bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophilic Fe-Oxidizing Bacteria Are Abundant at the Loihi Seamount Hydrothermal Vents and Play a Major Role in Fe Oxide Deposition
Authors: Emerson David , Moyer Craig L.
Published: July 2002

A number of hydrothermal vent sites exist on the summit of the Loihi Seamount, a shield volcano that is part of the Hawaiian archipelago. The vents are 1,100 to 1,325 m below the surface and range in temperature from slightly above ambient (10°C) to high temperature (167°C). The vent fluid is characterized by high concentrations of CO
2
(up to 17 mM) and Fe(II) (up to 268 μM), but there is a general paucity of H
2
S. Most of the vents are surrounded by microbial mats that have a gelatinous texture and are heavily encrusted with rust-colored Fe oxides. Visually, the Fe oxides appeared homogeneous. However, light microscopy revealed that the oxides had different morphologies, which fell into three classes: (i) sheaths, (ii) twisted or irregular filaments, and (iii) amorphous oxides. A morphological analysis of eight different samples indicated that the amorphous oxides were overall the most abundant; however, five sites had >50% sheaths and filamentous oxides. These latter morphologies are most likely the direct result of microbial deposition. Direct cell counts revealed that all of the oxides had abundant microbial populations associated with them, from 6.9 × 10
7
to 5.3 × 10
8
cells per ml of mat material. At most sites, end point dilution series for lithotrophic Fe oxidizers were successful out to dilutions of 10
−6
and 10
−7
. A pure culture was obtained from a 10
−7
dilution tube; this strain, JV-1, was an obligate, microaerophilic Fe oxidizer that grew at 25 to 30°C. A non-cultivation-based molecular approach with terminal-restriction fragment length polymorphism also indicated the common presence of Fe-oxidizing bacteria at Loihi. Together, these results indicate that Fe-oxidizing bacteria are common at the Loihi Seamount and probably play a major role in Fe oxidation. A review of the literature suggests that microbially mediated Fe oxidation at hydrothermal vents may be important globally.
Analysis of the 16S rRNA Gene Sequence ofAnaplasma centraleand Its Phylogenetic Relatedness to Other Ehrlichiae
Authors: Inokuma Hisashi , Terada Yutaka , Kamio Tugihiko , Raoult Didier , Brouqui Philippe
Published: July 2002

The nucleotide sequence of theAnaplasma centrale16S rRNA gene was determined and compared with the sequences of ehrlichial bacteria. The sequence ofA. centralewas closely related toAnaplasma marginaleby both level-of-similarity (98.08% identical) and distance analysis. A species-specific PCR was developed based upon the alignment data. The PCR can detectA. centraleDNA extracted from 10 infected bovine red blood cells in a reaction mixture.A. centraleDNA was amplified in the reaction, but not other related ehrlichial species.
Adherence of Probiotic Bacteria to Human Intestinal Mucus in Healthy Infants and during Rotavirus Infection
Authors: Juntunen M. , Kirjavainen P. V. , Ouwehand A. C. , Salminen S. J. , Isolauri E.
Published: July 2002

The concentration of fecal mucin and the adhesion of specific probiotics and their combinations in the intestinal mucus of infants during and after rotavirus diarrhea and in healthy children were determined. Mucus was prepared from fecal samples from 20 infants during and after rotavirus diarrhea and from 10 healthy age-matched children. Mucin concentration was determined, and the adhesion of five probiotics—
Lactobacillus rhamnosus
GG,
Lactobacillus casei
Shirota,
Lactobacillus paracasei
F19,
Lactobacillus acidophilus
LA5, and
Bifidobacterium lactis
Bb12—and their combinations was tested in vitro. The mean concentrations of fecal mucin during and after rotavirus diarrhea, 15.2 and 14.1 mg/g, were comparable to that in healthy children, 14.9 mg/g. The adherence of probiotics ranged from 1 to 34% in healthy subjects as indicated for the following strains:
L. rhamnosus
GG, 34%;
B. lactis
Bb12, 31%;
L. acidophilus
LA5, 4%;
L. paracasei
F19, 3%; and
L. casei
Shirota, 1% (
P
= 0.0001). The distinctive pattern of probiotic adherence was not influenced by rotavirus diarrhea. The adhesion of Bb12 in the presence of GG increased from 31 to 39% in healthy infants (
P
= 0.018) and in episodes of diarrhea increased from 26 to 44% (
P
= 0.001). Rotavirus diarrhea does not decrease the production of fecal mucin or with respect to the adhesion of probiotic bacteria tested in vitro. Combination of specific probiotic strains may enhance adherence in a synergistic manner. Optimal clinical application of these interactions may offer novel therapeutic guidelines for the treatment and prevention of gastrointestinal infections.
Effect of Intranasal Administration ofLactobacillus caseiShirota on Influenza Virus Infection of Upper Respiratory Tract in Mice
Authors: Hori Tetsuji , Kiyoshima Junko , Shida Kan , Yasui Hisako
Published: July 2002

In mice administeredLactobacillus caseistrain Shirota (LcS) intranasally, potent induction of interleukin 12, gamma interferon, and tumor necrosis factor alpha, which play a very important role in excluding influenza virus (IFV), was evident in mediastinal lymph node cells. In this model of upper respiratory IFV infection, the titers of virus in the nasal wash of mice inoculated with 200 μg of LcS for three consecutive days (LcS 200 group) before infection were significantly (P< 0.01) lower than those of mice not inoculated with LcS (control group) (100.9 ± 0.6versus 102.1 ± 1.0). The IFV titer was decreased to about 1/10 of the control level. Using this infection model with modifications, we investigated whether the survival rate of mice was increased by intranasal administration of LcS. The survival rate of the mice in the LcS 200 group was significantly (P< 0.05) greater than that of the mice in the control group (69% versus 15%). It seems that the decrease in the titer of virus in the upper respiratory tract to 1/10 of the control level was important in preventing death. These findings suggest that intranasal administration of LcS enhances cellular immunity in the respiratory tract and protects against influenza virus infection.
Antibody Maturation in
Trypanosoma cruzi
-Infected Rats
Authors: Marcipar Iván S. , Risso Marikena G. , Silber Ariel M. , Revelli Silvia , Marcipar Alberto J.
Published: July 2002

The study of antibody avidity changes during infection has improved the understanding of the pathologic processes involved in several infectious diseases. In some infections, like toxoplasmosis, this information is being used for diagnostic purposes. Results of the evolution of antibody avidity for different specific antigens in
Trypanosome cruzi
-infected rats are presented. A Western blotting technique, combined with avidity analysis to identify antigens that elicit high-avidity antibodies, is suggested. In this system, antibodies showed high avidity values only during the chronic phase of infection and only in relation to antibodies against 21-, 33-, 41-, 42-, 56-, 58-, 66-, and 72-kDa antigens. Finally, a 97-kDa
T. cruzi
antigen, which was recognized by high-avidity antibodies and occurred in noninfected rats, was identified. These results allow us to evaluate the different antigens in chagasic infection. Our results show that with the correct choice of antigen it is possible to detect differences in maturation of antibodies and to discriminate, in an experimental model, between recent (acute) and chronic infections.
Treatment of Giardiasis
Authors: Gardner Timothy B. , Hill David R.
Published: July 2002

Giardia lamblia is both the most common intestinal parasite in the United States and a frequent cause of diarrheal illness throughout the world. In spite of its recognition as an important human pathogen, there have been relatively few agents used in therapy. This paper discusses each class of drugs used in treatment, along with their mechanism of action, in vitro and clinical efficacy, and side effects and contraindications. Recommendations are made for the preferred treatment in different clinical situations. The greatest clinical experience is with the nitroimidazole drugs, i.e., metronidazole, tinidazole, and ornidazole, which are highly effective. A 5- to 7-day course of metronidazole can be expected to cure over 90% of individuals, and a single dose of tinidazole or ornidazole will cure a similar number. Quinacrine, which is no longer produced in the United States, has excellent efficacy but may be poorly tolerated, especially in children. Furazolidone is an effective alternative but must be administered four times a day for 7 to 10 days. Paromomycin may be used during early pregnancy, because it is not systematically absorbed, but it is not always effective. Patients who have resistant infection can usually be cured by a prolonged course of treatment with a combination of a nitroimidazole with quinacrine.
Pandemic Threat Posed by Avian Influenza A Viruses
Authors: Horimoto Taisuke , Kawaoka Yoshihiro
Published: July 2002

Influenza pandemics, defined as global outbreaks of the disease due to viruses with new antigenic subtypes, have exacted high death tolls from human populations. The last two pandemics were caused by hybrid viruses, or reassortants, that harbored a combination of avian and human viral genes. Avian influenza viruses are therefore key contributors to the emergence of human influenza pandemics. In 1997, an H5N1 influenza virus was directly transmitted from birds in live poultry markets in Hong Kong to humans. Eighteen people were infected in this outbreak, six of whom died. This avian virus exhibited high virulence in both avian and mammalian species, causing systemic infection in both chickens and mice. Subsequently, another avian virus with the H9N2 subtype was directly transmitted from birds to humans in Hong Kong. Interestingly, the genes encoding the internal proteins of the H9N2 virus are genetically highly related to those of the H5N1 virus, suggesting a unique property of these gene products. The identification of avian viruses in humans underscores the potential of these and similar strains to produce devastating influenza outbreaks in major population centers. Although highly pathogenic avian influenza viruses had been identified before the 1997 outbreak in Hong Kong, their devastating effects had been confined to poultry. With the Hong Kong outbreak, it became clear that the virulence potential of these viruses extended to humans.
Antiviral Actions of Interferons
Authors: Samuel Charles E.
Published: July 2002

Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2′,5′-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2′-5′-oligoadenylate-dependent RNase L, respectively, and also in RNA editing by the IFN-inducible RNA-specific adenosine deaminase (ADAR1). IFN also induces a form of inducible nitric oxide synthase (iNOS2) and the major histocompatibility complex class I and II proteins, all of which play important roles in immune response to infections. Several additional genes whose expression profiles are altered in response to IFN treatment and virus infection have been identified by microarray analyses. The availability of cDNA and genomic clones for many of the components of the IFN system, including IFN-α, IFN-β, and IFN-γ, their receptors, Jak and Stat and IRF signal transduction components, and proteins such as PKR, 2′,5′-OAS, Mx, and ADAR, whose expression is regulated by IFNs, has permitted the generation of mutant proteins, cells that overexpress different forms of the proteins, and animals in which their expression has been disrupted by targeted gene disruption. The use of these IFN system reagents, both in cell culture and in whole animals, continues to provide important contributions to our understanding of the virus-host interaction and cellular antiviral response.
Differential Sensitivities ofChlamydia trachomatisStrains to Inhibitory Effects of Gamma Interferon
Authors: Morrison Richard P.
Published: July 2002

Gamma interferon (IFN-γ) is an important cytokine in host defense against chlamydial infection. An in vitro cell culture system was used to show that IFN-γ inhibition of chlamydial growth, as determined by diminished recovery of infectious elementary bodies, differed markedly among chlamydial strains. These differences in sensitivity among chlamydial strains to IFN-γ-mediated inhibition may profoundly influence the clinical outcome of infection.
Epithelial Cell Adherence Mediated by the Enterotoxigenic
Escherichia coli
Tia Protein
Authors: Mammarappallil Joseph G. , Elsinghorst Eric A.
Published: July 2002

In vitro studies have shown that enterotoxigenic
Escherichia coli
(ETEC) strains are capable of invading cultured epithelial cells derived from the human ileum and colon. Two separate invasion loci (
tia
and
tib
) have previously been isolated from the classical ETEC strain
H10407
. The
tia
locus has been shown to direct the synthesis of Tia, a 25-kDa outer membrane protein. Tia is sufficient to confer the adherence and invasion phenotypes on laboratory stains of
E. coli
, suggesting that this protein is an adhesin and invasin. Here we report the purification of Tia and characterize its biological activity. Tia was purified by electroelution of outer membrane proteins that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified Tia was labeled with biotin and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Polyclonal anti-Tia antiserum blocked this binding. These results show that Tia acts as an adhesin. Polyclonal anti-Tia antiserum also inhibited invasion of recombinant
E. coli
bearing
tia
clones, indirectly suggesting that Tia may also act as an invasin. We predict Tia to contain eight transmembrane amphipathic β-sheets with four loops that are exposed on the surface of the bacterial cell. A peptide corresponding to 19 residues in one of the four predicted surface-exposed loops inhibits Tia-mediated epithelial cell invasion. Seeding HCT8 cells on wells coated with purified Tia reduced Tia-mediated epithelial cell invasion. Together, these results indicate that Tia is an invasin and adhesin that binds a specific receptor on HCT8 cells.
OppA of
Listeria monocytogenes
, an Oligopeptide-Binding Protein Required for Bacterial Growth at Low Temperature and Involved in Intracellular Survival
Authors: Borezee Elise , Pellegrini Elisabeth , Berche Patrick
Published: July 2002

We identified a new oligopeptide permease operon in the pathogen
Listeria monocytogenes
. This
opp
operon consists of five genes (
oppA, oppB, oppC, oppD
, and
oppF
) and displays the same genetic organization as those of several bacterial species. The first gene of this operon,
oppA
, encodes a 62-kDa protein sharing 33% identity with OppA of
Bacillus subtilis
and is expressed predominantly during exponential growth. The function of
oppA
was studied by constructing an
oppA
deletion mutant. The phenotype analysis of this mutant revealed that OppA mediates the transport of oligopeptides and is required for bacterial growth at low temperature. The wild-type phenotype was restored by complementing the mutant with
oppA
. We also found that OppA is involved in intracellular survival in macrophages and in bacterial growth in organs of mice infected with
L. monocytogenes
, although the level of virulence was not altered in the mutant. These results show the major role of OppA in the uptake of oligopeptides and the pleiotropic effects of this oligopeptide-binding protein on the behavior of this pathogen in the environment and in its host.
Infectious CTXΦ and the Vibrio Pathogenicity Island Prophage in
Vibrio mimicus
: Evidence for Recent Horizontal Transfer between
V. mimicus
and
V. cholerae
Authors: Boyd E. Fidelma , Moyer Kathryn E. , Shi Lei , Waldor Matthew K.
Published: July 2002

Vibrio mimicus
differs from
Vibrio cholerae
in a number of genotypic and phenotypic traits but like
V. cholerae
can give rise to diarrheal disease. We examined clinical isolates of
V. mimicus
for the presence of CTXΦ, the lysogenic filamentous bacteriophage that carries the cholera toxin genes in epidemic
V. cholerae
strains. Four
V. mimicus
isolates were found to contain complete copies of CTXΦ. Southern blot analyses revealed that
V. mimicus
strain PT5 contains two CTX prophages integrated at different sites within the
V. mimicus
genome whereas
V. mimicus
strains PT48, 523-80, and 9583 each contain tandemly arranged copies of CTXΦ. We detected the replicative form of CTXΦ, pCTX, in all four of these
V. mimicus
isolates. The CTX prophage in strain PT5 was found to produce infectious CTXΦ particles. The nucleotide sequences of CTXΦ genes
orfU
and
zot
from
V. mimicus
strain PT5 and
V. cholerae
strain N16961 were identical, indicating contemporary horizontal transfer of CTXΦ between these two species. The receptor for CTXΦ, the toxin-coregulated pilus, which is encoded by another lysogenic filamentous bacteriophage, VPIΦ, was also present in the CTXΦ-positive
V. mimicus
isolates. The nucleotide sequences of VPIΦ genes
aldA
and
toxT
from
V. mimicus
strain PT5 and
V. cholerae
N16961 were identical, suggesting recent horizontal transfer of this phage between
V. mimicus
and
V. cholerae
. In
V. mimicus
, the vibrio pathogenicity island prophage was integrated in the same chromosomal attachment site as in
V. cholerae
. These results suggest that
V. mimicus
may be a significant reservoir for both CTXΦ and VPIΦ and may play an important role in the emergence of new toxigenic
V. cholerae
isolates.
Microgravity as a Novel Environmental Signal Affecting
Salmonella enterica
Serovar Typhimurium Virulence
Authors: Nickerson Cheryl A. , Ott C. Mark , Mister Sarah J. , Morrow Brian J. , Burns-Keliher Lisa , Pierson Duane L.
Published: July 2002

The effects of spaceflight on the infectious disease process have only been studied at the level of the host immune response and indicate a blunting of the immune mechanism in humans and animals. Accordingly, it is necessary to assess potential changes in microbial virulence associated with spaceflight which may impact the probability of in-flight infectious disease. In this study, we investigated the effect of altered gravitational vectors on
Salmonella
virulence in mice.
Salmonella enterica
serovar Typhimurium grown under modeled microgravity (MMG) were more virulent and were recovered in higher numbers from the murine spleen and liver following oral infection compared to organisms grown under normal gravity. Furthermore, MMG-grown salmonellae were more resistant to acid stress and macrophage killing and exhibited significant differences in protein synthesis than did normal-gravity-grown cells. Our results indicate that the environment created by simulated microgravity represents a novel environmental regulatory factor of
Salmonella
virulence.
Immune Response against a Cross-Reactive Epitope on the Heat Shock Protein 60 Homologue ofHelicobacter pylori
Authors: Yamaguchi Hiroyuki , Osaki Takako , Kai Masanori , Taguchi Haruhiko , Kamiya Shigeru
Published: July 2002

We previously established a monoclonal antibody (MAb), designated H9, which reacts with the heat shock protein 60 (HSP60) homologue ofHelicobacter pylorias well as with other bacterial and human HSP60s. To determine the importance of a cross-reactive epitope onH. pyloriHSP60 inH. pyloriimmunopathogenesis, we performed (i) mapping of an epitope onH. pyloriHSP60 recognized by the H9 MAb, (ii) analysis of immunoglobulin G responses of patients with or withoutH. pyloriinfection to its epitope region, and (iii) studies of the protective effect of immunization with its epitope region onH. pyloriinfection in mice. The epitope recognized by the H9 MAb was mapped to the sequence of amino acids 189 to 203 (VEGMQFDRGYLSPYF) on theH. pyloriHSP60 molecule. It was confirmed that the synthesized peptide designated pH9 was recognized by the H9 MAb. Enzyme-linked immunosorbent assay analysis showed that patients withH. pyloriinfection (n= 349) had significantly lower titers of pH9 antibody than did uninfected patients (n= 200) (P< 0.001), but this was not the case with purifiedH. pyloriHSP60 recombinantEscherichia coliGroEL, or recombinant human HSP60. In C57BL/6 mice immunized with the pH9 peptide with Freund's complete adjuvant (FCA), the number ofH. pyloriorganisms colonizing the stomach was significantly lower than that in mice immunized with pCont plus FCA (P< 0.0001) or FCA only (P< 0.005). The results suggest that the immune response to the cross-reactive epitope (pH9 region) onH. pyloriHSP60 is unique and might be associated with protection againstH. pyloriinfection.
Administration of Endotoxin Associated with Lipopolysaccharide Tolerance Protects Mice against Fungal Infection
Authors: Rayhane Naïma , Fitting Catherine , Lortholary Olivier , Dromer Francoise , Cavaillon Jean-Marc
Published: July 2002

Lipopolysaccharide (LPS) pretreatment of mice resulted in a significantly enhanced survival after disseminatedCryptococcus neoformansinfection. The survival was associated with reduced fungal burden in tissues. LPS-pretreated mice had lower levels of cytokines in blood, spleen, and lungs and higher levels in brain. Pentoxifylline abolished the beneficial effect of LPS pretreatment.
Improved Innate Immunity of Endotoxin-Tolerant Mice Increases Resistance toSalmonella entericaSerovar Typhimurium Infection despite Attenuated Cytokine Response
Authors: Lehner Martin D. , Ittner Josepha , Bundschuh Daniela S. , van Rooijen Nico , Wendel Albrecht , Hartung Thomas
Published: July 2002

During infection with gram-negative bacteria, exposure of immune cells to lipopolysaccharide (LPS) from the bacterial cell membrane induces a rapid cytokine response which is essential for the activation of host defenses against the invading pathogens. Administration of LPS to mice induces a state of hyporesponsiveness, or tolerance, characterized by reduced cytokine production upon subsequent LPS challenge. In the model of experimentalSalmonella entericaserovar Typhimurium infection of mice, we assessed the question of whether complete LPS tolerance induced by repetitive doses of LPS interfered with cytokine production and host defense against gram-negative bacteria. Although production of various cytokines in response to serovar Typhimurium was attenuated by LPS pretreatment, LPS-tolerant mice showed improved antibacterial activity, evidenced by a prolongation of survival and a continuously lower bacterial load. We attribute this protective effect to three independent mechanisms. (i) Peritoneal accumulation of leukocytes in the course of LPS pretreatment accounted for enhanced defense against serovar Typhimurium during the first 6 h of infection but not for decreased bacterial load in late-stage infection. (ii) LPS-tolerant mice had an increased capacity to recruit neutrophilic granulocytes during infection. (iii) LPS-tolerant mice showed threefold-increased Kupffer cell numbers, enhanced phagocytic activity of the liver, and strongly improved clearance of blood-borne serovar Typhimurium. These results demonstrate that despite attenuated cytokine response, acquired LPS tolerance is associated with enhanced resistance to infections by gram-negative bacteria and that this effect is mainly mediated by improved effector functions of the innate immune system.
Tick Salivary Gland Extract Inhibits Killing ofBorrelia afzeliiSpirochetes by Mouse Macrophages
Authors: Kuthejlová Marie , Kopecký Jan , Štěpánová Gabriela , Macela Aleš
Published: July 2002

Salivary gland extract (SGE) fromIxodes ricinusticks inhibited the killing ofBorrelia afzeliispirochetes by murine macrophages. SGE also reduced the production of two major defense molecules of phagocytes, superoxide and nitric oxide. It is likely that the suppression of macrophage microbicidal mechanisms contributes to the inhibitory effect of tick saliva on the killing ofB. afzeliispirochetes, thus facilitating the transmission of this important pathogen.
Urethral Cytokine and Immune Responses in
Chlamydia trachomatis
-Infected Males
Authors: Pate Mitchell S. , Hedges Spencer R. , Sibley Don A. , Russell Michael W. , Hook Edward W. , Mestecky Jiri
Published: July 2002

Penile urethral swabs collected from PCR-confirmed
Chlamydia trachomatis
-infected,
C. trachomatis
-uninfected, and non-
C. trachomatis
-infected, nongonococcal urethritis-infected males were analyzed for cytokine, total immunoglobulin (Ig), and specific antibody levels by enzyme-linked immunosorbent assay. Differential cellular components of the swab transport medium were also enumerated for the same groups. Although low, the levels of
C. trachomatis
-specific IgA and IgG antibodies and interleukin 8 cytokine were significantly higher in
C. trachomatis
-infected individuals. There were no significant differences in the levels of seven additional cytokines evaluated.
Characterization of the Adhesin of
Escherichia coli
F18 Fimbriae
Authors: Smeds A. , Hemmann K. , Jakava-Viljanen M. , Pelkonen S. , Imberechts H. , Palva A.
Published: July 2002

Previous research has suggested that the adhesin encoded by the F18 fimbrial operon in
Escherichia coli
is either the FedE or FedF protein. In this work, we show that anti-FedF antibodies, unlike anti-FedE serum, were able to inhibit
E. coli
adhesion to porcine enterocytes. Moreover, specific adhesion to enterocytes was shown with purified FedF-maltose binding protein.
Pneumolysin Is the Main Inducer of Cytotoxicity to Brain Microvascular Endothelial Cells Caused by
Streptococcus pneumoniae
Authors: Zysk Gregor , Schneider-Wald Barbara Katharina , Hwang Jae Hyuk , Bejo Levente , Kim Kwang Sik , Mitchell Timothy J. , Hakenbeck Regine , Heinz Hans-Peter
Published: July 2002

In pneumococcal meningitis it is assumed that bacteria cross the blood-brain barrier (BBB), which consists mainly of cerebral endothelial cells. The effect of
Streptococcus pneumoniae
on the BBB was investigated with an in vitro BBB model using a human brain microvascular endothelial cell line (HBMEC) and primary cultures of bovine brain microvascular endothelial cells (BBMEC). Within a few hours of incubation with pneumococci, rounding and detachment of the HBMEC were observed, and the transendothelial electrical resistance of the BBMEC monolayer decreased markedly. An
S. pneumoniae
mutant deficient in pneumolysin did not affect the integrity of the endothelial cell monolayer. Neither cell wall fragments nor isolated pneumococcal cell walls induced changes of endothelial cell morphology. However, purified pneumolysin caused endothelial cell damage comparable to that caused by the viable pneumococci. The cell detachment was dependent on de novo protein synthesis and required the activities of caspase and tyrosine kinases. The results show that pneumolysin is an important component for damaging the BBB and may contribute to the entry of pneumococci into the cerebral compartment and to the development of brain edema in pneumococcal meningitis.
Functional Characterization of Streptococcal Pyrogenic Exotoxin J, a Novel Superantigen
Authors: McCormick John K. , Pragman Alexa A. , Stolpa John C. , Leung Donald Y. M. , Schlievert Patrick M.
Published: July 2002

Streptococcal toxic shock syndrome (STSS) is a highly lethal, acute-onset illness that is a subset of invasive streptococcal disease. The majority of clinical STSS cases have been associated with the pyrogenic toxin superantigens (PTSAgs) streptococcal pyrogenic exotoxin A or C (SPE A or C), although cases have been reported that are not associated with either of these exotoxins. Recent genome sequencing projects have revealed a number of open reading frames that potentially encode proteins with similarity to SPEs A and C and to other PTSAgs. Here, we describe the cloning, expression, purification, and functional characterization of a novel exotoxin termed streptococcal pyrogenic exotoxin J (SPE J). Purified recombinant SPE J (rSPE J) expressed from
Escherichia coli
stimulated the expansion of both rabbit splenocytes and human peripheral blood lymphocytes, preferentially expanded human T cells displaying Vβ2, -3, -12, -14, and -17 on their T-cell receptors, and was active at concentrations as low as 5 × 10
−6
μg/ml. Furthermore, rSPE J induced fevers in rabbits and was lethal in two models of STSS. Biochemically, SPE J had a predicted molecular weight of 24,444 and an isoelectric point of 7.7 and lacked the ability to form the cystine loop structure characteristic of many PTSAgs. SPE J shared 19.6, 47.1, 38.8, 18.1, 19.6, and 24.4% identity with SPEs A, C, G, and H, streptococcal superantigen, and streptococcal mitogenic exotoxin Z-2, respectively, and was immunologically cross-reactive with SPE C. The characterization of a seventh functional streptococcal PTSAg raises important questions relating to the evolution of the streptococcal superantigens.
Signaling by Toll-Like Receptor 2 and 4 Agonists Results in Differential Gene Expression in Murine Macrophages
Authors: Hirschfeld Matthew , Weis Janis J. , Toshchakov Vladimir , Salkowski Cindy A. , Cody M. Joshua , Ward Dawn C. , Qureshi Nilofer , Michalek Suzanne M. , Vogel Stefanie N.
Published: July 2002

Lipopolysaccharide (LPS) derived from the periodontal pathogenPorphyromonas gingivalishas been reported to differ structurally and functionally from enterobacterial LPS. These studies demonstrate that in contrast to protein-free enterobacterial LPS, a similarly purified preparation ofP. gingivalisLPS exhibited potent Toll-like receptor 2 (TLR2), rather than TLR4, agonist activity to elicit gene expression and cytokine secretion in murine macrophages and transfectants. More importantly, TLR2 stimulation by thisP. gingivalisLPS preparation resulted in differential expression of a panel of genes that are normally induced in murine macrophages byEscherichia coliLPS. These data suggest that (i)P. gingivalisLPS does not signal through TLR4 and (ii) signaling through TLR2 and through TLR4 differs quantitatively and qualitatively. Our data support the hypothesis that the shared signaling pathways elicited by TLR2 and by TLR4 agonists must diverge in order to account for the distinct patterns of inflammatory gene expression.
Salmonella entericaSerovar Typhimurium Induces Cell Death in Bovine Monocyte-Derived Macrophages by EarlysipB-Dependent and DelayedsipB-Independent Mechanisms
Authors: Santos Renato L. , Tsolis Renée M. , Bäumler Andreas J. , Smith Roger , Adams L. Garry
Published: July 2002

It was previously demonstrated thatSalmonella entericaserovar Typhimurium induces cell death with features of apoptosis in murine macrophages. Mice infected withSalmonellaserovar Typhimurium develop systemic disease without diarrhea, whereas the infection in cattle and in humans is localized and characterized by diarrhea. Considering these clinical disease expression differences between mice and cattle, we investigated whether serovar Typhimurium is cytotoxic for bovine macrophages. Macrophages infected with serovar Typhimurium grown in the logarithmic phase quickly underwent cell death. Macrophages infected with stationary-phase cultures or with a mutant lackingsipBunderwent no immediate cell death but did develop delayed cytotoxicity, undergoing cell death between 12 and 18 h postinfection. Both pathways were temporarily blocked by the general caspase inhibitor Z-VAD-Fmk and by the caspase 1 inhibitor Z-YVAD-Fmk. Comparisons of macrophages from cattle naturally resistant or susceptible to intracellular pathogens indicated no differences between these two genetic backgrounds in terms of susceptibility to serovar Typhimurium-induced cell death. We conclude thatSalmonellaserovar Typhimurium induces cell death in bovine macrophages by two distinct mechanisms, earlysipB-mediated and delayedsipB-independent mechanisms.
Flow Cytometric Determination of Cellular Sources and Frequencies of Key Cytokine-Producing Lymphocytes Directed against Recombinant LACK and Soluble Leishmania Antigen in Human Cutaneous Leishmaniasis
Authors: Bottrel R. L. A. , Dutra W. O. , Martins F. A. , Gontijo B. , Carvalho E. , Barral-Netto M. , Barral A. , Almeida R. P. , Mayrink W. , Locksley R. , Gollob K. J.
Published: July 2002

Leishmaniasis, caused by infection with the protozoan parasiteLeishmania, affects millions of individuals worldwide, causing serious morbidity and mortality. This study directly determined the frequency of cells producing key immunoregulatory cytokines in response to the recombinant antigenLeishmaniahomolog of receptors for activated kinase C (LACK) and soluble leishmania antigen (SLA), and it determined relative contributions of these antigens to the overall cytokine profile in individuals infected for the first time withLeishmania braziliensis. All individuals presented with the cutaneous clinical form of leishmaniasis and were analyzed for proliferative responses to LACK antigen and SLA, frequency of lymphocyte subpopulations (analyzed ex vivo), and antigen-induced (LACK and SLA) cytokine production at the single-cell level (determined by flow cytometry). The following were determined. (i) The Th1-type response previously seen in patients with cutaneous leishmaniasis is due to gamma interferon (IFN-γ) production by several different sources, listed in order of contribution: CD4+T lymphocytes, CD4−, CD8−lymphocytes, and CD8+T lymphocytes. (ii) SLA induced a higher frequency of lymphocytes producing IFN-γ and tumor necrosis factor alpha (TNF-α) than did LACK. (iii) LACK induced an activation of monocyte populations as reflected by an increased percentage of CD14-positive cells. (iv) Neither SLA nor LACK induced detectable frequencies of cells producing interleukin-4 (IL-4) or IL-5. These data demonstrated a multifaceted immune response to SLA in human leishmaniasis involving Th1 CD4+T lymphocytes (IFN-γ+and IL-10−/IL-4−), Tc1 CD8+T cells (IFN-γ+, and IL-10−/IL-4−), and a high frequency of TNF-α-producing lymphocytes. Moreover, it was determined that the recombinant antigen LACK acts as a weak inducer of Th1-type lymphocyte responses compared to SLA.
Homogeneity of Antibody Responses in Tuberculosis Patients
Authors: Samanich K. , Belisle J. T. , Laal S.
Published: July 2002

The goals of the present study were twofold: (i) to compare the repertoires of antigens in culture filtrates of in vitro-grownMycobacterium tuberculosisthat are recognized by antibodies from noncavitary and cavitary tuberculosis (TB) patients and (ii) to determine the extent of variation that exists between the antigen profiles recognized by individual TB patients. Lipoarabinomannan-free culture filtrate proteins ofM. tuberculosiswere fractionated by one-dimensional (1-D) and 2-D polyacrylamide gel electrophoresis, and the Western blots were probed with sera from non-human immunodeficiency virus (non-HIV)-infected cavitary and noncavitary TB patients and from HIV-infected, noncavitary TB patients. In contrast to earlier studies based on recombinant antigens ofM. tuberculosiswhich suggested that antibody responses in TB patients were heterogeneous (K. Lyashchenko et al., 1998, Infect. Immun. 66:3936–3940, 1998), our studies with native culture filtrate proteins show that the antibody responses in TB patients show significant homogeneity in being directed against a well-defined subset of antigens. Thus, there is a well-defined subset of culture filtrate antigens that elicits antibodies during noncavitary and cavitary disease. In addition, another set of antigens is recognized primarily by cavitary TB patients. The mapping with individual patient sera presented here suggests that serodiagnostic tests based on the subset of antigens recognized during both noncavitary and cavitary TB will enhance the sensitivity of antibody detection in TB patients, especially in difficult-to-diagnose, smear-negative, noncavitary TB patients.
l-Tryptophan-l-Kynurenine Pathway Metabolism Accelerated byToxoplasmagondiiInfection Is Abolished in Gamma Interferon-Gene-Deficient Mice: Cross-Regulation between Inducible Nitric Oxide Synthase and Indoleamine-2,3-Dioxygenase
Authors: Fujigaki Suwako , Saito Kuniaki , Takemura Masao , Maekawa Naoya , Yamada Yasuhiro , Wada Hisayasu , Seishima Mitsuru
Published: July 2002

l-Tryptophan degradation by indoleamine 2,3-dioxygenase (IDO) might have an important role in gamma interferon (IFN-γ)-induced antimicrobial effects. In the present study, the effects ofToxoplasmagondiiinfection on IDO were investigated by using wild-type and IFN-γ-gene-deficient (knockout) (IFN-γ KO) mice. In wild-type C57BL/6J mice, enzyme activities and mRNA levels for IDO in both lungs and brain were markedly increased and lungl-tryptophan concentrations were dramatically decreased followingT.gondiiinfection. In contrast, these metabolic changes did not occur inT.gondii-infected IFN-γ KO mice or in uninfected IFN-γ KO mice. The levels of inducible nitric oxide synthase (iNOS) induction in infected IFN-γ KO mice were high in lungs and low in brain compared to those in infected wild-type mice. The extent of increased mRNA expression ofT.gondiisurface antigen gene 2 (SAG2) induced in lungs and brain byT.gondiiinfection was significantly enhanced in IFN-γ KO mice compared to wild-type mice on day 7 postinfection. Treatment withN-nitro-l-arginine methyl ester, an iNOS inhibitor, increased the levels of SAG2 mRNA in brain but not in lungs and of plasmal-kynurenine afterT. gondiiinfection. This in vivo study provides evidence thatl-tryptophan depletion caused byT. gondiiis directly mediated by IFN-γ in the lungs, where iNOS is not induced by IFN-γ. This study suggests that there is an antitoxoplasma mechanism of cross-regulation between iNOS and IDO and that the expression of the main antiparasite effector mechanisms for iNOS and/or IDO may vary among tissues.
Structural Decomposition and Heterogeneity of Commercial Lipoteichoic Acid Preparations
Authors: Morath Siegfried , Geyer Armin , Spreitzer Ingo , Hermann Corinna , Hartung Thomas
Published: July 2002

Fractionation of commercial preparations of lipoteichoic acids (LTA) by hydrophobic interaction chromatography (HIC) and nuclear magnetic resonance spectroscopy revealed very inhomogeneous compositions and decomposition of the LTA structure: LTA content of the preparations averaged 61% forStreptococcus pyogenes, 16% forBacillus subtilis, and 75% forStaphylococcus aureus. The decomposition was characterized by a loss of glycerophosphate units as well as alanine andN-acetylglucosamine substituents. All preparations contained—to varying degrees—non-LTA, non-lipopolysaccharide (LPS) immunostimulatory components as indicated by their elution profile in HIC, lack of phosphate, and negative Limulus amoebocyte lysate (LAL) test results. After purification, the commercial LTA fromBacillus subtilisandS. pyogenesbut not LTA fromS. aureusinduced the release of tumor necrosis factor alpha, interleukin 1 beta (IL-1β), IL-6, and IL-10 in human blood. While pure LTA are negative in the LAL assay, endotoxin equivalents of more than 10 ng of LPS/mg of LTA were found in the commercial preparations. Taken together, these data indicate that these crude preparations with relatively high endotoxin contamination are not suitable for characterizing the activation of immune cells by LTA.
Role of Flagella in Host Cell Invasion by
Burkholderia cepacia
Authors: Tomich Mladen , Herfst Christine A. , Golden Joseph W. , Mohr Christian D.
Published: July 2002

Burkholderia cepacia
is an important opportunistic human pathogen that affects immunocompromised individuals, particularly cystic fibrosis (CF) patients. Colonization of the lungs of a CF patient by
B. cepacia
can lead not only to a decline in respiratory function but also to an acute systemic infection, such as bacteremia. We have previously demonstrated that a CF clinical isolate of
B. cepacia
, strain J2315, can invade and survive within cultured respiratory epithelial cells. In order to further characterize the mechanisms of invasion of
B. cepacia
, we screened a transposon-generated mutant library of strain J2315 for mutants defective in invasion of A549 respiratory epithelial cells. Here we describe isolation and characterization of a nonmotile mutant of
B. cepacia
with reduced invasiveness due to disruption of
fliG
, which encodes a component of the motor-switch complex of the flagellar basal body. We also found that a defined null mutation in
fliI
, a gene encoding a highly conserved ATPase required for protein translocation via the flagellar type III secretion system, also resulted in loss of motility and a significant reduction in invasion. Both mutants lacked detectable intracellular flagellin and failed to export detectable amounts of flagellin into culture supernatants, suggesting that disruption of
fliG
and
fliI
impaired flagellar biogenesis. The reduction in invasion did not appear to be due to defective adherence of the flagellar mutants to A549 cells, suggesting that functional flagella and motility are required for full invasiveness of
B. cepacia
. Our findings indicate that flagellum-mediated motility may facilitate penetration of host epithelial barriers by
B. cepacia
, contributing to establishment of infection and systemic spread of the organism.
Regulation of the
Escherichia coli
K5 Capsule Gene Cluster: Evidence for the Roles of H-NS, BipA, and Integration Host Factor in Regulation of Group 2 Capsule Gene Clusters in Pathogenic
E. coli
Authors: Rowe Sonya , Hodson Nigel , Griffiths Gary , Roberts Ian S.
Published: July 2002

The expression of
Escherichia coli
group 2 capsules (K antigens) is temperature dependent, with capsules only being expressed at temperatures above 20°C. Thermoregulation is at the level of transcription, with no detectable transcription at 20°C. Using the
E. coli
K5 capsule gene cluster as a model system, we have shown that the nucleoid-associated protein H-NS plays a dual role in regulating transcription of group 2 capsule gene clusters at 37 and 20°C. At 37°C H-NS is required for maximal transcription of group 2 capsule gene clusters, whereas at 20°C H-NS functions to repress transcription. The BipA protein, previously identified as a tyrosine-phosphorylated GTPase and essential for virulence in enteropathogenic
E. coli
, was shown to play a similar role to H-NS in regulating transcription at 37 and 20°C. The binding of integration host factor (IHF) to the region 1 promoter was necessary to potentiate transcription at 37°C and IHF binding demonstrated by bandshift assays. The IHF binding site was 3′ to the site of transcription initiation, suggesting that sequences in the 5′ end of the first gene (
kpsF
) in region 1 may play a role in regulating transcription from this promoter at 37°C. Two additional
cis
-acting sequences, conserved in both the region 1 and 3 promoters, were identified, suggesting a role for these sequences in the coordinate regulation of transcription from these promoters. These results indicate that a complex regulatory network involving a number of global regulators exists for the control of expression of group 2 capsules in
E. coli
.
Interplay between Efflux Pumps May Provide Either Additive or Multiplicative Effects on Drug Resistance
Authors: Lee Angela , Mao Weimin , Warren Mark S. , Mistry Anita , Hoshino Kazuki , Okumura Ryo , Ishida Hiroko , Lomovskaya Olga
Published: July 2002

The effects of simultaneous expression of several efflux pumps on antibiotic resistance were investigated in
Escherichia coli
and
Pseudomonas aeruginosa
. Several combinations of efflux pumps have been studied: (i) simultaneous expression of a single-component efflux pump, which exports antibiotics into the periplasm, in combination with a multicomponent efflux pump that accomplishes efflux directly into the external medium; (ii) simultaneous expression of two single-component pumps; and (iii) simultaneous expression of two multicomponent pumps. It was found that when efflux pumps of different structural types were combined in the same cell (the first case), the observed antibiotic resistance was much higher than that conferred by each of the pumps expressed singly. Simultaneous expression of pairs of single-component or multicomponent efflux pumps (the second and third cases) did not produce strong increases in antibiotic resistance.
Initiation and Termination of DNA Transfer during Conjugation of IncI1 Plasmid R64: Roles of Two Sets of Inverted Repeat Sequences within
oriT
in Termination of R64 Transfer
Authors: Furuya Nobuhisa , Komano Teruya
Published: July 2002

Intercellular transfer of plasmid DNA during bacterial conjugation initiates and terminates at a specific origin of transfer,
oriT
. We have investigated the
oriT
structure of conjugative plasmid R64 with regard to the initiation and termination of DNA transfer. Using recombinant plasmids containing two tandemly repeated R64
oriT
sequences with or without mutations, the subregions required for initiation and termination were determined by examining conjugation-mediated deletion between the repeated
oriT
s. The
oriT
subregion required for initiation was found to be identical to the 44-bp
oriT
core sequence consisting of two units, the conserved nick region sequence and the 17-bp repeat A sequence, that are recognized by R64 relaxosome proteins NikB and NikA, respectively. In contrast, the nick region sequence and two sets of inverted repeat sequences within the 92-bp minimal
oriT
sequence were required for efficient termination. Mutant repeat A sequences lacking NikA-binding ability were found to be sufficient for termination, suggesting that the inverted repeat structures are involved in the termination process. A duplication of the DNA segment between the repeated
oriT
s was also found after mobilization of the plasmid carrying initiation-deficient but termination-proficient
oriT
and initiation-proficient but termination-deficient
oriT
, suggesting that the 3′ terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.
Positive Correlation between Virulence of
Pseudomonas aeruginosa
Mutants in Mice and Insects
Authors: Jander Georg , Rahme Laurence G. , Ausubel Frederick M.
Published: July 2002

Strain PA14, a human clinical isolate of
Pseudomonas aeruginosa
, is pathogenic in mice and insects (
Galleria mellonella
). Analysis of 32 different PA14 mutants in these two hosts showed a novel positive correlation in the virulence patterns. Thus,
G. mellonella
is a good model system for identifying mammalian virulence factors of
P. aeruginosa
.