bluuebunny/crossref_metadata_2025 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

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

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

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

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

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

Complement-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

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

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

Comparisons 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

Despite 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

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

In 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

Saccharomyces 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

Pertussis 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.
Attenuated
Shigella flexneri
2a Δ
guaBA
Strain CVD 1204 Expressing Enterotoxigenic
Escherichia coli
(ETEC) CS2 and CS3 Fimbriae as a Live Mucosal Vaccine against
Shigella
and ETEC Infection
Authors: Altboum Zeev , Barry Eileen M. , Losonsky Genevieve , Galen James E. , Levine Myron M.
Published: July 2002

To construct a prototype hybrid vaccine against
Shigella
and enterotoxigenic
Escherichia coli
(ETEC), the genes encoding the production of ETEC CS2 and CS3 fimbriae were isolated and expressed in attenuated
Shigella flexneri
2a
guaBA
strain CVD 1204. The CS2
cotA
to -
D
genes, isolated from ETEC strain C91F, and the CS3
cstA
to -
H
genes, subcloned from plasmid pCS100, were cloned into ∼15-copy-number-stabilized pGA1 behind the osmotically regulated
ompC
promoter, resulting in high expression of both fimbriae. Under nonselective in vitro growth conditions, pGA1-CS2 and pGA1-CS3 were stable in CVD 1204, exhibiting a plasmid loss of only approximately 1% per duplication. Expression of CS2 and CS3 reduced the invasiveness of
Shigella
for HeLa cells and slowed the intracellular growth rate. Guinea pigs immunized intranasally with CVD 1204(pGA1-CS2) or CVD 1204(pGA1-CS3), or with a mixture of these strains, developed secretory immunoglobulin A (IgA) in tears and serum IgG antibodies against
Shigella
lipopolysaccharide, CS2, and CS3 antigens. Moreover, the animals were protected against keratoconjunctivitis following conjunctival challenge with virulent
S. flexneri
2a strain 2457T. Animals immunized with
Shigella
expressing CS2 or CS3 developed serum antibodies that agglutinated
Shigella
as well as an ETEC strain bearing the homologous fimbriae, whereas animals immunized with combined CVD 1204(pGA1-CS2) and CVD 1204(pGA1-CS3) developed antibodies that agglutinated all three test strains. These observations support the feasibility of a multivalent vaccine against shigellosis and ETEC diarrhea consisting of multiple
Shigella
live vectors expressing relevant ETEC antigens.
Calprotectin Expression by Gingival Epithelial Cells
Authors: Ross Karen F. , Herzberg Mark C.
Published: July 2002

Calprotectin, a heterodimer of MRP8 and MRP14 with antimicrobial properties, is found in the cytosol of neutrophils, monocytes, and human gingival keratinocytes. During inflammation of the oral mucosa, the expression of immunoreactive calprotectin appears upregulated. Given the possible cell sources, we sought to learn if epithelial cells upregulate calprotectin in response to proinflammmatory agents. First, human gingival keratinocytes were maintained in primary culture until senescence. At each passage, cells were harvested and analyzed for quantitative expression of MRP8 and MRP14 subunit mRNA by RNase protection assays and calprotectin complex by enzyme-linked immunosorbent assay. Calprotectin expression was constitutive in the primary gingival keratinocytes, but calprotectin-specific mRNA and protein tended to increase as the cells neared senescence. To test whether calprotectin expression was inducible, immortalized gingival keratinocyte cultures were treated for 2 to 4 h with lipopolysaccharide (LPS) or interleukin-1β (IL-1β). As a positive control for inducible expression, immortalized keratinocytes were incubated with phorbol myristate acetate (PMA) (50 ng/ml) for 24 h. Incubation with PMA stimulated increased expression of MRP8 and MRP14 mRNA within 2 h, peaking within 5 h. MRP8- and MRP14-specific mRNA expression by immortalized keratinocytes appeared to be unaffected by LPS or IL-1β. In contrast, LPS, IL-1β, and PMA each upregulated IL-8. These data show that calprotectin mRNA is expressed constitutively in cultured keratinocytes, while expression by immortalized cells appears to be independent of the exogenous proinflammatory agents LPS and IL-1β.
Recruitment of Cytoskeletal and Signaling Proteins to Enteropathogenic and Enterohemorrhagic
Escherichia coli
Pedestals
Authors: Goosney Danika L. , DeVinney Rebekah , Finlay B. Brett
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC) is a human pathogen that attaches to intestinal epithelial cells and causes chronic watery diarrhea. A close relative, enterohemorrhagic
E. coli
(EHEC), causes severe bloody diarrhea and hemolytic-uremic syndrome. Both pathogens insert a protein, Tir, into the host cell plasma membrane where it binds intimin, the outer membrane ligand of EPEC and EHEC. This interaction triggers a cascade of signaling events within the host cell and ultimately leads to the formation of an actin-rich pedestal upon which the pathogen resides. Pedestal formation is critical in mediating EPEC- and EHEC-induced diarrhea, yet very little is known about its composition and organization. In EPEC, pedestal formation requires Tir tyrosine 474 phosphorylation. In EHEC Tir is not tyrosine phosphorylated, yet the pedestals appear similar. The composition of the EPEC and EHEC pedestals was analyzed by examining numerous cytoskeletal, signaling, and adapter proteins. Of the 25 proteins examined, only two, calpactin and CD44, were recruited to the site of bacterial attachment independently of Tir. Several others, including ezrin, talin, gelsolin, and tropomyosin, were recruited to the site of EPEC attachment independently of Tir tyrosine 474 phosphorylation but required Tir in the host membrane. The remaining proteins were recruited to the pedestal in a manner dependent on Tir tyrosine phosphorylation or were not recruited at all. Differences were also found between the EPEC and EHEC pedestals: the adapter proteins Grb2 and CrkII were recruited to the EPEC pedestal but were absent in the EHEC pedestal. These results demonstrate that although EPEC and EHEC recruit similar cytoskeletal proteins, there are also significant differences in pedestal composition.
Molecular and Genomic Analysis of Genes Encoding Surface-Anchored Proteins from
Clostridium difficile
Authors: Karjalainen Tuomo , Waligora-Dupriet Anne-Judith , Cerquetti Marina , Spigaglia Patrizia , Maggioni Andrea , Mauri Pierluigi , Mastrantonio Paola
Published: July 2002

The gene
slpA
, encoding the S-layer precursor protein in the virulent
Clostridium difficile
strains C253 and 79–685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79–685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins.
Influence of Cultivation Media on Genetic Regulatory Patterns in
Borrelia burgdorferi
Authors: Yang Xiaofeng , Popova Taissia G. , Goldberg Martin S. , Norgard Michael V.
Published: July 2002

Barbour-Stoenner-Kelly II (BSKII) medium and BSKH medium both are routinely used for the cultivation of
Borrelia burgdorferi
. However, heretofore there have been no studies to compare how these two media affect gene expression patterns in virulent
B. burgdorferi
. In the present study, we found that some
B. burgdorferi
strain 297 genes (e.g.,
ospA
,
mlp-7A
,
mlp-8
,
p22
, and
lp6.6
) that typically are regulated by temperature or pH displayed their predicted pattern of expression when
B. burgdorferi
was cultivated in BSKH medium; this was not true when spirochetes were cultivated in conventional BSKII medium. The results suggest that BSKH medium is superior to BSKII medium for gene expression studies with
B. burgdorferi
.
Malnutrition Alters the Innate Immune Response and Increases Early Visceralization followingLeishmania donovaniInfection
Authors: Anstead Gregory M. , Chandrasekar Bysani , Zhao Weiguo , Yang Jue , Perez Luis E. , Melby Peter C.
Published: July 2002

Malnutrition is a risk factor for the development of visceral leishmaniasis. However, the immunological basis for this susceptibility is unknown. We have developed a mouse model to study the effect of malnutrition on innate immunity and early visceralization followingLeishmania donovaniinfection. Three deficient diets were studied, including 6, 3, or 1% protein; these diets were also deficient in iron, zinc, and calories. The control diet contained 17% protein, was zinc and iron sufficient, and was provided ab libitum. Three days after infection withL. donovanipromastigotes, the total extradermal (lymph nodes, liver, and spleen) and skin parasite burdens were equivalent in the malnourished (3% protein) and control mice, but in the malnourished group, a greater percentage (39.8 and 4.0%, respectively;P= 0.009) of the extradermal parasite burden was contained in the spleen and liver. The comparable levels of parasites in the footpads in the two diet groups and the higher lymph node parasite burdens in the well-nourished mice indicated that the higher visceral parasite burdens in the malnourished mice were not due to a deficit in local parasite killing but to a failure of lymph node barrier function. Lymph node cells from the malnourished, infected mice produced increased levels of prostaglandin E2(PGE2) and decreased levels of interleukin-10. Inducible nitric oxide synthase activity was significantly lower in the spleen and liver of the malnourished mice. Thus, malnutrition causes a failure of lymph node barrier function afterL. donovaniinfection, which may be related to excessive production of PGE2and decreased levels of IL-10 and nitric oxide.
Chlamydia trachomatisPersistence in the Female Mouse Genital Tract: Inducible Nitric Oxide Synthase and Infection Outcome
Authors: Ramsey Kyle H. , Miranpuri Gurwattan S. , Sigar Ira M. , Ouellette Scott , Byrne Gerald I.
Published: July 2002

It was previously reported that female mice resolve a primaryChlamydia trachomatisurogenital infection independent of inducible nitric oxide synthase (iNOS). We now report that although iNOS-deficient (NOS2−/−) mice resolve culture-apparent infection in a fashion similar to that of normal control (NOS2+/+) mice, they sustain significantly increased rates of disease, as assessed by hydrosalpinx formation. PCR amplification ofompAfollowed by Southern blot detection of amplicands revealed the presence of chlamydial DNA in the lower genital tracts of both NOS2−/−and NOS2+/+mice at ≥120 days postinfection and in upper genital tract tissues at >120 days postinfection. However, only NOS2−/−mice shed low numbers of viable chlamydiae from the lower genital tract after immunosuppressive treatment at 120 days postinfection. When cultured primary murine lung fibroblasts were activated in the presence of gamma interferon (IFN-γ), inhibition of chlamydial growth occurred in both NOS2+/+and NOS2−/−cells, but the inhibition was reversible after removal of the cytokine in the NOS2−/−primary cell culture only. The iNOS-independent inhibition was microbistatic but was independent of 2,3-indoleamine dioxygenase activity. We conclude that chlamydial DNA and antigens persist in mice subsequent to culture-apparent resolution. In addition, IFN-γ induces in vivo inhibition of chlamydial growth through microbistatic mechanisms in the absence of iNOS activity, but in the presence of iNOS activity, IFN-γ is microbicidal and effects eradication.
Enteropathogenic
Escherichia coli
Activates Ezrin, Which Participates in Disruption of Tight Junction Barrier Function
Authors: Simonovic Ivana , Arpin Monique , Koutsouris Athanasia , Falk-Krzesinski Holly J. , Hecht Gail
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC) is an important human intestinal pathogen, especially in infants. EPEC adherence to intestinal epithelial cells induces the accumulation of a number of cytoskeletal proteins beneath the bacteria, including the membrane-cytoskeleton linker ezrin. Evidence suggests that ezrin can participate in signal transduction. The aim of this study was to determine whether ezrin is activated following EPEC infection and if it is involved in the cross talk with host intestinal epithelial cells. We show here that following EPEC attachment to intestinal epithelial cells there was significant phosphorylation of ezrin, first on threonine and later on tyrosine residues. A significant increase in cytoskeleton-associated ezrin occurred following phosphorylation, suggesting activation of this molecule. Nonpathogenic
E. coli
and EPEC strains harboring mutations in type III secretion failed to elicit this response. Expression of dominant-negative ezrin significantly decreased the EPEC-elicited association of ezrin with the cytoskeleton and attenuated the disruption of intestinal epithelial tight junctions. These results suggest that ezrin is involved in transducing EPEC-initiated signals that ultimately affect host physiological functions.
Delayed Invasion of the Kidney and Brain byBorrelia crociduraein Plasminogen-Deficient Mice
Authors: Nordstrand Annika , Shamaei-Tousi Alireza , Ny Annelii , Bergström Sven
Published: July 2002

Borrelia crociduraeis an etiologic agent of relapsing fever in Africa and is transmitted to humans by the bite of soft ticks of the genusOrnithodoros. The role of the plasminogen (Plg) activation system for the pathogenicity ofB. crociduraewas investigated by infection of Plg-deficient (plg−/−) and Plg wild-type (plg+/+) mice. No differences in spirochetemia were observed between theplg−/−andplg+/+mice. However, signs indicative of brain invasion, such as neurological symptoms and/or histopathological changes, were more common inplg+/+mice. Quantitative immunohistochemical analysis demonstrated infection of spirochetes in kidney interstitium and brain as soon as 2 days postinoculation. Lower numbers of extravascular spirochetes inplg−/−mice during the first days of infection suggested a less efficient invasion mechanism in these mice than in theplg+/+mice. The invasion of the kidneys inplg−/−mice produced no significant inflammation, as seen by quantitative immunohistochemistry of the CD45 common leukocyte marker. However, significant kidney inflammation was observed with infection in theplg+/+mice. In brain, inflammation was more severe inplg+/+mice than inplg−/−mice, and the numbers of CD45+cells increased significantly with duration of infection in theplg+/+mice. The results show that invasion of brain and kidney occurs as early as 2 days after inoculation. Also, Plg is not required for establishment of spirochetemia by the organism, whereas it is involved in the invasion of organs.
Molting, Ecdysis, and Reproduction of
Trichinella spiralis
Are Supported In Vitro by Intestinal Epithelial Cells
Authors: Gagliardo L. F. , McVay C. S. , Appleton J. A.
Published: July 2002

Trichinella spiralis
is an obligate parasite of animals that has an unusual intracellular life cycle. Investigation of parasitism at the cellular and molecular levels has been challenging because of a shortage of tools for in vitro cultivation of
T. spiralis.
We have found that
T. spiralis
larvae molt, ecdyse, develop to adulthood, and reproduce when they are inoculated onto cultured intestinal epithelial cells. Initially, larvae invade and migrate through cells in a monolayer (T. ManWarren, L. Gagliardo, J. Geyer, C. McVay, S. Pearce-Kelling, and J. Appleton, Infect. Immun. 65:4806-4812, 1997). During prolonged culture in Caco-2 epithelial cells, L1 larvae molted and ecdysed with efficiencies as high as 50%. Molting and ecdysis in vitro required entry of the parasite into cells; conditions that prevented entry into cells also prevented ecdysis. When larvae were inoculated at a low density and cultured for 5 to 9 days, as many as 50% of the larvae developed to adult stages. Low numbers of mature male worms with copulatory appendages were observed in these cultures. The majority of worms that survived for five or more days were unfertilized females. Low-density cultures supported development of female worms with embryos at rates of 4 to 5%. These results show that the intestinal life cycle of
T. spiralis
can be supported entirely by host epithelial cells. Our model should allow more detailed investigation of intracellular parasitism by
T. spiralis
.
Helicobacter pylori
Uses Motility for Initial Colonization and To Attain Robust Infection
Authors: Ottemann Karen M. , Lowenthal Andrew C.
Published: July 2002

Helicobacter pylori
has been shown to require flagella for infection of the stomach. To analyze whether flagella themselves or motility is needed by these pathogens, we constructed flagellated nonmotile mutants. This was accomplished by using both an insertion mutant and an in-frame deletion of the
motB
gene. In vitro, these mutants retain flagella (Fla
+
) but are nonmotile (Mot
−
). By using FVB/N mice, we found that these mutants had reduced ability to infect mice in comparison to that of their isogenic wild-type counterparts. When these mutants were coinfected with wild type, we were unable to detect any
motB
mutant. Finally, by analyzing the 50% infectious dose, we found that motility is needed for initial colonization of the stomach mucosa. These results support a model in which motility is used for the initial colonization of the stomach and also to attain full infection levels.
Specific Entry ofHelicobacter pyloriinto Cultured Gastric Epithelial Cells via a Zipper-Like Mechanism
Authors: Kwok Terry , Backert Steffen , Schwarz Heinz , Berger Jürgen , Meyer Thomas F.
Published: July 2002

AlthoughHelicobacter pylorihas generally been considered an extracellular pathogen, a number of in vitro infection experiments and biopsy examinations have shown that it is capable of occasionally entering mammalian host cells. Here, we characterized this entry process by using AGS cells as a host cell model. In gentamicin protection-invasion assays, the number ofH. pyloricolonies recovered was lower than that forSalmonella entericaserovar Typhimurium X22,Escherichia coliexpressing InvA, andYersinia enterocoliticaYO:9 grown at 25°C but higher than that forNeisseria gonorrhoeaeVP1 andY. enterocoliticaYO:9 grown at 37°C. At the ultrastructural level, the entry process was observed to occur via a zipper-like mechanism. InternalizedH. pyloriwas bound in tight LAMP-1-containing vacuoles in close association with condensed filamentous actin and tyrosine phosphorylation signals. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, and calphostin C, an inhibitor of protein kinase C, both inhibited the entry ofH. pyloriin a sensitive and dose-dependent manner; however, the level of entry was enhanced by sodium vanadate, an inhibitor of tyrosine phosphatases and ATPases. Furthermore, the cytokine tumor necrosis factor alpha antagonized the entry ofH. pyloriinto AGS cells. Collectively, these results demonstrate that the entry ofH. pyloriinto AGS cells occurs via a zipper-like mechanism which involves various host signal transduction events.
Afa, a Diffuse Adherence Fibrillar Adhesin Associated with Enteropathogenic
Escherichia coli
Authors: Keller Rogéria , Ordoñez Juana G. , de Oliveira Rosana R. , Trabulsi Luiz R. , Baldwin Thomas J. , Knutton Stuart
Published: July 2002

O55 is one of the most frequent enteropathogenic
Escherichia coli
(EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H
−
ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic
E. coli
. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.
Fc-Dependent and Fc-Independent Opsonization of
Cryptococcus neoformans
by Anticapsular Monoclonal Antibodies: Importance of Epitope Specificity
Authors: Netski Dale , Kozel Thomas R.
Published: July 2002

Monoclonal antibodies (MAbs) reactive with glucuronoxylomannan (GXM), the major capsular polysaccharide of the yeast
Cryptococcus neoformans
, produce distinct capsular reactions when viewed by differential interference contrast microscopy. These reactions depend on the epitope specificity of the antibody. Opsonic activities of immunoglobulin G1 (IgG1) MAbs that produce patterns termed rim and puffy were examined. Rim-pattern MAbs are reactive with an epitope shared by GXM serotypes A, B, C, and D. Puffy-pattern MAbs are reactive only with serotypes A and D. In phagocytosis assays, using serotype A cells and resident murine peritoneal macrophages, rim-pattern MAbs were markedly more opsonic than puffy-pattern MAbs. F(ab′)
2
fragments of rim-pattern MAbs were synergistic with heat-labile factors in normal human serum for opsonization of the yeast. F(ab′)
2
fragments of puffy-pattern MAbs were also synergistic with normal serum in opsonization but at a much lower level than fragments of rim-pattern MAbs. Normal serum alone was not opsonic. F(ab′)
2
fragments of rim-pattern MAbs, but not puffy-pattern MAbs, stimulated phagocytosis of encapsulated cryptococci in the absence of serum. This serum-independent opsonic action of F(ab′)
2
fragments was abrogated by pretreatment of macrophages with purified GXM, suggesting the involvement of a phagocyte GXM receptor. The results indicate that (i) there are multiple mechanisms by which anticapsular IgG MAbs facilitate phagocytosis of encapsulated cryptococci, (ii) some anti-GXM antibodies are opsonic in an Fc-independent manner, and (iii) opsonic activity correlates with the capsular reaction and occurs in an epitope-specific manner.
Induction of Interleukin-12 and Gamma Interferon Requires Tumor Necrosis Factor Alpha for Protective T1-Cell-Mediated Immunity to PulmonaryCryptococcus neoformansInfection
Authors: Herring Amy C. , Lee John , McDonald Roderick A. , Toews Galen B. , Huffnagle Gary B.
Published: July 2002

The development of T1-cell-mediated immunity is required to clear a pulmonaryCryptococcus neoformansinfection. The objective of these studies was to determine the mechanism by which tumor necrosis factor alpha (TNF-α) augments the development of pulmonary T1 immunity toC. neoformansinfection. TNF-α expression was detected in lavage sample cells at days 2, 3, and 7 followingC. neoformansinfection. The numbers of CFU in the lung were not different between control and anti-TNF-α-treated mice at any time point examined during the afferent phase of the response (days 0 to 7). However, neutralization of TNF-α prevented the initiation of pulmonary clearance during the efferent phase of the response (day 14). Administration of anti-TNF-α monoclonal antibody (day 0) diminished the lung levels of TNF-α, interleukin-12 (IL-12), and gamma interferon (IFN-γ) induced byC. neoformansat day 7 postinfection. Neutralization of TNF-α (day 0) also altered the IFN-γ/IL-4 ratio in the lung-associated lymph nodes at day 7 followingC. neoformansinfection. Anti-TNF-α-treated mice developed a pulmonary eosinophilia at day 14 postinfection. Consistent with the pulmonary eosinophilia, anti-TNF-α-treated mice exhibited elevated serum immunoglobulin E and inhibition of the anticryptococcal delayed-type hypersensitivity response, indicating a shift toward a T2 response. Neutralization of IL-12 also prevented lung leukocyte production of IFN-γ in response to the infection. These findings demonstrate that afferent-phase TNF-α production is essential for the induction of IL-12 and IFN-γ and neutralization of early TNF-α results in a T2 shift of the T1/T2 balance of antifungal immunity.
Cloning and Characterization of the
CSF1
Gene of
Saccharomyces cerevisiae
, Which Is Required for Nutrient Uptake at Low Temperature
Authors: Tokai Masaya , Kawasaki Hideki , Kikuchi Yasuhiro , Ouchi Kozo
Published: July 2002

We have isolated cold-sensitive fermentation mutants (Csf mutants) of a commercial baker's yeast that have practically no fermentation capacity at 5°C and return to their normal capacity at 25 to 40°C.
CSF1
was cloned by functional complementation of the Csf phenotype.
CSF1
contain an open reading frame of 8,874 nucleotides, encoding a protein of 2,958 amino acids. The nucleotide sequence was identical to that of the YLR087C gene in the
Saccharomyces
genome database, but there was no information about the function of the predicted CSF1 (YLR087C) protein. Gene disruption shows that
CSF1
is required for growth and fermentation only at low temperatures. Permeabilized cells of the disruptant showed nearly the same ethanol production rate as those of the parent strain, even at 10°C. The disruptant cells had the same glucose uptake rates as the parental cells at 30°C, but three- to fivefold-lower rates than the parental cells at 10°C. These findings suggest that
CSF1
associates with a new nutrient transport system which exists on the plasma membrane and is required only at low temperature.