Pathogens

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20 pages, 2027 KiB

Open AccessArticle

Clinical Strains of Mycobacterium tuberculosis Representing Different Genotype Families Exhibit Distinct Propensities to Adopt the Differentially Culturable State

by Bhavna Gowan Gordhan, Kiyasha Padarath, Astika Sewcharran, Amanda McIvor, Michael S. VanNieuwenhze, Ziyaad Waja, Neil Martinson and Bavesh Davandra Kana

Pathogens 2024, 13(4), 318; https://doi.org/10.3390/pathogens13040318 - 12 Apr 2024

Viewed by 740

Abstract

Growing evidence points to the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens from individuals with active tuberculosis (TB) disease. These bacteria are unable to grow on solid media but can resuscitate in liquid media. Given the epidemiological success of certain [...] Read more.

Growing evidence points to the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens from individuals with active tuberculosis (TB) disease. These bacteria are unable to grow on solid media but can resuscitate in liquid media. Given the epidemiological success of certain clinical genotype families of Mycobacterium tuberculosis, we hypothesize that different strains may have distinct mechanisms of adaptation and tolerance. We used an in vitro carbon starvation model to determine the propensity of strains from lineages 2 and 4 that included the Beijing and LAM families respectively, to generate DCTB. Beijing strains were associated with a greater propensity to produce DCTB compared to LAM strains. Furthermore, LAM strains required culture filtrate (CF) for resuscitation whilst starved Beijing strains were not dependent on CF. Moreover, Beijing strains showed improved resuscitation with cognate CF, suggesting the presence of unique growth stimulatory molecules in this family. Analysis of starved Beijing and LAM strains showed longer cells, which with resuscitation were restored to a shorter length. Cell wall staining with fluorescent D-amino acids identified strain-specific incorporation patterns, indicating that cell surface remodeling during resuscitation was distinct between clinical strains. Collectively, our data demonstrate that M. tuberculosis clinical strains from different genotype lineages have differential propensities to generate DCTB, which may have implications for TB treatment success. Full article

(This article belongs to the Special Issue Biology of Mycobacterial Pathogens)

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28 pages, 8229 KiB

Open AccessArticle

In Silico Drug Repurposing Studies for the Discovery of Novel Salicyl-AMP Ligase (MbtA)Inhibitors

by Gourav Rakshit, Abanish Biswas and Venkatesan Jayaprakash

Pathogens 2023, 12(12), 1433; https://doi.org/10.3390/pathogens12121433 - 9 Dec 2023

Cited by 2 | Viewed by 1275

Abstract

Tuberculosis (TB) continues to pose a global health challenge, exacerbated by the rise of drug-resistant strains. The development of new TB therapies is an arduous and time-consuming process. To expedite the discovery of effective treatments, computational structure-based drug repurposing has emerged as a [...] Read more.

Tuberculosis (TB) continues to pose a global health challenge, exacerbated by the rise of drug-resistant strains. The development of new TB therapies is an arduous and time-consuming process. To expedite the discovery of effective treatments, computational structure-based drug repurposing has emerged as a promising strategy. From this perspective, conditionally essential targets present a valuable opportunity, and the mycobactin biosynthesis pathway stands out as a prime example highlighting the intricate response of Mycobacterium tuberculosis (Mtb) to changes in iron availability. This study focuses on the repurposing and revival of FDA-approved drugs (library) as potential inhibitors of MbtA, a crucial enzyme in mycobactin biosynthesis in Mtb conserved among all species of mycobacteria. The literature suggests this pathway to be associated with drug efflux pumps, which potentially contribute to drug resistance. This makes it a potential target for antitubercular drug discovery. Herein, we utilized cheminformatics and structure-based drug repurposing approaches, viz., molecular docking, dynamics, and PCA analysis, to decode the intermolecular interactions and binding affinity of the FDA-reported molecules against MbtA. Virtual screening revealed ten molecules with significant binding affinities and interactions with MbtA. These drugs, originally designed for different therapeutic indications (four antiviral, three anticancer, one CYP450 inhibitor, one ACE inhibitor, and one leukotriene antagonist), were repurposed as potential MbtA inhibitors. Furthermore, our study explores the binding modes and interactions between these drugs and MbtA, shedding light on the structural basis of their inhibitory potential. Principal component analysis highlighted significant motions in MbtA-bound ligands, emphasizing the stability of the top protein–ligand complexes (PLCs). This computational approach provides a swift and cost-effective method for identifying new MbtA inhibitors, which can subsequently undergo validation through experimental assays. This streamlined process is facilitated by the fact that these compounds are already FDA-approved and have established safety and efficacy profiles. This study has the potential to lay the groundwork for addressing the urgent global health challenge at hand, specifically in the context of combating antimicrobial resistance (AMR) and tuberculosis (TB). Full article

(This article belongs to the Special Issue Biology of Mycobacterial Pathogens)

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13 pages, 2034 KiB

Open AccessArticle

Comparison of Ultrastructure, Extracellular Matrix, and Drug Susceptibility in M. avium subs. hominissuis Biofilms

by William R. McManus and Jeffrey S. Schorey

Pathogens 2023, 12(12), 1427; https://doi.org/10.3390/pathogens12121427 - 8 Dec 2023

Viewed by 884

Abstract

Pulmonary infections with Mycobacterium avium occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. Many methods have been used to generate biofilms of M. avium, and it is unknown whether different approaches generate [...] Read more.

Pulmonary infections with Mycobacterium avium occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. Many methods have been used to generate biofilms of M. avium, and it is unknown whether different approaches generate similar structures and cell phenotypes. To make a parallel comparison of in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and the drug susceptibility of biofilm resident bacteria, we used two published methods to generate M. avium biofilms: four-week incubation in M63 medium or 24 h exposure to dithiothreitol (DTT). Scanning electron microscopy revealed differences in the biofilm ultrastructure between the two methods, including variation in the appearance of ECM materials and morphology of resident cells, while light microscopy and staining with calcofluor white indicated that both biofilms contained polysaccharides characteristic of cellulose. Measuring the susceptibility of biofilms to degradation by enzymes suggested differences in structurally important ECM molecules, with DTT biofilms having important protein and, to a lesser extent, cellulose components, and M63 biofilms having moderate protein, cellulose, and DNA components. Both biofilms conferred resistance to the bactericidal effects of amikacin and clarithromycin, with resident cells being killed at greater than 10-fold lower rates than planktonic cells at almost all concentrations. These comparisons indicate differences in biofilm responses by M. avium under differing conditions, but also suggest common features of biofilm formation, including cellulose production and antimicrobial resistance. Full article

(This article belongs to the Special Issue Biology of Mycobacterial Pathogens)

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14 pages, 5210 KiB

Open AccessArticle

A Loss of Function in LprG−Rv1410c Homologues Attenuates Growth during Biofilm Formation in Mycobacterium smegmatis

by Lisa-Marie Nisbett, Mary L. Previti and Jessica C. Seeliger

Pathogens 2023, 12(12), 1375; https://doi.org/10.3390/pathogens12121375 - 21 Nov 2023

Viewed by 1209

Abstract

MmpL (mycobacterial membrane protein large) proteins are integral membrane proteins that have been implicated in the biosynthesis and/or transport of mycobacterial cell wall lipids. Given the cellular location of these proteins, however, it is unclear how cell wall lipids are transported beyond the [...] Read more.

MmpL (mycobacterial membrane protein large) proteins are integral membrane proteins that have been implicated in the biosynthesis and/or transport of mycobacterial cell wall lipids. Given the cellular location of these proteins, however, it is unclear how cell wall lipids are transported beyond the inner membrane. Moreover, given that mycobacteria grow at the poles, we also do not understand how new cell wall is added in a highly localized and presumably coordinated manner. Here, we examine the relationship between two lipid transport pathways associated with the proteins MmpL11 and LprG−Rv1410c. The lipoprotein LprG has been shown to interact with proteins involved in cell wall processes including MmpL11, which is required in biofilms for the surface localization of certain lipids. Here we report that deletion of mmpL11 (MSMEG_0241) or the lprG−rv1410c operon homologues MSMEG_3070−3069 in Mycobacterium smegmatis produced similar biofilm defects that were distinct from that of the previously reported mmpL11 transposon insertion mutant. Analysis of pellicle biofilms, bacterial growth, lipid profiles, and gene expression revealed that the biofilm phenotypes could not be directly explained by changes in the synthesis or localization of biofilm-related lipids or the expression of biofilm-related genes. Instead, the shared biofilm phenotype between ΔMSMEG_3070−3069 and ΔmmpL11 may be related to their modest growth defect, while the origins of the distinct mmpL11::Tn biofilm defect remain unclear. Our findings suggest that the mechanisms that drive pellicle biofilm formation in M. smegmatis are not connected to crosstalk between the LprG−Rv1410c and MmpL11 pathways and that any functional interaction between these proteins does not relate directly to their lipid transport function. Full article

(This article belongs to the Special Issue Biology of Mycobacterial Pathogens)

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13 pages, 2023 KiB

Open AccessArticle

Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling

by Santosh Kumar, Steven Sega, Jamie K. Lynn-Barbe, Dannika L. Harris, Jordan T. Koehn, Debbie C. Crans and Dean C. Crick

Pathogens 2023, 12(9), 1171; https://doi.org/10.3390/pathogens12091171 - 18 Sep 2023

Viewed by 1107

Abstract

In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆¹-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of pathogenic M. tuberculosis. Highly active [...] Read more.

In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆¹-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of pathogenic M. tuberculosis. Highly active enzymes were expressed and purified using a Mycobacterium smegmatis expression system. The purified enzymes were characterized using natural substrates and chemically synthesized analogs. The structural requirements of the quinone electron acceptor were examined. PruB displayed activity with all tested lipoquinone analogs (naphthoquinone or benzoquinone). In PruB assays utilizing analogs of the native naphthoquinone [MK-9 (II-H₂)] specificity constants K_cat/K_m were an order of magnitude greater for the menaquinone analogs than the benzoquinone analogs. In addition, mycobacterial PruA was enzymatically characterized for the first time using exogenous chemically synthesized P5C. A K_m value of 120 ± 0.015 µM was determined for P5C, while the K_m value for NAD⁺ was shown to be 33 ± 4.3 µM. Furthermore, proline competitively inhibited PruA activity and coupled enzyme assays, suggesting that the recombinant purified monofunctional PruB and PruA enzymes of M. tuberculosis channel substrate likely increase metabolic flux and protect the bacterium from methylglyoxal toxicity. Full article

(This article belongs to the Special Issue Biology of Mycobacterial Pathogens)

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13 pages, 1511 KiB

Open AccessArticle

A Transcriptional Analysis of Cattle Immune Cells Reveals a Central Role of Type 1 Interferon in the In Vitro Innate Immune Response against Mycobacterium bovis

by Federico Carlos Blanco, María Mercedes Bigi, Elizabeth Andrea García, María Teresa Elola, Cristina Lourdes Vázquez and Fabiana Bigi

Pathogens 2023, 12(9), 1159; https://doi.org/10.3390/pathogens12091159 - 14 Sep 2023

Cited by 1 | Viewed by 1084

Abstract

Bovine tuberculosis is a chronic infectious disease primarily caused by Mycobacterium bovis, a bacterium that affects cattle and other mammals, including humans. Despite the availability of vast research about the immune response mechanisms of human tuberculosis caused by Mycobacterium tuberculosis, the [...] Read more.

Bovine tuberculosis is a chronic infectious disease primarily caused by Mycobacterium bovis, a bacterium that affects cattle and other mammals, including humans. Despite the availability of vast research about the immune response mechanisms of human tuberculosis caused by Mycobacterium tuberculosis, the knowledge of bovine tuberculosis’s immunology, particularly regarding the innate immune response, still remains scarce. In this study, we compared the transcriptome of cell cultures containing lymphocytes and M. bovis infected-macrophages with two strains of variable virulence, the virulent Mb04-303 strain and the attenuated Mb534. To that end, we infected bovine macrophages at a multiplicity of infection of one, and co-cultured the infections with autologous lymphocytes. RNA obtained from the co-cultures was sequenced to identify differentially expressed gene pathways by using the database Reactome. The RNA-seq analysis showed that the Mb04-303 infection upregulated the type 1 interferon signalling pathway, while it downregulated the KEAP1-NFE2L2 pathway. According to the literature, this last pathway is involved in the activation of antioxidant genes and inflammasome. In addition, the macrophages infected with Mb04-303 recruited more Galectin 8 than those infected with Mb534. This result indicates that Mb04-303 induced higher phagosome membrane damage, with the possible concomitant release of bacterial compounds into the cytoplasm that activates the type I signalling pathway. Altogether, Mb04-303 repressed the antioxidant and anti-inflammatory responses, likely impairing interleukin-1β activation, and trigged the canonical type 1 interferon signalling. Although these responses led to the control of bacterial replication during early infection, the virulent strain eventually managed to establish a successful infection. Full article

(This article belongs to the Special Issue Biology of Mycobacterial Pathogens)

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22 pages, 3012 KiB

Open AccessEditor’s ChoiceArticle

Mycobacterium tuberculosis Central Metabolism Is Key Regulator of Macrophage Pyroptosis and Host Immunity

by Michelle E. Maxson, Lahari Das, Michael F. Goldberg, Steven A. Porcelli, John Chan and William R. Jacobs, Jr.

Pathogens 2023, 12(9), 1109; https://doi.org/10.3390/pathogens12091109 - 30 Aug 2023

Cited by 1 | Viewed by 1909

Abstract

Metabolic dysregulation in Mycobacterium tuberculosis results in increased macrophage apoptosis or pyroptosis. However, mechanistic links between Mycobacterium virulence and bacterial metabolic plasticity remain ill defined. In this study, we screened random transposon insertions of M. bovis BCG to identify mutants that induce pyroptotic [...] Read more.

Metabolic dysregulation in Mycobacterium tuberculosis results in increased macrophage apoptosis or pyroptosis. However, mechanistic links between Mycobacterium virulence and bacterial metabolic plasticity remain ill defined. In this study, we screened random transposon insertions of M. bovis BCG to identify mutants that induce pyroptotic death of the infected macrophage. Analysis of the transposon insertion sites identified a panel of fdr (functioning death repressor) genes, which were shown in some cases to encode functions central to Mycobacterium metabolism. In-depth studies of one fdr gene, fdr8 (BCG3787/Rv3727), demonstrated its important role in the maintenance of M. tuberculosis and M. bovis BCG redox balance in reductive stress conditions in the host. Our studies expand the subset of known Mycobacterium genes linking bacterial metabolic plasticity to virulence and also reveal that the broad induction of pyroptosis by an intracellular bacterial pathogen is linked to enhanced cellular immunity in vivo. Full article

(This article belongs to the Special Issue Biology of Mycobacterial Pathogens)

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30 pages, 4405 KiB

Open AccessArticle

Genetic Underpinnings of Carotenogenesis and Light-Induced Transcriptome Remodeling in the Opportunistic Pathogen Mycobacterium kansasii

by Niklas Janisch, Keith Levendosky, William C. Budell and Luis E. N. Quadri

Pathogens 2023, 12(1), 86; https://doi.org/10.3390/pathogens12010086 - 5 Jan 2023

Cited by 2 | Viewed by 2871

Abstract

Mycobacterium kansasii (Mk) causes opportunistic pulmonary infections with tuberculosis-like features. The bacterium is well known for its photochromogenicity, i.e., the production of carotenoid pigments in response to light. The genetics defining the photochromogenic phenotype of Mk has not been investigated and [...] Read more.

Mycobacterium kansasii (Mk) causes opportunistic pulmonary infections with tuberculosis-like features. The bacterium is well known for its photochromogenicity, i.e., the production of carotenoid pigments in response to light. The genetics defining the photochromogenic phenotype of Mk has not been investigated and defined pigmentation mutants to facilitate studies on the role of carotenes in the bacterium’s biology are not available thus far. In this study, we set out to identify genetic determinants involved in Mk photochromogenicity. We screened a library of ~150,000 transposon mutants for colonies with pigmentation abnormalities. The screen rendered a collection of ~200 mutants. Each of these mutants could be assigned to one of four distinct phenotypic groups. The insertion sites in the mutant collection clustered in three chromosomal regions. A combination of phenotypic analysis, sequence bioinformatics, and gene expression studies linked these regions to carotene biosynthesis, carotene degradation, and monounsaturated fatty acid biosynthesis. Furthermore, introduction of the identified carotenoid biosynthetic gene cluster into non-pigmented Mycobacterium smegmatis endowed the bacterium with photochromogenicity. The studies also led to identification of MarR-type and TetR/AcrR-type regulators controlling photochromogenicity and carotenoid breakdown, respectively. Lastly, the work presented also provides a first insight into the Mk transcriptome changes in response to light. Full article

(This article belongs to the Special Issue Biology of Mycobacterial Pathogens)

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