Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human gastrointestinal tract.

A novel lanC-like sequence was identified from the dominant human gut bacterium Blautia obeum strain A2-162. This sequence was extended to reveal a putative lantibiotic operon with biosynthetic and transport genes, two sets of regulatory genes, immunity genes, three identical copies of a nisin-like lanA gene with an unusual leader peptide, and a fourth putative lanA gene. Comparison with other nisin clusters showed that the closest relationship was to nisin U. B. obeum A2-162 demonstrated antimicrobial activity against Clostridium perfringens when grown on solid medium in the presence of trypsin. Fusions of predicted nsoA structural sequences with the nisin A leader were expressed in Lactococcus lactis containing the nisin A operon without nisA. Expression of the nisA leader sequence fused to the predicted structural nsoA1 produced a growth defect in L. lactis that was dependent upon the presence of biosynthetic genes, but failed to produce antimicrobial activity. Insertion of the nso cluster into L. lactis MG1614 gave an increased immunity to nisin A, but this was not replicated by the expression of nsoI. Nisin A induction of L. lactis containing the nso cluster and nisRK genes allowed detection of the NsoA1 pre-peptide by Western hybridization. When this heterologous producer was grown with nisin induction on solid medium, antimicrobial activity was demonstrated in the presence of trypsin against C. perfringens, Clostridium difficile and L. lactis. This research adds to evidence that lantibiotic production may be an important trait of gut bacteria and could lead to the development of novel treatments for intestinal diseases.

A Novel Tightly Regulated Gene Expression System for the Human Intestinal Symbiont Bacteroides thetaiotaomicron.

There is considerable interest in studying the function of Bacteroides species resident in the human gastrointestinal (GI)-tract and the contribution they make to host health. Reverse genetics and protein expression techniques, such as those developed for well-characterized Escherichia coli cannot be applied to Bacteroides species as they and other members of the Bacteriodetes phylum have unique promoter structures. The availability of useful Bacteroides-specific genetic tools is therefore limited. Here we describe the development of an effective mannan-controlled gene expression system for Bacteroides thetaiotaomicron containing the mannan-inducible promoter-region of an α-1,2-mannosidase gene (BT_3784), a ribosomal binding site designed to modulate expression, a multiple cloning site to facilitate the cloning of genes of interest, and a transcriptional terminator. Using the Lactobacillus pepI as a reporter gene, mannan induction resulted in an increase of reporter activity in a time- and concentration-dependent manner with a wide range of activity. The endogenous BtcepA cephalosporinase gene was used to demonstrate the suitability of this novel expression system, enabling the isolation of a His-tagged version of BtCepA. We have also shown with experiments performed in mice that the system can be induced in vivo in the presence of an exogenous source of mannan. By enabling the controlled expression of endogenous and exogenous genes in B. thetaiotaomicron this novel inducer-dependent expression system will aid in defining the physiological role of individual genes and the functional analyses of their products.

Elucidating pathways of Toxoplasma gondii invasion in the gastrointestinal tract: involvement of the tight junction protein occludin.

Toxoplasma gondii is an obligate intracellular parasite infecting one third of the world's population. The small intestine is the parasite's primary route of infection, although the pathway of epithelium transmigration remains unclear. Using an in vitro invasion assay and live imaging we showed that T. gondii (RH) tachyzoites infect and transmigrate between adjacent intestinal epithelial cells in polarized monolayers without altering barrier integrity, despite eliciting the production of specific inflammatory mediators and chemokines. During invasion, T. gondii co-localized with occludin. Reducing the levels of endogenous cellular occludin with specific small interfering RNAs significantly reduced the ability of T. gondii to penetrate between and infect epithelial cells. Furthermore, an in vitro invasion and binding assays using recombinant occludin fragments established the capacity of the parasite to bind occludin and in particular to the extracellular loops of the protein. These findings provide evidence for occludin playing a role in the invasion of T. gondii in small intestinal epithelial cells.

Cephalosporinases associated with outer membrane vesicles released by Bacteroides spp. protect gut pathogens and commensals against ß-lactam antibiotics.

OBJECTIVES: To identify ß-lactamase genes in gut commensal Bacteroides species and to assess the impact of these enzymes, when carried by outer membrane vesicles (OMVs), in protecting enteric pathogens and commensals. METHODS: A deletion mutant of the putative class A ß-lactamase gene (locus tag BT_4507) found in the genome of the human commensal Bacteroides thetaiotaomicron was constructed and a phenotypic analysis performed. A phylogenetic tree was built from an alignment of nine Bacteroides cephalosporinase protein sequences, using the maximum likelihood method. The rate of cefotaxime degradation after incubation with OMVs produced by different Bacteroides species was quantified using a disc susceptibility test. The resistance of Salmonella Typhimurium and Bifidobacterium breve to cefotaxime in liquid culture in the presence of B. thetaiotaomicron OMVs was evaluated by measuring bacterial growth. RESULTS: The B. thetaiotaomicron BT_4507 gene encodes a ß-lactamase related to the CepA cephalosporinase of Bacteroides fragilis. OMVs produced by B. thetaiotaomicron and several other Bacteroides species, except Bacteroides ovatus, carried surface-associated ß-lactamases that could degrade cefotaxime. ß-Lactamase-harbouring OMVs from B. thetaiotaomicron protected Salmonella Typhimurium and B. breve from an otherwise lethal dose of cefotaxime. CONCLUSIONS: The production of membrane vesicles carrying surface-associated ß-lactamases by Bacteroides species, which constitute a major part of the human colonic microbiota, may protect commensal bacteria and enteric pathogens, such as Salmonella Typhimurium, against ß-lactam antibiotics.

Luminal microbes promote monocyte-stem cell interactions across a healthy colonic epithelium.

The intestinal epithelium forms a vital barrier between luminal microbes and the underlying mucosal immune system. Epithelial barrier function is maintained by continuous renewal of the epithelium and is pivotal for gut homeostasis. Breaching of the barrier causes mobilization of immune cells to promote epithelial restitution. However, it is not known whether microbes at the luminal surface of a healthy epithelial barrier influence immune cell mobilization to modulate tissue homeostasis. Using a mouse colonic mucosal explant model, we demonstrate that close proximity of luminal microbes to a healthy, intact epithelium results in rapid mucus secretion and movement of Ly6C(+)7/4(+) monocytes closer to epithelial stem cells. These early events are driven by the epithelial MyD88-signaling pathway and result in increased crypt cell proliferation and intestinal stem cell number. Over time, stem cell number and monocyte-crypt stem cell juxtapositioning return to homeostatic levels observed in vivo. We also demonstrate that reduced numbers of tissue Ly6C+ monocytes can suppress Lgr5EGFP+ stem cell expression in vivo and abrogate the response to luminal microbes ex vivo. The functional link between monocyte recruitment and increased crypt cell proliferation was further confirmed using a crypt-monocyte coculture model. This work demonstrates that the healthy gut epithelium mediates communication between luminal bacteria and monocytes, and monocytes can modulate crypt stem cell number and promote crypt cell proliferation to help maintain gut homeostasis.

A bacterial homolog of a eukaryotic inositol phosphate signaling enzyme mediates cross-kingdom dialog in the mammalian gut.

Dietary InsP6 can modulate eukaryotic cell proliferation and has complex nutritive consequences, but its metabolism in the mammalian gastrointestinal tract is poorly understood. Therefore, we performed phylogenetic analyses of the gastrointestinal microbiome in order to search for candidate InsP6 phosphatases. We determined that prominent gut bacteria express homologs of the mammalian InsP6 phosphatase (MINPP) and characterized the enzyme from Bacteroides thetaiotaomicron (BtMinpp). We show that BtMinpp has exceptionally high catalytic activity, which we rationalize on the basis of mutagenesis studies and by determining its crystal structure at 1.9 Å resolution. We demonstrate that BtMinpp is packaged inside outer membrane vesicles (OMVs) protecting the enzyme from degradation by gastrointestinal proteases. Moreover, we uncover an example of cross-kingdom cell-to-cell signaling, showing that the BtMinpp-OMVs interact with intestinal epithelial cells to promote intracellular Ca(2+) signaling. Our characterization of BtMinpp offers several directions for understanding how the microbiome serves human gastrointestinal physiology.

Expression and delivery of an endolysin to combat Clostridium perfringens.

Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, we investigated the activity and delivery of a bacteriophage endolysin. We identified a new endolysin, designated CP25L, which shows similarity to an N-acetylmuramoyl-L-alanine amidase domain and is distinct from other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp25l gene was cloned and expressed in Escherichia coli, and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract. The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (P nisA ), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.

Spontaneous mutation reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.

As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

Defining the bacteroides ribosomal binding site.

The human gastrointestinal tract, in particular the colon, hosts a vast number of commensal microorganisms. Representatives of the genus Bacteroides are among the most abundant bacterial species in the human colon. Bacteroidetes diverged from the common line of eubacterial descent before other eubacterial groups. As a result, they employ unique transcription initiation signals and, because of this uniqueness, they require specific genetic tools. Although some tools exist, they are not optimal for studying the roles and functions of these bacteria in the human gastrointestinal tract. Focusing on translation initiation signals in Bacteroides, we created a series of expression vectors allowing for different levels of protein expression in this genus, and we describe the use of pepI from Lactobacillus delbrueckii subsp. lactis as a novel reporter gene for Bacteroides. Furthermore, we report the identification of the 3' end of the 16S rRNA of Bacteroides ovatus and analyze in detail its ribosomal binding site, thus defining a core region necessary for efficient translation, which we have incorporated into the design of our expression vectors. Based on the sequence logo information from the 5' untranslated region of other Bacteroidales ribosomal protein genes, we conclude that our findings are relevant to all members of this order.

Complete genome sequence of Lactobacillus johnsonii FI9785, a competitive exclusion agent against pathogens in poultry.

Lactobacillus johnsonii is a member of the acidophilus group of lactobacilli. Because of their probiotic properties, including attachment to epithelial cells, immunomodulation, and competitive exclusion of pathogens, representatives of this group are being intensively studied. Here we report the complete annotated genome sequence of Lactobacillus johnsonii FI9785, a strain which prevents the colonization of specific-pathogen-free chicks by Clostridium perfringens.

Enhanced secretion of biologically active murine interleukin-12 by Lactococcus lactis.

The novel signal peptide SLPmod was used for the secretion of murine interleukin-12 (mIL-12) by Lactococcus lactis. A >4-fold increase in secretion was observed when SLPmod was used instead of the Usp45-derived secretion signal. Oral delivery of this cytokine using the autoinducible host L. lactis FI5876 utilizing SLPmod resulted in a significant increase in mIL-12 plasma levels in mice.

Nisin-controlled extracellular production of interleukin-2 in Lactococcus lactis strains, without the requirement for a signal peptide sequence.

Secretion of the cytokine interleukin-2 (IL-2) was investigated in Lactococcus lactis using the secretory machinery of the bacteriocin lactococcin A. Surprisingly, the lcnCD transport genes were not essential for mouse IL-2 secretion. Furthermore, expression of a mature mouse IL-2 gene resulted in interleukin secretion without the requirement for a leader sequence.

Characterisation of a novel plasmid p9785S from Lactobacillus johnsonii FI9785.

Lactobacillus johnsonii FI9785, a strain originally isolated from poultry gastrointestinal tract for its probiotic function as a competitive excluder of pathogens, was found to contain two cryptic plasmids of 3.5 and 25.6 kb. Nucleotide sequence analysis of the entire small plasmid, designated p9785S (3471 bp), indicated a G+C content of 35.8%, and revealed two open reading frames (orfs). The product of orf1 exhibited similarity to the relaxases of mobilizable plasmids, whereas the product of orf2 displayed significant homology to replication proteins of plasmids which use the rolling circle mode of replication. A conserved double-strand origin of replication was also present in p9785S. A definite minus origin was not identified although a region with extensive intrastrand base pairing potential was revealed. A 1.4 kb fragment encoding the chloramphenicol resistance gene was cloned into p9785S and the resulting vector, pFI2431, was stably maintained when introduced into the parent Lactobacillus cells.

Nisin-controlled production of pediocin PA-1 and colicin V in nisin- and non-nisin-producing Lactococcus lactis strains.

The introduction of chimeric genes encoding the fusion leader of lactococcin A-propediocin PA-1 or procolicin V under the control of the inducible nisA promoter and the lactococcin A-dedicated secretion genes (lcnCD) into Lactococcus lactis strains, including a nisin producer, expressing the two component regulator NisRK led to the production or pediocin PA-1 or colicin V, respectively.

High-level coproduction of the bacteriocins nisin A and lactococcin A by Lactococcus lactis.

In this study, a two-plasmid system for enhanced and consistent biosynthesis of the model lactococcal bacteriocin lactococcin A in non-producing Lactococcus lactis hosts was developed. The system comprised a plasmid carrying the genes lcnA and lciA under the control of the nisin-inducible nisA promoter, and a second plasmid harbouring the lcnC and lcnD genes. The introduction of both plasmids into two strains containing the nisRK genes required for nisin-controlled expression, Lc. lactis FI5876 (a nisin A-producer strain) and FI7847, resulted in production of extracellular lactococcin A at a higher level than that for the parental strain, Lc. lactis WM4. In addition, transformation of the nisin-producing host with both plasmids led to a high-level production of both lactococcal bacteriocins, which may provide a means to exploit their complementary properties in cheese ripening.

A gene replacement strategy for engineering nisin.

A lactococcal expression system was developed which allows the exclusive production of novel nisins encoded by mutated pre-nisin (nisA) genes. This system is based on a combination of a specifically constructed host strain and vectors which facilitate the genetic manipulation of the nisA gene. The wild-type chromosomal gene is effectively replaced with a variant nisA gene, by the technique of gene replacement. The recovery of full nisin immunity was employed as a means of directly selecting strains that had acquired an intact nisA gene by the gene replacement process. With this approach the other genes required for pre-nisin maturation are not affected and any alterations to DNA sequences are restricted to only those specific mutations introduced in the nisA gene. The effectiveness of the system was demonstrated by the expression of a number of variant nisA genes leading to the successful production and characterization of nisins containing the substitutions Dha5A, Dha33A, Dha5, 33A, H27K, 130W and K12L. The enhanced yields of these engineered nisin molecules, when compared to their production in a plasmid-complementation system, underlines the improvement offered by this gene replacement strategy.