Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata.
Understanding how Candida glabrata is able to establish oral mucosal infections is particularly important since many C. glabrata strains are innately resistant to azole antifungal drugs used in treating mucosal and disseminated infections. The epidemiology of C. glabrata oral infections shows that C. glabrata is very often present as a co-infection with Candida albicans. Here we suggest a mechanism to explain this clinical finding. We show that C. glabrata is unable to colonize the oral mucosa in a murine oral infection model. However, prior or co-colonization by C. albicans allows C. glabrata to colonize and persist in the oral cavity. Mechanistically, we show that C. glabrata binds specifically to C. albicans hyphae, mediated by hyphally expressed ALS adhesins in C. albicans and cell surface proteins in C. glabrata that are transcriptionally up-regulated in the presence of C. abicans. In this sense, C. glabrata is a piggy-back fungus that relies upon binding to C. albicans hyphae for oral colonization. This finding has implications for treatment of oral candidiasis and may shed light on colonization mechanisms of other non-hyphae producing fungi.
Citation: Tati S, Davidow P, McCall A, Hwang-Wong E, Rojas IG, Cormack B, et al. (2016) Candida glabrata Binding to Candida albicans Hyphae Enables Its Development in Oropharyngeal Candidiasis. PLoS Pathog 12(3): e1005522. https://doi.org/10.1371/journal.ppat.1005522
Editor: Mairi C. Noverr, Louisiana State University Health Sciences Center, UNITED STATES
Received: August 25, 2015; Accepted: March 2, 2016; Published: March 30, 2016
Copyright: © 2016 Tati et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by grants from the National Institute of Dental and Craniofacial Research R01DE10641 (ME), R01DE022720 (ME) and by the National Institute for Allergy and Infectious Diseases R01046223 (BC), National Institutes of Health, USA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Oropharyngeal candidiasis (OPC) is an opportunistic mucosal infection caused by Candida species [1,2]. Candida albicans and Candida glabrata are the first and second major etiological agents of OPC, respectively . Although other Candida species, including C. parapsilosis, C. tropicalis, and C. krusei, may be isolated as the sole species from oral infection sites, single species infection by C. glabrata alone is rare [4,5]. C. glabrata is most frequently co-isolated along with C. albicans in mixed species oral infections [4,6,7]. Oral infections involving C. glabrata have increased by 17% over the past several years , and are particularly common in cancer patients, denture-wearers, or following prolonged use of broad spectrum antibiotics, steriods or following head and neck radiation therapy . These infections were often associated with multiple Candida species [3,4]. Oral infections with mixed C. albicans and C. glabrata were found to be more severe and difficult to treat  since many C. glabrata strains are innately resistant to azole antifungal agents used in treating mucosal infections. Prophylactic use of azole antifungal drugs has been implicated as a major cause for the increase in non-C. albicans fungemia . Fungemia caused by C. glabrata has high mortality especially in adult patients in intensive care units , and although fluconazole prophylaxis has reduced the incidence of invasive candidiasis in high-risk neonates and immunosuppressed patients, there has been little effect on the overall incidence of C. glabrata candidiasis. Given the frequency of C. glabrata and C. albicans co-infection, it is imperative to understand the mechanisms deployed by C. glabrata in co- infections with C. albicans.
C. albicans is a diploid, polymorphic fungus that exists in yeast, hyphal, and psuedohyphal forms . C. albicans hyphae express numerous proteins that enhance virulence by adhering to host cells or damaging host tissue . C. albicans hyphae are known to penetrate epithelial surfaces, damage endothelial cells, and aid in systemic infection by colonizing different organs such as kidneys, spleen and brain [10,12]. Als (Agglutinin Like Sequence proteins), Hwp1p (Hyphal wall protein), and Eap1 (Enhanced Adherence to Polystyrene) are well-characterized C. albicans hyphal wall adhesins that mediate C. albicans interaction with host epithelial, endothelial and host tissue proteins [13–15]. C. albicans adhesins contribute not only to its ability to adhere and colonize multiple types of host tissues, but also serve as binding moieties for other microbes such as Streptococcus gordonii, Pseudomonas aeruginosa, and Staphylococcus aureus [16–19]. It is therefore possible that one or more C. albicans hyphal-specific adhesins may play a role in C. glabrata interaction as well.
In terms of host tissue invasion, C. albicans has a fitness advantage over C. glabrata in terms of its ability to switch between yeast to hyphal forms. By contrast, C. glabrata virulence must be independent of its morphology, since it lacks the ability to form true hyphae. However, C. glabrata is likely to express specific adhesins in order to establish colonization [20,21]. Phylogenetic analysis of the C. glabrata genome showed 66 putative cell wall proteins, of which only a few have been well characterized in terms of host cell adhesion . Cell wall protein families known to be involved in adhesion to endothelial and epithelial cells include the EPA (Epithelial cell adhesin), AED (Adherence to endothelial cells), and PWP (PA14 domain containing Wall Protein) proteins . C. glabrata Epa1, 6, and 7 adhesins bind to both endothelial and epithelial host cells [22,23], while Pwp7p and Aed1p are known to interact with endothelial cells . Deletion of these Epa1 adhesins attenuated virulence in a murine model of disseminated candidiasis [22,23]. The role of C. glabrata adhesins, beyond their ability to mediate adherence to host tissues, is understudied. We hypothesize that one or more of these adhesins may promote interspecies interaction with C. albicans during mixed species OPC.
Co-adhesion is the basis for both single and multispecies colonization in the host . Co-adhesion in bacteria is well studied and it has been established that the expression of multiple bacterial adhesins drive interspecies oral bacterial colonization [24,25]. Although mixed infections of C. glabrata and C. albicans occur frequently, the mechanism of co-adhesion and interspecies colonization is not well understood . In our study, in spite of C. glabrata encoding several cell wall adhesins known to bind host epithelial and endothelial cells, we documented poor colonization in our murine OPC model. We hypothesized that co- infection or prior infection with C. albicans may facilitate C. glabrata infection. Here we characterize the co-colonization of C. glabrata and C. albicans in a murine model of OPC, and explore the role of cell wall proteins from both species in mediating cell-cell interaction and co-colonization.
C. glabrata and C. albicans show enhanced growth in dual species biofilm
We initially performed an in vitro biofilm assays to test whether C. albicans and C. glabrata have any cooperative growth effects. Two strains of C. glabrata (BG2 wild type, WT) and a GFP-expressing strain CgVSY55 (ura3Δ::hph ScPGKp-yEGFP-URA3-CEN-ARS) derived from a CgDSY562 WT  and two strains of C. albicans (CAI4 WT with URA replaced, URA+) or CAF2-yCherry strain  were used in biofilm experiments. In a static plate assay, C. albicans CAI4 and CgBG2 each formed single species biofilms with similar robustness. However, when grown together as a dual species biofilm, the total dry weight was significantly (P<0.001) higher compared to single species (Fig 1A). Fluorescent quantitation of co-culture of C. albicans CAF2-yCherry with C. glabrata CgVSY55 under static biofilm growth showed enhanced growth of both species occurred compared with single species (Fig 1B). In contrast, under dynamic flow conditions, C. glabrata (CgVSY55) alone was unable to form biofilms within the flow chamber, while C. albicans formed abundant biofilms. However, when both species were co-cultured under dynamic flow conditions, C. glabrata CgVSY55 cells (green) were found associated with C. albicans (red) nascent biofilm regions, and were concentrated along C. albicans hyphae (Fig 1C arrows).
(A) C. albicans CAI4 and C. glabrata strains CgBG2 and CgVSY55 were grown as biofilms for 24h in plastic wells. Both CgBG2 and CgVSY562 strains showed significantly (*P<0.02, ***P<0.001 by student’s t test) increased biomass in a dual species biofilm (black bars) as compared to single species biofilm (white and grey bars). Fluorescent quantitation of co-culture of C. albicans CAF2-ycherry with C. glabrata CgVSY55 showed enhanced growth of both species compared with single species (Fig 1B). When both species were co-cultured under dynamic flow conditions, C. glabrata (green) was able to bind C. albicans (red) biofilms and hyphae under conditions of flow (Fig 1C). Fluorescent images were merged with DIC images to enhance hyphal visibility. Hyphal binding is indicated by arrows. Scale bars represent 50 μm.
C. glabrata adheres to C. albicans hyphae
To further examine how these two Candida species might be interacting, we examined their association directly by fluorescence microscopy. C. albicans cells were grown in YNB + 1.25% glucose (for yeast phase cells) or in YNB + 1.25% N-acetyl glucosamine at 37°C (to induce hyphal cells) for 3 h. C. albicans cells were then incubated with C. glabrata cells at 1:1 ratio for 60 min. C. glabrata cells did not adhere with C. albicans yeast cells (Fig 2A, upper left); however they showed strong adhesion along the length of germinated C. albicans hyphae (Fig 2A left). Scanning Electron Microscopy (SEM) further illustrated this interaction showing that C. glabrata cells adhered along the entire length of C. albicans hyphae (Fig 2A right). We observed that C. glabrata cells formed rows of adherent cells along the length of hyphae, but did not adhere to other C. glabrata cells. Next, we quantified adhesion as defined by the number of C. glabrata cells adhering to 10 μm length of C. albicans hyphae in seven different strains of C. glabrata. Among the C. glabrata strains examined, CgDSY56 (the parent strain of CgVSY55) had significantly (P<0.0001) higher adherence (6.4 ± 0.2 cells / 10 μm hyphae, high adherence strain) when compared to other strains tested. CgBG2 and Cg960032 (4.2 ± 0.2 cells / 10 μm hyphae) showed medium adherence; and Cg931010, Cg932474, Cg148042, and Cg90030 showed low adherence (3.0 ± 0.2cells / 10 μm hyphae) (Fig 2B).
C. albicans germinated or yeast cells were incubated with C. glabrata cells at 1:1 ratio for 60 min. While little adherence of C. glabrata was found with C. albicans yeast, large numbers of C. glabrata CgVSY55 or CgBG2 were found adherent along the length of C. albicans hyphae using fluorescence microscopy (A left panel). Adhesion of along the length of C. albicans hyphae was also found in scanning electron micrograph images of CgVSY55 and CAF2-yCherry (A right panel). Magnifications are 500X (scale bar: 5μm, 20μm). C. glabrata cells adhesion to C. albicans hyphae (10μm length) was quantitated microscopically. Seven wild type strains of C. glabrata were screened for adherence to C. albicans hyphae and grouped as high (CgDSY562), medium (CgBG2, Cg60032, Cg931010) and low (Cg932474, Cg148042, Cg90030) binders. (B).
Yeast to hyphae transition in C. albicans induces expression of hyphal-specific proteins as well as altering mannans and glucans levels in the hyphal cell wall . To identify whether binding between C. albicans and C. glabrata was mediated by cell wall carbohydrates, we performed blocking experiments with C. albicans using concanavalin A (which binds cell wall mannans) and an antibody to β,1–3 glucan at concentrations that we previously showed provided good cell coverage . C. albicans hyphae were treated with concanavalin A or with β,1–3 glucan Ab for 30 min, washed, then incubated with C. glabrata; however C. glabrata adhesion to C. albicans hyphae was unchanged, suggesting that C. albicans adhesion is not mediated by binding to C. albicans mannose or β,1–3 glucan. This is consistent with the fact that we did not detect C. glabrata binding to other C. glabrata cells since the C. glabrata cell wall contains both mannan and β,1–3 glucan.
C. albicans hyphal wall Als adhesins are needed for C. glabrata adherence
We hypothesized that C. glabrata might bind C. albicans cell wall proteins directly. To test candidate C. albicans hyphal wall adhesins required for C. glabrata adhesion, we performed co-adhesion assays with ALS1 and ALS3 deficient C. albicans (Fig 3). We found that C. glabrata had significantly decreased adherence to hyphae of a C. albicans als3Δ/Δ mutant (72.3% reduction), an als1Δ/Δ mutant (28.8% reduction), and an als1/als3Δ/Δ double mutant (86% reduction). ALS1 and ALS3 complementation strains showed restoration of adherence to levels closer to that of wild type strain (Fig 3A). To further validate the role of C. albicans Als1 and Als3, we performed a quantitative adherence assay by direct microscopic observation using S. cerevisiae strains expressing C. albicans ALS1 and ALS3 with a GFP-tagged C. glabrata strain. Both Als1 and Als3 expressing S. cerevisiae strains showed significantly higher binding (Binding Index = 52.0 ± 3.0 and 58.2 ± 1.4, respectively) compared to S. cerevisiae expressing an empty vector (Binding Index = 19.7 ± 2.3) (Fig 3B).
(A) C. glabrata (CgVSY55) adherence to C. albicans Als1, Als3 and Als1/3 deficient strains was measured using fluorescent microscopy. C. glabrata showed a significant (**P<0.001,***P<0.0006) decrease in adherence to C. albicans hyphae of als3Δ/Δ, als1Δ/Δ, and als1/als3Δ/Δ mutants; while C. albicans Als1 and Als3 complementation strains had restored adherence. (B) S. cerevisiae strains expressing C. albicans Als1 and Als3 adhesins and an empty vector (control) were incubated with CgVSY55 and their adherence was quantifed by direct visualization. Both Als1 and Als3 expressing S. cerevisiae strains had a significant (***P<0.001) increase in Binding Index compared to control empty vector. Differences between groups were analyzed by a student’s t test.
C. albicans is required to establish C. glabrata infection in OPC
To determine whether our observed binding between C. albicans and C. glabrata has relevance in vivo, we examined the ability of C. glabrata to establish infection in our murine model of OPC. Since C. glabrata has not been used before in OPC infection models, we began with a single species oral infection of C57BL/6 mice with C. glabrata alone as we have previously done with C. albicans (Fig 4A). In this model, sublingual infection with a C. albicans inoculum of 1 X 106 cells/ml typically produces clinical symptoms and white tongue plaques 4–5 days post infection, and recovery of 1 X 107 CFU / gm tongue tissue at 5 days post infection. Surprisingly, in no infection experiments using C. glabrata did we observe the typical appearance of white tongue plaques indicative of clinical infection. We tried varying immunosuppressive agents (triampicinolone acetonide, cyclophosphamide), mouse strains (BALB/c, IL17RAk/o) and used inocula size of C. glabrata ranging from 1 X 107 to 1 X 1010 cells/ml. In all cases, infection with C. glabrata alone resulted in no clinical appearance of disease or weight loss in animals. Consistent with this lack of disease, the recoverable C. glabrata CFUs from the tongue were extremely low (4–7 X 102 CFU/g of tongue tissue).
(A) Colony Forming Units (CFU) per gram of tongue tissues were recovered from infections with C. albicans alone, C. glabrata alone or C. albicans and C. glabrata co-infected mice after 5 days. C. glabrata colonization increased by one log-fold upon co-infection with C. albicans. (B) Pre-establishing C. albicans infection (24 h or 48 h before C. glabrata infection) further increased subsequent C. glabrata colonization by two log-fold in mice tongue tissues. Differences between groups were analyzed by a student’s t test (**P<0.003, **P<0.001, ***P<0.0001). (C) C. glabrata infection alone did not result in mice weight loss, however the rate of weight loss was accelerated in the mixed infection (red arrow indicates initiation of C. glabrata infection). Animal weights (mean and SD of each group, n = 7) are shown for each group (black, C. glabrata infection only; blue C. albicans infection only; red, C. albicans infection for 48 h followed by C. glabrata infection).
Since our in vitro biofilm and adhesion assays showed enhanced adhesion and growth of C. glabrata when mixed with C. albicans, we next attempted a mixed infection with C. glabrata (1X109 cells/ml) either as a co-infection with C. albicans (5 X 107 cells/ml); or as a delayed infection with C. glabrata 24 or 48 h after infection of C. albicans (Fig 4). C. glabrata CFU were significantly (P<0.02) increased by ten-fold (3 X 103 CFU/g of tongue tissue) when mice were co-infected with C. albicans (Fig 4A). Co-infection with C. glabrata did not alter C. albicans infection levels (1.2 X 107 CFU/g of tongue tissue) compared with C. albicans infection alone (Fig 4A). However, delaying C. glabrata infection for 24 or 48 h after establishment of C. albicans infection further increased C. glabrata oral infection by a further 10-fold (3.5–4.5 X1 04 CFU/g of tongue tissue, P<0.0001) compared to C. glabrata single species infection (Fig 4B). Mean animal weights did not change upon C. glabrata infection only (Fig 4C). However, mice lost weight more rapidly following a mixed infection compared with infection by C. albicans alone, so that mice in the mixed infection group had to be sacrified one day sooner due to total weight loss compared with mice infected with C. albicans only (Fig 4C). Thus our data show that levels of oral infection of C. glabrata were signifcantly increased by an established C. albicans oral infection and the rate of weight loss was increased upon dual species infection.
Next, we examined tongues of mixed-infected mice histologically to determine whether C. glabrata alters C. albicans invasive properties and to identify the localization of C. glabrata infection within the mucosal epithelium. For these experiments we infected mice with fluorescent-tagged strains of C. albicans (CAF2-yCherry) on day 0 and C. glabrata (CgVSY55) on day 2; and collected tongue tissues on day 5. Tongues were sectioned and stained with either PAS to visualize fungal-tissue architecture or cryo-sectioned for visualization of yeast cell localization by fluorescence microscopy. Tongues from mice with mixed infection showed robust fungal plaque formation as well as extensive C. albicans hyphal penetration of the superficial epithelium (Fig 5A, boxed region) as well as invasion into some regions of the underlying epithelium and lamina propria (Fig 5A, arrows). Closer inspection of these regions showed widespread C. albicans hyphae; and in some areas yeast cells were observed both adherent to hyphae and as unattached cells that were likely to be C. glabrata (Fig 5B and 5C, arrows). Fluorescent imaging of these regions confirmed that the majority of tissue invasion was with C. albicans hyphae (Fig 5D, boxed region, red), however C. glabrata cells (green) were also observed within these tissues both associated with C. albicans hyphae as well as being unconnected and separate within the epithelium (Fig 5E and 5F, arrows). In contrast, mono-species C. glabrata infection resulted on only very small superficial plaques that were localized on the surface mucosa without any invasion. Thus, infection of oral epithelium with C. albicans and the presence of its hyphae were permissive for infection and tissue invasion by C. glabrata.
(A, B and C) PAS stained formalin-fixed and paraffin embedded sections (5 μm) from tongues of mixed C. albicans and C. glabrata infection at day 5 showed widespread fungal plaques and hyphal invasion (yeast are stained magenta) of superficial epithelium (box) and underlying epithelium (arrows). Dark blue cells are neutrophils, lighter blue cells are tongue epithelia. Magnification is 10x. (B) 40x and (C) 100x magnification show C. albicans hyphae with associated yeast cells within the mucosa. (D, E and F) Immunofluorescent and DIC merged images of C. albicans (red) and C. glabrata (green) from tongues at day 5 post-infection. (D) Arrowheads show hyphae penetration into the epithelium. (E and F) Arrowheads at left indicate C. glabrata in contact with hyphae, while other C. glabrata are within epithelium unassociated with C. albicans hyphae (E, lower arrow). Scale bars represent, in order (A-F), 50, 10, 5, 50, 10, and 10 μm.
To further confirm the requirement of C. albicans for C. glabrata for initial infection, we treated mice with fluconazole (Flu) after establishing mixed infection using Flu sensitive (CaFluS) or Flu resistant C. albicans (CaFluR) strains and Flu resistant C. glabrata (CgFluR) (Fig 6). Mice were treated with Flu for four days after an oral mixed infection was already established for four days. As expected, Flu treatment did not alter infection levels of either species in a mixed infection with CgFluR and CaFluR strains. However, for a mixed infection with C. glabrata CgFluR and C. albicans CaFluS strains, Flu treatment resulted in significant (by two logs, P<0.001) reduction of both C. glabrata and C. albicans. Flu treated animals infected with CaFluR strains in a mixed infection lost significantly (P<0.05) more weight (21.2 ± 0.2%) than mice infected with C. albicans CaFluS strains (18.9 ± 0.3%). Although we could not determine the co-locallization of C. albicans and C. glabrata histologically due to lack of fluorescent markers in Flu resistant strains, examination of tongues confirmed the reduction in superficial epithelial fungal burden and invasion upon Flu treatment (Fig 6). Thus, C. glabrata infection levels were proportional to those of C. albicans, showing that C. glabrata requires the presence of C. albicans for early infection in vivo.
Mixed species oral infection with fluconazole resistant (CaFluR, CgFluR) and fluconazole sensitive (CaFluS) strains showed a significant reduction in number C. glabrata fluconazole resistant CgFluR cells (solid circles) following fluconazole treatment (despite being fluconazole resistant) that was proportional with reduction in fluconazole sensitive C. albicans CaFluS (open circles) CFUs (right). Tongue tissues were stained with Periodic acid-Schiff stain and viewed at 10X magnification (left). CaFluS and CgFluR infected mice showed normal tongue histology with a reduced fungal burden following fluconazole treatment, while CaFluR and CgFluR (yeast cells are shown in pink) infected mice showed typical hyphal invasion of the superficial epithelium with high fungal burden.
C. glabrata requires C. albicans Als1 and Als3 adhesins in OPC
Since our in vitro data showed that C. albicans Als1 and Als3 adhesins were important for C. glabrata adherence, we next examined their role in mixed C. glabrata-C. albicans oral infection in vivo. A 48 h delayed infection of C. glabrata following infection with C. albicans wild type or Als adhesin deficient strains was performed (Fig 7). C. albicans als1Δ/Δ and als3Δ/Δ mutants were able to establish infection at the same levels as WT cells. However, C. glabrata tongue CFUs were significantly (P<0.05) decreased (2.8 X 104 CFU/g) following infection with the C. albicans als1Δ/Δ mutant; and were even further reduced (6.6 X 103 CFU/g, P< 0.001) following infection by C. albicans als3Δ/Δ. Infection of C. glabrata with C. albicans Als1 and Als3 complemented strains showed restoration of C. glabrata colonization to levels similar to those observed with the wildtype C. albicans strain (Fig 7). No differences in animal weights between the groups was found since levels of infection by C. albicans were similar between groups.
Infection levels of Als1 and Als3 deficient strains of C. albicans did not differ significantly from WT; however, C. glabrata infection levels were decreased significantly in mice upon co-infection with Ca als1Δ/Δ, als3Δ/Δ mutants compared to WT. Mixed infection of C. glabrata with C. albicans Als1 and Als3 complementation strains showed restoration of C. glabrata colonization. Differences between groups were analyzed by a student’s t test (*P<0.02, **P<0.001).
C. glabrata cell wall proteins are required for C. albicans adherence
To identify adhesion partners on C. glabrata, we screened 44 S. cerevisiae strains expressing C. glabrata cell wall proteins and identified five strains expressing CgEpa8, CgEpa19, CgAwp2, CgAwp7 or ORF CAGL0F00181 that were most adhesive (2–5 cells/10 μm C. albicans hyphae) (Fig 8A). Most other tested strains, including the S. cerevisiae parental strain, had no adhesion to C. albicans hyphae. Next, we examined comparative transcription levels of these five candidate genes in C. glabrata strains which have high adherence (CgDSY562), medium adherence (CgBG2), and low adherance (Cg90030) in vitro to C. albicans. We used C. glabrata EPA1 and EPA6 genes as a negative control since S. cerevisiae expressing C. glabrata Epa1 and Epa6 did not bind to C. albicans hyphae, although they are highly expressed major adhesins in C. glabrata. To confirm that these strains also had differential binding to C. albicans during infection, we compared infection levels in a mixed infection in OPC, and found that indeed, the low and high adherence strains had a significant (P<0.01) difference in infection levels (Fig 8B). Then, transcriptional levels of these candidate genes were measured by qPCR before and after incubation with germinated C. albicans. Although CgEPA8 and CgAWP7 were most highly expressed in the high adhesion strain compared to the lower adhesion strains, we did not find significant differences in basal expression levels among the three other candidate genes among the C. glabrata strains. However, transcriptional levels of four genes (CgEPA8, CgEPA19, CgAWP2, and ORF CAGL0F00181) were increased significantly by 6–7 fold, while CgAWP7 was increased by 2-fold in the high adherence strain (CgDSY562) upon incubation with C. albicans hyphae. This induction was less for CgEPA19, CgAWP2, and CgCAGL0F00181 in the intermediate adherent strain, while the low adhesion Cg90030 strain had the least induction by C. albicans for all five genes (Fig 8C). Expression of CgEPA6 and CgEPA1 genes, which serve as controls since they do not mediate adherence to C. albicans, were both modestly down-regulated in the presence of C. albicans. Taken together, these results show that C. glabrata cell wall genes EPA8, EPA19, AWP2, AWP7 and CAGL0F0018 are upregulated by C. albicans and may promote a dual species oral infection.
(A) Adherence of S. cerevisiae strains expressing C. glabrata cell wall adhesins to C. albicans hyphae was measured. Among 44 strains tested, only five strains were adherent to C. albicans hyphae, and adhesion levels were about half that of CgDSY562 (Cg) (B) Mixed species infection with CgDSY562 (High adherence), CgBG2 (Medium adherence) and Cg90030 (Low adherence) and C. albicans CAI4 showed significant reduction in infection levels (*P<0.01 student’s t test) between the High (CgDSY562), and Low (Cg90030) adherence C. glabrata strains. (C) Expression levels of C. glabrata EPA8, EPA19, AWP2, AWP7 and CAGL0F00181 genes in three wild type strains CgDSY562 (High adherence), CgBG2 (Medium adherence) and Cg90030 (Low adherence) were measured by qRT-PCR following incubation with germinated C. albicans. All five genes identified by screening (A) were induced in the presence of C. albicans hyphae proportionally with strain adherence and infection levels (B). In contrast, control C. glabrata EPA1 and EPA6 genes (far right) were not induced by C. albicans hyphae. The values plotted are means ± SEMs of n = 3 or 4 independent experiments.
Although clinical studies have shown that C. albicans and C. glabrata are common partners co-isolated from oral infections, C. glabrata alone rarely causes oral infection. This work identifies for the first time that C. glabrata adherence to C. albicans hyphae is the basis for this partnership and that it is mediated by specific adhesins on both species. Previous in vitro studies found that C. glabrata alone was unable to colonize or invade reconstituted human vaginal epithelium (RHVE)  or reconstituted human oral epithelium (RHOE) . Mixed infections using both C. glabrata and C. albicans increased tissue damage in RHOE  and were permissive for infection in RHVE  and in vivo in tongues of immunosuppressed mice , although others found no difference in host damage or inflammation in co-infected human oral epithelial . These and our own studies are in agreement that C. glabrata alone is non-invasive in respect to oral-esophageal mucosal epithelium, in contrast to its ability to penetrate gastric epithelium . The basis for this difference in tissue tropism is unknown, although it is possible that differences in the gut environment induces differential expression of C. glabrata adhesins.
We found that two major fungal hyphal wall adhesins Als3 and Als1 contribute to binding C. glabrata in vitro and to establish oral infection in vivo. C. albicans Als3 appears to make the major contribution towards binding with C. glabrata, with Als1 having a secondary role. Consistent with this, loss of Als1 on its own does not strongly reduce adherence to C. glabrata. However, in strains deleted for ALS3, additional loss of ALS1 further reduced adherence by an additional two-fold. Als3 is a well known multifunctional surface protein, however we have identified an additional novel function of this adhesin in binding C. glabrata. Since Als 3 proteins are very abundant on C. albicans hyphae, and we only find 2–6 C. glabrata cells per hyphae, we expect that substantial numbers of Als proteins would still be available on hyphae to carry out other functions in the context of oral infection. It is also possible that Als3 might have a similar role in binding other non-hyphal forming Candida species such as C. krusei that are frequently co-isolated along with C. albicans in OPC. C. albicans Als3 seems to be promiscuous in its binding partners since Als3 proteins have been shown to bind the oral bacteria S. gordonii through its SspB cell surface protein in a mixed species biofilm  and to S. aureus during polymicrobial biofilm growth . Hence Als3 may to be an excellent target for disruption of mixed species and inter-kingdom biofilms.
The C. glabrata Epa family consists of at least 20 GPI-anchored surface exposed adhesins whose expression of individual members is strain dependent . Epa proteins recognize host glycans, and C. glabrata Epa1 is the best characterized member that is involved in adhesion to mammalian epithelium. Epa1 preferentially recognizes Gal β1–3 glycans, and variations of its adhesion domain conferred promiscuity of ligand binding . Recently, Epa binding domains were functionally classified according to their ligand binding profiles, and interestingly our identified adhesins C. glabrata Epa8 and Epa19 were found to be very closely related and within the functional class III of Epa ligands that have weak binding to epithelial cells . Thus, we speculate that some Class III Epa adhesins may have ligand functions with other cell types including C. albicans.
Another similarlity among the C. glabrata adhesins we identified (EPA8, EPA19, AWP2, AWP7 and CAGL0F00181) is that their expression levels were all induced by incubation with C. albicans hyphae (Fig 8C). In contrast, C. glabrata EPA1 and EPA6 (both Class I ligands with high binding to epithelial and endothelial  cells, and highly expressed in log phase cells ), were not up-regulated following incubation with C. albicans hyphae. In agreement with our findings, no increase in expression levels of C. glabrata EPA1, EPA6 or EPA7 was found following co-infection with C. albicans in RHVE cells . Based on our results, we propose a role of these C. glabrata CWPs (EPA8, EPA19, AWP2, AWP7, and CAGL0F00181) in interspecies binding and further suggest that C. glabrata is able to transcriptionally regulate selected genes needed for its colonization and survival in a host. It is known that many C. glabrata EPA genes are transcriptional silenced. Since EPA1 and EPA6 (both of which are strongly silenced) are not up-regulated by co-culture with C. albicans (Fig 8), this suggests that the transcriptional regulation of C. glabrata EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 is not through general antagonism of sub-telomeric silencing . How C. glabrata regulates these genes in the presence of C. albicans remains to be determined.
C. glabrata alone was not competent to cause infection in our OPC model. Our data further suggest that while C. glabrata colonizes oral mucosa poorly (even in an immunosuppressed host), it has evolved to exploit the presence of hyphae-producing C. albicans to establish colonization and invasion of oral epithelium; and its presence enhanced the severity of OPC as measured by rate of weight loss of animals. Furthermore, co-infections treated with Fluconazole reduced levels of C. glabrata concomitantly with C. albicans over four days, showing its dependence upon the presence of C. albicans in early infections. However, our results show that C. glabrata is found both together and apart from C. albicans hyphae in tissues, suggesting that once it gains a foothold in oral epithelium by binding C. albicans hyphae, it can survive alone in mucosal tissues, albeit at low levels. These C. glabrata cells existing independently in oral mucosa may be a colonization reservoir for dissemination if the oral epithelium is breached by trauma, chemotherapy or other factors. In this regard, our preliminary experiments showed that mice with mixed C. glabrata and C. albicans oral infections had significantly higher stomach colonization of both species, suggesting that gut colonization might serve as such a reservoir. Also, these reservoirs may become clinically significant following long-term azole therapy providing an environment in which drug resistant C. glabrata could emerge.
Our data suggests a model whereby oral tissues that are inherently resistant to infection by C. glabrata, are colonized by piggybacking with C. albicans to establish a foothold of tissue infection. Of interest, and the subject of ongoing studies in our lab, is the role of oral and gut reservoirs of C. glabrata in subequent colonization of other tissues that have a naturally higher tropism for infection by C. glabrata, as well as their role in subsequent dessimination.
All Candida and S. cerevisiae strains used are listed in Table 1 and Table in S1 Table. C. albicans cells were maintained in yeast extract/peptone/dextrose (YPD; Difco) medium with the addition of uridine (50 mg/ml; Sigma) when required and stored as -80°C. S. cerevisiae containing pADH or pADH-ALS3 were maintained on synthetic medium lacking uracil (CSM-glu) (0.077% CSM-ura, 0.67% yeast nitrogen base [Difco], 1.25% glucose, and 2.5% agar). S cerevisiae strains expressing N-terminal domains of C. glabrata Cell Wall Proteins (CWP) were made as described , and are in preparation for publication elsewhere). The ORFs whose domains mediate adherence to Candida hyphae are shown in S1 Table.
C. albicans cells were cultured overnight in YPD broth, diluted to an OD600 = 0.3 in pre-warmed YNB medium supplemented with 1.25% GlcNAc, and incubated for 3 h at 37°C with gentle shaking to induce germination. C. glabrata or S. cerevisiae strains were grown similarly except in YNB + 1.25% of glucose. Cells were collected by centrifugation (100 X g), washed once in PBS, and then re-suspended in PBS. Germination of C. albicans cells was confirmed by microscopic observation. C. albicans cells were then incubated with C. glabrata cells at a 1:1 ratio for 60 min. Blocking experiments described previously , were carried out using washed C. albicans cells incubated with concanavalin A (100 ug/ml; mannan binding lectin, Sigma) or β,1–3 glucan Ab (10 μg/ml, Biosupplies) for 30 min (concentrations that gave high coverage of cells as determined by FACScan), then washed in PBS before assay. For adhesion assays of S. cerevisiae strains expressing C. albicans Als1 and Als3 adhesins, S. cerevisiae cells or an S. cerevisiae empty vector (control) were incubated with CgVSY55 for 1 h at 37°C (at cell ratio 1:1), then a Binding Index was calculated as the number of C. glabrata cells bound to S. cerevisiae cells divided by (number of bound C. glabrata cells plus unbound C. glabrata cells plus unbound S. cerevisiae cells) X 100 per field. At least 10 separate fields were used to obtain averages.
Each Candida strain was grown overnight to OD600~2.0, washed twice in Phosphate Buffered Saline (PBS), re-suspended in YNB without uridine, and 1 ml cells (1 X106 cells/ml) were added to polystyrene wells. For mixed species biofilms, 500 μl of each species (5 X 105 cells /ml) for a total of 1 ml was added to the well. After incubation for 3 h to allow adhesion, non-adherent cells were gently removed by aspiration and 1 ml of fresh media was added. Biofilms were grown for 24 h at 37°C on an orbital shaker and biofilm dry weight was measured as previously described . For fluorescence biofilm assays, single and dual species biofilms were grown on 96 well microtiter plates using a yCherry expressing strain of C. albicans and a GFP expressing C. glabrata strain. Fluorescent counts were recorded at 37°C using a Bio-Tek multifunction plate reader and analyzed using Gen5 software. Alternatively, we examined non-static dual species biofilms grown under flow conditions. For these experiments, YPD media containing the C. albicans WT strain CAF2 cells expressing the fluorescent protein mCherry and the C. glabrata WT strain VSY55 expressing GFP (both at 1 × 106 cells/ml) were circulated through a μ-Slide I 0.8 Luer family ibiTreat flow chamber (ibidi, Martinsried, Germany) for 2 h at 37°C and a shear force at the coverslip surface of 0.8 dynes/cm2. Images were obtained using a Zeiss LSM 510 confocal microscope, and analyzed using ZEN imaging software (Zeiss, Göttingen, Germany). Flow was maintained during image acquisition.
RNA extraction and qRT-PCR
Overnight cultures of C. albicans were diluted to an OD600 = 0.3 in pre-warmed YNB medium supplemented with 1.25% GlcNAc and incubated for 3 h at 37°C to induce germination, or diluted in YNB medium supplemented with 1.25% glucose at room temperature for yeast cells. C. glabrata CgDSY562, CgBG2, and Cg90030 overnight cultures were grown similarly using YNB + 1.25% of glucose. Cells were collected by centrifugation (100 X g), and re-suspended in PBS. C. glabrata cells were then incubated with germinated or yeast form C. albicans at a 1:1 ratio for 30 min. Total RNA was isolated from C. glabrata, C. glabrata and C. albicans 1 X 107 cells using an RNeasy minikit (Qiagen). Reverse transcription (RT) was performed using SuperScript III reverse transcriptase, and oligo(dT)20 primer (Invitrogen). cDNA was purified (Geneflow PCR purification kit) and quantified with a NanoDrop spectrophotometer (NanoDrop Technologies). Quantitative RT-PCR (qRT-PCR) was performed in triplicate for CgACT1, CgAWP2,7, CgEPA1,6,8,19 and CgCAGL0F00181 using gene-specific primers (Table in S2 Table). C. albicans cDNA was used as a negative control in all experiments to verify specifity of amplification. Genes were normalized to CgACT1 in each respective strain and condition as decribed previously .
Fluorescent microscopy was done using a yCherry expressing strain of C. albicans  and the GFP expressing C. glabrata (VSY55: ura3Δ::hph ScPGKp-yEGFP-URA3-CEN-ARS) derived from a C. glabrata DSY562 clinical isolate . Scanning electron microscope observations were carried out on C. albicans hyphae and C. glabrata cells. C. albicans cells were grown in YNB for yeast phase cells or in YNB + 1.25% GlcNAc at 37°C (to induce hyphae) for 3 h. C. albicans cells were then incubated with C. glabrata cells at 1:1 ratio for 30 min. Cells were incubated on a concavalin A (100ug/ml; Sigma) coated glass slide for 1 h at RT. Cells were washed twice with PBS, fixed with 2% glutaraldehyde (Sigma) for 30 min at 4°C, then washed twice with distilled water. Samples were dehydrated in 30%, 50%, 70%, 85%, and 95% ethanol for 15 min each and 100% ethanol twice for 15 min each. Samples were exchanged into 100% hexamethyldisilazane (HMDS) and allowed to dry in a hood before visualization. SEM observation was done under the following analytical condition: L = SE1 and EHT = 2.5 kV to study the binding of C. glabrata on C. albicans cells with Hitachi SU70 FESEM operating at 2.0 keV.
Mixed Candida infection in murine oral candidiasis
C. albicans murine OPC model [47,48] was used for infection with C. glabrata. Mice (BALB/c, C57BL/6, and IL17RAk/o) were immuno-suppressed with cortisone acetate (150–250 mg/kg), triampicinolone acetonide (100–150 mg/kg) or cyclophosphamide (100–150 mg/kg) one day before infection with C. glabrata (1 X 107 to 1 X 109 cells/ml). For mixed infections, mice (female C57BL/6, 4–6 weeks old) were immunosuppressed with cortisone acetate 225 mg/kg (Sigma) on day -1, +1, and +3, and then infected with C. albicans (5 X 107 cells/ml) on day 0; or infected with C. glabrata, (1 X 109 cells/ml) on day 2 after pre-establishing C. albicans infection on day 0. C. albicans and C. glabrata colonies from tongue tissues were differentiated on CHROMagar media. On the fifth or sixth day after infection, mice were euthanized by cervical dislocation under anesthesia (ketamine/xylazine); tongue tissues were excised and hemi-sectioned along the long axis with a scalpel. One half was weighed and homogenized for quantification of fungi, and the other half was processed for histopathological analysis. Tongue hemi-sections were fixed in 10% buffered-formalin for 24 h, paraffin embedded, and then cut into 5μm sections for Periodic Acid-Schiff (PAS) staining as we previously described . For histological co-localization experiments, animals were infected with C. albicans yCherry and the GFP expressing C. glabrata (VSY55) strains as described above. For these experiments, tongue hemi sections were fixed in 4% (w/v) paraformaldehyde (PFA) for 24 h, incubated in 30% sucrose for 3 days, snap frozen in OCT compound (Tissue-Tek, Sakura, Torrance, CA) with liquid nitrogen, and cut into 8μm cryosections.
For Fluconazole (Flu) treatment studies, ten mice were used for each group (drug treatment and controls using combinations of Flu resistant and sensitive strains of C. albicans and Flu resistant C. glabrata shown in Table 1). Sensitivities of each strain to Flu was verified using MIC assays. Immunosuppression was induced on days −1, +1 and +3 post-infection. Mice were infected sublingually with C. albicans (5 X 107 cells/ml) on day 0, and C. glabrata (1 X 109 cells/ml) on day 2, and were sacrificed on day +7. Mice received daily intraperitoneal injections of 100 mg/kg Fluconazole that was initiated 48 h after C. glabrata infection and continued through post-infection day 7.
Statistical analyses were performed using GraphPad Prism software version 5.0 (GraphPad Software, San Diego, CA, USA) using unpaired Student's t-tests. Differences of P<0.05 were considered significant. All experiments were performed at least thrice.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. This protocol was approved by the University of Buffalo Institutional Animal Care and Use Committee (Project Number: ORB06042Y).
S1 Table. List of S. cerevisiae strains expressing C. glabrata adhesins.
We acknowledge the assistance of Dr. Wade Sigurdson with Confocal and Fluorescence Microscopy. We acknowledge the assistance of Mr. Peter Bush with Scanning Electron Microscopy.
Conceived and designed the experiments: ST BC ME. Performed the experiments: ST EHW PD IGR AM. Analyzed the data: ST PD ME. Contributed reagents/materials/analysis tools: BC ME. Wrote the paper: ST BC ME.
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