Growth in and breakdown of purified rabbit small intestinal mucin by Yersinia enterocolitica.

The mucus lining of the gastrointestinal tract serves as a protective barrier over the epithelial surface that must be crossed by invading bacteria seeking entry into the mucosa. The gel-forming component of mucus is mucin, a large polymeric glycoprotein. The present study examined the growth of Yersinia enterocolitica (with and without its virulence plasmid) in purified rabbit small intestinal mucin and the ability of bacteria to degrade mucin. Both virulent and nonvirulent organisms showed enhanced growth in mucin-supplemented media compared with unsupplemented media, but only at 37 degrees C and not at 25 degrees C. The effects of mucin were not specific because medium supplemented with bovine serum albumin also enhanced bacterial growth at 37 degrees C. Purified mucin was broken down into lower-molecular-weight components (assessed by monitoring its elution profile on a Sepharose CL-2B column) by plasmid-bearing Y. enterocolitica but not by plasmid-cured organisms. Culturing virulent Y. enterocolitica at 25 degrees C completely suppressed its capacity to degrade mucin, suggesting that this activity depends on plasmid expression. These results were confirmed in similar studies with purified rabbit colonic mucin. Mucin-degrading activity could be demonstrated in spent culture media from virulent Y. enterocolitica incubated at 37 degrees C but not in bacterial membrane preparations. Changes in the elution profiles of small intestinal and colonic mucins exposed to plasmid-bearing Y. enterocolitica at 37 degrees C were consistent with proteolytic depolymerization. The ability to grow well in mucin may help Y. enterocolitica to colonize the intestine, while the production of a mucin-degrading enzyme(s) by plasmid-bearing organisms may assist pathogenic strains to solubilize and penetrate the mucus gel layer.     

Genetic analysis of virulence plasmid from a serogroup 9 Yersinia enterocolitica strain: role of outer membrane protein P1 in resistance to human serum and autoagglutination

Enteropathogenic strains of Yersinia enterocolitica harbor a virulence plasmid (70 kilobases) which specifies, at 37 degrees C, a calcium requirement for growth, autoagglutinability, resistance to the bactericidal activity of human serum, and the expression of some outer membrane proteins (OMPs). To map the genes encoding these properties, the virulence plasmid of a serogroup 9 strain (W22708) was subjected to transposon mutagenesis. A set of 68 independent mutations was obtained in Escherichia coli by transposon Tn813 (a tnpR mutant of Tn21)-mediated cointegration with the self-transmissible R388 plasmid. The resulting cointegrates were introduced and studied in Y. enterocolitica W22708. One mutant lost the calcium dependence property. Two other mutants presented a peculiar phenotype: they grew poorly at 37 degrees C, especially in the presence of calcium. Lastly, two mutants were affected in the properties of autoagglutination and resistance to human serum. Analysis of the OMP pattern of these two mutants revealed the absence of the largest OMP, called P1 (I. B?lin, and H. Wolf-Watz, Infect. Immun. 43:72-78, 1984). Complementation of one of these mutations with the cloned structural gene of OMP P1 restored the wild-type phenotype. However, OMP P1 was not sufficient by itself to specify the serum resistance property and a rapid autoagglutination of the host.     

Production of bacteriocin-like antagonism by clinical isolates of Yersinia enterocolitica.

Fourteen clinical isolates of Yersinia enterocolitica serotype O:3 and four well-documented virulent strains of serotypes O:3, O:8, and O:9 were biotyped and examined for plasmid-associated autoagglutination and calcium dependency and for epithelial cell adherence. These strains were tested for the production of bacteriocin-like antagonism by using tryptone soya blood agar at room temperature and at 37 degrees C. By using the cross-streaking method, three clinical isolates produced inhibitory substances at room temperature. These substances were active against a variety of clinical isolates and their plasmid-cured derivatives at both room temperature and 37 degrees C. The inhibition was easier to read after incubation of the cross-streaked plate at 37 degrees C. The inhibition patterns indicate that two of the three producer strains appear to recognize potentially virulent O:3 strains, with or without the virulence plasmid.     

Virulence factors of Haemophilus ducreyi

We investigated the susceptibility of virulent and avirulent strains of Haemophilus ducreyi to the bactericidal activity of normal human serum and to phagocytosis and killing by human polymorphonuclear leukocytes (PMNL). Strains were defined as virulent if intradermal inoculation into a rabbit produced a typical necrotic lesion. Nonvirulent strains produced no cutaneous lesions in rabbits. Virulent strains were resistant to the complement-mediated lethal action of normal human and rabbit sera, whereas avirulent strains were susceptible (greater than 95% kill, 60 min). Virulent strains were relatively resistant to phagocytosis and killing by human PMNL, in contrast to the avirulent strains. In past studies polymyxin resistance has been correlated with virulence in H. ducreyi. In our studies, polymyxin resistance could not be correlated with virulence, since polymyxin-sensitive mutants obtained from polymyxin-resistant parent strains remained virulent for rabbits and resistant to bactericidal action of normal serum and phagocytosis and killing by human PMNL. Similarly, polymyxin-resistant mutants obtained from polymyxin-sensitive parent strains remained avirulent for rabbits and susceptible to bactericidal action of normal serum and PMNL. The acquisition of polymyxin resistance was accompanied by the loss of a 47,000-molecular-weight protein. The association of serum resistance and resistance to phagocytosis and killing by human PMNL with virulent strains, as defined by the rabbit intradermal test, suggests that these factors may mediate the pathogenicity of H. ducreyi.     

Plasmid-associated cell surface charge and hydrophobicity of Yersinia enterocolitica

Virulent strains of Yersinia enterocolitica and their plasmidless, avirulent derivatives were examined for their cell surface properties. Increased surface charge and hydrophobicity of Y. enterocolitica were found to be associated with the possession of a 40- to 48-megadalton plasmid. These surface properties were expressed, as were other plasmid-associated properties, at 37 but not at 22 degrees C. The concentration of calcium in the growth medium had a moderate effect on the expression of the cell surface properties. These cell surface properties were greatly reduced among plasmid-bearing cells grown on tryptic soy agarose regardless of growth temperatures. These properties were also associated with the ability of Y. enterocolitica to colonize the gastrointestinal tract of mice.     

In vitro assessment of virulence in Yersinia enterocolitica and related species.

We have examined 136 isolates of Yersinia species, comprising 112 strains of Yersinia enterocolitica, 12 of Y. frederiksenii, 8 of Y. intermedia, and 5 of Y. kristensenii, for the presence of 40- to 50-megadalton virulence-associated plasmids and expression of the following plasmid-associated characteristics: Congo red pigmentation (CR), calcium dependence, autoagglutination, hydrophobicity, resistance to normal human serum, and pathogenicity in mice. All 136 strains yielded both pigmented (CR+) and nonpigmented (CR-) variants. Only CR+ variants, however, were virulent for iron-overloaded, desferrioxamine B-treated mice (R. M. Robins-Browne and J. K. Prpic, Infect. Immun. 47:744-779, 1985). Although the in vitro virulence-associated characteristics generally occurred together, each one could be expressed independently. Strains of Y. frederiksenii, Y. intermedia, and Y. kristensenii also expressed individual virulence-associated properties. Of 53 Y. enterocolitica strains which were virulent for iron-overloaded, desferrioxamine-treated mice, all but one expressed every virulence-associated characteristic. Several strains which were avirulent for mice, however, demonstrated these characteristics in various combinations. Because many Yersinia strains, particularly environmental isolates, carried plasmids of 40 to 50 megadaltons, detection of plasmids provided little information about bacterial pathogenicity unless virulence-associated properties were also sought. The best in vitro predictor of virulence was autoagglutination, followed by calcium dependence. Because only CR+ variants expressed virulence-associated determinants, Congo red pigmentation is useful for selecting potentially virulent strains.     

Interaction of monoclonal antibodies with pertussis toxin and its subunits

The pathogenicity of Shigella spp. involves the ability of the bacteria to penetrate and replicate within the epithelial cells of the large intestine. Model systems for examining the virulence of shigellae employ Henle intestinal epithelial cells in tissue culture and an in vivo assay for virulence in guinea pig eyes (Sereny test). Using these systems, we studied the genetic and physiological bases for the ability of shigellae to invade epithelial cells. We found that expression of virulence in Shigella spp. is dependent on the temperature at which the bacteria are grown. When grown at 37 degrees C, strains of Shigella flexneri 2a, Shigella sonnei, and Shigella dysenteriae 1 were fully virulent and invaded Henle cells. They also produced keratoconjunctivitis in guinea pigs. When grown at 30 degrees C, the bacteria neither penetrated Henle cells nor produced conjunctivitis in the Sereny test and were phenotypically avirulent. Strains grown at 33 degrees C were only partially invasive in the Henle assay, whereas strains grown at 35 degrees C were as invasive as strains grown at 37 degrees C. Using the Henle cell assay, we determined that the loss of ability to penetrate epithelial cells was completely reversed by shifting the growth temperature from 30 to 37 degrees C. The percentage of Henle cells invaded by bacteria increased with increasing time of growth at 37 degrees C. Restoration of invasiveness after growth at 30 degrees C required protein synthesis. When shigellae were grown at 30 degrees C and shifted to 37 degrees C for 2 h in the presence of chloramphenicol, the bacteria remained noninvasive. Similarly treated bacteria grown at 37 degrees C were still invasive. These results suggested that expression of one or more genes required for virulence of Shigella spp. are subject to regulation by growth temperature.     

Presence of a capsule in Erysipelothrix rhusiopathiae and its relationship to virulence for mice.

Three avirulent insertional mutants of Erysipelothrix rhusiopathiae were obtained by the technique of transposon mutagenesis with the self-conjugative transposon Tn916. The interactions between murine polymorphonuclear leukocytes and parent and mutant strains were studied in vitro. In the presence of normal serum, the virulent parent strain was resistant to phagocytosis, whereas the avirulent mutant strains were efficiently phagocytosed. In the presence of immune serum, the parent and the mutant strains were both efficiently phagocytosed. Electron microscopic examination of the parent strain demonstrated the presence of a structure resembling a capsule which was absent on the mutant strains, suggesting that a capsule may be involved in virulence. This was confirmed in studies in which an avirulent mutant strain reverted to virulence following acquisition of a capsule when the transposon was lost by spontaneous excision. These results strongly suggest that virulence of E. rhusiopathiae is associated, at least in part, with resistance to phagocytosis by polymorphonuclear leukocytes and that this antiphagocytic ability of the bacterium results from its possession of a capsule.     

An enterotoxin-negative strain of Yersinia enterocolitica serotype O:3 is capable of producing diarrhea in mice.

A strain of Yersinia enterocolitica serotype O:3 that consistently produced heat-stable enterotoxin at 22 but not at 37 degrees C and another strain of the same serotype which did not produce enterotoxin at 22 degrees C were both positive for autoagglutination at 35 degrees C, a test that has been related to virulence in yersiniae. Both strains were infective for HeLa cells and produced guinea pig conjunctivitis. Mice infected with either strain through their drinking water developed diarrhea and excreted the organism in high numbers in the feces. A control strain of serotype O:3 positive for enterotoxin and HeLa cell infectivity but negative for autoagglutination was avirulent. Extracts of feces and intestines from mice with diarrhea were negative for enterotoxin. The results indicate that the heat-stable enterotoxin produced in vitro by some strains of Y. enterocolitica and measured by the infant mouse assay plays no role in pathogenesis as described by the mouse diarrhea model.     

Identification of specific outer membrane polypeptides associated with virulent Yersinia enterocolitica

An antiserum (WA-SAA) that agglutinates specifically with mouse virulent but not avirulent strains of Yersinia enterocolitica was used to identify virulence-associated factors by Western blot techniques. Several outer membrane polypeptides were identified only in the virulent strains, which included serotypes O:8, O:3, O:9, O:4,32, O:5,27, and O:21. These included three, and possibly four, major outer membrane polypeptides. The prominent high-molecular-weight species was demonstrated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot, whereas the others were only revealed by the Western blot technique. Expression of these polypeptides correlated with antiserum agglutination reaction and the presence of a 42- and/or 82-megadalton plasmid. These polypeptides were highly temperature dependent and only slightly affected by the inclusion of 10 mM Ca2+ in the growth medium. These polypeptides were produced during both the logarithmic and stationary phases of growth at 37 degrees C. We suggest that the production of these specific polypeptides and calcium dependency may be coded for by the plasmid(s) but are regulated by independent mechanisms. These polypeptides appear to be novel markers specific for virulent strains of Y. enterocolitica and may be important to the pathogenicity of this organism.     

Expression of the temperature-inducible outer membrane proteins of yersiniae.

The expression of the temperature-inducible plasmid-coded outer membrane proteins (YOPs) of Yersinia pseudotuberculosis was studied. These proteins were not recovered in the outer membrane fraction when the strain was grown in minimal medium at 37 degrees C, but they were expressed under these conditions. A strict correlation was found between Ca2+ dependency in the virulent strain, YPIII(pIB1), and ability to express YOPs. Ca2+-independent plasmid mutants or RNA-polymerase mutants harboring the virulence plasmid were unable to express YOPs, in contrast to the wild-type strain. These strains were also found to be avirulent. Sera recovered from patients or animals undergoing infection with either Y. pseudotuberculosis, Y. pestis, or Y. enterocolitica possessed antibodies directed against YOPs, indicating that they were expressed in all three pathogenic Yersinia species during infection. The YOPs of the three different species showed high immunological relatedness.     

Evaluation of DNA colony hybridization and other techniques for detection of virulence in Yersinia species.

The virulence of yersiniae varies according to (i) species and biotype and (ii) possession of a 67- to 72-kilobase virulence plasmid. Y. pestis, Y. pseudotuberculosis, and biotypes 1B, 2, 3, 4, and 5 of Y. enterocolitica are inherently virulent but express full virulence only when in possession of a virulence plasmid. Other Yersinia species and biotypes 1A and 3B of Y. enterocolitica are seldom implicated in disease. In this study, we prepared DNA probes from eight nonoverlapping regions of the virulence plasmid of a strain of Y. enterocolitica and from the inv and ail chromosomal loci responsible for the invasive capacity of Y. enterocolitica and Y. pseudotuberculosis. The probes were used in colony hybridization experiments to investigate 156 yersiniae of various species and biotypes and of differing virulence. Probes prepared from the inv gene of Y. pseudotuberculosis hybridized with Y. pseudotuberculosis and Y. pestis only, whereas an analogous probe prepared from Y. enterocolitica hybridized with all species and biotypes of yersiniae (but not with other bacteria) regardless of virulence or potential virulence. Probes prepared from the ail region of Y. enterocolitica reacted almost exclusively with Y. enterocolitica strains of pathogenic biotypes. Probes prepared from the virulence plasmid of a serogroup O:8, biotype 1B isolate of Y. enterocolitica identified virulent yersiniae in all species with a high degree of sensitivity and specificity. These probes did not react with yersiniae of avirulent biotypes or species. Of the other assays of virulence evaluated (calcium dependence, binding of crystal violet, and pyrazinamidase activity), binding of crystal violet provided a simple means for identifying plasmid-bearing strains.     

Plasmid-mediated and temperature-regulated surface properties of Yersinia enterocolitica.

Enteropathogenic strains of Yersinia enterocolitica harbor a virulence plasmid which codes for a series of novel outer membrane proteins. The expression of these proteins on the outer membrane is temperature regulated: when cells are grown at 25 degrees C, these proteins are not exposed on the outer membrane, whereas they occur in high copy number when cells are grown at 37 degrees C. The majority of these proteins are externally exposed on the cell surface as evidenced by their susceptibility to proteolysis by exogenously added proteases. The expression of the plasmid-mediated proteins on the outer membrane does not favor adherence of the bacteria to intestinal epithelial cells in vitro. Cultures grown at 25 degrees C adhered to Henle cell monolayers, whereas those grown at 37 degrees C did so much less effectively. The presence of the proteins on the bacterial surface appears to be involved in rendering the cells resistant to the bactericidal effects of serum, i.e., 37 degrees C-grown cells were resistant to serum killing, and removal of the outer membrane proteins with pronase rendered them sensitive. Evidence is presented which strongly suggests that the plasmid-mediated proteins are synthesized and expressed on the cell surface either during or after transit of the ingested bacteria to the lamina propria. Some properties afforded to the cells by the outer membrane proteins are described.     

Interaction of Yersinia enterocolitica and Y. pseudotuberculosis with platelets.

Yersinia enterocolitica is a bacterium capable of growth at 4 degrees C in donated blood and has been responsible for many deaths following transfusion. Interaction of Y. enterocolitica with blood cells is of interest in understanding the mechanisms of survival and growth in blood. The closely related organism Y. pseudotuberculosis is known to invade platelets and cause platelet aggregation by a mechanism that involves expression of the chromosomal inv gene. Yersinia isolates were made to express green fluorescent protein (GFP) and their interaction with platelets was studied by flow cytometry, enterocolitica did not cause platelet aggregation or activation, not even when grown at 22 degrees C to maximise inv expression. Attachment of Y. enterocolitica O:9 to platelets occurred with virulence plasmid-bearing (pYV+) strains grown at 37 degrees C but not with pYV- strains nor with strains grown at 22 degrees C. Y. pseudotuberculosis containing inv did cause platelet activation and aggregation when grown at 22 degrees C, as has been shown before, but also showed enhanced attachment to platelets when grown at 37 degrees C. Electron microscopy studies confirmed that inv-expressing Y. pseudotuberculosis invaded platelets but Y. enterocolitica attached only to the outer surface of platelets. Interaction of Y. enterocolitica O:9 with platelets provided a modest protection against bacterial killing by human serum. Interaction of Y. enterocolitica O:9 with platelets does not lead to platelet invasion or activation, and is mediated through plasmid-coded factors, not inv.     

Serological relatedness of mouse-virulent Yersinia enterocolitica.

An antiserum (WA-SAA) was produced which agglutinated specifically with mouse-virulent but not with avirulent strains of Yersinia enterocolitica. Expression of the antigenic determinant(s) reaction with WA-SAA was temperature dependent; for growth temperatures of 20 to 40 degrees C, agglutination titers were lowest for cultures grown at 20 degrees C and highest for cultures grown at 35 to 40 degrees C. Addition of Ca2+ (2.5 to 10 mM) to the growth medium had little effect on the agglutination titer, and gel diffusion studies with monospecific anti-V serum indicated that V antigen was not likely to be the determinant reacting with WA-SAA. Immunohistological studies of Peyer's patches of mice infected with Y. enterocolitica WA revealed that the antigenic determinant(s) reacting with WA-SAA was expressed in vivo. The strong correlation of agglutination titer with mouse virulence and the expression in vivo of the antigenic determinant(s) reacting with WA-SAA suggest that the antigen(s) may be associated with the pathogenicity of Y. enterocolitica.     

Assay of crystal violet binding for rapid identification of virulent plasmid-bearing clones of Yersinia enterocolitica.

A rapid, reliable, and simple method based on the binding of crystal violet (CV) is described for differentiating virulence-plasmid-bearing strains of Yersinia enterocolitica from their plasmidless derivatives. As with other plasmid-mediated properties of this organism, the binding of CV occurs at 37 degrees C but not at 25 degrees C. The CV-binding technique provides a simple and efficient means of screening Y. enterocolitica for virulence and for identifying individual plasmid-bearing colonies.     

Virulence and phenotypic characterization of Yersinia enterocolitica isolated from humans in the United States

Yersinia enterocolitica was recently reclassified into Yersinia enterocolitica sensu stricto and three additional species. With this new classification, it was of interest to reexamine pathogenicity previously ascribed to Y. enterocolitica. All available clinical isolates of Y. enterocolitica sent to the Centers for Disease Control from 1970 through 1980 were selected for characterization and comparison. One-hundred such strains had been submitted, from 21 states. Most (85%) were biotype 1, and O:8 was the most common of the 24 serotypes encountered. All strains were examined by several virulence assays. Two strains caused conjunctivitis in guinea pigs, 7 were lethal for mice, 54 invaded HEp2 cells, 18 produced a heat-stable enterotoxin, 9 were calcium dependent, 20 autoagglutinated, and 34 had a distinctive colonial morphology at 37 degrees C. Ten isolates of each of the new species that had previously been grouped with Y. enterocolitica (Y. kristensenii, Y. intermedia, and Y. frederiksenii) were characterized and were generally negative in all assays. This study points out pathogenicity differences among Yersinia species, confirms the complex nature of virulence in Y. enterocolitica, and confirms that no single current assay correlates with virulence in Y. enterocolitica.     

Plasmids in Yersinia enterocolitica serotypes O:3 and O:9: correlation with epithelial cell adherence in vitro.

Human isolates of Yersinia enterocolitica serotypes O:3 (biotype 4) and O:9 (biotype 3) harbored plasmids sized approximately 47 and 44 megadaltons, respectively. No such plasmids were found in "apathogenic" strains of Y. enterocolitica belonging to biotype 1. There was a positive correlation among the presence of plasmid, autoagglutination, and adherence to and toxicity for HEp-2 cell cultures; all of these properties were lost by culturing at 37 degrees C in the absence of calcium. Strains of Y. enterocolitica O:3 and O:9 cured of the plasmids showed increased invasiveness in the HEp-2 cell culture model, but no invasiveness in guinea pig eye. It is suggested that the plasmids of Y. enterocolitica primarily determine epithelial cell adherence, but may also be associated with other pathogenic properties.     

Class-specific immune response to Yersinia enterocolitica serotype O9 antigens as determined by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) using lipopolysaccharide (S-LPS) as the antigen was used to analyze the antibody response in rabbits orogastrically and intravenously infected with virulent (plasmid-bearing) Yersinia enterocolitica O9 strains (pYV+) and with the avirulent (plasmid-cured) derivatives (pYV-). A significative response of immunoglobulin G (IgG), IgA, and IgM antibodies against the S-LPS antigen was evident in sera from the rabbits orogastrically infected with pYV+ strains. This immune response was stronger and persisted longer than those obtained with the corresponding pYV- strains. In contrast, few differences were observed in the titers and evolution of IgG, IgA, and IgM antibodies against the S-LPS antigen in rabbits intravenously infected with pYV+ and pYV- strains. These results suggest that the necessity of the virulence plasmid for the establishment of infection by Y. enterocolitica serotype O9 is conditioned by the infection route used. When the S-LPS ELISA was compared with the radial immunodiffusion test using the native hapten as the antigen, the results showed that the ELISA technique was more sensitive. However, only those sera obtained between 2 and 8 weeks postinfection from rabbits intravenously infected with plasmid-bearing strains were positive in the radial immunodiffusion test.