SummaryThe 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, a 2 -antiplasmin, and the bacteria were found to inactivate a 2 -antiplasmin. In contrast, only poor plasminogen activation and no cleavage of a 2 -antiplasmin was observed with recombinant bacteria expressing the homologous gene ompT from E. coli. A b-barrel topology model for Pla and OmpT predicted 10 transmembrane b-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 a 2 -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 a-Pla of 292 amino acids was processed into b-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 a 2 -antiplasmin. Cleavage of a 2 -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 a 2 -antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive character of plague.
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. Numerous proteinaceous adhesins have been detected inEscherichia coli (for recent reviews, see references 20 and 27). These adhesins occur in the form of fimbrial filaments or are nonfimbrial proteins of the outer membrane. The adhesins recognize different receptor molecules on the mammalian epithelia or extracellular matrices and function to enable colonization of E. coli at specific ecological niches. Many of the adhesins are associated with E. coli isolates from specific disease manifestations and contribute to the establishment of the infections. Examples of such disease-associated adhesins include the P fimbria of uropathogenic E. coli (UPEC) (55) and the various adhesin types detected in E. coli pathogroups causing diarrheal diseases (reviewed in references 13 and 36). Some adhesin genes, such as those encoding the mannosidebinding type 1 fimbriae (26) and th...
We developed a modified flagellar type III secretion apparatus to secrete heterologous polypeptides into the growth medium of Escherichia coli. The secretion was facilitated by fusing the 173-bp untranslated DNA fragment upstream of the gene fliC (encoding flagellin) as well as a transcriptional terminator from fliC, into the gene encoding the polypeptide of interest. The polypeptides secreted into the growth medium at concentrations ranging from 1 to 15 mg/l were from Campylobacter jejuni (262 residues in length), Streptococcus pneumoniae (434 residues), Staphylococcus aureus (115 residues), and N-terminal FliC hybrid proteins, for example, the eukaryotic green fluorescent protein (238 residues). The expressed proteins represented >50% of total secreted protein. Previously reported protein yields from extracellular secretion of foreign proteins in E. coli have been low, approximately 100 microg/l. The strengths of our method are the concentration and purity of the secreted proteins and its versatility with regard to the proteins' length and origin.
Regeneration processes in the periodontium occur by the interaction of different cell populations. It is known that these cells are also capable of forming new periodontal tissue after culture in vitro. The present study investigated whether replanted cultured cells from the periodontium could contribute to attachment formation. Primary cell cultures from alveolar bone and periodontal ligament were obtained from 11 minipigs. Experimentally induced furaction and interdental defects (n = 168) were treated in groups: (a) flap surgery, replantation of alveolar bone cells, and covering of the defects with Teflon membranes (ABC group); (b) flap surgery, replantation of periodontal ligament cells and membranes (PLC group); (c) flap surgery, bone gelatin (carrier material) and membranes (BG group); (d) flap surgery and membranes (NBG group); (e) flap surgery (FS group); and (f) no treatment (NT group). The defects were clinically and histologically (polyfluorochrome labeling) assessed after 10, 30, and 90 days. In the ABC group, initial calcified tissue formation at the roots was apparent after only 8 days. Marked new formation of cementum and alveolar bone and the development of a new attachment were observed after 90 days. In the BG and the NBG groups, wound healing varied depending on membrane healing and the morphology of the defects, which led to significantly poorer and variable results. Similar results were found in the PLC group, although some defects showed extensive cementum and bone formation. Defects in the FS and the NT groups healed largely by epithelialization. The study shows that replantation of cultured alveolar bone cells leads to formation of new cementum and bone, which, in turn, leads to formation of a new attachment. It is likely that the cells stabilize the tissue formation in the defect or on the root surface in the early phase of wound healing and prevent epithelial downgrowth. Results also show that regeneration in the periodontium is determined by the availability of (precursor) cells capable of forming calcified tissues.
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