bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoinducer 3 and Epinephrine Signaling in the Kinetics of Locus of Enterocyte Effacement Gene Expression in Enterohemorrhagic
Escherichia coli
Authors: Walters Matthew , Sperandio Vanessa
Published: September 2006

Enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 is responsible for causing outbreaks of bloody diarrhea and hemolytic-uremic syndrome throughout the world. The locus of enterocyte effacement (LEE) consists of five major operons and is required for the formation of attaching and effacing lesions that disrupt intestinal epithelial microvilli. We have previously reported that expression of EHEC LEE genes is regulated by the
luxS
quorum-sensing system. The
luxS
gene in EHEC affects the production of autoinducer 3 (AI-3), which activates the LEE. Epinephrine and norepinephrine also activate the LEE in a manner similar to that of AI-3. Previous studies of quorum-sensing regulation of LEE transcription have thus far been restricted to using reporter systems in an
E. coli
K-12 background. Here, we examined the kinetics of LEE gene transcription, protein expression, and function of the LEE type III secretion apparatus in wild-type (WT) EHEC and an isogenic
luxS
mutant. The results revealed that the
luxS
mutant had diminished transcription from the LEE promoters during the mid-exponential growth phase; decreased protein levels of EscJ, Tir, and EspA; and reduced secretion of EspA and EspB. The
luxS
mutation also caused a delay in the formation of attaching and effacing lesions on cultured epithelial cells compared to the wild type. Epinephrine enhanced LEE expression in both the WT and the
luxS
mutant, but the WT still exhibited greater LEE activation. The results suggest a possible synergistic relationship between AI-3 and epinephrine. The combined effects of these two signaling molecules may lead to greater LEE expression and a more efficient infection.
Escherichia coliPrevents Phagocytosis-Induced Death of Macrophages via Classical NF-κB Signaling, a Link to T-Cell Activation
Authors: Groesdonk Heinrich V. , Schlottmann Silke , Richter Friederike , Georgieff Michael , Senftleben Uwe
Published: September 2006

NF-κB is a crucial mediator of macrophage inflammatory responses, but its role in the context of pathogen-induced adaptive immune responses has yet to be elucidated. Here, we demonstrate that classical NF-κB activation delays phagocytosis-induced cell death (PICD) in Raw 264.7 and bone marrow-derived macrophages (BMDMs) upon ingestion of bacteria from theEscherichia colilaboratory strain Top10. By expression of a nondegradable form of IκBα (superrepressor) and pyrrolidine dithiocarbamate treatment, prolonged activation of NF-κB upon bacterial coculture is suppressed, whereas initial induction is only partially inhibited. This activation pattern results in partial inhibition of cellular activation and reduced expression of costimulatory CD86. Notably, suppression of classical NF-κB activation does not influence bacterial uptake rates but is followed by increased production of oxygen radicals and enhanced intracellular killing in Raw macrophages. This is associated with reduced expression of NF-κB-dependent antiapoptotic c-IAP-2 and a loss of the mitochondrial transmembrane potential. Accordingly, NF-κB inhibition in Raw cells and BMDMs causes increased apoptotic rates within 12 h of bacterial ingestion. Interestingly, accelerated eradication ofE. coliin NF-κB-inhibited macrophages is associated with reduced antigen-specific T-cell activation in macrophage-lymphocyte cocultures. These data suggest thatE. coliinhibits PICD of macrophages via classical, antiapoptotic NF-κB activation and thus facilitates signaling to T cells. Subsequently, a proper adaptive immune response is likely to be generated. Conclusively, therapeutic inhibition of classical NF-κB activation in macrophages may hamper the initiation of adaptive immunity.
Common and Divergent Immune Response Signaling Pathways Discovered in Peripheral Blood Mononuclear Cell Gene Expression Patterns in Presymptomatic and Clinically Apparent Malaria
Authors: Ockenhouse Christian F. , Hu Wan-chung , Kester Kent E. , Cummings James F. , Stewart Ann , Heppner D. Gray , Jedlicka Anne E. , Scott Alan L. , Wolfe Nathan D. , Vahey Maryanne , Burke Donald S.
Published: September 2006

Using genome-wide expression profiles from persons either experimentally challenged with malaria-infected mosquitoes or naturally infected withPlasmodium falciparummalaria, we present details of the transcriptional changes that occur with infection and that either are commonly shared between subjects with presymptomatic and clinically apparent malaria or distinguish these two groups. Toll-like receptor signaling through NF-κB pathways was significantly upregulated in both groups, as were downstream genes that function in phagocytosis and inflammation, including the cytokines tumor necrosis factor alpha, gamma interferon (IFN-γ), and interleukin-1β (IL-1β). The molecular program derived from these signatures illuminates the closely orchestrated interactions that regulate gene expression by transcription factors such asIRF-1in the IFN-γ signal transduction pathway. Modulation of transcripts in heat shock and glycolytic enzyme genes paralleled the intensity of infection. Major histocompatibility complex class I molecules and genes involved in class II antigen presentation are significantly induced in 90% of malaria-infected persons regardless of group. Differences between early presymptomatic infection and natural infection involved genes that regulate the induction of apoptosis through mitogen-activated protein (MAP) kinases and signaling pathways through the endogenous pyrogen IL-1β, a major inducer of fever. The induction of apoptosis in peripheral blood mononuclear cells from patients with naturally acquired infection impacted the mitochondrial control of apoptosis and the activation of MAP kinase pathways centered around MAPK14 (p38α and p38β). Our findings confirm and extend findings regarding aspects of the earliest responses to malaria infection at the molecular level, which may be informative in elucidating how innate and adaptive immune responses may be modulated in different stages of infection.
Pseudomonas aeruginosaInfection of Airway Epithelial Cells Modulates Expression of Kruppel-Like Factors 2 and 6 via RsmA-Mediated Regulation of Type III Exoenzymes S and Y
Authors: O'Grady Eoin P. , Mulcahy Heidi , O'Callaghan Julie , Adams Claire , O'Gara Fergal
Published: September 2006

Pseudomonas aeruginosais an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding protein, controls the expression of a large number of virulence-related genes inP. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection withP. aeruginosaPAO1 and anrsmAmutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors KLF2 and KLF6 as targets of theP. aeruginosatype III exoenzymes S and Y, with potential importance in host cell death.
Application of Mutated
Clostridium difficile
Toxin A for Determination of Glucosyltransferase-Dependent Effects
Authors: Teichert Matthias , Tatge Helma , Schoentaube Janett , Just Ingo , Gerhard Ralf
Published: September 2006

Mutation of tryptophan-101 in
Clostridium difficile
toxin A, a 308-kDa glucosyltransferase, resulted in a 50-fold-reduced cytopathic activity in cell culture experiments. The mutant toxin A was characterized and applied to distinguish between glucosyltransferase-dependent and -independent effects with respect to RhoB up-regulation as a cellular stress response.
Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins
Authors: Cai Fang , Adrion Carrie B. , Keller James E.
Published: September 2006

Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+withN,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+is not required for toxin entry into neurons, and removal of Zn2+from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.
Opsonic Antibodies to
Enterococcus faecalis
Strain 12030 Are Directed against Lipoteichoic Acid
Authors: Theilacker Christian , Kaczynski Zbigniew , Kropec Andrea , Fabretti Francesca , Sange Tatjana , Holst Otto , Huebner Johannes
Published: September 2006

A teichoic acid (TA)-like polysaccharide in
Enterococcus faecalis
has previously been shown to induce opsonic antibodies that protect against bacteremia after active and passive immunization. Here we present new data providing a corrected structure of the antigen and the epitope against which the opsonic antibodies are directed. Capsular polysaccharide isolated from
E. faecalis
strain 12030 by enzymatic digestion of peptidoglycan and chromatography (enzyme-TA) was compared with lipoteichoic acid (LTA) extracted using butanol and purified by hydrophobic-interaction chromatography (BuOH-LTA). Structural determinations were carried out by chemical analysis and nuclear magnetic resonance spectroscopy. Antibody specificity was assessed by enzyme-linked immunosorbent assay and the opsonophagocytosis assay. After alanine ester hydrolysis, there was structural identity between enzyme-TA and BuOH-LTA of the TA-parts of the two molecules. The basic enterococcal LTA structure was confirmed: 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at position C-2 of the glycerol residues with
d
-Ala and kojibiose. We also detected a novel substituent at position C-2, [
d
-Ala→6]-α-
d
-Glc
p
-(1→2-[
d
-Ala→6]-α-
d
-Glc
p
-1→). Antiserum raised against enzyme-TA bound equally well to BuOH-LTA and dealanylated BuOH-LTA as to the originally described enzyme-TA antigen. BuOH-LTA was a potent inhibitor of opsonophagocytic killing by the antiserum to enzyme-TA. Immunization with antibiotic-killed whole bacterial cells did not induce a significant proportion of antibodies directed against alanylated epitopes on the TA, and opsonic activity was inhibited completely by both alanylated and dealanylated BuOH-LTA. In summary, the
E. faecalis
strain 12030 enzyme-TA is structurally and immunologically identical to dealanylated LTA. Opsonic antibodies to
E. faecalis
12030 are directed predominantly to nonalanylated epitopes on the LTA molecule.
Characterization and Regulation of the Trehalose Synthesis Pathway and Its Importance in the Pathogenicity of
Cryptococcus neoformans
Authors: Petzold Elizabeth Wills , Himmelreich Uwe , Mylonakis Eleftherios , Rude Thomas , Toffaletti Dena , Cox Gary M. , Miller Jackie L. , Perfect John R.
Published: September 2006

The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of
Cryptococcus neoformans
identified the presence of a functioning trehalose pathway during infection and suggested its importance for
C. neoformans
survival in the host. Therefore, in
C. neoformans
we created null mutants of the trehalose-6-phosphate (T6P) synthase (
TPS1
), trehalose-6-phophate phosphatase (
TPS2
), and neutral trehalase (
NTH1
) genes. We found that both
TPS1
and
TPS2
are required for high-temperature (37°C) growth and glycolysis but that the block at
TPS2
results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the
tps1
and
tps2
mutants at 37°C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in
C. neoformans
. The essential nature of this pathway for disease was confirmed when a
tps1
mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate
C. elegans
, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the
tps1
mutant, and this supports the hypothesis that the trehalose pathway in
C. neoformans
is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in
C. neoformans
and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.
Interleukin-10 Anti-Inflammatory Response toBorrelia burgdorferi, the Agent of Lyme Disease: a Possible Role for Suppressors of Cytokine Signaling 1 and 3
Authors: Dennis Vida A. , Jefferson Ayanna , Singh Shree R. , Ganapamo Frédéric , Philipp Mario T.
Published: September 2006

It has been established that interleukin-10 (IL-10) inhibits inflammatory cytokines produced by macrophages in response toBorrelia burgdorferior its lipoproteins. The mechanism by which IL-10 exerts this anti-inflammatory effect is still unknown. Recent findings indicate that suppressors of cytokine signaling (SOCS) proteins are induced by cytokines and Toll-like receptor (TLR)-mediated stimuli, and in turn they can down-regulate cytokine and TLR signaling in macrophages. Because it is known that SOCS are induced by IL-10 and thatB. burgdorferiand its lipoproteins most likely interact via TLR2 or the heterodimers TLR2/1 and/or TLR2/6, we hypothesized that SOCS are induced by IL-10 andB. burgdorferiand its lipoproteins in macrophages and that SOCS may mediate the inhibition by IL-10 of concomitantly elicited cytokines. We report here that mouse J774 macrophages incubated with IL-10 and addedB. burgdorferispirochetes (freeze-thawed, live, or sonicated) or lipidated outer surface protein A (L-OspA) augmented their SOCS1/SOCS3 mRNA and protein expression, with SOCS3 being more abundant. Pam3Cys, a synthetic lipopeptide, also induced SOCS1/SOCS3 expression under these conditions, but unlipidated OspA was ineffective. Neither endogenous IL-10 nor the translation inhibitor cycloheximide blocked SOCS1/SOCS3 induction byB. burgdorferiand its lipoproteins, indicating that the expression of other genes is not required. This temporally correlated with the IL-10-mediated inhibition of the inflammatory cytokines IL-1β, IL-6, IL-12p40, IL-18, and tumor necrosis factor α. Our data are evidence to suggest that expression of SOCS is part of the mechanism of IL-10-mediated inhibition of inflammatory cytokines elicited byB. burgdorferiand its lipoproteins.
Cord Blood Dendritic Cell Subsets in African Newborns Exposed toPlasmodium falciparumIn Utero
Authors: Breitling Lutz P. , Fendel Rolf , Mordmueller Benjamin , Adegnika Ayola A. , Kremsner Peter G. , Luty Adrian J. F.
Published: September 2006

PlacentalPlasmodium falciparuminfection affects birth outcomes and sensitizes fetal lymphocytes to parasite antigens. We assessed the influence of maternalP. falciparuminfection on fetal myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC), analyzing the cord blood of offspring of Gabonese mothers with different infection histories. Cord blood from newborns of mothers with malarial infection at delivery had significantly more mDC than that from nonexposed newborns (P= 0.028) but mDC and pDC HLA-DR expression was unrelated to maternal infection history. Independently of these findings, cord blood mDC and pDC numbers declined significantly as a function of increasing maternal age (P= 0.029 andP= 0.033, respectively). The inducible antigen-specific interleukin-10-producing regulatory-type T-cell population that we have previously detected in cord blood of newborns with prolonged in utero exposure toP. falciparummay directly reflect the altered DC numbers in such neonates, while the maintenance of cord blood DC HLA-DR expression contrasts with that of DC fromP. falciparummalaria patients.
A New Immunoglobulin-Binding Protein, EibG, Is Responsible for the Chain-Like Adhesion Phenotype of Locus of Enterocyte Effacement-Negative, Shiga Toxin-Producing
Escherichia coli
Authors: Lu Yan , Iyoda Sunao , Satou Hiromi , Satou Hitomi , Itoh Kenichiro , Saitoh Takehito , Watanabe Haruo
Published: September 2006

Shiga toxin-producing
Escherichia coli
(STEC) are important enteropathogens causing severe diseases such as hemorrhagic colitis and hemolytic-uremic syndrome in humans. The majority of STEC strains of serogroups O157, O26, or O111 associated with severe cases of these diseases possess a pathogenicity island termed the locus of enterocyte effacement (LEE). LEE, which is responsible for the formation of attaching-and-effacing lesions on intestinal epithelial cells, is important for the full virulence of STEC. Nonetheless, LEE-negative STEC strains have repeatedly been reported to be associated with severe diseases in humans. In this study, we characterized adhesion to cultured epithelial cells of certain LEE-negative STEC isolated from humans with or without bloody diarrhea. Several LEE-negative STEC belonging to serogroup O91 showed an unusual, chain-like adhesion pattern to HEp-2 cells. Using Tn
5
-based transposon mutagenesis, we identified the gene essential for the chain-like adhesion phenotype of this O91 STEC strain. Sequence analysis of the Tn
5
-inserted allele identified a novel chromosomal open reading frame (ORF) encoding a polypeptide with a high degree of similarity to the
E. coli
immunoglobulin-binding (Eib) proteins EibA, -C, -D, -E, and -F. Therefore, the ORF was designated EibG. Laboratory
E. coli
strain MC4100 transformed with a multicopy plasmid carrying
eibG
showed chain-like adhesion to HEp-2 cells, and whole-cell lysates of the strain bound to human-derived immunoglobulin G (IgG) Fc and IgA. These results indicate that EibG acts as an IgG Fc- and IgA-binding protein, as well as an adhesin of LEE-negative STEC.
Effects of Temperature on Gene Expression Patterns in
Leptospira interrogans
Serovar Lai as Assessed by Whole-Genome Microarrays
Authors: Lo Miranda , Bulach Dieter M. , Powell David R. , Haake David A. , Matsunaga James , Paustian Michael L. , Zuerner Richard L. , Adler Ben
Published: September 2006

Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic
Leptospira
spp. from infected animals or contaminated water or soil. The availability of genome sequences for
Leptospira interrogans
, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression.
Leptospira
spp. can grow in artificial media at a range of temperatures reflecting conditions found in the environment and the mammalian host. Therefore, transcriptional changes were compared between cultures grown at 20°C, 30°C, 37°C, and 39°C to represent ambient temperatures in the environment, growth under laboratory conditions, and temperatures in healthy and febrile hosts. Data from direct pairwise comparisons of the four temperatures were consolidated to examine transcriptional changes at two generalized biological conditions representing mammalian physiological temperatures (37°C and 39°C) versus environmental temperatures (20°C and 30°C). Additionally, cultures grown at 30°C then shifted overnight to 37°C were compared with those grown long-term at 30°C and 37°C to identify genes potentially expressed in the early stages of infection. Comparison of data sets from physiological versus environmental experiments with upshift experiments provided novel insights into possible transcriptional changes at different stages of infection. Changes included differential expression of chemotaxis and motility genes, signal transduction systems, and genes encoding proteins involved in alteration of the outer membrane. These findings indicate that temperature is an important factor regulating expression of proteins that facilitate invasion and establishment of disease.
The Cryptococcal Enzyme Inositol Phosphosphingolipid-Phospholipase C Confers Resistance to the Antifungal Effects of Macrophages and Promotes Fungal Dissemination to the Central Nervous System
Authors: Shea John M. , Kechichian Talar B. , Luberto Chiara , Del Poeta Maurizio
Published: September 2006

In recent years, sphingolipids have emerged as critical molecules in the regulation of microbial pathogenesis. In fungi, the synthesis of complex sphingolipids is important for the regulation of pathogenicity, but the role of sphingolipid degradation in fungal virulence is not known. Here, we isolated and characterized the inositol phosphosphingolipid-phospholipase C1 (
ISC1
) gene from the fungal pathogen
Cryptococcus neoformans
and showed that it encodes an enzyme that metabolizes fungal inositol sphingolipids. Isc1 protects
C. neoformans
from acidic, oxidative, and nitrosative stresses, which are encountered by the fungus in the phagolysosomes of activated macrophages, through a Pma1-dependent mechanism(s). In an immunocompetent mouse model, the
C. neoformans Δisc1
mutant strain is almost exclusively found extracellularly and in a hyperencapsulated form, and its dissemination to the brain is remarkably reduced compared to that of control strains. Interestingly, the dissemination of the
C. neoformans Δisc1
strain to the brain is promptly restored in these mice when alveolar macrophages are pharmacologically depleted or when infecting an immunodeficient mouse in which macrophages are not efficiently activated. These studies suggest that Isc1 plays a key role in protecting
C. neoformans
from the intracellular environment of macrophages, whose activation is important for preventing fungal dissemination of the
Δisc1
strain to the central nervous system and the development of meningoencephalitis.
Growth Phase- and Nutrient Limitation-Associated Transcript Abundance Regulation inBordetella pertussis
Authors: Nakamura Mari M. , Liew Sin-Yee , Cummings Craig A. , Brinig Mary M. , Dieterich Christine , Relman David A.
Published: September 2006

To survive in a host environment, microbial pathogens must sense local conditions, including nutrient availability, and adjust their growth state and virulence functions accordingly. No comprehensive investigation of growth phase-related gene regulation inBordetella pertussishas been reported previously. We characterized changes in genome-wide transcript abundance ofB. pertussisas a function of growth phase and availability of glutamate, a key nutrient for this organism. Using aBordetellaDNA microarray, we discovered significant changes in transcript abundance for 861 array elements during the transition from log phase to stationary phase, including declining transcript levels of many virulence factor genes. The responses to glutamate depletion exhibited similarities to the responses induced by exit from log phase, including decreased virulence factor transcript levels. However, only 23% of array elements that showed at least a fourfold growth phase-associated difference in transcript abundance also exhibited glutamate depletion-associated changes, suggesting that nutrient limitation may be one of several interacting factors affecting gene regulation during stationary phase. Transcript abundance patterns of a Bvg+phase-locked mutant revealed that the BvgAS two-component regulatory system is a key determinant of growth phase- and nutrient limitation-related transcriptional control. Several adhesin genes exhibited lower transcript abundance during stationary phase and under glutamate restriction conditions. The predicted bacterial phenotype was confirmed: adherence to bronchoepithelial cells decreased 3.3- and 4.4-fold at stationary phase and with glutamate deprivation, respectively. Growth phase and nutrient availability may serve as cues by whichB. pertussisregulates virulence according to the stage of infection or the location within the human airway.
Characterization of
igaB
, a Second Immunoglobulin A1 Protease Gene in Nontypeable
Haemophilus influenzae
Authors: Fernaays Matthew M. , Lesse Alan J. , Cai Xueya , Murphy Timothy F.
Published: September 2006

Nontypeable
Haemophilus influenzae
is an important respiratory pathogen, causing otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Immunoglobulin A1 (IgA1) protease is a well-described protein and potential virulence factor in this organism as well as other respiratory pathogens. IgA1 proteases cleave human IgA1, are involved in invasion, and display immunomodulatory effects. We have identified a second IgA1 protease gene,
igaB
, in
H. influenzae
that is present in addition to the previously described IgA1 protease gene,
iga
. Reverse transcriptase PCR and IgA1 protease assays indicated that the gene is transcribed, expressed, and enzymatically active in
H. influenzae
. The product of this gene is a type 2 IgA1 protease with homology to the
iga
gene of
Neisseria
species. Mutants that were deficient in
iga
,
igaB
, and both genes were constructed in
H. influenzae
strain 11P6H, a strain isolated from a patient with COPD who was experiencing an exacerbation. Analysis of these mutants indicated that
igaB
is the primary mediator of IgA1 protease activity in this strain. IgA1 protease activity assays on 20 clinical isolates indicated that the
igaB
gene is associated with increased levels of IgA1 protease activity. Approximately one-third of 297 strains of
H. influenzae
of diverse clinical and geographic origin contained
igaB
. Significant differences in the prevalence of
igaB
were observed among isolates from different sites of isolation (sputum > middle ear > nasopharynx). These data support the hypothesis that the newly discovered
igaB
gene is a potential virulence factor in nontypeable
H. influenzae
.
Porphyromonas gingivalis
Minor Fimbriae Are Required for Cell-Cell Interactions
Authors: Lin Xinghua , Wu Jie , Xie Hua
Published: September 2006

Two distinctive types of fimbriae have been identified in
Porphyromonas gingivalis
. In this report, we demonstrate that minor fimbriae are involved in
P. gingivalis
autoaggregation and colonization. A mutant with a deficiency in minor fimbriae can bind to a saliva-coated surface but does not form microcolonies as the wild-type strain does.
The
mgtC
Gene of
Burkholderia cenocepacia
Is Required for Growth under Magnesium Limitation Conditions and Intracellular Survival in Macrophages
Authors: Maloney Kendra E. , Valvano Miguel A.
Published: September 2006

Burkholderia cenocepacia
, a bacterium commonly found in the environment, is an important opportunistic pathogen in patients with cystic fibrosis (CF). Very little is known about the mechanisms by which
B. cenocepacia
causes disease, but chronic infection of the airways in CF patients may be associated, at least in part, with the ability of this bacterium to survive within epithelial cells and macrophages. Survival in macrophages occurs in a membrane-bound compartment that is distinct from the lysosome, suggesting that
B. cenocepacia
prevents phagolysosomal fusion. In a previous study, we employed signature-tagged mutagenesis and an agar bead model of chronic pulmonary infection in rats to identify
B. cenocepacia
genes that are required for bacterial survival in vivo. One of the most significantly attenuated mutants had an insertion in the
mgtC
gene. Here, we show that
mgtC
is also needed for growth of
B. cenocepacia
in magnesium-depleted medium and for bacterial survival within murine macrophages. Using fluorescence microscopy, we demonstrated that
B. cenocepacia mgtC
mutants, unlike the parental isolate, colocalize with the fluorescent acidotropic probe LysoTracker Red. At 4 h postinfection,
mgtC
mutants expressing monomeric red fluorescent protein cannot retain this protein within the bacterial cytoplasm. Together, these results demonstrate that, unlike the parental strain, an
mgtC
mutant does not induce a delay in phagolysosomal fusion and the bacterium-containing vacuoles are rapidly targeted to the lysosome, where bacteria are destroyed.
Structural Characterization of Peptide-Mediated Inhibition of
Porphyromonas gingivalis
Biofilm Formation
Authors: Daep Carlo Amorin , James DeAnna M. , Lamont Richard J. , Demuth Donald R.
Published: September 2006

Porphyromonas gingivalis
is a periodontal pathogen whose primary niche is the anaerobic environment of subgingival dental plaque, but initial colonization of the oral cavity is likely to occur on supragingival surfaces that already support robust biofilm communities. Our studies have shown that
P. gingivalis
adheres to
Streptococcus gordonii
through interaction of the minor fimbrial antigen Mfa1 with a specific region of the streptococcal SspB polypeptide (residues 1167 to 1193) designated BAR. We show that a synthetic peptide comprising the BAR sequence potently inhibits
P. gingivalis
adherence to
S. gordonii
(50% inhibitory concentration = 1.3 μM) and prevents the development of
P. gingivalis
biofilms. However, a retroinverso peptide that possessed the same side chain topology as that of BAR was inactive, suggesting that interactions of Mfa1 with the peptide backbone of BAR are important for binding. A conformationally constrained analog of BAR inhibited
P. gingivalis
adherence and biofilm formation but at a lower specific activity than that of BAR. Therefore, to further define the structural features of the Mfa1-BAR interaction, we functionally screened combinatorial libraries of BAR in which active site residues (Asn
1182
, Thr
1184
, and Val
1185
) were replaced with each of the 19 common amino acids. Peptides containing positively charged amino acids at position 1182 or hydrophobic residues at position 1185 bound
P. gingivalis
more efficiently than did control peptides containing Asn and Val at these positions, suggesting that electrostatic and hydrophobic interactions may contribute to Mfa1-SspB binding. In contrast, replacement of Pro or Gly at these positions was detrimental to adherence, suggesting that perturbation of the BAR secondary structure influences activity. The net effect of substitutions for Thr
1184
was less pronounced either positively or negatively than that at the other sites. These results define physicochemical characteristics of the interacting interface of Mfa1 and SspB and suggest that peptides or peptidomimetics with greater specific inhibitory activity than that of BAR can be developed. These compounds may represent potential therapeutics that target some of the first molecular interactions that allow
P. gingivalis
to colonize the oral cavity.
Expression of the Lipopolysaccharide-Modifying Enzymes PagP and PagL Modulates the Endotoxic Activity ofBordetella pertussis
Authors: Geurtsen Jeroen , Steeghs Liana , Hamstra Hendrik-Jan , ten Hove Jan , de Haan Alex , Kuipers Betsy , Tommassen Jan , van der Ley Peter
Published: September 2006

Lipopolysaccharide (LPS) is one of the major constituents of the gram-negative bacterial cell envelope. Its endotoxic activity causes the relatively high reactogenicity of whole-cell vaccines. Several bacteria harbor LPS-modifying enzymes that modulate the endotoxic activity of the LPS. Here we evaluated whether two such enzymes, i.e., PagP and PagL, could be useful tools for the development of an improved and less reactogenic whole-cell pertussis vaccine. We showed that expression of PagP and PagL inBordetella pertussisleads to increased and decreased endotoxic activity of the LPS, respectively. As expected, PagP activity also resulted in increased endotoxic activity of whole bacterial cells. However, more unexpectedly, this was also the case for PagL. This paradoxical result may be explained, in part, by an increased release of LPS, which we observed in the PagL-expressing cells.
Multivalent Recombinant Protein Vaccine against Coccidioidomycosis
Authors: Tarcha Eric J. , Basrur Venkatesha , Hung Chiung-Yu , Gardner Malcolm J. , Cole Garry T.
Published: September 2006

Coccidioidomycosis is a human respiratory disease that is endemic to the southwestern United States and is caused by inhalation of the spores of a desert soilborne fungus. Efforts to develop a vaccine against this disease have focused on identification of T-cell-reactive antigens derived from the parasitic cell wall which can stimulate protective immunity againstCoccidioides posadasiiinfection in mice. We previously described a productive immunoproteomic/bioinformatic approach to the discovery of vaccine candidates which makes use of the translated genome ofC. posadasiiand a computer-based method of scanning deduced sequences of seroreactive proteins for epitopes that are predicted to bind to human major histocompatibility (MHC) class II-restricted molecules. In this study we identified a set of putative cell wall proteins predicted to contain multiple, promiscuous MHC II binding epitopes. Three of these were expressed byEscherichia coli, combined in a vaccine, and tested for protective efficacy in C57BL/6 mice. Approximately 90% of the mice survived beyond 90 days after intranasal challenge, and the majority cleared the pathogen. We suggest that the multicomponent vaccine stimulates a broader range of T-cell clones than the single recombinant protein vaccines and thereby may be capable of inducing protection in an immunologically heterogeneous human population.
Mutations within the Catalytic Motif of DNA Adenine Methyltransferase (Dam) of
Aeromonas hydrophila
Cause the Virulence of the Dam-Overproducing Strain To Revert to That of the Wild-Type Phenotype
Authors: Erova Tatiana E. , Fadl Amin A. , Sha Jian , Khajanchi Bijay K. , Pillai Lakshmi L. , Kozlova Elena V. , Chopra Ashok K.
Published: September 2006

In this study, we demonstrated that the methyltransferase activity associated with Dam was essential for attenuation of
Aeromonas hydrophila
virulence. We mutated aspartic acid and tyrosine residues to alanine within the conserved DPPY catalytic motif of Dam and transformed the pBAD/
dam
D/A
, pBAD/
dam
Y/A
, and pBAD/
dam
AhSSU
(with the native
dam
gene) recombinant plasmids into the
Escherichia coli
GM33 (
dam
-deficient) strain. Genomic DNA (gDNA) isolated from either of the
E. coli
GM33 strains harboring the pBAD vector with the mutated
dam
gene was resistant to DpnI digestion and sensitive to DpnII restriction endonuclease cutting. These findings were contrary to those with the gDNA of
E. coli
GM33 strain containing the pBAD/
dam
AhSSU
plasmid, indicating nonmethylation of
E. coli
gDNA with mutated Dam. Overproduction of mutated Dam in
A. hydrophila
resulted in bacterial motility, hemolytic and cytotoxic activities associated with the cytotoxic enterotoxin (Act), and protease activity similar to that of the wild-type (WT) bacterium, which harbored the pBAD vector and served as a control strain. On the contrary, overproduction of native Dam resulted in decreased bacterial motility, increased Act-associated biological effects, and increased protease activity. Lactone production, an indicator of quorum sensing, was increased when the native
dam
gene was overexpressed, with its levels returning to that of the control strain when the
dam
gene was mutated. These effects of Dam appeared to be mediated through a regulatory glucose-inhibited division A protein. Infection of mice with the mutated Dam-overproducing strains resulted in mortality rates similar to those for the control strain, with 100% of the animals dying within 2 to 3 days with two 50% lethal doses (LD
50
s) of the WT bacterium. Importantly, immunization of mice with a native-Dam-overproducing strain at the same LD
50
did not result in any lethality and provided protection to animals after subsequent challenge with a lethal dose of the control strain.
Gingipains from
Porphyromonas gingivalis
W83 Synergistically Disrupt Endothelial Cell Adhesion and Can Induce Caspase-Independent Apoptosis
Authors: Sheets Shaun M. , Potempa Jan , Travis James , Fletcher Hansel M. , Casiano Carlos A.
Published: September 2006

We have shown previously that gingipains from
Porphyromonas gingivalis
W83 can induce cell detachment, cell adhesion molecule (CAM) cleavage, and apoptosis in endothelial cells; however, the specific roles of the individual gingipains are unclear. Using purified gingipains, we determined that each of the gingipains can cleave CAMs to varying degrees with differing kinetics. Kgp and HRgpA work together to quickly detach endothelial cells. Interestingly, in the absence of active caspases, both gingipain-active W83 extracts and purified HRgpA and RgpB induce apoptotic morphology, suggesting that the gingipains can induce both caspase-dependent and caspase-independent apoptosis. Using z-VAD-FMK to inhibit Kgp activity and leupeptin to inhibit Rgp activity in gingipain-active W83 extracts, we investigated the relative significance of the synergistic role of the gingipains. z-VAD-FMK or leupeptin delayed, but did not inhibit, cell detachment induced by gingipain-active W83 extracts or purified gingipains. There was partial cleavage of N-cadherin and cleavage of VE-cadherin was not inhibited. Degradation of integrin β1 was inhibited only in the presence of z-VAD-FMK. These results further clarify the role
P. gingivalis
plays in tissue destruction occurring in the periodontal pocket.
Mapping of the
Plasmodium chabaudi
Resistance Locus
char2
Authors: Lin Enmoore , Pappenfuss Tony , Tan Rachel B. , Senyschyn Danielle , Bahlo Melanie , Speed Terence P. , Foote Simon J.
Published: September 2006

Animals congenic for the
char2
host response locus to the murine malarial parasite
Plasmodium chabaudi
have been bred, and they demonstrated a phenotypic difference from the parental lines. These congenic lines have been crossed back to the parental line to generate recombinants across the congenic intervals. The recombinants were inbred, and the subcongenic intervals were fixed. These lines were then challenged with parasites and assessed as being either resistant or susceptible. From the analysis of many subcongenic lines, it has become obvious that there are at least two loci underlying the
char2
locus and that both of these mediate resistance when the haplotype derives from the resistant C57BL/6 strain.
Induction of Neutrophil Chemotaxis by the Quorum-Sensing Molecule
N
-(3-Oxododecanoyl)-
l
-Homoserine Lactone
Authors: Zimmermann Sabine , Wagner Christof , Müller Wencke , Brenner-Weiss Gerald , Hug Friederike , Prior Birgit , Obst Ursula , Hänsch Gertrud Maria
Published: September 2006

Acyl homoserine lactones are synthesized by
Pseudomonas aeruginosa
as signaling molecules which control production of virulence factors and biofilm formation in a paracrine manner. We found that
N
-(3-oxododecanoyl)-
l
-homoserine lactone (3OC12-HSL), but not its 3-deoxo isomer or acyl-homoserine lactones with shorter fatty acids, induced the directed migration (chemotaxis) of human polymorphonuclear neutrophils (PMN) in vitro. By use of selective inhibitors a signaling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, and mitogen-activated protein kinase C, could be delineated. In contrast to the well-studied chemokines complement C5a and interleukin 8, the chemotaxis did not depend on pertussis toxin-sensitive G proteins, indicating that 3OC12-HSL uses another signaling pathway. Strong evidence for the presence of a receptor for 3OC12-HSL on PMN was derived from uptake studies; by use of radiolabeled 3OC12-HSL, specific and saturable binding to PMN was seen. Taken together, our data provide evidence that PMN recognize and migrate toward a source of 3OC12-HSL (that is, to the site of a developing biofilm). We propose that this early attraction of PMN could contribute to prevention of biofilm formation.
Sixty years on: the price of assembling military gas masks in 1940
Authors: McDonald J C , Harris J M , Berry G
Published: September 2006

Background:Between 1940 and 1944 military gas masks with filter pads containing 20% crocidolite were assembled in a Nottingham factory.

Methods:Records supplied by the late Professor Stephen Jones were of 1154 persons, mainly women, who had worked in the factory during this period; they included many deaths from mesothelioma. A systematic effort was therefore made to establish causes of death for the whole cohort.

Results:Of 640 employees with full name and sex recorded, 567 (89%) were traced. Of these, 491 had died, including 65 from mesothelioma, though only 54 were certified as such. After exclusion of these 54, standardised mortality ratios were significantly raised for respiratory cancer (SMR 2.5) and carcinomatosis (SMR 3.2). The pattern of mortality in the remaining 514 employees without full identification was similar, but a low tracing rate (40%) did not justify their further analysis. The first death from mesothelioma was in 1963 (22 years after first exposure) and the last in 1994, whereas a further 5.0 cases would have been expected between 1996 and 2003 (p = 0.0065).

Conclusion:These findings in a cohort followed over 60 years after brief exposure to crocidolite confirm a high and specific risk of mesothelioma (28% peritoneal) and perhaps of lung cancer some 20–50 years later. The statistically significant absence of further mesothelioma cases during the past eight years suggests that crocidolite, though durable, is slowly removed.
Spin transfer in ferromagnetic systems
Authors: Kim Wonkee , Marsiglio F
Published: September 2006

We illustrate spin transfer from spin-polarized electrons to ferromagnetic layers. Unlike the field-induced Zeeman interaction, which explains only the precession of a magnetic moment, the spin flip interaction describes the spin torque as well as the precession. The detailed dynamics of a magnetic moment depends on important parameters such as the incoming energy of electrons. In the case of multilayer systems, we demonstrate quantum phenomena ranging from spin transmission resonance to magnetoelectric spin echo that depend on the interlayer coupling between the two ferromagnetic thin films. We also show the possibility of a negative spin torque acting on the second moment in a bilayer system. PACS Nos.: 75.70.Ak,72.25.-b,85.75.-d
Is entanglement entropy proportional to area?
Authors: Ahmadi Morteza , Das Saurya , Shankaranarayanan S
Published: September 2006

It is known that the entanglement entropy of a scalar field, found by tracing over its degrees of freedom inside a sphere of radius [Formula: see text], is proportional to the area of the sphere (and not its volume). This suggests that the origin of black-hole entropy, also proportional to its horizon area, may lie in the entanglement between the degrees of freedom inside and outside the horizon. We examine this proposal carefully by including excited states, to check probable deviations from the area law.PACS Nos.: 04.60.m, 04.62, 04.70.s, 03.65.Ud
Relativistic field theories in a strong magnetic background asnoncommutative field theories
Authors: Miransky V A
Published: September 2006

The connection between the dynamics in relativistic field theories in a strong magnetic field and the dynamics of noncommutative field theories (NCFTs) is discussed. It is shown that this connection is rather sophisticated. In fact, the corresponding NCFTs are different from the conventional ones considered in the literature. In particular, the UV/IR mixing is absent in these theories. The reason for this is an inner structure (i.e., dynamical form factors) of neutral composites that plays an important role in providing consistency for NCFTs. NCFTs could find interesting applications in condensed-matter systems and string theories. PACS Nos.: 11.10.Nx, 11.30.Qc, 11.30.Rd
What if θ13 is small?
Authors: Lam C S
Published: September 2006

In the basis where the charged lepton mass matrix is diagonal, the left-handed neutrino mass matrix is invariant under the permutation of the second and third generations if, and only if, the reactor angle θ13 is zero and the atmospheric mixing angle θ23 is maximal. In the presence of the seesaw mechanism, this symmetry leads to an inverted hierarchy, with m3 = 0. This inverted mass spectrum is doubly protected if the right-handed neutrinos also have a 2–3 symmetry.PACS No.: 14.60Pq
Star-quantization of an infinite wall
Authors: Kryukov S , Walton M A
Published: September 2006

In deformation quantization (a.k.a. the WignerWeylMoyal formulation of quantum mechanics), we consider a single quantum particle moving freely in one dimension, except for the presence of one infinite potential wall. Dias and Prata pointed out that, surprisingly, its stationary-state Wigner function does not obey the naive equation of motion, i.e., the naive stargenvalue (*-genvalue) equation. We review our recent work on this problem that treats the infinite wall as the limit of a Liouville potential. Also included are some new results: (i) we show explicitly that the Wigner-Weyl transform of the usual density matrix is the physical solution, (ii) we prove that an effective-mass treatment of the problem is equivalent to the Liouville one, and (iii) we point out that self-adjointness of the operator Hamiltonian requires a boundary potential, but one apparently different from that proposed by Dias and Prata. PACS Nos.: 03.65.w, 03.65.Ca, 03.65.Ge