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Pathogenic fungi populate a wide range of environments and infect a diversity of host species. Despite this substantial biological flexibility, the impact of interactions between fungi and their hosts on the evolution of pathogenicity remains unclear. We studied how repeated interactions between the fungus Cryptococcus neoformans and relevant environmental and mammalian host cells—amoeba and mouse macrophages—shape the evolution of this model fungal pathogen. First, using a collection of clinical and environmental isolates of C. neoformans, we characterized a range of survival phenotypes for these strains when exposed to host cells of different species. We then performed serial passages of an environmentally isolated C. neoformans strain through either amoeba or macrophages for ~75 generations to observe how these interactions select for improved replication within hosts. In an adapted population, we identified a single point mutation in the adenylate cyclase gene, CAC1, that swept to fixation and confers a strong competitive advantage for growth inside of macrophages. Strikingly, this growth advantage in macrophages is inversely correlated with disease severity during mouse infections, suggesting that adaptations to specific host niches can markedly reduce the pathogenicity of these fungi. These results raise intriguing questions about the influence of cAMP signaling on pathogenicity and highlight the role of seemingly small adaptive changes in promoting fundamental shifts in the intracellular behavior and virulence of these important human pathogens.

Purpose Newborn screening disorders increasingly require genetic variant analysis as part of second-tier or confirmatory testing. Sanger sequencing and gene-specific next-generation sequencing (NGS)-based tests, the current methods of choice, are costly and lack scalability when expanding to new conditions. We describe a scalable, exome sequencing-based NGS pipeline with a priori analysis restriction that can be universally applied to any NBS disorder. Methods De-identified abnormal newborn screening specimens representing severe combined immune deficiency (SCID), cystic fibrosis (CF), VLCAD deficiency, metachromatic leukodystrophy (MLD), and in silico sequence read data sets were used to validate the pipeline. To support interpretation and clinical decision-making within the bioinformatics pipeline, variants from multiple databases were curated and validated. Results CFTR variant panel analysis correctly identified all variants. Concordance compared with diagnostic testing results for targeted gene analysis was between 78.6% and 100%. Validation of the bioinformatics pipeline with in silico data sets revealed a 100% detection rate. Varying degrees of overlap were observed between ClinVar and other databases ranging from 3% to 65%. Data normalization revealed that 11% of variants across the databases required manual curation. Conclusion This pipeline allows for restriction of analysis to variants within a single gene or multiple genes, and can be readily expanded to full exome analysis if clinically indicated and parental consent is granted.

Environmental pathogens move from ecological niches to mammalian hosts, requiring adaptation to dramatically different environments. Microbes that disseminate farther, including the fungal meningitis pathogen Cryptococcus neoformans, require additional adaptation to diverse tissues. We demonstrate that the formation of a small C. neoformans morphotype-called "seed" cells due to their colonizing ability-is critical for extrapulmonary organ entry. Seed cells exhibit changes in fungal cell size and surface expression that result in an enhanced macrophage update. Seed cell formation is triggered by environmental factors, including C. neoformans' environmental niche, and pigeon guano with phosphate plays a central role. Seed cells show the enhanced expression of phosphate acquisition genes, and mutants unable to acquire phosphate fail to adopt the seed cell morphotype. Additionally, phosphate can be released by tissue damage, potentially establishing a feed-forward loop of seed cell formation and dissemination. Thus, C. neoformans' size variation represent inducible morphotypes that change host interactions to facilitate microbe spread.

PURPOSE OF REVIEW: Environmental fungi such as Cryptococcus neoformans and Aspergillus fumigatus must survive many different and changing environments as they transition from their environmental niches to human lungs and other organs. Fungi alter their cell surfaces and secreted macromolecules to respond to and manipulate their surroundings. RECENT FINDINGS: This review focuses on exo-polysaccharides, chains of sugars that transported out of the cell and spread to the local environment. Major exo-polysaccharides for C. neoformans and A. fumigatus are glucuronylxylomannan (GXM) and galactosaminogalactan (GAG), respectively, which accumulate at high concentrations in growth medium and infected patients SUMMARY: Here we discuss GXM and GAG synthesis and export, their immunomodulatory properties, and their roles in biofilm formation. We also propose areas of future research to address outstanding questions in the field that could facilitate development of new disease treatments.

SUMMARYEnvironmental pathogens, which move from ecological niches to mammalian hosts, must adapt to dramatically different environments. Microbes that disseminate farther, including the fungal meningitis pathogen Cryptococcus neoformans, require additional adaptation to diverse tissues. When C. neoformans enters the lungs, infecting cells (30 μm diameter), then form a heterogeneous population. The brain contains uniformly small cells (∼7 μm). We demonstrate that formation of a small C. neoformans morphotype – called “seed” cells due to their disseminating ability – is critical for extrapulmonary organ entry. Seed cell formation is triggered by environmental factors, including C. neoformans’ environmental niche, pigeon guano. The underlying trigger, phosphate, can be released by tissue damage, potentially establishing a feed-forward loop of seed cell formation and dissemination. We demonstrate that C. neoformans’ size variation is not just a continuum but inducible subpopulations that change host interactions to facilitate microbe survival and spread.

Volume 201, no. 2, e00635-18, 2019, [https://doi.org/10.1128/JB.00635-18][1]. Page 10, line 14: “Acyl-carrier-protein” should read “Activator of cylindrical proteases.” [1]: /lookup/doi/10.1128/JB.00635-18

Chlamydiais an obligate intracellular bacterium that differentiates between two distinct functional and morphological forms during its developmental cycle: elementary bodies (EBs) and reticulate bodies (RBs). EBs are non-dividing, small electron dense forms that infect host cells. RBs are larger, non-infectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other.Chlamydiahas five uncharacterizedclpgenes:clpX,clpC, twoclpPparalogs, andclpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined these genes are expressed mid-cycle. Bioinformatic analyses of these proteins identified key residues important for activity. Over-expression of inactiveclpPmutants inChlamydiasuggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, ClpP2, but not ClpP1, protease activity was detectedin vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.ImportanceChlamydia trachomatisis the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated, obligate intracellular pathogens that alternate between two functional and morphologic forms with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression inChlamydia, their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.