bluuebunny/crossref_metadata_2025 huggingface dataset:
Site-specific mutagenesis of the catalytic subunit of cholera toxin: substituting lysine for arginine 7 causes loss of activity
Authors: Burnette W N , Mar V L , Platler B W , Schlotterbeck J D , McGinley M D , Stoney K S , Rohde M F , Kaslow H R
Published: January 2020

Cholera and pertussis toxins each contain a subunit with ADP-ribosyltransferase activity, sharing a region of nearly identical amino acid sequence near the NH2 terminus. Previous investigations have shown that substitution of a lysine residue for Arg-9 in the catalytic A subunit of pertussis toxin substantially eliminates its enzyme activity. We now report that substitution of lysine for the position-equivalent Arg-7 of cholera toxin subunit A leads to a similar loss of catalytic activity. This result suggests a correlation of function with structure between the sequence-related cholera and pertussis toxin A subunits and may contribute to the design of a vaccine containing an enzymatically inert analog of cholera toxin.
The eae gene of enteropathogenic Escherichia coli encodes a 94-kilodalton membrane protein, the expression of which is influenced by the EAF plasmid
Authors: Jerse A E , Kaper J B
Published: January 2020

The production of a characteristic intestinal histopathology called attaching and effacing (A/E) lesions by enteropathogenic Escherichia coli (EPEC) is a major characteristic of EPEC pathogenesis. We previously identified a chromosomal gene (eae) of EPEC necessary for the production of A/E lesions on human tissue culture cells. Using antiserum raised to an Eae-PhoA fusion protein, we found that the eae gene encodes a 94-kDa membrane protein. This antiserum recognized a 94-kDa membrane protein in parent strain E2348/69 and a protein of similar size in E. coli HB101 carrying eae on a plasmid but did not recognize any proteins in E. coli HB101 carrying a plasmid with an internal deletion in the eae gene. Antigenically related proteins of ca. 94 kDa were detected in a collection of EPEC strains representing seven EPEC serogroups and in two EHEC strains of serotype O26:H11. Volunteer sera drawn 28 days after but not before ingestion of strain E2348/69 recognized the 94-kDa Eae protein as well as a 128-kDa Eae-PhoA fusion protein, suggesting that the Eae protein is likely to be a previously reported 94-kDa protein shown to be immunogenic in volunteers. The amount of detectable Eae protein was increased in the presence of a high-molecular-weight plasmid which is associated with the ability to produce localized adherence to tissue culture cells. These data suggest that the virulence plasmid of EPEC strain E2348/69 may have a regulatory role in the production of A/E activity.
Correlation of carotenoid production, decreased membrane fluidity, and resistance to oleic acid killing in Staphylococcus aureus 18Z
Authors: Chamberlain N R , Mehrtens B G , Xiong Z , Kapral F A , Boardman J L , Rearick J I
Published: January 2020

Staphylococcus aureus is susceptible to killing by host-derived fatty acids. Studies were performed to test for a correlation between carotenoid production by S. aureus and protection against oleic acid. Oleic acid killing of cells grown in carotenoid expression medium was determined as the dosage of oleic acid in 2 M NaCl-2 mM EDTA that would kill 20% of the cells in 60 min at 37 degrees C (i.e., the 20% lethal dose). Compared with the wild-type strain (18Z), a carotenoid-deficient mutant strain (18Z-76) and strain 18Z grown in a medium that suppressed carotenoid production both showed increased sensitivity to oleic acid. Spontaneous revertants of strain 18Z-76 that regained the ability to produce carotenoids were as resistant to oleic acid as the wild-type strain. Oleic acid was shown by fluorescence polarization to decrease polarization values. Lower polarization values indicate a more-fluid membrane. To determine whether protection against oleic acid killing might depend on carotenoid stabilization of membranes, fluorescence polarization values were determined for strains showing different levels of carotenoid production. An indirect correlation was found between membrane fluidity and carotenoid production. We were able to conclude that there is a direct correlation between carotenoid production (i.e., cell pigmentation), cell membrane stability, and resistance to oleic acid-induced cell killing.
Relation between structure and immunologic properties of the Vi capsular polysaccharide
Authors: Szu S C , Li X R , Stone A L , Robbins J B
Published: January 2020

The Vi capsular polysaccharide of Salmonella typhi is a linear homopolymer of poly-alpha(1----4)GalNAcp variably O acetylated at the C-3 position. Serum antibodies elicited by this antigen confer protective immunity against typhoid fever. The relation between the immunologic properties and structure of Vi was investigated by carboxyl reduction, O deacetylation, and acid hydrolysis. The immunogenicity of Vi was closely related to its degree of O acetylation. Partial O deacetylation slightly increased immunogenicity; complete O deacetylation eliminated the immunogenicity of Vi. O-deacetylated Vi, however, still reacted with antisera prepared by injection of whole bacteria. Carboxyl reduction, in contrast, had a comparatively slight effect upon both the immunogenicity and antigenicity of Vi. Retention levels of antigenicity after acid treatment were greater for both the native and carboxyl-reduced Vi than for the O-deacetylated product. The Courtauld-Koltun space-filling model of a pentamer of Vi demonstrated that the bulky nonpolar O-acetyls, which protrude in rows on both sides, make up most of the surface. The carboxyls are less exposed and are partially shielded by the O-acetyls. The molecular model thus provides an explantation for the dominant role of the O-acetyls, as well as the lesser effect of carboxyl reduction, upon the immunologic properties of Vi.
Evidence for polymorphonuclear leukocyte collagenase and 92-kilodalton gelatinase in gingival crevicular fluid
Authors: Overall C M , Sodek J , McCulloch C A , Birek P
Published: January 2020

Analysis of inflammatory exudate collected from sites of experimental periodontitis in cynomolgus monkeys has revealed the presence of collagenase and a 92-kDa gelatinase that comigrated after electrophoresis with the 92-kDa gelatinase released from polymorphonuclear leukocytes. Since neutralizing antibodies to fibroblast collagenase had no effect on the collagenase activity and bacterial collagenases could not be detected, polymorphonuclear leukocytes appear to be the major source of collagenolytic proteinases in inflammatory fluid from gingiva.
Localization of translocating Escherichia coli, Proteus mirabilis, and Enterococcus faecalis within cecal and colonic tissues of monoassociated mice
Authors: Wells C L , Erlandsen S L
Published: January 2020

Normal bacteria can translocate (migrate) across an intact intestinal mucosa, but the anatomical site of bacterial translocation has not been defined. Gastrointestinal (GI) cross-sections were obtained from mice monoassociated with high cecal concentrations of Escherichia coli, Proteus mirabilis, or Enterococcus faecalis. As previously reported (C. L. Wells, R. P. Jechorek, and K. J. Gillingham, Arch. Surg. 126:247-252, 1991), these mice had viable translocating bacteria recovered from mesenteric lymph nodes and livers. No abnormal GI histology was noted. Immunofluorescence was used to localize GI bacteria, and similar observations were made with each of the three bacterial species. Smaller numbers (P less than 0.01) of bacteria were observed in stomach and small intestinal tissues than in cecal and colonic tissues, suggesting that the preferred site of tissue penetration for intestinal E. coli, P. mirabilis, and E. faecalis might be the cecum or colon as opposed to the stomach or small intestine.
Limited clonal heterogeneity of antigen-specific T cells localizing in the pleural space during mycobacterial infection
Authors: Manca F , Rossi G , Valle M T , Lantero S , Li Pira G , Fenoglio D , De Bruin J , Costantini M , Damiani G , Balbi B
Published: January 2020

To detect possible differences in phenotype and fine specificity for mycobacterial antigens between CD4-positive T cells from peripheral blood (PB) and from inflammatory sites, we identified four patients presenting with a mycobacterial pleural exudate (PE) rich in PPD-specific lymphocytes and with a negative skin test to tuberculin purified protein derivative (PPD) and a negative proliferative response of PB lymphocytes to PPD at the same time. Several weeks after chemotherapy, these patients converted to PPD responsiveness in the periphery, and PPD-specific clones could be generated from PB at this stage. The phenotypic comparison of PE lymphocytes and concomitant PB lymphocytes obtained before treatment showed an increase of CD8 cells and a high frequency of HLA-DR-positive activated T cells in PE. The frequency of tetanus toxoid-specific and Candida albicans-specific proliferating T cells was lower than that of PPD-specific cells in PE but not in PB. PPD-specific clones were derived initially from PE and from PB once the patients had converted to PPD responsiveness. The two sets of clones from each patient were compared for proliferative response to mycobacterial antigen clusters of defined molecular weight ranges. A large number of PE-derived clones (36%) responded to a fraction of 27 to 35 kDa, whereas only one clone from PB responded to the same fraction. The purified antigen P32 (32 kDa), a soluble mycobacterial protein, stimulated PE-derived clones that were responsive to the 37- to 27-kDa fraction but did not stimulate PB-derived clones. The data demonstrate that PE- and PB-derived lymphocytes differ both in phenotype and in fine specificity, suggesting a limited clonal heterogeneity of T cells localizing at the inflammatory site in tuberculous patients without a PPD response in the periphery. Therefore T cells compartmentalized at inflammatory sites provide information that is different from that provided by T cells in the periphery.
Properties of pertussis toxin mutant PT-9K/129G after formaldehyde treatment
Authors: Nencioni L , Volpini G , Peppoloni S , Bugnoli M , De Magistris T , Marsili I , Rappuoli R
Published: January 2020

Formaldehyde treatment is a method routinely used to detoxify diphtheria, tetanus, and pertussis toxins as well as other molecules suitable for vaccine production. To investigate whether chemical detoxification alters the immunological properties of vaccine components, we have treated the pertussis toxin mutant PT-9K/129G with formaldehyde and tested the properties of the resulting molecules. Very low concentrations of formaldehyde stabilize the molecule without affecting the physicochemical and immunological parameters. Increasing doses of formaldehyde abolish the mitogenic and hemagglutinating activities of PT-9K/129G. At the same time, the molecule loses the ability to be recognized by a monoclonal antibody specific for a major protective epitope on the S1 subunit of pertussis toxin and its affinity for anti-pertussis toxin polyclonal antibodies is also reduced. In marked contrast, the ability of PT-9K/129G to be recognized by human T-cell clones is not affected by Formalin treatment. In vivo, the formaldehyde-treated molecules induce amounts of specific antibodies comparable with those of untreated molecules but significantly lower levels of toxin-neutralizing antibodies. Furthermore, the formaldehyde-treated molecules also show a reduced protective activity in the intracerebral challenge assay.
Attachment of Trypanosoma cruzi to mammalian cells requires parasite energy, and invasion can be independent of the target cell cytoskeleton
Authors: Schenkman S , Robbins E S , Nussenzweig V
Published: January 2020

We have previously shown that the binding of Trypanosoma cruzi trypomastigotes to glutaraldehyde-fixed mammalian cells has the characteristics of a receptor-mediated process and that it mimics the attachment step of the invasion of live cells by this parasite. In this study we examined the metabolic requirements for the attachment of trypomastigotes to glutaraldehyde-fixed fibroblasts. The attachment of trypomastigotes to fixed cells is prevented when the energy conservation mechanisms are inhibited with the drugs 2-deoxyglucose, sodium azide, antimycin, crystal violet, oligomycin, N,N'-dicyclohexylcarbodiimide, and carbonyl cyanide 3-chlorophenylhydrazone. However, under the same experimental conditions, the movement of parasites is not significantly affected. Several of these drugs totally inhibit the penetration of the parasite into live target cells. We conclude that the attachment of trypomastigotes to mammalian cells is an active process that requires trypomastigote energy. In addition, we present evidence that penetration into nonphagocytic cells can also be an active process. Trypomastigotes can be seen in scanning electron micrographs traversing extended lamellipodia and entering paraformaldehyde-fixed epithelial cells. Cytochalasin D, a drug that disrupts microfilaments and prevents the formation of plasma membrane extensions mediated by actin, had little or no effect on trypomastigote invasion, while it inhibited Salmonella entry into epithelial cells.
Mutations at rfc or pmi attenuate Salmonella typhimurium virulence for mice
Authors: Collins L V , Attridge S , Hackett J
Published: January 2020

Insertion mutations were constructed in cloned pmi and rfc genes of Salmonella typhimurium, and these mutations were recombined (singly) into the chromosome of mouse-virulent S. typhimurium C5, displacing the wild-type alleles. Phage sensitivity profiles, lipopolysaccharide analysis, and DNA blotting all confirmed that the replacement events had occurred. The mutations were complemented by plasmid-borne wild-type alleles, as judged by the restoration of wild-type phage plaquing profiles and lipopolysaccharide production (both mutants) and the restoration of pmi-encoded enzyme production (pmi mutant). The virulence, persistence, and immunizing capacities of the mutants fed to mice were compared with those of the wild-type strain and complemented mutants. Both mutants were much reduced in virulence, with the rfc mutant being avirulent even at 10(9) bacteria per mouse. This mutant was also avirulent at up to 10(6) bacteria per mouse when administered intraperitoneally. Both the rfc and pmi mutant strains persisted in the Peyer's patches of the gut after feeding and were capable of colonizing the deeper tissues of the mice from such initial infective foci. Both mutant strains were effective as live oral vaccines (10(7) bacteria or more) against oral S. typhimurium challenge (10(4) 50% lethal doses; 6 x 10(8) bacteria) in mice.
Humoral and cellular defense against intestinal murine infection with Yersinia enterocolitica
Authors: Hanski C , Naumann M , Grützkau A , Pluschke G , Friedrich B , Hahn H , Riecken E O
Published: January 2020

The role of phagocytes and the complement system as potential host defense mechanisms against bacterial infection were studied in mice with two isogenic strains of Yersinia enterocolitica serotype O8 differing in pathogenicity because of differences in plasmid content. Complement depletion in mice by intraperitoneal injection of cobra venom factor did not affect the course of colonization of the intestinal tissue by each strain, indicating that in mice complement is not essential for the elimination of these bacteria. This conclusion is supported by the fact that fresh murine serum had no bactericidal effect in vitro either on the pathogenic or on the nonpathogenic strain. However, in the intestinal tissue as well as in the peritoneal cavity, only the pathogenic, plasmid-bearing Y. enterocolitica strain survived, while the nonpathogenic, plasmidless strain was rapidly eliminated. Since elimination from the peritoneal cavity is due to phagocytosis by polymorphonuclear leukocytes and macrophages, resistance to phagocytosis in vivo seems to be the decisive factor determining the virulence of pathogenic Y. enterocolitica strains.
An interleukin-1 receptor antagonist blocks lipopolysaccharide-induced colony-stimulating factor production and early endotoxin tolerance
Authors: Henricson B E , Neta R , Vogel S N
Published: January 2020

In this report, administration of a recombinant interleukin-1 receptor antagonist protein to mice was found to inhibit induction of colony-stimulating factor as well as induction of early endotoxin tolerance by lipopolysaccharide. These findings provide direct evidence that interleukin-1 is an intermediate in these two lipopolysaccharide-induced phenomena.
Cell and receptor requirements for streptococcal pyrogenic exotoxin T-cell mitogenicity
Authors: Leonard B A , Lee P K , Jenkins M K , Schlievert P M
Published: January 2020

Streptococcal pyrogenic exotoxins (SPEs) A, B, and C, like other members of the pyrogenic toxin family, are able to cause toxic shock-like syndromes. One of the major properties of these toxins is the ability to induce T-cell proliferation. Characterization of T cell mitogenicity associated with SPEs A, B, and C was undertaken. SPEs A, B, and C were mitogenic for C57BL10/SnJ and BALB/cWAT T cells, with activities differing in intensity depending on the mouse strain and toxin employed. SPE-induced, T-cell-proliferative activity was dependent on class II major histocompatibility complex molecules expressed on antigen-presenting cells. The abilities of SPEs A, B, and C to preferentially stimulate murine cells with certain T-cell receptor V beta s were investigated by fluorescence-activated cell sorter analysis. SPE A preferentially activated T cells expressing V beta 8 but not V beta 3, 6, or 11, while SPEs B and C preferentially stimulated T cells which did not express any of the tested V beta s.
Experimental infection of newborn pigs with an attaching and effacing Escherichia coli O45:K"E65" strain
Authors: Helie P , Morin M , Jacques M , Fairbrother J M
Published: January 2020

The ability of a nonenterotoxigenic, K88-negative porcine Escherichia coli strain of serogroup O45:K"E65" to induce attaching-effacing lesions was investigated in newborn pigs. Typical attaching-effacing lesions, characterized by intimate adherence of bacteria to mature enterocyte brush borders with effacement of the microvilli, were observed on light and electron microscopy. Bacteria were also seen in intracytoplasmic vacuoles of mature enterocytes and, in areas of heavier colonization, in the lamina propria of the intestinal mucosa. A moderate inflammatory response with mild focal ulceration of the intestinal mucosa was observed. In a sequential study, we observed that the attaching-effacing lesions were well established in the duodenum, jejunum, and ileum at 12 h postinoculation but did not develop in the cecum and colon until 24 to 48 h postinoculation, although bacteria had colonized the latter areas as early as 12 h postinoculation. Initially, bacteria were very intimately attached, with an irregular arrangement on the enterocyte apical cell membrane, and subsequently reoriented to form a typical palisade arrangement with a narrow regular gap between the bacterial cell wall and the enterocyte apical cell membrane. This phenomenon of early intimate attachment of irregularly disposed bacteria has not been reported for human enteropathogenic attaching and effacing E. coli and could represent a new and different mechanism of attachment and effacement to intestinal epithelial cells.
Analysis of adherence of Streptococcus defectivus and endocarditis-associated streptococci to extracellular matrix
Authors: Tart R C , van de Rijn I
Published: January 2020

Pathogenesis of nutritionally variant streptococcal (NVS) endocarditis initiates with bacterial attachment to and colonization of the damaged heart valve surface. Underlying extracellular matrix (ECM) exposed to the environment during damage to cardiac endothelium provides additional receptors that could be involved in bacterial adherence. The ability of NVS and endocarditis-associated streptococci to bind ECM was investigated by using an enzyme-linked immunosorbent assay system that incorporated ECM secreted by baby hamster kidney and human umbilical vein endothelial cells in culture. Streptococcus defectivus, the major species isolated from NVS endocarditis cases, bound ECM of fibroblasts and endothelial cells, indicating that the ECM molecule involved in the binding was a common constituent of diverse matrices. The specific binding of S. defectivus to ECM was demonstrated by saturation binding and specific antibody inhibition studies. Of the 15 S. defectivus strains analyzed, 13 bound ECM, whereas Streptococcus adjacens and NVS serotype III strains were unable to bind the matrix. This selective binding suggested that S. defectivus binds to heart valves through a mechanism different from those of other NVS in subacute bacterial endocarditis. A survey of non-NVS streptococcal endocarditis isolates demonstrated that S. mutans, S. mitis, S. sanguis, and S. faecalis also bound ECM, whereas other viridans species were unable to bind the matrix.
Binding kinetics of Clostridium difficile toxins A and B to intestinal brush border membranes from infant and adult hamsters
Authors: Rolfe R D
Published: January 2020

This study was undertaken to determine if the relative resistance of neonates and infants to Clostridium difficile-associated intestinal disease can be related to age-dependent differences in intestinal receptors for C. difficile toxins A and B. Brush border membranes (BBMs) from the small intestines of adult and infant hamsters were examined for their ability to bind radiolabeled toxins A and B. [125I]toxin A bound to both infant and adult hamster BBMs at physiological temperature, whereas [125I]toxin B did not bind to the BBMs under any of the conditions examined. The number of [125I]toxin A molecules bound at saturation was approximately 4 x 10(10) per micrograms of membrane protein for adult BBMs and 1 x 10(11) per micrograms of membrane protein for infant BBMs. Scatchard plot analysis suggested the presence of a single class of toxin A binding sites on both infant and adult hamster BBMs. Maximal binding capacity and Kd values were 0.63 pmol/mg of protein and 66.7 nM, respectively, for the infant BBMs, and 0.24 pmol/mg of protein and 27 nM, respectively, for the adult BBMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of extracted BBM proteins revealed differences in the proteins of infant and adult BBMs. However, there were not any detectable differences in the protein bands which bound [125I]toxin A between infant and adult hamsters. The results from these investigations indicate that differences in the binding kinetics of toxins A and/or B to infant and adult hamster BBMs do not account for the observed differences in their susceptibility to C. difficile-associated intestinal disease.
Protective immunity induced in Aotus monkeys by recombinant SERA proteins of Plasmodium falciparum
Authors: Inselburg J , Bzik D J , Li W B , Green K M , Kansopon J , Hahm B K , Bathurst I C , Barr P J , Rossan R N
Published: January 2020

We describe the vaccination of Panamanian monkeys (Aotus sp.) with two recombinant blood stage antigens that each contain a portion of the N-terminal region of the SERA (serine repeat antigen) protein of the malaria parasite Plasmodium falciparum. We immunized with either a 262-amino-acid SERA fragment (SERA I) that contains amino acids 24 to 285 of the 989-amino-acid protein or a 483-amino-acid SERA fragment (SERA N) that contains amino acids 24 to 506 as part of a fusion protein with human gamma interferon. The recombinant proteins were shown to stimulate protective immunity when administered with complete and incomplete Freund adjuvant. Four of six immunized monkeys challenged by intravenous inoculation with blood stage P. falciparum developed parasitemias that were reduced by at least 1,000-fold. Two of six immunized monkeys developed parasitemias which were comparable to the lowest parasitemia in one of four controls and were 50- to 1,000-fold lower than in the other three controls.
Protection of immunosuppressed mice against infection with Pseudomonas aeruginosa by recombinant P. aeruginosa lipoprotein I and lipoprotein I-specific monoclonal antibodies
Authors: Finke M , Muth G , Reichhelm T , Thoma M , Duchêne M , Hungerer K D , Domdey H , von Specht B U
Published: January 2020

Outer membrane protein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. The protective effect of OprI vaccination and that of three OprI-specific monoclonal antibodies (MAbs) against infection with P. aeruginosa were tested in immunosuppressed mice. The combination of Oprl and MAb 2A1 protected the mice against a challenge with a 96-fold 50% lethal dose. The binding site of MAb 2A1 was mapped, resulting in the identification of a protective epitope (amino acids 7 to 20).
Effect of urease on HeLa cell vacuolation induced by Helicobacter pylori cytotoxin
Authors: Cover T L , Puryear W , Perez-Perez G I , Blaser M J
Published: January 2020

Concentrated broth culture supernatants from 50 to 60% of Helicobacter pylori strains induce eukaryotic cell vacuolation in vitro. A quantitative assay for cell vacuolation was developed on the basis of the rapid uptake of visibly vacuolated HeLa cells was significantly greater than that of nonvacuolated cells. By using the rapid NRU assay, we sought to determine the roles of H. pylori cytotoxin, urease, and ammonia in the vacuolation of HeLa cells. The NRU of HeLa cells incubated in medium containing ammonium chloride or ammonium sulfate was significantly greater than that of cells incubated in medium alone. In addition, ammonium salts augmented the NRU induced by H. pylori supernatants. The NRU induced by jack bean urease was augmented by the addition of urea to cell culture medium; this suggests that urease-mediated NRU occurs via the generation of ammonia. Acetohydroxamic acid blocked the NRU induced by jack bean urease and urea but failed to block the uptake induced by H. pylori supernatants. Supernatant from a non-urease-producing H. pylori mutant strain induced NRU identical to that of the urease-positive parental strain. These observations indicate that the vacuolating activity in H. pylori supernatants is not mediated solely by urease activity but that it may be potentiated by urease-mediated ammonia production.
Purification and immunological characterization of a GroEL-like protein from Bordetella pertussis
Authors: Burns D L , Gould-Kostka J L , Kessel M , Arciniega J L
Published: January 2020

A GroEL-like protein from Bordetella pertussis was purified. This protein was found to have the tetradecameric subunit structure typical of the GroEL family of proteins and to contain epitopes similar to those of other members of this family, including a human GroEL-like protein. Active immunization of neonatal mice with the B. pertussis GroEL-like protein provided little protection against an aerosol challenge with B. pertussis. Antibodies to this protein were elicited in mice by a standard diphtheria-tetanus-pertussis (DTP) vaccine but not by an experimental acellular pertussis vaccine. Since the Bordetella GroEL-like protein was found to contain epitopes similar to those on the mammalian analog, the potential exists that vaccination with standard DTP vaccines may induce antibodies which react with the mammalian GroEL analog.
T cell-mediated immunity in the lung: a Cryptococcus neoformans pulmonary infection model using SCID and athymic nude mice
Authors: Huffnagle G B , Yates J L , Lipscomb M F
Published: January 2020

T cells are important in systemic anticryptococcal defenses, but a role in controlling an initial pulmonary infection has not been demonstrated. A murine model with intratracheal inoculation was developed to study the acquisition and expression of pulmonary T cell-mediated immunity against Cryptococcus neoformans. Infections with four strains of C. neoformans (305, 68A, 613D, and 52D) in two strains of mice (BALB/c and C57BL/6) were examined. Unencapsulated strain 305 and slowly growing strain 68A were readily controlled apparently by nonimmune pulmonary defenses, and no extrapulmonary dissemination was detected. Strain 613D grew progressively in the lungs and disseminated to the brain and spleen. Strain 52D initially grew rapidly in the lungs and disseminated to the spleen, but a clearance mechanism developed in the lungs after day 7 postinfection and in the spleen after day 28. SCID and athymic nude mice were unable to clear a strain 52D pulmonary infection, and a lethal disseminated infection occurred. Pulmonary clearance could be adoptively transferred into SCID mice infected with strain 52D by use of immune T cells from the spleen and lungs and hilar lymph nodes of infected immunocompetent donors. Furthermore, pulmonary clearance was almost 100-fold better in SCID mice that received immune T cells from the lungs and hilar lymph nodes than in those that received immune T cells from the spleen, even though equivalent levels of delayed-type hypersensitivity were transferred by both cell populations. These adoptive transfer studies suggested that the lung and hilar lymph node T cells from immune animals either are enriched in such a way as to mediate protective immunity or home to the lungs better than do splenic T cells.
Induction of antibody response to Chlamydia trachomatis in the genital tract by oral immunization
Authors: Cui Z D , Tristram D , LaScolea L J , Kwiatkowski T , Kopti S , Ogra P L
Published: January 2020

Groups of BALB/c mice were orally immunized with Chlamydia trachomatis serovar L2/434/Bu in order to characterize the nature and kinetics of the chlamydial antibody response in the cervix and other mucosal sites. These animals were subsequently challenged intravaginally to determine whether oral immunization offers protection against chlamydial antigen shedding in the genital tract. Following oral immunization, immunoglobulin A antibody activity was detected in the genital tract as well as other mucosal sites. Subsequent intravaginal challenges exhibited booster effects on preexisting antibody activity in the genital tract. Significant protection against challenge infection in the genital tract was observed by oral immunization. This was indicated by the absence of any chlamydial antigen shedding in cervical secretions. On the other hand, passively administered chlamydial-specific serum immunoglobulin G antibody did not significantly influence the course of cervical shedding of the organism and did not confer any protection against a subsequent intravaginal challenge. It is concluded that prior oral immunization can induce a secretory antibody response in the genital tract and provide protection against subsequent infection.
Molecular cloning and characterization of a 35.5-kilodalton lipoprotein of Treponema pallidum
Authors: Hubbard C L , Gherardini F C , Bassford P J , Stamm L V
Published: January 2020

A clone expressing a 35.5-kDa recombinant treponemal protein was isolated from a genomic DNA library constructed from Treponema pallidum street strain 14. Polyclonal antiserum raised against the recombinant protein reacted with a corresponding native protein of comparable size in T. pallidum that is specific to the pathogenic treponemes. Radiolabeling of the recombinant protein with [3H]palmitate demonstrated that it is lipid modified. Like other recently characterized T. pallidum lipoproteins, the 35.5-kDa lipoprotein partitioned into the detergent phase from T. pallidum cells fractionated with Triton X-114, suggesting that it is an integral membrane protein. Processing of the recombinant 35.5-kDa lipoprotein from a precursor form to a smaller mature form was not evident in pulse-chase experiments. However, pretreatment of Escherichia coli cells expressing the 35.5-kDa lipoprotein with inhibitors of protein processing or translocation revealed the existence of a higher-molecular-mass precursor. Gene fusion studies with the transposon TnphoA demonstrated the presence of an export signal in the 35.5-kDa lipoprotein that promotes the extracytoplasmic localization of a 35.5-kDa lipoprotein-PhoA hybrid.
Heat shock response of spirochetes
Authors: Stamm L V , Gherardini F C , Parrish E A , Moomaw C R
Published: January 2020

We examined the heat shock response of the pathogenic spirochetes Treponema pallidum, Borrelia burgdorferi, and Leptospira interrogans and certain saprophytic spirochetes. Cellular proteins synthesized after shifts to higher temperatures were [35S]methionine labeled and analyzed by gel electrophoresis and fluorography. Only T. pallidum failed to exhibit an obvious heat shock response. GroEL and DnaK homologs were identified in the various species, although these proteins were not thermoinducible in T. pallidum or Treponema denticola. DNA hybridization studies indicate that spirochetal groEL and dnaK genes are highly conserved.
In vitro inhibition of adhesion of Candida albicans clinical isolates to human buccal epithelial cells by Fuc alpha 1----2Gal beta-bearing complex carbohydrates
Authors: Brassart D , Woltz A , Golliard M , Neeser J R
Published: January 2020

The role of cell surface glycoconjugates as possible adhesion receptors for Candida albicans yeasts on human buccal epithelial cells was investigated by using a quantitative radiometric assay involving 14C-metabolically labeled microorganisms. Various structurally defined soluble glycopeptides and oligosaccharides were tested at a low concentration (1 mg/ml) for their ability to competitively inhibit yeast adhesion to such exfoliated cells. Comparisons were also made with various molecular species previously proposed to act as adhesion molecules. A preparation of glycopeptides derived from pooled human newborn meconiums inhibited the attachment (up to 55%) of all three clinical isolates examined. The mild hydrolysis of fucosyl residues from the above mixture totally abolished its inhibitory potency. By using human milk oligosaccharide probes, the minimal structural requirement for activity was found to be the Fuc alpha 1----2Gal beta determinant (the H sugar sequence found on all blood group substances of the ABO [H] system). By contrast, the fucosylated determinants of the Lewis blood group system were found to be totally inactive. Total adhesion inhibitions were never obtained in the present experiments, suggesting that H disaccharide-bearing cell surface glycoconjugates could act as host receptors for C. albicans on human buccal epithelial cells as a part of a mechanism involving multireceptor specificities.
Comparison of properties of virulent, avirulent, and interferon-resistant Rickettsia prowazekii strains
Authors: Turco J , Winkler H H
Published: January 2020

Several properties of virulent, avirulent, and interferon-resistant Rickettsia prowazekii strains were compared. All of the interferon-resistant rickettsial strains (which were derived from the avirulent Madrid E strain) resembled the virulent Breinl strain in that they grew well in untreated mouse macrophagelike RAW264.7 cells. In contrast, the avirulent Madrid E strain grew poorly in untreated RAW264.7 cells. Pretreatment of interferon-resistant rickettsiae or R. prowazekii Breinl with antirickettsial serum or immunoglobulin G suppressed the ability of the rickettsiae to grow in untreated RAW264.7 cells. Interferon-resistant R. prowazekii strains, like the Madrid E and Breinl strains, rapidly killed a substantial proportion of RAW264.7 cells that had been treated with gamma interferon or very high concentrations of alpha/beta interferon. Untreated infected RAW264.7 cells and interferon-treated mock-infected RAW264.7 cells were not killed during the same period. In cultures of RAW264.7 cells treated with either alpha/beta interferon (120 to 1,200 U/ml) or a subsaturating concentration of gamma interferon (0.5 U/ml), R. prowazekii Breinl organisms killed a higher percentage of the cells than did comparable numbers of R. prowazekii Madrid E organisms or interferon-resistant rickettsiae. Although R. prowazekii Breinl (like R. prowazekii Madrid E) was quite sensitive to gamma interferon in mouse L929 cells, the Breinl strain was resistant to murine alpha/beta interferon compared with the Madrid E strain and the two strains selected for resistance to murine gamma interferon. One of the interferon-resistant strains (strain 60P, which was selected for resistance to murine alpha/beta interferon) differed from the other R. prowazekii strains in that it induced little or no detectable interferon in L929 cell cultures.
Lectinophagocytosis of encapsulated Klebsiella pneumoniae mediated by surface lectins of guinea pig alveolar macrophages and human monocyte-derived macrophages
Authors: Athamna A , Ofek I , Keisari Y , Markowitz S , Dutton G G , Sharon N
Published: January 2020

Macrophages express a mannose/N-acetylglucosamine-specific lectin which serves as a receptor for nonopsonic phagocytosis of mannose-coated particles. We have examined the binding to guinea pig alveolar macrophages in a serum-free medium of 16 Klebsiella pneumoniae serotypes and of the capsular polysaccharides isolated from 7 of these serotypes. Only five polysaccharides containing the repeating sequence Man alpha 2/3Man or L-Rha alpha 2/3-L-Rha bound to the macrophages. Of the 11 bacterial serotypes expressing such disaccharides in their capsular polysaccharides, 7 bound efficiently, 2 bound poorly, and 2 did not bind at all. No binding occurred with five serotypes lacking these disaccharides. Binding of the bacteria was inhibited by homologous and heterologous capsular polysaccharides that contain the disaccharide sequences, by mannan, and by (Man)25BSA (where BSA is bovine serum albumin). Man alpha 2/3Man-containing oligosaccharides were potent inhibitors compared with monosaccharides. Binding was dependent on Ca2+, modulated by cultivating the macrophages on mannan-coated surfaces, and increased in human monocyte-derived macrophages compared with monocytes. The bulk of the bacteria bound to the macrophages was internalized and killed. The data taken together suggest that Klebsiella pneumoniae cells undergo lectinophagocytosis mediated by capsular disaccharides recognized by the mannose/N-acetylglucosamine-specific lectin of macrophages. This may enhance clearance of the organisms from the serum-poor environment of the lung.
Hemolytic and sphingomyelinase activities of Clostridium perfringens alpha-toxin are dependent on a domain homologous to that of an enzyme from the human arachidonic acid pathway
Authors: Titball R W , Leslie D L , Harvey S , Kelly D
Published: January 2020

The N-terminal domain of Clostridium perfringens alpha-toxin, homologous with the nontoxic phospholipase C of Bacillus cereus, was expressed in Escherichia coli and shown to retain all of the phosphatidylcholine hydrolyzing activity of the alpha-toxin, but not the sphingomyelinase, hemolytic, or lethal activities. The C-terminal domain of alpha-toxin showed sequence and predicted structural homologies with the N-terminal region of arachidonate 5-lipoxygenase, an enzyme from the human arachidonic acid pathway which plays a role in inflammatory and cardiovascular diseases in humans.
Serum antibody response to Pseudomonas aeruginosa antigens during corneal infection
Authors: Preston M J , Berk J M , Hazlett L D , Berk R S
Published: January 2020

Previous studies in our laboratory have indicated that naturally resistant, inbred DBA/2J mice mount a greater serum antibody response to Pseudomonas aeruginosa 19660 than susceptible C57BL/6J mice. However, the specificity of the antibody produced was not known. The present study examines the specificity and kinetics of the humoral response of these mouse strains to potential virulence factors produced by the organism during both a primary and a secondary corneal infection administered 4 weeks after the primary infection. Serum antibody levels specific for lipopolysaccharide (LPS), exotoxin A, phospholipase C (PLC), alkaline protease, elastase, and flagella were measured by enzyme-linked immunosorbent assay. Little or no antibody to either alkaline protease or elastase was detected during either primary or secondary infection. Immunoglobulin G (IgG) antibodies specific to exotoxin A, PLC, and flagella were detected 2 weeks after primary infection, and a rapid response to these antigens was measured 1 week after secondary infection. During primary infection, detectable LPS-specific antibody was only IgM, while IgG appeared only after secondary infection. The kinetics of the humoral response in susceptible C57BL/6J mice were similar to those in resistant DBA/2J mice, although the magnitude of the response varied according to the antigen tested. These results indicate that LPS, exotoxin A, PLC, and flagella are present or produced in amounts that are immunogenic during corneal infection by P. aeruginosa 19660 in the mouse strains tested.
Two novel virulence loci, mxiA and mxiB, in Shigella flexneri 2a facilitate excretion of invasion plasmid antigens
Authors: Andrews G P , Hromockyj A E , Coker C , Maurelli A T
Published: January 2020

A bank of over 4,200 lacZ protein fusions in Shigella flexneri 2a was screened for fusions to temperature-regulated promoters. One mutant, BS260, was completely noninvasive on HeLa cells and mapped to a region on the 220-kb virulence plasmid in which we had previously localized several avirulent temperature-regulated operon fusions (A.E. Hromockyj and A.T. Maurelli, Infect. Immun. 57:2963-2970, 1989). The phenotype of BS260 was similar to that of the previously identified mxi (membrane expression of invasion plasmid antigens) mutants, since it made wild-type intracellular levels of the invasion plasmid antigens (Ipa) but was deficient in the surface expression of IpaB and IpaC. Six kilobases of DNA upstream of the BS260 fusion end joint were cloned, but no temperature-regulated promoter was found, whereas the fusion end joint clone of the noninvasive mxi operon fusion mutant BS226 contained a temperature-regulated promoter. The locus defined by BS260 was designated mxiA, and that defined by BS226 was designated mxiB. Closer analysis of the mxiA and mxiB phenotypes by a cell-free enzyme-linked immunosorbent assay revealed that the mutants failed to excrete IpaB and IpaC into the culture medium, whereas wild-type cells actively released these antigens. Excretion of the ipa polypeptides from wild-type bacteria was confirmed by Western blot analysis of culture supernatants. Protease protection experiments revealed that wild-type S. flexneri 2a actually had much lower levels of surface-exposed IpaB and IpaC relative to those in the total antigen pool. In addition, examination of cellular fractions showed that, although there was no IpaB or IpaC in the outer membrane of BS260 and BS226, the antigens did accumulate in the cytoplasmic membrane. A 76-kDa temperature-regulated polypeptide in wild-type S. flexneri was identified as the putative mxiA gene product. These results strongly suggest that IpaB and IpaC represent truly excreted proteins of S. flexneri and that the mxiA and mxiB loci on the plasmid code for accessory proteins required to facilitate their export through the bacterial outer membrane. These data also suggest that mxiA is part of an operon that specifies additional mxi genes. The products of this operon may constitute a unique multicomponent protein secretion apparatus involved in the transport of Shigella virulence determinants.
Binding of Escherichia coli and Salmonella strains to members of the carcinoembryonic antigen family: differential binding inhibition by aromatic alpha-glycosides of mannose
Authors: Leusch H G , Drzeniek Z , Markos-Pusztai Z , Wagener C
Published: January 2020

Various Escherichia coli and Salmonella strains bound to glycoproteins of the family of carcinoembryonic antigens (CEA). As judged from plateau regions of the binding curves, CEA, nonspecific cross-reacting antigen of Mr 55,000 (NCA-55), and biliary glycoprotein of Mr 85,000 (BGP-85) showed similar binding activities. The binding to ovalbumin was significantly lower and the binding to fetuin was insignificant under identical experimental conditions. The binding of E. coli and S. typhi to the different glycoproteins was similar as judged from the binding curves. In comparison with alpha-methyl-D-mannopyranoside, aromatic alpha-glycosides of mannose were more potent binding inhibitors of E. coli but not of salmonellae to CEA and NCA-55. These results are similar to those previously obtained with intestinal epithelial cells and yeast cells (N. Firon, S. Ashkenazi, D. Mirelman, I. Ofek, and N. Sharon, Infect. Immun. 55:472-476, 1987). The binding of E. coli to CEA was inhibited by purified type 1 fimbriae. On the basis of the distribution of CEA-like glycoproteins in tissues and body fluids, the results indicate that glycoproteins of the CEA family may be involved in the recognition of bacteria and the regulation of bacterial colonization.
The carboxyl-terminal region of staphylococcal enterotoxin type A is required for a fully active molecule
Authors: Hufnagle W O , Tremaine M T , Betley M J
Published: January 2020

Staphylococcal enterotoxin type A (SEA) gene (sea+) mutations were constructed by exonuclease III digestion or cassette mutagenesis. Five different sea mutations that had 1, 3, 7, 39, and 65 codons deleted from the 3' end of sea+ were identified and confirmed by restriction enzyme and nucleotide sequence analyses. Each of these sea mutations was constructed in Escherichia coli and transferred to Staphylococcus aureus by using the plasmid vector pC194. Culture supernatants from the parent S. aureus strain that lacked an enterotoxin gene (negative controls) and from derivatives that contained either sea+ (positive control) or a sea mutation were examined for in vitro sensitivity to degradation by monkey stomach lavage fluid, the ability to cause emesis when administered by an intragastric route to rhesus monkeys, and the ability to induce T-cell proliferation and by Western immunoblot analysis and a gel double-diffusion assay with polyclonal antibodies prepared against SEA. Altered SEAs corresponding to the predicted sizes were visualized by Western blot analysis of culture supernatants for each of the staphylococcal derivatives that contained a sea mutation. The altered SEA that lacked the C-terminal amino acid residue behaved like SEA in all of the assays performed. The altered SEA that lacked the three C-terminal residues of SEA caused T-cell proliferation but was not emetic; this altered SEA was degraded in vitro by monkey stomach lavage fluid and did not reach in the gel double diffusion assay. Altered SEAs that lacked 7, 39, or 65 carboxyl-terminal residues were degraded by stomach lavage fluid in vitro, did not produce an emetic response, and did not induce T-cell proliferation or form a visible reaction in the gel double-diffusion assay.