Fungal NOX is an essential factor for induction of TG2 in human hepatocytes.

NADPH oxidases (Nox) generate reactive oxygen species (ROS) such as superoxide anion radical (O2-) and hydrogen peroxide (H2O2). The pathogenic fungi Candida albicans and Candida glabrata enhance cellular transglutaminase 2 (TG2) activity levels in co-cultured human hepatic cells in a ROS-mediated manner. Deletion of NOX1 (CgNOX1) in C. glabrata blocks the ability of C. glabrata to induce TG2 activity. Here, we investigated whether Nox proteins from C. albicans and Saccharomyces cerevisiae are related with induction of TG2 activity in hepatic cells. C. albicans CFL11 (CaCFL11) was identified as a key factor in this fungus for hepatic TG2 induction in the co-cultures. The cfl11 mutant of C. albicans did not induce TG2 activity in hepatocytes. In addition, overexpression of YNO1, a homolog of CgNOX1, in S. cerevisiae led to induction of ROS generation and TG2 activity in hepatic cells in co-incubation experiments. These findings indicated that a fungal Nox plays a role in enhancing TG2 activity in human hepatocytes and leads to apoptosis.

Identification and functional characterization of Candida albicans mannose-ethanolamine phosphotransferase (Mcd4p).

Glycosylphosphatidylinositol (GPI) is an important compound for the growth of fungi, because GPI-anchored proteins including glycosyltransferases and adhesins are involved in cell-wall integrity, adhesion, and nutrient uptake in this organism. In this study, we examined orf19.5244 in the genome database of the pathogenic fungus Candida albicans, a homologue of the Saccharomyces cerevisiae mannose-ethanolamine phosphotransferase gene, MCD4, which plays a role in GPI synthesis. Expression of this homologue, designated CaMCD4, restored cell growth in a defective conditional mcd4 mutant of S. cerevisiae, Scmcd4t, in which expression of native MCD4 was repressed in the presence of doxycycline (Dox). Analysis of radiolabeled lipids showed that the accumulation of abnormal GPI anchor precursors in Scmcd4t decreased markedly upon expression of CaMCD4. Moreover, we constructed a single mutant (Camcd4/CaMCD4) and a conditional double mutant (Camcd4/Camcd4t) at the MCD4 locus of C. albicans. Repression of CaMCD4 expression by Dox led to a decrease in growth and appearance of abnormal morphology in C. albicans, both in vitro and in a silkworm infection model. These results suggest that CaMcd4p is indispensable for growth of C. albicans both in vitro and in infected hosts and a candidate target for the development of new antifungals.

Role of major facilitator superfamily transporter Qdr2p in biofilm formation by Candida glabrata.

Candida glabrata represents the second-most frequent cause of candidiasis infections of the mucosa, bloodstream and genito-urinary tract in immunocompromised individuals. The incidence of C glabrata infection has increased significantly in the last two decades, mainly due to this species' abilities to resist various antifungal drugs and to form biofilms. We focused on the relationship between biofilm formation and the product of QDR2, a C glabrata member of the major facilitator superfamily (MFS) gene family, given that fungal biofilm formation limits drug penetration and is associated with persistent infection. The fungal cells in biofilms were compared between a C glabrata ∆qdr2 mutant and its wild-type strain. Cells were analysed for metabolism activity and drug susceptibility (using tetrazolium assay), adhesion activity, growth assay and intracellular pH (using flow cytometry). Compared to the wild type, the C glabrata ∆qdr2 showed lower adhesion activity and higher fluconazole susceptibility when assessed as a biofilm. The mutant also showed decreased metabolic activity during biofilm formation. Furthermore, the mutant grew more slowly under neutral-basic pH conditions. The qdr2 deletion in C glabrata resulted in an impaired ability to maintain pH homeostasis, which led in turn to a reduction of cell growth and of adherence to an artificial matrix. These results suggested that the Qdr2p function is needed for proper biofilm formation and biofilm maintenance in C glabrata as well as biofilm drug resistance towards fluconazole. Qdr2p may play an important role in C glabrata's ability to form biofilms on implanted medical devices in human bodies.

Candida albicans fatty acyl-CoA synthetase, CaFaa4p, is involved in the uptake of exogenous long-chain fatty acids and cell activity in the biofilm.

Fatty acyl-CoA synthetase (Faa) activates fatty acid (FA) by converting the FA into the CoA ester in the cell. In the present study, we characterized a FAA homologue (CaFAA4) from the opportunistic pathogen Candida albicans. Most organisms can not only synthesize long-chain fatty acyl-CoAs (LCFA-CoAs) endogenously using a fatty acid synthase (Fas) activity but also can uptake long-chain fatty acids (LCFAs) from the extracellular environment and convert them into LCFA-CoAs via a vectorial acylation system. The budding yeast Saccharomyces cerevisiae possesses two LCFA-CoA synthetases, ScFaa1p and ScFaa4p. The disruption of ScFAA1 and ScFAA4 leads to synthetic lethality in the presence of a fatty acid synthesis inhibitor-cerulenin. The homologue-CaFAA4-rescued the lethality of an S. cerevisiae Scfaa1-Scfaa4 double mutant in the presence of cerulenin. On the other hand, a C. albicans faa4 mutant was unable to grow in the presence of cerulenin even if LCFAs were provided exogenously. Moreover, a biofilm analysis showed that the metabolic activity of the Cafaa4 mutant was approximately 40% lower than that of the wild-type parent, even though there was no significant difference in cell number or cell morphology between these strains. Notably, the Cafaa4 mutant showed increased susceptibility to micafungin during biofilm formation, a phenotype that presumably can be attributed to the impaired metabolism of the mutant strain. These results indicated that CaFaa4p is the unique C. albicans Faa protein responsible for activating LCFAs and is involved in the metabolism of biofilms.

The role of Bgl2p in the transition to filamentous cells during biofilm formation by Candida albicans.

The fungal pathogen Candida albicans undergoes a transition from yeast cells to filamentous cells that is related to its pathogenicity. The complex multicellular processes involved in biofilm formation by this fungus also include this transition. In this work, we investigated the morphological role of the Bgl2 protein (Bgl2p) in the transition to filamentous cells during biofilm formation by C. albicans. Bgl2p has been identified as a ß-1, 3-glucosyltransferase, and transcription of the CaBGL2 gene is upregulated during biofilm formation. We used scanning electron microscopy to observe the microstructure of a bgl2 null mutant during biofilm formation and found a delay in the transition to filamentous cells in the premature phase (24 hours) of biofilm formation. Deletion of the CaBGL2 gene led to a decrease in the expression of CPH2 and TEC1, which encode transcription factors required for the transition to the filamentous form. These findings indicate that Bgl2p plays a role in the transition to filamentous cells during biofilm formation by C. albicans.

Identification and characterization of Candida utilis multidrug efflux transporter CuCdr1p.

The edible, nitrate assimilating, yeast Candida utilis is a commercial food additive, and it is a potentially useful host for heterologous protein expression. A number of ATP-binding cassette (ABC) transporters are multidrug efflux pumps that can cause multidrug resistance in opportunistic pathogens. In order to develop optimal novel antimicrobial agents it is imperative to understand the structure, function and expression of these transporters. With the ultimate aim of developing an alternative yeast host for the heterologous expression of eukaryotic membrane transporters, and to identify ABC transporters potentially associated with C. utilis multidrug resistance, we classified the entire repertoire of 30 C. utilis ABC proteins. We named the open reading frame most similar to the archetype multidrug efflux pump gene C. albicans CDR1 as CuCDR1 Overexpression of CuCDR1 in Saccharomyces cerevisiae ADΔ caused multidrug resistance similar to that of cells overexpressing CaCDR1 Unlike CaCdr1p, however, the C-terminally green fluorescent protein (GFP) tagged CuCdr1p-GFP was functionally impaired and did not properly localize to the plasma membrane. CuCdr1p function could be recovered however by adding a 15 amino acid linker -GAGGSAGGSGGAGAG- between CuCdr1p and the C-terminal GFP tag.

Identification and functional characterization of Penicillium marneffei pleiotropic drug resistance transporters ABC1 and ABC2.

Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump.

Toll-like Receptor 10 in Helicobacter pylori Infection.

Innate immunity plays important roles in the primary defense against pathogens, and epidemiological studies have suggested a role for Toll-like receptor 1 (TLR1) in Helicobacter pylori susceptibility. Microarray analysis of gastric biopsy specimens from H. pylori-positive and uninfected subjects showed that TLR10 messenger RNA (mRNA) levels were upregulated approximately 15-fold in infected subjects; these findings were confirmed by real-time quantitative polymerase chain reaction analysis. Immunohistochemical investigation showed increased TLR10 expression in the gastric epithelial cells of infected individuals. When H. pylori was cocultured with NCI-N87 gastric cells, both TLR10 and TLR2 mRNA levels were upregulated. We compared the ability of TLR combinations to mediate nuclear factor-κB (NF-κB) activation. Compared with other TLR2 subfamily heterodimers, the TLR2/TLR10 heterodimer mediated the greatest NF-κB activation following exposure to heat-killed H. pylori or H. pylori lipopolysaccharide. We conclude that TLR10 is a functional receptor involved in the innate immune response to H. pylori infection and that the TLR2/TLR10 heterodimer functions in H. pylori lipopolysaccharide recognition.

Discovery of novel mutations for clarithromycin resistance in Helicobacter pylori by using next-generation sequencing.

OBJECTIVES: Resistance to clarithromycin is the most important factor causing failure of Helicobacter pylori eradication. Although clarithromycin resistance is mainly associated with three point mutations in the 23S rRNA genes, it is unclear whether other mutations are associated with this resistance. METHODS: Two types of clarithromycin-resistant strains (low- and high-resistance strains) were obtained from clarithromycin-susceptible H. pylori following exposure to low clarithromycin concentrations. The genome sequences were determined with a next-generation sequencer. Natural transformation was used to introduce the candidate mutations into strain 26695. Etest and an agar dilution method were used to determine the MICs. RESULTS: High-resistance strains contained the mutation A2143G in the 23S rRNA genes, whereas low-resistance strains did not. There were seven candidate mutations in six genes outside of the 23S rRNA genes. The mutated sequences in hp1048 (infB), hp1314 (rpl22) and the 23S rRNA gene were successfully transformed into strain 26695 and the transformants showed an increased MIC of and low resistance to clarithromycin. The transformants containing a single mutation in infB or rpl22 (either a 9 bp insertion or a 3 bp deletion) or the 23S rRNA gene showed low MICs (0.5, 2.0, 4.0 and 32 mg/L, respectively) while the transformants containing double mutations (mutation in the 23S rRNA genes and mutation in infB or rpl22) showed higher MICs (>256 mg/L). CONCLUSIONS: Next-generation sequencing can be a useful tool for screening mutations related to drug resistance. We discovered novel mutations related to clarithromycin resistance in H. pylori (infB and rpl22), which have synergic effects with 23S rRNA resulting in higher MICs.

Gene cluster responsible for secretion of and immunity to multiple bacteriocins, the NKR-5-3 enterocins.

Enterococcus faecium NKR-5-3, isolated from Thai fermented fish, is characterized by the unique ability to produce five bacteriocins, namely, enterocins NKR-5-3A, -B, -C, -D, and -Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). Genetic analysis with a genome library revealed that the bacteriocin structural genes (enkA [ent53A], enkC [ent53C], enkD [ent53D], and enkZ [ent53Z]) that encode these peptides (except for Ent53B) are located in close proximity to each other. This NKR-5-3ACDZ (Ent53ACDZ) enterocin gene cluster (approximately 13 kb long) includes certain bacteriocin biosynthetic genes such as an ABC transporter gene (enkT), two immunity genes (enkIaz and enkIc), a response regulator (enkR), and a histidine protein kinase (enkK). Heterologous-expression studies of enkT and ΔenkT mutant strains showed that enkT is responsible for the secretion of Ent53A, Ent53C, Ent53D, and Ent53Z, suggesting that EnkT is a wide-range ABC transporter that contributes to the effective production of these bacteriocins. In addition, EnkIaz and EnkIc were found to confer self-immunity to the respective bacteriocins. Furthermore, bacteriocin induction assays performed with the ΔenkRK mutant strain showed that EnkR and EnkK are regulatory proteins responsible for bacteriocin production and that, together with Ent53D, they constitute a three-component regulatory system. Thus, the Ent53ACDZ gene cluster is essential for the biosynthesis and regulation of NKR-5-3 enterocins, and this is, to our knowledge, the first report that demonstrates the secretion of multiple bacteriocins by an ABC transporter.

Bifunctional gene cluster lnqBCDEF mediates bacteriocin production and immunity with differential genetic requirements.

A comprehensive gene disruption of lacticin Q biosynthetic cluster lnqQBCDEF was carried out. The results demonstrated the necessity of the complete set of lnqQBCDEF for lacticin Q production, whereas immunity was flexible, with LnqEF (ABC transporter) being essential for and LnqBCD partially contributing to immunity.

Uptake of Levilactobacillus brevis JCM 1059 by THP-1 Cells via Interaction between SlpB and CAP-1 Promotes Cytokine Production.

Several probiotic lactic acid bacteria (LAB) exert immunomodulatory effects on the host. However, the reasons for the different effects of LAB have not been fully elucidated. To understand the different immunomodulatory effects of LAB, we evaluated the levels of critical molecules in differentiated monocytic THP-1 and dendritic cells (DCs) following the uptake of various LAB strains. Lactobacillus helveticus JCM 1120, Lactobacillus acidophilus JCM 1132, Levilactobacillus brevis JCM 1059, and Lentilactobacillus kefiri JCM 5818 showed significantly higher uptake among the 12 LAB species tested. The uptake of microbeads by THP-1 DC increased when coupled with the surface layer proteins (Slps) from the tested strains. SlpB was mainly observed in the L. brevis JCM 1059 Slps extract. The expected cell surface receptor for SlpB on THP-1 DC was purified using SlpB-coupled affinity resin and identified as adenylyl cyclase-associated protein 1 (CAP-1). SlpB binding to THP-1 DC decreased after the addition of anti-CAP-1 and anti-DC-SIGN antibodies but not after the addition of anti-macrophage-inducible C-type lectin (Mincle) antibody. These results suggest that SlpB on L. brevis JCM 1059 plays preferentially binds to CAP-1 on THP-1 DC and plays a crucial role in bacterial uptake by THP-1 cells as well as in subsequent interleukin-12 (IL-12) production.

Identification of the genes involved in the secretion and self-immunity of lacticin Q, an unmodified leaderless bacteriocin from Lactococcus lactis QU 5.

Lacticin Q (LnqQ) produced by Lactococcus lactis QU 5 is an unmodified linear bacteriocin, which is synthesized without an N-terminal leader peptide. In vitro synthesis and in vivo expression of LnqQ have revealed the intracellular toxicity of this leaderless peptide, as well as the necessity of a dedicated secretion and self-immunity system of producer cells. Further DNA sequencing and analysis have discovered 11 putative orf genes at the LnqQ locus. None of the orf genes showed similarities to any of the bacteriocin biosynthetic genes characterized to date; however, six orf genes (orf2q-7q), not including the structural gene (lnqQ), were highly conserved at the lacticin Z locus (orf2z-7z), which is a LnqQ homologue produced by L. lactis QU 14. ORF2q (ORF2z), the gene of which is located upstream of the structural gene, is a putative transcriptional regulator, whereas ORF6q and ORF7q (ORF6z and ORF7z) form a putative ATP-binding cassette transporter. The ORF3q-5q (ORF3z-5z) are all predicted to be membrane proteins with no clear functions. Co-expression of LnqQ and ORF3q-7q in a heterologous host allowed the extracellular production of LnqQ; additionally, the expression of ORF3q-7q rendered the host cells immune to LnqQ. This self-immunity was facilitated possibly by two means; firstly, by secreting the active LnqQ peptides, thus reducing the intracellular toxicity, and secondly, by protecting the host cells from extracellularly released LnqQ. This is the first report, to our knowledge, that describes intracellular toxicity of a leaderless bacteriocin and provides a rare example of biosynthetic genes that are required for bacteriocin secretion and immunity.

Lactobacillus johnsonii enhances the gut barrier integrity via the interaction between GAPDH and the mouse tight junction protein JAM-2.

Commensal intestinal microbiota interacts with gut epithelial cells in the host by binding to specific host receptors. Several pattern recognition receptors on the gut that sense conserved microbial-associated molecular patterns have been reported; however, many of the gut receptor molecules involved in bacterial binding have not yet been identified. In this study, commensal intestinal bacteria interacting with mouse gut surface proteins were screened from fecal bacterial samples, to identify novel receptors on the epithelial cells in the mouse gut. Among the screened intestinal lactic acid bacteria, the frequently isolated Lactobacillus johnsonii MG was used for the purification of gut receptor proteins. An approximately 30 kDa protein was purified using affinity resin coupled surface layer proteins isolated from L. johnsonii MG. The purified gut protein was identified as a member of the tight junction protein family, junctional adhesion molecule-2 (JAM-2). As expected, the tight junctions of Caco-2 cells damaged by H2O2 were repaired by incubation with L. johnsonii MG. RNA sequence analysis showed significant upregulation of the expression of genes for tight junctions, anti-inflammatory effects, transcriptional regulation, and apoptosis in Caco-2 cells, following L. johnsonii MG treatment. In L. johnsonii MG, the surface layer 40 kDa protein was purified with gut protein-coupled affinity resin and identified as the moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These results suggest that L. johnsonii MG promotes the barrier function integrity in Caco-2 cells via GAPDH-JAM-2 binding. Here, we propose a promising approach to identify novel gut receptor molecules based on commensal bacterial interactions and understand host-bacterial communication in a mouse model.

Release of an anti-anxiety peptide in casein hydrolysate with Aspergillus oryzae protease.

Food protein-derived peptides with agonistic effects on receptors have great potential for treating anxiety, hypertension, and stress. In the present study, opioid peptides with agonistic activities for δ-receptor-expressing HEK293 cells were screened from casein hydrolysates prepared with five types of food grade proteolytic enzymes, among which casein hydrolysate with Aspergillus oryzae protease ASD showed the highest opioid activity. Eluted fractions showing potent opioid activity were further purified for active peptides by reverse phase-HPLC. The peptide in the active fraction was identified as YPFPGPIPNS, a member of ß-casomorphin (CM-10) (ß-casein 60-69). Various CM-10 derivative peptides were synthesized and their characteristic features for specificities towards δ- and µ-receptors were determined. Peptides 5 to 12 amino acids long showed relatively higher opioid activities for δ- and µ-receptors. CM-10 was docked into the optimized δ-receptor model. The CDOCKER energies of the CM-10 derivatives were consistent with their opioid activities. In the elevated plus-maze study, CM-10 showed a significant anti-anxiety effect in BALB/c mice at a dose of 10 mg per kg body weight when administered orally, but not via intravenous injection. Furthermore, intravital imaging revealed that Ca2+ signaling was induced in the small intestinal villi of a Yellow Cameleon 3.60 (YC3.60)-expressing mouse upon injection with CM-10. However, this decreased in the presence of δ- or µ-receptor antagonists. These results suggest that the opioid peptide CM-10 prepared from casein with ASD has an anti-anxiety effect through interaction with gut δ- and/or µ-opioid receptors in the mouse gut.

Dietary Bacillus subtilis C-3102 Supplementation Enhances the Exclusion of Salmonella enterica from Chickens.

Among the reported probiotic Bacillus strains, B. subtilis C-3102 has the unique potential to improve feed uptake under stress conditions in the broilers, piglets, and cows. In this study, we sought to evaluate the protective effect of feed additive probiotic Bacillus subtilis C-3102 against Salmonella enterica infection of specific pathogen-free (SPF) chicks in floor pens in two experiments. In the experiment-1, the chicks in the control group (n=32) were fed a basal diet and those in the C-3102 group (n=32) were fed a basal diet supplemented with 1×106 CFU/g of feed for 28 days. On day 7 post-challenge with S. enterica, there was no significant change in the body weight between both the groups throughout the test period, whereas detection rates of S. enterica in the C-3102 group were significantly lower in the cecum and liver on days 21 and 14 post-challenge, respectively. In the experiment-2, minimum dosage of C-3102 cells required to protect Salmonella infection was evaluated using 3 dosages. Chicks were divided into four groups, fed with different dosages of C-3102 (1×106, 5×105, 3×105, and 0 CFU/g of feed), and challenged with S. enterica (2.8×108 CFU/chicken). S. enterica infection was completed within 7 days post- challenge and was almost excluded from the liver and spleen on day 21 post- challenge in the control group. Average values showed a trend for higher infection rates in the control group >3×105>5×105>1×106 CFU/g on days 14 and 21 post-challenge. These results suggest that B. subtilis C-3102 supplementation has the potential to reduce S. enterica infection rates and/or to accelerate the exclusion of S. enterica from the chicks.

The Lack of SNARE Protein Homolog Syn8 Influences Biofilm Formation of Candida glabrata.

Biofilm formation of Candida species is considered to be a pathogenic factor of host infection. Since biofilm formation of Candida glabrata has not been as well studied as that of Candida albicans, we performed genetic screening of C. glabrata, and three candidate genes associated with biofilm formation were identified. Candida glabrata SYN8 (CAGL0H06325g) was selected as the most induced gene in biofilm cells for further research. Our results indicated that the syn8Δ mutant was defective not only in biofilm metabolic activity but also in biofilm morphological structure and biomass. Deletion of SYN8 seemed to have no effect on extracellular matrix production, but it led to a notable decrease in adhesion ability during biofilm formation, which may be linked to the repression of two adhesin genes, EPA10 and EPA22. Furthermore, hypersensitivity to hygromycin B and various ions in addition to the abnormal vacuolar morphology in the syn8Δ mutant suggested that active vacuolar function is required for biofilm formation of C. glabrata. These findings enhance our understanding of biofilm formation in this fungus and provide information for the development of future clinical treatments.

Probiotic Bacillus subtilis C-3102 Improves Eggshell Quality after Forced Molting in Aged Laying Hens.

This study was carried out to evaluate the effects of probiotic Bacillus subtilis C-3102 feed additive on quality characteristics including strength, thickness, and weight of eggshells of Boris Brown laying hens. The control group (n=64) was fed a basal diet comprised of maize and feed rice, whereas the experimental group (n=64) was fed a basal diet supplemented with B. subtilis C-3102 (3×105 CFU/g) starting at 49 weeks of age. From 67 to 69 weeks, all hens were induced to molt using an anorexic program; then, the birds in both groups returned to their respective diets (from 69 to 82 weeks). Eggshell strength, measured six times with 60 eggs selected before the molting treatment, was significantly greater in the C-3102 group than in the control group at 51, 59, 63, and 66 weeks (3.45, 3.44, 3.28, and 3.13 kg/cm2; P<0.05, 0.05, 0.01, and 0.01, respectively). Moreover, eggshell strength-measured three times after the molting treatment-was significantly greater in the C-3102 group than in the control group at 73 and 77 weeks (3.79 and 3.65 kg/cm2; P<0.01 and 0.01, respectively). Eggshell thickness was also significantly greater in the C-3102 group than in the control group at 73 and 77 weeks (0.400 and 0.390 mm; P<0.01 and 0.01, respectively). Fecal samples collected from eight hens of each group at 70 weeks of age after forced molting, showed a significantly higher proportion of Lactobacillus spp. in the C-3102 group (8.94 log CFU/g) (P<0.05) than in the control group (8.63 log CFU/g). Clostridium spp. abundance was significantly lower in the C-3102 group (2.92 log CFU/g) than in the control group (4.3 log CFU/g). These results suggest that C-3102 supplementation improves eggshell quality in aged laying hens, particularly after forced molting.

Candida albicans Bgl2p, Ecm33p, and Als1p proteins are involved in adhesion to saliva-coated hydroxyapatite.

Introduction: Candida albicans is an opportunistic pathogen that causes oral candidiasis. A previous study showed that Bgl2p and Ecm33p may mediate the interaction between the yeast and saliva-coated hydroxyapatite (SHA; a model for the tooth surface). This study investigated the roles of these cell wall proteins in the adherence of C. albicans to SHA beads. Methods: C. albicans BGL2 and ECM33 null mutants were generated from wild-type strain SC5314 by using the SAT1-flipper gene disruption method. A novel method based on labelling the yeast with Nile red, was used to investigate the adherence. Results: Adhesion of bgl2Δ and ecm33Δ null mutants to SHA beads was 76.4% and 64.8% of the wild-type strain, respectively. Interestingly, the adhesion of the bgl2Δ, ecm33Δ double mutant (87.7%) was higher than that of both single mutants. qRT-PCR analysis indicated that the ALS1 gene was over-expressed in the bgl2Δ, ecm33Δ strain. The triple null mutant showed a significantly reduced adherence to the beads, (37.6%), compared to the wild-type  strain. Conclusion: Bgl2p and Ecm33p contributed to the interaction between C. albicans and SHA beads. Deletion of these genes triggered overexpression of the ALS1 gene in the bgl2Δ/ecm33Δ mutant strain, and deletion of all three genes caused a significant decrease in adhesion.