RNA Interference Screening Reveals Host CaMK4 as a Regulator of Cryptococcal Uptake and Pathogenesis

Abstract

Cryptococcus neoformans, the causative agent of cryptococcosis, is an opportunistic fungal pathogen that kills over 200,000 individuals annually. This yeast may grow freely in body fluids, but it also flourishes within host cells. Despite extensive research on cryptococcal pathogenesis, host genes involved in the initial engulfment of fungi and subsequent stages of infection are woefully understudied. To address this issue, we combined short interfering RNA silencing and a high-throughput imaging assay to identify host regulators that specifically influence cryptococcal uptake. Of 868 phosphatase and kinase genes assayed, we discovered 79 whose silencing significantly affected cryptococcal engulfment. For 25 of these, the effects were fungus specific, as opposed to general alterations in phagocytosis. Four members of this group significantly and specifically altered cryptococcal uptake; one of them encoded CaMK4, a calcium/calmodulin-dependent protein kinase. Pharmacological inhibition of CaMK4 recapitulated the observed defects in phagocytosis. Furthermore, mice deficient in CaMK4 showed increased survival compared to wild-type mice upon infection with C. neoformans This increase in survival correlated with decreased expression of pattern recognition receptors on host phagocytes known to recognize C. neoformans Altogether, we have identified a kinase that is involved in C. neoformans internalization by host cells and in host resistance to this deadly infection.

Keywords: Cryptococcus neoformans; fungal pathogenesis; image-based screen.

FIG 1

RNAi screening for regulators of fungal uptake. (A) Fungal uptake by THP-1 cells treated with siRNA pools targeting CamK4 (orange) or 867 other kinases and phosphatases (dark blue symbols), no siRNA (light blue symbols), or scrambled siRNA (white symbols); see Materials and Methods for additional controls. The red lines indicate the upper and lower boundaries of the plate averages using the quartile-based threshold method. Candidate genes outside those bounds (79) advanced to the next round of screening. (B) A second round of screening identified gene depletions that altered uptake of cryptococcal cells but not of an inert particle control (latex beads). Results were normalized to no-siRNA control assays with the appropriate particle and assessed by Student's t test. Controls (as described for panel A) fitted to the diagonal (red line) had an R² value of 0.76. (C) THP-1 cells were treated with siRNA targeting CaMK4, and uptake of C. neoformans (Cn), C. albicans (Ca), S. cerevisiae (Sc), E. coli (Ec), or latex beads (LB) was assayed. Phagocytosis of C. neoformans was significantly decreased compared to that of the other particles (P < 0.1 for C. albicans and P < 0.05 for all other particles). Red line, normalized uptake value of 1.

FIG 2

Inhibition or knockout of CamK4 influences fungal uptake and pathogenesis. (A) THP-1 cells were assayed for uptake of the organisms indicated after treatment with PBS alone (blue) or 10 nM KN93 in PBS (orange). The averages and standard errors of the means (SEM) from five independent studies are shown (*, P < 0.05 by Student's t test). (B) Survival of C57BL/6 mice treated with either PBS alone (blue) or with 250 μg/g body weight KN93 in PBS (orange). Mice were treated 1 day prior to infection with 5 × 10⁴ cryptococci and three times a week postinfection. Results are representative of two independent experiments with 6 mice per group; P < 0.05 by log-rank test. (C) Peritoneal macrophages from WT (blue) and CaMK4^−/− (orange) mice were assayed for uptake of C. neoformans. The averages and SEM from four independent studies are shown (***, P < 0.0001 by Student's t test). (D) Wild-type (WT; n = 16) and CaMK4 knockout (n = 9) mice were infected with C. neoformans and monitored as detailed in Materials and Methods. Results are representative of four independent experiments; P < 0.05 by log-rank test.

FIG 3

CaMK4 knockout mice exhibit no difference in leukocyte infiltration in response to C. neoformans infection. Shown are leukocyte profiles of WT (blue circles) and CaMK4 knockout mice (orange squares) infected with 10⁴ C. neoformans KN99α for the times shown. Each symbol represents data from an individual mouse. Results from 2 independent experiments with 4 to 5 mice per group per time point are shown; means ± SEM are also plotted. Leukocytes were labeled with antibodies to identify B cells (CD45⁺ and CD19⁺), CD4⁺ T cells (CD45⁺, CD4⁺, and CD8⁻), CD8⁺ T cells (CD45⁺, CD8⁺, and CD4⁻), DCs (CD45⁺, CD11b⁺, CD11c⁺, and F4/80⁻), eosinophils (CD45⁺, CD11b⁺, and SiglecF⁺), exudate or recruited macrophages (CD45⁺, CD11b⁺, CD11c⁺, and F4/80⁺), macrophages (CD45⁺, CD11b⁺, CD11c⁻, and F4/80⁺), and polymorphonuclear leukocytes (PMNs; CD45⁺, CD11b⁺, and Ly6G^hi) and then analyzed by flow cytometry (see Table S3 in the supplemental material for antibodies and Fig. S4 for gating strategy).

FIG 4

Leukocytes from CaMK4^−/− mice show reduced expression of PRRs implicated in cryptococcal recognition. PRR profiling of leukocytes from WT (blue circles) and CaMK4 knockout (orange squares) mice 7 days after infection with 10⁴ C. neoformans KN99α. Antibody labeling and flow cytometry was used to classify pulmonary leukocytes as DCs (CD45⁺, CD11b⁺, CD11c⁺, and F4/80⁻), exudate or recruited macrophages (Ex Mϕ) (CD45⁺, CD11b⁺, CD11c⁺, and F4/80⁺), or macrophages/monocytes (CD45⁺, CD11b⁺, CD11c⁻, and F4/80⁺) and to assess PRR expression. Symbols indicate data from individual mice in 2 independent experiments with 4 to 5 mice per group; means ± SEM are also shown (*, P < 0.05; **, P < 0.01 by Student's t test).