MOLECULAR MECHANISMS OF DRUG RESISTANCE IN CANDIDA ALBICANS: ROLE OF TRANSCRIPTION FACTORS MCM1 AND ADA2

Candida albicans is an opportunistic fungal pathogen responsible for localized as well as disseminated infections. C. albicans is the most commonly isolated specie from blood cultures, accounting for over 60% of all Candida isolates. The fungistatic drug fluconazole is the most widely used to treat fungal infections, thanks to its favorable bio-availability and to its low toxicity. The frequent and widespread use of this antifungal has led to the outcome of drug resistant clinical isolates. Various drug resistance mechanisms are known that render C. albicans resistant to fluconazole, among those, the overexpression of efflux pumps, which extrude the drug out of the fungal cell, and the overexpression of the fluconazole target, the Erg11 enzyme. Overexpression of drug resistance genes is often associated to mutations in trans-acting transcription factors. The present study aimed at better understanding the role of known transcription factors involved in fluconazole resistance in C. albicans. In particular the role of TFs Mcm1 and Ada2 in resistance gene promoter activation has been evaluated. The transcription factors Mrr1 and Cap1 mediate MDR1 upregulation in response to inducing stimuli, and gain-of-function mutations in Mrr1 or Cap1, which render the transcription factors hyperactive, result in constitutive MDR1 overexpression. The essential MADS box transcription factor Mcm1 also binds to the MDR1 promoter, but its role in inducible or constitutive MDR1 upregulation is unknown. Using a conditional mutant in which Mcm1 can be depleted from the cells, the importance of Mcm1 for MDR1 expression was investigated. The results obtained indicated that Mcm1 was dispensable for MDR1 upregulation by H2O2, but required for full MDR1 induction by benomyl. A C-terminally truncated, hyperactive Cap1 could upregulate MDR1 expression both in the presence and absence of Mcm1. In contrast, a hyperactive Mrr1 containing a gain-of-function mutation depended on Mcm1 to cause MDR1 overexpression. These results demonstrate a differential requirement of the co-regulator Mcm1 for Cap1- and Mrr1-mediated MDR1 upregulation. When activated by oxidative stress or a gain-of-function mutation, Cap1 could induce MDR1 expression independently of Mcm1, whereas Mrr1 required either Mcm1 or an active Cap1 to cause overexpression of the MDR1 efflux pump. Other transcription factors that mediate drug resistance gene regulation are Tac1, which regulates CDR1 and CDR2 expression, and Upc2, regulating ERG11 gene expression. Also in this case, gain-of-function mutations, which render these transcription factors hyperactive, result in constitutive overexpression of the target genes. Ada2 is part of the SAGA/ADA complex and has been shown to be recruited to 200 promoters upstream of genes involved in different stress-response functions and metabolic processes. As for Mcm1, the importance of Ada2 for MDR1 expression was investigated, as well as for CDR2 and ERG11 expression. Ada2 was found to be dispensable for MDR1 upregulation by H2O2, but required for MDR1 activation by hyperactive Cap1. When activated by benomyl or a gain-of-function mutation, Mrr1 induced MDR1 expression even better in the absence of Ada2. CDR2 expression by hyperactive Tac1 was facilitated in the presence of Ada2 and an opposite behaviour was observed when CDR2 expression was stimulated by the presence of fluphenazine in the medium. Finally, further experiments are required to better understand the role of Ada2 in the expression of ERG11. Overall, these findings provide a more detailed insight into the molecular mechanisms of drug resistance in this important human fungal pathogen

Genotypic and phenotypic properties of Candida parapsilosis sensu strictu strains isolated from different geographic regions and body sites

<p>Abstract</p> <p>Background</p> <p><it>Candida parapsilosis </it>is known to show limited genetic variability, despite different karyotypes and phenotypes have been described. To further investigate this aspect, a collection of 62 <it>sensu strictu </it><it>C. parapsilosis </it>independent isolates from 4 geographic regions (Italy, n = 19; New Zealand, n = 15; Argentina, n = 14; and Hungary, n = 14) and different body sites (superficial and deep seated) were analysed for their genetic and phenotypic traits. Amplification fragment length polymorphism (AFLP) analysis was used to confirm species identification and to evaluate intraspecific genetic variability. Phenotypic characterisation included clinically relevant traits, such as drug susceptibility, in vitro biofilm formation and aspartyl protease secretion.</p> <p>Results</p> <p>AFLP genotyping showed little variation among isolates, when the presence/absence of bands was considered. However, when AFLP profiles were compared by relative intensity for each fragment, a significant level of variation and geographical clustering was observed. All isolates were found to be susceptible to commonly used antifungals, although a reduced susceptibility to echinocandins was observed in all isolates. <it>C. parapsilosis </it>isolates from different geographic origins varied in the number of biofilm producers, with a higher prevalence of producers isolated in Hungary and Argentina. The frequency of secreted proteinase producers also varied in isolates obtained from different areas, with a higher number of proteinase producers found in Italy and New Zealand. Interestingly, biofilm production and proteinase secretion were negatively correlated. This finding could be explained by assuming that proteinase activity plays a role in detachment and release from a mature biofilm, via degradation of <it>C. parapsilosis </it>adhesins and/or extracellular matrix components, as observed for other microorganisms.</p> <p>Conclusions</p> <p>The low number of polymorphic AFLP bands (18 out of 80) obtained for <it>C. parapsilosis </it>isolates is in agreement with the limited sequence variability described for this species. However, when band intensity was included in the analysis, geographical clustering was observed. Expression of virulence factors varied among strains isolated from different geographical regions, with biofilm and proteinase producers more frequently isolated from Hungary and Italy, respectively.</p

The Transcription Factor Ndt80 Does Not Contribute to Mrr1-, Tac1-, and Upc2-Mediated Fluconazole Resistance in Candida albicans

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80Δ mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80Δ mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors

Supplementary material for the article: Lazić, J.; Ajdačić, V.; Vojnovic, S.; Zlatović, M.; Pekmezovic, M.; Mogavero, S.; Opsenica, I.; Nikodinovic-Runic, J. Bis-Guanylhydrazones as Efficient Anti-Candida Compounds through DNA Interaction. Appl Microbiol Biotechnol 2018, 102 (4), 1889–1901. https://doi.org/10.1007/s00253-018-8749-3

Supplementary material for: [https://doi.org/10.1007/s00253-018-8749-3]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2078

Supplementary material for the article: Lazić, J.; Ajdačić, V.; Vojnovic, S.; Zlatović, M.; Pekmezovic, M.; Mogavero, S.; Opsenica, I.; Nikodinovic-Runic, J. Bis-Guanylhydrazones as Efficient Anti-Candida Compounds through DNA Interaction. Appl Microbiol Biotechnol 2018, 102 (4), 1889–1901. https://doi.org/10.1007/s00253-018-8749-3

Supplementary material for: [https://doi.org/10.1007/s00253-018-8749-3]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2078

Human albumin enhances the pathogenic potential of Candida glabrata on vaginal epithelial cells

Funding: M.P., H.H., T.G., and B.H. received funding from the European Union Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642095 (OPATHY). A.K. and B. H. received support from the European Union Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 812969 (FunHoMic). S.A. and B.H. were supported by funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 847507 (HDM-FUN). B.H. is further supported by the DFG within the Collaborative Research Centre (CRC)/Transregio (TRR) 124 “FungiNet” project C1 (DFG project number 210879364) and the Balance of the Microverse Cluster (Germany´s Excellence Strategy – EXC 2051 – Project-ID 390713860). M.S.G. was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft - DFG) Emmy Noether Program (project no. 434385622 / GR 5617/1-1), and a Research Grant 2019 from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. M.P. and H.H. received salary from grant agreement No 642095 (OPATHY) (2016-2019). A.K. received salary from grant agreement No 812969 (FunHoMic) (2019-2022).Peer reviewedPublisher PD

Bevacizumab plus XELOX as first-line treatment of metastatic colorectal cancer: The OBELIX study

AIM: To confirm the efficacy and safety of bevacizumab/XELOX combination for the treatment of locally advanced or metastatic colorectal cancer (CRC) in Italy. METHODS: This multicentric, prospective, open-label study included patients with CRC previously untreated with chemotherapy. Patients were administered bevacizumab in combination with XELOX. The primary efficacy end-point was progression-free survival (PFS). Secondary end-points included time to overall response (TOR), duration of response (DOR), time to treatment failure (TTF) and overall survival (OS). The incidence and type of adverse events AEs and severe AEs were evaluated. Also, the mutational status of BRAF and KRAS was assessed by high resolution melting and direct sequencing, and quality of life (QoL) was measured by the EuroQoL EQ-5D questionnaire at baseline and at the last visit. RESULTS: The intention-to-treat population included 197 patients (mean age: 62.3 ± 9.9 years, 56.4% males). At baseline, 16/34 evaluable subjects (47.1%) harbored a KRAS and/or a BRAF mutation; the mean QoL index was 80.2 ± 14.3. First-line therapy was given for 223.7 ± 175.9 d, and after a mean follow-up of 387.7 ± 238.8 d all patients discontinued from the study mainly for disease progression (PD, 45.4%) and AEs (25.4%). Median PFS was 9.7 mo (95%CI: 8.4-10.5) and the median values for secondary end-points were: TOR = 3.9 mo (95%CI: 2.6-4.7), DOR = 8.5 mo (95%CI: 7.3-10.3), TTF = 6.7 mo (95%CI: 6.0-7.7) and OS = 23.2 mo (95%CI: 20.1-27.2). Patients carrying at least one lesion had a lower overall response rate (66.7% vs 88.9%) and a lower probability of achieving complete or partial response than those without mutations, but the difference in relative risk was not statistically significant (P = 0.2). Mean EQ-5D-3L raw index score significantly decreased to 74.9 ± 19.1 at the last visit (signed-rank test, P = 0.0076), but in general the evaluation on QoL perceived by patients was good. CONCLUSION: The efficacy of bevacizumab in combination with XELOX in terms of PFS in patients with aCRC or mCRC in Italy was confirmed, with acceptable toxicity

A New Phenotype in Candida-Epithelial Cell Interaction Distinguishes Colonization- versus Vulvovaginal Candidiasis- Associated Strains

Vulvovaginal candidiasis (VVC) affects nearly 3/4 of women during their lifetime, and its symptoms seriously reduce quality of life. Although Candida albicans is a common commensal, it is unknown if VVC results from a switch from a commensal to pathogenic state, if only some strains can cause VVC, and/or if there is displacement of commensal strains with more pathogenic strains. We studied a set of VVC and colonizing C. albicans strains to identify consistent in vitro phenotypes associated with one group or the other. We find that the strains do not differ in overall genetic profile or behavior in culture media (i.e., multilocus sequence type [MLST] profile, rate of growth, and filamen- tation), but they show strikingly different behaviors during their interactions with vaginal epithelial cells. Epithelial infections with VVC-derived strains yielded stronger fungal prolif- eration and shedding of fungi and epithelial cells. Transcriptome sequencing (RNA-seq) analysis of representative epithelial cell infections with selected pathogenic or commensal isolates identified several differentially activated epithelial signaling pathways, including the integrin, ferroptosis, and type I interferon pathways; the latter has been implicated in damage protection. Strikingly, inhibition of type I interferon signaling selectively increases fungal shedding of strains in the colonizing cohort, suggesting that increased shedding correlates with lower interferon pathway activation. These data suggest that VVC strains may intrinsically have enhanced pathogenic potential via differential elicitation of epithelial responses, including the type I interferon pathway. Therefore, it may eventually be possible to evaluate pathogenic potential in vitro to refine VVC diagnosis

Allestimento di un microarray per la diagnostica molecolare di miceti patogeni umani

Le infezioni opportunistiche di natura fungina costituiscono un problema di rilevante importanza medica, risultando tra le principali cause di mortalità nonché di morbidità negli individui immunocompromessi. Nonostante numerosi studi abbiano contribuito ad approfondire le attuali conoscenze sulle infezioni fungine, la diagnosi di queste ultime è attualmente basata sull’isolamento colturale e successiva identificazione mediante analisi delle caratteristiche macro e microscopiche della colonia, del profilo biochimico e sull’analisi morfologica delle strutture riproduttive. Tali metodi richiedono lunghi tempi di esecuzione e non sempre permettono, per le specie di recente definizione, una accurata discriminazione. Pertanto, è molto sentita l’esigenza di allestire nuove strategie di identificazione per una rapida diagnosi delle infezioni micotiche. In tale ottica, in questo lavoro di tesi, è stato allestito un pannello di identificazione su vetrino (micro-array) per la diagnostica molecolare dei principali miceti patogeni per l’uomo, basato sull’impiego di sonde molecolari specie-specifiche, complementari a regioni conservate nel genoma di ciascuna specie fungina in esame (regione ITS1-rRNA 5,8S –ITS2). Questo sistema prevede l’estrazione del DNA genomico, l’amplificazione della regione ITS e successiva ibridazione del prodotto di PCR sul vetrino contenente sonde specifiche costruite sulla regione ITS. Il sistema di rilevazione del segnale è basato sulla nuova tecnica molecolare, denominata APEX (arrayed primer extension), che prevede che ogni frammento ibridi specificamente con l’oligonucleotide complementare spottato sul vetrino in posizione nota e che la DNA polimerasi estenda la base complementare immediatamente successiva in posizione 3’. L’estensione si arresta immediatamente in quanto la base incorporata è un terminatore che reca un fluorocromo specifico, per cui la lettura della fluorescenza emessa da ogni spot indicherà l’avvenuta ibridazione del frammento con la sonda e, quindi, la corretta identificazione del microorganismo. Per la messa a punto della metodica sono stati utilizzati ceppi di riferimento e isolati clinici di 24 specie di lieviti, tra i quali Candida albicans, Candida spp., Cryptococcus neoformans, Saccharomyces cerevisiae, e di ifomiceti come Aspergillus fumigatus, Aspergillus terreus, Microsporum canis, Trichosporon cutaneum. In conclusione, questo lavoro di tesi ha permesso di dimostrare l’utilità dell’ applicazione della metodologia APEX alla diagnostica dei funghi patogeni, consentendo una inequivocabile discriminazione anche di specie correlate molto vicine geneticamente, come C. albicans e C. dubliniensis o C. parapsilosis, C. orthopsilosis e C. metapsilosis, o come A. fumigatus e A. terreus. I dati ottenuti indicano che questo sistema consente un’identificazione non soggettiva, rapida, estremamente sensibile e specifica di varie specie fungine, risultando di potenziale applicazione direttamente su materiale clinico