bluuebunny/crossref_metadata_2025 huggingface dataset:
Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry
Authors: Sonnenberg M G , Belisle J T
Published: January 2020

A number of the culture filtrate proteins secreted by Mycobacterium tuberculosis are known to contribute to the immunology of tuberculosis and to possess enzymatic activities associated with pathogenicity. However, a complete analysis of the protein composition of this fraction has been lacking. By using two-dimensional polyacrylamide gel electrophoresis, detailed maps of the culture filtrate proteins of M. tuberculosis H37Rv were generated. In total, 205 protein spots were observed. The coupling of this electrophoretic technique with Western blot analysis allowed the identification and mapping of 32 proteins. Further molecular characterization of abundant proteins within this fraction was achieved by N-terminal amino acid sequencing and liquid chromatography-mass spectrometry. Eighteen proteins were subjected to N-group analysis; of these, only 10 could be sequenced by Edman degradation. Among the most interesting were a novel 52-kDa protein demonstrating significant homology to an alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708, a 25-kDa protein corresponding to open reading frame 28 of the M. tuberculosis cosmid MTCY1A11, and a 31-kDa protein exhibiting an amino acid sequence identical to that of antigen 85A and 85B. This latter product migrated with an isoelectric point between those of antigen 85A and 85C but did not react with the antibody specific for this complex, suggesting that there is a fourth member of the antigen 85 complex. Novel N-terminal amino acid sequences were obtained for three additional culture filtrate proteins; however, these did not yield significant homology to known protein sequences. A protein cluster of 85 to 88 kDa, recognized by the monoclonal antibodies IT-57 and IT-42 and known to react with sera from a large proportion of tuberculosis patients, was refractory to N-group analysis. Nevertheless, mass spectrometry of peptides obtained from one member of this complex identified it as the M. tuberculosis KatG catalase/peroxidase. Thus, the detailed mapping of M. tuberculosis proteins, combined with state-of-the-art analytical techniques such as mass spectrometry, provides a basis for further analysis and rapid identification of biologically relevant molecules.
A novel component different from endotoxin extracted from Prevotella intermedia ATCC 25611 activates lymphoid cells from C3H/HeJ mice and gingival fibroblasts from humans
Authors: Iki K , Kawahara K , Sawamura S , Arakaki R , Sakuta T , Sugiyama A , Tamura H , Sueda T , Hamada S , Takada H
Published: January 2020

A novel immunobiologically active fraction was prepared from a phenol-water extract of Prevotella intermedia ATCC 25611 by Sephadex G-100 column chromatography. The fraction consisted mainly of carbohydrate and protein and was devoid of fatty acid. The fraction showed high-molecular-weight bands (10,000 to 12,000) on deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) and was scarcely active in a Limulus test. We designated the fraction Prevotella glycoprotein (PGP). The PGP fraction showed strong mitogenicity on splenocytes and cytokine-inducing activities on peritoneal macrophages from both C3H/HeJ and C3H/HeN mice, and it stimulated human gingival fibroblasts to produce cytokines. The activities of the PGP fraction were resistant to heat inactivation (100 degrees C for 1 h) and protease treatments and were scarcely inhibited by polymyxin B. In contrast, the purified lipopolysaccharide fraction (LPS-PCP) extracted from the same bacterium with a phenol-chloroform-petroleum ether mixture, which showed strong Limulus activity and a single low-molecular-weight band (approximately 3,000) on DOC-PAGE, lacked the activities on splenocytes and macrophages from C3H/HeJ mice and human gingival fibroblasts. The activities of the LPS-PCP fraction on cells from C3H/HeN mice were completely inhibited by polymyxin B. The LPS extracted from the same bacterium with hot phenol-water (LPS-PW) exhibited the properties of both the PGP fraction and the LPS-PCP fraction. These findings suggest that the unique bioactivities of the LPS-PW fraction of oral black-pigmented bacteria reported to date, which differed from those of the classical endotoxin, were derived from the PGP fraction and not from the LPS itself.
Failure to block adhesion of Plasmodium falciparum-infected erythrocytes to ICAM-1 with soluble ICAM-1
Authors: Craig A G , Pinches R , Khan S , Roberts D J , Turner G D , Newbold C I , Berendt A R
Published: January 2020

The adhesion of Plasmodium falciparum-infected erythrocytes is thought to play a central role in the pathogenesis of severe malaria. ICAM-1 has been identified as one of the host receptors for parasitized erythrocytes and has been implicated as being involved in progression to cerebral malaria. Thus, intervention strategies based on the reversal of this interaction could potentially be used to reduce morbidity and mortality. We have investigated the inhibition of the interaction between ICAM-1 and infected erythrocytes by using recombinant soluble ICAM-1 as competitor and find that we are unable to reduce adhesion to ICAM-1 in vitro.
Induction of an immune response by oral administration of recombinant botulinum toxin
Authors: Kiyatkin N , Maksymowych A B , Simpson L L
Published: January 2020

A gene encoding the full-size botulinum neurotoxin serotype C was reconstructed in vector pQE-30 and expressed at high levels in Escherichia coli. Three amino acid mutations (H229-->G, E230-->T, and H233-->N) were generated in the zinc-binding motif, resulting in complete detoxification of the modified recombinant holotoxin. The PCR-amplified wild-type light chain of botulinum neurotoxin serotype C was also expressed in E. coli and used as a control in all experiments. Modified recombinant holotoxin and light chain contained a histidine affinity tag at the amino terminus, which was used for detection and purification. Recombinant proteins were purified on nickel affinity resin and analyzed by Western blotting with the anti-histidine tag and anti-neurotoxin C antibodies. The results indicated that the 150-kDa molecule of modified recombinant holotoxin and the 50-kDa recombinant light chain were synthesized without degradation; however, E. coli did not provide for efficient nicking of modified recombinant toxin. Modified recombinant holotoxin was not toxic to mice, had no effect on nerve-evoked muscle twitch in vitro, and was not able to cleave syntaxin in crude synaptosome preparations. The recombinant light chain was also nontoxic in vivo, had no effect on evoked muscle twitch, but was able to cleave syntaxin. Modified recombinant neurotoxin and light chain were administered to animals either orally or subcutaneously. Both oral administration and subcutaneous administration of modified recombinant neurotoxin evoked high levels of serum antibodies and protective immunity. Oral administration of recombinant light chain evoked no systemic response, whereas subcutaneous administration evoked antibody production and immunity.
Contribution of the Mn-cofactored superoxide dismutase (SodA) to the virulence of Yersinia enterocolitica serotype O8
Authors: Roggenkamp A , Bittner T , Leitritz L , Sing A , Heesemann J
Published: January 2020

Enteric pathogens harbor a set of enzymes (e.g., superoxide dismutases [SOD]) for detoxification of endogenous and exogenous reactive oxygen species which are encountered during infection. To analyze the role of the Mn-cofactored SOD (SodA) in the pathogenicity of yersiniae, we cloned the sodA gene of Yersinia enterocolitica serotype O8 by complementation of an Escherichia coli sodA sodB mutant and subsequently constructed an isogenic mutant by allelic exchange. Sequence analysis revealed an open reading frame that enabled the deduction of a sequence of 207 amino acids with 85% identity to SodA of E. coli. In a mouse infection model, the sodA null mutant was strongly attenuated in comparison to its parental strain. After intravenous infection, the survival and multiplication of the mutant in the spleen and liver were markedly reduced. In contrast, inactivation of sodA had only minor effects on survival and multiplication in the gut and Peyer's patches, as could be demonstrated in the orogastric infection model. The reduction in virulence was accompanied by a low but significant increase of susceptibility of the soda mutant to bacterial killing by polymorphonuclear leukocytes (PMN) and an alteration of the intracellular chemiluminescence response of PMN. These results suggest that the resistance of Y. enterocolitica to exogenous oxygen radicals produced by phagocytes involves the Mn-cofactored SOD. The important role of sodA for the pathogenicity of Y. enterocolitica could also be due to detoxification of endogenous, metabolically produced oxygen radicals which are encountered by extracellular enteric pathogens during the invasion of the host.
Interaction of Neisseria meningitidis with a polarized monolayer of epithelial cells
Authors: Pujol C , Eugène E , de Saint Martin L , Nassif X
Published: January 2020

An important step in the pathogenesis of Neisseria meningitidis is the crossing of two cellular barriers, one in the nasopharynx and one in the brain. To approach the mechanisms by which this bacterium can achieve these goals, we studied the interactions between N. meningitidis and a monolayer of polarized tight junction-forming T84 cells grown on filter units. A capsulated, piliated, Opa-, and Opc- N. meningitidis strain is shown to be capable of adhering to and crossing this monolayer several orders of magnitude more efficiently than an isogenic nonpiliated derivative. This bacterial interaction does not affect the barrier function of tight junctions, as assessed by (i) the absence of modification of the transepithelial resistance, (ii) the lack of increase of [3H]inulin penetration across the monolayer, and (iii) the absence of delocalization of ZO-1, a tight junction protein. Electron microscopy studies and confocal examinations demonstrated that N. meningitidis (i) induces cytoskeletal rearrangements with actin polymerization beneath adherent bacteria, (ii) is intimately attached to the apical membrane of the cells, and (iii) can be internalized inside cells. Immunofluorescent staining with antipilus antibodies showed evidence that meningococcal piliation was dramatically reduced at later time points of bacterial cell interaction compared to the early phase of this interaction. In addition, adhesive bacteria recovered from an infected monolayer are piliated, capsulated, Opa-, and Opc-, a phenotype similar to that of the parental strain. Taken together, these data demonstrate that following pilus-mediated adhesion, N. meningitidis is involved in an intimate attachment which requires a bacterial component different from Opa and Opc and that meningococci cross a monolayer of tight-junction-forming epithelial cells by using a transcellular pathway rather than a paracellular route.
Bacterial phospholipase C upregulates matrix metalloproteinase expression by cultured epithelial cells
Authors: Firth J D , Putnins E E , Larjava H , Uitto V J
Published: January 2020

Phospholipase C (PLC) is a putative virulence factor of several pathogenic bacteria. We studied if exogenous PLC would perturb epithelial behavior in infected tissues. Gelatin and casein zymography of cell culture medium indicated that the broad-spectrum PLC of Bacillus cereus induced matrix metalloproteinase (MMP) production in epithelial cells of human skin (NHEK), human gingiva (HGE), and porcine periodontal ligament (PLE). In all three cell types, the strongest increase (ninefold) at 0.1 U/ml was seen in the MMP-9 (92-kDa gelatinase) activity, and the effect was dose dependent in the range of 0.1 to 1.0 U/ml. A relatively weaker increase (twofold) in MMP-2 (72-kDa gelatinase) was also observed in each cell type. PLC induction of MMP-3 (48-kDa stromelysin) was also seen in NHEK and HGE on gelatin and more sensitively for PLE by casein zymography (fivefold). Total gelatinolytic activity as measured by degradation of 14C-labeled denatured type I collagen increased by about 18-fold (NHEK), 12-fold (HGE), and 14-fold (PLE). Northern analysis showed a clear increase in the MMP-9, and a minor increase in MMP-3 mRNA levels but no significant increase in MMP-2 mRNA levels. Further studies with PLE revealed that MMP-9 induction by PLC progressively increased with the length of cell culture time in the absence of serum. PLC induction of MMPs was polar, with MMP-9 and MMP-3 secreted primarily in the apical direction and MMP-2 secreted mainly in the basal direction. The PLC effect was blocked by neomycin, an inhibitor of the phosphoinositol signal pathway. No significant effects were observed in MMP expression with the calcium ionophore A23187 or phospholipase A2. Morphologically, PLC treatment resulted in reduced contacts between the cultured cells and loss of the cell surface microvilli. These results suggest that PLC secreted by bacterial pathogens may disrupt epithelium of infected tissue and increase the subepithelial tissue destruction through induction of MMPs.
MTC28, a novel 28-kilodalton proline-rich secreted antigen specific for the Mycobacterium tuberculosis complex
Authors: Manca C , Lyashchenko K , Colangeli R , Gennaro M L
Published: January 2020

Proteins that are actively secreted by Mycobacterium tuberculosis serve as major targets of immune responses in the infected host. To identify and purify novel proteins in the filtrates of M. tuberculosis cultures, a bacteriophage lambda library of M. tuberculosis H37Rv DNA was immunoscreened by using an anti-culture filtrate rabbit antiserum. Of 20 positive clones isolated, 6 were analyzed and found to express the genes for two known components of the early culture filtrate, the secreted 45/47-kDa antigen complex and the KatG protein, and two novel genes. Here we report the molecular cloning and nucleotide sequence of one of the new genes encoding a culture filtrate protein of 310 amino acid (aa) residues. We called this gene mtc28. The deduced polypeptide sequence contained an NH2-terminal, highly hydrophobic 32-aa region having properties of a secretion signal peptide. The putative 278-aa mature MTC28 protein was characterized at its NH2 and COOH termini by a high content of proline and alanine residues organized in an (AP)n motif. Thus, MTC28 is a new member of a group of proline-rich antigens found in M. tuberculosis and Mycobacterium leprae. As shown by DNA hybridization experiments, the mtc28 gene was present only in species of the M. tuberculosis complex. Purified recombinant MTC28 antigen evoked strong delayed-type hypersensitivity and antibody responses in guinea pigs immunized with Mycobacterium bovis BCG, but not in guinea pigs immunized with Mycobacterium avium. The strong immunological activity of MTC28 and the absence of B- and T-cell epitopes cross-reactive with a common environmental mycobacterial species, such as M. avium, make this novel antigen an attractive reagent for immunodiagnosis of tuberculosis.
Isolation, cloning, and expression of a 70-kilodalton plasminogen binding protein of Borrelia burgdorferi
Authors: Hu L T , Pratt S D , Perides G , Katz L , Rogers R A , Klempner M S
Published: January 2020

Surface receptors for plasminogen are expressed by many gram-positive and gram-negative bacteria and may play a role in the dissemination of organisms by binding plasminogen, which upon conversion to plasmin can digest extracellular matrix proteins. Two plasminogen binding proteins have been identified for Borrelia burgdorferi, outer surface protein A and a 70-kDa protein (BPBP). We purified BPBP by plasminogen affinity chromatography and obtained its amino acid sequence by Edman degradation of a tryptic digest. The gene coding for BPBP was isolated from a lambda-ZAP II genomic library with probes developed from sequenced portions of the protein. This gene was expressed in Escherichia coli; the recombinant product was seen by antibody raised against native BPBP and also bound 125I-labeled plasminogen. The experimentally derived amino acid sequences corresponded to the predicted sequence encoded by the BPBP gene. The deduced amino acid sequence for BPBP revealed significant similarity to p30, a 30-kDa protein of B. burgdorferi (54% identity and 65% similarity), to a 60-kDa protein in Borrelia coriaceae (66% identity and 80% similarity), to oligopeptide binding protein A of E. coli (34% identity and 57% similarity), and, more generally, to the periplasmic oligopeptide binding family of proteins.
Structural and antigenic types of cell wall polysaccharides from viridans group streptococci with receptors for oral actinomyces and streptococcal lectins
Authors: Cisar J O , Sandberg A L , Reddy G P , Abeygunawardana C , Bush C A
Published: January 2020

Lectin-mediated interactions between oral viridans group streptococci and actinomyces may play an important role in microbial colonization of the tooth surface. The presence of two host-like motifs, either GalNAc beta1-->3Gal (Gn) or Gal beta1-->3GalNAc (G), in the cell wall polysaccharides of five streptococcal strains accounts for the lactose-sensitive coaggregations of these bacteria with Actinomyces naeslundii. Three streptococcal strains which have Gn-containing polysaccharides also participate in GalNAc-sensitive coaggregations with strains of Streptococcus gordonii and S. sanguis. Each Gn- or G-containing polysaccharide is composed of a distinct phosphodiester-linked hexa- or heptasaccharide repeating unit. The occurrence of these polysaccharides on 19 additional viridans group streptococcal strains that participate in lactose-sensitive coaggregations with actinomyces was examined. Negatively charged polysaccharides that reacted with Bauhinia purpurea agglutinin, a Gal and GalNAc binding plant lectin, were isolated from 17 strains by anion exchange column chromatography of mutanolysin-cell wall digests. Results from nuclear magnetic resonance and immunodiffusion identified each of 16 polysaccharides as a known Gn- or G-containing structural type and one polysaccharide as a new but closely related Gn-containing type. Unlike the reactions of lectins, the cross-reactions of most rabbit antisera with these polysaccharides were correlated with structural features other than the host-like motifs. Gn-containing polysaccharides occurred primarily on the strains of S. sanguis and S. oralis while G-containing polysaccharides were more common among the strains of S. gordonii and S. mitis examined. The findings strongly support the hypothesis that lectin-mediated recognition of these streptococci by other oral bacteria depends on a family of antigenically diverse Gn- and G-containing cell wall polysaccharides, the occurrence of which may differ between streptococcal species.
A specific cell surface antigen of Streptococcus gordonii is associated with bacterial hemagglutination and adhesion to alpha2-3-linked sialic acid-containing receptors
Authors: Takahashi Y , Sandberg A L , Ruhl S , Muller J , Cisar J O
Published: January 2020

A Ca2+-independent lectin activity for alpha2-3-linked sialic acid-containing receptors is associated with Streptococcus gordonii DL1 (Challis) but not with a spontaneous mutant, strain D102, that specifically lacks hemagglutinating activity. Comparison of crossed-immunoelectrophoresis patterns of parent and mutant sonicated cell extracts identified a unique antigen (Hs antigen) in the parent cell extract that was purified by DEAE Sephacel column chromatography and by a wheat germ agglutinin (WGA) lectin affinity column. The purified antigen formed a single arc in crossed immunoelectrophoresis with anti-DL1 serum and migrated as a diffuse band above the 200-kDa marker in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy with specific anti-Hs antibody revealed labeling of structures in the fibrillar layer of strain DL1 and no labeling of fibrillar structures on strain D102. Rabbit anti-DL1 serum and anti-Hs Fab inhibited the hemagglutinating activity of strain DL1, and the inhibition was specifically neutralized by purified Hs antigen. Anti-Hs Fab did not inhibit the hemagglutinating activities of several heterologous S. gordonii strains; however, these bacteria were agglutinated by anti-Hs immunoglobulin G and also by WGA. In contrast, two S. gordonii strains that lacked hemagglutinating activity did not react with anti-Hs antibody or with WGA. These findings associate the sialic acid-binding lectin activity of S. gordonii DL1 with a specific fibrillar antigen, which is composed of protein and WGA reactive carbohydrate, and indicate that cross-reactive antigens occur on other strains of this species that possess hemagglutinating activity.
Oligoclonality of serum immunoglobulin G antibody responses to Streptococcus pneumoniae capsular polysaccharide serotypes 6B, 14, and 23F
Authors: Lucas A H , Granoff D M , Mandrell R E , Connolly C C , Shan A S , Powers D C
Published: January 2020

Serum antibodies (Abs) specific for the capsular polysaccharides of Streptococcus pneumoniae provide protection against invasive pneumococcal disease. Previous studies indicate that Abs to pneumococcal polysaccharide (PPS) serotypes 1 and 6B have limited clonal diversity. To determine if restricted diversity was a feature common to other PPS specificities, we examined the light (L)-chain expression and isoelectric heterogeneity of type 6B, 14, and 23F Abs elicited in 15 adults following PPS vaccination. At the population level, both PPS-6B and PPS-14 Abs expressed kappa and lambda chains, although 6B Abs more frequently expressed lambda chains lambda and 14 Abs more frequently expressed kappa chains. In individual sera, Abs were generally skewed towards either kappa or lambda expression. 23F-specific Abs had predominantly kappa chains. Isoelectric focusing analyses showed that sera contained one or at most a few immunoglobulin G Ab spectrotypes to all three respective capsular serotypes, a result indicative of oligoclonality. A sequence analysis of a purified PPS-14-specific Ab having a single spectrotype gave uniform amino-terminal sequences for both the heavy chain (V(H)III subgroup) and the L chain (kappaIII-A27 V region). From these results we conclude that within individual adults, serum Ab responses to PPS serotypes 6B, 14, and 23F derive from a small number of dominant B-cell clones, and consequently variable-region expression is probably individually limited as well. Oligoclonality appears to be a general characteristic of human PPS-specific Ab repertoires, and we suggest that this property could lead to individual differences in Ab fine specificity and/or functional activity against encapsulated pneumococci.
Cellular changes and cytokine expression in the ilea of gnotobiotic piglets resulting from peroral Salmonella typhimurium challenge
Authors: Trebichavský I , Dlabac V , Reháková Z , Zahradnícková M , Splíchal I
Published: January 2020

Two stable rough mutants of Salmonella spp. were studied as live peroral vaccines. The SF1591 mutant of S. typhimurium (Ra chemotype) protected germ-free piglets against subsequent infection with virulent smooth S. typhimurium LT2, whereas a deep-rough mutant of S. minnesota mR595 (Re chemotype) did not. We investigated cytokine and leukocyte profiles in the ilea of gnotobiotic piglets colonized for 1 week either with rough mutants alone or with rough mutants followed by S. typhimurium LT2. The ileal mucosae of piglets associated with strain SF1591 alone were not inflamed. Villi contained activated macrophages, and enterocytes expressed transforming growth factor beta (TGF-beta). Subsequent infection of piglets with S. typhimurium LT2 resulted in immigration of alphabeta T cells and immunoglobulin A (IgA) response. In contrast, the ileal mucosae of piglets associated with strain mR595 alone expressed heat shock proteins and inflammatory cytokines but not TGF-beta. Acellular villi contained numerous gammadelta T cells but no alphabeta T cells. After subsequent challenge with the LT2 strain, most piglets died of sepsis. Intestinal mucosae contained IgG but no IgA. These findings suggest the importance of cytokine signals in the regulation of intestinal responses against Salmonella infection.
Apoptosis of human monocytes and macrophages by Mycobacterium avium sonicate
Authors: Hayashi T , Catanzaro A , Rao S P
Published: January 2020

Mycobacterium avium is an intracellular organism which multiplies predominantly within human macrophages. This organism has previously been shown to induce apoptosis in human macrophages. With a view to identifying M. avium components that induce cell death in infected host cells, sonicated extracts of M. avium as well as individual components isolated from the M. avium sonicate were tested in various assays with a human monocytic cell line (THP-1). THP-1 cells incubated with M. avium sonicate showed significantly reduced viability after a 2-day exposure compared to control cells incubated with media alone. This effect was dose dependent, with only 6.6% +/- 5.2% and 48.8% +/- 10.3% of the cells being viable by trypan blue exclusion at 600 and 300 microg/ml, respectively. Control cells, on the other hand, exhibited a viability of 98.8% +/- 1.0%. In addition, an 80% ammonium sulfate fraction of the M. avium sonicate and the previously characterized 68-kDa protein were found to have similar effects on THP-1 cells. In both cases, the reduction in viability was due to apoptosis characterized by chromatin condensation, DNA fragmentation by agarose gel electrophoresis, or terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL) and release of nuclear matrix protein (NMP) into the culture medium. M. avium sonicate-induced apoptosis of THP-1 cells was completely inhibited by the commonly used antioxidants pyrrolidinedithiocarbamate (PDTC) and butylated hydroxyanisole (BHA), indicating that the generation of free oxygen radicals may be responsible for inducing cell death. M. avium sonicate was found to induce apoptosis of monocyte-derived macrophages (MDMs) as well. This effect was not reversed in the presence of PDTC and was not accompanied with DNA fragmentation when determined by agarose gel electrophoresis, as seen in the case of THP-1 cells. However, these MDMs were found to contain fragmented DNA by TUNEL. These findings suggest that the mechanism of cell death in MDMs may be different from that observed with THP-1 cells. Furthermore, these results provide new insight into the effect of M. avium components on host cell responses during M. avium infection.
Expression of thin aggregative fimbriae promotes interaction of Salmonella typhimurium SR-11 with mouse small intestinal epithelial cells
Authors: Sukupolvi S , Lorenz R G , Gordon J I , Bian Z , Pfeifer J D , Normark S J , Rhen M
Published: January 2020

The factors that mediate binding of Salmonella typhimurium to small intestinal epithelial cells have not been fully characterized. In this paper we demonstrate that elimination of production of thin aggregative fiber by a transposon insertion within the gene encoding the subunit protein of the fiber reduced binding of S. typhimurium SR-11 to a conditionally immortalized proximal small intestinal epithelial cell line established from transgenic mice. This binding defect could be overcome by transcomplementation with a wild-type allele. The conditionally immortalized cell line should prove useful in identifying the epithelial cell receptor for bacterial attachment since expression of its bacterial binding activity can be induced by manipulating the line's proliferative status.
A systemic downregulation of gamma interferon production is associated with acute shigellosis
Authors: Raqib R , Gustafsson A , Andersson J , Bakhiet M
Published: January 2020

Production of cytokines by peripheral blood mononuclear cells from Shigella-infected patients was assessed. The frequencies of tumor necrosis factor alpha (TNF-alpha), TNF-beta, and transforming growth factor beta mRNA-expressing cells were persistently upregulated during the course of shigellosis in comparison to those of healthy controls. In contrast, the frequency of gamma interferon (IFN-gamma) mRNA-expressing cells was significantly reduced during the acute stage compared to that during the convalescent stage and to that of healthy Bangladeshi controls (P < 0.01). Constitutive IFN-gamma production in Bangladeshi controls was significantly upregulated compared to that in Swedish controls.
Quantification of conserved antigens in Helicobacter pylori during different culture conditions
Authors: Lindholm C , Osek J , Svennerholm A M
Published: January 2020

In this study, we raised monoclonal antibodies (MAbs) against three conserved Helicobacter pylori antigens, i.e., the N-acetylneuraminyllactose-binding fibrillar hemagglutinin, HpaA; the flagellin subunits, FlaA and FlaB; and a species-specific 26-kDa protein. The MAbs were used for the development of sensitive inhibition enzyme-linked immunosorbent assays for quantification of these antigens in H. pylori during various culture conditions. The quantities of these antigens varied considerably (up to 8-fold) during different culture procedures and between strains (up to 10-fold).
Identification of the gene family encoding the 160-kilodalton Trypanosoma cruzi complement regulatory protein
Authors: Norris K A , Schrimpf J E , Szabo M J
Published: January 2020

Trypanosoma cruzi trypomastigotes are exquisitely resistant to the lytic effects of vertebrate complement, and this characteristic contributes to the survival of the parasites in the host bloodstream. Trypomastigotes avoid complement-mediated lysis by the production of a surface glycoprotein that inhibits the formation of the alternative and classical C3 convertase, thus preventing activation and amplification of the complement cascade at the parasite surface. We have developed a monoclonal antibody to the 160-kDa T. cruzi complement regulatory protein (CRP) and describe a one-step immunoaffinity purification procedure. The CRP was purified to homogeneity and subjected to amino-terminal peptide sequence analysis. Based on the protein sequence obtained, the CRP was identified as a member of a large family of trypomastigote-specific genes, and a complete cDNA was isolated and sequenced. The complete coding sequence was cloned in Escherichia coli, and antibodies raised against the full-length recombinant protein reacted specifically with a 160-kDa protein in trypomastigote membrane protein preparations as well as with native, purified CRP. Indirect immunofluorescence revealed that the protein is uniformly expressed at the cell surfaces of trypomastigotes.
Nramp1 transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on antigen processing and presentation
Authors: Lang T , Prina E , Sibthorpe D , Blackwell J M
Published: January 2020

The natural resistance-associated macrophage protein (Nramp1) regulates macrophage activation. One of its pleiotropic effects on macrophage function is to regulate expression of major histocompatibility class II molecules. In this study macrophages stably transfected with the wild-type (infection-resistant) or the natural mutant (infection-susceptible) allele of the Nramp1 gene were used to study class II expression and processing and presentation of recombinant protein antigens to CD4+ T-cell hybridomas. As demonstrated previously for macrophages from Nramp1-resistant and -susceptible congenic mouse strains, transfected macrophage clones carrying the wild-type allele showed enhanced upregulation of class II molecules in response to gamma interferon compared to that shown by macrophage clones carrying an endogenous mutant allele or transfected with the mutant allele expressed under a viral long terminal repeat promoter. The wild-type allele-transfected macrophage clones also demonstrated an enhanced, lipopolysaccharide-dependent ability to process the recombinant leishmanial antigen LACK-delta 1 (the Leishmania homolog of receptors for activated C kinase) for presentation to LACK-specific CD4+ T cells. An influence on antigen processing must therefore be added to the growing list of pleiotropic effects of the Nramp1 gene potentially contributing to its role in infectious and autoimmune disease susceptibility. These results also have important implications for analysis of T-cell responses to vaccination, especially where antigens are presented to the immune system using live Salmonella species or Mycobacterium bovis BCG as a vaccine vehicle.
Role of the carboxyl-terminal region of Porphyromonas gingivalis fimbrillin in binding to salivary proteins
Authors: Nagata H , Sharma A , Sojar H T , Amano A , Levine M J , Genco R J
Published: January 2020

Porphyromonas gingivalis fimbriae are considered to play an important role in the adherence and colonization of the bacteria in the oral cavity. In this study, we generated and purified three carboxyl-terminal variants of recombinant fimbrillin (r-FimA 224-337, r-FimA 266-337, and r-FimA 287-337, corresponding to amino acid residues 224 to 337, 266 to 337, and 287 to 337, respectively, of the 43-kDa fimbrillin of P. gingivalis 2561). They were used as inhibitors of P. gingivalis cell binding to human salivary protein-coated hydroxyapatite (HAP) beads. All of the carboxyl-terminal region polypeptides inhibited binding in a dose-dependent manner; however, the inhibitory effect of r-FimA 287-337 was less than that of the other two polypeptides when HAP beads were coated with whole saliva or purified salivary proline-rich protein 1 (PRP1). Assays of binding of a synthetic peptide corresponding to amino acid residues 266 to 286 of P. gingivalis 2561 fimbrillin to salivary proteins showed that this peptide bound strongly to whole saliva or PRP1 but only weakly to statherin. These results suggest that the carboxyl-terminal region corresponding to amino acid residues 266 to 337 of P. gingivalis fimbrillin plays an important role in binding to salivary proteins and that the domain corresponding to amino acids 266 to 286 is likely a major binding site for PRP1s and the domain corresponding to amino acids 287 to 337 is likely a major binding site for statherin.
Identification of neutralizing epitopes on Pseudomonas aeruginosa elastase and effects of cross-reactions on other thermolysin-like proteases
Authors: Kooi C , Hodges R S , Sokol P A
Published: January 2020

Monoclonal antibodies (MAbs) to a Burkholderia (Pseudomonas) cepacia 36-kDa protease (PSCP) which neutralize PSCP and Pseudomonas aeruginosa elastase but not P. aeruginosa alkaline protease have been isolated (C. Kooi et al., Infect. Immun. 62:2811-2817, 1994). These MAbs, designated 36-6-6 and 36-6-8, react with N-chlorosuccinimide cleavage products of P. aeruginosa elastase, consistent with the recognition of a 13.9-kDa fragment which contains the active site. Overlapping 9-mer peptides that span this region were synthesized. Neutralizing MAbs to PSCP reacted strongly with two peptides (341HGFTEQNSG349 and 395RYM DQPSRD403). Peptide 341HGFTEQNSG349 overlaps the motif 337HEXXH341, which has been found in many zinc-dependent endopeptidases. Peptide 395RYMDQPSRD403 lies between E361, which binds a zinc atom, and H420, which acts as a proton donor at the active site. Polyclonal rabbit sera raised against these peptides reacted with elastase on Western immunoblots and by enzyme-linked immunosorbent assay. With hide powder azure as the substrate, antisera to either HGFTEQNG and RYMDQPSRD completely neutralized the activities of elastase, thermolysin, Vibrio cholerae hemagglutinin/protease, and PSCP but had no effect on P. aeruginosa alkaline protease or the Serratia marcescens major protease. These results suggest that the MAbs recognize two different epitopes on P. aeruginosa elastase and that antibodies raised against synthetic peptides corresponding to either of these epitopes neutralize proteolytic activity.
Biochemical comparison of the Cu,Zn superoxide dismutases of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii
Authors: Hamilton A J , Holdom M D
Published: January 2020

Cu,Zn superoxide dismutases (SODs) have been purified to homogeneity from the two varieties of Cryptococcus neoformans, C. neoformans var. neoformans and var. gattii. The N-terminal amino acid sequences of the two enzymes were similar, though not identical, and demonstrated homology with Cu,Zn SODs from other organisms. SOD activity was present in supernatants from stationary-phase cultures of isolates of C. neoformans var. neoformans and was also present from the mid-log phase onwards in cultures of an acapsular mutant of C. neoformans var. neoformans. SOD activity was practically undetectable in culture supernatants from isolates of C. neoformans var. gattii. The C. neoformans var. neoformans SOD had a reduced relative molecular mass of 19 kDa, and in its nonreduced form the enzyme was present as a 125-kDa species. Isoelectric focusing indicated that four species with pIs of 5.9, 6.15, 6.35, and 6.6 were present. The equivalent reduced molecular mass of the C. neoformans var. gattii enzyme was 19 kDa, with a single species present under nonreducing conditions (relative molecular mass of 145 kDa) with a pI of 7.5. The activities of the enzymes from both varieties were inhibited by KCN; however, the copper chelator diethyldithiocarbamate was inhibitory only against the C. neoformans var. gattii enzyme, as was sodium azide. The C. neoformans var. neoformans SOD was not affected by preincubation for 1 h at 70 degrees C, and it also retained most of its activity when incubated at 37 degrees C relative to its activity when incubated at 20 degrees C, in contrast to the C. neoformans var. gattii enzyme. The pronounced differences in the physical and biochemical characteristics of the Cu,Zn SODs from the two Cryptococcus varieties complement recent reports illustrating the biochemical and genetic differences between C. neoformans var. neoformans and C. neoformans var. gattii, and the successful purification of the two enzymes comprises the first step in determining what role, if any, the cryptococcal Cu,Zn SODs might have in protection against externally generated superoxide.
Pseudomonas aeruginosa-mediated cytotoxicity and invasion correlate with distinct genotypes at the loci encoding exoenzyme S
Authors: Fleiszig S M , Wiener-Kronish J P , Miyazaki H , Vallas V , Mostov K E , Kanada D , Sawa T , Yen T S , Frank D W
Published: January 2020

Pseudomonas aeruginosa, an opportunistic pathogen, is capable of establishing both chronic and acute infections in compromised hosts. Previous studies indicated that P. aeruginosa displays either a cytotoxic or an invasive phenotype in corneal epithelial cells. In this study, we used polarized MDCK cells for in vitro infection studies and confirmed that P. aeruginosa isolates can be broadly differentiated into two groups, expressing either a cytotoxic or an invasive phenotype. In vivo infection studies were performed to determine if cytotoxic and invasive strains displayed differential pathology. Invasion was assayed in vivo by in situ infection of mouse tracheal tissue followed by electron microscopy. Both cytotoxic and invasive strains entered mouse tracheal cells in situ; however, more necrosis was associated with the cytotoxic strain. In an acute lung infection model in rats, cytotoxic strains were found to damage lung epithelium more than invasive strains during the short infection period of this assay. The expression of cytotoxicity requires a functional exsA allele. In the strains tested, the ability to invade epithelial cells in vitro appears to be independent of exsA expression. Since ExsA is a transcriptional regulator of the exoenzyme S regulon, chromosomal preparations from invasive and cytotoxic strains were screened for their complement of exoenzyme S structural genes, exoS, encoding the 49-kDa ADP-ribosyltransferase (ExoS), and exoT, encoding the 53-kDa form of the enzyme (Exo53). Invasive strains possess both exoS and exoT, while cytotoxic strains appear to have lost exoS and retained exoT. These data indicate that the expression of cytotoxicity may be linked to the expression of Exo53, deletion of exoS and perhaps other linked loci, or expression of other ExsA-dependent virulence determinants. In the absence of a functional cytotoxicity pathway (exsA::omega strains), invasion of eukaryotic cells is detectable.
Cytokine and adhesion molecule expression in human monocytes and endothelial cells stimulated with bacterial heat shock proteins
Authors: Galdiero M , de l'Ero G C , Marcatili A
Published: January 2020

Bacterial heat shock proteins (HSPs) from Escherichia coli (GroES, GroEL, and DNAk) were tested for their ability to induce by themselves the expression and release of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and granulocyte-monocyte colony-stimulating factor (GM-CSF) by human monocytes and GM-CSF, IL-6, E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) by human umbilical vein endothelial cells (HUVEC). Our study demonstrated that treatment of monocytes with DNAk increased IL-6, TNF-alpha, and GM-CSF release in a dose-dependent manner. The same effect was elicited by GroEL but at a lower rate. Treatment of HUVEC cultures with DNAk and GroEL also increased GM-CSF, IL-6, E-selectin, ICAM-1, and VCAM-1 release in a dose-dependent fashion. In any case, the greatest release was obtained by using DNAk and GroEL at a concentration of 1 microg/ml. DNAk and GroEL were also able to up-regulate the surface expression of E-selectin, ICAM-1, and VCAM-1. As detected by reverse transcription-PCR analysis, DNAk and GroEL also increased the steady-state levels of cytokines and adhesion molecules in human monocytes and endothelial cells. In our study GroES showed a significant activity only on the release, surface expression, and mRNA transcription of E-selectin. Adhesion molecule expression seems to be a direct effect of HSPs and not via cytokines. Furthermore, these effects are due to HSPs properties because they are inhibited by specific monoclonal antibodies. These findings support the potential role of HSPs in modulating cell interactions during immunological and inflammatory responses.
Reactivity patterns and epitope specificities of anti-Cryptococcus neoformans monoclonal antibodies by enzyme-linked immunosorbent assay and dot enzyme assay
Authors: Belay T , Cherniak R , Kozel T R , Casadevall A
Published: January 2020

Cryptococcus neoformans glucuronoxylomannans (GXM) are capsular polysaccharides important for virulence in cryptococcosis. This study used dot enzyme assays (DEA) and enzyme-linked immunosorbent assays (ELISA) to determine the reactivity patterns of 21 murine monoclonal antibodies (MAbs) with structurally defined GXMs from five serotypes. The MAbs were categorized into eight groups on the basis of DEA and five groups on the basis of ELISA. MAbs 302, 339, and 439 were studied extensively for their binding to various native and chemically modified GXMs. Quantitative variation in the inhibitory effects of GXMs on the binding of MAbs 302, 339, and 439 were observed by competitive ELISA. O-Deacetylation of serotype A, B, and D GXM resulted in the complete loss of their inhibitory properties. Carboxyl group reduction of GXMs from serotypes A and D resulted in a significant decrease of inhibitory activity for MAb. Xylomannans and methyl glycosides exhibited no detectable inhibitory activity on MAb binding to GXM. The results indicate (i) the existence of five to eight MAb-defined distinct epitopes in C. neoformans GXM that can elicit antibody responses, (ii) MAb detection of antigenic variation within GXMs assigned to a particular serotype, (iii) good correspondence between the patterns of MAb reactivities and polyclonal rabbit factor sera, (iv) good agreement between MAb molecular structure and serotype reactivity, and (v) a dependence of the serotype reactivity profile for a given MAb on the technique used to measure binding.
Inhibition of Helicobacter pylori binding to gastrointestinal epithelial cells by sialic acid-containing oligosaccharides
Authors: Simon P M , Goode P L , Mobasseri A , Zopf D
Published: January 2020

Helicobacterpylori, the ulcer pathogen residing in the human stomach, binds to epithelial cells of the gastric antrum. We have examined binding of 13 bacterial isolates to epithelial cell lines by use of a sensitive microtiter plate method in which measurement of bacterial urease activity provides the means for quantitation of bound organisms. Several established human gastrointestinal carcinoma cell lines grown as monolayers were compared for suitability in these assays, and the duodenum-derived cell line HuTu-80 was selected for testing bacterial binding inhibitors. When bacteria are pretreated with oligosaccharides, glycoproteins, and glycolipids, a complex picture of bacterial-epithelial adherence specificities emerges. Among the monovalent inhibitors tested, 3'-sialyllactose (NeuAc alpha2-3Gal beta1-4Glc; 3'SL) was the most active oligosaccharide, inhibiting adherence for recent clinical isolates of H. pylori with a millimolar 50% inhibitory concentration (IC50). Its alpha2-6 isomer (6'SL) was less active. Most of the recent clinical isolates examined were inhibited by sialyllactose, whereas long-passaged isolates were insensitive. Among the long-passaged bacterial strains whose binding was not inhibited by 3'SL was the strain ATCC 43504, also known as NCTC 11637 and CCUG 17874, in which the proposed sialyllactose adhesin was recently reported to lack surface expression (P. G. O'Toole, L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. H. Trust, J. Bacteriol. 177:6049-6057, 1995). Pretreatment of the epithelial monolayer with neuraminidase reduced the extent of binding by those bacteria that are sensitive to inhibition by 3'SL. Other potent inhibitors of bacterial binding are the glycoproteins alpha1-acid glycoprotein, fetuin, porcine gastric and bovine submaxillary mucins, and the glycolipid sulfatide, all of which present multivalent sialylated and/or sulfated galactosyl residues under the conditions of the binding assay. Consistent with this pattern, a multivalent neoglycoconjugate containing 20 mol of 3'SL per mol of human serum albumin inhibited bacterial binding with micromolar IC50. The H. pylori isolate most sensitive to inhibition by 3'SL was least sensitive to inhibition by sulfatide, gastric mucin, and other sulfated oligosaccharides. Bacteria that have been allowed to bind epithelial cells are also effectively detached by 3'SL. These results describe a heterogeneous adherence repertoire for these bacteria, but they also confirm the critical role of the 3'SL structure on human gastric epithelial cells as an adherence ligand for recent isolates of H. pylori.