bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional Control of the
rhuIR-bhuRSTUV
Heme Acquisition Locus in
Bordetella avium
Authors: King Natalie D. , Kirby Amy E. , Connell Terry D. 
Published: February 2005

ABSTRACT

Iron (Fe) is an essential nutrient for most bacterial pathogens. In these organisms, a variety of regulatory systems that respond to specific Fe complexes found within their vertebrate hosts have evolved. In
Bordetella avium
, the heme utilization locus encoded by
rhuIR-bhuRSTUV
mediates efficient acquisition of Fe from heme and hemoproteins. Control of
bhuRSTUV
expression is promulgated at two levels. When Fe is abundant, expression is repressed in a Fur-dependent manner which is partially relieved when Fe is limiting. In the presence of heme or hemoproteins, expression of the
bhuRSTUV
operon is induced via a three-component signal transduction cascade composed of RhuI, RhuR, and BhuR. Herein, we report the identification of two promoters (P

rhuI

and P

bhuR

) that control expression of the
rhuIR-bhuRSTUV
cluster. Primer extension analysis identified the transcriptional start site of P

rhuI

within a putative Fur box. Transcriptional initiation of P

bhuR

mapped within the
rhuR-bhuR
intergenic region. Maximal transcription from P

bhuR

required Fe-limiting conditions, the presence of heme (or hemoglobin), and
rhuI
; however, analysis of transcripts produced from the
rhuIR-bhuRSTUV
locus revealed a pattern of low-level
bhuR
transcription in the absence of heme which originated from both P

bhuR

and P

rhuI

. Transcription from P

rhuI

was repressed by Fe in the presence of
fur
and somewhat enhanced by the addition of hemin to Fe-limited media. The nature of this hemin-associated P

rhuI

stimulation was
rhuI
independent and therefore not induced by heme via the BhuR-RhuR-RhuI signal cascade. Fe also repressed transcription from P

bhuR

in a
fur
-dependent manner; however, activation from this promoter, in the presence or absence of heme, did not occur without
rhuI
.
Interactions of OspA Monoclonal Antibody C3.78 withBorrelia burgdorferiwithin Ticks
Authors: Gipson Clay L. , de Silva Aravinda M. 
Published: February 2005

ABSTRACTTheBorrelia burgdorferiouter surface protein A (OspA) vaccine induces antibodies that prevent transmission from the tick to the host. Here we describe studies with an OspA monoclonal antibody (C3.78) to understand the mechanism by which antibodies entering the tick blockBorreliatransmission. Host complement in the tick's blood meal did not contribute to protection because the antibody was equally effective whether infected ticks fed on normal or complement-deficient mice. Antibody-mediated cross-linking of bacteria or cross-linking of OspA molecules was not required for protection because C3.78 Fab′ fragments were as effective as whole antibody molecules. At low C3.78 concentrations, transmission was blocked despite the presence of many live spirochetes within the tick, confirming that clearance ofBorreliaorganisms was not required to block transmission. We propose that OspA antibody binding to the surface of spirochetes blocks transmission by a mechanism that does not require bacterial killing.
Role of the Type III Secreted Exoenzymes S, T, and Y in Systemic Spread of
Pseudomonas aeruginosa
PAO1 In Vivo
Authors: Vance Russell E. , Rietsch Arne , Mekalanos John J. 
Published: February 2005

ABSTRACT

Pseudomonas aeruginosa
uses a dedicated type III secretion system to deliver toxins directly into the cytoplasm of host cells. While progress has been made in elucidating the function of type III-secreted toxins in vitro, the in vivo functions of the type III-secreted exoenzymes are less well understood, particularly for the sequenced strain PAO1. Therefore, we have systematically deleted the genes for the three known type III effector molecules (
exoS
,
exoT
, and
exoY
) in
P. aeruginosa
PAO1 and assayed the effect of the deletions, both singly and in combination, on cytotoxicity in vitro and in vivo. We found that the type III secretion system acts differently on different cell types, causing an
exoST
-dependent rounding of a lung epithelial-like cell line in contrast to causing an
exoSTY
-independent but translocase (
popB
)-dependent lysis of a macrophage cell line. We utilized an in vivo competitive infection model to test each of our mutants, examining replication in the lung and spread to secondary sites such as the blood and spleen. Type III mutants inoculated intranasally exhibited only a minor defect in replication and survival in the lung, but
popB
and
exoSTY
triple mutants were profoundly defective in their ability to spread systemically. Intravenous injection of the mutants indicated that the type III secretion machinery is required for survival in the blood. Furthermore, our findings suggest that the effector-independent
popB
-dependent cytotoxicity that we and others have observed in vitro in macrophage cell lines may not be of great importance in vivo.
Plasmodium falciparumMalaria: Reduction of Endothelial Cell Apoptosis In Vitro
Authors: Hemmer Christoph Josef , Lehr Hans Anton , Westphal Kathi , Unverricht Marcus , Kratzius Manja , Reisinger Emil Christian 
Published: February 2005

ABSTRACTOrgan failure inPlasmodium falciparummalaria is associated with neutrophil activation and endothelial damage. This study investigates whether neutrophil-induced endothelial damage involves apoptosis and whether it can be prevented by neutralization of neutrophil secretory products. Endothelial cells from human umbilical veins were coincubated with neutrophils from healthy donors and with sera from eight patients withP. falciparummalaria, three patients withP. vivaxmalaria, and three healthy controls. Endothelial apoptosis was demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and annexin V staining. The rate of apoptosis of cells was markedly increased after incubation with patient serum compared to that with control serum. Apoptosis was most pronounced after incubation with sera from two patients with fatal cases ofP. falciparummalaria, followed by sera of survivors with severeP. falciparummalaria and, finally, by sera of patients with mildP. falciparumandP. vivaxmalaria. Ascorbic acid, tocopherol, and ulinastatin reduced the apoptosis rate, but gabexate mesilate and pentoxifylline did not. Furthermore, in fatalP. falciparummalaria, apoptotic endothelial cells were identified in renal and pulmonary tissue by TUNEL staining. These findings show that apoptosis caused by neutrophil secretory products plays a major role in endothelial cell damage in malaria. The antioxidants ascorbic acid and tocopherol and the protease inhibitor ulinastatin can reduce malaria-associated endothelial apoptosis in vitro.
Phenotypic Switching ofCryptococcus neoformansCan Produce Variants That Elicit Increased Intracranial Pressure in a Rat Model of Cryptococcal Meningoencephalitis
Authors: Fries B. C. , Lee S. C. , Kennan R. , Zhao W. , Casadevall A. , Goldman D. L. 
Published: February 2005

ABSTRACTIncreased intracranial pressure (ICP) plays an important role in the morbidity and mortality of cryptococcal meningoencephalitis. The microbial and host factors that contribute to the development of increased ICP are poorly understood. We found that phenotypic switch variants ofCryptococcus neoformans(smooth and mucoid) differed in their abilities to promote increased ICP in a rat model of cryptococcal meningitis. Rats infected with the mucoid variant developed increased ICP, whereas rats infected with the smooth parent did not. This trend correlated with a shorter survival time and a higher cerebrospinal fluid (CSF) fungal burden for mucoid variant-infected rats, although brain fungal burdens were comparable between mucoid variant- and smooth parent-infected rats. Magnetic resonance imaging revealed enhanced T2 signal intensity over the surfaces of the brains of mucoid variant-infected rats. In addition, more polysaccharide accumulated in the CSF and brains of mucoid variant-infected rats. The accumulation of glucorunoxylomannan was associated with elevated levels of MCP-1 (CCL2) and, accordingly, a more pronounced but ineffective monocytic inflammatory response in the meninges of mucoid variant-infected rats. In summary, these findings suggest that strain-specific characteristics can influence the development of increased ICP and indicate a manner in which phenotypic switching could influence the outcome of a central nervous system infection.
The Gamma Interferon Receptor Is Required for the Protective Pulmonary Inflammatory Response toCryptococcus neoformans
Authors: Chen Gwo-Hsiao , McDonald Roderick A. , Wells Jason C. , Huffnagle Gary B. , Lukacs Nicholas W. , Toews Galen B. 
Published: February 2005

ABSTRACTMice with a null deletion mutation in the gamma interferon (IFN-γ) receptor gene were used to study the role of IFN-γ responsiveness during experimental pulmonary cryptococcosis.Cryptococcus neoformanswas inoculated intratracheally into mice lacking the IFN-γ receptor gene (IFN-γR−/−) and into control mice (IFN-γR+/+). The numbers of CFU in lung, spleen, and brain were determined to assess clearance; cytokines produced by lung leukocytes were measured, and survival curves were generated. In the present study, we demonstrate the following points. (i) IFN-γR−/−mice are markedly more susceptible toC. neoformansinfection than IFN-γR+/+mice. (ii) In the absence of IFN-γ signaling, pulmonary CFU continue to increase over the course of infection, and the infection disseminates to the brain. (iii) In the absence of IFN-γ receptor, recruitment of inflammatory cells in response to pulmonary cryptococcal infection is not impaired. (iv) At week 5 postinfection, IFN-γR−/−mice have recruited greater numbers of leukocytes into their lungs, with neutrophils, eosinophils, and lymphocytes accounting for this cellular increase. (v) IFN-γ signaling is required for the development of a T1 over a T2 immune response in the lung following cryptococcal infection. These results indicate that in the absence of IFN- γ responsiveness, even though the recruitment of pulmonary inflammatory cells is not impaired and the secretion of IFN-γ is not affected, IFN-γR−/−mice do not have the ability to resolve the cryptococcal infection. In conclusion, our data suggest that proper functional IFN-γ signaling, possibly through a mechanism which inhibits the potentially disease-promoting T2 response, is required for mice to confine the cryptococcal infection.
Use of Genome-Wide Expression Profiling and Mutagenesis To Study the Intestinal Lifestyle of
Campylobacter jejuni
Authors: Stintzi Alain , Marlow Denver , Palyada Kiran , Naikare Hemant , Panciera Roger , Whitworth Lisa , Clarke Cyril 
Published: February 2005

ABSTRACT

Campylobacter jejuni
is the most common bacterial cause of diarrhea worldwide. To colonize the gut and cause infection,
C. jejuni
must successfully compete with endogenous microbes for nutrients, resist host defenses, persist in the intestine, and ultimately infect the host. These challenges require the expression of a battery of colonization and virulence determinants. In this study, the intestinal lifestyle of
C. jejuni
was studied using whole-genome microarray, mutagenesis, and a rabbit ileal loop model. Genes associated with a wide range of metabolic, morphological, and pathological processes were expressed in vivo. The in vivo transcriptome of
C. jejuni
reflected its oxygen-limited, nutrient-poor, and hyperosmotic environment. Strikingly, the expression of several
C. jejuni
genes was found to be highly variable between individual rabbits. In particular, differential gene expression suggested that
C. jejuni
extensively remodels its envelope in vivo by differentially expressing its membrane proteins and by modifying its peptidoglycan and glycosylation composition. Furthermore, mutational analysis of seven genes,
hspR
,
hrcA
,
spoT
, Cj0571, Cj0178, Cj0341, and
fliD
, revealed an important role for the stringent and heat shock response in gut colonization. Overall, this study provides new insights on the mechanisms of gut colonization, as well as possible strategies employed by
Campylobacter
to resist or evade the host immune responses.
Biofilm Formation,
icaADBC
Transcription, and Polysaccharide Intercellular Adhesin Synthesis by Staphylococci in a Device-Related Infection Model
Authors: Fluckiger Ursula , Ulrich Martina , Steinhuber Andrea , Döring Gerd , Mack Dietrich , Landmann Regine , Goerke Christiane , Wolz Christiane 
Published: February 2005

ABSTRACT

Biofilm formation of
Staphylococcus epidermidis
and
S. aureus
is mediated by the polysaccharide intercellular adhesin (PIA) encoded by the
ica
operon. We used a device-related animal model to investigate biofilm formation, PIA expression (immunofluorescence), and
ica
transcription (quantitative transcript analysis) throughout the course of infection by using two prototypic
S. aureus
strains and one
S. epidermidis
strain as well as corresponding
ica
mutants. During infection, the
ica
mutants were growth attenuated when inoculated in competition with the corresponding wild-type strains but not when grown singly. A typical biofilm was observed at the late course of infection. Only in
S. aureus
RN6390, not in
S. aureus
Newman, were PIA and
ica-
specific transcripts detectable after anaerobic growth in vitro. However, both
S. aureus
strains were PIA positive in vivo by day 8 of infection.
ica
transcription preceded PIA expression and biofilm formation in vivo. In
S. epidermidis
, both PIA and
ica
expression levels were elevated compared to those in the
S. aureus
strains in vitro as well as in vivo and were detectable throughout the course of infection. In conclusion, in
S. aureus
, PIA expression is dependent on the genetic background of the strain as well as on strong inducing conditions, such as those dominating in vivo. In
S. epidermidis
, PIA expression is elevated and less vulnerable to environmental conditions.
Both CD1d Antigen Presentation and Interleukin-12 Are Required To Activate Natural Killer T Cells duringTrypanosoma cruziInfection
Authors: Duthie Malcolm S. , Kahn Maria , White Maria , Kapur Raj P. , Kahn Stuart J. 
Published: February 2005

ABSTRACTMechanisms of natural killer T (NKT)-cell activation remain unclear. Here, we report that duringTrypanosoma cruziinfection, interleukin-12 (IL-12) deficiency or anti-CD1d antibody treatment prevents normal activation. The required IL-12 arises independently of MyD88. The data support a model of normal NKT-cell activation that requires IL-12 and TCR stimulation.
Signature for Long-Term Vaccine-Mediated Control of a Simian and Human Immunodeficiency Virus 89.6P Challenge: Stable Low-Breadth and Low-Frequency T-Cell Response Capable of Coproducing Gamma Interferon and Interleukin-2
Authors: Sadagopal Shanmugalakshmi , Amara Rama Rao , Montefiori David C. , Wyatt Linda S. , Staprans Silvija I. , Kozyr Natalia L. , McClure Harold M. , Moss Bernard , Robinson Harriet L. 
Published: February 2005

ABSTRACTIn 2001, we reported 20 weeks of control of challenge with the virulent 89.6P chimera of simian and human immunodeficiency viruses (SHIV-89.6P) by a Gag-Pol-Env vaccine consisting of DNA priming and modified vaccinia virus Ankara boosting. Here we report that 22 out of 23 of these animals successfully controlled their viremia until their time of euthanasia at 200 weeks postchallenge. At euthanasia, all animals had low to undetectable viral loads and normal CD4 counts. During the long period of viral control, gamma interferon (IFN-γ)-producing antiviral T cells were present at unexpectedly low breadths and frequencies. Most animals recognized two CD8 and one CD4 epitope and had frequencies of IFN-γ-responding T cells from 0.01 to 0.3% of total CD8 or CD4 T cells. T-cell responses were remarkably stable over time and, unlike responses in most immunodeficiency virus infections, maintained good functional characteristics, as evidenced by coproduction of IFN-γ and interleukin-2. Overall, high titers of binding and neutralizing antibody persisted throughout the postchallenge period. Encouragingly, long-term control was effective in macaques of diverse histocompatibility types.
Functional Interaction of Heterogeneous Nuclear Ribonucleoprotein C with Poliovirus RNA Synthesis Initiation Complexes
Authors: Brunner Jo Ellen , Nguyen Joseph H. C. , Roehl Holger H. , Ho Tri V. , Swiderek Kristine M. , Semler Bert L. 
Published: February 2005

ABSTRACT

We had previously demonstrated that a cellular protein specifically interacts with the 3′ end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione
S
-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D
pol
, as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1ΔC) diminished these protein-protein interactions. When GST/hnRNP C1ΔC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.
Single-Amino-Acid Substitutions in Open Reading Frame (ORF) 1b-nsp14 and ORF 2a Proteins of the Coronavirus Mouse Hepatitis Virus Are Attenuating in Mice
Authors: Sperry Steven M. , Kazi Lubna , Graham Rachel L. , Baric Ralph S. , Weiss Susan R. , Denison Mark R. 
Published: February 2005

ABSTRACT

A reverse genetic system was recently established for the coronavirus mouse hepatitis virus strain A59 (MHV-A59), in which cDNA fragments of the RNA genome are assembled in vitro into a full-length genome cDNA, followed by electroporation of in vitro-transcribed genome RNA into cells with recovery of viable virus. The “in vitro-assembled” wild-type MHV-A59 virus (icMHV-A59) demonstrated replication identical to laboratory strains of MHV-A59 in tissue culture; however, icMHV-A59 was avirulent following intracranial inoculation of C57BL/6 mice. Sequencing of the cloned genome cDNA fragments identified two single-nucleotide mutations in cloned genome fragment F, encoding a Tyr6398His substitution in open reading frame (ORF) 1b p59-nsp14 and a Leu94Pro substitution in the ORF 2a 30-kDa protein. The mutations were repaired individually and together in recombinant viruses, all of which demonstrated wild-type replication in tissue culture. Following intracranial inoculation of mice, the viruses encoding Tyr6398His/Leu94Pro substitutions and the Tyr6398His substitution alone demonstrated log
10
50% lethal dose (LD
50
) values too great to be measured. The Leu94Pro mutant virus had reduced but measurable log
10
LD
50
, and the “corrected” Tyr6398/Leu94 virus had a log
10
LD
50
identical to wild-type MHV-A59. The experiments have defined residues in ORF 1b and ORF 2a that attenuate virus replication and virulence in mice but do not affect in vitro replication. The results suggest that these proteins serve roles in pathogenesis or virus survival in vivo distinct from functions in virus replication. The study also demonstrates the usefulness of the reverse genetic system to confirm the role of residues or proteins in coronavirus replication and pathogenesis.
Architecture of Replication Compartments Formed during Epstein-Barr Virus Lytic Replication
Authors: Daikoku Tohru , Kudoh Ayumi , Fujita Masatoshi , Sugaya Yutaka , Isomura Hiroki , Shirata Noriko , Tsurumi Tatsuya 
Published: February 2005

ABSTRACT

Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1
oriLyt
binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.