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Frontiers in Cellular and Infection... 2023() is a non-tuberculous (NTM) that can cause infectious diseases in aquatic animals and humans. Culture-based pathogen detection is the gold standard for diagnosing...
() is a non-tuberculous (NTM) that can cause infectious diseases in aquatic animals and humans. Culture-based pathogen detection is the gold standard for diagnosing NTM infection. However, this method is time-consuming and has low positivity rates for fastidious organisms. Oxford Nanopore MinION sequencing is an emerging third-generation sequencing technology that can sequence DNA or RNA directly in a culture-independent manner and offers rapid microbial identification. Further benefits include low cost, short turnaround time, long read lengths, and small equipment size. Nanopore sequencing plays a crucial role in assessing drug resistance, clinical identification of microbes, and monitoring infectious diseases. Some reports on (MTB) using nanopore sequencing have been published, however, there are few reports on NTM, such as . Here, we report the use of nanopore sequencing for the diagnosis of .
Topics: Animals; Humans; Nanopore Sequencing; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Communicable Diseases; High-Throughput Nucleotide Sequencing
PubMed: 37965260
DOI: 10.3389/fcimb.2023.1238872 -
Frontiers in Microbiology 2023Despite the great diversity of malonate semialdehyde decarboxylases (MSADs), one of five subgroups of the tautomerase superfamily (TSF) found throughout the biosphere,...
Phylogenetic distribution of malonate semialdehyde decarboxylase (MSAD) genes among strains within the genus : evidence of MSAD gene loss in the evolution of pathogenic mycobacteria.
Despite the great diversity of malonate semialdehyde decarboxylases (MSADs), one of five subgroups of the tautomerase superfamily (TSF) found throughout the biosphere, their distribution among strains within the genus remains unknown. In this study, we sought to investigate the phylogenetic distribution of MSAD genes of mycobacterial species via genome analysis of 192 different reference species or subspecies retrieved from NCBI databases. We found that in a total of 87 of 192 strains (45.3%), MSAD-1 and MSAD-2 were distributed in an exclusive manner among species except for 12 strains, including members, with both in their genome. Of note, strains better adapted to the host and of high virulence potential, such as the complex, , , , and subsp. , had no orthologs of MSAD in their genome, suggesting MSAD loss during species differentiation in pathogenic slow-growing . To investigate the MSAD distribution among strains of subspecies, the genome sequences of a total of 255 reference strains from the four subspecies of (43 of subspecies , 162 of subspecies , 49 of subspecies , and 1 of subspecies ) were further analyzed. We found that only 121 of 255 strains (47.4%) had MSADs in their genome, with none of the 49 subsp. strains having MSAD genes. Even in 13 of 121 strains with the MSAD-1 gene in their genome, deletion mutations in the 98th codon causing premature termination of MSAD were found, further highlighting the occurrence of MSAD pseudogenization during species or subspecies differentiation of In conclusion, our data indicated that there are two distinct types of MSADs, MSAD-1 and MSAD-2, among strains in the genus, but more than half of the strains, including pathogenic mycobacteria, and , have no orthologs in their genome, suggesting MSAD loss during host adaptation of pathogenic mycobacteria. In the future, the role of two distinct MSADs, MSAD-1 and MSAD-2, in mycobacterial pathogenesis or evolution should be investigated.
PubMed: 37901833
DOI: 10.3389/fmicb.2023.1275616 -
Microorganisms Oct 2023Hydroxamic acid (HA) derivatives display antibacterial and antifungal activities. HA with various numbers of carbon atoms (C, C, C, C, C and C), complexed with different...
Hydroxamic acid (HA) derivatives display antibacterial and antifungal activities. HA with various numbers of carbon atoms (C, C, C, C, C and C), complexed with different metal ions, including Fe(II/III), Ni(II), Cu(II) and Zn(II), were evaluated for their antimycobacterial activities and their anti-biofilm activities. Some derivatives showed antimycobacterial activities, especially in biofilm growth conditions. For example, 20-100 µM of HA10Fe2, HA10FeCl, HA10Fe3, HA10Ni2 or HA10Cu2 inhibited , BCG and biofilm development. HA10Fe2, HA12Fe2 and HA12FeCl could even attack pre-formed biofilms at higher concentrations (around 300 µM). The phthiocerol dimycocerosate (PDIM)-deficient H37Ra was more sensitive to the ion complexes of HA compared to other mycobacterial strains. Furthermore, HA10FeCl could increase the susceptibility of BCG to vancomycin. Proteomic profiles showed that the potential targets of HA10FeCl were mainly related to mycobacterial stress adaptation, involving cell wall lipid biosynthesis, drug resistance and tolerance and siderophore metabolism. This study provides new insights regarding the antimycobacterial activities of HA and their complexes, especially about their potential anti-biofilm activities.
PubMed: 37894269
DOI: 10.3390/microorganisms11102611 -
Frontiers in Immunology 2023In this work, we aimed at investigating cell and tissue responses of the apple snail , following the inoculation of the zoonotic pathogen . Different doses were tested...
In this work, we aimed at investigating cell and tissue responses of the apple snail , following the inoculation of the zoonotic pathogen . Different doses were tested (10, 20, 65, and 100 M CFU) and the mortality rate was negligible. The histopathogenesis was followed at 4, 9, and 28 days after inoculation. Overt histopathological lesions were consistently observed after the two largest doses only. In the lung, marked hemocyte aggregations, including intravascular nodule formation, were observed within the large blood veins that run along the floor and roof of this organ. Hemocyte aggregations were found occluding many of the radial sinuses supplying the respiratory lamina. Acid-fast bacilli were contained in the different hemocyte aggregations. In addition, hemocytes were observed infiltrating the storage tissue, which makes up most of the lung wall, and the connective tissue of the mantle edge. Additionally, signs of degradation in the storage tissue were observed in the lung wall on day 28. In the kidney, nodules were formed associated with the constitutive hemocyte islets and with the subpallial hemocoelic space, in whose hemocytes the acid-fast structures were found. Electron microscopy analysis revealed the presence of bacteria-containing phagosomes within hemocytes located in the surface zone of the islets. Additionally, electron-dense spheroidal structures, which are likely remnants of digested mycobacteria, were observed in close proximity to the hemocytes' nuclei. The size attained by the hemocyte nodules varied during the observation period, but there was no clear dependence on dose or time after inoculation. Nodules were also formed subpallially. Some of these nodules showed 2-3 layers with different cellular composition, suggesting they may also form through successive waves of circulating cells reaching them. Nodular cores, including those formed intravascularly in the lung, would exhibit signs of hemocyte dedifferentiation, possibly proliferation, and death. Hemocyte congestion was observed in the hemocoelic spaces surrounding the pallial ends of the renal crypts, and the renal crypts themselves showed de-epithelization, particularly on day 28. The diverse cellular responses of to inoculation and the high resilience of this snail to the pathogen make it a suitable species for studying mycobacterial infections and their effects on cellular and physiological processes.
Topics: Animals; Snails; Hemocytes; Microscopy, Electron; Phagosomes; Lung
PubMed: 37876924
DOI: 10.3389/fimmu.2023.1253099 -
BioRxiv : the Preprint Server For... Jan 2024( ), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS)...
UNLABELLED
( ), the pathogenic bacterium that causes tuberculosis, has evolved sophisticated defense mechanisms to counteract the cytotoxicity of reactive oxygen species (ROS) generated within host macrophages during infection. The gene in and ( ) plays a crucial role in defense mechanisms against ROS generated during infection. We demonstrate that encodes an epoxide hydrolase and contributes to ROS detoxification. Deletion of in resulted in a mutant with increased sensitivity to oxidative stress, increased accumulation of aldehyde species, and decreased production of mycothiol and ergothioneine. This heightened vulnerability is attributed to the increased expression of , a universal stress sensor. The absence of also resulted in reduced intracellular levels of NAD , NADH, and ATP. Bacterial growth was impaired, even in the absence of external stressors, and the impairment was carbon-source-dependent. Initial MelH substrate specificity studies demonstrate a preference for epoxides with a single aromatic substituent. Taken together, these results highlight the role of in mycobacterial bioenergetic metabolism and provide new insights into the complex interplay between redox homeostasis and generation of reactive aldehyde species in mycobacteria.
IMPORTANCE
This study unveils the pivotal role played by the gene in and in combatting the detrimental impact of oxidative conditions during infection. This investigation revealed notable alterations in the level of cytokinin-associated aldehyde, -hydroxybenzaldehyde, as well as the redox buffer ergothioneine, upon deletion of . Moreover, changes in crucial cofactors responsible for electron transfer highlighted 's crucial function in maintaining a delicate equilibrium of redox and bioenergetic processes. MelH prefers epoxide small substrates with a phenyl substituted substrate. These findings collectively emphasize the potential of as an attractive target for the development of novel antitubercular therapies that sensitize mycobacteria to host stress, offering new avenues for combating tuberculosis.
PubMed: 37873194
DOI: 10.1101/2023.10.02.560593 -
ImmunoHorizons Oct 2023The amphibian Xenopus laevis tadpole provides a unique comparative experimental organism for investigating the roles of innate-like T (iT) cells in tolerogenic immunity...
The amphibian Xenopus laevis tadpole provides a unique comparative experimental organism for investigating the roles of innate-like T (iT) cells in tolerogenic immunity during early development. Unlike mammals and adult frogs, where conventional T cells are dominant, tadpoles rely mostly on several prominent distinct subsets of iT cells interacting with cognate nonpolymorphic MHC class I-like molecules. In the present study, to investigate whole T cell responsiveness ontogenesis in X. laevis, we determined in tadpoles and adult frogs the capacity of splenic T cells to proliferate in vivo upon infection with two different pathogens, ranavirus FV3 and Mycobacterium marinum, as well as in vitro upon PHA stimulation using the thymidine analogous 5-ethynyl-2'-deoxyuridine and flow cytometry. We also analyzed by RT-quantitative PCR T cell responsiveness upon PHA stimulation. In vivo tadpole splenic T cells showed limited capacity to proliferate, whereas the in vitro proliferation rate was higher than adult T cells. Gene markers for T cell activation and immediate-early genes induced upon TCR activation were upregulated with similar kinetics in tadpole and adult splenocytes. However, the tadpole T cell signature included a lower amplitude in the TCR signaling, which is a hallmark of mammalian memory-like T cells and iT or "preset" T cells. This study suggests that reminiscent of mammalian neonatal T cells, tadpole T cells are functionally different from their adult counterpart.
Topics: Animals; Xenopus laevis; T-Lymphocytes; Larva; Cell Differentiation; Receptors, Antigen, T-Cell; Mammals
PubMed: 37870488
DOI: 10.4049/immunohorizons.2300081 -
Evolution, Medicine, and Public Health 2023Sex and reproductive status of the host have a major impact on the immune response against infection. Our aim was to understand their impact on host tolerance or...
Sex and reproductive status of the host have a major impact on the immune response against infection. Our aim was to understand their impact on host tolerance or resistance in the systemic infection of . We measured host survival and bacillary load at time of death, as well as expression by quantitative real-time polymerase chain reaction of immune genes (diptericin and drosomycin). We also assessed the impact of metabolic and hormonal regulation in the protection against infection by measuring expression of upd3, impl2 and ecR. Our data showed increased resistance in actively mating flies and in mated females, while reducing their tolerance to infection. Data suggests that Toll and immune deficiency (Imd) pathways determine tolerance and resistance, respectively, while higher basal levels of ecR favours the stimulation of the Imd pathway. A dual role has been found for upd3 expression, linked to increased/decreased mycobacterial load at the beginning and later in infection, respectively. Finally, impl2 expression has been related to increased resistance in non-actively mating males. These results allow further assessment on the differences between sexes and highlights the role of the reproductive status in to face infections, demonstrating their importance to determine resistance and tolerance against infection.
PubMed: 37868078
DOI: 10.1093/emph/eoad029 -
Acta Crystallographica. Section D,... Nov 2023Haloalkane dehalogenases (HLDs) are a family of α/β-hydrolase fold enzymes that employ S2 nucleophilic substitution to cleave the carbon-halogen bond in diverse...
Haloalkane dehalogenases (HLDs) are a family of α/β-hydrolase fold enzymes that employ S2 nucleophilic substitution to cleave the carbon-halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Å resolution. The structures show a canonical αβα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.
Topics: Mycobacterium marinum; Aspartic Acid; Stereoisomerism; Hydrolases; Substrate Specificity
PubMed: 37860958
DOI: 10.1107/S2059798323006642 -
The Journal of Steroid Biochemistry and... Dec 2023The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as...
The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as the initial activating step in their catabolism. However, several bacterial species contain multiple genes encoding CYP125 enzymes and other CYP enzymes which catalyze cholesterol/cholest-4-en-3-one hydroxylation. An important question is why these bacterium have more than one enzyme with overlapping substrate ranges capable of catalyzing the terminal oxidation of the alkyl chain of these sterols. To further understand the role of these enzymes we investigated CYP125A6 and CYP125A7 from Mycobacterium marinum with various cholesterol analogues. These have modifications on the A and B rings of the steroid and we assessed the substrate binding and catalytic activity of these with each enzyme. CYP125A7 gave similar results to those reported for the CYP125A1 enzyme from M. tuberculosis. Differences in the substrate binding and catalytic activity with the cholesterol analogues were observed with CYP125A6. For example, while cholesteryl sulfate could bind to both enzymes it was only oxidized by CYP125A6 and not by CYP125A7. CYP125A6 generated higher levels of metabolites with the majority of C-3 and C-7 substituted cholesterol analogues such 7-ketocholesterol. However, 5α-cholestan-3β-ol was only oxidized by CYP125A7 enzyme. The cholest-4-en-3-one and 7-ketocholesterol-bound forms of the CYP125A6 and CYP125A7 enzymes were modelled using AlphaFold. The structural models highlighted differences in the binding modes of the steroid derivatives within the same enzyme. Significant changes in the binding mode of the steroids between these CYP125 enzymes and other bacterial cholesterol oxidizing enzymes, CYP142A3 and CYP124A1, were also seen. Despite this, all these models predicted the selectivity for terminal methyl hydroxylation, in agreement with the experimental data.
Topics: Mycobacterium marinum; Oxidation-Reduction; Cytochrome P-450 Enzyme System; Steroids; Sterols; Mycobacterium tuberculosis
PubMed: 37793577
DOI: 10.1016/j.jsbmb.2023.106406 -
ACS Infectious Diseases Oct 2023By illuminating key 6-azasteroid-protein interactions in both () and the closely related model organism (), we sought to improve the antimycobacterial potency of...
By illuminating key 6-azasteroid-protein interactions in both () and the closely related model organism (), we sought to improve the antimycobacterial potency of 6-azasteroids and further our understanding of the mechanisms responsible for their potentiation of the antituberculosis drug bedaquiline. We selected a newly developed 6-azasteroid analog and an analog reported previously ( , (7), 1239-1251) to study their phenotypic effects on and , both alone and in combination with bedaquiline. The 6-azasteroid analog, 17β-[-(4-trifluoromethoxy-diphenylmethyl)carbamoyl]-6-propyl-azaandrostan-3-one, robustly potentiated bedaquiline-mediated antimycobacterial activity, with a nearly 8-fold reduction in bedaquiline minimal inhibitory concentration (85 nM alone versus 11 nM with 20 μM 6-azasteroid). This analog displayed minimal inhibitory activity against recombinant mycobacterial 3β-hydroxysteroid dehydrogenase, a previously identified target of several 6-azasteroids. Dose-dependent potentiation of bedaquiline by this analog reduced mycobacterial intracellular ATP levels and impeded the ability of to neutralize exogenous oxidative stress in culture. We developed two 6-azasteroid photoaffinity probes to investigate azasteroid-protein interactions in whole cells. Using bottom-up mass spectrometric profiling of the cross-linked proteins, we identified eight potential / protein targets for 6-azasteroids. The nature of these potential targets indicates that proteins related to oxidative stress resistance play a key role in the BDQ-potentiating activity of azasteroids and highlights the potential impact of inhibition of these targets on the generation of drug sensitivity.
Topics: Azasteroids; Antitubercular Agents; Mycobacterium tuberculosis; Bacterial Proteins; Mycobacterium marinum
PubMed: 37774412
DOI: 10.1021/acsinfecdis.3c00296