bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of sensitivity of blood forms of Trypanosoma cruzi to antibody-mediated, complement-dependent lysis
Authors: Kierszenbaum F , Ramirez M A
Published: January 2020

The numerous reports on lysis of blood (trypomastigote) forms of Trypanosoma cruzi by specific antibodies plus complement have systematically shown that a certain proportion of parasites survives. However, it is not known whether the insensitive organisms represent a subpopulation (or clones) or a certain developmental phase of otherwise morphologically identical parasites. In this work, we established that partial lysis was not due to the use of insufficient amounts of lytic reagents. Thus, supernatants of lytic reaction mixtures killed the same proportion of T. cruzi as previously unused reagents. Moreover, in parallel tests in which the trypomastigote concentration was up to four times greater than that used in standard lysis tests, the percentages of lysis were comparable. Incubation periods as long as 4 h did not increase the extent of lysis beyond the value observed after only 1 h, indicating that the routinely used 1-h incubation was appropriate. The extent of lysis was not increased by additional amounts of antibody, complement, or both. Instead, trypomastigotes surviving immune lysis, washed, and incubated with fresh diluent for 45 to 120 min before being used in new lysis tests did manifest additional sensitivity to immune lysis. Three successive infections in mice with parasites which had survived immune lysis led to the production of trypanosomes that displayed the same level of resistance to immune lysis as the original, untreated parasite population. Of interest, the average parasitemias of these groups of mice did not evidence a tendency to increase, as might have occurred if an immune-lysis-resistant subpopulation had been selected. Since trypomastigotes exhibiting resistance to immune lysis can eventually become sensitive, resistance to immune lysis does not represent an insensitive parasite subpopulation. This resistance appears to be modulated by the presence of the lytic reagents and might involve expression of as yet unidentified surface components playing a role in complement activation.
Cloning and expression of the phospholipase D gene from Corynebacterium pseudotuberculosis in Escherichia coli
Authors: Songer J G , Libby S J , Iandolo J J , Cuevas W A
Published: January 2020

A toxic phospholipase D (PLD) is putatively involved in pathogenesis of Corynebacterium pseudotuberculosis infections. We report here the cloning and expression of the PLD gene (pld) in Escherichia coli. A cosmid library of DNA from C. pseudotuberculosis biovar ovis isolate Whetten 1 was constructed and screened for PLD-producing recombinants by plating them on LB agar containing sheep erythrocytes and equi factors. One recombinant, designated pCpO1, yielded a gene product which displayed synergistic hemolytic and sphingomyelinase D activities, both of which are characteristic of PLD. Subcloning into pUC19 yielded a recombinant, pCpO50, which contained a 1.8-kilobase insert. Analysis of supernatant fluids and cell extracts of cultures of E. coli(pCpO50) revealed sphingomyelinase activity and a protein of about 31,000 Mr, neither of which were detected in E. coli(pUC19). The 31-kilodalton protein also reacted with antibodies in serum from a sheep naturally infected with C. pseudotuberculosis, serum which also contained PLD-neutralizing antibodies. When Southern blots of BamHI digests of DNA from biovar ovis and biovar equi isolates of C. pseudotuberculosis were probed with pCpO50, bands of 4.8 and 1.9 kilobases, respectively, were seen, suggesting that the genome organization of pld is different for isolates from the two biovars.
Passive immunization prevents induction of Lyme arthritis in LSH hamsters
Authors: Schmitz J L , Schell R F , Hejka A G , England D M
Published: January 2020

We determined that sera obtained from hamsters infected with Borrelia burgdorferi could prevent the induction of Lyme arthritis. When irradiated hamsters were administered immune serum and subsequently challenged with B. burgdorferi, no evidence of infection was detected. Recipients failed to develop swelling of the hind paws, and no histopathologic changes were detected. In addition, B. burgdorferi was not recovered from tissues of hamsters that were passively immunized. By contrast, irradiated hamsters that were administered normal hamster serum or saline and infected with the Lyme spirochete developed arthritis. Extensive histopathologic changes occurred in the hind paws and knee joints, and spirochetes were recovered from most of the tissues examined. These results show that immune serum can confer complete protection on recipient hamsters to challenge with B. burgdorferi.
The capsular polysaccharide is a major determinant of serum resistance in K-1-positive blood culture isolates of Escherichia coli
Authors: Leying H , Suerbaum S , Kroll H P , Stahl D , Opferkuch W
Published: January 2020

Serum resistance is a major virulence factor of gram-negative bacteria, and K-1 polysaccharide has been shown to contribute to serum resistance in selected strains. To obtain further information about the role of K-1 in serum resistance and to find out whether loss of the ability to produce K-1 can induce loss of serum resistance, we studied the serum resistance of mutants derived from completely serum-resistant, K-1-positive blood culture isolates of Escherichia coli by selection for resistance to infection with K-1 specific bacteriophages. The amounts of K-1 polysaccharide produced by wild-type strains and mutants were measured, and outer membrane protein and lipopolysaccharide (LPS) patterns were analyzed. In each group of mutants, several highly serum-sensitive strains were found. All mutant strains expressed less K-1 than did the corresponding wild-type strains. Mutants that became highly serum sensitive always had less K-1 than did mutants with less-pronounced changes of serum resistance. A few mutants derived from different wild-type strains showed increased expression of outer membrane proteins with molecular weights of about 46,000 and 67,000. All of the wild-type strains examined had smooth-type LPS, and only two mutants had altered LPS structures; alterations of mutants in outer membrane proteins and LPS could not be correlated with alterations of serum resistance. The results indicate that for K-1-positive blood culture strains of E. coli, K-1 expression is a prerequisite for serum resistance, and loss of ability to synthesize K-1 leads to loss of serum resistance.
Differences in microbicidal activities of human macrophages against Toxoplasma gondii and Trypanosoma cruzi
Authors: Israelski D M , Araujo F G , Wachtel J S , Heinrichs L , Remington J S
Published: January 2020

The microbicidal activities of normal human pelvic macrophages against infection by the intracellular protozoa Toxoplasma gondii and Trypanosoma cruzi were examined. Macrophages allowed infection by T. gondii and yet possessed remarkable microbicidal activity against this organism. In contrast, human macrophages were not microbicidal against T. cruzi.
The Dr hemagglutinin, afimbrial adhesins AFA-I and AFA-III, and F1845 fimbriae of uropathogenic and diarrhea-associated Escherichia coli belong to a family of hemagglutinins with Dr receptor recognition
Authors: Nowicki B , Labigne A , Moseley S , Hull R , Hull S , Moulds J
Published: January 2020

The receptor specificities of four Escherichia coli cloned hemagglutinins, AFA-I, AFA-III, F1845 fimbriae, and the Dr hemagglutinin were studied. Evidence is provided that all four hemagglutinins recognize as their receptor the Dr blood group antigen. However, results of experiments using enzyme-treated erythrocytes and monoclonal antibodies indicate that the four adhesins recognize different epitopes on the Dr antigen and thus constitute a family of Dr receptor-recognizing bacterial adhesins. Furthermore, the same results suggest that the Dr antigen itself may be divided into subcomponents on the basis of bacterial adhesins.
Bacterial lipopolysaccharide potentiates gamma interferon-induced cytotoxicity for normal mouse and rat fibroblasts
Authors: Dijkmans R , Van Damme J , Cornette F , Heremans H , Billiau A
Published: January 2020

Gamma interferon (IFN-gamma) can be cytolytic for normal mouse fibroblasts isolated from embryonic or adult tissue (R. Dijkmas, B. Decock, H. Heremans, J. Van Damme, and A. Billiau, Lymphokine Res. 8:25-34, 1989). This cytotoxicity has been shown to be transcription and translation dependent, thereby suggesting involvement of a suicidelike mechanism. The dose of IFN-gamma required for cytotoxicity is higher than that needed for antiviral and macrophage activation but can be reduced 10- to 100-fold by cotreatment of the cells with tumor necrosis factor or interleukin-1 (IL-1) or both, two cytokines that by themselves are not toxic for these cells. Here, we show that bacterial lipopolysaccharide (LPS), which alone has no effect on the viability of mouse fibroblasts, stimulates cell suicide induced by IFN-gamma. The effect was observed in cultures that were virtually free of nonfibroblastoid cells. LPS showed its toxicity-enhancing effect only if applied on the cells simultaneously with or immediately after treatment with IFN-gamma. Pretreatment of the cells with LPS was ineffective. Inclusion of antibodies directed against tumor necrosis factor alpha or IL-1 alpha in the culture medium did not block the cytotoxic effect of combined IFN-gamma plus LPS treatment. The time courses of cell toxicity appearance in fibroblasts treated with combined IFN-gamma plus LPS or IFN-gamma plus IL-1 were similar. In addition to LPS, heat-killed gram-negative (Escherichia coli) but also gram-positive (Staphylococcus aureus, Listeria monocytogenes) bacteria were found to enhance IFN-gamma-induced cell death. These findings suggest that IFN-gamma formed in vivo during infectious processes directly aggravates tissue destruction.
Bordetella pertussis filamentous hemagglutinin: evaluation as a protective antigen and colonization factor in a mouse respiratory infection model
Authors: Kimura A , Mountzouros K T , Relman D A , Falkow S , Cowell J L
Published: January 2020

Filamentous hemagglutinin (FHA) is a cell surface protein of Bordetella pertussis which functions as an adhesin for this organism. It is a component of many new acellular pertussis vaccines. The proposed role of FHA in immunity to pertussis is based on animal studies which have produced some conflicting results. To clarify this situation, we reexamined the protective activity of FHA in an adult mouse respiratory infection model. Four-week-old BALB/c mice were immunized with one or two doses of 4 or 8 micrograms of FHA and then aerosol challenged with B. pertussis Tohama I. In control mice receiving tetanus toxoid, the CFU in the lungs increased from 10(5) immediately following infection to greater than 10(6) by days 5 and 9 after challenge. Mice immunized with FHA by the intraperitoneal or intramuscular route had significantly reduced bacterial colonization in the lungs. A decrease in colonization of the trachea was also observed in FHA-immunized mice. Evaluation of antibody responses in these mice revealed high titers of immunoglobulin G (IgG) and IgM to FHA in sera and of IgG to FHA in lung lavage fluids. No IgA to FHA was detected. BALB/c mice were also passively immunized intravenously with either goat or rat antibodies to FHA and then aerosol challenged 24 h later, when anti-FHA antibodies were detected in the respiratory tract. Lung and tracheal colonization was markedly reduced in mice immunized with FHA-specific antibodies compared with those receiving control antibodies. In additional studies, the role of FHA in the colonization of the mouse respiratory tract was evaluated by using strain BP101, an FHA mutant of B. pertussis. FHA was important in the initial colonization of the mouse trachea, but was not required for colonization of the trachea later in the infection. FHA was not a factor in colonization of the lungs. Collectively, these experiments demonstrate (i) that systemic immunization with FHA can provide significant protection against B. pertussis infection in both the lower and upper respiratory tract of mice as defined by the lungs and trachea, respectively; (ii) that this protection is mediated primarily by serum antibodies to FHA, which transudate into respiratory secretions; and (iii) that FHA is an important upper respiratory tract colonization factor. These studies provide further evidence for the role of FHA in pertussis pathogenesis and immunity.
Glycoprotein glycans that inhibit adhesion of Escherichia coli mediated by K99 fimbriae: treatment of experimental colibacillosis
Authors: Mouricout M , Petit J M , Carias J R , Julien R
Published: January 2020

Calf diarrhea due to infection by enterotoxigenic Escherichia coli was treated by administration of glycoprotein glycans derived from bovine plasma. The glycan moieties of the nonimmunoglobulin fraction of plasma mimicked the oligosaccharide moiety of intestinal receptors recognized by K99 pili. These glycoprotein glycans inhibited adhesion of E. coli K99+ ST+ to erythrocyte glycoconjugates in vitro, and they protected colostrum-deprived newborn calves against lethal doses of enterotoxigenic E. coli (10(10) bacteria). Adhesion of bacteria to the intestines (duodenum, jejunum, and ileum) was significantly reduced (by 2 orders of magnitude) in treated calves.
Cloning and nucleotide sequence of a heat-stable enterotoxin gene from Vibrio cholerae non-O1 isolated from a patient with traveler's diarrhea
Authors: Ogawa A , Kato J , Watanabe H , Nair B G , Takeda T
Published: January 2020

We determined the nucleotide sequence of the heat-stable enterotoxin (STa) gene of the Vibrio cholerae non-O1 strain NRT36 isolated from a patient with traveler's diarrhea. The gene is chromosomally encoded and the presumed product is 78 amino acids in length, with a molecular weight of 8,814, though the genes of Escherichia coli STa(s) (STh and STp) are encoded in plasmid DNA and both products are 72 amino acids in length. The first 18 amino acids at the NH2 terminus were hydrophobic, suggesting that this region of the polypeptide acts as a signal sequence for the toxin. The last 17 amino acids at the COOH terminus were identical to those deduced from the toxin (NAG-ST) produced by V. cholerae non-O1 strain A-5 isolated from a frozen shrimp. The deduced amino acid sequence of the NAG-ST precursor had 50 and 46% homology to those of E. coli STh and STp, respectively. The hydropathy plot analysis of each predicted protein revealed similar profiles between them, suggesting that the NAG-ST precursor has structural similarity to those of E. coli STa(s).
Development of an in vitro model for study of non-O1 Vibrio cholerae virulence using Caco-2 cells
Authors: Panigrahi P , Tall B D , Russell R G , Detolla L J , Morris J G
Published: January 2020

Non-O1 Vibrio cholerae strains have been reported as a causative agent of diarrhea throughout the world. We recently reported that non-O1 V. cholerae strains cause diarrhea in human volunteers. In this study we evaluated the virulence of three strains of non-O1 V. cholerae in a Caco-2 cell adherence assay by light and electron microscopy. A-5 is an environmental isolate which failed to colonized volunteers and did not cause diarrhea. It exhibited low numbers of organisms adherent to Caco-2 cells, leaving the microvilli intact. Strain 2076-79, isolated from a patient with diarrhea, colonized human volunteers without producing disease. It adhered to Caco-2 cells in moderate numbers without producing any damage to the microvilli. Strain NRT36S, a clinical isolate, colonized human volunteers and produced significant diarrhea disease. This strain adhered in very large numbers to Caco-2 cells and caused damage to the brush borders. Membrane-bound bacteria were also seen within the cytoplasm of these cells. Scanning electron microscopy confirmed the generalized adherence of NRT36S to the microvilli of Caco-2 cells. The three strains did not appear to compete with each other for binding sites on Caco-2 cells and were not adherent when assays were conducted at 4 degrees C. Our results with strains A-5, 2076-79, and NRT36S correlate well with observations in human volunteer studies, suggesting that Caco-2 cells provide an appropriate in vitro system for further investigation of the pathogenesis of non-O1 V. cholerae gastroenteritis.
Characterization of a monoclonal antibody that selectively recognizes a subset of Entamoeba histolytica isolates
Authors: Bhattacharya A , Ghildyal R , Bhattacharya S , Diamond L S
Published: January 2020

Monoclonal antibody 2D7.10 recognized an antigen present in seven of nine isolates of axenically cultured Entamoeba histolytica and absent in all other Entamoeba isolates studied. The antigen was absent in two isolates: 200:NIH and Rahman. All nine isolates belonged to pathogenic zymodeme II. Western blot (immunoblot) analysis and treatment with periodate and the proteolytic enzyme trypsin suggest that the antigen recognized by 2D7.10 is a carbohydrate moiety.
Role of lipopolysaccharides in adherence of Actinobacillus pleuropneumoniae to porcine tracheal rings
Authors: Bélanger M , Dubreuil D , Harel J , Girard C , Jacques M
Published: January 2020

The ability of 17 Actinobacillus pleuropneumoniae isolates, representing serotypes 1, 2, 5, and 7, to adhere to tracheal rings maintained in culture was examined. Porcine tracheal rings were infected, and 8 h after inoculation, adherent bacterial cells were evaluated. A. pleuropneumoniae adhered to tracheal rings, and marked variations were observed between and even within serotypes, suggesting that adherence of this microorganism is not primarily related to the serotype of the isolate. No relationship was found between adherence to porcine tracheal rings and plasmid profiles, virulence in mice, hemagglutination, capsular material thickness, or whole-cell protein profiles. On the other hand, we observed that all isolates of serotypes 1 and 5 had a semirough-type lipopolysaccharide (LPS), whereas isolates of serotypes 2 and 7 had a smooth-type LPS (75%) or a semirough-type LPS (25%). Results showed that 83% of isolates with a smooth-type LPS adhered in large numbers to tracheal rings, whereas 80% of isolates with a semirough-type LPS adhered poorly (P less than 0.007). Our data indicated that the degree of adherence of A. pleuropneumoniae to porcine tracheal rings appeared to be related, at least in part, to LPS profiles. Furthermore, LPS seemed to be the adhesin of A. pleuropneumoniae, since purified LPS blocked adherence of this microorganism to porcine tracheal rings.
Gene encoding the major subunit of CS1 pili of human enterotoxigenic Escherichia coli
Authors: Perez-Casal J , Swartley J S , Scott J R
Published: January 2020

Some enterotoxigenic strains of Escherichia coli (ETEC) utilize the CS1 pilus for colonization of human intestinal epithelium. We have cloned the gene which encodes the major CS1 subunit and called it cooA (for coli surface antigen one). Hybridization showed that the ETEC strain from which it was cloned carried cooA on a plasmid different from the one encoding its positive regulator, rns. Based on the cooA DNA sequence, cleavage with signal peptidase would be expected to produce a mature protein of 15.2 kDa; a 16-kDa polypeptide that reacted with CS1-specific antiserum was observed on electrophoresis. At the protein level, there was 92% similarity and 55% identity between cooA and cfaB, the major colonization factor antigen I (CFA/I) antigen. However, CS1-specific antisera did not react with CfaB. No hybridization was seen between either of two different cooA probes and total DNA from ETEC strains expressing AFA-1, CFA/I, CS2, CS3, CS4, CS5, or CS6.
Genetic relationships among strains of avian Escherichia coli associated with swollen-head syndrome
Authors: White D G , Wilson R A , Gabriel A S , Saco M , Whittam T S
Published: January 2020

Genetic diversity among 22 Escherichia coli strains isolated from chickens with swollen-head syndrome (SHS), an acute respiratory disease of domestic poultry, and 93 strains isolated from birds with colibacillosis was assessed on the basis of allelic variation at 20 enzyme-encoding loci detected by multilocus enzyme electrophoresis. SHS isolates from Spain and Canada were polymorphic at 14 loci and were classified into 19 multilocus genotypes, defining clones that differed on average at 34% of the loci. In most cases, SHS isolates of different clonal genotypes were distinct in O:H serotype and expressed different fimbrial antigens. Comparisons with 93 isolates obtained from birds with colibacillosis revealed enzyme polymorphisms at 17 of 20 loci, with an average of 3.5 alleles per locus. In the total sample, 56 clonal genotypes were distinguished, with 27 (23%) of the isolates belonging to one of three common clones. Both SHS and colibacillosis isolates were genetically diverse, with an average single-locus diversity of 0.36, indicating that a wide variety of naturally occurring bacterial clones is associated with these acute avian infections. Six previously defined groups of clones identified in diseased birds from the United States were represented in isolates from Spain, indicating that similar clones occur in widely separated geographic areas. In addition, one group of SHS isolates was closely related to a recognized widespread clone complex incriminated in human septicemia and meningitis. The results suggest that certain strains implicated in SHS infections belong to a clone complex whose members have special attributes that promote involvement in invasive diseases in humans and animals.
Adenylate cyclase toxin of Bordetella pertussis: production, purification, and partial characterization
Authors: Leusch M S , Paulaitis S , Friedman R L
Published: January 2020

Bordetella pertussis produces a number of virulence determinants which contribute to its pathogenicity. One factor, the adenylate cyclase toxin (ACT), has been suggested to directly penetrate human phagocytes and disrupt their normal function by direct production of intracellular cyclic AMP (cAMP). Experiments evaluating the production of cell-associated ACT in liquid cultures of B. pertussis 504 demonstrated that the greatest activity was observed during mid-log-phase growth. Urea extracts of cells harvested during the time of maximal ACT production have been used to purify the toxin with both biological and enzymatic activities. ACT is a protein with an apparent molecular mass of 220 kDa and an isoelectric point of 7.0. The specific activity of purified ACT is 17,000 mumol of cAMP formed per mg per min. The the biological specific activity of purified ACT is 6,250 nmol of intracellular cAMP formed per mg per min in 2 x 10(6) S49 lymphoma cells per ml. Preparations containing 8 micrograms of ACT completely abrogated the chemiluminescence response of 2 x 10(6) human neutrophils per ml.
Generation and characterization of murine antiflagellum monoclonal antibodies that are protective against lethal challenge with Pseudomonas aeruginosa
Authors: Rosok M J , Stebbins M R , Connelly K , Lostrom M E , Siadak A W
Published: January 2020

Two murine monoclonal antibodies, IIG5 (IgG3) and IVE8 (IgG2a), that bind to Pseudomonas aeruginosa type a flagella and type b flagella, respectively, were prepared by conventional hybridoma methodology. Specificity of each monoclonal antibody for type a or type b flagella was demonstrated by enzyme-linked immunosorbent assay, indirect immunofluorescence, and immunoblotting. The percentage of P. aeruginosa isolates recognized by each monoclonal antibody was analyzed by enzyme-linked immunosorbent assay. Among a panel of 257 flagellated P. aeruginosa clinical isolates, IIG5 bound to 67.7% of the isolates and IVE8 bound to another 30.7%, for a combined coverage of 98.4%. Inhibition of motility of P. aeruginosa by the monoclonal antibodies was observed in vitro in a soft agar assay and was dose dependent. The protective efficacy of IIG5 and IVE8 was examined in a mouse burn wound sepsis model. The antiflagellum monoclonal antibodies provided specific and significant prophylactic and therapeutic protection against lethal challenge with P. aeruginosa strains.
Human phagocytes have multiple lipid A-binding sites
Authors: Golenbock D T , Hampton R Y , Raetz C R , Wright S D
Published: January 2020

Bacterial lipopolysaccharide (LPS) is a potent stimulus of cells, yet a target protein for LPS has not been defined. We used two approaches to define LPS-binding sites on cell surfaces: one assay measured binding of LPS-coated erythrocytes (ELPS) to cultured human cells, and a second measured binding of a radiolabeled probe, [32P]lipid IVA, to intact leukocytes. The first approach identified the CD11-CD18 family of integrins as lipid A-binding sites in human phagocytes, and the latter approach demonstrated saturable lipid A binding to intact murine macrophages, as well as to an approximately 95-kDa binding protein in purified membrane preparations. Because CD18 has a known molecular mass of 95 kDa, we sought to determine whether the [32P]lipid IVA-binding site was CD18. Binding of ELPS and [32P]lipid IVA to human macrophages was found to differ with respect to temperature, divalent cation dependence, cellular specificity, and susceptibility to competition by polyanions. To determine whether the previously described 95-kDa lipid A-binding protein was CD18, nitrocellulose-immobilized RAW264.7 membrane proteins were probed with [32P]lipid IVA and subsequently immunoblotted with a monoclonal antibody to murine CD18. The lipid A-binding protein has an electrophoretic mobility slightly different from that of CD18. Moreover, monoclonal antibodies and polyclonal antiserum to the CD11-CD18 family of proteins did not inhibit lipid IVA binding to intact human macrophages. Finally, mononuclear cells from two patients with CD18 deficiency failed to form rosettes with ELPS but bound [32P]lipid IVA normally. Thus, different LPS preparations may bind to cells in a CD18-dependent or -independent manner. Since ELPS is particulate and lipid IVA is a fine dispersion, the identity of the binding site may depend on the physical state of the LPS.
Identification of major Babesia divergens polypeptides that induce protection against homologous challenge in gerbils
Authors: Gorenflot A , Precigout E , Bissuel G , Lecointre O , Brasseur P , Vidor E , L'Hostis M , Schrevel J
Published: January 2020

[35S]methionine-radiolabeled proteins from the Babesia divergens Rouen 1987 isolate were immunoprecipitated with immune sera from three potential hosts: human, ox, and gerbil. The results showed a constant humoral response against major babesial antigens. Similarly, immunoprecipitation of radiolabeled in vitro culture supernatant demonstrated that the exoantigens of 37, 46, 70, and 90 kDA were the immunodominant polypeptides, whatever the host. The effects of vaccination with concentrated supernatant from B. divergens Rouen 1987 in vitro cultures (30 to 40% parasitemia) were examined in gerbils inoculated with the homologous B. divergens isolate. Gerbils having received two or three injections of a whole vaccine dose (1.5 ml of parasitized culture supernatant equivalent [PCSE]) or of a 1:5 diluted vaccine dose (0.3 ml of PCSE) showed 100% survival after intraperitoneal challenge with 10(6) B. divergens-infected gerbil erythrocytes. Moreover, two or three injections of a 1:25 diluted vaccine dose (0.06 ml of PCSE) or 9% NaCl or 1.5 ml of unparasitized culture supernatant equivalent resulted in a mortality rate of 80 to 90% of the infected gerbils. Immunoprecipitation and immunofluorescence assays performed with antisera from vaccinated and control gerbils demonstrated that a single vaccine injection induced a humoral response, which increased slightly after the second or third injection. After challenge, antibody levels increased significantly, although the immunoprecipitation did not display any modification of Babesia antigen patterns.
Localization of the cleavage site specificity determinant of Haemophilus influenzae immunoglobulin A1 protease genes
Authors: Grundy F J , Plaut A G , Wright A
Published: January 2020

Immunoglobulin A1 (IgA1) proteases are produced by a number of different species of bacteria which cause infection at human mucosal surfaces. The sole substrate of these proteases is human IgA1. Cleavage is within the hinge region of IgA1, although there is variability in the exact peptide bond within the hinge region that is cut by a particular protease. The cleavage site of the Haemophilus influenzae type 1 protease is located four amino acids from the cleavage site of the type 2 enzyme. In this study, the region of the H. influenzae IgA1 protease gene (iga) that determines the cleavage site specificity was localized through the comparison of the type 1 and type 2 genes and the construction and analysis of type 1-type 2 hybrid genes. The hybrid genes were generated by in vivo and in vitro techniques which facilitated the selection and screening of randomly generated hybrids. The cleavage site determinant was found to be within a 370-base-pair region near the amino-terminal coding region, in one of two large areas of nonhomology between the two types of H. influenzae iga genes. DNA sequence analysis of the cleavage site determinant and surrounding regions did not reveal a simple mechanism whereby one enzyme type could be converted to the other type. Comparison of the type 2 gonococcal IgA1 protease gene to the two Haemophilus genes revealed a significant amount of homology around the cleavage site determinant, with the two type 2 genes showing greater homology.
Interferon production by Shigella flexneri-infected fibroblasts depends upon intracellular bacterial metabolism
Authors: Hess C B , Niesel D W , Holmgren J , Jonson G , Klimpel G R
Published: January 2020

The role of bacterial invasion and subsequent intracellular metabolism or replication, or both, in the induction of interferon (IFN) production in primary cultures of murine embryo fibroblasts (MEFs) was examined. IFN production appeared to be dependent upon bacterial invasion. MEFs that were challenged with Shigella flexneri cultured at 30 degrees C to inhibit the temperature-dependent virulence gene expression that is essential for invasion failed to produce IFN. Furthermore, inhibition of S. flexneri invasion by pretreatment of MEFs with cytochalasin B resulted in a reduction in IFN production. Intracellular bacterial residence alone, however, was not sufficient for the induction of IFN production since an avirulent isogenic variant of S. flexneri which invades but fails to grow intracellularly did not induce IFN production. In fact, the blocking of bacterial RNA synthesis immediately after cellular uptake of S. flexneri by rifampin inhibited IFN production by MEFs. Transfer of the invasion-encoding plasmid to a noninvasive Escherichia coli strain conferred upon the bacteria the ability to invade MEFs and induce IFN production. These results suggest that the induction of IFN production in S. flexneri-infected fibroblasts requires bacterial invasion and intracellular bacterial metabolism or replication, or both.
Immunocytochemical detection of chitin in Pneumocystis carinii
Authors: Walker A N , Garner R E , Horst M N
Published: January 2020

Polyclonal antisera against chitin and chitin oligomers were used to stain Pneumocystis carinii by the immunoperoxidase technique in Formalin-fixed, paraffin-embedded sections of four human lung biopsies and in alcohol-fixed, paraffin-embedded cell blocks of two bronchioloalveolar lavage specimens from infected human patients. In all cases, the antisera bound P. carinii but did not bind the host tissue elements. Moreover, the antisera bound not only to the cyst forms of P. carinii but also to the intracystic bodies and to the trophic forms. Preadsorption of the anti-chitin antiserum with purified chitin abolished all staining of P. carinii. Our results indicate that P. carinii produces chitin at more than one stage of its life cycle in the infected human host.
Regulation of expression of Streptococcus mutans genes important to virulence
Authors: Hudson M C , Curtiss R
Published: January 2020

Studies were initiated to investigate the regulation of Streptococcus mutans genes which are believed to be important to virulence. Operon fusions were constructed between S. mutans gene regulatory regions and a promoterless chloramphenicol acetyltransferase gene (cat) found on the plasmid pMH109. Specifically, fusions were generated between cat and the S. mutans genes encoding fructosyltransferase (ftf) and the glucosyltransferase B/C (gtfB/C) operon. Constructs were confirmed by restriction enzyme analysis, and the fusions were subcloned into the integration vehicle pVA891. Following generation of multimeric DNA, recombinant plasmids were introduced into the s. mutans genome by Campbell-type insertion, resulting in single-copy operon fusions. Chloramphenicol acetyltransferase specific activities were used to monitor the expression of the S. mutans gtfB/C operon and ftf determinants. The expression of these genes is increased by the presence of sucrose and is followed by a rapid decline in expression over time. Additionally, expression of the gtfB/C operon is increased in S. mutans cells bound to artificial tooth pellicles.
Identification of lectin-binding proteins in Chlamydia species
Authors: Swanson A F , Kuo C C
Published: January 2020

Lectin-binding proteins of chlamydiae were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. All three Chlamydia species tested expressed two proteins when whole-elementary-body lysates were reacted with the biotinylated lectin Dolichos biflorus agglutinin. The protein with a molecular mass of 18 kilodaltons (kDa) responded strongly compared with a higher-molecular-mass protein that varied from 27 to 32 kDa with each chlamydia strain tested. Among six lectins tested, including concanavalin A, D. biflorus agglutinin, Ulex europaeus agglutinin, soybean agglutinin, peanut agglutinin, and wheat germ agglutinin, the latter was the only lectin that did not recognize any chlamydial protein. For each lectin that reacted against the elementary body of serovar L2 of Chlamydia trachomatis, the same two peptides, an 18-kDa peptide and a 32-kDa peptide, were revealed. These two polypeptides adhered to HeLa cell surface components. Binding of a lectin to the L2 reticulate body resulted in a reduced response at the 18-kDa peptide. The 18- and 32-kDa peptides were purified from L2 serovar elementary bodies by affinity chromatography. The two proteins isolated from a concanavalin A-agarose column maintained their lectin-binding capacities and elicited hemagglutinating properties against mouse erythrocytes. Periodate oxidation abolished the abilities of the peptides to adhere to any of the lectins tested. These results suggest that these lectin-binding proteins are glycoproteins that may be an essential factor for attachment of chlamydial organisms to host cells.
Relative permissiveness of macrophages from black and white people for virulent tubercle bacilli
Authors: Crowle A J , Elkins N
Published: January 2020

Epidemiological, clinical, and histopathological evidence suggests that black people are more susceptible to tuberculosis than are white people. The cellular basis of this putative susceptibility was investigated in vitro by comparing responses of blood-derived macrophages from black and white donors to experimental infection with virulent tubercle bacilli. Phagocytes from pairs of black and white donors were infected. The uptake and replication of the tubercle bacilli in these cells were measured by microscopic counts and by CFU counts of bacilli at 0, 4, and 7 days. The effects of donor serum, of 1,25-(OH)2-vitamin D3, and gamma interferon on the infection also were studied. Black-donor phagocytes killed more bacilli during phagocytosis than white-donor phagocytes did. However, the bacilli grew consistently and significantly faster in successfully infected macrophages from black than from white donors, especially in the presence of black-donor serum. 1,25-(OH)2-vitamin D3 gave significantly less protection against tubercle bacilli to macrophages from black donors than to macrophages from white donors. The permissiveness of the macrophages from the two races was affected equally by gamma interferon. These results demonstrate some inherent and environmental liabilities in the monocytic phagocytes and serum of black people compared with white people, which may contribute to their greater susceptibility to tuberculosis.
Cryptosporidiosis in guinea pigs: an animal model
Authors: Chrisp C E , Reid W C , Rush H G , Suckow M A , Bush A , Thomann M J
Published: January 2020

Cryptosporidia from natural cryptosporidiosis in guinea pigs were experimentally transmitted to both adult and juvenile guinea pigs. Cryptosporidia were associated with the villi of the ileum, jejunum, and duodenum. Both juveniles and adults were equally susceptible to cryptosporidia, as determined by decreases in villus height, increases in crypt depth, and decreases in villus height/crypt depth ratios, when compared with uninoculated animals. When multiple paired comparisons were made between 2 and 10 days postinoculation, there were significant decreases in villus height/crypt depth ratios with time. A dose study showed that 6-week-old guinea pigs were all infected with doses as low as 325 oocysts per animal. When sampled at weekly intervals postinoculation, guinea pigs had significant evidence of infection up to 2 weeks but had recovered completely by 4 weeks. Guinea pigs mounted a specific humoral immune response against cryptosporidia, as measured by an immunoperoxidase technique. Guinea pigs challenged by reinoculation with cryptosporidial oocysts were completely refractory to reinfection. These studies show that cryptosporidiosis in guinea pigs is a useful small animal model of this disease.
The 96-kilodalton antigen as an integral membrane protein in pathogenic Entamoeba histolytica: potential differences in pathogenic and nonpathogenic isolates
Authors: Torian B E , Reed S L , Flores B M , Creely C M , Coward J E , Vial K , Stamm W E
Published: January 2020

A surface antigen (EH-96) of Entamoeba histolytica was demonstrated to be a plasma membrane antigen by immunoprecipitation of metabolically 35S-labeled antigen from live trophozoites, Triton X-114 detergent extracts, and plasma membrane-enriched fractions prepared by concanavalin A membrane stabilization and differential centrifugation. In addition, the antigen was localized to the plasma membrane by electron microscopy with colloidal gold. Antigen from E. histolytica strains immunoprecipitated with specific immunoglobulin M (IgM) or IgG2b monoclonal antibody was identical by one-dimensional peptide mapping with N-chlorosuccinimide. Additionally, antigen from different axenically cultivated amebae was demonstrated to be identical by N-chlorosuccinimide peptide mapping, as were peptide maps of IgG and IgM monoclonal antibody-purified antigen. The 96-kilodalton (kDa) surface antigen was identified on four axenically cultivated pathogenic isolates and on three polyxenically cultivated pathogenic isolates (zymodeme II) of E. histolytica but was absent or present in lesser quantity on six nonpathogenic polyxenically cultivated isolates. The 96-kDa antigen was detected in liver abscess fluid from four patients with amebic abscesses by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation. Two-dimensional gel electrophoresis profiles of the 96-kDa antigen purified from abscess material or from polyxenically cultivated trophozoites demonstrated that the antigens were related to the 96-kDa antigen found in axenically cultivated organisms.
Effect of environmental conditions on production of toxic shock syndrome toxin 1 by Staphylococcus aureus
Authors: Wong A C , Bergdoll M S
Published: January 2020

The kinetics of toxic shock syndrome toxin 1 (TSST-1) production by Staphylococcus aureus was studied in a fermentor in which aeration rate, atmospheric composition, pH, and temperature were controlled. The toxin was synthesized at a maximal rate during the exponential phase. High bacterial populations were not necessarily accompanied by high TSST-1 yields. Aerobiosis increased TSST-1 production, but excessive aeration had an adverse effect. Addition of CO2 enhanced TSST-1 yield by increasing toxin production rate and efficiency. Cultures with no pH control made more TSST-1 than those maintained at pH 5.5 to 7.5. Maximum TSST-1 yields were obtained when cultures were supplied with air (20 cm3/min) and CO2 (5 cm3/min) via a sintered glass sparger.
Isolation, characterization, and inhibition kinetics of enolase from Streptococcus rattus FA-1
Authors: Hüther F J , Psarros N , Duschner H
Published: January 2020

One aspect in a broad spectrum of possible mechanisms of cariostatic reactions of fluoride is its interaction with the metabolism of oral bacteria. Information on the mechanisms and kinetics of fluoride inhibition of essential enzymes of the glycolytic pathway of the relevant bacteria is lacking. In this work, the isolation and purification of enolase from Streptococcus rattus and its characterization are described. The enzyme has been isolated in a monomeric (22 kilodaltons) and dimeric (49 kilodaltons) form. The Km for 2-phosphoglycerate is 4.35 mM. Fluoride inhibition kinetics have competitive character, while phosphate in concentrations above 2 mM and in the presence of 0.5 mM fluoride alters the inhibition kinetics from competitive to noncompetitive. Without fluoride, 2 mM phosphate has a slight stimulatory effect on the enzyme. Monofluorophosphate has a noncompetitive inhibiting effect on the enzyme. This finding suggests that the effect of phosphate may be due to an additional binding of fluoride to the enolase, resulting in a conformational change of the enzyme.
Monoclonal antibodies to outer membrane protein PII block interactions of Neisseria gonorrhoeae with human neutrophils
Authors: Elkins C , Rest R F
Published: January 2020

Nonopsonic binding of gonococci to human neutrophils appears to be mediated by a family of heat-modifiable outer membrane proteins termed protein IIs (PIIs). We studied the ability of a wide variety of antigonococcal monoclonal antibodies (MAbs) to inhibit the interactions of nonpiliated PII+ gonococci with human neutrophils by measuring gonococcal adherence to neutrophils and subsequent luminol-enhanced neutrophil chemiluminescence. From one set of 95 MAbs reacting with whole gonococci, only two, 7VA2 and 7B9, inhibited the ability of gonococci to induce neutrophil chemiluminescence. 7VA2 and 7B9 both reacted only with PII. MAb 53C4, from a smaller set of anti-PII MAbs, inhibited adherence to neutrophils of PII variants that bound 53C4, but not of PII variants that did not. It also inhibited gonococcus-induced neutrophil chemiluminescence. Using a whole-cell binding assay and Western blotting (immunoblotting), we showed that MAb 53C4 bound to one PII (PII4) of strain F62 and to two PIIs (PIIb and PIId) of strain FA1090. The present studies confirm and extend the role of PII in gonococcal adherence to and stimulation of human neutrophils and show intrastrain conservation of PII epitopes. The results indicate that PII is the only outer membrane component involved in adherence of nonpiliated gonococci to human neutrophils.
Borrelia burgdorferi contains repeated DNA sequences that are species specific and plasmid associated
Authors: Simpson W J , Garon C F , Schwan T G
Published: January 2020

Borrelia burgdorferi, the causative agent of Lyme borreliosis, contains linear and supercoiled circular (SC) plasmids. Because SC plasmids are present in multiple copies, these plasmids were examined for species-specific sequences that could serve as high-copy-number target DNAs for a diagnostic probe. Three EcoRI fragments (4.3, 4.2, and 3.5 kilobase pairs [kb]) that hybridized with multiple DNA fragments from B. burgdorferi were identified and cloned from a SC plasmid-enriched fraction. The 4.2- and 3.5-kb fragments were similar in that they hybridized with each other and with similar-sized EcoRI fragments from two unrelated strains of B. burgdorferi. The 4.3-kb fragment did not hybridize with the other two cloned sequences. Both types of sequences hybridized with most of the SC plasmids in seven B. burgdorferi isolates, whereas only a single 49-kb linear plasmid, found in two of the seven strains tested, hybridized with the cloned sequences. None of the cloned sequences hybridized with chromosomal DNA from B. burgdorferi or with total DNA or SC plasmids from Borrelia hermsii, B. turicatae, B. coriaceae, B. parkeri, or B. anserina. These data indicate that the repeated DNA sequences described in this study appear to be plasmid associated and specific to B. burgdorferi. Heteroduplexes formed from the 4.2- and 3.5-kb fragments showed that hybridizing regions in each fragment comprise a 1.8-kb conserved region that is adjacent to a 1.5-kb region that exhibits greater sequence variability. The sequence divergence seen in the variable region is likely the result of genetic drift and may mean that these regions represent closely related genes that encode functionally similar but antigenically distinct proteins.
Characteristics of binding of Escherichia coli serotype O157:H7 strain CL-49 to purified intestinal mucin
Authors: Sajjan S U , Forstner J F
Published: January 2020

Purified rat intestinal mucin was used as a model mucin to study the binding of Escherichia coli serotype O157:H7, a human pathogen associated with outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. Of six O157:H7 strains, only one strain (designated CL-49) bound to rat (and other) intestinal mucins by a specific and saturable process. Binding was observed only after the bacteria were serially passaged to promote the expression of type 1 pili (fimbriae). Several other type 1-piliated E. coli strains, however, did not bind to mucin. Binding of E. coli CL-49 was inhibited by D-mannose and short oligomannosyl derivatives, particularly Man-alpha-1,3-Man, Man-alpha-1,2-Man, and Man-alpha-1,3-Man-beta-1,4-N-acetylglucosamine. Other inhibitors of binding included p-nitrophenol (10(-4) M), heating at 60 degrees C (to remove pili), an antibody to type 1 pili, and purified type 1 pili of E. coli CL-49 used as hapten inhibitors. A comparison of the hydrophobicity of piliated E. coli CL-49 with other type 1-piliated E. coli strains indicated that the former strain was much more hydrophobic than the others. These findings indicate that highly purified intestinal mucins possess specific mannosyl receptor sites for bacterial type 1 pili on E. coli CL-49, but that strong hydrophobic interactions between the mucin and the pili stabilize the mannose-dependent binding process. We speculate that the mucin receptors for type 1 pili reside in oligosaccharides of the 118-kilodalton "link" glycopeptide, since this is the only mucin component known to contain mannose.