Gastrointestinal (GI) diseases, and in particular those caused by bacterial infections, are a major cause of morbidity and mortality worldwide. Treatment is becoming increasingly difficult due to the increase in number of species that have developed resistance to antibiotics. Probiotic lactic acid bacteria (LAB) have considerable potential as alternatives to antibiotics, both in prophylactic and therapeutic applications. Several studies have documented a reduction, or prevention, of GI diseases by probiotic bacteria. Since the activities of probiotic bacteria are closely linked with conditions in the host's GI-tract (GIT) and changes in the population of enteric microorganisms, a deeper understanding of gut-microbial interactions is required in the selection of the most suitable probiotic. This necessitates a deeper understanding of the molecular capabilities of probiotic bacteria. In this review, we explore how probiotic microorganisms interact with enteric pathogens in the GIT. The significance of probiotic colonization and persistence in the GIT is also addressed.
Lanthipeptides are ribosomally synthesized and post translationally modified peptides, with modifications that are incorporated during biosynthesis by dedicated enzymes. Various modifications of the peptides are possible resulting in a highly diverse group of bioactive peptides that offer a potential reservoir for use in the fight against a plethora of diseases. Their activities range from the antimicrobial properties of lantibiotics, especially against antibiotic-resistant strains, to antiviral activity, immunomodulatory properties, antiallodynic effects and the potential to alleviate cystic fibrosis symptoms. Lanthipeptide biosynthetic genes are widespread within bacterial genomes providing a substantial repository for novel bioactive peptides. Using genome mining tools novel bioactive lanthipeptides can be identified and coupled with rapid screening and heterologous expression technologies the lanthipeptide drug discovery pipeline can be significantly sped up. Lanthipeptides represents a group of bioactive peptides that holds great potential as biotherapeutics, especially at a time when novel and more effective therapies are required. With this review we provide insight into the latest developments made towards the therapeutic applications and production of lanthipeptides, specifically looking at heterologous expression systems.
Lactic acid bacteria (LAB) are natural inhabitants of the gastrointestinal tract (GIT) of humans and animals, and some LAB species receive considerable attention due to their health benefits. Although many papers have been published on probiotic LAB, only a few reports have been published on the migration and colonization of the cells in the GIT. This is due mostly to the lack of efficient reporter systems. In this study, we report on the application of the fluorescent mCherry protein in the in vivo tagging of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423. The mCherry gene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome of L. plantarum 423 by homologous recombination. In the case of E. mundtii ST4SA, the mCherry gene was cloned into the pGKV223D LAB/Escherichia coli expression vector. Expression of the mCherry gene did not alter the growth rate of the two strains and had no effect on bacteriocin production. Both strains colonized the cecum and colon of mice. Lactic acid bacteria (LAB) are common inhabitants of a healthy human gastrointestinal tract (GIT). Lactobacillus spp. are used as starter cultures in many fermented foods and are well known for their probiotic properties and the exclusion of pathogens from the GIT (1-6). Some strains have been closely associated with the treatment of gastrointestinal disorders, lactose intolerance, and stimulation of the immune system (7,8).Despite the increasing consumer interest in probiotic LAB, the mechanisms whereby these bacteria exert their beneficial effects in the GIT are not well understood (9). Although simulated gastrointestinal models (10-14) produced valuable data on the colonization of probiotic LAB and the exclusion of pathogens, the method remains an in vitro approach and is not a true reflection of in situ conditions. Labeling of probiotic bacteria with genes expressing fluorophores allows studying colonization and competition between gut microorganisms in vivo (15)(16)(17)(18)(19).Since the discovery of the green fluorescent protein (GFP) by Shimomura et al. (20), a number of genetic variants that emit light at longer wavelengths, and that are more suited for in vivo animal studies, have been described (21-23). The mCherry red fluorescence protein (RFP), a variant of the Discosoma red (DsRed) protein (24,25), is excited at wavelengths longer than 600 nm and is photostable (26-28). Studies conducted with Bacillus subtilis (29) and Escherichia coli (30) showed that expression of the mCherry protein, even at high levels, had no effect on the cell's physiology. Tauer and coworkers (31) used the mCherry gene to study recombinant protein expression in Lactobacillus plantarum. Mohedano and coworkers (32) adapted the broad-host-range vector pNZ8048 to study gene expression in Lactobacillus and showed that the mCherry construct could be used to study complex promoter induction mechanisms, such as bacteriocin production by Lactobacillus acidophilus. The role that lactococcin 972 (...
Listeria monocytogenes is an opportunistic food-borne pathogen and is life-threatening to individuals with a weakened immune system. The aim of this study was to determine if Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA could prevent colonisation of L. monocytogenes in the gastro-intestinal tract (GIT). Mice were gavaged with L. plantarum 423, E. mundtii ST4SA, and a combination of the two strains, for 6 consecutive days and orally infected with a bioluminescent strain of L. monocytogenes (strain EGDe) on the last day of treatment. 30 min after infection, high cell numbers of L. plantarum 423, E. mundtii ST4SA and L. monocytogenes EGDe were isolated from faeces. L. monocytogenes EGDe cells were absent from the small intestine of L. plantarum 423-treated mice 4 h after infection and from the large intestine 2 h later. No bioluminescent, and thus metabolically active, cells of L. monocytogenes EGDe were recorded in the GIT of mice treated with E. mundtii ST4SA, suggesting that their growth was repressed. L. plantarum 423 and E. mundtii ST4SA colonised the colon the strongest. These strains may be considered for the competitive exclusion of L. monocytogenes from the GIT.
Probiotics play an important role in maintaining a healthy and stable intestinal microbiota, primarily by preventing infection. Probiotic lactic acid bacteria (LAB) are known to be inhibitory to many bacterial enteric pathogens, including antibiotic-resistant strains. Whilst the positive role that probiotics have on human physiology, specifically in the treatment or prevention of specific infectious diseases of the gastro-intestinal tract (GIT) is known, the precise mechanistic basis of these effects remains a major research goal. In this study, molecular evidence to underpin the protective and anti-listerial effect of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA against orally administered Listeria monocytogenes EGDe in the GIT of mice is provided. Bacteriocins plantaricin 423 and mundticin ST4SA, produced by L. plantarum 423 and E. mundtii ST4SA, respectively, inhibited the growth of L. monocytogenes in vitro and in vivo. Bacteriocin-negative mutants of L. plantarum 423 and E. mundtii ST4SA failed to exclude L. monocytogenes EGDe from the gastrointestinal tract (GIT) of mice. Furthermore, L. plantarum 423 and E. mundtii ST4SA failed to inhibit recombinant strains of L. monocytogenes EGDe in vivo that expressed the immunity proteins of the two bacteriocins. These results confirmed that bacteriocins plantaricin 423 and mundticin ST4SA acted as anti-infective mediators in vivo. Compared to wild type strains, mutants of L. plantarum 423 and E. mundtii ST4SA, in which the adhesion genes were knocked out, were less effective in the exclusion of L. monocytogenes EGDe from the GIT of mice. This work demonstrates the importance of bacteriocin and adhesion genes as probiotic anti-infective mechanisms.
Background The underlying mechanisms by which probiotic lactic acid bacteria (LAB) enhance the health of the consumer have not been fully elucidated. Verification of probiotic modes of action can be achieved by using single- or multiple-gene knockout analyses of bacterial mutants in in vitro or in vivo models. We developed a novel system based on an inducible toxin counter-selection system, allowing for rapid and efficient isolation of LAB integration or deletion mutants. The Lactococcus lactis nisin A inducible promoter was used for expression of the Escherichia coli mazF toxin gene as counter-selectable marker. Results The flippase (FLP)/flippase recognition target (FRT) recombination system and an antisense RNA transcript were used to create markerless chromosomal gene integrations/deletions in LAB. Expression of NisR and NisK signalling proteins generated stable DNA integrations and deletions. Large sequences could be inserted or deleted in a series of steps, as demonstrated by insertion of the firefly bioluminescence gene and erythromycin resistance marker into the bacteriocin operons or adhesion genes of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA. Conclusions The system was useful in the construction of L. plantarum 423 and E. mundtii ST4SA bacteriocin and adhesion gene mutants. This provides the unique opportunity to study the role of specific probiotic LAB genes in complex environments using reverse genetics analysis. Although this work focuses on two probiotic LAB strains, L . plantarum 423 and E. mundtii ST4SA, the system developed could be adapted to most, if not all, LAB species. Electronic supplementary material The online version of this article (10.1186/s12867-019-0127-x) contains supplementary material, which is available to authorized users.
BackgroundLactic acid bacteria (LAB) are major inhabitants and part of the normal microflora of the gastrointestinal tract (GIT) of humans and animals. Despite substantial evidence supporting the beneficial properties of LAB, only a few studies have addressed the migration and colonization of probiotic bacteria in the GIT. The reason for this is mostly due to the limitations, or lack of, efficient reporter systems. Here we describe the development and application of a non-invasive in vivo bioluminescence reporter system to study, in real-time, the spatial and temporal persistence of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA in the intestinal tract of mice.ResultsThis study reports on the application of the firefly luciferase gene (ffluc) from Photinus pyralis to develop luciferase-expressing L. plantarum 423 and E. mundtii ST4SA, using a Lactococcus lactis NICE system on a high copy number plasmid (pNZ8048) and strong constitutive lactate dehydrogenase gene promoters (Pldh and STldh). The reporter system was used for in vivo and ex vivo monitoring of both probiotic LAB strains in the GIT of mice after single and multiple oral administrations. Enterococcus mundtii ST4SA reached the large intestine 45 min after gavage, while L. plantarum 423 reached the cecum/colon after 90 min. Both strains predominantly colonized the cecum and colon after five consecutive daily administrations. Enterococcus mundtii ST4SA persisted in faeces at higher numbers and for more days compared to L. plantarum 423.ConclusionsOur findings demonstrate the efficiency of a high-copy number vector, constitutive promoters and bioluminescence imaging to study the colonization and persistence of L. plantarum 423 and E. mundtii ST4SA in the murine GIT. The system allowed us to differentiate between intestinal transit times of the two strains in the digestive tract. This is the first report of bioluminescence imaging of a luciferase-expressing E. mundtii strain to study colonization dynamics in the murine model. The bioluminescence system developed in this study may be used to study the in vivo colonization dynamics of other probiotic LAB.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1315-4) contains supplementary material, which is available to authorized users.
Bioluminescence (BLI) and fluorescence imaging (FI) allow for non-invasive detection of viable microorganisms from within living tissue and are thus ideally suited for in vivo probiotic studies. Highly sensitive optical imaging techniques detect signals from the excitation of fluorescent proteins, or luciferase-catalyzed oxidation reactions. The excellent relation between microbial numbers and photon emission allow for quantification of tagged bacteria in vivo with extreme accuracy. More information is gained over a shorter period compared to traditional pre-clinical animal studies. The review summarizes the latest advances in in vivo bioluminescence and fluorescence imaging and points out the advantages and limitations of different techniques. The practical application of BLI and FI in the tracking of lactic acid bacteria in animal models is addressed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.