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 C ryptococcus neoformans is a ubiquitous encapsulated fungal pathogen that causes pneumonia and meningitis in immunocompromised individuals. Cryptococcal infection is responsible for significant morbidity and mortality in AIDS patients and is the third most common invasive fungal infection in organ transplant recipients (1, 2), causing an estimated one million cases of meningitis and over 200,000 deaths each year (3-5). Current antifungal therapy is hampered by toxicity, the emergence of drug-resistant organisms, and the inability of the host's immune system to aid in resolution of the disease; treatment is further limited by drug cost and availability in the resource-limited settings where this disease is rampant (6, 7).Cryptococcosis is primarily an opportunistic infection, highlighting the vital role of host immune responses in control of this infection. Once C. neoformans enters the host, the first line of defense it encounters is the innate immune system. Phagocytic cells, including macrophages and dendritic cells (DCs), engulf C. neoformans and present antigen to initiate the adaptive immune response.Fungal internalization by host phagocytes may benefit either the pathogen or the host, depending on microbial virulence factors, host species, host immune status, and the stage of infection. Cryptococci can survive and even proliferate in macrophage phagosomes (8, 9), potentially using these host cells to disseminate. This idea is supported by the reduced C. neoformans ...

Cdk8 and Ssn801 Regulate Oxidative Stress Resistance and Virulence in Cryptococcus neoformans

“…Tissue culture medium was chosen as a “host-like” condition that resembles the context under which C. neoformans would be most likely to experience oxidative stress (26). Morphology was categorized as diffuse, where the mitochondria are distributed in a thin, reticular network without strong focal regions of fluorescence; fragmented, where the dominant impression is of individual puncta scattered throughout the cell; or tubular, where large fused tubules may be observed (see Fig.…”

“…In parallel, cryptococcal strains were grown overnight in YPD and washed as above, opsonized in 40% human serum in PBS (30 min at 37°C at 1 × 10 7 cells/ml), washed in PBS, and resuspended in prewarmed (37°C) RPMI. Opsonized fungi were added to differentiated THP-1 cells at an MOI of 0.1 in triplicate wells in three parallel plates (26, 56). The plates were incubated for 1 h to permit uptake of opsonized cells, washed twice with sterile prewarmed PBS, and refilled with prewarmed THP-1 medium.…”

Sac1 links phosphoinositide turnover to cryptococcal virulence

“…In one example, Chun and colleagues subjected cryptococcal cells to insertional mutagenesis and identified mutations that affected their uptake by RAW264.7 macrophages (18). Srikanta et al used RNA interference and high-throughput imaging to find host regulatory genes which govern the interactions of C. neoformans with THP-1 cells and discovered 25 fungal-specific regulators of phagocytosis (19). By applying a similar high-throughput imaging assay to a C. neoformans gene deletion collection (20), Santiago-Tirado et al identified 56 fungal genes that affect uptake by THP-1 cells (10).…”

An Automated Assay to Measure Phagocytosis of Cryptococcus neoformans

“…The method presented here allows quantitation of the initial engulfment of C. neoformans. By using an automated assay to measure phagocytosis (Srikanta, Yang, Williams, & Doering, 2011), we can efficiently study fungal (Santiago-Tirado, Peng, Yang, Hang, & Doering, 2015), host (Srikanta, Hole, Williams, Khader, & Doering, 2017), or environmental factors that influence the avidity of uptake.…”