bluuebunny/crossref_metadata_2025 huggingface dataset:
T-Cell Recognition of Mycobacterial GroES Peptides in Thai Leprosy Patients and Contacts
Authors: Chua-Intra Boosbun , Peerapakorn Somchai , Davey Nick , Jurcevic Stipo , Busson Marc , Vordermeier H. Martin , Pirayavaraporn Charoon , Ivanyi Juraj
Published: December 2019

We report here the mapping of T-cell-stimulatory determinants of the GroES 10-kDa heat shock protein homologues fromMycobacterium lepraeandMycobacterium tuberculosis, which are known as major immunogens in mycobacterial infections. Peripheral blood mononuclear cells (PBMC) from treated tuberculoid leprosy or lepromatous leprosy patients and from healthy household or hospital staff contacts of the patients were cultured with 20 16-mer peptides covering the entire sequences of bothM. lepraeandM. tuberculosisGroES. The total number of recognized peptides was found to be the largest in family contacts, while responder frequencies to the individual tested peptides varied (5 to 80%) with specificity between the patient and contact groups. Proliferative responses to some peptides showed positive or negative associations of low statistical significance with DR and DQ alleles, though responses to most GroES peptides were genetically permissive. Notably, the sequence of the 25–40 peptide ofM. leprae, but not that ofM. tuberculosis, was more frequently stimulatory in tuberculoid leprosy patients than in either group of sensitized healthy contacts. This peptide bound to a number of HLA-DR molecules, of which HLA-DRB5*0101 had the strongest affinity. The epitope core binding to this allele was localized to the 29-to-37 sequence, and its key residue was localized to theM. leprae-specific glutamic acid at position 32. This epitope may be of interest for the development of a blood test- or skin test-based diagnostic reagent for tuberculoid leprosy, subject to further clinical evaluation in untreated patients.
Effects of Orally Administered Epidermal Growth Factor on Enteropathogenic
Escherichia coli
Infection in Rabbits
Authors: Buret Andre , Olson Merle E. , Gall D. Grant , Hardin James A.
Published: December 2019

The increased intestinal absorption induced by epidermal growth factor (EGF) is associated with diffuse lengthening of brush border microvilli. The aim of this study was to examine the in vivo effects of oral administration of EGF during infection with enteropathogenic
Escherichia coli
. New Zealand White rabbits (4 weeks old) received orogastric EGF daily starting 3 days prior to infection with enteropathogenic
E. coli
RDEC-1 and were compared with sham-treated infected animals and uninfected controls. Weight gain, food intake, fecal
E. coli
, and stool consistency were assessed daily. On day 10, segments of jejunum, ileum, proximal, and distal colon were assessed for gram-negative bacterial colonization, disaccharidase activities, and epithelial ultrastructure. Effects of EGF on
E. coli
RDEC-1 proliferation were studied in vitro.
E. coli
RDEC-1 caused diarrhea and reduced weight gain. Seven days postinfection, the small and large intestines were colonized with numerous bacteria, brush border microvilli were disrupted, and maltase and sucrase activities were significantly reduced in the jejunum. Daily treatment with EGF prevented the occurrence of diarrhea and reduction of weight gain. These effects were associated with significant inhibition of
E. coli
colonization in the small and large intestine, improved jejunal maltase and sucrase activities and reduced microvillous injury. EGF did not affect the proliferation of
E. coli
in vitro. The findings suggest that EGF protects the gastrointestinal tract against colonization by enteropathogenic
E. coli.
Interleukin-12 Has a Role in Mediating Resistance of Murine Strains to Tyzzer’s Disease
Authors: Van Andel Roger A. , Hook Reuel R. , Franklin Craig L. , Besch-Williford Cynthia L. , Riley Lela K.
Published: December 2019

Clostridium piliformeinduces enterohepatic disease in many domestic and laboratory animal species. Susceptibility to infection is known to vary with the immune status and strain of the host, but little is known about specific immune mechanisms that regulate this disease. To evaluate host control ofC. piliformeinfection, we examined the role of interleukin-12 (IL-12) both in the control of and in the response to murineC. piliformeinfection. For this study, 3-week-oldC. piliforme-resistant C57BL/6 or -susceptible DBA/2 mice were infected intravenously with either the toxic H1 or the nontoxic M1C. piliformeisolate. Serum and liver samples were collected prior toC. piliformeinoculation (day 0) and at days 1, 3, 7, 14, and 28 postinoculation. Evaluation of hepatic IL-12 p40 mRNA expression by reverse transcription-PCR and of total-IL-12 protein levels in serum by enzyme-linked immunosorbent assay revealed thatC. piliformeinduced elevations in both hepatic p40 mRNA and serum total-IL-12 levels at all times postinoculation. Elevations were similar with both toxic and nontoxicC. piliformeisolates. Levels of total IL-12 in serum were significantly (P< 0.05) higher in C57BL/6 mice than in DBA/2 mice. Additional experiments were performed in which polyclonal antibody treatment was used to neutralize IL-12 in mice of both strains prior to intravenous inoculation with toxicC. piliformeH1. IL-12 neutralization increased the severity of Tyzzer’s disease at day 3 postinoculation in both mouse strains, but the degree of increase was greater in C57BL/6 mice than in DBA/2 mice.
Production of Interleukin-12 by Murine Macrophages in Response to Bacterial Peptidoglycan
Authors: Lawrence Christine , Nauciel Charles
Published: December 2019

Peptidoglycan (PG), a component of the bacterial cell wall, has various immunomodulating activities, including the capacity to induce delayed-type hypersensitivity reactions to antigens administered in Freund’s adjuvant. We report that PG induces interleukin-12 (IL-12) mRNA production and IL-12 secretion by mouse macrophages. The capacity of PG to induce IL-12 production, like its previously reported immunomodulating activities, was dependent on the structure of its peptide subunit. PG fromBacillus megateriumandStaphylococcus aureusinduced IL-12 production, whereas PG fromMicrococcus luteusandCorynebacterium poinsettiaedid not. The ability of most bacterial PGs to induce IL-12 production suggests that they play an important role in triggering host defense mechanisms against bacterial infections.
A
Chlamydia pneumoniae
Component That Induces Macrophage Foam Cell Formation Is Chlamydial Lipopolysaccharide
Authors: Kalayoglu Murat V. , Byrne Gerald I.
Published: December 2019

Chlamydia pneumoniae
infection is associated with atherosclerotic heart and vessel disease, but a causal relationship between this pathogen and the disease process has not been established. Recently, it was reported that
C. pneumoniae
induces human macrophage foam cell formation, a key event in early atheroma development, suggesting a role for the organism in atherogenesis. This study further examines
C. pneumoniae
-induced foam cell formation in the murine macrophage cell line RAW-264.7. Infected RAW cells accumulated cholesteryl esters when cultured in the presence of low-density lipoprotein in a manner similar to that described for human macrophages. Exposure of
C. pneumoniae
elementary bodies to periodate, but not elevated temperatures, inhibited cholesteryl ester accumulation, suggesting a role for chlamydial lipopolysaccharide (cLPS) in macrophage foam cell formation. Purified cLPS was found to be sufficient to induce cholesteryl ester accumulation and foam cell formation. Furthermore, the LPS antagonist lipid X inhibited
C. pneumoniae
and cLPS-induced lipid uptake. These data indicate that cLPS is a
C. pneumoniae
component that induces macrophage foam cell formation and suggest that infected macrophages chronically exposed to cLPS may accumulate excess cholesterol to contribute to atheroma development.
New Scavenger Receptor-Like Receptors for the Binding of Lipopolysaccharide to Liver Endothelial and Kupffer Cells
Authors: van Oosten Marijke , van de Bilt Erika , van Berkel Theo J. C. , Kuiper Johan
Published: December 2019

Lipopolysaccharide (LPS) is cleared from the blood mainly by the liver. The Kupffer cells are primarily responsible for this clearance; liver endothelial and parenchymal cells contribute to a lesser extent. Although several binding sites have been described, only CD14 is known to be involved in LPS signalling. Among the other LPS binding sites that have been identified are scavenger receptors. Scavenger receptor class A (SR-A) types I and II are expressed in the liver on endothelial cells and Kupffer cells, and a 95-kDa receptor, identified as macrosialin, is expressed on Kupffer cells. In this study, we examined the role of scavenger receptors in the binding of LPS by the liver in vivo and in vitro. Fucoidin, a scavenger receptor ligand, significantly reduced the clearance of125I-LPS from the serum and decreased the liver uptake of125I-LPS about 40%. Within the liver, the in vivo binding of125I-LPS to Kupffer and liver endothelial cells was decreased 72 and 71%, respectively, while the binding of125I-LPS to liver parenchymal cells increased 34% upon fucoidin preinjection. Poly(I) inhibited the binding of125I-LPS to Kupffer and endothelial cells in vitro 73 and 78%, respectively, while poly(A) had no effect. LPS inhibited the binding of acetylated low-density lipoprotein (acLDL) to Kupffer and liver endothelial cells 40 and 55%, respectively, and the binding of oxidized LDL (oxLDL) to Kupffer and liver endothelial cells 65 and 61%, respectively. oxLDL and acLDL did not significantly inhibit the binding of LPS to these cells. We conclude that on both endothelial cells and Kupffer cells, LPS binds mainly to scavenger receptors, but SR-A and macrosialin contribute to a limited extent to the binding of LPS.
Expression of the
tpr
Protease Gene of
Porphyromonas gingivalis
Is Regulated by Peptide Nutrients
Authors: Lu Biqing , McBride Barry C.
Published: December 2019

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

Lipoprotein (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

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

Interleukin-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

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

Helicobacter 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

People 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

We 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

Bovine 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

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

Several 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

Mice 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

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

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.
Translocated Intimin Receptors (Tir) of Shiga-Toxigenic
Escherichia coli
Isolates Belonging to Serogroups O26, O111, and O157 React with Sera from Patients with Hemolytic-Uremic Syndrome and Exhibit Marked Sequence Heterogeneity
Authors: Paton Adrienne W. , Manning Paul A. , Woodrow Matthew C. , Paton James C.
Published: December 2019

The capacity to form attaching and effacing (A/E) lesions on the surfaces of enterocytes is an important virulence trait of several enteric pathogens, including enteropathogenic
Escherichia coli
(EPEC) and Shiga-toxigenic
E. coli
(STEC). Formation of such lesions depends upon an interaction between a bacterial outer membrane protein (intimin) and a bacterially encoded receptor protein (Tir) which is exported from the bacterium and translocated into the host cell membrane. Intimin, Tir, and several other proteins necessary for generation of A/E lesions are encoded on a chromosomal pathogenicity island termed the locus for enterocyte effacement (LEE). Reports of sequence heterogeneity and antigenic variation in the region of intimin believed to be responsible for receptor binding raise the possibility that the receptor itself is also heterogeneous. We have examined this by cloning and sequencing
tir
genes from three different STEC strains belonging to serogroups O26, O111, and O157. The deduced amino acid sequences for the Tir homologues from these strains varied markedly, exhibiting only 65.4, 80.2, and 56.7% identity, respectively, to that recently reported for EPEC Tir. STEC Tir is also highly immunogenic in humans. Western blots of
E. coli
DH5α expressing the various STEC
tir
genes cloned in pBluescript [but not
E. coli
DH5α(pBluescript)] reacted strongly with convalescent sera from patients with hemolytic-uremic syndrome (HUS) caused by known LEE-positive STEC. Moreover, no reaction was seen when the various clone lysates were probed with serum from a patient with HUS caused by a LEE-negative STEC or with serum from a healthy individual. Covariation of exposed epitopes on both intimin and Tir may be a means whereby STEC avoid host immune responses without compromising adhesin-receptor interaction.
Morphological Transition of
Paracoccidioides brasiliensis
Conidia to Yeast Cells: In Vivo Inhibition in Females
Authors: Aristizabal Beatriz H. , Clemons Karl V. , Stevens David A. , Restrepo Angela
Published: December 2019

Clinical paracoccidioidomycosis is 13 times more common in men than in women. Estrogen inhibits the transition of mycelia or conidia (the saprophytic form of
Paracoccidoides brasiliensis
) to yeasts (the parasitic form) in vitro. Here, we show that, in male mice that were infected intranasally (mimicking natural infection) the transition of conidia in bronchoalveolar lavage fluids to intermediate forms and yeasts occurred over 24 to 96 h; CFU and yeasts (shown by histopathology) increased subsequently. In females, transition did not occur and infection cleared. These events in vivo are consistent with epidemiological and in vitro observations, suggesting that female hormones block transition and are responsible for resistance.
Genetic and Immunologic Analyses of PlpE, a Lipoprotein Important in Complement-Mediated Killing ofPasteurella haemolyticaSerotype 1
Authors: Pandher Karamjeet , Confer Anthony W. , Murphy George L.
Published: December 2019

Pasteurella haemolyticaserotype 1 is the bacterium most commonly associated with bovine shipping fever. The presence of antibodies againstP. haemolyticaouter membrane proteins (OMPs) correlates statistically with resistance to experimentalP. haemolyticachallenge in cattle. Until now, specificP. haemolyticaOMPs which elicit antibodies that function in host defense mechanisms have not been identified. In this study, we have cloned and sequenced the gene encoding one such protein, PlpE. Analysis of the deduced amino acid sequence revealed that PlpE is a lipoprotein and that it is similar to anActinobacillus pleuropneumoniaelipoprotein, OmlA. Affinity-purified, anti-PlpE antibodies recognize a protein in all serotypes ofP. haemolyticaexcept serotype 11. We found that intactP. haemolyticaand recombinantE. coliexpressing PlpE are capable of absorbing anti-PlpE antibodies from bovine immune serum, indicating that PlpE is surface exposed inP. haemolyticaand assumes a similar surface-exposed conformation inE. coli. In complement-mediated killing assays, we observed a significant reduction in killing ofP. haemolyticawhen bovine immune serum that was depleted of anti-PlpE antibodies was used as the source of antibody. Our data suggest that PlpE is surface exposed and immunogenic in cattle and that antibodies against PlpE contribute to host defense againstP. haemolytica.
Sulfatide from the Pig Jejunum Brush Border Epithelial Cell Surface Is Involved in Binding of
Escherichia coli
Enterotoxin b
Authors: Rousset Élodie , Harel Josée , Dubreuil J. Daniel
Published: December 2019

Using a quantitative dot blot overlay assay of polyvinylidene difluoride membranes, we investigated the ability of
Escherichia coli
heat-stable enterotoxin b (STb) to bind to various glycolipids of defined structure. STb bound strongly to acidic glycosphingolipids, including sulfatide (or 3′-sulfogalactosylceramide) and several gangliosides, but not significantly to their derivatives, galactosylceramide and asialogangliosides, respectively. STb exhibited the highest binding affinity for sulfatide. STb bound to pure sulfatide in a dose-dependent and saturable manner, with a detection level of a few nanograms. The binding was not inhibited by tetramethylurea, which is a strong disrupter of hydrophobic interactions, or by the anionic sulfated polymer of glucose, dextran sulfate, indicating that the binding is not due solely to either hydrophobic or ionic interactions via the sulfate group of the sulfatide. The specificity of the binding was confirmed by the finding that a 500-fold molar excess of sulfatide inhibited STb binding by approximately 45%, whereas no competition was obtained with galactosylceramide under the same conditions. Taken together, our data indicated that a galactose residue linked to a sulfate group is required for the binding specificity of STb. Then, total lipids extracted either from the mucous layer or from the epithelial cells of the pig jejunum brush border, the natural target of STb, were analyzed by thin-layer chromatography (TLC). Both extracts contained a lipidic molecule with a relative mobility on a TLC plate similar to that of the sulfatide standard. The migrated lipid extracted directly from a preparative TLC plate was confirmed to be sulfatide, as it was recognized by laminin, a sulfated glycolipid binding protein, and by a monoclonal antibody directed against sulfatide. In an overlay assay on PVDF membranes, STb bound to the sulfatide prepared from porcine jejunum as well as to the sulfatide standard. Thus, these findings suggest that the terminal oligosaccharide sequence Gal(3SO
4
)β1- on sulfatide could mediate binding of STb to its target cells and, in support of a recent report (E. Rousset, J. Harel, and J. D. Dubreuil, Microb. Pathog. 24:277–288, 1998), probably terminal sialic acid residue on another glycosphingolipid. Moreover, pretreatment in the ligated intestinal loop assay with laminin or sulfatase altered the biological activity of STb. In summary, we present data indicating that sulfatide represents a functional receptor for the STb toxin.
Interleukin-15 May Be Responsible for Early Activation of Intestinal Intraepithelial Lymphocytes after Oral Infection withListeria monocytogenesin Rats
Authors: Hirose Kenji , Suzuki Hirohiko , Nishimura Hitoshi , Mitani Akio , Washizu Junji , Matsuguchi Tetsuya , Yoshikai Yasunobu
Published: December 2019

Exogenous interleukin-15 (IL-15) stimulates intestinal intraepithelial lymphocytes (i-IEL) from mice to proliferate and produce gamma interferon (IFN-γ) in vitro. To determine whether endogenous IL-15 is involved in activation of i-IEL during intestinal infection, we examined IL-15 synthesis by intestinal epithelial cells (i-EC) after infection withListeria monocytogenesin rats. In in vitro experiments, invasion ofL. monocytogenesinto IEC-6 cells, a rat small intestine epithelial cell line, evidently induced IL-15 mRNA expression coincident with nuclear factor κB (NF-κB) activation, which is essential for IL-15 gene expression. IL-15 synthesis was detected in rat i-EC on day 1 after an oral inoculation ofL. monocytogenesin vivo. The numbers of T-cell receptor (TCR) γδ+T cells, NKR.P1+cells, and CD3+CD8+αα cells in i-IEL were significantly increased on day 1 after oral infection. The i-IEL from infected rats produced larger amounts of IFN-γ upon stimulation with immobilized anti-TCR γδ or anti-NKR.P1 monoclonal antibodies. These results suggest that IL-15 produced by i-EC may stimulate significant fractions of i-IEL to produce IFN-γ at an early phase of oral infection withL. monocytogenes.
Complete DNA Sequence and Detailed Analysis of the
Yersinia pestis
KIM5 Plasmid Encoding Murine Toxin and Capsular Antigen
Authors: Lindler Luther E. , Plano Gregory V. , Burland Valerie , Mayhew George F. , Blattner Frederick R.
Published: December 2019

Yersinia pestis
, the causative agent of plague, harbors at least three plasmids necessary for full virulence of the organism, two of which are species specific. One of the
Y. pestis
-specific plasmids, pMT1, is thought to promote deep tissue invasion, resulting in more acute onset of symptoms and death. We determined the entire nucleotide sequence of
Y. pestis
KIM5 pMT1 and identified potential open reading frames (ORFs) encoded by the 100,990-bp molecule. Based on codon usage for known yersinial genes, homology with known proteins in the databases, and potential ribosome binding sites, we determined that 115 of the potential ORFs which we considered could encode polypeptides in
Y. pestis
. Five of these ORFs were genes previously identified as being necessary for production of the classic virulence factors, murine toxin (MT), and the fraction 1 (F1) capsule antigen. The regions of pMT1 encoding MT and F1 were surrounded by remnants of multiple transposition events and bacteriophage, respectively, suggesting horizontal gene transfer of these virulence factors. We identified seven new potential virulence factors that might interact with the mammalian host or flea vector. Forty-three of the remaining 115 putative ORFs did not display any significant homology with proteins in the current databases. Furthermore, DNA sequence analysis allowed the determination of the putative replication and partitioning regions of pMT1. We identified a single 2,450-bp region within pMT1 that could function as the origin of replication, including a RepA-like protein similar to RepFIB, RepHI1B, and P1 and P7 replicons. Plasmid partitioning function was located ca. 36 kb from the putative origin of replication and was most similar to the
parABS
bacteriophage P1 and P7 system.
Y. pestis
pMT1 encoded potential genes with a high degree of similarity to a wide variety of organisms, plasmids, and bacteriophage. Accordingly, our analysis of the pMT1 DNA sequence emphasized the mosaic nature of this large bacterial virulence plasmid and provided implications as to its evolution.
PseudomonasPyocyanin Increases Interleukin-8 Expression by Human Airway Epithelial Cells
Authors: Denning Gerene M. , Wollenweber Laura A. , Railsback Michelle A. , Cox Charles D. , Stoll Lynn L. , Britigan Bradley E.
Published: December 2019

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects ofP. aeruginosaare mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1α. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n -acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed inPseudomonas-associated lung disease.
Evaluation of Immunoglobulin A1 (IgA1) Protease and IgA1 Protease-Inhibitory Activity in Human Female Genital Infection withNeisseria gonorrhoeae
Authors: Hedges Spencer R. , Mayo Matthew S. , Kallman Lisa , Mestecky Jiri , Hook Edward W. , Russell Michael W.
Published: December 2019

Immunoglobulin A1 (IgA1) protease, an enzyme that selectively cleaves human IgA1, may be a virulence factor for pathogenic organisms such asNeisseria gonorrhoeae. Host protection from the effects of IgA1 protease includes antibody-mediated inhibition of IgA1 protease activity, and it is believed that the relative balance between IgA1 protease and inhibitory antibodies contributes to the pathogenesis of disease caused by IgA1 protease-producing organisms. We have examined the levels of these two opposing factors in genital tract secretions and sera from women with uncomplicated infection withN. gonorrhoeae. When IgA1 in cervical mucus was examined by Western blotting, no evidence of cleavage fragments characteristic of IgA1 protease activity was seen in gonococcus-infected or control patients. Cleavage fragments typical of IgA1 protease were detected, however, after the addition of exogenous IgA1 protease to cervical mucus. Degraded IgA1 was detected in some vaginal wash samples, but the fragment pattern was not typical of IgA1 protease activity. AllN. gonorrhoeaeisolates from the infected patients produced IgA1 protease in vitro. All but two serum samples and 16 of 65 cervical mucus samples displayed inhibitory activity against gonococcal IgA1 protease, but there was no significant difference in the level of inhibitory activity between gonococcus-infected and noninfected patients in either cervical mucus or serum. There was no difference in the levels of IgA1 protease-inhibitory activity in serum or cervical mucus collected from patients at recruitment and 2 weeks later. These results suggest that cleavage of IgA1 by gonococcal IgA1 protease within the lumen of the female lower genital tract is unlikely to be a significant factor in the pathogenesis of infections byN. gonorrhoeae.