Project description:Candida albicans, a major opportunistic fungal pathogen is frequently found together with Streptococcus mutans in dental biofilms associated with severe childhood tooth-decay, a prevalent pediatric oral disease. Previous studies have demonstrated that S. mutans and C. albicans synergizes virulence of plaque-biofilms in vivo. However, the nature of this bacterial-fungal relationship in this cross-kingdom biofilm remains largely uncharacterized. Using iTRAQ based quantitative proteomics, we found that proteins associated with carbohydrate metabolism such as alpha-1,4 glucan phosphorylase, Hexokinase-2, Isocitrate lyase and malate synthase were significantly upregulated in C. albicans in the mixed-species biofilms (P<0.05). C. albicans proteins associated with growth/morphogenesis such as pH-responsive protein-2, Fma1p and Hsp21 were also induced. Conversely, S. mutans proteins in the tricarboxylic acid cycle such as citrate synthase and in the pentose phosphate pathway such as Ribose-5-phosphate isomerase A as well as proteins associated with sugar transport systems were upregulated indicating enhanced carbohydrate metabolism. Interestingly mixed-species biofilm microenvironment had a lower pH than S. mutans single-species biofilms. This observation was supported by proteomics, wherein proteins associated with lactate and formate assimilation such as Glyoxalase and putative NADPH-dependent methylglyoxal reductase proteins were significantly upregulated in the mixed-species biofilms (P<0.05). Furthermore, we unexpectedly found that S. mutans derived glucosyltransferase B (GtfB), responsible for co-adhesion via glucans, can also contribute to C. albicans growth and carbohydrate metabolism by providing glucose and fructose from sucrose breakdown. These findings demonstrate synergistic bacterial-fungal interactions within mixed-species biofilms and a novel GtfB cross-feeding role. Taken together, quantitative proteomics provides new insights into this virulent cross-kingdom oral biofilm.
Project description:Fungal-bacterial interactions generate unique biofilms that cause many infections in humans. Candida albicans interact with Streptococcus mutans in dental biofilms associated with severe childhood tooth-decay, a prevalent pediatric oral disease. Current modalities are ineffective and primarily based on antimicrobial monotherapies despite the polymicrobial nature of the infection. Here, we show that the combination of clinically used topical antifungal fluconazole with povidone iodine (PI) can completely suppress C. albicans carriage and mixed-biofilm formation without increasing bacterial killing activity in vivo. We unexpectedly found that the inclusion of PI enhanced fluconazole efficacy by potently disrupting the assembly of a protective bacterial exopolysaccharide (EPS) matrix through inhibition of α-glucan synthesis by S. mutans exoenzyme (GtfB) bound on the fungal surface. Further analyses revealed that the EPS produced in situ directly bind and sequester fluconazole, reducing uptake and intracellular transportation of the drug. Conversely, inhibition of GtfB activity by PI, enzymatic degradation of the α-glucan matrix or co-culturing with gtfB-defective S. mutans re-established antifungal susceptibility. Hence, topical antifungal has limitations in mixed oral biofilms due to enhanced C. albicans tolerance to fluconazole afforded by the shielding effect of bacterial-derived EPS. The data provide new insights for treatment of C. albicans in cross-kingdom biofilms, indicating that EPS inhibitors may be required for enhanced killing efficacy and optimal anti-biofilm activity.
Project description:MicroRNAs (miRNAs) are endogenous, noncoding, smallRNAs that regulate gene expression at the post-transcriptional level during plant development, growth and seed germination. Among all medicinal plants, Moringa oleifera is one of the most useful trees for which, despite its diffusion, no information about its miRNAs and their respective target genes is available. In this research, we report results obtained from a high-throughput sequencing analysis performed with the Illumina platform. A total of 31,290,964 raw reads were produced from M. oleifera seed smallRNA library. First, we found 99 conserved miRNAs and 43 novel ones that we partially validated by qRT- PCR. Second, by comparing their expression abundances with those of other common plants, we identified 20 conserved M. oleifera miRNAs. For both these results an in silico analysis allowed us to predict some of their targets which in turn allowed us to link them to a wide range of physiological processes. Based on qRT-PCR expression analyses, we reported the expression profile of some selected conserved miRNAs in different M. oleifera tissues (roots, stems and leafs). We compared the most conserved miRNAs found in Moringa with those of other edible plants, such as Olea europaea and Brassica rapa. Furthermore, by taking advantage of a recently developed web- application based on an algorithm that compares plants and mammalian miRNAs, we identify a few possible plant miRNAs with functional homologies with mammalian ones. We used the 20 most abundant M. oleifera miRNAs to conduct a preliminary analysis to investigate potential cross-kingdom gene regulation. To our knowledge, this is the first report of M. oleifera miRNAs that uses high-throughput sequencing analysis. Our analysis increases the amount of information about plant miRNAs currently available and it can help us understanding the molecular mechanism of this medical plant. microRNA profile of M. oleifera seed, germinated on paper soaked in sterile water.
Project description:MicroRNAs (miRNAs) are endogenous, noncoding, smallRNAs that regulate gene expression at the post-transcriptional level during plant development, growth and seed germination. Among all medicinal plants, Moringa oleifera is one of the most useful trees for which, despite its diffusion, no information about its miRNAs and their respective target genes is available. In this research, we report results obtained from a high-throughput sequencing analysis performed with the Illumina platform. A total of 31,290,964 raw reads were produced from M. oleifera seed smallRNA library. First, we found 99 conserved miRNAs and 43 novel ones that we partially validated by qRT- PCR. Second, by comparing their expression abundances with those of other common plants, we identified 20 conserved M. oleifera miRNAs. For both these results an in silico analysis allowed us to predict some of their targets which in turn allowed us to link them to a wide range of physiological processes. Based on qRT-PCR expression analyses, we reported the expression profile of some selected conserved miRNAs in different M. oleifera tissues (roots, stems and leafs). We compared the most conserved miRNAs found in Moringa with those of other edible plants, such as Olea europaea and Brassica rapa. Furthermore, by taking advantage of a recently developed web- application based on an algorithm that compares plants and mammalian miRNAs, we identify a few possible plant miRNAs with functional homologies with mammalian ones. We used the 20 most abundant M. oleifera miRNAs to conduct a preliminary analysis to investigate potential cross-kingdom gene regulation. To our knowledge, this is the first report of M. oleifera miRNAs that uses high-throughput sequencing analysis. Our analysis increases the amount of information about plant miRNAs currently available and it can help us understanding the molecular mechanism of this medical plant.
Project description:Candida albicans is known to form polymicrobial biofilms with various Streptococcus spp., including mitis and mutans group streptococci. Streptococcus gordonii (mitis group) has been shown to bind avidly to C. albicans hyphae via direct cell-to-cell interaction, while the cariogenic pathogen Streptococcus mutans (mutans group) interacts with the fungal cells via extracellular glucans. However, the biophysical properties of these cross-kingdom interactions at the single-cell level during the early stage of biofilm formation remain understudied. Here, we examined the binding forces between S. mutans (or S. gordonii) and C. albicans in the presence and absence of in situ glucans on the fungal surface using single-cell atomic force microscopy and their influence on biofilm initiation and subsequent development under cariogenic conditions. The data show that S. gordonii binding force to the C. albicans surface is significantly higher than that ofS. mutans to the fungal surface (~2-fold). However, S. mutans binding forces are dramatically enhanced when the C. albicans cell surface is locally coated with extracellular glucans (~6-fold vs. uncoated C. albicans), which vastly exceeds the forces between S. gordonii andC. albicans. The enhanced binding affinity of S. mutans to glucan-coated C. albicans resulted in a larger structure during early biofilm initiation compared to S. gordonii-C. albicans biofilms. Ultimately, this resulted in S. mutans dominance composition in the 3-species biofilm model under cariogenic conditions. This study provides a novel biophysical aspect of Candida-streptococcal interaction whereby extracellular glucans may selectively favor S. mutans binding interactions with C. albicans during cariogenic biofilm development.