Toxicity of recombinant toxic shock syndrome toxin 1 and mutant toxins produced by Staphylococcus aureus in a rabbit infection model of toxic shock syndrome.

Menstrually associated toxic shock syndrome (TSS) is attributed primarily to the effects of staphylococcal exotoxin toxic shock syndrome toxin 1 (TSST-1). A region of the 194-amino-acid toxin spanning residues 115 through 144 constitutes a biologically active site. Several point mutations in the TSST-1 gene in that region result in gene products with reduced mitogenic activity for murine T cells. In this study we evaluated the toxicity of recombinant TSST-1 and several mutants of TSST-1 made by transformed Staphylococcus aureus during in vivo growth in a rabbit infection model of TSS. The toxicities of the transformed strains of S. aureus for rabbits correlated with the mitogenic activities of the recombinant toxins. An isolate originally obtained from a patient with a confirmed case of TSS (S. aureus 587) implanted in a subcutaneous chamber served as a positive control. TSST-1 produced in vivo led to lethal shock within 48 h, and a TSST-1-neutralizing antibody (monoclonal antibody 8-5-7) administered to rabbits challenged with S. aureus 587 prevented fatal illness. Rabbits infected with transformed S. aureus RN4220 expressing wild-type toxin (p17) or mutant toxins retaining mitogenic activity for T cells succumbed within a similar time frame. Blood chemistries of samples obtained from infected animals before death indicated abnormalities in renal and hepatic functions similar to those induced by parenteral injection of purified staphylococcal TSST-1. Mutant toxin 135 (histidine modified to alanine at residue 135) possessed only 5 to 10% of the mitogenic activity of wild-type toxin. Rabbits challenged with transformed S. aureus RN4220 expressing mutant toxin 135 exhibited only mild transient illness. Mutant toxin 135 retained reactivity with monoclonal antibody 8-5-7 and by several criteria was conformationally intact. Toxin from a double mutant, 141.144, with alanine substitutions at residues 141 (histidine) and 144 (tyrosine), also was devoid of mitogenic activity. In this case, antibody recognition was lost. Mutant toxins 115 and 141 were found to possess approximately half-maximal mitogenic activity. Rabbits challenged with S. aureus RN4220 expressing either 115 or 141 toxin succumbed to lethal shock. We conclude that the ability of TSST-1 to activate murine T cells in vitro and its expression of toxicity leading to lethal shock in rabbits are related phenomena.

Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli.

The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated. No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes. However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR. Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain. For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX. Four cysteine residues in the amino-terminal region are also conserved. The promoter region of hlyX is very similar to that of fnr. It has a putative -10 sequence which closely resembles the E. coli -10 consensus sequence. This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence. Functional homology between HlyX and FNR was also demonstrated. Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E. coli. These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E. coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded.

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

This article describes the molecular cloning and expression of a hemolysin gene from a serotype 1 strain of Actinobacillus pleuropneumoniae. The hemolysin was a thermolabile protein with an apparent molecular weight of 29,500 (29.5K hemolysin). Unlike expression of the recently described 105K hemolysin of A. pleuropneumoniae (J. Frey and J. Nicolet, FEMS Microbiol. Lett. 55:41-46, 1988), expression of this hemolysin was not regulated by Ca2+. Antiserum prepared against the 105K hemolysin did not neutralize the activity of the 29.5K hemolysin; conversely, antiserum prepared against the 29.5K hemolysin did not neutralize the activity of the 105K hemolysin. The hemolytic activity was not neutralized with antisera against hemolytic Escherichia coli, Streptococcus agalactiae, or purified streptolysin O, but antisera prepared against recombinants containing the 29.5K gene and convalescent pig sera abrogated hemolytic activity. Although hemolytic activity could be detected in several strains of E. coli K-12 and in minicells expressing several different constructs encoding the 29.5K hemolysin, we could not rigorously exclude the possibility that the gene which we have isolated encodes a regulator of hemolytic activity rather than a hemolysin per se.

Invasiveness of Yersinia enterocolitica lacking the virulence plasmid: an in-vivo study.

Rabbits were given, by the intra-gastric route, two isogenic strains of Yersinia enterocolitica that differed only in the presence or absence of the virulence plasmid. Clinical illness and characteristic morphological lesions of Y. enterocolitica infection were seen only in rabbits infected with the plasmid-bearing strain (MCH700S). Although rabbits infected with a strain lacking the plasmid (MCH700L) remained healthy, mild histological changes in the small intestine, consisting of epithelial-cell damage, dilatation of lymphatics and a slight increase in neutrophil polymorphonuclear leukocytes in lamina propria, were seen in the first 12 h after inoculation. Bacteria, which were identified as Y. enterocolitica by indirect fluorescent antibody staining, were seen in dilated lymphatics. These early lesions tended to abate quickly and were no longer detectable at 24 h. Strain MCH700L was recovered from the mesenteric lymph nodes in increasing numbers until 24 h after inoculation; the number then began to decrease rapidly. In contrast, the early lesions in rabbits given strain MCH700S progressed to micro-abscesses, focal destruction of villi, and ulcerations beginning 24 h after inoculation; the number of bacteria recovered from the lymph nodes continued to increase beyond 24 h after inoculation. Bacteria were also recovered from the liver and spleen. These results suggest that both plasmid-bearing and non-bearing strains of Y. enterocolitica are capable of penetrating the intestinal mucosa. However, the virulence plasmid is required for invading bacteria to proliferate in the host tissue and to establish infection.

Plasmid-mediated resistance to phagocytosis in Yersinia enterocolitica.

Results of our previous studies have shown that the chemiluminescence response of human neutrophils (polymorphonuclear leukocytes [PMNs]) is inhibited by plasmid-mediated cell surface components from Yersinia enterocolitica. In this study we examined the susceptibility to phagocytosis of Y. enterocolitica cells with or without plasmid-mediated surface structure and the effect of isolated outer membrane fragments on phagocytosis of Escherichia coli by PMNs in vitro. Y. enterocolitica cells with expressed plasmid-mediated surface structure were much less sensitive to ingestion by PMNs than those without it, and the resistance to phagocytosis was readily eliminated in a dose-dependent fashion by pronase treatment of whole cells, which was shown to remove plasmid-encoded outer membrane proteins. Ingestion and intracellular killing of E. coli were inhibited significantly in the presence of isolated outer membrane fragments derived from plasmid-bearing Y. enterocolitica cells. To assess the interaction of Y. enterocolitica with phagocytic cells in vivo, two isogenic strains of Y. enterocolitica, differing only in the presence or absence of the virulence plasmid, were inoculated intradermally into the backs of rabbits; and tissue sections obtained at 12 h postinoculation were examined by light and electron microscopy. The plasmidless strain was found almost entirely in PMNs or mononuclear cells. In contrast, the plasmid-bearing strain was found to be surrounded by, or interspersed with, PMNs and mononuclear cells; but most bacteria were extracellular, with little evidence of phagocytosis. These results suggest that plasmid-mediated cell surface components of Y. enterocolitica act as antiphagocytic factors, thus facilitating the survival and proliferation of the organism in the host tissue.

Clinical, morphological, and biochemical alterations in acute intestinal Yersiniosis.

The aim of this study was to examine the intestinal response to Yersinia enterocolitica (YE) infection. Growing New Zealand white rabbits (450-800 g) were infected with 10(10) organisms of a human pathogenic strain (n = 43) or NaHCO3 for controls (n = 30) and studied 3, 6, 10, and 14 days after infection. In a separate experiment infected (n = 6) and pair-fed controls (n = 6) were studied 6 days after infection. Weight gain, excretion of YE, and diarrhea were examined daily. At sacrifice segments of proximal and mid- and distal small intestine, cecum, and colon were obtained for histologic examination and mucosa of small intestine and colon for enzyme determinations. Infection with YE resulted in weight loss and diarrhea within 48-72 h. Microabscesses were present in all sections of small and large intestine by day 3 but became more severe in the ileocecal region by day 6. In infected animals at day 6 there was crypt hyperplasia throughout the small intestine and villus atrophy in the ileum. Disaccharidases were decreased in all regions by day 3 but returned to normal by day 14 in proximal and mid-, but not distal, small intestine. The pair-fed controls experienced a similar weight loss to infected animals, but showed only minor morphologic changes and no mucosal enzyme abnormalities. Our findings demonstrate that infection of weanling rabbits with YE causes diarrhea and weight loss and that, while the weight loss is largely due to reduced food intake, the morphologic and mucosal enzyme alterations are due to intestinal injury by the organism.

Inhibition of human neutrophil chemiluminescence by plasmid-mediated outer membrane proteins of Yersinia enterocolitica.

Recent studies have shown that the cell surface properties of Yersinia enterocolitica are altered by the presence of the virulence plasmid, which mediates temperature-inducible outer membrane proteins (OMP). We investigated the interaction of Y. enterocolitica with human polymorphonuclear leukocytes by monitoring luminol-enhanced chemiluminescence (CL) responses. A plasmid-bearing strain grown at 37 degrees C induced four- to sixfold less CL than did the same strain grown at 25 degrees C or a plasmidless, isogenic strain grown at either temperature. Inhibition of CL responses by whole cells was related to plasmid-mediated expression of OMP. The OMP alone could inhibit the CL response of polymorphonuclear leukocytes stimulated by either opsonized zymosan or whole cells of Y. enterocolitica. Pronase treatment of whole cells, which removed the plasmid-mediated OMP, resulted in partial but significant elimination of CL inhibition by whole cells and by OMP derived from them. Incubation with Y. enterocolitica for 60 min did not affect the viability of polymorphonuclear leukocytes. Our results suggest that the interaction of Y. enterocolitica with human polymorphonuclear leukocytes is directly affected by the plasmid-mediated OMP.

The use of specific antibodies to demonstrate the glycocalyx and spatial relationships of a K99-, F41- enterotoxigenic strain of Escherichia coli colonizing the ileum of colostrum-deprived calves.

Electron microscopy was used to study the interaction between the glycocalyx of enterotoxigenic Escherichia coli strain 210 (09:K30+;K99-;F41-:H-) and the glycocalyx of epithelial cells in then ileum of experimentally infected newborn colostrum-deprived calves. Fixation of tissues in anti-K30 antibody and ruthenium red was used to stabilize the bacterial glycocalyx so that the spatial relationship between the bacteria and the intestinal epithelial cells could be characterized. When strain 210 was grown in vitro and reacted with anti-K30 antibody prior to staining with ruthenium red, the extensive glycocalyx could be clearly visualized surrounding the bacterial cells. By negative staining, an unidentified pilus was also seen. Sections of ileum from infected calves, which were not fixed in antibody nor stained with ruthenium red, revealed attached bacteria which were surrounded by an electron-translucent zone and no visible bacterial glycocalyx. When ruthenium red staining was used, the bacterial glycocalyx partially collapsed during the dehydration steps of fixation, but could be seen as either a fibrous capsule or an electron-dense accretion on the bacterial cell surface. When ileal tissue was reacted for one hour in anti-K30 antibody before staining with ruthenium red, the bacterial glycocalyx was seen as a discrete electron-dense structure up to 1.0microm thick which was in intimate contact with the glycocalyx of the epithelial cells. The importance of the bacterial exopolysaccharide to microcolony formation on the villi could be clearly visualized.

Some properties of the human erythrocyte receptors for Neisseria gonorrhoeae.

The nature of the receptors for T1 and T4 Neisseria gonorrhoeae on erythrocytes and other cells was investigated. In general, cells of nonprimate origin contained few receptors for gonococci. Receptors for T4 gonococci were only uncovered when host cells were pretreated with trypsin. Trypsinization, while unnecessary for T1 adherence to erythrocytes, enhanced attachment in inverse proportion to original erythrocyte sensitivity. Receptors for T1 and T4 organisms on trypsinized and trypsin-neuraminidase-treated erythrocytes were blocked by concanavalin A and peanut lectins, respectively, but a distinction could be made between them with wheat germ lectin and galactose oxidase. Of a number of sugars tested as inhibitors, only D-galactose blocked adherence of T4 but was without effect on T1. While the identity of erythrocyte receptors is uncertain, likely candidates are "band 3" protein and glycophorin, by virtue of their galactose content, lectin binding capacity, and partial exposure on the outer surface of the erythrocyte.

Antimicrobial susceptibility of Haemophilus ducreyi.

The susceptibility of 19 isolates of Haemophilus ducreyi from a recent chancroid outbreak and four reference strains was determined in vitro to 13 antimicrobial agents. The rabbit intradermal test for virulence was positive for all of the local isolates, but not for the reference strains. The "nonvirulent" reference strains were inhibited by lower minimum inhibitory concentrations (MICs) of most agents tested. For the virulent isolates, the range of MICs (in micrograms per milliliter) of the following were: of vancomycin, 8 to 128; of polymyxin, 32 to 128; of cloxacillin, 32 to 64; of tetracycline, 0.5 to 32; of cephalothin, 4 to 8; of doxycycline, 0.25 to 8; and of kanamycin, 1 to 8. Three strains were resistant to penicillin and ampicillin (MIC >/= 128 mug/ml), and these three strains produced beta-lactamase. The remainder were susceptible to 4 mug/ml. All strains were susceptible to rifampin (MIC

Determination of the hemin requirement of Haemophilus ducreyi: evaluation of the porphyrin test and media used in the satellite growth test.

Gonococcal (GC) agar supplemented with glucose and glutamine was found to be superior to Eugonagar and Trypticase soy agar in demonstrating the hemin requirement of 23 strains of Haemophilus ducreyi by the satellite growth test. The porphyrin test confirmed the requirement for exogenous hematin. With the agar dilution technique, using supplemented GC agar, the hemin concentration required to initiate growth was 10 microgram/ml, and the optimal hemin concentration to produce growth equivalent to that on chocolate agar was between 200 and 500 microgram/ml. On GC agar with added glucose and glutamine, the lowest hemin concentration impregnated in paper disks able to initiate satellite growth was 50 microgram/ml. The hemin requirements of these H. ducreyi were much higher than that reported for other Haemophilus species.

Comparison of specimen collection and laboratory techniques for isolation of Haemophilus ducreyi.

Sixteen patients with clinical chancroid were studied prospectively; different culture media and sampling techniques from genital lesions were evaluated. Technique A was aspiration of a saline wash from the ulcer which was pooled and inoculated into rabbit blood, rabbit blood + vancomycin (5 microgram/ml), and semisolid chocolate agar + vancomycin (3 microgram/ml). Each primary culture medium was subcultured to chocolate agar with 1% IsoVitaleX (CA), CA with vancomycin (3 microgram/ml) plus polymyxin (7.5 microgram/ml; CA + vp). Technique B was the use of a cotton swab, plated directly on CA, CA + v, and CA + vp. Nine strains of Haemophilus ducreyi were obtained. Technique A yielded seven strains, whereas technique B yielded eight strains; with each technique, five strains were isolated only after use of selective antibiotic media. CA + v medium yielded the largest number of isolates. Direct inoculation by swab to CA + v from chancroidal ulcers is effective as an isolation technique for growth of H. ducreyi.