bluuebunny/crossref_metadata_2025 huggingface dataset:
Design Optimization of Hierarchically Decomposed Multilevel Systems Under Uncertainty
Authors: Kokkolaras Michael , Mourelatos Zissimos P. , Papalambros Panos Y.
Published: February 2006

This paper presents a methodology for design optimization of hierarchically decomposed systems under uncertainty. We propose an extended, probabilistic version of the deterministic analytical target cascading (ATC) formulation by treating uncertain quantities as random variables and posing probabilistic design constraints. A bottom-to-top coordination strategy is used for the ATC process. Given that first-order approximations may introduce unacceptably large errors, we use a technique based on the advanced mean value method to estimate uncertainty propagation through the multilevel hierarchy of elements that comprise the decomposed system. A simple yet illustrative hierarchical bilevel engine design problem is used to demonstrate the proposed methodology. The results confirm the applicability of the proposed probabilistic ATC formulation and the accuracy of the uncertainty propagation technique.
Quantum Computation as Geometry
Authors: Nielsen Michael A. , Dowling Mark R. , Gu Mile , Doherty Andrew C.
Published: February 2006

Quantum computers hold great promise for solving interesting computational problems, but it remains a challenge to find efficient quantum circuits that can perform these complicated tasks. Here we show that finding optimal quantum circuits is essentially equivalent to finding the shortest path between two points in a certain curved geometry. By recasting the problem of finding quantum circuits as a geometric problem, we open up the possibility of using the mathematical techniques of Riemannian geometry to suggest new quantum algorithms or to prove limitations on the power of quantum computers.
Comment on "On the Regulation of Populations of Mammals, Birds, Fish, and Insects" II
Authors: Ross Joshua V.
Published: February 2006

Sibly
et al
. (Reports, 22 July 2005, p. 607) recently estimated the relationship between population size and growth rate for 1780 time series of various species. I explain why some aspects of their analysis are questionable and, therefore, why their results and estimation procedure should be used with care.
A Swimming Mammaliaform from the Middle Jurassic and Ecomorphological Diversification of Early Mammals
Authors: Ji Qiang , Luo Zhe-Xi , Yuan Chong-Xi , Tabrum Alan R.
Published: February 2006

A docodontan mammaliaform from the Middle Jurassic of China possesses swimming and burrowing skeletal adaptations and some dental features for aquatic feeding. It is the most primitive taxon in the mammalian lineage known to have fur and has a broad, flattened, partly scaly tail analogous to that of modern beavers. We infer that docodontans were semiaquatic, convergent to the modern platypus and many Cenozoic placentals. This fossil demonstrates that some mammaliaforms, or proximal relatives to modern mammals, developed diverse locomotory and feeding adaptations and were ecomorphologically different from the majority of generalized small terrestrial Mesozoic mammalian insectivores.
MyD88 Deficiency Results in Tissue-Specific Changes in Cytokine Induction and Inflammation in Interleukin-18-Independent Mice Infected withBorrelia burgdorferi
Authors: Behera Aruna K. , Hildebrand Ethan , Bronson Roderick T. , Perides George , Uematsu Satoshi , Akira Shizuo , Hu Linden T.
Published: February 2006

Toll-like receptors (TLRs) play an important role in the control of infection withBorrelia burgdorferi. Deficiencies in TLR-2 or the shared TLR adapter molecule MyD88 have been shown to result in greatly increased bacterial burdens in mice. However, although in vitro studies have shown that the activation of TLR pathways byB. burgdorferiresults in the release of inflammatory cytokines, studies in deficient mice have shown either no change or increased rather than decreased inflammation in infected animals. In this study, we looked at mechanisms to explain the increase in inflammation in the absence of MyD88. We found that MyD88-deficient mice infected withB. burgdorferidid not show increased inflammation at sites typically associated with Lyme disease (joints and heart). However, there was markedly increased inflammation in the muscles, kidneys, pancreas, and lungs of deficient animals. Muscle inflammation was typically seen perivascularly and perineuronally similar to that seen in infected humans. Chemotactic chemokines and cytokines were greatly increased in the muscle and kidneys of MyD88-deficient animals but not in the joints or heart tissue, suggesting that MyD88-independent pathways for recognizingB. burgdorferiand inducing these chemokines are present in the muscle and kidneys. Interleukin-18 signaling through MyD88 does not appear to play a role in either control of infection or inflammation.
Profiling of Human Antibody Responses toChlamydia trachomatisUrogenital Tract Infection Using Microplates Arrayed with 156 Chlamydial Fusion Proteins
Authors: Sharma Jyotika , Zhong Youmin , Dong Feng , Piper Jeanna M. , Wang Guqi , Zhong Guangming
Published: February 2006

The available chlamydial genome sequences have made it possible to comprehensively analyze host responses to all chlamydial proteins, which is essential for further understanding of chlamydial pathogenesis and development of effective chlamydial vaccines. Microplates arrayed with 156Chlamydia trachomatisfusion proteins were used to evaluate antibody responses in women urogenitally infected withC. trachomatis. Based on both the antibody recognition frequency and titer, seven chlamydial antigens encoded by open reading frames (ORFs) CT089, CT147, CT226, CT681, CT694, CT795, and CT858, respectively, were identified as relatively immunodominant; six of these are encoded by hypothetical ORFs. Antibody binding to these chlamydial fusion proteins was blocked byC. trachomatis-infected but not by normal HeLa cell lysates or irrelevant bacterial lysates. These results have revealed novel immune-reactive chlamydial antigens, not only indicating that the hypothetical ORF-encoded proteins are expressed during chlamydial infection in humans but also providing the proof of principle that the fusion protein-based approach can be used to profile human immune responses to chlamydial infection at the whole-genome scale.
Estimating the Relative Contributions of Virulence Factors for Pathogenic Microbes
Authors: McClelland Erin E. , Bernhardt Paul , Casadevall Arturo
Published: February 2006

Many pathogenic microbes have multiple virulence factors that can cause damage to the host and thus contribute to an overall virulence phenotype for that organism. Although current techniques are suitable for demonstrating that a particular microbial characteristic contributes to virulence, no formal approach for defining the relative contributions of multiple virulence factors to overall virulence has been proposed. This paper describes the use of multivariate linear regression to estimate the relative contributions of virulence factors to the overall phenomenon of virulence. The approach is illustrated here with sample calculations of the relative contributions of individual
Cryptococcus neoformans
and
Bacillus anthracis
virulence factors to the overall virulence phenotype. These calculations were derived from a small underpowered experimental data set for the fungus and two larger sets of randomly generated data for both microbes. The major limitation of this method is a requirement for large data sets of microbial strains that differ in virulence and virulence factor expression. Multivariate linear regression can be used to identify the relative levels of importance of virulence factors in virulence studies, and this information can be used to prioritize antigen identification for vaccine development and the design of antimicrobial strategies that target virulence mechanisms.
The Proinflammatory Response Induced by Wild-TypeYersinia pseudotuberculosisInfection Inhibits Survival ofyopMutants in the Gastrointestinal Tract and Peyer's Patches
Authors: Logsdon Lauren K. , Mecsas Joan
Published: February 2006

Single-strain infections and coinfections are frequently used to assess roles of virulence factors in infected tissues. After oral inoculation of mice,Yersinia pseudotuberculosis yopEandyopHmutants colonize the intestines and Peyer's patches in single-strain infections but fail to persist in competition with wild-typeY. pseudotuberculosis, indicating that these two infection models provide different insights into the roles of Yops. To determine how wild-typeY. pseudotuberculosishindersyopmutant survival,yopmutant colonization and host responses were investigated in several different infection models that isolated specific features of wild-typeY. pseudotuberculosisinfection. Infection with wild-typeY. pseudotuberculosiscaused significantly more inflammation thanyopmutants. Results from coinfections of gamma interferon (IFN-γ)−/−mice revealed that IFN-γ-regulated defenses target these mutants, suggesting that YopE and YopH protectY. pseudotuberculosisfrom these defenses in BALB/c mice. We developed an oral-intraperitoneal infection model to evaluate the effects of spleen and liver colonization byY. pseudotuberculosisonyopmutants in the intestines. Spleen and liver infection increased inflammation and decreasedyopmutant survival in the intestines, indicating that infection of these organs has consequences in intestinal tissues. Finally, competition infections withY. pseudotuberculosismutants with various abilities to induce inflammation demonstrated that survival of theyopE, but not theyopH, mutant was consistently decreased in inflamed tissues. In summary, infection withY. pseudotuberculosisin intestinal and systemic sites induces intestinal inflammation, which decreasesyopmutant survival. Thus, competition studies with wild-type yersiniae reveal critical roles of Yops in combating host responses to a normal virulent infection.
The Onset of CD8+-T-Cell Contraction Is Influenced by the Peak ofListeria monocytogenesInfection and Antigen Display
Authors: Porter Brandon B. , Harty John T.
Published: February 2006

The CD8+-T-cell response to infection withListeria monocytogenesconsists of expansion, contraction, and memory phases. The transition between expansion and contraction is reported to occur on different days postinfection with virulent (8 to 9 days) and attenuated (ΔactA) (7 days)L. monocytogenesstrains. We hypothesized that differences in the infectious courses, and therefore antigen (Ag) display, determine the precise time of the expansion/contraction transition in response to these infections. To test this, we infected BALB/c mice with 0.1 50% lethal dose of ΔactAor virulentL. monocytogenesand measured bacterial numbers, Ag display, and Ag-specific CD8+-T-cell responses on various days after infection. We found that bacterial numbers and Ag display peaked between 12 and 36 h and between 36 and 60 h after infection with ΔactAand virulentL. monocytogenesstrains, respectively. Infection with ΔactA L. monocytogenesresulted in a sharp peak in the Ag-specific CD8+-T-cell response on day 7, while infection with virulentL. monocytogenesyielded a prolonged peak with equivalent numbers of Ag-specific CD8+T cells on days 6, 7, and 8 after infection. Truncating virulent infection with antibiotics on day 1 or 2 after infection resulted in a shift in the expansion/contraction transition from day 8 to day 7 after infection. However, antibiotic treatment beginning on day 3, after the peak of virulentL. monocytogenesinfection and Ag display, had no effect upon the magnitude or timing of the CD8+-T-cell response. These results demonstrate a direct relationship between the course of infection and Ag display and that the timing of these events is important in shaping the T-cell response to infection.
The Outer Core Lipopolysaccharide of
Salmonella enterica
Serovar Typhi Is Required for Bacterial Entry into Epithelial Cells
Authors: Hoare Anilei , Bittner Mauricio , Carter Javier , Alvarez Sergio , Zaldívar Mercedes , Bravo Denisse , Valvano Miguel A. , Contreras Inés
Published: February 2006

Salmonella enterica
serovar Typhi causes typhoid fever in humans. Central to the pathogenicity of serovar Typhi is its capacity to invade intestinal epithelial cells. The role of lipopolysaccharide (LPS) in the invasion process of serovar Typhi is unclear. In this work, we constructed a series of mutants with defined deletions in genes for the synthesis and polymerization of the O antigen (
wbaP
,
wzy
, and
wzz
) and the assembly of the outer core (
waaK
,
waaJ
,
waaI
,
waaB
, and
waaG
). The abilities of each mutant to associate with and enter HEp-2 cells and the importance of the O antigen in serum resistance of serovar Typhi were investigated. We demonstrate here that the presence and proper chain length distribution of the O-antigen polysaccharide are essential for serum resistance but not for invasion of epithelial cells. In contrast, the outer core oligosaccharide structure is required for serovar Typhi internalization in HEp-2 cells. We also show that the outer core terminal glucose residue (Glc II) is necessary for efficient entry of serovar Typhi into epithelial cells. The Glc I residue, when it becomes terminal due to a polar insertion in the
waaB
gene affecting the assembly of the remaining outer core residues, can partially substitute for Glc II to mediate bacterial entry into epithelial cells. Therefore, we conclude that a terminal glucose in the LPS core is a critical residue for bacterial recognition and internalization by epithelial cells.
Trypanosoma cruziInfection and Nuclear Factor Kappa B Activation Prevent Apoptosis in Cardiac Cells
Authors: Petersen Christine A. , Krumholz Katherine A. , Carmen John , Sinai Anthony P. , Burleigh Barbara A.
Published: February 2006

Studies of cardiac pathology and heart failure have implicated cardiomyocyte apoptosis as a critical determinant of disease. Recent evidence indicates that the intracellular protozoan parasiteTrypanosoma cruzi, which causes heart disease in chronically infected individuals, impinges on host apoptotic pathways in a cell type-dependent manner.T. cruziinfection of isolated neuronal cells and cardiomyocytes protects against apoptotic cell death, whereas apoptosis is triggered in T cells inT. cruzi-infected animals. In this study, we demonstrate that the ability ofT. cruzito protect cardiac cells in vitro from apoptosis triggered by a combination of tumor necrosis factor alpha and serum reduction correlates with the presence of intracellular parasites and involves activation of host cell NF-κB. We further demonstrate that the apoptotic block diminishes activation of caspase 3. The ability ofT. cruzito prevent apoptosis of infected cardiomyocytes is likely to play an important role in establishment of persistent infection in the heart while minimizing potential damage and remodeling that is associated with cardiomyocyte apoptosis in cardiovascular disease.
Vaccination of Mice with Gonococcal TbpB Expressed In Vivo from Venezuelan Equine Encephalitis Viral Replicon Particles
Authors: Thomas Christopher E. , Zhu Weiyan , Van Dam Cornelius N. , Davis Nancy L. , Johnston Robert E. , Sparling P. Frederick
Published: February 2006

We investigated the immunogenicity of gonococcal transferrin binding protein B (TbpB) expressed with and without a eukaryotic secretion signal from a nonpropagating Venezuelan equine encephalitis virus replicon particle (VRP) delivery system. TbpB was successfully expressed in baby hamster kidney (BHK) cells, and the presence of the eukaryotic secretion signal not only apparently increased the protein's expression but also allowed for extracellular localization and glycosylation. Mice immunized with VRPs produced significant amounts of serum antibody although less than the amounts produced by mice immunized with recombinant protein. The response of mice immunized with VRPs encoding TbpB was consistently more Th1 biased than the response of mice immunized with recombinant protein alone. Boosting with recombinant protein following immunization with TbpB VRPs resulted in higher specific-antibody levels without altering the Th1/Th2 bias. Most of the immunization groups produced significant specific antibody binding to the intact surface of the homologousNeisseria gonorrhoeaestrain. Immunization with TbpB VRPs without a eukaryotic secretion signal generated no measurable specific antibodies on the genital mucosal surface, but inclusion of a eukaryotic secretion signal or boosting with recombinant protein resulted in specific immunoglobulin G (IgG) and IgA in mucosal secretions after TbpB VRP immunization. The TbpB VRP system has potential for anN. gonorrhoeaevaccine.
Role of Phagosomes and Major Histocompatibility Complex Class II (MHC-II) Compartment in MHC-II Antigen Processing ofMycobacterium tuberculosisin Human Macrophages
Authors: Torres Martha , Ramachandra Lakshmi , Rojas Roxana E. , Bobadilla Karen , Thomas Jeremy , Canaday David H. , Harding Clifford V. , Boom W. Henry
Published: February 2006

Mycobacterium tuberculosisresides in phagosomes inside macrophages. In this study, we analyzed the kinetics and location ofM. tuberculosispeptide-major histocompatibility complex class II (MHC-II) complexes inM. tuberculosis-infected human macrophages.M. tuberculosispeptide-MHC-II complexes were detected with polyclonal autologousM. tuberculosis-specific CD4+T cells or F9A6 T hybridoma cells specific forM. tuberculosisantigen (Ag) 85B (96-111). Macrophages processed heat-killedM. tuberculosismore rapidly and efficiently than liveM. tuberculosis. To determine whereM. tuberculosispeptide-MHC-II complexes were formed intracellularly, macrophages incubated with heat-killedM. tuberculosiswere homogenized, and subcellular compartments were separated on Percoll density gradients analyzed with T cells. In THP-1 cells,M. tuberculosisAg 85B (96- 111)-DR1 complexes appeared initially in phagosomes, followed by MHC class II compartment (MIIC) and the plasma membrane fractions. In monocyte-derived macrophages,M. tuberculosispeptide-MHC-II complexes appeared only in MIIC fractions and subsequently on the plasma membrane. Although phagosomes from both cell types acquired lysosome-associated membrane protein 1 (LAMP-1) and MHC-II, THP-1 phagosomes that support formation ofM. tuberculosispeptide-MHC-II complexes had increased levels of both LAMP-1 and MHC-II. Thus,M. tuberculosisphagosomes with high levels of MHC-II and LAMP-1 and MIIC both have the potential to form peptide-MHC-II complexes fromM. tuberculosisantigens in human macrophages.
Gingipains ofPorphyromonas gingivalisModulate Leukocyte Adhesion Molecule Expression Induced in Human Endothelial Cells by Ligation of CD99
Authors: Yun Peter L. W. , DeCarlo Arthur A. , Hunter Neil
Published: February 2006

Porphyromonas gingivalishas been implicated as a key etiologic agent in the pathogenesis of destructive chronic periodontitis. Among virulence factors of this organism are cysteine proteinases, or gingipains, that have the capacity to modulate host inflammatory defenses. Intercellular adhesion molecule expression by vascular endothelium represents a crucial process for leukocyte transendothelial migration into inflamed tissue. Ligation of CD99 on endothelial cells was shown to induce expression of endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and major histocompatibility complex class II molecules and to increase adhesion of leukocytes. CD99 ligation was also found to induce nuclear translocation of NF-κB. These results indicate that endothelial cell activation by CD99 ligation may lead to the up-regulation of adhesion molecule expression via NF-κB activation. However, pretreatment of endothelial cells with gingipains caused a dose-dependent reduction of adhesion molecule expression and leukocyte adhesion induced by ligation of CD99 on endothelial cells. The data provide evidence that the gingipains can reduce the functional expression of CD99 on endothelial cells, leading indirectly to the disruption of adhesion molecule expression and of leukocyte recruitment to inflammatory foci.
Identification of
Legionella pneumophila
-Specific Genes by Genomic Subtractive Hybridization with
Legionella micdadei
and Identification of
lpnE
, a Gene Required for Efficient Host Cell Entry
Authors: Newton Hayley J. , Sansom Fiona M. , Bennett-Wood Vicki , Hartland Elizabeth L.
Published: February 2006

Legionella pneumophila
is a ubiquitous environmental organism and a facultative intracellular pathogen of humans. To identify genes that may contribute to the virulence of
L. pneumophila
, we performed genomic subtractive hybridization between
L. pneumophila
serogroup 1 strain 02/41 and
L. micdadei
strain 02/42. A total of 144
L. pneumophila
-specific clones were sequenced, revealing 151 genes that were absent in
L. micdadei
strain 02/42. Low-stringency Southern hybridization was used to determine the distribution of 41 sequences, representing 40 open reading frames (ORFs) with a range of putative functions among
L. pneumophila
isolates of various serogroups as well as strains of
Legionella longbeachae
,
L. micdadei
,
Legionella gormanii
, and
Legionella jordanis
. Twelve predicted ORFs were
L. pneumophila
specific, including the gene encoding the
dot/icm
effector,
lepB
, as well as several genes predicted to play a role in lipopolysaccharide biosynthesis and cell wall synthesis and several sequences with similarity to virulence-associated determinants. A further nine predicted ORFs were in all
L. pneumophila
serotypes tested and an isolate of
L. gormanii
. These included
icmD
, the 5′ end of a
pilMNOPQ
locus, and two genes known to be upregulated during growth within macrophages,
cadA2
and
ceaA
. Disruption of an
L. pneumophila
-specific gene (lpg2222 locus tag) encoding a putative protein with eight tetratricopeptide repeats resulted in reduced entry into the macrophage-like cell line, THP-1, and the type II alveolar epithelial cell line, A549. The gene was subsequently renamed
lpnE
, for “
L. pneumophila
entry.” In summary, this investigation has revealed important genetic differences between
L. pneumophila
and other
Legionella
species that may contribute to the phenotypic and clinical differences observed within this genus.
Induction of a Novel Chicken Toll-Like Receptor following
Salmonella enterica
Serovar Typhimurium Infection
Authors: Higgs Rowan , Cormican Paul , Cahalane Sarah , Allan Brenda , Lloyd Andrew T. , Meade Kieran , James Tharappel , Lynn David J. , Babiuk Lorne A. , O'Farrelly Cliona
Published: February 2006

Toll-like receptors (TLRs) are a group of highly conserved molecules that initiate the innate immune response to pathogens by recognizing structural motifs expressed by microbes. We have identified a novel TLR, TLR15, by bioinformatic analysis of the chicken genome, which is distinct from any known vertebrate TLR and thus appears to be avian specific. The gene for TLR15 was sequenced and is found on chromosome 3, and it has archetypal TIR and transmembrane domains and a distinctive arrangement of extracellular leucine-rich regions. mRNA for TLR15 was detected in the spleen, bursa, and bone marrow of healthy chickens, suggesting a role for this novel receptor in constitutive host defense. Following in vivo
Salmonella enterica
serovar Typhimurium infection, quantitative real-time PCR demonstrated significant upregulation of TLR15 in the cecum of infected chickens. Interestingly, similar induction of TLR2 expression following infection was also observed. In vitro studies revealed TLR15 upregulation in chicken embryonic fibroblasts stimulated with heat-killed
S. enterica
serovar Typhimurium. Collectively, these results suggest a role for the TLR in avian defense against bacterial infection. We hypothesize that TLR15 may represent an avian-specific TLR that has been either retained in chicken and lost in other taxa or gained in the chicken.
CpG-Modified Plasmid DNA Encoding Flagellin Improves Immunogenicity and Provides Protection againstBurkholderia pseudomalleiInfection in BALB/c Mice
Authors: Chen Yao-Shen , Hsiao Yu-Shan , Lin Hsi-Hsun , Liu Yin , Chen Ya-Lei
Published: February 2006

The plasmid DNA encoding thefliCgene ofBurkholderia pseudomalleicombined with CpG oligodeoxynucleotide (ODN) was injected intramuscularly into BALB/c mice, resulting in the increased production of certain humoral antibodies and flagellin-specific spleen cell clonal expansion. CpG ODN, as an immunoadjuvant, was added to the plasmid containing thefliCgene in order to obtain ongoing expression in muscle for a long period. Functional expression of flagellin from the constructed CpG-modified plasmid in transfected peritoneal exudate cells of BALB/c mice was shown by reverse transcription-PCR and Western blotting. Furthermore, BALB/c mice immunized with the modified plasmid had relatively higher resistance toB. pseudomalleiinfection in vivo than did mice immunized with unmodified plasmid DNA. The time course of restricted bacterial growth in spleen and liver and changes in the cytokine profiles of immunized mice suggested that the stimulated phagocytic cells would be able to kill the bacteria eventually, possibly as a consequence of the induction of Th-1-type immune polarization in vivo. Th-1-type immune polarization was detected in response to flagellin induction in mice immunized with CpG-modified plasmid DNA by the appearance of increased levels of immunoglobulin G2a antibodies and gamma interferon-secreting cells specific to flagellin. The exogenous CpG motifs added to thefliCgene would contribute to an adjuvant-like response that enhances the flagellin-specific immunogenicity and provides protection againstB. pseudomalleiinfection. This CpG-modified plasmid DNA vaccination is an important potential strategy that should be developed to protect against melioidosis.
Mycobacterium bovis
BCG Substrains Confer Different Levels of Protection against
Mycobacterium tuberculosis
Infection in a BALB/c Model of Progressive Pulmonary Tuberculosis
Authors: Castillo-Rodal Antonia Isabel , Castañón-Arreola Mauricio , Hernández-Pando Rogelio , Calva Juan José , Sada-Díaz Eduardo , López-Vidal Yolanda
Published: February 2006

Mycobacterium bovis
BCG is the only available vaccine against tuberculosis. Reasons for why diverse BCG substrains induce different levels of protection in clinical trials remain unclear. The aim of this study was to compare the effectiveness of 10 BCG substrains in a mouse model of pulmonary tuberculosis. BALB/c mice were subcutaneously vaccinated and 2 months later were challenged with
Mycobacterium tuberculosis
H37Rv by intratracheal injection. Two and 4 months after challenge, delayed-type hypersensitivity (DTH) response, lung tissue affected by pneumonia, CFU, T-cell counts, and cytokine expression (interleukin-2 [IL-2], IL-4, IL-10, and gamma interferon) were determined. A differential protective effect of the diverse BCG substrains was found. BCG Phipps led to the largest and most persistent reduction of CFU counts and of the area of pneumonia at 2 and 4 months after challenge. This protection was accompanied by reduced IL-10-producing T cells. Contemporary BCG substrains induce a wide range of protection in this animal model. These data can help in the selection of the best vaccine for human immunization and for the development of novel recombinant BCG-based vaccine.
Heme Transport Contributes to In Vivo Fitness of
Bordetella pertussis
during Primary Infection in Mice
Authors: Brickman Timothy J. , Vanderpool Carin K. , Armstrong Sandra K.
Published: February 2006

Bordetella pertussis
, the causative agent of whooping cough or pertussis, is an obligate human pathogen with multiple high-affinity iron transport systems. Maximal expression of the dedicated heme utilization functions encoded by the
hurIR bhuRSTUV
genes requires an iron starvation signal to relieve Fur repression at the
hurIR
promoter-operator and an inducing signal supplied by heme for HurI-mediated transcriptional activation at the
bhuRSTUV
promoter. The BhuR outer membrane receptor protein is required for heme uptake and for heme sensing for induction of
bhuRSTUV
transcription. It was hypothesized that heme utilization contributed to the success of
B. pertussis
as a pathogen. In this study, virulence attenuation resulting from inactivation of the
B. pertussis
heme system was assessed using mixed infection competition experiments in a mouse model. As a measure of in vivo fitness, the ability of a
B. pertussis
heme utilization mutant to colonize and persist was determined relative to that of an isogenic coinfecting wild-type strain. Relative fitness of the mutant strain declined significantly after 7 days postinfection and continued to decline throughout the remainder of the 28-day infection time course. In parallel infections using inocula supplemented with an inducing 2 μM concentration of hemin chloride, hemin coadministration augmented the competitive advantage of the wild-type strain over the mutant. The results confirm that heme utilization contributes to the pathogenesis of
B. pertussis
in the mouse infection model and indicate that heme utilization may be most important for adaptation to host conditions existing during the later stages of infection.
The Twin Arginine Translocation System Is Essential for Virulence ofYersinia pseudotuberculosis
Authors: Lavander Moa , Ericsson Solveig K. , Bröms Jeanette E. , Forsberg Åke
Published: February 2006

Yersiniaspecies pathogenic to humans have been extensively characterized with respect to type III secretion and its essential role in virulence. This study concerns the twin arginine translocation (Tat) pathway utilized by gram-negative bacteria to secrete folded proteins across the bacterial inner membrane into the periplasmic compartment. We have shown that theYersiniaTat system is functional and required for motility and contributes to acid resistance. AYersinia pseudotuberculosismutant strain with a disrupted Tat system (tatC) was, however, not affected in in vitro growth or more susceptible to high osmolarity, oxidative stress, or high temperature, nor was it impaired in type III secretion. Interestingly, thetatCmutant was severely attenuated via both the oral and intraperitoneal routes in the systemic mouse infection model and highly impaired in colonization of lymphoid organs like Peyer's patches and the spleen. Our work highlights that Tat secretion plays a key role in the virulence ofY. pseudotuberculosis.
Identification of Fibronectin-Binding Proteins inMycoplasma gallisepticumStrain R
Authors: May Meghan , Papazisi Leka , Gorton Timothy S. , Geary Steven J.
Published: February 2006

We have determined that virulentMycoplasma gallisepticumstrain Rlowis capable of binding the extracellular matrix protein fibronectin. Fibronectin was found to be present inM. gallisepticumRlowprotein extracts by Western blotting and peptide sequencing.Mycoplasma gallisepticumRhigh, the attenuated, high-passage derivative of Rlow, is deficient in this ability. MGA_1199, theM. gallisepticumhomologue of the cytadherence-associated protein P65 fromMycoplasma pneumoniae, and MGA_0928, theM. gallisepticumhomologue of theM. pneumoniaecytoskeletal protein HMW3, were identified as fibronectin-binding proteins. Peptides from the regions of MGA_1199 and MGA_0928 exhibiting the highest degree of homology with known fibronectin-binding proteins were shown to bind the gelatin/heparin-binding domain of fibronectin. MGA_1199 and MGA_0928 were shown to be absent and aberrant, respectively, in Rhigh, explaining its lack of fibronectin-binding capability. Consistent with itsM. pneumoniaecounterpart, MGA_1199 (renamed PlpA) was demonstrated to be surface exposed, despite a lack of classical membrane-spanning domains. Due to its demonstrated topology and the strength of interaction between its binding peptide and fibronectin, we propose that PlpA functions as a fibronectin-binding protein in vivo and may possess atypical transmembrane domains.
A
Helicobacter pylori
Vacuolating Toxin Mutant That Fails To Oligomerize Has a Dominant Negative Phenotype
Authors: Genisset Christophe , Galeotti Cesira L. , Lupetti Pietro , Mercati David , Skibinski David A. G. , Barone Silvia , Battistutta Roberto , de Bernard Marina , Telford John L.
Published: February 2006

Most
Helicobacter pylori
strains secrete a toxin (VacA) that causes massive vacuolization of target cells and which is a major virulence factor of
H. pylori
. The VacA amino-terminal region is required for the induction of vacuolization. The aim of the present study was a deeper understanding of the critical role of the N-terminal regions that are protected from proteolysis when VacA interacts with artificial membranes. Using a counterselection system, we constructed an
H. pylori
strain, SPM 326-Δ49-57, that produces a mutant toxin with a deletion of eight amino acids in one of these protected regions. VacA Δ49-57 was correctly secreted by
H. pylori
but failed to oligomerize and did not have any detectable vacuolating cytotoxic activity. However, the mutant toxin was internalized normally and stained the perinuclear region of HeLa cells. Moreover, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. This loss of activity was correlated with the disappearance of oligomers in electron microscopy. These findings indicate that the deletion in VacA Δ49-57 disrupts the intermolecular interactions required for the oligomerization of the toxin.
Interaction of
Chlamydia trachomatis
with Mammalian Cells Is Independent of Host Cell Surface Heparan Sulfate Glycosaminoglycans
Authors: Stephens Richard S. , Poteralski Jesse M. , Olinger Lynn
Published: February 2006

The hypothesis that host cell surface heparan sulfate is required to promote chlamydial infection was tested using a cell line (CHO-18.4) containing a single retroviral insertion and the concomitant loss of heparan sulfate biosynthesis. Tests of chlamydial infectivity of heparan sulfate-deficient CHO-18.4 cells and parental cells, CHO-22, demonstrated that both were equally sensitive to infection by
Chlamydia trachomatis
serovars L2 and D. These data do not support the hypothesis and demonstrate that host cell surface heparan sulfate does not serve an essential functional role in chlamydial infectivity.
Role for the CD28 Molecule in the Control ofCoxiella burnetiiInfection
Authors: Honstettre Amélie , Meghari Soraya , Nunès Jacques A. , Lepidi Hubert , Raoult Didier , Olive Daniel , Mege Jean-Louis
Published: February 2006

Q fever is an infectious disease caused byCoxiella burnetii, an obligate intracellular bacterium that replicates in macrophages. As cell-mediated immune response to microbial pathogens requires signals mediated by T-cell receptors and costimulatory molecules such as CD28, we wondered if CD28 is involved in protection againstC. burnetiiinfection. CD28-deficient (CD28−/−) mice were inoculated withC. burnetiiby intraperitoneal and intravenous routes. With both wild-type and CD28−/−mice,C. burnetiiorganisms were detected exclusively in spleen and liver. The antibody response againstC. burnetiiwas impaired in CD28−/−animals, but, surprisingly, the lack of CD28 decreasedC. burnetiiburden in the infected tissues, whatever the manner of inoculation of bacteria. The CD28 deficiency had no effect on either granuloma formation, which reflects cell-mediated immunity againstC. burnetii, or the production of gamma interferon and tumor necrosis factor, two cytokines known to be involved in granuloma formation. On the other hand, the production of interleukin-10 (IL-10) by peritoneal macrophages was highly impaired in CD28−/−mice. The results suggest that CD28 initiates a signal that favorsC. burnetiireplication through the modulation of the IL-10 pathway.
Conditioned Medium from EnterohemorrhagicEscherichia coli-Infected T84 Cells Inhibits Signal Transducer and Activator of Transcription 1 Activation by Gamma Interferon
Authors: Jandu Narveen , Ceponis Peter J. M. , Kato Seiichi , Riff Jason D. , McKay Derek M. , Sherman Philip M.
Published: February 2006

Gamma interferon (IFN-γ) is a cytokine important to host defense which can signal through signal transducer and activator of transcription 1 (Stat1). EnterohemorrhagicEscherichia coli(EHEC) modulates host cell signal transduction to establish infection, and EHEC serotypes O113:H21 and O157:H7 both inhibit IFN-γ-induced Stat1 tyrosine phosphorylation in vitro. The aim of this study was to delineate both bacterial and host cell factors involved in the inhibition of Stat1 tyrosine phosphorylation. Human T84 colonic epithelial cells were challenged with direct infection, viable EHEC separated from T84 cells by a filter, sodium orthovanadate, isolated flagellin, bacterial culture supernatants, and conditioned medium treated with proteinase K, trypsin, or heat inactivation. Epithelial cells were then stimulated with IFN-γ and protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-inducible Stat1 tyrosine phosphorylation was inhibited when EHEC adhered to T84 cells, but not by bacterial culture supernatants or bacteria separated from the epithelial monolayer. Conditioned medium from T84 cells infected with EHEC O157:H7 suppressed Stat1 activation, and this was not reversed by treatment with proteinases or heat inactivation. Use of pharmacological inhibitors showed that time-dependent bacterial, but not epithelial, protein synthesis was involved. Stat1 inhibition was also independent of bacterial flagellin, host proteasome activity, and protein tyrosine phosphatases. Infection led to altered IFN-γ receptor domain 1 subcellular distribution and decreased expression in cholesterol-enriched membrane microdomains. Thus, suppression of host cell IFN-γ signaling by production of a contact-dependent, soluble EHEC factor may represent a novel mechanism for this pathogen to evade the host immune system.
Induced Biliary Excretion ofListeria monocytogenes
Authors: Hardy Jonathan , Margolis Jeffrey J. , Contag Christopher H.
Published: February 2006

Listeria monocytogenesis a ubiquitous gram-positive bacterium that can cause systemic and often life-threatening disease in immunocompromised hosts. This organism is largely an intracellular pathogen; however, we have determined that it can also grow extracellularly in animals, in the lumen of the gallbladder. The significance of growth in the gallbladder with respect to the pathogenesis and spread of listeriosis depends on the ability of the bacterium to leave this organ and be disseminated to other tissues and into the environment. Should this process be highly inefficient, growth in the gallbladder would have no impact on pathogenesis or spread, but if it occurs efficiently, bacterial growth in this organ may contribute to listeriosis and dissemination of this organism. Here, we use whole-body imaging to determine the efficacy and kinetics of food- and hormone-induced biliary excretion ofL. monocytogenesfrom the murine gallbladder, demonstrating that transit through the bile duct into the intestine can occur within 5 min of induction of gallbladder contraction by food or cholecystokinin and that movement of bacteria through the intestinal lumen can occur very rapidly in the absence of fecal material. These studies demonstrate thatL. monocytogenesbacteria replicating in the gallbladder can be expelled from the organ efficiently and that the released bacteria move into the intestinal tract, where they pass into the environment and may possibly reinfect the animal.
Biofilm Growth Increases Phosphorylcholine Content and Decreases Potency of NontypeableHaemophilus influenzaeEndotoxins
Authors: West-Barnette Shayla , Rockel Andrea , Swords W. Edward
Published: February 2006

NontypeableHaemophilus influenzae(NTHI) is a common respiratory commensal and opportunistic pathogen. NTHI is normally contained within the airways by host innate defenses that include recognition of bacterial endotoxins by Toll-like receptor 4 (TLR4). NTHI produces lipooligosaccharide (LOS) endotoxins which lack polymeric O side chains and which may contain host glycolipids. We recently showed that NTHI biofilms contain variants with sialylated LOS glycoforms that are essential to biofilm formation. In this study, we show that NTHI forms biofilms on epithelial cell layers. Confocal analysis revealed that sialylated variants were distributed throughout the biofilm, while variants expressing phosphorylcholine (PCho) were found within the biofilm. Consistent with this observation,PCho content of LOS purified from NTHI biofilms was increased compared to LOS from planktonic cultures. Hypothesizing that the observed changes in endotoxin composition could affect bioactivity, we compared inflammatory responses to NTHI LOS purified from biofilm and planktonic cultures. Our results show that endotoxins from biofilms induced weaker host innate responses. While we observed a minimal effect of sialylation on LOS bioactivity, there was a significant decrease in bioactivity associated withPCho substitutions. We thus conclude that biofilm growth increases the proportion ofPCho+variants in an NTHI population, resulting in a net decrease in LOS bioactivity. Thus, in addition to their well-documented resistance phenotypes, our data show that biofilm communities of NTHI bacteria contain variants that evoke less potent host responses.
Role of Tumor Necrosis Factor Alpha (TNF-α) and Interleukin-10 in the Pathogenesis of Severe Murine Monocytotropic Ehrlichiosis: Increased Resistance of TNF Receptor p55- and p75-Deficient Mice to Fatal Ehrlichial Infection
Authors: Ismail Nahed , Stevenson Heather L. , Walker David H.
Published: February 2006

Intraperitoneal (i.p.) infection with a high dose of a highly virulentEhrlichiastrain (IOE) results in a toxic shock-like syndrome characterized by severe liver injury and systemic overproduction of tumor necrosis factor alpha (TNF-α) by CD8+T cells. We examined the role of TNF-α and TNF receptors in high-dose-IOE-induced shock/liver injury. TNF receptor (TNFR) I/II−/−mice lacking both the p55 and p75 receptors for this cytokine were more resistant to IOE-induced liver injury than their wild-type background controls. TNFR I/II−/−mice survived longer, dying between 15 and 18 days, with evidence of mild liver necrosis/apoptosis. In contrast, wild-type mice were not rescued from the lethal effect of IOE by TNF-α neutralization. TNF-α-depleted mice developed severe liver injury and succumbed to disease between days 9 and 11 postinfection, similar to sham-treated, infected wild-type mice. Although IFN-γ production in the spleens of IOE-infected TNFR I/II−/−and TNF-α-depleted mice was higher than that detected in wild-type controls, these mice had higher bacterial burdens than infected controls. Following high-dose IOE challenge, TNFR I/II−/−and TNF-α-depleted mice have an early increase in IL-10 levels in sera and spleens, which was produced mainly by adherent spleen cells. In contrast, a late burst of interleukin-10 (IL-10) was observed in control mice. Nonadherent spleen cells were the major source of IL-10 in IOE-infected wild-type mice. We conclude that TNFR I/II and TNF-α participate inEhrlichia-induced shock and host defense by regulating liver injury and controlling ehrlichial burden. Our data suggest that fatal ehrlichiosis could be a multistep process, where TNF-α is not solely responsible for mortality.
Two Monoclonal Antibodies with Defined Epitopes of P44 Major Surface Proteins NeutralizeAnaplasma phagocytophilumby Distinct Mechanisms
Authors: Wang Xueqi , Kikuchi Takane , Rikihisa Yasuko
Published: February 2006

Anaplasma phagocytophilumis an obligatory intracellular bacterium that causes human granulocytic anaplasmosis. The polymorphic 44-kDa major outer membrane proteins ofA. phagocytophilumare dominant antigens recognized by patients and infected animals. However, the ability of anti-P44 antibody to neutralize the infection has been unclear due to a mixture of P44 proteins with diverse hypervariable region amino acid sequences expressed by a given bacterial population and lack of epitope-defined antibodies. Monoclonal antibodies (MAbs) 5C11 and 3E65 are directed to different domains of P44 proteins, the N-terminal conserved region and P44-18 central hypervariable region, respectively. Passive immunization with either MAb 5C11 or 3E65 partially protects mice from infection withA. phagocytophilum. In the present study, we demonstrated that the two monoclonal antibodies recognize bacterial surface-exposed epitopes of naturally folded P44 proteins and mapped these epitopes to specific peptide sequences. The two MAbs almost completely blocked the infection of theA. phagocytophilumpopulation that predominantly expressed P44-18 in HL-60 cells by distinct mechanisms: MAb 5C11 blocked the binding, but MAb 3E65 did not block binding or internalization. Instead, MAb 3E65 inhibited internalizedA. phagocytophilumto develop into microcolonies called morulae. Some plasma from experimentally infected horses and mice reacted with these two epitopes. Taken together, these data indicate the presence of at least two distinct bacterial surface-exposed neutralization epitopes in P44 proteins. The results indicate that antibodies directed to certain epitopes of P44 proteins have a critical role in inhibitingA. phagocytophiluminfection of host cells.
Hypophysectomy and Neurointermediate Pituitary Lobectomy Reduce Serum Immunoglobulin M (IgM) and IgG and Intestinal IgA Responses toSalmonella entericaSerovar Typhimurium Infection in Rats
Authors: Campos-Rodríguez Rafael , Quintanar-Stephano Andrés , Jarillo-Luna Rosa Adriana , Oliver-Aguillón Gabriela , Ventura-Juárez Javier , Rivera-Aguilar Victor , Berczi Istvan , Kovacs Kalman
Published: February 2006

The influence of anterior pituitary hormones on the gastrointestinal tract of humans and animals has been reported. Hypophysectomy (HYPOX) in the rat causes atrophy of the intestinal mucosa, reduction of gastric secretion and intestinal absorption, and increased susceptibility to infections. To our knowledge, there are no studies on the humoral immune response of the gut-associated lymphoid tissue after HYPOX. We have reported that decreased secretion of vasopressin and oxytocin due to neurointermediate pituitary lobectomy (NIL) diminishes humoral and cell-mediated immune responses. However, no data have been published on whether NIL can affect intestinal immune responses. We analyzed the effects of HYPOX and NIL on bacterial colonization of the intestinal lumen, Peyer's patches, and spleen as well as the serum immunoglobulin G (IgG) and IgM and specific intestinal IgA levels in response toSalmonella entericaserovar Typhimurium oral infection. Results showed the following: (i)Salmonellaserovar Typhimurium was eliminated from the intestinal lumen at the same rate in rats that underwent a sham operation, HYPOX, and NIL; (ii)Salmonellaserovar Typhimurium colonization of Peyer's patches and spleen was significantly higher in both HYPOX and NIL rats than in sham-operated rats; (iii) serum IgG and IgM and intestinal IgA against surface proteins ofSalmonellaserovar Typhimurium were significantly lower in HYPOX and NIL rats than in sham-operated rats; and (iv) compared to NIL rats, higher Peyer's patch and spleen bacterial colonization and decreased IgG, IgM, and IgA production were observed in HYPOX rats. We conclude that hormones from each pituitary lobe affect the systemic and gastrointestinal humoral immune responses through different mechanisms.