Biology subjects

Alanio, A.

Publications and source records attributed to Alanio, A..

3 recordsLinked to original sources

bioRxiv2017

Outbreak of invasive wound mucormycosis in a burn unit due to multiple strains of Mucor circinelloides f. circinelloides resolved by whole genome sequencing

Mucorales are ubiquitous environmental molds responsible for mucormycosis in diabetic, immunocompromised, and severely burned patients. Small outbreaks of invasive wound mucormycosis (IWM) have already been reported in burn units without extensive microbiological investigations. We faced an outbreak of IWM in our center and investigated the clinical isolates with whole genome sequencing (WGS) analysis.\n\nWe analyzed M. circinelloides isolates from patients in our burn unit (BU1) together with non-outbreak isolates from burn unit 2 (BU2, Paris area) and from France over a two-year period (2013-2015). For each isolate, WGS and a de novo genome assembly was performed from read data extracted from the aligned contig sequences of the reference genome (1006PhL).\n\nA total of 21 isolates were sequenced including 14 isolates from six BU1 patients. Phylogenetic classification showed that the clinical isolates clustered in four highly divergent clades. Clade1 contained at least one of the strains from the six epidemiologically-linked BU1 patients. The clinical isolates seemed specific to each patient. Two patients were infected with more than two strains from different clades suggesting that an environmental reservoir of clonally unrelated isolates was the source of contamination. Only two patients shared one strain in BU1, suggesting direct transmission or contamination with the same environmental source.\n\nWGS coupled with precise epidemiological data and analysis of several isolates per patients revealed in our study a complex situation with both potential cross-transmission and multiple contaminations with a heterogeneous pool of strains from a cryptic environmental reservoir.\n\nImportanceInvasive wound mucormycosis (IWM) is a severe infection due to the environmental molds belonging to the order Mucorales. Severely burned patients are particularly at risk for IWM. Here, we used Whole Genome Sequencing (WGS) analysis to resolve an outbreak of IWM due to Mucor circinelloides that occurred in our hospital (BU1). We sequenced 21 clinical isolates, including 14 from BU1 and 7 unrelated isolates, and compared them to the reference genome (1006PhL). This analysis revealed that the outbreak was mainly due to multiple strains that seemed patient-specific, suggesting that the patients were more likely infected from a pool of diverse strains from the environment rather than from direct transmission between the patients. This study revealed the complexity of a Mucorales outbreak in the settings of IWM in burn patients, which has been highlighted based on whole genome sequencing and careful sampling.

microbiology↗

bioRxiv2017

Identification of environmental and genetic factors important for Cryptococcus neoformans titan cell formation using new in vitro inducing conditions

The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 {micro}m cells and large titan cells (> 10 {micro}m and up to 100 {micro}m). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 {micro}m), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.\n\nAuthor SummaryCryptococcus neoformans is a yeast that is capable of morphological change upon interaction with the host. Particularly, in the lungs of infected mice, a subpopulation of yeast enlarges, producing cells up to 100 {micro}m in cell body diameter - referred to as titan cells. Along with their large size, the titan cells have other unique characteristics such as thickened cell wall, dense capsule, polyploidization, large vacuole with peripheral nucleus and cellular organelles. The generation of a large number of such cells outside the lungs of mice has been described but was not reproducible nor standardized. Here we report standardized, reproducible, robust conditions for generation of titan cells and explored the environmental and genetic factors underlying the genesis of these cells. We showed that titan cells were generated upon stresses such as change in the incubation medium, nutrient deprivation, hypoxia and low pH. Using collections of well characterized reference strains and clinical isolates, we validated with our model that the cAMP/PKA/Rim101 pathway is a major genetic determinant of titan cell formation. This study opens the way for a more comprehensive picture of the ontology of morphological changes in Cryptococcus neoformans and its impact on pathobiology of this deadly pathogen.

microbiology↗

bioRxiv2017

Population Genomics Of Cryptococcus neoformans var. grubii Reveals New Biogeographic Relationships And Finely Maps Hybridization

Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically HIV/AIDS patients from developing countries. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. Here we sequence the genomes of 188 diverse isolates and characterized the major subdivisions, their relative diversity and the level of genetic exchange between them. While most isolates of C. neoformans var. grubii belong to one of three major lineages (VNI, VNII, and VNB), some haploid isolates show hybrid ancestry including some that appear to have recently interbred, based on the detection of large blocks of each ancestry across each chromosome. Many isolates display evidence of aneuploidy, which was detected for all chromosomes. In diploid isolates of C. neoformans var. grubii (serotype A/A) and of hybrids with C. neoformans var. neoformans (serotype A/D) such aneuploidies have resulted in loss of heterozygosity, where a chromosomal region is represented by the genotype of only one parental isolate. Phylogenetic and population genomic analyses of isolates from Brazil revealed that the previously African VNB lineage occurs naturally in the South American environment. This suggests migration of the VNB lineage between Africa and South America prior to its diversification, supported by finding ancestral recombination events between isolates from different lineages and regions. The results provide evidence of substantial population structure, with all lineages showing multi-continental distributions demonstrating the highly dispersive nature of this pathogen.\n\nAuthor SummaryCryptococcus neoformans var. grubii is a human fungal pathogen of immunocompromised individuals that has global clinical impact, causing half a million deaths per year. Substantial genetic substructure exists for this pathogen, with two lineages found globally (VNI, VNII) whereas a third has appeared confined to sub-Saharan Africa (VNB). Here, we utilized genome sequencing of a large set of global isolates to examine the genetic diversity, hybridization, and biogeography of these lineages. We found that while the three major lineages are well separated, recombination between the lineages has occurred, notably resulting in hybrid isolates with segmented ancestry across the genome. In addition, we showed that isolates from South America are placed within the VNB lineage, formerly thought to be confined to Africa, and that there is phylogenetic separation between these geographies that substantially expands the diversity of these lineages. Our findings provide a new framework for further studies of the dynamics of natural populations of C. neoformans var. grubii.

genomics↗