tacrolimus and Cryptococcosis

Cryptococcus neoformans infections cause approximately 15% of AIDS-related deaths owing to a combination of limited antifungal therapies and drug resistance. A collection of clinical and environmental C. neoformans isolates were assayed for increased mutation rates via fluctuation analysis, and we identified two hypermutator C. neoformans clinical isolates with increased mutation rates when exposed to the combination of rapamycin and FK506. Sequencing of drug target genes found that Cnl1 transposon insertions conferred the majority of resistance to rapamycin and FK506 and could also independently cause resistance to 5-fluoroorotic acid and the clinically relevant antifungal 5-flucytosine. Whole-genome sequencing revealed both hypermutator genomes harbour a nonsense mutation in the RNA-interference component ZNF3 and hundreds of Cnl1 elements organized into massive subtelomeric arrays on each of the fourteen chromosomes. Quantitative trait locus mapping in 28 progeny derived from a cross between a hypermutator and wild-type identified a locus associated with hypermutation that included znf3. CRISPR editing of the znf3 nonsense mutation abolished hypermutation and restored small-interfering-RNA production. We conclude that hypermutation and drug resistance in these clinical isolates result from RNA-interference loss and accumulation of Cnl1 elements.

Topics: Antifungal Agents; Codon, Nonsense; Cryptococcosis; Cryptococcus neoformans; Drug Resistance, Fungal; Humans; RNA Interference; Sirolimus; Tacrolimus

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.

Topics: Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Binding Sites; Calcineurin; Calcineurin Inhibitors; Candida albicans; Cells, Cultured; Coccidioides; Cryptococcosis; Cryptococcus neoformans; Crystallography, X-Ray; Drug Discovery; Female; Male; Mice; Mice, Inbred A; Mice, Inbred C57BL; Molecular Dynamics Simulation; Tacrolimus; Tacrolimus Binding Protein 1A

Topics: Antifungal Agents; Bronchiolitis; Cellulitis; Cryptococcosis; Female; Humans; Immunocompromised Host; Lupus Erythematosus, Systemic; Middle Aged; Prednisolone; Tacrolimus

FK506 (tacrolimus) is an FDA-approved immunosuppressant indicated for the prevention of allograft rejections in patients undergoing organ transplants. In mammals, FK506 inhibits the calcineurin-nuclear factor of activated T cells (NFAT) pathway to prevent T-cell proliferation by forming a ternary complex with its binding protein, FKBP12, and calcineurin. FK506 also exerts antifungal activity by inhibiting calcineurin, which is essential for the virulence of human-pathogenic fungi. Nevertheless, FK506 cannot be used directly as an antifungal drug due to its immunosuppressive action. In this study, we analyzed the cytotoxicity, immunosuppressive activity, and antifungal activity of four FK506 analogs, 31-

Topics: Animals; Antifungal Agents; Aspergillosis; Aspergillus fumigatus; Calcineurin; Calcineurin Inhibitors; Candida albicans; Candidiasis; Cells, Cultured; Cryptococcosis; Cryptococcus neoformans; Female; Fluconazole; Immunosuppressive Agents; Male; Mice; Microbial Sensitivity Tests; Tacrolimus; Tacrolimus Binding Protein 1A

Synergistic interactions were observed between CIs and antifungal agents against 53 (90%) of 59 Cryptococcus neoformans isolates from solid organ transplant recipients with cryptococcosis and may account for better outcomes in patients with cryptococcosis receiving these immunosuppressive agents.

Topics: Antifungal Agents; Calcineurin Inhibitors; Cryptococcosis; Cryptococcus neoformans; Drug Synergism; Humans; Immunosuppressive Agents; Microbial Sensitivity Tests; Organ Transplantation; Tacrolimus; Treatment Outcome

The immunosuppressants tacrolimus (FK506) and cyclosporine A inhibit calcineurin and have potent antifungal activity. In this study, 24% of Cryptococcus neoformans isolates from solid-organ transplant patients exhibited altered sensitivity to these drugs, which may have an impact on the infectious course but does not appear to be the consequence of immunosuppressive therapy.

Topics: Calcineurin; Calcineurin Inhibitors; Cryptococcosis; Cryptococcus neoformans; Cyclosporine; Drug Resistance, Fungal; Humans; Immunosuppressive Agents; Microbial Sensitivity Tests; Organ Transplantation; Tacrolimus

We describe an immune reconstitution syndrome (IRS)-like entity in the course of evolution of Cryptococcus neoformans infection in organ transplant recipients.. The study population comprised a cohort of 83 consecutive organ transplant recipients with cryptococcosis who were observed for a median of 2 years in an international, multicenter study.. In 4 (4.8%) of the 83 patients, an IRS-like entity was observed a median of 5.5 weeks after the initiation of appropriate antifungal therapy. Worsening of clinical manifestations was documented, despite cultures being negative for C. neoformans. These patients were significantly more likely to have received tacrolimus, mycophenolate mofetil, and prednisone as the regimen of immunosuppressive therapy than were all other patients (P = .007). The proposed basis of this phenomenon is reversal of a predominantly Th2 response at the onset of infection to a Th1 proinflammatory response as a result of receipt of effective antifungal therapy and a reduction in or cessation of immunosuppressive therapy.. This study demonstrated that an IRS-like entity occurs in organ transplant recipients with C. neoformans infection. Furthermore, this entity may be misconstrued as a failure of therapy. Immunomodulatory agents may have a role as adjunctive therapy in such cases.

Topics: Adult; Aged; Antifungal Agents; Cohort Studies; Cryptococcosis; Female; Humans; Immune System Diseases; Immunosuppressive Agents; Male; Middle Aged; Mycophenolic Acid; Organ Transplantation; Prednisone; Tacrolimus

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Adaptation, Physiological; Animals; Antifungal Agents; Cryptococcosis; Cryptococcus neoformans; Cyclosporine; Fluconazole; Gene Deletion; Gene Expression Regulation, Fungal; Genes, Fungal; Homocysteine; Hot Temperature; Melanins; Methionine; Mice; Microbial Sensitivity Tests; Mutation; RNA, Fungal; RNA, Messenger; Tacrolimus; Transcription, Genetic; Virulence; Virulence Factors

Calcineurin is a Ca2+-calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37 degrees C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12-FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12-FK506-calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.

Topics: Amino Acid Sequence; Animals; Base Sequence; Calcineurin; Cryptococcosis; Cryptococcus neoformans; Disease Models, Animal; DNA, Fungal; Fungal Proteins; Genes, Fungal; Heating; Humans; Mice; Mice, Inbred DBA; Molecular Sequence Data; Mutagenesis; Protein Structure, Secondary; Recombination, Genetic; Sequence Homology, Amino Acid; Tacrolimus; Tacrolimus Binding Protein 1A; Virulence

Invasive cryptococcal infections have been reported in 0.3-1% of the patients undergoing liver transplantation in the previous reports. In contrast, invasive cryptococcosis developed in 6% of 102 consecutive liver transplants at our institution receiving tacrolimus as primary immunosuppression. Cutaneous and/or osteoarticular infections due to cryptococcus were detected in 67% of the patients with cryptococcosis, whereas meningitis was present only in 17%. One of the six patients with cryptococcosis presented with refractory shock and multiorgan system failure attributable solely to cryptococcosis. Patients with cryptococcal infections were significantly older than all other liver transplant recipients (p = 0.017), suggesting reactivation as opposed to primary infection as pathogenesis of cryptococcosis. 100% of the patients with cryptococcosis resided on the Eastern coast of the United States as compared to 59% of the patients without cryptococcosis (p = 0.08). There was no difference in the severity of underlying liver disease, degree of immunosuppression or CMV infection or disease between patients who did and did not develop cryptococcosis. Atypical manifestations, e.g. cutaneous diseases or sepsis syndrome, as opposed to subclinical meningitis, may be a clinical feature of cryptococcal infection in liver transplant recipients.

Topics: Adult; Aged; Antibiotic Prophylaxis; Cryptococcosis; Female; Humans; Immunosuppressive Agents; Liver Transplantation; Male; Middle Aged; Opportunistic Infections; Prospective Studies; Risk Factors; Survival Rate; Tacrolimus; United States

The microbial origin, timing, risk factors, and outcome of bloodstream infections (bacteremia and fungemia) were prospectively analyzed in 130 consecutive liver transplant recipients receiving tacrolimus-based immunosuppression; median followup was 3 yr. 22% (29/130) of the patients developed 36 episodes of bloodstream infections (0.28 episodes/patient). Bloodstream infections accounted for 36% (36/100) of all major infections. 81% (29/36) of bloodstream infections were due to bacteremia and 19% (7/36) due to fungemia (candidemia 14% and cryptococcemia 5%). Intravascular catheters were the most frequent source and methicillin-resistant Staphylococcus aureus was the most frequent pathogen causing bloodstream infections. 70% of the catheter related and all bacteremias due to intra-abdominal infections occurred < or = 90 d, whereas 75% of the bacteremias due to biliary source occurred > 90 d after transplantation. Length of initial post-transplant intensive care unit stay (p = 0.014) and readmission to the intensive care unit (p = 0.003) were independently significant predictors of bloodstream infections. 40% of the candidemias occurred within 30 d of transplantation and were of unknown portal, whereas the portal in all candidemias occurring > 30 d post-transplant was known (catheter, hepatic abscess, urinary tract). Mortality in patients with bloodstream infections was 52% (15/29) vs. 9% (9/101) in patients without bloodstream infections (p = 0.0001). In conclusion, intravascular catheters (and not intra-abdominal infections) have emerged as the most common source of bloodstream infections, and gram-positive cocci (S. aureus) as the predominant pathogens in bloodstream infections after liver transplantation.

Topics: Abdomen; Adult; Aged; Bacteremia; Biliary Tract; Candidiasis; Catheterization, Peripheral; Critical Care; Cryptococcosis; Equipment Contamination; Female; Follow-Up Studies; Forecasting; Fungemia; Humans; Immunosuppressive Agents; Length of Stay; Liver Abscess; Liver Transplantation; Male; Methicillin Resistance; Middle Aged; Patient Readmission; Prospective Studies; Risk Factors; Staphylococcal Infections; Staphylococcus aureus; Survival Rate; Tacrolimus; Time Factors; Treatment Outcome; Urinary Tract Infections