bluuebunny/crossref_metadata_2025 huggingface dataset:
gfp
-Based
N
-Acyl Homoserine-Lactone Sensor Systems for Detection of Bacterial Communication
Authors: Andersen Jens Bo , Heydorn Arne , Hentzer Morten , Eberl Leo , Geisenberger Otto , Christensen Bjarke Bak , Molin So/ren , Givskov Michael
Published: July 2002

In order to perform single-cell analysis and online studies of
N
-acyl homoserine lactone (AHL)-mediated communication among bacteria, components of the
Vibrio fischeri
quorum sensor encoded by

luxR-P
luxI

have been fused to modified versions of
gfp
mut3∗ genes encoding unstable green fluorescent proteins. Bacterial strains harboring this green fluorescent sensor detected a broad spectrum of AHL molecules and were capable of sensing the presence of 5 nM
N
-3-oxohexanoyl-
l
-homoserine lactone in the surroundings. In combination with epifluorescent microscopy, the sensitivity of the sensor enabled AHL detection at the single-cell level and allowed for real-time measurements of fluctuations in AHL concentrations. This green fluorescent AHL sensor provides a state-of-the-art tool for studies of communication between the individuals present in mixed bacterial communities.
Genetic Evidence for a Defective Xylan Degradation Pathway in
Lactococcus lactis
Authors: Erlandson Karn A. , Delamarre Soazig C. , Batt Carl A.
Published: July 2002

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains,
Lactococcus lactis
210,
L. lactis
IO-1, and
L. lactis
NRRL B-4449. Immediately downstream of the xylulose kinase gene (
xylB
) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974–3980, 1999) are two open reading frames encoding a mutarotase (
xylM
) and a xyloside transporter (
xynT
) and a partial open reading frame encoding a β-xylosidase (
xynB
). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative
xylM
gene product was confirmed by overexpression of the
L. lactis
enzyme in
Escherichia coli
and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the
xylM
and
xynTB
genes are cotranscribed with the
xylRAB
genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and
L. lactis
IO-1 and
L. lactis
NRRL B-4449 had functional mutarotases.
Occurrence and Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production Facilities
Authors: Chee-Sanford J. C. , Aminov R. I. , Krapac I. J. , Garrigues-Jeanjean N. , Mackie R. I.
Published: July 2002

In this study, we used PCR typing methods to assess the presence of tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying two swine farms. All eight classes of genes encoding this mechanism of resistance [
tet
(O),
tet
(Q),
tet
(W),
tet
(M),
tetB
(P),
tet
(S),
tet
(T), and
otrA
] were found in total DNA extracted from water of two lagoons. These determinants were found to be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the
tet
(M) gene, which was not predominant in the environmental samples and was identical to
tet
(M) from the lagoons. The presence of this gene in some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study demonstrated that
tet
genes occur in the environment as a direct result of agriculture and suggested that groundwater may be a potential source of antibiotic resistance in the food chain.
Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?
Authors: Lebaron Philippe , Servais Pierre , Agogué Helene , Courties Claude , Joux Fabien
Published: July 2002

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.
Genotypes Associated with Virulence in Environmental Isolates of
Vibrio cholerae
Authors: Rivera Irma N. G. , Chun Jongsik , Huq Anwar , Sack R. Brad , Colwell Rita R.
Published: July 2002

Vibrio cholerae
is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of
V. cholerae
were examined for the presence of
ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR
, and
zot
genes, using multiplex PCR. Based on
tcpA
and
hlyA
gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The
toxR, hlyA
, and
ompU
genes were present in 100, 98.6, and 87.0% of the
V. cholerae
isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (
tcpA
ET) gene were present in all toxigenic
V. cholerae
O1 and
V. cholerae
O139 strains examined in this study. Three of four nontoxigenic
V. cholerae
O1 strains contained
tcpA
ET. Interestingly, among the isolates of
V. cholerae
non-O1/non-O139, two had
tcpA
Classical, nine contained
tcpA
El Tor, three showed homology with both biotype genes, and four carried the
ctxA
gene. The
stn/sto
genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic
V. cholerae
O1, and in 14.3% of the O139 serogroups. Except for
stn/sto
genes, all of the other genes studied occurred with high frequency in toxigenic
V. cholerae
O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139
V. cholerae
in the aquatic environment, combined with genotype monitoring using
ctxA, stn/sto
, and
tcpA
ET genes, could be valuable in human health risk assessment.
Quantitation of Viral DNA by Real-Time PCR Applying Duplex Amplification, Internal Standardization, and Two-Color Fluorescence Detection
Authors: Gruber Franz , Falkner Falko G. , Dorner Friedrich , Hämmerle Thomas
Published: July 2002

A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 10
2
to at least 5 × 10
6
copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.
Improvement of Cellulolytic Properties of
Clostridium cellulolyticum
by Metabolic Engineering
Authors: Guedon Emmanuel , Desvaux Mickaël , Petitdemange Henri
Published: July 2002

Cellulolytic clostridia have evolved to catabolize lignocellulosic materials at a seasonal biorhythm, so their biotechnological exploitation requires genetic improvements. As high carbon flux leads to pyruvate accumulation, which is responsible for the cessation of growth of
Clostridium cellulolyticum
, this accumulation is decreased by heterologous expression of pyruvate decarboxylase and alcohol dehydrogenase from
Zymomonas mobilis
. In comparison with that of the wild strain, growth of the recombinant strain at the same specific rate but for 145 h instead of 80 h led to a 150% increase in cellulose consumption and a 180% increase in cell dry weight. The fermentation pattern was shifted significantly: lactate production decreased by 48%, whereas the concentrations of acetate and ethanol increased by 93 and 53%, respectively. This study demonstrates that the fermentation of cellulose, the most abundant and renewable polymer on earth, can be greatly improved by using genetically engineered
C. cellulolyticum
.
Transcript Levels of the Eukaryotic Translation Initiation Factor 5A Gene Peak at Early G
1
Phase of the Cell Cycle in the Dinoflagellate
Crypthecodinium cohnii
Authors: Chan K. L. , New D. , Ghandhi S. , Wong F. , Lam C. M. C. , Wong J. T. Y.
Published: July 2002

A cDNA encoding a eukaryotic translation initiation factor 5A (eIF-5A) homolog in heterotrophic dinoflagellate
Crypthecodinium cohnii
(CceIF-5A) was isolated through random sequencing of a cDNA library. The predicted amino acid sequence possesses the 12 strictly conserved amino acids around lysine 52 (equivalent to lysine 50 or 51 in other eukaryotes). A single 1.2-kb band was detected in Northern blot analysis. In synchronized
C. cohnii
cells, the transcript level peaked at early G
1
and decreased dramatically on the entry to S phase. Although this has not been previously reported, studies of budding yeast (
Saccharomyces cerevisiae
) and certain mammalian cell types suggest a role for eIF-5A in the G
1
/S transition of the eukaryotic cell cycle. Phylogenetic trees constructed with 26 other published eIF-5A sequences suggest that CceIF-5A, while falling within the eukaryotic branches, forms a lineage separate from those of the plants, animals, and archaebacteria. The posttranslational modification of eIF-5A by a transfer of a 4-aminobutyl moiety from spermidine to conserved lysine 50 or 51, forming amino acid hypusine, is the only demonstrated specific function of polyamines in cell proliferation. It has been suggested that polyamines stimulate population growth of bloom-forming dinoflagellates in the sea. We demonstrate here putrescine-stimulated cell proliferation. Furthermore, ornithine decarboxylase inhibitor
d
-difluoromethylornithine and the specific hypusination inhibitor
N
-guanyl-1,7-diaminoheptane exhibited inhibitory effects in two species of dinoflagellates. The possible links of polyamines and saxitoxin synthesis to the arginine cycle are also discussed.
Sequence Variation in the
porA
Gene of a Clone of
Neisseria meningitidis
during Epidemic Spread
Authors: Jelfs J. , Munro R. , Wedege E. , Caugant D. A.
Published: July 2002

The ET-15 clone within the electrophoretic type (ET)-37 complex of
Neisseria meningitidis
was first detected in Canada in 1986 and has since been associated with outbreaks of meningococcal disease in many parts of the world. While the majority of the strains of the ET-37 complex are serosubtype P1.5,2, serosubtype determination of ET-15 strains may often be incomplete, with either only one or none of the two variable regions (VRs) of the serosubtype PorA outer membrane protein reacting with monoclonal antibodies. DNA sequence analysis of the
porA
gene from ET-15 strains with one or both unidentified serosubtype determinants was undertaken to identify the genetic basis of the lack of reaction with the monoclonal antibodies. Fourteen different
porA
alleles were identified among 38 ET-15 strains from various geographic origins. The sequences corresponding to subtypes P1.5a,10d, P1.5,2, P1.5,10d, P1.5a,10k, and P1.5a,10a were identified in 18, 11, 2, 2, and 1 isolate, respectively. Of the remaining four strains, which all were nonserosubtypeable, two had a stop codon within the VR1 and the VR2, respectively, while in the other two the
porA
gene was interrupted by the insertion element, IS
1301
. Of the strains with P1.5,2 sequence, one had a stop codon between the VR1 and VR2, one had a four-amino-acid deletion outside the VR2, and another showed no expression of PorA on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results reveal that numerous genetic events have occurred in the
porA
gene of the ET-15 clone in the short time of its epidemic spread. The magnitude of microevolutionary mechanisms available in meningococci and the remarkable genetic flexibility of these bacteria need to be considered in relation to PorA vaccine development.
Close Association between Pulmonary Disease Manifestation inMycoplasma pneumoniaeInfection and Enhanced Local Production of Interleukin-18 in the Lung, Independent of Gamma Interferon
Authors: Narita Mitsuo , Tanaka Hiroshi , Abe Shosaku , Yamada Satoshi , Kubota Mitsuru , Togashi Takehiro
Published: July 2002

To investigate pathophysiologies ofMycoplasma pneumoniaeinfection from an immunological point of view, we measured the levels of interleukin-18 (IL-18) (originally designated gamma interferon [IFN-γ]-inducing factor) in 19 serum samples from 10 patients with pneumonia without pleural effusion (ages 1 to 16 years), 3 serum and 13 pleural fluid samples from 11 patients with pleural effusions (ages 11 months to 15 years), and 18 serum and 27 cerebrospinal fluid samples from 24 patients with central nervous system complications (ages 1 to 15 years). IL-18 was measured by a commercially available enzyme-linked immunosorbent assay kit (MBL, Nagoya, Japan). In addition, the levels of tumor necrosis factor alpha, IFN-γ, IL-6, IL-12, and KL-6 (a mucin-like glycoprotein expressed on type 2 pneumocytes) were measured in selected samples. The results concerning pleural effusions showed that elevated levels of IL-18 in pleural fluid, but not in serum, were solely associated with a sustained fibrotic change of the lung on chest roentgenography which might represent a pathological feature of intraluminal organization. All the pleural fluid samples with elevated levels of IL-18 were positive by PCR forM. pneumoniaeDNA. There was no association between IL-18 and IFN-γ levels in serum or in the pleural fluid. On the other hand, elevated levels of IL-18 in serum, but not in cerebrospinal fluid samples, were observed in the cases complicated by central nervous system involvement, including profound brain dysfunction with seizures. Our study demonstrated thatM. pneumoniaecan induce IL-18 and that the enhanced local production of IL-18 in the lung is closely associated with pulmonary disease manifestation.
Quantitative Analyses of Variations in the Injury of Endothelial Cells Elicited by 11 Isolates ofRickettsia rickettsii
Authors: Eremeeva Marina E. , Dasch Gregory A. , Silverman David J.
Published: July 2002

Eleven isolates of spotted fever group rickettsiae from the blood of patients or ixodid ticks from North and South America were characterized. All isolates were identified asRickettsia rickettsiiusing restriction fragment length polymorphism analysis of a 532-bp rOmpA gene fragment obtained by PCR. The ability of theR. rickettsiiisolates to elicit cytopathic effects and parameters of oxidative injury were examined in cultured human EA.hy 926 endothelial cells. Cytopathic effects were determined by direct observation of infected cultures, by measuring the release of cytoplasmic lactate dehydrogenase (LDH), and by determination of intracellular pools of peroxide and reduced glutathione. Four biotypes ofR. rickettsiiwere defined. Group I included two highly cytopathic isolates from Montana, Bitterroot and Sheila Smith, and three isolates from Maryland, North Carolina, and Brazil. These isolates rapidly damaged cells, released large amounts of cytoplasmic LDH, caused accumulation of intracellular peroxide, and depleted intracellular pools of reduced glutathione. Group II contained three isolates, two from Montana, Hlp#2 and Lost Horse Canyon, and an isolate from Colombia, which were similar to group I but caused either lower responses in LDH release or smaller changes in intracellular peroxide levels. The group III isolates, Sawtooth from Montana and 84JG from North Carolina, caused lower cellular injury by all measures. Group IV isolate Price T from Montana was the least cytopathic and caused minimal alterations of all parameters measured. Understanding the molecular basis for the varied cellular injury caused by different isolates ofR. rickettsiimay contribute to improved treatment of Rocky Mountain spotted fever and to the rapid identification of those isolates which are more likely to cause fulminant disease.
Periodontal Disease as a Specific, albeit Chronic, Infection: Diagnosis and Treatment
Authors: Loesche Walter J. , Grossman Natalie S.
Published: July 2002

Periodontal disease is perhaps the most common chronic infection in adults. Evidence has been accumulating for the past 30 years which indicates that almost all forms of periodontal disease are chronic but specific bacterial infections due to the overgrowth in the dental plaque of a finite number of mostly anaerobic species such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola. The success of traditional debridement procedures and/or antimicrobial agents in improving periodontal health can be associated with the reduction in levels of these anaerobes in the dental plaque. These findings suggest that patients and clinicians have a choice in the treatment of this overgrowth, either a debridement and surgery approach or a debridement and antimicrobial treatment approach. However, the antimicrobial approach, while supported by a wealth of scientific evidence, goes contrary to centuries of dental teaching that states that periodontal disease results from a “dirty mouth.” If periodontal disease is demonstrated to be a risk factor for cardiovascular disease and stroke, it will be a modifiable risk factor since periodontal disease can be prevented and treated. Since the antimicrobial approach may be as effective as a surgical approach in the restoration and maintenance of a periodontally healthy dentition, this would give a cardiac or stroke patient and his or her physician a choice in the implementation of treatment seeking to improve the patient's periodontal condition so as to reduce and/or delay future cardiovascular events.
Clinical Aspects and Pathophysiology of Inflammatory Bowel Disease
Authors: Hendrickson Barbara A. , Gokhale Ranjana , Cho Judy H.
Published: July 2002

The chronic inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, are recognized as important causes of gastrointestinal disease in children and adults. In this review we delineate the clinical manifestations and diagnostic features of IBD. In addition, we summarize important recent advances in our understanding of the immune mediators of intestinal inflammation. This information has led to new therapeutic approaches in IBD. Further, we discuss the considerable data that point to the significance of genetic factors in the development of IBD and the genetic loci which have been implicated through genomewide searches. The commensal bacterial flora also appears to be a critical element, particularly in regards to Crohn's disease, although the precise role of the bacteria in the disease manifestations remains unclear. Current investigations promise to yield fresh insights in these areas.
Molecular Variation among Type IV Pilin (
bfpA
) Genes from Diverse Enteropathogenic
Escherichia coli
Strains
Authors: Blank T. Eric , Zhong Hailang , Bell Alison L. , Whittam Thomas S. , Donnenberg Michael S.
Published: July 2002

Typical enteropathogenic
Escherichia coli
(EPEC) strains produce bundle-forming pili (BFP), type IVB fimbriae that have been implicated in EPEC virulence, antigenicity, autoaggregation, and localized adherence to epithelial cells (LA). BFP are polymers of bundlin, a pilin protein that is encoded by the
bfpA
gene found on a large EPEC plasmid. Striking sequence variation has previously been observed among type IV pilin genes of other gram-negative bacterial pathogens (e.g.,
Pseudomonas
and
Neisseria
spp.). In contrast, the established sequences of
bfpA
genes from two distantly related prototype EPEC strains vary by only a single base pair. To determine whether bundlin sequences vary more extensively, we used PCR to amplify the
bfpA
genes from 19 EPEC strains chosen for their various serotypes and sites and years of isolation. Eight different
bfpA
alleles were identified by sequencing of the PCR products. These alleles can be classified into two major groups. The α group contains three alleles derived from strains carrying O55, O86, O111, O119, O127, or O128 somatic antigens. The β group contains five alleles derived from strains carrying O55, O110, O128ab, O142, or nontypeable antigens. Sequence comparisons show that bundlin has highly conserved and variable regions, with most of the variation occurring in the C-terminal two-thirds of the protein. The results of multilocus enzyme electrophoresis support the hypothesis that
bfpA
sequences have spread horizontally across distantly related clonal lineages. Strains with divergent bundlin sequences express bundlin protein, produce BFP, and carry out autoaggregation and LA. However, four strains lack most or all of these phenotypes despite having an intact
bfpA
gene. These results have important implications for our understanding of bundlin structure, transmission of the
bfp
gene cluster among EPEC strains, and the role of bundlin variation in the evasion of host immune system responses.
Disease Outcome Subsequent to Primary and Secondary Urogenital Infection with Murine or Human Biovars ofChlamydia trachomatis
Authors: Ramsey Kyle H. , DeWolfe Jennifer L. , Salyer Rena D.
Published: July 2002

A susceptible strain of mice infected intravaginally with the mouse pneumonitis biovar ofChlamydia trachomatisbecame infertile and sustained high rates of hydrosalpinx formation regardless of prior infection with a human serovar. Conversely, susceptible mice infected with human serovars remained fertile unless challenged with a homologous human serovar.
Distribution of Core Oligosaccharide Types in Lipopolysaccharides from
Escherichia coli
Authors: Amor Karen , Heinrichs David E. , Frirdich Emilisa , Ziebell Kim , Johnson Roger P. , Whitfield Chris
Published: July 2002

In the lipopolysaccharides of
Escherichia coli
there are five distinct core oligosaccharide (core OS) structures, designated K-12 and R1 to R4. The objective of this work was to determine the prevalences of these core OS types within the species. Unique sequences in the
waa
(core OS biosynthesis) gene operon were used to develop a PCR-based system that facilitated unequivocal determination of the core OS types in isolates of
E. coli
. This system was applied to the 72 isolates in the
E. coli
ECOR collection, a compilation of isolates that is considered to be broadly representative of the genetic diversity of the species. Fifty (69.4%) of the ECOR isolates contained the R1 core OS, 8 (11.1%) were representatives of R2, 8 (11.1%) were R3, 2 (2.8%) were R4, and only 4 (5.6%) were K-12. R1 is the only core OS type found in all four major phylogenetic groups (A, B1, B2, and D) in the ECOR collection. Virulent extraintestinal pathogenic
E. coli
isolates tend to be closely related to group B2 and, to a lesser extent, group D isolates. All of the ECOR representatives from the B2 and D groups had the R1 core OS. In contrast, commensal
E. coli
isolates are more closely related to group A, which contains isolates representing each of the five core OS structures. R3 was the only core OS type found in 38 verotoxigenic
E. coli
(VTEC) isolates from humans and cattle belonging to the common enterohemorrhagic
E. coli
serogroups O157, O111, and O26. Although isolates from other VTEC serogroups showed more core OS diversity, the R3 type (83.1% of all VTEC isolates) was still predominant. When non-VTEC commensal isolates from cattle were analyzed, it was found that most possessed the R1 core OS type.
Regulation of Immunoglobulin G2 Production by Prostaglandin E2and Platelet-Activating Factor
Authors: Ishihara Yuichi , Zhang Ji-Bo , Quinn Steve M. , Schenkein Harvey A. , Best Al M. , Barbour Suzanne E. , Tew John G.
Published: July 2002

Patients with localized juvenile periodontitis (LJP) have elevated levels of immunoglobulin G2 (IgG2) in their sera. This is also observed in vitro when peripheral blood leukocytes from LJP patients are stimulated with pokeweed mitogen. In previous studies, we showed that lymphocytes from subjects with no periodontitis (NP subjects) produced substantial amounts of IgG2 when they were cultured with monocytes from LJP patients (LJP monocytes). These observations indicate that monocytes or monocyte-derived mediators are positive regulators of the production of IgG2. The present study was initiated to determine if secreted factors from LJP monocytes were capable of enhancing IgG2 production and to determine if prostaglandin E2 (PGE2), which LJP monocytes produce at elevated levels, enhances IgG2 production. Experiments in a transwell system and with monocyte-conditioned media indicated that cell-cell contact was not necessary for LJP monocytes to augment the production of IgG2 by T and B cells from NP subjects. Moreover, the production of IgG2 was selectively induced by the addition of PGE2or platelet-activating factor (PAF), another lipid cytokine, which can elevate PGE2synthesis. Furthermore, IgG2 production was abrogated when cells were treated with indomethacin, a cyclooxygenase inhibitor that blocks the synthesis of PGE2, or the PAF antagonists CV3988 and TEPC-15. The effects of indomethacin were completely reversed by PGE2, indicating that this is the only prostanoid that is essential for the production of IgG2. Similarly, PGE2reversed the effects of a PAF antagonist, suggesting that the effects of PAF are mediated through the induction of PGE2synthesis. Together, these data indicate that PGE2and PAF are essential for the production of IgG2.
Developmental Expression of a Tandemly Repeated, Glycine- and Serine-Rich Spore Wall Protein in the Microsporidian Pathogen
Encephalitozoon cuniculi
Authors: Bohne Wolfgang , Ferguson David J. P. , Kohler Karoline , Gross Uwe
Published: July 2002

Microsporidia are intracellular organisms of increasing importance as opportunistic pathogens in immunocompromised patients. Host cells are infected by the extrusion and injection of polar tubes located within spores. The spore is surrounded by a rigid spore wall which, in addition to providing mechanical resistance, might be involved in host cell recognition and initiation of the infection process. A 51-kDa outer spore wall protein was identified in
Encephalitozoon cuniculi
with the aid of a monoclonal antibody, and the corresponding gene,
SWP1
, was cloned by immunoscreening of a cDNA expression library. The cDNA encodes a protein of 450 amino acids which displays no significant similarities to known proteins in databases. The carboxy-terminal region consists of five tandemly arranged glycine- and serine-rich repetitive elements.
SWP1
is a single-copy gene that is also present in the genomes of
Encephalitozoon intestinalis
and
Encephalitozoon hellem
as demonstrated by Southern analysis. Indirect immunofluorescence and immunoelectron microscopy revealed that SWP1 is differentially expressed during the infection cycle. The protein is absent in replicative meronts until 24 h postinfection, and its expression is first induced in early sporonts at a time when organisms translocate from the periphery to the center of the parasitophorous vacuole. Expression of SWP1 appears to be regulated at the mRNA level, as was shown by reverse transcriptase PCR analysis. Further identification and characterization of stage-specific genes might help to unravel the complex intracellular differentiation process of microsporidia.
Vervet Monkeys Vaccinated with KilledLeishmania majorParasites and Interleukin-12 Develop a Type 1 Immune Response but Are Not Protected against Challenge Infection
Authors: Gicheru Michael M. , Olobo Joseph O. , Anjili Christopher O. , Orago Alloys S. , Modabber Farrokh , Scott Phillip
Published: July 2002

Leishmania majoris a protozoan parasite that causes chronic cutaneous lesions that often leave disfiguring scars. Infections in mice have demonstrated that leishmanial vaccines that include interleukin-12 (IL-12) as an adjuvant are able to induce protective immunity. In this study, we assessed the safety, immunopotency, and adjuvant potential of two doses of IL-12 when used with a killedL. majorvaccine in vervet monkeys. The induction of cell-mediated immunity following vaccination was determined by measuring delayed-type hypersensitivity, in vitro lymphocyte proliferation, and gamma interferon (IFN-γ) production. Protection was assessed by challenging the animals withL. majorparasites and monitoring the course of infection. At low doses of IL-12 (10 μg), a small increase in the parameters of cell-mediated immunity was observed, relative to those in animals that received antigen without IL-12. However, none of these animals were protected against a challenge infection. At higher doses of IL-12 (30 μg), a substantial increase inLeishmania-specific immune responses was observed, and monkeys immunized with antigen and IL-12 exhibited an IFN-γ response that was as great as that in animals that had resolved a primary infection and were immune. Nevertheless, despite the presence of correlates of protection, the disease course was only slightly altered, and protection was low compared to that in self-cured monkeys. These data suggest that protection against leishmaniasis may require more than the activation ofLeishmania-specific IFN-γ-producing T cells, which has important implications for designing a vaccine against leishmaniasis.
Salmonella enterica
Serovar Typhimurium Response Involved in Attenuation of Pathogen Intracellular Proliferation
Authors: Cano David A. , Martı́nez-Moya Marina , Pucciarelli M. Graciela , Groisman Eduardo A. , Casadesús Josep , Garcı́a-Del Portillo Francisco
Published: July 2002

Salmonella enterica
serovar Typhimurium proliferates within cultured epithelial and macrophage cells. Intracellular bacterial proliferation is, however, restricted within normal fibroblast cells. To characterize this phenomenon in detail, we investigated the possibility that the pathogen itself might contribute to attenuating the intracellular growth rate.
S
.
enterica
serovar Typhimurium mutants were selected in normal rat kidney fibroblasts displaying an increased intracellular proliferation rate. These mutants harbored loss-of-function mutations in the virulence-related regulatory genes
phoQ
,
rpoS
,
slyA
, and
spvR
. Lack of a functional PhoP-PhoQ system caused the most dramatic change in the intracellular growth rate.
phoP-
and
phoQ
-null mutants exhibited an intracellular growth rate 20- to 30-fold higher than that of the wild-type strain. This result showed that the PhoP-PhoQ system exerts a master regulatory function for preventing bacterial overgrowth within fibroblasts. In addition, an overgrowing clone was isolated harboring a mutation in a previously unknown serovar Typhimurium open reading frame, named
igaA
for intracellular growth attenuator. Mutations in other serovar Typhimurium virulence genes, such as
ompR
,
dam
,
crp
,
cya
,
mviA
,
spiR (ssrA)
,
spiA
, and
rpoE
, did not result in pathogen intracellular overgrowth. Nonetheless, lack of either SpiA or the alternate sigma factor RpoE led to a substantial decrease in intracellular bacterial viability. These results prove for the first time that specific serovar Typhimurium virulence regulators are involved in a response designed to attenuate the intracellular growth rate within a nonphagocytic host cell. This growth-attenuating response is accompanied by functions that ensure the viability of intracellular bacteria.
Regulation of
Streptococcus gordonii sspB
by the
sspA
Gene Product
Authors: El-Sabaeny Azza , Demuth Donald R. , Lamont Richard J.
Published: July 2002

Streptococcus gordonii
expresses two related adhesins, SspA and SspB, the genes for which are adjacent on the chromosome and are regulated independently. Although the adhesins are functionally similar, the
sspA
promoter is more active than that of
sspB
. In this study we show an additional role for SspA in the control of
sspB
activity. Gel shift and DNA footprinting assays demonstrate that the SspA protein binds to the
sspB
promoter and protects a region 233 to 264 bp upstream of the predicted −35 promoter element. The responsiveness of the
sspB
promoter to SspA was investigated with a promoter-
cat
reporter. Expression of the
sspB
promoter was reduced by over 60% in an SspA-deficient mutant of
S. gordonii
. These results indicate that expression of
S. gordonii sspB
is positively regulated by the
sspA
gene product.
Microsporidian Invasion Apparatus: Identification of a Novel Polar Tube Protein and Evidence for Clustering of
ptp1
and
ptp2
Genes in Three
Encephalitozoon
Species
Authors: Delbac Frédéric , Peuvel Isabelle , Metenier Guy , Peyretaillade Eric , Vivares Christian P.
Published: July 2002

Microsporidia are unicellular eukaryotes occuring as obligate intracellular parasites which produce resistant spores. A unique motile process is represented by the sudden extrusion of the sporal polar tube for initiating entry of the parasite into a new host cell. The complete sequence of an acidic proline-rich polar tube protein (renamed PTP1) has been previously reported for
Encephalitozoon cuniculi
and
E. hellem
. Our immunological investigations provided evidence for an additional PTP in
E. cuniculi
, termed PTP2. The corresponding gene was sequenced and then expressed in
Escherichia coli
. As expected, mouse antibodies raised against the recombinant protein reacted specifically with the polar tube. The singlecopy
ptp1
and
ptp2
genes of
E. cuniculi
were tandemly arranged on chromosome VI. Polyadenylation of the mRNAs was demonstrated. Identification and sequencing of homologous genes in the two other human-infecting
Encephalitozoon
species (
ptp2
in
E. hellem
and
ptp1
and
ptp2
in
E. intestinalis
) were facilitated by conserved gene clustering. PTP2 appears as a novel structural protein (30 kDa) with a basic lysine-rich core and an acidic tail. Unlike PTP1, this protein is devoid of large tandem repeats. The interspecies conservation of cysteine residues supports a major role of disulfide bridges in polar tube assembly. The two PTPs should serve as both molecular markers of spore differentiation and diagnostic tools.
Differential Biological and Adjuvant Activities of Cholera Toxin andEscherichia coliHeat-Labile Enterotoxin Hybrids
Authors: Bowman Christal C. , Clements John D.
Published: July 2002

Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains ofVibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains ofEscherichia coli. Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique. The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules. Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin. These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed. Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay. The results demonstrated that LT-A–CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid. Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice. Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A. Specifically, LT-A–CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-γ) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A–LT-B and native CT both produced lower levels of antigen-specific IFN-γ. Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.
Multilocus Sequence Typing ofStreptococcus pyogenesand the Relationships betweenemmType and Clone
Authors: Enright Mark C. , Spratt Brian G. , Kalia Awdhesh , Cross John H. , Bessen Debra E.
Published: July 2002

Multilocus sequence typing (MLST) is a tool that can be used to study the molecular epidemiology and population genetic structure of microorganisms. A MLST scheme was developed forStreptococcus pyogenesand the nucleotide sequences of internal fragments of seven selected housekeeping loci were obtained for 212 isolates. A total of 100 unique combinations of housekeeping alleles (allelic profiles) were identified. The MLST scheme was highly concordant with several other typing methods. Theemmtype, corresponding to a locus that is subject to host immune selection, was determined for each isolate; of the >150 distinctemmtypes identified to date, 78 are represented in this report. For a givenemmtype, the majority of isolates shared five or more of the seven housekeeping alleles. Stable associations betweenemmtype and MLST were documented by comparing isolates obtained decades apart and/or from different continents. For the 33emmtypes for which more than one isolate was examined, only fiveemmtypes were present on widely divergent backgrounds, differing at four or more of the housekeeping loci. The findings indicate that the majority ofemmtypes examined define clones or clonal complexes. In addition, an MLST database is made accessible to investigators who seek to characterize other isolates of this species via the internet (http://www.mlst.net).
Analysis of Chicken Mucosal Immune Response toEimeria tenellaandEimeria maximaInfection by Quantitative Reverse Transcription-PCR
Authors: Laurent Fabrice , Mancassola Roselyne , Lacroix Sonia , Menezes Rita , Naciri Muriel
Published: July 2002

The recent cloning of chicken genes coding for interleukins, chemokines, and other proteins involved in immune regulation and inflammation allowed us to analyze their expression during infection withEimeria. The expression levels of different genes in jejunal and cecal RNA extracts isolated from uninfected chickens and chickens infected withEimeria maximaorE. tenellawere measured using a precise quantitative reverse transcription-PCR technique. Seven days afterE. tenellainfection, expression of the proinflammatory cytokine interleukin-1β (IL-1β) mRNA was increased 80-fold. Among the chemokines analyzed, the CC chemokines K203 (200-fold) and macrophage inflammatory factor 1β (MIP-1β) (80-fold) were strongly upregulated in the infected ceca, but the CXC chemokines IL-8 and K60 were not. However, the CXC chemokines were expressed at very high levels in uninfected cecal extracts. The levels of gamma interferon (IFN-γ) (300-fold), inducible nitric oxide synthase (iNOS) (200-fold), and myelomonocytic growth factor (MGF) (50-fold) were also highly upregulated during infection withE. tenella, whereas cyclooxygenase 2 showed a more modest (13-fold) increase. The genes upregulated duringE. tenellainfection were generally also upregulated duringE. maximainfection but at a lower magnitude except for those encoding MIP-1β and MGF. For these two cytokines, no significant change in expression levels was observed afterE. maximainfection. CD3+intraepithelial lymphocytes may participate in the IFN-γ upregulation observed after infection, since both recruitment and upregulation of the IFN-γ mRNA level were observed in the infected jejunal mucosa. Moreover, in the chicken macrophage cell line HD-11, CC chemokines, MGF, IL-1β, and iNOS were inducible by IFN-γ, suggesting that macrophages may be one of the cell populations involved in the upregulation of these cytokines observed in vivo during infection withEimeria.
Recombinant Urease and Urease DNA ofCoccidioides immitisElicit an Immunoprotective Response against Coccidioidomycosis in Mice
Authors: Li Kun , Yu Jieh-Juen , Hung Chiung-Yu , Lehmann Paul F. , Cole Garry T.
Published: July 2002

Coccidioides immitisantigens which stimulate a T helper cell 1 (Th1) pathway of host immune response are considered to be essential components of a vaccine against coccidioidomycosis. Recombinant urease (rURE) and recombinant heat shock protein 60 (rHSP60) ofC. immitiswere expressed inEscherichia coliand tested as vaccine candidates in BALB/c mice. A synthetic oligodeoxynucleotide which contained unmethylated CpG dinucleotides and was previously shown to enhance a murine Th1 response was used as an immunoadjuvant. T cells isolated from the spleens and lymph nodes of the rURE- and rHSP60-immune mice showed in vitro proliferative responses to the respective recombinant protein, but only those T lymphocytes from rURE-immunized mice revealed markedly elevated levels of expression of selected Th1-type cytokine genes. BALB/c mice immunized subcutaneously with rURE and subsequently challenged by the intraperitoneal (i.p.) route with a lethal inoculum ofC. immitisarthroconidia demonstrated a significant reduction in the level ofC. immitisinfection compared to control animals. rHSP60 was much less effective as a protective antigen. Evaluation of cytokine gene expression in lung tissue and levels of recombinant urease-specific immunoglobulins (immunoglobulin G1 [IgG1] versus IgG2a) in murine sera at 12 days after challenge provided additional evidence that immunization with rURE stimulated a Th1 response to the pathogen. Urease was further evaluated by expression of theUREgene in a mammalian plasmid vector (pSecTag2A.URE) which was used to immunize mice by the intradermal route. In this case, 82% of the vector construct-immunized animals survived more than 40 days after i.p. infection, compared to only 10% of the mice immunized with the vector alone. In addition, 87% of the pSecTag2A.URE-immunized survivors had sterile lungs and spleens. These data support the need for further evaluation of theC. immitisurease as a candidate vaccine against coccidioidomycosis.
Identification and Functional Analysis of an Immunoreactive DsbA-Like Thio-Disulfide Oxidoreductase of
Ehrlichia
spp
Authors: McBride Jere W. , Ndip Lucy M. , Popov Vsevolod L. , Walker David H.
Published: July 2002

Novel homologous DsbA-like disulfide bond formation (Dsb) proteins of
Ehrlichia chaffeensis
and
Ehrlichia canis
were identified which restored DsbA activity in complemented
Escherichia coli dsbA
mutants. Recombinant
Ehrlichia
Dsb (eDsb) proteins were recognized by sera from
E. canis-
infected dogs but not from
E. chaffeensis
-infected patients. The eDsb proteins were observed primarily in the periplasm of
E. chaffeensis
and
E. canis
.
In Vivo Expression and Immunological Studies of the 42-Kilodalton Carboxyl-Terminal Processing Fragment ofPlasmodium falciparumMerozoite Surface Protein 1 in the Baculovirus-Silkworm System
Authors: Pang Alan L. Y. , Hashimoto Caryn N. , Tam Leslie Q. , Meng Z. Q. , Hui George S. N. , Ho Walter K. K.
Published: July 2002

The 42-kDa carboxyl-terminal processing fragment ofPlasmodium falciparummerozoite surface protein 1 (MSP-142) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-142was expressed by using theBombyx morinuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 μg/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-142were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freund's adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-119proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-119. Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-142. Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-142at elevated levels; thus, it is an attractive alternative for producing a protective MSP-142vaccine for human use.
A Slow-Motility Phenotype Caused by Substitutions at Residue Asp31 in the PomA Channel Component of a Sodium-Driven Flagellar Motor
Authors: Kojima Seiji , Shoji Tomokazu , Asai Yukako , Kawagishi Ikuro , Homma Michio
Published: July 2002

PomA is thought to be a component of the ion channel in the sodium-driven polar-flagellar motor of
Vibrio alginolyticus
. We have found that some cysteine substitutions in the periplasmic region of PomA result in a slow-motility phenotype, in which swarming and swimming speeds are reduced even in the presence of high concentrations of NaCl. Most of the mutants showed a sodium ion dependence similar to that of the wild type but with significantly reduced motility at all sodium ion concentrations. By contrast, motility of the D31C mutant showed a sharp dependence on NaCl concentration, with a threshold at 38 mM. The motor of the D31C mutant rotates stably, as monitored by laser dark-field microscopy, suggesting that the mutant PomA protein is assembled normally into the motor complex. Mutational studies of Asp31 suggest that, although this residue is not essential for motor rotation, a negative charge at this position contributes to optimal speed and/or efficiency of the motor.