bluuebunny/crossref_metadata_2025 huggingface dataset:
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

In 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

Bacterial 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

Toxoplasma 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

The 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

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

We 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

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

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

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

Mice 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

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

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

Subclinical 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.
Intrapulmonary Delivery of Tumor Necrosis Factor Agonist Peptide Augments Host Defense in Murine Gram-Negative Bacterial Pneumonia
Authors: Laichalk Lauri L. , Bucknell Kathy A. , Huffnagle Gary B. , Wilkowski Jodi M. , Moore Thomas A. , Romanelli Robert J. , Standiford Theodore J.
Published: December 2019

Tumor necrosis factor alpha (TNF) has been shown to be an essential cytokine mediator of innate immunity inKlebsiellapneumonia. Recently, a TNF agonist peptide consisting of the 11-amino-acid TNF binding site (TNF70-80) has been shown to possess many of the leukocyte-activating properties of TNF without the associated toxicity when administered locally or systemically. Given the beneficial effects of TNF in gram-negative pneumonia, we hypothesize that the intratracheal (i.t.) administration of TNF70-80would augment lung innate immunity in mice challenged with intrapulmonaryKlebsiella pneumoniae. The administration of TNF70-80i.t. to CBA/J mice 7 days prior to, but not concomitantly with, the i.t. delivery of 3 × 103CFU ofK. pneumoniaeresulted in a marked increase in survival compared to that of animals receiving a control peptide i.t. In addition, pretreatment with TNF70-80resulted in improved bacterial clearance, which occurred in association with enhanced lung myeloperoxidase activity (as a measure of lung polymorphonuclear leukocyte influx), and increased expression of the important activating cytokines TNF, macrophage inflammatory protein-2, interleukin-12, and gamma interferon compared that for animals receiving control peptide. Finally, the administration of TNF70-80intraperitoneally resulted in enhanced rather than decreased lethality ofKlebsiellapneumonia compared to that for animals receiving either TNF70-80or control peptide i.t. Our studies suggest that the intrapulmonary, but not systemic, administration of the TNF agonist peptide may serve as an important immunoadjuvant in the treatment of murineKlebsiellapneumonia.
Characterization of a
sar
Homolog of
Staphylococcus epidermidis
Authors: Fluckiger Ursula , Wolz Christiane , Cheung Ambrose L.
Published: December 2019

Coagulase-negative staphylococci are common nosocomial pathogens. A regulatory element, designated
sar
, partially controls exoprotein synthesis in coagulase-positive
Staphylococcus aureus
by modulating the expression of another regulatory locus, called
agr
. We report here the cloning of a
sar
homolog in
S. epidermidis
. The major open reading frame within
sar
in
S. epidermidis
is highly homologous (84%) to the
S. aureus
SarA protein. Primer extension studies revealed three
sar
transcripts (0.64, 0.76, and 0.85 kb) initiated from three distinct promoters. The interpromoter region in
S. epidermidis
differs from its
S. aureus
counterpart, possibly suggesting target gene differences and a disparate pattern for
sar
activation. Remarkably, the
S. epidermidis sar
homolog interacts with an
agr
promoter fragment of
S. aureus
in gel shift assays. Additionally,
S. epidermidis sar
fragments could restore hemolysin production in an
S. aureus sar
mutant. As typical virulence determinants controlled by
sar
in
S. aureus
are not present in
S. epidermidis
, an examination of functional and structural similarities and divergence of
sar
in staphylococci will be of major interest.
Assimilation of Xylose, Mannose, and Mannitol for Synthesis of Glucuronoxylomannan of
Cryptococcus neoformans
Determined by
13
C Nuclear Magnetic Resonance Spectroscopy
Authors: Cherniak Robert , O’Neill Eric B. , Sheng Shuqun
Published: December 2019

Cryptococcus neoformans
NIH 409 was cultured in a defined medium containing
d
-[1-
13
C]xylose (Xyl),
d
-[1-
13
C]mannose (Man), or
d
-[1-
13
C]mannitol as the sole carbon source. The distribution of
13
C in the Man, Xyl, glucuronic acid (GlcA), and O-acetyl constituents of native and de-O-acetylated glucuronoxylomannan (GXM) was determined by one-dimensional
13
C nuclear magnetic resonance spectroscopy. The carbon chain of Man was incorporated intact into GXM since
13
C was observed only in carbon 1 of Man, GlcA, and Xyl. The carbon chain of mannitol was incorporated intact into GXM since
13
C was observed only in carbons 1 and 6. This was expected since mannitol has an axis of symmetry. The carbon chain of Xyl was identified only in carbons 1 and 3 of Man, GlcA, and Xyl. This pattern of labeling is consistent with the assimilation of Xyl through the pentophosphate pathway.
IS
195
, an Insertion Sequence-Like Element Associated with Protease Genes in
Porphyromonas gingivalis
Authors: Lewis Janina P. , Macrina Francis L.
Published: December 2019

Porphyromonas gingivalis
is recognized as an important etiologic agent in adult and early-onset periodontal disease. Proteases produced by this organism contribute to its virulence in mice. Protease-encoding genes have been shown to contain multiple copies of repeated nucleotide sequences. These conserved sequences have also been found in hemagglutinin genes. In the process of studying the genetic loci containing the conserved repeated sequences, we have characterized a
prtP
gene homolog from
P. gingivalis
W83 encoding a cysteine protease with Lys-X specificity. However, this
prtP
gene was interrupted by an insertion sequence-like element which we designated IS
195
. Furthermore, IS
195
and another element, IS
1126
, were present downstream of
prtP
gene homologs (
kgp
) found in
P. gingivalis
H66 and 381. IS
195
, a 1,068-bp insertion sequence-like element, contained 11-bp inverted repeats at its termini and was bordered by 9-bp direct repeats presumed to be a transposition-mediated target site duplication. Its central region contained one large open reading frame encoding a predicted 300-amino-acid protein which appeared to be a transposase. We isolated two naturally occurring variants of
P. gingivalis
W83, one carrying IS
195
within the coding region of the
prtP
gene and another containing an intact
prtP
gene. Biochemical characterization revealed a lack of trypsin-like Lys-X specific proteolytic activity in the
P. gingivalis
W83 variant carrying the disrupted
prtP
gene. Studies using a mouse model revealed a reduction of virulence resulting from insertion of IS
195
into the coding region of the
prtP
gene. An allelic-exchange mutant defective in the
prtP
gene also was constructed and tested in vivo. It displayed intermediate virulence compared to that of the wild-type and
prtP
::IS
195
mutant strains. We conclude that the Lys-X cysteine protease contributes to virulence in soft tissue infections.
Pseudomonas aeruginosaExoenzyme S Is a Mitogen but Not a Superantigen for Human T Lymphocytes
Authors: Bruno Tony F. , Buser Deborah E. , Syme Rachel M. , Woods Donald E. , Mody Christopher H.
Published: December 2019

Virtually all cystic fibrosis (CF) patients become infected withPseudomonas aeruginosa, and once the infection is established, the organism is rarely cleared. One of theP. aeruginosavirulence factors, exoenzyme S, has been shown to correlate with increased morbidity and mortality both in rat models of chronic pulmonary inflammation and in human CF patients. It has previously been shown that exoenzyme S is a potent stimulus for the proliferation of T cells in greater than 95% of adults, which could contribute to the pathogenesis of CF. The goal of this study was to determine the mechanism of T-cell stimulation by exoenzyme S in an effort to shed light on the immune response and contribute to understanding its role inP. aeruginosapathogenesis. The current studies demonstrate that exoenzyme S stimulates naive T cells, since fetal blood lymphocytes proliferated and adult lymphocytes that expressed CD45RA proliferated. The percentage of T cells activated by exoenzyme S after a 4-h culture (as measured by CD69 surface expression) was intermediate in magnitude compared to levels induced by a panel of superantigens and mitogens. To determine the mechanism of activation, the requirement for accessory cells was investigated. The proliferative response to exoenzyme S was dependent on the presence of accessory cells but was not blocked by an anti-DR antibody. Exoenzyme S activated both CD4+and CD8+T cells, but CD4+T cells were preferentially activated. The Vβ repertoire of donor T cells showed no preferential activation or preferential expansion after stimulation by exoenzyme S, suggesting that it is not a superantigen. Taken together, our data suggest that exoenzyme S is a T-cell mitogen but not a superantigen. Activation of a large percentage of T lymphocytes by exoenzyme S may produce a lymphocyte-mediated inflammatory response that should be considered in the pathogenesis of CF.
Immunization with
Pneumocystis carinii
gpA Is Immunogenic but Not Protective in a Mouse Model of
P. carinii
Pneumonia
Authors: Gigliotti Francis , Wiley James A. , Harmsen Allen G.
Published: December 2019

Immunization with whole
Pneumocystis carinii
has been shown to protect mice from the development of
P. carinii
pneumonia (PCP) when they are subsequently immunosuppressed and challenged with viable organisms. To determine whether these results could be duplicated by using a subunit vaccine, we examined the immunogenicity and efficacy of an immunization strategy based on
P. carinii
gpA. This antigen was chosen for study because passive immunoprophylaxis, based on gpA, has been shown to be partially protective in various animal models of infection. Immunization with gpA produced an anti-gpA specific antibody response comparable to that resulting from immunization with whole organisms. However, in contrast to immunization with whole
P. carinii
, which was protective, immunization with gpA did not protect T-cell-depleted mice from the development of PCP. These studies suggest that other antigens in addition to gpA need to be evaluated for their role in protective immunity against
P. carinii.
Identification of New Cytotoxic T-Cell Epitopes on the 38-Kilodalton Lipoglycoprotein ofMycobacterium tuberculosisby Using Lipopeptides
Authors: da Fonseca Dora P. A. J. , Joosten Dianne , van der Zee Ruurd , Jue Danny L. , Singh Mahavir , Vordermeier Hans M. , Snippe Harm , Verheul André F. M.
Published: December 2019

Induction of cytotoxic T lymphocytes (CTLs) by vaccination has been shown to protect against bacterial, viral, and tumoral challenge. The aim of this study was to identify CTL epitopes on the 38-kDa lipoglycoprotein fromMycobacterium tuberculosis. The identification of these CTL epitopes was based on synthesizing peptides designed from the 38-kDa lipoglycoprotein, with known major histocompatibility complex class I (MHC-I) binding motifs (H-2Db), and studying their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S. To improve the capacity of the identified peptides to induce CTL responses in mice, palmitic acid with a cysteine-serine-serine spacer amino acid sequence was attached to the amino terminus of the peptide. Two of five peptides with H-2Dbbinding motifs and their corresponding lipopeptides up-regulated and stabilized the H-2Dbmolecules on RMA-S cells. Both lipopeptides, in combination with incomplete Freund’s adjuvant, induced CTL responses in C57BL/6 (H-2b) mice. Moreover, the lipopeptide induced stronger CTL responses than the peptide. The capacity of the various lipopeptides to induce CTL displayed a good relationship with the ability of the (lipo)peptide to up-regulate and to stabilize H-2Dbmolecules. The capacity of the peptides and lipopeptides to up-regulate and stabilize MHC-I expression can therefore be used to predict their potential to function as a CTL epitope. The newly identified CTL epitopes and their lipid derivatives provide us with important information for futureM. tuberculosisvaccine design.
Potential Role of Epithelial Cell-Derived Histone H1 Proteins in Innate Antimicrobial Defense in the Human Gastrointestinal Tract
Authors: Rose F. R. A. J. , Bailey K. , Keyte J. W. , Chan W. C. , Greenwood D. , Mahida Y. R.
Published: December 2019

In the human gastrointestinal tract, microorganisms are present in large numbers in the colon but are sparse in the proximal small intestine. In this study, we have shown that acid extracts of fresh human terminal ileal mucosal samples mediate antimicrobial activity. Following cation-exchange chromatography, one of the eluted fractions demonstrated antibacterial activity against bacteria normally resident in the human colonic lumen. This activity was further fractionated by reverse-phase high-performance liquid chromatography and identified as histone H1 and its fragments. We have also shown that in tissue sections, immunoreactive histone H1 is present in the cytoplasm of villus epithelial cells. In vitro culturing of detached (from the basement membrane) villus epithelial cells led to the release of antimicrobial histone H1 proteins, while the cells demonstrated ultrastructural features of programmed cell death. Our studies suggest that cytoplasmic histone H1 may provide protection against penetration by microorganisms into villus epithelial cells. Moreover, intestinal epithelial cells released into the lumen may mediate antimicrobial activity by releasing histone H1 proteins and their fragments.
Identification and Characterization of Staphylococcal Enterotoxin Types G and I from
Staphylococcus aureus
Authors: Munson Sibyl H. , Tremaine Mary T. , Betley Marsha J. , Welch Rodney A.
Published: December 2019

Staphylococcal enterotoxins are exotoxins produced by
Staphylococcus aureus
that possess emetic and superantigenic properties. Prior to this research there were six characterized enterotoxins, staphylococcal enterotoxin types A to E and H (referred to as SEA to SEE and SEH). Two new staphylococcal enterotoxin genes have been identified and designated
seg
and
sei
(staphylococcal enterotoxin types G and I, respectively).
seg
and
sei
consist of 777 and 729 nucleotides, respectively, encoding precursor proteins of 258 (SEG) and 242 (SEI) deduced amino acids. SEG and SEI have typical bacterial signal sequences that are cleaved to form toxins with 233 (SEG) and 218 (SEI, predicted) amino acids, corresponding to mature proteins of 27,043 Da (SEG) and 24,928 Da (SEI). Biological activities for SEG and SEI were determined with recombinant
S. aureus
strains. SEG and SEI elicited emetic responses in rhesus monkeys upon nasogastric administration and stimulated murine T-cell proliferation with the concomitant production of interleukin 2 (IL-2) and gamma interferon (IFN-γ), as measured by cytokine enzyme-linked immunoassays. SEG and SEI are related to other enterotoxins of
S. aureus
and to streptococcal pyrogenic exotoxin A (SpeA) and streptococcal superantigen (SSA) of
Streptococcus pyogenes
. Phylogenetic analysis and comparisons of amino acid and nucleotide sequence identities were performed on related staphylococcal and streptococcal protein toxins to group SEG and SEI among the characterized toxins. SEG is most similar to SpeA, SEB, SEC, and SSA (38 to 42% amino acid identity), while SEI is most similar to SEA, SEE, and SED (26 to 28% amino acid identity). Polyclonal antiserum was generated against purified histidine-tagged SEG and SEI (HisSEG and HisSEI). Immunoblot analysis of the enterotoxins, toxic-shock syndrome toxin 1, and SpeA with antiserum prepared against HisSEG and HisSEI revealed that SEG shares some epitopes with SEC1 while SEI does not.
Susceptibility toSalmonella typhimuriumInfection and Effectiveness of Vaccination in Mice Deficient in the Tumor Necrosis Factor Alpha p55 Receptor
Authors: Everest Paul , Roberts Mark , Dougan Gordon
Published: December 2019

Mice defective in the ability to produce the tumor necrosis factor alpha p55 receptor (TNFαp55R) were orally challenged with a number ofSalmonella typhimuriumHWSH derivatives that differ in virulence. In comparison to TNFαp55R+/+mice, TNFαp55R−/−mice succumbed earlier to challenge with wild-typeS. typhimuriumHWSH andS. typhimuriumHWSHpurE. In contrast, TNFαp55R−/−mice were able to control anS. typhimuriumHWSHaroAchallenge, although greater numbers ofSalmonellaorganisms were present in the tissues for a longer time period than was observed with TNFαp55R+/+mice. Vaccination of normal and TNFαp55R knockout animals withS. typhimuriumHWSHaroAshowed that TNFαp55R−/−mice, unlike TNFαp55R+/+mice, were not protected against a virulentS. typhimuriumHWSH challenge. Splenocytes from TNFαp55R−/−mice exhibited a reduced ability to proliferate in the presence ofS. typhimuriumantigen compared to TNFαp55R+/+mice. Thus, TNFαp55R is essential for controllingSalmonellagrowth in tissues and for recall of immunity in murine salmonellosis.
Study of Immunization against Anthrax with the Purified Recombinant Protective Antigen of
Bacillus anthracis
Authors: Singh Yogendra , Ivins Bruce E. , Leppla Stephen H.
Published: December 2019

Protective antigen (PA) of anthrax toxin is the major component of human anthrax vaccine. Currently available human vaccines in the United States and Europe consist of alum-precipitated supernatant material from cultures of toxigenic, nonencapsulated strains of
Bacillus anthracis
. Immunization with these vaccines requires several boosters and occasionally causes local pain and edema. We previously described the biological activity of a nontoxic mutant of PA expressed in
Bacillus subtilis
. In the present study, we evaluated the efficacy of the purified mutant PA protein alone or in combination with the lethal factor and edema factor components of anthrax toxin to protect against anthrax. Both mutant and native PA preparations elicited high anti-PA titers in Hartley guinea pigs. Mutant PA alone and in combination with lethal factor and edema factor completely protected the guinea pigs from
B. anthracis
spore challenge. The results suggest that the mutant PA protein may be used to develop an effective recombinant vaccine against anthrax.
Two-Dimensional Electrophoresis for Analysis ofMycobacterium tuberculosisCulture Filtrate and Purification and Characterization of Six Novel Proteins
Authors: Weldingh Karin , Rosenkrands Ida , Jacobsen Susanne , Rasmussen Peter Birk , Elhay Martin J. , Andersen Peter
Published: December 2019

Culture filtrate fromMycobacterium tuberculosiscontains molecules which promote high levels of protective immunity in animal models of subunit vaccination against tuberculosis. We have used two-dimensional electrophoresis for analysis and purification of six novelM. tuberculosisculture filtrate proteins (CFPs): CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28. The proteins were tested for recognition byM. tuberculosis-reactive memory cells from different strains of inbred mice and for their capacity to induce a skin test response inM. tuberculosis-infected guinea pigs. CFP17, CFP20, CFP21 and CFP25 induced both a high gamma interferon release and a strong delayed-type hypersensitivity response, and CFP21 was broadly recognized by different strains of inbred mice. N-terminal sequences were obtained for the six proteins, and the corresponding genes were identified in the SangerM. tuberculosisgenome database. In parallel we established a two-dimensional electrophoresis reference map of short-term culture filtrate components and mapped novel proteins as well as already-known CFP.
Mapping of Specific and Promiscuous HLA-DR-Restricted T-Cell Epitopes on thePlasmodium falciparum27-Kilodalton Sexual Stage-Specific Antigen
Authors: Contreras Carmen E. , Ploton Isabelle N. , Siliciano Robert F. , Karp Christopher L. , Viscidi Raphael , Kumar Nirbhay
Published: December 2019

We have characterized HLA-DR-restricted T-cell epitopes on the 27-kDa protein (Pfg27), a sexual stage-specific antigen, of the human malaria parasitePlasmodium falciparumin subjects with a history of malaria. Pfg27, expressed early in the sexual stages, is recognized by monoclonal antibodies capable of reducing the infectivity of gametocytes in mosquitoes. By using 16 Pfg27-specific CD4+-T-cell clones derived from three donors, seven different T-cell epitopes were identified. Among them, P11 (amino acids 191 to 210 of the Pfg27 sequence, IDVVDSYIIKPIPALPVTPD) was found to contain a previously described binding motif for multiple HLA-DR allotypes. Indeed, P11 was found to be promiscuous in that it could be recognized by T cells in the context of at least five different HLA-DR molecules. The cytokine profile of the clones was mixed. Seven of nine T-cell clones exhibited a Th0-like cytokine profile, producing high levels of gamma interferon (IFN-γ) and interleukin-4 (IL-4) upon stimulation with specific peptides and mitogens. The other two clones had a Th1-like cytokine profile with high expression of IFN-γ and no IL-4. Identification of a promiscuous epitope in Pfg27 could play a significant role in the design of a subunit vaccine for suppressing malaria transmission.
Phylogenetic Analysis of theChlamydia trachomatisMajor Outer Membrane Protein and Examination of Potential Pathogenic Determinants
Authors: Stothard Diane R. , Boguslawski George , Jones Robert B.
Published: December 2019

Phylogenetic analysis was utilized to investigate biological relationships (tissue tropism, disease presentation, and epidemiologic success), as evidenced by coevolution, among human strains ofChlamydia trachomatis. Nucleotide sequences ofomp1, the gene encoding the major outer membrane protein (MOMP) ofC. trachomatis, were determined for 40 strains representing 11 serovars. These data were combined with availableomp1sequences from GenBank for an analysis encompassing a total of 69 strains representing 17 serovars infecting humans. Phylogenetic analysis of the nucleotide and inferred amino acid sequences showed no evolutionary relationships among serovars that corresponded to biological or pathological phenotypes (tissue tropism, disease presentation, and epidemiologic success). In addition, no specific residues that may have evolved to play a role in determining biologically relevant characteristics of chlamydia, such as tissue specificity, disease presentation, and epidemiologic success, were apparent in the MOMP. These results suggest that variation in MOMP may have arisen from a need to be diverse in the presence of immune pressure rather than as a function of pathogenicity. Therefore, the role of MOMP in disease pathogenesis and infection may be passive, and it may not be the major ligand responsible for directing infection of various human cell types.
Major Antigenic Proteins of the Agent of Human Granulocytic Ehrlichiosis Are Encoded by Members of a Multigene Family
Authors: Murphy Cheryl I. , Storey James R. , Recchia Joanne , Doros-Richert Linda A. , Gingrich-Baker Cindy , Munroe Kenneth , Bakken Johan S. , Coughlin Richard T. , Beltz Gerald A.
Published: December 2019

Western blot analysis of proteins from a cell culture isolate (USG3) of the human granulocytic ehrlichiosis (HGE) agent has identified a number of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa. Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were used to amplify DNA from USG3. Sequencing of a 550-bp PCR product revealed that it encodes a protein homologous to the MSP-2 proteins of
Anaplasma marginale
. Concurrently, an expression library made from USG3 genomic DNA was screened with granulocytic
Ehrlichia
(GE)-positive immune sera. Analysis of two clones showed that they contain one partial and three full-length highly related genes, suggesting that they are part of a multigene family. Amino acid alignment showed conserved amino- and carboxy-terminal regions which flank a variable region. The conserved regions of these proteins are also homologous to the MSP-2 proteins of
A. marginale
; thus, they were designated GE MSP-2A (45 kDa), MSP-2B (34 kDa), and MSP-2C (38 kDa). The PCR fragment obtained as a result of peptide sequencing was completely contained within the
msp-2A
clone, and all of the sequenced peptides were found in the GE MSP-2 proteins. Recombinant MSP-2B protein and an MSP-2A fusion protein were expressed in
Escherichia coli
and reacted with human sera positive for the HGE agent by immunofluorescence assay. These data suggest that the 43- and 45-kDa proteins of the HGE agent are encoded by members of the GE MSP-2 multigene family.
Only Viable Parasites Are Detected by PCR following Clearance of Rodent Malarial Infections by Drug Treatment or Immune Responses
Authors: Jarra William , Snounou Georges
Published: December 2019

Detection and analysis of pathogens by PCR plays an important role in infectious disease research. The value of these studies would be diminished if nuclear material from dead parasites were found to remain in circulation for extended periods and thus result in positive amplification. This possibility was tested in experimental rodent malaria infections. Blood samples were obtained from infected mice during and following drug or immune clearance of
Plasmodium chabaudi chabaudi
parasitemias. Detection of parasite DNA by a sensitive
Plasmodium
-specific PCR amplification assay was associated with the presence of viable parasites, as detected by subinoculation. No parasite DNA could be detected by PCR 48 h after the injection of killed parasites into mice. Nuclear material from parasites removed by drug or immune responses is rapidly cleared from the circulation and does not contribute significantly to amplification. Thus, results from PCR analysis of malaria-infected blood accurately reflect the presence of live parasites.