bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface Zn-proteinase as a molecule for defense of Leishmania mexicana amazonensis promastigotes against cytolysis inside macrophage phagolysosomes
Authors: Seay M B , Heard P L , Chaudhuri G
Published: January 2020

The role of the surface membrane Zn-proteinase in protecting the cellular integrity of the macrophage parasite Leishmania mexicana amazonensis from intraphagolysosomal cytolysis was studied. These cells lose their infectivity to host macrophages after prolonged cultivation in axenic growth medium. The virulent and attenuated variants of the parasite cells were cloned. Failure of these attenuated parasite cells to survive inside macrophage phagolysosomes is associated with 20- to 50-fold reduction in the expression of surface gp63 protein. In situ inhibition of gp63 proteinase activity inside Leishmania-infected macrophage phagolysosomes with targeted delivery of an inhibitor of gp63 proteinase activity, 1,10-phenanthroline, selectively eliminated intracellular Leishmania amastigotes, further suggesting the importance of this proteinase in phagolysosomal survival of the parasite. An upstream sequence (US) of the gp63 gene was cloned in front of the bacterial chloramphenicol acetyltransferase (CAT) gene in plasmid pCATbasic. Transfection of L. mexicana amazonensis cells with this recombinant plasmid showed that expression of the CAT gene from this US is 15- to 20-fold higher in virulent clones than in avirulent clones of the parasite. Band shift analysis with the cloned US also showed that binding of protein(s) was 15- to 20-fold higher in virulent cell extract than in avirulent cell extract. Coating of attenuated cells or liposomes with proteolytically active gp63 protects them from degradation inside macrophage phagolysosomes. These results suggest a novel mechanism of survival of this phagolysosomal parasite with the help of its surface Zn-proteinase.
Purification and characterization of a pilus of a Vibrio cholerae strain: a possible colonization factor
Authors: Yamashiro T , Iwanaga M
Published: January 2020

A new flexible type of pilus was purified from Vibrio cholerae non-O1, non-0139 strain NAGV14 and characterized. The molecular mass of the pilin was estimated to be 20 kDa, and the antigenicity differed from that of known pili such as toxin-coregulated pili, mannose-sensitive hemagglutinating pili, V10 pili, and Al-1841 pili. The NAGV14 pilus was regarded as a colonization factor because the purified pili adhered to rabbit intestine and adhesion was inhibited by treating the organisms with the Fab fraction of an antipilus antibody. An intestinal receptor blockade using purified pili failed to inhibit adhesion of the organisms. The NAGV14 pili adhered to the surface of live V. cholerae. An antigen cross-reacting with the NAGV14 pili was widely and specifically distributed among V. cholerae strains irrespective of serotype and biotype. The amino acid sequence of the pilin was homologous with that of MshA. The NAGV14 pili did not agglutinate human and rabbit erythrocytes.
Mesophilic Aeromonas sp. serogroup O:11 resistance to complement-mediated killing
Authors: Merino S , Rubires X , Aguilar A , Albertí S , Hernandez-Allés S , Benedí V J , Tomas J M
Published: January 2020

The complement activation by and resistance to complement-mediated killing of Aeromonas sp. strains from serogroup O:11 were investigated by using different wild-type strains (with an S-layer characteristic of this serogroup) and their isogenic mutants characterized for their surface components (S-layer and lipopolysaccharide [LPS]). All of the Aeromonas sp. serogroup O:11 wild-type strains are unable to activate complement, which suggested that the S-layer completely covered the LPS molecules. We found that the classical complement pathway is involved in serum killing of susceptible Aeromonas sp. mutant strains of serogroup O11, while the alternative complement pathway seems not to be involved, and that the complement activation seems to be independent of antibody. The smooth mutant strains devoid of the S-layer (S-layer isogenic mutants) or isogenic LPS mutant strains with a complete or rather complete LPS core (also without the S-layer) are able to activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b is rapidly degraded, and therefore the lytic membrane attack complex (C5b-9) is not formed. Isogenic LPS rough mutants with an incomplete LPS core are serum sensitive because they bind more C3b than the resistant strains, the C3b is not completely degraded, and therefore the lytic complex (C5b-9) is formed.
Molecular characterization of a major serotype M49 group A streptococcal DNase gene (sdaD)
Authors: Podbielski A , Zarges I , Flosdorff A , Weber-Heynemann J
Published: January 2020

Group A streptococci (GAS) express up to four types of secreted DNases. Although GAS infections are correlated with the production of anti-DNase B antibodies, the roles of DNases in the pathogenesis of GAS infections remain unclear. From a lambda library of serotype M49 strain CS101 GAS genome, a 2,147-bp fragment expressing DNase activity on an indicator agar was identified and sequenced. One 1,155-bp open reading frame (ORF) was identified in this fragment. This ORF was found to be 48% identical on the amino acid level to group C streptococcal DNase (Sdc). The regions of highest homology corresponded to amino acid residues that were also identified as part of the active site in staphylococcal nuclease. Transcription analysis revealed a specific 1.3-kb mRNA, which corresponded to the size predicted by the promoter and transcription termination signature sequences and indicated a monocistronic mode of transcription. Allelic replacement of the ORF rendered a M49 mutant devoid of extracellular DNase activity when cultured on indicator agar. Virulence parameters such as resistance to phagocytosis were not affected by the mutation. The sda gene was cloned and expressed in Escherichia coli as a thioredoxin fusion. By performing Ouchterlony immunodiffusion on the recombinant protein and by using protein preparations from culture supernatants of wild-type bacteria and the DNase mutant, the results of immunoreactivity with DNase type-specific polyclonal rabbit antisera classified the DNase as a type D enzyme. Fifty percent of patients with sera exhibiting high titers of antistreptolysin or anti-DNase B antibodies also had SdaD-reactive antibodies in comparison with <10% of serologically normal controls. While the value of recombinant SdaD for diagnostic purposes needs to be clarified, the isogenic DNase mutant pair of M49 should allow the significance of GAS DNase D as a bacterial virulence factor to be determined.
Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells
Authors: Cywes C , Godenir N L , Hoppe H C , Scholle R R , Steyn L M , Kirsch R E , Ehlers M R
Published: January 2020

Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis is likely important in the establishment of a primary infection in the lung. M. tuberculosis binds to a variety of phagocyte receptors, of which the mannose receptor and complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a beta2 integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tuberculosis H37Rv to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesized complement, which are inherent in studies with mononuclear phagocytes. The surface expression of CD11b and CD18 was assessed by immunofluorescence, immunobead binding, flow cytometry, and immunoprecipitation with anti-CD11b and anti-CD18 monoclonal antibodies (MAbs). The functional activity of the surface-expressed CD11b/CD18 (CR3) heterodimer was confirmed by rosetting with C3bi-coated microspheres. We found that M. tuberculosis bound four- to fivefold more avidly to CR3-expressing CHO cells than to wild-type cells and, importantly, that this binding was at similar levels in the presence of fresh or heat-inactivated human or bovine serum or no serum. In contrast, Mycobacterium smegmatis bound poorly to CR3-expressing CHO cells in the absence of serum, but after opsonization in serum, binding was comparable to that of M. tuberculosis. The binding of M. tuberculosis to the transfected CHO cells was CR3 specific, as it was inhibited by anti-CR3 MAbs, particularly the anti-CD11b MAbs LM2/1 (I domain epitope) and OKM1 (C-terminal epitope), neither of which inhibit C3bi binding. MAb 2LPM19c, which recognizes the C3bi-binding site on CD11b, had little or no effect on M. tuberculosis binding. The converse was found for the binding of opsonized M. smegmatis, which was strongly inhibited by 2LPM19c but unaffected by LM2/1 or OKM1. CR3-specific binding was also evidenced by the failure of M. tuberculosis to bind to CHO cells transfected with an irrelevant surface protein (angiotensin-converting enzyme) in the presence or absence of serum. We conclude that the binding of M. tuberculosis H37Rv to CR3 expressed in CHO cells is predominantly nonopsonic and that the organism likely expresses a ligand that binds directly to CR3.
Effect of in vitro infection of human monocytes with low numbers of Mycobacterium tuberculosis bacteria on monocyte apoptosis
Authors: Dürrbaum-Landmann I , Gercken J , Flad H D , Ernst M
Published: January 2020

The effect of Mycobacterium tuberculosis H37Rv on spontaneous apoptosis of human monocytes from healthy donors in vitro was investigated. Infection with low numbers of viable M. tuberculosis bacteria prevented spontaneously occurring apoptosis in monocytes. Our data suggest that apoptosis of monocytes appears to be negatively associated with the presence of tumor necrosis factor alpha.
Sequence analysis of the chromosomal region around and within the V-1-encoding gene of Mycoplasma pulmonis: evidence for DNA inversion as a mechanism for V-1 variation
Authors: Simmons W L , Zuhua C , Glass J I , Simecka J W , Cassell G H , Watson H L
Published: January 2020

Although the variation of V-1 antigens of Mycoplasma pulmonis has been correlated with variable expression of the cytadherence properties of this organism and has been implicated as a virulence determining factor in M. pulmonis-induced murine respiratory disease, the precise function of these antigens remains unknown. We have cloned and characterized genes encoding V-1 from two M. pulmonis UAB CT V-1 variants that differ in hemadsorption properties. A comparison of the nucleotide sequences revealed that these two variant genes were identical in the 5'-most 724 nucleotides. Regions of extensive divergence that contained repeated sequences were found 3' to this conserved region. On the basis of their deduced amino acid sequences, one variant expressed a V-1 protein of 94.2 kDa presumptively containing 40 repeats of 17 amino acids and the other expressed a protein of 27.4 kDa consisting 2 direct, noncontiguous 9-amino-acid repeats. These general properties, as well as the presence of a prokaryotic lipoprotein acylation sequence (L-X-Y-C), indicated that the genes encoding V-1 were similar in structure to genes encoding other mycoplasma surface lipoproteins. Further analysis of sequences flanking these genes revealed that these variants arose via an inversion event which provided an interchange of the two variable regions as well as for the conserved region of these genes and immunoblot analyses using rabbit polyclonal antibodies specific for synthetic peptides derived from the sequences of the different variable regions indicated that DNA inversion acted as a switch which allowed only one of the two different genes to be expressed at any given time. This inversion model clearly provides a mechanism by which M. pulmonis can alter its surface architecture and also strongly suggests that the as-yet-undefined function of V-1 residues in the variable carboxy region of these proteins.
Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque streptococcus
Authors: Chen Y Y , Clancy K A , Burne R A
Published: January 2020

The hydrolysis of urea by urease enzyme of oral bacteria is believed to have a major impact on oral microbial ecology and to be intimately involved in oral health and diseases. To begin to understand the biochemistry and genetics of oral ureolysis, a study of the urease of Streptococcus salivarius, a highly ureolytic organism which is present in large numbers on the soft tissues of the oral cavity, has been initiated. By using as a probe a 0.6-kpb internal fragment of the S. salivarius 57.I ureC gene, two clones from subgenomic libraries of S. salivarius 57.I in an Escherichia coli plasmid vector were identified. Nucleotide sequence analysis revealed the presence of one partial and six complete open reading frames which were most homologous to ureIAB-CEFGD of other ureolytic bacteria. Plasmid clones were generated to construct a complete gene cluster and used to transform E. coli and Streptococcus gordonii DL1, a nonureolytic, dental plaque microorganism. The recombinant organisms expressed high levels of urease activity when the growth medium was supplemented with NiCl2. The urease enzyme was purified from E. coli, and its biochemical properties were compared with those of the urease produced by S. salivarius and those of the urease produced by S. gordonii carrying the plasmid-borne ure genes. In all cases, the enzyme had a Km of 3.5 to 4.1 mM, a pH optimum near 7.0, and a temperature optimum near 60 degrees C. S. gordonii carrying the urease genes was then demonstrated to have a significant capacity to temper glycolytic acidification in vitro in the presence of concentrations of urea commonly found in the oral cavity. The ability to genetically engineer plaque bacteria that can modulate environmental pH through ureolysis will open the way to using recombinant ureolytic organisms to test hypotheses regarding the role of oral ureolysis in dental caries, calculus formation, and periodontal diseases. Such recombinant organisms may eventually prove useful for controlling dental caries by replacement therapy.
Rapid cyclic changes in density and accessibility of endometrial ligands for Escherichia coli Dr fimbriae
Authors: Kaul A K , Kumar D , Nagamani M , Goluszko P , Nowicki S , Nowicki B J
Published: January 2020

The mechanisms of developing infection in young, noncompromised individuals are well understood. Colonization is prerequisite for the development of infection. In human, ligands serving bacterial colonization belong to common antigens. Consequently, a majority of individuals should be sensitive to infection at all times. We hypothesize that the temporal patterns of some infections and sensitivity to them are associated with sudden changes in the density and accessibility of common receptors. Endometrial samples from women having normal menstrual cycles were examined for histological location, receptor density, and in situ hybridization of Dr (decaying-accelerating factor) ligands for Escherichia coli Dr fimbriae. Significant up-regulation and luminal expression of Dr ligands occurred during the secretory phase, whereas receptors were expressed in the basement membrane and in smaller quantities during the proliferative phase. This observation agrees with our hypotheses that some ligands recognized by bacterial adhesins change their compartmentalization and, most importantly, that they up-regulate expression at specific times.
Role of sulfatides in adhesion of Helicobacter pylori to gastric cancer cells
Authors: Kamisago S , Iwamori M , Tai T , Mitamura K , Yazaki Y , Sugano K
Published: January 2020

We have demonstrated that clinical isolates of Helicobacter pylori preferentially bind to sulfatides (I3SO3-GalCer) and GM3 gangliosides (II3NeuAcLacCer), two predominant acidic glycosphingolipids in the human gastric mucosa, on thin-layer chromatography plates. However, it has not yet been clarified that these glycospingolipids truly serve as adhesion receptors for H. pylori in live cells. In this study, we used a gastric cancer cell line, KATO III, as a cellular model of H. pylori adhesion and examined the role of sulfatides in attachment. The adhesion of H. pylori (i.e., a standard strain of H. pylori, NCTC 11637) to KATO III cells and the effects of various substances on this adhesion were monitored and semiquantitated by flow cytometric analysis. Sulfated glycoconjugates, such as heparin and gastric mucin, significantly inhibited H. pylori adhesion to KATO III cells. Membrane preparations from KATO III cells strongly inhibited this adhesion. In the membrane preparations, sulfatides were present as a major acidic glycosphinoglipid. With the exception of sulfatides, no distinct adhesion of H. pylori to glycospingolipids from KATO III cells were observed. Moreover, H. pylori did not bind to any membrane proteins of KATO III cells. Finally, a monoclonal anti-sulfatide antibody markedly reduced H. pylori adhesion to KATO III cells. These results suggest that sulfatides, and possibly related sulfated compounds, serve as a major receptor for cell adhesion by H. pylori.
Signal transduction in Pneumocystis carinii: characterization of the genes (pcg1) encoding the alpha subunit of the G protein (PCG1) of Pneumocystis carinii carinii and Pneumocystis carinii ratti
Authors: Smulian A G , Ryan M , Staben C , Cushion M
Published: January 2020

Pneumocystis carinii is a eukaryotic organism that causes pneumonia in immunocompromised hosts. The cell biology and life cycle of the organism are poorly understood primarily because of the lack of a continuous in vitro cultivation system. These limitations have prevented investigation of the organism's infectious cycle and hindered the rational development of new antimicrobial therapies and implementation of measures to prevent exposure to the organism or transmission. The interaction of P. carinii with its host and its environment may be critical determinants of pathogenicity and life cycle. Signal transduction pathways are likely to be critical in regulating these processes. G proteins are highly conserved members of the pathways important in many cellular events, including cell proliferation and environmental sensing. To characterize signal transduction pathways in P. carinii, we cloned a G-protein alpha subunit (G-alpha) of P. carinii carinii and P. carinii ratti by PCR amplification and hybridization screening. The gene encoding the G-alpha was present in single copy on a 450-kb chromosome of P.c. ratti. The 1,062-bp G-alpha open reading frame is interrupted by nine introns. The predicted polypeptide showed 29 to 53% identity with known fungal G-alpha proteins with greatest homology to Neurospora crassa Gna-2. Northern (RNA) blot analysis and immunoprecipitation demonstrated expression of the G-alpha mRNA and protein P. carinii isolated from heavily infected animals. Some alteration in the level of transcription was noted in short-term maintenance in starvation or rich medium. Characterization of signal transduction in P. carinii will permit a better understanding of the reproductive capacity and other cellular processes in this family or organisms that cannot be cultured continuously.
Interleukins 6 and 11 protect mice from mortality in a staphylococcal enterotoxin-induced toxic shock model
Authors: Barton B E , Shortall J , Jackson J V
Published: January 2020

BALB/By mice given doses of D-galactosamine plus Staphylococcus aureus enterotoxin B die within 48 h of administration. The cause of death is a syndrome much like toxic shock syndrome in humans. We used this model to investigate the role of two cytokines, interleukin 6 and interleukin 11, which share the signal transducing subunit, gp130, of their respective receptors. We observed that pretreatment of mice with antibody to interleukin 6 increased mortality from 55% to nearly 90% (P < 0.001), while pretreatment with either cytokine reduced death. The protection was dose dependent; however, interleukin 6 was about 10-fold more potent that interleukin 11. These data indicate that endogenous interleukin 6 plays a protective role in attenuating acute inflammatory responses; furthermore, interleukin 6 and interleukin 11 can abrogate T-cell activation due to triggering by superantigen. A possible clinical role for these cytokines in the treatment of toxic shock merits further investigation.
Induction of differential T-helper-cell responses in mice infected with variants of the parasitic nematode Trichuris muris
Authors: Bellaby T , Robinson K , Wakelin D
Published: January 2020

The resistance or susceptibility of mice to infection with the intestinal nematode parasite Trichuris muris is closely correlated with polarization of T helper (Th) cell responses to the type 2 (Th2) or type 1 (Th1) subset. Comparison of infections with three isolates of T. muris (E/K, E/N, and S) in three inbred strains of mice (CBA, C57BL/10, and B10.BR) has shown that host Th response phenotype can be parasite determined. Although the mouse strains used show genetically determined variation in ability to respond to T. muris (CBA > C57BL/10 > B10.BR), the speed of worm expulsion in a given strain depended upon the isolate used for infection (E/K > E/N > S). The two isolates that induced the most effective resistance (E/K and E/N) elicited parasite-specific host antibody responses that were dominated by immunoglobulin G1 (IgG1), and antigen-stimulated T cells from infected mice released interleukin-5 in vitro. With the isolate that induced the least host resistance (S), the dominant antibody response was IgG2a, and T cells released gamma interferon in vitro. These data show clearly that parasite variant-specific factors play a major role in Th subset polarization during infection.
Cloning and characterization of the galE locus of Pasteurella haemolytica A1
Authors: Potter M D , Lo R Y
Published: January 2020

The enzyme UDP-galactose 4-epimerase (GalE) is involved in one of the major steps of galactose metabolism in bacteria. In many cases, GalE is required for the biosynthesis of extracellular polysaccharide materials such as lipopolysaccharide (LPS) and capsule. Mutants defective in galE have been shown to exhibit reduced virulence. Here we describe the cloning and characterization of the galE gene from the bovine pathogen Pasteurella haemolytica A1. This was achieved by the complementation of a Salmonella typhimurium galE mutant with a P. haemolytica A1 plasmid bank. Analysis of six clones recovered on minimal media with galactose as the carbon source showed that they all contained the same recombinant plasmid with a 5-kbp DNA insert. The galE-complementing activity was localized to a 2.2-kbp DNA region by subcloning. Biochemical, immunological, and phage sensitivity analyses of the recombinant LPS in S. typhimurium showed that it is essentially identical to that of the wild type. In vivo expression studies showed that a 37-kDa protein is expressed from the complementing plasmids, and the presence of GalE activity was confirmed by an assay for epimerase activity. Nucleotide sequence analysis of the cloned DNA identified the galE gene. Comparison of the deduced amino acid sequence analysis of P. haemolytica A1 GalE with published data showed high-level homology, 81.6%, with the GalE of Haemophilus influenzae type b. However, the sequences flanking galE do not show similarity with any other gal gene, suggesting that P. haemolytica A1 galE is not linked to the other genes of the gal operon, as is the case for Neisseria meningitidis, Neisseria gonorrhoeae, and H. influenzae. The separation of galE from the classical gal operon genes was confirmed by Southern blot hybridization studies, and a physical map showing the relative positions of galE, galT, and galK was constructed.
Pasteurella multocida toxin is a mitogen for bone cells in primary culture
Authors: Mullan P B , Lax A J
Published: January 2020

The effect of recombinant Pasteurella multocida toxin (PMT) on primary cultures of embryonic chick bone-derived osteoblastic cells was investigated. It was found that PMT was a potent mitogen for primary derived chicken osteoblasts. The toxin stimulated DNA synthesis and cell proliferation in quiescent osteoblasts at the first passage and accelerated cell growth in subconfluent cultures. Cell viability was not affected by PMT, even at relatively high concentrations. Osteoblast numbers increased in a dose-dependent manner in response to PMT. Intracellular inositol phosphates were elevated in response to PMT, but no elevation in cyclic AMP (cAMP) levels was evident. Indeed, PMT inhibited cAMP elevation in osteoblasts in response to cholera toxin at a stage before other PMT-mediated events take place. In addition to increased cell turnover, PMT down-regulated the expression of several markers of osteoblast differentiation. Both alkaline phosphatase and type I collagen were reduced, but osteonectin was not affected. The in vitro deposition of mineral in cultures of primary osteoblasts and osteoblast-like osteosarcoma cells was also inhibited by the presence of PMT. This suggests that PMT interferes with differentiation at a preosteoblastic stage.
Differentiation-associated toxin receptor modulation, cytokine production, and sensitivity to Shiga-like toxins in human monocytes and monocytic cell lines
Authors: Ramegowda B , Tesh V L
Published: January 2020

Infections with Shiga toxin-producing Shigella dysenteriae type 1 or Shiga-like toxin (SLT)-producing Escherichia coli cause bloody diarrhea and are associated with an increased risk of acute renal failure and severe neurological complications. Histopathological examination of human and animal tissues suggests that the target cells for toxin action are vascular endothelial cells. Proinflammatory cytokines regulate endothelial cell membrane expression of the glycolipid globotriaosylceramide (Gb(3)) which serves as the toxin receptor, suggesting that the host response to the toxins or other bacterial products may contribute to pathogenesis by regulating target cell sensitivity to the toxins. We examined the effects of purified SLTs on human peripheral blood monocytes (PBMn) and two monocytic cell lines. Undifferentiated THP-1 cells were sensitive to SLTs. Treatment of the cells with a number of differentiation factors resulted in increased toxin resistance which was associated with decreased toxin receptor expression. U-937 cells, irrespective of maturation state, and PBMn were resistant to the toxins. U-937 cells expressed low levels of GB(3), and toxin receptor expression was not altered during differentiation. Treatment of monocytic cells with tumor necrosis factor alpha (TNF-alpha) did not markedly increase sensitivity or alter toxin receptor expression. Undifferentiated monocytic cells failed to synthesize TNF and interleukin 1beta when treated with sublethal concentrations of SLT type I (SLT-I), whereas cells treated with 12-0-tetradecanoylphorbol-13-acetate acquired the ability to produce cytokines when stimulated with SLT-I. When stimulated with SLT-I, U-937 cells produced lower levels of TNF than PBMn and THP-1 cells did.
Acquired resistance against a secondary infection with Listeria monocytogenes in mice is not dependent on reactive nitrogen intermediates
Authors: Samsom J N , Langermans J A , Groeneveld P H , van Furth R
Published: January 2020

During an infection, inflammatory mediators can induce the production of nitric oxide, a reactive nitrogen intermediate (RNI) which plays a role in antimicrobial activity against a wide variety of pathogens. In vitro experiments have shown that release of RNI by macrophages is mediated by tumor necrosis factor alpha (TNF). Since TNF is essential for acquired resistance during a secondary Listeria monocytogenes infection in mice, the aim of the present study was to determine whether RNI are also involved in the course of such an infection. Mice which had recovered from a sublethal primary infection with 0.1 50% lethal dose of (LD50) L. monocytogenes were infected intravenously with 10LD50 of L. monocytogenes. During a primary infection, the number of bacteria in the liver and spleen, as well as the concentration of RNI in plasma, increased. During a secondary infection, the number of bacteria in the liver and spleen decreased whereas no significant increase in the concentration of RNI in plasma was observed. Neutralization of endogenously produced TNF and gamma interferon by subcutaneous injection of alginate-encapsulated monoclonal antibody-forming cells during a secondary infection resulted in an increase in the number of bacteria in the liver and spleen an increase in the concentration of RNI in plasma. When the production of RNI was inhibited by treatment of mice with competitive NO-synthase inhibitor N omega-nitro-L-arginine methyl ester hydrochloride (L-Name) and an iota-arginine-deficient diet during a secondary infection, the proliferation of L. monocytogenes in the liver and spleen was not affected whereas the concentration of RNI in plasma of these mice was significantly reduced. Our findings that inhibition of RNI formation during a secondary infection does not affect the proliferation of L. monocytogenes in the liver and spleen and that enhanced elimination of bacteria from these organs is not accompanied by an increase in the concentration of RNI in plasma led to the conclusion that resistance against a secondary infection with L. monocytogenes is not dependent on RNI.
Characterization of the O-polysaccharide structure of lipopolysaccharide from Actinobacillus actinomycetemcomitans serotype b
Authors: Perry M B , MacLean L L , Gmür R , Wilson M E
Published: January 2020

We previously reported that the serotype b antigen of Actinobacillus actinomycetemcomitans is a constituent of the polysaccharide region of lipopolysaccharide (LPS) and contains significant amount of the neutral sugars rhamnose and fucose (M. Wilson and R. Schifferle, Infect. Immun. 59:1544-1551, 1991). In the present study, we determined the structure of the O antigen of A. actinomycetemcomitans Y4 (serotype b) LPS. Aqueous phase LPS was obtained from a phenol-water extract of A. actinomycetemcomitans Y4. This material was found to react with rabbit polyclonal antiserum to serotype b but not with antisera specific for other A. actinomycetemcomitans serotypes. Analyses revealed that the O polysaccharide of Y4 LPS consists of a polymer of trisaccharide repeating units composed of D-Fuc, AL-Rha, and D-GalNAc residues. An identical structure was obtained for the O polysaccharide of LPS from A. actinomycetemcomitans JP2, another serotype b strain. These results indicate that the serotype b antigen of A. actinomycetemcomitans is defined by a trisaccharide repeating unit present in the O polysaccharide of LPS.
Molecular characterization, genomic arrangement, and expression of bmpD, a new member of the bmp class of genes encoding membrane proteins of Borrelia burgdorferi
Authors: Ramamoorthy R , Povinelli L , Philipp M T
Published: January 2020

An expression library made with Borrelia burgdorferi DNA in the vector lambda ZapII was screened with serum from a monkey infected with the Lyme disease agent. This serum killed B. burgdorferi in vitro by an antibody-dependent, complement-mediated mechanism and contained antibodies to at least seven spirochetal antigens, none of which were the major outer surface proteins OspA or OspB. Among several positive clones, a clone containing the B. burgdorferi bmpA gene encoding the immunodominant antigen P39 was obtained. Chromosome walking and DNA sequence analysis permitted the identification of two additional upstream genes homologous to the bmpA gene and its related companion, bmpB. The first of these was the recently characterized bmpC gene, and adjacent to it was the fourth and new member of this class, which has been designated bmpD. The gene product encoded by bmpD is 34l residues long, contains a signal sequence with a potential signal peptidase II cleavage site, and has 26% identity with TmpC of Treponema pallidum. Southern blotting confirmed the tandem arrangement of all four bmp genes in the chromosome of B. burgdorferi JD1. However, Northern (RNA) blotting revealed that bmpD is expressed as a monocistronic transcript, which indicates that it is not part of an operon at the bmp locus. The bmpD gene was found to be conserved in representative members of the three species of the B. burgdorferi sensu lato complex, suggesting that it serves an important biological function in the spirochete.
Identification and characterization of epitopes on the 120-kilodalton surface protein antigen of Rickettsia prowazekii with synthetic peptides
Authors: Ching W M , Wang H , Jan B , Dasch G A
Published: January 2020

The 120-kDa surface protein antigens (SPAs) of typhus rickettsiae are highly immunogenic and have been shown to be responsible for the species-specific serological reactions of the typhus group rickettsiae. To study the immunochemistry of these proteins, overlapping decapeptides encompassing the whole protein were synthesized on derivatized polyethylene pins. A modified enzyme-linked immunosorbent assay was used to identify epitopes recognized by rabbit hyperimmune antisera to Rickettsia prowazekii SPA. Eight distinct epitopes were mapped by this method in three regions. Four of the epitopes, which were located in the carboxyterminus of mature processed SPA, were strongly competitively inhibited by native folded SPA but not by intact rickettsiae, suggesting that they were on the SPA surface but not exposed on the rickettsial surface. Three of these epitopes were present on both R. prowazekii and Rickettsia typhi SPAs. The immunoreactivities of five epitopes were further characterized by synthesizing modified peptides. Glycine substitution experiments determined the critical residues in the epitopes. The dependence of binding of the peptide epitopes to the polyclonal antisera was mapped to single residues. The limited number and weak reactivity of linear peptide epitopes observed with human and rabbit sera, possibly due to a lack of the methylated amino acids which are present in rickettsia-derived SPA, suggest that the present approach will not provide useful synthetic antigens for diagnosis of typhus infections.
Characterization of neuraminidases produced by various serotypes of Pasteurella multocida
Authors: Straus D C , Jolley W L , Purdy C W
Published: January 2020

Neuraminidases produced by 16 strains of Pasteurella multocida (serotypes 1 to 16) were characterized by molecular weight, substrate specificity, and antigenic identity. After growth in a chemically defined medium, stage I (lyophilized) culture supernatants were assayed for activity with N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Neuraminidase produced by P. multocida A:3 was purified by a combination of salt fractionation, ion-exchange chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. Purified P. multocida A:3 neuraminidase was employed to immunize rabbits, and the resulting antiserum reduced the activity of the P. multocida A:3 enzyme by 40.3%. This antiserum also reduced the activities of the neuraminidases produced by other serotypes by between 30.8 and 59.6%. Molecular weight estimates of the neuraminidases produced by the various serotypes were obtained by gel filtration chromatography on Sephadex G-200. Each of the 16 serotypes examined produced a neuraminidase with a molecular weight of approximately 500,000. In addition, all 16 high-molecular-weight neuraminidases showed similar substrate specificities. On the basis of these data, it appears that the high-molecular-weight neuraminidases produced by different P. multocida serotypes are quite similar.
The pertussis toxin liberation genes of Bordetella pertussis are transcriptionally linked to the pertussis toxin operon
Authors: Ricci S , Rappuoli R , Scarlato V
Published: January 2020

The DNA sequence of the pertussis toxin operon (ptx) of Bordetella pertussis predicts that transcription of the operon ends downstream from the ptxS3 gene at a possible stem-loop structure. Secretion of the assembled pertussis toxin into the culture medium required the expression of 8 genes arranged in an operon (ptl) and lying 55 bp downstream from the ptx and ptl operons are cotranscribed and coregulated by the P(TOX) promoter. Deletion of the 55-bp DNA region caused an increase in the amount of the ptl transcripts. It is likely that this DNA region is involved in regulation of the ptx-pti expression.
In vivo induction of apoptosis and immune responses in mice by administration of lipopolysaccharide from Porphyromonas gingivalis
Authors: Isogai E , Isogal H , Kimura K , Fujii N , Takagi S , Hirose K , Hayashi M
Published: January 2020

In vivo administration of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) to mice induced apoptosis before a specific immune response. Apoptosis was associated with the expression of immunoglobulin and Ia on B cells and of CD5 and several markers on T cells. Apoptosis peaked in the spleen and lymph nodes on day 2, and the second peak occurred in the thymus on day 9. Tumor necrosis factor alpha (TNF-alpha) could mediate apoptosis, because the serum TNF-alpha levels were significantly higher than those of controls at 1 day before apoptosis and recombinant murine TNF-alpha induced apoptosis. The apoptosis induced by Pg-LPS was similar to that induced by Escherichia coli LPS in its basic manner, but it was unique in the response of thymus T cells. It was suggested that Pg-LPS could induce apoptosis for the elimination of early nonspecific activated lymphocytes.
Chronic proliferative hepatitis in A/JCr mice associated with persistent Helicobacter hepaticus infection: a model of helicobacter-induced carcinogenesis
Authors: Fox J G , Li X , Yan L , Cahill R J , Hurley R , Lewis R , Murphy J C
Published: January 2020

Helicobacter hepaticus causes hepatitis in selected strains of mice and in A/JCr mice is linked to liver cancer. To analyze whether H. hepaticus persists in specified ecological niches, to determine whether biomarkers of infection exist, and to analyze the influence of H. hepaticus on hepatocyte proliferation, a longitudinal study of H. hepaticus-infected A/JCr mice was undertaken. A/JCr mice were serially euthanatized from 3 through 18 months and surveyed by enzyme-linked immunosorbent assay; bacterial culture of liver, colon, and cecum; histology; electron microscopy; hepatocyte proliferation indices determined by using 5-bromo-2'-deoxyuridine; and measurement of the liver enzyme alanine aminotransferase. In infected animals throughout the 18-month study, H. hepaticus was consistently isolated from the lower bowel but only sporadically from the liver. By electron microscopy, H. hepaticus was noted infrequently and only in bile canaliculi. Infected mice, particularly males, showed chronic inflammation; oval cell, Kupffer cell, and Ito cell hyperplasia; hepatocytomegaly; and bile duct proliferation. The inflammatory and necrotizing lesion was progressive and involved the hepatic parenchyma, portal triads, and intralobular venules. Hepatic adenomas were noted only in male mice, whereas 5-bromo-2'-deoxyuridine proliferation indices were markedly increased in both sexes, but especially in males, compared to control A/J mice. Infected mice also developed sustained anti-H. hepaticus serum immunoglobulin G antibody responses and elevated alanine aminotransferase levels. H. hepaticus, which persists in the lower bowels and livers of A/JCr mice, is associated with a chronic proliferative hepatitis, and hepatomas in selected male mice indicate that this novel bacterium may cause an increased risk of hepatic cancer induction in susceptible strains of mice. This murine model should prove useful in dissecting the molecular events operable in the development of neoplasms induced by bacteria belonging to this expanding genera of pathogenic Helicobacter species.
gamma Interferon gene expression and release in human lymphocytes directly activated by Cryptococcus neoformans and Candida albicans
Authors: Levitz S M , North E A
Published: January 2020

Previous studies in our laboratory and others have demonstrated that T and/or NK cells can directly bind to and inhibit the growth of the medically important fungal pathogens Cryptococcus neoformans and Candida albicans by apparently non-major histocompatibility complex-restricted mechanisms. Here, we examined whether this direct interaction between lymphocytes and fungi also results in cytokine gene expression and release. Nonadherent lymphocytes (NAL), isolated from human peripheral blood mononuclear cells by depletion of cells adherent to plastic and nylon wool, released gamma interferon (IFN-gamma), but not interleukin-4 (IL-4) and IL-10, following stimulation with C. neoformans yeast cells and C. albicans yeast cells, hyphae, and supernatants. The fungal stimuli also induced IFN-gamma mRNA, with peak gene expression seen at or after 18 h. IFN-gamma release was still seen even when either NK cells or T lymphocytes were depleted by negative selection, suggesting that both cell types can be stimulated by fungi to produce IFN-gamma. Release of IFN-gamma from fungus-stimulated NAL occurred in the absence of an intact complement system and was not especially enhanced by culture with IL-2 or IL-12. These data expand the mechanisms by which the direct interaction of NAL with fungal targets can lead to immune activation. Moreover, to our knowledge, this is the first demonstration of direct stimulation of T-cell cytokine release by microbial pathogens.
Defining antibody targets in Streptococcus oralis infection
Authors: Burnie J P , Brooks W , Donohoe M , Hodgetts S , al-Ghamdi A , Matthews R C
Published: January 2020

Immunoblotting of sera from 12 neutropenic patients with Streptococcus oralis septicemia and 18 patients with endocarditis due to viridans group streptococci revealed immunodominant S. oralis antigens at 85 and 180 kDa. The former cross-reacted with a mouse monoclonal antibody to hsp90. The latter was identified by sequencing positive clones obtained by screening a genomic expression library of S. oralis with pooled sera from patients who had been infected with S. oralis. Antibody eluted from one of these clones reacted with the 180-kDa antigen of S. oralis. Southern blotting confirmed the origin of the clone from S. oralis. The derived amino acid sequence showed 76.2% homology with the PAc protein precursor of Streptococcus mutans and 73.8% homology with the SpaA protein precursor of Streptococcus sobrinus. Epitope mapping of the derived amino acid sequence with sera from patients with viridans group streptococcal endocarditis delineated nine epitopes. Peptides 1 (TMYPNRQPGSGWDSS) and 2 (WYSLNGKIRAVDVPK), representing two of these epitopes, and peptide 3 (YEVEKPLEPAPVAPS), representing the repeat proline region, were synthesized. These three peptides were used to screen a phage antibody display library derived from a patient who had recovered from S. oralis infection. Two of the human recombinant antibodies produced (SORAL 3 and SORAL 4 against peptide 3) and a human recombinant antibody (B3.7) against the conserved epitope (LKVIRK) of hsp90 gave statistically significant protection, compared with control groups, in a mouse model of lethal S. oralis infection.
Attenuated vaccinia virus-circumsporozoite protein recombinants confer protection against rodent malaria
Authors: Lanar D E , Tine J A , de Taisne C , Seguin M C , Cox W I , Winslow J P , Ware L A , Kauffman E B , Gordon D , Ballou W R , Paoletti E , Sadoff J C
Published: January 2020

NYVAC-based vaccinia virus recombinants expressing the circumsporozoite protein (CSP) were evaluated in the Plasmodium berghei rodent malaria model system. Immunization of mice with a NYVAC-based CSP recombinant elicited a high level of protection (60 to 100%). Protection did not correlate with CS repeat-specific antibody responses and was abrogated by in vivo CD8+ T-cell depletion. Protection was not enhanced by modification of the subcellular localization of CSP. These results suggest the potential of poxvirus-based vectors for the development of vaccine candidates for human malaria.
A cloned major Schistosoma mansoni egg antigen with homologies to small heat shock proteins elicits Th1 responsiveness
Authors: Cai Y , Langley J G , Smith D I , Boros D L
Published: January 2020

In schistosomiasis mansoni, soluble egg antigens of the worm induce chronic T-cell-mediated granulomatous tissue responses. Since the first preparation of crude soluble egg antigen extract, a dearth of highly purified antigens has hampered the identification of granuloma inducer molecules. Here we report that a cloned 38-kDa egg polypeptide (r38) with homologies to small heat shock proteins is a strong immunogen. The recombinant and the sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated and eluted native 38-kDa (p38) polypeptides, used in microgram amounts and unaided by adjuvant, sensitized mice for a Th1-type immune response, with strong interleukin-2 (IL-2) and gamma interferon secretion but no IL-4 and IL-10 secretion. Extensive cross-reactivity between these two polypeptides was evident. THis pattern was confirmed by reverse transcription-PCR that showed strong IL-2 and gamma interferon message expression but trace amounts of IL-4 message expression in r38-sensitized splenocytes. In mice, the polypeptide induced pulmonary mononuclear granuloma formation around antigen-coupled beads or worm eggs. We propose that the superior immunogenicity of r38 is linked to its relatedness to small heat shock proteins and that the 38-kDa polypeptide may induce the Th1 cytokine responses observed during the early development phase of the egg-induced granuloma.
Pathogenicity of the diffusely adhering strain Escherichia coli C1845: F1845 adhesin-decay accelerating factor interaction, brush border microvillus injury, and actin disassembly in cultured human intestinal epithelial cells
Authors: Bernet-Camard M F , Coconnier M H , Hudault S , Servin A L
Published: January 2020

The diffusely adhering Escherichia coli strain C1845 harboring the fimbrial F1845 adhesin can infect cultured human intestinal epithelial cells. The mechanism by which E. coli C1845 induces diarrheal illness remains unknown. This study investigated the injuries of cultured human intestinal cells promoted by E. coli C1845. Membrane-associated decay accelerating factor was identified as the intestinal receptor for the F1845 fimbrial adhesin of the E. coli C1845 strain by using purified F1845 adhesin, antibody directed against the F1845 adhesin, and monoclonal antibodies directed against the decay accelerating factor. Using monolayers of Caco-2 cells apically infected with E. coli C1845 and examined by scanning and transmission electron microscopy, we observed that strain C1845 induced injury to microvilli (MV) characterized by elongation and nucleation of the MV. We observed that infection of T84 and Caco-2 cells by E. coli C1845 was followed by disassembly of the actin network in the apical and basal cell domains. MV injury was differentiation related: E. coli C1845 promoted MV injury only when the cells were fully differentiated. The disassembly of the actin network occurred in poorly differentiated and fully differentiated Caco-2 cells but not in undifferentiated cells. Moreover, apical actin disassembly was observed in fully differentiated Caco-2 cells infected with the laboratory strain E. coli HB101(pSSS1) expressing the F1845 adhesin. In conclusion, E. coli C1845 promotes MV lesion in human epithelial intestinal cells, resulting from disassembly of the actin network.