bluuebunny/crossref_metadata_2025 huggingface dataset:
Multilevel Control of Competence Development and Stress Tolerance in
Streptococcus mutans
UA159
Authors: Ahn Sang-Joon , Wen Zezhang T. , Burne Robert A.
Published: February 2006

Genetic competence appears to be important in establishment of biofilms and tolerance of environmental insults. We report here that the development of competence is controlled at multiple levels in a complex network that includes two signal-transducing two-component systems (TCS). Using
Streptococcus mutans
strain UA159, we demonstrate that the histidine kinase CiaH, but not the response regulator CiaR, causes a dramatic decrease in biofilm formation and in transformation efficiency. Inactivation of
comE
or
comD
had no effect on stress tolerance, but transformability of the mutants was poor and was not restored by addition of competence-stimulating peptide (CSP). Horse serum (HS) or bovine serum albumin (BSA) had no impact on transformability of any strains. Interestingly, though, the presence of HS or BSA in combination with CSP was required for efficient induction of
comD
,
comX
, and
comYA
, and induction was dependent on ComDE and CiaH, but not CiaR. Inactivation of
comC
, encoding CSP, had no impact on transformation, and CiaH was shown to be required for optimal
comC
expression. This study reveals that
S. mutans
integrates multiple environmental signals through CiaHR and ComDE to coordinate induction of
com
genes and that CiaH can exert its influence through CiaR and as-yet-unidentified regulators. The results highlight critical differences in the role and regulation of CiaRH and
com
genes in different
S. mutans
isolates and between
S. mutans
and
Streptococcus pneumoniae
, indicating that substantial divergence in the role and regulation of TCS and competence genes has occurred in streptococci.
Silencing of the Laminin γ-1 Gene Blocks
Trypanosoma cruzi
Infection
Authors: Nde Pius N. , Simmons Kaneatra J. , Kleshchenko Yuliya Y. , Pratap Siddharth , Lima Maria F. , Villalta Fernando
Published: February 2006

It is thought that
Trypanosoma cruzi
, the protozoan that causes Chagas' disease, modulates the extracellular matrix network to facilitate infection of human cells. However, direct evidence to document this phenomenon is lacking. Here we show that the
T. cruzi
gp83 ligand, a cell surface
trans
-sialidase-like molecule that the parasite uses to attach to host cells, increases the level of laminin γ-1 transcript and its expression in mammalian cells, leading to an increase in cellular infection. Stable RNA interference (RNAi) with host cell laminin γ-1 knocks down the levels of laminin γ-1 transcript and protein expression in mammalian cells, causing a dramatic reduction in cellular infection by
T. cruzi
. Thus, host laminin γ-1, which is regulated by the parasite, plays a crucial role in the early process of infection. This is the first report showing that knocking down the expression of a human gene by RNAi inhibits the infection of an intracellular parasite.
Salmonella enterica
Highly Expressed Genes Are Disease Specific
Authors: Rollenhagen Claudia , Bumann Dirk
Published: February 2006

During in vitro broth culture, bacterial gene expression is typically dominated by highly expressed factors involved in protein biosynthesis, maturation, and folding, but it is unclear if this also applies to conditions in natural environments. Here, we used a promoter trap strategy with an unstable green fluorescent protein reporter that can be detected in infected mouse tissues to identify 21
Salmonella enterica
promoters with high levels of activity in a mouse enteritis model. We then measured the activities of these and 31 previously identified
Salmonella
promoters in both the enteritis and a murine typhoid fever model. Surprisingly, the data reveal that instead of protein biosynthesis genes, disease-specific genes such as
Salmonella
pathogenicity island 1 (SPI-1)-associated genes and genes involved in anaerobic respiration (enteritis) or SPI-2-associated genes and genes of the PhoP regulon (typhoid fever), respectively, dominate
Salmonella
in vivo gene expression. The overall functional profile of highly expressed genes suggests a marked shift in major transcriptional activities to nutrient utilization during enteritis or to fighting against the host during typhoid fever. The large proportion of known and novel essential virulence factors among the identified genes suggests that high expression levels during infection may correlate with functional relevance.
Quorum Quenching by anN-Acyl-Homoserine Lactone Acylase fromPseudomonas aeruginosaPAO1
Authors: Sio Charles F. , Otten Linda G. , Cool Robbert H. , Diggle Stephen P. , Braun Peter G. , Bos Rein , Daykin Mavis , Cámara Miguel , Williams Paul , Quax Wim J.
Published: February 2006

The virulence of the opportunistic human pathogenPseudomonas aeruginosaPAO1 is controlled by anN-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in theP. aeruginosaPAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of theN-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3′ position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules ofP. aeruginosaPAO1,N-butanoyl-l-homoserine lactone (C4-HSL) andN-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein toP. aeruginosaPAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed inP. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enableP. aeruginosaPAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.
Protection against HeterologousBurkholderia pseudomalleiStrains by Dendritic Cell Immunization
Authors: Elvin Stephen J. , Healey Gareth D. , Westwood Angie , Knight Stella C. , Eyles James E. , Williamson E. Diane
Published: February 2006

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium which can cause either chronic infections or acute lethal sepsis in infected individuals. The disease is endemic in Southeast Asia and northern Australia, but little is known about the mechanisms of protective immunity to the bacterium. In this study, we have developed a procedure to utilize dendritic cells in combination with CpG oligodeoxynucleotides as a vaccine delivery vector to induce protective immune responses to various strains ofB. pseudomallei. Our results show that strong cell-mediated immune responses were generated, while antibody responses, although low, were detectable. Upon virulent challenge withB. pseudomalleistrain K96243, NCTC 4845, or 576, animals immunized with dendritic cells that were pulsed with heat-killed K96243 and matured in the presence of CpG 1826 showed significant levels of protection. These results show that a vaccine strategy that actively targets dendritic cells can evoke protective immune responses.
Endoproteolytic Processing of RhoA by Rce1 Is Required for the Cleavage of RhoA by
Yersinia enterocolitica
Outer Protein T
Authors: Fueller Florian , Bergo Martin O. , Young Stephen G. , Aktories Klaus , Schmidt Gudula
Published: February 2006

The bacterial toxin
Yersinia
outer protein T (YopT) is a cysteine protease that cleaves Rho GTPases immediately upstream of a carboxyl-terminal isoprenylcysteine. By clipping off the lipid anchor, YopT releases Rho GTPases from membranes, resulting in rounding up of mammalian cells in culture. The proteolytic activity of YopT depends on the isoprenylation of the cysteine within the carboxyl-terminal CaaX motif, a reaction carried out by geranylgeranyltransferase type I. The CaaX motif (where “a” indicates aliphatic amino acids) of Rho proteins undergoes two additional processing steps: endoproteolytic removal of the last three amino acids (i.e., -aaX) by Rce1 (Ras-converting enzyme 1) and methylation of the geranylgeranylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). In in vitro experiments, RhoA retaining -aaX cannot be cleaved by YopT. Nothing is known, however, about the influence of Rce1-mediated removal of -aaX on the activity of YopT in living cells. We hypothesized that Rce1-deficient mouse fibroblasts, in which the geranylgeranylated Rho proteins are not endoproteolytically processed, would be resistant to YopT. Indeed, this was the case. Microinjection of recombinant YopT into Rce1-deficient fibroblasts had no impact on the subcellular localization of RhoA and no impact on cell morphology. To determine if carboxyl methylation is also required for YopT action, we microinjected YopT into
Icmt
-deficient fibroblasts. In contrast to the results with
Rce1
-deficient cells, YopT cleaved RhoA and caused rounding up of the
Icmt
-deficient cells. Our data demonstrate that
Rce1
-mediated removal of -aaX from isoprenylated Rho GTPases is required for the proteolytic activity of YopT in living cells, whereas carboxyl methylation by Icmt is not.
Identification of a Meningococcall-Glutamate ABC Transporter Operon Essential for Growth in Low-Sodium Environments
Authors: Monaco Caterina , Talà Adelfia , Spinosa Maria Rita , Progida Cinzia , De Nitto Eleanna , Gaballo Antonio , Bruni Carmelo B. , Bucci Cecilia , Alifano Pietro
Published: February 2006

GdhR is a meningococcal transcriptional regulator that was previously shown to positively control the expression ofgdhA, encoding the NADP-specificl-glutamate dehydrogenase (NADP-GDH), in response to the growth phase and/or to the carbon source. In this study we used reverse transcriptase-PCR-differential display (to identify additional GdhR-regulated genes. The results indicated that GdhR, in addition to NADP-GDH, controls the expression of a number of genes involved in glucose catabolism by the Entner-Doudoroff pathway and inl-glutamate import by an unknown ABC transport system. The genes encoding the putative periplasmic substrate-binding protein (NMB1963) and the permease (NMB1965) of the ABC transporter were genetically inactivated. Uptake experiments demonstrated an impairment ofl-glutamate import in the NMB1965-defective mutant in the absence or in the presence of a low sodium ion concentration. In contrast, at a sodium ion concentration above 60 mM, the uptake defect disappeared, possibly because the activity of a sodium-driven secondary transporter became predominant. Indeed, the NMB1965-defective mutant was unable to grow at a low sodium ion concentration (<20 mM) in a chemically defined medium containingl-glutamate and four other amino acids that supported meningococcal growth, but it grew when the sodium ion concentration was raised to higher values (>60 mM). The same growth phenotype was observed in the NMB1963-defective mutant. Cell invasion and intracellular persistence assays and expression data during cell invasion provided evidence that thel-glutamate ABC transporter, tentatively named GltT, was critical for meningococcal adaptation in the low-sodium intracellular environment.
Immunoprotection of Recombinant Leptospiral Immunoglobulin-Like Protein A against
Leptospira interrogans
Serovar Pomona Infection
Authors: Palaniappan Raghavan U. M. , McDonough Sean P. , Divers Thomas J. , Chen Chia-Sui , Pan Ming-Jeng , Matsumoto Mitsuharu , Chang Yung-Fu
Published: February 2006

We previously reported the cloning and characterization of leptospiral immunoglobulin-like proteins LigA and LigB of
Leptospira interrogans
. LigA and LigB are conserved at the amino-terminal region but are variable at the carboxyl-terminal region. Here, we evaluate the potential of recombinant LigA (rLigA) as a vaccine candidate against infection by
L. interrogans
serovar Pomona in a hamster model. rLigA was truncated into conserved (rLigAcon) and variable (rLigAvar) regions and expressed in
Escherichia coli
as a fusion protein with glutathione-
S
-transferase (rLigA). Golden Syrian hamsters were immunized at 3 and 6 weeks of age with rLigA (rLigAcon and rLigAvar) with aluminum hydroxide as an adjuvant. Hamsters given recombinant glutathione-
S
-transferase (rGST)-adjuvant and phosphate-buffered saline-adjuvant served as nonvaccinated controls. Three weeks after the last vaccination, all animals were challenged intraperitoneally with 10
8
L. interrogans
serovar Pomona bacteria (NVSL 1427-35-093002). All hamsters immunized with recombinant LigA survived after challenge and had no significant histopathological changes. In contrast, nonimmunized and rGST-immunized hamsters were subjected to lethal doses, and the hamsters that survived showed severe tubulointerstitial nephritis. All vaccinated animals showed a rise in antibody titers against rLigA. Results from this study indicate that rLigA is a potential vaccine candidate against
L. interrogans
serovar Pomona infection.
Cytosolic Phospholipase A
2
Enzymes Are Not Required by Mouse Bone Marrow-Derived Macrophages for the Control of
Mycobacterium tuberculosis
In Vitro
Authors: Vandal Omar H. , Gelb Michael H. , Ehrt Sabine , Nathan Carl F.
Published: February 2006

During the course of infection
Mycobacterium tuberculosis
predominantly resides within macrophages, where it encounters and is often able to resist the antibacterial mechanisms of the host. In this study, we assessed the role of macrophage phospholipases A
2
(PLA
2
s) in defense against
M. tuberculosis
. Mouse bone marrow-derived macrophages (BMDMs) expressed cPLA
2
-IVA, cPLA
2
-IVB, iPLA
2
-VI, sPLA
2
-IIE, and sPLA
2
-XIIA. The expression of cPLA
2
-IVA was increased in response to
M. tuberculosis
, gamma interferon, or their combination, and cPLA
2
-IVA mediated the release of arachidonic acid, which was stimulated by
M. tuberculosis
in activated, but not unactivated, macrophages. We confirmed that arachidonic acid is highly mycobactericidal in a concentration- and pH-dependent manner in vitro. However, when
M. tuberculosis
-infected macrophages were treated with PLA
2
inhibitors, intracellular survival of
M. tuberculosis
was not affected, even in inducible nitric oxide synthase-deficient macrophages, in which a major bactericidal mechanism is removed. Moreover, intracellular survival of
M. tuberculosis
was similar in cPLA
2
-IVA-deficient and wild-type macrophages. Our results demonstrate that the cytosolic PLA
2
s are not required by murine BMDMs to kill
M. tuberculosis
.
A Mycobacterial Operon Essential for Virulence In Vivo and Invasion and Intracellular Persistence in Macrophages
Authors: Gao Lian-Yong , Pak Melissa , Kish Rabab , Kajihara Kimberly , Brown Eric J.
Published: February 2006

The ability to invade and grow in macrophages is necessary for
Mycobacterium tuberculosis
to cause disease. We have found a
Mycobacterium marinum
locus of two genes that is required for both invasion and intracellular survival in macrophages. The genes were designated
iipA
(mycobacterial invasion and intracellular persistence) and
iipB
. The
iip
mutant, which was created by insertion of a kanamycin resistance gene cassette at the 5′ region of
iipA
, was completely avirulent to zebra fish. Expression of the
M. tuberculosis
orthologue of
iipA
, Rv1477, fully complemented the
iip
mutant for infectivity in vivo, as well as for invasion and intracellular persistence in macrophages. In contrast, the
iipB
orthologue, Rv1478, only partially complemented the
iip
mutant in vivo and restored invasion but not intracellular growth in macrophages. While IipA and IipB differ at their N termini, they are highly similar throughout their C-terminal NLPC_p60 domains. The p60 domain of Rv1478 is fully functional to replace that of Rv1477, suggesting that the N-terminal sequence of Rv1477 is required for full virulence in vivo and in macrophages. Further mutations demonstrated that both Arg-Gly-Asp (RGD) and Asp-Cys-Ser-Gly (DCSG) sequences in the p60 domain are required for function. The
iip
mutant exhibited increased susceptibility to antibiotics and lysozyme and failed to fully separate daughter cells in liquid culture, suggesting a role for
iip
genes in cell wall structure and function. Altogether, these studies demonstrate an essential role for a p60-containing protein, IipA, in the pathogenesis of
M. marinum
infection.
γδ T Cells Regulate the Early Inflammatory Response toBordetella pertussisInfection in the Murine Respiratory Tract
Authors: Zachariadis O. , Cassidy J. P. , Brady J. , Mahon B. P.
Published: February 2006

The role of γδ T cells in the regulation of pulmonary inflammation followingBordetella pertussisinfection was investigated. Using a well-characterized murine aerosol challenge model, inflammatory events in mice with targeted disruption of the T-cell receptor δ-chain gene (γδ TCR−/−mice) were compared with those in wild-type animals. Early following challenge withB. pertussis, γδ TCR−/−mice exhibited greater pulmonary inflammation, as measured by intra-alveolar albumin leakage and lesion histomorphometry, yet had lower contemporaneous bacterial lung loads. The larger numbers of neutrophils and macrophages and the greater concentration of the neutrophil marker myeloperoxidase in bronchoalveolar lavage fluid from γδ TCR−/−mice at this time suggested that differences in lung injury were mediated through increased leukocyte trafficking into infected alveoli. Furthermore, flow cytometric analysis found the pattern of recruitment of natural killer (NK) and NK receptor+T cells into airspaces differed between the two mouse types over the same time period. Taken together, these findings suggest a regulatory influence for γδ T cells over the early pulmonary inflammatory response to bacterial infection. The absence of γδ T cells also influenced the subsequent adaptive immune response to specific bacterial components, as evidenced by a shift from a Th1 to a Th2 type response against theB. pertussisvirulence factor filamentous hemagglutinin in γδ TCR−/−mice. The findings are relevant to the study of conditions such as neonatalB. pertussisinfection and acute respiratory distress syndrome where γδ T cell dysfunction has been implicated in the inflammatory process.
Toll-Like Receptor 2 Mediates Alveolar Macrophage Response toPneumocystis murina
Authors: Zhang Chen , Wang Shao-Hung , Lasbury Mark E. , Tschang Dennis , Liao Chung-Ping , Durant Pamela J. , Lee Chao-Hung
Published: February 2006

The innate immune response toPneumocystisinfection is not well understood. In this study, normal C57BL/6 mouse alveolar macrophages were found to respond toPneumocystis murinaorganisms through Toll-like receptor 2 (TLR2), leading to the nuclear translocation of NF-κB and the production of proinflammatory cytokine tumor necrosis factor alpha (TNF-α) and chemokine macrophage inflammatory protein 2 (MIP-2).P. murinastimulation of normal alveolar macrophages from C57BL/6 mice resulted in increased TLR2 transcription but not increased TLR4 transcription. In gain-of-function studies with HEK293 cells expressing TLR2 or TLR4, only TLR2 was found to stimulate an NF-κB response toP. murina. TNF-α and MIP-2 production in response toP. murinaby mouse alveolar macrophages was inhibited by a monoclonal antibody that specifically blocked the ligand-binding ability of TLR2. Alveolar macrophages from TLR2 knockout (TLR2−/−) mice showed little increase in TNF-α and MIP-2 mRNA levels uponP. murinastimulation. An in vivo study showed that TLR2−/−mice challenged withP. murinahad reduced cytokine responses. These results indicate that TLR2 plays a major role in the innate immune response toP. murina.
Protein Expression Profiling of
Coccidioides posadasii
by Two-Dimensional Differential In-Gel Electrophoresis and Evaluation of a Newly Recognized Peroxisomal Matrix Protein as a Recombinant Vaccine Candidate
Authors: Orsborn Kris I. , Shubitz Lisa F. , Peng Tao , Kellner Ellen M. , Orbach Marc J. , Haynes Paul A. , Galgiani John N.
Published: February 2006

Coccidioides posadasii
and
Coccidioides immitis
are dimorphic, soil-dwelling pathogenic ascomycetes endemic to the southwestern United States. Infection can result from inhalation of a very few arthroconidia, but following natural infection, long-lived immunity is the norm. Previous work in the field has shown that spherule-derived vaccines afford more protection than those from mycelia. We have used two-dimensional differential in-gel electrophoresis coupled with nano-high-performance liquid chromatography-tandem mass spectrometry to directly assess both absolute abundance and differential expression of proteins in the spherule and the mycelial phases of
C. posadasii
with the intent to identify potential vaccine candidates. Peptides derived from 40 protein spots were analyzed and a probable identity was assigned to each. One spherule-abundant protein, identified as Pmp1, showed homology to allergens from
Aspergillus fumigatus
and other fungi, all of which exhibit similarity to yeast thiol peroxidases. Recombinant Pmp1 was reactive with serum from individuals with both acute and protracted disease, and evoked protection in two murine models of infection with
C. posadasii
. These results demonstrate the utility of proteomic analysis as a point of discovery for protective antigens for possible inclusion in a vaccine candidate to prevent coccidioidomycosis.
Genome-Wide Expression Analysis of Lipopolysaccharide-Induced Mastitis in a Mouse Model
Authors: Zheng Jiamao , Watson Anjanette D. , Kerr David E.
Published: February 2006

To better understand the acute host response to
Escherichia coli
mastitis, we analyzed gene expression patterns of approximately 23,000 transcripts 4 h after an intramammary infusion of lipopolysaccharide (LPS) in a mouse model. A total of 489 genes were significantly affected, of which 391 were induced and 98 were repressed. Gene ontology analysis demonstrated that most of the induced genes were associated with the innate immune response, apoptosis, and cell proliferation. Substantial induction of the chemokines CXCL1, CXCL2, and S100A8; the acute-phase protein SAA3; and the LPS binding protein CD14 were confirmed by Northern blot analysis. A subsequent time course experiment revealed CXCL1 induction prior to that of CD14 and SAA3. Mammary epithelial cell cultures also showed marked expression of these factors in response to LPS. The expression of immune-related genes in mammary epithelial cells indicates the importance of this cell type in initiating the inflammatory responses. Repressed genes include several carbohydrate and fatty acid metabolic enzymes and potassium transporters, which may contribute to milk composition changes during mastitis. Therefore, the overall transcription profile, in conjunction with gene ontology analysis, provides a detailed picture of the molecular mechanisms underlying the complex biological processes that occur during LPS-induced mastitis.
Amelioration of Influenza-Induced Pathology in Mice by Coinfection withTrichinella spiralis
Authors: Furze Rebecca C. , Hussell Tracy , Selkirk Murray E.
Published: February 2006

Illness due to respiratory virus infection is often induced by excessive infiltration of cells into pulmonary tissues, leading to airway occlusion. We show here that infection withTrichinella spiralisresults in lower levels of tumor necrosis factor in bronchoalveolar lavage fluid and inhibits cellular recruitment into the airways of mice coinfected with influenza A virus. Infiltration of neutrophils and CD4+and CD8+lymphocytes was reduced, resulting in animals gaining weight more rapidly following the initial phase of infection. Influenza resulted in a generalized increase in vascular permeability in pulmonary tissues, and this was suppressed by parasite infection, although the effects were restricted to the early phase of trichinosis. Moreover, the number of cells producing interleukin-10 (IL-10), and the local levels of this cytokine, were reduced, suggesting that amelioration of pulmonary pathology by parasite infection occurs independently of IL-10 production.
A CpG Oligonucleotide Can Protect Mice from a Low Aerosol Challenge Dose ofBurkholderia mallei
Authors: Waag David M. , McCluskie Michael J. , Zhang Ningli , Krieg Arthur M.
Published: February 2006

Treatment with an oligodeoxynucleotide (ODN) containing CPG motifs (CpG ODN 7909) was found to protect BALB/c mice from lung infection or death after aerosol challenge withBurkholderia mallei. Protection was associated with enhanced levels of gamma interferon (IFN-γ)-inducible protein 10, interleukin-12 (IL-12), IFN-γ, and IL-6. Preexposure therapy with CpG ODNs may protect victims of a biological attack from glanders.
Routine Markerless Gene Replacement in
Bacillus anthracis
Authors: Janes Brian K. , Stibitz Scott
Published: February 2006

An improved genetic tool suitable for routine markerless allelic exchange in
Bacillus anthracis
has been constructed. Its utility was demonstrated by the introduction of insertions, deletions, and missense mutations on the chromosome and plasmid pXO1 of the Sterne strain of
B. anthracis
.
Monoclonal Antibodies Passively Protect BALB/c Mice against
Burkholderia mallei
Aerosol Challenge
Authors: Treviño Sylvia R. , Permenter Amy R. , England Marilyn J. , Parthasarathy Narayanan , Gibbs Paul H. , Waag David M. , Chanh Tran C.
Published: February 2006

Glanders is a debilitating disease with no vaccine available. Murine monoclonal antibodies were produced against
Burkholderia mallei
, the etiologic agent of glanders, and were shown to be effective in passively protecting mice against a lethal aerosol challenge. The antibodies appeared to target lipopolysaccharide. Humoral antibodies may be important for immune protection against
B. mallei
infection.
Reduced Virulence of an
fliC
Mutant of Shiga-Toxigenic
Escherichia coli
O113:H21
Authors: Rogers Trisha J. , Paton James C. , Wang Hui , Talbot Ursula M. , Paton Adrienne W.
Published: February 2006

The contribution of flagellin to the virulence of the O113:H21 Shiga-toxigenic
Escherichia coli
(STEC) strain 98NK2 was investigated in the streptomycin-treated mouse model. Groups of mice were challenged with either the wild-type STEC or a
fliC
deletion derivative thereof. There was no difference in the level of gut colonization by the two strains, but the
fliC
mutant was significantly less virulent than its parent; the overall survival rates were 43.7% and 81.2%, respectively (
P
< 0.025). This is the first report of a nontoxic accessory virulence factor contributing to a fatal outcome of STEC infection in this model. Although H21 FliC is known to be a potent inducer of CXC chemokines, including interleukin 8, there was no obvious difference in the recruitment of polymorphonuclear leukocytes to the intestinal epithelium of mice challenged with either strain. However, immunofluorescence microscopy suggested that the
fliC
mutant was less capable of forming a close association with the colonic epithelium. This may have reduced the uptake of Stx2 by mice infected with the mutant.
Selective Binding of
Borrelia burgdorferi
OspE Paralogs to Factor H and Serum Proteins from Diverse Animals: Possible Expansion of the Role of OspE in Lyme Disease Pathogenesis
Authors: Hovis Kelley M. , Tran Emily , Sundy Christina M. , Buckles Eric , McDowell John V. , Marconi Richard T.
Published: February 2006

The binding of
Borrelia burgdorferi
OspE, OspF, and family 163 (Elp) proteins to factor H/factor H-like protein 1 (FHL-1) and other serum proteins from different animals was assessed. OspE paralogs bound factor H and unidentified serum proteins from a subset of animals, while OspF and Elp proteins did not. These data advance our understanding of factor H binding, the host range of the Lyme spirochetes, and the expanding role of OspE in pathogenesis.
Commensal Bacteria Influence
Escherichia coli
O157:H7 Persistence and Shiga Toxin Production in the Mouse Intestine
Authors: Gamage Shantini D. , Patton Angela K. , Strasser Jane E. , Chalk Claudia L. , Weiss Alison A.
Published: February 2006

The presence of commensal flora reduced colonization of
Escherichia coli
O157:H7 and production of Shiga toxin (Stx) in the murine intestine. Stx production was not detected in mice colonized with
E. coli
that were resistant to the Shiga toxin phage, but it was detected in mice colonized with phage-susceptible
E. coli
.
Preclinical Evaluation of Two Neutralizing Human Monoclonal Antibodies against Hepatitis C Virus (HCV): a Potential Treatment To Prevent HCV Reinfection in Liver Transplant Patients
Authors: Eren Rachel , Landstein Dorit , Terkieltaub Dov , Nussbaum Ofer , Zauberman Arie , Ben-Porath Judith , Gopher Judith , Buchnick Rachel , Kovjazin Riva , Rosenthal-Galili Ziva , Aviel Sigal , Ilan Ehud , Shoshany Yariv , Neville Lewis , Waisman Tal , Ben-Moshe Ofer , Kischitsky Alberto , Foung Steven K. H. , Keck Zhen-Yong , Pappo Orit , Eid Ahmed , Jurim Oded , Zamir Gidi , Galun Eithan , Dagan Shlomo
Published: February 2006

Passive immunotherapy is potentially effective in preventing reinfection of liver grafts in hepatitis C virus (HCV)-associated liver transplant patients. A combination of monoclonal antibodies directed against different epitopes may be advantageous against a highly mutating virus such as HCV. Two human monoclonal antibodies (HumAbs) against the E2 envelope protein of HCV were developed and tested for the ability to neutralize the virus and prevent human liver infection. These antibodies, designated HCV-AB 68 and HCV-AB 65, recognize different conformational epitopes on E2. They were characterized in vitro biochemically and functionally. Both HumAbs are immunoglobulin G1 and have affinity constants to recombinant E2 constructs in the range of 10
−10
M. They are able to immunoprecipitate HCV particles from infected patients' sera from diverse genotypes and to stain HCV-infected human liver tissue. Both antibodies can fix complement and form immune complexes, but they do not activate complement-dependent or antibody-dependent cytotoxicity. Upon complement fixation, the monoclonal antibodies induce phagocytosis of the immune complexes by neutrophils, suggesting that the mechanism of viral clearance includes endocytosis. In vivo, in the HCV-Trimera model, both HumAbs were capable of inhibiting HCV infection of human liver fragments and of reducing the mean viral load in HCV-positive animals. The demonstrated neutralizing activities of HCV-AB 68 and HCV-AB 65 suggest that they have the potential to prevent reinfection in liver transplant patients and to serve as prophylactic treatment in postexposure events.
Replication and Clearance of Venezuelan Equine Encephalitis Virus from the Brains of Animals Vaccinated with Chimeric SIN/VEE Viruses
Authors: Paessler Slobodan , Ni Haolin , Petrakova Olga , Fayzulin Rafik Z. , Yun Nadezhda , Anishchenko Michael , Weaver Scott C. , Frolov Ilya
Published: February 2006

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.
Immune Suppression Uncovers Endogenous Cytopathic Effects of the Hepatitis B Virus
Authors: Meuleman Philip , Libbrecht Louis , Wieland Stefan , De Vos Rita , Habib Nagy , Kramvis Anna , Roskams Tania , Leroux-Roels Geert
Published: February 2006

It is generally accepted that the host's immune response rather than the virus itself is causing the hepatocellular damage seen in acute and chronic hepatitis B virus (HBV) infections. However, in situations of severe immune suppression, chronic HBV patients may develop a considerable degree of liver disease. To examine whether HBV has direct cytopathic effects in severely immune compromised hosts, we have infected severe combined immune deficient mice (uPA-SCID), harboring human liver cells, with HBV. Serologic analysis of the plasma of HBV-infected animals revealed the presence of extremely high amounts of viral genomes and proteins. Histological analysis of the livers of uPA-SCID chimeras infected with HBV for more than 2 months showed that the majority of human hepatocytes had a ground-glass appearance, stained intensely for viral proteins, and showed signs of considerable damage and cell death. This histopathologic pattern closely resembles the picture observed in the livers of immunosuppressed HBV patients. These lesions were not observed in animals infected with HBV for less than 1 month. Ultrastructural analysis of long-term-infected hepatocytes showed a highly increased presence of cylindrical HBsAg structures, core particles, and Dane particles compared to short-term-infected hepatocytes. These long-term-infected hepatocytes also contained elevated amounts of HBV cccDNA. In conclusion, HBV causes dramatic intracellular changes and hepatocellular damage in the human hepatocytes that reside in a severely immune deficient mouse. These lesions show much resemblance to the ones encountered in immunosuppressed chronic HBV patients. Our observations indicate that HBV may be directly cytopathic in conditions of severe immune suppression.
Cyclophilin A and TRIM5α Independently Regulate Human Immunodeficiency Virus Type 1 Infectivity in Human Cells
Authors: Sokolskaja Elena , Berthoux Lionel , Luban Jeremy
Published: February 2006

Cyclophilin A (CypA), a cytoplasmic, human immunodeficiency virus type 1 (HIV-1) CA-binding protein, acts after virion membrane fusion with human cells to increase HIV-1 infectivity. HIV-1 CA is similarly greeted by CypA soon after entry into rhesus macaque or African green monkey cells, where, paradoxically, the interaction decreases HIV-1 infectivity by facilitating TRIM5α-mediated restriction. These observations conjure a model in which CA recognition by the human TRIM5α orthologue is precluded by CypA. Consistent with the model, selection of a human cell line for decreased restriction of the TRIM5α-sensitive, N-tropic murine leukemia virus (N-MLV) rendered HIV-1 transduction of these cells independent of CypA. Additionally, HIV-1 virus-like particles (VLPs) saturate N-MLV restriction activity, particularly when the CA-CypA interaction is disrupted. Here the effects of CypA and TRIM5α on HIV-1 restriction were examined directly. RNA interference was used to show that endogenous human TRIM5α does indeed restrict HIV-1, but the magnitude of this antiviral activity was not altered by disruption of the CA-CypA interaction or by elimination of CypA protein. Conversely, the stimulatory effect of CypA on HIV-1 infectivity was completely independent of human TRIM5α. Together with previous reports, these data suggest that CypA protects HIV-1 from an unknown antiviral activity in human cells. Additionally, target cell permissivity increased after loading with heterologous VLPs, consistent with a common saturable target that is epistatic to both TRIM5α and the putative CypA-regulated restriction factor.
Inhibition of Human Immunodeficiency Virus Type 1 Replication with Artificial Transcription Factors Targeting the Highly Conserved Primer-Binding Site
Authors: Eberhardy Scott R. , Goncalves Joao , Coelho Sofia , Segal David J. , Berkhout Ben , Barbas Carlos F.
Published: February 2006

The human immunodeficiency virus type 1 (HIV-1) primer-binding site (PBS) is a highly conserved region in the HIV genome and represents an attractive target for the development of new anti-HIV therapies. In this study, we designed four artificial zinc finger transcription factors to bind at or adjacent to the PBS and repress transcription from the HIV-1 long terminal repeat (LTR). These proteins bound to the LTR in vivo, as demonstrated by the chromatin immunoprecipitation assay. In transient reporter assays, three of the four proteins repressed transcription of a reporter driven by the HIV-1 LTR. Only one of these proteins, however, designated KRAB-PBS2, was able to prevent virus production when transduced into primary lymphocytes. We observed >90% inhibition of viral replication over the course of several weeks compared to untransduced cells, and no significant cytotoxicity was observed. Long-term exposure of HIV-1 to KRAB-PBS2 induced mutations in the HIV-1 PBS that reduced the effectiveness of the repressor, but these mutations also resulted in decreased rates of viral replication. These results show that KRAB-PBS2 has the potential to be used in antiviral therapy for AIDS patients and might complement other gene-based strategies.
Inhibition of Receptor Binding Stabilizes Newcastle Disease Virus HN and F Protein-Containing Complexes
Authors: McGinnes L. W. , Morrison T. G.
Published: February 2006

Receptor binding of paramyxovirus attachment proteins and the interactions between attachment and fusion (F) proteins are thought to be central to activation of the F protein activity; however, mechanisms involved are unclear. To explore the relationships between Newcastle disease virus (NDV) HN and F protein interactions and HN protein attachment to sialic acid receptors, HN and F protein-containing complexes were detected and quantified by reciprocal coimmunoprecipitation from extracts of transfected avian cells. To inhibit HN protein receptor binding, cells transfected with HN and F protein cDNAs were incubated with neuraminidase from the start of transfection. Under these conditions, no fusion was observed, but amounts of HN and F protein complexes increased twofold over amounts detected in extracts of untreated cells. Stimulation of attachment by incubation of untransfected target cells with neuraminidase-treated HN and F protein-expressing cells resulted in a twofold decrease in amounts of HN and F protein complexes. In contrast, high levels of complexes containing HN protein and an uncleaved F protein (F-K115Q) were detected, and those levels were unaffected by neuraminidase treatment of cell monolayers or by incubation with target cells. These results suggest that HN and F proteins reside in a complex in the absence of receptor binding. Furthermore, the results show that not only receptor binding but also F protein cleavage are necessary for disassociation of the HN and F protein-containing complexes.
A Single Codon in the Nucleocapsid Protein C Terminus Contributes to In Vitro and In Vivo Fitness of Edmonston Measles Virus
Authors: Carsillo Thomas , Zhang Xinsheng , Vasconcelos Daphne , Niewiesk Stefan , Oglesbee Michael
Published: February 2006

The major inducible 70-kDa heat shock protein (hsp72) increases measles virus (MV) transcription and genome replication. This stimulatory effect is attributed to hsp72 interaction with two highly conserved hydrophobic domains in the nucleocapsid protein (N) C terminus of Edmonston MV. These domains are known as Box-2 and Box-3. A single amino acid substitution in Box-3 of Edmonston MV (i.e., N522D) disrupts hsp72 binding. The prevalence of the N522D substitution in contemporary wild-type MV isolates suggests that this sequence has been positively selected. The present work determined if the N522D substitution enhances viral fitness and the degree to which any fitness advantage is influenced by hsp72 levels. Both parent Edmonston MV (Ed N) and an N522D substitution mutant (Ed N-522D) exhibited similar growth on Vero and murine neuroblastoma cells and in cotton rat lung, although Ed N-522D virus exhibited an attenuated in vitro response to hsp72 overexpression. In contrast, mixed infections showed a significantly reduced in vitro and in vivo fitness of Ed N-522D virus. Results support the involvement of additional selectional pressures that maintain the circulation of virus containing N-522D despite the cost to viral fitness.
A Hydrophobic Binding Surface on the Human Immunodeficiency Virus Type 1 Nef Core Is Critical for Association with p21-Activated Kinase 2
Authors: Agopian Kristin , Wei Bangdong L. , Garcia J. Victor , Gabuzda Dana
Published: February 2006

The interaction of human immunodeficiency virus type 1 (HIV-1) Nef with p21-activated kinase 2 (Pak2) has been proposed to play an important role in T-cell activation and disease progression during viral infection. However, the mechanism by which Nef activates Pak2 is poorly understood. Mutations in most Nef motifs previously reported to be required for Pak2 activation (G
2
, PxxP
72
, and RR
105
) also affect other Nef functions, such as CD4 or major histocompatibility complex class I (MHC-I) downregulation. To better understand Nef interactions with Pak2, we performed mutational analysis of three primary HIV-1 Nef clones that exhibited similar capacities for downregulation of CD4 and MHC-I but variable abilities to associate with activated Pak2. Our results demonstrate that Nef amino acids at positions 85, 89, 187, 188, and 191 (L, H, S, R, and F in the clade B consensus, respectively) are critical for Pak2 association. Mutation of these Nef residues dramatically altered association with Pak2 without affecting Nef expression levels or CD4 and MHC-I downregulation. Furthermore, compensation occurred at positions 89 and 191 when both amino acids were substituted. Since residues 85, 89, 187, 188, and 191 cluster on the surface of the Nef core domain in a region distinct from the dimerization and SH3-binding domains, we propose that these Nef residues form part of a unique binding surface specifically involved in association with Pak2. This binding surface includes exposed and recessed hydrophobic residues and may participate in an as-yet-unidentified protein-protein interaction to facilitate Pak2 activation.
Analysis of the pH Requirement for Membrane Fusion of Different Isolates of the Paramyxovirus Parainfluenza Virus 5
Authors: Bissonnette Mei Lin Z. , Connolly Sarah A. , Young Daniel F. , Randall Richard E. , Paterson Reay G. , Lamb Robert A.
Published: February 2006

Paramyxoviruses enter cells by fusing their envelopes with the plasma membrane, a process that occurs at neutral pH. Recently, it has been found that there is an exception to this dogma in that a porcine isolate of the paramyxovirus parainfluenza virus 5 (PIV5), known as SER, requires a low-pH step for fusion (S. Seth, A. Vincent, and R. W. Compans, J. Virol.
77:
6520-6527, 2003). As a low-pH activation mechanism for fusion would greatly facilitate biophysical studies of paramyxovirus-mediated membrane fusion, we have reexamined the triggering of the PIV5 SER fusion protein. Using multiple assays, we could not find a requirement for low-pH triggering of PIV5 SER fusion. The challenge of discovering how the paramyxovirus receptor binding protein (HN, H, or G) activates the metastable fusion protein to cause membrane fusion at neutral pH remains.
Reovirus Delays Diabetes Onset but Does Not Prevent Insulitis in Nonobese Diabetic Mice
Authors: Wetzel J. Denise , Barton Erik S. , Chappell James D. , Baer Geoffrey S. , Mochow-Grundy Michelle , Rodgers Steven E. , Shyr Yu , Powers Alvin C. , Thomas James W. , Dermody Terence S.
Published: February 2006

Mice infected with reovirus develop abnormalities in glucose homeostasis. Reovirus strain type 3 Abney (T3A) was capable of systemic infection of nonobese diabetic (NOD) mice, an experimental model of autoimmune diabetes. Reovirus antigen was detected in pancreatic islets of T3A-infected mice, and primary cultures of pancreatic islets from NOD mice supported T3A growth. Significantly fewer T3A-infected animals compared to uninfected controls developed diabetes. However, despite the alteration in diabetes penetrance, insulitis was evident in T3A-infected mice. These results suggest that viral infection of NOD mice alters autoimmune responses to β-cell antigens and thereby delays development of diabetes.
Sequential CD134-CXCR4 Interactions in Feline Immunodeficiency Virus (FIV): Soluble CD134 Activates FIV Env for CXCR4-Dependent Entry and Reveals a Cryptic Neutralization Epitope
Authors: de Parseval Aymeric , Grant Chris K. , Sastry K. Jagannadha , Elder John H.
Published: February 2006

Recombinant soluble CD134 (sCD134) facilitated feline immunodeficiency virus (FIV) entry into CXCR4-positive, cell surface CD134-negative target cells. sCD134-activated entry was dose dependent and CXCR4 dependent. We used the sCD134 activation system to explore the neutralization by four anti-V3 monoclonal antibodies (MAbs). V3 MAbs weakly neutralized FIV infection using target cells expressing both CD134 and CXCR4 but potently inhibited sCD134-activated entry into target cells expressing CXCR4 alone. These findings provide direct evidence for a sequential interaction of FIV Env with CD134 and CXCR4 and reveal the presence of a cryptic epitope in V3 that is masked in the mature envelope oligomers.
In Vitro Characterization of a Koala Retrovirus
Authors: Oliveira Nidia M. , Farrell Karen B. , Eiden Maribeth V.
Published: February 2006

Recently, a new endogenous koala gammaretrovirus, designated KoRV, was isolated from koalas. The KoRV genome shares 78% nucleotide identity with another gammaretrovirus, gibbon ape leukemia virus (GALV). KoRV is endogenous in koalas, while GALV is exogenous, suggesting that KoRV predates GALV and that gibbons and koalas acquired the virus at different times from a common source. We have determined that subtle adaptive differences between the KoRV and GALV envelope genes account for differences in their receptor utilization properties. KoRV represents a unique example of a gammaretrovirus whose envelope has evolved to allow for its expanded host range and zoonotic potential.