Wanliang Shi

Pyrazinamide Inhibits Trans-Translation in Mycobacterium tuberculosis

The target of a first-line tuberculosis drug that acts against persister bacteria is identified. Pyrazinamide (PZA) is a first-line tuberculosis drug that plays a unique role in shortening the duration of tuberculosis chemotherapy. PZA is hydrolyzed intracellularly to pyrazinoic acid (POA) by pyrazinamidase (PZase, encoded by pncA), an enzyme frequently lost in PZA-resistant strains, but the target of POA in Mycobacterium tuberculosis has remained elusive. Here, we identify a previously unknown target of POA as the ribosomal protein S1 (RpsA), a vital protein involved in protein translation and the ribosome-sparing process of trans-translation. Three PZA-resistant clinical isolates without pncA mutation harbored RpsA mutations. RpsA overexpression conferred increased PZA resistance, and we confirmed that POA bound to RpsA (but not a clinically identified ΔAla mutant) and subsequently inhibited trans-translation rather than canonical translation. Trans-translation is essential for freeing scarce ribosomes in nonreplicating organisms, and its inhibition may explain the ability of PZA to eradicate persisting organisms.

Genetic Basis of Virulence Attenuation Revealed by Comparative Genomic Analysis of Mycobacterium tuberculosis Strain H37Ra versus H37Rv

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading infectious disease despite the availability of chemotherapy and BCG vaccine. The commonly used avirulent M. tuberculosis strain H37Ra was derived from virulent strain H37 in 1935 but the basis of virulence attenuation has remained obscure despite numerous studies. We determined the complete genomic sequence of H37Ra ATCC25177 and compared that with its virulent counterpart H37Rv and a clinical isolate CDC1551. The H37Ra genome is highly similar to that of H37Rv with respect to gene content and order but is 8,445 bp larger as a result of 53 insertions and 21 deletions in H37Ra relative to H37Rv. Variations in repetitive sequences such as IS6110 and PE/PPE/PE-PGRS family genes are responsible for most of the gross genetic changes. A total of 198 single nucleotide variations (SNVs) that are different between H37Ra and H37Rv were identified, yet 119 of them are identical between H37Ra and CDC1551 and 3 are due to H37Rv strain variation, leaving only 76 H37Ra-specific SNVs that affect only 32 genes. The biological impact of missense mutations in protein coding sequences was analyzed in silico while nucleotide variations in potential promoter regions of several important genes were verified by quantitative RT-PCR. Mutations affecting transcription factors and/or global metabolic regulations related to in vitro survival under aging stress, and mutations affecting cell envelope, primary metabolism, in vivo growth as well as variations in the PE/PPE/PE-PGRS family genes, may underlie the basis of virulence attenuation. These findings have implications not only for improved understanding of pathogenesis of M. tuberculosis but also for development of new vaccines and new therapeutic agents.

Mutations in panD encoding aspartate decarboxylase are associated with pyrazinamide resistance in Mycobacterium tuberculosis

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug susceptible and multidrug-resistant tuberculosis (MDR-TB). Resistance to PZA is most commonly associated with mutations in the pncA gene encoding nicotinamidase/pyrazinamidase which converts the prodrug PZA to the active form pyrazinoic acid (POA). RpsA (ribosomal protein S1) involved in trans-translation was recently shown to be a target of PZA and mutations in RpsA are found in some PZA-resistant TB strains. However, some other PZA-resistant strains lack mutations in either pncA or rpsA. To identify potential new mechanisms of PZA resistance, we isolated 174 in vitro mutants of M. tuberculosis H37Rv resistant to PZA to search for resistant isolates that do not have pncA or rpsA mutations. DNA sequencing revealed that 169 of the 174 (97.1%) PZA-resistant mutants had pncA mutations but 5 mutants lacked pncA or rpsA mutations. Whole genome sequencing analyses revealed that the 5 PZA-resistant mutants had different mutations all occurring in the same gene panD encoding aspartate decarboxylase, which is involved in synthesis of β-alanine that is a precursor for pantothenate and co-enzyme A biosynthesis. panD mutations were identified in naturally PZA-resistant Mycobacterium canetti strain and a PZA-resistant MDR-TB clinical isolate. Future studies are needed to address the role of panD mutations in PZA resistance and confirm PanD as a new target of PZA.

Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.

Identification of novel activity against Borrelia burgdorferi persisters using an FDA approved drug library

Although antibiotic treatment for Lyme disease is effective in the majority of cases, especially during the early phase of the disease, a minority of patients suffer from post-treatment Lyme disease syndrome (PTLDS). It is unclear what mechanisms drive this problem, and although slow or ineffective killing of Borrelia burgdorferi has been suggested as an explanation, there is a lack of evidence that viable organisms are present in PTLDS. Although not a clinical surrogate, insight may be gained by examining stationary-phase in vitro Borrelia burgdorferi persisters that survive treatment with the antibiotics doxycycline and amoxicillin. To identify drug candidates that can eliminate B. burgdorferi persisters more effectively, we screened an Food and Drug Administration (FDA)-approved drug library consisting of 1524 compounds against stationary-phase B. burgdorferi by using a newly developed high throughput SYBR Green I/propidium iodide (PI) assay. We identified 165 agents approved for use in other disease conditions that had more activity than doxycycline and amoxicillin against B. burgdorferi persisters. The top 27 drug candidates from the 165 hits were confirmed to have higher anti-persister activity than the current frontline antibiotics. Among the top 27 confirmed drug candidates from the 165 hits, daptomycin, clofazimine, carbomycin, sulfa drugs (e.g., sulfamethoxazole), and certain cephalosporins (e.g. cefoperazone) had the highest anti-persister activity. In addition, some drug candidates, such as daptomycin and clofazimine (which had the highest activity against non-growing persisters), had relatively poor activity or a high minimal inhibitory concentration (MIC) against growing B. burgdorferi. Our findings may have implications for the development of a more effective treatment for Lyme disease and for the relief of long-term symptoms that afflict some Lyme disease patients.

Identification of novel mutations associated with clofazimine resistance in Mycobacterium tuberculosis

OBJECTIVES Although clofazimine has been traditionally used to treat leprosy, there is recent interest in using clofazimine for the treatment of MDR-TB and drug-susceptible TB. However, the mechanisms of resistance to clofazimine are poorly understood. Here, we investigated the molecular basis of clofazimine resistance using resistant mutants isolated in vitro. METHODS We isolated 96 mutants of Mycobacterium tuberculosis resistant to clofazimine and performed WGS and Sanger sequencing to identify possible mutations associated with clofazimine resistance. RESULTS We found that 97% (93/96) of clofazimine-resistant mutants had a mutation in rv0678 encoding a transcription repressor for efflux pump MmpL5. Two mutational hot spots at nucleotide positions 193 and 466 in rv0678 accounted for 43.8% (42/96) and 11.5% (11/96) of the mutations, respectively. The previously reported A202G mutation (S68G) in rv0678 occurred less frequently, in 5 of 96 mutants. The remaining 34 mutations were scattered along the entire rv0678 gene. We discovered two new genes (rv1979c and rv2535c) associated with clofazimine resistance in mutants without rv0678 mutations. CONCLUSIONS Mutations in rv0678 are a major mechanism of clofazimine resistance. Our findings provide useful information for the design of new molecular tests for rapid detection of clofazimine resistance. Further studies are needed to address the role of rv1979c and rv2535c in clofazimine resistance and mechanisms of action.

An Optimized SYBR Green I/PI Assay for Rapid Viability Assessment and Antibiotic Susceptibility Testing for Borrelia burgdorferi

Lyme disease caused by Borrelia burgdorferi is the most common tick-borne disease in the US and Europe. Unlike most bacteria, measurements of growth and viability of B. burgdorferi are challenging. The current B. burgdorferi viability assays based on microscopic counting and PCR are cumbersome and tedious and cannot be used in a high throughput format. Here, we evaluated several commonly used viability assays including MTT and XTT assays, fluorescein diacetate assay, Sytox Green/Hoechst 33342 assay, the commercially available LIVE/DEAD BacLight assay, and SYBR Green I/PI assay by microscopic counting and by automated 96-well plate reader for rapid viability assessment of B. burgdorferi. We found that the optimized SYBR Green I/PI assay based on green to red fluorescence ratio is superior to all the other assays for measuring the viability of B. burgdorferi in terms of sensitivity, accuracy, reliability, and speed in automated 96-well plate format and in comparison with microscopic counting. The BSK-H medium which produced a high background for the LIVE/DEAD BacLight assay did not affect the SYBR Green I/PI assay, and the viability of B. burgdorferi culture could be directly measured using a microtiter plate reader. The SYBR Green I/PI assay was found to reliably assess the viability of planktonic as well as biofilm B. burgdorferi and could be used as a rapid antibiotic susceptibility test. Thus, the SYBR Green I/PI assay provides a more sensitive, rapid and convenient method for evaluating viability and antibiotic susceptibility of B. burgdorferi and can be used for high-throughput drug screens.

Critical Role of Toll-Like Receptor 9 in Morphine and Mycobacterium tuberculosis–Induced Apoptosis in Mice

Background Although it is established that opioid and Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive. Methodology/Principal Findings We report here that mice subjected to chronic morphine administration and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines (TNF-α, IL-1β, and IL-6) in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3β in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3β in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration. Conclusions/Significance These data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection.

A Drug Combination Screen Identifies Drugs Active against Amoxicillin-Induced Round Bodies of In Vitro Borrelia burgdorferi Persisters from an FDA Drug Library

Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10–20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi persisters in vitro, even when pre-treated with amoxicillin. These findings may have implications for improved treatment of Lyme disease.

Mutation in clpC1 encoding an ATP-dependent ATPase involved in protein degradation is associated with pyrazinamide resistance in Mycobacterium tuberculosis

Emerging Microbes & Infections (2017) 6, e8; doi:10.1038/emi.2017.1; published online 15 February 2017