Biodiversity-based identification and functional characterization of the mannose-specific adhesin of Lactobacillus plantarum

Lactobacillus plantarum is a frequently encountered inhabitant of the human intestinal tract, and some strains are marketed as probiotics. Their ability to adhere to mannose residues is a potentially interesting characteristic with regard to proposed probiotic features such as colonization of the intestinal surface and competitive exclusion of pathogens. In this study, the variable capacity of 14 L. plantarum strains to agglutinate Saccharomyces cerevisiae in a mannose-specific manner was determined and subsequently correlated with an L. plantarum WCFS1-based genome-wide genotype database. This led to the identification of four candidate mannose adhesin-encoding genes. Two genes primarily predicted to code for sortase-dependent cell surface proteins displayed a complete gene-trait match. Their involvement in mannose adhesion was corroborated by the finding that a sortase (srtA) mutant of L. plantarum WCFS1 lost the capacity to agglutinate S. cerevisiae. The postulated role of these two candidate genes was investigated by gene-specific deletion and overexpression in L. plantarum WCFS1. Subsequent evaluation of the mannose adhesion capacity of the resulting mutant strains showed that inactivation of one candidate gene (lp_0373) did not affect mannose adhesion properties. In contrast, deletion of the other gene (lp_1229) resulted in a complete loss of yeast agglutination ability, while its overexpression quantitatively enhanced this phenotype. Therefore, this gene was designated to encode the mannose-specific adhesin (Msa; gene name, msa) of L. plantarum. Domain homology analysis of the predicted 1,000-residue Msa protein identified known carbohydrate-binding domains, further supporting its role as a mannose adhesin that is likely to be involved in the interaction of L. plantarum with its host in the intestinal tract.     

A mannose-specific adherence mechanism in Lactobacillus plantarum conferring binding to the human colonic cell line HT-29.

Two Lactobacillus plantarum strains of human intestinal origin, strains 299 (= DSM 6595) and 299v (= DSM 9843), have proved to be efficient colonizers of the human intestine under experimental conditions. These strains and 17 other L. plantarum strains were tested for the ability to adhere to cells of the human colonic cell line HT-29.L.plantarum 299 and 299v and nine other L. plantarum strains, including all six strains that belong to the same genetic subgroup as L. plantarum 299 and 299v, adhered to HT-29 cells in a manner that could be inhibited by methyl-alpha-D-mannoside. The ability to adhere to HT-29 cells correlated with an ability to agglutinate cells of Saccharomyces cerevisiae and erythrocytes in a mannose-sensitive manner and with adherence to D-mannose-coated agarose beads. L. plantarum 299 and 299v adhered to freshly isolated human colonic and ileal enterocytes, but the binding was not significantly inhibited by methyl-alpha-D-mannoside. Periodate treatment of HT-29 cells abolished mannose-sensitive adherence, confirming that the cell-bound receptor was of carbohydrate nature. Proteinase K treatment of the bacteria also abolished adherence, indicating that the binding involved protein structures on the bacterial cell surface. Thus, a mannose-specific adhesin has been identified in L. plantarum; this adhesin could be involved in the ability to colonize the intestine.     

TNF-alpha sensitizes HT-29 colonic epithelial cells to intestinal lactobacilli

The ability of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) to influence epithelial interleukin (IL)-8 responses to the intestinal bacterium Lactobacillus plantarum 299v was analyzed in the human HT-29 colonic epithelial cell line. In the absence of TNF-alpha, IL-8 mRNA expression was not detectable by Northern blot analysis in HT-29 cells alone or in HT-29 cells co-cultured with L. plantarum 299v. However, TNF-alpha induced IL-8 mRNA expression, and co-culture of TNF-alpha-treated HT-29 cells with L. plantarum 299v significantly increased IL-8 mRNA expression above levels induced by TNF-alpha alone in an adhesion-dependent manner. The increase in IL-8 mRNA expression was not observed in TNF-alpha-treated HT-29/L. plantarum 299v co-cultures using heat-killed lactobacilli or when L. plantarum adhesion was prevented using mannoside or a trans-well membrane. Paradoxically, IL-8 secretion was decreased in TNF-alpha-treated HT-29 cells with L. plantarum 299v relative to cells treated with TNF-alpha alone. TNF-alpha-mediated responsiveness to L. plantarum 299v was further investigated by analyzing expression of a coreceptor for bacterial cell wall products CD14. HT-29 cells expressed CD14 mRNA and cell-surface CD14; however, TNF-alpha did not alter CD14 mRNA or cell-surface expression, and blockade of CD14 with monoclonal antibody MY4 did not alter the IL-8 response to L. plantarum 299v in TNF-alpha-treated HT-29 cells. These results indicate that although TNF-alpha sensitizes HT-29 epithelial cells to intestinal lactobacilli, the bacteria exert a protective effect by downregulating IL-8 secretion.     

Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat-labile (LT) enterotoxins are cause of post-weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac(+), LT(+) STa(+) STb(+) ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.     

Use of the alr gene as a food-grade selection marker in lactic acid bacteria

Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.     

Lactobacillus Plantarum 299v Changes miRNA Expression in the Intestines of Piglets and Leads to Downregulation of LITAF by Regulating ssc-miR-450a

Lactiplantibacillus plantarum subsp. plantarum 299v (L. plantarum 299v) is one of the most important probiotic strains in animal health, but the molecular mechanisms of how it exerts health benefits remain unclear. The purpose of this study was to explore the changes in miRNA expression profiles in the intestinal tissues of piglets by L. plantarum 299v and to explore its possible molecular regulatory mechanism in intestinal function. Neonatal piglets were orally administered L. plantarum 299v daily from 1 to 20 days old, and high-throughput sequencing was conducted to analyse the changes in miRNA expression in the jejunum and ileum. The results showed that 370 known porcine miRNAs were identified from eight libraries. Five miRNAs (ssc-miR-21-5p, -143-3p, -194b-5p, -192, and -126-3p) were highly expressed in the intestinal tissues. There were 15 differentially expressed miRNAs between the control group and the L. plantarum group, and only miR-450a was expressed differentially in both intestinal tissues. KEGG analysis revealed that the target genes of the 15 differentially expressed miRNAs were involved in 37 significantly enriched pathways (P < 0.01). Then, quantitative polymerase chain reaction confirmed that the miRNA expression was corresponded well with those from the sequencing. Luciferase reporter assays verified that lipopolysaccharide-induced TNF-α factor is a target of miR-450a. Our results also showed L. plantarum 299v could influence intestinal function by changing the levels of cytokines via miRNA expression. This is the first study to analyse differential expression miRNA profiles in intestinal tissue after L. plantarum 299v treatment and investigate the molecular regulatory mechanism of functional miRNA.

     

Amino acid transport and metabolism in mycobacteria: cloning, interruption, and characterization of an L-Arginine/gamma-aminobutyric acid permease in Mycobacterium bovis BCG

Genes encoding L-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter from Mycobacterium bovis BCG and the effects of its deletion on L-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to the rocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using (14)C-labeled substrates. Greatly reduced uptake of L-arginine and gamma-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow), L-[(14)C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenous L-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium. L-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.     

The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii

To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoS mutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in the rpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS in L. pneumophila is very different from what has previously been reported for E. coli rpoS.     

健康仔猪肠道乳杆菌黑龙江地方株的鉴定与种属分析

为了从健康仔猪肠道中分离筛选用于研制微生态制剂的乳酸杆菌菌株,选择黑龙江省大庆、哈尔滨及宝泉岭地区部分猪场,采集12-60日龄健康仔猪肠道粪样64份。选用MRS乳酸杆菌专用培养基分离培养乳酸杆菌,通过分离株的形态特征和培养特性筛选出革兰阳性,厌氧,无芽胞杆菌48株。再通过生化试验鉴定和PCR种属分析,确定18株为乳酸杆菌。其中,罗伊乳杆菌7株,嗜酸乳杆菌5株,约氏乳杆菌2株,短乳杆菌1株,干酪乳杆菌假植物亚种1株,植物乳杆菌1株,詹氏乳杆菌1株。     

Development of an amylolytic Lactobacillus plantarum silage strain expressing the Lactobacillus amylovorus alpha-amylase gene.

An amylolytic Lactobacillus plantarum silage strain with the starch-degrading ability displayed by Lactobacillus amylovorus was developed. An active fragment of the gene coding for alpha-amylase production in L. amylovorus was cloned and integrated into the chromosome of the competitive inoculant strain L. plantarum Lp80 at the cbh locus. The alpha-amylase gene fragment was also introduced into L. plantarum Lp80 on an autoreplicative plasmid. Both constructions were also performed in the laboratory strain L. plantarum NCIB8826. All four recombinant strains secreted levels of amylase ranging from 23 to 69 U/liter, compared with 47 U/liter for L. amylovorus. Secretion levels were higher in L. plantarum NCIB8826 than in L. plantarum Lp80 derivatives and were higher in recombinant strains containing autoreplicative plasmids than in the corresponding integrants. The L. plantarum Lp80 derivative containing the L. amylovorus alpha-amylase gene fragment integrated into the host chromosome secreted alpha-amylase to a level comparable to that of L. amylovorus and was stable over 50 generations of growth under nonselective conditions. It grew to a higher cell density than either the parent strain or L. amylovorus in MRS medium containing a mixture of starch and glucose as the fermentable carbohydrate source. This recombinant alpha-amylolytic L. plantarum strain would therefore seem to have considerable potential as a silage inoculant for crops such as alfalfa, in which water-soluble carbohydrate levels are frequently low but starch is present as an alternative carbohydrate source.     

Genetic modification of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> by heterologous gene integration in a not functional region of the chromosome

This report describes the vector-free engineering of Lactobacillus plantarum by chromosomal integration of an exogenous gene without inactivation of physiological traits. The integrative plasmid vector pP7B6 was derived from pGIP73 by replacing the cbh site, encoding the L. plantarum conjugated bile salt hydrolase, with the prophage fragment P7B6, from L. plantarum Lp80 (DSM 4229). Plasmid pP7B6NI was obtained by inserting the nisin immunity gene nisI of Lactococcus lactis subsp. lactis DSM 20729, preceded by the constitutive promoter P32 from the same strain, in a unique XbaI site of fragment P7B6 and was used to electrotransform L. plantarum Lp80. A food grade recombinant L. plantarum Lp80NI, with 480-fold higher immunity to nisin than the wild type, was derived by integration of pP7B6NI followed by the excision of pP7B6. Polymerase chain reaction tests demonstrated that the integration of nisI in the prophage region had occurred and that the erythromycin resistance marker from pP7B6 was lost. Fifteen among 31 L. plantarum strains tested hybridized with P7B6, indicating that the integration of pP7B6-derived vectors might occur in some other L. plantarum strains. This was experimentally confirmed by constructing the recombinant strain L. plantarum LZNI from the dairy isolate L. plantarum LZ (LMG 24600).     

A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i.

A new suicide vector, pRVS1, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denitrificans. The vector was derived from suicide vector pGRPd1, which was equipped with the lacZ gene encoding beta-galactosidase. The reporter gene was found to be a successful screening marker for the discrimination between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPd1 and pRVS1 were used in gene replacement techniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the periplasmic cytochromes c550, c551i, and c553i, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the correctness of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type levels of methylamine dehydrogenase activities. cycA mutant strains, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow on methanol and showed wild-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum specific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes.     

OmpD but not OmpC is involved in adherence of Salmonella enterica serovar typhimurium to human cells

Conflicting reports exist regarding the role of porins OmpC and OmpD in infections due to Salmonella enterica serovar Typhimurium. This study investigated the role of these porins in bacterial adherence to human macrophages and intestinal epithelial cells. ompC and ompD mutant strains were created by transposon mutagenesis using P22-mediated transduction of Tn10 and Tn5 insertions, respectively, into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with host cells at various bacteria to cell ratios for 1 h at 37 degrees C and analyzed by flow cytometry. The mean fluorescence intensity of cells with associated wild-type and mutant bacteria was used to estimate the number of bacteria bound per host cell. Adherence was also measured by fluorescence microscopy. Neither assay showed a significant difference in binding of the ompC mutant and wild-type strains to the human cells. In contrast, the ompD mutant exhibited lowered binding to both cell types. Our findings suggest that OmpD but not OmpC is involved in the recognition of Salmonella serovar Typhimurium by human macrophages and intestinal epithelial cells.     

The Lactobacillus plantarum strain ACA-DC287 isolated from a Greek cheese demonstrates antagonistic activity in vitro and in vivo against Salmonella enterica serovar Typhimurium

AIMS: The purpose of this study was to investigate the antibacterial activity of the Xynotyri cheese isolate Lactobacillus plantarum ACA-DC287 using a set of in vitro and in vivo assays. METHODS AND RESULTS: The co-culture of L. plantarum strain ACA-DC287 and Salmonella enterica serovar Typhimurium strain SL1344 results in the killing of the pathogen. The killing activity was produced mainly by non-lactic acid molecule(s) that were present in the cell-free culture supernatant of the L. plantarum strain ACA-DC287. The culture of the L. plantarum strain ACA-DC287 inhibited the penetration of S. typhimurium SL1344 into cultured human enterocyte-like Caco-2/TC7 cells. In conventional mice infected with S. typhimurium SL1344, the intake of L. plantarum strain ACA-DC287 results in a decrease in the levels of Salmonella associated with intestinal tissues or those present in the intestinal contents. In germ-free mice, the L. plantarum strain ACA-DC287 colonized the gastrointestinal tract. CONCLUSIONS: The L. plantarum strain ACA-DC287 strain exerts anti-Salmonella activity similar that of the established probiotic strains Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029 and Lactobacillus johnsonii La1. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation that a selected cheese Lactobacillus strain exerted antibacterial activity that was similar to those of probiotic Lactobacillus strains, is of interest for the use of this strain as an adjunct strain for the production of health-giving cheeses.     

Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.     

Exploring Lactobacillus plantarum genome diversity by using microarrays

Lactobacillus plantarum is a versatile and flexible species that is encountered in a variety of niches and can utilize a broad range of fermentable carbon sources. To assess if this versatility is linked to a variable gene pool, microarrays containing a subset of small genomic fragments of L. plantarum strain WCFS1 were used to perform stringent genotyping of 20 strains of L. plantarum from various sources. The gene categories with the most genes conserved in all strains were those involved in biosynthesis or degradation of structural compounds like proteins, lipids, and DNA. Conversely, genes involved in sugar transport and catabolism were highly variable between strains. Moreover, besides the obvious regions of variance, like prophages, other regions varied between the strains, including regions encoding plantaricin biosynthesis, nonribosomal peptide biosynthesis, and exopolysaccharide biosynthesis. In many cases, these variable regions colocalized with regions of unusual base composition. Two large regions of flexibility were identified between 2.70 and 2.85 and 3.10 and 3.29 Mb of the WCFS1 chromosome, the latter being close to the origin of replication. The majority of genes encoded in these variable regions are involved in sugar metabolism. This functional overrepresentation and the unusual base composition of these regions led to the hypothesis that they represented lifestyle adaptation regions in L. plantarum. The present study consolidates this hypothesis by showing that there is a high degree of gene content variation among L. plantarum strains in genes located in these regions of the WCFS1 genome. Interestingly, based on our genotyping data L. plantarum strains clustered into two clearly distinguishable groups, which coincided with an earlier proposed subdivision of this species based on conventional methods.