scholarly journals Mutants of Mycobacterium tuberculosis Lacking Three of the Five rpf-Like Genes Are Defective for Growth In Vivo and for Resuscitation In Vitro

2005 ◽  
Vol 73 (5) ◽  
pp. 3038-3043 ◽  
Author(s):  
Katrina J. Downing ◽  
Vladimir V. Mischenko ◽  
Margarita O. Shleeva ◽  
Danielle I. Young ◽  
Michael Young ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Micrococcus Luteus ◽  
Most Probable Number ◽  
Infection Model ◽  
Term Survival ◽  
Probable Number ◽  
Mouse Infection Model ◽  
Nonculturable State

ABSTRACT Mycobacterium tuberculosis contains five genes, rpfA through rpfE, that bear significant homology to the resuscitation-promoting factor (rpf) gene of Micrococcus luteus, whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf-like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf-like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the three strains tested (the ΔrpfB, ΔrpfD, and ΔrpfE strains). In contrast, two multiple strains, KDT8 (ΔrpfA-mutation ΔrpfC ΔrpfB) and KDT9 (ΔrpfA ΔrpfC ΔrpfD), which lack three of the five rpf-like genes, were significantly yet differentially attenuated in a mouse infection model. These mutants were also unable to resuscitate spontaneously in vitro, demonstrating the importance of the Rpf-like proteins of M. tuberculosis in resuscitation from the nonculturable state. These results strongly suggest that the biological functions of the five rpf-like genes of M. tuberculosis are not wholly redundant and underscore the potential utility of these proteins as targets for therapeutic intervention.

scholarly journals An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism

2019 ◽  
Vol 116 (32) ◽  
pp. 15907-15913 ◽  
Author(s):  
Hua Wang ◽  
Alexander A. Fedorov ◽  
Elena V. Fedorov ◽  
Debbie M. Hunt ◽  
Angela Rodgers ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Resistance Mechanism ◽  
Infection Model ◽  
Enzymatic Assays ◽  
X Ray Crystallography ◽  
Mouse Infection Model ◽  
Leucine Catabolism ◽  
Global Population

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional β-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.

scholarly journals Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo

2004 ◽  
Vol 72 (1) ◽  
pp. 515-526 ◽  
Author(s):  
JoAnn M. Tufariello ◽  
William R. Jacobs, ◽  
John Chan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Stationary Phase ◽  
Essential Gene ◽  
Micrococcus Luteus ◽  
Expression Patterns ◽  
Active Growth ◽  
Prolonged Incubation ◽  
Aerosol Infection

ABSTRACT Mycobacterium tuberculosis possesses five genes with significant homology to the resuscitation-promoting factor (Rpf) of Micrococcus luteus. The M. luteus Rpf is a secreted ∼16-kDa protein which restores active growth to cultures of M. luteus rendered dormant by prolonged incubation in stationary phase. More recently, the Rpf-like proteins of M. tuberculosis have been shown to stimulate the growth of extended-stationary-phase cultures of Mycobacterium bovis BCG. These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function of M. tuberculosis Rpf homologues in vivo. To address these questions, we have disrupted each of the five rpf-like genes in M. tuberculosis Erdman, and analyzed the mutants for their growth in vitro and in vivo. In contrast to M. luteus, for which rpf is an essential gene, we find that all of the M. tuberculosis rpf deletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection. Analysis of rpf expression in M. tuberculosis cultures from early log phase through late stationary phase indicates that expression of the rpf-like genes is growth phase-dependent, and that the expression patterns of the five M. tuberculosis rpf genes, while overlapping to various degrees, are not uniform. We also provide evidence that mycobacterial rpf genes are expressed in vivo in the lungs of mice acutely infected with virulent M. tuberculosis.

scholarly journals Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model

2020 ◽  
Vol 75 (5) ◽  
pp. 1218-1227
Author(s):  
Samantha A Michaels ◽  
Han-Wei Shih ◽  
Bailin Zhang ◽  
Edelmar D Navaluna ◽  
Zhongsheng Zhang ◽  
...  
Keyword(s):  
Giardia Lamblia ◽  
Trna Synthetase ◽  
Luciferase Reporter ◽  
Infection Model ◽  
Cell Assay ◽  
Mouse Infection Model ◽  
Mouse Infection ◽  
Methionyl Trna Synthetase

Abstract Background Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. Objectives To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. Methods Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model. Results Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays. Conclusions The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

scholarly journals The Mycobacterium tuberculosis Extracytoplasmic-Function Sigma Factor SigL Regulates Polyketide Synthases and Secreted or Membrane Proteins and Is Required for Virulence

2005 ◽  
Vol 187 (20) ◽  
pp. 7062-7071 ◽  
Author(s):  
Mi-Young Hahn ◽  
Sahadevan Raman ◽  
Mauricio Anaya ◽  
Robert N. Husson
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Stress Responses ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Infection Model ◽  
Transcription Start Sites ◽  
Promoter Sequences ◽  
Extracytoplasmic Function Sigma Factor

ABSTRACT Mycobacterium tuberculosis sigL encodes an extracytoplasmic function (ECF) sigma factor and is adjacent to a gene for a membrane protein (Rv0736) that contains a conserved HXXXCXXC sequence. This motif is found in anti-sigma factors that regulate several ECF sigma factors, including those that control oxidative stress responses. In this work, SigL and Rv0736 were found to be cotranscribed, and the intracellular domain of Rv0736 was shown to interact specifically with SigL, suggesting that Rv0736 may encode an anti-sigma factor of SigL. An M. tuberculosis sigL mutant was not more susceptible than the parental strain to several oxidative and nitrosative stresses, and sigL expression was not increased in response to these stresses. In vivo, sigL is expressed from a weak SigL-independent promoter and also from a second SigL-dependent promoter. To identify SigL-regulated genes, sigL was overexpressed and microarray analysis of global transcription was performed. Four small operons, sigL (Rv0735)-Rv0736, mpt53 (Rv2878c)-Rv2877c, pks10 (Rv1660)-pks7 (Rv1661), and Rv1139c-Rv1138c, were among the most highly upregulated genes in the sigL-overexpressing strain. SigL-dependent transcription start sites of these operons were mapped, and the consensus promoter sequences TGAACC in the −35 region and CGTgtc in the −10 region were identified. In vitro, purified SigL specifically initiated transcription from the promoters of sigL, mpt53, and pks10. Additional genes, including four PE_PGRS genes, appear to be regulated indirectly by SigL. In an in vivo murine infection model, the sigL mutant strain showed marked attenuation, indicating that the sigL regulon is important in M. tuberculosis pathogenesis.

scholarly journals Simultaneous Administration of 2-Aminoethyl Diphenylborinate and Chloroquine Reverses Chloroquine Resistance in Malaria Parasites

2015 ◽  
Vol 59 (5) ◽  
pp. 2890-2892 ◽  
Author(s):  
Ehab Mossaad ◽  
Wakako Furuyama ◽  
Masahiro Enomoto ◽  
Satoru Kawai ◽  
Katsuhiko Mikoshiba ◽  
...  
Keyword(s):  
Chloroquine Resistance ◽  
Infection Model ◽  
Simultaneous Administration ◽  
Plasmodium Chabaudi ◽  
Content Type ◽  
Mouse Infection Model ◽  
Complete Reversal ◽  
The Mean

ABSTRACTA nearly complete reversal of chloroquine (CQ) resistance in the CQ-resistantPlasmodium falciparumK-1 strain, with a significant decrease in the mean ± standard deviation (SD) 50% inhibitory concentration (IC50) from 1,050 ± 95 nM to 14 ± 2 nM, was achievedin vitroby the simultaneous administration of 2-aminoethyl diphenylborinate (2-APB). The CQ resistance-reversing activity of 2-APB, which showed the same efficacy as verapamil, was also observed in anin vivomouse infection model with the CQ-resistantPlasmodium chabaudiAS(30CQ) strain.

scholarly journals In Vitro and In Vivo Activity of AS101 against Carbapenem-Resistant Acinetobacter baumannii

2021 ◽  
Vol 14 (8) ◽  
pp. 823
Author(s):  
Tsung-Ying Yang ◽  
Sung-Pin Tseng ◽  
Heather Nokulunga Dlamini ◽  
Po-Liang Lu ◽  
Lin Lin ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Antibacterial Activities ◽  
Treated Group ◽  
Infection Model ◽  
High Dose ◽  
Mouse Infection Model ◽  
Carbapenem Resistant ◽  
Time Kill

The increasing trend of carbapenem-resistant Acinetobacter baumannii (CRAB) worldwide has become a concern, limiting therapeutic alternatives and increasing morbidity and mortality rates. The immunomodulation agent ammonium trichloro (dioxoethylene-O,O′-) tellurate (AS101) was repurposed as an antimicrobial agent against CRAB. Between 2016 and 2018, 27 CRAB clinical isolates were collected in Taiwan. The in vitro antibacterial activities of AS101 were evaluated using broth microdilution, time-kill assay, reactive oxygen species (ROS) detection and electron microscopy. In vivo effectiveness was assessed using a sepsis mouse infection model. The MIC range of AS101 for 27 CRAB isolates was from 0.5 to 32 µg/mL, which is below its 50% cytotoxicity (approximately 150 µg/mL). Bactericidal activity was confirmed using a time-kill assay. The antibacterial mechanism of AS101 was the accumulation of the ROS and the disruption of the cell membrane, which, in turn, results in cell death. The carbapenemase-producing A. baumannii mouse sepsis model showed that AS101 was a better therapeutic effect than colistin. The mice survival rate after 120 h was 33% (4/12) in the colistin-treated group and 58% (7/12) in the high-dose AS101 (3.33 mg/kg/day) group. Furthermore, high-dose AS101 significantly decreased bacterial population in the liver, kidney and spleen (all p < 0.001). These findings support the concept that AS101 is an ideal candidate for further testing in future studies.

scholarly journals Discovery of (3-Benzyl-5-hydroxyphenyl)carbamates as New Antitubercular Agents with Potent In Vitro and In Vivo Efficacy

Molecules ◽  
2019 ◽  
Vol 24 (10) ◽  
pp. 2021 ◽  
Author(s):  
Ya-Juan Cheng ◽  
Zhi-Yong Liu ◽  
Hua-Ju Liang ◽  
Cui-Ting Fang ◽  
Niu-Niu Zhang ◽  
...  
Keyword(s):  
Oral Administration ◽  
Inhibitory Activity ◽  
Multidrug Resistant ◽  
Infection Model ◽  
Antitubercular Agents ◽  
In Vivo Efficacy ◽  
Mouse Infection Model ◽  
Good Potential

A series of 3-amino-5-benzylphenol derivatives were designed and synthesized. Among them, (3-benzyl-5-hydroxyphenyl)carbamates were found to exert good inhibitory activity against M. tuberculosis H37Ra, H37Rv and clinically isolated multidrug-resistant M. tuberculosis strains (MIC = 0.625–6.25 μg/mL). The privileged compounds 3i and 3l showed moderate cytotoxicity against cell line A549. Compound 3l also exhibited potent in vivo inhibitory activity on a mouse infection model via the oral administration. The results demonstrated 3-hydroxyphenylcarbamates as a class of new antitubercular agents with good potential.

scholarly journals In Vitro and In Vivo Activities of SCH 56592 (Posaconazole), a New Triazole Antifungal Agent, againstAspergillus and Candida

2000 ◽  
Vol 44 (8) ◽  
pp. 2017-2022 ◽  
Author(s):  
Anthony Cacciapuoti ◽  
David Loebenberg ◽  
Erik Corcoran ◽  
Fred Menzel ◽  
Eugene L. Moss ◽  
...  
Keyword(s):  
Body Weight ◽  
Antifungal Agent ◽  
Systemic Infection ◽  
Infection Model ◽  
Mouse Infection Model ◽  
Immunocompromised Mouse ◽  
Excellent Activity ◽  
Dose Dependent

ABSTRACT SCH 56592 (posaconazole), a new triazole antifungal agent, was tested in vitro, and its activity was compared to that of itraconazole against 39 Aspergillus strains and to that of fluconazole against 275 Candida and 9 Cryptococcus strains. The SCH 56592 MICs for Aspergillus ranged from ≤0.002 to 0.5 μg/ml, and those of itraconazole ranged from ≤0.008 to 1 μg/ml. The SCH 56592 MICs for Candida andCryptococcus strains ranged from ≤0.004 to 16 μg/ml, and those of fluconazole ranged from ≤0.062 to >64 μg/ml. SCH 56592 showed excellent activity against Aspergillus fumigatus andAspergillus flavus in a pulmonary mouse infection model. When administered therapeutically, the 50% protective doses (PD50s) of SCH 56592 ranged from 3.6 to 29.9 mg/kg of body weight, while the PD50s of SCH 56592 administered prophylactically ranged from 0.9 to 9.0 mg/kg; itraconazole administered prophylactically was ineffective (PD50s, >75 mg/kg). SCH 56592 was also very efficacious against fluconazole-susceptible, -susceptible dose-dependent, or -resistantCandida albicans strains in immunocompetent or immunocompromised mouse models of systemic infection. The PD50s of SCH 56592 administered therapeutically ranged from 0.04 to 15.6 mg/kg, while the PD50s of SCH 56592 administered prophylactically ranged from 1.5 to 19.4 mg/kg. SCH 56592 has excellent potential for therapy against seriousAspergillus or Candida infections.

scholarly journals Preclinical Testing of the Nitroimidazopyran PA-824 for Activity against Mycobacterium tuberculosis in a Series of In Vitro and In Vivo Models

2005 ◽  
Vol 49 (6) ◽  
pp. 2294-2301 ◽  
Author(s):  
Anne J. Lenaerts ◽  
Veronica Gruppo ◽  
Karen S. Marietta ◽  
Christine M. Johnson ◽  
Diane K. Driscoll ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Load ◽  
Multidrug Resistant ◽  
Infection Model ◽  
Significant Activity ◽  
Dependent Manner ◽  
In Vivo Models ◽  
Long Term Treatment

ABSTRACT This study extends earlier reports regarding the in vitro and in vivo efficacies of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis. PA-824 was tested in vitro against a broad panel of multidrug-resistant clinical isolates and was found to be highly active against all isolates (MIC < 1 μg/ml). The activity of PA-824 against M. tuberculosis was also assessed grown under conditions of oxygen depletion. PA-824 showed significant activity at 2, 10, and 50 μg/ml, similar to that of metronidazole, in a dose-dependent manner. In a short-course mouse infection model, the efficacy of PA-824 at 50, 100, and 300 mg/kg of body weight formulated in methylcellulose or cyclodextrin/lecithin after nine oral treatments was compared with those of isoniazid, rifampin, and moxifloxacin. PA-824 at 100 mg/kg in cyclodextrin/lecithin was as active as moxifloxacin at 100 mg/kg and isoniazid at 25 mg/kg and was slightly more active than rifampin at 20 mg/kg. Long-term treatment with PA-824 at 100 mg/kg in cyclodextrin/lecithin reduced the bacterial load below 500 CFU in the lungs and spleen. No significant differences in activity between PA-824 and the other single drug treatments tested (isoniazid at 25 mg/kg, rifampin at 10 mg/kg, gatifloxacin at 100 mg/kg, and moxifloxacin at 100 mg/kg) could be observed. In summary, its good activity in in vivo models, as well as its activity against multidrug-resistant M. tuberculosis and against M. tuberculosis isolates in a potentially latent state, makes PA-824 an attractive drug candidate for the therapy of tuberculosis. These data indicate that there is significant potential for effective oral delivery of PA-824 for the treatment of tuberculosis.

scholarly journals Expression Analysis of the Yersiniabactin Receptor GenefyuA and the Heme Receptor hemR ofYersinia enterocolitica In Vitro and In Vivo Using the Reporter Genes for Green Fluorescent Protein and Luciferase

2001 ◽  
Vol 69 (12) ◽  
pp. 7772-7782 ◽  
Author(s):  
Christoph A. Jacobi ◽  
Sebastian Gregor ◽  
Alexander Rakin ◽  
Jürgen Heesemann
Keyword(s):  
Green Fluorescent Protein ◽  
Expression Analysis ◽  
Peritoneal Cavity ◽  
Fluorescent Protein ◽  
Reporter Genes ◽  
Infection Model ◽  
Mouse Infection Model ◽  
Green Fluorescent

ABSTRACT The enteropathogenic Yersinia enterocolitica strains have several systems for scavenging iron from their environment. We have studied the expression of the fyuA gene, which encodes the outer membrane receptor for the siderophore yersiniabactin (Ybt), and the hemR gene, which encodes the receptor for heme, using the reporter genes gfp (encoding green fluorescent protein) and luc (encoding firefly luciferase). To study gene expression in vitro as well as in vivo, we have constructed several translational reporter gene fusions to monitor simultaneously expression of fyuA andhemR or expression of one gene by agfp-luc tandem reporter. Results of the in vitro expression analysis (liquid media) indicated that fyuA and hemR are strongly derepressed under iron starvation conditions, resulting in strong fluorescence and/or luminescence at 27°C. In the in vivo BALB/C mouse infection model, tissue-specific expression of fyuA andhemR reporter fusions was observed. Surprisingly,fyuA and hemR reporter constructs were weakly expressed by yersiniae located in the liver and intestinal lumen, whereas strong expression was found for yersiniae in the peritoneal cavity and moderate expression was found in the spleen. Strikingly, yersiniae carrying fyuA orhemR reporter fusions exhibited threefold-stronger signals when grown in the peritoneal cavity of mice than those growing under iron derepression in vitro. This hyperexpression suggests that besides Fur derepression, additional activators may be involved in the enhanced expression of fyuA and hemRunder peritoneal growth conditions. Differential expression of thefyuA and hemR reporter fusions could not be observed, suggesting similar regulation of fyuA andhemR in the mouse infection model.

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