bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Behavior of a
Helicobacter pylori
SS1
nixA
Mutant with Reduced Urease Activity
Authors: Nolan Kylie J. , McGee David J. , Mitchell Hazel M. , Kolesnikow Tassia , Harro Janette M. , O'Rourke Jani , Wilson John E. , Danon Stephen J. , Moss Nathan D. , Mobley Harry L. T. , Lee Adrian 
Published: July 2002

ABSTRACT

Helicobacter pylori
mutants devoid of urease activity fail to colonize the gastric mucosa of mice; however, the effect of decreased levels of urease on colonization has not been examined. The
nixA
gene, required for full urease activity, encodes a cytoplasmic membrane nickel transporter that imports nickel ions and leads to incorporation of nickel ions into apourease. A
nixA
mutant of the Sydney strain of
H. pylori
(SS1) was constructed by disruption of the
nixA
gene with a kanamycin resistance cassette. This mutant retained only half the urease activity of the wild-type (wild-type) SS1 strain. C57BL/6j (
n
= 75) and BALB/c (
n
= 75) mice were inoculated independently with the wild-type or the
nixA
strain. The level and distribution of colonization were assessed by bacterial colony counts and histological grading at 4, 12, and 24 weeks postinfection. Colonization levels of the
nixA
strain in BALB/c mice were significantly lower compared with SS1 (
P
= 0.005), while colonization in C57BL/6j mice was similar for both the wild-type and mutant strains. Subtle differences in colonization of the different regions of the stomach, determined by microscopic grading, were observed between wild-type SS1 and the
nixA
strain in BALB/c mice. On the contrary, when C57BL/6j (
n
= 35) and BALB/c (
n
= 35) mice were coinfected with the wild-type and
nixA
strains simultaneously, the
nixA
mutant failed to colonize and was outcompeted by the wild-type SS1 strain, which established normal levels of colonization. These results demonstrate the importance of the
nixA
gene for increasing the fitness of
H. pylori
for gastric colonization. Since
nixA
is required for full urease activity, the decreased fitness of the
nixA
mutant is likely due to reduced urease activity; however, pleiotropic effects of the mutation cannot be completely ruled out.
Lactoferrin Inhibits the Lipopolysaccharide-Induced Expression and Proteoglycan-Binding Ability of Interleukin-8 in Human Endothelial Cells
Authors: Elass Elisabeth , Masson Maryse , Mazurier Joël , Legrand Dominique 
Published: July 2002

ABSTRACTInterleukin-8 (IL-8), a C-X-C chemokine bound to endothelium proteoglycans, initiates the activation and selective recruitment of leukocytes at inflammatory foci. We demonstrate that human lactoferrin, an antimicrobial lipopolysaccharide (LPS)-binding protein, decreases both IL-8 mRNA and protein expression induced by the complexEscherichia coli055:B5 LPS/sCD14 in human umbilical vein endothelial cells. The use of recombinant lactoferrins mutated in the LPS-binding sites indicates that this inhibitory effect is mediated by an interaction of lactoferrin with LPS and CD14s that suppresses the endotoxin biological activity. Furthermore, since dimeric IL-8 and lactoferrin are both proteoglycan-binding molecules, the competition between these proteins for heparin binding was investigated. Lactoferrin strongly inhibited the interaction of radiolabeled IL-8 to immobilized heparin, whereas a lactoferrin variant lacking the amino acid residues essential for heparin binding was not inhibitory. Moreover, this process is specific, since serum transferrin, a glycoprotein whose structure is close to that of lactoferrin, did not prevent the interaction of IL-8 with heparin. These results suggest that the anti-inflammatory properties of lactoferrin during septicemia are related, at least in part, to the regulation of IL-8 production and also to the ability of lactoferrin to compete with chemokines for their binding to proteoglycans.
Dendritic Cells as Effector Cells: Gamma Interferon Activation of Murine Dendritic Cells Triggers Oxygen-Dependent Inhibition ofToxoplasma gondiiReplication
Authors: Aline F. , Bout D. , Dimier-Poisson I. 
Published: July 2002

ABSTRACTToxoplasma gondiiis an obligate intracellular parasite that infects a wide variety of nucleated cells in its numerous intermediate hosts, including humans. Much interest has focused on the ability of gamma interferon (IFN-γ)-activated macrophages to prevent intracellular replication, but some other cells (e.g., fibroblasts, endothelial cells, microglial cells, astrocytes, enterocytes and retinal pigment cells) can also be activated to induce this inhibition of proliferation. Dendritic cells are generally known to be involved in the induction of immune responses, but no previous study had investigated the possibility that dendritic cells may act as effector cells of this system. Our results show that IFN-γ-activation inhibits the replication ofT. gondiiin dendritic cells, with the inhibition being dose dependent. Neither nitrogen derivatives nor tryptophan starvation appears to be involved in the inhibition of parasite replication by IFN-γ. Experiments with oxygen scavengers indicate that intracellularT. gondiireplication is oxygen dependent. Our findings suggest that, in addition to their essential role in stimulating the immune system, dendritic cells probably act as effector cells in the first line of defense against pathogen invasion.
Rickettsia-Macrophage Interactions: Host Cell Responses toRickettsia akariandRickettsia typhi
Authors: Radulovic S. , Price P. W. , Beier M. S. , Gaywee J. , Macaluso J. A. , Azad A. 
Published: July 2002

ABSTRACTThe existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akariand -Rickettsia typhiinteractions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated thatR. akariandR. typhicaused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-6. Furthermore, macrophage infection withR. akariandR. typhiresulted in differential synthesis and expression of IL-β and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-γ), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed betweenR. akari-andR. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.
Chlamydia trachomatis
Disrupts N-Cadherin-Dependent Cell-Cell Junctions and Sequesters β-Catenin in Human Cervical Epithelial Cells
Authors: Prozialeck Walter C. , Fay Michael J. , Lamar Peter C. , Pearson Celeste A. , Sigar Ira , Ramsey Kyle H. 
Published: July 2002

ABSTRACT

The cadherin/catenin complex serves as an important structural component of adherens junctions in epithelial cells. Under certain conditions, β-catenin can be released from this complex and interact with transcription factors in the nucleus to stimulate the expression of genes that regulate apoptosis and cell cycle control. While studying the effects of the bacterial pathogen
Chlamydia trachomatis
on human cervical epithelial cells in culture, we observed that
C. trachomatis
caused the epithelial cells to separate from each other without detaching from their growing surface. The objective of the present study was to determine if this effect might involve the disruption of the cadherin/catenin complex. Primary cultures of human cervical epithelial cells or HeLa cells were infected with
C. trachomatis
serovar E. Cadherin-like immunoreactive materials and β-catenin were visualized by immunofluorescence. Preliminary studies showed that N-cadherin was the primary cadherin expressed in both the primary cultures and the HeLa cells. In noninfected cells, N-cadherin and β-catenin were colocalized at the intercellular junctional complexes. By contrast, the infected cells showed a marked loss of both N-cadherin and β-catenin labeling from the junctional complexes and the concomitant appearance of intense β-catenin labeling associated with the chlamydial inclusion. The results of Western blot analyses of extracts of
C. trachomatis
showed no evidence of cross-reactivity with the β-catenin antibody. These results indicate that
C. trachomatis
causes the breakdown of the N-cadherin/β-catenin complex and that the organism can sequester β-catenin within the chlamydial inclusion. This could represent an important mechanism by which
C. trachomatis
alters epithelial cell function.
Role of Toll-Like Receptors in Changes in Gene Expression and NF-κB Activation in Mouse Hepatocytes Stimulated with Lipopolysaccharide
Authors: Liu Shubing , Gallo David J. , Green Angela M. , Williams Debra L. , Gong Xiaoyan , Shapiro Richard A. , Gambotto Andrea A. , Humphris Elisabeth L. , Vodovotz Yoram , Billiar Timothy R. 
Published: July 2002

ABSTRACTThe liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-κB activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.
Biosynthesis of Lipoteichoic Acid in
Lactobacillus rhamnosus
: Role of DltD in
d
-Alanylation
Authors: Debabov Dmitri V. , Kiriukhin Michael Y. , Neuhaus Francis C. 
Published: July 2002

ABSTRACT

The
dlt
operon (
dltA
to
dltD
) of
Lactobacillus rhamnosus
7469 encodes four proteins responsible for the esterification of lipoteichoic acid (LTA) by
d
-alanine. These esters play an important role in controlling the net anionic charge of the poly (GroP) moiety of LTA.
dltA
and
dltC
encode the
d
-alanine–
d
-alanyl carrier protein ligase (Dcl) and
d
-alanyl carrier protein (Dcp), respectively. Whereas the functions of DltA and DltC are defined, the functions of DltB and DltD are unknown. To define the role of DltD, the gene was cloned and sequenced and a mutant was constructed by insertional mutagenesis of
dltD
from
Lactobacillus casei
102S. Permeabilized cells of a
dltD
::
erm
mutant lacked the ability to incorporate
d
-alanine into LTA. This defect was complemented by the expression of DltD from pNZ123/
dlt
. In in vitro assays, DltD bound Dcp for ligation with
d
-alanine by Dcl in the presence of ATP. In contrast, the homologue of Dcp, the
Escherichia coli
acyl carrier protein (ACP), involved in fatty acid biosynthesis, was not bound to DltD and thus was not ligated with
d
-alanine. DltD also catalyzed the hydrolysis of the mischarged
d
-alanyl–ACP. The hydrophobic N-terminal sequence of DltD was required for anchoring the protein in the membrane. It is hypothesized that this membrane-associated DltD facilitates the binding of Dcp and Dcl for ligation of Dcp with
d
-alanine and that the resulting
d
-alanyl–Dcp is translocated to the primary site of
d
-alanylation.
Growth Phase-Coupled Changes of the Ribosome Profile in Natural Isolates and Laboratory Strains of
Escherichia coli
Authors: Wada Akira , Mikkola Riitta , Kurland Charles G. , Ishihama Akira 
Published: July 2002

ABSTRACT

The growth phase-dependent change in sucrose density gradient centrifugation patterns of ribosomes was analyzed for both laboratory strains of
Escherichia coli
and natural isolates from the ECOR collection. All of the natural isolates examined formed 100S ribosome dimers in the stationary phase, and ribosome modulation factor (RMF) was associated with the ribosome dimers in the ECOR strains as in the laboratory strain W3110. The ribosome profile (70S monomers versus 100S dimers) follows a defined pattern over time during lengthy culture in both the laboratory strains and natural isolates. There are four discrete stages: (i) formation of 100S dimers in the early stationary phase; (ii) transient decrease in the dimer level; (iii) return of dimers to the maximum level; and (iv) dissociation of 100S dimers into 70S ribosomes, which are quickly degraded into subassemblies. The total time for this cycle of ribosome profile change, however, varied from strain to strain, resulting in apparent differences in the ribosome profiles when observed at a fixed time point. A correlation was noted in all strains between the decay of 100S ribosomes and the subsequent loss of cell viability. Two types of
E. coli
mutants defective in ribosome dimerization were identified, both of which were unable to survive for a prolonged period in stationary phase. The W3110 mutant, with a disrupted
rmf
gene, has a defect in ribosome dimerization because of lack of RMF, while strain Q13 is unable to form ribosome dimers due to a ribosomal defect in binding RMF.
Fermentative Metabolism of
Bacillus subtilis
: Physiology and Regulation of Gene Expression
Authors: Cruz Ramos Hugo , Hoffmann Tamara , Marino Marco , Nedjari Hafed , Presecan-Siedel Elena , Dreesen Oliver , Glaser Philippe , Jahn Dieter 
Published: July 2002

ABSTRACT

Bacillus subtilis
grows in the absence of oxygen using nitrate ammonification and various fermentation processes. Lactate, acetate, and 2,3-butanediol were identified in the growth medium as the major anaerobic fermentation products by using high-performance liquid chromatography. Lactate formation was found to be dependent on the
lctEP
locus, encoding lactate dehydrogenase and a putative lactate permease. Mutation of
lctE
results in drastically reduced anaerobic growth independent of the presence of alternative electron acceptors, indicating the importance of NADH reoxidation by lactate dehydrogenase for the overall anaerobic energy metabolism. Anaerobic formation of 2,3-butanediol via acetoin involves acetolactate synthase and decarboxylase encoded by the
alsSD
operon. Mutation of
alsSD
has no significant effect on anaerobic growth. Anaerobic acetate synthesis from acetyl coenzyme A requires phosphotransacetylase encoded by
pta
. Similar to the case for
lctEP
, mutation of
pta
significantly reduces anaerobic fermentative and respiratory growth. The expression of both
lctEP
and
alsSD
is strongly induced under anaerobic conditions. Anaerobic
lctEP
and
alsSD
induction was found to be partially dependent on the gene encoding the redox regulator Fnr. The observed
fnr
dependence might be the result of Fnr-induced
arfM
(
ywiD
) transcription and subsequent
lctEP
and
alsSD
activation by the regulator ArfM (YwiD). The two-component regulatory system encoded by
resDE
is also involved in anaerobic
lctEP
induction. No direct
resDE
influence on the redox regulation of
alsSD
was observed. The alternative electron acceptor nitrate represses anaerobic
lctEP
and
alsSD
transcription. Nitrate repression requires
resDE
- and
fnr
-dependent expression of
narGHJI
, encoding respiratory nitrate reductase. The gene
alsR
, encoding a regulator potentially responding to changes of the intracellular pH and to acetate, is essential for anaerobic
lctEP
and
alsSD
expression. In agreement with its known aerobic function, no obvious oxygen- or nitrate-dependent
pta
regulation was observed. A model for the regulation of the anaerobic fermentation genes in
B. subtilis
is proposed.
Evidence that SpoIVFB Is a Novel Type of Membrane Metalloprotease Governing Intercompartmental Communication during
Bacillus subtilis
Sporulation
Authors: Yu Yuen-Tsu Nicco , Kroos Lee 
Published: July 2002

ABSTRACT

Processing of pro-ς
K
in the mother cell compartment of sporulating
Bacillus subtilis
involves SpoIVFB and is governed by a signal from the forespore. SpoIVFB has an HEXXH motif characteristic of metalloproteases embedded in one of its transmembrane segments. Several conservative single amino acid changes in the HEXXH motif abolished function. However, changing the glutamic acid residue to aspartic acid, or changing the isoleucine residue that precedes the motif to proline, permitted SpoIVFB function. Only one other putative metalloprotease, site 2 protease has been shown to tolerate aspartic acid rather than glutamic acid in its HEXXH sequence. Site 2 protease and SpoIVFB share a second region of similarity with a family of putative membrane metalloproteases. A conservative change in this region of SpoIVFB abolished function. Interestingly, SpoIVFA increased the accumulation of certain mutant SpoIVFB proteins but was unnecessary for accumulation of wild-type SpoIVFB.
Target Site Selection by Tn
7
:
att
Tn
7
Transcription and Target Activity
Authors: DeBoy Robert T. , Craig Nancy L. 
Published: July 2002

ABSTRACT

The bacterial transposon Tn
7
inserts at high frequency into a specific site called
att
Tn
7
, which is present in the chromosomes of many bacteria. We show here that transcription of a nearby gene,
glmS
, decreases the frequency of Tn
7
insertion into
att
Tn
7
, thus providing a link between Tn
7
transposition and host cell metabolism.
Megaplasmid pRme2011a of
Sinorhizobium meliloti
Is Not Required for Viability
Authors: Oresnik Ivan J. , Liu Shu-Lin , Yost Christopher K. , Hynes Michael F. 
Published: July 2002

ABSTRACT

We report the curing of the 1,360-kb megaplasmid pRme2011a from
Sinorhizobium meliloti
strain Rm2011. With a positive selection strategy that utilized Tn
5
B12-S containing the
sacB
gene, we were able to cure this replicon by successive rounds of selecting for deletion formation in vivo. Subsequent Southern blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were consistent with the hypothesis that the resultant strain was indeed missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number of substrates as a sole source of carbon on defined media.
Eight of Fourteen
gvp
Genes Are Sufficient for Formation of Gas Vesicles in Halophilic Archaea
Authors: Offner Sonja , Hofacker Annette , Wanner Gerhard , Pfeifer Felicitas 
Published: July 2002

ABSTRACT

The minimal number of genes required for the formation of gas vesicles in halophilic archaea has been determined. Single genes of the 14
gvp
genes present in the p-vac region on plasmid pHH1 of
Halobacterium salinarum
(p-
gvpACNO
and p-
gvpDEFGHIJKLM
) were deleted, and the remaining genes were tested for the formation of gas vesicles in
Haloferax volcanii
transformants. The deletion of six
gvp
genes (p-
gvpCN
, p-
gvpDE
, and p-
gvpHI
) still enabled the production of gas vesicles in
H. volcanii
. The gas vesicles formed in some of these
gvp
gene deletion transformants were altered in shape (ΔI, ΔC) or strength (ΔH) but still functioned as flotation devices. A minimal p-vac region (minvac) containing the eight remaining genes (
gvpFGJKLM-gvpAO
) was constructed and tested for gas vesicle formation in
H. volcanii
. The minvac transformants did not form gas vesicles; however, minvac/gvpJKLM double transformants contained gas vesicles seen as light refractile bodies by phase-contrast microscopy. Transcript analyses demonstrated that minvac transformants synthesized regular amounts of
gvpA
mRNA, but the transcripts derived from
gvpFGJKLM
were mainly short and encompassed only
gvpFG
(
J
), suggesting that the
gvpJKLM
genes were not sufficiently expressed. Since
gvpAO
and
gvpFGJKLM
are the only
gvp
genes present in minvac/JKLM transformants containing gas vesicles, these
gvp
genes represent the minimal set required for gas vesicle formation in halophilic archaea. Homologs of six of these
gvp
genes are found in
Anabaena flos-aquae
, and homologs of all eight minimal halobacterial
gvp
genes are present in
Bacillus megaterium
and in the genome of
Streptomyces coelicolor
.
Elevated Levels of Synthesis of over 20 Proteins Results after Mutation of the
Rhizobium leguminosarum
Exopolysaccharide Synthesis Gene
pssA
Authors: Guerreiro Nelson , Ksenzenko Vladimir N. , Djordjevic Michael A. , Ivashina Tanya V. , Rolfe Barry G. 
Published: July 2002

ABSTRACT

The protein expression profiles of
Rhizobium leguminosarum
strains in response to specific genetic perturbations in exopolysaccharide (EPS) biosynthesis genes were examined using two-dimensional gel electrophoresis. Lesions in either
pssA
,
pssD
, or
pssE
of
R. leguminosarum
bv. viciae VF39 or in
pssA
of
R. leguminosarum
bv. trifolii ANU794 not only abolished the capacity of these strains to synthesize EPS but also had a pleiotropic effect on protein synthesis levels. A minimum of 22 protein differences were observed for the two
pssA
mutant strains. The differences identified in the
pssD
and
pssE
mutants of strain VF39 were a distinct subset of the same protein synthesis changes that occurred in the
pssA
mutant. The
pssD
and
pssE
mutant strains shared identical alterations in the proteins synthesized, suggesting that they share a common function in the biosynthesis of EPS. In contrast, a
pssC
mutant that produces 38% of the EPS level of the parental strain showed no differences in its protein synthesis patterns, suggesting that the absence of EPS itself was contributing to the changes in protein synthesis and that there may be a complex interconnection of the EPS biosynthetic pathway with other metabolic pathways. Genetic complementation of
pssA
can restore wild-type protein synthesis levels, indicating that many of the observed differences in protein synthesis are also a specific response to a dysfunctional PssA. The relevance of these proteins, which are grouped as members of the
pssA
mutant stimulon, remains unclear, as the majority lacked a homologue in the current sequence databases and therefore possibly represent a novel functional network(s). These findings have illustrated the potential of proteomics to reveal unexpected higher-order processes of protein function and regulation that arise from mutation. In addition, it is evident that enzymatic pathways and regulatory networks are more interconnected and more sensitive to structural changes in the cell than is often appreciated. In these cases, linking the observed phenotype directly to the mutated gene can be misleading, as the phenotype could be attributable to downstream effects of the mutation.