RhoGAP19D inhibits Cdc42 laterally to control epithelial cell shape and prevent invasion

Cdc42-GTP is required for apical domain formation in epithelial cells, where it recruits and activates the Par-6–aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically is unclear. We used sequence analysis and 3D structural modeling to determine which Drosophila GTPase-activating proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the only high-probability Cdc42GAP required for polarity in the follicular epithelium. RhoGAP19D is recruited by α-catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of precancerous breast lesions. Thus, RhoGAP19D couples lateral cadherin adhesion to the apical localization of active Cdc42, thereby suppressing epithelial invasion.

RhoGAP19D inhibits Cdc42 laterally to control epithelial cell shape and prevent invasion

SummaryCdc42-GTP is required for apical domain formation in epithelial cells where it recruits and activates the Par-6/aPKC polarity complex, but how the activity of Cdc42 itself is restricted apically unclear. We used sequence analysis and 3D structure modelling to determine which Drosophila GTPase Activating Proteins (GAPs) are likely to interact with Cdc42 and identified RhoGAP19D as the sole Cdc42GAP required for polarity in the follicle cell epithelium. RhoGAP19D is recruited by α-catenin to lateral E-cadherin adhesion complexes, resulting in exclusion of active Cdc42 from the lateral domain. rhogap19d mutants therefore lead to lateral Cdc42 activity, which expands the apical domain through increased Par-6/aPKC activity and stimulates lateral contractility through the myosin light chain kinase, Genghis khan (MRCK). This causes buckling of the epithelium and invasion into the adjacent tissue, a phenotype resembling that of pre-cancerous breast lesions. Thus, RhoGAP19D couples lateral Cadherin adhesion to the apical localisation of active Cdc42, thereby suppressing epithelial invasion.

Cooperative Role of MAPK Pathways in the Interaction of Candida albicans with the Host Epithelium

Candida albicans is an important human fungal pathogen responsible for tens of millions of infections as well as hundreds of thousands of severe life-threatening infections each year. MAP kinase (MAPK) signal transduction pathways facilitate the sensing and adaptation to external stimuli and control the expression of key virulence factors such as the yeast-to-hypha transition, the biogenesis of the cell wall, and the interaction with the host. In the present study, we have combined molecular approaches and infection biology to analyse the role of C. albicans MAPK pathways during an epithelial invasion. Hog1 was found to be important for adhesion to abiotic surfaces but was dispensable for damage to epithelial cells. The Mkc1 cell wall integrity (CWI) and Cek1 pathways, on the other hand, were both required for oral epithelial damage. Analysis of the ability to penetrate nutrient-rich semi-solid media revealed a cooperative role for Cek1 and Mkc1 in this process. Finally, cek2Δ (as well as cek1Δ) but not mkc1Δ or hog1Δ mutants, exhibited elevated β-glucan unmasking as revealed by immunofluorescence studies. Therefore, the four MAPK pathways play distinct roles in adhesion, epithelial damage, invasion and cell wall remodelling that may contribute to the pathogenicity of C. albicans.

Candida albicans and Pseudomonas aeruginosa Interact To Enhance Virulence of Mucosal Infection in Transparent Zebrafish

ABSTRACT Polymicrobial infections often include both fungi and bacteria and can complicate patient treatment and resolution of infection. Cross-kingdom interactions among bacteria, fungi, and/or the immune system during infection can enhance or block virulence mechanisms and influence disease progression. The fungus Candida albicans and the bacterium Pseudomonas aeruginosa are coisolated in the context of polymicrobial infection at a variety of sites throughout the body, including mucosal tissues such as the lung. In vitro, C. albicans and P. aeruginosa have a bidirectional and largely antagonistic relationship. Their interactions in vivo remain poorly understood, specifically regarding host responses in mediating infection. In this study, we examine trikingdom interactions using a transparent juvenile zebrafish to model mucosal lung infection and show that C. albicans and P. aeruginosa are synergistically virulent. We find that high C. albicans burden, fungal epithelial invasion, swimbladder edema, and epithelial extrusion events serve as predictive factors for mortality in our infection model. Longitudinal analyses of fungal, bacterial, and immune dynamics during coinfection suggest that enhanced morbidity is associated with exacerbated C. albicans pathogenesis and elevated inflammation. The P. aeruginosa quorum-sensing-deficient ΔlasR mutant also enhances C. albicans pathogenicity in coinfection and induces extrusion of the swimbladder. Together, these observations suggest that C. albicans-P. aeruginosa cross talk in vivo can benefit both organisms to the detriment of the host.

Enterococcus faecalisbacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence ofCandida albicans

Enterococcus faecalis, a Gram-positive bacterium, andCandida albicans, a fungus, occupy overlapping niches as ubiquitous constituents of the gastrointestinal and oral microbiome. Both species also are among the most important and problematic, opportunistic nosocomial pathogens. Surprisingly, these two species antagonize each other’s virulence in both nematode infection and in vitro biofilm models. We report here the identification of theE. faecalisbacteriocin, EntV, produced from theentV(ef1097) locus, as both necessary and sufficient for the reduction ofC. albicansvirulence and biofilm formation through the inhibition of hyphal formation, a critical virulence trait. A synthetic version of the mature 68-aa peptide potently blocks biofilm development on solid substrates in multiple media conditions and disrupts preformed biofilms, which are resistant to current antifungal agents. EntV68is protective in three fungal infection models at nanomolar or lower concentrations. First, nematodes treated with the peptide at 0.1 nM are completely resistant to killing byC. albicans. The peptide also protects macrophages and augments their antifungal activity. Finally, EntV68reduces epithelial invasion, inflammation, and fungal burden in a murine model of oropharyngeal candidiasis. In all three models, the peptide greatly reduces the number of fungal cells present in the hyphal form. Despite these profound effects, EntV68has no effect onC. albicansviability, even in the presence of significant host-mimicking stresses. These findings demonstrate that EntV has potential as an antifungal agent that targets virulence rather than viability.