bluuebunny/crossref_metadata_2025 huggingface dataset:
Mouse β-Defensin 3 Is an Inducible Antimicrobial Peptide Expressed in the Epithelia of Multiple Organs
Authors: Bals Robert , Wang Xiaorong , Meegalla Rupalie L. , Wattler Sigrid , Weiner Daniel J. , Nehls Michael C. , Wilson James M.
Published: December 2019

One component of host defense at mucosal surfaces is epithelium-derived peptides with antimicrobial activity called defensins. We describe in this report the isolation and characterization of a murine homologue of human β-defensin 2 (hBD-2) called mouse β-defensin 3 (mBD-3). The predicted amino acid sequence shows the hallmark features of other known epithelial defensins, including the ordered array of six cysteine residues. Analysis of a genomic clone of mBD-3 revealed two exons separated by a 1.7-kb intron. The mBD-3 gene is localized at the proximal portion of chromosome 8, the site where genes for mouse α- and β-defensins are found. Under basal condition, mBD-3 transcripts were detected at low levels in epithelial cells of surface organs, such as the intestine and lung. After instillation of
Pseudomonas aeruginosa
PAO1 into mouse airways, mBD-3-specific mRNA was upregulated significantly not only in large airways but also in the small bowel and liver. Recombinant mBD-3 peptide, produced from a baculovirus expression system, showed antimicrobial activity against
P. aeruginosa
PAO1 (MIC of 8 μg/ml) and
Escherichia coli
D31 (MIC of 16 μg/ml) in a salt-dependent manner. This study demonstrates that a murine homologue of hBD-2 is present in the respiratory system and other mucosal surfaces. These similarities between murine and human host defense apparatus provide further impetus to evaluate the mouse as a model for studying the human innate host defense system.
Fatal Granuloma Necrosis without Exacerbated Mycobacterial Growth in Tumor Necrosis Factor Receptor p55 Gene-Deficient Mice Intravenously Infected withMycobacterium avium
Authors: Ehlers Stefan , Benini Jochen , Kutsch Stefanie , Endres Robert , Rietschel Ernst T. , Pfeffer Klaus
Published: December 2019

The pathogenesis of mycobacterial infections is associated with the formation of granulomas in which both antibacterial protection and tissue damage take place concomitantly. We used murineMycobacterium aviuminfection to compare the development of granulomatous lesions in intravenously infected tumor necrosis factor receptor p55 (TNFRp55) gene-deficient (p55−/−) mice to the development of granulomatous lesions inM. avium-infected syngeneic C57BL/6 (p55+/+) mice. Up to 5 weeks after infection with either the highly virulentM. aviumstrain TMC724 or the intermediately virulentM. aviumstrain SE01, bacterial counts in the liver, spleen, and lung of p55−/−mice were identical to or lower than those in infected p55+/+mice. However, the formation of mononuclear cell foci in the liver was delayed by approximately 2 to 3 weeks in p55−/−mice compared to the results obtained for infected p55+/+mice. Despite comparable bacterial loads, granulomas in p55−/−mice underwent progressive necrosis, causing damage to the surrounding liver tissue. The appearance of necrotizing granulomas was associated with the death of all infected p55−/−mice, regardless of the virulence of theM. aviumstrain used for infection. Granulomatous lesions in the liver contained three times as many CD3+cells in p55−/−mice yet appeared more diffuse than in p55+/+mice. Semiquantitative reverse transcription-PCR studies revealed that prior to mouse death, interleukin-12 (IL-12) and gamma interferon mRNA levels were up regulated in the livers of infected p55−/−mice, while mRNA levels for tumor necrosis factor, the inducible isoform of nitric-oxide synthase (iNOS), and IL-10 were similar to those found in infected p55+/+mice. In response to persistent mycobacterial infection, the absence of TNFRp55 causes the disregulation of T-cell–macrophage interactions and results in fatal granuloma necrosis even when adequate antibacterial functions are maintained.
Invasive Lesions Containing Filamentous Forms Produced by a
Candida albicans
Mutant That Is Defective in Filamentous Growth in Culture
Authors: Riggle Perry J. , Andrutis Karl A. , Chen Xi , Tzipori Saul R. , Kumamoto Carol A.
Published: December 2019

A
Candida albicans efg1 cph1
double mutant is nonfilamentous under standard laboratory conditions and avirulent in mice. However, this mutant produced filaments in the tongues of immunosuppressed gnotobiotic piglets and when embedded in agar, demonstrating that an Efg1p- and Cph1p-independent pathway for promotion of filamentous growth exists.
Pneumolysin, a Protein Toxin of
Streptococcus pneumoniae
, Induces Nitric Oxide Production from Macrophages
Authors: Braun Johann S. , Novak Rodger , Gao Geli , Murray Peter J. , Shenep Jerry L.
Published: December 2019

Nitric oxide (NO) production by inducible NO synthase (iNOS) during inflammation is an essential element of antimicrobial immunity but can also contribute to host-induced tissue damage. Under conditions of bacterial sepsis, large amounts of NO are produced, causing hypotension, a critical pathological feature of septic shock. In sepsis caused by gram-positive organisms, the bacterial factors contributing to host NO production are poorly characterized. We show that a soluble toxin of
Streptococcus pneumoniae
, pneumolysin (Pln), is a key component initiating NO production from macrophages. In contrast to wild-type bacteria, a mutant of
S. pneumoniae
lacking Pln failed to elicit NO production from murine macrophages. Purified recombinant Pln induced NO production at low concentrations and independently of exogenous gamma interferon (IFN-γ) priming of RAW 264.7 macrophages. However, IFN-γ was essential for Pln-induced NO production, since primary macrophages from mice lacking the IFN-γ receptor or interferon regulatory factor 1, a transcription factor essential for iNOS expression, failed to produce NO when stimulated with Pln. In addition, Pln acts as an agonist of tumor necrosis factor alpha and interleukin 6 production in macrophages. The properties of Pln, previously identified as a pore-forming hemolysin, also include a role as a general inflammatory agonist.
Cytotoxic T-Cell-Mediated Response againstYersinia pseudotuberculosisin HLA-B27 Transgenic Rat
Authors: Falgarone Géraldine , Blanchard Hervé S. , Riot Bertrand , Simonet Michel , Breban Maxime
Published: December 2019

Yersinia-induced reactive arthritis is highly associated with HLA-B27, the role of which in defense against the triggering bacteria remains unclear. The aim of this study was to examine the capacity of rats transgenic for HLA-B27 to mount a cytotoxic T-lymphocyte (CTL) response againstY. pseudotuberculosisand to determine the influence of the HLA-B27 transgene on this response. Rats transgenic for HLA-B*2705 and human β2-microglobulin of the 21-4L line, which do not spontaneously develop disease, and nontransgenic syngeneic Lewis (LEW) rats were infected withY. pseudotuberculosis. Lymph node cells were restimulated in vitro, and the presence of forY. pseudotuberculosis-specific CTLs against infected targets was determined. Infection of 21-4L rats triggered a CD8+T cell-mediated cytotoxic response specific forY. pseudotuberculosis. Analysis of this response demonstrated restriction by an endogenous major histocompatibility complex molecule. However, no restriction by HLA-B27 was detected. In addition, kinetics studies revealed a weaker anti-YersiniaCTL response in 21-4L rats than in nontransgenic LEW rats, and the level of cytotoxicity against 21-4L lymphoblast targets sensitized with Y. pseudotuberculosis was lower than that against nontransgenic LEW targets. We conclude that HLA-B27 transgenic rats mount a CTL response againstY. pseudotuberculosisthat is not restricted by HLA-B27. Yet, HLA-B27 exerts a negative effect on the level of this response, which could contribute to impaired defense againstYersinia.
Adhesion of
Tritrichomonas foetus
to Bovine Vaginal Epithelial Cells
Authors: Singh B. N. , Lucas J. J. , Beach D. H. , Shin S. T. , Gilbert R. O.
Published: December 2019

An in vitro culture system of bovine vaginal epithelial cells (BVECs) was developed to study the cytopathogenic effects of
Tritrichomonas foetus
and the role of lipophosphoglycan (LPG)-like cell surface glycoconjugates in adhesion of parasites to host cells. Exposure of BVEC monolayers to
T. foetus
resulted in extensive damage of monolayers. Host cell disruption was measured quantitatively by a trypan blue exclusion assay and by release of
3
H from [
3
H]thymidine-labeled host cells. Results indicated contact-dependent cytotoxicity of host cells by
T. foetus
. The cytopathogenic effect was a function of
T. foetus
density. Metronidazole- or periodate-treated
T. foetus
showed no damage to BVEC monolayers. A related human trichomonad,
Trichomonas vaginalis
, showed no cytotoxic effects, indicating species-specific host-parasite interactions. A direct binding assay was developed and used to investigate the role of a major cell surface LPG-like molecule in host-parasite adhesion. The results of competition experiments showed that the binding to BVECs was displaceable, was saturable, and yielded a typical binding curve, suggesting that specific receptor-ligand interactions mediate the attachment of
T. foetus
to BVECs. Progesterone-treated BVECs showed enhanced parasite binding.
T. foetus
LPG inhibited the binding of
T. foetus
to BVECs; the LPG from
T. vaginalis
and a variety of other glycoconjugates did not. These data imply specificity of LPG on host-parasite adhesion. Periodate-treated parasites showed no adherence to host cells, indicating the involvement of carbohydrate containing molecules in the adhesion process.
Multiple Genes in the Left Half of thecagPathogenicity Island ofHelicobacter pyloriAre Required for Tyrosine Kinase-Dependent Transcription of Interleukin-8 in Gastric Epithelial Cells
Authors: Li Shude D. , Kersulyte Dangeruta , Lindley Ivan J. D. , Neelam Beena , Berg Douglas E. , Crabtree Jean E.
Published: December 2019

Helicobacter pyloristrains that contain thecagpathogenicity island (PAI) elicit increased synthesis of gastric C-X-C chemokines, promote neutrophilic infiltration into the gastric epithelium, and stimulate the synthesis of interleukin-8 (IL-8) in cultured gastric epithelial cells. To investigate the effects ofcagPAI genes on the transcription of the IL-8 gene, the Kato-3 gastric epithelial cell line was stably transfected with plasmid DNA containing the IL-8 gene promoter fused to a luciferase reporter gene. The resulting reporter cell line, L5F11, was used to monitor the effects of infection in cell culture byH. pylori26695 and isogenic derivatives with null mutations in genes in thecagPAI on transcription of the IL-8 gene. We found that null mutations in eight open reading frames, including homologs of theAgrobacterium virB9,virB10, andvirB11genes, in the left half of thecagPAI abrogated the induction of IL-8 gene transcription. Further studies with the L5F11 cell line showed that IL-8 gene transcription induced byH. pyloriwas blocked by the protein tyrosine kinase inhibitor herbimycin A but not by the protein kinase C inhibitor calphostin C or by the protein kinase G inhibitor KT5823. IL-8 gene transcription in L5F11 cells could also be induced by the cytokine tumor necrosis factor alpha (TNF-α) without exposure toH. pylori. This TNF-α-induced IL-8 transcription was inhibited by the protein kinase A inhibitor H7, which had no significant effect onH. pylori-induced IL-8 transcription. These studies show that multiple genes in the left half of thecagPAI are essential for the transcription of the IL-8 gene in gastric epithelial cells and that this depends on protein tyrosine kinase activation.
Limited Local and Systemic Antibody Responses toNeisseria gonorrhoeaeduring Uncomplicated Genital Infections
Authors: Hedges Spencer R. , Mayo Matthew S. , Mestecky Jiri , Hook Edward W. , Russell Michael W.
Published: December 2019

Repeated infections withNeisseria gonorrhoeaeare common among patients attending sexually transmitted disease clinics. We examined whether previous infections or site of infection altered the local and systemic antigonococcal antibody levels in males and females. Antibodies againstN. gonorrhoeaeMS11 and the patients’ homologous infecting isolates were measured by enzyme-linked immunosorbent assay. In general, the local and systemic immune responses to gonococci were extremely modest. There was a slight increase in serum immunoglobulin G (IgG) against the MS11 strain and the homologous isolates in infected males. Levels of serum IgA1 antibodies against MS11 were slightly higher in infected than in uninfected females. A history of previous infections withN. gonorrhoeaedid not alter the antibody levels in patients with a current infection, suggesting that immunological memory is not induced by uncomplicated gonococcal infections. Antibody responses to infected subjects’ homologous isolates were observed in cervical mucus; IgA1 levels increased while IgG levels decreased. The decline in mucosal IgG against the homologous isolates was less common in subjects having both rectal and cervical infections; otherwise, no effect of rectal involvement was observed. The absence of substantially higher antibody levels to gonococci where there is infection at a site known to contain organized lymphoid tissue suggests that the low levels of responses to uncomplicated infections may not be due simply to an absence of inductive sites in the genital tract. We propose that in addition to its potential ability to avoid the effects of an immune response,N. gonorrhoeaedoes not elicit strong humoral immune responses during uncomplicated genital infections.
Early Acidification of Phagosomes ContainingBrucella suisIs Essential for Intracellular Survival in Murine Macrophages
Authors: Porte Françoise , Liautard Jean-Pierre , Köhler Stephan
Published: December 2019

Brucella suisis a facultative intracellular pathogen of mammals, residing in macrophage vacuoles. In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication ofB. suis. Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence intensity of opsonized, carboxyfluorescein-rhodamine- and Oregon Green 488-rhodamine-labeled bacteria. Compartments containing liveB. suisacidified to a pH of about 4.0 to 4.5 within 60 min. Acidification ofB. suis-containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A1, a specific inhibitor of vacuolar proton-ATPases. This neutralization at 1 h postinfection resulted in a 2- to 34-fold reduction of opsonized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfection, respectively. Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability. Addition of ammonium chloride at 7 h after the beginning of infection, however, did not affect intracellular multiplication ofB. suis, in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. Thus, we conclude that phagosomes withB. suisacidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage.
Nine-Year Longitudinal Study of Antibodies to Variant Antigens on the Surface ofPlasmodium falciparum-Infected Erythrocytes
Authors: Giha Haider A. , Staalsoe Trine , Dodoo Daniel , Elhassan Ibrahim M. , Roper Cally , Satti Gwiria M. H. , Arnot David E. , Theander Thor G. , Hviid Lars
Published: December 2019

PfEMP1 is an antigenically variable molecule which mediates the adhesion of parasitized erythrocytes to a variety of cell types and which is believed to constitute an important target for naturally acquired protective immune responses in malaria. For 9 years we have monitored individuals living in an area of low-intensity, seasonal, and unstable malaria transmission in eastern Sudan, and we have used this database to study the acquisition, specificity, and duration of the antibody response to variant parasitized erythrocyte surface antigens. Both the levels and the spectrum of reactivity of these antibodies varied considerably among individuals, ranging from low levels of antibodies recognizing only few parasitized erythrocyte surface antigens to high levels of broad-specificity antibodies. In general, episodes of clinical malaria were associated with increases in the levels of parasitized erythrocyte surface-specific antibodies that subsided within months of the attack. This response was often, but not always, specific for the antigenic variants expressed by the parasite isolate causing disease. Our study provides evidence thatPalciparum falciparummalaria is associated with a short-lived, variant-specific antibody response to PfEMP1-like antigens exposed on the surface of parasitized erythrocytes. Furthermore, our data suggest that the antigenic repertoires of variant antigens expressed by different parasite isolates show considerable overlapping, at least under Sahelian conditions of low-intensity, seasonal, and unstable malaria transmission. Finally, we demonstrate the existence of persistent differences among individuals in the capacity to mount antibody responses to variant surface antigens.
Citrobacter freundii
Invades and Replicates in Human Brain Microvascular Endothelial Cells
Authors: Badger Julie L. , Stins Monique F. , Sik Kim Kwang
Published: December 2019

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

To 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

Adenylate 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

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

The 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

Intravenous 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

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

Surfactant 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

Protective 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

The 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

Cell-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

The 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.
Fibronectin Binding Protein and Host Cell Tyrosine Kinase Are Required for Internalization ofStaphylococcus aureusby Epithelial Cells
Authors: Dziewanowska Katarzyna , Patti Joseph M. , Deobald Claudia F. , Bayles Kenneth W. , Trumble William R. , Bohach Gregory A.
Published: December 2019

Staphylococcus aureusexpresses several surface proteins that promote adherence to host cell extracellular matrix proteins, including fibronectin (Fn). Since this organism has recently been shown to be internalized by nonprofessional phagocytes, a process that typically requires high-affinity binding to host cell receptors, we investigated the role of its Fn binding proteins (FnBPs) and other surface proteins in internalization by the bovine mammary gland epithelial cell line (MAC-T). Efficient internalization ofS. aureus8325-4 required expression of FnBPs; an isogenic mutant (DU5883), not expressing FnBPs, was reduced by more than 95% in its ability to invade MAC-T cells. Moreover, D3, a synthetic peptide derived from the ligand binding domain of FnBP, inhibited the internalization of the 8325-4 strain in a dose-dependent fashion and the efficiency of staphylococcal internalization was partially correlated with Fn binding ability. Interestingly, Fn also inhibited the internalization and adherence ofS. aureus8325-4 in a dose-dependent manner. In contrast to internalization, adherence of DU5883 to MAC-T was reduced by only approximately 40%, suggesting that surface binding proteins, other than FnBPs, can mediate bacterial adherence to cells. Adherence via these proteins, however, does not necessarily result in internalization of the staphylococci. An inhibitor of protein tyrosine kinase, genistein, reduced MAC-T internalization ofS. aureusby 95%, indicating a requirement for a host signal transduction system in this process. Taken together, these results indicate thatS. aureusinvades nonprofessional phagocytes by a mechanism requiring interaction between FnBP and the host cell, leading to signal transduction and subsequent rearrangement of the host cell cytoskeleton.
Pneumococcal Surface Protein A Inhibits Complement Activation byStreptococcus pneumoniae
Authors: Tu Anh-Hue T. , Fulgham Robert L. , McCrory Mark A. , Briles David E. , Szalai Alexander J.
Published: December 2019

Pneumococcal surface protein A (PspA) is a surface-exposed protein virulence factor forStreptococcus pneumoniae. In this study, no significant depletion of serum complement was observed for the serum of mice infected with pneumococci that express PspA. In contrast, in mice infected with an isogenic strain of pneumococci lacking PspA, significant activation of serum complement was detected within 30 min after infection. Also, the PspA-deficient strain but not the PspA-expressing strain was cleared from the blood within 6 h. The contribution of PspA to pneumococcal virulence was further investigated by using mice deficient for C5, C3, or factor B. In mice deficient for C3 or factor B, PspA-negative pneumococci became fully virulent. In contrast, in C5-deficient mice as in wild-type mice, PspA-deficient pneumococci were avirulent. These in vivo data suggest that, in nonimmune mice infected with pneumococci, PspA interferes with complement-dependent host defense mechanisms mediated by factor B. Immunoblots of pneumococci opsonized in vitro suggested that more C3b was deposited on PspA-negative than on PspA-positive pneumococci. This was observed with and without anticapsular antibody. Furthermore, processing of the α chain of C3b was reduced in the presence of PspA. We propose that PspA exerts its virulence function by interfering with deposition of C3b onto pneumococci and/or by inhibiting formation of a fully functional alternative pathway C3 convertase. By blocking recruitment of the alternative pathway, PspA reduces the amount of C3b deposited onto pneumococci, thereby reducing the effectiveness of complement receptor-mediated pathways of clearance.
Interactions ofPenicillium marneffeiwith Human Leukocytes In Vitro
Authors: Rongrungruang Yong , Levitz Stuart M.
Published: December 2019

Penicillium marneffei, a dimorphic fungus endemic in parts of Asia, causes disease in those with impaired cell-mediated immunity, especially persons with AIDS. The histopathology of penicilliosis marneffei features the intracellular infection of macrophages. We studied the interactions between human leukocytes and heat-killed yeast-phaseP. marneffei. Monocyte-derived macrophages bound and internalizedP. marneffeiin the presence of complement-sufficient pooled human serum (PHS). Binding and phagocytosis were still seen if PHS was heat inactivated or omitted altogether. The binding of unopsonizedP. marneffeito monocyte-derived macrophages occurred in the absence of divalent cations and was not affected by inhibitors of mannose and β-glucan receptors or monoclonal antibodies directed against CD14 and CD11/CD18. Binding was profoundly inhibited by wheat germ agglutinin. A vigorous respiratory burst was seen in peripheral blood mononuclear cells (PBMC) stimulated withP. marneffei, regardless of whether the fungi were opsonized. However, tumor necrosis factor alpha (TNF-α) release from PBMC stimulated withP. marneffeioccurred only if serum was present. These data demonstrate that (i) monocyte-derived macrophages bind and phagocytoseP. marneffeieven in the absence of opsonization, (ii) binding is divalent cation independent but is inhibited by wheat germ agglutinin, suggesting that the major receptor(s) recognizingP. marneffeiis a glycoprotein with exposedN-acetyl-β-d-glucosaminyl groups, (iii)P. marneffeistimulates the respiratory burst regardless of whether opsonins are present, and (iv) serum factors are required forP. marneffeito stimulate TNF-α release. The ability of unopsonizedP. marneffeito parasitize mononuclear phagocytes without stimulating the production of TNF-α may be critical for the virulence of this intracellular parasite.
The Gene Locus
yijP
Contributes to
Escherichia coli
K1 Invasion of Brain Microvascular Endothelial Cells
Authors: Wang Ying , Huang Sheng-He , Wass Carol A. , Stins Monique F. , Kim Kwang Sik
Published: December 2019

Most cases of
Escherichia coli
meningitis develop as a result of hematogenous spread, but it is not clear how circulating
E. coli
crosses the blood-brain barrier. A Tn
phoA
mutant of
E. coli
K1 RS218 was shown to be significantly less invasive than its parent strain in bovine and human brain microvascular endothelial cells (BMEC), which constitute the blood-brain barrier. More importantly, traversal of the blood-brain barrier was significantly less with this mutant than with the parent strain in newborn rats with experimental hematogenous meningitis. A DNA segment containing the Tn
phoA
insertion site was cloned from RS218, and the cloned DNA complemented the Tn
phoA
mutant in invasion of BMEC. Nucleotide sequence revealed a near identity to that of a hypothetical
yijP
gene (also called
f577
) in the
E. coli
K-12 genome. Sequence analysis indicated that the
E. coli
K1
yijP
gene likely encodes a 66.6-kDa membrane protein. Deletion and complementation experiments indicated that the
yijP
gene was involved in
E. coli
K1 invasion of BMEC, i.e., the invasive ability of
E. coli
K1 was significantly reduced after
yijP
was deleted and was restored by complementation with a plasmid containing the
yijP
open reading frame. This is the first demonstration that the
yijP
gene locus plays a role in the pathogenesis of
E. coli
K1 meningitis.