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PubMed1990

Secretion of the Bordetella pertussis adenylate cyclase from Escherichia coli containing the hemolysin operon.

The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H.R., & Storm, D.R. (1989) biochemistry 28, 438-442]. The gene encoding this enzyme, cyaA, is part of the cya operon that also includes the genes cyaB, cyaD, and cyaE. A comparison of the predicted amino acid sequences encoded by cyaA, cyaB, and cyaD with the amino acid sequences encoded by hlyA, hlyB, and hlyD genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997-4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of HlyA (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, CyaB, CyaD, and CyaE are required for the secretion of CyaA from Bordetella pertussis. We placed the cyaA gene and a truncated cyaA gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cyaA gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1994

Binding to and killing of human renal epithelial cells by hemolytic P-fimbriated E. coli.

Acute pyelonephritis is a common invasive infection frequently caused by E. coli that possess P-fimbriae and secrete hemolysin. We have examined the role of P fimbriae and hemolysin in the killing of putative target cells of acute pyelonephritis, that is, human renal epithelial cells (HRPTEC). Cultures of HRPTEC were overlaid with (1) a prototypic pyelonephritogenic E. coli (CFT073) which expresses both P fimbriae and hemolysin; (2) its hemolysin-negative isogenic mutant (CFT073hlyD::TnphoA); or (3) a prototypic nonpyelonephritogenic fecal E. coli (FN414) which is negative for both P fimbriae and hemolysin. CFT073 and CFT073hlyD::TnphoA but not FN414 adhered to HRPTEC, as demonstrated by electron microscopy and direct counting. Adherence was diminished by antisera directed against P fimbriae and by a monoclonal antibody recognizing the epithelial receptor for P fimbriae. CFT073 was significantly more cytolethal for HRPTEC than its hemolysin-negative mutant. The bacteria-free filtrate of CFT073 was both hemolytic and cytolethal whereas that of CFT073hyD::TnphoA was not hemolytic and was significantly less cytolethal. Finally, we demonstrated that CFT073 passed through monolayers of HRPTEC at a higher rate than CFT073hlyD::TnphoA, indicating that hemolysin damages HRPTEC, facilitating passage of bacteria through the epithelial barrier. With HRPTEC and a pyelonephritogenic strain of E. coli we have reproduced in vitro bacterial attachment and toxin delivery by P fimbriae and hemolysin, factors epidemiologically associated with acute pyelonephritis in patients.

Bacterial Adhesion↗
PubMed1989

Cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae.

Neutralizing antisera to the leukotoxin secreted by Pasteurella haemolytica neutralized the hemolysin of Actinobacillus pleuropneumoniae and recognized a 110-kD antigen in cell-free culture supernatants from this organism. A series of nine overlapping recombinant phage clones carrying the gene for this 110-kD antigen were identified using affinity-purified anti-hemolysin antibody and a DNA probe containing sequences from the P. haemolytica lktCA genes. Eight of the nine clones expressed a 110-kD protein recognized by both anti-leukotoxin and anti-hemolysin antisera. The remaining clone expressed a truncated 80-kD antigen which was also recognized by both antisera. Sequence analysis of a region of the cloned DNA revealed two open reading frames encoding proteins with predicted masses of 18.5 and 102.5 kD. These genes, which we designate appC and appA, respectively, are similar in sequence to the hlyCA genes of Escherichia coli and the lktCA genes of P. haemolytica. Hemolytic activity could be detected in lysates of E. coli harboring plasmids containing the appCa genes.

Actinobacillus↗
PubMed2001

Comparative genetic characterization of Listeria monocytogenes isolates from human and animal listeriosis cases.

Listeria monocytogenes isolates from human sporadic and epidemic cases (n=119) and from animal cases (n=76) were characterized by automated ribotyping and PCR-restriction fragment length polymorphism (PCR-RFLP) typing of the virulence genes actA and hly. This combination of typing methods differentiated 39 distinctive strains, each reflecting a unique combination of ribotypes, hly and actA alleles. Simpson's index of discrimination indicated a high discriminatory ability of ribotyping for both animal (0.867) and human isolates (0.857), which was further increased by the addition of hly and actA typing (0.916 and 0.904, respectively). Ribotype and hly allele data were further used to group isolates into three genetically distinct lineages. Each lineage is composed of several ribotype fragment subsets, each of which contains multiple ribotypes characterized by common ribotype fragments. To determine whether certain clones of L. monocytogenes show indications for unique pathogenic potential or host specificity, frequency distributions for five genetic characteristics (i.e. lineage, ribotype, ribotype fragment subset and hly and actA allele) were calculated for isolates from animal cases, human epidemic cases and human sporadic cases. Lineage III isolates were found less frequently in human cases (1 of 119 isolates) than in animal cases (8 of 76 isolates; P=0.003). These results suggest the possibility of host specificity for non-primate mammals among lineage III strains. In addition, lineage I strains were found more frequently among human cases than among animal cases (P<0.001). Among the eight hly alleles observed, hly allele 1 was more common among human isolates as compared to animal isolates (P=0.002). We also identified one ribotype (DUP-1030) which was significantly more common among animal isolates (P=0.005) and one ribotype (DUP-1038; lineage I) which was significantly more common among human epidemic isolates as compared to human sporadic isolates (P<0.001). These findings confirm the presence of clonal groups of L. monocytogenes, which appear to be characterized by unique virulence or host specificity patterns. This study also establishes baseline data describing the genetic diversity of human and animal L. monocytogenes isolates which can be utilized in future surveillance programmes to track the emergence of new strains.

Alleles↗
PubMed1989

Specificity of insertion of IS91, an insertion sequence present in alpha-haemolysin plasmids of Escherichia coli.

We have determined the DNA sequences of eight different insertions of IS91 in a specifically engineered recipient plasmid of known DNA sequence (pSU300). The sequences at the termini of IS91 are 5'-CGAGTAGG...CCTATCGAT. IS91 inserts specifically 5' to either one of the tetranucleotides 5'-GAAC or 5'-CAAG, and always in the same relative orientation with respect to the sequence of the target. Except in one special case, no duplications of the recipient DNA were produced at the site of insertion.

Bacterial Proteins↗
PubMed1983

Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice.

After intraperitoneal injection of mice with Escherichia coli strains isolated from patients with urinary tract infections, the mortality due to hemolytic (Hly+) and nonhemolytic (Hly-) isolates was 77 and 40%, respectively. Deletion of the chromosomal hemolysin (hly) determinant in an E. coli O6: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 pHly152-encoded hly determinant into the Hly- E. coli O6 mutant strain increased toxicity by only a marginal degree, transformation with the cloned chromosomal hly determinants from two E. coli strains of serotypes O18ac:K5:H- and O75:K95:H? resulted in markedly greater toxicity, even exceeding that of the original Hly+ E. coli O6 wild-type strain.

Animals↗
PubMed1980

Hemolysis determinant common to Escherichia coli hemolytic plasmids of different incompatibility groups.

By using cloned deoxyribonucleic acid fragments from the hemolysis determinant of the hemolytic plasmid pHly152 as hybridization probes, a deoxyribonucleic acid segment of about 3.8 megadaltons was identified as a common sequence in several hemolytic (Hly) plasmids of Escherichia coli belonging in four different incompatibility groups. This segment contained the genetic information for the synthesis and secretion of the extracellular toxin alpha-hemolysin of E. coli. With the exception of pSU5, representing a composite plasmid, one part of which seems to be very similar to pHly152, the overall sequence homology of these Hly plasmids with pHly152 seems to be rather restricted. However, the Hly plasmid pSU316 showed sequence homology with pHly152 that did not extend beyond the hemolysis determinant. The two other plasmids, pSU233 and pSU105, also shared homology with pHly152 in the hemolysis determinant as well as in various other parts of this plasmid which did not seem to be directly linked to the hemolysis determinant. This suggests that the hemolysis determinant has spread to presumably unrelated plasmids of E. coli.

Base Sequence↗
PubMed1981

Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli.

The alpha-hemolytic Escherichia coli strain PM152 harbors three transmissible plasmids, which have molecular weights of 65 X 10(6) (pA152), 41 X 10(6) pHly152), and 32 X 10(6) (pC152). Plasmids pHly152 and pC152 belong to incompatibility groups J2 and N, respectively. By transforming E. coli K-12 with isolated plasmids, we showed that the genetic determinant required for hemolysis was located entirely on plasmid pHly152, and a physical map of this plasmid was constructed. By transposon mutagenesis, a deoxyribonucleic acid segment of about 3.5 X 10(6) daltons was identified as being essential for hemolysis. Most of the EcoRI and HindIII fragments of the hemolytic plasmid pHly152 were cloned by using pACYC184 and RSF2124 as vectors. Two classes of Tn3-induced hemolysis-negative mutants could be complemented by recombinant plasmids carrying fragments from the hemolysis region of pHly152, whereas a third class could be restored to hemolytic activity only by recombination between the mutant plasmids and a suitable recombinant deoxyribonucleic acid. These data suggest that there are at least three clustered cistrons which are required for hemolysis. Other EcoRI and HindIII fragments of pHly152 were identified as being essential for replication, incompatibility, transfer, and restriction.

Cloning, Molecular↗
PubMed1982

Analysis of hemolytic determinants of plasmid pHly185 by Tn5 mutagenesis.

A nonconjugative hemolysin plasmid, pHly185, was isolated from Escherichia coli 185. Tn5 mutagenesis produced nonhemolytic derivatives that either expressed no hemolytic activity or did not secrete activity. These Tn5 insertions were located in three contiguous EcoRI fragments. Insertions inactivating hemolysin structural gene(s) were identified on all three EcoRI fragments. Mutations affecting the secretory function were clustered in one portion of a single EcoRI fragment.

Bacteriophage lambda↗
PubMed1983

Spontaneous deletions and flanking regions of the chromosomally inherited hemolysin determinant of an Escherichia coli O6 strain.

The hemolytic Escherichia coli strain 536 (O6) 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-/Hlyin-) and in addition shows no mannose-resistant hemagglutination (Mrh-), whereas the other type (type II) is Hlyex-/Hlyin+ 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 II 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.

Base Sequence↗
PubMed1998

A homolog of CcpA mediates catabolite control in Listeria monocytogenes but not carbon source regulation of virulence genes.

Readily utilizable sugars down-regulate virulence gene expression in Listeria monocytogenes, which has led to the proposal that this regulation may be an aspect of global catabolite regulation (CR). We recently demonstrated that the metabolic enzyme alpha-glucosidase is under CR in L. monocytogenes. Here, we report the cloning and characterization from L. monocytogenes of an apparent ortholog of ccpA, which encodes an important mediator of CR in several low-G+C-content gram-positive bacteria. L. monocytogenes ccpA (ccpALm) is predicted to encode a 335-amino-acid protein with nearly 65% identity to the gene product of Bacillus subtilis ccpA (ccpABs). Southern blot analysis with a probe derived from ccpALm revealed a single strongly hybridizing band and also a second band of much lower intensity, suggesting that there may be other closely related sequences in the L. monocytogenes chromosome, as is the case in B. subtilis. Disruption of ccpALm resulted in the inability of the mutant to grow on glucose-containing minimal medium or increase its growth rate in the presence of preferred sugars, and it completely eliminated CR of alpha-glucosidase activity in liquid medium. However, alpha-glucosidase activity was only partially relieved from CR on solid medium. These results suggest that ccpA is an important element of carbon source regulation in L. monocytogenes. Nevertheless, utilizable sugars still down-regulate the expression of hly, which encodes the virulence factor hemolysin, in a ccpALm mutant, indicating that CcpA is not involved in carbon source regulation of virulence genes.

Amino Acid Sequence↗
PubMed1988

Active and inactive forms of hemolysin (HlyA) from Escherichia coli.

The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W. & Hedgpeth, J. (1982) J. Bacteriol. 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle. However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase. External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication. The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished. On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA. Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes. Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein. An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product. This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase.

Chromatography↗
PubMedDate not supplied

Virulence factors of avian pathogenic Escherichia coli isolated from broilers from the south of Brazil.

Sixty-three Escherichia coli strains isolated from broilers with respiratory problems were examined for virulence factors, hemolysin synthesis ability, motility, hemagglutination capacity, operon pap presence, colicin production, and serum resistance. The capacity to hemagglutinate guinea pig erythrocytes was found in 53 (84.1%) of the samples, but only 30 (47.6%) agglutinated chicken erythrocytes. D-mannose-sensitive hemagglutination against guinea pig erythrocytes was found in 19 (30.2%) samples and against chicken erythrocytes, in 15 (23.8%) samples, whereas the D-mannose-resistant hemagglutination with guinea pig erythrocytes was found in 34 (54%) samples, and 13 of these (20.6%) showed this characteristic against chicken erythrocytes. Operon pap, P fimbria codifier, was detected in 26 samples in a total of 34 D-mannose-resistant samples. Colicin production was observed in 55 (87.3%) of the strains, and 41.8% presented V colicin production. Of the samples analyzed, 56 (88.9%) presented serum resistance, six (9.5%) were intermediate, and only one (1.6%) was sensitive to the action of the complement. The diversity of virulence profiles detected in the samples in this study explains in part the multifactorial characteristics of avian colibacillosis.

Animals↗
PubMed1991

Clonal distribution of K1 and K5 antigen possessing Escherichia coli isolates.

A significant difference was observed in the occurrence of the examined markers (Col+, ColV+, Hly+, Aer+, AbR) and in the plasmid carrier state between strains with and without K1 and K5 antigens. Plasmids of the same size were harboured by serotypes possessing K1 and K5 antigens, e.g. among O1: K1: H- strains plasmids of 60-79 Md, among O1: K1: H7, O18ac: K1: H7, O45: K1: H7 and O83: K1: H- strains plasmids of 80-95 Md were frequent. The average plasmid number was higher in K1 strains than in K5 strains. In serogroup O1 the frequency of the plasmid carrier state was associated with the O serogroup and not with the K antigen. The plasmid number in K5 of serogroups O6 and O18 was lower than in K5- strains. Plasmids of 80-95 Md were predominant among the strains derived from blood and cerebrospinal fluid, whereas these plasmids were rare among the K1 and K5 strains isolated from other sources. Plasmids of 60-79 Md were frequent among strains derived from different sources. The 30-40 Md plasmids were relatively frequent among strains isolated from urine. In contrast with literary data, O1: K1: H-, O1: K1: H7 and other frequent serotypes consisted of different clones. Different clones were found within a single serotype, too.

Anti-Bacterial Agents↗
PubMed1991

Activator proteins for glycosphingolipid hydrolysis by endoglycoceramidases. Elucidation of biological functions of cell-surface glycosphingolipids in situ by endoglycoceramidases made possible using these activator proteins.

Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). Recently, by extensive purification, it was separated from cell-lytic factor (hemolysin) and found to consist of three molecular species each with its own specificity (EGCases I, II, and III) (Ito, M., and Yamagata, T. (1989) J. Biol. Chem. 264, 9510-9519). A detergent was required for EGCases to express full activity, possibly due to their hydrophobic nature, and thus EGCases cannot be used for research on live cells. This paper presents findings on activator proteins in the culture supernatant of Rhodococcus sp. M-777 regarding the stimulation of EGCase activity in the absence of detergents. The activator protein, exhaustively purified and designated as activator II in this study, showed a single protein band on sodium dodecyl sulfate-, native-, and isoelectrofocussing-polyacrylamide slab gel electrophoresis after being stained with Coomassie Brilliant Blue. Its molecular weight and pI were 69,200 and 4.0, respectively. The activator protein enhanced the hydrolysis of glycosphingolipids in vitro and on the cell-surface by EGCase II in the absence of detergents in a concentration-dependent manner. Interestingly, activator II stimulated the activity of EGCase II much more than that of EGCase I on using asialo-GM1 as the substrate. This activator protein was found nonspecific to substrates susceptible to hydrolysis with EGCase II. Besides activator II, strain M-777 produced a second minor molecular species of activator protein designated as activator I which appeared specific for stimulating the activity of EGCase I in contrast to activator II. Following the addition of activator II, EGCase II hydrolyzed cell-surface glycosphingolipids quite efficiently at neutral pH at which hydrolysis hardly occurred at all in its absence. When using activator II in place of Triton X-100 for stimulating EGCase II activity, it was also noted to cause no damage to intact cells. It is thus possible by activator proteins to elucidate the biological functions of endogenous glycosphingolipids in situ by EGCases.

Amino Acid Sequence↗
PubMed1990

[Distribution and transcription of determinants homologous to the alpha-hemolysin (hlY) gene in the genomes of HLY- and HLY+ staphylococci].

The molecular organization of the alpha-hemolysin gene from Staphylococcus aureus strain O15 has been studied. Hybridization of the DNA from this strain with the [32P]-labeled cloned fragment containing alpha-hemolysin gene has shown the studied gene to be unique in the genome of the strain. Sequences homologous to the gene were not found to be dispersed along the genome of Staphylococcus aureus O15. The presence of alpha-hemolysin gene in some Staphylococcus epidermidis strains has been monitored. The strains 1413, 303 have been demonstrated to contain no gene for alpha-hemolysin. The hemolysin genes were found in the genomes of the strains 159, 169, 180 by DNA hybridization technique. In the genome of the strain 180 the long region including the right end of alpha-hemolysin gene is deleted. Hybridization with the net RNA of these strains shows the absence of transcription of intact as well as deleted alpha-hemolysin genes.

Cloning, Molecular↗