Exposure of Cryptococcus neoformans to Seven Commonly Used Agricultural Azole Fungicides Induces Resistance to Fluconazole as Well as Cross-Resistance to Voriconazole, Posaconazole, Itraconazole and Isavuconazole.

BACKGROUND: Cross-resistance to medical azoles by exposure to azole pesticides is well documented for Aspergillus family fungi but is poorly evaluated for other environmental pathogen fungi, particularly for yeasts belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. METHODS: One thousand C. neoformans yeast were exposed to various concentrations of seven different commonly used azole pesticides. Clones surviving exposure were picked randomly, and their minimal inhibitory concentrations (MICs) of fluconazole, voriconazole, posaconazole, itraconazole and isavuconazole were assessed. RESULTS: Depending on the pesticide used for exposure, up to 13.3% of selected Cryptococcus colonies showed a phenotype of resistance to fluconazole, and among them, several showed cross-resistance to another or several other medical azoles. Molecular mechanisms involved in the resistance setups seem to be dependent on ERG11 and AFR1 gene overexpression. CONCLUSION: Exposure to any of the seven azole pesticides tested is capable of increasing the MIC of fluconazole in C. neoformans, including up to the level of the fluconazole-resistant phenotype, as well as generating cross-resistance to other medical azoles in some cases.

A targeted immunomic approach identifies diagnostic antigens in the human pathogen Babesia microti.

BACKGROUND: Babesia microti is a protozoan parasite responsible for the majority of reported cases of human babesiosis and a major risk to the blood supply. Laboratory screening of blood donors may help prevent transfusion-transmitted babesiosis but there is no Food and Drug Administration-approved screening method yet available. Development of a sensitive, specific, and highly automated B. microti antibody assay for diagnosis of acute babesiosis and blood screening could have an important impact on decreasing the health burden of B. microti infection. STUDY DESIGN AND METHODS: Herein, we take advantage of recent advances in B. microti genomic analyses, field surveys of the reservoir host, and human studies in endemic areas to apply a targeted immunomic approach to the discovery of B. microti antigens that serve as signatures of active or past babesiosis infections. Of 19 glycosylphosphatidylinositol (GPI)-anchored protein candidates (BmGPI1-19) identified in the B. microti proteome, 17 were successfully expressed, printed on a microarray chip, and used to screen sera from uninfected and B. microti-infected mice and humans to determine immune responses that are associated with active and past infection. RESULTS: Antibody responses to various B. microti BmGPI antigens were detected and BmGPI12 was identified as the best biomarker of infection that provided high sensitivity and specificity when used in a microarray antibody assay. CONCLUSION: BmGPI12 alone or in combination with other BmGPI proteins is a promising candidate biomarker for detection of B. microti antibodies that might be useful in blood screening to prevent transfusion-transmitted babesiosis.

Whole genome mapping and re-organization of the nuclear and mitochondrial genomes of Babesia microti isolates.

Babesia microti is the primary causative agent of human babesiosis, an emerging pathogen that causes a malaria-like illness with possible fatal outcome in immunocompromised patients. The genome sequence of the B. microti R1 strain was reported in 2012 and revealed a distinct evolutionary path for this pathogen relative to that of other apicomplexa. Lacking from the first genome assembly and initial molecular analyses was information about the terminal ends of each chromosome, and both the exact number of chromosomes in the nuclear genome and the organization of the mitochondrial genome remained ambiguous. We have now performed various molecular analyses to characterize the nuclear and mitochondrial genomes of the B. microti R1 and Gray strains and generated high-resolution Whole Genome maps. These analyses show that the genome of B. microti consists of four nuclear chromosomes and a linear mitochondrial genome present in four different structural types. Furthermore, Whole Genome mapping allowed resolution of the chromosomal ends, identification of areas of misassembly in the R1 genome, and genomic differences between the R1 and Gray strains, which occur primarily in the telomeric regions. These studies set the stage for a better understanding of the evolution and diversity of this important human pathogen.

Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti.

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.

Hydrophobic moeties in recombinant proteins are crucial to generate efficient saponin-based vaccine against Apicomplexan Babesia divergens.

Throughout Europe, bovine babesiosis is mainly caused by Babesia divergens, an Apicomplexan parasite transmitted by tick bites. The intra-erythrocytic development of B. divergens merozoites leads to haemolytic anaemia, and bovine babesiosis is responsible for economic losses in the agro-business industry. A totally efficient recombinant vaccine based on the merozoite surface protein Bd37 and saponin QuilA was recently described. In the present study we determined that protective immunity elicited by the Bd37 recombinant protein was related to the presence of hydrophobic residues in the protein. Using polymeric fusion of Bd37 as well as cell-free in vitro protein expression, we successfully expressed recombinant proteins containing hydrophobic sequences without the need of GST fusion. We used different hydrophobic sequences and different recombinant Bd37 proteins to demonstrate that antigen hydrophobicity affects the immune system, turning an inefficient protein into a 100% protective vaccine. Some hypotheses about the hydrophobic effect and its potential application to other parasitic protozoa vaccine are also discussed.