bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional Activation of APETALA1 by LEAFY
Authors: Wagner Doris , Sablowski Robert W. M. , Meyerowitz Elliot M.
Published: July 2002

Plants produce new appendages reiteratively from groups of stem cells called shoot apical meristems. LEAFY (LFY) and APETALA1 (AP1) are pivotal for the switch to the reproductive phase, where instead of leaves the shoot apical meristem produces flowers. Use of steroid-inducible activation of LFY demonstrated that early expression of
AP1
is a result of transcriptional induction by LFY. This
AP1
induction is independent of protein synthesis and occurs specifically in the tissues and at the developmental stage in which floral fate is assumed. Later expression of
AP1
appears to be only indirectly affected by LFY.
Whither the Future of Controlling Quantum Phenomena?
Authors: Rabitz Herschel , de Vivie-Riedle Regina , Motzkus Marcus , Kompa Karl
Published: July 2002

This review puts into perspective the present state and prospects for controlling quantum phenomena in atoms and molecules. The topics considered include the nature of physical and chemical control objectives, the development of possible quantum control rules of thumb, the theoretical design of controls and their laboratory realization, quantum learning and feedback control in the laboratory, bulk media influences, and the ability to utilize coherent quantum manipulation as a means for extracting microscopic information. The preview of the field presented here suggests that important advances in the control of molecules and the capability of learning about molecular interactions may be reached through the application of emerging theoretical concepts and laboratory technologies.
Direct Condensation of Carboxylic Acids with Alcohols Catalyzed by Hafnium(IV) Salts
Authors: Ishihara Kazuaki , Ohara Suguru , Yamamoto Hisashi
Published: July 2002

In order to promote atom efficiency in synthesis and to avoid the generation of environmental waste, the use of stoichiometric amounts of condensing reagents or excess substrates should be avoided. In esterification, excesses of either carboxylic acids or alcohols are normally needed. We show that the direct condensation of equimolar amounts of carboxylic acids and alcohols can be achieved with the use of hafnium(IV) salts, such as commercially available hafnium(IV) chloride and hafnium(IV)
tert
-butoxide. The present method can be applied to direct polyesterification and may be suitable for large-scale operations.
Effects of
cis
Arrangement of Chromatin Insulators on Enhancer-Blocking Activity
Authors: Cai Haini N. , Shen Ping
Published: July 2002

Chromatin boundary elements or insulators are believed to regulate gene activity in complex genetic loci by organizing specialized chromatin structures
.
Here, we report that the enhancer-blocking activity of the
Drosophila
suHw insulator is sensitive to insulator copy number and position. Two tandem copies of suHw were ineffective in blocking various enhancers from a downstream promoter
.
Moreover, an enhancer was blocked more effectively from a promoter by two flanking suHw insulators than by a single intervening one. Thus, insulators may modulate enhancer-promoter interactions by interacting with each other and facilitating the formation of chromatin loop domains.
Flow Cytometry Antifungal Susceptibility Testing of Pathogenic Yeasts other than
Candida albicans
and Comparison with the NCCLS Broth Microdilution Test
Authors: Ramani Rama , Chaturvedi Vishnu
Published: July 2002

Candida
species other than
Candida albicans
frequently cause nosocomial infections in immunocompromised patients. Some of these pathogens have either variable susceptibility patterns or intrinsic resistance against common azoles. The availability of a rapid and reproducible susceptibility-testing method is likely to help in the selection of an appropriate regimen for therapy. A flow cytometry (FC) method was used in the present study for susceptibility testing of
Candida glabrata
,
Candida guilliermondii
,
Candida krusei
,
Candida lusitaniae
,
Candida parapsilosis
,
Candida tropicalis
, and
Cryptococcus neoformans
based on accumulation of the DNA binding dye propidium iodide (PI). The results were compared with MIC results obtained for amphotericin B and fluconazole using the NCCLS broth microdilution method (M27-A). For FC, the yeast inoculum was prepared spectrophotometrically, the drugs were diluted in either RPMI 1640 or yeast nitrogen base containing 1% dextrose, and yeast samples and drug dilutions were incubated with amphotericin B and fluconazole, respectively, for 4 to 6 h. Sodium deoxycholate and PI were added at the end of incubation, and fluorescence was measured with a FACScan flow cytometer (Becton Dickinson). The lowest drug concentration that showed a 50% increase in mean channel fluorescence compared to that of the growth control was designated the MIC. All tests were repeated once. The MICs obtained by FC for all yeast isolates except
C. lusitaniae
were in very good agreement (within 1 dilution) of the results of the NCCLS broth microdilution method. Paired
t
test values were not statistically significant (
P
= 0.377 for amphotericin B;
P
= 0.383 for fluconazole). Exceptionally,
C. lusitaniae
isolates showed higher MICs (2 dilutions or more) than in the corresponding NCCLS broth microdilution method for amphotericin B. Overall, FC antifungal susceptibility testing provided rapid, reproducible results that were statistically comparable to those obtained with the NCCLS method.
New TEM Variant (TEM-92) Produced by
Proteus mirabilis
and
Providencia stuartii
Isolates
Authors: de Champs Christophe , Monne Claire , Bonnet Richard , Sougakoff Wladimir , Sirot Danielle , Chanal Catherine , Sirot Jacques
Published: July 2002

The sequences of the
bla
TEM
genes encoding TEM-92 in
Proteus mirabilis
and
Providencia stuartii
isolates were determined and were found to be identical. Except for positions 218 (Lys-6) and 512 (Lys-104), the nucleotide sequence of
bla
TEM-92
was identical to that of
bla
TEM-20
, including the sequence of the promoter region harboring a 135-bp deletion combined with a G-162→T substitution. The deduced amino acid sequence of TEM-92 differed from that of TEM-52 by the presence of a substitution (Gln-6→Lys) in the peptide signal.
In Vitro Activities of Four Novel Triazoles against
Scedosporium
spp
Authors: Carrillo A. J. , Guarro J.
Published: July 2002

In order to develop new approaches to the treatment of the severe and usually fatal infections caused by
Scedosporium
spp., the in vitro antifungal activities of four novel triazoles (posaconazole, ravuconazole, voriconazole, and UR-9825) and some current antifungals (amphotericin B, ketoconazole, itraconazole, and nystatin) were determined. The latter group was clearly ineffective against the two species tested. The four new antifungals showed activity against
Scedosporium apiospermum
, and UR-9825 and voriconazole were active against
S. prolificans
.
Cloning and Biochemical Characterization of a Class A β-Lactamase from
Prevotella intermedia
Authors: Madinier I. , Fosse T. , Giudicelli J. , Labia R.
Published: July 2002

The gene encoding a β-lactamase of
Prevotella intermedia
was cloned and sequenced. This gene, called
cfxA2
, shared 98% identity with
cfxA
, the structural gene of a β-lactamase previously described in
Bacteroides vulgatus
. The deduced protein sequence had a K272E substitution. CfxA2 had the characteristics of class A, group 2e β-lactamases.
Structural Analysis of Chloroquine Resistance Reversal by Imipramine Analogs
Authors: Bhattacharjee Apurba K. , Kyle Dennis E. , Vennerstrom Jonathan L.
Published: July 2002

For imipramine, desipramine, and eight analogs of these well-known drugs, an N-5-aminoalkyl substitution was a minimum but insufficient structural feature associated with chloroquine resistance reversal. Although a second distal aliphatic nitrogen atom was unnecessary for resistance reversal, the direction of the dipole moment vector was critical.
Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB
Authors: Heddle Jonathan , Maxwell Anthony
Published: July 2002

DNA gyrase is a prokaryotic type II topoisomerase and a major target of quinolone antibacterials. The majority of mutations conferring resistance to quinolones arise within the quinolone resistance-determining region of GyrA close to the active site (Tyr
122
) where DNA is bound and cleaved. However, some quinolone resistance mutations are known to exist in GyrB. Present structural data suggest that these residues lie a considerable distance from the quinolone resistance-determining region, and it is not obvious how they affect quinolone action. We have made and purified two such mutant proteins, GyrB(Asp
426
→Asn) and GyrB(Lys
447
→Glu), and characterized them in vitro. We found that the two proteins behave similarly to GyrA quinolone-resistant proteins. We showed that the mutations exert their effect by decreasing the amount of quinolone bound to a gyrase-DNA complex. We suggest that the GyrB residues form part of a quinolone-binding pocket that includes DNA and the quinolone resistance-determining region in GyrA and that large conformational changes during the catalytic cycle of the enzyme allow these regions to come into close proximity.
(1→6)-β-d-Glucan as Cell Wall Receptor forPichia membranifaciensKiller Toxin
Authors: Santos A. , Marquina D. , Leal J. A. , Peinado J. M.
Published: July 2002

The killer toxin fromPichia membranifaciensCYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1→6)-β-d-glucan of the cell wall of a sensitive yeast (Candida boidiniiIGC 3430). The (1→6)-β-d-glucan was purified from cell walls ofC. boidiniiby alkali and hot-acetic acid extraction, a procedure which solubilizes glucans. The major fraction of receptor activity remained with the alkali-insoluble (1→6)-β- and (1→3)-β-d-glucans. The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and1H nuclear magnetic resonance) analyses of the fractions obtained showed that (1→6)-β-d-glucan was a receptor. Adsorption of most of the killer toxin to the (1→6)-β-d-glucan was complete within 2 min. Killer toxin adsorption to the linear (1→6)-β-d-glucan, pustulan, and a glucan fromPenicillium allahabadensewas observed. Other polysaccharides with different linkages failed to bind the killer toxin. The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines. This toxin shows its maximum killer activity in the presence of NaCl. This report is the first to identify the (1→6)-β-d-glucan as a receptor for this novel toxin.
Mutants with Enhanced Nitrogenase Activity in HydroponicAzospirillum brasilense-Wheat Associations
Authors: Pereg Gerk Lily , Gilchrist Kate , Kennedy Ivan R.
Published: July 2002

The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression byAzospirillum brasilenseis described. The geneflcAof strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZandnifH-lacZreporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants andflcATn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-inducedflcAmutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis betweenAzospirillumand wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism.
Growth and Phylogenetic Properties of Novel Bacteria Belonging to the Epsilon Subdivision of the
Proteobacteria
Enriched from
Alvinella pompejana
and Deep-Sea Hydrothermal Vents
Authors: Campbell Barbara J. , Jeanthon Christian , Kostka Joel E. , Luther George W. , Cary S. Craig
Published: July 2002

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of
Proteobacteria
(epsilon
Proteobacteria
). Here, we report the first enrichments and characterizations of four epsilon
Proteobacteria
that are directly associated with
Alvinella pompejana
, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45°C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to
Sulfurospirillum arcachonense
, an epsilon
Proteobacteria
isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon
Proteobacteria
, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon
Proteobacteria
likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.
Quantitative Detection of Microbial Genes by Using DNA Microarrays
Authors: Cho Jae-Chang , Tiedje James M.
Published: July 2002

To quantify target genes in biological samples using DNA microarrays, we employed reference DNA to normalize variations in spot size and hybridization. This method was tested using nitrate reductase (
nirS
), naphthalene dioxygenase (
nahA
), and
Escherichia coli
O157 O-antigen biosynthesis genes as model genes and lambda DNA as the reference DNA. We observed a good linearity between the log signal ratio and log DNA concentration ratio at DNA concentrations above the method's detection limit, which was approximately 10 pg. This approach for designing quantitative microarrays and the inferred equation from this study provide a simple and convenient way to estimate the target gene concentration from the hybridization signal ratio.
Highly Active Antiretroviral Therapy (HAART) and Circulating Markers of Immune Activation: Specific Effect of HAART on Neopterin
Authors: Amirayan-Chevillard Nathalie , Tissot-Dupont Herve , Obadia Yolande , Gallais Herve , Mege Jean-Louis , Capo Christian
Published: July 2002

The circulating levels of immune activation markers, including neopterin, tumor necrosis factor receptor type II, and interleukin-2 receptors, are increased in human immunodeficiency virus-infected patients. We show here that highly active antiretroviral therapy significantly decreased neopterin levels. This effect is reversible, since neopterin levels increased after the arrest of treatment. Their determination may be useful in the evaluation of the efficacy of antiretroviral therapy.
Chemokine C10 Promotes Disease Resolution and Survival in an Experimental Model of Bacterial Sepsis
Authors: Steinhauser M. L. , Hogaboam C. M. , Matsukawa A. , Lukacs N. W. , Strieter R. M. , Kunkel S. L.
Published: July 2002

Previous studies have suggested that the C-C chemokine C10 is involved in the chronic stages of host defense reactions. The present study addressed the role of C10 in a murine model of septic peritonitis, induced by cecal ligation and puncture (CLP). Unlike other C-C chemokines, C10 levels in the peritoneal wash were increased approximately 30-fold above baseline levels at 48 h after CLP surgery. Immunoneutralization of peritoneal C10 levels with polyclonal anti-C10 antiserum during CLP-induced peritonitis negatively impacted mouse survival over 4 days. In contrast, when 500 ng of recombinant murine C10 was administered immediately after CLP surgery, the 4-day survival rate increased from 20% to over 60%. The C10 therapy appeared to facilitate a rapid and significant enhancement of the levels of tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) and a later increase in interleukin-13 (IL-13) levels in the peritoneal cavity. In vitro studies showed that the combination of IL-1β and C10 markedly augmented TNF-α synthesis by peritoneal macrophages and that C10 synthesis was induced in these cells following their exposure to IL-13. At 24 h after CLP surgery, only 25% of C10-treated mice were bacteremic versus 85% of the control group that exhibited dissemination of bacteria into the circulation. The lack of bacteremia in C10-treated mice appeared to be related, in part, to in vitro evidence that C10 significantly enhanced the bacterial phagocytic activity of peritoneal macrophages. In addition, in vivo evidence suggested that C10 therapy significantly reduced the amount of material that leaked from the damaged gut. Taken together, the results of this study demonstrate that the C10 chemokine rapidly promotes disease resolution in the CLP model through its direct effects on the cellular events critically involved in host defense during septic peritonitis.
Phagocytosis and Protein Processing Are Required for Presentation ofCryptococcus neoformansMitogen to T Lymphocytes
Authors: Syme Rachel M. , Spurrell Jason C. L. , Ma Ling Ling , Green Francis H. Y. , Mody Christopher H.
Published: July 2002

In addition to eliciting antigen specific T-cell-mediated immunity,Cryptococcus neoformanspossesses a mitogen (CnM) that activates naive T cells to proliferate. This mechanism of T-cell activation is accessory cell dependent and major histocompatibility complex unrestricted. CnM-induced T-cell proliferation correlates with internalization of the organism, suggesting that intracellular processing is required to liberate CnM prior to presentation to T cells. To determine whether phagocytosis and processing are required, various inhibitors of accessory cell uptake and processing were used.C. neoformanswas observed within the accessory cells. Paraformaldehyde fixation of the accessory cell abrogated presentation of CnM to T cells, indicating that a dynamic accessory cell surface was required. A lysosomotropic agent abrogated the response to CnM but had no effect on a control stimulus that did not require processing. Both aspartic acid and cysteine protease inhibitors blocked effective processing of CnM, so that it was unable to stimulate T cells. Finally, an inhibitor of microfilament polymerization abrogated proliferation to CnM. These results indicate that the mitogenic activity ofC. neoformansrequires phagocytosis of the organism, lysosomal or endosomal processing, proteolytic activity, and microfilament polymerization and intracellular transport as a prerequisite for T-cell proliferation.
Identification of Polymorphonuclear Leukocyte and HL-60 Cell Receptors for Adhesins ofStreptococcus gordoniiandActinomyces naeslundii
Authors: Ruhl Stefan , Cisar John O. , Sandberg Ann L.
Published: July 2002

Interactions of oral streptococci and actinomyces with polymorphonuclear leukocytes (PMNs), mediated by sialic acid- and Gal/GalNAc-reactive adhesins, respectively, result in activation of the PMNs and thereby may contribute to the initiation of oral inflammation. Sialidase treatment of PMNs or HL-60 cells abolished adhesion ofStreptococcus gordoniibut was required for adhesion ofActinomyces naeslundii. The same effects of sialidase were noted for adhesion of these bacteria to a major 150-kDa surface glycoprotein of either PMNs or undifferentiated HL-60 cells and to a 130-kDa surface glycoprotein of differentiated HL-60 cells. These glycoproteins were both identified as leukosialin (CD43) by immunoprecipitation with a specific monoclonal antibody (MAb). Adhesion of streptococci and actinomyces to a 200-kDa minor PMN surface glycoprotein was also detected by bacterial overlay of untreated and sialidase-treated nitrocellulose transfers, respectively. This glycoprotein was identified as leukocyte common antigen (CD45) by immunoprecipitation with a specific MAb. CD43 and CD45 both possess extracellular mucinlike domains in addition to intracellular domains that are implicated in signal transduction. Consequently, the interactions of streptococci and actinomyces with the mucinlike domains of these mammalian cell surface glycoproteins result not only in adhesion but, in addition, may represent the initial step in PMN activation by these bacteria.
Construction and Characterization of a Nonproliferative El Tor Cholera Vaccine Candidate Derived from Strain 638
Authors: Valle Edgar , Ledón Talena , Cedré Bárbara , Campos Javier , Valmaseda Tania , Rodríguez Boris , García Luis , Marrero Karen , Benítez Jorge , Rodríguez Sandra , Fando Rafael
Published: July 2002

In recent clinical assays, our cholera vaccine candidate strain,
Vibrio cholerae
638 El Tor Ogawa, was well tolerated and immunogenic in Cuban volunteers. In this work we describe the construction of 638T, a thymidine auxotrophic version of improved environmental biosafety. In so doing, the
thyA
gene from
V. cholerae
was cloned, sequenced, mutated in vitro, and used to replace the wild-type allele. Except for its dependence on thymidine for growth in minimal medium, 638T is essentially indistinguishable from 638 in the rate of growth and morphology in complete medium. The two strains showed equivalent phenotypes with regard to motility, expression of the
celA
marker, colonization capacity in the infant mouse cholera model, and immunogenicity in the adult rabbit cholera model. However, the ability of this new strain to survive environmental starvation was limited with respect to that of 638. Taken together, these results suggest that this live, attenuated, but nonproliferative strain is a new, promising cholera vaccine candidate.
Hemolytic and Hemoxidative Activities inMycoplasma penetrans
Authors: Kannan T. R. , Baseman Joel B.
Published: July 2002

Mycoplasma penetransis a newly isolatedMollicutefrom the urine of patients infected with human immunodeficiency virus that demonstrates the capacity to adhere to and invade human cells. A previous report, based on assays with mouse red blood cells (RBCs), indicated thatM. penetranslacked hemolytic activity. In our studies, we incubated different isolates ofM. penetranswith various RBC species and observed hemolytic zones surrounding individual mycoplasma colonies. AllM. penetransstrains displayed hemolysis after 2 to 3 days of incubation. Hemolytic activity diffused from single colonies, eventually causing complete lysis. Hemolysis was most pronounced with sheep RBCs, followed by horse, chicken, and human cells. Furthermore, hemolytic activity was demonstrable in both intact mycoplasma cell preparations and spent culture supernatant. However, unlike intact mycoplasmas, the hemolytic activity in the supernatant was dependent on the reducing agent, cysteine. In addition to hemolysis, a brown precipitate was closely associated with mycoplasma colonies, suggesting oxidation of hemoglobin. Absorption spectra indicated that hemoglobin was oxidized to methemoglobin, and the addition of catalase demonstrated H2O2-mediated hemoxidation. Other experiments suggested that hemoxidation enhanced total hemolysis, providing the first evidence of both hemolytic and hemoxidative activities inM. penetrans.
Temporal Pore Formation-Mediated Egress from Macrophages and Alveolar Epithelial Cells byLegionella pneumophila
Authors: Alli O. A. Terry , Gao Lian-Yong , Pedersen Lisa L. , Zink Steven , Radulic Marina , Doric Miljenko , Abu Kwaik Yousef
Published: July 2002

Legionella pneumophiladoes not induce apoptosis in the protozoan host, but induces pore formation-mediated cytolysis after termination of intracellular replication (L.-Y. Gao and Y. Abu Kwaik, Environ. Microbiol. 2:79–90, 2000). In contrast to this single mode of killing of protozoa, we have recently proposed a biphasic model by whichL. pneumophilakills macrophages, in which the first phase is manifested through the induction of apoptosis during early stages of the infection, followed by an independent and temporal induction of necrosis during late stages of intracellular replication. Here we show that, similar to the protozoan host, the induction of necrosis and cytolysis of macrophages byL. pneumophilais mediated by the pore-forming toxin or activity. This activity is temporally and maximally expressed only upon termination of bacterial replication and correlates with cytolysis of macrophages and alveolar epithelial cells in vitro. We have identified fiveL. pneumophilamutants defective in the pore-forming activity. The phagosomes harboring the mutants do not colocalize with the late endosomal or lysosomal marker Lamp-1, and the mutants replicate intracellularly similar to the parental strain. Interestingly, despite their prolific intracellular replication, the mutants are defective in cytotoxicity and are “trapped” within and fail to lyse and egress from macrophages and alveolar epithelial cells upon termination of intracellular replication. However, the mutants are subsequently released from the host cell, most likely due to apoptotic death of the host cell. Data derived from cytotoxicity assays, confocal laser scanning microscopy, and electron microscopy confirm the defect in the mutants to induce necrosis of macrophages and the failure to egress from the host cell. Importantly, the mutants are completely defective in acute lethality (24 to 48 h) to intratracheally inoculated A/J mice. We conclude that the pore-forming activity ofL. pneumophilais not required for phagosomal trafficking or for intracellular replication. This activity is expressed upon termination of bacterial replication and is essential to induce cytolysis of infected macrophages to allow egress of intracellular bacteria. In addition, this activity plays a major role in pulmonary immunopathology in vivo.
Expression of Peptidoglycan-Associated Lipoprotein Is Required for Virulence in the Human Model ofHaemophilus ducreyiInfection
Authors: Fortney Kate R. , Young Royden S. , Bauer Margaret E. , Katz Barry P. , Hood Antoinette F. , Munson Robert S. , Spinola Stanley M.
Published: July 2002

Haemophilus ducreyiexpresses a peptidoglycan-associated lipoprotein (PAL) that exhibits extensive homology toHaemophilus influenzaeprotein 6. We constructed an isogenic PAL mutant (35000HP-SMS4) by the use of a suicide vector that containslacZas a counterselectable marker.H. ducreyi35000HP-SMS4 and its parent, 35000HP, had similar growth rates in broth and similar lipooligosaccharide profiles. 35000HP-SMS4 formed smaller, more transparent colonies than 35000HP and, unlike its parent, was hypersensitive to antibiotics. Complementation of the mutant intransrestored the parental phenotypes. To test whether expression of PAL is required for virulence, nine human volunteers were experimentally infected. Each subject was inoculated with two doses (41 to 89 CFU) of live 35000HP and one dose of heat-killed bacteria on one arm and with three doses (ranging from 28 to 800 CFU) of live 35000HP-SMS4 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent but were significantly smaller at mutant-inoculated sites than at parent-inoculated sites. The pustule formation rate was 72% (95% confidence interval [CI], 46.5 to 90.3%) at 18 parent sites and 11% (95% CI, 2.4 to 29.2%) at 27 mutant sites (P< 0.0001). The rates of recovery ofH. ducreyifrom surface cultures were 8% (n= 130; 95% CI, 4.3 to 14.6%) for parent-inoculated sites and 0% (n= 120; 95% CI, 0.0 to 2.5%) for mutant-inoculated sites (P< 0.001).H. ducreyiwas recovered from six of seven biopsied parent-inoculated sites and from one of three biopsied mutant-inoculated sites. Confocal microscopy confirmed that the bacteria present in a mutant inoculation site pustule lacked a PAL-specific epitope. Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of PAL facilitates the ability ofH. ducreyito progress to the pustular stage of disease.
Identification of an Antigen Localized to an Apparent Septum within Dividing Chlamydiae
Authors: Brown W. J. , Rockey D. D.
Published: July 2002

The process of chlamydial cell division has not been thoroughly investigated. The lack of detectable peptidoglycan and the absence of an FtsZ homolog within chlamydiae suggest an unusual mechanism for the division process. Our laboratory has identified an antigen (SEP antigen) localized to a ring-like structure at the apparent septum within dividing chlamydial reticulate bodies (RB). Antisera directed against SEP show similar patterns of antigen distribution in
Chlamydia trachomatis
and
Chlamydia psittaci
RB. In contrast to localization in RB, SEP in elementary bodies appears diffuse and irregular, suggesting that the distribution of the antigen is developmental-stage specific. Treatment of chlamydiae with inhibitors of peptidoglycan synthesis or culture of chlamydiae in medium lacking tryptophan leads to the formation of nondividing, aberrant RB. Staining of aberrant RB with anti-SEP reveals a marked redistribution of the antigen. Within
C. trachomatis
-infected cells, ampicillin treatment leads to high levels of SEP accumulation at the periphery of aberrant RB, while in
C. psittaci
, treatment causes SEP to localize to distinct punctate sites within the bacteria. Aberrancy produced via tryptophan depletion results in a different pattern of SEP distribution. In either case, the reversal of aberrant formation results in the production of normal RB and a redistribution of SEP to the apparent plane of bacterial division. Collectively these studies identify a unique chlamydial-genus-common and developmental-stage-specific antigen that may be associated with RB division.
Intracellular Infection by the Human Granulocytic Ehrlichiosis Agent Inhibits Human Neutrophil Apoptosis
Authors: Yoshiie Kiyotaka , Kim Hyung-Yong , Mott Jason , Rikihisa Yasuko
Published: July 2002

In patients with human granulocytic ehrlichiosis (HGE), the HGE agent has been seen only in the peripheral blood granulocytes, which have a life span too short for ehrlichial proliferation. To determine if the HGE agent delays the apoptosis of human peripheral blood neutrophils for its advantage, peripheral blood granulocytes consisting mostly of neutrophils were incubated with freshly freed host cell-free HGE agent in vitro. The HGE agent induced a significant delay in morphological apoptosis and the cytoplasmic appearance of histone-associated DNA fragments in the granulocytes. This antiapoptotic effect was dose dependent. Although much weaker than the HGE agent freshly freed from the host cells, noninfectious purified HGE agent stored frozen and thawed also had antiapoptotic effect, which was lost with proteinase K treatment but not with periodate treatment. Treatment of neutrophils with a transglutaminase inhibitor, monodansylcadaverine, blocked the antiapoptotic effect of the HGE agent. Addition of oxytetracycline, however, did not prevent or reverse the antiapoptotic effect of the HGE agent. These results suggest that binding of a protein component(s) of the HGE agent to neutrophils and subsequent cross-linking and/or internalization of the receptor and ehrlichiae are required for antiapoptotic signaling, but ehrlichial protein synthesis and/or proliferation is not required. MG-132, a proteasome inhibitor, and cycloheximide accelerated the apoptosis of neutrophils and overrode the antiapoptotic effect of the HGE agent. Studies with specific inhibitors suggest that protein kinase A, NF-κB, and interleukin 1β are not involved in the antiapoptotic mechanism of the HGE agent.
Interleukin-5 Is Essential for Vaccine-Mediated Immunity but Not Innate Resistance to a Filarial Parasite
Authors: Le Goff Laetitia , Loke P'ng , Ali H. Fahimeda , Taylor David W. , Allen Judith E.
Published: July 2002

The study of protective immune mechanisms effective against filarial nematodes has been hampered by the inability of these important human pathogens to infect laboratory mice. Recently,Litomosoides sigmodontis, a natural parasite of rats, has been developed as a valuable model for the study of filarial infection. BALB/c mice are fully susceptible to infection withL. sigmodontisthird-stage larvae and develop patent infection. In contrast, mice on the C57BL background are resistant, and parasites undergo only a single molt and do not mature to adulthood. We used interleukin-5 (IL-5)-deficient mice on the C57BL/6 background to address the role of IL-5 and eosinophils in the innate resistance of C57BL/6 mice. We found no differences in parasite survival between IL-5-deficient and C57BL/6 mice. However, when these mice were used for the analysis of vaccine-mediated immunity, a critical role for IL-5 was elucidated. Mice genetically deficient in IL-5 were unable to generate a protective immune response when vaccinated with irradiated larvae, whereas C57BL/6 mice were fully protected from challenge infection. These studies help to clarify the highly controversial role of eosinophils in filarial infection.
In Vitro Resistance of
Staphylococcus aureus
to Thrombin-Induced Platelet Microbicidal Protein Is Associated with Alterations in Cytoplasmic Membrane Fluidity
Authors: Bayer Arnold S. , Prasad Rajendra , Chandra Jyotsna , Koul Anjni , Smriti M. , Varma Archana , Skurray Ronald A. , Firth Neville , Brown Melissa H. , Koo Su-Pin , Yeaman Michael R.
Published: July 2002

Platelet microbicidal proteins (PMPs) are small, cationic peptides which possess potent microbicidal activities against common bloodstream pathogens, such as
Staphylococcus aureus
. We previously showed that
S. aureus
strains exhibiting resistance to thrombin-induced PMP (tPMP-1) in vitro have an enhanced capacity to cause human and experimental endocarditis (T. Wu, M. R. Yeaman, and A. S. Bayer, Antimicrob. Agents Chemother. 38:729–732, 1994; A. S. Bayer et al., Antimicrob. Agents Chemother. 42:3169–3172, 1998; V. K. Dhawan et al., Infect. Immun. 65:3293–3299, 1997). However, the mechanisms mediating tPMP-1 resistance in
S. aureus
are not fully delineated. The
S. aureus
cell membrane appears to be a principal target for the action of tPMP-1. To gain insight into the basis of tPMP-1 resistance, we compared several parameters of membrane structure and function in three tPMP-1-resistant (tPMP-1
r
) strains and their genetically related, tPMP-1-susceptible (tPMP-1
s
) counterpart strains. The tPMP-1
r
strains were derived by three distinct methods: transposon mutagenesis, serial passage in the presence of tPMP-1 in vitro, or carriage of a naturally occurring multiresistance plasmid (pSK1). All tPMP-1
r
strains were found to possess elevated levels of longer-chain, unsaturated membrane lipids, in comparison to their tPMP-1
s
counterparts. This was reflected in corresponding differences in cell membrane fluidity in the strain pairs, with tPMP-1
r
strains exhibiting significantly higher degrees of fluidity as assessed by fluorescence polarization. These data provide further support for the concept that specific alterations in the cytoplasmic membrane of
S. aureus
strains are associated with tPMP-1 resistance in vitro.
Synthetic Polysaccharide Type 3-Related Di-, Tri-, and Tetrasaccharide–CRM
197
Conjugates Induce Protection against
Streptococcus pneumoniae
Type 3 in Mice
Authors: Benaissa-Trouw Barry , Lefeber Dirk J. , Kamerling Johannis P. , Vliegenthart Johannes F. G. , Kraaijeveld Kees , Snippe Harm
Published: July 2002

Di-, tri-, and tetrasaccharides, synthesized according to the chemical structure of pneumococcal polysaccharide type 3 (PS3), were coupled to the cross-reactive material (CRM
197
) of modified diphtheria toxin in different molar carbohydrate/protein ratios using the squarate coupling method. To study protective immunity, female BALB/c mice were subcutaneously immunized twice (with a 3-week interval) using the amount of conjugates corresponding to 2.5 μg of oligosaccharide per mouse. The conjugates evoked PS3 binding immunoglobulin G antibodies that lasted for at least 7 weeks after the booster. Immunogenicity was not influenced by the carbohydrate/protein ratio. All mice with PS3-specific antibodies survived the intraperitoneal challenge with
Streptococcus pneumoniae
type 3. Therefore, synthetic oligosaccharide-protein conjugates might have potential as vaccines.
Effect of O Acetylation of
Neisseria meningitidis
Serogroup A Capsular Polysaccharide on Development of Functional Immune Responses
Authors: Berry David S. , Lynn Freyja , Lee Che-Hung , Frasch Carl E. , Bash Margaret C.
Published: July 2002

The importance of O-acetyl groups to the immunogenicity of
Neisseria meningitidis
serogroup A polysaccharide (PS) was examined in studies using human sera and mouse immunization. In 17 of 18 postimmunization human sera, inhibition enzyme-linked immunosorbent assay indicated that the majority of antibodies binding to serogroup A PS were specific for epitopes involving O-acetyl groups. Studies with mice also showed an essential role for O-acetyl groups, where serum bactericidal titers following immunization with de-O-acetylated (de-O-Ac) conjugate vaccine were at least 32-fold lower than those following immunization with O-Ac PS-conjugate vaccine and 4-fold lower than those following immunization with native capsular PS. Inhibition studies using native and de-O-Ac PS confirmed the specificity of murine antibodies to native PS. The dramatic reduction in immunogenicity associated with removal of O-acetyl groups indicates that O acetylation is essential to the immunogenic epitopes of serogroup A PS. Since levels of bactericidal antibodies are correlated with protection against disease, O-acetyl groups appear to be important in protection.
Characterization of a
Bacteroides
Mobilizable Transposon, NBU2, Which Carries a Functional Lincomycin Resistance Gene
Authors: Wang Jun , Shoemaker Nadja B. , Wang Gui-Rong , Salyers Abigail A.
Published: July 2002

The mobilizable
Bacteroides
element NBU2 (11 kbp) was found originally in two
Bacteroides
clinical isolates,
Bacteroides fragilis
ERL and
B. thetaiotaomicron
DOT. At first, NBU2 appeared to be very similar to another mobilizable
Bacteroides
element, NBU1, in a 2.5-kbp internal region, but further examination of the full DNA sequence of NBU2 now reveals that the region of near identity between NBU1 and NBU2 is limited to this small region and that, outside this region, there is little sequence similarity between the two elements. The integrase gene of NBU2,
intN2
, was located at one end of the element. This gene was necessary and sufficient for the integration of NBU2. The integrase of NBU2 has the conserved amino acids (R-H-R-Y) in the C-terminal end that are found in members of the lambda family of site-specific integrases. This was also the only region in which the NBU1 and NBU2 integrases shared any similarity (28% amino acid sequence identity and 49% sequence similarity). Integration of NBU2 was site specific in
Bacteroides
species. Integration occurred in two primary sites in
B. thetaiotaomicron
. Both of these sites were located in the 3′ end of a serine-tRNA gene NBU2 also integrated in
Escherichia coli
, but integration was much less site specific than in
B. thetaiotaomicron
. Analysis of the sequence of NBU2 revealed two potential antibiotic resistance genes. The amino acid sequences of the putative proteins encoded by these genes had similarity to resistances found in gram-positive bacteria. Only one of these genes was expressed in
B. thetaiotaomicron
, the homolog of
linA
, a lincomycin resistance gene from
Staphylococcus aureus
. To determine how widespread elements related to NBU1 and NBU2 are in
Bacteroides
species, we screened 291
Bacteroides
strains. Elements with some sequence similarity to NBU2 and NBU1 were widespread in
Bacteroides
strains, and the presence of

linA
N

in
Bacteroides
strains was highly correlated with the presence of NBU2, suggesting that NBU2 has been responsible for the spread of this gene among
Bacteroides
strains. Our results suggest that the NBU-related elements form a large and heterogeneous family, whose members have similar integration mechanisms but have different target sites and differ in whether they carry resistance genes.
Viability of an
Escherichia coli pgsA
Null Mutant Lacking Detectable Phosphatidylglycerol and Cardiolipin
Authors: Kikuchi Shin , Shibuya Isao , Matsumoto Kouji
Published: July 2002

Phosphatidylglycerol, the most abundant acidic phospholipid in
Escherichia coli
, has been considered to play specific roles in various cellular processes and is believed to be essential for cell viability. It is functionally replaced in some cases by cardiolipin, another abundant acidic phospholipid derived from phosphatidylglycerol. However, we now show that a null
pgsA
mutant is viable, if the major outer membrane lipoprotein is deficient. The
pgsA
gene normally encodes phosphatidylglycerophosphate synthase that catalyzes the committed step in the biosynthesis of these acidic phospholipids. In the mutant, the activity of this enzyme and both phosphatidylglycerol and cardiolipin were not detected (less than 0.01% of total phospholipid, both below the detection limit), although phosphatidic acid, an acidic biosynthetic precursor, accumulated (4.0%). Nonetheless, the null mutant grew almost normally in rich media. In low-osmolarity media and minimal media, however, it could not grow. It did not grow at temperatures over 40°C, explaining the previous inability to construct a null
pgsA
mutant (W. Xia and W. Dowhan, Proc. Natl. Acad. Sci. USA 92:783–787, 1995). Phosphatidylglycerol and cardiolipin are therefore nonessential for cell viability or basic life functions. This notion allows us to formulate a working model that defines the physiological functions of acidic phospholipids in
E. coli
and explains the suppressing effect of lipoprotein deficiency.
Mutational Analysis of Ligand Recognition by Tcp, the Citrate Chemoreceptor of
Salmonella enterica
Serovar Typhimurium
Authors: Iwama Tomonori , Nakao Ko-Ichiro , Nakazato Hiroshi , Yamagata Shuzo , Homma Michio , Kawagishi Ikuro
Published: July 2002

The chemoreceptor Tcp mediates taxis to citrate. To identify citrate-binding residues, we substituted cysteine for seven basic or polar residues that are chosen based on the comparison of Tcp with the well-characterized chemoreceptors. The results suggest that Arg-63, Arg-68, Arg-72, Lys-75, and Tyr-150 (and probably other unidentified residues) are involved in the recognition of citrate.
Identification and Characterization of a Monofunctional Glycosyltransferase from
Staphylococcus aureus
Authors: Wang Q. May , Peery Robert B. , Johnson Robert B. , Alborn William E. , Yeh Wu-Kuang , Skatrud Paul L.
Published: July 2002

A gene (
mgt
) encoding a monofunctional glycosyltransferase (MGT) from
Staphylococcus aureus
has been identified. This first reported gram-positive MGT shared significant homology with several MGTs from gram-negative bacteria and the N-terminal glycosyltransferase domain of class A high-molecular-mass penicillin-binding proteins from different species.
S. aureus
MGT contained an N-terminal hydrophobic domain perhaps involved with membrane association. It was expressed in
Escherichia coli
cells as a truncated protein lacking the hydrophobic domain and purified to homogeneity. Analysis by circular dichroism revealed that secondary structural elements of purified truncated
S. aureus
MGT were consistent with predicted structural elements, indicating that the protein might exhibit the expected folding. In addition, purified
S. aureus
MGT catalyzed incorporation of UDP-
N
-acetylglucosamine into peptidoglycan, proving that it was enzymatically active. MGT activity was inhibited by moenomycin A, and the reaction product was sensitive to lysozyme treatment. Moreover, a protein matching the calculated molecular weight of
S. aureus
MGT was identified from an
S. aureus
cell lysate using antibodies developed against purified MGT. Taken together, our results suggest that this enzyme is natively present in
S. aureus
cells and that it may play a role in bacterial cell wall biosynthesis.
In
Staphylococcus aureus
, Fur Is an Interactive Regulator with PerR, Contributes to Virulence, and Is Necessary for Oxidative Stress Resistance through Positive Regulation of Catalase and Iron Homeostasis
Authors: Horsburgh Malcolm J. , Ingham Eileen , Foster Simon J.
Published: July 2002

The
Staphylococcus aureus
genome encodes three ferric uptake repressor (Fur) homologues: Fur, PerR, and Zur. To determine the exact role of Fur in
S. aureus
, we inactivated the
fur
gene by allelic replacement using a tetracycline resistance cassette, creating strain MJH010 (
fur
). The mutant had a growth defect in rich medium, and this defect was exacerbated in metal-depleted CL medium. This growth defect was partially suppressed by manganous ion, a metal ion with known antioxidant properties. This suggests that the
fur
mutation leads to an oxidative stress condition. Indeed, MJH010 (
fur
) has reduced levels of catalase activity resulting from decreased
katA
transcription. Using a
katA-lacZ
fusion we have determined that Fur functions, either directly or indirectly, as an iron-dependent positive regulator of
katA
expression. Transcription of
katA
is coregulated by Fur and PerR, since in MJH010 (
fur
) transcription was still repressed by manganese while transcription in MJH201 (
fur perR
) was unresponsive to the presence of iron or manganese. Siderophore biosynthesis was repressed by iron in 8325-4 (wild-type) but in MJH010 (
fur
) was constitutive. A number of putative Fur-regulated genes were identified in the incomplete genome databases using known
S. aureus
Fur box sequences. Of those tested, the
sstABCD
and
sirABC
operons and the
fhuD2
and
orf4
genes were found to have Fur-regulated expression. MJH010 (
fur
) was attenuated (
P
< 0.04) in a murine skin abscess model of infection, as was double-mutant MJH201 (
fur perR
) (
P
< 0.03). This demonstrates the importance in vivo of iron homeostasis and oxidative stress resistance regulation in
S. aureus.