Recipient mucosal-associated invariant T cells control GVHD within the colon

Mucosal-associated invariant T (MAIT) cells are a unique innate-like T cell subset that responds to a wide array of bacteria and yeast through recognition of riboflavin metabolites presented by the MHC class I–like molecule MR1. Here, we demonstrate using MR1 tetramers that recipient MAIT cells are present in small but definable numbers in graft-versus-host disease (GVHD) target organs and protect from acute GVHD in the colon following bone marrow transplantation (BMT). Consistent with their preferential juxtaposition to microbial signals in the colon, recipient MAIT cells generate large amounts of IL-17A, promote gastrointestinal tract integrity, and limit the donor alloantigen presentation that in turn drives donor Th1 and Th17 expansion specifically in the colon after BMT. Allogeneic BMT recipients deficient in IL-17A also develop accelerated GVHD, suggesting MAIT cells likely regulate GVHD, at least in part, by the generation of this cytokine. Indeed, analysis of stool microbiota and colon tissue from IL-17A–/– and MR1–/– mice identified analogous shifts in microbiome operational taxonomic units (OTU) and mediators of barrier integrity that appear to represent pathways controlled by similar, IL-17A–dependent mechanisms. Thus, MAIT cells act to control barrier function to attenuate pathogenic T cell responses in the colon and, given their very high frequency in humans, likely represent an important population in clinical BMT

Characterization of an Nmr Homolog That Modulates GATA Factor-Mediated Nitrogen Metabolite Repression in Cryptococcus neoformans

Nitrogen source utilization plays a critical role in fungal development, secondary metabolite production and pathogenesis. In both the Ascomycota and Basidiomycota, GATA transcription factors globally activate the expression of catabolic enzyme-encoding genes required to degrade complex nitrogenous compounds. However, in the presence of preferred nitrogen sources such as ammonium, GATA factor activity is inhibited in some species through interaction with co-repressor Nmr proteins. This regulatory phenomenon, nitrogen metabolite repression, enables preferential utilization of readily assimilated nitrogen sources. In the basidiomycete pathogen Cryptococcus neoformans, the GATA factor Gat1/Are1 has been co-opted into regulating multiple key virulence traits in addition to nitrogen catabolism. Here, we further characterize Gat1/Are1 function and investigate the regulatory role of the predicted Nmr homolog Tar1. While GAT1/ARE1 expression is induced during nitrogen limitation, TAR1 transcription is unaffected by nitrogen availability. Deletion of TAR1 leads to inappropriate derepression of non-preferred nitrogen catabolic pathways in the simultaneous presence of favoured sources. In addition to exhibiting its evolutionary conserved role of inhibiting GATA factor activity under repressing conditions, Tar1 also positively regulates GAT1/ARE1 transcription under non-repressing conditions. The molecular mechanism by which Tar1 modulates nitrogen metabolite repression, however, remains open to speculation. Interaction between Tar1 and Gat1/Are1 was undetectable in a yeast two-hybrid assay, consistent with Tar1 and Gat1/Are1 each lacking the conserved C-terminus regions present in ascomycete Nmr proteins and GATA factors that are known to interact with each other. Importantly, both Tar1 and Gat1/Are1 are suppressors of C. neoformans virulence, reiterating and highlighting the paradigm of nitrogen regulation of pathogenesis

Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence

Genomic comparisons between var. <i>grubii</i> and var. <i>neoformans</i> reveal relatively few rearrangements.

<p>Since divergence 18.5 million years ago, the two varieties of <i>C. neoformans</i> have maintained a largely syntenic genome structure with only a few inversions specific to each variety (marked with arrows). Comparisons also uncovered several strain-specific events: a translocation in the case of the var. <i>grubii</i> type strain H99 and telomere-telomere fusion and introgression in the case of var. <i>neoformans</i> JEC21.</p

Comparative genomics of non-pseudomonal bacterial species colonising paediatric cystic fibrosis patients

The genetic disorder cystic fibrosis is a life-limiting condition affecting similar to 70,000 people worldwide. Targeted, early, treatment of the dominant infecting species, Pseudomonas aeruginosa, has improved patient outcomes; however, there is concern that other species are now stepping in to take its place. In addition, the necessarily long-term antibiotic therapy received by these patients may be providing a suitable environment for the emergence of antibiotic resistance. To investigate these issues, we employed whole-genome sequencing of 28 non-Pseudomonas bacterial strains isolated from three paediatric patients. We did not find any trend of increasing antibiotic resistance (either by mutation or lateral gene transfer) in these isolates in comparison with other examples of the same species. In addition, each isolate contained a virulence gene repertoire that was similar to other examples of the relevant species. These results support the impaired clearance of the CF lung not demanding extensive virulence for survival in this habitat. By analysing serial isolates of the same species we uncovered several examples of strain persistence. The same strain of Staphylococcus aureus persisted for nearly a year, despite administration of antibiotics to which it was shown to be sensitive. This is consistent with previous studies showing antibiotic therapy to be inadequate in cystic fibrosis patients, which may also explain the lack of increasing antibiotic resistance over time. Serial isolates of two naturally multi-drug resistant organisms, Achromobacter xylosoxidans and Stenotrophomonas maltophilia, revealed that while all S. maltophilia strains were unique, A. xylosoxidans persisted for nearly five years, making this a species of particular concern. The data generated by this study will assist in developing an understanding of the non-Pseudomonas species associated with cystic fibrosis

Polyploid Titan Cells Produce Haploid and Aneuploid Progeny To Promote Stress Adaptation

Cryptococcus neoformans is a major life-threatening fungal pathogen. In response to the stress of the host environment, C. neoformans produces large polyploid titan cells. Titan cell production enhances the virulence of C. neoformans, yet whether the polyploid aspect of titan cells is specifically influential remains unknown. We show that titan cells were more likely to survive and produce offspring under multiple stress conditions than typical cells and that even their normally sized daughters maintained an advantage over typical cells in continued exposure to stress. Although polyploid titan cells generated haploid daughter cell progeny upon in vitro replication under nutrient-replete conditions, titan cells treated with the antifungal drug fluconazole produced fluconazole-resistant diploid and aneuploid daughter cells. Interestingly, a single titan mother cell was capable of generating multiple types of aneuploid daughter cells. The increased survival and genomic diversity of titan cell progeny promote rapid adaptation to new or high-stress conditions

A fluorogenic C. neoformans reporter strain with a robust expression of m-cherry expressed from a safe haven site in the genome

C. neoformans is an encapsulated fungal pathogen with defined asexual and sexual life cycles. Due to the availability of genetic and molecular tools for its manipulation, it has become a model organism for studies of fungal pathogens, even though it lacks a reliable system for maintaining DNA fragments as extrachromosomal plasmids. To compensate for this deficiency, we identified a genomic gene-free intergenic region where heterologous DNA could be inserted by homologous recombination without adverse effects on the phenotype of the recipient strain. Since such a site in the C. neoformans genome at a different location has been named previously as "safe haven", we named this locus second safe haven site (SH2). Insertion of DNA into this site in the genome of the KN99 congenic strain pair caused minimal change in the growth of the engineered strain under a variety of in vitro and in vivo conditions. We exploited this 'safe' locus to create a genetically stable highly fluorescent strain expressing mCherry protein (KN99mCH); this strain closely resembled its wild-type parent (KN99α) in growth under a variety of in vitro stress conditions and in the expression of virulence traits. The efficiency of phagocytosis and the proliferation of KN99mCH inside human monocyte-derived macrophages were comparable to those of KN99α, and the engineered strain showed the expected organ dissemination after inoculation, although there was a slight reduction in virulence. The mCherry fluorescence allowed us to measure specific association of cryptococci with leukocytes in the lungs and mediastinal lymph nodes of infected animals and, for the first-time, to assess their live/dead status in vivo. These results highlight the utility of KN99mCH for elucidation of host-pathogen interactions in vivo. Finally, we generated drug-resistant KN99 strains of both mating types that are marked at the SH2 locus with a specific drug resistant gene cassette; these strains will facilitate the generation of mutant strains by mating