bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cholera Toxin B Subunit Activates Arachidonic Acid Metabolism
Authors: Peterson Johnny W. , Finkelstein Richard A. , Cantu Juan , Gessell Deborah L. , Chopra Ashok K. 
Published: December 2019

ABSTRACT

Cholera toxin (CT) increases intestinal secretion of water and electrolytes and modulates the mucosal immune response by stimulating cellular synthesis of arachidonic acid (AA) metabolites (e.g., prostaglandin E
2
), as well as the intracellular second messenger cyclic AMP (cAMP). While much is known about the mechanism of CT stimulation of adenylate cyclase, the toxin’s activation of phospholipase A
2
, which results in increased hydrolysis of AA from membrane phospholipids, is not well understood. To determine whether CT activation of AA metabolism requires CT’s known enzymatic activity (i.e., ADP-ribosylation of G
Sα
), we used native CT and a mutant CT protein (CT-2*) lacking ADP-ribose transferase activity in combination with S49 wild-type (WT) and S49 cyc
−
murine Theta (Th)1.2-positive lymphoma cells deficient in G
Sα
. The experimental results showed that native CT stimulated the release of [
3
H[AA from S49 cyc
−
cells at a level similar to that for S49 WT cells, indicating that G
Sα
is not essential for this process. Further, levels of cAMP in the CT-treated cyc
−
cells remained the same as those in the untreated control cells. The ADP-ribosyltransferase-deficient CT-2* protein, which was incapable of increasing synthesis of cAMP, displayed about the same capacity as CT to evoke the release of [
3
H]AA metabolites from both S49 WT and cyc
−
cells. We concluded that stimulation of arachidonate metabolism in S49 murine lymphoma cells by native CT does not require enzymatically functional CT, capable of catalyzing the ADP-ribosylation reaction. These results demonstrated for the first time that stimulation of adenylate cyclase by CT and stimulation of AA metabolism by CT are not necessarily coregulated. In addition, the B subunits purified from native CT and CT-2* both simulated the release of [
3
H]AA from S49 cyc
−
cells and murine monocyte/macrophage cells (RAW 264.7), suggesting a receptor-mediated cell activation process of potential importance in enhancing immune responses to vaccine components.
In Vivo Blockage of Nitric Oxide with Aminoguanidine Inhibits Immunosuppression Induced by an Attenuated Strain ofSalmonella typhimurium, PotentiatesSalmonellaInfection, and Inhibits Macrophage and Polymorphonuclear Leukocyte Influx into the Spleen
Authors: MacFarlane Amanda Shearer , Schwacha Martin G. , Eisenstein Toby K. 
Published: December 2019

ABSTRACTOur laboratory has previously shown that after immunization with a strain ofSalmonella typhimurium, SL3235, made avirulent by a blockage in the pathway of aromatic synthesis, murine splenocytes were profoundly suppressed in their capacity to mount an in vitro antibody plaque-forming cell (PFC) response to sheep erythrocytes. Evidence indicated that suppression was mediated by nitric oxide (NO), since the in vitro addition ofNG-monomethyl-l-arginine blocked suppression. The present studies examined the effect of blocking NO production onSalmonella-induced immunosuppression by in vivo administration of aminoguanidine hemisulfate (AG). AG was administered to C3HeB/FeJ mice in their drinking water (2.5% solution) for 7 days prior to intraperitoneal inoculation with SL3235. AG treatment inhibited the increase in nitrate and nitrite levels in plasma and nitrite levels in the spleen seen in immunized mice. Importantly, AG treatment completely blocked suppression of the splenic PFC response and markedly attenuated the suppression of the response to concanavalin A in immunized mice, providing further evidence thatSalmonella-induced immunosuppression is mediated by NO. AG treatment also alleviated the majority of the splenomegaly associated with SL3235 inoculation, which correlated with a blockage of influx of neutrophils and macrophages into spleens, as assessed by flow cytometry. AG treatment unexpectedly resulted in 90% mortality in mice injected with the highly attenuated vaccine strain ofSalmonella, SL3235. Increased mortality in AG-treated mice correlated with inability to clear organisms from the spleen by day 15 postinoculation and with persistent bacteremia, compared with control mice. Collectively, these in vivo results underscore the dual biological consequences of NO production followingSalmonellainfection, with NO being necessary for host defense, but also having the potentially adverse effect of immunosuppression. A unifying hypothesis to explain how these seemingly paradoxical effects could both result from NO production is presented.
Regulation of ExoS Production and Secretion by
Pseudomonas aeruginosa
in Response to Tissue Culture Conditions
Authors: Vallis Amy J. , Yahr Timothy L. , Barbieri Joseph T. , Frank Dara W. 
Published: December 2019

ABSTRACT

This study was initiated to characterize the regulation and secretion of ExoS by
Pseudomonas aeruginosa
during contact with eukaryotic cells. The production of ExoS was monitored by a sensitive ADP-ribosyltransferase activity assay, and specific activities were calculated for supernatant and cell-associated fractions. Time course analysis indicated that ExoS was produced after a lag period, suggesting that induction of the regulon is necessary for the expression of detectable amounts of enzyme activity. Under tissue culture growth conditions, ExoS was induced when
P. aeruginosa
was in contact with Chinese hamster ovary (CHO) cells or after growth in tissue culture medium with serum. The serum induction of ExoS appeared to result in generalized type III secretion, while induction by contact with CHO cells appeared to result in polarized type III secretion. Mutants in the type III secretory system that express a null phenotype for ExoS production in bacteriological medium produced but did not secrete the enzyme when
P. aeruginosa
was grown under inducing conditions in tissue culture medium. These results suggest that both induction and secretion of ExoS may differ when the bacteria are exposed to different growth environments. The putative type III translocation proteins and secretion apparatus of
P. aeruginosa
were required for translocation of bacterial factors that mediate changes in CHO cell morphology during infection.
Fas-FasL Interaction Involved in Pathogenesis of Ocular Toxoplasmosis in Mice
Authors: Hu Mark S. , Schwartzman Joseph D. , Yeaman Grant R. , Collins Jane , Seguin Rosanne , Khan Imtiaz A. , Kasper Lloyd H. 
Published: December 2019

ABSTRACTOcular toxoplasmosis is a potentially blinding intraocular inflammation. The intent of this study was to investigate the role of Fas-FasL interaction in a murine model of acquired ocular toxoplasmosis induced by intracameral inoculation ofToxoplasma gondii. Intraocular inflammation, Fas and FasL expression on lymphocytes and on ocular tissues, the occurrence of apoptosis, and the frequency of CD8+and CD4+T cells in the infected eyes were analyzed in C57BL/6 (B6) mice. Susceptibility to parasite-induced intraocular inflammation was observed in Fas-deficient (B6-lpr) and FasL-deficient (B6-gld) mice. Inoculation of 5,000T. gondiitachyzoites induced significant intraocular inflammation associated with increase of Fas and FasL expression in the inoculated eyes of wild-type B6 mice. Flow cytometry demonstrated a significant increase of Fas and FasL expression on the splenocytes from naive mice incubated in vitro with the parasite and on the splenocytes harvested from the infected mice at day 8 after parasite inoculation. Apoptosis of inflammatory cells and cells in ocular tissues was seen, and a greater frequency of CD8+than CD4+T cells was observed in the infected eyes. The intensity of intraocular inflammation was greater in B6-lprand B6-gldmice than in wild-type B6 mice (P< 0.05). The results suggest that Fas-FasL interaction associated with apoptosis is involved in the pathogenesis of acquired ocular toxoplasmosis in mice.
Functional Analysis of the Carboxy-Terminal Domain of
Bacillus anthracis
Protective Antigen
Authors: Brossier Fabien , Sirard Jean-Claude , Guidi-Rontani Chantal , Duflot Edith , Mock Michele 
Published: December 2019

ABSTRACT

Protective antigen (PA) is the common receptor-binding component of the two anthrax toxins. We investigated the involvement of the PA carboxy-terminal domain in the interaction of the protein with cells. A deletion resulting in removal of the entire carboxy-terminal domain of PA (PA608) or part of an exposed loop of 19 amino acids (703 to 722) present within this domain was introduced into the
pag
gene. PA608 did not induce the lethal-factor (LF)-mediated cytotoxic effect on macrophages because it did not bind to the receptor. In contrast, PA711- and PA705-harboring lethal toxins (9- and 16-amino-acid deletions in the loop, starting after positions 711 and 705, respectively) were 10 times less cytotoxic than wild-type PA. After cleavage by trypsin, the mutant PA proteins formed heptamers and bound LF. The capacity of PA711 and PA705 to interact with cells was 1/10 that of wild-type PA. In conclusion, truncation of the carboxy-terminal domain or deletions in the exposed loop resulted in PA that was less cytotoxic or nontoxic because the mutated proteins did not efficiently bind to the receptor.
Role of DnaK in In Vitro and In Vivo Expression of Virulence Factors ofVibrio cholerae
Authors: Chakrabarti Sabyasachi , Sengupta Nilanjan , Chowdhury Rukhsana 
Published: December 2019

ABSTRACTThednaKgene ofVibrio choleraewas cloned, sequenced, and used to construct adnaKinsertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathogen. The central regulator of several virulence genes ofV. choleraeis ToxR, a transmembrane DNA binding protein. TheV. cholerae dnaKmutant grown in standard laboratory medium exhibited phenotypes characteristic of cells deficient in ToxR activity. Using Northern blot analysis andtoxRtranscriptional fusions, we demonstrated a reduction in expression of thetoxRgene in thednaKmutant strain together with a concomitant increase in expression of ahtpG-like heat shock gene that is located immediately upstream and is divergently transcribed fromtoxR. This may be due to increased heat shock induction in thednaKmutant. In vivo, however, although expression from heat shock promoters in thednaKmutant was similar to that observed in vitro, expression of bothtoxRandhtpGwas comparable to that by the parental strain. In both strains, in vivo expression oftoxRwas significantly higher than that observed in vitro, but no reciprocal decrease inhtpGexpression was observed. These results suggest that the modulation oftoxRexpression in vivo may be different from that observed in vitro.
The
Pseudomonas aeruginosa
Secretory Product Pyocyanin Inactivates α
1
Protease Inhibitor: Implications for the Pathogenesis of Cystic Fibrosis Lung Disease
Authors: Britigan Bradley E. , Railsback Michelle A. , Cox Charles D. 
Published: December 2019

ABSTRACT

α
1
Protease inhibitor (α
1
PI) modulates serine protease activity in the lung. Reactive oxygen species inactivate α
1
PI, and this process has been implicated in the pathogenesis of a variety of forms of lung injury. An imbalance of protease-antiprotease activity is also detected in the airways of patients with cystic fibrosis-associated lung disease who are infected with
Pseudomonas aeruginosa. P. aeruginosa
secretes pyocyanin, which, through its ability to redox cycle, induces cells to generate reactive oxygen species. We tested the hypothesis that redox cycling of pyocyanin could lead to inactivation of α
1
PI. When α
1
PI was exposed to NADH and pyocyanin, a combination that results in superoxide production, α
1
PI lost its ability to form an inhibitory complex with both porcine pancreatic elastase (PPE) and trypsin. Similarly, addition of pyocyanin to cultures of human airway epithelial cells to which α
1
PI was also added resulted in a loss of the ability of α
1
PI to form a complex with PPE or trypsin. Neither superoxide dismutase, catalase, nor dimethylthiourea nor depletion of the media of O
2
to prevent formation of reactive oxygen species blocked pyocyanin-mediated inactivation of α
1
PI. These data raise the possibility that a direct interaction between reduced pyocyanin and α
1
PI is involved in the process. Consistent with this possibility, pretreatment of α
1
PI with the reducing agent β-mercaptoethanol also inhibited binding of trypsin to α
1
PI. These data suggest that pyocyanin could contribute to lung injury in the
P. aeruginosa
-infected airway of cystic fibrosis patients by decreasing the ability of α
1
PI to control the local activity of serine proteases.
Treponema denticola
Outer Membrane Enhances the Phagocytosis of Collagen-Coated Beads by Gingival Fibroblasts
Authors: Battikhi Tulin , Lee Wilson , McCulloch Christopher A. G. , Ellen Richard P. 
Published: December 2019

ABSTRACT

Human gingival fibroblasts (HGFs) degrade collagen fibrils in physiological processes by phagocytosis. Since
Treponema denticola
outer membrane (OM) extract perturbs actin filaments, important structures in phagocytosis, we determined whether the OM affects collagen phagocytosis in vitro by HGFs. Phagocytosis was measured by flow cytometric assessment of internalized collagen-coated fluorescent latex beads. Confluent HGFs pretreated with
T. denticola
ATCC 35405 OM exhibited an increase in the percentage of collagen phagocytic cells (phagocytosis index [PI]) and in the number of beads per phagocytosing cell (phagocytic capacity [PC]) compared with untreated controls. The enhancement was swift (within 15 min) and was still evident after 1 day. PI and PC of HGFs for bovine serum albumin (BSA)-coated beads were also increased, indicating a global increase in phagocytic processes. These results contrasted those for control OM from
Veillonella atypica
ATCC 17744, which decreased phagocytosis. The
T. denticola
OM-induced increase in bead uptake was eliminated by heating the OM and by depolymerization of actin filaments by cytochalasin D treatment of HGFs. Fluid-phase accumulation of lucifer yellow was enhanced in a saturable, concentration-dependent, transient manner by the
T. denticola
OM. Our findings were not due to HGF detachment or cytotoxicity in response to the
T. denticola
OM treatment since the HGFs exhibited minimal detachment from the substratum; they did not take up propidium iodide; and there was no change in their size, granularity, or content of sub-G
1
DNA. We conclude that a heat-sensitive component(s) in
T. denticola
OM extract stimulates collagen phagocytosis and other endocytic processes such as nonspecific phagocytosis and pinocytosis by HGFs.
Association of a Myosin Immunoanalogue with Cell Envelopes of
Aspergillus fumigatus
Conidia and Its Participation in Swelling and Germination
Authors: Esnault Karine , el Moudni Brahim , Bouchara Jean-Philippe , Chabasse Dominique , Tronchin Guy 
Published: December 2019

ABSTRACT

A myosin immunoanalogue was identified in conidia of
Aspergillus fumigatus
by Western blotting, indirect immunofluorescence assay, and gold immunoelectron microscopy with two different antimyosin antibodies. The distribution pattern of this protein was followed during the early stages of germination. A single 180-kDa polypeptide, detected predominantly in a cell envelope extract, was found to cross-react with monoclonal and polyclonal antibodies raised against vertebrate muscle myosin. Immunoelectron microscopy permitted precise localization of this polypeptide, indicating that myosin analogue was mainly distributed along the plasma membrane of resting and swollen conidia. In germinating conidia, indirect immunofluorescence microscopy revealed myosin analogue at the periphery of germ tubes, whereas actin appeared as dispersed punctate structures in the cytoplasm that were more concentrated at the site of germ tube emergence. A myosin ATPase inhibitor, butanedione monoxime, greatly reduced swelling and blocked germination. In contrast, when conidia were treated with cytochalasin B, an inhibitor of actin polymerization, swelling was not affected and germination was only partially reduced. Butanedione monoxime-treated conidia showed accumulation of cytoplasmic vesicles and did not achieve cell wall reorganization, unlike swollen conidia. Collectively, these results suggest an essential role for this myosin analogue in the deposition of cell wall components during germination of
A. fumigatus
conidia and therefore in host tissue colonization.
Noncompetitive Expansion of Cytotoxic T Lymphocytes Specific for Different Antigens during Bacterial Infection
Authors: Vijh Sujata , Pilip Ingrid M. , Pamer Eric G. 
Published: December 2019

ABSTRACTListeria monocytogenesis an intracellular bacterium that elicits complex cytotoxic T-lymphocyte (CTL) responses in infected mice. The responses of CTL populations that differ in antigen specificity range in magnitude from large, dominant responses to small, subdominant responses. To test the hypothesis that dominant T-cell responses inhibit subdominant responses, we eliminated the two dominant epitopes ofL. monocytogenesby anchor residue mutagenesis and measured the T-cell responses to the remaining subdominant epitopes. Surprisingly, the loss of dominant T-cell responses did not enhance subdominant responses. While mice immunized with bacteria lacking dominant epitopes developedL. monocytogenes-specific immunity, their ability to respond to dominant epitopes upon rechallenge with wild-type bacteria was markedly diminished. Recall responses in mice immunized with wild-type or epitope-deficientL. monocytogenesshowed that antigen presentation during recall infection is sufficient for activating memory cells yet insufficient for optimal priming of naive T lymphocytes. Our findings suggest that T-cell priming to different epitopes duringL. monocytogenesinfection is not competitive. Rather, T-cell populations specific for different antigens but the same pathogen expand independently.
Hyperproduction of Alpha-Hemolysin in a
sigB
Mutant Is Associated with Elevated SarA Expression in
Staphylococcus aureus
Authors: Cheung Ambrose L. , Chien Yueh-tyng , Bayer Arnold S. 
Published: December 2019

ABSTRACT

To evaluate the role of SigB in modulating the expression of virulence determinants in
Staphylococcus aureus
, we constructed a
sigB
mutant of RN6390, a prototypic
S. aureus
strain. The mutation in the
sigB
gene was confirmed by the absence of the SigB protein in the mutant on an immunoblot as well as the failure of the mutant to activate ςB-dependent promoters (e.g., the
sarC
promoter) of
S. aureus
. Phenotypic analysis indicated that both alpha-hemolysin level and fibrinogen-binding capacity were up-regulated in the mutant strain compared with the parental strain. The increase in fibrinogen-binding capacity correlated with enhanced expression of clumping factor and coagulase on immunoblots. The effect of the
sigB
mutation on the enhanced expression of the alpha-hemolysin gene (
hla
) was primarily transcriptional. Upon complementation with a plasmid containing the
sigB
gene,
hla
expression returned to near parental levels in the mutant. Detailed immunoblot analysis as well as a competitive enzyme-linked immunosorbent assay of the cell extract of the
sigB
mutant with anti-SarA monoclonal antibody 1D1 revealed that the expression of SarA was higher in the mutant than in the parental control. Despite an elevated SarA level, the transcription of RNAII and RNAIII of the
agr
locus remained unaltered in the
sigB
mutant. Because of a lack of perturbation in
agr
, we hypothesize that inactivation of
sigB
leads to increased expression of SarA which, in turn, modulates target genes via an
agr
-independent but SarA-dependent pathway.
Role of Iron inNramp1-Mediated Inhibition of Mycobacterial Growth
Authors: Zwilling Bruce S. , Kuhn Donald E. , Wikoff Lisa , Brown David , Lafuse William 
Published: December 2019

ABSTRACTInnate resistance to mycobacterial growth is mediated by a gene,Nramp1. We have previously reported thatNramp1mRNA from macrophages ofMycobacterium bovisBCG-resistant (Bcgr) mice is more stable thanNramp1mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely relatedNramp2gene is a metal ion transporter, we evaluated the effect of iron on the growth ofMycobacterium aviumwithin macrophages as well as on the stability ofNramp1mRNA. The addition of iron to macrophages fromBcgsmice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages fromBcgrmice to control the growth ofM. avium. When we treated recombinant gamma interferon (IFN-γ)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-γ-activated macrophages fromBcgsmice but that it did not affect the capacity of macrophages fromBcgrmice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages fromBcgrmice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages fromBcgrmice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth.
Molecular and Evolutionary Analysis of
Borrelia burgdorferi
297 Circular Plasmid-Encoded Lipoproteins with OspE- and OspF-Like Leader Peptides
Authors: Akins Darrin R. , Caimano Melissa J. , Yang Xiaofeng , Cerna Felix , Norgard Michael V. , Radolf Justin D. 
Published: December 2019

ABSTRACT

We previously described two OspE and three OspF homologs in
Borrelia burgdorferi
297 (D. R. Akins, S. F. Porcella, T. G. Popova, D. Shevchenko, S. I. Baker, M. Li, M. V. Norgard, and J. D. Radolf, Mol. Microbiol. 18:507–520, 1995; D. R. Akins, K. W. Bourell, M. J. Caimano, M. V. Norgard, and J. D. Radolf, J. Clin. Investig. 101:2240–2250, 1998). In this study, we characterized four additional lipoproteins with OspE/F-like leader peptides (Elps) and demonstrated that all are encoded on plasmids homologous to cp32 and cp18 from the B31 and N40 strains, respectively. Statistical analysis of sequence similarities using the binary comparison algorithm revealed that the nine lipoproteins from strain 297, as well as the OspE, OspF, and Erp proteins from the N40 and B31 strains, fall into three distinct families. Based upon the observation that these lipoproteins all contain highly conserved leader peptides, we now propose that the ancestors of each of the three families arose from gene fusion events which joined a common N terminus to unrelated proteins. Additionally, further sequence analysis of the strain 297 circular plasmids revealed that rearrangements appear to have played an important role in generating sequence diversity among the members of these three families and that recombinational events in the downstream flanking regions appear to have occurred independently of those within the lipoprotein-encoding genes. The association of hypervariable regions with genes which are differentially expressed and/or subject to immunological pressures suggests that the Lyme disease spirochete has exploited recombinatorial processes to foster its parasitic strategy and enhance its immunoevasiveness.