The commensal fungus Candida albicans secretes a considerable number of proteins and, as in different fungal pathogens, extracellular vesicles (EVs) have also been observed. Our report contains the first proteomic analysis of EVs in C. albicans and a comparative proteomic study of the soluble secreted proteins. With this purpose, cell-free culture supernatants from C. albicans were separated into EVs and EV-free supernatant and analyzed by LC-MS/MS. A total of 96 proteins were identified including 75 and 61 proteins in EVs and EV-free supernatant, respectively. Out of these, 40 proteins were found in secretome by proteomic analysis for the first time. The soluble proteins were enriched in cell wall and secreted pathogenesis related proteins. Interestingly, more than 90% of these EV-free supernatant proteins were classical secretory proteins with predicted N-terminal signal peptide, whereas all the leaderless proteins involved in metabolism, including some moonlighting proteins, or in the exocytosis and endocytosis process were exclusively cargo of the EVs. We propose a model of the different mechanisms used by C. albicans secreted proteins to reach the extracellular medium. Furthermore, we tested the potential of the Bgl2 protein, identified in vesicles and EV-free supernatant, to protect against a systemic candidiasis in a murine model.

An in vitro biofilm model was developed and validated, demonstrating a pattern of bacterial colonization and maturation similar to the in vivo development of the subgingival biofilm.

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The opportunistic human fungal pathogen Candida albicans causes a wide variety of infections including deep systemic syndromes. The C. albicans plasma membrane is an important interface in the host-pathogen relationship. The plasma membrane proteins mediate a variety of functions, including sensing and signalling to the external environment, in which the glycosylphosphatidylinositol (GPI)-anchored membrane proteins play a crucial role. A subproteomic approach to obtain a global picture of the protein composition of the C. albicans plasma membrane was developed, and different strategies were tested in order to extract the largest number of yeast plasma membrane proteins and GPI-anchored membrane proteins. These methods involved: (i) protoplast generation, (ii) mechanical disruption, (iii) ultracentrifugation in sucrose gradients, and (iv) Na(2)CO(3) treatments. To isolate GPI-anchored proteins two additional steps were performed: two-phase separation and phosphatidylinositol-phospholipase C treatment. After LC-MS/MS analysis using both a MALDI-TOF/TOF and a linear ion trap quadrupole, a total of 214 membrane proteins were identified, including 41 already described as plasma membrane proteins, 20 plasma membrane associated proteins, and 22 proteins with unknown membrane localisation. Bioinformatic analysis revealed that this set of C. albicans membrane proteins is highly enriched in proteins involved in biopolymer biosynthesis or transport processes. Furthermore, after phosphatidylinositol-phospholipase C treatment, 12 GPI-anchored membrane proteins were released and identified; most of them are associated with cell wall beta-glucan synthesis and maintenance or are virulence factors, such as phospholipases or aspartyl proteinases.

Our results suggest that we have a highly reproducible system for multispecies oral biofilm formation and that it is a useful tool for assessing antibacterial molecules before their clinical evaluation. It also has great potential to be used in basic research on supragingival and subgingival biofilms.

SummaryWe have studied the role of acidic pH as a barrier for the colonization of the plant apoplast by Erwinia chrysanthemi . A minitransposon containing a promoterless reporter gene, gus , was used for random mutagenesis of the bacterial genome. An acid-sensitive mutant, named BT119, was isolated and had the following differential features with respect to the wildtype strain: (i) inability to grow at pH £ £ £ £ 5.5; (ii) decreased survival at acid pH and in plant tissues; (iii) increased susceptibility to antimicrobial peptides; (iv) decreased virulence in chicory leaves and pear fruits; (v) reduced polygalacturonase production; and (vi) reduced ability to alkalinize chicory tissues after infection. The sequence of the interrupted gene was highly similar to the phoQ gene, which is involved in environmental sensing in several bacteria, such as Yersinia pseudotuberculosis , Erwinia carotovora , Salmonella typhimurium and Escherichia coli and thus, this designation was used for the E. chrysanthemi system. This gene was induced at low Mg 2+ + + + concentrations and in planta . These results suggest that E. chrysanthemi PhoP-PhoQ system plays an important role in bacterial survival in plant tissues during the initial infection stages.

We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Escherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species.

Objectives To describe the early biofilm formation over whole dental implants with its micro‐ and macrostructure, using an in vitro multispecies biofilm model. Material and methods Six bacterial strains (Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans) were used to develop in vitro biofilms over whole titanium implants (growth times 12, 24, 48, 72, 96, and 120 hr). The morphology of biofilms was studied by confocal laser scanning microscopy and scanning electron microscopy, and the bacterial dynamics through quantitative polymerase chain reaction. As primary outcome variable, numbers of each species [colony‐forming units per milliliter (CFU/ml)] at different incubation times were compared using the one‐way analysis of variance and post hoc testing with Bonferroni's correction. Furthermore, implants were fixed in methacrylate stents to reproduce the clinical situation and biofilms were developed and analyzed by scanning electron microscopy. Results Bacteria colonized implants in a short period of time. Biofilms reached a mature state at 96 hr, exhibiting different ratios of live/dead cells depending on their location, being the peaks of the threads the areas harboring more live bacteria. The densities of each bacteria fluctuated in time, reaching its maximum at 96 hr. Even though the coefficients of variation were high, percentages were similar to those published previously using implant surface specimens, rather than whole implants. Conclusion Dental implants can be colonized by different bacterial species, developing into a mature and well‐structured biofilm, which depending on the location may exhibit different degree of bacterial vitality.