bluuebunny/crossref_metadata_2025 huggingface dataset:
A Gaseous Metal Disk Around a White Dwarf
Authors: Gänsicke B. T. , Marsh T. R. , Southworth J. , Rebassa-Mansergas A.
Published: December 2006

The destiny of planetary systems through the late evolution of their host stars is very uncertain. We report a metal-rich gas disk around a moderately hot and young white dwarf. A dynamical model of the double-peaked emission lines constrains the outer disk radius to just 1.2 solar radii. The likely origin of the disk is a tidally disrupted asteroid, which has been destabilized from its initial orbit at a distance of more than 1000 solar radii by the interaction with a relatively massive planetesimal object or a planet. The white dwarf mass of 0.77 solar mass implies that planetary systems may form around high-mass stars.
Untemplated Oligoadenylation Promotes Degradation of RISC-Cleaved Transcripts
Authors: Ibrahim Fadia , Rohr Jennifer , Jeong Won-Joong , Hesson Jennifer , Cerutti Heriberto
Published: December 2006

Double-stranded RNA, processed to small interfering RNAs (siRNAs) by Dicer and incorporated into the RNA-induced silencing complex (RISC), triggers gene silencing by a variety of pathways in eukaryotes. RNA interference involving the degradation of homologous transcripts is the best-characterized mechanism. However, the fate of the RNA fragments resulting from siRNA-directed cleavage is poorly understood. We have identified a gene (
MUT68
) in the unicellular green alga
Chlamydomonas reinhardtii
that is required for the efficient decay of siRNA-targeted transcripts.
MUT68
encodes a noncanonical polyadenylate polymerase that adds untemplated adenines to the 5′ RNA fragments after siRNA-mediated cleavage and appears to stimulate their exosome-dependent degradation.
Relating Three-Dimensional Structures to Protein Networks Provides Evolutionary Insights
Authors: Kim Philip M. , Lu Long J. , Xia Yu , Gerstein Mark B.
Published: December 2006

Most studies of protein networks operate on a high level of abstraction, neglecting structural and chemical aspects of each interaction. Here, we characterize interactions by using atomic-resolution information from three-dimensional protein structures. We find that some previously recognized relationships between network topology and genomic features (e.g., hubs tending to be essential proteins) are actually more reflective of a structural quantity, the number of distinct binding interfaces. Subdividing hubs with respect to this quantity provides insight into their evolutionary rate and indicates that additional mechanisms of network growth are active in evolution (beyond effective preferential attachment through gene duplication).
Increase in Nalidixic Acid Resistance among Non-Typhi
Salmonella enterica
Isolates in the United States from 1996 to 2003
Authors: Stevenson Jennifer E. , Gay Kathryn , Barrett Timothy J. , Medalla Felicita , Chiller Tom M. , Angulo Frederick J.
Published: December 2006

Fluoroquinolones commonly are used to treat adult
Salmonella
infections. Fluoroquinolone treatment has failed for persons infected with nalidixic acid-resistant
Salmonella
. From 1996 to 2003, state public health laboratories forwarded 12,252 non-Typhi
Salmonella enterica
isolates to the Centers for Disease Control and Prevention for antimicrobial susceptibility testing; 203 (1.6%) of the isolates were nalidixic acid resistant, and 14 (7%) of those were ciprofloxacin resistant. Resistance to nalidixic acid significantly increased from 0.4% in 1996 to 2.3% in 2003. All ciprofloxacin-resistant isolates had at least one point mutation in the quinolone resistance determining region (QRDR) of
gyrA
and did not harbor
qnr
or have point mutations in the QRDR of
gyrB
,
parC
, or
parE
. Continued surveillance of antimicrobial resistance among non-Typhi
S. enterica
isolates is needed to mitigate the increasing prevalence of nalidixic acid resistance.
Combination of Amphotericin B with Flucytosine Is Active In Vitro against Flucytosine-Resistant Isolates of
Cryptococcus neoformans
Authors: Schwarz Patrick , Janbon Guilhem , Dromer Françoise , Lortholary Olivier , Dannaoui Eric
Published: December 2006

The combination of flucytosine and amphotericin B was tested against 10 flucytosine-resistant isolates of
Cryptococcus neoformans
by checkerboard, killing curves, and Etest. Although differences were observed depending on the technique used, antagonism was never observed. The synergistic interaction was related to the mechanism of flucytosine resistance of the isolates.
High-Level Vancomycin-ResistantStaphylococcus aureusIsolates Associated with a Polymicrobial Biofilm
Authors: Weigel Linda M. , Donlan Rodney M. , Shin Dong Hyeon , Jensen Bette , Clark Nancye C. , McDougal Linda K. , Zhu Wenming , Musser Kimberlee A. , Thompson Jill , Kohlerschmidt Donna , Dumas Nellie , Limberger Ronald J. , Patel Jean B.
Published: December 2006

Glycopeptides such as vancomycin are the treatment of choice for infections due to methicillin-resistantStaphylococcus aureus. This study describes the identification of high-level vancomycin-resistantS. aureus(VRSA) isolates in a polymicrobial biofilm within an indwelling nephrostomy tube in a patient in New York.S. aureus,Enterococcus faecalis,Enterococcus faecium,Micrococcusspecies,Morganella morganii, andPseudomonas aeruginosawere isolated from the biofilm. For VRSA isolates, vancomycin MICs ranged from 32 to >128 μg/ml. VRSA isolates were also resistant to aminoglycosides, fluoroquinolones, macrolides, penicillin, and tetracycline but remained susceptible to chloramphenicol, linezolid, rifampin, and trimethoprim-sulfamethoxazole. ThevanAgene was localized to a plasmid of ∼100 kb in VRSA andE. faeciumisolates from the biofilm. Plasmid analysis revealed that the VRSA isolate acquired the 100-kbE. faeciumplasmid, which was then maintained without integration into the MRSA plasmid. The tetracycline resistance genestet(U) andtet(S), not previously detected inS. aureusisolates, were identified in the VRSA isolates. Additional resistance elements in the VRSA isolate included a multiresistance gene cluster,ermB-aadE-sat4-aphA-3,msrA(macrolide efflux), and the bifunctional aminoglycoside resistance geneaac(6′)-aph(2")-Ia. Multiple combinations of resistance genes among the various isolates of staphylococci and enterococci, includingvanA,tet(S), andtet(U), illustrate the dynamic nature of gene acquisition and loss within and between bacterial species throughout the course of infection. The potential for interspecies transfer of antimicrobial resistance genes, including resistance to vancomycin, may be enhanced by the microenvironment of a biofilm.
Transcriptional Profiles of the Response to Ketoconazole and Amphotericin B in
Trichophyton rubrum
Authors: Yu Lu , Zhang Wenliang , Wang Lingling , Yang Jian , Liu Tao , Peng Junping , Leng Wenchuan , Chen Lihong , Li Ruoyu , Jin Qi
Published: December 2006

Trichophyton rubrum
is a pathogenic filamentous fungus of increasing medical concern. Two antifungal agents, ketoconazole (KTC) and amphotericin B (AMB), have specific activity against dermatophytes. To identify the mechanisms of action of KTC and AMB against
T. rubrum
, a cDNA microarray was constructed from the expressed sequence tags of the cDNA library from different developmental stages, and transcriptional profiles of the responses to KTC and AMB were determined.
T. rubrum
was exposed to subinhibitory concentrations of KTC and AMB for 12 h, and microarray analysis was used to examine gene transcription. KTC exposure induced transcription of genes involved in lipid, fatty acid, and sterol metabolism, including
ERG11
,
ERG3
,
ERG25
,
ERG6
,
ERG26
,
ERG24
,
ERG4
,
CPO
,
INO1
, DW700960,
CPR
, DW696584, DW406350, and
ATG15
. KTC also increased transcription of the multidrug resistance gene
ABC1
. AMB exposure increased transcription of genes involved in lipid, fatty acid, and sterol metabolism (DW696584, EB801458,
IVD
, DW694010, DW688343, DW684992), membrane transport (
Git1
, DW706156, DW684040,
DMT
, DW406136,
CCH1
, DW710650), and stress-related responses (
HSP70
,
HSP104
,
GSS
,
AOX
, EB801455, EB801702,
TDH1
,
UBI4
) but reduced transcription of genes involved in maintenance of cell wall integrity and signal transduction pathways (
FKS1
,
SUN4
, DW699324,
GAS1
, DW681613,
SPS1
, DW703091,
STE7
, DW703091, DW695308) and some ribosomal proteins. This is the first report of the use of microarray analysis to determine the effects of drug action in
T. rubrum
.
Sublethal Concentrations of the Aminoglycoside Amikacin Interfere with Cell Division without Affecting Chromosome Dynamics
Authors: Possoz Christophe , Newmark Jason , Sorto Nohemy , Sherratt David J. , Tolmasky Marcelo E.
Published: December 2006

Aminoglycosides bind to the 16S rRNA at the tRNA acceptor site (A site) and disturb protein synthesis by inducing codon misreading. We investigated
Escherichia coli
cell elongation and division, as well as the dynamics of chromosome replication and segregation, in the presence of sublethal concentrations of amikacin (AMK). The fates of the chromosome
ori
and
ter
loci were monitored by visualization by using derivatives of LacI and TetR fused to fluorescent proteins in
E. coli
strains that carry operator arrays at the appropriate locations. The results showed that cultures containing sublethal concentrations of AMK contained abnormally elongated cells. The chromosomes in these cells were properly located, suggesting that the dynamics of replication and segregation were normal. FtsZ, an essential protein in the process of cell division, was studied by using an ectopic FtsZ-cyan fluorescent protein fusion. Consistent with a defect in cell division, we revealed that the Z ring failed to properly assemble in these elongated cells.
Posaconazole Prophylaxis in Experimental Systemic Zygomycosis
Authors: Barchiesi Francesco , Spreghini Elisabetta , Santinelli Alfredo , Fothergill Annette W. , Pisa Eleonora , Giannini Daniele , Rinaldi Michael G. , Scalise Giorgio
Published: December 2006

Three isolates of zygomycetes belonging to two different genera (
Rhizopus oryzae
and
Absidia corymbifera
) were used to produce a systemic infection in neutropenic mice. On days −2 and −1 and at 2 h prior to infection, the mice received either posaconazole (POS) at doses ranging from 20 to 80 mg/kg of body weight/day or amphotericin B (AMB) at 1 mg/kg/day. Antifungal drug efficacy was assessed by determination of the prolongation of survival, determination of the percentage of infected organs (brain, lung, spleen, and kidney), and histological examination for the number of infection foci and their sizes in brain and kidney tissues. AMB significantly prolonged the survival of mice infected with all isolates. POS significantly prolonged the survival of mice infected with zygomycetes. Cultured organs from mice infected with
R. oryzae
were all positive, while treated mice challenged with
A. corymbifera
generally showed lower percentages of infected organs compared with the percentages for the controls. Zygomycete isolates established an active infection (the presence of hyphae) in the brains and the kidneys of all controls. In mice challenged with
R. oryzae
, both antifungal drugs were effective at reducing the number and the size of infection foci in the kidneys. Only AMB reduced the numbers, but not the sizes, of infection foci in the brain. Finally, both drugs significantly reduced the numbers and the sizes of infection foci in both tissues of mice infected with
A. corymbifera
. Our data suggest that prophylaxis with POS has some potential to prevent zygomycosis.
Simple Colorimetric Inhibition Assay of Heme Crystallization for High-Throughput Screening of Antimalarial Compounds
Authors: Huy Nguyen Tien , Uyen Dinh Thanh , Maeda Atsushi , Trang Dai Thi Xuan , Oida Tatsuo , Harada Shigeharu , Kamei Kaeko
Published: December 2006

Current assays for screening new antimalarials need initiators of β-hematin formation that require laborious preparation, special devices, and substrates. In this study, based on reduction of heme absorption in β-hematin formation, we developed a simple colorimetric assay using Tween 20 as an initiator and a microplate reader for high-throughput screening of inhibitors of β-hematin formation.
Signature-Tagged Transposon Mutagenesis Identifies Novel
Mycobacterium tuberculosis
Genes Involved in the Parasitism of Human Macrophages
Authors: Rosas-Magallanes Vania , Stadthagen-Gomez Gustavo , Rauzier Jean , Barreiro Luis B. , Tailleux Ludovic , Boudou Frédéric , Griffin Ruth , Nigou Jérome , Jackson Mary , Gicquel Brigitte , Neyrolles Olivier
Published: December 2006

Using signature-tagged transposon mutagenesis, we isolated 23
Mycobacterium tuberculosis
mutants, corresponding to 21 genes or genetic regions, attenuated in their ability to parasitize human macrophages. Mutants disrupted in the ABC transporter-encoding genes Rv0986 and Rv0987 were further characterized as being impaired in their ability to bind to host cells.
Cellular Recognition of
Mycobacterium tuberculosis
ESAT-6 and KatG Peptides in Systemic Sarcoidosis
Authors: Drake Wonder P. , Dhason Mary S. , Nadaf Michele , Shepherd Bryan E. , Vadivelu Sangeetha , Hajizadeh Rana , Newman Lee S. , Kalams Spyros A.
Published: December 2006

Sarcoidosis is an enigmatic disease with a pathology similar to that of tuberculosis. We detected Th-1 immune responses to
Mycobacterium tuberculosis
ESAT-6 and KatG peptides from peripheral blood mononuclear cells from 15/26 sarcoidosis, 1/24 purified-protein-derivative-negative (PPD−) (
P
< 0.0001, Fisher's exact test), and 7/8 PPD-positive (PPD+) subjects (
P
= 0.21). This finding provides immunologic links between mycobacteria and systemic sarcoidosis.
Proteome and Antigen Profiling of
Coxiella burnetii
Developmental Forms
Authors: Coleman Sherry A. , Fischer Elizabeth R. , Cockrell Diane C. , Voth Daniel E. , Howe Dale , Mead David J. , Samuel James E. , Heinzen Robert A.
Published: December 2006

A biphasic developmental cycle whereby highly resistant small-cell variants (SCVs) are generated from large-cell variants (LCVs) is considered fundamental to the virulence of
Coxiella burnetii
, the causative agent of human Q fever. In this study a proteome analysis of
C. burnetii
developmental forms was conducted to provide insight into their unique biological and immunological properties. Silver-stained gels of SCV and LCV lysates separated by two-dimensional (2-D) gel electrophoresis resolved over 675 proteins in both developmental forms. Forty-eight proteins were greater than twofold more abundant in LCVs than in SCVs, with six proteins greater than twofold more abundant in SCVs than in LCVs. Four and 15 upregulated proteins of SCVs and LCVs, respectively, were identified by mass spectrometry, and their predicted functional roles are consistent with a metabolically active LCV and a structurally resistant SCV. One-dimensional and 2-D immunoblots of cell form lysates probed with sera from infected/vaccinated guinea pigs and convalescent-phase serum from human patients who had recovered from acute Q fever, respectively, revealed both unique SCV/LCV antigens and common SCV/LCV antigens that were often differentially synthesized. Antigens recognized during human infection were identified by mass spectroscopy and included both previously described immunodominant proteins of
C. burnetii
and novel immunogenic proteins that may be important in the pathophysiology of clinical Q fever and/or the induction of protective immunity.
AMycobacterium marinum mel2Mutant Is Defective for Growth in Macrophages That Produce Reactive Oxygen and Reactive Nitrogen Species
Authors: Subbian Selvakumar , Mehta Parmod K. , Cirillo Suat L. G. , Bermudez Luiz E. , Cirillo Jeffrey D.
Published: December 2006

Macrophages produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to bacterial infections. Mycobacteria are relatively resistant to ROS, but RNS inhibit growth of, and possibly even kill, mycobacteria in activated macrophages. We recently constructed aMycobacterium marinum mel2locus mutant, which is known to affect macrophage infection. We found previously that themel2locus confers resistance to ROS and RNS in laboratory medium, suggesting that this locus might play a similar role during growth in macrophages. Since J774A.1 murine macrophages produce high levels of ROS and RNS upon activation with gamma interferon (IFN-γ), we examined the effects of IFN-γ on ROS and RNS production by these cells as well as the effects on growth ofM. marinumin these cells. We found that anM. marinummutant with mutation of the first gene in themel2locus,melF, is defective for growth in IFN-γ-plus-lipopolysaccharide-treated J774A.1 cells and that this defect is abrogated by the presence of either inhibitors of nitric oxide synthase or ROS scavengers. Furthermore, theM. marinum melFmutant displays a defect at late stages in the mouse footpad model of infection. These phenotypic characteristics could be complemented fully by the entiremel2locus but only partially by the presence ofmelFalone, supporting data suggesting that this insertion mutation has polar effects on downstream genes in themel2locus. These observations demonstrate that theM. marinum mel2locus plays a role in resistance to ROS and RNS produced by activated macrophages.
Identification of Genes Involved in the Expression of Atypical Lipooligosaccharide Structures from a Second Class of
Haemophilus ducreyi
Authors: Post Deborah M. B. , Munson Robert S. , Baker Beth , Zhong Huachun , Bozue Joel A. , Gibson Bradford W.
Published: December 2006

Haemophilus ducreyi
is a gram-negative bacterium that is the causative agent of chancroid. Strain 35000HP has been well characterized and is representative of the majority of
H. ducreyi
strains. Strain 35000HP produces a lipooligosaccharide (LOS) that contains
d
-
glycero
-
d
-
manno
-heptose in the main oligosaccharide chain extension; the
lbgB
gene has been shown to encode the
dd
-heptosyltransferase. The
lbgB
gene is found in a gene cluster together with the
lbgA
gene, which encodes for the galactosyltransferase I. These two genes are flanked by two housekeeping genes,
rpmE
and
xthA
, encoding the ribosomal protein L31 and the exonuclease III, respectively. Recently, a second group of
H. ducreyi
strains have been identified. Strain 33921, a representative of the class II strains, produces an LOS that lacks
dd
-heptose in the oligosaccharide portion of its LOS. To better understand the biosynthesis of the
dd
-heptose-deficient 33921 LOS, we cloned and sequenced the corresponding
lbgAB
genomic region from strain 33921. Similar to strain 35000HP, the 33921 genome contains
xthA
and
rpmE
. However, between these two genes we identified genes encoding two putative glycosyltransferases that were not highly homologous to the 35000HP
lbgAB
genes. In this study, we demonstrate that the product of one of these genes encodes a galactosyltransferase. In addition, dot blot hybridization determined that 3 of 35 strains tested had the atypical transferases present, as did 4 strains characterized as class II strains by other criterion. These data indicate that the
lbgAB
genes can serve as one indicator of the classification of
H. ducreyi
strains.
Mimotopes of Apical Membrane Antigen 1: Structures of Phage-Derived Peptides Recognized by the Inhibitory Monoclonal Antibody 4G2dc1 and Design of a More Active Analogue
Authors: Sabo Jennifer K. , Keizer David W. , Feng Zhi-Ping , Casey Joanne L. , Parisi Kathy , Coley Andrew M. , Foley Michael , Norton Raymond S.
Published: December 2006

Apical membrane antigen 1 (AMA1) of the malaria parasite
Plasmodium falciparum
is an integral membrane protein that plays a key role in merozoite invasion of host erythrocytes. A monoclonal antibody, 4G2dc1, recognizes correctly folded AMA1 and blocks merozoite invasion. Phage display was used to identify peptides that bind to 4G2dc1 and mimic an important epitope of AMA1. Three of the highest-affinity binders—J1, J3, and J7—were chosen for antigenicity and immunogenicity studies. J1 and J7 were found to be true antigen mimics since both peptides generated inhibitory antibodies in rabbits (J. L. Casey et al., Infect. Immun. 72:1126-1134, 2004). In the present study, the solution structures of all three mimotopes were investigated by nuclear magnetic resonance spectroscopy. J1 adopted a well-defined region of structure, which can be attributed in part to the interactions of Trp11 with surrounding residues. In contrast, J3 and J7 did not adopt an ordered conformation over the majority of residues, although they share a region of local structure across their consensus sequence. Since J1 was the most structured of the peptides, it provided a template for the design of a constrained analogue, J1cc, which shares a structure similar to that of J1 and has a disulfide-stabilized conformation around the Trp11 region. J1cc binds with greater affinity to 4G2dc1 than does J1. These peptide structures provide the foundation for a better understanding of the complex conformational nature of inhibitory epitopes on AMA1. With its greater conformational stability and higher affinity for AMA1, J1cc may be a better in vitro correlate of immunity than the peptides identified by phage display.
Resistance of Primary Murine CD4
+
T Cells to
Helicobacter pylori
Vacuolating Cytotoxin
Authors: Algood Holly M. Scott , Torres Victor J. , Unutmaz Derya , Cover Timothy L.
Published: December 2006

Persistent colonization of the human stomach by
Helicobacter pylori
is a risk factor for the development of gastric cancer and peptic ulcer disease.
H. pylori
secretes a toxin, VacA, that targets human gastric epithelial cells and T lymphocytes and enhances the ability of
H. pylori
to colonize the stomach in a mouse model. To examine how VacA contributes to
H. pylori
colonization of the mouse stomach, we investigated whether murine T lymphocytes were susceptible to VacA activity. VacA inhibited interleukin-2 (IL-2) production by a murine T-cell line (LBRM-33), similar to its effects on a human T-cell line (Jurkat), but did not inhibit IL-2 production by primary murine splenocytes or CD4
+
T cells. VacA inhibited activation-induced proliferation of primary human CD4
+
T cells but did not inhibit the proliferation of primary murine CD4
+
T cells. Flow cytometry studies indicated that the levels of VacA binding to primary murine CD4
+
T cells were significantly lower than levels of VacA binding to human CD4
+
T cells. This suggests that the resistance of primary murine CD4
+
T cells to VacA is attributable, at least in part, to impaired VacA binding to these cells.
The Immunologically Distinct O Antigens fromFrancisella tularensisSubspeciestularensisandFrancisella novicidaAre both Virulence Determinants and Protective Antigens
Authors: Thomas Rebecca M. , Titball Richard W. , Oyston Petra C. F. , Griffin Kate , Waters Emma , Hitchen Paul G. , Michell Stephen L. , Grice I. Darren , Wilson Jennifer C. , Prior Joann L.
Published: December 2006

We have determined the sequence of the gene cluster encoding the O antigen inFrancisella novicidaand compared it to the previously reported O-antigen cluster inFrancisella tularensissubsp.tularensis. Immunization with purified lipopolysaccharide (LPS) fromF. tularensissubsp.tularensisorF. novicidaprotected against challenge withFrancisella tularensissubsp.holarcticaandF. novicida, respectively. The LPS fromF. tularensissubsp.tularensisdid not confer protection against challenge withF. novicida, and the LPS fromF. novicidadid not confer protection against challenge withF. tularensissubsp.holarctica. Allelic replacement mutants ofF. tularensissubsp.tularensisorF. novicidawhich failed to produce O antigen were attenuated, but exposure to these mutants did not induce a protective immune response. The O antigen ofF. tularensissubsp.tularensisappeared to be important for intracellular survival whereas the O antigen ofF. novicidaappeared to be critical for serum resistance and less important for intracellular survival.
The Combined CTA1-DD/ISCOMs Vector Is an Effective Intranasal Adjuvant for Boosting PriorMycobacterium bovisBCG Immunity toMycobacterium tuberculosis
Authors: Andersen Claire Swetman , Dietrich Jes , Agger Else Marie , Lycke Nils Y. , Lövgren Karin , Andersen Peter
Published: December 2006

Infection withMycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains one of the leading causes of mortality worldwide. The current “gold standard” vaccineMycobacterium bovisBCG has a limited efficacy that wanes over time. The development of a vaccine to boost BCG-induced immunity is therefore a highly active area of research. Mucosal administration of vaccines is believed to provide better protection against pathogens, such asM. tuberculosis, that invade the host via mucosal surfaces. In this study we demonstrate that an intranasal vaccine, comprising the antigenic fusion protein Ag85B-ESAT-6 and the mucosal combined adjuvant vector CTA1-DD/ISCOMs, strongly promotes a Th1-specific immune response, dominated by gamma interferon-secreting CD4-positive T cells. Mucosal administration of Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs strongly boosted prior BCG immunity, leading to a highly increased recruitment of antigen-specific cells to the site of infection. Most importantly, we observed a significantly (P< 0.001) reduced bacterial burden in the lung compared to nonboosted control animals. Thus, the results demonstrate the effectiveness of mucosal vaccination with Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs as adjuvant for stimulating TB-specific protective immunity in the lung.
The TolC Homologue of
Brucella suis
Is Involved in Resistance toAntimicrobial Compounds and Virulence
Authors: Posadas Diana M. , Martín Fernando A. , Sabio y García Julia V. , Spera Juan M. , Delpino M. Victoria , Baldi Pablo , Campos Eleonora , Cravero Silvio L. , Zorreguieta Angeles
Published: December 2006

Brucella
spp., like other pathogens, must cope with the environment of diverse
host niches during the infection process. In doing this, pathogens
evolved different type of transport systems to help them survive and
disseminate within the host. Members of the TolC family have been shown
to be involved in the export of chemically diverse molecules ranging
from large protein toxins to small toxic compounds. The role of
proteins from the TolC family in
Brucella
and otherα
-2-proteobacteria has been explored little. The gene encoding
the unique member of the TolC family from
Brucella suis
(BepC)
was cloned and expressed in an
Escherichia coli
mutant
disrupted in the gene encoding TolC, which has the peculiarity of being
involved in diverse transport functions. BepC fully complemented the
resistance to drugs such as chloramphenicol and acriflavine but was
incapable of restoring hemolysin secretion in the
tolC
mutant
of
E. coli
. An insertional mutation in the
bepC
gene
strongly affected the resistance phenotype of
B. suis
to bile
salts and toxic chemicals such as ethidium bromide and rhodamine and
significantly decreased the resistance to antibiotics such as
erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the
B. suis bepC
mutant was attenuated in the mouse model of
infection. Taken together, these results suggest that BepC-dependent
efflux processes of toxic compounds contribute to
B. suis
survival inside the
host.
Identification of a Glycosylated
Ehrlichia canis
19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope
Authors: McBride Jere W. , Doyle C. Kuyler , Zhang Xiaofeng , Cardenas Ana Maria , Popov Vsevolod L. , Nethery Kimberly A. , Woods Michael E.
Published: December 2006

Ehrlichia canis
has a small subset of major immunoreactive proteins that includes a 19-kDa protein that elicits an early
Ehrlichia
-specific antibody response in infected dogs. We report herein the identification and molecular characterization of this highly conserved 19-kDa major immunoreactive glycoprotein (gp19) ortholog of the
Ehrlichia chaffeensis
variable-length PCR target (VLPT) protein.
E. canis
gp19 has substantial carboxyl-terminal amino acid homology (59%) with
E. chaffeensis
VLPT and the same chromosomal location; however, the
E. chaffeensis
VLPT gene (594 bp) has tandem repeats that are not present in the
E. canis
gp19 gene (414 bp). Consistent with other ehrlichial glycoproteins, the gp19 protein exhibited a larger-than-predicted mass (∼3 kDa), O-linked glycosylation sites were predicted in an amino-terminal serine/threonine/glutamate (STE)-rich patch (26 amino acids), carbohydrate was detected on the recombinant gp19 protein, and the neutral sugars glucose and galactose were detected on the recombinant amino-terminal polypeptide.
E. canis
gp19 composition consists of five predominant amino acids, cysteine, glutamate, tyrosine, serine, and threonine, concentrated in the STE-rich patch and a carboxyl-terminal domain predominated by cysteine and tyrosine (55%). The amino-terminal STE-rich patch contained a major species-specific antibody epitope strongly recognized by serum from an
E. canis-
infected dog. The recombinant glycopeptide epitope was substantially more reactive with antibody than the synthetic (nonglycosylated) peptide, and periodate treatment of the recombinant glycopeptide epitope reduced its immunoreactivity, demonstrating the importance of a carbohydrate immunodeterminant(s). The gp19 protein was present on reticulate and dense-cored cells, and it was found extracellularly in the fibrillar matrix and associated with the morula membrane, the host cell cytoplasm, and the nucleus.
A Genotype-Independent Real-Time PCR Assay for Quantification of Hepatitis B Virus DNA
Authors: Liu Ying , Hussain Munira , Wong Stephen , Fung Scott K. , Yim Hyung Joon , Lok Anna S. F.
Published: December 2006

Accurate quantification of hepatitis B virus (HBV) DNA levels is important for monitoring patients with chronic HBV infection and for assessing their responses to antiviral therapy. This study aimed to develop a real-time PCR assay that is sensitive and can accurately quantify a wide range of HBV DNA levels across the known HBV genotypes. An “in-house” real-time PCR assay using primers and a TaqMan probe in a highly conserved region of the HBV surface gene was designed. The assay was standardized against a WHO standard and validated against plasmids of HBV genotypes A through H. The linear quantification range was approximately 5 × 10
0
to 2.0 × 10
9
IU/ml. Results of samples from patients infected with HBV genotypes A through H tested using our real-time “in-house” PCR assay showed an excellent correlation with those of the Cobas Amplicor HBV Monitor (
R
2
= 0.9435) and the Cobas TaqMan HBV (
R
2
= 0.9873) tests. We have established a real-time PCR assay that is genotype independent and can accurately quantify a wide range of HBV DNA levels. Further studies of additional samples are ongoing to validate the genotype independence of our assay.
Comparative Study of Kaposi's Sarcoma-Associated Herpesvirus Serological Assays Using Clinically and Serologically Defined Reference Standards and Latent Class Analysis
Authors: Nascimento Maria Claudia , de Souza Vanda Akico , Sumita Laura Masami , Freire Wilton , Munoz Fernando , Kim Joseph , Pannuti Claudio S. , Mayaud Philippe
Published: December 2006

Accurate determination of infection with Kaposi's sarcoma-associated herpesvirus (KSHV) has been hindered by the lack of a “gold standard” for comparison of serological assays used to estimate KSHV prevalence in serosurveys conducted in different settings. We have evaluated the performance of five in-house (developed at University College London [UCL], United Kingdom, and at the virology laboratory of the Instituto de Medicine Tropical [IMT] in Sao Paulo, Brazil) and two commercial (ABI and DIAVIR) serological assays to detect antibodies to latency-associated nuclear antigen (LANA) and to lytic KSHV antigens. We used a variety of serum samples assembled to represent populations likely to be at high, intermediate, and low risk of KSHV infection in Brazil. Composite reference standard panels were prepared based on clinical and serological parameters, against which assay performances were assessed using conventional Bayesian statistics and latent class analysis (LCA). Against the clinical reference standard, in-house immunofluorescence assays to detect anti-LANA antibodies (IFA-LANA) produced at UCL and IMT had similar performances, with sensitivities of 61% (95% confidence interval [CI], 48% to 74%) and 72% (95% CI, 58% to 83%) and specificities of 99% (95% CI, 94% to 100%) and 100% (95% CI, 96% to 100%), respectively, and only the IMT IFA-LANA was included in LCA, together with the IMT IFA-lytic and four enzyme-linked immunosorbent assays (ELISAs). The LCA indicated that the IMT whole-virus ELISA performed best (sensitivity, 87% [95% CI, 81% to 91%]; and specificity, 100% [95% CI, 98% to 100%]), confirming the results obtained with the conventional statistical approach. Commercially available ELISA-based tests yielded the lowest specificities using a spectrum of serum samples. The evaluation of KSHV serological assays is warranted before planning serosurveys in various settings.
Rapid and Highly Sensitive Pathotyping of Avian Influenza A H5N1 Virus by Using Real-Time Reverse Transcription-PCR
Authors: Hoffmann Bernd , Harder Timm , Starick Elke , Depner Klaus , Werner Ortrud , Beer Martin
Published: December 2006

Rapid typing of the pathogenicity of avian influenza A viruses (AIV) of subtypes H5 and H7 is crucial to initiate adequate protective measures preventing the spread of highly pathogenic AIV (HPAIV). Here, a new real-time reverse transcription-PCR assay which enables sensitive and specific detection and cleavage site analysis of HPAIV H5N1 of the Qinghai lineage is described.
Use of New T-Cell-Based Cell Lines Expressing Two Luciferase Reporters for Accurately Evaluating Susceptibility to Anti-Human Immunodeficiency Virus Type 1 Drugs
Authors: Chiba-Mizutani Tomoko , Miura Hideka , Matsuda Masakazu , Matsuda Zene , Yokomaku Yoshiyuki , Miyauchi Kosuke , Nishizawa Masako , Yamamoto Naoki , Sugiura Wataru
Published: December 2006

Two new T-cell-based reporter cell lines were established to measure human immunodeficiency virus type 1 (HIV-1) infectivity. One cell line naturally expresses CD4 and CXCR4, making it susceptible to X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line HPB-Ma, which demonstrates high susceptibility to HIV-1, with genomes expressing two different luciferase reporters, HIV-1 long terminal repeat-driven firefly luciferase and cytomegalovirus promoter-driven renilla luciferase. Upon HIV infection, the cells expressed firefly luciferase at levels that were highly correlated (r2= 0.91 to 0.98) with the production of the capsid antigen p24. The cells also constitutively expressed renilla luciferase, which was used to monitor cell numbers and viability. The reliability of the cell lines for two in vitro applications, drug resistance phenotyping and drug screening, was confirmed. As HIV-1 efficiently replicated in these cells, they could be used for multiple-round replication assays as an alternative method to a single-cycle replication protocol. Coefficients of variation for drug susceptibility evaluated with the cell lines ranged from 17 to 41%. The new cell lines were beneficial for evaluating antiretroviral drug resistance. Firefly luciferase gave a wider dynamic range for evaluating virus infectivity, and the introduction of renilla luciferase improved assay reproducibility. The cell lines were also beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test compounds was easily detected by monitoring renilla luciferase activity.
GeneChip Resequencing of the Smallpox Virus Genome Can Identify Novel Strains: a Biodefense Application
Authors: Sulaiman Irshad M. , Tang Kevin , Osborne John , Sammons Scott , Wohlhueter Robert M.
Published: December 2006

We developed a set of seven resequencing GeneChips, based on the complete genome sequences of 24 strains of smallpox virus (variola virus), for rapid characterization of this human-pathogenic virus. Each GeneChip was designed to analyze a divergent segment of approximately 30,000 bases of the smallpox virus genome. This study includes the hybridization results of 14 smallpox virus strains. Of the 14 smallpox virus strains hybridized, only 7 had sequence information included in the design of the smallpox virus resequencing GeneChips; similar information for the remaining strains was not tiled as a reference in these GeneChips. By use of variola virus-specific primers and long-range PCR, 22 overlapping amplicons were amplified to cover nearly the complete genome and hybridized with the smallpox virus resequencing GeneChip set. These GeneChips were successful in generating nucleotide sequences for all 14 of the smallpox virus strains hybridized. Analysis of the data indicated that the GeneChip resequencing by hybridization was fast and reproducible and that the smallpox virus resequencing GeneChips could differentiate the 14 smallpox virus strains characterized. This study also suggests that high-density resequencing GeneChips have potential biodefense applications and may be used as an alternate tool for rapid identification of smallpox virus in the future.
Vibrio cholerae
Strain Typing and Phylogeny Study Based on Simple Sequence Repeats
Authors: Danin-Poleg Yael , Cohen Lyora A. , Gancz Hanan , Broza Yoav Y. , Goldshmidt Hanoh , Malul Elinor , Valinsky Lea , Lerner Larisa , Broza Meir , Kashi Yechezkel
Published: December 2006

Vibrio cholerae
is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of
V. cholerae
is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of
V. cholerae
. In silico screening of the
V. cholerae
genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32
V. cholerae
strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for
V. cholerae
typing.
Vancomycin-Resistant
Enterococcus faecalis
Endocarditis: Linezolid Failure and Strain Characterization of Virulence Factors
Authors: Tsigrelis Constantine , Singh Kavindra V. , Coutinho Thais D. , Murray Barbara E. , Baddour Larry M.
Published: December 2006

Infective endocarditis due to vancomycin-resistant (VR)
Enterococcus faecalis
has only rarely been reported. We report a case of VR
E. faecalis
endocarditis that failed to respond to linezolid therapy, outline the virulence traits of the isolate, and review previously published cases of VR
E. faecalis
endocarditis.
Characterization of Primary and Memory CD8 T-Cell Responses against Ranavirus (FV3) inXenopus laevis
Authors: Morales Heidi D. , Robert Jacques
Published: December 2006

In mammals, resistance to primary and secondary viral infections critically involves major histocompatibility complex class I-restricted cytotoxic CD8+T lymphocytes (CTLs). Although many gene homologues involved in CTL function have been identified in all vertebrate classes, antiviral CTL responses have been poorly characterized for ectothermic vertebrates. Because of the threat of emerging wildlife viral diseases to global biodiversity, fundamental research on comparative viral immunity has become crucial. Ranaviruses (familyIridoviridae) are double-stranded DNA viruses possibly implicated in the worldwide decline of amphibian populations. We used the frogXenopus laevisas a model to evaluate adaptive immune responses to the ranavirus frog virus 3 (FV3). FV3 infects the kidneys of adults but is cleared within 4 weeks, with faster clearance upon secondary infections. In vivo depletion of CD8+T cells markedly decreases the survival of adults after viral infection. To further investigate the involvement of anti-FV3 CD8+T-cell effectors in host resistance in vivo, we determined the proliferation kinetics of CD8+T cells in the spleen by bromodeoxyuridine incorporation and their infiltration of kidneys by immunohistology. Upon primary infection, CD8+T cells significantly proliferate in the spleen and accumulate in infected kidneys from day 6 onward, in parallel with virus clearance. Earlier proliferation and infiltration associated with faster viral clearance were observed during a secondary infection. These results provide in vivo evidence of protective antigen-dependent CD8+T-cell proliferation, recognition, and memory in fighting a natural pathogen inXenopus.
Reduced Maximal Inhibition in Phenotypic Susceptibility Assays Indicates that Viral Strains Resistant to the CCR5 Antagonist Maraviroc Utilize Inhibitor-Bound Receptor for Entry
Authors: Westby Mike , Smith-Burchnell Caroline , Mori Julie , Lewis Marilyn , Mosley Michael , Stockdale Mark , Dorr Patrick , Ciaramella Giuseppe , Perros Manos
Published: December 2006

Maraviroc is a CCR5 antagonist in clinical development as one of a new class of antiretrovirals targeting human immunodeficiency virus type 1 (HIV-1) coreceptor binding. We investigated the mechanism of HIV resistance to maraviroc by using in vitro sequential passage and site-directed mutagenesis. Serial passage through increasing maraviroc concentrations failed to select maraviroc-resistant variants from some laboratory-adapted and clinical isolates of HIV-1. However, high-level resistance to maraviroc was selected from three of six primary isolates passaged in peripheral blood lymphocytes (PBL). The SF162 strain acquired resistance to maraviroc in both treated and control cultures; all resistant variants were able to use CXCR4 as a coreceptor. In contrast, maraviroc-resistant virus derived from isolates CC1/85 and RU570 remained CCR5 tropic, as evidenced by susceptibility to the CCR5 antagonist SCH-C, resistance to the CXCR4 antagonist AMD3100, and an inability to replicate in CCR5 Δ32/Δ32 PBL. Strain-specific mutations were identified in the V3 loop of maraviroc-resistant CC1/85 and RU570. The envelope-encoding region of maraviroc-resistant CC1/85 was inserted into an NL4-3 background. This recombinant virus was completely resistant to maraviroc but retained susceptibility to aplaviroc. Reverse mutation of gp120 residues 316 and 323 in the V3 loop (numbering from HXB2) to their original sequence restored wild-type susceptibility to maraviroc, while reversion of either mutation resulted in a partially sensitive virus with reduced maximal inhibition (plateau). The plateaus are consistent with the virus having acquired the ability to utilize maraviroc-bound receptor for entry. This hypothesis was further corroborated by the observation that a high concentration of maraviroc blocks the activity of aplaviroc against maraviroc-resistant virus.
Enhancement of Hepatitis B Virus Replication by the Regulatory X Protein In Vitro and In Vivo
Authors: Keasler Victor V. , Hodgson Amanda J. , Madden Charles R. , Slagle Betty L.
Published: December 2006

The 3.2-kb hepatitis B virus (HBV) genome encodes a single regulatory protein termed HBx. While multiple functions have been identified for HBx in cell culture, its role in virus replication remains undefined. In the present study, we combined an HBV plasmid-based replication assay with the hydrodynamic tail vein injection model to investigate the function(s) of HBx in vivo. Using a greater-than-unit-length HBV plasmid DNA construct (payw1.2) and a similar construct with a stop codon at position 7 of the HBx open reading frame (payw1.2*7), we showed that HBV replication in transfected HepG2 cells was reduced 65% in the absence of HBx. These plasmids were next introduced into the livers of outbred ICR mice via hydrodynamic tail vein injection. At the peak of virus replication, at 4 days postinjection, intrahepatic markers of HBV replication were reduced 72% to 83% in mice injected with HBx-deficient payw1.2*7 compared to those measured in mice receiving wild-type payw1.2. A second plasmid encoding HBx was able to restore virus replication from payw1.2*7 to wild-type levels. Finally, viremia was monitored over the course of acute virus replication, and at 4 days postinjection, it was reduced by nearly 2 logs in the absence of HBx. These studies establish that the role for HBx in virus replication previously shown in transfected HepG2 cells is also apparent in the mouse liver within the context of acute hepatitis. Importantly, the function of HBx can now be studied in an in vivo setting that more closely approximates the cellular environment for HBV replication.
Cytosolic Prion Protein Toxicity Is Independent of Cellular Prion Protein Expression and Prion Propagation
Authors: Norstrom Eric M. , Ciaccio Mark F. , Rassbach Benjamin , Wollmann Robert , Mastrianni James A.
Published: December 2006

Prion diseases are transmissible neurodegenerative diseases caused by a conformational isoform of the prion protein (PrP), a host-encoded cell surface sialoglycoprotein. Recent evidence suggests a cytosolic fraction of PrP (cyPrP) functions either as an initiating factor or toxic element of prion disease. When expressed in cultured cells, cyPrP acquires properties of the infectious conformation of PrP (PrP
Sc
), including insolubility, protease resistance, aggregation, and toxicity. Transgenic mice (2D1 and 1D4 lines) that coexpress cyPrP and PrP
C
exhibit focal cerebellar atrophy, scratching behavior, and gait abnormalities suggestive of prion disease, although they lack protease-resistant PrP. To determine if the coexpression of PrP
C
is necessary or inhibitory to the phenotype of these mice, we crossed Tg1D4(
Prnp

+/+

) mice with PrP-ablated mice (Tg
Prnp
o/o
) to generate Tg1D4(
Prnp
o/o
) mice and followed the development of disease and pathological phenotype. We found no difference in the onset of symptoms or the clinical or pathological phenotype of disease between Tg1D4(
Prnp

+/+

) and Tg1D4(
Prnp
o/o
) mice, suggesting that cyPrP and PrP
C
function independently in the disease state. Additionally, Tg1D4(
Prnp
o/o
) mice were resistant to challenge with mouse-adapted scrapie (RML), suggesting cyPrP is inaccessible to PrP
Sc
. We conclude that disease phenotype and cellular toxicity associated with the expression of cyPrP are independent of PrP
C
and the generation of typical prion disease.