bluuebunny/crossref_metadata_2025 huggingface dataset:
Penetration of Atazanavir in Seminal Plasma of Men Infected with Human Immunodeficiency Virus Type 1
Authors: van Leeuwen E. , ter Heine R. , van der Veen F. , Repping S. , Beijnen J. H. , Prins J. M.
Published: December 2006

In this exploratory study, the human immunodeficiency virus (HIV) protease inhibitor atazanavir was detected in seminal plasma in 15 out of 15 HIV-infected men taking an atazanavir-containing regimen. However, this penetration was limited and variable, and the median seminal/blood plasma ratio was only 0.1.
Activity of Ribavirin against Hantaan Virus Correlates with Production of Ribavirin-5′-Triphosphate, Not with Inhibition of IMP Dehydrogenase
Authors: Sun Yanjie , Chung Dong-Hoon , Chu Yong-Kyu , Jonsson Colleen B. , Parker William B.
Published: December 2006

Ribavirin (RBV) is a broad-spectrum antiviral agent that inhibits the production of infectious Hantaan virus (HTNV). Although the mechanism of action of RBV against HTNV is not understood, RBV is metabolized in human cells to both RBV-5′-monophosphate, which inhibits IMP dehydrogenase, resulting in a decrease in intracellular GTP levels, and RBV-5′-triphosphate (RBV-TP), which could selectively interact with the viral RNA polymerase. To elucidate which activity of RBV was most important to its anti-HTNV activity, the mechanism of action of RBV was studied in Vero E6 cells. Incubation with 10 to 40 μg/ml RBV resulted in a small decrease in GTP levels that was not dose dependent. Increasing the RBV concentration from 10 to 40 μg/ml resulted in a decrease in viral RNA (vRNA) levels and an increase in RBV-TP formation. Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, also resulted in a decrease in vRNA levels; however, treatment with MPA resulted in a much greater decrease in GTP levels than that seen with RBV. Treatment with both MPA and RBV resulted in increased reduction of vRNA levels but did not result in enhanced depression of GTP levels. Although guanosine prevented the depression in GTP levels caused by RBV, guanosine only partially prevented the effect of RBV on vRNA levels. These results suggest that the inhibition of IMP dehydrogenase by RBV is of secondary importance to the inhibition of vRNA replication by RBV and that the interaction of RBV-TP with the viral polymerase is the primary action of RBV.
Inhibitors of Nonhousekeeping Functions of the Apicoplast Defy Delayed Death in
Plasmodium falciparum
Authors: Ramya T. N. C. , Mishra Satyendra , Karmodiya Krishanpal , Surolia Namita , Surolia Avadhesha
Published: December 2006

Targeting of apicoplast replication and protein synthesis in the apicomplexan
Toxoplasma gondii
has conventionally been associated with the typical “delayed death” phenotype, characterized by the death of parasites only in the generation following drug intervention. We demonstrate that antibiotics like clindamycin, chloramphenicol, and tetracycline, inhibitors of prokaryotic protein synthesis, invoke the delayed death phenotype in
Plasmodium falciparum
, too, as evident from a specific reduction of apicoplast genome copy number. Interestingly, however, molecules like triclosan, cerulenin, fops, and NAS-91, inhibitors of the recently discovered fatty acid synthesis pathway, and succinyl acetone, an inhibitor of heme biosynthesis that operates in the apicoplast of the parasite, display rapid and striking parasiticidal effects. Our results draw a clear distinction between apicoplast functions per se and the apicoplast as the site of metabolic pathways, which are required for parasite survival, and thus subserve the development of novel antimalarial therapy.
Molecular Mechanism of Flucytosine Resistance in
Candida lusitaniae
: Contribution of the
FCY2
,
FCY1
, and
FUR1
Genes to 5-Fluorouracil and Fluconazole Cross-Resistance
Authors: Papon Nicolas , Noël Thierry , Florent Martine , Gibot-Leclerc Stéphanie , Jean Dorothée , Chastin Christiane , Villard Jean , Chapeland-Leclerc Florence
Published: December 2006

Inactivation of the
FCY2
(cytosine permease),
FCY1
(cytosine deaminase), and
FUR1
(uracil phosphoribosyltransferase) genes in
Candida lusitaniae
produced two patterns of resistance to flucytosine. Mutant
fur1
demonstrated resistance to 5-fluorouracil, whereas mutants
fcy1
and
fcy2
demonstrated fluconazole resistance in the presence of subinhibitory flucytosine concentrations.
Markers of Sulfadoxine-Pyrimethamine-Resistant
Plasmodium falciparum
in Placenta and Circulation of Pregnant Women
Authors: Mockenhaupt Frank P. , Bedu-Addo George , Junge Claudia , Hommerich Lena , Eggelte Teunis A. , Bienzle Ulrich
Published: December 2006

Placental sequestration of
Plasmodium falciparum
in pregnancy may impair the usefulness of molecular markers of sulfadoxine-pyrimethamine resistance. In 300 infected, delivering women, the concordance of PCR-restriction fragment length polymorphism-derived parasite resistance alleles in matched samples from placenta and circulation was 83 to 98%. Sulfadoxine-pyrimethamine resistance typing in peripheral blood is reasonably representative of
P. falciparum
infecting pregnant women.
Susceptibility of
Acinetobacter
Strains Isolated from Deployed U.S. Military Personnel
Authors: Hawley Joshua S. , Murray Clinton K. , Griffith Matthew E. , McElmeel M. Leticia , Fulcher Letitia C. , Hospenthal Duane R. , Jorgensen James H.
Published: December 2006

The susceptibilities of 142
Acinetobacter baumannii-calcoaceticus
complex isolates (95 from wounded U.S. soldiers deployed overseas) to 13 antimicrobial agents were determined by broth microdilution. The most active antimicrobial agents (≥95% of isolates susceptible) were colistin, polymyxin B, and minocycline.
Structure and Biosynthesis of Heat-Stable Antifungal Factor (HSAF), a Broad-Spectrum Antimycotic with a Novel Mode of Action
Authors: Yu Fengan , Zaleta-Rivera Kathia , Zhu Xiangcheng , Huffman Justin , Millet Jeffrey C. , Harris Steven D. , Yuen Gary , Li Xing-Cong , Du Liangcheng
Published: December 2006

A screen for antifungal compounds from
Lysobacter enzymogenes
strain C3, a bacterial biological control agent of fungal diseases, has previously led to the isolation of heat-stable antifungal factor (HSAF). HSAF exhibits inhibitory activities against a wide range of fungal species and shows a novel mode of antifungal action by disrupting the biosynthesis of a distinct group of sphingolipids. We have now determined the chemical structure of HSAF, which is identical to that of dihydromaltophilin, an antifungal metabolite with a unique macrocyclic lactam system containing a tetramic acid moiety and a 5,5,6-tricyclic skeleton. We have also identified the genetic locus responsible for the biosynthesis of HSAF in strain C3. DNA sequencing of this locus revealed genes for a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), a sterol desaturase, a ferredoxin reductase, and an arginase. The disruption of the PKS-NRPS gene generated C3 mutants that lost the ability to produce HSAF and to inhibit fungal growth, demonstrating a hybrid PKS-NRPS that catalyzed the biosynthesis of the unique macrolactam system that is found in many biologically active natural products isolated from marine organisms. In addition, we have generated mutants with disrupted sterol desaturase, ferredoxin reductase, and arginase and examined the metabolites produced in these mutants. The work represents the first study of the genetic basis for the biosynthesis of the tetramic acid-containing macrolactams. The elucidation of the chemical structure of HSAF and the identification of the genetic locus for its biosynthesis establish the foundation for future exploitation of this group of compounds as new fungicides or antifungal drugs.
Activities of Naphthylisoquinoline Alkaloids and Synthetic Analogs againstLeishmania major
Authors: Ponte-Sucre Alicia , Faber Johan H. , Gulder Tanja , Kajahn Inga , Pedersen Sarah E. H. , Schultheis Martina , Bringmann Gerhard , Moll Heidrun
Published: December 2006

The current treatments for leishmaniasis are unsatisfactory due to their toxic side effects, high costs, and increasing problems with drug resistance. Thus, there is an urgent need for alternative drugs against leishmaniasis. Different approaches have been used to identify novel pharmacophores againstLeishmaniasp. parasites, and one strategy has been the analysis of naturally occurring plant-derived compounds, including naphthylisoquinoline alkaloids. In the present study, we examined the abilities of these alkaloids to inhibit the growth ofLeishmania majorpromastigotes and evaluated their effects on macrophages, dendritic cells, and fibroblasts. Furthermore, we determined the efficacy of selected compounds in decreasing the infection rate of macrophages and regulating their production of cytokines and nitric oxide. Our results demonstrate that the naphthylisoquinoline alkaloids ancistrocladiniums A and B (compounds 10 and 11) and the synthetic isoquinolinium salt (compound 14) were effective against intracellular amastigotes in the low submicromolar range, while toxicity against mammalian cells was observed at concentrations that were significantly higher than those needed to impair parasite replication. The activities of compounds 11 and 14 were mainly directed against the amastigote stage ofL. major. This effect was not associated with the stimulation of host macrophages to produce nitric oxide or secrete cytokines relevant for the leishmanicidal function. In conclusion, our data suggest that ancistrocladiniums A and B (compounds 10 and 11) and the synthetically prepared isoquinolinium salt (compound 14) are promising candidates to be considered as lead compounds for leishmanicidal drugs.
Variability of Voriconazole Plasma Levels Measured by New High-Performance Liquid Chromatography and Bioassay Methods
Authors: Pascual Andres , Nieth Valérie , Calandra Thierry , Bille Jacques , Bolay Saskia , Decosterd Laurent A. , Buclin Thierry , Majcherczyk Paul A. , Sanglard Dominique , Marchetti Oscar
Published: December 2006

Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible
Candida albicans
mutant lacking multidrug efflux transporters (
cdr1
Δ/
cdr1
Δ,
cdr2
Δ/
cdr2
Δ,
flu1
Δ/
flu1
Δ, and
mdr1
Δ/
mdr1
Δ) and calcineurin subunit A (
cna
Δ/
cna
Δ) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% ± 5.0%/96.5% ± 2.4% (HPLC) and 94.9% ± 6.1%/94.7% ± 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% ± 1.5%/5.4% ± 0.9% and 6.5% ± 2.5%/4.0% ± 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg · h/liter (range, 12.9 to 71.1) on the first and 27.4 mg · h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg · h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg · h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.
Inhibition of Anthrax Protective Antigen Outside and Inside the Cell
Authors: Backer Marina V. , Patel Vimal , Jehning Brian T. , Claffey Kevin P. , Karginov Vladimir A. , Backer Joseph M.
Published: December 2006

In the course of
Bacillus anthracis
infection,
B. anthracis
lethal factor (LF) and edema factor bind to a protective antigen (PA) associated with cellular receptors ANTXR1 (TEM8) or ANTXR2 (CMG2), followed by internalization of the complex via receptor-mediated endocytosis. A new group of potential antianthrax drugs, β-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-β-cyclodextrin (AmPrβCD), was shown to inhibit the toxicity of LF in vitro and in vivo. In order to determine which steps in lethal factor trafficking are inhibited by AmPrβCD, we developed two targeted fluorescent tracers based on LFn, a catalytically inactive fragment of LF: (i) LFn site specifically labeled with the fluorescent dye AlexaFluor-594 (LFn-Al), and (ii) LFn-decorated liposomes loaded with the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (LFn-Lip). Both tracers retained high affinity to PA/ANTXR complexes and were readily internalized via receptor-mediated endocytosis. Using fluorescent microscopy, we found that AmPrβCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrβCD works outside the cell. Moreover, AmPrβCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrβCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrβCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrβCD. Notably, internalized LFn-Al and LFn-Lip protected cells that overexpressed anthrax receptor TEM8 from PA-induced, LF-independent toxicity, suggesting an independent mechanism for PA inhibition inside the cell. These data suggest the potential for the use of β-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets.
Characterization of Squalene Epoxidase ofSaccharomyces cerevisiaeby Applying Terbinafine-Sensitive Variants
Authors: Ruckenstuhl Christoph , Lang Silvia , Poschenel Andrea , Eidenberger Armin , Baral Pravas Kumar , Kohút Peter , Hapala Ivan , Gruber Karl , Turnowsky Friederike
Published: December 2006

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies top-hydroxybenzoate hydroxylase (PHBH) fromPseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of theERG1gene, which codes for theSaccharomyces cerevisiaeSE, we isolatederg1alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed inerg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G25S, D335X (W, F, P), and G210A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G30S and L37P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase inerg1mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R269G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines.
Development of a Novel Dicistronic Reporter-Selectable Hepatitis C Virus Replicon Suitable for High-Throughput Inhibitor Screening
Authors: Hao Weidong , Herlihy Koleen J. , Zhang Noelle Jie , Fuhrman Shella A. , Doan Chau , Patick Amy K. , Duggal Rohit
Published: December 2006

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized
Renilla
luciferase (hRLuc) gene separated from the selectable Neo
r
marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated
Z
′ value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 μM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals ≥50 and ≤40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.
Quinupristin-Dalfopristin Nonsusceptibility in Pneumococci from Sickle Cell Disease Patients
Authors: Obert Caroline A. , Miller Martha L. , Montgomery Jeremy , Adamkiewicz Thomas , Tuomanen Elaine I.
Published: December 2006

Sickle cell disease (SCD) is a risk factor for fatal pneumococcal infection. Nonsusceptibilty to quinupristin-dalfopristin (Q-D) was absent from 105 non-SCD-associated pneumococcal isolates but was present in 33/148 (22%) SCD-associated isolates. One-third of the isolates harbored a known resistance mechanism. Q-D is not optimal for use for the treatment of pneumococcal infection in SCD patients.
Phenotypic and Molecular Analysis of Penicillin-Nonsusceptible
Streptococcus pneumoniae
Isolates in Poland
Authors: Sadowy Ewa , Izdebski Radosław , Skoczyńska Anna , Grzesiowski Paweł , Gniadkowski Marek , Hryniewicz Waleria
Published: December 2006

β-Lactams are the drugs of choice for the treatment of infections caused by the important bacterial pathogen
Streptococcus pneumoniae
. The recent growth of resistance of this organism to penicillin observed worldwide is of the highest concern. In this study, using 887 surveillance pneumococcal isolates recovered in Poland from 1998 to 2002, we observed the increase in penicillin nonsusceptibility from 8.7% to 20.3%. All of the 109 penicillin-nonsusceptible
S. pneumoniae
(PNSP) isolates identified, together with 22 archival PNSP isolates from 1995 to 1997, were subsequently analyzed by susceptibility testing, serotyping, profiling of
pbp
genes, pulsed-field gel electrophoresis, and multilocus sequence typing (MLST). Four predominant serotypes, serotypes 6B, 9V, 14, and 23F, characterized 85.5% of the isolates. MLST revealed the presence of 34 sequence types, 15 of which were novel types. Representatives of seven multiresistant international clones (Spain
23F
-1, Spain
6B
-2, Spain
9V
-3, Taiwan
23F
-15, Poland
23F
-16, Poland
6B
-20, and Sweden
15A
-25) or their closely related variants comprised the majority of the study isolates. The spread of Spain
9V
-3 and its related clone of serotype 14/ST143 has remarkably contributed to the recent increase in penicillin resistance in pneumococci in the country.
SGI1-K, a Variant of the SGI1 Genomic Island Carrying a Mercury Resistance Region, in
Salmonella enterica
Serovar Kentucky
Authors: Levings Renee S. , Partridge Sally R. , Djordjevic Steven P. , Hall Ruth M.
Published: December 2006

A multiple-antibiotic-resistant
Salmonella enterica
serovar Kentucky strain was found to contain SGI1-K, a variant form of the
Salmonella
genomic island 1 (SGI1) with an In4-type class 1 integron that contains only one cassette array,
aacCA5-aadA7
, and an adjacent mercury resistance module. Part of the 3′-conserved segment (3′-CS) of the integron, together with the inverted short segment from the right-hand end of the integron transposition module normally found between the 3′-CS and IS
6100
in In4 family integrons, has been removed by an IS
6100
-mediated deletion. IRt, the right-hand inverted repeat found at the outer end of the integron, abuts a mercury resistance region instead of the usual SGI1 backbone segment. The
mer
module is a hybrid of those found in Tn
501
and Tn
21
. This
mer
region and a further uncharacterized segment of at least 10 kb appear to have been incorporated between IRt and the SGI1 backbone. These findings demonstrate that the multidrug resistance region in SGI1 can incorporate new DNA segments in the same way as multiple antibiotic resistance regions in plasmids.
Identification of theActinobacillus pleuropneumoniaeLeucine-Responsive Regulatory Protein and Its Involvement in the Regulation of In Vivo-Induced Genes
Authors: Wagner Trevor K. , Mulks Martha H.
Published: December 2006

Actinobacillus pleuropneumoniaeis a gram-negative bacterial pathogen that causes a severe hemorrhagic pneumonia in swine. We have previously shown that the limitation of branched-chain amino acids (BCAAs) is a cue that induces the expression of a subset ofA. pleuropneumoniaegenes identified as specifically induced during infection of the natural host animal by using an in vivo expression technology screen. Leucine-responsive regulatory protein (Lrp) is a global regulator and has been shown inEscherichia colito regulate many genes, including genes involved in BCAA biosynthesis. We hypothesized thatA. pleuropneumoniaecontains a regulator similar to Lrp and that this protein is involved in the regulation of a subset of genes important during infection and recently shown to have increased expression in the absence of BCAAs. We report the identification of anA. pleuropneumoniaeserotype 1 gene encoding a protein with similarity to amino acid sequence and functional domains of other reported Lrp proteins. We further show that purifiedA. pleuropneumoniaeHis6-Lrp binds in vitro to theA. pleuropneumoniaepromoter regions forilvI, antisensecps1AB,lrp, andnqr. A genetically definedA. pleuropneumoniae lrpmutant was constructed using an allelic replacement and sucrose counterselection method. Analysis of expression from theilvIand antisensecps1ABpromoters in wild-type,lrpmutant, and complementedlrpmutant strains indicated that Lrp is required for induction of expression ofilvIunder BCAA limitation.
Essential Role of CCL2 in Clustering of Splenic ERTR-9+Macrophages during Infection of BALB/c Mice byListeria monocytogenes
Authors: Jablonska Jadwiga , Dittmar Kurt E. , Kleinke Tanja , Buer Jan , Weiss Siegfried
Published: December 2006

Early interactions between pathogens and host cells are often decisive for the subsequent course of infection. Here we investigated early events during infection byListeria monocytogenes, a ubiquitously occurring facultative intracellular microorganism that exhibits severe pathogenicity, mainly in immunocompromised individuals. We show that the inflammatory chemokine CCL2 is highly up-regulated early afterListeriainfection in spleens of BALB/c mice. ERTR-9+macrophages of the marginal zone were identified as the only infected cells and exclusive producers of CCL2 at the early time point. Consequently, clusters of different cell types were formed around infected ERTR-9+cells. Metallophilic MOMA-1+marginal zone macrophages were, however, excluded from the clusters and migrated into the B-cell follicles. Depletion of CCL2 during infection resulted in a different composition of cell clusters in the spleen and increased the mortality rate of treated mice. Interestingly, ERTR-9+macrophages no longer were part of clusters in such mice but remained at their original location in the marginal zone.
The YfgL Lipoprotein Is Essential for Type III Secretion System Expression and Virulence of
Salmonella enterica
Serovar Enteritidis
Authors: Fardini Yann , Chettab Kamel , Grépinet Olivier , Rochereau Sandrine , Trotereau Jérôme , Harvey Philippa , Amy Maïté , Bottreau Elisabeth , Bumstead Nat , Barrow Paul A. , Virlogeux-Payant Isabelle
Published: December 2006

Salmonella enterica
, like many gram-negative pathogens, uses type three secretion systems (TTSS) to infect its hosts. The three TTSS of
Salmonella
, namely, TTSS-1, TTSS-2, and flagella, play a major role in the virulence of this bacterium, allowing it to cross the intestinal barrier and to disseminate systemically. Previous data from our laboratory have demonstrated the involvement of the chromosomal region harboring the
yfgL
,
engA
, and
yfgJ
open reading frames in
S. enterica
serovar Enteritidis virulence. Using microarray analysis and real-time reverse transcription-PCR after growth of bacterial cultures favorable for either TTSS-1 or TTSS-2 expression, we show in this study that the deletion in
S. enterica
serovar Enteritidis of
yfgL
, encoding an outer membrane lipoprotein, led to the transcriptional down-regulation of most
Salmonella
pathogenicity island 1 (SPI-1), SPI-2, and flagellar genes encoding the TTSS structural proteins and effector proteins secreted by these TTSS. In line with these results, the virulence of the Δ
yfgL
mutant was greatly attenuated in mice. Moreover, even if YfgL is involved in the assembly of outer membrane proteins, the regulation of TTSS expression observed was not due to an inability of the Δ
yfgL
mutant to assemble TTSS in its membrane. Indeed, when we forced the transcription of SPI-1 genes by constitutively expressing HilA, the secretion of the TTSS-1 effector protein SipA was restored in the culture supernatant of the mutant. These results highlight the crucial role of the outer membrane lipoprotein YfgL in the expression of all
Salmonella
TTSS and, thus, in the virulence of
Salmonella
. Therefore, this outer membrane protein seems to be a privileged target for fighting
Salmonella
.
Generation of Cross-Protective Antibodies against
Plasmodium falciparum
Sequestration by Immunization with an Erythrocyte Membrane Protein 1-Duffy Binding-Like 1α Domain
Authors: Moll Kirsten , Pettersson Fredrik , Vogt Anna M. , Jonsson Cathrine , Rasti Niloofar , Ahuja Sanjay , Spångberg Mats , Mercereau-Puijalon Odile , Arnot David E. , Wahlgren Mats , Chen Qijun
Published: December 2006

The
Plasmodium falciparum
erythrocyte membrane protein 1 (PfEMP1) is an important virulence factor on the surface of infected erythrocytes. Naturally acquired antibodies to PfEMP1 expressed by parasites causing severe malaria are suggested to be protective and of major interest for the development of a vaccine against severe disease. In this study, the PfEMP1 expressed by a parasite clone displaying a multiadhesive phenotype associated with severe malaria was well recognized by sera of malaria semi-immune children. The efficiency of the Duffy binding-like 1α (DBL1α) domain of this PfEMP1 was therefore, alone or in combination with two additional DBL1α domains, evaluated as a potential vaccine candidate using both a rodent model and a primate model. Antibodies against the DBL1α domain were generated by immunization with recombinant DBL1α-Semliki Forest virus particles and recombinant protein and analyzed in vitro. The immunized animals were challenged in vivo with various parasite strains or clones. Immunization with the PfEMP1-DBL1α domain abolished the PfEMP1-dependent sequestration of the homologous strain in immunized rats and substantially inhibited parasite adhesion in immunized monkeys. Protection against sequestration of heterologous parasite strains was also confirmed by direct or indirect challenge in the rat model. These results strongly support the use of the DBL1α domain in the development of a vaccine targeting severe malaria.
Absence of Toll-Like Receptor 4 Signaling Results in Delayed
Yersinia enterocolitica
YopP-Induced Cell Death of Dendritic Cells
Authors: Gröbner Sabine , Schulz Sebastian , Soldanova Irena , Gunst Dani S. J. , Waibel Michaela , Wesselborg Sebastian , Borgmann Stefan , Autenrieth Ingo B.
Published: December 2006

In an initial period (≤4 h) Toll-like receptor 4 (TLR4) signaling is required for
Yersinia enterocolitica
YopP-induced dendritic cell (DC) death. Later (>4 h), DC die independent of TLR4 signaling. In TLR4-deficient DC caspase 8 cleavage is delayed, indicating that TLR4 signaling accelerates caspase 8 activation, leading to DC death.
Secretion of Functional Monocyte Chemotactic Protein 3 by RecombinantMycobacterium bovisBCG Attenuates Vaccine Virulence and Maintains Protective Efficacy againstM. tuberculosisInfection
Authors: Ryan Anthony A. , Spratt Joanne M. , Britton Warwick J. , Triccas James A.
Published: December 2006

A strain ofMycobacterium bovisBCG that secretes high levels of functional murine monocyte chemotactic protein 3 (BCGMCP-3) was developed. Mice vaccinated with BCGMCP-3displayed increased lymphocyte migration in vivo and augmented antigen-specific T-cell responses compared to mice vaccinated with BCG alone. The level of protection afforded by BCGMCP-3was equivalent to that with control BCG; however, immunodeficient mice infected with BCGMCP-3survived significantly longer than mice infected with the control BCG strain. Therefore, BCGMCP-3may be a safer alternative than conventional BCG for vaccination of immunocompromised individuals.
Proteolytic Processing of the
Cryptosporidium
Glycoprotein gp40/15 by Human Furin and by a Parasite-Derived Furin-Like Protease Activity
Authors: Wanyiri Jane W. , O'Connor Roberta , Allison Geneve , Kim Kami , Kane Anne , Qiu Jiazhou , Plaut Andrew G. , Ward Honorine D.
Published: December 2006

The apicomplexan parasite
Cryptosporidium
causes diarrheal disease worldwide. Proteolytic processing of proteins plays a significant role in host cell invasion by apicomplexan parasites. In previous studies, we described gp40/15, a
Cryptosporidium
sp. glycoprotein that is proteolytically cleaved to yield two surface glycopeptides (gp40 and gp15), which are implicated in mediating infection of host cells. In the present study, we showed that biosynthetically labeled gp40/15 is processed in
Cryptosporidium parvum
-infected HCT-8 cells. We identified a putative furin cleavage site RSRR↓ in the deduced amino acid sequence of gp40/15 from
C. parvum
and from all
Cryptosporidium hominis
subtypes except subtype 1e. Both human furin and a protease activity present in a
C. parvum
lysate cleaved recombinant
C. parvum
gp40/15 protein into 2 peptides, identified as gp40 and gp15 by size and by immunoreactivity with specific antibodies.
C. hominis
gp40/15 subtype 1e, in which the RSRR sequence is replaced by ISKR, has an alternative furin cleavage site (KSISKR↓) and was also cleaved by both furin and the
C. parvum
lysate. Site-directed mutagenesis of the
C. parvum
RSRR sequence to ASRR resulted in inhibition of cleavage by furin and the
C. parvum
lysate. Cleavage of recombinant gp40/15 and a synthetic furin substrate by the
C. parvum
lysate was inhibited by serine protease inhibitors, by the specific furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-cmk), and by calcium chelators, suggesting that the parasite expresses a Ca
2+
dependent, furin-like protease activity. The furin inhibitor Dec-RVKR-cmk decreased
C. parvum
infection of HCT-8 cells, suggesting that a furin-like protease activity may be involved in mediating host-parasite interactions.
A Bile Salt Hydrolase of
Brucella abortus
Contributes to the Establishment of a Successful Infection through the Oral Route in Mice
Authors: Delpino M. Victoria , Marchesini María I. , Estein Silvia M. , Comerci Diego J. , Cassataro Juliana , Fossati Carlos A. , Baldi Pablo C.
Published: December 2006

Choloylglycine hydrolase (CGH), a bile salt hydrolase, has been annotated in all the available genomes of
Brucella
species. We obtained the
Brucella
CGH in recombinant form and demonstrated in vitro its capacity to cleave glycocholate into glycine and cholate.
Brucella abortus
2308 (wild type) and its isogenic Δ
cgh
deletion mutant exhibited similar growth rates in tryptic soy broth in the absence of bile. In contrast, the growth of the Δ
cgh
mutant was notably impaired by both 5% and 10% bile. The bile resistance of the complemented mutant was similar to that of the wild-type strain. In mice infected through the intragastric or the intraperitoneal route, splenic infection was significantly lower at 10 and 20 days postinfection in animals infected with the Δ
cgh
mutant than in those infected with the wild-type strain. For both routes, no differences in spleen CFU were found between animals infected with the wild-type strain and those infected with the complemented mutant. Mice immunized intragastrically with recombinant CGH mixed with cholera toxin (CGH+CT) developed a specific mucosal humoral (immunoglobulin G [IgG] and IgA) and cellular (interleukin-2) immune responses. Fifteen days after challenge by the same route with live
B. abortus
2308 cells, splenic CFU counts were 10-fold lower in mice immunized with CGH+CT than in mice immunized with CT or phosphate-buffered saline. This study shows that CGH confers on
Brucella
the ability to resist the antimicrobial action of bile salts. The results also suggest that CGH may contribute to the ability of
Brucella
to infect the host through the oral route.
Analysis ofBartonellaAdhesin A Expression Reveals Differences between VariousB. henselaeStrains
Authors: Riess Tanja , Raddatz Günter , Linke Dirk , Schäfer Andrea , Kempf Volkhard A. J.
Published: December 2006

Bartonella henselaecauses cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis in humans. One of the best known pathogenicity factors ofB. henselaeisBartonellaadhesin A (BadA), which is modularly constructed, consisting of head, neck/stalk, and membrane anchor domains. BadA is important for the adhesion ofB. henselaeto extracellular-matrix proteins and endothelial cells (ECs). In this study, we analyzed differentB. henselaestrains for BadA expression, autoagglutination, fibronectin (Fn) binding, and adhesion to ECs. We found that theB. henselaestrains Marseille, ATCC 49882, Freiburg 96BK3 (FR96BK3), FR96BK38, and G-5436 express BadA. Remarkably, BadA expression was lacking in aB. henselaeATCC 49882 variant, in strains ATCC 49793 and Berlin-1, and in the majority of bacteria of strain Berlin-2. Adherence ofB. henselaeto ECs and Fn reliably correlated with BadA expression.badAwas present in all tested strains, although the length of the gene varied significantly due to length variations of the stalk region. Sequencing of the promoter, head, and membrane anchor regions revealed only minor differences that did not correlate with BadA expression, apart from strain Berlin-1, in which a 1-bp deletion led to a frameshift in the head region of BadA. Our data suggest that, apart from the identified genetic modifications (frameshift deletion and recombination), other so-far-unknown regulatory mechanisms influence BadA expression. Because of variations between and within differentB. henselaeisolates, BadA expression should be analyzed before performing infection experiments withB. henselae.
Compartmentalization of the Broad-Range Phospholipase C Activity to the Spreading Vacuole Is Critical forListeria monocytogenesVirulence
Authors: Yeung P. S. Marie , Na Yoojin , Kreuder Amanda J. , Marquis Hélène
Published: December 2006

Listeria monocytogenesis a bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell by using an actin-based mechanism of motility. The broad-range phospholipase C (PC-PLC) ofL. monocytogenescontributes to bacterial escape from vacuoles formed upon cell-to-cell spread. PC-PLC is made as an inactive proenzyme whose activation requires cleavage of an N-terminal propeptide. During infection, PC-PLC is activated specifically in acidified vacuoles. To assess the importance of compartmentalizing PC-PLC activity during infection, we created a mutant that makes constitutively active PC-PLC (theplcBΔpro mutant). Results from intracellular growth and cell-to-cell spread assays showed that theplcBΔpro mutant was sensitive to gentamicin, suggesting that unregulated PC-PLC activity causes damage to host cell membranes. This was confirmed by the observation of a twofold increase in staining of live infected cells by a non-membrane-permeant DNA fluorescent dye. However, membrane damage was not sufficient to cause cell lysis and was dependent on bacterial cell-to-cell spread, suggesting that damage was localized to bacterium-containing filopodia. Using an in vivo competitive infection assay, we observed that theplcBΔpro mutant was outcompeted up to 200-fold by the wild-type strain in BALB/c mice. Virulence attenuation was greater when mice were infected orally than when they were infected intravenously, presumably because theplcBΔpro mutant was initially outcompeted in the intestines, reducing the number of mutant bacteria reaching the liver and spleen. Together, these results emphasize the importance forL. monocytogenesvirulence of compartmentalizing the activity of PC-PLC during infection.
Role of Toll-Like Receptor 9 in
Legionella pneumophila
-Induced Interleukin-12 p40 Production in Bone Marrow-Derived Dendritic Cells and Macrophages from Permissive and Nonpermissive Mice
Authors: Newton Cathy A. , Perkins Izabella , Widen Raymond H. , Friedman Herman , Klein Thomas W.
Published: December 2006

The progression of
Legionella pneumophila
infection in macrophages is controlled by the
Lgn1
gene locus, which expresses the nonpermissive phenotype in cells from BALB/c mice but the permissive phenotype in cells from A/J mice. Activation of dendritic cells and macrophages by
L. pneumophila
is mediated by the pathogen recognition receptor Toll-like receptor 2 (TLR2); furthermore,
Legionella
induces innate and adaptive immune cytokines by the MyD88-dependent pathway. TLR9 is coupled to MyD88 and mediates the production of interleukin-12 (IL-12) in dendritic cells infected with other facultatively intracellular pathogens. In the current study,
L. pneumophila
growth in dendritic cells from BALB/c and A/J mice was examined along with the role of TLR9 in the induction of IL-12 in these cells. Dendritic cells from both strains were nonpermissive for
L. pneumophila
intracellular growth, suggesting that the products of the
Lgn1
gene locus that control intracellular growth in macrophages do not control the growth of
Legionella
in dendritic cells. In addition, chloroquine treatment suppressed IL-12 p40 production in response to
Legionella
treatment in dendritic cells and macrophages from BALB/c and A/J mice. Furthermore, the TLR9 inhibitor ODN2088 suppressed the
Legionella
-induced IL-12 production in dendritic cells from both mouse strains. These results suggest that
L. pneumophila
is similar to other intracellular bacteria in that it stimulates the production of immune-transitioning cytokines, such as IL-12, through activation of TLR9 and that this receptor provides a common mechanism for sensing these types of microbes and inducing innate and adaptive immunity.
Role of Complement in Defense of the Middle Ear Revealed by Restoring the Virulence of NontypeableHaemophilus influenzae siaBMutants
Authors: Figueira Marisol A. , Ram Sanjay , Goldstein Richard , Hood Derek W. , Moxon E. Richard , Pelton Stephen I.
Published: December 2006

Nontypeable (NT)Haemophilus influenzaeis an important cause of otitis media in children. We have shown previously that NTH. influenzaemutants defective in their ability to sialylate lipopolysaccharide (LPS), calledsiaBmutants, show attenuated virulence in a chinchilla model of experimental otitis media (EOM). We show that complement is a key arm of host innate immunity against NTH. influenzae-induced EOM. Depleting complement in chinchillas by use of cobra venom factor (CoVF) rendered two otherwise avirulentsiaBmutants fully virulent and able to cause EOM with severity similar to that of wild-type strains. Clearance of infection caused bysiaBmutants in CoVF-treated animals coincided with reappearance of C3. Wild-type strains were more resistant to direct complement-mediated killing than theirsiaBmutants. The serum-resistant strain bound less C3 and C4 than the serum-sensitive strain. Neither NTH. influenzaestrain tested bound factor H (alternative complement pathway regulator). Selective activation of the alternative pathway resulted in more C3 binding tosiaBmutants. LPS sialylation had a more profound impact on the amount of alternative-pathway-mediated C3 binding (∼5-fold decrease in fluorescence) when LPS was the main C3 target, as occurred on the more serum-resistant strain. In contrast, only an ∼1.5-fold decrease in fluorescence intensity of C3 binding was seen with the serum-sensitive strain, where surface proteins predominantly bound C3. Differences in binding sites for C3 and C4 may account for variations in serum resistance between NTH. influenzaestrains, which in turn may impact their virulence. These data demonstrate a central role for complement in innate immune defenses against NTH. influenzaeinfections and specifically EOM.
Development of a Vaccine against Invasive Pneumococcal Disease Based on Combinations of Virulence Proteins of
Streptococcus pneumoniae
Authors: Ogunniyi Abiodun D. , Grabowicz Marcin , Briles David E. , Cook Jan , Paton James C.
Published: December 2006

Current global efforts are focused on exploring alternative pneumococcal vaccine strategies, aimed at addressing the shortcomings of existing formulations, without compromising efficacy. One such strategy involves the use of one or more pneumococcal protein antigens common to all serotypes, to provide cheap, non-serotype-dependent protection. In this study, we evaluated the protective efficacy of immunization of mice with PdB (a pneumolysin toxoid), PspA, PspC (CbpA), PhtB, and PhtE in an invasive-disease model. The antigens were administered in alum adjuvant, either alone or in various combinations. Protection against intraperitoneal challenge with virulent type 2 and 6A strains was assessed in two murine strains. Our findings show that in some situations, different individual proteins gave the best (and worst) protection. However, in many cases, a synergistic/additive effect was seen by using multiple proteins even where the individual proteins showed little value by themselves. For instance, the median survival times for mice immunized with combinations of PdB and PspA, PdB and PspC, or PspA and PspC were significantly longer than those for mice immunized with any of the single antigens. To date, the combination of PdB, PspA, and PspC offers the best protection.
Cerebrospinal Fluid-Infiltrating CD4+T Cells RecognizeBorrelia burgdorferiLysine-Enriched Protein Domains and Central Nervous System Autoantigens in Early Lyme Encephalitis
Authors: Lünemann Jan D. , Gelderblom Harald , Sospedra Mireia , Quandt Jacqueline A. , Pinilla Clemencia , Marques Adriana , Martin Roland
Published: December 2006

Neurological manifestations of Lyme disease are usually accompanied by inflammatory changes in the cerebrospinal fluid (CSF) and the recruitment of activated T cells into the CSF compartment. In order to characterize the phenotype and identify target antigens of CSF-infiltrating T cells in early neuroborreliosis with central nervous system (CNS) involvement, we combined T-cell cloning, functional testing of T-cell responses with positional scanning synthetic combinatorial peptide libraries, and biometric data analysis. We demonstrate that CD4+gamma interferon-producing T cells specifically responding toBorrelia burgdorferilysate were present in the CSF of a patient with acute Lyme encephalitis. Some T-cell clones recognized previously uncharacterizedB. burgdorferiepitopes which show a specific enrichment for lysine, such as the heat shock-induced chaperone HSP90. Degenerate T-cell recognition that included T-cell responses to borrelia-specific and CNS-specific autoantigens derived from the myelin protein 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) could be demonstrated for one representative clone. Our results show that spirochetal antigen-specific and Th1-polarized CD4+lymphocytes infiltrate the CSF during monophasic CNS symptoms of Lyme disease and demonstrate that cross-recognition of CNS antigens byB. burgdorferi-specific T cells is not restricted to chronic and treatment-resistant manifestations.
Quantitative Analysis oftprGene Expression inTreponema pallidumIsolates: Differences among Isolates and Correlation with T-Cell Responsiveness in Experimental Syphilis
Authors: Giacani Lorenzo , Molini Barbara , Godornes Charmie , Barrett Lynn , Van Voorhis Wesley , Centurion-Lara Arturo , Lukehart Sheila A.
Published: December 2006

Transcriptional analysis of thetprgenes inTreponema pallidumsubsp.pallidum(referred to here as simplyT. pallidum) has been limited to date, and yet the expression of members of this gene family is likely relevant to the pathogenesis of syphilis. Recently, immunological studies and semiquantitative mRNA analysis led to the hypothesis of the modulation oftprgene transcription during infection and suggested that various strains ofT. pallidummight differentially express these genes. In this study we developed a real-time amplification assay to quantify thetprmRNAs with respect to the 47-kDa lipoprotein message and to compare transcript levels among four different strains ofT. pallidum. In addition, we analyzed the lymphocyte responsiveness pattern toward the Tpr antigens in late experimental syphilis to identifytprgenes that had been expressed during the course of infection. The T-cell response has been implicated in clearance of treponemes from early lesions, and some of the Tprs were identified as strong targets of the cellular immune response. We show that message for many of thetprgenes can be detected in treponemes harvested at the peak of early infection. Interestingly,tprKseems to be preferentially expressed in almost every strain, and it is uniformly the target of the strongest cellular immune response. These studies demonstrate the differential expression of certaintprgenes among strains ofT. pallidum, and further studies are needed to explore the relationship betweentprgene expression and the clinical course of syphilis in infected individuals.