bluuebunny/crossref_metadata_2025 huggingface dataset:
Listeria monocytogenes infects human endothelial cells by two distinct mechanisms
Authors: Drevets D A , Sawyer R T , Potter T A , Campbell P A
Published: January 2020

Infection of endothelial cells by bacteria may be an important component of the bacteria's ability to escape host defenses and cause disease. Listeria monocytogenes cause sepsis and central nervous system infection in domesticated animals and immunocompromised humans, suggesting that this bacterium interacts with endothelial cells in a significant fashion. The experiments presented here tested the hypothesis that L. monocytogenes can invade and replicate within human endothelial cells. We found that L. monocytogenes grows readily in umbilical vein endothelial cells and that its intracellular life cycle involves phagosomal escape, F-actin-based motility, and cell-to-cell spread. We found that L. monocytogenes invaded endothelial cells by cell-to-cell spread from adherent mononuclear phagocytes which were previously infected by this bacterium. Interestingly, L. monocytogenes mutants lacking the invasion protein, internalin, bound less well to endothelial cells than did wild-type bacteria in the absence, but not the presence, of serum, and their invasion of endothelial cells was diminished under both conditions. Thus, endothelial cell infection by L. monocytogenes can occur by two distinct mechanisms: direct bacterial invasion of the endothelial cells in an internalin-mediated fashion or cell-to-cell spread from adherent, infected mononuclear phagocytes. These data support the idea that endothelial cell infection by L. monocytogenes is an important event in the pathogenesis of listeriosis.
Adherence of Streptococcus pneumoniae to immobilized fibronectin
Authors: van der Flier M , Chhun N , Wizemann T M , Min J , McCarthy J B , Tuomanen E I
Published: January 2020

Adherence to extracellular matrix proteins, such as fibronectin, affords pathogens with a mechanism to invade injured epithelia. Streptococcus pneumoniae was found to adhere to immobilized fibronectin more avidly than other streptococci and staphylococci do. Binding was dose, time, and temperature dependent. Trypsin treatment of the bacteria resulted in decreased binding, suggesting that the bacterial adhesive component was a protein. Fragments of fibronectin generated by proteolysis or by expression of recombinant gene segments were compared for the ability to bind pneumococci and to compete against bacterial binding to immobilized fibronectin. Fragments from the carboxy-terminal heparin binding domain were consistently active, suggesting that this region contains the pneumococcal binding site, a region distinct from that supporting the attachment of most other bacteria.
Molecular characterization of a carboxy-terminal eukaryotic-cell-binding domain of intimin from enteropathogenic Escherichia coli
Authors: Frankel G , Candy D C , Fabiani E , Adu-Bobie J , Gil S , Novakova M , Phillips A D , Dougan G
Published: January 2020

A eukaryotic cell-binding domain from the intimin (Int) polypeptide of enteropathogenic Escherichia coli O127 (EPEC) was investigated. Derivatives of the carboxy-terminal 280-amino-acid domains of Int (Int-EPEC280) and the Int homolog invasin (Inv) from Yersinia pseudotuberculosis (InvYP280) were fused to the E. coli maltose-binding protein (MBP), expressed, and purified. The smallest MBP-IntEPEC fusion protein that efficiently mediated binding to HEp-2 cells, monitored by using purified fusion proteins in fluorescence activated cell sorter analysis or by using fluorescent Covaspheres coated with purified fusions, contained the carboxy-terminal 150 amino acids of Int. Replacement of Cys-937 with Ser (IntEPEC280CS) destroyed the cell-binding activity of IntEPEC280. Covaspheres coated with MBP-IntEPEC280 were associated with HEp-2 cell microvilli but failed to induce actin accumulation underneath bound particles or cell spreading on coated plastic surfaces. MBP-IntEPEC280, but not MBP, MBP-IntEPEC280CS, or MBP-InvYP280, inhibited EPEC entry into HEp-2 cells.
Onchocerca volvulus larval antigen, OvB20, induces partial protection in a rodent model of onchocerciasis
Authors: Taylor M J , Abdel-Wahab N , Wu Y , Jenkins R E , Bianco A E
Published: January 2020

OvB20 is an antigen of Onchocerca volvulus preferentially recognized by sera from cattle vaccinated with irradiated infective larvae of Onchocerca lienalis. Antibodies raised against the recombinant protein were used to characterize the expression of the native protein in different developmental stages of O. volvulus and the rodent filaria Acanthocheilonema viteae. In O. volvulus, antibodies reacted to a polypeptide of 42 kDa in microfilariae and with proteins of 52 and 65 kDa in third-stage larvae. No products were detected in adult stages. Immunogold electron microscopy localized the native protein to discrete patches of the hypodermis and cuticle of infective larvae. Characterization of a homologous protein in A. viteae confirmed the stage-specific expression in infective larvae of the 65-kDa protein, which was secreted during in vitro culture. Vaccination of rodents against A. viteae with a B20-maltose-binding-protein fusion protein resulted in a 49 to 60% reduction in adult worm recoveries with a corresponding 97% reduction in microfilaremia.
Attenuation of gamma interferon-induced tyrosine phosphorylation in mononuclear phagocytes infected with Leishmania donovani: selective inhibition of signaling through Janus kinases and Stat1
Authors: Nandan D , Reiner N E
Published: January 2020

The induction of gene transcription in response to gamma interferon is impaired in mononuclear phagocytes infected with Leishmania donovani, and the mechanisms involved are not fully understood. The changes in gene expression brought about by gamma interferon are thought to involve transient increases in the activities of cellular protein tyrosine kinases, including the Janus kinases Jak1 and Jak2, leading to tyrosine phosphorylation of the transcription factor Stat1. To investigate the mechanisms accounting for the impaired responses to gamma interferon, a model system for examining overall changes in protein tyrosine phosphorylation, activation of Jak1 and Jak2 and phosphorylation of Stat1 was developed in phorbol 12-myristate 13-acetate-differentiated U-937 cells. Analysis of whole-cell lysates by antiphosphotyrosine immunoblotting showed that incubation with gamma interferon brought about specific increases in phosphotyrosine labeling of several proteins. Increased labeling of these proteins occurred to similar extents in control cells and in cells that had been infected with L. donovani for 16 h. Jak1, Jak2, and Stat1 were immunoprecipitated from control and interferon-treated cells, and tyrosine phosphorylation of these proteins, detected by antiphosphotyrosine immunoblotting was used to measured their activation. Tyrosine phosphorylation of Jak1, Jak2, and Stat1 increased markedly, in a dose-dependent manner, in U-937 cells incubated with gamma interferon. In contrast, in cells infected with L. donovani, tyrosine phosphorylation of Jak1, Jak2, and Stat1 was markedly impaired. This effect was dependent upon the duration of exposure to L. donovani and was maximal and complete at 16 h. Results similar to those observed with U-937 cells were also obtained with human peripheral blood monocytes. These findings indicate that infection of human mononuclear phagocytes with L. donovani leads to impaired gamma interferon-mediated tyrosine phosphorylation and selective effects on the Jak-Stat1 pathway. Unresponsiveness to gamma interferon for activation of this pathway may explain impaired transcriptional responses in leishmania-infected cells.
Changes to the ocular biota with time in extended- and daily-wear disposable contact lens use
Authors: Stapleton F , Willcox M D , Fleming C M , Hickson S , Sweeney D F , Holden B A
Published: January 2020

Gram-negative bacteria may play a role in the etiology of certain soft contact lens (SCL)-related diseases. Contact lens (CL) wear may modify the normal ocular biota, providing a more favorable environment for potential pathogens. This study reports temporal changes in ocular biota in daily-wear (DW) and extended-wear (EW) disposable SCL use in experienced and neophyte wearers. Lid margin and bulbar conjunctival biota were sampled prior to CL fitting in 26 previous DW SCL users, 18 previous EW SCL users, and 26 neophytes. Wearers were fitted with an etafilcon A CL in one eye and a polymacon CL in the fellow eye. Lenses were worn on a daily basis by the 26 previous DW SCL wearers and on an EW basis by the remaining 44 subjects. The ocular biota was further sampled after 1, 3, 6, 9, and 12 months of wear. The ocular biota consisted of coagulase-negative staphylococci, Corynebacterium spp., Micrococcus spp., and Propionibacterium spp. Potential pathogens were rarely isolated at baseline. No significant trend of increasing ocular colonization was shown for extended CL wear. Lid and conjunctival colonization increased with DW SCL use (P < 0.001), although this increase occurred for nonpathogenic species only. Fewer potential pathogens were isolated from DW SCL than from EW SCL users (P < 0.05). The lid margin consistently showed greater colonization than the conjunctiva and may be a source of potential pathogens during CL wear. Hydrogel CL wear appears to modify the ocular biota. An increased number of commensal organisms were present in DW SCL use. EW SCL use altered the spectrum of organisms isolated. These alterations may suppress the normal ocular defense mechanisms and may be relevant in the pathogenesis of CL-related disease.
Binding and accumulation of hemin in Neisseria gonorrhoeae
Authors: Desai P J , Nzeribe R , Genco C A
Published: January 2020

The ability to utilize hemin and hemin-containing compounds for nutritional iron (Fe) uptake has been documented for several pathogenic bacteria. Neisseria gonorrhoeae can utilize free hemin as a source of Fe for growth; however, little is known concerning the mechanisms involved in hemin transport. In this study we have characterized the binding and accumulation of hemin by N. gonorrhoeae and defined the specificity of the gonococcal hemin receptor. N. gonorrhoeae F62 was grown in a chemically defined medium containing the iron chelator Desferal, and hemin transport was initiated by the addition of [59Fe]hemin (4.0 or 8.0 microM; specific activity, 7.0 Ci/mol). 59Fe uptake from radiolabeled hemin by N. gonorrhoeae was energy dependent, and 59Fe was shown to accumulate in the cell at a constant rate during logarithmic growth. However, we observed a decrease in the uptake of 59Fe from radiolabeled hemin when inorganic iron was present in the growth medium. Binding of 59Fe from radiolabeled hemin was inhibited by the addition of either cold hemin, hematoporphyrin, or hemoglobin, but not by ferric citrate. Although [14C]hemin was found to support the growth of N. gonorrhoeae, we did not detect the uptake of 14C from radiolabeled hemin. Extraction of the gonococcal periplasmic ferric binding protein (Fbp) from cultures grown with [59Fe]hemin indicated that a majority of the 59Fe was associated with the Fbp. Taken together, the results presented here indicate that hemin binds to a gonococcal outer membrane receptor through the protoporphyrin portion of the molecule and that following binding, iron is removed and transported into the cell, where it is associated with the gonococcal periplasmic ferric binding protein, Fbp.
Gene knockout mice establish a primary protective role for major histocompatibility complex class II-restricted responses in Chlamydia trachomatis genital tract infection
Authors: Morrison R P , Feilzer K , Tumas D B
Published: January 2020

Mice with disrupted beta 2-microglobulin (beta 2m-/-), I-A (class II-/-), or CD4 (CD4-/-) genes were examined for their capacity to resolve Chlamydia trachomatis genital tract infection. C57BL/6 and beta 2m-/- mice resolved infection similarly and were culture negative by 4 to 5 weeks following infection. Conversely, major histocompatibility complex (MHC) class II-/- mice failed to resolve infection, and CD4-/- mice showed a significant delay (2 weeks). Secondary challenge of C57BL/6, beta 2m-/-, and CD4-/- mice established that acquired protective immunity, which was characterized by an infection of shortened duration and reduced shedding of infectious organisms, developed. Serological analysis of C57BL/6 and beta 2m-/- mice by enzyme-linked immunosorbent assays revealed no striking differences in the immunoglobulin subclass specificity of the anti-Chlamydia response, although some differences were observed in the magnitude of the immunoglobulin G2a (IgG2a) and IgG2b responses. Class II-/- mice produced lower-titered serum anti-Chlamydia antibodies of all isotypes. The serum antibody responses of CD4-/- mice were similar to those of C57BL/6 mice, except that the anti-Chlamydia IgA response was delayed by approximately 3 weeks. Analysis of vaginal washes for Chlamydia-reactive antibodies revealed the presence of IgG2a, IgG2b, and IgA in C57BL/6 and beta 2m-/- mice and primarily of IgA in CD4-/- mice. Vaginal washes from class II-/- mice were consistently antibody negative. Interestingly, the Chlamydia-specific IgA response in the vaginal washes of CD4-/- mice was delayed, but its appearance coincided with decreased shedding of infectious organisms and resolution of infection. Our results demonstrate that MHC class II-restricted T-cell responses are necessary for the development of protective immunity to Chlamydia genital tract infection and that local (vaginal) anti-Chlamydia IgA antibody coincides with the resolution of infection. A substantive role for MHC class I-restricted T-cell responses in protective immunity to Chlamydia genital tract infection was not confirmed.
Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis
Authors: Moreillon P , Entenza J M , Francioli P , McDevitt D , Foster T J , François P , Vaudaux P
Published: January 2020

The pathogenic role of staphylococcal coagulase and clumping factor was investigated in the rat model of endocarditis. The coagulase-producing and clumping factor-producing parent strain Staphylococcus aureus Newman and a series of mutants defective in either coagulase, clumping factor, or both were tested for their ability (i) to attach in vitro to either rat fibrinogen or platelet-fibrin clots and (ii) to produce endocarditis in rats with catheter-induced aortic vegetations. In vitro, the clumping factor-defective mutants were up to 100 times less able than the wild type strain to attach to fibrinogen and also significantly less adherent than the parents to platelet-fibrin clots. Coagulase-defective mutants, in contrast, were not altered in their in vitro adherence phenotype. The rate of in vivo infection was inoculum dependent. Clumping factor-defective mutants produced ca. 50% less endocarditis than the parent organisms when injected at inoculum sizes infecting, respectively, 40 and 80% (ID40 and ID80, respectively) of rats with the wild-type strain. This was a trend at the ID40 but was statistically significant at the ID80 (P < 0.05). Coagulase-defective bacteria were not affected in their infectivity. Complementation of a clumping factor-defective mutant with a copy of the wild-type clumping factor gene restored both its in vitro adherence and its in vivo infectivity. These results show that clumping factor plays a specific role in the pathogenesis of S. aureus endocarditis. Nevertheless, the rate of endocarditis with clumping factor-defective mutants increased with larger inocula, indicating the contribution of additional pathogenic determinants in the infective process.
Borrelia burgdorferi vesicle production occurs via a mechanism independent of immunoglobulin M involvement
Authors: Shoberg R J , Thomas D D
Published: January 2020

Borrelia burgdorferi produces extracellular vesicles containing various borrelial protein antigens when propagated in vitro in culture media. Commonly observed components of borrelial vesicle preparations are borrelial surface antigens, bovine serum albumin, and the heavy chains of rabbit immunoglobulin G and immunoglobulin M. This study employed ultracentrifugation to harvest borrelial vesicles and analyzed these preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. We demonstrated that the rabbit mu heavy-chain band observed was devoid of OspA or at most levels below those detectable by immunoblot. We also demonstrated the recovery of borrelial vesicles at relative centrifugal forces as low as 25,000 x g, compared with the force of > 200,000 x g normally employed. Further, the mu heavy-chain band was recovered from uninoculated growth media processed at 25,000 x g, suggesting that it behaves as a particle rather than as a soluble molecule under these conditions. Lastly, vesicles were demonstrated to be present in preparations harvested from growth media supplemented with fetal calf serum, suggesting that vesicle production by B. burgdorferi can occur in the absence of immunoglobulins.
Effect of free and vesicle-bound cysteine proteinases of Porphyromonas gingivalis on plasma clot formation: implications for bleeding tendency at periodontitis sites
Authors: Imamura T , Potempa J , Pike R N , Moore J N , Barton M H , Travis J
Published: January 2020

Infection by Porphyromonas gingivalis is strongly associated with adult periodontitis, with proteinases from this bacterium now considered to be important virulence factors. In order to investigate possible pathological functions of these enzymes, we examined the effect of both free and vesicle-bound forms of the two major cysteine proteinases (gingipains) of P. gingivalis on plasma clot formation by using thrombin time (TT) measurements. Both Lys-gingipain (gingipain-K) and Arg-gingipain (gingipain-R) prolonged plasma TT in a dose- and time-dependent manner, and this was also found with vesicles which are the biological carriers of P. gingivalis proteinases. The increase in plasma TT by vesicles could be completely reversed by treatment with nonspecific cysteine proteinase inhibitors but only partially by compounds selective for either gingipain-K or gingipain-R. Preincubation of vesicles with a gingipain-K-specific inhibitor (z-FK-ck) reduced plasma TT more than a gingipain-R-specific inhibitor (leupeptin), suggesting that under physiological conditions gingipain-K was more effective in fibrinogen destruction. Each purified enzyme also markedly increased fibrinogen TT, gingipain-R being fourfold more potent than gingipain-K. However, in plasma, gingipain-R was ineffective because of the inhibitory effect of albumin. These results imply that cysteine proteinases, especially gingipain-K, abrogate the clotting potential of fibrinogen and, therefore, may contribute to the bleeding tendency and to persistent inflammation in periodontitis sites infected with P. gingivalis.
A tissue culture bilayer model to study the passage of Neisseria meningitidis
Authors: Birkness K A , Swisher B L , White E H , Long E G , Ewing E P , Quinn F D
Published: January 2020

A tissue culture bilayer system has been developed as a model to study the mechanisms of attachment and invasion involved in the pathogenesis of Neisseria meningitidis. The model incorporates epithelial and endothelial cell layers separated by a microporous membrane and makes it possible to observe and quantify the passage of bacteria through the multiple layers and to study the mechanisms by which they make this passage. This model is adaptable to a wide variety of microbial pathogens and can be modified by substituting any physiologically relevant eucaryotic cells for the component layers. The system's makeup of cells of human origin and its reproducibility give it advantages over animal and primary organ culture models, while the added complexity of multiple layers allowing cell-to-cell communication makes it a more realistic human tissue model than standard cell monolayers.
Staphylococcal enterotoxin A and toxic shock syndrome toxin compete with CD4 for human major histocompatibility complex class II binding
Authors: Bavari S , Ulrich R G
Published: January 2020

We have examined the role of the CD4 molecule in primary T-lymphocyte responses to the staphylococcal enterotoxins SEA, SEB, SEC1, and the toxic shock syndrome toxin TSST-1. Proliferating cells were predominantly CD4+; however, the responses to SEA and TSST-1 were most sensitive to inhibition by the anti-CD4 antibody Leu-3a. T-lymphocyte responses to the bacterial superantigens were inhibited by site-directed mutations of residues in the DR beta membrane-proximal domain (DR beta 2) that are also known to be important for interactions with CD4. SEA and TSST-1 binding to DR was reduced by the DR beta 2 mutations and by competition with soluble recombinant CD4. We propose that bacterial superantigens sequentially, or simultaneously with CD4, stabilize complexes of T-cell antigen receptors and major histocompatibility complex class II molecules. The superantigen qualities of these toxins may be due, in part, to a molecular mimicry of CD4 and other adhesion molecules.
Response to stimulation with recombinant cytokines and synthesis of cytokines by murine intestinal macrophages infected with the Mycobacterium avium complex
Authors: Hsu N , Young L S , Bermudez L E
Published: January 2020

Current evidence suggests that the gut is the chief portal of entry for organisms of the Mycobacterium avium complex (MAC) in AIDS patients. Bacterial invasion of intestinal mucosa presumably occurs through epithelial cells, and M cells in the Peyer's patches, where the bacteria have contact with immunocompetent cells such as macrophages and T and B lymphocytes. As mucosal macrophages are probably the first line of defense against MAC, we examined their ability to inhibit intracellular growth of MAC when properly stimulated. Mouse intestinal macrophages were purified, infected with MAC 101, serovar 1, and MAC 86-2686, serovar 16, and subsequently stimulated with recombinant tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage colony-stimulating factor (M-CSF). Viable intracellular bacteria were quantitated at 24 h after infection and again after 4 days of infection. Stimulation with TNF-alpha, IFN-gamma, and GM-CSF, but not M-CSF, was associated with mycobacteriostatic and/or mycobactericidal activity in macrophages. Treatment with 10(3) U of TNF-alpha, GM-CSF, and IFN-gamma per ml at 24 h prior to infection with MAC resulted in a significant enhancement in killing of MAC at 4 days after infection, compared with that observed for macrophages exposed to cytokines after infection. When stimulated with lipopolysaccharide or live MAC, intestinal macrophages had produced significantly less TNF-alpha and transforming growth factor beta than had splenic and peritoneal macrophages, although the levels of production of interleukin 6 and interleukin 10 among the three populations of cells were similar. Intestinal macrophages can be stimulated with cytokines to inhibit the intracellular growth of MAC, but they have differentiated abilities to produce cytokines which can modulate the anti-MAC immune response.
Mice immunized by primary vaginal Candida albicans infection develop acquired vaginal mucosal immunity
Authors: Fidel P L , Lynch M E , Conaway D H , Tait L , Sobel J D
Published: January 2020

It has been postulated that systemic cell-mediated immunity (CMI) is an important host defense mechanism against Candida infections of the vagina. However, in an estrogen-dependent murine model of experimental vaginal candidiasis, we recently showed that systemic Candida-specific Th1-type CMI induced by immunization with Candida culture filtrate antigen had no effect on vaginal Candida population levels during the course of a vaginal infection. In the present study, mice given a second vaginal inoculation in the presence of peripheral Candida-specific Th1-type CMI induced by prior vaginal infection had anamnestic-type increased delayed-type hypersensitivity (DTH) responses, concomitant with significantly fewer Candida organisms in the vagina than in primary-infected mice. In addition, organisms in secondary-infected mice were fragmented and superficial penetration into the epithelium was reduced. The systemic presence of Candida-specific T suppressor (Ts) cells that significantly suppressed the infection-derived anamnestic DTH reactivity did not abrogate the protective effect in the vagina. Additional experiments showed that vaginally immunized mice were not protected from gastrointestinal or systemic candidiasis and, in contrast to mice with a second vaginal infection, did not demonstrate anamnestic DTH reactivity. These results suggest that a moderate level of local protection against a Candida vaginal infection can be achieved by vaginal immunization but that the protective role of acquired peripheral Candida-specific Th1-type reactivity at the vaginal mucosa appears to be limited.
Antigenic and structural similarities between Mycobacterium tuberculosis 50- to 55-kilodalton and Mycobacterium bovis BCG 45- to 47-kilodalton antigens
Authors: Espitia C , Espinosa R , Saavedra R , Mancilla R , Romain F , Laqueyrerie A , Moreno C
Published: January 2020

The relationship between Mycobacterium tuberculosis 50- to 55-kDa protein and Mycobacterium bovis BCG 45- to 47-kDa antigen was examined by using immunological and biochemical criteria. Reciprocal cross-reactivity with a rabbit polyclonal antiserum against the M. bovis BCG protein and with a monoclonal antibody raised against the M. tuberculosis antigen was observed. The epitope recognized by this antibody was apparently present only in proteins of M. tuberculosis and M. bovis BCG among the 11 mycobacterial species tested. The amino-terminal sequences and total amino acid contents of these proteins showed strong similarities. Both antigens are glycoproteins as assessed by binding of concanavalin A, labeling of carbohydrate moieties with biotin-hydrazide, and digestion of carbohydrates with jack bean alpha-D-mannosidase, which produced a reduction of the molecular weights of the proteins and totally eliminated concanavalin A binding. Both M. tuberculosis and M. bovis BCG proteins are secreted, since they were found mainly in the culture medium. Analysis of M. tuberculosis 50- to 55-kDa antigen by two-dimensional gel electrophoresis showed at least seven different components, as previously described for the M. bovis BCG antigen. Solid-phase immunoassays showed that the purified M. tuberculosis 50- to 55-kDa protein was recognized by serum specimens from 70% of individuals with pulmonary tuberculosis from a total of 77 Mexican patients examined.
Relationship of secretion pattern and MPB70 homology with osteoblast-specific factor 2 to osteitis following Mycobacterium bovis BCG vaccination
Authors: Ulstrup J C , Jeansson S , Wiker H G , Harboe M
Published: January 2020

Significant homology was found between MPB70 and each of four repeat domains of osteoblast-specific factor 2 (OSF-2). Two internal homology regions within each repeat domain of OSF-2 presumed to be related to the active site(s) of this bone adhesion molecule showed the highest homology. A literature search concerning osteitis after Mycobacterium bovis BCG vaccination in neonates revealed that MPB70-high-producer substrains were associated with an increased incidence of osteitis following vaccination. These observations indicate that the function of MPB70 is related to the interaction between bacilli and the host following vaccination or infection with mycobacteria.
Inhibition of the Fc receptor-mediated oxidative burst in macrophages by the Yersinia pseudotuberculosis tyrosine phosphatase
Authors: Bliska J B , Black D S
Published: January 2020

Suppression of host-cell-mediated immunity is a hallmark feature of Yersinia pseudotuberculosis infection. To better understand this process, the interaction of Y. pseudotuberculosis with macrophages and the effect of the virulence plasmid-encoded Yersinia tyrosine phosphatase (YopH) on the oxidative burst was analyzed in a chemiluminescence assay. An oxidative burst was generated upon infection of macrophages with a plasmid-cured strain of Y. pseudotuberculosis opsonized with immunoglobulin G antibody. Infection with plasmid-containing Y. pseudotuberculosis inhibited the oxidative burst triggered by secondary infection with opsonized bacteria. The tyrosine phosphatase activity of YopH was necessary for this inhibition. These results indicate that YopH inhibits Fc receptor-mediated signal transduction in macrophages in a global fashion. In addition, bacterial protein synthesis was not required for macrophage inhibition, suggesting that YopH export and translocation are controlled at the posttranslational level.
Diphosphoryl lipid A from Rhodobacter sphaeroides transiently activates NF-kappa B but inhibits lipopolysaccharide induction of kappa light chain and Oct-2 in the B-cell lymphoma line 70Z/3
Authors: Lawrence O , Rachie N , Qureshi N , Bomsztyk K , Sibley C H
Published: January 2020

Lipopolysaccharide (LPS) is implicated in much of the pathophysiology associated with gram-negative septic shock. One approach to this serious clinical problem is to develop new drugs that antagonize the action of toxic LPS. A model system to study LPS action and test for potential antagonists is readily provided by LPS regulation of the kappa gene in the murine B-cell line 70Z/3. Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) effectively blocked toxic LPS induction of kappa light-chain immunoglobulin expression in 70Z/3 cells. Induction of kappa expression by LPS is dependent on the activation of at least two transcription factors, Oct-2 and NF-kappa B. RsDPLA completely repressed the long-term activation of NF-kappa B observed after 24 h of Salmonella typhosa LPS treatment and antagonized activation of oct-2 mRNA expression. However, RsDPLA was not an inert competitor of LPS. RsDPLA alone strongly activated NF-kappa B binding activity by 30 min but not beyond 9 h of treatment. It also induced a small increase in oct-2 mRNA levels. RsDPLA is not simply a weak agonist; we found no graded increase in kappa expression with increasing RsDPLA concentrations up to 50 micrograms/ml. The NF-kappa B complexes activated by RsDPLA and S. typhosa LPS were both composed of the p50-p65 heterodimer. These results suggest that the physiological LPS receptor(s) on B cells transmits qualitatively different signals depending on the nature of the binding ligand and that the fatty acyl groups of LPS play an important role in activating signal transduction.
Globotriaosylceramide, Gb3, is an alternative functional receptor for Shiga-like toxin 2e
Authors: Keusch G T , Jacewicz M , Acheson D W , Donohue-Rolfe A , Kane A V , McCluer R H
Published: January 2020

We reexamined the binding specificity of the Shiga-like toxin variant associated with porcine edema disease, SLT2e, which is reported to be more cytotoxic for Vero cells than for HeLa cells, by using receptor-deficient cells and a liposomal insertion system for purified glycolipids. We found that SLT2e preferentially uses globotetraosylceramide as a receptor but can also cause cytotoxicity by using globotriaosylceramide, the SLT2 receptor. We conclude that the differential cytotoxicity of SLT2e on HeLa and Vero cells is a function of both the receptor preference of the toxin and the specific glycolipid content of the target cells being used.
Roles of gamma interferon and other cytokines in suppression of the spleen cell proliferative response to concanavalin A and toxoplasma antigen during acute toxoplasmosis
Authors: Candolfi E , Hunter C A , Remington J S
Published: January 2020

Suppressed splenocyte proliferation in response to mitogen and toxoplasma lysate antigen (TLA) is observed in mice acutely infected with Toxoplasma gondii. Recently, we reported that NG-monomethyl-L-arginine (NMMA), an inhibitor of reactive nitrogen intermediate (RNI) production, partially restored proliferative responses of splenocytes from infected mice. In the present study we have examined the effect of NMMA on production of cytokines by splenocytes from mice acutely infected with T. gondii and assessed the role of gamma interferon (IFN-gamma) and interleukin-10 (IL-10) in the RNI-mediated suppression. Stimulation with concanavalin A (ConA) or TLA of splenocytes from CBA/Ca mice infected for 7 days resulted in increased production of IFN-gamma, IL-4, and IL-10 but reduced levels of IL-2 when compared with cultures of splenocytes from uninfected mice. Whereas addition of NMMA did not alter levels of cytokines produced by splenocytes from uninfected mice, splenocytes from infected mice stimulated with ConA produced significantly higher levels of IL-10 and reduced levels of IL-2 and IL-4. Addition of anti-IFN-gamma monoclonal antibodies to cultures of spleen cells from mice infected for 7 or 14 days remarkably decreased the levels of nitrite and resulted in a 47- and 4-fold increase in proliferation induced by stimulation with ConA or TLA, respectively. Anti-IL-10 did not reduce levels of nitrite produced in culture but did result in a fourfold increase in the proliferative response of splenocytes from mice infected for 14 days. In vivo administration of anti-IFN-gamma or anti-IL-10 monoclonal antibodies to infected mice partially restored ex vivo spleen cell proliferative responses by approximately 40 and 15%, respectively. Our data indicate that IFN-gamma is important in inducing the RNI-mediated immunosuppression, which, in turn, affects production of cytokines by splenocytes. Our data also demonstrate that IL-10 is involved in the suppression observed but that this activity is independent of RNI.
Intravascular cryptococcal culture filtrate (CneF) and its major component, glucuronoxylomannan, are potent inhibitors of leukocyte accumulation
Authors: Dong Z M , Murphy J W
Published: January 2020

Disseminated cryptococcosis is characterized by high titers of cryptococcal polysaccharides in serum and minimal cellular infiltrates in infected tissues of patients. The main objective of this study was to determine whether the circulating cryptococcal polysaccharides could contribute to the lack of cellular infiltration into infected tissues. To assess this possibility, a gelatin sponge implantation model was used. We found that intravenous (i.v.) injection of mice with cryptococcal culture filtrate antigen (CneF) inhibited migration of leukocytes (neutrophils, lymphocytes, and monocytes) into the intrasponge sites of acute inflammation induced by CneF, tumor necrosis factor alpha, or formylmethionyl leucyl phenylalanine. In addition, i.v. administration of CneF inhibited leukocyte migration into the intrasponge sites of a cell-mediated immune reaction irrespective of whether the delayed-type hypersensitivity response was to CneF or the mycobacterial antigen purified protein derivative. Glucuronoxylomannan, a major constituent of CneF and a major cryptococcal antigen detected in the sera of patients with disseminated cryptococcosis, when given i.v. to mice, inhibited leukocyte migration into the sponges. Our results suggest that the minimal cellular infiltrates observed in infected tissues of cryptococcosis patients may be due, in part, to the circulating cryptococcal polysaccharide functioning as we have demonstrated in the mouse model. Furthermore, the high titers of cryptococcal antigen in the sera of patients may diminish leukocyte migration in response to stimuli other than Cryptococcus neoformans, a point that may be relevant in AIDS patients with cryptococcosis.
Hemagglutination and proteoglycan binding by the Lyme disease spirochete, Borrelia burgdorferi
Authors: Leong J M , Morrissey P E , Ortega-Barria E , Pereira M E , Coburn J
Published: January 2020

The ability of the Lyme disease spirochete to attach to host components may contribute to its ability to infect diverse tissues. We present evidence that the Lyme disease spirochete expresses a lectin activity that promotes agglutination of erythrocytes and bacterial attachment to glycosaminoglycans. Among a diverse collection of 21 strains of Lyme disease spirochete, hemagglutinating activity was easily detected in all but 3 strains, and these three strains were noninfectious. The ability to agglutinate erythrocytes was associated with the ability of the spirochete to bind to the sulfated polysaccharide dextran sulfate and to mammalian cells. Soluble dextran sulfate was a potent inhibitor of both hemagglutination and attachment to mammalian cells, while dextran had no effect on either activity, suggesting that dextran sulfate may inhibit attachment by mimicking host cell glycosaminoglycans. Consistent with this, the spirochete bound to immobilized heparin, and soluble heparin inhibited bacterial adhesion to mammalian cells. The bacterium did not bind efficiently to Vero cells treated with heparinase or heparitinase or to mutant CHO cell lines that are deficient in proteoglycan synthesis. Sulfation of glycosaminoglycans was critical for efficient bacterial recognition, as Vero cells treated with an inhibitor of sulfation, or a mutant CHO cell line that produces undersulfated heparan sulfate, did not mediate maximal spirochetal binding. Binding of the spirochete to extracellular matrix also appeared to be dependent upon this attachment pathway. These findings suggest that a glycosaminoglycan-binding activity which can be detected by hemagglutination contributes to the attachment of the Lyme disease spirochete to host cells and matrix.
Impact of microfilaremia on maintenance of a hyporesponsive cellular immune response in Brugia-infected gerbils (Meriones unguiculatus)
Authors: Bosshardt S C , Coleman S U , McVay C S , Jones K L , Klei T R
Published: January 2020

The purpose of these experiments was to define the significance of the microfilarial stage to the hyporesponsive condition seen in lymphatic filariasis. Two types of experiments were conducted with Brugia pahangi-infected gerbils. In one, in vitro lymphocyte blastogenesis and in vivo granuloma formation in response to parasite antigen were correlated to microfilaremia in chronically infected individuals. In a second set of experiments, the level of in vivo granuloma formation was assessed following chemotherapeutic removal of microfilariae with ivermectin. The results indicated that the microfilarial stage alone is not responsible for the maintenance of the low cellular responses seen during chronic infections in this model. Furthermore, the data suggest that the degree of downregulation of these responses may be related to parasite burden.
Cloning and expression of a neutral phosphatase gene from Treponema denticola
Authors: Ishihara K , Kuramitsu H K
Published: January 2020

We have isolated and characterized a neutral phosphatase gene, phoN, from Treponema denticola ATCC 35405. The gene was isolated from a T. denticola clone bank constructed in the medium-copy-number plasmid vector pMCL19. Subcloning and nucleotide sequencing of the DNA insert from one phosphatase clone, pTph14, revealed that the activity corresponded to an open reading frame consisting of 1,027 bp coding for a 37.9-kDa protein. Hydrophobicity analysis indicated that the protein exhibits some hydrophobic regions. Indeed, partial purification of the phosphatase suggested that the enzyme was membrane associated both in T. denticola and in the Escherichia coli clone. The pH optimum of the enzyme, approximately pH 6.4, indicated that it corresponded to a neutral phosphatase activity from T. denticola. An examination of possible natural substrates for the enzyme suggested that this enzyme hydrolyzes nucleoside di- and triphosphates. Northern (RNA) blot analysis revealed that this phosphatase gene is not likely to be present in an operon structure.
Binding, oligomerization, and pore formation by streptolysin O in erythrocytes and fibroblast membranes: detection of nonlytic polymers
Authors: Walev I , Palmer M , Valeva A , Weller U , Bhakdi S
Published: January 2020

Streptolysin O (SLO) is a representative of the family of cholesterol-binding cytolysins that form large pores in target cell membranes. Aggregation of the toxin to polymeric structures is required for pore formation. However, it is not known whether, vice versa, polymers may under certain circumstances remain nonfunctional, and whether this might be the cause underlying the relative resistance of certain cells towards toxin action. In the present study, we applied radioiodinated, functionally active SLO to human, rabbit, and mouse erythrocytes and to human fibroblasts and keratinocytes. Binding and polymerization were quantified and correlated with membrane damage. At low toxin concentrations, human and rabbit but not mouse erythrocytes were lysed, but binding and polymerization of SLO were essentially identical in all cases. Nonlytic polymers were also detected on human fibroblasts and keratinocytes treated with subcytotoxic concentrations of SLO, and quantitative estimates indicated that nonpermeabilized cells could carry hundreds of polymers on their surface. When applied at low concentrations to fibroblasts, much of the toxin remained in monomer form and was subsequently shed from the cells. This was shown by monitoring the fate of radioiodinated toxin and also by using a sensitive cell enzyme-linked immunosorbent assay that permitted immunological detection of surface-exposed SLO. Thus, relative resistance of cells towards the permeabilizing action of SLO may be due to their ability to tolerate formation of a limited number of SLO polymers and to shedding of nonoligomerized toxin from their surface.
Effect of oral immunization with recombinant urease on murine Helicobacter felis gastritis
Authors: Pappo J , Thomas W D , Kabok Z , Taylor N S , Murphy J C , Fox J G
Published: January 2020

The ability of oral immunization to interfere with the establishment of infection with Helicobacter felis was examined. Groups of Swiss Webster mice were immunized orally with 250 micrograms of Helicobacter pylori recombinant urease (rUrease) and 10 micrograms of cholera toxin (CT) adjuvant, 1 mg of H. felis sonicate antigens and CT, or phosphate-buffered saline (PBS) and CT. Oral immunization with rUrease resulted in markedly elevated serum immunoglobulin G (IgG), serum IgA, and intestinal IgA antibody responses. Challenge with live H. felis further stimulated the urease-specific intestinal IgA and serum IgG and IgA antibody levels in mice previously immunized with rUrease but activated primarily the serum IgG compartment of PBS-treated and H. felis-immunized mice. Intestinal IgA and serum IgG and IgA anti-urease antibody responses were highest in rUrease-immunized mice at the termination of the experiment. Mice immunized with rUrease were significantly protected (P < or = 0.0476) against infection when challenged with H. felis 2 or 6 weeks post-oral immunization in comparison with PBS-treated mice. Whereas H. felis-infected mice displayed multifocal gastric mucosal lymphoid follicles consisting of CD45R+ B cells surrounded by clusters of Thy1.2+ T cells, gastric tissue from rUrease-immunized mice contained few CD45R+ B cells and infrequent mucosal follicles. These observations show that oral immunization with rUrease confers protection against H. felis infection and suggest that gastric tissue may function as an effector organ of the mucosal immune system which reflects the extent of local antigenic stimulation.
Role of Pseudomonas aeruginosa pili in acute pulmonary infection
Authors: Tang H , Kays M , Prince A
Published: January 2020

The role of piliation in the development and course of acute pulmonary infection was examined using infant BALB/cByJ mice inoculated by intranasal instillation of isogenic Pil+ and Pil- mutants of Pseudomonas aeruginosa PA1244, PAK, and PAO1. The piliated strains caused more cases of pneumonia, bacteremia, and mortality than the nonpiliated strains (chi-square analysis, alpha = 0.001). The piliated strains were more often associated with severe diffuse pneumonias, while the nonpiliated organisms resulted in less severe, focal pneumonias, although these differences did not achieve statistical significance. Purified pilin protein used to inoculate the mice resulted in local inflammatory changes. The nonpiliated strain PA1244-NP was as virulent as the piliated strain PAO1, suggesting that expression of other virulence factors are also important in the development of acute pneumonia. This infant mouse model of pulmonary infection appears to be a useful system for the analysis of P. aeruginosa virulence factors involved in the pathogenesis of pneumonia.
Phospholipid composition of Pneumocystis carinii carinii and effects of methylprednisolone immunosuppression on rat lung lipids
Authors: Guo Z , Kaneshiro E S
Published: January 2020

The phospholipid class composition of Pneumocystis carinii carinii freshly isolated from infected lungs generally resembled that of the host lung, suggesting that the parasite scavenges lung alveolar lipids. However, subtle quantitative differences were demonstrated, indicating that the pathogen has the metabolic capacity to de novo synthesize, or at least tailor, its lipids. The concentration of phosphatidylcholine, the major lung surfactant lipid, in the organism was lower than that in lungs of normal and immunosuppressed uninfected rats, and the concentration of phosphatidylinositol was higher. Phosphonolipids were not detected in the organism by chemical analysis and nuclear magnetic resonance spectrometry. The immunosuppressive regimen alone caused increases in both surfactant protein A and the lipid content of the whole lung. The lungs of rats that were subjected to corticosteroid immunosuppression and had heavy parasite loads had dramatically elevated surfactant protein A levels, whereas the lipid contents of these lungs were not different from lipid contents in whole lungs of immunosuppressed uninfected rats. P. carinii was found to concentrate lipids, indicating that a large amount of the lipids in the whole infected rat lung was within the parasites residing in the organ. These observations have important implications relevant to the use of corticosteroid therapy for P. carinii pneumonitis.
Safety and immunogenicity of a live oral bivalent typhoid fever (Salmonella typhi Ty21a)-cholera (Vibrio cholerae CVD 103-HgR) vaccine in healthy adults
Authors: Cryz S J , Que J U , Levine M M , Wiedermann G , Kollaritsch H
Published: January 2020

The safety and immunogenicity of the live oral attenuated vaccine strains vibrio cholerae CVD 103-HgR and Salmonella typhi Ty21a were evaluated alone or in a combined bivalent formulation in four groups composed of 185 healthy European adults. All presentations were well tolerated. The serum anti-S. typhi lipopolysaccharide immunoglobulin G and immunoglobulin A antibody responses were comparable for all groups (66 to 72% seroconversion). The serum vibriocidal antibody seroconversion rate ranged from 78 to 92.5% (P > 0.05) among the groups. However, the peak and geometric mean vibriocidal antibody titers were significantly higher (P < 0.005) in the groups which received the bivalent formulation along with two doses of Ty21a than in the group which received CVD 103-HgR followed by two doses of killed Escherichia coli K-12 placebo. The ingestion of a placebo shortly after CVD 103-HgR may have suppressed the magnitude of the immune response. These findings demonstrate the feasibility of producing multivalent live oral attenuated vaccines.