Pushing the Limits of MALDI-TOF Mass Spectrometry: Beyond Fungal Species Identification

Matrix assisted laser desorption ionization time of flight (MALDI–TOF) is a powerful analytical tool that has revolutionized microbial identification. Routinely used for bacterial identification, MALDI-TOF has recently been applied to both yeast and filamentous fungi, confirming its pivotal role in the rapid and reliable diagnosis of infections. Subspecies-level identification holds an important role in epidemiological investigations aimed at tracing virulent or drug resistant clones. This review focuses on present and future applications of this versatile tool in the clinical mycology laboratory

Studio del ruolo svolto dal gene CPAG_05056 nella virulenza e patogenicità di Candida parapsilosis

Candida parapsilosis è un lievito patogeno opportunista considerato ad oggi la seconda causa più comune di candidemia dopo Candida albicans. Poco è noto riguardo ai meccanismi di virulenza utilizzati da C. parapsilosis durante l’instaurarsi del processo infettivo, tuttavia, la capacità di aderire alle superfici dell’ospite è ritenuta un evento chiave nelle prime fasi dell’infezione. In C. albicans un ruolo cruciale nell’adesione a superfici biotiche è svolto da una classe di glicoproteine di superficie, chiamate proteine Als (Agglutinin-Like Sequence proteins), codificate da una famiglia multi-genica composta da 8 membri (ALS1-8). La recente pubblicazione dell’intera sequenza del genoma di C. parapsilosis e le prime analisi di omologia hanno evidenziato la presenza di 5 possibili omologhi dei geni ALS (CpALS1-5). Il presente lavoro di tesi si è inserito all’interno di un progetto più ampio volto alla comprensione del contributo di ciascuno dei geni ALS nell’adesione di C. parapsilosis a superfici biotiche e abiotiche, mediante l’allestimento di ceppi mutanti privi dei geni CpALS1-5. In particolare, il gene CPAG_05056 (successivamente indicato come CpALS3), il cui omologo in C. albicans svolge un ruolo di primaria importanza nella virulenza di questo lievito, è stato scelto come primo target per la delezione mediante ricombinazione omologa. Durante la prima parte dell’internato di tesi è stata condotta una caratterizzazione fenotipica del ceppo di riferimento di C. parapsilosis ATCC 22019 (wild type, WT), e di mutanti privi di una (CpALS3H) o di entrambe le copie (CpALS3KO) del gene CpALS3 ottenuti mediante la strategia di delezione basata sull’ utilizzo della cassetta SAT1-flipper. L’analisi della capacità replicativa della collezione di mutanti in terreno YPD e in terreno contenente agenti perturbanti la parete, ha indicato che la delezione di CpALS3 non compromette la vitalità dei ceppi in studio, almeno per quanto concerne le condizioni di crescita utilizzate. Sia il ceppo wild type che i ceppi mutanti, se sottoposti a crescita in condizioni inducenti, sono risultati in grado di produrre pseudoife, a dimostrazione che la delezione di CpALS3 non altera la capacità di C. parapsilosis di andare incontro a morfogenesi. Successivamente, mediante l’allestimento di un saggio di adesione in vitro a cellule buccali umane (HBEC), sono state saggiate le capacità adesive della collezione di mutanti a seguito di co-incubazione per 45 minuti a 37°C di una sospensione cellulare contenente HBEC e lieviti (rapporto 1:1000). Dai dati ottenuti è stato possibile osservare una drastica diminuzione nelle capacità adesive del ceppo mutante privo di entrambe le copie del gene CpALS3. Allo scopo di confermare il contributo di tale gene nell’adesione di C. parapsilosis, nel corso di questo lavoro di tesi è stato creato un ceppo complementato, tramite la re- introduzione di una copia wild type del gene CpALS3 nel background del mutante nullo CpALS3KOb. I risultati ottenuti hanno confermato il ripristino della funzione di adesione alle cellule HBEC da parte del ceppo complementato. La citotossicità del pannello di ceppi in studio è stata, inoltre, valutata mediante quantificazione del rilascio di lattato deidrogenasi (LDH) da parte della linea tumorale epiteliale umana A549. Il saggio non ha permesso di evidenziare alcun rilascio di LDH da parte dei ceppi mutanti e/o WT, mentre un danno quantificabile (44%) era indotto dal ceppo di C. albicans SC5314, utilizzato come controllo positivo. Infine, una prima analisi del potenziale patogenico è stata valutata nel lepidottero Galleria mellonella. La percentuale di sopravvivenza delle larve infettate con il ceppo mutante non differiva significativamente rispetto al ceppo WT o ricostituito, in accordo con il fatto che CpALS3 possa svolgere un ruolo primario nelle fasi di colonizzazione, ma non sia direttamente coinvolto nel danno tissutale

Gene editing and knock-out strategies to dissect the role of ALS genes in Candida species

The ability to adhere to biotic and abiotic surfaces represents an essential trait during the early stages of microbial infection. Agglutinin like sequence (Als) cell-wall proteins play a key role in the adhesion of Candida species. In silico analysis led to identification of 5 ALS members in C. parapsilosis genome and 3 ALS members in C. orthopsilosis. The present PhD project was aimed at extending the current knowledge on the opportunistic pathogens C. parapsilosis and C. orthopsilosis, by dissecting the contribution of Als proteins through the generation of mutant strains using conventional gene disruption techniques (SAT1-flipper cassette) and gene editing approaches (episomal CRISPR/Cas9 system). Furthermore, an accurate genome assembly of C. orthopsilosis was generated providing a first insight into the ALS gene family in C. orthopsilosis and the basics for their functional characterization. Analysis of the phenotypic traits of the mutant strains generated in both species revealed an involvement of Als proteins in biofilm formation, adhesion on human buccal epithelial cells (HBECs) and in the pathogenic potential when tested in models of murine candidiasis

A ribonucleoprotein transfection strategy for CRISPR/Cas9‐mediated gene editing and single cell cloning in rainbow trout cells

Background: The advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the genome of every living cell. Since its discovery, different gene editing approaches based on the CRISPR/Cas9 technology have been widely established in mammalian cell lines, while limited knowledge is available on genetic manipulation in fish cell lines. In this work, we developed a strategy to CRISPR/Cas9 gene edit rainbow trout (Oncorhynchus mykiss) cell lines and to generate single cell clone-derived knock-out cell lines, focusing on the phase I biotransformation enzyme encoding gene, cyp1a1, and on the intestinal cell line, RTgutGC, as example. Results: Ribonucleoprotein (RNP) complexes, consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting the cyp1a1 gene, were delivered via electroporation. A T7 endonuclease I (T7EI) assay was performed on flow cytometry enriched transfected cells in order to detect CRISPR-mediated targeted mutations in the cyp1a1 locus, revealing an overall gene editing efficiency of 39%. Sanger sequencing coupled with bioinformatic analysis led to the detection of multiple insertions and deletions of variable lengths in the cyp1a1 region directed by CRISPR/Cas9 machinery. Clonal isolation based on the use of cloning cylinders was applied, allowing to overcome the genetic heterogeneity created by the CRISPR/Cas9 gene editing. Using this method, two monoclonal CRISPR edited rainbow trout cell lines were established for the first time. Sequencing analysis of the mutant clones confirmed the disruption of the cyp1a1 gene open reading frame through the insertion of 101 or 1 base pair, respectively. Conclusions: The designed RNP-based CRISPR/Cas9 approach, starting from overcoming limitations of transfection to achieving a clonal cell line, sets the stage for exploiting permanent gene editing in rainbow trout, and potentially other fish cells, for unprecedented exploration of gene function

Pushing the Limits of MALDI-TOF Mass Spectrometry: Beyond Fungal Species Identification

Matrix assisted laser desorption ionization time of flight (MALDI-TOF) is a powerful analytical tool that has revolutionized microbial identification. Routinely used for bacterial identification, MALDI-TOF has recently been applied to both yeast and filamentous fungi, confirming its pivotal role in the rapid and reliable diagnosis of infections. Subspecies-level identification holds an important role in epidemiological investigations aimed at tracing virulent or drug resistant clones. This review focuses on present and future applications of this versatile tool in the clinical mycology laboratory

A ribonucleoprotein transfection strategy for CRISPR/Cas9‐mediated gene editing and single cell cloning in rainbow trout cells

Background The advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the genome of every living cell. Since its discovery, different gene editing approaches based on the CRISPR/Cas9 technology have been widely established in mammalian cell lines, while limited knowledge is available on genetic manipulation in fish cell lines. In this work, we developed a strategy to CRISPR/Cas9 gene edit rainbow trout (Oncorhynchus mykiss) cell lines and to generate single cell clone-derived knock-out cell lines, focusing on the phase I biotransformation enzyme encoding gene, cyp1a1, and on the intestinal cell line, RTgutGC, as example. Results Ribonucleoprotein (RNP) complexes, consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting the cyp1a1 gene, were delivered via electroporation. A T7 endonuclease I (T7EI) assay was performed on flow cytometry enriched transfected cells in order to detect CRISPR-mediated targeted mutations in the cyp1a1 locus, revealing an overall gene editing efficiency of 39%. Sanger sequencing coupled with bioinformatic analysis led to the detection of multiple insertions and deletions of variable lengths in the cyp1a1 region directed by CRISPR/Cas9 machinery. Clonal isolation based on the use of cloning cylinders was applied, allowing to overcome the genetic heterogeneity created by the CRISPR/Cas9 gene editing. Using this method, two monoclonal CRISPR edited rainbow trout cell lines were established for the first time. Sequencing analysis of the mutant clones confirmed the disruption of the cyp1a1 gene open reading frame through the insertion of 101 or 1 base pair, respectively. Conclusions The designed RNP-based CRISPR/Cas9 approach, starting from overcoming limitations of transfection to achieving a clonal cell line, sets the stage for exploiting permanent gene editing in rainbow trout, and potentially other fish cells, for unprecedented exploration of gene function.ISSN:2045-370

Use of Amplification Fragment Length Polymorphism to genotype Pseudomonas stutzeri strains following exposure to ultraviolet light A

Changes in ultraviolet light radiation can act as a selective force on the genetic and physiological traits of a microbial community. Two strains of the common soil bacterium Pseudomonas stutzeri, isolated from aquifer cores and from human spinal fluid were exposed to ultraviolet light. Amplification length polymorphism analysis (AFLP) was used to genotype this bacterial species and evaluate the effect of UVA-exposure on genomic DNA extracted from 18 survival colonies of the two strains compared to unexposed controls. AFLP showed a high discriminatory power, confirming the existence of different genotypes within the species and presence of DNA polymorphisms in UVA-exposed colonies

CORT0C04210 is required for Candida orthopsilosis adhesion to human buccal cells

Candida orthopsilosis is a human fungal pathogen belonging to the Candida parapsilosis sensu lato species complex. C. orthopsilosis annotated genome harbors 3 putative agglutinin-like sequence (ALS) genes named CORT0B00800, CORT0C04210 and CORT0C04220. The aim of this study was to investigate the role played by CORT0C04210 (CoALS4210) in the virulence and pathogenicity of this opportunistic yeast. Heterozygous and null mutant strains lacking one or both copies of CoALS4210 were obtained using the SAT1-flipper cassette strategy and were characterized in in vitro, ex vivo and in vivo models. While no differences between the mutant and the wild-type strains were observed in in vitro growth or in the ability to undergo morphogenesis, the CoALS4210 null mutant showed an impaired adhesion to human buccal epithelial cells compared to heterozygous and wild type strains. When the pathogenicity of CoALS4210 mutant and wild type strains was evaluated in a murine model of systemic candidiasis, no statistically significant differences were observed in fungal burden of target organs. Since gene disruption could alter chromatin structure and influence transcriptional regulation of other genes, two independent CRISPR/Cas9 edited mutant strains were generated in the same genetic background used to create the deleted strains. CoALS4210-edited strains were tested for their in vitro growing ability, and compared with the deleted strain for adhesion ability to human buccal epithelial cells. The results obtained confirmed a reduction in the adhesion ability of C. orthopsilosis edited strains to buccal cells. These findings provide the first evidence that CRISPR/Cas9 can be successfully used in C. orthopsilosis and demonstrate that CoALS4210 plays a direct role in the adhesion of C. orthopsilosis to human buccal cells but is not primarily involved in the onset of disseminated candidiasis

CoERG11 A395T mutation confers azole resistance in Candida orthopsilosis clinical isolates

SYNOPSIS Background: Candida orthopsilosis is a human fungal pathogen responsible for a wide spectrum of symptomatic infections. Evidence suggests that C. orthopsilosis is mainly susceptible to azoles, the most extensively used antifungals for treatment of these infections. However, fluconazole-resistant clinical isolates are reported. Objectives: This study evaluated the contribution of a single amino acid substitution in the azole target CoErg11 to the development of azole resistance in C. orthopsilosis. Methods: C. orthopsilosis clinical isolates (n = 40) were tested for their susceptibility to azoles, and their CoERG11 genes were sequenced. We used a SAT1 flipper driven transformation to integrate a mutated CoERG11 allele in the genetic background of a fluconazole-susceptible isolate. Results: Susceptibility testing revealed that 16 of 40 C. orthopsilosis clinical isolates were resistant to fluconazole and to at least another azole. We identified an A395T mutation in the CoERG11 coding sequence of azole-resistant isolates only that resulted in the non-synonymous amino acid substitution Y132F. The SAT1 flipper cassette strategy led to the creation of C. orthopsilosis mutants, which carried the A395T mutation in one or both CoERG11 alleles (heterozygous or homozygous mutant, respectively) in an azole susceptible genetic background. We tested mutant strains for azole susceptibility and for hot-spot locus heterozygosity. Either heterozygous or homozygous mutant strains exhibited an azole-resistant phenotype. Conclusions: These findings provide the first evidence that CoErg11 Y132F substitution confers multi-azole resistance in C. orthopsilosis