title: Distribution dynamics of recombinant Lactobacillus in the gastrointestinal tract of neonatal rats.
abstract: One approach to deliver therapeutic agents, especially proteins, to the gastro-intestinal (GI) tract is to use commensal bacteria as a carrier. Genus Lactobacillus is an attractive candidate for use in this approach. However, a system for expressing exogenous proteins at a high level has been lacking in Lactobacillus. Moreover, it will be necessary to introduce the recombinant Lactobacillus into the GI tract, ideally by oral administration. Whether orally administered Lactobacillus can reach and reside in the GI tract has not been explored in neonates. In this study, we have examined these issues in neonatal rats. To achieve a high level of protein expression in Lactobacillus, we tested the impact of three promoters and two backbones on protein expression levels using mRFP1, a red fluorescent protein, as a reporter. We found that a combination of an L-lactate dehydrogenase (ldhL) promoter of Lactobacillus sakei with a backbone from pLEM415 yielded the highest level of reporter expression. When this construct was used to transform Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus acidophilus, high levels of mRFP1 were detected in all these species and colonies of transformed Lactobacillus appeared pink under visible light. To test whether orally administered Lactobacillus can be retained in the GI tract of neonates, we fed the recombinant Lactobacillus casei to neonatal rats. We found that about 3% of the bacteria were retained in the GI tract of the rats at 24 h after oral feeding with more recombinant Lactobacillus in the stomach and small intestine than in the cecum and colon. No mortality was observed throughout this study with Lactobacillus. In contrast, all neonatal rats died within 24 hours after fed with transformed E. coli. Taken together, our results indicate that Lactobacillus has the potential to be used as a vehicle for the delivery of therapeutic agents to neonates.
authors.affiliation: Saint James School of Medicine, Bonaire, The Netherlands Antilles. baosujin@yahoo.com
authors.forename: Sujin
authors.initials: S
authors.lastname: Bao
authors.forename: Libin
authors.initials: L
authors.lastname: Zhu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhuang
authors.forename: Lucia
authors.initials: L
authors.lastname: Wang
authors.forename: Pin-Xian
authors.initials: PX
authors.lastname: Xu
authors.forename: Keiji
authors.initials: K
authors.lastname: Itoh
authors.forename: Ian R
authors.initials: IR
authors.lastname: Holzman
authors.forename: Jing
authors.initials: J
authors.lastname: Lin
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D004274:DNA, Recombinant / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000382:microbiology*; D005822:Genetic Vectors / Q000235:genetics; D007778:Lactobacillus / Q000502:physiology*; D008164:Luminescent Proteins / Q000378:metabolism; D051381:Rats; D000097573:Red Fluorescent Protein
chemical_list: D004274:DNA, Recombinant; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
references.citation: N Engl J Med. 2011 Jan 20;364(3):255-64
references.pmid: 21247316
references.citation: Plasmid. 1997;37(3):199-203
references.pmid: 9200223
references.citation: Appl Environ Microbiol. 1987 Oct;53(10):2452-7
references.pmid: 2447829
references.citation: Ann N Y Acad Sci. 2006 Aug;1072:176-86
references.pmid: 17057198
references.citation: Am J Surg. 1979 Dec;138(6):845-50
references.pmid: 389076
references.citation: Inflamm Allergy Drug Targets. 2012 Apr;11(2):79-89
references.pmid: 22280243
references.citation: Annu Rev Biochem. 1998;67:509-44
references.pmid: 9759496
references.citation: Pediatrics. 2010 May;125(5):921-30
references.pmid: 20403939
references.citation: Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7877-82
references.pmid: 12060735
references.citation: FEMS Microbiol Lett. 2001 Jan 15;194(2):127-33
references.pmid: 11164296
references.citation: FEMS Microbiol Lett. 2010 Jul 1;308(1):8-15
references.pmid: 20455948
references.citation: J Bacteriol. 1997 Jul;179(14):4473-9
references.pmid: 9226255
references.citation: J Pediatr Gastroenterol Nutr. 2011 Dec;53 Suppl 2:S32-4
references.pmid: 22235463
references.citation: FEMS Microbiol Rev. 1993 Sep;12(1-3):207-20
references.pmid: 8398215
references.citation: Pediatrics. 2008 Oct;122(4):693-700
references.pmid: 18829790
references.citation: Nat Rev Microbiol. 2008 May;6(5):349-62
references.pmid: 18345021
references.citation: Am J Obstet Gynecol. 2007 Feb;196(2):147.e1-8
references.pmid: 17306659
references.citation: J Biosci Bioeng. 2010 Feb;109(2):189-92
references.pmid: 20129106
references.citation: J Pediatr Gastroenterol Nutr. 2000;30 Suppl 2:S34-8
references.pmid: 10749399
references.citation: Nat Methods. 2012 Jul;9(7):671-5
references.pmid: 22930834
references.citation: J Med Microbiol. 2005 Nov;54(Pt 11):1093-1101
references.pmid: 16192442
references.citation: FEMS Microbiol Lett. 2007 Oct;275(2):207-13
references.pmid: 17714484
references.citation: Appl Environ Microbiol. 2001 Oct;67(10):4939-42
references.pmid: 11571208