Biology subjects

Li, S.-Y.

Publications and source records attributed to Li, S.-Y..

24 records · Page 2Linked to original sources

bioRxiv2020

Differential in vitro activity of individual drugs and bedaquiline-rifabutin combinations against actively multiplying and nutrient-starved Mycobacterium abscessus

Current treatment options for lung disease caused by Mycobacterium abscessus complex infections have limited effectiveness. To maximize the use of existing antibacterials and to help inform regimen design for treatment, we assessed the in vitro bactericidal activity of single drugs against actively multiplying and net non-replicating M. abscessus populations in nutrient-rich and nutrient starvation conditions, respectively. As single drugs, bedaquiline and rifabutin exerted bactericidal activity only against nutrient-starved and actively growing M. abscessus, respectively. However, when combined, both bedaquiline and rifabutin were able to specifically contribute bactericidal activity at relatively low, clinically relevant concentrations against both replicating and non-replicating bacterial populations. The addition of a third drug, amikacin, further enhanced the bactericidal activity of the bedaquiline-rifabutin combination against nutrient-starved M. abscessus. Overall, these in vitro data suggest that bedaquiline-rifabutin may be a potent backbone combination to support novel treatment regimens for M. abscessus infections. This rich dataset of differential time-and concentration-dependent activity of drugs, alone and together, against M. abscessus also highlights several issues affecting interpretation and translation of in vitro findings.

microbiology↗

bioRxiv2020

Mutations in fbiD(Rv2983) as a novel determinant of resistance to pretomanid and delamanid in Mycobacterium tuberculosis

The nitroimidazole pro-drugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10-5 CFU. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 91% of which occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance: fbiC (56%), fbiA (15%), ddn (12%), fgd (4%) and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983, a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance, but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

microbiology↗

bioRxiv2019

Contribution of pretomanid to novel regimens containing bedaquiline with either linezolid or moxifloxacin and pyrazinamide in murine models of tuberculosis

Novel regimens combining bedaquiline and pretomanid with either linezolid (BPaL regimen) or moxifloxacin and pyrazinamide (BPaMZ regimen) shorten the treatment duration needed to cure TB in BALB/c mice compared to the first-line regimen and have yielded promising results in initial clinical trials. However, the independent contribution of the investigational new drug pretomanid to the efficacy of BPaMZ has not been examined and its contribution to BPaL has been examined only over the first 2 months of treatment. In the present study, the addition of pretomanid to BL increased bactericidal activity, prevented emergence of bedaquiline resistance, and shortened the duration needed to prevent relapse with drug-susceptible isolates by at least 2 months in BALB/c mice. Addition of pretomanid to BMZ resulted in a 1 log10 greater CFU reduction after 1 month of treatment and/or reduced the number of mice relapsing in each of 2 experiments in BALB/c mice and in immunocompromised nude mice. Bedaquiline-resistant isolates were found at relapse in only one BMZ-treated nude mouse. Treatment of infection with a pyrazinamide-resistant mutant in BALB/c mice with BPaMZ prevented selection of bedaquiline-resistant mutants and reduced the proportion of mice relapsing compared to BMZ alone. Among severely ill C3HeB/FeJ mice with caseous pneumonia and cavitation, BPaMZ increased median survival ([≥]60 vs. 21 days) and reduced median lung CFU by 2.4 log10 at 1 month compared to BMZ. In conclusion, in 3 different mouse models, pretomanid contributed significantly to the efficacy of the BPaMZ and BPaL regimens, including restricting the selection of bedaquiline-resistant mutants.

pharmacology and toxicology↗

bioRxiv2018

Mutations in Rv2983 as a novel determinant of resistance to nitroimidazole drugs in Mycobacterium tuberculosis

Delamanid represents one of two novel antimicrobial classes approved to treat tuberculosis in over 40 years. Pretomanid is another promising nitroimidazole pro-drug in clinical development. Characterization of the full spectrum of mutations conferring resistance to nitroimidazoles and their related phenotypes in Mycobacterium tuberculosis will inform development of suitable genotypic and phenotypic drug susceptibility tests. Here, we used a range of pretomanid doses to select pretomanid-resistant mutants in two pathologically distinct murine TB models. The frequency of spontaneous pretomanid resistance mutations was approximately 10-5 CFU. Pretomanid demonstrated dose-dependent bactericidal activity and selective amplification of resistant mutants. Whole genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, 90% of which were found in 1 of 5 genes previously associated with nitroimidazole activation and resistance. The remaining 10% harbored isolated mutations in Rv2983. Complementing an Rv2983 mutant with a wild-type copy of Rv2983 restored wild-type susceptibility to pretomanid and delamanid, confirming that loss of Rv2983 function causes nitroimidazole resistance. By quantifying F420 and its precursor Fo in Mycobacterium smegmatis overexpressing Rv2983 and an M. tuberculosis Rv2983 mutant, we provide evidence that Rv2983 is necessary for F420 biosynthesis and nitroimidazole activation, perhaps as the guanylyltransferase CofC. F420H2-deficient mutants displayed hypersusceptibility to malachite green (MG), a selective decontaminant present in solid media used to isolate and propagate mycobacteria from clinical samples. The wide diversity of mutations causing high-level pretomanid resistance and MG hypersusceptibility of most mutants poses significant challenges to clinical detection of nitroimidazole resistance using either genotypic or phenotypic methods.\n\nSignificanceNitroimidazole pro-drugs represent a promising new class of anti-tuberculosis drugs. Reliable methods to assure nitroimidazole susceptibility are critical to assure their optimal use. Yet, the spectrum of nitroimidazole resistance mutations remains incompletely characterized. Using 161 pretomanid-resistant Mycobacterium tuberculosis isolates selected in pretomanid-treated mice, we discovered a novel resistance determinant, Rv2983, required for cofactor F420 biosynthesis and characterized the remarkable diversity of mutations in this and 5 other genes involved in nitroimidazole activation. We show that F420H2-deficient nitroimidazole-resistant mutants are hypersusceptible to the selective decontaminant malachite green used in solid media to isolate mycobacteria and may evade detection on such media. These results have important implications for development and clinical use of genotypic and phenotypic methods for nitroimidazole susceptibility testing.

microbiology↗

bioRxiv2018

High-dose rifamycins enable shorter oral treatment in a murine model of Mycobacterium ulcerans disease

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is a neglected tropical skin and soft tissue infection that is associated with disability and social stigma. The mainstay of BU treatment is an eight-week course of 10 mg/kg rifampin (RIF) and 150 mg/kg streptomycin (STR). Recently, the injectable STR has been shown to be replaceable with oral clarithromycin (CLR) for smaller lesions for the last four weeks of treatment. A shorter, all-oral, highly efficient regimen for BU is needed, as the long treatment duration and indirect costs currently burden patients and health systems. Increasing the dose of RIF or replacing it with the more potent rifamycin drug rifapentine (RPT) could provide such a regimen. Here, we performed a dose-ranging experiment of RIF and RPT in combination with CLR over four weeks of treatment in a mouse model of M. ulcerans disease. A clear dose-dependent effect of RIF on both clinical and microbiological outcomes was found, with no ceiling effect observed with tested doses up to 40 mg/kg. RPT-containing regimens were more effective on M. ulcerans. All RPT-containing regimens achieved culture negativity after only four weeks while only the regimen with the highest RIF dose (40 mg/kg) did so. We conclude that there is dose-dependent efficacy of both RIF and RPT and that a ceiling effect is not reached with the current standard regimen used in the clinic. A regimen based on higher rifamycin doses that are currently being evaluated against tuberculosis in clinical trials could shorten and improve therapy of Buruli ulcer.

microbiology↗

bioRxiv2018

DNMT1 in Six2 progenitor cells is essential for transposable element silencing and kidney development

Cytosine methylation (5mC) plays a key role in maintaining progenitor cell self-renewal and differentiation. Here, we analyzed the role of 5mC in kidney development by genome-wide methylation, expression profiling, and by systematic genetic targeting of DNA methyltransferases (Dnmt) and Ten-eleven translocation methylcytosine hydroxylases (Tet).\n\nIn mice, nephrons differentiate from Six2+ progenitor cells, therefore we created animals with genetic deletion of Dnmt1, 3a, 3b, Tet1, and Tet2 in the Six2+ population (Six2Cre/Dnmt1flox/flox, Six2Cre/Dnmt3aflox/flox, Six2Cre/Dnmt3bflox/flox, Six2Cre/Tet2flox/flox and Tet1-/-). Animals with conditional deletion of Dnmt3a, 3b, Tet1 and Tet2 showed no significant structural or functional renal abnormalities. On the other hand, Six2Cre/Dnmt1flox/flox mice died within 24hrs of birth. Dnmt1 knock-out animals had small kidneys and significantly reduced nephron number. Genome-wide methylation analysis indicated marked loss of methylation mostly on transposable elements. RNA sequencing detected endogenous retroviral (ERV) gene transcripts and early embryonic genes. Increase in levels of interferon (and RIG-I signaling) and apoptosis (Trp53) in response to ERV activity likely contributed to the phenotype development. Once epithelial differentiation was established, loss of Dnmt1, 3a, 3b, Tet1 or Tet2 in glomerular epithelial cells did not lead to functional or structural differences at baseline or following toxic glomerular injury.\n\nGenome-wide cytosine methylation and gene expression profiling showed that Dnmt1-mediated DNA methylation is essential for kidney development by preventing regression of progenitor cells into a primitive undifferentiated state and demethylation of transposable elements.\n\nSignificanceCytosine methylation of regulatory regions (promoters and enhancers) has been proposed to play a key role in establishing gene expression and thereby cellular phenotype. DNMT1 is the key enzyme responsible for maintaining methylation patterns during DNA replication. While the role of Dnmt1 has been described in multiple organs, here we identified a novel, critically important mechanism how Dnmt1 controls tissue progenitors. The greatest methylation difference in Dnmt1 knock-out mice was observed on transposable elements (TE), which resulted in increase of endogenous retroviruses and cell death. We believe that release of TE was a critically overlooked component of phenotype development in previous studies that our comprehensive genome wide methylation analysis allowed us to identify.\n\nCompeting interestsThe Susztak lab receives research support from Biogen, Boehringer Ingelheim, Celgene, GSK, Merck, Regeneron and ONO Pharma for work not related to this manuscript.

developmental biology↗