Biomedical subjects

S Kathariou

Publications and source records attributed to S Kathariou.

36 records · Page 2Linked to original sources

PubMed1995

Differentiation of epidemic-associated strains of Listeria monocytogenes by restriction fragment length polymorphism in a gene region essential for growth at low temperatures (4 degrees C).

The growth of Listeria monocytogenes in food stored in the cold has often been implicated in outbreaks of listeriosis. Many subtyping schemes have suggested that epidemic-associated strains belong to a unique genetic group. It has not yet been possible, however, to identify molecular or bacteriologic markers unique to epidemic-associated strains. Recently we cloned three genes of L. monocytogenes, ltrA, ltrB, and ltrC, which are essential for growth at low temperatures (4 degrees C). The use of a 1.2-kb PstI fragment derived from ltrB as a probe in Southern blots of HindIII-digested DNA revealed three hybridization patterns: the first (a 5.0-kb band) was observed in strains of serotypes 4b, 1/2b, and 3b; the second (a 3.1-kb band) was seen in strains of serotypes 1/2a, 3a, 1/2c, and 3c; and the third (a 9.5-kb band) was characteristic of epidemic-associated serotype 4b strains. These and other data suggest that probes derived from this gene region that is essential for growth at low temperatures can be useful molecular tools for the subtyping of strains implicated in food-borne listeriosis.

Base Sequence↗

PubMed1995

Repression of motility and flagellin production at 37 degrees C is stronger in Listeria monocytogenes than in the nonpathogenic species Listeria innocua.

Listeria monocytogenes and Listeria innocua differ markedly in virulence but are indistinguishable by classical taxonomic criteria. Both species are actively motile and produce abundant flagellin at 22 degrees C. We have found, however, noticeable differences between L. monocytogenes and L. innocua in motility and flagellin production at 37 degrees C. At this temperature, L. monocytogenes strains were virtually nonmotile and produced little or no detectable flagellin, whereas strains of L. innocua were frequently motile and produced substantial amounts of flagellin. This flagellin was recognized by a Listeria genus-specific monoclonal antibody that also recognized flagellin produced at 22 degrees C. These results suggest differential regulation of flagellin production between L. monocytogenes and L. innocua at 37 degrees C.

Antibodies, Bacterial↗

PubMed1994

Transposon-induced mutants of Listeria monocytogenes incapable of growth at low temperature (4 degrees C).

Transposon mutagenesis was used to pursue the molecular basis of cold resistance in Listeria and its possible involvement in pathogenesis. We have generated transposon-induced mutants of Listeria monocytogenes which were unable to grow at 4 degrees C but grew at 8 degrees C, 22 degrees C and 37 degrees C. The transposon was localized in the same 1.8-kb EcoRI fragment in two independently derived mutants. Mutants which carried single transposon insertions had normal hemolytic activity, motility and invasion of cultured fibroblasts. These results indicate that in L. monocytogenes cold sensitivity can be single transposon insertions and that loss of cold resistance may not interfere with other phenotypes, including invasion in a cell culture model.

Cold Temperature↗

PubMed1994

Monoclonal antibodies with a high degree of specificity for Listeria monocytogenes serotype 4b.

Strains of Listeria monocytogenes serotype 4b account for a large fraction of sporadic listeriosis cases, as well as all major food-borne epidemics attributed to this pathogen. We have identified a set of three monoclonal antibodies which showed a high degree of specificity for strains of L. monocytogenes serotype 4b. Two of these antibodies (c74.33 and c74.180, isotypes immunoglobulin M [IgM] and IgG3, respectively) recognized all serotype 4b strains, whereas antibody c74.22 (isotype IgG1) failed to recognize certain epidemic-associated strains. The corresponding antigens were located on the surface of the bacteria and were expressed following bacterial growth in different media and over a wide range of temperatures (4, 22, and 37 degrees C). Heating L. monocytogenes cells at 80,90, or 100 degrees C abolished reactivity for c74.22 but not for c74.33 MAb. These MAbs were negative for all of the non-Listeria strains tested, including representatives of several gram-negative and gram-positive species. The surface antigen recognized by c74.22 appeared to be associated with the ability of the bacteria to enter (invade) mammalian cells in culture.

Animals↗

PubMed1992

A filamentous-like mutant of Listeria monocytogenes with reduced expression of a 60-kilodalton extracellular protein invades and grows in 3T6 and Caco-2 cells.

We describe a spontaneous rough mutant of Listeria monocytogenes that produces reduced amounts of a 60-kilodalton major extracellular polypeptide (p60) as shown by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and Western blot analysis. The cells of this mutant are filamentous, do not give rise to smooth wild-type colonies, and produce listeriolysin O in amounts equal to that of the wild-type cells, but they show a reduced virulence in the mouse LD50 model and in the Caco-2 tissue culture virulence assay. Light and electron microscopic studies show that this mutant invades and remains filamentous during in vivo growth in both Caco-2 and 3T6 tissue culture monolayers. The reduced virulence of the rough mutant is not due to the inability of its filamentous forms to invade or to grow in nonprofessional phagocytes since invasion and growth of the smooth wild-type and the rough mutants are comparable in both Caco-2 and 3T6 monolayers.

Animals↗

PubMed1991

The type strain(s) of Listeria monocytogenes: a source of continuing difficulties.

The type strain of Listeria monocytogenes differs from wild-type L. monocytogenes strains in more characteristics than just the previously reported deficiency in hemolytic activity and virulence in the murine infection model. The type strain from the American Type Culture Collection (strain ATCC 15313) produces lecithinase, is hemolytic on rabbit (but not sheep) blood agar, lacks motility, and shows limited cytopathogenic effects on Caco-2 monolayers, whereas the type strain from the Special Listeria Culture Collection (strain SLCC 53) is unable to produce lecithinase, is nonhemolytic on rabbit or sheep blood agar, is motile, and shows no cytopathogenic effects on Caco-2 monolayers.

Hemolysis↗

PubMed1991

Insertion of Tn916 in Neisseria meningitidis resulting in loss of group B capsular polysaccharide.

We recently found that the 16.4-kb conjugative transposon Tn916 could be introduced into Neisseria meningitidis by transformation and that it appeared to transpose to many different sites in the chromosome of recipient meningococci. In order to identify transposon-induced alterations of specific meningococcal virulence determinants, a library of meningococcal Tetr transformants containing Tn916 was made and screened for those altered in the production of group B capsular polysaccharide. A capsule-defective mutant, M7, was identified by using monoclonal and polyclonal antisera to group B polysaccharide in immunoblot and agar antiserum procedures. Growth of M7 was similar to that of the parent strain. M7 produced no group B capsular polysaccharide by rocket immunoelectrophoresis, and the mutation was stable during laboratory passage. The capsule-defective phenotype was linked to Tetr, as demonstrated by immunoblot and Southern blot analysis of progeny Tetr transformants (transformants of the parent strain obtained with DNA from M7). A capsule-deficient mutant, O8, was identified by using a similar approach. Analysis of the Tn916 insertions in M7 and O8 indicated that a significant portion of the transposon on either side of the tetM determinant had been lost. The ability of Tn916 to generate defined, stable mutations in meningococcal virulence determinants is demonstrated by our study.

Antigens, Bacterial↗

PubMed1991

Cytopathogenic effects in enterocytelike Caco-2 cells differentiate virulent from avirulent Listeria strains.

We have developed a simple test that differentiates between virulent and avirulent Listeria species as defined by the mouse 50% lethal doses (LD50S). The assay is based on trypan blue-revealed cytopathogenic effects that are produced during the infection of the human enterocytelike cell line Caco-2. These effects were elicited only by Listeria strains that had an intraperitoneal mouse LD50 less than 10(8) and were not produced by nonhemolytic, avirulent strains of Listeria monocytogenes generated spontaneously or by Tn916 mutagenesis or by avirulent Listeria species. A negative test was also obtained with hemolysin-producing, avirulent L. monocyotogenes NCTC5105 and Listeria ivanovii KC1786. The test was negative with avirulent L. monocytogenes strains which are strong inducers of opacity in egg yolk agar. However, a strain which has a low LD50, such as 10(4), may show less severe cytopathogenic effects than a strain having a higher LD50, such as 10(6). The test has been effectively used to screen for virulent listerial isolates, spontaneous mutants, and transposon-induced mutants.

Animals↗

PubMed1990

Transposition of Tn916 to different sites in the chromosome of Neisseria meningitidis: a genetic tool for meningococcal mutagenesis.

The difficulty in obtaining mutants in pathogenic Neisseria has limited the ability to genetically define determinants responsible for virulence as well as the ability to generate a genetic map. We show that the 16.5kb conjugative transposon Tn916 can be introduced into Neisseria meningitidis on the suicide vectors pAM120 and pAM170. After introduction, Tn916 transposed to different sites in the chromosome of recipient meningococci, apparently at random, and was stably incorporated. Following its integration into the meningococcal chromosome, Tn916 did not appear to readily express its conjugative and transpositional functions. However, chromosomal DNA from Tn916-carrying meningococci could be used to transform other meningococcal strains to tetracycline resistance. These studies indicate that Tn916 may be an important tool for genetic analysis of N. meningitidis.

Chromosomes↗

PubMed1990

Nonhemolytic Listeria monocytogenes mutants that are also noninvasive for mammalian cells in culture: evidence for coordinate regulation of virulence.

We identified nonhemolytic mutants of Listeria monocytogenes that were severely deficient in their ability to invade mammalian nonprofessional phagocytes. These mutants were generated spontaneously or by means of transposon Tn916 mutagenesis. In terms of their extracellular proteins, the noninvasive mutants were deficient not only in the sulfhydryl-activated hemolysin (listeriolysin) but also in an antigenically unrelated extracellular protein with an apparent molecular weight of 32,000 which could induce opacity in egg yolk and is considered to be a phospholipase. Our results suggest the existence of a common genetic control between the expression of listeriolysin and that of other determinants, including a phospholipase and determinants involved in the ability of L. monocytogenes to enter mammalian cells.

Animals↗

PubMed1988

Hemolysin from Listeria--biochemistry, genetics and function in pathogenesis.

Thiol-activated hemolysins (listeriolysins) from Listeria monocytogenes (Sv4b) and Listeria ivanovii were purified to homogeneity. The N-terminal amino acid sequences of the 58 kDa listeriolysin of L. ivanovii and of a 24 kDa protein which may represent the CAMP-factor of L. ivanovii were determined. Antibodies raised against the L. ivanovii listeriolysin and anti-streptolysin O antibodies were used in Western blot analyses to detect listeriolysin(s) in virulent and avirulent Listeria strains. It was found that all virulent strains of L. monocytogenes synthesize and secrete listeriolysin (Mr 58-59 kDa), albeit in significantly variable quantities. No protein cross-reaction with anti-listeriolysin antibodies or anti-streptolysin O-antibodies was present in the supernatant of Listeria innocua, Listeria welshimeri, Listeria grayi and Listeria murrayi strains. Furthermore, the avirulent but hemolytic Listeria seeligeri did not cross-react with these antibodies. In a L. monocytogenes (strain EGD) gene bank constructed in Escherichia coli two types of hemolytic clones were identified. The first type carried recombinant plasmids with a common 2.0 kb fragment coding for a 23 kDa protein. This hemolytic activity was not activated by DTT and the 23 kDa protein did not cross react with anti-listeriolysin or anti-streptolysin antibodies. The other type of hemolytic clones was detected by using anti-streptolysin O antibodies to screen the gene bank. Some of these clones synthesized a protein of 61 kDa which cross reacted with anti-streptolysin O (or anti-listeriolysin) antibodies. By transposon Tn916 mutagenesis of L. monocytogenes two types of nonhemolytic mutants were obtained. Type I produced no extracellular protein that cross reacted with anti-listeriolysin (or anti SLO) antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

Inhibitory effect of tetracycline and doxycycline on resistance of mice to infection with a tetracycline-resistant strain of Listeria monocytogenes.

By transposon mutagenesis a tetracycline-susceptible strain of Listeria monocytogenes (MIC 1 mg/l. for tetracycline and 0.25 mg/l. for doxycycline) was rendered resistant (MIC 64 mg/l. for tetracycline and 16 mg/l. for doxycycline). Infection of mice with this resistant strain led to an acute infection. Treatment with 2 x 2 mg tetracycline per day did not influence the course of infection during the first 3 days, indicating that the nonspecific resistance, mediated mainly by macrophages and granulocytes, was not affected by this treatment. The second phase of infection, characterized by a continuous resistance to infection due to macrophages activated by T-lymphocytes was, however, definitely hampered. Even acquired immunity to a secondary infection was impaired by treatment with tetracycline, indicating that cell-mediated immunity can be blocked. The course of infection of athymic, nude mice which are unable to build up a cell-mediated immune response, was not affected by tetracycline treatment. Doxycycline expressed the same activities as tetracycline.

Animals↗

PubMed1988

Hemolysin supports survival but not entry of the intracellular bacterium Listeria monocytogenes.

The gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen. The only known property of L. monocytogenes which has been shown to be involved in virulence is a hemolysin, listeriolysin (J. L. Gaillard, P. Berche, and P. Sansonetti, Infect. Immun. 52:50-55, 1986; S. Kathariou, P. Metz, H. Hof, and W. Goebel, J. Bacteriol. 169:1291-1297, 1987). Using our previously obtained transposon Tn916-induced hemolysin-negative mutants of L. monocytogenes Sv1/2a (Mackaness strain), we demonstrated that the loss of hemolysin reduced significantly the rate of survival of the bacteria in mouse peritoneal macrophages but did not reduce their uptake. It was further shown that virulent L. monocytogenes strains could invade the mouse embryo fibroblast 3T6 cell line, i.e., mammalian cells which are nonprofessional phagocytes. This uptake was inhibited by cytochalasin B and hence seems to be accomplished by parasite-induced endocytosis. Hemolysin was not essential for this step. Strains of other Listeria species could not efficiently penetrate the 3T6 cells.

Animals↗

PubMed1988

Levels of Listeria monocytogenes hemolysin are not directly proportional to virulence in experimental infections of mice.

The sulfhydryl-activated hemolysin of Listeria monocytogenes has been implicated in the virulence of the bacteria. Although loss of hemolytic activity by means of transposon mutagenesis is accompanied by loss of virulence in the mouse infection model, a direct relationship between in vitro production of hemolysin and virulence was not observed. Noticeable deviations in the extent of hemolysin production appeared to leave virulence unaffected.

Animals↗

PubMed1987

Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes.

A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.

DNA Transposable Elements↗

PubMed1987

Listeria monocytogenes ATCC 35152 and NCTC 7973 contain a nonhemolytic, nonvirulent variant.

Listeria monocytogenes NCTC 7973 and this same strain deposited as ATCC 35152 contain two phenotypes: hemolytic virulent colonies and nonvirulent colonies that show no zones of hemolysis when streaked on heart infusion agar containing 5% rabbit blood. Results of examinations of these virulent and nonvirulent strains by investigators at the Centers for Disease Control, Atlanta, Ga., the Pasteur Institute, Paris, France, and the University of Würzburg, Federal Republic of Germany, support the conclusion that the avirulent strain is a nonhemolytic mutant of the virulent strain and that hemolysin is a virulence factor for L. monocytogenes.

Animals↗

PubMed1983

Chemoattractants elicit methylation of specific polypeptides in Spirochaeta aurantia.

On the basis of this investigation, chemotaxis in Spirochaeta aurantia correlates with methylation of specific polypeptides which are presumed to be analogous to the methyl-accepting chemotaxis proteins (MCPs) in bacteria such as Escherichia coli. The polypeptides exhibited apparent molecular weights in the range of 55,000 to 65,000. Generally, two major presumptive MCP bands and three minor bands were observed on sodium dodecyl sulfate-polyacrylamide gels. Upon addition of D-glucose to S. aurantia cells, methylation of the presumptive MCPs increased for 10 to 12 min to a level greater than 4 times the level of methylation in the absence of D-glucose. Removal of D-glucose resulted in a decrease in methylation of the presumptive MCPs to a level similar to that in unstimulated cells. All attractants tested, including a non-metabolizable attractant (alpha-methyl-D-glucoside) stimulated methylation of the presumptive MCPs (from 1.7 to 4.3 times the level of methylation in unstimulated cells). D-Mannitol, a metabolizable sugar which is not an attractant for S. aurantia, did not stimulate methylation. Stimulation of methylation by D-galactose occurred in cells induced for D-galactose taxis but not in uninduced cells. These data are indicative of an evolutionary relationship between the chemotaxis systems of spirochetes and of flagellated bacteria.

Bacterial Proteins↗

PubMedDate not supplied

Molecular analysis of bacterial cytolysins.

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 hyl determinants of one nephropathogenic E. coli strain revealed the existence of specific, large chromosomal insertions 75 kb and 100 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.

Aeromonas↗