bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tumor Necrosis Factor Alpha Is a Key Mediator in the Regulation of ExperimentalTrypanosoma bruceiInfections
Authors: Magez Stefan , Radwanska Magdalena , Beschin Alain , Sekikawa Kenji , De Baetselier Patrick 
Published: December 2019

ABSTRACTIn order to evaluate during experimentalTrypanosoma bruceiinfections the potential role of tumor necrosis factor alpha (TNF-α) in the host-parasite interrelationship, C57BL/6 TNF-α knockout mice (TNF-α−/−) as well as C57BL/6 wild-type mice were infected with pleomorphicT. bruceiAnTat 1.1 E parasites. In the TNF-α−/−mice, the peak levels of parasitemia were strongly increased compared to the peak levels recorded in wild-type mice. The increased parasite burden did not reflect differences in clearance efficacy or in production ofT. brucei-specific immunoglobulin M (IgM) and IgG antibodies. Trypanosome-mediated immunopathological features, such as lymph node-associated immunosuppression and lipopolysaccharide hypersensitivity, were found to be greatly reduced in infected TNF-α−/−mice. These results demonstrate that, during trypanosome infections, TNF-α is a key mediator involved in both parasitemia control and infection-associated pathology.
Differential Roles of Segmented Filamentous Bacteria and Clostridia in Development of the Intestinal Immune System
Authors: Umesaki Yoshinori , Setoyama Hiromi , Matsumoto Satoshi , Imaoka Akemi , Itoh Kikuji 
Published: December 2019

ABSTRACTThe presence of microflora in the digestive tract promotes the development of the intestinal immune system. In this study, to evaluate the roles of two types of indigenous microbe, segmented filamentous bacteria (SFB) and clostridia, whose habitats are the small and large intestines, respectively, in this immunological development, we analyzed three kinds of gnotobiotic mice contaminated with SFB, clostridia, and both SFB and clostridia, respectively, in comparison with germfree (GF) or conventionalized (Cvd) mice associated with specific-pathogen-free flora. In the small intestine, the number of αβ T-cell receptor-bearing intraepithelial lymphocytes (αβIEL) increased in SFB-associated mice (SFB-mice) but not in clostridium-associated mice (Clost-mice). There was no great difference in Vβ usage among GF mice, Cvd mice, and these gnotobiotic mice, although the association with SFB decreased the proportion of Vβ6+cells in CD8β−subsets to some extent, compared to that in GF mice. The expression of major histocompatibility complex class II molecules on the epithelial cells was observed in SFB-mice but not in Clost-mice. On the other hand, in the large intestine, the ratio of the number of CD4−CD8+cells to that of CD4+CD8−cells in αβIEL increased in Clost-mice but not in SFB-mice. On association with both SFB and clostridia, the numbers and phenotypes of IEL in the small and large intestines changed to become similar to those in Cvd mice. In particular, the ratio of the number of CD8αβ+cells to that of CD8αα+cells in αβIEL, unusually elevated in the small intestines of SFB-mice, decreased to the level in Cvd mice on contamination with both SFB and clostridia. The number of immunoglobulin A (IgA)-producing cells in the lamina propria was more elevated in SFB-mice than in Clost-mice, not only in the ileum but also in the colon. The number of IgA-producing cells in the colons of Clost-mice was a little increased compared to that in GF mice. Taken together, SFB and clostridia promoted the development of both IEL and IgA-producing cells in the small intestine and that of only IEL in the large intestine, respectively, suggesting the occurrence of compartmentalization of the immunological responses to the indigenous bacteria between the small and large intestines.
Dynamics of Actin-Based Movement by
Rickettsia rickettsii
in Vero Cells
Authors: Heinzen Robert A. , Grieshaber Scott S. , Van Kirk Levi S. , Devin Clinton J. 
Published: December 2019

ABSTRACT

Actin-based motility (ABM) is a virulence mechanism exploited by invasive bacterial pathogens in the genera
Listeria
,
Shigella
, and
Rickettsia
. Due to experimental constraints imposed by the lack of genetic tools and their obligate intracellular nature, little is known about rickettsial ABM relative to
Listeria
and
Shigella
ABM systems. In this study, we directly compared the dynamics and behavior of ABM of
Rickettsia rickettsii
and
Listeria monocytogenes
. A time-lapse video of moving intracellular bacteria was obtained by laser-scanning confocal microscopy of infected Vero cells synthesizing β-actin coupled to green fluorescent protein (GFP). Analysis of time-lapse images demonstrated that
R. rickettsii
organisms move through the cell cytoplasm at an average rate of 4.8 ± 0.6 μm/min (mean ± standard deviation). This speed was 2.5 times slower than that of
L. monocytogenes
, which moved at an average rate of 12.0 ± 3.1 μm/min. Although rickettsiae moved more slowly, the actin filaments comprising the actin comet tail were significantly more stable, with an average half-life approximately three times that of
L. monocytogenes
(100.6 ± 19.2 s versus 33.0 ± 7.6 s, respectively). The actin tail associated with intracytoplasmic rickettsiae remained stationary in the cytoplasm as the organism moved forward. In contrast, actin tails of rickettsiae trapped within the nucleus displayed dramatic movements. The observed phenotypic differences between the ABM of
Listeria
and
Rickettsia
may indicate fundamental differences in the mechanisms of actin recruitment and polymerization.
Expression and Immunogenicity of a Mutant Diphtheria Toxin Molecule, CRM
197
, and Its Fragments in
Salmonella typhi
Vaccine Strain CVD 908-
htrA
Authors: Orr Nadav , Galen James E. , Levine Myron M. 
Published: December 2019

ABSTRACT

Mutant diphtheria toxin molecule CRM
197
and fragments thereof were expressed in attenuated
Salmonella typhi
CVD 908-
htrA
, and the constructs were tested for their ability to induce serum antitoxin. Initially, expressed proteins were insoluble, and the constructs failed to induce neutralizing antitoxin. Soluble CRM
197
was expressed at low levels by utilizing the hemolysin A secretion system from
Escherichia coli.
Cloning and Molecular Characterization of Plasmid-Encoded Antigens of
Borrelia burgdorferi
Authors: Skare Jonathan T. , Foley Denise M. , Hernandez Santiago R. , Moore Deanna C. , Blanco David R. , Miller James N. , Lovett Michael A. 
Published: December 2019

ABSTRACT

Thirteen independent clones that encode
Borrelia burgdorferi
antigens utilizing antiserum from infection-immune rabbits were identified. The serum was adsorbed against noninfectious
B. burgdorferi
B31 to enrich for antibodies directed against either infection-associated antigens of
B. burgdorferi
B31 or proteins preferentially expressed during mammalian infection. The adsorption efficiency of the immune rabbit serum (IRS) was assessed by Western immunoblot analysis with protein lysates derived from infectious and noninfectious
B. burgdorferi
B31. The adsorbed IRS was used to screen a
B. burgdorferi
expression library to identify immunoreactive phage clones. Clones were then expressed in
Escherichia coli
and subsequently analyzed by Western blotting to determine the molecular mass of the recombinant
B. burgdorferi
antigens. Southern blot analysis of the 13 clones indicated that 10 contained sequences unique to infectious
B. burgdorferi
. Nucleotide sequence analysis indicated that the 13 clones were composed of 9 distinct genetic loci and that all of the genes identified were plasmid encoded. Five of the clones carried
B. burgdorferi
genes previously identified, including those encoding decorin binding proteins A and B (
dbpAB
), a
rev
homologue present on the 9-kb circular plasmid (cp9), a
rev
homologue from the 32-kb circular plasmid (cp32-6),
erpM
, and
erpX
. Additionally, four previously uncharacterized loci with no known homologues were identified. One of these unique clones encoded a 451-amino-acid lipoprotein with 21 consecutive, invariant 9-amino-acid repeats near the amino terminus that we have designated VraA (for “virulent strain-associated repetitive antigen A”). Since all the antigens identified are recognized by serum from infection immune rabbits, these antigens represent potential vaccine candidates and, based on the identification of
dbpAB
in this screen, may also be involved in pathogenic processes operative in Lyme borreliosis.
Staphylococcus aureusIsolates from Patients with Kawasaki Disease Express High Levels of Protein A
Authors: Wann Elisabeth R. , Fehringer Amy P. , Ezepchuk Yuri V. , Schlievert Patrick M. , Bina Paul , Reiser Raoul F. , Höök Magnus M. , Leung Donald Y. M. 
Published: December 2019

ABSTRACTKawasaki disease (KD) is an acute vasculitis of young children that can be complicated by coronary artery abnormalities. Recent findings suggest that a superantigen(s) may play an important role in stimulating the immune activation associated with the disease, although the origin of this superantigen(s) is unclear.Staphylococcus aureus, isolated from the rectum or pharynx of patients with KD, secretes toxic shock syndrome toxin 1 (TSST-1). The KD isolates express low levels of other exoproteins compared to isolates from patients with toxic shock syndrome (TSS). Thus, it was previously suggested that the KD isolates may be defective in the global regulatory locusagr(for accessory gene regulator), which positively regulates these factors (D. Y. M. Leung et al., Lancet 342:1385–1388, 1993). Here we describe another characteristic of KD isolates. When considered collectively, the KD isolates were found to express higher levels of staphylococcal protein A than the TSS isolates, another characteristic of anagr-defective phenotype. This correlated with a higher level ofspamRNA in these isolates. In contrast, the KD and TSS isolates expressed comparable levels of TSST-1, consistent with previous findings (D. Y. M. Leung et al., Lancet 342:1385–1388, 1993). Analysis of RNAIII transcript levels and nucleotide sequence analysis of the RNAIII-coding region suggested that the KD isolates are not defective in RNAIII, the effector molecule of theagrregulatory system. However, induction of RNAIII transcription in the KD isolates did not result in a dramatic decrease in the amount ofspamRNA, as has been reported for other strains (F. Vandenesch, J. Kornblum, and R. P. Novick, J. Bacteriol. 173:6313–6320, 1991).
Activation of Caspase 3 duringLegionella pneumophila-Induced Apoptosis
Authors: Gao Lian-Yong , Abu Kwaik Yousef 
Published: December 2019

ABSTRACTThe hallmark of Legionnaires’ disease is replication ofLegionella pneumophilawithin cells in the alveolar spaces. The mechanisms by whichL. pneumophilareplicates intracellularly and kills the host cell are largely not understood. We have recently shown that within 3 h of initiation of the infection and prior to intracellular replication,L. pneumophilainduces apoptosis in macrophages, alveolar epithelial cells, and peripheral blood monocytes, which correlates with cytopathogenicity (L.-Y. Gao and Y. Abu Kwaik, Infect. Immun. 67:862–870, 1999). In this report, we show that the ability ofL. pneumophilato induce apoptosis is, largely, not growth phase regulated. We demonstrate that the induction of apoptosis byL. pneumophilain macrophages is mediated through the activation of caspase 3. The enzymatic activity of caspase 3 to cleave a specific synthetic substrate in vitro is detected inL. pneumophila-infected macrophages at 2 h after infection and is maximal at 3 h, with over 900% increase in activity. The activity of caspase 3 to cleave a specific substrate [poly(ADP-ribose) polymerase, or PARP] in vivo is also detected at 2 h and is maximal at 3 h postinfection. The activity of caspase 3 to cleave the synthetic substrate in vitro and PARP in vivo is blocked by a specific inhibitor of caspase 3. The kinetics of caspase 3 activation correlates with that ofL. pneumophila-induced nuclear apoptosis. Inhibition of caspase 3 activity blocksL. pneumophila-induced nuclear apoptosis and cytopathogenicity during early stages of the infection. Consistent with the ability to induce apoptosis, extracellularL. pneumophilaalso activates caspase 3. ThreedotA/icmWXYZmutants ofL. pneumophilathat are defective in inducing apoptosis do not induce caspase 3 activation, suggesting that expression and/or export of the apoptosis-inducing factor(s) is regulated by thedot/icmvirulence system. This is the first description of the role of caspase 3 activation in induction of nuclear apoptosis in the host cell infected by a bacterial pathogen.
Purification and Characterization of Thin Pili of IncI1 Plasmids ColIb-P9 and R64: Formation of PilV-Specific Cell Aggregates by Type IV Pili
Authors: Yoshida Tetsu , Furuya Nobuhisa , Ishikura Masayuki , Isobe Toshiaki , Haino-Fukushima Kazu , Ogawa Toshio , Komano Teruya 
Published: December 2019

ABSTRACT

Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili. They were sedimented by ultracentrifugation from culture medium in which
Escherichia coli
cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation. In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope. Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein. Pilin was demonstrated to be the product of the
pilS
gene. Pilin was first synthesized as a 22-kDa prepilin from the
pilS
gene and subsequently processed to a 19-kDa protein by the function of the
pilU
product. The N-terminal amino group of the processed protein was shown to be modified. The C-terminal segments of the
pilV
products vary among six or seven different types, as a result of shufflon DNA rearrangements of the
pilV
gene. These PilV proteins were revealed to comprise a minor component of thin pili. Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.
Promoter Recognition by
Bacillus subtilis
ς
W
: Autoregulation and Partial Overlap with the ς
X
Regulon
Authors: Huang Xuejun , Fredrick Kurt L. , Helmann John D. 
Published: December 2019

ABSTRACT

The
Bacillus subtilis
genome encodes at least 17 distinct sigma factors, including seven members of the extracytoplasmic function (ECF) subfamily. We have investigated the expression and regulation of the ECF ς factor encoded by the
sigW
gene. A ς
W
-dependent promoter (P
W
) precedes
sigW
, demonstrating that this transcription factor is positively autoregulated. Expression of
sigW
is regulated by both growth phase and medium composition. Maximal expression is attained in early-stationary-phase cells grown in rich medium. We previously reported that
sigW
mutants have elevated transcription of some ς
X
-controlled genes, and we now report that the converse is also true: in a
sigX
mutant, P
W
is derepressed during logarithmic growth. Thus, these two regulons are mutually antagonistic. Reconstituted ς
W
holoenzyme faithfully recognizes the P
W
preceding
sigW
but does not recognize the P
X
promoter preceding the
sigX
gene. Autoregulation of
sigX
is also highly specific: ς
X
holoenzyme initiates transcription from P
X
but recognizes P
W
poorly if at all. In contrast, several promoters that are at least partially under ς
X
control are active with both the ς
X
and ς
W
holoenzymes in vitro. This finding supports the suggestion that the ς
W
and ς
X
regulons overlap. Sequence comparisons suggest that promoters recognized by these two ς factors have similar −35 elements but are distinguished by different base preferences at two key positions within the −10 element.
Induced Levels of Heat Shock Proteins in a
dnaK
Mutant of
Lactococcus lactis
Authors: Koch Birgit , Kilstrup Mogens , Vogensen Finn K. , Hammer Karin 
Published: December 2019

ABSTRACT

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and proteases, including the DnaK-GrpE-DnaJ and the GroELS chaperone complexes. In order to investigate the importance of the DnaK chaperone complex for growth and heat shock response regulation in
Lactococcus lactis
, we have constructed two
dnaK
mutants with C-terminal deletions in
dnaK
. The minor deletion of 65 amino acids in the
dnaKΔ2
mutant resulted in a slight temperature-sensitive phenotype. BK6, containing the larger deletion of 174 amino acids (
dnaKΔ1
), removing the major part of the inferred substrate binding site of the DnaK protein, exhibited a pronounced temperature-sensitive phenotype and showed altered regulation of the heat shock response. The expression of the heat shock proteins was increased at the normal growth temperature, measured as both protein synthesis rates and mRNA levels, indicating that DnaK could be involved in the regulation of the heat shock response in
L. lactis
. For
Bacillus subtilis
, it has been found (A. Mogk, G. Homuth, C. Scholz, L. Kim, F. X. Schmid, and W. Schumann, EMBO J. 16:4579–4590, 1997) that the activity of the heat shock repressor HrcA is dependent on the chaperone function of the GroELS complex and that a
dnaK
insertion mutant has no effect on the expression of the heat shock proteins. The present data from
L. lactis
suggest that the DnaK protein could be involved in the maturation of the homologous HrcA protein in this bacterium.
Identification of OmpT as the Protease That Hydrolyzes the Antimicrobial Peptide Protamine before It Enters Growing Cells of
Escherichia coli
Authors: Stumpe Stefan , Schmid Roland , Stephens Daren L. , Georgiou George , Bakker Evert P. 
Published: December 2019

ABSTRACT

The influence of extracytoplasmic proteases on the resistance of
Escherichia coli
to the antimicrobial peptide protamine was investigated by testing strains with deletions in the protease genes
degP
,
ptr
, and
ompT
. Only Δ
ompT
strains were hypersusceptible to protamine. This effect was abolished by plasmids carrying
ompT
. Both at low and at high Mg
2+
concentrations,
ompT
+
strains cleared protamine from the medium within a few minutes. By contrast, at high Mg
2+
concentrations, protamine remained present for at least 1 h in the medium of an
ompT
strain. These data indicate that OmpT is the protease that degrades protamine and that it exerts this function at the external face of the outer membrane.
Genetic Analysis of the Role of the Transfer Gene,
traN
, of the F and R100-1 Plasmids in Mating Pair Stabilization during Conjugation
Authors: Klimke William A. , Frost Laura S. 
Published: December 2019

ABSTRACT

Mating pair stabilization occurs during conjugative DNA transfer whereby the donor and recipient cells form a tight junction which requires pili as well as TraN and TraG in the donor cell. The role of the outer membrane protein, TraN, during conjugative transfer was examined by introduction of a chloramphenicol resistance cassette into the
traN
gene on an F plasmid derivative, pOX38, to produce pOX38
N1
::CAT. pOX38
N1
::CAT was greatly reduced in its ability to transfer DNA, indicating that TraN plays a greater role in conjugation than previously thought. F and R100-1
traN
were capable of complementing pOX38
N1
::CAT transfer equally well when wild-type recipients were used. F
traN
, but not R100-1
traN
, supported a much lower level of transfer when there was an
ompA
mutation or lipopolysaccharide (LPS) deficiency in the recipient cell, suggesting receptor specificity. The R100-1
traN
gene was sequenced, and the gene product was found to exhibit 82.3% overall similarity with F TraN. The differences were mainly located within a central region of the proteins (amino acids 162 to 333 of F and 162 to 348 of R100-1). Deletion analysis of F
traN
suggested that this central portion might be responsible for the receptor specificity displayed by TraN. TraN was not responsible for TraT-dependent surface exclusion. Thus, TraN, and not the F pilus, appears to interact with OmpA and LPS moieties during conjugation, resulting in mating pair stabilization, the first step in efficient mobilization of DNA.
Tetrachloroethene Dehalogenase from
Dehalospirillum multivorans
: Cloning, Sequencing of the Encoding Genes, and Expression of the
pceA
Gene in
Escherichia coli
Authors: Neumann Anke , Wohlfarth Gert , Diekert Gabriele 
Published: December 2019

ABSTRACT

The genes encoding tetrachloroethene reductive dehalogenase, a corrinoid-Fe/S protein, of
Dehalospirillum multivorans
were cloned and sequenced. The
pceA
gene is upstream of
pceB
and overlaps it by 4 bp. The presence of a ς70-like promoter sequence upstream of
pceA
and of a ρ-independent terminator downstream of
pceB
indicated that both genes are cotranscribed. This assumption is supported by reverse transcriptase PCR data. The
pceA
and
pceB
genes encode putative 501- and 74-amino-acid proteins, respectively, with calculated molecular masses of 55,887 and 8,354 Da, respectively. Four peptides obtained after trypsin treatment of tetrachloroethene (PCE) dehalogenase were found in the deduced amino acid sequence of
pceA
. The N-terminal amino acid sequence of the PCE dehalogenase isolated from
D. multivorans
was found 30 amino acids downstream of the N terminus of the deduced
pceA
product. The
pceA
gene contained a nucleotide stretch highly similar to binding motifs for two Fe
4
S
4
clusters or for one Fe
4
S
4
cluster and one Fe
3
S
4
cluster. A consensus sequence for the binding of a corrinoid was not found in
pceA
. No significant similarities to genes in the databases were detected in sequence comparisons. The
pceB
gene contained two membrane-spanning helices as indicated by two hydrophobic stretches in the hydropathic plot. Sequence comparisons of
pceB
revealed no sequence similarities to genes present in the databases. Only in the presence of pUBS 520 supplying the recombinant bacteria with high levels of the rare
Escherichia coli
tRNA
4
Arg
was
pceA
expressed, albeit nonfunctionally, in recombinant
E. coli
BL21 (DE3).
Gene Transfer between Related Bacteria by Electrotransformation: Mapping
Salmonella typhi
Genes in
Salmonella typhimurium
Authors: Toro Cecilia S. , Mora Guido C. , Figueroa-Bossi Nara 
Published: December 2019

ABSTRACT

Transfer of newly isolated mutations into a fresh background is an essential step of genetic analysis and strain construction. Gene transfer is hampered in
Salmonella typhi
and in other pathogenic bacteria by the lack of a generalized transduction system. We show here that this problem can be partially circumvented by using electrotransformation as a means for delivering
S. typhi
DNA into suitable
S. typhi
or
Salmonella typhimurium
recipients. Transferred DNA can recombine with the homologous region in the host chromosome. In one application of the method, mutations isolated in
S. typhi
were genetically mapped in
S. typhimurium
.