bluuebunny/crossref_metadata_2025 huggingface dataset:
Cryptococcal polysaccharides bind to CD18 on human neutrophils
Authors: Dong Z M , Murphy J W
Published: January 2020

CD18, the beta chain of the beta 2 integrin family of adhesion molecules, is associated with three different alpha chains (CD11a, -b, and -c) and is expressed on the surface of all types of leukocytes. CD18-containing molecules are up-regulated on the surface of neutrophils (polymorphonuclear cells [PMN]) in response to chemotactic agents and are implicated in mediating adhesion to an inflamed endothelium, which is a prerequisite to migration of PMN into infected tissues. In a previous study, we found that a cryptococcal culture filtrate (CneF), when injected into the bloodstream of mice to simulate the antigenemia in cryptococcosis, inhibits PMN accumulation at the site of an inflammatory stimulus. In the present study, we assessed the ability of CneF and its individual components, i.e., glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoprotein (MP), to interact with CD18 on human PMN. CneF labeled with 14C was shown to bind to human PMN in a dose-dependent manner. Pretreatment of PMN with anti-CD18, but not an isotype-matched control monoclonal antibody (MAb) or anti-CD11a MAb, blocked the binding of 14C-labeled CneF to PMN. In addition, CneF, GXM, and GalXM but not MP significantly blocked the binding of the anti-CD18 MAb to CD18 on the surface of unactivated and formyl methionyl leucyl phenylalanine-activated PMN as determined by indirect immunofluorescence staining and flow cytometric analysis. In the same experiments, the cryptococcal polysaccharides did not affect the binding of an anti-CD11a or anti-L-selectin MAb to the surface of PMN at 4 degrees C. The results suggest that CneF and its components GXM and GalXM bind to CD18 on human PMN. Based on our findings, we propose that CD18 is a possible molecular target of cryptococcal polysaccharides and that binding of the polysaccharides to CD18 has the potential to inhibit leukocyte infiltration into inflammatory sites.
Stabilized expression of mRNA is associated with mycobacterial resistance controlled by Nramp1
Authors: Brown D H , Lafuse W P , Zwilling B S
Published: January 2020

Control of innate resistance to the growth of mycobacteria is mediated by a gene termed Nramp1. Although the role of the protein product of Nramp1 in mediating resistance to mycobacterial growth is not known, the effect of the gene is pleiotropic and it has been suggested that the gene controls macrophage priming for activation. We have found that the functional capacity of macrophages from Mycobacterium bovis BCG-susceptible mice can be suppressed by corticosterone, while the function of macrophages from BCG-resistant mice remains unaffected. In this study, we show that corticosterone differentially affects the stability of mRNAs of several recombinant gamma interferon (rIFN-gamma)-induced genes. Treatment of macrophages from BCG-susceptible mice with corticosterone accelerates the decay of Nramp1 mRNA. The mRNA of IFN-gamma-induced genes of macrophages from BCG-resistant mice was more stable than the mRNA of macrophages from BCG-susceptible mice in the presence or absence of corticosterone. The results of this investigation suggest that Nramp1 acts by stabilizing the mRNA of genes associated with macrophage activation, thus accounting for the functional differences that have been attributed to these macrophage populations.
Adjuvant modulation of immune responses to tuberculosis subunit vaccines
Authors: Lindblad E B , Elhay M J , Silva R , Appelberg R , Andersen P
Published: January 2020

Mice were immunized with experimental subunit vaccines based on secreted antigens from Mycobacterium tuberculosis in a series of adjuvants, comprising incomplete Freund's adjuvant (IFA), dimethyl dioctadecyl ammoniumbromide (DDA), RIBI adjuvant, Quil-A saponin, and aluminum hydroxide. Immune responses induced by these vaccines were characterized by in vitro culture of primed cells, PCR analysis for cytokine mRNA, detection of specific immunoglobulin G isotypes induced, and monitoring of protective immunity to tuberculosis (TB). The study demonstrated marked differences in the immune responses induced by the different adjuvants and identified both IFA and DDA as efficient adjuvants for a TB subunit vaccine. Aluminum hydroxide, on the other hand, induced a Th2 response which increased the susceptibility of the animals to a subsequent TB challenge. DDA was further coadjuvanted with either the Th1-stimulating polymer poly(I-C) or the cytokines gamma interferon, interleukin 2 (IL-2), and IL-12. The addition of IL-12 was found to amplify a Th1 response in a dose-dependent manner and promoted a protective immune response against a virulent challenge. However, if the initial priming in the presence of IL-12 was followed by two booster injections of vaccine without IL-12, no improvement in long-term efficacy was found. This demonstrates the efficacy of DDA to promote an efficient immune response and suggests that IL-12 may accelerate this development, but not change the final outcome of a full vaccination regime.
Cloning and sequence analysis of the gbpC gene encoding a novel glucan-binding protein of Streptococcus mutans
Authors: Sato Y , Yamamoto Y , Kizaki H
Published: January 2020

We have isolated dextran-aggregation-negative mutants of Streptococcus mutans following random mutagenesis with plasmid pVA891 clone banks. A chromosomal region responsible for this phenotype was characterized in one of the mutants. A 2.2-kb fragment from the region was cloned in Escherichia coli and sequenced. A gene specifying a putative protein of 583 amino acid residues with a calculated molecular weight of 63,478 was identified. The amino acid sequence deduced from the gene exhibited no similarity to the previously identified S. mutans 74-kDa glucan-binding protein or to glucan-binding domains of glucosyltransferases but exhibited similarity to surface protein antigen (Spa)-family proteins from streptococci. Extract from an E. coli clone of the gene exhibited glucan-binding activity. Therefore, the gene encoded a novel glucan-binding protein.
Characterization of hemin binding activity of Streptococcus pneumoniae
Authors: Tai S S , Wang T R , Lee C J
Published: January 2020

Streptococcus pneumoniae is a causative agent of bacterial pneumonia, otitis media, meningitis, and bacteremia. It causes considerable morbidity and mortality throughout the world, especially among children, the elderly, and immunocompromised individuals. We have demonstrated previously that the growth of S. pneumoniae is limited under iron-depleted conditions and can be restored by the addition of either hemin or hemoglobin. In the present study, we showed that S. pneumoniae had the ability to bind hemin and that the level of hemin binding activity was not affected by supplementation of the growth medium with iron. Approximately 70 to 80% of the hemin binding activity was mediated by proteinase-resistant components, and the remainder was mediated by proteins. Hemin binding proteins were located in both soluble extract and envelope fractions of pneumococcal cells. By batch affinity chromatography, a major hemin binding polypeptide with an apparent molecular mass of 43 kDa was identified in the cell lysate of S. pneumoniae. Polyclonal antibodies against this polypeptide were raised. By immunoblot analysis, this hemin binding polypeptide was localized in the envelope and did not exhibit any variation in molecular weight among all serotypes tested. The subcellular distribution of hemin binding activity may have functional implications.
Role of pertussis toxin A subunit in neutrophil migration and vascular permeability
Authors: Brito G A , Souza M H , Melo-Filho A A , Hewlett E L , Lima A A , Flores C A , Ribeiro R A
Published: January 2020

The anti-inflammatory activity of pertussis toxin (Ptx) was compared to that of a noncatalytic mutant of pertussis toxin (9K/129G; Ptxm), which contains two amino acid substitutions in the A protomer, by using a rat model of inflammation. The toxins were administered intravenously 1 h prior to the injection of inflammatory stimuli. Ptx, but not Ptxm, inhibited neutrophil migration into peritoneal cavities in response to formyl-methionyl-leucyl-phenylalanine and lipopolysaccharide. The inhibitory effect of Ptx on neutrophil migration could not be explained by the ability of the toxin to induce leukopenia or neutropenia. The increase in skin vascular permeability induced by leukotriene B4, a powerful neutrophil chemotactic agent, was also inhibited only by Ptx. On the other hand, the increase in skin vascular permeability induced by histamine was potentiated by both toxins. These data show that Ptx inhibits neutrophil-mediated inflammation in vivo and that this effect is dependent on the ADP-ribosyltransferase activity of the A protomer.
Protective immunity against Salmonella typhimurium elicited in mice by oral vaccination with phosphorylcholine encapsulated in poly(DL-lactide-co-glycolide) microspheres
Authors: Allaoui-Attarki K , Pecquet S , Fattal E , Trollé S , Chachaty E , Couvreur P , Andremont A
Published: January 2020

Encapsulation of vaccines in biodegradable microspheres provides excellent mucosal immunogens with a high potential for immunization against bacterial infections. We tested the protective immunity elicited by intragastric vaccination with phosphorylcholine (PC) encapsulated in poly(DL-lactide-co-glycolide) (DL-PLG) microspheres against Salmonella typhimurium in a mouse model of invasive intestinal infection. We chose PC as the antigen because it was found to elicit an immune response after intestinal exposure of mice to PC-bearing S. typhimurium and because anti-PC immunity protects mice against Streptococcus pneumoniae, another PC-bearing microorganism. Mice were primed intragastrically on days 1, 2, and 3 and boosted on days 28, 29, and 30 with PC (280 microg) coupled to porcine thyroglobulin (PC-thyr) encapsulated in DL-PLG microspheres, free PC-thyr, or blank microspheres. A significant rise in anti-PC immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent assay, was observed in the intestinal secretions after immunization with PC-loaded microspheres, compared to the titers of mice immunized with free PC-thyr or blank microspheres. This antibody response peaked 14 days after the last boost and correlated with a highly significant resistance to oral challenge by S. typhimurium C5 (P < 10(-3)). Control mice were primed intraperitoneally on day 1 with 15 microg of PC in complete Freund's adjuvant and boosted on days 10, 14, and 20 with the same dose without adjuvant but via the same route. In these mice, the levels of anti-PC IgA in intestinal secretions were equivalent to those of the mice intragastrically immunized with PC-loaded microspheres, but protection was significantly weaker, suggesting that either the IgAs were not functional or that other immune mechanisms are important in protection. Taken together, our results highlight the potential of antigen encapsulation in DL-PLG microspheres for eliciting protective immunity against invasive intestinal bacterial diseases and suggest that a similar strategy could be used against diseases caused by other PC-bearing microorganisms.
Specific binding of soluble peptidoglycan and muramyldipeptide to CD14 on human monocytes
Authors: Weidemann B , Schletter J , Dziarski R , Kusumoto S , Stelter F , Rietschel E T , Flad H D , Ulmer A J
Published: January 2020

Previously, we were able to show that soluble peptidoglycan (sPG)-induced monokine production in human peripheral monocytes is inhibited by anti-CD14 monoclonal antibodies and by lipid A partial structures. This suggested but did not prove that monocytic surface protein CD14 is involved in the activation of human monocytes not only by cell wall components of gram-negative bacteria such as lipopolysaccharide (LPS) but also by cell wall components of gram-positive bacteria such as sPG. In the present study, we provide experimental evidence that CD14 indeed constitutes a binding site for sPG recognition and activation of human monocytes. The results show that fluorescein isothiocyanate-sPG (FITC-sPG) binds to human monocytes in a saturable, dose-dependent, and specific manner. For maximal binding, 2 to 3 microg of FITC-sPG per ml was sufficient, and this binding is completed within 90 min; about 40% of the binding is completed within the first 3 min. The FITC-sPG binding is considered specific because unlabeled sPG and also muramyldipeptide (MDP), the minimal bioactive structure of sPG, inhibit the binding of sPG to monocytes in a dose-dependent manner. This specific binding was also inhibited by an anti-CD14 monoclonal antibody, LPS, and lipid A partial structure compound 406. Direct evidence for an interaction of sPG with CD14 is provided by experiments involving native polyacrylamide gel electrophoresis that showed a shift of the electrophoretic mobility of CD14 by LPS as well as by sPG. These results allow the conclusion that sPG binds directly to CD14, that MDP represents the active substructure of sPG, and that CD14 may be a lectin-like receptor which plays a key role in cellular stimulation by bioactive components of not only gram-negative but also gram-positive bacteria.
Intrathecal production of interleukin-12 and gamma interferon in patients with bacterial meningitis
Authors: Kornelisse R F , Hack C E , Savelkoul H F , van der Pouw Kraan T C , Hop W C , van Mierlo G , Suur M H , Neijens H J , de Groot R
Published: January 2020

To assess the role of interleukin-12 (IL-12) and gamma interferon (IFN-gamma) in children with bacterial meningitis, bioactive IL-12 (p70) and the inactive subunit p40 and IFN-gamma were measured in serum and cerebrospinal fluid (CSF) from 35 children with bacterial meningitis and 10 control subjects. The production of IFN-gamma is induced by IL-12 with tumor necrosis factor alpha (TNF-alpha) as a costimulator and inhibited by IL-10. CSF concentrations of IL-12 p40 as well as those of IFN-gamma were markedly elevated, whereas IL-12 p70 was hardly detectable. Detectable CSF levels of IFN-gamma correlated positively with IL-12 p40 (r = 0.40, P = 0.02) and TNF-alpha (r = 0.46, P = 0.04) but not with IL-6, IL-8, or IL-10. In contrast to CSF levels of TNF-alpha, IL-12, and IL-10, those of IFN-gamma were significantly higher in patients with pneumococcal meningitis than in children with meningitis caused by Haemophilus influenzae and Neisseria meningitidis, presumably because of a high CSF TNF-alpha/IL-10 ratio in the former. We suggest that IL-12- and TNF-alpha-induced IFN-gamma production may contribute to the natural immunity against microorganisms in the CSF compartment during the acute phase of bacterial meningitis.
Interleukin-12 modulates the protective immune response in SCID mice infected with Histoplasma capsulatum
Authors: Zhou P , Sieve M C , Tewari R P , Seder R A
Published: January 2020

Infection with Histoplasma capsulatum results in a subclinical infection in immunocompetent hosts due to an effective cellular immune response. By contrast, immunodeficient individuals can have a severe disseminated and potentially fatal disease. In a previous study, it was demonstrated that normal mice infected intravenously with H. capsulatum and treated with interleukin-12 (IL-12) at the time of infection were protected from a fatal outcome. In this study, we examined the immunomodulatory effects of IL-12 on disseminated histoplasmosis in immunodeficient SCID mice. SCID mice infected with H. capsulatum and treated with IL-12 showed an increase in survival and a reduction in the colony counts of H. capsulatum in internal organs at 14 days after infection. The protective effect of IL-12 was abrogated if animals were also treated with a neutralizing antibody to gamma interferon (IFN-gamma). IL-12 treatment also resulted in an increase in mRNA expression and protein production for IFN-gamma, tumor necrosis factor alpha (TNF-alpha), and nitric oxide from spleen cells. When IL-12 was combined with amphotericin B (AmB) treatment, there was a significant increase in survival compared with either modality alone. Moreover, combined treatment resulted in an increase in both IFN-gamma and TNF-alpha production, as well as in a substantial reduction in H. capsulatum burden at 35 and 90 days postinfection. This study demonstrates that IL-12 modulates the protective immune response to histoplasmosis in SCID mice and also suggests that IL-12 in combination with AmB may be useful as a treatment for H. capsulatum in immunodeficient hosts.
Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival
Authors: Broug-Holub E , Toews G B , van Iwaarden J F , Strieter R M , Kunkel S L , Paine R , Standiford T J
Published: January 2020

To study the in vivo role of alveolar macrophages (AM) in gram-negative bacterial pneumonia in mice, AM were eliminated by the intratracheal (i.t.) administration of dichloromethylene diphosphonate encapsulated liposomes. Subsequently, the AM-depleted mice were infected i.t. with 100 CFU of Klebsiella pneumoniae, and the effects of AM depletion on survival, bacterial clearance, and neutrophil (polymorphonuclear leukocyte [PMN]) recruitment were assessed. It was shown that depletion of AM decreases survival dramatically, with 100% lethality at day 3 postinfection, versus 100% long-term survival in the control group. This increased mortality was accompanied by 20- to 27- and 3- to 10-fold increases in the number of K. pneumoniae CFU in lung and plasma, respectively, compared to those in nondepleted animals. This decreased bacterial clearance was not due to an impaired PMN recruitment; on the contrary, the K. pneumoniae-induced PMN recruitment in AM-depleted lungs was sevenfold greater 48 h postinfection than that in control infected lungs. Together with an increased PMN infiltration, 3- and 10-fold increases in lung homogenate tumor necrosis factor alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) levels, respectively, were measured. Neutralization of TNF-alpha or MIP-2, 2 h before infection, reduced the numbers of infiltrating PMN by 41.6 and 64.2%, respectively, indicating that these cytokines mediate PMN influx in infected lungs, rather then just being produced by the recruited PMN themselves. Our studies demonstrate, for the first time, the relative importance of the AM in the containment and clearance of bacteria in the setting of Klebsiella pneumonia.
Cell-density-dependent expression of Borrelia burgdorferi lipoproteins in vitro
Authors: Indest K J , Ramamoorthy R , Solé M , Gilmore R D , Johnson B J , Philipp M T
Published: January 2020

Previously, we had identified non-OspA-OspB surface proteins of Borrelia burgdorferi that are targeted by the antibody-dependent complement-mediated killing mechanism. Here we demonstrate by Western blotting that one of these proteins, P35, is upregulated at the onset of stationary phase in vitro. Northern analysis revealed that the upregulation of P35 is at the level of transcription. In addition, the expression of an open reading frame (ORF) located downstream of the p35 gene was found to be regulated in the same fashion as that of P35. This ORF encodes a 7.5-kDa lipoprotein. The transcriptional start sites for both of these genes were determined, to aid in the identification of the putative promoter regions. Additional sequencing of the 5' flanking region of the p35 gene revealed a region of dyad symmetry 52 bp upstream of the transcription start site. Southern analysis demonstrated that the expression of these genes was not due to a cell-density-dependent rearrangement in the genome of B. burgdorferi. These findings provide an in vitro model for studying mechanisms of gene regulation in B. burgdorferi.
Molecular characterization of pldA, the structural gene for a phospholipase A from Campylobacter coli, and its contribution to cell-associated hemolysis
Authors: Grant K A , Belandia I U , Dekker N , Richardson P T , Park S F
Published: January 2020

A gene (pldA) encoding a 35.0-kDa protein with significant homology to the Escherichia coli outer membrane phospholipase was identified upstream of an operon encoding an enterochelin transport system in Campylobacter coli. The results of this study suggest that this gene encodes an outer membrane phospholipase A in C. coli. First, expression of the pldA gene product in a PldA-deficient mutant of E. coli led to the restoration of phospholipase A activity. The recombinant product also partitioned to the outer membrane, suggesting that it may be similarly located in C. coli. Second, heterologous overexpression in E. coli, followed by in vitro folding and purification of C. coli PldA, resulted in pure protein which displayed calcium-dependent lysophospholipase and phospholipase A activities in vitro. Finally, mutants of C. coli in which the pldA gene had been inactivated by allelic exchange were deficient in phospholipase A activity. Phospholipases are associated with lysis of erythrocytes by a number of bacterial pathogens. The pldA mutant was shown to have a reduced hemolytic activity compared to the wild-type strain, suggesting a role for the phospholipase A in the lysis of erythrocytes by C. coli. Since hemolysins are intimately associated with the disease-causing potential of a number of bacterial pathogens, it is likely that the phospholipase A plays some role in Campylobacter virulence.
Production of interleukin-8 (IL-8) by cultured endothelial cells in response to Borrelia burgdorferi occurs independently of secreted [corrected] IL-1 and tumor necrosis factor alpha and is required for subsequent transendothelial migration of neutrophils
Authors: Burns M J , Sellati T J , Teng E I , Furie M B
Published: January 2020

Previous studies have shown that Borrelia burgdorferi, the spirochetal agent of Lyme disease, promotes inflammation by stimulating endothelial cells to upregulate adhesion molecules for leukocytes and to produce a soluble agent that is chemotactic for neutrophils. We determined that interleukin-8 (IL-8) was the chemotactic agent for neutrophils present in conditioned media from cultured human umbilical vein endothelial cells stimulated with B. burgdorferi. As few as one spirochete per endothelial cell stimulated production of IL-8 within 8 h of coincubation. When 10 spirochetes per endothelial cell were added, IL-8 was detected after 4 h of coculture. Production of IL-8 continued in a linear fashion for at least 24 h. Neutralizing antibodies against IL-8 reduced migration of neutrophils across spirochete-stimulated endothelial monolayers by 93%. In contrast, pretreatment of neutrophils with antagonists of platelet-activating factor did not inhibit migration. Increases in production of IL-8 and expression of the adhesion molecule E-selectin by endothelial cells in response to B. burgdorferi were not inhibited by IL-1 receptor antagonist or a neutralizing monoclonal antibody directed against tumor necrosis factor alpha, used either alone or in combination. These results suggest that activation of endothelium by B. burgdorferi is not mediated through the autocrine action of secreted IL-1 or tumor necrosis factor alpha. Rather, it appears that B. burgdorferi must stimulate endothelium either by a direct signaling mechanism or by induction of a novel host-derived proinflammatory cytokine.
Differences in virulence for mice among Streptococcus pneumoniae strains of capsular types 2, 3, 4, 5, and 6 are not attributable to differences in pneumolysin production
Authors: Benton K A , Paton J C , Briles D E
Published: January 2020

We observed that differences in the in vivo growth kinetics of pneumococcal strains of capsular types 3, 4, 5, and 6 were reminiscent of differences that we had previously reported for type 2 strain D39 and its pneumolysin-deficient mutant, PLN. Capsular type 2 Streptococcus pneumoniae D39 exhibits exponential growth in the blood of XID mice until the death of the mice at 24 to 36 h. In contrast, PLN reaches a plateau in growth that is maintained for several days. Capsular type 3 and 5 strains exhibited exponential growth and caused rapid death of XID mice following intravenous challenge, similar to the observation with D39. Strains of capsular types 4 and 6 exhibited growth kinetics reminiscent of PLN. Since the observed differences in the pathogenesis of types 3 and 5 compared to 4 and 6 were reminiscent of the effects of pneumolysin deficiency in type 2, we examined the levels of in vitro pneumolysin production for the entire panel of strains. The onset of pneumolysin production in most strains was rapid and occurred near the end of log-phase growth. Differences in in vivo growth patterns of capsular type 2, 3, 4, 5, and 6 strains were not found to be associated with differences in the levels of pneumolysin.
The fibronectin-binding protein of Streptococcus pyogenes, SfbI, is involved in the internalization of group A streptococci by epithelial cells
Authors: Molinari G , Talay S R , Valentin-Weigand P , Rohde M , Chhatwal G S
Published: January 2020

Streptococcus pyogenes organisms (group A streptococci) are considered to be highly adhesive extracellular pathogens. However, it has recently been reported that S. pyogenes has the capacity to efficiently invade eukaryotic cells. In this study, we demonstrate that the interaction of S. pyogenes fibronectin-binding protein (SfbI) with fibronectin on nonphagocytic HEp-2 cells triggers bacterial internalization. Blocking of the SfbI adhesin by either antibodies against the whole protein or antibodies against the fibronectin-binding domains of SfbI, as well as pretreatment of HEp-2 cells with purified SfbI protein, prevents both S. pyogenes attachment and internalization. Inert latex beads precoated with the purified SfbI protein are ingested by eukaryotic cells, demonstrating that SfbI is per se enough to trigger the internalization process. Experiments performed with a recombinant SfbI domain encompassing the two fibronectin-binding regions of the SfbI molecule demonstrated that these binding regions are essential and sufficient to activate uptake by HEp-2 cells. These results demonstrate that the fibronectin-binding protein SfbI is involved in both S. pyogenes' attachment to and ingestion by HEp-2 cells and contribute to elucidation of the underlying molecular events leading to eukaryotic cell invasion by S. pyogenes.
Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196
Authors: Perelle S , Gibert M , Bourlioux P , Corthier G , Popoff M R
Published: January 2020

A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M. R. Popoff, E. J. Rubin, D. M. Gill, and P. Boquet, Infect. Immun. 56:2299-2306, 1988). The CDT locus from C. difficile CD196 was cloned and sequenced. It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene. The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin. CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins. The lower level of production of binary toxin by CD196 than of iota toxin by C. perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes. The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains. One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb. These results indicate that some C. difficile strains synthesize a binary toxin that could be an additional virulence factor.
Effect of carbon source on localized adherence of enteropathogenic Escherichia coli
Authors: Vanmaele R P , Armstrong G D
Published: January 2020

Enteropathogenic Escherichia coli (EPEC) strains attach to epithelial cells as discrete clusters of bacteria which are localized at a few sites on the cell surface. Previously, it was shown that this localized-adherence (LA) phenotype is induced by specific growth conditions. We found that wild-type EPEC attached to HEp-2 cells in an LA pattern when the bacteria were grown in Dulbecco's modified Eagle medium (DMEM) containing glucose as the carbon source. In contrast, bacteria incubated in DMEM containing galactose did not adhere to epithelial cells. The latter results were similar to those observed when JPN15, an LA-negative strain, was grown under conditions which promoted bacterial binding. The differences in attachment of wild-type EPEC were independent of the stage of log-phase growth of the cultures and of the number of CFU incubated with the HEp-2 monolayers. Expression of the adherence phenotype by organisms grown in glucose was associated with increased expression of intimin and bundle-forming pilin. In contrast, bacteria grown in medium containing galactose expressed these proteins at levels similar to those observed when JPN15 was grown in medium containing glucose.
The Bacteroides fragilis toxin fragilysin disrupts the paracellular barrier of epithelial cells
Authors: Obiso R J , Azghani A O , Wilkins T D
Published: January 2020

Bacteroides fragilis is a member of the normal colonic microflora of most mammals and is the most commonly isolated anaerobe from human clinical specimens. Some strains produce a toxin (fragilysin, a zinc-metalloproteinase) implicated as a cause of diarrheal disease in farm animals and humans. Studies in our laboratory confirm that the proteolytic activity of this toxin is responsible for the fluid secretion and tissue damage observed in vivo. In this study, we investigated the effects of fragilysin on the paracellular barrier of epithelial cells. Researchers suggest that, since the toxin rapidly intoxicates HT-29 cells, it may be internalized. However, we could not prevent cell rounding by using inhibitors of receptor-mediated endocytosis, which indicates that the toxin may act outside the cell. Based on these observations, we studied the effects of the highly purified B. fragilis fragilysin on the barrier function of cultured epithelial cells. Fragilysin rapidly increased the permeability of the paracellular barrier of epithelial cells to ions (decrease in electrical resistance across monolayers) and to larger molecules (increase in mannitol flux across monolayers). We tested a human colon cell line and cell lines from the lung and the kidney; the human colon cell line was most sensitive, but all three were affected in the same manner. Our studies show that B. fragilis fragilysin alters the barrier function of the epithelial lining, possibly by degrading the tight junction proteins, such as ZO-1. The proteolytic activity is required to cause this effect. The toxin's action has been assumed to be limited to the intestine; however, our studies show that fragilysin could also contribute to the pathogenesis of B. fragilis in extraintestinal infections.
Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56
Authors: Seong S Y , Huh M S , Jang W J , Park S G , Kim J G , Woo S G , Choi M S , Kim I S , Chang W H
Published: January 2020

Although the 56-kDa protein of Rickettsia tsutsugamushi has been presumed to play important roles in generating protective immunity against scrub typhus, studies of this protein have been impeded. We used the recombinant 56-kDa protein of R. tsutsugamushi Boryong fused with the maltose-binding protein of Escherichia coli (MBP-Bor56) to analyze its ability to induce protective immunity in a C3H/HeDub murine model. Intraperitoneal immunization of mice with MBP-Bor56 resulted in an increase in the 50% minimal lethal dose of more than 160 times compared with that for the control mice. Splenic mononuclear cells from the mice immunized with MBP-Bor56 showed a dose-dependent pattern of lymphocyte proliferation response and secreted gamma interferon and interleukin-2 when stimulated with irradiated R. tsutsugamushi Boryong, which is a cytokine profile of Th1 cells. High titers of antibody to R. tsutsugamushi were also demonstrated by indirect immunofluorescent-antibody testing. These findings suggest that the 56-kDa protein of R. tsutsugamushi is one of the candidates for a vaccine against scrub typhus.
Genetic organization of the yersiniabactin biosynthetic region and construction of avirulent mutants in Yersinia pestis
Authors: Bearden S W , Fetherston J D , Perry R D
Published: January 2020

We have identified an approximately 22-kb region of the pgm locus of Yersinia pestis KIM6+ which encodes a number of iron-regulated proteins involved in the biosynthesis of the Y. pestis cognate siderophore, yersiniabactin (Ybt), and which is located immediately upstream of the pesticin/yersiniabactin receptor gene (psn). Sequence analysis and the construction of insertion and deletion mutants allowed us to determine the putative location of the irp1 gene and the positions of irp2, ybtT, and ybtE within the ybt operon. Mutations in the irp1, irp2, or ybtE gene yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from yersiniabactin-producing strains of Y. pestis grown under iron-limiting conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell extracts from Ybt+ and Ybt- strains grown in iron-deficient media revealed that expression of ybt-encoded proteins is not only iron regulated but also influenced by the presence of the siderophore itself. Finally, Y. pestis strains with mutations in either the psn or irp2 gene were avirulent in mice when inoculated subcutaneously.
Role of interleukin-10 in regulation of T-cell-dependent and T-cell-independent mechanisms of resistance to Toxoplasma gondii
Authors: Neyer L E , Grunig G , Fort M , Remington J S , Rennick D , Hunter C A
Published: January 2020

Interleukin-10 (IL-10) is a cytokine which can inhibit T-cell and natural killer (NK) cell functions associated with cell-mediated immunity to intracellular infections. The production of IL-10 by mice infected with Toxoplasma gondii has been implicated in the suppression of lymphocyte proliferation observed during acute toxoplasmosis, as well as susceptibility to infection with this parasite. We have used C57BL/6 mice which lack a functional IL-10 gene (IL-10(-/-) mice) to investigate the role of IL-10 in acute toxoplasmosis. Intraperitoneal infection of IL-10(-/-) mice with T. gondii resulted in 100% mortality by day 13, whereas wild-type C57BL/6 (WT) mice survived acute infection. IL-10(-/-) mice infected with T. gondii had significantly higher serum levels of IL-12 and gamma interferon (IFN-gamma) than WT mice. Early mortality of infected IL-10(-/-) mice was prevented by treatment with IL-10 and significantly delayed by neutralizing antibodies to IL-12 and IFN-gamma. Further studies revealed that SCID/IL-10(-/-) mice infected with T. gondii had delayed time to death compared to IL-10(-/-) mice, indicating that lymphocytes contributed to death of IL-10(-/-) mice. In addition, infected SCID/IL-10(-/-) mice survived longer than infected SCID mice. These latter data indicate that in mice lacking lymphocytes, endogenous IL-10 is associated with increased susceptibility to T. gondii. However, the lack of IL-10 does not alter the infection-induced suppression of T cell and NK cell functions. Our experiments reveal that IL-10 is associated with protection or increased susceptibility to infection with T. gondii, depending on whether mice possess lymphocytes, and demonstrate the important roles of IL-12 and IFN-gamma in the early infection-induced mortality observed in the IL-10(-/-) mice.
Evidence for a methyl-accepting chemotaxis protein gene (mcp1) that encodes a putative sensory transducer in virulent Treponema pallidum
Authors: Hagman K E , Porcella S F , Popova T G , Norgard M V
Published: January 2020

The clinical and histopathological manifestations of syphilis and the invasive behavior of Treponema pallidum in tissue culture systems reflect the propensity for treponemes to migrate through skin, hematogenously disseminate, and invade targeted tissues. Treponemal motility is believed to be essential to this process and thereby an important facet of syphilis pathogenesis. By analogy with other bacterial pathogens, it is plausible that treponemal motility and tissue invasion are modulated by sensory transduction events associated with chemotactic responses. Recent studies have demonstrated the existence in T. pallidum of accessory molecules typically associated with sensory transduction events involving methyl-accepting chemotaxis proteins (MCPs). Intrinsic radiolabeling of T. pallidum in vitro with L-[methyl-3H] methionine revealed one methylated treponemal polypeptide with an apparent molecular mass of 64 kDa. A degenerate oligonucleotide probe corresponding to a highly conserved C-terminal domain within Bacillus subtilis and Escherichia coli MCPs was used in Southern blotting of T. pallidum DNA to identify and subsequently clone a putative T. pallidum MCP gene (mcp1). Computer analyses predicted a near-consensus promoter upstream of mcp1, and primer extension analysis employing T. pallidum RNA revealed a transcriptional initiation site. T. pallidum mcp1 encoded a 579-amino-acid (64.6-kDa) polypeptide which was highly homologous to at least 69 other known or putative sensory transducer proteins, with the highest degrees of homology existing between the C terminus of mcp1 and the C-terminal (signaling) domains of the other bacterial MCPs. Other salient features of Mcp1 included (i) six potential membrane-spanning domains at the N terminus, (ii) two predicted alpha-helical coiled coil regions containing at least three putative methylation sites, and (iii) homologies with two ligand-binding domains (LI-1 and LI-2) of the E. coli MCPs Trg and Tar. This study is the first to provide both metabolic and genetic evidence for an MCP sensory transducer in T. pallidum. The combined findings prompt key questions regarding the relationship(s) among sensory transduction, regulation of endoflagellar rotation, and chemotactic responses (in particular, the role of glucose) during virulence expression by T. pallidum.
Alveolar macrophage priming by intravenous administration of chitin particles, polymers of N-acetyl-D-glucosamine, in mice
Authors: Shibata Y , Foster L A , Metzger W J , Myrvik Q N
Published: January 2020

Intravenous (i.v.) administration of phagocytosable chitin particles (1 to 10 microm) in C57BL/6 mice and SCID mice primed alveolar macrophages (Mphi) within 3 days to yield up to a 50-fold increase in their oxidative burst when elicited in vitro with phorbol myristate acetate (PMA). C57BL/6 mice pretreated with monoclonal antibodies (MAbs) against mouse gamma interferon (IFN-gamma) or NK1.1 showed a markedly decreased level of alveolar Mphi priming following injection of chitin particles. To confirm IFN-gamma production in vitro, spleen cells isolated from normal C57BL/6 mice and SCID mice were cultured with chitin particles. Significant IFN-gamma production was observed following stimulation with chitin but not with chitosan or latex beads. When spleen cells were treated with anti-NK1.1 MAb, IFN-gamma production was significantly inhibited. Another set of experiments showed that when C57BL/6 mice were pretreated i.v. with a small dose IFN-gamma, a higher level of priming was induced with not only phagocytosable chitin particles but also phagocytosable chitosan and even latex beads. Likewise, the spleen cell cultures preconditioned with IFN-gamma provided an up-regulation of IFN-gamma production by these phagocytosable particles. Taken together, the in vivo and in vitro results suggest that (i) the alveolar Mphi priming mechanism is due, at least in part, to direct activation of Mphi by IFN-gamma, which is produced by NK1.1+ CD4- cells; (ii) IFN-gamma would have an autocrine-like effect on Mphi and make them more responsive to particle priming; and (iii) phagocytosis of particulates, probably by a postmembrane event such as interiorization, appears to be important for the up-regulation of alveolar Mphi priming and IFN-gamma production.
The role of nitric oxide in experimental murine sepsis due to pyrogenic exotoxin A-producing Streptococcus pyogenes
Authors: Sriskandan S , Moyes D , Buttery L K , Wilkinson J , Evans T J , Polak J , Cohen J
Published: January 2020

Nitric oxide (NO) produced by inducible NO synthase (iNOS) mediates hypotension in endotoxemia. In this study, NO induction by a toxin-producing Streptococcus pyogenes isolate, H250, and by recombinant streptococcal pyrogenic exotoxin A (rSPEA) has been examined, both in vitro and in vivo. Streptococcal supernatants, but not rSPEA, induce production of nitrite by murine macrophages when both are coincubated with gamma interferon. Intraperitoneal injection of rSPEA did not cause significant production of NO. However, an elevated level of nitrate in serum was detected in a model of streptococcal fasciitis due to live H250. iNOS was localized to Kupffer cells, hepatocytes, and renal tubular cells by immunostaining. Administration of a NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA), reduced peak concentrations of nitrate in serum but did not affect survival. NO is induced by H250, both in vitro and in vivo, mainly via SPEA-independent mechanisms. In this model, iNOS is expressed predominantly in the liver. Furthermore, in this model L-NMMA is not protective.
Activation, binding, and processing of complement component 3 (C3) by Blastomyces dermatitidis
Authors: Zhang M X , Klein B
Published: January 2020

Complement plays a key role in phagocyte recognition and killing of Blastomyces dermatitidis, but little is known about how complement components interact with the yeast. We report the characteristics of activation, binding, and processing of C3 by B. dermatitidis. In pooled normal human serum (NHS), deposition of C3 on the yeast was detectable within 2 min, whereas in NHS containing MgEGTA, deposition was delayed by 6 min, indicating that the yeast activates C3 by both classical and alternative pathways. When both pathways were operative, maximal binding of 4.5 x 10(6) C3 molecules/cell was achieved in less than 30 min. In the absence of the classical pathway, yeast cells bound 80% of the maximum C3, indicating that the yeast intrinsically activates the alternative pathway. Delayed deposition of C3 in NHS-MgEGTA was similar to that in NHS preabsorbed by the yeast or by immobilized protein A/G to remove serum immunoglobulin. Purified immunoglobulin restored C3 binding to NHS preabsorbed by the yeast, suggesting that antibody in nonimmune serum initiates the classical pathway. beta-Glucan absorption of NHS abolished the classical pathway, suggesting that cell wall beta-glucan is a target of initiating antibodies. Hydroxylamine treatment of NHS-opsonized yeast cells showed that 76% of C3 was bound to yeast cells by ester linkage, supporting a role for hydroxyl groups on cell wall polysaccharides. Hydroxylamine-cleaved fragments were chiefly C3b and iC3b; 70% of hydroxylamine-sensitive C3b was converted to iC3b within 1 min of opsonization, and the ratio was stable over 1 h. Our data predict that C3b and iC3b on opsonized yeast cells direct binding to CR1 and CR3 receptors on human phagocytes, which, in turn, may influence the fate of this host-pathogen interaction.
Expression of the Yersinia pestis capsular antigen (F1 antigen) on the surface of an aroA mutant of Salmonella typhimurium induces high levels of protection against plague
Authors: Titball R W , Howells A M , Oyston P C , Williamson E D
Published: January 2020

The caf operon from Yersinia pestis encoding the structural subunit (caf1), the molecular chaperone (caf1M), the outer membrane anchor (caf1A), and the regulatory protein (caf1R) was cloned into Salmonella typhimurium SL3261 aroA. The recombinant Salmonella organisms were encapsulated when cultured at 37 degrees C but not when cultured at 28 degrees C. Oral inoculation of mice with the recombinant Salmonella induced predominantly an immunoglobulin G2a response to F1 antigen, and isolated T cells showed a recall response to soluble or Salmonella-associated F1 antigen. Mice immunized with S. typhimurium SL3261 aroA expressing F1 antigen intracellularly developed lower antibody responses to F1 antigen and showed a T-cell recall response only to Salmonella-associated F1 antigen. Mice immunized orally with two doses of the recombinant Salmonella which expressed F1 antigen on the surface were protected against 10(7) 50% lethal doses (LD50) of virulent Y. pestis given by the subcutaneous route of challenge, whereas mice immunized with the recombinant Salmonella expressing F1 antigen intracellularly were only partially protected against 10(5) LD50 of Y. pestis.
Production of tumor necrosis factor and nitric oxide by macrophages infected with live and dead mycobacteria and their suppression by an interleukin-10-secreting recombinant
Authors: Marshall B G , Chambers M A , Wangoo A , Shaw R J , Young D B
Published: January 2020

We have analyzed mycobacterium-induced cytokine secretion in the J774A.1 macrophage-like cell line. Tumor necrosis factor alpha (TNF-alpha) was preferentially induced by live organisms, both slow and rapid growing. Expression of interleukin-10 by a recombinant strain of Mycobacterium smegmatis caused reduced production of TNF-alpha and nitric oxide during the early stages of infection.
Siderophore production by Staphylococcus aureus and identification of iron-regulated proteins
Authors: Courcol R J , Trivier D , Bissinger M C , Martin G R , Brown M R
Published: January 2020

Siderophore activity of Staphylococcus aureus was detected in an iron-restricted chemically defined medium. The molecular mass of this siderophore, called aureochelin, was 577 Da. Surface-associated proteins of 120, 88, 57, 35, and 33 kDa were mainly expressed under iron restriction conditions. Results showed a relationship between siderophore production and the existence of the 120- and 88-kDa proteins. Western blotting of surface-associated proteins revealed that these proteins were recognized both by patients sera and polyclonal rabbit serum.
Arabinofuranosyl-terminated and mannosylated lipoarabinomannans from Mycobacterium tuberculosis induce different levels of interleukin-12 expression in murine macrophages
Authors: Yoshida A , Koide Y
Published: January 2020

Lipoarabinomannan (LAM) is a major surface lipoglycan of Mycobacterium tuberculosis. In the present study, we demonstrated that arabinofuranosyl-terminated LAM (AraLAM) derived from a rapidly growing Mycobacterium sp., but not extensively mannosylated LAM derived from the Erdman strain, is capable of inducing interleukin-12 (IL-12) expression in murine macrophages. Since IL-12 is known to drive the differentiation of naive T cells toward T-helper type 1 (Th1) cell development, AraLAM may be an effective adjuvant in vaccines and immunotherapies that need Th1 responses.
Temporal production of streptococcal erythrogenic toxin B (streptococcal cysteine proteinase) in response to nutrient depletion
Authors: Chaussee M S , Phillips E R , Ferretti J J
Published: January 2020

The effects of various growth conditions on the production of streptococcal erythrogenic toxin B (streptococcal pyrogenic exotoxin B [SPE B]) by Streptococcus pyogenes were analyzed. SPE B was detected in broth culture supernatant fluid only during the stationary phase of growth when glucose and other potential carbon sources were depleted from the medium. Additionally, SPE B production was inhibited when the concentration of glucose in the medium was maintained. These results suggest that SPE B is secreted under conditions of starvation and may be involved in nutrient acquisition.