Bastiaan P. Krom

Diazirine based photoaffinity labeling.

Diazirines are among the smallest photoreactive groups that form a reactive carbene upon light irradiation. This feature has been widely utilized in photoaffinity labeling to study ligand-receptor, ligand-enzyme and protein-protein interactions, and in the isolation and identification of unknown proteins. This review summarizes recent advances in the use of diazirines in photoaffinity labeling.

Farnesol-Induced Apoptosis in Candida albicans

ABSTRACT Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival.

Role of Extracellular DNA in Initial Bacterial Adhesion and Surface Aggregation

ABSTRACT Extracellular DNA (eDNA) is an important component of the biofilm matrix. We show that removal of eDNA from Gram-positive bacteria reduces initial adhesion to and aggregation of bacteria on surfaces. Thermodynamic analyses indicated that eDNA introduces favorable acid-base interactions, explaining the effect of eDNA on aggregation and adhesion to the surface.

The Two-Component Signal Transduction Protein Chk1p Regulates Quorum Sensing in Candida albicans

ABSTRACT Regulation of hyphal morphogenesis in Candida albicans can occur through quorum sensing (QS). A QS signal, farnesol, is produced during high-density growth and inhibits morphogenesis. However, the signal transduction pathway that regulates QS is unknown. Here, we show that a C. albicans mutant lacking Chk1p but not either the Sln1p or the Nik1p histidine kinase is refractory to the inhibitory effect of farnesol both in cell suspension and during the formation of a biofilm. This study is the first to demonstrate a role for a two-component signal transduction protein in QS by a eukaryotic organism.

Macromolecular Inhibition of Quorum Sensing : Enzymes, Antibodies, and Beyond

Department of Chemistry and National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be’er Sheva, Israel; Department of Biomedical Engineering, The Kolff Institute, University Medical Center Groningen and the University of Groningen, Groningen, The Netherlands; and Departments of Chemistry and Immunology & Microbial Science, The Scripps Research Institute, La Jolla, California, USA

Effect of Cinnamon Oil on icaA Expression and Biofilm Formation by Staphylococcus epidermidis

ABSTRACT Staphylococcus epidermidis is notorious for its biofilm formation on medical devices, and novel approaches to prevent and kill S. epidermidis biofilms are desired. In this study, the effect of cinnamon oil on planktonic and biofilm cultures of clinical S. epidermidis isolates was evaluated. Initially, susceptibility to cinnamon oil in planktonic cultures was compared to the commonly used antimicrobial agents chlorhexidine, triclosan, and gentamicin. The MIC of cinnamon oil, defined as the lowest concentration able to inhibit visible microbial growth, and the minimal bactericidal concentration, the lowest concentration required to kill 99.9% of the bacteria, were determined using the broth microdilution method and plating on agar. A checkerboard assay was used to evaluate the possible synergy between cinnamon oil and the other antimicrobial agents. The effect of cinnamon oil on biofilm growth was studied in 96-well plates and with confocal laser-scanning microscopy (CLSM). Biofilm susceptibility was determined using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Real-time PCR analysis was performed to determine the effect of sub-MIC concentrations of cinnamon oil on expression of the biofilm-related gene, icaA. Cinnamon oil showed antimicrobial activity against both planktonic and biofilm cultures of clinical S. epidermidis strains. There was only a small difference between planktonic and biofilm MICs, ranging from 0.5 to 1% and 1 to 2%, respectively. CLSM images indicated that cinnamon oil is able to detach and kill existing biofilms. Thus, cinnamon oil is an effective antimicrobial agent to combat S. epidermidis biofilms.

Streptococcus mutans, Candida albicans, and the human mouth: a sticky situation

The human mouth with its diverse niches and ample supply of nutrients is undoubtedly conducive for the unrestricted formation of natural microbial biofilms. The oral microbial communities are some of the most complex microbial floras in the human body, consisting of more than 700 different bacterial species [1], [2]. Occurrence of disease results from disturbance of the equilibrium of this complex ecosystem, where population shifts lead to overrepresentation of pathogenic species which contribute to the onset and progression of the most common oral diseases, caries and periodontal disease [3]. Culture-independent molecular methods such as proteomics and 16S rRNA sequencing aiming to determine the bacterial diversity in the human oral cavity have demonstrated that in the supragingival plaque, S. mutans was the dominant species, with elevated levels of other streptococci including S. sanguinis, S. mitis, and S. salivarius in addition to lactobacilli and Veillonella. In contrast, the subgingival plaque was made up primarily of Gram-negative anaerobic bacteria such as Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia which are known to be periodontal pathogens [3]–[5]. The dental tissues—enamel, dentin, and cementum—constitute the oral solid surfaces coated by a pellicle to which the microbial cells attach. The primary colonizers and secondary organisms stick to each other on the surface of teeth and generate a matrix of exopolysaccharide within which cells grow, forming a community with a collective physiology [6]. The resulting biofilm formed, known as dental plaque, subjects the teeth and gingival tissues to high concentrations of microbial metabolites which result in dental disease [2], [7]. The interactions between the various species in these mixed biofilms can be synergistic in that the presence of one microorganism generates a niche for other pathogenic microorganisms which can serve to facilitate the retention of organisms, an oral phenomenon known as coaggregation [3], [8]. The bacteria in the biofilm are always metabolically active which causes fluctuations in pH and loss of minerals from the tooth, ultimately resulting in dissolution of the dental hard tissues and formation of lesions known as dental caries [6], [9]. Interestingly, metabolic communications among oral bacteria may occur where the excretion of a metabolite by one organism is used as a nutrient by other organisms and breakdown of a substrate by enzymatic activity of one organism creates available substrates for different organisms [10], [11].

Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p

The bacterium Staphylococcus (St.) aureus and the opportunistic fungus Candida albicans are currently among the leading nosocomial pathogens, often co-infecting critically ill patients, with high morbidity and mortality. Previous investigations have demonstrated preferential adherence of St. aureus to C. albicans hyphae during mixed biofilm growth. In this study, we aimed to characterize the mechanism behind this observed interaction. C. albicans adhesin-deficient mutant strains were screened by microscopy to identify the specific receptor on C. albicans hyphae recognized by St. aureus. Furthermore, an immunoassay was developed to validate and quantify staphylococcal binding to fungal biofilms. The findings from these experiments implicated the C. albicans adhesin agglutinin-like sequence 3 (Als3p) in playing a major role in the adherence process. This association was quantitatively established using atomic force microscopy, in which the adhesion force between single cells of the two species was significantly reduced for a C. albicans mutant strain lacking als3. Confocal microscopy further confirmed these observations, as St. aureus overlaid with a purified recombinant Als3 N-terminal domain fragment (rAls3p) exhibited robust binding. Importantly, a strain of Saccharomyces cerevisiae heterologously expressing Als3p was utilized to further confirm this adhesin as a receptor for St. aureus. Although the parental strain does not bind bacteria, expression of Als3p on the cell surface conferred upon the yeast the ability to strongly bind St. aureus. To elucidate the implications of these in vitro findings in a clinically relevant setting, an ex vivo murine model of co-infection was designed using murine tongue explants. Fluorescent microscopic images revealed extensive hyphal penetration of the epithelium typical of C. albicans mucosal infection. Interestingly, St. aureus bacterial cells were only seen within the epithelial tissue when associated with the invasive hyphae. This differed from tongues infected with St. aureus alone or in conjunction with the als3 mutant strain of C. albicans, where bacterial presence was limited to the outer layers of the oral tissue. Collectively, the findings generated from this study identified a key role for C. albicans Als3p in mediating this clinically relevant fungal-bacterial interaction.

Influence of Culture Heterogeneity in Cell Surface Charge on Adhesion and Biofilm Formation by Enterococcus faecalis

Biofilm formation is an increasing problem in medicine, due to the intrinsic resistance of microorganisms in the biofilm mode of growth against the host immune system and antimicrobial therapy. Adhesion is an important step in biofilm formation, influenced, among other factors, by the surface hydrophobicities and charges of both the substratum and the adhering microorganisms. Enterococcus faecalis strains generally display subpopulations with different surface charges, expressed as bimodal zeta potential distributions. Two-thirds of E. faecalis strains isolated from clogged biliary stents displayed such heterogeneity of surface charges in culture. In this study, the influence of this culture heterogeneity on initial adhesion and subsequent biofilm formation was investigated. Heterogeneous strains were retained in higher numbers on polystyrene than homogeneous strains. Also, biofilm formation was much more pronounced for heterogeneous strains than for homogeneous strains. In a population enriched to display only one subpopulation, fewer bacteria were retained than in its original heterogeneous culture. Also, the enriched subpopulation formed less biofilm than its original heterogeneous culture. The presence of ox bile during adhesion resulted in fewer retained bacteria, although heterogeneous strains were still retained in significantly higher numbers than were homogeneous strains, and, in general, the presence of ox bile reduced biofilm formation. The initial adhesion and biofilm formation were independent of the presence of the gene encoding the enterococcal surface protein (esp) or the expression of gelatinase (GelE). It is concluded that heterogeneity in cell surface charge represents an advantage for bacteria in the colonization of surfaces.

Complementary Metal Ion Specificity of the Metal-Citrate Transporters CitM and CitH of Bacillus subtilis

Citrate uptake in Bacillus subtilis is stimulated by a wide range of divalent metal ions. The metal ions were separated into two groups based on the expression pattern of the uptake system. The two groups correlated with the metal ion specificity of two homologous B. subtilis secondary citrate transporters, CitM and CitH, upon expression in Escherichia coli. CitM transported citrate in complex with Mg(2+), Ni(2+), Mn(2+), Co(2+), and Zn(2+) but not in complex with Ca(2+), Ba(2+), and Sr(2+). CitH transported citrate in complex with Ca(2+), Ba(2+), and Sr(2+) but not in complex with Mg(2+), Ni(2+), Mn(2+), Co(2+), and Zn(2+). Both transporters did not transport free citrate. Nevertheless, free citrate uptake could be demonstrated in B. subtilis, indicating the expression of at least a third citrate transporter, whose identity is not known. For both the CitM and CitH transporters it was demonstrated that the metal ion promoted citrate uptake and, vice versa, that citrate promoted uptake of the metal ion, indicating that the complex is the transported species. The results indicate that CitM and CitH are secondary transporters that transport complexes of divalent metal ions and citrate but with a complementary metal ion specificity. The potential physiological function of the two transporters is discussed.