bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Divergence and Evolutionary Instability in
ospE
-Related Members of the Upstream Homology Box Gene Family in
Borrelia burgdorferi
Sensu Lato Complex Isolates
Authors: Sung Shian-Ying , Lavoie Crystal P. , Carlyon Jason A. , Marconi Richard T. 
Published: December 2019

ABSTRACT

A series of related genes that are flanked at their 5′ ends by a conserved upstream sequence element called the upstream homology box (UHB) have been identified in
Borrelia burgdorferi
. These genes have been referred to as the UHB or
erp
gene family. We previously demonstrated that among a limited number of
B. burgdorferi
isolates, the UHB gene family is variable in composition and organization. Prior to this report the UHB gene family in other species of the
B. burgdorferi
sensu lato complex had not been studied, and if this family is important in the pathogenesis or biology of the Lyme disease spirochetes, then a wide distribution among species and isolates of the
B. burgdorferi
sensu lato complex would be expected. To assess this, we screened for the UHB element by Southern hybridization and determined its restriction fragment length polymorphism (RFLP) patterns. The UHB element was found to be carried by all
B. burgdorferi
sensu lato complex species tested (
B. burgdorferi
,
B. garinii
,
B. afzelii
,
B. japonica
,
B. valaisiana
sp. nov., and
B. andersonii
), but the RFLP patterns varied widely at both the inter- and intraspecies levels. Variation in both the number and size of the hybridizing restriction fragments was evident. PCR analyses also revealed the presence of polymorphic,
ospE
-related alleles in many isolates. Sequence analyses identified the molecular basis of the polymorphisms as being primarily insertions and deletions. Sequence variation and the insertions and deletions were found to be clustered in two distinct domains (variable domains 1 and 2). In many isolates variable domain 1 is flanked by direct repeat elements, some as long as 38 bp. Computer analyses of the deduced amino acid sequences encoded within variable domain 1 predict them to be hydrophilic, surface exposed, and antigenic. The analyses conducted here suggest that the UHB gene family, as evidenced by the variable UHB RFLP patterns, is not evolutionarily stable and that the polymorphic
ospE
alleles are derived from a common ancestral gene which has been modified through mutation or recombination events. The characterization of
ospE
-related genes of the UHB gene family among
B. burgdorferi
sensu lato species will prove important in attempts to construct a model for UHB gene family organization and in deciphering the role of the UHB gene family in the biology and pathogenesis of the Lyme disease spirochetes.
Functional Analyses of a Conserved Region in Glucosyltransferases of
Streptococcus mutans
Authors: Chia Jean-San , Yang Czau-Siung , Chen Jen-Yang 
Published: December 2019

ABSTRACT

Streptococcus mutans
glucosyltransferases (GTFs; GtfB, -C, and -D) synthesize water-soluble and -insoluble glucan polymers from sucrose. We have identified previously a conserved region of 19 amino acids (aa) (Gtf-P1; aa 409 to 427 of GtfB and aa 435 to 453 of GtfC) which is functionally important for both enzymatic activity and bacterial adherence. Monoclonal antibodies directed against Gtf-P1 selectively inhibited insoluble glucan synthesis by GtfB and -C but had no effect on soluble glucan synthesis by GtfD, suggesting that despite an apparent near identity of sequence, corresponding residues may function differently in these enzymes. To test this hypothesis, we used different strategies of mutagenesis to analyze amino acid residues of GtfB and GtfC in Gtf-P1. In-frame insertion of 6 amino acids preceding, or deletion of 14 amino acids within, this conserved region abolished the enzymatic activities of both GtfB and GtfC. Substitution of several residues in combination by random mutagenesis resulted in GtfB, but not GtfC, enzymes exhibiting decreased glucan synthesis and reduced rates of sucrose hydrolysis. Amino acid substitutions of Asp residues in GtfB or GtfC were found to be more critical for enzymatic activity than at other positions of this region. Interestingly, single mutation at Asp411 or Asp413 of GtfB resulted in enzymes retaining about 20% of wild-type activity, whereas mutagenesis of the corresponding Asp at position 437 or 439 in GtfC resulted in complete loss of enzymatic activity. Furthermore, single amino acid substitution of a Val residue between the two Asp residues enhanced the sucrase- and glucan-synthesizing activities of GtfB and GtfC. These results confirmed the report from another laboratory that Asp residues in the Gtf-P1 region are essential for enzymatic catalysis and provide new evidence that identical residues may function differently in closely related Gtf enzymes.
Experimental Infection of Mongolian Gerbils with Wild-Type and Mutant
Helicobacter pylori
Strains
Authors: Wirth Hans-Peter , Beins Michael H. , Yang Manqiao , Tham Kyi T. , Blaser Martin J. 
Published: December 2019

ABSTRACT

Experimental
Helicobacter pylori
infection was studied in Mongolian gerbils with fresh human isolates that carry or do not carry
cagA
(
cagA
-positive or
cagA
-negative, respectively), multiply passaged laboratory strains, wild-type strain G1.1, or isogenic
ureA
,
cagA
, or
vacA
mutants of G1.1. Animals were sacrificed 1 to 32 weeks after challenge, the stomach was removed from each animal for quantitative culture, urease test, and histologic testing, and blood was collected for antibody determinations. No colonization occurred after ≥20 in vitro passages of wild-type strain G1.1 or with the
ureA
mutant of G1.1. In contrast, infection occurred in animals challenged with wild-type G1.1 (99 of 101 animals) or the
cagA
(25 of 25) or
vacA
(25 of 29) mutant of G1.1. Infection with G1.1 persisted for at least 8 months. All 15 animals challenged with any of three fresh human
cagA
-positive isolates became infected, in contrast to only 6 (23%) of 26 animals challenged with one of four fresh human
cagA
-negative isolates (
P
< 0.001). Similar to infection in humans,
H. pylori
colonization of gerbils induced gastric inflammation and a systemic antibody response to
H. pylori
antigens. These data confirm the utility of gerbils as an animal model of
H. pylori
infection and indicate the importance of bacterial strain characteristics for successful infection.
Intestinal Secretory Factor Released by Macrophages Stimulated with
Clostridium difficile
Toxin A: Role of Interleukin 1β
Authors: Rocha M. F. G. , Soares A. M. , Flores C. A. , Steiner T. S. , Lyerly D. M. , Guerrant R. L. , Ribeiro R. A. , Lima A. A. M. 
Published: December 2019

ABSTRACT

Clostridium difficile
toxin A is associated with enterocolitis in animals and humans. However, the mechanisms of its secretory and damaging effects are not totally understood. In this work, we examined the intestinal secretion of electrolytes and water caused by supernatants from macrophages stimulated with toxin A in rabbit ileal mucosa mounted in Üssing chambers. We also investigated the mechanism by which the intestinal secretory factor (ISF) is released from stimulated macrophages. Supernatants from macrophages stimulated with toxin A caused potent intestinal secretion (change in short-circuit current [ΔIsc], 76 μA · cm
−2
;
P
< 0.01). The release of the ISF was pertussis toxin sensitive (reduction, 61%;
P
< 0.01) and was also reduced (
P
< 0.05) by a protein synthesis inhibitor (67%), protease inhibitors (57%), a phospholipase A
2
inhibitor (54%), a cyclo-oxygenase inhibitor (62%), a dual cyclo- and lipoxygenase inhibitor (48%), a platelet-activating factor (PAF) receptor antagonist (55%), and tumor necrosis factor alpha (TNF-α) synthesis inhibitors (48%). However, this release was not inhibited by a lipo-oxygenase inhibitor. Monoclonal anti-interleukin 1β (IL-1β) but not anti-IL-1α antibody blocked (72%;
P
< 0.01) the secretory action of the ISF, as did recombinant human IL-1 receptor antagonist (80%;
P
< 0.01). High levels of IL-1β (3,476 pg/ml) were detected by an enzyme-linked immunosorbent assay in the above supernatants. Furthermore, the addition of IL-1β to the serosal side caused a potent secretory effect (ΔIsc, 80 μA · cm
−2
;
P
< 0.01). These results show that macrophages stimulated with toxin A release an ISF capable of provoking intestinal secretion. The regulation of this factor is dependent upon the activation of the G protein. In addition, prostaglandins, PAF, and TNF-α are involved in the release of the ISF. We conclude that IL-1β is probably the ISF released by macrophages in response to toxin A.
Cloning and Molecular Characterization of a cDNA Clone Coding for
Trichomonas vaginalis
Alpha-Actinin and Intracellular Localization of the Protein
Authors: Addis Maria Filippa , Rappelli Paola , Delogu Giuseppe , Carta Franco , Cappuccinelli Piero , Fiori Pier Luigi 
Published: December 2019

ABSTRACT

We have identified and sequenced a cDNA clone coding for
Trichomonas vaginalis
alpha-actinin. Analysis of the obtained sequence revealed that the 2,857-nucleotide-long cDNA contained an open reading frame encoding 849 amino acids which showed consistent homology with alpha-actinins of different species. Such homology was particularly significant in regions which have been reported to represent the actin-binding and Ca
2+
-binding domains in other alpha-actinins. The deduced protein was also characterized by the presence of a divergent central region thought to play a role in its high immunogenicity. A study of protein localization performed by immunofluorescence revealed that the protein is diffusely distributed throughout the
T. vaginalis
cytoplasm when the cell is pear shaped. When parasites adhere and transform into the amoeboid morphology, the protein is located only in areas close to the cytoplasmic membrane and colocalizes with actin. Concomitantly with transformation into the amoeboid morphology, alpha-actinin mRNA expression is upregulated.
Identification of Two Laminin-Binding Fimbriae, the Type 1 Fimbria of
Salmonella enterica
Serovar Typhimurium and the G Fimbria of
Escherichia coli
, as Plasminogen Receptors
Authors: Kukkonen Maini , Saarela Sirkku , Lähteenmäki Kaarina , Hynönen Ulla , Westerlund-Wikström Benita , Rhen Mikael , Korhonen Timo K. 
Published: December 2019

ABSTRACT

Escherichia coli
strains carrying recombinant plasmids encoding either the type 1 fimbria of
Salmonella enterica
serovar Typhimurium or the G fimbria of
E. coli
exhibited binding of human
125
I-Glu-plasminogen and enhanced the tissue-type plasminogen activator-catalyzed conversion of plasminogen to plasmin. Purified type 1 or G fimbriae similarly bound plasminogen and enhanced its activation. The binding of plasminogen did not involve the characteristic carbohydrate-binding property of the fimbriae but was inhibited at low concentrations by the lysine analog ɛ-aminocaproic acid. Because these fimbrial types bind to laminin of basement membranes (M. Kukkonen et al., Mol. Microbiol. 7:229–237, 1993; S. Saarela et al., Infect. Immun. 64:2857–2860, 1996), the results demonstrate a structural unity in the creation and targeting of bacterium-bound proteolytic plasmin activity to basement membranes.
Effect of Cytokines on Growth ofToxoplasma gondiiin Murine Astrocytes
Authors: Halonen S. K. , Chiu F.-C. , Weiss L. M. 
Published: December 2019

ABSTRACTCytokines play a significant role in the regulation ofToxoplasma gondiiin the central nervous system. Cytokine-activated microglia are important host defense cells in central nervous system infections. Recent evidence indicates that astrocytes can also be activated by cytokines to inhibit intracellular pathogens. In this study, we examined the effect of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1 on the growth ofT. gondiiin a primary murine astrocyte culture. Pretreatment of astrocytes with IFN-γ resulted in 65% inhibition ofT. gondiigrowth. Neither TNF-α, IL-1, nor IL-6 alone had any effect onT. gondiigrowth. IFN-γ in combination with either TNF-α, IL-1, or IL-6 caused a 75 to 80% inhibition of growth. While nitric oxide was produced by astrocytes treated with these cytokines, inhibition ofT. gondiigrowth was not reversed by the addition of the nitric oxide synthase inhibitorNG-monomethyl-l-arginine. Furthermore, IFN-γ in combination with IL-1, IL-6, or TNF-α also induced inhibition in astrocytes derived from syngeneic mice deficient in the enzyme inducible nitric oxide synthase. This finding suggests that the mechanism of cytokine inhibition is not nitric oxide mediated. Similarly, the addition of tryptophan had no effect on inhibition, indicating that the mechanism was not mediated via induction of the enzyme indoleamine 2,3-dioxygenase. The mechanism of inhibition remains to be elucidated. Results from this study demonstrate that cytokine-activated astrocytes are capable of significantly inhibiting the growth ofT. gondii. These data indicate that astrocytes may be important host defense cells in controlling toxoplasmosis in the brain.
Effective, Nonsensitizing Vaccination with Culture Filtrate Proteins against VirulentMycobacterium bovisInfections in Mice
Authors: Bosio Catharine M. , Orme Ian M. 
Published: December 2019

ABSTRACTVaccination of mice withMycobacterium bovisculture filtrate proteins (CFP), prepared in a variety of adjuvants (aluminum hydroxide, Quil-A, and dimethyldioctyldecyl ammonium bromide [DDA]), provided significant protection against an aerosol challenge of virulentM. bovis. Additionally, vaccination with CFP in DDA or Quil-A did not sensitize mice toM. bovispurified protein derivative.
Local Immune Responses to
Chlamydia pneumoniae
in the Lungs of BALB/c Mice during Primary Infection and Reinfection
Authors: Penttilä Jenni M. , Anttila Marjukka , Puolakkainen Mirja , Laurila Aino , Varkila Kari , Sarvas Matti , Mäkelä P. Helena , Rautonen Nina 
Published: December 2019

ABSTRACT

Cell-mediated immune (CMI) responses play a major role in protection as well as pathogenesis of many intracellular bacterial infections. In this study, we evaluated the infection kinetics and assessed histologically the lymphoid reactions and local, in vitro-restimulated CMI responses in lungs of BALB/c mice, during both primary infection and reinfection with
Chlamydia pneumoniae
. The primary challenge resulted in a self-restricted infection with elimination of culturable bacteria by day 27 after challenge. A mild lymphoid reaction characterized the pathology in the lungs. In vitro CMI responses consisted of a weak proliferative response and no secretion of gamma interferon (IFN-γ). The number of lung-derived mononuclear cells increased substantially during the primary infection; the largest relative increase was observed in B cells (B220
+
). After reinfection, the number of lung-derived mononuclear cells increased further, and the response consisted mainly of T cells. The reinfection was characterized in vivo by significant protection from infection (fewer cultivable bacteria in the lungs for a shorter period of time) but increased local lymphoid reaction at the infection site. In vitro, as opposed to the response in naive mice, acquired immunity was characterized by a strongly Th1-biased (IFN-γ) CMI response. These results suggest that repeated infections with
C. pneumoniae
may induce Th1-type responses with similar associated tissue reactions, as shown in
C. trachomatis
infection models.
Differential Expression of
Borrelia burgdorferi
Proteins during Growth In Vitro
Authors: Ramamoorthy Ramesh , Philipp Mario T. 
Published: December 2019

ABSTRACT

In an earlier paper we described the transcriptionally regulated differential levels of expression of two lipoproteins of
Borrelia burgdorferi
, P35 and P7.5, during growth of the spirochetes in culture from logarithmic phase to stationary phase (K. J. Indest, R. Ramamoorthy, M. Solé, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165–1171, 1997). Here we further assess this phenomenon by investigating whether the expression of other antigens of
B. burgdorferi
, including some well-characterized ones, are also regulated in a growth-phase-dependent manner in vitro. These studies revealed 13 additional antigens, including OspC, BmpD, and GroEL, that were upregulated 2- to 66-fold and a 28-kDa protein that was downregulated 2- to 10-fold, during the interval between the logarithmic- and stationary-growth phases. Unlike with these in vitro-regulated proteins, the levels of expression of OspA, OspB, P72, flagellin, and BmpA remained unchanged throughout growth of the spirochetes in culture. Furthermore,
ospAB
,
bmpAB
,
groEL
, and
fla
all exhibited similar mRNA profiles, which is consistent with the constitutive expression of these genes. By contrast, the mRNA and protein profiles of
ospC
and
bmpD
indicated regulated expression of these genes. While
bmpD
exhibited a spike in mRNA expression in early stationary phase,
ospC
maintained a relatively higher level of mRNA throughout culture. These findings demonstrate that there are additional genes besides
P7.5
and
P35
whose regulated expression can be investigated in vitro and which may thus serve as models to facilitate the study of regulatory mechanisms in an organism that cycles between an arthropod and a vertebrate host.
Disruption of an Internal Membrane-Spanning Region in Shiga Toxin 1 Reduces Cytotoxicity
Authors: Suhan Michelle L. , Hovde Carolyn J. 
Published: December 2019

ABSTRACT

Shiga toxin type 1 (Stx1) belongs to the Shiga family of bipartite AB toxins that inactivate eukaryotic 60S ribosomes. The A subunit of Stxs are N-glycosidases that share structural and functional features in their catalytic center and in an internal hydrophobic region that shows strong transmembrane propensity. Both features are conserved in ricin and other ribosomal inactivating proteins. During eukaryotic cell intoxication, holotoxin likely moves retrograde from the Golgi apparatus to the endoplasmic reticulum. The hydrophobic region, spanning residues I224 through N241 in the Stx1 A subunit (Stx1A), was hypothesized to participate in toxin translocation across internal target cell membranes. The TMpred computer program was used to design a series of site-specific mutations in this hydrophobic region that disrupt transmembrane propensity to various degrees. Mutations were synthesized by PCR overlap extension and confirmed by DNA sequencing. Mutants StxAF226Y, A231D, G234E, and A231D-G234E and wild-type Stx1A were expressed in
Escherichia coli
SY327 and purified by dye-ligand affinity chromatography. All of the mutant toxins were similar to wild-type Stx1A in enzymatic activity, as determined by inhibition of cell-free protein synthesis, and in susceptibility to trypsin digestion. Purified mutant or wild-type Stx1A combined with Stx1B subunits in vitro to form a holotoxin, as determined by native polyacrylamide gel electrophoresis immunoblotting. StxA mutant A231D-G234E, predicted to abolish transmembrane propensity, was 225-fold less cytotoxic to cultured Vero cells than were the wild-type toxin and the other mutant toxins which retained some transmembrane potential. Furthermore, compared to wild-type Stx1A, A231D-G234E Stx1A was less able to interact with synthetic lipid vesicles, as determined by analysis of tryptophan fluorescence for each toxin in the presence of increasing concentrations of lipid membrane vesicles. These results provide evidence that this conserved internal hydrophobic motif contributes to Stx1 translocation in eukaryotic cells.
Molecular Analysis of Sequence Heterogeneity among Genes Encoding Decorin Binding Proteins A and B of
Borrelia burgdorferi
Sensu Lato
Authors: Roberts William C. , Mullikin Brian A. , Lathigra Raju , Hanson Mark S. 
Published: December 2019

ABSTRACT

Immunization of mice with
Borrelia burgdorferi
decorin binding protein A (DbpA), one of two gene products of the
dbpBA
locus, has been shown recently to confer protection against challenge. Hyperimmune DbpA antiserum killed a large number of
B. burgdorferi
sensu lato isolates of diverse phylogeny and origin, suggesting conservation of the protective epitope(s). In order to evaluate the heterogeneity of DbpA and DbpB and to facilitate defining the conserved epitope(s) of these antigens, the sequences of the
dbpA
genes from 29
B. burgdorferi
sensu lato isolates and of the
dbpB
genes from 15
B. burgdorferi
sensu lato isolates were determined. The predicted DbpA sequences were fairly heterogeneous among the isolates (58.3 to 100% similarity), but DbpA sequences with the highest similarity tended to group into species previously defined by well-characterized chromosomal markers. In contrast, the predicted DbpB sequences were highly conserved (96.3 to 100% similarity). Substantial diversity in DbpA sequence was seen among isolates previously shown to be killed by antiserum against a single DbpA, suggesting that one or more conserved protective epitopes are composed of noncontiguous amino acids. The observation of individual
dbpA
alleles with sequence elements characteristic of more than one
B. burgdorferi
sensu lato species was consistent with a role for genetic recombination in the generation of
dbpA
diversity.
Mechanisms Involved in the Pathogenesis of Sepsis Are Not Necessarily Reflected by In Vitro Cell Activation Studies
Authors: Amura Claudia R. , Silverstein R. , Morrison D. C. 
Published: December 2019

ABSTRACTIt is thought that lipopolysaccharide (LPS) from gram-negative bacteria contributes significantly to the pathogenesis of septic shock. In vitro studies to address the mechanisms involved in this process have often investigated human monocytes or mouse macrophages, since these cells produce many of the mediators found in septic patients. Targeting of these mediators, especially tumor necrosis factor alpha (TNF-α), has been pursued as a means of reducing mortality in sepsis. Two experimental approaches were designed to test the assumption that in vitro studies with macrophages accurately predict in vivo mechanisms of LPS pathogenesis. In the first approach, advantage was taken of the fact that on consecutive days after injection of thioglycolate into mice, increased numbers of macrophages could be harvested from the peritoneum. These cells manifested markedly enhanced levels of in vitro TNF-α, interleukin 6 (IL-6), and nitric oxide production in response to LPS. Ind-galactosamine-sensitized mice, however, thioglycolate treatment significantly decreased mortality due to LPS, as well as levels of circulating TNF-α and IL-6. Anti-TNF-α treatment confirmed this cytokine’s role in the observed lethality. In a second experimental approach, we compared the mouse macrophage-stimulating potencies of different LPS preparations with their lethalities to mice. In these studies, the in vitro macrophage-stimulating profiles presented by rough-LPS and smooth-LPS preparations were the reverse of their relative lethal potencies in vivo. In conclusion, peritoneal macrophages appear not to be the major cells responsible for the overall host response during endotoxic shock. These findings underscore the importance of verifying the correlation of in vivo systems with in vitro systems when attributing specific functions to a cell type.