S-Layer Proteins In Lactobacillus Crispatus ZJ001: Gene Structure, Adhesion And Anchor Functions Of Their Expression Products

Lactobacillus spp is one of the normal microflora in human and animal intestinal and vaginal tracts, and can exert probiotic effects through competitive colonization, improvement of intestine microecological environments, and enhancement of immune functions. The proteinaceous surface layers (S-layers) consist of subunits of single protein or glycoprotein covering the entire cell as the outermost envelope with molecular masses ranging from 40 to 200 kDa. The S-layer protein represents 10-15% of the total protein of the bacterial cells and is weakly acidic with pI value ranging from 4 to 6. The S-layer proteins characterized from the genus Lactobacillus range from 25 to 71 kDa in size, and are the smallest among the known for the S-layer proteins. The S-layer proteins of lactobacilli are highly basic proteins with calculated pI values above 9. Only a few lactobacilli are known for the primary structures of their S-layer proteins, and the functions are poorly characterized. Currently, little information in China is available on the expression, structure, functions and application of Lactobacillus S-layer proteins. The present research was aimed to examine the in vitro probiotic activities of Lactobacillus crispatus ZJ001 isolated from pig intestines and characterize the gene structure encoding the S-layer protein, as well as its functional domains involved in anchoring to the bacterial cell wall and adhering to the host cells.L. crispatus ZJ001 was resistant to acidic condition and bile salt. Its antagonistic properties such as adhesion, inhibition of the pathogen growth and competitive exclusion against Eshcerichia coli O157:H7 and Salmonella typhimurium was apparently advantageous over L. acidophilus ATCC 4356. The more inhibitory effect of L. crispatus strain ZJ001 against S. typhimurium and E. coli O157:H7 might be due to more acid production by strain ZJ001 than that of ATCC 4356. The differences of competitive exclusion between the two strains used in this experiment were related to variations of their adhering ability, strian ZJ001 was more adherent to HeLa cells than strain ATCC 4356 with statistical difference at P＜0.01. These results suggest that L. crispatus ZJ001 possesses probiotic properties and may be a potential candidate of strains for probiotic application.SDS-PAGE analysis revealed the presence of S-layer proteins in L. crispatus ZJ001 at about 42 kDa upon LiCl treatment. The S-layer proteins were stably expressed on the surface of strain ZJ001. The S-layer proteins were easily removed by 5 mol/L LiCl and sensitive to enzymatic treatment with proteinase K. The S-layer proteins was formed again after removal by LiCl, when the treated cells were placed in a fresh growth medium for 8 h. Removal of the S-layer proteins reduced autoaggregation, adhesion to HeLa cells and inhibition of pathogens adhesion. The functional role of the S-layer proteins in adhesion was also confirmed by the antibody mediated inhibition assay using the polyclonal antibody against the S-layer protein. L. crispatus ZJ001 was able to compete against adhesion of S. typhimurium and E. coli O157:H7 to HeLa cells, but could not displace the pathogens already adhered to the cells. Removal of S-layer proteins showed reduced competitive inhibition against E. coli O157:H7. The S-layer proteins from L. crispatus ZJ001 was more inhibitory to E. coli O157:H7 adhesion than S. typhimurium. Adhesion of E. coli O157:H7 to HeLa cells was reduced by 87.0±5.30% in the presence of 100μg S-layer proteins, higher than that of S. typhimurium. These results suggest that S-layer proteins are involved in L. crispatus ZJ001 adhesion and competitive exclusion of pathogens to HeLa cells.L. crispatus ZJ001 harbours two S-layer genes slp A and slpB, and their encoded proteins have typical fetures of lactobacillial S-layer proteins. The slpA gene (1329bp) encodes a polypeptide of 443 amino acids including a signal sequence of 30 amino acids. The calculated molecular weight is 44.3 kDa and the isoelectric point 9.57 as mature protein. The ORF of slpB (1386bp) encodes a predicted protein of 461 amino acids. The first 30 N-terminal amino acids are signal sequence identical to that of slpA, and the predicted mature slpB has a theoretical molecular mass of 45.1 kDa with an isoelectric point of 9.57. The amino acid compositions of mature slpA and slpB proteins are of high similarity. They have typical features of an S-layer protein such as high content of hydrophobic amino acid residues as well as of charged residues and polar amino acids, and no cysteine residues. According to the secondary structure predictions, the slpA protein comprises 37%α-helical regions and 49.7% β-sheet domains, and slpB consists of 27.9%α-helical regions and 58.9%β-sheet domains. Proteins slpA and slpB exhibit 47% similarity in amino acid sequences, and have high homology in the C-terminal region, but substantial variability in N-terminal and middle segments (30%). Proteins slpA and slpB of L. crispatus ZJ001 share considerable sequence similarity to other seven Lactobacillus S-layer proteins at the C-terminal regions in the protein database (77%-95%). The N-terminal regions are more variable. The protein slpA from strain ZJ001 exhibits highest identity to CbsA of L. crispatus JCM5810 (82%). There was strong cross-reactivity between slpA and slpB as revealed by Western blot and indirect ELISA. Unlike the natural S-layer proteins from ZJ001, His-slpA expressed in E. coli self-assembled into sheet-like crystalline layers, whilst His-slpB exhibited disk-like structures. HeLa cells were efficiently adhered to nitrocellulose membranes coated with His-slpA and His-slpB though these two proteins had different structures.Long-range PCR and sequence analysis indicate that slpA and slpB genes are located on a 9kb chromosomal segment in opposite orientation and interspaced with a 4.5kb region. There is a putative promoter upstream of slpA but was absent from slpB. Only slp A gene was expressed in strain ZJ001 as revealed by RT-PCR and immunoassays. The 5'-identity regions of slpA and slpB suggested that slpB might be translocated to the downstream of slpA promoter via an inversion of a chromosomal segment by site-specific recombination system, which had been identified in L. acidophilus ATCC 4356. Under aerobic and anaerobic growth conditions, L. crispatus ZJ001 was found to form three colony types, rough, smooth and flat, respectively all expressing the same S-layer protein. The interchange of slpA and slpB did not happen, and only slpA gene was transcribed and expressed. These results indicated that oxygen content did not show any effect on the expression of S-layer genes in L. crispatus ZJ001, nor on the interchange of of slpA and slpB.The presence of two domains in slpA proteins of L. crispatus ZJ001 was inferred from an alignment of S-layer protein sequences from related lactobacilli. SlpA encompasses an N-terminal region of about 290aa (SAN) that is highly variable among the S-layer proteins and consists of alternating regions of hydrophilic and hydrophobic amino acids. The C-terminal region of about 122aa (SAC) is highly conserved and is almost exclusively comprised of hydrophilic, mainly basic, amino acid residues. To obtain more information about the function of SAN and SAC, His-SAN-eGFP and His-eGFP-SAC were expressed in E. coli and purified. Using the antibody against S-layer proteins from L. crispatus ZJ001 as the probe, strong signals could be seen with peptide His-SAN-eGFP, while faint signals was observed with His-eGFP-SAC. His-SAN-eGFP efficiently adhered to HeLa cells, and His-eGFP-SAC could bind to L. crispatus ZJ001 and L. acidophilus ATCC 4356 cells from which the S-layer had been stripped by LiCl, but not to non-stripped ZJ001 and ATCC 4356 cells. His-eGFP-SAC could bind to Lactobacillus casei M4 with low efficiency, but not to Lactobacillus johnsonii R20 cells. These results suggested that SAN might mediate the adhesion of slpA to host cells, and SAC was responsible for the anchoring of slpA subunits to the bacterial cell wall.In summary, the present studies have laid good foundation for further exploration of L. crispatus ZJ001 as potential vaccine vectors for other passenger antigens.