bluuebunny/crossref_metadata_2025 huggingface dataset:
Habitat Association in Two Genetic Groups of the Insect-Pathogenic FungusMetarhizium anisopliae: Uncovering Cryptic Species?
Authors: Bidochka Michael J. , Kamp Andrena M. , Lavender T. Michael , Dekoning Jason , De Croos J. N. Amritha
Published: July 2002

Strains of insect-pathogenic fungi with high virulence toward certain pest insects have great potential for commercial biological control applications. Identifying such strains has been a central theme in using fungi for biological control. This theme is supported by a persistent paradigm in insect pathology which suggests that the host insect is the predominant influence on the population genetics of insect-pathogenic fungi. In this study, a population genetics analysis of the insect-pathogenic fungusMetarhizium anisopliaefrom forested and agricultural habitats in Ontario, Canada, showed a nonrandom association of alleles between two distinct, reproductively isolated groups (index of multilocus association = 1.2). Analyses of the mitochondrial DNA showed no differences between the groups. The two groups were associated with different habitat types, and associations with insect hosts were not found. The group from forested areas showed an ability for cold-active growth (i.e., 8°C), while the group from the agricultural area showed an ability for growth at high temperatures (i.e., 37°C) and resilience to UV exposure. These results represent a significant paradigm shift; habitat selection, not host insect selection, drives the population structure of these insect-pathogenic deuteromycetous fungi. With each group we observed recombining population structures as well as clonally reproducing lineages. We discuss whether these groups may represent cryptic species. Worldwide,M. anisopliaemay be an assembly of cryptic species, each adapted to certain environmental conditions. The association of fungal genotypes with habitat but not with host insects has implications on the criteria for utility of this, and perhaps other, fungal biocontrol agents.
Comparison ofnifHGene Pools in Soils and Soil Microenvironments with Contrasting Properties
Authors: Poly Franck , Ranjard Lionel , Nazaret Sylvie , Gourbière François , Monrozier Lucile Jocteur
Published: July 2002

The similarities and differences in the structures of thenifHgene pools of six different soils (Montrond, LCSA-p, Vernon, Dombes, LCSA-c, and Thysse Kaymor) and five soil fractions extracted from LCSA-c were studied. Bacterial DNA was directly extracted from the soils, and a region of thenifHgene was amplified by PCR and analyzed by restriction. Soils were selected on the basis of differences in soil management, plant cover, and major physicochemical properties. Microenvironments differed on the basis of the sizes of the constituent particles and the organic carbon and clay contents. Restriction profiles were subjected to principal-component analysis. We showed that the composition of the diazotrophic communities varied both on a large scale (among soils) and on a microscale (among microenvironments in LCSA-c soil). Soil management seemed to be the major parameter influencing differences in thenifHgene pool structure among soils by controlling inorganic nitrogen content and its variation. However, physicochemical parameters (texture and total C and N contents) were found to correlate with differences amongnifHgene pools on a microscale. We hypothesize that the observednifHgenetic structures resulted from the adaptation to fluctuating conditions (cultivated soil, forest soil, coarse fractions) or constant conditions (permanent pasture soil, fine fractions). We attempted to identify a specific band within the profile of the clay fraction by cloning and sequencing it and comparing it with the gene databases. Unexpectedly, thenifHsequences of the dominant bacteria were most similar to sequences of unidentified marine eubacteria.
Active Subtilisin-Like Protease from a Hyperthermophilic Archaeon in a Form with a Putative Prosequence
Authors: Kannan Yuji , Koga Yuichi , Inoue Yohei , Haruki Mitsuru , Takagi Masahiro , Imanaka Tadayuki , Morikawa Masaaki , Kanaya Shigenori
Published: July 2002

The gene encoding subtilisin-like protease
T. kodakaraensis
subtilisin was cloned from a hyperthermophilic archaeon
Thermococcus kodakaraensis
KOD1.
T. kodakaraensis
subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins,
T. kodakaraensis
subtilisin was overproduced in
Escherichia coli
in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding,
T. kodakaraensis
subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant
T. kodakaraensis
subtilisin which is composed of 398 amino acid residues (Gly
−82
to Gly
316
), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that
T. kodakaraensis
subtilisin exists in a monomeric form.
T. kodakaraensis
subtilisin hydrolyzed the synthetic substrate
N
-succinyl-Ala-Ala-Pro-Phe-
p
-nitroanilide only in the presence of the Ca
2+
ion with an optimal pH and temperature of pH 9.5 and 80°C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80°C, 20 min at 90°C, and 7 min at 100°C.
Detection of
Lactobacillus, Pediococcus, Leuconostoc
, and
Weissella
Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis
Authors: Walter Jens , Hertel Christian , Tannock Gerald W. , Lis Claudia M. , Munro Karen , Hammes Walter P.
Published: July 2002

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera
Lactobacillus, Pediococcus, Leuconostoc
, and
Weissella
in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as
Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides
, and
Pediococcus pentosaceus
. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain
Lactobacillus rhamnosus
DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that
L. rhamnosus
was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.
The Functional
ccpA
Gene Is Required for Carbon Catabolite Repression in
Lactobacillus plantarum
Authors: Muscariello Lidia , Marasco Rosangela , De Felice Maurilio , Sacco Margherita
Published: July 2002

We report the characterization of the
ccpA
gene of
Lactobacillus plantarum
, coding for catabolite control protein A. The gene is linked to the
pepQ
gene, encoding a proline peptidase, in the order
ccpA-pepQ
, with the two genes transcribed in tandem from the same strand as distinct transcriptional units. Two
ccpA
transcription start sites corresponding to two functional promoters were found, expression from the upstream promoter being autogenously regulated through a catabolite-responsive element (
cre
) sequence overlapping the upstream +1 site. During growth on ribose, the upstream promoter showed maximal expression, while growth on glucose led to transcription from the downstream promoter. In a
ccpA
mutant strain, the gene was transcribed mainly from the upstream promoter in both repressing and non repressing conditions. Expression of two enzyme activities, β-glucosidase and β-galactosidase, was relieved from carbon catabolite repression in the
ccpA
mutant strain. In vivo footprinting analysis of the catabolite-controlled
bglH
gene regulatory region in the
ccpA
mutant strain showed loss of protection of the
cre
under repressing conditions.
Effect of Turbulent-Flow Pasteurization on Survival ofMycobacterium aviumsubsp.paratuberculosisAdded to Raw Milk
Authors: Pearce Lindsay E. , Truong H. Tuan , Crawford Robert A. , Yates Gary F. , Cavaignac Sonia , de Lisle Geoffrey W.
Published: July 2002

A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity ofMycobacterium aviumsubsp.paratuberculosisadded to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72°C in duplicate trials. No strains survived at 72°C for 15 s; and only one strain survived at 69°C. Means of pooledDvalues (decimal reduction times) at 63 and 66°C were 15.0 ± 2.8 s (95% confidence interval) and 5.9 ± 0.7 s (95% confidence interval), respectively. The mean extrapolatedD72°Cwas <2.03 s. This was equivalent to a >7 log10kill at 72°C for 15 s (95% confidence interval). The meanZvalue (degrees required for the decimal reduction time to traverse one log cycle) was 8.6°C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72°C for 15 s, the minimumM. aviumsubsp.paratuberculosiskill was >4 log10. Properly maintained and operated equipment should ensure the absence of viableM. aviumsubsp.paratuberculosisin retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2° above 72°C.
Two-Dimensional Electrophoresis Study of
Lactobacillus delbrueckii
subsp.
bulgaricus
Thermotolerance
Authors: Gouesbet Gwenola , Jan Gwenael , Boyaval Patrick
Published: July 2002

The response of
Lactobacillus delbrueckii
subsp.
bulgaricus
cells to heat stress was studied by use of a chemically defined medium. Two-dimensional electrophoresis (2-DE) analysis was used to correlate the kinetics of heat shock protein (HSP) induction with cell recovery from heat injury. We demonstrated that enhanced viability, observed after 10 min at 65°C, resulted from the overexpression of HSP and from mechanisms not linked to protein synthesis. In order to analyze the thermoadaptation mechanisms involved, thermoresistant variants were selected. These variants showed enhanced constitutive tolerance toward heat shock. However, contrary to the wild-type strain, these variants were poorly protected after osmotic or heat pretreatments. This result suggests that above a certain threshold, cells reach a maximum level of protection that cannot be easily exceeded. A comparison of protein patterns showed that the variants were able to induce more rapidly their adaptive mechanisms than the original strain. In particular, the variants were able to express constitutively more HSP, leading to the higher level of thermoprotection observed. This is the first report of the study by 2-DE of the heat stress response in
L. delbrueckii
subsp.
bulgaricus
.
Temporal Changes in Archaeal Diversity and Chemistry in a Mid-Ocean Ridge Subseafloor Habitat
Authors: Huber Julie A. , Butterfield David A. , Baross John A.
Published: July 2002

The temporal variation in archaeal diversity in vent fluids from a midocean ridge subseafloor habitat was examined using PCR-amplified 16S rRNA gene sequence analysis and most-probable-number (MPN) cultivation techniques targeting hyperthermophiles. To determine how variations in temperature and chemical characteristics of subseafloor fluids affect the microbial communities, we performed molecular phylogenetic and chemical analyses on diffuse-flow vent fluids from one site shortly after a volcanic eruption in 1998 and again in 1999 and 2000. The archaeal population was divided into particle-attached (>3-μm-diameter cells) and free-living fractions to test the hypothesis that subseafloor microorganisms associated with active hydrothermal systems are adapted for a lifestyle that involves attachment to solid surfaces and formation of biofilms. To delineate between entrained seawater archaea and the indigenous subseafloor microbial community, a background seawater sample was also examined and found to consist only of Group I
Crenarchaeota
and Group II
Euryarchaeota
, both of which were also present in vent fluids. The indigenous subseafloor archaeal community consisted of clones related to both mesophilic and hyperthermophilic
Methanococcales
, as well as many uncultured
Euryarchaeota
, some of which have been identified in other vent environments. The particle-attached fraction consistently showed greater diversity than the free-living fraction. The fluid and MPN counts indicate that while culturable hyperthermophiles represent less than 1% of the total microbial community, the subseafloor at new eruption sites does support a hyperthermophilic microbial community. The temperature and chemical indicators of the degree of subseafloor mixing appear to be the most important environmental parameters affecting community diversity, and it is apparent that decreasing fluid temperatures correlated with increased entrainment of seawater, decreased concentrations of hydrothermal chemical species, and increased incidence of seawater archaeal sequences.
Effect of Growth Conditions and Staining Procedure upon the Subsurface Transport and Attachment Behaviors of a Groundwater Protist
Authors: Harvey Ronald W. , Mayberry Naleen , Kinner Nancy E. , Metge David W. , Novarino Franco
Published: July 2002

The transport and attachment behaviors of
Spumella guttula
(Kent), a nanoflagellate (protist) found in contaminated and uncontaminated aquifer sediments in Cape Cod, Mass., were assessed in flowthrough and static columns and in a field injection-and-recovery transport experiment involving an array of multilevel samplers. Transport of
S. guttula
harvested from low-nutrient (10 mg of dissolved organic carbon per liter), slightly acidic, granular (porous) growth media was compared to earlier observations involving nanoflagellates grown in a traditional high-nutrient liquid broth. In contrast to the highly retarded (retardation factor of ∼3) subsurface transport previously reported for
S. guttula
, the peak concentration of porous-medium-grown
S. guttula
traveled concomitantly with that of a conservative (bromide) tracer. About one-third of the porous-medium-grown nanoflagellates added to the aquifer were transported at least 2.8 m downgradient, compared to only ∼2% of the broth-grown nanoflagellates. Flowthrough column studies revealed that a vital (hydroethidine [HE]) staining procedure resulted in considerably less attachment (more transport) of
S. guttula
in aquifer sediments than did a staining-and-fixation procedure involving 4′,6′-diamidino-2-phenylindole (DAPI) and glutaraldehyde. The calculated collision efficiency (∼10
−2
for porous-medium-grown, DAPI-stained nanoflagellates) was comparable to that observed earlier for the indigenous community of unattached groundwater bacteria that serve as prey. The attachment of HE-labeled
S. guttula
onto aquifer sediment grains was independent of pH (over the range from pH 3 to 9) suggesting a primary attachment mechanism that may be fundamentally different from that of their prey bacteria, which exhibit sharp decreases in fractional attachment with increasing pH. The high degree of mobility of
S. guttula
in the aquifer sediments has important ecological implications for the protistan community within the temporally changing plume of organic contaminants in the Cape Cod aquifer.
Identification of NovelHelicobacterspp. from a Beluga Whale
Authors: Harper Claudia M. G. , Xu Shilu , Feng Yan , Dunn J. Lawrence , Taylor Nancy S. , Dewhirst Floyd E. , Fox James G.
Published: July 2002

The gastric fluid and feces of three belugas from the Mystic Aquarium were assessed for the presence ofHelicobacterspp. Gastric fluid and feces from the two clinically healthy belugas were negative for helicobacter, and endoscopy performed on these animals revealed no lesions. However, a helicobacter isolate and PCR product similar to helicobacter strains previously recovered from dolphins were identified, respectively, from the feces and gastric fluid of a beluga manifesting intermittent inappetence and lethargy. Esophageal and forestomach ulcers were noted on endoscopy. This is the first report of novelHelicobacterspp. being identified from whales.
Novel Plant-Microbe Rhizosphere Interaction Involving
Streptomyces lydicus
WYEC108 and the Pea Plant (
Pisum sativum
)
Authors: Tokala Ranjeet K. , Strap Janice L. , Jung Carina M. , Crawford Don L. , Salove Michelle Hamby , Deobald Lee A. , Bailey J. Franklin , Morra M. J.
Published: July 2002

A previously undescribed plant-microbe interaction between a root-colonizing
Streptomyces
species,
S. lydicus
WYEC108, and the legume
Pisum sativum
is described. The interaction is potentially of great importance to the health and growth in nature of this nodulating legume. The root-colonizing soil actinomycete
S. lydicus
WYEC108 influences pea root nodulation by increasing root nodulation frequency, possibly at the level of infection by
Rhizobium
spp.
S. lydicus
also colonizes and then sporulates within the surface cell layers of the nodules. Colonization leads to an increase in the average size of the nodules that form and improves the vigor of bacteroids within the nodules by enhancing nodular assimilation of iron and possibly other soil nutrients. Bacteroid accumulation of the carbon storage polymer, poly-β-hydroxybutyrate, is reduced in colonized nodules. Root nodules of peas taken from agricultural fields in the Palouse hills of northern Idaho were also found to be colonized by actinomycete hyphae. We hypothesize that root and nodule colonization is one of several mechanisms by which
Streptomyces
acts as a naturally occurring plant growth-promoting bacterium in pea and possibly other leguminous plants.
Salmonella entericaInfections in Market Swine with and without Transport and Holding
Authors: Hurd H. S. , McKean J. D. , Griffith R. W. , Wesley I. V. , Rostagno M. H.
Published: July 2002

The objective of this study was to compare, by using identical sample types, theSalmonella entericaprevalences and serovar diversities between pigs necropsied on the farm and those necropsied at the abattoir after transport and holding. We necropsied 567 market weight pigs (>70 kg) from six herds. Pigs were alternately assigned to be necropsied on the farm or at the abattoir. One-half of the group was sent in clean, disinfected trailers to slaughter at a commercial abattoir. After transport (mean distance, 169 km) and 2 to 3 h of holding in antemortem pens, these pigs were necropsied. The 50 pigs remaining on the farm were necropsied the following day. The same sample types and amounts were collected forS. entericaculture at both locations. Results show a sevenfold-higher (P< 0.001)S. entericaisolation rate from pigs necropsied at the abattoir (39.9%; 114 of 286) than from those necropsied on the farm (5.3%; 15 of 281). This difference was also observed for each individual herd. All sample types showed a significantly higher prevalence when comparing abattoir to on-farm collection, respectively: lymph nodes, 9.15 versus 3.6%; cecal contents, 13.6 versus 1.8%; 1 g of fecal matter, 25.2 versus 0.7%. Recovery of additional serovars at the abattoir suggests the pigs are receivingS. entericafrom extra-farm sources. This study demonstrates that rapid infection during transport, and particularly during holding, is a major reason for increasedS. entericaprevalence in swine. This finding identifies the holding pen as an importantS. entericacontrol point in the pork production chain.
Apoptosis in T-Lymphocyte Subsets in Human Immunodeficiency Virus-Infected Children Measured Immediately Ex Vivo and following In Vitro Activation
Authors: Niehues Tim , McCloskey Thomas W. , Ndagijimana Jennifer , Horneff Gerd , Wahn Volker , Pahwa Savita
Published: July 2002

Phosphatidylserine molecules are translocated to the outer plasma membrane of lymphocytes undergoing apoptosis and can be detected by the binding of fluorochrome-conjugated annexin V. Using the annexin V assay, we examined CD4 and CD8 T cells from human immunodeficiency virus (HIV)-infected children for apoptosis upon isolation or following in vitro culture. Immediate ex vivo analysis or overnight culture showed significantly higher levels of apoptosis in CD8 cells than in CD4 cells. Following culture with the activating stimulus phytohemagglutinin or anti-CD3 monoclonal antibody, we observed an increase in the percentage of apoptotic CD4 cells, whereas there was no change in the rate of CD8 cell death. These results demonstrate that in HIV-infected children, CD8 apoptosis may occur at a greater rate than CD4 apoptosis in vivo; greater CD4 depletion may be observed due to more efficient mechanisms for peripheral lymphocyte replacement in the CD8 compartment. Furthermore, our data suggest that CD8 lymphocytes may be maximally activated in vivo, a condition which may lead to the exhaustion of CD8-mediated immunity. These findings clarify the differences between the CD4 and CD8 apoptotic responses to HIV.
Expression of Urokinase Plasminogen Activator Receptor on Monocytes from Patients with Relapsing-Remitting Multiple Sclerosis: Effect of Glatiramer Acetate (Copolymer 1)
Authors: Balabanov Roumen , Lisak Deena , Beaumont Thomas , Lisak Robert P. , Dore-Duffy Paula
Published: July 2002

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system in which peripheral blood monocytes play an important role. We have previously reported that patients with chronic progressive MS (CPMS) have significantly increased numbers of circulating monocytes which express the urokinase plasminogen activator receptor (uPAR). In the present study, we examined the expression of uPAR on monocytes in patients with relapsing-remitting multiple sclerosis (RRMS) not currently participating in a clinical trial and in patients with RRMS who were enrolled in a double-blind multicenter clinical trial designed to examine the effect of glatiramer acetate (copolymer 1; Copaxone) on relapsing disease. Patients with CPMS have sustained high levels of circulating uPAR-positive (uPAR+) monocytes. In comparison, patients with RRMS displayed variable levels of circulating uPAR+monocytes. Mean values for uPAR in patients with RRMS were above those seen for controls but were not as high as those observed for patients with secondary progressive MS. Patients with RRMS in the clinical trial also had variable levels of monocyte uPAR. However, patients in the treatment group displayed lower levels following 2 years of treatment. In both placebo-treated and glatiramer acetate-treated patients, the percentage of circulating uPAR+monocytes, as well as the density of uPAR expressed per cell (mean linear fluorescence intensity), increased just prior to the onset of a clinically documented exacerbation. Values fell dramatically with the development of clinical symptoms. uPAR levels in all groups correlated with both clinical activity and severity. Results indicate that monocyte activation is impatient in MS and that glatiramer acetate may have a significant effect on monocyte activation in patients with RRMS.
Interpretation of the Gamma Interferon Test for Diagnosis of Subclinical Paratuberculosis in Cattle
Authors: Jungersen G. , Huda A. , Hansen J. J. , Lind P.
Published: July 2002

A group of 252 cattle without clinical signs of paratuberculosis (paraTB) in 10 herds infected with paraTB and a group of 117 cattle in 5 herds without paraTB were selected. Whole-blood samples were stimulated with bovine, avian, and johnin purified protein derivative (PPD) and examined for gamma interferon (IFN-γ) release. For diagnosis of paraTB, satisfactory estimated specificities (95 to 99%) could be obtained by johnin PPD stimulation irrespective of interpretation relative to bovine PPD or no-antigen stimulation alone, but numbers of test positives in the infected herds varied from 64 to 112 with different interpretation criteria. For a limited number of test-positive animals, no change in the test results could be observed with increasing antigen concentrations but IFN-γ responses were significantly reduced (P< 0.0001) and four out of seven reactors tested negative when stimulation was performed on day-old samples. Denmark is free of bovine tuberculosis, but cross-reactivity with paraTB could be documented for cattle more than 14 months old in paraTB-infected herds compared with those in non-paraTB-infected herds. In both paraTB-free and paraTB-infected herds, false positives were observed when the test was applied to calves less than 15 months of age. Until novel antigen formulations more specific for these diseases are available, interpretation of the IFN-γ test must be individually adjusted to fit specific needs and the context within which the test is applied and, for paraTB, the test seems most appropriate for use as a supportive tool for evaluation of disease-preventive measures in young stock.
Cytokine Induction byStreptococcus mutansand Pulpal Pathogenesis
Authors: Hahn Chin-Lo , Best Al M. , Tew John G.
Published: July 2002

Chronic pulpal inflammation under caries appears to be elicited by bacterial antigens that diffuse into the pulp through dentinal tubules. This prompted the hypothesis that cytokines elicited by antigens fromStreptococcus mutans, which frequently dominates shallow lesions, could play a major role in eliciting the initial T-cell response in the pulp. To test this, we examined the ability ofS. mutansto stimulate T cells and elicit cytokines and usedLactobacillus casei, which often predominates in deep carious lesions where B cells and plasma cells predominate, as a control. In addition, the presence of cytokines in the pulp was analyzed at the mRNA level.S. mutanselicited potent gamma interferon (IFN-γ) responses in peripheral blood mononuclear cell cultures and reduced the CD4/CD8 ratio by promoting CD8+T cells. Multiple inflammatory cytokine mRNAs (IFN-γ, interleukin 4 [IL-4], and IL-10) were detected in human dental pulp. A higher prevalence of IFN-γ (67%) than IL-4 (19%) or IL-10 (29%) was obtained in shallow caries, suggesting a type 1 cytokine mechanism in early pulpitis whereS. mutanspredominates. In contrast, in deep caries no differences in cytokine frequency were observed. Furthermore, the presence of IFN-γ in the pulp correlated with the presence ofS. mutans. The extraordinary induction of type 1 cytokines and the preferential activation of CD8+T cells byS. mutansoffers an explanation for the etiology of the CD8+T-cell-dominant lesion in early pulpitis and suggests thatS. mutansmay have a major impact on the initial lesion and pulpal pathology.
Surface Protein Variation by Expression Site Switching in the Relapsing Fever Agent
Borrelia hermsii
Authors: Barbour Alan G. , Carter Carol J. , Sohaskey Charles D.
Published: July 2002

Borrelia hermsii
, an agent of relapsing fever, undergoes antigenic variation of serotype-specifying membrane proteins during mammalian infections. When
B. hermsii
is cultivated in broth medium, one serotype, 33, eventually predominates in the population. Serotype 33 has also been found to be dominant in ticks but not in mammalian hosts. We investigated the biology and genetics of two independently derived clonal populations of serotype 33 of
B. hermsii
. Both isolates infected immunodeficient mice, but serotype 33 cells were limited in number and were only transiently present in the blood. Probes for
vsp33
, which encodes the serotype-specifying Vsp33 outer membrane protein, revealed that the gene was located on a 53-kb linear plasmid and that there was only one locus for the gene in serotype 33. The
vsp33
probe and probes for other variable membrane protein genes showed that expression of Vsp33 was determined at the level of transcription and that when the
vsp33
expression site was active, an expression site for other variable proteins was silent. The study confirmed that serotype 33 is distinct from other serotypes of
B. hermsii
in its biology and demonstrated that
B. hermsii
can change its major surface protein through switching between two expression sites.
Nonpolar Inactivation of the Hypervariable Streptococcal Inhibitor of Complement Gene (
sic
) in Serotype M1
Streptococcus pyogenes
Significantly Decreases Mouse Mucosal Colonization
Authors: Lukomski Slawomir , Hoe Nancy P. , Abdi Iman , Rurangirwa Jacqueline , Kordari Parichher , Liu Mengyao , Dou Shu-Jun , Adams Gerald G. , Musser James M.
Published: July 2002

Group A
Streptococcus
(GAS) is a human pathogen that commonly infects the upper respiratory tract. GAS serotype M1 strains are frequently isolated from human infections and contain the gene encoding the hypervariable streptococcal inhibitor of complement protein (Sic). It was recently shown that Sic variants were rapidly selected on mucosal surfaces in epidemic waves caused by M1 strains, an observation suggesting that Sic participates in host-pathogen interactions on the mucosal surface (N. P. Hoe, K. Nakashima, S. Lukomski, D. Grigsby, M. Liu, P. Kordari, S.-J. Dou, X. Pan, J. Vuopio-Varkila, S. Salmelinna, A. McGeer, D. E. Low, B. Schwartz, A. Schuchat, S. Naidich, D. De Lorenzo, Y.-X. Fu, and J. M. Musser, Nat. Med. 5:924–929, 1999). To test this idea, a new nonpolar mutagenesis method employing a spectinomycin resistance cassette was used to inactivate the
sic
gene in an M1 GAS strain. The isogenic Sic-negative mutant strain was significantly (
P
< 0.019) impaired in ability to colonize the mouse mucosal surface after intranasal infection. These results support the hypothesis that the predominance of M1 strains in human infections is related, in part, to a Sic-mediated enhanced colonization ability.
Mannose-Binding Lectin Binds to a Range of Clinically Relevant Microorganisms and Promotes Complement Deposition
Authors: Neth Olaf , Jack Dominic L. , Dodds Alister W. , Holzel Helen , Klein Nigel J. , Turner Malcolm W.
Published: July 2002

Mannose-binding lectin (MBL) is a collagenous serum lectin believed to be of importance in innate immunity. Genetically determined low levels of the protein are known to predispose to infections. In this study the binding of purified MBL to pathogens isolated from immunocompromised children was investigated by flow cytometry. DiverseCandidaspecies,Aspergillus fumigatus,Staphylococcus aureus, and beta-hemolytic group A streptococci exhibited strong binding of MBL, whereasEscherichia coli,Klebsiellaspecies, andHaemophilus influenzaetype b were characterized by heterogeneous binding patterns. In contrast, beta-hemolytic group B streptococci,Streptococcus pneumoniae, andStaphylococcus epidermidisshowed low levels of binding. Bound MBL was able to promote C4 deposition in a concentration-dependent manner. We conclude that MBL may be of importance in first-line immune defense against several important pathogens.
Role of Tir and Intimin in the Virulence of Rabbit EnteropathogenicEscherichia coliSerotype O103:H2
Authors: Marchès Olivier , Nougayrède Jean-Philippe , Boullier Séverine , Mainil Jacques , Charlier Gérard , Raymond Isabelle , Pohl Pierre , Boury Michèle , De Rycke Jean , Milon Alain , Oswald Eric
Published: July 2002

Attaching and effacing (A/E) rabbit enteropathogenicEscherichia coli(REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenictirnull mutant together with a previously describedeaenull mutant. When human HeLa epithelial cells were infected, thetirmutant was still able to induce the formation of stress fibers as previously reported for theeaenull mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both theeaeandtirmutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for theeaenull mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.
Cloning and Sequencing of the
Klebsiella pneumoniae
O5
wb
Gene Cluster and Its Role in Pathogenesis
Authors: Merino Susana , Altarriba Maria , Izquierdo Luis , Nogueras María Mercé , Regué Miguel , Tomás Juan M.
Published: July 2002

One representative recombinant clone encoding
Klebsiella pneumoniae
O5-antigen lipopolysaccharide (LPS) was found upon screening for serum resistance in a cosmid-based genomic library of
K. pneumoniae
KT769 (O5:K57) introduced into
Escherichia coli
DH5α. A total of eight open reading frames (
wb
O5
gene cluster) were necessary to produce
K. pneumoniae
O5-antigen LPS in
E. coli
K-12. The enzymatic activities proposed for the
wb
O5
gene cluster are in agreement with the activities proposed for the biosynthesis of
K. pneumoniae
O5-antigen LPS. Using the complete DNA sequence of the
K. pneumoniae wb
O5
gene cluster, we obtained (by single or double recombination) genetically well-characterized mutants devoid only of this O5-antigen LPS. Finally, using these O5
−
mutants and the corresponding wild-type strains or complemented mutants with the
wb
O5
gene cluster (O5
+
strains), we found that the presence of
K. pneumoniae
O5-antigen LPS is essential for some pathogenic features like serum resistance, adhesion to uroepithelial cells, and colonization (experimental infections) of the urinary tract in rats.
Interactions between Periodontal Bacteria and Human Oral Epithelial Cells:
Fusobacterium nucleatum
Adheres to and Invades Epithelial Cells
Authors: Han Yiping W. , Shi Wenyuan , Huang George T.-J. , Kinder Haake Susan , Park No-Hee , Kuramitsu Howard , Genco Robert J.
Published: July 2002

Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including
Bacteroides forsythus
,
Campylobacter curvus
,
Eikenella corrodens
,
Fusobacterium nucleatum
,
Porphyromonas gingivalis
, and
Prevotella intermedia
, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that
F. nucleatum
adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of
F. nucleatum
were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a “zipping” mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by
F. nucleatum
. A spontaneous mutant,
lam
, of
F. nucleatum
, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of
F. nucleatum
to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of
F. nucleatum
in periodontal infections.
Bile Mediates Intestinal Pathology in Endotoxemia in Rats
Authors: Jackson Graham D. F. , Dai Yung , Sewell William A.
Published: July 2002

Intestinal pathology frequently accompanies experimental endotoxic shock and is mediated by proinflammatory cytokines. Our hypotheses are that hepatobiliary factors operating from the luminal side of the gut make a major contribution to this damage and that tumor necrosis factor alpha (TNF-α) is involved in the pathology. We treated rats with lipopolysaccharide (LPS) intravenously and found that external drainage of bile totally protected the gastrointestinal tract, macroscopically and microscopically, 4 h after LPS administration and dramatically improved survival of the animals for 48 h after LPS administration. The concentration of TNF-α in bile increased markedly after LPS administration and was over 30 times higher in bile than in serum. Tissue damage and the biliary TNF-α response were abrogated when animals were pretreated with gadolinium chloride to eliminate Kupffer cells. TNF-α infusion into the duodenal lumen caused intestinal damage similar to that elicited by intravenous LPS. In rats treated with LPS, survival was significantly increased during the first 36 h in animals given an infusion of anti-TNF-α antibody into the duodenum. These results demonstrate that in endotoxemia, intestinal damage is mediated by factors derived from the bile. The findings indicate that luminally acting TNF-α contributes to the intestinal damage.
Enhancement of Clearance of Bacteria from Murine Lungs by Immunization with Detoxified Lipooligosaccharide fromMoraxella catarrhalisConjugated to Proteins
Authors: Hu Wei-Gang , Chen Jing , Battey James F. , Gu Xin-Xing
Published: July 2002

Moraxella catarrhalisstrain 25238 detoxified lipooligosaccharide (dLOS)-protein conjugates induced a significant rise of bactericidal anti-LOS antibodies in animals. This study reports the effect of active or passive immunization with the conjugates or their antiserum on pulmonary clearance ofM. catarrhalisin an aerosol challenge mouse model. Mice were injected subcutaneously with dLOS-tetanus toxoid (dLOS-TT), dLOS–high-molecular-weight proteins (dLOS-HMP) from nontypeableHaemophilus influenzae(NTHi), or nonconjugated materials in Ribi adjuvant and then challenged withM. catarrhalisstrain 25238 or O35E or NTHi strain 12. Immunization with dLOS-TT or dLOS-HMP generated a significant rise of serum anti-LOS immunoglobulin G and 68% and 35 to 41% reductions of bacteria in lungs compared with the control (P< 0.01) following challenge with homologous strain 25238 and heterologous strain O35E, respectively. Serum anti-LOS antibody levels correlated with its bactericidal titers againstM. catarrhalisand bacterial CFU in lungs. Additionally, immunization with dLOS-HMP generated a 54% reduction of NTHi strain 12 compared with the control (P< 0.01). Passive immunization with a rabbit antiserum against dLOS-TT conferred a significant reduction of strain 25238 CFU in lungs in a dose- and time-dependent pattern compared with preimmune serum-treated mice. Kinetic examination of lung tissue sections demonstrated that antiserum-treated mice initiated and offset inflammatory responses more rapidly than preimmune serum-treated mice. These data indicate that LOS antibodies (whether active or passive) play a major role in the enhancement of pulmonary clearance of different test strains ofM. catarrhalisin mice. In addition, dLOS-HMP is a potential candidate for a bivalent vaccine againstM. catarrhalisand NTHi infections.
Enterococcus faecalis
Adhesin, Ace, Mediates Attachment to Extracellular Matrix Proteins Collagen Type IV and Laminin as well as Collagen Type I
Authors: Nallapareddy Sreedhar R. , Qin Xiang , Weinstock George M. , Höök Magnus , Murray Barbara E.
Published: July 2002

Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most
Enterococcus faecalis
isolates, after growth at 46°C, to ECM proteins collagen types I and IV and laminin; identified an
E. faecalis
-specific gene,
ace
, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an
ace
disruption mutant from
E. faecalis
strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46°C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single ∼105-kDa band of mutanolysin extracts from OG1RF grown at 46°C, while no band was detected in extracts from OG1RF grown at 37°C, nor from the OG1RF
ace
mutant grown at 37 or 46°C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46°C-grown
E. faecalis
OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 μg/ml, and also inhibited the 46°C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of
E. faecalis
OG1RF to collagen type IV and laminin in addition to collagen type I.
Induction of Immune Response in BALB/c Mice with a DNA Vaccine Encoding Bacterioferritin or P39 ofBrucellaspp
Authors: Al-Mariri Ayman , Tibor Anne , Mertens Pascal , De Bolle Xavier , Michel Patrick , Godfroid Jacques , Walravens Karl , Letesson Jean-Jacques
Published: July 2002

In this study, we evaluated the ability of DNA vaccines encoding the bacterioferritin (BFR) or P39 proteins ofBrucellaspp. to induce cellular and humoral immune responses and to protect BALB/c mice against a challenge withB. abortus544. We constructed eukaryotic expression vectors called pCIBFR and pCIP39, encoding BFR or P39 antigens, respectively, and we verified that these proteins were produced after transfection of COS-7 cells. PCIBFR or pCIP39 was injected intramuscularly three times, at 3-week intervals. pCIP39 induced higher antibody responses than did the DNA vector encoding BFR. Both vectors elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the specific antigens orBrucellaextract. In this report, we also demonstrat that animals immunized with these plasmids elicited a strong and long-lived memory immune response which persisted at least 3 months after the third vaccination. Furthermore, pCIBFR and pCIP39 induced a typical T-helper 1-dominated immune response in mice, as determined by cytokine or immunoglobulin G isotype analysis. The pCIP39 delivered by intramuscular injection (but not the pCIBFR or control vectors) induced a moderate protection in BALB/c mice challenged withB. abortus544 compared to that observed in positive control mice vaccinated with S19.
Phenotypic and Genomic Analyses of the
Mycobacterium avium
Complex Reveal Differences in Gastrointestinal Invasion and Genomic Composition
Authors: McGarvey Jeffery A. , Bermudez Luiz E.
Published: July 2002

Mycobacterium avium
and
Mycobacterium intracellulare
are closely related organisms and comprise the
Mycobacterium avium
complex. These organisms share many common characteristics, including the ability to cause life-threatening respiratory infections in people with underlying lung pathology or immunological defects and occasionally in those with no known predisposing conditions. However, the ability to invade the mucosa of the gastrointestinal tract and cause disseminated disease in AIDS patients has not been epidemiologically linked to
M. intracellulare
and appears to be unique to
M. avium
. We compared the abilities of
M. avium
and
M. intracellulare
to tolerate the acidic conditions of the stomach, to resist the membrane-disrupting activity of cationic peptides, and to invade intestinal epithelial cells in vitro and in vivo. We observed that
M. avium
and
M. intracellulare
were both tolerant to the acidic conditions encountered in the stomach and resistant to cationic peptides. However, when strains of
M. avium
and
M. intracellulare
were examined for their ability to enter cultured human intestinal cells or mouse intestinal mucosa, we observed that
M. avium
could invade more efficiently than
M. intracellulare
. To elucidate the basis of this pathogenic difference and identify genes involved in the invasion of the intestinal mucosa, we performed chromosomal DNA subtractive hybridization using
M. avium
and
M. intracellulare
chromosomal DNAs. In all, 21 genes that were present in
M. avium
but absent in
M. intracellulare
were identified, including some that may be associated with the ability of
M. avium
to invade the intestinal mucosa.