# Probiotic Interference ofLactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 with the Opportunistic Fungal Pathogen Candida albicans

Authors: Gerwald A. Köhler; Senait Assefa; Gregor Reid
Journal: Infectious Diseases in Obstetrics and Gynecology
 (2012)

---

## Abstract

Candida albicans is the most important Candida species causing vulvovaginal candidiasis (VVC). VVC has significant medical and economical impact on women’s health and wellbeing. While current antifungal treatment is reasonably effective, supportive and preventive measures such as application of probiotics are required to reduce the incidence of VVC. We investigated the potential of the probiotics Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 towards control of C. albicans. In vitro experiments demonstrated that lactic acid at low pH plays a major role in suppressing fungal growth. Viability staining following cocultures with lactobacilli revealed that C. albicans cells lost metabolic activity and eventually were killed. Transcriptome analyses showed increased expression of stress-related genes and lower expression of genes involved in fluconazole resistance, which might explain the increased eradication of Candida in a previous clinical study on conjoint probiotic therapy. Our results provide insights on the impact of probiotics on C. albicans survival.


---

## Body

## 1. Introduction

Mucosal infections byCandida albicans and related non-albicans Candidaspecies are some of the most frequently encountered infections in modern medicine. Oral thrush and vulvovaginal candidiasis (VVC) amass the largest numbers of incidences among mucocutaneous Candida infections. While most forms of candidiasis affect immunocompromised individuals, VVC is commonly encountered by millions of otherwise healthy women. Estimates on the occurrence of VVC point to at least one episode of VVC in 75% of all women [1–3]. Clinical signs of vaginitis appear when the balance between the host and fungal colonization is disrupted, for example, by antibiotic suppression of beneficial components of the bacterial vaginal microbiota. Of note, unlike bacterial vaginosis (BV), the condition is not associated with a depletion of lactobacilli per se. Recurrent VVC, defined as at least four episodes in one year, affects a subpopulation, the treatment of whom requires long-term antifungal therapy [4]. After BV, VVC is the second most common cause of vaginitis, affecting about 40% of women with vaginal complaints [5]. In the USA, diagnosis and treatment of VVC and the concomitant loss of productivity was last estimated in 1995 to cost US$ 1.8 billion [6].While the treatment of VVC is relatively effective, it has been suggested that patients could benefit from restoration of the vaginal microbiota and/or modulation of the local mucosal immune response via supplementation with probiotics. Furthermore, some non-albicansCandidaspecies are inherently more resistant to antifungal agents and therefore can pose a greater challenge to treatment. Establishment of a healthy vaginal microbiota using beneficial Lactobacillus strains might be a supportive and preventive measure against VVC. A recent randomized, double-blind and placebo-controlled trial by Martinez and coworkers has shown that Lactobacillus rhamnosusGR-1 and L. reuteriRC-14 increased the efficacy of antifungal agents in women diagnosed with VVC [7]. In a related in vitrostudy it was also noted that these probiotics had the potential to inhibit C. albicans, the most common cause of VVC [8].In the present study, the objective was to learn more about the molecular mechanisms ofC. albicans growth inhibition by the probiotic strains using microbiological and genome-wide expression analyses.


## 2. Material and Methods

### 2.1. Strains and Culture Conditions

TheCandida albicans and bacterial strains used in this study are listed in Table 1. Stock cultures of all strains were stored at −80°C with 15% v/v glycerol as cryopreservative. C. albicans was routinely grown in YPD media (10 g/L yeast extract, 20 g/L tryptone, and 20 g/L dextrose). For coculture assays, C. albicanswas grown in the same media as lactobacilli and staphylococci, that is, Difco Lactobacilli MRS broth (BD Diagnostics, Franklin Lakes, NJ, USA). Regular agar plates contained 15 g/L Agar (Fisher BioReagents, Pittsburgh, PA, USA) while 7 g/L of agar were used in soft agar overlays. Standard incubation conditions for pure yeast cultures were 30°C and ambient aeration. Bacteria were grown at 37°C under a 5% CO2 atmosphere in a CO2 incubator. Bacteria-Candidacocultures were incubated as indicated in a CO2 incubator or in ambient air at 37°C growth temperature.Table 1
Bacterial andCandida  albicans strains used in this study.
StrainOriginReferenceCandida albicansSC5314Wild-type isolate[9]Lactobacillus johnsoniiPV016Intestinal isolateThis studyLactobacillus rhamnosusGR-1Vaginal isolate[10, 11]Lactobacillus reuteriRC-14Vaginal isolate[10, 11]Staphylococcus aureusATCC 25923Clinical isolatehttp://www.atcc.org/


### 2.2. Growth Inhibition Studies

The effects of the presence ofLactobacillus strains on the growth of C. albicans were determined in vitro on agar plates and in liquid cultures. The intestinal Lactobacillus johnsoniistrain PV016 and the non-lactic acid bacterium Staphylococcus aureus ATCC 25923 were used as controls. Plate assays with deferred inoculation of C. albicans were performed on MRS or MRS-MOPS (MRS medium buffered with 0.165 M 3-morpholinopropane-1-sulfonic acid to pH 7.0) media. For the plate overlay assays, overnight cultures of the bacteria were diluted to an OD600nm of 1 and 4 μL of the dilution were spotted on MRS or MRS-MOPS agar plates. After incubation for 2 days at 37°C in a CO2 incubator, plates were overlayed with a C. albicans suspension (OD600nm=0.01) in MRS soft agar (0.7% agar) that was prepared after cooling the autoclaved agar medium to 45°C. Following solidification of the soft agar overlay, plates were incubated for one day at 37°C in normal aerobic atmosphere. Overlay plates were inspected for clear zones with no C. albicans growth surrounding bacterial colonies capable of inhibiting growth of the fungi. Disk diffusion assays were used to detect inhibition of C. albicansgrowth by lactic acid. Briefly, 20 μL of lactic acid dilutions were pipetted onto antibiotic assay disks (diameter 6 mm, Whatman, GE Health Care, Piscataway, NJ, USA) and the disks were placed on MRS or MRS-MOPS agar plates inoculated for dense growth of C. albicans. Clear zones around the disks after 24 hr incubation at 37°C indicated fungal growth inhibition by lactic acid.


### 2.3.Candida albicans Viability Assay

Viability ofC. albicans cells during coculture with lactobacilli was qualitatively assessed using the FUN 1 viability indicator (LIVE/DEAD Yeast Viability Kit; Invitrogen, Carlsbad, CA, USA) and fluorescence microscopy. After a 24 hr incubation of C. albicans cells in MRS at 37°C in presence or absence (control) of lactobacilli, cells were washed with GH buffer (10 mM Na-HEPES buffer pH 7.2 containing 2% glucose). FUN 1 cell stain was diluted in the same buffer to a concentration of 5 μM and the solution was added to the cells. Following incubation for 30 min in the dark, a Biorad MRC-1024 confocal laser scanning microscope was used to determine the metabolic activity of the yeast cells. Excitation and emission filter sets for green and red fluorescence of FUN 1 were used according to the manufacturer’s directions. Intact metabolically active C. albicans cells converted the green-fluorescent intracellular FUN 1 dye to red fluorescent intravacuolar structures. Cells with little or no metabolic activity but still intact membranes just show diffuse cytoplasmic green fluorescence and no red intravacuolar bodies. In contrast, diffuse and extremely bright green-yellow fluorescence indicates dead cells. For semiquantitative assessment of fungal viability, the FUN 1 viability assay was adapted for fluorometric measurement of metabolic activity according to the manufacturer’s recommendations. Following overnight incubation of C. albicans cells (starting OD600nm 0.1) in MRS with bacteria (starting OD600nm 0.05) or in the presence of lactic acid, the viability of C. albicans cells was determined by kinetic readings of red (excitation filter 485/20, emission filter 590/35) and green (excitation filter 485/20, emission filter 528/20) fluorescence in a BioTek Synergy 2 multimode microplate reader (BioTek, Winooski, VT, USA). For this purpose, aliquots from cultured cells were washed in GH buffer, adjusted to OD600nm 0.5 in 200 μL GH + 5 μM FUN 1 and subsequently incubated for 1 hr in the microplate reader at 30°C. Red and green fluorescence readings were taken every 5 minutes and red/green ratios were determined to monitor the viability of fungal cells. An increase in the red/green ratio over time indicated the presence of the metabolic activity required for formation of the red fluorescent intravacuolar structures. Bacteria did not convert the dye (data not shown).


### 2.4. Transcriptional Profiling

For genome-wide transcriptional analysis ofC. albicans challenged by the presence of lactobacilli, cocultures of both microorganisms were established in 6-well plates with ThinCert (Greiner Bio-One, Monroe, NC, USA) inserts that kept both cell populations separated by a PET membrane with a pore-size of 0.45 μm. Fungal cells were inoculated in the lower compartment and pregrown for 6 hrs at 37°C in 5% CO2 atmosphere without shaking. Lactobacilli were then inoculated in the ThinCert compartment and the cocultures were incubated up to 24 hrs total incubation time (i.e., 18 hrs in coculture). Parallel C. albicans cultures without addition of lactobacilli served as controls. At various time points, the pH of the medium was measured and the cell density of both bacterial and fungal cells was determined by spectrophotometry at 600 nm. C. albicans cells were harvested and total RNA was isolated using a FastPrep FP120A instrument in conjunction with the FastRNA Pro Red kit (MP Biomedicals, Solon, OH, USA). Isolated total RNAs underwent further purification with the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA). Integrity and concentration of the total RNAs were determined by agarose gel electrophoresis and spectrophotometry, respectively. For preparation of Affymetrix GeneChip (Affymetrix, Santa Clara, CA, USA) hybridization targets, 50 ng of the highly-purified total RNAs were used for cDNA generation, Ribo-SPIA amplification, and biotin-labeling using the NuGEN Ovation Biotin system (NuGEN Technologies Inc., San Carlos, CA, USA). The resulting biotin-labeled single-stranded cDNAs were used in C. albicans GeneChip hybridizations according to the manufacturers’ directions. The custom-made C. albicans GeneChips were described previously [12]. Following hybridization in a GeneChip Hybridization Oven 640 and a GeneChip Fluidics Station 400 (Affymetrix), the microarrays were scanned using a GeneArray Scanner (Hewlett-Packard). The resulting image files were processed for absolute and comparative expression analysis using MICROARRAY SUITE 5.0 (MAS 5.0; Affymetrix). Global scaling to a target intensity of 500 was used to correct for variations between arrays. Experimental data were stored in MICRODB 3.0 and analyzed in DATA MINING TOOL 3.0 (Affymetrix) as well as MICROSOFT ACCESS. Default parameters were used for the statistical algorithms implemented in MAS 5.0 to calculate probe set signals, signal log ratios (SLR) as well as detection and change P values. For determination of differential gene expression between Lactobacillus-Candida coculture experiments and Candida-only controls, we also employed the SNOMAD software for standardization and normalization of microarray data (http://pevsnerlab.kennedykrieger.org/snomadinput.html [13]). Here, signals (intensities) of experiment-control sets are log-transformed and the log ratios are normalized by calculation of a local mean across the element signal intensities (LOESS fit). Furthermore, a local variance correction is applied and a Z score is calculated for each probe set as a measure for differential expression. Transcripts with Z scores ≥3 were considered significantly increased and transcripts with Z scores ≤−3 were considered significantly decreased in comparative analyses. In order to group genes with similar regulation, cluster analyses were performed using the programs Cluster and TreeView [14].


## 2.1. Strains and Culture Conditions

TheCandida albicans and bacterial strains used in this study are listed in Table 1. Stock cultures of all strains were stored at −80°C with 15% v/v glycerol as cryopreservative. C. albicans was routinely grown in YPD media (10 g/L yeast extract, 20 g/L tryptone, and 20 g/L dextrose). For coculture assays, C. albicanswas grown in the same media as lactobacilli and staphylococci, that is, Difco Lactobacilli MRS broth (BD Diagnostics, Franklin Lakes, NJ, USA). Regular agar plates contained 15 g/L Agar (Fisher BioReagents, Pittsburgh, PA, USA) while 7 g/L of agar were used in soft agar overlays. Standard incubation conditions for pure yeast cultures were 30°C and ambient aeration. Bacteria were grown at 37°C under a 5% CO2 atmosphere in a CO2 incubator. Bacteria-Candidacocultures were incubated as indicated in a CO2 incubator or in ambient air at 37°C growth temperature.Table 1
Bacterial andCandida  albicans strains used in this study.
StrainOriginReferenceCandida albicansSC5314Wild-type isolate[9]Lactobacillus johnsoniiPV016Intestinal isolateThis studyLactobacillus rhamnosusGR-1Vaginal isolate[10, 11]Lactobacillus reuteriRC-14Vaginal isolate[10, 11]Staphylococcus aureusATCC 25923Clinical isolatehttp://www.atcc.org/


## 2.2. Growth Inhibition Studies

The effects of the presence ofLactobacillus strains on the growth of C. albicans were determined in vitro on agar plates and in liquid cultures. The intestinal Lactobacillus johnsoniistrain PV016 and the non-lactic acid bacterium Staphylococcus aureus ATCC 25923 were used as controls. Plate assays with deferred inoculation of C. albicans were performed on MRS or MRS-MOPS (MRS medium buffered with 0.165 M 3-morpholinopropane-1-sulfonic acid to pH 7.0) media. For the plate overlay assays, overnight cultures of the bacteria were diluted to an OD600nm of 1 and 4 μL of the dilution were spotted on MRS or MRS-MOPS agar plates. After incubation for 2 days at 37°C in a CO2 incubator, plates were overlayed with a C. albicans suspension (OD600nm=0.01) in MRS soft agar (0.7% agar) that was prepared after cooling the autoclaved agar medium to 45°C. Following solidification of the soft agar overlay, plates were incubated for one day at 37°C in normal aerobic atmosphere. Overlay plates were inspected for clear zones with no C. albicans growth surrounding bacterial colonies capable of inhibiting growth of the fungi. Disk diffusion assays were used to detect inhibition of C. albicansgrowth by lactic acid. Briefly, 20 μL of lactic acid dilutions were pipetted onto antibiotic assay disks (diameter 6 mm, Whatman, GE Health Care, Piscataway, NJ, USA) and the disks were placed on MRS or MRS-MOPS agar plates inoculated for dense growth of C. albicans. Clear zones around the disks after 24 hr incubation at 37°C indicated fungal growth inhibition by lactic acid.


## 2.3.Candida albicans Viability Assay

Viability ofC. albicans cells during coculture with lactobacilli was qualitatively assessed using the FUN 1 viability indicator (LIVE/DEAD Yeast Viability Kit; Invitrogen, Carlsbad, CA, USA) and fluorescence microscopy. After a 24 hr incubation of C. albicans cells in MRS at 37°C in presence or absence (control) of lactobacilli, cells were washed with GH buffer (10 mM Na-HEPES buffer pH 7.2 containing 2% glucose). FUN 1 cell stain was diluted in the same buffer to a concentration of 5 μM and the solution was added to the cells. Following incubation for 30 min in the dark, a Biorad MRC-1024 confocal laser scanning microscope was used to determine the metabolic activity of the yeast cells. Excitation and emission filter sets for green and red fluorescence of FUN 1 were used according to the manufacturer’s directions. Intact metabolically active C. albicans cells converted the green-fluorescent intracellular FUN 1 dye to red fluorescent intravacuolar structures. Cells with little or no metabolic activity but still intact membranes just show diffuse cytoplasmic green fluorescence and no red intravacuolar bodies. In contrast, diffuse and extremely bright green-yellow fluorescence indicates dead cells. For semiquantitative assessment of fungal viability, the FUN 1 viability assay was adapted for fluorometric measurement of metabolic activity according to the manufacturer’s recommendations. Following overnight incubation of C. albicans cells (starting OD600nm 0.1) in MRS with bacteria (starting OD600nm 0.05) or in the presence of lactic acid, the viability of C. albicans cells was determined by kinetic readings of red (excitation filter 485/20, emission filter 590/35) and green (excitation filter 485/20, emission filter 528/20) fluorescence in a BioTek Synergy 2 multimode microplate reader (BioTek, Winooski, VT, USA). For this purpose, aliquots from cultured cells were washed in GH buffer, adjusted to OD600nm 0.5 in 200 μL GH + 5 μM FUN 1 and subsequently incubated for 1 hr in the microplate reader at 30°C. Red and green fluorescence readings were taken every 5 minutes and red/green ratios were determined to monitor the viability of fungal cells. An increase in the red/green ratio over time indicated the presence of the metabolic activity required for formation of the red fluorescent intravacuolar structures. Bacteria did not convert the dye (data not shown).


## 2.4. Transcriptional Profiling

For genome-wide transcriptional analysis ofC. albicans challenged by the presence of lactobacilli, cocultures of both microorganisms were established in 6-well plates with ThinCert (Greiner Bio-One, Monroe, NC, USA) inserts that kept both cell populations separated by a PET membrane with a pore-size of 0.45 μm. Fungal cells were inoculated in the lower compartment and pregrown for 6 hrs at 37°C in 5% CO2 atmosphere without shaking. Lactobacilli were then inoculated in the ThinCert compartment and the cocultures were incubated up to 24 hrs total incubation time (i.e., 18 hrs in coculture). Parallel C. albicans cultures without addition of lactobacilli served as controls. At various time points, the pH of the medium was measured and the cell density of both bacterial and fungal cells was determined by spectrophotometry at 600 nm. C. albicans cells were harvested and total RNA was isolated using a FastPrep FP120A instrument in conjunction with the FastRNA Pro Red kit (MP Biomedicals, Solon, OH, USA). Isolated total RNAs underwent further purification with the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA, USA). Integrity and concentration of the total RNAs were determined by agarose gel electrophoresis and spectrophotometry, respectively. For preparation of Affymetrix GeneChip (Affymetrix, Santa Clara, CA, USA) hybridization targets, 50 ng of the highly-purified total RNAs were used for cDNA generation, Ribo-SPIA amplification, and biotin-labeling using the NuGEN Ovation Biotin system (NuGEN Technologies Inc., San Carlos, CA, USA). The resulting biotin-labeled single-stranded cDNAs were used in C. albicans GeneChip hybridizations according to the manufacturers’ directions. The custom-made C. albicans GeneChips were described previously [12]. Following hybridization in a GeneChip Hybridization Oven 640 and a GeneChip Fluidics Station 400 (Affymetrix), the microarrays were scanned using a GeneArray Scanner (Hewlett-Packard). The resulting image files were processed for absolute and comparative expression analysis using MICROARRAY SUITE 5.0 (MAS 5.0; Affymetrix). Global scaling to a target intensity of 500 was used to correct for variations between arrays. Experimental data were stored in MICRODB 3.0 and analyzed in DATA MINING TOOL 3.0 (Affymetrix) as well as MICROSOFT ACCESS. Default parameters were used for the statistical algorithms implemented in MAS 5.0 to calculate probe set signals, signal log ratios (SLR) as well as detection and change P values. For determination of differential gene expression between Lactobacillus-Candida coculture experiments and Candida-only controls, we also employed the SNOMAD software for standardization and normalization of microarray data (http://pevsnerlab.kennedykrieger.org/snomadinput.html [13]). Here, signals (intensities) of experiment-control sets are log-transformed and the log ratios are normalized by calculation of a local mean across the element signal intensities (LOESS fit). Furthermore, a local variance correction is applied and a Z score is calculated for each probe set as a measure for differential expression. Transcripts with Z scores ≥3 were considered significantly increased and transcripts with Z scores ≤−3 were considered significantly decreased in comparative analyses. In order to group genes with similar regulation, cluster analyses were performed using the programs Cluster and TreeView [14].


## 3. Results and Discussion

### 3.1.In Vitro Growth Inhibition

The rich MRS medium was chosen for these experiments because it supported vigorous growth by theLactobacillus, Staphylococcus aureus,and C. albicans strains. Agar overlays of C. albicanson MRS plates inoculated with the probiotic lactobacilli and control bacteria represented a relatively simple screening method for growth inhibitory activities of the bacteria towards this yeast. The deferred assay regimen with a 2-day preincubation of the bacteria was the method of choice because simultaneous inoculation often led to no or very small bacterial colonies due to rapid overgrowth by the fungal cells.After 48 hrs of incubation, the probioticLactobacillus—C. albicansoverlays revealed clearly visible zones of fungal growth inhibition around the Lactobacillus GR-1 and RC-14 colonies (see Figure 1(a)) on MRS plates. Very weak or no inhibition zones were found around the colonies of the control strains L. johnsoniiPV016 and S. aureusATCC 25923, respectively. PV016 was isolated by us from the prairie vole intestine and classified by molecular methods as L. johnsonii (data not shown). This strain appears to have only limited anti-Candidaactivity. Despite the vigorous growth of the S. aureus strain on MRS, no antifungal activity was detected in the overlay assay. S. aureus is not considered a lactic acid bacterium and appears to produce only limited amounts of lactic acid under aerobic conditions [15]. On buffered MRS-MOPS agar plates the clear areas around the probiotic lactobacilli were much smaller or absent. Thus, the inhibitory activities of lactobacilli towards C. albicansappear to be decreased when the media is buffered with MOPS to an initial pH of 7.0 before inoculation. The pH of unbuffered MRS media was 6.5 before inoculation. The formation of lactic acid and other organic acids by the growing probiotic lactobacilli is likely to cause a substantial decrease in the pH of unbuffered MRS.Agar inhibition assays with bacteria, lactic acid, andC. albicans. The images in (a) show the results of deferred agar overlay assays on bacterial colonies (L. rhamnosusGR-1, L. reuteriRC-14, L. johnsoniiPV016,and S. aureus ATCC 25923) and C. albicansas the tester organism. The bacteria were spotted on MRS or buffered MRS-MOPS (pH 7.0) plates. After incubation for 48 hrs, bacterial colonies were overlayed with soft agar containing live C. albicans cells and incubated for another 24 hrs. For details see Section 2. A C. albicanslawn developed, but fungal growth was inhibited around the Lactobacilluscolonies (clear zones), most predominantly around the probiotic strains GR-1 and RC-14. No inhibition was found with S. aureus. On MRS-MOPS buffered to pH 7 inhibition zones are much smaller or absent indicating reduced effectiveness of the lactobacilli.   (b) Shows the results of disk diffusion assays using lactic acid in the indicated molarities (20 μL per disk) as the inhibitory compound. Plates were inoculated to produce a lawn of C. albicans and disks with lactic acid were immediately placed on the inoculated plates. Following 24 hrs incubation, only the 6 M concentration of lactic acid showed an inhibition zone on MRS. Similar to the overlay assays, the inhibition zone on the buffered MRS-MOPS medium was largely reduced.
(a)(b)In comparison to the probiotics, theL. johnsoniiand S. aureuscontrol strains apparently were not able to produce sufficient acid or other metabolic products to inhibit the fungi. On the other hand, the control results suggest that potential glucose or other nutrient exhaustion around the bacterial colonies is not a likely cause for fungal growth inhibition in the overlay assay. C. albicans also produces acids during aerobic growth and can drastically reduce the pH of batch cultures in some media [16, 17]. However, in an overnight culture of C. albicans grown in MRS at 37°C, we observed a pH reduction to only pH 6.0. In a disk diffusion assay with lactic acid, the potential inhibition of C. albicansgrowth by the acid was confirmed (Figure 1(b)). Clear zones around the disks were visible if the concentration of the acid was sufficiently high. However, performing the disk diffusion assay on MRS-MOPS revealed that buffering diminished the antifungal activity, similar to the overlay assays with bacteria.For a more detailed assessment of the acid tolerance ofC. albicansin MRS broth, we conducted growth assays with MRS containing lactic acid at different pH levels (not pH adjusted or adjusted to pH 7.0 and 4.5), and MRS adjusted to pH 4.5 using hydrochloric acid (Figure 2). These experiments revealed that C. albicans SC5314 growth is decreased at lower pH levels. The strongest growth inhibition was detected in MRS that contained lactic acid (120 mM) with no pH adjustment. Addition of 120 mM lactic acid to MRS resulted in an actual pH of 4.2. The growth inhibitory effect of this high concentration of lactic acid was reduced by pH adjustment to 4.5 and completely abrogated by neutralization to pH 7.0 with sodium hydroxide. Adjustment of the pH of MRS to 4.5 using hydrochloric acid led to intermediate growth reduction which was significantly lower than the reduction achieved by 120 mM lactic acid at pH 4.5. These results indicate that C. albicansappears to be sensitive to low pH and lactic acid.Figure 2
Inhibition ofC. albicans growth by lactic acid at low pH. Microplate growth assays were used to determine the effect of lactic acid on the growth of C. albicans. MRS medium was supplemented with 120 mM lactic acid and the pH was adjusted to pH 4.5 or 7.0. MRS with 120 mM lactic acid had a pH of 4.2. As a control for low pH, MRS was adjusted to pH 4.5 using hydrochloric acid (MRS (HCL)). While acidic pH slows growth of C. albicans, the strongest growth inhibition was achieved with lactic acid at low pH. This inhibitory effect was completely lost by neutralization. Error bars show the standard deviations of two experiments with quadruplicate measurements. Statistical significant (P<0.01) differences to all other conditions as calculated by ANOVA are indicated by asterisks.In conjunction with acid stress, the increased concentration of membrane-permeable, undissociated lactic acid (pKa = 3.86) at low pH could have an inhibitory effect on the growth of the pathogenic yeast. Undissociated lactic acid permeates the plasma membrane by diffusion and subsequently dissociates into protons (H+) and lactic acid counterions. The charged ions are unable to cross the membrane bilayer, accumulate, and interfere with cell metabolism by acidification of the cytosol. Inhibition of fungal growth through lactic acid production by lactobacilli might therefore be most efficient at low pH.Growth inhibition assays using culture filtrates fromL. rhamnosus GR-1 and L. reuteri RC-14 indeed revealed that C. albicans growth was suppressed at low pH by the bacterial culture supernatants. Neutralization of the culture filtrates, however, completely abrogated the inhibitory effects of these supernatants (Figure 3). While the contribution of additional antifungal compounds produced by the lactobacilli in inhibiting C. albicansis unknown at present, our results indicate that lactic acid at low pH plays a major role in keeping fungal growth in check. Hydrogen peroxide production by L. reuteri RC-14 could be an additional anti-Candida factor. In contrast to L. rhamnosus GR-1, L. reuteri RC-14 produces H2O2 constitutively on MRS (data not shown). However, the relatively high resistance of C. albicansto this potential growth inhibitor likely diminishes its efficacy. The growth assays conducted in this study did not show that L. reuteri RC-14 was more potent in inhibiting C. albicans, despite the strain’s ability to generate H2O2.Figure 3
Growth inhibition ofC. albicansby culture supernatants from probiotic lactobacilli. Cell-free culture supernatants of L. rhamnosusGR-1 and L. reuteriRC-14 were collected after 48 hrs of growth and adjusted to pH 4.5 and pH 6.8 using sodium hydroxide. Varying amounts of the supernatants were added to MRS media inoculated with C. albicansin microtiter plates (total volume per well 200 μL). After incubation at 37°C for 24 hrs, the cell densities were determined by OD600nm readings. The inhibitory effect of the supernatants at low pH was lost at pH 6.8. Error bars show the standard deviations of at least triplicate experiments.


### 3.2. Fungicidal Activities of Lactobacilli

A microscopic approach was taken to visualize whether the probiotic lactobacilli are in fact able to kill the fungal cells. FUN-1 LIVE/DEAD staining following cocultures of the bacteria withC. albicansrevealed that fungal cells lost metabolic activity in the presence of the lactobacilli and eventually were killed (Figure 4(a)). Semiquantitative analyses of C. albicanscell viability following exposure to low pH, lactic acid, and the bacterial strains used in this study revealed again that lactic acid at low pH and the probiotic strains GR-1 and RC-14 exert the most potent antifungal properties. The microplate-based assay system was used to determine cell viability by kinetic measurement of the intracellular conversion of the green fluorescent FUN-1 dye to red fluorescent intravacuolar structures. Only viable and metabolically active cells are able to carry out this conversion which can be monitored by determination of red/green fluorescence ratios in a fluorometer. Figure 4(b) shows the effect of MRS with lactic acid at pH 4.5 compared to MRS with HCl-adjusted pH 4.5 on C. albicanscell viability after overnight incubation. Presence of lactic acid under these conditions appears to affect fungal cell viability substantially more than just the low pH. The efficacy of the probiotic strains GR-1 and RC-14 to compete with C. albicansgrowth is also confirmed by the fluorometric viability assay results shown in Figure 4(c). Both strains generated the flattest red/green ratio curves indicating the strongest antagonistic effects on the fungi in this assay system. L. johnsoniiPV016 appeared to have an intermediate effect on fungal cell viability while S. aureusonly produced a minor reduction in the conversion rate from green to red fluorescence when compared to the C. albicans control (see Figure 4(c)). Overall, these viability assay results provide strong evidence that the probiotics are not only competing for nutrients and have fungistatic properties—under suitable conditions they can indeed exert fungicidal effects.Viability assays ofC. albicans cells following coculture with bacteria and exposure to lactic acid. (a) Microscopic viability assay: C. albicans cells were stained with the FUN 1 cell stain (Invitrogen) for viability after coculture with L. rhamnosus GR-1 (Ca + Lb-GR-1). A C. albicans culture without lactobacilli served as control (Ca). Cell images were obtained by confocal laser scanning microscopy. Results for the vital staining procedure are depicted above the images. White arrows in the zoomed insert image on the left indicate CIVS formation in metabolically active cells of the control culture. The presence of lactobacilli increases the number of metabolically inactive and dead cells (right image). (b) Fluorometric assay of C. albicanscells viability following 16 hr incubation in MRS, MRS (HCl) pH 4.5, and MRS (lactic acid) pH 4.5. Cell viability was measured by monitoring the conversion of FUN 1 to red fluorescent intravacuolar structures in a microplate assay. The red/green fluorescence ratio increases in the presence of viable, metabolically active cells, particularly within the first 30 min of the assay. Metabolically inactive cells show no increase of the red/green ratio. For assay details see Section 2. The figure shows the results of a representative experiment. Incubation of C. albicans cells in MRS at pH 4.5 in the presence of lactic acid appears to reduce cell viability much more than incubation in MRS that was adjusted to pH 4.5 using HCl. (c) Example of a fluorometric FUN 1 cell viability assay of C. albicans following coculture with L. rhamnosus GR-1, L. reuteri RC-14, L. johnsonii, or S. aureus. A C. albicansculture without bacteria served as control (Ca + MRS). GR-1 and RC-14 consistently caused the strongest reduction in C. albicansviability. All fluorometric experiments were conducted in triplicate. Error bars indicate standard deviations.
(a)(b)(c)


### 3.3. Transcriptional Profiling ofC. albicans in Coculture with Lactobacilli

The 0.45μm porous membrane was effective at separating the fungi from the lactobacilli cells (see Figure 5(a)). Using this approach, it was possible to isolate total fungal RNA without contaminating bacterial RNA. Similar to previous experiments, presence of lactobacilli in the culture reduced C. albicans growth as determined by OD600nm measurements (Figure 5(b)). Despite physical separation of the microorganisms, the competition for nutrients and the presence of diffusible inhibiting substances (e.g., lactic acid) produced by the lactobacilli led to fungal growth retardation. Transcriptome analysis of C. albicans using custom-made CandidaAffymetrix GeneChip expression arrays revealed that genes involved in lactic acid utilization were induced early in cocultures with lactobacilli in contrast to control cultures without the bacteria. Table 2 lists genes that were differentially expressed in at least three of the seven coculture experiments. Surprisingly, the lactate importer gene JEN1in C. albicans[18] was not induced and remained at relatively low expression levels in all conditions tested. JEN1is reportedly induced on lactate as the sole carbon source, but subject to glucose repression [18]. Thus, presence of glucose (MRS contains 2% glucose) might have affected the lactate transporter expression at least in the early stage cocultures. Interestingly, the related JEN2 gene encoding a dicarboxylic acid transport protein [19] was transiently induced in two experiments (data not shown). Whether increased acidification of the cytosol at later growth stages also suppressed the expression of the lactate/proton symporter gene JEN1is unknown.Table 2
Differentially expressedC. albicansgenes in cocultures with L. rhamnosusGR-1 or L. reuteriRC-14. Z values for genes with at least three observed incidences of significant differential regulation in the tested conditions are shown. Z-values ≥3 and ≤−3 were considered significant (see Section 2). Values followed by the symbol † indicate gene induction when compared to C. albicanscultures without addition of lactobacilli. Downregulation values are followed by symbol #. Differential gene expression data from coculture experiments with L. rhamnosusGR-1 (GR1_a, b, c, d, e) and L. reuteriRC-14 (RC14_a, b) are grouped according to the C. albicans growth phase (log phase and late log to early stationary phase). Genes are ordered in functional groupings adapted from gene ontology (GO) annotations shown in the Candida Genome Database (http://www.candidagenome.org/). Differentially expressed genes are also divided into upregulated genes (a), genes with mixed regulation (b), and downregulated genes (c). Genes followed by the symbol * are further discussed in the text.
(a)
UpregulatedC. albicansgenes
ORFaGeneMolecular functionbLog phase coculturesLate log to early stationary phase coculturesGR1_aRC14_aGR1_bGR1_cGR1_dRC14_bGR1_eAmino acid metabolismcorf19.5610ARG3Ornithine carbamoyltransferase0.492.43−0.531.693.54†4.36†3.20†Carbohydrate metabolismorf19.3675GAL7Putative galactose-1-phosphate uridyl transferase0.692.121.556.12†3.40†4.11†0.47orf19.1788XKS1Putative xylulokinase0.392.260.373.24†2.914.33†3.06†Cell wall biosynthesisorf19.3688Putative chromatin DNA binding protein7.69†1.660.350.1910.34†4.25†−0.09orf19.3674GAL102UDP-glucose 4,6-dehydratase with role in mannosylation of cell wall proteins0.111.70−0.063.63†4.48†4.75†0.21Fatty acid metabolismorf19.5640PEX5Putative peroxisomal targeting receptor0.14−2.143.31†5.21†3.34†4.03†1.03orf19.5215TES15Putative acyl-CoA thioesterase−0.91−0.761.913.58†5.17†5.66†−2.00Glycogen metabolismorf19.3302Putative type-1 protein phosphatase targeting subunit−0.61−0.980.583.03†6.26†3.28†1.32Glycolysisorf19.6116GLK4Glucokinase−0.850.481.250.824.13†3.05†6.62†Intracellular transportorf19.3233HSE1Putative SH3-domain-containing protein−1.94−0.812.303.34†5.24†3.66†−0.24orf19.7086Putative protein transporter0.54−1.18−1.101.354.74†4.15†3.27†orf19.3841Putative protein serine/threonine kinase−2.91−0.82−0.684.07†4.94†4.92†−1.58Oxidation/Reductionorf19.5805*DLD1
*D-lactate dehydrogenase*1.340.724.57†3.63†6.23†5.90†8.57†orf19.1048IFD6Aldo-keto reductase−0.092.071.36−0.925.16†3.41†6.71†orf19.742ALD6Putative aldehyde dehydrogenase−1.270.96−0.194.46†4.77†5.36†3.16†orf19.5288IFE2Alcohol dehydrogenase1.03−0.07−0.665.77†6.75†6.04†0.33Protein catabolismorf19.1191Possible ubiquitin-protein ligase1.12−0.913.14†2.253.64†4.31†−0.45orf19.5752Possible ubiquitin-protein ligase−1.59−0.660.174.35†3.16†3.29†2.38Repairorf19.1331HSM3Protein may be involved in DNA mismatch repair−0.74−2.27−0.144.18†8.78†6.68†5.39†Respirationorf19.4551CTN1Predicted carnitine acetyl transferase1.211.970.0811.85†3.64†7.52†2.61RNA catabolismorf19.5614RNH1Putative ribonuclease H1−1.330.38−0.073.06†4.11†2.963.22†Signalingorf19.399Putative serine/threonine protein kinase−2.21−2.59−0.104.08†4.88†4.44†−0.22orf19.5531CDC37Chaperone for Crk1 protein kinase−0.450.950.802.135.46†3.19†6.05†Stress responseorf19.882*HSP78
*Heat-shock protein*0.00−1.350.064.21†4.52†5.81†4.66†orf19.3861*SIS1
*Putative Type II HSP40 co-chaperone*−0.91−0.400.791.653.92†4.78†3.10†orf19.5348*TPS3
*Trehalose-phosphate synthase subunit*0.59−0.26−0.994.23†8.95†7.42†5.40†Transcriptional regulationorf19.3047SIP3Protein kinase-related protein1.230.770.663.62†3.69†4.84†4.52†orf19.909STP4Putative transcription factor−0.52−1.281.296.75†5.15†7.01†2.41orf19.3190HAL9Putative transcription factor involved in salt tolerance0.981.832.823.87†4.99†5.03†−0.59Translationorf19.154Possible 90S pre-ribosome complex protein9.04†1.52−0.034.19†3.22†2.770.90Transportorf19.5079*CDR4
*ABC transporter*2.773.79†−0.063.22†2.893.51†1.78orf19.4802FTH1Putative high affinity iron transporter (vacuolar)−0.450.761.104.54†5.95†7.85†−0.38orf19.3668HGT2Putative glucose transporter1.171.351.739.56†4.30†3.16†−2.49orf19.5447HGT19Putative glucose/myo-inositol transporter2.513.11†2.554.67†2.955.05†5.41†orf19.3526ITR1Inositol transporter1.82−0.201.693.52†5.21†5.25†2.91orf19.1979GIT1Putative glycerophosphoinositol permease0.976.75†−1.943.73†5.82†8.01†1.64orf19.1027PDR16Phosphatidylinositol transfer protein−0.47−1.630.612.193.61†4.23†3.05†orf19.4737*TPO3
*Possible polyamine transporter*0.08−0.023.65†4.30†4.59†7.16†3.48†orf19.700*SEO1
*Similar to permeases (↑weak acid stress)*−0.79−1.344.50†4.90†2.885.02†−0.14Unknownorf19.2498Unknown−2.12−1.080.293.03†3.05†3.99†−0.55orf19.5245Unknown−0.36−1.741.590.845.03†5.24†4.78†orf19.6321PGA48Putative GPI-anchored protein of unknown function−1.55−0.790.71−1.465.14†3.50†3.62†orf19.5295Unknown−0.761.02−2.035.83†3.06†3.51†−1.02orf19.3448Unknown1.020.86−0.595.89†2.513.32†3.52†(b)
C. albicansgenes with mixed regulation
ORFaGeneMolecular functionbLog phase coculturesLate log to early stationary phase coculturesGR1_aRC14_aGR1_bGR1_cGR1_dRC14_bGR1_eAmino acid metabolismcorf19.3974PUT21-Pyrroline-5-carboxylate dehydrogenase−4.32#−0.86−0.393.07†−3.25#−6.02#−1.68orf19.105HAL22Put. phosphoadenosine-5′-phosphate phosphatase−2.16−4.51#3.44†3.62†−0.793.67†−3.38#orf19.5811MET1Putative uroporphyrin-3 C-methyltransferase0.370.590.674.93†−1.933.53†−4.15#Carbohydrate metabolismorf19.7481MDH1Malate dehydrogenase−1.68−2.141.193.97†−3.66#−3.34#−1.21Cell morphogenesisorf19.4892TPK1Subunit of cAMP-dependent protein kinase (PKA)0.191.68−3.02#−1.852.693.01†−3.77#Cell wall assemblyorf19.377PHR3Putative beta-1,3-glucanosyltransferase−1.110.743.09†3.46†1.671.60−3.52#Cytoskeleton maintenanceorf19.3505Similar toS. cerevisiae phosphoinositide binding protein Slm2−0.04−4.18#−1.260.595.35†3.91†0.07Filamentous growthorf19.4246Similar toS. cerevisiae Ykr070w protein−1.10−4.36#0.894.67†3.16†2.663.93†orf19.860BMT8Putative beta-mannosyltransferase−4.31#1.933.88†1.90−3.35#−2.13−0.42Oxidation/reductionorf19.7323CBP1Corticosteroid binding protein−1.743.42†0.830.26−3.66#−3.27#−1.81orf19.6139FRE7Putative ferric reductase−1.250.174.56†−2.56−3.14#−4.12#−0.42orf19.6531NUC2NADH-ubiquinone oxidoreductase−2.950.143.33†−2.10−3.89#−4.12#−1.28pH responseorf19.101RIM9Unknown, involved in cellular response to pH−0.73−0.50−0.842.433.69†3.75†−3.08#Stress responseorf19.3707*YHB1
*Nitric oxide dioxygenase*−0.61−1.541.074.53†2.615.15†−3.06#orf19.6232*NPR1
*Predicted serine/threonine protein kinase*−3.75#−1.634.49†3.98†−0.211.51−0.30Transcriptionorf19.2831RPC31Putative RNA polymerase III subunit C31−0.703.18†2.42−3.33#−3.63#−2.32−1.66orf19.5908TEC1TEA/ATTS transcription factor2.312.201.746.57†1.814.84†−4.97#orf19.5992WOR2Transcriptional regulator of white-opaque switching−1.25−3.19#−0.426.58†6.05†8.17†−3.00#Transportorf19.6578Predicted membrane transporter0.28−3.37#−0.564.20†6.39†3.27†0.01tRNA aminoacylationorf19.5239similarity to alanyl-tRNA synthases0.30−4.73#−3.55#0.740.583.76†0.69Unknownorf19.925−1.03−1.593.42†−0.81−4.07#−3.38#0.11orf19.5992.894.04†4.02†−3.40#0.510.332.02orf19.4765PGA6Putative GPI-anchored cell wall adhesin-like protein−0.675.78†−3.74#−0.92−0.13−4.12#2.61orf19.63422.064.26†−3.63#−3.94#1.702.111.27(c)
DownregulatedC. albicansgenes
ORFaGeneMolecular functionbLog phase coculturesLate log to early stationary phase coculturesGR1_aRC14_aGR1_bGR1_cGR1_dRC14_bGR1_eAdhesioncorf19.2122*ALS12
*ALS family protein; role in adhesion, biofilm formation, germ tube induction*−1.84−3.38#0.641.51−3.68#−0.73−3.71#Amino acid metabolismorf19.646GLN1Putative glutamate synthase−3.16#−0.500.451.56−1.62−3.20#−3.08#orf19.3846LYS4Homoaconitase−2.68−1.540.63−4.04#−5.51#−4.80#−0.52orf19.2360URA2Putative bifunctional carbamoylphosphate synthetase-aspartate transcarbamylase−0.58−5.46#−0.75−1.48−3.62#−4.30#−0.13orf19.6257GLT1Putative glutamate synthase−0.80−3.46#0.19−3.07#−5.30#−3.61#0.29Cell wall organizationorf19.386SAM4Putative S-adenosylmethionine-homocysteine methyltransferase−1.87−1.600.64−3.11#−3.40#−4.50#−2.44orf19.3829*PHR1
*Glycosidase of cell surface*−4.84#−4.20#−3.95#−7.74#−3.17#−6.66#−5.39#orf19.5171PMT1Protein mannosyltransferase,−0.75−0.200.42−4.18#−3.67#−4.02#−0.86Cytoskeleton organizationorf19.2286Putative deoxyhypusine hydroxylase−2.970.36−1.06−1.51−3.07#−3.08#−3.49#DNA replicationorf19.5183*POL3
*Large subunit of DNA polymerase III*−2.60−4.03#0.98−0.12−4.07#−3.39#−2.58orf19.6078POL93Putative gypsy-like reverse transcriptase−3.48#−0.48−0.43−6.24#−1.66−4.44#0.28orf19.2885*PRI2
*Putative DNA primase*0.27−3.17#−3.27#−3.20#0.35−2.050.31orf19.3322DUT1dUTP pyrophosphatase−3.24#−4.14#−0.28−2.23−3.38#−4.36#−0.44Ergosterol biosynthesisorf19.1631*ERG6
*Delta(24)-sterol C-methyltransferase*−0.670.20−3.61#−2.84−6.05#−4.09#−1.34orf19.2016*Possible role in ergosterol biosynthesis*−2.260.162.13−3.22#−3.69#−3.14#1.05orf19.922*ERG11
*Lanosterol 14-alpha-demethylase*−1.29−0.44−0.73−5.19#−4.14#−5.50#−2.26Filamentous growthorf19.815DCK1Putative guanine nucleotide exchange factor required for embedded filamentous growth−1.76−4.90#−0.070.47−4.22#−1.11−4.89#orf19.3669SHA3Putative serine/threonine kinase−1.04−2.35−0.77−3.24#−3.27#−1.55−3.89#orf19.3575*CDC19
*Putative pyruvate kinase; mutation affects filamentation*−2.04−0.070.20−3.09#−3.12#−3.97#0.49Gluconeogenesisorf19.7514*PCK1*Phosphoenolpyruvate carboxykinase*−0.22−3.90#2.821.21−3.89#−3.66#−2.91Glutathione biosynthetic processorf19.5059GCS1Gamma-glutamylcysteine synthetase−2.60−3.06#−3.10#1.00−2.27−0.52−3.85#Intracellular transportorf19.4805Putative membrane protein−1.191.35−0.56−3.69#−3.87#−3.64#−2.47Lipoate biosynthetic processorf19.819Possible role in lipoate biosynthesis−1.06−3.54#1.910.27−4.15#−3.69#−0.94NAD biosynthetic processorf19.1460Putative glutamine-dependent NAD synthetase−2.41−2.490.05−0.79−3.60#−3.23#−3.51#Oxidation/Reductionorf19.5517Similar to alcohol dehydrogenases−2.94−0.19−0.85−4.83#−6.56#−8.21#−0.07orf19.2525LYS12Mitochondrial homoisocitrate dehydrogenase−2.171.04−1.19−5.01#−5.72#−7.22#0.65RNA processingorf19.7215Ortholog(s) have snoRNA binding activity−2.83−0.06−0.38−0.16−5.20#−4.12#−3.23#orf19.3159UTP20Similar toS. cerevisiae nucleolar protein Utp10p−1.27−0.48−0.20−2.29−5.60#−3.26#−3.15#Signalingorf19.3453Possible role in signal transduction−1.58−0.210.89−4.27#−3.25#−4.07#0.09orf19.6906*ASC1
*Protein described as part of 40S ribosomal subunit*−3.30#−2.92−1.910.37−3.74#−4.70#−2.62TCA cycleorf19.6632ACO2Aconitate hydratase 2−1.14−0.250.55−3.34#−6.53#−4.39#0.05Translationorf19.6253*RPS23A
*Putative ribosomal protein*−1.19−3.68#−0.63−3.51#−2.82−2.86−3.17#orf19.4152*CEF3
*Translation elongation factor 3*−2.13−3.35#0.540.12−3.18#−1.03−3.86#Transportorf19.4599PHO89Putative phosphate permease0.73−0.06−2.63−11.05#−6.49#−5.80#−6.50#orf19.5170ENA21Possible sodium transporter sodium transporters−0.77−2.46−1.35−3.59#−4.30#−5.16#−1.38orf19.6000*CDR1
*ABC transporter involved in multidrug resistance*−0.09−0.21−2.97−1.76−5.00#−5.04#−3.82#Unknownorf19.7413MMS21-SMC5-SMC6 complex component−3.01#−3.22#1.62−3.10#−1.53−1.750.55orf19.1549Unknown−0.42−1.89−3.17#−3.96#−3.83#−2.95−1.10orf19.4651PGA53Putative GPI-anchored protein−0.84−1.33−1.16−3.73#−3.33#−3.75#0.35orf19.5799Unknown0.24−0.13−1.23−3.54#−4.95#−5.27#−1.11orf19.915Unknown−0.561.05−0.590.19−3.60#−3.15#−3.21#orf19.5760IHD1Putative GPI-anchored protein−2.64−5.84#−0.54−5.21#−3.27#−5.36#−5.36#orf19.3548Unknown−3.48#−1.88−0.441.89−3.13#−3.08#−0.96aFor further information on the open reading frames (ORFs) see the CandidaGenome Database (CGD: [20]) (http://www.candidagenome.org/).bInformation on the molecular function is abridged. More information is available at the CGD website.cFunctional groupings derived from Biological Process GO annotations. Due to space restrictions, the groupings are simplified. Individual genes can be involved in multiple biological processes.Lactobacillus-C. albicans cocultures for transcriptome analysis. The experimental setup employing a 0.45 μm membrane for separation of the bacterial and fungal cultures in 6-well tissue culture plates is represented schematically in (a). Results of a typical Lactobacillus-C. albicans coculture and C. albicans-only control experiment are shown in (b). Growth of C. albicans and Lactobacillus(Lb) in the respective compartments was determined by OD600nm readings (left y-axis). The pH of the culture media is depicted in dashed lines using the right y-axis. For transcriptome analysis, total RNA was isolated from C. albicans cells harvested from cocultures and controls.
(a)(b)The upregulation of the L-lactate cytochrome-c oxidoreductase geneCYB2 (orf19.5000) in two experiments in conjunction with the continued induction of the putative D-lactate dehydrogenase gene DLD1(orf19.5805, see Table 2(a)) indicated that the fungal cells indeed metabolized lactate despite the lack of strong induction of lactate transporter genes. It remains to be determined whether additional transport systems facilitate the transport of lactic acid or whether undissociated lactic acid enters the fungal cell by simple diffusion. Transcriptional profiling of the C. albicanscells during coculture with the probiotic lactobacilli also indicated that the fungal cells came under increased stress. The increased expression of stress-related genes (e.g., SIS1, TPS3, HSP78, TPO3, SEO1)in progressed cocultures might signify that the fungal cells were challenged by acid production of the lactobacilli and the concomitant lowering of the pH.The mixed expression profile of the stress-related genesYHB1 and NPR1(Table 2(b)) might indicate transient induction of components of the stress response, however further studies are necessary for detailed analysis of stress responses induced by the lactobacilli. Interesting aspects of the interaction of the microorganisms can also be gleaned from the list of downregulated genes (see Table 2(c)): For example, ergosterol biosynthetic genes (ERG6, ERG11, orf19.2016) were repressed. It remains to be elucidated, whether this repression was caused simply by growth retardation or specific interactions with products of the lactobacilli. Several important antifungals target the ergosterol biosynthetic pathway in fungi. The ERG11gene product lanosterol 14-alpha-demethylase is the target enzyme for fluconazole and other azoles [21–23]. The CDR1 gene encoding an important drug efflux pump involved in fluconazole resistance [21, 24, 25] appears also on the list of genes with significantly lower expression (Table 2(c)). In contrast, a related ABC transporter gene, CDR4, showed increased expression in the present study. CDR4is not involved in fluconazole resistance, but appears to be induced in the core stress response of C. albicans [26, 27]. Thus, downregulation of the target enzyme Erg11p and the drug efflux pump Cdr1p could render the fungal cells more susceptible to the antifungal drug. These findings are especially interesting in light of recent results in a randomized, double-blind and placebo-controlled trial on the effect of L. rhamnosus GR-1 and L. reuteri RC-14 application in fluconazole-treated women with vulvovaginal candidiasis [7]. The probiotics significantly improved the outcome of the treatment.Production of lactic acid and other short-chain fatty acids by the lactobacilli leads to acidification of the surrounding microenvironment such as the vaginal ecosystem or anin vitro culture vessel. Low pH favors the yeast form of C. albicansand inhibits the invasive hyphal form of these fungi [28, 29]. The transcriptome analysis in this study confirmed at the gene expression level that the fungi were in an increasingly acidic environment. For example, acidic culture conditions were indicated in all cocultures by the observed repression of the cell wall beta-(1,3)-glucanosyltransferase encoded by the PHR1 gene. This pH-responsive gene is only induced under high pH (in vitropH > 5.5) or conditions supporting hyphal growth of C. albicans [30, 31]. Interestingly, phr1/phr1 null mutants of C. albicans show defects in adhesion to abiotic and epithelial surfaces [32] indicating that repression of this gene could affect biofilm formation of C. albicans. Probiotic lactobacilli such as L. rhamnosusGR-1 are able to suppress biofilm formation of C. albicans on abiotic surfaces [33]. This is presumably achieved by combining growth inhibition and repression of genes involved in biofilm formation (e.g., PHR1, ALS12; see Table 2(c)).The list of downregulated genes in the cocultures experiments also reflects the growth inhibitory effects of the lactobacilli. Key genes involved in DNA replication (POL3, PRI2), translation (CEF3, RPS23A, ASC1), glycolysis (CDC19), and gluconeogenesis (PCK1) are expressed at lower levels. Overall, the exploratory C. albicanstranscriptome analysis presented in this study has revealed first indications on the molecular mechanisms of probiotic interference instigated by L. rhamnosusGR-1 and L. reuteriRC-14 towards the opportunistic fungal pathogen C. albicans. Elucidation of the specific inhibitory mechanisms employed by the probiotic strains will require further analysis. To this end, the development of continuous coculture systems mimicking the vaginal environment as close as possible in conjunction with genomic and proteomic analyses will further improve our understanding of the molecular basis of probiotic effects.


## 3.1.In Vitro Growth Inhibition

The rich MRS medium was chosen for these experiments because it supported vigorous growth by theLactobacillus, Staphylococcus aureus,and C. albicans strains. Agar overlays of C. albicanson MRS plates inoculated with the probiotic lactobacilli and control bacteria represented a relatively simple screening method for growth inhibitory activities of the bacteria towards this yeast. The deferred assay regimen with a 2-day preincubation of the bacteria was the method of choice because simultaneous inoculation often led to no or very small bacterial colonies due to rapid overgrowth by the fungal cells.After 48 hrs of incubation, the probioticLactobacillus—C. albicansoverlays revealed clearly visible zones of fungal growth inhibition around the Lactobacillus GR-1 and RC-14 colonies (see Figure 1(a)) on MRS plates. Very weak or no inhibition zones were found around the colonies of the control strains L. johnsoniiPV016 and S. aureusATCC 25923, respectively. PV016 was isolated by us from the prairie vole intestine and classified by molecular methods as L. johnsonii (data not shown). This strain appears to have only limited anti-Candidaactivity. Despite the vigorous growth of the S. aureus strain on MRS, no antifungal activity was detected in the overlay assay. S. aureus is not considered a lactic acid bacterium and appears to produce only limited amounts of lactic acid under aerobic conditions [15]. On buffered MRS-MOPS agar plates the clear areas around the probiotic lactobacilli were much smaller or absent. Thus, the inhibitory activities of lactobacilli towards C. albicansappear to be decreased when the media is buffered with MOPS to an initial pH of 7.0 before inoculation. The pH of unbuffered MRS media was 6.5 before inoculation. The formation of lactic acid and other organic acids by the growing probiotic lactobacilli is likely to cause a substantial decrease in the pH of unbuffered MRS.Agar inhibition assays with bacteria, lactic acid, andC. albicans. The images in (a) show the results of deferred agar overlay assays on bacterial colonies (L. rhamnosusGR-1, L. reuteriRC-14, L. johnsoniiPV016,and S. aureus ATCC 25923) and C. albicansas the tester organism. The bacteria were spotted on MRS or buffered MRS-MOPS (pH 7.0) plates. After incubation for 48 hrs, bacterial colonies were overlayed with soft agar containing live C. albicans cells and incubated for another 24 hrs. For details see Section 2. A C. albicanslawn developed, but fungal growth was inhibited around the Lactobacilluscolonies (clear zones), most predominantly around the probiotic strains GR-1 and RC-14. No inhibition was found with S. aureus. On MRS-MOPS buffered to pH 7 inhibition zones are much smaller or absent indicating reduced effectiveness of the lactobacilli.   (b) Shows the results of disk diffusion assays using lactic acid in the indicated molarities (20 μL per disk) as the inhibitory compound. Plates were inoculated to produce a lawn of C. albicans and disks with lactic acid were immediately placed on the inoculated plates. Following 24 hrs incubation, only the 6 M concentration of lactic acid showed an inhibition zone on MRS. Similar to the overlay assays, the inhibition zone on the buffered MRS-MOPS medium was largely reduced.
(a)(b)In comparison to the probiotics, theL. johnsoniiand S. aureuscontrol strains apparently were not able to produce sufficient acid or other metabolic products to inhibit the fungi. On the other hand, the control results suggest that potential glucose or other nutrient exhaustion around the bacterial colonies is not a likely cause for fungal growth inhibition in the overlay assay. C. albicans also produces acids during aerobic growth and can drastically reduce the pH of batch cultures in some media [16, 17]. However, in an overnight culture of C. albicans grown in MRS at 37°C, we observed a pH reduction to only pH 6.0. In a disk diffusion assay with lactic acid, the potential inhibition of C. albicansgrowth by the acid was confirmed (Figure 1(b)). Clear zones around the disks were visible if the concentration of the acid was sufficiently high. However, performing the disk diffusion assay on MRS-MOPS revealed that buffering diminished the antifungal activity, similar to the overlay assays with bacteria.For a more detailed assessment of the acid tolerance ofC. albicansin MRS broth, we conducted growth assays with MRS containing lactic acid at different pH levels (not pH adjusted or adjusted to pH 7.0 and 4.5), and MRS adjusted to pH 4.5 using hydrochloric acid (Figure 2). These experiments revealed that C. albicans SC5314 growth is decreased at lower pH levels. The strongest growth inhibition was detected in MRS that contained lactic acid (120 mM) with no pH adjustment. Addition of 120 mM lactic acid to MRS resulted in an actual pH of 4.2. The growth inhibitory effect of this high concentration of lactic acid was reduced by pH adjustment to 4.5 and completely abrogated by neutralization to pH 7.0 with sodium hydroxide. Adjustment of the pH of MRS to 4.5 using hydrochloric acid led to intermediate growth reduction which was significantly lower than the reduction achieved by 120 mM lactic acid at pH 4.5. These results indicate that C. albicansappears to be sensitive to low pH and lactic acid.Figure 2
Inhibition ofC. albicans growth by lactic acid at low pH. Microplate growth assays were used to determine the effect of lactic acid on the growth of C. albicans. MRS medium was supplemented with 120 mM lactic acid and the pH was adjusted to pH 4.5 or 7.0. MRS with 120 mM lactic acid had a pH of 4.2. As a control for low pH, MRS was adjusted to pH 4.5 using hydrochloric acid (MRS (HCL)). While acidic pH slows growth of C. albicans, the strongest growth inhibition was achieved with lactic acid at low pH. This inhibitory effect was completely lost by neutralization. Error bars show the standard deviations of two experiments with quadruplicate measurements. Statistical significant (P<0.01) differences to all other conditions as calculated by ANOVA are indicated by asterisks.In conjunction with acid stress, the increased concentration of membrane-permeable, undissociated lactic acid (pKa = 3.86) at low pH could have an inhibitory effect on the growth of the pathogenic yeast. Undissociated lactic acid permeates the plasma membrane by diffusion and subsequently dissociates into protons (H+) and lactic acid counterions. The charged ions are unable to cross the membrane bilayer, accumulate, and interfere with cell metabolism by acidification of the cytosol. Inhibition of fungal growth through lactic acid production by lactobacilli might therefore be most efficient at low pH.Growth inhibition assays using culture filtrates fromL. rhamnosus GR-1 and L. reuteri RC-14 indeed revealed that C. albicans growth was suppressed at low pH by the bacterial culture supernatants. Neutralization of the culture filtrates, however, completely abrogated the inhibitory effects of these supernatants (Figure 3). While the contribution of additional antifungal compounds produced by the lactobacilli in inhibiting C. albicansis unknown at present, our results indicate that lactic acid at low pH plays a major role in keeping fungal growth in check. Hydrogen peroxide production by L. reuteri RC-14 could be an additional anti-Candida factor. In contrast to L. rhamnosus GR-1, L. reuteri RC-14 produces H2O2 constitutively on MRS (data not shown). However, the relatively high resistance of C. albicansto this potential growth inhibitor likely diminishes its efficacy. The growth assays conducted in this study did not show that L. reuteri RC-14 was more potent in inhibiting C. albicans, despite the strain’s ability to generate H2O2.Figure 3
Growth inhibition ofC. albicansby culture supernatants from probiotic lactobacilli. Cell-free culture supernatants of L. rhamnosusGR-1 and L. reuteriRC-14 were collected after 48 hrs of growth and adjusted to pH 4.5 and pH 6.8 using sodium hydroxide. Varying amounts of the supernatants were added to MRS media inoculated with C. albicansin microtiter plates (total volume per well 200 μL). After incubation at 37°C for 24 hrs, the cell densities were determined by OD600nm readings. The inhibitory effect of the supernatants at low pH was lost at pH 6.8. Error bars show the standard deviations of at least triplicate experiments.


## 3.2. Fungicidal Activities of Lactobacilli

A microscopic approach was taken to visualize whether the probiotic lactobacilli are in fact able to kill the fungal cells. FUN-1 LIVE/DEAD staining following cocultures of the bacteria withC. albicansrevealed that fungal cells lost metabolic activity in the presence of the lactobacilli and eventually were killed (Figure 4(a)). Semiquantitative analyses of C. albicanscell viability following exposure to low pH, lactic acid, and the bacterial strains used in this study revealed again that lactic acid at low pH and the probiotic strains GR-1 and RC-14 exert the most potent antifungal properties. The microplate-based assay system was used to determine cell viability by kinetic measurement of the intracellular conversion of the green fluorescent FUN-1 dye to red fluorescent intravacuolar structures. Only viable and metabolically active cells are able to carry out this conversion which can be monitored by determination of red/green fluorescence ratios in a fluorometer. Figure 4(b) shows the effect of MRS with lactic acid at pH 4.5 compared to MRS with HCl-adjusted pH 4.5 on C. albicanscell viability after overnight incubation. Presence of lactic acid under these conditions appears to affect fungal cell viability substantially more than just the low pH. The efficacy of the probiotic strains GR-1 and RC-14 to compete with C. albicansgrowth is also confirmed by the fluorometric viability assay results shown in Figure 4(c). Both strains generated the flattest red/green ratio curves indicating the strongest antagonistic effects on the fungi in this assay system. L. johnsoniiPV016 appeared to have an intermediate effect on fungal cell viability while S. aureusonly produced a minor reduction in the conversion rate from green to red fluorescence when compared to the C. albicans control (see Figure 4(c)). Overall, these viability assay results provide strong evidence that the probiotics are not only competing for nutrients and have fungistatic properties—under suitable conditions they can indeed exert fungicidal effects.Viability assays ofC. albicans cells following coculture with bacteria and exposure to lactic acid. (a) Microscopic viability assay: C. albicans cells were stained with the FUN 1 cell stain (Invitrogen) for viability after coculture with L. rhamnosus GR-1 (Ca + Lb-GR-1). A C. albicans culture without lactobacilli served as control (Ca). Cell images were obtained by confocal laser scanning microscopy. Results for the vital staining procedure are depicted above the images. White arrows in the zoomed insert image on the left indicate CIVS formation in metabolically active cells of the control culture. The presence of lactobacilli increases the number of metabolically inactive and dead cells (right image). (b) Fluorometric assay of C. albicanscells viability following 16 hr incubation in MRS, MRS (HCl) pH 4.5, and MRS (lactic acid) pH 4.5. Cell viability was measured by monitoring the conversion of FUN 1 to red fluorescent intravacuolar structures in a microplate assay. The red/green fluorescence ratio increases in the presence of viable, metabolically active cells, particularly within the first 30 min of the assay. Metabolically inactive cells show no increase of the red/green ratio. For assay details see Section 2. The figure shows the results of a representative experiment. Incubation of C. albicans cells in MRS at pH 4.5 in the presence of lactic acid appears to reduce cell viability much more than incubation in MRS that was adjusted to pH 4.5 using HCl. (c) Example of a fluorometric FUN 1 cell viability assay of C. albicans following coculture with L. rhamnosus GR-1, L. reuteri RC-14, L. johnsonii, or S. aureus. A C. albicansculture without bacteria served as control (Ca + MRS). GR-1 and RC-14 consistently caused the strongest reduction in C. albicansviability. All fluorometric experiments were conducted in triplicate. Error bars indicate standard deviations.
(a)(b)(c)


## 3.3. Transcriptional Profiling ofC. albicans in Coculture with Lactobacilli

The 0.45μm porous membrane was effective at separating the fungi from the lactobacilli cells (see Figure 5(a)). Using this approach, it was possible to isolate total fungal RNA without contaminating bacterial RNA. Similar to previous experiments, presence of lactobacilli in the culture reduced C. albicans growth as determined by OD600nm measurements (Figure 5(b)). Despite physical separation of the microorganisms, the competition for nutrients and the presence of diffusible inhibiting substances (e.g., lactic acid) produced by the lactobacilli led to fungal growth retardation. Transcriptome analysis of C. albicans using custom-made CandidaAffymetrix GeneChip expression arrays revealed that genes involved in lactic acid utilization were induced early in cocultures with lactobacilli in contrast to control cultures without the bacteria. Table 2 lists genes that were differentially expressed in at least three of the seven coculture experiments. Surprisingly, the lactate importer gene JEN1in C. albicans[18] was not induced and remained at relatively low expression levels in all conditions tested. JEN1is reportedly induced on lactate as the sole carbon source, but subject to glucose repression [18]. Thus, presence of glucose (MRS contains 2% glucose) might have affected the lactate transporter expression at least in the early stage cocultures. Interestingly, the related JEN2 gene encoding a dicarboxylic acid transport protein [19] was transiently induced in two experiments (data not shown). Whether increased acidification of the cytosol at later growth stages also suppressed the expression of the lactate/proton symporter gene JEN1is unknown.Table 2
Differentially expressedC. albicansgenes in cocultures with L. rhamnosusGR-1 or L. reuteriRC-14. Z values for genes with at least three observed incidences of significant differential regulation in the tested conditions are shown. Z-values ≥3 and ≤−3 were considered significant (see Section 2). Values followed by the symbol † indicate gene induction when compared to C. albicanscultures without addition of lactobacilli. Downregulation values are followed by symbol #. Differential gene expression data from coculture experiments with L. rhamnosusGR-1 (GR1_a, b, c, d, e) and L. reuteriRC-14 (RC14_a, b) are grouped according to the C. albicans growth phase (log phase and late log to early stationary phase). Genes are ordered in functional groupings adapted from gene ontology (GO) annotations shown in the Candida Genome Database (http://www.candidagenome.org/). Differentially expressed genes are also divided into upregulated genes (a), genes with mixed regulation (b), and downregulated genes (c). Genes followed by the symbol * are further discussed in the text.
(a)
UpregulatedC. albicansgenes
ORFaGeneMolecular functionbLog phase coculturesLate log to early stationary phase coculturesGR1_aRC14_aGR1_bGR1_cGR1_dRC14_bGR1_eAmino acid metabolismcorf19.5610ARG3Ornithine carbamoyltransferase0.492.43−0.531.693.54†4.36†3.20†Carbohydrate metabolismorf19.3675GAL7Putative galactose-1-phosphate uridyl transferase0.692.121.556.12†3.40†4.11†0.47orf19.1788XKS1Putative xylulokinase0.392.260.373.24†2.914.33†3.06†Cell wall biosynthesisorf19.3688Putative chromatin DNA binding protein7.69†1.660.350.1910.34†4.25†−0.09orf19.3674GAL102UDP-glucose 4,6-dehydratase with role in mannosylation of cell wall proteins0.111.70−0.063.63†4.48†4.75†0.21Fatty acid metabolismorf19.5640PEX5Putative peroxisomal targeting receptor0.14−2.143.31†5.21†3.34†4.03†1.03orf19.5215TES15Putative acyl-CoA thioesterase−0.91−0.761.913.58†5.17†5.66†−2.00Glycogen metabolismorf19.3302Putative type-1 protein phosphatase targeting subunit−0.61−0.980.583.03†6.26†3.28†1.32Glycolysisorf19.6116GLK4Glucokinase−0.850.481.250.824.13†3.05†6.62†Intracellular transportorf19.3233HSE1Putative SH3-domain-containing protein−1.94−0.812.303.34†5.24†3.66†−0.24orf19.7086Putative protein transporter0.54−1.18−1.101.354.74†4.15†3.27†orf19.3841Putative protein serine/threonine kinase−2.91−0.82−0.684.07†4.94†4.92†−1.58Oxidation/Reductionorf19.5805*DLD1
*D-lactate dehydrogenase*1.340.724.57†3.63†6.23†5.90†8.57†orf19.1048IFD6Aldo-keto reductase−0.092.071.36−0.925.16†3.41†6.71†orf19.742ALD6Putative aldehyde dehydrogenase−1.270.96−0.194.46†4.77†5.36†3.16†orf19.5288IFE2Alcohol dehydrogenase1.03−0.07−0.665.77†6.75†6.04†0.33Protein catabolismorf19.1191Possible ubiquitin-protein ligase1.12−0.913.14†2.253.64†4.31†−0.45orf19.5752Possible ubiquitin-protein ligase−1.59−0.660.174.35†3.16†3.29†2.38Repairorf19.1331HSM3Protein may be involved in DNA mismatch repair−0.74−2.27−0.144.18†8.78†6.68†5.39†Respirationorf19.4551CTN1Predicted carnitine acetyl transferase1.211.970.0811.85†3.64†7.52†2.61RNA catabolismorf19.5614RNH1Putative ribonuclease H1−1.330.38−0.073.06†4.11†2.963.22†Signalingorf19.399Putative serine/threonine protein kinase−2.21−2.59−0.104.08†4.88†4.44†−0.22orf19.5531CDC37Chaperone for Crk1 protein kinase−0.450.950.802.135.46†3.19†6.05†Stress responseorf19.882*HSP78
*Heat-shock protein*0.00−1.350.064.21†4.52†5.81†4.66†orf19.3861*SIS1
*Putative Type II HSP40 co-chaperone*−0.91−0.400.791.653.92†4.78†3.10†orf19.5348*TPS3
*Trehalose-phosphate synthase subunit*0.59−0.26−0.994.23†8.95†7.42†5.40†Transcriptional regulationorf19.3047SIP3Protein kinase-related protein1.230.770.663.62†3.69†4.84†4.52†orf19.909STP4Putative transcription factor−0.52−1.281.296.75†5.15†7.01†2.41orf19.3190HAL9Putative transcription factor involved in salt tolerance0.981.832.823.87†4.99†5.03†−0.59Translationorf19.154Possible 90S pre-ribosome complex protein9.04†1.52−0.034.19†3.22†2.770.90Transportorf19.5079*CDR4
*ABC transporter*2.773.79†−0.063.22†2.893.51†1.78orf19.4802FTH1Putative high affinity iron transporter (vacuolar)−0.450.761.104.54†5.95†7.85†−0.38orf19.3668HGT2Putative glucose transporter1.171.351.739.56†4.30†3.16†−2.49orf19.5447HGT19Putative glucose/myo-inositol transporter2.513.11†2.554.67†2.955.05†5.41†orf19.3526ITR1Inositol transporter1.82−0.201.693.52†5.21†5.25†2.91orf19.1979GIT1Putative glycerophosphoinositol permease0.976.75†−1.943.73†5.82†8.01†1.64orf19.1027PDR16Phosphatidylinositol transfer protein−0.47−1.630.612.193.61†4.23†3.05†orf19.4737*TPO3
*Possible polyamine transporter*0.08−0.023.65†4.30†4.59†7.16†3.48†orf19.700*SEO1
*Similar to permeases (↑weak acid stress)*−0.79−1.344.50†4.90†2.885.02†−0.14Unknownorf19.2498Unknown−2.12−1.080.293.03†3.05†3.99†−0.55orf19.5245Unknown−0.36−1.741.590.845.03†5.24†4.78†orf19.6321PGA48Putative GPI-anchored protein of unknown function−1.55−0.790.71−1.465.14†3.50†3.62†orf19.5295Unknown−0.761.02−2.035.83†3.06†3.51†−1.02orf19.3448Unknown1.020.86−0.595.89†2.513.32†3.52†(b)
C. albicansgenes with mixed regulation
ORFaGeneMolecular functionbLog phase coculturesLate log to early stationary phase coculturesGR1_aRC14_aGR1_bGR1_cGR1_dRC14_bGR1_eAmino acid metabolismcorf19.3974PUT21-Pyrroline-5-carboxylate dehydrogenase−4.32#−0.86−0.393.07†−3.25#−6.02#−1.68orf19.105HAL22Put. phosphoadenosine-5′-phosphate phosphatase−2.16−4.51#3.44†3.62†−0.793.67†−3.38#orf19.5811MET1Putative uroporphyrin-3 C-methyltransferase0.370.590.674.93†−1.933.53†−4.15#Carbohydrate metabolismorf19.7481MDH1Malate dehydrogenase−1.68−2.141.193.97†−3.66#−3.34#−1.21Cell morphogenesisorf19.4892TPK1Subunit of cAMP-dependent protein kinase (PKA)0.191.68−3.02#−1.852.693.01†−3.77#Cell wall assemblyorf19.377PHR3Putative beta-1,3-glucanosyltransferase−1.110.743.09†3.46†1.671.60−3.52#Cytoskeleton maintenanceorf19.3505Similar toS. cerevisiae phosphoinositide binding protein Slm2−0.04−4.18#−1.260.595.35†3.91†0.07Filamentous growthorf19.4246Similar toS. cerevisiae Ykr070w protein−1.10−4.36#0.894.67†3.16†2.663.93†orf19.860BMT8Putative beta-mannosyltransferase−4.31#1.933.88†1.90−3.35#−2.13−0.42Oxidation/reductionorf19.7323CBP1Corticosteroid binding protein−1.743.42†0.830.26−3.66#−3.27#−1.81orf19.6139FRE7Putative ferric reductase−1.250.174.56†−2.56−3.14#−4.12#−0.42orf19.6531NUC2NADH-ubiquinone oxidoreductase−2.950.143.33†−2.10−3.89#−4.12#−1.28pH responseorf19.101RIM9Unknown, involved in cellular response to pH−0.73−0.50−0.842.433.69†3.75†−3.08#Stress responseorf19.3707*YHB1
*Nitric oxide dioxygenase*−0.61−1.541.074.53†2.615.15†−3.06#orf19.6232*NPR1
*Predicted serine/threonine protein kinase*−3.75#−1.634.49†3.98†−0.211.51−0.30Transcriptionorf19.2831RPC31Putative RNA polymerase III subunit C31−0.703.18†2.42−3.33#−3.63#−2.32−1.66orf19.5908TEC1TEA/ATTS transcription factor2.312.201.746.57†1.814.84†−4.97#orf19.5992WOR2Transcriptional regulator of white-opaque switching−1.25−3.19#−0.426.58†6.05†8.17†−3.00#Transportorf19.6578Predicted membrane transporter0.28−3.37#−0.564.20†6.39†3.27†0.01tRNA aminoacylationorf19.5239similarity to alanyl-tRNA synthases0.30−4.73#−3.55#0.740.583.76†0.69Unknownorf19.925−1.03−1.593.42†−0.81−4.07#−3.38#0.11orf19.5992.894.04†4.02†−3.40#0.510.332.02orf19.4765PGA6Putative GPI-anchored cell wall adhesin-like protein−0.675.78†−3.74#−0.92−0.13−4.12#2.61orf19.63422.064.26†−3.63#−3.94#1.702.111.27(c)
DownregulatedC. albicansgenes
ORFaGeneMolecular functionbLog phase coculturesLate log to early stationary phase coculturesGR1_aRC14_aGR1_bGR1_cGR1_dRC14_bGR1_eAdhesioncorf19.2122*ALS12
*ALS family protein; role in adhesion, biofilm formation, germ tube induction*−1.84−3.38#0.641.51−3.68#−0.73−3.71#Amino acid metabolismorf19.646GLN1Putative glutamate synthase−3.16#−0.500.451.56−1.62−3.20#−3.08#orf19.3846LYS4Homoaconitase−2.68−1.540.63−4.04#−5.51#−4.80#−0.52orf19.2360URA2Putative bifunctional carbamoylphosphate synthetase-aspartate transcarbamylase−0.58−5.46#−0.75−1.48−3.62#−4.30#−0.13orf19.6257GLT1Putative glutamate synthase−0.80−3.46#0.19−3.07#−5.30#−3.61#0.29Cell wall organizationorf19.386SAM4Putative S-adenosylmethionine-homocysteine methyltransferase−1.87−1.600.64−3.11#−3.40#−4.50#−2.44orf19.3829*PHR1
*Glycosidase of cell surface*−4.84#−4.20#−3.95#−7.74#−3.17#−6.66#−5.39#orf19.5171PMT1Protein mannosyltransferase,−0.75−0.200.42−4.18#−3.67#−4.02#−0.86Cytoskeleton organizationorf19.2286Putative deoxyhypusine hydroxylase−2.970.36−1.06−1.51−3.07#−3.08#−3.49#DNA replicationorf19.5183*POL3
*Large subunit of DNA polymerase III*−2.60−4.03#0.98−0.12−4.07#−3.39#−2.58orf19.6078POL93Putative gypsy-like reverse transcriptase−3.48#−0.48−0.43−6.24#−1.66−4.44#0.28orf19.2885*PRI2
*Putative DNA primase*0.27−3.17#−3.27#−3.20#0.35−2.050.31orf19.3322DUT1dUTP pyrophosphatase−3.24#−4.14#−0.28−2.23−3.38#−4.36#−0.44Ergosterol biosynthesisorf19.1631*ERG6
*Delta(24)-sterol C-methyltransferase*−0.670.20−3.61#−2.84−6.05#−4.09#−1.34orf19.2016*Possible role in ergosterol biosynthesis*−2.260.162.13−3.22#−3.69#−3.14#1.05orf19.922*ERG11
*Lanosterol 14-alpha-demethylase*−1.29−0.44−0.73−5.19#−4.14#−5.50#−2.26Filamentous growthorf19.815DCK1Putative guanine nucleotide exchange factor required for embedded filamentous growth−1.76−4.90#−0.070.47−4.22#−1.11−4.89#orf19.3669SHA3Putative serine/threonine kinase−1.04−2.35−0.77−3.24#−3.27#−1.55−3.89#orf19.3575*CDC19
*Putative pyruvate kinase; mutation affects filamentation*−2.04−0.070.20−3.09#−3.12#−3.97#0.49Gluconeogenesisorf19.7514*PCK1*Phosphoenolpyruvate carboxykinase*−0.22−3.90#2.821.21−3.89#−3.66#−2.91Glutathione biosynthetic processorf19.5059GCS1Gamma-glutamylcysteine synthetase−2.60−3.06#−3.10#1.00−2.27−0.52−3.85#Intracellular transportorf19.4805Putative membrane protein−1.191.35−0.56−3.69#−3.87#−3.64#−2.47Lipoate biosynthetic processorf19.819Possible role in lipoate biosynthesis−1.06−3.54#1.910.27−4.15#−3.69#−0.94NAD biosynthetic processorf19.1460Putative glutamine-dependent NAD synthetase−2.41−2.490.05−0.79−3.60#−3.23#−3.51#Oxidation/Reductionorf19.5517Similar to alcohol dehydrogenases−2.94−0.19−0.85−4.83#−6.56#−8.21#−0.07orf19.2525LYS12Mitochondrial homoisocitrate dehydrogenase−2.171.04−1.19−5.01#−5.72#−7.22#0.65RNA processingorf19.7215Ortholog(s) have snoRNA binding activity−2.83−0.06−0.38−0.16−5.20#−4.12#−3.23#orf19.3159UTP20Similar toS. cerevisiae nucleolar protein Utp10p−1.27−0.48−0.20−2.29−5.60#−3.26#−3.15#Signalingorf19.3453Possible role in signal transduction−1.58−0.210.89−4.27#−3.25#−4.07#0.09orf19.6906*ASC1
*Protein described as part of 40S ribosomal subunit*−3.30#−2.92−1.910.37−3.74#−4.70#−2.62TCA cycleorf19.6632ACO2Aconitate hydratase 2−1.14−0.250.55−3.34#−6.53#−4.39#0.05Translationorf19.6253*RPS23A
*Putative ribosomal protein*−1.19−3.68#−0.63−3.51#−2.82−2.86−3.17#orf19.4152*CEF3
*Translation elongation factor 3*−2.13−3.35#0.540.12−3.18#−1.03−3.86#Transportorf19.4599PHO89Putative phosphate permease0.73−0.06−2.63−11.05#−6.49#−5.80#−6.50#orf19.5170ENA21Possible sodium transporter sodium transporters−0.77−2.46−1.35−3.59#−4.30#−5.16#−1.38orf19.6000*CDR1
*ABC transporter involved in multidrug resistance*−0.09−0.21−2.97−1.76−5.00#−5.04#−3.82#Unknownorf19.7413MMS21-SMC5-SMC6 complex component−3.01#−3.22#1.62−3.10#−1.53−1.750.55orf19.1549Unknown−0.42−1.89−3.17#−3.96#−3.83#−2.95−1.10orf19.4651PGA53Putative GPI-anchored protein−0.84−1.33−1.16−3.73#−3.33#−3.75#0.35orf19.5799Unknown0.24−0.13−1.23−3.54#−4.95#−5.27#−1.11orf19.915Unknown−0.561.05−0.590.19−3.60#−3.15#−3.21#orf19.5760IHD1Putative GPI-anchored protein−2.64−5.84#−0.54−5.21#−3.27#−5.36#−5.36#orf19.3548Unknown−3.48#−1.88−0.441.89−3.13#−3.08#−0.96aFor further information on the open reading frames (ORFs) see the CandidaGenome Database (CGD: [20]) (http://www.candidagenome.org/).bInformation on the molecular function is abridged. More information is available at the CGD website.cFunctional groupings derived from Biological Process GO annotations. Due to space restrictions, the groupings are simplified. Individual genes can be involved in multiple biological processes.Lactobacillus-C. albicans cocultures for transcriptome analysis. The experimental setup employing a 0.45 μm membrane for separation of the bacterial and fungal cultures in 6-well tissue culture plates is represented schematically in (a). Results of a typical Lactobacillus-C. albicans coculture and C. albicans-only control experiment are shown in (b). Growth of C. albicans and Lactobacillus(Lb) in the respective compartments was determined by OD600nm readings (left y-axis). The pH of the culture media is depicted in dashed lines using the right y-axis. For transcriptome analysis, total RNA was isolated from C. albicans cells harvested from cocultures and controls.
(a)(b)The upregulation of the L-lactate cytochrome-c oxidoreductase geneCYB2 (orf19.5000) in two experiments in conjunction with the continued induction of the putative D-lactate dehydrogenase gene DLD1(orf19.5805, see Table 2(a)) indicated that the fungal cells indeed metabolized lactate despite the lack of strong induction of lactate transporter genes. It remains to be determined whether additional transport systems facilitate the transport of lactic acid or whether undissociated lactic acid enters the fungal cell by simple diffusion. Transcriptional profiling of the C. albicanscells during coculture with the probiotic lactobacilli also indicated that the fungal cells came under increased stress. The increased expression of stress-related genes (e.g., SIS1, TPS3, HSP78, TPO3, SEO1)in progressed cocultures might signify that the fungal cells were challenged by acid production of the lactobacilli and the concomitant lowering of the pH.The mixed expression profile of the stress-related genesYHB1 and NPR1(Table 2(b)) might indicate transient induction of components of the stress response, however further studies are necessary for detailed analysis of stress responses induced by the lactobacilli. Interesting aspects of the interaction of the microorganisms can also be gleaned from the list of downregulated genes (see Table 2(c)): For example, ergosterol biosynthetic genes (ERG6, ERG11, orf19.2016) were repressed. It remains to be elucidated, whether this repression was caused simply by growth retardation or specific interactions with products of the lactobacilli. Several important antifungals target the ergosterol biosynthetic pathway in fungi. The ERG11gene product lanosterol 14-alpha-demethylase is the target enzyme for fluconazole and other azoles [21–23]. The CDR1 gene encoding an important drug efflux pump involved in fluconazole resistance [21, 24, 25] appears also on the list of genes with significantly lower expression (Table 2(c)). In contrast, a related ABC transporter gene, CDR4, showed increased expression in the present study. CDR4is not involved in fluconazole resistance, but appears to be induced in the core stress response of C. albicans [26, 27]. Thus, downregulation of the target enzyme Erg11p and the drug efflux pump Cdr1p could render the fungal cells more susceptible to the antifungal drug. These findings are especially interesting in light of recent results in a randomized, double-blind and placebo-controlled trial on the effect of L. rhamnosus GR-1 and L. reuteri RC-14 application in fluconazole-treated women with vulvovaginal candidiasis [7]. The probiotics significantly improved the outcome of the treatment.Production of lactic acid and other short-chain fatty acids by the lactobacilli leads to acidification of the surrounding microenvironment such as the vaginal ecosystem or anin vitro culture vessel. Low pH favors the yeast form of C. albicansand inhibits the invasive hyphal form of these fungi [28, 29]. The transcriptome analysis in this study confirmed at the gene expression level that the fungi were in an increasingly acidic environment. For example, acidic culture conditions were indicated in all cocultures by the observed repression of the cell wall beta-(1,3)-glucanosyltransferase encoded by the PHR1 gene. This pH-responsive gene is only induced under high pH (in vitropH > 5.5) or conditions supporting hyphal growth of C. albicans [30, 31]. Interestingly, phr1/phr1 null mutants of C. albicans show defects in adhesion to abiotic and epithelial surfaces [32] indicating that repression of this gene could affect biofilm formation of C. albicans. Probiotic lactobacilli such as L. rhamnosusGR-1 are able to suppress biofilm formation of C. albicans on abiotic surfaces [33]. This is presumably achieved by combining growth inhibition and repression of genes involved in biofilm formation (e.g., PHR1, ALS12; see Table 2(c)).The list of downregulated genes in the cocultures experiments also reflects the growth inhibitory effects of the lactobacilli. Key genes involved in DNA replication (POL3, PRI2), translation (CEF3, RPS23A, ASC1), glycolysis (CDC19), and gluconeogenesis (PCK1) are expressed at lower levels. Overall, the exploratory C. albicanstranscriptome analysis presented in this study has revealed first indications on the molecular mechanisms of probiotic interference instigated by L. rhamnosusGR-1 and L. reuteriRC-14 towards the opportunistic fungal pathogen C. albicans. Elucidation of the specific inhibitory mechanisms employed by the probiotic strains will require further analysis. To this end, the development of continuous coculture systems mimicking the vaginal environment as close as possible in conjunction with genomic and proteomic analyses will further improve our understanding of the molecular basis of probiotic effects.


## 4. Conclusions

The results of the present study confirm that the probiotic strainsL. rhamnosusGR-1 and L. reuteriRC-14 are able to suppress the growth of VVC-causing C. albicans and can even kill the fungus. The probiotics were effective at low pH levels, similar to those found in a healthy vaginal environment. The transcriptome analysis elucidated some of the molecular mechanisms of probiotic interference.



---

*Source: 636474-2012-07-01.xml*