bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterium bovis-Infected Cervine Alveolar Macrophages Secrete Lymphoreactive Lipid Antigens
Authors: Aldwell Frank E. , Dicker Bridget L. , Da Silva Tatley Fernanda M. , Cross Martin F. , Liggett Simon , Mackintosh Colin G. , Griffin J. Frank T.
Published: July 2002

Tuberculosis is caused by intracellular bacteria belonging to the genusMycobacterium, includingM. tuberculosisandM. bovis. Alveolar macrophages (AMs) are the primary host cell for inhaled mycobacteria. However, little is known about the mechanisms by which infected AMs can process and present mycobacterial antigens to primed lymphocytes and how these responses may affect ensuing protection in the host. In the present study, we sought to determine whether AMs from a naturally susceptible host forMycobacterium bovis(red deer) could produce and secrete soluble immunoreactive antigens following mycobacterial infection in vitro. Confluent monolayers of deer AMs were infected with either heat-killed or live virulentM. bovisorM. bovisBCG at a multiplicity of infection of 5:1 and cultured for 48 h. Culture supernatants were collected, concentrated, and tested for the presence of mycobacterial antigens in a lymphocyte proliferation assay by using peripheral blood mononuclear cells fromM. bovis-sensitized or naive deer. Supernatants derived from macrophages which had been infected with live bacilli stimulated the proliferation of antigen-sensitized, but not naive, lymphocytes. Supernatants derived from uninoculated AMs or AMs inoculated with heat-killed bacilli failed to stimulate lymphocyte proliferation. The lymphoproliferative activity was retained following lipid extraction of the supernatants, which were free of amino groups as determined by thin-layer chromatography. These results demonstrate that mycobacteria which are actively growing within AMs produce lipids which are secreted into the extracellular milieu and that these lipids are recognized by lymphocytes from mycobacterium-primed hosts. We suggest that mycobacterial lipids are released from AMs following aerosol infection in vivo and that they play an important role in the early immune response to tuberculosis.
A Peptide Permease Mutant of
Mycobacterium bovis
BCG Resistant to the Toxic Peptides Glutathione and
S
-Nitrosoglutathione
Authors: Green Renee M. , Seth Anjali , Connell Nancy D.
Published: July 2002

Oligopeptides play important roles in bacterial nutrition and signaling. Using sequences from the available genome database for
Mycobacterium tuberculosis
H37Rv, the oligopeptide permease operon (
oppBCDA
) of
Mycobacterium bovis
BCG was cloned from a cosmid library. An
opp
mutant strain was constructed by homologous recombination with an allele of
oppD
interrupted by kanamycin and streptomycin resistance markers. The deletion was complemented with a wild-type copy of the
opp
operon. Two approaches were taken to characterize the peptide transporter defect in this mutant strain. First, growth of wild-type and mutant strains was monitored in media containing a wide variety of peptides as sole source of carbon and/or nitrogen. Among 25 peptides ranging from two to six amino acids in length, none was capable of supporting measurable growth as the sole carbon source in either wild-type or mutant strains. The second approach exploited the resistance of permease mutants to toxic substrates. The tripeptide glutathione (γ-glutamyl-
l
-cyteinylglycine [GSH]) is toxic to wild-type BCG and was used successfully to characterize peptide uptake in the
opp
mutant. In 2 mM GSH, growth of the wild-type strain is inhibited, whereas the
opp
mutant is resistant to concentrations as high as 10 mM. Similar results were found with the tripeptide
S
-nitrosoglutathione (GSNO), thought to be a donor of NO in mammalian cells. Using incorporation of [
3
H]uracil to monitor the effects of GSH and GSNO on macromolecular synthesis in growing cells, it was demonstrated that the
opp
mutant is resistant, whereas the wild type and the mutant complemented with a wild-type copy of the operon are sensitive to both tripeptides. In uptake measurements, incorporation of [
3
H]GSH is reduced in the mutant compared with wild type and the complemented mutant. Finally, growth of the three strains in the tripeptides suggests that GSH is bacteriostatic, whereas GSNO is bacteriocidal.
GAA Trinucleotide Repeat Region Regulates M9/pMGA Gene Expression in
Mycoplasma gallisepticum
Authors: Liu Li , Dybvig Kevin , Panangala Victor S. , van Santen Vicky L. , French Christopher T.
Published: July 2002

Mycoplasma gallisepticum
, the cause of chronic respiratory infections in the avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) associated with hemagglutination. Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes. The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlation between gene expression and the number of tandem GAA repeat motifs located upstream of the putative promoter. In this study, transposon Tn
4001
was used as a vector with the
Escherichia coli lacZ
gene as the reporter system to examine the role of the GAA repeats in M9/pMGA gene expression in
M. gallisepticum
. A 336-bp M9 gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligated with a
lacZ
gene from
E. coli
, and inserted into the Tn
4001
-containing plasmid pISM2062. This construct was transformed into
M. gallisepticum
PG31. Transformants were filter cloned on agar supplemented with 5-bromo-4-chloro-3-indolyl-β-
d
-galactopyranoside (X-Gal) to monitor
lacZ
gene expression on the basis of blue/white color selection. Several cycles of filter cloning resulted in cell lineages in which
lacZ
gene expression alternated between the On and Off states in successive generations of progeny clones. The promoter regions of the M9-
lacZ
hybrid genes of individual progeny clones were amplified by PCR and sequenced. The only differences between the promoter regions of the blue and white colonies were in the number of GAA repeats. Clones that expressed
lacZ
had exactly 12 tandem copies of the GAA repeat. Clones that did not express
lacZ
invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats. Southern analysis of
M. gallisepticum
chromosomal DNA confirmed that the phase-variable expression of the
lacZ
reporter gene was not caused by Tn
4001
transposition. These data strongly indicate that changes in the length of the GAA repeat region are responsible for regulating M9/pMGA gene expression.
Differential Regulation of CD4 Lymphocyte Recruitment between the Upper and Lower Regions of the Genital Tract duringChlamydia trachomatisInfection
Authors: Kelly Kathleen A. , Walker Jennifer C. , Jameel Shimul H. , Gray Heather L. , Rank Roger G.
Published: July 2002

Genital infection withChlamydia trachomatisresults in both the local recruitment of protective immune responses and an inflammatory infiltrate that may also participate in tubal pathology. As a beginning to understanding the etiology of immune system-mediated tubal pathology, we evaluated the regional recruitment of lymphocyte subsets to different areas of the female genital tract (GT) over the course of a murine infection with the mouse pneumonitis agent ofChlamydia trachomatis(MoPn). Using flow cytometric techniques we found that the CD4 lymphocyte subset was preferentially recruited to the upper GT (oviduct and uterine horn) over the lower GT (cervical-vaginal region) throughout the course of MoPn infection. The influx of CD4 cells also correlated with the expression of endothelial cell adhesion molecules (ECAMs) and in vitro lymphocyte adherence in the upper GT. Interestingly, the expression of ECAMs in the lower GT was not maintained longer than 7 days after infection, even in the presence of viable chlamydiae. Taken together, these data suggest that regulatory mechanisms of lymphocyte recruitment differ between the upper and lower regions of the GT and may influence the clearance of chlamydiae and the development of tubal pathology.
pH-Induced Conformational Changes in
Clostridium difficile
Toxin B
Authors: Qa'Dan Maen , Spyres Lea M. , Ballard Jimmy D.
Published: July 2002

Toxin B from
Clostridium difficile
is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells. In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B. Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation. Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment. Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents. Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(
p
-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease. As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B. The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence. Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments. Toxin B also demonstrated variable susceptibility to
Staphylococcus aureus
V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein.
Characterization of Recombinant, Ureolytic
Streptococcus mutans
Demonstrates an Inverse Relationship between Dental Plaque Ureolytic Capacity and Cariogenicity
Authors: Clancy K. Anne , Pearson Sylvia , Bowen William H. , Burne Robert A.
Published: July 2002

Dental caries results from prolonged plaque acidification that leads to the establishment of a cariogenic microflora and demineralization of the tooth. Urease enzymes of oral bacteria hydrolyze urea to ammonia, which can neutralize plaque acids. To begin to examine the relationship between plaque ureolytic activity and the incidence of dental caries, recombinant, ureolytic strains of
Streptococcus mutans
were constructed. Specifically, the
ureABCEFGD
operon from
Streptococcus salivarius
57.I was integrated into the
S. mutans
chromosome in such a way that the operon was transcribed from a weak, cognate promoter in
S. mutans
ACUS4 or a stronger promoter in
S. mutans
ACUS6. Both strains expressed NiCl
2
-dependent urease activity, but the maximal urease levels in ACUS6 were threefold higher than those in ACUS4. In vitro pH drop experiments demonstrated that the ability of the recombinant
S. mutans
strains to moderate a decrease in pH during the simultaneous metabolism of glucose and urea increased proportionately with the level of urease activity expressed. Specific-pathogen-free rats that were infected with ACUS6 and fed a cariogenic diet with drinking water containing 25 mM urea and 50 μM NiCl
2
had relatively high levels of oral urease activity, as well as dramatic decreases in the prevalence of smooth-surface caries and the severity of sulcal caries, relative to controls. Urease activity appears to influence plaque biochemistry and metabolism in a manner that reduces cariogenicity, suggesting that recombinant, ureolytic bacteria may be useful to promote dental health.
Protection Elicited by Native Outer Membrane Protein Oms66 (p66) against Host-AdaptedBorrelia burgdorferi: Conformational Nature of Bactericidal Epitopes
Authors: Exner Maurice M. , Wu Xiaoyang , Blanco David R. , Miller James N. , Lovett Michael A.
Published: July 2002

Oms66 is aBorrelia burgdorferiouter membrane porin protein whose role in Lyme disease pathogenesis and immunity has not been well established. Oms66 was solubilized from whole-cell lysates of strain B313 (which is derived from B31 but lacks OspA, -B, -C, and -D) and purified to homogeneity by fast-protein liquid chromatography. Purified native Oms66 (nOms66), which retained the ability to form large channels in a planar lipid bilayer model membrane system, and denatured Oms66 (hOms66) were used to immunize New Zealand White rabbits. The resulting Oms66 antisera were tested in a complement-dependent borreliacidal assay in parallel with basal serum and with serum from rabbits immune to reinfection withB. burgdorferi(IRS). IRS showed high-titer complement-dependent killing of both strains B31 and B313. Sera from animals immunized with nOms66 showed high-titer complement-dependent killing activity against strain B313 but exhibited no killing of B31. By comparison, serum generated from immunizations with hOms66 showed no killing activity against either strain. Following adsorption of antiserum to nOms66 with recombinant Oms66 (rOms66), the serum antibodies no longer bound to rOms66 or to nOms66 that had been denatured with 8 M urea. However, the antibodies still bound to nOms66 and killing activity against B313 was retained, thus suggesting that native, conformational epitopes are targets of this bactericidal activity. Six C3H HeJ mice were immunized with nOms66 and were challenged using “host-adapted”B. burgdorferiB31 by skin implantation of infected mouse ear tissue. Four of the six mice were protected against both localized and disseminated infection. These findings indicate that native Oms66 can elicit potent bactericidal activity and significant protective immunity against host-adapted organisms.
Parenteral Adjuvant Activities ofEscherichia coliHeat-Labile Toxin and Its B Subunit for Immunization of Mice against GastricHelicobacter pyloriInfection
Authors: Weltzin Richard , Guy Bruno , Thomas William D. , Giannasca Paul J. , Monath Thomas P.
Published: July 2002

The heat-labile toxin (LT) ofEscherichia coliis a potent mucosal adjuvant that has been used to induce protective immunity againstHelicobacter felisandHelicobacter pyloriinfection in mice. We studied whether recombinant LT or its B subunit (LTB) has adjuvant activity in mice when delivered withH. pyloriurease antigen via the parenteral route. Mice were immunized subcutaneously or intradermally with urease plus LT, recombinant LTB, or a combination of LT and LTB prior to intragastric challenge withH. pylori. Control mice were immunized orally with urease plus LT, a regimen shown previously to protect againstH. pylorigastric infection. Parenteral immunization using either LT or LTB as adjuvant protected mice againstH. pylorichallenge as effectively as oral immunization and enhanced urease-specific immunoglobulin G (IgG) responses in serum as effectively as aluminum hydroxide adjuvant. LT and LTB had adjuvant activity at subtoxic doses and induced more consistent antibody responses than those observed with oral immunization. A mixture of a low dose of LT and a high dose of LTB stimulated the highest levels of protection and specific IgG in serum. Urease-specific IgG1 and IgG2a antibody subclass responses were stimulated by all immunization regimens tested, but relative levels were dependent on the adjuvant used. Compared to parenteral immunization with urease alone, LT preferentially enhanced IgG1, while LTB or the LT-LTB mixture preferentially enhanced IgG2a. Parenteral immunization using LT or LTB as adjuvant also induced IgA to urease in the saliva of some mice. These results show that LT and LTB stimulate qualitatively different humoral immune responses to urease but are both effective parenteral adjuvants for immunization of mice againstH. pyloriinfection.
Inducible Expression of Human β-Defensin 2 byFusobacterium nucleatumin Oral Epithelial Cells: Multiple Signaling Pathways and Role of Commensal Bacteria in Innate Immunity and the Epithelial Barrier
Authors: Krisanaprakornkit Suttichai , Kimball Janet R. , Weinberg Aaron , Darveau Richard P. , Bainbridge Brian W. , Dale Beverly A.
Published: July 2002

Human gingival epithelial cells (HGE) express two antimicrobial peptides of the β-defensin family, human β-defensin 1 (hBD-1) and hBD-2, as well as cytokines and chemokines that contribute to innate immunity. In the present study, the expression and transcriptional regulation of hBD-2 was examined. HBD-2 mRNA was induced by cell wall extract ofFusobacterium nucleatum, an oral commensal microorganism, but not by that ofPorphyromonas gingivalis, a periodontal pathogen. HBD-2 mRNA was also induced by the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) and phorbol myristate acetate (PMA), an epithelial cell activator. HBD-2 mRNA was also expressed in 14 of 15 noninflamed gingival tissue samples. HBD-2 peptide was detected by immunofluorescence in HGE stimulated withF. nucleatumcell wall, consistent with induction of the mRNA by this stimulant. Kinetic analysis indicates involvement of multiple distinct signaling pathways in the regulation of hBD-2 mRNA; TNF-α andF. nucleatumcell wall induced hBD-2 mRNA rapidly (2 to 4 h), while PMA stimulation was slower (∼10 h). In contrast, each stimulant induced interleukin 8 (IL-8) within 1 h. The role of TNF-α as an intermediary inF. nucleatumsignaling was ruled out by addition of anti-TNF-α that did not inhibit hBD-2 induction. However, inhibitor studies show thatF. nucleatumstimulation of hBD-2 mRNA requires both new gene transcription and new protein synthesis. Bacterial lipopolysaccharides isolated fromEscherichia coliandF. nucleatumwere poor stimulants of hBD-2, although they up-regulated IL-8 mRNA. Collectively, our findings show inducible expression of hBD-2 mRNA via multiple pathways in HGE in a pattern that is distinct from that of IL-8 expression. We suggest that different aspects of innate immune responses are differentially regulated and that commensal organisms have a role in stimulating mucosal epithelial cells in maintaining the barrier that contributes to homeostasis and host defense.
Blood Group A Antigen Is a Coreceptor in
Plasmodium falciparum
Rosetting
Authors: Barragan Antonio , Kremsner Peter G. , Wahlgren Mats , Carlson Johan
Published: July 2002

The malaria parasite
Plasmodium falciparum
utilizes molecules present on the surface of uninfected red blood cells (RBC) for rosette formation, and a dependency on ABO antigens has been previously shown. In this study, the antirosetting effect of immune sera was related to the blood group of the infected human host. Sera from malaria-immune blood group A (or B) individuals were less prone to disrupt rosettes from clinical isolates of blood group A (or B) patients than to disrupt rosettes from isolates of blood group O patients. All fresh clinical isolates and laboratory strains exhibited distinct ABO blood group preferences, indicating that utilization of blood group antigens is a general feature of
P. falciparum
rosetting. Soluble A antigen strongly inhibited rosette formation when the parasite was cultivated in A RBC, while inhibition by glycosaminoglycans decreased. Furthermore, a soluble A antigen conjugate bound to the cell surface of parasitized RBC. Selective enzymatic digestion of blood group A antigen from the uninfected RBC surfaces totally abolished the preference of the parasite to form rosettes with these RBC, but rosettes could still form. Altogether, present data suggest an important role for A and B antigens as coreceptors in
P. falciparum
rosetting.
Characterization of FimY as a Coactivator of Type 1 Fimbrial Expression in
Salmonella enterica
Serovar Typhimurium
Authors: Tinker Juliette K. , Clegg Steven
Published: July 2002

Type 1 fimbriae of
Salmonella enterica
serovar Typhimurium are surface appendages that carry adhesins specific for mannosylated host glycoconjugates. Regulation of the major fimbrial subunit is thought to be controlled by a number of ancillary
fim
genes, including
fimZ
,
fimY
,
fimW
, and
fimU
. Previous studies using a FimZ mutant have indicated that this protein is necessary for
fimA
expression, and in vitro DNA binding assays determined that FimZ is a transcriptional activator that binds directly to the
fimA
promoter. To determine the role of FimY as a potential regulator of fimbrial expression, a
fimY
mutant of serovar Typhimurium was generated by allelic exchange. This mutant was found to be phenotypically nonfimbriate. No transcription from the
fimA
promoter was detected in a
fimY
mutant containing a
fimA-lacZ
reporter construct located on the chromosome. In addition, transcription from the cloned
fimY
promoter was not detected in
Escherichia coli
unless both FimZ and FimY were present, indicating that these proteins also act as coactivators of
fimY
expression. Consistent with these results, there is no transcription from a
fimY-lacZ
reporter construct within a serovar Typhimurium
fimY
or
fimZ
mutant. Studies using the
fimY-lacZ
construct reveal that expression of this gene varies with environmental conditions in a manner similar to
fimA
expression. Extensive in vitro DNA binding assays using extracts from
E. coli
that overexpress FimY, as well as partially purified FimY, were unable to identify a specific interaction between FimY and the
fimA
or
fimY
promoter. The results indicate that FimY is a positive regulator of fimbrial expression and that this protein acts in cooperation with FimZ to regulate the expression of
Salmonella
type 1 fimbrial appendages.
Schistosoma mansoniPhosphoenolpyruvate Carboxykinase, a Novel Egg Antigen: Immunological Properties of the Recombinant Protein and Identification of a T-Cell Epitope
Authors: Asahi Hiroko , Osman Ahmed , Cook Rosemary M. , LoVerde Philip T. , Stadecker Miguel J.
Published: July 2002

In schistosomiasis mansoni, hepatic granulomatous inflammation surrounding parasite eggs is mediated by CD4+T helper (Th) cells sensitized to schistosomal egg antigens (SEA). We previously showed that a prominent lymphoproliferative response of CD4+Th cells from schistosome-infected C57BL/6 (BL/6) mice was directed against a 62-kDa component of SEA. A partial amino acid sequence of the 62-kDa component was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Based on this sequence, a cDNA clone containing the entire coding region of PEPCK was identified, and the full recombinantSchistosoma mansoniPEPCK (rSm-PEPCK) of 626 amino acids was purified from a prokaryotic expression system. rSm-PEPCK strongly stimulated a specific T-cell hybridoma, 4E6, as well as CD4+Th cells from SEA-immunized BL/6 mice and from infected BL/6, CBA, and BALB/c mice. In the infected mice, rSm-PEPCK elicited significant gamma interferon production as well as, to a lesser extent, production of interleukin-2 and -5. In BL/6 and BALB/c mice, the CD4+Th cell response to rSm-PEPCK was greater than that directed against the egg antigen Sm-p40; conversely, CBA mice responded better to Sm-p40 than to Sm-PEPCK. A 12-amino-acid region (residues 398 to 409: DKSKDPKAHPNS) was demonstrated to contain a T-cell epitope; synthetic peptides containing this epitope significantly stimulated specific hybridoma 4E6 and polyclonal CD4+Th cells. The identification and characterization of immunogenic egg components will contribute to the understanding and possible control of T-cell-mediated schistosomal disease.
In Vivo Administration of Mycobacterial Cord Factor (Trehalose 6,6′-Dimycolate) Can Induce Lung and Liver Granulomas and Thymic Atrophy in Rabbits
Authors: Hamasaki Naoko , Isowa Ko-Ichi , Kamada Kohachi , Terano Yoshitake , Matsumoto Takayuki , Arakawa Tetsuo , Kobayashi Kazuo , Yano Ikuya
Published: July 2002

Trehalose 6,6′-dimycolate (TDM) is a cell surface molecule of
Mycobacterium tuberculosis
. TDM induced a loss of body weight and prominent granulomas in the liver and lungs by the intravenous injection of TDM into rabbits. TDM also induced atrophy of the thymus and spleen due to apoptosis. By contrast, sulfolipid (2,3,6,6′-tetraacyl trehalose 2′-sulfate) induced neither toxicity, nor granuloma formation, nor atrophy of the thymus and spleen. In rabbits the histopathological changes were more dramatic than in mice. The rabbit model may be more sensitive and may provide more information on the beneficial or pathological effects of TDM.
Ruminant Gastrointestinal Cell Proliferation and Clearance of
Escherichia coli
O157:H7
Authors: Magnuson Bernadene A. , Davis Margaret , Hubele Suzanna , Austin Paula R. , Kudva Indira T. , Williams Christopher J. , Hunt Carl W. , Hovde Carolyn J.
Published: July 2002

Human infections with
Escherichia coli
O157:H7 cause hemorrhagic colitis that can progress to a life-threatening sequelae. The most common mode of disease transmission is ingestion of contaminated bovine food products, and it is well established that
E. coli
O157:H7 is a transient member of the bovine microbiota. However, the conditions that induce acquisition and subsequent clearance of this bacterium from the ruminant gastrointestinal tract (GIT) are not understood. Evidence that the rates of epithelial cell proliferation in the lower GIT of cattle are associated with the duration animals remained
E. coli
O157:H7 culture positive is presented. Cattle with slower rates of intestinal cell proliferation in the cecum and the distal colon were culture positive significantly longer than cohort cattle with faster cell proliferation rates. Cell death rates (apoptotic indices) between the short- and long-term culture-positive animals were not different. Typical grain-based finishing diets and forage-based growing diets did not effect GIT cell proliferation or the duration animals remained
E. coli
O157:H7 culture positive. To identify a dietary intervention that would effect GIT cell proliferation, we used sheep as a model ruminant. A fasting-refeeding regime that increased the rate of GIT cell proliferation was developed. The fasting-refeeding protocol was used in cattle to test the hypothesis that feeding interventions that increase the rate of GIT cell proliferation induce the clearance of
E. coli
O157:H7 from the bovine GIT.
Clostridium perfringens
Iota-Toxin Requires Activation of Both Binding and Enzymatic Components for Cytopathic Activity
Authors: Gibert Maryse , Petit Laetitia , Raffestin Stephanie , Okabe Akinobu , Popoff Michel R.
Published: July 2002

Iota-toxin is produced by
Clostridium perfringens
type E strains and consists of two independent components, the enzymatic and binding components, referred to as Ia and Ib, respectively. A recombinant
C. perfringens
strain, strain 667/pMRP147, produced processed Ia and partially processed Ib, while a recombinant
C. perfringens
type A strain, strain TS133/pMRP147, in which the VirR-VirS two-component system is inactivated, produced only precursor forms of Ia and Ib. This suggests that iota-toxin is processed by a VirR-VirS-responsive protease, although not completely in the recombinant type A strain. The precursor forms of Ia and Ib were purified from cultures of the latter strain, and their proteolytic activation was examined. Treatment with proteases cleaved off small peptides (9 to 13 amino acid residues) and a 20-kDa peptide from the N termini of the Ia and Ib precursors, respectively, leading to their active forms. They were activated efficiently by α-chymotrypsin, pepsin, proteinase K, subtilisin, and thermolysin but only weakly by trypsin, as demonstrated by the cell-rounding assay. λ-Protease from the
C. perfringens
type E strain, which was found to be a zinc-dependent protease related to thermolysin, activated iota-toxin as efficiently as did α-chymotrypsin. These results suggest that λ-protease is most responsible for the activation of iota-toxin in type E strains.
Low Interleukin-12 Activity in SeverePlasmodium falciparumMalaria
Authors: Luty Adrian J. F. , Perkins Douglas J. , Lell Bertrand , Schmidt-Ott Ruprecht , Lehman Leopold G. , Luckner Doris , Greve Bernhard , Matousek Peter , Herbich Klaus , Schmid Daniela , Weinberg J. Brice , Kremsner Peter G.
Published: July 2002

We compared interleukin-12 (IL-12) and other cytokine activities during and after an acute clinical episode in a matched-pair case-control study of young African children who presented with either mild or severePlasmodium falciparummalaria. The acute-phase, pretreatment plasma IL-12 and alpha interferon (IFN-α) levels, as well as the acute-phase mitogen-stimulated whole-blood production capacity of IL-12, were significantly lower in children with severe rather than mild malaria. IL-12 levels, in addition, showed strong inverse correlations both with parasitemia and with the numbers of circulating malaria pigment-containing neutrophils. Acute-phase plasma tumor necrosis factor (TNF) and IL-10 levels were significantly higher in those with severe malaria, and the concentrations of both of these cytokines were positively correlated both with parasitemia and with the numbers of pigment-containing phagocytes in the blood. Children with severe anemia had the highest levels of TNF in plasma. In all the children, the levels in plasma and production capacities of all cytokines normalized when they were healthy and parasite free. The results indicate that severe but not mildP. falciparummalaria in young, nonimmune African children is characterized by down-regulated IL-12 activity, contrasting markedly with the up-regulation of both TNF and IL-10 in the same children. A combination of disturbed phagocyte functions resulting from hemozoin consumption, along with reduced IFN-γ responses, may contribute to these differential effects.
Identification and Subcellular Localization of the
Legionella pneumophila
IcmX Protein: a Factor Essential for Establishment of a Replicative Organelle in Eukaryotic Host Cells
Authors: Matthews Miguelina , Roy Craig R.
Published: July 2002

The gram-negative respiratory pathogen
Legionella pneumophila
infects and grows within mammalian macrophages and protozoan host cells. Upon uptake into macrophages,
L. pneumophila
establishes a replicative organelle that avoids fusion with endocytic vesicles. There are 24
dot/icm
genes on the
L. pneumophila
chromosome required for biogenesis of this vacuole. Many of the Dot/Icm proteins are predicted to be components of a membrane-bound secretion apparatus similar to type IV conjugal transfer systems. We have been investigating the function of
L. pneumophila dot/icm
gene products that do not have obvious orthologs in other type IV transfer systems, since these determinants could govern processes unique to phagosome biogenesis. The
icmX
gene product falls into this category. To understand the role of the IcmX protein in pathogenesis, we have detailed interactions between an
L. pneumophila icmX
deletion mutant and murine bone marrow-derived macrophages. These data demonstrate that
icmX
is required for biogenesis of the
L. pneumophila
replicative organelle. Immunoblot analysis indicates that the
icmX
gene product is a polypeptide with an estimated molecular mass of 50 kDa. The IcmX protein was localized to the bacterial periplasm, and periplasmic translocation was mediated by an N-terminal
sec
-dependent leader peptide. A truncated IcmX product was secreted into culture supernatants by wild-type
L. pneumophila
growing extracellularly in liquid media; however, transport of the IcmX protein into eukaryotic host cells was not detected. Proteins similar in molecular weight to IcmX were identified in other
Legionella
species by immunoblot analysis using a monoclonal antibody specific for
L. pneumophila
IcmX protein. From these data, we conclude that the IcmX protein is an essential component of the
dot/icm
secretion apparatus, and that a conserved mechanism of host cell parasitism exists for members of the
Legionellaceae
family.
The Human Cationic Antimicrobial Protein (hCAP-18) Is Expressed in the Epithelium of Human Epididymis, Is Present in Seminal Plasma at High Concentrations, and Is Attached to Spermatozoa
Authors: Malm Johan , Sørensen Ole , Persson Terese , Frohm-Nilsson Margareta , Johansson Bengt , Bjartell Anders , Lilja Hans , Ståhle-Bäckdahl Mona , Borregaard Niels , Egesten Arne
Published: July 2002

Innate immunity is important for the integrity of the host against potentially invasive pathogenic microorganisms in the environment. Antibiotic peptides with broad antimicrobial activity are part of the innate immune system. We investigated the presence of the cathelicidin, human cationic antimicrobial protein (hCAP-18), in the male reproductive system. We found strong expression of the hCAP-18 gene by in situ hybridization and hCAP-18 protein, as detected by immunohistochemistry, in the epithelium of the epididymis, but not in the testis. The highest expression in the epididymis was in the caudal part. Western blotting showed a doublet band, the upper part corresponding to the size of hCAP-18 in plasma and neutrophils. Using a specific enzyme-linked immunosorbent assay (ELISA), levels of 86.5 ± 37.8 μg/ml (mean ± standard deviation; range, 41.8 to 142.8 μg/ml;n= 10) were detected in seminal plasma from healthy donors, which is 70-fold higher than the level in blood plasma. Flow cytometry and immunocytochemistry revealed the presence of hCAP-18 on spermatozoa. ELISA measurement showed levels of 196 ng/106spermatozoa, corresponding to 6.6 × 106molecules of hCAP-18 per spermatozoon. Our results suggest a key role for hCAP-18 in the antibacterial integrity of the male reproductive system. The attachment of hCAP-18 to spermatozoa may implicate a role for hCAP-18 in conception.
Aggregation Substance Promotes Adherence, Phagocytosis, and Intracellular Survival ofEnterococcus faecaliswithin Human Macrophages and Suppresses Respiratory Burst
Authors: Süßmuth Sigurd D. , Muscholl-Silberhorn Albrecht , Wirth Reinhard , Susa Milorad , Marre Reinhard , Rozdzinski Eva
Published: July 2002

The aggregation substance (AS) ofEnterococcus faecalis, encoded on sex pheromone plasmids, is a surface-bound glycoprotein that mediates aggregation between bacteria thereby facilitating plasmid transfer. Sequencing of the pAD1-encoded Asa1 revealed that this surface protein contains two RGD motifs which are known to ligate integrins. Therefore, we investigated the influence of AS on the interaction ofE. faecaliswith human monocyte-derived macrophages which constitutively express β2integrins (e.g., CD18). AS was found to cause a greater-than-fivefold increase in enterococcal adherence to macrophages and a greater-than-sevenfold increase in phagocytosis. Adherence was mediated by an interaction between the RGD motif and the integrin CD11b/CD18 (complement receptor type 3) as demonstrated by inhibition studies with monoclonal antibodies and RGD peptide. AS-bearing enterococci were significantly more resistant to macrophage killing during the first 3 h postinfection, probably due to inhibition of the respiratory burst as indicated by reduced concentrations of superoxide anion.
Role of Ribosomal Protein L12 in Gonococcal Invasion of Hec1B Cells
Authors: Spence Janice M. , Clark Virginia L.
Published: July 2002

Previous studies led to the development of a model of contact-induced enhanced gonococcal invasion of human reproductive cells that utilizes the lutropin receptor (LHr) as both the induction signal for conversion to this enhanced-gonococcal-invasion phenotype (Inv
+
GC) and as the specific Inv
+
GC uptake mechanism. This model proposes that gonococci express a surface feature that mimics human chorionic gonadotropin (hCG), the cognate ligand for LHr, and that this structure is responsible for the specific and productive interaction of GC with LHr. In this report, we identify a 13-kDa gonococcal protein with immunological similarities to hCG. The antiserum reactivity is specific since interaction with the 13-kDa gonococcal protein can be blocked by the addition of highly purified hCG. This gonococcal “hCG-like” protein, purified from two-dimensional gels and by immunoprecipitation, was determined by N-terminal sequencing to be the ribosomal protein L12. We present evidence that gonococcal L12 is membrane associated and surface exposed in gonococci, as shown by immunoblot analysis of soluble and insoluble gonococcal protein and antibody adsorption studies with fixed GC. Using highly purified recombinant gonococcal L12, we show that preincubation of Inv
−
GC with micromolar amounts of rL12 leads to a subsequent five- to eightfold increase in invasion of the human endometrial cell line, Hec1B. In addition, nanomolar concentrations of exogenous L12 inhibits gonococcal invasion to approximately 70% of the level in controls. Thus, we propose a novel cellular location for the gonococcal ribosomal protein L12 and concomitant function in LHr-mediated gonococcal invasion of human reproductive cells.
Enhancement of Neonatal Innate Defense: Effects of Adding an N-Terminal Recombinant Fragment of Bactericidal/Permeability-Increasing Protein on Growth and Tumor Necrosis Factor-Inducing Activity of Gram-Negative Bacteria Tested in Neonatal Cord Blood Ex Vivo
Authors: Levy Ofer , Sisson Richard B. , Kenyon Jonathan , Eichenwald Eric , Macone Ann B. , Goldmann Donald
Published: July 2002

Innate defense against microbial infection requires the action of neutrophils, which have cytoplasmic granules replete with antibiotic proteins and peptides. Bactericidal/permeability-increasing protein (BPI) is found in the primary granules of adult neutrophils, has a high affinity for lipopolysaccharides (or “endotoxins”), and exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria. We have previously reported that neutrophils derived from newborn cord blood are deficient in BPI (O. Levy et al., Pediatrics 104:1327–1333, 1999). The relative deficiency in BPI of newborns raised the possibility that supplementing the levels of BPI in plasma might enhance newborn antibacterial defense. Here we determined the effects of addition of recombinant 21-kDa N-terminal BPI fragment (rBPI
21
) on the growth and tumor necrosis factor (TNF)-inducing activity of representative gram-negative clinical isolates. Bacteria were tested in citrated newborn cord blood or adult peripheral blood. Bacterial viability was assessed by plating assay, and TNF-α release was measured by enzyme-linked immunosorbent assay. Whereas adult blood limited the growth of all isolates except
Klebsiella pneumoniae
, cord blood also allowed logarithmic growth of
Escherichia coli
K1/r and
Citrobacter koseri
. Bacteria varied in their susceptibility to rBPI
21
's bactericidal action:
E. coli
K1/r was relatively susceptible (50% inhibitory concentration [IC
50
], ∼10 nM),
C. koseri
was intermediate (IC
50
, ∼1,000 nM),
Klebsiella pneumoniae
was resistant (IC
50
, ∼10,000 nM), and
Enterobacter cloacae
and
Serratia marcescens
were highly resistant (IC
50
, >10,000 nM). All isolates were potent inducers of TNF-α activity in both adult and newborn cord blood. In contrast to its variable antibacterial activity, rBPI
21
consistently inhibited the TNF-inducing activity of all strains tested (IC
50
, 1 to 1,000 nM). The antibacterial effects of rBPI
21
were additive with those of a combination of conventional antibiotics typically used to treat bacteremic newborns (ampicillin and gentamicin). Whereas ampicillin and gentamicin demonstrated little inhibition of bacterially induced TNF release, addition of rBPI
21
either alone or together with ampicillin and gentamicin profoundly inhibited release of this cytokine. Thus, supplementing newborn cord blood with rBPI
21
potently inhibited the TNF-inducing activity of a variety of gram-negative bacterial clinical pathogens and, in some cases, enhanced bactericidal activity. These results suggest that administration of rBPI
21
may be of clinical benefit to neonates suffering from gram-negative bacterial infection and/or endotoxemia.
Effect ofHelicobacter pylorion Polymorphonuclear Leukocyte Migration across Polarized T84 Epithelial Cell Monolayers: Role of Vacuolating Toxin VacA andcagPathogenicity Island
Authors: Hofman Véronique , Ricci Vittorio , Galmiche Antoine , Brest Patrick , Auberger Patrick , Rossi Bernard , Boquet Patrice , Hofman Paul
Published: July 2002

Helicobacter pyloriinfection can induce polymorphonuclear leukocyte (PMNL) infiltration of the gastric mucosa, which characterizes acute chronic gastritis. The mechanisms underlying this process are poorly documented. The lack of an in vitro model has considerably impaired the study of transepithelial migration of PMNL induced byH. pylori. In the present work, we used confluent polarized monolayers of the human intestinal cell line T84 grown on permeable filters to analyze the epithelial PMNL response induced by broth culture filtrates (BCFs) and bacterial suspensions from different strains ofH. pylori. We have evaluated the role of the vacuolating cytotoxin VacA and of thecagpathogenicity island (PAI) ofH. pyloriin PMNL migration via their effects on T84 epithelial cells. We noted no difference in the rates of PMNL transepithelial migration after epithelial preincubation with bacterial suspensions or with BCFs of VacA-negative or VacA-positiveH. pyloristrains. In contrast, PMNL transepithelial migration was induced after incubation of the T84 cells withcagPAI-positive andcagE-positiveH. pyloristrains. Finally, PMNL migration was correlated with a basolateral secretion of interleukin-8 by T84 cells, thus creating a subepithelial chemotactic gradient for PMNL. These data provide evidence that the vacuolating cytotoxin VacA is not involved in PMNL transepithelial migration and that thecagPAI, with a pivotal role for thecagEgene, provokes a transcellular signal across T84 monolayers, inducing a subepithelial PMNL response.
Evidence for Contribution of Tripartite Hemolysin BL, Phosphatidylcholine-Preferring Phospholipase C, and Collagenase to Virulence of
Bacillus cereus
Endophthalmitis
Authors: Beecher Douglas J. , Olsen Timothy W. , Somers Eileen B. , Wong Amy C. L.
Published: July 2002

Bacillus cereus
causes a highly fulminant endophthalmitis which usually results in blindness. We previously concluded that hemolysin BL (HBL), a tripartite necrotizing pore-forming toxin, is a probable endophthalmitis virulence factor because it is highly toxic to retinal tissue in vitro and in vivo. We also determined that
B. cereus
produces additional retinal toxins that might contribute to virulence. Here we fractionated crude
B. cereus
culture supernatant by anion-exchange chromatography and found that in vitro retinal toxicity was also associated with phosphatidylcholine-preferring phospholipase C (PC-PLC). The pure enzyme also caused retinal necrosis in vivo. We showed that phosphatidylinositol-specific PLC and sphingomyelinase were nontoxic and that two hemolysins, cereolysin O and a novel hemolysin designated hemolysin IV, were marginally toxic in vitro. The histopathology of experimental septic endophthalmitis in rabbits mimicked the pathology produced by pure HBL, and both HBL and PC-PLC were detected at toxic concentrations in infected vitreous fluid. Bacterial cells were first seen associated with the posterior margin of the lens and eventually were located throughout the lens cortex. Detection of collagenase in the vitreous humor suggested that infiltration was facilitated by the breakdown of the protective collagen lens capsule by that enzyme. This work supports our conclusion that HBL contributes to
B. cereus
virulence and implicates PC-PLC and collagenase as additional virulence factors.
Locus of Enterocyte Effacement from
Citrobacter rodentium
: Sequence Analysis and Evidence for Horizontal Transfer among Attaching and Effacing Pathogens
Authors: Deng Wanyin , Li Yuling , Vallance Bruce A. , Finlay B. Brett
Published: July 2002

The family of attaching and effacing (A/E) bacterial pathogens, which includes diarrheagenic enteropathogenic
Escherichia coli
(EPEC) and enterohemorrhagic
E
.
coli
(EHEC), remains a significant threat to human and animal health. These bacteria intimately attach to host intestinal cells, causing the effacement of brush border microvilli. The genes responsible for this phenotype are encoded in a pathogenicity island called the locus of enterocyte effacement (LEE).
Citrobacter rodentium
is the only known murine A/E pathogen and serves as a small animal model for EPEC and EHEC infections. Here we report the full DNA sequence of
C
.
rodentium
LEE and provide a comparative analysis with the published LEEs from EPEC, EHEC, and the rabbit diarrheagenic
E
.
coli
strain RDEC-1. Although
C
.
rodentium
LEE shows high similarities throughout the entire sequence and shares all 41 open reading frames with the LEE from EPEC, EHEC, and RDEC-1, it is unique in its location of the
rorf1
and
rorf2/espG
genes and the presence of several insertion sequences (IS) and IS remnants. The LEE of EPEC and EHEC is inserted into the
selC
tRNA gene. In contrast, the
Citrobacter
LEE is flanked on one side by an operon encoding an ABC transport system, and an IS element and sequences homologous to
Shigella
plasmid R100 and EHEC pO157 flank the other. The presence of plasmid sequences next to
C
.
rodentium
LEE suggests that the prototype LEE resided on a horizontally transferable plasmid. Additional sequence analysis reveals that the 3-kb plasmid in
C
.
rodentium
is nearly identical to p9705 in EHEC O157:H7, suggesting that horizontal plasmid transfer among A/E pathogens has occurred. Our results indicate that the LEE has been acquired by
C
.
rodentium
and A/E
E
.
coli
strains independently during evolution.
Patterns of Chemokine Expression in Models ofSchistosoma mansoniInflammation and Infection Reveal Relationships between Type 1 and Type 2 Responses and Chemokines In Vivo
Authors: Park Matthew K. , Hoffmann Karl F. , Cheever Allen W. , Amichay Doron , Wynn Thomas A. , Farber Joshua M.
Published: July 2002

To explore the roles of chemokines in type 1 and type 2 responses in vivo, we examined mRNA expression for a panel of up to 17 chemokines in experimental mouse models usingSchistosoma mansoni. These studies revealed that Mig (monokine induced by gamma interferon), cytokine-responsive gene 2/10-kDa interferon-inducible protein, RANTES, lymphotactin, macrophage inflammatory protein 1β (MIP-1β), JE/monocyte chemoattractant protein 1, and MIP-2 are associated with type 1 egg-induced responses and that thymus-derived chemotactic agent 3 (TCA3), eotaxin, MIP-1α, and MIP-1γ are associated with type 2 egg-induced responses. After cercarial infection, both type 1-associated and type 2-associated chemokines were elevated in the livers of infected mice presensitized with eggs and recombinant interleukin-12 (rIL-12), a regimen that diminishes pathology. Neutralization of IL-12 or gamma interferon during egg deposition reversed the effects of prior treatment with rIL-12, leading to a return to larger granulomas; persistently elevated expression of TCA3, eotaxin, and MIP-1α; and a marked reduction in the expression of type 1-associated chemokines despite the maintenance of a dominant type 1 cytokine response in the draining lymph nodes. Our findings suggest that there are patterns of coordinate chemokine expression characteristic of type 1 and type 2 responses in vivo; that the cells recruited by a given pattern of chemokines may differ, depending on the composition of peripheral populations; and that patterns of tissue expression of chemokines may determine the character of an inflammatory response independently of the dominant pattern of differentiation of antigen-specific T cells. Our data reveal new relationships between chemokines and polarized immune responses and suggest that end organ inflammation might be altered by chemokine blockade without necessitating reversal of the phenotype of the majority of differentiated T cells.