bluuebunny/crossref_metadata_2025 huggingface dataset:
Current Status of Nonculture Methods for Diagnosis of Invasive Fungal Infections
Authors: Yeo Siew Fah , Wong Brian
Published: July 2002

The incidence of invasive fungal infections has increased dramatically in recent decades, especially among immunocompromised patients. However, the diagnosis of these infections in a timely fashion is often very difficult. Conventional microbiologic and histopathologic approaches generally are neither sensitive nor specific, and they often do not detect invasive fungal infection until late in the course of disease. Since early diagnosis may guide appropriate treatment and prevent mortality, there has been considerable interest in developing nonculture approaches to diagnosing fungal infections. These approaches include detection of specific host immune responses to fungal antigens, detection of specific macromolecular antigens using immunologic reagents, amplification and detection of specific fungal nucleic acid sequences, and detection and quantitation of specific fungal metabolite products. This work reviews the current status and recent developments as well as problems in the design of nonculture diagnostic methods for invasive fungal infections.
LeishmaniaPriming of Human Dendritic Cells for CD40 Ligand-Induced Interleukin-12p70 Secretion Is Strain and Species Dependent
Authors: McDowell Mary Ann , Marovich Mary , Lira Rosalia , Braun Michael , Sacks David
Published: July 2002

A major question in the study of leishmaniasis is what dictates clinical disease expression produced by differentLeishmaniaspecies, i.e., cutaneous versus systemic and healing versus nonhealing. Animal models using aLeishmaniaspecies associated with self-limiting cutaneous disease (L. major)have revealed that protective immunity requires CD40/CD40 ligand (CD40L)-dependent, interleukin-12 (IL-12)-driven Th1 responses. We recently showed thatL. majorcan prime human dendritic cells (DCs) for CD40L-triggered IL-12p70 secretion and that these cells can drive a Th1 response in autologous T cells from sensitized individuals. Here we show that in contrast toL. major,Leishmaniaspecies responsible for visceral disease (L. donovani), as well as species associated with persistent, cutaneous lesions and occasional systemic disease (L. tropica), did not induce CD40L-dependent IL-12p70 production, despite comparable levels of uptake by DCs. Up-regulated surface expression of CD40 did not correlate with IL-12p70 production, and appreciable CD40L-induced IL-12p40 secretion was observed in uninfected as well as infected DCs, regardless of species. Reverse transcription-PCR analysis confirmed that the production of heterodimeric IL-12 was limited by expression of IL-12p35 mRNA, which was dependent on both a microbial priming signal and CD40 engagement for its high-level induction. The intrinsic differences in the ability ofLeishmaniaspecies to prime DCs for CD40L-dependent IL-12p70 secretion may account, at least in part, for the evolution of healing and nonhealing forms of leishmanial disease.
Salmonella entericaSerovar Typhi Live Vector Vaccines Delivered Intranasally Elicit Regional and Systemic Specific CD8+Major Histocompatibility Class I-Restricted Cytotoxic T Lymphocytes
Authors: Pasetti Marcela F. , Salerno-Gonçalves Rosangela , Sztein Marcelo B.
Published: July 2002

We investigated the ability of live attenuatedSalmonella entericaserovar Typhi strains delivered to mice intranasally to induce specific cytotoxic T-lymphocyte (CTL) responses at regional and systemic levels. Mice immunized with two doses (28 days apart) ofSalmonellaserovar Typhi strain Ty21a, the licensed oral typhoid vaccine, and genetically attenuated mutants CVD 908 (ΔaroCΔaroD), CVD 915 (ΔguaBA), and CVD 908-htrA(ΔaroCΔaroDΔhtrA) induced CTL specific forSalmonellaserovar Typhi-infected cells in spleens and cervical lymph nodes. CTL were detected in effector T cells that had been expanded in vitro for 7 days in the presence ofSalmonella-infected syngeneic splenocytes. A second round of stimulation further enhanced the levels of specific cytotoxicity. CTL activity was observed in sorted αβ+CD8+T cells, which were remarkably increased after expansion, but not in CD4+T cells. CTL from both cervical lymph nodes and spleens failed to recognizeSalmonella-infected major histocompatibility complex (MHC)-mismatched cells, indicating that the responses were MHC restricted. Studies in which MHC blocking antibodies were used showed that H-2Ldwas the restriction element. This is the first demonstration thatSalmonellaserovar Typhi vaccines delivered intranasally elicit CD8+MHC class I-restricted CTL. The results further support the usefulness of the murine intranasal model for evaluating the immunogenicity of typhoid vaccine candidates at the preclinical level.
Phosphoantigen Presentation by Macrophages toMycobacterium tuberculosis-Reactive Vγ9Vδ2+T Cells: Modulation by Chloroquine
Authors: Rojas Roxana E. , Torres Martha , Fournié Jean-Jacques , Harding Clifford V. , Boom W. Henry
Published: July 2002

Vγ9Vδ2+T cells (γδ T cells) are activated byMycobacterium tuberculosisand recognize mycobacterial nonpeptide phosphoantigens. The role of antigen-presenting cells in the processing and presentation of phosphoantigens to Vγ9Vδ2+T cells is not understood. We analyzed the role of macrophages for activation of γδ T cells by a new synthetic phosphoantigen bromohydrin pyrophosphate (BrHPP) andM. tuberculosis. Macrophages greatly increased γδ T-cell activation by both BrHPP andM. tuberculosis. Fixation of macrophages before infection demonstrated that uptake ofM. tuberculosiswas required for presentation to γδ T cells. Antigens ofM. tuberculosisremained stably associated with macrophage surface and were not removed by paraformaldehyde fixation or washing. Macrophages processedM. tuberculosisfor γδ T cells through a brefeldin A-insensitive pathway, suggesting that transport through the endoplasmic reticulum and Golgi complex of a putative presenting molecule is not important in the early processing ofM. tuberculosisantigens for γδ T cells. Processing ofM. tuberculosiswas not eliminated by chloroquine, indicating that processing of γδ antigens is not dependent on acidic pH in the lysosomes. Chloroquine treatment of BrHPP-pulsed macrophages increased activation of γδ T cells. Ammonium chloride treatment of macrophages did not increase reactivity of γδ T cells to BrHPP, indicating that the effect of chloroquine was independent of pH changes in endosomes. Chloroquine, by inhibiting membrane traffic, may increase association and retention of phosphoantigens with cell surface membrane molecules on macrophages.
Bacterium-Induced CXCL10 Secretion by Osteoblasts Can Be Mediated in Part through Toll-Like Receptor 4
Authors: Gasper Nancy A. , Petty Cynthia C. , Schrum Laura W. , Marriott Ian , Bost Kenneth L.
Published: July 2002

Two common pathogens known to cause bone infection,SalmonellaandStaphylococcus aureus, were investigated to determine their abilities to induce chemokine expression in cultured mouse and human osteoblasts. While these cells are responsible for bone formation, we were surprised to find that they could respond to bacterial infection by upregulating expression of the chemokine CXCL10 (IP-10). However, there were significant differences in the abilities of the gram-negative bacteriumSalmonellaand the gram-positive bacteriumS. aureusto induce expression of CXCL10. Reverse transcription-PCR and enzyme-linked immunosorbent assay analyses showed high levels ofSalmonella-induced CXCL10 mRNA and protein expression, respectively, whereas the osteoblast response toS. aureuswas significantly less. Consistent with these findings,Salmonella-derived lipopolysaccharide (LPS), but notS. aureus-derived peptidoglycan, could induce expression of CXCL10. An antibody against toll-like receptor 4 (TLR4) could block the LPS-induced CXCL10 production, demonstrating the functional expression of TLR4 by osteoblasts. Despite the inducible nature of TLR2 mRNA expression by bacterium-infected osteoblasts, peptidoglycan failed to stimulate CXCL10 secretion. Immunofluorescent staining of bacterium-infected calvaria (i.e., skull bone) demonstrated the presence of CXCL10 in osteoblasts. The fact that osteoblasts did not express CXCR3 mRNA, whereas T lymphocytes can express high levels of this receptor, suggests that osteoblast-derived CXCL10 may recruit T lymphocytes to the sites of bone infections.
Modification of the Structure and Activity of Lipid A inYersinia pestisLipopolysaccharide by Growth Temperature
Authors: Kawahara Kazuyoshi , Tsukano Hiroko , Watanabe Haruo , Lindner Buko , Matsuura Motohiro
Published: July 2002

Yersinia pestisstrain Yreka was grown at 27 or 37°C, and the lipid A structures (lipid A-27°C and lipid A-37°C) of the respective lipopolysaccharides (LPS) were investigated by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Lipid A-27°C consisted of a mixture of tri-acyl, tetra-acyl, penta-acyl, and hexa-acyl lipid A's, of which tetra-acyl lipid A was most abundant. Lipid A-37°C consisted predominantly of tri- and tetra-acylated molecules, with only small amounts of penta-acyl lipid A; no hexa-acyl lipid A was detected. Furthermore, the amount of 4-amino-arabinose was substantially higher in lipid A-27°C than in lipid A-37°C. By use of mouse and human macrophage cell lines, the biological activities of the LPS and lipid A preparations were measured via their abilities to induce production of tumor necrosis factor alpha (TNF-α). In both cell lines the LPS and the lipid A from bacteria grown at 27°C were stronger inducers of TNF-α than those from bacteria grown at 37°C. However, the difference in activity was more prominent in human macrophage cells. These results suggest that in order to reduce the activation of human macrophages, it may be more advantageous forY. pestisto produce less-acylated lipid A at 37°C.
Immunization with a Polyprotein Vaccine Consisting of the T-Cell Antigens Thiol-Specific Antioxidant,Leishmania majorStress-Inducible Protein 1, andLeishmaniaElongation Initiation Factor Protects against Leishmaniasis
Authors: Coler Rhea N. , Skeiky Yasir A. W. , Bernards Karen , Greeson Kay , Carter Darrick , Cornellison Charisa D. , Modabber Farrokh , Campos-Neto Antonio , Reed Steven G.
Published: July 2002

Development of an effective vaccine againstLeishmaniainfection is a priority of tropical disease research. We have recently demonstrated protection againstLeishmania majorin the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant,L. majorstress-inducible protein 1 (LmSTI1), andLeishmaniaelongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using theL. majorchallenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use.
Effect of Acid Adaptation on the Fate of
Listeria monocytogenes
in THP-1 Human Macrophages Activated by Gamma Interferon
Authors: Conte Maria Pia , Petrone Gloria , Di Biase Assunta Maria , Longhi Catia , Penta Michela , Tinari Antonella , Superti Fabiana , Fabozzi Giulia , Visca Paolo , Seganti Lucilla
Published: July 2002

In
Listeria monocytogenes
the acid tolerance response (ATR) takes place through a programmed molecular response which ensures cell survival under unfavorable conditions. Much evidence links ATR with virulence, but the molecular determinants involved in the reactivity to low pHs and the behavior of acid-exposed bacteria within host cells are still poorly understood. We have investigated the effect of acid adaptation on the fate of
L. monocytogenes
in human macrophages. Expression of genes encoding determinants for cell invasion and intracellular survival was tested for acid-exposed bacteria, and invasive behavior in the human myelomonocytic cell line THP-1 activated with gamma interferon was assessed. Functional approaches demonstrated that preexposure to an acidic pH enhances the survival of
L. monocytogenes
in activated human macrophages and that this effect is associated with an altered pattern of expression of genes involved in acid resistance and cell invasion. Significantly decreased transcription of the
plcA
gene, encoding a phospholipase C involved in vacuolar escape and cell-to-cell spread, was observed in acid-adapted bacteria. This effect was due to a reduction in the quantity of the bicistronic
plcA-prfA
transcript, concomitant with an increase in the level(s) of the monocistronic
prfA
mRNA(s). The transcriptional shift from distal to proximal
prfA
promoters resulted in equal levels of the
prfA
transcript (and, as a consequence, of the
inlA
,
hly
, and
actA
transcripts) under neutral and acidic conditions. In contrast, the
sodC
and
gad
genes, encoding a cytoplasmic superoxide dismutase and the glutamate-based acid resistance system, respectively, were positively regulated at a low pH. Morphological approaches confirmed the increased intracellular survival and growth of acid-adapted
L. monocytogenes
cells both in vacuoles and in the cytoplasm of interferon gamma-activated THP-1 macrophages. Our data indicate that preexposure to a low pH has a positive impact on subsequent challenge of
L. monocytogenes
with macrophagic cells.
Molecular Cloning and Characterization of Genes for
Shigella sonnei
Form I O Polysaccharide: Proposed Biosynthetic Pathway and Stable Expression in a Live
Salmonella
Vaccine Vector
Authors: Xu De-Qi , Cisar John O. , Ambulos Nicholas , Burr Donald H. , Kopecko Dennis J.
Published: July 2002

The gene region for biosynthesis of
Shigella sonnei
form I O polysaccharide (O-Ps) and flanking sequences, totaling >18 kb, was characterized by deletion analysis to define a minimal construct for development of
Salmonella
-based live vaccine vector strains. Lipopolysaccharide (LPS) expression and DNA sequence studies of plasmid deletion derivatives indicated form I O-Ps expression from a 12.3-kb region containing a putative promoter and 10 contiguous open reading frames (ORFs), one of which is the transposase of IS
630
. A detailed biosynthetic pathway, consistent with the predicted functions of eight of the nine essential ORFs and the form I O-Ps structure, is proposed. Further sequencing identified partial IS elements (i.e., IS
91
and IS
630
) and
wzz
upstream of the form I coding region and a fragment of
aqpZ
and additional full or partial IS elements (i.e., IS
629
, IS
91
, and IS
911
) downstream of this region. The stability of plasmid-based form I O-Ps expression was greater from low-copy vectors than from high-copy vectors and was enhanced by deletion of the downstream IS
91
from plasmid inserts. Both core-linked (i.e., LPS) and non-core-linked (i.e., capsule-like) surface expression of form I O-Ps were detected by Western blotting and silver staining of polyacrylamide gel electrophoresis-separated
Shigella
and
Escherichia coli
extracts. However, salmonellae, which have a core that is chemically dissimilar to that of shigellae, expressed only non-core-linked surface-associated form I O-Ps. Finally, attenuated
Salmonella enterica
serovar Typhi live vaccine vector candidates, containing minimal-sized form I operon constructs, elicited immune protection in mice against virulent
S. sonnei
challenge, thereby supporting the promise of live, oral vaccines for the prevention of shigellosis.
Dissemination ofMycobacterium tuberculosisIs Influenced by Host Factors and Precedes the Initiation of T-Cell Immunity
Authors: Chackerian Alissa A. , Alt Jennifer M. , Perera Thushara V. , Dascher Christopher C. , Behar Samuel M.
Published: July 2002

We report that dissemination ofMycobacterium tuberculosisin the mouse is under host control and precedes the initiation of T-cell immunity. Nine to eleven days after aerosol inoculation,M. tuberculosisdisseminates to the pulmonary lymph nodes (LN), whereM. tuberculosis-specific T cells are detected 2 to 3 days thereafter. This indicates that the initial spread of bacteria occurs via lymphatic drainage and that the acquired T-cell immune response is generated in the draining LN. Dissemination to peripheral sites, such as the spleen and the liver, occurs 11 to 14 days postinfection and is followed by the appearance ofM. tuberculosis-specific T cells in the lung and the spleen. In all cases studied, dissemination to the LN or the spleen preceded activation ofM. tuberculosis-specific T cells in that organ. Interestingly, bacteria disseminate earlier from the lungs of resistant C57BL/6 mice than from the lungs of susceptible C3H mice, and consequently, C57BL/6 mice generate an immune response toM. tuberculosissooner than C3H mice generate an immune response. Thus, instead of spreading infection, early dissemination ofM. tuberculosismay aid in the initiation of an appropriate and timely immune response. We hypothesize that this early initiation of immunity following inoculation withM. tuberculosismay contribute to the superior resistance of C57BL/6 mice.
Theomp-1Major Outer Membrane Multigene Family ofEhrlichia chaffeensisIs Differentially Expressed in Canine and Tick Hosts
Authors: Unver Ahmet , Rikihisa Yasuko , Stich Roger W. , Ohashi Norio , Felek Suleyman
Published: July 2002

Sixteen of 22omp-1paralogs encoding 28-kDa-range immunodominant outer membrane proteins ofEhrlichia chaffeensiswere transcribed in blood monocytes of dogs throughout a 56-day infection period. Only one paralog was transcribed byE. chaffeensisin three developmental stages ofAmblyomma americanumticks before or afterE. chaffeensistransmission to naïve dogs.
Role of Polyphosphate Kinase in Biofilm Formation by
Porphyromonas gingivalis
Authors: Chen Wen , Palmer Robert J. , Kuramitsu Howard K.
Published: July 2002

In order to assess the role of polyphosphate kinase (PPK) in the physiology of
Porphyromonas gingivalis
, a
ppk
gene mutant, CW120, was constructed and characterized.
P. gingivalis
was demonstrated to synthesize short-chain polyphosphate (polyP) but not long-chain polyP. CW120 failed to survive in the stationary phase as well as the parental cell did, and it was attenuated in biofilm formation on polyvinylchloride and glass surfaces. Furthermore, the complementation by insertion of an intact copy of the
ppk
gene into the mutant CW120 restored its biofilm formation and stationary-phase survival. These results suggest that PPK may be important for incorporation of these organisms into subgingival plaque in the human oral cavity.
Alternative Splicing of
LYT1
Transcripts in
Trypanosoma cruzi
Authors: Manning-Cela Rebeca , González Antonio , Swindle John
Published: July 2002

As a result of alternative
trans
splicing, three distinct
LYT1
mRNAs are produced in
Trypanosoma cruzi
, two encoding the full-length LYT1 protein and the third encoding a truncated LYT1 protein lacking a possible signal sequence. Analysis of the three mRNAs in different developmental forms of the parasite revealed that the alternative processing events were regulated differently during the parasite life cycle.
Functional Analysis and Regulation of the Divergent
spuABCDEFGH-spuI
Operons for Polyamine Uptake and Utilization in
Pseudomonas aeruginosa
PAO1
Authors: Lu Chung-Dar , Itoh Yoshifumi , Nakada Yuji , Jiang Ying
Published: July 2002

A multiple-gene locus for polyamine uptake and utilization was discovered in
Pseudomonas aeruginosa
PAO1. This locus contained nine genes designated
spuABCDEFGHI
(
spu
for spermidine and putrescine utilization). The physiological functions of the
spu
genes in utilization of two polyamines (putrescine and spermidine) were analyzed by using Tn
5
transposon-mediated
spu
knockout mutants. Growth and uptake experiments support that the
spuDEFGH
genes specify components of a major ABC-type transport system for spermidine uptake, and enzymatic measurements indicated that
spuC
encodes putrescine aminotransferase with pyruvate as the amino group receptor. Although
spuA
and
spuB
mutants showed an apparent defect in spermidine utilization, the biochemical functions of the gene products have yet to be elucidated. Assays of
lacZ
fusions demonstrated the presence of agmatine-, putrescine-, and spermidine-inducible promoters for the
spuABCDEFGH
operon and the divergently transcribed
spuI
gene of unknown function. Since the observed induction effect of agmatine was abolished in an
aguA
mutant where conversion of agmatine into putrescine was blocked, putrescine or spermidine, but not agmatine, serves as the inducer molecule of the
spuA-spuI
divergent promoters. S1 nuclease mappings confirmed further the induction effects of the polyamines on transcription of the divergent promoters and localized the transcription initiation sites. Gel retardation assays with extracts from the cells grown on putrescine or spermidine demonstrated the presence of a polyamine-responsive regulatory protein interacting with the divergent promoter region. Finally, the absence of the putrescine-inducible
spuA
expression and putrescine aminotransferase (
spuC
) formation in the
cbrB
mutant indicated that the
spu
operons are regulated by the global CbrAB two-component system perhaps via the putative polyamine-responsive transcriptional activator.
Novel DNA-Binding Proteins in the Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Koksharova Olga A. , Wolk C. Peter
Published: July 2002

As an approach towards elucidation of the biochemical regulation of the progression of heterocyst differentiation in
Anabaena
sp. strain PCC 7120, we have identified proteins that bind to a 150-bp sequence upstream from
hepC
, a gene that plays a role in the synthesis of heterocyst envelope polysaccharide. Such proteins were purified in four steps from extracts of vegetative cells of
Anabaena
sp. Two of these proteins (Abp1 and Abp2) are encoded by neighboring genes in the
Anabaena
sp. chromosome. The genes that encode the third (Abp3) and fourth (Abp4) proteins are situated at two other loci in that chromosome. Insertional mutagenesis of
abp2
and
abp3
blocked expression of
hepC
and
hepA
and prevented heterocyst maturation and aerobic fixation of N
2
.
In Vivo and In Vitro Studies of
Bacillus subtilis
Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway
Authors: Olsson Ulf , Billberg Annika , Sjövall Sara , Al-Karadaghi Salam , Hansson Mats
Published: July 2002

Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium
Bacillus subtilis
, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix α3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of
B. subtilis
and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in
V
max
. The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.
The
fixA
and
fixB
Genes Are Necessary for Anaerobic Carnitine Reduction in
Escherichia coli
Authors: Walt Angelique , Kahn Michael L.
Published: July 2002

In
Escherichia coli
, the use of carnitine as a terminal electron acceptor depends on a functional
caiTABCDE
operon. It had been suggested that the adjacent but divergent
fixABCX
operon is also required for carnitine metabolism, perhaps to provide electrons for carnitine reduction. We have constructed
E. coli fixA
and
fixB
mutants and find that they are unable to reduce carnitine to γ-butyrobetaine under anaerobic conditions.
Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen
Authors: Ge Yong , Vaillancourt Frédéric H. , Agar Nathalie Y. R. , Eltis Lindsay D.
Published: July 2002

Toluate dioxygenase (TADO) of
Pseudomonas putida
mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with
m
-toluate, and each component had a full complement of their respective Fe
2
S
2
centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for
m
-toluate, displaying apparent parameters of
KmA = 9 ± 1 μM
,
k
cat
= 3.9 ± 0.2 s
−1
, and
K

m

O
2
= 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°C), where
K

m

O
2
represents the
K

m

for O
2
and
KmA
represents the
K

m

for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity:
m
-toluate > benzoate ≃ 3-chlorobenzoate >
p
-toluate ≃ 4-chlorobenzoate ≫
o
-toluate ≃ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O
2
utilization and yielded the corresponding 1,2-
cis
-dihydrodiol. In contrast, the transformation of
ortho
-substituted benzoates was poorly coupled to O
2
utilization, with >10 times more O
2
being consumed than benzoate. However, the apparent
K

m

of TADO for these benzoates was >100 μM, indicating that they do not effectively inhibit the turnover of good substrates.
DNA-Binding Activities of the HilC and HilD Virulence Regulatory Proteins ofSalmonella entericaSerovar Typhimurium
Authors: Olekhnovich Igor N. , Kadner Robert J.
Published: July 2002

The HilC and HilD proteins ofSalmonella entericaserovar Typhimurium are members of the AraC/XylS family of transcription regulators. They are encoded onSalmonellapathogenicity island 1 (SPI1) and control expression of thehilAgene, which encodes the major transcriptional activator for many genes encoded on SPI1 and elsewhere that contribute to invasion of host cells. Gel electrophoretic shift and DNase footprinting assays revealed that purified HilC and HilD proteins can bind to multiple regions in thehilAandhilCpromoters and to a single region in thehilDpromoter. Although both HilC and -D proteins can bind to the same DNA regions, they showed different dependencies on the sequence and lengths of their DNA targets. To identify the binding-sequence specificity of HilC and HilD, a series of single base substitutions changing each position in a DNA fragment corresponding to positions −92 to −52 of thehilCpromoter was tested for binding to HilC and HilD in a gel shift DNA-binding assay. This mutational analysis in combination with sequence alignments allowed deduction of consensus sequences for binding of both proteins. The consensus sequences overlap but differ so that HilC can bind to both types of sites but HilD only to one. ThehilAandhilCpromoters contain multiple binding sites of each type, whereas thehilDpromoter contains a site that binds HilC but not HilD without additional binding elements. The HilC and HilD proteins had no major effect on transcription from thehilAorhilDpromoters using purified proteins in vitro but changed the choice of promoter athilC. These results are consistent with a model derived from analysis oflacZfusions stating that HilC and HilD enhancehilAexpression by counteracting a repressing activity.
Construction, Characterization, and Use of TwoListeria monocytogenesSite-Specific Phage Integration Vectors
Authors: Lauer Peter , Chow Man Yin Nora , Loessner Martin J. , Portnoy Daniel A. , Calendar Richard
Published: July 2002

Two site-specific shuttle integration vectors were developed with two different chromosomal bacteriophage integration sites to facilitate strain construction inListeria monocytogenes. The first vector, pPL1, utilizes the listeriophage U153 integrase and attachment site within thecomKgene for chromosomal insertion. pPL1 contains a useful polylinker, can be directly conjugated fromEscherichia coliintoL. monocytogenes, forms stable, single-copy integrants at a frequency of ∼10−4per donor cell, and can be used in theL. monocytogenes1/2 and 4b serogroups. Methods for curing endogenous prophages from thecomKattachment site in 10403S-derived strains were developed. pPL1 was used to introduce thehlyandactAgenes atcomK-attBB′in deletion strains derived from 10403S and SLCC-5764. These strains were tested for second-site complementation in hemolysin assays, plaquing assays, and cell extract motility assays. Unlike plasmid-complemented strains, integrated pPL1-complemented strains were fully virulent in the mouse 50% lethal dose assay. Additionally, the PSA phage attachment site on theL. monocytogeneschromosome was characterized, and pPL1 was modified to integrate at this site. The listeriophage PSA integrates in the 3′ end of an arginine tRNA gene. There are 17 bp of DNA identity between the bacterial and phage attachment sites. The PSA prophage DNA sequence reconstitutes a complete tRNAArggene. The modified vector, pPL2, was integration proficient at the same frequency as pPL1 in common laboratory serotype 1/2 strains as well as serotype 4b strains.
Functional Characterization of Gne (UDP-
N
-Acetylglucosamine- 4-Epimerase), Wzz (Chain Length Determinant), and Wzy (O-Antigen Polymerase) of
Yersinia enterocolitica
Serotype O:8
Authors: Bengoechea José Antonio , Pinta Elise , Salminen Tiina , Oertelt Clemens , Holst Otto , Radziejewska-Lebrecht Joanna , Piotrowska-Seget Zofia , Venho Reija , Skurnik Mikael
Published: July 2002

The lipopolysaccharide (LPS) O-antigen of
Yersinia enterocolitica
serotype O:8 is formed by branched pentasaccharide repeat units that contain
N
-acetylgalactosamine (GalNAc),
l
-fucose (Fuc),
d
-galactose (Gal),
d
-mannose (Man), and 6-deoxy-
d
-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called
galE
encodes a UDP-
N
-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed
gne
. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an
Escherichia coli
K-12
galE
mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of
E. coli
GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the
N
-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-
N
-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the
E. coli galE
mutant. Finally, we report that
Yersinia
Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.
Characterization of a
Bacillus subtilis
Thermosensitive Teichoic Acid-Deficient Mutant: Gene
mnaA
(
yvyH
) Encodes the UDP-
N
-Acetylglucosamine 2-Epimerase
Authors: Soldo Blazenka , Lazarevic Vladimir , Pooley Harold M. , Karamata Dimitri
Published: July 2002

The
Bacillus subtilis
thermosensitive mutant ts-21 bears two C-G→T-A transitions in the
mnaA
gene. At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content. MnaA converts UDP-
N
-acetylglucosamine into UDP-
N
-acetylmannosamine, a precursor of the teichoic acid linkage unit.
Rapid Detection and Quantification of RNA of Ebola and Marburg Viruses, Lassa Virus, Crimean-Congo Hemorrhagic Fever Virus, Rift Valley Fever Virus, Dengue Virus, and Yellow Fever Virus by Real-Time Reverse Transcription-PCR
Authors: Drosten Christian , Göttig Stephan , Schilling Stefan , Asper Marcel , Panning Marcus , Schmitz Herbert , Günther Stephan
Published: July 2002

Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum
Taq
polymerase enzyme mixture. Novel primers and/or 5′-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5′-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5′-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were thoroughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The ≥95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients.
Horizontal Transmission of
Candida parapsilosis
Candidemia in a Neonatal Intensive Care Unit
Authors: Lupetti Antonella , Tavanti Arianna , Davini Paola , Ghelardi Emilia , Corsini Valerio , Merusi Ilaria , Boldrini Antonio , Campa Mario , Senesi Sonia
Published: July 2002

This report describes the nosocomial acquisition of
Candida parapsilosis
candidemia by one of the six premature newborns housed in the same room of a neonatal intensive care unit at the Ospedale Santa Chiara, Pisa, Italy. The infant had progeria, a disorder characterized by retarded physical development and progressive senile degeneration. The infant, who was not found to harbor
C. parapsilosis
at the time of his admission to the intensive care unit, had exhibited symptomatic conjunctivitis before the onset of a severe bloodstream infection. In order to evaluate the source of infection and the route of transmission, two independent molecular typing methods were used to determine the genetic relatedness among the isolates recovered from the newborn, the inanimate hospital environment, hospital personnel, topically and intravenously administered medicaments, and indwelling catheters. Among the isolates collected, only those recovered from the hands of two nurses attending the newborns and from both the conjunctiva and the blood of the infected infant were genetically indistinguishable. Since
C. parapsilosis
was never recovered from indwelling catheters or from any of the drugs administered to the newborn, we concluded that (i) horizontal transmission of
C. parapsilosis
occurred through direct interaction between nurses and the newborn and (ii) the conjunctiva was the site through which
C. parapsilosis
entered the bloodstream. This finding highlights the possibility that a previous
C. parapsilosis
colonization and/or infection of other body sites may be a predisposing condition for subsequent
C. parapsilosis
hematogenous dissemination in severely ill newborns.
Occurrence of a Multidrug-Resistant
Pseudomonas aeruginosa
Clone in Different Hospitals in Rio de Janeiro, Brazil
Authors: Pellegrino Flávia Lúcia Piffano Costa , Teixeira Lúcia Martins , Carvalho Maria da Glória Siqueira , Aranha Nouér Simone , Pinto de Oliveira Márcia , Mello Sampaio Jorge Luiz , D'Ávila Freitas Andrea , Ferreira Adriana Lucia Pires , Amorim Efigenia de Lourdes Teixeira , Riley Lee W. , Moreira Beatriz Meurer
Published: July 2002

Multidrug-resistant
Pseudomonas aeruginosa
nosocomial infections are increasingly recognized worldwide. The existence of metallo-β-lactamase- and extended-spectrum β-lactamase-producing isolates exhibiting resistance to most β-lactam antimicrobial agents greatly complicates the clinical management of patients infected with such isolates. Since 1998,
P. aeruginosa
isolates resistant to all commercially available antimicrobial agents have been detected at a university-affiliated public hospital in Rio de Janeiro, Brazil. The present study was designed to characterize the antimicrobial resistance profiles and the genetic diversity of the
P. aeruginosa
strains isolated at this hospital and four private hospitals in Rio de Janeiro. Between April 1999 and March 2000, 200 consecutive isolates were obtained and analyzed for antimicrobial resistance. The genetic diversity of a selected number of them was evaluated by pulsed-field gel electrophoresis and PCR with the ERIC-2 primer. A predominant genotype, designated genotype A, was identified among isolates from four of the five hospitals evaluated. Eighty-four ceftazidime-resistant isolates were evaluated for metallo-β-lactamase production, which was detected in 20 (91%) of 22 genotype A isolates and 11 (18%) of 62 isolates belonging to other genotypes (
P
< 0.05). Two metallo-β-lactamase-producing genotype A isolates also produced an extended-spectrum β-lactamase. The occurrence of multidrug-resistant
P. aeruginosa
strains belonging to a unique genotype in different hospitals in Rio de Janeiro underscores the importance of the contribution of a single clone to the increase in the incidence of multidrug-resistant
P. aeruginosa
nosocomial infections.
VIDAS Test for Avidity of
Toxoplasma
-Specific Immunoglobulin G for Confirmatory Testing of Pregnant Women
Authors: Montoya Jose G. , Liesenfeld Oliver , Kinney Sandra , Press Cynthia , Remington Jack S.
Published: July 2002

Because congenital toxoplasmosis is almost solely the result of maternal infection acquired during gestation, it is critical to determine whether infection during pregnancy has occurred. In the United States, definitive diagnosis of the acute infection and the time of its occurrence have been compromised by a lack of systematic screening and the fact that only a single serum sample is submitted for testing. In studies in Europe, and depending on the method used, the demonstration of high-avidity immunoglobulin G (IgG) toxoplasma antibodies has been shown to exclude infection having occurred in the first 3 to 5 months of pregnancy. We investigated the usefulness of determining the avidity of IgG toxoplasma antibodies with a VIDAS kit (herein referred to as the VIDAS Toxo-IgG avidity kit, the VIDAS kit essentially rules out acute infection having occurred within the 4 prior months) in the setting of a reference serology laboratory in the United States. Sera (132 samples) from 132 women in the first 16 weeks of pregnancy were chosen because at least one test in the toxoplasma serological profile (TSP) suggested or was equivocal for a recently acquired infection. High-avidity antibodies were demonstrated in 75% of 99 sera positive with the IgM enzyme-linked immunosorbent assay (ELISA) and 31.3% of 16 sera with acute TSP results. A significant percentage of sera with equivocal results wtih the IgM ELISA or TSP also had high-avidity test results. Of 39 women for whom treatment with spiramycin had been suggested to attempt to prevent congenital transmission, 19 (48.7%) had high-avidity antibodies. These findings highlight the value of the VIDAS IgG avidity kit when used in combination with the TSP to exclude recent infection, especially when only a single serum sample is available.
Detection of Isoniazid-Resistant
Mycobacterium tuberculosis
Strains by a Multiplex Allele-Specific PCR Assay Targeting
katG
Codon 315 Variation
Authors: Mokrousov Igor , Otten Tatiana , Filipenko Maxim , Vyazovaya Anna , Chrapov Eugeny , Limeschenko Elena , Steklova Lidia , Vyshnevskiy Boris , Narvskaya Olga
Published: July 2002

We describe a simple multiplex allele-specific (MAS)-PCR assay to detect mutations in the second base of the
katG
gene codon 315, including AGC→ACC and ACA (Ser→Thr) substitutions that confer resistance to isoniazid (INH) in
Mycobacterium tuberculosis
clinical isolates. The 315 ACC allele is found in the majority of Inh
r
strains worldwide, especially in areas with a high incidence of tuberculosis. The 315 ACA allele is characteristic of the New York City multidrug-resistant (MDR) strain W and its progenies in the United States. The mutations in
katG315
are revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. Initially optimized on the purified DNA samples, the assay was then tested on crude cell lysates and auramine-stained sputum slide preparations with the same reproducibility and interpretability of profiles generated by agarose gel electrophoresis. The MAS-PCR assay can be used for the detection of resistance to INH in clinical laboratories in regions with a high prevalence of MDR
M. tuberculosis
strains.
Detection of Duck Hepatitis B Virus DNA on Filter Paper by PCR and SYBR Green Dye-Based Quantitative PCR
Authors: Wang Chi-Young J. , Giambrone Joseph J. , Smith Bruce F.
Published: July 2002

Duck hepatitis B virus
(DHBV) belongs to the
Hepadnaviridae
family, which includes human
Hepatitis B virus
(HBV) and
Woodchuck hepatitis virus
. It is widely distributed in wild and domestic ducks due to congenital transmission. HBV is a worldwide health problem, with carriers at risk of developing cirrhosis and liver cancer. Medical staff and scientists working with HBV must be vaccinated because of its contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Collection of serum and blood samples on filter paper has been used to screen for metabolic disorders, genetic diseases, and viral infection and for evolutionary studies of the genome. In this study, DHBV from serum and blood dried on filters was detected by PCR. A 0.1-μl sample was sufficient for detection. The immobilization potential of filter papers for DHBV was examined, and the highest yield of PCR products was observed with Whatman paper. Dried serum was stable under different storage temperatures for 4 weeks, but the yields of PCR products decreased when the temperature was ⩾4°C. The optimal condition for storage was −70°C. A newly developed quantitative PCR based on monitoring the amplification by measuring the increase in fluorescence caused by the binding of SYBR green dye to double-stranded products was applied herein. DHBV genomic DNA cloned in a plasmid was used for the generation of standard DHBV DNA for quantitative PCR. It validated results from PCR in terms of the copy number of DHBV particles. The specificity of PCR was demonstrated by melting curve analysis, and the differentiation of two DHBV isolates amplified from dried serum was demonstrated based on their melting temperatures determined by GC contents and sequence. It was easier and simpler than other PCR-based DNA techniques. The use of serum dried on filters allows samples from distant field for which cold storage and transportation are a problem to be mailed to the diagnostic laboratory. Samples can be archived for comparison and used as a source of DNA for cloning and sequencing.
Use of the Sensititre Colorimetric Microdilution Panel for Antifungal Susceptibility Testing of Dermatophytes
Authors: Pujol I. , Capilla J. , Fernández-Torres B. , Ortoneda M. , Guarro J.
Published: July 2002

The Sensititre YeastOne antifungal panel was used to test 49 dermatophytes belonging to the species
Epidermophyton floccosum
,
Microsporum gypseum
,
Microsporum canis
,
Trichophyton tonsurans
,
Trichophyton rubrum
, and
Trichophyton mentagrophytes
. The MICs of four antifungals obtained with the Sensititre YeastOne antifungal panel were compared with those obtained by the reference NCCLS microdilution method. The levels of agreement between the two methods (≤2 dilutions) were 81.6% with amphotericin B, 87.7% with itraconazole, 67.3% with fluconazole, and 69.4% with ketoconazole.
Prevalence of
Helicobacter pylori
in Sri Lanka as Determined by PCR
Authors: Fernando N. , Holton J. , Vaira D. , DeSilva M. , Fernando D.
Published: July 2002

Fifty-seven Sinhalese patients were investigated for the presence of
Helicobacter pylori
by PCR. A prevalence of 70.1%, with 47.5% positive for
cagA
, was demonstrated. The most common
vacA
allele was s1am1. There was no significant association between either the s1 allele or the
cagA
allele and severe gastroduodenal disease. There was an association between the s1 allele and the
cagA
locus.
Spoligotyping of
Mycobacterium tuberculosis
Isolates from Multiple-Drug-Resistant Tuberculosis Patients from Bombay, India
Authors: Mistry Nerges F. , Iyer Anand M. , D'souza Desirée T. B. , Taylor G. Michael , Young Douglas B. , Antia Noshir H.
Published: July 2002

Spoligotyping was undertaken in 65 multiple-drug-resistant
Mycobacterium tuberculosis
isolates from Bombay, India. The spoligotype patterns showed seven closely related clusters, a cluster with 2 Beijing-like isolates, and unique spoligotypes (43%). Of the clusters, one with 29% of all the isolates suggested transmission of a dominant resistant clone.