-down of MSMEG_4272 resulted in a growth defect under standard culture conditions, which was exacerbated in mineral-defined media. The knockdown strain displayed reduced intracellular iron levels under iron-replete conditions and increased susceptibility to clofazimine, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and isoniazid, while the activity of the Fe-S containing enzymes, succinate dehydrogenase, and aconitase were not affected. This study suggests that MSMEG_4272 plays a role in the regulation of intracellular iron levels and is required for in vitro growth of M. smegmatis, particularly during exponential growth.

Prioritizing health: Churches response to the COVID-19 pandemic.

Churches serve as a source of connection and support for spiritual wellbeing. More recently, church communities recognize the importance of extending support beyond spirituality and taking a holistic approach that includes mental and physical health. How each church goes about providing support varies among denominations and the needs of their communities. This exploratory study examines how churches of various denominations in the Tri-City region (Pomona, La Verne, and Claremont) of Los Angeles County perceive the seriousness of COVID-19, their responses to the pandemic, and the potential impact on their congregations. Results indicated that the majority (84%) of spiritual community participants view COVID-19 as a threat to personal health, and are taking steps to minimize the threat to their congregations' health and surrounding communities. Implications for church leadership to consider when planning continued operations and congregant support in response to COVID-19 are discussed.

Host and Bacterial Iron Homeostasis, an Underexplored Area in Tuberculosis Biomarker Research.

Mycobacterium tuberculosis (Mtb) "a human adapted pathogen" has found multiple ways to manipulate the host immune response during infection. The human immune response to Mtb infection is a highly complex cascade of reactions, with macrophages as preferred intracellular location. Interaction with the host through infection gives rise to expression of specific gene products for survival and multiplication within the host. The signals that the pathogens encounter during infection cause them to selectively express genes in response to signals. One strategy to identify Mtb antigens with diagnostic potential is to identify genes that are specifically induced during infection or in specific disease stages. The shortcomings of current immunodiagnostics include the failure to detect progression from latent infection to active tuberculosis disease, and the inability to monitor treatment efficacy. This highlights the need for new tuberculosis biomarkers. These biomarkers should be highly sensitive and specific diagnosing TB infection, specifically distinguishing between latent infection and active disease. The regulation of iron levels by the host plays a crucial role in the susceptibility and outcome of Mtb infection. Of interest are the siderophore biosynthetic genes, encoded by the mbt-1 and mbt-2 loci and the SUF (mobilization of sulphur) operon (sufR-sufB-sufD-sufC-csd-nifU-sufT), which encodes the primary iron-sulphur cluster biogenesis system. These genes are induced during iron limitation and intracellular growth of Mtb, pointing to their importance during infection.

Interprofessional Education Activities for Students in Physician Assistant, Clinical Psychology, and Athletic Training Programs Utilizing Aspects of Team-Based and Problem-Based Learning Practices.

Interprofessional education (IPE) allows two or more professionals to learn from one another through partnership to improve patient outcomes. Implementation of IPE varies within health profession programs and universities, requiring programs to develop IPE activities that adhere to specific learning objectives or accreditation standards. These activities were a preliminary investigation on the feasibility of IPE activities at an institution with no substantial IPE infrastructure. Students integrated aspects of team-based learning (TBL), problem-based learning (PBL), and didactic components into diverse simulated patient cases with health profession students to develop skills in interdisciplinary patient-centered care. Lessons learned and future directions are discussed.

Genetic Manipulation of Non-tuberculosis Mycobacteria.

Non-tuberculosis mycobacteria (NTMs) comprise a large group of organisms that are phenotypically diverse. Analysis of the growing number of completed NTM genomes has revealed both significant intra-genus genetic diversity, and a high percentage of predicted genes that appear to be unique to this group. Most NTMs have not been studied, however, the rise in NTM infections in several countries has prompted increasing interest in these organisms. Mycobacterial research has recently benefitted from the development of new genetic tools and a growing number of studies describing the genetic manipulation of NTMs have now been reported. In this review, we discuss the use of both site-specific and random mutagenesis tools in NTMs, highlighting the challenges that exist in applying these techniques to this diverse group of organisms.

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

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 ß-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 ß-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.

SufT is required for growth of Mycobacterium smegmatis under iron limiting conditions.

Iron-sulphur (FeS) clusters are versatile cofactors required for a range of biological processes within cells. Due to the reactive nature of the constituent molecules, assembly and delivery of these cofactors requires a multi-protein machinery in vivo. In prokaryotes, SufT homologues are proposed to function in the maturation and transfer of FeS clusters to apo-proteins. This study used targeted gene deletion to investigate the role of SufT in the physiology of mycobacteria, using Mycobacterium smegmatis as a model organism. Deletion of the sufT gene in M. smegmatis had no impact on growth under standard culture conditions and did not significantly alter activity of the FeS cluster dependent enzymes succinate dehydrogenase (SDH) and aconitase (ACN). Furthermore, the ΔsufT mutant was no more sensitive than the wild-type strain to the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), or the anti-tuberculosis drugs isoniazid, clofazimine or rifampicin. In contrast, the ΔsufT mutant displayed a growth defect under iron limiting conditions, and an increased requirement for iron during biofilm formation. This data suggests that SufT is an accessory factor in FeS cluster biogenesis in mycobacteria which is required under conditions of iron limitation.

Rv1460, a SufR homologue, is a repressor of the suf operon in Mycobacterium tuberculosis.

Iron-sulphur (Fe-S) clusters are ubiquitous co-factors which require multi-protein systems for their synthesis. In Mycobacterium tuberculosis, the Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466 operon (suf operon) encodes the primary Fe-S cluster biogenesis system. The first gene in this operon, Rv1460, shares homology with the cyanobacterial SufR, which functions as a transcriptional repressor of the sufBCDS operon. Rv1460's function in M. tuberculosis has however not been determined. In this study, we demonstrate that M. tuberculosis mutants lacking a functional Rv1460 protein are impaired for growth under standard culture conditions. Elevated expression of Rv1460 and Rv1461 was observed in the mutant, implicating Rv1460 in the regulation of the suf operon. Binding of an Fe-S cluster to purified recombinant Rv1460 was confirmed by UV-visible spectroscopy and circular dichroism. Furthermore, three conserved cysteine residues, C203, C216 and C244, proposed to provide ligands for the coordination of an Fe-S cluster, were shown to be required for the function of Rv1460 in M. tuberculosis. Rv1460 therefore seems to be functionally analogous to cyanobacterial SufR.

Mycobacterial nucleoid associated proteins: An added dimension in gene regulation.

Nucleoid associated proteins (NAPs) are known organisers of chromosomal structure and regulators of transcriptional expression. The number of proposed NAPs in mycobacteria are significantly lower than the number identified in other organisms. An interesting feature of mycobacterial NAPs is their low sequence similarity with those in other species, a property that has hindered their identification. In this review, we discuss the current evidence for the proposed classification of six mycobacterial proteins, Lsr2, EspR, mIHF, HupB, MDP2 and NapM, as NAPs in mycobacterial species with an emphasis on their roles in modulating chromosome structure and transcriptional regulation. In addition, we highlight the technical difficulties associated with investigating and providing evidence for the classification of proteins as NAPs in mycobacteria. We also address the role of mycobacterial NAPs as mediators of stress responses and highlight the recent developments aimed at targeting NAP-DNA interactions for the development of novel anti-TB drugs.

bis-Molybdopterin guanine dinucleotide is required for persistence of Mycobacterium tuberculosis in guinea pigs.

Mycobacterium tuberculosis is able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo to bis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of the mobA homolog in M. tuberculosis was confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in a mobA deletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or more bis-MGD-dependent enzymes in the persistence of M. tuberculosis in guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen.

Ergothioneine is a secreted antioxidant in Mycobacterium smegmatis.

Ergothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle of Mycobacterium smegmatis revealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant of M. smegmatis lacking egtD (MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion of egtD from wild-type M. smegmatis and an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protecting M. smegmatis against oxidative stress and that ERG is able to partly compensate for the loss of MSH.

Functional analysis of molybdopterin biosynthesis in mycobacteria identifies a fused molybdopterin synthase in Mycobacterium tuberculosis.

Most mycobacterial species possess a full complement of genes for the biosynthesis of molybdenum cofactor (MoCo). However, a distinguishing feature of members of the Mycobacterium tuberculosis complex is their possession of multiple homologs associated with the first two steps of the MoCo biosynthetic pathway. A mutant of M. tuberculosis lacking the moaA1-moaD1 gene cluster and a derivative in which moaD2 was also deleted were significantly impaired for growth in media containing nitrate as a sole nitrogen source, indicating a reduced availability of MoCo to support the assimilatory function of the MoCo-dependent nitrate reductase, NarGHI. However, the double mutant displayed residual respiratory nitrate reductase activity, suggesting that it retains the capacity to produce MoCo. The M. tuberculosis moaD and moaE homologs were further analyzed by expressing these genes in mutant strains of M. smegmatis that lacked one or both of the sole molybdopterin (MPT) synthase-encoding genes, moaD2 and moaE2, and were unable to grow on nitrate, presumably as a result of the loss of MoCo-dependent nitrate assimilatory activity. Expression of M. tuberculosis moaD2 in the M. smegmatis moaD2 mutant and of M. tuberculosis moaE1 or moaE2 in the M. smegmatis moaE2 mutant restored nitrate assimilation, confirming the functionality of these genes in MPT synthesis. Expression of M. tuberculosis moaX also restored MoCo biosynthesis in M. smegmatis mutants lacking moaD2, moaE2, or both, thus identifying MoaX as a fused MPT synthase. By implicating multiple synthase-encoding homologs in MoCo biosynthesis, these results suggest that important cellular functions may be served by their expansion in M. tuberculosis.