bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of acid secretion from parietal cells by non-human-infecting Helicobacter species: a factor in colonization of gastric mucosa?
Authors: Vargas M , Lee A , Fox J G , Cave D R
Published: January 2020

Helicobacter pylori has been shown to produce a protein that inhibits acid secretion from parietal cells. We have examined other non-human-infecting Helicobacter species for this property by measuring the uptake of [14C]aminopyrine into rabbit parietal cells as an indirect assessment of acid secretion. Helicobacter felis and an isolate from a rhesus monkey were shown to inhibit acid secretion. Isolates of Helicobacter mustelae gave variable responses. Whole bacteria and cell-free sonicates impaired the uptake of [14C]aminopyrine. We also tested other bacteria, including Escherichia coli, Proteus vulgaris, Klebsiella oxytoca, and Campylobacter jejuni. As whole organisms, these control bacteria had little effect on acid secretion, but sonicates caused pronounced inhibition that was partially heat labile. Pronase treatment of H. pylori destroyed its inhibitory effect. These results suggest that most Helicobacter species, but not all isolates, are able to inhibit acid secretion from rabbit parietal cells. This property may be a factor in the establishment of long-term infection by these species.
Antibodies and DNA probes used to analyze variant populations of the Indochina-1 strain of Plasmodium falciparum
Authors: Hommel M , Hughes M , Bond P , Crampton J M
Published: January 2020

Ten variant populations derived from the Indochina-1 strain of Plasmodium falciparum were analyzed by using (i) hyperimmune serum raised against some of these populations in squirrel monkeys and (ii) an oligonucleotide probe based on the rep-20 sequence, which had previously been shown to be a useful marker of diversity. Although all 10 subpopulations had an identical fingerprint pattern on Southern blots probed with the oligonucleotide, thus demonstrating a homogeneous genetic makeup, they all had a different phenotype for erythrocyte-associated antigens, thus confirming serological variant-specific differences. Antibodies to erythrocyte-associated antigens were measured with a new technique including immunogold and silver enhancement. The results of this study indicate that antigenic variation can occur without major genomic reorganization.
Virulence-related protein synthesis in Naegleria fowleri
Authors: Hu W N , Band R N , Kopachik W J
Published: January 2020

Protein synthesis patterns of the low-virulence Naegleria fowleri LEE strain from axenic culture, the same strain after mouse brain passage to increase virulence, and the same strain after growth on bacteria were studied. Comparisons of accumulated proteins, in vivo-synthesized proteins, and in vitro-synthesized proteins translated from poly(A)+ mRNA were made. Differences between amoebae from the different treatments were noted. After 6 months in axenic culture, pathogenic protein synthesis patterns were lost and there was a decrease in virulence. Therefore, the increase in virulence is correlated with numerous specific changes in protein synthesis.
Immunogenicity of Salmonella typhi Ty21a vaccine for young children
Authors: Murphy J R , Grez L , Schlesinger L , Ferreccio C , Baqar S , Muñoz C , Wasserman S S , Losonsky G , Olson J G , Levine M M
Published: January 2020

An attenuated Salmonella typhi Ty21a vaccine was administered to 18 infants and toddlers (less than or equal to 24 months old) to determine its safety and immunogenicity. The vaccination (10(9) CFU per dose, three doses) was well tolerated. However, after the vaccination there was no evidence of a humoral or cellular immune response to S. typhi. The vaccine used was known to be immunogenic for older children and adults. The results support the view that the immunogenicity of Ty21a is age dependent.
Characterization of immunodominant surface antigens of Haemophilus somnus
Authors: Corbeil L B , Kania S A , Gogolewski R P
Published: January 2020

An immunodominant Haemophilus somnus outer membrane protein with an apparent molecular mass of 40 kDa on Western blots (immunoblots) of gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was characterized because a monospecific antibody against this antigen was protective. This monospecific antibody was used for immunoaffinity purification of the antigen. The immunoaffinity-purified antigen reacted with a polyclonal antibody to the 40-kDa antigen but not with a monoclonal antibody (3G9) which reacted with the 40-kDa antigen in gradient gels. On 8 or 10% gels, the approximately 40-kDa antigen was resolved as two bands, a 40-kDa band which reacted with the protective monospecific polyclonal antibody (p40) and a band of lower molecular mass which reacted with monoclonal antibody 3G9. The latter antigen was designated p39. Both antigens were conserved in all H. somnus isolates tested. The specific antibodies were also used to detect cross-reacting antigens in other gram-negative bacteria. Antibody to p40 reacted with proteins of 55 to 28 kDa, with the greatest intensity shown among proteins from other members of the family Pasteurellaceae. Antibody to p40 was reduced by absorption with live H. somnus or other members of the family Pasteurellaceae, so the antigen appears to be surface exposed. Antibody to p39 only cross-reacted with a broad band (38 to 40 kDa) in Haemophilus agni. Since H. agni is not a bovine pathogen and since convalescent-phase serum from H. somnus-infected animals did recognize p39, the latter may be a good immunodiagnostic antigen, if the lack of cross-reactivity with antigens in other gram-negative bacteria is confirmed with a polyclonal antibody to p39. The cross-reactivity of antiserum to p40 with antigens of members of the family Pasteurellaceae and the ability of this antiserum to protect against H. somnus pneumonia indicate that p40 may be a useful vaccine antigen for H. somnus disease and perhaps even diseases caused by other members of the family Pasteurellaceae.
Stimulation of human neutrophil oxidative metabolism by nonopsonized Neisseria gonorrhoeae
Authors: Naids F L , Rest R F
Published: January 2020

Nonopsonized gonococci possessing opacity-associated (Opa; previously PII) outer membrane proteins stimulate neutrophils to undergo a vigorous oxidative response when measured by luminol-dependent chemiluminescence (LDCL). In these studies, we characterized the mechanism of this stimulation. No gonococci that we tested induced measurable release of neutrophil superoxide anion (O2-) or hydrogen peroxide (H2O2) as measured by reduction of cytochrome c or the oxidation of scopoletin, respectively. Neutrophils pretreated with gonococci and then exposed to phorbol myristate acetate, the chemotactic peptide formylmethionylleucylphenylalanine, or opsonized zymosan released levels of neutrophil O2- and H2O2 comparable to controls, indicating that gonococci were not preventing or inhibiting neutrophil O2- or H2O2 release. To ascertain a possible explanation for these seemingly contradictory observations (i.e., induction of LDCL, but no release of O2- or H2O2), we further characterized the ability of Opa+ gonococci to stimulate LDCL. By using 1 mM azide and 4 U of horseradish peroxidase to monitor extracellular LDCL selectively and 2,000 U of catalase to monitor intracellular LDCL selectively, we determined that greater than 80% of total gonococcus-induced neutrophil LDCL occurred intracellularly. In addition, neutrophils stimulated with Opa+ gonococci showed a marked increase in O2 uptake and hexose monophosphate shunt activity. We conclude that Neisseria gonorrhoeae induces neutrophil oxidative metabolism without causing release of detectable amounts of reactive oxygen intermediates into the surrounding milieu. The gonococcus apparently directs oxidase assembly and activity to the phagolysosomal membrane. This could be a mechanism by which extracellular gonococci persist for extended periods in vivo in the presence of high concentrations of neutrophils.
Protective role of bovine neutrophils in Pasteurella haemolytica-mediated endothelial cell damage
Authors: Breider M A , Kumar S , Corstvet R E
Published: January 2020

The purpose of this study was to determine if Pasteurella haemolytica can directly injure bovine pulmonary endothelial cells (EC) and if neutrophils have a beneficial or detrimental role in bacterium-EC interaction. Various combinations of live P. haemolytica, heat-killed P. haemolytica, anti-P. haemolytica immune serum, polymyxin B, and bovine neutrophils were added to confluent monolayers of bovine EC. Monitoring included determination of 51Cr release from EC, phase microscopy, and transmission electron microscopy. Although toxic changes were not evident at 5 h postinoculation, both live and heat-killed P. haemolytica produced extensive EC damage by 22 h postinoculation. Damage by live P. haemolytica was prevented only when both neutrophils and immune serum were used. Polymyxin B effectively prevented the toxic effect of heat-killed P. haemolytica, suggesting that lipopolysaccharide was the major toxic factor. Morphological studies showed close apposition of P. haemolytica to EC membranes, neutrophil activation, and adherence to EC but no evidence of neutrophil-associated EC membrane damage. These studies demonstrate that neutrophils and immune serum in combination are effective in preventing EC damage mediated by live P. haemolytica.
Nuclease-sensitive binding of an Actinobacillus actinomycetemcomitans leukotoxin to the bacterial cell surface
Authors: Ohta H , Kato K , Kokeguchi S , Hara H , Fukui K , Murayama Y
Published: January 2020

A leukotoxin of Actinobacillus actinomycetemcomitans 301-b was solubilized from cell-associated membrane vesicles by treatment with externally added DNase and RNase and was further purified by a procedure which included ammonium sulfate fractionation, gel filtration chromatography, and ion-exchange chromatography. The purified toxin had a molecular mass of 113,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a high isoelectric point (approximately 8.8). From these characteristics, it was to be expected that the membrane vesicle toxin was almost identical to the leukotoxin extracted with polymyxin B in an earlier study (C.-C. Tsai, B. J. Shenker, J. M. DiRienzo, D. Malamud, and N. S. Taichman, Infect, Immun. 43:700-705, 1984). The treatment with DNase and RNase was also highly effective for solubilizing the leukotoxin directly from whole cells, suggesting that the toxin is secreted extracellularly but retained in nucleic acids on the outermost surface of bacterial cells.
Detection of immunoglobulin A1 protease-induced Fab alpha fragments on dental plaque bacteria
Authors: Ahl T , Reinholdt J
Published: January 2020

The mechanisms by which immunoglobulin A1 (IgA1) protease activity may enable bacteria to evade the effect of specific secretory IgA (S-IgA) antibodies are not clear. A possibility which has received indirect experimental support is that bacteria, as a consequence of the protease activity, become coated with incompetent Fab alpha fragments instead of with intact antibody molecules. Using a combination of nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, we detected Fab alpha fragments not only on oral streptococci (Streptococcus sanguis and Streptococcus gordonii) incubated in saliva but also on the bacteria in incipient dental plaque. These results are of relevance to our previous observation that IgA1 protease activity may neutralize the ability of S-IgA antibodies to inhibit the adherence of oral streptococci to saliva-coated hydroxyapatite.
Antigen-specific suppressor T cells respond to recombinant interleukin-2 and other lymphokines
Authors: Taylor C E , Fauntleroy M B , Stashak P W , Baker P J
Published: January 2020

Previous studies have shown that transfer of whole spleen cell populations obtained from primed donors or transfer of purified T cells enriched for suppressor activity (Ts) to recipient mice decreased the antibody response to pneumococcal polysaccharide type III (SSS-III) when the animals were simultaneously immunized with SSS-III. In the present studies, such suppression of the antibody response was transferred with 10- to 100-fold fewer primed spleen cells when the cells were treated in vitro with recombinant interleukin-2 (rIL-2) before transfer; spleen cells from naive mice or mice primed with an unrelated antigen (dextran) and then treated with rIL-2 did not cause suppression of the antibody response to SSS-III, thereby eliminating the possibility of nonspecific carryover effects induced by rIL-2. In vivo administration of rIL-2 at the time of immunization with an optimally immunogenic dose of SSS-III resulted in significant (P less than 0.05) suppression of the antibody response relative to that of control animals, suggesting that IL-2 augments the clonal expansion of Ts cells in vivo. Further, the ability of passively administered anti-IL-2 receptor antibody to inhibit generation of Ts cells in vivo is consistent with such a view. Spleen cells from primed animals treated with rIL-4, rIL-5, or gamma interferon--but not those from primed animals treated with rIL-6--likewise were able to transfer suppression of the antibody response with fewer cells than those required when primed cells not treated with lymphokines were used. Thus, these studies indicate that Ts cell activity is greatly influenced by lymphokines produced by helper T cells. The studies also suggest that these lymphokines are required during activation and/or clonal expansion of Ts cells.
Efficacy of recombinant leukotoxin in protection against pneumonic challenge with live Pasteurella haemolytica A1
Authors: Conlon J A , Shewen P E , Lo R Y
Published: January 2020

The recombinant leukotoxin (rLKT) of the bacterium Pasteurella haemolytica A1 was examined for its ability to protect cattle from experimental challenge with logarithmic-phase P. haemolytica. Six different vaccines were utilized in the experiment: P. haemolytica culture supernatant, P. haemolytica culture supernatant enriched with rLKT, rLKT alone, P. haemolytica culture supernatant enriched with Escherichia coli supernatant not containing LKT, E. coli supernatant alone, and phosphate-buffered saline. rLKT alone showed no protective capacity against development of clinical signs of respiratory disease or against development of postmortem lung lesions after experimental challenge. It was, however, shown to enhance the efficacy of the culture supernatant vaccine and decrease clinical signs and pneumonic lesions. The complexity of protective immunity in this disease is emphasized in this study, and, although LKT is an important virulence factor of the organism, an immune response to LKT alone does not protect animals against disease.
Pseudomonas aeruginosa recognizes carbohydrate chains containing type 1 (Gal beta 1-3GlcNAc) or type 2 (Gal beta 1-4GlcNAc) disaccharide units
Authors: Ramphal R , Carnoy C , Fievre S , Michalski J C , Houdret N , Lamblin G , Strecker G , Roussel P
Published: January 2020

The adhesion of Pseudomonas aeruginosa to type 1 (Gal beta 1-3GlcNAc) and type 2 (Gal beta 1-4GlcNAc) disaccharide determinants was studied in a microtiter adhesion assay and a thin-layer chromatography bacterial overlay assay. The oligosaccharides were prepared from human breast milk and human urine and were conjugated to hexadecylaniline to form neoglycolipids that were used in were used in the assays. Both the mucoid and the nonmucoid strains that were studied recognized the disaccharide determinants Sialylation of the oligosaccharides did not suppress binding in the thin-layer chromatography assay, but alpha 2-6-linked sialic acid blocked binding in the microtiter assay. The use of bovine serum albumin instead of gelatin as a blocking agent against nonspecific binding completely suppressed binding in the thin-layer chromatography assay. Isogenic nonpiliated mutants of nonmucoid strains constructed by interrupting the pilin gene retained their adhesive capacity for the disaccharide units, indicating that binding to the disaccharides was mediated by a nonpilus adhesin(s). Furthermore, two monoclonal antibodies that recognize the type 2 disaccharide determinant (Gal beta 1-4GlcNAc) partially inhibited adhesion of a pair of piliated and nonpiliated isogenic strains to mucin. This study suggests that P. aeruginosa utilizes a nonpilus adhesin(s) to bind to disaccharide units commonly found in mucins, in addition to pili and alginate, two previously described adhesins.
Susceptibility of mast cell-deficient W/Wv mice to Cryptosporidium parvum
Authors: Harp J A , Moon H W
Published: January 2020

Mast cell-deficient W/Wv infant mice were similar to normal mice in their susceptibility to and recovery from infection with the intestinal protozoan Cryptosporidium parvum. W/Wv adult mice were significantly more susceptible to primary infection than were normal adult mice, but both groups recovered at a similar rate.
Adherence of Bacteroides fragilis group species
Authors: Brook I , Myhal M L
Published: January 2020

The ability of piliated and capsulated Bacteroides fragilis and Bacteroides ovatus to adhere to intestinal cells and mucus was investigated. The adherence of piliated and capsulated strains was at least five times greater than the adherence of their nonpiliated and noncapsulated or capsulated only counterparts. These data illustrate the importance of pili as promoters of adherence of B. fragilis group species to the gastrointestinal mucosa.
Virulence of Staphylococcus aureus mutants altered in type 5 capsule production
Authors: Albus A , Arbeit R D , Lee J C
Published: January 2020

Most clinical isolates of Staphylococcus aureus produce microcapsules (uronic acid-containing extracellular polysaccharides) that are detectable by serologic methods but are not visible by negative staining. Among the 11 reported serotypes, capsule types 5 and 8 comprise approximately 75% of all isolates. Transposon mutagenesis was performed on S. aureus to create mutants altered in capsule expression. Tn918 was introduced into the capsule type 5 strain Reynolds by filter mating, and a capsule-deficient transconjugate, JL236, was isolated. The wild-type strain was transformed with JL236 chromosomal DNA to confirm that transfer of the appropriate-size chromosomal fragment containing Tn918 generated a capsule-deficient transformant. Strain Reynolds was mutagenized with ethyl methanesulfonate to obtain a capsule-negative mutant (strain JL240). Capsular phenotypes were determined by colony immunoblots, antibody adsorption experiments, and transmission electron microscopy. The virulences of the parental and mutant strains in mice were compared. The 50% lethal doses for strains Reynolds, JL236, and JL240 were similar (10(8.59), 10(8.98), and 10(8.93) CFU, respectively). Animals injected intraperitoneally with either wild-type or mutant strains had comparable levels of bacteremia at 3 and 24 h after challenge. Quantitative cultures of blood and kidneys from animals challenged intravenously with sublethal doses of the S. aureus strains also showed no differences in bacterial clearance or renal abscess formation. These studies indicate that the type 5 S. aureus microcapsule does not promote bacterial virulence in the animal models tested.
Augmented production of granulocyte-macrophage colony-stimulating factor and alpha/beta interferon in mice inoculated with heat-killed Corynebacterium liquefaciens
Authors: Yoshida T , Hotta T , Shimokata K , Ichihara M , Isobe K , Nakashima I
Published: January 2020

We demonstrated that heat-killed Corynebacterium liquefaciens bacteria, as a known potent host immune activity modulator, stimulate spleen cells to produce granulocyte-macrophage (GM) colony-stimulating factor (CSF) and another CSF with similar activity, as well as alpha/beta interferon, when injected intravenously into mice. Alpha/beta interferon was shown to be produced by C. liquefaciens-activated plastic-G-10 column-adherent cells (A cells) in a thymus-independent manner. In contrast, augmented production of GM-CSF required the action of C. liquefaciens-activated T lymphocytes that collaborated with normal A cells. Non-T spleen cells from C. liquefaciens-stimulated athymic mice, however, produced an alternative CSF that partially replaced GM-CSF. Correspondingly, the numbers of GM-producing CFU developing in cultures of spleen cells from C. liquefaciens-treated euthymic or athymic mice were 10 to 30 times higher than those in cultures of spleen cells from untreated mice. These results suggest that gram-positive rods such as C. liquefaciens activate T and A cells for production of multiple cytokines and that potential cooperative actions of these cytokines underlie the known immunomodulatory action of coryneforms.
Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases
Authors: Mengaud J , Geoffroy C , Cossart P
Published: January 2020

The region flanking the transposon in a Tn1545-induced lecithinase-negative mutant of Listeria monocytogenes EGD was cloned and sequenced. The transposon had inserted in ORF D, the open reading frame previously identified downstream from hlyA, the gene encoding listeriolysin O. The complete sequence of ORF D from strain EGD has been determined as well as that of two other strains: LO28, a clinical isolate; and LM8, an epidemic strain. ORF D is 1,533 bp long and encodes a protein highly homologous to metalloproteases of bacilli, Serratia sp., Legionella pneumophila, and Pseudomonas aeruginosa. It was renamed prtA. Northern RNA blot analysis indicated that prtA is the first gene of a 6-kb operon, suggesting that the lecithinase-negative phenotype of the mutant might be due to a polar effect of the transposon insertion.
Effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of osteoblastic clone MC3T3-e1 cells
Authors: Horiguchi Y , Nakai T , Kume K
Published: January 2020

The effects of Bordetella bronchiseptica dermonecrotic toxin on the structure and function of a clonal osteoblastic cell line, MC3T3-E1, were investigated. The toxin induced a morphological change in the cells from a spindle shape to a spherical form with many blebs. The toxin-treated cells were viable and grew to form confluent cell layers composed of irregularly shaped cells and multinuclear cells. The toxin inhibited elevation of alkaline phosphatase activity in the cells in a dose-dependent manner at concentrations from 10 pg to 10 ng/ml. The accumulation of type I collagen in the cells was also reduced by the toxin. Since high alkaline phosphatase activity and accumulation of collagen are closely linked to differentiation of the cells into osteoblasts, it is considered likely that B. bronchiseptica dermonecrotic toxin impairs the ability of the cells to differentiate.
Siderophore production by Pseudomonas pseudomallei
Authors: Yang H M , Chaowagul W , Sokol P A
Published: January 2020

Eighty-four strains of Pseudomonas pseudomallei isolated from patients with melioidosis were examined for siderophore production. All the strains were shown to produce siderophore both on chrome azurol S agar plates and in liquid medium under iron-deficient conditions. Chemical assays indicated that the siderophore belongs to the hydroxamate class. Addition of iron to the culture medium resulted in increased culture growth with markedly decreased yield of siderophore. Siderophore produced by strain U7 was purified by gel filtration chromatography, and the molecular weight was estimated to be 1,000. When this partially purified siderophore was added to culture medium, it promoted iron uptake by P. pseudomallei in the presence of EDTA and enhanced growth of the organism in the presence of transferrin. We have given this siderophore the trivial name malleobactin.
Differential adherence of hydrophobic and hydrophilic Candida albicans yeast cells to mouse tissues
Authors: Hazen K C , Brawner D L , Riesselman M H , Jutila M A , Cutler J E
Published: January 2020

Using an ex vivo binding assay, we previously demonstrated that yeast cells grown at 37 degrees C display binding specificity in mouse spleen, lymph node, and kidney tissues. In spleen and lymph node tissues, binding was predominantly in regions rich in macrophages. Here, we tested the possibility that hydrophobic and hydrophilic cells bind differentially to host tissues. Hydrophobic and hydrophilic yeast cells of four Candida albicans strains were incubated for 15 min at 4 degrees C with cryostat sections of organs that had been rapidly frozen after removal from BALB/cByJ mice. Unattached cells were removed by washing, and the sections were examined. Hydrophobic cells bound diffusely and abundantly to all tissues, while hydrophilic cell attachment was restricted to specific sites. For example, hydrophobic cells bound to the white and red pulp and the marginal zones in spleens, whereas hydrophilic cells attached primarily to the marginal zones. Hydrophobic yeast cells attached throughout lymph node tissue including paracortical areas, but hydrophilic cell attachment occurred primarily at the subcapsular and trabecular sinuses, EDTA inhibited the adherence of hydrophilic cells but not hydrophobic cells to mouse tissues. Hydrophobic C. albicans strains displaying similar levels of hydrophobicity differed quantitatively in their levels of attachment to kidney and spleen tissues, confirming our earlier observation that surface hydrophobicity is not the sole determinant in adherence to host cells. Other studies have shown that hydrophobic and hydrophilic cells display different virulence characteristics related to their surface properties and that hydrophobic cells are more virulent than hydrophilic cells. Taken together, the present results suggest that the enhanced virulence of hydrophobic cells over hydrophilic cells may be due, in part, to the potential of hydrophobic cells to bind throughout various organs following clearance from the bloodstream.
Adhesion of Pseudomonas aeruginosa pilin-deficient mutants to mucin
Authors: Ramphal R , Koo L , Ishimoto K S , Totten P A , Lara J C , Lory S
Published: January 2020

Attachment of Pseudomonas aeruginosa to epithelial cells or tracheobronchial mucin is mediated by surface adhesins. Pili, composed of monomeric pilin subunits, make up one such class of adhesins. The formation of pili and flagella in P. aeruginosa is under the control of the alternative sigma factor rpoN. Isogenic mutant strains with insertionally inactivated rpoN genes were constructed with strains PAK, 1244, and CF613 and were tested for their ability to adhere to respiratory mucin. All rpoN mutants showed significant reduction of adherence to mucin relative to that of their wild-type parents. In contrast, the adherence of pilin structural gene mutants was similar to the adherence of wild types. These results provide suggestive evidence that P. aeruginosa also binds to mucin via adhesins that are distinct from pilin and are still under the genetic control of rpoN. Unlike the laboratory strain PAK, the clinical strains 1244 and CF613 are capable of agglutinating erythrocytes. The rpoN mutation had a minimal effect on the interaction of bacteria with erythrocytes, indicating that the transcription of a gene(s) specifying the agglutination phenomenon does not utilize rpoN. These findings collectively indicate the existence of several classes of adhesins on the surface of P. aeruginosa that may play an important role in colonization of the human respiratory tract.
Localized adherence and attaching-effacing properties of nonenteropathogenic serotypes of Escherichia coli
Authors: Albert M J , Alam K , Ansaruzzaman M , Montanaro J , Islam M , Faruque S M , Haider K , Bettelheim K , Tzipori S
Published: January 2020

Traditional enteropathogenic Escherichia coli serotypes demonstrate a plasmid-mediated localized adherence in cultured HeLa or HEp-2 cells and induce an attaching-effacing intestinal lesion, both of which are considered pathognomonic and causes of diarrhea. This study describes three E. coli strains from infantile diarrhea which share these properties but belong to serotypes (O2:H2, O2:H25 and O15:H2) not considered enteropathogenic.
Congenital syphilis in newborn rabbits: immune functions and susceptibility to challenge infection at 2 and 5 weeks of age
Authors: Fitzgerald T J , Froberg M K
Published: January 2020

Experiments were performed to further elaborate on our congenital syphilis rabbit model. Attempts were made to determine whether in utero exposure to Treponema pallidum would stimulate immune reactivity and whether this activity would, in turn, affect lesion development upon challenge infection. Newborn rabbits aged 2 or 5 weeks were obtained from control does or from does infected intravenously with T. pallidum during pregnancy. Congenitally infected newborns exhibited increased immunologic functions. Concanavalin A-induced T-lymphocyte proliferation was elevated at both 2 and 5 weeks. In addition, macrophage Ia expression and RPR antibody titers were increased at 5 weeks. In separate experiments, newborn rabbits from control does or from does infected during pregnancy were challenged intradermally with viable organisms at either 2 or 5 weeks of age. Subsequent lesion severity was markedly increased in those newborns previously exposed to treponemes in utero. These observations further strengthen our model for congenital transmission of T. pallidum during pregnancy. We propose that at least some of the tissue pathology in syphilitic infection is associated with activated host defenses.
Mapping antibody-binding domains of the major outer surface membrane protein (OspA) of Borrelia burgdorferi
Authors: Schubach W H , Mudri S , Dattwyler R J , Luft B J
Published: January 2020

The major outer surface membrane protein of Borrelia burgdorferi, OspA, is one of several antigens recognized by sera from some patients in the chronic phase of Lyme borreliosis. We have expressed the OspA open reading frame in Escherichia coli and generated a series of deletion constructs of the gene and expressed them as trpE fusion proteins in E. coli. These constructs were used to identify antibody-binding sites of both rabbit antiserum and mouse monoclonal antibodies (MAbs) directed against OspA. All antibodies tested failed to bind to a fusion protein containing the first 61 amino acids of OspA, suggesting that the amino-terminal domain of OspA is unexposed to the cell surface. The binding site for one MAb, 184.1, was identified in a region centered around amino acid 61, while the binding site for MAb 105.5 was identified in a region centered around amino acids 214 to 217. Sera from two patients which were reactive to OspA identified distinct epitopes that lie between those recognized by our MAbs.
Evidence that the major outer membrane protein of Chlamydia trachomatis is glycosylated
Authors: Swanson A F , Kuo C C
Published: January 2020

The major outer membrane protein (MOMP) of Chlamydia trachomatis was determined to be a glycoprotein on the basis of susceptibility to glycosidase digestion and the presence of carbohydrate by staining and radiolabeling. The MOMP of the serovar L2 organisms was isolated by electroelution from the protein band excised from the gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incubation of MOMP with N-glycosidase F, an endoglycosidase that cleaves the N-glycan, and periodate resulted in two new molecular weight species. While MOMP treated with N-glycosidase F showed a lower-molecular-weight mobility, the periodate-treated MOMP increased in molecular weight. Both treatments abolished the ability of the MOMP to bind to HeLa cell components. In the immunoblot, the reactivity to the monoclonal antibody specific against the C. trachomatis species was preserved. The endoglycosidase specific to O-linked glycan, endo-alpha-N-acetylgalactosaminidase, had no visible effect on the isolated MOMP. Carbohydrate was detected in the MOMP by p-phenylenediamine staining of the protein band in the gel following SDS-PAGE. Autoradiograms of proteins of chlamydial organisms metabolically labeled with [3H]galactose or [3H]glucosamine and separated by SDS-PAGE revealed the MOMP band. The isolated MOMP was shown to bind specifically to concanavalin A, wheat germ agglutinin, and Dolichos biflorus agglutinin in the lectin binding assay. No binding was observed with Ulex europaeus agglutinin I, soybean agglutinin, or Ricinus communis agglutinin.
Intramacrophage growth of Mycobacterium avium during infection of mice
Authors: Frehel C , de Chastellier C , Offredo C , Berche P
Published: January 2020

Growth of the virulent Mycobacterium avium strain TMC 724 in host tissues during persistent infection of mice was studied. Following intravenous infection of C57BL/6 mice, the kinetics of bacterial growth was biphasic in the spleen and liver, with a significant reduction of the multiplication rate after day 21 to 28 of infection. An electron-microscopic study of the liver and spleen of infected mice showed that the bacteria were strictly intracellular. They were observed within inflammatory macrophages populating granulomas disseminated in host tissues. The bacteria were confined to the phagosome compartment, and they were encapsulated. Phagosome-lysosome fusions were encountered, but the bacteria showed no visible signs of degradation and continued to multiply. These results are the first in vivo evidence that virulent M. avium multiplies exclusively intracellularly and that encapsulated bacteria resist the microbicidal mechanisms of macrophages inside the phagosomal compartment.
Lack of cleavage of immunoglobulin A (IgA) from rhesus monkeys by bacterial IgA1 proteases
Authors: Reinholdt J , Kilian M
Published: January 2020

Bacterial immunoglobulin A1 (IgA1) proteases cleaving IgA1 and secretory IgA1 molecules in the hinge region are believed to be important virulence factors. Previous studies have indicated that IgA of humans, gorillas, and chimpanzees are the exclusive substrates of these enzymes. In a recent study, IgA from the rhesus monkey was found to be susceptible to the IgA1 protease activity of Streptococcus pneumoniae. In an attempt to reproduce this observation, we found that neither five isolates of S. pneumoniae nor other IgA1 protease-producing bacteria representing different cleavage specificities caused cleavage of rhesus monkey IgA. Hence, the rhesus monkey does not appear to be a suitable animal model for studies of IgA1 proteases as virulence factors.
Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1
Authors: Weiser J N , Gotschlich E C
Published: January 2020

We examined whether outer membrane protein A (OmpA) contributes to gram-negative pathogenesis by determining the effect of mutagenesis of ompA in a virulent Escherichia coli K-1 isolate. An OmpA mutant was generated by insertion of the transposon TnphoA, which was genetically modified to increase the efficiency of its delivery by conjugation. The mutant was less virulent than its parent strain in two models of E. coli K-1 infection. Equal inocula of the OmpA+ and OmpA- strains fed to neonatal rats resulted in a sevenfold-greater incidence of bacteremia at 72 h from the OmpA+ strain. The lethal effect of the OmpA- mutant was significantly less than that of the OmpA+ parent strain when inoculated onto the chorioallantoic membrane of 10-day embryonated chick eggs. There was, however, no difference between strains in growth characteristics under physiologic conditions, either in rat serum or in unembryonated chick eggs. In the presence of a 10-day chick embryo, there was a 10-fold increase in the survival and growth of the OmpA+ strain. Correction of the mutation in ompA with an E. coli K-12 ompA gene restored a level of virulence equivalent to that of the parent strain. The ompA mutant was more sensitive to the bactericidal effect of pooled human serum by the classical pathway of complement activation. These results suggest that OmpA contributes to E. coli K-1 pathogenesis by a mechanism which may involve increased serum resistance.
Significance of flagella in colonization resistance of rabbits immunized with Campylobacter spp
Authors: Pavlovskis O R , Rollins D M , Haberberger R L , Green A E , Habash L , Strocko S , Walker R I
Published: January 2020

Cross-protection among different Lior and Penner serogroups of Campylobacter spp. was studied. Rabbits were orally immunized by gastric feeding with Campylobacter spp., and 27 to 30 days later, they were challenged with matched or unmatched serogroups by the removable intestinal tie adult rabbit diarrhea (RITARD) procedure. When immunized animals were challenged with different Lior serotypes, no protection against colonization was seen; however, when challenged with homologous Lior serogroups, protection was demonstrated. Immune animals were colonized for an average of 1 day or less versus at least 6 days for nonimmune animals. Rabbits challenged with matched Penner-unmatched Lior strains showed only marginal protection. Our study also demonstrated that flagella are important in initiating colonization and eliciting protective immunity. Campylobacter coli VC167B3, an isogenic, nonflagellated mutant, did not colonize rabbits regardless of the route of administration. Single feeding of the mutant strain did not protect the host, whereas three feedings, 48 h apart, resulted in complete protection against the flagellated parent strain. When mutant strain immunized rabbits were challenged with other strains of the same Lior serotype, marginal protection was obtained. Immunogold labeling indicated that there is one or more antigens on the cell surface of the nonflagellated mutant which reacts with a polyclonal antiserum from organisms of the same Lior serogroup. These data implicated the flagellum as the cross-strain protective component of the Lior antigen complex.
The heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans: relationship to OmpA proteins
Authors: Wilson M E
Published: January 2020

The outer membrane of Actinobacillus actinomycetemcomitans contains a 29-kDa protein which exhibits heat modifiability on sodium dodecyl sulfate-polyacrylamide gels and represents a major target for immunoglobulin G antibody in sera of periodontitis patients colonized by this organism. In the present study, the N-terminal amino acid sequence of the 29-kDa outer membrane protein was determined and compared with reported sequences for other known proteins. The heat-modifiable outer membrane protein of A. actinomycetemcomitans was found to exhibit significant N-terminal homology with the OmpA proteins of other gram-negative bacteria. Moreover, this protein reacted with antiserum raised against the purified OmpA protein of Escherichia coli K-12. Whether the heat-modifiable OMP of A. actinomycetemcomitans also shares functional properties of OmpA proteins, particularly with respect to bacteriophage receptor activity, is presently under investigation.
Neutralization of Chlamydia trachomatis: kinetics and stoichiometry
Authors: Peeling R W , Brunham R C
Published: January 2020

Monoclonal antibodies to the major outer membrane protein of Chlamydia trachomatis were used to neutralize C. trachomatis infectivity in HeLa 229 cells and to determine the kinetics and stoichiometry of the reaction. In vitro neutralization of C. trachomatis infectivity proceeded as a first-order reaction and required an activation energy of approximately 20 kcal/mol (ca. 84 kJ/mol). The rate of neutralization was linear with respect to antibody concentration and reaction temperature. The efficiency of neutralization decreased exponentially as the ratio of noninfective to infective chlamydiae increased in the antigen preparation. The neutralization assay was also significantly affected by reaction parameters such as the reaction volume and the duration of incubation. Stoichiometric calculations showed that an average ratio of 10(3) and 10(4) immunoglobulin molecules per chlamydial particle was required to yield 50% neutralization by monoclonal antibodies specifying serovar-specific and species-specific epitopes, respectively. The implications of these findings for vaccine design and for the role of the major outer membrane protein in the pathogenesis of chlamydial infections are discussed.
Roles of complement and complement receptor type 3 in phagocytosis of Listeria monocytogenes by inflammatory mouse peritoneal macrophages
Authors: Drevets D A , Campbell P A
Published: January 2020

Listeria monocytogenes is a facultative intracellular bacterium that is phagocytosed by and can proliferate within cells of the mononuclear phagocyte system. However, the receptors used by macrophages to internalize this organism have not been identified. In the experiments described here, the contributions of serum complement component C3 and macrophage complement receptor type 3 (CR3) to opsonization and phagocytosis of L. monocytogenes by mouse inflammatory peritoneal macrophages were studied. An assay which allowed the distinction of adherent versus internalized bacteria was used to show that following mixing of L. monocytogenes with inflammatory macrophages, greater than 95% of the bacteria bound were internalized by these phagocytes. When immunofluorescent antibodies to C3 and immunoglobulin were used, C3 but not immunoglobulin was detected on L. monocytogenes following incubation in normal serum or ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetracetic acid-Mg(2+)-chelated serum. When macrophages were incubated with 5% serum and L. monocytogenes in a standard assay, approximately 80% of the phagocytosis was inhibited by heat-inactivated serum or by the addition of F(ab')2 anti-C3 antibody. The role of macrophage CR3 was demonstrated by the ability of anti-CR3 monoclonal antibody M1/70 to decrease phagocytosis to the same levels as those seen with heat-inactivated serum. These experiments indicated that in the presence of normal serum, L. monocytogenes is phagocytosed by inflammatory macrophages primarily because CR3 on these cells binds to C3 deposited on the bacterial surface.
Sequence and structural analysis of surface protein antigen I/II (SpaA) of Streptococcus sobrinus
Authors: LaPolla R J , Haron J A , Kelly C G , Taylor W R , Bohart C , Hendricks M , Pyati J P , Graff R T , Ma J K , Lehner T
Published: January 2020

Streptococcal antigen I/II or the surface protein antigen A (SpaA) of Streptococcus sobrinus is an adhesin which mediates binding of the organism to tooth surfaces. The complete sequence of the gene which encodes SpaA has been determined. The gene consists of 4,584 bp and encodes a protein of 1,528 amino acid residues. The deduced amino acid sequence shows extensive homology with those of the cell surface adhesins from Streptococcus mutans serotypes c and f and from Streptococcus sanguis. Structural analysis of the N-terminal region (residues 50 to 550), which is rich in alanine and includes four tandem repeats of an 82-residue sequence, suggests that it adopts an alpha-helical coiled-coil conformation. Cell surface hydrophobicity may be associated with this region. The C-terminal region is more conserved and includes two tandem repeats of a 39-residue proline-rich sequence. A further proline-rich sequence in this region is predicted to span the cell wall. Although a hydrophobic sequence is present in the C-terminal region, it appears to be too short to span the cell membrane. Anchoring of SpaA in the cell membrane may therefore require some form of posttranslational modification or association with another membrane protein.
Evidence for invasion of a human oral cell line by Actinobacillus actinomycetemcomitans
Authors: Meyer D H , Sreenivasan P K , Fives-Taylor P M
Published: January 2020

Actinobacillus actinomycetemcomitans, an oral bacterial species associated with periodontal disease, was found to invade human cell lines. Invasion was demonstrated by recovery of viable organisms from gentamicin-treated KB cell monolayers and by light and electron microscopy. Internalization occurred through a cytochalasin D-sensitive process. Invasion efficiencies of some A. actinomycetemcomitans strains were comparable to those of invasive members of the family Enterobacteriaceae. Differences in invasiveness were correlated with bacterial colonial morphology. Smooth variants invaded more proficiently than rough variants. A. actinomycetemcomitans can undergo a smooth-to-rough colonial morphology shift which results in the loss of invasiveness. Coordinated regulation of genes involved in the rough-to-smooth phenotypic transitions may play a role in the episodic nature of periodontal disease.
Role of tumor necrosis factor in macrophage leishmanicidal activity in vitro and resistance to cutaneous leishmaniasis in vivo
Authors: Theodos C M , Povinelli L , Molina R , Sherry B , Titus R G
Published: January 2020

Recombinant human tumor necrosis factor (TNF) and purified murine TNF were both able to activate macrophages to destroy intracellular Leishmania major in vitro. In addition, parasitizing macrophages with L. major markedly increased the ability of the cells to produce TNF. Finally, when mice were vaccinated with an avirulent form of L. major, the animals produced large amounts of TNF but no gamma interferon in response to infection with virulent L. major. Treating these mice with a neutralizing anti-TNF antibody led to partial but not complete inhibition of the resistant state, which suggests that factors other than TNF and gamma interferon contribute to resistance to L. major.
Live vaccine strain of Francisella tularensis: infection and immunity in mice
Authors: Fortier A H , Slayter M V , Ziemba R , Meltzer M S , Nacy C A
Published: January 2020

The live vaccine strain (LVS) of Francisella tularensis caused lethal disease in several mouse strains. Lethality depended upon the dose and route of inoculation. The lethal dose for 50% of the mice (LD50) in four of six mouse strains (A/J, BALB/cHSD, C3H/HeNHSD, and SWR/J) given an intraperitoneal (i.p.) inoculation was less than 10 CFU. For the other two strains tested, C3H/HeJ and C57BL/6J, the i.p. log LD50 was 1.5 and 2.7, respectively. Similar susceptibility was observed in mice inoculated by intravenous (i.v.) and intranasal (i.n.) routes: in all cases the LD50 was less than 1,000 CFU. Regardless of the inoculation route (i.p., i.v., or i.n.), bacteria were isolated from spleen, liver, and lungs within 3 days of introduction of bacteria; numbers of bacteria increased in these infected organs over 5 days. In contrast to the other routes of inoculation, mice injected with LVS intradermally (i.d.) survived infection: the LD50 of LVS by this route was much greater than 10(5) CFU. This difference in susceptibility was not due solely to local effects at the dermal site of inoculation, since bacteria were isolated from the spleen, liver, and lungs within 3 days by this route as well. The i.d.-infected mice were immune to an otherwise lethal i.p. challenge with as many as 10(4) CFU, and immunity could be transferred with either serum, whole spleen cells, or nonadherent spleen cells (but not Ig+ cells). A variety of infectious agents induce different disease syndromes depending on the route of entry. Francisella LVS infection in mice provides a model system for analysis of locally induced protective effector mechanisms.