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  • American Society for Microbiology  (34)
  • Singh, Amit  (34)
  • 1
    Online Resource
    Online Resource
    Mucin Protein of Aedes aegypti Interacts with Dengue Virus 2 and Influences Viral Infection
    American Society for Microbiology ; 2023
    In:  Microbiology Spectrum Vol. 11, No. 2 ( 2023-04-13)
    Details
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 11, No. 2 ( 2023-04-13)
    Abstract: Dengue, caused by dengue virus (DENV), is the most prevalent vector-borne viral disease, posing a serious health concern to 2.5 billion people worldwide. DENV is primarily transmitted among humans by its mosquito vector Aedes aegypti ; hence, the identification of a novel dengue virus receptor in mosquitoes is critical for the development of new anti-mosquito measures. In the current study, we have identified peptides which potentially interact with the surface of the virion particles and facilitate virus infection and movement during their life cycle in the mosquito vector. To identify these candidate proteins, we performed phage-display library screening against domain III of the envelope protein (EDIII), which plays an essential role during host cell receptor binding for viral entry. The mucin protein, which shared sequence similarity with the peptide identified in the screening, was cloned, expressed, and purified for in vitro interaction studies. Using in vitro pulldown and virus overlay protein-binding assay (VOPBA), we confirmed the positive interaction of mucin with purified EDIII and whole virion particles. Finally, blocking of mucin protein with anti-mucin antibodies partially reduced DENV titers in infected mosquitos. Moreover, mucin protein was found to be localized in the midgut of Ae. aegypti . IMPORTANCE Identification of interacting protein partners of DENV in the insect vector Aedes aegypti is crucial for designing vector control-based strategies and for understanding the molecular mechanism DENV uses to modulate the host, gain entry, and survive successfully. Similar proteins can be used in generating transmission-blocking vaccines.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    URL: Article
    DOI: 10.1128/spectrum.02503-22
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 2807133-5
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  • 2
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    Performance of Xpert MTB/RIF Assay in Diagnosis of Pleural Tuberculosis by Use of Pleural Fluid Samples
    American Society for Microbiology ; 2015
    In:  Journal of Clinical Microbiology Vol. 53, No. 11 ( 2015-11), p. 3636-3638
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 53, No. 11 ( 2015-11), p. 3636-3638
    Abstract: Prospectively, 162 pleural fluid samples from patients with probable tuberculous pleural effusion were tested by the Xpert MTB/RIF assay and the Bactec MGIT-960 culture system. Of these, 43 (26.5%) were positive in the MGIT-960 culture, and 23 (14.2%), in the Xpert MTB/RIF assay. The sensitivity and specificity of the Xpert MTB/RIF compared with the MGIT-960 culture were 54.8% and 100%, respectively.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/JCM.02182-15
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2015
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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  • 3
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    Role of Mycobacterium tuberculosis Ser/Thr Kinase PknF: Implications in Glucose Transport and Cell Division
    American Society for Microbiology ; 2005
    In:  Journal of Bacteriology Vol. 187, No. 10 ( 2005-05-15), p. 3415-3420
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 187, No. 10 ( 2005-05-15), p. 3415-3420
    Abstract: Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF , one of the 11 kinases of Mycobacterium tuberculosis , encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis . Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis . Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3 H-labeled and 14 C-labeled substrates. A significant increase in the uptake of d -glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis .
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.187.10.3415-3420.2005
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2005
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 4
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    Moxifloxacin-Mediated Killing of Mycobacterium tuberculosis Involves Respiratory Downshift, Reductive Stress, and Accumulation of Reactive Oxygen Species
    American Society for Microbiology ; 2022
    In:  Antimicrobial Agents and Chemotherapy Vol. 66, No. 9 ( 2022-09-20)
    Details
    In: Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Vol. 66, No. 9 ( 2022-09-20)
    Abstract: Moxifloxacin is central to treatment of multidrug-resistant tuberculosis. Effects of moxifloxacin on the Mycobacterium tuberculosis redox state were explored to identify strategies for increasing lethality and reducing the prevalence of extensively resistant tuberculosis. A noninvasive redox biosensor and a reactive oxygen species (ROS)-sensitive dye revealed that moxifloxacin induces oxidative stress correlated with M. tuberculosis death. Moxifloxacin lethality was mitigated by supplementing bacterial cultures with an ROS scavenger (thiourea), an iron chelator (bipyridyl), and, after drug removal, an antioxidant enzyme (catalase). Lethality was also reduced by hypoxia and nutrient starvation. Moxifloxacin increased the expression of genes involved in the oxidative stress response, iron-sulfur cluster biogenesis, and DNA repair. Surprisingly, and in contrast with Escherichia coli studies, moxifloxacin decreased expression of genes involved in respiration, suppressed oxygen consumption, increased the NADH/NAD + ratio, and increased the labile iron pool in M. tuberculosis . Lowering the NADH/NAD + ratio in M. tuberculosis revealed that NADH-reductive stress facilitates an iron-mediated ROS surge and moxifloxacin lethality. Treatment with N -acetyl cysteine (NAC) accelerated respiration and ROS production, increased moxifloxacin lethality, and lowered the mutant prevention concentration. Moxifloxacin induced redox stress in M. tuberculosis inside macrophages, and cotreatment with NAC potentiated the antimycobacterial efficacy of moxifloxacin during nutrient starvation, inside macrophages, and in mice, where NAC restricted the emergence of resistance. Thus, NADH-reductive stress contributes to moxifloxacin-mediated killing of M. tuberculosis , and the respiration stimulator (NAC) enhances lethality and suppresses the emergence of drug resistance.
    Type of Medium: Online Resource
    ISSN: 0066-4804 , 1098-6596
    URL: Article
    DOI: 10.1128/aac.00592-22
    RVK:
    XA 24552
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 1496156-8
    SSG: 12
    SSG: 15,3
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  • 5
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    Mycobacterium tuberculosis Reactivates HIV-1 via Exosome-Mediated Resetting of Cellular Redox Potential and Bioenergetics
    American Society for Microbiology ; 2020
    In:  mBio Vol. 11, No. 2 ( 2020-04-28)
    Details
    In: mBio, American Society for Microbiology, Vol. 11, No. 2 ( 2020-04-28)
    Abstract: The synergy between Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) interferes with therapy and facilitates the pathogenesis of both human pathogens. Fundamental mechanisms by which M. tuberculosis exacerbates HIV-1 infection are not clear. Here, we show that exosomes secreted by macrophages infected with M. tuberculosis , including drug-resistant clinical strains, reactivated HIV-1 by inducing oxidative stress. Mechanistically, M. tuberculosis -specific exosomes realigned mitochondrial and nonmitochondrial oxygen consumption rates (OCR) and modulated the expression of host genes mediating oxidative stress response, inflammation, and HIV-1 transactivation. Proteomics analyses revealed the enrichment of several host factors (e.g., HIF-1α, galectins, and Hsp90) known to promote HIV-1 reactivation in M. tuberculosis -specific exosomes. Treatment with a known antioxidant—N-acetyl cysteine (NAC)—or with inhibitors of host factors—galectins and Hsp90—attenuated HIV-1 reactivation by M. tuberculosis - specific exosomes. Our findings uncover new paradigms for understanding the redox and bioenergetics bases of HIV- M. tuberculosis coinfection, which will enable the design of effective therapeutic strategies. IMPORTANCE Globally, individuals coinfected with the AIDS virus (HIV-1) and with M. tuberculosis (causative agent of tuberculosis [TB]) pose major obstacles in the clinical management of both diseases. At the heart of this issue is the apparent synergy between the two human pathogens. On the one hand, mechanisms induced by HIV-1 for reactivation of TB in AIDS patients are well characterized. On the other hand, while clinical findings clearly identified TB as a risk factor for HIV-1 reactivation and associated mortality, basic mechanisms by which M. tuberculosis exacerbates HIV-1 replication and infection remain poorly characterized. The significance of our research is in identifying the role of fundamental mechanisms such as redox and energy metabolism in catalyzing HIV- M. tuberculosis synergy. The quantification of redox and respiratory parameters affected by M. tuberculosis in stimulating HIV-1 will greatly enhance our understanding of HIV- M. tuberculosis coinfection, leading to a wider impact on the biomedical research community and creating new translational opportunities.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.03293-19
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
    detail.hit.zdb_id: 2557172-2
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  • 6
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    In Vitro Resistance against DNA Gyrase Inhibitor SPR719 in Mycobacterium avium and Mycobacterium abscessus
    American Society for Microbiology ; 2022
    In:  Microbiology Spectrum Vol. 10, No. 1 ( 2022-02-23)
    Details
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 10, No. 1 ( 2022-02-23)
    Abstract: The aminobenzimidazole SPR719 targets DNA gyrase in Mycobacterium tuberculosis . The molecule acts as inhibitor of the enzyme’s ATPase located on the Gyrase B subunit of the tetrameric Gyrase A 2 B 2 protein. SPR719 is also active against non-tuberculous mycobacteria (NTM) and recently entered clinical development for lung disease caused by these bacteria. Resistance against SPR719 in NTM has not been characterized. Here, we determined spontaneous in vitro resistance frequencies in single step resistance development studies, MICs of resistant strains, and resistance associated DNA sequence polymorphisms in two major NTM pathogens Mycobacterium avium and Mycobacterium abscessus . A low-frequency resistance (10 −8/ CFU) was associated with missense mutations in the ATPase domain of the Gyrase B subunit in both bacteria, consistent with inhibition of DNA gyrase as the mechanism of action of SPR719 against NTM. For M. abscessus , but not for M. avium , a second, high-frequency (10 −6/ CFU) resistance mechanism was observed. High-frequency SPR719 resistance was associated with frameshift mutations in the transcriptional repressor MAB_4384 previously shown to regulate expression of the drug efflux pump system MmpS5/MmpL5. Our results confirm DNA gyrase as target of SPR719 in NTM and reveal differential resistance development in the two NTM species, with M. abscessus displaying high-frequency indirect resistance possibly involving drug efflux. IMPORTANCE Clinical emergence of resistance to new antibiotics affects their utility. Characterization of in vitro resistance is a first step in the profiling of resistance properties of novel drug candidates. Here, we characterized in vitro resistance against SPR719, a drug candidate for the treatment of lung disease caused by non-tuberculous mycobacteria (NTM). The identified resistance associated mutations and the observed differential resistance behavior of the two characterized NTM species provide a basis for follow-up studies of resistance in vivo to further inform clinical development of SPR719.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    URL: Article
    DOI: 10.1128/spectrum.01321-21
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 2807133-5
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  • 7
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    Respiratory Quinone Switches from Menaquinone to Polyketide Quinone during the Development Cycle in Streptomyces sp. Strain MNU77
    American Society for Microbiology ; 2023
    In:  Microbiology Spectrum Vol. 11, No. 1 ( 2023-02-14)
    Details
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 11, No. 1 ( 2023-02-14)
    Abstract: Type III polyketide synthases (PKSs) found across Streptomyces species are primarily known for synthesis of a vast repertoire of clinically and industrially relevant secondary metabolites. However, our understanding of the functional relevance of these bioactive metabolites in Streptomyces physiology is still limited. Recently, a role of type III PKS harboring gene cluster in producing alternate electron carrier, polyketide quinone (PkQ) was established in a related member of the Actinobacteria , Mycobacteria , highlighting the critical role these secondary metabolites play in primary cellular metabolism of the producer organism. Here, we report the developmental stage-specific transcriptional regulation of homologous type III PKS containing gene cluster in freshwater Streptomyces sp. strain MNU77. Gene expression analysis revealed the type III PKS gene cluster to be stringently regulated, with significant upregulation observed during the dormant sporulation stage of Streptomyces sp. MNU77. In contrast, the expression levels of only known electron carrier, menaquinone biosynthetic genes were interestingly found to be downregulated. Our liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis of a metabolite extract from the Streptomyces sp. MNU77 spores also showed 10 times more metabolic abundance of PkQs than menaquinones. Furthermore, through heterologous complementation studies, we demonstrate that Streptomyces sp. MNU77 type III PKS rescues a respiratory defect of the Mycobacterium smegmatis type III PKS deletion mutant. Together, our studies reveal that freshwater Streptomyces sp. MNU77 robustly produces novel PkQs during the sporulation stage, suggesting utilization of PkQs as alternate electron carriers across Actinobacteria during dormant hypoxic conditions. IMPORTANCE The complex developmental life cycle of Streptomyces sp. mandates efficient cellular respiratory reconfiguration for a smooth transition from aerated nutrient-rich vegetative hyphal growth to the hypoxic-dormant sporulation stage. Polyketide quinones (PkQs) have recently been identified as a class of alternate electron carriers from a related member of the Actinobacteria , Mycobacteria , that facilitates maintenance of membrane potential in oxygen-deficient niches. Our studies with the newly identified freshwater Streptomyces sp. strain MNU77 show conditional transcriptional upregulation and metabolic abundance of PkQs in the spore state of the Streptomyces life cycle. In parallel, the levels of menaquinones, the only known Streptomyces electron carrier, were downregulated, suggesting deployment of PkQs as universal electron carriers in low-oxygen, unfavorable conditions across the Actinobacteria family.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    URL: Article
    DOI: 10.1128/spectrum.02597-22
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 2807133-5
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  • 8
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    Secondary Bacterial Infections in Mucormycosis-COVID-19 Cases: Experience during the Second COVID-19 Wave in India
    American Society for Microbiology ; 2022
    In:  Microbiology Spectrum Vol. 10, No. 6 ( 2022-12-21)
    Details
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 10, No. 6 ( 2022-12-21)
    Abstract: In the second wave of COVID-19 in India, there was a new challenge in the form of mucormycosis. Coinfection with mucormycosis was perilous as both conditions required a prolonged hospital stay, thus serving as an ideal platform for secondary infections. Using a retrospective observational study, we studied secondary infections and their impact on the outcome in COVID-19 patients with mucormycosis. The outcome in these patients was evaluated and compared with COVID-19 patients with mucormycosis but without any secondary infection. SPSS V-20 was used for data analysis. Fifty-five patients tested positive for mucormycosis (55/140; 39.28). Twelve out of these 55 (21.8%) developed secondary infections during their hospital stay. Bloodstream infection was the most common (42.86%) secondary infection. The Gram-negative (GN) organisms were more common (11/16; 68.75%) compared with the Gram-positives (GP) (5/16; 31.25%). But the most common isolate was Enterococcus faecium (5/16; 31.25%). A high percentage of microorganisms isolated were multidrug-resistant (15/16; 93.75%). Two out of five (40%) isolates of Enterococcus faecium were vancomycin-resistant (VRE). High resistance to carbapenems was noted in the GN isolates (9/11; 81.81%). The comparison of length of stay in both subgroups was statistically significant ( P value 〈 0.001). When compared, the length of stay in people with adverse outcomes was also statistically significant ( P value 〈 0.001). Procalcitonin (PCT) had a positive predictive value for the development of secondary bacterial infections ( P value 〈 0.001). Antimicrobial stewardship and strict infection control practices are the need of the hour. IMPORTANCE Although our knowledge about COVID-19 and secondary infections in patients is increasing daily, little is known about the secondary infections in COVID-19-mucormycosis patients. Thus, we have intended to share our experience regarding this subgroup. The importance of this study is that it brings to light the type of secondary infections seen in COVID-19-mucormycosis patients. These secondary infections were partially responsible for the mortality and morbidity of the unfortunate ones. We, as health care workers, can learn the lesson and disseminate the knowledge so that in similar situations, health care workers, even in other parts of the world, know what to expect.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    URL: Article
    DOI: 10.1128/spectrum.00919-22
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 2807133-5
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  • 9
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    Comparison of Xpert MTB/RIF with Line Probe Assay for Detection of Rifampin-Monoresistant Mycobacterium tuberculosis
    American Society for Microbiology ; 2014
    In:  Journal of Clinical Microbiology Vol. 52, No. 6 ( 2014-06), p. 1846-1852
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 52, No. 6 ( 2014-06), p. 1846-1852
    Abstract: The MTBDR plus line probe assay (LPA) and Xpert MTB/RIF have been endorsed by the World Health Organization for the rapid diagnosis of drug-resistant tuberculosis. However, there is no clarity regarding the superiority of one over the other. In a double-blinded prospective study, we evaluated the efficacy of the Xpert MTB/RIF on samples that were first tested by LPA under the revised national tuberculosis control program of India. A total of 405 sputum samples from suspected drug-resistant tuberculosis patients were included. Of these, 285 smear-positive samples were subjected to LPA. Seventy-two (25.8%) samples showed multidrug resistance, 62 (22.2%) showed rifampin monoresistance, 29 (10.3%) showed isoniazid monoresistance, and 116 (41.5%) were pan-susceptible. Six (2.1%) of the samples gave invalid results. Of the 62 rifampin-monoresistant samples by LPA, 38 (61.4%) showed rifampin resistance, while 21 (33.8%) were found susceptible to rifampin by Xpert MTB/RIF using cartridge version G4. Three (4.8%) samples gave an error. Of the 116 pan-susceptible samples, only 83 were available for Xpert MTB/RIF testing; 4 (5.1%) were rifampin resistant, 74 (94.8%) were susceptible, and 5 (6.0%) showed an error. The 25 discrepant samples were further subjected to MGIT960 drug susceptibility testing. The MGIT960 results showed 100% agreement with LPA results but only 64.4% agreement with Xpert MTB/RIF results. Sequencing analysis of discrepant samples showed 91.3% concordance with LPA but only 8.7% concordance with the Xpert MTB/RIF assay. These findings indicate that by using Xpert MTB/RIF testing we might be underestimating the burden of drug-resistant tuberculosis and indicate that country-specific probes need to be designed to increase the sensitivity of the Xpert MTB/RIF.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/JCM.03005-13
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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  • 10
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    Characterization of Phosphopantetheinyl Hydrolase from Mycobacterium tuberculosis
    American Society for Microbiology ; 2021
    In:  Microbiology Spectrum Vol. 9, No. 2 ( 2021-10-31)
    Details
    In: Microbiology Spectrum, American Society for Microbiology, Vol. 9, No. 2 ( 2021-10-31)
    Abstract: Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from Mycobacterium tuberculosis that removes 4′-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis . We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis , was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis ’s phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium’s cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB.
    Type of Medium: Online Resource
    ISSN: 2165-0497
    URL: Article
    DOI: 10.1128/Spectrum.00928-21
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2021
    detail.hit.zdb_id: 2807133-5
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