Elevated infant fecal concentrations of the bacterial-derived lipid 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME) increase the risk for childhood atopy and asthma. However, the mechanisms by which this lipid contributes to disease development are largely unknown. We hypothesized that macrophages, which are key to both antimicrobial and antigen responses, are functionally and epigenetically modified by 12,13-diHOME leading to short- and long-term dysfunction with consequences for both antimicrobial and antigenic responses. Macrophages exposed to 12,13-diHOME are skewed toward inflammatory IL-1βhighCD206low cells, a phenomenon that is further amplified in the presence of common microbial-, aero-, and food-allergens. These IL-1βhighCD206low macrophages also exhibit reduced bacterial phagocytic capacity. In primary immune cell coculture assays involving peanut allergen stimulation, 12,13-diHOME promotes both IL-1β and IL-6 production, memory B cell expansion, and increased IgE production. Exposure to 12,13-diHOME also induces macrophage chromatin remodeling, specifically diminishing access to interferon-stimulated response elements resulting in reduced interferon-regulated gene expression upon bacterial lipopolysaccharide stimulation. Thus 12,13-diHOME reprograms macrophage effector function, B-cell interactions and promotes epigenetic modifications that exacerbate inflammatory response to allergens and mutes antimicrobial response along the interferon axis. These observations offer plausible mechanisms by which this lipid promotes early-life pathogenic microbiome development and innate immune dysfunction associated with childhood allergic sensitization.

The fungal pathogen Candida auris represents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in healthcare settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targeting C. auris and narrowed our focus to five Food and Drug Administration (FDA)-approved compounds with inhibitory concentrations under 10 µM for C. auris and significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors of C. auris. To pursue these compounds further, we exposed C. auris to clioquinol in an extended experimental evolution study and found that C. auris developed only twofold to fivefold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulator CAP1 (causing upregulation of the drug transporter MDR1) and in the drug transporter CDR1. These mutations had only modest effects on resistance to traditional antifungal agents, and the CDR1 mutation rendered C. auris more susceptible to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might prevent C. auris from developing resistance to this established antifungal agent. IMPORTANCE The rapidly emerging fungal pathogen Candida auris represents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated 8-hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains of C. auris. Although this compound has been identified in previous screens, we extended the analysis by showing that C. auris developed only modest twofold to fivefold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500-fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated 8-hydroxyquinolines are working inside the fungal cell and should be developed further to combat C. auris and other fungal pathogens. Lohse and colleagues characterize a class of compounds that inhibit the fungal pathogen C. auris. Unlike many other antifungal drugs, C. auris does not readily develop resistance to this class of compounds.

AbstractThe fungal pathogenCandida aurisrepresents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in health-care settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targetingC. aurisand narrowed our focus to five FDA-approved compounds with inhibitory concentrations under 10 µM forC. aurisand significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors ofC. auris. To pursue these compounds further, we exposedC. auristo clioquinol in an extended experimental evolution study and found thatC. aurisdeveloped only 2- to 5-fold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulatorCAP1(causing upregulation of the drug transporterMDR1) and in the drug transporterCDR1. These mutations had only modest effects on resistance to traditional antifungal agents, and theCDR1mutation renderedC. aurismore sensitive to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might preventC. aurisfrom developing resistance to this established antifungal agent.Abstract ImportanceThe rapidly emerging fungal pathogenCandida aurisrepresents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains ofC. auris. Although this compound has been identified in previous screens, we extended the analysis by showing thatC. aurisdeveloped only modest 2- to 5-fold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500- fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated hydroxyquinolines are working inside the fungal cell and should be developed further to combatC. aurisand other fungal pathogens.TweetLohse and colleagues characterize a class of compounds that inhibit the fungal pathogenC. auris. Unlike many other antifungal drugs,C. aurisdoes not readily develop resistance to this class of compounds.

As many as one in five survivors of COVID-19 often have persistent pulmonary, neurologic, and mental health symptoms, prompting high health care utilization and high morbidity and mortality.1 During the COVID-19 pandemic, many institutions started multidisciplinary post-COVID-19 clinics based on earlier models for post-ICU care.2 How post-COVID-19 clinics impact longitudinal health care utilization is unclear. The American Thoracic Society/European Respiratory Society statement on post-COVID-19 care noted insufficient evidence to recommend these clinics, and starting a new clinic may have high costs.

The fungal pathogen Candida auris represents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in health-care settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targeting C. auris and narrowed our focus to five FDA-approved compounds with inhibitory concentrations under 10 µM for C. auris and significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors of C. auris . To pursue these compounds further, we exposed C. auris to clioquinol in an extended experimental evolution study and found that C. auris developed only 2- to 5-fold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulator CAP1 (causing upregulation of the drug transporter MDR1 ) and in the drug transporter CDR1 . These mutations had only modest effects on resistance to traditional antifungal agents, and the CDR1 mutation rendered C. auris more sensitive to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might prevent C. auris from developing resistance to this established antifungal agent.Abstract Importance:The rapidly emerging fungal pathogen Candida auris represents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains of C. auris . Although this compound has been identified in previous screens, we extended the analysis by showing that C. auris developed only modest 2- to 5-fold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500- fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated hydroxyquinolines are working inside the fungal cell and should be developed further to combat C. auris and other fungal pathogens.Tweet:Lohse and colleagues characterize a class of compounds that inhibit the fungal pathogen C. auris . Unlike many other antifungal drugs, C. auris does not readily develop resistance to this class of compounds.

Early-life gut microbiome perturbation and metabolic dysfunction precede atopy and asthma development in childhood. However, mechanisms by which gut-microbial metabolites drive long-lasting immune dysfunction remain elusive. We previously showed that elevated concentrations of the gut bacteria-derived lipid, 12,13-diHOME, is a risk factor for development of atopy and asthma in childhood, and that it suppresses regulatory T cells by altering dendritic cell function.

Secondary bacterial infections, including ventilator-associated pneumonia (VAP), lead to worse clinical outcomes and increased mortality following viral respiratory infections including in patients with coronavirus disease 2019 (COVID-19). Using a combination of tracheal aspirate bulk and single-cell RNA sequencing (scRNA-seq) we assessed lower respiratory tract immune responses and microbiome dynamics in 28 COVID-19 patients, 15 of whom developed VAP, and eight critically ill uninfected controls. Two days before VAP onset we observed a transcriptional signature of bacterial infection. Two weeks prior to VAP onset, following intubation, we observed a striking impairment in immune signaling in COVID-19 patients who developed VAP. Longitudinal metatranscriptomic analysis revealed disruption of lung microbiome community composition in patients with VAP, providing a connection between dysregulated immune signaling and outgrowth of opportunistic pathogens. These findings suggest that COVID-19 patients who develop VAP have impaired antibacterial immune defense detectable weeks before secondary infection onset.

Author(s): Crawford, Emily D; Acosta, Irene; Ahyong, Vida; Anderson, Erika C; Arevalo, Shaun; Asarnow, Daniel; Axelrod, Shannon; Ayscue, Patrick; Azimi, Camillia S; Azumaya, Caleigh M; Bachl, Stefanie; Bachmutsky, Iris; Bhaduri, Aparna; Brown, Jeremy Bancroft; Batson, Joshua; Behnert, Astrid; Boileau, Ryan M; Bollam, Saumya R; Bonny, Alain R; Booth, David; Borja, Michael Jerico B; Brown, David; Buie, Bryan; Burnett, Cassandra E; Byrnes, Lauren E; Cabral, Katelyn A; Cabrera, Joana P; Caldera, Saharai; Canales, Gabriela; Castaneda, Gloria R; Chan, Agnes Protacio; Chang, Christopher R; Charles-Orszag, Arthur; Cheung, Carly; Chio, Unseng; Chow, Eric D; Citron, Y Rose; Cohen, Allison; Cohn, Lillian B; Chiu, Charles; Cole, Mitchel A; Conrad, Daniel N; Constantino, Angela; Cote, Andrew; Crayton-Hall, Tre'Jon; Darmanis, Spyros; Detweiler, Angela M; Dial, Rebekah L; Dong, Shen; Duarte, Elias M; Dynerman, David; Egger, Rebecca; Fanton, Alison; Frumm, Stacey M; Fu, Becky Xu Hua; Garcia, Valentina E; Garcia, Julie; Gladkova, Christina; Goldman, Miriam; Gomez-Sjoberg, Rafael; Gordon, M Grace; Grove, James CR; Gupta, Shweta; Haddjeri-Hopkins, Alexis; Hadley, Pierce; Haliburton, John; Hao, Samantha L; Hartoularos, George; Herrera, Nadia; Hilberg, Melissa; Ho, Kit Ying E; Hoppe, Nicholas; Hosseinzadeh, Shayan; Howard, Conor J; Hussmann, Jeffrey A; Hwang, Elizabeth; Ingebrigtsen, Danielle; Jackson, Julia R; Jowhar, Ziad M; Kain, Danielle; Kim, James YS; Kistler, Amy; Kreutzfeld, Oriana; Kulsuptrakul, Jessie; Kung, Andrew F

Caloric restriction (CR) is the leading non-pharmaceutical dietary intervention to improve health- and lifespan in most model organisms. A wide array of cellular pathways is induced in response to CR and CR-mimetics, including the transcriptional activator Nuclear factor erythroid-2-related factor 2 (Nrf2), which is essential in the upregulation of multiple stress-responsive and mitochondrial enzymes. Nrf2 is necessary in tumor protection but is not essential for the lifespan extending properties of CR in outbred mice. Here, we sought to study Nrf2-knockout (KO) mice and littermate controls in male C57BL6/J, an inbred mouse strain. Deletion of Nrf2 resulted in shortened lifespan compared to littermate controls only under ad libitum conditions. CR-mediated lifespan extension and physical performance improvements did not require Nrf2. Metabolic and protein homeostasis and activation of tissue-specific cytoprotective proteins were dependent on Nrf2 expression. These results highlight an important contribution of Nrf2 for normal lifespan and stress response.

Caloric restriction (CR) is the most potent nonpharmacological intervention known to both protect against carcinogenesis and delay aging in laboratory animals. There is a growing number of anticarcinogens and CR mimetics that activate NAD(P)H:quinone oxidoreductase 1 (NQO1). We have previously shown that NQO1, an antioxidant enzyme that acts as an energy sensor through modulation of intracellular redox and metabolic state, is upregulated by CR. Here, we used NQO1-knockout (KO) mice to investigate the role of NQO1 in both the aging process and tumor susceptibility, specifically in the context of CR. We found that NQO1 is not essential for the beneficial effects of CR on glucose homeostasis, physical performance, metabolic flexibility, life-span extension, and (unlike our previously observation with Nrf2) chemical-induced tumorigenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neonates at risk of childhood atopy and asthma are characterized by gut microbiome perturbation and fecal enrichment of 12,13 DiHOME([1][1]); however, the underlying mechanism and source of this metabolite remain poorly understood. Here we show that 12,13 DiHOME treatment of human dendritic cells altered peroxisome proliferator-activated receptor γ regulated gene expression and decreased immune tolerance. In mice, 12,13 DiHOME treatment prior to airway challenge exacerbated pulmonary inflammation and decreased lung regulatory T cells. Neonatal fecal metagenomic sequencing revealed putative bacterial sources of 12,13 DiHOME. In our cohort, three bacterial genes and their product, 12,13 DiHOME, are associated with increased odds of childhood atopy or asthma, suggesting that early-life gut-microbiome risk factors may shape immune tolerance and identify high-risk neonates years in advance of clinical symptoms.One Sentence Summary Early-life gut-microbiome risk factors may shape immune tolerance and identify neonates at high-risk of disease. [1]: #ref-1

12,13 DiHOME is enriched in the stool of neonates with a perturbed gut microbiota, who are at high risk of developing multi-sensitized atopy at age 2. This linoleic acid-derivative is structurally similar to known ligands of peroxisome proliferator-activated receptor gamma (PPARγ), is generated by an epoxide hydrolase (EH) and can suppress regulatory T cells (Treg) ex vivo. Therefore, we hypothesize that gut microbiome-derived 12,13 DiHOME contributes to allergic sensitization in childhood via PPARγ signaling on human dendritic cells (DCs), resulting in suppression of Tregs. Human DC/T cell co-culture assays, qPCR, and a mouse model of cockroach antigen (CRA) allergic airway sensitization were used to evaluate the effects of 12,13 DiHOME. Metagenomics and LC-MS were used to quantify EH genes and 12,13 DiHOME in stool samples from the Wayne County Health, Environment, Allergy, and Asthma Longitudinal Study. Human DCs treated with 12,13 DiHOME exhibited significantly decreased IL-10 secretion (p=0.0009), altered expression of PPARγ-regulated genes, CD36 (p=0.002) and CD1a (p=0.002), and decreased Treg frequency following co-culture (p=0.0004). CRA-sensitized mice, treated with peritoneal-delivered 12,13 DiHOME, exhibited an increase in circulating IgE (p=0.055) and a significant decrease in lung Tregs (p=0.019). Neonates who developed multi-sensitized atopy at age 2 years exhibited a significantly increased abundance of fecal bacterial EH genes (p=0.0017) and of 12,13 DiHOME (p=0.002). Early-life 12,13 DiHOME synthesis by the gut microbiome may contribute to loss of immune tolerance and the development of allergic disease in childhood.

12,13 DiHOME is enriched in neonates with a perturbed gut microbiota, who are at significantly higher relative-risk of developing predominantly multi-sensitized atopy at age two and parental reported doctor-diagnosed asthma at age four. This oxylipin can suppress regulatory T cell (Treg) populations ex vivo1 and is structurally similar to known ligands of peroxisome proliferator-activated receptor gamma (PPARγ). Loss of PPARγ impairs immune tolerance and prevents Treg maturation in mice. Therefore, we hypothesize that 12,13 DiHOME contributes to allergic sensitization in humans via inhibition of PPARγ signaling on human dendritic cells (DCs) resulting in suppression of Treg populations. Human DC/T cell co-culture assays and a murine model of allergic airway sensitization were used to evaluate the effects of 12,13 DiHOME treatment. Flow cytometry, a cytometric bead array, and qPCR were used to quantify Treg frequency and function. Linear mixed-effects model was used for data analysis. Treatment of human DCs with 130 uM 12,13 DiHOME did not reduce cell viability but significantly decreased IL-10 secretion (p=0.0009) and Treg frequency (p=0.0004). This was consistent with the decrease observed using an established PPARγ inhibitor, GW9662. C57b6 mice, treated (via peritoneal injection) with 30 mg/kg 12,13 DiHOME and subjected to allergic airway sensitization using cockroach antigen, exhibited a significant decrease in lung Tregs (p=0.019). Current efforts are examining PPARγ-regulated gene expression. These data indicate that 12,13 DiHOME suppresses Treg populations in a manner consistent with inhibition of PPARγ signaling, indicating a potential mechanism for loss of immune tolerance in early life.

Differences in the composition of the gut microbiota of infants associate with relative risk of atopy in childhood, and metabolites linked with these distinct microbial states alter T cell differentiation ex vivo. Gut microbiota bacterial depletions and altered metabolic activity at 3 months are implicated in childhood atopy and asthma1. We hypothesized that compositionally distinct human neonatal gut microbiota (NGM) exist, and are differentially related to relative risk (RR) of childhood atopy and asthma. Using stool samples (n = 298; aged 1–11 months) from a US birth cohort and 16S rRNA sequencing, neonates (median age, 35 d) were divisible into three microbiota composition states (NGM1–3). Each incurred a substantially different RR for multisensitized atopy at age 2 years and doctor-diagnosed asthma at age 4 years. The highest risk group, labeled NGM3, showed lower relative abundance of certain bacteria (for example, Bifidobacterium, Akkermansia and Faecalibacterium), higher relative abundance of particular fungi (Candida and Rhodotorula) and a distinct fecal metabolome enriched for pro-inflammatory metabolites. Ex vivo culture of human adult peripheral T cells with sterile fecal water from NGM3 subjects increased the proportion of CD4+ cells producing interleukin (IL)-4 and reduced the relative abundance of CD4+CD25+FOXP3+ cells. 12,13-DiHOME, enriched in NGM3 versus lower-risk NGM states, recapitulated the effect of NGM3 fecal water on relative CD4+CD25+FOXP3+ cell abundance. These findings suggest that neonatal gut microbiome dysbiosis might promote CD4+ T cell dysfunction associated with childhood atopy.

Significant gut microbiota heterogeneity exists among ulcerative colitis (UC) patients, though the clinical implications of this variance are unknown. We hypothesized that ethnically distinct UC patients exhibit discrete gut microbiotas with unique metabolic programming that differentially influence immune activity and clinical status. Using parallel 16S rRNA and internal transcribed spacer 2 sequencing of fecal samples (UC, 30; healthy, 13), we corroborated previous observations of UCassociated bacterial diversity depletion and demonstrated significant Saccharomycetales expansion as characteristic of UC gut dysbiosis. Furthermore, we identified four distinct microbial community states (MCSs) within our cohort, confirmed their existence in an independent UC cohort, and demonstrated their coassociation with both patient ethnicity and disease severity. Each MCS was uniquely enriched for specific amino acid, carbohydrate, and lipid metabolism pathways and exhibited significant luminal enrichment of the metabolic products of these pathways. Using a novel ex vivo human dendritic cell and T-cell coculture assay, we showed that exposure to fecal water from UC patients caused significant Th2 skewing in CD4(+) T-cell populations compared to that of healthy participants. In addition, fecal water from patients in whom their MCS was associated with the highest level of disease severity induced the most dramatic Th2 skewing. Combined with future investigations, these observations could lead to the identification of highly resolved UC subsets based on defined microbial gradients or discrete microbial features that may be exploited for the development of novel, more effective therapies.IMPORTANCE Despite years of research, the etiology of UC remains enigmatic. Diagnosis is difficult and the patient population heterogeneous, which represents a significant barrier to the development of more effective, tailored therapy. In this study, we demonstrate the clinical utility of the gut microbiome in stratifying UC patients by identifying the existence of four distinct interkingdom pathogenic microbiotas within the UC patient population that are compositionally and metabolically distinct, covary with clinical markers of disease severity, and drive discrete CD4(+) T-cell expansions ex vivo. These findings offer new insight into the potential value of the gut microbiome as a tool for subdividing UC patients, opening avenues to the development of more personalized treatment plans and targeted therapies.

Bacterial pathogens secrete a variety of effector molecules to support colonization of the human host. Included in this list are pore-forming toxins (PFTs), which kill host cells by selectively targeting and disrupting the plasma membrane. To improve the efficiency of cell lysis, many PFTs contain recognition motifs that bind protein, lipid, and carbohydrate molecules on the cell surface. The human pathogen Vibrio cholerae secretes a PFT (VCC) that forms heptameric channels in membranes and lyses cells at picomolar concentrations. The toxin is thought to serve as a defensive agent against innate immune cells thus facilitating colonization of the host. To achieve high potency, VCC contains two structural domains with lectin-like folds that target carbohydrate molecules found on mammalian cell surfaces. Disruptive mutations within one of these domains significantly reduces the activity of the toxin against model cell membranes suggesting that carbohydrate-binding is important for targeting membranes at low concentrations. To better understand this process, we conducted screens against a variety of simple and complex carbohydrate ligands and identified glycans recognized by VCC lectin-like domains. Several of these ligands are potential physiological targets of the toxin and we show that they bind VCC with low nanomolar affinity. These glycans decrease toxin activity when added in trans and may serve as a starting point for therapeutic intervention. To aid in this goal, we are pursuing structural characterization of toxin-glycan complexes to better understand the nature of these interactions.

Pathogens selectively target host cells using adhesion molecules and secreted virulence factors that may utilize protein, lipid, or carbohydrate ligands on the cell surface. The human intestinal pathogen Vibrio cholerae secretes a pore-forming toxin, V. cholerae cytolysin (VCC), which contains two domains that are structurally similar to known carbohydrate-binding proteins. These tandem domains are attached to the carboxy-terminus of the cytolytic domain and contain a β-trefoil fold and a β-prism fold. VCC has been shown to bind glycosylated proteins, and removal of the β-prism domain leads to a large decrease in lytic activity against rabbit erythrocytes. Despite these clues, the identity of the glycan receptors of VCC and the role of glycan binding in toxin activity remain unknown. To better understand this specificity, we used a combination of structural and functional approaches to characterize the carbohydrate-binding activity of the VCC toxin. We first probed the monosaccharide-binding activity of VCC and demonstrated that the toxin exhibits millimolar affinity for aldohexoses. To understand this specificity, we solved the crystal structure of the VCC β-prism domain bound to methyl-α-mannose. Next, we utilized a mammalian glycan screen to determine that the β-prism domain preferentially binds complex N-glycans with a heptasaccharide GlcNAc4Man3 core (NGA2). Fluorescence anisotropy and surface plasmon resonance indicated an approximately 100-nM affinity of the β-prism domain for the heptasaccharide core. Our results suggest that carbohydrate-binding domains on the VCC toxin facilitate high-affinity targeting of mammalian cell membranes, which may contribute to the ability of VCC to lyse cells at picomolar concentrations.

Vibrio cholerae cytolysin (VCC) is a secreted pore-forming toxin that contains two putative sugar-binding domains: the β-trefoil domain and the β-prism domain. Previous hemagglutinating studies suggest that VCC binds glycoproteins with terminal β1-galactosyl moieties [1]; however, crystallization of monomeric VCC with β-octyl glucoside suggests the β-prism domain may have an affinity for glucosides. We aim to characterize the carbohydrate-binding specificity of the β-trefoil and β-prism domains. The crystal structure of VCC indicates that Asp617 within the β-prism domain forms hydrogen bonds with the bound glucoside moeity. Hemolysis assays demonstrated that the removal of the β-prism domain causes a 1000-fold decrease in lytic activity toward rabbit erythrocytes, while conversion of Asp617 to alanine and lysine decreases the hemolytic activity100-fold, indicating that the sugar-binding activity of VCC may play a role in cell lysis. The β-prism domain was expressed as a separate construct and isothermal titration calorimetry (ITC) was used to determine the binding affinity for the interaction of the β-prism domain with monosaccharides. The binding of the isolated β-prism domain reflected the binding affinities observed using ITC for full length VCC. The β-prism domain showed an affinity for mannose, but not galactose, indicating that recognition of terminal β1-galactosyl moieties does not occur at the β-prism domain. The carbohydrate-binding activity of the β-prism domain may explain its role in cell lysis. [1] Saha, N; Banerjee, K.K. Carbohydrate-mediated Regulation of Interaction of Vibrio cholerae Hemolysin with Erythrocyte and Phospholipid Vesicle, J. Biol. Chem. (1997) 272, 162-167.