Biology subjects

Bolejack, M.

Publications and source records attributed to Bolejack, M..

3 recordsLinked to original sources

bioRxiv2024

Enolase inhibitors as therapeutic leads for Naegleria fowleri infection

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate that these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 value of 0.14 {+/-} 0.04 {micro}M) that was toxic to trophozoites (EC50 value of 0.21 {+/-} 0.02 {micro}M) while the reported CC50 was >300 {micro}M. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the conclusion of the experiment, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. Brains of six of the eight survivors were positive for amoebae, suggesting the agent at the tested dose suppressed, but did not eliminate, infection. These findings suggest that HEX is a promising lead for the treatment of PAM.

biochemistry↗

bioRxiv2022

Regulation of multiple dimeric states of E-cadherin by adhesion activating antibodies revealed through Cryo-EM and X-ray crystallography

E-cadherin adhesion is regulated at the cell surface, a process that can be replicated by activating antibodies. We use cryo-EM and X-ray crystallography to examine functional states of the cadherin adhesive dimer. This dimer is mediated by N-terminal beta strand-swapping involving Trp2, and forms via a different transient X-dimer intermediate. X-dimers are observed in cryo-EM along with monomers and strand-swap dimers, indicating that X-dimers form stable interactions. A novel EC4-mediated dimer was also observed. Activating Fab binding caused no gross structural changes in E- cadherin monomers but can facilitate strand swapping. Moreover, activating Fab binding is incompatible with the formation of the X-dimer. Both cryo-EM and X-ray crystallography reveal a distinctive twisted strand-swap dimer conformation caused by an outward shift in the N-terminal beta strand that may represent a strengthened state. Thus, regulation of adhesion involves changes in cadherin dimer configurations.

biochemistry↗

bioRxiv2020

Horizontal gene transfer in the human and skin commensal Malassezia: a bacterially-derived flavohemoglobin is required for NO resistance and host interaction

The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions. Malassezia are the most abundant fungal skin inhabitant of warm-blooded animals, and have been implicated in skin diseases and systemic disorders, including Crohns disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the Malassezia genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in M. sympodialis, we demonstrated that bacterially-derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNAseq analysis revealed that endogenous accumulation of nitric oxide resulted in upregulation of genes involved in stress response, and downregulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of Malassezia flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of Malassezia flavohemoglobin. Lastly, we identified additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin. Significance statementMalassezia species are the main fungal components of the mammalian skin microbiome and are associated with a number of skin disorders. Recently, Malassezia has also been found in association with Crohns Disease and with pancreatic cancer. The elucidation of the molecular bases of skin adaptation by Malassezia is critical to understand its role as commensal and pathogen. In this study we employed evolutionary, molecular, biochemical, and structural analyses to demonstrate that the bacterially-derived flavohemoglobins acquired by Malassezia through horizontal gene transfer resulted in a gain of function critical for nitric oxide detoxification and resistance to nitrosative stress. Our study underscores horizontal gene transfer as an important force modulating Malassezia evolution and niche adaptation.

microbiology↗