Genetic Interaction Analysis Reveals that Cryptococcus neoformans Utilizes Multiple Acetyl-CoA-Generating Pathways during Infection

Abstract

Cryptococcus neoformans is an important human fungal pathogen for which the external environment is its primary niche. Previous work has shown that two nonessential acetyl-CoA metabolism enzymes, ATP-citrate lyase (ACL1) and acetyl-CoA synthetase (ACS1), play important roles in C. neoformans infection. Here, we took a genetic interaction approach to studying the interplay between these two enzymes along with an enzyme initially called ACS2 but which we have found is an acetoacetyl-CoA synthetase; we have renamed the gene 2-ketobutyryl CoA synthetase 1 (KBC1) based on its biochemical activity and the systematic name of its substrate. ACL1 and ACS1 represent a synthetic lethal pair of genes based on our genetic interaction studies. Double mutants of KBC1 with either ACS1 or ACL1 do not have significant synthetic phenotypes in vitro, although we find that deletion of any one of these enzymes reduces fitness within macrophages. Importantly, the acs1Δ kbc1Δ double mutant has significantly reduced fitness in the CNS relative to either single mutant as well as WT (~2 log₁₀ CFU reduction in fungal burden), indicating the important role these enzymes play during infection. The expression of both ACS1 and KBC1 is increased in vivo relative to in vitro conditions. The acs1Δ mutant is hypersusceptible to fluconazole in vivo despite its minimal in vitro phenotypes. These data not only provide insights into the in vivo mechanism of action for a new class of antifungal Acs inhibitors but also into metabolic adaptations of C. neoformans to the host environment. IMPORTANCE The adaptation of environmental fungal pathogens to the mammalian host is critical to pathogenesis. Of these adaptations, the remodeling of carbon metabolism is particularly important. Here, we generated a focused set of double mutants of nonessential genes (ACL1, ACS1, KBC1) involved in acetyl-CoA metabolism and examined their fitness in vitro and during CNS infection. From these studies, we found that all three enzymes play important roles during infection but that the role of ACS1/KBC1 was minimal in vitro. Consistent with these observations, the expression of ACS1 and KBC1 was increased in vivo relative to standard in vitro conditions. Furthermore, strains lacking both ACL1 and ACS1 were not viable. These data clearly show that C. neoformans employs multiple carbon metabolism pathways to adapt to the host environment. They also provide insights into the potential mechanism of action for anti-cryptococcal Acs inhibitors.

Keywords: Cryptococcus neoformans; acetyl CoA; carbon metabolism; fungal pathogenesis.

FIG 1

Sequence and structure homology indicate CNAG_02045 encodes a 3-keto-butanoyl-CoA synthetase, KBC1. Phylogenetic relationship of ANL-family acyl-CoA synthetases generated using iTOL from a multiple sequence alignment using COBALT (A). Reaction diagram of ANL-family acyl-CoA synthetases (B). AlphaFold2 model of C. neoformans Kbc1 generated using the Colab server and colored by spectrum (red-to-blue with increasing level of confidence) of predicted LDDT (local distance difference test) per residue. Image rendered using PyMol v.2.4.0a0 (C). Overlay of AlphaFold2 model of C. neoformans Kbc1 (green), S. lividans Kbc1 (PDB 4WD1, orange), and key components from C. neoformas Acs1 (PDB 7KNO, cyan). Ethyl-AMP inhibitor from CnAcs1 shown to demonstrate relative position of the active site. CnAcs1 “Trp wall” residue W439 side chain occupies same position as the backbone atoms for CnKbc1 (G434) and SlKbc1 (G422), effectively closing off the pocket to larger substrates (D).

FIG 2

Biochemical characterization validates acetoacetate substrate preference of Kbc1. Kbc1 activity using different substrate substitutions for acetoacetate; all substrates at 10 mM except for acetoacetate at 1 mM.

FIG 3

Acetyl-CoA production requires ACL1 or ACS1, but not KBC1 in vitro. Diagram of major acetyl-CoA sources in C. neoformans (A). Different single and double deletion mutants made, spots on YPD were imaged after 48 h at 30°C (B). Spot dilutions of all mutants on YNB supplemented with either 2% of glucose, acetate, or leucine as the carbon source, imaged after 72 h at 30°C (C). MICs of fluconazole in RPMI, 72 h at 37°C (D).

FIG 4

ACL1, ACS1, and KBC1 are all required for full fitness in phagocytosed cells. Phagocytosis of different mutants by J774 (MOI 5:1) macrophages normalized to H99 parent strain (A). Survival of phagocytosed mutants in J774 cells 24 h after uptake, normalized to each strain’s phagocytosis levels (B). Spot dilutions of aclΔ mutants show a growth defect on RPMI-MOPS with 5% CO₂, plates imaged after 96 h at 37°C ± 5% CO₂ (C). Growth curves show aclΔ mutants have 48 h lag phase in RPMI-MOPS after YPD overnights; however, when hour 69 samples were taken and back diluted, there were no growth differences between any strain (D). Analysis done using a one-way ANOVA; *, P < 0.05 compared to H99.

FIG 5

ACS1 and KBC1 expression is increased in vivo and in vitro in the presence of fatty acid. In vivo ACS1 and KBC1 expression from lungs recovered 17 days after intranasal inoculation and brains recovered 8 days after intravenous inoculation compared to in vitro cultures grown to early log phase in RPMI at 30°C (A). ACS1 and KBC1 expression is induced by the addition of a soluble lipid (Tween 20) to RPMI (B).

FIG 6

Alternative Pathways for acetyl-CoA generation are required for full fitness during brain infection and affect in vivo fluconazole susceptibility. CD-1 mice were inoculated with either the acs1Δ or kbc1Δ mutant and treated with either 125 mg/kg fluconazole or vehicle for 2 days when their brains were harvested, homogenized, plated, and growth at 30°C for 48 h before counting (A). The experiment was repeated with the acs1kbc1 double mutant (B). Analysis done using a two-way ANOVA; ***, 0.0001 < P < 0.001; ****, P < 0.0001.