bluuebunny/crossref_metadata_2025 huggingface dataset:
AttenuatedYersinia pseudotuberculosisCarrier Vaccine for Simultaneous Antigen-Specific CD4 and CD8 T-Cell Induction
Authors: Rüssmann Holger , Gerdemann Ulrike , Igwe Emeka I. , Panthel Klaus , Heesemann Jürgen , Garbom Sara , Wolf-Watz Hans , Geginat Gernot
Published: May 2003

Yersinia pseudotuberculosisemploys a type III secretion system for targeting of several virulence factors directly to the cytosol of eukaryotic cells. This protein translocation mechanism mediates the ability ofYersiniato resist phagocytosis and is required for sustained extracellular bacterial replication. In the present study, theYersiniaouter protein E (YopE) was used as a carrier molecule for type III-dependent secretion and translocation of listeriolysin O (LLO) fromListeria monocytogenes. In comparison to wild-typeYersinia, an attenuatedY.pseudotuberculosis yopK-null mutant strain hypertranslocates chimeric YopE/LLO into the cytosol of macrophages, resulting in enhanced major histocompatibility complex (MHC) class I-restricted antigen presentation of an LLO-derived CD8 T-cell epitope. Remarkably, T-cell activation assays also revealed a superior ability of translocated over secreted LLO to induce MHC class II-restricted antigen presentation. These in vitro observations were confirmed after immunization of mice with a single dose of theyopK-null mutant strain. Animals orally inoculated with recombinantYersiniaexpressing translocated chimeric YopE/LLO revealed high numbers of gamma interferon-producing LLO-specific CD4 and CD8 T cells. For the first time, it is shown that cytosolic antigen display mediated by an extracellular bacterial carrier vaccine results in simultaneous CD4 and CD8 T-cell priming, conferring protection against an intracellular pathogen.
Deletion of the Gene Encoding p60 inListeria monocytogenesLeads to Abnormal Cell Division and Loss of Actin-Based Motility
Authors: Pilgrim Sabine , Kolb-Mäurer Annette , Gentschev Ivaylo , Goebel Werner , Kuhn Michael
Published: May 2003

Protein p60 encoded by theiapgene is regarded as an essential gene product ofListeria monocytogenes. Here we report, however, the successful construction of a viableiapdeletion mutant ofL. monocytogenesEGD. The mutant, which produces no p60, shows abnormal septum formation and tends to form short filaments and hooked forms during logarithmic growth. These abnormal bacterial cells break into almost normal sized single bacteria in the late-stationary-growth phase. Theiapmutant is strongly attenuated in a mouse model after intravenous injection, demonstrating the importance of p60 during infection, and the invasiveness of the Δiapmutant for 3T6 fibroblasts and Caco-2 epithelial cells is slightly reduced. Upon uptake by epithelial cells and macrophages, theiapmutant escapes from the phagosome into the cytosol with the same efficiency as the wild-type strain, and the mutant bacteria also grow intracellularly at a rate similar to that of the wild-type strain. Intracellular movement and cell-to-cell spread are drastically reduced in various cell lines, since theiap-negative bacteria fail to induce the formation of actin tails. However, the bacteria are covered with actin filaments. Most intracellular bacteria show a nonpolar and uneven distribution of ActA around the cell, in contrast to that for the wild-type strain, where ActA is concentrated at the old pole. In aniap+revertant strain that produces wild-type levels of p60, intracellular movement, cell-to-cell spread, and polar distribution of ActA are fully restored. In vitro analysis of ActA distribution on the filaments of the Δiapstrain shows that the loss of bacterial septum formation leads to ActA accumulation at the presumed division sites. In the light of data presented here and elswhere, we propose to renameiap(invasion-associated protein)cwhA(cell wall hydrolase A).
Genetic Diversity and Antigenic Polymorphism in
Plasmodium falciparum
: Extensive Serological Cross-Reactivity between Allelic Variants of Merozoite Surface Protein 2
Authors: Franks Simon , Baton Luke , Tetteh Kevin , Tongren Eric , Dewin David , Akanmori Bartholomew D. , Koram Kojo A. , Ranford-Cartwright Lisa , Riley Eleanor M.
Published: May 2003

Diversity in the surface antigens of malaria parasites is generally assumed to be a mechanism for immune evasion, but there is little direct evidence that this leads to evasion of protective immunity. Here we show that alleles of the highly polymorphic merozoite surface protein 2 (MSP-2) can be grouped (within the known dimorphic families) into distinct serogroups; variants within a serogroup show extensive serological cross-reactivity. Cross-reactive epitopes are immunodominant, and responses to them may be boosted at the expense of responses to novel epitopes (original antigenic sin). The data imply that immune selection explains only some of the diversity in the
msp-2
gene and that MSP-2 vaccines may need to include only a subset of the known variants in order to induce pan-reactive antibodies.
Helicobacter pylori
Activates Toll-Like Receptor 4 Expression in Gastrointestinal Epithelial Cells
Authors: Su Bin , Ceponis Peter J. M. , Lebel Sylvie , Huynh Hien , Sherman Philip M.
Published: May 2003

Helicobacter pylori
activates the transcription factor NF-κB, leading to proinflammatory cytokine production by gastric epithelial cells. However, the receptors for the initial bacterial interaction with host cells which activate downstream signaling events have not been completely defined. Recently, it has been shown that microbial components activate Toll-like receptors (TLRs), thereby leading to AP-1- and NF-κB-dependent transcription and resulting in the production of proinflammatory cytokines. The aim of this study was to determine whether
H. pylori
activates TLR4. Reverse transcription-PCR showed that both type I and type II
H. pylori
clinical isolates induced TLR4 mRNA expression in AGS cells compared with that by uninfected controls.
H. pylori
upregulated TLR4 protein expression in two gastric epithelial cell lines (AGS and MKN45) and one intestinal epithelial cell line (T84). Monoclonal TLR4 antibody inhibited lipopolysaccharide-induced interleukin-8 secretion from THP-1 macrophages but not from gastric epithelial cells infected with
H. pylori
.
H. pylori
demonstrated increased adherence to CHO TLR4-transfected cells compared with that to both CHO TLR2-transfected and nontransfected CHO cells (
P
< 0.01). These results indicate that
H. pylori
activates TLR4 expression in epithelial cells and that TLR4 can serve as a receptor for
H. pylori
binding.
Gamma Interferon Augments the Intracellular Pathway for Lipopolysaccharide (LPS) Recognition in Human Intestinal Epithelial Cells through Coordinated Up-Regulation of LPS Uptake and Expression of the Intracellular Toll-Like Receptor 4-MD-2 Complex
Authors: Suzuki Manabu , Hisamatsu Tadakazu , Podolsky Daniel K.
Published: May 2003

Although some intestinal epithelial cell lines are known to respond to lipopolysaccharide (LPS), understanding of the relationship between LPS responsiveness and the expression of LPS receptors or factors regulating LPS responsiveness of intestinal epithelial cell lines is incomplete. In this study, we demonstrate that commonly studied human intestinal epithelial cell lines can be classified into at least three different types on the basis of LPS responsiveness, Toll-like receptor-4 (TLR4) expression, and the effects of gamma interferon (IFN-γ) on LPS responsiveness. The first phenotype, which includes the HCT-116 and Caco-2 cell lines, is characterized by relative hyporesponsiveness to LPS and diminished expression of TLR4 protein. In these cells, IFN-γ does not induce LPS responsiveness. The second phenotype, which includes cell line SW480, exhibits a highly LPS-responsive phenotype and surface expression of TLR4 protein even in unprimed conditions. These lines are functionally similar to cells of monocytic lineage. In the third phenotype, which includes the HT-29 and Colo205 cell lines, TLR4 protein is largely present in the cytoplasmic fraction and the cells are hyporesponsive to LPS in an unprimed condition. However, priming of these cells with IFN-γ can induce LPS responsiveness through augmentation of LPS uptake and expression of MD-2 mRNA and intracellular TLR4 proteins. Finally, these findings suggest that the Th1 cytokine IFN-γ modulates LPS responsiveness through several mechanisms in intestinal epithelial cells and that these cells may comprise different subpopulations with distinct roles in innate immune responses.
The
rovA
Mutant of
Yersinia enterocolitica
Displays Differential Degrees of Virulence Depending on the Route of Infection
Authors: Dube Peter H. , Handley Scott A. , Revell Paula A. , Miller Virginia L.
Published: May 2003

Yersinia enterocolitica
is an invasive enteric pathogen that causes significant inflammatory disease. Recently, we identified and characterized a global regulator of virulence (
rovA
). When mice are infected orally with the
rovA
mutant they are attenuated by 50% lethal dose (LD
50
) analysis and have altered kinetics of infection. Most significantly, mice orally infected with the
rovA
mutant have greatly reduced inflammation in the Peyer's patches compared to those infected with wild-type
Y. enterocolitica.
However, we present data here indicating that when the
rovA
mutant bacteria are delivered intraperitoneally (i.p.), they are significantly more virulent than when delivered orally. The i.p. LD
50
for the
rovA
mutant is only 10-fold higher than that of the wild-type
Y. enterocolitica
, and there are significant inflammatory responses to the
rovA
mutant that are evident in the liver and spleen. Altogether, these data suggest that the RovA regulon may be required for the early events of the infection that occur in the Peyer's patches. Furthermore, these data suggest that the RovA regulon may be dispensable for
Y. enterocolitica
systemic disease and inflammatory responses if the Peyer's patches are bypassed.
CD40 Contributes to Lethality in Acute Sepsis: In Vivo Role for CD40 in Innate Immunity
Authors: Gold Jeffrey A. , Parsey Merdad , Hoshino Yoshihiko , Hoshino Satomi , Nolan Anna , Yee Herman , Tse Doris B. , Weiden Michael D.
Published: May 2003

Sepsis induces an early inflammatory cascade initiated by the innate immune response. This often results in the development of multisystem organ failure. We examined the role of CD40, a costimulatory molecule that is integral in adaptive immunity, by using a murine model of polymicrobial sepsis. CD40 knockout (KO) mice had delayed death and improved survival after cecal ligation and puncture (CLP). In addition, they had less remote organ injury as manifested by reduced pulmonary capillary leakage. The improvements in survival and remote organ dysfunction in CD40 KO mice were associated with reduced interleukin-6 (IL-6) and IL-10 levels in serum and bronchoalveolar lavage fluid compared to the levels in wild-type (WT) controls. Furthermore, in contrast to WT mice, CD40 KO mice had no induction of the Th1 cytokines IL-12 and gamma interferon in serum or lungs after CLP. The alterations in cytokine production in CD40 KO mice were associated with similar changes in transcription factor activity. After CLP, CD40 KO mice had attenuated activation of nuclear factor κB and signal transducer and activator of transcription 3 in both the lung and the liver. Finally, WT mice had increased expression of CD40 on their alveolar macrophages. These data highlight the importance of CD40 activation in the innate immune response during polymicrobial sepsis and the subsequent development of remote organ dysfunction.
pH-Regulated Gene Expression of the Gastric PathogenHelicobacter pylori
Authors: Merrell D. Scott , Goodrich Maria L. , Otto Glen , Tompkins Lucy S. , Falkow Stanley
Published: May 2003

Colonization by the gastric pathogenHelicobacter pylorihas been shown to be intricately linked to the development of gastritis, ulcers, and gastric malignancy. Little is known about mechanisms employed by the bacterium that help it adapt to the hostile environment of the human stomach. In an effort to extend our knowledge of these mechanisms, we utilized spotted-DNA microarrays to characterize the response ofH. pylorito low pH. Expression of approximately 7% of the bacterial genome was reproducibly altered by shift to low pH. Analysis of the differentially expressed genes led to the discovery that acid exposure leads to profound changes in motility ofH. pylori, as a larger percentage of acid-exposed bacterial cells displayed motility and moved at significantly higher speeds. In contrast to previous publications, we found that expression of the bacterial virulence genecagAwas strongly repressed by acid exposure. Furthermore, this transcriptional repression was reflected at the level of protein accumulation in theH. pyloricell.
Drosophila melanogaster
Is a Genetically Tractable Model Host for
Mycobacterium marinum
Authors: Dionne Marc S. , Ghori Nafisa , Schneider David S.
Published: May 2003

Mycobacterium marinum
is a pathogenic mycobacterial species that is closely related to
Mycobacterium tuberculosis
and causes tuberculosis-like disease in fish and frogs. We infected the fruit fly
Drosophila melanogaster
with
M. marinum
. This bacterium caused a lethal infection in the fly, with a 50% lethal dose (LD
50
) of 5 CFU. Death was accompanied by widespread tissue damage.
M. marinum
initially proliferated inside the phagocytes of the fly; later in infection, bacteria were found both inside and outside host cells. Intracellular
M. marinum
blocked vacuolar acidification and failed to colocalize with dead
Escherichia coli
, similar to infections of mouse macrophages.
M. marinum
lacking the
mag24
gene were less virulent, as determined both by LD
50
and by death kinetics. Finally, in contrast to all other bacteria examined, mycobacteria failed to elicit the production of antimicrobial peptides in
Drosophila
. We believe that this system should be a useful genetically tractable model for mycobacterial infection.
Catalases of
Aspergillus fumigatus
Authors: Paris Sophie , Wysong Deborah , Debeaupuis Jean-Paul , Shibuya Kazutoshi , Philippe Bruno , Diamond Richard D. , Latgé Jean-Paul
Published: May 2003

Upon infection of a host, the pathogenic fungus
Aspergillus fumigatus
is attacked by the reactive oxygen species produced by phagocytic cells. Detoxification of hydrogen peroxide by catalases was proposed as a way to overcome this host response.
A. fumigatus
produces three active catalases; one is produced by conidia, and two are produced by mycelia. The mycelial catalase Cat1p was studied previously. Here we characterized the two other catalases, their genes, and the phenotypes of gene-disrupted mutants. CatAp, a spore-specific monofunctional catalase, is resistant to heat, metal ions, and detergent. This enzyme is a dimeric protein with 84.5-kDa subunits. The 749-amino-acid polypeptide exhibits high levels of similarity to the
Aspergillus nidulans
CatA catalase and to bacterial catalase HPII of
Escherichia coli
. In spite of increased sensitivity to H
2
O
2
, killing of Δ
catA
conidia by alveolar macrophages and virulence in animals were similar to the killing of conidia by alveolar macrophages and virulence in animals observed for the wild type. In contrast to the Cat1p and CatAp catalases, the mycelial Cat2p enzyme is a bifunctional catalase-peroxidase and is sensitive to heat, metal ions, and detergent. This enzyme, an 82-kDa monomer, is homologous to catalase-peroxidases of several fungi and bacteria. Surprisingly, mycelium of the double Δ
cat1
Δ
cat2
mutant with no catalase activity exhibited only slightly increased sensitivity to H
2
O
2
and was as sensitive to killing by polymorphonuclear neutrophils as mycelium of the wild-type strain. However, this mutant exhibited delayed infection in the rat model of aspergillosis compared to infection by the wild-type strain. These results indicate that conidial catalase is not a virulence factor and that mycelial catalases transiently protect the fungus from the host.
Interleukin-12 p40 Secretion by Cutaneous CD11c+and F4/80+Cells Is a Major Feature of the Innate Immune Response in Mice That Develop Th1-Mediated Protective Immunity toSchistosomamansoni
Authors: Hogg Karen G. , Kumkate Supeecha , Anderson Sonia , Mountford Adrian P.
Published: May 2003

Radiation-attenuated (RA) schistosome larvae are potent stimulators of innate immune responses at the skin site of exposure (pinna) that are likely to be important factors in the development of Th1-mediated protective immunity. In addition to causing an influx of neutrophils, macrophages, and dendritic cells (DCs) into the dermis, RA larvae induced a cascade of chemokine and cytokine secretion following in vitro culture of pinna biopsy samples. While macrophage inflammatory protein 1α and interleukin-1β (IL-1β) were produced transiently within the first few days, the Th1-promoting cytokines IL-12 and IL-18 were secreted at high levels until at least day 14. Assay of C3H/HeJ mice confirmed that IL-12 secretion was not due to lipopolysaccharide contaminants binding Toll-like receptor 4. Significantly, IL-12 p40 secretion was sustained in pinnae from vaccinated mice but not in those from nonprotected infected mice. In contrast, IL-10 was produced from both vaccinated and infected mice. This cytokine regulates IL-12-associated dermal inflammation, since in vaccinated IL-10−/−mice, pinna thickness was greatly increased concurrent with elevated levels of IL-12 p40. A significant number of IL-12 p40+cells were detected as emigrants from in vitro-cultured pinnae, and most were within a population of rare large granular cells that were Ia+, consistent with their being antigen-presenting cells. Labeling of IL-12+cells for CD11c, CD205, CD8α, CD11b, and F4/80 indicated that the majority were myeloid DCs, although a proportion were CD11c−F4/80+, suggesting that macrophages were an additional source of IL-12 in the skin.
Helicobacter-Induced Chronic Active Lymphoid Aggregates Have Characteristics of Tertiary Lymphoid Tissue
Authors: Shomer Nirah H. , Fox James G. , Juedes Amy E. , Ruddle Nancy H.
Published: May 2003

Susceptible strains of mice that are naturally or experimentally infected with murine intestinal helicobacter species develop hepatic inflammatory lesions that have previously been described as chronic active hepatitis. The inflammatory infiltrates in some models of chronic autoimmunity or inflammation resemble tertiary lymphoid organs hypothesized to arise by a process termed lymphoid organ neogenesis. To determine whether hepatic inflammation caused by infection with helicobacter could give rise to tertiary lymphoid organs, we used fluorescence-activated cell sorting, immunohistochemistry, and in situ hybridization techniques to identify specific components characteristic of lymphoid organs in liver tissue sections and liver cell suspensions from helicobacter-infected mice. Small venules (high endothelial venules [HEVs]) in inflammatory lesions inHelicobacterspecies-infected livers were positive for peripheral node addressin. Mucosal addressin cell adhesion molecule also stained HEVs and cells with a staining pattern consistent with scattered stromal cells. The chemokines SLC (CCL 21) and BLC (CXCL13) were present, as were B220-positive B cells and T cells. The latter included a naïve (CD45lo-CD62Lhi) population. These findings suggest that helicobacter-induced chronic active hepatitis arises through the process of lymphoid organ neogenesis.
Intracellular Replication of
Mycobacterium marinum
within
Dictyostelium discoideum
: Efficient Replication in the Absence of Host Coronin
Authors: Solomon Jonathan M. , Leung Grace S. , Isberg Ralph R.
Published: May 2003

Mycobacterium marinum
causes tuberculosis-like disease in fish and amphibians and has been used as a model mycobacterial species because of its rapid growth and less stringent containment requirements relative to other mycobacterial species. We demonstrate here that
M. marinum
grows within
Dictyostelium discoideum
cells, allowing the genetic analysis of host factors that may modulate the replication of mycobacterial species. Intracellular growth of
M. marinum
was shown to mimic the properties previously observed for growth within cultured phagocytes. A defined bacterial mutant defective for growth within phagocytic cells was shown to be similarly defective for growth within
D. discoideum
. To test the role of host coronin, which was previously hypothesized to positively modulate mycobacterial growth within mouse macrophages, a defined
D. discoideum
coronin mutant was analyzed. Surprisingly, the absence of coronin resulted in enhanced intracellular replication of
M. marinum
relative to the control wild-type strain. Consistent with previous observations, some phagosomes showed persistence of coronin about the surface of the compartment, but colocalization of the protein was far from uniform. We conclude that in
D. discoideum
factors other than coronin support intracellular replication of
M. marinum
.
Analysis of the OspE Determinants Involved in Binding of Factor H and OspE-Targeting Antibodies Elicited during
Borrelia
burgdorferi
Infection in Mice
Authors: Metts Michael S. , McDowell John V. , Theisen Michael , Hansen Paul Robert , Marconi Richard Thomas
Published: May 2003

Immune evasion by Lyme spirochetes is a multifactorial process involving numerous mechanisms. The OspE protein family undergoes antigenic variation during infection and binds factor H (fH) and possibly FHL-1/reconectin. In
Borrelia burgdorferi
B31MI, the OspE family consists of three paralogs: BBL39 (ErpA), BBP38, and BBN38 (ErpP). BBL39 and BBP38 are identical and therefore are referred to here as BBL39. The goals of this study were to assess the specificity of the antibody (Ab) response to the OspE paralogs and to identify the domains or determinants of OspE that are required for the binding of fH and OspE-targeting Abs that develop during infection. Here we demonstrate that at least some of the anti-OspE Abs produced during infection are paralog specific and that Ab binding requires conformational determinants whose formation requires both the N- and C-terminal domains of OspE. The binding of fH to OspE was also found to be dependent on conformational determinants. It is also demonstrated here that all of the OspE paralogs expressed by
B. burgdorferi
B31MI are capable of binding fH. The binding of fH to members of the OspF protein family was also assessed. In contrast to an earlier report, no binding of BBO39 or BBR42 to human fH was detected. Lastly, a series of competitive binding enzyme-linked immunosorbent assay analyses, designed to determine if fH and infection serum Abs bind to the same sites on OspE, revealed that these ligands interact with different regions of OspE.
Comprehensive Analysis of the Factor H Binding Capabilities of
Borrelia
Species Associated with Lyme Disease: Delineation of Two Distinct Classes of Factor H Binding Proteins
Authors: McDowell John V. , Wolfgang Jill , Tran Emily , Metts Michael S. , Hamilton Duncan , Marconi Richard T.
Published: May 2003

Some Lyme disease spirochete isolates can bind complement regulatory protein factor H (fH), a process that may allow evasion of complement-mediated killing. Here we demonstrate significant differences in the fH binding capabilities of species of the
Borrelia
burgdorferi
sensu lato complex. The percentages of
B
.
burgdorferi
,
B
.
afzelii
, and
B
.
garinii
bacteria that bound fH in either enzyme-linked immunosorbent assays or affinity ligand binding immunoblot assays were 100, 83, and 29%, respectively. The fH binding protein profiles were examined and found to exhibit variability among isolates and to form two distinct classes. Differences in fH binding ability may contribute to the differences in pathogenesis and clinical course observed upon infection with different species of the
B
.
burgdorferi
sensu lato complex.
A Dominant Role for Extracellular Glutathione
S
-Transferase from
Onchocerca volvulus
Is the Production of Prostaglandin D
2
Authors: Sommer Alexandra , Rickert Rainer , Fischer Peter , Steinhart Hans , Walter Rolf D. , Liebau Eva
Published: May 2003

The extracellular glutathione
S
-transferase from the filarial parasite
Onchocerca volvulus
(
Ov
-GST1) is a glutathione-dependent prostaglandin D synthase.
Ov
-GST1, located in the outer hypodermal lamellae and in parts of the cuticle, produces prostaglandin D
2
directly at the parasite-host interface.
Ov
-GST1 therefore has the potential to participate in the modulation of the host immune response by contributing to the production of prostanoids; this supports the predominant hypothesis that parasite-derived eicosanoids influence host inflammatory and immune cells.
Visualization of
Proteus mirabilis
Morphotypes in the Urinary Tract: the Elongated Swarmer Cell Is Rarely Observed in Ascending Urinary Tract Infection
Authors: Jansen Angela M. , Lockatell C. Virginia , Johnson David E. , Mobley Harry L. T.
Published: May 2003

Proteus mirabilis
, a common cause of nosocomial and catheter-associated urinary tract infection, colonizes the bladder and ascends the ureters to the proximal tubules of the kidneys, leading to the development of acute pyelonephritis.
P
.
mirabilis
is capable of swarming, a form of multicellular behavior in which bacteria differentiate from the short rod typical of members of the family
Enterobacteriaceae
, termed the swimmer cell, into hyperflagellated elongated bacteria capable of rapid and coordinated population migration across surfaces, called the swarmer cell. There has been considerable debate as to which morphotype predominates during urinary tract infection.
P
.
mirabilis
(pBAC001), which expresses green fluorescent protein in both swimming and swarming morphotypes, was constructed to quantify the prevalence of each morphotype in ascending urinary tract infection. Transurethral inoculation of
P
.
mirabilis
(pBAC001) resulted in ascending urinary tract infection and kidney pathology in mice examined at both 2 and 4 days postinoculation. Using confocal microscopy, we were able to investigate the morphotypes of the bacteria in the urinary tract. Of 5,087 bacteria measured in bladders, ureters, and kidneys, only 7 (0.14%) were identified as swarmers. MR/P fimbria expression, which correlates with the swimmer phenotype, is prevalent on bacteria in the ureters and bladder. We conclude that, by far, the predominant morphotype present in the urinary tract during ascending infection is the short rod-the swimmer cell.
Listeriolysin O-Mediated Calcium Influx Potentiates Entry ofListeria monocytogenesinto the Human Hep-2 Epithelial Cell Line
Authors: Dramsi Shaynoor , Cossart Pascale
Published: May 2003

To investigate factors which modulate the entry ofListeria monocytogenesinto mammalian cells, we have analyzed the role of Ca2+. We show thatL. monocytogenesinduced Ca2+transients into the human Hep-2 epithelial cell line. The nonpathogenic speciesL. innocuaor aL. monocytogenesmutant strain defective in listeriolysin O (LLO) production was unable to induce these calcium fluxes. Addition of plasma membrane calcium channel antagonists or chelation of extracellular calcium markedly reducedL. monocytogenesentry. In contrast, chelation of host cytosolic Ca2+or blockade of Ca2+release from intracellular stores did not affect invasion. These results indicate thatL. monocytogenes-induced mobilization of extracellular Ca2+by LLO and activation of downstream Ca2+-dependent signaling are required for efficient cell invasion.
The FimH A27V Mutation Is Pathoadaptive for Urovirulence in
Escherichia coli
B2 Phylogenetic Group Isolates
Authors: Hommais Florence , Gouriou Stéphanie , Amorin Christine , Bui Hung , Rahimy Mohamed Chérif , Picard Bertrand , Denamur Erick
Published: May 2003

Correlations between FimH mutations and virulence were established by studying a collection of human commensal and extraintestinal pathogenic
Escherichia coli
natural isolates. Pathoadaptive (A27V and, to a lesser extent, A119V) and “commensal-adaptive” (A202V) mutations were evidenced in B2 phylogenetic group strains.
fimH
phylogenetic analysis indicates that these pathoadaptive mutations occurred several times.
Helicobacter pylori
Supernatants Cause Epithelial Cytoskeletal Disruption That Is Bacterial Strain and Epithelial Cell Line Dependent but Not Toxin VacA Dependent
Authors: Bebb James R. , Letley Darren P. , Rhead Joanne L. , Atherton John C.
Published: May 2003

We show here that
Helicobacter pylori
broth culture supernatants disrupt the actin cytoskeleton of epithelial cell lines, leading to cell rounding and apoptosis through anoikis. We demonstrate that there are marked quantitative differences between strains and that there are different cell line sensitivities. By constructing VacA null isogenic mutants, we show that the effect is not due to the vacuolating cytotoxin.
Vaccine-Induced Protection againstHelicobacter pyloriin Mice Lacking Both Antibodies and Interleukin-4
Authors: Garhart Christine A. , Nedrud John G. , Heinzel Frederick P. , Sigmund Norma E. , Czinn Steven J.
Published: May 2003

To test the hypothesis that a Th2 response toHelicobacter pyloriis necessary for protection and to address the possibility that humoral and Th2 cellular responses may compensate for each other, we generated mice deficient in both interleukin-4 (IL-4) and antibodies. The immunized double-knockout mice were protected fromH. pylorichallenge, as were the parental strains and wild-type C57BL/6 mice. Neutralization of IL-4 in B-cell-deficient mice did not prevent protection. Immunized IL-5-deficient mice were also protected. Thus, IL-4 and IL-5 are not essential for protection.
Cytolethal Distending Toxin Gene Cluster in Enterohemorrhagic
Escherichia
coli
O157:H
−
and O157:H7: Characterization and Evolutionary Considerations
Authors: Janka Andreas , Bielaszewska Martina , Dobrindt Ulrich , Greune Lilo , Schmidt M. Alexander , Karch Helge
Published: May 2003

We identified a cytolethal distending toxin (
cdt
) gene cluster in 87, 6, and 0% of sorbitol-fermenting (SF) enterohemorrhagic
Escherichia
coli
(EHEC) O157:H
−
, EHEC O157:H7, and
E
.
coli
O55:H7/H
−
strains, respectively. The toxin was expressed by the wild-type EHEC O157 strains and by a
cdt
-containing cosmid from a library of SF EHEC O157:H
−
strain 493/89. The
cdt
flanks in strain 493/89 were homologous to bacteriophages P2 and lambda. Our data demonstrate that
cdt
, encoding a potential virulence factor, is present in the EHEC O157 complex and suggest that
cdt
may have been acquired by phage transduction.
Complete Sequence of the
cap
Locus of
Haemophilus influenzae
Serotype b and Nonencapsulated b Capsule-Negative Variants
Authors: Satola Sarah W. , Schirmer Patricia L. , Farley Monica M.
Published: May 2003

The complete capsule (
cap
) loci from three
Haemophilus influenzae
strains, one serotype b (Hib) and two nonencapsulated b capsule-negative variants, were sequenced. Two new open reading frames,
hcsA
and
hcsB
, were identified in region III and thought to be involved in postpolymerization modification of the capsule. The location of the
cap
locus in the
Haemophilus influenzae
chromosome was identified within section 97 of the Rd genome (chromosomal coordinates 1074542 to 1086327) and found to be the same for the Hib and two Hib
−
strains as well as some other encapsulated division I
H. influenzae
strains.
Involvement of Nicotinic Acetylcholine Receptors in Controlling
Chlamydia
pneumoniae
Growth in Epithelial HEp-2 Cells
Authors: Yamaguchi Hiroyuki , Friedman Herman , Yamamoto Yoshimasa
Published: May 2003

Nicotinic acetylcholine receptors (nAChRs) play an essential role in neurotransmission. Recent studies have indicated that nAChRs may be involved in the regulation of some bacterial infections through immunological mechanisms in macrophages. However, the regulation of infection with
Chlamydia
pneumoniae
, which is a ubiquitous pneumonia-causing bacterium, by an nAChR-mediated mechanism is still unclear. In the present study, it was found that stimulation of nAChRs with ligands such as nicotine and acetylcholine altered the growth of
C
.
pneumoniae
in epithelial HEp-2 cells. Thus, the results revealed a possible pathophysiological role of nAChRs in the regulation of intracellular bacterial infection.
Chemokine Receptor CCR2 Is Not Essential for the Development of Experimental Cerebral Malaria
Authors: Belnoue Elodie , Costa Fabio T. M. , Vigário Ana M. , Voza Tatiana , Gonnet Françoise , Landau Irène , van Rooijen Nico , Mack Matthias , Kuziel William A. , Rénia Laurent
Published: May 2003

Infection withPlasmodium bergheiANKA induces cerebral malaria in susceptible mice. Brain-sequestered CD8+T cells are responsible for this pathology. We have evaluated the role of CCR2, a chemokine receptor expressed on CD8+T cells. Infected CCR2-deficient mice were as susceptible to cerebral malaria as wild-type mice were, and CD8+T-cell migration to the brain was not abolished.
Actinobacillus actinomycetemcomitans
Serotype b Lipopolysaccharide Mediates Coaggregation with
Fusobacterium nucleatum
Authors: Rosen Graciela , Nisimov Ira , Helcer Monica , Sela Michael N.
Published: May 2003

Purified
Actinobacillus actinomycetemcomitans
serotype b lipopolysaccharide (LPS) was found to be able to bind
Fusobacterium nucleatum
cells and to inhibit binding of
F. nucleatum
to
A. actinomycetemcomitans
serotype b. Sugar binding studies showed that the requirements for binding of
A. actinomycetemcomitans
serotype b LPS to the
F. nucleatum
lectin are the presence of a metal divalent ion, an axial free hydroxyl group at position 4, and free equatorial hydroxyl groups at positions 3 and 6 of
d
-galactose, indicating that the β-
N
-acetyl-
d
-galactosamine in the serotype b LPS trisaccharide repeating unit is the monosaccharide residue recognized by the
F. nucleatum
lectin. These data strongly suggest that
A. actinomycetemcomitans
serotype b LPS is one of the receptors responsible for the lactose-inhibitable coaggregation of
A. actinomycetemcomitans
to fusobacteria.
Physical Map and Genome Sequencing Survey ofMycoplasma haemofelis(Haemobartonella felis)
Authors: Berent Linda M. , Messick Joanne B.
Published: May 2003

Mycoplasma haemofelisis an uncultivable red-cell pathogen of cats. IsolatedM. haemofelisDNA was used to create a bacterial artificial chromosome library and physical map. Random sequencing of this material revealed 75 genes that had not been previously reported forM. haemofelisor any other hemotrophic mycoplasma.
The Stereochemistry of the Amino Acid Side Chain Influences the Inflammatory Potential of Muramyl Dipeptide in Experimental Meningitis
Authors: Cottagnoud P. , Gerber C. M. , Majcherczyk P. A. , Acosta F. , Cottagnoud M. , Neftel K. , Moreillon P. , Täuber M. G.
Published: May 2003

Intrathecal injections of 50 to 100 μg of (
N
-acetylmuramyl-
l
-alanyl-
d
-isoglutamine) muramyl dipeptide (MDP)/rabbit dose-dependently triggered tumor necrosis factor alpha (TNF-α) secretion (12 to 40,000 pg/ml) preceding the influx of leukocytes in the subarachnoid space of rabbits. Intrathecal instillation of heat-killed unencapsulated R6 pneumococci produced a comparable leukocyte influx but only a minimal level of preceding TNF-α secretion. The stereochemistry of the first amino acid (
l
-alanine) of the MDP played a crucial role with regard to its inflammatory potential. Isomers harboring
d
-alanine in first position did not induce TNF-α secretion and influx of leukocytes. This stereospecificity of MDPs was also confirmed by measuring TNF-α release from human peripheral mononuclear blood cells stimulated in vitro. These data show that the inflammatory potential of MDPs depends on the stereochemistry of the first amino acid of the peptide side chain and suggest that intact pneumococci and MDPs induce inflammation by different pathways.
Natural History of
Helicobacter hepaticus
Infection in Conventional A/J Mice, with Special Reference to Liver Involvement
Authors: Avenaud Philippe , Le Bail Brigitte , Mayo Kathryn , Marais Armelle , Fawaz Rabia , Bioulac-Sage Paulette , Megraud Francis
Published: May 2003

It has been reported that
Helicobacter hepaticus
infection of mice leads to chronic hepatitis and hepatocarcinoma. Our aim was to monitor a cohort of 80 conventional A/J mice in which half of the mice were infected by
H
.
hepaticus
in order to study the evolution of the infection and the pathological changes in comparison to uninfected mice.
H
.
hepaticus
was detected by culture only in some colon and cecum specimens after 17 months of age, while PCR detected
H
.
hepaticus
in the intestines of all inoculated mice after only 5 months of infection. The percentage of mice in which
H
.
hepaticus
was detected in the gallbladder, bile ducts, and liver by PCR, as well as the number of bacteria present in the liver, tended to increase with increasing age and longer infection time. Anti-
H
.
hepaticus
immunoglobulin G antibodies were positive by enzyme-linked immunosorbent assay only in inoculated mice. Pathological findings were also more frequent as the mice grew older: fibrosis was present (especially in the peripheral part of the liver), and significant portal inflammation including lymphoid nodules was present in almost all infected animals. Biliary lesions of neutrophilic acute cholangitis or lymphocytic cholangitis were noted. However, lesions were also observed in uninfected animals, although at a significantly lower level, and the only hepatocellular carcinoma occurred in an uninfected mouse. The evolution towards hepatocarcinoma is not always the endpoint and may depend on the bacterial strain and on the environmental conditions.
Microsatellite analysis of genetic diversity in four populations ofPopulus tremuloidesin Quebec
Authors: Wyman Janet , Bruneau Anne , Tremblay M -Francine
Published: May 2003

To determine overall genetic variability in aspen (Populus tremuloides Michx.) along the clay belt of northwestern Quebec, four populations were sampled at the transition zone between mixed-wood and conifer-dominated forests using a hierarchical strategy. Our objectives were (i) to estimate gene diversity among and within populations using microsatellite markers and (ii) to examine whether clonal structuring in aspen could be detected. For each population, three stands at least 1 km apart were sampled. Within each stand, five putative clones were sampled with a distance of 50 m between each clone. To examine potential genetic diversity within clones, for three of the putative clones sampled per stand, the three trees closest to this individual (potential ramets) were also sampled. All samples were analysed at four microsatellite loci. Measures of genetic variation all indicated that most of the variation occurred within rather than among the populations. Rst, based on a stepwise mutation model, was 0.0409, slightly higher than Fst, which was based on infinite allele model (0.0323). Within each stand, DNA fingerprint of five putative clones allowed the differentiation of genetically distinct individuals within these putative clones, and from 1.62 to 2.2 times more genetically distinct individuals were identified than by morphological identification. In the present study there was no correlation between the mean distance of the potential ramet to the central trunk (potential ramets were often within 23 m of the central trunk) and the possibility of the central trunk and ramet sharing the same genotype. The number of unique genotypes for the three putative clones sampled per stand varied from 11 genotypes per 15 samples to 14 per 15 samples. Consequently, it was not possible to identify clones by either distance from a given central trunk or morphological characteristics. Thus, after stand disturbance, suckers from different genotypes are likely to be closely mixed.Key words: genetic diversity, Populus tremuloides, microsatellites, clone size.
Detection and phylogenetic analysis of mating type genes ofOphiosphaerella korrae
Authors: Hsiang Tom , Chen Fajun , Goodwin Paul H
Published: May 2003

Portions of the mating type genes from Ophiosphaerella korrae (J. Walker & A.M. Smith) R.A. Shoemaker (=Leptosphaeria korrae J. Walker & A.M. Smith), a pathogenic fungus of grasses, were examined by PCR (polymerase chain reaction). For nine isolates of O. korrae from North America, both mating type genes were amplified, demonstrating that both MAT idiomorphs are detectable in this homothallic ascomycete. Amplified fragments from three isolates were sequenced, and parsimony analyses of MAT1 nucleotide and protein sequences placed O. korrae in the basal position of a clade of Phaeosphaeriaceae and Pleosporaceae, whereas the MAT2 nucleotide and protein data placed O. korrae in a clade with Pleosporaceae. The internal transcribed spacer (ITS) and 18S ribosomal DNA of O. korrae were also sequenced. The 18S sequences had insufficient variability to resolve the placement of O. korrae, whereas the ITS data placed it in Phaeosphaeriaceae. A total evidence analysis of Dothideomycetes with 18S, ITS, and MAT data placed O. korrae alongside Phaeosphaeria species, with moderate bootstrap support. However, the KishinoHasegawa test did not demonstrate this topology to be significantly different from one where O. korrae was placed with Pleosporales. Although O. korrae does not belong in Leptosphaeria based on ITS data, MAT data do not strongly support its placement in Phaeosphaeriaceae.Key words: ascomycetes, mating type genes, ribosomal genes, taxonomy.
Competitive and facilitative interactions within and between two species of coastal dune perennials
Authors: Franks Steven J
Published: May 2003

While there is substantial evidence for facilitation, the effects of such factors as stress and species identity on positive interactions remain controversial. At two coastal dune sites, I tested the hypotheses that facilitative interactions increase with increasing stress and disturbance along an environmental gradient and that facilitative interactions are stronger among heterospecific than among conspecific individuals. I transplanted Uniola paniculata and Iva imbricata plants into plots along with four conspecific neighbors, four heterospecific neighbors, or no neighbors across an environmental gradient. Neighbors increased target plant survival, suggesting facilitation, but biomass of targets was reduced by the presence of neighbors, suggesting competition. Unexpectedly, competition was greatest in the purportedly most stressful and disturbed zone. In this study, the outcome of neighbor interactions differed for biomass and survival and depended on position along the environmental gradient, but was independent of neighbor identity.Key words: competition, disturbance, facilitation, Iva imbricata, stress, Uniola paniculata.
Identity and phylogenetic relationships of ophiostomatoid fungi associated with invasive and native Tetropium species (Coleoptera: Cerambycidae) in Atlantic Canada
Authors: Jacobs Karin , Seifert Keith A , Harrison Ken J , Kirisits Thomas
Published: May 2003

In 1999, red spruce (Picea rubens Sarg.) trees in a small area of the Halifax Regional Municipality in Nova Scotia, Atlantic Canada, were found to be infested and killed by Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae), a Eurasian species of longhorn beetle. We isolated and identified the fungi associated with T. fuscum in Halifax and compared them with ophiostomatoid fungi associated with Tetropium spp. on spruce in Europe. Ophiostoma species associated with Tetropium cinnamopterum Kirby (indigenous to and transcontinental in Canada) were also compared with those associated with T. fuscum. Strains were identified based on morphological and molecular characters. Ophiostoma piceae (Münch) Syd. & P. Syd., Pesotum fragrans Mathiesen-Käärik, and Ophiostoma tetropii Mathiesen were the most frequently isolated species. Lack of type material for O. tetropii and discrepancies between the ex-type strain of O. tetropii and its original description prompted us to lectotypify this species and designate epitype material for it. The results suggest that O. tetropii was introduced with T. fuscum from Europe into Atlantic Canada.Key words: Ophiostoma tetropii, Tetropium fuscum, molecular phylogeny, invasive species.
Accuracy and precision of population estimates of Verticillium dahliae on growth media in quantitative soil assays
Authors: Mpofu S I , Hall R
Published: May 2003

Verticillium dahliae Kleb. is a serious pathogen of many plant species. Growth media used to measure population density of V. dahliae in soil were evaluated for high recovery of the pathogen, as well as accuracy and precision of population estimates from naturally and artificially infested sandy loam soil using the soil dilution method. Recovery of V. dahliae from naturally infested field soil was highest on soil pectate Tergitol agar (SPT), soil extract agar + sodium polypectate (SEAP), modified pectate agar (MPA), potato dextrose agar + streptomycin sulphate (PDAS), and Talboys' prune lactose agar (TPA); however, PDAS and TPA were overgrown with contaminating fungi, making enumeration difficult. Use of SPT medium resulted in the most precise population estimate with a standard error (SE) of 12.6% of the mean followed by use of pectate agar (PA) (SE = 14.8%) and SEAP (SE = 19.1%). Ethanol, biotin, and Dox salts enhanced recovery of V. dahliae from naturally infested soil, but combining Dox salts with ethanol and biotin significantly reduced population density. Soil extract had no significant effects on population density. Accuracy of V. dahliae population estimates from sterile artificially inoculated soil was highest on modified soil extract agar (MSEA) (64%) followed by SPT (58%). However, accuracy of V. dahliae population estimates from nonsterile artificially inoculated soil was highest on SPT (36%). Soil extract is not an essential ingredient and biotin may increase recovery of V. dahliae from soil.Key words: Verticillium dahliae, verticillium wilt, population density, recovery, accuracy, precision.
Crown development of a clone of Populus tremuloides exhibiting "crooked" architecture and a comparison with wild-type trees
Authors: Remphrey William R , Pearn Linda P
Published: May 2003

Populus tremuloides Michx. (trembling aspen) is a tree species native to much of North America and is normally ascribed to the architectural model of Rauh, characterized by an excurrent crown structure with a central main stem and orthotropic branches. A mutant clone of trembling aspen is located near Hafford, Saskatchewan, exhibiting an architecture with crooked and twisted tree trunks. It was the objective of the present study to determine how the architectural development of the crooked clone differed from the wild type. In a study conducted over a 5-year period, four mutant trees were compared with four young wild-type aspen in the Winnipeg, Manitoba, area. Based on detailed quantitative data, it was determined that the architecture of the crooked clone of aspen differs greatly from the wild type. The trees are built by the continuous superposition of vigorous relay shoots with a mixed orientation, that is, shoots that take over the main growth of the tree, and have a more or less upright basal part and a horizontal to pendulous distal part. The development of the crookedness starts with the bending of the relay shoots, mostly in relation to the gravitational direction, which is followed in the subsequent years by various gravimorphic responses. In particular, the longest lateral shoots on a parent relay shoot occur in the middle regions, and the tip of the parent relay shoot generally loses vigour over time. The parent shoot may die back to the junction with a daughter relay shoot, causing a sharp bend at that point. Moreover, the divergence angles of relay shoots with the parent shoots were shown to be greater than in wild-type aspen, and this appears to exacerbate the crookedness. The new relay shoot may actually grow back towards the centre of the crown, opposite to the direction of growth of the parent. The results of this study demonstrate how a quantitative change in one architectural character can set in motion a series of developmental processes that result in a vastly different crown structure from the wild type.Key words: Populus tremuloides, trembling aspen, architecture, crooked clone.