bluuebunny/crossref_metadata_2025 huggingface dataset:
A Quorum-Sensing Signaling System Essential for Genetic Competence in
Streptococcus mutans
Is Involved in Biofilm Formation
Authors: Li Yung-Hua , Tang Nan , Aspiras Marcelo B. , Lau Peter C. Y. , Lee Janet H. , Ellen Richard P. , Cvitkovitch Dennis G.
Published: July 2002

In a previous study, a quorum-sensing signaling system essential for genetic competence in
Streptococcus mutans
was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of
S. mutans
to initiate biofilm formation. To test this hypothesis,
S. mutans
wild-type strain NG8 and its knockout mutants defective in
comC
,
comD
,
comE
, and
comX
, as well as a
comCDE
deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The
comC
mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the
comD
and
comE
mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type
comC
gene into the cultures restored the wild-type biofilm architecture of
comC
mutants but showed no effect on the
comD
,
comE
, or
comX
mutant biofilms. The fact that biofilms formed by
comC
mutants differed from the
comD
,
comE
, and
comX
mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type
comC
gene on a shuttle vector into the
comCDE
deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in
S. mutans
is important for the formation of biofilms by this gram-positive organism.
Formation of dTDP-Rhamnose Is Essential for Growth of Mycobacteria
Authors: Ma Yufang , Pan Fei , McNeil Michael
Published: July 2002

It was determined that the dTDP-rhamnose synthesis gene,
rmlD
, could be inactivated in
Mycobacterium smegmatis
only in the presence of a rescue plasmid carrying functional
rmlD.
Hence, dTDP-rhamnose biosynthesis is essential for the growth of mycobacteria and the targeting of dTDP-rhamnose synthesis for new tuberculosis drugs is supported.
Quorum Sensing Is Not Required for Twitching Motility in
Pseudomonas aeruginosa
Authors: Beatson Scott A. , Whitchurch Cynthia B. , Semmler Annalese B. T. , Mattick John S.
Published: July 2002

It has been reported that mutations in the quorum-sensing genes
lasI
and
rhlI
in
Pseudomonas aeruginosa
result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of
lasI
and
rhlI
and the corresponding regulatory genes
lasR
and
rhlR
and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of
lasI
and
rhlI
mutants. Further analysis showed that the stable twitching-defective variants of
lasI
and
rhlI
mutants had arisen as a consequence of secondary mutations in
vfr
and
algR
, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture,
lasI
and
rhlI
mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in
P. aeruginosa
strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.
Point Mutations in HpuB Enable Gonococcal HpuA Deletion Mutants To Grow on Hemoglobin
Authors: Chen Ching-Ju , Mclean Dalton , Thomas Christopher E. , Anderson James E. , Sparling P. Frederick
Published: July 2002

Neisseria gonorrhoeae
ordinarily requires both HpuA and HpuB to use hemoglobin (Hb) as a source of iron for growth. Deletion of HpuA resulted in reduced Hb binding and failure of growth on Hb. We identified rare Hb-utilizing colonies (Hb
+
) from an
hpuA
deletion mutant of FA1090, which fell into two phenotypic classes. One class of the Hb
+
revertants required expression of both TonB and HpuB for growth on Hb, while the other class required neither TonB nor HpuB. All TonB/HpuB-dependent mutants had single amino acid alterations in HpuB, which occurred in clusters, particularly near the C terminus. The point mutations in HpuB did not restore normal Hb binding. Human serum albumin inhibited Hb-dependent growth of HpuB point mutants lacking HpuA but did not inhibit growth when expression of HpuA was restored. Thus, HpuB point mutants internalized heme in the absence of HpuA despite reduced binding of Hb. HpuA facilitated Hb binding and was important in allowing use of heme from Hb for growth.
Lateral Flagella and Swarming Motility in
Aeromonas
Species
Authors: Kirov Sylvia M. , Tassell Bronwen C. , Semmler Annalese B. T. , O’Donovan Lisa A. , Rabaan Ali A. , Shaw Jonathan G.
Published: July 2002

Swarming motility, a flagellum-dependent behavior that allows bacteria to move over solid surfaces, has been implicated in biofilm formation and bacterial virulence. In this study, light and electron microscopic analyses and genetic and functional investigations have shown that at least 50% of
Aeromonas
isolates from the species most commonly associated with diarrheal illness produce lateral flagella which mediate swarming motility.
Aeromonas
lateral flagella were optimally produced when bacteria were grown on solid medium for ≈8 h. Transmission and thin-section electron microscopy confirmed that these flagella do not possess a sheath structure. Southern analysis of
Aeromonas
reference strains and strains of mesophilic species (
n
= 84, varied sources and geographic regions) with a probe designed to detect lateral flagellin genes (
lafA1
and
lafA2
) showed there was no marked species association of
laf
distribution. Approximately 50% of these strains hybridized strongly with the probe, in good agreement with the expression studies. We established a reproducible swarming assay (0.5% Eiken agar in Difco broth, 30°C) for
Aeromonas
spp. The
laf
-positive strains exhibited vigorous swarming motility, whereas
laf
-negative strains grew but showed no movement from the inoculation site. Light and scanning electron microscopic investigations revealed that lateral flagella formed bacterium-bacterium linkages on the agar surface. Strains of an
Aeromonas caviae
isolate in which lateral flagellum expression was abrogated by specific mutations in flagellar genes did not swarm, proving conclusively that lateral flagella are required for the surface movement. Whether lateral flagella and swarming motility contribute to
Aeromonas
intestinal colonization and virulence remains to be determined.
Identification and Functional Characterization of
flgM
, a Gene Encoding the Anti-Sigma 28 Factor in
Pseudomonas aeruginosa
Authors: Frisk A. , Jyot J. , Arora S. K. , Ramphal R.
Published: July 2002

We describe here the functional characterization of the putative
flgM
gene of
Pseudomonas aeruginosa
. FlgM of
P. aeruginosa
is most similar to FlgM of
Vibrio parahaemolyticus
. A conserved region is present in the C-terminal half of the FlgM of
P. aeruginosa
and in FlgM homologues of other organisms that includes the σ
28
binding domain. A role for the
flgM
gene of
P. aeruginosa
in motility was demonstrated by its inactivation. The β-galactosidase activity of a transcriptional fusion of the
fliC
promoter to
lacZ
was upregulated in the
flgM
mutant, suggesting that the activity of FliA, the sigma factor that regulates
fliC
, was increased. Consistent with these results, an increased amount of flagellin was demonstrated in the
flgM
mutant of
P. aeruginosa
strain PAK by Western blot, suggesting that FlgM negatively regulates transcription of
fliC
by inhibiting the activity of FliA. Direct interaction of the
P. aeruginosa
FlgM with the alternative sigma factor σ
28
was demonstrated by utilizing the yeast two-hybrid system. Three putative consensus σ
54
recognition sites and one σ
28
site were found in the
flgM
upstream region. However, analysis of the transcriptional fusion of the
flgM
promoter to
lacZ
in different mutant backgrounds showed that the
flgM
promoter was not entirely dependent on either σ
28
or σ
54
. A transcript was detected by primer extension that was 8 bp downstream of the consensus σ
28
-binding site. Thus, a system for the control of flagellin synthesis by FlgM exists in
P. aeruginosa
that is different from that in the enteric bacteria and seems to be most similar to that of
V. cholerae
where both σ
28
-dependent and -independent mechanisms of transcription exist.
Frequency of
rpoB
Mutations Inside and Outside the Cluster I Region in Rifampin-Resistant Clinical
Mycobacterium tuberculosis
Isolates
Authors: Heep Markus , Brandstätter Barbara , Rieger Ulrich , Lehn Norbert , Richter Elvira , Rüsch-Gerdes Sabine , Niemann Stefan
Published: July 2002

The prevalence of recently described mutation V176F, located in the beginning of the
rpoB
gene and associated with rifampin resistance and the wild-type cluster I sequence, was determined by analyzing the distribution of
rpoB
mutations among 80 rifampin (RIF)-resistant
Mycobacterium tuberculosis
strains isolated in Germany during 1997. The most frequent
rpoB
mutations were changes in codon 456 (52 isolates, 65%), followed by changes in codon 441 (13 isolates, 16%) and codon 451 (11 isolates, 14%). The V176F mutation was detected in one isolate of the study population and in 5 of 18 RIF-resistant strains with no cluster I mutation from six previously published studies. In three isolates, a mixture of resistant and susceptible subpopulations (heteroresistance) prohibited the detection of
rpoB
mutations in the initial analysis; however, in these isolates, cluster I mutations could be verified after a passage on RIF-containing medium. IS
6110
DNA fingerprinting of 76 strains revealed eight clusters comprising 27 strains with identical restriction fragment length polymorphism patterns that mainly also show identical
rpoB
mutations and identical or similar drug resistance patterns. In conclusion, our results indicate that the V176F mutation should be included in molecular tests for prediction of RIF resistance in
M. tuberculosis
. We further demonstrated that heteroresistance caused by a mixture of mycobacterial subpopulations with different susceptibilities to RIF may influence the sensitivity of molecular tests for detection of resistance.
Evaluation of Etest Method for Determining Posaconazole MICs for 314 Clinical Isolates of
Candida
Species
Authors: Pfaller M. A. , Messer S. A. , Mills K. , Bolmström A. , Jones R. N.
Published: July 2002

The performance of the Etest for posaconazole (SCH 56592) susceptibility testing of 314 isolates of
Candida
spp. was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. MICs were determined by Etest for all 314 isolates with RPMI agar containing 2% glucose (RPG agar) and were read after incubation for 48 h at 35°C. The
Candida
isolates included
C. albicans
(
n
= 174),
C. glabrata
(
n
= 57),
C. tropicalis
(
n
= 31),
C. parapsilosis
(
n
= 39),
C. krusei
(
n
= 5),
C. guilliermondii
(
n
= 6), and
C. lusitaniae
(
n
= 2). The Etest results correlated well with reference MICs. Overall agreement was 95%, and agreements for individual species were as follows:
C. krusei
, 100%;
C. albicans
, 98%;
C. tropicalis
, 97%;
C. glabrata
, 93%;
C. parapsilosis
, 85%;
C. guilliermondii
, 83%; and
C.
lusitaniae
, 50%. The problem of trailing end points was minimized with RPG agar, and good agreement with broth dilution MICs was obtained when discernible growth within an established ellipse was ignored. The Etest method using RPG agar appears to be a useful method for determining posaconazole susceptibilities of
Candida
species.
Evaluation of the NucliSens Basic Kit for Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in Genital Tract Specimens Using Nucleic Acid Sequence-Based Amplification of 16S rRNA
Authors: Mahony J. B. , Song X. , Chong S. , Faught M. , Salonga T. , Kapala J.
Published: July 2002

We evaluated a new RNA amplification and detection kit, the NucliSens Basic Kit (Organon Teknika), for the detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in genitourinary specimens. The Basic Kit provides an open platform for RNA amplification and detection and contains isolation reagents for nucleic acid extraction, nucleic acid sequence-based amplification (NASBA) reagents (enzymes and buffers), and a generic ruthenium-labeled probe for electrochemiluminescent (ECL) detection of amplified product. Using freshly purified and titrated stocks of
C. trachomatis
and
N. gonorrhoeae
and in vitro-generated RNA transcripts for sensitivity determinations, the Basic Kit detected 1 inclusion-forming unit of
C. trachomatis
, 1 CFU of
N. gonorrhoeae
, and 100 RNA molecules of 16S rRNA for both bacteria. The clinical performance of the Basic Kit was evaluated by testing a total of 250 specimens for
N. gonorrhoeae
by culture and NASBA and a total of 96 specimens for
C. trachomatis
by PCR and NASBA. The Basic Kit detected 139 of 142
N. gonorrhoeae
culture-positive specimens and gave a negative result for 73 of 74 culture-negative specimens, for a sensitivity and specificity of 97.9 and 98.7%, respectively. For
C. trachomatis
, the Basic Kit detected 24 of 24 PCR-positive specimens and gave a negative result for 71 of 72 PCR-negative specimens, for a sensitivity and specificity of 100 and 98.6%, respectively. The Basic Kit also detected specimens containing both
N. gonorrhoeae
and
C. trachomatis
, using a multiplex NASBA assay using primers for both bacteria. The NucliSens Basic Kit offers a versatile platform for the development of sensitive RNA detection assays for sexually transmitted diseases.
Assignment of CDC Weak Oxidizer Group 2 (WO-2) to the Genus
Pandoraea
and Characterization of Three New
Pandoraea
Genomospecies
Authors: Daneshvar Maryam I. , Hollis Dannie G. , Steigerwalt Arnold G. , Whitney Anne M. , Spangler Laura , Douglas Michael P. , Jordan Jean G. , MacGregor John P. , Hill Bertha C. , Tenover Fred C. , Brenner Don J. , Weyant Robbin S.
Published: July 2002

CDC weak oxidizer group 2 (WO-2) consists of nine phenotypically similar human clinical isolates received by the Centers for Disease Control and Prevention between 1989 and 1998. Four of the isolates were from blood, three were from sputum, and one each was from bronchial fluid and maxillary sinus. All are aerobic nonfermentative, motile gram-negative rods with one to eight polar flagella per cell. All grew at 25 and 35°C and were positive for catalase, urease (usually delayed 3 to 7 days), citrate, alkalinization of litmus milk, oxidization of glycerol (weakly), and growth on MacConkey agar and in nutrient broth without NaCl. All except one strain were oxidase positive with the Kovács method, and all except one isolate weakly oxidized
d
-glucose. All were negative for oxidation of
d
-xylose,
d
-mannitol, lactose, sucrose, maltose, and 20 other carbohydrates, esculin hydrolysis, indole production, arginine dihydrolase, and lysine and ornithine decarboxylase. Only two of nine isolates reduced nitrate. Broth microdilution susceptibilities were determined for all strains against 13 antimicrobial agents. Most of the strains were resistant to ampicillin, extended-spectrum cephalosporins, and aminoglycosides, including gentamicin, tobramycin, and amikacin, but they varied in their susceptibility to fluoroquinolones. High-performance liquid chromatographic and mass spectrometric analyses of the WO-2 group identified ubiquinone-8 as the major quinone component. The percent G+C of the WO-2 strains ranged from 65.2 to 70.7% (thermal denaturation method). All shared a common cellular fatty acid (CFA) profile, which was characterized by relatively large amounts (7 to 22%) of 16:1ω7c, 16:0, 17:0cyc, 18:1ω7c, and 19:0cyc
11-12
; small amounts (1 to 3%) of 12:0 and 14:0; and eight hydroxy acids, 2-OH-12:0 (4%), 2-OH-14:0 (trace), 3-OH-14:0 (12%), 2-OH-16:1 (1%), 2-OH-16:0 (3%), 3-OH-16:0 (4%), 2-OH-18:1 (2%), and 2-OH-19:0cyc (3%). This profile is similar to the CFA profile of
Pandoraea
, a recently described genus associated with respiratory infections in cystic fibrosis patients (T. Coenye et al., Int. J. Syst. Evol. Microbiol., 50:887–899, 2000). Sequencing of the 16S rRNA gene (1,300 bp) for all nine strains indicated a high level (≥98.8%) of homogeneity with
Pandoraea
spp. type strains. DNA-DNA hybridization analysis (hydroxyapatite method; 70°C) confirmed the identity of WO-2 with the genus
Pandoraea
and assigned three strains to
Pandoraea apista
and three to
Pandoraea pnomenusa
, and identified three additional new genomospecies containing one strain each (ATCC BAA-108, ATCC BAA-109, ATCC BAA-110). This study also shows that
Pandoraea
isolates may be encountered in blood cultures from patients without cystic fibrosis.
Comparison of
Flavivirus
Universal Primer Pairs and Development of a Rapid, Highly Sensitive Heminested Reverse Transcription-PCR Assay for Detection of Flaviviruses Targeted to a Conserved Region of the NS5 Gene Sequences
Authors: Scaramozzino Natale , Crance Jean-Marc , Jouan Alain , DeBriel Dominique A. , Stoll Françoise , Garin Daniel
Published: July 2002

Arthropod-transmitted flaviviruses are responsible for considerable morbidity and mortality, causing severe encephalitic, hemorrhagic, and febrile illnesses in humans. Because there are no specific clinical symptoms for infection by a determined virus and because different arboviruses could be present in the same area, a genus diagnosis by PCR would be a useful first-line diagnostic method. The six published
Flavivirus
genus primer pairs localized in the NS1, NS3, NS5, and 3′ NC regions were evaluated in terms of specificity and sensitivity with flaviviruses (including the main viruses pathogenic for humans) at a titer of 10
5
50% tissue culture infectious doses (TCID
50
s) ml
−1
with a common identification step by agarose gel electrophoresis. Only one NS5 primer pair allowed the detection of all tested flaviviruses with the sensitivity limit of 10
5
TCID
50
s ml
−1
. Using a heminested PCR with new primers designed in the same region after an alignment of 30 different flaviviruses, the sensitivity of reverse transcription-PCR was improved and allowed the detection of about 200 infectious doses ml
−1
with all of the tick- and mosquito-borne flaviviruses tested. It was confirmed that the sequenced amplified products in the NS5 region allowed predictability of flavivirus species by dendrogram, including the New York 99 West Nile strain. This technique was successfully performed with a cerebrospinal fluid sample from a patient hospitalized with West Nile virus encephalitis.
Phenotypic Switching and Genetic Diversity of
Cryptococcus neoformans
Authors: Sukroongreung Samaniya , Lim Shirlene , Tantimavanich Srisurang , Eampokalap Boonchaoy , Carter Dee , Nilakul Churairatana , Kulkeratiyut Suriyaphongse , Tansuphaswadikul Somsit
Published: July 2002

Niger seed agar was used as a primary plating medium for the isolation of
Cryptococcus neoformans
from cerebrospinal fluid specimens from AIDS patients with untreated primary cryptococcosis. The medium was used as the primary means to detect variations in the colony morphology of the yeast. To search for phenotypic and genetic variations, nine patients individually harboring two or three types of colony morphology were studied. Intraindividual isolates from nine patients had minor variations in the API 20C profile, and the MICs of one or more antifungal agents (amphotericin B, fluconazole, and itraconazole) for isolates from three patients were significantly different. Intraindividual isolates from three patients had minor karyotype differences, and one showed a dramatic chromosomal length polymorphism. In addition, three serial isolates from a patient with two episodes of infection showed similar karyotypes, confirming persistent infection by the same strain. Random amplified polymorphic DNA products were identical for all isolates (including three isolates from a relapse case). Our results provided evidence suggesting that (i) in humans,
C. neoformans
may undergo phenotypic and genetic changes during early infection prior to antifungal agent administration; (ii) dramatic variations in electrophoretic karyotypes and in phenotypes, as demonstrated during the early infection of one patient, may be due to infection by different strains; and (iii) the use of niger seed agar as a primary plating medium is useful for studying antifungal susceptibility, phenotypic switching, genetic diversity, and multiple-strain infections.
Evaluation of Methods for Detection of Toxins in Specimens of Feces Submitted for Diagnosis of
Clostridium difficile
- Associated Diarrhea
Authors: O'Connor Don , Hynes Pearl , Cormican Martin , Collins Edward , Corbett-Feeney Geraldine , Cassidy Michael
Published: July 2002

Clostridium difficile
is the principal pathogen associated with hospital-acquired acute diarrheal disease. We have evaluated the performances of six approaches for diagnosis of
C. difficile
-associated diarrhea (CDAD). Consecutive stool specimens (
n
= 200) from 133 patients were examined by cytotoxin assay, by culture of
C. difficile
on cycloserine-cefoxitin-fructose agar, and by toxin detection using four rapid immunoassay systems (Oxoid Toxin A test, ImmunoCard Toxin A test, TechLab Tox A/B II test, and Premier Toxins A&B test). A diagnosis of CDAD was established for 35 (27%) patients (representing 29% of specimens). The adjusted sensitivity and specificity of the methods were, respectively, 98 and 99% for the cytotoxin assay, 54 and 99% for ImmunoCard, 50 and 98% for Oxoid, 79 and 98% for TechLab, 80 and 98% for Premier, and 57 and 100% for culture. The TechLab and Premier assays are acceptable tests for diagnosis of CDAD but are not equivalent to the cytotoxin assay.
Burkholderia cepacia
Complex Infection in Italian Patients with Cystic Fibrosis: Prevalence, Epidemiology, and Genomovar Status
Authors: Agodi Antonella , Mahenthiralingam Eshwar , Barchitta Martina , Gianninò Viviana , Sciacca Agata , Stefani Stefania
Published: July 2002

The prevalence, epidemiology, and genomovar status of
Burkholderia cepacia
complex strains recovered from Italian cystic fibrosis (CF) patients were investigated using genetic typing and species identification methods. Four CF treatment centers were examined: two in Sicily, one in central Italy, and one in northern Italy.
B. cepacia
complex bacteria were isolated from 59 out of 683 CF patients attending these centers (8.6%). For the two geographically related treatment centers in Sicily, there was a high incidence of infection caused by a single epidemic clone possessing the
cblA
gene and belonging to
B. cepacia
genomovar III,
recA
group III-A, closely related to the major North America-United Kingdom clone, ET12; instability of the
cblA
sequence was also demonstrated for clonal isolates. In summary, of all the strains of
B. cepacia
encountered in the Italian CF population, the genomovar III,
recA
group III-A strains were the most prevalent and transmissible. However, patient-to-patient spread was also observed with several other genomovars, including strains of novel taxonomic status within the
B. cepacia
complex. A combination of genetic identification and molecular typing analysis is recommended to fully define specific risks posed by the genomovar status of strains within the
B. cepacia
complex.
Use of Serum Immune Complexes in a New Test That Accurately Confirms Early Lyme Disease and Active Infection with
Borrelia burgdorferi
Authors: Brunner Michael , Sigal Leonard H.
Published: July 2002

The present recommendation for serologic confirmation of Lyme disease (LD) calls for immunoblotting in support of positive or equivocal ELISA.
Borrelia burgdorferi
releases large quantities of proteins, suggesting that specific antibodies in serum might be trapped in immune complexes (ICs), rendering the antibodies undetectable by standard assays using unmodified serum. Production of ICs requires ongoing antigen production, so persistence of IC might be a marker of ongoing or persisting infection. We developed an immunoglobulin M (IgM) capture assay (EMIBA) measuring IC-derived IgM antibodies and tested it using three well-defined LD populations (from an academic LD referral center, a well-described Centers for Disease Control and Prevention (CDC) serum bank, and a group of erythema migrans patients from whose skin lesions
B. burgdorferi
was grown) and controls (non-Lyme arthritis inflammatory joint disease, syphilis, multiple sclerosis, and nondisease subjects from a region where LD is endemic, perhaps the most relevant comparison group of all). Previous studies demonstrated that specific antigen-antibody complexes in the sera of patients with LD could be precipitated by polyethylene glycol and could then be disrupted with maintenance of the immunoreactivity of the released antibodies, that specific anti-
B. burgdorferi
IgM was concentrated in ICs, and that occasionally IgM to specific
B. burgdorferi
antigens was found in the IC but not in unprocessed serum. EMIBA compared favorably with commercial and CDC flagellin-enhanced enzyme-linked immunosorbent assays and other assays in confirming the diagnosis of LD. EMIBA confirmed early
B. burgdorferi
infection more accurately than the comparator assays. In addition, EMIBA more accurately differentiated seropositivity in patients with active ongoing infection from seroreactivity persisting long after clinically successful antibiotic therapy; i.e., EMIBA identified seroreactivity indicating a clinical circumstance requiring antibiotic therapy. Thus, EMIBA is a promising new assay for accurate serologic confirmation of early and/or active LD.
Quantitative Analysis of Mycobacterial and Propionibacterial DNA in Lymph Nodes of Japanese and European Patients with Sarcoidosis
Authors: Eishi Yoshinobu , Suga Moritaka , Ishige Ikuo , Kobayashi Daisuke , Yamada Tetsuo , Takemura Tamiko , Takizawa Touichiro , Koike Morio , Kudoh Shoji , Costabel Ulrich , Guzman Josune , Rizzato Gianfranco , Gambacorta Marcello , du Bois Ronald , Nicholson Andrew G. , Sharma Om P. , Ando Masayuki
Published: July 2002

The cause(s) of sarcoidosis is unknown.Mycobacteriumspp. are suspected in Europe andPropionibacteriumspp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes ofPropionibacterium acnes,Propionibacterium granulosum,Mycobacterium tuberculosis,Mycobacterium aviumsubsp.paratuberculosis, andEscherichia coli(as the control) were counted by quantitative real-time PCR. EitherP. acnesorP. granulosumwas found in all but two of the sarcoid samples.M. aviumsubsp.paratuberculosiswas found in no sarcoid sample.M. tuberculosiswas found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes ofP. acnesorP. granulosumwere far more than those ofM. tuberculosis. P. acnesorP. granulosumwas found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes ofP. acnesorP. granulosumin such samples were less than those in sarcoid samples.Propionibacteriumspp. are more likely thanMycobacteriaspp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.
Comparative Performance of Herpes Simplex Virus Type 1-Specific Serologic Assays from MRL and Meridian Diagnostics
Authors: Ribes Julie A. , Smith Angela , Hayes Marie , Baker Doris J. , Winters Jeffrey L.
Published: July 2002

Two companies, MRL and Meridian Diagnostics, have developed Food and Drug Administration-approved herpes simplex virus type 1 type-specific enzyme immunoassays. The sensitivity, specificity, and overall testing efficiency of these assays were 98.2, 93.8, and 96.6% for MRL and 98.8, 99.0, and 98.1% for Meridian, making both of these kits suitable for use in the clinical lab.
Evaluation of Two Nested PCR Assays for Detection of
Histoplasma capsulatum
DNA in Human Tissue
Authors: Bialek Ralf , Feucht Antje , Aepinus Christian , Just-Nübling Gudrun , Robertson Valerie J. , Knobloch Jürgen , Hohle Rolf
Published: July 2002

In order to evaluate the diagnostic relevance of two nested PCR assays for diagnosis of histoplasmosis in clinical specimens, 100 paraffin-embedded biopsy specimens were examined. Upon microscopy of tissue, 50 biopsy specimens were histoplasma positive and 50 were negative. Due to destruction by formalin fixation, successful extraction of amplifiable human DNA was limited to 29 and 33 samples, respectively. A product of the
Histoplasma capsulatum
nested PCR assay targeting the gene encoding the unique fungal 100-kDa-like protein was detected in 20 histopathologically positive biopsy specimens but in none of the microscopically negative samples. Sequencing revealed that all 20 products of 210 bp were identical to the sequence of
H. capsulatum
in the GenBank database. In contrast, the nested PCR assay targeting the fungal 18S rRNA genes amplified products in 26 histopathologically positive but also in 18 microscopically negative biopsy specimens. However, sequencing revealed that only 20 of these 44 PCR products (231 bp) were identical to the sequence of
H. capsulatum
. The remaining 24 sequences were homologous to those of several
Euascomycetes
. These PCR products were detected only in tissues possibly colonized by nonpathogenic fungi, possibly causing these nonspecific amplifications. The detection limit of both
H. capsulatum
nested PCR assays was 1 to 5 fungal cells per sample. The two assays were similarly sensitive in identifying
H. capsulatum
. In this preliminary study, the novel 100-kDa-like-protein gene nested PCR revealed a specificity of 100% without requiring sequencing, which was necessary for identification of the 18S ribosomal DNA nested PCR products in order to avoid a high rate of false-positive results.
Genomic Determinants of Cardiovirulence in Coxsackievirus B3 Clinical Isolates: Localization to the 5′ Nontranslated Region
Authors: Dunn James J. , Chapman Nora M. , Tracy Steven , Romero JoséR.
Published: July 2002

Coxsackievirus B3 (CVB3) infections can cause myocarditis in humans and are implicated in the pathogenesis of dilated cardiomyopathy. The natural genetic determinants of cardiovirulence for CVB3 have not been identified, although using strains engineered in the laboratory, cardiovirulence determinants have been identified in the CVB3 5′ nontranslated region (5′NTR) and capsid. The myocarditic phenotypes of two CVB3 clinical isolates were determined using an established murine model of inflammatory heart disease. The 5′NTRs and capsid proteins of the noncardiovirulent CVB3/CO strain and cardiovirulent CVB3/AS strain were examined to determine their influence on the cardiovirulence phenotype. Six intratypic chimeric viruses were constructed in which 5′NTR and capsid sequences of the infectious cDNA copy of the cardiovirulent CVB3/20 genome were replaced by homologous sequences from CVB3/CO or CVB3/AS. Chimeric strains were tested for cardiovirulence by inoculation of C3H/HeJ mice. Sections of hearts removed at 10 days postinoculation were examined for evidence of myocarditis by light microscopy and assayed for the presence of virus. Replacement of the CVB3/20 capsid coding region by that from the homologous region of CVB3/CO resulted in no change in the cardiovirulent CVB3/20 phenotype, with virus recoverable from the heart at 10 days postinoculation. However, recombinant virus containing the CVB3/CO 5′NTR alone or the 5′NTR and capsid sequences together were not myocarditic, and infectious virus was not recovered from the myocardium. Chimeric viruses containing the CVB3/AS 5′NTR alone, capsid sequence alone, or both together preserved the myocarditic phenotype. These data support the 5′NTR as the primary site in the determination of the natural cardiovirulence phenotype of CVB3.
Leaky Scanning Is the Predominant Mechanism for Translation of Human Papillomavirus Type 16 E7 Oncoprotein from E6/E7 Bicistronic mRNA
Authors: Stacey Simon N. , Jordan Deborah , Williamson Andrew J. K. , Brown Michael , Coote Joanna H. , Arrand John R.
Published: July 2002

Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5′ untranslated region (UTR) was shown to promote translation as efficiently as a UTR fromXenopusβ-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5′ end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region betweenRenillaand firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5′ end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did theRenillaopen reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.
Initial Interactions of Subgenus D Adenoviruses with A549 Cellular Receptors: Sialic Acid versus α
v
Integrins
Authors: Arnberg Niklas , Kidd Alistair H. , Edlund Karin , Olfat Farzad , Wadell Göran
Published: July 2002

Selected members of the adenovirus family have been shown to interact with the coxsackie adenovirus receptor, α
v
integrins, and sialic acid on target cells. Initial interactions of subgenus D adenoviruses with target cells have until now been poorly characterized. Here, we demonstrate that adenovirus type 8 (Ad8), Ad19a, and Ad37 use sialic acid as a functional cellular receptor, whereas the Ad9 and Ad19 prototypes do not.
Early Alterations of the Receptor-Binding Properties of H1, H2, and H3 Avian Influenza Virus Hemagglutinins after Their Introduction into Mammals
Authors: Matrosovich Mikhail , Tuzikov Alexander , Bovin Nikolai , Gambaryan Alexandra , Klimov Alexander , Castrucci Maria R. , Donatelli Isabella , Kawaoka Yoshihiro
Published: July 2002

Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3′SL-PAA and 6′SLN-PAA, which contained, respectively, 3′-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6′-sialyl(
N
-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6′SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q→L, increased binding to 6′SLN-PAA, while among H1 swine viruses, the 190E→D and 225G→E mutations in the HA appeared important for the increased affinity of the viruses for 6′SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.
The DNA of a Plant Retroviroid-Like Element Is Fused to Different Sites in the Genome of a Plant Pararetrovirus and Shows Multiple Forms with Sequence Deletions
Authors: Vera Antonio , Daròs José-Antonio , Flores Ricardo , Hernández Carmen
Published: July 2002

Carnation small viroid-like RNA (CarSV RNA) and its homologous DNA are the two forms of a unique plant retroviroid-like system. CarSV RNA is a 275-nucleotide noninfectious viroid-like RNA, present in certain carnation plants, which can adopt hammerhead structures in both polarity strands and self-cleave accordingly. CarSV DNA is organized as a series of head-to-tail multimers forming part of extrachromosomal elements in which CarSV DNA sequences are fused to sequences of carnation etched ring virus (CERV), a plant pararetrovirus. Analysis of more than 30 CarSV-CERV DNA junctions showed that distinct regions of the viral genome seem able to interact with CarSV DNA. All these junctions were short nucleotide stretches common to both CarSV and CERV DNAs. This suggests a polymerase-driven mechanism for their origin involving an enzyme with low processivity, most likely the viral reverse transcriptase. This view was further supported by the observation that most of CarSV sequences forming part of the junctions correspond either to strong secondary structure motifs in the conformation proposed for CarSV RNA or to its self-cleavage sites, which may have facilitated polymerase jumping. Accompanying the most-abundant CarSV RNA, a series of CarSV RNAs with sequence deletions were previously characterized. Here we have identified some of their corresponding DNA forms, together with other CarSV DNA forms with deletions not found in any CarSV RNA species identified so far. Some of these CarSV DNA forms have also been detected fused to CERV sequences. The existence of these shortened CarSV DNA versions may provide a continuous input of their corresponding transcripts and explain the persistence of CarSV RNAs with defective hammerhead structures for which an RNA-RNA model of amplification seems unlikely.
The Epstein-Barr Virus Promoter Initiating B-Cell Transformation Is Activated by RFX Proteins and the B-Cell-Specific Activator Protein BSAP/Pax5
Authors: Tierney Rosemary , Kirby Helen , Nagra Jasdeep , Rickinson Alan , Bell Andrew
Published: July 2002

Epstein-Barr virus (EBV)-induced B-cell growth transformation, a central feature of the virus' strategy for colonizing the human B-cell system, requires full virus latent gene expression and is initiated by transcription from the viral promoter Wp. Interestingly, when EBV accesses other cell types, this growth-transforming program is not activated. The present work focuses on a region of Wp which in reporter assays confers B-cell-specific activity. Bandshift studies indicate that this region contains three factor binding sites, termed sites B, C, and D, in addition to a previously characterized CREB site. Here we show that site C binds members of the ubiquitously expressed RFX family of proteins, notably RFX1, RFX3, and the associated factor MIBP1, whereas sites B and D both bind the B-cell-specific activator protein BSAP/Pax5. In reporter assays with mutant Wp constructs, the loss of factor binding to any one of these sites severely impaired promoter activity in B cells, while the wild-type promoter could be activated in non-B cells by ectopic BSAP expression. We suggest that Wp regulation by BSAP helps to ensure the B-cell specificity of EBV's growth-transforming function.
The ORF1 Products of Tombusviruses Play a Crucial Role in Lethal Necrosis of Virus-Infected Plants
Authors: Burgyán József , Hornyik Csaba , Szittya György , Silhavy Dániel , Bisztray György
Published: July 2002

Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms ofNicotiana benthamianainfected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided intransin transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector inN. benthamianacaused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.
A Functional Complex of Adenovirus Proteins E1B-55kDa and E4orf6 Is Necessary To Modulate the Expression Level of p53 but Not Its Transcriptional Activity
Authors: Cathomen Toni , Weitzman Matthew D.
Published: July 2002

In adenovirus-infected cells, binding of E1B-55kDa and E4orf6 to the tumor suppressor protein p53 inhibits its transcriptional activity and causes rapid turnover of the protein. To investigate the requirements of the E1B-E4orf6 complex to modulate p53 function, we generated an E4orf6 mutant that failed to associate functionally and physically with E1B-55kDa but still interacted with p53. We confirm that E4orf6 and E1B-55kDa reduce p53 transactivation individually and show that their combined inhibition is additive rather than synergistic. Furthermore, we found that downregulation of p53's expression level, but not transcriptional inhibition of p53, depends on a functional E1B-E4 complex. A functional interaction of E1B-55kDa with p53, on the other hand, is a prerequisite for both transcriptional repression and downregulation of p53. The separation of these two functions will enable further dissection of the requirements for oncogenicity by the E4orf6 protein.
Wide Range of Viral Load in Healthy African Green Monkeys Naturally Infected with Simian Immunodeficiency Virus
Authors: Goldstein Simoy , Ourmanov Ilnour , Brown Charles R. , Beer Brigitte E. , Elkins William R. , Plishka Ronald , Buckler-White Alicia , Hirsch Vanessa M.
Published: July 2002

The distribution and levels of simian immunodeficiency virus (SIV) in tissues and plasma were assessed in naturally infected African green monkeys (AGM) of the vervet subspecies (
Chlorocebus pygerythrus
) by limiting-dilution coculture, quantitative PCR for viral DNA and RNA, and in situ hybridization for SIV expression in tissues. A wide range of SIV RNA levels in plasma was observed among these animals (<1,000 to 800,000 copies per ml), and the levels appeared to be stable over long periods of time. The relative numbers of SIV-expressing cells in tissues of two monkeys correlated with the extent of plasma viremia. SIV expression was observed in lymphoid tissues and was not associated with immunopathology. Virus-expressing cells were observed in the lamina propria and lymphoid tissue of the gastrointestinal tract, as well as within alveolar macrophages in the lung tissue of one AGM. The range of plasma viremia in naturally infected AGM was greater than that reported in naturally infected sooty mangabeys. However, the degree of viremia in some AGM was similar to that observed during progression to AIDS in human immunodeficiency virus-infected individuals. Therefore, containment of viremia is an unlikely explanation for the lack of pathogenicity of SIVagm in its natural host species, AGM.
Hepatitis B Virus X Protein Colocalizes to Mitochondria with a Human Voltage-Dependent Anion Channel, HVDAC3, and Alters Its Transmembrane Potential
Authors: Rahmani Zohra , Huh Kyung-Won , Lasher Robert , Siddiqui Aleem
Published: July 2002

Understanding the mechanism(s) of action of the hepatitis B virus (HBV)-encoded protein HBx is fundamental to elucidating the underlying mechanisms of chronic liver disease and hepatocellular carcinoma caused by HBV infection. In our continued attempts to identify cellular targets of HBx, we have previously reported the identification of a novel cellular protein with the aid of a yeast two-hybrid assay. This cellular gene was identified as a third member of the family of human genes that encode the voltage-dependent anion channel (HVDAC3). In the present study, physical interaction between HBx and HVDAC3 was established by standard in vitro and in vivo methods. Confocal laser microscopy of transfected cells with respective expression vectors colocalized HVDAC3 and HBx to mitochondria. This novel, heretofore unreported subcellular distribution of HBx in mitochondria implies a functional role of HBx in functions associated with mitochondria. Using a stable cationic fluorophore dye, CMXRos, we show that HBx expression in cultured human hepatoma cells leads to alteration of mitochondrial transmembrane potential. Such functional roles of HBx in affecting mitochondrial physiology have implications for HBV-induced liver injury and the development of hepatocellular carcinoma.