RESEARCH PAPER Virulence 5:8, 810--818; November/December 2014; © 2014 Taylor & Francis Group, LLC

Candida albicans VPS4 contributes differentially to epithelial and mucosal pathogenesis Hallie S Rane1, Sarah Hardison2,3, Claudia Botelho4, Stella M Bernardo1,5, Floyd Wormley Jr2,3, and Samuel A Lee1,5,* 1

Division of Infectious Diseases; University of New Mexico Health Science Center; Albuquerque, NM USA; 2South Texas Center for Emerging Infectious Diseases; San Antonio, TX ogica; Universidade do Minho; Campus de Gualtar; Braga, Portugal; USA; 3The University of Texas at San Antonio; San Antonio, TX USA; 4Departamento de Engenharia Biol
5 Section of Infectious Diseases; New Mexico Veterans Healthcare System; Albuquerque, NM USA

Keywords: Candida albicans, protein secretion, secreted aspartyl proteases, vacuole, virulence, VPS4 Abbreviations: LDH, lactate dehydrogenase; OEMS, oral epithelial model system; RHE, reconstituted human epithelium; SAP, secreted aspartyl protease

We have previously demonstrated that the C. albicans pre-vacuolar protein sorting gene VPS4 is required for extracellular secretion of the secreted aspartyl proteases Sap2p and Saps4–6p. Furthermore, the vps4D null mutant has been shown to be markedly hypovirulent in a murine tail vein model of disseminated candidiasis. In these experiments, we sought to further define the role of the pre-vacuolar secretion pathway mediated by the pre-vacuolar sorting gene VPS4 in the pathogenesis of epithelial and mucosal infection using a broad range of virulence models. The C. albicans vps4D mutant demonstrates reduced tolerance of cell wall stresses compared to its isogenic, complemented control strain. In an in vitro oral epithelial model (OEM) of tissue invasion, the vps4D mutant caused reduced tissue damage compared to controls. Further, the vps4D mutant was defective in macrophage killing in vitro, and was attenuated in virulence in an in vivo Caenorhabditis elegans model representative of intestinal epithelial infection. In contrast, the vps4D mutant caused a similar degree of tissue damage in an in vitro uroepithelial model of Candida infection compared with controls. Furthermore, in an in vivo murine model of vaginal candidiasis there was no reduction in fungal colony burden and no differences in vaginal histopathology compared to wild-type and complemented controls. These results suggest that VPS4 contributes to several key aspects of oral epithelial but not uroepithelial infection, and in contrast to systemic infection, plays no major role in the pathogenesis of Candida vaginitis. By using a wide range of virulence models, we demonstrate that C. albicans VPS4 contributes to virulence according to the specific tissue that is infected. Thus, in order to gain a full understanding of C. albicans virulence in relation to a particular gene or pathway of interest, a selected range of infection models may need to be utilized.

Introduction C. albicans secretes aspartyl proteases (Saps)1-3 extracellular phospholipases4 and secreted lipases5 in order to assist in pathogenesis. In vivo, C. albicans sap1D, sap2D, and sap3D null mutants are attenuated in virulence, and a triple sap4D, sap5D, and sap6D null mutant is greatly attenuated in virulence in a mouse model of disseminated candidiasis.3,6 In early studies, it was demonstrated that Sap2p secretion in C. albicans requires the rab-like GTPases YPT1 and SEC4, which regulate trafficking in the pre- and post-Golgi general secretory pathway, respectively.7,8 We subsequently demonstrated that the C. albicans prevacuolar secretory pathway gene VPS4 is required for secretion of Sap2p and Saps4–6p in vitro, thus suggesting that these Sap enzymes may be trafficked via a pre-vacuolar branch of exocytosis.9 Also supporting this hypothesis is evidence that Sap2p secretion is reduced in a C. albicans tet-regulated VPS1 mutant,10 since Vps1p is a late-Golgi protein mediating pre-vacuolar

trafficking. Similarly, we have demonstrated that Sap2p secretion is reduced in the pre-vacuolar pep12D mutant, which lacks the t-SNARE proteins required for docking of vesicles to the prevacuolar compartment.11 In addition, the vps4D mutant is defective in phospholipase and lipase secretion. Furthermore, the vps4D mutant also displays differential global secretion of a wide range of extracellularly secreted proteins.12 In vivo, we have previously shown that the C. albicans vps4D null mutant is markedly attenuated in virulence in a standard mouse tail vein of disseminated candidiasis, thus demonstrating a clear contribution of the pre-vacuolar secretory pathway to pathogenesis.13 Work in the model yeast Saccharomyces cerevisiae has demonstrated that exocytic cargo is sorted and transported by at least 2 different routes, namely the general secretory pathway, and a prevacuolar sorting pathway.14-16 C. albicans VPS4 encodes a key AAA-type ATPase that mediates vesicle budding from the prevacuolar compartment for trafficking to the vacuole, or alternatively for exocytosis via a pre-vacuolar secretory pathway.

*Correspondence to: Samuel A Lee; Email: [email protected] Submitted: 04/14/2014; Revised: 06/25/2014; Accepted: 08/17/2014 http://dx.doi.org/10.4161/21505594.2014.956648

810

Virulence

Volume 5 Issue 8

C. albicans vps4D mutants accumulate a distinctive, aberrant prevacuolar compartment and enlarged vacuole, termed a class "E" vacuolar compartment.9 In S. cerevisiae, some cell wall maintenance enzymes are sorted via the general secretory pathway, whereas inducible, environmentally-regulated proteins are sorted via a pre-vacuolar pathway. Analogously, in the pathogenic yeast C. albicans, it appears that environmentally-regulated virulence proteins such as the Saps may also follow a pre-vacuolar route of exocytosis. In order to further define additional contributions of pre-vacuolar secretion to virulence-related attributes, we first investigated the role of VPS4 in tolerance of cell wall and antifungal stressors, and in macrophage killing in an in vitro model. We further characterized the role of VPS4 in virulence by utilizing a Caenorhabditis elegans intestinal model of infection. Next, we sought to determine the specific role of VPS4 in epithelial and mucosal infection by utilizing in vitro models of oral epithelial and uroepithelial infection, and in an in vivo model of murine vaginal candidiasis. Thus, we surveyed the contribution of pre-vacuolar secretion to virulence mediated by C. albicans VPS4, using a wide range of virulence models.

C. albicans must adapt to a wide range of host temperatures, pH, and osmotic stresses. Thus, in order to gain a more thorough understanding of the role of C. albicans VPS4 in stress tolerance, we assayed the ability of the vps4D null mutant to tolerate cell wall stresses and antifungal agents in vitro, which we had not previously studied (Fig. 1). Although there was only a modest reduction in growth on media containing the detergent SDS, there was a substantial reduction in growth on media containing the cell wall perturbing agents Congo Red or Calcofluor White compared to controls. The vps4D null mutant was also much more susceptible than control strains to sub-inhibitory concentrations of the cell wall active agent caspofungin, and to a modest extent the ergosterol synthesis inhibitor, fluconazole.

The vps4D null mutant is defective in macrophage killing and has attenuated virulence in a C. elegans model of infection We then assayed the ability of the vps4D mutant to kill macrophages in an in vitro macrophage killing assay (Fig. 2). Compared to both DAY185 and the VPS4 reintegrant, the vps4D null mutant displayed a significant reduction in its ability to kill macrophages following phagocytosis. The VPS4 reintegrant showed slightly reduced macrophage killing compared to DAY185 (Fig. 2A); however, this difference was not statistically significant. Next, we studied the role of VPS4 in virulence in a C. elegans Results intestinal model of infection (Fig. 3). C. elegans is a widely-studied The vps4D null mutant is hyper-susceptible to cell wall stress soil-dwelling nematode that has been utilized as a model host for a variety of different pathogens,17 including C. albicans.18-20 C. eleand has reduced adherence In order to successfully survive in a wide range of host envi- gans infection by C. albicans has been shown to involve both C. albi20 ronments, including colonization or infection of mucosal sites, cans hyphal formation and a specific C. elegans immune 21 invasion of kidneys and other organs, and infection of blood, response, highlighting the robust utility of this model. In the C. elegans model system, which has been used to study virulence in a broad range of pathogenic yeast and bacterial species, the normal laboratory food source is replaced with the pathogen of interest as the food source.22 Upon ingestion of C. albicans cells, the nematodes develop a persistent intestinal infection cumulating in C. albicans hyphal cells penetrating the worm cuticle. Therefore, we infected C. elegans nematodes with C. albicans DAY185, the vps4D mutant, or the VPS4 reintegrant strain and compared them with nematodes fed non-pathogenic E. coli OP50 as a negative control (Fig. 3A). Median survival of worms infected with DAY185 was 42 h. Virulence of Figure 1. The C. albicans vps4D mutant is more sensitive to cell wall stress and challenge with antifungals. the vps4D mutant against C. elegans C. albicans DAY185, vps4D, and VPS4 reintegrant strains were grown on agar plates containing cell-wall pernematodes was attenuated, with a turbing agents or antifungal drugs. Strains are indicated on the left. Cell densities decrease from left to right median survival of 66 h. The VPS4 (1 £ 107, 2 £ 106, 4 £ 105, and 8 £ 104 cells per mL). Normal growth on YPD medium is shown on the top right. YPD medium containing cell wall perturbing agents including 0.02% SDS, 200 mg/mL Congo Red, and reintegrant had an intermediate 50 mg/mL Calcofluor White, as well as medium containing the antifungal drugs caspofungin (0.1 mg/mL) and phenotype, with a median survival fluconazole (4 mg/mL), are shown. of 45 h. All survival curves were

www.landesbioscience.com

Virulence

811

Effects of the vps4D mutation on tissue damage in an uroepithelial invasion model We next sought to assess the role of VPS4 in the pathogenesis of urinary epithelial invasion. Therefore, we infected an uroepithelial cell line with C. albicans DAY185, vps4D, and the VPS4 reintegrant cells for 24 h, and performed colony counts of adherent C. albicans cells after 2 h and 24 h of contact with uroepithelial cells (Fig. 5A). There was no difference in the adherence of C. albicans DAY185, vps4D, or the VPS4 reintegrant strains after 2 h. After 24 h, there was a modest but statistically significant reduction in adherence by the vps4D strain compared to both DAY185 and the VPS4 reintegrant. Next, we assessed the role of

Figure 2. The C. albicans vps4D mutant is attenuated in macrophage killing. C. albicans cells were co-incubated with macrophage cells in unbuffered DMEMC5%FCS at an MOI of 2. (A) Counts of live macrophage cells from 12 separate fields after 24 h of co-incubation with C. albicans strains. Asterisk (*) denotes statistical significance, P < 0.05, compared to all other treatments. Experiment was performed in quadruplicate; the average of all 4 experiments is shown. (B) Live (green) and dead (red) macrophage cells were co-stained with calcein AM and ethidium bromide homodimer, respectively, and visualized by fluorescence microscopy. Representative images from the 24 h timepoint are shown.

significantly different from one another as determined by the Mantel-Cox test and the log-rank test for trend (GraphPad Prism v. 6.0). Worms scored as dead were further analyzed via light microscopy (Fig. 3B). Nematodes infected with C. albicans vps4D had less hyphae visible protruding from the worm cuticle.

The vps4D null mutant causes less tissue damage in an oral epithelial invasion model We next sought to assess the role of VPS4 in the ability to cause invasion and tissue damage in a commercial reconstituted oral epithelial model (SkinEthic, Lyon, France) (Fig. 4). After 10 h, the vps4D null mutant exhibited a significant reduction in its ability to cause tissue damage, as measured by LDH release, compared to its controls (data not shown). After 24 h, the vps4D null mutant exhibited significantly reduced tissue damage compared to DAY185 or the VPS4 reintegrant strain (Fig. 4).

812

Figure 3. The C. albicans vps4D mutant is attenuated in virulence in a Caenorhabditis elegans model of infection. C. albicans DAY185, vps4D, and the VPS4 reintegrant were tested in a C. elegans model of intestinal epithelial infection. C. elegans N2 nematodes were incubated with C. albicans cells for 4 hours, then monitored twice daily for survival. (A) Survival curve of C. elegans nematodes infected with C. albicans strains of interest. Survival of nematodes infected with C. albicans DAY185, vps4D, or VPS4 reintegrant cells, or fed nonpathogenic E. coli OP50 as a negative control, is shown as percent survival relative to day zero. (B) Light microscopy of nematodes killed by C. albicans DAY185, vps4D, or VPS4 reintegrant. After worms were scored as dead, worms were visualized via light microscopy. Extensive hyphae are seen piercing the cuticle of worms infected with C. albicans DAY185 and VPS4 reintegrant cells. Less extensive hyphae (indicated by black arrows) are visible protruding from the intestine of worms infected with the C. albicans vps4D null mutant.

Virulence

Volume 5 Issue 8

VPS4 in C. albicans vaginitis in an in vivo standard murine model of vaginal candidiasis.23 After establishment of vaginal infection, we assessed vaginal fungal colony burden at prespecified time points (Fig. 6A). At early time points, C. albicans vps4D vaginal fungal burden was reduced compared to DAY185 or the VPS4 reintegrant, although not significantly. However, by day 14, total vaginal fungal burden was similar in all 3 strains. Histopathological examination of fungal invasion showed no major differences in filamentation or tissue damage (Fig. 6B).

Discussion It has been increasingly recognized that there are multiple aspects of pathogenesis required for the different types of

Figure 4. The C. albicans vps4D mutant causes less LDH release by oral epithelial cells in an oral epithelial model of infection. C. albicans DAY185, vps4D, and the VPS4 reintegrant were tested in an oral epithelial model of tissue invasion. C. albicans cells were co-incubated with oral epithelial cells for 24 h and then assayed for lactate dehydrogenase (LDH) release. LDH units were calculated by extrapolation from a standard curve and the number of LDH units released by the untreated positive control was subtracted from the total LDH units released by the infected oral epithelial cells.

VPS4 in uroepithelial cell invasion and tissue damage, as measured by LDH release, after 2 and 24 h of infection (Fig. 5B). After 2 h, the amount of LDH released by uninfected uroepithelial cells was 17.3 § 4.6, and the amount of LDH released by uroepithelial cells infected with C. albicans DAY185, vps4D, or the VPS4 reintegrant was not significantly different from the uninfected control. After 24 h, the amount of LDH released by uninfected uroepithelial cells was 14.46 § 8.8. At this timepoint, uroepithelial cells infected with each C. albicans strain exhibited a statistically significant increase in LDH release as compared to the uninfected control. These results indicate that infection with C. albicans results in significant uroepithelial cell tissue damage after 24 h. However, the vps4D null mutant did not cause a significant difference in tissue damage as measured by LDH release compared to either DAY185 or the VPS4 reintegrant at either timepoint. Effects of the vps4D mutation on virulence in a mouse model of vaginal candidiasis Previously, we had shown that pre-vacuolar secretion mediated by VPS4 is a key contributor to virulence in a disseminated mouse model of infection.13 The current in vitro studies indicated reduced ability to kill macrophages and decreased tissue damage in an oral epithelial model of C. albicans infection. Furthermore, since Sap2p is expressed during mucosal forms of Candida infection, we next sought to directly determine the role of

www.landesbioscience.com

Figure 5. The C. albicans vps4D mutant is less adherent to uro-epithelial cells in a uro-epithelial model of infection. C. albicans DAY185, vps4D, and the VPS4 reintegrant were tested in a monolayer uro-epithelial model. (A) Adherence of C. albicans cells to the uro-epithelial monolayer after 2 h and 24 h of co-incubation was assessed by CFU counts. Asterisk (*) denotes statistical significance, P < 0.05, compared to all other treatments. (B) C. albicans cells were co-incubated with uro-epithelial cells for 2 h and 24 h and then assayed for lactate dehydrogenase (LDH) release.% LDH release was calculated as a percentage as compared to a maximum LDH release control.

Virulence

813

infection by the opportunistic fungal pathogen Candida albicans,24 which has led to the concomitant development and use of a wide range of infection models.25 In prior studies, we have demonstrated that the pre-vacuolar secretory pathway mediated

by the vacuolar protein sorting gene VPS4 is required for secretion of Sap2 and Saps4–6 in vitro,9 and that mutants lacking VPS4 are dramatically reduced in virulence in a mouse tail vein model of disseminated infection.13 Thus, the major goals of this study were to determine if the pre-vacuolar branch of the secretory pathway mediated by VPS4 is required for superficial (i.e., epithelial and mucosal) infection. We therefore sought to further define the role of pre-vacuolar secretion in epithelial and mucosal virulence using a series of in vitro assays, and an in vivo model of Candida vaginitis. More recently, the contribution of Saps to virulence has come into question, as the influence of the URA3 positional effect on virulence may have served as a confounder of these earlier studies. For example, in reconstituted epithelial models26,27 and a systemic mouse model,28 Sap1–6 mutants were of similar virulence to wild-type C. albicans. However, Saps4–6 have recently been shown to induce apoptosis of oral epithelial cells.29 Nonetheless, in addition to deficient Sap2p and Sap4–6p secretion, we had previously demonstrated that the vps4D mutant was deficient in lipase and phospholipase secretion in vitro, despite having an intact general secretory pathway.9,13 Furthermore, we also determined that global secretion of a wide range of extracellularly secreted proteins was altered in the vps4D mutant.12 Thus, we hypothesized that the vps4D mutant would be defective in epithelial virulence, given these demonstrated alterations in secretion of virulence-related and other canonically secreted proteins. The oro-epithelial, but not the uroepithelial, model of infection showed reduced tissue damage caused by the vps4D mutant during experimental in vitro infection. The vps4D mutant also was less able to tolerate cell wall stressors and antifungal agents, particularly the cell wall active agent caspofungin. It should also be noted that the VPS4 re-integrant strain (with a single copy of VPS4) tended to display an intermediate phenotype between the diploid wild-type strain DAY185 and the null mutant;13 this gene dosage effect is not an uncommon finding in C. albicans genetics.11,30 Thus, these findings suggest that pre-vacuolar secretion is required for some, but not all, epithelial forms of infection. Recent elegant work on Candida – epithelial interactions has underlined the complexity of the epithelial response to C. albicans infection.29,31-34 However, immuno-pathologic differences between oral and uroepithelial cells upon infection with C. albicans have yet to be elucidated. Because the vps4D mutant is impaired in trafficking, we hypothesize that C. albicans vps4D mislocalizes a protein (or proteins) required for virulence in oral epithelial, but not uroepithelial, infection. Furthermore, we have Figure 6. The C. albicans vps4D mutant is virulent in a mouse model of vaginal candidiasis. C. albicans DAY185, vps4D, and the VPS4 reintegrant were tested for virulence in a mouse model of vaginal candidiasis. Mice were infected with 5 £ 105 C. albicans cells or with a PBS-only control. (A) Fungal burden after 3, 7 and 10 days of C. albicans infection. Fungal burden is indicated by CFUs per mL. (B) Vaginal histopathology comparing tissue invasion and damage caused by (a) wild-type SC5314, (b) wildtype DAY185, (c) vps4D and (d) the VPS4 reintegrant strain. Paraffin sections of vaginas from randomly selected mice were prepared and stained with hematoxylin.

814

Virulence

Volume 5 Issue 8

previously identified global alterations in extracellularly secreted proteins in the vps4D null mutant; most notably, the extracellular proteins Cht3p, Pra1p, Mp65p and Sun41p are reduced.12 Given the differences in environmental pH (pH 7.4 of the RPMI-1640 used in the oral epithelial model, and pH 5.8 of the artificial urine used in the uroepithelial model), it is possible that the ability of the vps4D mutant to cause infection is affected by environmental pH. Specifically, Pra1p is a pH-sensitive zinc scavenger protein; a pra1D null mutant causes reduced damage in a HUVEC endothelial monolayer infection model at alkaline pH.35 We conjecture that at the higher pH of the oral epithelial model used in these studies, diminished expression of Pra1p may reduce virulence further in the vps4D mutant compared to its controls. In our animal model of Candida vaginitis, we did not detect any differences in tissue damage. We did observe a modest decrease in fungal burden at 3 and 7 days post-infection; however, by day 14, fungal burden in mice infected with C. albicans vps4D was statistically indistinguishable from mice infected with wild-type C. albicans. Given the clear in vitro findings regarding reduced cell wall integrity and decreased tissue damage in the oral epithelial model of infection, as well as decreased macrophage killing and attenuated virulence in the C. elegans intestinal epithelial model of infection, the minimal contribution of VPS4 to Candida vaginitis infection in this model was unexpected. It is quite possible that more dramatic in vivo findings could be seen with an oral model of Candida infection. Nonetheless, there are clear examples of genes for which null mutations lead to attenuated virulence in systemic infection but not in vaginitis. Examples of these genes include the filamentation-related transcription factors EFG1, CPH1, and NRG1.36 These results indicate that the contribution of VPS4 to pathogenesis is a complex phenomenon, where abnormal vacuolar structure and function differentially impact virulence according to the specific tissue that is infected. Importantly, in order to fully understand the contribution of a particular gene or pathway of interest to virulence, use of a wide range of pathogenesis models is required.

Methods Strains and media C. albicans strains SC5314 (from W. Fonzi, Georgetown University), DAY185 (from A. Mitchell, Carnegie Mellon University), vps4D null mutant, and vps4DCVPS4 reintegrant12 were grown at 30 C in YPD (1% yeast extract, 2% peptone, 2% glucose), or in minimal glucose (0.67% yeast nitrogen base without amino acids [YNB], 2% glucose). Solid media were prepared by adding 2% agar. Cell wall stress was assayed on complete synthetic media (CSM) (0.67% YNB, 0.079% complete synthetic mixture, 2% glucose, 2% agar) containing either 0.02% SDS, 200 mg/mL Congo red (Sigma-Aldrich, #860956), or 50 mg/mL calcofluor white (Sigma-Aldrich, #18909). The ability of strains to respond to challenge with antifungals was tested on YPD with 0.1 mg/mL caspofungin (Merck) or 4 mg/mL fluconazole (Sigma-Aldrich, #F8929) added. For cell wall stress and

www.landesbioscience.com

antifungal tolerance assays, serial dilutions of overnight cultures (1 £ 107, 2 £ 106, 4 £ 105, and 8 £ 104 cells per mL) were spotted onto agar media and incubated 48 h at 30 C. Statistical analyses For all experiments, results were analyzed for statistical differences using one-way ANOVA, followed by the Tukey’s multiple comparison test (GraphPad Prism 5.01). A result was considered significant when P < 0.05 compared to all other treatments. Macrophage killing assay The macrophage killing assay was performed as previously described.37 Briefly, the murine macrophage cell line J774A.1 (ATCC) was grown for 18 h in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) C5% fetal calf serum (FCS) at 37 C, 10% CO2. Next, macrophages cells were co-incubated with the C. albicans control strain DAY185, the vps4D null, and the isogenic vps4DCVPS4 reintegrant at a multiplicity of infection (MOI) of 2 for 24 h in high-glucose DMEM C 5% FCS at 37 C, 10% CO2. Macrophage killing was then assessed using the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, L-3224) which fluorescently stains live and dead macrophage cells green and red, respectively. Cells were visualized via fluorescence microscopy. Live macrophage cells were counted for 5 to 6 fields per treatment. The experiment was completed 4 times independently, and the average of all 4 experiments is presented. C. elegans model of infection The C. elegans infection model was adapted from a previously described assay,21 with one key modification. C. elegans nematodes reproduce rapidly, producing progeny that can interfere with the infection assay and that can cause matricidal killing of worms that is unrelated to infection.18 Rather than genetically or chemically inhibiting reproduction as has been done previously,18-21 we utilized a temperature-dependent inhibition strategy where wild-type C. elegans nematodes were maintained at 30 C rather than 25 C post-infection, as 30 C is a temperature at which worm reproduction but not viability is inhibited.38 Uninfected nematodes were maintained on standard worm growth medium (Nematode growth media [NGM] C Escherichia coli OP50)39 at 30 C as a negative control to ensure that the higher temperature did not affect worm survival. C. elegans N2 nematodes were obtained from the Caenorhabditis Genetics Center. Standard nematode maintenance was performed at 15 C on NGM C E. coli plates as described previously.39 To prepare C. albicans infection plates, 50 mL cells from overnight cultures C. albicans strains DAY185, vps4D null mutant, and vps4DCVPS4 reintegrant was spread onto YPD plates in a square lawn, then incubated at 30 C for 18 h. C. elegans nematodes were synchronized to the same life-stage via bleaching as described previously.39 Then, once nematodes reached the L4 life-stage, worms were washed from NGM plates using 7 mL M9 buffer (3 g/L KH2PO4, 6 g/L Na2HPO4, 5 g/L NaCl, 1 mM MgSO4)39 and

Virulence

815

collected via centrifugation at 6000 rpm for 1 minute. Collected nematodes were added to the center of C. albicans lawns on YPD plates and incubated at 30 C for 4 hours to infect nematodes. After 4 hours, worms were washed from infection plates with 7 mL M9 buffer, taking care to avoid the C. albicans lawn. Then, worms in M9 were collected via centrifugation and added to the center of unseeded NGM plates to allow the M9 buffer to dry. Then, 80–100 nematodes per treatment were transferred to unseeded NGM plates via picking with a platinum-wire pick. As a negative control, collected nematodes were added to the center of an unseeded YPD plate and incubated at 30 C for 4 hours. After 4 hours, 100–150 uninfected nematodes were transferred via picking with a platinum-wire pick to NGM C E. coli OP50 plates. Plates were incubated at 30 C. Nematode survival was monitored at selected intervals up to 120 h post-infection; worms were scored as dead if they did not respond to stimulation with a platinum wire pick. Nematodes that crawled off the plate were excluded from analysis. The experiment was conducted twice independently. Survival curves and statistical analyses were completed using GraphPad Prism v. 6.0. Oral epithelial model of infection The capacity for oral epithelial tissue invasion of the C. albicans strains was assessed in a reconstituted human epithelium (RHE) oral epithelial model system (OEMS) of tissue invasion (SkinEthic Laboratories) as previously described.40 Briefly, oral epithelial tissue was added to a 6-well plate containing 1 mL of SkinEthic Maintenance Medium and grown at 37 C, 5% CO2 for 24 h. C. albicans cells were synchronized cells to the same growth phase, as described previously.40 Then, 2 £ 106 phasesynchronized C. albicans cells were co-incubated with epithelial cells for 10 h–24 h at 37 C with 5% CO2. 50 mL PBS was added to epithelial cells to serve as an untreated control. At selected time points, culture medium was transferred to 1.5 mL microcentrifuge tubes and centrifuged at 10,000 £ g for 5 min at 4 C. The supernatant was transferred to a fresh 1.5 mL microcentrifuge tube. Then, lactate dehydrogenase (LDH) release was assayed using the CytoTox 96Ò Non-Radioactive Cytotoxicity Assay (Promega, G1780) according to manufacturer’s instructions. The LDH positive control (CytoTox 96Ò , Promega) was used to generate a standard curve using known concentrations of LDH; LDH units of experimental samples were calculated by extrapolation from the standard curve. The number of LDH units released by the untreated control was subtracted from the total LDH units released by the oral epithelial cells in each of the experimental samples. Uroepithelial model of infection To study the contribution of VPS4 to uroepithelial infection, we utilized a Candida-uroepithelial infection model that has been previously described.41,42 Cells from overnight cultures of C. albicans DAY185, vps4D null mutant, and vps4DCVPS4 reintegrant strains were harvested by centrifugation for 5 min and washed twice with PBS (0.1 mol l¡1, pH 7). A human urinary epithelial cell line (TCC-SUP) was cultured at 37 C, 5% CO2 in DMEM C 10% FCS for 24 h. A suspension of 1 £ 107 yeast

816

cells/mL in DMEMC10%FCS was prepared and 500 ml of that suspension was added to the confluent layer of urinary epithelial cells. After 2 h and 24 h of contact between the urinary epithelial cells and yeast cells at 37 C, 5% CO2, the culture medium was removed and cells were washed with PBS to remove non-adherent cells. 100 ml of Trypsin was added to each well for 15 min at 37 C. After incubation, 900 ml PBS C 10% FCS were added and the adherent cells at the bottom of the plate were scraped. After serial dilutions, 25 ml of the resultant suspension were plated on SDA plates and incubated at 37 C. CFUs were counted after 24 h. Next, we measured LDH release by uroepithelial cells after 2 h and 24 h of infection with C. albicans. TCC-SUP cells were cultured at 37 C, 5% CO2 in DMEMC10% FCS for 24 h. Cells from overnight cultures of C. albicans SC5314, vps4D null mutant, and vps4DCVPS4 reintegrant strains were harvested by centrifugation for 5 min and washed twice with PBS (0.1 mol l¡1, pH 7). A suspension of 1 £ 107 yeast cells/ mL in DMEM C 10% FCS was prepared and 500 ml of that suspension was added to the confluent layer of urinary epithelial cells. After 2 h and 24 h, the release of LDH in the medium was measured using the CytoTox-ONETM kit according to the manufacturer’s instructions. A positive control using a lysis solution to ensure a maximum LDH release was performed. The effect of C. albicans SC5314, vps4D, or vps4DCVPS4 on epithelial cells was expressed as the percentage of LDH released compared to the maximum LDH release control. The experiment was repeated in triplicate in 3 independent assays. Murine model of vaginal candidiasis Female BALB/c (H-2d) mice, 4 to 6 weeks of age (National Cancer Institute/Charles River Laboratories, Wilmington, MA), were used in these experiments. Mice were housed at The University of Texas at San Antonio Small Animal Laboratory vivarium and handled according to guidelines approved by the institutional animal care and use committee. The virulence of the vps4D null mutant and controls was assessed utilizing a standard murine model of vaginal candidiasis.23 In this model, 72 h prior to vaginal initiation of candidal infection, mice were treated subcutaneously with 0.5 mg of estradiol valerate and weekly thereafter, since vaginitis in rodents is inducible only under conditions of pseudoestrus. Next, C. albicans blastoconidia (5 £ 105) from the wild-type, vps4D null, and vps4D C VPS4 reintegrant strains obtained from a fresh stationary-phase culture in 20 mL of PBS were inoculated into the vaginas of estrogen-treated mice. Control animals were inoculated with 20 mL of PBS only. At weekly intervals, mice were sacrificed, followed immediately by vaginal lavage with 100 mL of PBS and gentle scraping of vaginal tissue. Fungal burden was compared by performing colony counts on serial 1:10 dilutions of the lavage fluid, followed by plating on Sabouraud dextrose agar plates and incubating for 48–72 h at 30 C. Next, vaginal histopathology was assessed in order to compare the degree of tissue invasion and damage. Paraffin sections of vaginas from randomly selected mice were prepared and stained with hematoxylin for histological examination.

Virulence

Volume 5 Issue 8

which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

We thank William Fonzi (Georgetown University) for providing strain SC5314, and Aaron Mitchell (Columbia University) for providing strain DAY185. The C. elegans N2 strain was obtained from the Caenorhabditis Genetics Center (CGC), References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

Hoegl L, Ollert M, Korting HC. The role of Candida albicans secreted aspartic proteinase in the development of candidoses. J Mol Med Berl Ger 1996; 74:135-42; http://dx.doi.org/10.1007/BF01575445 Hube B, Sanglard D, Odds FC, Hess D, Monod M, Sch€afer W, Brown AJ, Gow NA. Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence. Infect Immun 1997; 65:3529-38; PMID:9284116 Sanglard D, Hube B, Monod M, Odds FC, Gow NA. A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infect Immun 1997; 65:3539-46; PMID:9284117 Ghannoum MA. Potential role of phospholipases in virulence and fungal pathogenesis. Clin Microbiol Rev 2000; 13:122-43; PMID:10627494; http://dx. doi.org/10.1128/CMR.13.1.122-143.2000 Hube B, Stehr F, Bossenz M, Mazur A, Kretschmar M, Sch€afer W. Secreted lipases of Candida albicans: cloning, characterisation and expression analysis of a new gene family with at least ten members. Arch Microbiol 2000; 174:362-74; PMID:11131027; http://dx.doi.org/10.1007/s002030000218 Hube B, Monod M, Schofield DA, Brown AJ, Gow NA. Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol 1994; 14:87-99; PMID:7830564; http://dx.doi.org/10.1111/j.13652958.1994.tb01269.x Lee SA, Mao Y, Zhang Z, Wong B. Overexpression of a dominant-negative allele of YPT1 inhibits growth and aspartyl protease secretion in Candida albicans. Microbiol Read Engl 2001; 147:1961-70 Mao Y, Kalb VF, Wong B. Overexpression of a dominant-negative allele of SEC4 inhibits growth and protein secretion in Candida albicans. J Bacteriol 1999; 181:7235-42; PMID:10572126 Lee SA, Jones J, Khalique Z, Kot J, Alba M, Bernardo S, Seghal A, Wong B. A functional analysis of the Candida albicans homolog of Saccharomyces cerevisiae VPS4. FEMS Yeast Res 2007; 7:973-85; PMID:17506830; http://dx.doi.org/10.1111/j.15671364.2007.00253.x Bernardo SM, Khalique Z, Kot J, Jones JK, Lee SA. Candida albicans VPS1 contributes to protease secretion, filamentation, and biofilm formation. Fungal Genet Biol 2008; 45:861-77; PMID:18296085; http://dx.doi.org/10.1016/j.fgb.2008.01.001 Palanisamy SKA, Ramirez MA, Lorenz M, Lee SA. Candida albicans PEP12 is required for biofilm integrity and in vivo virulence. Eukaryot Cell 2010; 9:26677; PMID:20023068; http://dx.doi.org/10.1128/ EC.00295-09 Thomas DP, Lopez-Ribot JL, Lee SA. A proteomic analysis of secretory proteins of a pre-vacuolar mutant of Candida albicans. J Proteomics 2009; 73:342-51; PMID:19819358; http://dx.doi.org/ 10.1016/j.jprot.2009.10.003 Lee SA, Jones J, Hardison S, Kot J, Khalique Z, Bernardo SM, Lazzell A, Monteagudo C, Lopez-Ribot J. Candida albicans VPS4 is required for secretion of

www.landesbioscience.com

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Funding

This work was supported by grants from the Department of Veterans Affairs (Merit Award 5I01BX001130 to SAL) and the Biomedical Research Institute of New Mexico (to SAL).

aspartyl proteases and in vivo virulence. Mycopathologia 2009; 167:55-63; PMID:18814053; http://dx.doi. org/10.1007/s11046-008-9155-7 Gurunathan S, David D, Gerst JE. Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast. EMBO J 2002; 21:60214; PMID:11847108; http://dx.doi.org/10.1093/ emboj/21.4.602 Harsay E, Schekman R. A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway. J Cell Biol 2002; 156:271-85; PMID:11807092; http://dx.doi.org/10.1083/ jcb.200109077 Harsay E, Bretscher A. Parallel secretory pathways to the cell surface in yeast. J Cell Biol 1995; 131:297310; PMID:7593160; http://dx.doi.org/10.1083/ jcb.131.2.297 Mylonakis E, Ausubel FM, Tang RJ, Calderwood SB. The art of serendipity: killing of Caenorhabditis elegans by human pathogens as a model of bacterial and fungal pathogenesis. Expert Rev Anti Infect Ther 2003; 1:167-73; PMID:15482109; http://dx.doi. org/10.1586/14787210.1.1.167 Breger J, Fuchs BB, Aperis G, Moy TI, Ausubel FM, Mylonakis E. Antifungal chemical compounds identified using a C. elegans pathogenicity assay. PLoS Pathog 2007; 3:e18; PMID:17274686; http://dx.doi. org/10.1371/journal.ppat.0030018 Okoli I, Coleman JJ, Tampakakis E, An WF, Holson E, Wagner F, Conery AL, Larkins-Ford J, Wu G, Stern A, et al. Identification of antifungal compounds active against Candida albicans using an improved high-throughput Caenorhabditis elegans assay. PloS One 2009; 4:e7025; PMID:19750012; http://dx.doi.org/10.1371/ journal.pone.0007025 Pukkila-Worley R, Peleg AY, Tampakakis E, Mylonakis E. Candida albicans hyphal formation and virulence assessed using a Caenorhabditis elegans infection model. Eukaryot Cell 2009; 8:1750-8; PMID:19666778; http://dx.doi.org/10.1128/ EC.00163-09 Pukkila-Worley R, Ausubel FM, Mylonakis E. Candida albicans infection of Caenorhabditis elegans induces antifungal immune defenses. PLoS Pathog 2011; 7:e1002074; PMID:21731485; http://dx.doi. org/10.1371/journal.ppat.1002074 Sifri CD, Begun J, Ausubel FM. The worm has turned–microbial virulence modeled in Caenorhabditis elegans. Trends Microbiol 2005; 13:119-27; PMID:15737730; http://dx.doi.org/ 10.1016/j.tim.2005.01.003 Fidel PL Jr, Lynch ME, Sobel JD. Candida-specific cell-mediated immunity is demonstrable in mice with experimental vaginal candidiasis. Infect Immun 1993; 61:1990-5; PMID:8097493 Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms. Virulence 2013; 4:11928; PMID:23302789; http://dx.doi.org/10.4161/ viru.22913 Maccallum DM. Hosting infection: experimental models to assay Candida virulence. Int J Microbiol 2012; 2012:363764; PMID:22235206; http://dx. doi.org/10.1155/2012/363764

Virulence

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Lermann U, Morschh€auser J. Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans. Microbiology 2008; 154:3281-95; PMID:18957582; http://dx.doi. org/10.1099/mic.0.2008/022525-0 Naglik JR, Moyes D, Makwana J, Kanzaria P, Tsichlaki E, Weindl G, Tappuni AR, Rodgers CA, Woodman AJ, Challacombe SJ, et al. Quantitative expression of the Candida albicans secreted aspartyl proteinase gene family in human oral and vaginal candidiasis. Microbiol Read Engl 2008; 154:3266-80; http://dx.doi.org/10.1099/ mic.0.2008/022293-0 Correia A, Lermann U, Teixeira L, Cerca F, Botelho S, da Costa RMG, Sampaio P, G€artner F, Morschh€auser J, Vilanova M, et al. Limited role of secreted aspartyl proteinases Sap1 to Sap6 in Candida albicans virulence and host immune response in murine hematogenously disseminated candidiasis. Infect Immun 2010; 78:4839-49; PMID:20679440; http://dx.doi.org/10.1128/IAI.00248-10 Wu H, Downs D, Ghosh K, Ghosh AK, Staib P, Monod M, Tang J. Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism. FASEB J Off Publ Fed Am Soc Exp Biol 2013; 27:2132-44; PMID:23430844; http://dx.doi.org/10.1096/fj.12214353 Uhl MA, Biery M, Craig N, Johnson AD. Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the diploid human fungal pathogen C. albicans. EMBO J 2003; 22:2668-78; PMID:12773383; http://dx.doi.org/10.1093/emboj/ cdg256 Moyes DL, Shen C, Murciano C, Runglall M, Richardson JP, Arno M, Aldecoa-Otalora E, Naglik JR. Protection against epithelial damage during Candida albicans infection is mediated by PI3K/Akt and mammalian target of rapamycin signaling. J Infect Dis 2014; 209:1816-26; PMID:24357630; http:// dx.doi.org/10.1093/infdis/jit824 W€achtler B, Citiulo F, Jablonowski N, F€orster S, Dalle F, Schaller M, Wilson D, Hube B. Candida albicansepithelial interactions: dissecting the roles of active penetration, induced endocytosis and host factors on the infection process. PloS One 2012; 7:e36952; PMID:22606314; http://dx.doi.org/10.1371/journal. pone.0036952 Wagener J, Weindl G, de Groot PWJ, de Boer AD, Kaesler S, Thavaraj S, Bader O, Mail€ander-Sanchez D, Borelli C, Weig M, et al. Glycosylation of Candida albicans cell wall proteins is critical for induction of innate immune responses and apoptosis of epithelial cells. PloS One 2012; 7:e50518; PMID:23226301; http://dx.doi.org/10.1371/ journal.pone.0050518 Yano J, Kolls JK, Happel KI, Wormley F, Wozniak KL, Fidel PL Jr. The acute neutrophil response mediated by S100 alarmins during vaginal Candida infections is independent of the Th17-pathway. PloS One 2012; 7:e46311; PMID:23050010; http://dx.doi. org/10.1371/journal.pone.0046311 Citiulo F, Jacobsen ID, Miram
on P, Schild L, Brunke S, Zipfel P, Brock M, Hube B, Wilson D.

817

36.

37.

818

Candida albicans scavenges host zinc via Pra1 during endothelial invasion. PLoS Pathog 2012; 8: e1002777; PMID:22761575; http://dx.doi.org/ 10.1371/journal.ppat.1002777 Peters BM, Palmer GE, Nash AK, Lilly EA, Fidel PL, Noverr MC. Fungal morphogenetic pathways are required for the hallmark inflammatory response during Candida albicans vaginitis. Infect Immun 2014; 82:532-43; PMID:24478069; http://dx.doi.org/ 10.1128/IAI.01417-13 Palmer GE, Kelly MN, Sturtevant JE. The Candida albicans vacuole is required for differentiation and efficient macrophage killing. Eukaryot Cell 2005;

38.

39.

40.

4:1677-86; PMID:16215175; http://dx.doi.org/ 10.1128/EC.4.10.1677-1686.2005 McMullen PD, Aprison EZ, Winter PB, Amaral LAN, Morimoto RI, Ruvinsky I. Macro-level modeling of the response of C. elegans reproduction to chronic heat stress. PLoS Comput Biol 2012; 8:e1002338; PMID: 22291584; http://dx.doi.org/10.1371/journal.pcbi.1002338 Stiernagle T. Maintenance of C. elegans. In WormBook (ed.), The C. elegans Research Community. 2006. http://dx.doi.org/10.1895/wormbook.1.101.1 Schaller M, Zakikhany K, Naglik JR, Weindl G, Hube B. Models of oral and vaginal candidiasis based on in vitro reconstituted human epithelia. Nat Protoc

Virulence

41.

42.

2006; 1:2767-73; PMID:17406503; http://dx.doi. org/10.1038/nprot.2006.474 Negri M, Silva S, Breda D, Henriques M, Azeredo J, Oliveira R. Candida tropicalis biofilms: effect on urinary epithelial cells. Microb Pathog 2012; 53:95-9; PMID:22627049; http://dx.doi.org/10.1016/j. micpath.2012.05.006 Negri M, Botelho C, Silva S, Lopes LMRH, Henriques M, Azeredo J, Oliveira R. An in vitro evaluation of Candida tropicalis infectivity using human cell monolayers. J Med Microbiol 2011; 60:1270-5; PMID:21566089; http://dx.doi.org/10.1099/ jmm.0.031195-0

Volume 5 Issue 8

Copyright of Virulence is the property of Landes Bioscience and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.