Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens.

The human intestine, colonized by a dense community of resident microbes, is a frequent target of bacterial pathogens. Undisturbed, this intestinal microbiota provides protection from bacterial infections. Conversely, disruption of the microbiota with oral antibiotics often precedes the emergence of several enteric pathogens. How pathogens capitalize upon the failure of microbiota-afforded protection is largely unknown. Here we show that two antibiotic-associated pathogens, Salmonella enterica serovar Typhimurium (S. typhimurium) and Clostridium difficile, use a common strategy of catabolizing microbiota-liberated mucosal carbohydrates during their expansion within the gut. S. typhimurium accesses fucose and sialic acid within the lumen of the gut in a microbiota-dependent manner, and genetic ablation of the respective catabolic pathways reduces its competitiveness in vivo. Similarly, C. difficile expansion is aided by microbiota-induced elevation of sialic acid levels in vivo. Colonization of gnotobiotic mice with a sialidase-deficient mutant of Bacteroides thetaiotaomicron, a model gut symbiont, reduces free sialic acid levels resulting in C. difficile downregulating its sialic acid catabolic pathway and exhibiting impaired expansion. These effects are reversed by exogenous dietary administration of free sialic acid. Furthermore, antibiotic treatment of conventional mice induces a spike in free sialic acid and mutants of both Salmonella and C. difficile that are unable to catabolize sialic acid exhibit impaired expansion. These data show that antibiotic-induced disruption of the resident microbiota and subsequent alteration in mucosal carbohydrate availability are exploited by these two distantly related enteric pathogens in a similar manner. This insight suggests new therapeutic approaches for preventing diseases caused by antibiotic-associated pathogens.

A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3).

Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 µM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by ∼50-60%, heparan sulfate by ∼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by ∼74 and ∼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by ∼33%. Although the analog was readily transformed in cells into the uridine 5'-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis.

Impact of COVID-19 on ophthalmic surgical procedures in sub-Saharan Africa: a multicentre study.

BACKGROUND: The COVID-19 pandemic had a profound impact on healthcare and ophthalmology services globally. Numerous studies amongst various medical and surgical specialties showed a reduction in patient attendance and surgical procedures performed. Prior published ophthalmic literature focused on specific types of procedures and were usually single centre. The current study attempts to quantify the impact on a larger scale, namely that of sub-Saharan Africa, and to include all ophthalmic subspecialties. METHODS: This is a retrospective analysis of the surgical records from 17 ophthalmology centres in seven countries located in East, Central, West and Southern Africa. The date of declaration of the first lockdown was used as the beginning of the pandemic and the pivot point to compare theatre records one year prior to the pandemic and the first year of the pandemic. We examined the total number of surgical procedures over the two year period and categorized them according to ophthalmic subspecialty and type of procedure performed. We then compared the pre-pandemic and pandemic surgical numbers over the two year period. RESULTS: There were 26,357 ophthalmic surgical procedures performed with a significant decrease in the first year of the pandemic (n = 8942) compared to the year prior to the pandemic (n = 17,415). The number of surgical procedures performed was lower in the first year of the pandemic compared to the year prior to the pandemic by 49% [Incidence rate ratio (IRR) 0.51, 95% CI 0.41-0.64), 27% (0.73, 0.55-0.99), 46% (0.54, 0.30-0.99), 40% (0.60, 0.39-0.92) and 59% (0.41, 0.29-0.57) in sub-Saharan Africa (4 regions combined), West, Central, East and Southern Africa, respectively]. The number of surgical procedures in the different sub-specialty categories in sub-Saharan Africa (4 regions combined) was significantly lower in the first year of the pandemic compared to the year prior to the pandemic, except for glaucoma (IRR 0.72, 95% CI 0.52-1.01), oncology (0.71, 0.48-1.05), trauma (0.90, 0.63-1.28) and vitreoretinal (0.67, 0.42-1.08) categories. CONCLUSION: This study provides insight into the impact of the COVID-19 pandemic in multiple regions and countries on the African continent. The identification of which surgical subspecialty was most affected by the COVID-19 pandemic in each region allows for better planning and resource allocation to address these backlogs.

The human milk oligosaccharide disialyllacto-N-tetraose prevents necrotising enterocolitis in neonatal rats.

BACKGROUND: Necrotising enterocolitis (NEC) is one of the most common and fatal intestinal disorders in preterm infants. Breast-fed infants are at lower risk for NEC than formula-fed infants, but the protective components in human milk have not been identified. In contrast to formula, human milk contains high amounts of complex glycans. OBJECTIVE: To test the hypothesis that human milk oligosaccharides (HMO) contribute to the protection from NEC. METHODS: Since human intervention studies are unfeasible due to limited availability of HMO, a neonatal rat NEC model was used. Pups were orally gavaged with formula without and with HMO and exposed to hypoxia episodes. Ileum sections were scored blindly for signs of NEC. Two-dimensional chromatography was used to determine the most effective HMO, and sequential exoglycosidase digestions and linkage analysis was used to determine its structure. RESULTS: Compared to formula alone, pooled HMO significantly improved 96-hour survival from 73.1% to 95.0% and reduced pathology scores from 1.98 ± 1.11 to 0.44 ± 0.30 (p<0.001). Within the pooled HMO, a specific isomer of disialyllacto-N-tetraose (DSLNT) was identified to be protective. Galacto-oligosaccharides, currently added to formula to mimic some of the effects of HMO, had no effect. CONCLUSION: HMO reduce NEC in neonatal rats and the effects are highly structure specific. If these results translate to NEC in humans, DSLNT could be used to prevent or treat NEC in formula-fed infants, and its concentration in the mother's milk could serve as a biomarker to identify breast-fed infants at risk of developing this disorder.

Imaging the sialome during zebrafish development with copper-free click chemistry.

The sialome comprises sialylated glycoproteins and glycolipids that play essential roles in cell-cell communication. Using azide-modified molecular precursors of sialic acids and copper-free click chemistry, we visualized the spatiotemporal dynamics of the sialome in live zebrafish embryos.

Genomic and metabolic profiling of nonulosonic acids in Vibrionaceae reveal biochemical phenotypes of allelic divergence in Vibrio vulnificus.

Nonulosonic acids (NulOs) encompass a large group of structurally diverse nine-carbon backbone α-keto sugars widely distributed among the three domains of life. Mammals express a specialized version of NulOs called sialic acids, which are displayed in prominent terminal positions of cell surface and secreted glycoconjugates. Within bacteria, the ability to synthesize NulOs has been demonstrated in a number of human pathogens and is phylogenetically widespread. Here we examine the distribution, diversity, evolution, and function of NulO biosynthesis pathways in members of the family Vibrionaceae. Among 27 species of Vibrionaceae examined at the genomic level, 12 species contained nab gene clusters. We document examples of duplication, divergence, horizontal transfer, and recombination of nab gene clusters in different Vibrionaceae lineages. Biochemical analyses, including mass spectrometry, confirmed that many species do, in fact, produce di-N-acetylated NulOs. A library of clinical and environmental isolates of Vibrio vulnificus served as a model for further investigation of nab allele genotypes and levels of NulO expression. The data show that lineage I isolates produce about 20-fold higher levels of NulOs than lineage II isolates. Moreover, nab gene alleles found in a subset of V. vulnificus clinical isolates express 40-fold higher levels of NulOs than nab alleles associated with environmental isolates. Taken together, the data implicate the family Vibrionaceae as a "hot spot" of NulO evolution and suggest that these molecules may have diverse roles in environmental persistence and/or animal virulence.

A multi-site study confirms abnormal glycosylation in the Tamm-Horsfall protein of patients with interstitial cystitis.

PURPOSE: We confirm the single site observation of decreased sialylation and abnormal glycosylation of Tamm-Horsfall protein in patients with interstitial cystitis compared to control subjects. MATERIALS AND METHODS: Urine samples from 41 controls and 48 patients with interstitial cystitis from a total of 5 North American sites were obtained in blinded fashion as to participant status. Tamm-Horsfall protein was isolated from urine samples by salt precipitation. Protein content was determined by size exclusion chromatography and normalized to creatinine. Sialic acid was quantified by 1,2-diamino-4,5-methylene dioxybenzene (Sigma®) high performance liquid chromatography with fluorescence detection. Neutral and amino sugars were determined by high pH anion exchange chromatography with pulsed amperometric detection. N-glycans were labeled with 2-aminobenzamide and profiled using high pH anion exchange chromatography with fluorescence detection. Samples were also analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry. Permethylated N-glycans were analyzed in the mass-to-charge ratio range of 3,000 to 6,000. RESULTS: There was no difference in the protein-to-creatinine ratio of Tamm-Horsfall protein from patients with interstitial cystitis vs controls (49.12 vs 46.4 mg/gm, p = 0.26). Sialic acid content (67 vs 77 nmol/mg Tamm-Horsfall protein, p = 0.025) and total monosaccharide content (590.9 vs 680.6 nmol/mg Tamm-Horsfall protein, p = 0.003) were significantly decreased in patients with interstitial cystitis vs controls. Results were supported by 2-aminobenzamide N-glycan profiling and mass spectrometry, which showed a 45% decrease in a major tetra-sialylated peak (mass-to-charge ratio 4,590) in Tamm-Horsfall protein from patients with interstitial cystitis compared to controls. CONCLUSIONS: These multisite data validate that abnormal glycosylation of Tamm-Horsfall protein occurs in patients with interstitial cystitis and may have a role in interstitial cystitis causation.

Galactose residues on the lipooligosaccharide of Moraxella catarrhalis 26404 form the epitope recognized by the bactericidal antiserum from conjugate vaccination.

Lipooligosaccharide (LOS) from Moraxella catarrhalis has the potential to elicit bactericidal antibodies against the pathogen. We generated LOS-based conjugate vaccines that elicited bactericidal antibodies in animal models. However, epitopes on the LOS recognized by the functional anti-LOS antibodies remain unidentified. In this study, a mutant strain, D4, which lost the recognition by a bactericidal anti-LOS rabbit serum in Western blotting was generated from a serotype C strain 26404 by random transposon mutagenesis. Sequence analysis revealed there was an insertion of a kanamycin resistance gene in the lgt2 gene of D4, which encodes beta(1-4)-galactosyltransferase. An isogenic lgt2 mutant, 26404lgt2, was constructed. Structural analysis indicated that the mutant strain produced a truncated LOS lacking terminal galactoses from 4- and 6-linked oligosaccharide chains of strain 26404. Further studies showed that the antiserum lost the recognition of both mutant cells and LOSs in Western blotting, an enzyme-linked immunosorbent assay (ELISA), or a flow cytometry assay. The antiserum also lost the ability to kill the mutant strain in a bactericidal assay, whereas it showed a bactericidal titer of 1:80 to strain 26404. In an inhibition ELISA, d-(+)-galactose or 26404lgt2 LOS showed no inhibition. However, the 26404 LOS and a serotype A O35E LOS with terminal galactoses on its 6-linked oligosaccharide chain showed >90% inhibition, while a serotype B 26397 LOS showed >60% inhibition. These studies suggest that the terminal alpha-Gal-(1-->4)-beta-Gal on the 6-linked oligosaccharide chain of 26404 LOS plays a critical role in forming the epitope recognized by the bactericidal antiserum induced by immunization with our conjugate vaccine.

Treatment response in Kawasaki disease is associated with sialylation levels of endogenous but not therapeutic intravenous immunoglobulin G.

OBJECTIVES: Although intravenous immunoglobulin (IVIG) is highly effective in Kawasaki disease (KD), mechanisms are not understood and 10-20% of patients are treatment-resistant, manifesting a higher rate of coronary artery aneurysms. Murine models suggest that α2-6-linked sialic acid (α2-6Sia) content of IVIG is critical for suppressing inflammation. However, pro-inflammatory states also up-regulate endogenous levels of ß-galactoside:α2-6 sialyltransferase-I (ST6Gal-I), the enzyme that catalyzes addition of α2-6Sias to N-glycans. We asked whether IVIG failures correlated with levels of α2-6Sia on infused IVIG or on the patient's own endogenous IgG. METHODS: We quantified levels of α2-6Sia in infused IVIG and endogenous IgG from 10 IVIG-responsive and 10 resistant KD subjects using multiple approaches. Transcript levels of ST6GAL1, in patient whole blood and B cell lines were evaluated by RT-PCR. Plasma soluble (s)ST6Gal-I levels were measured by ELISA. RESULTS: There was no consistent difference in median sialylation levels of infused IVIG between groups. However, α2-6Sia levels in endogenous IgG, ST6GAL1 transcript levels, and ST6Gal-I protein in serum from IVIG-resistant KD subjects were lower than in responsive subjects at both pre-treatment and one-year time points (p <0.001, respectively). CONCLUSIONS: Our data indicate sialylation levels of therapeutic IVIG are unrelated to treatment response in KD. Rather, lower sialylation of endogenous IgG and lower blood levels of ST6GALI mRNA and ST6Gal-I enzyme predict therapy resistance. These differences were stable over time, suggesting a genetic basis. Because IVIG-resistance increases risk of coronary artery aneurysms, our findings have important implications for the identification and treatment of such individuals.

Interfering with UDP-GlcNAc metabolism and heparan sulfate expression using a sugar analogue reduces angiogenesis.

Heparan sulfate (HS), a long linear polysaccharide, is implicated in various steps of tumorigenesis, including angiogenesis. We successfully interfered with HS biosynthesis using a peracetylated 4-deoxy analogue of the HS constituent GlcNAc and studied the compound's metabolic fate and its effect on angiogenesis. The 4-deoxy analogue was activated intracellularly into UDP-4-deoxy-GlcNAc, and HS expression was inhibited up to ∼96% (IC50 = 16 µM). HS chain size was reduced, without detectable incorporation of the 4-deoxy analogue, likely due to reduced levels of UDP-GlcNAc and/or inhibition of glycosyltransferase activity. Comprehensive gene expression analysis revealed reduced expression of genes regulated by HS binding growth factors such as FGF-2 and VEGF. Cellular binding and signaling of these angiogenic factors was inhibited. Microinjection in zebrafish embryos strongly reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish and chicken model systems. All of these data identify 4-deoxy-GlcNAc as a potent inhibitor of HS synthesis, which hampers pro-angiogenic signaling and neo-vessel formation.