bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorus-31 Nuclear Magnetic Resonance Study of the Effect of Pentachlorophenol (PCP) on the Physiologies of PCP-Degrading Microorganisms
Authors: Lohmeier-Vogel Elke M. , Leung Kam T. , Lee Hung , Trevors Jack T. , Vogel Hans J.
Published: July 2002

Free and agarose-encapsulated pentachlorophenol (PCP)-degradingSphingomonassp. isolates UG25 and UG30 were compared toSphingomonas chlorophenolicaATCC 39723 with respect to the ability to degrade PCP. Pretreatment of the UG25 and UG30 strains with 50 μg of PCP per ml enabled the cells to subsequently degrade higher levels of this environmental pollutant. Similar treatment of ATCC 39723 cells had no effect on the level of PCP degraded by this strain. Phosphorus-31 nuclear magnetic resonance spectra of agarose-immobilized strains UG25 and UG30 grown in the absence of PCP showed that there was marked deenergization of the cells upon exposure to a nonlethal concentration of PCP (120 μg/ml). For example, no transmembrane pH gradient was observed, and the ATP levels were lower than the levels obtained in the absence of PCP. The transmembrane pH gradient and ATP levels were restored once the immobilized cells had almost completely degraded the PCP in the perfusion medium. PCP-pretreated cells, on the other hand, maintained their transmembrane pH gradient and ATP levels even in the presence of high levels of PCP. The ability of PCP-pretreated strain UG25 and UG30 cells to remain energized in the presence of PCP was shown to correlate with an altered membrane phospholipid profile; these cells had a higher concentration of cardiolipin than cells cultured in the absence of PCP. Strain ATCC 39723, which did not degrade higher levels of PCP after PCP pretreatment, did not show this response.
Influence of Soil on Fecal Indicator Organisms in a Tidally Influenced Subtropical Environment
Authors: Desmarais Timothy R. , Solo-Gabriele Helena M. , Palmer Carol J.
Published: July 2002

The potential regrowth of fecal indicator bacteria released into coastal environments in recreational water bodies has been of concern, especially in tropical and subtropical areas where the number of these bacteria can be artificially elevated beyond that from fecal impacts alone. The task of determining the factors that influence indicator bacterial regrowth was addressed though a series of field sampling and laboratory experiments using in situ densities ofEscherichia coli, enterococci, andClostridium perfringensin river water, sediment, and soil. Field sampling efforts included the collection of surface sediments along the cross section of a riverbank, a 20-cm-deep soil core, and additional surface soils from remote locations. In addition to field sampling, two types of laboratory experiments were conducted. The first experiment investigated the survival of bacteria already present in river water with the addition of sterile and unsterile sediment. The second experiment was designed to simulate the wetting and drying effects due to tidal cycles. The results from the sampling study found elevated numbers ofE. coliandC. perfringensin surficial sediments along the riverbank near the edge of the water.C. perfringenswas found in high numbers in the subsurface samples obtained from the soil core. Results from laboratory experiments revealed a significant amount of regrowth for enterococci andE. coliwith the simulation of tides and addition of sterile sediment. Regrowth was not observed forC. perfringens.This study demonstrates the need to further evaluate the characteristics of indicator microbes within tropical and subtropical water systems where natural vegetation, soil embankments, and long-term sediment accumulation are present. In such areas, the use of traditional indicator microbes to regulate recreational uses of a water body may not be appropriate.
Proline Reverses the Abnormal Phenotypes of
Colletotrichum trifolii
Associated with Expression of Endogenous Constitutively Active Ras
Authors: Memmott Stephen D. , Ha Young-sil , Dickman Martin B.
Published: July 2002

Colletotrichum trifolii
is the causative organism of alfalfa anthracnose. We previously cloned and characterized the small prototypical G protein, Ras, of
C. trifolii
, which is involved in the signaling pathways that mediate interaction between the pathogen and its host. Transformants expressing constitutively active forms of Ras have growth medium-dependent phenotypes. In nutrient-rich media (e.g., yeast extract and peptone), the phenotype of the transformants was indistinguishable from that of the wild type. However, during nutrient starvation, the transformants lose polarity, have distended hyphae, and fail to sporulate and produce appressoria. Since peptone caused the phenotype to revert, amino acids were tested singly and in combination to identify the responsible amino acid(s). We found that 1.6 mM proline in the medium reverses the constitutively active Ras phenotype.
Linking Central Metabolism with Increased Pathway Flux:
l
-Valine Accumulation by
Corynebacterium glutamicum
Authors: Radmacher Eva , Vaitsikova Adela , Burger Udo , Krumbach Karin , Sahm Hermann , Eggeling Lothar
Published: July 2002

Mutants of
Corynebacterium glutamicum
were made and enzymatically characterized to clone
ilvD
and
ilvE
, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with
ilvBN
(which encodes acetohydroxy acid synthase) and
ilvC
(which encodes isomeroreductase) in the wild type, which does not excrete
l
-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since
l
-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes,
ilvBNCD
, overexpressed in an
ilvA
deletion mutant which is unable to synthesize
l
-isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when
panBC
was deleted, making the resulting mutant auxotrophic for
d
-pantothenate. When the resulting strain,
C. glutamicum
13032ΔilvAΔpanBC with
ilvBNCD
overexpressed, was grown under limiting conditions it accumulated 91 mM
l
-valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the
l
-valine biosynthesis pathway.
Mechanisms for Accessing Insoluble Fe(III) Oxide during Dissimilatory Fe(III) Reduction by
Geothrix fermentans
Authors: Nevin Kelly P. , Lovley Derek R.
Published: July 2002

Mechanisms for Fe(III) oxide reduction were investigated in
Geothrix fermentans
, a dissimilatory Fe(III)-reducing microorganism found within the Fe(III) reduction zone of subsurface environments. Culture filtrates of
G. fermentans
stimulated the reduction of poorly crystalline Fe(III) oxide by washed cell suspensions, suggesting that
G. fermentans
released one or more extracellular compounds that promoted Fe(III) oxide reduction. In order to determine if
G. fermentans
released electron-shuttling compounds, poorly crystalline Fe(III) oxide was incorporated into microporous alginate beads, which prevented contact between
G. fermentans
and the Fe(III) oxide.
G. fermentans
reduced the Fe(III) within the beads, suggesting that one of the compounds that
G. fermentans
releases is an electron-shuttling compound that can transfer electrons from the cell to Fe(III) oxide that is not in contact with the organism. Analysis of culture filtrates by thin-layer chromatography suggested that the electron shuttle has characteristics similar to those of a water-soluble quinone. Analysis of filtrates by ion chromatography demonstrated that there was as much as 250 μM dissolved Fe(III) in cultures of
G. fermentans
growing with Fe(III) oxide as the electron acceptor, suggesting that
G. fermentans
released one or more compounds capable of chelating and solubilizing Fe(III). Solubilizing Fe(III) is another strategy for alleviating the need for contact between cells and Fe(III) oxide for Fe(III) reduction. This is the first demonstration of a microorganism that, in defined medium without added electron shuttles or chelators, can reduce Fe(III) derived from Fe(III) oxide without directly contacting the Fe(III) oxide. These results are in marked contrast to those with
Geobacter metallireducens
, which does not produce electron shuttles or Fe(III) chelators. These results demonstrate that phylogenetically distinct Fe(III)-reducing microorganisms may use significantly different strategies for Fe(III) reduction. Thus, it is important to know which Fe(III)-reducing microorganisms predominate in a given environment in order to understand the mechanisms for Fe(III) reduction in the environment of interest.
Mechanisms of Induction of Germination of
Bacillus subtilis
Spores by High Pressure
Authors: Paidhungat Madan , Setlow Barbara , Daniels William B. , Hoover Dallas , Papafragkou Efstathia , Setlow Peter
Published: July 2002

Spores of
Bacillus subtilis
lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.
Measurement of T-Lymphocyte Responses in Whole-Blood Cultures Using Newly Synthesized DNA and ATP
Authors: Sottong P. R. , Rosebrock J. A. , Britz J. A. , Kramer T. R.
Published: July 2002

The proliferative response is most frequently determined by estimating the amount of [
3
H]thymidine incorporated into newly synthesized DNA. The [
3
H]thymidine procedure requires the use of radioisotopes as well as lengthy periods of incubation (>72 h). An alternative method of assessing T-lymphocyte activation in whole-blood cultures involves the measurement of the nucleotide ATP instead of [
3
H]thymidine incorporation. In addition, the Luminetics assay of T-cell activation measures specific T-lymphocyte subset responses through the use of paramagnetic particles coated with monoclonal antibodies against CD antigens. This assay permits rapid (24 h) analysis of lymphocyte subset activation responses to mitogens and recall antigens in small amounts of blood.
Jasplakinolide Induces Apoptosis in Various Transformed Cell Lines by a Caspase-3-Like Protease-Dependent Pathway
Authors: Odaka Chikako , Sanders Miranda L. , Crews Phillip
Published: July 2002

To clarify the mechanisms underlying the antiproliferative effects of jasplakinolide, a cyclic depsipeptide from marine sponges, we examined whether jasplakinolide induces apoptosis in a variety of transformed and nontransformed cells. Jasplakinolide inhibited proliferation of human Jurkat T cells, resulting in cell death. This was accompanied by chromatin condensation and DNA cleavage at the linker regions between the nucleosomes. When caspase-3-like activity in the cytosolic extracts of Jurkat T cells was examined with a fluorescent substrate, DEVD-MAC (
N
-acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide), the activity in the cells treated with jasplakinolide was remarkably increased in a time-dependent manner. Pretreatment of Jurkat T cells with the caspase inhibitor zVAD [benzyloxycarbonyl(Cbz)-Val-Ala-β-Asp(OMe)-fluoromethylketone] or DEVD-CHO (
N
-acetyl-Asp-Glu-Val-Asp-1-aldehyde) prevented the induction of apoptosis by jasplakinolide. Moreover, exposure of various murine transformed cell lines to jasplakinolide resulted in cell death, which was inhibited by zVAD. Although it has been well established that murine immature thymocytes are sensitive to apoptosis when exposed to various apoptotic stimuli, these cells as well as mature T lymphocytes were resistant to jasplakinolide-induced apoptosis. The results suggest that jasplakinolide induces apoptotic cell death through a caspase-3-like protease-dependent pathway. Another important outcome is that transformed cell lines were more susceptible to jasplakinolide-induced apoptosis than normal nontransformed cells.
Immunodiagnosis of Human Fascioliasis by an Enzyme-Linked Immunosorbent Assay (ELISA) and a Micro-ELISA
Authors: Carnevale Silvana , Rodrı́guez Mónica I. , Santillán Graciela , Labbé Jorge H. , Cabrera Marta G. , Bellegarde Enrique J. , Velásquez Jorge N. , Trgovcic Jorge E. , Guarnera Eduardo A.
Published: July 2002

Enzyme-linked immunosorbent assay (ELISA) and micro-ELISA were evaluated for their ability to detect anti-
Fasciola hepatica
antibodies in humans by using excretory-secretory antigen. The sensitivity of each method was 100%, but the specificity was 100% for ELISA and 97% for micro-ELISA. The micro-ELISA could be used as a screening assay and ELISA could be used as a confirmatory method for the serodiagnosis of human fascioliasis.
New Coupled-Particle Light-Scattering Assay for Detection of Ro/SSA (52 and 60 Kilodaltons) and La/SSB Autoantibodies in Connective Tissue Diseases
Authors: Bizzaro Nicola , Bonelli Fabrizio , Tonutti Elio , Tozzoli Renato , Villalta Danilo
Published: July 2002

The diagnostic and analytical performance of the coupled-particle light-scattering assay in detecting anti-Ro/SSA autoantibodies (the 60-kDa [Ro60] and the 52-kDa [Ro52] antibodies) and anti-La/SSB autoantibodies was evaluated. The antigens were obtained by recombinant DNA procedures to include the most immunogenic epitopes for each protein by using a prokaryotic expression system. Serum samples from 151 patients with connective tissue diseases and 52 control subjects (including patients with viral infections, patients with Lyme disease, and healthy subjects) were studied. Sensitivities for detection of anti-Ro/SSA and anti-La/SSB were 88.2 and 95.2%, respectively; specificities were 97.6 and 98.1%, respectively. The intra-assay coefficient of variation (CV) ranged from 4.3 to 10.9% for anti-Ro/SSA and from 2.8 to 12.5% for anti-La/SSB; interassay CVs ranged from 6.5 to 13.2% and from 8.2 to 14.5%, respectively. Among the anti-Ro/SSA-positive samples, Ro60 was recognized by 66% of the test sera and Ro52 was recognized by 95% of the test sera. Thirty-four percent of the Ro/SSA-positive sera were reactive only with the Ro52 antigen, indicating that anti-Ro52 is the most common antibody specificity recognized by anti-Ro/SSA autoantibodies. No differences were found between the prevalences of anti-Ro60 and anti-Ro52 in relation to systemic lupus erythematosus or Sjögren's syndrome. The results of the present study indicate that this new immunoassay is an efficient diagnostic tool for the detection of anti-Ro/SSA and anti-La/SSB antibodies in patients with autoimmune disorders.
Determinants of
Staphylococcus
aureus
Nasal Carriage
Authors: Cole Alexander M. , Tahk Samuel , Oren Ami , Yoshioka Dawn , Kim Yong-Hwan , Park Albert , Ganz Tomas
Published: July 2002

Nasal carriage of
Staphylococcus
aureus
has been identified as a risk factor for community-acquired and nosocomial infections. We screened 230 donors of diverse ethnic and socioeconomic backgrounds and identified 62 (27%) whose nasal secretions were colonized by
S
.
aureus
. In 18 donors in whom the various regions of the nasal luminal surface were separately sampled, the predominant region of
S
.
aureus
colonization was the moist squamous epithelium on the septum adjacent to the nasal ostium. Nasal fluid from carriers was defective in killing endogenous
S
.
aureus
and nasal carrier isolates of
S
.
aureus
but not a laboratory
S
.
aureus
strain. Transmission electron microscopy revealed that
S
.
aureus
isolates incubated in nasal fluid from carriers for 2 h at 37°C were less damaged than those incubated in noncarrier fluid and were coated with an electron-dense layer. Compared with that from healthy donors and patients with acute rhinitis, nasal fluid from carriers contained elevated concentrations of the neutrophil-derived defensins human neutrophil peptides 1 to 3 (47- and 4-fold increases, respectively), indicative of a neutrophil-mediated inflammatory host response to
S
.
aureus
colonization. The concentration of the inducible epithelial antimicrobial peptide human β-defensin 2 was also highly elevated compared to that in healthy donors, in whom the level was below the detection limit, or patients with acute rhinitis (sixfold increase). Thus, nasal carriage of
S
.
aureus
takes hold in nasal fluid that is permissive for colonization and induces a local inflammatory response that fails to clear the colonizing bacteria.
Use of Immunoglobulin M Cross-Reactions in Differential Diagnosis of Human Flaviviral Encephalitis Infections in the United States
Authors: Martin Denise A. , Biggerstaff Brad J. , Allen Becky , Johnson Alison J. , Lanciotti Robert S. , Roehrig John T.
Published: July 2002

To define the virus specificity of the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) among the medically important members of the Japanese encephalitis (JE) virus serocomplex of flaviviruses, 103 IgM-positive human serum samples from patients with confirmed West Nile (WN) virus, St. Louis encephalitis (SLE) virus, or JE virus infections were assembled and simultaneously tested against all three viral antigens in a standardized MAC-ELISA. Of the serum samples tested, 96 (93%) showed higher positive-to-negative absorbance ratios (P/Ns) with the infecting virus antigen compared to those obtained with the other two virus antigens. Of the seven specimens with higher P/Ns with heterologous virus antigens, six were from patients with SLE virus infections (the serum samples had higher levels of reactivity with WN virus antigen) and one was from a patient with a JE virus infection (this serum sample also had a higher level of reactivity with WN virus antigen). Not surprisingly, similar virus specificity was observed with WN virus-elicited IgM in cerebrospinal fluid. As shown in previous studies, a subset of these specimens was even less reactive in the MAC-ELISA with dengue virus, a member of a different flavivirus serocomplex. The degree of virus cross-reactivity did not appear to be related to days postonset, at least during the first 40 days of infection. Infections with WN virus could be correctly distinguished from infections with SLE virus on the basis of the observed anti-viral IgM cross-reactivities alone 92% of the time. Infections with SLE virus resulted in antibody that was more cross-reactive, so identification of SLE virus as the infecting agent by use of MAC-ELISA cross-reactivity alone was more problematic.
Evaluation of the In Vitro Pyrogen Test System Based on Proinflammatory Cytokine Release from Human Monocytes: Comparison with a Human Whole Blood Culture Test System and with the Rabbit Pyrogen Test
Authors: Nakagawa Yukari , Maeda Hideko , Murai Toshimi
Published: July 2002

The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG),Staphylococcus aureusCowan 1 (SAC), and poly(I ·  C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I · C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans.
Mycobacterium aviumsubsp.paratuberculosisin Veterinary Medicine
Authors: Harris N. Beth , Barletta Raúl G.
Published: July 2002

Mycobacterium avium subsp. paratuberculosis (basonym M. paratuberculosis) is the etiologic agent of a severe gastroenteritis in ruminants known as Johne's disease. Economic losses to the cattle industry in the United States are staggering, reaching $1.5 billion annually. A potential pathogenic role in humans in the etiology of Crohn's disease is under investigation. In this article, we review the epidemiology, pathogenesis, diagnostics, and disease control measures of this important veterinary pathogen. We emphasize molecular genetic aspects including the description of markers used for strain identification, diagnostics, and phylogenetic analysis. Recent important advances in the development of animal models and genetic systems to study M. paratuberculosis virulence determinants are also discussed. We conclude with proposals for the applications of these models and recombinant technology to the development of diagnostic, control, and therapeutic measures.
Moraxella catarrhalis: from Emerging to Established Pathogen
Authors: Verduin Cees M. , Hol Cees , Fleer André , van Dijk Hans , van Belkum Alex
Published: July 2002

Moraxella catarrhalis (formerly known as Branhamella catarrhalis) has emerged as a significant bacterial pathogen of humans over the past two decades. During this period, microbiological and molecular diagnostic techniques have been developed and improved for M. catarrhalis, allowing the adequate determination and taxonomic positioning of this pathogen. Over the same period, studies have revealed its involvement in respiratory (e.g., sinusitis, otitis media, bronchitis, and pneumonia) and ocular infections in children and in laryngitis, bronchitis, and pneumonia in adults. The development of (molecular) epidemiological tools has enabled the national and international distribution of M. catarrhalis strains to be established, and has allowed the monitoring of nosocomial infections and the dynamics of carriage. Indeed, such monitoring has revealed an increasing number of Β-lactamase-positive M. catarrhalis isolates (now well above 90%), underscoring the pathogenic potential of this organism. Although a number of putative M. catarrhalis virulence factors have been identified and described in detail, their relationship to actual bacterial adhesion, invasion, complement resistance, etc. (and ultimately their role in infection and immunity), has been established in a only few cases. In the past 10 years, various animal models for the study of M. catarrhalis pathogenicity have been described, although not all of these models are equally suitable for the study of human infection. Techniques involving the molecular manipulation of M. catarrhalis genes and antigens are also advancing our knowledge of the host response to and pathogenesis of this bacterial species in humans, as well as providing insights into possible vaccine candidates. This review aims to outline our current knowledge of M. catarrhalis, an organism that has evolved from an emerging to a well-established human pathogen.
Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms
Authors: Donlan Rodney M. , Costerton J. William
Published: July 2002

Though biofilms were first described by Antonie van Leeuwenhoek, the theory describing the biofilm process was not developed until 1978. We now understand that biofilms are universal, occurring in aquatic and industrial water systems as well as a large number of environments and medical devices relevant for public health. Using tools such as the scanning electron microscope and, more recently, the confocal laser scanning microscope, biofilm researchers now understand that biofilms are not unstructured, homogeneous deposits of cells and accumulated slime, but complex communities of surface-associated cells enclosed in a polymer matrix containing open water channels. Further studies have shown that the biofilm phenotype can be described in terms of the genes expressed by biofilm-associated cells. Microorganisms growing in a biofilm are highly resistant to antimicrobial agents by one or more mechanisms. Biofilm-associated microorganisms have been shown to be associated with several human diseases, such as native valve endocarditis and cystic fibrosis, and to colonize a wide variety of medical devices. Though epidemiologic evidence points to biofilms as a source of several infectious diseases, the exact mechanisms by which biofilm-associated microorganisms elicit disease are poorly understood. Detachment of cells or cell aggregates, production of endotoxin, increased resistance to the host immune system, and provision of a niche for the generation of resistant organisms are all biofilm processes which could initiate the disease process. Effective strategies to prevent or control biofilms on medical devices must take into consideration the unique and tenacious nature of biofilms. Current intervention strategies are designed to prevent initial device colonization, minimize microbial cell attachment to the device, penetrate the biofilm matrix and kill the associated cells, or remove the device from the patient. In the future, treatments may be based on inhibition of genes involved in cell attachment and biofilm formation.
Aggregation Substance Increases Adherence and Internalization, but Not Translocation, of
Enterococcus faecalis
through Different Intestinal Epithelial Cells In Vitro
Authors: Sartingen S. , Rozdzinski E. , Muscholl-Silberhorn A. , Marre R.
Published: July 2002

The aggregation substance of
Enterococcus faecalis
increased bacterial adherence to and internalization by epithelial cells originating from the colon and duodenum but not by cells derived from the ileum. However, enterococcal translocation through monolayers of intestinal epithelium was not observed.
Vibrio cholerae
Requires
rpoS
for Efficient Intestinal Colonization
Authors: Merrell D. Scott , Tischler Anna D. , Lee Sang Ho , Camilli Andrew
Published: July 2002

Vibrio cholerae
is a facultative intestinal pathogen that lives in aquatic environments, often in association with planktonic species. In the suckling mouse, oral inoculation with
V. cholerae
leads to intestinal colonization and symptoms of diarrheal disease. Results reported here indicate a role for the alternative sigma factor, RpoS, in intestinal colonization in this model of cholera. We constructed within
rpoS
multiple independent mutations which consistently resulted in a fivefold decrease in colonization ability as assessed by competition assays. These mutations had no detectable effect on the in vitro growth of
V. cholerae
in a rich medium. The occurrence of spontaneous suppressor mutations potentially required for viability of
rpoS
strains was ruled out by determination of the frequency of insertional inactivation of
rpoS
in comparison to two other nonessential loci. Finally, both the in vitro and in vivo mutant phenotypes of
rpoS
strains were fully complemented by providing
rpoS
in
trans
or by allelic reversion, indicating that the observed decrease in colonization fitness was indeed due to the loss of functional RpoS.
A Major Cell Surface Antigen of
Coccidioides immitis
Which Elicits Both Humoral and Cellular Immune Responses
Authors: Hung Chiung-Yu , Ampel Neil M. , Christian Lara , Seshan Kalpathi R. , Cole Garry T.
Published: July 2002

Multinucleate parasitic cells (spherules) of
Coccidioides immitis
isolates produce a membranous outer wall component (SOW) in vitro which has been reported to be reactive with antibody from patients with coccidioidal infection, elicits a potent proliferative response of murine immune T cells, and has immunoprotective capacity in a murine model of coccidioidomycosis. To identify the antigenic components of SOW, the crude wall material was first subjected to Triton X-114 extraction, and a water-soluble fraction derived from this treatment was examined for protein composition and reactivity in humoral and cellular immunoassays. Protein electrophoresis revealed that the aqueous fraction of three different isolates of
C. immitis
each contained one or two major glycoproteins (SOWgps), distinguished by their molecular sizes, which ranged from 58 to 82 kDa. The SOWgps, however, showed identical N-terminal amino acid sequences, and each was recognized by sera from patients with
C. immitis
infection. Antibody raised against the purified 58-kDa glycoprotein (SOWgp58) of the Silveira isolate was used for Western blot and immunolocalization analyses. Expression of SOWgp was shown to be parasitic phase specific, and the antigen was localized to the membranous SOW. The water-soluble fraction of SOW and the purified SOWgp58 were tested for the ability to stimulate proliferation of human peripheral monocytic cells (PBMC). The latter were obtained from healthy volunteers with positive skin test reaction to spherulin, a parasitic-phase antigen of
C. immitis
, and from volunteers who showed no skin test reaction to the same antigen. The SOW preparations stimulated proliferation of PBMC from skin test-positive but not skin test-negative donors, and the activated cells secreted gamma interferon, which is indicative of a T helper 1 pathway of immune response. Results of this study suggest that SOWgp is a major parasitic cell surface-expressed antigen that elicits both humoral and cellular immune responses in patients with coccidioidal infection.
Helicobacter felisInfection Is Associated with Lymphoid Follicular Hyperplasia and Mild Gastritis but Normal Gastric Secretory Function in Cats
Authors: Simpson Kenneth W. , Strauss-Ayali Dalit , Scanziani Eugenio , Straubinger Reinhard K. , McDonough Patrick L. , Straubinger Alix F. , Chang Yung-Fu , Domeneghini Cynzia , Arebi Naila , Calam John
Published: July 2002

The relationship ofHelicobacter felis, a bacterium observed in the stomachs of cats, to gastric disease is unclear. The objective of this study was to determine ifH. felisinfection alters gastric histopathology, proinflammatory cytokine expression, and secretory function and evokes a humoral immune response in cats. Five specific-pathogen-free (SPF)Helicobacter-free cats were studied before and for 1 year after oral inoculation withH. felis(ATCC 49179). Four SPFH. felis-uninfected cats served as controls. The stomachs of all fiveH. felis-inoculated cats became colonized, as determined by urease activity, histopathology, PCR, culture, and transmission electron microscopy of serial gastric biopsies at 0, 3, 5, 8, and 12 months. Uninoculated cats remainedHelicobacterfree. Lymphoid follicular hyperplasia, atrophy, and fibrosis were observed primarily in the pylorus of infected cats. Mild mononuclear inflammation was detected in both infected and uninfected cats, but was more extensive in infected cats, with pangastric inflammation, eosinophilic infiltrates, and cardia gastritis observed only in infected cats. No upregulation of antral mucosal interleukin 1α (IL-1α), IL-1β, or tumor necrosis factor alpha was detected by reverse transcription-PCR in any cat. The gastric secretory axes, assessed by fasting plasma gastrin, antral mucosal gastrin and somatostatin immunoreactivity, and pentagastrin-stimulated gastric acid secretion, were similar in both infected and uninfected cats. Gradual seroconversion (immunoglobulin G) was observed in four of five infected cats, with enzyme-linked immunosorbent assay values reaching 4× to 12× baseline 12 months postinfection. These findings indicate thatH. felisinfection in cats induces lymphoid follicular hyperplasia, mild gastritis, and seroconversion, but is associated with normal gastric secretory function.
Immunity Reduces Reservoir Host Competence ofPeromyscus leucopusforEhrlichia phagocytophila
Authors: Levin Michael L. , Fish Durland
Published: July 2002

Infection withEhrlichia phagocytophilain white-footed mice is transient and followed by a strong immune response. We investigated whether the presence of acquired immunity againstE. phagocytophilaprecludes white-footed mice from further maintenance of this agent in nature. Mice were infected withE. phagocytophilavia tick bite and challenged either 12 or 16 weeks later byIxodes scapularisnymphs infected with the same agent. Xenodiagnostic larvae fed upon each mouse simultaneously with challenging nymphs and 1 week thereafter. Ticks were tested for the agent by PCR, and the prevalence of infection was compared to that in ticks that fed upon nonimmune control mice. Only 30% of immunized mice sustained cofeeding transmission ofE. phagocytophilabetween simultaneously feeding infected and uninfected ticks, compared to 100% of control mice. An average of 6.3% of xenodiagnostic ticks acquiredEhrlichiafrom previously immunized mice when fed 1 week after the challenge, compared to 82.5% infection in the control group. Although an immune response to a single infection withE. phagocytophilain white-footed mice provided only partial protection against reinfection with the same agent, the majority of mice were rendered reservoir incompetent for at least 12 to 16 weeks. Immunity acquired by mice duringI. scapularisnymphal activity in early summer may exclude a large proportion of the mouse population from maintainingE. phagocytophiladuring the period of larval activity later in the season.
Role of
Bordetella bronchiseptica
Fimbriae in Tracheal Colonization and Development of a Humoral Immune Response
Authors: Mattoo Seema , Miller Jeff F. , Cotter Peggy A.
Published: July 2002

Fimbriae are filamentous, cell surface structures which have been proposed to mediate attachment of
Bordetella
species to respiratory epithelium.
Bordetella bronchiseptica
has four known fimbrial genes:
fim2
,
fim3
,
fimX
, and
fimA
. While these genes are unlinked on the chromosome, their protein products are assembled and secreted by a single apparatus encoded by the
fimBCD
locus. The
fimBCD
locus is embedded within the
fha
operon, whose genes encode another putative adhesin, filamentous hemagglutinin (FHA). We have constructed a Fim
−
B. bronchiseptica
strain, RB63, by introducing an in-frame deletion extending from
fimB
through
fimD
. Western blot analysis showed that RB63 is unable to synthesize fimbriae but is unaffected for FHA expression. Using this mutant, we assessed the role of fimbriae in pathogenesis in vitro and in vivo in natural animal hosts. Although RB63 was not significantly defective in its ability to adhere to various tissue culture cell lines, including human laryngeal HEp-2 cells, it was considerably altered in its ability to cause respiratory tract infections in rats. The number of Δ
fimBCD
bacteria recovered from the rat trachea at 10 days postinoculation was significantly decreased compared to that of wild-type
B. bronchiseptica
and was below the limit of detection at 30 and 60 days postinoculation. The number of bacteria recovered from the nasal cavity and larynx was not significantly different between RB63 and the wild-type strain at any time point. The ability of fimbriae to mediate initial attachment to tracheal tissue was tested in an intratracheal inoculation assay. Significantly fewer RB63 than wild-type bacteria were recovered from the tracheas at 24 h after intratracheal inoculation. These results demonstrate that fimbriae are involved in enhancing the ability of
B. bronchiseptica
to establish tracheal colonization and are essential for persistent colonization at this site. Interestingly, anti-
Bordetella
serum immunoglobulin M (IgM) levels were significantly lower in animals infected with RB63 than in animals infected with wild-type
B. bronchiseptica
at 10 days postinoculation. Even at 30 days postinoculation, RB63-infected animals had lower serum anti-
Bordetella
antibody titers in general. This disparity in antibody profiles suggests that fimbriae are also important for the induction of a humoral immune response.
Identification of Regions of the
Escherichia coli
Chromosome Specific for Neonatal Meningitis-Associated Strains
Authors: Bonacorsi Stéphane Pierre Paul , Clermont Olivier , Tinsley Colin , Le Gall Isabelle , Beaudoin Jean-Christophe , Elion Jacques , Nassif Xavier , Bingen Edouard
Published: July 2002

Specific virulence factors associated with the pathogenesis of
Escherichia coli
strains causing neonatal meningitis (ECNM), such as the K1 capsular polysaccharide, the S fimbriae, and the Ibe10 protein, have been previously identified. However, some other yet unidentified factors are likely to be involved in the pathogenesis of ECNM. To identify specialized unique DNA regions associated with ECNM virulence, we used the representational difference analysis technique. The genomes of two strains belonging to nonpathogenic phylogenetic group A of the ECOR reference collection were subtracted from
E. coli
strain C5, isolated from a case of neonatal meningitis. Strain C5 belongs to the phylogenetic group B2 as do the majority of ECNM. We have isolated and mapped 64 DNA fragments which are specific for strain C5 and not found in nonpathogenic strains. Of these clones, 44 were clustered in six distinct regions on the chromosome. The
sfa
and
ibe10
genes were located in regions 2 and 6, respectively. A group of genes (
cnf1
,
hra
,
hly
, and
prs
) known to be present in a pathogenicity island of the uropathogenic strain
E. coli
J96 colocalized with region 6. The occurrence of these DNA regions was tested in a set of meningitis-associated strains and in a control group composed of non-meningitis-associated strains belonging to the same B2 group. Regions 1, 3, and 4 were present in 91, 82, and 81%, respectively, of the meningitis strains and in 40, 13, and 47% of the control strains. Together, these data suggest that regions 1, 3, and 4 code for factors associated with the ability of
E. coli
to invade the meninges of neonates.
Four Different Genes Responsible for Nonimmune Immunoglobulin-Binding Activities within a Single Strain of
Escherichia coli
Authors: Sandt Carol H. , Hill Charles W.
Published: July 2002

Certain
Escherichia coli
strains bind the Fc fragment of immunoglobulin G (IgG) at the bacterial cell surface. Previous work established that this nonimmune Ig binding depends on several large proteins with apparent molecular masses that can exceed 200 kDa. For
E. coli
strain ECOR-9, four distinct genes (designated
eibA
,
eibC
,
eibD
, and
eibE
) are responsible for Ig binding. Two
eib
genes are linked to
eaa
genes, which are homologous to genes for the autotransporter family of secreted proteins. With reference to the
E. coli
K-12 chromosome, the
eibA-eaaA
cluster is adjacent to
trpA
(min 28.3) while the
eibC-eaaC
cluster is adjacent to
aspS
(min 42.0). Sequence adjacent to the
eibA-eaaA
cluster converges with that of strain K-12 precisely as observed for the Atlas family of prophages, suggesting that
eibA
is part of one of these. All four
eib
genes, when cloned into plasmid vectors, impart IgG binding to
E. coli
K-12 strains, and three impart IgA binding also. The IgG binding occurs at the bacterial cell surface, and its expression increases survival in serum by up to 3 orders of magnitude. The
eib
sequences predict a C-terminal peptide motif that is characteristic of outer membrane proteins, and the protein sequences show significant similarity near the C terminus to both the YadA virulence factor of
Yersinia
species and the universal surface protein A II of
Moraxella catarrhalis
. The sizes predicted for Eib proteins from DNA sequence are much smaller than their apparent sizes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, possibly reflecting stable oligomerization.
CpG Oligodeoxynucleotides and Interleukin-12 Improve the Efficacy ofMycobacterium bovisBCG Vaccination in Mice Challenged withM. tuberculosis
Authors: Freidag Brenda L. , Melton Genevieve B. , Collins Frank , Klinman Dennis M. , Cheever Allen , Stobie Laura , Suen Winnie , Seder Robert A.
Published: July 2002

Mycobacterium bovisbacillus Calmette-Guérin (BCG) is the only vaccine approved for prevention of tuberculosis. It has been postulated that serial passage of BCG over the years may have resulted in attenuation of its effectiveness. Because interleukin-12 (IL-12) and oligodeoxynucleotides (ODN) containing cytidine phosphate guanosine (CpG) motifs have been shown to enhance Th1 responses in vivo, they were chosen as adjuvants to increase the effectiveness of BCG vaccination. In this report, mice were vaccinated with BCG with or without IL-12 or CpG ODN and then challenged 6 weeks later via the aerosol route with the Erdman strain ofM. tuberculosis. Mice vaccinated with BCG alone showed a 1- to 2-log reduction in bacterial load compared with control mice that did not receive any vaccination prior toM. tuberculosischallenge. Moreover, the bacterial loads of mice vaccinated with BCG plus IL-12 or CpG ODN were a further two- to fivefold lower than those of mice vaccinated with BCG alone. As an immune correlate, the antigen-specific production IFN-γ and mRNA expression in spleen cells prior to challenge were evaluated. Mice vaccinated with BCG plus IL-12 or CpG ODN showed enhanced production of IFN-γ compared with mice vaccinated with BCG alone. Finally, granulomas in BCG-vaccinated mice were smaller and more lymphocyte rich than those in unvaccinated mice; however, the addition of IL-12 or CpG ODN to BCG vaccination did not alter granuloma formation or result in added pulmonary damage. These observations support a role for immune adjuvants given with BCG vaccination to enhance its biologic efficacy.
R1 Region of P97 Mediates Adherence ofMycoplasma hyopneumoniaeto Swine Cilia
Authors: Minion F. Chris , Adams Cary , Hsu Tsungda
Published: July 2002

Adherence ofMycoplasma hyopneumoniaeto the swine respiratory tract is mediated by the membrane protein P97. This protein is located on the outer membrane surface, and its role in adherence has been firmly established. The general region of P97 that mediates adherence to swine cilia is thought to be the R1 region near the carboxy terminus of the protein, but it was not clear if this region could mediate adherence to swine cilia independently of other P97 sequences. To examine this in more detail, a series of R1 repeat sequences containing different numbers of repeating units cloned in frame withlacZwas used to produce R1–β-galactosidase fusion proteins. These proteins were then tested for adherence to swine cilia and for reactivity to the adherence-blocking monoclonal antibody F2G5 and convalescent-phase swine sera. In this way it was possible to accurately define the cilium binding epitope of P97 and the minimal epitope recognized by antibody. Our results indicate that eight R1 repeating units are required for cilium binding and that three repeating units are needed for antibody recognition. These results could lead to more effective therapeutic measures against this important swine pathogen.
Interaction of
Listeria monocytogenes
with Human Brain Microvascular Endothelial Cells: an Electron Microscopic Study
Authors: Greiffenberg Lars , Goebel Werner , Kim Kwang Sik , Daniels Justin , Kuhn Michael
Published: July 2002

Internalization of
Listeria monocytogenes
into human brain microvascular endothelial cells (HBMEC) has recently been demonstrated to be dependent upon the
inlB
gene. In the present scanning electron microscopic study we show that
L. monocytogenes
efficiently interacts with the surface of HBMEC in an
inlB
-independent manner which is also different from invasion. The
inlB
-dependent invasion of HBMEC by
L. monocytogenes
is accompanied by intracellular multiplication, movement, and production of bacterium-containing protrusions. These protrusions extend from the cell surface without perturbation of any adjacent cellular membrane.
Characterization of Hemolysin ofMoraxella bovisUsing a Hemolysis-Neutralizing Monoclonal Antibody
Authors: Billson F. Mark , Harbour Colin , Michalski Wojtek P. , Tennent Jan M. , Egerton John R. , Hodgson Jennifer L.
Published: July 2002

A concentrated bacterial culture supernatant from the hemolyticMoraxella bovisstrain UQV 148NF was used to immunize mice and generate monoclonal antibodies (MAbs). One, MAb G3/D7, neutralized the hemolytic activity ofM. bovisand recognized a 94-kDa protein by Western blot analysis in hemolyticM. bovisstrains representing each of the different fimbrial serogroups. Exposure of corneal epithelial cells toM. bovisconcentrated culture supernatants demonstrated a role for an exotoxin in the pathogenesis of infectious bovine keratoconjunctivitis, while neutralization of hemolytic and cytotoxic activities by MAb G3/D7 implies that these activities are related or have common epitopes. The action ofM. bovishemolysin was further characterized in sheep erythrocyte preparations with a binding step and Ca2+required for lysis to proceed, similar to the RTX family of bacterial exotoxins. Neutralization of lytic activity in vitro is evidence for the presence ofM. bovisantigens, which may be capable of protecting cattle from the development of infectious bovine keratoconjunctivitis.
Cytocidal and Apoptotic Effects of the ClyA Protein from
Escherichia coli
on Primary and Cultured Monocytes and Macrophages
Authors: Lai Xin-He , Arencibia Ignacio , Johansson Anders , Wai Sun Nyunt , Oscarsson Jan , Kalfas Sotos , Sundqvist Karl-Gösta , Mizunoe Yoshimitsu , Sjöstedt Anders , Uhlin Bernt Eric
Published: July 2002

Cytolysin A (ClyA) is a newly discovered cytolytic protein of
Escherichia coli
K-12 that mediates a hemolytic phenotype. We show here that highly purified ClyA and ClyA-expressing
E. coli
were cytotoxic and apoptogenic to fresh as well as cultured human and murine monocytes/macrophages.