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Pseudomonas-Candida Interactions: An Ecological Role for Virulence Factors
Deborah A. Hogan and Roberto Kolter

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Materials and Methods

Growth conditions. Unless otherwise stated, all experiments were performed with P. aeruginosa PA14, C. albicans SC5314, or C. albicans tup1 (S1) grown in M63 medium (S2) with 0.2% glucose with shaking at either 30°C or 37°C depending on the experiment. Yeast-form C. albicans SC5314 was grown at 30°C. C. albicans filamentation was induced by growth at 37°C; the majority of C. albicans cells were filamentous under these conditions. Coincubation of P. aeruginosa and C. albicans was performed in conditioned medium, which is defined as sterile filtered supernatant from a wild-type P. aeruginosa culture grown to early stationary phase (OD600 = 1.2). The use of conditioned medium created a nutrient-limiting environment that favored biofilm formation on the fungal filaments. This medium also allowed us to dilute P. aeruginosa to low densities suitable for microscopy while maintaining the nutrient-limiting environment.

Microscopy. For microscopy studies, 1 ml of C. albicans was transferred from an overnight culture to a 35-mm Petri dish with a 0.1-mm-thick glass coverslip covering a hole in the bottom of the dish (MatTek Corp., Ashland, MA). Dishes were spun in a swinging bucket centrifuge at 3000 rpm for 15 min, then gently washed once to remove cells that did not sediment to the bottom. Bacteria from an overnight culture were diluted in 2 ml of conditioned medium to a final density of 5 Multiplication Symbol 106 CFU/ml, then added to the dish containing C. albicans. Cultures were incubated statically at 37°C. Phase-contrast images were captured with a 100Multiplication Symbol objective on a Nikon Eclipse TE300 inverted microscope equipped with Metamorph imaging software (Universal Imaging Corporation, Downingtown, PA.) For transmission electron microscopy, P. aeruginosa and C. albicans were incubated together in suspension for 10 min prior to negative staining with 1% uranyl acetate; scanning electron microscopy samples were coincubated for 48 h prior to fixation with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. Samples were prepared for scanning electron microscopy according to standard protocols by the Electron Microscopy Facility in Cell Biology at Harvard Medical School.

P. aeruginosa mutants. The origins of the P. aeruginosa PA14 mutants are as follows: lasR (S3); rhlR (made in PA14 using an rhlR::tetR construct (pSB224.10A) described in (S4); gacA, plcS, toxA (S5); phnAB (S6); plcR (constructed by L. Rahme); rpoN (S7); pilB, pilC, flgK (S8). Other than the rpoN mutant, all strains have similar growth rates in M63 medium.

Attachment assays. For attachment assays, bacteria were diluted in 2 ml of conditioned medium to an OD600 = 0.025. The bacterial suspension was transferred to a 35-mm dish containing washed fungal filaments as described above. Plate counts were performed on the diluted P. aeruginosa cultures to assure that equivalent numbers of cells were used. To count the average number of bacteria per single filamentous cell, P. aeruginosa and C. albicanstup1 were incubated together for exactly 10 min prior to fixation by the addition of formaldehyde (0.02% final concentration). Because the C. albicanstup1 mutant grows predominantly in the pseudohyphal form with septal invaginations between cells, individual cells of roughly equivalent lengths were easily identified for the purposes of counting the number of bacteria per filament. To determine the number of filaments colonized by P. aeruginosa, the two organisms were coincubated for 20 min, fixed with formaldehyde, then scored as either positive (with more than 1 bacterium attached per filamentous cell) or negative. In both experiments, cells were counted using a 100Multiplication Symbol objective lens on an inverted microscope. For each sample, 150 filaments were examined; duplicate samples were analyzed for enumeration assays, and triplicate samples were included for the percent colonization experiments. Each experiment was repeated at least twice and the sample identities were revealed to the experimenter only after the analyses were completed.

Assessing virulence of P. aeruginosa mutants toward C. albicans filaments. For viability assays, C. albicans filaments were incubated with P. aeruginosa (5 Multiplication Symbol 106 CFU/ml) in 2 ml conditioned medium in a 13 Multiplication Symbol 100 mm tube. Tubes were incubated with shaking at 37°C. C. albicans titers were determined by plating on YPD medium supplemented with tetracycline (60 Greek Letter Mulg/ml), gentamicin (30 Greek Letter Mulg/ml), and chloramphenicol (30 Greek Letter Mulg/ml) to suppress the growth of P. aeruginosa.

Alternative media conditions. Biofilm formation by P. aeruginosa and killing of the fungal filaments occurred with similar kinetics in conditioned medium from P. aeruginosa cultures grown in either MOPS medium (S9) and M63 at both 30°C and 37°C. C. albicanstup1 filaments were killed to below the limit of detection by P. aeruginosa in Luria-Bertani medium in less than 20 hours at 37°C. C. albicans SC5314 and tup1 grow well in M63, MOPS, and Luria-Bertani as pure cultures.


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