bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of
Streptococcus pneumoniae clp
Genes and Their Role in Competence Development and Stress Survival
Authors: Chastanet Arnaud , Prudhomme Marc , Claverys Jean-Pierre , Msadek Tarek
Published: July 2002

In vitro
mariner
transposon mutagenesis of
Streptococcus pneumoniae
chromosomal DNA was used to isolate regulatory mutants affecting expression of the
comCDE
operon, encoding the peptide quorum-sensing two-component signal transduction system controlling competence development. A transposon insertion leading to increased
comC
expression was found to lie directly upstream from the
S. pneumoniae clpP
gene, encoding the proteolytic subunit of the Clp ATP-dependent protease, whose expression in
Bacillus subtilis
is controlled by the CtsR repressor. In order to examine
clp
gene regulation in
S. pneumoniae
, a detailed analysis of the complete genome sequence was performed, indicating that there are five likely CtsR-binding sites located upstream from the
clpE
,
clpP
, and
clpL
genes and the
ctsR
-
clpC
and
groESL
operons. The
S. pneumoniae ctsR
gene was cloned under the control of an inducible promoter and used to demonstrate regulation of the
S. pneumoniae clpP
and
clpE
genes and the
clpC
and
groESL
operons by using
B. subtilis
as a heterologous host. The CtsR protein of
S. pneumoniae
was purified and shown to bind specifically to the
clpP, clpC, clpE
, and
groESL
regulatory regions.
S. pneumoniae
Δ
ctsR
, Δ
clpP
, Δ
clpC
, and Δ
clpE
mutants were constructed by gene deletion/replacement. ClpP was shown to act as a negative regulator, preventing competence gene expression under inappropriate conditions. Phenotypic analyses also indicated that ClpP and ClpE are both required for thermotolerance. Contrary to a previous report, we found that ClpC does not play a major role in competence development, autolysis, pneumolysin production, or growth at high temperature of
S. pneumoniae
.
Genetic Analysis of Assembly of theSalmonella entericaSerovar Typhimurium Type III Secretion-Associated Needle Complex
Authors: Sukhan Anand , Kubori Tomoko , Wilson James , Galán Jorge E.
Published: July 2002

Several pathogenic bacteria have evolved a specialized protein secretion system termed type III to secrete and deliver effector proteins into eukaryotic host cells.Salmonella entericaserovar Typhimurium uses one such system to mediate entry into nonphagocytic cells. This system is composed of more than 20 proteins which are encoded within a pathogenicity island (SPI-1) located at centisome 63 of its chromosome. A subset of these components form a supramolecular structure, termed the needle complex, that resembles the flagellar hook-basal body complex. The needle complex is composed of a multiple-ring cylindrical base that spans the bacterial envelope and a needle-like extension that protrudes from the bacterial outer surface. Although the components of this structure have been identified, little is known about its assembly. In this study we examined the effect of loss-of-function mutations in each of the type III secretion-associated genes encoded within SPI-1 on the assembly of the needle complex. This analysis indicates that the assembly of this organelle occurs in discrete, genetically separable steps. A model for the assembly pathway of this important organelle is proposed that involves asec-dependent step leading to the assembly of the base substructure followed by asec-independent process resulting in the assembly of the needle portion.
Selective mRNA Degradation by Polynucleotide Phosphorylase in Cold Shock Adaptation in
Escherichia coli
Authors: Yamanaka Kunitoshi , Inouye Masayori
Published: July 2002

Upon cold shock,
Escherichia coli
cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A
pnp
mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15°C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.
Mosaic Structure and Molecular Evolution of the Leukotoxin Operon (
lktCABD
) in
Mannheimia
(
Pasteurella
) haemolytica,
Mannheimia glucosida
, and
Pasteurella trehalosi
Authors: Davies Robert L. , Campbell Susan , Whittam Thomas S.
Published: July 2002

The mosaic structure and molecular evolution of the leukotoxin operon (
lktCABD
) was investigated by nucleotide sequence comparison of the
lktC
,
lktB
, and
lktD
genes in 23
Mannheimia
(
Pasteurella
)
haemolytica
, 6
Mannheimia glucosida
, and 4
Pasteurella trehalosi
strains. Sequence variation in the lktA gene has been described previously (R. L. Davies et al., J. Bacteriol. 183:1394–1404, 2001). The leukotoxin operon of M. haemolytica has a complex mosaic structure and has been derived by extensive inter- and intraspecies horizontal DNA transfer and intragenic recombination events. However, the pattern of recombination varies throughout the operon and among the different evolutionary lineages of
M. haemolytica
. The lktA and lktB genes have the most complex mosaic structures with segments derived from up to four different sources, including
M. glucosida
and
P. trehalosi
. In contrast, the
lktD
gene is highly conserved in
M. haemolytica
. The
lktC
,
lktA
, and
lktB
genes of strains representing the major ovine lineages contain recombinant segments derived from bovine or bovine-like serotype A2 strains. These findings support the previous conclusion that host switching of bovine A2 strains from cattle to sheep has played a major role in the evolution of the leukotoxin operon in ovine strains of
M. haemolytica
. Homologous segments of donor and recipient alleles are identical, or nearly identical, indicating that the recombinational exchanges occurred relatively recent in evolutionary terms. The 5′ and 3′ ends of the operon are highly conserved in
M. haemolytica
, which suggests that multiple horizontal exchanges of the complete operon have occurred by a common mechanism such as transduction. Although the
lktA
and
lktB
genes both have complex mosaic structures and high nucleotide substitution rates, the amino acid diversity of LktB is significantly lower than that of LktA due to a higher degree of evolutionary constraint against amino acid replacement. The recombinational exchanges within the leukotoxin operon have had greatest effect on LktA and probably provide an adaptive advantage against the host antibody response by generating novel antigenic variation at surface-exposed sites.
Substrate Specificity of Nickel/Cobalt Permeases: Insights from Mutants Altered in Transmembrane Domains I and II
Authors: Degen Olaf , Eitinger Thomas
Published: July 2002

HoxN, a high-affinity, nickel-specific permease ofRalstonia eutrophaH16, and NhlF, a nickel/cobalt permease ofRhodococcus rhodochrousJ1, are structurally related members of the nickel/cobalt transporter (NiCoT) family. These transporters have an eight-helix structure and are characterized by highly conserved segments with polar or charged amino acid residues in transmembrane domains (TMDs) II, III, V, and VI. Two histidine residues in a Ni2+binding motif, the signature sequence of NiCoTs, in TMD II of HoxN have been shown to be crucial for activity. Replacement of the corresponding His residues in NhlF affected both Co2+and Ni2+uptake, demonstrating that NhlF employs a HoxN-like mechanism for transport of the two cations. Multiple alignments of bacterial NiCoT sequences identified a striking correlation between a hydrophobic residue (Val or Phe) in TMD II and a position in the center of TMD I occupied by either an Asn (as in HoxN) or a His (as in NhlF). Introducing an isoleucine residue at the latter position strongly reduced HoxN activity and abolished NhlF activity, suggesting that a Lewis base N-donor moiety is important. The Asn-to-His exchange had no effect on HoxN, whereas the converse replacement reduced NhlF-mediated Ni2+uptake significantly. Replacement of the entire TMD I of HoxN by the respective NhlF segment resulted in a chimera that transported Ni2+and Co2+with low capacity. The Val-to-Phe exchange in TMD II of HoxN led to a considerable rise in Ni2+uptake capacity and conferred to the variant the ability to transport Co2+. NhlF activity dropped in response to the converse mutation. Our data predict that TMDs I and II in NiCoTs spatially interact to form a critical part of the selectivity filter. As seen for the V64F variant of HoxN, modification of this site can increase the velocity of transport and concomitantly reduce the specificity.
An Antimicrobial Peptide Is Produced by Extracellular Processing of a Protein from
Propionibacterium jensenii
Authors: Faye Therese , Brede Dag Anders , Langsrud Thor , Nes Ingolf F. , Holo Helge
Published: July 2002

A protease-activated antimicrobial peptide (PAMP) and its inactive precursor were purified from the culture supernatant of
Propionibacterium jensenii
LMG 3032 and characterized at the molecular level. PAMP is a 64-amino-acid cationic peptide of 6,383 Da with physicochemical features similar to those of bacteriocins from gram-positive bacteria. This peptide displayed bactericidal activity against several propionibacteria and lactobacilli. DNA sequencing indicated that the PAMP-encoding gene (
pamA
) is translated as a proprotein of 198 amino acids with an N-terminal signal peptide of 27 amino acids and that PAMP constitutes the C-terminal part of this precursor. The amino acid sequence of pro-PAMP showed no similarity to those of other known proteins. By using activity tests and mass spectrometry, we showed that PAMP was formed upon protease treatment of the precursor protein. The propionibacteria produced the PAMP precursor constitutively during growth up to a level of ∼4 mg/liter, but the producing bacteria were unable to activate the precursor. The requirement for an external protease represents a novel strategy for generating antimicrobial peptides.
Transcription Factor FnrP from
Paracoccus denitrificans
Contains an Iron-Sulfur Cluster and Is Activated by Anoxia: Identification of Essential Cysteine Residues
Authors: Hutchings Matthew I. , Crack Jason C. , Shearer Neil , Thompson Benjamin J. , Thomson Andrew J. , Spiro Stephen
Published: July 2002

The
Paracoccus denitrificans
transcription factor FnrP has been characterized using artificial FNR-dependent promoter-
lacZ
fusion plasmids in
Escherichia coli
. FnrP can activate both class I and class II FNR-dependent promoters in response to anoxia but shows a marked preference for the class II promoter, where the FNR binding site is centered at −41.5 with respect to the transcription start site. FnrP was found to be inactive in an
iscS
mutant in vivo, demonstrating a requirement for cysteine desulfurase activity to assemble an iron-sulfur cluster in FnrP. Accordingly, an iron-sulfur cluster could be reconstituted into the purified protein in vitro using cysteine desulfurase, ferrous ions, and cysteine. Thus, FnrP is a true orthologue of FNR from
E. coli
and switches on target genes in response to anoxia. Inactivation of FnrP by oxygen very likely involves the oxidative disassembly of an iron-sulfur cluster. Possible ligands for the iron-sulfur cluster were identified by substituting each of the seven cysteine residues with serine and characterizing the altered proteins in vivo. Four substituted proteins showed activities less than 5% of the wild type, and so identify the four cysteines (Cys-14, Cys-17, Cys-25, and Cys-113) that are most likely to be involved in cluster ligation. The effects of N-oxides, NO-releasing compounds and a nitrosating agent on FNR and FnrP activity were investigated in vivo using the reporter system. Both proteins are very sensitive to the inclusion of sodium nitroprusside (a source of NO
+
) in defined growth media but are only moderately sensitive to those sources of NO that were tested.
Role of the RecBCD Recombination Pathway in
Salmonella
Virulence
Authors: Cano David A. , Pucciarelli M. Graciela , García-del Portillo Francisco , Casadesús Josep
Published: July 2002

Mutants of
Salmonella enterica
lacking the RecBC function are avirulent in mice and unable to grow inside macrophages (N. A. Buchmeier, C. J. Lipps, M. Y. H. So, and F. Heffron, Mol. Microbiol. 7:933–936, 1993). The virulence-related defects of RecBC
−
mutants are not suppressed by
sbcB
and
sbcCD
mutations, indicating that activation of the RecF recombination pathway cannot replace the virulence-related function(s) of RecBCD. Functions of the RecF pathway such as RecJ and RecF are not required for virulence. Since the RecBCD pathway, but not the RecF pathway, is known to participate in the repair of double-strand breaks produced during DNA replication, we propose that systemic infection by
S. enterica
may require RecBCD-mediated recombinational repair to prime DNA replication inside phagocytes. Mutants lacking both RecD and RecJ are also attenuated in mice and are unable to proliferate in macrophages, suggesting that exonucleases V and IX provide alternative functions for RecBCD-mediated recombinational repair during
Salmonella
infection.
Nonisotopic Detection of Human Papillomavirus DNA in Clinical Specimens Using a Consensus PCR and a Generic Probe Mix in an Enzyme-Linked Immunosorbent Assay Format
Authors: Kornegay J. R. , Shepard A. P. , Hankins C. , Franco E. , Lapointe N. , Richardson H. ,   The Canadian Women's HIV Study Group, Coutleé F.
Published: July 2002

We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR–enzyme-linked immunosorbent assay format to screen for the presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated β-globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses more objective interpretation criteria, making it useful in studies of HPV natural history.
V3 Serological Subtyping of Human Immunodeficiency Virus Type 2 Infection Is Not Relevant
Authors: Plantier Jean-Christophe , Damond Florence , Souquières Sandrine , Brun-Vézinet Françoise , Simon François , Barin Francis
Published: July 2002

V3 enzyme immunoassays have been shown to discriminate effectively between human immunodeficiency virus type 1 (HIV-1) subtypes. The aim of this study was to investigate the feasibility of V3 serotyping for HIV-2 infection. We serotyped 29 sera with three peptides, corresponding to the V3 loop of subtypes A, B, and D of HIV-2. Sera were collected from HIV-2-infected patients, whose infecting strains were sequenced and subjected to phylogenetic analysis. Our results indicate that HIV-2 serotyping using V3 peptides is not relevant. V3 serotyping data were not consistent with genotyping results. The V3-A and V3-D peptides displayed poor discrimination, and the V3-B peptide was not representative of circulating viruses. Comparison of amino acid sequences and serotype reactivities demonstrated the importance of positions 309 and 314, located on either side of the tip of the V3 loop, in antibody binding.
Use of Spoligotyping To Study the Evolution of the Direct Repeat Locus by IS
6110
Transposition in
Mycobacterium tuberculosis
Authors: Legrand Eric , Filliol Ingrid , Sola Christophe , Rastogi Nalin
Published: July 2002

Based on the variability of 43 spacers within the direct repeat (DR) locus of
Mycobacterium tuberculosis
complex organisms, spoligotyping is a rapid method that aids in the study of the epidemiology of tuberculosis. It was recently hypothesized that despite its presence in the DR locus, spacer 31 could not be amplified in
M. tuberculosis
clinical isolates belonging to spoligotype 50 due to the insertion of an extra copy of IS
6110
between spacers 31 and 32 that could lead to an asymmetrical split of the primer targets (I. Filliol, C. Sola, and N. Rastogi, J. Clin. Microbiol. 38:1231–1234, 2000). In the present investigation, previous observations were extended to 25 clinical isolates of type 50 showing that the primer set IS6-DRb that selectively amplified the left and central DR regions was indeed able to demonstrate the presence of spacer 31. IS
6110
-restriction fragment length polymorphism (RFLP) and DR-RFLP showed that type 50 isolates were characterized by the presence of two copies of IS
6110
associated with the DR locus and an additional double IS
6110
band of 1.4 kb. The primer set IS3-IS6 was then used to selectively amplify a 750-bp inter-IS
6110
fragment within the DR locus. The sequencing of the central DR region corroborated our previous findings and showed that the absence of spacer 31 among the type 50 isolates was due to the asymmetric insertion of an extra copy of IS
6110
between spacers 31 and 32, leading to an unequal split of the DRa-DRb target into two portions, of 6 and 30 bp, respectively. These results show that the DR locus constitutes an ideal IS
6110
preferential locus (
ipl
), permitting the insertion of two or more copies of IS
6110
, and provide new clues for epidemiological and phylogenetic interpretation of changes in IS
6110
-RFLP and spoligotyping profiles.
Characterization of Human Cytomegalovirus Strains by Analysis of Short Tandem Repeat Polymorphisms
Authors: Walker Adrianne , Petheram Susan J. , Ballard Linda , Murph Jody R. , Demmler Gail J. , Bale James F.
Published: July 2002

Human cytomegalovirus (HCMV) strains display genetic polymorphisms, and these polymorphisms can be analyzed to study viral transmission and pathogenesis. Recently, short tandem repeat (STR) length polymorphisms have been identified in the HCMV genome. We assessed the utility of STRs in characterizing HCMV strains and found that a multiplexed PCR assay using primers based upon these STRs accurately maps HCMV strains. Using primers for 10 microsatellite regions, the STR profiles of 44 wild-type and 2 laboratory strains of HCMV were characterized. The results of STR analysis were compared with those for strain characterization using nucleotide sequencing and restriction fragment length polymorphism analysis. In each instance, STR analysis accurately and specifically identified strains that were indistinguishable or distinct by conventional molecular analysis. Analysis of short tandem repeats also detected polymorphisms that supported simultaneous excretion of two HCMV strains. These results indicate that STR analysis allows rapid, precise molecular characterization of HCMV strains.
Identification of
Aspergillus fumigatus
and Related Species by Nested PCR Targeting Ribosomal DNA Internal Transcribed Spacer Regions
Authors: Zhao Jun , Kong Fanrong , Li Ruoyu , Wang Xiaohong , Wan Zhe , Wang Duanli
Published: July 2002

Aspergillus fumigatus
is the most common species that causes invasive aspergillosis. In order to identify
A. fumigatus
, partial ribosomal DNA (rDNA) from two to six strains of five different
Aspergillus
species was sequenced. By comparing sequence data from GenBank, we designed specific primer pairs targeting rDNA internal transcribed spacer (ITS) regions of
A. fumigatus
. A nested PCR method for identification of other
A. fumigatus
-related species was established by using the primers. To evaluate the specificities and sensitivities of those primers, 24 isolates of
A. fumigatus
and variants, 8 isolates of
Aspergillus nidulans
, 7 isolates of
Aspergillus flavus
and variants, 8 isolates of
Aspergillus terreus
, 9 isolates of
Aspergillus niger
, 1 isolate each of
Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus versicolor, Aspergillus wangduanlii, Aspergillus qizutongii, Aspergillus beijingensis
, and
Exophiala dermatitidis
, 4 isolates of
Candida
, 4 isolates of bacteria, and human DNA were used. The nested PCR method specifically identified the
A. fumigatus
isolates and closely related species and showed a high degree of sensitivity. Additionally, four
A. fumigatus
strains that were recently isolated from our clinic were correctly identified by this method. Our results demonstrate that these primers are useful for the identification of
A. fumigatus
and closely related species in culture and suggest further studies for the identification of
Aspergillus fumigatus
species in clinical specimens.
Serological Expression Cloning and Immunological Evaluation of MTB48, a NovelMycobacterium tuberculosisAntigen
Authors: Lodes Michael J. , Dillon Davin C. , Mohamath Raodoh , Day Craig H. , Benson Darin R. , Reynolds Lisa D. , McNeill Patricia , Sampaio Diana Pedral , Skeiky Yasir A. W. , Badaro Roberto , Persing David H. , Reed Steven G. , Houghton Raymond L.
Published: July 2002

Improved diagnostics are needed for the detection ofMycobacterium tuberculosis,especially for patients with smear-negative disease. To address this problem, we have screenedM. tuberculosis(H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity withM. tuberculosislysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of theM. tuberculosisH37Rv isolate and showed 99% identity to theMycobacterium bovisandM. bovisBCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing theesat-6gene that is deleted in theM. bovisBCG isolate. Themtb481,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in theM. tuberculosisandM. bovisisolates tested but not in other mycobacterial species tested, includingMycobacterium lepraeandMycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and theM. tuberculosis38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity forM. tuberculosisinfection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.
Clarithromycin-Susceptible and -Resistant
Helicobacter pylori
Isolates with Identical Randomly Amplified Polymorphic DNA-PCR Genotypes Cultured from Single Gastric Biopsy Specimens Prior to Antibiotic Therapy
Authors: van der Ende A. , van Doorn L.-J. , Rooijakkers S. , Feller M. , Tytgat G. N. J. , Dankert J.
Published: July 2002

Of the
Helicobacter pylori
populations from 976 patients, six contained clarithromycin-resistant as well as -susceptible colonies. In each heterogeneous
H. pylori
population, resistant
H. pylori
colonies harbored identical 23S ribosomal DNA (rDNA) mutations associated with clarithromycin resistance, while the susceptible
H. pylori
colonies all had wild-type 23S rDNA. The resistant and susceptible colonies of each of the heterogeneous
H. pylori
populations had identical randomly amplified polymorphic DNA-PCR genotypes. In conclusion, evaluation of antimicrobial susceptibility can be misinterpreted if only a single colony from the primary
H. pylori
population is used to test for clarithromycin susceptibility.
Antibody-Secreting Cell Responses to Rotavirus Proteins in Gnotobiotic Pigs Inoculated with Attenuated or Virulent Human Rotavirus
Authors: Chang K. O. , Vandal O. H. , Yuan L. , Hodgins D. C. , Saif L. J.
Published: July 2002

Because of their similarities to infants in mucosal immune responses and their susceptibility to human rotavirus (HRV) diarrhea, gnotobiotic pigs provide a useful model for rotaviral disease. In this study, we performed quantitative enzyme-linked immunospot (ELISPOT) assays to measure local and systemic isotype-specific antibody-secreting cell (ASC) responses to individual structural (VP4, VP6, and VP7) and nonstructural (NSP3 and NSP4) proteins of Wa HRV. The
Spodoptera frugiperda
cells expressing each recombinant baculovirus HRV protein were formalin fixed and used as antigen for ELISPOT assays. Neonatal gnotobiotic pigs were orally inoculated once with virulent Wa (WaV) or three times with attenuated Wa (WaA) HRV or mock inoculated (Mock) and then were challenged with virulent Wa (WaV/PC) 28 days after the first inoculation. The ASCs from intestinal and systemic lymphoid tissues of pigs from each group were quantitated by ELISPOT assay at the day of challenge, at postinoculation day 28 (WaV, WaA, and Mock) or at postchallenge day (PCD) 7 (WaV+WaV/PC, WaA+WaV/PC, and Mock+WaV/PC). In all virus-inoculated pigs, regardless of the inoculum, lymphoid tissue, or isotype, VP6 induced the highest numbers of ASCs, followed by VP4; ASCs specific for VP7, NSP3, and NSP4 were less numerous. At challenge, total HRV- and HRV protein-specific immunoglobulin A (IgA) and IgG ASCs in intestinal lymphoid tissues were significantly greater in WaV- than in WaA-inoculated pigs, and WaV pigs were fully protected against diarrhea postchallenge, whereas the WaA pigs were partially protected. At PCD 7, there were no significant differences in ASC numbers for any HRV proteins between the WaV+WaV/PC and WaA+WaV/PC groups.
Sequence Typing Confirms that
Campylobacter jejuni
Strains Associated with Guillain-Barré and Miller-Fisher Syndromes Are of Diverse Genetic Lineage, Serotype, and Flagella Type
Authors: Dingle K. E. , Van Den Braak N. , Colles F. M. , Price Lawrence J. , Woodward David L. , Rodgers Frank G. , Endtz H. P. , Van Belkum A. , Maiden M. C. J.
Published: July 2002

Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) are correlated with prior infection by
Campylobacter jejuni
in up to 40% of cases. Nucleotide sequence-based typing of 25
C. jejuni
isolates associated with neuropathy permitted robust comparisons with equivalent data from approximately 800
C. jejuni
isolates not associated with neuropathy. A total of 13 genetic lineages and 20
flaA
short variable region nucleotide sequences were present among the 25 isolates. A minority of isolates (4 of 25) had the
flaA
short variable region nucleotide sequences that were previously proposed as a marker for GBS-associated isolates. These 4 isolates probably represented the Penner serotype 19 lineage, which has been proposed to have an association with GBS.
Identification of
Streptococcus pneumoniae
Revisited
Authors: Kellogg James A. , Bankert David A. , Elder Carol J. , Gibbs Joanne L. , Smith Marie C.
Published: July 2002

The sensitivities and specificities of several different diagnostic assays for
Streptococcus pneumoniae
were assessed using 99 clinical isolates of
S. pneumoniae
and 101 viridans streptococci and were as follows: Pneumoslide, 99 and 87%, respectively; Directigen, 100 and 85%, respectively; Phadebact, 100 and 98%, respectively; deoxycholate drop test, 99 and 98%, respectively; deoxycholate tube test, 100 and 99%, respectively; optochin, 99 and 98%, respectively; and Gram Positive Identification Card, 90 and 96%, respectively. Identification of clinical isolates of
S. pneumoniae
should be confirmed using one or more diagnostic assays with well-documented high (e.g., ≥95%) sensitivities and specificities.
Association of
cagA
and
vacA
Genotypes of
Helicobacter pylori
with Gastric Diseases in Estonia
Authors: Andreson Helena , Lõivukene Krista , Sillakivi Toomas , Maaroos Heidi-Ingrid , Ustav Mart , Peetsalu Ants , Mikelsaar Marika
Published: July 2002

Gastric biopsy specimens from 156 adult patients from southern Estonia suffering from chronic gastritis, peptic ulcer disease, and perforated peptic ulcer were analyzed by PCR. The
cagA
gene was evenly distributed throughout 87% of the specimens from the patients with the different gastric diseases. The presence of the
cagA
gene correlated with that of
vacA
signal sequence type s1a (99%). However, no clear differences were found in the distribution of
cagA
and
vacA
genotypes among patients in Estonia with severe perforated peptic ulcer, uncomplicated peptic ulcer, or chronic gastritis.
Immunofluorescence Technique Using HeLa Cells Expressing Recombinant Nucleoprotein for Detection of Immunoglobulin G Antibodies to Crimean-Congo Hemorrhagic Fever Virus
Authors: Saijo Masayuki , Qing Tang , Niikura Masahiro , Maeda Akihiko , Ikegami Tetsuro , Sakai Koji , Prehaud Christophe , Kurane Ichiro , Morikawa Shigeru
Published: July 2002

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections.
Molecular Characterization of Extended-Spectrum β-Lactamases Produced by Nosocomial Isolates of
Enterobacteriaceae
from an Italian Nationwide Survey
Authors: Perilli Mariagrazia , Dell'Amico Emanuela , Segatore Bernardetta , de Massis Maria Rosaria , Bianchi Ciro , Luzzaro Francesco , Rossolini Gian Maria , Toniolo Antonio , Nicoletti Giuseppe , Amicosante Gianfranco
Published: July 2002

Extended-spectrum β-lactamases (ESBLs) are widespread in hospital settings worldwide. The present investigation was undertaken to assess the distribution and prevalence of ESBLs belonging to the TEM and SHV families in 448 ESBL-producing clinical isolates of
Enterobacteriaceae
collected from 10 different Italian hospitals. The natures of TEM and SHV determinants were identified by direct sequencing of PCR-amplified genes. TEM-52 and SHV-12 were the most common variants, and they were found in most hospitals and in several different species. Other less frequent variants included TEM-5, TEM-12, TEM-15, TEM-19, TEM-20, TEM-24, TEM-26, TEM-43, TEM-60, TEM-72, TEM-87, SHV-2a, SHV-5, and SHV-11.
Proteus mirabilis
was the most common producer of TEM-type ESBLs, while
Klebsiella pneumoniae
was the most common producer of SHV-type ESBLs. The distribution of TEM- and SHV-type ESBL variants in
Enterobacteriaceae
from Italian hospitals exhibited notable differences from those from other geographical settings.
Diffusely Adherent
Escherichia coli
as a Cause of Acute Diarrhea in Young Children in Northeast Brazil: a Case-Control Study
Authors: Scaletsky Isabel C. A. , Fabbricotti Sandra H. , Carvalho Rozane L. B. , Nunes Claudia R. , Maranhão Helcio S. , Morais Mauro B. , Fagundes-Neto Ulysses
Published: July 2002

In a prospective study carried out in two urban centers in northeastern Brazil, 195 HEp-2-adherent
Escherichia coli
strains were isolated; 110 were identified as the only pathogen in stools of children with diarrhea, and 85 were from controls. Enteropathogenic
E. coli
isolates were identified in 21 children with diarrhea (8.9%) and 7 children without diarrhea (3.0%), and they were significantly associated with diarrhea (
P
< 0.01). Enteroaggregative
E. coli
strains were isolated from 40 children with diarrhea (16.9%) and 38 children without diarrhea (16.4%) and showed no correlation with diarrhea (
P
> 0.5). In 49 children with diarrhea (20.7%) and 40 children without diarrhea (17.3%), diffusely adherent
E. coli
(DAEC) isolates were detected and were not found to be associated with diarrhea (
P
= 0.41). However, after stratification, for children older than 12 months of age a significant correlation between DAEC infection and diarrhea was detected (
P
= 0.01). These results suggest that DAEC isolates should be considered potential pathogens in northeastern Brazil and also confirm the association of DAEC with age-dependent diarrhea.
Genotyping of Clinical Methicillin-Susceptible
Staphylococcus aureus
Isolates in a Dutch Teaching Hospital
Authors: Van Dijk Yvonne , Wielders Camiel L. C. , Fluit Ad C. , Paauw Armand , Diepersloot Rob J. A. , Mascini Ellen M.
Published: July 2002

Methicillin-susceptible
Staphylococcus aureus
isolates, recovered from 204 patients in our hospital in a 22-month period, were characterized by pulsed-field gel electrophoresis. Among the multiple
S. aureus
types six clonal lineages dominated, comprising isolates from 158 patients. Despite the limited genetic variation, cross-transmission was made plausible only sporadically.
Susceptibility Testing of Fluconazole by the NCCLS Broth Macrodilution Method, E-Test, and Disk Diffusion for Application in the Routine Laboratory
Authors: Vandenbossche Inge , Vaneechoutte Mario , Vandevenne Marleen , De Baere Thierry , Verschraegen Gerda
Published: July 2002

Antifungal susceptibility testing may be an important aid in the treatment of patients with life-threatening yeast infections. In order to establish the suitability of different susceptibility test methods for fluconazole with yeasts, the Rosco tablet and the E-test were compared with the gold standard NCCLS broth macrodilution method for 106 yeast strains. These included 102 clinical isolates of
Candida
spp., including
Candida glabrata
(
n
= 30),
Candida albicans
(
n
= 20),
Candida tropicalis
(
n
= 13),
Candida parapsilosis
(
n
= 10),
Candida krusei
(
n
= 8), plus
Cryptococcus neoformans
(
n
= 3),
Saccharomyces cerevisiae
(
n
= 2), and 16 strains belonging to other
Candida
spp. Four American Type Culture Collection strains of
Candida
were included as quality controls. The NCCLS method was found to be too complex and labor-intensive for routine testing. The E-test is an accurate alternative, but experience in determining MICs and careful attention to procedural details are critically important. The Rosco tablet showed the best agreement with the NCCLS reference method, especially when newly established breakpoints of
R
≤ 10 mm and
S
≥ 21 mm were used.
Molecular Characterization of Vancomycin-Resistant Enterococci Repopulating the Gastrointestinal Tract following Treatment with a Novel Glycolipodepsipeptide, Ramoplanin
Authors: Baden L. R. , Critchley I. A. , Sahm D. F. , So W. , Gedde M. , Porter S. , Moellering R. C. , Eliopoulos G.
Published: July 2002

We characterized baseline and repopulating stool isolates recovered during a phase II trial of ramoplanin for the treatment of patients with stool carriage of vancomycin-resistant enterococci (VRE). Repopulation with a strain with a related genotype was found in 74, 60, and 53% of individuals in groups treated with placebo, 100 mg of ramoplanin, and 400 mg of ramoplanin, respectively. All ramoplanin-treated patients with a culture positive for VRE at day 7 had a relapse caused by a genotypically related isolate. In ramoplanin-treated patients, antibiotics with activities against anaerobic organisms were associated with positive cultures on day 7 (relative risk [RR] = 8.8;
P
= 0.004), and the avoidance of such antibiotics was significantly associated with culture negativity through day 21 (RR = 0.16;
P
= 0.02).
Real-Time PCR Coupled with Automated DNA Extraction and Detection of Galactomannan Antigen in Serum by Enzyme-Linked Immunosorbent Assay for Diagnosis of Invasive Aspergillosis
Authors: Costa Catherine , Costa Jean-Marc , Desterke Christophe , Botterel Françoise , Cordonnier Catherine , Bretagne Stéphane
Published: July 2002

To improve the diagnosis of invasive aspergillosis (IA), we developed a LightCycler PCR assay targeted to
Aspergillus fumigatus
and
A. flavus
mitochondrial DNA. To avoid contamination, fully automated nucleic acid extraction with the MagNA Pure LC apparatus was used. The linearity of the results was achieved over a 6-log range of input
A. fumigatus
DNA, from 0.3 ng to 3 fg/10 μl of water. We retrospectively compared the LightCycler PCR and an enzyme-linked immunosorbent assay for the detection of galactomannan (GM) in serum from 14 patients with IA. The GM assay was more frequently positive (57 of 109; 52%) than the PCR assay (49 of 109; 45%). The LightCycler PCR assay, combined with automated DNA extraction, could be used in association with the GM assay to improve the reliability of IA diagnosis.
H-2Db−/−Mice Are Susceptible to Persistent Infection by Theiler's Virus
Authors: Azoulay-Cayla Arièle , Dethlefs Sven , Pérarnau Béatrice , Larsson-Sciard Eva-Lotta , Lemonnier François A. , Brahic Michel , Bureau Jean-François
Published: July 2002

H-2bmice are resistant to persistent infection of the central nervous system by Theiler's virus. They clear the infection 7 to 10 days after intracranial inoculation. Resistance maps to theH-2Dgene and not to theH-2Kgene and is associated with a potent antiviral cytotoxic T-lymphocyte (CTL) response. We usedH-2bmice in which theH-2Dor theH-2Kgene had been inactivated to dissect the respective roles of these genes in resistance. We report thatH-2D−/−but notH-2K−/−mice were susceptible to persistent infection. Furthermore, whereasH-2K−/−mice mounted a vigorous virus-specific CTL response, similar to that of control C57BL/6 mice, the CTL response ofH-2D−/−mice was nil or minimal. Using target cells transfected with theH-2Dbor theH-2Kbgene, we showed that theH-2K-restricted CTL response against the virus was minimal inH-2D−/−mice. These results demonstrate that theH-2DbandH-2Kbgenes play nonredundant roles in the resistance to this persistent infection.
Mutations in the E1 Hydrophobic Domain of Rubella Virus Impair Virus Infectivity but Not Virus Assembly
Authors: Qiu Zhiyong , Yao Jiansheng , Cao Hanwei , Gillam Shirley
Published: July 2002

Rubella virus (RV) virions contain three structural proteins, a capsid protein that interacts with viral genomic RNA to form a nucleocapsid and two membrane glycoproteins, E2 and E1. We found that substitution of either an aspartic acid residue at Gly93 (G93D) or a glycine residue at Pro104 (P104G) in the internal hydrophobic domain of E1 affected virus infectivity but not virus assembly. Viruses carrying G93D and P104G mutations had impaired infectivity, reduced 1,000-fold and 10-fold, respectively. A revertant was isolated from the G93D mutant. Sequencing analysis showed that the substituted aspartic acid residue in G93D mutant had reverted to the original glycine residue, suggesting the involvement of Gly93 in membrane fusion during viral entry.
Efficient Translation of Rotavirus mRNA Requires Simultaneous Interaction of NSP3 with the Eukaryotic Translation Initiation Factor eIF4G and the mRNA 3′ End
Authors: Vende Patrice , Piron Maria , Castagné Nathalie , Poncet Didier
Published: July 2002

In contrast to the vast majority of cellular proteins, rotavirus proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically binds the 3′-end consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G. Here we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between the cap structure and the 3′ end of rotavirus mRNA was observed in NSP3-expressing cells. The enhancement of viral mRNA translation by NSP3 was also observed in a rabbit reticulocyte lysate translation system supplemented with recombinant NSP3. The use of NSP3 mutants indicates that its RNA- and eIF4G-binding domains are both required to enhance the translation of viral mRNA. The results reported here show that NSP3 forms a link between viral mRNA and the cellular translation machinery and hence is a functional analogue of cellular poly(A)-binding protein.
Characterization of a Novel Respiratory Syncytial Virus-Specific Human Cytotoxic T-Lymphocyte Epitope
Authors: Goulder Philip J. R. , Lechner Franziska , Klenerman Paul , McIntosh Kenneth , Walker Bruce D.
Published: July 2002

Respiratory syncytial virus (RSV) infection is a major cause of morbidity in childhood worldwide. The first human RSV-specific cytotoxic T-lymphocyte epitope to be defined is described. This HLA B7-restricted epitope in nucleoprotein (NP) was detectable in four healthy, B7-positive adult subjects using B7-RSV-NP tetrameric complexes to stain CD8+T cells.
Coinfection of SCID-hu Thy/Liv Mice with Human Herpesvirus 6 and Human Immunodeficiency Virus Type 1
Authors: Gobbi Alberto , Stoddart Cheryl A. , Locatelli Giuseppe , Santoro Fabio , Bare Christopher , Linquist-Stepps Valerie , Moreno Mary E. , Abbey Nancy W. , Herndier Brian G. , Malnati Mauro S. , McCune Joseph M. , Lusso Paolo
Published: July 2002

Human herpesvirus 6 (HHV-6) has been proposed as a potential cofactor in the progression of human immunodeficiency virus type 1 (HIV-1) disease. We used the SCID-hu Thy/Liv mouse model to evaluate the in vivo interactions between HHV-6 and HIV-1. Our results demonstrate that HHV-6 and HIV-1 can simultaneously replicate in the human thymus in vivo. In this model, however, the presence of one virus appears not to modify the replication or cytopathicity of the other.
Viral Vascular Endothelial Growth Factor Plays a Critical Role in Orf Virus Infection
Authors: Savory Loreen J. , Stacker Steven A. , Fleming Stephen B. , Niven Brian E. , Mercer Andrew A.
Published: July 2002

Infection by the parapoxvirus orf virus causes proliferative skin lesions in which extensive capillary proliferation and dilation are prominent histological features. This infective phenotype may be linked to a unique virus-encoded factor, a distinctive new member of the vascular endothelial growth factor (VEGF) family of molecules. We constructed a recombinant orf virus in which the VEGF-like gene was disrupted and show that inactivation of this gene resulted in the loss of three VEGF activities expressed by the parent virus: mitogenesis of vascular endothelial cells, induction of vascular permeability, and activation of VEGF receptor 2. We used the recombinant orf virus to assess the contribution of the viral VEGF to the vascular response seen during orf virus infection of skin. Our results demonstrate that the viral VEGF, while recognizing a unique profile of the known VEGF receptors (receptor 2 and neuropilin 1), is able to stimulate a striking proliferation of blood vessels in the dermis underlying the site of infection. Furthermore, the data demonstrate that the viral VEGF participates in promoting a distinctive pattern of epidermal proliferation. Loss of a functional viral VEGF resulted in lesions with markedly reduced clinical indications of infection. However, viral replication in the early stages of infection was not impaired, and only at later times did it appear that replication of the recombinant virus might be reduced.
Molecular Correlates of Influenza A H5N1 Virus Pathogenesis in Mice
Authors: Katz Jacqueline M. , Lu Xiuhua , Tumpey Terrence M. , Smith Catherine B. , Shaw Michael W. , Subbarao Kanta
Published: July 2002

Highly pathogenic avian influenza A H5N1 viruses caused an outbreak of human respiratory illness in Hong Kong. Of 15 human H5N1 isolates characterized, nine displayed a high-, five a low-, and one an intermediate-pathogenicity phenotype in the BALB/c mouse model. Sequence analysis determined that five specific amino acids in four proteins correlated with pathogenicity in mice. Alone or in combination, these specific residues are the likely determinants of virulence of human H5N1 influenza viruses in this model.
Glycoprotein Exchange Vectors Based on Vesicular Stomatitis Virus Allow Effective Boosting and Generation of Neutralizing Antibodies to a Primary Isolate of Human Immunodeficiency Virus Type 1
Authors: Rose Nina F. , Roberts Anjeanette , Buonocore Linda , Rose John K.
Published: July 2002

Live recombinant vesicular stomatitis viruses (VSVs) expressing foreign antigens are highly effective vaccine vectors. However, these vectors induce high-titer neutralizing antibody directed at the single VSV glycoprotein (G), and this antibody alone can prevent reinfection and boosting with the same vector. To determine if efficient boosting could be achieved by changing the G protein of the vector, we have developed two new recombinant VSV vectors based on the VSV Indiana serotype but with the G protein gene replaced with G genes from two other VSV serotypes, New Jersey and Chandipura. These G protein exchange vectors grew to titers equivalent to wild-type VSV and induced similar neutralizing titers to themselves but no cross-neutralizing antibodies to the other two serotypes. The effectiveness of these recombinant VSV vectors was illustrated in experiments in which sequential boosting of mice with the three vectors, all encoding the same primary human immunodeficiency virus (HIV) envelope protein, gave a fourfold increase in antibody titer to an oligomeric HIV envelope compared with the response in animals receiving the same vector three times. In addition, only the animals boosted with the exchange vectors produced antibodies neutralizing the autologous HIV primary isolate. These VSV envelope exchange vectors have potential as vaccines in immunizations when boosting of immune responses may be essential.