bluuebunny/crossref_metadata_2025 huggingface dataset:
Cosecretion of Chaperones and Low-Molecular-Size Medium Additives Increases the Yield of Recombinant Disulfide-Bridged Proteins
Authors: Schäffner Jörg , Winter Jeannette , Rudolph Rainer , Schwarz Elisabeth
Published: July 2002

Attempts were made to engineer the periplasm of
Escherichia coli
to an expression compartment of heterologous proteins in their native conformation. As a first approach the low-molecular-size additive
l
-arginine and the redox compound glutathione (GSH) were added to the culture medium. Addition of 0.4 M
l
-arginine and 5 mM reduced GSH increased the yield of a native tissue-type plasminogen activator variant (rPA), consisting of the kringle-2 and the protease domain, and a single-chain antibody fragment (scFv) up to 10- and 37-fold, respectively. A variety of other medium additives also had positive effects on the yield of rPA. In a second set of experiments, the effects of cosecreted ATP-independent molecular chaperones on the yields of native therapeutic proteins were investigated. At optimized conditions, cosecretion of
E. coli
DnaJ or murine Hsp25 increased the yield of native rPA by a factor of 170 and 125, respectively. Cosecretion of DnaJ also dramatically increased the amount of a second model protein, native proinsulin, in the periplasm. The results of this study are anticipated to initiate a series of new approaches to increase the yields of native, disulfide-bridged, recombinant proteins in the periplasm of
E. coli
.
gly
Gene Cloning and Expression and Purification of Glycinecin A, a Bacteriocin Produced by
Xanthomonas campestris
pv. glycines 8ra
Authors: Heu Sunggi , Oh Jonghee , Kang Youngsung , Ryu Sangryeol , Cho Somi K. , Cho Youngsup , Cho Moonjae
Published: July 2002

Glycinecin A, a bacteriocin produced by
Xanthomonas campestris
pv. glycines, inhibits the growth of
X. campestris
pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in
Escherichia coli
. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60°C. Glycinecin A is a heterodimer consisting of 39- and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes,
glyA
and
glyB
, encoding the 39- and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences of
glyA
and
glyB
, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in
E. coli
, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from
Xanthomonas
was found in the culture media.
E. coli
transformed with either
glyA
or
glyB
separately did not show the bacteriocin activity.
Dehalogenation of Chlorinated Hydroxybiphenyls by Fungal Laccase
Authors: Schultz Asgard , Jonas Ulrike , Hammer Elke , Schauer Frieder
Published: July 2002

We have investigated the transformation of chlorinated hydroxybiphenyls by laccase produced by
Pycnoporus cinnabarinus
. The compounds used were transformed to sparingly water-soluble colored precipitates which were identified by gas chromatography-mass spectrometry as oligomerization products of the chlorinated hydroxybiphenyls. During oligomerization of 2-hydroxy-5-chlorobiphenyl and 3-chloro-4-hydroxybiphenyl, dechlorinated C—C-linked dimers were formed, demonstrating the dehalogenation ability of laccase. In addition to these nonhalogenated dimers, both monohalogenated and dihalogenated dimers were identified.
Occurrence of Beauvericin and Enniatins in Wheat Affected by
Fusarium avenaceum
Head Blight
Authors: Logrieco A. , Rizzo A. , Ferracane R. , Ritieni A.
Published: July 2002

We evaluated
Fusarium
contamination and the levels of hexadepsipeptide mycotoxins in 13 wheat samples affected by head blight in Finland.
Fusarium avenaceum
was the dominant species (91%) isolated from all samples, but isolates of
F. culmorum
(4%),
F. tricinctum
(3%), and
F. poae
(2%) also were recovered. Beauvericin (0.64 to 3.5 μg/g) was detected in all 13 samples. Enniatin B (trace to 4.8 μg/g) was detected in 12 samples, enniatin B
1
(trace to 1.9 μg/g) was detected in 8 samples, and enniatin A
1
(trace to 6.9 μg/g) was detected in 10 samples. Ten of 13 strains of
F. avenaceum
and 2 strains of
F. poae
and
F. tricinctum
produced beauvericin in culture on rice (trace to 70, 9.4, and 33 μg/g, respectively). All strains also produced enniatins (trace to 2,700 μg/g). This is the first report on the natural cooccurence of beauvericin and enniatins in wheat infected predominantly by
F. avenaceum
.
Molecular Method To Assess the Diversity of
Burkholderia
Species in Environmental Samples
Authors: Salles Joana Falcão , De Souza Francisco Adriano , van Elsas Jan Dirk
Published: July 2002

In spite of the importance of many members of the genus
Burkholderia
in the soil microbial community, no direct method to assess the diversity of this genus has been developed so far. The aim of this work was the development of soil DNA-based PCR-denaturing gradient gel electrophoresis (DGGE), a powerful tool for studying the diversity of microbial communities, for detection and analysis of the
Burkholderia
diversity in soil samples. Primers specific for the genus
Burkholderia
were developed based on the 16S rRNA gene sequence and were evaluated in PCRs performed with genomic DNAs from
Burkholderia
and non-
Burkholderia
species as the templates. The primer system used exhibited good specificity and sensitivity for the majority of established species of the genus
Burkholderia
. DGGE analyses of the PCR products obtained showed that there were sufficient differences in migration behavior to distinguish the majority of the 14
Burkholderia
species tested. Sequence analysis of amplicons generated with soil DNA exclusively revealed sequences affiliated with sequences of
Burkholderia
species, demonstrating that the PCR-DGGE method is suitable for studying the diversity of this genus in natural settings. A PCR-DGGE analysis of the
Burkholderia
communities in two grassland plots revealed differences in diversity mainly between bulk and rhizosphere soil samples; the communities in the latter samples produced more complex patterns.
Bacterial Cell Surface Display of Organophosphorus Hydrolase for Selective Screening of Improved Hydrolysis of Organophosphate Nerve Agents
Authors: Cho Catherine Mee-Hie , Mulchandani Ashok , Chen Wilfred
Published: July 2002

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface ofEscherichia coliusing the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow productp-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.
Melanin Production and Use as a Soluble Electron Shuttle for Fe(III) Oxide Reduction and as a Terminal Electron Acceptor byShewanella algaeBrY
Authors: Turick Charles E. , Tisa Louis S. , Caccavo, Frank
Published: July 2002

Dissimilatory metal-reducing bacteria (DMRB) utilize numerous compounds as terminal electron acceptors, including insoluble iron oxides. The mechanism(s) of insoluble-mineral reduction by DMRB is not well understood. Here we report that extracellular melanin is produced byShewanella algaeBrY. The extracted melanin served as the sole terminal electron acceptor. Upon reduction the reduced, soluble melanin reduced insoluble hydrous ferric oxide in the absence of bacteria, thus demonstrating that melanin produced byS. algaeBrY is a soluble Fe(III)-reducing compound. In the presence of bacteria, melanin acted as an electron conduit to Fe(III) minerals and increased Fe(III) mineral reduction rates. Growth ofS. algaeBrY occurred in anaerobic minimal medium supplemented with melanin extracted from previously grown aerobic cultures ofS. algaeBrY. Melanin produced byS. algaeBrY imparts increased versatility to this organism as a soluble Fe(III) reductant, an electron conduit for iron mineral reduction, and a sole terminal electron acceptor that supports growth.
Substrate Interactions during the Biodegradation of Benzene, Toluene, Ethylbenzene, and Xylene (BTEX) Hydrocarbons by the Fungus
Cladophialophora
sp. Strain T1
Authors: Prenafeta-Boldú F. X. , Vervoort J. , Grotenhuis J. T. C. , van Groenestijn J. W.
Published: July 2002

The soil fungus
Cladophialophora
sp. strain T1 (= ATCC MYA-2335) was capable of growth on a model water-soluble fraction of gasoline that contained all six BTEX components (benzene, toluene, ethylbenzene, and the xylene isomers). Benzene was not metabolized, but the alkylated benzenes (toluene, ethylbenzene, and xylenes) were degraded by a combination of assimilation and cometabolism. Toluene and ethylbenzene were used as sources of carbon and energy, whereas the xylenes were cometabolized to different extents.
o
-Xylene and
m
-xylene were converted to phthalates as end metabolites;
p
-xylene was not degraded in complex BTEX mixtures but, in combination with toluene, appeared to be mineralized. The metabolic profiles and the inhibitory nature of the substrate interactions indicated that toluene, ethylbenzene, and xylene were degraded at the side chain by the same monooxygenase enzyme. Our findings suggest that soil fungi could contribute significantly to bioremediation of BTEX pollution.
Pneumococcal Type 22F Polysaccharide Absorption Improves the Specificity of a Pneumococcal-Polysaccharide Enzyme-Linked Immunosorbent Assay
Authors: Concepcion Nelydia F. , Frasch Carl E.
Published: July 2002

The specificity of the immune response to the 23-valent pneumococcal-polysaccharide (PS) vaccine in healthy adults and to a pneumococcal conjugate vaccine in infants was examined by measuring immunoglobulin G (IgG) antibody titers by enzyme-linked immunosorbent assay (ELISA) and the opsonophagocytosis assay. ELISA measures total antipneumococcal IgG titers including the titers of functional and nonfunctional antibodies, while the opsonophagocytosis assay measures only functional-antibody titers. Twenty-four pairs of pre- and post-pneumococcal vaccination sera from adults were evaluated (ELISA) for levels of IgG antibodies against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Twelve of the pairs were also examined (opsonophagocytosis assay) for their functional activities. The correlation coefficients between assay results for most types ranged from 0.75 to 0.90, but the correlation coefficient was only about 0.6 for serotypes 4 and 19F. The specificities of these antibodies were further examined by the use of competitive ELISA inhibition. A number of heterologous polysaccharides (types 11A, 12F, 15B, 22F, and 33A) were used as inhibitors. Most of the sera tested showed cross-reacting antibodies, in addition to those removed by pneumococcal C PS absorption. Our data suggest the presence of a common epitope that is found on most pneumococcal PS but that is not absorbed by purified C PS. Use of a heterologous pneumococcal PS (22F) to adsorb the antibodies to the common epitope increased the correlation between the IgG ELISA results and the opsonophagocytosis assay results. The correlation coefficient improve from 0.66 to 0.92 for type 4 and from 0.63 to 0.80 for type 19F. These common-epitope antibodies were largely absent in infants at 7 months of age, suggesting the carbohydrate nature of the epitope.
Quantification of Bacterial Transcripts during Infection Using Competitive Reverse Transcription-PCR (RT-PCR) and LightCycler RT-PCR
Authors: Goerke Christiane , Bayer Manfred G. , Wolz Christiane
Published: July 2002

Bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments. In order to monitor bacterial gene expression and regulation in the host, methods for direct transcript analysis from clinical specimens are needed. For most bacterial infections, amplification of the mRNAs of interest is necessary due to the low numbers of cells present and the low levels of specific transcripts. Here we compare two methods of quantitative reverse transcription-PCR (RT-PCR)—competitive RT-PCR using a one-tube system followed by standard gel analysis and the real-time detection of PCR product formation by fluorescence resonance energy transfer technology using the LightCycler unit. We isolated
Staphylococcus aureus
RNA directly from clinical specimens obtained from cystic fibrosis patients with chronic
S. aureus
lung infection and from an animal model of foreign-body infection with no further cultivation of the bacteria. Competitive RT-PCR and LightCycler RT-PCR were tested for their ability to quantify the transcription of a constitutively expressed gyrase gene (
gyr
) and a highly regulated α-toxin gene (
hla
) of
S. aureus
. Reproducible results were obtained with both methods. A sensitivity of 10
4
(
gyr
) and 10
3
(
hla
) copies, respectively, was reached, which was sufficient for the quantification of transcripts during bacterial infection. Overall, the competitive RT-PCR is a robust technique which does not need special RNA purification. On the negative side, it is labor intensive and time consuming, thus limiting the numbers of samples which can be analyzed at a given time. LightCycler RT-PCR is very susceptible to even traces of inhibitors, but it allows high-throughput processing of samples.
Functional Antibody Activity Elicited by Fractional Doses ofHaemophilus influenzaeType b Conjugate Vaccine (Polyribosylribitol Phosphate–Tetanus Toxoid Conjugate)
Authors: Romero-Steiner Sandra , Fernandez Josefina , Biltoft Christel , Wohl Melissa E. , Sanchez Jacqueline , Feris Jesus , Balter Sharon , Levine Orin S. , Carlone George M.
Published: July 2002

We evaluated the functional activities of antibodies, serum bactericidal activity (SBA), and immunoglobulin G (IgG) antibody avidity indices, using sodium thiocyanate (NaSCN) elution, elicited after vaccination with fractional doses of theHaemophilus influenzaetype b conjugate (polyribosylribitol phosphate [PRP] conjugated to tetanus toxoid [PRP-T]) vaccine. A cohort of 600 infants from the Dominican Republic were randomized to receive one of three regimens of the PRP-T vaccine at ages 2, 4, and 6 months: full doses (10 μg of PRP antigen), one-half doses (5.0 μg), and one-third doses (3.3 μg) (J. Fernandez et al., Am. J. Trop. Med. Hyg. 62:485–490, 2000). Sixty serum samples, collected at age 7 months, with ≥2.0 μg of anti-PRP IgG per ml were randomly selected for avidity determinations. Geometric mean IgG concentrations were 13, 14, and 17 μg/ml for infants who received the full-dose (n= 19), one-half-dose (n= 19), and one-third-dose (n= 22) regimens, respectively. SBA geometric mean titers (1/dilution) were 85.0, 82.0, and 76.1 in sera from infants receiving the full-, one-half-, and one-third-dose regimens, respectively. Avidity indices (mean ± standard error weighted average of NaSCN molar concentration × serum dilution factor) were 71.9 ± 9.4, 123.6 ± 26.8, and 150.9 ± 24.9 for the full-, one-half-, and one-third-dose regimens, respectively. Upon comparison, the only significant difference (P= 0.024) found was a greater avidity index for sera from infants receiving the one-third-dose regimen than for sera from infants receiving the the full-dose regimen. We conclude that fractional doses elicit similar functional antibody activities in infants with ≥2 μg of anti-PRP IgG per ml, corresponding to 89, 90, and 97% of infants receiving three doses of either the full concentration or one-half or one-third of the labeled concentration, respectively. This approach offers an alternative strategy for the prevention ofH. influenzaetype b disease in countries with limited resources.
Improved Assessment of T-Cell Receptor (TCR) VB Repertoire in Clinical Specimens: Combination of TCR-CDR3 Spectratyping with Flow Cytometry-Based TCR VB Frequency Analysis
Authors: Pilch H. , Höhn H. , Freitag K. , Neukirch C. , Necker A. , Haddad P. , Tanner B. , Knapstein P. G. , Maeurer M. J.
Published: July 2002

Antigen-specific T-cell responses may be described by combining three categories: (i) the specificity and effector functions of a T-cell population, (ii) the quantity of T-cell responses (i.e., the number of responding T cells within the CD4/CD8 population), and (iii) the “quality” of T cells (defined by the T-cell receptor [TCR] structure). Several methods to measure T-cell responses are now available including evaluation of T-cell precursors using limiting dilution, the enzyme-linked immunospot assay, ex vivo TCR variable (v)-segment analysis determined by flow cytometry, and TCR-CDR3 length analysis (spectratyping), as well as identification of peptide-specific T cells using major histocompatibility complex (MHC) class I tetramers containing appropriate peptides. Until now, only a limited set of MHC-peptide complexes have been available as tetramer complexes. We demonstrate that CD8+or CD4+T cells in patients with cancer can be molecularly defined using a combination of spectratyping (TCR structure and “molecular composition”) plus the implementation of an antibody panel directed against 21 individual VB TCR chains (“quantity” of T-cell families). This approach is instrumental in defining and comparing the magnitudes of CD4+or CD8+T-cell responses over time in individual patients, in comparing the TCR VA and VB repertoire in different anatomic compartments, and in comparing the TCR VA-VB diversity with that in normal healthy controls. This method provides the means of objectively defining and comparing the TCR repertoire in patients undergoing vaccination protocols and underlines the necessity to calibrate the TCR-CDR3 analysis with a qualitative assessment of individual TCR VB families.
Cytotoxic Necrotizing Factor Type 1 of Uropathogenic
Escherichia coli
Kills Cultured Human Uroepithelial 5637 Cells by an Apoptotic Mechanism
Authors: Mills Melody , Meysick Karen C. , O'Brien Alison D.
Published: July 2002

Pathogenic
Escherichia coli
associated with urinary tract infections (UTIs) in otherwise healthy individuals frequently produce cytotoxic necrotizing factor type 1 (CNF1), a member of the family of bacterial toxins that target the Rho family of small GTP-binding proteins. To gain insight into the function of CNF1 in the development of
E. coli
-mediated UTIs, we examined the effects of CNF1 intoxication on a panel of human cell lines derived from physiologically relevant sites (bladder, ureters, and kidneys). We identified one uroepithelial cell line that exhibited a distinctly different CNF1 intoxication phenotype from the prototypic one of multinucleation without cell death that is seen when HEp-2 or other epithelial cells are treated with CNF1. The 5637 bladder cell line detached from the growth surface within 72 h of CNF1 intoxication, a finding that suggested frank cytotoxicity. To determine the basis for the unexpected toxic effect of CNF1 on 5637 cells, we compared the degree of toxin binding, actin fiber formation, and Rho modification with those CNF1-induced events in HEp-2 cells. We found no apparent difference in the amount of CNF1 bound to 5637 cells and HEp-2 cells. Moreover, CNF1 modified Rho, in vivo and in vitro, in both cell types. In contrast, one of the classic responses to CNF1 in HEp-2 and other epithelial cell lines, the formation of actin stress fibers, was markedly absent in 5637 cells. Indeed, actin stress fiber induction by CNF1 did not occur in any of the other human bladder cell lines that we tested (J82, SV-HUC-1, or T24). Furthermore, the appearance of lamellipodia and filopodia in 5637 cells suggested that CNF1 activated the Cdc42 and Rac proteins. Finally, apoptosis was observed in CNF1-intoxicated 5637 cells. If our results with 5637 cells reflect the interaction of CNF1 with the transitional uroepithelium in the human bladder, then CNF1 may be involved in the exfoliative process that occurs in that organ after infection with uropathogenic
E. coli
.
Phenotypic Switching in
Candida glabrata
Involves Phase-Specific Regulation of the Metallothionein Gene
MT-II
and the Newly Discovered Hemolysin Gene
HLP
Authors: Lachke Salil A. , Srikantha Thyagarajan , Tsai Luong K. , Daniels Karla , Soll David R.
Published: July 2002

Although
Candida glabrata
has emerged in recent years as a major fungal pathogen, there have been no reports demonstrating that it undergoes either the bud-hypha transition or high-frequency phenotypic switching, two developmental programs believed to contribute to the pathogenic success of other
Candida
species. Here it is demonstrated that
C. glabrata
undergoes reversible, high-frequency phenotypic switching between a white (Wh), light brown (LB), and dark brown (DB) colony phenotype discriminated on an indicator agar containing 1 mM CuSO
4
. Switching regulates the transcript level of the
MT-II
metallothionein gene(s) and a newly discovered gene for a hemolysin-like protein,
HLP
. The relative
MT-II
transcript levels in Wh, LB, and DB cells grown in the presence of CuSO
4
are 1:27:81, and the relative transcript levels of
HLP
are 1:20:35. The relative
MT-II
and
HLP
transcript levels in cells grown in the absence of CuSO
4
are 1:20:30 and 1:20:25, respectively. In contrast, switching has little or no effect on the transcript levels of the genes
MT-I
,
AMT-I
,
TRPI
,
HIS3
,
EPAI
, and
PDHI
. Switching of
C. glabrata
is not associated with microevolutionary changes identified by the DNA fingerprinting probe Cg6 and does not involve tandem amplification of the
MT-IIa
gene, which has been shown to occur in response to elevated levels of copper. Finally, switching between Wh, LB, and DB occurred in all four clinical isolates examined in this study. As in
Candida albicans
, switching in
C. glabrata
may provide colonizing populations with phenotypic plasticity for rapid responses to the changing physiology of the host, antibiotic treatment, and the immune response, through the differential regulation of genes involved in pathogenesis. More importantly, because
C. glabrata
is haploid, a mutational analysis of switching is now feasible.
Mechanisms of the Proinflammatory Response of Endothelial Cells toCandida albicansInfection
Authors: Orozco Alison S. , Zhou Xiang , Filler Scott G.
Published: July 2002

Endothelial cells can influence significantly the host inflammatory response against blood-borne microbial pathogens. Previously, we found that endothelial cells respond to in vitro infection withCandida albicansby secreting interleukin 8 (IL-8) and expressing E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). We have now examined the mechanisms mediating this endothelial cell response. We determined thatC. albicansstimulated endothelial cells to synthesize tumor necrosis factor alpha (TNF-α), which in turn induced these infected cells to secrete IL-8 and express E-selectin by an autocrine mechanism. Expression of VCAM-1 was mediated not only by TNF-α but also by IL-1α and IL-1β, all of which were synthesized by endothelial cells in response toC. albicans. These three cytokines remained primarily cell associated rather than being secreted. Candidal induction of ICAM-1 expression was independent of TNF-α, IL-1α, and IL-1β. These observations demonstrate that different proinflammatory endothelial cell responses toC. albicansare induced by distinct mechanisms. A clear understanding of these mechanisms is important for therapeutically modulating the endothelial cell response toC. albicansand perhaps other opportunistic pathogens that disseminate hematogenously.
Interaction between
Burkholderia pseudomallei
and
Acanthamoeba
Species Results in Coiling Phagocytosis, Endamebic Bacterial Survival, and Escape
Authors: Inglis Timothy J. J. , Rigby Paul , Robertson Terry A. , Dutton Nichole S. , Henderson Mandy , Chang Barbara J.
Published: July 2002

Burkholderia pseudomallei
causes melioidosis, a potentially fatal disease whose clinical outcomes include rapid-onset septicemia and relapsing and delayed-onset infections. Like other facultative intracellular bacterial pathogens,
B. pseudomallei
is capable of survival in human phagocytic cells, but unlike mycobacteria,
Listeria monocytogenes
, and
Salmonella
serovar Typhimurium, the species has not been reported to survive as an endosymbiont in free-living amebae. We investigated the consequences of exposing
Acanthamoeba astronyxis
,
A. castellani
, and
A. polyphaga
to
B. pseudomallei
NCTC 10276 in a series of coculture experiments. Bacterial endocytosis was observed in all three
Acanthamoeba
species. A more extensive range of cellular interactions including bacterial adhesion, incorporation into amebic vacuoles, and separation was observed with
A. astronyxis
in timed coculture experiments. Amebic trophozoites containing motile intravacuolar bacilli were found throughout 72 h of coculture. Confocal microscopy was used to confirm the intracellular location of endamebic
B. pseudomallei
cells. Transmission electron microscopy of coculture preparations revealed clusters of intact bacilli in membrane-lined vesicles inside the trophozoite cytoplasm; 5 × 10
2
CFU of bacteria per ml were recovered from lysed amebic trophozoites after 60 min of coculture. Demonstration of an interaction between
B. pseudomallei
and free-living acanthamebae in vitro raises the possibility that a similar interaction in vivo might affect environmental survival of
B. pseudomallei
and subsequent human exposure. Endamebic passage of
B. pseudomallei
warrants further investigation as a potential in vitro model of intracellular
B. pseudomallei
infection.
Intestinal Epithelial Cell Apoptosis followingCryptosporidium parvumInfection
Authors: McCole Declan F. , Eckmann Lars , Laurent Fabrice , Kagnoff Martin F.
Published: July 2002

Cryptosporidium parvuminduces moderate levels of apoptosis of cultured human intestinal epithelial cells, which are maximal at 24 h after infection. Apoptosis is further increased inC. parvum-infected cells by inhibition of NF-κB.C. parvuminfection also attenuates epithelial apoptosis induced by strongly proapoptotic agents. The data suggestC. parvumhas developed strategies to limit apoptosis in order to facilitate its growth and maturation in the early period after epithelial cell infection.
T-Cell Epitopes in Variable Segments ofChlamydia trachomatisMajor Outer Membrane Protein Elicit Serovar-Specific Immune Responses in Infected Humans
Authors: Ortiz Linette , Angevine Mark , Kim Seon-Kyeong , Watkins David , DeMars Robert
Published: July 2002

We previously identified 18 stimulatoryChlamydia trachomatismajor outer membrane protein (MOMP) peptides containing at least 23 epitopes presented with various HLA class II allotypes. Only one peptide contained an epitope localized in a variable segment (VS2). Continued studies reported here identified a total of five VS peptides containing T-cell epitopes that are distributed among MOMPs VS1, VS2, and VS4. Only MOMP-primed T-cell cultures from subjects infected with serovar E responded to the serovar E VS peptides, while the response of such cultures to constant-segment peptides was independent of the infecting serovar. Furthermore, MOMP-primed T cells proliferated in response only to the VS peptides encoded in serovar E but not to the corresponding peptides derived from serovar F, I, or J, confirming that these responses were serovar specific.
Identification of a New Repetitive Element in
Staphylococcus aureus
Authors: Cramton Sarah E. , Schnell Norbert F. , Götz Friedrich , Brückner Reinhold
Published: July 2002

The
Staphylococcus aureus
repeat (STAR) element is a sequence identified in two intergenic regions in
S. aureus
. The element is found in 13 to 21 copies in individual
S. aureus
strains, and elements in the homologous intergenic location are variable in length. The element sequence consists of several small and unusually GC-rich direct repeats with recurring intervening sequences. In addition, STAR-like elements may be present in related staphylococcal species.
Attenuation of and Protection Induced by a Leucine Auxotroph ofMycobacterium tuberculosis
Authors: Hondalus Mary K. , Bardarov Stoyan , Russell Robert , Chan John , Jacobs William R. , Bloom Barry R.
Published: July 2002

Attenuated mutants ofMycobacterium tuberculosisrepresent potential vaccine candidates for the prevention of tuberculosis. It is known that auxotrophs of a variety of bacteria are attenuated in vivo and yet provide protection against challenge with wild-type organisms. A leucine auxotroph ofM. tuberculosiswas created by allelic exchange, replacing wild-typeleuD(Rv2987c), encoding isopropyl malate isomerase, with a mutant copy of the gene in which 359 bp had been deleted, creating a strain requiring exogenous leucine supplementation for growth in vitro. The frequency of reversion to prototrophy was <10−11. In contrast to wild-typeM. tuberculosis, the ΔleuDmutant was unable to replicate in macrophages in vitro. Its attenuation in vivo and safety as a vaccine were established by the fact that it caused no deaths in immunodeficient SCID mice. Complementation of the mutant with wild-typeleuDabolished the requirement for leucine supplementation and restored the ability of the strain to grow both in macrophages and in SCID mice, thus confirming that the attenuated phenotype was due to the ΔleuDmutation. As a test of the vaccine potential of the leucine auxotroph, immunocompetent BALB/c mice, susceptible to fatal infection with wild-typeM. tuberculosis, were immunized with the ΔleuDmutant and subsequently challenged with virulentM. tuberculosisby both the intravenous and aerosol routes. A comparison group of mice was immunized with conventionalMycobacterium bovisBCG vaccine. Whereas all unvaccinated mice succumbed to intravenous infection within 15 weeks, mice immunized with either BCG or the ΔleuDmutant ofM. tuberculosisexhibited enhanced and statistically equivalent survival curves. However, theleuDauxotroph was less effective than live BCG in reducing organ burdens and tissue pathology of mice challenged by either route. We conclude that attenuation and protection againstM. tuberculosischallenge can be achieved with a leucine auxotroph and suggest that to induce optimal protection, attenuated strains ofM. tuberculosisshould persist long enough and be sufficiently metabolically active to synthesize relevant antigens for an extended period of time.
Interleukin-6 Induces Early Gamma Interferon Production in the Infected Lung but Is Not Required for Generation of Specific Immunity toMycobacterium tuberculosisInfection
Authors: Saunders Bernadette M. , Frank Anthony A. , Orme Ian M. , Cooper Andrea M.
Published: July 2002

Immunity toMycobacterium tuberculosisis dependent upon the generation of a protective gamma interferon (IFN-γ)-producing T-cell response. Recent studies have suggested that interleukin-6 (IL-6) is required for the induction of a protective T-cell response and that IL-4 may suppress the induction of IFN-γ. To evaluate the role of the cytokines IL-6 and IL-4 in the generation of pulmonary immunity toM. tuberculosis, IL-6 and IL-4 knockout mice were infected withM. tuberculosisvia aerosol. The absence of IL-6 led to an early increase in bacterial load with a concurrent delay in the induction of IFN-γ. However, mice were able to contain and control bacterial growth and developed a protective memory response to secondary infection. This demonstrates that while IL-6 is involved in stimulating early IFN-γ production, it is not essential for the development of protective immunity againstM. tuberculosis. In contrast, while the absence of IL-4 resulted in increased IFN-γ production, this had no significant effect upon bacterial growth.
Prevalence of Virulence Genes and Clonality in
Escherichia coli
Strains That Cause Bacteremia in Cancer Patients
Authors: Hilali Farida , Ruimy Raymond , Saulnier Patrick , Barnabé Christian , Lebouguénec Chantal , Tibayrenc Michel , Andremont Antoine
Published: July 2002

Phenotypic analysis of
Escherichia coli
strains causing bacteremia in cancer patients suggests that they possess specific virulence properties. To investigate this hypothesis, we compared the frequency of the virulence-related genes
cnf1
,
cnf2
,
papC
,
hlyC
, and
iut
in 155
E. coli
strains isolated from hospitalized cancer patients with epidemiologically unrelated cases of bacteremia to their frequency in 70
E. coli
strains isolated from the feces of healthy unrelated volunteers. Of the blood isolates, 24, 37, and 26% were positive for
cnf1
,
papC
, and
hlyC
, respectively, versus only 6, 17, and 6% of the fecal isolates (
P
< 0.05 in all instances). By contrast, 47% of both isolates carried the
iut
gene. The patients' clinical characteristics did not significantly influence these frequencies. The presence on various pathogenicity islands (PAIs) of a combination of the
cnf1
,
papC
, and
hlyC
genes on the chromosome was strongly suggested by Southern blotting of pulsed-field gel electrophoresis (PFGE) patterns with specific DNA probes. The phylogenetic relatedness among 60 strains carrying three, two, one, or no virulence genes and 6 ECOR strains included as references was determined by neighbor joining, the unweighted pair-group method with arithmetic mean, and Wagner analysis of the randomly amplified polymorphic DNA (RAPD) patterns generated by 11 primers. Identification of a major cluster including 96.4% of the strains carrying the
cnf1
,
papC
, and
hlyC
genes and ECOR subgroup B2 strains suggested that the virulent
E. coli
strains causing bacteremia in cancer patients are closely related to ECOR B2 strains. The presence in the
E. coli
population surveyed of a strong linkage disequilibrium, and especially of a highly significant correlation between PFGE and RAPD genetic distances, confirms that clonal propagation has a major impact on the
E. coli
population structure. Nevertheless, low bootstrap values in the phylogenetic tree suggested that frequent genetic exchange inhibits the individualization of discrete genetic lineages, which are stable on an evolutionary scale.
Granulocyte-Macrophage Colony-Stimulating Factor-Deficient Mice Have Impaired Resistance to Blood-Stage Malaria
Authors: Riopel Julie , Tam MiFong , Mohan Karkada , Marino Michael W. , Stevenson Mary M.
Published: July 2002

The contribution of granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting GM-CSF-deficient (knockout [KO]) mice withPlasmodium chabaudiAS. KO mice were more susceptible to infection than wild-type (WT) mice, as evidenced by higher peak parasitemia, recurrent recrudescent parasitemia, and high mortality.P. chabaudiAS-infected KO mice had impaired splenomegaly and lower leukocytosis but equivalent levels of anemia compared to infected WT mice. Both bone marrow and splenic erythropoiesis were normal in infected KO mice. However, granulocyte-macrophage colony formation was significantly decreased in these tissues of uninfected and infected KO mice, and the numbers of macrophages in the spleen and peritoneal cavity were significantly lower than in infected WT mice. Serum levels of gamma interferon (IFN-γ) were found to be significantly higher in uninfected KO mice, and the level of this cytokine was not increased during infection. In contrast, IFN-γ levels were significantly above normal levels in infected WT mice. During infection, tumor necrosis factor alpha (TNF-α) levels were significantly increased in KO mice and were significantly higher than TNF-α levels in infected WT mice. Our results indicate that GM-CSF contributes to resistance toP. chabaudiAS infection and that it is involved in the development of splenomegaly, leukocytosis, and granulocyte-macrophage hematopoiesis. GM-CSF may also regulate IFN-γ and TNF-α production and activity in response to infection. The abnormal responses seen in infected KO mice may be due to the lack of GM-CSF during development, to the lack of GM-CSF in the infected mature mice, or to both.
Impaired Pulmonary NF-κB Activation in Response to Lipopolysaccharide in NADPH Oxidase-Deficient Mice
Authors: Koay M. Audrey , Christman John W. , Segal Brahm H. , Venkatakrishnan Annapurna , Blackwell Thomas R. , Holland Steven M. , Blackwell Timothy S.
Published: July 2002

Reactive oxygen species (ROS) are thought to be involved in intracellular signaling, including activation of the transcription factor NF-κB. We investigated the role of NADPH oxidase in the NF-κB activation pathway by utilizing knockout mice (p47phox−/−) lacking the p47phoxcomponent of NADPH oxidase. Wild-type (WT) controls and p47phox−/−mice were treated with intraperitoneal (i.p.)Escherichia colilipopolysaccharide (LPS) (5 or 20 μg/g of body weight). LPS-induced NF-κB binding activity and accumulation of RelA in nuclear protein extracts of lung tissue were markedly increased in WT compared to p47phox−/−mice 90 min after treatment with 20 but not 5 μg of i.p. LPS per g. In another model of lung inflammation, RelA nuclear translocation was reduced in p47phox−/−mice compared to WT mice following treatment with aerosolized LPS. In contrast to NF-κB activation in p47phox−/−mice, LPS-induced production of macrophage inflammatory protein 2 in the lungs and neutrophilic lung inflammation were not diminished in these mice compared to WT mice. We conclude that LPS-induced NF-κB activation is deficient in the lungs of p47phox−/−mice compared to WT mice, but this abnormality does not result in overt alteration in the acute inflammatory response.
Immunization with theChlamydia trachomatisMouse Pneumonitis Major Outer Membrane Protein Can Elicit a Protective Immune Response against a Genital Challenge
Authors: Pal Sukumar , Theodor Ida , Peterson Ellena M. , de la Maza Luis M.
Published: July 2002

Infertility, ectopic pregnancy, and chronic abdominal pain are frequent complications of genital infections withChlamydia trachomatis. In an attempt to produce a vaccine to protect against this pathogen we purified and refolded theC. trachomatismouse pneumonitis (MoPn) major outer membrane protein (MOMP). This preparation, mixed with Freund's adjuvant using vortexing or sonication, was used to immunize BALB/c mice that were subsequently challenged in the upper genital tract. Vaginal cultures were taken on a weekly basis, and mice were mated 6 weeks after the challenge. Gels of the vortexed MOMP showed a predominant band with a molecular size of 62 kDa and weaker bands at 42 and 132 kDa, while the sonicated MOMP had a single band with a molecular size of 42 kDa. Following immunization with these two preparations, strong humoral and cell-mediated immune responses were detected in the mice inoculated with the vortexed MOMP. On the other hand, mice immunized with the sonicated MOMP had a strong humoral immune response but a relatively weak cell-mediated immune response, as determined by a T-cell lymphoproliferative assay and level of cytokine production by splenocytes. Vaginal cultures showed that the mice immunized with the vortexed MOMP were significantly protected, as determined by a decrease in the number of animals with positive cultures, the length of time the mice shed viable organisms, and the number of inclusion-forming units recovered per mouse. Animals immunized with the sonicated MOMP, on the other hand, showed a weaker level of protection based on the same three parameters. After mating, the number of fertile animals and number of embryos per mouse were significantly higher for the mice immunized with vortexed MOMP, but not for the mice immunized with sonicated MOMP, compared to those of the control groups. In conclusion, immunization with a purified and refolded preparation of theC. trachomatisMoPn MOMP confers a significant level of protection in mice against a genital challenge.
Heme Utilization in
Bordetella avium
Is Regulated by RhuI, a Heme-Responsive Extracytoplasmic Function Sigma Factor
Authors: Kirby Amy E. , Metzger Daniel J. , Murphy Erin R. , Connell Terry D.
Published: July 2002

Efficient utilization of heme as an iron (Fe) source by
Bordetella avium
requires
bhuR
, an Fe-regulated gene which encodes an outer membrane heme receptor. Upstream of
bhuR
is a 507-bp open reading frame, hereby designated
rhuI
(for regulator of heme uptake), which codes for a 19-kDa polypeptide. Whereas the 19-kDa polypeptide had homology to a subfamily of alternative sigma factors known as the extracytoplasmic function (ECF) sigma factors, it was hypothesized that
rhuI
encoded a potential in-
trans
regulator of the heme receptor gene in
trans
. Support for the model was strengthened by the identification of nucleotide sequences common to ECF sigma-dependent promoters in the region immediately upstream of
bhuR
. Experimental evidence for the regulatory activities of
rhuI
was first revealed by recombinant experiments in which overproduction of
rhuI
was correlated with a dramatically increased expression of BhuR. A putative
rhuI
-dependent
bhuR
promoter was identified in the 199-bp region located proximal to
bhuR
. When a transcriptional fusion of the 199-bp region and a promoterless
lacZ
gene was introduced into
Escherichia coli
, promoter activity was evident, but only when
rhuI
was coexpressed in the cell. Sigma competition experiments in
E
.
coli
demonstrated that
rhuI
conferred biological properties on the cell that were consistent with RhuI having sigma factor activity. Heme, hemoglobin, and several other heme-containing proteins were shown to be the extracellular inducers of the
rhuI
-dependent regulatory system. Fur titration assays indicated that expression of
rhuI
was probably Fur dependent.
Characterization of Auxotrophic Mutants of
Mycobacterium tuberculosis
and Their Potential as Vaccine Candidates
Authors: Smith Debbie A. , Parish Tanya , Stoker Neil G. , Bancroft Gregory J.
Published: July 2002

Auxotrophic mutants of
Mycobacterium tuberculosis
have been proposed as new vaccine candidates. We have analyzed the virulence and vaccine potential of
M. tuberculosis
strains containing defined mutations in genes involved in methionine (
metB
), proline (
proC
), or tryptophan (
trpD
) amino acid biosynthesis. The
metB
mutant was a prototrophic strain, whereas the
proC
and
trpD
mutants were auxotrophic for proline and tryptophan, respectively. Following infection of murine bone marrow-derived macrophages, H37Rv and the
metB
mutant strain survived intracellularly for over 10 days, whereas over 90% of
proC
and
trpD
mutants were killed during this time. In SCID mice, both H37Rv and the
metB
mutant were highly virulent, with mouse median survival times (MST) of 28.5 and 42 days, respectively. The
proC
mutant was significantly attenuated (MST, 130 days), whereas the
trpD
mutant was essentially avirulent in an immunocompromised host. Following infection of immunocompetent DBA mice with H37Rv, mice survived for a median of 83.5 days and the
metB
mutant now showed a clear reduction in virulence, with two of five infected mice surviving for 360 days. Both
proC
and
trpD
mutants were avirulent (MST of >360 days). In vaccination studies, prior infection with either the
proC
or
trpD
mutant gave protection equivalent (
proC
mutant) to or better (
trpD
mutant) than BCG against challenge with
M. tuberculosis
H37Rv. In summary,
proC
and
trpD
genes are essential for the virulence of
M. tuberculosis
, and mutants with disruptions in either of these genes show strong potential as vaccine candidates.
Comparison of
Vibrio cholerae
Pathogenicity Islands in Sixth and Seventh Pandemic Strains
Authors: Karaolis David K. R. , Lan Ruiting , Kaper James B. , Reeves Peter R.
Published: July 2002

Epidemic
Vibrio cholerae
strains possess a large cluster of essential virulence genes on the chromosome called the
Vibrio
pathogenicity island (VPI). The VPI contains the
tcp
gene cluster encoding the type IV pilus toxin-coregulated pilus colonization factor which can act as the cholera toxin bacteriophage (CTXΦ) receptor. The VPI also contains genes that regulate virulence factor expression. We have fully sequenced and compared the VPI of the seventh-pandemic (El Tor biotype) strain N16961 and the sixth-pandemic (classical biotype) strain 395 and found that the N16961 VPI is 41,272 bp and encodes 29 predicted proteins, whereas the 395 VPI is 41,290 bp. In addition to various nucleotide and amino acid polymorphisms, there were several proteins whose predicted size differed greatly between the strains as a result of frameshift mutations. We hypothesize that these VPI sequence differences provide preliminary evidence to help explain the differences in virulence factor expression between epidemic strains (i.e., the biotypes) of
V. cholerae
.
Local and Systemic Immune Responses to Rectal Administration of Recombinant Cholera Toxin B Subunit in Humans
Authors: Jertborn Marianne , Nordström Inger , Kilander Anders , Czerkinsky Cecil , Holmgren Jan
Published: July 2002

The induction of immune responses to rectally administered recombinant cholera toxin B subunit (CTB) in humans was studied. Three immunizations induced high levels of CTB-specific antibody-secreting cells, particular of the immunoglobulin A isotype, in both rectum and peripheral blood. Antitoxin antibody responses in rectal secretions and serum were also found.
Cloning and Expression of Two Novel Hemin Binding Protein Genes fromTreponema denticola
Authors: Xu Xiaoping , Holt Stanley C. , Kolodrubetz David
Published: July 2002

Treponema denticoladoes not appear to produce siderophores, so it must acquire iron by other pathways. Indeed,T. denticolahas been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability. To characterize the HbpA protein, its gene was cloned from genomic DNA libraries ofT. denticola. Sequence analysis of thehbpAopen reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide. HbpA has no significant homology to any proteins in the databases. Southern blot analysis demonstrated thathbpAis present in severalT. denticolaATCC strains and clinical isolates, but not inTreponema pectinovorum, Treponema socranskii, orEscherichia coli. HbpA, expressed as a recombinant protein inE. coliand purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining. Northern blot analysis showed that there were twohbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology tohbpA. This downstream gene, calledhbpB, was cloned and sequenced and its product was expressed as a fusion protein inE. coli. ThehbpBgene product is 49% identical to HbpA and binds hemin. Thus,T. denticolahas two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway.