bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combinatorial Effect of Two Framework Mutations (E119V and I222L) in the Neuraminidase Active Site of H3N2 Influenza Virus on Resistance to Oseltamivir
Authors: Richard Mathilde , Ferraris Olivier , Erny Alexandra , Barthélémy Mendy , Traversier Aurélien , Sabatier Murielle , Hay Alan , Lin Yi Pu , Russell Rupert J. , Lina Bruno 
Published: March 2011

ABSTRACTNeuraminidase (NA) inhibitors (NIs) are the first line of defense against influenza virus. Reverse genetics experiments allow the study of resistance mechanisms by anticipating the impacts of mutations to the virus. To look at the possibility of an increased effect on the resistance phenotype of a combination of framework mutations, known to confer resistance to oseltamivir or zanamivir, with limited effect on virus fitness, we constructed 4 viruses by reverse genetics in the A/Moscow/10/99 H3N2 background containing double mutations in their neuraminidase genes: E119D+I222L, E119V+I222L, D198N+I222L, and H274Y+I222L (N2 numbering). Among the viruses produced, the E119D+I222L mutant virus was not able to grow without bacterial NA complementation and the D198N+I222L mutant and H274Y+I222L mutant were not stable after passages in MDCK cells. The E119V+I222L mutant was stable after five passages in MDCK cells. This E119V-and-I222L combination had a combinatorial effect on oseltamivir resistance. The total NA activity of the E119V+I222L mutant was low (5% compared to that of the wild-type virus). This drop in NA activity resulted from a decreased NA quantity in the virion in comparison to that of the wild-type virus (1.4% of that of the wild type). In MDCK-SIAT1 cells, the E119V+I222L mutant virus did not present a replicative advantage over the wild-type virus, even in the presence of oseltamivir. Double mutations combining two framework mutations in the NA gene still have to be monitored, as they could induce a high level of resistance to NIs, without impairing the NA affinity. Our study allows a better understanding of the diversity of the mechanisms of resistance to NIs.
Wild-Type MIC Distributions and Epidemiological Cutoff Values for Caspofungin and Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 Document)
Authors: Espinel-Ingroff A. , Fothergill A. , Fuller J. , Johnson E. , Pelaez T. , Turnidge J. 
Published: March 2011

ABSTRACT

Clinical breakpoints have not been established for mold testing. Epidemiologic cutoff values (ECVs) are available for six
Aspergillus
spp. and the triazoles, but not for caspofungin. Wild-type (WT) minimal effective concentration (MEC) distributions (organisms in a species-drug combination with no acquired resistance mechanisms) were defined in order to establish ECVs for six
Aspergillus
spp. and caspofungin. The number of available isolates was as follows: 1,691
A. fumigatus
, 432
A. flavus
, 192
A. nidulans
, 440
A. niger
, 385
A. terreus
, and 75
A. versicolor
isolates. CLSI broth microdilution MEC data gathered in five independent laboratories in Canada, Europe, and the United States were aggregated for the analyses. ECVs expressed in μg/ml that captured 95% and 99% of the modeled wild-type population were for
A. fumigatus
0.5 and 1,
A. flavus
0.25 and 0.5,
A. nidulans
0.5 and 0.5,
A. niger
0.25 and 0.25,
A. terreus
0.25 and 0.5, and
A. versicolor
0.25 and 0.5. Although caspofungin ECVs are not designed to predict the outcome of therapy, they may aid in the detection of strains with reduced antifungal susceptibility to this agent and acquired resistance mechanisms.
Dynamics of the Action of Biocides in Pseudomonas aeruginosa Biofilms
Authors: Bridier A. , Dubois-Brissonnet F. , Greub G. , Thomas V. , Briandet R. 
Published: March 2011

ABSTRACT

The biocidal activity of peracetic acid (PAA) and benzalkonium chloride (BAC) on
Pseudomonas aeruginosa
biofilms was investigated by using a recently developed confocal laser scanning microscopy (CLSM) method that enables the direct and real-time visualization of cell inactivation within the structure. This technique is based on monitoring the loss of fluorescence that corresponds to the leakage of a fluorophore out of cells due to membrane permeabilization by the biocides. Although this approach has previously been used with success with various Gram-positive species, it is not directly applicable to the visualization of Gram-negative strains such as
P. aeruginosa
, particularly because of limitations regarding fluorescence staining. After adapting the staining procedure to
P. aeruginosa
, the action of PAA and BAC on the biofilm formed by strain ATCC 15442 was investigated. The results revealed specific inactivation patterns as a function of the mode of action of the biocides. While PAA treatment triggered a uniform loss of fluorescence in the structure, the action of BAC was first localized at the periphery of cell clusters and then gradually spread throughout the biofilm. Visualization of the action of BAC in biofilms formed by three clinical isolates then confirmed the presence of a delay in penetration, showing that diffusion-reaction limitations could provide a major explanation for the resistance of
P. aeruginosa
biofilms to this biocide. Biochemical analysis suggested a key role for extracellular matrix characteristics in these processes.
Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, Belonging to the “phiKMV-Like Viruses”
Authors: Adriaenssens Evelien M. , Ceyssens Pieter-Jan , Dunon Vincent , Ackermann Hans-Wolfgang , Van Vaerenbergh Johan , Maes Martine , De Proft Maurice , Lavigne Rob 
Published: March 2011

ABSTRACTPantoea agglomeransis a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight ofP. agglomeransare lytic phages, isolated from soil samples, belonging to thePodoviridaeand are the firstPantoeaphages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of theAutographivirinae, within the genus of the “phiKMV-like viruses.” Phylogenetic analysis of all the sequenced members of theAutographivirinaesupports the classification of phages LIMElight and LIMEzero as members of the “phiKMV-like viruses” and corroborates the subdivision into the different genera. These data expand the knowledge ofPantoeaphages and illustrate the wide host diversity of phages within the “phiKMV-like viruses.”
Targeted Allele Replacement Mutagenesis of Corynebacterium pseudotuberculosis
Authors: Walker Caray A. , Donachie Willie , Smith David G. E. , Fontaine Michael C. 
Published: March 2011

ABSTRACT

A two-step allele replacement mutagenesis procedure, using a conditionally replicating plasmid, was developed to allow the creation of targeted, marker-free mutations in
Corynebacterium pseudotuberculosis
. The relationship between homologous sequence length and recombination frequency was determined, and enhanced plasmid excision was observed due to the rolling-circle replication of the mutagenesis vector. Furthermore, an antibiotic enrichment procedure was applied to improve the recovery of mutants. Subsequently, as proof of concept, a marker-free,
cp40
-deficient mutant of
C. pseudotuberculosis
was constructed.
Functional Analysis of the
Trichoderma harzianum nox1
Gene, Encoding an NADPH Oxidase, Relates Production of Reactive Oxygen Species to Specific Biocontrol Activity against Pythium ultimum
Authors: Montero-Barrientos M. , Hermosa R. , Cardoza R. E. , Gutiérrez S. , Monte E. 
Published: March 2011

ABSTRACT

The synthesis of reactive oxygen species (ROS) is one of the first events following pathogenic interactions in eukaryotic cells, and NADPH oxidases are involved in the formation of such ROS. The
nox1
gene of
Trichoderma harzianum
was cloned, and its role in antagonism against phytopathogens was analyzed in
nox1
-overexpressed transformants. The increased levels of
nox1
expression in these transformants were accompanied by an increase in ROS production during their direct confrontation with
Pythium ultimum
. The transformants displayed an increased hydrolytic pattern, as determined by comparing protease, cellulase, and chitinase activities with those for the wild type. In confrontation assays against
P. ultimum
the
nox1
-overexpressed transformants were more effective than the wild type, but not in assays against
Botrytis cinerea
or
Rhizoctonia solani
. A transcriptomic analysis using a
Trichoderma
high-density oligonucleotide (HDO) microarray also showed that, compared to gene expression for the interaction of wild-type
T. harzianum
and
P. ultimum
, genes related to protease, cellulase, and chitinase activities were differentially upregulated in the interaction of a
nox1
-overexpressed transformant with this pathogen. Our results show that
nox1
is involved in
T. harzianum
ROS production and antagonism against
P. ultimum
.
Genome Signatures of Escherichia coli O157:H7 Isolates from the Bovine Host Reservoir
Authors: Eppinger Mark , Mammel Mark K. , LeClerc Joseph E. , Ravel Jacques , Cebula Thomas A. 
Published: March 2011

ABSTRACT

Cattle comprise a main reservoir of Shiga toxin-producing
Escherichia coli
O157:H7 (STEC). The significant differences in host prevalence, transmissibility, and virulence phenotypes among strains from bovine and human sources are of major interest to the public health community and livestock industry. Genomic analysis revealed divergence into three lineages: lineage I and lineage I/II strains are commonly associated with human disease, while lineage II strains are overrepresented in the asymptomatic bovine host reservoir. Growing evidence suggests that genotypic differences between these lineages, such as polymorphisms in Shiga toxin subtypes and synergistically acting virulence factors, are correlated with phenotypic differences in virulence, host ecology, and epidemiology. To assess the genomic plasticity on a genome-wide scale, we have sequenced the whole genome of strain EC869, a bovine-associated
E. coli
O157:H7 isolate. Comparative phylogenomic analysis of this key isolate enabled us to place accurately bovine lineage II strains within the genetically homogenous
E. coli
O157:H7 clade. Identification of polymorphic loci that are anchored both in the chromosomal backbone and horizontally acquired regions allowed us to associate bovine genotypes with altered virulence phenotypes and host prevalence. This study catalogued numerous novel lineage II-specific genome signatures, some of which appear to be associated intimately with the altered pathogenic potential and niche adaptation within the bovine rumen. The presented extended list of polymorphic markers is valuable in the development of a robust typing system critical for forensic, diagnostic, and epidemiological studies of this emerging human pathogen.
Grx4 Monothiol Glutaredoxin Is Required for Iron Limitation-Dependent Inhibition of Fep1
Authors: Jbel Mehdi , Mercier Alexandre , Labbé Simon 
Published: March 2011

ABSTRACTThe expression of iron transport genes inSchizosaccharomyces pombeis controlled by the Fep1 transcription factor. When iron levels exceed those needed by the cells, Fep1 represses iron transport genes. In contrast, Fep1 is unable to bind chromatin under low-iron conditions, and that results in activation of genes involved in iron acquisition. Studies of fungi have revealed that monothiol glutaredoxins are required to inhibit iron-dependent transcription factors in response to high levels of iron. Here, we show that the monothiol glutaredoxin Grx4 plays an important role in the negative regulation of Fep1 activity in response to iron deficiency. Deletion of thegrx4+gene led to constitutive promoter occupancy by Fep1 and caused an invariable repression of iron transport genes. We found that Grx4 and Fep1 physically interact with each other. Grx4 contains an N-terminal thioredoxin (TRX)-like domain and a C-terminal glutaredoxin (GRX)-like domain. Deletion mapping analysis revealed that the TRX domain interacts strongly and constitutively with the C-terminal region of Fep1. As opposed to the TRX domain, the GRX domain associates weakly and in an iron-dependent manner with the N-terminal region of Fep1. Further analysis showed that Cys35 of Grx4 is required for the interaction between the Fep1 C terminus and the TRX domain, whereas Grx4 Cys172 is necessary for the association between the Fep1 N terminus and the GRX domain. Our results describe the first example of a monothiol glutaredoxin that acts as an inhibitory partner for an iron-regulated transcription factor under conditions of low iron levels.
A Chlamydia trachomatis OmcB C-Terminal Fragment Is Released into the Host Cell Cytoplasm and Is Immunogenic in Humans
Authors: Qi Manli , Gong Siqi , Lei Lei , Liu Quanzhong , Zhong Guangming 
Published: March 2011

ABSTRACT

The
Chlamydia trachomatis
outer membrane complex protein B (OmcB) is an antigen with diagnostic and vaccine relevance. To further characterize OmcB, we generated antibodies against OmcB C-terminal (OmcBc) and N-terminal (OmcBn) fragments. Surprisingly, the anti-OmcBc antibody detected dominant signals in the host cell cytosol, while the anti-OmcBn antibody exclusively labeled intrainclusion signals in
C. trachomatis
-infected cells permeabilized with saponin. Western blot analyses revealed that OmcB was partially processed into OmcBc and OmcBn fragments. The processed OmcBc was released into host cell cytosol, while the OmcBn and remaining full-length OmcB were retained within the chlamydial inclusions. The organism-associated OmcB epitopes became detectable only after the
C. trachomatis
-infected cells were permeabilized with strong detergents such as SDS. However, the harsh permeabilization conditions also led to the leakage of the already secreted OmcBc and chlamydia-secreted protease (CPAF) out of the host cells. The OmcBc processing and release occurred in all biovars of
C. trachomatis
. Moreover, the released OmcBc but not the retained OmcBn was highly immunogenic in
C. trachomatis
-infected women, which is consistent with the concept that exposure of chlamydial proteins to host cell cytosol is accompanied by increased immunogenicity. These observations have provided important information for further exploring/optimizing OmcB as a target for the development of diagnosis methods and vaccines.
Vitamin D-Mediated Induction of Innate Immunity in Gingival Epithelial Cells
Authors: McMahon Laura , Schwartz Kyell , Yilmaz Ozlem , Brown Eleith , Ryan Lisa K. , Diamond Gill 
Published: March 2011

ABSTRACTHuman gingival epithelial cells (GEC) produce peptides, such as β-defensins and the cathelicidin LL-37, that are both antimicrobial and that modulate the innate immune response. In myeloid and airway epithelial cells, the active form of vitamin D3[1,25(OH)2D3] increases the expression and antibacterial activity of LL-37. To examine the activity of vitamin D on the innate immune defense of the gingival epithelium, cultured epithelial cells were treated with either 10−8M 1,25(OH)2D3or ethanol for up to 24 h. A time-dependent induction of LL-37 mRNA up to 13-fold at 24 h in both standard monolayer and three-dimensional cultures was observed. Induction of the vitamin D receptor and the 1-α-hydroxylase genes was also observed. The hydroxylase was functional, as LL-37 induction was observed in response to stimulation by 25(OH)D3. Through microarray analysis of other innate immune genes, CD14 expression increased 4-fold, and triggering receptor expressed on myeloid cells-1 (TREM-1) was upregulated 16-fold after 24 h of treatment with 1,25(OH)2D3. TREM-1 is a pivotal amplifier of the innate immune response in macrophages, leading to increased production by inflammatory response genes. Activation of TREM-1 on the GEC led to an increase in interleukin-8 (IL-8) mRNA levels. Incubation of three-dimensional cultures with 1,25(OH)2D3led to an increase in antibacterial activity against the periodontal pathogenAggregatibacter actinomycetemcomitanswhen the bacteria were added to the apical surface. This study is the first to demonstrate the effect of vitamin D on antibacterial defense of oral epithelial cells, suggesting that vitamin D3could be utilized to enhance the innate immune defense in the oral cavity.
Role of Fibronectin-Binding Proteins A and B inIn VitroCellular Infections andIn VivoSeptic Infections by Staphylococcus aureus
Authors: Shinji Hitomi , Yosizawa Yukio , Tajima Akiko , Iwase Tadayuki , Sugimoto Shinya , Seki Keiko , Mizunoe Yoshimitsu 
Published: March 2011

ABSTRACTFibronectin-binding protein A (FnBPA) and FnBPB are important adhesins forStaphylococcus aureusinfection. We constructedfnbAand/orfnbBmutant strains fromS. aureusSH1000, which possesses intactrsbU, and studied the role of these adhesins inin vitroandin vivoinfections. In intravenous infection, allfnbmutants caused a remarkable reduction in the colonization rate in kidneys and the mortality rate of mice.fnbBmutant caused a more severe decrease in body weight than that caused byfnbAmutant. Serum levels of interleukin-6 and nuclear factor κB (NF-κB) activation in spleen cells were remarkably reduced infnbAorfnbA fnbBmutant infections; however, there was no significant reduction infnbBmutant infections. Inin vitrocellular infection, FnBPA was shown to be indispensable for adhesion to and internalization by nonprofessional phagocytic cells upon ingestion by inflammatory macrophages and NF-κB activation. However, both FnBPs were required for efficient cellular responses. The results showed that FnBPA is more important forin vitroandin vivoinfections; however, cooperation between FnBPA and FnBPB is indispensable for the induction of severe infection resulting in septic death.
MyD88 Signaling Is Required for Efficient Innate and Adaptive Immune Responses to Paracoccidioides brasiliensis Infection
Authors: Loures Flávio V. , Pina Adriana , Felonato Maíra , Feriotti Claudia , de Araújo Eliseu F. , Calich Vera L. G. 
Published: March 2011

ABSTRACTThe mechanisms that govern the initial interaction betweenParacoccidioides brasiliensis, a primary dimorphic fungal pathogen, and cells of the innate immunity need to be clarified. Our previous studies showed that Toll-like receptor 2 (TLR2) and TLR4 regulate the initial interaction of fungal cells with macrophages and the pattern of adaptive immunity that further develops. The aim of the present investigation was to assess the role of MyD88, an adaptor molecule used by TLRs to activate genes of the inflammatory response in pulmonary paracoccidioidomycosis. Studies were performed with normal and MyD88−/−C57BL/6 mice intratracheally infected withP. brasiliensisyeast cells. MyD88−/−macrophages displayed impaired interaction with fungal yeast cells and produced low levels of IL-12, MCP-1, and nitric oxide, thus allowing increased fungal growth. Compared with wild-type (WT) mice, MyD88−/−mice developed a more severe infection of the lungs and had marked dissemination of fungal cells to the liver and spleen. MyD88−/−mice presented low levels of Th1, Th2, and Th17 cytokines, suppressed lymphoproliferation, and impaired influx of inflammatory cells to the lungs, and this group of cells comprised lower numbers of neutrophils, activated macrophages, and T cells. Nonorganized, coalescent granulomas, which contained high numbers of fungal cells, characterized the severe lesions of MyD88−/−mice; the lesions replaced extensive areas of several organs. Therefore, MyD88−/−mice were unable to control fungal growth and showed a significantly decreased survival time. In conclusion, our findings demonstrate that MyD88 signaling is important in the activation of fungicidal mechanisms and the induction of protective innate and adaptive immune responses againstP. brasiliensis.
Serine Protease PrtA from Streptococcus pneumoniae Plays a Role in the Killing of S. pneumoniae by Apolactoferrin
Authors: Mirza Shaper , Wilson Landon , Benjamin William H. , Novak Jan , Barnes Stephen , Hollingshead Susan K. , Briles David E. 
Published: March 2011

ABSTRACT

It is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, including
Streptococcus pneumoniae
. Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease,
prtA
, lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin (
P
< 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin kills
Streptococcus pneumoniae
requires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci.
The Collagen-Binding Protein Cnm Is Required for Streptococcus mutans Adherence to and Intracellular Invasion of Human Coronary Artery Endothelial Cells
Authors: Abranches Jacqueline , Miller James H. , Martinez Alaina R. , Simpson-Haidaris Patricia J. , Burne Robert A. , Lemos José A. 
Published: March 2011

ABSTRACT

Streptococcus mutans
is considered the primary etiologic agent of dental caries, a global health problem that affects 60 to 90% of the population, and a leading causative agent of infective endocarditis. It can be divided into four different serotypes (
c
,
e
,
f
, and
k
), with serotype
c
strains being the most common in the oral cavity. In this study, we demonstrate that in addition to OMZ175 and B14, three other strains (NCTC11060, LM7, and OM50E) of the less prevalent serotypes
e
and
f
are able to invade primary human coronary artery endothelial cells (HCAEC). Invasive strains were also significantly more virulent than noninvasive strains in the
Galleria mellonella
(greater wax worm) model of systemic disease. Interestingly, the invasive strains carried an additional gene,
cnm
, which was previously shown to bind to collagen and laminin
in vitro
. Inactivation of
cnm
rendered the organisms unable to invade HCAEC and attenuated their virulence in
G. mellonella
. Notably, the
cnm
knockout strains did not adhere to HCAEC as efficiently as the parental strains did, indicating that the loss of the invasion phenotype observed for the mutants was linked to an adhesion defect. Comparisons of the invasive strains and their respective
cnm
mutants did not support a correlation between biofilm formation and invasion. Thus, Cnm is required for
S. mutans
invasion of endothelial cells and possibly represents an important virulence factor of
S. mutans
that may contribute to cardiovascular infections and pathologies.