Host-pathogen interaction and signaling molecule secretion are modified in the dpp3 knockout mutant of Candida lusitaniae

Abstract

Candida lusitaniae is an emerging opportunistic yeast and an attractive model to discover new virulence factors in Candida species by reverse genetics. Our goal was to create a dpp3Δ knockout mutant and to characterize the effects of this gene inactivation on yeast in vitro and in vivo interaction with the host. The secretion of two signaling molecules in Candida species, phenethyl alcohol (PEA) and tyrosol, but not of farnesol was surprisingly altered in the dpp3Δ knockout mutant. NO and reactive oxygen species (ROS) production as well as tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) secretion were also modified in macrophages infected with this mutant. Interestingly, we found that the wild-type (WT) strain induced an increase in IL-10 secretion by zymosan-activated macrophages without the need for physical contact, whereas the dpp3Δ knockout mutant lost this ability. We further showed a striking role of PEA and tyrosol in this modulation. Last, the DPP3 gene was found to be an essential contributor to virulence in mice models, leading to an increase in TNF-α secretion and brain colonization. Although reinsertion of a WT DPP3 copy in the dpp3Δ knockout mutant was not sufficient to restore the WT phenotypes in vitro, it allowed a restoration of those observed in vivo. These data support the hypothesis that some of the phenotypes observed following DPP3 gene inactivation may be directly dependent on DPP3, while others may be the indirect consequence of another genetic modification that systematically arises when the DPP3 gene is inactivated.

FIG 1

Measurement of intracellular pyrophosphate. Intracellular PPi was measured in WT, KO, dbKO, and REC yeast lysates by fluorimetry assay. Results are expressed as mean values ± standard deviations (SD) of data from three independent experiments. Means with different letters are significantly different (P < 0.05).

FIG 2

Production of aromatic alcohols. PEA and tyrosol produced in WT, KO, and REC yeast cell cultures were quantified by GC/MS. Results are expressed as mean values ± SD of data from eight independent experiments. Means with different letters are significantly different (P < 0.05).

FIG 3

Macrophage killing of intracellular yeasts and ROS production. (A) Infected macrophages were lysed after 24 h, and WT, KO, and REC yeast mortality was assessed. (B) Fluorimetric measurement of intracellular ROS production by macrophages infected with the WT, the WT plus 10 μM tyrosol (Tyr), the KO, the KO plus 10 μM PEA, or the REC strain. Noninfected macrophages produced basal levels of ROS production that were designated “0.” Results are expressed as mean values ± SD of data from three independent experiments. Means with different letters are significantly different (P < 0.05). A.F.U., arbitrary fluorescence units.

FIG 4

Cytokine secretion by macrophages. (A) TNF-α and IL-10 measurement by ELISA at 6 h postinfection. (B) IL-10 secretion by macrophages after 6 h of coincubation with yeasts in transwells. Zymosan was added to stimulate macrophages under every set of conditions. Uninfected macrophages (Mac+Z) were used as a control. Basal levels of cytokine secretion corresponding to noninfected and nonstimulated macrophages were designated “0.” Results are expressed as mean values ± SD of data from three independent experiments. Means with different letters are significantly different (P < 0.05).

FIG 5

Effects of yeast infection on DBA2/J mouse survival rates, TNF-α levels, and brain colonization. (A) Mouse survival rates after infection with the WT, the KO, and the REC strains. For each strain, 17 to 19 mice were used. (B) TNF-α secretion in mouse sera after 4 days of infection with the WT, the KO, and the REC strains. Mice injected with PBS served as controls. Six to eight mice per strain were used for serum collection. (C) Brain colonization by the WT, the KO, and the REC strains. After 4 days, the brains of six mice from each infection group were collected and pulverized and the suspension was plated on YPD agar for CFU counting. Means with different letters are significantly different (P < 0.05).

FIG 6

RT-PCR and Western blot analyses showing the transcription and translation of the DPP3 gene. (A) DPP3 mRNA presence was detected by RT-PCR. The ACT1 housekeeping gene was used as a positive control. DNA contamination of RNA preparations was verified by PCR without the prior use of a reverse transcriptase (RT). (B) Dpp3 protein (30-kDa) presence was detected by Western blot analysis. α-Tubulin protein (50-kDa) detection was used as a positive control.