Three-dimensional organotypic models of human colonic epithelium to study the early stages of enteric salmonellosis.

In vitro cell culture models used to study how Salmonella initiates disease at the intestinal epithelium would benefit from the recognition that organs and tissues function in a three-dimensional (3-D) environment and that this spatial context is necessary for development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3-D models of human colonic epithelium and apply them to study the early stages of enteric salmonellosis. The human colonic cell line HT-29 was cultured in 3-D and characterized by immunohistochemistry, histology, and scanning electron microscopy. Wild-type Salmonella typhimurium and an isogenic SPI-1 type three secretion system (TTSS) mutant derivative (invA) were used to compare the interactions with 3-D cells and monolayers in adherence/invasion, tissue pathology, and cytokine expression studies. The results showed that 3-D culture enhanced many characteristics normally associated with fully differentiated, functional intestinal epithelia in vivo, including better organization of junctional, extracellular matrix, and brush-border proteins, and highly localized mucin production. Wild-type Salmonella demonstrated increased adherence, but significantly lower invasion for 3-D cells. Interestingly, the SPI-I TTSS mutant showed wild-type ability to invade into the 3-D cells but did not cause significant structural changes to these cells. Moreover, 3-D cells produced less interleukin-8 before and after Salmonella infection. These results suggest that 3-D cultures of human colonic epithelium provide valuable alternative models to study human enteric salmonellosis with potential for novel insight into Salmonella pathogenesis.

FELINES: a utility for extracting and examining EST-defined introns and exons.

FELINES (Finding and Examining Lots of Intron 'N' Exon Sequences) is a utility written to automate construction and analysis of high quality intron and exon sequence databases produced from EST (expressed sequence tag) to genomic sequence alignments. We demonstrated the various programs of the FELINES utility by creating intron and exon sequence databases for the fungal organism Schizosaccharomyces pombe from alignments of EST to genomic sequences. In addition, we analyzed our constructed S.pombe sequence databases and the well-established Saccharomyces cerevisiae intron database from Manuel Ares' Laboratory for conserved sequence motifs. FELINES was shown to be useful for characterizing branchsites, polypyrimidine tracts and 5' and 3' splice sites in the intron databases and exonic splicing enhancers (ESEs) in S.pombe exons. FELINES is available at http://www.genome.ou.edu/informatics.html.

Spaceflight enhances cell aggregation and random budding in Candida albicans.

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans-induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public.

Media ion composition controls regulatory and virulence response of Salmonella in spaceflight.

The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth.

Role of protein O-mannosyltransferase Pmt4 in the morphogenesis and virulence of Cryptococcus neoformans.

Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen Cryptococcus neoformans. Disruption of the C. neoformans PMT4 gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the pmt4 mutant compared to wild-type cells. The pmt4 mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the pmt4 mutant, as well as a global decrease in protein mannosylation. Heterologous expression of C. neoformans PMT4 in a Saccharomyces cerevisiae pmt1pmt4 mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in C. neoformans was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of C. neoformans.

Role of CD4+ T cells in a protective immune response against Cryptococcus neoformans in the central nervous system.

Cryptococcosis is a life-threatening disease caused by the encapsulated yeast, Cryptococcus neoformans. Although infection with C. neoformans is initiated in the lungs, morbidity and mortality is mostly associated with infections of the central nervous system (CNS). Individuals with deficiencies in cell-mediated immunity, such as patients with AIDS, are more susceptible to disseminated cryptococcosis, highlighting the importance of cell-mediated immunity and CD4+ T cells in host resistance against C. neoformans. Using a mouse model of cryptococcal meningoencephalitis, we have shown that immunization of mice with a cryptococcal antigen induced a protective immune response that crossed the blood-brain barrier and initiated an immune response directly in the CNS if C. neoformans was present. The regional protective response was characteristic of a Type-1 (Th1) response in the types of cells present at the site of infection and in the cytokines and chemokines expressed. Here, we extend those findings and report that CD4+ T cells are required for survival of immune mice infected directly in the brain with C. neoformans and sensitized CD4 + T cells can transfer partial protection to naive mice infected intracerebrally with C. neoformans. Furthermore, CD4 + T cells were also important for optimal infiltration of inflammatory cells at the site of infection and in the expression of cytokines and chemokines associated with protection in the brain. Lastly, CD4+ T cells were required for optimal regional production and secretion of IFNgamma and in the significantly increased expression of iNOS in C. neoformans-infected brains of immune mice.

Cytokine and chemokine expression in the central nervous system associated with protective cell-mediated immunity against Cryptococcus neoformans.

Cryptococcus neoformans is a yeast that causes cryptococcosis, a life-threatening disease that develops following inhalation and dissemination of the organisms. C. neoformans has a predilection for the central nervous system (CNS) and mortality is most frequently associated with meningoencephalitis. Susceptibility to cryptococcosis is increased in patients with deficiencies in cell-mediated immunity (CMI). Because cryptococcal CNS infections are associated with mortality and diagnosis of cryptococcosis is often not made until after dissemination to the CNS, a better understanding of host defense mechanisms against C. neoformans in the CNS is needed to design improved therapies for immunocompromised individuals suffering from cryptococcosis. Using a mouse model, we previously described a protective cell-mediated immune response induced in the periphery that limited the growth of C. neoformans in the CNS. In the current investigation, we examined cytokine and chemokine expression in the CNS to identify factors important in achieving protective immunity. We observed increased expression of IL-1beta, TNF-alpha, IFN-gamma, MCP-1, RANTES, and IP-10 in C. neoformans-infected brains of immune mice compared to control mice suggesting that these cytokines and chemokines are associated with the protective immune response. Furthermore, the Th1-type cytokines TNF-alpha and IFN-gamma, but not the Th2 cytokines IL-4 and IL-5, were secreted at significantly higher levels in C. neoformans-infected brains of immune mice compared to control mice. Our results demonstrate that cytokines and chemokines associated with CMI are produced following infection in the CNS of immunized mice, and the expression of these factors correlates with protection against C. neoformans in the CNS.

Introns and splicing elements of five diverse fungi.

Genomic sequences and expressed sequence tag data for a diverse group of fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Neurospora crassa, and Cryptococcus neoformans) provided the opportunity to accurately characterize conserved intronic elements. An examination of large intron data sets revealed that fungal introns in general are short, that 98% or more of them belong to the canonical splice site (ss) class (5'GU...AG3'), and that they have polypyrimidine tracts predominantly in the region between the 5' ss and the branch point. Information content is high in the 5' ss, branch site, and 3' ss regions of the introns but low in the exon regions adjacent to the introns in the fungi examined. The two yeasts have broader intron length ranges and correspondingly higher intron information content than the other fungi. Generally, as intron length increases in the fungi, so does intron information content. Homologs of U2AF spliceosomal proteins were found in all species except for S. cerevisiae, suggesting a nonconventional role for U2AF in the absence of canonical polypyrimidine tracts in the majority of introns. Our observations imply that splicing in fungi may be different from that in vertebrates and may require additional proteins that interact with polypyrimidine tracts upstream of the branch point. Theoretical protein homologs for Nam8p and TIA-1, two proteins that require U-rich regions upstream of the branch point to function, were found. There appear to be sufficient differences between S. cerevisiae and S. pombe introns and the introns of two filamentous members of the Ascomycota and one member of the Basidiomycota to warrant the development of new model organisms for studying the splicing mechanisms of fungi.