bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Analysis of
Bacillus anthracis
Protective Antigen by Using Neutralizing Monoclonal Antibodies
Authors: Brossier Fabien , Lévy Martine , Landier Annie , Lafaye Pierre , Mock Michèle
Published: October 2004

Protective antigen (PA) is central to the action of the lethal and edema toxins produced by
Bacillus anthracis
. It is the common cell-binding component, mediating the translocation of the enzymatic moieties (lethal factor [LF] and edema factor) into the cytoplasm of the host cell. Monoclonal antibodies (MAbs) against PA, able to neutralize the activities of the toxins in vitro and in vivo, were screened. Two such MAbs, named 7.5 and 48.3, were purified and further characterized. MAb 7.5 binds to domain 4 of PA and prevents the binding of PA to its cell receptor. MAb 48.3 binds to domain 2 and blocks the cleavage of PA into PA63, a step necessary for the subsequent interaction with the enzymatic moieties. The epitope recognized by this antibody is in a region involved in the oligomerization of PA63; thus, MAb 48.3 does not recognize the oligomer form. MAbs 7.5 and 48.3 neutralize the activities of anthrax toxins produced by
B. anthracis
in mice. Also, there is an additive effect between the two MAbs against PA and a MAb against LF, in protecting mice against a lethal challenge by the Sterne strain. This work contributes to the functional analysis of PA and offers immunotherapeutic perspectives for the treatment of anthrax disease.
Antimycobacterial Calixarenes Enhance Innate Defense Mechanisms in Murine Macrophages and Induce Control ofMycobacterium tuberculosisInfection in Mice
Authors: Colston M. Joseph , Hailes Helen C. , Stavropoulos Evangelos , Hervé Anne C. , Hervé Gwenaelle , Goodworth Kerry J. , Hill Alison M. , Jenner Peter , Hart Philip D. , Tascon Ricardo E.
Published: October 2004

Tuberculosis remains the leading cause of death among infectious diseases, accounting for more than two million deaths annually. The incidence of the disease is increasing globally, partially because of the resurgence of drug-resistant strains ofMycobacterium tuberculosis. Calixarenes are macrocyclic oligomers, some of which are able to modify the growth ofM. tuberculosisin infected cells. Most experimental work has been carried out with Macrocyclon, also known as HOC 12.5EO. In this study, we demonstrate that Macrocyclon is effective in controllingM. tuberculosisinfections, and we provide evidence that its effect is partially mediated by anl-arginine-dependent mechanism of macrophage activation that involves the activity of the inducible nitric oxide synthase. We also show that Macrocyclon is effective in athymic and major histocompatibility complex class II−/−mice and synthesized a number of structurally related calixarenes expressing significant antimycobacterial activity.
Glycosylation of the Major Polar Tube Protein ofEncephalitozoon hellem, a Microsporidian Parasite That Infects Humans
Authors: Xu Yanji , Takvorian Peter M. , Cali Ann , Orr George , Weiss Louis M.
Published: October 2004

The microsporidia are ubiquitous, obligate intracellular eukaryotic spore-forming parasites infecting a wide range of invertebrates and vertebrates, including humans. The defining structure of microsporidia is the polar tube, which forms a hollow tube through which the sporoplasm is transferred to the host cell. Research on the molecular and cellular biology of the polar tube has resulted in the identification of three polar tube proteins: PTP1, PTP2, and PTP3. The major polar tube protein, PTP1, accounts for at least 70% of the mass of the polar tube. In the present study, PTP1 was found to be posttranslationally modified. Concanavalin A (ConA) bound to PTP1 and to the polar tube of several different microsporidia species. Analysis of the glycosylation ofEncephalitozoon hellemPTP1 suggested that it is modified by O-linked mannosylation, and ConA binds to these O-linked mannose residues. Mannose pretreatment of RK13 host cells decreased their infection byE. hellem, consistent with an interaction between the mannosylation of PTP1 and some unknown host cell mannose-binding molecule. A CHO cell line (Lec1) that is unable to synthesize complex-type N-linked oligosaccharides had an increased susceptibility toE. helleminfection compared to wild-type CHO cells. These data suggest that the O-mannosylation of PTP1 may have functional significance for the ability of microsporidia to invade their host cells.
Cytokine Response to Infection with
Bacillus anthracis
Spores
Authors: Pickering Alison K. , Osorio Manuel , Lee Gloria M. , Grippe Vanessa K. , Bray Mechelle , Merkel Tod J.
Published: October 2004

Bacillus anthracis
, the etiological agent of anthrax, is a gram-positive, spore-forming bacterium. The inhalational form of anthrax is the most severe and is associated with rapid progression of the disease and the outcome is frequently fatal. Transfer from the respiratory epithelium to regional lymph nodes appears to be an essential early step in the establishment of infection. This transfer is believed to occur by means of carriage within alveolar macrophages following phagocytosis. Therefore, the ability of
B. anthracis
to transit through the host macrophage or dendritic cell appears to be an early and critical step in
B. anthracis
pathogenesis. In this work, we examined the cytokine responses to spore infection in mouse primary peritoneal macrophages, in primary human dendritic cells, and during a spore aerosol infection model utilizing the susceptible A/J mouse strain. We demonstrated that both mouse peritoneal macrophages and human dendritic cells exhibited significant intracellular bactericidal activity during the first hours following uptake, providing the necessary time to mount a cytokine response prior to cell lysis. Strong tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) responses were seen in mouse peritoneal macrophages. In addition to TNF-α and IL-6, human dendritic cells produced the cytokines IL-1β, IL-8, and IL-12. A mixture of Th1 and Th2 cytokines were detected in sera obtained from infected animals. In this study, we provide further evidence of an acute cytokine response when cells in culture and mice are infected with
B. anthracis
spores.
In Vivo Compartmentalization of Functionally Distinct, Rapidly Responsive Antigen-Specific T-Cell Populations in DNA-Immunized orSalmonella entericaSerovar Typhimurium-Infected Mice
Authors: Kirby Alun C. , Sundquist Malin , Wick Mary Jo
Published: October 2004

The location and functional properties of antigen-specific memory T-cell populations in lymphoid and nonlymphoid compartments following DNA immunization or infection withSalmonellawere investigated. Epitope-specific CD8+-T-cell expansion and retention during the memory phase were analyzed for DNA-immunized mice by use of a 5-h peptide restimulation assay. These data revealed that epitope-specific gamma interferon (IFN-γ)-positive CD8+T cells occur at higher frequencies in the spleen, liver, and blood than in draining or peripheral lymph nodes during the expansion phase. Moreover, this distribution is maintained into long-term memory. The location and function of both CD4+and CD8+Salmonella-specific memory T cells in mice who were given a single dose ofSalmonella entericaserovar Typhimurium was also quantitated by an ex vivo restimulation with bacterial lysate to detect the totalSalmonella-specific memory pool. Mice immunized up to 6 months previously withS. entericaserovar Typhimurium had bacterium-specific CD4+T cells that were capable of producing IFN-γ or tumor necrosis factor alpha (TNF-α) at each site analyzed. Similar findings were observed for CD8+T cells that were capable of producing IFN-γ, while a much lower frequency and more restricted distribution were associated with TNF-α-producing CD8+T cells. This study is the first to assess the frequencies, locations, and functions of both CD4+and CD8+memory T-cell populations in the sameSalmonella-infected individuals and demonstrates the organ-specific functional compartmentalization of memory T cells afterSalmonellainfection.
Novel Human In Vitro System for Evaluating Antimycobacterial Vaccines
Authors: Kampmann Beate , Tena Gwen N. , Mzazi Shumikazi , Eley Brian , Young Douglas B. , Levin Michael
Published: October 2004

Major research efforts are directed towards the development of a better antimycobacterial vaccine. But progress in the field of tuberculosis vaccine development has been hampered by the lack of human in vitro models to assess vaccine immunogenicity and efficacy. New candidate vaccines will have to be evaluated against the existingMycobacterium bovisBCG “gold standard.” It is therefore important to understand the type of immune responses elicited by BCG vaccination to enable comparisons with potential new candidates. We used a novel human in vitro whole-blood model, which measures immune responses to mycobacteria by use of reporter gene-tagged BCG (BCGlux), to study immune responses to BCG vaccination in 50 neonates in a setting in Cape Town, Republic of South Africa, where tuberculosis is endemic. BCG vaccination significantly reduced growth of BCGluxin whole blood (prevaccination median growth ratio [GR], 9.6; range, 1.3 to 24; postvaccination median GR, 3.9; range, 0.6 to 12.2 [P< 0.0001]). Growth of BCGluxwas better restricted in vaccinated infants than in unvaccinated age-matched controls (n= 4). BCG vaccination induced significantly higher gamma interferon production in response to BCGlux(P< 0.0001) and to purified protein derivative (P= 0.0001). No significant changes in either growth of BCGluxor cytokine production occurred in an adult control group (n= 6) over the study period. The whole-blood luminescence model detects changes in cellular immune responses to mycobacteria induced by BCG vaccination. It is therefore a useful new tool in studying the immunogenicity of newly developed vaccine candidates prior to large field trials assessing efficacy.
Gonococcal Porin IB Activates NF-κB in Human Urethral Epithelium and Increases the Expression of Host Antiapoptotic Factors
Authors: Binnicker Matthew J. , Williams Richard D. , Apicella Michael A.
Published: October 2004

Infection of human urethral epithelial cells (UECs) with
Neisseria gonorrhoeae
increases the transcription of several host antiapoptotic genes, including
bfl-1
,
cox-2
, and
c-IAP-2
. In order to identify the bacterial factor(s) responsible for eliciting these changes, the transcriptional status of apoptotic machinery was monitored in UECs challenged with certain gonococcal membrane components. Initially, we observed that infection of UECs with gentamicin-killed gonococci increased the expression of the antiapoptotic Bcl-2 family member,
bfl-1
. This observation indicated that viable, replicating bacteria are not required for induction of antiapoptotic gene expression. Confirming this observation, treatment of UECs with purified gonococcal membrane increased the expression of
bfl-1
,
cox-2
, and
c-IAP-2
. This finding suggested that a factor or multiple factors present in the outer membrane (OM) are responsible for altering UEC antiapoptotic gene expression. Interestingly, treatment of UECs with gonococcal porin IB (PorB IB), a major constituent of the OM, significantly increased the transcription of
bfl-1
,
cox-2
, and
c-IAP-2
. The upregulation of these genes by PorB IB was determined to be dependent on NF-κB activation, as inhibiting NF-κB blocked induced expression of these genes. This work demonstrates the altered expression of host apoptotic factors in response to gonococcal PorB IB and supports a model whereby UEC cell death may be modulated as a potential mechanism of bacterial survival and proliferation.
Identification of a Hemin Utilization Protein of
Moraxella catarrhalis
(HumA)
Authors: Furano Kristin , Campagnari Anthony A.
Published: October 2004

Moraxella catarrhalis
is a major cause of acute otitis media in young children and has also been implicated as an important cause of exacerbations in adults with underlying pulmonary disease. Due to the considerable level of antibiotic resistance and the high degree of carriage rates in young children, it is likely that the incidence of
M. catarrhalis
infections will continue to rise.
M. catarrhalis
is a strict human respiratory pathogen, and this bacterium uses both transferrin and lactoferrin receptors to fulfill the essential iron requirement for survival in vivo. However, these are the only described iron acquisition systems for this organism. In this report we have demonstrated that
M. catarrhalis
can also utilize hemin as a sole source of iron for growth. In addition, we have identified and characterized an outer membrane protein with homology (26 to 28% similarity) to other known hemin binding and uptake proteins in related gram-negative organisms (i.e.,
Bordetella
and
Yersinia
spp.). This newly described
M. catarrhalis
protein, termed HumA, is capable of directly binding to hemin coupled to a solid-phase matrix.
M. catarrhalis
HumA expressed on the surface of an
Escherichia coli hemA
-deficient strain (K-12 EB53) is fully capable of complementing the defect and thus restoring the ability of this strain to grow in the presence of hemin. When
M. catarrhalis
is grown in the presence of hemin, HumA expression is clearly increased as shown by Western blotting with polyclonal antiserum developed against a HumA peptide. In addition, growth analyses revealed that a HumA-deficient mutant of
M. catarrhalis
(7169::
humA
) is restricted for growth in the presence of hemin as the sole iron source compared to the wild-type strain. We conclude that HumA is an essential component of a hemin uptake and utilization system previously undescribed for
M. catarrhalis
, thus providing another mechanism of iron acquisition that may facilitate persistent colonization of the mucosal surface.
RpoS Is Not Central to the General Stress Response inBorrelia burgdorferibut Does Control Expression of One or More Essential Virulence Determinants
Authors: Caimano Melissa J. , Eggers Christian H. , Hazlett Karsten R. O. , Radolf Justin D.
Published: October 2004

Borrelia burgdorferi, the Lyme disease spirochete, undergoes dramatic changes in antigenic composition as it cycles between its arthropod and mammalian hosts. A growing body of evidence suggests that these changes reflect, at least in part, the need for spirochetes to adapt to the physiological stresses imposed by abrupt changes in environmental conditions and nutrient availability. In many microorganisms, global responses are mediated by master regulators such as alternative sigma factors, withEscherichia coliRpoS (σS) serving as a prototype. The importance of this transcriptional activator in other bacteria, coupled with the report by Hübner et al. (A. Hübner, X. Yang, D. M. Nolen, T. G. Popova, F. C. Cabello, and M. V. Norgard, Proc. Natl. Acad. Sci. USA98:12724-12729, 2001) demonstrating that the borrelial RpoS ortholog controls expression of OspC and decorin-binding protein A (DbpA), prompted us to examine more closely the roles of RpoS-dependent and -independent differential gene expression in physiological adaptation by the Lyme disease spirochete. We observed thatB. burgdorferi rpoS(rpoSBb) was induced following temperature shift and transcript levels were further enhanced by reduced pH (pH 6.8). Using quantitative real-time reverse transcription-PCR (RT-PCR), we demonstrated that, in contrast to its ortholog (rpoSEc) inEscherichia coli,rpoSBbwas expressed at significant levels inB. burgdorferithroughout all phases of growth following temperature shift. By comparing aB. burgdorferistrain 297rpoSBbmutant to its wild-type counterpart, we determined that RpoSBbwas not required for survival following exposure to a wide range of environmental stresses (i.e., temperature shift, serum starvation, increased osmolality, reactive oxygen intermediates, and increased or reduced oxygen tension), although the mutant was more sensitive to extremes of pH. WhileB. burgdorferistrains lacking RpoS were able to survive within intraperitoneal dialysis membrane chambers at a level equivalent to that of the wild type, they were avirulent in mice. Lastly, RT-PCR analysis of theospE-ospF-elpparalogous lipoprotein families complements earlier findings that many temperature-inducible borrelial loci are controlled in an RpoSBb-independent manner. Together, these data point to fundamental differences between the role(s) of RpoS inB. burgdorferiand that inE. coli. Rather than functioning as a master regulator, RpoSBbappears to serve as a stress-responsive activator of a subset of virulence determinants that, together with the RpoS-independent, differentially expressed regulon, encompass the spirochete's genetic programs required for mammalian host adaptation.
Differential Immune Modulatory Activity of
Pseudomonas aeruginosa
Quorum-Sensing Signal Molecules
Authors: Hooi Doreen S. W. , Bycroft Barrie W. , Chhabra Siri Ram , Williams Paul , Pritchard David I.
Published: October 2004

Pseudomonas aeruginosa
releases a spectrum of well-regulated virulence factors, controlled by intercellular communication (quorum sensing) and mediated through the production of small diffusible quorum-sensing signal molecules (QSSM). We hypothesize that QSSM may in fact serve a dual purpose, also allowing bacterial colonization via their intrinsic immune-modulatory capacity. One class of signal molecule, the
N
-acylhomoserine lactones, has pleiotropic effects on eukaryotic cells, particularly those involved in host immunity. In the present study, we have determined the comparative effects of two chemically distinct and endobronchially detectable QSSM,
N
-(3-oxododecanoyl)-
l
-homoserine lactone (3-oxo-C
12
-HSL) and 2-heptyl-3-hydroxy-4 (1
H
)-quinolone or the
Pseudomonas
quinolone signal (PQS), on human leukocytes exposed to a series of stimuli designed to detect differential immunological activity in vitro. 3-Oxo-C
12
-HSL and PQS displayed differential effects on the release of interleukin-2 (IL-2) when human T cells were activated via the T-cell receptor and CD28 (a costimulatory molecule). 3-Oxo-C
12
-HSL inhibited cell proliferation and IL-2 release; PQS inhibited cell proliferation without affecting IL-2 release. Both molecules inhibited cell proliferation and the release of IL-2 following mitogen stimulation. Furthermore, in the presence of
Escherichia coli
lipopolysaccharide, 3-oxo-C
12
-HSL inhibited tumor necrosis factor alpha release from human monocytes, as reported previously (K. Tateda et al., Infect. Immun. 64:37-43, 1996), whereas PQS did not inhibit in this assay. These data highlight the presence of two differentially active immune modulatory QSSM from
P. aeruginosa
, which are detectable endobronchially and may be active at the host/pathogen interface during infection with
P. aeruginosa
, should the bronchial airway lymphoid tissues prove to be accessible to QSSM.
Antibodies to the N-Terminal Block 2 ofPlasmodium falciparumMerozoite Surface Protein 1 Are Associated with Protection against Clinical Malaria
Authors: Cavanagh David R. , Dodoo Daniel , Hviid Lars , Kurtzhals Jørgen A. L. , Theander Thor G. , Akanmori Bartholomew D. , Polley Spencer , Conway David J. , Koram Kojo , McBride Jana S.
Published: October 2004

This longitudinal prospective study shows that antibodies to the N-terminal block 2 region of thePlasmodium falciparummerozoite surface protein 1 (MSP-1) are associated with protection against clinical malaria in an area of stable but seasonal malaria transmission of Ghana. Antibodies to the block 2 region of MSP-1 were measured in a cohort of 280 children before the beginning of the major malaria transmission season. The cohort was then actively monitored for malaria, clinically and parasitologically, over a period of 17 months. Evidence is presented for an association between antibody responses to block 2 and a significantly reduced risk of subsequent clinical malaria. Furthermore, statistical survival analysis provides new information on the duration of the effect over time. The results support a conclusion that the block 2 region of MSP-1 is a target of protective immunity againstP. falciparumand, thus, a promising new candidate for the development of a malaria vaccine.
Phase I Testing of a Malaria Vaccine Composed of Hepatitis B Virus Core Particles ExpressingPlasmodium falciparumCircumsporozoite Epitopes
Authors: Nardin Elizabeth H. , Oliveira Giane A. , Calvo-Calle J. Mauricio , Wetzel Kristiane , Maier Carolin , Birkett Ashley J. , Sarpotdar Pramod , Corado Michael L. , Thornton George B. , Schmidt Annette
Published: October 2004

We report the first phase I trial to assess the safety and immunogenicity of a malaria vaccine candidate, ICC-1132 (Malarivax), composed of a modified hepatitis B virus core protein (HBc) containing minimal epitopes of thePlasmodium falciparumcircumsporozoite (CS) protein. When expressed inEscherichia coli, the recombinant ICC-1132 protein forms virus-like particles that were found to be highly immunogenic in preclinical studies of mice and monkeys. Twenty healthy adult volunteers received a 20- or a 50-μg dose of alum-adsorbed ICC-1132 administered intramuscularly at 0, 2, and 6 months. The majority of volunteers in the group receiving the 50-μg dose developed antibodies to CS repeats as well as to HBc. Malaria-specific T cells that secreted gamma interferon were also detected after a single immunization with ICC-1132-alum. These studies support ICC-1132 as a promising malaria vaccine candidate for further clinical testing using more-potent adjuvant formulations and confirm the potential of modified HBc virus-like particles as a delivery platform for vaccines against other human pathogens.
The
Streptococcus gordonii
Surface Proteins GspB and Hsa Mediate Binding to Sialylated Carbohydrate Epitopes on the Platelet Membrane Glycoprotein Ibα
Authors: Bensing Barbara A. , López José A. , Sullam Paul M.
Published: October 2004

Platelet binding by
Streptococcus gordonii
strain M99 is dependent on expression of the cell wall-anchored glycoprotein GspB. This large cell surface protein is exported from the M99 cytoplasm via a dedicated transport system that includes SecA2 and SecY2. GspB is highly similar to Hsa, a protein expressed by
S. gordonii
Challis that has been characterized as a sialic acid binding hemagglutinin. In this study, we compared the contribution of GspB and Hsa to the adherence of
S. gordonii
to selected glycoproteins. Our results indicate that GspB can mediate binding to a variety of sialylated glycoproteins. GspB facilitates binding to carbohydrates bearing sialic acid in either α(2-3) or α(2-6) linkages, with a slight preference for α(2-3) linkages. Furthermore, GspB readily mediates binding to sialic acid residues on immobilized glycocalicin, the extracellular portion of the platelet membrane glycoprotein (GP) Ibα (the ligand binding subunit of the platelet von Willebrand factor receptor complex GPIb-IX-V). Although Hsa is required for the binding of
S. gordonii
Challis to sialic acid, most of the Hsa expressed by Challis is retained in the cytoplasm. The deficiency in export is due, at least in part, to a nonsense mutation in
secA2
. Hsa export can be enhanced by complementation with
secA2
from M99, which also results in significantly greater binding to sialylated glycoproteins, including glycocalicin. The combined results indicate that GspB and Hsa contribute similar binding capabilities to M99 and Challis, respectively, but there may be subtle differences in the preferred epitopes to which these adhesins bind.
Association of
iss
and
iucA
, but Not
tsh
, with Plasmid-Mediated Virulence of Avian Pathogenic
Escherichia coli
Authors: Tivendale Kelly A. , Allen Joanne L. , Ginns Carol A. , Crabb Brendan S. , Browning Glenn F.
Published: October 2004

Avian pathogenic
Escherichia coli
(APEC) is an economically important respiratory pathogen of chickens worldwide. Factors previously associated with the virulence of APEC include adhesins, iron-scavenging mechanisms, the production of colicin V (ColV), serum resistance, and temperature-sensitive hemagglutination, but virulence has generally been assessed by parenteral inoculation, which does not replicate the normal respiratory route of infection. A large plasmid, pVM01, is essential for virulence in APEC strain E3 in chickens after aerosol exposure. Here we establish the size of pVM01 to be approximately 160 kb and show that the putative virulence genes
iss
(increased serum survival) and
tsh
(temperature-sensitive hemagglutinin) and the aerobactin operon are on the plasmid. These genes were not clustered on pVM01 but, rather, were each located in quite distinct regions. Examination of APEC strains with defined levels of respiratory pathogenicity after aerosol exposure showed that both the aerobactin operon and
iss
were associated with high levels of virulence in APEC but that the possession of either gene was sufficient for intermediate levels of virulence. In constrast, the presence of
tsh
was not necessary for high levels of virulence. Thus, both the aerobactin operon and
iss
are associated with virulence in APEC after exposure by the natural route of infection. The similarities between APEC and extraintestinal
E. coli
infection in other species suggests that they may be useful models for definition of the role of these virulence genes and of other novel virulence genes that may be located on their virulence plasmids.
Tpr Homologs inTreponema paraluiscuniculiCuniculi A Strain
Authors: Giacani Lorenzo , Sun Eileen S. , Hevner Karin , Molini Barbara J. , Van Voorhis Wesley C. , Lukehart Sheila A. , Centurion-Lara Arturo
Published: October 2004

Treponema paraluiscuniculi, the etiologic agent of rabbit venereal syphilis, is morphologically indistinguishable fromTreponema pallidumsubsp.pallidum(T. pallidum), the human syphilis treponeme, and induces similar immune responses and histopathologic changes in the infected host. Because of their high degree of relatedness, comparative studies are likely to identify genetic determinants that contribute to pathogenesis or virulence in human syphilis. Thetpr(Treponema pallidumrepeat) genes are believed to code for potential virulence factors. In this study, we identified 10tprhomologs inTreponema paraluiscuniculiCuniculi A strain and determined their sequence architecture. Half of this group of paralogous genes were predicted to be nonfunctional due to the presence of frameshifts and premature stop codons. Furthermore, the immune response against theT. paraluiscuniculiTpr homologs in long-term-infected rabbits was studied by enzyme-linked immunosorbent assay and lymphocyte proliferation assay, showing that TprK is the only target of the antibody and T-cell responses during experimental infection and emphasizing the importance of this putative virulence factor in venereal treponematosis.
Effects of
vlsE
Complementation on the Infectivity of
Borrelia burgdorferi
Lacking the Linear Plasmid lp28-1
Authors: Lawrenz Matthew B. , Wooten R. Mark , Norris Steven J.
Published: October 2004

The loss of linear plasmid lp28-1, which contains the
vls
antigenic variation locus, is associated with reduced infectivity of
Borrelia burgdorferi
in immunocompetent mice. The recombinant shuttle vector pBBE22, which includes the virulence determinant BBE22 from lp25 and restores infectivity to readily transformable
B. burgdorferi
lacking lp25 and lp56, was used to determine the effect of
trans
expression of
vlsE
on virulence. Spirochetes lacking lp28-1 were complemented with the plasmid pBBE22:vlsE, containing both BBE22 and
vlsE
. VlsE protein produced by this construct was expressed and surface accessible in in vitro-cultured
B. burgdorferi
, as determined by surface proteolysis and immunoblot analysis. Clones lacking lp25 but containing lp28-1 and either pBBE22 or pBBE22:vlsE were reisolated consistently from immunocompetent mice 8 weeks after infection. In contrast, a clone lacking both lp25 and lp28-1 and complemented with pBBE22:vlsE was isolated from only a single tissue of one of six C3H/HeN mice 8 weeks postinfection. These results indicate that either an intact
vls
antigenic variation locus or another determinant on lp28-1 is required to restore complete infectivity. In addition, an isogenic clone that retained lp28-1 was complemented with the
vlsE
shuttle plasmid and was examined for
vlsE
sequence variation and infectivity. Sequence variation was not observed for the shuttle plasmid, indicating that the
cis
arrangement of
vlsE
and the
vls
silent cassettes in lp28-1 facilitate
vlsE
gene conversion. Lack of
vlsE
sequence variation on the shuttle plasmid thus did not result in clearance of the
trans
-complemented strain in immunocompetent mice under the conditions tested.
Distinct Trafficking and Localization of STEVOR Proteins in Three Stages of the
Plasmodium falciparum
Life Cycle
Authors: McRobert Louisa , Preiser Peter , Sharp Sarah , Jarra William , Kaviratne Mallika , Taylor Martin C. , Renia Laurent , Sutherland Colin J.
Published: October 2004

The genome of
Plasmodium falciparum
harbors three extensive multigene families,
var
,
rif
, and
stevor
(for subtelomeric variable open reading frame), located mainly in the subtelomeric regions of the parasite's 14 chromosomes. STEVOR variants are known to be expressed in asexual parasites, but no function has as yet been ascribed to this protein family. We have examined the expression of STEVOR proteins in intraerythrocytic sexual stages, gametocytes, and extracellular sporozoites isolated from infected
Anopheles
mosquitoes. In gametocytes,
stevor
transcripts appear transiently early in development but STEVOR proteins persist for several days and are transported out of the parasite, travel through the host cell cytoplasm, and localize to the erythrocyte plasma membrane. In contrast to asexual parasites, gametocytes move STEVOR to the periphery via a trafficking pathway independent of Maurer's clefts. In sporozoites, STEVOR appear dispersed throughout the cytoplasm in vesicle-like structures. The pattern of STEVOR localization we have observed in gametocytes and sporozoites differs significantly from that in asexual parasite stages. STEVOR variants are therefore likely to perform different functions in each stage of the parasites life cycle in which they occur.
Prolonged Toll-Like Receptor Signaling byMycobacterium tuberculosisand Its 19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Induced Regulation of Selected Genes in Macrophages
Authors: Pai Rish K. , Pennini Meghan E. , Tobian Aaron A. R. , Canaday David H. , Boom W. Henry , Harding Clifford V.
Published: October 2004

Infection of macrophages withMycobacterium tuberculosisor exposure toM. tuberculosis19-kDa lipoprotein for >16 h inhibits gamma interferon (IFN-γ)-induced major histocompatibility complex class II (MHC-II) expression by a mechanism involving Toll-like receptors (TLRs).M. tuberculosiswas found to inhibit murine macrophage MHC-II antigen (Ag) processing activity induced by IFN-γ but not by interleukin-4 (IL-4), suggesting inhibition of IFN-γ-induced gene regulation. We designed an approach to test the ability ofM. tuberculosis-infected cells to respond to IFN-γ. To model chronic infection withM. tuberculosiswith accompanying prolonged TLR signaling, macrophages were infected withM. tuberculosisor incubated withM. tuberculosis19-kDa lipoprotein for 24 h prior to the addition of IFN-γ. Microarray gene expression studies were then used to determine whether prolonged TLR signaling byM. tuberculosisbroadly inhibits IFN-γ regulation of macrophage gene expression. Of 347 IFN-γ-induced genes,M. tuberculosisand 19-kDa lipoprotein inhibited induction of 42 and 36%, respectively. Key genes or gene products were also examined by quantitative reverse transcription-PCR and flow cytometry, confirming and extending the results obtained by microarray studies.M. tuberculosisinhibited IFN-γ induction of genes involved in MHC-II Ag processing, Ag presentation, and recruitment of T cells. These effects were largely dependent on myeloid differentiation factor 88, implying a role for TLRs. Thus, prolonged TLR signaling byM. tuberculosisinhibits certain macrophage responses to IFN-γ, particularly those related to MHC-II Ag presentation. This inhibition may promoteM. tuberculosisevasion of T-cell responses and persistence of infection in tuberculosis.
Toll-Like Receptor 4-Dependent Early Elicited Tumor Necrosis Factor Alpha Expression Is Critical for Innate Host Defense againstBordetella bronchiseptica
Authors: Mann Paul B. , Elder Kelly D. , Kennett Mary J. , Harvill Eric T.
Published: October 2004

Toll-like receptor 4 (TLR4) mediates the response to lipopolysaccharide, and its activation induces the expression of a large number of inflammatory genes, many of which are also induced by other pathogen-associated molecular patterns. Interestingly, the subset of genes that are dependent on TLR4 for optimal expression during gram-negative bacterial infection has not been determined. We have previously shown that TLR4-deficient mice rapidly develop acute pneumonia after inoculation withBordetella bronchiseptica, suggesting that TLR4 is required for expression of early elicited gene products in this model. Microarray analysis with macrophages derived from wild-type and TLR4-deficient mice was used to identify genes whose expression, within 1 h of bacterial exposure, is dependent on TLR4. The results of this investigation suggest that TLR4 is not required for the majority of the transcriptional response toB. bronchiseptica. However, early tumor necrosis factor alpha (TNF-α) mRNA expression is primarily dependent on TLR4 and in vitro and in vivo protein levels substantiate this finding. TLR4-deficient mice and TNF-α−/−mice are similarly susceptible to infection with relatively low doses ofB. bronchisepticaand in vivo neutralization studies indicate that it is the TLR4-dependent early elicited TNF-α response that is critical for preventing severe pneumonia and limiting bacterial growth. These results suggest that one critical role for TLR4 is the generation of a robust but transient TNF-α response that is critical to innate host defense during acute gram-negative respiratory infection.
Mucosal Administration of Flagellin Induces Innate Immunity in the Mouse Lung
Authors: Honko Anna N. , Mizel Steven B.
Published: October 2004

Nonsurgical intratracheal instillation of 1 μg of purified, recombinant flagellin in several strains of mice stimulated a transient innate immune response in the lung characterized by the infiltration of neutrophils and the rapid production of tumor necrosis factor alpha, interleukin 6, granulocyte colony-stimulating factor, and the chemokines keratinocyte-derived chemokine, MIP1α, and MIP-2.
LcrV Synthesis Is Altered by DNA Adenine Methylase Overproduction in
Yersinia pseudotuberculosis
and Is Required To Confer Immunity in Vaccinated Hosts
Authors: Badie Golnaz , Heithoff Douglas M. , Mahan Michael J.
Published: October 2004

Yersinia pseudotuberculosis
mutants that overproduce the DNA adenine methylase (Dam
OP
Yersinia
) are attenuated, confer robust protective immune responses, and synthesize or secrete several
Yersinia
outer proteins (Yops) under conditions that are nonpermissive for synthesis and secretion in wild-type strains. To understand the molecular basis of immunity elicited by Dam
OP
Yersinia
, we investigated the effects of Dam overproduction on the synthesis and localization of a principal
Yersinia
immunogen, LcrV, a low-calcium-responsive virulence factor involved in Yop synthesis, localization, and suppression of host inflammatory activities. Dam overproduction relaxed the stringent temperature and calcium regulation of LcrV synthesis. Moreover, the LcrV-dependent synthesis and localization of the actin cytotoxin, YopE, were shown to be relaxed in Dam
OP
cells, suggesting that the synthesis and localization of Yops can occur via both LcrV-dependent and -independent mechanisms. Last, the immunity conferred by Dam
OP
Yersinia
was strictly dependent on the presence of LcrV, which may result from its role (i) as an immunogen, (ii) as an immunomodulator of host anti-inflammatory activities, or (iii) in the altered synthesis and localization of Yops that could contribute to immunogen repertoire expansion.
Comparative Analysis of Locus of Enterocyte Effacement Pathogenicity Islands of Atypical Enteropathogenic
Escherichia coli
Authors: Gärtner Julia F. , Schmidt M. Alexander
Published: October 2004

The pathogenicity of enteropathogenic
Escherichia coli
(EPEC) is linked to the locus of enterocyte effacement, or LEE, encoding a type III secretion system (T3SS) that directly transfers bacterial effector proteins into eukaryotic cells. Atypical diffusely adhering EPEC (DA-EPEC) strains that harbor homologues of the LEE but lack the EPEC adherence factor plasmid have been increasingly associated with outbreaks of diarrhea. In this study, we have completely sequenced and functionally characterized LEE pathogenicity islands derived from the clinical DA-EPEC isolates 3431 (O8:H
−
) and 0181 (O119:H9:K61). LEE
3431
and LEE
0181
exhibit genetic organization analogous to that of the prototype LEE
E2348/69
. Genes constituting the T3SS apparatus are highly conserved. However, LEE-encoded effector proteins exhibit major differences. Transfer and functional expression of LEE
0181
in an
E. coli
XL1 blue MR background demonstrated that LEE
0181
contains all the information for signal transduction and pedestal formation.
Lack of Stage-Specific Proteins in Coccoid
Helicobacter pylori
Cells
Authors: Bumann Dirk , Habibi Hajar , Kan Biao , Schmid Monika , Goosmann Christian , Brinkmann Volker , Meyer Thomas F. , Jungblut Peter R.
Published: October 2004

Helicobacter pylori
exists in two distinct forms, rod shaped or coccoid, in stomachs of infected patients. Based on in vitro proteome comparisons, there are no detectable coccoid-specific proteins, which argues against the specific adaptation of coccoid
Helicobacter
to distinct biological functions, such as enhanced persistence or transmission to other hosts.
The Cauliflower Mosaic Virus 35S Promoter Extends into the Transcribed Region
Authors: Pauli Sandra , Rothnie Helen M. , Chen Gang , He Xiaoyuan , Hohn Thomas
Published: October 2004

A 60-nucleotide region (S1) downstream of the transcription start site of the cauliflower mosaic virus 35S RNA can enhance gene expression. By using transient expression assays with plant protoplasts, this activity was shown to be at least partially due to the effect of transcriptional enhancers within this region. We identify sequence motifs with enhancer function, which are normally masked by the powerful upstream enhancers of the 35S promoter. A repeated CT-rich motif is involved both in enhancer function and in interaction with plant nuclear proteins. The S1 region can also enhance expression from heterologous promoters.
Expression and Localization of the Epstein-Barr Virus-Encoded Protein Kinase
Authors: Gershburg E. , Marschall M. , Hong K. , Pagano J. S.
Published: October 2004

The protein kinase (PK) encoded by the Epstein-Barr Virus (EBV) BGLF4 gene is the only EBV protein kinase. The expression pattern of EBV PK during the reactivation of the viral lytic cycle and the subcellular localization of the protein were analyzed with a polyclonal antiserum raised against a peptide corresponding to the N terminus of EBV PK. Based on previously published data (E. Gershburg and J. S. Pagano, J. Virol.
76:
998-1003, 2002) and the expression pattern described here, we conclude that EBV PK is an early protein that requires viral-DNA replication for maximum expression. By biochemical fractionation, the protein could be detected mainly in the nuclear fraction 4 h after viral reactivation in Akata cells. Nuclear localization could be visualized by indirect immunofluorescence in HeLa cells transiently expressing EBV BGLF4 in the absence of other viral products. Transient expression of 3′-terminal deletion mutants of EBV BGLF4 resulted in cytoplasmic localization, confirming the presence of a nuclear localization site in the C-terminal region of the protein. In contrast to the wild-type EBV PK, all of the mutants were unable to hyperphosphorylate EA-D during coexpression or to phosphorylate ganciclovir, as measured by an in-cell activity assay. Thus, the results demonstrate that the nuclear localization, as well as the kinase activity, of BGFL4 is dependent on an intact C-terminal region.
A Retroviral Promoter and a Cellular Enhancer Define a Bipartite Element Which Controls
env
ERVWE1 Placental Expression
Authors: Prudhomme Sarah , Oriol Guy , Mallet François
Published: October 2004

The HERV-W family contains hundreds of loci diversely expressed in several physiological and pathological contexts. A unique locus termed ERVWE1 encodes an envelope glycoprotein (syncytin) involved in hominoid placental physiology. Here we show that syncytin expression is regulated by a bipartite element consisting of a cyclic AMP (cAMP)-inducible long terminal repeat (LTR) retroviral promoter adjacent to a cellular enhancer conferring a high level of expression and placental tropism. Deletion mutant analysis showed that the ERVWE1 5′ LTR contains binding sites essential for basal placental activity in the region from positions +1 to +125. The region from positions +125 to +310 represents a cAMP-responsive core HERV-W promoter active in all cell types. Site-directed mutagenesis analysis highlighted the complexity of U3 regulation. ERVWE1 placenta-specific positive (e.g., T240) and negative (e.g., G71) regulatory sites were identified, as were essential sites required for basic activity (e.g., A247). The flanking sequences of the ERVWE1 provirus contain several putative regulatory elements. The upstream HERV-H and HERV-P LTRs were found to be inactive. Conversely, the 436-bp region located between the HERV-P LTR and ERVWE1 was shown to be an upstream regulatory element (URE) which is significantly active in placenta cells. This URE acts as a tissue-specific enhancer. Genetic and functional analyses of hominoid UREs revealed large differences between UREs of members of the Hominidae and the Hylobatidae. These data allowed the identification of a positive regulatory region from positions −436 to −128, a mammalian apparent LTR retrotransposon negative regulatory region from positions −128 to −67, and a trophoblast-specific enhancer (TSE) from positions −67 to −35. Putative AP-2, Sp-1, and GCMa binding sites are essential constituents of the 33-bp TSE.
De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities
Authors: Ranjith-Kumar C. T. , Sarisky R. T. , Gutshall L. , Thomson M. , Kao C. C.
Published: October 2004

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol.
76:
3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a

K
m

for GTP of 3.5 μM, all of the mutations except one had

K
m

s that were three- to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.
Conditionally Replicating Adenoviruses Kill Tumor Cells via a Basic Apoptotic Machinery-Independent Mechanism That Resembles Necrosis-Like Programmed Cell Death
Authors: Abou El Hassan Mohamed A. I. , van der Meulen-Muileman Ida , Abbas Saman , Kruyt Frank A. E.
Published: October 2004

Conditionally replicating adenoviruses (CRAds) represent a promising class of novel anticancer agents that are used for virotherapy. The E1AΔ24 mutation-based viruses, Ad5-
Δ
24 [CRAd(E3−); E3 region deleted] and infectivity-enhanced Ad5-
Δ
24RGD [CRAd(E3+)] have been shown to potently eradicate tumor cells. The presence of the E3 region in the latter virus is known to improve cell killing that can be attributed to the presence of the oncolysis-enhancing Ad death protein. The more precise mechanism by which CRAds kill tumor cells is unclear, and the role of the host cell apoptotic machinery in this process has been addressed only in a limited way. Here, we examine the role of several major apoptotic pathways in the CRAd-induced killing of non-small-cell lung cancer H460 cells. As expected, CRAd(E3+) was more potent than CRAd(E3−). No evidence for the involvement of the p53-Bax apoptotic pathway was found. Western blot analyses demonstrated strong suppression of p53 expression and unchanged Bax levels during viral replication, and stable overexpression of human papillomavirus type 16-E6 in H460 cells did not affect killing by both CRAds. CRAd activity was also not hampered by stable overexpression of anti-apoptotic Bcl2 or BclXL, and endogenous Bcl2/BclXL protein levels remained constant during the oncolytic cycle. Some evidence for caspase processing was obtained at late time points after infection; however, the inhibition of caspases by the X-linked inhibitor of apoptosis protein overexpression or cotreatment with zVAD-fmk did not inhibit CRAd-dependent cell death. Analyses of several apoptotic features revealed no evidence for nuclear fragmentation or DNA laddering, although phosphatidylserine externalization was detected. We conclude that despite the known apoptosis-modulating abilities of individual Ad proteins, Ad5-
Δ
24-based CRAds trigger necrosis-like cell death. In addition, we propose that deregulated apoptosis in cancer cells, a possible drug resistance mechanism, provides no barrier for CRAd efficacy.
Multivesicular Bodies as a Platform for Formation of the Marburg Virus Envelope
Authors: Kolesnikova Larissa , Berghöfer Beate , Bamberg Sandra , Becker Stephan
Published: October 2004

The Marburg virus (MARV) envelope consists of a lipid membrane and two major proteins, the matrix protein VP40 and the glycoprotein GP. Both proteins use different intracellular transport pathways: GP utilizes the exocytotic pathway, while VP40 is transported through the retrograde late endosomal pathway. It is currently unknown where the proteins combine to form the viral envelope. In the present study, we identified the intracellular site where the two major envelope proteins of MARV come together as peripheral multivesicular bodies (MVBs). Upon coexpression with VP40, GP is redistributed from the trans-Golgi network into the VP40-containing MVBs. Ultrastructural analysis of MVBs suggested that they provide the platform for the formation of membrane structures that bud as virus-like particles from the cell surface. The virus-like particles contain both VP40 and GP. Single expression of GP also resulted in the release of particles, which are round or pleomorphic. Single expression of VP40 led to the release of filamentous structures that closely resemble viral particles and contain traces of endosomal marker proteins. This finding indicated a central role of VP40 in the formation of the filamentous structure of MARV particles, which is similar to the role of the related Ebola virusVP40. In MARV-infected cells, VP40 and GP are colocalized in peripheral MVBs as well. Moreover, intracellular budding of progeny virions into MVBs was frequently detected. Taken together, these results demonstrate an intracellular intersection between GP and VP40 pathways and suggest a crucial role of the late endosomal compartment for the formation of the viral envelope.