Timo K. Korhonen

Bacterial proteins binding to the mammalian extracellular matrix.

Pathogenic bacteria frequently express surface proteins with affinity for components of the mammalian extracellular matrix, i.e. collagens, laminin, fibronectin or proteoglycans. This review summarizes our current knowledge on the mechanisms of bacterial adherence to extracellular matrices and on the biological significance of these interactions. The best‐characterized bacterial proteins active in these interactions are the mycobacterial fibronectin‐binding proteins, the fibronectin‐ and the collagen‐binding proteins of staphylococci and streptococci, specific enterobacterial fimbrial types, as well as the polymeric surface proteins YadA of yersinias and the A‐protein of Aeromonas. Some of these bacterial proteins are highly specific for an extracellular matrix protein, some are multifunctional and express binding activities towards a number of target proteins. The interactions can be based on a protein‐protein or on a protein‐carbohydrate interaction, or on a bridging mechanism mediated by a bivalent soluble target protein. Many of the interactions have also been demonstrated on tissue sections or in vivo, and adherence to the extracellular matrix has been shown to promote bacterial colonization of damaged tissues.

MANNOSE-RESISTANT HAEMAGGLUTINATION AND P ANTIGEN RECOGNITION ARE CHARACTERISTIC OF ESCHERICHIA COLI CAUSING PRIMARY PYELONEPHRITIS

Thirty-two Escherichia coli strains from 30 children with pyelonephritis were examined for their haemagglutination patterns and O and K serotypes. 29 (91%) of the strains showed mannose-resistant haemagglutination (MRHA). By use of well-defined target cells, these MRHA+ strains could be shown to recognise human cells either in a P-specific manner (recognising a specific galactosyl-galactose structure which is part of P blood groups antigens) or in a separate, X-specific manner. Both recognition mechanisms could occur separately or together on the same bacteria, the frequencies of P and X specificity being 81 and 19%, respectively. Both MRHA and P specificity were significantly associated with the O antigens 01, 04, 06, 016, and 018, and the capsular antigen K1, which have previously been associated with pyelonephritis. However, the association of MRHA and P specificity with upper urinary tract infection in children is greater than that of any other laboratory-defined bacterial characteristic.

A Controlled Trial on the Effects of Patient Education in the Treatment of Insulin-dependent Diabetes

The effect of patient education on diabetic control in insulin-treated diabetic adults was studied in 77 subjects randomized into two groups: intensive patient education (group A) and control (group B). The subjects in group A received intensive patient instruction, both individually and in small groups, from a team of physicians, teaching nurses, and a dietitian. The patients in group B received a short instruction course consisting mainly of printed material. A highly significant improvement in diabetic control was observed in both groups immediately after the education programs, with gradual return to the original level during the following 3–6 mo. No difference was observed between the two groups in any of the measured parameters during the 18-mo investigation. Factors related to good control during the study included the length of school education, the quality of the control at the beginning of the study, and the high degree of self-confidence and lack of signs of anxiety in the psychological tests. The results demonstrate that the effects of educational programs are of limited value if they do not lead to permanent changes in attitudes and motivation, which are critical factors affecting long-term diabetic control.

Hydrophobic domains affect the collagen‐binding specificity and surface polymerization as well as the virulence potential of the YadA protein of Yersinia enterocolitica

The YadA surface protein of enteropathogenic Yersinia species contains two highly hydrophobic regions: one close to the amino terminal, and the other at the carboxy‐terminal end of the YadA polypeptide. To study the role of these hydrophobic regions, we constructed 66 bp deletion mutants of the yadA genes of Yersinia enterocolitica serotype O:3 strain 6471/76 (YeO3) and of O:8 strain 8081 (YeO8). The mutant proteins, YadAYeO3‐Δ83–104 and YadAYeO8‐Δ80–101, lacked 22 amino acids from the amino‐terminal hydrophobic region, formed fibrillae and were expressed on the cell surface. Bacteria expressing the mutated protein lost their auto‐agglutination potential as well as their collagen‐binding property. Binding to fibronectin and laminin was affected differently in the YeO3 and the YeO8 constructs. The deletion did not influence YadA‐mediated complement inhibition. Loss of the collagen‐binding property was associated with loss of virulence in mice. We also constructed a number of YadAYeO3 deletion mutants lacking the hydrophobic carboxy‐terminal end of the protein. Deletions ranging from 19 to 79 amino acids from the carboxy terminus affected polymerization of the YadA subunits, and also resulted in the loss of the YadA expression on the cell surface. This suggests that the carboxy terminus of YadA is involved in transport of the protein to the bacterial outer surface.

Characterization of the Collagen-Binding S-Layer Protein CbsA of Lactobacillus crispatus

The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

Lack of O‐antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica

The O‐antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express β‐barrel surface proteases of the omptin family that activate human plasminogen. Plasminogen activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O‐antigen repeats on wild‐type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents plasminogen activation by PgtE of S. enterica and Pla of Y. pestis; the O‐antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6‐Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla‐mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O‐antigen prevented PgtE‐mediated bacterial adhesion to basement membrane. Substitution of Arg‐138 and Arg‐171 of the motif for protein binding to lipid A 4′‐phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O‐antigen sterically prevents recognition of large‐molecular‐weight substrates. Loss of O‐antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders plasminogen activator cryptic in S. enterica.

Domains in the S‐layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self‐assembly

The protein regions in the S‐layer protein CbsA of Lactobacillus crispatus JCM 5810, needed for binding to collagens and laminin, anchoring to bacterial cell wall, as well as self‐assembly, were mapped by deletion analysis of His‐tagged peptides isolated from Escherichia coli and by heterologous expression on Lactobacillus casei. Mature CbsA is 410 amino acids long, and stepwise genetic truncation at both termini revealed that the region 32–271 carries the infor‐mation for self‐assembly of CbsA into a periodic structure. The lactobacillar S‐layer proteins exhibit sequence variation in their assembly domain, but the border regions 30–34 and 269–274 in CbsA are conserved in valine‐rich short sequences. Short deletions or substitutions at these regions affected the morphology of His‐CbsA polymers, which varied from sheet‐like to cylindrical tubular polymers, and further truncation beyond the DNA encoding residues 32 and 271 leads to a non‐periodic aggregation. The self‐assembly of the truncated peptides, as seen by electron microscopy, was correlated with their behaviour in a cross‐linking study. The shorter peptides not forming a regular polymer were observed by the cross‐linking study and mass spectrometry to form dimers, trimers and tetramers, whereas the other peptides were cross‐linked to large multimers only. Binding of solubilized type I and IV collagens was observed with the His‐CbsA peptides 1–274 and 31–287, but not with the smaller peptides regardless of their ability to form regular polymers. Strain JCM 5810 also adheres to immobilized laminin and, in order to analyse the possible laminin binding by CbsA, cbsA and its fragments were expressed on the surface of L. casei. Expression of the CbsA peptides 1–274, 1–287, 28–287 and 31–287 on L. casei conferred adhesiveness to both laminin and collagen immobilized on glass as well as to laminin‐ and collagen‐containing regions in chicken colon and ileum. The C‐terminal peptides 251–410 and 288–410 bound to L. crispatus JCM 5810 cells from which the S‐layer had been depleted by chemical extraction, whereas no binding was seen with the His‐CbsA peptides 1–250 or 1–269 or to cells with an intact S‐layer. The His‐CbsA peptides 251–410 and 288–410 bound to teichoic acids of several bacterial species. The results show that CbsA is an adhesive complex with an N‐terminal assembly domain exhibiting affinity for pericellular tissue components and a cationic C‐terminal domain binding to negatively charged cell wall components.

Amino acid residue Ala‐62 in the FimH fimbrial adhesin is critical for the adhesiveness of meningitis‐associated Escherichia coli to collagens

Adhesion of meningitis‐associated Escherichia coli O18acK1H7 to collagens was characterized. The E. coli strain IHE 3034 adhered to type IV and type I collagens but not to type III collagen immobilized on glass. Collagens lack terminal mannosyl units, yet the bacterial adhesion was completely abolished in the presence of α‐methyl‐D‐mannoside. A cat cassette was introduced into the fimA gene of IHE 3034, and the resulting mutant strain IHE 3034‐2 failed to adhere to collagens. In contrast, insertion of a Gm cassette into the sfaA gene of IHE 3034, encoding the S‐fimbrillin, had no significant effect on the adhesiveness. The fim cluster from IHE 3034 was cloned and expressed in trans in the fimA::cat mutant strain IHE 3034‐2. The complemented strain IHE 3034‐2(pRPO‐1) exhibited adhesiveness to type IV and type I collagens, confirming the function of the type 1 fimbria in the adhesion. We have previously shown that the type 1 fimbria from E. coli K‐12 strain PC31 does not confer bacterial adhesiveness to collagens. The fimH genes from E. coli IHE 3034 as well as from PC31 were expressed in the fimH‐null strain MS4. The FimH from IHE 3034 potentiated collagen adherence, whereas the FimH from PC31 was inactive. Sequence comparison of fimH from IHE 3034 and PC31 revealed five amino‐acid differences in the predicted mature FimH proteins: at residues 27, 62, 70, 78 and 201. Each of these residues in the IHE 3034‐FimH were individually substituted to the corresponding amino acid in the PC31‐FimH. The substitution S62→A completely abolished collagen adhesiveness. The reverse substitution A62→S in the PC31‐FimH as well as in the FimH from another E. coli strain induced collagen adhesiveness to the level seen with IHE 3034‐FimH. Out of nine fimH genes analysed from isolates of E. coli, collagen adhesiveness as well as alanine at position 62 in FimH were found only in two O18acK1H7 isolates with the isoenzyme profile ET type 1. Our results demonstrate that the amino‐acid residue Ala‐62 in the FimH lectin is critical for the adhesion to collagens by a highly virulent clonal group of E. coli.

Protein regions important for plasminogen activation and inactivation of α2‐antiplasmin in the surface protease Pla of Yersinia pestis

The plasminogen activator, surface protease Pla, of the plague bacterium Yersinia pestis is an important virulence factor that enables the spread of Y. pestis from subcutaneous sites into circulation. Pla‐expressing Y. pestis and recombinant Escherichia coli formed active plasmin in the presence of the major human plasmin inhibitor, α2‐antiplasmin, and the bacteria were found to inactivate α2‐antiplasmin. In contrast, only poor plasminogen activation and no cleavage of α2‐antiplasmin was observed with recombinant bacteria expressing the homologous gene ompT from E. coli. A β‐barrel topology model for Pla and OmpT predicted 10 transmembrane β‐strands and five surface‐exposed loops L1–L5. Hybrid Pla–OmpT proteins were created by substituting each of the loops between Pla and OmpT. Analysis of the hybrid molecules suggested a critical role of L3 and L4 in the substrate specificity of Pla towards plasminogen and α2‐antiplasmin. Substitution analysis at 25 surface‐located residues showed the importance of the conserved residues H101, H208, D84, D86, D206 and S99 for the proteolytic activity of Pla‐expressing recombinant E. coli. The mature α‐Pla of 292 amino acids was processed into β‐Pla by an autoprocessing cleavage at residue K262, and residues important for the self‐recognition of Pla were identified. Prevention of autoprocessing of Pla, however, had no detectable effect on plasminogen activation or cleavage of α2‐antiplasmin. Cleavage of α2‐antiplasmin and plasminogen activation were influenced by residue R211 in L4 as well as by unidentified residues in L3. OmpT, which is not associated with invasive bacterial disease, was converted into a Pla‐like protease by deleting residues D214 and P215, by substituting residue K217 for R217 in L4 of OmpT and also by substituting the entire L3 with that from Pla. This simple modification of the surface loops and the substrate specificity of OmpT exemplifies the evolution of a housekeeping protein into a virulence factor by subtle mutations at critical protein regions. We propose that inactivation of α2‐antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive character of plague.

The O75X adhesin of uropathogenic Escherichia coli is a type IV collagen-binding protein

Interaction of the basement‐membrane binding O75X adhesin of uropathogenic Escherichia coli with various extracellular matrix proteins was studied. The adhesin showed strong binding to type IV collagen immobilized on microtitre plates, whereas other collagens, laminin and fibronectin, were only weakly recognized. Similarly, specific binding of [125I]‐labelled type IV collagen to 075X‐positive bacteria was shown. Interaction of the two proteins was also demonstrated by affinity chromatography of the O75X adhesin on immobilized type IV collagen. The adhesin bound strongly to the immobilized N‐terminal 7S domain of type IV collagen, and the binding of [125I]‐labelled type IV collagen to O75X‐positive bacteria was inhibited by the soluble 7S domain. Binding of O75X to type IV collagen and to its 7S domain was specifically inhibited by chloramphenicol but was not affected by periodate or endoglycosidase‐H treatment of the glycoproteins. Our results show that the 7S domain of type IV collagen is the basement membrane receptor for the O75X adhesin and suggest an interaction based on protein‐protein recognition. Inhibition of the interaction by chloramphenicol favours the supposition that a modified tyrosine is involved in the binding site.