Pseudomonas aeruginosa inhibits the growth of Cryptococcus species

Abstract

Pseudomonas aeruginosa is a ubiquitous and opportunistic bacterium that inhibits the growth of different microorganisms, including Gram-positive bacteria and fungi such as Candida spp. and Aspergillus fumigatus. In this study, we investigated the interaction between P. aeruginosa and Cryptococcus spp. We found that P. aeruginosa PA14 and, to a lesser extent, PAO1 significantly inhibited the growth of Cryptococcus spp. The inhibition of growth was observed on solid medium by the visualization of a zone of inhibition of yeast growth and in liquid culture by viable cell counting. Interestingly, such inhibition was only observed when P. aeruginosa and Cryptococcus were co-cultured. Minimal inhibition was observed when cell-cell contact was prevented using a separation membrane, suggesting that cell contact is required for inhibition. Using mutant strains of Pseudomonas quinoline signaling, we showed that P. aeruginosa inhibited the growth of Cryptococcus spp. by producing antifungal molecules pyocyanin, a redox-active phenazine, and 2-heptyl-3,4-dihydroxyquinoline (PQS), an extracellular quorum-sensing signal. Because both P. aeruginosa and Cryptococcus neoformans are commonly found in lung infections of immunocompromised patients, this study may have important implication for the interaction of these microbes in both an ecological and a clinical point of view.

Fig. 1

Pa inhibited the growth of Cryptococcus spp. PAO1 and PA14 were inoculated on paper disks, on YNB plates with C. neoformans var. grubii serotype A (Cn serotype A H99), C. gattii serotypes B (Cg serotype B MMRL 1336), C. gattii serotypes C (Cg serotype C MMRL1343) and C. neoformans var. neoformans serotype D (Cn serotype D JEC21). Control disks containing LB medium were placed on the plates (indicated with ctrl on the plates). The diameters of the zone of inhibition were determined after 72 h at 30°C

Fig. 2

Pa has fungicidal effect against C. neoformans in co-culture. Different C. neoformans (Cn) dilutions were mixed with Pa strains and incubated in 96-well plates. The viability of Cn in co-culture with PAO1 (a) and PA14 (c) was determined by CFU counting on YNB plates. The viability of PAO1 (b) and PA14 (d) in co-culture with Cn was determined by CFU counting on LB plates. The inhibitory effect of Pa on Cn is fungicidal and is dependent on the relative cell density

Fig. 3

Enhanced inhibition of C. neoformans by Pa in co-culture supernatants. Dried co-culture supernatants [Sup(Cn H99 + PAO1), Sup(Cn H99 + PA14)] (a, c) and dried Pa supernatants (SupPAO1, SupPA14) (b, d), resuspended in YPD broth, were inoculated into 8-mm wells on YPD plates, which contained a lawn of C. neoformans (Cn H99). The diameters of the zone of inhibition were determined

Fig. 4

Cell–cell contact is important for Pa to inhibit C. neoformans. An assay in YPD media using a U-tube was performed. A sterile membrane was used to separate the two sides of the U-tube, in which Pa strains and C. neoformans (Cn) were cultured. Separately, Erlenmeyer flasks were used for controls, including co-cultures of Cn with PAO1 or PA14, Cn with E. coli and cultures of Cn alone and PAO1 or PA14 alone. The inhibitory effect of Pa on Cn, in the U-tube and in co-culture, was determined by CFU counting (a, c). When E. coli and Cn were co-cultured, no inhibitory effects of Cn were observed (a, c). The growth of PAO1 and PA14 was not affected by Cn (b, d). P values were calculated by Student’s t test, *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

Pa inhibited C. neoformans in physiological condition. PAO1 and PA14 were co-cultured with C. neoformans (Cn) in DMEM for 24 and 48 h. Viable Cn and Pa were measured by CFU on YNB and LB plates, respectively. Cn inhibition was observed with both Pa strains (a, c). Pa cells number counting showed no significant increase in the number of cells in DMEM (b, d). P-values were calculated by Student’s t test, *P < 0.05

Fig. 6

Effects of pqs mutants of PAO1 on the growth of C. neoformans. The pyocyanin production was measured for all mutants (a). pqsB mutant secreted pyocyanin at a level that was about 50% of the wild-type PAO1 (a). PAO1, pqsAB, pqsB and pqsE were inoculated on paper disks, on YNB plates, which contained a lawn of C. neoformans (Cn). The diameters of the zone of inhibition were determined after 72 h at 30°C (b)

Fig. 7

Antifungal activity of pyocyanin and PQS against C. neoformans. Chemical structures of 3,4-dihydroxy-2-heptylquinoline (PQS), 2,4-dihydroxyquinoline (DHQ), 4-hydroxy-2-heptylquinoline (HHQ) and pyocyanin (a). C. neoformans (Cn) was incubated in microtiter plates with different concentration of PQS, DHQ, HHQ and PYO. The growth of yeast cells was assessed by monitoring optical density at 495 nm (b)