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Serum antibody profile during colonization of the mouse gut by Candida albicans: relevance for protection during a systemic infection. Blanca Huertas, Daniel Prieto, Aida Pitarch, Concha Gil, Jesus Pla, and Rosalia Diez-Orejas J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00383 • Publication Date (Web): 18 Aug 2016 Downloaded from http://pubs.acs.org on August 21, 2016

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Journal of Proteome Research

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Serum antibody profile during colonization of the mouse gut by Candida albicans: relevance for protection during systemic infection. Blanca Huertas‡, Daniel Prieto‡, Aida Pitarch, Concha Gil, Jesús Pla, and Rosalía DíezOrejas* Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid and Ramón y Cajal Institute of Health Research (IRYCIS), Spain.

ABSTRACT

Candida albicans is a commensal microorganism in the oral cavity, gastrointestinal and urogenital tracts of most individuals that acts like as an opportunistic pathogen when the host immune responses is reduced. Here, we settled down different immunocompetent murine models, to analyze the antibody responses to the C. albicans proteome during commensalism, commensalism followed by infection, and infection (C, C+I and I models, respectively). Serum anti-C. albicans IgG antibody levels were higher in colonized mice than in infected mice. The antibody responses during gut commensalism (up to 55 days of colonization) mainly focused on C. albicans proteins involved in stress response and metabolism, and differed in both models of commensalism. Different serum IgG antibody-reactivity profiles were also found along the time


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among the three murine models. C. albicans gut colonization protected mice from an intravenous lethal fungal challenge, emphasizing the benefits of fungal gut colonization. This work highlights the importance of fungal gut colonization for future immune prophylactic therapies.

TEXT 1. INTRODUCTION The polymorphic fungus Candida albicans is a pathogen of humans, and is associated with infections that are often difficult to treat and may result in high mortality and morbidity despite antifungal therapy.1 This opportunistic pathogen can grow as a commensal microorganism in the oral cavity, gastrointestinal (GI) and urogenital tracts of most individuals,2 but when the host immune response is diminished it behaves like a true pathogen.3 This transition from commensalism to infection may involve the translocation of the fungus from different niches to the bloodstream, reaching essential organs and causing very severe invasive candidiasis. Furthermore, high fungal GI levels may be an important predisposing factor for acquired Candida infections,4 which can mainly come from endogenous origin.5 The complex interactions between fungal and bacterial commensals, either directly or through the participation of the host immune system, have impact on the pathophysiology of a number of inflammatory diseases that, in turn, may lead to secondary fungal infections. An increased understanding of the importance of microbiota in shaping the host's immune and metabolic activities has rendered fungal interactions with their hosts more complex than previously appreciated. It is now clear that a three-way interaction between host, fungi, and microbiota dictates the types of host-fungus relationship. Indeed, microbial dysbiosis predisposes to a variety of fungal infections and diseases at local and distant sites.6 Previous studies have suggested that a better knowledge on


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the C. albicans commensalism could provide novel insight into the development of a future vaccine against this opportunistic fungal pathogen.7, 8 Most invasive fungal infections occur in individuals with defective cellular immunity, indicating the relevance of this type of immunity in the control of the progression of the disease.9 In contrast to initial studies suggesting an uncertain role of antibodies in host protection,10, 11 recent works have highlighted the importance of the humoral immunity for defense against C. albicans.12-14 Furthermore, due to the nephrotoxicity of some antifungal drugs and the increasing fungal resistance to azole and echinocandin treatments,15, 16 new antifungal therapies, such as those based on antibodies, are necessary.17 To date, there are few antibody molecules that have shown to be relatively efficient for the treatment of invasive candidiasis in murine models1820

with the exception of a human recombinant antibody directed against C. albicans Hsp90 that

has gone into in a multinational phase III clinical trials.21, 22 Proteome analysis is an important approach for a comprehensive characterization of dynamic variations of complex biological systems. Proteomics could have a major role in creating a predictive, preventative, and personalized approach to medicine in future.23 Immunoproteomics or serological proteome analysis (SERPA), based on the combination of 2-DE with Western blotting and MS, has shown to be a useful tool for profiling the antibody responses to C. albicans24-28 and for other human pathogens.29-31 In this work, we describe different murine models to analyze the antibody responses against C. albicans during commensalism and a sub-lethal systemic infection. We employed these models in conjunction with SERPA to define and compare the serologic responses in these conditions, ranging from commensalism (early and late colonization) to infection. Finally, we


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investigated whether colonization could protect mice from an intravenous lethal fungal challenge, and provide experimental support for future immune prophylactic therapies.

2. MATERIALS AND METHODS 2.1. Strains and growth conditions The C. albicans strain used in this work was a wild type variant (CAF2) derived from SC531432 that produce the ovalbumin protein (CAF2-OVA). CAF2-OVA strain was constructed as follows: The codons from the OVA gene coding DNA sequence CDS from Gallus gallus (GenBank NM_205152) were re-codified to the codon usage of C. albicans, avoiding CTG codon, as reported with other heterologous genes33 and a hemagglutinin (HA) tag was added in the 3’ end. Two restrictions sequences were placed in the 5’ and 3’ ends (SalI and BglII, respectively). The whole sequence was chemically synthesized (GenScript, NJ, US). The OVA gene was cloned through SalI and BglII cuts in the doxycycline regulated plasmid pNIM1R-GFP 34

to generate pNIM1R-OVA-HA. Transformation of the CAF2 strain (through electroporation)

with the fragment KpnI-KspI of the pNIM1R-OVA-HA and selection with nourseothricin (200 mg/mL) led to the CAF2-OVA strain. Yeast strains were grown at 37 ºC in YPD medium (2% glucose, 2% peptone, and 1% yeast extract) or SD (synthetic dextrose) medium (2% glucose, 0.5% ammonium sulfate, 0.17% yeast nitrogen base) plus amino acids. 2.2. In vivo procedures Any experiment that implied the use of animals was performed in strict accordance with the regulations in the “Real Decreto 1201/2005, BOE 252” for the Care and Use of Laboratory


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Animals of the “Ministerio de la Presidencia,” Spain. The protocols were approved by the Animal Experimentation Committee of Complutense University of Madrid (Permit Number: CEA 25/2012, and BIO2012-31839-1). Mouse euthanasia was performed by CO2 inhalation following standard protocols (AVMA Guidelines for the Euthanasia of Animals: 2013 Edition). Female mice C57BL/6 of 7–10 weeks-old (Harlan Laboratories Inc., Italy) were used for colonization and infection experiments. Mice housing and non-invasive procedures took place in the animal facility from the Medicine Faculty of Complutense University of Madrid. 2.2.1. Colonization and infection models Harmless colonization was established using a protocol previously described in other works34, 35. Briefly, a single gavage of 107 C. albicans cells was inoculated in a group of 6 mice after 4 days of antibiotic pretreatment (2 mg/mL streptomycin, 1 mg/mL bacitracin, and 0.1 mg/mL gentamicin) in drinking water. Antibiotics-based therapy was maintained along 55 days of colonization. A sub-lethal systemic infection was generated after injecting a low inoculum of 105 C. albicans cells in the lateral vein of 6 non-colonized mice and 6 colonized mice (at day 10 of colonization) and maintained along 30 days. 2.2.2. Fungal burden Fungal levels of C. albicans colonizing mouse gut along the experiment were determined as follows. Fresh stool samples were collected periodically from every mouse in each group, mechanically homogenized in sterile PBS, and serially diluted before plating in YPDchloramphenicol agar plates.34 After incubation at 37ºC for 48 h, colony forming units (CFU) were counted.


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For quantification of yeast cells in kidneys, liver and mesenteric lymph nodes (MLNs), mouse euthanasia was performed by CO2 inhalation. Organs from individual mice were removed aseptically and placed in a tissue homogenizer with sterile PBS. The number of viable yeast units in these specimens (including two mice per group) was determined by the plate dilution method using YPD-chloramphenicol.36 After incubation at 37ºC for 48 h, colony forming units (CFU) were counted. 2.2.3. Serum samples Serum samples were collected from the submaxillary vein of all the mice in each of the three models (6 mice per group) at different days (see Fig. 1). Serum obtained from mice on day 0 was used as a control serum. Pooled sera for each specific group and time point were obtained through mixing of individual samples. This non-invasive procedure rendered 100% of mice viability. 2.2.4. Protection assays A group of 5 C. albicans gut colonized mice were injected in their lateral vein with a high inoculum of 106 C. albicans cells after 40 days of colonization. Another group of 5 C. albicans gut colonized mice were sub-lethally infected at day 10 with 105 C. albicans cells as described above, and then lethally infected at day 40 with 106 C. albicans cells. A control group of 3 untreated mice were lethally infected with a high inoculum of 106 C. albicans cells.36 2.3. Preparation of C. albicans protein extracts Yeast cells were grown in YPD medium at 37 °C up to an A600

nm

of 1 and washed with water.

Cells were resuspended in cold lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM


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EDTA, 1 mM DTT, 0.5 mM PMSF, and protease inhibitors (complete Mini, EDTA-free, Roche, Germany)]. Mechanical lysis was then performed in centrifuge tubes with an equal volume of glass beads in a fast-prep cell breaker (Q-Biogene, Carlsbad, CA). Protein precipitation was obtained in 15% TCA. The clarified supernatant was stored at -80 °C. Protein concentration was measured with the Bradford assay (Bio-Rad, Hercules, CA). 2.4. ELISA Pooled sera from each group of mice (C, C+I and I models) were evaluated using an indirect ELISA. Wells of polystyrene microtiter plates (Nunc-Immuno Plate) were coated with 400 ng of CAF2-OVA strain protein extracts overnight at 4 ºC. All subsequent steps were carried out at room temperature. After blocking non-specific sites with 4% non-fat milk powder in PBS for 1 h, two-fold serial dilutions of each serum were incubated in the plate wells for 2 h. After extensive washing with PBS containing 0.05% Tween 20 (PBS-T), a secondary horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham Biosciences, Stressgen and Cultek, Germany) was added at a 1:1000 dilution. Plates were incubated for 1 h, and then washed three times with PBS-T. A solution of 0.4 mg/mL o-phenylenediamine (Sigma, Germany) and 0.04% (v/v) H2O2 was added to develop the reaction. It was stopped by addition of 3 N H2SO4. The colorimetric change was measured at 492 nm in a microplate reader (model 680, BioRad). 2.5. SERPA C. albicans protein extracts (150 µg) were incubated in a rehydration buffer [7 M urea, 2 M thiourea, 2% CHAPS, 65 mM DTE, 0.5% immobilized pH gradient (IPG) buffer pH 3−10 (GE Healthcare, Buckinghamshire, UK), and 0.002% bromophenol blue], and then separated by 2-DE as described elsewhere,25, 37 using immobilized, nonlinear pH 3–11 gradient strips (18 cm; GE


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Healthcare) for isoelectric focusing, and 10% SDS-polyacrylamide gels (10% T; 1.6% C) for the second-dimension separation. The 2-DE-separated proteins were then either visualized with colloidal Coomassie Brilliant Blue or transferred to nitrocellulose membranes (HyBond ECL; GE Healthcare).37 Blots were blocked with 5% nonfat dry milk in PBS for 2 h. After rinsing with PBS, blots were incubated with pooled sera from control (day 0), colonized, colonized plus infected or infected mice at a 1:500 dilution for 2 h. After washing four times with PBS containing 0.01% Tween-20, blots were incubated again with the IRDye 800 CW conjugated goat (polyclonal) anti-mouse IgG antibodies (1:4000 dilution; LI-COR Biosciences, Germany). They were rinsed again three times with PBS-T and then with PBS. The blots were scanned using the Odyssey infrared imaging system (LI-COR Biosciences). Protein spots of interest were identified using our reference 2-DE map of C. albicans immunogenic proteins (Fig. S1), which were characterized previously by peptide mass fingerprinting24, 27, 38 and MS/MS.24, 39 This map is

also

available

on

our

COMPLUYEAST-2DPAGE

database

at

http://compluyeast2dpage.dacya.ucm.es/cgi-bin/2d/2d.cgi.40, 41 2.6 Statistical analysis Differences in antibody titters among groups in the ELISA assay were evaluated using the Student’s two-tailed unpaired t-test. Kaplan-Mayer survival curves were examined with the logrank (Mantel-Cox) test. 3. RESULTS 3.1. Analysis of the C. albicans CAF-OVA strain


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In order to study the serologic response elicited by the host after the interaction with the opportunistic pathogen C. albicans, we developed cells that expressed a model antigen. We chose a highly immunogenic protein like chicken ovoalbumin (OVA). Therefore, we adapted the DNA sequence from the OVA gene and HA tag to the codon bias of C. albicans as determined previously.34 Then, it was introduced in the wild type C. albicans strain (CAF2) under the regulation of the Tet-OFF system, developing a yeast strain (CAF2-OVA) that is able to produce the protein OVA (tagged with HA) intracellularly in the absence of tetracycline and repress it in the presence of this antibiotic. The conditional expression and recognition of the model antigen OVA were validated by Western-blot using C. albicans total protein extracts obtained after growth in the presence or absence of doxycycline (20 µg/mL). Both anti-HA and anti-OVA antibodies detected specifically the protein under the different inducing conditions (Fig. S2 A). 3.2. Establishment of different murine models We set up three experimental models to analyze and compare the serologic responses of immunocompetent mice after C. albicans GI colonization, colonization followed by sub-lethal infection and sub-lethal systemic infection. CAF2-OVA cells suspended in PBS were inoculated by gavage or intravenously in these groups of mice to simulate three different host-fungus interactions: (i) gut commensalism (C); (ii) commensalism plus systemic infection (C+I), and (iii) systemic infection (I). We followed the scheme displayed in Fig. 1, where the time course of the experiment is depicted. The groups C and C+I were inoculated intragastrically on day 0 and maintained colonized for 55 and 40 days, respectively. A sub-lethal infection was produced through


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intravenous inoculation in I model (at day 0) and C+I model (once colonization was established on day 10) and maintained during 30 days to avoid animal suffering. Pooled serum samples were obtained at different time points to evaluate antibody production in each model and along the experiment (Fig. 1). We first tested the feasibility of the models to evaluate the behavior of mice. For this purpose, we measured the weight of the mice at different time points along the experiment. All the mice were healthy as reflected by the maintenance of a similar weight evolution independent of the model employed (Fig. 2 A). 3.2.1. Gut colonization (C model) We employed a murine model of GI commensalism described previously by our group.34 In this model, it is necessary to diminish bacterial colonization to allow fungal establishment in the gut,7 thus colonization assays began with a four days-antibiotic pretreatment followed by a single gavage of 107 yeast cells in 100 mL of sterile PBS. To confirm that a steady and high level of fungal colonization was established, we determined C. albicans loads in stools every 2-4 days (Fig. 2 B). High fungal levels detected by colony forming units (CFU) were similar in both groups of colonized mice along the experiment (107108 CFU/g stool) and in line with those detected in previous works.34 3.2.2. Gut colonization followed by sub-lethal systemic infection (C+I model) Previous works have described that to incite fungal dissemination after C. albicans colonization, it is necessary to induce immunosuppression and disruption of the mucosal integrity of the animals.42 However, we have settled down a model of fungal commensalism and dissemination


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through the bloodstream employing immunocompetent mice, and thus making possible an accurate antibody response. In this model, after confirmation of fungal gut establishment (Fig. 2 B), colonized mice were then infected in the lateral vein with a sub-lethal dose of the fungus (see below). To check out gut colonization of these mice, we measured the fungal presence in the stools as reported previously (Fig. 2 B). We observed similar levels of gut colonization in both murine models (C and C+I). The fungal gut colonization level was similar in all the models and independent of the parallel systemic infection. After 15 days of infection, fungal dissemination was detected in kidneys (around 3x106 CFU/g of tissue), as well as in the liver and MLNs with 20 and 105 CFU/g of tissue, respectively (Fig. 2 C). CFUs in these organs confirmed the development of a systemic infection in these immunocompetent mice, as revealed by the close correlation between fungal burdens and infection rate. 3.2.3. Sub-lethal systemic infection (I model) We used the murine model of disseminated candidiasis because this closely resembles, in nature and distribution, the lesions reported in the human disease, and has proved to be a useful tool for studying the humoral response.43-45 We induced sub-lethal infection using a low fungal dose (intravenously injecting 105 yeast cells) with the aim of generating a chronic infection and inducing a robust humoral response in the mice. To confirm fungal systemic infection in these mice, we first measured the C. albicans load in their kidneys, liver and MLNs after 15 days as described above (Fig. 2 C). The fungal burden detected in the kidneys from the infected group (I model) and the colonized plus infected group (C+I model) was similar. This finding was in agreement with data obtained previously in similar


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experiments36,

46

as well as with the establishment of a chronic fungal infection. No fungal

infection was detected in the liver or MLNs from only infected mice, as expected due to the close relationship of both organs to a systemic infection from gut and the low dose given. 3.3. Anti-Candida IgG antibody titers in sera from colonized and infected mice We first quantified the antibody response produced against C. albicans in each murine model by ELISA, using either the commercially available OVA or CAF2-OVA protein total extract as an antigen source. When employing OVA as an antigen we could not detect specific antibodies by ELISA (data not shown) or Western-blot (Fig. S2 B). However, we detected C. albicans-specific antibodies using CAF2-OVA extracts (Fig. 3), suggesting that the humoral response of the mice depends on the model of host-fungus interaction employed. Serum titers increased along the time independently of the murine model. No antibodies were detected in the control serum from animals on day 0. Unsurprisingly, when comparing the same time points, the highest levels of total serum anti-Candida IgG antibodies were observed in the group C+I while the lowest levels were detected in the group I. On day 40, the group C+I showed a 6.5-fold increase in serum antiCandida IgG antibody titers as compared with the group C, and ~30-fold increase as compared with the group I (Fig. 3). Although these differences were also found on day 25 of the experiment, they were notably higher on day 40. 3.4. Serum IgG antibody-reactivity profiles to the C. albicans intracellular proteome in colonized and infected mice To examine the serologic responses in our murine models, C. albicans protein species from the CAF2-OVA strain were immunodetected on 2-DE blots using pooled sera from each


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experimental model at different time points. Different immunoreactivity patterns were observed among the three murine models and along the time course (Figs. 4 and 5, and Table 1). 3.4.1. Time course of serum anti-C. albicans IgG antibody-reactivity profiles in colonized mice Serum samples from colonized mice were analyzed by SERPA at 10, 25, 40 and 55 days to compare antibody responses along the time course of colonization. No antibody response was detected in the control serum from mice on day 0, while increasing serum IgG antibodyreactivity levels were observed along the time course of colonization (Fig. 4 and Table 1). A total of seven immunoreactive C. albicans proteins involved in stress response and metabolism were detected on 2-DE blots from the murine sera obtained at the early colonization stage (Table 1). We observed a weak immunoreactivity against Hsp90, Pdc11 and Cdc19, but a moderate immunoreactivity against Pgk1 and Met6, and a strong immunoreactivity against Hsp70 and Eno1 (mainly, against one of its protein species) (Fig. 4 B). In the serum samples obtained after long gut colonization, a higher immunoreactivity against most of the previous detected C. albicans proteins (Pdc11, Pgk1, Met6, Hsp70 and Eno1) was found (Fig. 4 C), an expected finding because of the high titers obtained in our ELISA (Fig. 3). Serum IgG antibodies to some other immunogenic C. albicans proteins were also detected. In particular, a strong immunoreactivity against Hxk2 and a weak immunoreactivity against Asc1 were also found. Therefore, at least nine immunogenic proteins were detected in long term colonization. 3.4.2. Comparison of serum IgG antibody-reactivity profiles to the C. albicans intracellular proteome among the different murine models


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The different 2-DE immunoreactivity patterns observed among the three murine models (C, C+I and I) at day 40 (Fig. 1) are depicted in Fig. 5 (A, B and C, respectively). In the colonization model, moderate immunoreactivity against C. albicans Pgk1 and Met6, a strong reaction against Hsp70 and Eno1 and a faint reaction against Hsp90, Asc1, Cdc19 and Pdc11 were found (Fig. 5 A). In the murine model of gut colonization followed by sub-lethal systemic infection (C+I model), which showed the highest serum titers in our ELISA (Fig. 3), a robust immunoreactivity against Eno1, Pdc11, Pgk1, Met6, Hsp70 and Ssa2, and a lower reaction against Adh1 and Cdc19 were observed. The lowest immunoreactivity was shown against Asc1, Fba1 and Atp1 (Fig. 5 B). In the sub-lethal infection model, there was a faint antibody response to Ssa2 and Cdc19, and a higher reaction to Hsp70, Pdc11, Pgk1 and Eno1 (Fig. 5 C). This low immunoreactivity was in agreement with the serum titers detected in our ELISA (Fig. 3). The immunoreactivity against some C. albicans proteins observed in the different murine models along the time is summarized in Table 1. Briefly, colonized mice exhibited a strong reaction against Hsp70 and Eno1, which increased along the time. A moderate reaction against Met6, Pdc11, and Pgk1, and a faint, but maintained along the time, reaction against Hsp90, Asc1 and Cdc19 were also detected, which increased and maintained along the time, respectively. It is also noticeable that in this C model immunoreactivity against Hxk2 was detected at the latest time point (55 days; Fig. 4 C). The immunoreactivity profile of the C+I model was related not only to the long gut colonization, but also to the intravenous inoculation (on day 10). A total of eleven proteins were immunodetected in sera from the C+I group at the comparison time point. In general, serum levels of antibodies were higher than in colonization. Serum levels of IgG antibodies to Eno1, Hsp70, Ssa2, Pgk1, Adh1, Pdc11, Cdc19 and Met6 increased, while those to Fba1, Asc1 and Atp1 faintly appeared. For the I model, the immunoreactivity of the sera along


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the time was lower when compared with the C and C+I models, in line with our previous results (Fig. 3). Only seven proteins were immunodetected (Eno1, Hsp70, Pdc11, Pgk1, Cdc19 and Ssa2), although three of them (Met6, Cdc19 and Ssa2) were very weakly immunorecognized. We detected differences among the three murine models. Serum IgG antibodies to Hxk2 were detected in the long term colonization state. In contrast, high reactivity levels of IgG antibodies to several metabolic and heat shock proteins like Eno1, Met6, Hsp70, Pcd11, Pgk1 and Cdc19 were found in colonization and infection (C, C+I and I models). Serum IgG antibodies to Ssa2 were found very faintly in the I model, but increased in the C+I model. Serum IgG antibodies to Adh1, Atp1 and Fba1 were only detected in the C+I model, probably due to high immunoreactivity of this serum in quantity and composition terms. Serum IgG antibodies to Eno1 were detected (with a very high intensity) in all the sera studied, even in early colonization and infection (Table 1). Serum IgG antibodies to Pdc11 were also detected in all the models studied, but in lower intensity than anti-Eno1 IgG antibodies. IgG antibodies to Hsp70 were also found in high concentration in most sera, but they were undetectable at early infection times. IgG antibodies to Met6 were detected with an increased intensity along the time in colonized mice (C and C+I models). 3.5. Protective antibody response against a lethal C. albicans challenge in the different murine models We next evaluated whether the immune response generated after C. albicans gut commensalism could induce protection against a fungal systemic infection. For this purpose, mice from our two colonization models (C and C+I) were challenged with a high dose of C. albicans to induce a lethal systemic infection (Fig. 6 A). This lethal infection was induced after 40 days from the


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beginning of the experiments thus allowing a suitable immune response to C. albicans in the different murine models. A strong antibody response was detected in our ELISA at this time point in C and C+I models (Fig. 3). A group of untreated mice was also employed as a control of the lethality of the inoculum. We also tested the healthy state of the different groups of mice both before (data not shown) and after the lethal fungal challenge in the same way as described above. The weight of the mice after the lethal challenge is displayed in Fig. 6 B. A group of untreated mice (without initial contact with the fungus) was also analyzed. All the mice (independent of the group) showed a decrease in weight, reflecting the burden induced by the lethal infection. After 10 days, mice recovered their initial weight and after 15 days the colonized mice showed a weight similar to the beginning of the assay. The untreated mice group exhibited a weight loss from the beginning of the experiment until day 8 where the damage was up to 50% of the initial weight, thus reflecting the lethality of the challenge. The survival rate of the mice from both models (C and C+I) after the lethal challenge is shown in Fig. 6 C. These data suggested that colonized mice rendered protection against a lethal fungal challenge as reflected by a significant increase in the survival percentage compared with the untreated mice group (C vs. untreated group; p < 0.05, and C+I vs. untreated group; p < 0.05).

4. DISCUSSION The opportunistic pathogen C. albicans exists as a commensal member of the mammalian microbiome of the GI tract from healthy humans. However, under certain circumstances this


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fungus can disseminate to virtually any internal organs. Previous studies of the importance of microbiota in shaping the host immune and metabolic activities suggest that these fungal-host interactions should be more complex than previously appreciated.47 It is well-known that the microbial inhabitants of the intestine peacefully coexist due to the elaborated and interconnected regulatory mechanisms that maintain immune homeostasis limiting the face of potential infection and tissue damage by pathogenic microorganisms.48 Physical barriers, antimicrobial factors and antibodies act in concert to keep microbes at a distance from the epithelium and initiate repair mechanisms in the event of damage. Because of this dual relationship with the host, in this work, we studied some aspects (basically the antibody response) of this association with the aim of gaining further insight into the meaning of fungal commensalism to the host. One of the novelties of this work is the description of the description of the antibody response during a long term gut commensalism using SERPA. Another originality of this study is that we have developed similar murine models that reproduce different conditions of host-fungus interactions and we have compared their antibody profiles. The murine model of commensalism used in this work is the same as that previously settled down in our group to study C. albicans behavior in a situation of harmless interaction with the host.34,

35

We have chosen this murine model of commensalism due to the availability of

modified murine and yeast strains and reagents that make possible numerous studies of host response. Although we are conscious that the murine and human microbiotas are different and C. albicans is not normally found in mice, this model has been considered the best model so far.34, 49, 50 We have also employed the murine model of systemic infection because it closely resembles candidiasis situations in the human being.36, 51 In previous works of our group we have used this murine model for studying the antibody response of different strains of mice after sub-


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lethal systemic C. albicans infection25 as well as for describing the different antibody response induced by diverse C. albicans strains during systemic infection.44 Taking into account our experience, we have combined these two murine models, and, settled down a new model of commensalism plus infection. This is a new approach using immunocompetent and healthy mice when comparing with the methods described in the literature because all of them employed immunosuppressed mice.7,

42

These models have proved to be a useful tool for studying the

immune response induced after C. albicans colonization in the face of immunodeficiency.52 We observed that serum anti-C. albicans antibody levels were significantly higher in colonized mice (C and C+I models) than in the control serum obtained on day 0. An intensification of almost seven times was also found in these levels when comparing with only colonized mice. These results are in agreement with an earlier study showing host immune responses during commensalism where mice responded to the presence of C. albicans colonization by producing antibodies.53 With the establishment of our two colonization models using immunocompetent mice (able to develop a robust antibody production), we could study the humoral response induced by the fungus during its commensal state and infection, and thus confirm those earlier findings. This study has highlighted the serologic response against C. albicans gut commensalism, describing, for the first time, the serum antibody composition using SERPA. We have analyzed serum IgG antibody-reactivity profiles along the time course up to 55 days of colonization. After an extended fungal presence in the gut, serum IgG antibody composition was maintained but IgG antibody levels increased, reflecting an increase of the immune responses along the time. It is noteworthy the high levels of several serum IgG antibodies along colonization state (in


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particular, those against Eno1, Met6, Hsp70, Pgk1 and Pdc11), as well as low levels of serum IgG antibodies against Hsp90, Asc1 and Cdc19 along colonization. When evaluating the antibody responses to the C. albicans proteome in both models of commensalism (C and C+I models), we found higher serum levels of IgG antibodies in the C+I model than in the C model. We have described some differences in the IgG antibody-reactivity patterns against C. albicans heat shock proteins and metabolic enzymes. Faint, but detectable, levels of IgG antibodies to Fba1, Atp1, Adh1, as well as high anti-Ssa2 antibody levels appeared, while no detectable levels of IgG antibodies to Hsp90 were observed. The unconventional location of proteins related to heat stress, like Hsp90, Hsp70 and Ssa2, at the C. albicans cell wall has been described previously.54-56 This is a privileged site for antibody recognition, in some way explaining the elevated immunogenicity of Hsp7057 and the high levels of IgG antibodies to these immunogenic proteins in all tested sera (particularly very high IgG those to Hsp70 in colonized mice). Strikingly, one main difference between sera from both models was the absence of detectable levels of serum IgG antibodies to Hsp90 in C+I mice. Previous studies have described that patients with high anti-Hsp90 IgG antibody concentrations exhibited greater risk for suffering invasive candidiasis.58-62 Hsp90 represents an important target for protective antibodies in disseminated candidiasis,21 and plays an important role in mediating Candida resistance to echinocandins, giving to its elicited antibodies more attractive biological properties.17 In contrast, a strong antibody response was detected against Ssa2 in the C+I model. This C. albicans antigen has been described previously as a human histatin-binding protein63 involved in adherence to epithelial and endothelial cells as well as endocytosis.64-67 The presence of these antibodies during commensalism could prevent fungal binding to histatins, thus limiting host cell adhesion and endocytosis.


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Some of the metabolism-involved proteins strongly recognized by sera from our C+I model (such as Met6, Eno1 and Pgk1) have already been described as immunogenic proteins25, 44 as well as useful for immunodiagnostic reagents for invasive candidiasis.58,

68-70

Some other

immunoreactive proteins (such as Fba1, Adh1 and Atp1) were only found in the C+I model. The detection of IgG antibodies against some glycolytic enzymes, such as Eno1, Pgk1, Cdc19, Fba1 and Hxk2 (being stronger recognized after a long fungal exposure), reflects that the glycolytic transcriptional regulation is important for the metabolic flexibility of C.albicans and other pathogens in their attempts to colonize diverse niches.71 The detection of a strong humoral response against enolase in our murine model is in agreement with the description of high serum anti-Eno1 antibody levels both in patients and in mice with invasive candidiasis.43, 44, 58, 69 The location of Eno1 on the cell surface might facilitate the production of antibodies by C. albicansinfected hosts.54,

72, 73

In addition, Eno1 is a metabolic protein and one of the most abundant

cytosolic enzymes in many organisms,74 and is considered a multifunctional protein and an immunodominant antigen,75 with a variable gene expression over the course of infection.76 The relationship between Eno1 and gut epithelia has been described previously, showing that pretreatment of GI epithelia with this protein blocks adhesion.77, 78 Once we have settled down two murine models of commensalism and commensalism followed by infection, we compared these data with serum IgG antibody profiles obtained after systemic infection. The employment of a sub-lethal infection model has often been used to establish a chronic infection, and study the course of the infection monitored by fungal load in the most representative organs (like the kidney).79 The finding of low serum anti-C. albicans antibody levels in sub-lethally infected mice is in agreement with the low dose of inoculum and induced immune response.25,

36

On the other hand, when comparing serum antibody patterns from


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colonized or infected mice, our results revealed differences in the serum composition. These findings suggest that the immune system can discriminate between GI tract colonization and infection. Taking into account our results, we propose that during commensalism the host immune system could selectively induce IgG antibodies to several abundant immunogenic proteins from C. albicans (such as Eno1, Pgk1, Cdc19, Met6, Pdc11 and Hsp70) to limit C. albicans colonization during the permanent host−pathogen interplay in the commensal state.80 After a fungal challenge in colonized mice, the levels of these IgG antibodies could increase as a result from a second antibody response of the immune system to these cell surface-exposed proteins5456

and other cell wall-associated and abundant immunogenic proteins could further be recognized

(such as Fba1, Atp1, Adh1 and Ssa2). This secondary antibody response against the fungal challenge would therefore reflect the host defense response against the fungal pathogen. In contrast, the lower antibody response observed when only infection was induced as compared when previous colonization took place suggests that a previous contact with a commensal microorganism favors a stronger immune response against this opportunistic pathogen. To our best knowledge, in this work we have settled down three murine models for the first time to make possible a comparative study of the serum composition after different C. albicans–host interactions. These murine models have allowed us to analyze the antibody composition by SERPA at the same time points and using the same conditions of commensalism and/or infection. In order to discern the possible role of C. albicans gut commensalism for protection against a systemic infection, we employed these two models of commensalism (C and C+I). The presence


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of high levels of antibodies in the sera of colonized/infected mice suggests that these animals could develop a robust antibody response against Candida after systemic infection. This is in line with the ability of Candida–colonized mice to respond to a vaccine formulation against candidiasis.8 Antibodies with defined specificities could act with different degrees showing protection against systemic and mucosal candidiasis. In a similar way to our previous works of protection capability using different strains of C. albicans,43,

45

we settled down a vaccination

assay using gut colonized and colonized/infected mice. Our results indicate that colonized mice may generate a significant protective immune response against a lethal challenge of this fungal pathogen. A weak but significant protection was also detected when C+I mice were employed. These findings point out that these mice colonized and infected with a low dose may also produce a moderate protection. The ability of our C. albicans colonization model to protect against an intravenous infection points to the benefits of being colonized by this fungal commensal microorganism in order to stimulate (or even prepare) the antibody response against future fungal contacts (like intravenous acquisition through a contaminated catheter) increasing the survival fate. This pre-colonization may also increase the antibody response after vaccination procedures. Due to the susceptibility of candidiasis in neutropenic patients, pre-colonization experiments should be done in future research to analyze the antibody response after vaccination assays and elucidate its potential role in protection. Our study about the composition of the different sera has revealed several important immunogenic C. albicans proteins that could be employed for future vaccine development. Protective responses using antibodies against Fba1, Hsp90, and Met6, among others, have already been described.22, 27, 68, 81, 82 The protective role of antibodies directed against Eno1 has also been reported in a murine model of systemic candidiasis.72, 83, 84 Supporting this idea, mice


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vaccinated with Eno1 have shown higher survival time than mice that were not vaccinated.84 Furthermore, a synthetic glycopeptide vaccine employing Fba1, Met6, Hwp1, Eno1, or Pgk1 has been employed to induce protective immune responses against invasive candidiasis.81, 82 C. albicans colonization is required for invasive infection. Unlike humans, adult mice with mature intact gut microbiota are resistant to C. albicans GI colonization. In addition to bacteria, there appears to be a possible link between the commensal yeast C. albicans and disease development, making the yeast a possible initiator of the inflammatory process observed in inflammatory bowel disease.85 This work supports the notion that microorganisms with a potential role in intestinal homeostasis and inflammation could have specific impacts on the host.

5. CONCLUSIONS We have highlighted the importance of gut microbiota in shaping the host immune response. Using two models of commensalism and commensalism followed by an intravenous fungal infection in immunocompetent mice, we have detected high titers of anti-C. albicans antibodies in their sera. Serum composition in commensalism has revealed the presence of certain antibodies against several proteins that are important for diagnosis and future vaccine development against invasive candidiasis. In adition, we have detected reactivity levels of serum IgG antibodies from mice once colonized and sub-lethally infected, which points towards the advantage of being fungal gut colonized. We have also found that colonized mice produce a protective immune response against a lethal


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fungal infection, suggesting that fungal colonization could be an advantage for a future exogenous fungal infection (like systemic infection due to a contaminated catheter).

7. CONFLICT OF INTEREST DISCLOSURE The authors declare no competing financial interest.

FIGURES Figure 1. Schematic diagram of the time course of the three experimental murine models established. C. albicans GI colonization is represented with a black line, the sub-lethal systemic infection is shown with a red line and colonization followed by sub-lethal infection on day 10 is depicted with black and red discontinuous lines. The time point for comparison of the three models was on day 40 (or 30 for infection) and is labelled with a blue rectangle. Figure 2. Mouse weight and fungal colonization and infection of the three murine models. Colonized mice are represented with black circles, infected mice with red squares, and colonized and infected mice with black and red triangles. Vertical lines show standard deviation. A. Weight of the mice from the three models at different time points along the experiment. B. Time course of GI fungal colonization. C. albicans loads in stools were determined by CFUs along the time. C. Fungal systemic infection after 15 days of C. albicans inoculation as evaluated by counting of CFUs in kidneys, liver and MLNs.


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Figure 3. Total serum titers of IgG antibodies against C. albicans cell extracts in C, C+I and I mice. Pooled sera from each specific group were tested at different time points. Serum IgG antibody titers were measured using an indirect ELISA. * p < 0.05, when colonized mice were compared with infected mice; ** p < 0.01, when the C model was compared with the C+I model; and *** p < 0.001, when the I model was compared with the C+I model. Figure 4. Time course of serum anti-C.albicans IgG antibody-reactivity profiles in colonized mice (C model). Immunoreactive protein spots were detected using (i) the control serum from animals at day 0, (A) and (ii) pooled sera from colonized mice along the time: at day 10 (B) and at day 55 (C). Figure 5. Comparison of serum IgG antibody-reactivity profiles from mice of the C, C+I and I models. Pooled sera from each specific group were analyzed by SERPA at the comparison time point shown in Fig. 1. A. Colonized mice at day 40 B. Colonized and infected mice at day 40. C. Infected mice at day 30. Figure 6. Time course scheme of fungal colonization (C and C+I models) and subsequent lethal C. albicans challenge. A. C. albicans GI colonization is represented with a black line and colonization followed by sub-lethal infection on day 10 is shown with black and red discontinuous lines. Untreated mice were used as a control of the lethality of the inoculum. On day 40 of both models, a lethal challenge of C. albicans was inoculated. B. Weight of the mice at different time points along the experiment. C. Survival of colonized mice and colonized plus infected mice. Survival of both murine models was compared with control untreated mice after infection with a lethal dose (106 cells) of C. albicans SC5314 strain using Kaplan-Meier curves.


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* p < 0.05 in long-rank analysis. †One out of five survival data of the C+I model was a missing value. SUPLEMENTAL FIGURE LEGENDS Figure S1. Reference 2-DE map of C. albicans immunogenic proteins. Protein spots were previously characterized by peptide mass fingerprinting 24, 27, 38 and MS/MS.24, 39 Figure S2. A. Analysis of conditional expression of ovalbumin in C. albicans-OVA strain. CAF2-OVA total protein extracts in the presence or absence of doxycycline (20 µg/mL). Different amounts of protein were loaded to detect OVA-HA and ovalbumin (as a positive control) by Western-blot analysis. B. Western-blot analysis of antibody response against OVA from pooled sera from the C, C+I and I models at different time points

TABLES. Table 1. Recognition patterns of C. albicans immunogenic proteins in the three models assessed. Colonization + Protein

Description

CGD accession number

Colonization

Infection Infection

10

25

40

55

25

40

15

30

Eno1

Enolase I

C1_08500C

++

+++

+++

++++

+++

+++++

+

++

Pgk1

Phosphoglycerate kinase

C6_00750C

+

++

++

+++

++

+++

-

++

Cdc19

Pyruvate kinase

C2_05460W

+/-

+/-

+

+

+

++

-

+/-

Fba1

Fructosebiphosphate aldolase

C4_01750C

-

-

-

-

+

+

-

-


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Hxk2

Hexokinase II

CR_04510W

-

-

-

++

-

-

-

-

Met6

Methionine synthase

CR_01620C

+

++

++

+++

+

+++

-

+/-

Pdc11

Pyruvate decarboxylase

C4_06570C

+

+

+

+++

+++

+++

++

+++

Asc1

Protein of the 40S ribosomal subunit

C7_01250W

-

+/-

+

+

+

+

-

-

Atp1

F1F0-ATPase alpha subunit

C1_04610W

-

-

-

-

+

+

-

-

Adh1

Alcohol dehydrogenase

C5_05050W

-

-

-

-

+

++

-

-

Hsp90

Heat protein 90

shock

C7_02030W

+

+

+

+

-

-

-

-

Hsp70

Heat protein 70

shock

C1_13480W

++

++

+++

++++

+++

++++

-

++

Ssa2

Heat shock protein of HSP70 family

C1_04300C

-

-

-

-

+++

++++

-

+

AUTHOR INFORMATION Corresponding Author * To whom correspondence should be addressed: Rosalía Díez-Orejas, Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid, Plaza Ramón y Cajal s/n, 28040-Madrid, Spain. Phone: +34-91-394-1888. Fax: +34-91-394-1745. E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources


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This work was supported in part by grants from the Ministry of Economy and Competitiveness BIO2012-31839, BIO-2012-31767, BIO2015-64777-P, BIO2015-65147-R and PCIN-2014-052 (INFECT-ERA). ACKNOWLEDGMENT This work was supported in part by grants from the Ministry of Economy and Competitiveness BIO2012-31839, BIO-2012-31767, BIO2015-64777-P, BIO2015-65147-R and PCIN-2014-052 (INFECT-ERA). ABBREVIATIONS GI, gastrointestinal; SERPA, serological proteome analysis; HA, hemaglutinin; OVA, ovalbumin; group C, gut colonization; group I, sub-lethal systemic infection; group C+I, gut colonization followed by sub-lethal systemic infection; PBS, phosphate buffered saline; CFU, colony forming units. MLNs, mesenteric lymphoid nodes; YPD: yeast extract, peptone and dextrose; PMSF, phenylmethylsulfonyl fluoride.

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Figure 1. Schematic diagram of the time course of the three experimental murine models established. C. albicans GI colonization is represented with a black line, the sub-lethal systemic infection is shown with a red line and colonization followed by sub-lethal infection on day 10 is depicted with black and red discontinuous lines. The time point for comparison of the three models was on day 40 (or 30 for infection) and is labelled with a blue rectangle. Fig. 1 87x44mm (300 x 300 DPI)


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Figure 2. Mouse weight and fungal colonization and infection of the three murine models. Colonized mice are represented with black circles, infected mice with red squares, and colonized and infected mice with black and red triangles. Vertical lines show standard deviation. A. Weight of the mice from the three models at different time points along the experiment. B. Time course of GI fungal colonization. C. albicans loads in stools were determined by CFUs along the time. C. Fungal systemic infection after 15 days of C. albicans inoculation as evaluated by counting of CFUs in kidneys, liver and MLNs. Fig. 2 201x325mm (300 x 300 DPI)



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Figure 3. Total serum titers of IgG antibodies against C. albicans cell extracts in C, C+I and I mice. Pooled sera from each specific group were tested at different time points. Serum IgG antibody titers were measured using an indirect ELISA. * p < 0.05, when colonized mice were compared with infected mice; ** p < 0.01, when the C model was compared with the C+I model; and *** p < 0.001, when the I model was compared with the C+I model. Fig. 3 91x88mm (300 x 300 DPI)


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Figure 4. Time course of serum anti-C.albicans IgG antibody-reactivity profiles in colonized mice (C model). Immunoreactive protein spots were detected using (i) the control serum from animals at day 0, (A) and (ii) pooled sera from colonized mice along the time: at day 10 (B) and at day 55 (C). Fig. 4 54x15mm (300 x 300 DPI)



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Figure 5. Comparison of serum IgG antibody-reactivity profiles from mice of the C, C+I and I models. Pooled sera from each specific group were analyzed by SERPA at the comparison time point shown in Fig. 1. A. Colonized mice at day 40 B. Colonized and infected mice at day 40. C. Infected mice at day 30. Fig. 5 55x16mm (300 x 300 DPI)


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Figure 6. Time course scheme of fungal colonization (C and C+I models) and subsequent lethal C. albicans challenge. A. C. albicans GI colonization is represented with a black line and colonization followed by sublethal infection on day 10 is shown with black and red discontinuous lines. Untreated mice were used as a control of the lethality of the inoculum. On day 40 of both models, a lethal challenge of C. albicans was inoculated. B. Weight of the mice at different time points along the experiment. C. Survival of colonized mice and colonized plus infected mice. Survival of both murine models was compared with control untreated mice after infection with a lethal dose (106 cells) of C. albicans SC5314 strain using Kaplan-Meier curves. * p < 0.05 in long-rank analysis. †One out of five survival data of the C+I model was a missing value. Fig. 6 225x373mm (300 x 300 DPI)


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