bluuebunny/crossref_metadata_2025_split huggingface dataset:
Application of a Seven-Target Pyrosequencing Assay To Improve the Detection of Neuraminidase Inhibitor-Resistant Influenza A(H3N2) Viruses
Authors: Tamura Daisuke , Okomo-Adhiambo Margaret , Mishin Vasiliy P. , Guo Zhu , Xu Xiyan , Villanueva Julie , Fry Alicia M. , Stevens James , Gubareva Larisa V. 
Published: February 2015

ABSTRACT

National U.S. influenza antiviral surveillance incorporates data generated by neuraminidase (NA) inhibition (NI) testing of isolates supplemented with NA sequence analysis and pyrosequencing analysis of clinical specimens. A lack of established correlates for clinically relevant resistance to NA inhibitors (NAIs) hinders interpretation of NI assay data. Nonetheless, A(H3N2) viruses are commonly monitored for moderately or highly reduced inhibition in the NI assay and/or for the presence of NA markers E119V, R292K, and N294S. In 2012 to 2013, three drug-resistant A(H3N2) viruses were detected by NI assay among isolates (
n
= 1,424); all showed highly reduced inhibition by oseltamivir and had E119V. In addition, one R292K variant was detected among clinical samples (
n
= 1,024) by a 3-target pyrosequencing assay. Overall, the frequency of NAI resistance was low (0.16% [4 of 2,448]). To screen for additional NA markers previously identified in viruses from NAI-treated patients, the pyrosequencing assay was modified to include Q136K, I222V, and deletions encompassing residues 245 to 248 (del245-248) and residues 247 to 250 (del247-250). The 7-target pyrosequencing assay detected NA variants carrying E119V, Q136, and del245-248 in an isolate from an oseltamivir-treated patient. Next, this assay was applied to clinical specimens collected from hospitalized patients and submitted for NI testing but failed cell culture propagation. Of the 27 clinical specimens tested, 4 (15%) contained NA changes: R292K (
n
= 2), E119V (
n
= 1), and del247-250 (
n
= 1). Recombinant NAs with del247-250 or del245-248 conferred highly reduced inhibition by oseltamivir, reduced inhibition by zanamivir, and normal inhibition by peramivir and laninamivir. Our results demonstrated the benefits of the 7-target pyrosequencing assay in conducting A(H3N2) antiviral surveillance and testing for clinical care.
A Fluorescent Microplate Assay Quantifies Bacterial Efflux and Demonstrates Two Distinct Compound Binding Sites in AcrB
Authors: Iyer Ramkumar , Ferrari Annette , Rijnbrand R. , Erwin Alice L. 
Published: February 2015

ABSTRACT

A direct assay of efflux by
Escherichia coli
AcrAB-TolC and related multidrug pumps would have great value in discovery of new Gram-negative antibiotics. The current understanding of how efflux is affected by the chemical structure and physical properties of molecules is extremely limited, derived from antibacterial data for compounds that inhibit growth of wild-type
E. coli
. We adapted a previously described fluorescent efflux assay to a 96-well microplate format that measured the ability of test compounds to compete for efflux with Nile Red (an environment-sensitive fluor), independent of antibacterial activity. We show that Nile Red and the lipid-sensitive probe DiBAC
4
-(3) [bis-(1,3-dibutylbarbituric acid)-trimethine oxonol] can quantify efflux competition in
E. coli
. We extend the previous findings that the tetracyclines compete with Nile Red and show that DiBAC
4
-(3) competes with macrolides. The extent of the competition shows a modest correlation with the effect of the
acrB
deletion on MICs within the compound sets for both dyes. Crystallographic studies identified at least two substrate binding sites in AcrB, the proximal and distal pockets. High-molecular-mass substrates bound the proximal pocket, while low-mass substrates occupied the distal pocket. As DiBAC
4
-(3) competes with macrolides but not with Nile Red, we propose that DiBAC
4
-(3) binds the proximal pocket and Nile Red likely binds the distal site. In conclusion, competition with fluorescent probes can be used to study the efflux process for diverse chemical structures and may provide information as to the site of binding and, in some cases, enable rank-ordering a series of related compounds by efflux.
IRGM3 Contributes to Immunopathology and Is Required for Differentiation of Antigen-Specific Effector CD8+T Cells in Experimental Cerebral Malaria
Authors: Guo Jintao , McQuillan James A. , Yau Belinda , Tullo Gregory S. , Long Carole A. , Bertolino Patrick , Roediger Ben , Weninger Wolfgang , Taylor Gregory A. , Hunt Nicholas H. , Ball Helen J. , Mitchell Andrew J. 
Published: February 2015

ABSTRACTGamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection withPlasmodiumspecies. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used thePlasmodium bergheiANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA forIrgm1andIrgm3was found in the brains and spleens of infected mice at times of peak IFN-γ production.Irgm3−/−but notIrgm1−/−mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8+T cells within the brain. Although antigen-specific proliferation of transferred CD8+T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred intoIrgm3−/−recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens ofIrgm3−/−mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology inIrgm3−/−mice occurs due to impaired generation of CD8+effector function. This defect is nonintrinsic to CD8+T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.
Mutant Brucella abortus Membrane Fusogenic Protein Induces Protection against Challenge Infection in Mice
Authors: de Souza Filho Job Alves , Martins Vicente de Paulo , Campos Priscila Carneiro , Alves-Silva Juliana , Santos Nathalia V. , de Oliveira Fernanda Souza , Menezes Gustavo B. , Azevedo Vasco , Cravero Silvio Lorenzo , Oliveira Sergio Costa 
Published: February 2015

ABSTRACTBrucellaspecies can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence ofBrucellaspp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of theBrucellamembrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed thatB. abortusΔmfp::kanand Δomp19::kandeletion mutant strains have reduced persistencein vivoin C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kanor Δomp19::kanstrain expressing green fluorescent protein (GFP) approximately 80% or 65% ofBrucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments.B. abortusΔomp19::kanwas attenuatedin vivobut had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kanstrain had a lower virulence in these same mouse models. Furthermore, Δmfp::kanand Δomp19::kanstrains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kanstrain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kanstrain induced protection similar to that of RB51. Thus, these results demonstrate thatBrucellaMfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kanmutant may serve as a potential vaccine candidate in future studies.
Dendritic Cell-Based Immunization Ameliorates Pulmonary Infection with Highly Virulent Cryptococcus gattii
Authors: Ueno Keigo , Kinjo Yuki , Okubo Yoichiro , Aki Kyoko , Urai Makoto , Kaneko Yukihiro , Shimizu Kiminori , Wang Dan-Ni , Okawara Akiko , Nara Takuya , Ohkouchi Kayo , Mizuguchi Yuki , Kawamoto Susumu , Kamei Katsuhiko , Ohno Hideaki , Niki Yoshihito , Shibuya Kazutoshi , Miyazaki Yoshitsugu 
Published: February 2015

ABSTRACT

Cryptococcosis due to a highly virulent fungus,
Cryptococcus gattii
, emerged as an infectious disease on Vancouver Island in Canada and surrounding areas in 1999, causing deaths among immunocompetent individuals. Previous studies indicated that
C. gattii
strain R265 isolated from the Canadian outbreak had immune avoidance or immune suppression capabilities. However, protective immunity against
C. gattii
has not been identified. In this study, we used a gain-of-function approach to investigate the protective immunity against
C. gattii
infection using a dendritic cell (DC)-based vaccine. Bone marrow-derived dendritic cells (BMDCs) efficiently engulfed acapsular
C. gattii
(Δ
cap60
strain), which resulted in their expression of costimulatory molecules and inflammatory cytokines. This was not observed for BMDCs that were cultured with encapsulated strains. When Δ
cap60
strain-pulsed BMDCs were transferred to mice prior to intratracheal R265 infection, significant amelioration of pathology, fungal burden, and the survival rate resulted compared with those in controls. Multinucleated giant cells (MGCs) that engulfed fungal cells were significantly increased in the lungs of immunized mice. Interleukin 17A (IL-17A)-, gamma interferon (IFN-γ)-, and tumor necrosis factor alpha (TNF-α)-producing lymphocytes were significantly increased in the spleens and lungs of immunized mice. The protective effect of this DC vaccine was significantly reduced in IFN-γ knockout mice. These results demonstrated that an increase in cytokine-producing lymphocytes and the development of MGCs that engulfed fungal cells were associated with the protection against pulmonary infection with highly virulent
C. gattii
and suggested that IFN-γ may have been an important mediator for this vaccine-induced protection.
Uncoupling of Pro- and Anti-Inflammatory Properties of Staphylococcus aureus
Authors: Peres Adam G. , Stegen Camille , Li Junbin , Xu An Qi , Levast Benoit , Surette Michael G. , Cousineau Benoit , Desrosiers Martin , Madrenas Joaquín 
Published: February 2015

ABSTRACTStaphylococcus aureusis a Gram-positive bacterium that is carried by a quarter of the healthy human population and that can cause severe infections. This pathobiosis has been linked to a balance between Toll-like receptor 2 (TLR2)-dependent pro- and anti-inflammatory responses. The relationship between these two types of responses is unknown. Analysis of 16 nasal isolates ofS. aureusshowed heterogeneity in their capacity to induce pro- and anti-inflammatory responses, suggesting that these two responses are independent of each other. Uncoupling of these responses was corroborated by selective signaling through phosphoinositol 3-kinase (PI3K)-Akt-mTOR and extracellular signal-regulated kinase (ERK) for the anti-inflammatory response and through p38 for the proinflammatory response. Uncoupling was also observed at the level of phagocytosis and phagosomal processing ofS. aureus, which were required solely for the proinflammatory response. Importantly, the anti-inflammatory properties of anS. aureusisolate correlated with its ability to modulate T cell immunity. Our results suggest the presence of anti-inflammatory TLR2 ligands in the staphylococcal cell wall, whose identification may provide templates for novel immunomodulatory drugs.
Identification and Characterization of Clostridium perfringens Beta Toxin Variants with Differing Trypsin Sensitivity and
In Vitro
Cytotoxicity Activity
Authors: Theoret James R. , Uzal Francisco A. , McClane Bruce A. 
Published: February 2015

ABSTRACT

By producing toxins,
Clostridium perfringens
causes devastating diseases of both humans and animals.
C. perfringens
beta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure-function relationship. Amino acid sequence comparisons of the CPBs made by 8 randomly selected isolates identified two natural variant toxins with four conserved amino acid changes, including a switch of E to K at position 168 (E168K) that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate whether this potential trypsin cleavage site affects sensitivity to trypsin, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. The results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch by using site-directed recombinant CPB (rCPB) mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant that possessed more cytotoxic activity. Mapping of sequence variations on a CPB structure modeled using related toxins suggests that the E168K substitution is surface localized and so can interact with trypsin and that the A300V substitution is located in a putative binding domain of the CPB toxin.
Rab14 Regulates Maturation of Macrophage Phagosomes Containing the Fungal Pathogen Candida albicans and Outcome of the Host-Pathogen Interaction
Authors: Okai Blessing , Lyall Natalie , Gow Neil A. R. , Bain Judith M. , Erwig Lars-Peter 
Published: February 2015

ABSTRACT

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogen
Candida albicans
undergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated by
C. albicans
hyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment of
C. albicans
cells. Phagosomes containing live
C. albicans
cells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages toward
C. albicans
cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing by
C. albicans
. Therefore, Rab14 activity promotes phagosome maturation during
C. albicans
infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.
Binding of Complement Factor H to PorB3 and NspA Enhances Resistance of Neisseria meningitidis to Anti-Factor H Binding Protein Bactericidal Activity
Authors: Giuntini Serena , Pajon Rolando , Ram Sanjay , Granoff Dan M. 
Published: February 2015

ABSTRACTAmong 25 serogroup BNeisseria meningitidisclinical isolates, we identified four (16%) with high factor H binding protein (FHbp) expression that were resistant to complement-mediated bactericidal activity of sera from mice immunized with recombinant FHbp vaccines. Two of the four isolates had evidence of human FH-dependent complement downregulation independent of FHbp. Since alternative complement pathway recruitment is critical for anti-FHbp bactericidal activity, we hypothesized that in these two isolates binding of FH to ligands other than FHbp contributes to anti-FHbp bactericidal resistance. Knocking out NspA, a known meningococcal FH ligand, converted both resistant isolates to anti-FHbp susceptible isolates. The addition of a nonbactericidal anti-NspA monoclonal antibody to the bactericidal reaction also increased anti-FHbp bactericidal activity. To identify a role for FH ligands other than NspA or FHbp in resistance, we created double NspA/FHbp knockout mutants. Mutants from both resistant isolates bound 10-fold more recombinant human FH domains 6 and 7 fused to Fc than double knockout mutants prepared from two sensitive meningococcal isolates. In light of recent studies showing functional FH-PorB2 interactions, we hypothesized that PorB3 from the resistant isolates recruited FH. Allelic exchange ofporB3from a resistant isolate to a sensitive isolate increased resistance of the sensitive isolate to anti-FHbp bactericidal activity (and vice versa). Thus, some PorB3 variants functionally bind human FH, which in the presence of NspA enhances anti-FHbp resistance. Combining anti-NspA antibodies with anti-FHbp antibodies can overcome resistance. Meningococcal vaccines that target both NspA and FHbp are likely to confer greater protection than either antigen alone.
Protein A Is Released into the Staphylococcus aureus Culture Supernatant with an Unprocessed Sorting Signal
Authors: O'Halloran Dara P. , Wynne Kieran , Geoghegan Joan A. 
Published: February 2015

ABSTRACT

The immunoglobulin binding protein A (SpA) of
Staphylococcus aureus
is synthesized as a precursor with a C-terminal sorting signal. The sortase A enzyme mediates covalent attachment to peptidoglycan so that SpA is displayed on the surface of the bacterium. Protein A is also found in the extracellular medium, but the processes involved in its release are not fully understood. Here, we show that a portion of SpA is released into the supernatant with an intact sorting signal, indicating that it has not been processed by sortase A. Release of SpA was reduced when the native sorting signal of SpA was replaced with the corresponding region of another sortase-anchored protein (SdrE). Similarly, a reporter protein fused to the sorting signal of SpA was released to a greater extent than the same polypeptide fused to the SdrE sorting signal. Released SpA protected bacteria from killing in human blood, indicating that it contributes to immune evasion.
The Bacteroides thetaiotaomicron Protein Bacteroides Host Factor A Participates in Integration of the Integrative Conjugative Element CTnDOT into the Chromosome
Authors: Ringwald Kenneth , Gardner Jeffrey 
Published: February 2015

ABSTRACT

CTnDOT is a conjugative transposon found in
Bacteroides
species. It encodes multiple antibiotic resistances and is stimulated to transfer by exposure to tetracycline. CTnDOT integration into the host chromosome requires IntDOT and a previously unknown host factor. We have identified a protein, designated BHFa (
Bacteroides
host factor A), that participates in integrative recombination. BHFa is the first host factor identified for a site-specific recombination reaction in the CTnDOT family of integrative and conjugative elements. Based on the amino acid sequence of BHFa, the ability to bind specifically to 4 sites in the
attDOT
DNA, and its activity in the integration reaction, BHFa is a member of the IHF/HU family of nucleoid-associated proteins. Other DNA bending proteins that bind DNA nonspecifically can substitute for BHFa in the integration reaction.

IMPORTANCE
Bacteroides
species are normal members of the human colonic microbiota. These species can harbor and spread self-transmissible genetic elements (integrative conjugative elements [ICEs]) that contain antibiotic resistance genes. This work describes the role of a protein, BHFa, and its importance in the integration reaction required for the element CTnDOT to persist in
Bacteroides
host cells.
The Enhancer Binding Protein Nla6 Regulates Developmental Genes That Are Important for Myxococcus xanthus Sporulation
Authors: Giglio Krista M. , Zhu Chengjun , Klunder Courtney , Kummer Shelley , Garza Anthony G. 
Published: February 2015

ABSTRACTIn the bacteriumMyxococcus xanthus, starvation triggers the formation of multicellular fruiting bodies containing thousands of stress-resistant spores. Recent work showed that fruiting body development is regulated by a cascade of transcriptional activators called enhancer binding proteins (EBPs). The EBP Nla6 is a key component of this cascade; it regulates the promoters of other EBP genes, including a downstream-functioning EBP gene that is crucial for sporulation. In recent expression studies, hundreds of Nla6-dependent genes were identified, suggesting that the EBP gene targets of Nla6 may be part of a much larger regulon. The goal of this study was to identify and characterize genes that belong to the Nla6 regulon. Accordingly, a direct repeat [consensus, C(C/A)ACGNNGNC] binding site for Nla6 was identified usingin vitroandin vivomutational analyses, and the sequence was subsequently used to find 40 potential developmental promoter (88 gene) targets. We showed that Nla6 binds to the promoter region of four new targets (asgE,exo, MXAN2688, and MXAN3259)in vitroand that Nla6 is important for their normal expressionin vivo. Phenotypic studies indicate that all of the experimentally confirmed targets of Nla6 are primarily involved in sporulation. These targets include genes involved in transcriptional regulation, cell-cell signal production, and spore differentiation and maturation. Although sporulation occurs late in development, all of the developmental loci analyzed here show an Nla6-dependent burst in expression soon after starvation is induced. This finding suggests that Nla6 starts preparing cells for sporulation very early in the developmental process.IMPORTANCEBacterial development yields a remarkable array of complex multicellular forms. One such form, which is commonly found in nature, is a surface-associated aggregate of cells known as a biofilm. Mature biofilms are structurally complex and contain cells that are highly resistant to antibacterial agents. When starving, the model bacteriumMyxococcus xanthusforms a biofilm containing a thin mat of cells and multicellular structures that house a highly resistant cell type called a myxospore. Here, we identify the promoter binding site of the transcriptional activator Nla6, identify genes in the Nla6 regulon, and show that several of the genes in the Nla6 regulon are important for production of stress-resistant spores in starvation-inducedM. xanthusbiofilms.