bluuebunny/crossref_metadata_2025_split huggingface dataset:
Salmonella enterica
Serovar Typhimurium Response Involved in Attenuation of Pathogen Intracellular Proliferation
Authors: Cano David A. , Martı́nez-Moya Marina , Pucciarelli M. Graciela , Groisman Eduardo A. , Casadesús Josep , Garcı́a-Del Portillo Francisco 
Published: July 2002

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

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

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

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

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

ABSTRACT

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

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

ABSTRACT

PomA is thought to be a component of the ion channel in the sodium-driven polar-flagellar motor of
Vibrio alginolyticus
. We have found that some cysteine substitutions in the periplasmic region of PomA result in a slow-motility phenotype, in which swarming and swimming speeds are reduced even in the presence of high concentrations of NaCl. Most of the mutants showed a sodium ion dependence similar to that of the wild type but with significantly reduced motility at all sodium ion concentrations. By contrast, motility of the D31C mutant showed a sharp dependence on NaCl concentration, with a threshold at 38 mM. The motor of the D31C mutant rotates stably, as monitored by laser dark-field microscopy, suggesting that the mutant PomA protein is assembled normally into the motor complex. Mutational studies of Asp31 suggest that, although this residue is not essential for motor rotation, a negative charge at this position contributes to optimal speed and/or efficiency of the motor.
Identification and Characterization of Two Chemotactic Transducers for Inorganic Phosphate in
Pseudomonas aeruginosa
Authors: Wu Hong , Kato Junichi , Kuroda Akio , Ikeda Tsukasa , Takiguchi Noboru , Ohtake Hisao 
Published: July 2002

ABSTRACT

Two chemotactic transducers for inorganic phosphate (P
i
), designated CtpH and CtpL, have been identified in
Pseudomonas aeruginosa
. The corresponding genes (
ctpH
and
ctpL
) were inactivated by inserting kanamycin and tetracycline resistance gene cassettes into the wild-type genes in the
P. aeruginosa
PAO1 genome. Computer-assisted capillary assays showed that the
ctpH
single mutant failed to exhibit P
i
taxis when the concentration of P
i
in the capillary was higher than 5 mM. Conversely, the
ctpL
single mutant could not respond to P
i
at the concentration of 0.01 mM. The
ctpH ctpL
double mutant was defective in P
i
taxis at any concentration ranging from 0.01 to 10 mM. To investigate regulation of P
i
taxis, the
ctpH
and
ctpL
genes were also disrupted individually in the
P. aeruginosa phoU
and
phoB
single mutants. The
ctpH phoU
and
ctpH phoB
double mutants were defective in P
i
taxis, regardless of whether the cells were starved for P
i
. The
ctpL phoU
double mutant was constitutive for P
i
taxis, whereas the
ctpL phoB
double mutant was induced by P
i
limitation for P
i
taxis. The region upstream of
ctpL
, but not
ctpH
, contained a putative
pho
box sequence. Expression of
ctpL
::
lacZ
was induced by P
i
limitation in PAO1, while it was constitutive in the
phoU
mutant. In contrast, the
phoB
mutant showed only background levels of
ctpL
::
lacZ
expression. These results showed that
ctpL
is involved in the
pho
regulon genes in
P. aeruginosa
. The
ctpH phoU
mutant, which failed to exhibit P
i
taxis, was constitutive for
ctpL
::
lacZ
expression, suggesting that the P
i
detection by CtpL requires PhoU. Like PAO1, the
phoB
and
phoU
single mutants were constitutive for expression of
ctpH
::
lacZ
. Thus, the evidence that the
ctpL phoU
mutant, but not the
ctpL phoB
mutant and PAO1, was constitutive for P
i
taxis raised the possibility that PhoU exerts a negative control on P
i
detection by CtpH at the posttranscriptional level.
Genetic Characterization of
Escherichia coli
Type 1 Pilus Adhesin Mutants and Identification of a Novel Binding Phenotype
Authors: Hamrick Terri S. , Harris Sandra L. , Spears Patricia A. , Havell Edward A. , Horton John R. , Russell Perry W. , Orndorff Paul E. 
Published: July 2002

ABSTRACT

Five
Escherichia coli
type 1 pilus mutants that had point mutations in
fimH
, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts)
fimH
mutant that agglutinated guinea pig erythrocytes after growth at 31°C but not at 42°C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a
fimH
insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42°C but sensitive after growth at 31°C. The ts mutant thus binds macrophages with one receptor specificity at 31°C and another at 42°C.
SprE Levels Are Growth Phase Regulated in a ς
S
-Dependent Manner at the Level of Translation
Authors: Gibson Katherine E. , Silhavy Thomas J. 
Published: July 2002

ABSTRACT

SprE regulates ς
S
levels in response to nutrient availability by promoting ClpXP-mediated degradation. Paradoxically, we observe that SprE is similarly regulated, accumulating preferentially upon starvation. This regulation of SprE levels is ς
S
dependent, altering SprE synthesis at the level of translation. Thus, we demonstrate that SprE and ς
S
function within a regulatory feedback loop.
Escherichia coli
DNA Polymerase IV Mutator Activity: Genetic Requirements and Mutational Specificity
Authors: Wagner Jérôme , Nohmi Takehiko 
Published: July 2002

ABSTRACT

The
dinB
gene of
Escherichia coli
is known to be involved in the untargeted mutagenesis of λ phage. Recently, we have demonstrated that this damage-inducible and SOS-controlled gene encodes a novel DNA polymerase, DNA Pol IV, which is able to dramatically increase the untargeted mutagenesis of F′ plasmid. At the amino acid level, DNA Pol IV shares sequence homologies with
E. coli
UmuC (DNA Pol V), Rev1p, and Rad30p (DNA polymerase η) of
Saccharomyces cerevisiae
and human Rad30A (XPV) proteins, all of which are involved in translesion DNA synthesis. To better characterize the Pol IV-dependent untargeted mutagenesis, i.e., the DNA Pol IV mutator activity, we analyzed the genetic requirements of this activity and determined the forward mutation spectrum generated by this protein within the
c
II gene of λ phage. The results indicated that the DNA Pol IV mutator activity is independent of
polA
,
polB
,
recA
,
umuDC
,
uvrA
, and
mutS
functions. The analysis of more than 300 independent mutations obtained in the wild-type or
mutS
background revealed that the mutator activity clearly promotes single-nucleotide substitutions as well as one-base deletions in the ratio of about 1:2. The base changes were strikingly biased for substitutions toward G:C base pairs, and about 70% of them occurred in 5′-GX-3′ sequences, where X represents the base (T, A, or C) that is mutated to G. These results are discussed with respect to the recently described biochemical characteristics of DNA Pol IV.