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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.
S fimbrial adhesins (Sfa) enable pathogenic Escherichia coli strains to bind to sialic acid-containing eucaryotic receptor molecules. In order to determine the inftuence of culture conditions on the expression of the sfa determinant in a wild-type strain, we fused the gene lacZ, coding for the enzyme ß-galactosidase, to the sfaA gene, responsible for the major protein subunit of S fimbriae. By using a plasmid which carries an R6K origin, the sfaA-Iac hybrid construct was site-specifically integrated into the chromosome of the uropathogenic E. coli strain S36WT. The expression of lacZ, which was under the control of the sfa wild-type promoters, was now equivalent to the sfa expression of strain S36WT. With the help of this particular wild-type construct, it was demonstrated that the sfa determinant is better expressed on solid media than in liquid broth. The growth rate bad a strong inftuence on Sfa expression under aerobic but not under anaerobic conditions. Production of Sfa was further regulated by catabolite repression, osmolarity, and temperature.
The S fimbrial adhesin (sfa) determinant of E. co/i comprises nine genes situated on a stretch of 7.9 kilobases (kb) DNA. Here the nucleotide sequence of the genes sfa B and sfaC situated proximal to the main structural gene sfaA is described. Sfa-LacZ fusions show that the two genes are transcribed in opposite directions. The isolation of mutants in the proximal region of the sfa gene cluster, the construction of sfa-phoA gene fusions and subsequent transcomplementation sturlies indicated that the genes sfaB and sfaC play a role in regulation of the sfa determinant. ln addition the nucleotide sequence of the genes sfa D, sfa E and sfa F situated between the genes sfaA and sfaG responsible for S subunit proteins, were determined. lt is suggested that these genes are involved in transport and assembly of fimbrial subunits. Thus the entire genetic organization of the sfa determinant is presented and compared with the gene clusters coding for P fimbriae (pap), F1 C fimbriae (foc) and type I fimbriae ( fim). The evolutionary relationship of fimbrial adhesin determinants is discussed.
The S flmbrial adhesln (Sfa) enables Esch richla colito attach to slalfc acld-containing receptor molecules of eukaryotJc cells. As prevlously reported, the genetlc determinant coding for the Sfa of an E. co/1 06 strain was cloned, the gene codlng for the major fimbrfal subunit was ldentlfled and sequenced and th.e S speclflc adhesin was detected. Here we present evidence that ln addltlon to the major subunit proteln SfaA three other minor subunit proteins, SfaG (17 kD), SfaS (14kD) and SfaH (31 kD) can be isolated from the S..speclfic flmbrial adhesln complex. The genes coding for these minor subunits were ldenblied, mutagenlzed separately and sequenced. Using haemagglutlnatton tests. electron-microscopy and quantitative ELISA assays with monoclonal anti-SfaA and anti-SfaS antlbodles the functlons of the minor subunlts were determined. lt was determlned that SfaS ls ldentlcal to the S-specific adhesln; whlch also plays a role ln deterrninatlon of the degree of fimbri· ation ofthe cell. The mlnor subunit SfaH also had some Jnfluence on the Ievei of fimbrlation of the cell. while StaG ls necessary for full expression of S·specific binding. lt was further shown that the amino-terminal proteln sequence of the isolated SfaS profein was identJcal to the proteln sequence calculated from the DNA sequence of the sfaS gene locus.
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.
We investigated the role of bacterial adherence and hemolysin production from Escherichia coli parent and genetically cloned strains as to their eft'ects on bistaJidne release from rat mast cells and leukotriene generation from human polymorphonuclear granulocytes. These mediators were involved in the induction of inftammatory disease processes and led, for example, to enhancement of vascular permeability, chemotaxis (leukotriene 84 [LTB4]), chemoaggregation, lysosomal enzyme release, and smooth muscle contraction, (LTC4, LTD4 , and LTE4). Washed bacteria (E. coli K-12 Ms+ my=; E. coli 536 Ms+ MR= my=) as weil as their culture supematants were analyzed. Washed E. coli K-12 (Hiy+), unlike Hly- strains, induced high amounts of histamine release from rat mast cells and chemotactic activity from human polymorphonuclear granulocytes. Significant leukotriene releasewas obtained with washed E. coli K-12 my+ strains and their bacterial culture supematants. Leukotriene induction was dependent on the amount of hemolysin activity present in the supematant. However, additional soluble factors should also be considered. The presence of hemolysin appeared to aceeierate and enhance the rate of phagocytosis of bacteria by neutrophUs. When E. coli 536 (MS+ MR= Hly=) strains were analyzed, the simultaneous presence of MR+ pili and hemolysin production led to an increase in histamine release as compared with MR- my+ strains. The genetically cloned MR+ my+ E. coli 536 strain induced higher amounts of IeukotrieDes as compared with the wUd-type strain. Our data soggest a potent role for adhesins and hemolysin as virulence factors in inducing the release of inftammatory mediators.
Tbe genetic organization of tbe foc gene duster bas been studied; six genes involved in tbe biogenesis of Fl C fimbriae were identifi.ed.focA encodes tbe major fimbrial subunit, focC encodes a product tbat is indispensable for fimbria formation,focG andjocH encode minor ftmbrial subunits, andfocl encodes a protein wbicb sbows similarities to the subunit protein FocA. Apart from tbe FocA major subunits, purified FlC fimbriae contain at least two minor subunits, FocG and FocH. Minor proteins of similar size were observed in purified S fimbriae. Remarkably, some mutations in tbe foc gene duster result in an altered 6mbrial morpbology, i.e., rigid stubs or long, curly ftmbriae.
The Escherichia coli blood culture isolate BK658 (07S:K1:H7) expresses F1A and F1B fimbriae as weil as a third fimbrial type which reacts with anti-S-fimbrial antiserum but fails to show S-specific binding properlies (i.e., agglutination of bovine erythrocytes). To characterize these fimbriae, we cloned the respective genetic determinant in E. coli K-12. The resulting recombinant clone HB101(pMMP658-6) expresses fimbriae of 1.2-p.m length and a diameter of approximately 7 nm. The determinant codes for the fimbrillin subunit, a protein of 17 kUodaltons in size, and for at least five other proteins of 87, 31, 23, 14.3, and 13.8 kUodaltons. By restriction analysis and by DNA-DNA hybridization, it could be shown that the cloned fimbrial determinant of strain BK658 exhibits a high degree of sequence homology to the gene clusters coding for S fimbrial adhesins (sfa) and F1C fimbriae (/oc). By using the Western blot (immunoblot) technique and a quantitative enzyme-linked immunosorbent assay, it could be further demonstrated that the cloned fimbriae of BK658, S fimbriae, and FlC fimbriae share cross-reactive epitopes as weil as antigenic determinants specific for each fimbrial type. No antigenic cross-reactivity with F1C fimbriae could be detected. The results indicate a genetical and serological relatedness of the cloned fimbriae toS fimbriae and F1C fimbriae. Therefore, this new type of fimbriae is preliminarily termed SIF1C-related fimbriae (Sfr).
The effect of Escherichia coli strains isolated from blood and cerebrospinal fluid of septic infants on plasminogen activation was studied. These strains typically carry a filamentous surface protein, S fimbria, that has formerly been shown to bind to endothelial cells and interact with plasminogen. The bacteria effectively promoted plasminogen activation by tissue plasminogen activator (t-PA) which was inhibited by e-aminocaproic acid. A recombinant strain expressing S fimbriae accelerated t-PAcatalyzed plasminogen activation to a similar extent as did the wild-type strains whereas the nonfimbriate recipient strain had no effect. After incubation with t-PA and plasminogen, the S-fimbriate strain displayed bacterium-bound plasmin activity whereas the nonfimbriate strain did not. Bacterium-associated plasmin generation was also observed with a strain expressing mutagenized S fimbriae that Iack the cell-binding subunit SfaS but not with a strain lacking the major subunit SfaA. Both t-PA and plasminogen bound to purified S fimbriae in a lysine-dependent manner and purified S fimbriae accelerated t-PA-catalyzed plasminogen activation. The results indicate that E. coli S fimbriae form a complex with t-PA and plasminogen which enhances the rate of plasminogen activation and generates bacterium-bound plasmin. This may promote bacterial invasion and persistence in tissues and contribute to the systemic activation of fibrinolysis in septicaemia.
Binding sites in the rat brain for Escherichia coli S fimbriae associated with neontal meningitis
(1988)
Escherichia coli strains that cause sepsis and meningitis in neonatal infants carry S fimbriae that bind to sialyl galactoside units of cell surface glycoproteins. To investigate the possible role of S fimbriae in determining the tissue tropism of neonatal menlngitis, we have studied the preselice of binding sites for S fimbriae in different tissues of the neonatal rat which is susceptible to meningitis caused by S-fimbriated E. coli. Purified S fimbriae were incubated on cryostat sections of different rat oipns and their bindina was assessed by indirect immunofluorescence. In the bnin of the neonatal rat, S fimbriae specifically bound to the luminal surfaces of the vascular endothelium and of the epithelium lining the choroid plexuses and bnin ventricles. The · bindlog W.s completely inhibited by the trisaccharide NeuAca2-3Ga)ßl-4Gic, a receptor analogue of S fimbriae, and by a preceding neuraminidase treatment of the sections. A recombinant E. coli strain expressina S fimbriae adhered in large numbers to the same tissue sites in the neonatal brain sections as did the purified fimbriae, · whereas the nonfimbriated host strahi and a recombiiuuit strain expresslog P fi.mbriae did not adhere to brain tissues. The results soggest that adhesion of S-fimbriated bacteria to the binding sites observed in the neonatai bnin has a pathogenetic roJe durlog bacterial Invasion from cii'culation into the cerebrospinal fluid.
DNA probes specific for different regions of the S-fimbrial adhesin (sja) determinant were constructed and hybridized with DNA sequences coding for P (F8 and F13), mannose-sensitive hemagglutinating type 1 (FlA), and FlC fimbriae. While the sfa and F1C DNA determinants exhibited homology along their entire lengths, the P-fimbrial and type 1-fimbrial determinants exhibited homology to regions of the sfa duster responsible for the control of transcription and, to a minor extent, to regions coding for proteins involved in biogenesis and/or adhesion of the fimbriae and for the N-terminal part of the fimbrillin subunit.
Legionel/a pneumophila, the causative agent of Legionnaires' disease, was analysed by electron microscopy for production of surface structures. Crystalline surface (S-) layers and fimbriae were not detected, but monotrichous flagellation was seen. Polyclonal antibodies specific for the 47 kDa ftagellin subunit of L. pneumophila Philadelphia I were used in Western blots to confirm the presence of flagella subunits in various L. pneumophila strains tested, but the antiserumalso reacted with flagellin subunits of L. micdlulei, L. hackelia (serogroup (SG) l and SG21 and L./ongbetichae (SG2). Flagellation of Legionellae was shown to be temperature regulated. When the growth temperature of virulent and avirulent variants of strain L. pneumophila Philadelphia I was shifted from 30 oc to either 37 or 41 oc, a decrease in the percentage offtagellated bacteria within the populationwas observed.
Fimbrial 8dhesins en8ble b8cteria to 8ttach t9 eucaryotic ceU~. The genetic determin8nts for S fimbrial 8dhesins (sja) an.d for FlC ("pseudotype I") fimbri8e ifoc) were compared. Sfa and FlC represent functionally distinct 8dbesins in tbeir receptor specificities. Nevertheless, 8 high degree of bomology between both determin8nts was found on the basis of DNA-DNA hybridizations. Characteristic difl'erences in the restriCtion maps of tbe corresponding gene clusters, bowever, were visible in regions coding for the fimbrial subunits and for the S-specific 8dhesin. While a plasmid carrying the geneiic deternlinant for FlC fimbri8e was 8ble to complement transposon-induced sfa mutants, 8 plasmid carrying tbe genetic determin8nt for 8 tbird 8dht$in type, termed P fimbriae, was un8ble to do so. Proximal sfa-specific sequences carrying the S fimbrial st'"uctural gene were fused to sequences representing tbe di$tal part of the foc gene cluster to form 8 hybrid cluster, and tbe foc proxim~ region coding for tbe structural protein was Iigated to sfa distal sequences to form 8 second hybrid. Botb hybrid clones produced intact fimbriae. Anti-FlC monoclonal8ntibodies (MAbs) only recognized clones which produced FlC fimbriae, and an ~ti-S 8dhesin MAb marked clones whicb expressed the S adhesin. Bowever, one of four other anti-S fimbri8e-specific MAbs reacted witb both fimbrial structures, S and FlC, indicating 8 common epitope on both antigens. The results presented bere ~upport tbe view th8t sfa and foc determinants code for fimbri8e tb8t 8re simil8r in several aspects, wbile the P fimbri8e are members of 8 more distantly rel8ted group.
Recently we have described the molecular cloning of the genetic determinant coding for the S-fimbrial adhesin (Sfa), a sialic acid-recognizing pilus frequently found among extraintestinal Eschenchili coli isolates. Fimbriae from the resulting Sfa + E. coli K-12 clone were isolated, and an Sfa-specific antiserum was prepared. Western blots indicate that S fimbriae isolated from different uropathogenic and meningitis-associated E. coli strains, including 083:Kl isolates, were serologically related. The Sfa-specific antibodies did not cross-react with P fimbriae, but did cross-react with FlC fimbriae. Furthermore the sja+ recombinant DNAs and some cloned s/a-flanking regions were used as probes in Southem experiments. Chromosomal DNAs isolated from 018:Kl and 083:Kl meningitis strains with and without S fimbriae and from uropathogenic 06:K + strains were hybridized against these sfa-specific probes. Only one copy of the sfa determinant was identified on the chromosome of these strains. No sfa-specific sequences were observed on the chromosome of E. coli K-12 strains and an 07:Kl isolate. With the exception of small alterations in the sfa-coding region the genetic determinants for S fimbriae were identical in uropathogenic 06:K + and meningitis 018:Kl and 083:Kl strains. The sfa determinant was also detected on the chromosome of Kl isolates with an Sfa-negative phenotype, and specific cross-hybridization signals were visible after blotting against FlC-specific DNA. In addition homology among the different strains was observed in the sfa-flanking regions.
The uropathogenic Escherichia coli wiJd..:type strain 536 produces S-fimbriae, P-related fimbriae and type I fimbriae. Using immuno-colony dot and ELISA techniques, variants were detected showing an increased degree of S-fimbrial production. It was demonstrated by itrtmunofluorescence microscopy that in noimal (wild-type) and hyperS- fimbriated E. coli populaiions non-fimbriated cells also · exist, and that the percentage of Sfinibrlated and non-fimbriated bacteria was roughly identica1 in either population. Hyper-Sfimbriated variants could be stably maintained. The transition from wild-type to hyper-S-fimbriation, which occurs spontaneously, is markedly higher than vice versa. Southern blot analysis of the S fimbrial adhesin (sfa) determinants of normal and hyper-fimbriated strains revealed no marked difference in the gene structure.
Ten Legionella pneumophUa strains isolated from dift'erent sources were analyzed according to their restriction fragment patterils obtained by cle~vage of gen.omic DNA With Notl and Sftl and separation by pulsed field electrophoresis. Three L. pneumophila isolate~ from a nosocomial outbreak in Lü~k (Germany) and three other L. prreumophilll stralns independently isolated from a water tap located in the care unit where tbe patients were bospitalized 'xhibited identical restricti9n fragment profiles. Therefore, we concluded that these environment81 spee~ens were the source of the Legionnatres dlsease. Anotber two isolates from patients and two strains from the environment, all unrelated to the outJlreak described, sbowed different cleavage patterns.
A hospital warm water system was monitored for the prcsence and distribution of lcgionellac. Subtyping of ten scletled Legionella pneumophiltl isolates. originating from four different sites in the system by using serogroup spccific antisera in an indircct immunofluorcscence tcst, rcvcalcd that nine of the tcn isolatcs belonged to scrogroup 6, while the remaining one was serogroup I 0. Two monoclonal antibodics (mAbs) spccific for a subgroup of serogroup 6 strains were further used for characterization. None of the strains reactcd with these mAbs. Genome analysis by elaborating Not I profiles using the pulscd field gel electrophoresis (PFGE) technique revealed that nearly all serogroup 6 isolates dcrived from different sites, including a new building connected hy a ring pipe. wcrc identical according to restriction fragment pattems. The patterns were distinguishable from those of the two L. pnewnophi/a serogroup 6 rcfcrencc strains, and ftom that of thc L. pneumophila scrogroup 10 isolate. These data arguc for a relatively homogeneaus L. pneunwpltila serogroup 6 population in the entire watcr system.
The protein PpiA (19 kD) cloned from a genomic library of Legionella pneumophila, Philadelphia 1, represents a peptido-glycan associated outer membrane protein in recombinant E. coli K-12 and L. pneumophila. lt exhibits distinct sequence homology to Iipoproteins of Haemophilus influenzae and E. coli. A ppiA specific DNA probe generated by PCR was used in Southern hybridizations of chromosomal DNA of Legionella strains and other Gram-negative pathogens. Under conditions of high stringency, hybridization could only be observed in L. pneumophila isolates, but alt other Legionella strains tested displayed hybridization under lower stringency. No signals appeared after hybridization of chromosomal DNA from a variety of other bacteria. Using anti-PpiA monospecific polyclonal antibodies in Western blots, it was demonstrated that PpiA related proteins of nearly the same size are found in all L. pneumophila isolates and in a variety of, but not alt, the Legionella species analysed here.
A total of 127 extraintestinal Escherichia coli strains of the capsule serotypes Kl, KS, and KlOO from human and animal sources were analyzed for DNA sequences specific for the genes for various adhesins (P fimbriae fpap] and P-related sequences fprs], S fimbriae [s/a)/FlC fimbriae [foc], and type I fimbriae lfim]), aerobactin (aer), and hemolysin (hly). The expression of corresponding virulence factors was also tested. Twenty-four selected strains were analyzed by long-range DNA mapping to evaluate their genetic relationships. DNA sequences for the adhesins were often found in strains not expressing them, while strains with hemolysin and aerobactin genes usually did express them. Different isolates of the same serotype orten expressed different virulence patterns. The use of virulence-associated gene probes for Southern hybridization with genomic DNA fragments separated by pulsed-field gel electrophoresis revealed that a highly heterogeneous restriction fragment length and hybridization pattern existed even within strains of the same serotype. Long-range DNA mapping is therefore useful for the evaluation of genetic relatedness among individual isolates and facilitates the performance of .precise molecular epidemiology.
We analyzed an Escherichia coli strain which harbours a chromosomal mutation that blocks the hemolysin excretion. Compartmentation studies showed that hemolysin accumulates in the cytoplasm and not in the periplasm. The mutation did not affect the SDS-PAGE protein pattern of the outer membrane, although some alterations were apparent in the periplasmic protein pattern. The mutant strain, E. coli Hsb-1 also failed to export a cloned fimbrial adhesin. The mutation maps in the min. 3.5 of the E. coli genetic map.
Characterization of a monoclonal antibody against the fimbrial F8 antigen of Escherichia coli
(1986)
A monoclonal lgG 1 antibody against F8 fimbriae was obtained with the hybridoma technique using spieen cells from C3H/f rnice immunised with a fimbrial preparation of Escherichia coli 2980 (018ac: K5: H-: FIC, F8) and Sp 2/0 Ag8 myeloma cells. The hybrid cells were cloned twice by lirniting dilution and grown in tissue culture. The monoclonal antibody was purified from culture supernatants on Protein A Sepharose. lt reacted with F8 fimbriae in colony blot, enzyme-linked immunosorbent assay (ELISA) and immunoblot after electrotransfer from sodium dodecyl sulphate-polyacrylarnide gel electrophoresis (SOS-PAGE) of fimbrial preparations. The antibody bound to and agglutinated F8-fimbriated bacteria.
S fimbriae are able to recognize receptor molecules containing sialic acid and are produced by pathogenic E. coli strains causing urinary tract infection and menigitis. In order to characterize the corresponding genetic determinant, termed S fimbrial adhesin ( sfa) gene duster, we have cloned the S-specific genes from a urinary pathogen and from a meningitis isolate. Nine genes are involved in the production of S fimbriae, two of these, sfaB and sfaC code for regulatory proteins being necessary for the expression of S fimbriae. Two promoters, PB and Pc, are located in front of these genes. Transcription of the sfa determinant is influenced by activation of the promotersvia SfaB and SfaC, the action of the H-NS protein and an RNaseE-specific mRNA processing. In addition, a third promoter, P A• located in front of the major subunit gene sfaA, can be activated under special circumstances. Four genes of the sfa determinant code for the subunit-specific proteins, SfaA (16 kda), SfaG (17 kda), SfaS (14 kda) and SfaH (29 kda). It was demonstrated that the protein SfaA is the major subunit protein while SfaS is identical to the sialic-acid-specific adhesin of S fimbriae. The introduction of specific mutations into sfaS revealed that a region of six amino acids of the adhesin which includes two lysine and one arginine residues is involved in the receptor specific interaction of S fimbriae. Additionally, it has been shown that SfaS is necessary for the induction of fimbriation while SfaH plays a role in the stringency of binding of S fimbriae to erythrocytes.
The sfa determinant codes for S fimbrial adhesins which constitute adherence factors of pathogenic Escherichia coli strains. Wehave recently shown that the sfa determinant is transcribed from three prömoters, pA, pB, and pC. In comparison with the promoters pB and pC, promoter pA, which is located in front of the structural gene sfaA, showed very weak activity. Herewe have determined the exact positions ofthe mRNA start points by primer extension studies. We have also shown that mRNAs of 500, 700 and 1400 bases can be detected using oligonucleotide probes specific for the genes sfaB, sfaC and sfaA. SfaB and SfaC arepositive regulators infiuencing fimbriation and the production of the S-specific adhesin which is encoded by the gene sfaS Iocated in the distal half of the determinant. In addition, it is demonstrated that SfaB and SfaC interfere with the regulatory effect of the histone-like protein H-NS, encoded by a locus termed drdX or osmZ. In a drdx+ strain the regulators are necessary for transcription of the sfa determinant. In contrast, sfa expression is activator-independent in a drdx- strain. In this latter genetic background, a substantial fraction of the sfa transcripts is initiated from promoter pA. On the basis of these data we discuss a model for the regulation of this adhesin-specific determinant.
The gene coding for the sialic acid-specific adhesin SfaS produced by the S fimbrial adhesin (sfa) determinant of Escherichia coli has been modified by oligonucleotide-directed, site-specific mutagenesis. Lysine 116, arginine 118, and Iysine 122 were replaced by threonine, serine, and threonine, respectively. The mutagenized gene dusters were able to produce S fimbrial adhesin complexes consisting of the S-specific subunit proteins including the adhesin SfaS. The mutant clones were further characterized by hemagglutination and by enzyme-linked immunoassay tests with antifimbria- and anti-adhesin-specific monoclonal antibodies, one of which is able to block S-specific binding (Moch et al., Proc. Natl. Acad. Sei. USA 84:3462-3466, 1987). The lysine-122 mutantclone was indistinguishable from the wild-type clone in these assays. Replacement of Iysine 116 and ai'ginine 118, however, abolished hemagglutination and resulted in clones which showed a weak (Iysine 116) or a negative (arginine 118) reaction with the antiadhesin-specific antibody Al. We therefore suggest that Iysine 116 and arginine 118 have an inßuence on binding of SfaS to the sialic acid residue of the receptor molecule. Substitution of arginine 118 by serine also had a negative efl"ect on the amount of SfaS adhesin proteins isolated from the S fimbrial adhesin complex.
The \(\alpha\)-Sialyl-\(\beta\) 2-3-Galactosyl-specific adhesin (S adhesin) was isolated from cells of a recombinant Escherichia coli K-12 strain expressing the S-flmbrial adhesin complex. A crude cell extract was partiaUy dissociated into fimbriae and an adhesin-enriched fraction by heating to 7O°C. From the latter, adhesin was purified to apparent homogeneity (by fast protein liquid chromatography, immunoblot, and NaDodSO\(_4\)/PAGE) by differential ammonium sulfate precipitation, dissociation in 8 M guanidine hydrochloride, and high-resolution anion-exchange chromatography in 8 M urea. The purified adhesin formed an aggregate of M\(_r\)\(\approx\)10\(^6\) that was made up of one type of 12-kDa polypeptide (fimbrillin is 16.5 kDa). It had pI value of 4.7 (fimbriae has a pI value of 6). Adhesin and fimbrillin had different amino add compositions. The purified adhesins agglutinated human and bovine erythrocytes with the same speclfkity as the whole bacteria; purified fimbriae were not adhesive. Monoclonal anti-adhesin and anti-fimbriae antibodies were obtained. Monoclonal antiadhesin, but none of the anti-fimbriae, antibodies inhibited the agglutination of erythrocytes. The anti-adhesive antibodies were used in immuno-gold electron microscopy to localize adhesin exclusively on the fimbriae, with a possible preference to their tips.
Escherichia coU K-12 strains producing S-fimbrial adhesins, FlC fimbriae, and mutagenized fimbriae were tested in a binding assay with a renal tubular cell line. S-fimbrial adhesins and FlC fimbriae mediated bindlog to tubular cells. The SfaA, SfaG, and SfaS subunits of S fimbriae contributed to attachment. Site-specific mutations in the sfaS gene reduced binding. The Inhibitionprofile of FlC fimbriae resembled that of S fimbriae.
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.
Escherichia coli 536 (06:K15:H31), which was isolated from a case of urinary tract infection, determines high nephropathogenicity in a rat pyelonephritis system as measured by renal bacterial counts 7 days after infection. The loss of S fimbrial adhesin formation (Sfa-) (mannose-resistant hemagglutination [Mrh-] and fimbria production [Fim-]), serum resistance (Sre-), and hemolysin production (Hly-) in the mutaßt 536-21 led to a dramatic reduction of bacterial counts from almost tOS to only 40 cells per g of kidney. The reintroduction of the cloned S fimbrial adhesin determinant (sfa) increases the virulence of the avirulent mutant strain by a factor of 20; almost the same eß'ect was observed after restoration of serum resistance by Integration of an sja+ recombinant cosmid into the chromosome. Additional reintroduction of the my+ phenotype by Iransformation of two hly determinants increased the virulence of the strains. Demolysin production determined increased renal elimination of leukocytes and erythrocytes. Thus all three determinants investigated, S fimbriae, serum resistance, and hemolysin, contribute to the multifactorial phenomenon of E. coli nephropathogenicity.
Over a period of 3 years, Legionella pneumophila serogroup 6 strains were isolated from warm water outlets and dental units in the Dental Faculty and from the Surgery and Internal Medicine Clinics at the University of Dresden, Dresden, Germany. In the bacteriological unit of the above-mentioned facility, L. pneumophila serogroups 3 and 12 were grown frl,)m warm water specimens. The medical facilities are located in separate buildings connected with a ring pipe warm water system. All L. pneumophila serogroup 6 strains isolated from the warm water supply reacted with a serogroup-specific monoclonal antibody, but not with two other monoclonal antibodies which are subgroup specific, reacting with other serogroup 6 strains. The NolI genomic profiles obtained by pulsed-field gel electrophoresis of 25 serogroup 6 strains isolated from the Dental Faculty over a 3-year period, 1 isolate from the Internal Medicine Clinic, and 4 strains from the Surgery Clinic were identical. Furthermore, all these strains hybridized with a 3OO-kb NolI fragment when a legiolysin (lIy)-specific DNA probe was used. The NolI pattern, however, differed from those of six serogroup 6 strains of other origins, one serogroup 12 strain from the bacteriological unit, and another six unrelated strains of serogroups other than serogroup 6. L. pneumophila serogroup 6 strains which can be divided into only two subgroups by the use of monoclonal antibodies are differentiated in at least six Noli cleavage types obtained by pulsed-field electrophoresis.
A genomic library of Legionello pneumophihz, the causative agent of Legionnaires disease in humans, was constructed in Escherichill coli K-12, and the recombinant clones were screened by immuno-colony blots with im antiserum raised against heat-killed L. pneumophilo. Twenty-three clones coding for a LegioneUa-specific protein of 19 kDa were isolated. The 19-kDa protein, which represents an outer membrane protein, was found tobe associated with the peptidoglycan layer bothin L. pneumophilo andin the recombinant E. coli clones. This was shown by electrophoresis and Western immunoblot analysis of bacterial cell membrane fractions witb a monospecific polyclonal 19-kDa protein-specific antiserum. Tbe protein was termed peptidoglycan-associated protein of L. pneumophilo (Ppl). The corresponding genetic determinant, ppl, was subcloned on a 1.8-kb Clol fragment. DNA sequence studies revealed that two open reading frames, pplA and pplB, coding for putative proteins of 18~9 and 16.8 kDa, respectively, were located on the Clol fragment. Exonuclease 111 digestion studies confirmed tbat pplA is the gene coding for the peptidoglycan.;.associated 19-kDa protein of L. pneumophilo. The amino acid sequence of PpiA exhibits a high degree of homology to the sequences of the Pal Iipoproteins of E. coli K-12 and liaemophilus injluenvze.
Thirtccn StttJ1hylococcus dw·eus and s: <'pid<'l'· midis strains ohtaincd from nnsc and hand nf twn cmployccs and onc paticnt uf a mcdical ward as weil as two S. hemol.\"licus strains wcrc analyscd according to thcir rcstrktion fmgmcnt lcngth pattcrns ( RFLP) hy pulscd-ficld gcl clcctrophorcsis (PFGE) using thc rcslriction cnzymcs SmaJ and s.. .· tll. Spccics idcntification nf thc isolatcs was pcrformcd hy a systcm which includcs :!O hiochcmical rc"ctions. Furthcrmorc. thc antillintic resistancc pattcrns of thc stmins wcrc dctcrmincd. Whilc scvcral isolatcs cxhihitcd idcnticaf antihiotic susccptihilitics and hiochcmical prnfilcs. diffcrences in thc RFLP wcrc ohtaincd. ln thrcc cascs, S. epidermülis strains colonizing thc skin showcd an idcntical rcstriction profilc as isollltcs from thc mucous mcmhrancs of thc samc pcrson. Wc C(mcludcd that thc analysis of staphylococcal strains hy PFGE is an important cpidcmiolngical tnnl with high discrimination power.
Genetically cloned E. co/i strains expressing cloned virulence factors were studied with regard to their capability to induce inflammatory mediator release from various target cells. Among the strains were E. co/i strains with mannose-resistant haemagglutination (MRH +) and mannose-resistant adhesins, e.g. E. coli 536/21 pANN 80 I /4, E. coli 536/21 pANN 921 and E. coli 536/21 pANN 801-1. In comparison, E. coli 536/21, E. coli 536/21 pGB 30 int and E. coli Kl2, without and with mannosesensitive haemagglutination (MSH±), and adhesins were studied. The properties of the various strains for human PMN with regard to adherence and phagocytosis, chemiluminescence, 5-lipoxygenase activation of arachidonic acid, leukotriene formation, granular enzyme release and release of histamine from rat mast cells were analysed. It is evident that the various 'biochemical processes of cell activation are dissociated events. The highest chemiluminescence response is obtained with strains expressing MSH+, P-M RH+ or S-M RH+; the presence of S-adhesins suppressed the response. Highest leukotriene formation is obtained with E. coli 536/21 pANN 801-4, while E. coli with MSH was inactive. The concomitant presence of haemolysin secretion enhanced mediator release significantly. Our data suggest a potent role for mannose-resistant haemagglutination (MRH), adhesins and haemolysin as virulence factors in inducing the release of inflammatory mediators.
We investigated the role of bacterial mannose-resistant fimbriation of S fimbriae (Firn), mannose-resistant hemagglutination (S-Mrh), and hemolysin (Hiy) production by an Escherichitl coli parent and genetically cloned strains as regards (i) their eß'ect on histamine release from rat mast ceUs and (ii) generation of the chemiluminescence response, leukotriene, and enzyme release from human polymorphonuclear granulocytes. These mediators are involved in the induction of inftammatory disease processes and Iead, e.g., to the enhancement of vascular permeability, chemotaxis, aggregation of granulocytes (leukotriene 8 4), lysosomal enzyme release, and smooth-muscle contraction (leukotrienes C4, D4, and E4). The content of azurophilic and specific granules in polymorphonuclear granulocytes consists of highly reactive enzymes which amplify inflammatory reactions. Washed bacteria (E. coli 764 my:t:, E. coli 21085 Hly:t:, E. coli 536 Hly:t: Firn:~: Mrh:t:), as weil as their culture supernatants, were analyzed at various times during their growth cycle. No differences exist between parent and cloned or mutant strains with respect to their outer . membrane proteins and lipopolysaccharide pattern. Washed bacteria [E. coli 764 and 21085(pANN202-312)] which produced hemolysin, unlike my- strains, induced high Ievels of histamine release from rat mast ceUs and led to a significant chemiluminescence response and enzyme and leukotriene release from human polymorphonuclear granulocytes. Bacterial culture supernatants from Hly+ and secreting strains showed similar results with the exception of E. coli 21085(pANN202-312), which is a hemolysin-producing bot not a secretory strain. Our data soggest a potent role for hernolysin as a stimulus for noncytotoxic mediator release from various cells. Furthermore, we showed that the presence of Firn and S Mrh potentiales mediator release. The simultaneous presence of Mrh and Firn [E. coli 535/2l(pANN801-4)] increased mediator release compared with Mrh+ Firn- strains [E. coli 536/21(pANN801-1)]. E. coli 536/21 (Msh- Mrh- Firn- Hly-) did not induce mediator release. Escherichia coli alpha-hemolysin is a protein that causes in vitro Iysis of erythrocytes from several species of animals (6, 12, 1~18, 23). Hemolysin-producing E. coli strains occur only infrequently in the normal fecal ftora of humans but are often isolated from patients with extraintestinal infections such as urinary tract infections, bacteremia, and septicemia (13, 22, 25, 36-38, 46-48). The high percentage of Hly+ E. coli strains among isolates from patients with urinary tract infections suggested that hemolysin contributes to the virulence of E. coli strains. The role of hemolysin as a virulence factor has been recently demonstrated by using various animal models and cell cultures. Alpha-hemolysin is one of the very few proteins produced by members of the family Enterobacteriaceae that is released extracellulary. The genetic control of alpha-hemolysin production, transport, and release from cells is complex (24, 26, 30). At least four genes located on the bacterial chromosome or on ]arge transmissible plasmids are required to elicit a cell-free hemolytic phenotype. Bobach and Snyder (6) suggested that the existence of alpha-hemolysin complexed with lipopolysaccharide may have important implications in the understanding of its biological effects. In addition to hemolysin production, a variety of factors, e.g., fimbriae, expression of specific hemagglutination, and • Corresponding author. 886 0 and K antigens, may contribute to the vi
During a study of the nutritional requirements of clinical isolates of Escherichia coli, we found that 21 (7.0%) of 301 strains required nicotinamide to grow in minimal medium. The nicotinamide- requiring strains were present in 16 (15.8%) of 101 cultures of urine from young women with acute cystitis, in 5 (5.0%) of 100 stool specimens from healthy adults, and in none of 100 blood samples from adult patients with bacteremia. Most of the strains belonged to serogroup OI8:KI:H7, were hemolytic, possessed type I fimbriae, and exhibited similar patterns of antibiotic susceptibility. Two of the urinary isolates expressed S fimbriae, and all 16 urinary isolates contained the s/aS homologue gene on their chromosomes. One of the stool isolates contained the s/aS gene. The urinary isolates closely resembled a large clone of E. coli that is reportedly associated with neonatal meningitis and sepsis. It may be possible to detect this and related clones by their requirement for nicotinamide and to screen strains for S fimbriae by relatively inexpensive hemagglutination methods, including the use of avian PI antigens to detect mannose- resistant, non-P-fimbriated E. coli; the agglutination of bovine erythrocytes; and the use of bovine mucin to detect sialyl galactosides in S fimbriae.
DNA hybridization experiments demonstrated that the gene clusters encoding the F8 fimbriae (fei) as well as the type I fimbriae (pi/) exist in a single copy on the chromosome of E. coli 018:K5 strain 2980. In conjugation experiments with appropriate donors, the chromosomal site of these gene clusters was determined. The pil genes were mapped close to the gene clusters thr and Jeu controlling the biosynthesis of threonine and leucine, respectively. The fei genes were found to be located close to the galactose operon (gal) between the position 17 and 21 of the E. coli chromosomallinkage map.
Purified S fimbriae and an Escherichia coli strain carrying the recombinant plasmid pANN801-4 that encodes S fimbriae were tested for adhesion to frozen sections of human kidney. The fimbrlae and the bacteria bound to the same tissue domains, and in both cases the binding was specifically inhibited by the receptor analog of S fimbria, sialyl(a2-3)1actose. S fimbriae bound specifically to the epithelial elements in the kidneys; to the epithelial cells of proximal and distal tubules as weil as of the collecting ducts and to the visceral and parietal glomerular epithelium. In addition, they bound to the vascular endothelium of glomerull and of the renal Interstitium. No blnding to connective tissue elements was observed. The results suggest that the biological functlon of S fimbriae is to mediate the adheslon of E. coli to human epithelial and vascular endothellal ceUs.
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 haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstream of the hlyC gene. The sequence at the other (right-hand) end (RS) also shows multiple hybridization with the plasmid pHly152 and the chromosome of some Escherichia coli strains but the nucleotide sequence of this region does not reveal the typical properties of an IS element. Similar arrangements in the regions flanking the hly determinant are also found on various Hly plasmids from uropathogenic E. coli strains. Chromosomal hly determinants Iack both flanking sequences (IS2 and RS) in the immediate vicinity of the hly genes. The sequences immediately upstream of the hlyC gene have been determined from several chromosomal hly determinants and compared with the corresponding sequence of the hly determinant of the plasmid pHly152. We show that these sequences, which contain one promoter (left promoter, phlyL) in all hly determinants tested, vary considerably although common sequence elements can still be identified. In contrast, only relatively few nucleotide exchanges have been detected in the adjacent structural hlyC genes. The A + T content of the 200 bp sequence upstream of hlyC is very high (72 mol% A + T) but even the structural hly genes show a considerably higher A + T content (about 60 mol%) than the E. coli chromosome on average (50 mol% A+T) suggesting that the hly determinant may not have originated in E. coli.
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.
While clear evidence exists for the direct involvement of cytolysins in the pathogenesis of Gram-positive bacteria, the significance of Gram-negative haemolysins remains unclear. This paper presents briefly data indicating a role for haemolysin production in infections caused by Escherichia coli and also experiments which have allowed an analysis of the molecular basis of the haemolysis among pathogenic and non-pathogenic strains of this species.
Potential virulence, as defined by combined Ievels of adhesion to urinary epithelial cells, serum resistance, and mouse toxicity, was assessed for Escherichia coli strains causing symptomatic and asymptomatic urinary tract infections in relation to the carriage of hemolysin and other suspected virulence determinants. Hemolysin production (Hly), associated with certain 0 (04, 06, 018, and 075), K (5), and hemagglutination (VI and VII) antigenic types but not colicin V production (Cva), was evident in 83 and 60% ofisolates in groups possessing high potential virulence andin only 11 and 6% of those with low virulence. Strains of particular 0-types were not more virulent per se, but among the serotypes, specific combinations of virulence factors appeared decisive, e.g., 018 HAVI B/D/G Hly+ K5+t- and 018 HAIIIIIVBN Hly- Cva +t- Kl +t- strains were, respectively, of high and low potential virulence. Isolates with high potential virulence were found to a similar extent in symptomatic and asymptomatic infections.
The role of macrophages in primary and secondary infection of mice with Salmonella typhimurium
(1982)
Elimination of macrophages with high-molecular dextran sulphate (OS) markedly impairs resistance of mice to primary infection with smooth, virulent strains of Salmonella typhimurium, whereas stimulation of this system by killed Bordetella pertussis organisms increases resistance. In infection with rough, avirulent strains of S. iyphimurium the elimination of macro phages was not followed by an essential loss of resistance, and it appears that other non-specific defence mechanisms, for example the complement system, may have compensated for the lack of macrophages. Macrophages, therefore, play an important role in defence during primary infection with virulent strains. In immunity to challenge infection with S. typhimurium, macrophages play an even more significant role. Treatment with OS completely removes immunity, and both humoral and cell-mediated immune mechanisms seem to require the participation of macrophages.
A new mouse model for systemic infection with Escherichia coli is presented. Whereas in other models 107_108 bacteria have to be injected into an animal to induce toxic effects resulting in death within 24 hours, now, only 103_104 bacteria of an appropriate strain are required to produce a genuine infection characterized by an increase in the bacterial load over several days. The quantitative determination of bacterial counts per liver allows a more sensitive measurement than recording death rates. Furthermore, few animals are required for a definite result in contrast to the LDso determination of other models. The salient point regarding this new model is that conditioning of animals has to be achieved by incorporating the inoculum into agar which is injected subcutaneously. The resulting infection is completely dependent on the E. colicondistrain used. Whereas a hemolytic, uropathogenic strain is so virulent that an overwhelming infection develops within 48 hours after the injection of 103 bacterial cells, a non-hemolytic variant of this strain is completely avirulent, being unable to multiply in spite of the potentiating agar. The hemolytic E. coli strain ATCC 25922 is intermediate in virulence. The bacterial counts per liver increase steadily until death occurs five to seven days after the injection of 104 bacteria. This bacterial infection can be therapeutically influenced by daily treatment with various drugs. Ciprofloxacin, ceftriaxone and co-trimoxazole are able to cure the infection, whereas amoxicillin given orally is only moderately active against this ATCC strain, which is relatively resistant to amoxicillin.
Isolation and characterization of coliphage Omega18A specific for Escherichia coli O18ac strains
(1987)
The bactedophage Q18A, specific for Escherichia coli 018ac srrains, was isolated frorn sewage. The results of host range and conjugation experiments showed that the sensitivity of bacteria to the phage is associated with rhe presence of 018ac antigens. With sorne of rhe 018 strains rhe phage Q18A produces clear Iysis on bacterial lawns only when applied at a high multiplicity and moreover the phage does not multiply. With rhe help of the phage Ql8A, E. coli 0 18ac strains could be divided inro rwo serologically clistinct subgroups called 018A and 018A1• E. coli strains belanging to the sugroup 0 ISAare sensitive to phage Q t8A wheteas bacteria of subgroup A1 are resistanr.
E. coli stcains isolated from patients with urinary tcact infecrions (UTn very often possess mannose"sensitive (MS) and mannose-resistant (MR) adherence facmrs (fimbriae). According to their receptor specificity the mannose-resistant adhesins can be divided inm several types, P, S, M and X. We have cloned rhe determinants of rhree groups of UTI E. coli adhesins, MS, p and S, and prepared specific aorisera against the fimbriae antigens. 189 hernagglutination (HA+) -positive stcains, 96 fecal isolates and 93 strains isoJated from UTI . have been tesred with rhese specific antisera and further characterized by receptor specific : HA, HA parteras and further of rhe "common 0 serogroups" 01, 02, 04, 06, 07, 08, 018, ' 025, 075, most prevalenr in UTI, and hemolysin production. · 68 (73 %) of the UTI srrains a.nd 50 (52%) of the fecal isolates showed P-receptor specificiry; 16 (17%) of the uropathogenic bacteria and 33 (34%) of the fecal strains exhibited S, M or X-fimbriae antigens. 24% of rhe P-hemagglutinating (P+) strains reacted wirb P (F8)-specific antiserum. In contrast, more than three quaner of the s+-srrains were agglutinated by S-specific antiserum. HA-pattern VJ and 018 amigen were found to be associared with P-fimbriae strains, wbereas HA-pattern V and VII and the 0 anrigens 02 (M-type), 06 and 018 (5-type) occurred most frequently in p- -strains. A high percentage of P-fimbriated strains showed mannose-sensitive hemagglurination and hemolysin production.
The Qropathogenic Escherichia coli strain 536 (06:K15:H31) exhibits a mannose-resistant hemagglutination phenotype (Mrh) with bovine erythrocytes and delayed Mrh with human and guinea pig erythrocytes. Neuraminidase treatment of the erythrocytes abolishes mannose resistant hemagglutination, which is typical for X fimbriae. E. coli strain 536 synthesizes two different fimbriae (Fim phenotype) prQtein subunits, 16.5 and 22 kilodaltons in size. In addition the strain shows mannose-sensitive hemagglutination and common type I (Fl) fimbriae. The cosmid clone E. coli K-12(pANN801) and another nine independently isolated Mrh+ cosmid clones derived from a cosmid gene bank of strain 536 express the 16.5-kilodalton protein band, bot not the 22-kilodalton protein, indicating an association of the Mrh+ property with the "16.5-kilodalton fimbriae." All cosmid clones were fimbriated, and they reacted with antiserum produced against Mrh+ fimbriae of the E. coli strain HB101(pANN801) and lacked mannose-sensitive hemagglutination (Fl) funbriae. From the Mrh fim cosmid DNA pANN801, several subclones coding for hemagglutination and X fimbriae were constructed. Subclones that express both hemagglutination and fimbriae and subclones that only code for the hemagglutination antigen were isolated; subclones that only produce fimbriae were not detected. By transposon Tn5 mutagenesis we demonstrated that about 6.5 kilobases of DNA is required for the Mrh+ Fim+ phenotype, and the 1.5- to 2-kilobase DNA region coding for the structural proteiil of the fimbriae has been mapped adjacent to the region responsible for the Mrh+ phenotype. Two different regions can thus be distinguished in the adhesion determinant, one coding for hemagglutination and the other coding for fimbria formation. Transformation of plasmid DNA from these subclones into a Mrh- Fim- mutant of E. coli 536 and into a galE (rough) strain of Salmonella typhimurium yielded transformants that expressed both hemagglutination and fimbria production.
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.
Legionella pneumophila, the causative agent of Legionnaires' disease is a facultative intracellular bacterium, which in the course of human infection multiplies in lung macrophages predominantly manifesting as pneumonia. The natural habitat of Legionella is found in sweet water reservoirs and man-made water systems. Virulent L. pneumophila spontaneously convert to an avirulent status at a high frequency. Genetic approaches have led to the identification of various L. pneumophila genes. The mip (macrophage infectivity potentiator) determinant remains at present the sole established virulence factor. The Mip protein exhibits activity of a peptidyl prolyl cis trans isomerase (PPiase), an enzyme which is able to bind the immunosuppressant FK506 and is involved in protein folding. The recently cloned major outer membrane protein (MOMP) could play a role in the uptake of legionellae by macrophages. Cellular models are useful in studying the intracellular replication of legionellae in eukaryotic cells. Human celllines and protozoan models are appropriate for this purpose. By using U 937 macrophage-like cells and Acanthamoeba castellanii as hosts, we could discriminate virulent and avirulent L. pneumophila variants since only the virulent strain was capable of intracellular growth at 37 oc. By using these systems we further demonstrated that a hemolytic factor cloned and characterized in our laboratory, legiolysin (lly), had no influence on the intracellular growth of L. pneumophila.
E. coli strain 536 (06: K15: H31) isolated from a case of acute pyelonephritis, expresses S-fimbrial adhesins, P-related fimbriae, common type I fimbriae, and hemolysins. The respective chromosomally encoded determinants were cloned by constructing a genomic library of this strain. Furthermore, the strain produces the iron uptake substance, enterocheline, damages HeLa cells, and behaves in a serum-resistant mode. Genetic analysis of spontaneously arising non-hemolytic variants revealed that some of the virulence genes were physically linked to large unstable DNA regions, termed "pathogenicity islands", which were mapped in the respective positions on the E. coli K-12linkage map. By comparing the wild type strain and mutants in in vitro and in vivo assays, virulence features have been evaluated. In addition, a regulatory cross talk between adhesin determinants was found for the wild-type isolate. This particular mode of virulence regulation is missing in the mutant strain.
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.
Legionella pneumophila, the causative agent of Legionnaires' disease is able to live and multiply within macrophages as weil as within protozoan organisms. Legionella strains inhibit phagosome-lysosome fusion and phagosome acidification. By using two different cell culture systems, one derived from human macrophages and the other from human.embryo lung fibro:blastic cells, it is demonstrated that Legionella strains lose their virulence following cultivation in the laboratory. In order to study the mechanisms involved in intracellular survival of Legionella a genomic library of strain Legionella pneumophila Philadelphia I was established in Escherichia coli K-12. By cosmid cloning technique we were able to clone five putative virulence factors, two of which exhibit hemolytic activities and three of which represent membrane-associated proteins of 19, 26 and 60 kilodalton. One of the hemolytic proteins, termed legiolysin, represents a new toxin which specifically lyses human erythrocytes. The other hemolysin exhibits proteolytic properties in addition and is cytolytic for Vero and CHO cells. Further sturlies will be necessary to determine the exact role of the cloned proteins in the pathogenesis of Legionella. Zusammenfassung: Intrazelluläres Überleben
The hemolytic Escherichia coli strain 536 (06) propagates spontaneous hemolysin- negative mutants at relatively high rates (10-3 to 10-4 ). One type of mutant (type I) lacks both secreted (external) and periplasmic (internal) hemolysin activity (HlYex - IHlYin -) and in addition shows no mannose-resistant hemagglutination (Mrh -), whereas the other type (type II) is HlYex -IHIYin + and Mrh +. The genetic determinants for hemolysin production (hly) and for mannose-resistant hemagglutination (mrh) of this strain are located on the chromosome. Hybridization experiments with DNA probes specific for various parts of the hly determinant reveal that mutants of type I have lost the total hly determinant, whereas those of type 11 lack only part of the hlyB that is essential for transport of hemolysin across the outer membrane. Using a probe that contains the end sequence of the plasmid pHly152-encoded hly determinant (adjacent to hlyB), we determined that a related sequence flanks also the hlyB-distal end of the chromosomal hly determinant of E. coli 536. In addition several other similar or even identical sequences are found in the vicinity of the hlyC- and the hlyB-distal ends of both the chromosomal and the plasmid hly determinants.
S fimbrial adbesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newbom meningitis. We recently described tbe cloning and molecular cbaracterization of a determinant, termed sftJI, from the chromosome of an E. coli urinary tract infection strain. Herewe present data conceming a S fimbria-specific gene duster, designated sfall, of an E. coli newbom meningitis strain. Like tbe Sfal complex, Sfall consists of tbe major subunit protein SfaA (16 kDa) and the minor subunit proteins SfaG (17 kDa), SfaS (15 kDa), and SfaH (29 kDa). The genes encoding tbe subunit proteins of Sfall were identified and sequenced. Their protein sequences were calculated from the DNA sequences and compared with tbose of the Sfal complex subunits. Altbough the sequences ofthe two major SfaA subunits ditf'ered markedly, tbe sequences ofthe minor subunits sbowed only a few amino acid exchanges (SfaG, SfaH) or were completely identical (SfaS). The introduction of a site-specific mutation into the gene sfaSII and subsequent analysis of an SfaS-negative clone indicated that sfaSII codes for the sialic acid-specific adhesin of tbe meninigitis isolate. These data were confirmed by tbe isolation and characterization of tbe SfaSII protein and the determination of its N-terminal amino acid sequence. The identity between the sialic acid-specific adhesins of Sfal and Sfall revealed that difl'erences between the two Sfa complexes with respect to tbeir capacities to agglutinate erythrocytes must result from sequence alterations of subunit proteins other tban SfaS.
After intraperitoneal injection of mice with Escherichia coli strains isolated from patients with urinary tract infections, the mortality due to hemolytic (Hly+) and nonhemolytic (Hiy-) isolates was 77 and 40%, respectively. Deletion of the chromosomal hemolysin (h/y) determinant in an E. co/i 06:K15:H31 urinary tract infection strain led to a significant reduction in toxicity for mice, and its reintroduction on a recombinant plasmid partially restored the original toxicity. Although introduction of the cloned plasmid pHiy152-encoded hly determinant into the Hly- E. coli 06 mutant strain increased toxicity by only a marginal degree, transformation with the cloned chromosomal hly determinants from two E. coli strains of serotypes 018ac:K5:H- and 075:K95:H? resulted in markedly greater toxicity, even exceeding that of the original Hly+ E. coli 06 wild-type strain.
Like all other Salmonella typhimurium strains examined, the smooth variants SF1397 (L T2) and 1366 and also their semi-rough and rough derivatives are non-haemolytic. Nevertheless, two haemolysin (Hly) plasmids of E. coli belonging to the inc groups incFllI,lv (pSU316) and incIz (pHly152) were able to be introduced into these strains by conjugation and stably maintained. A considerable percentage of the Hly+ transconjugants obtained had lost parts of their O-side chains, a result of selection for the better recipient capability of « semi-rough» variants rather than the direct influence of the Hly+ plasmids themselves. In contrast to the incF1lI1V plasmid pSU316, which exhibited higher conjugation rates with rough recipients, the incIz plasmid pHly152 was accepted best by smooth strains. Transformation with cloned E. coli haemolysin (hly) determinant was inefficient ( <10-8) for smooth strains, but 102-103 times higher for rough recipients, and was increased by the use of Salmonella-modified DNA. The transform ants and transconjugants were relatively stable and showed the same haemolytic activity as the E. coli donor strains. The virulence of the Hly+ smooth, semi-rough and rough S. typhimurium strains was tested in two mouse models, and neither the mortality rate nor the ability to multiply within the mouse spleen was influenced by the hly determinants.
The virulence of the uropathogenic E. coli strain 536 (06: K 1 5: H31) which produces the S-fimbrial adhesin (Sfa•), is serum-resistant (Sre+) and hemolytic (Hiy+) and its derivatives were assessed in five different animal models. Cloned hemolysin (h/y) determinants from the Chromosomes of 06,018 and 075 E. colistrains and from the plasmid pHiy152 were introduced into the spontaneaus Sfa-, Sre-, Hly- mutant 536-21 and its Sfa+, Sre+, Hly- variant 536-31. As already demonstrated for the 536-21 strains {lnfect. Immun. 42: 57-63) the 018-hly determinant but not the plasmid-encoded hly determinant of pHiy 1 52 transformed into 536-31 contribute to lethality in a mouse peritonitis modal. Similar results were obtained with both Hlyhost strains and their Hly+ transformants in a chicken embryo test and in a mouse nephropathogenicity assay in which the renal bacterial counts were measured 1 5 min to 8 hours after i.v. infection. S-fimbriae and serum resistance had only a marginal influence in these three in vivo systems. ln centrast all three factors, S-fimbriae, serum resistance and hemolysin, were necessary for full virulence in a respiratory mouse infection assay. ln a subcutaneously-induced sepsis model in the mouse restoration of S-fimbriae and serum resistance and separately chromosomally-encoded hemolysis increased virulence to a Ievel comparable to that of the parental 536 strain.
Role of alpha-Hemolysin for the in vitro Phagocytosis and intracellular killing of Escherichia coli
(1989)
The_role of a-hemolysin for the elimination of Eschericbia coli by phagocyres in vitro was investigated using sets of isogenic strains which included wild-type a -hemolyric srrains, derived strains with a reduced production of a-hemolysin and derived nonhemolytic strains. Phagocyrosis and intracellular killing of the bacteria by human blood granulocytes or monocytes were measured using growth inhibition rechniques. a-hemolytic strains were phagocytosed and killed ro a Jesser extent than isogenic strains with a reduced production of o:hemoJysin and isogenic nonhemolytic strains. The results obrained with granulocyres were similar to rhose obtained with monocyres although the elimination of bacteria by monocytes was less than that by granulocytes. These resulcs strongJy suggest that production of ahemolysin is a means by which E. coli counteracrs the activity of phagocytes by injuring these cells with the toxin.
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.
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.
A total of 16 Escherichia coli 06 strains isolated from cases of extraintestinal infections were analysed for the genetic presence and phenotypic expression of fimbrial adhesins ( P, S/FIC, type I), aerobactin and hemolysin. ln addition restriction fragment length polymorphisms (RFLPs) of Xbal-cleaved genomic DNA of seven selected strains, separated by orthogonal field alternation gel electrophoresis {OFAGE) were determined and virulence-associated DNA probes were used for Southern hybridization studies of the Xbal-cleaved genomic DNAs. The virulence characteristics and hybridization patterns obtained differed between the various isolates. ln three isolates hemolysin genes and P fimbrial determinants were located on the same Xbal fragments. Furthermore, multiple copies of FIC determinants (foc) could be detected in two strains. Our data show that the new technique of pulse field electrophoresis tagether with Southern hybridization represents a powerful tool for the genetic analysis of pathogenic bacteria.
We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes 04, 06, 018, and 075, The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further sub cloned either as Sail fragments in pACYC184 or as BamHI-SaLI fragments in a recombinant plasmid (pANN202) containing cistron C (hlye) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure, These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.
Genome analysis of Legionella spp. by orthogonal field alternation gel electrophoresis (OFAGE)
(1990)
Various Legionella isolates from different sources and origins were analysed by orthogonal field alternation gel electrophoresis of Not I cleaved genomic DNA. The genome of L pneumophila Philadelphia I, the original isolate of the epidemics in 1976, exhibits only five Not I fragments. Two virulent derivatives. derived from L pneumophila Philadelphia I. which were obtained by prolonged passage on artificial cuhure media, did not differ from their isogenic virulent strain according the Not I fragment pattern. By summing the lengths of the Notl fragments, the genome size of L. pneumophila Philadelphia I was calculated as approximately 3.9 Mb. Environmental L pneumophila strains exhibited different Not I pattems, as did Legionella strains not belongi'ng to the species pneumophila. The usefulness of DNA long range mapping of Legionella ssp. with Notl for epidemiology and evaluation of their evolutionary rela· tionships is discussed.
The legiolysin gene (lly) cloned from Legionella pneumophila Philadelphia 1 confers the phenotypes of hemolysis and browning of the culture medium. An internal Uy-specific DNA probe was used in Southern hybridizations for the detection of Uy-specific DNA in the genomes of legioneUae and other gram-negative pathogenic bacteria. Under conditi9ns of high stringency, tlie Uy DNA probe specifically reacted with DNA fragments fr9m L. pneumophiüz isolates; by reducing stringency, hybridization was also observed for all other Legionella strains tested. No hybridization occurred with DNAs isolated from bact~ria of other genera. The Uy genewas mapped by pulsed-field gel electrophoresis to the respective genomic Notl fragments of Legionelltz isolates. By using antilegiolysin monospecific polyclonal antibodies in Western blots (immunoblots), Lly proteins could be detected only in L. pneumophila isolates.