bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-10 Modulates Proinflammatory Cytokines in the Human Monocytic Cell Line THP-1 Stimulated withBorrelia burgdorferiLipoproteins
Authors: Murthy P. K. , Dennis Vida A. , Lasater Barbara L. , Philipp Mario T.
Published: July 2002

We determined previously that lipoproteins ofBorrelia burgdorferistimulate inflammatory and anti-inflammatory cytokines (interleukin-10 [IL-10]) in monocytes. IL-10 could have an effect on innate and acquired immune responses toB. burgdorferiand influence the magnitude of the infectious inoculum and disease outcome. To understand the mechanism(s) of IL-10 action during early infection, when innate immunity expressed chiefly by skin macrophages is key, we investigated the effect of exogenous and endogenous IL-10 on the production of the macrophage-derived cytokines IL-6, IL-1β, IL-12, and tumor necrosis factor alpha (TNF-α). We used the THP-1 human monocytic cell line and recombinant lipidated OspA (L-OspA) as the model target cell and stimulant, respectively. To determine the kinetics of cytokine production by THP-1 cells, we stimulated them with L-OspA and also with heat-killedB. burgdorfericells (HBb) and lipopolysaccharide (LPS). Exogenous IL-10 dampened production of inflammatory cytokines, as elicited by lipoproteins. The inhibition of endogenous IL-10 function by anti-IL-10 antibody reduced the production of IL-12 and IL-6 but not that of IL-1β and TNF-α. An inspection of the kinetics of cytokine production clarified this finding. TNF-α was produced prior to, and IL-β was produced at the same time as, IL-10, whereas IL-6 and IL-12 were produced later. HBb, LPS, and L-OspA yielded similar kinetics of cytokine production. This result reinforces the notion that lipoproteins are the functional molecules in HBb and perhaps in vivo. It indicates also that signaling pathways utilized by LPS and lipoproteins may be extensively shared. The results are consistent with a major role played by IL-10 in controlling the initial phase of infection with this spirochete.
Phagocytic Uptake ofEncephalitozoon cuniculiby Nonprofessional Phagocytes
Authors: Couzinet Sabine , Cejas Elisabeth , Schittny Johannes , Deplazes Peter , Weber Rainer , Zimmerli Stefan
Published: July 2002

Encephalitozoon cuniculiis an obligate intracellular, spore-forming parasite belonging to the microsporidia that can cause disseminated infection in immunocompromised persons.E. cuniculispores infect host cells by germination, i.e., by explosively everting the polar filament, through which the spore contents (sporoplasms) are subsequently injected into the cytoplasm. In addition, we observed intracellular, nongerminated spores in various nonprofessional phagocytes. In MRC5 cells, the number of internalized spores was approximately 10-fold higher than the number of injected sporoplasms. Compared to the rate of uptake by human monocyte-derived macrophages, internalization rates by A549 cells, MRC5 cells, and 293 cells were 0.6, 4.4, and 22.2%, respectively. The mechanism of uptake was studied in MRC5 cells. Killed spores were internalized at the same rate as live spores, indicating that nongerminated parasites do not actively participate in cell entry. Cytochalasin D inhibited uptake of spores by 95%, demonstrating an actin-dependent process. By electron and epifluorescence microscopy, intracellular spores were found in a tightly fitting membrane-bound compartment. The vacuole containing the spores was positive for the lysosomal membrane protein LAMP-1 and colocalized with the late endosomal-lysosomal content marker rhodamine dextran. Our results show that, in addition to the unique way in which microsporidia infect cells,E. cuniculispores enter nonprofessional phagocytes by phagocytosis and traffic into a late endosomal-lysosomal compartment.
Comparative Analysis of Antibody Responses against HSP60, Invariant Surface Glycoprotein 70, and Variant Surface Glycoprotein Reveals a Complex Antigen-Specific Pattern of Immunoglobulin Isotype Switching during Infection byTrypanosoma brucei
Authors: Radwanska Magdalena , Magez Stefan , Michel Alain , Stijlemans Benoît , Geuskens Maurice , Pays Etienne
Published: July 2002

DuringTrypanosoma bruceiinfections, the response against the variant surface glycoprotein (VSG) of the parasite represents a major interaction between the mammalian host immune system and the parasite surface. Since immune recognition of other parasite derived factors also occurs, we examined the humoral host response against trypanosome heat shock protein 60 (HSP60), a conserved antigen with an autoimmune character. During experimentalT. bruceiinfection in BALB/c mice, the anti-HSP60 response was induced when parasites differentiated into stumpy forms. This response was characterized by a stage-specific immunoglobulin isotype switching as well as by the induction of an autoimmune response. Specific recognition of trypanosome HSP60 was found to occur during the entire course of infection. Immunoglobulin G2a (IgG2a) and IgG2b antibodies, induced mainly in a T-cell-independent manner, were observed during the first peak of parasitemia, whereas IgG1 and IgG3 antibodies were found at the end of the infection, due to a specific T-cell-mediated response. Comparative analysis of the kinetics of anti-HSP60, anti-invariant surface glycoprotein 70 (ISG70), and anti-VSG antibody responses indicated that the three trypanosome antigens give rise to specific and independent patterns of immunoglobulin isotype switching.
Acquired Immunity toChlamydia pneumoniaeIs Dependent on Gamma Interferon in Two Mouse Strains That Initially Differ in This Respect after Primary Challenge
Authors: Vuola Jenni M. , Puurula Vuokko , Anttila Marjukka , Mäkelä P. Helena , Rautonen Nina
Published: July 2002

The role of gamma interferon (IFN-γ) in aChlamydia pneumoniaemouse model was studied by in vivo neutralization in two inbred mouse strains. During primaryC. pneumoniaeinfection, neutralization of IFN-γ increased both the numbers of bacteria and the pneumonia score in the lungs of C57BL/6 mice but not BALB/c mice. During reinfection, the bacterial counts in the lungs were increased by IFN-γ neutralization in both mouse strains. Thus, the effect of IFN-γ neutralization was dependent on the genetic background in primary infection. However, IFN-γ appeared to be equally important in both mouse strains during reinfection.
Macrophage Class A Scavenger Receptor-Mediated Phagocytosis ofEscherichia coli: Role of Cell Heterogeneity, Microbial Strain, and Culture Conditions In Vitro
Authors: Peiser Leanne , Gough Peter J. , Kodama Tatsuhiko , Gordon Siamon
Published: July 2002

Macrophage class A scavenger receptors (SR-AI and SR-AII) contribute to host defense by binding polyanionic ligands such as lipopolysaccharide and lipoteichoic acid. SR-A knockout (SR-A−/−) mice are more susceptible to endotoxic shock andListeria monocytogenesinfection in vivo, possibly due to decreased clearance of lipopolysaccharide and microorganisms, respectively. We have used flow cytometry to analyze the role of SR-A and other scavenger-like receptors in phagocytosis of bacteria in vitro. Chinese hamster ovary cells stably transfected with human SR-A boundEscherichia coliandStaphylococcus aureusbut ingested few organisms. Primary human monocyte-derived macrophages (Mφ) bound and ingestedE. colimore efficiently, and this was partially but selectively blocked by the general SR inhibitor, poly(I). A specific and selective role for SR-A was shown, since bone marrow culture-derived Mφ from SR-A−/−mice ingested fewerE. coliorganisms than did wild-type cells, while uptake of antibody-opsonizedE. coliwas unaffected. SR-A-dependent uptake ofE. colivaried with the bacterial strain; ingestion of DH5α and K1 by SR-A−/−Mφ was reduced by 30 to 60% and 70 to 75%, respectively. Phagocytosis and endocytosis via SR-A were markedly down-modulated when Mφ were plated on serum-coated tissue culture plastic compared to bacteriologic plastic, where cell adhesion is mediated by SR-A and CR3, respectively. This paper demonstrates that SR-A can bind and ingest bacteria directly, consistent with a role in host defense in vivo, and highlights the importance of the source of the Mφ, bacterial strain, and culture conditions on receptor function in vitro.
Human Immune Responses toSchistosoma mansoniVaccine Candidate Antigens
Authors: Ribeiro de Jesus Amélia , Araújo Ilma , Bacellar Olívia , Magalhães Andréa , Pearce Edward , Harn Donald , Strand Mette , Carvalho Edgar M.
Published: July 2002

To determine the naturally occurring immunological responses to theSchistosoma mansoniantigens paramyosin, IrV-5, Sm-23 (MAP-3), and triose phosphate isomerase (MAP-4), a total of 119 subjects from an area of endemicity for schistosomiasis, including “resistant” subjects (n= 17) were evaluated. Specific immunoglobulin G1 (IgG1), IgG2, IgG3, IgG4, and IgA levels for each of the antigens and the cytokine profile in culture supernatants from antigen-stimulated peripheral blood mononuclear cells (PBMC) were determined. Although all the subjects had a high degree of contaminated water exposure, their infection levels were variable (0 to 1,128 eggs/g of stool). There were direct correlations between infection levels and levels of SWAP- and paramyosin-specific IgG1 and IgG4 (P< 0.05). However, an inverse correlation between infection levels and specific IgG2 to IrV-5 (P< 0.01) was observed. The evaluation of the cytokine profile (interleukin 5 [IL-5], IL-10, gamma interferon [IFN-γ], and tumor necrosis factor alpha) in response to these antigens showed inverse correlations between the degree of infection and IFN-γ levels in PBMC supernatants stimulated with paramyosin (P< 0.05) and IrV-5 (P< 0.01). Additionally, inverse correlations between the degree of infection and IL-5 levels in MAP-3- and MAP-4-stimulated PBMC supernatants (P< 0.01) were found. Logistic regression analysis was performed to adjust the results of cytokine profile by age. IL-5 production in MAP-3-stimulated PBMC supernatants was associated with lower infection levels (odds ratio = 11.2 [95% confidence interval, 2.7 to 45.8]).
CS22, a Novel Human Enterotoxigenic
Escherichia coli
Adhesin, Is Related to CS15
Authors: Pichel Mariana , Binsztein Norma , Viboud Gloria
Published: July 2002

Enterotoxigenic
Escherichia coli
(ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H− strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of
Salmonella enterica
serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from the
nfaA
sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.
Antibody Is Required for Protection against Virulent but Not AttenuatedSalmonella entericaSerovar Typhimurium
Authors: McSorley Stephen J. , Jenkins Marc K.
Published: July 2002

Resolution of infection with attenuatedSalmonellais an active process that requires CD4+T cells. Here, we demonstrate that costimulation via the surface molecule CD28, but not antibody production by B cells, is required for clearance of attenuatedaroA Salmonella entericaserovartyphimurium. In contrast, specific antibody is critical for vaccine-induced protection against virulent bacteria. Therefore, CD28+CD4+T cells are sufficient for clearance of avirulentSalmonellain naive hosts, whereas CD4+T cells and specific antibodies are required for protection from virulentSalmonellain immune hosts.
Antibodies against Thrombospondin-Related Anonymous Protein Do Not InhibitPlasmodiumSporozoite Infectivity In Vivo
Authors: Gantt Soren , Persson Cathrine , Rose Keith , Birkett Ashley J. , Abagyan Ruben , Nussenzweig Victor
Published: July 2002

Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required forPlasmodiumsporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain ofPlasmodium yoeliiand located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve.
Toxoplasma gondiiUses Sulfated Proteoglycans for Substrate and Host Cell Attachment
Authors: Carruthers Vern B. , Håkansson Sebastian , Giddings Olivia K. , Sibley L. David
Published: July 2002

Toxoplasma gondiiis an obligate intracellular parasite that actively invades a wide variety of vertebrate cells, although the basis of this pervasive cell recognition is not understood. We demonstrate here that binding to the substratum and to host cells is partially mediated by interaction with sulfated glycosaminoglycans (GAGs). Addition of excess soluble GAGs blocked parasite attachment to serum-coated glass, thereby preventing gliding motility of extracellular parasites. Similarly, excess soluble GAGs decreased the attachment of parasites to human host cells from a variety of lineages, including monocytic, fibroblast, endothelial, epithelial, and macrophage cells. The inhibition of parasite attachment by GAGs was observed with heparin and heparan sulfate and also with chondroitin sulfates, indicating that the ligands for attachment are capable of recognizing a broad range of GAGs. The importance of sulfated proteoglycan recognition was further supported by the demonstration that GAG-deficient mutant host cells, and wild-type cells treated enzymatically to remove GAGs, were partially resistant to parasite invasion. Collectively, these studies reveal that sulfated proteoglycans are one determinant used for substrate and cell recognition byToxoplasma. The widespread distribution of these receptors may contribute to the broad host and tissue ranges of this highly successful intracellular parasite.
Plasmodium chabaudi
-Infected Erythrocytes Adhere to CD36 and Bind to Microvascular Endothelial Cells in an Organ-Specific Way
Authors: Mota Maria M. , Jarra William , Hirst Elizabeth , Patnaik Pradeep K. , Holder Anthony A.
Published: July 2002

Adherence of erythrocytes infected with
Plasmodium falciparum
to microvascular endothelial cells (sequestration) is considered to play an important role in parasite virulence and pathogenesis. However, the real importance of sequestration for infection and disease has never been fully assessed. The absence of an appropriate in vivo model for sequestration has been a major barrier. We have examined the rodent malaria parasite
Plasmodium chabaudi chabaudi
AS in mice as a potential model. Erythrocytes infected with this parasite adhere in vitro to purified CD36, a critical endothelium receptor for binding
P. falciparum
-infected erythrocytes.
P. c. chabaudi
-infected erythrocytes adhere in vitro to endothelial cells in a gamma interferon-dependent manner, suggesting the involvement of additional adhesion molecules in the binding process, as is also the case with
P. falciparum
-infected cells. Furthermore, plasma or sera from infected and hyperimmune mice, respectively, have the ability to block binding of infected erythrocytes to endothelial cells. In vivo, erythrocytes containing mature
P. c. chabaudi
parasites are sequestered from the peripheral circulation. Sequestration is organ specific, occurring primarily in the liver, although intimate contact between infected erythrocytes and endothelial cells is also observed in the spleen and brain. The results are discussed in the context of the use of this model to study (i) the relationship between endothelial cell activation and the level of sequestration and (ii) the primary function of sequestration in malaria infection.
Ultrastructure of
Rickettsia rickettsii
Actin Tails and Localization of Cytoskeletal Proteins
Authors: Van Kirk Levi S. , Hayes Stanley F. , Heinzen Robert A.
Published: July 2002

Actin-based motility (ABM) is a mechanism for intercellular spread that is utilized by vaccinia virus and the invasive bacteria within the genera
Rickettsia
,
Listeria
, and
Shigella
. Within the
Rickettsia
, ABM is confined to members of the spotted fever group (SFG), such as
Rickettsia rickettsii
, the agent of Rocky Mountain spotted fever. Infection by each agent induces the polymerization of host cell actin to form the typical F (filamentous)-actin comet tail. Assembly of the actin tail propels the pathogen through the host cytosol and into cell membrane protrusions that can be engulfed by neighboring cells, initiating a new infectious cycle. Little is known about the structure and morphogenesis of the
Rickettsia rickettsii
actin tail relative to
Shigella
and
Listeria
actin tails. In this study we examined the ultrastructure of the rickettsial actin tail by confocal, scanning electron, and transmission electron microscopy. Confocal microscopy of rhodamine phalloidin-stained infected Vero cells revealed the typhus group rickettsiae,
Rickettsia prowazekii
and
Rickettsia typhi
, to have no actin tails and short (∼1- to 3-μm) straight or hooked actin tails, respectively. The SFG rickettsia,
R. rickettsii
, displayed long actin tails (>10 μm) that were frequently comprised of multiple, distinct actin bundles, wrapping around each other in a helical fashion. Transmission electron microscopy, in conjunction with myosin S1 subfragment decoration, revealed that the individual actin filaments of
R. rickettsii
tails are >1 μm long, arranged roughly parallel to one another, and oriented with the fast-growing barbed end towards the rickettsial pole. Scanning electron microscopy of intracellular rickettsiae demonstrated
R. rickettsii
to have polar associations of cytoskeletal material and
R. prowazekii
to be devoid of cytoskeletal interactions. By indirect immunofluorescence, both
R. rickettsii
and
Listeria monocytogenes
actin tails were shown to contain the cytoskeletal proteins vasodilator-stimulated phosphoprotein profilin, vinculin, and filamin. However, rickettsial tails lacked ezrin, paxillin, and tropomyosin, proteins that were associated with actin tails of cytosolic or protrusion-bound
Listeria
. The unique ultrastructural and compositional characteristics of the
R. rickettsii
actin tail suggest that rickettsial ABM is mechanistically different from previously described microbial ABM systems.
Initial Characterization of CST1, a
Toxoplasma gondii
Cyst Wall Glycoprotein
Authors: Zhang Yi Wei , Halonen Sandra K. , Ma Yan Fen , Wittner Murray , Weiss Louis M.
Published: July 2002

Toxoplasma gondii
is an important protozoan pathogen of humans that can cause encephalitis in immunocompromised individuals such as those with AIDS. This encephalitis is due to reactivation of latent infection in
T. gondii
-seropositive patients. Latent organisms survive within tissue cysts, which are specialized parasitophorous vacuoles containing bradyzoites. The cyst wall of this structure is produced by modification of the parasitophorous vacuole by the parasite and is important in cyst survival. The components of the cyst wall have been poorly characterized. By using immunofluorescence and immunoelectron microscopy, we have identified a monoclonal antibody (MAb 93.18) that reacts with the cyst wall. This antibody recognizes a 116-kDa glycoprotein, which we have termed CST1, containing sugar residues that bind
Dolichos biflorans
lectin (DBA). CST1 is distinct from
T. gondii
antigen labeled with succinyl
Triticum vulgare
lectin (S-WGA) and represents the major DBA-binding component in
T. gondii
. The carbohydrate components of the tissue cyst, such as CST1, are probably important in both providing stability and facilitating persistence in its host. As is seen in the carbohydrate capsules of fungi, glycoproteins in the
T. gondii
cyst wall may protect cysts from the immune response of the host. Further characterization of the formation of the cyst wall and its components should lead to insights into the mechanism of tissue cyst persistence and may suggest novel therapeutic approaches to eliminate tissue cysts of this organism.
Cytotoxic Necrotizing Factor Type 1-PositiveEscherichia coliCauses Increased Inflammation and Tissue Damage to the Prostate in a Rat Prostatitis Model
Authors: Rippere-Lampe Karen E. , Lang Michael , Ceri Howard , Olson Merle , Lockman Hank A. , O'Brien A. D.
Published: July 2002

Infection of rat prostates with cytotoxic necrotizing factor type 1 (CNF1)-positive uropathogenicEscherichia colicaused more inflammation-mediated morphological and histological tissue damage than did infection with isogenic CNF1-negative mutants. These striking differences occurred despite the finding that bacterial counts for the strain pairs were indistinguishable. We conclude that CNF1 contributes toE.colivirulence in a model of acute prostatitis. To our knowledge, the results of this study provide the first demonstration of a role for any uropathogenicE.colivirulence factor in acute prostatitis.
RecombinantYersiniaYopT Leads to Uncoupling of RhoA-Effector Interaction
Authors: Sorg Isabel , Goehring Udo-Michael , Aktories Klaus , Schmidt Gudula
Published: July 2002

Yersinia enterocolitica, Yersinia pseudotuberculosis, andYersinia pestisdeliver different Yop (Yersiniaouter proteins) effector proteins into mammalian cells by a type III secretion mechanism. Recently, it was shown thatYersiniaproducing YopT leads to disruption of the actin cytoskeleton of HeLa cells (M. Iriarte and G. R. Cornelis, Mol. Microbiol. 29:915–929, 1998). To analyze the molecular mechanism of YopT, we cloned and expressed YopT as a glutathioneS-transferase fusion protein. Recombinant YopT caused rounding up of embryonic bovine lung cells and redistribution of the actin cytoskeleton rapidly after microinjection. TheEscherichia colicytotoxic necrotizing factor (CNF1), which constitutively activates Rho proteins, was not able to inhibit or revert YopT-induced cell rounding. YopT caused release of RhoA from embryonic bovine lung membranes and released recombinant isoprenylated RhoA from artificial PE or PE/PIP2 vesicles. Incubation of lysate or cytosol with YopT caused inhibition of the RhoA-rhotekin interaction but led to increased RhoA-RhoGDI interaction. It is suggested that inhibition of the interaction between RhoA and effectors is the underlying mechanism of the YopT action on the cytoskeleton.
Expression of Chemokine Genes in Human Dermal Microvascular Endothelial Cell Lines Infected withOrientia tsutsugamushi
Authors: Cho Nam-Hyuk , Seong Seung-Yong , Choi Myung-Sik , Kim Ik-Sang
Published: July 2002

Scrub typhus, caused byOrientia tsutsugamushi, is characterized by local as well as systemic inflammatory manifestations. The main pathologic change is focal or disseminated multiorgan vasculitis, which is caused by the destruction of endothelial cells and perivascular infiltration of leukocytes. We investigated the regulation of chemokine induction in transformed human dermal microvascular endothelial cells (HMEC-1) in response toO. tsutsugamushiinfection. The monocyte chemoattractant protein-1 (MCP-1) and interleukin 8 (IL-8) mRNAs were induced, and their levels showed a transitory peak at 3 and 6 h, respectively. The RANTES transcript was detected at 6 h after infection, with increased levels evident by 48 h. The induction of the MCP-1 and IL-8 genes was not blocked by cycloheximide, suggesting that de novo protein synthesis of host cell proteins is not required for their transcriptional activation. Heat- or UV-inactivatedO. tsutsugamushiinduced a similar extent of MCP-1 and IL-8 responses. The induction of MCP-1 and IL-8 transcripts in the endothelial cells byO. tsutsugamushiwas not blocked by the inhibitors of NF-κB. Furthermore, the activation of NF-κB was not detected in HMEC-1 stimulated withO. tsutsugamushi. These results demonstrate that heat-stable molecules ofO. tsutsugamushiinduce the MCP-1 and IL-8 genes and the induction of the chemokine genes may be mediated by an NF-κB independent mechanism. We also showed that another major transcription factor, activator protein-1 (AP-1), was up-regulated in HMEC-1 afterO. tsutsugamushiinfection. This suggests the possible involvement of AP-1 in the chemokine gene expression.
Streptococcus parasanguis
Fimbria-Associated Adhesin Fap1 Is Required for Biofilm Formation
Authors: Froeliger Eunice H. , Fives-Taylor Paula
Published: July 2002

The sanguis streptococci are primary colonizers of the tooth surface and thus form the foundation for the complex multiple species biofilm known as dental plaque. In addition, these bacteria can colonize native and prosthetic heart valves and are a common cause of endocarditis. Little is known about the molecular mechanisms governing multiple or single species biofilm development within this group of organisms. Using an in vitro assay for biofilm formation, we determined that (i)
Streptococcus parasanguis
FW213 can form biofilms on inert surfaces such as polystyrene and (ii) environmental and nutritional factors, such as glucose, affect
S. parasanguis
biofilm formation. Several isogenic mutants of FW213 were tested in the biofilm assay. Strains containing mutations in
fap1
, a gene encoding a protein required for assembly of fimbriae, were deficient in biofilm formation. Mutants defective in
recA
, PepO endopeptidase activity, or the production of a fimbriae-associated protein, FimA, were still capable of biofilm formation. Phase-contrast microscopy was used to follow biofilm development by wild-type and
fap1
mutant strains on plastic coverslips over time. Wild-type FW213 attached to the surface, formed aggregates of cells, and eventually formed a dense layer of cells that included microcolonies. In contrast, few
fap1
mutant cells were observed attached to the surface, and no cell aggregates or microcolonies were formed. These results suggest that the long peritrichous fimbriae of FW213 are critical for the formation of biofilms on solid surfaces.
Curli Fibers Mediate Internalization of
Escherichia coli
by Eukaryotic Cells
Authors: Gophna U. , Barlev M. , Seijffers R. , Oelschlager T. A. , Hacker J. , Ron E. Z.
Published: July 2002

Curli fibers are adhesive surface fibers expressed by
Escherichia coli
and
Salmonella enterica
that bind several host extracellular matrix and contact phase proteins and were assumed to have a role in pathogenesis. The results presented here suggest that one such role is internalization into host cells. An
E. coli
K-12 strain transformed with a low-copy vector containing the gene cluster encoding curli fibers (
csg
operon) was internalized by several lines of eukaryotic cells. The internalization could be correlated with a high level of curli fiber expression and was abolished by disruption of the
csg
operon. The ability to be internalized by eukaryotic cells could be conferred even by the curli fiber gene cluster of a noninvasive K-12 strain, but the homologous
csg
cluster from a virulent septicemic
E. coli
isolate mediated a higher level of internalization. The finding that curli fibers promote bacterial internalization indicates a new role for curli fibers in pathogenesis.
Gallinacin-3, an Inducible Epithelial β-Defensin in the Chicken
Authors: Zhao Chengquan , Nguyen Tung , Liu Lide , Sacco Randy E. , Brogden Kim A. , Lehrer Robert I.
Published: July 2002

Gallinacin-3 and gallopavin-1 (GPV-1) are newly characterized, epithelial β-defensins of the chicken (Gallus gallus) and turkey (Meleagris gallopavo), respectively. In normal chickens, the expression of gallinacin-3 was especially prominent in the tongue, bursa of Fabricius, and trachea. It also occurred in other organs, including the skin, esophagus, air sacs, large intestine, and kidney. Tracheal expression of gallinacin-3 increased significantly after experimental infection of chickens withHaemophilus paragallinarum, whereas its expression in the tongue, esophagus, and bursa of Fabricius was unaffected. The precursor of gallinacin-3 contained a long C-terminal extension not present in the prepropeptide. By comparing the cDNA sequences of gallinacin-3 and GPV-1, we concluded that a 2-nucleotide insertion into the gallinacin-3 gene had induced a frameshift that read through the original stop codon and allowed the chicken propeptide to lengthen. The striking structural resemblance of the precursors of β-defensins to those of crotamines (highly toxic peptides found in rattlesnake venom) supports their homology, even though defensins are specialized to kill microorganisms and crotamines are specialized to kill much larger prey.
Shear Stress Prevents Fibronectin Binding Protein-Mediated
Staphylococcus aureus
Adhesion to Resting Endothelial Cells
Authors: Reddy Kesav , Ross Julia M.
Published: July 2002

Fibronectin binding proteins (FnBP) on the surface of
Staphylococcus aureus
have previously been shown to mediate adherence of the organism to resting endothelial cells in static adhesion assays. However, in this study using well-defined flow assays, we demonstrate that physiologic levels of shear stress prevent FnBP-mediated adhesion of
S. aureus
8325-4 to resting endothelial cells. This result suggests that mechanical forces present in vivo may influence the ability of staphylococci to bind endothelial cell surfaces.
Requirement for Capsule in Colonization by
Streptococcus pneumoniae
Authors: Magee Ashalla D. , Yother Janet
Published: July 2002

Nasopharyngeal colonization is a necessary first step in the pathogenesis of
Streptococcus pneumoniae
. Using isolates containing defined mutations in the
S. pneumoniae
capsule locus, we found that expression of the capsular polysaccharide is essential for colonization by the type 2 strain D39 and the type 3 strains A66 and WU2. Nonencapsulated derivatives of each of these strains were unable to colonize BALB/cByJ mice. Similarly, type 3 mutants that produced <6% of the parental amounts of capsule could not colonize. Capsule production equivalent to that of the parent strain was not required for efficient colonization, however, as type 3 mutants producing approximately 20% of the parental amounts of capsule colonized as effectively as the parent. This 80% reduction in capsule level had only a minimal effect on intraperitoneal virulence but caused a significant reduction in virulence via the intravenous route. In the X-linked immunodeficient CBA/N mouse, the type 3 mutant producing ∼20% of the parental amount of capsule (AM188) was diminished in its ability to cause invasive disease and death following intranasal inoculation. Following intravenous or intraperitoneal challenge, however, only extended survival times were observed. Our results demonstrate an additional role for capsule in the pathogenesis of
S. pneumoniae
and show that isolates producing reduced levels of capsule can remain highly virulent.
Complete DNA Sequence of
Yersinia enterocolitica
Serotype 0:8 Low-Calcium-Response Plasmid Reveals a New Virulence Plasmid-Associated Replicon
Authors: Snellings Norma J. , Popek Michael , Lindler Luther E.
Published: July 2002

The complete nucleotide sequence and organization of the
Yersinia enterocolitica
serotype 0:8 low-calcium-response (LCR) plasmid, pYVe8081, were determined. The 67,720-bp plasmid encoded all the genes known to be part of the LCR stimulon except for
ylpA
. Eight of 13 intact open reading frames of unknown function identified in pYVe8081 had homologues in
Yersinia pestis
plasmid pCD1 or in
Y. enterocolitica
serotype 0:9 plasmid pYVe227. A region of approximately 17 kbp showed no DNA identity to pCD1 or pYVe227 and contained six potential new genes, a possible new replicon, and two intact insertion sequence (IS) elements. One intact IS element, IS
Yen1
, was a new IS belonging to the IS
256
family. Several vestigial IS elements appeared different from the IS distribution seen in the other LCR plasmids. The RepA proteins encoded by
Y. enterocolitica
serotype 0:8 pYVeWA and pYVe8081 were identical. The putative pYVe8081 replicon showed significant homology to the IncL/M replicon of pMU407.1 but was only distantly related to the replicons of pCD1 and pYVe227. In contrast, the putative partitioning genes of pYVe8081 showed 97% DNA identity to the
spy/sopABC
loci of pCD1 and pYVe227. Sequence analysis suggests that
Yersinia
LCR plasmids are from a common ancestor but that
Y. enterocolitica
serotype 0:8 plasmid replicons may have evolved independently via cointegrate formation following a transposition event. The change in replicon structure is predicted to change the incompatibility properties of
Y. enterocolitica
serotype 0:8 plasmids from those of
Y. enterocolitica
serotype 0:9 and
Y. pestis
LCR plasmids.
Cytolethal Distending Toxin Demonstrates Genotoxic Activity in a Yeast Model
Authors: Hassane Duane C. , Lee Robert B. , Mendenhall Michael D. , Pickett Carol L.
Published: July 2002

Cytolethal distending toxins (CDTs) are multisubunit proteins produced by a variety of bacterial pathogens that cause enlargement, cell cycle arrest, and apoptosis in mammalian cells. While their function remains uncertain, recent studies suggest that they can act as intracellular DNases in mammalian cells. Here we establish a novel yeast model for understanding CDT-associated disease. Expression of the CdtB subunit in yeast causes a G2/M arrest, as seen in mammalian cells. CdtB toxicity is not circumvented in yeast genetically altered to lack DNA damage checkpoint control or that constitutively promote cell cycle progression via mutant Cdk1, because CdtB causes a permanent type of damage that results in loss of viability. Finally, we establish that CDTs are likely to be potent genotoxins, as indicated by in vivo degradation of chromosomal DNA associated with expression of CdtB—suggesting that the varied distribution of CDT in bacteria implicates many human pathogens as possessors of genotoxic activity.
Vibrio cholerae
OmpU and OmpT Porins Are Differentially Affected by Bile
Authors: Wibbenmeyer Jamie A. , Provenzano Daniele , Landry Candice F. , Klose Karl E. , Delcour Anne H.
Published: July 2002

OmpT and OmpU are pore-forming proteins of the outer membrane of
Vibrio cholerae
, a pathogen that colonizes the intestine and produces cholera. Expression of the
ompU
and
ompT
genes is under the regulation of ToxR, a transmembrane transcriptional activator that also controls expression of virulence factors. It was recently shown that bile stimulates the ToxR-mediated transcription of
ompU
and that
ompU
-expressing strains are more resistant to bile and anionic detergents than
ompT
-expressing cells. In order to further understand the role of the OmpT and OmpU porins in the ability of
V. cholerae
to survive and colonize the host intestine, we examined the outer membrane permeability of cells expressing only
ompU
or only
ompT
or both genes in the absence and in the presence of bile. By comparing various strains in terms of the rate of degradation of the β-lactam antibiotic cephaloridine by the periplasmic β-lactamase, we found that the permeation of the antibiotic through the outer membrane of OmpU-containing cells was slower than the permeation in OmpT-containing cells. In addition, the OmpU-mediated outer membrane permeability was not affected by external bile, while the OmpT-mediated antibiotic flux was reduced by bile in a concentration-dependent manner. Our results confirm that OmpT and OmpU provide a passageway for hydrophilic solutes through the outer membrane and demonstrate that bile might interfere with this traffic in OmpT-producing cells by functionally inhibiting the OmpT pore. The insensitivity of OmpU to bile may be due to its small pore size and may provide an explanation for the resistance of OmpU-producing cells to bile in vivo.
Serum Stimulates Growth of and Proteinase Secretion byAspergillus fumigatus
Authors: Gifford Anna H. T. , Klippenstein Jodine R. , Moore Margo M.
Published: July 2002

Serum contains iron-binding proteins, which inhibit the growth of most pathogenic microorganisms, including fungi. The purpose of this research was to investigate the effect of serum on growth of the opportunistic fungal pathogenAspergillus fumigatus. Supplementing minimal essential medium (MEM) with up to 80% human serum or up to 80% fetal bovine serum (FBS) stimulated growth and increased the amount ofA. fumigatusdry biomass approximately fourfold. In addition, a 100-fold increase in proteinase secretion, as measured by azocasein hydrolysis, was observed when 10% human serum or 10% FBS was added to MEM. The fungal proteinases secreted in serum-containing media were shown to degrade3H-labeled basal lamina proteins. The factor in serum that stimulated proteinase secretion was larger than 10 kDa and was 85% inactivated when the serum was heated for 30 min at 66°C. The proportions of proteinases of each catalytic class secreted byA. fumigatusin the presence of serum were different from the proportions secreted in media containing single proteins. Proteinase secretion did not result from increased protein concentration in the medium per se because bovine serum albumin (BSA) at a concentration equivalent to the concentration of serum produced only 20% of the proteinase activity per milligram (dry weight) that was produced by FBS. Addition of BSA plus 100 μM FeCl3to MEM resulted in the same level of growth as addition of serum, indicating that a combination of nutritional factors in serum may stimulate growth. However, the level of proteinase secretion was still only 30% of the level observed with FBS. These data indicate that serum does not inhibit the growth ofA. fumigatusand that the nutrients in serum result in high levels of proteinase secretion, potentially increasing the invasiveness of this species.
Increased CD8+-T-Cell Expression and a Type 2 Cytokine Pattern during the Muscular Phase ofTrichinellaInfection in Humans
Authors: Gomez Morales Maria Angeles , Mele Raffaella , Sanchez Massimo , Sacchini Daria , De Giacomo Marzia , Pozio Edoardo
Published: July 2002

Cell-mediated immunity during the muscular phase ofTrichinellainfection in humans was studied. Cell proliferation, the phenotypic changes in the T-cell population, and expression and production of cytokines were examined by using peripheral blood mononuclear cells (PBMC) collected at different times postinfection from 10 individuals who had acquiredTrichinella spiralisand five individuals who had acquiredTrichinella britoviin two distinct outbreaks.T. spiralisandT. britovicrude worm extracts induced proliferation of PBMC fromT. spiralis- andT. britovi-infected donors. Cytokine gene expression showed a predominant type 2 pattern for the entire period of infection studied, although gamma interferon (IFN-γ) was expressed. Interleukin-2 (IL-2), IL-5, IL-10, and IFN-γ production was found in PBMC of all donors. There was a good correspondence between the cytokine expression and production patterns. Changes in PBMC composition, with a trend toward an increase in CD8+lymphocyte counts, were observed.
In Vivo Complementation of
ureB
Restores the Ability of
Helicobacter pylori
To Colonize
Authors: Eaton Kathryn A. , Gilbert Joanne V. , Joyce Elizabeth A. , Wanken Amy E. , Thevenot Tracy , Baker Patrick , Plaut Andrew , Wright Andrew
Published: July 2002

The objective of this study was to determine (i) if complementation of
ureB
-negative
Helicobacter pylori
restores colonization and (ii) if urease is a useful reporter for promoter activity in vivo. Strains used were M6, M6Δ
ureB
, and 10 recombinant derivatives of M6 or M6Δ
ureB
in which urease expression was under the control of different
H. pylori
promoters. Mice were orally inoculated with either the wild type or one of the mutant strains, and colonization, in vivo urease activity, and extent of gastritis were determined. Of eight M6Δ
ureB
recombinants tested, four colonized mice. Of those, three had the highest in vitro urease activity of any of the recombinants, significantly different from that of the noncolonizing mutants. The fourth colonizing recombinant, with
ureB
under control of the
cag-15
promoter, had in vitro urease activity which did not differ significantly from the noncolonizing strains. In vivo, urease activities of the four colonizing transformants and the wild-type control were indistinguishable. There were no differences in gastritis or epithelial lesions between mice infected with M6 and those infected with the transformants. These results demonstrate that recovery of urease activity can restore colonizing ability to urease-negative
H. pylori
. They also suggest that
cag-15
is upregulated in vivo, as was previously suggested by demonstrating that it is upregulated upon contact with epithelial cells. Finally, our results suggest that total urease activity and colonization density do not contribute to gastritis due to
H. pylori.
Role of Cholesterol and the Ganglioside GM
1
in Entry and Short-Term Survival of
Brucella suis
in Murine Macrophages
Authors: Naroeni Aroem , Porte Françoise
Published: July 2002

Brucella
species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. We have previously shown that the maturation inhibition of the
Brucella
-containing phagosome appears to be restricted at the phagosomal membrane, but the precise molecular mechanisms and factors involved in this inhibition have yet to be identified. Interestingly, recent studies have revealed that caveolae or lipid rafts are implicated in the entry of some microorganisms into host cells and mediate an endocytic pathway avoiding fusion with lysosomes. In this study, we investigated the role of cholesterol and the ganglioside GM
1
, two components of lipid rafts, in entry and short-term survival of
Brucella suis
in murine macrophages, by using cholesterol-sequestering (filipin and β-methyl cyclodextrin) and GM
1
-binding (cholera toxin B) molecules. Our results suggest that lipid rafts may provide a portal for entry of
Brucella
into murine macrophages under nonopsonic conditions, thus allowing phagosome-lysosome fusion inhibition, and provide further evidence to support the idea that the phagosome maturation inhibition is restricted at the phagosomal membrane.
Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 ofChlamydia trachomatis
Authors: Dessus-Babus Sophie , Darville Toni L. , Cuozzo Francis P. , Ferguson Kaethe , Wyrick Priscilla B.
Published: July 2002

The inflammatory response associated withChlamydia trachomatisgenital infections is thought to be initiated by the release of proinflammatory cytokines from infected epithelial cells. This study focuses on the interactions betweenC. trachomatis-infected HeLa cells and immune cells involved in the early stages of infection, i.e., neutrophils and macrophages. First, we showed that the expression of interleukin-11 (IL-11), an anti-inflammatory cytokine mainly active on macrophages, was upregulated at the mRNA level in the genital tracts of infected mice. Second, incubation of differentiated THP-1 (dTHP-1) cells or monocyte-derived macrophages (MdM) with basal culture supernatants fromC. trachomatisserovar E- or serovar L2-infected HeLa cells resulted in macrophage activation with a differential release of tumor necrosis factor alpha (TNF-α) and upregulation of indoleamine 2,3-deoxygenase (IDO) but not of Toll-like receptor 2 and 4 mRNA expression. Third, coculture of infected HeLa cells with dTHP-1 cells resulted in a reduction in chlamydial growth, which was more dramatic for serovar E than for L2 and which was partially reversed by the addition of anti-TNF-α antibodies for serovar E or exogenous tryptophan for both serovars but was not reversed by the addition of superoxide dismutase or anti-IL-8 or anti-IL-1β antibodies. A gamma interferon-independent IDO mRNA upregulation was also detected in dTHP-1 cells from infected cocultures. Lastly, with a two-stage coculture system, we found that (i) supernatants from neutrophils added to the apical side of infected HeLa cell cultures were chlamydicidal and induced MdM to express antichlamydial activity and (ii) although polymorphonuclear leukocytes released more proinflammatory cytokines in response to serovar E- than in response to L2-infected cells, MdM were strongly activated by serovar L2 infection, indicating that the early inflammatory response generated with a nondisseminating or a disseminating strain is different.
Reevaluation of the Role of
HWP1
in Systemic Candidiasis by Use of
Candida albicans
Strains with Selectable Marker
URA3
Targeted to the
ENO1
Locus
Authors: Sundstrom Paula , Cutler Jim E. , Staab Janet F.
Published: July 2002

Previous evaluation of
HWP1
in systemic candidiasis in CBA/J mice was done with
Candida albicans
strains with differing genetic locations of
URA3
as a result of Ura-blaster mutagenesis. In this study, the presence of
HWP1
and the location of
URA3
contributed to the severity of murine systemic candidiasis in BALB/c mice.
Cytokines Induce Indoleamine 2,3-Dioxygenase Expression in Human Atheroma-Associated Cells: Implications for PersistentChlamydophila pneumoniaeInfection
Authors: Sakash Jessica B. , Byrne Gerald I. , Lichtman Andrew , Libby Peter
Published: July 2002

This study shows that vascular smooth muscle cells express significantly higher levels of gamma interferon-inducible indoleamine 2,3-dioxygenase (IDO) activity than endothelium or mononuclear cells. Since IDO activity is linked to persistentChlamydophila pneumoniaeinfection, our results suggest that smooth muscle cells may be an important reservoir of that organism in atherosclerosis.