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Crawford, C. J.

Publications and source records attributed to Crawford, C. J..

4 recordsLinked to original sources

Polyelectrolyte mannan from diatoms reshapes sunlit ocean microbiome

Diatoms are a keystone phylum in Earths ecosystems, specializing in oxygen production and carbohydrate fixation that fuels global food webs. Diatoms host a microbiome, but how they preferentially collect bacteria with complementary traits remains unknown. Here we show that diatoms exude a C6-sulfated -1,3-mannan that serves as a selective carbon source for adapted bacteria. Its structure was resolved by NMR spectroscopy, chromatography, chemical synthesis, and enzymatic dissection. Biochemical, physiological, and structural analyses revealed that specialized Bacteroidota employ a four-enzyme pathway to metabolize this mannan. Metagenomic and transcriptomic data indicate that the mannan globally selects for bacteria carrying these enzymes and associated traits. Because the mannan provides only carbon, oxygen, sulfur, and hydrogen, bacteria must obtain other essential elements from alternative sources, reinforcing metabolic interdependence. We propose that diatoms use sulfated mannans to attract beneficial partners and exclude competitors, thereby engineering a microbiome that enhances their productivity and underpins carbon cycling. Significance statementEukaryotes host microbial partners that shape their health, yet how they selectively assemble beneficial microbes remains unclear. Using diatom microalgae as a model, we show they exude a sulfated mannan that nourishes highly adapted bacteria tracking them across the global ocean. Our findings suggest that single-celled eukaryotes can "domesticate" prokaryotes--analogous to how humans have domesticated animals--albeit on a microscopic scale. Dominating much of Earths aquatic surface, diatoms drive [~]20% of global photosynthesis. We propose that sulfated mannan contributes to this success by helping diatoms shape microbial partnerships that underpin planetary energy balance and atmospheric chemistry.

ecology↗

Novel sulfatase cancer therapeutics negatively impact Bacteroidota of the colonic microbiota in a non-sulfatase dependent manner

Excessive degradation of the colonic mucin layer by Bacteroides within the human gut microbiota drives inflammatory bowel disease in mice. Bacterial carbohydrate sulfatases are key enzymes in gut colonization, as they are elevated in human inflammatory bowel disease and correlate with disease severity. Selective inhibitors of carbohydrate sulfatases could function as sulfatase-selective drugs, allowing precise control of sulfatase activity while preserving these otherwise beneficial bacteria. Arylsulfamates are covalent inhibitors that target a catalytic formylglycine residue of steroid sulfatases, a residue that is also conserved in carbohydrate sulfatases. Here, we find that a library of aryl- and carbohydrate sulfamates is ineffective against Bacteroides carbohydrate sulfatases, yet can inhibit human gut microbiota species grown on sulfated glycans. Leveraging thermal proteome profiling, we identify a lipid kinase as the target responsible for these effects. This work highlights the imperative for developing specific inhibitors targeting carbohydrate sulfatases and reveals the adverse effects that arylsulfamates have on Bacteroides species of the human gut microbiota. Significance statementArylsulfamates are currently the only effective class of sulfatase inhibitors available and offer a potential strategy to treat inflammatory bowel disease driven by gut microbiota carbohydrate sulfatases. Although arylsulfamates inhibit the growth of microbiota Bacteroides species on sulfated glycans, this is not mediated through carbohydrate sulfatases but, via a conserved lipid kinase. Carbohydrate sulfatases are resistant to arylsulfamates whilst steroid sulfatases are susceptible despite a conserved active site. Finally, selected complex plant glycans confer a resistant/protective phenotype against the harmful effects of arylsulfamates. These data guide the future development of targeted carbohydrate sulfatase inhibitors and potential drug-prebiotic pairings.

biochemistry↗

Semi-synthetic glycoconjugate vaccine candidate against Cryptococcus neoformans

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, posing a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semi-synthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semi-synthetic glycoconjugate vaccines contain the identical synthetic decasaccharide (M2 motif) antigen. This motif is present in serotype A strains, which constitute 95% of clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity towards M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). While these findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. It could serve as a component in a multi-valent GXM motif vaccine, enhancing both strength and breadth of immune responses.

immunology↗

The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: a combined NMR and MD study

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans capsular polysaccharide and shed exopolysaccharide functions both as a key virulence factor and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this combined NMR and MD study, experimentally determined NOEs and J-couplings support a structure of the synthetic decasaccharide, GXM10-Ac3, obtained by MD. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is characterized by a 6-residue -mannan backbone repeating unit, consisting of a triad of -(1[->]3)-mannoses, modified by {beta}-(1[->]2)-xyloses on the first two mannoses and a {beta}-(1[->]2)-glucuronic acid on the third mannose. The combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with xylose/glucuronic acid branches alternating sides along the -mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This experimentally consistent GXM model displays high flexibility while maintaining a structural identity, yielding new insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture. Significance StatementThis study utilized a combined NMR and MD approach to elucidate the structure of a Cryptococcus neoformans GXM synthetic decasaccharide (GXM10-Ac3), recognized by protective anti-GXM mAbs. The data revealed an extended structure in which the xylose/glucuronic acid branches and pairs of 6-O-acetyl esters predominantly alternate sides along the -mannan backbone. MD analysis of a GXM polysaccharide predicts that the decasaccharide structure is uniformly represented in the polysaccharide. Additionally, the GXM exhibits high flexibility while maintaining structural identity. These findings lay the foundation for future studies aimed at understanding anti-GXM antibody-polysaccharide interactions.

biochemistry↗