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The avirulent Salmonella typhimurium F885 was transformed with a plasmid carrying the cloned S fimbriae genes of a uropathogenic Escherichia coli. The resulting transformant (F885-1) produced efficiently E. coli S fimbriae and was used for live oral vaccination of rats. For comparison rats were immunized subcutaneously with isolated S fimbriae. Both routes of vaccination resulted in a significant lgG antibody response to S fimbriae. In addition live oral vaccination induced a serum lgA response against S fimbriae. After transurethral infection of rats with a S fimbriae producing E. coli a 10-fold reduction of bacterial counts in the kidney was observed in rats orally vaccinated with F885-1 as compared to unvaccinated controls. This study suggests that the avirulent Salmonella F885 may be used as a fimbrial antigen carrier for oral vaccination against renal infections.
Nucleotide sequence of the sfaA gene coding for the S fimbrial protein subunit of Escherichia coli
(1987)
The sfaA gene of the uropathogenic Escherichia coli 06 strain 536, which is responsible for the determination of the S fimbrial protein subunit, was sequenced. The structural gene codes for a polypeptide of 180 amino acids including a 24-residue N-terminal signal sequence. A size of 15.95 kDa was calculated for the processed SfaA protein. The nucleotide and deduced amino acid sequences show significant homology to those of the F1C fimbria and, to a lesser extent, of the mannose- sensitive hemagglutinating fimbria (FimA, PilA). Only week homology toP fimbriae subunits (F72 , Pap) was found.
F 1 C fimbriae allow uropathogenic Escherichia coli to adhere to specific epithelial surfaces. This adhesive property is probably due to the presence of minor fimbrial components in F1C fimbriae. The foe gene cluster encoding F1C fimbriae has been cloned, as described previously. Here we present the nucleotide sequence (2081 bp) coding for the F 1 C minor fimbria I subunits. The structural genes code for polypeptides of 175 (FocF), 166 (FocG), and 300 (FocH) amino acids. The deduced amino acids of the F 1 C minor subunits were compared with the reported sequences of the minor subunits of other types of fimbriae. The data show that the Foc minor subunits are highly homologous to the corresponding Sfa proteins, whereas homology to the minor subunits of type 1 and P fimbriae is much lower.
The 06 serogroup Escherichia coli strain 536 carries two hemolysin (hly) determinants integrated into the chromosome. The two hly determinants are not completely identical, either functionally or structurally, as demonstrated by spontaneous deletion mutants carrying only one of them and by cloning each of the two determinants separately into cosmid vectors. Each hly determinant is independently deleted at a frequency of 10-4 , leading to variants which exhibit similar levels of internal hemolysin but different amounts of secreted hemolysin. The two hly determinants were also identified in the 04 E. coli strain 519. The three E. coli strains 251, 764, and 768, which belong to the serogroup 018, and the 04 strain 367 harbor a single chromosomal hly determinant, as demonstrated by hybridization with hly-gene-specific probes. However, a hybridization probe derived from a sequence adjacent to the hlyC-proximal end of the plasmid pHlyl52-encoded hly determinant hybridizes with several additional chromosomal bands in hemolytic 018 and 06 E. coli strains and even in E. coli K-12. The size ofthe probe causing the multiple hybridization suggests a 1,500- to 1,800-base pair sequence directly flanking hlyC. Spontaneous hemolysin-negative mutants were isolated from strains 764 and 768, which had lost the entire hly determinant but retained all copies of the hlyC-associated sequence. This sequence is not identical to a previously identified (J. Hacker, S. Knapp, and W. Goebel, J. Bacteriol. 154:1145-1154, 1983) somewhat smaller (about 850 base pairs) sequence flanking the other (hlyBb-proximal) end of the plasmid pHlyl52-encoded hly determinant which, as shown here, exists also in multiple copies in these hemolytic E. coli strains and in at least two copies in E. coli K-12. In contrast to the plasmid-encoded hly determinant which is directly flanked at both ends by these two diJJerent sequences, the chromosomal hly determinants are not immediately flanked by such sequences.
The genetic determinant coding for the Pspecific F8 fimbriae was cloned from · the chromosome of the Escherichia coli wild-type strain 2980 (018: K5: H5: FlC, F8). The F8 determinant was further subcloned into the Pstl site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned Counterpart was demonstrated. The cloned F8 fimbriäe and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepi thellal cells. The cloned F8 determinant was weil expressed in a variety of host strains.
Results of molecular and pathogenic studies of three different bacterial hemolysins (cytolysins) are presented. These exoproteins derive from the two gram-negative bacteria Escherichia coli and Aeromonas hydrophila and from the gram-positive pathogen Listeria monocytogenes. The hemolysin of E. coli is determined by an 8-kilobase (kb) region that includes four clustered genes (hlyC, hlyA, hlyB, and hlyD). This hemolysin determinant is part either of large transmissible plasmids or of the chromosome. The genes located chromosomally are found predominantly in E. coli strains that can cause pyelonephritis and/or other extraintestinal infections. A detailed analysis of the chromosomal hly determinants of one nephropathogenic E. coli strain revealed the existence of specific, large chromosomal insertions 75 kb and lOO kb in size that carry the hly genes but that also influence the expression of other virulence properties, i.e., adhesion and serum resistance. The direct involvement of E. coli hemolysin in virulence could be demonstrated in several model systems. The genetic determinants for hemolysin (cytolysin) formation in , A. hydrophila (aerolysin) and L. monocytogenes (listeriolysin) are less complex. Both cytolysins seem to be encoded by single genes, although two loci (aerB and aerC) that affect the expression and activity of aerolysin have been identified distal and proximal to the structural gene for aerolysin (aerA). Cytolysin-negative mutants of both bacteria were obtained by site-specific deletion and/or transposon mutagenesis. These mutants show a drastic reduction in the virulence of the respective bacteria.
Background:
A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs) - including pathogenicity islands (PAIs) - in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial) islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT). Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far.
Results:
To study mobilisation of such large genomic regions in prototypic uropathogenic E. coli (UPEC) strain 536, PAI II(536) was supplemented with the mob(RP4) region, an origin of replication (oriV(R6K)), an origin of transfer (oriT(RP4)) and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II(536) construct occured from strain 536 into an E. coli K-12 recipient. In transconjugants, PAI II(536) existed either as a cytoplasmic circular intermediate (CI) or integrated site-specifically into the recipient's chromosome at the leuX tRNA gene. This locus is the chromosomal integration site of PAI II(536) in UPEC strain 536. From the E. coli K-12 recipient, the chromosomal PAI II(536) construct as well as the CIs could be successfully remobilised and inserted into leuX in a PAI II(536) deletion mutant of E. coli 536.
Conclusions:
Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands.
Mip protein of Legionella pneumophila exhibits peptidyl-prolyl cis-trans-isomerase (PPIase) activity
(1992)
Legfonells pneumoph/la is an intracellular paraslte which ts able to survtve and multipJy in human monocytes and alveolar macrophages. The Mtp (macrophage lnfectiv1ty potentlator) protein has been shown to be an essential virulente factor. A search of translated nuclelt .acld data ba.ses has shown that the Mip proteJn from strain Wadsworth possesses reglons homologaus to those found in the FK.506-bindfng proteins (FKBPs) of several different eukaryotlc organisms. FKBPs are abte to bind to the fmmunosuppressant macrollde FK506 and possess peptidyf .. prolyl cisltrans Isomerase (PPiase) activlty. The gene coding for the Mlp proteln was cloned from the ehromo. some of L. pneumophila straln Philadelph·a I and sequenced. II was synthesl%ed in Escherichla coll ·K- 12 and alter purlfication it exhibited PPiase activity catalyslng the slow clsltrans lsomerization of prolyl peptlde bonds. ln ollgopeptides. Mip ls inhibi~ted by FK506 and fully reslstant to cyclosporln A, as was also found for the recently characterlzed FKBP-type PPiases of eukaryotes. However, the N-terminal extenslon of Mip and/or the substltutrons of the vari· ab1e amlno acrds ln the C-termlnal FKBP core Iead to variatlons,. when compared with eukaryotlc FKBPs, Jn substrate specfflclty wlth the Oligopeptide substrates of' type Suc-Aia-Xaa-Pro-Phe·4·nitroanUide. Never· theless, the Legionella Mip factor represents a bacte· rial gene product whtch shares some characteristics normally found in eukaryotic proteins. ln view of the activity of PPiases in protein-folding reactlonsf such prokaryotic FKBP analogues may represent a new class of bacterial. pathogenicity factors.
Legionella pneumophila generares exotoxins, cytolysins, proteases oc hemolysins that darnage host cells llke erythrocytes or rissue cu lrure cells. The gene for a new L. pneumophila hemolysin withour a proteolytic activiry was idemified, cloned in E. coli and sequenced. The gene producr was analysed by SDS-Polyacrylamide-gel-electrophoresis.
The hemolytic, uropathogenic Escherichia coli 536 (06:K15:H31) contains two inserts in its chromosome (insert I and insert II), both of which carried hly genes, were rather unstable, and were deleted spontaneously with a frequen~y of 10-3 to 10-4• These inserts were not found in the chromosome of two nonhemolytic E. coli strains, whereas the chromosomal ~equences adjacent to these inserts appeared tobe again homologous in the uropathogenic and two other E. coü strains. Insert I was 75 kilobases in size and was ftanked at both ends by 16 base pairs (bp) (TTCGACTCCTGTGATC) which were arranged in direct orientation. For insert I it was demonstrated that deletion occurred by recombination between the two 16-bp ftanking sequences, since mutants lacking this insert still carried a single copy of the 16-bp sequence in the chromosome. 8oth inserts contained a functional hemolysin determinant. However, the loss of the inserts not only atfected the hemolytic phenotype bot led to a considerable reduction in serum resistance and the loss of mannose-resistant hemagglutination, caused by the presence of S-type funbriae (sja). lt is shown that the Sfa-negative phenotype is due to a block in transcription of the sfa genes. Mutants of strain 536 which lacked both inserts were entirely avirulent when tested in several animal model systems.