Monoclonal antibodies specific for Candida albicans Als3 that immunolabel fungal cells in vitro and in vivo and block adhesion to host surfaces

Coleman, David; Oh, Soo Hee; Zhao, Xiaomin; Zhao, Hongyuan; Hutchins, Jeff; Vernachio, John; Patti, Joseph M.; Hoyer, Lois L.

doi:10.1016/j.mimet.2009.05.002

Cited by 63 publications

(139 citation statements)

References 22 publications

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“…However, this mutant has normal adherence to fibronectin, possibly due to the compensatory effects of other Als proteins, such as Als1, that also bind to this extracellular matrix protein. As expected, both full-length monoclonal antibodies and singlechain variable fragments of human antibodies against Als3 block adherence to both endothelial and oral epithelial cells (6,14,28). These antibodies are directed against the N-terminal region of Als3, consistent with the model that this region contains the substrate binding domain.…”

Section: Als3 Functionsupporting

confidence: 80%

“…As expected from the multifunctional nature of Als3, mutant strains of C. albicans that lack this protein have prominent defects in assays of host cell interactions, biofilm formation, and iron acquisition in vitro. In addition, Als3 is strongly expressed by hyphae in the kidneys of mice with disseminated candidiasis (14), and high levels of ALS3 mRNA are present in oral scrapings of patients with oropharyngeal candidiasis (68). Thus, one would expect that an als3⌬/⌬ mutant would have significantly attenuated virulence in experimental animal models of candidiasis.…”

Section: Thus C Albicans Als3 Functions As a Molecular Mimic Of Mammentioning

confidence: 99%

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Candida albicans Als3, a Multifunctional Adhesin and Invasin

2011

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2Candida albicans is part of the normal human flora, and it grows on mucosal surfaces in healthy individuals. In susceptible hosts, this organism can cause both mucosal and hematogenously disseminated disease. For C. albicans to persist in the host and induce disease, it must be able to adhere to biotic and abiotic surfaces, invade host cells, and obtain iron. The C. albicans hypha-specific surface protein Als3 is a member of the agglutininlike sequence (Als) family of proteins and is important in all of these processes. Functioning as an adhesin, Als3 mediates attachment to epithelial cells, endothelial cells, and extracellular matrix proteins. It also plays an important role in biofilm formation on prosthetic surfaces, both alone and in mixed infection with Streptococcus gordonii. Als3 is one of two known C. albicans invasins. It binds to host cell receptors such as E-cadherin and N-cadherin and thereby induces host cells to endocytose the organism. Als3 also binds to host cell ferritin and enables C. albicans to utilize this protein as a source of iron. Because of its multiple functions and its high expression level in vivo, Als3 is a promising target for vaccines that induce protective cell-mediated and antibody responses. This review will summarize the multiple functions of this interesting and multifunctional protein.Candida spp. are the fourth most common cause of nosocomial bloodstream infections, and Candida albicans accounts for approximately 50% of cases of candidemia (20,67). This organism also causes at least 80% of cases of oropharyngeal and vulvovaginal candidiasis (57, 66). The predominance of C. albicans as a cause of both hematogenously disseminated and mucosal disease suggests that this organism possesses unique virulence factors compared to other species of Candida. One such factor, which is present only in C. albicans, is Als3. This protein plays a key role in multiple processes that are necessary for the organism to colonize the host and cause disease. These processes include adherence to host cells, biofilm formation, host cell invasion, and iron acquisition. Moreover, because Als3 is highly expressed in vivo, it is a promising target for therapeutic antibody and vaccine development. This review will discuss Als3 structure and function, as well as its utility as a therapeutic target. GENETIC AND BIOCHEMICAL CHARACTERISTICS OF Als3The ALS gene family. Als3 is encoded by the ALS3 gene, which is a member of the agglutinin-like sequence (ALS) gene family (23). This family was discovered by Hoyer et al. (24) based on its similarity to Saccharomyces cerevisiae SAG1, which codes for ␣ agglutinin. The ALS gene family has eight members (ALS1 to ALS7 and ALS9) (Fig. 1). These genes encode cell surface proteins that are predicted to share the same overall structure (Fig. 2). At the N terminus is a signal peptide followed by a 300-amino-acid immunoglobulin-like domain and a 104-amino-acid threonine-rich domain that contains ␤-sheets (18,22,47,55). Most Als proteins have adhesin function (18,55,72), an...

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Section: Als3 Functionsupporting

confidence: 80%

Section: Thus C Albicans Als3 Functions As a Molecular Mimic Of Mammentioning

confidence: 99%

Candida albicans Als3, a Multifunctional Adhesin and Invasin

2011

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“…Following expression, the protein of interest was purified by column chromatography and its identity was confirmed as Als3 by MS. The N-terminal domain of Als1p (amino acids 18-329 of the protein) was produced in Pichia pastoris as described previously and used as a control (Coleman et al, 2009). …”

Section: Methodsmentioning

confidence: 99%

Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p

et al. 2012

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The bacterium Staphylococcus (St.) aureus and the opportunistic fungus Candida albicans are currently among the leading nosocomial pathogens, often co-infecting critically ill patients, with high morbidity and mortality. Previous investigations have demonstrated preferential adherence of St. aureus to C. albicans hyphae during mixed biofilm growth. In this study, we aimed to characterize the mechanism behind this observed interaction. C. albicans adhesin-deficient mutant strains were screened by microscopy to identify the specific receptor on C. albicans hyphae recognized by St. aureus. Furthermore, an immunoassay was developed to validate and quantify staphylococcal binding to fungal biofilms. The findings from these experiments implicated the C. albicans adhesin agglutinin-like sequence 3 (Als3p) in playing a major role in the adherence process. This association was quantitatively established using atomic force microscopy, in which the adhesion force between single cells of the two species was significantly reduced for a C. albicans mutant strain lacking als3. Confocal microscopy further confirmed these observations, as St. aureus overlaid with a purified recombinant Als3 N-terminal domain fragment (rAls3p) exhibited robust binding. Importantly, a strain of Saccharomyces cerevisiae heterologously expressing Als3p was utilized to further confirm this adhesin as a receptor for St. aureus. Although the parental strain does not bind bacteria, expression of Als3p on the cell surface conferred upon the yeast the ability to strongly bind St. aureus. To elucidate the implications of these in vitro findings in a clinically relevant setting, an ex vivo murine model of co-infection was designed using murine tongue explants. Fluorescent microscopic images revealed extensive hyphal penetration of the epithelium typical of C. albicans mucosal infection. Interestingly, St. aureus bacterial cells were only seen within the Abbreviations: AFM, atomic force microscopy; CLSM, confocal laser scanning microscopy; ConA-Texas red, concanavalin A conjugated to Texas red; H&E, haematoxylin and eosin; IFA, indirect immunofluorescence; PNA-FISH, peptide nucleic acid probes for fluorescent in situ hybridization; SEM, scanning electron microscopy. A supplementary figure is available with the online version of this paper. Microbiology

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“…To analyze Als3 expression during hyphal growth, cells cultured in YPG medium plus 10% serum were fixed with 3% formaldehyde (Sigma) in PBS for 30 min and added to poly-L-lysine (Sigma)-coated glass coverslips. After 30 min, cells blocked with 3% bovine serum albumin were stained with an Als3 monoclonal antibody (36) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Molecular Probes). Photographs were taken using an Olympus BX61 microscope equipped with differential interference contrast (DIC) optics, appropriate filters, and a camera (Olympus DP71).…”

Section: Methodsmentioning

confidence: 99%

The NDR Kinase Cbk1 Downregulates the Transcriptional Repressor Nrg1 through the mRNA-Binding Protein Ssd1 in Candida albicans

et al. 2015

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NDR (nuclear Dbf2-related) kinases are essential components for polarized morphogenesis, cytokinesis, cell proliferation, and apoptosis. The NDR kinase Cbk1 is required for the hyphal growth of Candida albicans; however, the molecular functions of Cbk1 in hyphal morphogenesis are largely unknown. Here, we report that Cbk1 downregulates the transcriptional repressor Nrg1 through the mRNA-binding protein Ssd1, which has nine Cbk1 phosphorylation consensus motifs. We found that deletion of SSD1 partially suppressed the defective hyphal growth of the C. albicans cbk1⌬/⌬ mutant and that Ssd1 physically interacts with Cbk1. Cbk1 was required for Ssd1 localization to polarized growth sites. The phosphomimetic SSD1 allele (ssd1-9E) allowed the cbk1⌬/⌬ mutant to form short hyphae, and the phosphodeficient SSD1 allele (ssd1-9A) resulted in shorter hyphae than did the wild-type SSD1 allele, indicating that Ssd1 phosphorylation by Cbk1 is important for hyphal morphogenesis. Furthermore, we show that the transcriptional repressor Nrg1 does not disappear during hyphal initiation in the cbk1⌬/⌬ mutant but is completely absent in the cbk1⌬/⌬ ssd1⌬/⌬ double mutant. Deletion of SSD1 also increased Als3 expression and internalization of the cbk1⌬/⌬ mutant in the human embryonic kidney cell line HEK293T. Collectively, our results suggest that one of the functions of Cbk1 in the hyphal morphogenesis of C. albicans is to downregulate Nrg1 through Ssd1.C ell polarity, a structural and functional asymmetry of cellular organization, is observed in diverse animal cell types, such as neurons and epithelial cells, and a variety of unicellular organisms, including yeast, fungi, protozoa, and prokaryotes (1-3). Cell polarity is established and maintained by directional movement and an unbalanced distribution of proteins, mRNAs, and subcellular organelles (4-7). Directional movement of cellular components requires delicately coordinated spatial and temporal regulation of polarity proteins, actin and tubulin cytoskeletons, and endomembranes (3, 4). In yeast, polarized cell growth occurs during budding, mating, and filamentous growth. Although the polarization events differ in spatial cues and spatiotemporal controls, the events are generally initiated and established from local accumulation of active GTP-bound Cdc42 and scaffold proteins, as well as the directed orientation of cytoskeletal elements (8-10).Candida albicans is an important opportunistic fungal pathogen that causes not only superficial infection but also systemic or life-threatening infections in immunocompromised hosts (11,12). C. albicans switches between yeast and hyphal forms to rapidly disseminate inside the host and escape from host defense systems, thus resulting in fatal infection (13,14). The morphological shape of C. albicans is determined by multiple signaling pathways that respond to the numerous environmental challenges C. albicans encounters in the host. The known major signaling pathways include the cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway via Efg...

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Monoclonal antibodies specific for Candida albicans Als3 that immunolabel fungal cells in vitro and in vivo and block adhesion to host surfaces

Cited by 63 publications

References 22 publications

Candida albicans Als3, a Multifunctional Adhesin and Invasin

Candida albicans Als3, a Multifunctional Adhesin and Invasin

Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p

The NDR Kinase Cbk1 Downregulates the Transcriptional Repressor Nrg1 through the mRNA-Binding Protein Ssd1 in Candida albicans

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