Donald C. Sheppard

Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells

Candida albicans is the most common cause of hematogenously disseminated and oropharyngeal candidiasis. Both of these diseases are characterized by fungal invasion of host cells. Previously, we have found that C. albicans hyphae invade endothelial cells and oral epithelial cells in vitro by inducing their own endocytosis. Therefore, we set out to identify the fungal surface protein and host cell receptors that mediate this process. We found that the C. albicans Als3 is required for the organism to be endocytosed by human umbilical vein endothelial cells and two different human oral epithelial lines. Affinity purification experiments with wild-type and an als3Δ/als3Δ mutant strain of C. albicans demonstrated that Als3 was required for C. albicans to bind to multiple host cell surface proteins, including N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. Furthermore, latex beads coated with the recombinant N-terminal portion of Als3 were endocytosed by Chinese hamster ovary cells expressing human N-cadherin or E-cadherin, whereas control beads coated with bovine serum albumin were not. Molecular modeling of the interactions of the N-terminal region of Als3 with the ectodomains of N-cadherin and E-cadherin indicated that the binding parameters of Als3 to either cadherin are similar to those of cadherin–cadherin binding. Therefore, Als3 is a fungal invasin that mimics host cell cadherins and induces endocytosis by binding to N-cadherin on endothelial cells and E-cadherin on oral epithelial cells. These results uncover the first known fungal invasin and provide evidence that C. albicans Als3 is a molecular mimic of human cadherins.

Complementary adhesin function in C. albicans biofilm formation.

BACKGROUND Biofilms are surface-associated microbial communities with significant environmental and medical impact. Here, we focus on an adherence mechanism that permits biofilm formation by Candida albicans, the major invasive fungal pathogen of humans. RESULTS The Als surface-protein family has been implicated in biofilm formation, and we show that Als1 and Als3 have critical but redundant roles. Overexpression of several other Als proteins permits biofilm formation in a biofilm-defective als1/als1 als3/als3 strain, thus arguing that the function of Als proteins in this process is governed by their respective expression levels. The surface protein Hwp1 is also required for biofilm formation, and we find that a mixture of biofilm-defective hwp1/hwp1 and als1/als1 als3/als3 strains can form a hybrid biofilm both in vitro and in vivo in a catheter infection model. Complementary function of Hwp1 and Als1 and 3 seems to reflect their interaction because expression of Hwp1 in the heterologous host S. cerevisiae permits adherence to wild-type C. albicans, but not to an als1/als1 als3/als3 strain. CONCLUSIONS The complementary roles of Hwp1 and Als1 and Als3 in biofilm formation are analogous to the roles of sexual agglutinins in mating reactions. This analogy suggests that biofilm-adhesin complementarity may promote formation of monospecies biofilms.

Candida albicans Als1p: an adhesin that is a downstream effector of the EFG1 filamentation pathway.

Filamentation and adherence to host cells are critical virulence factors of Candida albicans. Multiple filamentation regulatory pathways have been discovered in C. albicans using Saccharomyces cerevisiae as a model. In S. cerevisiae, these pathways converge on Flo11p, which functions as a downstream effector of filamentation and also mediates cell–cell adherence (flocculation). In C. albicans, such effector(s) have not yet been identified. Here, we demonstrate that the cell surface protein Als1p is an effector of filamentation in C. albicans. We show that Als1p expression is controlled by the transcription factor Efg1p, which is known to be a key regulator of filamentation in C. albicans. Further, disruption of ALS1 inhibited filamentation, and autonomous expression of Als1p restored filamentation in an efg1 homozygous null mutant. Thus, Als1p functions as a downstream effector of the EFG1 filamentation pathway. In addition, we found that Als1p mediates both flocculation and adherence of C. albicans to endothelial cells in vitro. As a cell surface glycoprotein that mediates filamentation and adherence, Als1p has both structural and functional similarity to S. cerevisiae Flo11p. Consistent with our in vitro results, Als1p was required for both normal filamentation and virulence in the mouse model of haematogenously disseminated candidiasis.

Fungal Invasion of Normally Non-Phagocytic Host Cells

Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research.

Role of the fungal Ras‐protein kinase A pathway in governing epithelial cell interactions during oropharyngeal candidiasis

Tpk1p, Tpk2p and Efg1p are members of the Ras‐protein kinase A pathway that governs the yeast‐to‐hyphal transition in Candida albicans. We used tpk1Δ/tpk1Δ, tpk2Δ/tpk2Δ and efg1Δ/efg1Δ mutants to investigate the role of these proteins in regulating the interactions of C. albicans with oral epithelial cell lines in vitro and virulence in murine models of oropharyngeal candidiasis (OPC) and haematogenously disseminated candidiasis (HDC). The tpk1Δ/tpk1Δ strain adhered to, invaded and damaged oral epithelial cells in vitro similarly to the wild‐type strain. In contrast, both the tpk2Δ/tpk2Δ and efg1Δ/efg1Δ strains had reduced capacity to invade and damage oral epithelial cells, and the efg1Δ/efg1Δ strain also exhibited decreased adherence to these cells. Consistent with these in vitro findings, the tpk2Δ/tpk2Δ and efg1Δ/efg1Δ strains also had significantly attenuated virulence during OPC. Therefore, Tpk2p and Efg1p both govern factors that enable C. albicans to invade and damage oral epithelial cells in vitro and cause OPC. These results also suggest that hyphal formation mediated by the Ras‐protein kinase A pathway is a key virulence mechanism during OPC. Interestingly, the efg1Δ/efg1Δ strain, but not the tpk2Δ/tpk2Δ had reduced virulence during HDC. Thus, Tpk2p may be more important for governing virulence during OPC than HDC.

International expert opinion on the management of infection caused by azole-resistant Aspergillus fumigatus

An international expert panel was convened to deliberate the management of azole-resistant aspergillosis. In culture-positive cases, in vitro susceptibility testing should always be performed if antifungal therapy is intended. Different patterns of resistance are seen, with multi-azole and pan-azole resistance more common than resistance to a single triazole. In confirmed invasive pulmonary aspergillosis due to an azole-resistant Aspergillus, the experts recommended a switch from voriconazole to liposomal amphotericin B (L-AmB; Ambisome(®)). In regions with environmental resistance rates of ≥10%, a voriconazole-echinocandin combination or L-AmB were favoured as initial therapy. All experts recommended L-AmB as core therapy for central nervous system aspergillosis suspected to be due to an azole-resistant Aspergillus, and considered the addition of a second agent with the majority favouring flucytosine. Intravenous therapy with either micafungin or L-AmB given as either intermittent or continuous therapy was recommended for chronic pulmonary aspergillosis due to a pan-azole-resistant Aspergillus. Local and national surveillance with identification of clinical and environmental resistance patterns, rapid diagnostics, better quality clinical outcome data, and a greater understanding of the factors driving or minimising environmental resistance are areas where research is urgently needed, as well as the development of new oral agents outside the azole drug class.

The role of mast cells in the defence against pathogens.

Although mast cells are best known for their role in mediating allergic diseases, recent studies have highlighted the important role that these cells play in the protection against infection with a variety of organisms.

Novel Inhalational Murine Model of Invasive Pulmonary Aspergillosis

ABSTRACT We developed a novel model of invasive aspergillosis (IA) that recapitulates human disease. Mice were immunosuppressed with cyclophosphamide and cortisone acetate and then infected in an aerosol chamber. This procedure reproducibly delivered 1 × 103 to 3 × 103 conidia to the lungs. Lethal pulmonary IA developed over 2 weeks and was prevented by amphotericin B.

Aspergillus Galactosaminogalactan Mediates Adherence to Host Constituents and Conceals Hyphal β-Glucan from the Immune System

Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall β-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.

Entamoeba histolytica and Entamoeba dispar: Epidemiology and Comparison of Diagnostic Methods in a Setting of Nonendemicity

Recent studies suggest that stool antigen assays are more sensitive and specific than microscopy for the diagnosis of Entamoeba histolytica infection. One hundred twelve patients presenting at 3 centers with symptoms or risk factors of E. histolytica infection were prospectively enrolled in this study to evaluate new diagnostic tests for infections with E. histolytica and Entamoeba dispar. Four ELISA-based stool antigen kits for detecting E. histolytica or E. dispar were blindly compared with stool microscopy. Amebic serology was assessed by indirect hemagglutination. When antigen assays were used as the reference standard, microscopy performed at referral centers was more specific (68.4% vs. 9.5%) but less sensitive (70.4% vs. 92.1%) than microscopy performed in community laboratories. Diagnosis with the E. histolytica test and Merlin Optimun S ELISA indicated that only 3 (4.2%) of 72 coproantigen-positive stools were positive for E. histolytica. Indirect hemagglutination was a good predictor of E. histolytica infection when titers of antibody to ameba were >/=1:512.