bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of the
tpr
Protease Gene of
Porphyromonas gingivalis
Is Regulated by Peptide Nutrients
Authors: Lu Biqing , McBride Barry C. 
Published: December 2019

ABSTRACT

The Tpr protease of
Porphyromonas gingivalis
W83 is a membrane-associated enzyme capable of hydrolyzing chromogenic substrates for trypsin and bacterial collagenases. A previous study by us indicated that Tpr expression was increased under conditions of nutrient limitation. In the present study, we further characterized expression of the
tpr
gene using a
tpr
::
lacZ
reporter gene construct under a range of nutrient conditions. In
P. gingivalis
, transcription of
tpr
was initiated 215 bp upstream of the coding region and regulation of
tpr
expression was at the level of transcription. Deletion mutations in the
tpr
upstream region identified the promoter region immediately upstream of the transcription start site, determined by primer extension analysis. Three identical 17-bp direct repeats identified within the 5′ end of
tpr
mRNA were involved in
tpr
regulation. In an
Escherichia coli
background,
tpr
transcription was initiated after an AT-rich region upstream of
tpr
but not at the
P. gingivalis
start site. Tpr expression in
P. gingivalis
was suppressed by the addition of peptide and protein nutrients to a peptide-limited growth medium but was only slightly affected by addition of free amino acids. Low-molecular-weight fractions of brain heart infusion rich in phenylalanine, proline, and alanine had the greatest inhibitory effects on expression of the
tpr
::
lacZ
construct. Addition of the dipeptide phenylalanyl-phenylalanine to the growth medium resulted in a 10-fold decrease in
tpr
expression. This suggests that specific phenylalanine-containing peptides are a major factor controlling Tpr expression. Neither hemin starvation, heat shock, nor pH change had significant effects on Tpr expression.
Lipoprotein Release by Bacteria: Potential Factor in Bacterial Pathogenesis
Authors: Zhang Hongwei , Niesel David W. , Peterson Johnny W. , Klimpel Gary R. 
Published: December 2019

ABSTRACTLipoprotein (LP) is a major component of the outer membrane of bacteria in the familyEnterobacteriaceae. LP induces proinflammatory cytokine production in macrophages and lethal shock in LPS-responsive and -nonresponsive mice. In this study, the release of LP from growing bacteria was investigated by immuno-dot blot analysis. An immuno-dot blot assay that could detect LP at levels as low as 100 ng/ml was developed. By using this assay, significant levels of LP were detected in culture supernatants of growingEscherichia colicells. During mid-logarithmic growth, approximately 1 to 1.5 μg of LP per ml was detected in culture supernatants fromE. coli. In contrast, these culture supernatants contained 5 to 6 μg/ml of lipopolysaccharide (LPS). LP release was not unique toE. coli. Salmonella typhimurium,Yersinia enterocolitica, and two pathogenicE. colistrains also released LP during in vitro growth. Treatment of bacteria with the antibiotic ceftazidime significantly enhanced LP release. Culture supernatants from 5-h cultures ofE. coliwere shown to induce in vitro production of interleukin-6 (IL-6) by macrophages obtained from LPS-nonresponsive C3H/HeJ mice. In contrast, culture supernatants from anE. coliLP-deletion mutant were significantly less efficient at inducing IL-6 production in C3H/HeJ macrophages. These results suggest, for the first time, that LP is released from growing bacteria and that this released LP may play an important role in the induction of cytokine production and pathologic changes associated with gram-negative bacterial infections.
Neisseria gonorrhoeae
Heme Biosynthetic Mutants Utilize Heme and Hemoglobin as a Heme Source but Fail To Grow within Epithelial Cells
Authors: Turner Paul C. , Thomas Christopher E. , Elkins Christopher , Clary Susan , Sparling P. F. 
Published: December 2019

ABSTRACT

Many bacterial pathogens, including pathogenic neisseriae, can use heme as an iron source for growth. To study heme utilization by
Neisseria gonorrhoeae
, two heme biosynthetic mutants were constructed, one with a mutation in
hemH
(the gene encoding ferrochelatase) and one with a mutation in
hemA
(the gene encoding γ-glutamyl tRNA reductase). The
hemH
mutant failed to grow without an exogenous supply of heme or hemoglobin, whereas the
hemA
mutant failed to grow unless heme, hemoglobin, or heme precursors were present. Growth of the mutants with hemoglobin required expression of the hemoglobin receptor (HpuAB) and was TonB dependent. However, growth with heme required neither HpuAB nor TonB. An
fbpA
mutant grew normally when either heme or hemoglobin was present in the medium. The heme biosynthetic mutants showed reduced intracellular survival, compared to the parent strain, within A-431 endocervical epithelial cell cultures. These studies demonstrate that in addition to synthesizing their own heme,
N. gonorrhoeae
strains are able to internalize and utilize exogenous heme independently of FbpA but appear unable to obtain heme from within epithelial cells for growth.
Acquired Resistance but Not Innate Resistance toMycobacterium bovisBacillus Calmette-Guérin Is Compromised by Interleukin-12 Ablation
Authors: Thompson-Snipes LuAnn , Skamene Emil , Radzioch Danuta 
Published: December 2019

ABSTRACTInterleukin-12 (IL-12) is one of the first cytokines produced by macrophages, key mediators of innate resistance, during the host’s immune response to infections. Therefore, in this study we propose that IL-12 has an important role in the early phase of the immune response toMycobacterium bovisBCG. IL-12 has been shown to enhance the maturation of protective Th1 cells and gamma interferon (IFN-γ) production during mycobacterial infection. Therefore, it may play a crucial role during the immune phase of infection as well. To examine the role of IL-12 in both the innate and the immune phase of infection, we compared BCG-resistant mice, B10.A (Bcgr), to the susceptible congenic strain B10.A (Bcgs) following administration of a blocking monoclonal antibody to IL-12 (10F6). Anti-IL-12-treated susceptible animals exhibited a two- to threefold increase in spleen CFU by day 21. In contrast, anti-IL-12 treatment had little or no effect on the response of the genetically resistant animals to infection. The B10.A (Bcgr) but not the B10.A (Bcgs) mice had an increase in IFN-γ mRNA relative to baseline levels as early as day 1 of infection irrespective of anti-IL-12 treatment. By day 14, B10.A (Bcgr) mice showed a decrease in IFN-γ mRNA while the B10.A (Bcgs) mice showed a significant increase in IFN-γ mRNA levels. Thus, during BCG infection, the B10.A (Bcgr) mice mount an early IFN-γ response against BCG whereas the B10.A (Bcgs) mice have a delayed IFN-γ response correlating with their genetic permissiveness expressed as an increased mycobacterial load by day 21. Overall, our data demonstrate that the inherent resistance of B10.A (Bcgr) mice to mycobacteria does not depend on optimal levels of IL-12 to maintain effective control of the bacteria, whereas IL-12 is important for the susceptible animals’ response to BCG during the peak of infection.
Subcellular Localization and Cytotoxic Activity of the GroEL-Like Protein Isolated from
Actinobacillus actinomycetemcomitans
Authors: Goulhen F. , Hafezi A. , Uitto V.-J. , Hinode D. , Nakamura R. , Grenier D. , Mayrand D. 
Published: December 2019

ABSTRACT

The subcellular locations, ultrastructure, and cytotoxic activity of the GroEL-like protein from
Actinobacillus actinomycetemcomitans
were investigated. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that synthesis of the GroEL-like protein is substantially increased after a thermal shock. Analysis of the purified native GroEL-like protein by transmission electron microscopy revealed the typical 14-mer cylindrical molecule, which had a diameter of about 12 nm.
A. actinomycetemcomitans
cells grown at 35°C and heat shocked at 43°C were fractionated, and fractions were separated by SDS-PAGE and analyzed by Western immunoblotting using antibodies to GroEL- and DnaK-like proteins. The GroEL-like protein was found in both the soluble and membrane fractions, whereas the DnaK-like protein was mostly found in the cytoplasm. An increase in specific proteins, including the GroEL- and DnaK-like proteins, was found in heat-shocked cells. The subcellular localization of the GroEL-like protein was examined by immunoelectron microscopy of whole cells. More GroEL-like protein was detected in stressed cells than in unstressed cells, and most of it was found not directly associated with outer membranes but rather in extracellular material. The native GroEL-like protein was assessed for cytotoxic activities. The GroEL-like protein increased the proliferation of periodontal ligament epithelial cells at concentrations between 0.4 and 1.0 μg/ml. The number of cells in the culture decreased significantly at higher concentrations. A cell viability assay using HaCaT epithelial cells indicated that the GroEL-like protein was strongly toxic for the cells. These studies suggest the extracellular nature of the GroEL-like protein and its putative role in disease initiation.
Helicobacter pyloriLipopolysaccharide Binds to CD14 and Stimulates Release of Interleukin-8, Epithelial Neutrophil-Activating Peptide 78, and Monocyte Chemotactic Protein 1 by Human Monocytes
Authors: Bliss Charles M. , Golenbock Douglas T. , Keates Sarah , Linevsky Joanne K. , Kelly Ciarán P. 
Published: December 2019

ABSTRACTHelicobacter pylorigastritis is characterized by leukocyte infiltration of the gastric mucosa. The aims of this study were to determine whetherH. pylori-derived factors stimulate chemokine release from human monocytes and to ascertain whetherH. pylorilipopolysaccharide (LPS) may be responsible for this effect. Human peripheral blood monocytes were exposed to anH. pyloriwater extract (HPE) or to purifiedH. pyloriLPS. Levels of the chemokines interleukin-8 (IL-8), epithelial neutrophil-activating peptide 78 (ENA-78), and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. The contribution ofH. pyloriLPS to monocyte activation was determined by using the LPS antagonistRhodobacter sphaeroideslipid A (RSLA) and a blocking monoclonal antibody to CD14 (60bca). HPE increased monocyte secretion of IL-8, ENA-78, and MCP-1. Heat treatment of HPE did not reduce its ability to activate monocytes. PurifiedH. pyloriLPS also stimulated monocyte chemokine production but was 1,000-fold less potent thanSalmonella minnesotalipid A. RSLA blockedH. pyloriLPS-induced monocyte IL-8 release in a dose-dependent fashion (maximal inhibition 82%,P< 0.001). RSLA also inhibited HPE-induced IL-8 release (by 93%,P< 0.001). The anti-CD14 monoclonal antibody 60bca substantially inhibited IL-8 release from HPE-stimulated monocytes (by 88%,P< 0.01), whereas the nonblocking anti-CD14 monoclonal antibody did not. These experiments with potent and specific LPS inhibitors indicate that the main monocyte-stimulating factor in HPE is LPS.H. pyloriLPS, acting through CD14, stimulates human monocytes to release the neutrophil-activating chemokines IL-8 and ENA-78 and the monocyte-activating chemokine MCP-1. Despite its low relative potency,H. pyloriLPS may play an important role in the pathogenesis ofH. pylorigastritis.
Immunogenicity and Protective Efficacy of the WI-1 Adhesin ofBlastomyces dermatitidis
Authors: Wüthrich Marcel , Chang Wun-ling , Klein Bruce S. 
Published: December 2019

ABSTRACTPeople infected withBlastomyces dermatitidisdevelop strong immunity to the yeast surface adhesin WI-1, including antibody responses to the adhesive domain, a 25-amino-acid repeat, and cellular responses to the N terminus. We studied the immunogenicity of WI-1 and the ability of anti-WI-1 immune responses to protect against lethal pulmonary infection in mice. WI-1 immunization, given in Freund’s adjuvant subcutaneously in two doses 2 weeks apart, evoked delayed hypersensitivity responses in a concentration-dependent manner. Immunized mice also had anti-WI-1 antibody responses, with titers reaching an endpoint dilution of approximately 1:800,000. Anti-WI-1 immunoglobulin G (IgG) antibody subclasses were IgG1 > IgG2b > IgG2a > IgG3, indicating a mixed T helper 1 and T helper 2 immune response. In protection experiments, WI-1 immunization significantly prolonged the survival of C57BL/6 and BALB/c mice compared to controls following intranasal administration of a lethal dose ofB. dermatitidisyeasts (Kaplan-Meier survival curvePvalues of 0.027 to 0.0002) and also protected a proportion of the animals from death due to progressive pulmonary blastomycosis. Taken together, our results suggest that administration of WI-1 raises antibody and cell-mediated immune responses, which enhance resistance against pulmonary infection withB. dermatitidis. Mechanisms of vaccine-induced resistance require further investigation.
Effect ofClostridium difficileToxin A on Human Colonic Lamina Propria Cells: Early Loss of Macrophages Followed by T-Cell Apoptosis
Authors: Mahida Y. R. , Galvin A. , Makh S. , Hyde S. , Sanfilippo L. , Borriello S. P. , Sewell H. F. 
Published: December 2019

ABSTRACTWe have previously shown thatClostridium difficiletoxin A induces detachment of human colonic epithelial cells from the basement membrane and subsequent cell death by apoptosis. Because these cells require adhesion-dependent signalling from the extracellular matrix for survival, their detachment from the basement membrane by other means also induces apoptosis. The role of toxin A in the induction of apoptosis therefore remains to be determined. In addition, sensitivities toC. difficiletoxin A of lamina propria lymphocytes, macrophages, and eosinophils, which lie below the surface epithelium, are not known. In contrast to epithelial cells, these lamina propria cells do not require adhesion-dependent signalling from the extracellular matrix for survival, and this may allow the mechanisms of toxin A-induced cell death to be further investigated. The aim of this study was to investigate the effect of purifiedC. difficiletoxin A on human colonic lamina propria T cells, macrophages, and eosinophils. We show thatC. difficiletoxin A induces loss of viability in isolated colonic lamina propria cell preparations containing the three different cell types in a dose- and time-dependent fashion. Exposure to high concentrations of the toxin led to loss of macrophages within 72 h. T-lymphocyte and eosinophil cell death was prominent at later time points and occurred by apoptosis. Exposure to toxin A also induced the production of tumor necrosis factor alpha by the isolated colonic lamina propria cells. However, the presence of neutralizing antibodies to this cytokine did not influenceC. difficiletoxin A-induced T-cell apoptosis. Moreover, purified T cells also underwent apoptosis following exposure to toxin A, implying that apoptosis occurred as a consequence of a direct interaction between T cells and the toxin. Our studies suggest thatC. difficiletoxin A is capable of suppressing human colonic mucosal immune responses by inducing early loss of macrophages followed by T-cell apoptosis.
Oral Immunization with aSalmonella typhimuriumVaccine Vector Expressing Recombinant EnterotoxigenicEscherichia coliK99 Fimbriae Elicits Elevated Antibody Titers for Protective Immunity
Authors: Ascón Miguel A. , Hone David M. , Walters Nancy , Pascual David W. 
Published: December 2019

ABSTRACTBovine enterotoxigenicEscherichia coli(ETEC) continues to cause mortality in piglets and newborn calves. In an effort to develop a safe and effective vaccine for the prevention of F5+ETEC infections, a balanced lethalasd+plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd+. Introduction of this plasmid into an attenuatedSalmonella typhimurium Δaro Δasdstrain, H683, resulted in strain AP112, which stably expressesE. coliK99 fimbriae. A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks. IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer’s patches (PP), and spleens of vaccinated and control BALB/c mice. This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen. These antibodies were important for protective immunity. One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a controlSalmonellavector. These results confirm that oralSalmonellavaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity.
The EspB Protein of Enteropathogenic
Escherichia coli
Is Targeted to the Cytoplasm of Infected HeLa Cells
Authors: Taylor Kathleen A. , O’Connell Colin B. , Luther Paul W. , Donnenberg Michael S. 
Published: December 2019

ABSTRACT

The EspB protein of enteropathogenic
Escherichia coli
(EPEC) is exported via a type III secretion apparatus. EspB is critical for signaling the host cell and for the development of the attaching and effacing lesion characteristic of EPEC infection. We used cellular fractionation and confocal laser scanning microscopy to determine the cellular location of EspB during infection of HeLa cells. Both methods indicated that EspB is targeted to the cytoplasm of infected cells. Using mutants, we found that EspB targeting to the host cell cytoplasm requires the type III secretion apparatus and the secreted proteins EspA and EspD, but not intimin. These results provide insights into the function of the type III secretion apparatus of EPEC and the functions of the Esp proteins.
Role of γδ T Cells in Immunopathology of PulmonaryMycobacterium aviumInfection in Mice
Authors: Saunders Bernadette M. , Frank Anthony A. , Cooper Andrea M. , Orme Ian M. 
Published: December 2019

ABSTRACTSeveral studies have shown that γδ T cells influence granuloma development after infection with intracellular pathogens. The role of γδ T cells in controlling the influx of inflammatory cells into the lung afterMycobacterium aviuminfection was therefore examined with gene-disrupted mice (K/O). The mice were infected with eitherM. avium724, a progressively replicating highly virulent strain ofM. avium, or withM. avium2-151 SmT, a virulent strain that induces a chronic infection. γδ-K/O mice infected withM. avium2-151 SmT showed early enhanced bacterial growth within the lung compared to the wild-type mice, although granuloma formation was similar in both strains. γδ-K/O mice infected withM. avium724 showed identical bacterial growth within the lung compared to the wild-type mice, but they developed more-compact lymphocytic granulomas and did not show the extensive neutrophil influx and widespread tissue necrosis seen in wild-type mice. These data support the hypothesis that isolates ofM. aviumthat induce protective T-cell-specific immunity are largely unaffected by the absence of γδ T cells. Whereas with bacterial strains that induce poor protective immunity, the absence of γδ T cells led to significant reductions in both the influx of neutrophils and tissue damage within the lungs of infected mice.
Protection by CD4 or CD8 T Cells against PulmonaryMycobacterium bovisBacillus Calmette-Guérin Infection
Authors: Xing Zhou , Wang Jun , Croitoru Kenneth , Wakeham Julia 
Published: December 2019

ABSTRACTMice deficient in CD8 T cells demonstrated levels of Th1 cytokines and granulomatous responses in the lungs very similar to those demonstrated by normal control mice and were fully capable of controlling pulmonary mycobacterial infection byMycobacterium bovisBCG as assessed at day 37 postinfection. In comparison, mice deficient in CD4 T cells had similar levels of interleukin-12 (IL-12) and tumor necrosis factor alpha but lower levels of gamma interferon in the lungs and were still able to mount tissue granulomatous responses and control pulmonary mycobacterial infection. In contrast, IL-12−/−mice with impaired CD4 and CD8 T-cell responses had a markedly weakened control of infection, whereas SCID mice deficient in all T cells succumbed to such pulmonary mycobacterial infections.
Structural and Antigenic Characteristics of
Streptococcus sobrinus
Glucan Binding Proteins
Authors: Smith Daniel J. , King William F. , Wu Christine D. , Shen Bella I , Taubman Martin A. 
Published: December 2019

ABSTRACT

Three purified glucan binding proteins (GBP-2, GBP-3, and GBP-5) from
Streptococcus sobrinus
6715 were compared structurally by mass spectroscopy of tryptic fragments and antigenically by Western blot analysis with rat antisera to each GBP or to peptides containing putative glucan binding epitopes of mutans streptococcal glucosyltransferases. Structural and antigenic analyses indicated that GBP-3 and GBP-5 are very similar but that both are essentially unrelated to GBP-2. None of these
S. sobrinus
GBPs appeared to have a strong antigenic relationship with GBPs from
Streptococcus mutans
. Thus,
S. sobrinus
GBP-2 and GBP-3 appear to be distinct proteins with potentially different functions.
S. sobrinus
GBP-5 may be a proteolytic fragment of GBP-3, or, alternatively, the genes coding for these proteins may be closely related.
Characterization of a 34-Kilodalton Protein of
Mycobacterium leprae
That Is Isologous to the Immunodominant 34-Kilodalton Antigen of
Mycobacterium paratuberculosis
Authors: Silbaq Fauzi S. , Cho Sang-Nae , Cole Stewart T. , Brennan Patrick J. 
Published: December 2019

ABSTRACT

During DNA sequence analysis of cosmid L373 from the
Mycobacterium leprae
genome, an open reading frame of 1.4 kb encoding a protein with some homology to the immunodominant 34-kDa protein of
Mycobacterium paratuberculosis
, but lacking significant serological activity, was detected. The DNA sequence predicted a signal peptide with a modified lipoprotein consensus sequence, but the protein proved to be devoid of lipid attachment.