bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Herpes Simplex Virus Thymidine Kinase inToxoplasma gondiiAttenuates Tachyzoite Virulence in Mice
Authors: Radke Jay R. , White Michael W.
Published: December 2019

We tested the virulence in mice ofToxoplasma gondiiRH strain tachyzoites containing various copies of the chloramphenicol acetyl transferase-herpes simplex virus thymidine kinase fusion sequence (CAT-HSTK). Tachyzoite isolates containing ≥five copies of the fusion sequence were not lethal to female CD-1 outbred or BALB/c inbred mice, at doses up to 106parasites, while the parental RH strain caused 100% mortality within 2 weeks at doses as low as 10 parasites. Mice infected with CTK11, an isolate containing five copies of the fusion sequence, showed no overt symptoms of disease and were protected from lethal challenge with the parental RH strain. The CTK11 isolate showed no difference in growth rate, the rate of host cell invasion, or extracellular viability in cell culture compared with parental RH parasites, demonstrating that the CAT-HSTK fusion protein does not affect the normal viability of this isolate. B11, B11C, and D1 isolates contained one or two copies of the CAT-HSTK coding sequence, were not sensitive to thymidine in cell culture, and caused 100% mortality in CD-1 outbred mice in <12 days. A fourth isolate, D1C, contained seven copies of the CAT-HSTK fusion sequence and was sensitive to exogenous thymidine (50% inhibitory concentration = 5.5 μM). Mice infected with D1C showed no symptoms of disease and survived beyond 90 days, thus correlating increased CAT-HSTK gene copies with thymidine sensitivity in cell culture and attenuated virulence in mice. BALB/c mice containing a targeted disruption of the gamma interferon gene (gko) were also susceptible to infection with CTK11 parasites but could be rescued by administration of subcutaneous thymidine once each day for 5 or 10 days following infection. These results suggest that the attenuation of CAT-HSTK+isolates in mice is directly due to active thymidine kinase that likely alters the pyrimidine biosynthetic pathway in these parasites.
Analysis of Receptor for
Vibrio cholerae
El Tor Hemolysin with a Monoclonal Antibody That Recognizes Glycophorin B of Human Erythrocyte Membrane
Authors: Zhang Dongyan , Takahashi Junko , Seno Taiko , Tani Yoshihiko , Honda Takeshi
Published: December 2019

El Tor hemolysin (ETH), a pore-forming toxin secreted by
Vibrio cholerae
O1 biotype El Tor and most
Vibrio cholerae
non-O1 isolates, is able to lyse erythrocytes and other mammalian cells. To study the receptor for this toxin or the related molecule(s) on erythrocyte, we first isolated a monoclonal antibody, B1, against human erythrocyte membrane, which not only blocks the binding of ETH to human erythrocyte but also inhibits the hemolytic activity of ETH. Biochemical characterization and immunoblotting revealed that this antibody recognized an epitope on the extracellular domain of glycophorin B, a sialoglycoprotein of erythrocyte membrane. Erythrocytes lacking glycophorin B but not glycophorin A were less sensitive to the toxin than were normal human erythrocytes. These results indicate that glycophorin B is a receptor for ETH or at least an associated molecule of the receptor for ETH on human erythrocytes.
Virulence Role of V Antigen ofYersinia pestisat the Bacterial Surface
Authors: Fields Kenneth A. , Nilles Matthew L. , Cowan Clarissa , Straley Susan C.
Published: December 2019

Yersinia pestis, the etiologic agent of plague, secretes a set of environmentally regulated, plasmid pCD1-encoded virulence proteins termed Yops and V antigen (LcrV) by a type III secretion mechanism (Ysc). LcrV is a multifunctional protein that has been shown to act at the level of secretion control by binding the Ysc inner-gate protein LcrG and to modulate the host immune response by altering cytokine production. LcrV also is essential for the unidirectional targeting of Yops to the cytosol of infected eukaryotic cells. In this study, we constructed an in-frame deletion withinlcrG(ΔlcrG3) to further analyze the requirement of LcrV in Yop targeting. We confirmed the essentiality of LcrV and found that LcrG may have a facilitative role, perhaps by promoting efficient secretion of LcrV. We also constructed mutants oflcrVexpressing LcrV truncated at the N or C terminus. Both the N and C termini of LcrV were required for the secretion of LcrV into the medium and targeting of Yops. LcrV was detected in punctate zones on the surface of fixedY. pestisby laser-scanning confocal microscopy, and this localization required a functional Ysc. However, the truncated LcrV proteins were not found on the bacterial surface. Finally, we tested the ability of LcrV-specific Fab antibody fragments or full-length antibody to interfere with Yop targeting and found no interference, even though this antibody protects mice against plague. These results indicate that LcrV may function in Yop targeting at the extracellular surface of yersiniae and that the protective efficacy of LcrV-specific antibodies can be manifested without blocking Yop targeting.
Conformational Nature of the
Borrelia burgdorferi
B31 Outer Surface Protein C Protective Epitope
Authors: Gilmore Robert D. , Mbow M. Lamine
Published: December 2019

Active immunization with
Escherichia coli
-expressed recombinant outer surface protein C (OspC) of
Borrelia burgdorferi
has been demonstrated to confer protection against a tick-transmitted infection on laboratory animals. A previous study in this laboratory showed that OspC antibody raised against a denatured immunogen isolated from
B. burgdorferi
cells failed to provide protective immunity. Therefore, to determine whether the protective epitope of the recombinant antigen was sensitive to denaturation, recombinant OspC preparations were subjected to heat and chemical treatments prior to animal immunization. Following seroconversion to OspC, the animals were challenged with an infectious dose of
B. burgdorferi
B31 by tick bite. Whereas mice immunized with a soluble, nondenatured form continued to show protection rates close to 100%, mice that had been immunized with denatured antigen were not protected. Furthermore, mice that were immunized with an insoluble (rather than a soluble), nondenatured form of the recombinant OspC showed a protection rate of only 40%. Protective epitope localization experiments showed that either the amino or the carboxy end of the recombinant protein was required to react with a protective OspC-specific monoclonal antibody. The data from these experiments demonstrate that a conformational organization of the protein is essential for the protective capability of the strain B31 OspC immunogen.
An OspC-Specific Monoclonal Antibody Passively Protects Mice from Tick-Transmitted Infection by
Borrelia burgdorferi
B31
Authors: Mbow M. Lamine , Gilmore Robert D. , Titus Richard G.
Published: December 2019

A murine monoclonal antibody directed against
Borrelia burgdorferi
B31 outer surface protein C (OspC) antigen was generated by a method whereby borreliae were inoculated into the mouse via the natural transmission mode of tick feeding. Passive immunization with this antibody resulted in protection of C3H/HeJ and outbred mice from a tick-transmitted challenge infection. Immunofluorescence staining of borrelia cells indicated surface exposure of the OspC epitope reactive with the monoclonal antibody.
Tumor Necrosis Factor (TNF) and a TNF-Mimetic Peptide Modulate the Granulomatous Response toMycobacterium bovisBCG Infection In Vivo
Authors: Roach D. R. , Briscoe H. , Baumgart K. , Rathjen D. A. , Britton W. J.
Published: December 2019

Tumor necrosis factor (TNF) is a critical mediator in the immune response to mycobacteria, particularly in the formation and maintenance of granulomas. Treatment ofMycobacterium bovisBCG-infected mice with TNF and a TNF-mimetic peptide (TNF70–80) altered the number and cellular composition of granulomas. This change was associated with a moderate decrease in the bacterial burden.
Evidence ThatCryptococcus neoformansIs Melanized in Pigeon Excreta: Implications for Pathogenesis
Authors: Nosanchuk Joshua D. , Rudolph Joseph , Rosas Angel L. , Casadevall Arturo
Published: December 2019

Structures similar to the melanin “ghosts” of melanized cryptococcal cells were isolated from pigeon excreta contaminated withCryptococcus neoformans, and their growth in pigeon excreta supported melanization. The results suggest that environmentalC. neoformanscells are melanized and imply that initial infection may involve exposure to melanized cells.
Growth Rate of Mycobacteria in Mice as an Unreliable Indicator of Mycobacterial Virulence
Authors: North Robert J. , Ryan Lynn , LaCource Ronald , Mogues Tirsit , Goodrich Mariam E.
Published: December 2019

The CDC1551 strain ofMycobacterium tuberculosiswas compared with the H37Rv strain ofM. tuberculosisand the Ravenel strain ofMycobacterium bovisfor virulence in mice. Although all three strains gave rise to the same level of stationary infection in major organs, mice infected with the Ravenel strain died much earlier from lung disease.
Expression of
Chlamydia psittaci
- and Human Immunodeficiency Virus-Derived Antigens on the Cell Surface of
Lactobacillus fermentum
BR11 as Fusions to BspA
Authors: Turner Mark S. , Giffard Philip M.
Published: December 2019

The basic surface protein, BspA, has been used as a fusion partner to direct peptide antigens from the human immunodeficiency virus gp41 protein and the
Chlamydia psittaci
OmpA protein to the cell surface of
Lactobacillus fermentum
BR11. BspA has potential utility in the construction of live vaccines and diagnostic reagents.
Growth-Phase-Dependent Expression of Virulence Factors in an M1T1 Clinical Isolate of
Streptococcus pyogenes
Authors: Unnikrishnan Meera , Cohen Jonathan , Sriskandan Shiranee
Published: December 2019

The effect of growth phase on expression of virulence-associated factors was studied by Northern hybridization in an M1T1 clinical isolate of
Streptococcus pyogenes
. Expression of M protein, C5a peptidase, and capsule was maximal in the exponential phase of growth, while streptococcal pyrogenic exotoxins A and B and mitogenic factor were maximally expressed in later phases of growth.
Role of Gamma Interferon in Controlling Murine Chlamydial Genital Tract Infection
Authors: Ito James I. , Lyons Joseph M.
Published: December 2019

Earlier investigations have not shown an important role for gamma interferon (IFN-γ) in the early clearance of chlamydial infection from the murine female genital tract. In a model using a human genital isolate ofChlamydia trachomatisin IFN-γ and IFN-γ receptor knockout mice, we were able to demonstrate a major role for IFN-γ in mediating control of infection throughout the course of infection.
Type III Secretion-Dependent Hemolytic Activity of Enteropathogenic
Escherichia coli
Authors: Warawa Jonathan , Finlay B. Brett , Kenny Brendan
Published: December 2019

Enteropathogenic
Escherichia coli
(EPEC) was found to exhibit a type III secretion-dependent, contact-mediated, hemolytic activity requiring the EspA, EspB, and EspD secreted proteins. EspB and EspD display homology to pore-forming molecules. Our data suggest a mechanism to explain the requirement for all three Esp proteins in the transfer of EPEC proteins, such as Tir, into target cells.
Alpha-Toxin Damages the Air-Blood Barrier of the Lung in a Rat Model ofStaphylococcus aureus-Induced Pneumonia
Authors: McElroy Mary C. , Harty Helen R. , Hosford Gayle E. , Boylan Gráinne M. , Pittet Jean-François , Foster Timothy J.
Published: December 2019

We have shown that injury to alveolar epithelial type I cells may account, in part, for damage to the air-blood barrier of the lung in a rat model ofStaphylococcus aureuspneumonia. We have also shown that alpha-toxin is an important cause of damage to the air-blood barrier; however, our data suggest that the toxin is not acting directly on alveolar type I cells.
Control ofLeishmania infantumInfection Is Associated with CD8+and Gamma Interferon- and Interleukin-5-Producing CD4+Antigen-Specific T Cells
Authors: Mary Charles , Auriault Valérie , Faugère Bernard , Dessein Alain J.
Published: December 2019

Visceral leishmaniasis is a severe and lethal disease caused by the protozoan parasites of the genusLeishmania. In areas where leishmaniasis is endemic, most infected individuals control the infection and remain asymptomatic; chemotherapy of visceral leishmaniasis restores some immunity which protects against relapses. In the present study,Leishmania-specific T-cell clones were established from six asymptomatic and five cured patients. Cytokines production by these clones was analyzed. A large fraction of the parasite-specific T-cell clones from asymptomatic patients were CD8+and produced high amounts of gamma interferon (IFN-γ). Most CD4+T-cell clones from two asymptomatic subjects exhibited an unusual phenotype: production of high levels of IFN-γ low levels of interleukin-4, (IL-4), but high levels of IL-5. In contrast, only few parasite-specific CD8+T-cell clones were obtained from cured patients after chemotherapy; moreover, CD4+T-cell clones from these patients exhibited an heterogeneous profile of cytokines from Th1-like to Th2-like phenotypes. These results point to CD8+T cells and to IL-5- and IFN-γ-producing CD4+T cells as possible contributors to human resistance toLeishmaniainfection. They should stimulate new immunological approaches in the control of this disease.
Interleukin-1 Inhibits Gamma Interferon-Induced Bacteriostasis in Human Uroepithelial Cells
Authors: Däubener Walter , Hucke Christian , Seidel Kerstin , Hadding Ulrich , MacKenzie Colin R.
Published: December 2019

The most prominent gamma interferon (IFN-γ)-induced antimicrobial effector mechanisms are the induction of nitric oxide (NO) synthase (NOS) and of indoleamine 2,3-dioxygenase (IDO) activity. We have recently found that human glioblastoma cells and human macrophages inhibit the growth of group B streptococci after stimulation with IFN-γ. In this report, we show that in addition, human RT4 (uroepithelial) cells can inhibit the growth of enterococci. Murine macrophages (RAW cells) are unable to inhibit bacterial growth after IFN-γ stimulation. Stimulation of human glioblastoma cells, macrophages, and RT4 cells with human IFN-γ results in a strong expression of IDO activity; however, NO production remains undetectable. In strong contrast, murine RAW cells produce large amounts of NO when stimulated with murine IFN-γ and IDO activity is not detectable. Interleukin-1 (IL-1) induces NO synthase in human RT4 cells when the cells are costimulated with IFN-γ. We found that IL-1 inhibits IFN-γ-stimulated IDO activity and antimicrobial effects in RT4 cells, while in human glioblastoma cells, which lack detectable NO synthase activity, neither of these effects was altered by costimulation with IFN-γ and IL-1. The IL-1-mediated inhibition of IDO activity and of subsequent antibacterial effect is due to the production of NO. This conclusion was supported by evidence thatNG-monomethyl-l-arginine, a competitive inhibitor of inducible NOS activity, is able to block the inhibitory action of IL-1 on IFN-γ-induced bacteriostasis. We therefore conclude that NO production does not inhibit the growth of enterococci but might be involved in the regulation of IDO activity in some human cells.
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

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

PknB 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

Two 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

This 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

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

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

We 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

To 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

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

Coxiella 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

Candida 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

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

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.
Pregenomic Comparative Analysis betweenBordetella bronchisepticaRB50 andBordetella pertussisTohama I in Murine Models of Respiratory Tract Infection
Authors: Harvill Eric T. , Cotter Peggy A. , Miller Jeff F.
Published: December 2019

We describe here a side-by-side comparison of murine respiratory infection byBordetella pertussisandBordetella bronchisepticastrains whose genomes are currently being sequenced (Tohama I and RB50, respectively).B. pertussisandB. bronchisepticaare most appropriately classified as subspecies. Their high degree of genotypic and phenotypic relatedness facilitates comparative studies of pathogenesis. RB50 and Tohama I differ in their abilities to grow in the nose, trachea, and lungs of BALB/c mice and to induce apoptosis, lung pathology, and an antibody response. To focus on the interactions between the bacteria and particular aspects of the host immune response, we used mice with specific immune defects. Mice lacking B cells and T cells were highly susceptible toB. bronchisepticaand were killed by intranasal inoculation with doses as low as 500 CFU. These mice were not killed byB. pertussis, even when doses as high as 105CFU were delivered to the lungs.B. bronchiseptica, which was highly resistant to naive serum in vitro, caused bacteremia in these immunodeficient mice, whileB. pertussis, which was highly sensitive to naive serum, did not cause bacteremia.B. bronchisepticawas, however, killed by immune serum in vitro, and adoptive transfer of anti-Bordetellaantibodies protected SCID-beige mice fromB. bronchisepticalethal infection. Neutropenic mice were similarly killed byB. bronchisepticabut notB. pertussisinfection, suggesting neutrophils are critical to the early inflammatory response to the former but not the latter.B. bronchisepticawas dramatically more active thanB. pertussisin mediating the lysis of J774 cells in vitro and in inducing apoptosis of inflammatory cells in mouse lungs. This side-by-side comparison describes phenotypic differences that may be correlated with genetic differences in the comparative analysis of the genomes of these two highly related organisms.
Bacterial Species- and Strain-Dependent Induction of Tissue Factor in Human Vascular Endothelial Cells
Authors: Veltrop M. H. A. M. , Beekhuizen H. , Thompson J.
Published: December 2019

A cardinal process in bacterial endocarditis (BE) is the activation of the clotting system and the formation of a fibrin clot on the inner surface of the heart, the so-called endocardial vegetation. The processes that lead to the activation of the clotting system on endothelial surfaces upon exposure to bacteria are largely unknown. In the present study, we investigated in an in vitro model whether infection of human endothelial cells (EC) with bacteria that are relevant to BE, such asStaphylococcus aureus,Streptococcus sanguis, andStaphylococcus epidermidis, leads to induction of tissue factor (TF)-dependent procoagulant activity (TFA) and whether this process is influenced by host factors, such as interleukin-1 (IL-1), that are produced in response to the bacteremia in vivo. The results show thatS. aureusbinds to and is internalized by EC, resulting in expression of TF mRNA and TF surface protein as well as generation of TFA within 4 to 8 h after infection. No TFA was found when EC were exposed to UV-irradiatedS. aureusor bacterial cell wall fragments.S. sanguisandS. epidermidis, although also binding to EC, did not induce endothelial TFA. This indicates a species and strain dependency. EC also expressed TFA after exposure to IL-1. The enhanced TFA of EC after exposure toS. aureuswas not prevented by IL-1 receptor antagonist, arguing against an auto- or paracrine contribution of endogenous IL-1. When IL-1 was applied together with bacteria, this had a synergistic effect on the induction of EC TFA. This was found in particular withS. aureusbut also, although to a lesser degree, withS. sanguisandS. epidermidis. This influence of IL-1 on the species- and strain-dependent induction of EC TFA suggests that bacterial factors as well as host factors orchestrate the induction of coagulation in an early stage in the pathogenesis of endovascular disease, such as BE.
Analysis of
Pneumocystis carinii
Introns
Authors: Thomas Charles F. , Leof Edward B. , Limper Andrew H.
Published: December 2019

Pneumocystis carinii
is an ascomycete phylogenetically related to
Schizosaccharomyces pombe
. Little is known about gene regulation in
P. carinii
. The removal of introns from pre-mRNA requires spliceosomal recognition of the intron-exon boundary. In
S. pombe
and higher eukaryotes, this boundary and a branch site within the intron are conserved. We recently demonstrated that
P. carinii cdc2
cDNA can complement
S. pombe
containing conditional mutations of
cdc2
, an essential gene involved in cell cycle regulation. We next tested whether
P. carinii
genomic
cdc2
(with six introns) could also complement
S. pombe cdc2
mutants and found genomic sequences incapable of this activity. Reverse transcriptase PCR confirmed the inability of the
S. pombe cdc2
mutants to splice the
P. carinii
genomic
cdc2
. Analysis of 83 introns from 19
P. carinii
protein-encoding genes demonstrated that the sequence GTWWDW functions as a donor consensus in
P. carinii
, whereas YAG serves as an acceptor consensus. These sequences are similar in
S. pombe
; however, a branch site sequence was not found in the
P. carinii
genes studied.
The Outer Membrane ofBrucella ovisShows Increased Permeability to Hydrophobic Probes and Is More Susceptible to Cationic Peptides than Are the Outer Membranes of Mutant RoughBrucella abortusStrains
Authors: Freer Enrique , Pizarro-Cerdá Javier , Weintraub Andrej , Bengoechea José-Antonio , Moriyón Ignacio , Hultenby Kjell , Gorvel Jean-Pierre , Moreno Edgardo
Published: December 2019

The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural roughBrucella ovisand for mutant roughBrucella abortusstrains. The OM ofB. ovisdisplayed an abrupt and faster kinetic profile than roughB. abortusduring the uptake of the hydrophobic probeN-phenyl-naphthylamine.B. oviswas more sensitive than roughB. abortusto the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both roughBrucellastrains.B. ovislipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smoothB. abortusstrains. Polymyxin B, poly-l-lysine, and poly-l-ornithine produced a thick coating on the surfaces of both strains, which was more evident inB. ovisthan in roughB. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells.
Syntheses and Immunologic Properties of
Escherichia coli
O157 O-Specific Polysaccharide and Shiga Toxin 1 B Subunit Conjugates in Mice
Authors: Konadu Edward , Donohue-Rolfe Arthur , Calderwood Stephen B. , Pozsgay Vince , Shiloach Joseph , Robbins John B. , Szu Shousun C.
Published: December 2019

Escherichia coli
O157 is the major cause of diarrhea-associated hemolytic uremic syndrome (HUS). Strains causing HUS contain either Shiga toxin 1 (Stx1) or Stx2, or both. In adult volunteers, conjugate vaccines of detoxified lipopolysaccharide (LPS) elicited bactericidal antibodies to
E. coli
O157. Here, the detoxified LPS was conjugated with improved schemes to the nontoxic B subunit of Stx1. Mice injected with these bivalent conjugates elicited both bactericidal antibodies to
E. coli
O157 and neutralization antibodies to Stx1.
Urease-Based Mucosal Immunization againstHelicobacter heilmanniiInfection Induces Corpus Atrophy in Mice
Authors: Dieterich Christine , Bouzourène Hanifa , Blum André L. , Corthésy-Theulaz Irène E.
Published: December 2019

Mucosal immunization withHelicobacter heilmanniiurease B orHelicobacter pyloriurease, given nasally with cholera toxin, protects BALB/c mice againstH. heilmanniiinfection and significantly reduces a preexisting infection. However, immunization aggravates gastric corpus atrophy. Our results underline the necessity of defining immunization regimens that do not enhance mucosal damage.