bluuebunny/crossref_metadata_2025_split huggingface dataset:
Xanthine Oxidase Contributes to Host Defense againstBurkholderia cepaciain the p47phox−/−Mouse Model of Chronic Granulomatous Disease
Authors: Segal Brahm H. , Sakamoto Nobuaki , Patel Mayur , Maemura Kosei , Klein Andrew S. , Holland Steven M. , Bulkley Gregory B. 
Published: July 2002

ABSTRACTChronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide and downstream microbicidal reactive oxidants, leading to recurrent life-threatening bacterial and fungal infections. Xanthine oxidase (XO) is another enzyme known to produce superoxide in many tissues. Using the p47phox−/−mouse model of CGD, we evaluated the residual antibacterial activity of XO. Clearance ofBurkholderia cepacia, a major pathogen in CGD, was reduced in p47phox−/−mice compared to that in wild-type mice and was further inhibited in p47phox−/−mice by pretreatment with the specific XO inhibitor allopurinol. HepaticB. cepaciaburden was similar in the two genotypes, but allopurinol significantly reduced net hepatic killing and killing efficiency only in p47phox−/−mice. Clearance and killing of intravenousEscherichia coliwas intact in p47phox−/−mice and was unaffected by pretreatment with allopurinol. In CGD, XO may contribute to host defense against a subset of reactive oxidant-sensitive pathogens.
Serological Expression Cloning of Novel Immunoreactive Antigens of
Babesia microti
Authors: Lodes Michael J. , Houghton Raymond L. , Bruinsma Elizabeth S. , Mohamath Raodoh , Reynolds Lisa D. , Benson Darin R. , Krause Peter J. , Reed Steven G. , Persing David H. 
Published: July 2002

ABSTRACT

Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent,
Babesia microti
. To develop more sensitive and specific assays for
B. microti
, we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique
B. microti
antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to
Plasmodium
sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during
B. microti
infection in humans.
Actinobacillus pleuropneumoniaeIron Transport and Urease Activity: Effects on Bacterial Virulence and Host Immune Response
Authors: Baltes Nina , Tonpitak Walaiporn , Gerlach Gerald-F. , Hennig-Pauka Isabel , Hoffmann-Moujahid Astrid , Ganter Martin , Rothkötter Hermann-J. 
Published: July 2002

ABSTRACTActinobacillus pleuropneumoniae, a porcine respiratory tract pathogen, has been shown to express transferrin-binding proteins and urease during infection. Both activities have been associated with virulence; however, their functional role for infection has not yet been elucidated. We used two isogenicA. pleuropneumoniaesingle mutants (ΔexbBand ΔureC) and a newly constructedA. pleuropneumoniaedouble (ΔureCΔexbB) mutant in aerosol infection experiments. Neither theA. pleuropneumoniaeΔexbBmutant nor the double ΔureCΔexbBmutant was able to colonize sufficiently long to initiate a detectable humoral immune response. These results imply that the ability to utilize transferrin-bound iron is required for multiplication and persistence ofA. pleuropneumoniaein the porcine respiratory tract. TheA. pleuropneumoniaeΔureCmutant and the parent strain both caused infections that were indistinguishable from one another in the acute phase of disease; however, 3 weeks postinfection theA. pleuropneumoniaeΔureCmutant, in contrast to the parent strain, could not be isolated from healthy lung tissue. In addition, the local immune response—as assessed by fluorescence-activated cell sorter and enzyme-linked immunosorbent spot analyses—revealed a significantly higher number ofA. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with theA. pleuropneumoniaeΔureCmutant than in the BALF of those infected with the parent strain. These results imply thatA. pleuropneumoniaeurease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positiveA. pleuropneumoniaeparent strain.
Purification of Anthrax Edema Factor from
Escherichia coli
and Identification of Residues Required for Binding to Anthrax Protective Antigen
Authors: Kumar Praveen , Ahuja Nidhi , Bhatnagar Rakesh 
Published: July 2002

ABSTRACT

The structural gene for anthrax edema factor (EF) was expressed in
Escherichia coli
under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography. From 1 liter of culture, 2.5 mg of biologically active EF was easily purified. This is the first report of purification of anthrax EF from
E. coli
. EF purified from
E. coli
was biologically and functionally as active as its
Bacillus anthracis
counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen. The adenylate cyclase activity of the mutants remained unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells.
Identification of an Antigenic Epitope in
Helicobacter pylori
Urease That Induces Neutralizing Antibody Production
Authors: Hirota Kaoru , Nagata Kumiko , Norose Yoshihiko , Futagami Seiji , Nakagawa Yohko , Senpuku Hidenobu , Kobayashi Masafumi , Takahashi Hidemi 
Published: July 2002

ABSTRACT

We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for
Helicobacter pylori
urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of
H. pylori
, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of
H. pylori
infection.
Chlamydia pneumoniae
Infects and Multiplies in Lymphocytes In Vitro
Authors: Haranaga Shusaku , Yamaguchi Hiroyuki , Friedman Herman , Izumi Shin-ichi , Yamamoto Yoshimasa 
Published: July 2002

ABSTRACT

The obligate intracellular pathogen
Chlamydia (Chlamydophila) pneumoniae
is known to be associated with some chronic inflammatory diseases, such as atherosclerosis. Interaction between
C. pneumoniae
and immune cells is important in the development of such diseases. However, susceptibility of immune cells, particularly lymphocytes, to
C. pneumoniae
infection has not been reported, even though lymphocytes play a pivotal role in the development of the diseases caused by this bacterium. In this regard, we examined the susceptibility of lymphocytes to
C. pneumoniae
infection in vitro. The results demonstrated that human peripheral blood lymphocytes as well as mouse spleen lymphocytes could be infected with
C. pneumoniae
. Furthermore, purified T lymphocytes as well as established T-lymphocyte cell line cells showed an obvious susceptibility to
C. pneumoniae
infection, indicating that T cells could be one of the host cells for this bacterial infection. These findings reveal a new infection site for
C. pneumoniae
, i.e., lymphocytes.
Salmonella
Pathogenicity Island 2-Encoded Proteins SseC and SseD Are Essential for Virulence and Are Substrates of the Type III Secretion System
Authors: Klein Joanna R. , Jones Bradley D. 
Published: July 2002

ABSTRACT

Survival of
Salmonella enterica
serovar Typhimurium within host phagocytic cells is a critical step in establishing systemic infection in mice. Genes within
Salmonella
pathogenicity island 2 (SPI-2) encode a type III secretion system that is required for establishment of systemic infection. Several proteins encoded by SPI-2 have homology to type III secreted proteins from enteropathogenic
Escherichia coli
and
Yersinia
and, based on that homology, are predicted to be secreted through the SPI-2 type III secretion system. We have investigated the roles of two of these proteins, SseC and SseD. We demonstrate here that the SseD protein is required for systemic
Salmonella
infection of the mouse, and we confirmed the virulence requirement for the SseC protein. Experiments were performed, using cellular fractionation and immunoblotting, to identify the subcellular location of the SseC and SseD proteins. Both proteins were found to localize predominantly to the bacterial cell membrane. In addition, our work revealed that SseC and SseD are exposed to the extracellular environment and are loosely associated with the bacterial membrane. Furthermore, localization of SseC and SseD to the bacterial membrane was found to require a functional SPI-2 type III secretion system. Collectively, these results indicate that the SseC and SseD proteins are secreted by the SPI-2 type III secretion system to the bacterial membrane in order to perform their virulence functions.
Conserved DegP Protease in Gram-Positive Bacteria Is Essential for Thermal and Oxidative Tolerance and Full Virulence in
Streptococcus pyogenes
Authors: Jones C. Hal , Bolken Tove' C. , Jones Kevin F. , Zeller Gloria O. , Hruby Dennis E. 
Published: July 2002

ABSTRACT

The DegP protease, a multifunctional chaperone and protease, has been shown to be essential for virulence in gram-negative pathogens such as
Salmonella enterica
serovar Typhimurium,
Brucella abortus, Yersinia enterocolitica
, and
Pseudomonas aeruginosa
. The function of DegP in pathogenesis appears to be the degradation of damaged proteins that accumulate as a result of the initial host response to infection, which includes the release of reactive oxygen intermediates. Additionally, the DegP protease plays a major role in monitoring and maintaining the
Escherichia coli
periplasm and influences
E. coli
pilus biogenesis. We report here the identification of highly homologous enzymes in
Streptococcus pyogenes, Streptococcus gordonii, Streptococcus mutans, Staphylococcus aureus
, and
Enterococcus faecalis
. Moreover, the phenotype of an insertionally inactivated
degP
allele in
S. pyogenes
is similar to that reported for
E. coli
, with temperature sensitivity for growth and enhanced sensitivity to reactive oxygen intermediates. Virulence studies in a mouse model of streptococcal infection indicate that a functional DegP protease is required for full virulence. These results suggest DegP as an attractive broad-spectrum target for future anti-infective drug development.
Absence of All Components of the Flagellar Export and Synthesis Machinery Differentially Alters Virulence ofSalmonella entericaSerovar Typhimurium in Models of Typhoid Fever, Survival in Macrophages, Tissue Culture Invasiveness, and Calf Enterocolitis
Authors: Schmitt Clare K. , Ikeda Jack S. , Darnell Stephen C. , Watson Patricia R. , Bispham Jennifer , Wallis Timothy S. , Weinstein Debra L. , Metcalf Eleanor S. , O'Brien Alison D. 
Published: July 2002

ABSTRACTIn this study, we constructed anflhD(the master flagellar regulator gene) mutant ofSalmonella entericaserovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that theflhDmutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, theflhDmutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on anfljB fliCmutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.
Conformationally Correct Expression of Membrane-AnchoredToxoplasma gondiiSAG1 in the Primitive ProtozoanGiardia duodenalis
Authors: Marti Matthias , Li Yajie , Köhler Peter , Hehl Adrian B. 
Published: July 2002

ABSTRACTTo explore the possibility of expressing membrane-anchored exodomains of heterologous surface antigens inGiardia, a chimeric construct containing theToxoplasma gondii SAG1gene was made. TheGiardiasystem is shown here to provide a means of generating correctly folded chimeric surface proteins in a native and unmodified form.
Urease of Enterohemorrhagic
Escherichia coli
: Evidence for Regulation by Fur and a
trans
-Acting Factor
Authors: Heimer Susan R. , Welch Rod A. , Perna Nicole T. , Pósfai György , Evans Peter S. , Kaper James B. , Blattner Fred R. , Mobley Harry L. T. 
Published: July 2002

ABSTRACT

Recent genomic analyses of
Escherichia coli
O157:H7 strain EDL933 revealed two loci encoding urease gene homologues (
ureDABCEFG
), which are absent in nonpathogenic
E. coli
strain K-12. This report demonstrates that the cloned EDL933
ure
gene cluster is capable of synthesizing urease in an
E. coli
DH5α background. However, when the gene fragment is transformed back into the native EDL933 background, the enzymatic activity of the cloned determinants is undetectable. We speculate that an unidentified
trans
-acting factor in enterohemorrhagic
E. coli
(EHEC) is responsible for this regulation of
ure
expression. In addition, Fur-like recognition sites are present in three independent O157:H7 isolates upstream of
ureD
and
ureA
. Enzymatic assays confirmed a difference in urease expression of cloned EHEC
ure
clusters in
E. coli
MC3100Δ
fur
. Likewise, interruption of
fur
in O157:H7 isolate IN1 significantly diminished urease activity. We propose that, similar to the function of Fur in regulating the acid response of
Salmonella enterica
serovar Typhimurium, it modulates urease expression in EHEC, perhaps contributing to the acid tolerance of the organism.
Identification and Characterization of the ESAT-6 Homologue ofMycobacterium lepraeand T-Cell Cross-Reactivity withMycobacterium tuberculosis
Authors: Geluk Annemieke , van Meijgaarden Krista E. , Franken Kees L. M. C. , Subronto Yanri W. , Wieles Brigitte , Arend Sandra M. , Sampaio Elizabeth P. , de Boer Tjitske , Faber William R. , Naafs Ben , Ottenhoff Tom H. M. 
Published: July 2002

ABSTRACTIn this paper we describe identification and characterization ofMycobacterium lepraeESAT-6 (L-ESAT-6), the homologue ofM. tuberculosisESAT-6 (T-ESAT-6). T-ESAT-6 is expressed by all pathogenic strains belonging to theM. tuberculosiscomplex but is absent from virtually all other mycobacterial species, and it is a promising antigen for immunodiagnosis of tuberculosis (TB). Therefore, we analyzed whether L-ESAT-6 is a similarly powerful tool for the study of leprosy by examining T-cell responses against L-ESAT-6 in leprosy patients, TB patients, and exposed or nonexposed healthy controls from areas where leprosy and TB are endemic and areas where they are not endemic. L-ESAT-6 was recognized by T cells from leprosy patients, TB patients, individuals who had contact with TB patients, and healthy individuals from an area where TB and leprosy are endemic but not by T cells from individuals who were not exposed toM. tuberculosisandM. leprae. Moreover, leprosy patients who were not responsive toM. lepraefailed to respond to L-ESAT-6. A very similar pattern was obtained with T-ESAT-6. These results show that L-ESAT-6 is a potentM. lepraeantigen that stimulates T-cell-dependent gamma interferon production in a large proportion of individuals exposed toM. leprae. Moreover, our results suggest that there is significant cross-reactivity between T-ESAT-6 and L-ESAT-6, which has implications for the use of ESAT-6 as tool for diagnosis of leprosy and TB in areas where both diseases are endemic.
Mechanisms of Monophosphoryl Lipid A Augmentation of Host Responses to Recombinant HagB fromPorphyromonas gingivalis
Authors: Yang Qiu-Bo , Martin Michael , Michalek Suzanne M. , Katz Jannet 
Published: July 2002

ABSTRACTPorphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Our laboratory has shown the induction of protection toP. gingivalisinfection after subcutaneous immunization with recombinant hemagglutinin B (rHagB). The purpose of this study was to determine if humoral antibody responses are induced after intranasal (i.n.) immunization of rHagB and if monophosphoryl lipid A (MPL), a nontoxic derivative of the lipid A region of lipopolysaccharide, acts as a mucosal adjuvant and potentiates responses to rHagB. Further, the effects of MPL on the nature of the response to HagB and on the costimulatory molecules B7-1 and B7-2 on different antigen-presenting cells (APC) were evaluated. Groups of BALB/c mice were immunized three times (2-week intervals) by the i.n. route with HagB (20 μg) alone or with MPL (25 μg). A group of nonimmunized mice served as control. Serum and saliva samples were collected prior to immunization and at approximately 2-week intervals and evaluated for serum immunoglobulin G (IgG) and IgG subclass and for salivary IgA antibody activity by enzyme-linked immunosorbent assay. Mice immunized with rHagB plus MPL had significantly higher salivary IgA (P< 0.05) and serum IgG (P< 0.05) anti-HagB responses than mice immunized with rHagB alone. The IgG1 and IgG2a subclass responses seen in mice immunized with rHagB plus MPL were significantly higher (P< 0.05) than those seen in mice immunized with rHagB only. Further, the IgG2a/IgG1 ratio in the latter group was ≈1, whereas in mice immunized with rHagB plus MPL the ratio was <1. These results provide evidence for the participation of T helper (Th) 1 and Th2 cells in responses to rHagB and that MPL potentiates a type 2 response to HagB. MPL was also shown to preferentially up-regulate B7-2 expression on B cells, whereas a preferential increase in B7-1 costimulatory molecule was seen on macrophages and dendritic cells. These results provide evidence that MPL exerts a differential regulation in the expression of costimulatory molecules on APC.
Essential Role of Ferritin Pfr in
Helicobacter pylori
Iron Metabolism and Gastric Colonization
Authors: Waidner Barbara , Greiner Stefan , Odenbreit Stefan , Kavermann Holger , Velayudhan Jyoti , Stähler Frank , Guhl Johannes , Bissé Emmanuel , van Vliet Arnoud H. M. , Andrews Simon C. , Kusters Johannes G. , Kelly David J. , Haas Rainer , Kist Manfred , Bereswill Stefan 
Published: July 2002

ABSTRACT

The reactivity of the essential element iron necessitates a concerted expression of ferritins, which mediate iron storage in a nonreactive state. Here we have further established the role of the
Helicobacter pylori
ferritin Pfr in iron metabolism and gastric colonization. Iron stored in Pfr enabled
H. pylori
to multiply under severe iron starvation and protected the bacteria from acid-amplified iron toxicity, as inactivation of the
pfr
gene restricted growth of
H. pylori
under these conditions. The lowered total iron content in the
pfr
mutant, which is probably caused by decreased iron uptake rates, was also reflected by an increased resistance to superoxide stress. Iron induction of Pfr synthesis was clearly diminished in an
H. pylori feoB
mutant, which lacked high-affinity ferrous iron transport, confirming that Pfr expression is mediated by changes in the cytoplasmic iron pool and not by extracellular iron. This is well in agreement with the recent discovery that iron induces Pfr synthesis by abolishing Fur-mediated repression of
pfr
transcription, which was further confirmed here by the observation that iron inhibited the in vitro binding of recombinant
H. pylori
Fur to the
pfr
promoter region. The functions of
H. pylori
Pfr in iron metabolism are essential for survival in the gastric mucosa, as the
pfr
mutant was unable to colonize in a Mongolian gerbil-based animal model. In summary, the
pfr
phenotypes observed give new insights into prokaryotic ferritin functions and indicate that iron storage and homeostasis are of extraordinary importance for
H. pylori
to survive in its hostile natural environment.