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 with C. albicans revealed that fungal cells lost metabolic activity in the presence of the lactobacilli and eventually were killed (Figure 4(a)). Semiquantitative analyses of C. albicans cell 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. albicans cell 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. johnsonii PV016 appeared to have an intermediate effect on fungal cell viability while S. aureus only 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.

Figure 4

Viability assays of C. 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 ...

3.3. Transcriptional Profiling of C. albicans in Coculture with Lactobacilli

The 0.45 μm 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. albicansgrowth as determined by OD_{600 nm} 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 Candida Affymetrix 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 JEN1 in C. albicans [18] was not induced and remained at relatively low expression levels in all conditions tested. JEN1 is 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 JEN1 is unknown.

Figure 5

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). ...

Table 2

Differentially expressed C. albicans genes in cocultures with L. rhamnosus GR-1 or L. reuteri RC-14. Z values for genes with at least three observed incidences of significant differential regulation in the tested conditions are shown. Z-values ≥3 ...

The upregulation of the L-lactate cytochrome-c oxidoreductase gene CYB2 (orf19.5000) in two experiments in conjunction with the continued induction of the putative D-lactate dehydrogenase geneDLD1 (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. albicans cells 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 genes YHB1 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. TheERG11 gene 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 an in vitro culture vessel. Low pH favors the yeast form of C. albicans and 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 vitro pH > 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. rhamnosus GR-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. albicans transcriptome analysis presented in this study has revealed first indications on the molecular mechanisms of probiotic interference instigated by L. rhamnosus GR-1 and L. reuteri RC-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.

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Conclusions

The results of the present study confirm that the probiotic strains L. rhamnosus GR-1 and L. reuteri RC-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.

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Acknowledgments

This paper was supported partially by funds from the California HIV/AIDS Research Program (former University-wide AIDS Research Program) of the University of California, Grant ID04-SF-030 to G. Köhler. Additional funding to G. Köhler was provided by the Oklahoma State University Center for Health Sciences. For annotation information on Candida genes, the authors are grateful to the CandidaGenome Database (CGD) which is funded by the National Institute of Dental & Craniofacial Research at the US National Institutes of Health. Funding of the Reid lab was provided by NSERC. They also would like to thank R. Tom Glass and Jay Bullard, OSU Center for Health Sciences, for providing the S. aureusstrain.

Date: 2016-01-03; view: 861