scholarly journals A high-frequency single nucleotide polymorphism in the MtrB sensor kinase in clinical strains of Mycobacterium tuberculosis alters its biochemical and physiological properties

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0256664
Author(s):  
Uchenna Watson Waturuocha ◽  
Athira P. J. ◽  
Krishna Kumar Singh ◽  
Vandana Malhotra ◽  
M. S. Krishna ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pathogenic Bacteria ◽  
Physiological Role ◽  
Dna Polymorphisms ◽  
Membrane Properties ◽  
Sensor Kinase ◽  
Bacterial Cell Division ◽  
Clinical Strains ◽  
The Impact

The DNA polymorphisms found in clinical strains of Mycobacterium tuberculosis drive altered physiology, virulence, and pathogenesis in them. Although the lineages of these clinical strains can be traced back to common ancestor/s, there exists a plethora of difference between them, compared to those that have evolved in the laboratory. We identify a mutation present in ~80% of clinical strains, which maps in the HATPase domain of the sensor kinase MtrB and alters kinase and phosphatase activities, and affects its physiological role. The changes conferred by the mutation were probed by in-vitro biochemical assays which revealed changes in signaling properties of the sensor kinase. These changes also affect bacterial cell division rates, size and membrane properties. The study highlights the impact of DNA polymorphisms on the pathophysiology of clinical strains and provides insights into underlying mechanisms that drive signal transduction in pathogenic bacteria.

scholarly journals A Low-Prevalence Single-Nucleotide Polymorphism in the Sensor Kinase PhoR in Mycobacterium tuberculosis Suppresses Its Autophosphatase Activity and Reduces Pathogenic Fitness: Implications in Evolutionary Selection

2021 ◽  
Vol 12 ◽  
Author(s):  
Uchenna Watson Waturuocha ◽  
M. S. Krishna ◽  
Vandana Malhotra ◽  
Narendra M. Dixit ◽  
Deepak Kumar Saini
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cells ◽  
Infection Model ◽  
Causative Organism ◽  
Sensor Kinase ◽  
Single Nucleotide ◽  
Clinical Strains ◽  
Evolutionary Forces ◽  
Low Prevalence ◽  
The Impact

The genome sequencing of Mycobacterium tuberculosis, the causative organism of tuberculosis, has significantly improved our understanding of the mechanisms that drive the establishment of infection and disease progression. Several clinical strains of M. tuberculosis exhibit single-nucleotide polymorphisms (SNPs), the implications of which are only beginning to be understood. Here, we examined the impact of a specific polymorphism in PhoR, the sensor kinase of the PhoPR two-component system. Biochemical analysis revealed reduced autophosphatase/ATPase activity, which led to enhanced downstream gene expression. We complemented M. tuberculosis H37Ra with the wild-type and mutant phoPR genes and characterized the strains in a cell line infection model. We provide an explanation for the low prevalence of the SNP in clinical strains (∼1%), as the mutation causes a survival disadvantage in the host cells. The study provides a rare example of selection of a signaling node under competing evolutionary forces, wherein a biochemically superior mutation aids bacterial adaptation within-host but has low fitness for infection and hence is not selected. Our study highlights the importance of accounting for such SNPs to test therapeutic and co-therapeutic methods to combat TB.

scholarly journals Staphylococcus aureus Serves as an Iron Source for Pseudomonas aeruginosa during In Vivo Coculture

2005 ◽  
Vol 187 (2) ◽  
pp. 554-566 ◽  
Author(s):  
Lauren M. Mashburn ◽  
Amy M. Jett ◽  
Darrin R. Akins ◽  
Marvin Whiteley
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Iron Acquisition ◽  
Low Oxygen ◽  
Dialysis Membrane ◽  
Opportunistic Human Pathogen ◽  
The Impact

ABSTRACT Pseudomonas aeruginosa is a gram-negative opportunistic human pathogen often infecting the lungs of individuals with the heritable disease cystic fibrosis and the peritoneum of individuals undergoing continuous ambulatory peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this study, a rat dialysis membrane peritoneal model was used to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo compared to in vitro controls. Included in this analysis are genes important for iron acquisition and growth in low-oxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in this environment. We propose a model where P. aeruginosa lyses S. aureus and uses released iron for growth in low-iron environments.

scholarly journals Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo

2016 ◽  
Vol 213 (5) ◽  
pp. 809-825 ◽  
Author(s):  
Yancheng Liu ◽  
Shumin Tan ◽  
Lu Huang ◽  
Robert B. Abramovitch ◽  
Kyle H. Rohde ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Cell ◽  
Stress Responses ◽  
Drug Action ◽  
Host Cells ◽  
Transcriptional Analysis ◽  
Drug Pressure ◽  
Cytokine Activation ◽  
The Impact

Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb’s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs’ primary targets. Mtb’s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb’s response to drug pressure and suggest novel routes for future drug discovery programs.

scholarly journals Mycofactocin Is Associated with Ethanol Metabolism in Mycobacteria

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Gopinath Krishnamoorthy ◽  
Peggy Kaiser ◽  
Laura Lozza ◽  
Karin Hahnke ◽  
Hans-Joachim Mollenkopf ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Treatment Options ◽  
Physiological Role ◽  
Acetic Acid Bacteria ◽  
Ethanol Metabolism ◽  
Ethanol Treatment ◽  
Primary Alcohols ◽  
Poor Growth ◽  
Content Type

ABSTRACTMycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in manyActinobacteria.Mycobacterium tuberculosisassimilates cholesterol during chronic infection, and itsin vitrogrowth in the presence of cholesterol requires most of the MFT biosynthesis genes (mftA,mftB,mftC,mftD,mftE, andmftF), although the reasons for this requirement remain unclear. To identify the function of MFT, we characterized MFT biosynthesis mutants constructed inMycobacterium smegmatis,M. marinum, andM. tuberculosis. We found that the growth deficit ofmftdeletion mutants in medium containing cholesterol—a phenotypic basis for gene essentiality prediction—depends on ethanol, a solvent used to solubilize cholesterol. Furthermore, functionality of MFT was strictly required for growth of free-living mycobacteria in ethanol and other primary alcohols. Among other genes encoding predicted MFT-associated dehydrogenases,MSMEG_6242was indispensable forM. smegmatisethanol assimilation, suggesting that it is a candidate catalytic interactor with MFT. Despite being a poor growth substrate, ethanol treatment resulted in a reductive cellular state with NADH accumulation inM. tuberculosis. During ethanol treatment,mftCmutant expressed the transcriptional signatures that are characteristic of respirational dysfunction and a redox-imbalanced cellular state. Counterintuitively, there were no differences in cellular bioenergetics and redox parameters inmftCmutant cells treated with ethanol. Therefore, further understanding of the function of MFT in ethanol metabolism is required to identify the cause of growth retardation of MFT mutants in cholesterol. Nevertheless, our results establish the physiological role of MFT and also provide new insights into the specific functions of MFT homologs in other actinobacterial systems.IMPORTANCETuberculosis is caused byMycobacterium tuberculosis, and the increasing emergence of multidrug-resistant strains renders current treatment options ineffective. Although new antimycobacterial drugs are urgently required, their successful development often relies on complete understanding of the metabolic pathways—e.g., cholesterol assimilation—that are critical for persistence and for pathogenesis ofM. tuberculosis. In this regard, mycofactocin (MFT) function appears to be important because its biosynthesis genes are predicted to be essential forM. tuberculosisin vitrogrowth in cholesterol. In determining the metabolic basis of this genetic requirement, our results unexpectedly revealed the essential function of MFT in ethanol metabolism. The metabolic dysfunction thereof was found to affect the mycobacterial growth in cholesterol which is solubilized by ethanol. This knowledge is fundamental in recognizing the bona fide function of MFT, which likely resembles the pyrroloquinoline quinone-dependent ethanol oxidation in acetic acid bacteria exploited for industrial production of vinegar.

scholarly journals Discovery of a novel stereospecific β-hydroxyacyl-CoA lyase/thioesterase shared by three metabolic pathways in Mycobacterium tuberculosis

2018 ◽  
Author(s):  
Hua Wang ◽  
Alexander A. Fedorov ◽  
Elena V. Fedorov ◽  
Deborah M. Hunt ◽  
Angela Rodgers ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Metabolic Pathways ◽  
Resistance Mechanism ◽  
Physiological Role ◽  
Vast Number ◽  
X Ray Crystallography ◽  
Leucine Catabolism ◽  
Orphan Enzyme

AbstractThe vast number of poorly characterised enzymes in Mycobacterium tuberculosis (Mtb) is one of the key barriers precluding a better understanding of the biology that underpins pathogenesis. Here, we investigated the Mtb orphan enzyme Rv2498c to delineate its physiological role. Our results from in vitro enzymatic assays, phylogenetic analysis, X-ray crystallography and in vivo Mtb experiments, de-orphan Rv2498c as a multi-functional β-hydroxyacyl-CoA lyase/thioesterase (β-HAClyase/thioesterase) that participates in three different metabolic pathways: L-leucine catabolism, itaconate dissimilation, and glyoxylate shunt. Moreover, the deletion of the rv2498c gene from the Mtb genome resulted in attenuation in the mouse model compared to infection with the parent strain. To the best of our knowledge, this is the first report of an (R)-3-hydroxyl-3-methylglutaryl-CoA for leucine catabolism and an itaconate-specific resistance mechanism in Mtb.

scholarly journals Contribution of rpoB Mutations to Development of Rifamycin Cross-Resistance in Mycobacterium tuberculosis

1998 ◽  
Vol 42 (7) ◽  
pp. 1853-1857 ◽  
Author(s):  
D. L. Williams ◽  
L. Spring ◽  
L. Collins ◽  
L. P. Miller ◽  
L. B. Heifets ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cross Resistance ◽  
In Vitro Mutagenesis ◽  
Missense Mutations ◽  
Resistance Mutations ◽  
Development Of Resistance ◽  
High Level ◽  
The Impact ◽  
Level Resistance

ABSTRACT The contributions of 23 insertion, deletion, or missense mutations within an 81-bp fragment of rpoB, the gene encoding the β-subunit of the DNA-dependent RNA polymerase of Mycobacterium tuberculosis, to the development of resistance to rifamycins (rifampin, rifabutin, rifapentine, and KRM-1648) in 29 rifampin-resistant clinical isolates were defined. Specific mutantrpoB alleles led to the development of cross-resistance to all rifamycins tested, while a subset of mutations were associated with resistance to rifampin and rifapentine but not to KRM-1648 or rifabutin. To further study the impact of specific rpoBmutant alleles on the development of rifamycin resistance, mutations were incorporated into the rpoB gene of M. tuberculosis H37Rv, contained on a mycobacterial shuttle plasmid, by in vitro mutagenesis. Recombinant M. tuberculosis clones containing plasmids with specific mutations in either codon 531 or 526 of rpoB exhibited high-level resistance to all rifamycins tested, whereas clones containing a plasmid with a mutation in codon 516 exhibited high-level resistance to rifampin and rifapentine but were susceptible to both rifabutin and KRM-1648. These results provided additional proof of the association of specificrpoB mutations with the development of rifamycin resistance and corroborate previous reports of the usefulness of rpoB genotyping for predicting rifamycin-resistant phenotypes.

scholarly journals DNA Gyrase Inhibition Assays Are Necessary To Demonstrate Fluoroquinolone Resistance Secondary togyrBMutations in Mycobacterium tuberculosis

2011 ◽  
Vol 55 (10) ◽  
pp. 4524-4529 ◽  
Author(s):  
Alix Pantel ◽  
Stéphanie Petrella ◽  
Stéphanie Matrat ◽  
Florence Brossier ◽  
Sylvaine Bastian ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Susceptibility Testing ◽  
Dna Gyrase ◽  
Fluoroquinolone Resistance ◽  
Wild Type ◽  
Content Type ◽  
Clinical Strains ◽  
Highly Active ◽  
The Impact

ABSTRACTThe main mechanism of fluoroquinolone (FQ) resistance inMycobacterium tuberculosisis mutation in DNA gyrase (GyrA2GyrB2), especially ingyrA. However, the discovery of unknown mutations ingyrBwhose implication in FQ resistance is unclear has become more frequent. We investigated the impact on FQ susceptibility of eightgyrBmutations inM. tuberculosisclinical strains, three of which were previously identified in an FQ-resistant strain. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineeredgyrBalleles by mutagenesis were overexpressed inEscherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. We demonstrated that the eight substitutions in GyrB (D473N, P478A, R485H, S486F, A506G, A547V, G551R, and G559A), recently identified in FQ-resistant clinical strains or encountered inM. tuberculosisstrains isolated in France, are not implicated in FQ resistance. These results underline that, as opposed to phenotypic FQ susceptibility testing, the DNA gyrase inhibition assay is the only way to prove the role of a DNA gyrase mutation in FQ resistance. Therefore, the use of FQ in the treatment of tuberculosis (TB) patients should not be ruled out only on the basis of the presence of mutations ingyrB.

THE ANTIBACTERIAL SPECTRUM OF USTILAGIC ACID

1953 ◽  
Vol 31 (6) ◽  
pp. 505-511
Author(s):  
R. W. Reed ◽  
M. A. Holder
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Oral Administration ◽  
Pathogenic Bacteria ◽  
Antibacterial Effect ◽  
Gram Negative ◽  
In Vitro Inhibition ◽  
Urine Levels ◽  
Antibacterial Spectrum ◽  
Serum And Urine

Ustilagic acid is shown to be relatively inactive against common Gram-positive and Gram-negative pathogenic bacteria and against Mycobacterium tuberculosis. Serum and urine levels in rabbits following oral administration are much lower than the concentration required for in vitro inhibition of most bacteria tested. Human serum depresses the antibacterial effect of ustilagic acid in vitro. The drug had no effect on the course of experimental infection in mice.

scholarly journals Inhibition of Response to Alpha Interferon by Mycobacterium tuberculosis

2003 ◽  
Vol 71 (5) ◽  
pp. 2487-2497 ◽  
Author(s):  
Savita Prabhakar ◽  
Yaming Qiao ◽  
Yoshihiko Hoshino ◽  
Michael Weiden ◽  
Antony Canova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tyrosine Phosphorylation ◽  
Pathogenic Bacteria ◽  
Binding Activity ◽  
Regulation Of Transcription ◽  
Dna Binding Activity ◽  
Transcription Factor Activation ◽  
Monocytes And Macrophages ◽  
The Impact ◽  
Stimulation Of

ABSTRACT We previously reported that infection by Mycobacterium tuberculosis, the causative agent of tuberculosis, leads to secretion of alpha/beta interferon (IFN-α/β). While IFN-α/β ordinarily stimulates formation of signal transducer and stimulator of transcription-1 (STAT-1) homodimers and IFN-stimulated gene factor-3 (ISGF-3), only ISGF-3 is found in infected human monocytes and macrophages. We have now investigated the basis for this unusual profile of transcription factor activation and its consequences for regulation of transcription, as well as the impact of infection on response to IFN-α. After infection, IFN-α stimulation of STAT-1 homodimers is inhibited in monocytes and macrophages, while stimulation of ISGF-3 increases in monocytes but tends to decline in macrophages. Effects of infection on the abundance of ISGF-3 subunits, STAT-1, STAT-2, and interferon regulatory factor 9, and on tyrosine phosphorylation of STAT-1 and STAT-2 explain the observed changes in DNA-binding activity, which correlate with increased or inhibited transcription of genes regulated by ISGF-3 and STAT-1. Infection by Mycobacterium bovis BCG does not inhibit IFN-α-stimulated tyrosine phosphorylation of STAT-1, formation of homodimers, or transcription of genes regulated by STAT-1 homodimers, suggesting that inhibition of the response to IFN-α/β by M. tuberculosis is an aspect of pathogenicity. Thus, this well-known feature of infection by pathogenic viruses may also be a strategy employed by pathogenic bacteria.

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