Biology subjects

Lodge, J. K.

Publications and source records attributed to Lodge, J. K..

4 recordsLinked to original sources

bioRxiv2026

Mitochondrial cardiolipin sequestration of caspofungin underlies Cryptococcus neoformans inherent resistance and may contribute to cardiotoxicity

Cryptococcus exhibits inherent resistance to the echinocandin, caspofungin, which inhibits the synthesis of (1,3)-{beta}-D-glucan, a key component of the polysaccharide cell wall. The essential FKS1 gene encodes the catalytic subunit of (1,3)-{beta}-D-glucan synthase and caspofungin effectively inhibits its activity in vitro, yet the drug remains ineffective against Cryptococcus, suggesting mechanisms beyond target insensitivity. The underlying mechanisms of caspofungin resistance remain unknown, although altered regulation of cell-wall remodeling genes, plasma membrane modifications, drug efflux pathways, and melanin biosynthesis have been suggested. Using boron dipyrromethene (BD-) and fluorescein (F-) labelled caspofungin, we demonstrate that caspofungin enters the cryptococcal cell and primarily accumulates in the mitochondrial inner membrane rather than the plasma membrane. We further establish that this mitochondrial accumulation is driven by a specific interaction between caspofungin and cardiolipin, a phospholipid found in mitochondrial membranes. We demonstrate that this unforeseen localization indicates that mitochondrial sequestration diminishes the effective drug concentrations at the intended target. Notably, the interaction between caspofungin and cardiolipin also takes place in human cells, establishing a mechanistic connection to caspofungin-related cardiotoxicity. Our findings reveal a previously unrecognized mechanism of echinocandin resistance in Cryptococcus and emphasize cardiolipin as an important factor in caspofungin effectiveness.

microbiology↗

bioRxiv2019

Cryptococcus neoformans Chitin Synthase 3 (Chs3) Plays a Critical Role in Dampening Host Inflammatory Responses

Cryptococcus neoformans infections are significant causes of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. One of the main interfaces between the fungus and the host is the fungal cell wall. The cryptococcal cell wall is unusual among human pathogenic fungi in that the chitin is predominantly deacetylated to chitosan. Chitosan deficient strains of C. neoformans were found to be avirulent and rapidly cleared from the murine lung. Moreover, infection with a chitosan deficient C. neoformans lacking three chitin deacetylases (cda1{Delta}2{Delta}3{Delta}) was found to confer protective immunity to a subsequent challenge with a virulent wild type counterpart. In addition to the chitin deacetylases, it was previously shown that chitin synthase 3 (Chs3) is also essential for chitin deacetylase mediated formation of chitosan. Mice inoculated with chs3{Delta} at a dose previously shown to induce protection with cda1{Delta}2{Delta}3{Delta} die within 36 hours after installation of the organism. Mortality was not dependent on viable fungi as mice inoculated with heat-killed preparation of chs3{Delta} died at the same rate as mice inoculated with live chs3{Delta}, suggesting the rapid onset of death was host mediated likely caused by an over exuberant immune response. Histology, cytokine profiling, and flow cytometry indicates a massive neutrophil influx in the mice inoculated with chs3{Delta}. Mice depleted of neutrophils survived chs3{Delta} inoculation indicating that death was neutrophil mediated. Altogether, these studies lead us to conclude that Chs3, along with chitosan, plays critical roles in dampening cryptococcal induced host inflammatory responses.\n\nIMPORTANCECryptococcus neoformans is the most common disseminated fungal pathogen in AIDS patients, resulting in [~]200,000 deaths each year. There is a pressing need for new treatments for this infection, as current antifungal therapy is hampered by toxicity and/or the inability of the hosts immune system to aid in resolution of the disease. An ideal target for new therapies is the fungal cell wall. The cryptococcal cell wall is different than many other pathogenic fungi in that it contains chitosan. Strains that have decreased chitosan are less pathogenic and strains that are deficient in chitosan are avirulent and can induce protective responses. In this study we investigated the host responses to chs3{Delta}, a chitosan-deficient strain, and found mice inoculated with chs3{Delta} all died within 36 hours and death was associated with an aberrant hyperinflammatory immune response driven by neutrophils, indicating that chitosan is critical in modulating the immune response to Cryptococcus.

immunology↗

bioRxiv2019

Chitosan biosynthesis and virulence in the human fungal pathogen Cryptococcus gattii

Cryptococcus gattii R265 is a hyper-virulent fungal strain responsible for the major outbreak of cryptococcosis in Vancouver Island of British Columbia in 1999. It differs significantly from C. neoformans in its natural environment, its preferred site in the mammalian host, and in the nature and mode of pathogenesis. Our previous studies in C. neoformans have shown that the presence of chitosan, the deacetylated form of chitin, in the cell wall attenuates inflammatory responses in the host, while its absence induces robust immune responses, which in turn facilitate clearance of the fungus and induces a protective response. The results of the present investigation reveal that the cell wall of C. gattii R265 contains 2-3-fold higher amount of chitosan compared to that of C. neoformans. The genes responsible for the biosynthesis of chitosan are highly conserved in the R265 genome; the roles of the three chitin deacetylases (CDA) have however, been modified. To deduce their roles, single, double and a triple CDA deletion strains were constructed in a R265 background and were subjected to mammalian infection studies. Unlike C. neoformans where Cda1 has a discernible role in fungal pathogenesis, in R265 Cda3 is critical for virulence. Deletion of either CDA3 alone (cda3{Delta}) or in combination with either CDA1 (cda1{Delta}3{Delta}) or CDA2 (cda2{Delta}3{Delta}) or both (cda1{Delta}2{Delta}3{Delta}) rendered the yeast cells avirulent and were cleared from the infected host. Moreover, the cda1{Delta}2{Delta}3{Delta} strain of R265 induced a protective response to a subsequent infection with R265. These studies shed more light into the regulation of chitosan biosynthesis of C. gattii and its subsequent effect on fungal virulence.\n\nImportanceThe fungal cell wall is an essential organelle whose components provide the first line of defense against host-induced antifungal activity. Chitosan is one of the carbohydrate polymers in the cell wall that significantly affects the outcome of host-pathogen interaction. Chitosan-deficient strains are avirulent, implicating chitosan as a critical virulence factor. C. gattii R265 is an important fungal pathogen of concern due to its ability to cause infections in individuals with no apparent immune dysfunction and an increasing geographical distribution. Characterization of the fungal cell wall and understanding the contribution of individual molecules of the cell wall matrix to fungal pathogenesis offers new therapeutic avenues for intervention. In this report, we show that the C. gattii R265 strain has evolved alternate regulation of chitosan biosynthesis under both laboratory growth conditions and during mammalian infection compared to that of C. neoformans.

microbiology↗

bioRxiv2019

The aminoalkylindole, BML-190, negatively regulates chitosan synthesis via the cAMP/PKA1 pathway in Cryptococcus neoformans

Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immune-compromising conditions. Current antifungals are suboptimal to treat this disease, therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall, is required for survival in the mammalian host, and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis.\n\nTo identify these compounds we developed a robust and novel cell-based flow cytometry screening method to identify small molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans. We used flow cytometry-based counter and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits.\n\nOne of the confirmed hits that reduced chitosan content was the aminoalkylindole, BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein coupled receptor, Gpr4, and via the cAMP/PKA signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis, and could lead to potential novel therapeutics against C. neoformans.\n\nImportanceCryptococcus neoformans is a fungal pathogen that kills [~]200,000 people every year. The cell wall is an essential organelle that protects fungus from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of cryptococcal cells wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in C. neoformans. Two of these compounds were validated using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan in C. neoformans, and validates that this screening approach could be used to find potential antifungal agents.

microbiology↗