bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interleukin-12 Is Required for Control of the Growth of Attenuated Aromatic-Compound-Dependent Salmonellae in BALB/c Mice: Role of Gamma Interferon and Macrophage Activation
Authors: Mastroeni Pietro , Harrison J. A. , Robinson J. H. , Clare S. , Khan S. , Maskell D. J. , Dougan G. , Hormaeche C. E. 
Published: December 2019

ABSTRACTThe attenuatedS. typhimuriumSL3261 (aroA) strain causes mild infections in BALB/c mice. We were able to exacerbate the disease by administering anti-interleukin-12 (IL-12) antibodies, resulting in bacterial counts in the spleens and livers of anti-IL-12-treated mice that were 10- to 100-fold higher than the ones normally observed in premortem mice; yet the animals showed only mild signs of illness. Nevertheless, they eventually died of a slow, progressive disease. Mice infected with salmonellae become hypersusceptible to endotoxin. We found that IL-12 neutralization prevented the death of infected mice following subcutaneous injection of lipopolysaccharide. Granulomatous lesions developed in the spleens and livers of control animals, as opposed to a widespread infiltration of mononuclear cells seen in the organs of anti-IL-12-treated mice. In the latter (heavily infected), salmonellae were seen within mononuclear cells, indicating an impairment of the bactericidal or bacteriostatic ability of the phagocytes in the absence of biologically active IL-12. Gamma interferon (IFN-γ) levels were reduced in the sera and tissue homogenates from anti-IL-12-treated mice compared to those in control animals. Furthermore, fluorescence-activated cell sorter analysis on spleen cells showed that IL-12 neutralization impaired the upregulation of I-Ad/I-Edantigens on macrophages from infected mice. Inducible nitric oxide synthase and IFN-γ mRNA production was down-regulated in anti-IL-12-treated mice, which also showed an increased production of IL-10 mRNA and a decrease in nitric oxide synthase activity in the tissues. Administration of recombinant IFN-γ to anti-IL-12-treated mice was able to restore host resistance, granuloma formation, and expression of major histocompatibility complex class II antigens in F4/80+and CD11b+spleen cells.
Contact-Dependent Protein Secretion in
Porphyromonas gingivalis
Authors: Park Yoonsuk , Lamont Richard J. 
Published: December 2019

ABSTRACT

Porphyromonas gingivalis
can induce its uptake by host epithelial cells; however, the nature and role of the
P. gingivalis
molecules involved in this invasion process have yet to be determined. In this study, modulation of secreted
P. gingivalis
proteins following association with gingival epithelial cells was investigated. Western immunoblot analysis showed that contact with epithelial cells or epithelial cell growth media induces
P. gingivalis
33277 to secrete several proteins with molecular masses between 35 and 95 kDa. Secretion of the Arg-gingipain and Lys-gingipain proteases was repressed under these conditions. The contact-induced secreted protein profile was altered in Arg-gingipain-deficient and Lys-gingipain-deficient mutants, indicating a possible role for these proteases in the secretion pathway. The
P. gingivalis
contact-dependent protein secretion pathway differs to some extent from type III protein secretion pathways in enteric pathogens, as a gene homologous to the
invA
family genes was not detected in
P. gingivalis
. The secreted proteins of
P. gingivalis
may play a role in the interactions of the organism with host cells.
Characterization of
Clostridium botulinum
Type B Neurotoxin Associated with Infant Botulism in Japan
Authors: Kozaki Shunji , Kamata Yoichi , Nishiki Tei-ichi , Kakinuma Hiroaki , Maruyama Hiromi , Takahashi Hiroaki , Karasawa Tadahiro , Yamakawa Kiyotaka , Nakamura Shinichi 
Published: December 2019

ABSTRACT

The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that
125
I-labeled 111/NT bound to a single binding site with a dissociation constant (

K
d

) of 2.5 nM; the value was rather lower than that (0.42 nM) of
125
I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b,
125
I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the

K
d

values accorded with those of the low- and high-affinity binding sites in synaptosomes. However,
125
I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity.
Modulation of Enzymatic Activity and Biological Function ofListeria monocytogenesBroad-Range Phospholipase C by Amino Acid Substitutions and by Replacement with theBacillus cereusOrtholog
Authors: Zückert Wolfram R. , Marquis Hélène , Goldfine Howard 
Published: December 2019

ABSTRACTThe secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) ofListeria monocytogenesplays a role in the bacterium’s ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs,Clostridium perfringensα-toxin andBacillus cereusPLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBcactive site, while a gene replacement resulted inL. monocytogenessecreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBcexpressed byL. monocytogeneswas highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBcwas significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells.
Surface Structure, Hydrophobicity, Phagocytosis, and Adherence to Matrix Proteins of
Bacillus cereus
Cells with and without the Crystalline Surface Protein Layer
Authors: Kotiranta Anja , Haapasalo Markus , Kari Kirsti , Kerosuo Eero , Olsen Ingar , Sorsa Timo , Meurman Jukka H. , Lounatmaa Kari 
Published: December 2019

ABSTRACT

Nonopsonic phagocytosis of
Bacillus cereus
by human polymorphonuclear leukocytes (PMNs) with particular attention to bacterial surface properties and structure was studied. Two reference strains (ATCC 14579
T
and ATCC 4342) and two clinical isolates (OH599 and OH600) from periodontal and endodontic infections were assessed for adherence to matrix proteins, such as type I collagen, fibronectin, laminin, and fibrinogen. One-day-old cultures of strains OH599 and OH600 were readily ingested by PMNs in the absence of opsonins, while cells from 6-day-old cultures were resistant. Both young and old cultures of the reference strains of
B. cereus
were resistant to PMN ingestion. Preincubation of PMNs with the phagocytosis-resistant strains of
B. cereus
did not affect the phagocytosis of the sensitive strain. Negatively stained cells of OH599 and OH600 studied by electron microscopy had a crystalline protein layer on the cell surface. In thin-sectioned cells of older cultures (3 to 6 days old), the S-layer was observed to peel off from the cells. No S-layer was detected on the reference strains. Extraction of cells with detergent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major 97-kDa protein from the strains OH599 and OH600 but only a weak 97-kDa band from the reference strain ATCC 4342. One-day-old cultures of the clinical strains (hydrophobicity, 5.9 to 6.0%) showed strong binding to type I collagen, laminin, and fibronectin. In contrast, reference strains (hydrophobicity, −1.0 to 4.2%) as well as 6-day-old cultures of clinical strains (hydrophobicity, 19.0 to 53.0%) bound in only low numbers to the proteins. Gold-labelled biotinylated fibronectin was localized on the S-layer on the cell surface as well as on fragments of S-layer peeling off the cells of a 6-day-old culture of
B. cereus
OH599. Lactose, fibronectin, laminin, and antibodies against the S-protein reduced binding to laminin but not to fibronectin. Heating the cells at 84°C totally abolished binding to both proteins. Benzamidine, a noncompetitive serine protease inhibitor, strongly inhibited binding to fibronectin whereas binding to laminin was increased. Overall, the results indicate that changes in the surface structure, evidently involving the S-layer, during growth of the clinical strains of
B. cereus
cause a shift from susceptibility to PMN ingestion and strong binding to matrix and basement membrane proteins. Furthermore, it seems that binding to laminin is mediated by the S-protein while binding to fibronectin is dependent on active protease evidently attached to the S-layer.
Expression of the Virulence Plasmid-Carried Apyrase Gene (
apy
) of Enteroinvasive
Escherichia coli
and
Shigella flexneri
Is under the Control of H-NS and the VirF and VirB Regulatory Cascade
Authors: Berlutti Francesca , Casalino Mariassunta , Zagaglia Carlo , Fradiani Piera Assunta , Visca Paolo , Nicoletti Mauro 
Published: December 2019

ABSTRACT

The transcription of the virulence plasmid (pINV)-carried invasion genes of
Shigella flexneri
and enteroinvasive
Escherichia coli
(EIEC) is induced at 37°C and repressed at 30°C. In this work, we report that the O135: K−:H− EIEC strain HN280 and
S. flexneri
SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the
apy
gene. In addition, the
S. flexneri
strains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the
phoN-Sf
gene. Both
apy
and
phoN-Sf
are pINV-carried loci whose contribution to the pathogenicity of enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both the
apy
and the
phoN-Sf
genes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of
virB
transcription) expressed the
apy
gene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Δ
hns
deletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of
virB
transcription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb
−
) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of
virF
or
virB
and by the presence of the intact
apy
locus or intact
apy
and
phoN-Sf
loci were detected among Crb
−
mutants of HN280 and SFZM53, respectively. While all Crb
−
apy
+
mutants of HN280 failed to produce apyrase, Crb
−

apy
+
phoN-Sf

+
mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying cloned
virF
(pMYSH6504) or
virB
(pBN1) into Crb
−
mutants of HN280 and SFZM53 lacking
virF
or
virB
, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes,
apy
is another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of enteroinvasive bacteria.
Plasminogen Binding and Activation at the Surface of
Helicobacter pylori
CCUG 17874
Authors: Pantzar Martina , Ljungh Åsa , Wadström Torkel 
Published: December 2019

ABSTRACT

The binding of iodine-labelled plasminogen to
Helicobacter pylori
CCUG 17874 was characterized. Inhibition of the binding was observed after preincubation of
H. pylori
cells with nonradiolabelled plasminogen, lysine, or the lysine analogue ɛ-aminocaproic acid. Fragments of plasminogen, kringles 1 to 3, kringle 4, and mini-plasminogen, were also studied as potential inhibitors. Mini-plasminogen caused total inhibition of the plasminogen binding, while the other fragments caused only partial inhibition. These findings suggest that
H. pylori
binds specifically the fifth kringle structure of the plasminogen molecule. Plasminogen binding to
H. pylori
seems to be independent of culture media and independent of the presence of the cytotoxin-associated CagA antigen. Immunoblot analysis identified two plasminogen binding proteins of 57 and 42 kDa. Scatchard plot analysis revealed one binding mechanism with a

K
d

value of 7 × 10
−7
M. Conversion of
H. pylori
cell-bound plasminogen to plasmin in the presence of a tissue-type plasminogen activator was demonstrated by digestion of the chromogenic substrate S-2251. No activation was noted when plasminogen or tissue-type plasminogen activator was incubated with
H. pylori
cells alone. Formation of
H. pylori
cell surface-bound plasmin may be important to provide a powerful proteolytic mechanism for gastric tissue penetration in type B gastritis and peptic ulcer disease, since plasmin degrades not only fibrin but also extracellular matrix proteins such as various collagens and fibronectin.
Mutational Analysis of Superantigen Activity Responsible for the Induction of Skin Erythema by Streptococcal Pyrogenic Exotoxin C
Authors: Yamaoka Junichi , Nakamura Eijiro , Takeda Yoshifumi , Imamura Sadao , Minato Nagahiro 
Published: December 2019

ABSTRACTStreptococcal pyrogenic exotoxin C (SPEC), when injected intradermally, induces erythema in unsensitized rabbits. In the present study, we examined whether this erythema induction is due to the T-cell stimulatory activity of SPEC as a superantigen. Analysis by using single-residue mutant SPECs indicated that mutant SPECs Y15I, A16E, and Y17I, in which tyrosine 15, alanine 16, and tyrosine 17 were replaced with isoleucine, glutamic acid, and isoleucine, respectively, exhibited significantly reduced mitogenic activity for Vβ2+human T cells in vitro, and Y15I showed as much as a 1,000-fold reduction. Y15I mutant SPEC, however, retained the ability to bind to major histocompatibility complex class II antigen and to form a homodimer, implying that residue 15 is critically important for the interaction of SPEC with T-cell antigen receptor β chains. When injected intradermally into normal rabbits, wild-type SPEC induced a characteristic erythema after 3 h in a dose-dependent fashion, which was associated with polymorphonuclear and mononuclear cell infiltration. This erythema formation was found to be severely suppressed by systemic pretreatment with cyclosporin A, suggesting the involvement of host T cells. Y15I mutant SPEC exhibited nearly 1,000-fold less erythema induction in vivo than wild-type SPEC. Altogether, the present results strongly suggest that erythema induction in rabbits by SPEC is attributable mostly to its T-cell stimulatory activity as a superantigen.
CD95 Expression in Aged Mice Infected with Tuberculosis
Authors: Roberts A. D. , Orme Ian M. 
Published: December 2019

ABSTRACTThe interaction between CD95 and its ligand is an important homeostatic mechanism that leads to the induction of apoptosis in activated T cells. In view of recent evidence that this pathway might be defective in aged mice, this study investigated CD95 expression on T cells in old mice activated by infection withMycobacterium tuberculosis. The results of the study do not support the hypothesis that CD95 is poorly expressed on CD4 T cells from old mice; instead, it was found that similar numbers of T cells from young and old mice expressed CD95, with the intensity of expression if anything higher on the cells from the old mice. In addition, the study demonstrated that changes in CD44 and CD45RB expression previously observed in young infected mice proceeded in a similar fashion in old animals and, as would be predicted, that CD95hiexpression was primarily associated with CD4 T cells expressing the activated CD44hiCD45RBhiphenotype.
Acquisition of Iron byGardnerella vaginalis
Authors: Jarosik Gregory P. , Land Carol Beth , Duhon Patrice , Chandler Roderick , Mercer Tammy 
Published: December 2019

ABSTRACTSixGardnerella vaginalisstrains were examined for the ability to utilize various iron-containing compounds as iron sources. In a plate bioassay, all six strains acquired iron from ferrous chloride, ferric chloride, ferrous sulfate, ferric ammonium citrate, ferrous ammonium sulfate, bovine and equine hemin, bovine catalase, and equine, bovine, rabbit, and human hemoglobin. All six strains also acquired iron from human lactoferrin, but not from human transferrin, as determined by a liquid broth growth assay. Siderophore production was detected in eightG. vaginalisstrains by the chrome azurol S universal chemical assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cytoplasmic membrane proteins isolated fromG. vaginalis594 grown under iron-replete and iron-restricted conditions revealed several iron-regulated proteins ranging in molecular mass from 33 to 94 kDa. These results indicate thatG. vaginalismay acquire iron from iron salts and host iron compounds.
Expression of CD14 by Hepatocytes: Upregulation by Cytokines during Endotoxemia
Authors: Liu Shubing , Khemlani Lajwanti S. , Shapiro Richard A. , Johnson Mark L. , Liu Kaihong , Geller David A. , Watkins Simon C. , Goyert Sanna M. , Billiar Timothy R. 
Published: December 2019

ABSTRACTStudies were undertaken to examine hepatocyte CD14 expression during endotoxemia. Our results show that lipopolysaccharide (LPS) treatment in vivo caused a marked upregulation in CD14 mRNA and protein levels in rat hepatocytes. Detectable increases in mRNA were seen as early as 1.5 h after LPS treatment; these increases peaked at 20-fold by 3 h and returned to baseline levels by 24 h. In situ hybridization localized the CD14 mRNA expression to hepatocytes both in vitro and in vivo. Increases in hepatic CD14 protein levels were detectable by 3 h and peaked at 12 h. Hepatocytes from LPS-treated animals expressed greater amounts of cell-associated CD14 protein, and more of the soluble CD14 was released by hepatocytes from LPS-treated rats in vitro. The increases in hepatocyte CD14 expression during endotoxemia occurred in parallel to increases of CD14 levels in plasma. To provide molecular identification of the hepatocyte CD14, we cloned the rat liver CD14 cDNA. The longest clone consists of a 1,591-bp insert containing a 1,116-bp open reading frame. The deduced amino acid sequence is 372 amino acids long, has 81.8 and 62.8% homology to the amino acid sequences of mouse and human CD14, respectively, and is identical to the rat macrophage CD14. The expressed CD14 protein from this clone was functional, as indicated by NF-κB activation in response to LPS and fluorescein isothiocyanate-LPS binding in CHO cells stably transfected with rat CD14. A nuclear run-on assay showed that CD14 transcription rates were significantly increased in hepatocytes from LPS-treated animals, indicating that the upregulation in CD14 mRNA levels observed in rat hepatocytes after LPS treatment is dependent, in part, on increased transcription. In vitro and in vivo experiments indicated that interleukin-1β and/or tumor necrosis factor α participate in the upregulation of CD14 mRNA levels in hepatocytes. Our data indicate that hepatocytes express CD14 and that hepatocyte CD14 mRNA and protein levels increase rapidly during endotoxemia. Our observations also support the idea that soluble CD14 is an acute-phase protein and that hepatocytes could be a source for soluble CD14 production.
Differential Growth Characteristics and Streptomycin Susceptibility of Virulent and Avirulent
Mycobacterium tuberculosis
Strains in a Novel Fibroblast-Mycobacterium Microcolony Assay
Authors: Byrd Thomas F. , Green Gary M. , Fowlston Sharon E. , Lyons C. Rick 
Published: December 2019

ABSTRACT

The ability to spread from cell to cell may be an important virulence determinant of
Mycobacterium tuberculosis
. An in vitro assay was developed to characterize this ability among four strains of
M. tuberculosis
: the attenuated strain H37Ra, the virulent strains H37Rv and Erdman, and a virulent clinical isolate (Stew). Confluent monolayers of human skin fibroblasts were infected with these strains and overlaid with agar-medium.
M. tuberculosis
infection developed over 21 days as microcolonies originating within the plane of the fibroblasts. Microcolonies of the virulent strains had an elongated appearance and exhibited extensive cording. The cords appeared to invade adjacent cells within the plane of the monolayer. Microcolony diameter of the Erdman strain was significantly larger than that of the other virulent strains, indicating that virulent strains can have distinguishing phenotypes in this assay. In contrast, avirulent H37Ra microcolonies were rounded and noncorded. H37Ra microcolonies were significantly smaller than those of the virulent strains. Microcolony diameter of the virulent strains was not reduced by the extracellularly acting antibiotic streptomycin at concentrations of up to 5.0 μg/ml. In contrast, H37Ra microcolony size was reduced at concentrations as low as 0.5 μg/ml. Growth of all strains was similarly inhibited by 1.0 μg of streptomycin per ml in fibroblast-conditioned tissue culture medium alone. When fibroblasts were infected with the
M. tuberculosis
strains without an agar overlay, with and without streptomycin, numbers of CFU mirrored the changes observed in the microcolony assay. There was a statistically significant decrease in H37Ra CFU compared to virulent strains after treatment with streptomycin. These differences between H37Ra and virulent strains in human fibroblasts suggest that H37Ra may be lacking a virulence determinant involved in cell-to-cell spread of
M. tuberculosis
.