Ritva Virkola

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.

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.

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.

Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin

Adherence of type‐1‐fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type‐1‐fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non‐fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside‐inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(plSF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae‐coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type‐1 ‐fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type‐1‐fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type‐1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(plSF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type‐1 fimbriae of S. enterica and E. coli bind to oMgomannoside chains of the lamjnjn network in basement membranes.

matB, a Common Fimbrillin Gene of Escherichia coli, Expressed in a Genetically Conserved, Virulent Clonal Group

A novel fimbrial type in Escherichia coli was identified and characterized. The expression of the fimbria was associated with the O18acK1H7 clonal group of E. coli, which cause newborn meningitis and septicemia when grown at low temperature; hence, it was named the Mat (meningitis associated and temperature regulated) fimbria. The fimbriae were purified from a fimA::cat sfaA::Gm fliC::St derivative of the O18K1H7 isolate E. coli IHE 3034. The purified Mat fimbrillin had an apparent molecular mass of 18 kDa and did not serologically cross-react with the type 1 or S fimbria of the same strain. The matB gene encoding the major fimbrillin was cloned from the genomic DNA of the fimA::cat sfaA::Gm fliC::St derivative of IHE 3034. The predicted MatB sequence was of 195 amino acids, contained a signal sequence of 22 residues, and did not show significant homology to any of the previously characterized fimbrial proteins. The DNA sequence of matB was 97.8% identical to a region from nucleotides 17882 to 18469 in the 6- to 8-min region of the E. coli K-12 chromosome, reported to encode a hypothetical protein. The 7-kb DNA fragment containing matB of IHE 3034 was found by restriction mapping and partial DNA sequencing to be highly similar to the corresponding region in the K-12 chromosome. Trans complementation of the matB::cat mutation in the IHE 3034 chromosome showed that matB in combination with matA or matC restored surface expression of the Mat fimbria. A total of 27 isolates representing K-12 strains and the major pathogroups of E. coli were analyzed for the presence of a matB homolog as well as for expression of the Mat fimbria. A conserved matB homolog was found in 25 isolates; however, expression of the Mat fimbriae was detected only in the O18acK1H7 isolates. Expression of the Mat fimbria was temperature regulated, with no or a very small amount of fimbriae or intracellular MatB fimbrillin being detected in cells cultivated at 37(o)C. Reverse transcriptase PCR and complementation assays with mat genes controlled by the inducible trc promoter indicated that regulation of Mat fimbria expression involved both transcriptional and posttranscriptional events.

Multifunctional nature of P fimbriae of uropathogenic Escherichia coli: mutations in fsoE and feoF influence fimbrial binding to renal tubuli and immobilized fibronectin

P fimbriae of the F71 serotype of Escherichia coli are composed of a major subunit, FsoA, and of three minor proteins named FsoG, FsoE, and FsoF. FsoG is the Galα(1–4)Gal‐specific lectin. We assessed mutated recombinant strains each deficient in one fimbrial component for adhesion to frozen sections of rat cortical kidney and to fibronectin immobilized on glass. Rat kidney lacks the Galα(1–4)Gal‐containing glycolipids. The fsoG mutant strain was as adhesive to sections of rat kidney and to fibronectin‐coated glass as was the recombinant strain expressing the complete fso gene cluster. The fsoA mutant strain was highly adhesive to fibronectin and to kidney sections. In the rat kidney, the adhesion of these strains was predominantly localized to sites of basolateral membranes of tubuli. The fsoE and the fsoF mutant strains were slightly less adhesive to kidney structures and failed to adhere to fibronectin. The fsoE, fsoF double mutant strain adhered neither to fibronectin nor to kidney sections. None of the fso recombinant strains reacted with soluble fibronectin, suggesting that the interaction is dependent on the conformation of the fibronectin molecules. Recombinant strains expressing the F72, F8, F11, F13, and F14 serovariants of the P fimbria also showed adherence to immobilized fibronectin. The results show that in addition to binding to globoseries of glycolipids via the G protein, the P fimbriae of uropathogenic E. coli exhibit a tissue‐binding property influenced by fsoE and fsoF gene products and with affinity for basolateral membranes and fibronectin.

Using Every Trick in the Book: The Pla Surface Protease of Yersinia pestis

The Pla surface protease of Yersinia pestis, encoded by the Y. pestis-specific plasmid pPCP1, is a versatile virulence factor. In vivo studies have shown that Pla is essential in the establishment of bubonic plague, and in vitro studies have demonstrated various putative virulence functions for the Pla molecule. Pla is a surface protease of the omptin family, and its proteolytic targets include the abundant, circulating human zymogen plasminogen, which is activated by Pla to the serine protease plasmin. Plasmin is important in cell migration, and Pla also proteolytically inactivates the main circulating inhibitor of plasmin, alpha2-antiplasmin. Pla also is an adhesin with affinity for laminin, a major glycoprotein of mammalian basement membranes, which is degraded by plasmin but not by Pla. Together, these functions create uncontrolled plasmin proteolysis targeted at tissue barriers. Other proteolytic targets for Pla include complement proteins. Pla also mediates bacterial invasion into human endothelial cell lines; the adhesive and invasive charateristics of Pla can be genetically dissected from its proteolytic activity. Pla is a 10-stranded antiparallel beta-barrel with five surface-exposed short loops, where the catalytic residues are oriented inwards at the top of the beta-barrel. The sequence of Pla contains a three-dimensional motif for protein binding to lipid A of the lipopolysaccharide. Indeed, the proteolytic activity of Pla requires rough lipopolysaccharide but is sterically inhibited by the O antigen in smooth LPS, which may be the selective advantage of the loss of O antigen in Y. pestis. Members of the omptin family are highly similar in structure but differ in functions and virulence association. The catalytic residues of omptins are conserved, but the variable substrate specificities in proteolysis by Pla and other omptins are dictated by the amino acid sequences near or at the surface loops, and hence reflect differences in substrate binding. The closest orthologs of Pla are PgtE of Salmonella and Epo of Erwinia, which functionally differ from Pla. Pla gives a model of how a horizontally transferred protein fold can diverge into a powerful virulence factor through adaptive mutations.

Interaction of Fimbriae of Haemophilus influenzae Type B with Heparin-Binding Extracellular Matrix Proteins

ABSTRACT The interaction of the fimbriae of Haemophilus influenzae type b (Hib) with two heparin-binding extracellular matrix proteins, human fibronectin (Fn) and heparin-binding growth-associated molecule (HB-GAM) from mouse, were studied. The fimbriated Hib strain 770235 fim+, as well as the recombinant strainE. coli HB101(pMH140), which expressed Hib fimbriae, adhered strongly to Fn and HB-GAM immobilized on glass. Purified Hib fimbriae bound to Fn and HB-GAM, and within the Fn molecule, the binding was localized to the N-terminal 30,000-molecular-weight (30K) and 40K fragments, which contain heparin-binding domains I and II, respectively. Fimbrial binding to Fn, HB-GAM, and the 30K and the 40K fragments was inhibited by high concentrations of heparin. The results show that fimbriae of Hib interact with heparin-binding extracellular matrix proteins. The nonfimbriated Hib strain 770235 fim− exhibited a low level of adherence to Fn but did not react with HB-GAM, indicating that Hib strains also possess a fimbria-independent mechanism to interact with Fn.

Casein glycomacropeptide in the diet may reduce Escherichia coli attachment to the intestinal mucosa and increase the intestinal lactobacilli of early weaned piglets after an enterotoxigenic E. coli K88 challenge

Casein glycomacropeptide (CGMP), a glycoprotein originating during cheese manufacture, has shown promising effects by promoting the growth of some beneficial bacteria in vitro, although its activity has not been well explored. The present study was designed to evaluate the effects of CGMP against enterotoxigenic Escherichia coli (ETEC) K88 in vitro (Trial 1) and in vivo (Trial 2). In Trial 1, increasing concentrations of CGMP (0, 0.5, 1.5 or 2.5 mg/ml) were tested regarding its ability to block the attachment of ETEC K88 to ileal mucosa tissues obtained from piglets. Increasing the concentration of CGMP resulted in a gradual decrease in ETEC K88 attachment to the epithelial surface. In Trial 2, seventy-two piglets were distributed in a 2 × 2 factorial combination including or omitting CGMP in the diet (control diet v. CGMP) and challenged or not with ETEC K88 (yes v. no). Inclusion of CGMP increased crude protein, ammonia and isoacid concentrations in colon digesta. CGMP also increased lactobacilli numbers in ileum and colon digesta, and reduced enterobacteria counts in mucosa scrapings and the percentage of villi with E. coli adherence measured by fluorescence in situ hybridisation. The inclusion of CGMP in the diets of challenged animals also prevented the increase of enterobacteria in ileal digesta. We can conclude that CGMP may improve gut health by diminishing the adhesion of ETEC K88 to the intestinal mucosa, by increasing the lactobacilli population in the intestine and by reducing the overgrowth of enterobacteria in the digestive tract of piglets after an ETEC K88 challenge.

Tissue interactions of Escherichia coli adhesins

ConclusionsThe E. coli adhesions show a remarkable tissue tropism in the human urinary tract. This obviously relates to the known compartmentation of glycoconjugates in the kidney. To function as a virulence factor in human urinary tract infections, an adhesin must evidently recognize such receptors at uroepithelia that are not excreted in soluble form in urine. This prerequisite is filled by P fimbriae but not by type-1 or S fimbriae. Most of the tissue interactions of E.coli adhesins involve binding to carbohydrate receptors, whereas the binding of the 075X adhesin to type IV collagen appears to rely on protein-protein interactions. Binding of P fimbriae to immobilized fibronectin is independent of the lectin activity of the fimbriae and suggests of an additional function for the fimbrillin in mediating interaction with matrix and basement membrane proteins. Such interaction might be useful after colonization and disruption of epithelial surfaces, when the lectin activity of the fimbriae is not any more important.