bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of Siderophore Production on
Pseudomonas aeruginosa
Infections in Immunosuppressed Mice
Authors: Takase Hiroyuki , Nitanai Hironobu , Hoshino Kazuki , Otani Tsuyoshi 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
produces siderophores, pyoverdin and pyochelin, for high-affinity iron uptake. To investigate their contribution to
P. aeruginosa
infections, we constructed allelic exchange mutants from strain PAO1 which were deficient in producing one or both of the siderophores. When inoculated into the calf muscles of immunosuppressed mice, pyochelin-deficient and pyoverdin-deficient mutants grew and killed the animals as efficiently as PAO1. In contrast, the pyochelin- and pyoverdin-deficient (double) mutant did not show lethal virulence, although it did infect the muscles. On the other hand, when inoculated intranasally, all mutants grew in the lungs and killed immunosuppressed mice. Compared with PAO1, however, the pyoverdin-deficient mutant and the double mutant grew poorly in the lungs, and the latter was significantly attenuated for virulence. Irrespective of the inoculation route, the pyoverdin-deficient and doubly deficient mutants detected in the blood were significantly less numerous than PAO1. Additionally, in vitro examination demonstrated that the growth of the double mutant was extremely reduced under a free-iron-restricted condition with apotransferrin but that the growth reduction was completely canceled by supplementation with hemoglobin as a heme source. These results suggest that both pyoverdin and pyochelin are required for efficient bacterial growth and full expression of virulence in
P. aeruginosa
infection, although pyoverdin may be comparatively more important for bacterial growth and dissemination. However, the siderophores were not always required for infection. It is possible that non-siderophore-mediated iron acquisition, such as via heme uptake, might also play an important role in
P. aeruginosa
infections.
The
galE
Gene of
Campylobacter jejuni
Is Involved in Lipopolysaccharide Synthesis and Virulence
Authors: Fry Benjamin N. , Feng Shi , Chen Yuen-Yuen , Newell Diane G. , Coloe Peter J. , Korolik Victoria 
Published: July 2002

ABSTRACT

Lipopolysaccharide (LPS) is one of the main virulence factors of gram-negative bacteria. The LPS from
Campylobacter
spp. has endotoxic properties and has been shown to play a role in adhesion. We previously cloned a gene cluster (
wla
) which is involved in the synthesis of the
Campylobacter jejuni
81116 LPS molecule. Sequence alignment of the first gene in this cluster indicated similarity with
galE
genes. These genes encode a UDP-glucose 4-epimerase, which catalyzes the interconversion of UDP-galactose and UDP-glucose. A
Salmonella galE
mutant was transformed with the
galE
gene from
C. jejuni
. The LPS analysis of wild-type,
galE
, and complemented
galE Salmonella
strains showed that the
C. jejuni galE
gene could restore the smooth wild-type
Salmonella
LPS. A UDP-glucose 4-epimerase assay was used to demonstrate that the
galE
gene from
C. jejuni
encoded this epimerase. We constructed a
C. jejuni galE
mutant which expressed a lipid A-core molecule of reduced molecular weight that did not react with antiserum raised against the parental strain. These results show an essential role for the
galE
gene in the synthesis of
C. jejuni
LPS. The
galE
mutant also showed a reduction in its ability to adhere to and invade INT407 cells. However, it was still able to colonize chickens to the same level as the wild-type strain. The serum resistance and hemolytic activity of this mutant were not changed compared to the parent strain. The ability of the mutant to take up DNA and integrate it in its genome was reduced 20-fold. These results show that LPS of
C. jejuni
is an important virulence factor.
Immune Response to an 18-Kilodalton Outer Membrane Antigen Identifies Lipoprotein 20 as a
Helicobacter pylori
Vaccine Candidate
Authors: Keenan Jacqueline , Oliaro Jane , Domigan Neil , Potter Howard , Aitken Geoff , Allardyce Randall , Roake Justin 
Published: July 2002

ABSTRACT

Experiments were performed using the standardized murine model of
Helicobacter pylori
infection to determine the immunogenicity of
H. pylori
outer membrane vesicles in immune protection. These vesicles, which are naturally shed from the surface of the bacterium, induce a protective response when administered intragastrically to mice in the presence of cholera holotoxin, despite the absence of the urease enzyme and associated Hsp54 chaperonin. Immunoblotting identified a specific serum immunoglobulin G (IgG) response to an 18-kDa outer membrane protein in a significant number of immunized animals. This commonly expressed, immunodominant protein was subsequently identified as lipoprotein 20 (Lpp20). Hybridoma backpacks secreting an IgG1 subclass monoclonal antibody to Lpp20 were generated in
H. pylori
-infected mice and were found to significantly reduce bacterial numbers, providing evidence that this surface-exposed antigen is a true vaccine candidate and not merely an antigenic marker for successful, protective immunization.
Cooperation between Reactive Oxygen and Nitrogen Intermediates in Killing ofRhodococcus equiby Activated Macrophages
Authors: Darrah Patricia A. , Hondalus Mary K. , Chen Quiping , Ischiropoulos Harry , Mosser David M. 
Published: July 2002

ABSTRACTRhodococcus equiis a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense againstR. equiby using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-γ), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO−), which killsR. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS−/−) or the oxidative burst (gp91phox−/−) are more susceptible to lethalR. equiinfection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O2−) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays,R. equiremains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO−efficiently kills virulentR. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO−in situ. Inhibition of nitric oxide production by activated macrophages by usingNG-monomethyl-l-arginine blocks their production of ONOO−and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing ofR. equiby IFN-γ-activated macrophages.
Shigella flexneriIpaH7.8Facilitates Escape of Virulent Bacteria from the Endocytic Vacuoles of Mouse and Human Macrophages
Authors: Fernandez-Prada Carmen M. , Hoover David L. , Tall Ben D. , Hartman Antoinette B. , Kopelowitz June , Venkatesan Malabi M. 
Published: July 2002

ABSTRACTThe behavior ofShigella flexneri ipaHmutants was studied in human monocyte-derived macrophages (HMDM), in 1-day-old human monocytes, and in J774 mouse macrophage cell line. In HMDM, strain pWR700, anipaH7.8deletion mutant ofS. flexneri2a strain 2457T, behaved like the wild-type strain 2457T. This strain caused rapid host cell death by oncosis, and few bacterial CFU were recovered after incubation in the presence of gentamicin as previously described for 2457T-infected HMDM. However, analysis of bacterial compartmentalization within endocytic vacuoles with gentamicin and chloroquine indicated that more pWR700 than 2457T was present within the endocytic vacuoles of HMDM, suggesting thatipaH7.8deletion mutant transited more slowly from the vacuoles to the cytoplasm. In contrast to findings with HMDM, CFU recovered from pWR700-infected mouse J774 cells were 2 to 3 logs higher than CFU from 2457T-infected J774 cells. These values exceeded CFU recovered after infection of J774 cells with plasmid-cured avirulent strain M4243A1. Incubation with gentamicin and chloroquine clearly showed that pWR700 within J774 cells was mostly present within the endocytic vacuoles. This distribution pattern was similar to that seen with M4243A1 and contrasted with the pattern seen with 2457T. Complementation of pWR700 with a recombinant clone expressingipaH7.8restored the intracellular distribution of bacteria to that seen with the wild-type strain. Strains with deletions inipaH4.5oripaH9.8, however, behaved like 2457T in both HMDM and J774 cells. The distribution profile of pWR700 in 1-day-old monocytes was similar to that seen in J774 cells. Like infected J774 cells, 1-day-old human monocytes demonstrated apoptosis upon infection with virulentShigella. These results suggest that a role of theipaH7.8gene product is to facilitate the escape of the virulent bacteria from the phagocytic vacuole of monocytes and macrophages.
Characterization of a Streptococcal Endopeptidase with Homology to Human Endothelin-Converting Enzyme
Authors: Oetjen Joyce , Fives-Taylor Paula , Froeliger Eunice 
Published: July 2002

ABSTRACT

A gene encoding an endopeptidase from
Streptococcus parasanguis
FW213 has been cloned and shown to have high sequence homology to genes encoding mammalian metalloendopeptidases. The gene, designated
S. parasanguis pepO
, was cloned into the pET28a expression vector, resulting in a fusion of vector sequences encoding a hexahistidine tag at the carboxyl terminus. The recombinant PepO (rPepO) was expressed in
Escherichia coli
and purified using an Ni
2+
affinity column. Polyclonal antiserum to rPepO was raised in rabbits and used to localize FW213 PepO to the cytosol. Southern hybridization and immunoblot analysis revealed that other oral streptococci contain regions of DNA with homology to
pepO
and produce a protein with antigenic properties similar to that of FW213 PepO. Enzymatic activity assays indicated that only
S. parasanguis
species possess the ability to cleave metenkephalin, the natural substrate of the human neutral endopeptidase (NEP). Inhibition assays revealed that
S. parasanguis
PepO is a member of the M13 category of metalloendopeptidases, which includes NEP and endothelin-converting enzyme 1 (ECE-1), an enzyme involved in the maintenance of vascular tone. Thiorphan and phosphoramidon, two specific inhibitors of this category of endopeptidases, were used to determine that
S. parasanguis
PepO is more similar to ECE-1 than to NEP.
Gonococcal Genes Encoding Transferrin-Binding Proteins A and B Are Arranged in a Bicistronic Operon but Are Subject to Differential Expression
Authors: Ronpirin Chalinee , Jerse Ann E. , Cornelissen Cynthia Nau 
Published: July 2002

ABSTRACTNeisseria gonorrhoeaeis capable of utilizing host iron-binding proteins, such as transferrin, lactoferrin, and hemoglobin, as the sole source of iron. The receptor involved in transferrin iron acquisition is composed of two distinct transferrin-binding proteins, TbpA and TbpB. The genes that encode these proteins are linked on the chromosome in the ordertbpB-tbpAbut are separated by an inverted repeat of unknown function. In this study, we sought to understand the transcriptional organization and regulation of thetbpgenes, using a combination oflacZtranscriptional fusion analysis and reverse transcriptase PCR (RT-PCR). First, we demonstrated thattbpBandtbpAare cotranscribed and coregulated from the common upstream promoter that precedestbpB. Using β-galactosidase activity as a surrogate fortbp-specific transcription, we found thattbpB-specific transcripts were more prevalent thantbpA-specific transcripts after 2 h of growth under iron stress conditions. We confirmed the results obtained by fusion analysis by using RT-PCR applied to native RNA isolated from wild-type gonococci. Three different varieties of RT-PCR were employed: relative, competitive, and real time quantitative. The results of all analyses indicated thattbpB-specific transcripts were approximately twofold more prevalent thantbpA-specific transcripts at steady state. In iron-stressed cultures, the ratio oftbpB- totbpA-specific message was approximately 2; however, in iron-replete cultures, this ratio dropped to 1. Using these techniques, we also quantitated the effects of iron, external pH, and presence of ligand ontbpmRNA levels.
Mouse Strain-Dependent Chemokine Regulation of the Genital Tract T Helper Cell Type 1 Immune Response
Authors: Darville Toni , Andrews Charles W. , Sikes James D. , Fraley Patrick L. , Braswell Leah , Rank Roger G. 
Published: July 2002

ABSTRACTVaginal infection with the mouse pneumonitis agent ofChlamydia trachomatis(MoPn) produces shorter courses of infection in C57BL/6 and BALB/c mice than in C3H/HeN mice, while C57BL/6 mice are more resistant to oviduct pathology. A robust Th1 response is extremely important in host defense against chlamydia. In this study we examined gamma interferon (IFN-γ), interleukin 10 (IL-10), and the T-cell-regulatory chemokines macrophage inflammatory protein-1α (MIP-1α) and monocyte chemoattractant protein-1 (MCP-1) to determine if differences in these responses were associated with the differential courses of infection seen in these three strains of mice. Increased and prolonged IFN-γ responses and lower IL-10 responses were observed in the C57BL/6 strain compared to BALB/c and C3H. Examination of genital tract chemokines revealed a marked predominance of MIP-1α over MCP-1 only in the C57 strain. Thus, a pattern of high MIP-1α and low MCP-1 levels during the first week of infection is associated with an increased Th1 response and a shorter, more benign chlamydial infection. Inhibition of the MCP-1 response in C3H mice increased their later T-cell production of IFN-γ but decreased their early IFN-γ response and had no effect on the course or outcome of infection. Inhibition of MCP-1 is not beneficial in chlamydial infection because of its pleiotropic effects.
Molecular Characterization of a NovelStaphylococcus aureusSerine Protease Operon
Authors: Reed Samantha B. , Wesson Carla A. , Liou Linda E. , Trumble William R. , Schlievert Patrick M. , Bohach Gregory A. , Bayles Kenneth W. 
Published: July 2002

ABSTRACTThe present study identified and characterized a unique operon (spl) encoding six serine protease-like proteins. In addition, native Spl proteins were isolated and characterized. Typical of most exoproteins, thesplgene products contain putative 35- or 36-amino-acid signal peptides. The Spl proteins share 44 to 95% amino acid sequence identity with each other and 33 to 36% sequence identity with V8 protease. They also contain amino acids found in catalytic triads of enzymes in the trypsin-like serine protease family, and SplB and SplC were shown to degrade casein. Thesploperon is transcribed on a 5.5-kb transcript, but several nonrandom degradation products of this transcript were also identified. Similar to otherS. aureusexoprotein genes, thesploperon is maximally expressed during the transition into stationary phase and is positively controlled by the Agr virulence factor regulator. The Sar regulatory system did not affectsploperon expression. PCR analysis revealed the presence of thesploperon in 64% of theS. aureusisolates tested, although onesploperon-negative isolate was shown to contain at least two of thesplgenes. Finally, intraperitoneal injection of ansploperon deletion mutant revealed no major differences in virulence compared to the parental strain.
Role of Alginate O Acetylation in Resistance of MucoidPseudomonas aeruginosato Opsonic Phagocytosis
Authors: Pier Gerald B. , Coleman Fadie , Grout Martha , Franklin Michael , Ohman Dennis E. 
Published: July 2002

ABSTRACTEstablishment and maintenance of chronic lung infections with mucoidPseudomonas aeruginosain patients with cystic fibrosis (CF) require that the bacteria avoid host defenses. Elaboration of the extracellular, O-acetylated mucoid exopolysaccharide, or alginate, is a major microbial factor in resistance to immune effectors. Here we show that O acetylation of alginate maximizes the resistance of mucoidP. aeruginosato antibody-independent opsonic killing and is the molecular basis for the resistance of mucoidP. aeruginosato normally nonopsonic but alginate-specific antibodies found in normal human sera and sera of infected CF patients. O acetylation of alginate appears to be critical forP. aeruginosaresistance to host immune effectors in CF patients.
Evidence that the
Campylobacter fetus sap
Locus Is an Ancient Genomic Constituent with Origins before Mammals and Reptiles Diverged
Authors: Tu Zheng-Chao , Dewhirst Floyd E. , Blaser Martin J. 
Published: July 2002

ABSTRACT

Campylobacter fetus
bacteria, isolated from both mammals and reptiles, may be either subsp.
fetus
or subsp.
venerealis
and either serotype A or serotype B. Surface layer proteins, expressed and secreted by genes in the
sap
locus, play an important role in
C. fetus
virulence. To assess whether the
sap
locus represents a pathogenicity island and to gain further insights into
C. fetus
evolution, we examined several
C. fetus
genes in 18 isolates. All of the isolates had 5 to 9
sapA
or
sapB
homologs. One strain (85-387) possessed both
sapA
and
sapB
homologs, suggesting a recombinational event in the
sap
locus between
sapA
and
sapB
strains. When we amplified and analyzed nucleotide sequences from portions of housekeeping gene
recA
(501 bp) and
sapD
(450 bp), a part of the 6-kb
sap
invertible element, the phylogenies of the genes were highly parallel. Among the 15 isolates from mammals, serotype A and serotype B strains generally had consistent positions. The fact that the serotype A
C. fetus
subsp.
fetus
and subsp.
venerealis
strains were on the same branch suggests that their differentiation occurred after the type A-type B split. Isolates from mammals and reptiles formed two distinct tight phylogenetic clusters that were well separated. Sequence analysis of 16S rRNA showed that the reptile strains form a distinct phylotype between mammalian
C. fetus
and
Campylobacter hyointestinalis
. The phylogenies and sequence results showing that
sapD
and
recA
have similar G + C contents and substitution rates suggest that the
sap
locus is not a pathogenicity island but rather is an ancient constituent of the
C. fetus
genome, integral to its biology.
Cloning, Expression, and Catalytic Activity of
Helicobacter hepaticus
Urease
Authors: Beckwith Catherine S. , McGee David J. , Mobley Harry L. T. , Riley Lela K. 
Published: July 2002

ABSTRACT

Helicobacter hepaticus
causes disease in the liver and lower intestinal tract of mice. It is strongly urease positive, although it does not live in an acidic environment. The
H. hepaticus
urease gene cluster was expressed in
Escherichia coli
with and without coexpression of the
Helicobacter pylori
nickel transporter NixA. As for
H. pylori
, it was difficult to obtain enzymatic activity from recombinant
H. hepaticus
urease; special conditions including NiCl
2
supplementation were required. The
H. hepaticus
urease cluster contains a homolog of each gene in the
H. pylori
urease cluster, including the urea transporter gene
ureI
. Downstream genes were homologs of the
nik
nickel transport operon of
E. coli
. Nongastric
H. hepaticus
produces urease similar to that of
H. pylori
.
Enhanced Delivery of Exogenous Peptides into the Class I Antigen Processing and Presentation Pathway
Authors: de Haan Lolke , Hearn Arron R. , Rivett A. Jennifer , Hirst Timothy R. 
Published: July 2002

ABSTRACTCurrent immunization strategies, using peptide or protein antigens, generally fail to elicit cytotoxic-T-lymphocyte responses, since these antigens are unable to access intracellular compartments where loading of major histocompatibility complex class I (MHC-I) molecules occurs. In an attempt to circumvent this, we investigated whether the GM1 receptor-binding B subunit ofEscherichia coliheat-labile toxin (EtxB) could be used to deliver class I epitopes. When a class I epitope was conjugated to EtxB, it was delivered into the MHC-I presentation pathway in a GM1-binding-dependent fashion and resulted in the appearance of MHC-I-epitope complexes at the cell surface. Importantly, we show that the efficiency of EtxB-mediated epitope delivery could be strikingly enhanced by incorporating, adjacent to the class I epitope, a 10-amino-acid segment from the C terminus of the DNA polymerase (Pol) of herpes simplex virus. The replacement of this 10-amino-acid segment by a heterologous sequence or the introduction of specific amino acid substitutions within this segment either abolished or markedly reduced the efficiency of class I epitope delivery. If the epitope was extended at its C terminus, EtxB-mediated delivery into the class I presentation pathway was found to be completely dependent on proteasome activity. Thus, by combining the GM1-targeting function of EtxB with the 10-amino-acid Pol segment, highly efficient delivery of exogenous epitopes into the endogenous pathway of class I antigen processing and presentation can be achieved.
Tol1, a Fission Yeast Phosphomonoesterase, Is an In Vivo Target of Lithium, and Its Deletion Leads to Sulfite Auxotrophy
Authors: Miyamoto Rumi , Sugiura Reiko , Kamitani Shinya , Yada Tomoko , Lu Yabin , Sio Susie O. , Asakura Masahiro , Matsuhisa Akio , Shuntoh Hisato , Kuno Takayoshi 
Published: July 2002

ABSTRACT

Lithium is the drug of choice for the treatment of bipolar affective disorder. The identification of an in vivo target of lithium in fission yeast as a model organism may help in the understanding of lithium therapy. For this purpose, we have isolated genes whose overexpression improved cell growth under high LiCl concentrations. Overexpression of
tol1
+
, one of the isolated genes, increased the tolerance of wild-type yeast cells for LiCl but not for NaCl.
tol1
+
encodes a member of the lithium-sensitive phosphomonoesterase protein family, and it exerts dual enzymatic activities, 3′(2′),5′-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase.
tol1
+
gene-disrupted cells required high concentrations of sulfite in the medium for growth. Consistently, sulfite repressed the sulfate assimilation pathway in fission yeast. However,
tol1
+
gene-disrupted cells could not fully recover from their growth defect and abnormal morphology even when the medium was supplemented with sulfite, suggesting the possible implication of inositol polyphosphate 1-phosphatase activity for cell growth and morphology. Given the remarkable functional conservation of the lithium-sensitive dual-specificity phosphomonoesterase between fission yeast and higher-eukaryotic cells during evolution, it may represent a likely in vivo target of lithium action across many species.
Cloning of the Gene Coding for
Staphylococcus hyicus
Exfoliative Toxin B and Its Expression in
Escherichia coli
Authors: Watanabe Takao , Sato Hisaaki , Hatakeyama Yu , Matsuzawa Takeshi , Kawai Masanori , Aizawa Chikara , Danbara Hirofumi , Maehara Nobutoshi 
Published: July 2002

ABSTRACT

The
Staphylococcus hyicus
exfoliative toxin B (SHETB) gene was cloned into pUC118 and expressed in
Escherichia coli
. The nucleotide sequence of the SHETB gene consists of a coding region of 804 bp specifying a polypeptide of 268 amino acid residues, which included a putative 20-residue signal sequence.