bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Protein Expression in Phenotypic Variants of
Streptococcus pneumoniae
Authors: Overweg Karin , Pericone Chris D. , Verhoef Gerridina G. C. , Weiser Jeffrey N. , Meiring Hugo D. , De Jong Ad P. J. M. , De Groot Ronald , Hermans Peter W. M. 
Published: July 2002

ABSTRACT

Streptococcus pneumoniae
undergoes spontaneous phase variation resulting in opaque and transparent colony forms. Differences in colony opacity correlate with differences in virulence: the transparent variants are more capable of colonizing the nasopharynx, whereas the opaque variants show increased virulence during systemic infections. To gain insight into the pathogenesis of pneumococcal disease at the molecular level, protein expression patterns of the phenotypic variants of two pneumococcal strains were compared by high-resolution two-dimensional protein electrophoresis. In comparison with transparent variants, the opaque variants reduced the expression of two proteins and overexpressed one protein. The proteins were identified by mass spectrometric analysis. The protein overexpressed in the opaque phenotype revealed significant homology to elongation factor Ts of
Helicobacter pylori
. One of the two proteins that were underexpressed in the opaque variants revealed significant homology to the proteinase maturation protein PrtM of
Lactocobacillus paracasei
, a member of the family of peptidyl-prolyl
cis/trans
isomerases. A consensus lipoprotein signal sequence suggests that the putative proteinase maturation protein A, designated PpmA, is located at the surface of the pneumococcus and may play a role in the maturation of surface or secreted proteins. The second underexpressed protein was identified as pyruvate oxidase, SpxB. The lower SpxB expression in opaque variants most probably explains the reduced production of hydrogen peroxide, a reaction product of SpxB, in this variant. Since a
spxB
-defective pneumococcal mutant has decreased ability to colonize the nasopharynx (B. Spellerberg, D. R. Cundell, J. Sandros, B. J. Pearce, I. Idanpaan-Heikkila, C. Rosenow, and H. R. Masure, 1996. Mol. Microbiol. 19:803–813, 1996), our data suggest that SpxB plays an important role in enhancing the ability of transparent variants to efficiently colonize the nasopharynx.
Cyclic Nucleotide Signaling in
Toxoplasma gondii
Bradyzoite Differentiation
Authors: Kirkman Laura A. , Weiss Louis M. , Kim Kami 
Published: July 2002

ABSTRACT

The ability of
Toxoplasma gondii
tachyzoites to differentiate into latent bradyzoite forms is essential for pathogenesis of clinical disease. We examined the effects of cyclic nucleotides on
T. gondii
bradyzoite differentiation in vitro. Differentiation of tachyzoites to bradyzoites was measured in an immunofluorescence assay using ME49 or its clonal derivative PLK, two well-characterized
T. gondii
strains. Treatment of human fibroblast cultures infected with
T. gondii
with 8-(4-chlorophenylthio)-cyclic GMP (CPT-cGMP), a membrane-permeable, nonhydrolyzable analogue of cGMP, resulted in an increased percentage of bradyzoite-positive vacuoles. Cyclic AMP (cAMP) also induced in vitro conversion of PLK, but the method of cAMP elevation was critical. Forskolin raises cAMP levels transiently and induced bradyzoites, whereas agents predicted to cause sustained elevation of cAMP were inhibitory to parasite conversion. Levels of cAMP were measured in host cells and extracellular tachyzoites. Forskolin, CPT-cGMP, and agents known to induce bradyzoite formation elevated cAMP in host cells and PLK parasites. These data suggest cyclic nucleotide signaling pathways are important in the stress-induced conversion of
T. gondii
tachyzoites to bradyzoites. Furthermore, because cAMP elevation was seen in PLK but not RH, a
T. gondii
strain that did not differentiate well in our assay, cAMP signaling within the parasite is likely to be critical.
Description of Staphylococcus Serine Protease (
ssp
) Operon in
Staphylococcus aureus
and Nonpolar Inactivation of
sspA
-Encoded Serine Protease
Authors: Rice Kelly , Peralta Robert , Bast Darrin , de Azavedo Joyce , McGavin Martin J. 
Published: July 2002

ABSTRACT

Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of
Staphylococcus aureus
in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the
ssp
locus of
S. aureus
RN6390. The
ssp
gene encoding V8 protease is designated as
sspA
, and is followed by
sspB
, which encodes a 40.6-kDa cysteine protease, and
sspC
, which encodes a 12.9-kDa protein of unknown function.
S. aureus
SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22- to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of
sspA
exerted a pleiotropic effect,
S. aureus
SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.
Interaction of
Bartonella henselae
with the Murine Macrophage Cell Line J774: Infection and Proinflammatory Response
Authors: Musso Tiziana , Badolato Raffaele , Ravarino Daniela , Stornello Sarah , Panzanelli Patrizia , Merlino Chiara , Savoia Dianella , Cavallo Rossana , Ponzi Alessandro Negro , Zucca Mario 
Published: July 2002

ABSTRACT

Bartonella henselae
is the causative agent of cat scratch disease (CSD), a self-limiting condition characterized by a subacute regional lymphadenopathy that may develop into disseminated bartonellosis in immunocompromised subjects. Mice experimentally infected with
B. henselae
display typical liver and spleen granulomas rich in T cells and macrophages. So far there are no data on the interaction between bartonellae and macrophages. In order to clarify this topic, we investigated the interaction of
B. henselae
with J774, a mouse macrophage cell line. Analysis of bacterial uptake by functional assays and transmission electron microscopy indicates that bartonellae can enter and survive inside J774. Entry occurred within 30 min postinfection and reached a plateau at 160 min. Infection of J774 was followed by a dose-dependent release of the proinflammatory cytokines tumor necrosis factor alpha, interleukin 1β (IL-1β), and IL-6. Bartonellae persisted intracellularly without loss of viability for at least 8 h, and their number slightly decreased 24 h postinfection. Gamma interferon (IFN-γ) treatment of J774 significantly decreased the number of recoverable bacteria at 8 and 24 h. This enhancement of macrophage bactericidal activity was associated with nitric oxide (NO) release and was prevented by the addition of the competitive inhibitor of NO synthesis

N
G

-monomethyl
l
-arginine. These findings suggest that IFN-γ-mediated activation of macrophages may be important for the clearing of
B. henselae
infection and that anti-
B. henselae
microbicidal activity of IFN-γ-activated macrophages is mediated to a large extent by NO production.
Uropathogenic
Escherichia coli
Potentiates Type 1 Pilus-Induced Apoptosis by Suppressing NF-κB
Authors: Klumpp David J. , Weiser Adam C. , Sengupta Shomit , Forrestal Sarah G. , Batler Robert A. , Schaeffer Anthony J. 
Published: July 2002

ABSTRACT

Urinary tract infections (UTIs) are among the most common inflammatory diseases. Acute UTIs are typically caused by type 1-piliated
Escherichia coli
and result in urothelial apoptosis, local cytokine release, and neutrophil infiltration. To examine the urothelial apoptotic response, a human urothelial cell line was incubated with various
E. coli
isolates and was then characterized by flow cytometry. Uropathogenic
E. coli
(UPEC) induced rapid urothelial apoptosis that was strictly dependent upon interactions mediated by type 1 pili. Interestingly, nonpathogenic HB101
E. coli
expressing type 1 pili induced apoptosis at approximately 50% of the level induced by UPEC, suggesting that pathogenic strains contribute to apoptosis by pilus-independent mechanisms. Consistent with this possibility, UPEC blocked activity of an NF-κB-dependent reporter in response to inflammatory stimuli, yet this effect was independent of functional type 1 pili and was not mediated by laboratory strains of
E. coli
. UPEC suppressed NF-κB by stabilizing IκBα, and UPEC rapidly altered cellular signaling pathways. Finally, blocking NF-κB activity increased the level of piliated HB101-induced apoptosis to the level of apoptosis induced by UPEC. These results suggest that UPEC blocks NF-κB and thereby enhances type 1 pili-induced apoptosis as a component of the uropathogenic program.
Capsule Production and Growth Phase Influence Binding of Complement toStaphylococcus aureus
Authors: Cunnion K. M. , Lee J. C. , Frank M. M. 
Published: July 2002

ABSTRACTComplement-mediated opsonization of bacteria by C3 binding is an important component of the host innate immune system. Little information is available concerning the interaction between complement proteins and capsule type 5 and 8Staphylococcus aureusstrains, even though these isolates are responsible for ∼70% of human staphylococcal infections. To investigate the importance of an intact complement pathway in an experimental staphylococcal infection, control and C3-depleted mice were challenged intravenously with 107CFU of a serotype 5S. aureusisolate. Whereas only 8% of the control mice succumbed to the infection, 64% of the complemented-depleted animals died. In vitro parameters of C3 binding to two heavily encapsulated (CP++) strains, three encapsulated (CP+) strains, and an isogenic capsule-negative (CP−) mutant were examined. The alternative pathway contributed 90% of C3 binding in 20% serum at 30 min, whereas it accounted for only 13% of C3 binding in 2% serum. Stationary-phase organisms bound only 10% as much C3 as mid-log-phase organisms; this was only in part due to capsule. When theS. aureusstrains were cultivated on solid medium, the CP++ isolates bound 50% less C3 than CP+ strains; a CP+ strain bound 42% less C3 than the CP− mutant. Both C3b and iC3b fragments of C3 bound toS. aureuscells, and about one-third of the bound C3 was shed from the staphylococcal surface as iC3b, regardless of the CP phenotype of the strain. Thus, the phase of growth and presence of capsule are critical to opsonization.
Streptococcus pneumoniae
Causes Experimental Meningitis following Intranasal and Otitis Media Infections via a Nonhematogenous Route
Authors: Marra Andrea , Brigham Daniel 
Published: July 2002

ABSTRACT

Using two different animal models of
Streptococcus pneumoniae
infection, we have demonstrated that this organism is able to spread to the central nervous system and cause meningitis by bypassing the bloodstream. Following respiratory tract infection induced via intranasal inoculation, bacteria were rapidly found in the bloodstream and brains in the majority of infected mice. A similar pattern of dissemination occurred following otitis media infection via transbullar injection of gerbils. However, a small percentage of animals infected by either route showed no bacteria in the blood and yet did have significant numbers of bacteria in brain tissue. Subsequent experiments using a
galU
mutant of
S. pneumoniae
, which is impaired in its ability to disseminate to the bloodstream following infection, showed that this organism is able to spread to the brain and cerebrospinal fluid. These results demonstrate that, unlike many bacterial pathogens that cause meningitis,
S. pneumoniae
is able to do so independent of bloodstream involvement upon different routes of infection. This may address the difficulty in treating human infections caused by this organism.
Application of High-Density Array-Based Signature-Tagged Mutagenesis To Discover Novel
Yersinia
Virulence-Associated Genes
Authors: Karlyshev A. V. , Oyston P. C. F. , Williams K. , Clark G. C. , Titball R. W. , Winzeler E. A. , Wren B. W. 
Published: July 2002

ABSTRACT

Yersinia pestis
, the causative agent of plague, and the enteropathogen
Yersinia pseudotuberculosis
have nearly identical nucleotide similarity yet cause markedly different diseases. To investigate this conundrum and to study
Yersinia
pathogenicity, we developed a high-density oligonucleotide array-based modification of signature-tagged mutagenesis (STM).
Y. pseudotuberculosis
YPIII mutants constructed with the tagged transposons were evaluated in the murine yersiniosis infection model. The DNA tags were amplified using biotinylated primers and hybridized to high-density oligonucleotide arrays containing DNA complementary to the tags. Comparison of the hybridization signals from input pools and output pools identified a mutant whose relative abundance was significantly reduced in the output pool. Sequence data from 31 transposon insertion regions was compared to the complete
Y. pestis
CO92 genome sequence. The 26 genes present in both species were found to be almost identical, but five
Y. pseudotuberculosis
genes identified through STM did not have counterparts in the
Y. pestis
genome and may contribute to the different tropisms in these closely related pathogens. Potential virulence genes identified include those involved in lipopolysaccharide biosynthesis, adhesion, phospholipase activity, iron assimilation, and gene regulation. The phospholipase A (PldA) mutant exhibited reduced phospholipase activity compared to the wild-type strain and in vivo attenuation of the mutant was confirmed. The combination of optimized double tag sequences and high-density array hybridization technology offers improved performance, efficiency, and reliability over classical STM and permits quantitative analysis of data.
Differential Patterns of Human Immunoglobulin G Subclass Responses to Distinct Regions of a Single Protein, the Merozoite Surface Protein 1 ofPlasmodium falciparum
Authors: Cavanagh David R. , Dobaño Carlota , Elhassan Ibrahim M. , Marsh Kevin , Elhassan Ahmed , Hviid Lars , Khalil E. At Tahir G. , Theander Thor G. , Arnot David E. , McBride Jana S. 
Published: July 2002

ABSTRACTComparisons of immunoglobulin G (IgG) subclass responses to the major polymorphic region and to a conserved region of MSP-1 in three cohorts of African villagers exposed toPlasmodium falciparumrevealed that responses to Block 2 are predominantly IgG3 whereas antibodies to MSP-119are mainly IgG1. The striking dominance of IgG3 to Block 2 may explain the short duration of this response and also the requirement for continuous stimulation by malaria infection to maintain clinical immunity.
Human Natural Killer Cells Mediate Killing of IntracellularMycobacterium tuberculosisH37Rv via Granule-Independent Mechanisms
Authors: Brill Kevin J. , Li Qing , Larkin Rhonda , Canaday David H. , Kaplan David R. , Boom W. Henry , Silver Richard F. 
Published: July 2002

ABSTRACTDespite the continued importance of tuberculosis as a world-wide threat to public health, little is known about the mechanisms used by human lymphocytes to contain and kill the intracellular pathogenMycobacterium tuberculosis. We previously described an in vitro model of infection of human monocytes (MN) with virulentM. tuberculosisstrain H37Rv in which the ability of peripheral blood lymphocytes to limit intracellular growth of the organism could be measured. In the current study, we determined that lymphocyte-mediated killing of intracellularM. tuberculosisoccurs within the first 24 h of coculture with infected MN. Natural killer (NK) cells isolated from both purified protein derivative (PPD)-positive and PPD-negative subjects were capable of mediating this early killing of intracellular H37Rv. NK cell-mediated killing of intracellularM. tuberculosiswas not associated with the production of gamma interferon. Transferred supernatants of cocultured NK cells andM. tuberculosis-infected MN could not mediate the killing of intracellularM. tuberculosis, and Transwell studies indicated that direct cell-to-cell contact was required for NK cells to mediate the killing of the organism. Killing was not dependent upon exocytosis of NK cell cytotoxic granules. NK cells induced apoptosis of mycobacterium-infected MN, but neither killing of intracellularM. tuberculosisby NK cells nor NK cell-induced apoptosis of infected MN was inhibited by blocking the interaction of FasL and Fas. Thus, human NK cells may mediate killing of intracellularM. tuberculosisvia alternative apoptotic pathways.
Cloning and Expression of the Gene Which Encodes a Tube Precipitin Antigen and Wall-Associated β-Glucosidase of
Coccidioides immitis
Authors: Hung Chiung-Yu , Yu Jieh-Juen , Lehmann Paul F. , Cole Garry T. 
Published: July 2002

ABSTRACT

We report the structure and expression of the
Coccidioides immitis BGL2
gene which encodes a previously characterized 120-kDa glycoprotein of this fungal respiratory pathogen. The glycoprotein is recognized by immunoglobulin M tube precipitin (TP) antibody present in sera of patients with coccidioidomycosis, a reaction which has been used for serodiagnosis of early coccidioidal infection. The deduced amino acid sequence of BGL2 shows 12 potential N glycosylation sites and numerous serine-threonine-rich regions which could function as sites for O glycosylation. In addition, the protein sequence includes a domain which is characteristic of family 3 glycosyl hydrolases. Earlier biochemical studies of the purified 120-kDa TP antigen revealed that it functions as a β-glucosidase (EC
3.2.1.21
). Its amino acid sequence shows high homology to several other reported fungal β-glucosidases which are members of the family 3 glycosyl hydrolases. Results of previous studies have also suggested that the 120-kDa β-glucosidase participates in wall modification during differentiation of the parasitic cells (spherules) of
C. immitis
. In this study we showed that expression of the
BGL2
gene is elevated during isotropic growth of spherules and the peak of wall-associated BGL2 enzyme activity correlates with this same phase of parasitic cell differentiation. These data support our hypothesis that the 120-kDa β-glucosidase plays a morphogenetic role in the parasitic cycle of
C. immitis
.
Different Regions of the Malaria Merozoite Surface Protein 1 ofPlasmodium chabaudiElicit Distinct T-Cell and Antibody Isotype Responses
Authors: Quin Stuart J. , Langhorne Jean 
Published: July 2002

ABSTRACTIn this study we have investigated the antibody and CD4 T-cell responses to the well-characterized malaria vaccine candidate MSP-1 during the course of a primaryPlasmodium chabaudi chabaudi(AS) infection. Specific antibody responses can be detected within the first week of infection, and CD4 T cells can be detected after 3 weeks of infection. The magnitude of the CD4 T-cell response elicited during a primary infection depended upon the region of MSP-1. In general, the highest precursor frequencies were obtained when a recombinant MSP-1 fragment corresponding to amino acids 900 to 1507 was used as the antigen in vitro. By contrast, proliferative and cytokine responses against amino acids 1508 to 1766 containing the C-terminal 21-kDa region of the molecule were low. The characteristic interleukin 4 (IL-4) switch that occurs in the CD4 T-cell population after an acute blood stageP. c. chabaudiinfection was only consistently observed in the response to the amino acid 900 to 1507 MSP1 fragment. A lower frequency of IL-4-producing cells was seen in response to other regions. Although the magnitudes of the immunoglobulin G antibody responses to the different regions of MSP-1 were similar, the isotype composition of each response was distinct, and there was no obvious relationship with the type of T helper cells generated. Interestingly, a relatively high antibody response to the C-terminal region of MSP-1 was observed, suggesting that T-cell epitopes outside of this region may provide the necessary cognate help for specific antibody production.
Saccharomyces boulardiiStimulates Intestinal Immunoglobulin A Immune Response toClostridium difficileToxin A in Mice
Authors: Qamar Amir , Aboudola Samer , Warny Michel , Michetti Pierre , Pothoulakis Charalabos , LaMont J. Thomas , Kelly Ciarán P. 
Published: July 2002

ABSTRACTSaccharomyces boulardiiis a nonpathogenic yeast that protects against antibiotic-associated diarrhea and recurrentClostridium difficilecolitis. The administration ofC. difficiletoxoid A by gavage toS. boulardii-fed BALB/c mice caused a 1.8-fold increase in total small intestinal immunoglobulin A levels (P= 0.003) and a 4.4-fold increase in specific intestinal anti-toxin A levels (P< 0.001). Enhancing host intestinal immune responses may be an important mechanism forS. boulardii-mediated protection against diarrheal illnesses.
Reversal of the CD4+/CD8+T-Cell Ratio in Lymph Node Cells upon In Vitro Mitogenic Stimulation by Highly Purified, Water-Soluble S3-S4 Dimer of Pertussis Toxin
Authors: Latif Rauf , de Rosbo Nicole Kerlero , Amarant Tany , Rappuoli Rino , Sappler Gregor , Ben-Nun Avraham 
Published: July 2002

ABSTRACTPertussis toxin (PT), a holomer consisting of a catalytic S1 subunit and a B oligomer composed of S2-S4 and S3-S4 dimers, held together by the S5 subunit, exerts profound effects on immune cells, including T-cell mitogenicity. While the mitogenic activity of PT was shown to reside fully within the B oligomer, it could not be assigned to any particular B-oligomer component. In this study, we purified the S3-S4 dimer to homogeneity under conditions propitious to maintenance of the native conformation. In contrast to previous reports which suggested that both S3-S4 and S2-S4 dimers are necessary for mitogenic activity, our preparation of the highly purified S3-S4 dimer was as strongly mitogenic as the B oligomer, suggesting that the S3-S4 dimer accounts for the mitogenic activity of the B oligomer. Moreover, in vitro stimulation of naive lymphocytes by the S3-S4 dimer resulted in reversal of the normal CD4+/CD8+T-cell ratio from approximately 2:1 to 1:2. The reversal of the CD4+/CD8+T-cell ratio is unlikely to be due to preferential apoptosis-necrosis of CD4+T cells, as indicated by fluorescence-activated cell sorter analysis of annexin-stained T-cell subsets, or to preferential stimulation of CD8+T cells. The mechanism underlying the reversal requires further investigation. Nevertheless, the data presented indicate that the S3-S4 dimer may have potential use in the context of diseases amenable to immunological modulation.