bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Sequential Changeover of Expressed
p44
Genes during the Acute Phase of
Anaplasma phagocytophilum
Infection in Horses
Authors: Wang Xueqi , Rikihisa Yasuko , Lai Tzung-Hui , Kumagai Yumi , Zhi Ning , Reed Stephen M. 
Published: November 2004

ABSTRACT

Anaplasma phagocytophilum
immunodominant polymorphic major surface protein P44s have been hypothesized to go through antigenic variation, but the within-host dynamics of
p44
expression has not been demonstrated. In the present study we investigated the composition and changes of
p44
transcripts in the blood during the acute phase of well-defined laboratory
A. phagocytophilum
infections in naïve equine hosts. Three traveling waves of sequential population changeovers of the
p44
transcript species were observed within a single peak of rickettsemia of less than 1 month. During the logarithmic increase, the rapid switch-off of the initial dominant transcript
p44-18
occurred regardless of whether the bacterium was transmitted by ticks or by intravenous inoculation. Each of the subsequently dominant
p44
transcript species was phylogenetically dissimilar from
p44-18
. Development of antibody to the hypervariable region of P44-18 during the rickettsemia suggests the suppression of dominance of immuno-cross-reactive
p44
populations. When
A. phagocytophilum
was preincubated with plasma from the infected horse and then coincubated with HL-60 cells, the dominance of the
p44-18
transcript was rapidly suppressed in vitro and most of the newly emerged
p44
transcript species were previously undetected in this horse. This work provides experimental evidence of within-host
p44
antigenic variation. Results suggest that the rapid and synchronized switch of expression is an intrinsic property of
p44
s reinitiated after transmission to naïve mammalian hosts and shaped upon exposure to immune plasma.
Identification of TwoMycobacterium marinumLoci That Affect Interactions with Macrophages
Authors: El-Etr Sahar H. , Subbian Selvakumar , Cirillo Suat L. G. , Cirillo Jeffrey D. 
Published: November 2004

ABSTRACTMycobacterium marinumis closely related toMycobacterium tuberculosis, the cause of tuberculosis in humans.M. marinumhas become an important model system for the study of the molecular mechanisms involved in causing tuberculosis in humans. Through molecular genetic analysis of the differences between pathogenic and nonpathogenic mycobacteria, we identified two loci that affect the ability ofM. marinumto infect macrophages, designatedmel1andmel2. In silico analyses of the 11 putative genes in these loci suggest thatmel1encodes secreted proteins that include a putative membrane protein and two putative transglutaminases, whereasmel2is involved in secondary metabolism or biosynthesis of fatty acids. Interestingly,mel2is unique toM. marinumand theM. tuberculosiscomplex and not present in any other sequenced mycobacterial species.M. marinummutants with mutations inmel1andmel2, constructed by allelic exchange, are defective in the ability to infect both murine and fish macrophage cell lines. These data suggest that the genes inmel1andmel2are important for the ability ofM. marinumto infect host cells.
Nitric Oxide Production and Nitric Oxide Synthase Activity in Malaria-Exposed Papua New Guinean Children and Adults Show Longitudinal Stability and No Association with Parasitemia
Authors: Boutlis Craig S. , Weinberg J. Brice , Baker Joanne , Bockarie Moses J. , Mgone Charles S. , Cheng Qin , Anstey Nicholas M. 
Published: November 2004

ABSTRACTIndividuals in areas of intense malaria transmission exhibit resistance (or tolerance) to levels of parasitemia in their blood that would normally be associated with febrile illness in malaria-naïve subjects. The resulting level of parasitemia associated with illness (the pyrogenic threshold) is highest in childhood and lowest in adulthood. Clinical parallels between malarial and bacterial endotoxin tolerance have led to the supposition that both share common physiological processes, with nitric oxide (NO) proposed as a candidate mediator. The hypotheses that NO mediates tolerance and blood stage parasite killing in vivo were tested by determining its relationship to age and parasitemia cross-sectionally and longitudinally in a population of 195 children and adults from Papua New Guinea encountering intense malaria exposure. Despite pharmacological clearance of asymptomatic parasitemia, NO production and mononuclear cell NO synthase (NOS) activity were remarkably stable within individuals over time, were not influenced by parasitemia, and varied little with age. These results contrast with previous smaller cross-sectional studies. Baseline NO production and NOS activity did not protect against recurrent parasitemia, consistent with previous data suggesting that NO does not have antiparasitic effects against blood stage infection in vivo. The NO indices studied were markedly higher in specimens from study subjects than in samples from Australian controls, and NOS activity was significantly associated with plasma immunoglobulin E levels, consistent with induction of NO by chronic exposure to other infections and/or host genetic factors. These results suggest that NO is unlikely to mediate killing of blood stage parasites in this setting and is unlikely to be the primary mediator in the acquisition or maintenance of malarial tolerance.
A Heterologous DNA Priming-
Mycobacterium bovis
BCG Boosting Immunization Strategy Using Mycobacterial Hsp70, Hsp65, and Apa Antigens Improves Protection against Tuberculosis in Mice
Authors: Ferraz Jose C. , Stavropoulos Evangelos , Yang Min , Coade Steve , Espitia Clara , Lowrie Douglas B. , Colston M. Joseph , Tascon Ricardo E. 
Published: November 2004

ABSTRACT

Tuberculosis is responsible for >2 million deaths a year, and the number of new cases is rising worldwide. DNA vaccination combined with
Mycobacterium bovis
bacillus Calmette Guérin (BCG) represents a potential strategy for prevention of this disease. Here, we used a heterologous prime-boost immunization approach using a combination of DNA plasmids and BCG in order to improve the efficacy of vaccination against
Mycobacterium tuberculosis
infection in mice. As model antigens, we selected the
M. tuberculosis
Apa (for alanine-proline-rich antigen) and the immunodominant Hsp65 and Hsp70 mycobacterial antigens combined with BCG. We demonstrated that animals injected with a combination of DNA vectors expressing these antigens, when boosted with BCG, showed increased specific antimycobacterial immune responses compared to animals vaccinated with BCG alone. More importantly, the protection achieved with this regimen was also significantly better than with BCG alone.
The C-Terminal Fragment of the Internal 110-Kilodalton Passenger Domain of the Hap Protein of NontypeableHaemophilus influenzaeIs a Potential Vaccine Candidate
Authors: Liu Dai-Fang , Mason Kathryn W. , Mastri Maria , Pazirandeh Mehran , Cutter David , Fink Doran L. , St. Geme Joseph W. , Zhu Duzhang , Green Bruce A. 
Published: November 2004

ABSTRACTNontypeableHaemophilus influenzaeis a major causative agent of bacterial otitis media in children.H. influenzaeHap autotransporter protein is an adhesin composed of an outer membrane Hapβ region and a moiety of an extracellular internal 110-kDa passenger domain called HapS. The HapSmoiety promotes adherence to human epithelial cells and extracellular matrix proteins, and it also mediates bacterial aggregation and microcolony formation. A recent work (D. L. Fink, A. Z. Buscher, B. A. Green, P. Fernsten, and J. W. St. Geme, Cell. Microbiol.5:175-186, 2003) demonstrated that HapSadhesive activity resides within the C-terminal 311 amino acids (the cell binding domain) of the protein. In this study, we immunized mice subcutaneously with recombinant proteins corresponding to the C-terminal region of HapSfromH. influenzaestrains N187, P860295, and TN106 and examined the resulting immune response. Antisera against the recombinant proteins from all three strains not only recognized native HapSpurified from strain P860295 but also inhibitedH. influenzaeHap-mediated adherence to Chang epithelial cells. Furthermore, when mice immunized intranasally with recombinant protein plus mutant cholera toxin CT-E29H were challenged with strain TN106, they were protected against nasopharyngeal colonization. These observations demonstrate that the C-terminal region of HapSis capable of eliciting cross-reacting antibodies that reduce nasopharyngeal colonization, suggesting utility as a vaccine antigen for the prevention of nontypeableH. influenzaediseases.
Relative Contributions of
Pseudomonas aeruginosa
ExoU, ExoS, and ExoT to Virulence in the Lung
Authors: Shaver Ciara M. , Hauser Alan R. 
Published: November 2004

ABSTRACT

Pseudomonas aeruginosa
uses a type III secretion system to promote development of severe disease, particularly in patients with impaired immune defenses. While the biochemical and enzymatic functions of ExoU, ExoS, and ExoT, three effector proteins secreted by this system, are well defined, the relative roles of each protein in the pathogenesis of acute infections is not clearly understood. Since ExoU and ExoS are usually not secreted by the same strain, it has been difficult to directly compare the effects of these proteins during infection. In the work described here, several isogenic mutants of a bacterial strain that naturally secretes ExoU, ExoS, and ExoT were generated to carefully evaluate the relative contribution of each effector protein to pathogenesis in a mouse model of acute pneumonia. Measurements of mortality, bacterial persistence in the lung, and dissemination indicated that secretion of ExoU had the greatest impact on virulence while secretion of ExoS had an intermediate effect and ExoT had a minor effect. It is of note that these results conclusively show for the first time that ExoS is a virulence factor. Infection with isogenic mutants secreting wild-type ExoS, ExoS defective in GTPase-activating protein (GAP) activity, or ExoS defective in ADP-ribosyltransferase activity demonstrated that the virulence of ExoS was largely dependent on its ADP-ribosyltransferase activity. The GAP activity of this protein had only a minor effect in vivo. The relative virulence associated with each of these type III effector proteins may have important prognostic implications for patients infected with
P. aeruginosa
.
Induction of Cationic Chicken Liver-Expressed Antimicrobial Peptide 2 in Response to
Salmonella enterica
Infection
Authors: Townes Claire L. , Michailidis Georgios , Nile Christopher J. , Hall Judith 
Published: November 2004

ABSTRACT

Cationic antimicrobial peptides constitute part of the innate immune system and provide an essential role in the defense against infection. At present there is a paucity of information regarding the antimicrobial profile of the chicken (
Gallus gallus
). Using in silico studies, an expressed sequence tag (EST) clone was identified which encodes a novel cationic antimicrobial peptide, chicken liver-expressed antimicrobial peptide 2 (cLEAP-2). The predicted amino acid sequence composed a prepropeptide, and the active peptide contained four conserved cysteine amino acids. The gene was localized to chromosome 13, and analysis of the genome revealed three exons separated by two introns. The cLEAP-2 gene was expressed in a number of chicken epithelial tissues including the small intestine, liver, lung, and kidney. Northern analysis identified liver-specific cLEAP-2 splice variants, suggesting some degree of tissue-specific regulation. To investigate whether cLEAP-2 expression was constitutive or induced in response to microbial infection, 4-day-old birds were orally infected with
Salmonella
. Analyses of cLEAP-2 expression by semiquantitative reverse transcription-PCR indicated that cLEAP-2 mRNA was upregulated significantly in the small intestinal tissues and the liver, indicative of direct and systemic responses. The antimicrobial activity of cLEAP-2 against
Salmonella
was analyzed in vitro with a time-kill assay and recombinant cLEAP-2. Interestingly
Salmonella enterica
serovar Typhimurium SL1344 showed increased susceptibility to the active cationic peptide (amino acids 37 to 76) compared to
S. enterica
serovar Typhimurium C5 and
Salmonella enteritidis
. Taken together, these data suggest that cationic cLEAP-2 is part of the innate host defense mechanisms of the chicken.
Rhodococcus equi
-Infected Macrophages Are Recognized and Killed by CD8
+
T Lymphocytes in a Major Histocompatibility Complex Class I-Unrestricted Fashion
Authors: Patton Kristin M. , McGuire Travis C. , Fraser Darrilyn G. , Hines Stephen A. 
Published: November 2004

ABSTRACT

The goal of this research was to examine the role of cytotoxic T lymphocytes (CTL) in the control of
Rhodococcus equi
and specifically to determine if
R. equi
-specific CD8
+
CTL occurred in the blood of immune horses. Equine peripheral blood mononuclear cells stimulated with antigen-presenting cells either infected with
R. equi
or exposed to soluble
R. equi
antigen lysed
R. equi
-infected target cells. Lysis was decreased to background by depletion of either CD2
+
or CD3
+
cells, indicating that the effector cell had a T-lymphocyte, but not NK cell, phenotype. Stimulation induced an increased percentage of CD8
+
T cells in the effector population, and depletion of CD8
+
T cells resulted in significantly decreased lysis of infected targets. Killing of
R. equi
-infected macrophages by effector cells was equally effective against autologous and equine leukocyte antigen A (classical major histocompatibility complex [MHC] class I) mismatched targets. To evaluate potential target antigens, target cells were infected with either virulent (80.6-kb plasmid-containing) or avirulent (plasmid-cured)
R. equi
. The degree of lysis was not altered by the presence of the plasmid, providing evidence that the virulence plasmid, which is required for survival within macrophages, was not necessary for recognition and killing of
R. equi
-infected cells. These data indicate that immunocompetent adult horses develop
R. equi-
specific CD8
+
CTL, which may play a role in immunity to
R. equi
. The apparent lack of restriction via classical MHC class I molecules suggests a novel or nonclassical method of antigen processing and presentation, such as presentation by CD1 or other nonclassical MHC molecules.
Adaptation of the Endogenous
Salmonella enterica
Serovar Typhi
clyA
-Encoded Hemolysin for Antigen Export Enhances the Immunogenicity of Anthrax Protective Antigen Domain 4 Expressed by the Attenuated Live-Vector Vaccine Strain CVD 908-
htrA
Authors: Galen James E. , Zhao Licheng , Chinchilla Magaly , Wang Jin Yuan , Pasetti Marcela F. , Green Jeffrey , Levine Myron M. 
Published: November 2004

ABSTRACT

Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of
Salmonella enterica
serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated
Salmonella
serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in
Salmonella
serovar Typhi CVD 908-
htrA
, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of
Bacillus anthracis
protective antigen (PA). A total of 11 of 15 mice immunized intranasally with
Salmonella
serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (
P
= 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines.
Truncation of Fibronectin-Binding Proteins in
Staphylococcus aureus
Strain Newman Leads to Deficient Adherence and Host Cell Invasion Due to Loss of the Cell Wall Anchor Function
Authors: Grundmeier Matthias , Hussain Muzaffar , Becker Petra , Heilmann Christine , Peters Georg , Sinha Bhanu 
Published: November 2004

ABSTRACT

Staphylococcus aureus
fibronectin-binding proteins (FnBPs) play a critical role in
S. aureus
pathogenesis. FnBPs mediate adhesion to fibronectin and invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, by fibronectin bridging to the host cell fibronectin receptor integrin (α
5
)β
1
. Strain Newman is a laboratory strain frequently used for genetic, functional, and in vivo studies. However, despite pronounced production of FnBPs, strain Newman is only weakly adherent to immobilized Fn and weakly invasive. We examined whether these effects are due to a structural difference of FnBPs. Here, we show that both
fnbA
Newman
and
fnbB
Newman
contain a centrally located point mutation resulting in a stop codon. This leads to a truncation of both FnBPs at the end of the C domain at identical positions. Most likely, the stop codon occurred first in
fnbB
Newman
and was subsequently transferred to
fnbA
Newman
by replacement of the entire region encompassing the C, D, and W domains with the respective sequence of
fnbB
Newman
. Using heterologous expression in
Staphylococcus carnosus
, we found that truncated FnBPs were completely secreted into the culture medium and not anchored to the cell wall, since they lack the sortase motif (LPETG). Consequently, this led to a loss of FnBP-dependent functions, such as strong adhesion to immobilized fibronectin, binding of fibrinogen, and host cell invasion. This mutation may explain some of the earlier reported conflicting data with strain Newman. Thus, care should be taken when drawing negative conclusions about the role of FnBPs as a virulence factor in a given model.
Identification of a Novel Virulence Factor in
Burkholderia cenocepacia
H111 Required for Efficient Slow Killing of
Caenorhabditis elegans
Authors: Huber Birgit , Feldmann Friederike , Köthe Manuela , Vandamme Peter , Wopperer Julia , Riedel Kathrin , Eberl Leo 
Published: November 2004

ABSTRACT

Burkholderia cenocepacia
H111, which was isolated from a cystic fibrosis patient, employs a quorum-sensing (QS) system, encoded by
cep
, to control the expression of virulence factors as well as the formation of biofilms. The QS system is thought to ensure that pathogenic traits are expressed only when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. While the wild-type strain effectively kills the nematode
Caenorhabditis elegans
, the pathogenicity of mutants with defective quorum sensing is attenuated. To date, very little is known about the
cep
-regulated virulence factors required for nematode killing. Here we report the identification of a
cep
-regulated gene, whose predicted amino acid sequence is highly similar to the QS-regulated protein AidA of the plant pathogen
Ralstonia solanacearum
. By use of polyclonal antibodies directed against AidA, it is demonstrated that the protein is expressed in the late-exponential phase and accumulates during growth arrest. We show that
B. cenocepacia
H111 AidA is essential for slow killing of
C. elegans
but has little effect on fast killing, suggesting that the protein plays a role in the accumulation of the strain in the nematode gut. Thus, AidA appears to be required for establishing an infection-like process rather than acting as a toxin. Furthermore, evidence is provided that AidA is produced not only by
B. cenocepacia
but also by many other strains of the
Burkholderia cepacia
complex.
Characterization of Immunodominant and Potentially Protective Epitopes ofMannheimia haemolyticaSerotype 1 Outer Membrane Lipoprotein PlpE
Authors: Ayalew Sahlu , Confer Anthony W. , Blackwood Emily R. 
Published: November 2004

ABSTRACTMannheimia haemolyticaserotype 1 (S1) is the most common bacterial isolate found in shipping fever pneumonia in beef cattle. Currently used vaccines againstM. haemolyticado not provide complete protection against the disease. Research withM. haemolyticaouter membrane proteins (OMPs) has shown that antibodies to one particular OMP from S1, PlpE, may be important in immunity. In a recently published work, members of our laboratory showed that recombinant PlpE (rPlpE) is highly immunogenic when injected subcutaneously into cattle and that the acquired immunity markedly enhanced resistance to experimental challenge (A. W. Confer, S. Ayalew, R. J. Panciera, M. Montelongo, L. C. Whitworth, and J. D. Hammer, Vaccine 21:2821-2829, 2003). The objective of this work was to identify epitopes of PlpE that are responsible for inducing the immune response. Western blot analysis of a series of rPlpE with nested deletions on both termini with bovine anti-PlpE hyperimmune sera showed that the immunodominant region is located close to the N terminus of PlpE. Fine epitope mapping, in which an array of overlapping 13-mer synthetic peptides attached to a derivatized cellulose membrane was probed with various affinity-purified anti-PlpE antibodies, identified eight highly reactive regions, of which region 2 (R2) was identified as the specific epitope. The R2 region is comprised of eight imperfect repeats of a hexapeptide (QAQNAP) and is located between residues 26 and 76. Complement-mediated bactericidal activity of affinity-purified anti-PlpE bovine antibodies confirmed that antibodies directed against the R2 region are effective in killingM. haemolytica.