bluuebunny/crossref_metadata_2025 huggingface dataset:
Low Levels of Antigenic Variability in Fluconazole-Susceptible and -Resistant
Candida albicans
Isolates from Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis
Authors: Lopez-Ribot Jose L. , McAtee Robert K. , Kirkpatrick William R. , La Valle Roberto , Patterson Thomas F.
Published: December 2019

Three serial isolates of
Candida albicans
were obtained by direct swab or by oral saline rinses from each of five human immunodeficiency virus-infected patients with recurrent oropharyngeal candidiasis. Genotyping techniques confirmed the presence of a persistent strain in multiple episodes from the same patient, which was different from the strains isolated from other patients. Fluconazole susceptibility was determined by both an agar dilution method and the National Committee for Clinical Laboratory Standards macrobroth procedure. In four of these patients the strains developed fluconazole resistance, and in one patient the strain remained susceptible. The different isolates were propagated as yeast cells on a synthetic medium, and their cell wall proteinaceous components were extracted by treatment with β-mercaptoethanol. Protein and mannoprotein components present in the extracts were analyzed by electrophoresis, immunoblotting, and lectin-blotting techniques. The analysis showed a similar composition, with only minor qualitative and quantitative differences in the polypeptidic and antigenic patterns associated with the cell wall extracts from serial isolates from the same patient, as well as those from different strains isolated from different patients. Use of monospecific antibodies generated against two immunodominant antigens during candidiasis (enolase and the 58-kDa fibrinogen-binding mannoprotein) demonstrated their expression in all isolates tested. Overall, the antigenic makeup of
C. albicans
strains remained constant during the course of infection and was not affected by development of fluconazole resistance. In contrast to previous reports, the low degree of antigenic variability observed in this study may be due to the fact that the isolates were obtained from a highly homogeneous population of patients and to the uniformity in techniques used for the isolation, storage, and culture of the different strains, as well as extraction methodologies.
Immune Response of Children Who Develop Persistent Diarrhea following Rotavirus Infection
Authors: Azim Tasnim , Ahmad S. Meshbahuddin , Khuda Sefat-e- , Sarker M. Safiullah , Unicomb Leanne E. , De Soma , Hamadani Jena D. , Salam Mohammed A. , Wahed M. A. , Albert M. John
Published: December 2019

A prospective study was conducted with Bangladeshi children with rotavirus (RV) diarrhea to assess whether nutritional and clinical parameters, RV serotypes, levels of interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ), and RV-specific antibody titers in plasma and stool were associated with the development of persistent diarrhea. Children with watery diarrhea for 6 to 8 days, selected from the Dhaka Hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), were enrolled in the study and monitored until diarrhea improved. Children were classified as having acute diarrhea (AD) if diarrhea resolved within 14 days of onset and as having persistent diarrhea (PD) if diarrhea persisted for more than 14 days after onset. Uninfected, control children (
n
= 13) from the Nutrition Follow-Up Unit of ICDDR,B were also enrolled. Of the 149 children with diarrhea enrolled, 29 had diarrhea with RV alone, of which 19 had AD and 10 developed PD. Samples of stool and blood were collected from all children on enrollment. Stool samples were collected again from children when they developed PD. Of the 10 children who had an initial RV infection and then developed PD, only one had persistent RV infection. Plasma levels of IL-10 and TNF-α were higher in children with diarrhea compared to uninfected controls but were similar in children with AD and PD. Plasma IFN-γ levels were higher in children who developed PD than in those with AD (
P
= 0.008) or uninfected controls (
P
= 0.001). In stools, the levels of TNF-α, the only cytokine detected, were similar in the three groups of children. RV-specific immunoglobulin G (IgG) titers in plasma were higher in uninfected children than in those with AD (
P
< 0.001) or PD (
P
= 0.024) but titers were similar in children with AD and PD. RV-specific IgA titers in plasma and stool were similar in the three groups of children. From all observed parameters, only elevated plasma IFN-γ levels were associated with subsequent development of PD. However, a larger sample size is necessary to substantiate this observation.
Detection of Antibodies to
Brucella
Cytoplasmic Proteins in the Cerebrospinal Fluid of Patients with Neurobrucellosis
Authors: Baldi Pablo C. , Araj George F. , Racaro Graciela C. , Wallach Jorge C. , Fossati Carlos A.
Published: December 2019

The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to
Brucella
lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) of
Brucella
spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12,800) and IgG to LPS (800 to 6,400) were found in the CSF of these patients, these antibodies were not detected in CSF samples from two patients who had systemic brucellosis without neurological involvement. The latter, however, had serum IgG and IgM to both LPS and CP. No reactivity to these antigens was found in CSF samples from 14 and 20 patients suffering from nonbrucellar meningitis and noninfectious diseases, respectively. These findings suggest that, in addition to its usefulness in the serological diagnosis of human systemic brucellosis, the ELISA with CP antigen can be used for the specific diagnosis of human neurobrucellosis.
Comparative Analysis of Two Meningococcal Immunotyping Monoclonal Antibodies by Resonant Mirror Biosensor and Antibody Gene Sequencing
Authors: Charalambous Bambos M. , Evans Janet , Feavers Ian M. , Maiden Martin C. J.
Published: December 2019

Lipooligosaccharide (LOS) is a major surface component of the cell walls of
Neisseria meningitidis
, which is important for its roles in pathogenesis and antigenic variation, as a target for immunological typing, and as a possible vaccine component. Although the structures of many antigenic variants have been determined, routine immunological typing of these molecules remains problematic. Resonant mirror analysis was combined with gene sequencing to characterize two monoclonal antibodies (MAbs) used in typing panels that were raised against the same LOS immunotype, L3,7,9. The two MAbs (MAb 4A8-B2 and MAb 9-2-L379) were of the same immunoglobulin subtype, but while MAb 9-2-L379 was more than a 1,000-fold more sensitive in immunotyping assays of both whole meningococcal cells and purified LOS, MAb 4A8-B2 was more specific for immunotype L3,7,9. The differences in sensitivity were a consequence of MAb 9-2-L379 having a 44-fold-faster association constant than MAb 4A8-B2. Comparison of the amino acid sequences of the variable chains of the MAbs revealed that they had very similar heavy chains (81% amino acid sequence identity) but diverse light chains (54% sequence identity). The differential binding kinetics and specificities observed with these MAbs were probably due to differences in the epitopes recognized, and these were probably a consequence of the different immunization protocols used in their production.
Longitudinal Analysis of Lymphocyte Function and Numbers in the First Year of Life in Chromosome 22q11.2 Deletion Syndrome (DiGeorge Syndrome/Velocardiofacial Syndrome)
Authors: Sullivan Kathleen E. , McDonald-McGinn Donna , Driscoll Deborah A. , Emanuel Beverly S. , Zackai Elaine H. , Jawad Abbas F.
Published: December 2019

Chromosome 22q11.2 deletion syndrome is a common syndrome typically consisting of variable cardiac defects, hypoparathyroidism, developmental delay, and immunodeficiency. The hemizygous deletion has variable effects on the immune system even within the same kindred, and the extent of the immunodeficiency is difficult to predict. Some patients have shown improvement over time; however, this is the first prospective longitudinal study of the dynamic nature of the immunodeficiency. Nineteen patients were studied prospectively between 1994 and 1997. The results of the newborn immunologic studies in the chromosome 22q11.2 deletion group were significantly different from those of a group of newborns with cardiac disease due to other causes. Peripheral blood T-cell numbers were decreased in the chromosome 22q11.2 deletion group, although T-cell function was largely preserved. The group as a whole demonstrated few changes in the first year of life, but a subset of patients with markedly diminished T-cell numbers did demonstrate improvement. Therefore, improvement in peripheral blood T-cell counts is variable in chromosome 22q11.2 deletion syndrome. The patients with the lowest T-cell counts improved the most in the first year of life.
Successful Treatment of Fluconazole-Resistant Oropharyngeal Candidiasis by a Combination of Fluconazole and Terbinafine
Authors: Ghannoum Mahmoud A. , Elewski Boni
Published: December 2019

Increasing incidence of resistance to conventional antifungal therapy has demanded that novel therapies be introduced. Recent in vitro studies have shown that combinations involving azoles and allylamines may be effective in inhibiting fluconazole-resistant fungi. In this report, we describe the case of a 39-year-old woman who presented with white patches on her buccal mucosa, tongue, and palate with a bright erythematous erosive base. A fungal culture revealed
Candida albicans
. The patient failed to respond to the initially prescribed fluconazole therapy. Failure of therapy can be attributed to a developed resistance to fluconazole from the patient’s intermittent use of this antifungal agent at varying dosages for the preceding 2 years due to a diagnosis of onychomycosis. In vitro testing of the culture from the patient showed elevated MICs of fluconazole, itraconzole, and terbinafine (MICs were 32, 0.5, and 64 μg/ml, respectively). Our goal was to combine therapies of fluconazole and terbinafine in an attempt to clear the fungal infection. Impressively, this combination resulted in the clearing of the clinical symptoms and the patient has successfully been asymptomatic for more than 12 months posttreatment.
Antigenic Homology of the Inducible Ferric Citrate Receptor (FecA) of Coliform Bacteria Isolated from Herds with Naturally Occurring Bovine Intramammary Infections
Authors: Lin Jun , Hogan Joseph S. , Smith K. Larry
Published: December 2019

Expression of ferric citrate receptor FecA by
Escherichia coli
and
Klebsiella pneumoniae
isolated from bovine mastitis was investigated. Transformant
E. coli
UT5600/pSV66, which produces large quantities of FecA in the presence of citrate, was constructed. The FecA of
E. coli
UT5600/pSV66 was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to prepare polyclonal antiserum in rabbits. All coliform isolates of
E. coli
(
n
= 18) and
K. pneumoniae
(
n
= 17) from naturally occurring bovine intramammary infections in five herds induced iron-regulated outer membrane proteins when grown in Trypticase soy broth containing 200 μM α-α′-dipyridyl and 1 mM citrate. Polyclonal antiserum against FecA was used in conjunction with an immunoblot technique to determine the degree of antigenic homology of FecA among isolates. In the presence of citrate, each isolate expressed FecA that reacted with the anti-FecA polyclonal antiserum. The molecular mass of FecA (∼80.5 kDa) was also highly conserved among isolates. Therefore, the ferric citrate iron transport may be induced in coliform bacteria and utilized to acquire iron in milk for survival and growth. The FecA is an attractive vaccine component for controlling coliform mastitis during the lactation period.
Identification of a Genetic Locus Essential for Serotype b-Specific Antigen Synthesis in
Actinobacillus actinomycetemcomitans
Authors: Yoshida Yasuo , Nakano Yoshio , Yamashita Yoshihisa , Koga Toshihiko
Published: December 2019

A large gene cluster associated with the biosynthesis of the serotype-specific polysaccharide antigen (SPA) of
Actinobacillus actinomycetemcomitans
Y4 (serotype b) was cloned and characterized. Western blot analysis showed that
Escherichia coli
DH5α, containing a plasmid carrying this cluster, produced a polysaccharide which reacted with a monoclonal antibody directed against the SPA of
A. actinomycetemcomitans
Y4. High-performance liquid chromatography analysis indicated that the polysaccharide produced by an
E. coli
transformant, as well as
A. actinomycetemcomitans
Y4 SPA, was composed of rhamnose and fucose. Furthermore, using various derivatives of the plasmid, we demonstrated that the cloned 13-kb
Bss
HII-
Bsp
HI fragment was indispensable for SPA synthesis in
E. coli
DH5α. The 24,909-bp nucleotide sequence, which included this fragment and its flanking regions, was determined. In the sequenced area, 24 open reading frames (ORFs) with the same orientation were found. Most of these were located sequentially within a short distance of each other. Many of the deduced amino acid sequences were similar to the gene products of the polysaccharide synthetic genes of other bacteria. The average G+C content (37.7%) of all 24 ORFs in the sequenced area was lower than that (45.6%) of the whole chromosome of
A. actinomycetemcomitans
Y4. It is noteworthy the average G+C content of the nine ORFs in the 8.5-kb central region of the 13-kb
Bss
HII-
Bsp
HI fragment indispensable for SPA synthesis in
E. coli
was found to be especially low (27.0%).
Role of BfpF, a Member of the PilT Family of Putative Nucleotide-Binding Proteins, in Type IV Pilus Biogenesis and in Interactions between Enteropathogenic
Escherichia coli
and Host Cells
Authors: Anantha Ravi P. , Stone Kelly D. , Donnenberg Michael S.
Published: December 2019

Adherence of enteropathogenic
Escherichia coli
(EPEC) to epithelial cells is dependent on a type IV fimbria, termed the bundle-forming pilus (BFP). A cluster of 14 genes is required for expression of BFP. The eighth gene in the cluster,
bfpF
, encodes a putative nucleotide-binding protein which resembles the PilT protein of
Pseudomonas aeruginosa
. It has been proposed that PilT is required for the retraction of the
P. aeruginosa
pilus, which results in twitching motility. To test the role of BfpF in BFP function and EPEC pathogenesis, two different mutations were constructed in the
bfpF
gene, one in the cloned gene cluster in a laboratory
E. coli
strain and one in wild-type EPEC. Neither mutation affected prepilin synthesis, leader sequence processing, or pilus biogenesis. However, both mutations resulted in increased localized adherence. In addition, the EPEC
bfpF
mutant displayed increased aggregation. The EPEC
bfpF
mutant was not deficient in attaching and effacing activity or invasion capacity. These results suggest that BfpF decreases aggregation and adherence by EPEC but that subsequent steps in EPEC pathogenesis do not require this protein.
Immunodominant Major Outer Membrane Proteins of
Ehrlichia chaffeensis
Are Encoded by a Polymorphic Multigene Family
Authors: Ohashi Norio , Zhi Ning , Zhang Yilan , Rikihisa Yasuko
Published: December 2019

Several immunodominant major proteins ranging from 23 to 30 kDa were identified in the outer membrane fractions of
Ehrlichia chaffeensis
and
Ehrlichia canis
. The N-terminal amino acid sequence of a 28-kDa protein of
E. chaffeensis
(one of the major proteins) was determined. The gene (
p28
), almost full length, encoding the 28-kDa protein was cloned by PCR with primers designed based on the N-terminal sequence of the
E. chaffeensis
28-kDa protein and the consensus sequence between the C termini of the
Cowdria ruminantium
MAP-1 and
Anaplasma marginale
MSP-4 proteins. The
p28
gene was overexpressed, and antibody to the recombinant protein was raised in a rabbit. The antibody and serum from a patient infected with
E. chaffeensis
reacted with the recombinant protein, three proteins (29, 28, and 25 kDa) of
E. chaffeensis
, and a 30-kDa protein of
E. canis
. Immunoelectron microscopy with the rabbit antibody revealed that the antigenic epitope of the 28-kDa protein was exposed on the surface of
E. chaffeensis
. Southern blot analysis with a
32
P-labeled
p28
gene probe revealed multiple copies of genes homologous to
p28
in the
E. chaffeensis
genome. Six copies of the
p28
gene were cloned and sequenced from the genomic DNA by using the same probe. The open reading frames of these gene copies were tandemly arranged with intergenic spaces. They were nonidentical genes and contained a semivariable region and three hypervariable regions in the predicted protein molecules. One of the gene copies encoded a protein with an internal amino acid sequence identical to the chemically determined N-terminal amino acid sequence of a 23-kDa protein of
E. chaffeensis
. Immunization with the recombinant P28 protein protected mice from infection with
E. chaffeensis
. These findings suggest that the 30-kDa-range proteins of
E. chaffeensis
represent a family of antigenically related homologous proteins encoded by a single gene family.
Cytokine Profiles for Peripheral Blood Lymphocytes from Patients with Active Pulmonary Tuberculosis and Healthy Household Contacts in Response to the 30-Kilodalton Antigen ofMycobacterium tuberculosis
Authors: Torres Martha , Herrera Teresa , Villareal Hector , Rich Elizabeth A. , Sada Eduardo
Published: December 2019

Patients with active tuberculosis (TB) have a stronger humoral but a poorer cellular immune response to the secreted 30-kDa antigen (Ag) ofMycobacterium tuberculosisthan do healthy household contacts (HHC), who presumably are more protected against disease. The basis for this observation was studied by examining the Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-γ])- and Th2 (IL-10 and IL-4)-type cytokines produced in response to the 30-kDa Ag by peripheral blood mononuclear cells (PBMC) from patients with active pulmonary TB (n= 7) and from HHC who were tuberculin (purified protein derivative) skin test positive (n= 12). Thirty-kilodalton-Ag-stimulated PBMC from TB patients produced significantly lower levels of IFN-γ (none detectable) than did those from HHC (212 ± 73 pg/ml, mean ± standard error) (P< 0.001). Likewise, 30-kDa-Ag-stimulated PBMC from TB patients failed to express IFN-γ mRNA by reverse transcription-PCR, whereas cells from HHC expressed the IFN-γ gene. In contrast, 30-kDa-Ag-stimulated PBMC from TB patients produced significantly higher levels of IL-10 (403 ± 80 pg/ml) than did those from HHC (187 ± 66 pg/ml) (P< 0.013), although cells from both groups expressed the IL-10 gene. IL-2 and IL-4 were not consistently produced, and their genes were not expressed by 30-kDa-Ag-stimulated cells from either TB patients or HHC. After treatment with antituberculous drugs, lymphocytes from four of the seven TB patients proliferated and three of them expressed IFN-γ mRNA in response to the 30-kDa Ag and produced decreased levels of IL-10.
Endothelial Cell Injury Caused by
Candida albicans
Is Dependent on Iron
Authors: Fratti Rutilio A. , Belanger Paul H. , Ghannoum Mahmoud A. , Edwards John E. , Filler Scott G.
Published: December 2019

Although it is known that
Candida albicans
causes endothelial cell injury, in vitro and in vivo, the mechanism by which this process occurs remains unknown. Iron is critical for the induction of injury in many types of host cells. Therefore, we investigated the role of iron in
Candida
-induced endothelial cell injury. We found that pretreatment of endothelial cells with the iron chelators phenanthroline and deferoxamine protected them from candidal injury, even though the organisms germinated and grew normally. Loading endothelial cells with iron reversed the cytoprotective effects of iron chelation. Moreover, chelation of endothelial cell iron significantly reduced phagocytosis of
C. albicans
by these cells, while candidal adherence to chelator-treated endothelial cells was slightly enhanced. Since endothelial cell phagocytosis of
C. albicans
is required for endothelial cell injury to occur, inhibition of phagocytosis is likely the principal mechanism of the cytoprotective effects of iron chelation. The production of toxic reactive oxygen intermediates by host cells is known to be inhibited by iron chelation. Therefore, we investigated whether treating endothelial cells with antioxidants could mimic the cytoprotective effects of iron chelation. Neither extracellular nor membrane-permeative antioxidants reduced candidal injury of endothelial cells. Furthermore, depleting endothelial cells of the endogenous antioxidant glutathione did not render them more susceptible to damage by
C. albicans
. These results suggest that candidal injury of endothelial cells is independent of the production of reactive oxygen intermediates and that the cytoprotective effects of iron chelation are not due to inhibition of the synthesis of these toxic intermediates.
Levels of Expression and Immunogenicity of Attenuated
Salmonella enterica
Serovar Typhimurium Strains Expressing
Escherichia coli
Mutant Heat-Labile Enterotoxin
Authors: Covone M. Giuseppina , Brocchi Marcelo , Palla Emanuela , da Silveira W. Dias , Rappuoli Rino , Galeotti Cesira L.
Published: December 2019

The effects of heterologous gene dosage as well as
Salmonella typhimurium
strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the
Escherichia coli
heat-labile enterotoxin LTK63, and the carrier
Salmonella
strain have been analyzed. Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in
S. typhimurium Δcya Δcrp Δasd
strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different
Salmonella
isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-
Salmonella
immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated Δ
cya Δcrp
UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in
S. typhimurium
strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated
S. typhimurium
strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the
S. typhimurium
strain in deep tissues has only marginal influence.
Activated Pulmonary Macrophages Are Insufficient for Resistance againstPneumocystis carinii
Authors: Hanano Ralph , Reifenberg Kurt , Kaufmann Stefan H. E.
Published: December 2019

CD4+T cells are pivotal for elimination ofPneumocystis cariniifrom infected lungs, and alveolar macrophages are considered the main effector cells clearing the infected host ofP. cariniiorganisms. To investigate this issue, several mutant mouse strains were used in a previously established experimental setup which facilitates natural acquisition of disease through inhalation of airborne fungal organisms. Mutant mice deficient in major histocompatibility complex class II molecules (Aβ−/−), T-cell receptor αβ cells (TCRβ−/−), or all mature T and B lymphocytes (RAG-1−/−) were naturally susceptible toP. carinii, whereas mouse mutants lacking the gamma interferon (IFN-γ) receptor (IFN-γ-R−/−) or tumor necrosis factor alpha (TNF-α) type I receptor (p55) (TNF-α-RI−/−) resisted disease acquisition. Analysis of pulmonary cytokine patterns and free radical expression revealed the presence of superoxide, nitric oxide, and interleukin-1 (IL-1) mRNA and elevated levels of IFN-γ, TNF-α, and IL-12 in diseased TCRβ−/−and RAG-1−/−mice. Pulmonary macrophages of all diseased mouse mutants expressed scavenger and mannose receptors. Morbid Aβ−/−mutants displayed significant NO levels and IL-1 mRNA only, whereas heterozygous controls did not exhibit any signs of disease. Interestingly, neither IFN-γ nor TNF-α appeared to be essential for resisting natural infection withP. carinii, nor were these cytokines sufficient for mediating resistance during established disease in the absence of CD4+T lymphocytes. Taken together, the results indicated that an activated phagocyte system, as evidenced by cytokine and NO secretion, in diseased mutants was apparently operative but did not suffice for parasite clearance in the absence of CD4+TCRαβ cells. Therefore, additional pathways, possibly involving interactions of inflammatory cytokines with CD4+T lymphocytes, must contribute to successful resistance againstP. carinii.
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

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

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

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

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

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

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

Cytokines 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

Vaccination 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

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

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

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

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

It 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.