Biology subjects

Staker, B.

Publications and source records attributed to Staker, B..

7 recordsLinked to original sources

bioRxiv2026

Relatedness of Gardnerella prophages and their endolysins

The Gardnerella genus includes a complex sub-taxonomy of bacterial species and subgroups which have been implicated in bacterial vaginosis. We analyzed G. leopoldii, G. swidsinski, G. piotii, and G. vaginalis genomes available in public databases and predicted proviral sequences using Cenote-Taker3. We clustered 266 prophage sequences into 140 viral genera level groupings that encode four distinct gene clusters and 19 different lysin proteins. Analysis of the bacterial and prophage sequence phylogenies reveals that while there were no specific prophage sequence lineages that infected each bacterial host, there is clustering of the prophage sequences by bacterial species within the Gardnerella genus.

microbiology↗

bioRxiv2026

Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK)

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and crystal structures of TvPPi-PFK are reported. TvPPi-PFK is the first reported eukaryotic PPi-PFK structure. TvPPi-PFK retains the overall PPi-PFK topology observed in bacterial PPi-PFK including conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. In addition to the catalytic PPi-PFK binding sites, TvPPi-PFK has two additional ligand binding sites. The first binds AMP usurped during protein production and helps stabilize the TvPPi-PFK tetramer. A second ligand binding site was observed in proximity to the AMP-binding site and accommodates sugar phosphates soaked into preformed crystals. This sugar phosphates binding site is distinct from the TvPPi-PFK active site that binds fructose-6-phosphate. Future mutagenesis and activity studies are planned to determine the relevance of both sites. SynopsisThe production, crystallization, and crystal structures of a pyrophosphate-dependent phosphofructokinase from Trichomonas vaginalis (TvPPi-PFK) are reported. TvPPi-PFK has a prototypical PPi-PFK active site as well as unexpected AMP and sugar-phosphate binding sites at the dimer interface.

biochemistry↗

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↗

bioRxiv2022

Molecular mechanism for strengthening E-cadherin adhesion using a monoclonal antibody

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using x-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif - the strand-swap dimer interface. Molecular dynamics simulations and single molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes, one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped {beta}-strand and its complimentary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.

biophysics↗

bioRxiv2020

Naegleria fowleri: protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brains frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogens high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. N. fowleri genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

molecular biology↗

bioRxiv2020

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N=80 samples from persons with RT-PCR confirmed SARS-CoV2 infection), and a specificity of 97.2% (N=106 control samples).

bioinformatics↗