scholarly journals Transposon mobilization in the human fungal pathogen Cryptococcus deneoformans is mutagenic during infection and promotes drug resistance in vitro

2020 ◽  
Author(s):  
Asiya Gusa ◽  
Jonathan D. Williams ◽  
Jang-Eun Cho ◽  
Anna Floyd-Averette ◽  
Sheng Sun ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Murine Model ◽  
Target Genes ◽  
Opportunistic Infections ◽  
Antifungal Drugs ◽  
Rnai Pathway ◽  
Human Populations ◽  
Human Pathogens

ABSTRACTWhen transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeast-like fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNAi pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host.SIGNIFICANCE STATEMENTThe incidence of infections due to fungal pathogens has dramatically increased in the past few decades with similar increases in human populations with weakened or suppressed immune systems. Understanding the mechanisms by which organisms rapidly adapt during human infection to enhance virulence and evolve drug resistance is important for developing effective treatments. We find that transposon mobilization in the human pathogen Cryptococcus causes genomic mutations in a murine model of infection and promotes resistance to antifungal drugs in vitro. Thermotolerance is a key virulence determinant for pathogenic fungi during the environment-to-host transition, and we demonstrate that a temperature increase is sufficient to trigger transposon mobilization in vitro. The link between temperature stress and transposon-associated mutations may significantly impact adaptation to the host during infection, including the acquisition of drug resistance.

scholarly journals Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro

2020 ◽  
Vol 117 (18) ◽  
pp. 9973-9980 ◽  
Author(s):  
Asiya Gusa ◽  
Jonathan D. Williams ◽  
Jang-Eun Cho ◽  
Anna Floyd Averette ◽  
Sheng Sun ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Target Genes ◽  
Opportunistic Infections ◽  
Temperature Shift ◽  
Antifungal Drugs ◽  
Rnai Pathway ◽  
Human Pathogens ◽  
Human Fungal Pathogen

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 (rap/FK506) and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNA interference (RNAi) pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host.

scholarly journals Maximal killing of lymphoma cells by DNA damage–inducing therapy requires not only the p53 targets Puma and Noxa, but also Bim

Blood ◽  
2010 ◽  
Vol 116 (24) ◽  
pp. 5256-5267 ◽  
Author(s):  
Lina Happo ◽  
Mark S. Cragg ◽  
Belinda Phipson ◽  
Jon M. Haga ◽  
Elisa S. Jansen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Drug Resistance ◽  
Target Genes ◽  
B Cell Lymphoma ◽  
Chemotherapeutic Agents ◽  
Lymphoid Cells ◽  
Lymphoma Cells ◽  
P53 Target Genes

Abstract DNA-damaging chemotherapy is the backbone of cancer treatment, although it is not clear how such treatments kill tumor cells. In nontransformed lymphoid cells, the combined loss of 2 proapoptotic p53 target genes, Puma and Noxa, induces as much resistance to DNA damage as loss of p53 itself. In Eμ-Myc lymphomas, however, lack of both Puma and Noxa resulted in no greater drug resistance than lack of Puma alone. A third B-cell lymphoma-2 homology domain (BH)3-only gene, Bim, although not a direct p53 target, was up-regulated in Eμ-Myc lymphomas incurring DNA damage, and knockdown of Bim levels markedly increased the drug resistance of Eμ-Myc/Puma−/−Noxa−/− lymphomas both in vitro and in vivo. Remarkably, c-MYC–driven lymphoma cell lines from Noxa−/−Puma−/−Bim−/− mice were as resistant as those lacking p53. Thus, the combinatorial action of Puma, Noxa, and Bim is critical for optimal apoptotic responses of lymphoma cells to 2 commonly used DNA-damaging chemotherapeutic agents, identifying Bim as an additional biomarker for treatment outcome in the clinic.

scholarly journals The celecoxib derivative AR-12 has broad spectrum antifungal activity in vitro and improves the activity of fluconazole in a murine model of cryptococcosis

2016 ◽  
pp. AAC.01061-16 ◽  
Author(s):  
Kristy Koselny ◽  
Julianne Green ◽  
Louis DiDone ◽  
Justin P. Halterman ◽  
Annette W. Fothergill ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Small Molecules ◽  
Broad Spectrum ◽  
Murine Model ◽  
Antifungal Drugs ◽  
Dimorphic Fungi ◽  
New Class ◽  
Cancer Agent

Only one new class of antifungal drugs has been introduced into clinical practice in the last thirty years and, thus, the identification of small molecules with novel mechanisms of action is an important goal of current anti-infective research. Here, we describe the characterization of the spectrum of in vitro activity and in vivo activity of AR-12, a celecoxib-derivative which has been tested in a Phase I clinical trial as an anti-cancer agent. AR-12 inhibits fungal acetyl CoA synthetase in vitro and is fungicidal at concentrations similar to those achieved in human plasma. AR-12 has a broad spectrum of activity including active against yeasts (e.g.,C. albicans, non-albicansCandidaspp.,C. neoformans); molds (e.g.,Fusarium,Mucor), and dimorphic fungi (Blastomyces,Histoplasma, andCoccidioides) with minimum inhibitory concentrations of 2-4 μg/mL. AR-12 is also active against azole- and echinocandin-resistantCandidaisolates and sub-inhibitory AR-12 concentrations increase susceptibility of fluconazole- and echinocandin-resistantCandidaisolates. Finally, AR-12 also increases the activity of fluconazole in a murine model of cryptococcosis. Taken together, these data indicate that AR-12 represents a promising class of small molecules with broad spectrum antifungal activity.

scholarly journals Fungal biofilm architecture produces hypoxic microenvironments that drive antifungal resistance

2020 ◽  
Vol 117 (36) ◽  
pp. 22473-22483 ◽  
Author(s):  
Caitlin H. Kowalski ◽  
Kaesi A. Morelli ◽  
Daniel Schultz ◽  
Carey D. Nadell ◽  
Robert A. Cramer
Keyword(s):  
Drug Resistance ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Drug Resistant ◽  
Antifungal Drug Resistance ◽  
Oxygen Gradients ◽  
Biofilm Architecture

Human fungal infections may fail to respond to contemporary antifungal therapies in vivo despite in vitro fungal isolate drug susceptibility. Such a discrepancy between in vitro antimicrobial susceptibility and in vivo treatment outcomes is partially explained by microbes adopting a drug-resistant biofilm mode of growth during infection. The filamentous fungal pathogenAspergillus fumigatusforms biofilms in vivo, and during biofilm growth it has reduced susceptibility to all three classes of contemporary antifungal drugs. Specific features of filamentous fungal biofilms that drive antifungal drug resistance remain largely unknown. In this study, we applied a fluorescence microscopy approach coupled with transcriptional bioreporters to define spatial and temporal oxygen gradients and single-cell metabolic activity withinA. fumigatusbiofilms. Oxygen gradients inevitably arise duringA. fumigatusbiofilm maturation and are both critical for, and the result of,A. fumigatuslate-stage biofilm architecture. We observe that these self-induced hypoxic microenvironments not only contribute to filamentous fungal biofilm maturation but also drive resistance to antifungal treatment. Decreasing oxygen levels toward the base ofA. fumigatusbiofilms increases antifungal drug resistance. Our results define a previously unknown mechanistic link between filamentous fungal biofilm physiology and contemporary antifungal drug resistance. Moreover, we demonstrate that drug resistance mediated by dynamic oxygen gradients, found in many bacterial biofilms, also extends to the fungal kingdom. The conservation of hypoxic drug-resistant niches in bacterial and fungal biofilms is thus a promising target for improving antimicrobial therapy efficacy.

scholarly journals Distinct Roles of Candida albicans Drug Resistance Transcription FactorsTAC1,MRR1, andUPC2in Virulence

2013 ◽  
Vol 13 (1) ◽  
pp. 127-142 ◽  
Author(s):  
Andrea Lohberger ◽  
Alix T. Coste ◽  
Dominique Sanglard
Keyword(s):  
Candida Albicans ◽  
Target Genes ◽  
Negative Impact ◽  
Antifungal Drugs ◽  
Wild Type ◽  
Drug Pressure ◽  
Content Type ◽  
Negative Effect

ABSTRACTAzoles are widely used in antifungal therapy in medicine. Resistance to azoles can occur inCandida albicansprincipally by overexpression of multidrug transporter geneCDR1,CDR2, orMDR1or by overexpression ofERG11, which encodes the azole target. The expression of these genes is controlled by the transcription factors (TFs)TAC1(involved in the control ofCDR1andCDR2),MRR1(involved in the control ofMDR1), andUPC2(involved in the control ofERG11). Several gain-of-function (GOF) mutations are present in hyperactive alleles of these TFs, resulting in the overexpression of target genes. While these mutations are beneficial toC. albicanssurvival in the presence of the antifungal drugs, their effects could potentially alter the fitness and virulence ofC. albicansin the absence of the selective drug pressure. In this work, the effect of GOF mutations onC. albicansvirulence was addressed in a systemic model of intravenous infection by mouse survival and kidney fungal burden assays. We engineered a set of strains with identical genetic backgrounds in which hyperactive alleles were reintroduced in one or two copies at their genomic loci. The results obtained showed that neitherTAC1norMRR1GOF mutations had a significant effect onC. albicansvirulence. In contrast, the presence of two hyperactiveUPC2alleles inC. albicansresulted in a significant decrease in virulence, correlating with diminished kidney colonization compared to that by the wild type. In agreement with the effect on virulence, the decreased fitness of an isolate withUPC2hyperactive alleles was observed in competition experiments with the wild typein vivobut notin vitro. Interestingly,UPC2hyperactivity delayed filamentation ofC. albicansafter phagocytosis by murine macrophages, which may at least partially explain the virulence defects. Combining theUPC2GOF mutation with another hyperactive TF did not compensate for the negative effect ofUPC2on virulence. In conclusion, among the major TFs involved in azole resistance, onlyUPC2had a negative impact on virulence and fitness, which may therefore have consequences for the epidemiology of antifungal resistance.

scholarly journals The Alternative Sigma Factor RpoE2 Is Involved in the Stress Response to Hypochlorite and in vivo Survival of Haemophilus influenzae

2021 ◽  
Vol 12 ◽  
Author(s):  
Marufa Nasreen ◽  
Aidan Fletcher ◽  
Jennifer Hosmer ◽  
Qifeng Zhong ◽  
Ama-Tawiah Essilfie ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sigma Factor ◽  
Target Genes ◽  
Cell Envelope ◽  
Sigma Factors ◽  
Methionine Sulfoxide Reductase ◽  
Respiratory Pathogen ◽  
Human Pathogens

Extracytoplasmic function (ECF) sigma factors underpin the ability of bacteria to adapt to changing environmental conditions, a process that is particularly relevant in human pathogens that inhabit niches where human immune cells contribute to high levels of extracellular stress. Here, we have characterized the previously unstudied RpoE2 ECF sigma factor from the human respiratory pathogen H. influenzae (Hi) and its role in hypochlorite-induced stress. Exposure of H. influenzae to oxidative stress (HOCl, H2O2) increased rpoE2 gene expression, and the activity of RpoE2 was controlled by a cytoplasmic 67-aa anti-sigma factor, HrsE. RpoE2 regulated the expression of the periplasmic MsrAB peptide methionine sulfoxide reductase that, in H. influenzae, is required for HOCl resistance, thus linking RpoE2 to HOCl stress. Interestingly, a HiΔrpoE2 strain had wild-type levels of resistance to oxidative stress in vitro, but HiΔrpoE2 survival was reduced 26-fold in a mouse model of lung infection, demonstrating the relevance of this sigma factor for H. influenzae pathogenesis. The HiRpoE2 system has some similarity to the ECF sigma factors described in Streptomyces and Neisseria sp. that also control the expression of msr genes. However, HiRpoE2 regulation extended to genes encoding other periplasmic damage repair proteins, an operon containing a DoxX-like protein, and also included selected OxyR-controlled genes. Based on our results, we propose that the highly conserved HiRpoE2 sigma factor is a key regulator of H. influenzae responses to oxidative damage in the cell envelope region that controls a variety of target genes required for survival in the host.

scholarly journals In vivo activity of anprocide alone, and in vitro activity in combination with conventional antibiotics against Staphylococcus aureus and Staphylococcus epidermidis biofilms

2009 ◽  
Vol 58 (9) ◽  
pp. 1203-1206 ◽  
Author(s):  
Robin K. Pettit ◽  
Christine A. Weber ◽  
Stacey B. Lawrence ◽  
George R. Pettit ◽  
Melissa J. Kean ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Drug Resistance ◽  
Murine Model ◽  
Staphylococcus Epidermidis ◽  
Multiple Drug Resistance ◽  
Multiple Drug ◽  
In Vitro Activity ◽  
Conventional Antibiotics

The alarming spread of multiple drug resistance in Staphylococcus aureus, combined with the frequent occurrence of S. aureus and Staphylococcus epidermidis in biofilm-type infections, indicates a growing need for new therapies. The experimental steroidal amide anprocide [3β-acetoxy-17β-(l-prolyl)amino-5α-androstane] significantly reduced c.f.u. ml−1 per suture (P <0.0001) in a murine model of topical S. aureus infection. In chequerboard assays with planktonic-grown S. aureus and S. epidermidis, anprocide was synergistic with bacitracin, oxacillin, clindamycin or ceftriaxone. Anprocide was also synergistic in combination with bacitracin or oxacillin against some isolates of biofilm-grown S. aureus and S. epidermidis.

scholarly journals The Notch Target Genes Hey1 and Hes7 Transcriptionally Suppress Gli1 Expression and Hedgehog Signaling in Hodgkin-Reed/Sternberg Cells of Classical Hodgkin Lymphoma: A Novel Mechanism of Drug Resistance

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 275-275
Author(s):  
Rolf Schwarzer ◽  
Julia Godau ◽  
Hermann Einsele ◽  
Franziska Jundt
Keyword(s):  
Drug Resistance ◽  
Tumor Growth ◽  
Tumor Cell ◽  
Hodgkin Lymphoma ◽  
Target Genes ◽  
Classical Hodgkin Lymphoma ◽  
Notch Inhibition ◽  
Hh Signaling

Abstract Tumor cell proliferation and survival of Hodgkin/Reed-Sternberg (HRS) cells are triggered through Jagged1 ligand-induced Notch1 signaling via homotypic and heterotypic cell-cell interactions in classical Hodgkin lymphoma. The developmental pathway Notch partly mediates its effects in HRS cells by stimulation of alternative NF-kB signaling. We further demonstrated that high-level expression of the essential Notch coactivator Mastermind-like 2 and downregulation of the Notch inhibitor Deltex1 contribute to aberrant activation of Notch signaling in HRS cells. Our data suggested that targeting the Notch pathway is a rational treatment strategy in classical Hodgkin lymphoma. In this study we analyzed Notch inhibition by use of the gamma secretase inhibitor GSI XII in a Hodgkin lymphoma xenotransplantation model. To this end the HRS cell line L540cy (1 x 107 cells/per mouse) was transplanted into NOD/SCID mice. After tumor growth (0.3 cm³ mean tumor volume) mice were treated daily with increasing doses of GSI XII (5-10 mg/kg). Surprisingly, L540cy cells were completely drug-resistant in vivo in contrast to high GSI XII sensitivity in vitro. To dissect potential mechanisms of drug resistance we performed human StellARrayTM quantitative polymerase chain reaction (qPCR) arrays to analyze Notch target genes in GSI XII-treated compared to untreated L540cy cells. Interestingly, inhibition of Notch activity resulted in strong mRNA upregulation of the transcription factor glioma-associated oncogene 1 (Gli1), a final effector of the developmental signaling pathway Hedgehog (HH). Chromatin immunoprecipitation (ChIP) further revealed that both negative regulatory Notch target proteins Hey1 and Hes7 directly bind three different N-boxes present in the GLI1 first intron to suppress GLI1 mRNA expression in untreated L540cy cells. In general, the HH pathway is activated through ligand binding of secreted Sonic Hedgehog (SHH). As a result Gli transcription factors translocate to the nucleus and induce target gene expression such as GLI1 or CCND1. Despite high secretion of SHH by HRS cells after two days in culture (conditioned medium), HH signaling was inactive in untreated L540cy cells. Only after release of the negative regulatory Notch targets of the hairy and enhancer of split (HES) family through Notch inhibition and concomitant increase of Gli1 expression, HH signaling was activated by SHH. HH signaling mediated drug resistance of L540cy cells in conditioned medium compared to fresh medium (SHH negative) and thereby compensated for reduced Notch activity in vitro. We hypothesized that this mechanism might contribute to GSI XII drug resistance in vivo. To proof our hypothesis we coinhibited the Notch and HH pathways in L540cy cells. As expected inhibition of the HH pathway alone by use of cyclopamine did not significantly reduce growth of L540cy cells. However, simultaneous targeting of L540cy tumors through GSI XII and cyclopamine efficiently controlled tumor cell growth. Our data indicate a first molecular link between Notch and HH in HRS cells mediating drug resistance. We suggest inhibition of both developmental pathways for effective HRS tumor growth control. Disclosures No relevant conflicts of interest to declare.

scholarly journals In VitroActivity of Miltefosine against Candida albicans under Planktonic and Biofilm Growth Conditions andIn VivoEfficacy in a Murine Model of Oral Candidiasis

2015 ◽  
Vol 59 (12) ◽  
pp. 7611-7620 ◽  
Author(s):  
Taissa Vieira Machado Vila ◽  
Ashok K. Chaturvedi ◽  
Sonia Rozental ◽  
Jose L. Lopez-Ribot
Keyword(s):  
Candida Albicans ◽  
Murine Model ◽  
Biofilm Formation ◽  
Pathogenic Fungus ◽  
Oral Candidiasis ◽  
Antifungal Drugs ◽  
Drug Candidate ◽  
Content Type

ABSTRACTThe generation of a new antifungal againstCandida albicansbiofilms has become a major priority, since biofilm formation by this opportunistic pathogenic fungus is usually associated with an increased resistance to azole antifungal drugs and treatment failures. Miltefosine is an alkyl phospholipid with promising antifungal activity. Here, we report that, when tested under planktonic conditions, miltefosine displays potentin vitroactivity against multiple fluconazole-susceptible and -resistantC. albicansclinical isolates, including isolates overexpressing efflux pumps and/or with well-characterized Erg11 mutations. Moreover, miltefosine inhibitsC. albicans biofilm formation and displays activity against preformed biofilms. Serial passage experiments confirmed that miltefosine has a reduced potential to elicit resistance, and screening of a library ofC. albicanstranscription factor mutants provided additional insight into the activity of miltefosine againstC. albicansgrowing under planktonic and biofilm conditions. Finally, we demonstrate thein vivoefficacy of topical treatment with miltefosine in the murine model of oropharyngeal candidiasis. Overall, our results confirm the potential of miltefosine as a promising antifungal drug candidate, in particular for the treatment of azole-resistant and biofilm-associated superficial candidiasis.

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