bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction between complement subcomponent C1q and the Klebsiella pneumoniae porin OmpK36
Authors: Albertí S , Marqués G , Hernández-Allés S , Rubires X , Tomás J M , Vivanco F , Benedí V J
Published: January 2020

The interaction between C1q, a subcomponent of the complement classical pathway component C1, and OmpK36, a porin protein from Klebsiella pneumoniae, was studied in a solid-phase direct-binding assay, inhibition assays with the purified globular and collagen-like regions of C1q, and cross-linking experiments. We have shown that the binding of C1q to the OmpK36 porin of the serum-sensitive strain K. pneumoniae KT707 occurs in an in vivo situation and that this binding leads to activation of the complement classical pathway and the subsequent deposition of complement components C3b and C5b-9 on the OmpK36 porin. Scatchard analysis of the binding of [125I]C1q to the OmpK36 porin showed two binding sites with dissociation constants of 1.5 and 75 nM. The decrease of [125I]C1q binding to the OmpK36 porin in buffer with increasing salt concentrations and the pIs of the C1q subcomponent (10.3) and OmpK36 porin (4.5) suggest that charged amino acids are involved in the binding phenomenon. In inhibition assays, only the globular regions of C1q inhibited the interaction between C1q and OmpK36 porin, demonstrating that C1q binds to porin through its globular region and not through the collagen-like stalks.
Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease
Authors: Burns E H , Marciel A M , Musser J M
Published: January 2020

Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection.
T84 cells in culture as a model for enteroaggregative Escherichia coli pathogenesis
Authors: Nataro J P , Hicks S , Phillips A D , Vial P A , Sears C L
Published: January 2020

Enteroaggregative Escherichia coli (EAEC) is an important cause of persistent diarrhea in many developing parts of the world, yet the pathogenetic mechanisms of EAEC diarrhea are unknown. Experiments with animal models suggest that EAEC strains damage the intestinal mucosa, and a putative cytotoxin has been described. To characterize the mucosal effects of EAEC, we studied strain 042, which we have shown to cause diarrhea in adult volunteers. Strain 042 was incubated in an in vitro organ culture model with biopsy-derived normal intestinal mucosa from pediatric patients. Strain 042 adhered strongly to samples of jejunal, ileal, and colonic mucosa. In addition, scanning electron microscopic examination of in vitro-infected intestinal biopsies revealed cytotoxic effects marked by exfoliation of mucosal epithelial cells. To develop an in vitro model to study these effects, we incubated 042 with polarized monolayers of the human intestinal epithelial cell lines Caco-2 and T84. Strain 042 adhered strongly to T84 cells but not to Caco-2 cells. T84 cells infected with 042 displayed marked toxic effects, most prominently in areas where bacteria were adhering. The apical membrane of damaged cells exhibited vesiculation and shedding of microvilli. The cytoplasm of affected cells displayed subnuclear vacuolization, and in some cases, nuclei of affected cells became separated from the surrounding cytoplasm. Severely affected cells ruptured, releasing their nuclei. Vacuolated remnant cells were seen throughout the monolayer. Strain 042 was not internalized by T84 cells. We concluded that EAEC strain 042 alters intestinal cell morphology, ultimately leading to cell death. Although the factor(s) required for this effect remains to be elucidated, T84 cells may serve as a valuable model in EAEC pathogenesis studies.
Lung phagocyte bactericidal function in strains of mice resistant and susceptible to Pseudomonas aeruginosa
Authors: Morissette C , Francoeur C , Darmond-Zwaig C , Gervais F
Published: January 2020

The host response to Pseudomonas aeruginosa lung infection varies among inbred mouse strains. Mice of the BALB/c strain are resistant to P. aeruginosa lung infection, whereas mice of the DBA/2 strain are susceptible. This phenotypic variation correlates with a difference in the magnitude of the inflammatory response induced early following infection. In order to determine whether the ability of lung phagocytic cells to kill P. aeruginosa plays a role in the host response to the infection, we measured the in vitro bactericidal activity of resident and inflammatory alveolar and interstitial macrophages, using a temperature-sensitive mutant of P. aeruginosa. Lung macrophages obtained from resistant and susceptible animals displayed similar bactericidal activities, suggesting that the ability of phagocytes to kill P. aeruginosa does not play a crucial role in the outcome of infection. The bactericidal activity of lung phagocytes was also assessed in vivo following endobronchial infection with the temperature-sensitive mutant of P. aeruginosa. Resistant mice showed a rapid influx of polymorphonuclear leukocytes (PMNs) to the bronchoalveolar space which was shortly followed by an efficient clearance of the bacteria. Susceptible mice had a delay in both the inflammatory response to P. aeruginosa and the initiation of bacterial clearance. Susceptible mice have been shown to have a defect in tumor necrosis factor alpha production when infected intratracheally with P. aeruginosa. Intratracheal instillation of tumor necrosis factor alpha to susceptible mice at the time of infection significantly improved the recruitment of PMNs to the site of infection without affecting the process of bacterial clearance. Overall, these results suggest that both recruitment of a high number of PMNs to the lungs and an efficient activation process of the phagocytes are crucial for the prompt clearance of P. aeruginosa.
Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae
Authors: Chen C J , Sparling P F , Lewis L A , Dyer D W , Elkins C
Published: January 2020

The majority of in vitro-grown Neisseria gonorrhoeae strains were unable to use hemoglobin as the sole source of iron for growth (Hgb-), but a minor population was able to do so (Hgb+). The ability of Hgb+ gonococci to utilize hemoglobin as the iron source was associated with the expression of an iron-repressible 89-kDa hemoglobin-binding protein in the outer membrane. The N-terminal amino acid sequence of this protein revealed amino acids, from positions 2 to 16, identical to those of HpuB, an 85 kDa iron-regulated hemoglobin-haptoglobin utilization outer membrane protein of Neisseria meningitidis. Isogenic mutants constructed by allelic replacement with a meningococcal hpu::mini-Tn3erm construct no longer expressed the 89-kDa protein. Mutants could not utilize hemoglobin to support growth but still grew on heme. Thus, the gonococcal HpuB homolog is a functional hemoglobin receptor and is essential for growth with hemoglobin.
Human intestinal epithelial cells swell and demonstrate actin rearrangement in response to the metalloprotease toxin of Bacteroides fragilis
Authors: Koshy S S , Montrose M H , Sears C L
Published: January 2020

Enterotoxigenic Bacteroides fragilis (ETBF) cells produce a 20-kDa heat-labile metalloprotease toxin which is potentially important in the pathogenesis of diarrhea associated with this infection. Previous studies indicate that subconfluent HT29/C1 cells treated with the B. fragilis toxin (BFT) develop morphologic changes with dissolution of tight clusters and apparent swelling. Such alterations suggest toxin-stimulated reorganization of the cellular cytoskeleton. The purpose of the current study was to evaluate the effect of BFT on actin microfilaments (F-actin) and cell volume. As assessed by fluorescent phallicidin staining which detects F-actin, BFT treatment of HT29/C1 cells resulted in redistribution of F-actin with loss of stress fibers, a floccular staining pattern, and cellular membrane blebbing without quantitative changes in F-actin fluorescence intensity. The F-actin redistribution was time and concentration dependent. In contrast to the cell shrinkage observed in response to the F-actin-depolymerizing agents cytochalasin D and Clostridium difficile toxin A, BFT stimulated an increase in HT29/C1 cell volume of 10 to 25% (compared with control cells) over a 24-h time course. Only 10 to 30 ng of BFT per ml was necessary to stimulate a maximal increase in HT29/C1 cell volume. The effect of BFT on cell volume was persistent and dependent on the proteolytic activity of BFT. In agreement with cell viability assays indicating that BFT did not injure HT29/C1 cells, intoxicated cells exhibited regulatory volume decrease, suggesting that toxin-treated cells remain physiologically dynamic. We conclude that BFT acts on the intestinal epithelial cell cytoskeleton to alter F-actin structure and to stimulate an increase in HT29/C1 cell volume. Although these two activities of BFT appear to be linked, the precise sequence of cellular events following intoxication of HT29/C1 cells with BFT remains unclear. We hypothesize that these F-actin and cell volume changes may lead to an alteration in tight junction function in the polarized intestinal epithelium, contributing to the pathogenesis of diarrhea in ETBF infections.
In utero exposure to Onchocerca volvulus: relationship to subsequent infection intensity and cellular immune responsiveness
Authors: Elson L H , Days A , Calvopiña M , Paredes W , Araujo E , Guderian R H , Bradley J E , Nutman T B
Published: January 2020

Afro-Ecuadorian individuals from an area where Onchocerca volvulus is hyperendemic have been monitored for infection over the past 16 years. To determine whether in utero exposure to O. volvulus biases a child's subsequent immune responses, children (9 to 16 years old) for whom the mother's infection status was known were chosen for study. Children of infected mothers (n = 19) had significantly higher levels of skin microfilariae than children of uninfected mothers (n = 13; P = 0.021). While the serum levels of O. volvulus-specific immunoglobulin G (IgG), IgG subclasses, and IgE showed no significant differences between the two groups of children, peripheral blood mononuclear cells of children of infected mothers produced higher levels of Th2-type cytokines to several parasite antigens and lower levels of Th1-type cytokines to nonparasite antigens than those of children of uninfected mothers. Thus, in utero exposure to O. volvulus has a long-term effect on the child's subsequent cellular immune response that may render the child more susceptible to O. volvulus infection postnatally.
Migration of the fungal pathogen Candida albicans across endothelial monolayers
Authors: Zink S , Nass T , Rösen P , Ernst J F
Published: January 2020

Migration of the fungal pathogen Candida albicans across the endothelial cell layer is considered a prerequisite for the invasion of multiple organs occurring in systemic candidiasis. We developed an experimental system in which C. albicans migrates from a luminal compartment across a monolayer of bovine aortic endothelial cells on a porous filter support to an abluminal compartment. In this system, a C. albicans wild-type strain (ATCC 10261) traverses the endothelial monolayer in a time-, glucose-, and cell concentration-dependent manner. A mutant derivative unable to grow and form hyphae (SGY-243) migrates at a reduced rate. Concomitant to transendothelial migration, the permeability of the endothelial monolayer for dextran diffusion markers is significantly increased. This increase in transendothelial exchange occurs before fungal cells are detectable in the abluminal compartment and is time, glucose, and cell concentration dependent. A mutant strain (hOG301) unable to interact with endothelial cells does not alter endothelial permeability. Thus, transendothelial migration of C. albicans is able to damage the barrier function of an endothelial monolayer. Our experimental system, which reflects key stages of transendothelial migration of C. albicans including adherence and passage across endothelial cells and the extracellular matrix, may be a useful model for comparisons of transendothelial migration characteristics of Candida strains.
Secretion of type II phospholipase A2 and cryptdin by rat small intestinal Paneth cells
Authors: Qu X D , Lloyd K C , Walsh J H , Lehrer R I
Published: January 2020

We examined the secretion of antimicrobial proteins and peptides into surgically isolated and continuously perfused segments of rat small intestine. Up to nine discrete antimicrobial molecules appeared in the intestinal perfusates following intravenous administration of bethanechol, a cholinergic agonist, or intralumenal instillation of lipopolysaccharide (LPS). Among them were three markers of Paneth cell secretion: lysozyme; type II (secretory) phospholipase A2; and at least one intestinal defensin, RIP-3, that appeared to be an alternatively processed variant of the rat neutrophil defensin RatNP-3. Both bethanechol- and LPS-stimulated intestinal lumenal perfusates (washings) contained molecules that killed Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes in vitro. These molecules were more active against the avirulent S. typhimurium strain 7953S (phoP) than against its virulent parent, S. typhimurium 14028S. These data demonstrate that small intestinal Paneth cells secrete antimicrobial peptides in vivo, that this secretion is regulated by the autonomic (parasympathetic) cholinergic nervous system, and that the release of antimicrobial molecules can be triggered by the presence of bacterial LPS in the intestinal lumen.
Acute virulence in mice is associated with markers on chromosome VIII in Toxoplasma gondii
Authors: Howe D K , Summers B C , Sibley L D
Published: January 2020

Toxoplasma gondii has an unusual population structure consisting of three widely distributed clonal lineages. Acute virulence in mice is strictly observed in type I strains, indicating that a genetic determinant(s) unique to this lineage controls acute pathogenesis. We have analyzed several naturally occurring recombinant strains of T. gondii that carry allele 1 at the SAG1 locus; this allele is characteristic of the type I strains and was previously found to be 100% correlated with the acute virulence phenotype. Recombinant strains G622-M and ROD both had a predominantly type III genotype, with the significant exception of allele 1 at the SAG1 locus. Although these two strains had virtually identical multilocus genotypes, they differed in their virulence in mice. Strain ROD was virulent whereas strain G622-M was nonvirulent, thus demonstrating that the presence of allele 1 at SAG1 is not alone sufficient to confer acute virulence. Several sequence polymorphisms upstream of SAG1 were found to be highly correlated with the acutely virulent lineages. Collectively, these results suggest that acute virulence is regulated by a region linked to the SAG1 locus on chromosome VIII in T. gondii.
Lack of the extracellular 19-kilodalton fibrinogen-binding protein from Staphylococcus aureus decreases virulence in experimental wound infection
Authors: Palma M , Nozohoor S , Schennings T , Heimdahl A , Flock J I
Published: January 2020

A mutant deficient for the 19-kDa extracellular fibrinogen-binding protein (Fib) from Staphylococcus aureus has been constructed. The gene was inactivated by allele replacement. A 2.0-kb fragment from transposon Tn4001 carrying the gene for gentamicin resistance was inserted into the gene encoding Fib (fib). The genotype was verified by PCR analysis, and the loss of Fib was demonstrated by Western blotting (immunoblotting). The mutation has not altered the ability of the strain to bind to fibrinogen or fibronectin compared with that of the isogenic parental strain, FDA486. The mutant, designated K4.3, was compared with strain FDA486 in a wound infection model in rats. Sixty-eight percent of the rats challenged with parental strain FDA486 developed severe clinical signs of wound infection, whereas only 29% of the animals challenged with isogenic mutant K4.3 showed severe symptoms (P < 0.01). The weight loss of animals infected with the wild type was also significantly different from that of animals infected with the mutant strain. The result demonstrates that the extracellular 19-kDa fibrinogen-binding protein from S. aureus contributes to the virulence in wound infection and delays the healing process.
In vivo apoptosis in Shigella flexneri infections
Authors: Zychlinsky A , Thirumalai K , Arondel J , Cantey J R , Aliprantis A O , Sansonetti P J
Published: January 2020

Shigella flexneri, an etiological agent of bacillary dysentery, causes apoptosis in vitro. Here we show that it also induces apoptosis in vivo. We were able to quantify the number of apoptotic cells in rabbit Peyer's patches infected with S. flexneri by detecting cells with fragmented DNA. Infection with virulent S. flexneri results in massive numbers of apoptotic cells within the lymphoid follicles. In contrast, neither an avirulent strain nor an avirulent strain capable of colonizing Peyer's patches increases the background level of apoptotic cells. Macrophages, T cells, and B cells are shown to undergo apoptosis in vivo. These results indicate that apoptosis may play a crucial role in the pathogenesis of shigellosis.
Vesicular interactions of the Chlamydia trachomatis inclusion are determined by chlamydial early protein synthesis rather than route of entry
Authors: Scidmore M A , Rockey D D , Fischer E R , Heinzen R A , Hackstadt T
Published: January 2020

Chlamydiae replicate intracellularly within a vacuole that has recently been characterized as intersecting an exocytic pathway. One of the initial events during chlamydial infection is the expression of a chlamydial early gene product(s) that effectively isolates the inclusion from the endocytic-lysosomal pathway and makes it fusogenic with sphingomyelin-containing exocytic vesicles. Associated with this change in vesicular interaction is the delivery of the vacuole to the peri-Golgi region of the host cell. Inhibition of chlamydial early transcription or translation causes Chlamydia trachomatis-containing vesicles to remain dispersed throughout the cytoplasm, where they eventually fuse with lysosomes. Chlamydiae that have been internalized by Fc-mediated endocytosis also avoid lysosomal digestion by a mechanism that requires chlamydial protein synthesis. These results suggest that the vesicular interactions of the chlamydial inclusion are defined by parasite-directed modification of the endocytic vesicle rather than by the route of internalization.
Role of endotoxemia in cardiovascular dysfunction and lethality: virulent and nonvirulent Escherichia coli challenges in a canine model of septic shock
Authors: Hoffman W D , Danner R L , Quezado Z M , Banks S M , Elin R J , Hosseini J M , Natanson C
Published: January 2020

We investigated whether the severity of septic shock is determined by virulence factors associated with or the levels of endotoxemia produced by two Escherichia coli strains. Canines were challenged intraperitoneally with an E. coli strain (O6:H1:K2) that has virulence factors associated with human disease or with an equal dose of a nonvirulent strain (O86:H8) that lacks these factors. Both strains were administered in viable, heat-killed, and purified endotoxin forms. Median survival times with the virulent strain compared with the nonvirulent strain were shorter with viable bacteria (5 x 10(10) CFU/kg) (144 h versus > 672 h; Wilcoxon, P = 0.03), longer with heat-killed bacteria (5 x 10(9) CFU/kg) ( > 676 h versus 26 h; P = 0.03), and similar with purified endotoxin (15 mg/kg) (28 h versus 48 h; P = 0.71). However, whether the challenge contained viable bacteria, heat-killed bacteria, or purified endotoxin, the virulent strain produced less endotoxemia (P = 0.001). Hence, the changing outcomes with differing forms of the two strains cannot be attributed solely to endotoxin levels. The viable virulent strain caused less endotoxemia but more harm, and this does not appear to be explained by a more potent endotoxin or other heat-stable component. This study suggests that circulating endotoxin levels per se are less important in the outcome of septic shock than virulence factors associated with E. coli strains. Furthermore, the data call into question the significance of the endotoxin concentration in the blood in predicting the severity of shock and the lethality of gram-negative infections.
Identification of murine protective epitopes on the Porphyromonas gingivalis fimbrillin molecule
Authors: Deslauriers M , Haque S , Flood P M
Published: January 2020

Fimbriae from Porphyromonas gingivalis are believed to play an important role in the pathogenesis of periodontal diseases. The aim of the present study was to identify the fimbrial protective T-cell epitopes in CBA/J mice. A truncated protein corresponding to amino acids 1 to 198, PgF1-198, was generated and allowed us to demonstrate that the N terminus of the protein contains T-cell epitopes. With synthetic peptides, an immunodominant sequence was identified between amino acids 103 and 122. The corresponding peptide, PgF-P8, induced T-cell proliferation after in vitro restimulation of in vivo-primed cells, giving a stimulation index comparable to the one obtained with r-fimbrillin, and induced production of both Th1 and Th2 cytokines. Growth supernatant contained significant levels of interleukin 2 (IL-2), gamma interferon, IL-4 (28 pg/ml), and tumor necrosis factor alpha. Immunization of mice with r-fimbrillin, PgF1-198, and PgF-P8 induced production of antibodies specific to r-fimbrillin and PgF-P8. In addition, by using the mouse chamber model we found that mice immunized with PgF-P8 were dramatically protected against a normally lethal injection of P. gingivalis. Animals immunized with PgF-P8 40 days prior to challenge showed a 60% survival rate when challenged with P. gingivalis, compared with just 25% survival in control animals and just 5% survival in mice immunized with PgF-P8 only 21 days prior to challenge. Although the protection depended on the time of immunization before the bacterial challenge, it did not correlate with in vivo local cytokine production (IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon), specific antibody levels, or the isotype of anti-PgF-P8 antibodies produced.
Influence of monocytes and antibiotic treatment on tissue factor activity of endocardial vegetations in rabbits infected with Streptococcus sanguis
Authors: Bancsi M J , Veltrop M H , Bertina R M , Thompson J
Published: January 2020

A main feature in the pathogenesis of bacterial endocarditis is the activation of the coagulation system via the extrinsic pathway, resulting in the formation of infected endocardial vegetations. Earlier studies gave indirect evidence that monocytes play an important role in the procoagulant response during the course of the disease. In this study, we assessed the role of monocytes more directly. We compared weights and tissue factor activities (TFA) of endocardial vegetations of normal rabbits infected with Streptococcus sanguis with those of rabbits which were treated with the cytostatic drug etoposide (Vepesid; Bristol-Myers Squibb B.V.) to induce a selective monocytopenia. Furthermore, the importance of the presence of bacteria was determined through the influence of antibiotic treatment on TFA, vegetational weight, and infection of the vegetations. The TFA of the vegetations was measured chromogenically by monitoring the factor VII-dependent activation of factor X with an amidolytic assay for factor Xa. We found that the degree of infection and the weight of vegetations of rabbits treated with the cytostatic drug etoposide did not differ from that of untreated rabbits. Their TFA, however, was significantly lower than the TFA of vegetations of rabbits not treated with etoposide. We also found that, as with the monocytopenic rabbits, the weight of the vegetations was not reduced in penicillin G-treated rabbits. The degree of infection and TFA, however, were significantly lower. We conclude that monocytes indeed are involved in the activation of the coagulation system during the course of bacterial endocarditis and that the degree of infection is positively correlated to the TFA of the vegetations.
Physical linkage of the Vibrio cholerae mannose-sensitive hemagglutinin secretory and structural subunit gene loci: identification of the mshG coding sequence
Authors: Marsh J W , Sun D , Taylor R K
Published: January 2020

Vibrio cholerae O1 expresses a variety of cell surface factors which mediate bacterial adherence and colonization at the intestinal epithelium. The mannose-sensitive hemagglutinin (MSHA), a type IV pilus, is a potential attachment factor of the V. cholerae El Tor biotype. We describe a TnphoA mutant that is defective in its ability to hemagglutinate mouse erythrocytes. The TnphoA insertion maps to a recently identified genetic locus that encodes products that are predicted to be essential for assembly and export of the MSHA pilus. Insertional disruption at this locus in a mshA-phoA reporter strain provides evidence for a role of this locus in the latter stages of pilus assembly and/or export. These constructs have provided physical markers by which we have established close physical linkage of this secretion locus to a set of genes that includes the mshA structural gene. Sequence analysis of the intervening region between these two loci has revealed the presence of an open reading frame with homology to pilus biogenesis genes of several gram-negative bacteria. This genetic organization suggests an entire operon encoding the MSHA pilus and the components necessary for its assembly and secretion to the bacterial cell surface. The nomenclature of the MSHA structural and secretory locus has been redefined accordingly.
Pyoverdin is essential for virulence of Pseudomonas aeruginosa
Authors: Meyer J M , Neely A , Stintzi A , Georges C , Holder I A
Published: January 2020

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.
Furin regulates both the activation of Pseudomonas exotoxin A and the Quantity of the toxin receptor expressed on target cells
Authors: Gu M , Gordon V M , Fitzgerald D J , Leppla S H
Published: January 2020

Pseudomonas exotoxin A (PE) binds and enters mammalian cells via the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). The toxin then requires proteolytic cleavage to generate an enzymatically active fragment with translocates to the cell cytosol and inhibits protein synthesis. To assess the role of furin in determining toxin susceptibility, CHO cells were transfected with a mouse furin gene (CHO+fur cells) and maintained under neomycin selection. Cells expressing the transfected gene were about two- to threefold more sensitive to PE than were cells expressing only a neomycin resistance gene (CHO+neo cells). Possible reasons for the increased toxin sensitivity include the cleavage of a greater number of PE molecules and/or the conversion of more single-chain LRP to the processed, two-chain form. Processing of LRP appears to be necessary to allow the surface display of this receptor. Results of ligand binding studies indicated that the CHO+fur cells displayed about twofold more surface-expressed LRP than did CHO+neo cells. In addition, the in vitro cleavage of PE by recombinant furin enhanced toxin potency about threefold for CHO+neo cells but enhanced it very little for CHO+fur cells. This suggested that CHO+fur cells were processing PE at close to the maximum usable rate. Together these findings suggest that furin is involved in at least two separate protein processing pathways that each contribute to the sensitivity of cells to PE.
A Th1-associated increase in tumor necrosis factor alpha expression in the spleen correlates with resistance to blood-stage malaria in mice
Authors: Jacobs P , Radzioch D , Stevenson M M
Published: January 2020

We investigated the kinetics of tissue-specific mRNA expression and systemic production of tumor necrosis factor alpha (TNF-alpha) and the kinetics of splenic expression of mRNAs of gamma interferon (INF-gamma) and interleukin-4 (IL-4), cytokines that may regulate TNF-alpha production, during the early phase of blood-stage infection with Plasmodium chabaudi AS. Northern blot analysis revealed that resistant C57BL/6 mice, which clear the infection by 4 weeks, had higher levels of TNF-alpha mRNA in the spleen and liver early during infection that did susceptible A/J mice, which succumb to the disease 10 days after initiation of infection. Treatment of resistant mice with a polyclonal anti-TNF-alpha antibody confirmed the protective role of TNF-alpha early during the course of infection. Furthermore, resistant C57BL/6 mice also expressed high levels of mRNA of IFN-gamma (a Th1 marker) and low levels of mRNA of IL-4 (a Th2 marker) in the spleen, whereas susceptible A/J mice had low levels of IFN-gamma mRNA but high levels of TNF-alpha mRNA in the liver and had high levels of TNF-alpha protein in serum, as measured by enzyme-linked immunosorbent assay, later during infection just before death occurred. These results demonstrate that a Th1-associated increase in TNF-alpha mRNA expression in the spleen early during infection correlates with resistance to P. chabaudi AS, whereas increased TNF-alpha mRNA levels in the liver and excessive levels of the TNF-alpha protein in serum later during infection correlate with susceptibility. Thus, the role of the TNF-alpha during malaria appears to depend on the timing and site of its expression and the presence of cytokines regulating its production.
Mycoplasma membrane lipoproteins induced proinflammatory cytokines by a mechanism distinct from that of lipopolysaccharide
Authors: Rawadi G , Roman-Roman S
Published: January 2020

To gain a clear understanding of the mechanisms by which mycoplasmas induced the expression of proinflammatory cytokines in monocytic cells, we have studied the induction of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, and IL-6 by mycoplasmas in three distinct human myelomonocytic cell lines in comparison with induction by lipopolysaccharide (LPS). HL-60 cell line did not release cytokines when induced with either LPS or mycoplasmas. In contrast to LPS, mycoplasmas failed to increase the weak levels of tumor necrosis factor alpha secreted by phorbol myristate acetate-differentiated U937 cells. In addition, Northern (RNA) blot analysis of cytokine expression in these cells showed that the induction of IL-1 beta by mycoplasmas involves, unlike that by LPS, posttranscriptional events. Interestingly, in THP-1 cells, cytokine induction pathways triggered by mycoplasmas remained operational under conditions where LPS pathways were abolished, suggesting functional independence. The study of cytokine-inducing activity displayed by distinct fractions derived from a series of different mycoplasma species demonstrated that lipid membrane constituents were largely responsible for these effects. Finally, we have demonstrated that tyrosine phosphorylation is a crucial event in the mycoplasma-mediated induction of proinflammatory cytokines in either THP-1 cells or human monocytes.
Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion
Authors: Bozue J A , Johnson W
Published: January 2020

Legionella pneumophila is a facultative intracellular parasite able to survive within both human monocytes and amoebae. We have demonstrated that processing of L. pneumophila by the free-living amoeba Acanthamoeba castellanii shows many similarities to the processing of L. pneumophila by monocytes. These similarities include uptake of L. pneumophila by coiling phagocytosis and the subsequent confinement of L. pneumophila in a ribosome-studded phagosome. In addition, as in monocytes, inhibition of lysosomal fusion with phagosomes containing L. pneumophila was detected in amoebae. With all clinical isolates, inhibition of phagosomes-lysosome fusion correlated with virulence. However, with one of the environmental isolates tested, no significant difference in phagosome-lysosome fusion was observed between the virulent and avirulent forms. These results indicate that the avirulent form of this isolate differed from the virulent form in some other respect critical to intracellular survival. Therefore, intracellular multiplication of L. pneumophila within A. castellanii may not be solely dependent upon the inhibition of lysosomal fusion.
Identification and purification of novel internalin-related proteins in Listeria monocytogenes and Listeria ivanovii
Authors: Lingnau A , Chakraborty T , Niebuhr K , Domann E , Wehland J
Published: January 2020

Monoclonal antibodies were generated against a 30-kDa protein fraction derived from culture supernatants of a Listeria monocytogenes strain complemented with additional copies of the prfA regulator gene. Several of the antibodies reacted specifically with a hitherto unidentified, secreted 30-kDa polypeptide. By immunoblot analysis, the expression of this 30kDa polypeptide was found to be dependent on the presence of the PrfA regulator protein. Microsequencing of peptides derived from the partially purified 30-kDa protein revealed homologies to the InlA and InlB polypeptides of L. monocytogenes, which are required for the internalization of the bacteria into nonphagocytic cell lines. This prompted us to term the 30-kDa polypeptide internalin-related protein (Irp). Irp-specific monoclonal antibodies cross-reacted with a 24-kDa polypeptide present in culture supernatants of Listeria ivanovii, indicating the existence of an Irp-related protein in this pathogenic Listeria species.
Platelet-activating-factor-mediated pathogenesis in Lyme disease
Authors: Isogai E , Kimura K , Fujii N , Nishikawa T , Ishii N , Postic D , Baranton G , Isogai H
Published: January 2020

This study describes a role of platelet-activating factor (PAF) as a potential inducer of inflammation in infection with Borrelia burgdorferi. Two approaches were taken. The first involved the use of a PAF antagonist to show the lack of an inflammatory response in skin lesions. The second was to show that the PAF antagonist reduced platelet aggregation when the spirochetes were incubated with polymorphonuclear leukocytes.
The capsular polysaccharide complex of Bacteroides fragilis induces cytokine production from human and murine phagocytic cells
Authors: Gibson F C , Tzianabos A O , Onderdonk A B
Published: January 2020

To stimulate early-phase immunologic events following Bacteroides fragilis infection in the peritoneal cavity, we examined the cytokine response of several cell types to purified capsular polysaccharide complex (CPC) and lipopolysaccharide (LPS) of this organism. Cytokines were produced from murine resident peritoneal (MRP) cells as well as human peripheral blood leukocytes. MRP cells cocultured with either B. fragilis CPC of LPS in vitro produced tumor necrosis factor alpha and interleukin-1alpha (IL-1alpha). In addition, MRP cells challenged with CPC produced IL-10. Human peripheral blood monocytes and polymorphonuclear leukocytes secreted IL-8 when cultured in the presence of CPC.
Immunoglobulin A response in murine schistosomiasis: stimulatory role of egg antigens
Authors: Poulain-Godefroy O , Gaubert S , Lafitte S , Capron A , Grzych J M
Published: January 2020

The immunoglobulin G (IgG) and IgA antibody responses to different Schistosoma mansoni antigens have been determined in chronically infected mice as well as in unisexually infected animals. With a panel of enzyme-linked immunosorbent assays (ELISAs), soluble antigens from furcocercariae, adult worms, and eggs were probed with sera collected at 3-week intervals. Bisexually infected animals developed significant IgG and IgA antibody responses to the antigens tested, which increased after egg deposition. In unisexual infections no significant differences were recorded in the IgG antibody profile for furocercaria and adult worm antigens, whereas the IgA antibody response was impaired. Both the IgA and IgG antibody responses toward egg antigens were reduced compared with those in a bisexual infection. Furthermore, a specific mucosal IgA antibody response was observed only in the bisexually infected animals. Histological analysis performed on bisexually infected mice led to the observation of eggs and granulomatous lesions within the Peyer's patch follicles, which are essential sites for the induction of mucosal immunity in the intestine. These data suggest a relationship between egg deposition and the induction of the IgA antibody response toward schistosomes.
Paclitaxel (Taxol)-induced NF-kappaB translocation in murine macrophages
Authors: Perera P Y , Qureshi N , Vogel S N
Published: January 2020

Interaction of bacterial lipopolysaccharide (LPS) with macrophages results in the induction of a cascade of cytokines that mediate the varied effects of LPS. An early intracellular signaling event that follows receptor engagement is the activation of transcription factor NF-kappaB. Nf-kappaB has been shown to be important for the induction of many LPS-inducible cytokine genes, including tumor necrosis factor alpha, interleukin-1beta, and interleukin-6. Previously, we and others have shown that the antitumor agent paclitaxel (Taxol) is able to mimic bacterial LPS in its ability to activate murine macrophages. In this report, we have extended these findings by demonstrating that paclitaxel, like LPS, is able to stimulate the translocation of primarily p50-p65 heterodimers of NF-kappaB to the nucleus. This activation is dose dependent and requires a concentration of > or =5 microM paclitaxel. The kinetics of NF-kappaB activation by paclitaxel are slower than those of LPS: by 15 min poststimulation, LPS-induced NF-kappaB activation was readily detected, whereas the paclitaxel-induced NF-kappaB activation was minimal. Moreover, paclitaxel- and protein-free LPS-induced translocation of NF-kappaB was seen only in macrophages derived from LPS-responsive C3H/OuJ mice and not from the LPS-hyporesponsive C3H/HeJ mice, a finding that is consistent with those of previous genetic studies linking paclitaxel responsiveness to the Lps gene. Finally, the LPS structural antagonist Rhodobacter sphaeroides diphosphoryl lipid A inhibited both LPS-and paclitaxel-induced NF-kappaB activation, suggesting a common receptor component in this activation.
A recombinant protein based on the Trypanosoma cruzi metacyclic trypomastigote 82-kilodalton antigen that induces and effective immune response to acute infection
Authors: Santori F R , Paranhos-Bacalla G S , Franco DA Silveira J , Yamauchi L M , Araya J E , Yoshida N
Published: January 2020

To further investigate the immunological properties of the stage-specific 82-kDa glycoprotein (gp82) of Trypanosoma cruzi metacyclic trypomastigotes, previously shown to induce antigen-specific humoral and T-cell responses in mice, we performed a series of experiments with recombinant proteins containing sequences of gp82 fused to glutathione S-transferase. Of five fusion proteins tested, only J18b and J18b1, the carboxyproximal peptides containing amino acids 224 to 516 and 303 to 516, respectively, were recognized by monoclonal antibody 3F6 as well as by various anti-T. cruzi antisera and, when administered to mice, were capable of eliciting antibodies directed to the native gp82. The amino-terminal peptide and other carboxyterminal recombinant proteins lacking the central domain of gp82 (amino acids 224 to 356), which is exposed on the surface of live metacyclic forms, did not display any of these properties. Spleen cells derived from mice immunized with any of the five recombinant proteins proliferated in vitro in the presence of native gp82.J18b was the most stimulatory, whereas J18b3, the peptide containing amino acids 408 to 516, elicited the weakest response. When BALB/c mice immunized with J18b antigen plus A1(OH)3 as adjuvant were challenged 10 5 metacyclic trypomastigotes, 85% of them resisted acute infection, in comparison with control mice that received glutathione S-transferase plus adjuvant. Antibodies induced by J18b protein lacked agglutinating or complement-dependent lytic activity and failed to neutralize parasite infectivity. On the other hand, CD4+T cells from the spleens of J18b-immunized mice displayed an intense proliferative activity upon stimulation with 1.25 microgram of native gp82 per ml, which resulted in increased production of gamma interferon, a cytokine associated with resistance to T. cruzi infection.
Differential production of interleukin-12 mRNA by murine macrophages in response to viable or killed Salmonella spp
Authors: Chong C , Bost K L , Clements J D
Published: January 2020

The use of attenuated Salmonella spp. as live oral vaccine carriers fo r foreign antigens has been extensively studied. We have shown that appropriately prepared nonviable organisms are as effective as viable organisms in eliciting humoral immune responses against a foreign antigen delivered by these vectors. It is not clear how strain viability affects the development of a cell-mediated immune response. In the present study, we demonstrate that BALB/c mice orally immunized with viable attenuated Salmonella spp. were protected against subsequent challenge while animals immunized with killed organisms were not. Protection was correlated with increased production of interleukin-12 (IL-12) p40 mRNA in the Peyer's patches within hours of oral administration. Peritoneal macrophages from lipopolysaccharide (LPS)-responsive and LPS-unresponsive mice were also examined for production of IL-12 p40 mRNA following exposure to the viable or killed attenuated Salmonella carrier. There was dramatic upregulation of IL-12 p40 mRNA following exposure of macrophages to either viable or killed organisms. By 4 h postexposure, viable organisms had induced a 27-fold increase in IL-12 p40 mRNA levels while killed organisms had induced a 9-fold increase in IL-12 p40 mRNA levels. This was observed in macrophages isolated from both LPS-responsive and unresponsive mice. The higher levels of IL-12 induced by viable Salmonella spp. may result in the development of a Th1 response and cell mediated immunity, while the lower levels of IL-12 induced by killed Salmonella spp. may not be sufficient to promote a Th1 response.
Antigen recognition during progression from acute to chronic infection with a cagA-positive strain of Helicobacter pylori
Authors: Mitchell H M , Hazell S L , Kolesnikow T , Mitchell J , Frommer D
Published: January 2020

We have previously published two reports on acute infection with Helicobacter pylori, one of an adult male and one of a family of four. In the present study, we have isolated H.pylori from each of twin boys in the family and compared these by use of random amplified polymorphic DNA PCR. In addition, we have monitored the antibody response over time of the family and the adult male by Western blotting (immunoblotting) with two different strains of H. pylori as the antigen and by use of a commercial kit. The acutely infected twin boys were infected by an identical strain of H. pylori. The twin boys responded to antigens of 19, 26.5, and 29 kDa 30 days after the initial diagnosis, with recognition of 43-to 49-, 66-, 69-, and 87-kDa antigens by day 63. One twin responded to the CagA antigen on day 63, whereas the other responded on day 857. Antibody to the CagA antigen was not detected by use of the infecting strain, UNSW-RU1. Investigation of UNSW-RU1 revealed the presence of cagA. In two acutely infected adults (one, the father of the boys), the initial response to a 45-kDA antigen was later followed by responses to 19-, 29-, 49-, 60-, 77-, and 84-kDa antigens. Sera from the twins' younger sister, born 17 months after the twins acute episode, indicated that she also had become infected. This report supports intrafamilial transmission of H. pylori. Initial antibody responses in the children were to small-molecular-size antigens; in the adults, the initial responses were to larger-molecular-size antigens. The pattern of the serological response differs according to the antigen used. This has implications in regard to international data comparisons.