Progress 10/01/04 to 09/30/10
Outputs
OUTPUTS: Manuscripts describing data from this reporting period are in progress. PARTICIPANTS: Ann Benefiel, Shikha Bhatia, Franck Carbonero, and Jennifer Croix. TARGET AUDIENCES: Members of the general public, biomedical professionals, and the animal health industry. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our efforts have focused on both microbial and host aspects of the sulfomucin niche in the terminal ileum and colon. Our use of the term niche does not follow the strict definitions used in ecological theory but falls within a similar framework and refers to syntrophic interactions between two functional groups of bacteria, glycosulfatase harboring bacteria and sulfate reducing bacteria that are dependent on a substrate provided by the host, in this case by intestinal goblet cells. Glycosulfatases represent a large and diverse enzymatic group present in eukaryotic and prokaryotic cells. A preliminary screening of glycosulfatase sequences in the Bacteroides thetaiotaomicron genome revealed the presence of 8 sulfatase encoding genes clearly defined by their use of sulfated mucins and 9 putative sulfatase genes. The presence of numerous glycosulfatase encoding genes in this dominant intestinal microorganism presumably indicates the importance of this mode of sulfate metabolism in the intestinal ecosystem. A search in GenBank for bacterial glycosulfatases revealed a predominance of sequences obtained from Bacteroides and Prevotella, which formed three coherent clusters. Other clusters containing microorganisms from non-intestinal environments were also observed. This greater genetic diversity among non-intestinal microbes can imply either that only a limited diversity of the intestinal glycosulfatases repertoire has been revealed by Bacteroides and Prevotella genomes or that evolutionary pressures in the mucin niche select specific enzymes. Regarding the host's contribution to the sulfomucin niche, we have focused our studies on mucin sulfation, which occurs within the trans-Golgi as a late step in glycoprotein synthesis when specific sulfotransferases transfer sulfate from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to mucin O-glycans at the C-3 position of galactose residues or the C-6 position of N-acetylglucosamine. Of the known galactose 3-O-sulfotransferases and N-acetylglucosamine-6-O sulfotransferases, galactose-3-O-sulfotransferase 2 (GAL3ST2) is the sulfotransferase responsible for sulfate addition to the C-3 position of Gal in human colonic mucins and carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5 (CHST5) the most likely candidate for sulfation of the C-6 position of N-acetylglucosamine. Evidence for the involvement of these two sulfotransferases in colonic sulfomucin synthesis includes the downregulation of GAL3ST2 expression in non-mucinous adenocarcinomas, corresponding to a decrease in sulfomucin expression, and the abundance and specificity of CHST5 for human small intestine and colon. In addition, CHST5 shows a preference for sulfating O-linked chains of the mucin-type, suggesting that it is involved in transfer of sulfate to the C-6 position of N- acetylglucosamine in sulfomucins. LS174T cells produce sulfomucins abundantly and this was further enhanced by exposure to both flagellin and IL-13, but not TNFα. One of the important features of LS174T cells is the expression and induction of both CHST5 and GAL3ST2 in response to inflammatory stimuli including bacterial flagellin, IL-13 and TNFα.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Studies were completed that focused on molecular mechanisms that underlie the development and functions of mucus-producing intestinal goblet cells. The data were presented as abstracts at the scientific meetings Experimental Biology 2009 (April 18-22, New Orleans, LA) and Digestive Diseases Week (May 30 - June 4, 2009, Chicago, IL). Manuscripts are in preparation. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Biomedical and veterinary scientists concerned with gut health. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We examined the effects of differentiation on goblet cell responsiveness to IL-13 using the LS174T and HT-29Cl.16E goblet cell lines. Expression of goblet cell secretory product genes mucin 2 (MUC2), resistin-like molecular beta (RETNLB), and trefoil Factor 3 (TFF3) and the sulfotransferase genes carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5 (CHST5) and beta-galactose-3-O-sulfotransferase 2 (GAL3ST2) were measured at different stages of differentiation and after stimulation with IL-13 by quantitative RT-PCR. Sialo- and sulfomucin chemotype expression was also assessed with HID/AB histochemistry. Expression of MUC2, TFF3, and RETNLB increased in HT-29Cl.16E cells within days post-confluence, consistent with goblet cell differentiation, and with the addition of IL-13 to pre-confluent cells. However, MUC2 and RETNLB expression decreased while TFF3 expression remained unchanged in the more differentiated, post-confluent HT-29Cl.16E cells treated with IL-13. CHST5 expression was increased 20-fold in preconfluent cells, but only 10-fold in post-confluent cells treated with IL-13. Expression of CHST5 but not GAL3ST2 was significantly increased in LS174T cells in response to IL-13. HID/AB histochemical analysis of sialo-and sulfomucins corroborated an increase in sulfomucin production in LS174T and HT-29Cl.16E cells in response to IL-13. Together the data demonstrate that IL-13 modulation of goblet cell lines is dependent on extent of differentiation. Increased expression of the sulfotransferase genes and sulfomucin data indicate that IL-13 may also stimulate mucin sulfation via effects predominately on the CHST5 sulfotransferase gene. A major finding was the novel observation that cysteine dioxygenase (CDO) expression is restricted to the secretory lineage in the small intestine and colon. Cysteine dioxygenase catalyzes the oxidation of cysteine to cysteine sulphinic acid, the first and rate limiting step for cysteine utilization. Mammals regulate cysteine metabolism through CDO to maintain sufficient cysteine for protein synthesis and production of other essential molecules such as glutathione, taurine, pyruvate and inorganic sulfate, but below the threshold for cytotoxicity. CDO expression in the intestine was previously uncharacterized. We examined CDO expression and localization in mouse small and large intestine and human colon using immunohistochemical and two-color immunofluorescence with antibodies against CDO, matrix metalloproteinase 7 (MMP-7), a marker of Paneth cells, and chromagranin A (ChrA), a marker of enteroendocrine cells. The data revealed CDO staining in goblet cells, Paneth cells and enteroendocrine cells while CDO staining was notably absent in absorptive epithelium. CDO expression was also observed in colonic goblet cells rather than absorptive coloncytes. Dual immunofluorescent staining for CDO and ChrA or MMP-7 confirmed CDO expression in enteroendocrine cells and Paneth cells, respectively. This striking difference in CDO expression between the absorptive and secretory cell lineages parallels a high cysteine requirement in goblet, Paneth, and enteroendocrine cells relative to absorptive epithelial cells.
Publications
- Croix, J.A., Ueki, I., Stipanuk, M.H., Greenberg, E. and Gaskins H.R. 2009. Cysteine dioxygenase expression is restricted to the secretory lineage in the small intestine and colon. Gastroenterology 136(5 Suppl):A-730.
- Croix, J.A., Collier, C.T., Khan, T.A. and Gaskins, H.R. 2009. Interleukin (IL)-13 modulation of goblet cell function is dependent on state of differentiation FASEB J. 23(4 Suppl):570.11.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Funding was withdrawn and research was not pursued in the previous calendar year relating specifically to the goals and objectives of the project. PARTICIPANTS: Funding was withdrawn and research was not pursued in the previous calendar year relating specifically to the goals and objectives of the project. TARGET AUDIENCES: Funding was withdrawn and research was not pursued in the previous calendar year relating specifically to the goals and objectives of the project. PROJECT MODIFICATIONS: Funding was withdrawn and research was not pursued in the previous calendar year relating specifically to the goals and objectives of the project.
Impacts
Funding was withdrawn and research was not pursued in the previous calendar year relating specifically to the goals and objectives of the project.
Publications
- Collier, C.T., Hofacre, C.L., Payne, A.M., Anderson, D.B., Kaiser, P., Mackie, R.I. and Gaskins, H.R. 2008. Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth. Vet. Immunol. Immunopathol. 122(1-2): 104-15.
- Gaskins, H.R., Croix, J.A., Nakamura, N. and Nava, G.M. 2008. Impact of the intestinal microbiota on the development of mucosal defense. Clin. Infect. Dis. 46 Suppl 2:S80-6; Discussion S144-51.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: A study was completed that focused on the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). The work was published in the referred scientific journal Veterinary Immunology and Immunopathology.
PARTICIPANTS: C.T. Collier, C.L. Hofacre, A.M. Payne, D.B. Anderson, P. Kaiser, R.I. Mackie, and H.R. Gaskins.
TARGET AUDIENCES: Researchers focused on enteric disorders as well as the animal health industry.
Impacts
Project ILLU-538-387 has focused on the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation-, and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent
increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, goblet cell number, and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as α toxin production were greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-γ expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE. Intestinal
health impacts the efficiency of animal growth and is a key factor underlying a number of enteric disorders in both pigs and poultry. The purpose of the work is to identify cellular and molecular mechanisms underlying host mucogenic responses to inflammatory cues and determining the extent to which they impact the normal microbiota. Such knowledge may have important implications for a wide variety of chronic intestinal inflammatory disorders of both animals and humans, given that mucogenesis appears to be a stereotypical response to local inflammation.
Publications
- Collier, C.T., Hofacre, C.L., Payne, A.M., Anderson, D.B., Kaiser, P., Mackie, R.I. and Gaskins, H.R. 2008. Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth. Veterinary Immunology and Immunopathology (In Press). Available online 27 October 2007.
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Progress 01/01/06 to 12/31/06
Outputs
Project ILLU-538-387 seeks to define the mucolytic bacterial groups that normally reside in the small and large intestine of the pig and chick and understand better the genetic basis of bacterial mucolysis. Based on our previous studies with a piglet model of total parenteral nutrition (TPN) and a chick model of necrotic enteritis (NE), it is postulated that a significant portion of commensal bacterial groups are able to grow on mucus as a substrate. Further, we hypothesize that more soluble nutrients are preferred substrates and that mucus-supported growth becomes particularly important in inflammatory conditions that characteristically activate host mucogenesis. This hypothesis was derived from evidence from both animal models that the growth of Clostridium perfringens, a bacterium present normally at low concentrations, is selected in response to increased mucus production by the host. The ability to utilize mucus coupled with a rapid doubling rate provides a
selective growth advantage to C. perfringens supporting the frequent classification of this organism as an opportunistic pathogen. We will take advantage of the fact that the C. perfringens genome has been fully sequenced to define the genetic pathways contributing to bacterial mucolysis. Research in 2006 overlaps with Project ILLU-538-910 and continued to focus on the set-up and validation of semi-continuous chemostats that will be used to compare the ability of a pure C. perfringens culture to grow and produce toxin A in a standard growth medium versus a medium in which mucin is the sole carbon source. The chemostat will be crucial most especially for the second objective of defining the genes required for C. perfringens to metabolize mucin for growth as well as genes associated with quorum sensing and toxin production. Additional work was also performed to address reviewer comments for a manuscript submitted, which describes a broiler chick model of NE we developed with researchers
at the College of Veterinary Medicine at The University of Georgia.
Impacts
Intestinal health impacts the efficiency of animal growth and is a key factor underlying a number of enteric disorders in both pigs and poultry. The purpose of the work is to identify normal gut bacteria in the piglet and broiler chick intestine that are capable of using host mucus as a substrate for growth and to define the molecular basis of mucolysis by Clostridium perfringens, the causative agent of necrotic enteritis in broiler chicks.
Publications
- Collier, C.T., Hofacre, C.L., Payne, A.M., Anderson, D.B., Kaiser, P., Mackie, R.I. and Gaskins, H.R. 2006. Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth. Immunology (Submitted).
- Krause, D.O., Gaskins, H.R. and Mackie, R.I. 2007. Prokaryotic diversity of gut mucosal biofilms in the human digestive tract. In: Biofilms in the Food Environment. (ed. H.P. Blascheck, H. Wang and M.E. Agle), Blackwell Publishing, pp. 125-50.
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Progress 01/01/05 to 12/31/05
Outputs
Project ILLU-538-387 seeks to define the mucolytic bacterial groups that normally reside in the small and large intestine of the pig and chick and understand better the genetic basis of bacterial mucolysis. Based on our previous studies with a piglet model of total parenteral nutrition (TPN) and a chick model of necrotic enteritis (NE), it is postulated that a significant portion of commensal bacterial groups are able to grow on mucus as a substrate. Further, we hypothesize that more soluble nutrients are preferred substrates and that mucus-supported growth becomes particularly important in inflammatory conditions that characteristically activate host mucogenesis. This hypothesis was derived from evidence from both animal models that the growth of Clostridium perfringens, a bacterium present normally at low concentrations, is selected in response to increased mucus production by the host. The ability to utilize mucus coupled with a rapid doubling rate provides a
selective growth advantage to C. perfringens supporting the frequent classification of this organism as an opportunistic pathogen. We will take advantage of the fact that the C. perfringens genome has been fully sequenced to define the genetic pathways contributing to bacterial mucolysis. The research in year 1 of this project overlaps with Project ILLU-538-910 and focused on 1) completing and submitting a manuscript that describes a broiler chick model of NE we developed with researchers at the College of Veterinary Medicine at The University of Georgia, and 2) initial set-up and validation of the semi-continuous chemostats that will be used to compare the ability of a pure C. perfringens culture to grow and produce toxin A in a standard growth medium versus a medium in which mucin is the sole carbon source. The chemostat will be crucial most especially for the second objective of defining the genes required for C. perfringens to metabolize mucin for growth as well as genes
associated with quorum sensing and toxin production.
Impacts
Intestinal health impacts the efficiency of animal growth and is a key factor underlying a number of enteric disorders in both pigs and poultry. The purpose of the work is to identify normal gut bacteria in the piglet and broiler chick intestine that are capable of using host mucus as a substrate for growth and to define the molecular basis of mucolysis by Clostridium perfringens, the causative agent of necrotic enteritis in broiler chicks.
Publications
- Collier, C.T., Hofacre, C.L., Payne, A.M., Anderson, D.B., Kaiser, P., Mackie, R.I. and Gaskins, H.R. 2006. Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth. Infection and Immunity (Submitted).
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