bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro and In Vivo Antifungal Activities of TAK-456, a Novel Oral Triazole with a Broad Antifungal Spectrum
Authors: Tsuchimori Noboru , Hayashi Ryogo , Kitamoto Naomi , Asai Kentaro , Kitazaki Tomoyuki , Iizawa Yuji , Itoh Katsumi , Okonogi Kenji
Published: July 2002

TAK-456 is a novel oral triazole compound with potent and broad-spectrum in vitro antifungal activity and strong in vivo efficacy against
Candida albicans
and
Aspergillus fumigatus
. TAK-456 inhibited sterol synthesis of
C. albicans
and
A. fumigatus
by 50% at 3 to 11 ng/ml. TAK-456 showed strong in vitro activity against clinical isolates of
Candida
spp.,
Aspergillus
spp., and
Cryptococcus neoformans
, except for
Candida glabrata
. The MICs at which 90% of the isolates tested were inhibited byTAK-456, fluconazole, itraconazole, voriconazole, and amphotericin B were 0.25, 4, 0.5, 0.13, and 0.5 μg/ml, respectively, for clinical isolates of
C. albicans
and 1, >64, 0.5, 0.5, and 0.5 μg/ml, respectively, for clinical isolates of
A. fumigatus
. Therapeutic activities of TAK-456 and reference triazoles against systemic lethal infections caused by
C. albicans
and
A. fumigatus
in mice were investigated by orally administering drugs once daily for 5 days, and efficacies of the compounds were evaluated by the prolongation of survival. In normal mice, TAK-456 and fluconazole were effective against infection caused by fluconazole-susceptible
C. albicans
at a dose of 1 mg/kg. In transiently neutropenic mice, therapeutic activity of TAK-456 at 1 mg/kg of body weight against infection with the same strain was stronger than those at 1 mg/kg of fluconazole. TAK-456 was effective against infections with two strains of fluconazole-resistant
C. albicans
at a dose of 10 mg/kg. TAK-456 also expressed activities similar to or higher than those of itraconazole against the infections caused by two strains of
A. fumigatus
in neutropenic mice at a dose of 10 mg/kg. These results suggest that TAK-456 is a promising candidate for development for the treatment of candidiasis and aspergillosis in humans.
Enhancement of Secretion and Extracellular Stability of Staphylokinase in
Bacillus subtilis
by
wprA
Gene Disruption
Authors: Lee Sang Jun , Kim Dong Min , Bae Kwang Hee , Byun Si Myung , Chung Jae Hoon
Published: July 2002

Staphylokinase (SAK), a polypeptide secreted by
Staphylococcus aureus
, is a plasminogen activator with a therapeutic potential in thrombosis diseases. A
Bacillus subtilis
strain which is multiply deficient in exoproteases was transformed by an expression plasmid carrying a promoter and a signal sequence of subtilisin fused in frame with the
sak
open reading frame. However, the amount of SAK secretion was marginal (45 mg/liter). In contrast, disruption of the
wprA
gene, which encodes a subtilisin-type protease, strongly promoted the production of SAK in the stationary phase (181 mg/liter). In addition, the extracellular stability of mature SAK was dramatically enhanced. These data indicate a significant role of the
wprA
gene product in degrading foreign proteins, both during secretion and in the extracellular milieu.
Pathogenic Role of SEF14, SEF17, and SEF21 Fimbriae in
Salmonella enterica
Serovar Enteritidis Infection of Chickens
Authors: Rajashekara Gireesh , Munir Shirin , Alexeyev Mikhail F. , Halvorson David A. , Wells Carol L. , Nagaraja Kakambi V.
Published: July 2002

Very little is known about the contribution of surface appendages of
Salmonella enterica
serovar Enteritidis to pathogenesis in chickens. This study was designed to clarify the role of SEF14, SEF17, and SEF21 fimbriae in serovar Enteritidis pathogenesis. Stable, single, defined
sefA
(SEF14),
agfA
(SEF17), and
fimA
(SEF21) insertionally inactivated fimbrial gene mutants of serovar Enteritidis were constructed. All mutant strains invaded Caco-2 and HT-29 enterocytes at levels similar to that of the wild type. Both mutant and wild-type strains were ingested equally well by chicken macrophage cell lines HD11 and MQ-NCSU. There were no significant differences in the abilities of these strains to colonize chicken ceca. The SEF14
−
strain was isolated in lower numbers from the livers of infected chickens and was cleared from the spleens faster than other strains. No significant differences in fecal shedding of these strains were observed.
Mycotoxins in Crude Building Materials from Water-Damaged Buildings
Authors: Tuomi Tapani , Reijula Kari , Johnsson Tom , Hemminki Kaisa , Hintikka Eeva-Liisa , Lindroos Outi , Kalso Seija , Koukila-Kähkölä Pirkko , Mussalo-Rauhamaa Helena , Haahtela Tari
Published: July 2002

We analyzed 79 bulk samples of moldy interior finishes from Finnish buildings with moisture problems for 17 mycotoxins, as well as for fungi that could be isolated using one medium and one set of growth conditions. We found the aflatoxin precursor, sterigmatocystin, in 24% of the samples and trichothecenes in 19% of the samples. Trichothecenes found included satratoxin G or H in five samples; diacetoxyscirpenol in five samples; and 3-acetyl-deoxynivalenol, deoxynivalenol, verrucarol, or T-2-tetraol in an additional five samples. Citrinine was found in three samples.
Aspergillus versicolor
was present in most sterigmatocystin-containing samples, and
Stachybotrys
spp. were present in the samples where satratoxins were found. In many cases, however, the presence of fungi thought to produce the mycotoxins was not correlated with the presence of the expected compounds. However, when mycotoxins were found, some toxigenic fungi usually were present, even if the species originally responsible for producing the mycotoxin was not isolated. We conclude that the identification and enumeration of fungal species present in bulk materials are important to verify the severity of mold damage but that chemical analyses are necessary if the goal is to establish the presence of mycotoxins in moldy materials.
A Comparison of Methods for Counting Viruses in Aquatic Systems
Authors: Bettarel Yvan , Sime-Ngando Telesphore , Amblard Christian , Laveran Henri
Published: July 2002

In this study, we compared different methods—including transmission electron microscopy—and various nucleic acid labeling methods in which we used the fluorochromes 4′,6′-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylmethyledene]-1-(3′-trimethyl ammoniumpropyl)-quinilinium diioide (YOPRO-1), and SYBR Green I, which can be detected by epifluorescence microscopy (EM), for counting viruses in samples obtained from freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results showed that the greatest efficiency for all ecosystems was obtained when we used the EM counting protocol in which YOPRO-1 was the label, as this fluorochrome exhibited strong and very stable fluorescence. A modification of the original protocol in which YOPRO-1 was used is recommended, because this modification makes the protocol faster and allows it to be used for routine analysis of fixed samples. Because SYBR Green I fades very quickly, the use of this fluorochrome is not recommended for systems in which the viral content is very high (>10
8
particles/ml), such as treated domestic sewage effluents. Experiments in which we used DNase and RNase revealed that the number of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids.
Alterations in the Proteome of the
Euprymna scolopes
Light Organ in Response to Symbiotic
Vibrio fischeri
Authors: Doino Lemus Judith , McFall-Ngai Margaret J.
Published: July 2002

During the onset of the cooperative association between the Hawaiian sepiolid squid
Euprymna scolopes
and the marine luminous bacterium
Vibrio fischeri
, the anatomy and morphology of the host's symbiotic organ undergo dramatic changes that require interaction with the bacteria. This morphogenetic process involves an array of tissues, including those in direct contact with, as well as those remote from, the symbiotic bacteria. The bacteria induce the developmental program soon after colonization of the organ, although complete morphogenesis requires 96 h. In this study, to determine critical time points, we examined the biochemistry underlying bacterium-induced host development using two-dimensional polyacrylamide gel electrophoresis. Specifically,
V. fischeri
-induced changes in the soluble proteome of the symbiotic organ during the first 96 h of symbiosis were identified by comparing the protein profiles of symbiont-colonized and uncolonized organs. Both symbiosis-related changes and age-related changes were analyzed to determine what proportion of the differences in the proteomes was the result of specific responses to interaction with bacteria. Although no differences were detected over the first 24 h, numerous symbiosis-related changes became apparent at 48 and 96 h and were more abundant than age-related changes. In addition, many age-related protein changes occurred 48 h sooner in symbiotic animals, suggesting that the interaction of squid tissue with
V. fischeri
cells accelerates certain developmental processes of the symbiotic organ. These data suggest that
V. fischeri
-induced modifications in host tissues that occur in the first 24 h of the symbiosis are independent of marked alterations in the patterns of abundant proteins but that the full 4-day morphogenetic program requires significant alteration of the host soluble proteome.
Non-Growth-Associated Demethylation of Dimethylsulfoniopropionate by (Homo)acetogenic Bacteria
Authors: Jansen Michael , Hansen Theo A.
Published: July 2002

The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied. Five
Eubacterium limosum
strains (including the type strain),
Sporomusa ovata
DSM 2662
T
,
Sporomusa sphaeroides
DSM 2875
T
, and
Acetobacterium woodii
DSM 1030
T
were shown to demethylate DMSP stoichiometrically to MTPA. The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass. The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens. In batch cultures of
E. limosum
PM31 DMSP and glycine betaine were demethylated simultaneously. In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol. In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h
−1
) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min
−1
mg of protein
−1
, both after growth in the presence of DMSP and after growth in its absence. In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min
−1
mg of protein
−1
were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower. A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero.
Gene Integration and Expression and Extracellular Secretion of
Erwinia chrysanthemi
Endoglucanase CelY (
celY
) and CelZ (
celZ
) in Ethanologenic
Klebsiella oxytoca
P2
Authors: Zhou Shengde , Davis F. C. , Ingram L. O.
Published: July 2002

The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) of
Klebsiella oxytoca
M5A1 with the native ability to metabolize cellobiose, the need for supplemental β-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. Genes
celY
and
celZ
from
Erwinia chrysanthemi
have been functionally integrated into the chromosome of P2 using surrogate promoters from
Zymomonas mobilis
for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.
EndB, a Multidomain Family 44 Cellulase from
Ruminococcus flavefaciens
17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another
R. flavefaciens
Protein via a Dockerin Domain
Authors: Rincón Marco T. , McCrae Sheila I. , Kirby James , Scott Karen P. , Flint Harry J.
Published: July 2002

The mechanisms by which cellulolytic enzymes and enzyme complexes in
Ruminococcus
spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene
endB
from
Ruminococcus flavefaciens
17 was purified as a His-tagged product after expression in
Escherichia coli
and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown
R. flavefaciens
that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945–1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.
Recovery and Analysis of Formyltetrahydrofolate Synthetase Gene Sequences from Natural Populations of Acetogenic Bacteria
Authors: Leaphart Adam B. , Lovell Charles R.
Published: July 2002

Primers for PCR amplification of partial (1,102 of 1,680 bp) formyltetrahydrofolate synthetase (FTHFS) gene sequences were developed and tested. Partial FTHFS sequences were successfully amplified from DNA from pure cultures of known acetogens, from other FTHFS-producing organisms, from the roots of the smooth cordgrass,
Spartina alterniflora
, and from fresh horse manure. The amplimers recovered were cloned, their nucleotide sequences were determined, and their translated amino acid sequences were used to construct phylogenetic trees. We found that FTHFS sequences from homoacetogens formed a monophyletic cluster that did not contain sequences from nonhomoacetogens and that FTHFS sequences appear to be informative regarding major physiological features of FTHFS-producing organisms.
Prevalence, Antibiotic Susceptibility, and Diversity of
Escherichia coli
O157:H7 Isolates from a Longitudinal Study of Beef Cattle Feedlots
Authors: Galland John C. , Hyatt Doreene R. , Crupper Scott S. , Acheson David W.
Published: July 2002

Prevalence, antibiotic susceptibility, and genetic diversity were determined for
Escherichia coli
O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of
E. coli
O157:H7 were identified by use of bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were
eaeA
gene positive.
Escherichia coli
O157:H7 was identified in at least one of the four feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections by PCR, but consecutive positive collections at a single feedlot were rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA, and 0.08% by PCR). No detectable differences in prevalence or antibiotic resistance were found between isolates collected from home pens and those from hospital pens, where antibiotic use is high. Resistant isolates were found for six of the eight antibiotics that could be used to treat
E. coli
infections in food animals, but few isolates were multidrug resistant. The high diversity of isolates as measured by random amplification of polymorphic DNA and other characteristics indicates that the majority of isolates were unique and did not persist at a feedlot, but probably originated from incoming cattle. The most surprising finding was the low frequency of virulence markers among
E. coli
isolates identified initially by C/LA as
E. coli
O157:H7. These results demonstrate that better ways of screening and confirming
E. coli
O157:H7 isolates are required for accurate determination of prevalence.
Interaction of the ΦHSIC Virus with Its Host: Lysogeny or Pseudolysogeny?
Authors: Williamson S. J. , McLaughlin M. R. , Paul J. H.
Published: July 2002

The marine phage ΦHSIC has been previously reported to enter into a lysogenic relationship with its host, HSIC, identified asListonella pelagia. This phage produces a variety of plaques on its host, including turbid and haloed plaques, from which lysogens were previously isolated. These lysogens were unstable during long-term storage at −80°C and were lost. When HSIC was reinfected with phage ΦHSIC, pseudolysogen-like interactions between the phage and its host were observed. The cells (termed HSIC-2 or HSIC-2e) produced high viral titers (1011ml−1) in the absence of inoculating phage and yet reached culture densities of nearly 109ml−1. Prophages were not induced by mitomycin C or the polyaromatic hydrocarbon naphthalene in cells harboring such infections. However, such cells were homoimmune to superinfection. Colonies hybridized strongly with a gene probe from a 100-bp fragment of the ΦHSIC genome, while the host did not. Analysis of chromosomal DNA preparations suggested the presence of a chromosomally integrated prophage. Phage adsorption experiments suggested that HSIC-2 was adsorption impaired. Because of the chromosomal prophage integration and homoimmunity, we interpret these results to indicate that ΦHSIC establishes a lysogenic relationship with its host that involves an extremely high level of spontaneous induction. This could be caused by a weak repressor of phage production. Additionally, poor phage adsorption of HSIC-2 compared to the wild type probably helped maintain this pseudolysogen-like relationship. In many ways, pseudolysogenic phage-host interactions may provide a paradigm for phage-host interactions in the marine environment.
Sequence Analysis of Insecticidal Genes from
Xenorhabdus nematophilus
PMFI296
Authors: Morgan J. Alun W. , Sergeant Martin , Ellis Debbie , Ousley Margaret , Jarrett Paul
Published: July 2002

Three strains of
Xenorhabdus nematophilus
showed insecticidal activity when fed to
Pieris brassicae
(cabbage white butterfly) larvae. From one of these strains (
X. nematophilus
PMFI296) a cosmid genome library was prepared in
Escherichia coli
and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in
E. coli
(50% lethal concentration [LC
50
] of 2 to 6 μg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (
xptA1, -A2, -B1, -C1
, and
-D1
) showed homology with up to 49% identity to insecticidal toxins identified in
Photorhabdus luminescens
, and also a smaller gene (
chi
) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes
xptA2, xptD1
, and
chi
were not important for the expression of insecticidal activity toward
P. brassicae
. One gene (
xptA1
) was found to be central for the expression of activity, and the genes
xptB1
and
xptC1
were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this
E. coli
clone. Although multiple genes may be needed for full activity,
E. coli
cells expressing the
xptA1
gene from the bacteriophage lambda
P
L
promoter were shown to have insecticidal activity (LC
50
of 112 μg of total protein/g of diet). This is contrary to the toxin genes identified in
P. luminescens
, which were not insecticidal when expressed individually in
E. coli
. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the
E. coli
clone expressing
xptA1
. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of
X. nematophilus
PMFI296.
Energy-Based Dynamic Model for Variable Temperature Batch Fermentation by
Lactococcus lactis
Authors: Dougherty Daniel P. , Breidt, Frederick , McFeeters Roger F. , Lubkin Sharon R.
Published: July 2002

We developed a mechanistic mathematical model for predicting the progression of batch fermentation of cucumber juice by
Lactococcus lactis
under variable environmental conditions. In order to overcome the deficiencies of presently available models, we use a dynamic energy budget approach to model the dependence of growth on present as well as past environmental conditions. When parameter estimates from independent experimental data are used, our model is able to predict the outcomes of three different temperature shift scenarios. Sensitivity analyses elucidate how temperature affects the metabolism and growth of cells through all four stages of fermentation and reveal that there is a qualitative reversal in the factors limiting growth between low and high temperatures. Our model has an applied use as a predictive tool in batch culture growth. It has the added advantage of being able to suggest plausible and testable mechanistic assumptions about the interplay between cellular energetics and the modes of inhibition by temperature and end product accumulation.
Clustering of Clinical Strains of
Helicobacter pylori
Analyzed by Two-Dimensional Gel Electrophoresis
Authors: Enroth Helena , Åkerlund Thomas , Sillén Anna , Engstrand Lars
Published: July 2002

Strain variations of
Helicobacter pylori
have been tested by numerous methods and compared among different patient groups. The aim of this study was to investigate whether
H. pylori
expresses disease-specific proteins that can be detected by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE).
H. pylori
strains isolated from duodenal ulcer, gastric cancer, and gastritis patients were analyzed. Extensive variation in spot patterns was observed between the strains, but a dendrogram analysis revealed that some strains within each disease group clustered together. Eight proteins were sequenced and found in the
H. pylori
genome sequence. 2-D PAGE is a useful method for studies of protein expression and for highlighting the extensive strain variation that
H. pylori
exhibits.
Production of a Mouse Antiserum toBacteroides fragilisEnterotoxin Using a Recombinant Enterotoxin Precursor
Authors: Sandini Silvia , d'Abusco Anna Scotto , La Valle Roberto , Pantosti Annalisa
Published: July 2002

The precursor of theBacteroides fragilismetalloprotease enterotoxin was cloned and expressed inEscherichia coli, which was not able to process the precursor into the biologically active enterotoxin. Mouse antiserum elicited to the recombinant precursor reacted with the purified enterotoxin and with a crude enterotoxin preparation from an enterotoxigenic strain. The antiserum neutralized the cytotoxic activity of the enterotoxin in HT-29 cells.
Type 1 Fimbriation and Its Phase Switching in Diarrheagenic
Escherichia coli
Strains
Authors: Iida Ken-Ichiro , Mizunoe Yoshimitsu , Wai Sun Nyunt , Yoshida Shin-Ichi
Published: July 2002

Type 1 fimbriae can be expressed by most
Escherichia coli
strains and mediate mannose-sensitive (MS) adherence to mammalian epithelial cells. However, the role of type 1 fimbriae in enteric pathogenesis has been unclear. Expression of type 1 fimbriae in
E. coli
is phase variable and is associated with the inversion of a short DNA element (
fim
switch). Forty-six strains of diarrheagenic
E. coli
were examined for the expression of type 1 fimbriae. Only four of these strains were originally type 1 fimbriated. Seventeen strains, originally nonfimbriated, expressed type 1 fimbriae in association with off-to-on inversion of the
fim
switch, after serial passages in static culture. The switching frequencies of these strains, from fimbriate to nonfimbriate, were greater than that of the laboratory strain
E. coli
K-12. None of the 16 strains of serovar O157:H7 or O157:H
−
expressed type 1 fimbriae after serial passages in static culture. The nucleotide sequence analysis of the
fim
switch region revealed that all of the O157:H7 and O157:H
−
strains had a 16-bp deletion in the invertible element, and the
fim
switch was locked in the “off” orientation. The results suggest that expression of type 1 fimbriae may be regulated differently in different
E. coli
pathogens causing enteric infections.
Identification ofAcinetobacterIsolates from Species Belonging to theAcinetobacter calcoaceticus-Acinetobacter baumanniiComplex with Monoclonal Antibodies Specific for O Antigens of Their Lipopolysaccharides
Authors: Pantophlet Ralph , Severin Juliëtte A. , Nemec Alexandr , Brade Lore , Dijkshoorn Lenie , Brade Helmut
Published: July 2002

The unambiguous identification ofAcinetobacterstrains, particularly those belonging to theAcinetobacter calcoaceticus-Acinetobacter baumanniicomplex, is often hindered by their close geno- and phenotypic relationships. In this study, monoclonal antibodies (MAbs) against the O antigens of the lipopolysaccharides from strains belonging to theA. calcoaceticus-A. baumanniicomplex were generated after the immunization of mice with heat-killed bacteria and shown by enzyme immunoassays and Western blotting to be specific for their homologous antigens. Since theA. calcoaceticus-A. baumanniicomplex comprises the most clinically relevant species, the MAbs were subsequently tested in dot and Western blots with proteinase K-treated lysates from a large collection ofAcinetobacterisolates (n= 631) to determine whether the antibodies could be used for the reliable identification of strains from this complex. Reactivity was observed with 273 of the 504 isolates (54%) from theA. calcoaceticus-A. baumanniicomplex which were included in this study. Isolates which reacted positively did so with only one antibody; no reactivity was observed with isolates not belonging to theA. calcoaceticus-A. baumanniicomplex (n= 127). To identify additional putative O serotypes, isolates from theA. calcoaceticus-A. baumanniicomplex which showed no MAb reactivity were subjected to a method that enables the detection of lipid A moieties in lipopolysaccharides with a specific MAb on Western blots following acidic treatment of the membrane. By this method, additional serotypes were indeed identified, thus indicating which strains to select for future immunizations. This study contributes to the completion of a serotype-based identification scheme forAcinetobacterspecies, in particular, those which are presently of the most clinical importance.
Tripeptidyl Peptidase II Promotes Maturation of Caspase-1 inShigella flexneri-Induced Macrophage Apoptosis
Authors: Hilbi Hubert , Puro Robyn J. , Zychlinsky Arturo
Published: July 2002

The invasive enteropathogenic bacteriumShigella flexneriactivates apoptosis in macrophages.Shigella-induced apoptosis requires caspase-1. We demonstrate here that tripeptidyl peptidase II (TPPII), a cytoplasmic, high-molecular-weight protease, participates in the apoptotic pathway triggered byShigella. The TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) andclasto-lactacystin β-lactone (lactacystin), an inhibitor of both TPPII and the proteasome, protected macrophages fromShigella-induced apoptosis. AAF-cmk was more potent than lactacystin and irreversibly blockedShigella-induced apoptosis by 95% at a concentration of 1 μM. Conversely, peptide aldehyde and peptide vinylsulfone proteasome inhibitors had little effect onShigella-mediated cytotoxicity. Both AAF-cmk and lactacystin prevented the maturation of pro-caspase-1 and its substrate pro-interleukin 1β inShigella-infected macrophages, indicating that TPPII is upstream of caspase-1. Neither of these compounds directly inhibited caspase-1. AAF-cmk and lactacystin did not impair macrophage phagocytosis or the ability ofShigellato escape the macrophage phagosome. TPPII was also found to be involved in apoptosis induced by ATP and the protein kinase inhibitor staurosporine. We propose that TPPII participates in apoptotic pathways.
Differential Host Inflammatory Responses to Viable Versus Antibiotic-Killed Bacteria in Experimental Microbial Sepsis
Authors: Silverstein Richard , Wood John G. , Xue Qiao , Norimatsu Mari , Horn David L. , Morrison David C.
Published: July 2002

Staphylococcus aureuskilled during imipenem or ceftazidime chemotherapy in mice elicited an early release of tumor necrosis factor alpha (TNF-α) into the systemic circulation. This response was coincident in time with an increase in leukocyte-endothelium adhesive interactions in the microvasculature. Equivalent responses were not observed without the antibiotic treatment (imipenem or ceftazidime). Protective efficacy of the same antibiotic treatment was markedly diminished ind-galactosamine-treated mice compared to controls; e.g., it dropped from 2,000-fold to 70-fold with 4 mg of imipenem per kg given at the time of challenge. Nevertheless, protection was quantitatively restored upon concurrent administration of neutralizing anti-TNF-α antibody or 4 mg of dexamethasone per kg to these TNF-α-hypersensitive mice. Importantly, protection afforded by dexamethasone was not seen when the animals were challenged with viable organisms but without the concurrent administration of antibiotic. An early TNF-α response could also be demonstrated upon challenge withEscherichia coli, but in this instance, neither the timing nor the magnitude of that response was influenced by treatment with these antibiotics. We conclude from these studies that the inflammatory response to viable versus killed bacteria may differ markedly depending on the particular bacterium, host sensitivity to TNF-α, and possibly the Gram stain classification.
Evaluation of an Isogenic Major Outer Membrane Protein-Deficient Mutant in the Human Model of
Haemophilus ducreyi
Infection
Authors: Throm Robert E. , Al-Tawfiq Jaffar A. , Fortney Kate R. , Katz Barry P. , Hood Antoinette F. , Slaughter Clive A. , Hansen Eric J. , Spinola Stanley M.
Published: July 2002

Haemophilus ducreyi
expresses 2 OmpA homologs, designated MOMP and OmpA2, whose genes are arranged in tandem on the chromosome. Northern blot analysis indicated that
momp
and
ompA2
are transcribed independently. Sequences of the
momp
open reading frame (ORF) lacking the transcriptional start site were amplified by PCR, and an Ω-Km2 cassette was ligated into the ORF. A plasmid containing this construction was electroporated into
H. ducreyi
35000HP, and an isogenic MOMP-deficient mutant (35000HP-SMS2) was generated by allele exchange. In Southern blotting, 35000HP-SMS2 contained one copy of the Ω-Km2 cassette in
momp
. 35000HP and 35000HP-SMS2 had similar outer membrane protein (OMP) and lipooligosaccharide profiles and growth rates except for up-regulation of a putative porin protein in the mutant. Five subjects were inoculated with three doses of live 35000HP-SMS2 on one arm and two doses of live 35000HP and one dose of a heat-killed control on the other arm in a double-blind escalating dose-response trial. Pustules developed at 7 of 10 sites inoculated with 35000HP and at 6 of 15 sites inoculated with 35000HP-SMS2 (
P
= 0.14). 35000HP and 35000HP-SMS2 were recovered at similar rates from daily surface cultures and semiquantitative cultures. The data suggest that expression of MOMP is not required for pustule formation by
H. ducreyi
in the human model of infection.
DNA Vaccination against Tuberculosis: Expression of a Ubiquitin-Conjugated Tuberculosis Protein Enhances Antimycobacterial Immunity
Authors: Delogu Giovanni , Howard Angela , Collins Frank M. , Morris Sheldon L.
Published: July 2002

Genetic immunization is a promising new technology for developing vaccines against tuberculosis that are more effective. In the present study, we evaluated the effects of intracellular turnover of antigens expressed by DNA vaccines on the immune response induced by these vaccines in a mouse model of pulmonary tuberculosis. The mycobacterial culture filtrate protein MPT64 was expressed as a chimeric protein fused to one of three variants of the ubiquitin protein (UbG, UbA, and UbGR) known to differentially affect the intracellular processing of the coexpressed antigens. Immunoblot analysis of cell lysates of in vitro-transfected cells showed substantial differences in the degradation rate of ubiquinated MPT64 (i.e., UbG64 < UbA64 < UbGR64). The specific immune response generated in mice correlated with the stability of the ubiquitin-conjugated antigen. The UbA64 DNA vaccine induced a weak humoral response compared to UbG64, and a mixed population of interleukin-4 (IL-4)- and gamma interferon (IFN-γ)-secreting cells. Vaccination with the UbGR64 plasmid generated a strong Th1 cell response (high IFN-γ, low IL-4) in the absence of a detectable humoral response. Aerogenic challenge of vaccinated mice withMycobacterium tuberculosisindicated that immunization with both the UbA64- and UbGR64-expressing plasmids evoked an enhanced protective response compared to the vector control. The expression of mycobacterial antigens from DNA vaccines as fusion proteins with a destabilizing ubiquitin molecule (UbA or UbGR) shifted the host response toward a stronger Th1-type immunity which was characterized by low specific antibody levels, high numbers of IFN-γ-secreting cells, and significant resistance to a tuberculous challenge.
Reduced Transendothelial Migration of Monocytes Infected byCoxiella burnetii
Authors: Dellacasagrande Jérôme , Moulin Pierre A. , Guilianelli Catherine , Capo Christian , Raoult Didier , Grau Georges E. , Mege Jean-Louis
Published: July 2002

The migratory properties of THP1 monocytes infected byCoxiella burnetiiwere determined in a transmigration assay across a human microvascular endothelial cell monolayer. Transendothelial migration of monocytes infected by virulent, but not avirulent,C. burnetiiwas inhibited. This inhibition was observed in spite of conserved adherence properties of infected monocytes.
Escherichia coli
K1
aslA
Contributes to Invasion of Brain Microvascular Endothelial Cells In Vitro and In Vivo
Authors: Hoffman Jill A. , Badger Julie L. , Zhang Yan , Huang Sheng-He , Kim Kwang Sik
Published: July 2002

Neonatal
Escherichia coli
meningitis remains a devastating disease, with unacceptably high morbidity and mortality despite advances in supportive care measures and bactericidal antibiotics. To further our ability to improve the outcome of affected neonates, a better understanding of the pathogenesis of the disease is necessary. To identify potential bacterial genes which contribute to
E. coli
invasion of the blood-brain barrier, a cerebrospinal fluid isolate of
E. coli
K1 was mutagenized with Tn
phoA
. Tn
phoA
mutant 27A-6 was found to have a significantly decreased ability to invade brain microvascular endothelial cells compared to the wild type. In vivo, 32% of the animals infected with mutant 27A-6 developed meningitis, compared to 82% of those infected with the parent strain, despite similar levels of bacteremia. The DNA flanking the Tn
phoA
insertion in 27A-6 was cloned and sequenced and determined to be homologous to
E. coli
K-12
aslA
(arylsulfatase-like gene). The deduced amino acid sequence of the
E. coli
K1
aslA
gene product shows homology to a well-characterized arylsulfatase family of enzymes found in eukaryotes, as well as prokaryotes. Two additional
aslA
mutants were constructed by targeted gene disruption and internal gene deletion. Both of these mutants demonstrated decreased invasion phenotypes, similar to that of Tn
phoA
mutant 27A-6. Complementation of the decreased-invasion phenotypes of these mutants was achieved when
aslA
was supplied in
trans
. This is the first demonstration that this locus contributes to invasion of the blood-brain barrier by
E. coli
K1.
Role of Inducible Nitric Oxide Synthase in Resistance toMycobacterium lepraein Mice
Authors: Adams Linda B. , Job Charles K. , Krahenbuhl James L.
Published: July 2002

The manifestation of leprosy in humans is largely determined by host immunity toMycobacterium lepraeand is a model for immunoregulation in a human disease. However, animal models available for exploration of the leprosy spectrum are inadequate. This study exploredM. lepraeinfection in mice deficient in inducible nitric oxide synthase, and this report describes elements resembling borderline tuberculoid leprosy in humans.
Biochemical and Biological Characterization of the ProtectiveLeishmania pifanoiAmastigote Antigen P-8
Authors: Colmenares M. , Tiemeyer M. , Kima P. , McMahon-Pratt D.
Published: July 2002

TheLeishmania pifanoiamastigote antigen P-8 has been previously shown to induce protective immunity in a murine model of cutaneous leishmaniasis (L. Soong, S. M. Duboise, P. Kima, and D. McMahon-Pratt, Infect. Immun. 63:3559–3566, 1995). As this antigen is of interest for further vaccine studies, the biochemical characterization of P-8 was undertaken. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot analysis, and gel filtration chromatography revealed that P-8 antigen consisted of two proteoglycolipid complexes. The P-8 epitope is associated with theL. pifanoiamastigote-specific glycolipid components found in the two complexes. The P-8 complex 1 (P-8c1) consists of a 56-kDa serine metalloproteinase, apolipoprotein E (derived from fetal bovine serum), and amastigote-specific glycolipids. The P-8 complex 2 (P-8c2) consists of a 31-kDa cysteine proteinase associated with amastigote glycolipids. Biochemical analyses suggest that the P-8 antigenic glycolipids may be distinct from previously describedLeishmaniaglycolipids (glycosylinositolphospholipids and sphingoglycolipids). Protective immunity studies revealed that P-8c1 (serine metalloproteinase-glycolipid complex) confers comparable protection against infection as immunopurified P-8. The isolated P-8c2 (cysteine proteinase-glycolipid complex) does not provide significant protection, nor does stimulation with P-8c2 result in significant T-cell activation in P-8- or P-8c2-vaccinated mice. Consequently, the P-8c1 complex appears to be the immunodominant component of P-8.
Mycobacterium ulceransCytotoxicity in an Adipose Cell Model
Authors: Dobos Karen M. , Small Pamela L. , Deslauriers Manon , Quinn Fredrick D. , King C. Harold
Published: July 2002

An adipose cell (SW872) model was developed to observe cellular necrosis and apoptosis uponMycobacterium ulceransinfection and treatment with mycobacterial exudate. Apoptosis was likely due to secreted proteins, while necrosis was likely due to mycolactone. Our data suggest that additional factors inM.ulceransmay be involved in Buruli ulcer pathogenesis.
Characterization of an Intestinal Epithelial Cell Receptor Recognized by theCryptosporidium parvumSporozoite Ligand CSL
Authors: Langer Rebecca C. , Schaefer Deborah A. , Riggs Michael W.
Published: July 2002

The protozoan parasiteCryptosporidium parvumis a leading cause of diarrhea in humans and neonatal calves. The absence of approved parasite-specific drugs, vaccines, and immunotherapies for cryptosporidiosis relates in part to limited knowledge on the pathogenesis of zoite attachment and invasion. We recently reported that theC. parvumapical complex glycoprotein CSL contains a zoite ligand for intestinal epithelial cells which is defined by monoclonal antibody (MAb) 3E2. In the present study, the host cell receptor for CSL was characterized. For these studies, a panel of epithelial and mesenchymal cell lines was examined for permissiveness toC. parvumand the ability to bind CSL. Cells of epithelial origin were significantly more permissive and bound significantly greater quantities of CSL than cells of mesenchymal origin. Caco-2 intestinal cells were selected from the epithelial panel for further characterization of the CSL receptor. Immunoelectron microscopy demonstrated that CSL bound initially to the surface of Caco-2 cells and was rapidly internalized. The molecule bound by CSL was identified as an 85-kDa Caco-2 cell surface protein by radioimmunoprecipitation and CSL affinity chromatography. Sporozoite incubation with the isolated 85-kDa protein reduced binding of MAb 3E2. Further, attachment and invasion were significantly inhibited when sporozoites were incubated with the 85-kDa protein prior to inoculation onto Caco-2 cells. These observations indicate that the 85-kDa protein functions as a Caco-2 cell receptor for CSL. CSL also bound specifically to intestinal epithelium from calves, indicating receptor expression in a second important host species. Molecular characterization of the CSL receptor may lead to novel avenues for disrupting ligand-receptor interactions in the pathogenesis ofC. parvuminfection.
Effect of Mycobacterial Phospholipids on Interaction ofMycobacterium tuberculosiswith Macrophages
Authors: Thorson Lisa M. , Doxsee Daniel , Scott Monisha G. , Wheeler Paul , Stokes Richard W.
Published: July 2002

This study demonstrates that pretreatment of macrophages with phosphatidylinositol, of either soya bean or mycobacterial origin, results in a down-regulation of the binding and uptake ofMycobacterium tuberculosisby the phagocytes. We also describe the novel observation that cardiolipin induces an increase in the binding and uptake ofM. tuberculosisby macrophages. Neither phospholipid interacts with macrophages via the 2F8 epitope of scavenger receptor A, and treatment of macrophages with either phospholipid results in a down-regulation of CR3 function and tumor necrosis factor alpha production by the phagocyte. We have also shown that the ability of macrophages to interact with mycobacteria is greatly affected by an as yet unidentified product from the interaction of chloroform and polypropylene tubes.
Subcytocidal Attack by Staphylococcal Alpha-Toxin Activates NF-κB and Induces Interleukin-8 Production
Authors: Dragneva Y. , Anuradha C. D. , Valeva A. , Hoffmann A. , Bhakdi S. , Husmann M.
Published: July 2002

Formation of transmembrane pores by staphylococcal alpha-toxin can provoke a spectrum of events depending on target cell species and toxin dose, and in certain cases, repair of the lesions has been observed. Here, we report that transcriptional processes are activated as a response of cells to low toxin doses. Exposure of monocytic (THP-1) or epithelial (ECV304) cells to 40 to 160 ng/ml alpha-toxin provoked a drop in cellular ATP level that was followed by secretion of substantial amounts of interleukin-8 (IL-8). Cells transfected with constructs comprising the proximal IL-8 promoter fused to luciferase or to green fluorescent protein cDNA exhibited enhanced reporter gene expression following toxin treatment. Electrophoretic mobility shift and immunofluorescence assays demonstrated that IL-8 secretion was preceded by activation of NF-κB. Transfection experiments conducted with p65/p50 double-deficient cells showed that activation of the IL-8 promoter/reporter by toxin was absolutely dependent on NF-κB. In contrast, this transcription factor was not required for lesion repair. Attack of cells by low doses of a pore-forming toxin can lead to transcriptional gene activation, which is followed by production of mediators that may contribute to the initiation and propagation of inflammatory lesions.
CD8+T Cells Have an Essential Role in Pulmonary Clearance of NontypeableHaemophilus influenzaefollowing Mucosal Immunization
Authors: Foxwell A. Ruth , Kyd Jennelle M. , Karupiah Guna , Cripps Allan W.
Published: July 2002

A rodent respiratory experimental model has proved useful for investigating the immune mechanisms responsible for clearance of bacteria from the lungs. Immunohistochemical studies in immune and nonimmune rats have identified the cellular kinetics of response to bacterial pulmonary infection for CD8+, CD4+, and γδ+T cells; B cells; and the expression of major histocompatibility complex class II (MHC-II). During the course of bacterial clearance, there was no apparent proliferation or extravasation of lymphocytes, nor was there increased expression of MHC-II in nonimmune animals despite an influx of polymorphonuclear leukocytes, whereas in immunized animals there was an early influx of CD8+and γδ+T cells, followed by enhanced expression of the MHC-II marker, cellular infiltration by polymorphonuclear leukocytes, and finally an increased number of CD4+T cells. Depletion of CD8+T cells confirmed their vital contribution in the preprimed immune response to pulmonary infection by significantly decreasing the animals' ability to clear bacteria following challenge.