The Role of Fatty Acid Metabolism in Drug Tolerance of Mycobacterium tuberculosis

Understanding the mechanisms underlying M. tuberculosis adaptive strategies to achieve drug tolerance is crucial for the identification of new targets and the development of new drugs. Here, we show that acetate medium triggers a drug-tolerant state in M. tuberculosis when challenged with antituberculosis (anti-TB) drugs.

Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5′-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that “man-made” fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.

Acquisition of cross-azole tolerance and aneuploidy in Candida albicans strains evolved to posaconazole

A number of in vitro studies have examined the acquisition of drug resistance to the triazole fluconazole, a first-line treatment for many Candida infections. Much less is known about posaconazole, a newer triazole. We conducted the first in vitro experimental evolution of replicates from eight diverse strains of C. albicans in a high level of the fungistatic drug posaconazole. Approximately half of the 132 evolved replicates survived 50 generations of evolution, biased towards some of the strain backgrounds. We found that although increases in drug resistance were rare, increases in drug tolerance (the slow growth of a subpopulation of cells in a level of drug above the resistance level) were common across strains. We also found that adaptation to posaconazole resulted in widespread cross-tolerance to other azole drugs. Widespread aneuploidy variation was also observed in evolved replicates from some strain backgrounds. Trisomy of chromosomes 3, 6, and R was identified in 11 of 12 whole-genome sequenced evolved SC5314 replicates. These findings document rampant evolved cross-tolerance among triazoles and highlight that increases in drug tolerance can evolve independently of drug resistance in a diversity of C. albicans strain backgrounds.

Tolerance to fluconazole in Candida albicans is regulated by temperature and aneuploidy

Candida albicans is a prevalent human fungal pathogen. Azoles are the most widely used antifungal drugs. Drug tolerance in bacteria is well defined and thoroughly studied, but in fungi, the definition of drug tolerance and the mechanism that drive it are not well understood. Here, we found that a large proportion of clinical isolates were intrinsically tolerant to fluconazole, and/or could be induced by high temperature (37°C) to become tolerant (conditionally tolerant). When treated with inhibitory doses of fluconazole, non-tolerant strains became tolerant by forming aneuploids involving different chromosomes, with chromosome R duplication as the most recurrent mechanism. Tolerance determines the ability to grow in the presence of fluconazole and other azoles, in a manner independent of the MIC. Both temperature conditional tolerance and the associated aneuploidy were sensitive to FK506, an inhibitor of calcineurin. Intrinsic and conditional tolerance were also abolished by deletions of genes encoding the calcineurin (CMP1 and CNB1). However, the dependence of tolerance on calcineurin could be bypassed by a different aneuploid chromosome. Thus, fluconazole tolerance in C. albicans is regulated by temperature and by aneuploidy and is dependent upon aneuploidy, but this dependence can be bypassed by an additional aneuploidy.

Drug tolerance facilitates the evolution of drug resistance in Candida albicans

Background For Candida albicans and Candidiasis, drug resistance is sometimes due to the pre-existence of genetic polymorphisms that bypass the mode of action of the drug, thus conferring a long-term survival benefit. In other cases, resistance is acquired via the evolution of de novo genetic polymorphisms. There is evidence that C. albicans possess a drug tolerance response which “buys time” for individuals to evolve beneficial mutations. Our goal here is to characterize this poorly understood epigenetic cytoprotective program at the single cell molecular level. Methods We developed a nano-litre droplet based Candida single cell sequencing platform capable of transcriptionally profiling several thousand individual cells in an efficient manner. We exploit this platform to profile both untreated and drug exposed (incl. fluconazole, caspofungin and nystatin) populations at early time points post-treatment (tolerance) and late time points (resistance) in order to understand survival trajectories. The profile are compared with the matched sequenced genomes. Results We show that untreated Candida populations exhibit “bet hedging”, stochastically expressing cytoprotective transcriptional programs, and drug tolerant individuals partition into distinct subpopulations, each with a unique survival strategy involving different transcriptional programs. We observe a burst of chromosomal aberrations at two days post-treatment that differ between survivor subpopulation. Discussion Our single cell approach highlights that survivor subpopulations pass through a tolerance phase that involves a multivariate transcriptional response including upregulation of efflux pumps, chaperones and transport mechanisms, and cell wall maintenance. Together this suggests that targeting the tolerance response concomitantly with standard therapies could represent an efficient approach to ablating clinical persistence.

Loss of GdpP function in Staphylococcus aureus leads to β-lactam tolerance and enhanced evolution of β-lactam resistance

Infections caused by Staphylococcus aureus are a leading cause of mortality. Treating infections caused by S. aureus is difficult due to resistance against most traditional antibiotics, including β-lactams. We previously reported the presence of mutations in gdpP among S. aureus strains that were obtained by serial passaging in β-lactam drugs. Similar mutations have recently been reported in natural S. aureus isolates that are either non-susceptible or resistant to β-lactam antibiotics. gdpP codes for a phosphodiesterase that cleaves cyclic-di-AMP (CDA), a newly discovered second messenger. In this study, we sought to identify the role of gdpP in β-lactam resistance in S. aureus . Our results showed that gdpP associated mutations caused loss of phosphodiesterase function, leading to increased CDA accumulation in the bacterial cytosol. Deletion of gdpP led to an enhanced ability of the bacteria to withstand a β-lactam challenge (two to three log increase in bacterial colony forming units) by promoting tolerance without enhancing MICs of β-lactam antibiotics. Our results demonstrated that increased drug tolerance due to loss of GdpP function can provide a selective advantage in acquisition of high-level β-lactam resistance. Loss of GdpP function thus increases tolerance to β-lactams that can lead to its therapy failure and can permit β-lactam resistance to occur more readily.

The Effects of Acute and Binge 3,4-methylenedioxymethamphetamine (MDMA) Exposure on Learning and Memory in Rats

<p>When rats are administered acute doses of MDMA they produce significantly more reference memory errors than working memory errors in the partially baited radial arm maze (Kay et al, 2009). The potential role of serotonin and dopamine in this effect was examined by administering the serotonin agonist Citalopram and the dopamine agonist GBR12909. GBR12909 produced significantly more reference memory errors, while Citalopram tended to produce more working memory errors. Administration of the D1 agonist A68930 and the D2 agonist Quinpirole predominantly produced reference memory errors, but to a lesser extent than acute MDMA administration. Low doses of both drugs produced a synergistic effect, more similar to that seen with acute MDMA administration. These findings suggest dopamine plays a role in the reference memory effect seen with MDMA exposure in the partially baited radial maze. In the second half of the thesis binge regimes of MDMA (4 x 10mg/kg) were administered to rats. When there was a gap of eight weeks between dosing and training the ability to acquire the radial arm maze was not significantly impaired. When this MDMA regime was repeated with a three-day gap between dosing and training it produced a significant but transient deficit in performance. When later challenged with acute doses of MDMA (4.0 mg/kg) the binge treated rats were less impaired than saline controls indicating drug tolerance. In an additional study that used a three-day delay between dosing and training a significant impairment in task acquisition was found. This deficit appeared to be long-term as the MDMA treated rats were impaired when the rules of task were changed suggesting a deficit in cognitive flexibility. Again when subjects were challenged with acute MDMA there was evidence of drug tolerance. The final study examined the effects of repeated MDMA exposure on task acquisition by administering acute doses of MDMA or saline once a week after rats had previously been treated with either a binge regime of MDMA or saline. MDMA exposure significantly impaired task acquisition and produced residual drug effects in the binge treated MDMA group the day after acute drug administration. However evidence of behavioural tolerance in this study was mixed due to a floor effect where performance of the binge MDMA group was so poor at the beginning of the study. In conclusion MDMA exposure impaired accuracy with reference memory processes were more affected than working memory processes. The underlying nature of this impairment remains unclear but it may be due to a long-term memory deficit, an impairment in understanding task rules or a perseverative pattern of responding. These findings imply human Ecstasy users may show deficits in acquiring information and may experience deficits in cognitive flexibility</p>

The Effects of Acute and Binge 3,4-methylenedioxymethamphetamine (MDMA) Exposure on Learning and Memory in Rats

<p>When rats are administered acute doses of MDMA they produce significantly more reference memory errors than working memory errors in the partially baited radial arm maze (Kay et al, 2009). The potential role of serotonin and dopamine in this effect was examined by administering the serotonin agonist Citalopram and the dopamine agonist GBR12909. GBR12909 produced significantly more reference memory errors, while Citalopram tended to produce more working memory errors. Administration of the D1 agonist A68930 and the D2 agonist Quinpirole predominantly produced reference memory errors, but to a lesser extent than acute MDMA administration. Low doses of both drugs produced a synergistic effect, more similar to that seen with acute MDMA administration. These findings suggest dopamine plays a role in the reference memory effect seen with MDMA exposure in the partially baited radial maze. In the second half of the thesis binge regimes of MDMA (4 x 10mg/kg) were administered to rats. When there was a gap of eight weeks between dosing and training the ability to acquire the radial arm maze was not significantly impaired. When this MDMA regime was repeated with a three-day gap between dosing and training it produced a significant but transient deficit in performance. When later challenged with acute doses of MDMA (4.0 mg/kg) the binge treated rats were less impaired than saline controls indicating drug tolerance. In an additional study that used a three-day delay between dosing and training a significant impairment in task acquisition was found. This deficit appeared to be long-term as the MDMA treated rats were impaired when the rules of task were changed suggesting a deficit in cognitive flexibility. Again when subjects were challenged with acute MDMA there was evidence of drug tolerance. The final study examined the effects of repeated MDMA exposure on task acquisition by administering acute doses of MDMA or saline once a week after rats had previously been treated with either a binge regime of MDMA or saline. MDMA exposure significantly impaired task acquisition and produced residual drug effects in the binge treated MDMA group the day after acute drug administration. However evidence of behavioural tolerance in this study was mixed due to a floor effect where performance of the binge MDMA group was so poor at the beginning of the study. In conclusion MDMA exposure impaired accuracy with reference memory processes were more affected than working memory processes. The underlying nature of this impairment remains unclear but it may be due to a long-term memory deficit, an impairment in understanding task rules or a perseverative pattern of responding. These findings imply human Ecstasy users may show deficits in acquiring information and may experience deficits in cognitive flexibility</p>

Temporal transcriptional response of Candida glabrata during macrophage infection reveals a multifaceted transcriptional regulator CgXbp1 important for macrophage response and drug resistance

Candida glabrata can thrive inside macrophages and tolerate high levels of azole antifungals. These innate abilities render infections by this human pathogen a clinical challenge. How C. glabrata reacts inside macrophages and what is the molecular basis of its drug tolerance are not well understood. Here, we mapped genome-wide RNA polymerase II (RNAPII) occupancy in C. glabrata to delineate its transcriptional responses during macrophage infection in high temporal resolution. RNAPII profiles revealed dynamic C. glabrata responses to macrophage with genes of specialized pathways activated chronologically at different times of infection. We identified an uncharacterized transcription factor (CgXbp1) important for the chronological macrophage response, survival in macrophages, and virulence. Genome-wide mapping of CgXbp1 direct targets further revealed its multi-faceted functions, regulating not only virulence-related genes but also genes associated with drug resistance. Finally, we showed that CgXbp1 indeed also affects azole resistance. Overall, this work presents a powerful approach for examining host-pathogen interaction and uncovers a novel transcription factor important for C. glabrata’s survival in macrophages and drug tolerance.