bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical and Biological Properties of Staphylococcal Enterotoxin K
Authors: Orwin Paul M. , Leung Donald Y. M. , Donahue Heather L. , Novick Richard P. , Schlievert Patrick M.
Published: July 2002

Staphylococcus aureus
is an important human pathogen which is implicated in a wide variety of diseases. Major determinants of the virulence of this organism include extracellular virulence factors. Staphylococcal enterotoxins (SEs) are important causative agents in staphylococcal toxic shock syndrome and food poisoning. Our study identified a novel enterotoxin, SEK, and examined its biochemical and biological properties. SEK had a molecular weight of 26,000 and an experimentally determined pI of between 7.0 and 7.5. SEK was secreted by clinical isolates of
S. aureus
. We demonstrated that SEK had many of the biological activities associated with the SEs, including superantigenicity, pyrogenicity, the ability to enhance the lethal effect of endotoxin, and lethality in a rabbit model when administered by subcutaneous miniosmotic pump. Recombinant SEK was shown to stimulate human CD4
+
and CD8
+
T cells in a Vβ-specific manner; T-cells bearing Vβ 5.1, 5.2, and 6.7 were significantly stimulated to proliferate.
Identification and Disruption of Two Discrete Loci Encoding Hyaluronic Acid Capsule Biosynthesis Genes
hasA
,
hasB
, and
hasC
in
Streptococcus uberis
Authors: Ward Philip N. , Field Terence R. , Ditcham William G. F. , Maguin Emmanuelle , Leigh James A.
Published: July 2002

The hyaluronic acid capsule of
Streptococcus uberis
has been implicated in conferring resistance to phagocytosis by bovine neutrophils. Construction of a bank of random insertion mutants of
S. uberis
(strain 0140J) was achieved using the pGh9::IS
S1
mutagenesis system (22). Phenotypic screening of approximately 5,000 clones enabled the isolation of 11 acapsular mutants. Southern hybridization indicated that two mutants carried a lesion within a group of genes similar to those involved in the assembly of the hyaluronic acid capsule found in the group A
Streptococcus
(GAS)
has
operon. The DNA sequence flanking the points of insertion confirmed the presence of homologues of GAS
hasA
and
hasB
in
S. uberis
. The DNA sequence flanking the IS
S1
insertion in another mutant identified a homologue of
hasC
in
S. uberis
. The GAS
hasABC
operon structure was not conserved in
S. uberis
, and two discrete loci comprising homologues of either
hasAB
or
hasC
were identified. Disruption of
S. uberis hasA
or
hasC
resulted in the complete cessation of hyaluronic acid capsule production. Correspondingly, these mutants were found to have lost their resistance to phagocytosis by bovine neutrophils. The bactericidal action of bovine neutrophils on
S. uberis
0140J was shown unequivocally to depend upon the capsule status of the bacterium.
Staphylococcus aureus agrGenotypes with Enterotoxin Production Capabilities Can Resist Neutrophil Bactericidal Activity
Authors: Mullarky I. K. , Su C. , Frieze N. , Park Y. H. , Sordillo L. M.
Published: July 2002

Staphylococcus aureuspathogenicity is mainly due to the production of a number of secreted and cell surface-associated proteins under the regulation of theagrgene. A region of theagrgene was used to subgroupS. aureusstrains according to restriction fragment length polymorphisms. Additionally, strains were subtyped according to the coagulase gene in order to strengthen discriminatory power. Virulence capabilities ofagrgenotype subgroups were evaluated using an in vitro neutrophil bactericidal assay, which showed that prevalent genotypes were significantly better at evading this primary host defense. Multiplex PCR was then used to detect enterotoxin genes among the genotype subgroups in order to determine possible virulence candidates that enable strains to combat neutrophil killing. The prevalent genotype strains were found to possess higher production capabilities for enterotoxin A than did low-prevalence strains. The significance of enterotoxin A production capabilities in affecting pathogenicity ofS. aureusstrains was evaluated and found to have a profound effect on neutrophil killing abilities. The use of a large epidemiological database as a tool for subgrouping strains with varying degrees of pathogenicity has allowed the identification of relevant and previously undefined virulence factors that affect a pathogen's capability to overcome host immune defenses.
Brucella suis
-Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications
Authors: Naroeni Aroem , Jouy Nicolas , Ouahrani-Bettache Safia , Liautard Jean-Pierre , Porte Françoise
Published: July 2002

Brucella
species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in
Brucella
maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of
Brucella
and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between
Brucella suis
-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live
Brucella suis
in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live
Brucella
in the cell. By in vitro reconstitution experiments, we observed a specific association between killed
B. suis
-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments.
Role of ADP-Ribosyltransferase Activity of Pertussis Toxin in Toxin-Adhesin Redundancy with Filamentous Hemagglutinin duringBordetella pertussisInfection
Authors: Alonso Sylvie , Pethe Kévin , Mielcarek Nathalie , Raze Dominique , Locht Camille
Published: July 2002

Pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two major virulence factors ofBordetella pertussis. FHA is the main adhesin, whereas PT is a toxin with an A-B structure, in which the A protomer expresses ADP-ribosyltransferase activity and the B moiety is responsible for binding to the target cells. Here, we show redundancy of FHA and PT during infection. Whereas PT-deficient and FHA-deficient mutants colonized the mouse respiratory tract nearly as efficiently as did the isogenic parent strain, a mutant deficient for both factors colonized substantially less well. This was not due to redundant functions of PT and FHA as adhesins, since in vitro studies of epithelial cells and macrophages indicated that FHA, but not PT, acts as an adhesin. An FHA-deficientB.pertussisstrain producing enzymatically inactive PT colonized as poorly as did the FHA-deficient, PT-deficient strain, indicating that the ADP-ribosyltransferase activity of PT is required for redundancy with FHA. Only strains producing active PT induced a local transient release of tumor necrosis factor alpha (TNF-α), suggesting that the pharmacological effects of PT are the basis of the redundancy with FHA, through the release of TNF-α. This may lead to damage of the pulmonary epithelium, allowing the bacteria to colonize even in the absence of FHA.
Identification of the Hemolysis-Associated Protein 1 as a Cross-Protective Immunogen ofLeptospira interrogansby Adenovirus-Mediated Vaccination
Authors: Branger C. , Sonrier C. , Chatrenet B. , Klonjkowski B. , Ruvoen-Clouet N. , Aubert A. , André-Fontaine G. , Eloit M.
Published: July 2002

New vaccine strategies are needed for the prevention of leptospirosis, a widespread human and animal disease caused by pathogenic leptospires. Our previous work determined that a protein leptospiral extract conferred cross-protection in a gerbil model of leptospirosis. The 31- to 34-kDa protein fraction ofLeptospira interrogansserovar autumnalis was shown sufficient for this purpose. In the present study, N-terminal sequencing of a 32-kDa fraction and Southern blotting of genomic DNA with corresponding degenerated oligonucleotide probes identified two of its constituents: hemolysis-associated protein 1 (Hap1) and the outer membraneLeptospiraprotein 1 (OmpL1). Adenovirus-mediated Hap1 vaccination induces significant protection against a virulent heterologousLeptospirachallenge in gerbils, whereas a similar OmpL1 construct failed to protect the animals. These data indicate that Hap1 could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease.
Sera from Adult Patients with Cystic Fibrosis Contain Antibodies to
Pseudomonas aeruginosa
Type III Apparatus
Authors: Moss Joel , Ehrmantraut Mary E. , Banwart Bruce D. , Frank Dara W. , Barbieri Joseph T.
Published: July 2002

Expression of type III proteins of
Pseudomonas aeruginosa
in patients with cystic fibrosis (CF) was investigated by measuring the immune response against components of the type III pathway. Twenty-three of the 33 sera contained antibodies against PcrV, a protein involved in translocation of type III cytotoxins into eukaryotic cells, and 11 of 33 had antibodies against ExoS, while most CF sera contained antibodies against PopB and PopD, components of the type III apparatus. These data indicate that
P. aeruginosa
commonly expresses components of the type III translocation apparatus in adult CF patients.
Cytotoxic T Lymphocyte Antigen Costimulation Influences T-Cell Activation in Response toCryptococcus neoformans
Authors: Pietrella Donatella , Perito Stefano , Bistoni Francesco , Vecchiarelli Anna
Published: July 2002

The kinetics of cytotoxic T lymphocyte antigen 4 (CTLA-4) expression on T cells responding toCryptococcus neoformansand its role in regulating the T-cell response were examined. Using peripheral blood mononuclear cells stimulated with encapsulated or acapsularC. neoformanswe showed that (i) the encapsulated strain augmented CTLA-4 expression on the T-cell surface while the acapsular strain was a weaker modulator, (ii) CTLA-4 molecules were rapidly up-regulated after the addition of encapsulatedC. neoformans, (iii) CTLA-4 was up-regulated predominantly in CD4+T cells responding toC. neoformans, and (iv) blockage of CTLA-4 with (Fab′)2of monoclonal antibody to CTLA-4 induced T-cell proliferation that paralleled the enhancement of interleukin-2 and gamma interferon production. These results suggest that capsular material, the major virulence factor ofC. neoformans, promotes synthesis and expression of CTLA-4 molecules predominantly in CD4+T cells. CTLA-4-mediated deactivation is due not to lack of costimulation but to specific recognition of CTLA-4 for B7 molecules. This appears to be a new mechanism by whichC. neoformansmay elude the host immune response.
Micrococcus luteusTeichuronic Acids Activate Human and Murine Monocytic Cells in a CD14- and Toll-Like Receptor 4-Dependent Manner
Authors: Yang Shuhua , Sugawara Shunji , Monodane Toshihiko , Nishijima Masahiro , Adachi Yoshiyuki , Akashi Sachiko , Miyake Kensuke , Hase Sumihiro , Takada Haruhiko
Published: July 2002

Teichuronic acid (TUA), a component of the cell walls of the gram-positive organismMicrococcus luteus(formerlyMicrococcus lysodeikticus), induced inflammatory cytokines in C3H/HeN mice but not in lipopolysaccharide (LPS)-resistant C3H/HeJ mice that have a defect in the Toll-like receptor 4 (TLR4) gene, both in vivo and in vitro, similarly to LPS (T. Monodane, Y. Kawabata, S. Yang, S. Hase, and H. Takada, J. Med. Microbiol. 50:4–12, 2001). In this study, we found that purified TUA (p-TUA) induced tumor necrosis factor alpha (TNF-α) in murine monocytic J774.1 cells but not in mutant LR-9 cells expressing membrane CD14 at a lower level than the parent J774.1 cells. The TNF-α-inducing activity of p-TUA in J774.1 cells was completely inhibited by anti-mouse CD14 monoclonal antibody (MAb). p-TUA also induced interleukin-8 (IL-8) in human monocytic THP-1 cells differentiated to macrophage-like cells expressing CD14. Anti-human CD14 MAb, anti-human TLR4 MAb, and synthetic lipid A precursor IVA, an LPS antagonist, almost completely inhibited the IL-8-inducing ability of p-TUA, as well as LPS, in the differentiated THP-1 cells. Reduced p-TUA did not exhibit any activities in J774.1 or THP-1 cells. These findings strongly suggested thatM. luteusTUA activates murine and human monocytic cells in a CD14- and TLR4-dependent manner, similar to LPS.
Micrococci and Peptidoglycan Activate TLR2→MyD88→IRAK→TRAF→NIK→IKK→NF-κB Signal Transduction Pathway That Induces Transcription of Interleukin-8
Authors: Wang Qiuling , Dziarski Roman , Kirschning Carsten J. , Muzio Marta , Gupta Dipika
Published: July 2002

This study was done to elucidate the signal transduction pathway of interleukin-8 (IL-8) induction by gram-positive bacteria. Bacteria (micrococci) and peptidoglycan (PGN) induced transcription of IL-8 in HEK293 cells expressing Toll-like receptor 2 (TLR2) and CD14 but not in those expressing TLR1 or TLR4. A mutation within the NF-κB site in the IL-8 promoter abrogated transcriptional induction of IL-8 by the two stimulants. Dominant negative myeloid differentiation protein (MyD88), IL-1 receptor-associated kinase (IRAK), NFκB-inducing kinase (NIK), and IκB kinase (IKK) mutant forms completely inhibited micrococcus- and PGN-induced activation of NF-κB and expression of the gene for IL-8. Induction of NF-κB was partially inhibited by dominant negative tumor necrosis factor receptor-associated kinase 6 (TRAF6) but not TRAF2, whereas induction of IL-8 gene was partially inhibited by both TRAF6 and TRAF2. These data indicate that micrococci and PGN induce TLR2-dependent activation of the gene for IL-8 and that this activation requires MyD88, IRAK, NIK, IKK, and NF-κB and may also utilize TRAF6 and, to a lesser extent, TRAF2.
Effective In Vitro Clearance ofPorphyromonas gingivalisby Fcα Receptor I (CD89) on Gingival Crevicular Neutrophils
Authors: Kobayashi Tetsuo , Yamamoto Kouji , Sugita Noriko , van Spriel Annemiek B. , Kaneko Susumu , van de Winkel Jan G. J. , Yoshie Hiromasa
Published: July 2002

Porphyromonas gingivalishas been implicated as a causative pathogen in periodontitis. Immunotherapeutic approaches have recently been suggested to aid in the clearance ofP. gingivalisfrom disease sites. Because antibody-Fc receptor (FcR) interactions play a role in the effector functions of polymorphonuclear neutrophils (PMN), we evaluated which FcR on PMN from gingival crevicular fluid (GCF) serves as an optimal target molecule for FcR-directed immunotherapy. GCF PMN and peripheral blood (PB) PMN from adult periodontitis patients were analyzed for their immunoglobulin G (IgG) and IgA FcR (FcγR and FcαR, respectively) expression and function by studying IgG- and IgA-mediated elimination ofP. gingivalis. GCF PMN exhibited higher FcαRI and FcγRI levels and lower FcγRIIa and FcγRIIIb levels than PB PMN. Functional studies revealed that GCF PMN exhibited less of a capacity to phagocytose and kill IgG1-opsonizedP. gingivalisthan PB PMN. IgA1-mediated phagocytosis and killing capacity was, however, comparable between GCF PMN and PB PMN. In summary, these in vitro results document that FcαRI represents a candidate target for FcR-directed immunotherapy for the clearance ofP. gingivalis.
Antigenic Diversity among
Helicobacter pylori
Vacuolating Toxins
Authors: Vinion-Dubiel Arlene D. , McClain Mark S. , Cao Ping , Mernaugh Raymond L. , Cover Timothy L.
Published: July 2002

Helicobacter pylori
vacuolating cytotoxin (VacA) is a secreted protein that induces vacuolation of epithelial cells. To study VacA structure and function, we immunized mice with purified type s1-m1 VacA from
H. pylori
strain 60190 and generated a panel of 10 immunoglobulin G1κ anti-VacA monoclonal antibodies. All of the antibodies reacted with purified native VacA but not with denatured VacA, suggesting that these antibodies react with conformational epitopes. Seven of the antibodies reacted with both native and acid-treated VacA, which suggests that epitopes present on both oligomeric and monomeric forms of the toxin were recognized. Two monoclonal antibodies, both reactive with epitopes formed by amino acids in the carboxy-terminal portion of VacA (amino acids 685 to 821), neutralized the cytotoxic activity of type s1-m1 VacA when toxin and antibody were mixed prior to cell contact but failed to neutralize the cytotoxic activity of type s1-m2 VacA. Only 3 of the 10 antibodies consistently recognized type s1-m1 VacA toxins from multiple
H. pylori
strains, and none of the antibodies recognized type s2-m2 VacA toxins. These results indicate that there is considerable antigenic diversity among VacA toxins produced by different
H. pylori
strains.
Primary and Booster Mucosal Immune Responses to Meningococcal Group A and C Conjugate and Polysaccharide Vaccines Administered to University Students in the United Kingdom
Authors: Zhang Q. , Lakshman R. , Burkinshaw R. , Choo S. , Everard J. , Akhtar S. , Finn A.
Published: July 2002

Meningococcal group A+C capsular polysaccharide (PS) conjugate vaccines may prime for serum immunoglobulin G (IgG) memory responses to meningococcal capsular PS. It is not known whether these vaccines induce immunological memory at the mucosal level, which may be important in reducing nasopharyngeal carriage. Mucosal immune responses to meningococcal conjugate and PS vaccines in young adults were investigated. Healthy university students were randomized to receive either a groups A+C meningococcal conjugate vaccine (MACconj,n= 100) or a group A+C meningococcal PS vaccine (MACPS,n= 95). One year after the primary immunization, both groups were randomized again to receive a MACconj or a MACPS booster vaccination. Saliva samples were collected before and 1 month after the primary and booster vaccinations. Anti-meningococcal A (MenA) and C (MenC) PS IgA and IgG antibody levels were measured by a standard enzyme-linked immunosorbent assay. After the primary vaccination, salivary MenA and MenC IgG and MenA IgA concentrations were significantly increased after immunization with both MACconj and MACPS vaccines, but the salivary Men C IgA level was increased only after MACPS vaccine (P< 0.01). IgA responses to both serogroups were greater for MACPS than MACconj vaccine (P< 0.05), whereas no significant differences were seen for IgG responses. MenA IgG titers were higher after the MACPS booster in MACconj-primed subjects than after the MACPS primary vaccination, suggesting the presence of IgG memory. Antibody responses to a dose of either MACPS or MACconj were not significantly reduced in those previously given MACPS compared to the primary responses to those vaccines. Meningococcal A+C conjugate and PS vaccines induce significant mucosal responses in young adults. MACconj priming may induce IgG memory at the mucosal level, which is likely to be a reflection of an anamnestic serum IgG response. No evidence of mucosal hyporesponsiveness was observed after MACPS priming in this study.
In Vitro and In Vivo Assessment ofSalmonella entericaSerovar Typhimurium DT104 Virulence
Authors: Allen Chris A. , Fedorka-Cray Paula J. , Vazquez-Torres Andrés , Suyemoto Mitsu , Altier Craig , Ryder L. Reeni , Fang Ferric C. , Libby Stephen J.
Published: July 2002

Multidrug-resistantSalmonella entericaserovar Typhimurium phage type DT104 has become a widespread cause of human and other animal infection worldwide. The severity of clinical illness inS. entericaserovar Typhimurium DT104 outbreaks has led to the suggestion that this strain possesses enhanced virulence. In the present study, in vitro and in vivo virulence-associated phenotypes of several clinical isolates ofS. entericaserovar Typhimurium DT104 were examined and compared toS. entericaserovar Typhimurium ATCC 14028s. The ability of these DT104 isolates to survive within murine peritoneal macrophages, invade cultured epithelial cells, resist antimicrobial actions of reactive oxygen and nitrogen compounds, and cause lethal infection in mice were assessed. Our results failed to demonstrate thatS. entericaserovar Typhimurium DT104 isolates are more virulent thanS. entericaserovar Typhimurium ATCC 14028s.
Contribution of Mn-Cofactored Superoxide Dismutase (SodA) to the Virulence of
Streptococcus agalactiae
Authors: Poyart Claire , Pellegrini Elisabeth , Gaillot Olivier , Boumaila Claire , Baptista Marina , Trieu-Cuot Patrick
Published: July 2002

Superoxide dismutases convert superoxide anions to molecular oxygen and hydrogen peroxide, which, in turn, is metabolized by catalases and/or peroxidases. These enzymes constitute one of the major defense mechanisms of cells against oxidative stress and hence play a role in the pathogenesis of certain bacteria. We previously demonstrated that group B streptococci (GBS) possess a single Mn-cofactored superoxide dismutase (SodA). To analyze the role of this enzyme in the pathogenicity of GBS, we constructed a
sodA
-disrupted mutant of
Streptococcus agalactiae
NEM316 by allelic exchange. This mutant was subsequently
cis
complemented by integration into the chromosome of pAT113/Sp harboring the wild-type
sodA
gene. The SOD specific activity detected by gel analysis in cell extracts confirmed that active SODs were present in the parental and complemented strains but absent in the
sodA
mutant. The growth rates of these strains in standing cultures were comparable, but the
sodA
mutant was extremely susceptible to the oxidative stress generated by addition of paraquat or hydrogen peroxide to the culture medium and exhibited a higher mutation frequency in the presence of rifampin. In mouse bone marrow-derived macrophages, the
sodA
mutant showed an increased susceptibility to bacterial killing by macrophages. In a mouse infection model, after intravenous injection the survival of the
sodA
mutant in the blood and the brain was markedly reduced in comparison to that of the parental and complemented strains whereas only minor effects on survival in the liver and the spleen were observed. These results suggest that SodA plays a role in GBS pathogenesis.
ADP-Ribosylation of Rab5 by ExoS of
Pseudomonas aeruginosa
Affects Endocytosis
Authors: Barbieri Alejandro M. , Sha Qun , Bette-Bobillo Pascale , Stahl Philip D. , Vidal Michel
Published: July 2002

Pseudomonas aeruginosa
exoenzyme S (ExoS) is an ADP-ribosyltransferase that modifies low-molecular-weight GTPases. Here we studied the effect of Rab5 ADP-ribosylation by ExoS on its cellular function, i.e., regulation of early endocytic events. Coculture of CHO cells with
P. aeruginosa
induced a marked decrease in horseradish peroxidase (HRP) uptake compared to noninfected cells, while coculture with a
P. aeruginosa
mutant strain that fails to produce ExoS did not lead to any change in HRP uptake. Microinjection of recombinant ExoS into
Xenopus
oocytes induced strong inhibition of basal HRP uptake by oocytes. Moreover, coinjection of recombinant ExoS with Rab5 abolished the typical stimulation of HRP uptake obtained after GTPase microinjection. Cytosols prepared from injected oocytes were used in an endosome-endosome fusion assay. Cytosol from ExoS-microinjected oocytes was ineffective in promoting endosome-endosome fusion. However, in these conditions, the addition of Rab5 to the assay led to fusion recovery. Finally, we found that the interaction of Rab5 with EEA1 was markedly diminished after Rab5 ADP-ribosylation by ExoS.
Isolation and Characterization of a Mutant Chinese Hamster Ovary Cell Line That Is Resistant to
Chlamydia trachomatis
Infection at a Novel Step in the Attachment Process
Authors: Carabeo Reynaldo A. , Hackstadt Ted
Published: July 2002

Host factors involved in
Chlamydia trachomatis
pathogenesis were investigated by random chemical mutagenesis of Chinese hamster ovary (CHO-K1) cells followed by selection for clones resistant to chlamydial infection. A clonal mutant cell line, D4.1–3, refractory to infection by the
C. trachomatis
L2 serovar was isolated. The D4.1–3 cell line appears to be lacking in a previously undescribed temperature-dependent and heparin-resistant binding step that occurs subsequent to engagement of cell surface heparan sulfate by L2 elementary bodies. This novel binding step differentiates the lymphogranuloma venereum (LGV) serovar from other serovars and may contribute the different pathologies associated with LGV and non-LGV strains.
Characterization of a Cytotoxic Factor in Culture Filtrates of
Serratia marcescens
Authors: Marty Kent B. , Williams Christopher L. , Guynn Linda J. , Benedik Michael J. , Blanke Steven R.
Published: July 2002

Serratia marcescens
culture filtrates have been reported to be cytotoxic to mammalian cells. Using biochemical and genetic approaches, we have identified a major source of this cytotoxic activity. Both heat and protease treatments abrogated the cytotoxicity of
S. marcescens
culture filtrates towards HeLa cells, suggesting the involvement of one or more protein factors. A screen for in vitro cytotoxic activity revealed that
S. marcescens
mutant strains that are deficient in production of a 56-kDa metalloprotease are significantly less cytotoxic to mammalian cells. Cytotoxicity was significantly reduced when culture filtrates prepared from wild-type strains were pretreated with either EDTA or 1,10-phenanthroline, which are potent inhibitors of the 56-kDa metalloprotease. Furthermore, cytotoxic activity was restored when the same culture filtrates were incubated with zinc divalent cations, which are essential for enzymatic activity of the 56-kDa metalloprotease. Finally, recombinant expression of the
S. marcescens
56-kDa metalloprotease conferred a cytotoxic phenotype on the culture filtrates of a nonpathogenic
Escherichia coli
strain. Collectively, these data suggest that the 56-kDa metalloprotease contributes significantly to the in vitro cytotoxic activity commonly observed in
S. marcescens
culture filtrates.
Extracellular Antigens fromSalmonella enteritidisInduce Effective Immune Response in Mice after Oral Vaccination
Authors: Strindelius Lena , Wikingsson Lena Degling , Sjöholm Ingvar
Published: July 2002

We have studied polyacryl starch microparticles as an adjuvant in oral vaccination in mice. Secreted antigens fromSalmonella entericaserovar Enteritidis were administered covalently conjugated to microparticles, or as free antigens, orally or intramuscularly and evaluated for their immunogenicity and ability to elicit protective immune response against an oral challenge with live serovar Enteritidis. The highest immunoglobulin M (IgM)-plus-IgG titers were obtained in the groups immunized with antigen-conjugated microparticles. The subclass profile switched to a stronger Th1 influence in the oral groups after booster, while the intramuscular group showed a constant Th1/Th2 profile. A strong specific IgA response was seen in feces in the oral groups, which was further confirmed in an enzyme-linked immunospot assay. The delayed-type hypersensitivity test, as a measure of the cellular response, showed a significant increase in ear thickness in all the immunized groups, except for the group that received free antigen orally, compared to the nonimmunized group. The cytokines released from in vitro-stimulated spleens showed a strong gamma interferon response in all immunized groups. A significant reduction in CFU in liver and spleen was seen in the orally immunized groups compared to the nonimmunized group after oral challenge with serovar Enteritidis. Western blotting analysis with both sera and feces revealed that antibodies against three bands, 53, 56, and 60 kDa, dominated the oral groups, and an electrospray-mass spectroscopy analysis of these bands showed amino acid sequences coinciding with those of phase-1 flagellin and hook-associated protein 2.
Role of Pneumococcal Surface Protein C in Nasopharyngeal Carriage and Pneumonia and Its Ability To Elicit Protection against Carriage of
Streptococcus pneumoniae
Authors: Balachandran Priya , Brooks-Walter Alexis , Virolainen-Julkunen Anni , Hollingshead Susan K. , Briles David E.
Published: July 2002

Previous studies suggested that PspC is important in adherence and colonization within the nasopharynx. In this study, we conducted mutational studies to further identify the role PspC plays in the pathogenesis of pneumococci.
pspC
and/or
pspA
was insertionally inactivated in a serotype 2
Streptococcus pneumoniae
strain and in a serotype 19
S. pneumoniae
strain. In the mouse colonization model, pneumococcal strains with mutations in
pspC
were significantly attenuated in their abilities to colonize. In a mouse pneumonia model, strains with mutations in
pspC
were unable to infect or multiply within the lung. Using reverse transcriptase PCR we were able to demonstrate that
pspC
is actively transcribed in vivo, when the bacteria are growing in the nasal cavity and in the lungs. In the bacteremia model, a strain mutated for
pspC
alone behaved like the wild type, but the absence of both
pspC
and
pspA
caused accelerated clearance of the bacteria. Intranasal immunization with PspC with cholera toxin subunit B as an adjuvant protected against intranasal challenge. Evidence was also obtained that revertants that spontaneously acquired PspC expression could multiply and colonize the nasal tissue. This latter finding strongly indicates that pneumococci are actively metabolizing and growing while in the nasopharynx.
Monocyte Differentiation in Localized Juvenile Periodontitis Is Skewed toward the Dendritic Cell Phenotype
Authors: Barbour Suzanne E. , Ishihara Yuichi , Fakher Mohammed , Al-Darmaki Salma , Caven Timothy H. , Shelburne C. P. , Best Al M. , Schenkein Harvey A. , Tew John G.
Published: July 2002

Several lines of evidence indicate that the monocytes of subjects with localized juvenile periodontitis (LJP) are functionally distinct from cells of age- and race-matched nonperiodontitis (NP) subjects. Among the abnormalities are the propensity to secrete large amounts of prostaglandin E2and the induction of immunoglobulin G2 (IgG2) antibodies. The experiments described here were performed to further characterize the LJP monocytes and to determine if these cells mature differently than NP monocytes. When adherent monocytes from LJP subjects were cultured in the presence of human serum, both macrophages and cells with the morphology of immature monocyte-derived dendritic cells (MDDC) were observed. Within 4 days the prevalence of the immature MDDC was approximately twofold higher in LJP cultures than in NP cultures. In addition to their dendritic morphology, these cells were CD11c+and CD14−or CD14lowand stimulated potent autologous mixed leukocyte reactions, consistent with differentiation to the MDDC phenotype. Like LJP monocytes, cultures of MDDC generated with interleukin-4 and granulocyte-macrophage colony-stimulating factor selectively induced IgG2 in cultures of pokeweed mitogen-stimulated NP leukocytes. Together, these data suggest that the monocytes of LJP subjects have a propensity to differentiate into MDDC and that this differentiation may be related to the high levels of IgG2 that are observed in the sera of LJP subjects. As high levels of circulating IgG2 are correlated with less severe disease, the propensity of LJP monocytes to differentiate into MDDC may have important implications for both the host response against oral pathogens and the progression of LJP.
Identification of a Disulfide Isomerase Protein ofLeishmania majoras a Putative Virulence Factor
Authors: Achour Y. Ben , Chenik M. , Louzir H. , Dellagi K.
Published: July 2002

Several approaches have been previously used to elucidate the genetic basis ofLeishmaniavirulence. In general, they were based on laboratoryLeishmaniaclones genetically modified or grown in the presence of selecting agents. In a previous study, we demonstrated thatLeishmania majorfreshly isolated from human cutaneous lesions showed significant differences in the severity of the experimental disease induced in BALB/c mice. Here, using the mRNA differential display technique, we analyzed gene expression inL. majorpromastigotes showing different levels of virulence. We have identified a novelLeishmaniagene encoding a 477-amino-acid protein exhibiting two distinct regions that are identical to the putative active-site sequence (CGHC) of the eukaryotic protein disulfide isomerase (PDI). The recombinant protein displayed a specific PDI enzymatic activity. ThisL. majordisulfide isomerase protein (LmPDI) is predominantly expressed, at both the mRNA and protein levels, in highly virulent strains. Specific PDI inhibitors abolished the enzymatic activity of the recombinant protein and profoundly affected parasite growth. These findings suggest that LmPDI may play an important role inLeishmanianatural pathogenicity and may constitute a new target for anti-Leishmaniachemotherapy.
Fluorescence Resonance Energy Transfer Microscopy of the
Helicobacter pylori
Vacuolating Cytotoxin within Mammalian Cells
Authors: Willhite David C. , Ye Dan , Blanke Steven R.
Published: July 2002

The
Helicobacter pylori
vacuolating cytotoxin (VacA) binds and enters mammalian cells to induce cellular vacuolation. To investigate the quaternary structure of VacA within the intracellular environment where toxin cytotoxicity is elaborated, we employed fluorescence resonance energy transfer (FRET) microscopy. HeLa cells coexpressing full-length and truncated forms of VacA fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) were analyzed for FRET to indicate direct associations. These studies revealed that VacA-CFP and VacA-YFP interact within vacuolated cells, supporting the belief that monomer associations at an intracellular site are important for the toxin's vacuolating activity. In addition, the two fragments of proteolytically nicked VacA, p37 and p58, interact when coexpressed within mammalian cells. Because p37 and p58 function in
trans
when expressed separately within mammalian cells, these data suggest that the mechanism by which these two fragments induce vacuolation requires direct association. FRET microscopy also demonstrated interactions between mutant forms of VacA, as well as wild-type VacA with mutant forms of the toxin within vacuolated cells. Finally, a dominant-negative form of the toxin directly associates with wild-type VacA in cells where vacuolation was not detectable, suggesting that the formation of complexes comprising wild-type and dominant-negative forms of toxin acts to block intracellular toxin function.
Modification of Type IV Pilus-Associated Epithelial Cell Adherence and Multicellular Behavior by the PilU Protein of
Neisseria gonorrhoeae
Authors: Park Hae-Sun Moon , Wolfgang Matthew , Koomey Michael
Published: July 2002

Expression of type IV pili (Tfp) correlates with the ability of
Neisseria gonorrhoeae
to colonize the human host, as well as with adherence to human epithelial tissue, twitching motility, competence for natural transformation, and autoagglutination.
N. gonorrhoeae
PilF (required for Tfp biogenesis) and PilT (required for twitching motility and transformation) share significant identities with members of a family of putative ATPases involved in membrane trafficking of macromolecules. An open reading frame downstream of the
pilT
locus encoding a 408-amino-acid protein with 33% identity with the gonococcal PilT protein and 45% identity with the PilU protein in
Pseudomonas aeruginosa
was characterized, and the corresponding gene was designated
pilU
. Unlike
N. gonorrhoeae pilT
mutants,
pilU
mutants express twitching motility and are competent for DNA transformation. However, loss-of-function mutations in
pilU
increased bacterial adherence to ME-180 human epithelial cells eightfold and disrupted in vitro Tfp-associated autoagglutination. Comparative alignment of
N. gonorrhoeae
PilU with other members of the TrbB-like family of traffic ATPases revealed a conserved carboxy-terminal domain unique to family members which are not essential for Tfp biogenesis but which specifically modify Tfp-associated phenotypes. Studies of the
pilT
-
pilU
locus by using Northern blotting, transcriptional fusions, and reverse transcription-PCR showed that the two genes encoding closely related proteins with dissimilar effects on Tfp phenotypes are transcribed from a single promoter.
Cyanobacterial Sulfide-Quinone Reductase: Cloning and Heterologous Expression
Authors: Bronstein Michal , Schütz Michael , Hauska Günter , Padan Etana , Shahak Yosepha
Published: July 2002

The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.′), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium
Oscillatoria limnetica
, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the
Rhodobacter capsulatus
SQR and in an open reading frame found in the genome of
Aquifex aeolicus
. SQR activity was also detected in the unicellular cyanobacterium
Aphanothece halophytica
following sulfide induction, with a
V
max
of 180 μmol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparent

K
m

values of 20 and 40 μM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding
A. halophytica
SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for
O. limnetica
SQR and 437 amino acids for
A. halophytica
SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and −14 for
O. limnetica
SQR and
A. halophytica
SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacterium
Anabaena
PCC7120 as well as the chemolithotrophic bacteria
Shewanella putrefaciens
and
Thiobacillus ferrooxidans
. All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in
Escherichia coli
under the control of the T7 promoter. Membranes isolated from
E. coli
cells expressing
A. halophytica
SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2
n
-nonyl-4-hydroxyquinoline-
N
-oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.
Virulence of the Phytopathogen
Pseudomonas syringae
pv. Maculicola Is
rpoN
Dependent
Authors: Hendrickson Erik L. , Guevera Pablo , Peñaloza-Vàzquez Alejandro , Shao Jing , Bender Carol , Ausubel Frederick M.
Published: July 2002

We cloned the
rpoN
(
ntrA
and
glnF
) gene encoding ς
54
from the phytopathogen
Pseudomonas syringae
pv. maculicola strain ES4326. The
P. syringae
ES4326
rpoN
gene complemented
Pseudomonas aeruginosa
,
Escherichia coli
, and
Klebsiella aerogenes rpoN
mutants for a variety of
rpoN
mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the
P. syringae
ES4326
rpoN
gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the ς
54
protein encoded by the
Pseudomonas putida rpoN
gene. A marker exchange protocol was used to construct an ES4326
rpoN
insertional mutation,
rpoN
::Km
r
. In contrast to wild-type ES4326, ES4326
rpoN
::Km
r
was nonmotile and could not utilize nitrate, urea, C
4
-dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources.
rpoN
was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326
rpoN
::Km
r
did not multiply or elicit disease symptoms when infiltrated into
Arabidopsis thaliana
leaves, did not elicit the accumulation of several
Arabidopsis
defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (
Nicotiana tabacum
) leaves. Furthermore, whereas
P. syringae
ES4326 carrying the avirulence gene
avrRpt2
elicited an HR when infiltrated into
Arabidopsis
ecotype Columbia leaves, ES4326
rpoN
::Km
r
carrying
avrRpt2
elicited no response. Constitutive expression of ES4326
hrpL
in ES4326
rpoN
::Km
r
partially restored defense-related mRNA accumulation, showing a direct role for the
hrp
cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression of
hrpL
in ES4326
rpoN
::Km
r
did not restore coronatine production, showing that coronatine biosynthesis requires factors other than
hrpL
.
Fur Positive Regulation of Iron Superoxide Dismutase in
Escherichia coli
: Functional Analysis of the
sodB
Promoter
Authors: Dubrac Sarah , Touati Danièle
Published: July 2002

In
Escherichia coli
, the expression of
sodB
, which encodes iron superoxide dismutase, has been suggested to be activated by Fur, the iron-responsive global regulator initially characterized as a transcriptional repressor. We investigated
sodB
regulation by functional analysis of the
sodB
promoter using
sodB-lac
fusions with various truncated and mutated promoters. Several
cis
- and
trans
-acting elements involved in
sodB
regulation have been identified. The β-galactosidase activity of
sodB-lacZ
reporter fusions and RNA analysis showed sevenfold iron-dependent, Fur-mediated activation of expression. A region just downstream from −10, including a large palindromic sequence encompassing the +1 position followed by a 14-bp AT-rich motif, is the site of Fur positive regulation, and the integrity of both sequences was required for full Fur-mediated activation. The life span of
sodB
mRNA was three times longer in a
fur
+
strain, indicating that Fur-mediated activation proceeds, at least in part, at the posttranscriptional level. The H-NS and IHF histone-like factors also affected
sodB
expression. IHF slightly repressed
sodB
expression independently of Fur regulation. In contrast, H-NS negative regulation operated only in the absence of Fur. Remarkably, p
sodB
behaved like a “pure extended -10” promoter. Deletion of the −35 region did not affect expression, whereas expression was totally abolished by a TG-to-CC mutation in the extended −10 sequence TGcTACCCT.
Identification, Expression, and Characterization of
Escherichia coli
Guanine Deaminase
Authors: Maynes Jason T. , Yuan Richard G. , Snyder Floyd F.
Published: July 2002

Using the human cDNA sequence corresponding to guanine deaminase, the
Escherichia coli
genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a

K
m

of 15 μM with guanine and a
k
cat
of 3.2 s
−1
. The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The
E. coli
guanine deaminase locus is 3′ from an open reading frame which shows homology to a bacterial purine base permease.
Plasmid pGS5 from the Hyperthermophilic Archaeon
Archaeoglobus profundus
Is Negatively Supercoiled
Authors: López-García Purificación , Forterre Patrick , van der Oost John , Erauso Gaël
Published: July 2002

We present evidence that, in contrast to plasmids from other hyperthermophilic archaea, which are in the relaxed to positively supercoiled state, plasmid pGS5 (2.8 kb) from
Archaeoglobus profundus
is negatively supercoiled. This might be due to the presence of a gyrase introducing negative supercoils, since gyrase genes are present in the genome of its close relative
A. fulgidus
, and suggests that gyrase activity predominates over reverse gyrase whenever the two topoisomerases coexist in cells.
Regioselectivity and Enantioselectivity of Naphthalene Dioxygenase during Arene
cis
-Dihydroxylation: Control by Phenylalanine 352 in the α Subunit
Authors: Parales Rebecca E. , Resnick Sol M. , Yu Chi-Li , Boyd Derek R. , Sharma Narain D. , Gibson David T.
Published: July 2002

The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by
Pseudomonas
sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including
cis
-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the α subunit of NDO altered the stereochemistry of naphthalene
cis
-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl
cis
-3,4-dihydrodiol [77 and 60% (−)-(3
S
,4
R
), respectively] to that formed by wild-type NDO [>98% (+)-(3
R
,4
S
)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene
cis
-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from
Sphingomonas yanoikuyae
B8/36. A recombinant
Escherichia coli
strain expressing the F352V variant of NDO and the enantioselective toluene
cis
-dihydrodiol dehydrogenase from
Pseudomonas putida
F1 was used to produce enantiomerically pure (−)-biphenyl
cis
-(3
S
,4
R
)-dihydrodiol and (−)-phenanthrene
cis
-(1
S
,2
R
)-dihydrodiol from biphenyl and phenanthrene, respectively.
The
Synechococcus
Strain PCC 7942
glnN
Product (Glutamine Synthetase III) Helps Recovery from Prolonged Nitrogen Chlorosis
Authors: Sauer Jörg , Dirmeier Ulrike , Forchhammer Karl
Published: July 2002

We report the cloning and sequencing of the
glnN
gene encoding a class III glutamine synthetase from the cyanobacterium
Synechococcus
strain PCC 7942. Mapping of the transcriptional start site revealed a DNA sequence in the promoter region that resembles an imperfect NtcA binding motif. Expression of
glnN
is impaired in NtcA- and P
II
-deficient mutants. The only parameter which was negatively affected in the
glnN
mutant compared to the wild type was the recovery rate of prolonged nitrogen-starved cells with low concentrations of combined nitrogen.