bluuebunny/crossref_metadata_2025 huggingface dataset:
Pathways for Extracellular Fenton Chemistry in the Brown Rot Basidiomycete
Gloeophyllum trabeum
Authors: Jensen Kenneth A. , Houtman Carl J. , Ryan Zachary C. , Hammel Kenneth E.
Published: July 2002

The brown rot fungus
Gloeophyllum trabeum
uses an extracellular hydroquinone-quinone redox cycle to reduce Fe
3+
and produce H
2
O
2
. These reactions generate extracellular Fenton reagent, which enables
G. trabeum
to degrade a wide variety of organic compounds. We found that
G. trabeum
secreted two quinones, 2,5-dimethoxy-1,4-benzoquinone (2,5-DMBQ) and 4,5-dimethoxy-1,2-benzoquinone (4,5-DMBQ), that underwent iron-dependent redox cycling. Experiments that monitored the iron- and quinone-dependent cleavage of polyethylene glycol by
G. trabeum
showed that 2,5-DMBQ was more effective than 4,5-DMBQ in supporting extracellular Fenton chemistry. Two factors contributed to this result. First,
G. trabeum
reduced 2,5-DMBQ to 2,5-dimethoxyhydroquinone (2,5-DMHQ) much more rapidly than it reduced 4,5-DMBQ to 4,5-dimethoxycatechol (4,5-DMC). Second, although both hydroquinones reduced ferric oxalate complexes, the predominant form of Fe
3+
in
G. trabeum
cultures, the 2,5-DMHQ-dependent reaction reduced O
2
more rapidly than the 4,5-DMC-dependent reaction. Nevertheless, both hydroquinones probably contribute to the extracellular Fenton chemistry of
G. trabeum
, because 2,5-DMHQ by itself is an efficient reductant of 4,5-DMBQ.
Bacteria Mediate Methylation of Iodine in Marine and Terrestrial Environments
Authors: Amachi Seigo , Kamagata Yoichi , Kanagawa Takahiro , Muramatsu Yasuyuki
Published: July 2002

Methyl iodide (CH
3
I) plays an important role in the natural iodine cycle and participates in atmospheric ozone destruction. However, the main source of this compound in nature is still unclear. Here we report that a wide variety of bacteria including terrestrial and marine bacteria are capable of methylating the environmental level of iodide (0.1 μM). Of the strains tested,
Rhizobium
sp. strain MRCD 19 was chosen for further analysis, and it was found that the cell extract catalyzed the methylation of iodide with
S
-adenosyl-
l
-methionine as the methyl donor. These results strongly indicate that bacteria contribute to iodine transfer from the terrestrial and marine ecosystems into the atmosphere.
Effects of Rice Seed Surface Sterilization with Hypochlorite on Inoculated
Burkholderia vietnamiensis
Authors: Miché Lucie , Balandreau Jacques
Published: July 2002

When a combination of hydrogen peroxide and hypochlorite was used to surface sterilize rice seeds, a 10
2
- to 10
4
-fold decrease in CFU was observed during the first 15 h after inoculation of the rice rhizosphere organism
Burkholderia vietnamiensis
TVV75. This artifact could not be eliminated simply by rinsing the seeds, even thoroughly, with sterile distilled water. When growth resumed, a significant increase in the frequency of rifampin- and nalidixic acid-resistant mutants in the population was observed compared to the control without seeds. This phenomenon was a specific effect of hypochlorite; it was not observed with hydrogen peroxide alone. It was also not observed when the effect of hypochlorite was counteracted by sodium thiosulfate. We hypothesized that the hypochlorite used for disinfection reacted with the rice seed surface, forming a chlorine cover which was not removed by rinsing and generated mutagenic chloramines. We studied a set of rifampin- and nalidixic acid-resistant mutants obtained after seed surface sterilization. The corresponding
rpoB
and
gyrA
genes were amplified and sequenced to characterize the induced mutations. The mutations in five of seven nalidixic acid-resistant mutants and all of the rifampin-resistant mutants studied were found to correspond to single amino acid substitutions. Hypochlorite surface sterilization can thus be a source of artifacts when the initial bacterial colonization of a plant is studied.
Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate
Authors: Sakamoto Takeshi , Joern John M. , Arisawa Akira , Arnold Frances H.
Published: July 2002

We are using directed evolution to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report on the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by laboratory evolution. The biotransformation of 4-picoline proceeds at only ∼4.5% of the rate of the natural reaction on toluene. Random mutagenesis, saturation mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding threonine.
Escherichia coli
-expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and ∼20% more activity towards toluene than wild-type TDO. The product of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no
cis
-diols of 4-picoline were observed.
Ruminococcin A, a New Lantibiotic Produced by a
Ruminococcus gnavu
s Strain Isolated from Human Feces
Authors: Dabard J. , Bridonneau C. , Phillipe C. , Anglade P. , Molle D. , Nardi M. , Ladiré M. , Girardin H. , Marcille F. , Gomez A. , Fons M.
Published: July 2002

When cultivated in the presence of trypsin, the
Ruminococcus gnavus
E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to
R. gnavus
. This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.
Comparative Genomic Analysis of Archaeal Genotypic Variants in a Single Population and in Two Different Oceanic Provinces
Authors: Béjà Oded , Koonin Eugene V. , Aravind L. , Taylor Lance T. , Seitz Heidi , Stein Jefferey L. , Bensen Daniel C. , Feldman Robert A. , Swanson Ronald V. , DeLong Edward F.
Published: July 2002

Planktonic crenarchaeotes are present in high abundance in Antarctic winter surface waters, and they also make up a large proportion of total cell numbers throughout deep ocean waters. To better characterize these uncultivated marine crenarchaeotes, we analyzed large genome fragments from individuals recovered from a single Antarctic picoplankton population and compared them to those from a representative obtained from deeper waters of the temperate North Pacific. Sequencing and analysis of the entire DNA insert from one Antarctic marine archaeon (fosmid 74A4) revealed differences in genome structure and content between Antarctic surface water and temperate deepwater archaea. Analysis of the predicted gene products encoded by the 74A4 sequence and those derived from a temperate, deepwater planktonic crenarchaeote (fosmid 4B7) revealed many typical archaeal proteins but also several proteins that so far have not been detected in archaea. The unique fraction of marine archaeal genes included, among others, those for a predicted RNA-binding protein of the bacterial cold shock family and a eukaryote-type Zn finger protein. Comparison of closely related archaea originating from a single population revealed significant genomic divergence that was not evident from 16S rRNA sequence variation. The data suggest that considerable functional diversity may exist within single populations of coexisting microbial strains, even those with identical 16S rRNA sequences. Our results also demonstrate that genomic approaches can provide high-resolution information relevant to microbial population genetics, ecology, and evolution, even for microbes that have not yet been cultivated.
Purification and Characterization of an Extracellular Protease from
Xenorhabdus nematophila
Involved in Insect Immunosuppression
Authors: Caldas C. , Cherqui A. , Pereira A. , Simões N.
Published: July 2002

Xenorhabdus nematophila
, a bacterium pathogenic for insects associated with the nematode
Steinernema carpocapsae
, releases high quantities of proteases, which may participate in the virulence against insects. Zymogram assays and cross-reactions of antibodies suggested that two distinct proteases were present. The major one, protease II, was purified and shown to have a molecular mass of 60 kDa and an estimated isoelectric point of 8.5. Protease II digested the chromogenic substrate
N
-tosyl-Gly-Pro-Arg-paranitroanilide (pNA) with
V
max
and

K
m

values of 0.0551 μM/min and 234 μM, respectively, and the substrate
dl
-Val-Leu-Arg-pNA with
V
max
and

K
m

values of 0.3830 μM/min and 429 μM, respectively. Protease II activity was inhibited 93% by Pefabloc SC and 45% by chymostatin. The optimum pH for protease II was 7, and the optimum temperature was 23°C. Proteolytic activity was reduced by 90% at 60°C for 10 min. Sequence analysis was performed on four internal peptides that resulted from the digestion of protease II. Fragments 29 and 45 are 75 and 68% identical to alkaline metalloproteinase produced by
Pseudomonas aeruginosa
. Fragment 29 is 79% identical to a metalloprotease of
Erwinia amylovora
and 75% identical to the protease C precursor of
Erwinia chrysanthemi
. Protease II showed no toxicity to hemocytes but destroyed antibacterial activity on the hemolymph of inoculated insects' larvae and reduced 97% of the cecropin A bacteriolytic activity.
Microbial Diversity of Hydrothermal Sediments in the Guaymas Basin: Evidence for Anaerobic Methanotrophic Communities
Authors: Teske Andreas , Hinrichs Kai-Uwe , Edgcomb Virginia , de Vera Gomez Alvin , Kysela David , Sylva Sean P. , Sogin Mitchell L. , Jannasch Holger W.
Published: July 2002

Microbial communities in hydrothermally active sediments of the Guaymas Basin (Gulf of California, Mexico) were studied by using 16S rRNA sequencing and carbon isotopic analysis of archaeal and bacterial lipids. The Guaymas sediments harbored uncultured euryarchaeota of two distinct phylogenetic lineages within the anaerobic methane oxidation 1 (ANME-1) group, ANME-1a and ANME-1b, and of the ANME-2c lineage within the
Methanosarcinales
, both previously assigned to the methanotrophic archaea. The archaeal lipids in the Guaymas Basin sediments included archaeol, diagnostic for nonthermophilic euryarchaeota, and
sn
-2-hydroxyarchaeol, with the latter compound being particularly abundant in cultured members of the
Methanosarcinales
. The concentrations of these compounds were among the highest observed so far in studies of methane seep environments. The δ-
13
C values of these lipids (δ-
13
C = −89 to −58‰) indicate an origin from anaerobic methanotrophic archaea. This molecular-isotopic signature was found not only in samples that yielded predominantly ANME-2 clones but also in samples that yielded exclusively ANME-1 clones. ANME-1 archaea therefore remain strong candidates for mediation of the anaerobic oxidation of methane. Based on 16S rRNA data, the Guaymas sediments harbor phylogenetically diverse bacterial populations, which show considerable overlap with bacterial populations of geothermal habitats and natural or anthropogenic hydrocarbon-rich sites. Consistent with earlier observations, our combined evidence from bacterial phylogeny and molecular-isotopic data indicates an important role of some novel deeply branching bacteria in anaerobic methanotrophy. Anaerobic methane oxidation likely represents a significant and widely occurring process in the trophic ecology of methane-rich hydrothermal vents. This study stresses a high diversity among communities capable of anaerobic oxidation of methane.
Organophosphonate Utilization by the Thermophile
Geobacillus caldoxylosilyticus
T20
Authors: Obojska Agnieszka , Ternan Nigel G. , Lejczak Barbara , Kafarski Pawel , McMullan Geoff
Published: July 2002

A strain of
Geobacillus caldoxylosilyticus
from central heating system water could utilize a number of organophosphonates as the sole phosphorus source for growth at 60°C. During growth on glyphosate, aminomethylphosphonate release to the medium was observed, and in cell extracts, a glyphosate oxidoreductase-type activity, producing stoichiometric amounts of aminomethylphosphonate and glyoxylate from glyphosate, was detectable.
Engineered Rhizosphere: the Trophic Bias Generated by Opine-Producing Plants Is Independent of the Opine Type, the Soil Origin, and the Plant Species
Authors: Mansouri Hounayda , Petit Annik , Oger Phil , Dessaux Yves
Published: July 2002

In a previous study, we demonstrated that transgenic
Lotus
plants producing opines (which are small amino acid and sugar conjugates) specifically favor growth of opine-degrading rhizobacteria. The opine-induced bias was repeated and demonstrated with another soil type and another plant species (
Solanum nigrum
). This phenomenon is therefore independent of both soil type and plant species.
Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for
Bacillus subtilis
Authors: Kaltwasser Marcus , Wiegert Thomas , Schumann Wolfgang
Published: July 2002

Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any
Bacillus subtilis
protein. These vectors are based on the backbone of pMUTIN2 and replace the
lacZ
gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3′ terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein
+
, yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.
Prevalence of Known P-Fimbrial G Alleles in
Escherichia coli
and Identification of a New Adhesin Class
Authors: Manning Shannon D. , Zhang Lixin , Foxman Betsy , Spindler Angela , Tallman Patricia , Marrs Carl F.
Published: July 2002

Screening a large
Escherichia coli
collection for P-fimbrial adhesin classes identified 20 unclassifiable strains. Cloning and sequencing of
papG
from an unclassifiable strain identified another G allele. The novel adhesin gene has 65% identity to the class I adhesin gene, 44% identity to the class II adhesin gene, and 43% identity to the class III adhesin gene.
Role of Alveolar Macrophages in
Candida
-Induced Acute Lung Injury
Authors: Kubota Yutaka , Iwasaki Yoshinobu , Harada Hidehiko , Yokomura Ichiro , Ueda Mikio , Hashimoto Shinichi , Nakagawa Masao
Published: July 2002

Recent studies have shown that alveolar macrophages (AMs) not only act as phagocytes but also play a central role as potent secretory cells in various lung diseases, including pneumonia and acute respiratory distress syndrome. The behavior of AMs during disseminated candidiasis, however, is insufficiently elucidated. This study is the first to report disseminated candidiasis in AM-depleted mice and to analyze the effect of AMs on
Candida
-induced acute lung injury. While all AM-sufficient mice died by day 2 after infection with
Candida albicans
, no mortality was observed among AM-depleted mice. Unexpectedly, the CFU numbers of
C. albicans
isolated from the lungs of AM-depleted mice were significantly higher than those for
C. albicans
isolated from AM-sufficient mice. The lung wet-to-dry weight ratio was lower for AM-depleted mice than for AM-sufficient mice, although this difference was not significant. We found that bronchoalveolar lavage fluid (BALF) from AM-depleted mice in candidemia contained fewer neutrophils than BALF from AM-sufficient mice. In addition, myeloperoxidase activities in lung homogenates of AM-depleted mice were significantly lower than those in homogenates of AM-sufficient mice. A significant decrease in levels of murine macrophage inflammatory protein 2 (MIP-2), a potent chemoattractant for neutrophils, was noted in lung homogenates from AM-depleted mice compared with levels in homogenates from AM-sufficient mice. Immunohistochemical studies using anti-MIP-2 antibodies revealed that AMs were the cellular source of MIP-2 within the lung during candidemia. We observed that AM depletion decreased levels of AM-derived neutrophil chemoattractant, alleviated acute lung injury during candidemia, and prolonged the survival of mice in candidemia, even though clearance of
C. albicans
from the lungs was reduced.
Bacterial Infection in Chronic Obstructive Pulmonary Disease in 2000: a State-of-the-Art Review
Authors: Sethi Sanjay , Murphy Timothy F.
Published: July 2002

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States. The precise role of bacterial infection in the course and pathogenesis of COPD has been a source of controversy for decades. Chronic bacterial colonization of the lower airways contributes to airway inflammation; more research is needed to test the hypothesis that this bacterial colonization accelerates the progressive decline in lung function seen in COPD (the vicious circle hypothesis). The course of COPD is characterized by intermittent exacerbations of the disease. Studies of samples obtained by bronchoscopy with the protected specimen brush, analysis of the human immune response with appropriate immunoassays, and antibiotic trials reveal that approximately half of exacerbations are caused by bacteria. Nontypeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae are the most common causes of exacerbations, while Chlamydia pneumoniae causes a small proportion. The role of Haemophilus parainfluenzae and gram-negative bacilli remains to be established. Recent progress in studies of the molecular mechanisms of pathogenesis of infection in the human respiratory tract and in vaccine development guided by such studies promises to lead to novel ways to treat and prevent bacterial infections in COPD.
Genetic Control of Susceptibility toPorphyromonas gingivalis-Induced Alveolar Bone Loss in Mice
Authors: Baker Pamela J. , Dixon Mark , Roopenian Derry C.
Published: July 2002

Periodontal disease affects a large percentage of the human population. Resorption of the alveolar bone of the jaw is a pivotal sequela of periodontal disease, because this bone is the attachment site for the periodontal ligaments that anchor the teeth. Using a murine model in which alveolar bone loss is induced by oral infection withPorphyromonas gingivalis, a gram-negative bacterium associated with human adult periodontal disease, we provide evidence suggesting that susceptibility to such bone loss is a genetically determined trait. AKR/J, DBA/2J, and BALB/cByJ or BALB/cJ mice were highly susceptible, while A/J, A/HeJ, 129/J, SJL/J, and C57BL/6J mice were much more resistant. When susceptible BALB/cJ and BALB/cByJ mice were crossed to resistant strains, two patterns were observed. (BALBc/ByJ × C57BL/6J)F1offspring were susceptible, suggesting C57BL/6J has recessive resistance alleles, while (BALB/cJ × A/J)F1mice were all resistant, suggesting that A/J mice have dominant resistance alleles. These results suggest a tractable genetic basis forP. gingivalis-induced alveolar bone loss and open the possibility of exploiting the mouse model to identify loci important for host susceptibility and resistance to periodontal disease.
Capsule Impedes Adhesion to and Invasion of Epithelial Cells by
Klebsiella pneumoniae
Authors: Sahly Hany , Podschun Rainer , Oelschlaeger Tobias A. , Greiwe Michael , Parolis Haralambos , Hasty David , Kekow Jörn , Ullmann Uwe , Ofek Itzhak , Sela Shlomo
Published: July 2002

The adhesion of K21a, K26, K36, and K50 capsulated
Klebsiella
strains to ileocecal (HCT-8) and bladder (T24) epithelial cell lines was significantly lower than that of their corresponding spontaneous noncapsulated variants K21a/3, K26/1, K36/3, and K50/3, respectively. Internalization of the bacteria by both epithelial cell lines was also significantly reduced. Similarly, a capsule-switched derivative, K2(K36), that exhibited a morphologically larger K36 capsule and formed more capsular material invaded the ileocecal epithelial cell line poorly compared to the corresponding K2 parent strain. None of the capsulated strains exhibited significant mannose-sensitive type 1 fimbriae, whereas two of the noncapsulated variants K21a/3 and K50/3 exhibited potent mannose-sensitive hemagglutinating activity. Although hemagglutinating activity that could be attributed to mannose-resistant
Klebsiella
type 3 fimbriae was weak in all strains, in several cases the encapsulated parent strains exhibited lower titers than their corresponding noncapsulated variants. Although the level of adhesion to the ileocecal cells is not different from adhesion to bladder cells, bacterial internalization by bladder cells was significantly lower than internalization by ileocecal cells, suggesting that bladder cells lack components required for the internalization of
Klebsiella.
Interleukin-18 (IL-18) Enhances Innate IL-12-Mediated Resistance toToxoplasma gondii
Authors: Cai Guifang , Kastelein Robert , Hunter Christopher A.
Published: July 2002

Innate resistance toToxoplasma gondiiis dependent on the ability of interleukin-12 (IL-12) to stimulate natural killer (NK) cell production of gamma interferon (IFN-γ). Since IL-18 is a potent enhancer of IL-12-induced production of IFN-γ by NK cells, SCID mice (which lack an adaptive immune response) were used to assess the role of IL-18 in innate resistance toT. gondii. Administration of anti-IL-18 to SCID mice infected withT. gondiiresulted in an early reduction in serum levels of IFN-γ but did not significantly decrease resistance to this infection. In contrast, administration of exogenous IL-18 to infected SCID mice resulted in increased production of IFN-γ, reduced parasite burden, and a delay in time to death. The protective effects of IL-18 treatment correlated with increased NK cell numbers and cytotoxic activity at the local site of administration and with elevated levels of inducible nitrous oxide synthose in the spleens of treated mice. In addition, in vivo depletion studies demonstrated that the ability of exogenous IL-18 to enhance resistance toT. gondiiwas dependent on IL-12, IFN-γ, and NK cells. Together, these studies demonstrate that although endogenous IL-18 appears to have a limited role in innate resistance toT. gondii, treatment with IL-18 can augment NK cell-mediated immunity to this pathogen.
Role of Toxin Functional Domains in Anthrax Pathogenesis
Authors: Brossier Fabien , Weber-Levy Martine , Mock Michele , Sirard Jean-Claude
Published: July 2002

We investigated the role of the functional domains of anthrax toxins during infection. Three proteins produced by
Bacillus anthracis
, the protective antigen (PA), the lethal factor (LF), and the edema factor (EF), combine in pairs to produce the lethal (PA+LF) and edema (PA+EF) toxins. A genetic strategy was developed to introduce by allelic exchange specific point mutations or in-frame deletions into
B. anthracis
toxin genes, thereby impairing either LF metalloprotease or EF adenylate cyclase activity or PA functional domains. In vivo effects of toxin mutations were analyzed in an experimental infection of mice. A tight correlation was observed between the properties of anthrax toxins delivered in vivo and their in vitro activities. The synergic effects of the lethal and edema toxins resulted purely from their enzymatic activities, suggesting that in vivo these toxins may act together. The PA-dependent antibody response to LF induced by immunization with live
B. anthracis
was used to follow the in vivo interaction of LF and PA. We found that the binding of LF to PA in vivo was necessary and sufficient for a strong antibody response against LF, whereas neither LF activity nor binding of lethal toxin complex to the cell surface was required. Mutant PA proteins were cleaved in mice sera. Thus, our data provide evidence that, during anthrax infection, PA may interact with LF before binding to the cell receptor. Immunoprotection studies indicated that the strain producing detoxified LF and EF, isogenic to the current live vaccine Sterne strain, is a safe candidate for use as a vaccine against anthrax.
Predictive Value of Nuclear Factor κB Activity and Plasma Cytokine Levels in Patients with Sepsis
Authors: Arnalich Francisco , Garcia-Palomero Esther , López Julia , Jiménez Manuel , Madero Rosario , Renart Jaime , Vázquez Juan José , Montiel Carmen
Published: July 2002

The relationship between fluctuating cytokine concentrations in plasma and the outcome of sepsis is complex. We postulated that early measurement of the activation of nuclear factor κB (NF-κB), a transcriptional regulatory protein involved in proinflammatory cytokine expression, may help to predict the outcome of sepsis. We determined NF-κB activation in peripheral blood mononuclear cells of 34 patients with severe sepsis (23 survivors and 11 nonsurvivors) and serial concentrations of inflammatory cytokines (interleukin-6, interleukin-1, and tumor necrosis factor) and various endogenous antagonists in plasma. NF-κB activity was significantly higher in nonsurvivors and correlated strongly with the severity of illness (APACHE II score), although neither was related to the cytokine levels. Apart from NF-κB activity, the interleukin-1 receptor antagonist was the only cytokine tested whose level in plasma was of value in predicting mortality by logistic regression analysis. These results underscore the prognostic value of early measurement of NF-κB activity in patients with severe sepsis.
Lipopolysaccharide-Binding Protein and Phospholipid Transfer Protein Release Lipopolysaccharides from Gram-Negative Bacterial Membranes
Authors: Vesy C. J. , Kitchens R. L. , Wolfbauer G. , Albers J. J. , Munford R. S.
Published: July 2002

Although animals mobilize their innate defenses against gram-negative bacteria when they sense the lipid A moiety of bacterial lipopolysaccharide (LPS), excessive responses to this conserved bacterial molecule can be harmful. Of the known ways for decreasing the stimulatory potency of LPS in blood, the binding and neutralization of LPS by plasma lipoproteins is most prominent. The mechanisms by which host lipoproteins take up the native LPS that is found in bacterial membranes are poorly understood, however, since almost all studies of host-LPS interactions have used purified LPS aggregates. Using nativeSalmonella entericaserovar Typhimurium outer membrane fragments (blebs) that contained3H-labeled lipopolysaccharide (LPS) and35S-labeled protein, we found that two human plasma proteins, LPS-binding protein (LBP) and phospholipid transfer protein (PLTP), can extract [3H]LPS from bacterial membranes and transfer it to human high-density lipoproteins (HDL). Soluble CD14 (sCD14) did not release LPS from blebs yet could facilitate LBP-mediated LPS transfer to HDL. LBP, but not PLTP, also promoted the activation of human monocytes by bleb-derived LPS. Whereas depleting or neutralizing LBP significantly reduced LPS transfer from blebs to lipoproteins in normal human serum, neutralizing serum PLTP had no demonstrable effect. Of the known lipid transfer proteins, LBP is thus most able to transfer LPS from bacterial membranes to the lipoproteins in normal human serum.
TheshdAGene Is Restricted to Serotypes ofSalmonella entericaSubspecies I and Contributes to Efficient and Prolonged Fecal Shedding
Authors: Kingsley Robert A. , van Amsterdam Karin , Kramer Naomi , Bäumler Andreas J.
Published: July 2002

Little is known about factors which enableSalmonellaserotypes to circulate within populations of livestock and domestic fowl. We have identified a DNA region which is present inSalmonellaserotypes commonly isolated from livestock and domestic fowl (S. entericasubspecies I) but absent from reptile-associatedSalmonellaserotypes (S. bongoriandS. entericasubspecies II to VII). This DNA region was cloned fromSalmonellaserotype Typhimurium and sequence analysis revealed the presence of a 6,105-bp open reading frame, designatedshdA, whose product's deduced amino acid sequence displayed homology to that of AIDA-I from diarrheagenicEscherichia coli, MisL of serotype Typhimurium, and IcsA ofShigella flexneri. TheshdAgene was located adjacent toxseAat 52 min, in a 30-kb DNA region which is not present inEscherichia coliK-12. A serotype TyphimuriumshdAmutant was shed with the feces in reduced numbers and for a shorter period of time compared to its isogenic parent. A possible role for theshdAgene during the expansion in host range ofS. entericasubspecies I to include warm-blooded vertebrates is discussed.
Curli Loci of
Shigella
spp
Authors: Sakellaris Harry , Hannink Nerissa K. , Rajakumar Kumar , Bulach Dieter , Hunt Meredith , Sasakawa Chihiro , Adler Ben
Published: July 2002

An unstable chromosomal element encoding multiple antibiotic resistance in
Shigella flexneri
serotype 2a was found to include sequences homologous to the
csg
genes encoding curli in
Escherichia coli
and
Salmonella enterica
serovar Typhimurium. As curli have been implicated in the virulence of serovar Typhimurium, we investigated the
csg
loci in all four species of
Shigella
. DNA sequencing and PCR analysis showed that the
csg
loci of a wide range of
Shigella
strains, of diverse serotypes and different geographical distributions, were almost universally disrupted by deletions or insertions, indicating the existence of a strong selective pressure against the expression of curli. Strains of enteroinvasive
E. coli
(EIEC), which share virulence traits with
Shigella
spp. and cause similar diseases in humans, also possessed insertions or deletions in the
csg
locus or were otherwise unable to produce curli. Since the production of curli is a widespread trait in environmental isolates of
E. coli
, our results suggest that genetic lesions that abolish curli production in the closely related genus
Shigella
and in EIEC are pathoadaptive mutations.
Expression of Cytokine Genes during Pneumococcal and NontypeableHaemophilus influenzaeAcute Otitis Media in the Rat
Authors: Melhus Åsa , Ryan Allen F.
Published: July 2002

Acute otitis media (AOM) elicits potent inflammatory responses from the cells of the middle ear mucosa as well as from infiltrating leukocytes. To explore host responses during experimental AOM induced byStreptococcus pneumoniaetype 3 and nontypeableHaemophilus influenzae(NTHi), otomicroscopy findings and expression of cytokine genes in the middle ear were monitored up to 1 month postinoculation. The mucosa and infiltrating cells responded rapidly to the bacterial challenge. Otomicroscopically, AOM appeared 1 day after NTHi inoculation and 3 days after pneumococcus inoculation. Pneumococcal AOM was more severe than NTHi otitis, but in general, lower transcript levels were detected in pneumococcus-infected than in NTHi-infected animals. Interleukin-6 (IL-6) mRNA levels peaked at 3 to 6 h for both pneumococcus-infected and NTHi-infected animals. IL-1α, tumor necrosis factor alpha, and IL-10 mRNA levels peaked at 6 h for NTHi otitis and 1 to 3 days for pneumococcal otitis. Comparing otomicroscopy with expression profiles, it would appear that the majority of cytokine mRNAs had passed their peak before the AOM diagnosis could be made clinically. Only transforming growth factor β mRNA followed a slower time course, peaking very late and continuing expression even after the AOM was otomicroscopically resolved. IL-2 and IL-4 mRNAs were not detected in any animal at any time. Most of the investigated cytokines are very early markers for AOM and may be involved in initiation of inflammation, but they would be poor targets for pharmacological manipulation since their levels decline before clinical signs appear.
Requirement of the
Pseudomonas aeruginosa tonB
Gene for High-Affinity Iron Acquisition and Infection
Authors: Takase Hiroyuki , Nitanai Hironobu , Hoshino Kazuki , Otani Tsuyoshi
Published: July 2002

To investigate the contribution of the TonB protein to high-affinity iron acquisition in
Pseudomonas aeruginosa
, we constructed
tonB
-inactivated mutants from strain PAO1 and its derivative deficient in producing the siderophores pyoverdin and pyochelin. The
tonB
mutants could not grow in a free-iron-restricted medium prepared by apotransferrin addition, even though the medium was supplemented with each purified siderophore or with a heme source (hemoglobin or hemin). The
tonB
inactivation was shown to make
P. aeruginosa
unable to acquire iron from the transferrin with either siderophore. Introduction of a plasmid carrying the intact
tonB
gene restored growth of the
tonB
mutant of PAO1 in the free-iron-restricted medium without any supplements and restored growth of the
tonB
mutant of the siderophore-deficient derivative in the medium supplemented with pyoverdin, pyochelin, hemoglobin, or hemin. In addition, animal experiments showed that, in contrast to PAO1, the
tonB
mutant of PAO1 could not grow in vivo, such as in the muscles and lungs of immunosuppressed mice, and could not kill any of the animals. The in vivo growth ability and lethal virulence were also restored by introduction of the
tonB
-carrying plasmid in the
tonB
mutant. These results indicate clearly that the intact
tonB
gene—and, therefore, the TonB protein encoded by it—is essential for iron acquisition mediated by pyoverdin and pyochelin and via heme uptake in
P. aeruginosa
and suggest that the TonB-dependent iron acquisition may be essential for
P. aeruginosa
to infect the animal host.
CD85/LIR-1/ILT2 and CD152 (Cytotoxic T Lymphocyte Antigen 4) Inhibitory Molecules Down-Regulate the Cytolytic Activity of Human CD4+T-Cell Clones Specific forMycobacterium tuberculosis
Authors: Merlo Andrea , Saverino Daniele , Tenca Claudya , Grossi Carlo Enrico , Bruno Silvia , Ciccone Ermanno
Published: July 2002

Antigen-specific cytolytic CD4+T lymphocytes controlMycobacterium tuberculosisinfection by secreting cytokines and by killing macrophages that have phagocytosed the pathogen. However, lysis of the latter cells promotes microbial dissemination, and other macrophages engulf the released bacteria. Subsequently, CD4+T-cell-mediated killing of macrophages goes on, and this persistent process may hamper control of infection, unless regulatory mechanisms maintain a subtle balance between lysis of macrophages by cytolytic CD4+cells and activation of cytolytic CD4+cells by infected macrophages. We asked whether inhibitory molecules expressed by CD4+cytolytic T lymphocytes could play a role in such a balance. To this end, human CD4+T-cell clones specific forM. tuberculosiswere produced that displayed an autologous major histocompatibility complex class II-restricted lytic ability against purified protein derivative (PPD)-pulsed antigen-presenting cells. All T-cell clones expressed CD152 (cytotoxic T-lymphocyte antigen 4 [CTLA-4]) and CD85/leukocyte immunoglobulin-like receptor 1 (LIR-1)/immunoglobulin-like transcript 2 (ILT2) inhibitory receptors, but not CD94 and the killer inhibitory receptor (or killer immunoglobulin-like receptor [KIR]) p58.2. CD3-mediated activation of the clones was inhibited in a redirected killing assay in which CD152 and CD85/LIR-1/ILT2 were cross-linked. Specific antigen-mediated proliferation of the clones was also sharply reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked by specific monoclonal antibody (MAb) followed by goat anti-mouse antiserum. In contrast, blockade of the receptors by specific MAb only increased their proliferation. Production of interleukin 2 (IL-2) and gamma interferon (IFN-γ) by the T-cell clones was also strongly reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked. The lytic activity of the T-cell clones against PPD-pulsed autologous monocytes or Epstein-Barr virus-activated B cells was increased by blockade and decreased by cross-linking of the receptors. These results indicate that CD152 and CD85/LIR-1/ILT2 play a role in the regulation of the antigen-specific activity of CD4+cytolytic T lymphocytes against PPD-presenting cells.
T Cells Augment Monocyte and Neutrophil Function in Host Resistance against Oropharyngeal Candidiasis
Authors: Farah C. S. , Elahi S. , Pang G. , Gotjamanos T. , Seymour G. J. , Clancy R. L. , Ashman R. B.
Published: July 2002

The purpose of this study was to identify the cell populations involved in recovery from oral infections withCandida albicans. Monoclonal antibodies specific for CD4+cells, CD8+cells, and polymorphonuclear leukocytes were used to deplete BALB/c and CBA/CaH mice of the relevant cell populations in systemic circulation. Monocytes were inactivated with the cytotoxic chemical carrageenan. Mice were infected with 108C. albicansyeast cells and monitored for 21 days. Systemic depletion of CD4+and CD8+T lymphocytes alone did not increase the severity of oral infection compared to that of controls. Oral colonization persisted in animals treated with head and neck irradiation and depleted of CD4+T cells, whereas infections in animals that received head and neck irradiation alone or irradiation and anti-CD8 antibody cleared the infection in a comparable fashion. The depletion of polymorphonuclear cells and the cytotoxic inactivation of mononuclear phagocytes significantly increased the severity of oral infection in both BALB/c and CBA/CaH mice. High levels of interleukin 12 (IL-12) and gamma interferon (IFN-γ) were produced by lymphocytes from the draining lymph nodes of recovering animals, whereas IL-6, tumor necrosis factor alpha, and IFN-γ were detected in the oral mucosae of both naı̈ve and infected mice. The results indicate that recovery from oropharyngeal candidiasis in this model is dependent on CD4+-T-cell augmentation of monocyte and neutrophil functions exerted by Th1-type cytokines such as IL-12 and IFN-γ.
Mycobacterial Protein HbhA Binds Human Complement Component C3
Authors: Mueller-Ortiz Stacey L. , Wanger Audrey R. , Norris Steven J.
Published: July 2002

Mycobacterium tuberculosisandMycobacterium aviumare facultative intracellular pathogens that are able to survive and replicate in mononuclear phagocytes. Human complement component C3 has previously been shown to mediate attachment and phagocytosis of these bacteria by mononuclear phagocytes. In this study, a C3 ligand affinity blot protocol was used to identify a 30-kDa C3-binding protein inM. tuberculosisandMycobacterium smegmatisand a 31-kDa C3-binding protein inM. avium. The C3-binding proteins inM. tuberculosisandM. aviumlocalized to the cell membrane fraction and partitioned to the detergent fraction during Triton X-114 phase partitioning. The C3-binding protein fromM. tuberculosiswas partially purified using a cation exchange column and was shown to bind concanavalin A. The N terminus and an internal fragment of the partially purified C3-binding protein were subjected to amino acid sequence analysis. The resulting amino acid sequences matched theM. tuberculosisheparin-binding hemagglutinin (HbhA) protein. Recombinant full-length HbhA and the C terminus of HbhA fused to maltose-binding protein, but not recombinant HbhA lacking the C-terminal region, bound human C3. Recombinant full-length HbhA coated on polystyrene beads, was found to enhance the adherence and/or phagocytosis of the coated beads to J774.A1 cells in both the presence and absence of human serum. The presence of complement-sufficient serum increased the adherence of the HbhA-coated beads to the J774.A1 cells in a C3-dependent manner. If HbhA within the bacterial cell membrane functions similarly to isolated HbhA, this protein may enhance the adherence and phagocytosis ofM. tuberculosisandM. aviumto mononuclear phagocytes through the binding of C3 and interaction with C3 receptors on mononuclear phagocytes.
Detection ofMycobacterium tuberculosisGroup Organisms in Human and Mouse Joint Tissue by Reverse Transcriptase PCR: Prevalence in Diseased Synovial Tissue Suggests Lack of Specific Association with Rheumatoid Arthritis
Authors: Kempsell Karen E. , Cox Charles J. , McColm Andrew A. , Bagshaw Julie A. , Reece Richard , Veale Douglas J. , Emery Paul , Isaacs John D. , Gaston J. S. Hill , Crowe J. Scott
Published: July 2002

Infection with mycobacterial species, includingMycobacterium tuberculosis, has long been implicated in the etiopathology of rheumatoid arthritis (RA) on the basis of clinical and pathological similarities between tuberculosis and RA. Despite evidence of immune responses to mycobacterial antigens in RA patient synovial fluid, cross-reactivity between these and host joint antigens, and the presence ofM. tuberculosisprotein antigen in RA synovial fluid, a definite causal association with RA has not been shown. Previous studies from our laboratory using reverse transcriptase PCR (RT-PCR) of bacterial rRNAs have shown RA synovium to be colonized by a diverse range of bacteria, most of commensal origin. However,M. tuberculosisgroup organism (MTG) RNA sequences were found in one RA patient tissue. Since this was considered of sufficient interest to warrant further investigation, we devised aM. tuberculosis-specific nested RT-PCR test which could be used for detection of MTG in a mixed pool of bacterial crDNAs. This test was used to investigate the distribution of MTG in RA synovial tissue and also non-RA arthritis and healthy control tissues and was also used to examine the tissue distribution of MTG in an acute and chronic model ofM. tuberculosisinfection in the BALB/c mouse. MTG sequences were found in a high proportion of RA patient synovial tissues but also in non-RA arthritis control tissues at lower frequency. This likely reflects trafficking of persistentM. bovisBCG to inflamed joint tissue, irrespective of cause. MTG were not found in healthy synovial tissue or the tissue of patients with undifferentiated arthritis. In both the acute and chronic models of infection in BALB/c mice,M. tuberculosiswas also found to have trafficked to joint tissues, however, no signs of inflammation, arthritis, or pathology associated withM. tuberculosisinfection was seen. These combined results would argue against a specific causal role of MTG in RA-like arthritis; however, their role as adjuvant in immune dysfunction in an innately susceptible host cannot be excluded.
Leukotriene B4Augments Neutrophil Phagocytosis ofKlebsiella pneumoniae
Authors: Mancuso Peter , Nana-Sinkam Patrick , Peters-Golden Marc
Published: July 2002

Neutrophils play a critical role in the clearance of bacteria from the lung and other organs by their capacity for phagocytosis and killing. Previously, we identified an important role for the leukotrienes in rat alveolar macrophage phagocytosis ofKlebsiella pneumoniae. In this report, we explored the possibility that the leukotrienes play an important role in phagocytosis by neutrophils as well. Inhibition of endogenous leukotriene synthesis by 5-lipoxygenase knockout in mice or by pharmacologic means in human peripheral blood neutrophils attenuated phagocytosis of opsonizedK. pneumoniae. Reduced phagocytosis was also observed in human neutrophils pretreated with a leukotriene B4receptor but not a cysteinyl-leukotriene receptor antagonist. While leukotriene B4reconstituted defective phagocytosis in leukotriene-deficient neutrophils and enhanced phagocytosis in neutrophils capable of leukotriene synthesis, leukotriene C4, leukotriene D4, 5-hydroperoxyeicosatetraenoic acid, and 5-oxo-eicosatetraenoic acid were ineffective. To determine the opsonin dependence of the leukotriene B4augmentation of phagocytosis, we assessed the ability of leukotriene B4to modulate neutrophil phagocytosis and the adherence of sheep erythrocytes opsonized with immunoglobulin G or the complement fragment C3bi. While leukotriene B4augmented both Fc receptor- and complement receptor-mediated phagocytosis, increased adherence to leukotriene B4-treated neutrophils was limited to complement opsonized targets. In conclusion, we have identified a novel role for leukotriene B4in the augmentation of neutrophil phagocytosis mediated by either the Fc or complement receptor.
Influence of Major Histocompatibility Complex on Bacterial Composition of Fecal Flora
Authors: Toivanen Paavo , Vaahtovuo Jussi , Eerola Erkki
Published: July 2002

Very little is known about how the host genome influences the composition of the gastrointestinal flora, largely due to the great number and diversity of bacteria present in the flora and the difficulties of using traditional methods of bacterial isolation and identification. We have approached the problem by studying bacterium-derived cellular fatty acids in the stool samples of six mouse strains congenic for the major histocompatibility complex (MHC). The results obtained indicate that the composition of the fecal flora is genetically regulated. In addition to undefined gene loci, MHC alone has a pronounced effect, since mice with different MHC in the same background have significantly different fecal floras. Demonstration of the genetic influence on the gastrointestinal flora opens a new approach to studying the pathogenesis of bacterially induced diseases.
Disruption ofPlasmodium falciparumChitinase Markedly Impairs Parasite Invasion of Mosquito Midgut
Authors: Tsai Yao-Lung , Hayward Rhian E. , Langer Rebecca C. , Fidock David A. , Vinetz Joseph M.
Published: July 2002

To initiate invasion of the mosquito midgut,Plasmodiumookinetes secrete chitinolytic activity to penetrate the peritrophic matrix surrounding the blood meal. While ookinetes of the avian malaria parasitePlasmodium gallinaceumappear to secrete products of two chitinase genes, to date only one chitinase gene,PfCHT1, has been identified in the nearly completedPlasmodium falciparumstrain 3D7 genome database. To test the hypothesis that the single identified chitinase ofP. falciparumis necessary for ookinete invasion, thePfCHT1gene was disrupted 39 bp upstream of the stop codon.PfCHT1-disrupted parasites had normal gametocytogenesis, exflagellation, and ookinete formation but were markedly impaired in their ability to form oocysts inAnopheles freebornimidguts. Confocal microscopy demonstrated that the truncated PfCHT1 protein was present in mutant ookinetes but that the concentration of mutant PfCHT1 within the apical end of the ookinetes was substantially reduced. These data suggest that full-length PfCHT1 is essential for intracellular trafficking and secretion and that thePfCHT1gene product is necessary for ookinetes to invade the mosquito midgut.