Pathogenic basidiomycetous yeast, Cryptococcus neoformans, causes fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug commonly administered to treat cryptococcosis. Unfortunately, FLC-resistant strains characterized by various degree of chromosomal instability were isolated from clinical patients. Importantly, the underlying mechanisms that lead to chromosomal instability in FLC-treated C. neoformans remain elusive. Previous studies in fungal and mammalian cells link chromosomal instability to the reactive oxygen species (ROS). This study provides the evidence that exposure of C. neoformans to FLC induces accumulation of intracellular ROS, which correlates with plasma membrane damage. FLC caused transcription changes of oxidative stress related genes encoding superoxide dismutase (SOD1), catalase (CAT3), and thioredoxin reductase (TRR1). Strikingly, FLC contributed to an increase of the DNA damage in vitro, when complexed with iron or copper in the presence of hydrogen peroxide. Strains with isogenic deletion of copper response protein metallothionein were more susceptible to FLC. Addition of ascorbic acid (AA), an anti-oxidant at 10 mM, reduced the inhibitory effects of FLC. Consistent with potential effects of FLC on DNA integrity and chromosomal segregation, FLC treatment led to elevated transcription of RAD54 and repression of cohesin-encoding gene SCC1. We propose that FLC forms complexes with metals and contributes to elevated ROS, which may lead to chromosomal instability in C. neoformans.

Despite intensive antimicrobial and anti-inflammatory therapies, cystic fibrosis (CF) patients are subjected to chronic infections due to opportunistic pathogens, including multidrug resistant (MDR) Pseudomonas aeruginosa. Macrophages from CF patients show many evidences of reduced phagocytosis in terms of internalization capability, phagosome maturation, and intracellular bacterial killing. In this study, we investigated if apoptotic body-like liposomes (ABLs) loaded with phosphatidylinositol 5-phosphate (PI5P), known to regulate actin dynamics and vesicular trafficking, could restore phagocytic machinery while limiting inflammatory response in in vitro and in vivo models of MDR P. aeruginosa infection. Our results show that the in vitro treatment with ABL carrying PI5P (ABL/PI5P) enhances bacterial uptake, ROS production, phagosome acidification, and intracellular bacterial killing in human monocyte-derived macrophages (MDMs) with pharmacologically inhibited cystic fibrosis transmembrane conductance regulator channel (CFTR), and improve uptake and intracellular killing of MDR P. aeruginosa in CF macrophages with impaired bactericidal activity. Moreover, ABL/PI5P stimulation of CFTR-inhibited MDM infected with MDR P. aeruginosa significantly reduces NF-κB activation and the production of TNF-α, IL-1β, and IL-6, while increasing IL-10 and TGF-β levels. The therapeutic efficacy of ABL/PI5P given by pulmonary administration was evaluated in a murine model of chronic infection with MDR P. aeruginosa . The treatment with ABL/PI5P significantly reduces pulmonary neutrophil infiltrate and the levels of KC and MCP-2 cytokines in the lungs, without affecting pulmonary bacterial load. Altogether, these results show that the ABL/PI5P treatment may represent a promising host-directed therapeutic approach to improve the impaired phagocytosis and to limit the potentially tissue-damaging inflammatory response in CF.

Accurate cellular replication balances the biogenesis and turnover of complex structures. Apicomplexan parasites such as Plasmodium and Toxoplasma replicate by forming daughter cells within an intact mother cell, creating additional challenges to ensuring fidelity of division. Critical to these parasites' infectivity is an intricate cytoskeleton structure called the apical complex. Before the daughter apical complex can be inserted into the plasma membrane, the maternal material must be turned over. We previously identified the kinase ERK7 as required for the maturation of the apical complex in Toxoplasma gondii. Here we define the Toxoplasma ERK7 interactome, and identify a putative E3 ligase, CSAR1, as the downstream effector responsible for the phenotype. Genetic disruption of CSAR1 fully suppresses loss of the apical complex upon ERK7 knockdown. Furthermore, we show that CSAR1 is normally responsible for turnover of maternal cytoskeleton during cytokinesis, and that its aberrant function is driven by a mislocalization from the parasite residual body to the maternal and daughter apical complexes. These data identify a protein homeostasis pathway critical for Toxoplasma replication and fitness and suggest an unappreciated role for the parasite residual body in compartmentalizing processes that threaten the fidelity of parasite development.

Accurate cellular replication balances the biogenesis and turnover of complex structures. In the apicomplexan parasite Toxoplasma gondii, daughter cells form within an intact mother cell, creating additional challenges to ensuring fidelity of division. The apical complex is critical to parasite infectivity and consists of apical secretory organelles and specialized cytoskeletal structures. We previously identified the kinase ERK7 as required for maturation of the apical complex in Toxoplasma. Here, we define the Toxoplasma ERK7 interactome, including a putative E3 ligase, CSAR1. Genetic disruption of CSAR1 fully suppresses loss of the apical complex upon ERK7 knockdown. Furthermore, we show that CSAR1 is normally responsible for turnover of maternal cytoskeleton during cytokinesis, and that its aberrant function is driven by mislocalization from the parasite residual body to the apical complex. These data identify a protein homeostasis pathway critical for Toxoplasma replication and fitness and suggest an unappreciated role for the parasite residual body in compartmentalizing processes that threaten the fidelity of parasite development.

While the canonical mitogen‐activated protein kinase (MAPK) pathways are well‐established and almost ubiquitous across the eukaryotic kingdoms, Toxoplasma gondii expresses a family of MAPK‐like proteins that deviate from classical kinase activation. One of these MAPK‐like proteins, MAPKL2, is exclusively found in alveolates, a superphylum that includes dinoflagellates, ciliates, and apicomplexa. T. gondii is an apicomplexan intracellular parasite that can infect nearly all warm‐blooded animals, including humans. MAPKL2 is an essential kinase without which the parasite dies, and thereby recommends itself as an ideal potential drug target for T. gondii, a pathogen for which treatment is limited, expensive, and often toxic to patients. Thus, the model organism provides both the context of an informative cellular biology and a medically relevant model for studying this non‐canonical MAP kinase. We used yeast‐two‐hybrid to identify potential binding partners and have narrowed the minimum sufficient binding region of the proteins to within 15–30 residues. By determining the minimum sufficient binding regions of the three proteins of interest, a consensus sequence may be developed for use in screening for more biochemical binders in T. gondii and other alveolates. Recent experiments suggesting localization and association with specific classes of proteins will also be discussed. Narrowing the category of proteins within which the consensus sequence is used to screen will provide a more focused direction for investigation of the role of MAPKL2 in biochemical and molecular mechanisms within the parasite and related species.