Nicole Marie Koropatkin

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone with low-affinity calcium-binding sites in its C-terminal domain. This region is altered by somatic mutations in the CRT gene ( CALR ), which drive a subset of myeloproliferative neoplasms (MPNs). Perturbations in ER calcium storage and signaling are reported for the MPN type I mutant, CRT _Del52 , and are linked to disease pathogenesis. Using recombinant CRT proteins, we found similar low-affinity calcium-binding characteristics for wild-type CRT and CRT _Del52 , as determined by isothermal titration calorimetry (ITC). Residues 340–349, conserved in both wild-type CRT and CRT _Del52 , contribute to binding. Furthermore, ER and cytosolic calcium levels, and store-operated calcium entry (SOCE), were comparable in CRT knockout (CRT-KO) cells reconstituted with wild-type CRT or CRT _Del52 . Notably, the CRT-KO induces expression of multiple ER proteins known to contain low-affinity calcium-binding sites or regulate ER calcium levels. Overall, these findings indicate that CRT _Del52 retains at least some low-affinity calcium-binding capacity and that CRT deficiency induces compensatory cellular changes that maintain ER calcium homeostasis.

Bacteroides are abundant gut bacteria with diverse polysaccharide metabolizing capabilities encoded by co-transcribed polysaccharide utilization loci (PULs). The starch utilization system (Sus) is required for starch and α-glucan metabolism in Bacteroides thetaiotaomicron and has served as a model for PUL studies. However, the mechanism of maltooligosaccharide (MOS) transport and the architecture of the SusCD complex remain incompletely understood. Here, we used cryo-electron microscopy to capture multiple transport-relevant conformations of the SusD lipoprotein and the TonB-dependent transporter SusC in both unliganded and ligand-bound states. In the absence of ligand, SusCD adopts three conformations including an open state in which SusD is displaced from SusC, a state containing the SusC plug, and a state lacking density for the SusC plug. Three-dimensional variability analysis of the structure where SusD is displaced from SusC reveals continuous SusD mobility coupled to movement of the SusC N-terminus. The maltoheptaose-bound SusCD structure reveals two binding sites, one at the SusD-SusC interface and a second within SusC, indicating how MOS stabilizes a closed SusCD assembly. Notably, all structures show that SusCD is a heterodimer, unlike other Sus-like transporters that assemble into SusCD heterotetramers. Native mass spectrometry confirms this heterodimeric stoichiometry, and comparisons with other SusCD-like complexes suggest a structural basis for this distinct assembly of SusCD. Together, these data define substrate binding and intermediate transport states in a canonical PUL system and reveal architectural divergence among Sus-like carbohydrate transport complexes in Bacteroides .

Bacteria from the Bacteroidota phylum are well-known for their exceptional carbohydrate degradation abilities, which are closely associated with their use of polysaccharide utilization loci (PULs). The native exchange of PULs between different Bacteroidota species via horizontal gene transfer has been confirmed experimentally, and imitating such transfers in vitro can enable detailed studies of PULs from unstudied species in readily tractable hosts and the creation of novel strains. Here, we present a new set of tools and workflow to transfer large genetic loci, such as PULs, into genetically tractable Bacteroides strains. Our method uses a newly designed pICKUP plasmid and derivatives that allow site-specific genome integration and subsequent removal of the plasmid backbone. Thus, an unlimited number of loci may be transferred using the same resistance markers. We validated the pICKUP-based workflow by transfer of a previously characterized mixed-linkage β -glucan utilization locus (MLGUL) into the genome of Bacteroides thetaiotaomicron, which enabled metabolism of mixed-linkage β -glucan by this strain. Subsequent transfer of a PUL from an uncultivated bacterium from the bovine rumen confers the ability to metabolize certain cellooligosaccharides and allows further scrutiny of various genes in the PUL and their involvement in cellooligosaccharide metabolism. The pICKUP-based method presented here expands the toolkit for investigation of PULs and other large loci, including those from intractable or uncharacterized members of the Bacteroidota. A robust method to transfer whole PULs between species also forms the basis for creating custom-tailored PULs and resulting strains, with potential applications in improved gut health and biomass valorization processes. IMPORTANCE: The gut environment is a highly competitive niche, where dietary fiber is a major source of carbon, and polysaccharide utilization loci (PULs) encoded by Bacteroidota species are linked to their dominance in this environment. Our proposed method to study such PULs, especially from non-cultivated or unculturable strains, by transferring them into genetically tractable hosts, represents a valuable alternative to typical studies of isolated PUL-derived proteins. By transferring PULs into culturable hosts, we enable both a better understanding of the PULs’ biological roles and modification of the individual encoded genes. Our results show how a single PUL transfer can confer hemicellulose-degrading ability to Bacteroides thetaiotaomicron , which is lacking in the wild-type strain. Transfer of a PUL from a distantly related strain, speculated to confer cellulose-degrading capacity, showcases that this annotation may be incorrect and indicates that the utilization of foreign genetic material depends on species relatedness in the same phylum.

Bacteroidota are prolific members of the human gut microbiota, influencing overall health through the degradation of various polysaccharides. To aid in this process, these bacteria deploy cell surface lipoproteins, proteins anchored to the membrane by a lipidated N-terminal cysteine. Despite their importance, lipoprotein synthesis and transport in Bacteroidota is not well defined, particularly how lipoproteins reach the outer membrane and cell surface. In Escherichia coli , lipoproteins are inserted into the outer membrane by LolB, the final step of the Lol pathway for localizing lipoproteins, previously thought to be confined to γ- and β-proteobacteria. Herein, we report a structural ortholog of LolB in Bacteroides that rescues the lethal phenotype associated with LolB depletion in E. coli . We demonstrate this ortholog’s LolB-like activity through mutagenesis studies and a lipoprotein insertion activity assay. We find that the gene cannot be deleted from Bacteroides , but that depletion does not significantly impact growth on polysaccharides, nor the surface localization of lipoproteins involved in starch degradation. Nonetheless, depletion significantly alters the composition of many proteins in both the inner and outer membranes, while others remain unchanged. Altogether, our findings contribute to elucidating the lipoprotein transport pathway in Bacteroides and how it impacts cell physiology.

Carbohydrate-binding modules (CBMs) play crucial roles in carbohydrate-active enzymes by promoting substrate recognition and proximity, particularly for insoluble polysaccharides. Here, we report the discovery and characterization of a novel β-trefoil structured CBM associated with a GH87 α-1,3-glucanase from Flavobacterium johnsoniae , which accordingly was designated Fj CBMXXX _GH87 . The full-length enzyme efficiently hydrolyzed α-1,3-glucan (mutan) and α-1,3/α-1,6-glucan (alternan), whereas the catalytic domain alone displayed reduced activity, indicating that Fj CBMXXX _GH87 enhances substrate interaction. Pull-down assays confirmed that Fj CBMXXX _GH87 binds α-1,3-linked glucans, and structural investigation together with site-directed mutagenesis identified two distinct binding sites essential for protein-ligand interactions. Phylogenetic analysis showed that CBMXXX homologs are present together with enzymes from families GH87, GH13, GH16, and GH99, and potentially may comprise up to three binding sites. Together, these findings establish Fj CBMXXX _GH87 as the founding member of a new CBM family, which may have broad functional versatility in polysaccharide recognition. This discovery expands the repertoire of β-trefoil CBMs and provides new insights into carbohydrate recognition strategies relevant to α-glucan degradation.

Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone with low affinity calcium binding sites in its C-terminal domain. This region is altered by somatic mutations in the CRT gene ( CALR ), which drive a subset of myeloproliferative neoplasms (MPN). Perturbations in ER calcium storage and signaling are reported for the MPN type I mutant, CRT _Del52 , and are linked to disease pathogenesis. Using recombinant CRT proteins, we found similar low affinity calcium binding characteristics for wild-type CRT and CRT _Del52 , as determined by isothermal titration calorimetry (ITC). Residues 340–349, conserved in both wild-type CRT and CRT _Del52 , contribute to binding. Furthermore, ER and cytosolic calcium levels and store-operated calcium entry (SOCE) were comparable in CRT knockout (CRT-KO) cells reconstituted with wild-type CRT or CRT _Del52 . Notably, the CRT-KO induces expression of multiple ER proteins known to contain low affinity calcium binding sites or regulate ER calcium levels. Overall, these findings indicate that CRT _Del52 retains at least some low affinity calcium binding capacity and that CRT-deficiency induces compensatory cellular changes that maintain ER calcium homeostasis. Summary: Pathogenic CRT _Del52 retains at least partial low-affinity calcium-binding capacity. Additionally, live-cell imaging and flow cytometry with ratiometric calcium probes indicate that ER and cytosolic calcium levels, as well as store-operated calcium entry (SOCE), are comparable in CRT-deficient cells rescued with either wild-type CRT or CRT _Del52.

The gut microbiota is a key modulator of human health and the status of major diseases including cancer, diabetes and inflammatory bowel disease. Central to microbiota survival is the ability to metabolise complex dietary and host-derived glycans, including intestinal mucins. The prominent human gut microbe Bacteroides thetaiotaomicron (B. theta) is a versatile and highly efficient complex glycan degrader thanks to the expansion of gene clusters termed polysaccharide utilisation loci (PULs). While the mechanism of action for several singular dietary glycan-induced PULs have been elucidated, studies on the unusually high number of mucin-inducible PULs in B. theta significantly lag behind. Here we show that a mucin inducible PUL BT4240-50 encodes activities consistent with the processing and metabolism of mucin O-glycoproteins and their core sugar N-acetylgalactosamine (GalNAc). PUL BT4240-50 was also shown to be important for competitive growth on mucins in vitro, encoding a kinase (BT4240) critical for GalNAc metabolism. Additionally, BT4240-kinase was shown to be essential for glycosaminoglycan metabolism, extending the PULs function beyond mucins. These data advance our understanding of glycoprotein metabolism at mucosal surfaces, highlighting GalNAc as a key metabolite for competitive microbial survival in the human gut.

α‐Amylases are the workhorse enzymes of starch degradation. They are central to human health, including as targets for anti‐diabetic compounds, but are also the key enzymes in the industrial processing of starch for biofuels, corn syrups, brewing and detergents. Dissection of the activity, specificity and stability of α‐amylases is crucial to understanding their biology and allowing their exploitation. Yet, functional characterization lags behind DNA sequencing and genomics; and new tools are required for rapid analysis of α‐amylase function. Here, we design, synthesize and apply new branched α‐amylase activity‐based probes. Using both α‐1,6 branched and unbranched α‐1,4 maltobiose activity‐based probes we were able to explore the stability and substrate specificity of both a panel of human gut microbial α‐amylases and a panel of industrially relevant α‐amylases. We also demonstrate how we can detect and annotate the substrate specificity of α‐amylases in the complex cell lysate of both a prominent gut microbe and a diverse compost sample by in‐gel fluorescence and mass spectrometry. A toolbox of starch‐active activity‐based probes will enable rapid functional dissection of α‐amylases. We envisage activity‐based probes contributing to better selection and engineering of enzymes for industrial application as well as fundamental analysis of enzymes in human health.

α‐Amylases are the workhorse enzymes of starch degradation. They are central to human health, including as targets for anti‐diabetic compounds, but are also the key enzymes in the industrial processing of starch for biofuels, corn syrups, brewing and detergents. Dissection of the activity, specificity and stability of α‐amylases is crucial to understanding their biology and allowing their exploitation. Yet, functional characterization lags behind DNA sequencing and genomics; and new tools are required for rapid analysis of α‐amylase function. Here, we design, synthesize and apply new branched α‐amylase activity‐based probes. Using both α‐1,6 branched and unbranched α‐1,4 maltobiose activity‐based probes we were able to explore the stability and substrate specificity of both a panel of human gut microbial α‐amylases and a panel of industrially relevant α‐amylases. We also demonstrate how we can detect and annotate the substrate specificity of α‐amylases in the complex cell lysate of both a prominent gut microbe and a diverse compost sample by in‐gel fluorescence and mass spectrometry. A toolbox of starch‐active activity‐based probes will enable rapid functional dissection of α‐amylases. We envisage activity‐based probes contributing to better selection and engineering of enzymes for industrial application as well as fundamental analysis of enzymes in human health.