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1.  Probiotic Interference of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 with the Opportunistic Fungal Pathogen Candida albicans 
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.
PMCID: PMC3395238  PMID: 22811591
2.  Microbiota Found in Protective Athletic Mouthguards 
Sports Health  2011;3(3):244-248.
Protective athletic mouthguards (PAM) produce oral mucosal injuries and may be associated with other systemic conditions.
With wear, PAM become contaminated by a range of microorganisms. The number of microorganisms in PAM can be reduced by daily use of an antimicrobial solution. Study Type: Case series.
Study Design and Methods:
Sixty-two division I football player volunteers were divided into 4 groups, using PAM for varying lengths of time before surrendering them for microbial analysis. One group had their PAM soaked in an antimicrobial solution between uses. The PAM were analyzed qualitatively and quantitatively for bacteria, yeasts, and fungi, using previously accepted methods.
The 62 football players surrendered a total of 81 PAM for microbial analysis. The PAM yielded 154 gram-positive cocci, 150 gram-positive bacilli, 21 gram-negative cocci, 31 gram-negative bacilli, 22 yeasts, and 107 molds. The most common species of gram-positive cocci were Staphylococcus spp. and Micrococcus spp. Only 3 PAM (4%) were positive for Staphylococcus aureus. The most common species of gram-positive bacilli were Brevibacterium spp. and Cellulomonas spp. The most common species of yeasts were Candida parapsilosis and Rhodotorula mucilaginosa, while the most common species of mold were Cochliobolus spp. and Penicillium chrysogenum. Soaking the PAM in an antimicrobial solution between uses substantially reduced the numbers of microorganisms.
Substantial microbial contamination of PAM occurs with use. The microbial load can be reduced by soaking in an antimicrobial solution between uses. Clinical Relevance: PAM are contaminated by microorganisms that have the potential to produce oral and systemic diseases. They should be sanitized daily and changed when they become sharp and/or jagged.
PMCID: PMC3445166  PMID: 23016013
protective athletic mouthguards; contamination; disease transmission; oral infections; systemic infections; microorganisms; mouthguard care
3.  Inactivation of the phospholipase B gene PLB5 in wild-type Candida albicans reduces cell-associated phospholipase A2 activity and attenuates virulence 
Phospholipases are critical for modification and redistribution of lipid substrates, membrane remodeling and microbial virulence. Among the many different classes of phospholipases, fungal phospholipase B (Plb) proteins show the broadest range of substrate specificity and hydrolytic activity, hydrolyzing acyl ester bonds in phospholipids and lysophospholipids and further catalyzing lysophospholipase-transacylase reactions. The genome of the opportunistic fungal pathogen Candida albicans encodes a PLB multigene family with five putative members; we present the first characterization of this group of potential virulence determinants. CaPLB5, the third member of this multigene family characterized herein is a putative secretory protein with a predicted GPI-anchor attachment site. Real-time RT-PCR gene expression analysis of CaPLB5 and the additional CaPLB gene family members revealed that filamentous growth and physiologically relevant environmental conditions are associated with increased phospholipase B gene activity. The phenotypes expressed by null mutant and revertant strains of CaPLB5 indicate that this lipid hydrolase plays an important role for cell-associated phospholipase A2 activity and in vivo organ colonization.
PMCID: PMC2481510  PMID: 16759910
GPI anchor; Phospholipase; Lysophospholipase; Candida; Selection marker; Virulence
4.  Phospholipase A2 and Phospholipase B Activities in Fungi 
Biochimica et biophysica acta  2006;1761(11):1391-1399.
As saprophytes or disease causing microorganisms, fungi acquire nutrients from dead organic material or living host organisms. Lipids as structural components of cell membranes and storage compartments play an important role as energy-rich food source. In recent years, it also has become clear that lipids have a wide range of bioactive properties including signal transduction and cell to cell communication. Thus, it is not surprising that fungi possess a broad range of hydrolytic enzymes that attack neutral lipids and phospholipids. Especially during infection of a mammalian host, phospholipase A2 (PLA2) enzymes released by fungi could play important roles not only for nutrient acquisition and tissue invasion, but for intricate modulation of the host’s immune response. Sequencing of fungal genomes has revealed a wide range of genes encoding PLA2 activities in fungi. We are just beginning to become aware of the significance these enzymes could have for the fungal cells and their interaction with the host.
PMCID: PMC2077850  PMID: 17081801
5.  Role of Calcineurin in Stress Resistance, Morphogenesis, and Virulence of a Candida albicans Wild-Type Strain  
Infection and Immunity  2006;74(7):4366-4369.
By generating a calcineurin mutant of the Candida albicans wild-type strain SC5314 with the help of a new recyclable dominant selection marker, we confirmed that calcineurin mediates tolerance to a variety of stress conditions but is not required for the ability of C. albicans to switch to filamentous growth in response to hypha-inducing environmental signals. While calcineurin was essential for virulence of C. albicans in a mouse model of disseminated candidiasis, deletion of CMP1 did not significantly affect virulence during vaginal or pulmonary infection, demonstrating that the requirement for calcineurin for a successful infection depends on the host niche.
PMCID: PMC1489686  PMID: 16790813
6.  A Human-Curated Annotation of the Candida albicans Genome 
PLoS Genetics  2005;1(1):e1.
Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.
Candida albicans is a commonly encountered fungal pathogen usually responsible for superficial infections (thrush and vaginitis). However, an estimated 30% of severe fungal infections, most due to Candida, result in death. Those who are most at risk include individuals taking immune-suppressive drugs following organ transplantation, people with HIV infection, premature infants, and cancer patients undergoing chemotherapy. Current therapies for this pathogen are made more difficult by the significant secondary effects of anti-fungal drugs that target proteins that are also found in the human host.
Recent sequencing and assembly of the genome for the fungal pathogen C. albicans used simple automated procedures for the identification of putative genes. Here, we report a detailed annotation of the 6,354 genes that are present in the genome sequence of this organism, essentially writing the dictionary of the C. albicans genome.
Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that are absent from the human genome and whose products might be targeted for antifungal therapy. The results of these efforts will thus ensure that the Candida research community has uniform and comprehensive genomic information for medical research, for the development of functional genomic tools as well as for future diagnostic and therapeutic applications.
PMCID: PMC1183520  PMID: 16103911
7.  Activation of the Multiple Drug Resistance Gene MDR1 in Fluconazole-Resistant, Clinical Candida albicans Strains Is Caused by Mutations in a trans-Regulatory Factor 
Journal of Bacteriology  2000;182(2):400-404.
Resistance of Candida albicans against the widely used antifungal agent fluconazole is often due to active drug efflux from the cells. In many fluconazole-resistant C. albicans isolates the reduced intracellular drug accumulation correlates with constitutive strong expression of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not detectably expressed in vitro in fluconazole-susceptible isolates. To elucidate the molecular changes responsible for MDR1 activation, two pairs of matched fluconazole-susceptible and resistant isolates in which drug resistance coincided with stable MDR1 activation were analyzed. Sequence analysis of the MDR1 regulatory region did not reveal any promoter mutations in the resistant isolates that might account for the altered expression of the gene. To test for a possible involvement of trans-regulatory factors, a GFP reporter gene was placed under the control of the MDR1 promoter from the fluconazole-susceptible C. albicans strain CAI4, which does not express the MDR1 gene in vitro. This MDR1P-GFP fusion was integrated into the genome of the clinical C. albicans isolates with the help of the dominant selection marker MPAR developed for the transformation of C. albicans wild-type strains. Integration was targeted to an ectopic locus such that no recombination between the heterologous and resident MDR1 promoters occurred. The transformants of the two resistant isolates exhibited a fluorescent phenotype, whereas transformants of the corresponding susceptible isolates did not express the GFP gene. These results demonstrate that the MDR1 promoter was activated by a trans-regulatory factor that was mutated in fluconazole-resistant isolates, resulting in deregulated, constitutive MDR1 expression.
PMCID: PMC94289  PMID: 10629186

Results 1-7 (7)