bluuebunny/crossref_metadata_2025_split huggingface dataset:
Integrated Physical and Genetic Mapping of
Bacillus cereus
and Other Gram-Positive Bacteria Based on IS
231
A Transposition Vectors
Authors: Léonard Catherine , Zekri Omar , Mahillon Jacques 
Published: December 2019

ABSTRACT

The genome structure of
Bacillus cereus
is relatively complex, its DNA being modulated between a size-varying chromosome and large plasmids. To study the genetic organization of the
B. cereus
type strain ATCC 14579, thermosensitive transposition vectors were designed on the basis of IS
231
A-derived cassettes containing uncommon restriction sites. A highly preferred insertion site for IS
231
A was detected in the chromosome by Southern blotting and pulsed-field gel electrophoresis (PFGE) analyses of independent insertion mutants. However, once this insertional hot spot was occupied, secondary IS
231
A insertions occurred randomly, as demonstrated by isolation of independent
B. cereus
auxotrophs at a frequency of approximately 0.6%. The hot-spot site, as well as several auxotrophic mutations, were mapped by using
Not
I,
Sfi
I, and
Asc
I PFGE restriction profiles. It was confirmed by sequencing that one of the insertions, generating an Ade
−
phenotype, had disrupted a gene of the purine synthesis pathway. These results showed that combined PFGE and sequencing analyses of mini-IS
231
A insertions enable the construction of integrated physical and genetic maps of
B. cereus
type strain. Moreover, the presence of the ultrarare I-
Sce
I restriction site in the mini-IS
231
A allowed the isolation, in double-insertion mutants, of contiguous and nonoverlapping large chromosomal fragments, convenient for direct sequencing. The system detailed in this report is therefore a powerful tool for comparative genetic studies among members of the
B. cereus
group (i.e.,
B. cereus
,
B. thuringiensis
,
B. mycoides
, and
B. anthracis
) and could also be applied to more distantly related gram-positive bacteria.
Structural Properties of Group B Streptococcal Type III Polysaccharide Conjugate Vaccines That Influence Immunogenicity and Efficacy
Authors: Wessels Michael R. , Paoletti Lawrence C. , Guttormsen Hilde-Kari , Michon Francis , D’Ambra Anello J. , Kasper Dennis L. 
Published: December 2019

ABSTRACTIn this study, we tested the hypothesis that the immunogenicity and protective efficacy of polysaccharide-protein conjugate vaccines are influenced by three variables: (i) molecular size of the conjugate, (ii) molecular size of the polysaccharide used for conjugation, and (iii) extent of polysaccharide-to-protein cross-linking. Type III group BStreptococcuscapsular polysaccharide was linked by reductive amination at multiple sites to tetanus toxoid to create a polysaccharide-protein conjugate (III-TT). A single lot of III-TT was fractionated into small, medium, and largeMrpools. Whereas all three conferred protection in a maternal immunization-neonatal challenge model in mice, the smallestMrconjugate evoked less polysaccharide-specific immunoglobulin G (IgG) than the two largerMrconjugates. To test whether the molecular size of the polysaccharide used for conjugation also affected the immunogenicity of the conjugate, vaccines were synthesized using capsular polysaccharides withMrs of 38,000, 105,000, and 349,000. Polysaccharide-specific IgG responses in mice increased with theMrof the polysaccharides, and protective efficacy was lower for the smallest polysaccharide conjugate compared to the other two vaccines. Immunogenicity testing of a series of vaccines prepared with different degrees of polysaccharide-to-protein cross-linking demonstrated higher polysaccharide-specific antibody responses as the extent of cross-linking increased. However, opsonic activity was greatest in mouse antiserum raised to a moderately cross-linked conjugate, suggesting that some antibodies evoked by highly cross-linked conjugates were directed to a nonprotective epitope. We conclude that conjugate size, polysaccharide size, and degree of polysaccharide-protein cross-linking influence the immunogenicity and protective efficacy of III-TT conjugate vaccines.
Changes in Murine Jejunal Morphology Evoked by the Bacterial SuperantigenStaphylococcus aureusEnterotoxin B Are Mediated by CD4+T Cells
Authors: Benjamin Michelle A. , Lu Jun , Donnelly Graeme , Dureja Parul , McKay Derek M. 
Published: December 2019

ABSTRACTBacterial superantigens (SAgs) are potent T-cell stimuli that have been implicated in the pathophysiology of autoimmune and inflammatory disease. We usedStaphylococcus aureusenterotoxin B (SEB) as a model SAg to assess the effects of SAg exposure on gut form and cellularity. BALB/c, SCID (lacking T cells) and T-cell-reconstituted SCID mice were treated with SEB (5 or 100 μg intraperitoneally), and segments of the mid-jejunum were removed 4, 12, or 48 h later and processed for histochemical or immunocytochemical analysis of gut morphology and major histocompatibility complex class II (MHC II) expression and the enumeration of CD3+T cells and goblet cells. Control mice received saline only. SEB treatment of BALB/c mice caused a time- and dose-dependent enteropathy that was characterized by reduced villus height, increased crypt depth, and a significant increase in MHC II expression. An increase in the number of CD3+T cells was observed 48 h after exposure to 100 μg of SEB. Enteric structural alterations were not apparent in SEB-treated SCID mice compared to saline-treated SCID mice. In contrast, SEB challenge of SCID mice reconstituted with a mixed lymphocyte population or purified murine CD4+T cells resulted in enteric histopathological changes reminiscent of those observed in SEB-treated BALB/c mice. These findings implicate CD4+T cells in this SEB-induced enteropathy. Our results show that SAg immune activation causes significant changes in jejunal villus-crypt architecture and cellularity that are likely to impact on normal physiological processes. We speculate that the elevated MHC II expression and increased number of T cells could allow for enhanced immune responsiveness to other SAgs or environmental antigens.
The Surface ofToxoplasmaTachyzoites Is Dominated by a Family of Glycosylphosphatidylinositol-Anchored Antigens Related to SAG1
Authors: Manger Ian D. , Hehl Adrian B. , Boothroyd John C. 
Published: December 2019

ABSTRACTToxoplasma gondiiis an Apicomplexan parasite with a complex life cycle that includes a rapidly dividing asexual stage known as the tachyzoite. The tachyzoite surface has been reported to comprise five major antigens, the most abundant of which is designated SAG1 (for surface antigen 1). At least one of the other four (SAG3) and another recently described minor antigen (SRS1 [for SAG1-related sequence 1]) have previously been shown to be structurally related to SAG1. To determine if further SAG1 homologs exist, we searched aToxoplasmaexpressed sequence tag (EST) database and found numerous ESTs corresponding to at least three new genes related toSAG1. LikeSAG1, these newSRSgenes encode apparently glycosylphosphatidylinositol-anchored proteins that share several motifs and a set of conserved cysteine residues. This family appears to have arisen by divergence from a common ancestor under selection for the conservation of overall topology. The products of two of these new genes (SRS2andSRS3) are shown to be expressed on the surface ofToxoplasmatachyzoites by immunofluorescence. We also identified strain-specific differences in relative expression levels. A total of 10 members of theSAG1gene family have now been identified, which apparently include three of the five major surface antigens previously described and one antigen expressed only in bradyzoites. The function of this family may be to provide a redundant system of receptors for interaction with host cells and/or to direct the immune responses that limit acuteT. gondiiinfections.
Disruption of the Cellular Inflammatory Response toListeria monocytogenesInfection in Mice with Disruptions in Targeted Genes
Authors: DiTirro J. , Rhoades E. R. , Roberts A. D. , Burke J. M. , Mukasa A. , Cooper A. M. , Frank A. A. , Born W. K. , Orme I. M. 
Published: December 2019

ABSTRACTThe results of this study to dissect the nature of the acquired immune response to infection withListeria monocytogenesin mice with targetted gene disruptions show that successful resolution of disease requires the essential presence of αβ T cells and the capacity to elaborate gamma interferon. In the absence of either of these entities, mice experience increasingly severe hepatitis and tissue necrosis and die within a few days. The data from this study support the hypothesis that the protective process is the efficient replacement of neutrophils in lesions by longer-lived mononuclear phagocytes; αβ-T-cell-knockout mice died from progressive infection before neutrophil replacement could occur, whereas in γδ-T-cell-knockout mice this replacement process in the liver has previously been shown to be much slower. In the present study we attribute this delay to reduced production of the macrophage-attracting chemokine MCP-1 in the γδ-T-cell-knockout animals. These data further support the hypothesis that γδ T cells are important in controlling the inflammatory process rather than being essential to the expression of protection.
Control of Ferredoxin and Gal/GalNAc Lectin Gene Expression in
Entamoeba histolytica
by a
cis
-Acting DNA Sequence
Authors: Gilchrist Carol A. , Mann Barbara J. , Petri William A. 
Published: December 2019

ABSTRACT

The ferredoxin (
fdx
) and lectin (
hgl
5) promoters of
Entamoeba histolytica
contain the DNA sequence motif TATTCTATT (URE3). Previously we showed that mutation of the URE3 motif in the
hgl
5 lectin promoter results in an increase in promoter reporter activity. Mutation of this motif in the
fdx
promoter led to a 40-to-50% decrease in
fdx
promoter activity as measured by reporter gene activity and abundance of mRNA.
E. histolytica
nuclear proteins exhibited sequence-specific binding to the URE3 motif in electrophoretic mobility shift assays. These results support the regulation of both ferredoxin and lectin promoters by a nuclear protein(s) which recognizes the URE3 motif.
γδ+T Cells Preferentially Respond to Live Rather than Killed Malaria Parasites
Authors: Waterfall Martin , Black Antony , Riley Eleanor 
Published: December 2019

ABSTRACTWe have compared the in vitro responses of peripheral blood T cells from malaria-unexposed donors to livePlasmodium falciparumschizonts, freeze-thawed schizont extracts (P. falciparumschizont extracts [PfSE]), and parasite culture supernatants. We show that the cells responding to PfSE and parasite culture supernatants are predominantly CD4+TCRαβ+while in the presence of live schizonts there is an additional activation of TCRγδ+cells. Activation of TCRγδ+cells in response to PfSE was seen only when irradiated autologous feeder cells or recombinant interleukin-2 (IL-2) was added to the cultures. Live schizonts but not PfSE induced significant IL-2 production in vitro in the first 5 days after stimulation, suggesting that induction of early IL-2 by live parasites may contribute to the marked activation of the TCRγδ+population.
Neurotoxicity of
Clostridium perfringens
Epsilon-Toxin for the Rat Hippocampus via the Glutamatergic System
Authors: Miyamoto Osamu , Minami Junzaburo , Toyoshima Tetsuhiko , Nakamura Takehiro , Masada Tetsuya , Nagao Seigo , Negi Tetsuro , Itano Toshifumi , Okabe Akinobu 
Published: December 2019

ABSTRACT

The neurotoxicity of epsilon-toxin, one of the major lethal toxins produced by
Clostridium perfringens
type B, was studied by histological examination of the rat brain. When the toxin was injected intravenously at a lethal dose (100 ng/kg), neuronal damage was observed in many areas of the brain. Injection of the toxin at a sublethal dose (50 ng/kg) caused neuronal damage predominantly in the hippocampus: pyramidal cells in the hippocampus showed marked shrinkage and karyopyknosis, or so-called dark cells. The dark cells lost the immunoreactivity to microtubule-associated protein-2, a postsynaptic somal and dendric marker, while acetylcholinesterase-positive fibers were not affected. Timm’s zinc staining revealed that zinc ions were depleted in the mossy layers of the CA3 subfield containing glutamate as a synaptic transmitter. The cerebral blood flow in the hippocampus was not altered significantly before or after administration of the toxin, as measured by laser-Doppler flowmetry, excluding the possibility that the observed histological change was due to a secondary effect of ischemia in the hippocampus. Prior injection of either a glutamate release inhibitor or a glutamate receptor antagonist protected the hippocampus from the neuronal damage caused by epsilon-toxin. These results suggest that epsilon-toxin acts on the glutamatergic system and evokes excessive release of glutamate, leading to neuronal damage.
Pseudomonas aeruginosa lasR
Transcription Correlates with the Transcription of
lasA
,
lasB
, and
toxA
in Chronic Lung Infections Associated with Cystic Fibrosis
Authors: Storey Douglas G. , Ujack Eva E. , Rabin Harvey R. , Mitchell Ian 
Published: December 2019

ABSTRACT

The role of
Pseudomonas aeruginosa
quorum-sensing systems in the lung infections associated with cystic fibrosis (CF) has not been examined. The purpose of this study was to determine if genes regulated by the LasR-LasI quorum-sensing system were coordinately regulated by the
P. aeruginosa
populations during the lung infections associated with CF. We also wanted to ascertain if there was a relationship between the expression of
lasR
, a transcriptional regulator, and some
P. aeruginosa
virulence factors during these infections. We extracted RNAs from the bacterial populations of 131 sputa taken from 23 CF patients. These RNAs were blotted and hybridized with probes to
P. aeruginosa lasA
,
lasB
, and
toxA
. The hybridization signals from each probe were ranked, and the rankings were analyzed by a Spearman rank correlation to determine if there was an association between the population transcript accumulations for the three genes. The correlations between the transcript accumulation patterns of pairs of the genes suggested that
lasA
,
lasB
, and
toxA
might be coordinately regulated during CF lung infections. To determine if this coordinate regulation might be due to regulation by LasR, we probed RNAs, extracted from 84 sputa, with the
lasR
,
lasA
,
lasB
,
toxA
, and
algD
probes. Statistical analysis indicated that
lasR
transcript accumulation correlated to
lasA
,
lasB
,
toxA
, and
algD
transcript accumulations. These results indicated that
lasR
may at least partially regulate or be coordinately regulated with
lasA
,
lasB
,
toxA
, and
algD
during the lung infections associated with CF. These results also suggested that the LasR-LasI quorum-sensing system may control the expression of at least some virulence factors in the lungs of patients with CF.
Molecular Characterization of
Mycoplasma arthritidis
Variable Surface Protein MAA2
Authors: Washburn Leigh Rice , Weaver Keith E. , Weaver Elizabeth J. , Donelan Wendy , Al-Sheboul Suhaila 
Published: December 2019

ABSTRACT

Earlier studies implied a role for
Mycoplasma arthritidis
surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability. Here we report the further analysis of MAA2 and the cloning and sequencing of the
maa2
gene from two
M. arthritidis
strains, 158p10p9 and H606, expressing two size variants of MAA2. Triton X-114 partitioning and metabolic labeling with [
3
H]palmitic acid suggested lipid modification of MAA2. Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y. The
maa2
genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats. The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide. The
maa2
gene was expressed in
Escherichia coli
from the
lacZ
promoter of vector pGEM-T. The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in
E. coli
. The
maa2
gene and upstream DNA sequences were cloned from
M. arthritidis
clonal variants differing in MAA2 expression state. Expression state correlated with the length of a poly(T) tract just upstream of a putative −10 box. Full-sized recombinant MAA2 was expressed in
E. coli
from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region.
Antigen-Induced Protective and Nonprotective Cell-Mediated Immune Components againstCryptococcus neoformans
Authors: Murphy Juneann W. , Schafer Fredda , Casadevall Arturo , Adesina Adekunle 
Published: December 2019

ABSTRACTMice immunized with two different cryptococcal antigen preparations, one a soluble culture filtrate antigen (CneF) in complete Freund’s adjuvant (CFA) and the other heat-killedCryptococcus neoformanscells (HKC), develop two different profiles of activated T cells. CneF-CFA induces CD4+T cells responsible for delayed-type hypersensitivity (DTH) reactivity and for amplification of the anticryptococcal DTH response, whereas HKC induce CD4+and CD8+T cells involved in anticryptococcal DTH reactivity and activated T cells which directly killC. neoformanscells. The main purpose of this study was to assess the level of protection afforded by each of the two different T-cell profiles against challenge with viableC. neoformanscells, thereby identifying which activated T-cell profile provides better protection. CBA/J mice immunized with CneF-CFA had significantly better protective responses, based on better clearance ofC. neoformansfrom tissues, on longer survival times, and on fewer and smaller lesions in the brain, than HKC-immunized mice or control mice similarly infected withC. neoformans. Both immunization protocols induced an anticryptococcal DTH response, but neither induced serum antibodies to glucuronoxylmannan, so the protection observed in the CneF-CFA immunized mice was due to the activated T-cell profile induced by that protocol. HKC-immunized mice, which displayed no greater protection than controls, did not have the amplifier cells. Based on our findings, we propose that the protective anticryptococcal T cells are the CD4+T cells which have been shown to be responsible for DTH reactivity and/or the CD4+T cells which amplify the DTH response and which have been previously shown to produce high levels of gamma interferon and interleukin 2. Our results imply that there are protective and nonprotective cell-mediated immune responses and highlight the complexity of the immune response toC. neoformansantigens.
Roles of the
Candida albicans
Mitogen-Activated Protein Kinase Homolog, Cek1p, in Hyphal Development and Systemic Candidiasis
Authors: Csank Csilla , Schröppel Klaus , Leberer Ekkehard , Harcus Doreen , Mohamed Othman , Meloche Sylvain , Thomas David Y. , Whiteway Malcolm 
Published: December 2019

ABSTRACT

Extracellular signal-regulated protein kinase (ERK, or mitogen-activated protein kinase [MAPK]) regulatory cascades in fungi turn on transcription factors that control developmental processes, stress responses, and cell wall integrity.
CEK1
encodes a
Candida albicans
MAPK homolog (Cek1p), isolated by its ability to interfere with the
Saccharomyces cerevisiae
MAPK mating pathway.
C. albicans
cells with a deletion of the
CEK1
gene are defective in shifting from a unicellular budding colonial growth mode to an agar-invasive hyphal growth mode when nutrients become limiting on solid medium with mannitol as a carbon source or on glucose when nitrogen is severely limited. The same phenotype is seen in
C. albicans
mutants in which the homologs (
CST20
,
HST7
, and
CPH1
) of the
S. cerevisiae STE20
,
STE7
, and
STE12
genes are disrupted. In
S. cerevisiae
, the products of these genes function as part of a MAPK cascade required for mating and invasiveness of haploid cells and for pseudohyphal development of diploid cells. Epistasis studies revealed that the
C. albicans CST20
,
HST7
,
CEK1
, and
CPH1
gene products lie in an equivalent, canonical, MAPK cascade. While Cek1p acts as part of the MAPK cascade involved in starvation-specific hyphal development, it may also play independent roles in
C. albicans
. In contrast to disruptions of the
HST7
and
CPH1
genes, disruption of the
CEK1
gene adversely affects the growth of serum-induced mycelial colonies and attenuates virulence in a mouse model for systemic candidiasis.
Neither the Bvg
−
Phase nor the
vrg6
Locus of
Bordetella pertussis
Is Required for Respiratory Infection in Mice
Authors: Martinez de Tejada Guillermo , Cotter Peggy A. , Heininger Ulrich , Camilli Andrew , Akerley Brian J. , Mekalanos John J. , Miller Jeff F. 
Published: December 2019

ABSTRACT

In
Bordetella
species, the BvgAS sensory transduction system mediates an alteration between the Bvg
+
phase, characterized by expression of adhesins and toxins, and the Bvg
−
phase, characterized by the expression of motility and coregulated phenotypes in
Bordetella bronchiseptica
and by the expression of
vrg
loci in
Bordetella pertussis
. Since there is no known environmental or animal reservoir for
B. pertussis
, the causative agent of whooping cough, it has been assumed that this phenotypic alteration must occur within the human host during infection. Consistent with this hypothesis was the observation that a
B. pertussis
mutant, SK6, containing a Tn
phoA
insertion mutation in a Bvg-repressed gene (
vrg6
) was defective for tracheal and lung colonization in a mouse model of respiratory infection (D. T. Beattie, R. Shahin, and J. Mekalanos, Infect. Immun. 60:571–577, 1992). This result was inconsistent, however, with the observation that a Bvg
+
phase-locked
B. bronchiseptica
mutant was indistinguishable from the wild type in its ability to establish a persistent respiratory infection in rabbits and rats (P. A. Cotter and J. F. Miller, Infect. Immun. 62:3381–3390, 1994; B. J. Akerley, P. A. Cotter, and J. F. Miller, Cell 80:611–620, 1995). To directly address the role of Bvg-mediated signal transduction in
B. pertussis
pathogenesis, we constructed Bvg
+
and Bvg
−
phase-locked mutants and compared them with the wild type for their ability to colonize the respiratory tracts of mice. Our results show that the Bvg
+
phase of
B. pertussis
is necessary and sufficient for respiratory infection. By constructing a strain with a deletion in the
bvgR
regulatory locus, we also show that ectopic expression of Bvg
−
phase phenotypes decreases the efficiency of colonization, underscoring the importance of Bvg-mediated repression of gene expression in vivo. Finally, we show that the virulence defect present in strain SK6 cannot be attributed to the
vrg6
mutation. These data contradict an in vivo role for the Bvg
−
phase of
B. pertussis.
Listeria monocytogenes-Infected Hepatocytes Are Targets of Major Histocompatibility Complex Class Ib-Restricted Antilisterial Cytotoxic T Lymphocytes
Authors: Bouwer H. G. Archie , Bai Ailin , Forman James , Gregory Stephen H. , Wing Edward J. , Barry Ronald A. , Hinrichs David J. 
Published: December 2019

ABSTRACTSubclinical infection of BALB/c mice with the intracellular bacterial pathogenListeria monocytogenesresults in the development of protective antilisterial immunity.L. monocytogenescan infect hepatocytes, and antilisterial cytotoxic T lymphocytes (CTL) lyseListeria-infected hepatocytes in a major histocompatibility complex (MHC) class Ia-restricted manner. It remained to be determined whetherL. monocytogenes-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis. In this study, we showed that hepatocytes express MHC class Ib molecule Qa-1bmRNA and protein. We further showed thatListeria-infected hepatocytes are susceptible to MHC class Ib-restricted cytolysis, since C57BL/6-derivedListeria-infected hepatocytes were lysed by BALB/c-derived antilisterial CTL. These results establish thatListeria-infected hepatocytes are susceptible to cytolysis by MHC class Ib restrictedListeria-specific CTL.