bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen-coated latex particles as a model system for probing monocyte responses in leprosy
Authors: Hasan R S , Dockrell H M , Jamil S , Chiang T J , Hussain R
Published: January 2020

To study responses to Mycobacterium leprae antigens, we developed an in vitro model system in which latex particles coated with M. leprae sonic extract (MLSON) antigen were presented to monocytes. Uptake and oxidative response as measured by superoxide production to these antigens were investigated. Phagocytosis of MLSON-coated particles was greater than that of control particles in monocytes from both leprosy patients and controls from leprosy-endemic areas; uptake of MLSON-coated particles was higher in monocytes from lepromatous leprosy patients than in cells from tuberculoid leprosy patients and controls. In both patients and controls, uptake of latex particles coated with leprosy antigens triggered very little reduction of nitroblue tetrazolium although the cells were capable of mounting a respiratory burst. Antigen-coated latex particles can therefore be used as a tool to investigate monocyte responses to M. leprae and individual recombinant antigens.
Borrelia burgdorferi outer surface lipoproteins OspA and OspB possess B-cell mitogenic and cytokine-stimulatory properties
Authors: Ma Y , Weis J J
Published: January 2020

Sonicated Borrelia burgdorferi was previously reported to possess both B-cell mitogenic and interleukin-6 (IL-6) stimulatory activities. In this report, two outer surface lipoproteins, OspA and OspB, were purified from B. burgdorferi and assessed for the presence of these functions. OspA was purified from two strains, an OspB-deficient variant of HB19 and N40, while OspB was purified from the N40 strain. All lipoprotein preparations were free of endotoxin contamination, and polymyxin B failed to inhibit responses, indicating that media contamination was not contributing to biological assays. All three preparations were able to stimulate proliferation of mononuclear cells from naive C3H/HeJ and BALB/c mice. Depletion experiments indicated that the responding cells were B lymphocytes and not T lymphocytes. Purified OspA and OspB stimulated immunoglobulin M production by splenocyte cultures from naive mice, a property also previously attributed to sonicated B. burgdorferi. OspA and OspB also stimulated the production of IL-6 and tumor necrosis factor alpha by bone marrow-derived macrophages from BALB/c and C3H/HeJ mice. Cytokine production was enhanced by the presence of gamma interferon in the cultures, indicating that the magnitude of responses to these lipoproteins may be modulated by cytokines in the microenvironment of infected tissues. Human endothelial cells produced IL-6 when incubated with OspA and OspB, indicating that non-hematopoietic lineage cells can respond to the lipoproteins. Purified OspA and OspB had approximately equal activity, with responses detected in the range of 10 ng of lipoprotein per ml to 1 microgram of lipoprotein per ml. Comparison with published dose responses for lipoproteins purified from Escherichia coli indicates that OspA and OspB purified from B. burgdorferi are much more potent. The high potency of the B. burgdorferi lipoproteins and the ability of the spirochete to invade tissues and persist argue that they could be important in the localized events contributing to the pathology of Lyme disease.
Similar proportions of immunoglobulin A1 (IgA1) protease-producing streptococci in initial dental plaque of selectively IgA-deficient and normal individuals
Authors: Reinholdt J , Friman V , Kilian M
Published: January 2020

By comparing the initial colonization of cleaned teeth in immunoglobulin A (IgA)-deficient, IgM-compensating individuals with that in normal individuals, no significant difference in the proportion of IgA1 protease-producing streptococci was found. Thus, as one of several bacterial means of immune evasion, the ability to cleave secretory IgA1 does not appear essential to the successful adherence of oral streptococci.
Characterization of a Mycobacterium leprae antigen related to the secreted Mycobacterium tuberculosis protein MPT32
Authors: Wieles B , van Agterveld M , Janson A , Clark-Curtiss J , Rinke de Wit T , Harboe M , Thole J
Published: January 2020

Secreted proteins may serve as major targets in the immune response to mycobacteria. To identify potentially secreted Mycobacterium leprae antigens, antisera specific for culture filtrate proteins of Mycobacterium tuberculosis were used to screen a panel of recombinant antigens selected previously by leprosy patient sera. Four potentially secreted antigens were identified by this approach, and one was recognized by antibodies specific for MPT32, a secreted M. tuberculosis protein. The DNA coding for the M. leprae antigen, which we have designated 43L, was isolated and characterized and found to encode a 25.5-kDa protein that is preceded by a consensus signal peptide of 39 amino acids. The N-terminal amino acid sequence of 43L shows 50% homology with the 20 known N-terminal amino acids of MPT32, and 47% homology was found with the N terminus of a 45/47-kDa antigen complex identified in Mycobacterium bovis BCG. These findings indicate that 43L represents an antigen related to MPT32 and the M. bovis BCG 45/47-kDa complex and that 43L is likely to be a protein secreted by M. leprae. Purified recombinant 43L protein is recognized by antibodies and T cells from healthy contacts and leprosy patients, illustrating that secreted proteins are of importance in the immune response to M. leprae.
Outer surface proteins E and F of Borrelia burgdorferi, the agent of Lyme disease
Authors: Lam T T , Nguyen T P , Montgomery R R , Kantor F S , Fikrig E , Flavell R A
Published: January 2020

We report the cloning and characterization of two outer surface proteins (Osps), designated OspE and OspF, from strain N40 of Borrelia burgdorferi, the spirochetal agent of Lyme disease. The ospE and ospF genes are structurally arranged in tandem as one transcriptional unit under the control of a common promoter. The ospE gene, located at the 5' end of the operon, is 513 nucleotides in length and encodes a 171-amino-acid protein with a calculated molecular mass of 19.2 kDa. The ospF gene, located 27 bp downstream of the stop codon of the ospE gene, consists of 690 nucleotides and encodes a protein of 230 amino acids with a calculated molecular mass of 26.1 kDa. Pulsed-field gel electrophoresis showed that the ospE and ospF genes are located on a 45-kb plasmid. Comparison of the leader sequences of OspE and OspF with those of the four known B. burgdorferi Osps (OspA, OspB, OspC, and OspD) reveals a hydrophobic domain and a consensus cleavage sequence (L-X-Y-C) recognized by signal peptidase II, and [3H]palmitate labeling shows that OspE and OspF are lipoproteins. Immunofluorescence studies demonstrated that both the OspE and OspF proteins are surface exposed. These features are consistent with the finding that OspE and OspF are B. burgdorferi surface lipoproteins.
Effects of cations on Helicobacter pylori urease activity, release, and stability
Authors: Pérez-Pérez G I , Gower C B , Blaser M J
Published: January 2020

The urease of Helicobacter pylori is an important antigen and appears critical for colonization and virulence. Several studies have indicated a superficial localization for the H. pylori urease, and the purpose of this study was to determine the effects of cations on the release and stability of urease activity from H. pylori cells. Incubation of partially purified H. pylori urease in water containing 1, 5, or 10 mM Ca2+, Mg2+, K+, Na+, EDTA, or EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] had little effect on activity. In contrast, 1 mM Fe3+, Cu2+, Co2+, or Zn2+ substantially (> 80%) inhibited activity, and 10 mM Fe2+, Mn2+, and Ni2+ inhibited about 30% of the activity. Addition of Ca2+ or Mg2+ markedly decreased extraction of urease from intact H. pylori cells by water, but 1 mM Na+, K+, EGTA, or EDTA each had minimal effects on release, suggesting that divalent cations have a role in attachment of urease to H. pylori cells. The stability of enzymatic activity at 4 degrees C was enhanced by addition of glycerol or 2-mercaptoethanol; however, even after loss of activity, full antigenicity for human serum was retained.
Antigenic variation of parasite-derived antigens on the surface of Babesia bovis-infected erythrocytes
Authors: Allred D R , Cinque R M , Lane T J , Ahrens K P
Published: January 2020

The hemoparasite Babesia bovis antigenically alters the bovine erythrocyte membrane surface by expression of isolate-specific, parasite-derived polypeptides. To determine whether antigenic variation also occurred on the infected erythrocyte surface, a calf was infected once with parasitized erythrocytes carrying the C9.1 clonal line of B. bovis. In vitro cultures then were established periodically from the peripheral blood and analyzed with sequentially collected sera from the same animal. The surface reactivity of infected erythrocytes cultured from the infected animal varied over time, on the basis of reactivity in live cell immunofluorescence, surface immunoprecipitation, and panning assays. Subclones C8 and H10, established from day 41 cultures, were analyzed immunochemically. A loss of immunoreactivity was observed in antigens corresponding to the 113- and 128-kDa parasite-derived antigens of clone C9.1, demonstrating epitopic variation in these antigens; the immunochemical recognition of these antigens paralleled the results of live cell immunofluorescence and panning assays. Concomitant size polymorphism suggested polypeptide structural variation of these antigens as well. Calves infected by inoculation of infected blood or by injection of cloned parasites from in vitro cultures rapidly developed antibodies which cross-reacted among the clonal variant lines, suggesting the presence of common as well as unique epitopes. These results demonstrate that antigenic variation occurs on the surface of B. bovis-infected erythrocytes and that the parasite-derived antigens of 113 and 128 kDa compose at least a part of the antigens undergoing variation.
Importance of ADP-ribosylation in the morphological changes of PC12 cells induced by cholera toxin
Authors: Glineur C , Locht C
Published: January 2020

Cholera toxin (CTX) is composed of two subunits, subunit A, which possesses ADP-ribosyltransferase activity, and subunit B, which is responsible for receptor binding. It has previously been shown that agents that increase cyclic AMP (cAMP) levels in cells induce differentiation of PC12 cells into neurite-like cells. In this report, we show that as little as 100 pg of CTX per ml induces such changes. CTX was found to ADP-ribosylate at least four membrane proteins of PC12 cells in vitro and in vivo and to increase intracellular cAMP levels. We have developed an inducible ctx gene expression system in Vibrio cholerae by using the tac promoter. The culture medium of the CTX-producing bacteria was able to induce the morphological changes and the ADP-ribosylation of the PC12 cell membrane proteins. We have constructed two CTX-cross-reactive mutant proteins (CTX-CRM) by site-directed mutagenesis. The choice of glutamic acid 29 as the target amino acid was based on sequence similarities with other bacterial toxins. CTX-CRM-E29 delta, in which the Glu-29 of the A subunit was deleted, showed strongly reduced ADP-ribosyltransferase activity and did not induce significant morphological changes of PC12 cells. In contrast, CTX-CRM-E29D, in which the Glu-29 was replaced by an aspartic acid, was as active as the wild-type protein. We conclude that the ADP-ribosylation activity of CTX is important for the toxin-induced differentiation of PC12 cells. Pertussis toxin, which had no visible effect on PC12 cell morphology, was also able to ADP-ribosylate a membrane-bound protein(s) in vitro and in vivo. Pertussis toxin alone did not significantly increase cAMP levels in PC12 cells, but it acted synergistically with CTX.
Characterization of hemagglutinating components on the Anaplasma marginale initial body surface and identification of possible adhesins
Authors: McGarey D J , Allred D R
Published: January 2020

Interaction of Anaplasma marginale initial bodies with the bovine erythrocyte surface was examined by a direct hemagglutination assay. Purified initial bodies were shown to specifically hemagglutinate bovine erythrocytes but not erythrocytes from nonhost animal species. Hemagglutination was inhibited by treatment of purified initial bodies with trypsin, alpha-chymotrypsin, or proteinase K but not by treatment with neuraminidase or sodium periodate. Treatment of bovine erythrocytes with alpha-chymotrypsin or neuraminidase partially inhibited hemagglutination of the treated cells by initial bodies. In contrast, no inhibition occurred after treatment of erythrocytes with trypsin, phospholipases, or sodium periodate or when monosaccharides and disaccharides were used as potential competitive inhibitors. Thus, the initial body receptor is probably a surface protein, whereas the bovine receptor may comprise both protein and carbohydrate. Hemagglutination was unaffected by treatment of initial bodies with monoclonal or polyclonal antibodies raised against the A. marginale 31-kDa (MSP4) major surface polypeptide or non-A. marginale proteins or by treatment with a monoclonal antibody to the A. marginale MSP1a neutralization-sensitive epitope. In contrast, antiserum raised against whole A. marginale initial bodies or monospecific antibodies raised against purified A. marginale major surface polypeptides with molecular sizes of 105 (MSP1a), 100 (MSP1b), 61, and 36 (MSP2) kDa completely or partially inhibited hemagglutination. These data confirm the proposed surface location of the proteins susceptible to inhibition and suggest that they mediate hemagglutination of bovine erythrocytes. We propose that these surface proteins are possible adhesins.
Host specificity of Salmonella infection in chickens and mice is expressed in vivo primarily at the level of the reticuloendothelial system
Authors: Barrow P A , Huggins M B , Lovell M A
Published: January 2020

By experimental infection, host-specific Salmonella serotypes were shown to demonstrate specificities for chickens, mice, and other laboratory animals. Following oral inoculation, four strains of Salmonella gallinarum and two S. pullorum strains, isolated from diseased poultry, were more virulent for chickens than for mice. By contrast, four strains each of S. choleraesuis and S. dublin, isolated from diseased pigs and cattle, respectively, were more virulent for mice than for chickens. These results were also reflected in the degree of virulence expressed after parenteral inoculation. In addition, S. choleraesuis, but not other serotypes, killed rats, guinea pigs, and rabbits. S. typhimurium strains varied widely in their virulence, and some strains were virulent for both mice and chickens. Four other serotypes isolated from poultry or human food poisoning cases and a nonpathogenic Escherichia coli strain were much less virulent for both experimental host species. Most of the host-specific Salmonella serotypes studied were able to colonize the distal alimentary tract and invade the tissues in both mice and chickens to various degrees. There was, however, a greater difference in the ability to survive and multiply in the visceral organs, particularly the spleen and the liver, once invasion had occurred which correlated with the virulence for the host species involved.
Serum Cohn fraction IV-1 supports the growth of Giardia lamblia in vitro
Authors: Lujan H D , Byrd L G , Mowatt M R , Nash T E
Published: January 2020

Bovine serum Cohn fractions were substituted for whole bovine serum in TYI-S-33 medium. Only fraction IV-1 supported attachment, cysteine uptake, and growth of Giardia lamblia. Among the mammalian sources tested, only goat and horse fractions showed activities comparable to that of the bovine fraction. Agglutinating immunoglobulins were identified as deleterious agents in human, rat, rabbit, and dog fractions.
In vivo production of neuraminidase by Pasteurella haemolytica A1 in goats after transthoracic challenge
Authors: Straus D C , Purdy C W
Published: January 2020

Nine goats were injected transthoracically with Pasteurella haemolytica A1 to determine if an extracellular bacterial enzyme, neuraminidase, was produced in vivo during infection with this organism. The principal group of goats (n = 9) each received 1 ml of 7.25 x 10(5) live P. haemolytica A1 cells in polyacrylate beads transthoracically in the left lung on days 0 and 21. Six goats were used as negative controls and received 0.3 g of polyacrylate beads subcutaneously in the right flank on days 0 and 21. Serum was obtained from all animals on days -4, 3, 7, 14, 21, 24, and 32. Preimmune serum from all animals showed no detectable antibody to P. haemolytica A1 neuraminidase in an enzyme neutralization assay. None of the sera from the negative control animals possessed a significant antibody concentration in response to the P. haemolytica A1 neuraminidase. On day 32, serum samples from the nine infected animals possessed enzyme neutralizing activity that ranged from 62% to 100%. Anti-neuraminidase antibody could be detected as early as day 14 by the enzyme neutralization assay. These data demonstrate that the enzyme neuraminidase is produced in vivo during an active P. haemolytica A1 lobar infection.
Invasiveness and persistence of Salmonella enteritidis, Salmonella typhimurium, and a genetically defined S. enteritidis aroA strain in young chickens
Authors: Cooper G L , Venables L M , Woodward M J , Hormaeche C E
Published: January 2020

Newly hatched chicks were dosed orally with a Salmonella typhimurium wild-type strain, an S. enteritidis wild-type strain, and a genetically defined S. enteritidis aroA vaccine candidate, strain CVL30. The S. typhimurium strain, 2391 Nalr, was virulent in newly hatched chicks and caused deaths in 7 of 20 chicks after an oral dose of 10(5) organisms. The S. enteritidis wild-type strain, LA5, caused death in 1 of 25 chicks and gross pathology including pericarditis and perihepatitis in 6 of the 24 survivors after an oral dose of 10(9) organisms. S. enteritidis aroA CVL30, attenuated by ca. 6.5 log10 in BALB/c mice, was nonvirulent when administered orally to chicks and did not cause morbidity. When newly hatched chicks were dosed, the pattern of invasion and colonization of the reticuloendothelial system by strain CVL30 was similar to that of its parent strain, LA5, irrespective of the dose. Oral inoculation of newly hatched chicks with < 10 organisms of S. enteritidis LA5 or CVL30 was followed by multiplication in the cecal contents. Within 3 days of hatching, the pH of the cecal contents was reduced from ca. 7 to 5. Samples of gut contents were inoculated in vitro. The S. enteritidis strains multiplied in samples taken from the ileum and duodenum irrespective of age but multiplied in the cecal samples from newly hatched chicks only. Invasion from the gut by S. enteritidis LA5 and CVL30 was both age and dose dependent.
Characterization and virulence analysis of catalase mutants of Haemophilus influenzae
Authors: Bishai W R , Howard N S , Winkelstein J A , Smith H O
Published: January 2020

In addition to detoxifying peroxides generated by aerobic metabolism, the catalases of pathogenic bacteria have also been hypothesized to serve as virulence factors by enabling microorganisms to resist the oxidative bursts of host inflammatory cells. Using transposon mutagenesis of the hktE gene, encoding the Haemophilus influenzae structural gene for catalase, we constructed defined catalase mutants of H. influenzae strains Rd- and Eagan b+. These mutants show no detectable catalase production during exponential or stationary phases or following induction with hydrogen peroxide or ascorbic acid, indicating that hktE is the only functional hydroperoxidase gene present in these two strains of H. influenzae. Exponential-phase cultures of hktE mutants are 8- to 25-fold more sensitive to hydrogen peroxide than the wild type. Using the infant rat model, hktE mutants of strain Eagan b+ were 2.3-fold less virulent than the wild type following intraperitoneal inoculation (P = 0.07). When administered intranasally, the Eagan b+ hktE mutant produced wild-type levels of bacteremia and nasal colonization. The results of this study show that while the H. influenzae hktE gene is important for survival in the presence of peroxides, deletion of the gene produces only a modest reduction in ability to cause lethal sepsis following parenteral challenge and no change in ability to colonize following intranasal inoculation in the infant rat model of infection.
Analysis of the role of M24 protein in group A streptococcal adhesion and colonization by use of omega-interposon mutagenesis
Authors: Courtney H S , Bronze M S , Dale J B , Hasty D L
Published: January 2020

We recently concluded that M protein mediates adherence of group A streptococci to HEp-2 tissue culture cells, because the N-terminal half of M protein blocked adherence and M+ strains attached in greater numbers than M- streptococci. To further assess the role of M protein in adhesion, an M-, isogenic mutant of M type M-, isogenic mutant of M type 24 group A streptococci was constructed by insertional inactivation of the emm24 gene with the omega-interposon flanked by emm24 gene sequences. Southern blot analysis confirmed that the omega-element inserted only into emm24. The M- isogenic mutant M24-omega 3 did not react with antiserum to M24 protein, not did it survive in whole human blood. Electron micrographs of M24-omega 3 showed a diminution of surface fibrillae and reduced binding of plasma components compared with the parent strain. The adhesion of the M+ parent to HEp-2 cells and to mouse oral epithelial cells was dramatically greater than the adhesion of the M24-omega 3 mutant, although there was no difference between the two in adhesion to human buccal cells. In addition, the parent strain was dramatically more effective than the M24-omega 3 mutant in colonizing the oral cavity of mice. These results indicate that the M24 protein can serve as an adhesin in streptococcal attachment to human cells in tissue culture and is important in the colonization of mouse mucosal surfaces.
Response of Leishmania chagasi promastigotes to oxidant stress
Authors: Wilson M E , Andersen K A , Britigan B E
Published: January 2020

At the onset of infection, Leishmania promastigotes are phagocytized by mammalian macrophages. They must survive despite exposure to toxic oxidants such as hydrogen peroxide (H2O2) and superoxide (.O2-) generated during phagocytosis. We investigated the effects of these oxidants on Leishmania chagasi promastigotes and promastigote mechanisms for oxidant resistance. According to spin trapping and electron paramagnetic resonance spectrometry, .O2- could be generated by exposure of promastigotes to the redox-cycling compound menadione. Incubation in either menadione or H2O2 caused a concentration-dependent loss of promastigote viability. However, incubation in sublethal concentrations of H2O2 or menadione caused a stress response in promastigotes. This oxidant-induced response was associated with an increase in the amount of heat shock protein hsp70. Induction of a stress response by exposure of promastigotes either to heat shock or to sublethal oxidants (H2O2 or menadione) caused promastigotes to become more resistant to H2O2 toxicity. Sublethal menadione also caused promastigotes to become more virulent in a BALB/c mouse model of leishmaniasis. We previously correlated H2O2 cytotoxicity for promastigotes with the formation of hydroxyl radical (.OH) from H2O2. However, according to electron paramagnetic resonance spectrometry, the increase in H2O2 resistance after exposure to sublethal oxidants was not associated with diminished generation (i.e., scavenging) of .OH. These data suggest that there is a cross-protective stress response that occurs after exposure of L. chagasi promastigotes to heat shock or to sublethal H2O2 or .O2-, exposures that also occur during natural infection. This response results in increased resistance to H2O2 toxicity and increased virulence for a mammalian host.
Endothelial cell proliferation associated with lesions of murine systemic candidiasis
Authors: Ashman R B , Papadimitriou J M
Published: January 2020

Neovascularization is associated with tumor growth and some inflammatory diseases but has not been reported to be induced by infectious agents. In a mouse model of systemic Candida albicans infection, extensive endothelial cell proliferation was seen in the periphery of brain abscesses and in the areas of fungal pyelonephritis in the kidney. This finding is important for an understanding of the pathogenesis of fungal infections and may contribute to an analysis of the mechanisms of angiogenesis.
Calcium-binding properties of SSP-5, the Streptococcus gordonii M5 receptor for salivary agglutinin
Authors: Duan Y , Fisher E , Malamud D , Golub E , Demuth D R
Published: January 2020

Streptococcus gordonii M5 expresses a lectin on its surface (SSP-5) which binds to human salivary agglutinin (SAG). This interaction requires sialic acid residues of SAG and divalent cations and may mediate the colonization of oral tissues by this organism. In this report, we show that the binding of SAG to SSP-5 requires calcium and that SSP-5 is a high-affinity calcium-binding protein. SAG-mediated aggregation of S. gordonii M5 was inhibited by 1 mM EDTA, and the restoration of aggregation occurred only upon the readdition of calcium. To ascertain the level at which calcium exerts its effects, the calcium-binding properties of SSP-5 were evaluated by using a 45Ca binding assay. In addition, a kinetic analysis of calcium binding was carried out by using fura2, a fluorescent calcium-binding dye. These analyses showed that SSP-5 is a high-affinity calcium-binding protein that binds 1 mol of calcium per mol of protein and has a dissociation constant of 0.45 +/- 0.2 microM. The calcium-binding capacity of SSP-5 was also calculated independently to be 1.0 +/- 0.2 mol of Ca per mol of SSP-5 by column chromatography on Sephadex G-25 equilibrated with 10 microM 45Ca. To localize the calcium binding site of SSP-5, a series of C-terminal deletion mutants were expressed in Escherichia coli and evaluated for calcium-binding activity. Deletion of the 250 C-terminal residues of SSP-5 had little effect on calcium binding. However, deletion of residues 1168 to 1250 resulted in the loss of calcium-binding activity, suggesting that this region is important for calcium binding by SSP-5.
An HLA-DRw53-restricted T-cell epitope from a novel Mycobacterium leprae protein antigen important to the human memory T-cell repertoire against M. leprae
Authors: Mustafa A S , Deggerdal A , Lundin K E , Meloen R M , Shinnick T M , Oftung F
Published: January 2020

Cellular immunity mediated by T cells plays a major role in protection against intracellular infections, including leprosy, a chronic disease caused by Mycobacterium leprae. In this work, we describe CD4+ T-cell clones, isolated from healthy humans immunized with M. leprae, which recognize a novel M. leprae protein antigen previously isolated from a lambda gt11 DNA expression library. On the basis of the deduced primary structure of the carboxyl-terminal part of the antigen, we have used a synthetic-peptide approach to exactly define the T-cell epitope recognized. Importantly, major histocompatibility complex restriction studies showed that the epitope is presented by an HLA-DRw53 molecule which is frequently expressed in many populations. In addition, we have demonstrated that a long-term cell-mediated immunity response against the peptide epitope is present after immunization with M. leprae. In conclusion, the M. leprae T-cell epitope described here fulfills the primary criteria for subunit vaccine candidates against leprosy.
Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O
Authors: Jones S , Portnoy D A
Published: January 2020

Listeriolysin O (LLO) is a pore-forming cytolysin that enables Listeria monocytogenes to escape from a host cell vacuole. The structural gene for the related cytolysin perfringolysin O (pfo) was cloned downstream from the promoter for hly, the gene encoding LLO, both on a plasmid and on the L. monocytogenes chromosome. Both strains secreted active PFO, although regulation was not identical to that of LLO. The chromosomal PFO-expressing strain was characterized for intracellular growth and cell-to-cell spread. It escaped from a host cell vacuole with 64% efficiency compared with the wild type as determined by immunofluorescent staining of bacteria for F-actin, a marker for entry into the cytoplasm. In addition, it replicated intracellularly with a doubling time similar to that of the wild type for 5 h, after which growth was aborted because of a cytotoxic effect on the host cell and influx of extracellular gentamicin. The chromosomal PFO strain was able to plaque in mouse L2 fibroblasts, but it did so at 20% efficiency compared with the wild type and the plaques were significantly smaller. Both strains expressing PFO were completely avirulent in mice. These results indicate that PFO can mediate escape from a host cell vacuole but cannot complement an hly deletion strain for virulence.
Human antibody response to Clostridium difficile toxin A in relation to clinical course of infection
Authors: Warny M , Vaerman J P , Avesani V , Delmée M
Published: January 2020

This study investigated whether differences in fecal and serum antitoxin A antibody levels may account for the duration of Clostridium difficile-associated diarrhea (CDAD) and the occurrence of relapses. By an enzyme linked-immunosorbent assay, we tested 40 patients with CDAD including 25 patients without immunodeficiency and 15 patients receiving antineoplastic drugs. Two hundred eighty serum samples and 80 normal stool samples were investigated as controls. In nonimmunocompromised patients, serum immunoglobulin (IgG) and fecal IgA antitoxin A antibody titers were significantly higher in patients who suffered a single episode (n = 21) than in those with relapsing CDAD (n = 4) whose titers were at control levels. Of these 25 patients, eight suffered from diarrhea which lasted for more than 2 weeks. These patients had significantly lower serum- and feces-specific antibody levels than the others who presented symptoms of shorter duration. In cytostatic-treated patients, antitoxin A antibody levels were similar to controls, but relapses occurred in a single case. These data suggest an association between a defective humoral response to toxin A and a more severe form of C. difficile infection. They also indicate that other host-related factors control the severity of CDAD and remain to be elucidated.
Fate of Listeria monocytogenes in murine macrophages: evidence for simultaneous killing and survival of intracellular bacteria
Authors: de Chastellier C , Berche P
Published: January 2020

The intracellular survival of the ubiquitous pathogen Listeria monocytogenes was studied in primary cultures of bone marrow-derived mouse macrophages. Bacteria were able to grow rapidly in these cells, with an apparent multiplication rate of about 40 min. Electron microscopy demonstrated that intracellular bacterial replication was the consequence of simultaneous intracellular killing and replication of bacteria in the same cells. Within the first hour following phagocytosis, most bacteria were destroyed in the phagosomal compartment to which they were confined. This was due to early transfer of hydrolytic enzymes to phagosomes, undoubtedly via phagosome-lysosome (P-L) fusion, as demonstrated by a quantitative analysis after staining for a lysosomal marker, acid phosphatase. One hour after infection, about 14% of the bacteria were free in the cytoplasm, in which they multiplied and induced actin polymerization and spreading to adjacent macrophages, as in epithelial cells. By using the 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine staining procedure, direct evidence is presented that all phagosomes were acidified immediately after phagocytosis, thus indicating that intraphagosomal bacteria were exposed to an acidic environment that might favor vacuolar lysis by listeriolysin O. Intracellular growth in macrophages, therefore, appears to be the result of a competition between the expression of the hydrolytic activity of these cells following P-L fusion and the capacity of L. monocytogenes to escape from the acidified phagosomal compartment before P-L fusion has occurred. The finding that concomitant intracellular killing and survival of L. monocytogenes occurs in the same macrophages might explain the high immunogenicity observed in vivo with live bacteria, as opposed to killed bacteria.
The Mycobacterium bovis 32-kilodalton protein antigen induces human cytotoxic T-cell responses
Authors: Munk M E , De Bruyn J , Gras H , Kaufmann S H
Published: January 2020

The 30-kDa protein (P32) is a mycobacterial secreted antigen which is homologous in Mycobacterium bovis and M. tuberculosis. In vitro, P32 induced T-cell proliferation. M. tuberculosis- or P32-stimulated T-cell lines lysed macrophages pulsed with P32 or M. tuberculosis, respectively. We conclude that P32 stimulates cytotoxic T cells specifically.
Amino acids in pneumolysin important for hemolytic activity identified by random mutagenesis
Authors: Hill J , Andrew P W , Mitchell T J
Published: January 2020

The structural gene of pneumolysin was randomly mutagenized. A screen, developed to identify mutants with reduced hemolytic activity, identified substitutions within the cysteine-containing region and also at residues toward the N terminus of the toxin. These are the first reported changes within the N-terminal region of the toxin that affect cytolytic activity.
Immunoblot analyses of chimpanzee sera after infection and after immunization and challenge with Mycoplasma pneumoniae
Authors: Franzoso G , Hu P C , Meloni G A , Barile M F
Published: January 2020

Consecutive weekly or biweekly serum specimens obtained during a 3- or 4-month study from 16 chimpanzees were examined by immunoblot analyses to identify the immunogenic components of Mycoplasma pneumoniae. Six experimentally infected chimpanzees showed significant signs of overt disease, including cough, pharyngitis, rhinitis, fever, and loss of appetite. The sera of these infected chimpanzees recognized from 17 to 20 protein bands. Two control chimpanzees that were not inoculated were included in the study. Three chimpanzees immunized with a formalin-inactivated OSU-1A vaccine and three chimpanzees immunized with an experimental acellular vaccine showed minimal signs of disease on challenge. After challenge, the serum immunoblot responses of the immunized chimpanzees were similar to those of the infected chimpanzees. Before challenge, the sera of two previously infected chimpanzees recognized protein bands of 169 (which comigrated with the P1 adhesin), 148, 130, 117, 86, 61, 44, 35, 30, and 29 kDa. After challenge, the previously infected chimpanzees showed the most intense serum immunoblot responses and were most protected against colonization and disease. The sera from each of the 16 chimpanzees examined recognized a large number of immunogenic components, and the serum immunoblot responses were virtually identical to those of patients. Sera from each chimpanzee and patient recognized 169-, 148-, 130-, 117-, 86-, 44-, and 35-kDa bands and many of them recognized 67-, 63-, 61-, 56-, 32-, 30-, and 29-kDa protein bands.
Binding studies of a monoclonal antibody specific for 3-deoxy-D-manno-octulosonic acid with a panel of Klebsiella pneumoniae lipopolysaccharides representing all of the O serotypes
Authors: van der Meer N M , Appelmelk B J , Verweij-van Vught A M , Nimmich W , Kosma P , Thijs L G , de Graaff J , MacLaren D M
Published: January 2020

A monoclonal antibody (MAb) raised against Salmonella minnesota R595 and specific for alpha-3-deoxy-D-manno-octulosonic acid (alpha-Kdo) of the inner core was tested for binding to lipopolysaccharides (LPS) of Klebsiella pneumoniae. The MAb was tested in several assay systems (enzyme-linked immunosorbent assay, passive hemolysis, and inhibition of passive hemolysis) with a large panel (n = 23) of K. pneumoniae LPS representing all nine currently known O serotypes. MAb 20 showed reactivity with almost all O serotypes of K. pneumoniae LPS, and this reactivity could be inhibited by synthetic Kdo. This suggests an epitope in the cores of these Klebsiella LPS much like that in the inner core of LPS of S. minnesota. Large differences in reactivity between LPS of different strains belonging to the same O serotype were observed. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPS followed by immunoblotting, reactivity of MAb 20 was observed only with the fast-moving fraction possibly representing the nonsubstituted core. No binding was seen with the high-molecular-weight fraction that contained core material substituted with several units of O-antigen building blocks. The chemical basis for these differences in reactivity remains to be established. As far as we know, this is the first report containing comprehensive immunochemical data on the LPS core of K. pneumoniae.
Cloning, sequencing, and expression of the gene coding for an antigenic 120-kilodalton protein of Rickettsia conorii
Authors: Schuenke K W , Walker D H
Published: January 2020

Several high-molecular-mass (above 100 kDa) antigens are recognized by sera from humans infected with spotted fever group rickettsiae and may be important stimulators of the host immune response. Molecular cloning techniques were used to make genomic Rickettsia conorii (Malish 7 strain) libraries in expression vector lambda gt11. The 120-kDa R. conorii antigen was identified by monospecific antibodies to the recombinant protein expressed on construct lambda 4-7. The entire gene DNA sequence was obtained by using this construct and two other overlapping constructs. An open reading frame of 3,068 bp with a calculated molecular mass of approximately 112 kDa was identified. Promoters and a ribosome-binding site were identified on the basis of their DNA sequence homology to other rickettsial genes and their relative positions in the sequence. The DNA coding region shares no significant homology with other spotted fever group rickettsial antigen genes (i.e., the R. rickettsii 190-, 135-, and 17-kDa antigen-encoding genes). The PCR technique was used to amplify the gene from eight species of spotted fever group rickettsiae. A 75-kDa portion of the 120-kDa antigen was overexpressed in and purified from Escherichia coli. This polypeptide was recognized by antirickettsial antibodies and may be a useful diagnostic reagent for spotted fever group rickettsioses.
The signal transduction mechanism responsible for gamma interferon-induced indoleamine 2,3-dioxygenase gene expression
Authors: Koide Y , Yoshida A
Published: January 2020

We examined the signal transduction mechanism responsible for the gamma interferon-induced indoleamine 2,3-dioxygenase (IDO) gene expression in a human monocytic cell line, THP-1. Our results suggest that gamma interferon-induced activation of protein tyrosine kinase is a prerequisite for gene expression and that activation of protein kinase C and another unknown signal(s), both of which are transduced by the protein tyrosine kinase, synergistically induce IDO gene expression. Neither Ca2+ influx nor cyclic nucleotide-dependent kinases were suggested to be involved in the signaling pathway.
Lactoferrin binds to porins OmpF and OmpC in Escherichia coli
Authors: Erdei J , Forsgren A , Naidu A S
Published: January 2020

Lactoferrin (Lf) is an iron-binding antimicrobial protein present in milk and on mucosal surfaces, with a suggested role in preimmune host defense. Certain strains of Escherichia coli (bacterial whole cells) demonstrate specific interaction with 125I-labeled Lf. A band with a mass of approximately 37 kDa, which was reactive with horseradish peroxidase-labeled Lf, was identified in the boiled cell envelope and outer membrane preparations of an Lf-binding E. coli strain, E34663, and a non-Lf-binding strain, HH45, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (immunoblotting). Such a band was not detected in the unboiled native cell envelope and outer membrane preparations. The molecular mass and the property of heat modifiability suggested that the Lf-binding proteins were porins. The native trimeric form of porin OmpF isolated from strain B6 and its dissociated monomeric form both reacted with horseradish peroxidase-labeled Lf and with monoclonal antibodies specific for OmpF. Furthermore, by using E. coli constructs with defined porin phenotypes, OmpF and OmpC were identified as the Lf-binding proteins by urea-SDS-PAGE and Western blotting and by 125I-Lf binding studies with intact bacteria. These data establish that Lf binds to porins, a class of well-conserved molecules common in E. coli and many other gram-negative bacteria. However, in certain strains of E. coli these pore-forming proteins are shielded from Lf interaction.
Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence
Authors: Pascopella L , Collins F M , Martin J M , Lee M H , Hatfull G F , Stover C K , Bloom B R , Jacobs W R
Published: January 2020

Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.
Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain
Authors: Provence D L , Curtiss R
Published: January 2020

In this article, we report the isolation and characterization of a gene that may be important in the adherence of avian pathogenic Escherichia coli to the avian respiratory tract. The E. coli strain HB101, which is unable to agglutinate chicken erythrocytes, was transduced with cosmid libraries from the avian pathogenic E. coli strain chi 7122. Enrichment of transductants that could agglutinate chicken erythrocytes yielded 19 colonies. These isolates contained cosmids that encompassed four nonoverlapping regions of the E. coli chromosome. Only one group of cosmids, represented by pYA3104, would cause E. coli CC118 to agglutinate chicken erythrocytes. A 10-kb fragment of this cosmid was subcloned in pACYC184. Transposon mutagenesis of this fragment with Tn5seq1 indicated that a contiguous 4.4-kb region of cloned DNA was required for hemagglutination. In vitro transcription/translation assays indicated that this 4.4-kb region of DNA encoded one protein of approximately 140 kDa. The nucleotide sequence of this region was determined and found to encode one open reading frame of 4,134 nucleotides that would encode a protein of 1,377 amino acids with a deduced molecular weight of 148,226. This gene confers on E. coli K-12 a temperature-sensitive hemagglutination phenotype that is best expressed when cells are grown at 26 degrees C, and we have designated this gene tsh and the deduced gene product Tsh. Insertional mutagenesis of the chromosomal tsh gene in chi 7122 had no effect on hemagglutination titers. The deduced protein was found to contain significant homology to the Haemophilus influenzae and Neisseria gonorrhoeae immunoglobulin A1 proteases. These data indicate that (i) a single gene isolated from the avian pathogenic E. coli strain chi 7122 will confer on E. coli K-12 a hemagglutination-positive phenotype, (ii) chi 7122 contains at least two distinct mechanisms to allow hemagglutination to occur, and (iii) the hemagglutinin Tsh has homology with a class of proteins previously not known to exist in E. coli.
Cytokine gene expression by cultures of human lymphocytes with autologous Mycobacterium tuberculosis-infected monocytes
Authors: Johnson B J , McMurray D N
Published: January 2020

In order to better understand the immunoregulation following Mycobacterium tuberculosis infection, cytokine mRNA induction in response to in vitro infection of human monocytes with live virulent M. tuberculosis H37Rv cocultured with autologous lymphocytes was quantitated by reverse transcriptase-PCR. Induced levels of interleukin 1 beta (IL-1 beta), IL-2, tumor necrosis factor alpha, and gamma interferon (IFN-gamma) were compared among groups of individuals representing three phases of immunity to infection with M. tuberculosis: naive normal control subjects, purified protein derivative (PPD)-reactive normal donors, and individuals with active tuberculosis (TB [diseased]). Levels of IL-1 beta and tumor necrosis factor alpha mRNA in cocultured cells from TB patients were 51 and 45%, respectively, of those obtained in cells from sensitized healthy volunteers and were comparable to those from naive normal donors. Lymphoproliferative responses to M. tuberculosis and induction of the T-cell cytokine IL-2 were predictably high in the cells of PPD-sensitized donors, low in normal naive individuals, and variable among TB patients. In contrast, the induced level of another lymphokine, IFN-gamma, did not follow the pattern seen in IL-2 induction. Infection with live M. tuberculosis induced high levels of IFN-gamma mRNA in lymphocytes of both PPD-sensitized and normal naive donors compared with those of TB patients. Interestingly, polyclonal stimulation with the mitogen concanavalin A induced similar IFN-gamma levels in cells from all three donor groups. The high level of IFN-gamma induced by the infection of monocytes from naive normal donors suggests a role for natural killer (NK) cells in the production of IFN-gamma in this coculture system. This response appears independent of the role performed by T cells.
Comparison of Vibrio cholerae O139 with V. cholerae O1 classical and El Tor biotypes
Authors: Calia K E , Murtagh M , Ferraro M J , Calderwood S B
Published: January 2020

Vibrio cholerae O139 is a recently identified non-O1 V. cholerae strain responsible for outbreaks of epidemic cholera in India, Bangladesh, and Thailand in the past 2 years. Other workers have demonstrated the presence of the cholera toxin genetic element in V. cholerae O139, unlike the situation for other non-O1 V. cholerae strains. We sought to compare further this strain with strains of V. cholerae O1, classical and El Tor biotypes, by classic microbiologic methods, Southern blot analysis for restriction fragment length polymorphisms with probes for iron-regulated genes of V. cholerae O1, and comparisons of outer membrane protein profiles. Our results were similar for V. cholerae O139 and the El Tor biotype of V. cholerae O1, with the exception of the constitutive expression in V. cholerae O139 of OmpS, an outer membrane protein that was maltose inducible in comparison strains of V. cholerae O1.