Studies On The Probiotic Properties And Adhesion Mechanisms Of Lactobacillus Crispatus

Lactobacilli are dominant inhabitants of humans and animals. They can maintain a balanced intestinal flora, improve immunity and promote the absorption of the nutrients. Also Lactobacillus can produce lactic acid, acetic acid, hydrogen peroxide, bacteriocins and have an inhibitory effect on enteric pathogens. Therefore, Lactobacillus has an important role of prebiotics on human and animals. Probiotic screening criteria include the adhesion and colonization ability, antagonistic to pathogens and no transferable antibiotc resistance gene. Adhesion and colonization determined by the composition and hydrophobic characteristics of surface layer protens is an important screening index. Besides, probiotics have an inhibitory effect on pathogens. The mechanisms of action include the inhibition of pathogen growth by competition for nutritional sources and adhesion sites, secretion of antimicrobial substances and toxin inactivation. Consequently, Lactobacillus was used in clinical to prevent and treat gastrointestinal infections and antibiotic-associated diarrhea diseases. But most of the researches just study the effects of prebiotics and lack of the mechanism of action of Lactobacillus. For the development of probiotics with a clear feature and high security, we studied mainly on the screening of the high adhesive Lactobacillus, the inhibition of pathogens adhesion to epithelia cells, the structure and function of the adhesion proteins, antibiotic resistance evaluation of intestinal Lactobacillus and construction of expression vectors for Lactobacillus, the results were described as follows:1. The isolation and in vitro evaluation of high adhesive Lactobacillus strainsCurrently, there is an increasing interest in the use of probiotics as an alternative strategy to antimicrobial compounds. But there are still many problems such as single strains and poor specificity. Thus, the breedings of the excellent probiotic strains are the key to the development of probiotics. In this study, six Lactobacillus strains isolated from chicken intestinal tract had the adhsion ability to HT-29cells. They were Lactobacillus crispatus K313, Lactobacillus crispatus K243, Lactobacillus helveticus2020T, Lactobacillus johnsonnii H31, Lactobacillus salivarius Z4and Lactobacillus salivarius K233. Lb. crispatus K313exhibited the highest adhesion capacity to HT-29cells (73.2bacteria/cell). The six Lactobacillus strains adherences to mucin and individual proteins of the mammalian extracellular matrix were tested. the high attachment to collagen type IV (1239bacteria/field) were observed with the strain Lb. crispatus K243. Lb. crispatus K243and K313showed srong tolerance to acid and bile salts. The two strains also had a cholesterol degradation ability and the rate were60.8%and51.4%. Both of strains showed the very high percentages adhered to hexadecane demonstrated hydrophobic properties in the cell surface of Lb. crispatus. A strong affinity to chloroform and a low adherence to ethyl acetate indicated the basic characteristics in the two strain surfaces. SDS-PAGE analysis revealed the presence of the potential S-proteins Slp A and SlpB in Lb. crispatus K243and K313. SlpB of the strain K313was sensitive to pepsin, but not to trypsin, while SlpA was sensitive to neither pepsin nor trypsin. The treatment with LiCl greatly reduced the adhesiveness of the two Lb. crispatus strains compared to the untreated bacterial cells, subsequently, purified S-proteins also exhibited the inhibition of Lb. crispatus adhesion to collagens and HT-29cells, which suggested the S-layer proteins might be involved in the adhesion process. After LiCl treatment, Lb. crispatus K313and K243become oval or irregular shape. Consequently, the S-protein play an important role in maintaining the normal morphology of the cells. After removal of S-proteins, the viability and tolerance of the two Lb. crispatus strains to simulated gastric and small intestinal juice were reduced, indicating the protective role of S-proteins against the hostile environments. All the results showed that the two strains have the excellent characteristics and can be used as probiotics to lauch a follow-up work.2. Lb. crispatus regulate the inflammation caused by pathogen infection in HT-29cellsProbiotics can prevent and treat diarrhea and other diseases caused by pathogens infections. The mechanism of action include reducing pathogen colonization, regulating the balance of intestinal flora and promoting the host immune response. Probiotics can inhibite the colonization of pathogen via coaggregation and competitive exclusion. In this work, the coaggregation between Lb. crispatus K313and Salmonella. braenderup H9812or Escherichia coli ATCC was57.7%and84.8%, however; in Lb. crispatus K243, the coaggregation was28.7%and26.2%, lower than K313. Lb. crispatus K313and K243exhibited the strong inhibitive activity against S. braenderup H9812and E. coli ATCC25922adhesion to HT-29cells. Exposure of polarized HT-29cells to S. braenderup increased the transcription yields of pro-inflammatory factors (IL-8, CXCL1and CCL20). The transcription of IL-8were down-regulated by42%and37%in the case of coinfection with both Lb. crispatus strains and S. braenderup H9812. CXCL1and CCL20transcription levels were down-regulated by63%and41%when HT-29cells were preincubated with the Lb. crispatus K313before infection with S. braenderup H9812. The production of IL-8was detected by IL-8ELISA. Lb. crispatus K243and K313inhibited the IL-8secretion triggered by S. braenderup H9812by32.8%and47.0%, indicating that the two isolates could attenuate the pro-inflammatory signaling induced by S. braenderup H9812, and have the potential for protecting the host against S. braenderup infection.3. The functional domains of an S-protein from Lactobacillus crispatus K313It was previously shown that the S-proteins covering the cell surface of Lactobacillus crispatus K313were involved in the adherence of this strain to human intestinal cell line HT-29. To further elucidate the structures and functions of S-layers, three putative S-protein genes(slpA, slpB, and slpC) of Lb. crispatus K313were amplified with two strategies. First, the conserved primers were synthesized based on the conserved part of the S-protein gene of Lb. crispatus, and PCR amplification was performed with the genomic DNA of Lb. crispatus K313as the template. The upstream and downstream regions of the conserved part of the S-protein gene were amplified using the ligation-anchored PCR. The DNA products were then purified, sequenced, and spliced, respectively. Subsequently, the obtained gene was named slp A. Second, the oligonucleotide complementary to the N-terminal amino acid sequence of S-protein isolated was synthesized. For cloning the upstream and downstream region, the Tail-PCR and ligation-anchored PCR was performed for chromosome walking. After splicing, two adjacent S-protein genes were obtained, and named slpB and slpC. Quantitative real time PCR analysis reveals that slpA was silent under the tested conditions, whereas the transcription of slpB was170-fold higher than that of slpC. Therefore, slpB was predominantly expressed in Lb. crispatus K313. Genetic truncation of the untranslated leader sequence (UTLS) of slpB results in a reduction in protein production, indicating that the UTLS contributed to the efficient S-protein expression. We obtained a set of N-and C-terminally truncated recombinant SlpB proteins by constructing a series of recombinant vectors in Escherichia coli. Collagen type I, collagen type IV and human plasma fibronectin were chosen as the extracellular matrix proteins to investigate the binding ability of SlpB using ELISA techniques. rSlpB1-501exhibited significant levels of attachment to collagen types IV and I, with levels4.8-fold and2.8-fold higher than the background level seen with the immobilized control protein BSA, respectively. No binding was observed to the plasma fibronectin. The N-terminal truncated peptides rSlpB1-190, rSlpB1-322, and rSlpB1-341and the C-terminal truncated peptides rSlpB323-501, rSlpB342-501, and rSlpB3go-501only exhibited a background level of binding to collagen types I and IV. However, binding of rSlpB1-359and rSlpB1-379to both collagen types was observed, suggesting that SlpB domain involved in the adherence to collagens is located at the N-terminus. The derivative rSlpB1-359was reduced in collagen binding, revealing the importance of the20aa-peptide of SlpB between359and379. The mutated rSlpB1-379peptides VTVNV364TTTNT had a reduced binding ability to collagens, indicating that the five amino acids "VTVNV" are critical for the adhsion of SlpB to collagens. The binding of truncated SlpB-GFP fusing proteins to CWFs isolated were tested. The results showed that the cell wall binding region was mapped to the C-terminus. The treated CWFs with10%TCA reduced the binding ability of SlpB by60%, suggesting that teichoic acid may be acting as the receptor of SlpB. Moreover, the binding ability of the C-terminus was variable among the Lactobacillus species (S-layer and non-S-layer producing strains). SlpB had the high binding capacity to the S-layer containing Lactobacillus. Moreover, LiCl pretreatment increased the binding level of rSlpB323-501-GFP. However, a low binding level of the rSlpB323-501-GFP peptide was detected with the native or LiCl treated cell, which do not express an S-layer. Therefore, the different binding capacities of SlpB depended on whether these strains possessed S-layers.4. The antibiotic resistance evaluation of Lb. crispatusIn addition to nutritional function and prebiotic effect, probiotics can not have any negative effects on human or animal. It needs to evaluate the safety of probiotics. One of the main principle is that the evaluation of the antibiotic resistance pattern. Antibiotic resistance genotype analysis showed that Lb. crispatus K243and K313harbored tetracycline resistance gene tetL and tetM. Lb. crispatus K313also harbored chloramphenicol resistance gene cat and erythromycin resistance gene ermB. tetL and tetM were cloned from Lb. crispatus K313. Phylogenetic tree of homologs analysis indicated that TetL are different from all previously described TetL and form an independent branch associated with a100%bootstrap value. TetM was shown to be identical to previously described TetM with a97%bootstrap value. Lb. crispatus K243and K313were used as the donor strains, and Enterococcus faecalis K9as the receptor strain. All the resistence genes could not transfer into Enterococcus faecalis K9, indicating that the resistence genes were stable in Lb. crispatus K243and K313.5. Construction of expression vectors for LactobacillusIn vitro evaluations of the probiotic role of Lb. crispatus K243and K313were performed in previous research. In order to study their colonizations and dynamic changes of growth in vivo, fluorescently labeled plasmid vector carrying the fluorescent protein gene was proposed to transform into Lactobacillus. The molecular tools for the genetic manipulation of lactobacilli are still not consummate. Based on this, a cryptic plasmid pD403was isolated from Lactobacillus plantarum D403derived from fermented dairy products in this work. It was2,791bp in size with a G+C content of37%. Nucleotide sequence analysis revealed two open reading frames, orfl and orf2. ORF1(318amino acids) was identified as a replication protein (RepA). ORF2(137amino acids) shared31%identity with the transcriptional regulator of Ralstonia pickettii12D. Functional investigation indicated that ORF2(Tra) is unessential for replication in different hosts, but it had the ability of improving the transformation efficiency. The origin of replication was predicted, suggesting that pD403was a rolling-circle-replication (RCR) plasmid, and The RepA had the high identity with the replication initiation protein belonged to group III family. To develop molecular tools on the basis of pD403, the replicon of pUC19, chloramphenicol resistant gene and the repA from pD403were ligated subsequently, yielding an E. coli/Lactobacillus shuttle vector pCD4032. The host range of the vector pCD4032was determined, and Lb. casei BL23, Lb. plantarum LPA, Lb. casei401, Lb. fermentum YB5and Lb. brevis CGMCC1.208could be transformed successfully. The transformation efficiencies were ranged from1.3×102to7×104transformants per microgram DNA. According to the genome sequence of the Lb. delbrueckii subsp. bulgaricus ATCC11842, the promoter and terminator sequences from lactate dehydrogenase were amplified and cloned into pCD4032, generating the expression vector pCD4033. Gfp gene was inserted into the downstream of the promoter pldh to generate the recombinant plasmid pCD4033-gfp. Then the plasmid pCD4033-gfp was electroporated into various lactobacilli cells. The green fluorescence in most of recombinant cells was observed. however, no fluorescence was observed in control strains. After optimizing the expression conditions, the highest amount of fluorescence was obtained when the transformants were incubated with aeration at30℃. These results proved that GFP was expressed successfully, and the vector pCD4033was feasible for heterologous proteins expression in Lactobacillus strains. Unfortunately, The vector pCD4033can not transform into Lb. crispatus K243and K313.