bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and Evaluation of an In-House Enzyme-Linked Immunosorbent Assay for Early Diagnosis and Monitoring of Human Pythiosis
Authors: Krajaejun Theerapong , Kunakorn Mongkol , Niemhom Sopaporn , Chongtrakool Piriyaporn , Pracharktam Roongnapa
Published: July 2002

Human pythiosis is an emerging, fatal, infectious disease caused by
Pythium insidiosum
and occurs in both tropical and subtropical countries. Thalassemic patients, farmers, and aquatic-habitat residents are predisposed to this disease. Delayed treatment due to the long time required for isolation and identification of the causative organism, as well as the difficulty in obtaining internal organ specimens, results in high morbidity and mortality. To facilitate rapid diagnosis, an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G antibodies against
P. insidiosum
was developed and evaluated for the diagnosis and monitoring of human pythiosis. Sixteen sera were collected from seven culture-proven human pythiosis cases. A total of 142 sera from thalassemic patients, from patients with other infectious diseases, and from healthy blood donors served as controls. All sera were tested in duplicate. By choosing a suitable cutoff point to maximize sensitivity and specificity, sera from pythiosis cases were all determined to be positive, whereas sera from control groups were all determined to be negative. ELISA signals from serial samples of sera taken from treated patients showed gradually declining levels of antibodies to
P. insidiosum
. The ELISA test was highly sensitive (100%) and specific (100%) and was useful for early diagnosis and for monitoring the treatment for pythiosis.
Newly Characterized Species-Specific ImmunogenicChlamydophila pneumoniaePeptide Reactive with Murine Monoclonal and Human Serum Antibodies
Authors: Marston Eric L. , James Andrea V. , Parker J. Todd , Hart John C. , Brown Teresa M. , Messmer Trudy O. , Jue Danny L. , Black Carolyn M. , Carlone George M. , Ades Edwin W. , Sampson Jacquelyn
Published: July 2002

A monoclonal antibody (MAb) directed against an unknownChlamydophila pneumoniaeepitope has been characterized, and the respective peptide mimotope has been identified. A murine MAb specific forC.pneumoniaewas used to select peptides from phage display libraries. The peptides identified from the phage display library clones reacted specifically with the respective target murine MAb and with human sera previously identified as having antibody titers toC. pneumoniae. The selected peptide mimotope sequences tended to be composed of charged residues surrounding a core of hydrophobic residues. The peptide with the best binding could inhibit >95% of binding to the MAb, suggesting that the selected peptide binds the paratope of the respective MAb. The peptide reacted with human sera previously determined by microimmunofluorescence to have anti-C. pneumoniaeantibodies. The peptide was competitively competed with the MAb against Renografin-purified, sonicatedC. pneumoniaein an enzyme-linked immunosorbent assay and with whole-cellC. pneumoniaein an indirect fluorescence assay format, demonstrating its potential utility in the development of diagnostics. The use of this novel peptide may allow investigators to establish standardized assays free from cross-reactiveChlamydia trachomatisandChlamydophila psittaciepitopes and immunoreactivity.
Expression of Mucosal Homing Receptor α4β7 Is Associated with Enhanced Migration to theChlamydia-Infected Murine Genital Mucosa In Vivo
Authors: Hawkins Raymond A. , Rank Roger G. , Kelly Kathleen A.
Published: July 2002

The CD4 T helper cell type 1 (Th1) response is essential for the resolution of chlamydial genital infection in mice. However, not all Th1 clones are equally protective in eradicating the infection. Since oral immunization regimens produce protective immunity, we evaluated the role of the mucosa-associated homing receptor, α4β7, in trafficking to the genital mucosa. Using a panel of CD4, Th1 cell lines and clones, we compared the lymphocyte homing patterns of aChlamydia-specific, protective clone (P-MoPn), a nonprotective clone (N-MoPn), and a keyhole limpet hemocyanin (KLH)-specific cell line (KLH-1). T cells were labeled with the fluorescent dye PKH-26, adoptively transferred intoChlamydia-infected mice, and monitored at different time points throughout the course of a genital infection. We found that clones P-MoPn and N-MoPn migrated to similar extents to the genital tract and in significantly greater numbers than the KLH-specific T-cell line. Both clones and the KLH-1 line expressed similar levels of the adhesion molecules α4, β1, CD44, and CD11a. However, clones P-MoPn and N-MoPn expressed higher levels of the mucosal homing receptor, α4β7. Also, clones P-MoPn and N-MoPn but not the KLH-1 line migrated to the mesenteric lymph node, suggesting a mucosal recirculation pattern. Moreover, blocking α4β7 adhesion interaction in vivo significantly reduced the recruitment of P-MoPn but not KLH-1 to the genital tract. These findings show that the mucosal homing receptor α4β7 is utilized by a subset of CD4 cells during migration to theChlamydia-infected genital tract.
Infection of Human Endothelial Cells withBartonella bacilliformisIs Dependent on Rho and Results in Activation of Rho
Authors: Verma Anita , Davis George E. , Ihler Garret M.
Published: July 2002

Bartonella bacilliformiswas continuously internalized into human endothelial cells beginning shortly after addition of the bacteria and continuing for at least 24 h after infection in vitro, with a major increase in uptake occurring between 16 and 24 h. Preincubation of endothelial cells with C3 exoenzyme, which inactivated intracellular Rho-GTPase, blocked internalization of the bacteria. Addition of C3 exoenzyme at any time after addition of the bacteria blocked further internalization of bacteria, including the major uptake of bacteria internalized at 16 to 24 h. Rho, a key signaling protein in pathways involving actin organization, was directly shown to be activated in endothelial cells undergoing infection withB. bacilliformis, with maximal activation and translocation to the plasma membrane at 12 to 16 h. At late times of infection, most of the bacteria were found in a perinuclear location. Staining of the Golgi complex with specific markers, anti-human Golgin-97, anti-KDEL receptor, and BODIPY-TR ceramide, showed colocalization of bacteria in the Golgi complex region. Disruption of the Golgi complex with brefeldin A scattered the bacteria from this perinuclear location and resulted in inhibition of internalization of the bacteria in endothelial cells.
Pseudomonas aeruginosa
ExoT Is a Rho GTPase-Activating Protein
Authors: Krall Rebecca , Schmidt Gudula , Aktories Klaus , Barbieri Joseph T.
Published: July 2002

Transient intracellular expression of ExoT in CHO cells stimulated cell rounding and actin reorganization. Biochemical studies showed that ExoT was a GTPase-activating protein for RhoA, Rac1, and Cdc42. Together, these data show that ExoT interferes with Rho signal transduction pathways, which regulate actin organization, exocytosis, cell cycle progression, and phagocytosis.
Multiple Roles forBordetellaLipopolysaccharide Molecules during Respiratory Tract Infection
Authors: Harvill Eric T. , Preston Andrew , Cotter Peggy A. , Allen Andrew G. , Maskell Duncan J. , Miller Jeff F.
Published: July 2002

Bordetella pertussis,Bordetella parapertussis, andBordetella bronchisepticaare closely related subspecies that cause respiratory tract infections in humans and other mammals and express many similar virulence factors. Their lipopolysaccharide (LPS) molecules differ, containing either a complex trisaccharide (B. pertussis), a trisaccharide plus an O-antigen-like repeat (B. bronchiseptica), or an altered trisaccharide plus an O-antigen-like repeat (B. parapertussis). Deletion of thewlblocus results in the loss of membrane-distal polysaccharide domains in the three subspecies of bordetellae, leaving LPS molecules consisting of lipid A and core oligosaccharide. We have usedwlbdeletion (Δwlb) mutants to investigate the roles of distal LPS structures in respiratory tract infection by bordetellae. Each mutant was defective compared to its parent strain in colonization of the respiratory tracts of BALB/c mice, but the location in the respiratory tract and the time point at which defects were observed differed significantly. Although the Δwlbmutants were much more sensitive to complement-mediated killing in vitro, they displayed similar defects in respiratory tract colonization in C5−/−mice compared with wild-type (wt) mice, indicating that increased sensitivity to complement-mediated lysis is not sufficient to explain the in vivo defects.B. pertussisandB. parapertussis Δwlbmutants were also defective compared to wt strains in colonization of SCID-beige mice, indicating that the defects were not limited to interactions with adaptive immunity. Interestingly, theB. bronchiseptica Δwlbstrain was defective, compared to the wt strain, in colonization of the respiratory tracts of BALB/c mice beginning 1 week postinoculation but did not differ from the wt strain in its ability to colonize the respiratory tracts of B-cell- and T-cell-deficient mice, suggesting thatwlb-dependent LPS modifications inB. bronchisepticamodulate interactions with adaptive immunity. These data show that biosynthesis of a full-length LPS molecule by these three bordetellae is essential for the expression of full virulence for mice. In addition, the data indicate that the different distal structures modifying the LPS molecules on these three closely related subspecies serve different purposes in respiratory tract infection, highlighting the diversity of functions attributable to LPS of gram-negative bacteria.
Involvement of CD14 and β2-Integrins in Activating Cells with Soluble and Particulate Lipopolysaccharides and Mannuronic Acid Polymers
Authors: Flo Trude H. , Ryan Liv , Kilaas Lars , Skjåk-Bræk Gudmund , Ingalls Robin R. , Sundan Anders , Golenbock Douglas T. , Espevik Terje
Published: July 2002

Lipopolysaccharide (LPS) and related bacterial products can be recognized by host inflammatory cells in a particulate, bacterium-bound form, as well as in various soluble, released forms. In the present study we have compared the mechanisms used by LPS, detoxified LPS (DLPS), and mannuronic acid polymers (M-polymers), in solution or covalently linked to particles, in stimulating monocytes to tumor necrosis factor (TNF) production. The addition of recombinant LPS binding protein (LBP) and/or soluble CD14 (sCD14) enhanced the production of TNF from monocytes stimulated with soluble LPS, DLPS, or M-polymer, but did not affect the response to M-polymer or DLPS attached to particles. Treatment of monocytes with antibody to CD14, CD18, or CD11b showed that CD14, but not CR3 (CD11b/CD18), mediated monocyte TNF production in response to the soluble antigens. In contrast, anti-CD14, anti-CD11b and anti-CD18 monoclonal antibodies all inhibited the response to the particulate stimuli. On the other hand, B975, a synthetic analog ofRhodobacter capsulatuslipid A, completely abrogated the monocyte TNF response induced by LPS but did not affect the TNF induction by DLPS or M-polymer, either in soluble or particulate forms. These data demonstrate that the engagement of immune receptors by bacterial products such as LPS, DLPS, and M-polymer is dependent upon the presentation form of their constituent carbohydrates, and that factors such as aggregation state, acylation, carbohydrate chain length, and solid versus liquid phase of bacterial ligands influence the mechanisms used by cells in mediating proinflammatory responses.
Impaired Bone Resorption by Lipopolysaccharide In Vivo in Mice Deficient in the Prostaglandin E Receptor EP4 Subtype
Authors: Sakuma Yoko , Tanaka Kiyoshi , Suda Michio , Komatsu Yasato , Yasoda Akihiro , Miura Masako , Ozasa Ami , Narumiya Shuh , Sugimoto Yukihiko , Ichikawa Atsushi , Ushikubi Fumitaka , Nakao Kazuwa
Published: July 2002

In a previous study we showed that the involvement of EP4 subtype of the prostaglandin E (PGE) receptor is crucial for lipopolysaccharide (LPS)-induced osteoclast formation in vitro. The present study was undertaken to test whether EP4 is actually associated with LPS-induced bone resorption in vivo. In wild-type (WT) mice, osteoclast formation in vertebrae and tibiae increased 5 days after systemic LPS injection, and urinary excretion of deoxypyridinoline, a sensitive marker for bone resorption, statistically increased 10 days after injection. In EP4 knockout (KO) mice, however, LPS injection caused no significant changes in these parameters throughout the experiment. LPS exposure for 4 h strongly induced osteoclast differentiation factor (ODF) mRNA expression in primary osteoblastic cells (POB) both from WT and EP4 KO mice, and this expression was not inhibited by indomethacin, suggesting prostaglandin (PG) independence. LPS exposure for 24 h further induced ODF expression in WT POB, but not in EP4 KO POB. Indomethacin partially inhibited ODF expression in WT POB, but not in EP4 KO POB. These data suggest that ODF is induced both PG dependently and PG independently. LPS exposure for 24 h induced slightly greater osteoclastgenesis inhibitory factor (OCIF) mRNA expression in EP4 KO than in WT POB. These findings suggest that the reduced ODF expression and apparently increased OCIF expression also are responsible for the markedly reduced LPS-induced osteoclast formation in EP4 KO mice. Our results show that the EP4 subtype of the PGE receptor is involved in LPS-induced bone resorption in vivo also. Since LPS is considered to be largely involved in bacterially induced bone loss, such as in periodontitis and osteomyelitis, our study is expected to help broaden our understanding of the pathophysiology of these conditions.
mig-14Is a Horizontally Acquired, Host-Induced Gene Required forSalmonella entericaLethal Infection in the Murine Model of Typhoid Fever
Authors: Valdivia Raphael H. , Cirillo Daniela M. , Lee Anthea K. , Bouley Donna M. , Falkow Stanley
Published: July 2002

We have characterized a host-induced virulence gene,mig-14, that is required for fatal infection in the mouse model of enteric fever.mig-14is present in allSalmonella entericasubspecies I serovars and maps to a region of the chromosome that appears to have been acquired by horizontal transmission. Amig-14mutant replicated in host tissues early after infection but was later cleared from the spleens and livers of infected animals. Bacterial clearance by the host occurred concomitantly with an increase in gamma interferon levels and recruitment of macrophages, but few neutrophils, to the infection foci. We hypothesize that themig-14gene product may repress immune system functions by interfering with normal cytokine expression in response to bacterial infections.
Secreted Enzymatic Activities of Wild-Type and
pilD
-Deficient
Legionella pneumophila
Authors: Aragon Virginia , Kurtz Sherry , Flieger Antje , Neumeister Birgid , Cianciotto Nicholas P.
Published: July 2002

Legionella pneumophila
, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the
Legionella pilD
gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by a
pilD
-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of
p
-nitrophenol (pNP) from
p
-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in
pilD
mutant supernatants. Mutant supernatants also did not release either pNP from
p
-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from
p
-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for
L. pneumophila
virulence.
Group B Streptococci and Other Gram-Positive Cocci Bind to Cytokeratin 8
Authors: Tamura Glen S. , Nittayajarn Aphakorn
Published: July 2002

Group B streptococci (GBS) adhere to surface receptors present on epithelial cells; these receptors include fibronectin and laminin. To identify other possible receptors, plasma membranes from A549 cells, a respiratory tract epithelial cell line, were prepared. These plasma membranes were tested in a protein blot analysis using radiolabeled GBS as a probe. GBS adhered to two species, with molecular masses of 50 kDa (p50) and 57 kDa (p57). We concluded that p50 and p57 correspond to two forms of cytokeratin 8 (CK8) on the basis of the following results: (i) protein blot results demonstrated that p50 and p57 exactly comigrated with two forms of CK8 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE); (ii) p50 and p57 exactly comigrated with CK8 after separation by two-dimensional PAGE; (iii) CK8 in solution bound to GBS, as demonstrated by immunoblot analysis of proteins from A549 lysates that bound to GBS in a liquid-phase assay; and (iv) radiolabeled GBS bound to A549 lysate-derived CK8 that had been captured in anti-CK8-coated microtiter wells. CK8 bound to COH1-13, an acapsular mutant of COH1, demonstrating that adherence is not mediated by capsular polysaccharide. Trypsin-treated GBS did not bind to CK8, indicating that adherence is mediated via a protein on the surface of GBS. Soluble CK8 bound to six of six GBS strains tested. Soluble CK8 also bound to
Staphylococcus aureus
,
Lactococcus lactis
,
Enterococcus faecalis
, and
Streptococcus pyogenes
. We hypothesize that adherence of GBS to cytokeratin may be important for maintenance of colonization at sites of keratinized epithelium, such as the vagina, or for adherence of these bacteria to damaged epithelial cells at other sites.
Immune Destruction of LarvalTaenia crassicepsin Mice
Authors: Mooney K. A. , Spolski R. J. , See E. J. , Kuhn R. E.
Published: July 2002

Immune destruction of larvalTaenia crassicepswas examined by first injecting BALB/cJ mice subcutaneously with larval buds and 30 to 60 days later challenging the mice with larvae injected into the peritoneal cavity. The larvae injected intraperitoneally (i.p.) secondarily are killed by host cells that completely encase the larvae in a thick sheath. The peritoneal exudate cells and the cytokines they produced were characterized by flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and reverse transcription PCR (RT-PCR). No changes in percentage of CD4+T cells, CD8+T cells, B1 cells, or macrophages were detected in the peritoneal cavities of mice that were killing larvae compared to mice with a primary 7-day infection i.p. Both RT-PCR and ELISA demonstrated a decrease in cytokines including gamma interferon (IFN-γ), interleukin-4 (IL-4), and IL-10 in mice that were killing the larvae compared to control mice infected for 30 to 60 days i.p. alone, although there was little difference compared to mice infected for 7 days i.p. alone. Serum cytokine levels in mice that were killing the larvae showed a decrease in IFN-γ and IL-4, an increase in IL-10 when compared to mice infected for 30 to 60 days i.p. alone, and increases in all cytokines compared to mice infected for 7 days i.p. alone. Inhibition of nitric oxide production did not significantly affect the number or the viability of larvae in the peritoneal cavity of mice that were killing larvae during secondary infection.
Characterization of an Isogenic Mutant of
Streptococcus pyogenes
Manfredo Lacking the Ability To Make Streptococcal Acid Glycoprotein
Authors: Degnan B. A. , Fontaine M. C. , Doebereiner A. H. , Lee J. J. , Mastroeni P. , Dougan G. , Goodacre J. A. , Kehoe M. A.
Published: July 2002

An isogenic mutant of
Streptococcus pyogenes
Manfredo that lacks the ability to make streptococcal acid glycoprotein (SAGP) has been constructed by inserting a deletion in the
sagp
gene using the method of allelic exchange. An assay of cell extracts (CE) prepared from the wild-type and mutant Manfredo strains for the enzyme arginine deiminase (AD) showed that significant activity was present in wild-type CE but none could be detected in mutant CE. These findings confirm our earlier conclusion that SAGP has AD activity (B. A. Degnan, J. M. Palmer, T. Robson, C. E. D. Jones, M. Fischer, M. Glanville, G. D. Mellor, A. G. Diamond, M. A. Kehoe, and J. A. Goodacre, Infect. Immun. 66:3050–3058, 1998). Wild-type CE but not mutant CE potently inhibited human peripheral blood mononuclear cell proliferation in response to phytohemagglutinin, and this inhibition was overcome by the addition of
l
-arginine to proliferation assay mixtures. Invasion assays showed that the isogenic mutant organisms lacking SAGP, and thus AD activity, were between three and five times less able to enter epithelial cells (Hep-2C and A549) than were the wild-type streptococci. Both wild-type and mutant
S. pyogenes
bacteria were extremely sensitive to low pH. However,
l
-arginine (1 mM or above) significantly increased the viability of the wild type but not the isogenic mutant organisms under acidic conditions. The difference in acid susceptibility between wild-type and mutant bacteria may explain the reduced capacity of the isogenic mutant bacteria to invade and survive intracellularly.
Characterization of Binding of
Candida albicans
to Small Intestinal Mucin and Its Role in Adherence to Mucosal Epithelial Cells
Authors: de Repentigny Louis , Aumont Francine , Bernard Karine , Belhumeur Pierre
Published: July 2002

In order to approximate and adhere to mucosal epithelial cells,
Candida
must traverse the overlying mucus layer. Interactions of
Candida
species with mucin and human buccal epithelial cells (BECs) were thus investigated in vitro. Binding of the
Candida
species to purified small intestinal mucin showed a close correlation with their hierarchy of virulence. Significant differences (
P
< 0.05) were found among three categories of
Candida
species adhering highly (
C. dubliniensis
,
C. tropicalis
, and
C. albicans
), moderately (
C. parapsilosis
and
C. lusitaniae
) or weakly (
C. krusei
and
C. glabrata
) to mucin. Adherence of
C. albicans
to BECs was quantitatively inhibited by graded concentrations of mucin. However, inhibition of adherence was reversed by pretreatment of mucin with pronase or
C. albicans
secretory aspartyl proteinase Sap2p but not with sodium periodate. Saturable concentration- and time-dependent binding of mucin to
C. albicans
was abrogated by pronase or Sap2p treatment of mucin but was unaffected by β-mercaptoethanol, sodium periodate, neuraminidase, lectins, or potentially inhibitory sugars. Probing of membrane blots of the mucin with
C. albicans
revealed binding of the yeast to the 66-kDa cleavage product of the 118-kDa C-terminal glycopeptide of mucin. Although no evidence was found for the participation of
C. albicans
cell surface mannoproteins in specific receptor-ligand binding to mucin, inhibition of binding by
p
-nitrophenol (1 mM) and tetramethylurea (0.36 M) revealed that hydrophobic interactions are involved in adherence of
C. albicans
to mucin. These results suggest that
C. albicans
may both adhere to and enzymatically degrade mucins by the action of Saps, and that both properties may act to modulate
Candida
populations in the oral cavity and gastrointestinal tract.
Induction of a Polarized Th1 Response by Insertion of Multiple Copies of a Viral T-Cell Epitope into Adenylate Cyclase ofBordetella pertussis
Authors: Dadaglio Gilles , Moukrim Zhora , Lo-Man Richard , Sheshko Valeria , Sebo Peter , Leclerc Claude
Published: July 2002

The adenylate cyclase (CyaA) ofBordetella pertussisdelivers the N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells, in particular, professional antigen-presenting cells. This allows the delivery of CD8+T-cell epitopes to the major histocompatibility complex class I presentation pathway. We have previously shown that immunization of mice with CyaA carrying a single CD8+T-cell epitope leads to antiviral protection as well as to protective and therapeutic antitumor immunity associated with the induction of specific cytotoxic T-lymphocyte (CTL) responses. Here, we evaluated the capacity of CyaA carrying one to four copies of the CD8+CD4+T-cell epitope from the nucleoprotein of the lymphocytic choriomeningitis virus to induce T-cell responses. Both CTL and Th1-like specific responses were detected in mice immunized with recombinant CyaA with or without adjuvant. Although the insertion of the larger peptides resulted in partial loss of the invasive capacity of recombinant CyaA, insertion of several copies of the same epitope led to a strong enhancement of Th1 responses and, to a lesser degree, CTL responses. These results underscore the potency of CyaA for vaccine design with a new impact on diseases in which the Th1 response has been described to have a beneficial effect.
Involvement of a Plasmid in Virulence of
Campylobacter jejuni
81-176
Authors: Bacon David J. , Alm Richard A. , Burr Don H. , Hu Lan , Kopecko Dennis J. , Ewing Cheryl P. , Trust Trevor J. , Guerry Patricia
Published: July 2002

Campylobacter jejuni
strain 81-176 contains two, previously undescribed plasmids, each of which is approximately 35 kb in size. Although one of the plasmids, termed pTet, carries a
tetO
gene, conjugative transfer of tetracycline resistance to another strain of
C. jejuni
could not be demonstrated. Partial sequence analysis of the second plasmid, pVir, revealed the presence of four open reading frames which encode proteins with significant sequence similarity to
Helicobacter pylori
proteins, including one encoded by the
cag
pathogenicity island. All four of these plasmid-encoded proteins show some level of homology to components of type IV secretion systems. Mutation of one of these plasmid genes,
comB3
, reduced both adherence to and invasion of INT407 cells to approximately one-third that seen with wild-type strain 81-176. Mutation of
comB3
also reduced the natural transformation frequency. A mutation in a second plasmid gene, a
virB11
homolog, resulted in a 6-fold reduction in adherence and an 11-fold reduction in invasion compared to the wild type. The isogenic
virB11
mutant of strain 81-176 also demonstrated significantly reduced virulence in the ferret diarrheal disease model. The
virB11
homolog was detected on plasmids in 6 out of 58 fresh clinical isolates of
C. jejuni
, suggesting that plasmids are involved in the virulence of a subset of
C. jejuni
pathogens.
Growth ofMycobacterium tuberculosisin a Defined Medium Is Very Restricted by Acid pH and Mg2+Levels
Authors: Piddington Debra L. , Kashkouli Ali , Buchmeier Nancy A.
Published: July 2002

Mycobacterium tuberculosisgrows within the phagocytic vacuoles of macrophages, where it encounters a moderately acidic and possibly nutrient-restricted environment. Other mycobacterial species encounter acidic conditions in soil and aquatic environments. We have evaluated the influence of pH and divalent cation levels on the growth ofM. tuberculosisand seven other mycobacterial species. In a defined medium, the growth ofM. tuberculosiswas very restricted by acidic pH. Higher levels of Mg2+were required for growth ofM. tuberculosisin mildly acidic media (pH 6.0 to 6.5) compared to pH 7.0 medium. The divalent cations Ca2+, Zn2+, or Mn2+could not replace Mg2+during growth at pH 6.25, but Ca2+could at least partially substitute for Mg2+during growth at pH 7.0. Among eight species of mycobacteria tested, there was a diversity of growth rates in media with acidic pH and low Mg2+levels.M. tuberculosiswas the most restricted in growth at pH 6.0, and all of this growth required elevated levels of Mg2+.M. kansasiiandM. smegmatisalso grew very poorly in acidic media with limiting Mg2+.M. fortuitum,M. marinum,M. scrofulaceum,M. avium, andM. chelonaegrew at pH 6.0 in an unrestricted manner. These results demonstrate thatM. tuberculosisis unique among the mycobacteria in its extreme sensitivity to acid and indicate thatM. tuberculosismust acquire sufficient Mg2+in order to grow in a mildly acidic environment such as within the phagosome of macrophages.
Novel Apoptosis-Inducing Activity in
Bacteroides forsythus
: a Comparative Study with Three Serotypes of
Actinobacillus actinomycetemcomitans
Authors: Arakawa Shinichi , Nakajima Takuma , Ishikura Hiroaki , Ichinose Shizuko , Ishikawa Isao , Tsuchida Nobuo
Published: July 2002

Bacteroides forsythus
, which has been reported to be associated with periodontitis but has not been recognized as a key pathogen, was found to induce cytolytic activity against HL-60 and other human leukemic cells. This cytolytic activity was demonstrated according to three different criteria: (i) loss of both mitochondrial membrane potential and membrane integrity in cells treated with bacterial extracts and then with Rh123 and propidium iodide, respectively, as demonstrated by flow cytometry; (ii) damage to cytoplasmic membrane, as revealed by scanning electron microscopy (SEM); and (iii) DNA ladder formation and activation of caspase-3. These results indicate that
B. forsythus
produced an apoptosis-inducing factor(s) found to be composed of protein as judged by heat and trypsin sensitivity. In addition to extracts from
B. forsythus
, the culture supernatant of this bacterium has the ability to induce a cytolytic effect against peripheral white blood cells, especially lymphocytes. For comparison with
B. forsythus
, the same analyses were applied to two strains with different serotypes of
Actinobacillus actinomycetemcomitans
, serotypes a (ATCC 43717) and c (ATCC 43719), in addition to previously reported apoptosis-inducing serotype b (ATCC 43718), which was used as a positive control. The strains of
A. actinomycetemcomitans
serotypes a and b induced apoptosis in HL-60 cells as judged by the above three criteria but to a slightly lesser extent than did
B. forsythus
, while the serotype c strain produced apoptosis to a negligible extent. Detailed SEM images showed that the
A. actinomycetemcomitans
serotype a strain induced large-pore formation and the serotype b strain produced small pores with typical blebbing, while
B. forsythus
induced severe membrane ruffling. Further DNA ladder formation and caspase-3 activation were observed in the serotype a and b strains but not in the serotype c strain. The present paper is the first report of a protein factor(s) from
B. forsythus
and the
A. actinomycetemcomitans
serotype a strain which induces apoptotic cell death.
Identification of Saliva-Regulated Genes of
Streptococcus gordonii
DL1 by Differential Display Using Random Arbitrarily Primed PCR
Authors: Dû Laurence D. , Kolenbrander Paul E.
Published: July 2002

Attachment of
Streptococcus gordonii
to the acquired pellicle of the tooth surface involves specific interactions between bacterial adhesins and adsorbed salivary components. To study saliva-regulated gene expression in
S. gordonii
, we used random arbitrarily primed PCR (RAP-PCR). Bacteria were incubated in either brain heart infusion medium or saliva. Total RNA from both conditions was purified and RAP fingerprinted and then PCR amplified with an arbitrary primer. The differentially displayed DNA fragments were cloned, sequenced, and analyzed using the BLAST search network service. Three DNA products were up-regulated. One was identified as that of the
sspA
and -
B
genes, which encode the salivary agglutinin glycoprotein-binding proteins SspA and SspB of
S. gordonii
; another had 79% identity with the
Lactococcus lactis clpE
gene, encoding a member of the Clp protease family; and the third product showed no significant homology to known genes. Five down-regulated genes were identified which encode proteins involved in bacterial metabolism. We have shown, for the first time, direct induction of
sspA
and -
B
in
S. gordonii
by human saliva.
In Vivo Genetic Analysis Indicates That PhoP-PhoQ and the
Salmonella
Pathogenicity Island 2 Type III Secretion System Contribute Independently to
Salmonella enterica
Serovar Typhimurium Virulence
Authors: Beuzón Carmen R. , Unsworth Kate E. , Holden David W.
Published: July 2002

Many virulence factors are required for
Salmonella enterica
serovar Typhimurium to replicate intracellularly and proliferate systemically within mice. In this work, we have carried out genetic analyses in vivo to determine the functional relationship between two major virulence factors necessary for systemic infection by
S. enterica
serovar Typhimurium: the
Salmonella
pathogenicity island 2 (SPI-2) type III secretion system (TTSS) and the PhoP-PhoQ two-component regulatory system. Although previous work suggested that PhoP-PhoQ regulates SPI-2 TTSS gene expression in vitro, in vivo competitive analysis of mutant strains indicates that these systems contribute independently to
S. typhimurium
virulence. Our results also suggest that mutation of
phoP
may compensate partially for defects in the SPI-2 TTSS by deregulating SPI-1 TTSS expression. These results provide an explanation for previous reports showing an apparent functional overlap between these two systems in vitro.
Synergistic Effects of Alpha-Toxin and Perfringolysin O in
Clostridium perfringens
-Mediated Gas Gangrene
Authors: Awad Milena M. , Ellemor Darren M. , Boyd Richard L. , Emmins John J. , Rood Julian I.
Published: July 2002

To examine the synergistic effects of alpha-toxin and perfringolysin O in clostridial myonecrosis, homologous recombination was used to construct an alpha-toxin deficient derivative of a perfringolysin O mutant of
Clostridium perfringens
. The subsequent strain was complemented with separate plasmids that carried the alpha-toxin structural gene (
plc)
, the perfringolysin O gene (
pfoA
), or both toxin genes, and the resultant isogenic strains were examined in a mouse myonecrosis model. Synergistic effects were clearly observed in these experiments. Infection with the control strain, which did not produce either toxin, resulted in very minimal gross pathological changes, whereas the isogenic strain that was reconstituted for both toxins produced a pathology that was clearly more severe than when alpha-toxin alone was reconstituted. These changes were most apparent in the rapid spread of the disease, the gross pathology of the footpad and in the rate at which the mice had to be euthanatized for ethical reasons. Elimination of both alpha-toxin and perfringolysin O production removed most of the histopathological features typical of clostridial myonecrosis. These effects were restored when the mutant was complemented with the alpha-toxin structural gene, but reconstituting only perfringolysin O activity produced vastly different results, with regions of coagulative necrosis, apparently enhanced by vascular disruption, being observed. Reconstitution of both alpha-toxin and perfringolysin O activity produced histopathology most similar to that observed with the alpha-toxin reconstituted strain. The spreading of myonecrosis was very rapid in these tissues, and coagulative necrosis appeared to be restricted to the lumen of the blood vessels. The results of these virulence experiments clearly support the hypothesis that alpha-toxin and perfringolysin O have a synergistic effect in the pathology of gas gangrene.
Innate Resistance toBabesiaInfection Is Influenced by Genetic Background and Gender
Authors: Aguilar-Delfin Irma , Homer Mary J. , Wettstein Peter J. , Persing David H.
Published: July 2002

Infection of severe combined immunodeficient mice withBabesiasp. strain WA1 was studied to assess the contributions of innate and adaptive immunity in resistance to acute babesiosis. Thescidmutation showed little effect in genetically susceptible C3H mice and did not decrease the inherent resistance of C57BL/6 mice to the infection, suggesting that innate immunity plays a central role in determining the course ofBabesiainfection in these strains. In contrast, thescidmutation dramatically impaired resistance in moderately susceptible BALB/c mice, suggesting that acquired immunity may play an important secondary role. In comparison to their female counterparts, male mice of different genetic backgrounds showed increased resistance to the infection, indicating that the gender of the host may influence protection against babesiosis.
Ammonia as an Accelerator of Tumor Necrosis Factor Alpha-Induced Apoptosis of Gastric Epithelial Cells in
Helicobacter pylori
Infection
Authors: Igarashi Muneki , Kitada Yukie , Yoshiyama Hironori , Takagi Atsushi , Miwa Takeshi , Koga Yasuhiro
Published: July 2002

The mechanism by which
Helicobacter pylori
induces apoptosis remains unclear. In a previous study using biopsy samples, we found a significant correlation between the urease activity of an
H. pylori
strain and the apoptosis level induced by this strain. Therefore, in this study, we investigated whether urease and/or the ammonia generated by urease can induce apoptosis. Human gastric epithelial cell lines were cocultured with
H. pylori
, and the levels of apoptosis and ammonia production were measured. The medium was supplemented (or not supplemented) with urea and cytokines. While a large amount of ammonia (>30 mM) accumulated in the coculture containing urease-positive
H. pylori
and urea, no significant degree of apoptosis occurred. In the presence of tumor necrosis factor alpha (TNF-α), however, a marked acceleration of apoptosis was found in this coculture. Such enhancement of apoptosis was also induced by the addition of 4 to 8 mM ammonia to the cell culture without either
H. pylori
or urea but containing TNF-α. These results suggested that ammonia accelerates cytokine-induced apoptosis in gastric epithelial cells, while ammonia or urease molecules alone are unable to induce a significant degree of apoptosis.
Interleukin-8 and Intercellular Adhesion Molecule 1 Regulation in Oral Epithelial Cells by Selected Periodontal Bacteria: Multiple Effects ofPorphyromonas gingivalisvia Antagonistic Mechanisms
Authors: Huang George T.-J. , Kim Daniel , Lee Jonathan K.-H. , Kuramitsu Howard K. , Haake Susan Kinder
Published: July 2002

Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced afterPorphyromonas gingivalisATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h followingFusobacterium nucleatuminfection whereas it rapidly decreased within 2 h afterP. gingivalisinfection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with eitherActinobacillus actinomycetemcomitans,F. nucleatum, orP. gingivalis. Attenuation of IL-8 secretion was facilitated by adherentP. gingivalisstrains. The IL-8 secreted from epithelial cells afterF. nucleatumstimulation could be down-regulated by subsequent infection withP. gingivalisor its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated withP. gingivalisproteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, whileP. gingivalisup-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge withP. gingivalisandF. nucleatumand vice versa were approximately identical and were lower than those followingF. nucleatumchallenge alone and higher than control levels or those followingP. gingivalischallenge alone. Thus, together with the protease effect,P. gingivalispossesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.
Molecular Cloning and Characterization of
WdPKS1
, a Gene Involved in Dihydroxynaphthalene Melanin Biosynthesis and Virulence in
Wangiella
(
Exophiala
)
dermatitidis
Authors: Feng Bin , Wang Xu , Hauser Melinda , Kaufmann Sarah , Jentsch Simone , Haase Gerhard , Becker Jeffery M. , Szaniszlo Paul J.
Published: July 2002

1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of
Wangiella dermatitidis
, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene,
WdPKS1
, in
W. dermatitidis
. The resulting
wdpks1
Δ disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact
WdPKS1
gene was then successfully recovered from two plasmids. The
WdPKS1
gene was also isolated independently by complementation of the
mel3
mutation in an albino mutant of
W. dermatitidis
using a cosmid library. Sequence analysis substantiated that
WdPKS1
encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a β-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all
wdpks1
Δ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a
wdpks1
Δ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.
Type II Protein Secretion Is a Subset of the PilD-Dependent Processes That Facilitate Intracellular Infection by
Legionella pneumophila
Authors: Rossier Ombeline , Cianciotto Nicholas P.
Published: July 2002

Previously, we had demonstrated that a
Legionella pneumophila
prepilin peptidase (
pilD
) mutant does not produce type IV pili and shows reduced secretion of enzymatic activities. Moreover, it displays a distinct colony morphology and a dramatic reduction in intracellular growth within amoebae and macrophages, two phenotypes that are not exhibited by a pilin (

pilE
L

) mutant. To determine whether these
pilD
-dependent defects were linked to type II secretion, we have constructed two new mutants of
L. pneumophila
strain 130b. Mutations were introduced into either
lspDE
, which encodes the type II outer membrane secretin and ATPase, or
lspFGHIJK
, which encodes the pseudopilins. Unlike the wild-type and

pilE
L

strains, both
lspDE
and
lspG
mutants showed reduced secretion of six
pilD
-dependent enzymatic activities; i.e., protease, acid phosphatase,
p
-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A. However, they exhibited a colony morphology different from that of the
pilD
mutant, suggesting that their surfaces are distinct. The
pilD, lspDE
, and
lspG
mutants were similarly and greatly impaired for growth within
Hartmannella vermiformis
, indicating that the intracellular defect of the peptidase mutant in amoebae is explained by the loss of type II secretion. When assessed for infection of U937 macrophages, both
lsp
mutants exhibited a 10-fold reduction in intracellular multiplication and a diminished cytopathic effect. Interestingly, the
pilD
mutant was clearly 100-fold more defective than the type II secretion mutants in U937 cells. These results suggest the existence of a novel
pilD
-dependent mechanism for promoting
L. pneumophila
intracellular infection of human cells.
Cellular Uptake of the
Clostridium perfringens
Binary Iota-Toxin
Authors: Blöcker Dagmar , Behlke Joachim , Aktories Klaus , Barth Holger
Published: July 2002

The binary iota-toxin is produced by
Clostridium perfringens
type E strains and consists of two separate proteins, the binding component iota b (98 kDa) and an actin-ADP-ribosylating enzyme component iota a (47 kDa). Iota b binds to the cell surface receptor and mediates the translocation of iota a into the cytosol. Here we studied the cellular uptake of iota-toxin into Vero cells. Bafilomycin A1, but not brefeldin A or nocodazole, inhibited the cytotoxic effects of iota-toxin, indicating that toxin is translocated from an endosomal compartment into the cytoplasm. Acidification (pH ≤ 5.0) of the extracellular medium enabled iota a to directly enter the cytosol in the presence of iota b. Activation by chymotrypsin induced oligomerization of iota b in solution. An average mass of 530 ± 28 kDa for oligomers was determined by analytical ultracentrifugation, indicating heptamer formation. The entry of iota-toxin into polarized CaCo-2 cells was studied by measuring the decrease in transepithelial resistance after toxin treatment. Iota-toxin led to a significant decrease in resistance when it was applied to the basolateral surface of the cells but not following application to the apical surface, indicating a polarized localization of the iota-toxin receptor.
Identification and Analysis of
Staphylococcus aureus
Components Expressed by a Model System of Growth in Serum
Authors: Wiltshire Michael D. , Foster Simon J.
Published: July 2002

A model system mimicking
Staphylococcus aureus
bacteremia was developed by growth in serum under microaerobic conditions. Eight genes induced by growth in serum were identified, including an antimicrobial peptide biosynthesis locus, amino acid biosynthetic loci, and genes encoding putative surface proteins. Nine independent insertions were found in the major lysine biosynthesis operon, which encodes eight genes, is repressed by lysine in vitro, and is expressed in vivo.
Escherichia coliCytotoxic Necrotizing Factor andPasteurella multocidaToxin Induce Focal Adhesion Kinase Autophosphorylation and Src Association
Authors: Thomas Warren , Pullinger Gillian D. , Lax Alistair J. , Rozengurt Enrique
Published: July 2002

Cytotoxic necrotizing factor 1 andPasteurella multocidatoxin induced dose- and time-dependent increases in focal adhesion kinase (FAK) Tyr397phosphorylation in Swiss 3T3 cells. FAK autophosphorylation was sensitive to inhibitors of p160/ROCK and coincided with the formation of stable complexes between FAK and Src family members.