bluuebunny/crossref_metadata_2025 huggingface dataset:
X‐ray structure of the AAC(6′)‐Ii antibiotic resistance enzyme at 1.8 Å resolution; examination of oligomeric arrangements in GNAT superfamily members
Authors: Burk David L. , Ghuman Navleen , Wybenga‐Groot Leanne E. , Berghuis Albert M.
Published: February 2003

The rise of antibiotic resistance as a public health concern has led to increased interest in studying the ways in which bacteria avoid the effects of antibiotics. Enzymatic inactivation by several families of enzymes has been observed to be the predominant mechanism of resistance to aminoglycoside antibiotics such as kanamycin and gentamicin. Despite the importance of acetyltransferases in bacterial resistance to aminoglycoside antibiotics, relatively little is known about their structure and mechanism. Here we report the three‐dimensional atomic structure of the aminoglycoside acetyltransferase AAC(6′)‐Ii in complex with coenzyme A (CoA). This structure unambiguously identifies the physiologically relevant AAC(6′)‐Ii dimer species, and reveals that the enzyme structure is similar in the AcCoA and CoA bound forms. AAC(6′)‐Ii is a member of the GCN5‐related N‐acetyltransferase (GNAT) superfamily of acetyltransferases, a diverse group of enzymes that possess a conserved structural motif, despite low sequence homology. AAC(6′)‐Ii is also a member of a subset of enzymes in the GNAT superfamily that form multimeric complexes. The dimer arrangements within the multimeric GNAT superfamily members are compared, revealing that AAC(6′)‐Ii forms a dimer assembly that is different from that observed in the other multimeric GNAT superfamily members. This different assembly may provide insight into the evolutionary processes governing dimer formation.
Molecular Fossil Record of Elevated Methane Levels in Late Pleistocene Coastal Waters
Authors: Hinrichs Kai-Uwe , Hmelo Laura R. , Sylva Sean P.
Published: February 2003

Accumulating evidence suggests that methane has been released episodically from hydrates trapped in sea floor sediments during many intervals of rapid climate warming. Here we show that sediments from the Santa Barbara Basin deposited during warm intervals in the last glacial period contain molecular fossils that are diagnostic of aerobic and anaerobic methanotrophs. Sediment intervals with high abundances of these compounds indicate episodes of vigorous methanotrophic activity in methane-laden water masses. Signals for anaerobic methanotrophy in 44,100-year-old sediment are evidence for particularly intense methane emissions and suggest that the basin's methane cycle can profoundly affect oxygen budgets in the water column.
Positional Cloning of the Human Quantitative Trait Locus Underlying Taste Sensitivity to Phenylthiocarbamide
Authors: Kim Un-kyung , Jorgenson Eric , Coon Hilary , Leppert Mark , Risch Neil , Drayna Dennis
Published: February 2003

The ability to taste the substance phenylthiocarbamide (PTC) has been widely used for genetic and anthropological studies, but genetic studies have produced conflicting results and demonstrated complex inheritance for this trait. We have identified a small region on chromosome 7q that shows strong linkage disequilibrium between single-nucleotide polymorphism (SNP) markers and PTC taste sensitivity in unrelated subjects. This region contains a single gene that encodes a member of the TAS2R bitter taste receptor family. We identified three coding SNPs giving rise to five haplotypes in this gene worldwide. These haplotypes completely explain the bimodal distribution of PTC taste sensitivity, thus accounting for the inheritance of the classically defined taste insensitivity and for 55 to 85% of the variance in PTC sensitivity. Distinct phenotypes were associated with specific haplotypes, which demonstrates that this gene has a direct influence on PTC taste sensitivity and that sequence variants at different sites interact with each other within the encoded gene product.
A Discrete Self-Assembled Metal Array in Artificial DNA
Authors: Tanaka Kentaro , Tengeiji Atsushi , Kato Tatsuhisa , Toyama Namiki , Shionoya Mitsuhiko
Published: February 2003

DNA has a structural basis to array functionalized building blocks. Here we report the synthesis of a series of artificial oligonucleotides, d(5′-G
H

n

C-3′) (
n
= 1 to 5), with hydroxypyridone nucleobases (
H
) as flat bidentate ligands. Right-handed double helices of the oligonucleotides,
n
Cu
2+
·d(5′-G
H

n

C-3′)
2
(
n
= 1 to 5), were quantitatively formed through copper ion (Cu
2+
)–mediated alternative base pairing (
H
-Cu
2+
-
H
), where the Cu
2+
ions incorporated into each complex were aligned along the helix axes inside the duplexes with the Cu
2+
-Cu
2+
distance of 3.7 ± 0.1 angstroms. The Cu
2+
ions were coupled ferromagnetically with one another through unpaired
d
electrons to form magnetic chains.
Synaptic Plasticity in Spinal Lamina I Projection Neurons That Mediate Hyperalgesia
Authors: Ikeda Hiroshi , Heinke Bernhard , Ruscheweyh Ruth , Sandkühler Jürgen
Published: February 2003

Inflammation, trauma, or nerve injury may cause enduring hyperalgesia, an enhanced sensitivity to painful stimuli. Neurons in lamina I of the spinal dorsal horn that express the neurokinin 1 receptor for substance P mediate this abnormal pain sensitivity by an unknown cellular mechanism. We report that in these, but not in other nociceptive lamina I cells, neurokinin 1 receptor–activated signal transduction pathways and activation of low-threshold (T-type) voltage-gated calcium channels synergistically facilitate activity- and calcium-dependent long-term potentiation at synapses from nociceptive nerve fibers. Thereby, memory traces of painful events are retained.
Gigahertz Electron Spin Manipulation Using Voltage-Controlled g-Tensor Modulation
Authors: Kato Y. , Myers R. C. , Driscoll D. C. , Gossard A. C. , Levy J. , Awschalom D. D.
Published: February 2003

We present a scheme that enables gigahertz-bandwidth three-dimensional control of electron spins in a semiconductor heterostructure with the use of a single voltage signal. Microwave modulation of the Landé g tensor produces frequency-modulated electron spin precession. Driving at the Larmor frequency results in g-tensor modulation resonance, which is functionally equivalent to electron spin resonance but without the use of time-dependent magnetic fields. These results provide proof of the concept that quantum spin information can be locally manipulated with the use of high-speed electrical circuits.
Encoding Pheromonal Signals in the Accessory Olfactory Bulb of Behaving Mice
Authors: Luo Minmin , Fee Michale S. , Katz Lawrence C.
Published: February 2003

Many mammalian species rely on pheromones—semiochemicals produced by other members of the same species—to communicate social status and reproductive readiness. To assess how the central nervous system integrates the complex repertoire of pheromones, we recorded from single neurons in the accessory olfactory bulb, a nucleus that processes pheromonal signals, of male mice engaged in natural behaviors. Neuronal firing was robustly modulated by physical contact with male and female conspecifics, with individual neurons activated selectively by specific combinations of the sex and strain of conspecifics. We infer that mammals encode social and reproductive information by integrating vomeronasal sensory activity specific to sex and genetic makeup.
Identification and Characterization of a
Streptococcus pyogenes
Operon Involved in Binding of Hemoproteins and Acquisition of Iron
Authors: Bates Christopher S. , Montañez Griselle E. , Woods Charles R. , Vincent Rebecca M. , Eichenbaum Zehava
Published: February 2003

The hemolytic
Streptococcus pyogenes
can use a variety of heme compounds as an iron source. In this study, we investigate hemoprotein utilization by
S. pyogenes
. We demonstrate that surface proteins contribute to the binding of hemoproteins to
S. pyogenes
. We identify an ABC transporter from the iron complex family named
sia
for streptococcal iron acquisition, which consists of a lipoprotein (
siaA
), membrane permease (
siaB
), and ATPase (
siaC
). The
sia
transporter is part of a highly conserved, iron regulated, 10-gene operon. SiaA, which was localized to the cell membrane, could specifically bind hemoglobin. The operon's first gene encodes a novel bacterial protein that bound hemoglobin, myoglobin, heme-albumin, and hemoglobin-haptoglobin (but not apo-haptoglobin) and therefore was named Shr, for streptococcal hemoprotein receptor. PhoZ fusion and Western blot analysis showed that Shr has a leader peptide and is found in both membrane-bound and soluble forms. An M1 SF370 strain with a polar mutation in
shr
was more resistant to streptonigrin and hydrogen peroxide, suggesting decreased iron uptake. The addition of hemoglobin to the culture medium increased cell resistance to hydrogen peroxide in SF370 but not in the mutant, implying the
sia
operon may be involved in hemoglobin-dependent resistance to oxidative stress. The
shr
mutant demonstrated reduced hemoglobin binding, though cell growth in iron-depleted medium supplemented with hemoglobin, whole blood, or ferric citrate was not affected, suggesting additional systems are involved in hemoglobin utilization. SiaA and Shr are the first hemoprotein receptors identified in
S. pyogenes
; their possible role in iron capture is discussed.
The Afa/Dr Adhesins of Diffusely AdheringEscherichia coliStimulate Interleukin-8 Secretion, Activate Mitogen-Activated Protein Kinases, and Promote Polymorphonuclear Transepithelial Migration in T84 Polarized Epithelial Cells
Authors: Bétis Fréderic , Brest Patrick , Hofman Véronique , Guignot Julie , Bernet-Camard Marie-Françoise , Rossi Bernard , Servin Alain , Hofman Paul
Published: February 2003

Afa/Dr diffusely adheringEscherichia coli(Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and theE. colilaboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody that recognized the short consensus repeat 3 domain of DAF (monoclonal antibody 1H4). Moreover, Afa/Dr DAEC strains induced tyrosine phosphorylation of several T84 proteins and activated the mitogen-activated protein kinases (ERK1/2 mitogen-activated protein, P38, and Jun-C kinases). These data demonstrated for the first time that, in vitro, Afa/Dr DAEC strains exert a proinflammatory signal in intestinal epithelial cells.
Production of the Type IV Secretion System Differs among
Brucella
Species as Revealed with VirB5- and VirB8-Specific Antisera
Authors: Rouot Bruno , Alvarez-Martinez Maria-Teresa , Marius Carine , Menanteau Pierrette , Guilloteau Laurence , Boigegrain Rose-Anne , Zumbihl Robert , O'Callaghan David , Domke Natalie , Baron Christian
Published: February 2003

Expression of the
virB
operon, encoding the type IV secretion system required for
Brucella suis
virulence, occurred in the acidic phagocytic vacuoles of macrophages and could be induced in minimal medium at acidic pH values. To analyze the production of VirB proteins, polyclonal antisera against
B. suis
VirB5 and VirB8 were generated. Western blot analysis revealed that VirB5 and VirB8 were detected after 3 h in acidic minimal medium and that the amounts increased after prolonged incubation. Unlike what occurs in the related organism
Agrobacterium tumefaciens
, the periplasmic sugar binding protein ChvE did not contribute to VirB protein production, and
B. suis
from which
chvE
was deleted was fully virulent in a mouse model. Comparative analyses of various
Brucella
species revealed that in all of them VirB protein production increased under acidic conditions. However, in rich medium at neutral pH,
Brucella canis
and
B. suis
, as well as the
Brucella abortus-
and
Brucella melitensis
-derived vaccine strains S19, RB51, and Rev.1, produced no VirB proteins or only small amounts of VirB proteins, whereas the parental
B. abortus
and
B. melitensis
strains constitutively produced VirB5 and VirB8. Thus, the vaccine strains were still able to induce
virB
expression under acidic conditions, but the VirB protein production was markedly different from that in the wild-type strains at pH 7. Taken together, the data indicate that VirB protein production and probably expression of the
virB
operon are not uniformly regulated in different
Brucella
species. Since VirB proteins were shown to modulate
Brucella
phagocytosis and intracellular trafficking, the differential regulation of the production of these proteins reported here may provide a clue to explain their role(s) during the infection process.
The Shufflon of
Salmonella enterica
Serovar Typhi Regulates Type IVB Pilus-Mediated Bacterial Self-Association
Authors: Morris Christina , Yip Cecilia M. C. , Tsui Inez S. M. , Wong Danny K.-H. , Hackett Jim
Published: February 2003

Previously, it was shown that type IVB pili encoded by the
Salmonella enterica
serovar Typhi
pil
operon are used to facilitate bacterial entry into human intestinal epithelial cells in vitro and that such entry is inhibited by purified prepilin (pre-PilS) protein (X.-L. Zhang, I. S. M. Tsui, C. M. C. Yip, A. W. Y. Fung, D. K.-H. Wong, X. Dai, Y. Yang, J. Hackett, and C. Morris, Infect. Immun. 68:3067-3073, 2000). The
pil
operon concludes with a simple shufflon, and a recombinase gene product (Rci) inverts DNA in the C-terminal region of the
pilV
gene to allow synthesis of two distinct PilV proteins, PilV1 and PilV2, which are presumptive minor pilus proteins. We show here that the type IVB pili mediate bacterial self-association, but only when the PilV1 and PilV2 proteins are not expressed. This may be achieved in wild-type serovar Typhi by rapid DNA inversion activity of the shufflon. We show that the inversion activity inhibits the expression of genes inserted between the 19-bp inverted repeats used for Rci-mediated recombination and that the activity of Rci increases when DNA is supercoiled. The data suggest that serovar Typhi self-associates under conditions (such as low oxygen tension in the gut) that favor DNA supercoiling. These results explain (i) the function of the serovar Typhi shufflon and (ii) why there are only two possible shufflon states, in contrast to the many possible states of other shufflon systems. The data further indicate that a very early step in serovar Typhi pathogenesis may be type IVB pilus-mediated self-association of bacteria in the anaerobic human small intestine prior to invasion of the human gut epithelium. The suggested type IVB pilus-dependent step in typhoid fever pathogenesis may partially explain the enhanced invasiveness of serovar Typhi for humans.
Expression and Immunogenicity ofMycoplasma hyopneumoniaeHeat Shock Protein Antigen P42 by DNA Vaccination
Authors: Chen Ya-Lei , Wang Shao-Ning , Yang Wen-Jen , Chen Yi-Jiun , Lin Hsi-Hsun , Shiuan David
Published: February 2003

Mycoplasma hyopneumoniaeis the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages. The goal of this study was to design DNA vaccines by constructing plasmid pcDNA3/P42, carrying the heat shock protein gene P42 ofM. hyopneumoniae, and to evaluate the immune responses elicited in BALB/c mice. The expression of P42 was first examined in transfected NIH 3T3 cells by reverse transcription-PCR to ensure that the construct was functional. The humoral and cell-mediated immune responses induced by the plasmid were further evaluated in BALB/c mice through intramuscular injection. Both immunoglobulin G1 (IgG1) and IgG2a levels were 64 times those of the control groups during the first 8 weeks. The levels of interleukin-2 (IL-2), IL-4, and gamma interferon mRNAs in the immunized animals were elevated, and the proliferation of spleen cells was also enhanced in the immunized animals. The results indicate that pcDNA3/P42 DNA immunization induces both Th1 and Th2 immune responses. In addition, antiserum from the immunized animals was found to inhibit the growth ofM. hyopneumoniae. The present study reveals that DNA vaccination could be a new strategy against infection byM. hyopneumoniaeand may have potential for developing vaccines for other infectious diseases as well.
Purification, Gene Cloning, Gene Expression, and Mutants of Dps from the Obligate Anaerobe
Porphyromonas gingivalis
Authors: Ueshima Junichi , Shoji Mikio , Ratnayake Dinath B. , Abe Kihachiro , Yoshida Shinichi , Yamamoto Kenji , Nakayama Koji
Published: February 2003

The periodontopathogen
Porphyromonas gingivalis
is an obligate anaerobe that is devoid of catalase but exhibits a relatively high degree of resistance to peroxide stress. In the present study, we demonstrate that
P. gingivalis
contains a Dps homologue that plays an important role in the protection of cells from peroxide stress. The Dps protein isolated from
P. gingivalis
displayed a ferritin-like spherical polymer consisting of 19-kDa subunits. Molecular cloning and sequencing of the gene encoding this protein revealed that it had a high similarity in nucleotide and amino acid sequences to Dps proteins from other species. The expression of Dps was significantly increased by exposure of
P. gingivalis
to atmospheric oxygen in an OxyR-dependent manner, indicating that it is regulated by the reactive oxygen species-regulating gene
oxyR
. The Dps-deficient mutants, including the
dps
single mutant and the
ftn dps
double mutant, showed no viability loss upon exposure to atmospheric oxygen for 6 h. In contrast to the wild type, however, these mutants exhibited the high susceptibility to hydrogen peroxide, thereby disrupting the viability. On the other hand, no significant difference in sensitivity to mitomycin C and metronidazole was observed between the wild type and the mutants. Furthermore, the
dps
single mutant, compared with the wild type, showed a lower viability in infected human umbilical vein endothelial cells.
Immunogenicity and Protective Immunity Induced by Synthetic Peptides Associated with Putative Immunodominant Regions ofStreptococcus mutansGlucan-Binding Protein B
Authors: Smith Daniel J. , King William F. , Barnes Leigh A. , Peacock Zachary , Taubman Martin A.
Published: February 2003

Glucan-binding protein B (GbpB) fromStreptococcus mutanshas been shown to induce protective immunity to dental caries in experimental models. Having recently sequenced thegbpBgene, our objective in this study was to identify immunogenic regions within the GbpB sequence for use in subunit vaccines. Potential regions of immunogenicity were sought by use of a matrix-based algorithm (EpiMatrix) to estimate the binding characteristics of peptides derived from the GbpB sequence by using a database of known major histocompatibility complex class II binding alleles. Screening the entire sequence revealed several peptides with estimated high binding probabilities. Two N-terminal 20-mer peptides (SYI and QGQ) subtending two of these regions were synthesized. A preliminary experiment, in which these peptides were synthesized in the multiple antigenic peptide format and were used to subcutaneously immunize Sprague-Dawley rats twice at a 21-day interval, revealed that the SYI peptide induced a higher percentage of responses to the inciting peptide as well as to intact GbpB, as measured by enzyme-linked immunosorbent assay. The effect of immunization with the SYI peptide construct on the cariogenicity ofS. mutanswas then investigated by immunizing weanling Sprague-Dawley rats twice at a 9-day interval with SYI or with phosphate-buffered saline. All rats were then orally infected withS. mutansstrain SJ. After a 78-day infection period, the SYI-immunized groups had significant reductions in dental caries on both smooth and occlusal surfaces compared with the sham-immunized group. Thus, these experiments indicated that at least one linear sequence, derived from the N-terminal third of GbpB, was sufficiently immunogenic to induce a protective immune response in this experimental rat model for dental caries.
Functional CD40 Expression Induced following Bacterial Infection of Mouse and Human Osteoblasts
Authors: Schrum Laura W. , Marriott Ian , Butler Betsy R. , Thomas Elaine K. , Hudson Michael C. , Bost Kenneth L.
Published: February 2003

Bacterially induced bone infections often result in significant local inflammatory responses which are coupled with loss of bone. However, the mechanisms necessary for the protective host response, or those responsible for pathogen-induced bone loss, are not clear. Recent evidence demonstrates that bacterially infected osteoblasts secrete chemokines and cytokines, suggesting that these cells may have an unappreciated role in supporting localized inflammation. In this study, mouse and human osteoblasts were investigated for their ability to express functional CD40 upon exposure to two important pathogens of bone,Staphylococcus aureusandSalmonella entericaserovar Dublin. Bacterial infection of cultured mouse or human osteoblasts resulted in increased CD40 mRNA and CD40 protein expression induced by either pathogen. Importantly, CD40 expression by osteoblasts was functional, as assessed by ligation of this molecule with recombinant, soluble CD154. CD40 activity was assessed by induction of interleukin-6 and granulocyte-macrophage colony-stimulating factor in osteoblasts following ligation. Cocultures of activated CD4+T lymphocytes and osteoblasts could interact via CD40 and CD154, since an antibody against CD40 could block macrophage inflammatory protein-1α secretion. Taken together, these studies conclusively demonstrate that infected osteoblasts can upregulate expression of functional CD40 molecules which mediate cytokine secretion. This surprising result further supports the notion that bone-forming osteoblasts can directly interact with CD154-expressing cells (i.e., T lymphocytes) and can contribute to the host response during bone infection.
Analysis of the Role ofBphs/Hrh1in the Genetic Control of Responsiveness to Pertussis Toxin
Authors: Gao Jian Feng , Call Stanford B. , Fillmore Parley D. , Watanabe Takeshi , Meeker Nathan D. , Teuscher Cory
Published: February 2003

In vivo intoxication withBordetella pertussistoxin (PTX) elicits a variety of physiological responses including a marked leukocytosis, disruption of glucose regulation, adjuvant activity, alterations in vascular function, hypersensitivity to vasoactive agents, and death. We recently identifiedBphs, the locus controlling PTX-induced hypersensitivity to the vasoactive amine histamine, as the histamine H1receptor (Hrh1). In this studyBphscongenic mice and mice with a disruptedHrh1gene were used to examine the role ofBphs/Hrh1in the genetic control of susceptibility to a number of phenotypes elicited following in vivo intoxication. We report that the contribution ofBphs/Hrh1to the overall genetic control of responsiveness to PTX is restricted to susceptibility to histamine hypersensitivity and enhancement of antigen-specific delayed-type hypersensitivity responses. Furthermore, the genetic contribution ofBphs/Hrh1to vasoactive amine sensitization is specific for histamine, since hypersensitivity to serotonin was unaffected byBphs/Hrh1. Bphs/Hrh1also did not significantly influence susceptibility to the lethal effects, the leukocytosis response, disruption of glucose regulation, and histamine-independent increases in vascular permeability associated with in vivo intoxication. Nevertheless, significant interstrain differences in susceptibility to the lethal effects of PTX and leukocytosis response were observed. These results indicate that the phenotypic variation in responsiveness to PTX reflects the genetic control of distinct intermediate phenotypes rather than allelic variation in genes controlling overall susceptibility to intoxication.
Human Immune Response to Outer Membrane Protein CD of
Moraxella catarrhalis
in Adults with Chronic Obstructive Pulmonary Disease
Authors: Murphy Timothy F. , Kirkham Charmaine , Liu Dai-Fang , Sethi Sanjay
Published: February 2003

Moraxella catarrhalis
is a common cause of lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). The antibody response to outer membrane protein (OMP) CD, a highly conserved surface protein of
M. catarrhalis
under consideration as a vaccine antigen, was studied in adults with COPD following 40 episodes of infection or colonization. Following infection or colonization, 9 of 40 patients developed new serum immunoglobulin G (IgG) to OMP CD, as measured by enzyme-linked immunosorbent assay. Adsorption assays revealed that a proportion of the serum IgG was directed toward surface-exposed epitopes on OMP CD in six of the nine patients who developed new IgG to OMP CD. Immunoblot assays with fusion peptide constructs indicated that the new antibodies that developed after infection or colonization recognized conformational epitopes, particularly in the carboxy region of the protein. Three of 28 patients developed new mucosal IgA to OMP CD in sputum supernatants. This study establishes that OMP CD is a target of a systemic and mucosal immune response following infection and colonization in some patients with COPD.
Complement Activation InfluencesStaphylococcus aureusAdherence to Endothelial Cells
Authors: Cunnion K. M. , Frank M. M.
Published: February 2003

The ability ofStaphylococcus aureusto adhere to endothelial cells (EC) is a critical step in the development of metastatic infection. The role of complement inS. aureusbinding to EC remains uninvestigated. Log-phaseS. aureus, expressing minimal capsule, was incubated with serum under various conditions, washed, and then incubated at 37°C for 30 min with cultured human umbilical vein EC (ATCC CRL-1730). Adherence was scored visually after staining with acridine orange. Incubation in 10% heat-inactivated human serum increased adherence to endothelial cells by 488% compared to organisms incubated in buffer. IncubatingS. aureusin complement-active normal human serum (NHS) decreased binding to EC by 58% compared to organisms incubated in heat-inactivated serum. The importance of active complement was confirmed by experiments using serum with added EDTA or cobra venom factor, a protein that depletes C3. The expression of capsule byS. aureusstrongly interfered with adherence. It has been shown that an important protein forS. aureusadhesion to EC is fibronectin.S. aureusadherence to purified fibronectin increased by 511% after incubation in heat-inactivated serum, compared to that of organisms incubated in buffer. This decreased by 56% in complement-active serum, suggesting that inhibition ofS. aureusadherence to EC is due, in part, to complement-mediated diminished binding to fibronectin. Interestingly, when EC were exposed toS. aureus-activated serum and then washed, binding byS. aureuswas 234% higher than that of EC exposed to NHS. Thus, complement-activated EC have increasedS. aureusbinding, while complement on the bacterial surface markedly reduces adherence.
ExperimentalPseudomonas aeruginosaKeratitis in Interleukin-10 Gene Knockout Mice
Authors: Cole Nerida , Krockenberger Mark , Stapleton Fiona , Khan Shamila , Hume Emma , Husband Alan J. , Willcox Mark
Published: February 2003

Pseudomonas aeruginosakeratitis is one of the most destructive diseases of the cornea. The host response to this infection is critical to the outcome. The cytokine interleukin-10 (IL-10) is thought to play an important role in modulating excessive inflammation and antimicrobial defenses. We have found that in IL-10−/−mice there is a significant decrease in bacterial load in corneas at 7 days postchallenge withP. aeruginosa. This decrease was accompanied by a reduction in neutrophil numbers in the cornea and changes in cytokine levels compared to those of wild-type mice. A characteristic increase in neovascularization in the cornea was found in the IL-10−/−mice. This increased angiogenesis correlated with an increased expression of KC, whereas the kinetics of macrophage inflammatory peptide 2 expression correlated with neutrophil numbers. This finding suggests that KC may play a role in corneal angiogenesis. The source of IL-10 in mouse corneas was identified as a subpopulation of infiltrating cells and keratocytes. This study demonstrates that IL-10 plays an important role in regulating the balance of inflammatory mediators duringP. aeruginosainfection of the cornea.
Localized Eosinophil Degranulation Mediates Disease in Tropical Pulmonary Eosinophilia
Authors: O'Bryan Laura , Pinkston Paula , Kumaraswami V. , Vijayan V. , Yenokida Gordon , Rosenberg Helene F. , Crystal Ronald , Ottesen Eric A. , Nutman Thomas B.
Published: February 2003

To explore the mechanisms underlying the eosinophil-mediated inflammation of tropical pulmonary eosinophilia (TPE), bronchoalveolar lavage (BAL) fluid, serum, and supernatants from pulmonary and blood leukocytes (WBC) from patients with acute TPE (n= 6) were compared with those obtained from healthy uninfected individuals (n= 4) and from patients with asthma (n= 4) or elephantiasis (n= 5). Although there were no significant differences in the levels of interleukin-4 (IL-4), IL-5, IL-13, eotaxin, granulocyte-macrophage colony-stimulating factor, RANTES, or eosinophil cationic protein, there was a marked increase in eosinophil-derived neurotoxin (EDN) both systemically and in the lungs of individuals with TPE compared to each of the control groups (P< 0.02). Moreover, there was a compartmentalization of this response, with EDN levels being higher in the BAL fluid than in the serum (P< 0.02). Supernatants from WBC from either whole blood or BAL cells were examined for chemokines, cytokines, eosinophil degranulation products, and arachidonic acid metabolites. Of the many mediators examined—particularly those associated with eosinophil trafficking—only EDN (in BAL fluid and WBC) and MIP-1α (in WBC) levels were higher for TPE patients than for the non-TPE control groups (P< 0.02). These data suggest it is the eosinophilic granular protein EDN, an RNase capable of damaging the lung epithelium, that plays the most important role in the pathogenesis of TPE.
Functional Genomics Approach to the Identification of Virulence Genes Involved in
Edwardsiella tarda
Pathogenesis
Authors: Srinivasa Rao Putanae S. , Lim Tit Meng , Leung Ka Yin
Published: February 2003

Edwardsiella tarda
is an important cause of hemorrhagic septicemia in fish and also of gastro- and extraintestinal infections in humans. Here, we report the identification of 14 virulence genes of pathogenic
E. tarda
that are essential for disseminated infection, via a genome-wide analysis. We screened 490 alkaline phosphatase fusion mutants from a library of 450,000 Tn
phoA
transconjugants derived from strain PPD130/91, using fish as an infection model. Compared to the wild type, 15 mutants showed significant decreases in virulence. Six mutants had insertions in the known virulence-related genes, namely,
fimA
,
gadB
,
katB
,
pstS
,
pstC
, and
ssrB
. Some mutants corresponded to known genes (
astA
,
isor
, and
ompS2
) that had not been previously shown to be involved in pathogenesis, and three had insertions in two novel genes. In vivo infection kinetics experiments confirmed the inability of these attenuated mutants to proliferate and cause fatal infection in fish. Screening for the presence of the above-described virulence genes in six virulent and seven avirulent strains of
E. tarda
indicated that seven of the genes were specific to pathogenic
E. tarda
. The genes identified here may be used to develop vaccines and diagnostic kits as well as for further studying the pathogenesis of
E. tarda
and other pathogenic bacteria.
Two Novel Superantigens Found in Both Group A and Group CStreptococcus
Authors: Proft Thomas , Webb Phillip D. , Handley Vanessa , Fraser John D.
Published: February 2003

Two novel streptococcal superantigen genes (speLSeandspeMSe) were identified from theStreptococcus equigenome database at the Sanger Center. Genotyping of 8S. equiisolates and 40Streptococcus pyogenesisolates resulted in the detection of the orthologous genesspeLandspeMin a restricted number ofS. pyogenesisolates (15 and 5%, respectively). Surprisingly, the novel superantigen genes could not be found in any of the analyzedS. equiisolates. The results suggest that both genes are located on a mobile element that enables gene transfer between individual isolates and between streptococci from different Lancefield groups.S. equipyrogenic exotoxin L (SPE-LSe)/streptococcal pyrogenic exotoxin L (SPE-L) and SPE-MSe/SPE-M are most closely related to SMEZ, SPE-C, SPE-G, and SPE-J, but build a separate branch within this group. Recombinant SPE-L (rSPE-L) and rSPE-M were highly mitogenic for human peripheral blood lymphocytes, with half-maximum responses at 1 and 10 pg/ml, respectively. The results from competitive binding experiments suggest that both proteins bind major histocompatibility complex class II at the β-chain, but not at the α-chain. The most common targets for both toxins were human Vβ1.1 expressing T cells. Seroconversion against SPE-L and SPE-M was observed in healthy blood donors, suggesting that the toxins are expressed in vivo. Interestingly, thespeLgene is highly associated withS. pyogenesM89, a serotype that is linked to acute rheumatic fever in New Zealand.
Primed Peritoneal B Lymphocytes Are Sufficient To Transfer Protection againstBrugia pahangiInfection in Mice
Authors: Paciorkowski Natalia , Shultz Leonard D. , Rajan T. V.
Published: February 2003

Lymphatic filariasis is a tropical disease caused by the nematode parasitesWuchereria bancroftiandBrugia malayi. Whereas the protective potential of T lymphocytes in filarial infection is well documented, investigation of the role of B lymphocytes in antifilarial immunity has been neglected. In this communication, we examine the role of B lymphocytes in antifilarial immunity, usingBrugia pahangiinfections in the murine peritoneal cavity as a model. We find that B lymphocytes are required for clearance of primary and challenge infections withB. pahangithird-stage larvae (L3). We assessed the protective potential of peritoneal B lymphocytes by adoptive transfer experiments. Primed but not naïve peritoneal B cells from wild-type mice that had been immunized withB. pahangiL3 protected athymic recipients from challenge infection. We evaluated possible mechanisms by which B cells mediate protection. Comparisons of cytokine mRNA expression between B-lymphocyte-deficient and immunocompetent mice followingB. pahangiinfection suggest that B cells are required for the early production of Th2-type cytokines by peritoneal cells. In addition, B-cell-deficient mice demonstrate a defect in inflammatory cell recruitment to the peritoneal cavity followingB. pahangiinfection. The data demonstrate a critical role of B lymphocytes in antifilarial immunity in naïve mice and in the memory response in primed mice.
Attenuated Virulence of a
Burkholderia cepacia
Type III Secretion Mutant in a Murine Model of Infection
Authors: Tomich Mladen , Griffith Adam , Herfst Christine A. , Burns Jane L. , Mohr Christian D.
Published: February 2003

Type III secretion systems are utilized by a number of gram-negative bacterial pathogens to deliver virulence-associated proteins into host cells. Using a PCR-based approach, we identified homologs of type III secretion genes in the gram-negative bacterium
Burkholderia cepacia
, an important pulmonary pathogen in immunocompromised patients and patients with cystic fibrosis. One of the genes, designated
bscN
, encodes a member of a family of ATP-binding proteins believed to generate energy driving virulence protein secretion. Genetic dissection of the regions flanking the
bscN
gene revealed a locus consisting of at least 10 open reading frames, predicted to encode products with significant homology to known type III secretion proteins in other bacteria. A defined null mutation was generated in the
bscN
gene, and the null strain and wild-type parent strain were examined by use of a murine model of
B. cepacia
infection. Quantitative bacteriological analysis of the lungs and spleens of infected C57BL/6 mice revealed that the
bscN
null strain was attenuated in virulence compared to the parent strain, with significantly lower bacterial recovery from the lungs and spleens at 3 days postinfection. Moreover, histopathological changes, including an inflammatory cell infiltrate, were more pronounced in the lungs of mice infected with the wild-type parent strain than in those of mice infected with the isogenic
bscN
mutant. These results implicate type III secretion as an important determinant in the pathogenesis of
B. cepacia
.
Expression of Toll-Like Receptor 2 on Human Schwann Cells: a Mechanism of Nerve Damage in Leprosy
Authors: Oliveira Rosane B. , Ochoa Maria T. , Sieling Peter A. , Rea Thomas H. , Rambukkana Anura , Sarno Euzenir N. , Modlin Robert L.
Published: February 2003

Nerve damage is a clinical hallmark of leprosy and a major source of patient morbidity. We investigated the possibility that human Schwann cells are susceptible to cell death through the activation of Toll-like receptor 2 (TLR2), a pattern recognition receptor of the innate immune system. TLR2 was detected on the surface of human Schwann cell line ST88-14 and on cultured primary human Schwann cells. Activation of the human Schwann cell line and primary human Schwann cell cultures with a TLR2 agonist, a synthetic lipopeptide comprising the N-terminal portion of the putativeMycobacterium leprae19-kDa lipoprotein, triggered an increase in the number of apoptotic cells. The lipopeptide-induced apoptosis of Schwann cells could be blocked by an anti-TLR2 monoclonal antibody. Schwann cells in skin lesions from leprosy patients were found to express TLR2. It was possible to identify in the lesions Schwann cells that had undergone apoptosis in vivo. The ability ofM. lepraeligands to induce the apoptosis of Schwann cells through TLR2 provides a mechanism by which activation of the innate immune response contributes to nerve injury in leprosy.
Intravenous Mouse Infection Model for Studying the Pathology ofEnterococcus faecalisInfections
Authors: Gentry-Weeks Claudia , Estay Monica , Loui Cindy , Baker Dale
Published: February 2003

An intravenous mouse infection model was used to compare the virulence ofEnterococcus faecalisstrains, to study bacterial localization and organ histopathology, and to examine the effects of Nramp1 and gamma interferon (IFN-γ) on the course of infection. Infection of BALB/c mice with 5 × 108CFU ofE. faecalisJH2-2, MGH-2, 418, DS16C2, or OG1X revealed the following virulence ranking (from highest to lowest): MGH-2, 418, DS16C2, JH2-2, and OG1X. Discernible differences in the number of MGH-2 and JH2-2 bacteria were observed at 7 days (168 h) in the blood (P= 0.037), at 72 h in the liver (P= 0.002), and at 8 h in the spleen (P= 0.036). At these time points, the number of MGH-2 bacteria was higher in the blood and liver while the number of JH2-2 bacteria was higher in the spleen. At 72 h, livers from MGH-2-infected mice had higher numbers of coalescing aggregates of leukocytes and a greater degree of caseous necrosis than those from JH2-2-infected mice. These results indicate a correlation between the virulence of theE. faecalisstrain, the number of bacteria in the liver, and the degree of histopathology of the liver at 72 h postinfection. IFN-γ was important inE. faecalisinfection, since IFN-γ gene knockout mice had reduced mortality and massive coagulative necrosis was observed in wild-type mice. The contribution of Nramp1 was unclear, sinceNramp1−/−mice and the respective control mice were innately resistant toE. faecalis. The mortality of mice in this model is probably due to induction of cytokine release and massive coagulative necrosis.
Role of the NF-κB Signaling Pathway and κBcis-Regulatory Elements on theIRF-1andiNOSPromoter Regions in Mycobacterial Lipoarabinomannan Induction of Nitric Oxide
Authors: Morris Kristin R. , Lutz Ryan D. , Choi Hyung-Seok , Kamitani Tetsu , Chmura Kathryn , Chan Edward D.
Published: February 2003

Nitric oxide (NO·) produced by inducible nitric oxide synthase (iNOS) is an important host defense molecule againstMycobacterium tuberculosisin mononuclear phagocytes. The objective of this study was to determine the role of the IκBα kinase-nuclear factor κB (IKK-NF-κB) signaling pathway in the induction of iNOS and NO·by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM) in mouse macrophages costimulated with gamma interferon (IFN-γ). NF-κB was activated by ManLAM as shown by electrophoretic mobility shift assay, by immunofluorescence of translocated NF-κB in intact cells, and by a reporter gene driven by four NF-κB-binding elements. Transduction of an IκBα mutant (Ser32/36Ala) significantly inhibited NO·expression induced by IFN-γ plus ManLAM. An activated SCF complex, a heterotetramer (Skp1, Cul-1, β-TrCP [F-box protein], and ROC1) involved with ubiquitination, is also required for iNOS-NO·induction. Two NF-κB-binding sites (κBI and κBII) present on the 5′-flanking region of theiNOSpromoter bound ManLAM-induced NF-κB similarly. By use of reporter constructs in which one or both sites are mutated, both NF-κB-binding positions were essential iniNOSinduction by IFN-γ plus ManLAM. IFN-γ-induced activation of the IRF-1 transcriptional complex is a necessary component in host defense against tuberculosis. Although the 5′-flanking region of theIRF-1promoter contains an NF-κB-binding site and ManLAM-induced NF-κB also binds to this site, ManLAM was unable to induce IRF-1 expression. The influence of mitogen-activated protein kinases on IFN-γ plus ManLAM induction of iNOS-NO·is not due to any effects on ManLAM induction of NF-κB.
Protection against FatalPseudomonas aeruginosaPneumonia in Mice after Nasal Immunization with a Live, AttenuatedaroADeletion Mutant
Authors: Priebe Gregory P. , Meluleni Gloria J. , Coleman Fadie T. , Goldberg Joanna B. , Pier Gerald B.
Published: February 2003

Studies of immunity toPseudomonas aeruginosahave indicated that a variety of potential immunogens can elicit protection in animal models, utilizing both antibody- and cell-mediated immune effectors for protection. To attempt to optimize delivery of multiple protective antigens and elicit a broad range of immune effectors, we produced anaroAdeletion mutant of theP. aeruginosaserogroup O2/O5 strain PAO1, designated PAO1ΔaroA. Previously, we reported that this strain elicits high levels of opsonic antibody directed against many serogroup O2/O5 strains after nasal immunization of mice and rabbits. Here, we assessed the protective efficacy of immunization with PAO1ΔaroAagainst acute fatal pneumonia in mice. After active immunization, high levels of protection were achieved against an ExoU-expressing cytotoxic variant of the parental strain PAO1 at doses up to 1,000-fold greater than the 50% lethal dose. Significant protection against PAO1 and two of four other serogroup O2/O5 strains was also found, but there was no protection against serogroup-heterologous strains. The serogroup O2/O5 strains not protected against were killed in opsonophagocytic assays as efficiently as the strains with which protection was seen, indicating a lack of correlation of protection and opsonic killing within the serogroup. In passive immunization experiments using challenge with wild-type PAO1 or other noncytotoxic members of the O2/O5 serogroup, there was no protection despite the presence of high levels of opsonic antibody in the mouse sera. However, passive immunization did prevent mortality from pneumonia due to the cytotoxic PAO1 variant at low-challenge doses. These data suggest that a combination of humoral and cellular immunity is required for protection againstP. aeruginosalung infections, that such immunity can be elicited by usingaroAdeletion mutants, and that a multivalentP. aeruginosavaccine composed ofaroAdeletion mutants of multiple serogroups holds significant promise.
Amino Acid Sequence Requirements in the Hinge of Human Immunoglobulin A1 (IgA1) for Cleavage by Streptococcal IgA1 Proteases
Authors: Batten Margaret R. , Senior Bernard W. , Kilian Mogens , Woof Jenny M.
Published: February 2003

The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species ofStreptococcuswere investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcα receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease fromStreptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases ofStreptococcus oralis,Streptococcus sanguis, andStreptococcus mitishad an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence inS. mitisthat proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors.
Venezuelan Equine Encephalitis Virus-Vectored Vaccines Protect Mice against Anthrax Spore Challenge
Authors: Lee John S. , Hadjipanayis Angela G. , Welkos Susan L.
Published: February 2003

Anthrax, a disease usually associated with herbivores, is caused by the bacterium
Bacillus anthracis
. The current vaccine licensed for human use requires a six-dose primary series and yearly boosters and causes reactogenicity in up to 30% of vaccine recipients. A minimally reactogenic vaccine requiring fewer inoculations is warranted. Venezuelan equine encephalitis (VEE) virus has been configured for use as a vaccine vector for a wide variety of immunogens. The VEE vaccine vector is composed of a self-replicating RNA (replicon) containing all of the VEE virus nonstructural genes and a multiple-cloning site in place of the VEE structural genes. Four different anthrax vaccines were constructed by cloning the protective antigen (PA) gene from
B. anthracis
into the VEE vaccine vector. The anthrax vaccines were produced by assembling the vectors into propagation-deficient VEE replicon particles in vitro. A/J mice inoculated subcutaneously with three doses of the mature 83-kDa PA vaccine were completely protected from challenge with the Sterne strain of
B. anthracis
. Similar results were obtained with vaccines composed of the PA gene fused to either the
B. anthracis
secretory sequence or to a tissue plasminogen activator secretory sequence in three additional mouse strains. Mice were unprotected from challenge after inoculation with the carboxy-terminal 63-kDa PA vaccine. These results suggest that these VEE-vectored vaccines may be suitable as candidate vaccines against anthrax.