bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino Acid Substitutions in GyrA ofBurkholderia glumaeAre Implicated in Not Only Oxolinic Acid Resistance but Also Fitness on Rice Plants
Authors: Maeda Yukiko , Kiba Akinori , Ohnishi Kouhei , Hikichi Yasufumi
Published: December 2006

Oxolinic acid (OA) resistance in field isolates ofBurkholderia glumae, a causal agent of bacterial grain rot, is dependent on an amino acid substitution at position 83 in GyrA (GyrA83). In the present study, among spontaneous in vitro mutants from the OA-sensitiveB. glumaestrain Pg-10, we selected OA-resistant mutants that emerged at a rate of 5.7 × 10−10. Nucleotide sequence analysis of the quinolone resistance-determining region in GyrA showed that Gly81Cys, Gly81Asp, Asp82Gly, Ser83Arg, Asp87Gly, and Asp87Asn are observed in these OA-resistant mutants. The introduction of each amino acid substitution into Pg-10 resulted in OA resistance, similar to what was observed for mutants with the responsible amino acid substitution. In vitro growth of recombinants with Asp82Gly was delayed significantly compared to that of Pg-10; however, that of the other recombinants did not differ significantly. The inoculation of each recombinant into rice spikelets did not result in disease. In inoculated rice spikelets, recombinants with Ser83Arg grew less than Pg-10 during flowering, and growth of the other recombinants was reduced significantly. On the other hand, the reduced growth of recombinants with Ser83Arg in spikelets was compensated for under OA treatment, resulting in disease. These results suggest that amino acid substitutions in GyrA ofB. glumaeare implicated in not only OA resistance but also fitness on rice plants. Therefore, GyrA83 substitution is thought to be responsible for OA resistance inB. glumaefield isolates.
Identification of the Mycobacterial Subcomponents Involved in the Release of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand from Human Neutrophils
Authors: Simons Mark P. , Moore Jill M. , Kemp Troy J. , Griffith Thomas S.
Published: December 2006

Intravesical administration ofMycobacterium bovisbacillus Calmette-Guérin (BCG) continues to be a successful immunotherapy for superficial bladder cancer. Recently, workers in our laboratory observed expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on neutrophils in voided urine following BCG therapy. Neutrophils released a soluble and functional form of TRAIL when they were stimulated in vitro with BCG, and the activity was localized predominantly to the cell wall fraction. In this study, we examined the ability of individual mycobacterial components to stimulate TRAIL release from neutrophils. Our results demonstrated that cell wall-derived lipoarabinomannan (LAM), mycolyl arabinogalactan-peptidoglycan complex, and a Triton X-114 (Tx114)-solubilized protein pool were effective agonists of TRAIL release from neutrophils. Mycobacterial DNA was also an agonist of TRAIL release from neutrophils. Furthermore, purified antigen 85 ABC complex and alpha-crystallin (HspX), two major cell wall antigens present in the Tx114 pool, induced TRAIL release from neutrophils. The Tx114 pool stimulated HEK-293 cells expressing either Toll-like receptor 2/1 (TLR2/1) or TLR2/6, but only HspX was able to stimulate TLR2/6-expressing cells. TLR4/MD2/CD14-expressing cells responded only to LAM. Collectively, these results suggested that TRAIL release from neutrophils was induced through the recognition of multiple mycobacterial components by TLR2 and TLR4.
A MyD88-Deficient Mouse Model Reveals a Role for Nramp1 in
Campylobacter jejuni
Infection
Authors: Watson Robert O. , Novik Veronica , Hofreuter Dirk , Lara-Tejero María , Galán Jorge E.
Published: December 2006

Campylobacter jejuni
is a major worldwide cause of enteric illnesses. Adult immunocompetent mice are not susceptible to
C. jejuni
infection. However, we show here that mice deficient in the adaptor protein myeloid differentiation factor 88 (MyD88), which is required for signaling through most Toll-like receptors, can be stably colonized by
C. jejuni
but not by isogenic derivatives carrying mutations in known virulence genes. We also found that Nramp1 deficiency increases the mouse susceptibility to
C. jejuni
infection when administered systemically. These results indicate that MyD88-deficient mice could be a useful model to study
C. jejuni
colonization and reveal a potential role for Nramp1 in the control of this bacterial pathogen.
“
Candidatus
Helicobacter heilmannii” from a Cynomolgus Monkey Induces Gastric Mucosa-Associated Lymphoid Tissue Lymphomas in C57BL/6 Mice
Authors: Nakamura Masahiko , Murayama Somay Yamagata , Serizawa Hiroshi , Sekiya Yukie , Eguchi Masahiro , Takahashi Shinichi , Nishikawa Kaori , Takahashi Tetsufumi , Matsumoto Tsukasa , Yamada Haruki , Hibi Toshifumi , Tsuchimoto Kanji , Matsui Hidenori
Published: December 2006

Both
Helicobacter pylori
and “
Candidatus
Helicobacter heilmannii” infections are associated with peptic ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas. However, good animal models of
H. pylori
clinical diseases are rare. In this study, we aimed to establish an animal model of “
Candidatus
Helicobacter heilmannii” gastric MALT lymphoma. We used a urease-positive gastric mucosal and mucus homogenate from a cynomolgus monkey maintained in C57BL/6 mouse stomachs. The bacterium in the homogenate was identified as “
Candidatus
Helicobacter heilmannii” based on a DNA sequence analysis of the 16S rRNA and urease genes. Mucosal and mucus homogenates were used to inoculate C57BL/6 mice, which were then examined for 24 months. We observed a gradual increase in the surface area of protrusive lesions in almost all infected C57BL/6 mouse fundic stomachs 6 months after infection. Light microscopic observations revealed an accumulation of B lymphocytes along with destruction of glandular elements and the presence of lymphoepithelial lesions consistent with low-grade MALT lymphomas. Electron microscopic observation revealed numerous “
Candidatus
Helicobacter heilmannii” bacilli in the fundic glandular lumen, the intracellular canaliculi, and the cytoplasm of intact cells, as well as damaged parietal cells. In conclusion, “
Candidatus
Helicobacter heilmannii” induced gastric MALT lymphomas in almost 100% of infected C57BL/6 mice after a 6-month period associated with the destruction of parietal cells.
Analysis of the Polysaccharide Capsule of the Systemic Pathogen
Streptococcus iniae
and Its Implications in Virulence
Authors: Lowe Beth A. , Miller Jesse D. , Neely Melody N.
Published: December 2006

Systemic pathogens have developed numerous strategies for evading the defenses of the host, permitting dissemination and multiplication in various tissues. One means of survival in the host, particularly in the bloodstream, has been attributed to the ability to avoid phagocytosis via capsular polysaccharide. To further define the virulence capacity of
Streptococcus iniae
, a zoonotic pathogen with the ability to cause severe systemic disease in both fish and humans, we performed an analysis of the capsule locus. The initial analysis included cloning and sequencing of the capsule synthesis operon, which revealed an approximately 21-kb region that is highly homologous to capsule operons of other streptococci. A genetic comparison of
S. iniae
virulent strain 9117 and commensal strain 9066 revealed that the commensal strain does not have the central region of the capsule operon composed of several important capsule synthesis genes. Four 9117 insertion or deletion mutants with mutations in the beginning, middle, or end of the capsule locus were analyzed to determine their capsule production and virulence. Virulence profiles were analyzed for each mutant using three separate criteria, which demonstrated the attenuation of each mutant in several tissue environments. These analyses also provided insight into the different responses of the host to each mutant strain compared to a wild-type infection. Our results demonstrate that capsule is not required for all host environments, while excess capsule is also not optimal, suggesting that for an “ideal” systemic infection, capsule production is most likely regulated while the bacterium is in different environments of the host.
Simultaneous Induction of Apoptotic and Survival Signaling Pathways in Macrophage-Like THP-1 Cells by Shiga Toxin 1
Authors: Lee Sang-Yun , Cherla Rama P. , Tesh Vernon L.
Published: December 2006

Shiga toxins have been shown to induce apoptosis in many cell types. However, Shiga toxin 1 (Stx1) induced only limited apoptosis of macrophage-like THP-1 cells in vitro. The mechanisms regulating macrophage death or survival following toxin challenge are unknown. Differentiated THP-1 cells expressed tumor necrosis factor receptors and membrane-associated tumor necrosis factor alpha (TNF-α) and produced soluble TNF-α after exposure to Stx1. However, the cells were refractory to apoptosis induced by TNF-α, although the cytokine modestly increased apoptosis in the presence of Stx1. Despite the partial resistance of macrophage-like THP-1 cells to Stx1-mediated killing, treatment of these cells with Stx1 activated a broad array of caspases, disrupted the mitochondrial membrane potential (ΔΨ
m
), and released cytochrome
c
into the cytoplasm. The ΔΨ
m
values were greatest in cells that had detached from plastic surfaces. Specific caspase inhibitors revealed that caspase-3, caspase-6, caspase-8, and caspase-9 were primarily involved in apoptosis induction. The antiapoptotic factors involved in macrophage survival following toxin challenge include inhibitors of apoptosis proteins and X-linked inhibitor of apoptosis protein. NF-κB and JNK mitogen-activated protein kinases (MAPKs) appeared to activate survival pathways, while p38 MAPK was involved in proapoptotic signaling. The JNK and p38 MAPKs were shown to be upstream signaling pathways which may regulate caspase activation. Finally, the protein synthesis inhibitors Stx1 and anisomycin triggered limited apoptosis and prolonged JNK and p38 MAPK activation, while macrophage-like cells treated with cycloheximide remained viable and showed transient activation of MAPKs. Collectively, these data suggest that Stx1 activates both apoptotic and cell survival signaling pathways in macrophage-like THP-1 cells.
Helicobacter pylori
CagA Induces AGS Cell Elongation through a Cell Retraction Defect That Is Independent of Cdc42, Rac1, and Arp2/3
Authors: Bourzac Kevin M. , Botham Crystal M. , Guillemin Karen
Published: December 2006

Helicobacter pylori
, which infects over one-half the world's population, is a significant risk factor in a spectrum of gastric diseases, including peptic ulcers and gastric cancer. Strains of
H. pylori
that deliver the effector molecule CagA into host cells via a type IV secretion system are associated with more severe disease outcomes. In a tissue culture model of infection, CagA delivery results in a dramatic cellular elongation referred to as the “hummingbird” phenotype, which is characterized by long, thin cellular extensions. These actin-based cytoskeletal rearrangements are reminiscent of structures that are regulated by Rho GTPases and the Arp2/3 complex. We tested whether these signaling pathways were important in the
H. pylori
-induced cell elongation phenotype. Contrary to our expectations, we found that these molecules are dispensable for cell elongation. Instead, time-lapse video microscopy revealed that cells infected by
cagA
+
H. pylori
become elongated because they fail to release their back ends during cell locomotion. Consistent with a model in which CagA causes cell elongation by inhibiting the disassembly of adhesive cell contacts at migrating cells' lagging ends, immunohistochemical analysis revealed that focal adhesion complexes persist at the distal tips of elongated cell projections. Thus, our data implicate a set of signaling molecules in the hummingbird phenotype that are different than the molecules previously suspected.
Extensive and Genome-Wide Changes in the Transcription Profile ofStaphylococcus aureusInduced by Modulating the Transcription of the Cell Wall Synthesis GenemurF
Authors: Sobral Rita G. , Jones Alison E. , Des Etages Shelley G. , Dougherty Thomas J. , Peitzsch Robert M. , Gaasterland Terry , Ludovice Ana Madalena , de Lencastre Hermínia , Tomasz Alexander
Published: December 2006

AmurFconditional mutant was used to evaluate the effect of suboptimal transcription of this gene on the transcriptome of the methicillin-resistantStaphylococcus aureusstrain COL. The mutant was grown in the presence of optimal and suboptimal concentrations of the inducer, and the relative levels of transcription of genes were evaluated genome wide with an Affymetrix DNA microarray that included all open reading frames (ORFs) as well as intergenic sequences derived from four sequencedS. aureusstrains. Using a sensitivity threshold value of 1.5, suboptimal expression ofmurFaltered the transcription of a surprisingly large number of genes, i.e., 668 out of the 2,740 ORFs (close to one-fourth of all ORFs), of the genome ofS. aureusstrain COL. The genes with altered transcription were distributed evenly around theS. aureuschromosome, and groups of genes involved with distinct metabolic functions responded in unique and operon-specific manners to modulation inmurFtranscription. For instance, all genes belonging to theisdoperon and all but 2 of the 35 genes of prophage L54a were down-regulated, whereas all but one of the 21 members of thevraSRregulon and most of the 79 virulence-related genes (those for fibronectin binding proteins A and B, clumping factor A, gamma hemolysin, enterotoxin B, etc.) were up-regulated in cells with suboptimal expression ofmurF. Most importantly, the majority of these altered gene expression profiles were reversible by resupplying the optimal concentration of IPTG (isopropyl-β-d-thiogalactopyranoside) to the culture. The observations suggest the coordinate regulation of a large sector of theS. aureustranscriptome in response to a disturbance in cell wall synthesis.
Characterization of Changes to the Cell Surface during the Life Cycle of
Streptomyces coelicolor
: Atomic Force Microscopy of Living Cells
Authors: Del Sol Ricardo , Armstrong Ian , Wright Chris , Dyson Paul
Published: December 2006

Cell surface changes that accompany the complex life cycle of
Streptomyces coelicolor
were monitored by atomic force microscopy (AFM) of living cells. Images were obtained using tapping mode to reveal that young, branching vegetative hyphae have a relatively smooth surface and are attached to an inert silica surface by means of a secreted extracellular matrix. Older hyphae, representing a transition between substrate and aerial growth, are sparsely decorated with fibers. Previously, a well-organized stable mosaic of fibers, called the rodlet layer, coating the surface of spores has been observed using electron microscopy. AFM revealed that aerial hyphae, prior to sporulation, possess a relatively unstable dense heterogeneous fibrous layer. Material from this layer is shed as the hyphae mature, revealing a more tightly organized fibrous mosaic layer typical of spores. The aerial hyphae are also characterized by the absence of the secreted extracellular matrix. The formation of sporulation septa is accompanied by modification to the surface layer, which undergoes localized temporary disruption at the sites of cell division. The characteristics of the hyphal surfaces of mutants show how various chaplin and rodlin proteins contribute to the formation of fibrous layers of differing stabilities. Finally, older spores with a compact rodlet layer develop surface concavities that are attributed to a reduction of intracellular turgor pressure as metabolic activity slows.
Transposon Tn
7
Is Widespread in Diverse Bacteria and Forms Genomic Islands
Authors: Parks Adam R. , Peters Joseph E.
Published: December 2006

We find that relatives of the bacterial transposon Tn
7
are widespread in disparate environments and phylogenetically diverse species. These elements form functionally diverse genomic islands at the specific site of Tn
7
insertion adjacent to
glmS
. This work presents the first example of genomic island formation by a DDE type transposon.
Colony Spreading in
Staphylococcus aureus
Authors: Kaito Chikara , Sekimizu Kazuhisa
Published: December 2006

Wild-type
Staphylococcus aureus
rapidly expands on the surface of soft agar plates. The rates of expansion and the shapes of the resultant giant colonies were distinct for different strains of laboratory stocks and clinical isolates. The colony spreading abilities did not correlate with the biofilm-forming abilities in these strains. Insertional disruption of the
dltABCD
operon, which functions at the step of
d
-alanine addition to teichoic acids, and of the
tagO
gene, which is responsible for the synthesis of wall teichoic acids, decreased the colony spreading ability. The results indicate that wall teichoic acids and
d
-alanylation of teichoic acids are required for colony spreading.
Effects of Phenotype and Genotype on Methods for Detection of Extended-Spectrum-β-Lactamase-Producing Clinical Isolates ofEscherichia coliandKlebsiella pneumoniaein Norway
Authors: Tofteland Ståle , Haldorsen Bjørg , Dahl Kristin H. , Simonsen Gunnar S. , Steinbakk Martin , Walsh Timothy R. , Sundsfjord Arnfinn ,   the Norwegian ESBL Study Group
Published: December 2006

Consecutive clinical isolates ofEscherichia coli(n= 87) andKlebsiella pneumoniae(n= 25) with reduced susceptibilities to oxyimino-cephalosporins (MICs > 1 mg/liter) from 18 Norwegian laboratories during March through October 2003 were examined forblaTEM/SHV/CTX-Mextended-spectrum-β-lactamase (ESBL) genes, oxyimino-cephalosporin MIC profiles, ESBL phenotypes (determined by the ESBL Etest and the combined disk and double-disk synergy [DDS] methods), and susceptibility to non-β-lactam antibiotics. Multidrug-resistant CTX-M-15-like (n= 23) and CTX-M-9-like (n= 15) ESBLs dominated among the 50 ESBL-positiveE. coliisolates. SHV-5-like (n= 9) and SHV-2-like (n= 4) ESBLs were the most prevalent in 19 ESBL-positiveK. pneumoniaeisolates. Discrepant ESBL phenotype test results were observed for one major (CTX-M-9) and several minor (TEM-128 and SHV-2/-28) ESBL groups and in SHV-1/-11-hyperproducing isolates. Negative or borderline ESBL results were observed when low-MIC oxyimino-cephalosporin substrates were used to detect clavulanic acid (CLA) synergy. CLA synergy was detected by the ESBL Etest and the DDS method but not by the combined disk method in SHV-1/-11-hyperproducing strains. The DDS method revealed unexplained CLA synergy in combination with aztreonam and cefpirome in threeE. colistrains. The relatively high proportion of ESBL-producingE. coliorganisms with a low ceftazidime MIC in Norway emphasizes that cefpodoxime alone or both cefotaxime and ceftazidime should be used as substrates for ESBL detection.
Laribacter hongkongensis
Isolated from a Patient with Community-Acquired Gastroenteritis in Hangzhou City
Authors: Ni Xiao-Ping , Ren Shu-Hua , Sun Jian-Rong , Xiang Hai-Qing , Gao Yan , Kong Qing-Xin , Cha Jie , Pan Jing-Chao , Yu Hua , Li Hua-Ming
Published: December 2006

We describe the isolation of
Laribacter hongkongensis
in Hangzhou City, People's Republic of China. One strain of bacterium, named LHHZ242, had many of the same phenotypic and genotypic characteristics as
Laribacter hongkongensis
described in previous publications. This discovery proves that
Laribacter hongkongensis
is also associated with community-acquired gastroenteritis outside Hong Kong.
Comparison of Serological Test Kits for Diagnosis of Typhoid Fever in the Philippines
Authors: Kawano Razel L. , Leano Susan A. , Agdamag Dorothy May A.
Published: December 2006

We evaluated four recent antibody-detection kits for typhoid fever by using 177 febrile patients from our hospital, in 75 of whom
Salmonella enterica
serotype Typhi grew. TUBEX performed best, achieving 94.7% sensitivity and 80.4% specificity. Typhidot, SD Bioline Typhoid, and Mega Salmonella were less specific and, in most cases, less sensitive.
Spoligotype Signatures in the
Mycobacterium tuberculosis
Complex
Authors: Streicher E. M. , Victor T. C. , van der Spuy G. , Sola C. , Rastogi N. , van Helden P. D. , Warren R. M.
Published: December 2006

Evolution of the direct repeat region in
Mycobacterium tuberculosis
has created unique spoligotype signatures specifically associated with IS
6110-
defined strain families. Spoligotyping signatures may enable the analysis of the strain population structure in different settings and will enable the rapid identification of strain families that acquire drug resistance or escape protective immunity in drug and vaccine trials.
Helicobacter cinaedi
Cellulitis and Bacteremia in Immunocompetent Hosts after Orthopedic Surgery
Authors: Kitamura Toshio , Kawamura Yoshiaki , Ohkusu Kiyofumi , Masaki Takayuki , Iwashita Hirofumi , Sawa Tomohiro , Fujii Shigemoto , Okamoto Tatsuya , Akaike Takaaki
Published: December 2006

At various times after orthopedic operations (more than a few weeks, with an average of 29.9 days), 11 patients had a sudden onset of high temperature (average 38.9°C) and local cellulitis at different sites on the operated sides. The wounds had completely healed, without complicated infections, when the cellulitis occurred. The clinical picture of cellulitis in all patients was atypical: diffuse salmon-pink skin color, local heat, swelling, spontaneous pain, and tenderness but no eruptions. No patient had any underlying immunocompromising conditions or had been given immunosuppressive agents. Gram-negative spiral bacteria were isolated from blood cultures and were identified as
Helicobacter cinaedi
on the basis of 16S rRNA gene sequencing and DNA-DNA hybridization using standard strains. By means of phylogenetic analysis, we divided these clinical isolates into two clones. The
H. cinaedi
strain isolated via fecal cultures from two patients without intestinal symptoms was the same clone as the blood isolate. All isolates were quite susceptible to various antibiotics, and clinical and inflammatory symptoms of bacteremia and cellulitis improved after treatment with penicillins and cephalosporins. A relatively high incidence of recurrence of the same disease was observed, however. Almost all patients responded immunologically to the infection, as evidenced by the production of serum antibody against
H. cinaedi
. We thus suggest that
H. cinaedi
should not be regarded as simply an opportunistic pathogen but that it may be a pathogen in immunocompetent hosts and may cause infections together with bacteremia and cellulitis.
Probe Hybridization Array Typing: a Binary Typing Method for
Escherichia coli
Authors: Srinivasan U. , Zhang L. , France A. M. , Ghosh D. , Shalaby W. , Xie J. , Marrs C. F. , Foxman B.
Published: December 2006

The ability to distinguish between
Escherichia coli
strains is critical for outbreak investigations. Binary typing, based on the presence or absence of genetic material, provides a high-throughput alternative to gel- and PCR-based typing techniques that generate complex banding patterns and lack uniform interpretation criteria. We developed, validated, and determined the discriminatory power of an
E. coli
binary typing method, probe hybridization array typing (PHAT). In PHAT, the absence or presence of genetic material is identified by using DNA hybridization to produce a reproducible and portable fingerprint for each genome. PHAT probes were generated from genome subtractive hybridization experiments. We PHAT typed the ECOR collection of strains from a variety of geographical locations, and 33 rectal
E. coli
strains selected from college-aged women with urinary tract infection. In the set of 33 human rectal strains, the discriminatory power of PHAT (98%) equaled that of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. However, for ECOR strains, which include nonhuman strains, the current set of PHAT probes was less discriminating than MLST, ribotyping, and enterobacterial repetitive intergenic consensus sequence PCR (80% versus 97, 92, and 97%, respectively). When we limited the analysis to ECOR strains of B2 and D lineage, which are associated with human infection, current PHAT probes were highly discriminatory (94%). PHAT can be applied in a high-throughput format (i.e., “library on a slide”), the discriminatory ability can be varied based on the probe set, and PHAT is readily adapted to other bacterial species with high variation in genetic content.
Use of Fluconazole as a Surrogate Marker To Predict Susceptibility and Resistance to Voriconazole among 13,338 Clinical Isolates of
Candida
spp. Tested by Clinical and Laboratory Standards Institute-Recommended Broth Microdilution Methods
Authors: Pfaller M. A. , Messer S. A. , Boyken L. , Rice C. , Tendolkar S. , Hollis R. J. , Diekema D. J.
Published: December 2006

Clinical laboratories frequently face the problem of delayed availability of commercially prepared approved reagents for performing susceptibility testing of new antimicrobials. Although this problem is encountered more often with antibacterial agents, it is also an issue with antifungal agents. A current example is voriconazole, a new triazole antifungal with an expanded spectrum and potency against
Candida
spp.,
Aspergillus
spp., and other opportunistic fungal pathogens. The present study addresses the use of fluconazole as a surrogate marker to predict the susceptibility of
Candida
spp. to voriconazole. Reference broth microdilution MIC results for 13,338 strains of
Candida
spp. isolated from more than 200 medical centers worldwide were used. Voriconazole MICs and interpretive categories (susceptible, ≤1 μg/ml; susceptible dose dependent, 2 μg/ml; resistant, ≥4 μg/ml) were compared with those of fluconazole by regression statistics and error rate bounding analyses. For all 13,338 isolates, the absolute categorical agreement was 91.6% (false susceptible or very major error [VME], 0.0%). Since voriconazole is 16- to 32-fold more potent than fluconazole, the performance of fluconazole as a surrogate marker for voriconazole susceptibility was improved by designating those isolates with fluconazole MICs of ≤32 μg/ml as being susceptible to voriconazole, resulting in a categorical agreement of 97% with 0.1% VME. Clinical laboratories performing antifungal susceptibility testing of fluconazole against
Candida
spp. can reliably use these results as surrogate markers until commercial FDA-approved voriconazole susceptibility tests become available.
Development of a Real-Time PCR Assay To Detect
Treponema pallidum
in Clinical Specimens and Assessment of the Assay's Performance by Comparison with Serological Testing
Authors: Leslie David E. , Azzato Franca , Karapanagiotidis Theo , Leydon Jennie , Fyfe Janet
Published: December 2006

The incidence of infectious syphilis in men who have sex with men and human immunodeficiency virus-infected patients has increased steadily in Victoria, Australia, since 2002. A TaqMan real-time PCR assay targeting the
polA
gene of
Treponema pallidum
(TpPCR) was developed. The analytical sensitivity of the assay was estimated to be 1.75 target copies per reaction. Initially, the assay was used to test a variety of specimens (excluding blood) from 598 patients. Of the 660 tests performed, positive PCR results were obtained for 55 patients. TpPCR results were compared with serology results for 301 patients being investigated for early syphilis. Of these patients, 41 were positive by both TpPCR and serology, 246 were negative by both TpPCR and serology, 4 were TpPCR positive but negative by serology, and 10 were TpPCR negative but showed evidence of recent or active infection by serology. Directly compared with serology, TpPCR showed 95% agreement, with a sensitivity of 80.39% and a specificity of 98.40%. Potential factors leading to the discrepant results are discussed. Concurrent serology on 21 patients with TpPCR-positive primary syphilitic lesions demonstrated that a panel of current syphilis serological tests has high sensitivity for the detection of early syphilis. We found that TpPCR is a useful addition to serology for the diagnosis of infectious syphilis. Direct comparison with other
T. pallidum
PCR assays will be required to fully assess the limitations of the assay.
Propionibacterium acnes
as a Cause of Prosthetic Valve Aortic Root Abscess
Authors: Hinestrosa Federico , Djurkovic Svetolik , Bourbeau Paul P. , Foltzer Michael A.
Published: December 2006

Propionibacterium acnes
isolates usually have relatively low virulence and are often classified as contaminants when isolated from blood and tissue cultures. We report a patient with
Propionibacterium acnes
bacteremia and late prosthetic valve endocarditis, complicated by an aortic root abscess.
Genetic Profiling of the Oral Microbiota Associated with Severe Early-Childhood Caries
Authors: Li Y. , Ge Y. , Saxena D. , Caufield P. W.
Published: December 2006

The determination of the composition of the microbial community in the oral cavity is usually based on cultivation methods; however, nearly half of the bacteria in the saliva and the dental plaque are not cultivable. In this study, we evaluated the difference in oral microbial diversity between children with severe early-childhood caries (S-ECC) and caries-free (CF) controls by means of a cultivation-independent approach called denaturing gradient gel electrophoresis (DGGE). Pooled dental plaque samples were collected from 20 children aged 2 to 8 years. Total microbial genomic DNA was isolated from those subjects, and a portion of the 16S rRNA gene locus was PCR amplified by using universal primers. We observed that the mean species richness of the bacterial population was greater in the CF children (
n
= 12) (42 ± 3.7) than in the S-ECC children (
n
= 8) (35 ± 4.3); the difference was statistically significant (
P
= 0.005). The overall diversity of plaque samples as measured by the Shannon index was 3.5 for the S-ECC group and 3.7 for the CF group (
P
= 0.004). Differences in DGGE profiles were distinguished on the basis of a cluster analysis. Sequence analysis of excised DGGE bands consisted of 2.7 phylotypes, on average. After adjusting for the number of observed bands, we estimated that the S-ECC group exhibited 94.5 total phylotypes and that the CF group exhibited 113.4. These results suggest that the microbial diversity and complexity of the microbial biota in dental plaque are significantly less in S-ECC children than in CF children.
Serological Cross-Reactivity between Different
Chlamydia
-Like Organisms
Authors: Casson Nicola , Entenza José M. , Greub Gilbert
Published: December 2006

The serological cross-reactivity between different recently described
Chlamydia
-related organisms was determined. Mouse sera exhibited a strong reactivity against autologous antigen and closely related heterologous antigen but no cross-reactivity with distantly related species. These results are important to better interpret serological studies and assess the pathogenic role of these obligate intracellular bacteria.
Rapid Molecular Strategy for Filovirus Detection and Characterization
Authors: Zhai Junhui , Palacios Gustavo , Towner Jonathan S. , Jabado Omar , Kapoor Vishal , Venter Marietjie , Grolla Allen , Briese Thomas , Paweska Janusz , Swanepoel Robert , Feldmann Heinz , Nichol Stuart T. , Lipkin W. Ian
Published: December 2006

Filoviruses have the capacity to cause lethal outbreaks of hemorrhagic fever in primates. Here we present a simple consensus reverse transcription-PCR method for filovirus recognition and characterization and demonstrate its utility with all known filovirus strains. Phylogenetic assignment is achieved by automated web-based sequence analysis of amplification products.
Role of Large Sequence Polymorphisms (LSPs) in Generating Genomic Diversity among Clinical Isolates of
Mycobacterium tuberculosis
and the Utility of LSPs in Phylogenetic Analysis
Authors: Alland David , Lacher David W. , Hazbón Manzour Hernando , Motiwala Alifiya S. , Qi Weihong , Fleischmann Robert D. , Whittam Thomas S.
Published: December 2006

Mycobacterium tuberculosis
strains contain different genomic insertions or deletions called large sequence polymorphisms (LSPs). Distinguishing between LSPs that occur one time versus ones that occur repeatedly in a genomic region may provide insights into the biological roles of LSPs and identify useful phylogenetic markers. We analyzed 163 clinical
M. tuberculosis
isolates for 17 LSPs identified in a genomic comparison of
M. tuberculosis
strains H37Rv and CDC1551. LSPs were mapped onto a single-nucleotide polymorphism (SNP)-based phylogenetic tree created using nine novel SNP markers that were found to reproduce a 212-SNP-based phylogeny. Four LSPs (group A) mapped to a single SNP tree segment. Two LSPs (group B) and 11 LSPs (group C) were inferred to have arisen independently in the same genomic region either two or more than two times, respectively. None of the group A LSPs but one group B LSP and five group C LSPs were flanked by IS
6110
sequences in the references strains. Genes encoding members of the proline-glutamic acid or proline-proline-glutamic acid protein families were present only in group B or C LSPs. SNP- versus LSP-based phylogenies were also compared. We classified each isolate into 58 LSP types by using a separate LSP-based phylogenetic analysis and mapped the LSP types onto the SNP tree. LSPs often assigned isolates to the correct phylogenetic lineage; however, significant mistakes occurred for 6/58 (10%) of the LSP types. In conclusion, most LSPs occur in genomic regions that are prone to repeated insertion/deletion events and were responsible for an unexpectedly high degree of genomic variation in clinical
M. tuberculosis.
Group B and C LSPs may represent polymorphisms that occur due to selective pressure and affect the phenotype of the organism, while group A LSPs are preferable phylogenetic markers.
A-to-G Hypermutation in the Genome of Lymphocytic Choriomeningitis Virus
Authors: Zahn Roland C. , Schelp Ina , Utermöhlen Olaf , von Laer Dorothee
Published: December 2006

The interferon-inducible adenosine deaminase that acts on double-stranded RNA (ADAR1-L) has been proposed to be one of the antiviral effector proteins within the complex innate immune response. Here, the potential role of ADAR1-L in the innate immune response to lymphocytic choriomeningitis virus (LCMV), a widely used virus model, was studied. Infection with LCMV clearly upregulated ADAR1-L expression and activity. The editing activity of ADAR1-L on an RNA substrate was not inhibited by LCMV replication. Accordingly, an adenosine-to-guanosine (A-to-G) and uracil-to-cytidine (U-to-C) hypermutation pattern was found in the LCMV genomic RNA in infected cell lines and in mice. In addition, two hypermutated clones with a high level of A-to-G or U-to-C mutations within a short stretch of the viral genome were isolated. Analysis of the functionality of viral glycoprotein revealed that A-to-G- and U-to-C-mutated LCMV genomes coded for nonfunctional glycoprotein at a surprisingly high frequency. Approximately half the GP clones with an amino acid mutation lacked functionality. These results suggest that ADAR1-L-induced mutations in the viral RNA lead to a loss of viral protein function and reduced viral infectivity. This study therefore provides strong support for the contribution of ADAR1-L to the innate antiviral immune response.
Attenuation of Herpes Simplex Virus Neurovirulence with Picornavirus
cis
-Acting Genetic Elements
Authors: Campbell Stephanie A. , Mulvey Matthew , Mohr Ian , Gromeier Matthias
Published: December 2006

Viral pathogenesis depends on a suitable milieu in target host cells permitting viral gene expression, propagation, and spread. In many instances, viral genomes can be manipulated to select for propagation in certain tissues or cell types. This has been achieved for the neurotropic poliovirus (PV) by exchange of the internal ribosomal entry site (IRES), which is responsible for translation of the uncapped plus-strand RNA genome. The IRES of human rhinovirus type 2 (HRV2) confers neuron-specific replication deficits to PV but has no effect on viral propagation in malignant glioma cells. We report here that placing the critical

γ
1

34.5
virulence genes of herpes simplex virus type 1 (HSV) under translation control of the HRV2 IRES results in neuroattenuation in mice. In contrast, IRES insertion permits HSV propagation in malignant glioma cell lines that do not support replication of HSV recombinants carrying

γ
1

34.5
deletions. Our observations indicate that the conditions for alternative translation initiation at the HRV2 IRES in malignant glioma cells differ from those in normal central nervous system (CNS) cells. Picornavirus regulatory sequences mediating cell type-specific gene expression in the CNS can be utilized to target cancerous cells at the level of translation regulation outside their natural context.
Vaccination of Cats with Attenuated Feline Immunodeficiency Virus Proviral DNA Vaccine Expressing Gamma Interferon
Authors: Gupta Soumi , Leutenegger Christian M. , Dean Gregg A. , Steckbeck Jonathan D. , Cole Kelly Stefano , Sparger Ellen E.
Published: December 2006

A feline immunodeficiency virus (FIV) provirus with a
vif
gene deletion (FIVΔvifATGγ) that coexpresses feline gamma interferon (IFN-γ) was tested as a proviral DNA vaccine to extend previous studies showing efficacy with an FIV-pPPRΔvif DNA vaccine. Cats were vaccinated with either FIVΔvifATGγ or FIV-pPPRΔvif proviral plasmid DNA or with both FIV-pPPRΔvif DNA and a feline IFN-γ expression plasmid (pCDNA-IFNγ). A higher frequency of FIV-specific T-cell proliferation responses was observed in cats immunized with either FIVΔvifATGγ or FIV-pPPRΔvif plus pCDNA-IFNγ, while virus-specific cytotoxic-T-lymphocyte responses were comparable between vaccine groups. Antiviral antibodies were not observed postvaccination. Virus-specific cellular and humoral responses were similar between vaccine groups after challenge with a biological FIV isolate (FIV-PPR) at 13 weeks postimmunization. All vaccinated and unvaccinated cats were infected after FIV-PPR challenge and exhibited similar plasma virus loads. Accordingly, inclusion of plasmids containing IFN-γ did not enhance the efficacy of FIV-pPPRΔvif DNA immunization. Interestingly, the lack of protection associated with FIV-pPPRΔvif DNA immunization contrasted with findings from a previous study and suggested that multiple factors, including timing of FIV-pPPRΔvif inoculations and challenge, as well as route of challenge virus delivery, may significantly impact vaccine efficacy.
The S Segment of Punta Toro Virus (
Bunyaviridae
,
Phlebovirus
) Is a Major Determinant of Lethality in the Syrian Hamster and Codes for a Type I Interferon Antagonist
Authors: Perrone Lucy A. , Narayanan Krishna , Worthy Melissa , Peters C. J.
Published: December 2006

Two
strains of Punta Toro virus (PTV), isolated from febrile humans in
Panama, cause a differential pathogenesis in Syrian hamsters, which
could be a useful model for understanding the virulence characteristics
and differential outcomes in other phleboviral infections such as Rift
Valley fever virus. Genetic reassortants produced between the lethal
Adames (A/A/A) and nonlethal Balliet (B/B/B) strains were used in this
study to investigate viral genetic determinants for pathogenesis and
lethality in the hamster model. The S segment was revealed to be a
critical genome segment, determining lethality with log
10
50% lethal doses for each PTV genotype as follows (L/M/S convention):
A/A/A, <0.7; B/A/A, <0.7; A/B/A, 1.5; B/B/A, 2.2;
B/A/B, 4.7; A/B/B, >4.7; A/A/B, >4.7; B/B/B,>
4.7. In addition, the Adames strain inhibits the induction of
alpha/beta interferon (IFN-α/β) in vivo and in vitro
and inhibits the activation of the IFN-β promoter. Expression
of the PTV Adames NSs protein, encoded by the S RNA segment, inhibited
the virus-mediated induction of an IFN-β promoter-driven
reporter gene, suggesting that PTV NSs functions as a type I IFN
antagonist. Taken together, these data indicate a mechanism of
pathogenesis in which the suppression of the type I IFN response early
during PTV infection leads to early and uncontrolled viral replication
and, ultimately, hamster death. This study contributes to our
understanding of
Phlebovirus
pathogenesis and identifies
potential targets for immune modulation to increase host
survival.
Inhibition of Retinoic Acid-Inducible Gene I-Mediated Induction of Beta Interferon by the NS1 Protein of Influenza A Virus
Authors: Mibayashi Masaki , Martínez-Sobrido Luis , Loo Yueh-Ming , Cárdenas Washington B. , Gale Michael , García-Sastre Adolfo
Published: December 2006

The retinoic acid-inducible gene I product (RIG-I) has been identified as a cellular sensor of RNA virus infection resulting in beta interferon (IFN-β) induction. However, many viruses are known to encode viral products that inhibit IFN-β production. In the case of influenza A virus, the viral nonstructural protein 1 (NS1) prevents the induction of the IFN-β promoter by inhibiting the activation of transcription factors, including IRF-3, involved in IFN-β transcriptional activation. The inhibitory properties of NS1 appear to be due at least in part to its binding to double-stranded RNA (dsRNA), resulting in the sequestration of this viral mediator of RIG-I activation. However, the precise effects of NS1 on the RIG-I-mediated induction of IFN-β have not been characterized. We now report that the NS1 of influenza A virus interacts with RIG-I and inhibits the RIG-I-mediated induction of IFN-β. This inhibition was apparent even when a mutant RIG-I that is constitutively activated (in the absence of dsRNA) was used to trigger IFN-β production. Coexpression of RIG-I, its downstream signaling partner, IPS-1, and NS1 resulted in increased levels of RIG-I and NS1 within an IPS-1-rich, solubilization-resistant fraction after cell lysis. These results suggest that RIG-I, IPS-1, and NS1 become part of the same complex. Consistent with this idea, NS1 was also found to inhibit IFN-β promoter activation by IPS-1 overexpression. Our results indicate that, in addition to sequestering dsRNA, the NS1 of influenza A virus binds to RIG-I and inhibits downstream activation of IRF-3, preventing the transcriptional induction of IFN-β.
The Varicella-Zoster Virus (VZV) ORF9 Protein Interacts with the IE62 Major VZV Transactivator
Authors: Cilloniz Cristian , Jackson Wallen , Grose Charles , Czechowski Donna , Hay John , Ruyechan William T.
Published: December 2006

The varicella-zoster virus (VZV) ORF9 protein is a member of the herpesvirus UL49 gene family but shares limited identity and similarity with the UL49 prototype, herpes simplex virus type 1 VP22. ORF9 mRNA is the most abundantly expressed message during VZV infection; however, little is known concerning the functions of the ORF9 protein. We have found that the VZV major transactivator IE62 and the ORF9 protein can be coprecipitated from infected cells. Yeast two-hybrid analysis localized the region of the ORF9 protein required for interaction with IE62 to the middle third of the protein encompassing amino acids 117 to 186. Protein pull-down assays with GST-IE62 fusion proteins containing N-terminal IE62 sequences showed that amino acids 1 to 43 of the acidic transcriptional activation domain of IE62 can bind recombinant ORF9 protein. Confocal microscopy of transiently transfected cells showed that in the absence of other viral proteins, the ORF9 protein was localized in the cytoplasm while IE62 was localized in the nucleus. In VZV-infected cells, the ORF9 protein was localized to the cytoplasm whereas IE62 exhibited both nuclear and cytoplasmic localization. Cotransfection of plasmids expressing ORF9, IE62, and the viral ORF66 kinase resulted in significant colocalization of ORF9 and IE62 in the cytoplasm. Coimmunoprecipitation experiments with antitubulin antibodies indicate the presence of ORF9-IE62-tubulin complexes in infected cells. Colocalization of ORF9 and tubulin in transfected cells was visualized by confocal microscopy. These data suggest a model for ORF9 protein function involving complex formation with IE62 and possibly other tegument proteins in the cytoplasm at late times in infection.
Protection against Vaccinia Virus Challenge by CD8 Memory T Cells Resolved by Molecular Mimicry
Authors: Cornberg Markus , Sheridan Brian S. , Saccoccio Frances M. , Brehm Michael A. , Selin Liisa K.
Published: December 2006

Live vaccinia virus (VV) vaccination has been highly successful in eradicating smallpox. However, the mechanisms of immunity involved in mediating this protective effect are still poorly understood, and the roles of CD8 T-cell responses in primary and secondary VV infections are not clearly identified. By applying the concept of molecular mimicry to identify potential CD8 T-cell epitopes that stimulate cross-reactive T cells specific to lymphocytic choriomeningitis virus (LCMV) and VV, we identified after screening only 115 peptides two VV-specific immunogenic epitopes that mediated protective immunity against VV. An immunodominant epitope, VV-e7r
130
, did not generate cross-reactive T-cell responses to LCMV, and a subdominant epitope, VV-a11r
198
, did generate cross-reactive responses to LCMV. Infection with VV induced strong epitope-specific responses which were stable into long-term memory and peaked at the time virus was cleared, consistent with CD8 T cells assisting in the control of VV. Two different approaches, direct adoptive transfer of VV-e7r-specific CD8 T cells and prior immunization with a VV-e7r-expressing ubiquitinated minigene, demonstrated that memory CD8 T cells alone could play a significant role in protective immunity against VV. These studies suggest that exploiting cross-reactive responses between viruses may be a useful tool to complement existing technology in predicting immunogenic epitopes to large viruses, such as VV, leading to a better understanding of the role CD8 T cells play during these viral infections.
Mathematical Modeling of Subgenomic Hepatitis C Virus Replication in Huh-7 Cells
Authors: Dahari Harel , Ribeiro Ruy M. , Rice Charles M. , Perelson Alan S.
Published: December 2006

Cell-based hepatitis C virus (HCV) replicon systems have provided a means for understanding HCV replication mechanisms and for testing new antiviral agents. We describe here a mathematical model of HCV replication that assumes that the translation of the HCV polyprotein occurs in the cytoplasm, that HCV RNA synthesis occurs in vesicular-membrane structures, and that the strategy of replication involves a double-stranded RNA intermediate. Our results shed light on the intracellular dynamics of subgenomic HCV RNA replication from transfection to steady state within Huh-7 cells. We predict the following: (i) about 6 × 10
3
ribosomes are involved in generating millions of HCV NS5B-polymerase molecules in a Huh-7 cell, (ii) the observed 10:1 asymmetry of plus- to minus-strand RNA levels can be explained by a higher-affinity (200-fold) interaction of HCV NS5B polymerase-containing replication complexes with HCV minus-strand RNA over HCV plus-strand RNA in order to initiate synthesis, (iii) the latter higher affinity can also account for the observed ∼6:1 plus-strand/minus-strand ratio in vesicular-membrane structures, and (iv) the introduction of higher numbers of HCV plus-strand RNA by transfection leads to faster attainment of steady-state but does not change the steady-state HCV RNA level. Fully permissive HCV replication systems have been developed, and the model presented here is a first step toward building a comprehensive model for complete HCV replication. Moreover, the model can serve as an important tool in understanding HCV replication mechanisms and should prove useful in designing and evaluating new antivirals against HCV.