bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inducible Promoter-Repressor System from the
Lactobacillus casei
Phage φFSW
Authors: Binishofer Bernhard , Moll Isabella , Henrich Bernhard , Bläsi Udo 
Published: October 2002

ABSTRACT

With the aim to extend the presently available inducible gene expression systems for lactobacilli, we have isolated a thermoinducible promoter-repressor cassette from the temperate
Lactobacillus casei
phage φFSW-TI in
Escherichia coli
. The φFSW-TI promoter fragment was abutted to the plasmid-borne promoterless β-glucuronidase (
gusA
) reporter gene and shown to direct its transcription in
L. casei
. In addition, the functionality of the promoter-repressor system was verified in the
L. casei
φFSW-TI lysogen by showing that the
gusA
reporter gene, controlled by the isolated φFSW-TI promoter, was repressed at 28°C and expressed at 42°C. Moreover, a homology search revealed that the C terminus of the isolated φFSW repressor shows a high similarity to the small
mutS
-related domain of the MutS2 protein family that is unprecedented for phage-encoded repressor proteins.
Inhibitory Effects of High Pressure and Heat on
Alicyclobacillus acidoterrestris
Spores in Apple Juice
Authors: Lee Sun-Young , Dougherty Richard H. , Kang Dong-Hyun 
Published: October 2002

ABSTRACT

The effectiveness of combined high pressure and heat treatment for reducing spore levels of
Alicyclobacillus acidoterrestris
, a thermoacidophilic spore-forming bacterium, in commercial pasteurized apple juice was investigated. Spores suspended in apple juice were successfully destroyed by combining high pressure with a mild or high temperature (45, 71, or 90°C).
Cryptococcal Glucuronoxylomannan Inhibits Adhesion of Neutrophils to Stimulated Endothelium In Vitro by Affecting Both Neutrophils and Endothelial Cells
Authors: Ellerbroek Pauline M. , Hoepelman Andy I. M. , Wolbers Floor , Zwaginga Jaap Jan , Coenjaerts Frank E. J. 
Published: October 2002

ABSTRACTCryptococcal infections are often characterized by a paucity of leukocytes in the infected tissues. Previous research has shown that the capsular polysaccharide glucuronoxylomannan (GXM) inhibits leukocyte migration. In this study we investigated whether the capsular polysaccharide GXM affects the migration of neutrophils (polymorphonuclear leukocytes [PMN]) through the endothelium by interfering with adhesion in a static adhesion model. Pretreatment of PMN with GXM inhibited PMN adhesion to tumor necrosis factor alpha (TNF-α)-stimulated endothelium up to 44%. Treatment of TNF-α-stimulated endothelium with GXM led to a 27% decrease in PMN adhesion. GXM treatment of both PMN and endothelium did not have an additive inhibitory effect. We demonstrated that GXM-induced L-selectin shedding does not play an important role in the detected inhibition of adhesion. L-selectin was still present on PMN in sufficient amounts after GXM treatment, since it could be further inhibited by blocking antibodies. Furthermore, blocking of GXM-related L-selectin shedding did not abolish the GXM-related inhibition of adhesion. GXM most likely exerts its effect on PMN by interfering with E-selectin-mediated binding. The use of blocking monoclonal antibodies against E-selectin, which was shown to decrease adhesion in the absence of GXM, did not cause additive inhibition of PMN adhesion after GXM pretreatment. The use of blocking antibodies also demonstrated that the inhibiting effect found after GXM treatment of endothelium probably involves interference with both intercellular adhesion molecule-1 and E-selectin binding.
Decreased Electroporation Efficiency in
Borrelia burgdorferi
Containing Linear Plasmids lp25 and lp56: Impact on Transformation of Infectious
B. burgdorferi
Authors: Lawrenz Matthew B. , Kawabata Hiroki , Purser Joye E. , Norris Steven J. 
Published: October 2002

ABSTRACT

The presence of the linear plasmids lp25 and lp56 of
Borrelia burgdorferi
B31 was found to dramatically decrease the rate of transformation by electroporation with the shuttle vector pBSV2, an autonomously replicating plasmid that confers kanamycin resistance (P. E. Stewart, R. Thalken, J. L. Bono, and P. Rosa, Mol. Microbiol. 39:714-721, 2001).
B. burgdorferi
B31 clones had transformation efficiencies that were either low, intermediate, or high, and this phenotype correlated with the presence or absence of lp25 and lp56. Under the conditions utilized in this study, no transformants were detected in clones that contained both lp25 and lp56; the few kanamycin-resistant colonies isolated did not contain pBSV2, indicating that the resistance was due to mutation. Intermediate electroporation rates (10 to 200 colonies per μg of DNA) were obtained with B31 clones that were either lp25
−
and lp56
+
or lp25
+
and lp56
−
. Clones in this group that initially contained lp25 lacked this plasmid in pBSV2 transformants, a finding consistent with selective transformation of lp25
−
variants. High transformation rates (>1,000 colonies per μg of DNA) occurred in clones that lacked both lp25 and lp56. Sequence analysis indicated that lp25 and lp56 contain genes that may encode restriction and/or modification systems that could result in the low transformation rates obtained with strains containing these plasmids. The previously reported correlation between lp25 and infectivity in mice, coupled with the barrier lp25 presents to transformation, may explain the difficulty in obtaining virulent transformants of
B. burgdorferi
.
Involvement of Lsp, a Member of the LraI-Lipoprotein Family inStreptococcus pyogenes, in Eukaryotic Cell Adhesion and Internalization
Authors: Elsner Andrea , Kreikemeyer Bernd , Braun-Kiewnick Andrea , Spellerberg Barbara , Buttaro Bettina A. , Podbielski Andreas 
Published: October 2002

ABSTRACTThree open reading frames (ORFs) were identified by a genome walking strategy in the genomes of serotype M49 group A streptococcal (GAS) strains CS101 and 591. These ORFs were located between themgacore regulon and the dipeptide permease operon. The deduced amino acid (aa) sequences contained signature sequences indicative of a lipoprotein (306 aa), an intracellular protein (823 aa), and a secreted peptide (66 aa), respectively. ORF1 (named Lsp for lipoprotein ofStreptococcus pyogenes) and ORF2 exhibited a high degree of homology to thelmb/ORF2 genes ofS. agalactiae(B. Spellerberg et al., Infect. Immun. 67:871-878, 1999). The three ORFs were found to be present in each of the 27 GAS serotype strains tested. Transcription analysis revealed a polycistroniclsp/ORF2 and a monocistronic ORF3 message that were detected primarily at the transition from exponential to stationary growth phase.lspand ORF2 mutants, ORF2- and ORF3-luciferase reporter fusions, and antiserum against recombinant Lsp were produced to examine the biological role of these genes. Although high Zn2+and Cu2+ion concentrations decreasedlspoperon expression, Lsp did not transport divalent cations as described for other LraI-type operons. Thelspmutant had reduced fibronectin binding. Although no direct binding of Lsp to fibronectin could be demonstrated, thelspmutant showed decreased transcription ofprtF2encoding the fibronectin-binding protein F2. Both thelspand ORF2 mutants showed decreased laminin binding. Adherence to and internalization into A549 epithelial cells of both mutants was reduced without a detectable effect on eukaryotic cell viability. The transcription of a number of virulence factors was altered in thelspmutants and ORF2 mutants. The changes in laminin binding and eukaryotic cell internalization could be explained by changes in transcription ofspeB(cysteine protease) and/or the global regulatorsmga,csrRS, andnra.
Nucleotidase Cascades Are Catalyzed by Secreted Proteins of the Parasitic NematodeTrichinella spiralis
Authors: Gounaris Kleoniki 
Published: October 2002

ABSTRACTExtracellular nucleotides are signaling molecules whose receptor-mediated effects are involved in a variety of physiological responses in mammalian tissues. An overwhelming body of data indicate that inflammatory and other immune responses can be modulated by the availability and local concentrations of nucleotides via nucleotide receptor signaling, but this is only just beginning to be investigated in the context of infectious disease. Evidence is provided here that the parasitic nematodeTrichinella spiraliscan catalyze the conversion and thus modulate both the availability and concentration of extracellular nucleotides by means of the following secreted exoenzymes: apyrase, 5′-nucleotidase, and adenosine deaminase. These enzymes were characterized in terms of substrate specificity, kinetic behavior, pH, divalent cation preferences, and response to a series of compounds. The secreted 5′-nucleotidase was identified as a protein with an apparent molecular mass of 67 kDa after N-terminal amino acid sequencing of the purified protein. The presence of adenosine deaminase was confirmed in the secreted products by Western blotting with an antibody against a mammalian enzyme, as a protein with an apparent molecular mass of 38 kDa. These secreted proteins constitute an enzymatic cascade which catalyzes the degradation of extracellular nucleotides, with a potential physiological role in the regulation of purinergic signaling.
Identification and Functional Mapping of theMycoplasma fermentansP29 Adhesin
Authors: Leigh Spencer A. , Wise Kim S. 
Published: October 2002

ABSTRACTInitial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein ofMycoplasma fermentans, binds human HeLa cells and inhibitsM. fermentansbinding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction ofM. fermentanswith a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells byMycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that byMycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).
Characterization of the Leptospiral Outer Membrane and Description of Three Novel Leptospiral Membrane Proteins
Authors: Haake David A. , Matsunaga James 
Published: October 2002

ABSTRACT

The outer membrane (OM) of the mammalian pathogen
Leptospira kirschneri
was isolated in the form of membrane vesicles by alkaline plasmolysis and separated from the protoplasmic cylinder by sucrose density gradient ultracentrifugation. All four components of the alkaline plasmolysis buffer, including 1.0 M NaCl, 27% sucrose (wt/vol), 2 mM EDTA, and 10 mM Tris (pH 9), were required for efficient OM release, as judged by recovery of leptospiral lipopolysaccharide. Two populations of OM vesicles (OMVs) were recovered, with peak concentrations found in the sucrose gradient at densities of 1.16 and 1.18 g/ml. Transmission electron microscopy revealed that the more buoyant OMV population was smaller (<0.1 μm in diameter) than the denser OMV population (0.2 to 0.3 μm in diameter). The densities of both populations of OMVs were distinct from that of the protoplasmic-cylinder material, which was found in the sucrose gradient at a density of 1.20 g/ml. The OMV fractions were free of protoplasmic-cylinder material, as judged by immunoblotting with antibodies to the endoflagellar sheath protein, heat shock protein GroEL, and two novel cytoplasmic membrane proteins, lipoprotein LipL31 and transmembrane protein ImpL63. The protein components of the OMVs were characterized by one- and two-dimensional immunoblotting and found to include previously described OM proteins (OMPs), including the porin OmpL1; the lipoproteins LipL32, LipL36, and LipL41; and the peripheral membrane protein P31
LipL45
. A number of less well-characterized OMPs were also identified, including those with molecular masses of 16, 21, 21.5, 22, 31, 36, 44, 48, 90, and 116 kDa. The 48-kDa OMP was identified as a novel OM lipoprotein designated LipL48. The use of membrane-specific markers in OM isolation techniques facilitates an accurate description of the leptospiral OM and its components.
Activation of p38 Mitogen-Activated Protein Kinase AttenuatesLeishmania donovaniInfection in Macrophages
Authors: Junghae Muthoni , Raynes John G. 
Published: October 2002

ABSTRACTLeishmania-induced macrophage dysfunctions have been correlated with altered signaling events. In this work, we report that SB203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), increasesLeishmania donovanisurvival in human peripheral blood mononuclear macrophages. Consistent with this finding, activation of p38 and c-junN-terminal kinase (JNK) MAPK signaling pathways by anisomycin significantly reduced parasite survival within these cells. However, the majority of the effect was seen in a 50% reduction in the percentage of macrophages infected, with little effect on the highly infected macrophages. The observed effect was likely to be due to the p38 MAPK pathway since SB203580 was able to completely reverse the effect of anisomycin. These findings suggest that the previously reported p38 MAPK inhibition byLeishmaniainfection may be partially overcome by anisomycin. Similar effects were observed in pretreated macrophages or in treatment of infected macrophages. These results suggests that p38 MAPK activation may have a potential therapeutic value in the treatment of visceral leishmaniasis.
The Human Polymeric Immunoglobulin Receptor Facilitates Invasion of Epithelial Cells byStreptococcus pneumoniaein a Strain-Specific and Cell Type-Specific Manner
Authors: Brock Sean C. , McGraw Patricia A. , Wright Peter F. , Crowe James E. 
Published: October 2002

ABSTRACTStreptococcus pneumoniaeis a gram-positive bacterial pathogen that causes invasive life-threatening disease worldwide. This organism also commonly colonizes the upper respiratory epithelium in an asymptomatic fashion. To invade, this pathogen must traverse the respiratory epithelial barrier, allowing it to cause disease locally or disseminate hematogenously throughout the body. Previous work has demonstrated thatS. pneumoniaecholine-binding protein A, a pneumococcal surface protein, interacts specifically with the human polymeric immunoglobulin receptor, which is expressed by cells in the respiratory epithelium. Choline-binding protein A is required for efficient colonization of the nasopharynx in vivo. Additionally, a recent study showed that the R6x laboratory strain ofS. pneumoniaeinvades a human pharyngeal cell line in a human polymeric immunoglobulin receptor-dependent manner. These findings raised the possibility that the interaction between choline-binding protein A and human polymeric immunoglobulin receptor may be a key determinant ofS. pneumoniaepathogenesis. However, the strain used in prior invasion studies, R6x, is an unencapsulated, nonpathogenic strain. In the present study we determined the relative ability of strain R6x or pathogenic strains to invade a variety of human polymeric immunoglobulin receptor-expressing epithelial cell lines. The results of this work suggest that human polymeric immunoglobulin receptor-dependent enhanced invasion of epithelial cells byS. pneumoniaeis a limited phenomenon that occurs in a strain-specific and cell type-specific manner.
Attenuated
Salmonella enterica
Serovar Typhi Expressing Urease Effectively Immunizes Mice against
Helicobacter pylori
Challenge as Part of a Heterologous Mucosal Priming-Parenteral Boosting Vaccination Regimen
Authors: Londoño-Arcila Patricia , Freeman Donna , Kleanthous Harry , O'Dowd Aisling M. , Lewis Susan , Turner Arthur K. , Rees Emma L. , Tibbitts Timothy J. , Greenwood Judith , Monath Thomas P. , Darsley Michael J. 
Published: October 2002

ABSTRACT

Recombinant vaccine strains of
Salmonella enterica
serovar Typhi capable of expressing
Helicobacter pylori
urease were generated by transforming strains CVD908 and CVD908-
htrA
with a plasmid harboring the
ureAB
genes under the control of an in vivo-inducible promoter. The plasmid did not interfere with the ability of either strain to replicate and persist in human monocytic cells or with their transient colonization of mouse lungs. When administered to mice intranasally, both recombinant strains elicited antiurease immune responses skewed towards a Th1 phenotype. Vaccinated mice exhibited strong immunoglobulin G2a (IgG2a)-biased antiurease antibody responses as well as splenocyte populations capable of proliferation and gamma interferon (IFNγ) secretion in response to urease stimulation. Boosting of mice with subcutaneous injection of urease plus alum enhanced immune responses and led them to a more balanced Th1/Th2 phenotype. Following parenteral boost, IgG1 and IgG2a antiurease antibody titers were raised significantly, and strong urease-specific splenocyte proliferative responses, accompanied by IFNγ as well as interleukin-4 (IL-4), IL-5, and IL-10 secretion, were detected. Neither immunization with urease-expressing
S. enterica
serovar Typhi alone nor immunization with urease plus alum alone conferred protection against challenge with a mouse-adapted strain of
H. pylori
; however, a vaccination protocol combining both immunization regimens was protective. This is the first report of effective vaccination against
H. pylori
with a combined mucosal prime-parenteral boost regimen in which serovar Typhi vaccine strains are used as antigen carriers. The significance of these findings with regard to development of a human vaccine against
H. pylori
and modulation of immune responses by heterologous prime-boost immunization regimens is discussed.
Increased
Trypanosoma cruzi
Invasion and Heart Fibrosis Associated with High Transforming Growth Factor β Levels in Mice Deficient in α
2
-Macroglobulin
Authors: Waghabi M. C. , Coutinho C. M. L. M. , Soeiro M. N. C. , Pereira M. C. S. , Feige J.-J. , Keramidas M. , Cosson A. , Minoprio P. , Van Leuven F. , Araújo-Jorge T. C. 
Published: October 2002

ABSTRACT

Trypanosoma cruzi
proteinases are involved in host cell invasion in human patients and in mouse models. In mice, murine α
2
-macroglobulin (MAM) and murinoglobulin are circulating plasma proteinase inhibitors that also have important roles in inflammation and immune modulation. To define their role in experimental Chagas disease, we investigated the susceptibility to
T. cruzi
infection of mice that are deficient only in α2-macroglobulins (AM-KO) or in both MAM and monomeric murinoglobulin-1 (MM-KO), relative to the wild type (WT). Despite the high parasite load, parasitemia was lower in AM-KO and MM-KO mice than in WT mice. Nevertheless, we observed a significantly higher parasite load in the hearts of AM-KO and MM-KO mice, i.e., more amastigote nests and inflammatory infiltrates than in WT mice. This result demonstrates a protective role for MAM in the acute phase of murine
T. cruzi
infection. We further demonstrated in vitro that human α2-macroglobulins altered the trypomastigote morphology and motility in a dose-dependent way, and that also impaired
T. cruzi
invasion in cardiomyocytes. Finally, we demonstrated that the levels of transforming growth factor β in AM-KO mice increased significantly in the third week postinfection, concomitant with high amastigote burden and important fibrosis. Combined, these in vivo and in vitro findings demonstrate that the MAM contribute to the resistance of mice to acute myocarditis induced by experimental
T. cruzi
infection.
Histoplasma capsulatumSynthesizes Melanin-Like Pigments In Vitro and during Mammalian Infection
Authors: Nosanchuk Joshua D. , Gómez Beatriz L. , Youngchim Sirida , Díez Soraya , Aisen Philip , Zancopé-Oliveira Rosely M. , Restrepo Angela , Casadevall Arturo , Hamilton Andrew J. 
Published: October 2002

ABSTRACTMelanin is made by several important pathogenic fungi and has been implicated in the pathogenesis of a number of fungal infections. This study investigated whether the thermally dimorphic fungal pathogenHistoplasma capsulatumvar. capsulatumproduced melanin or melanin-like compounds in vitro and during infection. Growth ofH. capsulatummycelia in chemically defined minimal medium produced pigmented conidia. Growth ofH. capsulatumyeast in chemically defined minimal medium withl-3,4-dihydroxyphenylalanine (DOPA) or (-)-epinephrine produced pigmented cells. Treatment of the pigmented cells with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were similar in size and shape to their respective propagules. Melanin-binding monoclonal antibodies (MAb) labeled pigmented conidia, yeast, and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that pigmented yeast cells and particles derived from pigmented cells were stable free radicals consistent with their identification as melanins. Tissues from mice infected withH. capsulatumand from biopsy specimens from a patient with histoplasmosis contained fungal cells that were labeled by melanin-binding MAb. Digestion of infected mouse tissues yielded dark particles that reacted with the melanin-binding MAb and were similar in appearance toH. capsulatumyeast cells. Additionally, sera from infected mice contained antibodies that bound melanin particles. Phenoloxidase activity capable of synthesizing melanin from L-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate thatH. capsulatumconidia and yeast can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role to play in the pathogenesis of histoplasmosis.