bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutagenesis of Asp-569 of Glucosyltransferase I Glucansucrase Modulates Glucan and Oligosaccharide Synthesis
Authors: Monchois Vincent , Vignon Michel , Russell Roy R. B.
Published: July 2002

Glucansucrases of oral streptococci and
Leuconostoc mesenteroides
are enzymes of medical and biotechnological interest that synthesize α-glucans. They can also synthesize oligosaccharides in the presence of a sugar acceptor. Previous reports have identified an amino acid residue that may affect the structure of the glucan product; therefore, random mutagenesis of the corresponding Asp-569 of
Streptococcus downei
glucosyltransferase I (GTF-I) was used to further understanding of its involvement in the catalytic mechanism and to evaluate how different amino acids can modulate glucan and oligosaccharide synthesis. GTF-I variants were obtained where Asp-569 was replaced by each of the different possible classes of amino acids. These were expressed in
Escherichia coli
and purified by means of a His
6
tag. The results showed that the amino acid in position 569 influences the structure of the glucan and the size of the oligosaccharides produced by GTF-I. The results suggest that the amino acid occupying this position is more likely to interact with the acceptor molecules (oligosaccharides or elongating glucan chain) than to be directly involved in glucosyl transfer from sucrose. Engineering of the equivalent position in glucansucrases thus appears to be a good target to expand the range of oligosaccharides synthesized.
Characterization of an Atrazine-DegradingPseudaminobactersp. Isolated from Canadian and French Agricultural Soils
Authors: Topp Edward , Zhu Hong , Nour Sarah M. , Houot Sabine , Lewis Melanie , Cuppels Diane
Published: July 2002

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Fourteen bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from soils obtained from two farms in Canada and two farms in France. These strains were indistinguishable from each other based on repetitive extragenic palindromic PCR genomic fingerprinting performed with primers ERIC1R, ERIC2, and BOXA1R. Based on 16S rRNA sequence analysis of one representative isolate, strain C147, the isolates belong to the genusPseudaminobacterin the familyRhizobiaceae. Strain C147 did not form nodules on the legumes alfalfa (Medicago sativaL.), birdsfoot trefoil (Lotus corniculatusL.), red clover (Trifolium pratenseL.), chickpea (Cicer arietinumL.), and soybean (Glycine maxL.). A number of chloro-substituteds-triazine herbicides were degraded, but methylthio-substituteds-triazine herbicides were not degraded. Based on metabolite identification data, the fact that oxygen was not required, and hybridization of genomic DNA to theatzABCgenes, atrazine degradation occurred via a series of hydrolytic reactions initiated by dechlorination and followed by dealkylation. Most strains could mineralize [ring-U-14C]atrazine, and those that could not mineralize atrazine lackedatzBoratzBC. TheatzABCgenes, which were plasmid borne in every atrazine-degrading isolate examined, were unstable and were not always clustered together on the same plasmid. Loss ofatzBwas accompanied by loss of a copy of IS1071. Our results indicate that an atrazine-degradingPseudaminobactersp. with remarkably little diversity is widely distributed in agricultural soils and that genes of the atrazine degradation pathway carried by independent isolates of this organism are not clustered, can be independently lost, and may be associated with a catabolic transposon. We propose that the widespread distribution of the atrazine-degradingPseudaminobactersp. in agricultural soils exposed to atrazine is due to the characteristic ability of this organism to utilize alkylamines, and therefore atrazine, as sole sources of carbon when theatzABCgenes are acquired.
Correlation of Activities of the Enzymes α-Phosphoglucomutase, UDP-Galactose 4-Epimerase, and UDP-Glucose Pyrophosphorylase with Exopolysaccharide Biosynthesis by
Streptococcus thermophilus
LY03
Authors: Degeest Bart , De Vuyst Luc
Published: July 2002

The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture
Streptococcus thermophilus
LY03. This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses. Lactose and glucose were fermented by
S. thermophilus
LY03 only when they were used as sole energy and carbohydrate sources. Fructose was also fermented when it was applied in combination with lactose or glucose. Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances. A combination of lactose and glucose resulted in the largest amounts of EPS. Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of α-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for
S. thermophilus
LY03.
Quantitative Approach in the Study of Adhesion of Lactic Acid Bacteria to Intestinal Cells and Their Competition with Enterobacteria
Authors: Lee Y. K. , Lim C. Y. , Teng W. L. , Ouwehand A. C. , Tuomola E. M. , Salminen S.
Published: July 2002

To describe the phenomena of bacterial adhesion to intestinal cells and the competition for adhesion between bacteria, mathematical equations based on a simple dissociation process involving a finite number of bacterial receptors on intestinal cell surface were developed. The equations allow the estimation of the maximum number of
Lactobacillus
sp. and
Escherichia coli
cells that can adhere to Caco-2 cells and intestinal mucus; they also characterize the affinity of the bacteria to Caco-2 cells and intestinal and fecal mucus and the theoretical adhesion ratio of two bacteria present in a mixed suspension. The competition for adhesion between
Lactobacillus rhamnosus
GG and
E. coli
TG1 appeared to follow the proposed kinetics, whereas the competition between
Lactobacillus casei
Shirota and
E. coli
TG1 may involve multiple adhesion sites or a soluble factor in the culture medium of the former. The displacement of the adhered
Lactobacillus
by
E. coli
TG1 seemed to be a rapid process, whereas the displacement of
E. coli
TG1 by the
Lactobacillus
took more than an hour.
First Evidence of Lysogeny in
Propionibacterium freudenreichii
subsp.
shermanii
Authors: Hervé C. , Coste A. , Rouault A. , Fraslin J. M. , Gautier M.
Published: July 2002

Dairy propionic acid bacteria, particularly the species
Propionibacterium freudenreichii
, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting
P. freudenreichii
have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of
P. freudenreichii
was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of
P. freudenreichii
was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in
P. freudenreichii
.
TaqMan PCR for Detection of
Vibrio cholerae
O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Oysters, and Synthetic Seawater
Authors: Lyon W. J.
Published: July 2002

Vibrio cholerae
is recognized as a leading human waterborne pathogen. Traditional diagnostic testing for
Vibrio
is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, a TaqMan PCR assay is presented for quantitative detection of
V. cholerae
in pure cultures, oysters, and synthetic seawater. Primers and probe were designed from the nonclassical hemolysin (
hly
A) sequence of
V. cholerae
strains. This probe was applied to DNA from 60 bacterial strains comprising 21 genera. The TaqMan PCR assay was positive for all of the strains of
V. cholerae
tested and negative for all other species of
Vibrio
tested. In addition, none of the other genera tested was amplified with the TaqMan primers and probe used in this study. The results of the TaqMan PCR with raw oysters and spiked with
V. cholerae
serotypes O1 and O139 were comparable to those of pure cultures. The sensitivity of the assay was in the range of 6 to 8 CFU g
−1
and 10 CFU ml
−1
in spiked raw oyster and synthetic seawater samples, respectively. The total assay could be completed in 3 h. Quantification of the
Vibrio
cells was linear over at least 6 log units. The TaqMan probe and primer set developed in this study can be used as a rapid screening tool for the presence of
V. cholerae
in oysters and seawater without prior isolation and characterization of the bacteria by traditional microbiological methods.
Persistence of an Occlusion-Negative Recombinant Nucleopolyhedrovirus in
Trichoplusia ni
Indicates High Multiplicity of Cellular Infection
Authors: Bull James C. , Godfray H. C. J. , O'Reilly David R.
Published: July 2002

We use data from the serial passage of co-occluded recombinant
Autographa californica
nuclear polyhedrosis virus (Ac
M
NPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of Ac
M
NPV was constructed with a deletion of its
polyhedrin
gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type Ac
M
NPV and used to infect fifth-instar
Trichoplusia ni
larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 ± 0.3 viral genomes per occlusion body-producing cell.
Citrate Metabolism by
Enterococcus faecalis
FAIR-E 229
Authors: Sarantinopoulos Panagiotis , Kalantzopoulos George , Tsakalidou Effie
Published: July 2002

Citrate metabolism by
Enterococcus faecalis
FAIR-E 229 was studied in various growth media containing citrate either in the presence of glucose or lactose or as the sole carbon source. In skim milk (130 mM lactose, 8 mM citrate), cometabolism of citrate and lactose was observed from the first stages of the growth phase. Lactose was stoichiometrically converted into lactate, while citrate was converted into acetate, formate, and ethanol. When de Man-Rogosa-Sharpe (MRS) broth containing lactose (28 mM) instead of glucose was used,
E. faecalis
FAIR-E 229 catabolized only the carbohydrate. Lactate was the major end product, and small amounts of ethanol were also detected. Increasing concentrations of citrate (10, 40, 70, and 100 mM) added to MRS broth enhanced both the maximum growth rate of
E. faecalis
FAIR-E 229 and glucose catabolism, although citrate itself was not catabolized. Glucose was converted stoichiometrically into lactate, while small amounts of ethanol were produced as well. Finally, when increasing initial concentrations of citrate (10, 40, 70, and 100 mM) were used as the sole carbon sources in MRS broth without glucose, the main end products were acetate and formate. Small amounts of lactate, ethanol, and acetoin were also detected. This work strongly supports the suggestion that enterococcal strains have the metabolic potential to metabolize citrate and therefore to actively contribute to the flavor development of fermented dairy products.
Phylogenetic Analysis of Culturable Dimethyl Sulfide-Producing Bacteria from a
Spartina
-Dominated Salt Marsh and Estuarine Water
Authors: Ansede John H. , Friedman Robert , Yoch Duane C.
Published: July 2002

Dimethylsulfoniopropionate (DMSP), an abundant osmoprotectant found in marine algae and salt marsh cordgrass, can be metabolized to dimethyl sulfide (DMS) and acrylate by microbes having the enzyme DMSP lyase. A suite of DMS-producing bacteria isolated from a salt marsh and adjacent estuarine water on DMSP agar plates differed markedly from the pelagic strains currently in culture. While many of the salt marsh and estuarine isolates produced DMS and methanethiol from methionine and dimethyl sulfoxide, none appeared to be capable of producing both methanethiol and DMS from DMSP. DMSP, and its degradation products acrylate and β-hydroxypropionate but not methyl-3-mecaptopropionate or 3-mercaptopropionate, served as a carbon source for the growth of all the α- and β- but only some of the γ-proteobacterium isolates. Phylogenetic analysis of 16S rRNA gene sequences showed that all of the isolates were in the group
Proteobacteria
, with most of them belonging to the α and γ subclasses. Only one isolate was identified as a β-proteobacterium, and it had >98% 16S rRNA sequence homology with a terrestrial species of
Alcaligenes faecalis
. Although bacterial population analysis based on culturability has its limitations, bacteria from the α and γ subclasses of the
Proteobacteria
were the dominant DMS producers isolated from salt marsh sediments and estuaries, with the γ subclass representing 80% of the isolates. The α-proteobacterium isolates were all in the
Roseobacter
subgroup, while many of the γ-proteobacteria were closely related to the pseudomonads; others were phylogenetically related to
Marinomonas, Psychrobacter
, or
Vibrio
species. These data suggest that DMSP cleavage to DMS and acrylate is a characteristic widely distributed among different phylotypes in the salt marsh-estuarine ecosystem.
Acetate Oxidation Coupled to Fe(III) Reduction in Hyperthermophilic Microorganisms
Authors: Tor Jason M. , Kashefi Kazem , Lovley Derek R.
Published: July 2002

No hyperthermophilic microorganisms have previously been shown to anaerobically oxidize acetate, the key extracellular intermediate in the anaerobic oxidation of organic matter. Here we report that two hyperthermophiles,
Ferroglobus placidus
and “
Geoglobus ahangari
,” grow at 85°C by oxidizing acetate to carbon dioxide, with Fe(III) serving as the electron acceptor. These results demonstrate that acetate could potentially be metabolized within the hot microbial ecosystems in which hyperthermophiles predominate, rather than diffusing to cooler environments prior to degradation as has been previously proposed.
Comparison of Different Approaches To Quantify
Staphylococcus aureus
Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese
Authors: Hein Ingeborg , Lehner Angelika , Rieck Petra , Klein Kurt , Brandl Ernst , Wagner Martin
Published: July 2002

Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of
Staphylococcus aureus
cells by targeting the thermonuclease (
nuc
) gene. Purified DNA extracts from pure cultures of
S. aureus
were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60
nuc
gene copies/μl) than using a fluorigenic TaqMan probe (6
nuc
gene copies/μl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify
S. aureus
cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 × 10
2
to 6.4 × 10
2
copies of the
nuc
gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and
nuc
gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of
S. aureus
in cheese by performing RTQ-PCR.
Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells
Authors: Lim Eelin L. , Tomita Aoy V. , Thilly William G. , Polz Martin F.
Published: July 2002

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate
Cafeteria roenbergensis
as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated
C. roenbergensis
cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 10
4
cells ml
−1
. The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells ml
−1
by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples.
Nitrous Oxide Formation in the Colne Estuary, England: the Central Role of Nitrite
Authors: Dong Liang F. , Nedwell David B. , Underwood Graham J. C. , Thornton Daniel C. O. , Rusmana Iman
Published: July 2002

Nitrate and nitrite concentrations in the water and nitrous oxide and nitrite fluxes across the sediment-water interface were measured monthly in the River Colne estuary, England, from December 1996 to March 1998. Water column concentrations of N2O in the Colne were supersaturated with respect to air, indicating that the estuary was a source of N2O for the atmosphere. At the freshwater end of the estuary, nitrous oxide effluxes from the sediment were closely correlated with the nitrite concentrations in the overlying water and with the nitrite influx into the sediment. Increases in N2O production from sediments were about 10 times greater with the addition of nitrite than with the addition of nitrate. Rates of denitrification were stimulated to a larger extent by enhanced nitrite than by nitrate concentrations. At 550 μM nitrite or nitrate (the highest concentration used), the rates of denitrification were 600 μmol N · m−2· h−1with nitrite but only 180 μmol N · m−2· h−1with nitrate. The ratios of rates of nitrous oxide production and denitrification (N2O/N2× 100) were significantly higher with the addition of nitrite (7 to 13% of denitrification) than with nitrate (2 to 4% of denitrification). The results suggested that in addition to anaerobic bacteria, which possess the complete denitrification pathway for N2formation in the estuarine sediments, there may be two other groups of bacteria: nitrite denitrifiers, which reduce nitrite to N2via N2O, and obligate nitrite-denitrifying bacteria, which reduce nitrite to N2O as the end product. Consideration of free-energy changes during N2O formation led to the conclusion that N2O formation using nitrite as the electron acceptor is favored in the Colne estuary and may be a critical factor regulating the formation of N2O in high-nutrient-load estuaries.
Bacillus stearothermophilus
Neopullulanase Selective Hydrolysis of Amylose to Maltose in the Presence of Amylopectin
Authors: Kamasaka Hiroshi , Sugimoto Kazuhisa , Takata Hiroki , Nishimura Takahisa , Kuriki Takashi
Published: July 2002

The specificity of
Bacillus stearothermophilus
TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 10
8
to 10
7
Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrolysis of amylose but not amylopectin, was also observed with cyclomaltodextrinase from alkaliphilic
Bacillus
sp. strain A2-5a and maltogenic amylase from
Bacillus licheniformis
ATCC 27811. These three enzymes hydrolyzed cyclomaltodextrins and amylose much faster than pullulan. Other amylolytic enzymes, such as bacterial saccharifying α-amylase, bacterial liquefying α-amylase, β-amylase, and neopullulanase from
Bacillus megaterium
, did not exhibit this distinct substrate specificity at all, i.e., the preference of amylose to amylopectin.
Taxis Response of Various Denitrifying Bacteria to Nitrate and Nitrite
Authors: Lee Dong Yun , Ramos Adela , Macomber Lee , Shapleigh James P.
Published: July 2002

The taxis response of
Rhodobacter sphaeroides
2.4.1 and 2.4.3,
Rhodopseudomonas palustris
, and
Agrobacterium tumefaciens
to nitrate and nitrite was evaluated by observing the macroscopic behavior of cells suspended in soft agar and incubated under various conditions.
R. sphaeroides
2.4.3, which is capable of both nitrate and nitrite reduction, showed a taxis response to both nitrate and nitrite.
R. sphaeroides
2.4.1, which contains nitrate reductase but not nitrite reductase, did not show a taxis response towards either nitrogen oxide. Insertional inactivation of the nitrite reductase structural gene or its transcriptional regulator, NnrR, in strain 2.4.3 caused a loss of a taxis response towards both nitrate and nitrite. An isolate of 2.4.1 carrying a copy of the nitrite reductase gene from 2.4.3 showed a taxis response to both nitrogen oxides. The taxis response of 2.4.3 was observed under anaerobic conditions, suggesting that the taxis response was due to nitrate and nitrite respiration, not to inhibition of oxygen respiration by respiration of nitrogen oxides. Strain 2.4.3 showed a taxis response to nitrate and nitrite under photosynthetic and aerobic conditions. Changing the carbon source in the culture medium caused an unexpected subtle shift in the taxis response of 2.4.3 to nitrite. A taxis response to nitrogen oxides was also observed in
R. palustris
and
A. tumefaciens. R. palustris
exhibited a taxis response to nitrite but not to nitrate, while
A. tumefaciens
exhibited a response to both compounds.
Cytoskeletal Alterations in Lipopolysaccharide-Induced Bovine Vascular Endothelial Cell Injury and Its Prevention by Sodium Arsenite
Authors: Chakravortty Dipshikha , Koide Naoki , Kato Yutaka , Sugiyama Tsuyoshi , Kawai Makoto , Fukada Masako , Yoshida Tomoaki , Yokochi Takashi
Published: July 2002

Morphological changes, especially cytoskeletal alterations, in lipopolysaccharide (LPS)-induced vascular endothelial cell injury were studied by using LPS-susceptible bovine aortic endothelial cells (BAEC). BAEC in cultures with LPS showed cell rounding, shrinking, and intercellular gap formation. In those cells, LPS caused the disorganization of actin, tubulin, and vimentin. LPS also induced a reduction in the F-actin pool and an elevation in the G-actin pool. Cytoskeletal disorganization affected transendothelial permeability across the endothelial monolayer. Pretreatment of BAEC with sodium arsenite (SA) prevented alterations in LPS-induced BAEC injury. However, posttreatment with SA had no protective effect on them. SA upregulated the expression of heat shock protein in the presence of LPS. The role of SA in prevention of LPS-induced BAEC injury is discussed.
Use of Synthetic Cardiolipin and Lecithin in the Antigen Used by the Venereal Disease Research Laboratory Test for Serodiagnosis of Syphilis
Authors: Castro Arnold R. , Morrill William E. , Shaw Walter A. , Gale David C. , Park Mahin M. , Peregrino-Ferreira Luiz A. , Bazzo Maria L. , Pope Victoria
Published: July 2002

The Venereal Disease Research Laboratory (VDRL) test is a microflocculation test for syphilis that uses an antigen containing cardiolipin, lecithin, and cholesterol. For more than 50 years, the preparation of natural cardiolipin and lecithin for this test has been based on the Pangborn method which involves isolating and purifying these components from beef hearts. This process is tedious and time-consuming and results in a variable purity range. In our studies, we found that a VDRL antigen using synthetic tetramyristoyl cardiolipin and synthetic 1-palmitoyl-2-oleoyl-
sn
-glycero-3-phosphocholine (lecithin) was as specific in detecting syphilis as a VDRL antigen made with natural components. In 85% of the cases, we obtained an endpoint titer of 1/2 or 1 dilution more than a titer obtained with a VDRL antigen made with natural components. The use of these pure synthetic compounds, with a purity of 99%, would offer advantages in the standardization and stability of the VDRL antigen. Because this antigen is the basic ingredient in the preparation of nontreponemal reagents such as the rapid plasma reagin, toluidine red unheated serum test, and the unheated serum reagin, the use of this synthetic VDRL antigen should also increase the reactivity of these reagents.
Evaluation of Antibodies against a Rubella Virus Neutralizing Domain for Determination of Immune Status
Authors: Cordoba Patricia , Lanoel Alejandra , Grutadauria Sergio , Zapata Marta
Published: July 2002

The protective immune responses against rubella virus (RV) are related to its neutralizing epitopes, an issue that is important to consider when assessing the immune status of patients with remote infection. In the present paper, we compare the antibodies detected by a synthetic-peptide-based enzyme immunoassay (EIA) with antibodies detected by the traditional technique of hemagglutination inhibition (HIA) in patients with remote RV infection. The synthetic peptide used as an antigen (SP15) represents a neutralizing epitope that corresponds to amino acids 208 to 239 of the E1 glycoprotein. The SP15-EIA was developed, all variables that affected the assay were standardized, and the test was validated using reference sera. Serum samples (
n
= 129) from patients with remote RV infection were tested by HIA and SP15-EIA. Discrepant sera were assayed by MEIA (IMX/Abbot). The comparison between HIA and SP15-EIA, taking HIA as the standard methodology for determining immune status, showed that SP15-EIA is very specific and sensitive for detecting protecting antibodies (specificity, 100%; sensitivity, 98.20%). This study demonstrates that antibodies against the neutralizing domain represented by SP15 would be important in the memory response after natural infection and may be a good tool in the determination of the true immune status of patients with remote infection with regard to RV.
Role of Genomic Typing in Taxonomy, Evolutionary Genetics, and Microbial Epidemiology
Authors: van Belkum Alex , Struelens Marc , de Visser Arjan , Verbrugh Henri , Tibayrenc Michel
Published: July 2002

Currently, genetic typing of microorganisms is widely used in several major fields of microbiological research. Taxonomy, research aimed at elucidation of evolutionary dynamics or phylogenetic relationships, population genetics of microorganisms, and microbial epidemiology all rely on genetic typing data for discrimination between genotypes. Apart from being an essential component of these fundamental sciences, microbial typing clearly affects several areas of applied microbiogical research. The epidemiological investigation of outbreaks of infectious diseases and the measurement of genetic diversity in relation to relevant biological properties such as pathogenicity, drug resistance, and biodegradation capacities are obvious examples. The diversity among nucleic acid molecules provides the basic information for all fields described above. However, researchers in various disciplines tend to use different vocabularies, a wide variety of different experimental methods to monitor genetic variation, and sometimes widely differing modes of data processing and interpretation. The aim of the present review is to summarize the technological and fundamental concepts used in microbial taxonomy, evolutionary genetics, and epidemiology. Information on the nomenclature used in the different fields of research is provided, descriptions of the diverse genetic typing procedures are presented, and examples of both conceptual and technological research developments for Escherichia coli are included. Recommendations for unification of the different fields through standardization of laboratory techniques are made.
Phagosome Acidification Has Opposite Effects on Intracellular Survival ofBordetella pertussisandB. bronchiseptica
Authors: Schneider Boris , Gross Roy , Haas Albert
Published: July 2002

Bordetella pertussisis readily killed after uptake by professional phagocytes, whereas its close relativeBordetella bronchisepticais not and can persist intracellularly for days. Phagocytosis of members of either species by a mouse macrophage cell line results in transport of the bacteria to a phagosomal compartment positive for the lysosome-associated membrane protein 1, the protease cathepsin D, and the late endosomal vacuolar proton-pumping ATPase but negative for the early endosome antigen 1 and the early endosomal transferrin receptor. In addition, we demonstrate thatBordetella-containing phagosomes rapidly acidify to pH 4.5 to 5.0. Taken together, these data demonstrate thatBordetella-containing phagosomes rapidly mature to an acidic late endosomal/lysosomal compartment. Following up on this observation, we determined thatB. pertussisdoes not survive in bacterial growth media adjusted to a pH of 4.5, whereas this pH has only minor effects on the growth ofB. bronchiseptica. Raising the intracellular pH in infected macrophages by the addition of bafilomycin A1, ammonium chloride, or monensin increases the survival of acid-sensitiveB. pertussisbut, surprisingly, decreases that of acid-tolerantB. bronchiseptica. In summary, we hypothesize that the differential survival ofB. pertussisandB. bronchisepticain macrophages is, at least in part, due to the differences in their acid tolerance.
Induction of Necrosis in Human Neutrophils byShigella flexneriRequires Type III Secretion, IpaB and IpaC Invasins, and Actin Polymerization
Authors: François Mathias , Le Cabec Véronique , Dupont Marie-Ange , Sansonetti Philippe J. , Maridonneau-Parini Isabelle
Published: July 2002

Infection byShigella flexneriis characterized by infiltration of neutrophils in the intestinal mucosa and by a strong inflammatory reaction. Although neutrophils are constitutively programmed to die by apoptosis, we show that isolated human neutrophils undergo necrosis 2 h after infection with virulentS. flexneristrain M90T but not with the virulence plasmid-cured strain BS176. This was demonstrated by the release of azurophil granule proteins concomitant with the release of lactate dehydrogenase (LDH), disruption of the plasma membrane, and absence of DNA fragmentation. Mutants with themxiD1gene, coding for an essential component of the secretion type III machinery, or the genes coding for IpaB or IpaC invasins deleted were not cytotoxic. Neutrophil necrosis occurred independently of the bacterial ability to leave phagosomes, and it involved actin polymerization, as the addition of cytochalasin D after phagocytosis ofShigellainhibited the release of LDH. In conclusion,Shigellakills neutrophils by necrosis, a process characterized by the release of tissue-injurious granular proteins. This probably contributes to disruption of the epithelial barrier, leading to the dysentery observed in shigellosis and allowingShigellato enter its host cells.
Identification of an Immunodominant ABC Transporter in Methicillin-Resistant
Staphylococcus aureus
Infections
Authors: Burnie James P. , Matthews Ruth C. , Carter Tracey , Beaulieu Elaine , Donohoe Michael , Chapman Caroline , Williamson Peter , Hodgetts Samantha J.
Published: July 2002

Immunoblotting sera from 26 patients with septicemia due to an epidemic strain of methicillin-resistant
Staphylococcus aureus
(EMRSA-15), 6 of whom died, revealed an immunodominant EMRSA-15 antigen at 61 kDa. There was a statistically significant correlate (
P
< 0.001) between survival and immunoglobulin G to the 61-kDa band. The antigen was identified by sequencing positive clones obtained by screening a genomic expression library of EMRSA-15 with pooled sera from patients taken after the septicemic episode. Eluted antibody reacted with the 61-kDa antigen on immunoblots. The amino terminus was obtained by searching the
S. aureus
NCTC 8325 and MRSA strain COL databases, and the whole protein was expressed in
Escherichia coli
TOP 10F′. The derived amino acid sequence showed homology with ABC transporters, with paired Walker A and Walker B motifs and 73% homology to YkpA from
Bacillus subtilis
. Epitope mapping of the derived amino acid sequence with sera from patients who had recovered from EMRSA-15 septicemia delineated seven epitopes. Three of these epitopes, represented by peptides 1 (KIKVYVGNYDFWYQS), 2 (TVIVVSHDRHFLYNNV), and 3 (TETFLRGFLGRMLFS), were synthesized and used to isolate human recombinant antibodies from a phage antibody display library. Recombinant antibodies against peptides 1 and 2 gave logarithmic reductions in organ colony counts, compared with control groups, in a mouse model of the infection. This study suggests the potential role of an ABC transporter as a target for immunotherapy.
Measurement of T-Cell-Derived Antigen Binding Molecules and Immunoglobulin G Specific toCandida albicansMannan in Sera of Patients with Recurrent Vulvovaginal Candidiasis
Authors: Little Colin H. , Georgiou George M. , Marceglia Alex , Ogedgebe Henry , Cone Robert E. , Mazza Danielle
Published: July 2002

Immunoglobulin G (IgG) and T-cell-derived antigen binding molecules (TABM) specific to wholeCandidaextract and toCandida-derived mannans prepared by both the cetryltrimethylammonium bromide (CTAB) and alkaline degradation (PEAT) methods were measured in the sera of women with vulvovaginal candidiasis and controls. In the patients there were significantly higher levels of IgG to both CTAB and PEAT mannans and of TABM to CTAB mannan. TABM specific to CTAB mannan was purified from the serum of a patient with a high titer of this TABM. The purified TABM bound specifically to CTAB mannan and to other yeast and mold extracts. This TABM preparation was associated with transforming growth factor β2 (TGF-β2), and on specific binding to mannan there was a marked increase in the level of detectable TGF-β2. This increase in TGF-β2 level was critically dependent on the relative concentrations of the purified TABM and mannan, being smallest when either was in excess. The TABM specific to CTAB mannan was also shown to inhibitCandida-stimulated gamma interferon production. The results suggest that CTAB mannan-specific TABM may increase susceptibility to vulvovaginal candidiasis in association with a shift in the immune response to the Th2 type.
Molecular Characterization of the
Mycoplasma gallisepticum pvpA
Gene Which Encodes a Putative Variable Cytadhesin Protein
Authors: Boguslavsky S. , Menaker D. , Lysnyansky I. , Liu T. , Levisohn S. , Rosengarten R. , García M. , Yogev D.
Published: July 2002

A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen
Mycoplasma gallisepticum
. The
pvpA
gene was cloned, expressed in
Escherichia coli
, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens
Mycoplasma pneumoniae
and
Mycoplasma genitalium
, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of
M. gallisepticum
and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of
M. pneumoniae
. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among
M. gallisepticum
strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of
M. gallisepticum
.
The Putative Proteinase Maturation Protein A of
Streptococcus pneumoniae
Is a Conserved Surface Protein with Potential To Elicit Protective Immune Responses
Authors: Overweg K. , Kerr A. , Sluijter M. , Jackson M. H. , Mitchell T. J. , de Jong A. P. J. M. , de Groot R. , Hermans P. W. M.
Published: July 2002

Surface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of
Streptococcus pneumoniae
. The opsonophagocytic activity of hyperimmune serum raised against this protein fraction was high and species specific. Moreover, the opsonophagocytic activity was independent of the capsular type and chromosomal genotype of the pneumococcus. Since the opsonophagocytic activity is presumed to correlate with in vivo protection, these data indicate that the protein fraction has the potential to elicit species-specific immune protection with cross-protection against various pneumococcal strains. Individual proteins in the extract were purified by two-dimensional gel electrophoresis. Antibodies raised against three distinct proteins contributed to the opsonophagocytic activity of the serum. The proteins were identified by mass spectrometry and N-terminal amino acid sequencing. Two proteins were the previously characterized pneumococcal surface protein A and oligopeptide-binding lipoprotein AmiA. The third protein was the recently identified putative proteinase maturation protein A (PpmA), which showed homology to members of the family of peptidyl-prolyl
cis/trans
isomerases. Immunoelectron microscopy demonstrated that PpmA was associated with the pneumococcal surface. In addition, PpmA was shown to elicit species-specific opsonophagocytic antibodies that were cross-reactive with various pneumococcal strains. This antibody cross-reactivity was in line with the limited sequence variation of
ppmA
. The importance of PpmA in pneumococcal pathogenesis was demonstrated in a mouse pneumonia model. Pneumococcal
ppmA
-deficient mutants showed reduced virulence. The properties of PpmA reported here indicate its potential for inclusion in multicomponent protein vaccines.
Central Role of Endogenous Gamma Interferon in Protective Immunity against Blood-StagePlasmodium chabaudiAS Infection
Authors: Su Zhong , Stevenson Mary M.
Published: July 2002

The role of endogenous gamma interferon (IFN-γ) in protective immunity against blood-stagePlasmodium chabaudiAS malaria was studied using IFN-γ gene knockout (GKO) and wild-type (WT) C57BL/6 mice. Following infection with 106parasitized erythrocytes, GKO mice developed significantly higher parasitemia during acute infection than WT mice and had severe mortality. In infected GKO mice, production of interleukin 12 (IL-12) p70 and tumor necrosis factor alpha in vivo and IL-12 p70 in vitro by splenic macrophages was significantly reduced compared to that in WT mice and the enhanced nitric oxide (NO) production observed in infected WT mice was completely absent. WT and GKO mice had comparable numbers of total nucleated spleen cells and B220+and Mac-1+spleen cells both before and after infection. Infected WT mice, however, had significantly more F4/80+, NK1.1+, and F4/80+Ia+spleen cells than infected GKO mice; male WT had more CD3+cells than male GKO mice. In comparison with those from WT mice, splenocytes from infected GKO mice had significantly higher proliferation in vitro in response to parasite antigen or concanavalin A stimulation and produced significantly higher levels of IL-10 in response to parasite antigen. Infected WT mice produced more parasite-specific immunoglobulin M (IgM), IgG2a, and IgG3 and less IgG1 than GKO mice. Significant gender differences in both GKO and WT mice in peak parasitemia levels, mortality, phenotypes of spleen cells, and proliferation of and cytokine production by splenocytes in vitro were apparent during infection. These results thus provide unequivocal evidence for the central role of endogenous IFN-γ in the development of protective immunity against blood-stageP. chabaudiAS.
Differences in Gamma Interferon Production In Vitro Predict the Pace of the In Vivo Response toLeishmania amazonensisin Healthy Volunteers
Authors: Pompeu M. M. L. , Brodskyn C. , Teixeira M. J. , Clarêncio J. , Van Weyenberg J. , Coelho I. C. B. , Cardoso S. A. , Barral A. , Barral-Netto M.
Published: July 2002

The initial encounter ofLeishmaniacells and cells from the immune system is fundamentally important in the outcome of infection and determines disease development or resistance. We evaluated the anti-Leishmania amazonensisresponse of naive volunteers by using an in vitro priming (IVP) system and comparing the responses following in vivo vaccination against the same parasite. In vitro stimulation allowed us to distinguish two groups of individuals, those who produced small amounts of gamma interferon (IFN-γ) (n= 16) (low producers) and those who produced large amounts of this cytokine (n= 16) (high producers). IFN-γ production was proportional to tumor necrosis factor alpha and interleukin 10 (IL-10) levels but did not correlate with IL-5 production. Volunteers who produced small amounts of IFN-γ in vitro remained low producers 40 days after vaccination, whereas high producers exhibited increased IFN-γ production. However, 6 months after vaccination, all individuals tested produced similarly high levels of IFN-γ upon stimulation of their peripheral blood mononuclear cells withLeishmaniapromastigotes, indicating that low in vitro producers respond slowly in vivo to vaccination. In high IFN-γ producers there was an increased frequency of activated CD8+T cells both in vitro and in vivo compared to the frequency in low producers, and such cells were positive for IFN-γ as determined by intracellular staining. Such findings suggest that IVP responses can be used to predict the pace of postvaccination responses of test volunteers. Although all vaccinated individuals eventually have a potent anti-Leishmaniacell-mediated immunity (CMI) response, a delay in mounting the CMI response may influence resistance against leishmaniasis.
Identification and Characterization of a Novel Family of Pneumococcal Proteins That Are Protective against Sepsis
Authors: Adamou John E. , Heinrichs Jon H. , Erwin Alice L. , Walsh William , Gayle Tony , Dormitzer Melissa , Dagan Ron , Brewah Yambasu A. , Barren Philip , Lathigra Raju , Langermann Solomon , Koenig Scott , Johnson Syd
Published: July 2002

Four pneumococcal genes (
phtA, phtB, phtD
, and
phtE
) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the
Streptococcus pneumoniae
genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four
pht
genes (
phtA, phtB, phtD
, and a truncated version of
phtE
) were expressed in
Escherichia coli
. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.
Haemophilus somnusInduces Apoptosis in Bovine Endothelial Cells In Vitro
Authors: Sylte Matt J. , Corbeil Lynette B. , Inzana Thomas J. , Czuprynski Charles J.
Published: July 2002

Haemophilus somnuscauses pneumonia, reproductive failure, infectious myocarditis, thrombotic meningoencephalitis, and other diseases in cattle. Although vasculitis is commonly seen as a result of systemicH. somnusinfections, the pathogenesis of vascular damage is poorly characterized. In this study, we demonstrated thatH. somnus(pathogenic isolates 649, 2336, and 8025 and asymptomatic carrier isolates 127P and 129Pt) induce apoptosis of bovine endothelial cells in a time- and dose-dependent manner, as determined by Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end labeling, DNA fragmentation, and transmission electron microscopy.H. somnusinduced endothelial cell apoptosis in as little as 1 h of incubation and did not require extracellular growth of the bacteria. ViableH. somnusorganisms induced greater endothelial cell apoptosis than heat-killed organisms. Since viableH. somnuscells release membrane fibrils and blebs, which contain lipooligosaccharide (LOS) and immunoglobulin binding proteins, we examined culture filtrates for their ability to induce endothelial cell apoptosis. Culture filtrates induced similar levels of endothelial cell apoptosis, as did viableH. somnusorganisms. Heat inactivation ofH. somnusculture filtrates partially reduced the apoptotic effect on endothelial cells, which suggested the presence of both heat-labile and heat-stable factors. We found thatH. somnusLOS, which is heat stable, induced endothelial cell apoptosis in a time- and dose-dependent manner and was inhibited by the addition of polymyxin B. These data demonstrate thatH. somnusand its LOS induce endothelial cell apoptosis, which may play a role in producing vasculitis in vivo.