bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a
Clostridium perfringens
Enterotoxin Region Required for Large Complex Formation and Cytotoxicity by Random Mutagenesis
Authors: Kokai-Kun John F. , Benton Kimberly , Wieckowski Eva U. , McClane Bruce A. 
Published: December 2019

ABSTRACT

Clostridium perfringens
enterotoxin (CPE), a single polypeptide of 319 amino acids, has a unique multistep mechanism of action. In the first step, CPE binds to claudin proteins and/or a 50-kDa eukaryotic membrane protein receptor, forming a small (∼90-kDa) complex. This small complex apparently then associates with a 70-kDa eukaryotic membrane protein, resulting in formation of a large complex that induces the onset of membrane permeability alterations. To better define the boundaries of CPE functional regions and to identify specific amino acid residues involved in various steps of CPE action, in this study we subjected the cloned
cpe
gene to random mutagenesis in XL-1 Red strains of
Escherichia coli
. Seven CPE random mutants with reduced cytotoxicity for Vero cells were phenotypically characterized for the ability to complete each step in CPE action. Five of these seven recombinant CPE (rCPE) random mutants (G49D, S59L, R116S, R137G, and S167P) exhibited binding characteristics similar to those of rCPE or native CPE, while the Y310C and W226Stop mutants showed reduced binding and no binding, respectively, to brush border membranes. Interestingly, two completely nontoxic mutants (G49D and S59L) were able to bind and form small complex but they did not mediate any detectable large complex formation. Another strongly attenuated mutant, R116S, formed reduced amounts of an anomalously migrating large complex. Collectively, these results provide further support for large complex formation being an essential step in CPE action and also identify the CPE region ranging from residues ∼45 to 116 as important for large complex formation. Finally, we also report that limited removal of extreme N-terminal CPE sequences, which may occur in vivo during disease, stimulates cytotoxic activity by enhancing large complex formation.
Expression and Characterization of theMycobacterium tuberculosisSerine/Threonine Protein Kinase PknB
Authors: Av-Gay Yossef , Jamil Sarwat , Drews Steven J. 
Published: December 2019

ABSTRACTPknB is a member of the newly discovered eukaryotic-like protein serine/threonine kinase (PSTK) family of proteins. ThepknBgene was cloned and expressed inEscherichia coli. The active recombinant protein was purified and shown to be reactive with antiphosphoserine antibodies, as well as with antibodies to the phosphorylated eukaryotic Ser/Thr kinases mitogen-activated protein kinase kinase 3 and 6, P38, and Creb. In vitro kinase assays demonstrated that PknB is a functional kinase that is autophosphorylated on serine/threonine residues and is also able to phosphorylate the peptide substrate myelin basic protein. Analysis ofpknBexpression inMycobacterium tuberculosisindicates the presence ofpknBmRNA in (i) organisms grown in vitro in bacteriological media, (ii) a murine macrophage in vitro infection model, and (iii) in vivo alveolar macrophages from a patient with tuberculosis.
Population Dynamics of a Naturally Occurring Heterogeneous Mixture ofBorrelia burgdorferiClones
Authors: Hofmeister Erik K. , Glass Gregory E. , Childs James E. , Persing David H. 
Published: December 2019

ABSTRACTTwo unique isolates ofBorrelia burgdorferi, differing in plasmid content and outer surface protein C expression, were cultured on sequential captures of a single free-livingPeromyscus leucopusmouse and were examined for differences in transmissibility. Both isolates were transmissible from inoculated C.B-17 mice to larvalIxodes scapularisticks and, subsequently, from infected nymphal ticks to C3H/HeJ mice. Plasmid and protein analyses suggested that the original isolates were a mixed population ofB. burgdorferi, and cloning by limiting dilution resulted in the identification of two clonal groups. In addition to being heterogeneous in plasmid and genomic macrorestriction analyses, the clones varied with respect to the electrophoretic mobilities and antigenicity of their OspC proteins, as shown by their reactivity to a panel of monoclonal antibodies. Plasmid analysis of sequential isolates from C3H mice experimentally infected with the primary isolate or various mixtures of its subclones showed an apparently random fluctuation in clonal dominance in the majority of mice. Surprisingly, mice infected with each subclone were permissive to superinfection with the heterologous subclone, despite the presence of anti-B. burgdorferiantibodies at the time of the secondary challenge. These results show conclusively that mice captured at Lyme disease enzootic sites may be infected by mixed populations of genetically and antigenically distinctB. burgdorfericlones and that these infections can be acquired by coinfection or by sequential infection. The lack of cross-immunization between clones existing within a naturally occurring population may play a role in the maintenance of the genetic heterogeneity ofB. burgdorferiin nature.
Lack of Humoral Immune Protection againstTreponema denticolaVirulence in a Murine Model
Authors: Kesavalu Lakshmyya , Holt Stanley C. , Ebersole Jeffrey L. 
Published: December 2019

ABSTRACTThis study investigated the characteristics of humoral immune responses toTreponema denticolafollowing primary infection, reinfection, and active immunization, as well as immune protection in mice. Primary infection withT. denticolainduced a significant (400-fold) serum immunoglobulin G (IgG) response compared to that in control uninfected mice. The IgG response to reinfection was 20,000-fold higher than that for control mice and 10-fold higher than that for primary infection. Mice actively immunized with formalin-killed treponemes developed serum antibody levels seven- to eightfold greater than those in animals after primary infection. Nevertheless, mice with this acquired antibody following primary infection or active immunization demonstrated no significant alterations of lesion induction or decreased size of the abscesses following a challenge infection. Mice with primary infection developed increased levels of IgG3, IgG2b, and IgG2a antibodies, with IgG1 being lower than the other subclasses. Reinfected mice developed enhanced IgG2b, IgG2a, and IgG3 and less IgG1. In contrast, immunized mice developed higher IgG1 and lower IgG3 antibody responses to infection. These IgG subclass distributions indicate a stimulation of both Th1 and Th2 activities in development of the humoral immune response to infection and immunization. Our findings also demonstrated a broad antigen reactivity of the serum antibody, which was significantly increased with reinfection and active immunization. Furthermore, serum antibody was effective in vitro in immobilizing and clumping the bacteria but did not inhibit growth or passively prevent the treponemal infection. These observations suggest that humoral immune responses, as manifested by antibody levels, isotype, and antigenic specificity, were not capable of resolving aT. denticolainfection.
Evaluation of a Truncated Recombinant Flagellin Subunit Vaccine against
Campylobacter jejuni
Authors: Lee Lanfong H. , Burg Edward , Baqar Shahida , Bourgeois A. L. , Burr Don H. , Ewing Cheryl P. , Trust Trevor J. , Guerry Patricia 
Published: December 2019

ABSTRACT

A recombinant protein comprising the maltose-binding protein (MBP) of
Escherichia coli
fused to amino acids 5 to 337 of the FlaA flagellin of
Campylobacter coli
VC167 was evaluated for immunogenicity and protective efficacy against challenge by a heterologous strain of campylobacter,
Campylobacter jejuni
81-176, in two murine models. The sequence of the
flaA
gene of strain 81-176 revealed a predicted protein which was 98.1% similar to that of VC167 FlaA over the region expressed in the fusion protein. Mice were immunized intranasally with two doses of 3 to 50 μg of MBP-FlaA, given 8 days apart, with or without 5 μg of the mutant
E. coli
heat-labile enterotoxin (LT
R192G
) as a mucosal adjuvant. The full range of MBP-FlaA doses were effective in eliciting antigen-specific serum immunoglobulin G (IgG) responses, and these responses were enhanced by adjuvant use, except in the highest dosing group. Stimulation of FlaA-specific intestinal secretory IgA (sIgA) responses required immunization with higher doses of MBP-FlaA (≥25 μg) or coadministration of lower doses with the adjuvant. When vaccinated mice were challenged intranasally 26 days after immunization, the best protection was seen in animals given 50 μg of MBP-FlaA plus LT
R192G
. The protective efficacies of this dose against disease symptoms and intestinal colonization were 81.1 and 84%, respectively. When mice which had been immunized with 50 μg of MBP-FlaA plus LT
R192G
intranasally were challenged orally with 8 × 10
10
, 8 × 10
9
, or 8 × 10
8
cells of strain 81-176, the protective efficacies against intestinal colonization at 7 days postinfection were 71.4, 71.4, and 100%, respectively.
Safety and Immunogenicity of Vi Conjugate Vaccines for Typhoid Fever in Adults, Teenagers, and 2- to 4-Year-Old Children in Vietnam
Authors: Kossaczka Zuzana , Lin Feng-Ying C. , Ho Vô Anh , Thuy Nguyen Thi Thanh , Bay Phan Van , Thanh Tran Cong , Khiem Ha Ba , Trach Dang Duc , Karpas Arthur , Hunt Steven , Bryla Dolores A. , Schneerson Rachel , Robbins John B. , Szu Shousun C. 
Published: December 2019

ABSTRACT

The capsular polysaccharide of
Salmonella typhi,
Vi, is an essential virulence factor and a protective vaccine for people older than 5 years. The safety and immunogenicity of two investigational Vi conjugate vaccines were evaluated in adults, 5- to 14-year-old children, and 2- to 4-year-old children in Vietnam. The conjugates were prepared with
Pseudomonas aeruginosa
recombinant exoprotein A (
r
EPA) as the carrier, using either
N
-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP; Vi-
r
EPA
1
) or adipic acid dihydrazide (ADH; Vi-
r
EPA
2
) as linkers. None of the recipients experienced a temperature of >38.5°C or significant local reactions. One injection of Vi-
r
EPA
2
into adults elicited a geometric mean (GM) increase in anti-Vi immunoglobulin G (IgG) from 9.62 enzyme-linked immunosorbent assay units/ml (EU) to 465 EU at 6 weeks; this level fell to 119 EU after 26 weeks. In the 5- to 14-year-old children, anti-Vi IgG levels at 6 weeks elicited by Vi-
r
EPA
2
, Vi-
r
EPA
1
, and Vi were 169, 22.8, and 18.9 EU, respectively (
P
= 0.0001 for Vi-
r
EPA
1
and Vi with respect to Vi-
r
EPA
2
). At 26 weeks, the anti-Vi IgG levels for recipients of Vi-
r
EPA
2
, Vi-
r
EPA
1
, and Vi were 30.0, 10.8, and 13.4 EU, respectively (
P
< 0.001 for Vi-
r
EPA
1
and Vi with respect to Vi-
r
EPA
2
); all were higher than the preinjection levels (
P
= 0.0001). Vi-
r
EPA
2
also elicited the highest anti-Vi IgM and IgA levels of the three vaccines. In the 2- to 4-year-old children at 6 weeks following the first injection, Vi-
r
EPA
2
elicited an anti-Vi IgG level of 69.9 EU compared to 28.9 EU for Vi-
r
EPA
1
(
P
= 0.0001). Reinjection increased Vi antibody levels from 69.9 to 95.4 EU for Vi-
r
EPA
2
and from 28.9 to 83.0 EU for Vi-
r
EPA
1
. At 26 weeks, anti-Vi IgG levels remained higher than those at preinjection (30.6 versus 0.18 for Vi-
r
EPA
2
and 12.8 versus 0.33 for Vi-
r
EPA
1
;
P
= 0.0001 for both). Vi vaccine is recommended for individuals of 5 years of age or older. In the present study, the GM level of anti-Vi IgG elicited by two injections of Vi-
r
EPA
2
in the 2- to 4-year-old children was higher than that elicited by Vi in the 5- to 14-year-old children (30.6 versus 13.4;
P
= 0.0001). The safety and immunogenicity of the Vi-
r
EPA
2
conjugate warrant further investigation.
Effect of Multiple Antigenic Exposures in the Gut on Oral Tolerance and Induction of Antibacterial Systemic Immunity
Authors: Garg Sanjay , Bal Vineeta , Rath Satyajit , George Anna 
Published: December 2019

ABSTRACTWe have analyzed oral tolerance of microbial antigens in an experimental model in which mice are treated orally with a single small dose of soluble antigen and challenged systemically with the antigen in complete Freund's adjuvant. We found that, while oral administration of sonicated extracts of eitherLeishmania major,Leishmania donovani, orStaphylococcus aureuswas tolerogenic, as was administration of the nominal antigen ovalbumin or conalbumin, oral administration ofEscherichia coliorSalmonella typhimuriumsonicated extract was not. SinceE. coliis an enteric commensal that colonizes the intestine soon after birth, these data suggested that lack of demonstrable oral tolerance may be related to the frequency of oral exposure to an antigen. In support of this, we found that multiple oral doses of ovalbumin orS. aureusorL. donovaniantigens did not increase systemic hyporesponsiveness beyond that achieved with a single oral dose. We have also tested the ability of mice fed with sonicates of the tolerogenicS. aureusor the nontolerogenicS. typhimuriumto clear a subsequent systemic infection with the homologous bacteria and found that, while clearance ofS. aureuswas unaffected by prior feeding, clearance ofS. typhimuriumwas actually enhanced. The data suggest that frequent oral antigenic exposure may eventually lead to induction of systemic immunity in tolerant mice.
Recognition of Schistosome Glycolipids by Immunoglobulin E: Possible Role in Immunity
Authors: van der Kleij Desiree , Tielens Aloysius G. M. , Yazdanbakhsh Maria 
Published: December 2019

ABSTRACTTo investigate the role of antibody responses to (glyco)lipids in immunity to schistosome infection, lipids extracted fromSchistosoma mansonieggs and adult worms were fractionated, and the antibody isotype profile reactive to the fractionated lipids in a well-characterizedS. haematobium-infected population was investigated. In tests of 10 plasma samples it was found that immunoglobulin G (IgG) reactivity was highest to the fraction containing ceramidepolyhexosides, whereas IgE reactivity was most prominent to both cerebroside- and ceramidepolyhexoside-containing fractions. The fraction containing ceramidepolyhexosides was then tested for reactivity with IgG subclasses and IgE in plasma samples from 66S. haematobium-infected patients. Considering IgG4 and IgE, isotypes of particular interest in helminth infections, we found that both isotypes recognized egg (glyco)proteins in more than 90% of the infected subjects. However, in the case of glycolipids, IgE reactivity was much more prominent than IgG4 reactivity (found in 80 and 41% of the subjects, respectively). Furthermore, worm glycolipid-specific IgE prior to treatment of the subjects with praziquantel was negatively correlated with egg counts at 2 years posttreatment, indicating that IgE directed towards glycolipids could play an important role in resistance to reinfection.
Experimental Gestational Pyelonephritis Induces Preterm Births and Low Birth Weights in C3H/HeJ Mice
Authors: Kaul Anil K. , Khan Shah , Martens Mark G. , Crosson John T. , Lupo Virginia R. , Kaul Rashmi 
Published: December 2019

ABSTRACT

Urinary tract infections (UTIs) are associated with approximately 27% of premature births.
Escherichia coli
is the most frequent causal agent of UTIs and expresses virulence factors, including surface adhesins that recognize specific host tissue receptors. We have reported that
E. coli
Dr adhesin recognizes decay-accelerating factor as the host tissue receptor and that these receptors are increased during pregnancy. Induction of pathogenesis is a cumulative effect of the host-pathogen relationship involving specific host factors and virulence characteristics of the invading organism. Recently, an experimental model of chronic pyelonephritis has been developed with
E. coli
bearing Dr adhesin (
E. coli
Dr
+
) in nonpregnant lipopolysaccharide hyporesponder C3H/HeJ mice. In this study, we investigated the role of
E. coli
Dr
+
on the outcome of pregnancy in C3H/HeJ mice. Groups of pregnant mice were infected with
E. coli
Dr
+
or its isogenic mutant which does not bear the Dr adhesin (
E. coli
Dr
−
) by urethral catheterization. Nearly 90% of pregnant mice infected with
E. coli
Dr
+
delivered preterm (before 90% gestation) compared to 10% of mice infected with
E. coli
Dr
−
and none of the mice treated with phosphate-buffered saline (PBS). Also, there was a significant reduction in fetal birth weight in the
E. coli
Dr
+
-infected group compared to the
E. coli
Dr
−
- and PBS-treated groups (
P
= 0.003). This experimental model of
E. coli
Dr
+
-induced preterm delivery in mice may help in understanding the molecular mechanisms involved in UTI-induced preterm labor involving bacterial adhesins.
Differential Expression of Translational Elements by Life Cycle Variants ofCoxiella burnetii
Authors: Seshadri Rekha , Hendrix Laura R. , Samuel James E. 
Published: December 2019

ABSTRACTCoxiella burnetiireplicates as distinct morphological forms, which may allow potential life cycle variants to survive the harsh environment of the phagolysosome. Monoclonal antibodies (MAbs) were compared by Western blotting for reactivity with large cell variant (LCV) and small cell variant (SCV) antigens to characterize proteins differentially expressed byC. burnetii. MAb NM7.3 reacted with a ∼32-kDa LCV-upregulated antigen, and MAb NM183 reacted with a ∼45-kDa LCV-specific antigen. MAb NM7.3 was used to screen a λZapIIC. burnetiiDNA expression library, and an immunoreactive clone was identified with sequence similarity to theEscherichia coli tsfgene, which encodes elongation factor Ts (EF-Ts). Since a similar screen with MAb NM183 did not identify immunoreactive clones, an alternate strategy was devised to clone the reactive antigen based on observations of cross-reactivity with the 45-kDa elongation factor Tu (EF-Tu) protein fromChlamydia trachomatis. The highly conserved nature of EF-Tu among eubacteria allowed PCR amplification of atufgene fragment (encoding ∼95% of the predicted EF-Tu open reading frame) fromC. burnetiiusing degenerate primers. The product of the clonedtufgene fragment reacted with MAb NM183 in Western blot analysis, confirming the identity of the 45-kDa LCV-specific antigen. Identification of two proteins differentially expressed byC. burnetii, EF-Tu and EF-Ts, both essential components of the translational machinery of the cell, supports the hypothesis that LCVs are metabolically more active than SCVs.
Overexpression of theCandida albicans ALA1Gene inSaccharomyces cerevisiaeResults in Aggregation following Attachment of Yeast Cells to Extracellular Matrix Proteins, Adherence Properties Similar to Those ofCandida albicans
Authors: Gaur Nand K. , Klotz Stephen A. , Henderson Ramona L. 
Published: December 2019

ABSTRACTCandida albicansmaintains a commensal relationship with human hosts, probably by adhering to mucosal tissue in a variety of physiological conditions. We show that adherence due to theC. albicansgeneALA1when transformed intoSaccharomyces cerevisiae, is comprised of two sequential steps. Initially,C. albicansrapidly attaches to extracellular matrix (ECM) protein-coated magnetic beads in small numbers (the attachment phase). This is followed by a relatively slower step in which cell-to-cell interactions predominate (the aggregation phase). Neither of these phases is observed inS. cerevisiae. However, expression of theC. albicans ALA1gene from a low-copy vector causesS. cerevisiaetransformants to attach to ECM-coated magnetic beads without appreciable aggregation. Expression ofALA1from a high-copy vector results in both attachment and aggregation. Moreover, transcriptional fusion ofALA1with the galactose-inducible promoters GALS, GALL, and GAL1, allowing for low, moderate, and high levels of inducible transcription, respectively, causes attachment and aggregation that correlates with the strength of the GAL promoter. The adherence ofC. albicansandS. cerevisiaeoverexpressingALA1to a number of protein ligands occurs over a broad pH range, is resistant to shear forces generated by vortexing, and is unaffected by the presence of sugars, high salt levels, free ligands, or detergents. Adherence is, however, inhibited by agents that disrupt hydrogen bonds. The similarities in the adherence and aggregation properties ofC. albicansandS. cerevisiaeoverexpressingALA1suggest a role in adherence and aggregation forALA1andALA1-like genes inC. albicans.
Augmentation of Innate Host Defense by Expression of a Cathelicidin Antimicrobial Peptide
Authors: Bals Robert , Weiner Daniel J. , Moscioni A. David , Meegalla Rupalie L. , Wilson James M. 
Published: December 2019

ABSTRACT

Antimicrobial peptides, such as defensins or cathelicidins, are effector substances of the innate immune system and are thought to have antimicrobial properties that contribute to host defense. The evidence that vertebrate antimicrobial peptides contribute to innate immunity in vivo is based on their expression pattern and in vitro activity against microorganisms. The goal of this study was to investigate whether the overexpression of an antimicrobial peptide results in augmented protection against bacterial infection. C57BL/6 mice were given an adenovirus vector containing the cDNA for LL-37/hCAP-18, a human cathelicidin antimicrobial peptide. Mice treated with intratracheal LL-37/hCAP-18 vector had a lower bacterial load and a smaller inflammatory response than did untreated mice following pulmonary challenge with
Pseudomonas aeruginosa
PAO1. Systemic expression of LL-37/hCAP-18 after intravenous injection of recombinant adenovirus resulted in improved survival rates following intravenous injection of lipopolysaccharide with galactosamine or
Escherichia coli
CP9. In conclusion, the data demonstrate that expression of an antimicrobial peptide by gene transfer results in augmentation of the innate immune response, providing support for the hypothesis that vertebrate antimicrobial peptides protect against microorganisms in vivo.
A New Rat Model of Otitis Media Caused by
Streptococcus pneumoniae
: Conditions and Application in Immunization Protocols
Authors: van der Ven Leo T. M. , van den Dobbelsteen Germie P. J. M. , Nagarajah Bhawani , van Dijken Harry , Dortant Paul M. , Vos Josephus G. , Roholl Paul J. M. 
Published: December 2019

ABSTRACT

Streptococcus pneumoniae
(pneumococcus [Pn]) can be cultured from up to 50% of acute otitis media (AOM) effusions, and these bacteria are the most common cause of AOM-related complications. With the recent advent of antibiotic-resistant Pn strains, treatment of Pn infections may meet with serious difficulties. Prevention through vaccination, notably for the four most common occurring Pn serotypes in humans (i.e., Pn 6B, Pn 14, Pn 19F, and Pn 23F), is a helpful alternative. Testing of vaccine efficacy should occur in an appropriate animal AOM model, which is presented here. The four involved Pn serotypes are not pathogenic to the rat, which was chosen as the experimental animal for practical reasons. To induce a natural infection (i.e., ascending through the eustachian tube), the mucociliary clearance of the eustachian tube was impaired by infusing histamine into the tympanic cavity on 2 consecutive days before intranasal inoculation of the bacteria. With this simple protocol, high and reproducible infection rates, as determined with bacterial cultures, of Pn-induced AOM (approximately 70%) with the two major Pn serotypes 14 and 19F (Pn 14 and Pn 19F) were obtained, whereas lower infection rates (25 to 50%) with Pn 6B and Pn 23F were obtained. In this model, intranasal priming with pneumococci, as well as subcutaneous vaccination with Pn 14 tetanus toxoid-conjugated polysaccharide, induced a protective effect against the induction of otitis media with these bacteria. This shows that immunity to Pn 14 AOM can be induced by both mucosal and systemic presentations of antigen. In conclusion, we have developed an animal model for Pn-induced AOM, which is suitable for the evaluation of the protecting effect of immunization.