bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous Extraction from Bacterioplankton of Total RNA and DNA Suitable for Quantitative Structure and Function Analyses
Authors: Weinbauer Markus G. , Fritz Ingo , Wenderoth Dirk F. , Höfle Manfred G.
Published: July 2002

The aim of this study was to develop a protocol for the simultaneous extraction from bacterioplankton of RNA and DNA suitable for quantitative molecular analysis. By using a combined mechanical and chemical extraction method, the highest RNA and DNA yield was obtained with sodium lauryl sarcosinate-phenol or DivoLab-phenol as the extraction mix. The efficiency of extraction of nucleic acids was comparatively high and varied only moderately in gram-negative bacterial isolates and bacterioplankton (RNA, 52 to 66%; DNA, 43 to 61%); significant amounts of nucleic acids were also obtained for a gram-positive bacterial isolate (RNA, 20 to 30%; DNA, 20 to 25%). Reverse transcription-PCR and PCR amplification products of fragments of 16S rRNA and its genes were obtained from all isolates and communities, indicating that the extracted nucleic acids were intact and pure enough for community structure analyses. By using single-strand conformation polymorphism of fragments of 16S rRNA and its gene, community fingerprints were obtained from pond bacterioplankton. mRNA transcripts encoding fragments of the enzyme nitrite reductase gene (
nir
gene) could be detected in a pond water sample, indicating that the extraction method is also suitable for studying gene expression. The extraction method presented yields nucleic acids that can be used to perform structural and functional studies of bacterioplankton communities from a single sample.
Diversity, Dynamics, and Activity of Bacterial Communities during Production of an Artisanal Sicilian Cheese as Evaluated by 16S rRNA Analysis
Authors: Randazzo Cinzia L. , Torriani Sandra , Akkermans Antoon D. L. , de Vos Willem M. , Vaughan Elaine E.
Published: July 2002

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and
Lactobacillus
group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of
Leuconostoc
,
Lactococcus lactis
, and
Macrococcus caseolyticus
were dominant in the raw milk, while
Streptococcus thermophilus
prevailed during lactic fermentation. Other thermophilic LAB, especially
Lactobacillus delbrueckii
and
Lactobacillus fermentum
, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the
Lactobacillus delbrueckii
species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.
Development and Characterization of a Green Fluorescent Protein-Based Bacterial Biosensor for Bioavailable Toluene and Related Compounds
Authors: Stiner Lawrence , Halverson Larry J.
Published: July 2002

A green fluorescent protein-based
Pseudomonas fluorescens
strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (
tbu
) pathway transcriptional activator TbuT from
Ralstonia pickettii
PKO1 and a transcriptional fusion of its promoter
PtbuA1
with a promoterless
gfp
gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (
nptII
) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of
gfp
expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of
gfp
expression per cell increases until saturation kinetics of the TbuT-PtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with
K
app
values that ranged from 3.3 ± 1.8 μM for toluene to 35.6 ± 16.6 μM for trichloroethylene (means ± standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di- and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants.
Fusarium Tri8
Encodes a Trichothecene C-3 Esterase
Authors: McCormick Susan P. , Alexander Nancy J.
Published: July 2002

Mutant strains of
Fusarium graminearum
Z3639 produced by disruption of
Tri8
were altered in their ability to biosynthesize 15-acetyldeoxynivalenol and instead accumulated 3,15-diacetyldeoxynivalenol, 7,8-dihydroxycalonectrin, and calonectrin.
Fusarium sporotrichioides
NRRL3299
Tri8
mutant strains accumulated 3-acetyl T-2 toxin, 3-acetyl neosolaniol, and 3,4,15-triacetoxyscirpenol rather than T-2 toxin, neosolaniol, and 4,15-diacetoxyscirpenol. The accumulation of these C-3-acetylated compounds suggests that
Tri8
encodes an esterase responsible for deacetylation at C-3. This gene function was confirmed by cell-free enzyme assays and feeding experiments with yeast expressing
Tri8.
Previous studies have shown that
Tri101
encodes a C-3 transacetylase that acts as a self-protection or resistance factor during biosynthesis and that the presence of a free C-3 hydroxyl group is a key component of
Fusarium
trichothecene phytotoxicity. Since
Tri8
encodes the esterase that removes the C-3 protecting group, it may be considered a toxicity factor.
Distribution of Membrane Lipids of Planktonic
Crenarchaeota
in the Arabian Sea
Authors: Sinninghe Damsté Jaap S. , Rijpstra W. Irene C. , Hopmans Ellen C. , Prahl Fredrick G. , Wakeham Stuart G. , Schouten Stefan
Published: July 2002

Intact core tetraether membrane lipids of marine planktonic
Crenarchaeota
were quantified in water column-suspended particulate matter obtained from four depth intervals (∼70, 500, 1,000 and 1,500 m) at seven stations in the northwestern Arabian Sea to investigate the distribution of the organisms at various depths. Maximum concentrations generally occurred at 500 m, near the top of the oxygen minimum zone, and the concentrations at this depth were, in most cases, slightly higher than those in surface waters. In contrast, lipids derived from eukaryotes (cholesterol) and from eukaryotes and bacteria (fatty acids) were at their highest concentrations in surface waters. This indicates that these crenarchaeotes are not restricted to the photic zone of the ocean, which is consistent with the results of recent molecular biological studies. Since the Arabian Sea has a strong oxygen minimum zone between 100 and 1,000 m, with minimum oxygen levels of <1 μM, the abundance of crenarchaeotal membrane lipids at 500 m suggests that planktonic
Crenarchaeota
are probably facultative anaerobes. The cell numbers we calculated from the concentrations of membrane lipids are similar to those reported for the Central Pacific Ocean, supporting the recent estimation of M. B. Karner, E. F. DeLong, and D. M. Karl (

Nature
409
:
507
-510,
2001

) that the world's oceans contain ca. 10
28
cells of planktonic
Crenarchaeota
.
Diversity of Hepatitis C Virus Quasispecies Evaluated by Denaturing Gradient Gel Electrophoresis
Authors: Harris K. A. , Teo C. G.
Published: July 2002

Denaturing gradient gel electrophoresis (DGGE) was used to study the diversity of hepatitis C virus (HCV) quasispecies. Optimized DGGE running conditions were applied to screen for variations in sequences cloned from amplicons originating from the nonstructural 5b (NS5b) gene of HCV in blood of hemophilia patients, intravenous drug users, and blood donors (five specimens from each study group, ca. 40 clones studied per specimen). Clones identified by DGGE as unique were sequenced. NS5b sequence entropy and mean genetic distance in hemophiliacs did not differ significantly from those in the other groups, pointing to a lack of correlation between HCV diversity and the multiplicity of past HCV exposures. DGGE was also applied to investigate variation in the HCV envelope 2/hypervariable region 1 (E2/HVR-1) in serum samples serially taken from two patients during the seroconversion phase of HCV infection. E2/HVR-1 sequence entropy changes were small and not correlated with rising anti-HCV antibody levels, reflecting mutational changes not mediated by antibody selection.
Human T-Cell Responses to the Glucosyltransferases ofStreptococcus mutans
Authors: Chia Jean-San , You Chiou-Mien , Hu Chung-Yi , Chiang Bor-Luen , Chen Jen-Yang
Published: July 2002

We previously reported differential humoral responses to glucosyltransferases (GTFs), with significantly higher saliva and serum antibody levels to GtfD than to GtfB or GtfC. To test the hypothesis that cellular immune responses to these molecules also may differ, peripheral blood mononuclear cell (PBMC) and T-cell proliferative responses in young adults and children with distinct genetic backgrounds were determined using purified recombinant GtfC and GtfD. PBMCs from all of the volunteers responded to GtfC and -D, but responses were directed predominantly towards GtfD and were major histocompatibility class II antigen dependent. A predominant T-cell response to GtfD, over GtfC, was detectable at various antigen concentrations ranging from 1 to 20 μg/ml and correlated with the differential serum immunoglobulin G (IgG) and salivary IgA antibody responses to the GTFs. Therefore, in naturally sensitized humans,Streptococcus mutansGTFs stimulate differential humoral and cellular immune responses, with the secreted form of GtfD eliciting a stronger response than the cell wall-associated form of GtfC.
Antigen-Driven T-Cell Selection in Patients with Cervical Cancer as Evidenced by T-Cell Receptor Analysis and Recognition of Autologous Tumor
Authors: Pilch H. , Höhn H. , Neukirch C. , Freitag K. , Knapstein P. G. , Tanner B. , Maeurer M. J.
Published: July 2002

We characterized the T-cell receptor (TCR) repertoire in freshly harvested tumor lesions, in short-term-expanded CD4+tumor infiltrating lymphocytes (TIL) as well as in CD4+and CD8+peripheral blood lymphocytes (PBL) from three patients with cervical cancer. Skewing of the T-cell repertoire as defined by measuring the length of the complementarity-determining region 3 (CDR3) of the TCR VA and VB chains was observed in CD8+PBL, in freshly harvested tumor tissue, as well as in CD4+TIL. Comparative analysis of the TCR repertoire revealed unique monoclonal TCR transcripts within the tumor lesion which were not present in PBL, suggesting selection of TCR clonotypes due to antigenic stimulation. TCR repertoire analysis of the short-term (7-day) CD4+TIL lines revealed that the TCR composition is markedly different from that in CD4+PBL or in the freshly harvested tumor tissue. Only one-third of CD4+TIL lines showed HLA-DR-restricted recognition of autologous tumor cells as defined by cytolysis. These data provide support for the antigen-driven selection of T cells within cervical cancer lesions and suggest that analysis of the TCR repertoire may aid in obtaining an objective description of the immune response in patients with cervical cancer who are undergoing epitope-based immunotherapy.
Endotoxin-Induced Gamma Interferon Production: Contributing Cell Types and Key Regulatory Factors
Authors: Varma Tushar K. , Lin Cheng Y. , Toliver-Kinsky Tracy E. , Sherwood Edward R.
Published: July 2002

Gamma interferon (IFN-γ) is an important mediator of endotoxin (lipopolysaccharide [LPS])-induced immune responses. However, the specific cell types that produce IFN-γ in response to LPS and the cellular factors that regulate LPS-induced IFN-γ production have not been fully determined. The present studies were undertaken to characterize the cell populations that produce IFN-γ after LPS challenge in the spleens of mice and to determine the regulatory factors that modulate LPS-induced production of IFN-γ. Our studies show that the levels of splenic IFN-γ mRNA and protein production peak at 6 and 8 h, respectively, after systemic LPS challenge. Approximately 60% of IFN-γ-producing cells are natural killer (NK) cells (CD3−DX5+) and 25% are NKT cells (CD3+DX5+). Most of the remaining IFN-γ-producing cells are T cells (CD3+DX5−), macrophages, and dendritic cells. Functionally, interleukin-12 (IL-12) is the major IFN-γ-stimulating factor after LPS challenge, with costimulation provided by IL-15, IL-18, and B7 proteins. IL-10 is a major inhibitor of LPS-induced IFN-γ production. Unlike intact heat-killed gram-negative and gram-positive bacteria, the class II major histocompatibility complex did not play a functional role in LPS-induced IFN-γ production. LPS is a potent stimulus for splenic IL-10, IL-12 p40, and IL-15 mRNA expression, whereas IL-12 p35 and IL-18 mRNAs, as well as B7 proteins, are constitutively expressed in the mouse spleen. Of the factors studied, IL-18 serves as the most potent costimulus with IL-12 for IFN-γ production, followed by IL-15 and B7 proteins. These data demonstrate that NK cells and NKT cells are the most abundant IFN-γ-producing cells in the mouse spleen after LPS challenge and that IL-10 and IL-12 are key functional regulators of LPS-induced IFN-γ production.
Comparison of O-Antigen Gene Clusters of
Escherichia coli
(
Shigella
) Sonnei and
Plesiomonas shigelloides
O17: Sonnei Gained Its Current Plasmid-Borne O-Antigen Genes from
P. shigelloides
in a Recent Event
Authors: Shepherd James G. , Wang Lei , Reeves Peter R.
Published: July 2002

Escherichia coli
Sonnei has an O antigen identical to that of
Plesiomonas shigelloides
O17, and its O-antigen gene cluster is located on a plasmid. By sequencing the chromosomal O-antigen gene cluster of
P. shigelloides
O17 and comparing it with that of Sonnei, we showed that Sonnei gained its O-antigen genes recently.
Generation of a Recombinant 65-Kilodalton Mannoprotein, a Major Antigen Target of Cell-Mediated Immune Response toCandida albicans
Authors: La Valle Roberto , Sandini Silvia , Gomez Maria Jesus , Mondello Francesca , Romagnoli Giulia , Nisini Roberto , Cassone Antonio
Published: July 2002

A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogenCandida albicans. An expression library ofC. albicanswas screened with serum from mice immunized with ScMp65 (ScW10), aSaccharomyces cerevisiaerecombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract ofC. albicans. After cloning and sequencing of the relevantC. albicanscDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designatedCaMP65) was localized to chromosome R ofC. albicansas determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His6-tagged protein (rCaMp65) was expressed inEscherichia coliunder an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4+T-cell clones were generated using aC. albicansmannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinantC. albicansMp65 but not to ScMp65. Thus, the recombinant Mp65 ofC. albicansretains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.
Modulation of Cytokine Profiles by the Mycoplasma SuperantigenMycoplasma arthritidisMitogen Parallels Susceptibility to Arthritis Induced byM. arthritidis
Authors: Mu Hong-Hua , Sawitzke Allen D. , Cole Barry C.
Published: July 2002

Mycoplasma arthritidismitogen (MAM) is a potent superantigen secreted byM. arthritidis, an agent of murine arthritis. Here we compare the abilities of MAM to induce a panel of cytokines in vitro and in vivo in BALB/c and C3H/HeJ mouse strains that differ in susceptibility to mycoplasmal arthritis. Splenocytes from both mouse strains produced high levels of all cytokines by 24 h following in vitro exposure to MAM. No differences in cytokine profiles were seen irrespective of the MAM dose. However, there were striking differences in cytokine profiles present in supernatants of splenocytes that had been collected from mice after intravenous (i.v.) injection of MAM and subsequently rechallenged with MAM in vitro. Splenocytes collected 24 and 72 h after i.v. injection of MAM and challenged in vitro with MAM showed the most marked divergence in the secreted cytokines. Type 1 cytokines were markedly elevated in C3H/HeJ cell supernatants, whereas they were depressed or remained low in BALB/c cell supernatants. In contrast, the levels of type 2 cytokines were all greatly increased in BALB/c cell cultures but were decreased or remained low in C3H/HeJ supernatants. Interleukin-12 mRNA and protein was also markedly elevated in C3H/HeJ mice, as were the levels of immunoglobulin G2a. The data indicate a major skewing in cytokine profiles to a type 1 inflammatory response in C3H/HeJ mice but to a protective type 2 response in BALB/c mice. These cytokine changes appear to be associated with the severe arthritis in C3H/HeJ mice following injection ofM. arthritidisin comparison to the mild disease seen in injected BALB/c mice.
Genetic Resistance to Experimental Infection with
Mycobacterium bovis
in Red Deer (
Cervus elaphus
)
Authors: Mackintosh Colin G. , Qureshi Tariq , Waldrup Ken , Labes Robert E. , Dodds Ken G. , Griffin J. Frank T.
Published: July 2002

Tuberculosis (Tb) caused by
Mycobacterium bovis
is a worldwide threat to livestock and humans. One control strategy is to breed livestock that are more resistant to
Mycobacterium bovis
. In a 3-year heritability study 6 farmed red deer stags were selected from 39 on the basis of their differing responses to experimental challenge via the tonsillar sac with approximately 500 CFU of
M. bovis
. Two stags remained uninfected, two were moderately affected, and two developed serious spreading Tb. Seventy offspring, bred from these six stags by artificial insemination using stored semen, were similarly challenged with
M. bovis
. The offspring showed patterns of response to
M. bovis
challenge similar to those of their sires, providing evidence for a strong genetic basis to resistance to Tb, with an estimated heritability of 0.48 (standard error, 0.096;
P
< 0.01). This is the first time the heritability of Tb resistance in domestic livestock has been measured. The breeding of selection lines of resistant and susceptible deer will provide an ideal model to study the mechanisms of Tb resistance in a ruminant and could provide an additional strategy for reducing the number and severity of outbreaks of Tb in farmed deer herds. Laboratory studies to identify genetic and immunological markers for resistance to Tb are under way. Preliminary studies showed no associations between NRAMP or DRB genes and resistance to Tb in deer. Patterns of immune responses seen in resistant animals suggest that both innate and acquired pathways of immunity are necessary to produce the resistant phenotype.
Enhanced Hematopoietic Activity Accompanies Parasite Expansion in the Spleen and Bone Marrow of Mice Infected withLeishmania donovani
Authors: Cotterell Sara E. J. , Engwerda Christian R. , Kaye Paul M.
Published: July 2002

In this study, we have analyzed hematopoietic activity in the spleen, bone marrow, and blood of BALB/c andscidmice infected withLeishmania donovani.Our analysis demonstrates that infection induces a rapid but transient mobilization of progenitor cells into the circulation, associated with elevated levels of granulocyte/macrophage colony-stimulating factor (GM-CSF) and MIP-1α. From 14 to 28 days postinfection, when parasite expansion begins in the spleen and bone marrow, both the frequency and cell cycle activity of hematopoietic progenitors, particulary CFU-granulocyte, monocyte, are dramatically increased in these organs. This is associated with increased accumulation of mRNA for GM-CSF, M-CSF, and G-CSF, but not interleukin-3. Our data also illustrate that hematopoietic activity, as assessed by changes in the frequency of progenitor cell populations and their levels of cell cycle activity, can be regulated in both a T-cell-independent and T-cell-dependent, as well as in an organ-specific, manner. Collectively, these data add to our knowledge of the long-term changes which occur in organs in whichL. donovaniis able to persist.
Immunity to Onchocerciasis: Cells from Putatively Immune Individuals Produce Enhanced Levels of Interleukin-5, Gamma Interferon, and Granulocyte-Macrophage Colony-Stimulating Factor in Response toOnchocerca volvulusLarval and Male Worm Antigens
Authors: Turaga Prasad S. D. , Tierney Tracy J. , Bennett Kristine E. , McCarthy Maggie C. , Simonek Scott C. , Enyong Peter A. , Moukatte Daniel W. , Lustigman Sara
Published: July 2002

Antigen-specific interleukin-5 (IL-5), gamma interferon (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) responses in individuals living in an area of hyperendemicity for onchocerciasis in Cameroon were examined. The responses against antigens prepared fromOnchocerca volvulusthird-stage larvae (L3), molting L3 (mL3), and crude extract from adult males (M-OvAg) were compared to the responses against antigens from adult female worms and skin microfilariae. Cytokine responses for the putatively immune individuals (PI) and the infected individuals (INF) were compared. A differential cytokine profile of IL-5 (Th2 phenotype) and IFN-γ (Th1 phenotype) was found in these individuals in response to the antigens. In both the PI and the INF, Th2 responses against all the antigens tested were dominant. However, in the PI group as a whole, there was an enhanced Th2 response against the larval antigens and the adult male and adult female antigens, and a Th1 response in a subgroup of the PI (27 to 54.5%) against L3, mL3, and M-OvAg antigens was present. While the PI produced significantly higher levels of GM-CSF against L3, mL3, and M-OvAg antigens than the INF, there was no difference in the GM-CSF responses of the groups against the other antigens. The present study indicated that, in comparison to the INF, the PI have distinct larva-specific and adult male-specific cytokine responses, thus supporting the premise that immunological studies of the PI would lead to the identification of immune mechanisms and the target genes that play a role in protective immunity.
Brucella abortusand Its Closest Phylogenetic Relative,Ochrobactrumspp., Differ in Outer Membrane Permeability and Cationic Peptide Resistance
Authors: Velasco J. , Bengoechea J. A. , Brandenburg K. , Lindner B. , Seydel U. , González D. , Zähringer U. , Moreno E. , Moriyón I.
Published: July 2002

The outer membrane (OM) of the intracellular parasiteBrucella abortusis permeable to hydrophobic probes and resistant to destabilization by polycationic peptides and EDTA. The significance of these unusual properties was investigated in a comparative study with the opportunistic pathogens of the genusOchrobactrum, the closest knownBrucellarelative.Ochrobactrumspp. OMs were impermeable to hydrophobic probes and sensitive to polymyxin B but resistant to EDTA. These properties were traced to lipopolysaccharide (LPS) because (i) insertion ofB. abortusLPS, but not ofEscherichia coliLPS, intoOchrobactrumOM increased its permeability; (ii) permeability and polymyxin B binding measured with LPS aggregates paralleled the results with live bacteria; and (iii) the predicted intermediate results were obtained withB. abortus-Ochrobactrum anthropiandE. coli-O. anthropiLPS hybrid aggregates. AlthoughOchrobactrumwas sensitive to polymyxin, self-promoted uptake and bacterial lysis occurred without OM morphological changes, suggesting an unusual OM structural rigidity.OchrobactrumandB. abortusLPSs showed no differences in phosphate, qualitative fatty acid composition, or acyl chain fluidity. However,OchrobactrumLPS, but notB. abortusLPS, contained galacturonic acid.B. abortusandOchrobactrumsmooth LPS aggregates had similar size and zeta potential (−12 to −15 mV). Upon saturation with polymyxin, zeta potential became positive (1 mV) forOchrobactrumsmooth LPS while remaining negative (−5 mV) forB. abortussmooth LPS, suggesting hindered access to inner targets. These results show that althoughOchrobactrumandBrucellashare a basic OM pattern, subtle modifications in LPS core cause markedly different OM properties, possibly reflecting the adaptive evolution ofB. abortusto pathogenicity.
In Vitro Cell Invasion of
Mycoplasma gallisepticum
Authors: Winner Florian , Rosengarten Renate , Citti Christine
Published: July 2002

The ability of the widespread avian pathogen
Mycoplasma gallisepticum
to invade cultured human epithelial cells (HeLa-229) and chicken embryo fibroblasts (CEF) was investigated by using the gentamicin invasion assay and a double immunofluorescence microscopic technique for accurate localization of cell-associated mycoplasmas. The presence of intracellular mycoplasmas in both cell lines was clearly demonstrated, with organisms entering the eukaryotic cells within 20 min. Internalized mycoplasmas have the ability to leave the cell, but also to survive within the intracellular space over a 48-h period. Frequencies of invasion were shown to differ between the two cell lines, but were also considerably dependent on the mycoplasma input population. Of the prototype strain R, a low-passage population in artificial medium, R
low
, was capable of active cell invasion, while a high-passage population, R
high
, showed adherence to but nearly no uptake into HeLa-229 and CEF. By passaging R
low
and R
high
multiple times through HeLa-229 cells, the invasion frequency was significantly increased. Taken together, these findings demonstrate that
M. gallisepticum
has the capability of entering nonphagocytic host cells that may provide this pathogen with the opportunity for resisting host defenses and selective antibiotic therapy, establishing chronic infections, and passing through the respiratory mucosal barrier to cause systemic infections.
Salmonella enterica
Serovar Typhimurium
waaP
Mutants Show Increased Susceptibility to Polymyxin and Loss of Virulence In Vivo
Authors: Yethon Jeremy A. , Gunn John S. , Ernst Robert K. , Miller Samuel I. , Laroche Line , Malo Danielle , Whitfield Chris
Published: July 2002

In
Escherichia coli
, the
waaP
(
rfaP
) gene product was recently shown to be responsible for phosphorylation of the first heptose residue of the lipopolysaccharide (LPS) inner core region. WaaP was also shown to be necessary for the formation of a stable outer membrane. These earlier studies were performed with an avirulent rough strain of
E. coli
(to facilitate the structural chemistry required to properly define
waaP
function); therefore, we undertook the creation of a
waaP
mutant of
Salmonella enterica
serovar Typhimurium to assess the contribution of WaaP and LPS core phosphorylation to the biology of an intracellular pathogen. The
S. enterica waaP
mutant described here is the first to be both genetically and structurally characterized, and its creation refutes an earlier claim that
waaP
mutations in
S. enterica
must be leaky to maintain viability. The mutant was shown to exhibit characteristics of the deep-rough phenotype, despite its ability to produce a full-length core capped with O antigen. Further, phosphoryl modifications in the LPS core region were shown to be required for resistance to polycationic antimicrobials. The
waaP
mutant was significantly more sensitive to polymyxin in both wild-type and polymyxin-resistant backgrounds, despite the decreased negative charge of the mutant LPSs. In addition, the
waaP
mutation was shown to cause a complete loss of virulence in mouse infection models. Taken together, these data indicate that WaaP is a potential target for the development of novel therapeutic agents.
Antigen Detection in Enteropathogenic
Escherichia coli
Using Secretory Immunoglobulin A Antibodies Isolated from Human Breast Milk
Authors: Manjarrez-Hernandez H. A. , Gavilanes-Parra S. , Chavez-Berrocal E. , Navarro-Ocaña A. , Cravioto A.
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC) produces a characteristic attaching and effacing (A/E) lesion in the small intestines of infected children. The immune response to EPEC infection remains poorly characterized. The molecular targets that elicit protective immunity against EPEC disease are unknown. In this study protein antigens from EPEC were identified using secretory immunoglobulin A (sIgA) antibodies isolated from milk from Mexican women by Western blot analysis. Purified sIgA antibodies, which inhibit the adherence of EPEC to cells, reacted to many EPEC proteins, the most prominent of which were intimin (a 94-kDa outer membrane protein) and two unknown proteins with apparent molecular masses of 80 and 70 kDa. A culture supernatant protein of 110 kDa also reacted strongly with the sIgA antibodies. The molecular size of this protein and its reactivity with specific anti-EspC antiserum suggest that it is EPEC-secreted protein C (EspC). These EPEC surface protein antigens were consistently recognized by all the different sIgA samples obtained from 15 women. Screening of clinical isolates of various O serogroups from cases of severe infantile diarrhea revealed that all EPEC strains able to produce the A/E lesion showed expression of intimin and the 80- and 70-kDa proteins. Such proteins reacted strongly with the purified sIgA pool. Moreover, nonvirulent
E. coli
strains were unable to generate a sIgA response. The immunogenic capacities of the 80- and 70-kDa proteins as virulence antigens have not been previously reported. The strong sIgA response to intimin and the 80- and 70-kDa proteins obtained in this study indicates that such antigens stimulate intestinal immune responses and may elicit protective immunity against EPEC disease.
Babesia bovis-Stimulated Macrophages Express Interleukin-1β, Interleukin-12, Tumor Necrosis Factor Alpha, and Nitric Oxide and Inhibit Parasite Replication In Vitro
Authors: Shoda Lisl K. M. , Palmer Guy H. , Florin-Christensen Jorge , Florin-Christensen Monica , Godson Dale L. , Brown Wendy C.
Published: July 2002

The tick-transmitted hemoparasiteBabesia boviscauses an acute infection that results in persistence and immunity against challenge infection in cattle that control the initial parasitemia. Resolution of acute infection with this protozoal pathogen is believed to be dependent on products of activated macrophages (Mφ), including inflammatory cytokines and nitric oxide (NO) and its derivatives.B. bovisstimulates inducible nitric oxide synthase (iNOS) and production of NO in bovine Mφ, and chemical donors of NO inhibit the growth ofB. bovisin vitro. However, the induction of inflammatory cytokines in Mφ by babesial parasites has not been described, and the antiparasitic activity of NO produced byB. bovis-stimulated Mφ has not been definitively demonstrated. We report that monocyte-derived Mφ activated byB. bovisexpressed enhanced levels of inflammatory cytokines interleukin-1β (IL-1β), IL-12, and tumor necrosis factor alpha that are important for stimulating innate and acquired immunity against protozoal pathogens. Furthermore, a lipid fraction ofB. bovis-infected erythrocytes stimulated iNOS expression and NO production by Mφ. Cocultures of Mφ andB. bovis-infected erythrocytes either in contact or physically separated resulted in reduced parasite viability. However, NO produced by bovine Mφ in response toB. bovis-infected erythrocytes was only partially responsible for parasite growth inhibition, suggesting that additional factors contribute to the inhibition ofB. bovisreplication. These findings demonstrate thatB. bovisinduces an innate immune response that is capable of controlling parasite replication and that could potentially result in host survival and parasite persistence.
Analysis of Genes Expressed at the Infective Larval Stage Validates Utility of
Litomosoides sigmodontis
as a Murine Model for Filarial Vaccine Development
Authors: Allen Judith E. , Daub Jennifer , Guiliano David , McDonnell Amanda , Lizotte-Waniewski Michelle , Taylor David W. , Blaxter Mark
Published: July 2002

We used an expressed sequence tag approach to analyze genes expressed by the infective larvae of the rodent filarial parasite
Litomosoides sigmodontis
. One hundred fifty two new genes were identified, including several proposed as vaccine candidates in studies with human filarial parasites. Our findings have important implications for the use of
L. sigmodontis
as a model for filarial infection.
espC
Pathogenicity Island of Enteropathogenic
Escherichia coli
Encodes an Enterotoxin
Authors: Mellies Jay L. , Navarro-Garcia Fernando , Okeke Iruka , Frederickson Julie , Nataro James P. , Kaper James B.
Published: July 2002

At least five proteins are secreted extracellularly by enteropathogenic
Escherichia coli
(EPEC), a leading cause of infant diarrhea in developing countries. However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa. Here we determine that the
espC
gene is located within a second EPEC pathogenicity island at 60 min on the chromosome of
E. coli
. We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers. In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative
E. coli
eliminated EspC enterotoxin activity. Like the EAF plasmid, the
espC
pathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.
Genetic Diversity of
Pseudomonas aeruginosa
Strains Isolated from Ventilated Patients with Nosocomial Pneumonia, Cancer Patients with Bacteremia, and Environmental Water
Authors: Ruimy Raymond , Genauzeau Emmanuel , Barnabe Christian , Beaulieu Arnaud , Tibayrenc Michel , Andremont Antoine
Published: July 2002

Random amplified polymorphic DNA typing was used to study the genetic diversity of
Pseudomonas aeruginosa
strains from (i) ventilated patients with nosocomial pneumonia who were hospitalized in intensive care units, (ii) cases of bacteremia in cancer patients with severe neutropenia, and (iii) rivers and swimming pools. Genetic diversity was determined by three phylogenetic methods and by statistical analysis of population genetics. The population studied undergoes epidemic clonality with a high rate of genetic recombination.
P. aeruginosa
bacteremia and pneumonia are not caused by specific clones within this species.
CD4+T Lymphocytes from Calves Immunized withAnaplasma marginaleMajor Surface Protein 1 (MSP1), a Heteromeric Complex of MSP1a and MSP1b, Preferentially Recognize the MSP1a Carboxyl Terminus That Is Conserved among Strains
Authors: Brown Wendy C. , Palmer Guy H. , Lewin Harris A. , McGuire Travis C.
Published: July 2002

Native major surface protein 1 (MSP1) of the ehrlichial pathogenAnaplasma marginaleinduces protective immunity in calves challenged with homologous and heterologous strains. MSP1 is a heteromeric complex of a single MSP1a protein covalently associated with MSP1b polypeptides, of which at least two (designated MSP1F1 and MSP1F3) in the Florida strain are expressed. Immunization with recombinant MSP1a and MSP1b alone or in combination fails to provide protection. The protective immunity in calves immunized with native MSP1 is associated with the development of opsonizing and neutralizing antibodies, but CD4+T-lymphocyte responses have not been evaluated. CD4+T lymphocytes participate in protective immunity to ehrlichial pathogens through production of gamma interferon (IFN-γ), which promotes switching to high-affinity immunoglobulin G (IgG) and activation of phagocytic cells to produce nitric oxide. Thus, an effective vaccine forA. marginaleand related organisms should contain both T- and B-lymphocyte epitopes that induce a strong memory response that can be recalled upon challenge with homologous and heterologous strains. This study was designed to determine the relative contributions of MSP1a and MSP1b proteins, which contain both variant and conserved amino acid sequences, in stimulating memory CD4+T-lymphocyte responses in calves immunized with native MSP1. Peripheral blood mononuclear cells and CD4+T-cell lines from MSP1-immunized calves proliferated vigorously in response to the immunizing strain (Florida) and heterologous strains ofA. marginale. The conserved MSP1-specific response was preferentially directed to the carboxyl-terminal region of MSP1a, which stimulated high levels of IFN-γ production by CD4+T cells. In contrast, there was either weak or no recognition of MSP1b proteins. Paradoxically, all calves developed high titers of IgG antibodies to both MSP1a and MSP1b polypeptides. These findings suggest that in calves immunized with MSP1 heteromeric complex, MSP1a-specific T lymphocytes may provide help to MSP1b-specific B lymphocytes. The data provide a basis for determining whether selected MSP1a CD4+T-lymphocyte epitopes and selected MSP1a and MSP1b B-lymphocyte epitopes presented on the same molecule can stimulate a protective immune response.
Structural Analysis and Antibody Response to the Extracellular GlutathioneS-Transferases fromOnchocerca volvulus
Authors: Sommer Alexandra , Nimtz Manfred , Conradt Harald S. , Brattig Norbert , Boettcher Kay , Fischer Peter , Walter Rolf D. , Liebau Eva
Published: July 2002

Onchocerca volvulusis a human pathogenic filarial parasite which, like other parasitic nematodes, is capable of surviving in an immunologically competent host by employing a variety of immune evasion strategies and defense mechanisms including the detoxification and repair mechanisms of the glutathioneS-transferases (GSTs). In this study we analyzed the glycosylation pattern and the immunological properties of extracellularO. volvulusGST1a and -1b (OvGST1a and -1b). The enzymes differ in only 10 amino acids, and both are glycoproteins that have cleavable signal peptides and unusual N-terminal extensions. These characteristics have not been described for other GSTs so far. Mass spectrometry analyses indicate that both enzymes carry high-mannose type oligosaccharides on at least four glycosylation sites. Glycosylation sites 1 to 3 ofOvGST1a (OvGST1b sites 2 to 4) are occupied by truncated N-glycans (Man2GlcNAc2 to Man5GlcNAc2), and N glycosylation site 4 ofOvGST1a (OvGST1b site 5) carries Man5GlcNAc2 to Man9GlcNAc2. To analyze the capacity of these secretory GSTs to stimulate host immune responses, we studied the antibody responses of onchocerciasis patients against the native affinity-purifiedOvGST1a and -1b. By enzyme-linked immunosorbent assay we showed thatOvGST1a and -1b are immunodominant antigens, with less than 7% nonresponder patients. A direct comparison of the antibody responses to the glycosylated and deglycosylated forms demonstrates the high immunogenicity of the N-glycans. Analyses of the antibody responses to the unusual N-terminal extension show an enhanced recognition of this portion by patients as opposed to recognition of the recombinant protein without extension.
Prevention ofPorphyromonas gingivalis-Induced Oral Bone Loss following Immunization with Gingipain R1
Authors: Gibson Frank C. , Genco Caroline Attardo
Published: July 2002

The arginine gingipains RgpA and RgpB ofPorphyromonas gingivalisare well-documented virulence factors of this organism. Structurally, RgpA and RgpB have nearly identical catalytic domains, while RgpA possesses an additional hemagglutinin domain. In this study, we examined the abilities of these proteins to elicit protection againstP. gingivalis-mediated oral bone loss in a murine oral challenge model. Mice immunized subcutaneously with heat-killedP. gingivalisor purified RgpA or RgpB possessed elevated levels ofP. gingivalis-specific immunoglobulin G; however, only the animals immunized withP. gingivaliswhole cells or RgpA were protected from maxillary bone loss. These data suggest that immunization with RgpA stimulates the production of hemagglutinin domain-specific antibodies, which contribute to the prevention ofP. gingivalis-mediated periodontal disease.
Toxin Synthesis and Mucin Breakdown Are Related to Swarming Phenomenon in
Clostridium septicum
Authors: Macfarlane Sandra , Hopkins Mark J. , Macfarlane George T.
Published: July 2002

Clostridium septicum
is responsible for several diseases in humans and animals. The bacterium is capable of a simple kind of multicellular behavior known as swarming. In this investigation, environmental and physiologic factors affecting growth and swarm cell formation in
C. septicum
were studied over a range of dilution rates (
D
= 0.02 to 0.65 h
−1
) in glucose-limited, glucose-excess, and mucin-limited chemostats. Cellular differentiation was observed at low specific growth rates, irrespective of the carbon and energy source, showing that swarming occurred in response to nutrient depletion. Differential expression of virulence determinants was detected in swarm cells. Hemolysin was secreted by short motile rods but not swarm cells, whereas in cultures grown with glucose, only swarm cells formed DNase, hyaluronidase, and neuraminidase. However, neuraminidase and, to a lesser degree, hyaluronidase were induced in short motile rods in mucin-limited cultures. Both swarm cells and short rods were cytotoxic to Vero cells. Mucin was chemotaxic to
C. septicum
, and large amounts of mucin-degrading enzymes (β-galactosidase,
N
-acetyl β-glucosaminidase, glycosulfatase, and neuraminidase) were produced. Synthesis of these enzymes was catabolite regulated. In chemostat experiments, glycosulfatase secretion occurred only in swarm cells at low dilution rates in mucin-limited cultures. Determinations of oligosaccharide utilization demonstrated that
N
-acetylglucosamine, galactose, and
N
-acetylgalactosamine were the main carbon sources for
C. septicum
in mucin. Neuraminic acid was not assimilated, showing that neuraminidase does not have a direct nutritional function in this pathogen.
Study of
Staphylococcus aureus
Pathogenic Genes by Transfer and Expression in the Less Virulent Organism
Streptococcus gordonii
Authors: Stutzmann Meier P. , Entenza J. M. , Vaudaux P. , Francioli P. , Glauser M. P. , Moreillon P.
Published: July 2002

Because
Staphylococcus aureus
strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific
S. aureus
genes in the less virulent organism
Streptococcus gordonii
and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive
S. gordonii
was more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (
P
< 0.05). Moreover, deleting
clfA
from
clfA
-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity of
clfA
-positive streptococci when both
clfA
and
coa
genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in
S. aureus
endovascular infections. This gain-of-function strategy might help solve the role of individual factors in the complex the
S. aureus
-host relationship.
Interleukin-4 Is Essential for the Control of Microfilariae in Murine Infection with the FilariaLitomosoides sigmodontis
Authors: Volkmann Lars , Saeftel Michael , Bain Odile , Fischer Kerstin , Fleischer Bernhard , Hoerauf Achim
Published: July 2002

Litomosoides sigmodontisis the only filaria which develops from infective larvae into microfilaria-producing adults in immunocompetent laboratory mice. In this study we report that interleukin-4 knockout (IL-4 KO) mice have an up to 100-fold-higher and a significantly prolonged microfilaremia compared to wild-type BALB/c mice, as well as 20 times more microfilariae in the thoracic cavity, the site of infection. While worm development and adult worm persistence were equivalent in IL-4 KO and wild-type mice, the fertility and length of adult female worms in IL-4 KO mice was clearly enhanced. The high susceptibility to microfilariae in IL-4 KO mice required the presence of adult worms in a full infection cycle since microfilariae loads did not differ much between IL-4 KO and wild-type mice when purified microfilariae were injected into mice. In addition, we found that eosinophilia was diminished and immunoglobulin E (IgE) was absent in IL-4 KO mice. IgE, however, does not seem to be the essential factor for microfilarial containment since microfilaremia was not elevated in B-cell KO mice. In conclusion, IL-4 is shown for the first time to be essential for the control of microfilarial loads but not of adult worm loads in a fully permissive murine filarial infection. IL-4 dependent effector pathways seem to operate on adult worms rather than directly on microfilariae.