Gnotobiotic piglets inoculated with Escherichia coli O157:H7, its luxS mutant derivative, or nonpathogenic E. coli were evaluated for attaching and effacing lesions. Although no differences in clinical symptoms were seen between pigs inoculated with the parent and those inoculated with the luxS mutant, the luxS mutant-inoculated pigs had a lower frequency of attaching and effacing lesions in the spiral colon than parent strain-inoculated pigs

Dean-Nystrom, Evelyn A.

Jordan, Dianna M.

Kaper, James B.

Moon, Harley W.

Sperandio, Vanessa

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 2-2005 Colonization of Gnotobiotic Piglets by a luxS Mutant Strain of Escherichia coli O157:H7 Dianna M. Jordan Iowa State University Vanessa Sperandio University of Maryland School of Medicine James B. Kaper University of Maryland School of Medicine Evelyn A. Dean-Nystrom Pre-Harvest Food Safety and Enteric Diseases Research Unit, evelyn.nystrom@ars.usda.gov Harley W. Moon Iowa State University Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub  Part of the Agricultural Science Commons Jordan, Dianna M.; Sperandio, Vanessa; Kaper, James B.; Dean-Nystrom, Evelyn A.; and Moon, Harley W., "Colonization of Gnotobiotic Piglets by a luxS Mutant Strain of Escherichia coli O157:H7" (2005). Publications from USDA-ARS / UNL Faculty. 935. https://digitalcommons.unl.edu/usdaarsfacpub/935 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. INFECTION AND IMMUNITY, Feb. 2005, p. 1214–1216 Vol. 73, No. 20019-9567/05/$08.000 doi:10.1128/IAI.73.2.1214–1216.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.Colonization of Gnotobiotic Piglets by a luxS Mutant Strain ofEscherichia coli O157:H7Dianna M. Jordan,1* Vanessa Sperandio,2† James B. Kaper,2Evelyn A. Dean-Nystrom,3 and Harley W. Moon1Department of Veterinary Pathology, Iowa State University,1 and Pre-Harvest Food Safety andEnteric Disease Research Unit, National Animal Disease Center, AgriculturalResearch Service, U.S. Department of Agriculture,3 Ames, Iowa, andCenter for Vaccine Development and Department of Microbiologyand Immunology, University of Maryland School of Medicine,Baltimore, Maryland2Received 12 July 2004/Returned for modification 20 August 2004/Accepted 7 October 2004Gnotobiotic piglets inoculated with Escherichia coli O157:H7, its luxSmutant derivative, or nonpathogenic E.coli were evaluated for attaching and effacing lesions. Although no differences in clinical symptoms were seenbetween pigs inoculated with the parent and those inoculated with the luxSmutant, the luxSmutant-inoculatedpigs had a lower frequency of attaching and effacing lesions in the spiral colon than parent strain-inoculatedpigs.Quorum sensing (QS) is a bacterial cell-to-cell signalingmechanism used by a wide variety of bacterial species forrecognition of the surrounding microbial community. The luxSQS system is responsible for production of a signaling com-pound called autoinducer 2 (AI-2) that can control gene ex-pression in a wide variety of gram-positive and gram-negativespecies (10). In Escherichia coli O157:H7, the LuxS enzymecontributes to the production of another signaling compound,AI-3, which is involved in the QS regulation of some entero-hemorrhagic E. coli (EHEC) virulence genes (11, 13). TheAI-3 signaling compound is also produced by the human res-ident intestinal flora and hypothesized to be involved in sig-naling to EHEC that it has reached the large intestine of thehost (4, 13). This QS signaling cascade activates transcriptionof the genes within the locus of enterocyte effacement (LEE)pathogenicity island, which encode a type III secretion system,bacterial effectors, and the bacterial adhesin intimin, essentialfor formation of the attaching and effacing (A/E) lesions inenterocytes (6, 11, 13). A luxS mutation in EHEC results indecreased transcription of the LEE region genes and a defi-ciency in type III secretion in broth cultures (11). Due to theinfluence of LuxS on the synthesis of AI-3, which modulatesEHEC colonization factors, it is hypothesized that LuxS facil-itates intestinal colonization of gnotobiotic pigs by E. coliO157:H7. The objective of this study was to determine if mu-tation of the luxS gene would impair the ability of E. coliO157:H7 to colonize gnotobiotic piglets.Germfree piglets, 24 h old, were orally inoculated with 105CFU of bacteria. Eleven pigs from four litters were inoculatedwith the parent strain, a spontaneous streptomycin- and nali-dixic acid-resistant derivative of E. coli O157:H7 86-24 (14).Fourteen pigs from five litters were inoculated with strainVS97, a luxS mutant derived from the parent strain by methodsdescribed by Sperandio et al. (12). Six pigs from two litterswere inoculated with nonpathogenic E. coli strain 123 (NPE123) (7) to serve as controls.Strains were grown as overnight cultures in tryptic soy brothwith appropriate antibiotics. The parent strain was grown with50 g of streptomycin/ml and 10 g of nalidixic acid/ml, theluxS mutant was grown with 10 g of tetracycline/ml, and NPE123 was grown without antibiotics. Inocula were prepared aspreviously described (1, 2, 9).Pigs inoculated with the parent strain and VS97 were nec-ropsied when neurologic signs were demonstrated or at 7 dayspostinoculation (p.i.). Control pigs were necropsied at 4 daysp.i., the average time of onset of clinical signs for pigs inocu-lated with the pathogenic strains. At necropsy, sections of ilea,ceca, spiral colons, cerebrums, cerebellums, and brain stemswere collected in neutral buffered formalin for histopathology,processed, and stained with hematoxylin and eosin. A specifichorseradish peroxidase immunohistochemical stain with theprimary antibody targeting the O157 antigen was used for theintestinal sections (3). Cross-sections from each intestinal seg-ment were examined microscopically in a blind fashion andscored for A/E lesions with intimately associated bacteria as ameasure of colonization (5, 8). Multiple sections of spiral co-lons from five pigs that were inoculated with the parent strainand the luxS mutant were prepared and evaluated by transmis-sion electron microscopy.Contingency analysis for ordinal data, followed by pairwisetesting, was done for the A/E scores. JMP (version 5.0.1a; SASInstitute, Inc.) was the statistical program utilized for analysis;a P value of 0.05 was used as the level of significance for allevaluations.Pigs from groups inoculated with the parent strain or its luxSmutant derivative showed clinical signs of ataxia, head-press-* Corresponding author. Present address: Veterinary Diagnosticand Production Animal Medicine, Iowa State University, Ames, IA50011. Phone: (515) 294-1950. Fax: (515) 294-3564. E-mail: dmjordan@iastate.edu.† Present address: Department of Microbiology, University of TexasSouthwestern Medical Center, Dallas, TX 75390-9048.1214ing, and/or recumbency due to the systemic effects of Shigatoxins (15). Eleven of 14 pigs inoculated with the luxS mutantdeveloped neurologic signs between 3.5 and 7 days p.i. Nine of11 pigs inoculated with the parent strain developed neurologicsigns between 3 and 7 days p.i. No clinical signs were observedin the control pigs necropsied at 4 days p.i.Upon necropsy, 10 of 14 luxS mutant-inoculated pigs and all11 parent strain-inoculated pigs had mesocolonic edema. Nogross enteric lesions were present in the NPE 123-inoculatedcontrol pigs.Histologically, the A/E lesions in the ilea, ceca, and spiralcolons from the luxS mutant-inoculated pigs were qualitativelysimilar to lesions from the pigs inoculated with the parentstrain. The distributions of the A/E lesion scores are summa-rized in Fig. 1. The parent strain-inoculated group had signif-icantly higher (P  0.0025) A/E scores (median, 2; range, 1 to3) for the spiral colon than did the luxS mutant-inoculated pigs(median, 1; range, 0 to 3). For the spiral colon sections, boththe parent strain- and the luxS mutant-inoculated pigs hadsignificantly higher (P 0.0001) A/E lesion scores than did theNPE 123-inoculated pigs. There was no significant difference(P  0.5) between the cecal A/E lesion scores for the parentstrain (median, 2; range, 0 to 3) and those for the luxS mutant-inoculated pigs (median, 1; range, 0 to 3); these two groups hadsignificantly higher cecal A/E lesion scores than did the NPE123-inoculated pigs (P  0.002). There were no A/E lesions inany intestinal segments from the pigs inoculated with NPE 123.There were no significant differences in A/E lesion scores forthe ileal sections from pigs inoculated with any of the strains.Classical A/E lesions (8) were identified with electron micros-copy in the spiral colons from all the parent strain- and luxSmutant-inoculated pigs examined. Microhemorrhages and ne-crotic vessels were detected in the brains of pigs inoculatedwith the parent strain or its luxS derivative; results are shownin Table 1. There was no statistically significant differencebetween the scores of these two groups of pigs for neurologicallesions. Pigs inoculated with NPE 123 had no neurologic le-sions.We hypothesized that LuxS was a critical enzyme in theproduction of autoinducer signals that are important for theexpression of virulence factors, such as those required for A/Elesion production by E. coli O157:H7 in vitro (11, 12). Thisstudy demonstrated that A/E lesions in spiral colons occurredless frequently in gnotobiotic piglets inoculated with the luxSmutant strain than in those inoculated with the parent strain.There were no significant differences in the frequencies oflesions in the ceca and ilea. Mutation in luxS did not eliminatethe ability of E. coli O157:H7 to produce A/E lesions or tocause clinical disease in piglets due to the effects of Shiga toxin.These results were consistent with a recent report by Speran-dio et al. (13) demonstrating that the eukaryotic hormonesepinephrine and norepinephrine activate the type III secretionsystem, LEE transcription, and, consequently, A/E lesion for-mation by the luxS mutant. Therefore, the host’s epinephrineand norepinephrine may be substituting for the AI-3 bacterialsignaling (13). In this study, the presence of these hormones orother host factors in the intestines of the gnotobiotic pigletspresumably may have signaled to the bacteria to express theLEE region genes for the development of A/E lesions; there-fore, compelling differences were not demonstrated in vivo asthey were in vitro with the luxS mutant of E. coli O157:H7.This work was supported by NIH grant 1R01DK58957, awarded toJ.B.K.We thank Lawayne Nusz and the NADC animal caretaker, RogerSpaete, for their assistance; Judy Stasko for transmission electronmicroscopy assistance; Richard Evans for statistical consultation; and,especially, Sheridan Booher for technical assistance in bacteriologyand animal handling.REFERENCES1. Booher, S. L., N. A. Cornick, and H. W. Moon. 2002. Persistence of Esche-richia coli O157:H7 in experimentally infected swine. Vet. Microbiol. 89:69–81.2. Cornick, N. A., S. L. Booher, T. A. Casey, and H. W. Moon. 2000. Persistentcolonization of sheep by Escherichia coli O157:H7 and other E. coli patho-types. Appl. Environ. Microbiol. 66:4926–4934.3. Dean-Nystrom, E. A., B. T. Bosworth, W. C. Cray, Jr., and H. W. Moon. 1997.Pathogenicity of Escherichia coli O157:H7 in the intestines of neonatalcalves. Infect. Immun. 65:1842–1848.4. Falcao, J. P., F. Sharp, and V. Sperandio. 2004. Cell-to-cell signaling inintestinal pathogens. Curr. Issues Intest. Microbiol. 5:9–17.5. Jordan, D. M., N. Cornick, A. G. Torres, E. A. Dean-Nystrom, J. B. Kaper,FIG. 1. A/E lesion scores in tissues of gnotobiotic piglets inocu-lated with nonpathogenic E. coli (NPE), E. coli O157:H7 strain VS97(luxS mutant), or 86-24 (Parent). The A/E lesion scores of the forma-lin-fixed intestinal sections were as follows: 0, no A/E lesions withdiscernable associated bacteria visible throughout the sections; 0.5,infrequent A/E lesions identified only with the immunohistochemicalstain; 1, discernable lesions on fewer than 10% of the enterocytes ofthe sections; 2, discernable lesions on more than 10% but fewer than50% of the enterocytes; 3, lesions present on 50% or more of theenterocytes. Contingency analysis detected statistically significant dif-ferences between the scores for the luxS mutant-inoculated tissues andthose for the parent-inoculated tissues from the spiral colons (P 0.03).TABLE 1. Clinical and microscopic findings for gnotobiotic pigletsinoculated with E. coli O157:H7 strain 86–24, VS97, or NPE 123Strain usedforinoculationNo. of samples positive for indicated finding/no. of pigsinoculatedClinicalsignsTissue colonization Brain histopathologyIleum Cecum Spiralcolon MicrohemorrhageVesselnecrosisParent 9/11 3/6 10/11 11/11 2/11 8/11luxS mutant 11/14 2/6 13/14 14/14 6/14 9/14NPE 123 0/6 0/6 0/6 0/6 0/6 0/6VOL. 73, 2005 NOTES 1215and H. W. Moon. 2004. Long polar fimbriae contribute to colonization byEscherichia coli O157:H7 in vivo. Infect. Immun. 72:6168–6171.6. McDaniel, T. K., K. G. Jarvis, M. S. Donnenberg, and J. B. Kaper. 1995. Agenetic locus of enterocyte effacement conserved among diverse enterobac-terial pathogens. Proc. Natl. Acad. Sci. USA 92:1664–1668.7. Moon, H. W., and S. C. Whipp. 1971. Systems for testing the enteropatho-genicity of Escherichia coli. Ann. N. Y. Acad. Sci. 176:197–211.8. Moon, H. W., S. C. Whipp, R. A. Argenzio, M. M. Levine, and R. A. Gian-nella. 1983. Attaching and effacing activities of rabbit and human entero-pathogenic Escherichia coli in pig and rabbit intestines. Infect. Immun. 41:1340–1351.9. Sarmiento, J. I., T. A. Casey, and H. W. Moon. 1988. Postweaning diarrheain swine: experimental model of enterotoxigenic Escherichia coli infection.Am. J. Vet. Res. 49:1154–1159.10. Schauder, S., K. Shokat, M. G. Surette, and B. L. Bassler. 2001. The LuxSfamily of bacterial autoinducers: biosynthesis of a novel quorum-sensingsignal molecule. Mol. Microbiol. 41:463–476.11. Sperandio, V., J. L. Mellies, W. Nguyen, S. Shin, and J. B. Kaper. 1999.Quorum sensing controls expression of the type III secretion gene transcrip-tion and protein secretion in enterohemorrhagic and enteropathogenic Esch-erichia coli. Proc. Natl. Acad. Sci. USA 96:15196–15201.12. Sperandio, V., A. G. Torres, J. A. Giro´n, and J. B. Kaper. 2001. Quorumsensing is a global regulatory mechanism in enterohemorrhagic Escherichiacoli O157:H7. J. Bacteriol. 183:5187–5197.13. Sperandio, V., A. G. Torres, B. Jarvis, J. P. Nataro, and J. B. Kaper. 2003.Bacteria-host communication: the language of hormones. Proc. Natl. Acad.Sci. USA 100:8951–8956.14. Tarr, P. I., M. A. Neill, C. R. Clausen, J. W. Newland, R. J. Neill, and S. L.Moseley. 1989. Genotypic variation in pathogenic Escherichia coli O157:H7isolated from patients in Washington, 1984–1987. J. Infect. Dis. 159:344–347.15. Tzipori, S., H. Karch, K. I. Wachsmuth, R. M. Robins-Browne, A. D.O’Brien, H. Lior, M. L. Cohen, J. Smithers, and M. M. Levine. 1987. Roleof a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis ofinfection caused by enterohemorrhagic Escherichia coli O157:H7 in gnoto-biotic piglets. Infect. Immun. 55:3117–3125.Editor: J. B. Bliska1216 NOTES INFECT. IMMUN.

Colonization of Gnotobiotic Piglets by a \u3ci\u3eluxS\u3c/i\u3e Mutant Strain of
\u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7

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Colonization of Gnotobiotic Piglets by a \u3ci\u3eluxS\u3c/i\u3e Mutant Strain of \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7

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