bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasmid-encoded outer membrane protein YadA mediates specific binding of enteropathogenic yersiniae to various types of collagen
Authors: Schulze-Koops H , Burkhardt H , Heesemann J , von der Mark K , Emmrich F
Published: January 2020

The plasmid-encoded outer membrane protein YadA of enteropathogenic yersiniae is associated with pathogenicity. Recently, collagen binding of YadA-positive yersiniae was reported without detailed characterization (L. Emödy, J. Heesemann, H. Wolf-Watz, M. Skurnik, G. Kapperud, P. O'Toole, and T. Wadström, J. Bacteriol. 171:6674-6679, 1989). To elucidate the nature of collagen binding to YadA, we used a recombinant Yersinia strain expressing the cloned YadA gene. Direct binding of YadA-positive yersiniae to collagens was demonstrated in affinity blot experiments on nitrocellulose filters. A spectrum of collagen types in a wide concentration range were tested for their ability to block binding of 125I-labeled collagen type II to YadA-positive yersiniae. The results indicate a specific binding site(s) for YadA in collagen types I, II, III, IV, V, and XI. In contrast, collagen type VI did not bind to YadA. To characterize the binding site(s) more precisely, isolated collagen chains and cyanogen bromide fragments were investigated. These studies revealed that binding of YadA to collagen type I is confined to the alpha 1(I) chain, whereas the binding site within collagen type XI is localized in the alpha 3(XI) chain. alpha 2(I), alpha 1(XI), and alpha 2(XI) did not bind to YadA. Most interestingly, in the alpha 1(II) chain the specific binding site for YadA resides in the cyanogen bromide fragment CB10. The latter might indicate a binding site that does not depend on conformation. Based on these findings, further fragmentation and the synthesis of peptides may allow definition of the peptide sequence(s) relevant for YadA binding.
Characterization of the epitope specificity of murine monoclonal antibodies directed against lipid A
Authors: Kuhn H M , Brade L , Appelmelk B J , Kusumoto S , Rietschel E T , Brade H
Published: January 2020

A series of monoclonal antibodies directed against lipid A was characterized by using synthetic lipid A analogs and partial structures. These compounds vary in phosphate substitution, acylation pattern (type, number, and distribution of fatty acids), and, in the case of monosaccharides, in their backbone glycosyl residue. The monoclonal antibodies tested could be subdivided into five groups according to their reactivity patterns. One group reacted exclusively with 1,4'-bisphosphoryl lipid A, and a second also reacted with 4'-monophosphoryl lipid A. Two further groups recognized either 4-phosphoryl or 1-phosphoryl monosaccharide partial structures of lipid A. The fifth group reacted with 4-phosphoryl monosaccharide structures and with phosphate-free compounds. Antibodies reactive with monosaccharide structures also recognized their epitopes in corresponding phosphorylated disaccharide compounds. Both groups of monosaccharide and monophosphoryl lipid A-recognizing antibodies have access to their epitopes in bisphosphoryl compounds as well. Because of this unidirectional reactivity with more complex structures, the various specificities cannot be distinguished by using bisphosphoryl lipid A (e.g., Escherichia coli lipid A) as a test antigen. The epitopes recognized by the various monoclonal antibodies all reside in the hydrophilic backbone of lipid A, and there was no indication that fatty acids were part of the epitopes recognized. Nevertheless, the reactivities of compounds in the different test systems are strongly influenced by their acylation patterns; i.e., acyl groups may modulate the exposure of lipid A epitopes.
Cytoadherence and ultrastructure of Plasmodium falciparum-infected erythrocytes from a splenectomized patient
Authors: Ho M , Bannister L H , Looareesuwan S , Suntharasamai P
Published: January 2020

In malarial infections of primates, the spleen has been shown to modulate parasite antigen expression on the surfaces of infected erythrocytes. The processes affected include cytoadherence, which is central to the pathophysiology of severe falciparum malaria, and the related phenomenon of rosette formation. In this study, the cytoadherence and rosette formation behaviors of Plasmodium falciparum-infected erythrocytes from a splenectomized patient were examined during the first erythrocytic cycle in vitro. Ultrastructural studies were also performed. Infected erythrocytes were found to cytoadhere to C32 melanoma cells via leukocyte differentiation antigen CD36 but not intercellular adhesion molecule 1. They also displayed on their surfaces electron-dense knobs similar in structure and density to those on infected erythrocytes from intact hosts. These findings may reflect a stable cytoadherent phenotype of the parasite isolate that is unaffected by the absence of the spleen. Alternatively, the modulating role of the spleen may have been assumed by other organs of the mononuclear phagocytic system in a previously infected individual. No rosette formation was observed, but as not all natural isolates form rosettes, this observation may or may not be related to the asplenic status of the patient. Parasite and host factors appear to be important in determining the effect of splenectomy on cytoadherence and rosette formation in human falciparum malaria.
Properties of pertussis toxin B oligomer assembled in vitro from recombinant polypeptides produced by Escherichia coli
Authors: Burnette W N , Arciniega J L , Mar V L , Burns D L
Published: January 2020

The subunits that make up the pentameric B oligomer of pertussis toxin (S2, S3, S4, and S5) were individually synthesized as recombinant polypeptides in Escherichia coli, isolated as insoluble inclusion bodies, and assembled into a multimeric form in vitro by spontaneous association following treatment with a chaotropic agent, reduction, and reoxidation. The recombinant B multimer, purified by fetuin-Sepharose affinity chromatography, contained all four of the individual subunits and possessed the mitogenic and hemagglutinating activities characteristic of the native B oligomer. Immunization of mice with the recombinant B oligomer elicited antibodies that neutralized pertussis toxin in vitro and, moreover, provided protection in vivo against the leukocytosis-promoting activity of the toxin. These results demonstrate the potential for assembly of complex multimeric proteins from recombinant DNA-derived polypeptides and provide a novel means for production of an acellular pertussis vaccine component.
Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes
Authors: Nataro J P , Deng Y , Maneval D R , German A L , Martin W C , Levine M M
Published: January 2020

Strains of enteroaggregative Escherichia coli (EAggEC) have been implicated in several studies as important agents of persistent diarrhea among infants in the developing world. We have previously shown that the aggregative adherence (AA) property of EAggEC is associated with the presence of a 60-MDa plasmid which confers AA when introduced into E. coli HB101. Here, we report the cloning of the AA determinant from EAggEC strain 17-2 into the 21.5-kb cosmid vector pCVD301. TnphoA mutagenesis of the AA cosmid clone pJPN31 implicated an AA region of approximately 12 kb. Transmission electron microscopy of HB101 (pJPN31) revealed the presence of bundle-forming fimbriae, which were absent in AA- TnphoA insertion mutants. The presence of these fimbriae, AA, and hemagglutination (HA) of human erythrocytes were all concurrently lost by single-insertion mutations. A 14-kDa protein was seen on polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of surface shear preparations from fimbriated clones. Twelve of nineteen volunteers fed EAggEC 17-2 developed rises in antibodies to the 14-kDa protein as determined by Western blot. We have termed the cloned bundle-forming fimbriae aggregative adherence fimbriae I (AAF/I); positivity with a previously described EAggEC probe and human erythrocyte HA appear to correlate with the presence of AAF/I.
Stage-specific expression and intracellular shedding of the cell surface trans-sialidase of Trypanosoma cruzi
Authors: Frevert U , Schenkman S , Nussenzweig V
Published: January 2020

We have used antibodies to the Trypanosoma cruzi trans-sialidase and to its product, the host cell invasion-related Ssp-3 epitope, to study the expression of the corresponding antigens during the intracellular development of the parasite and in the extracellular trypomastigotes. As soon as 2 h after host cell invasion, trans-sialidase was no longer detected, whereas the Ssp-3 epitope was still present on intracellular parasites. The amastigotes which subsequently developed remained nonreactive with the antibodies. Expression of enzymatically active T. cruzi trans-sialidase started again only after transformation of the amastigotes into trypomastigotes 72 h after host cell invasion. trans-Sialidase was shed from the trypanosomes into the host cell cytoplasm, where the enzyme accumulated until release of the parasites. All released trypomastigotes expressed trans-sialidase on their surfaces and in the flagellar pockets, but stumpy trypomastigotes were stained more intensely than slender trypomastigotes. Ssp-3, the sialylated reaction product of trans-sialidase, was assembled only after rupture of the host cell membrane and was detected on the plasma membranes and in the flagellar pockets of all trypomastigotes.
Molecular cloning of epithelial cell invasion determinants from enterotoxigenic Escherichia coli
Authors: Elsinghorst E A , Kopecko D J
Published: January 2020

Although penetration of the epithelial mucosa has not been identified as a virulence mechanism in enterotoxigenic Escherichia coli (ETEC), we have found that this pathogen is capable of invading human intestinal cell lines. Classical ETEC strain H10407 was most invasive for epithelial cell lines derived from ileocecal and colonic tissues. An ETEC cosmid library was screened for clones that could direct E. coli HB101 to invade cultured human ileocecal epithelial cells (HCT 8 cells). Three invasive recombinant cosmid clones were isolated. These cosmids could direct HB101 invasion at an efficiency that was equal to or greater than that of the parent ETEC strain. The invasion cosmids also allowed for enhanced HCT 8 cell adherence by HB101. Electron micrographs of ETEC and recombinant HB101 strains revealed intracellular bacteria contained within endocytic vacuoles. Restriction endonuclease mapping and hybridization analyses showed that the three ETEC clones represent two separate invasion systems present in the parent ETEC strain and that both systems are chromosomally encoded. The parent ETEC strain and one cloned invasion system did not invade HeLa cells. Interestingly, one cloned invasion system was capable of directing HB101 to invade HeLa cells. Invasion of HCT 8 cells by recombinant HB101 strains and the parent ETEC strain was inhibited by cytochalasin D, indicating that the wild-type and both cloned invasion systems trigger an actin polymerization-dependent uptake process. It is not known whether the invasive phenotype of ETEC is relevant for enterotoxigenic disease. However, the parent ETEC strain, as well as recombinant HB101 strains, was capable of transcytosis through polarized HCT 8 monolayers. This transcytosis suggests that ETEC may cross the gut epithelium in vivo and that this invasion may have a previously unrecognized role in the disease process.
Induction of tumor necrosis factor and interleukin-1 by purified staphylococcal toxic shock syndrome toxin 1 requires the presence of both monocytes and T lymphocytes
Authors: See R H , Kum W W , Chang A H , Goh S H , Chow A W
Published: January 2020

Highly purified staphylococcal toxic shock syndrome toxin 1 (TSST-1) was tested for its ability to induce the cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) from fractionated human peripheral blood mononuclear cells prepared from seven healthy donors. Highly purified monocytes alone or T lymphocytes alone did not produce TNF or IL-1 when incubated with TSST-1 at 37 degrees C for up to 72 h. However, the addition of 10 micrograms of TSST-1 per ml to a 1:1 mixture of monocytes and T cells resulted in significant TNF (predominantly TNF-alpha) and IL-1 beta production after 24 h at 37 degrees C. The nature of the monocyte/T-cell interaction did not appear to involve gamma interferon (IFN-gamma), since 10 micrograms of rabbit anti-IFN-gamma per ml did not neutralize TNF-alpha production after TSST-1 induction. Similarly, L243, a monoclonal antibody to HLA-DR which blocks TSST-1 binding to monocytes, did not inhibit TNF-alpha production following TSST-1 induction. However, direct contact between monocytes and T cells was required, since physical separation of cells in double-chamber culture wells abolished TNF-alpha secretion after TSST-1 stimulation. Furthermore, paraformaldehyde fixation of either monocytes or T cells prior to addition to viable T cells or monocytes, respectively, also abolished TNF-alpha secretion, suggesting that aside from cell contact, soluble factors were also involved. Our results suggest that cytokine production involves more than binding of TSST-1 to its receptor on monocytes alone and that cell contact with T cells and the release of a soluble factor(s) other than IFN-gamma may be essential for the induction of cytokines by this toxin.
Lactoferrin-binding proteins in Shigella flexneri
Authors: Tigyi Z , Kishore A R , Maeland J A , Forsgren A , Naidu A S
Published: January 2020

The ability of Shigella flexneri to interact with lactoferrin (Lf) was examined with a 125I-labeled protein-binding assay. The percent binding of human lactoferrin (HLf) and bovine lactoferrin (BLf) to 45 S. flexneri strains was 19 +/- 3 and 21 +/- 3 (mean +/- standard error of the mean), respectively. 125I-labeled HLf and BLf binding to strain M90T reached an equilibrium within 2 h. Unlabeled HLf and BLf displaced the 125I-HLf-bacteria interaction in a dose-dependent manner. The Lf-bacterium complex was uncoupled by KSCN or urea, but not by NaCl. The interaction was specific, and approximately 4,800 HLf binding sites (affinity constant [Ka], 690 nM) or approximately 5,700 BLf binding sites (Ka, 104 nM) per cell were estimated in strain M90T by a Scatchard plot analysis. The native cell envelope (CE) and outer membrane (OM) did not reveal Lf-binding components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, after being boiled, the CE and OM preparations showed three distinct horseradish peroxidase-Lf reactive bands of about 39, 22, and 16 kDa. The 39-kDa component was also reactive to a monoclonal antibody specific for porin (PoI) proteins of members of the family Enterobacteriaceae. The Lf-binding protein pattern was similar with BLf or HLf, for Crb+ and Crb- strains. The protein-Lf complex was dissociable by KSCN or urea and was stable after treatment with NaCl. Variation (loss) in the O chain of lipopolysaccharide (LPS) markedly enhanced the Lf-binding capacity in the isogenic rough strain SFL1070-15 compared with its smooth parent strain, SFL1070. These data establish that Lf binds to specific components in the bacterial OM; the heat-modifiable, anti-PoI-reactive, and LPS-associated properties suggested that the Lf-binding proteins are porins in S. flexneri.
Murine model of cutaneous infection with gram-positive cocci
Authors: Bunce C , Wheeler L , Reed G , Musser J , Barg N
Published: January 2020

Staphylococcus aureus has remained an important cause of nosocomial wound infections, but standardized or reproducible systems for analyzing cutaneous infections caused by S. aureus do not exist. A variety of foreign materials, variable inocula, and skin traumas have been used to promote infection. To minimize these variables and ensure reproducibility, we chose a model using subcutaneous injections of a fixed quantity of dextran microbeads (Cytodex) as the foreign material added to standardized broth suspensions of S. aureus. Suspensions (0.2 ml) injected into an outbred strain of immunocompetent hairless mice generated reproducible, measurable lesions. With S. aureus Smith Diffuse, fluctuant, erythematous lesions with a peak diameter of 15 mm were observed; these lesions yielded purulent material containing gram-positive cocci and neutrophils and yielded growth of S. aureus on culture. Lesion size was proportional to the bacterial inoculum size. Histologic examination of excised lesions revealed typical abscesses. A second strain of S. aureus (SLC3) produced dermonecrosis instead of abscesses at an inoculum size of 10(7) CFU. Control injections with a sterile Cytodex suspension regularly produced nondraining, nonerythematous nodules with maximum diameters of less than or equal to 5 mm. Streptococcus pyogenes produced late-onset necrotic lesions and abscesses. Using a foreign substance, this model generates easily observed and reproducible cutaneous infection with S. aureus and streptococci that can potentially discriminate between inter- and intrastrain differences. Such a model could be used to test the pathogenicity of isogeneic strains of these bacterial species and to evaluate the efficacy of antimicrobial agents.
pap-and pil-related DNA sequences and other virulence determinants associated with Escherichia coli isolated from septicemic chickens and turkeys
Authors: Dozois C M , Fairbrother J M , Harel J , Bossé M
Published: January 2020

Escherichia coli isolates from septicemic or healthy chickens and turkeys from Quebec were serotyped, examined genotypically by using DNA probes specific for the pil and pap fimbrial systems and the aerobactin siderophore system, and examined phenotypically for lethality in day-old chicks, hemagglutination, serum resistance, and aerobactin production. Serogroups O78 and O1 were most common in septicemic chickens and turkeys. pap+ isolates from chickens were associated with septicemia, and pap+ isolates from turkeys were associated with lethality in day-old chicks. Four of nine pap+ isolates from septicemic turkeys expressed P adhesin, whereas all pap+ isolates from septicemic chickens were negative for P adhesin. The pil+ genotype was associated with septicemia in chickens and with serum resistance in isolates from turkeys. Mannose-sensitive hemagglutination of guinea pig erythrocytes was associated with septicemia in chickens and turkeys, although this phenotype was not associated with pil+ isolates from turkeys. Serum resistance was associated with isolates from septicemic turkeys and with lethality in isolates from chickens. The aerobactin system was associated with isolates from septicemic chickens and turkeys. Overall, results indicated that (i) genotypic examination may reveal virulence-associated traits which differ from the typically expected phenotype and/or are not readily expressed in vitro, and (ii) certain phenotypic and genotypic traits associated with E. coli causing extraintestinal disease in humans and animals are also associated with E. coli causing avian septicemia.
Sera of patients with high titers of immunoglobulin G against Toxoplasma gondii induce secretion of tumor necrosis factor alpha by human monocytes
Authors: Pelloux H , Chumpitazi B F , Santoro F , Polack B , Vuillez J P , Ambroise-Thomas P
Published: January 2020

Toxoplasma gondii alone does not induce tumor necrosis factor alpha (TNF-alpha) secretion by human monocytes and macrophages. Nevertheless, sera from infected patients with high titers of specific immunoglobulin G antibodies against T. gondii induce TNF-alpha secretion, which is significantly higher than the corresponding induction by negative sera (P less than 0.05). After incubation with the positive serum, parasites also induce secretion of this cytokine, but TNF-alpha levels are lower (11.4 to 71.8%) than those obtained with positive serum alone. Therefore, this secretion seems to be elicited in part by antibody-T. gondii complexes and/or another unidentified factor(s), probably different from lipopolysaccharide, interleukin-1, TNF-alpha, and gamma interferon. In this study, monocytes secreted more TNF-alpha into the culture fluid than macrophages did (P less than 0.05), and no correlation was observed between secretion of this cytokine by the monocytes and the intracellular multiplication of the parasites, evaluated by [3H]uracil incorporation. Sera from patients with other infections diseases did not induce secretion of TNF-alpha; however, serum free of antibodies to T. gondii, obtained from patients with leishmaniosis, also stimulated secretion of the cytokine.
Cloning and DNA sequence analysis of a Serpulina (Treponema) hyodysenteriae gene encoding a periplasmic flagellar sheath protein
Authors: Koopman M B , de Leeuw O S , van der Zeijst B M , Kusters J G
Published: January 2020

A Serpulina (Treponema) hyodysenteriae expression library was constructed in vector lambda ZAP and screened with a polyclonal antiserum raised against S. hyodysenteriae periplasmic flagella. A single immunoreactive plaque was chosen for further analysis. The recombinant phage from this plaque contained a gene encoding the 44-kDa protein that is on the outer layer (or sheath) of the periplasmic flagella. DNA sequence analysis showed that the gene encodes a protein of 320 amino acids. The protein is homologous to the flagellar sheath proteins of Treponema pallidum and Spirochaeta aurantia but not to any other flagellar proteins. We designated the cloned S. hyodysenteriae flagellar sheath protein gene flaA and the encoded protein FlaA. The 19 N-terminal amino acid residues of FlaA constitute a signal peptide that is cleaved from the protein before assembly onto the flagella in the periplasm. Amino acid residues 20 to 38 correspond to the N-terminal amino acid sequence of the native protein. Upstream from the gene, DNA motifs that are similar to the consensus Escherichia coli -35 and -10 promoter sequences and a ribosome binding site were identified. Downstream from the gene, two inverted repeat sequences that may serve as a rho-independent transcription termination signal are present.
Effects of iron limitation on production of a siderophore, outer membrane proteins, and hemolysin and on hydrophobicity, cell adherence, and lethality for mice of Vibrio parahaemolyticus
Authors: Dai J H , Lee Y S , Wong H C
Published: January 2020

Vibrio parahaemolyticus is one of the most important enteropathogens in Taiwan, Japan, and other coastal regions. The pathogenesis of V. parahaemolyticus disease is not clearly understood. The expression of some factors by V. parahaemolyticus in iron-rich and iron-limited media was analyzed. In the clinical hemolytic strains, the production of a siderophore, two outer membrane proteins (77 and 80 kDa), and thermostable direct hemolysin was significantly enhanced in iron-limited culture, and hemolytic activities, cell hydrophobicity, HEp-2 cell adherence, and lethality for mice were also enhanced. The environmental nonhemolytic strain CCRC12958 that was cultured in iron-limited medium exhibited lethal activity for mice, and other factors except hemolysis were also enhanced like the responses of clinical strains were. These results suggested that a virulent factor(s) of V. parahaemolyticus may be induced or enhanced under iron-limited conditions. The iron-regulated factors reported in this paper may be important in the pathogenesis of V. parahaemolyticus disease.
Cloning of a locus involved in Streptococcus mutans intracellular polysaccharide accumulation and virulence testing of an intracellular polysaccharide-deficient mutant
Authors: Harris G S , Michalek S M , Curtiss R
Published: January 2020

The streptococcal transposon Tn916 (Tcr) was used to isolate mutants of Streptococcus mutans altered in glycogen accumulation to investigate whether glycogenlike intracellular polysaccharides (IPS) play an important role in S. mutans-induced caries formation. S. mutans UA130 (serotype c) was transformed with the Escherichia coli plasmid pAM620 (Tn916), and the resultant transposon libraries were screened for glycogen content by iodine staining. A transposon mutant, designated SMS201, demonstrated a glycogen-deficient phenotype on glucose-enriched medium. Quantitative electron microscopy confirmed that IPS concentrations were significantly reduced in SMS201 relative to the wild-type progenitor strain, UA130. Importantly, reversion to wild type correlated at all times with loss of the transposon. Transposon excisants were used to facilitate cloning of the streptococcal gene(s) involved in glycogen biosynthesis and storage. A 2.1-kb chromosomal determinant (glgR) which encodes a putative regulator of S. mutans glycogen accumulation was isolated. A stable deletion mutation (delta glgR) was subsequently generated in E. coli and introduced into S. mutans by allelic exchange. The resultant glycogen synthesis-deficient mutant, SMS203, demonstrated a significantly reduced cariogenic potential (P less than 0.01) on the buccal, sulcal, and proximal surfaces of teeth in germfree rats, relative to animals challenged with the glycogen synthesis-proficient progenitor strain, UA130. These observations confirm previous reports (J. M. Tanzer, M. L. Freedman, F. N. Woodiel, R. L. Eifert, and L. A. Rinehimer, p. 597-616, in H. M. Stiles, W. J. Loesche, and T. L. O'Brien, ed., Proceedings in Microbiology. Aspects of Dental Caries. Special Supplement to Microbiology Abstracts, vol. 3, 1976) which implicate IPS as significant contributors to the S. mutans cariogenic process.
Differential morphologic and metabolic alterations in permissive versus nonpermissive murine macrophages infected with Legionella pneumophila
Authors: Yamamoto Y , Klein T W , Brown K , Friedman H
Published: January 2020

Legionella pneumophila infection of macrophages from permissive guinea pigs and from A/J mice compared with infection of cells from nonpermissive BDF1 mice was studied by electron microscopy. The cells from the BDF1 mice were nonpermissive for legionella growth in vitro and showed few if any bacteria in phagosomes by electron microscopic examination. Similar electron micrographic examination of macrophages from A/J mice permissive for legionella growth showed numerous intact intracellular bacteria within 24 to 48 h of culture and the transition of intracellular bacteria from localization in a few large vacuoles early in the course of infection to later localization in areas surrounded and studded by ribosomes. These electron microscopic observations were similar to those seen in the case of guinea pig macrophages infected with legionellae. Biochemical studies of macrophages from permissive versus nonpermissive animals showed little or no differences in respiratory burst and lysosomal enzyme activity for macrophages from all animals tested. However, when zymosan was used as a stimulant, macrophages from the nonpermissive mouse strain produced a larger amount of H2O2 and O2- than did cells from permissive guinea pigs or A/J mice. However, legionella vaccine itself induced no detectable or very little H2O2 and O2- in macrophages tested from any source. These results suggest that permissiveness of A/J mouse macrophages to legionella growth may involve mechanisms similar to those occurring in guinea pig macrophages in terms of morphologic and possibly even biochemical events. The relatively higher production of reactive oxygens by BDF1 mouse macrophages in response to zymosan correlated with nonpermissiveness for legionella growth, although further analysis is necessary to link these observations.
Cross-reactivity of polyclonal serum antibodies generated against Cryptosporidium parvum oocysts
Authors: Ortega-Mora L M , Troncoso J M , Rojo-Vázquez F A , Gómez-Bautista M
Published: January 2020

Polyclonal antibodies raised against Cryptosporidium parvum oocysts were found to cross-react with Eimeria spp. oocyst antigens in an indirect immunofluorescence assay, and sera from Eimeria spp.-infected lambs reacted with some antigens from sonicated C. parvum oocysts (between 29 to 30 and 66 to 69 kDa) by Western blot (immunoblot). No cross-reaction was observed with cystozoites of Toxoplasma and Sarcocystis spp. These results show the existence of epitopes common to C. parvum and various Eimeria spp.
Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation
Authors: Karch H , Meyer T , Rüssmann H , Heesemann J
Published: January 2020

Forty-five consecutive patients with various gastrointestinal disorders were identified as having Shiga-like toxin (SLT)-producing Escherichia coli infections. This was shown by the cytotoxic effect of stool extracts in Vero cell cultures which was neutralizable by antibodies to SLTs and by isolation of E. coli that hybridized with DNA probes complementary to SLT-I and SLT-II sequences. When we tested the same strains for SLT genes after subcultivation, the isolates from 15 patients became negative by colony hybridization and polymerase chain reaction and failed to produce SLTs. The instability of SLT genes warrants direct screening methods for clinical material and the development of new culture methods to prevent the loss of SLT genes.
Role of the mononuclear phagocyte as an antigen-presenting cell for human gamma delta T cells activated by live Mycobacterium tuberculosis
Authors: Boom W H , Chervenak K A , Mincek M A , Ellner J J
Published: January 2020

gamma delta T cells, both human and murine, have been found to be highly responsive to mycobacterial antigens. However, the role and function of gamma delta T cells in the immune response to Mycobacterium tuberculosis remain largely unknown. In earlier studies, we demonstrated that monocytes infected with live M. tuberculosis were particularly effective inducers of human peripheral blood gamma delta T cells. The present studies were performed to further characterize the interaction between human mononuclear phagocytes, gamma delta T cells, and live M. tuberculosis, in comparison with CD4+ T cells. First, we found that resting gamma delta T cells expanded in vitro by live M. tuberculosis were specific for M. tuberculosis, and that heat killing and washing the mycobacteria removed the antigen(s) for gamma delta T cells. In contrast, the heat-killed mycobacteria retained significant antigenicity for CD4+ T cells. Second, live M. tuberculosis-expanded gamma delta T cells from healthy tuberculin-positive donors did not respond significantly to the antigens in M. tuberculosis culture filtrate, including the 65- and 71-kDa mycobacterial heat shock proteins. Third, the activation of gamma delta T cells by live mycobacteria was dependent on antigen-presenting cells, and mononuclear phagocytes were found to be very efficient antigen-presenting cells both for resting peripheral blood gamma delta T cells and for activated expanded gamma delta T cells. The mononuclear phagocyte carried the necessary costimulatory factors necessary for gamma delta T-cell proliferation. Fourth, the antigen repertoire and HLA requirements for CD4+ memory T cells and those for gamma delta T cells appear to be quite distinct from each other. CD4+ T cells recognized both soluble protein antigens and whole organisms in a class II major histocompatibility complex-restricted manner, whereas gamma delta T cells appeared to recognize only constituents associated with the whole organism and were not restricted by class I or class II major histocompatibility complex molecules. Finally, the assay system described to expand and purify responding CD4+ and gamma delta T cells after stimulation with live M. tuberculosis represented a simple approach to the direct comparison of these two T-cell populations in the interaction with mononuclear phagocytes infected with M. tuberculosis. Such studies provide insight not only into the relative roles of human CD4+ and gamma delta T cells in the human immune response to intracellular bacterial pathogens such as M. tuberculosis but also into the basic biologic role of human gamma delta T cells in antimicrobial immunity.
Staphylococcal alpha toxin: a study with chronically instrumented awake sheep
Authors: Harshman S , Lefferts P L , Snapper J R
Published: January 2020

The in vivo responses to staphylococcal alpha toxin are reported for 15 chronically instrumented awake yearling sheep. The data obtained from a total of 30 experiments are grouped into four categories of response: no response, noted in seven experiments done on 5 sheep; pressor response, obtained seven times in 4 sheep; fluid and solute exchange, noted on six occasions in 3 sheep; and acute heart failure and death, which occurred in 10 of the 15 sheep. "No response" denoted no change in any of the measured outcome variables. The group of sheep labeled as showing "pressor response" responded to alpha toxin infusion with an increase in pulmonary artery pressure, unaccompanied by changes either in lung lymph flow or in lung mechanics. "Changes in lung fluid and solute exchange" involve increases in lung lymph flow. The harbinger of the last category, acute left heart failure leading to death, was a marked elevation in left atrial pressure. The threshold response dose in sheep is approximately 21 micrograms/kg. A very steep dose-response curve is observed, with only a narrow window of doses, 15 to 25 micrograms/kg, between the group showing no response and the group showing death from acute heart failure. The data obtained in these studies indicate that the lethal effects of alpha toxin in sheep include acute heart failure, which may be due to direct toxicity to heart muscle and/or the coronary vasculature endothelium.
Presence of functional receptors for the Escherichia coli heat-stable enterotoxin in the gastrointestinal tract of the chicken
Authors: Katwa L C , White A A
Published: January 2020

Receptors for the Escherichia coli heat-stable enterotoxin (STa) were shown to be present throughout the digestive tract of the chicken, with binding activity present not only in the intestinal epithelium but also in the intestinal smooth muscle. Brush border membrane vesicles (BBMV) purified from chicken enterocyte homogenates and plasma membranes (SMPM) purified from intestinal smooth muscle homogenates were compared with pig enterocyte BBMV. All had similar 125I-STa binding affinities, but the 50% effective concentration for STa activation of guanylate cyclase was higher in SMPM than in BBMV. Maximal STa-stimulated guanylate cyclase activities were similar in chicken and pig BBMV and were seven- to eightfold higher than in SMPM, and the STa receptor density was five- to sixfold higher. Patterns unique to each membrane were demonstrated after affinity labelling of STa receptors with 125I-STa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The results demonstrated STa-stimulated guanylate cyclase activity in birds as well as mammals and suggested that there are different functional STa receptors in chicken BBMV and SMPM.
Contributions of complement and immunoglobulin to neutrophil-mediated killing of enterococci
Authors: Harvey B S , Baker C J , Edwards M S
Published: January 2020

Enterococci have become a frequent causative agent in neonatal sepsis. The relative contributions of antibody and complement and their interactions in the neutrophil-mediated bacterial killing of 11 Enterococcus strains from neonates were investigated. Polymorphonuclear leukocytes (PMNL) from adult and term newborn infants were tested with normal human serum, adult hypogammaglobulinemic serum, and normal newborn serum in a neutrophil bactericidal assay. Neutrophil bactericidal activity for enterococci was not influenced by the serum source but was essentially ablated after heat inactivation of complement in all sera. No differences were observed in the killing capacity of healthy newborn versus adult PMNL regardless of serum source. Representative Enterococcus strains were then tested with agammaglobulinemic serum or C4-deficient serum, resulting in neutrophil bactericidal activities consistently exceeding 90%. A neutrophil bactericidal assay performed with normal rabbit serum and hyperimmune rabbit serum against enterococci showed that antibodies to enterococci enhanced neutrophil-mediated killing of this organism. Thus, neutrophil killing of enterococci appears to be mediated primarily by complement, with antibody playing a less essential but potentially important role. PMNL from adult and healthy term infants functioned with equal efficiency in the neutrophil killing of enterococci.
Identification of a beta 1 integrin on Mycobacterium avium-Mycobacterium intracellulare
Authors: Rao S P , Gehlsen K R , Catanzaro A
Published: January 2020

Mycobacterium avium-Mycobacterium intracellulare (MAI) is an opportunistic intracellular pathogen responsible for the highest incidence of disseminated bacterial infection in patients with AIDS. Treatment of the infection is extremely difficult and has shown limited efficacy. A critical event in the initiation of a variety of bacterial infections involves the adherence of bacteria to host cell surfaces. In the present study, we have shown that MAI organisms bind avidly to extracellular matrix proteins such as laminin, collagen I, and fibronectin in an in vitro attachment assay. Immunoblot analysis of a sonicate of MAI with polyclonal antibodies against different integrin receptors indicated that the sonicate cross-reacts with polyclonal antibodies against a human laminin-binding integrin, alpha 3 beta 1, and a human fibronectin-binding integrin, alpha 5 beta 1, although it is reactive with only the beta 1 subunit in the case of both antisera. Antibodies against the alpha 3 beta 1 and alpha 5 beta 1 integrins specifically inhibited the binding of MAI to laminin, collagen I, and fibronectin by 70 to 97%, depending on the ligand, suggesting that the attachment of MAI to these extracellular matrix proteins may be mediated by a beta 1 integrin. Furthermore, the attachment of MAI to laminin, collagen I, and fibronectin was found to be cation dependent. MAI may use this and other beta 1-containing integrins to adhere and penetrate through basement membrane structures that underlie host cell linings. An understanding of the mechanism of attachment and a definition of the adhesive molecules on the surface of MAI may open up new approaches to the prevention of serious infection caused by this organism.
Characteristics of Helicobacter pylori variants selected for urease deficiency
Authors: Pérez-Pérez G I , Olivares A Z , Cover T L , Blaser M J
Published: January 2020

The urease of Helicobacter pylori is suspected to play a role in the pathogenesis of gastritis. Although all clinical isolates of H. pylori are urease positive (U+), we have selected and characterized several spontaneously arising urease-negative (U-) variants from wild-type strain 60190. Urease-negative variants were identified by growth in medium containing 60 mM urea and arose at a frequency of 10(-5) to 10(-6). The urease activity of the wild-type strain inhibited growth of this strain in the presence of 60 mM urea. U- variants retained the U- phenotype for more than 100 passages on medium with or without urea. The urease activities of the original U+ and derived U- cells were 9.55 to 16.7 and 0.01 to 0.17 U/mg of protein, respectively. Colonial growth and other biochemical characteristics were identical for the strains. U- variants showed three classes of whole-cell sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles: (i) identical to U+; (ii) change in the migration of the 61-kDa urease subunit; and (iii) lack of 61- and 30-kDa subunits. These differences were confirmed by immunoblotting and by protein separation using fast protein liquid chromatography. The U+ strain but not U- variants tolerated exposure to pH 4.0 for 60 min in the presence of urea. Supernatants of the U+ strain and U- variants contained vacuolating cytotoxin activity for HeLa cells in similar titers. By enzyme-linked immunosorbent assay, human serum samples recognized water extract from the U+ strain significantly better than extract from a U- variant lacking urease subunits. In conclusion, this study demonstrates that U- H. pylori variants may arise spontaneously, that urease activity enhances survival at acid pH, and that urease and cytotoxin activities are disparate phenotypes.
SCID mouse models of acute and relapsing chronic Toxoplasma gondii infections
Authors: Johnson L L
Published: January 2020

Lymphodeficient scid/scid (SCID) mice died from acute infection with a strain of Toxoplasma gondii that causes chronic infection with mild symptoms in immunocompetent non-SCID mice. However, most SCID mice reconstituted with spleen cells from immunocompetent mice 1 month prior to T. gondii infection survived in good health after a transient period during which they appeared ill. Unreconstituted SCID mice given sulfadiazine in their drinking water from day 10 of Toxoplasma infection onward survived the acute phase of infection and lived for many weeks without overt symptoms. Histological examination revealed Toxoplasma cysts in their brains. However, if sulfadiazine was withdrawn from the drinking water of these chronically infected SCID mice, the mice died within 1 week with large numbers of trophozoites throughout their brains. These findings establish SCID mice as a potentially useful resource with which to study various aspects of immunological control of T. gondii infection during either its acute or chronic phase. Furthermore, the ability to produce chronic infections with avirulent T. gondii in SCID mice and to cause acute relapsing infections at will suggests that SCID mice may be helpful in evaluating potential therapies for acute and chronic T. gondii infections in immunocompromised patients.
Mapping of Candida albicans oligomannosidic epitopes by using monoclonal antibodies
Authors: Trinel P A , Faille C , Jacquinot P M , Cailliez J C , Poulain D
Published: January 2020

Six monoclonal antibodies (MAbs) from various laboratory sources (EB-CA1, EB-CA2, H5, AF1, C6, and 5B2), reacting with the polysaccharidic moieties of Candida albicans mannoproteins, were used for epitope mapping by an enzyme-linked immunosorbent assay (ELISA) with neoglycolipids and by Western blotting (immunoblotting) of a C. albicans germ tube extract. The ELISA involved neoglycolipids constructed from three families of oligomannosides released by sequential depolymerization of C. albicans phosphopeptidomannan by acid hydrolysis (NGLH), beta-elimination (NGLO), and acetolysis (NGLA). All of the MAbs exhibited low reactivities against NGLO. MAbs EB-CA1, EB-CA2, and H5 reacted mainly against NGLA, and MAbs C6 and AF1 recognized mainly NGLH, whereas MAb 5B2 reacted with both families of neoantigens. When this method was compared with Western blotting, strong reactivity to NGLA was associated with the presence of epitopes shared by high-molecular-weight mannoproteins, whereas strong reactivity to NGLH was associated with a reactivity to a family of 14- to 18-kDa antigens. The reactivity of MAb 5B2 was associated with both high-molecular-weight mannoproteins and the 14- to 18-kDa antigens. In relation to the present knowledge about the structure of the C. albicans phosphopeptidomannan oligomannosidic repertoire, these results provide preliminary data concerning the molecular basis of the recognition of mannopyranosyl sequences by MAbs and their distribution among C. albicans mannoproteins.
Bacterial motility is a colonization factor in experimental urinary tract infection
Authors: Siitonen A , Nurminen M
Published: January 2020

In an experimental urinary tract infection of the mouse, colonization of the urinary bladder by isogenic strains of Salmonella enterica serovar Typhimurium was found to depend on the motility of the bacteria. Strains were obtained by genetic recombination between a highly motile O-6,7 and a poorly motile O-4,5,12 strain. The O antigen did not interfere with the colonization, whereas motility did; flagellated and motile O-6,7 and O-4,5,12 bacteria colonized the bladder equally well.
Comparison of aminopeptidase activities in four strains of mutans group oral streptococci
Authors: Cowman R A , Baron S S
Published: January 2020

In this study, native cells of Streptococcus mutans VA-29R and Streptococcus rattus FA-1 displayed significantly higher aminopeptidase activity than did cells of Streptococcus cricetus AHT or Streptococcus sobrinus 6715 toward the nitroanilide derivatives of leucine, alanine, methionine, arginine, and lysine. These differences in cellular aminopeptidase activity led us to investigate the subcellular localization of the aminopeptidase in these mutans group streptococci. Following conversion of native cells to protoplasts by treatment with lysozyme, most of the aminopeptidase activity detected in the native-cell preparations remained associated with the intact protoplasts. After lysis of protoplasts and differential centrifugation, most of the total cellular aminopeptidase activity was recovered with the cytoplasmic fraction. Membrane-associated aminopeptidases represented only minor activities in these mutans group streptococci. Although the four strains showed no differences with respect to a predominant cytoplasmic localization for the aminopeptidase activities, the levels of activity in the cytoplasmic fractions from S. cricetus AHT and S. sobrinus 6715 were significantly lower than those measurable in the corresponding fractions from S. mutans VA-29R and S. rattus FA-1. These results support the conclusion that the differences in aminopeptidase activity expressed by these streptococci reflect quantitative differences rather than differences in enzyme subcellular localization.
Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins
Authors: Brown W C , Zhao S , Woods V M , Tripp C A , Tetzlaff C L , Heussler V T , Dobbelaere D A , Rice-Ficht A C
Published: January 2020

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.