Genetic Manipulation as a Tool to Unravel Species Complex Virulence and Drug Resistance: State of the Art.

An increase in the rate of isolation of in the past decade, as well as increased identification of azole-resistant strains are concerning, and require better understanding of virulence-like factors and drug-resistant traits of these species. In this regard, the present review "draws a line" on the information acquired, thus far, on virulence determinants and molecular mechanisms of antifungal resistance in these opportunistic pathogens, mainly derived from genetic manipulation studies. This will provide better focus on where we stand in our understanding of the species complex-host interaction, and how far we are from defining potential novel targets or therapeutic strategies-key factors to pave the way for a more tailored management of fungal infections caused by these fungal pathogens.

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.

Role of and in Virulence: Evidence from a Murine Model of Vaginal Candidiasis.

The genome encodes for five agglutinin-like sequence (Als) cell-wall glycoproteins involved in adhesion to biotic and abiotic surfaces. The work presented here is aimed at analyzing the role of the two still uncharacterized genes in and , by the generation and characterization of C and single mutant strains. Phenotypic characterization showed that both mutant strains behaved as the parental wild type strain regarding growth rate in liquid/solid media supplemented with cell-wall perturbing agents, and in the ability to produce pseudohyphae.

TbD1 deletion as a driver of the evolutionary success of modern epidemic Mycobacterium tuberculosis lineages.

Mycobacterium tuberculosis (Mtb) strains are classified into different phylogenetic lineages (L), three of which (L2/L3/L4) emerged from a common progenitor after the loss of the MmpS6/MmpL6-encoding Mtb-specific deletion 1 region (TbD1). These TbD1-deleted "modern" lineages are responsible for globally-spread tuberculosis epidemics, whereas TbD1-intact "ancestral" lineages tend to be restricted to specific geographical areas, such as South India and South East Asia (L1) or East Africa (L7). By constructing and characterizing a panel of recombinant TbD1-knock-in and knock-out strains and comparison with clinical isolates, here we show that deletion of TbD1 confers to Mtb a significant increase in resistance to oxidative stress and hypoxia, which correlates with enhanced virulence in selected cellular, guinea pig and C3HeB/FeJ mouse infection models, the latter two mirroring in part the development of hypoxic granulomas in human disease progression.

CpALS4770 and CpALS4780 contribution to the virulence of Candida parapsilosis.

The ability of yeast to adhere to biotic and abiotic surfaces represents an essential trait during the early stages of infection. Agglutinin-like sequence (Als) cell-wall proteins play a key role in adhesion of Candida species. Candida parapsilosis genome encompasses 5 ALS members, of which only the role of CPAR2_404800 has been elucidated.

A CRISPR/Cas9-based strategy to simultaneously inactivate the entire gene family in .

In this study, the CRISPR gene-editing approach was used to simultaneously inactivate all three members of the gene family in the opportunistic pathogen . Using a single gRNA and repair template, CRISPR-edited clones were successfully generated in a one-step process in both reference and clinical strains. The phenotypic characterization of the triple-edited strains revealed no impact on growth in liquid or solid media.

Characterization of the Candida orthopsilosis agglutinin-like sequence (ALS) genes.

Agglutinin like sequence (Als) cell-wall proteins play a key role in adhesion and virulence of Candida species. Compared to the well-characterized Candida albicans ALS genes, little is known about ALS genes in the Candida parapsilosis species complex. Three incomplete ALS genes were identified in the genome sequence for Candida orthopsilosis strain 90-125 (GenBank assembly ASM31587v1): CORT0C04210 (named CoALS4210), CORT0C04220 (CoALS4220) and CORT0B00800 (CoALS800).

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.

CoERG11 A395T mutation confers azole resistance in Candida orthopsilosis clinical isolates.

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.

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.

Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection.

Candida parapsilosis is an emerging opportunistic pathogen, second in frequency only to C. albicans and commonly associated with both mucosal and systemic infections. Adhesion to biotic surfaces is a key step for the development of mycoses.

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.