Sexual reproduction of human fungal pathogens

We review here recent advances in our understanding of sexual reproduction in fungal pathogens that commonly infect humans, including Candida albicans, Cryptococcus neoformans/gattii, and Aspergillus fumigatus. Where appropriate or relevant, we introduce findings on other species associated with human infections. In particular, we focus on rapid advances involving genetic, genomic, and population genetic approaches that have reshaped our view of how fungal pathogens evolve. Rather than being asexual, mitotic, and largely clonal, as was thought to be prevalent as recently as a decade ago, we now appreciate that the vast majority of pathogenic fungi have retained extant sexual, or parasexual, cycles. In some examples, sexual and parasexual unions of pathogenic fungi involve closely related individuals, generating diversity in the population but with more restricted recombination than expected from fertile, sexual, outcrossing and recombining populations. In other cases, species and isolates participate in global outcrossing populations with the capacity for considerable levels of gene flow. These findings illustrate general principles of eukaryotic pathogen emergence with relevance for other fungi, parasitic eukaryotic pathogens, and both unicellular and multicellular eukaryotic organisms

Fig1 facilitates calcium influx and localizes to membranes destined to undergo fusion during mating in Candida albicans

Few mating-regulated genes have been characterized in Candida albicans. C. albicans FIG1 (CaFIG1) is a fungus-specific and mating-induced gene encoding a putative 4-transmembrane domain protein that shares sequence similarities with members of the claudin superfamily. In Saccharomyces cerevisiae, Fig1 is required for shmoo fusion and is upregulated in response to mating pheromones. Expression of CaFIG1 was also strongly activated in the presence of cells of the opposite mating type. CaFig1-green fluorescent protein (GFP) was visible only during the mating response, when it localized predominantly to the plasma membrane and perinuclear zone in mating projections and daughter cells. At the plasma membrane, CaFig1-GFP was visualized as discontinuous zones, but the distribution of perinuclear CaFig1-GFP was homogeneous. Exposure to pheromone induced a 5-fold increase in Ca(2+) uptake in mating-competent opaque cells. Uptake was reduced substantially in the fig1Δ null mutant. CaFig1 is therefore involved in Ca(2+) influx and localizes to membranes that are destined to undergo fusion during mating

Huntingtin regulates Ca2+ chemotaxis and K+-facilitated cAMP chemotaxis, in conjunction with the monovalent cation/H+ exchanger Nhe1, in a model developmental system: Insights into its possible role in Huntington׳s disease

AbstractHuntington׳s disease is a neurodegenerative disorder, attributable to an expanded trinucleotide repeat in the coding region of the human HTT gene, which encodes the protein huntingtin. These mutations lead to huntingtin fragment inclusions in the striatum of the brain. However, the exact function of normal huntingtin and the defect causing the disease remain obscure. Because there are indications that huntingtin plays a role in Ca2+ homeostasis, we studied the deletion mutant of the HTT ortholog in the model developmental system Dictyostelium discoideum, in which Ca2+ plays a role in receptor-regulated behavior related to the aggregation process that leads to multicellular morphogenesis. The D. discoideum htt−-mutant failed to undergo both K+-facilitated chemotaxis in spatial gradients of the major chemoattractant cAMP, and chemotaxis up a spatial gradient of Ca2+, but behaved normally in Ca2+-facilitated cAMP chemotaxis and Ca2+-dependent flow-directed motility. This was the same phenotypic profile of the null mutant of Nhel, a monovalent cation/H+exchanger. The htt−-mutant also failed to orient correctly during natural aggregation, as was the case for the Nhel mutant. Moreover, in a K+-based buffer the normal localization of actin was similarly defective in both htt− and nhe1− cells in a K+-based buffer, and the normal localization of Nhe1 was disrupted in the htt− mutant. These observations demonstrate that Htt and Nhel play roles in the same specific cation-facilitated behaviors and that Nhel localization is directly or indirectly regulated by Htt. Similar cation-dependent behaviors and a similar relationship between Htt and Nhe1 have not been reported for mammalian neurons and deserves investigation, especially as it may relate to Huntington׳s disease

Do systematic reviews on pediatric topics need special methodological considerations?

Abstract Background Systematic reviews are key tools to enable decision making by healthcare providers and policymakers. Despite the availability of the evidence based Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA-2009 and PRISMA-P 2015) statements that were developed to improve the transparency and quality of reporting of systematic reviews, uncertainty on how to deal with pediatric-specific methodological challenges of systematic reviews impairs decision-making in child health. In this paper, we identify methodological challenges specific to the design, conduct and reporting of pediatric systematic reviews, and propose a process to address these challenges. Discussion One fundamental decision at the outset of a systematic review is whether to focus on a pediatric population only, or to include both adult and pediatric populations. Both from the policy and patient care point of view, the appropriateness of interventions and comparators administered to pre-defined pediatric age subgroup is critical. Decisions need to be based on the biological plausibility of differences in treatment effects across the developmental trajectory in children. Synthesis of evidence from different trials is often impaired by the use of outcomes and measurement instruments that differ between trials and are neither relevant nor validated in the pediatric population. Other issues specific to pediatric systematic reviews include lack of pediatric-sensitive search strategies and inconsistent choices of pediatric age subgroups in meta-analyses. In addition to these methodological issues generic to all pediatric systematic reviews, special considerations are required for reviews of health care interventions’ safety and efficacy in neonatology, global health, comparative effectiveness interventions and individual participant data meta-analyses. To date, there is no standard approach available to overcome this problem. We propose to develop a consensus-based checklist of essential items which researchers should consider when they are planning (PRISMA-PC-Protocol for Children) or reporting (PRISMA-C-reporting for Children) a pediatric systematic review. Summary Available guidelines including PRISMA do not cover the complexity associated with the conduct and reporting of systematic reviews in the pediatric population; they require additional and modified standards for reporting items. Such guidance will facilitate the translation of knowledge from the literature to bedside care and policy, thereby enhancing delivery of care and improving child health outcomes

Cofilin determines the migration behavior and turning frequency of metastatic cancer cells

We have investigated the effects of inhibiting the expression of cofilin to understand its role in protrusion dynamics in metastatic tumor cells, in particular. We show that the suppression of cofilin expression in MTLn3 cells (an apolar randomly moving amoeboid metastatic tumor cell) caused them to extend protrusions from only one pole, elongate, and move rectilinearly. This remarkable transformation was correlated with slower extension of fewer, more stable lamellipodia leading to a reduced turning frequency. Hence, the loss of cofilin caused an amoeboid tumor cell to assume a mesenchymal-type mode of movement. These phenotypes were correlated with the loss of uniform chemotactic sensitivity of the cell surface to EGF stimulation, demonstrating that to chemotax efficiently, a cell must be able to respond to chemotactic stimulation at any region on its surface. The changes in cell shape, directional migration, and turning frequency were related to the re-localization of Arp2/3 complex to one pole of the cell upon suppression of cofilin expression

Candida parapsilosis Characterization in an Outbreak Setting

Candida parapsilosis is an important non-albicans species which infects hospitalized patients. No studies have correlated outbreak infections of C. parapsilosis with multiple virulence factors. We used DNA fingerprinting to determine genetic variability among isolates from a C. parapsilosis outbreak and from our clinical database. We compared phenotypic markers of pathogenesis, including adherence, biofilm formation, and protein secretion (secretory aspartic protease [SAP] and phospholipase). Adherence was measured as colony counts on silicone elastomer disks immersed in agar. Biofilms formed on disks were quantified by dry weight. SAP expression was measured by hydrolysis of bovine albumin; a colorimetric assay was used to quantitate phospholipase. DNA fingerprinting indicated that the outbreak isolates were clonal and genetically distinct from our database. Biofilm expression by the outbreak clone was greater than that of sporadic isolates (p < 0.0005). Adherence and protein secretion did not correlate with strain pathogenicity. These results suggest that biofilm production plays a role in C. parapsilosis outbreaks

Cooperation between a Coenzyme A-Independent Stand-Alone Initiation Module and an Iterative Type I Polyketide Synthase during Synthesis of Mycobacterial Phenolic Glycolipids

Several Mycobacterium tuberculosis strains, Mycobacterium leprae, and other mycobacterial pathogens produce a group of small-molecule virulence factors called phenolic glycolipids (PGLs). PGLs play key roles in pathogenicity and host−pathogen interaction. Thus, elucidation of the PGL biosynthetic pathway will not only expand our understanding of natural product biosynthesis, but may also illuminate routes to novel therapeutics to afford alternative lines of defense against mycobacterial infections. In this study, we report an investigation of the enzymatic requirements for the production of long-chain p-hydroxyphenylalkanoate intermediates of PGL biosynthesis. We demonstrate a functional cooperation between a coenzyme A-independent stand-alone didomain initiation module (FadD22) and a 6-domain reducing iterative type I polyketide synthase (Pks15/1) for production of p-hydroxyphenylalkanoate intermediates in in vitro and in vivo FadD22-Pks15/1 reconstituted systems. Our results suggest that Pks15/1 is an iterative type I polyketide synthase with a relaxed control of catalytic cycle iterations, a mechanistic property that explains the origin of a characteristic alkyl chain length variability seen in mycobacterial PGLs. The FadD22-Pks15/1 reconstituted systems lay an initial foundation for future efforts to unveil the mechanism of iterative catalysis control by which the structures of the final products of Pks15/1 are defined, and to scrutinize the functional partnerships of the FadD22-Pks15/1 system with downstream enzymes of the PGL biosynthetic pathway

N-Acetylglucosamine Induces White to Opaque Switching, a Mating Prerequisite in Candida albicans

To mate, the fungal pathogen Candida albicans must undergo homozygosis at the mating-type locus and then switch from the white to opaque phenotype. Paradoxically, opaque cells were found to be unstable at physiological temperature, suggesting that mating had little chance of occurring in the host, the main niche of C. albicans. Recently, however, it was demonstrated that high levels of CO2, equivalent to those found in the host gastrointestinal tract and select tissues, induced the white to opaque switch at physiological temperature, providing a possible resolution to the paradox. Here, we demonstrate that a second signal, N-acetylglucosamine (GlcNAc), a monosaccharide produced primarily by gastrointestinal tract bacteria, also serves as a potent inducer of white to opaque switching and functions primarily through the Ras1/cAMP pathway and phosphorylated Wor1, the gene product of the master switch locus. Our results therefore suggest that signals produced by bacterial co-members of the gastrointestinal tract microbiota regulate switching and therefore mating of C. albicans