bluuebunny/crossref_metadata_2025_split huggingface dataset:
Citrobacter freundii
Invades and Replicates in Human Brain Microvascular Endothelial Cells
Authors: Badger Julie L. , Stins Monique F. , Sik Kim Kwang 
Published: December 2019

ABSTRACT

Neonatal bacterial meningitis remains a disease with unacceptable rates of morbidity and mortality despite the availability of effective antimicrobial therapy.
Citrobacter
spp. cause neonatal meningitis but are unique in their frequent association with brain abscess formation. The pathogenesis of
Citrobacter
spp. causing meningitis and brain abscess is not well characterized; however, as with other meningitis-causing bacteria (e.g.,
Escherichia coli
K1 and group B streptococci), penetration of the blood-brain barrier must occur. In an effort to understand the pathogenesis of
Citrobacter
spp. causing meningitis, we have used the in vitro blood-brain barrier model of human brain microvascular endothelial cells (HBMEC) to study the interaction between
C. freundii
and HBMEC. In this study, we show that
C. freundii
is capable of invading and trancytosing HBMEC in vitro. Invasion of HBMEC by
C. freundii
was determined to be dependent on microfilaments, microtubules, endosome acidification, and de novo protein synthesis. Immunofluorescence microscopy studies revealed that microtubules aggregated after HBMEC came in contact with
C. freundii
; furthermore, the microtubule aggregation was time dependent and seen with
C. freundii
but not with noninvasive
E. coli
HB101 and meningitic
E. coli
K1. Also in contrast to other meningitis-causing bacteria,
C. freundii
is able to replicate within HBMEC. This is the first demonstration of a meningitis-causing bacterium capable of intracellular replication within BMEC. The important determinants of the pathogenesis of
C. freundii
causing meningitis and brain abscess may relate to invasion of and intracellular replication in HBMEC.
Fluorescent Labels Influence Phagocytosis ofBordetella pertussisby Human Neutrophils
Authors: Weingart Christine L. , Broitman-Maduro Gina , Dean Gary , Newman Simon , Peppler Mark , Weiss Alison A. 
Published: December 2019

ABSTRACTTo explore the role of neutrophil phagocytosis in host defense againstBordetella pertussis, bacteria were labeled extrinsically with fluorescein isothiocyanate (FITC) or genetically with green fluorescent protein (GFP) and incubated with adherent human neutrophils in the presence or absence of heat-inactivated human immune serum. In the absence of antibodies, FITC-labeled bacteria were located primarily on the surface of the neutrophils with few bacteria ingested. However, after opsonization, about seven times more bacteria were located intracellularly, indicating that antibodies promoted phagocytosis. In contrast, bacteria labeled intrinsically with GFP were not efficiently phagocytosed even in the presence of opsonizing antibodies, suggesting that FITC interfered with a bacterial defense. Because FITC covalently modifies proteins and could affect their function, we tested the effect of FITC on adenylate cyclase toxin activity, an important extracellular virulence factor. FITC-labeled bacteria had fivefold-less adenylate cyclase toxin activity than did unlabeled wild-type bacteria or GFP-expressing bacteria, suggesting that FITC compromised adenylate cyclase toxin activity. These data demonstrated that at least one extracellular virulence factor was affected by FITC labeling and that GFP is a more appropriate label forB. pertussis.
Characterization of Binding of Adenylate Cyclase Toxin to Target Cells by Flow Cytometry
Authors: Gray Mary C. , Ross William , Kim Keejun , Hewlett Erik L. 
Published: December 2019

ABSTRACTAdenylate cyclase (AC) toxin fromBordetella pertussisintoxicates eukaryotic cells by increasing intracellular cyclic AMP (cAMP) levels. In addition, insertion of AC toxin into the plasma membrane causes efflux of intracellular K+and, in a related process, hemolysis of sheep erythrocytes. Although intoxication, K+efflux, and hemolysis have been thoroughly investigated, there is little information on the nature of the interaction of this toxin with intact target cells. Using flow cytometry, we observe that binding of AC toxin to sheep erythrocytes and Jurkat T lymphocytes is dependent on posttranslational acylation of the toxin. Extracellular calcium is also necessary, with a steep calcium concentration dependence similar to that required for intoxication and hemolysis. Binding of AC toxin is concentration dependent but unsaturable up to 50 μg/ml, suggesting that if there is a specific receptor molecule with which the toxin interacts, it is not limiting. Visualization of cells by fluorescence microscopy supports the data obtained by flow cytometry and reveals a peripheral pattern of toxin distribution. AC toxin binds to erythrocytes at both 0 and 37°C; however, the total binding at 0°C is less than that at 37°C. In human erythrocytes, AC toxin does not cause an increase in K+efflux or hemolysis. While AC toxin exhibits reduced potency to increase cAMP in these cells than in sheep erythrocytes, there is only a modest reduction in the binding of the toxin as measured by flow cytometry. Further use of this technique will provide new approaches for dynamic and functional analysis of the early steps involved in intoxication, K+efflux, and hemolysis produced by AC toxin.
Role of Ornibactin Biosynthesis in the Virulence of
Burkholderia cepacia
: Characterization of
pvdA
, the Gene Encoding
l
-Ornithine
N
5
-Oxygenase
Authors: Sokol P. A. , Darling P. , Woods D. E. , Mahenthiralingam E. , Kooi C. 
Published: December 2019

ABSTRACT

Burkholderia cepacia
is a frequent cause of respiratory infections in cystic fibrosis patients.
B. cepacia
has been shown to produce at least four siderophores which may play a role in the virulence of this organism. To characterize genes involved in the synthesis of siderophores, Tn
5
-OT182 mutants were isolated in strain K56-2, which produces two siderophores, salicylic acid (SA) and ornibactins. Two mutants were characterized that did not produce zones on Chrome Azurol S agar in a commonly used assay to detect siderophore activity. These mutants were determined to produce sevenfold more SA than K56-2 yet did not produce detectable amounts of ornibactins. These mutants, designated I117 and T10, had a transposon insertion in genes with significant homology to pyoverdine biosynthesis genes of
Pseudomonas aeruginosa
. I117 contained an insertion in a
pvdA
homolog, the gene for the enzyme
l
-ornithine
N
5
-oxygenase, which catalyzes the hydroxylation of
l
-ornithine. Ornibactin synthesis in this mutant was partially restored when the precursor
l
-
N
5
-OH-Orn was added to the culture medium. T10 contained an insertion in a
pvdD
homolog, which is a peptide synthetase involved in pyoverdine synthesis. β-Galactosidase activity was iron regulated in both I117 and T10, suggesting that the transposon was inserted downstream of an iron-regulated promoter. Tn
5
-OT182 contains a
lacZ
gene that is expressed when inserted downstream of an active promoter. Both I117 and T10 were deficient in uptake of iron complexed to either ornibactins or SA, suggesting that transposon insertions in ornibactin biosynthesis genes also affected other components of the iron transport mechanism. The
B. cepacia pvdA
homolog was approximately 47% identical and 59% similar to
l
-ornithine
N
5
-oxygenase from
P. aeruginosa
. Three clones were identified from a K56-2 cosmid library that partially restored ornibactin production, SA production, and SA uptake to parental levels but did not affect the rate of
59
Fe-ornibactin uptake in I117. A chromosomal
pvdA
deletion mutant was constructed that had a phenotype similar to that of I117 except that it did not hyperproduce SA. The
pvdA
mutants were less virulent than the parent strain in chronic and acute models of respiratory infection. A functional
pvdA
gene appears to be required for effective colonization and persistence in
B. cepacia
lung infections.
Interaction ofLeishmaniagp63 with Cellular Receptors for Fibronectin
Authors: Brittingham Andrew , Chen Gang , McGwire Bradford S. , Chang Kwang-Poo , Mosser David M. 
Published: December 2019

ABSTRACTThe most abundant protein on the surface of the promastigote form of the protozoan parasitesLeishmaniaspp. is a 63-kDa molecule, designated gp63 or leishmanolysin. Because gp63 has been shown to possess fibronectin-like properties, we examined the interaction of gp63 with the cellular receptors for fibronectin. We measured the direct binding ofLeishmaniato human macrophages or to transfected mammalian cells expressing human fibronectin receptors.Leishmaniaexpressing gp63 exhibited modest but reproducible adhesion to human macrophages and to transfected CHO cells expressing α4/β1 fibronectin receptors. In both cases, this interaction depended on gp63 but occurred independently of the SRYD sequence of gp63, because parasites expressing gp63 with a mutated SRYD sequence bound to macrophages and α4/β1 receptor-expressing cells as well as did wild-type parasites. The contribution of gp63 to parasite adhesion was more pronounced when the assays were performed in the presence of complement, suggesting that the receptors for complement and fibronectin may cooperate to mediate the efficient adhesion of parasites to macrophages. The interaction of gp63 with fibronectin receptors may also play an important role in parasite internalization by macrophages. Erythrocytes to which gp63 was cross-linked were efficiently phagocytized by macrophages, whereas control erythrocytes opsonized with complement alone bound to macrophages but remained peripherally attached to the outside of the cell. Similarly, parasites expressing wild-type gp63 were rapidly and efficiently phagocytized by resting macrophages, whereas parasites lacking gp63 were internalized more slowly. This rapid internalization of gp63-expressing parasites was dependent on the β1 integrins, because pretreatment of macrophages with monoclonal antibodies to the β1 integrins decreased the internalization of gp63-expressing parasites. These observations indicate that complement receptors are the primary mediators of parasite adhesion; however, maximal parasite adhesion and internalization may require the participation of the β1 integrins, which recognize fibronectin-like molecules such as gp63 on the surface of the parasite.
Role of Tumor Necrosis Factor Alpha, Interleukin-1β, and Interleukin-6 in a Mouse Model of Group B Streptococcal Arthritis
Authors: Tissi Luciana , Puliti Manuela , Barluzzi Roberta , Orefici Graziella , von Hunolstein Christina , Bistoni Francesco 
Published: December 2019

ABSTRACTIntravenous inoculation of CD1 mice with 107CFU of type IV group BStreptococcus(GBS IV) results in a high incidence of diffuse septic arthritis. In this study the roles of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 in articular pathology were evaluated. Cytokine levels were quantified in the serum and joints by enzyme-linked immunosorbent assay in mice injected with GBS IV and tested or not tested with pentoxifylline (PTF), a methylxanthine that affects cytokine production. PTF was administered intraperitoneally at a dose of 1 mg/mouse (50 mg/kg of body weight) 1 h after GBS infection and then at 24-h intervals for 4 days. High levels of IL-1β and IL-6, but not TNF-α, were detected in the joints of mice injected with GBS IV from 5 to 15 days after infection, when articular lesions were most frequent and severe. IL-1β and IL-6 concentrations in the joints significantly (P< 0.001) exceeded those detected in the serum, confirming a strong local production. PTF treatment resulted in a strong reduction of cytokine production and in a marked decrease in both the incidence and severity of arthritis. Inoculation of exogenous murine recombinant IL-1β or IL-6 in mice treated with GBS IV plus PTF resulted in an incidence and severity of articular lesions similar to those obtained with inoculation of GBS IV alone. No significant effect was obtained with TNF-α administration. These data show a strong involvement of IL-1β and IL-6, but not TNF-α, in the pathogenesis of GBS arthritis.
Identification of a New Variable Sequence in the P1 Cytadhesin Gene of
Mycoplasma pneumoniae
: Evidence for the Generation of Antigenic Variation by DNA Recombination between Repetitive Sequences
Authors: Kenri Tsuyoshi , Taniguchi Rie , Sasaki Yuko , Okazaki Norio , Narita Mitsuo , Izumikawa Kinichi , Umetsu Masao , Sasaki Tsuguo 
Published: December 2019

ABSTRACT

A
Mycoplasma pneumoniae
cytadhesin P1 gene with novel nucleotide sequence variation has been identified. Four clinical strains of
M. pneumoniae
were found to carry this type of P1 gene. This new P1 gene is similar to the known group II P1 genes but possesses novel sequence variation of approximately 300 bp in the RepMP2/3 region. The position of the new variable region is distant from the previously reported variable regions known to differ between group I and II P1 genes. Two sequences closely homologous to this new variable region were found within the repetitive sequences outside the P1 gene of the
M. pneumoniae
M129 genome. This suggests that the new P1 gene was generated by DNA recombination between repetitive sequences and the P1 gene locus. The finding of this new type of P1 gene supports the hypothesis that the repetitive sequences of the
M. pneumoniae
genome serve as a reservoir to generate antigenic variation of the cytadhesin P1 gene.
Binding of Rat and Human Surfactant Proteins A and D toAspergillus fumigatusConidia
Authors: Allen Martin J. , Harbeck Ronald , Smith Bruce , Voelker Dennis R. , Mason Robert J. 
Published: December 2019

ABSTRACTSurfactant proteins A (SP-A) and D (SP-D) are thought to play important roles in pulmonary host defense. We investigated the interactions of rat and human SP-A and SP-D withAspergillus fumigatusconidia. Rat SP-D but not rat SP-A bound the conidia, and the binding was inhibited by EDTA, mannose, glucose, maltose, and inositol. Binding studies using a mutant recombinant rat SP-D with altered carbohydrate recognition but normal structural organization clearly established a role for the carbohydrate recognition domain in binding to conidia. However, neither rat SP-A nor SP-D increased the association of fluorescein isothiocyanate-labeled conidia with rat alveolar macrophages as determined by flow cytometry. Both human SP-A (isolated from normal and alveolar proteinosis lungs) and SP-D (recombinant protein and protein isolated from alveolar proteinosis lungs) bound the conidia. These data indicate that important differences exist between rat and human SP-A in binding to certain fungi. Human SP-A and SP-D binding to conidia was also examined in the presence of hydrophobic surfactant components (HSC), containing both the phospholipid and hydrophobic proteins of surfactant. We found that HSC inhibited but did not eliminate human SP-A binding toAspergillusconidia. In contrast, the SP-D binding to conidia was unaffected by HSC. These findings indicate that SP-D plays a major role in the recognition ofAspergillusconidia in alveolar fluid.
Assessment of Immunity to Mycobacterial Infection with Luciferase Reporter Constructs
Authors: Snewin Valerie A. , Gares Marie-Pierre , ÓGaora Peadar , Hasan Zahra , Brown Ivor N. , Young Douglas B. 
Published: December 2019

ABSTRACTProtective immunity to mycobacterial infection is incompletely understood but probably involves the coordinated interaction of multiple cell types and cytokines. With the aim of developing assays that might provide a surrogate measure of protective immunity, we have investigated the use of recombinant mycobacteria carrying luciferase reporter enzymes to assess the effectiveness of antimycobacterial immunity in model systems. Measurement of luminescence was shown to provide a rapid and simple alternative to the counting of CFU as a means of monitoring mycobacterial viability. We describe optimization of a luciferase reporter strain ofMycobacterium tuberculosisand demonstrate its application for the study of mycobacterial interactions with host cells in tissue culture and the rapid assessment of vaccine efficacy in a murine model.
Murine Splenocytes Induce Severe Gastritis and Delayed-Type Hypersensitivity and Suppress Bacterial Colonization inHelicobacter pylori-Infected SCID Mice
Authors: Eaton Kathryn A. , Ringler Susan R. , Danon Stephen J. 
Published: December 2019

ABSTRACTThe goal of this study was to evaluate the role of host immunity in gastritis and epithelial damage due toHelicobacter pylori. Splenocytes fromH. pylori-infected and uninfected C57BL/6 mice were adoptively transferred toH. pylori-infected and uninfected severe combined immunodeficient (SCID) mice. Transfer was verified by flow cytometry, and all mice were evaluated for the presence of delayed-type hypersensitivity (DTH) by footpad inoculation with sterileH. pylorisonicate and for humoral immunity by enzyme-linked immunosorbent assay. The severity of gastritis and gastric epithelial damage was quantified histologically, epithelial proliferation was determined by proliferating cell nuclear antigen staining, and colonization was quantified by culture. C57BL/6 mice, but not nonrecipient SCID mice, developed moderate gastritis in response toH. pylori. In contrast, recipient SCID mice developed severe gastritis involving 50 to 100% of the gastric mucosa and strong DTH responses not present in C57BL/6 mice. DTH, but not serum anti-H. pyloriimmunoglobulin G, correlated with adoptive transfer, gastritis, and bacterial clearance. Severe gastritis, but not bacterial colonization, was associated with epithelial metaplasia, erosions, and an elevated labeling index. This study demonstrates that (i) adaptive immunity is essential for development of gastritis due toH. pyloriin mice, (ii) T-cell-enriched lymphocytes in SCID mice induce DTH and gastritis, which is more severe than donor gastritis, and (iii) the host inflammatory response, not direct bacterial contact, causes epithelial damage. The greater severity of gastritis in recipient SCID mice than in donor C57BL/6 mice suggests that gastritis is due to specific T-cell subsets and/or the absence of regulatory cell subsets in the transferred splenocytes.
The Capsule ofCryptococcus neoformansReduces T-Lymphocyte Proliferation by Reducing Phagocytosis, Which Can Be Restored with Anticapsular Antibody
Authors: Syme Rachel M. , Bruno Tony F. , Kozel Thomas R. , Mody Christopher H. 
Published: December 2019

ABSTRACTCell-mediated immunity is critical for the host defense toCryptococcus neoformans, as demonstrated by numerous animal studies and the prevalence of the infection in AIDS patients. Previous studies have established that the polysaccharide capsule contributes to the virulence ofC. neoformansby suppressing T-lymphocyte proliferation, which reflects the clonal expansion of T lymphocytes that is a hallmark of cell-mediated immunity. The present studies were performed to identify the major mechanism by which polysaccharide impairs lymphocyte proliferation, since capsular polysaccharide has the potential to affect the development of T-lymphocyte responses by stimulating production of interleukin-10 (IL-10), inhibiting phagocytosis, and inducing shedding of cell surface receptors. We demonstrate that polysaccharide inhibits lymphocyte proliferation predominantly by blocking uptake ofC. neoformans, which is crucial for subsequent lymphocyte proliferation. In addition, we show that polysaccharide did not suppress lymphocyte proliferation via an IL-10-dependent mechanism, nor did it affect critical surface receptor interactions on the T cell or antigen-presenting cell. Having established that polysaccharide impairs phagocytosis, we performed studies to determine whether opsonization with human serum or with anticapsular antibody could reverse this effect. Impaired uptake and lymphocyte proliferation that were induced by polysaccharide can be enhanced through opsonization with monoclonal antibodies or human serum, suggesting that antipolysaccharide antibodies might enhance the host defense by restoring uptake of the organism and subsequent presentation to T lymphocytes. These studies support the therapeutic potential of stimulating cell-mediated immunity toC. neoformanswith anticapsular antibody.
Dominance of Immunoglobulin G2c in the Antiphosphorylcholine Response of Rats Infected withTrichinella spiralis
Authors: Peters Philip J. , Gagliardo Lucille F. , Sabin Elizabeth A. , Betchen Arin B. , Ghosh Kaya , Oblak Jeb B. , Appleton Judith A. 
Published: December 2019

ABSTRACTThe antibody response to the L1 stage ofTrichinella spiralishas been described as biphasic. Worms resident in the intestine during the first week of infection stimulate an antibody response against a subset of larval proteins. L1 larvae in the muscle at the end stage of infection stimulate a second antibody response against tyvelose-bearing glycoproteins. Antityvelose antibodies protect rats against challenge infection with larvae. The aim of this study was to characterize the rat B-cell response against larval antigens during the intestinal phase ofT. spiralisinfection and to test the antiparasitic effects of such antibodies. Strain PVG rats were infected orally with 500 larvae. Antibodies specific for phosphorylcholine-bearing proteins of L1 larvae first appeared in serum 9 days postinfection. Absorption experiments showed that the majority of antilarval antibodies produced in rats 16 days after infection withT. spiraliswere specific for phosphorylcholine-bearing proteins. A fraction of these antibodies bound to free phosphorylcholine. Immunoglobulin G2c (IgG2c) producing cells in the mesenteric lymph node dominated this early antibody response. IgG2c is associated with T-independent immune responses in the rat; however, a comparison of athymic rats with euthymic controls suggested that only a small fraction of the phosphorylcholine-related antibody response againstT. spiraliswas T independent. Phosphorylcholine is a common epitope in antigens of bacteria and nematode parasites and has been shown to be a target of protective immunity in certain bacteria. A monoclonal IgG2c antibody was prepared from infected rats and shown to be specific for phosphorylcholine. Monoclonal phosphorylcholine-specific IgG2c failed to protect rats against intestinal infection withT. spiralis. Therefore, our findings do not support a role for phosphorylcholine-bearing antigens in immune defense againstT. spiralis; however, the potency of the immune response induced suggests an immunomodulatory role for the lymphocytes involved.