The pathogenesis of candidiasis involves invasion of host tissues by filamentous forms of the opportunistic yeast Candida albicans. Morphology-specific gene products may confer proinvasive properties. A hypha-specific surface protein, Hwp1, with similarities to mammalian small proline-rich proteins was shown to serve as a substrate for mammalian transglutaminases. Candida albicans strains lacking Hwp1 were unable to form stable attachments to human buccal epithelial cells and had a reduced capacity to cause systemic candidiasis in mice. This represents a paradigm for microbial adhesion that implicates essential host enzymes.

Integration of the intestinal epithelium and the mucosal immune system is critical for gut homeostasis. The intestinal epithelium is a functional barrier that secludes luminal content, senses changes in the gut microenvironment, and releases immune regulators that signal underlying immune cells. However, interactions between epithelial and innate immune cells to maintain barrier integrity and prevent infection are complex and poorly understood. We developed and characterized a primary human macrophage-enteroid co-culture model for in-depth studies of epithelial and macrophage interactions. Human intestinal stem cell-derived enteroid monolayers co-cultured with human monocyte-derived macrophages were used to evaluate barrier function, cytokine secretion, and protein expression under basal conditions and following bacterial infection. Macrophages enhanced barrier function and maturity of enteroid monolayers as indicated by increased transepithelial electrical resistance and cell height. Communication between the epithelium and macrophages was demonstrated through morphological changes and cytokine production. Intraepithelial macrophage projections, efficient phagocytosis, and stabilized enteroid barrier function revealed a coordinated response to enterotoxigenic and enteropathogenic E. coli infections. In summary, we have established the first primary human macrophage-enteroid co-culture system, defined conditions that allow for a practical and reproducible culture model, and demonstrated its suitability to study gut physiology and host responses to enteric pathogens.

cDNA sequences encoding a cell wall protein have been isolated from the opportunistic pathogen, Candida albicans, an organism that can cause serious disease in immunocompromised patients such as those with AIDS. The cDNA encodes a peptide that is largely composed of an acidic, repeated motif 10 amino acids in length that is rich in proline and glutamine residues. The cDNA gene product was found to be present on hyphal surfaces by immunofluorescence assays using monospecific antisera raised to the recombinant protein produced in Pichia pastoris. The hyphae-specific surface location was also seen on organisms colonizing the gastrointestinal mucosa of mice, indicating that the antigen is produced and developmentally regulated during growth in host tissues. The cDNA clone hybridized to an abundant messenger RNA 2.3 kilobases in size that was present in hyphal but not yeast forms. These studies demonstrate that the bud-hypha transition is accompanied by the de novo synthesis of proteins that are targeted to hyphal surfaces. The primary sequence of the unique amino acid motif shares features with surface proteins of other lower eukaryotic microorganisms and with host acidic salivary proline-rich proteins.

Previous evaluation of HWP1 in systemic candidiasis in CBA/J mice was done with Candida albicans strains with differing genetic locations of URA3 as a result of Ura-blaster mutagenesis. In this study, the presence of HWP1 and the location of URA3 contributed to the severity of murine systemic candidiasis in BALB/c mice.In previous work (8), the HWP1 gene was required for virulence in systemic candidiasis. A perplexing observation from this study was the suggestion of a trend of increased survival, or slight loss of virulence, of mice given CAH7 (HWP1/hwp1) compared to that of mice given CAHR3 (HWP1/hwp1 revertant). The survival differences were apparently not related to differences in HWP1 gene expression as the amounts of HWP1 mRNA and protein were equivalent for the two strains. Moreover, the strains did not differ in growth rates. A difference between strains CAH7 and CAHR3 is the location of the selectable marker gene URA3. The URA3 gene is within the open reading frame of HWP1 in CAH7 but within ENO1 in CAHR3.The use of the Ura-blaster technique in Candida albicans (2) results in both inactivation of a gene of interest and ectopic placement of URA3 within the gene. The observation that C. albicans auxotrophic mutants that cannot produce orotidine 5Ј-monophosphate (OMP) decarboxylase because they lack the URA3 gene are not pathogenic (4) suggests that if reduced levels of OMP decarboxylase were to be produced in vivo, intermediate virulence might result. Placement of the C. albicans URA3 gene at a locus other than the URA3 locus during implementation of the Ura-blaster technique has been shown to result in reduced OMP decarboxylase activities in vitro, and the levels of reduction were different for different loci. However, a definitive relationship between OMP decarboxylase enzyme activity in in vitro cultures and virulence was not revealed (6). These studies raised the possibility that the genetic location of the URA3 gene affects differential in vivo growth rates of strains CAH7 and CAHR3, leading to a trend of decreased virulence of CAH7.The goals of the present study were twofold. The first was to make the expression of the URA3 gene independent of positional effects arising from placement at the HWP1 locus. The second goal was to be able to compare C. albicans strains that are identical with regard to the location of the URA3 gene. Two new C. albicans strains with disruptions at the HWP1 locus were created by introducing a DNA fragment with eno1::URA3