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Functional characterization of the Helicobacter hepaticus cytolethal distending toxin operon
AGAA323
April 2000
104 - 5
cecum and cfu/gm stool. Like the immunocompetent mice with defined flora, scm mice with the same defined GI flora became heavily colonized; 108-9 cfu/gm stool and large intestine/cecum tissue was recovered I week post-inoculation. In contrast to immunocompetent mice, however, recovery of C. jejuni from stool and large intestine/cecum tissue in scm mice remained at this high level beyond one month. CONCLUSIONS: Our results suggest that a limited enteric flora permits a much higher level of intestinal colonization in C3H mice by C.jejuni, suggesting the normal microbiological GI flora represents an important innate immune mechanism against establishment of Campylobacter infection. Furthermore, the importance of adaptive immunity in clearing an infection that does become established is suggested by the inability of scm mice to eliminate C. jejuni. Comparison of wild type and mutant C. jejuni strains in these three murine models should allow us to determine the roles of specific genes in overcoming both innate and adaptive immune functions.
1763 MURINE ANTIBODY RESPONSE TO A VACCINE FOR ENTEROTOXIGENIC ESCHERICHIA COLI. Arthur J. de Lorimier, Wyatt Byrd, Eric R. Hall, Zachary J. Roberts, Charles E. McQueen, Douglas Tang, William Vaughan, Frederick J. Cassels, Walter Reed Army Institute of Research, Washington, DC; Naval Med Research Ctr, Washington, DC. Introduction Enterotoxigenic Escherichia coli (ETEC) is the most common cause of bacterial diarrhea identified in deployed soldiers and an important cause of infant morbidity and mortality in developing nations. Our aim was to compare the immunogenicity of three dosing schedules of coli surface antigen 6 (CS6), an antigen expressed by 31% of ETEC worldwide, encapsulated in a biodegradable poly(DL-lactide-co-glycolide) (PLG) administered intranasally (IN) to mice with purified unencapsulated CS6 given alone. Methods We studied 49 female BALB/c mice given CS6 IN four weeks apart. The vaccine was administered as follows on days 0, 28 and 55: Group I (n=6) received 25p,g CS6 encapsulated in PLG (CS6-PLG) on day 0 only; Group II (n=6) received 25p,g CS6-PLG on days 0 and 28; Group III (n= 12) received 25p,g CS6-PLG on all 3 occasions; Group IV (n=5) received 25p,g CS6 unencapsulated (CS6) on day 0 only; Group V (n=5) received 25p,g CS6 on days 0 and 28; Group VI (n=12) received 25p,g CS6 on all 3 occasions; Group VII (n=3) (control) received 25p,g PLG-encapsulated bovine serum albumin (BSAPLG) on day 0 only. Mice were sacrificed on day 84. Antibody to CS6 was assayed from mucosal secretions and serum specimens that were collected from all animals within three days prior to each dose of vaccine and prior to sacrifice. Anti-CS6 antibody was measured by enzyme linked immunosorbent assay for serum IgG and secretory IgA (sIgA). The anti-CS6 titers for all groups were compared and analysis of variance was used to assess the data. Results Mice tolerated IN administration of CS6, CS6PLG and BSA-PLG well. Serum anti-CS6 IgG rose following all three dosing schedules of vaccines containing CS6. Mice vaccinated with three doses of CS6-PLG developed a >4 fold rise in serum anti-CS6 IgG titer values (I :338,000) than those immunized with three doses of CS6 alone (I :76,000). Two and three doses of CS6-PLG led to a 2 fold and 7 fold rise, respectively, in anti-CS6 sIgA geometric mean titer values over two and three doses ofCS6 alone (p
1764 FUNCTIONAL CHARACTERIZATION OF THE HELICOBACTER HEPATICUS CYTOLETHAL DISTENDING TOXIN OPERON. Zhongming Ge, Vincent B. Young. Chih Ching Chien, Nancy S. Taylor, David B. Schauer, James G. Fox, MIT, Cambridge, MA. Cytolethal distending toxin (CDT), which causes cell arrest in the G2/M phase of the cell cycle, is produced in a number of mucosal pathogens including certain strains of Escherichia coli, Shigella spp. and Campylobacter spp. The gene organization of the edt operon and the role of CDT in bacterial infection are poorly understood. We previously sequenced three genes (cdtA, cdtB and cdtC) involved in H. hepaticus CDT activity and demonstrated that the cdtB gene is present in several enterohepatic helicobacters including H. bilis, H. pullorum, and H. canis. In this study, the transcription initiation and termination sites of the H. hepaticus edt operon were determined by primer extension and RT-PCR. The edt operon transcription started at the site ~ 270 nucleotides upstream of the cdtA start codon and ended at the site approximately 90 nucleotides downstream of the cdtC stop codon. These results indicated that the H. hepaticus edt operon consists of cdtA, cdtB and cdtC genes. Isogenic cdtB and cdtC mutants were created by inserting the Cm' cassette into either cdtB or cdtC, followed by electrotransformation. These respective mutants lose eDT activity, demonstrating that both of the gene products are essential for CDT functioning. The on-going investigation of these edt mutants in mice will shed light on a role of CDT in colonization and pathogenesis of H. hepaticus.
1765 GENETIC DELETION OR INHIBITION OF NEUTRAL ENDOPEPTIDASE EXACERBATES INTESTINAL INFLAMMATION INDUCED BY CLOSTRIDIUM DIFFICILE TOXIN A. Kimberly S. Kirkwood, Ignazio Castagliuolo, John Maa, Jeff Zacks, Craig Gerard, Charlabos Pothoulakis, Eileen F. Grady, Univ of CA, San Francisco, CA; Beth Israel Deaconess Med Ctr, Boston, MA; UCSF, San Francisco, CA; Institute of Pathology, Boston, MA; Dept of Pediatric and Medicine, Harvard Med Sch, Boston, MA. Clostridium difficile toxin A (TxA) induces inflammation in the human intestine. In animal studies, the intestinal inflammatory response to TxA involves release of substance P and activation of sensory neurons. The cell surface enzyme neutral endopeptidase (NEP) inactivates substance P and may terminate its proinflammatory effects, but the role of NEP in enterotoxin-induced intestinal inflammation is unknown. We determined the role of NEP in TxA-mediated inflammation using NEP (-/-) and wild type (wt) mice, the NEP inhibitor phosphoramidon, and recombinant human NEP (rhNEP). TxA or buffer was injected into ileal loops (n=6-24/group), and after 3 hr, intestinal secretion (mg/cm of loop), myeloperoxidase activity (MPO, U/gm) and histologic damage (epithelial cell damage and neutrophil number, scored 0-3) were assessed. Baseline intestinal secretion was similar in wt and NEP (-/-) mice (33:t2 vs. 36:t2). In wt mice, 0.5 p,g, lug, 2 p,g, and 5p,g TxA stimulated a dose dependant increase in ileal secretion (46:t 6, 79:t II, 96:t 14, and 136:t 10), which dose for dose was significantly lower than that seen in NEP (-/-) mice (l01:t9, 138:t9, 146:t1l, and 185:t15, respectively). In the remaining experiments, 0.5p,g TxA was used since it yielded half-maximal responses in NEP (-/-) mice. Phosphoramidon (3mglkg) enhanced the response to TxA in wt mice (84:t1O, vs. 46:t6). rhNEP (3mglkg) diminished intestinal secretion in NEP (-/-) mice (64:t9 vs. 101:t9). Preinjection with carrier, BSA, or boiled rhNEP had no effect. MPO analysis confirmed these results. Baseline MPO was similar in wt (3.6:t2) and NEP (-/-) (I:t0.2) mice. In wt mice, 0.5 p,g TxA elevated MPO activity (l1.8:t3), and phosphoramidon significantly enhanced this response (30.5:t 12). In NEP (-/-) mice, TxA markedly increased MPO activity (32:t8), and rhNEP reduced MPO levels (9.2:t2). Similarly, in wt mice, phosphoramidon exacerbated TxA mediated epithelial damage (0.57:±:0.3 vs. 0) and neutrophil infiltration (2.43:t0.2 vs. I.7:±: 0.2). In NEP (-/-) mice, rhNEP attenuated epithelial cell damage (0 vs. 1.14:t0.4) and neutrophil infiltration (1.5:t0.2 vs. 2.71:t0.2). Thus, TxA-induced intestinal inflammation is exacerbated in NEP (-/-) mice and reduced to wt levels by rhNEP. Furthermore, NEP inhibition increases TxA responses in wt mice to that of the NEP (-/-). We conclude that NEP participates in the termination of intestinal inflammation induced by C. difficile TxA. NEP targeting may be a novel form of treatment for acute intestinal inflammation.
1766 EARLY MITOCHONDRIAL DAMAGE BY CLOSTRIDIUM DIFFI· CILE TOXIN A (TXA) REGULATES RELEASE OF INTERLEU· KIN-8 FROM HUMAN COLONOCYTES. Dan He, Charalabos Pothoulakis, Sara Keates, John T. LaMont, Beth Israel Deaconess Med Ctr & Harvard Med Sch, Boston, MA. Background and Aims: C. difficile TxA triggers acute colitis that involves activation of sensory nerves and mast cells and extravasation of neutrophils into the involved segment. These enterotoxic effects have been attributed to TxA s known inhibition of Rho proteins and actin filament formation. The purpose of this study was to explore the time-course and mechanism of release of IL-8 from enterocytes exposed to purified TxA. Methods: IL-8 release (ELISA), ATP production (luciferase assay) and reactive oxygen intermediates (ROIs) (dehydrorhodamine 123) were measured in HT 29 colon cancer cells exposed to 100 nM TxA with or without an antioxidant. Results: Within 10-30 min ofTxA exposure, ROIs were increased two-fold (p <0.007) and ATP concentration was reduced by 40% (from 762.6 to 460.6 nmol/mg protein, p
April 2000
104 - 5
cecum and cfu/gm stool. Like the immunocompetent mice with defined flora, scm mice with the same defined GI flora became heavily colonized; 108-9 cfu/gm stool and large intestine/cecum tissue was recovered I week post-inoculation. In contrast to immunocompetent mice, however, recovery of C. jejuni from stool and large intestine/cecum tissue in scm mice remained at this high level beyond one month. CONCLUSIONS: Our results suggest that a limited enteric flora permits a much higher level of intestinal colonization in C3H mice by C.jejuni, suggesting the normal microbiological GI flora represents an important innate immune mechanism against establishment of Campylobacter infection. Furthermore, the importance of adaptive immunity in clearing an infection that does become established is suggested by the inability of scm mice to eliminate C. jejuni. Comparison of wild type and mutant C. jejuni strains in these three murine models should allow us to determine the roles of specific genes in overcoming both innate and adaptive immune functions.
1763 MURINE ANTIBODY RESPONSE TO A VACCINE FOR ENTEROTOXIGENIC ESCHERICHIA COLI. Arthur J. de Lorimier, Wyatt Byrd, Eric R. Hall, Zachary J. Roberts, Charles E. McQueen, Douglas Tang, William Vaughan, Frederick J. Cassels, Walter Reed Army Institute of Research, Washington, DC; Naval Med Research Ctr, Washington, DC. Introduction Enterotoxigenic Escherichia coli (ETEC) is the most common cause of bacterial diarrhea identified in deployed soldiers and an important cause of infant morbidity and mortality in developing nations. Our aim was to compare the immunogenicity of three dosing schedules of coli surface antigen 6 (CS6), an antigen expressed by 31% of ETEC worldwide, encapsulated in a biodegradable poly(DL-lactide-co-glycolide) (PLG) administered intranasally (IN) to mice with purified unencapsulated CS6 given alone. Methods We studied 49 female BALB/c mice given CS6 IN four weeks apart. The vaccine was administered as follows on days 0, 28 and 55: Group I (n=6) received 25p,g CS6 encapsulated in PLG (CS6-PLG) on day 0 only; Group II (n=6) received 25p,g CS6-PLG on days 0 and 28; Group III (n= 12) received 25p,g CS6-PLG on all 3 occasions; Group IV (n=5) received 25p,g CS6 unencapsulated (CS6) on day 0 only; Group V (n=5) received 25p,g CS6 on days 0 and 28; Group VI (n=12) received 25p,g CS6 on all 3 occasions; Group VII (n=3) (control) received 25p,g PLG-encapsulated bovine serum albumin (BSAPLG) on day 0 only. Mice were sacrificed on day 84. Antibody to CS6 was assayed from mucosal secretions and serum specimens that were collected from all animals within three days prior to each dose of vaccine and prior to sacrifice. Anti-CS6 antibody was measured by enzyme linked immunosorbent assay for serum IgG and secretory IgA (sIgA). The anti-CS6 titers for all groups were compared and analysis of variance was used to assess the data. Results Mice tolerated IN administration of CS6, CS6PLG and BSA-PLG well. Serum anti-CS6 IgG rose following all three dosing schedules of vaccines containing CS6. Mice vaccinated with three doses of CS6-PLG developed a >4 fold rise in serum anti-CS6 IgG titer values (I :338,000) than those immunized with three doses of CS6 alone (I :76,000). Two and three doses of CS6-PLG led to a 2 fold and 7 fold rise, respectively, in anti-CS6 sIgA geometric mean titer values over two and three doses ofCS6 alone (p
1764 FUNCTIONAL CHARACTERIZATION OF THE HELICOBACTER HEPATICUS CYTOLETHAL DISTENDING TOXIN OPERON. Zhongming Ge, Vincent B. Young. Chih Ching Chien, Nancy S. Taylor, David B. Schauer, James G. Fox, MIT, Cambridge, MA. Cytolethal distending toxin (CDT), which causes cell arrest in the G2/M phase of the cell cycle, is produced in a number of mucosal pathogens including certain strains of Escherichia coli, Shigella spp. and Campylobacter spp. The gene organization of the edt operon and the role of CDT in bacterial infection are poorly understood. We previously sequenced three genes (cdtA, cdtB and cdtC) involved in H. hepaticus CDT activity and demonstrated that the cdtB gene is present in several enterohepatic helicobacters including H. bilis, H. pullorum, and H. canis. In this study, the transcription initiation and termination sites of the H. hepaticus edt operon were determined by primer extension and RT-PCR. The edt operon transcription started at the site ~ 270 nucleotides upstream of the cdtA start codon and ended at the site approximately 90 nucleotides downstream of the cdtC stop codon. These results indicated that the H. hepaticus edt operon consists of cdtA, cdtB and cdtC genes. Isogenic cdtB and cdtC mutants were created by inserting the Cm' cassette into either cdtB or cdtC, followed by electrotransformation. These respective mutants lose eDT activity, demonstrating that both of the gene products are essential for CDT functioning. The on-going investigation of these edt mutants in mice will shed light on a role of CDT in colonization and pathogenesis of H. hepaticus.
1765 GENETIC DELETION OR INHIBITION OF NEUTRAL ENDOPEPTIDASE EXACERBATES INTESTINAL INFLAMMATION INDUCED BY CLOSTRIDIUM DIFFICILE TOXIN A. Kimberly S. Kirkwood, Ignazio Castagliuolo, John Maa, Jeff Zacks, Craig Gerard, Charlabos Pothoulakis, Eileen F. Grady, Univ of CA, San Francisco, CA; Beth Israel Deaconess Med Ctr, Boston, MA; UCSF, San Francisco, CA; Institute of Pathology, Boston, MA; Dept of Pediatric and Medicine, Harvard Med Sch, Boston, MA. Clostridium difficile toxin A (TxA) induces inflammation in the human intestine. In animal studies, the intestinal inflammatory response to TxA involves release of substance P and activation of sensory neurons. The cell surface enzyme neutral endopeptidase (NEP) inactivates substance P and may terminate its proinflammatory effects, but the role of NEP in enterotoxin-induced intestinal inflammation is unknown. We determined the role of NEP in TxA-mediated inflammation using NEP (-/-) and wild type (wt) mice, the NEP inhibitor phosphoramidon, and recombinant human NEP (rhNEP). TxA or buffer was injected into ileal loops (n=6-24/group), and after 3 hr, intestinal secretion (mg/cm of loop), myeloperoxidase activity (MPO, U/gm) and histologic damage (epithelial cell damage and neutrophil number, scored 0-3) were assessed. Baseline intestinal secretion was similar in wt and NEP (-/-) mice (33:t2 vs. 36:t2). In wt mice, 0.5 p,g, lug, 2 p,g, and 5p,g TxA stimulated a dose dependant increase in ileal secretion (46:t 6, 79:t II, 96:t 14, and 136:t 10), which dose for dose was significantly lower than that seen in NEP (-/-) mice (l01:t9, 138:t9, 146:t1l, and 185:t15, respectively). In the remaining experiments, 0.5p,g TxA was used since it yielded half-maximal responses in NEP (-/-) mice. Phosphoramidon (3mglkg) enhanced the response to TxA in wt mice (84:t1O, vs. 46:t6). rhNEP (3mglkg) diminished intestinal secretion in NEP (-/-) mice (64:t9 vs. 101:t9). Preinjection with carrier, BSA, or boiled rhNEP had no effect. MPO analysis confirmed these results. Baseline MPO was similar in wt (3.6:t2) and NEP (-/-) (I:t0.2) mice. In wt mice, 0.5 p,g TxA elevated MPO activity (l1.8:t3), and phosphoramidon significantly enhanced this response (30.5:t 12). In NEP (-/-) mice, TxA markedly increased MPO activity (32:t8), and rhNEP reduced MPO levels (9.2:t2). Similarly, in wt mice, phosphoramidon exacerbated TxA mediated epithelial damage (0.57:±:0.3 vs. 0) and neutrophil infiltration (2.43:t0.2 vs. I.7:±: 0.2). In NEP (-/-) mice, rhNEP attenuated epithelial cell damage (0 vs. 1.14:t0.4) and neutrophil infiltration (1.5:t0.2 vs. 2.71:t0.2). Thus, TxA-induced intestinal inflammation is exacerbated in NEP (-/-) mice and reduced to wt levels by rhNEP. Furthermore, NEP inhibition increases TxA responses in wt mice to that of the NEP (-/-). We conclude that NEP participates in the termination of intestinal inflammation induced by C. difficile TxA. NEP targeting may be a novel form of treatment for acute intestinal inflammation.
1766 EARLY MITOCHONDRIAL DAMAGE BY CLOSTRIDIUM DIFFI· CILE TOXIN A (TXA) REGULATES RELEASE OF INTERLEU· KIN-8 FROM HUMAN COLONOCYTES. Dan He, Charalabos Pothoulakis, Sara Keates, John T. LaMont, Beth Israel Deaconess Med Ctr & Harvard Med Sch, Boston, MA. Background and Aims: C. difficile TxA triggers acute colitis that involves activation of sensory nerves and mast cells and extravasation of neutrophils into the involved segment. These enterotoxic effects have been attributed to TxA s known inhibition of Rho proteins and actin filament formation. The purpose of this study was to explore the time-course and mechanism of release of IL-8 from enterocytes exposed to purified TxA. Methods: IL-8 release (ELISA), ATP production (luciferase assay) and reactive oxygen intermediates (ROIs) (dehydrorhodamine 123) were measured in HT 29 colon cancer cells exposed to 100 nM TxA with or without an antioxidant. Results: Within 10-30 min ofTxA exposure, ROIs were increased two-fold (p <0.007) and ATP concentration was reduced by 40% (from 762.6 to 460.6 nmol/mg protein, p