bluuebunny/crossref_metadata_2025_split huggingface dataset:
Temperature-Regulated Microcolony Formation byBurkholderia pseudomalleiRequirespilAand Enhances Association with Cultured Human Cells
Authors: Boddey Justin A. , Flegg Cameron P. , Day Chris J. , Beacham Ifor R. , Peak Ian R. 
Published: August 2006

ABSTRACTBurkholderia pseudomalleiis the causative agent of melioidosis, a potentially fatal disease that is endemic to Northern Australia and Southeast Asia and is acquired from soil or water. Adherence ofB. pseudomallei08 to cultured cells increases dramatically following prior growth at 30°C or less compared to that following prior growth at 37°C. Here, we show that this occurs almost entirely as the result of microcolony formation (bacterium-bacterium interactions) following growth at 27°C but not at 37°C, which considerably enhances bacterial association with eukaryotic cells. Further, we demonstrate that the type IVA pilin-encoding gene,pilA, is essential for microcolony development byB. pseudomallei08, and thus optimum association with eukaryotic cells, but is not required for direct adherence (bacterium-cell interactions). In contrast, although theB. pseudomalleigenome sequence strain, K96243, also contains transcriptionally activepilA, microcolony formation rarely occurs following growth at either 27°C or 37°C and cell association occurs significantly less than with strain 08. Analysis ofpilAtranscription in 08 identified thatpilAis dramatically upregulated under microcolony-forming conditions, viz., growth at low temperature, and association with eukaryotic cells; the pattern of transcription ofpilAin K96243 differed from that in 08. Our study also suggests that biofilm formation byB. pseudomallei08 and K96243 on polyvinylchloride is not mediated bypilA. Adherence and microcolony formation, andpilAtranscription, vary between strains, consistent with known genomic variation inB. pseudomallei, and these phenotypes may be relevant to colonization from the environment.
Staphylococcus aureus
-Induced Corneal Inflammation Is Dependent on Toll-Like Receptor 2 and Myeloid Differentiation Factor 88
Authors: Sun Yan , Hise Amy G. , Kalsow Carolyn M. , Pearlman Eric 
Published: August 2006

ABSTRACT

Toll-like receptors (TLRs) expressed by the corneal epithelium represent a first line of host defense to microbial keratitis. The current study examined the role of TLR2, TLR4, and TLR9 and the common adaptor molecule myeloid differentiation factor 88 (MyD88) in a
Staphylococcus aureus
model of corneal inflammation. The corneal epithelia of C57BL/6, TLR2
−/−
, TLR4
−/−
, TLR9
−/−
, and MyD88
−/−
mice were abraded using a trephine and epithelial brush and were exposed to heat- or UV-inactivated
S. aureus
clinical strain 8325-4 and other clinical isolates. Corneal thickness and haze were measured by in vivo confocal microscopy, neutrophil recruitment to the corneal stroma was quantified by immunohistochemistry, and cytokine production was measured by enzyme-linked immunosorbent assay. The exposure of corneal epithelium to
S. aureus
induced neutrophil recruitment to the corneal stroma and increased corneal thickness and haze in control C57BL/6 mice but not in TLR2
−/−
or MyD88
−/−
mice. The responses of TLR4
−/−
and TLR9
−/−
mice were similar to those of C57BL/6 mice.
S. aureus
-induced cytokine production by corneal epithelial cells and neutrophils was also significantly reduced in TLR2
−/−
mice compared with that in C57BL/6 mice. These findings indicate that
S. aureus
-induced corneal inflammation is mediated by TLR2 and MyD88 in resident epithelial cells and infiltrating neutrophils.
Bartonella quintana
Variably Expressed Outer Membrane Proteins Mediate Vascular Endothelial Growth Factor Secretion but Not Host Cell Adherence
Authors: Schulte Berit , Linke Dirk , Klumpp Sandra , Schaller Martin , Riess Tanja , Autenrieth Ingo B. , Kempf Volkhard A. J. 
Published: August 2006

ABSTRACT

Bartonella quintana
causes trench fever, endocarditis, and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis in humans. Little is known about the interaction of this pathogen with host cells. We attempted to elucidate the interaction of
B. quintana
with human macrophages (THP-1) and epithelial cells (HeLa 229). Remarkably, only
B. quintana
strain JK-31 induced secretion of vascular endothelial growth factor (VEGF) from THP-1 and HeLa 229 cells upon infection similar to the secretion induced by
B. henselae
Marseille, whereas other strains (
B. quintana
2-D70,
B. quintana
Toulouse, and
B. quintana
Munich) did not induce such secretion. Immunofluorescence testing and electron microscopy revealed that the
B. quintana
strains unable to induce VEGF secretion did not express the variable outer membrane proteins (Vomps) on their surfaces. Surprisingly, the increase in VEGF secretion mediated by
B. quintana
JK-31 was not paralleled by elevated host cell adherence rates compared with the rates for Vomp-negative
B. quintana
strains. Our results suggest that the Vomps play a leading role in the angiogenic reprogramming of host cells by
B. quintana
but not in the adherence to host cells.
Constitutive Expression of Outer Surface Protein C Diminishes the Ability ofBorrelia burgdorferiTo Evade Specific Humoral Immunity
Authors: Xu Qilong , Seemanapalli Sunita V. , McShan Kristy , Liang Fang Ting 
Published: August 2006

ABSTRACTThe Lyme disease spirocheteBorrelia burgdorferireduces the expression of outer surface protein C (OspC) in response to the development of an anti-OspC humoral response, leading to the hypothesis that the ability to repress OspC expression is critical for the pathogen to proceed to chronic infection.B. burgdorferiwas genetically modified to constitutively express OspC by introducing an extraospCcopy fused with the borrelial flagellar gene (flaB) promoter. Such a genetic modification did not reduce infectivity or pathogenicity in severe combined immunodeficiency mice but resulted in clearance of infection by passively transferred OspC antibody. Spirochetes with constitutiveospCexpression were unable to establish chronic infections in immunocompetent mice unless they had undergone very destructive mutations in the introducedospCcopy. Two escape mutants were identified; one had all 7 bp deleted between the putative ribosome-binding site and the start codon, ATG, causing a failure in translational initiation, and the other mutant had an insertion of 2 bp between nucleotides 315 and 316, resulting in a nonsense mutation at codon 108. Thus, the ability ofB. burgdorferito repressospCexpression during mammalian infection allows the pathogen to avoid clearance and to preserve the integrity of the important gene for subsequent utilization during its enzootic life cycle.
Characterization of the Receptor-Ligand Pathways Important for Entry and Survival ofFrancisella tularensisin Human Macrophages
Authors: Balagopal Ashwin , MacFarlane Amanda Shearer , Mohapatra Nrusingh , Soni Shilpa , Gunn John S. , Schlesinger Larry S. 
Published: August 2006

ABSTRACTInhalational pneumonic tularemia, caused byFrancisella tularensis, is lethal in humans.F. tularensisis phagocytosed by macrophages followed by escape from phagosomes into the cytoplasm. Little is known of the phagocytic mechanisms forFrancisella, particularly as they relate to the lung and alveolar macrophages. Here we examined receptors on primary human monocytes and macrophages which mediate the phagocytosis and intracellular survival ofF. novicida. F. novicidaassociation with monocyte-derived macrophages (MDM) was greater than with monocytes. Bacteria were readily ingested, as shown by electron microscopy. Bacterial association was significantly increased in fresh serum and only partially decreased in heat-inactivated serum. A role for both complement receptor 3 (CR3) and Fcγ receptors in uptake was supported by studies using a CR3-expressing cell line and by down-modulation of Fcγ receptors on MDM, respectively. Consistent with Fcγ receptor involvement, antibody in nonimmune human serum was detected on the surface ofFrancisella. In the absence of serum opsonins, competitive inhibition of mannose receptor (MR) activity on MDM with mannan decreased the association ofF. novicidaand opsonization ofF. novicidawith lung collectin surfactant protein A (SP-A) increased bacterial association and intracellular survival. This study demonstrates that human macrophages phagocytose moreFrancisellathan monocytes with contributions from CR3, Fcγ receptors, the MR, and SP-A present in lung alveoli.
Vaccinations with Recombinant Variants of
Aspergillus fumigatus
Allergen Asp f 3 Protect Mice against Invasive Aspergillosis
Authors: Ito James I. , Lyons Joseph M. , Hong Teresa B. , Tamae Daniel , Liu Yi-Kuang , Wilczynski Sharon P. , Kalkum Markus 
Published: August 2006

ABSTRACT

A vaccine that effectively protects immunocompromised patients against invasive aspergillosis is a novel approach to a universally fatal disease. Here we present a rationale for selection and in vivo testing of potential protein vaccine candidates, based on the modification of an immunodominant fungal allergen for which we demonstrate immunoprotective properties. Pulmonary exposure to viable
Aspergillus fumigatus
conidia as well as vaccination with crude hyphal extracts protects corticosteroid-immunosuppressed mice against invasive aspergillosis (J. I. Ito and J. M. Lyons, J. Infect. Dis. 186:869-871, 2002). Sera from the latter animals contain antibodies with numerous and diverse antigen specificities, whereas sera from conidium-exposed mice contain antibodies predominantly against allergen Asp f 3 (and some against Asp f 1), as identified by mass spectrometry. Subcutaneous immunization with recombinant Asp f 3 (rAsp f 3) but not with Asp f 1 was protective. The lungs of Asp f 3-vaccinated survivors were free of hyphae and showed only a patchy low-density infiltrate of mononuclear cells. In contrast, the nonimmunized animals died with invasive hyphal elements and a compact peribronchial infiltrate of predominately polymorphonuclear leukocytes. Three truncated versions of rAsp f 3, spanning amino acid residues 15 to 168 [rAsp f 3(15-168)], 1 to 142, and 15 to 142 and lacking the known bipartite sequence required for IgE binding, were also shown to be protective. Remarkably, vaccination with either rAsp f 3(1-142) or rAsp f 3(15-168) drastically diminished the production of antigen-specific antibodies compared to vaccination with the full-length rAsp f 3(1-168) or the double-truncated rAsp f 3(15-142) version. Our findings point to a possible mechanism in which Asp f 3 vaccination induces a cellular immune response that upon infection results in the activation of lymphocytes that in turn enhances and/or restores the function of corticosteroid-suppressed macrophages to clear fungal elements in the lungs.
Campylobacter jejuniColonization of Mice with Limited Enteric Flora
Authors: Chang Christopher , Miller Jeff F. 
Published: August 2006

ABSTRACTWe have developed experimental murineCampylobacterinfection models which demonstrate efficient establishment and reproducible, high-level colonization. Following oral inoculation, wild-type C3H mice with normal enteric flora were colonized inconsistently and inefficiently byC. jejunistrain 81-176. However, C3H mice with a limited gut flora (LF) were efficiently colonized at high levels (108CFU/g of stool or large intestine tissue) followed by clearance after several weeks. Large intestine tissue showed minimal to mild inflammation at days 7 and 28 postinoculation. In striking contrast, C3H SCID mice with the same limited flora remained persistently colonized at a consistently high level until they were euthanized 8 months postinoculation. Lower gastrointestinal tract tissue from LF-SCID mice showed marked to severe inflammation in the colon and cecum at days 7 and 28 and intense inflammation of the stomach at day 28. These findings indicate that although the innate response alone cannot block colonization persistence, it is sufficient to orchestrate marked gut inflammation. Moreover, the adaptive immune response is critical to mediateC. jejuniclearance from the colonized gut. To validate our LF murine model, we verified that motility and chemotaxis are critical for colonization. Insertion-deletion mutations were generated inmotBandfliI, which encode products essential for motility and flagellar assembly, and in the presumptive chemotaxis genecheA(histidine kinase). All mutants failed to establish colonization in LF mice. Our limited flora murine colonization models serve as tractable, reproducible tools to define host responses toC. jejuniinfection and to identify and characterize virulence determinants required for colonization.
Mechanisms of Mortality in Early and Late Sepsis
Authors: Xiao Hongyan , Siddiqui Javed , Remick Daniel G. 
Published: August 2006

ABSTRACT
A recent hypothesis postulates that sepsis moves through different phases, with periods of enhanced inflammation alternating with periods of immune suppression. In this study we determined the levels of inflammation present during early and late septic deaths to examine whether death was due to hyperinflammation or immunosuppression. The murine model of sepsis induced by cecal ligation and puncture (CLP) was used. Complete blood counts, plasma interleukin-6 (IL-6) levels, and body weights were determined. Mice that died within the first 4 days had increased plasma levels of IL-6, indicating that there was activation of the immune system. Cecal resection on day 4 after CLP resulted in decreased abscess size, lower circulating neutrophil counts, decreased anemia, and improved survival compared to the results for mice that received only antibiotic and fluid therapy. All of the mice that died in the chronic phase of infection (after day 4) had positive peritoneal cultures containing significantly more bacteria than the cultures for surviving mice. After day 4, none of the surviving mice exhibited increases in the plasma levels of IL-6. Dying mice exhibited mixed IL-6 responses; for 41% of the mice there was never an increase in the IL-6 levels in the chronic phase, while for other mice the levels of IL-6 transiently increased prior to death. Peritoneal macrophages were obtained in the late phase of sepsis from moribund and healthy mice and were stimulated ex vivo. The cells from the moribund mice produced significantly less IL-6 than the cells obtained from healthy mice produced. These results indicate that in mice that die in the early phase there is uniformly increased inflammation. However, during the chronic phase of sepsis, some mice die with evidence of immunosuppression (increased bacterial growth and low IL-6 levels), while other mice die with immunostimulation (high IL-6 levels and bacterial growth). Determining the inflammatory status of individual patients may help guide appropriate, targeted therapy.
Intestinal Intraepithelial Lymphocytes Sustain the Epithelial Barrier Function againstEimeria vermiformisInfection
Authors: Inagaki-Ohara Kyoko , Dewi Fitriya Nurannisa , Hisaeda Hajime , Smith Adrian L. , Jimi Fumiko , Miyahira Maki , Abdel-Aleem Ayman Samir Farid , Horii Yoichiro , Nawa Yukifumi 
Published: August 2006

ABSTRACTEimeriaspp. are intracellular protozoa that infect intestinal epithelia of most vertebrates, causing coccidiosis. Intestinal intraepithelial lymphocytes (IEL) that reside at the basolateral site of epithelial cells (EC) have immunoregulatory and immunoprotective roles againstEimeriaspp. infection. However, it remains unknown how IEL are involved in the regulation of epithelial barrier duringEimeriasp. infection. Here, we demonstrated two distinct roles of IEL against infection withEimeria vermiformis, a murine pathogen: production of cytokines to induce protective immunity and expression of junctional molecules to preserve epithelial barrier. The number of IEL markedly increased when oocyst production reached a peak. During infection, IEL increased production of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and decreased transforming growth factor β (TGF-β) production. Addition of IFN-γ and TNF-α or supernatants obtained from cultured IEL fromE. vermiformis-infected mice reduced transepithelial electrical resistance (TER) in a confluent CMT93 cell monolayer, a murine intestine-derived epithelial line, but antibodies against these cytokines suppressed the decline of TER. Moreover, TGF-β attenuated the damage of epithelial monolayer and changes in TER caused by IFN-γ and TNF-α. The expression of junctional molecules by EC was decreased when IEL produced a high level of IFN-γ and TNF-α and a low level of TGF-β inE. vermiformis-infected mice. Interestingly, IEL constantly expressed junctional molecules and a coculture of EC with IEL increased TER. These results suggest that IEL play important multifunctional roles not only in protection of the epithelium againstE. vermiformis-induced change by cytokine production but also in direct interaction with the epithelial barrier when intra-EC junctions are down-regulated.
Methanosarcina acetivorans
Flap Endonuclease 1 Activity Is Inhibited by a Cognate Single-Stranded-DNA-Binding Protein
Authors: Lin Yuyen , Guzman Claudia E. , McKinney Mary C. , Nair Satish K. , Ha Taekjip , Cann Isaac K. O. 
Published: August 2006

ABSTRACT

The oligonucleotide/oligosaccharide-binding (OB) fold is central to the architecture of single-stranded- DNA-binding proteins, which are polypeptides essential for diverse cellular processes, including DNA replication, repair, and recombination. In archaea, single-stranded DNA-binding proteins composed of multiple OB folds and a zinc finger domain, in a single polypeptide, have been described. The OB folds of these proteins were more similar to their eukaryotic counterparts than to their bacterial ones. Thus, the archaeal protein is called replication protein A (RPA), as in eukaryotes. Unlike most organisms,
Methanosarcina acetivorans
harbors multiple functional RPA proteins, and it was our interest to determine whether the different proteins play different roles in DNA transactions. Of particular interest was lagging-strand DNA synthesis, where recently RPA has been shown to regulate the size of the 5′ region cleaved during Okazaki fragment processing. We report here that
M. acetivorans
RPA1 (MacRPA1), a protein composed of four OB folds in a single polypeptide, inhibits cleavage of a long flap (20 nucleotides) by
M. acetivorans
flap endonuclease 1 (MacFEN1). To gain a further insight into the requirement of the different regions of MacRPA1 on its inhibition of MacFEN1 endonuclease activity, N-terminal and C-terminal truncated derivatives of the protein were made and were biochemically and biophysically analyzed. Our results suggested that MacRPA1 derivatives with at least three OB folds maintained the properties required for inhibition of MacFEN1 endonuclease activity. Despite these interesting observations, further biochemical and genetic analyses are required to gain a deeper understanding of the physiological implications of our findings.
Regulation of
ppk
Expression and In Vivo Function of Ppk in
Streptomyces lividans
TK24
Authors: Ghorbel Sofiane , Smirnov Aleksey , Chouayekh Hichem , Sperandio Brice , Esnault Catherine , Kormanec Jan , Virolle Marie-Joelle 
Published: August 2006

ABSTRACT

The
ppk
gene of
Streptomyces lividans
encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol.
43:
919-930, 2002). In the present work, some regulatory features of the expression of
ppk
were established and the polyphosphate content of
S. lividans
TK24 and the
ppk
mutant was determined. In P
i
sufficiency, the expression of
ppk
was shown to be low but detectable. DNA gel shift experiments suggested that
ppk
expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the
ppk
mutant strain. The expression of
ppk
under P
i
-limiting conditions was shown to be much higher than that under P
i
-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the
ppk
mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in
S. lividans
TK24 is proposed.
Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis
Authors: Lee Jung-Kul , Ang Ee-Lui , Zhao Huimin 
Published: August 2006

ABSTRACT

Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from
Pseudomonas fluorescens
Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.
Superoxide Dismutase B Gene (sodB)-Deficient Mutants ofFrancisella tularensisDemonstrate Hypersensitivity to Oxidative Stress and Attenuated Virulence
Authors: Bakshi Chandra Shekhar , Malik Meenakshi , Regan Kevin , Melendez J. Andres , Metzger Dennis W. , Pavlov Vitaly M. , Sellati Timothy J. 
Published: August 2006

ABSTRACTAFrancisella tularensislive vaccine strain mutant (sodBFt) with reduced Fe-superoxide dismutase gene expression was generated and found to exhibit decreasedsodBactivity and increased sensitivity to redox cycling compounds compared to wild-type bacteria. ThesodBFtmutant also was significantly attenuated for virulence in mice. Thus, this study has identifiedsodBas an importantF. tularensisvirulence factor.
PBP5 Complementation of a PBP3 Deficiency inEnterococcus hirae
Authors: Leimanis S. , Hoyez N. , Hubert S. , Laschet M. , Sauvage Eric , Brasseur R. , Coyette J. 
Published: August 2006

ABSTRACTThe low susceptibility of enterococci to β-lactams is due to the activity of the low-affinity penicillin-binding protein 5 (PBP5). One important feature of PBP5 is its ability to substitute for most, if not all, penicillin-binding proteins when they are inhibited. That substitution activity was analyzed inEnterococcus hiraeSL2, a mutant whosepbp5gene was interrupted by thenisRKgenes and whose PBP3 synthesis was submitted to nisin induction. Noninduced SL2 cells were unable to divide except when plasmid-bornepbp5genes were present, provided that the PBP5 active site was functional. Potential protein-protein interaction sites of the PBP5 N-terminal module were mutagenized by site-directed mutagenesis. The T167-L184region (designated site D) appeared to be an essential intramolecular site needed for the stability of the protein. Mutations made in the two globular domains present in the N-terminal module indicated that they were needed for the suppletive activity. The P197-N209segment (site E) in one of these domains seemed to be particularly important, as single and double mutations reduced or almost completely abolished, respectively, the action of PBP5.