bluuebunny/crossref_metadata_2025 huggingface dataset:
In the Absence of Endogenous Gamma Interferon, Mice Acutely Infected withNeospora caninumSuccumb to a Lethal Immune Response Characterized by Inactivation of Peritoneal Macrophages
Authors: Nishikawa Yoshifumi , Tragoolpua Khajornsak , Inoue Noboru , Makala Levi , Nagasawa Hideyuki , Otsuka Haruki , Mikami Takeshi
Published: July 2002

Following infection withNeospora caninum, BALB/c mice were shown to be resistant to an acute infection but developed a latent chronic infection. However, BALB/c background gamma interferon (IFN-γ)-deficient mice were sensitive to the acute infection. Since the immune response in IFN-γ-deficient mice is scantly known, we examined the function of macrophages, major histocompatibility complex (MHC) class II expression, T-cell responses, and serum cytokine levels in the mice. All IFN-γ-deficient mice died within 9 days of infection withN. caninum, whereas those treated with exogenous IFN-γ lived longer. AlthoughN. caninuminvaded various organs in both types of mice at the early stage of infection, the parasite was not detected in the brains of resistant hosts until 21 days postinfection (dpi). Peritoneal macrophages from IFN-γ-deficient mice were activated by exogenous IFN-γ associated with inhibition of parasite growth and nitric oxide production as were those from BALB/c mice. IFN-γ-deficient mice failed to increase MHC class II expression on macrophages. Moreover, BALB/c mice induced T-cell proliferation while IFN-γ-deficient mice did not. However, in vivo treatment with exogenous IFN-γ induced up-regulated MHC class II expression in IFN-γ-deficient mice. BALB/c mice treated with an antibody to CD4 showed an increase in morbidity and mortality after parasite infection. In serum, significant levels of IFN-γ and interleukin-4 (IL-4) were detected in resistant hosts, whereas IL-10 was detected in IFN-γ-deficient mice. The levels of IL-12 in IFN-γ-deficient mice were higher than those in BALB/c mice at 7 dpi. The present study indicates that early IFN-γ production has a crucial role in the activation of peritoneal macrophages for the induction of protective immune responses againstN. caninum.
Stimulant-Dependent Modulation of Cytokines and Chemokines by Airway Epithelial Cells: Cross Talk between Pulmonary Epithelial and Peripheral Blood Mononuclear Cells
Authors: Krakauer Teresa
Published: July 2002

Staphylococcal exotoxins (SE) and lipopolysaccharide (LPS) stimulate cells of the immune system to produce proinflammatory cytokines and chemokines which mediate septic shock and acute lung inflammation. A coculture of human peripheral blood mononuclear cells (PBMC) and pulmonary A549 epithelial cells was used to investigate inflammatory responses triggered by staphylococcal enterotoxin B (SEB), toxic shock syndrome toxin 1, and LPS. The levels of interleukin 1β (IL-1β), IL-6, gamma interferon-inducible protein 10, monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1α, and RANTES were enhanced by 3.8-, 4.2-, 3.1-, 8.9-, 2-, and 2.9-fold, respectively, in cocultures of SEB-stimulated cells compared to in SEB-stimulated PBMC. In LPS-stimulated cocultures, only MCP-1 and RANTES levels were increased. These data suggest that the modulation of specific cytokines and chemokines is dependent on the stimulus and that there is bidirectional interaction between PBMC and lung epithelial cells to influence the immune response to these different stimuli.
Coinvasion of Dentinal Tubules by
Porphyromonas gingivalis
and
Streptococcus gordonii
Depends upon Binding Specificity of Streptococcal Antigen I/II Adhesin
Authors: Love Robert M. , McMillan Malcolm D. , Park Yoonsuk , Jenkinson Howard F.
Published: July 2002

Cell wall-anchored polypeptides of the antigen I/II family are produced by many species of oral streptococci. These proteins mediate adhesion of streptococci to salivary glycoproteins and to other oral microorganisms and promote binding of cells to collagen type I and invasion of dentinal tubules. Since infections of the root canal system have a mixed anaerobic bacterial etiology, we investigated the hypothesis that coadhesion of anaerobic bacteria with streptococci may facilitate invasive endodontic disease.
Porphyromonas gingivalis
ATCC 33277 cells were able to invade dentinal tubules when cocultured with
Streptococcus gordonii
DL1 (Challis) but not when cocultured with
Streptococcus mutans
NG8. An isogenic noninvasive mutant of
S. gordonii
, with production of SspA and SspB (antigen I/II family) polypeptides abrogated, was deficient in binding to collagen and had a 40% reduced ability to support adhesion of
P. gingivalis
. Heterologous expression of the
S. mutans
SpaP (antigen I/II) protein in this mutant restored collagen binding and tubule invasion but not adhesion to
P. gingivalis
or the ability to promote
P. gingivalis
coinvasion of dentin. An isogenic afimbrial mutant of
P. gingivalis
had 50% reduced binding to
S. gordonii
cells but was unaffected in the ability to coinvade dentinal tubules with
S. gordonii
wild-type cells. Expression of the
S. gordonii
SspA or SspB polypeptide on the surface of
Lactococcus lactis
cells endowed these bacteria with the abilities to bind
P. gingivalis
, penetrate dentinal tubules, and promote
P. gingivalis
coinvasion of dentin. The results demonstrate that collagen-binding and
P. gingivalis
-binding properties of antigen I/II polypeptides are discrete functions. Specificity of antigen I/II polypeptide recognition accounts for the ability of
P. gingivalis
to coinvade dentinal tubules with
S. gordonii
but not with
S. mutans
. This provides evidence that the specificity of interbacterial coadhesion may influence directly the etiology of pulpal and periapical diseases.
Colonic Bacteria Express an Ulcerative Colitis pANCA-Related Protein Epitope
Authors: Cohavy O. , Bruckner D. , Gordon L. K. , Misra R. , Wei B. , Eggena M. E. , Targan S. R. , Braun J.
Published: July 2002

Bacteria are a suspected pathogenic factor in inflammatory bowel disease, but the identity of the relevant microbial species remains unresolved. The pANCA autoantibody is associated with most cases of ulcerative colitis (UC) and hence reflects an immune response associated with the disease process. This study addresses the hypothesis that pANCA identifies an antigen(s) expressed by bacteria resident in the human colonic mucosa. Libraries of colonic bacteria were generated using aerobic and anaerobic microbiologic culture conditions, and bacterial pools and clonal isolates were evaluated for cross-reactive antigens by immunoblot analysis using the pANCA monoclonal antibody Fab 5-3. Two major species of proteins immunoreactive to pANCA monoclonal antibodies were detected in bacteria from the anaerobic libraries. Colony isolates of the expressing bacteria were identified asBacteroides caccaeandEscherichia coli. Isolation and partial sequencing of theB. caccaeantigen identified a 100-kDa protein without database homologous sequences. TheE. coliprotein was biochemically and genetically identified as the outer membrane porin OmpC. Enzyme-linked immunosorbent assay with human sera demonstrated elevated immunoglobulin G anti-OmpC in UC patients compared to healthy controls. These findings demonstrate that a pANCA monoclonal antibody detects a recurrent protein epitope expressed by colonic bacteria and implicates colonic bacterial proteins as a target of the disease-associated immune response.
Orally AdministeredGiardia duodenalisExtracts Enhance an Antigen-Specific Antibody Response
Authors: Dunn Linda A. , Upcroft Jacqueline A. , Fowler Elizabeth V. , Matthews Ben S. , Upcroft Peter
Published: July 2002

We have identified novel adjuvant activity in specific cytosol fractions from trophozoites ofGiardiaisolate BRIS/95/HEPU/2041 (J. A. Upcroft, P. A. McDonnell, and P. Upcroft, Parasitol. Today, 14:281–284, 1998). Adjuvant activity was demonstrated in the systemic and mucosal compartments whenGiardiaextract was coadministered orally with antigen to mice. Enhanced antigen-specific serum antibody responses were demonstrated by enzyme-linked immunosorbent assay to be comparable to those generated by the “gold standard,” mucosal adjuvant cholera toxin. A source of adjuvant activity was localized to the cytosolic component of the parasite. Fractionation of the cytosol produced fraction pools, some of which, when coadministered with antigen, stimulated an enhanced antigen-specific serum response. The toxic component of conventional mucosal adjuvants is associated with adjuvant activity; therefore, in a similar way, the toxin-like attributes of BRIS/95/HEPU/2041 may be responsible for its adjuvanticity. Complete characterization of the adjuvant is under way.
Administration of Superantigens Protects Mice from LethalListeria monocytogenesInfection by Enhancing Cytotoxic T Cells
Authors: Okamoto Shigefumi , Kawabata Shigetada , Nakagawa Ichiro , Hamada Shigeyuki
Published: July 2002

Superantigens stimulate T-cell-receptor Vβ-selective T-cell proliferation accompanying the release of cytokines, which may eventually protect the host from microbial infections. We investigated here whether superantigens can rescue the host from lethal bacterial infection. Mice were pretreated withStaphylococcus aureusenterotoxin B (SEB) 1 and 2 days before bacterial infection, and the mortality of infected mice was assessed. SEB pretreatment protected mice from lethal infection withListeria monocytogenesbut not from lethal infection withStreptococcus pyogenes. This enhanced protection was also observed upon pretreatment with recombinant streptococcal pyrogenic exotoxin A. Furthermore,L. monocytogenes-specific delayed-type hypersensitivity (DTH) due to type 1 helper T (Th1) cells and the cytotoxicity of CD8+T cells were significantly enhanced after SEB administration and bacterial infection. Depletion of either CD4+T cells or CD8+T cells in SEB-pretreated mice completely abolished this protection. This phenomenon was ascribed to the elimination ofL. monocytogenes-specific CD8+cytotoxic T lymphocytes (CTL). It was found that CD4+T cells contributed to the induction of the CTL populations. Furthermore, SEB pretreatment of heat-killedL. monocytogenes-immunized mice enhanced the protection from challenge ofL. monocytogenes. Taken together, these results indicated that administrations of superantigens protected mice from infection withL. monocytogenes, which was dependent on the enhancedL. monocytogenes-specific CTL activity in the presence of CD4+T cells, and superantigens exhibited adjuvant activity in the immunization against intracellular pathogens.
Immunomodulatory Effects of Anti-CD4 Antibody in Host Resistance against Infections and Tumors in Human CD4 Transgenic Mice
Authors: Herzyk Danuta J. , Gore Elizabeth R. , Polsky Rodd , Nadwodny Kimberly L. , Maier Curtis C. , Liu Susan , Hart Timothy K. , Harmsen Allen G. , Bugelski Peter J.
Published: July 2002

Anti-CD4 antibodies, which cause CD4+T-cell depletion, have been shown to increase susceptibility to infections in mice. Thus, development of anti-CD4 antibodies for clinical use raises potential concerns about suppression of host defense mechanisms against pathogens and tumors. The anti-human CD4 antibody keliximab, which binds only human and chimpanzee CD4, has been evaluated in host defense models using murine CD4 knockout-human CD4 transgenic (HuCD4/Tg) mice. In these mice, depletion of CD4+T cells by keliximab was associated with inhibition of anti-Pneumocystis cariniiand anti-Candida albicansantibody responses and rendered HuCD4/Tg mice susceptible toP. carinii, a CD4-dependent pathogen, but did not compromise host defense againstC. albicansinfection. Treatment of HuCD4/Tg mice with corticosteroids impaired host immune responses and decreased survival for both infections. Resistance to experimental B16 melanoma metastases was not affected by treatment with keliximab, in contrast to an increase in tumor colonization caused by anti-T cell Thy1.2 and anti-asialo GM-1 antibodies. These data suggest an immunomodulatory rather than an overt immunosuppressive activity of keliximab. This was further demonstrated by the differential effect of keliximab on type 1 and type 2 cytokine expression in splenocytes stimulated ex vivo. Keliximab caused an initial up-regulation of interleukin-2 (IL-2) and gamma interferon, followed by transient down-regulation of IL-4 and IL-10. Taken together, the effects of keliximab in HuCD4/Tg mice suggest that in addition to depleting circulating CD4+T lymphocytes, keliximab has the capability of modulating the function of the remaining cells without causing general immunosuppression. Therefore, keliximab therapy may be beneficial in controlling certain autoimmune diseases.
Expression of Tumor Necrosis Factor Alpha and Interleukin 1β in Jejuna of Volunteers after Experimental Challenge with
Cryptosporidium parvum
Correlates with Exposure but Not with Symptoms
Authors: Robinson Prema , Okhuysen Pablo C. , Chappell Cynthia L. , Lewis Dorothy E. , Shahab Imran , Janecki Andrzej , White A. Clinton
Published: July 2002

Jejunal biopsies from volunteers challenged with
Cryptosporidium parvum
were examined for tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β mRNA. Postchallenge biopsies from 15 of 28 (54%) volunteers expressed TNF-α; 14% expressed IL-1β. Cytokine expression did not correlate with enteric symptoms, suggesting that TNF-α and IL-1β are not key mediators of diarrhea in human cryptosporidiosis.
Cloning of a Beta-Hemolysin Gene ofBrachyspira(Serpulina)hyodysenteriaeand Its Expression inEscherichia coli
Authors: Hsu Tsungda , Hutto David L. , Minion F. Chris , Zuerner Richard L. , Wannemuehler Michael J.
Published: July 2002

Brachyspira(Serpulina)hyodysenteriaeinduces a mucohemorrhagic diarrheal disease in pigs. The production of a beta-hemolysin has been considered a major virulence attribute of this organism. Previous reports have failed to correlate a specific cloned gene sequence with a purified beta-hemolytic protein sequence. Thus, questions still remain concerning the structural gene sequence of the hemolysin. To answer this question unequivocally, the beta-hemolytic toxin was purified from extracts of log-phase spirochetes, and the N-terminal amino acid sequence was determined (K-D-V-V-A-N-Q-L-N-I-S-D-K) and compared with the translated sequences of previously cloned genes,tlyAtotlyC. The lack of homology betweentlyAtotlyCtranslated sequences and the purified beta-hemolytic toxin sequence resulted in the study that is reported here. A degenerate probe was designed based on the N-terminal amino acid sequence of the purified beta-hemolysin and used to screen aB. hyodysenteriaegenomic library. Three overlapping clones were identified, and one was sequenced to reveal an open reading frame coding for a putative 8.93-kDa polypeptide containing the N-terminal sequence of the purified beta-hemolysin. To distinguish this gene from thetlyAtotlyCgenes, it has been designatedhlyA. A hemolysis-negativeEscherichia colistrains containinghlyAwas beta-hemolytic on blood agar media. Also, the hemolytic activity of the recombinant protein had identical protease and lipase sensitivities and electrophoretic mobility to those of nativeB. hyodysenteriaebeta-hemolysin. Based on sequence analysis, the translated protein had a pI of 4.3, an α-helical structure, and a phosphopantetheine binding motif. Hybridization analysis of genomic DNA indicated that thehlyAgene was present inB. hyodysenteriaeandB. intermediabut was not detected inB. innocens,B. pilosicoli, orB. murdochiiunder high-stringency conditions. The location ofhlyAon the chromosomal map was distinct from the locations oftlyA,tlyB, andtlyC.
Recombinant PhpA Protein, a Unique Histidine Motif-Containing Protein fromStreptococcus pneumoniae, Protects Mice against Intranasal Pneumococcal Challenge
Authors: Zhang Ying , Masi Amy W. , Barniak Vicki , Mountzouros Ken , Hostetter Margaret K. , Green Bruce A.
Published: July 2002

The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the current conjugate vaccine may still cause pneumococcal disease. To address these serotypes and the remaining otitis media due toStreptococcus pneumoniae, we have been evaluating antigenically conserved proteins fromS. pneumoniaeas vaccine candidates. A previous report identified a 20-kDa protein with putative human complement C3-proteolytic activity. By utilizing the publicly released pneumococcal genomic sequences, we found the gene encoding the 20-kDa protein to be part of a putative open reading frame of approximately 2,400 bp. We recombinantly expressed a 79-kDa fragment (rPhpA-79) that contains a repeated HxxHxH motif and evaluated it for vaccine potential. The antibodies elicited by the purified rPhpA-79 protein were cross-reactive to proteins from multiple strains ofS. pneumoniaeand were against surface-exposed epitopes. Immunization with rPhpA-79 protein adjuvanted with monophosphoryl lipid A (for subcutaneous immunization) or a mutant cholera toxin, CT-E29H (for intranasal immunization), protected CBA/N mice against death and bacteremia, as well as reduced nasopharyngeal colonization, following intranasal challenge with a heterologous pneumococcal strain. In contrast, immunization with the 20-kDa portion of the PhpA protein did not protect mice. These results suggest that rPhpA-79 is a potential candidate for use as a vaccine against pneumococcal systemic disease and otitis media.
Gene Cluster for Assembly of Pilus Colonization Factor Antigen III of Enterotoxigenic
Escherichia coli
Authors: Taniguchi Tooru , Akeda Yukihiro , Haba Ayako , Yasuda Yoko , Yamamoto Koichiro , Honda Takeshi , Tochikubo Kunio
Published: July 2002

The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic
Escherichia coli
(ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including
cofA
and
cofP
. Several proteins encoded by
cof
genes were similar to previously described proteins, such as the toxin-coregulated pili of
Vibrio cholerae
and the bundle-forming pili of enteropathogenic
E. coli
. The G+C content of the
cof
gene cluster was 37%, which was significantly lower than the average for the
E. coli
genome (50%). The introduction of a recombinant plasmid containing the
cof
gene cluster into the
E. coli
K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.
Demonstration of Polysaccharide Capsule in
Campylobacter jejuni
Using Electron Microscopy
Authors: Karlyshev Andrey V. , McCrossan Maria V. , Wren Brendan W.
Published: July 2002

Recently, we reported that
Campylobacter jejuni
, an important gastrointestinal pathogen, has the genetic determinants to produce a capsular polysaccharide (Karlyshev et al., Mol. Microbiol. 35:529–541, 2000). Despite these data, the presence of a capsule in these bacteria has remained controversial. In this study we stain
C. jejuni
cells with the cationic dye Alcian blue and demonstrate for the first time by electron microscopy that
C. jejuni
cells produce a polysaccharide capsule that is retained in the coccoid form but is absent in a
kpsM
mutant.
Passive Intranasal Monoclonal Antibody Prophylaxis against MurinePneumocystis cariniiPneumonia
Authors: Gigliotti Francis , Haidaris Constantine G. , Wright Terry W. , Harmsen Allen G.
Published: July 2002

Passive antibody immunoprophylaxis is one method used to protect patients against infection if they are unable to mount an adequate active immune response. Topical application of antibody may be effective against infections at mucosal sites. Using a SCID mouse model ofPneumocystis cariniipneumonia, we were able to demonstrate protection against an airborne challenge withP. cariniiby intranasal administration of antibody. Immunoglobulin M (IgM) monoclonal antibodies to an epitope shared by mouse and humanP. cariniiorganisms reduced organism numbers by more than 99% under the conditions described. An IgG1 switch variant of one of the IgM monoclonal antibodies was also protective. These experiments provide a model for exploring the utility of this approach in protecting at-risk patients from infection withP. carinii.
Gender Is a Major Determinant of the Clinical Evolution and Immune Response in Hamsters Infected withLeishmaniaspp
Authors: Travi Bruno L. , Osorio Yaneth , Melby Peter C. , Chandrasekar Bysani , Arteaga Lourdes , Saravia Nancy G.
Published: July 2002

In regions where leishmaniasis is endemic, clinical disease is usually reported more frequently among males than females. This difference could be due to disparate risks of exposure of males and females, but gender-related differences in the host response to infection may also play a role. Experimental studies of the influence of gender onLeishmaniainfection have not included parasites of the subgenusViannia, which is the most common cause of cutaneous leishmaniasis in the Americas. Mice are not readily susceptible to infection byLeishmania(Viannia) spp., but cutaneous infection of hamsters withL.(V.)panamensisorL.(V.)guyanensisresulted in chronic lesions typical of the human disease caused by these parasites. Strikingly, infection of male hamsters resulted in significantly greater lesion size and severity, an increased rate of dissemination to distant cutaneous sites, and a greater parasite burden in the draining lymph node than infection in female animals. Two lines of evidence indicated this gender-related difference in disease evolution was determined at least in part by the sex hormone status of the animal. First, prepubertal male animals had smaller and/or less severe cutaneous lesions than adult male animals. Second, infection of testosterone-treated female animals resulted in significantly larger lesions than in untreated female animals. The increased severity of disease in male compared to female animals was associated with significantly greater intralesional expression of interleukin-4 (IL-4) (P= 0.04), IL-10 (P= 0.04), and transforming growth factor β (TGF-β) (P< 0.001), cytokines known to promote disease in experimental leishmaniasis. There was a direct correlation between the expression of TGF-β mRNA and lesion size (Spearman's correlation coefficient = 0.873;P< 0.001). These findings demonstrate an inherent risk of increased disease severity in male animals, which is associated with a more permissive immune response.
Characterization of a Novel Type IV Pilus Locus Encoded on the Large Plasmid of Locus of Enterocyte Effacement-Negative Shiga-Toxigenic
Escherichia coli
Strains That Are Virulent for Humans
Authors: Srimanote Potjanee , Paton Adrienne W. , Paton James C.
Published: July 2002

The majority of Shiga-toxigenic
Escherichia coli
(STEC) strains isolated from humans with gastrointestinal disease carry large (approximately 90-kb) plasmids. We have been analyzing the megaplasmid (designated pO113) from an O113:H21 STEC strain (98NK2). This strain lacks the locus for enterocyte effacement (LEE) and yet was responsible for an outbreak of hemolytic uremic syndrome. In the present study, we demonstrate that pO113 carries a novel type IV pilus biosynthesis locus (
pil
) related to those of the IncI plasmids R721, R64, and ColIb9. The pO113
pil
locus consists of 11 closely linked genes (
pilL
through
pilV
) with an additional separately transcribed upstream gene (
pilI
). It directs the expression of long thin pili on the 98NK2 surface and the hemagglutination of guinea pig erythrocytes. We also demonstrate that pO113 can be transferred by conjugation. However, the type IV pilus encoded by pO113 does not appear to be involved in the adherence of 98NK2 to HEp-2 or Hct-8 cells in vitro. Homologues of the pO113
pil
locus were present in several other LEE-negative STEC strains but not in LEE-positive STEC strains belonging to serogroup O26, O111, or O157.
Clearance ofHelicobacter pyloriInfection and Resolution of Postimmunization Gastritis in a Kinetic Study of Prophylactically Immunized Mice
Authors: Garhart Christine A. , Redline Raymond W. , Nedrud John G. , Czinn Steven J.
Published: July 2002

Patients infected withHelicobacter pylorimount an immune response which fails to clear the infection and may contribute to disease. Mice can be protected by immunization. To further characterize theH. pylori-mouse model, stomachs of unimmunized or intranasally immunized C57BL/6 mice were quantitatively cultured 3 days and 1, 2, 4, 8, 16, 32, and 52 weeks after challenge withH. pylori. At 3 days and 1 week after challenge, colonization was the same in the immunized and unimmunized mice. By 2 weeks after challenge, the immunized mice had a >2-log decrease in bacterial load, and at all later time points, they either were culture negative or had at least a 2-log decrease in bacterial load. Gastritis in the immunized mice peaked at 1 to 2 weeks after challenge and was characterized by a mixed inflammatory infiltrate and epithelial proliferation centered at the transition between corpus and antrum. By 52 weeks postchallenge, the gastric histology in the immunized mice was not different from that in control unchallenged mice. The unimmunized group began to show a reduction in bacterial load as early as 16 weeks after challenge, and by 52 weeks seven of eight unimmunized mice had developed gastritis and reduced bacterial loads. These results indicate that prophylactic immunization does not prevent colonization byH. pyloribut enables mice to clear the infection or significantly reduce the number of colonizing bacteria. The reduction in bacterial load is associated with gastric inflammation that subsides over time.
Characterization of the Distal Tail Fiber Locus and Determination of the Receptor for Phage AR1, Which Specifically Infects
Escherichia coli
O157:H7
Authors: Yu Sung-Liang , Ko Kai-Liang , Chen Chang-Shi , Chang Yu-Chung , Syu Wan-Jr
Published: July 2002

Phage AR1 is similar to phage T4 in several essential genes but differs in host range. AR1 infects various isolates of
Escherichia coli
O157:H7 but does not infect K-12 strains that are commonly infected by T4. We report here the determinants that confer this infection specificity. In T-even phages, gp37 and gp38 are components of the tail fiber that are critical for phage-host interaction. The counterparts in AR1 may be similarly important and, therefore, were characterized. The AR1 gp37 has a sequence that differs totally from those of T2 and T4, except for a short stretch at the N terminus. The gp38 sequence, however, has some conservation between AR1 and T2 but not between AR1 and T4. The sequences that are most closely related to the AR1 gp37 and gp38 are those of phage Ac3 in the T2 family. To identify the AR1-specific receptor,
E. coli
O157:H7 was mutated by Tn
10
insertion and selected for an AR1-resistant phenotype. A mutant so obtained has an insertion occurring at
ompC
that encodes an outer membrane porin. To confirm the role of OmpC in the AR1 infection, homologous replacement was used to create an
ompC
disruption mutant (RM). When RM was complemented with OmpC originated from an O157:H7 strain, but not from K-12, its AR1 susceptibility was fully restored. Our results suggest that the host specificity of AR1 is mediated at least in part through the OmpC molecule.
Insertional Inactivation of Genes Encoding Components of the Sodium-Type Flagellar Motor and Switch of
Vibrio parahaemolyticus
Authors: Boles Blaise R. , McCarter Linda L.
Published: July 2002

Vibrio parahaemolyticus
possesses two types of flagella, polar and lateral, powered by distinct energy sources, which are derived from the sodium and proton motive forces, respectively. Although proton-powered flagella in
Escherichia coli
and
Salmonella enterica
serovar Typhimurium have been extensively studied, the mechanism of torque generation is still not understood. Molecular knowledge of the structure of the sodium-driven motor is only now being developed. In this work, we identify the switch components, FliG, FliM, and FliN, of the sodium-type motor. This brings the total number of genes identified as pertinent to polar motor function to seven. Both FliM and FliN possess charged domains not found in proton-type homologs; however, they can interact with the proton-type motor of
E. coli
to a limited extent. Residues known to be critical for torque generation in the proton-type motor are conserved in the sodium-type motor, suggesting a common mechanism for energy transfer at the rotor-stator interface regardless of the driving force powering rotation. Mutants representing a complete panel of insertionally inactivated switch and motor genes were constructed. All of these mutants were defective in sodium-driven swimming motility. Alkaline phosphatase could be fused to the C termini of MotB and MotY without abolishing motility, whereas deletion of the unusual, highly charged C-terminal domain of FliM disrupted motor function. All of the mutants retained proton-driven, lateral motility over surfaces. Thus, although central chemotaxis genes are shared by the polar and lateral systems, genes encoding the switch components, as well as the motor genes, are distinct for each motility system.
TraG from RP4 and TraG and VirD4 from Ti Plasmids Confer Relaxosome Specificity to the Conjugal Transfer System of pTiC58
Authors: Hamilton Claire M. , Lee Hyewon , Li Pei-Li , Cook David M. , Piper Kevin R. , von Bodman Susanne Beck , Lanka Erich , Ream Walt , Farrand Stephen K.
Published: July 2002

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG
RP4
) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of
Agrobacterium tumefaciens
do not mobilize vectors containing the
oriT
of RP4, but these IncP1 plasmid derivatives lack the
trans
-acting Dtr functions and TraG
RP4
.
A. tumefaciens
donors transferred a chimeric plasmid that contains the
oriT
and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG
RP4
was expressed in the donors. Mutations in
traG
RP4
with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the
tra
system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG
RP4
nor VirD4 restored transfer to a
traG
mutant of the Ti plasmid. VirD4 also failed to complement a
traG
RP4
mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG
RP4
-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG
RP4
and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.
The Alkane Hydroxylase Gene of
Burkholderia cepacia
RR10 Is under Catabolite Repression Control
Authors: Marı́n Mercedes M. , Smits Theo H. M. , van Beilen Jan B. , Rojo Fernando
Published: July 2002

In many microorganisms the first step for alkane degradation is the terminal oxidation of the molecule by an alkane hydroxylase. We report the characterization of a gene coding for an alkane hydroxylase in a
Burkholderia cepacia
strain isolated from an oil-contaminated site. The protein encoded showed similarity to other known or predicted bacterial alkane hydroxylases, although it clustered on a separate branch together with the predicted alkane hydroxylase of a
Mycobacterium tuberculosis
strain. Introduction of the cloned
B. cepacia
gene into an alkane hydroxylase knockout mutant of
Pseudomonas fluorescens
CHAO restored its ability to grow on alkanes, which confirms that the gene analyzed encodes a functional alkane hydroxylase. The gene, which was named
alkB
, is not linked to other genes of the alkane oxidation pathway. Its promoter was identified, and its expression was analyzed under different growth conditions. Transcription was induced by alkanes of chain lengths containing 12 to at least 30 carbon atoms as well as by alkanols. Although the gene was efficiently expressed during exponential growth, transcription increased about fivefold when cells approached stationary phase, a characteristic not shared by the few alkane degraders whose regulation has been studied. Expression of the
alkB
gene was under carbon catabolite repression when cells were cultured in the presence of several organic acids and sugars or in a complex (rich) medium. The catabolic repression process showed several characteristics that are clearly different from what has been observed in other alkane degradation pathways.
Two Membrane-Associated NiFeS-Carbon Monoxide Dehydrogenases from the Anaerobic Carbon-Monoxide-Utilizing Eubacterium
Carboxydothermus hydrogenoformans
Authors: Svetlitchnyi Vitali , Peschel Christine , Acker Georg , Meyer Ortwin
Published: July 2002

Two monofunctional NiFeS carbon monoxide (CO) dehydrogenases, designated CODH I and CODH II, were purified to homogeneity from the anaerobic CO-utilizing eubacterium
Carboxydothermus hydrogenoformans
. Both enzymes differ in their subunit molecular masses, N-terminal sequences, peptide maps, and immunological reactivities. Immunogold labeling of ultrathin sections revealed both CODHs in association with the inner aspect of the cytoplasmic membrane. Both enzymes catalyze the reaction CO + H
2
O → CO
2
+ 2 e
−
+ 2 H
+
. Oxidized viologen dyes are effective electron acceptors. The specific enzyme activities were 15,756 (CODH I) and 13,828 (CODH II) μmol of CO oxidized min
−1
mg
−1
of protein (methyl viologen, pH 8.0, 70°C). The two enzymes oxidize CO very efficiently, as indicated by

k
cat
/K
m

values at 70°C of 1.3 · 10
9
M
−1
CO s
−1
(CODH I) and 1.7 · 10
9
M
−1
CO s
−1
(CODH II). The apparent

K
m

values at pH 8.0 and 70°C are 30 and 18 μM CO for CODH I and CODH II, respectively. Acetyl coenzyme A synthase activity is not associated with the enzymes. CODH I (125 kDa, 62.5-kDa subunit) and CODH II (129 kDa, 64.5-kDa subunit) are homodimers containing 1.3 to 1.4 and 1.7 atoms of Ni, 20 to 22 and 20 to 24 atoms of Fe, and 22 and 19 atoms of acid-labile sulfur, respectively. Electron paramagnetic resonance (EPR) spectroscopy revealed signals indicative of [4Fe-4S] clusters. Ni was EPR silent under any conditions tested. It is proposed that CODH I is involved in energy generation and that CODH II serves in anabolic functions.
Translocation-Specific Conformation of Adenylate Cyclase Toxin from
Bordetella pertussis
Inhibits Toxin-Mediated Hemolysis
Authors: Gray Mary C. , Lee Sang-Jin , Gray Lloyd S. , Zaretzky Franca R. , Otero Angela S. , Szabo Gabor , Hewlett Erik L.
Published: July 2002

Bordetella pertussis
adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three- to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, ΔAC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to ΔAC enhances its hemolytic activity three- to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, ΔN489 (missing amino acids 6 to 489) and ΔN518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while ΔN549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.
Characterization of Intragenic Recombination in a Hyperthermophilic Archaeon via Conjugational DNA Exchange
Authors: Reilly Michelle S. , Grogan Dennis W.
Published: July 2002

Sulfolobus acidocaldarius
is so far the only hyperthermophilic archaeon in which genetic recombination can be assayed by conjugation and simple selections. Crosses among spontanteous
pyr
mutants were able to resolve closely spaced chromosomal mutations, identify deletions and rearrangements, and map mutations to a given deletion interval. Frameshift mutations in
pyrE
exerted polar effects that depressed orotidine-5′-monophosphate decarboxylase activity (encoded by
pyrF
), whereas base pair substitutions and an 18-bp deletion had no effect.
Dynamics of Genome Architecture in
Rhizobium
sp. Strain NGR234
Authors: Mavingui Patrick , Flores Margarita , Guo Xianwu , Dávila Guillermo , Perret Xavier , Broughton William J. , Palacios Rafael
Published: July 2002

Bacterial genomes are usually partitioned in several replicons, which are dynamic structures prone to mutation and genomic rearrangements, thus contributing to genome evolution. Nevertheless, much remains to be learned about the origins and dynamics of the formation of bacterial alternative genomic states and their possible biological consequences. To address these issues, we have studied the dynamics of the genome architecture in
Rhizobium
sp. strain NGR234 and analyzed its biological significance. NGR234 genome consists of three replicons: the symbiotic plasmid pNGR234
a
(536,165 bp), the megaplasmid pNGR234
b
(>2,000 kb), and the chromosome (>3,700 kb). Here we report that genome analyses of cell siblings showed the occurrence of large-scale DNA rearrangements consisting of cointegrations and excisions between the three replicons. As a result, four new genomic architectures have emerged. Three consisted of the cointegrates between two replicons: chromosome-pNGR234
a
, chromosome-pNGR234
b
, and pNGR234
a
-pNGR234
b
. The other consisted of a cointegrate of the three replicons (chromosome-pNGR234
a
-pNGR234
b
). Cointegration and excision of pNGR234
a
with either the chromosome or pNGR234
b
were studied and found to proceed via a Campbell-type mechanism, mediated by insertion sequence elements. We provide evidence showing that changes in the genome architecture did not alter the growth and symbiotic proficiency of
Rhizobium
derivatives.
Cyclospora cayetanensis
in Three Populations at Risk in Guatemala
Authors: Pratdesaba Rafael A. , González Mario , Piedrasanta Evelyn , Mérida Claudia , Contreras Karen , Vela Carlos , Culajay Francisco , Flores Luis , Torres Olga
Published: July 2002

In 1996 and 1997, outbreaks of
Cyclospora cayetanensis
in North America were linked to Guatemalan raspberries. From April 1999 to April 2000, we undertook a survey for
C. cayetanensis
in raspberry farm workers, malnourished children, and human immunodeficiency virus and AIDS patients in Guatemala. Stool samples were analyzed using ethylacetate-formalin concentration, wet preparation, modified acid-fast staining method, and epifluorescence. Oocysts were found in 1.5% of the subjects, none of whom were raspberry farm workers.
E-Test Method for Testing Susceptibilities of
Aspergillus
spp. to the New Triazoles Voriconazole and Posaconazole and to Established Antifungal Agents: Comparison with NCCLS Broth Microdilution Method
Authors: Espinel-Ingroff Ana , Rezusta A.
Published: July 2002

NCCLS document M38-P describes standard parameters for testing the fungistatic activities (MICs) of established agents against filamentous fungi (molds). This study evaluated the in vitro susceptibilities of 15
Aspergillus flavus
isolates, 62
A. fumigatus
isolates, and 10 isolates each of
A. niger
,
A. nidulans
, and
A. terreus
to voriconazole, posaconazole, itraconazole, and amphotericin B by the E-test and NCCLS M38-P microdilution methods. The agreement (within 3 dilutions) between methods for voriconazole was independent of the E-test incubation time (93.3 to 100% for four of five species at both incubation times). In contrast, with amphotericin B, itraconazole, and posaconazole, E-test results were more dependent on the incubation time for certain species. For
A. fumigatus,
posaconazole E-test MICs had better concordance with reference values after 48 h (95.2%) than after 24 h (90%), while the highest agreement for itraconazole MICs was after 24 h (90.3 versus 74.2%) of incubation. Better agreement between the methods was also obtained with 24-h E-test amphotericin B MICs for
A. flavus
(73.3 versus 26.7%) and
A. fumigatus
(96.7 versus 64.5%). E-test MICs of the four agents had the lowest percentages of agreement with reference values for
A. nidulans
(60 to 80%). For isolates for which high MICs were obtained for the four agents by the reference method, high MICs were also obtained by E-test at both 24 and 48 h. The utility of in vitro results of either the E-test or the NCCLS broth microdilution (M38-P) method for
Aspergillus
spp. needs to be established in clinical trials.
Polymorphism in the Interleukin-4 Promoter Affects Acquisition of Human Immunodeficiency Virus Type 1 Syncytium-Inducing Phenotype
Authors: Nakayama Emi E. , Hoshino Yoshihiko , Xin Xiaomi , Liu Huanliang , Goto Mieko , Watanabe Nobukazu , Taguchi Hitomi , Hitani Akihiro , Kawana-Tachikawa Ai , Fukushima Masao , Yamada Kaneo , Sugiura Wataru , Oka Shin-Ichi , Ajisawa Atsushi , Sato Hironori , Takebe Yutaka , Nakamura Tetsuya , Nagai Yoshiyuki , Iwamoto Aikichi , Shioda Tatsuo
Published: July 2002

The emergence of syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) in infected individuals is an indicator of poor prognosis and is often correlated with faster CD4+cell depletion and rapid disease progression. Interleukin-4 (IL-4) is a pleiotropic cytokine with various immune-modulating functions including induction of immunoglobulin E (IgE) production in B cells, down-regulation of CCR5 (a coreceptor for HIV-1 non-SI [NSI] strains), and up-regulation of CXCR4 (a coreceptor for HIV-1 SI variants). Here we show that homozygosity of a polymorphism in the IL-4 promoter region, IL-4 −589T, is correlated with increased rates of SI variant acquisition in HIV-1-infected individuals in Japan. This mutation was also shown to be associated with elevated serum IgE levels in HIV-1-infected individuals, especially in those at advanced stages of disease. In contrast, neither a triallele polymorphism in IL-10, another Th2 cytokine, nor a biallele polymorphism in the RANTES promoter affected acquisition of the SI phenotype. This finding suggested that IL-4-589T increases IL-4 production in the human body and thus accelerates the phenotypic switch of HIV-1 from NSI to SI and possibly disease progression of AIDS.
The E1∧E4 Protein of Human Papillomavirus Type 16 Associates with a Putative RNA Helicase through Sequences in Its C Terminus
Authors: Doorbar John , Elston Robert C. , Napthine Sawsan , Raj Kenneth , Medcalf Elizabeth , Jackson Deborah , Coleman Nick , Griffin Heather M. , Masterson Philip , Stacey Simon , Mengistu Yohannes , Dunlop Julia
Published: July 2002

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1∧E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1∧E4.L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1∧E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1∧E4 (in vivo and in vitro) and could be immunoprecipitated as an E1∧E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1∧E4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1∧E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with threeEscherichia coliDEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein ofSaccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1∧E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1∧E4-associated protein indicates a possible role for E1∧E4 in virus synthesis.
Immune Responses following Neonatal DNA Vaccination Are Long-Lived, Abundant, and Qualitatively Similar to Those Induced by Conventional Immunization
Authors: Hassett Daniel E. , Zhang Jie , Slifka Mark , Whitton J. Lindsay
Published: July 2002

Virus infections are devastating to neonates, and the induction of active antiviral immunity in this age group is an important goal. Here, we show that a single neonatal DNA vaccination induces cellular and humoral immune responses which are maintained for a significant part of the animal's life span. We employ a sensitive technique which permits the first demonstration and quantitation, directly ex vivo, of virus-specific CD8+T cells induced by DNA immunization. One year postvaccination, antigen-specific CD8+T cells were readily detectable and constituted 0.5 to 1% of all CD8+T cells. By several criteria—including cytokine production, perforin content, development of lytic ability, and protective capacity—DNA vaccine-induced CD8+memory T cells were indistinguishable from memory cells induced by immunization with a conventional (live-virus) vaccine. Analyses of long-term humoral immune responses revealed that, in contrast to the strong immunoglobulin G2a (IgG2a) skewing of the humoral response seen after conventional vaccination, IgG1 and IgG2a levels were similar in DNA-vaccinated neonatal and adult animals, indicating a balanced T helper response. Collectively, these results show that a single DNA vaccination within hours or days of birth can induce long-lasting CD8+T- and B-cell responses; there is no need for secondary immunization (boosting). Furthermore, the observed immune responses induced in neonates and in adults are indistinguishable by several criteria, including protection against virus challenge.