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Biomedical subjects

H Lång

Publications and source records attributed to H Lång.

10 recordsLinked to original sources
PubMed2001

Protein regions important for plasminogen activation and inactivation of alpha2-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, alpha2-antiplasmin, and the bacteria were found to inactivate alpha2-antiplasmin. In contrast, only poor plasminogen activation and no cleavage of alpha2-antiplasmin was observed with recombinant bacteria expressing the homologous gene ompT from E. coli. A beta-barrel topology model for Pla and OmpT predicted 10 transmembrane beta-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 alpha2-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 alpha-Pla of 292 amino acids was processed into beta-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 alpha2-antiplasmin. Cleavage of alpha2-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 alpha2-antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive character of plague.

Amino Acid Sequence↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2000

Outer membrane proteins as surface display systems.

Outer membrane proteins (OMPs) of gram-negative bacteria can be used as carrier proteins to present foreign peptide epitopes on the bacterial cell surface. They all have a common structural motif of a beta-barrel that is composed of a variable number of transmembrane beta-strands connected at the periplasmic side with short turns and at the outside with long surface-accessible loops. Outer membrane proteins occur as monomers like OmpA, or assemble into trimers like the porins. Foreign gene products have been fused to surface-accessible regions of several outer membrane proteins including the porins OmpC, PhoE and LamB, lipoproteins as well as the OmpA protein. Short epitopes that are inserted into outer membrane proteins induce epitope-specific antibody responses, and are thus appealing candidates for live recombinant vaccines. Also large insertions, of more than 100 amino acids, are in some cases tolerated and do not affect the overall conformation of the carrier protein. The possible applications for outer membrane display include recombinant vaccines, peptide library screening, development of biocatalysts or whole-cell adsorbents, and adhesin-receptor interaction studies. It is expected that in the near future, development of new display systems will still increase the utilization of this emerging exciting technology.

Animals↗
PubMed2000

Characterization of adhesive epitopes with the OmpS display system.

OmpS is an outer membrane protein of Vibrio cholerae where it forms trimeric pores that function in the uptake of maltose and maltodextrins. Based on sequence similarity to LamB proteins, a model of OmpS folding in the outer membrane has been constructed. According to this model, OmpS contains 18 transmembrane beta-strands and nine surface-accessible loops. Adhesive epitopes can, when inserted into surface-accessible loop 4 (L4) and expressed in Escherichia coli, retain their functional characteristics. We inserted three D-repeats from the Staphylococcus aureus fibronectin-binding protein FnBPA into L4 of OmpS and showed that E. coli cells expressing these hybrids bind fibronectin. DNA fragments covering the N-terminal half of the globoside-binding P-fimbrial adhesin class II PapG of E. coli were cloned into the same surface accessible loop (L4) of OmpS. Fragments of papG encoding 53 or 186 amino acids from the N-terminal end of class II PapG adhesin were found to confer bacterial adhesiveness to globoside. Removal of 23 amino acids from the N-terminus of PapG did not affect receptor binding, but removal of 31 amino acids abolished it. The newly developed night sky image technique was also used to demonstrate the binding properties of membrane vesicles carrying the hybrid proteins. We raised antibodies against the purified hybrid protein containing 53 amino acids from PapG. This antiserum recognized the P-fimbriae on E. coli cells. These data provide evidence that the N-terminal first 53 amino acids of class II PapG contain the receptor-binding domain.

Adhesins, Bacterial↗
PubMed1997

The OmpS maltoporin of Vibrio cholerae as carrier of foreign epitopes.

Insertion of additional epitopes to outer membrane proteins can lead to display of the hybrid protein on the bacterial outer surface. OmpS is the maltoporin of Vibrio cholerae and forms trimeric pores which function in uptake of maltose and maltodextrins through the membrane. OmpS is present in all V. cholerae 01 and 0139 strains. Each monomer traverses the membrane 18 times and has thus 9 loops facing the outside world. We have developed an ompS-expression-plasmid based system where foreign epitopes can be inserted in one of its surface accessible loops leading to production of a hybrid protein which still has the normal OmpS folding and function. The immunogenic peptides tested as OmpS hybrids include the CTP3 epitope of cholera toxin B-subunit and the C3 epitope of poliovirus. These hybrids can be detected with epitope-specific antisera on the bacterial cell surface. OmpS hybrid proteins carrying 38, 76 or 115 aa of the fibronectin binding D1-D3 repeats of FnBPA of Staphylococcus aureus have been tested for binding characteristics.

Amino Acid Sequence↗
PubMed1995

Sequence alignment and structural modelling of the LamB glycoporin family.

lamB gene segments were obtained from Yersinia enterocolitica and Vibrio parahaemolyticus by the PCR and the DNA sequence determined. The deduced polypeptide sequences showed high similarity to six other LamB-related proteins and all contained typical signature sequences present in all members of the family but not other proteins. The aligned amino acid sequences permitted derivation of a model of LamB folding across the bacterial outer membrane using an approach successfully applied in the identification of structural features in other porins (Ferenci,T. (1994) Mol. Microbiol. 14:188-189). The alignment-based model differs from previous LamB structure predictions and is also more complex than that found for OmpF-related porins; more than 16 conserved stretches of amino acid sequence potentially corresponded to membrane-spanning segments.

Amino Acid Sequence↗
PubMed1994

The maltose regulon of Vibrio cholerae affects production and secretion of virulence factors.

The effects of maltose on production and secretion of virulence factors of Vibrio cholerae in strain X28214, classical biotype, and in maltose-defective transposon mutants constructed from this strain were characterized. Maltose was found to inhibit secretion of cholera toxin and to reduce production of the mannose-sensitive hemagglutinin and the soluble hemagglutinin-protease. In contrast, the amount of toxin-coregulated pilus was increased in the presence of maltose. The maltose effect was apparently mediated by genes of the maltose regulon, since inactivation of the malQ or malF gene of V. cholerae by transposon insertion was found to affect production and secretion of the same virulence factors that were responsive to maltose. The malQ and malF mutants showed, in addition, reduced virulence in an infant-mouse model. These results suggest that maltose may have a significant regulatory role in the production of virulence factors and that an intact maltose regulon is needed for full virulence of V. cholerae.

Animals↗
PubMed1993

The ompS gene of Vibrio cholerae encodes a growth-phase-dependent maltoporin.

The outer membrane of Vibrio cholerae contains a maltose-inducible major protein, OmpS (43 kDa), that is common to different isolates. Nucleotide sequence analysis of the corresponding structural gene, ompS, revealed an open reading frame encoding a 412-amino-acid polypeptide. The amino acid sequence of OmpS is similar to that of LamB, the Escherichia coli maltoporin, and to ScrY or Klebsiella pneumoniae, although the antigenic determinants of these proteins are different. The cloned ompS gene complemented an ompS mutation of V. cholerae and the corresponding polypeptide could function as a maltoporin in a LamB- mutant of E. coli. The promoter region of ompS is highly homologous to the malK-lamB promoter of E. coli and the ompS gene is controlled by MalT in E. coli. This indicates that the same kind of regulatory mechanism is used to activate the ompS expression in V. cholerae and malK-lamB expression in E. coli. An ompS-lacZ transcriptional fusion was used to demonstrate a dual control in ompS expression; the ompS gene is responsive to the inducers maltose and trehalose but in their absence it is also expressed in response to growth-phase. These different modes of induction might be of importance during different stages of V. cholerae infection.

Amino Acid Sequence↗