Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.

Bakteerien pintaproteiinit ovat tärkeitä bakteerien vuorovaikutuksessa isäntänsä, kuten ihmisen kanssa. Tässä työssä tutkittiin Lactobacillus crispatus bakteerin pintaproteiineja ja niiden merkitystä isäntä-bakteeri vuorovaikutuksessa, kuten bakteerin tarttumisessa suoliston limakalvoille. Laktobasillit kuuluvat maitohappobakteereihin, joita käytetään yleisesti lihan ja kasvisten valmistamisessa käymisreaktiolla sekä meijeriteollisuudessa mm. jogurtin valmistuksessa. Laktobasilleilla on havaittu monia terveyttä edistäviä vaikutuksia, ja niitä käytetäänkin yleisesti probioottisissa eli terveyttä edistävissä valmisteissa. Tutkimuksessani osoitettiin Lactobacillus crispatus bakteerien S-kerros -pintaproteiinin merkitys bakteerin tarttumisessa suolistoon sekä tutkittiin tarkoin proteiinin molekylaarinen rakenne. Proteiinin ensimmäiset 274 aminohappoa muodostavat polymeerisen S-kerros rakenteen ja ne tarvitaan bakteerin sitoutumiseen suoliston pintaan. Sen sijaan proteiinin loppupään 123 aminohappoa sitoutuvat bakteerin soluseinän ainesosiin, lipoteikohappoihin, ja siten kiinnittävät proteiinin bakteerin soluseinään. Laktobasillit tuottavat kasvaessaan maitohappoa, joka laskee ympäristön pH-arvoa. Työssäni osoitettiin, että kaksi muuta Lactobacillus crispatus bakteerin pintaproteiinia, enolaasi ja glyseraldehydi-3-fosfaatti-dehydrogenaasi (GAPDH), ovat sitoutuneet bakteerin soluseinään happamissa olosuhteissa, mutta irtoavat solusta korkeassa pH:ssa. Lisäksi työssä verrattiin enolaasi-proteiineja laktobasilleilla ja tautia aiheuttavilla bakteereilla, ja havaittiin, että enolaasi-proteiineilla on laktobasilleilla samanlaisia ominaisuuksia kuin tautia aiheuttavien bakteerien enolaaseilla. Tämä tutkimus valottaa pintaproteiinien roolia laktobasillien terveyttä edistävissä ominaisuuksissa sekä kuvaa mekanismin, joilla laktobasillit muokkaavat pintarakennettaan ympäristön muuttuessa.