bluuebunny/crossref_metadata_2025 huggingface dataset:
Epitope mapping and characterization of antigenic determinants of heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli by using monoclonal antibodies
Authors: Takeda T , Nair G B , Suzuki K , Zhe H X , Yokoo Y , De Mol P , Hemelhof W , Butzler J P , Takeda Y , Shimonishi Y
Published: January 2020

A panel of monoclonal antibodies (MAbs) specific for the heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli was produced. All four MAbs (8G7, 53-4, 11C, and SH1) bound to native STh in an enzyme-linked immunosorbent assay to various degrees, with clone SH1 showing the best affinity. The MAbs were screened for neutralizing and guanylate cyclase-inhibiting activities by the suckling mouse assay and the cyclic GMP assay using T84 cells, respectively. The contact amino acid residues governing the reactivity of the four MAbs were precisely determined by using several chemically synthesized analogs of the various heat-stable enterotoxins (STa's). Three distinct antigenic sites of STh sufficiently removed from each other, one near the N terminus, another in the core functional region of the toxin, and the third in the C-terminal region, were recognized by the different MAbs. MAb SH1, which recognized Asn at position 4 and Tyr at position 5 from the N terminus was 100 times more potent in neutralizing the bioactivity of STh in the suckling mouse assay than was MAb 11C, which recognized Thr at position 16 and Tyr at position 19 from the N terminus of the STh molecule. The MAbs which recognized Leu at position 9 from the N terminus (MAb 53-4) and Tyr at position 19 from the N terminus (MAb 8G7) showed intermediate activities in the neutralization assay. The guanylate cyclase-inhibiting activities of SH1 and 11C essentially paralleled the results for the neutralization of bioactivity, while MAbs 53-4 and 8G7 exhibited reverse activity. These results indicate that MAbs that recognize the N-terminal residues which have been shown not to be essential for toxic activity have a potent protective capacity. None of the MAbs reacted with reduced and carboxy-methylated native STh. This suggests that all of the MAbs mediate their effect by reacting with conformation-dependent antigenic determinants.
Interactions of Nocardia asteroides with murine glia cells in culture
Authors: Beaman L , Beaman B L
Published: January 2020

Astrocytes and microglia were obtained from brains of newborn mice and infected with Nocardia asteroides. Scanning electron micrographs showed nocardiae adhering to the astrocyte cell surface and entering the cytoplasm. After 6 h of incubation the intracellular nocardiae appeared as filaments, demonstrating that growth was occurring. In contrast, the microglia phagocytized nocardiae, but after 6 h the presence of coccoid cells indicated that nocardial growth was inhibited.
Acid resistance in enteric bacteria
Authors: Gorden J , Small P L
Published: January 2020

Shigella species require a uniquely small inoculum for causing dysentery. One explanation for the low infective dose is that Shigella species are better able to survive the acidic conditions encountered in the stomach than are other enteric pathogens. We have tested Shigella species, Escherichia coli, and Salmonella species for the ability to survive at pH 2.5 for at least 2 h. Most isolates of Shigella and E. coli survived this treatment, whereas none of the Salmonella isolates were able to do so. The ability of Shigella species to survive at low pHs does not require the presence of the large virulence plasmid or growth at 37 degrees C but is strikingly dependent on growth phase. We have also found that Shigella isolates exposed to acid lose the ability to invade epithelial cells.
Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a
Authors: Ofek I , Kabha K , Athamna A , Frankel G , Wozniak D J , Hasty D L , Ohman D E
Published: January 2020

The production of a capsular polysaccharide (CPS; K antigen) is characteristic of Klebsiella pneumoniae, but CPS structure varies among strains, and many different serotypes are now known. In this study, cps gene clusters encoding the elements of capsular polysaccharide biosynthesis were exchanged by homologous recombination between strains expressing different serotypes. The wild-type K. pneumoniae strains used for genetic exchange were KPA1 (cpsK2), expressing K2 CPS, and KPB1 (cpsK21a), expressing K21a CPS. Plasmid R68.45 was used to mobilize fragments of chromosomal DNA from auxotrophic derivatives of donor strains. Auxotrophic his alleles introduced into recipient strains provided selectable markers to coinherit the adjacent cps gene clusters from donors expressing a heterologous CPS. Each of the capsule-switched recombinants, KPA5 (cpsK21a) and KPB20 (cpsK2), was shown to have a CPS that was immunologically identical to the serotype of the respective donor. The recombinants retained their respective recipient strain background, as evidenced by a genetic marker and demonstration of a distinctive restriction fragment length polymorphism in genomic DNA. KPB1 CPS contained a sequence (mannose-alpha-2-mannose) that binds to a macrophage lectin and may be responsible for their higher susceptibility to macrophage binding and phagocytosis compared with KPA1, whose CPS lacked such sequences. The recombinant strains expressing heterologous cps genes inherited the macrophage-binding phenotype of the donor, thus confirming that relative susceptibility to phagocytosis was determined by the capsule type expressed. KPA1 was highly virulent in a mouse lethality assay, which is a feature typical of K2 strains, whereas KPB1 was not virulent in mice. Recombinant KPA5 retained relatively high virulence in mice, even though it produced the heterologous K21a CPS, which suggests that a virulence factor other than capsule biosynthesis is encoded by the KPA genomic strain background. In contrast, KPB20 gained marginal virulence in the mouse lethality assay through the inheritance and expression of the K2 CPS from the virulent strain. Thus, pathogenesis in K. pneumoniae may be multifactorial. Specific antibody was used to stabilize the CPS on the surface of K. pneumoniae, and the structural organization of the homologous and heterologous capsules was examined by electron microscopy. Recombinant KPB20, expressing heterologous K2 CPS, had a uniform layer of capsule surrounding the organism that was similar to that seen on the surfaces of the parental strains. However, KPA5, expressing the heterologous K21a CPS, was unusual in that the uniform capsular layer was physically separated from the cell wall by approximately 50 nm.(ABSTRACT TRUNCATED AT 400 WORDS)
Accelerated decay of C3b to iC3b when C3b is bound to the Cryptococcus neoformans capsule
Authors: Pfrommer G S , Dickens S M , Wilson M A , Young B J , Kozel T R
Published: January 2020

Incubation of encapsulated and nonencapsulated Cryptococcus neoformans in normal human serum (NHS) leads to activation and binding of potentially opsonic fragments of complement component C3 to the yeast cells. Analysis of the molecular forms of C3 after incubation of encapsulated cryptococci in NHS showed that the percentage of bound C3 occurring as iC3b approached 100% after 8 min. The percentage of bound C3 occurring as iC3b on nonencapsulated cryptococci never exceeded 70%, even after 60 min of incubation in NHS. Conversion of C3b to iC3b was assessed further by incubating C3b-coated cryptococci for various times with a mixture of complement factors H and I at 40% of their respective physiological concentrations. Most, if not all, of the C3b on encapsulated cryptococci was converted to iC3b at a single fast rate. Conversion of C3b to iC3b on nonencapsulated cryptococci did not follow a single rate constant and appeared to have a fast and a slow component. Studies of the requirements for factors H and I in cleavage of C3b to iC3b showed steep dose-response curves for both factors in the case of encapsulated cryptococci and shallow curves with C3b bound to nonencapsulated cryptococci. Taken together, our results indicate that C3b molecules bound to encapsulated cryptococci have a uniformly high susceptibility to conversion to iC3b by factors H and I. In contrast, a significant portion of the C3b bound to nonencapsulated cryptococci is very resistant to conversion to iC3b by factors H and I.
Isolation and preliminary characterization of the 14- to 18-kilodalton Candida albicans antigen as a phospholipomannan containing beta-1,2-linked oligomannosides
Authors: Trinel P A , Borg-von-Zepelin M , Lepage G , Jouault T , Mackenzie D , Poulain D
Published: January 2020

Western blot (immunoblot) analysis of Candida albicans germ tube extracts has demonstrated the probable presence of beta-1,2-linked oligomannosides acting as epitopes distributed over a 14- to 18-kDa antigen unreactive to concanavalin A. These conclusions about the existence of these non-mannan-associated oligomannoside species were reinforced in the present study by the demonstration of reactivity of factor serum 5 (Iatron Laboratories) with the same antigen. A monoclonal antibody which reacted in an enzyme immunoassay with beta-1,2-linked oligomannosides converted into neoglycolipids and in Western blotting with the 14- to 18-kDa antigen from yeast and germ tubes, through metaperiodate-sensitive epitopes, was used for further characterization of the molecule. Reducing agents and strong protease digestion, which have deleterious effects on C. albicans proteins and mannoproteins, affected neither the antigenicity nor the relative molecular weight of the molecule. Western blots performed after migration of protease-treated extracts in polyacrylamide gels without sodium dodecyl sulfate (SDS) showed that the 14- to 18-kDa antigen could be negatively charged, whereas metabolic radiolabeling demonstrated that these charges could originate, at least in part, from the presence of phosphorus within the molecule. Chloroform-methanol-water extraction of protease-resistant material led to purification of the 14- to 18-kDa antigen, as determined by SDS-polyacrylamide gel electrophoresis and Western blotting. Metabolic radiolabeling with mannose confirmed the presence of these sugar residues within the purified 14- to 18-kDa antigen (despite its nonreactivity to concanavalin A), whereas radiolabeling with palmitic acid demonstrated its lipopolysaccharidic nature. Together, these results led to the conclusion that the 14- to 18-kDa antigen is a phospholipomannan.
CVD110, an attenuated Vibrio cholerae O1 El Tor live oral vaccine strain
Authors: Michalski J , Galen J E , Fasano A , Kaper J B
Published: January 2020

The recent expansion of the seventh cholera pandemic into South America emphasizes the need for a safe, long-lasting, protective, and nonreactogenic vaccine for this disease. Since the predominant Vibrio cholerae O1 strains in the world today are of the El Tor biotype, a bivalent vaccine containing both classical and El Tor biotypes may be desirable. We have constructed a new oral vaccine candidate, V. cholerae CVD110 El Tor, Ogawa, from which all toxin genes so far identified in V. cholerae have been deleted. Three of these genes, those encoding cholera toxin (ctx), zonula occludens toxin (zot), and accessory cholera enterotoxin (ace), are located on a 4.5-kb virulence cassette flanked by repetitive sequences (RS1 elements). Homologous recombination between these RS1 elements resulted in the deletion of this virulence cassette to yield V. cholerae CVD109. Insertion of genes encoding mercury resistance (mer) and the cholera toxin B subunit (ctxB) into the hemolysin locus (hlyA) produced CVD110. This insertion serves three purpose. (i) It genetically tags the vaccine strain so as to distinguish it from wild-type V. cholerae O1. (ii) It produces cholera toxin B subunit in order to elicit antitoxic immunity. (iii) It inactivates the hemolysin gene, rendering the strain nonhemolytic on sheep erythrocyte plates. Supernatants from V. cholerae CVD110 cultures are nonreactogenic when assayed in Ussing chambers.
The RESA-2 gene of Plasmodium falciparum is transcribed in several independent isolates
Authors: Vazeux G , Le Scanf C , Fandeur T
Published: January 2020

The relevance of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum as a malaria vaccine candidate has been questioned of late because RESA-deficient parasites have been found to multiply normally in culture or in monkeys. The RESA-2 gene was recently described as a pseudogene highly homologous to RESA. In this report, we demonstrate that RESA-2 is not a pseudogene, because we were able to detect RESA-2 transcripts in asexual blood stages of multiple isolates by using polymerase chain reaction on reverse-transcribed mRNA. Transcription of RESA-2 was observed whether or not the isolates studied expressed the RESA protein.
Isotypic analysis of Plasmodium falciparum-specific antibodies and their relation to protection in Madagascar
Authors: Dubois B , Deloron P , Astagneau P , Chougnet C , Lepers J P
Published: January 2020

We previously reported that antibodies to the central repeat (DDEHVEEPTVA) of Pf155/RESA, a major Plasmodium falciparum antigen, were negatively related to antimalarial protection. We measured levels of isotype antibodies to this epitope and to P. falciparum in 76 Madagascan subjects. Immunoglobulin G (IgG) and isotype antibodies specific for P. falciparum had similar levels in individuals who were considered protected and those who were not (as determined during a longitudinal follow-up). The levels of IgG1 antibodies to (DDEHVEEPTVA)3 were higher in nonprotected subjects. The levels of total IgG and of other isotype antibodies had a similar tendency to be higher in nonprotected than in protected individuals.
Identification and cloning of a fur homolog from Neisseria gonorrhoeae
Authors: Berish S A , Subbarao S , Chen C Y , Trees D L , Morse S A
Published: January 2020

The promoter region of the major iron-regulated protein of Neisseria gonorrhoeae, Fbp, has two regions that exhibit homology with the Escherichia coli consensus Fur-binding sequences. Gel retardation assays suggested that purified E. coli Fur bound to two sites within the Fbp promoter. The presence of a gonococcal Fur homolog was suggested by Southern hybridization under conditions of low stringency, which revealed a DNA locus that exhibited homology to the E. coli fur gene. Oligonucleotides derived from the conserved regions of fur genes of extremely diverse bacteria were used to amplify a 140-bp fragment of a putative gonococcal fur gene. This fragment was used to identify clones containing the entire gonococcal fur gene. After sequencing the gonococcal fur gene and its promoter region, we found that gonococcal Fur exhibited 50% identity with E. coli Fur at the amino acid level; however, it complemented two E. coli Fur- mutants. The presence of a Fur homolog in N. gonorrhoeae suggests that Fur-regulated genes are widely distributed among extremely diverse bacteria.
The Escherichia coli heat-stable enterotoxin is a long-lived superagonist of guanylin
Authors: Carpick B W , Gariépy J
Published: January 2020

The mechanism by which bacterial heat-stable enterotoxins (ST I STA) cause diarrhea in humans and animals has been linked to the activation of an intestinal membrane-bound guanylate cyclase. Guanylin, a recently discovered rat intestinal peptide, is homologous in structure to ST I and can activate guanylate cyclase present on the human colonic carcinoma cell line T84. To directly test the mechanistic association of guanylate cyclase activation with diarrhea, we synthesized guanylin and a guanylin analog termed N9P10 guanylin and compared their biological activities with those of a synthetic ST I analog, termed ST Ib(6-18). We report that guanylin is able to inhibit the binding of a radiolabeled ST I analog to rat intestinal cells but causes diarrhea in infant mice only at doses at least 4 orders of magnitude higher than that of ST Ib(6-18). In contrast, N9P10 guanylin was enterotoxic in mice at much lower doses than guanylin but proved to be a weaker inhibitor of radiolabeled ST I than guanylin in the receptor binding assay. The pattern of guanylate cyclase activation observed for ST Ib(6-18) and the two guanylin analogs parallels the results observed in the receptor binding assay rather than those observed in the diarrheal assay. Treatment of guanylin with chymotrypsin or lumenal fluid derived from newborn mouse intestines resulted in a rapid loss of binding activity. Together, these results suggest that ST I enterotoxins may represent a class of long-lived superagonists of guanylin.
Cytokine profiles associated with induction of the anticryptococcal cell-mediated immune response
Authors: Murphy J W
Published: January 2020

Previous studies with a murine model have shown that immunization with cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund adjuvant (CFA) induces two populations of anticryptococcal reactive CD4+ T cells. One population (TDH cells) transfers anticryptococcal delayed-type hypersensitivity (DTH), and the other population (Tamp cells) amplifies the anticryptococcal DTH response of given to recipient mice at the time of immunization of the recipient. Treatment of mice with cyclosporin A (CsA) ablates the induction of Tamp cells but not TDH cells. The present study focused on assessing the cytokines produced by spleen cells taken from CsA-treated and control (solvent-treated) mice at days 1, 2, 4, and 6 after immunization. Supernatants from the spleen cells cultured in vitro for 24 or 48 h in medium alone or with CneF, concanavalin A, or phorbol 12-myristate 13-acetate plus calcium ionophore were assessed for the presence of interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-4, IL-5, and tumor necrosis factor. Spleen cells from CneF-CFA-treated mice produced IL-2 and IFN-gamma, but not IL-4 or IL-5, constitutively and in response to CneF, indicating that CneF-CFA induces a Th1 response. Tumor necrosis factor was not produced. Anticryptococcal TDH cells developed in spleens in which there were low levels of IFN-gamma and IL-2 (CsA-treated, immunized mice), whereas anticryptococcal Tamp cells along with TDH cells matured in spleens in which production of IFN-gamma and IL-2 was high (solvent-treated, immunized mice). The data also suggest that IL-2 and IFN-gamma produced by Tamp cells early after adoptive transfer are influential in the development of the amplified anticryptococcal DTH response that has been observed in Tamp cell-recipient mice.
Effects of multiplicity of infection, bacterial protein synthesis, and growth phase on adhesion to and invasion of human cell lines by Salmonella typhimurium
Authors: Kusters J G , Mulders-Kremers G A , van Doornik C E , van der Zeijst B A
Published: January 2020

Monolayers of intestine 407 (Int-407) cells were infected with the virulent Salmonella typhimurium strain C52, and the adhesion to and invasion of these cells were studied. The effects of the multiplicity of infection and growth phase of the bacteria (logarithmic versus stationary) on the interaction with eukaryotic cells were investigated. In contrast to other reports, we found no differences in the adhesive and invasive capacities of bacteria derived from logarithmic- or stationary-phase cultures. Invasion by S. typhimurium required bacterial protein synthesis and live Int-407 cells. Bacteria adhered equally well to dead or live Int-407 cells, which indicates that adhesion does not require metabolically active cells. Adhesion of S. typhimurium followed saturation kinetics, with a maximum of 10 adhesive bacteria per cell. This indicates that there is a limited number of bacterial adhesion sites (receptors) available on the surface of the host cell. Killed and live bacteria adhered equally well and competed with each other for cellular adhesion sites. This and adhesion experiments performed in the presence of chloramphenicol showed that bacterial protein synthesis is not required for adhesion. The general validity of the results obtained with S. typhimurium C52 was confirmed by comparing the invasion and adhesion data with those of the frequently used SL1344 and SR11 strains. In addition, we assayed the adhesion and invasion of S. typhimurium C52, SL1344, and SR11 and 27 S. typhimurium field isolates with Int-407, HeLa, and HEp-2 cells.
Mobility of human neutrophils in response to Cryptococcus neoformans cells, culture filtrate antigen, and individual components of the antigen
Authors: Dong Z M , Murphy J W
Published: January 2020

The mobility of human neutrophils (PMN) in response to encapsulated or nonencapsulated Cryptococcus neoformans cells or cryptococcal culture filtrate (CneF) and its components was studied by using a 48-well modified Boyden chamber. Encapsulated C. neoformans (isolate 184A) cells and CneF-184A stimulated directed migration of human PMN in the absence of serum (direct chemotactic activity) and activated a heat-labile component(s) in fresh human serum to become a chemoattractant(s) for human PMN (indirect chemotactic activity). At a 1:8 dilution (0.25 mg of carbohydrate per ml), CneF-184A displayed chemokinetic activity when assessed with a checkerboard assay. Nonencapsulated C. neoformans isolate 602 cells did not have direct chemotactic activity but did have indirect chemotactic activity. The capsule of C. neoformans is composed predominantly of glucuronoxylomannan (GXM). Purified GXM displayed both direct and indirect chemotactic activity. CneF-184A contains, in addition to GXM, a concanavalin A-binding mannoprotein (MP), whereas CneF-602 contains no GXM but does contain MP. CneF-184A showed direct chemotactic activity and CneF-602 did not. Both CneF-184A and CneF-602 displayed indirect chemotactic activity for human PMN. In addition, purified MP from CneF-184A, like CneF-602, showed only indirect chemotactic activity. These results indicate that GXM contributes to the direct chemotactic activity of PMN observed with the whole encapsulated yeast cells and the unfractionated CneF derived from the encapsulated cells. Both MP and GXM from encapsulated C. neoformans cells mediate indirect chemotactic activity on human PMN.
Chronic staphylococcal osteomyelitis: a new experimental rat model
Authors: Spagnolo N , Greco F , Rossi A , Ciolli L , Teti A , Posteraro P
Published: January 2020

A rat model of chronic staphylococcal osteomyelitis was developed. Fibrin glue (5 microliters) and Staphylococcus aureus (2 x 10(6) CFU/5 microliters) were inoculated into the proximal metaphysis of the tibia. The rats were killed at intervals of between 1 and 6 months, and the tibias were removed. Induced lesions were evaluated by radiographic, macroscopic, and histological examinations and bacterial counts. Roentgenograms revealed osteomyelitis in more than 90% of the tibias. Gross bone pathology revealed skeletal deformation, new bone formation, abscesses, and draining skin fistulas in more than 80% of cases. Histological examination revealed osteomyelitis in more than 90% of cases, and bacterial counts were positive in 86% of cases. Only fibrin glue (5 microliters) was inoculated into controls. Controls showed no osteomyelitic lesions, and counts were negative in seven of eight control tibias. The main feature of this model is the use of fibrin glue instead of the sclerosing agents and foreign bodies used in other models. The model reproduces lesions similar to those of human posttraumatic osteomyelitis and can be reliably used in pathophysiological and therapeutic studies.
Two different types of ADP-ribosyltransferase C3 from Clostridium botulinum type D lysogenized organisms
Authors: Moriishi K , Syuto B , Saito M , Oguma K , Fujii N , Abe N , Naiki M
Published: January 2020

We examined production of ADP-ribosyltransferase C3 in 11 strains of Clostridium botulinum type C and D and their nontoxigenic derivatives. Antisera to C3 proteins of type C organisms divided C3 proteins roughly into at least two groups, bearing no relation to their bacterial types. The C3 gene of type D strain South African was isolated from a toxigenic phage library, and the complete sequence of the C3 gene was determined. The C3 protein of type D strain South African had 98% homology to the C3 protein of type C strain 003-9 and 66% homology to that of type D strain 1873. These results indicate that there are two types of C3 protein in type D organisms, as there are in type C organisms.
Purification of Helicobacter pylori superoxide dismutase and cloning and sequencing of the gene
Authors: Spiegelhalder C , Gerstenecker B , Kersten A , Schiltz E , Kist M
Published: January 2020

The superoxide dismutase (SOD) of Helicobacter pylori, a pathogenic bacterium which colonizes the gastric mucosa, evoking a marked inflammatory response, was purified and characterized, and the N-terminal amino acid sequence was determined. The enzyme consists of two identical subunits each with an apparent molecular weight of 24,000. Analysis of the primary structure and inhibition studies revealed that H. pylori possesses a typical procaryotic iron-containing enzyme. No other isoenzymes could be detected. Indirect gold immunostaining of H. pylori SOD with a polyclonal antibody directed against the iron-containing SOD of Escherichia coli showed a surface-associated localization of the enzyme. The H. pylori SOD gene was cloned by functional complementation of a SOD-deficient E. coli mutant. Sequencing and alignment revealed striking homology to the following facultative intracellular human pathogens: Listeria ivanovii, Listeria monocytogenes, Coxiella burnetti, Porphyromonas gingivalis, Legionella pneumophila, and Entamoeba histolytica. An open reading frame of 642 bp encoding 214 amino acids was determined. There was no leader sequence detectable. Cloning of the H. pylori SOD gene is one of the prerequisites to investigation of its pathophysiological role in the defense against antimicrobial mechanisms of polymorphonuclear granulocytes.
Latency is not an inevitable outcome of infection with Pneumocystis carinii
Authors: Chen W , Gigliotti F , Harmsen A G
Published: January 2020

Severe combined immunodeficiency (SCID) mice resolve naturally acquired Pneumocystis carinii pneumonia after reconstitution with immunocompetent spleen cells and can therefore be used as a model to study latent P. carinii infection. Neither P. carinii nor amplified P. carinii DNA was detected in the lungs of SCID mice killed 21 days after spleen cell reconstitution. Furthermore, SCID mice that recovered from P. carinii infection failed to reactivate the infection after they were either depleted of CD4+ cells for up to 84 days or depleted of CD4+ cells and treated with corticosteroid for 35 days. These results indicate that an immune response to P. carinii can completely clear the organism from the host. This supports the hypothesis that P. carinii pneumonia that develops in immunocompromised patients may be a new infection resulting from exposure to an exogenous source of P. carinii and not necessarily from reactivation of latent infection.
Cloning and sequencing of Coxiella burnetii outer membrane protein gene com1
Authors: Hendrix L R , Mallavia L P , Samuel J E
Published: January 2020

The gene for an approximately 27-kDa outer membrane-associated, immunoreactive protein was cloned from the rickettsial pathogen Coxiella burnetii. The gene, designated com1 for Coxiella outer membrane protein 1, was expressed in Escherichia coli, presumably by its own promoter. The complete nucleotide sequence of the gene was determined. The deduced amino acid sequence of 252 residues includes a putative leader sequence. The leader sequence is recognized in and removed by E. coli on the basis of the difference in the molecular mass of the protein produced in an in vitro transcription-translation system (27.6 kDa) and that of the protein immunoprecipitated from an iodinated E. coli clone (25.7 kDa). The Com1 protein expressed in E. coli was proteinase K sensitive in nondisrupted cells and soluble in 1% Sarkosyl, suggesting a loose association with the outer membrane. While the complete predicted sequence of the Com1 protein does not show any overall similarity to those of previously described proteins, a region which includes the only two cysteines in Com1 is homologous to the catalytic site of protein disulfide oxidoreductases.
Bordetella pertussis and Bordetella parapertussis: two immunologically distinct species
Authors: Khelef N , Danve B , Quentin-Millet M J , Guiso N
Published: January 2020

Bordetella pertussis and Bordetella parapertussis are closely related species. Both are responsible for outbreaks of whooping cough in humans and produce similar virulence factors, with the exception of pertussis toxin, specific to B. pertussis. Current pertussis whole-cell vaccine will soon be replaced by acellular vaccines containing major adhesins (filamentous hemagglutinin and pertactin) and major toxin (pertussis toxin). All of these factors are antigens that stimulate a protective immune response in the murine respiratory model and in clinical assays. In the present study, we examined the protective efficacies of these factors, and that of adenylate cyclase-hemolysin, another B. pertussis toxin, against B. parapertussis infection in a murine respiratory model. As expected, pertussis toxin did not protect against B. parapertussis infection, since this bacterium did not express this protein, but the surprising result was that none of the other factors were protective against B. parapertussis infection. Furthermore, B. parapertussis adenylate cyclase-hemolysin, although it protected against B. parapertussis infection, did not protect against B. pertussis infection. Despite a high degree of homology between both B. pertussis and B. parapertussis species, no cross-protection was observed. Our results outline the fact that, as in other gram-negative bacteria, Bordetella surface proteins vary immunologically.
Suppression of lymphocyte and neutrophil functions by Pseudomonas aeruginosa mucoid exopolysaccharide (alginate): reversal by physicochemical, alginase, and specific monoclonal antibody treatments
Authors: Mai G T , Seow W K , Pier G B , McCormack J G , Thong Y H
Published: January 2020

The mucoid exopolysaccharide (MEP or alginate) of Pseudomonas aeruginosa is thought to be a virulence factor for this organism by virtue of its ability to suppress local host defense mechanisms. We purified MEP from clinical isolates of mucoid P. aeruginosa, subjected it to degradation by ultrasonication, heat, alkali, and alginase, and reacted it with monoclonal antibodies specific for MEP epitopes. Partial reversal or complete abrogation of the inhibitory effects of alginate on human neutrophil random migration, chemotaxis, and hexose monophosphate shunt activity and lymphocyte transformation were observed following most of these treatments. Physicochemical analysis of degraded MEP revealed a positive correlation between changes in molecular size and viscosity and loss of biological properties. The biological properties of MEP were also shown to be dependent on the structural integrity of the O-acetyl groups substituted for the mannuronic acid residues. The results show that the capacity of MEP to suppress neutrophil and lymphocyte functions is dependent on its acetyl content and the physical properties of large size and viscosity and may provide part of the explanation for the propensity of mucoid P. aeruginosa to persist in the airways of patients with cystic fibrosis. These findings highlight the important role of MEP as one of the virulence factors in the pathogenesis of inflammatory damage and subsequent pulmonary destruction in cystic fibrosis.
Modulating effect of dietary carbohydrate supplementation on Candida albicans colonization and invasion in a neutropenic mouse model
Authors: Vargas S L , Patrick C C , Ayers G D , Hughes W T
Published: January 2020

We studied the effect of dietary carbohydrate supplementation on Candida albicans colonization and invasion of the gastrointestinal tract in a neutropenic mouse model. Mice inoculated with C. albicans were allowed free access to standard chow and drinking water supplemented with either glucose or xylitol or no carbohydrates (control). On days 33 through 36 postinoculation, the mean +/- standard error log10 CFU of C. albicans per gram on the mucosal surface, determined by quantitating CFU dislodged in the first wash of the gastric wall, was significantly higher in mice given the glucose supplement: 7.20 +/- 0.09 (glucose) versus 5.38 +/- 0.28 (xylitol) and 5.11 +/- 0.33 (control) CFU/g (P < or = 0.05 for each comparison by Fisher's protected least-significant-difference test). Fecal cultures also yielded the highest quantities of C. albicans in the glucose group. Invasion of the gastric wall by C. albicans correlated well with surface colonization in glucose-supplemented animals. Eight of 10 mice in this group, all with > 10(6) CFU/g, showed extensive invasive growth, as compared with only 2 of 26 mice in the remaining groups (P = 0.00006 by Fisher's exact test). These results indicate that dietary glucose intake is a key determinant of C. albicans growth in the gastrointestinal tract. The data provide an experimental rationale for clinical trials to decrease the intake of glucose or its utilization by C. albicans in immunocompromised patients.
Neutralization of gamma interferon and tumor necrosis factor alpha blocks in vivo synthesis of nitrogen oxides from L-arginine and protection against Francisella tularensis infection in Mycobacterium bovis BCG-treated mice
Authors: Green S J , Nacy C A , Schreiber R D , Granger D L , Crawford R M , Meltzer M S , Fortier A H
Published: January 2020

Peritoneal cells from Mycobacterium bovis BCG-infected C3H/HeN mice produced nitrite (NO2-, an oxidative end product of nitric oxide [NO] synthesis) and inhibited the growth of Francisella tularensis, a facultative intracellular bacterium. Both NO2- production and inhibition of bacterial growth were suppressed by NG-monomethyl-L-arginine, a substrate inhibitor of nitrogen oxidation of L-arginine, and monoclonal antibodies (MAbs) to gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Intraperitoneal injection of mice with BCG increased urinary nitrate (NO3-) excretion coincident with development of activated macrophages capable of secreting nitrogen oxides and inhibiting F. tularensis growth in vitro. Eight days after BCG inoculation, mice survived a normally lethal intraperitoneal challenge with F. tularensis. Treatment of these BCG-infected mice with MAbs to IFN-gamma or TNF-alpha at the time of BCG inoculation reduced urinary NO3- levels to those found in normal uninfected mice for up to 14 days. The same anticytokine antibody treatment abolished BCG-mediated protection against F. tularensis: mice died within 4 to 6 days. Intraperitoneal administration of anti-IFN-gamma or anti-TNF-alpha antibody 8 days after BCG infection also reduced urinary NO3- and abolished protection against F. tularensis. Isotype control (immunoglobulin G) or anti-interleukin 4 MAbs had little effect on these parameters at any time of treatment. IFN-gamma and TNF-alpha were clearly involved in the regulation of macrophage activation by BCG in vivo. Protection against F. tularensis challenge by BCG depended upon the physiological generation of reactive nitrogen oxides induced by these cytokines.
Cloning and expression of the multiple sugar metabolism (msm) operon of Streptococcus mutans in heterologous streptococcal hosts
Authors: Tao L , Sutcliffe I C , Russell R R , Ferretti J J
Published: January 2020

The multiple sugar metabolism (msm) operon of Streptococcus mutans is responsible for the uptake and metabolism of a variety of sugars. In order to further characterize the substrate specificities of the transport system, a 12-kb region of DNA containing the entire msm operon was cloned, via a novel two-step integration strategy, into the chromosomes of two heterologous streptococcal strains, Streptococcus gordonii Challis and Streptococcus anginosus Is57, as well as the chromosome of a natural isolate of S. mutans with a deletion of the msm region. These strains are unable to transport or ferment melibiose, raffinose, or isomaltosaccharides, but the newly constructed recombinants gained the ability to ferment all of these sugars. The S. gordonii Challis construct containing msm was shown to transport radiolabelled melibiose, raffinose, isomaltotriose, and isomaltotetraose, and the transport function was also subjected to induction by raffinose, an inducer of the msm operon in S. mutans. The results confirm the role of the msm operon in the transport and metabolism of melibiose, raffinose, and isomaltosaccharides.
Aggregative adherence fimbria I expression in enteroaggregative Escherichia coli requires two unlinked plasmid regions
Authors: Nataro J P , Yikang D , Giron J A , Savarino S J , Kothary M H , Hall R
Published: January 2020

Adherence to HEp-2 cells by many enteroaggregative Escherichia coli (EAggEC) strains is associated with the expression of flexible, bundle-forming fimbriae 2 to 3 nm in diameter, designated aggregative adherence fimbriae I (AAF/I). We have previously reported the molecular cloning and TnphoA mutagenesis of AAF/I genes from the large plasmid of prototype EAggEC strain 17-2 (J. P. Nataro, Y. Deng, D. R. Maneval, A. L. German, W. C. Martin, and M. M. Levine, Infect. Immun. 60:2297-2304, 1992). Here, we report that further mapping and subcloning of AAF/I regions suggest that expression of the fimbriae requires two separate plasmid regions (designated regions 1 and 2). Approximately 9 kb of DNA unnecessary for fimbrial expression separates the two regions; this intervening segment encodes the EAggEC heat-stable enterotoxin (EAST1). Neither region was capable of conferring aggregative HEp-2 adherence (AA) when cloned individually; when the two regions were cloned as a single fragment or when each was cloned into a different vector and introduced into the same E. coli HB101 cell, AA was restored. AA-positive constructs also expressed human erythrocyte hemagglutination, autoagglutination in broth cultures, and the production of AAF/I as detected by immunogold electron microscopy.
Attaching-effacing lesions and intracellular penetration in HeLa cells and human duodenal mucosa by two Escherichia coli strains not belonging to the classical enteropathogenic E. coli serogroups
Authors: Pedroso M Z , Freymüller E , Trabulsi L R , Gomes T A
Published: January 2020

In the present study, we compared two strains of serotypes O88:H25 and O145:H45 with an enteropathogenic Escherichia coli (EPEC) adherence factor-positive (EAF+) strain of the classic enteropathogenic E. coli serotype O111ab:H2 for their association with HeLa cells and with biopsies of human duodenal mucosa. Both strains not belonging to the classic EPEC serotype showed virulence properties similar to those of the serotype O111ab:H2 strain, i.e., the production of attaching-effacing lesions and intracellular penetration in both systems. These virulence properties associated with the relatively high frequency at which the two serotypes had been detected in infant diarrhea in São Paulo, Brazil (T. A. T. Gomes, M. A. M. Vieira, I. K. Wachsmuth, P. A. Blake, and L. R. Trabulsi, J. Infect. Dis. 160:131-135, 1989) allowed us to suggest that strains of serotypes O88:H25 and O145:H45 should be included in the EAF+ EPEC category.
Tumor necrosis factor alpha binding to bacteria: evidence for a high-affinity receptor and alteration of bacterial virulence properties
Authors: Luo G , Niesel D W , Shaban R A , Grimm E A , Klimpel G R
Published: January 2020

Human and murine receptors for tumor necrosis factor alpha (TNF-alpha) are present on most somatic cells and have been characterized and cloned. In contrast, very little is currently known about whether TNF-alpha can bind to pathogens and whether such binding results in important biological consequences for the infected host. We now report that a number of gram-negative bacteria have receptors for TNF-alpha. Using 125I-labeled TNF-alpha, we show that Shigella flexneri has 276 receptors for TNF-alpha, with a Kd of 2.5 nM. The binding of labeled TNF-alpha to these bacterial receptors can be inhibited by cold TNF-alpha but not by cold TNF-beta. Binding of 125I-TNF-alpha to S. flexneri was inhibited by trypsin treatment of bacterial cells or incubation at 52 degrees C for 3 min. Monoclonal antibody to either the 55-kDa or the 75-kDa TNF-alpha receptor, which are present on different eukaryotic cells, had no effect on 125I-TNF-alpha binding to bacteria. A number of gram-negative bacteria were capable of binding 125I-TNF-alpha. Gram-positive bacteria bound significantly less 125I-TNF-alpha than gram-negative bacteria. Pretreatment of S. flexneri with TNF-alpha resulted in enhanced bacterial invasion of HeLa cells and enhanced uptake by human and murine macrophages. Pretreatment of HeLa cells with antibody to the 55-kDa TNF-alpha receptor abrogated enhanced invasion of HeLa cells by TNF-alpha-bacterium complexes. These results suggest that TNF-alpha-bacterium complexes can interact with TNF-alpha receptors present on eukaryotic cells. This report shows that gram-negative bacteria have receptors for TNF-alpha and that a virulence property of a bacterium is altered as a consequence of cytokine binding.
Conservation of antigen components from two recombinant hybrid proteins protective against malaria
Authors: Knapp B , Nau U , Hundt E
Published: January 2020

Recently, we have shown that two hybrid proteins carrying partial sequences of the blood-stage antigens SERP, HRPII, and MSAI from Plasmodium falciparum confer protective immunity on Aotus monkeys against an experimental parasite infection (B. Knapp, E. Hundt, B. Enders, and H. A. Küpper, Infect. Immun. 60:2397-2401, 1992). The malarial components of the hybrid proteins consist of amino acid residues 630 to 892 of SERP, amino acid residues 146 to 260 of MSAI, and the 189 C-terminal residues of HRPII. We have studied the diversity of these protein regions in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from two different areas where malaria is endemic. The gene regions of SERP and MSAI coding for the corresponding sequences of the protective hybrid proteins and the exon II region of the HRPII gene were amplified by polymerase chain reaction and sequenced. All three regions were found to be highly conserved. In the 262-amino-acid fragment of SERP, one single conservative amino acid substitution was found. The exon II region of HRPII showed only a slight variability in number and arrangement of the repeat units. The 115-amino-acid fragment of MSAI which is located within an N-terminal region known to be conserved among different parasite strains was shown to be the most variable among the vaccine components: amino acid substitutions were found in 14 different positions of this MSAI region when both laboratory strains and field isolates were compared.
Mammalian cell sialic acid enhances invasion by Trypanosoma cruzi
Authors: Schenkman R P , Vandekerckhove F , Schenkman S
Published: January 2020

We have used a Chinese hamster ovary cell mutant (Lec2) that express much less sialic acid on the surface than the parental cell line (Pro5) to investigate whether sialic acid plays a role during cell invasion by Trypanosoma cruzi. Trypomastigotes derived from a tissue culture (corresponding to bloodstream trypomastigotes) and metacyclic trypomastigotes (corresponding to infective stages of the insect vector) invaded the Lec2 mutant less efficiently than the parental cell line. Invasion of the Lec2 mutant cells could be restored to the Pro5 level by resialylation of the mutant cells with T. cruzi trans-sialidase and sialyllactose. Conversely, pretreatment of the Pro5 parental cells with bacterial neuraminidase decreased invasion. These results indicate that sialic acid associated with the host cell contributes to invasion by T. cruzi.
Characterization of specific binding of a human immunoglobulin M monoclonal antibody to lipopolysaccharide and its lipid A domain
Authors: Fujihara Y , Lei M G , Morrison D C
Published: January 2020

The human immunoglobulin M monoclonal antibody HA-1A was first described as an antibody which bound specifically to the lipid A region of lipopolysaccharide (LPS) (N. N. H. Teng, H. S. Kaplan, J. M. Herbert, C. Moore, H. Douglas, A. Wunderlich, and A. Braude, Proc. Natl. Acad. Sci. USA 82:1790-1794, 1985) and provided significant protection when administered to patients with gram-negative bacteremia and shock (E. J. Ziegler, C. J. Fisher, Jr., C. L. Sprung, R. C. Straube, J. C. Sadoff, G. E. Foulke, C. H. Wortel, M. P. Fink, R. P. Dellinger, N. N. H. Teng, I. E. Allen, H. J. Berger, G. L. Knatterud, A. F. LoBuglio, C. R. Smith, and the HA-1A Sepsis Study Group, New Engl. J. Med. 324:429-436, 1992). Since that original report, questions have arisen in the scientific literature concerning the specificity of this antibody in LPS and/or lipid A binding. Experiments have, therefore, been carried out with a variety of assay formats to determine the capacity of this HA-1A antibody to bind to lipid A and LPS. Direct binding experiments with a sensitive enzyme-linked immunosorbent assay (ELISA) system have established that HA-1A will bind to purified lipid A from both Escherichia coli and Salmonella spp. These results have been confirmed by using a fluid-phase antigen-antibody competitive inhibition assay with purified lipid A and an antibody-antibody competitive inhibition assay with a monoclonal antibody with known specificity for lipid A. The HA-1A monoclonal antibody has also been shown to bind to a panel of R-chemotype LPS by ELISA and, unlike many other previously reported anti-lipid A antibodies, binding of HA-1A to R-chemotype LPS and lipid A is comparable. Although binding of HA-1A to S-LPS (smooth, wild-type LPS) could not be detected by direct ELISA, competitive inhibition experiments with some preparations of S-LPS have been able to show specific HA-1A binding. Collectively, these data confirm the binding specificity of HA-1A for the lipid A component of LPS and provide evidence that this monoclonal antibody manifests a relatively uncommon profile in its capacity to bind lipid A and R-chemotype LPS as well as some preparations of S-LPS.
Cloning of the Streptococcus gordonii PK488 gene, encoding an adhesin which mediates coaggregation with Actinomyces naeslundii PK606
Authors: Andersen R N , Ganeshkumar N , Kolenbrander P E
Published: January 2020

Coaggregation between Streptococcus gordonii PK488 and Actinomyces naeslundii PK606 is mediated by a 38-kDa streptococcal protein, designated ScaA. The gene, scaA, which encodes this protein has been cloned into Escherichia coli. A genomic S. gordonii PK488 library (in Lambda ZAP II) was screened with anti-S. gordonii immunoglobulin G absorbed with S. gordonii PK1804, an isogenic coaggregation-defective mutant of strain PK488. A positive recombinant phage was isolated, and a phagemid designated pRA1 was obtained which contained a 6.6-kb insert. Expression of scaA from pRA1 and from a subcloned internal 2.1-kb fragment was observed. The absorbed antiserum cross-reacted with a 34.7-kDa protein, SsaB, from S. sanguis 12, also a coaggregation partner of A. naeslundii PK606. Absorbed antiserum to S. gordonii PK488 and antiserum to SsaB both reacted with 38-kDa proteins in supernatants from mildly sonicated preparations from 11 other coaggregation partners of A. naeslundii PK606. Putative adhesin genes were identified in each of these coaggregation partners by Southern analysis of their genomic DNA with the cloned 2.1-kb fragment as a probe. A 30-base oligonucleotide probe based on the sequence of ssaB of S. sanguis 12 hybridized in an identical manner. These data extend the notion that most of the viridans streptococci that coaggregate with actinomyces are capable of expressing ScaA-related proteins.