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.
Highlights
Probiotics are living microorganisms that confer a health benefit on the host upon ingestion in adequate amounts [1,2]
While the mechanisms remain unclear, clinical studies have demonstrated that supplementation of lactic acid bacteria as a probiotic can significantly reduce the incidence of neonatal necrotizing enterocolitis (NEC), a devastating disease affecting about 5–7% of infants with a birth weight less than 1500 grams and one of the significant causes of mortality and morbidity in premature infants [4,5,6,7]
To achieve a high level of protein expression in Lactobacillus, we tested the impact of three promoters and two backbones on protein expression since choices of the promoter and the backbone often have a significant impact on the expression level of a target gene
Summary
Probiotics are living microorganisms that confer a health benefit on the host upon ingestion in adequate amounts [1,2]. Lactic acid bacteria are a large group of bacteria that produce lactic acid as an end product of fermentation. They are commonly used as probiotics because they have long been used in food production and they are safe for human consumption [3]. In the past two decades, whereas significant advances have been made in using Lactococcus as a vehicle to deliver therapeutic agents to the GI tract, in part because expression of exogenous proteins in Lactococcus is well established [8,9], use of Lactobacillus in this aspect has lagged behind. We aimed at developing a Lactobacillus-based system to deliver therapeutic agents to the gastrointestinal tract of neonates. We used mRFP1 as a marker to label Lactobacillus and examined the retention rate of the recombinant Lactobacillus in the GI tract of neonatal rats
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