bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Rim Pathway Mediates Antifungal Tolerance in Candida albicans through Newly Identified Rim101 Transcriptional Targets, Including Hsp90 and Ipt1
Authors: Garnaud Cécile , García-Oliver Encar , Wang Yan , Maubon Danièle , Bailly Sébastien , Despinasse Quentin , Champleboux Morgane , Govin Jérôme , Cornet Muriel 
Published: January 2018

ABSTRACT

Invasive candidiasis (IC) is a major cause of morbidity and mortality despite antifungal treatment. Azoles and echinocandins are used as first-line therapies for IC. However, their efficacy is limited by yeast tolerance and the emergence of acquired resistance. Tolerance is a reversible stage created due to the yeast's capacity to counter antifungal drug exposure, leading to persistent growth. For
Candida albicans
, multiple stress signaling pathways have been shown to contribute to this adaptation. Among them, the pH-responsive Rim pathway, through its transcription factor Rim101p, was shown to mediate azole and echinocandin tolerance. The Rim pathway is fungus specific, is conserved among the members of the fungal kingdom, and plays a key role in pathogenesis and virulence. The present study aimed at confirming the role of Rim101p and investigating the implication of the other Rim proteins in antifungal tolerance in
C. albicans
, as well as the mechanisms underlying it. Time-kill curve experiments and colony formation tests showed that genetic inhibition of all the Rim factors enhances echinocandin and azole antifungal activity. Through RNA sequencing analysis of a
rim101
−/−
mutant, a strain constitutively overexpressing
RIM101
, and control strains, we discovered novel Rim-dependent genes involved in tolerance, including
HSP90
, encoding a major molecular chaperone, and
IPT1
, involved in sphingolipid biosynthesis. Rim mutants were also hypersensitive to pharmacological inhibition of Hsp90. Taken together, these data suggest that Rim101 acts upstream of Hsp90 and that targeting the Rim pathway in combination with existing antifungal drugs may represent a promising antifungal strategy to indirectly but specifically target Hsp90 in yeasts.
Mutant Prevention Concentration and Mutant Selection Window of Micafungin and Anidulafungin in Clinical Candida glabrata Isolates
Authors: Bordallo-Cardona María Ángeles , Marcos-Zambrano Laura Judith , Sánchez-Carrillo Carlos , de la Pedrosa Elia Gómez G. , Cantón Rafael , Bouza Emilio , Escribano Pilar , Guinea Jesús 
Published: January 2018

ABSTRACT

We report the mutant prevention concentration (MPC) and mutant selection window (MSW) for micafungin and anidulafungin administered to treat
Candida glabrata
. We also determine the mutation frequency. We studied 20 echinocandin-susceptible, fluconazole-intermediate, and
FKS
wild-type
C. glabrata
isolates. Adjusted inocula were stroked directly onto Sabouraud agar plates containing different concentrations of micafungin or anidulafungin and visually inspected daily for up to 5 days of incubation. Individual colonies growing on the plates containing echinocandins at 1 mg/liter were selected for antifungal susceptibility testing. The
FKS
genes of the resulting individual phenotypically resistant colonies were sequenced, and the MPC, MSW, and mutation frequency were determined. Biofilm was quantified, and the growth kinetics and virulence (
Galleria mellonella
model) of the resulting individual
FKS
mutant colonies were studied. For micafungin and anidulafungin, we found similar results for the MPC (0.06 to 2 mg/liter and 0.25 to 2 mg/liter, respectively), MSW (0.015 to 2 mg/liter for both echinocandins), and mutation frequency (3.7 × 10
−8
and 2.8 × 10
−8
, respectively). A total of 12 isolates were able to grow at 1 mg/liter on echinocandin-containing plates, yielding a total of 32 phenotypically resistant colonies; however,
FKS2
mutations (ΔF658, S663P, W715L, and E655A) were observed only in 21 colonies. We did not find differences in biofilm formation, the kinetic parameters studied, or the median survival of larvae infected by wild-type isolates and the resulting individual
FKS2
mutant colonies. Echinocandin concentrations lower than 2 mg/liter can lead to selection of resistance mutations in
C. glabrata
isolates
in vitro
.
Transcriptional Responses of Pseudomonas aeruginosa to Potable Water and Freshwater
Authors: English Erika L. , Schutz Kristin C. , Willsey Graham G. , Wargo Matthew J. 
Published: January 2018

ABSTRACT

Many
Pseudomonas aeruginosa
infections are derived from residential, recreational, or surface water sources; thus, these environments represent an important preinfection niche. To better understand
P. aeruginosa
biology in these environments, we quantified transcriptional changes by microarray after exposure to diluted LB, diluted R2B, potable tap water, and freshwater from a eutrophic pond. Quantitative reverse transcription-PCR (qRT-PCR) confirmed the conservation of these responses in other water sources, and competition experiments were used to test the importance of three implicated metabolic pathways. The global transcriptional responses in potable water and freshwater showed strong induction of genes involved in metabolism of the head groups and acyl tails of phospholipids, as well as nucleotide metabolism, with commensurate decreased transcript expression of genes encoding their synthetic pathways. These data suggest that phospholipids and nucleotides are part of the nutritional milieu of these two environments. A unique response in municipal-delivered potable water was to the metals in the piping system, particularly copper. To identify potential nutrient sources used by
P. aeruginosa
in these environments, we used competition assays between the wild-type and deletion mutant strains in three pathways induced under these conditions. For phospholipid head-group metabolism, ethanolamine utilization (
eutB
) was important for competition in potable water, while choline oxidation (
betBA
) was important for competition in freshwater. Nucleotide utilization, particularly pyrimidine metabolism (
dht
), showed a trend toward importance in freshwater but was not statistically significant. These findings provide new insights into the
P. aeruginosa
response to potable water and freshwater and led to the identification of potentially important nutrient sources in these environments.

IMPORTANCE
Much of our knowledge about
Pseudomonas aeruginosa
comes from the infection niche, and much less is known about its lifestyle in the environment.
P. aeruginosa
is an adaptable bacterium capable of growing in many environments but is particularly common in potable water systems and freshwater. We used the transcriptional responses of
P. aeruginosa
to these environments to identify important nutrient sources specific to either of these two environments. Additionally, these environments could provide experimental situations to understand gene function for the large number of transcripts with unknown functions induced under these conditions.
A Cytoplasmic Antiholin Is Embedded In Frame with the Holin in a Lactobacillus fermentum Bacteriophage
Authors: Guo Tingting , Xin Yongping , Zhang Chenchen , Kong Jian 
Published: January 2018

ABSTRACT

In double-stranded DNA bacteriophages, infection cycles are ended by host cell lysis through the action of phage-encoded endolysins and holins. The precise timing of lysis is regulated by the holin inhibitors, named antiholins. Sequence analysis has revealed that holins with a single transmembrane domain (TMD) are prevalent in
Lactobacillus
bacteriophages. A temperate bacteriophage of
Lactobacillus fermentum
, ϕPYB5, has a two-component lysis cassette containing endolysin Lyb5 and holin Hyb5. The
hyb5
gene is 465 bp long, encoding 154 amino acid residues with an N-terminal TMD and a large cytoplasmic C-terminal domain. However, the N terminus contains no dual-start motif, suggesting that Hyb5 oligomerization could be inhibited by a specific antiholin. Two internal open reading frames in
hyb5
,
hyb5
157–465
and
hyb5
209–328
, were identified as genes encoding putative antiholins for Hyb5 and were coexpressed in
trans
with
lyb5-hyb5
in
Escherichia coli
. Surprisingly, host cell lysis was delayed by Hyb5
157–465
but accelerated by abolishment of the translation initiation site of this protein, indicating that Hyb5
157–465
acts as an antiholin to holin Hyb5. Moreover, deletion of 45 amino acid residues at the C terminus of Hyb5 resulted in early cell lysis, even in the presence of Hyb5
157–465
, implying that the interaction between Hyb5
157–465
and Hyb5 occurs at the C terminus of the holin.
In vivo
and
in vitro
, Hyb5
157–465
and Hyb5 were detected in the cytoplasmic and membrane fractions, respectively, and pulldown assays confirmed direct interaction between Hyb5
157–465
and Hyb5. All the results suggest that Hyb5
157–465
is an antiholin of Hyb5 that is involved in lysis timing.

IMPORTANCE
Phage-encoded holins are considered to be the “molecular clock” of phage infection cycles. The interaction between a holin and its inhibitor antiholin precisely regulates the timing of lysis of the host cells. As a prominent biological group in dairy processes, phages of lactic acid bacteria (LAB) have been extensively genome sequenced. However, little is known about the antiholins of LAB phage holins and the holin-antiholin interactions. In this work, we identified an in-frame antiholin against the class III holin of
Lactobacillus fermentum
phage ϕPYB5, Hyb5, and demonstrated its interaction with the cognate holin, which occurred in the bacterial cytoplasm.
Detrimental Influence of Alveolar Macrophages on Protective Humoral Immunity during Francisella tularensis SchuS4 Pulmonary Infection
Authors: Steiner Donald J. , Furuya Yoichi , Metzger Dennis W. 
Published: January 2018

ABSTRACTOpsonizing antibody is a critical component of the host protective immune response against many respiratory pathogens. However, the role of antibodies in protection against pulmonary infection with highly virulentFrancisella tularensisstrain SchuS4 is unclear, and the mechanism that allowsF. tularensisto evade antibody-mediated bacterial clearance is not fully understood. We have now found that depletion of alveolar macrophages reveals an otherwise cryptic protective effect of opsonizing antibody. While antibody opsonization alone failed to confer any survival benefit against SchuS4 lung infection, significant protection was observed when mice were depleted of alveolar macrophages prior to infection. Blood immune signature analyses and bacterial burden measurements indicated that the treatment regimen blocked establishment of productive, systemic infection. In addition, protection was found to be dependent upon neutrophils. The results show for the first time a protective effect of opsonizing antibodies against highly virulentF. tularensisSchuS4 pulmonary infection through depletion of alveolar macrophages, the primary bacterial reservoir, and prevention of systemic dissemination. These findings have important implications for the potential use of therapeutic antibodies against intracellular pathogens that may escape clearance by residing within mucosal macrophages.
Multicenter Evaluation of the Accelerate PhenoTest BC Kit for Rapid Identification and Phenotypic Antimicrobial Susceptibility Testing Using Morphokinetic Cellular Analysis
Authors: Pancholi Preeti , Carroll Karen C. , Buchan Blake W. , Chan Raymond C. , Dhiman Neelam , Ford Bradley , Granato Paul A. , Harrington Amanda T. , Hernandez Diana R. , Humphries Romney M. , Jindra Matthew R. , Ledeboer Nathan A. , Miller Shelley A. , Mochon A. Brian , Morgan Margie A. , Patel Robin , Schreckenberger Paul C. , Stamper Paul D. , Simner Patricia J. , Tucci Nancy E. , Zimmerman Cynthia , Wolk Donna M. 
Published: January 2018

ABSTRACT

We describe results from a multicenter study evaluating the Accelerate Pheno system, a first of its kind diagnostic system that rapidly identifies common bloodstream pathogens from positive blood cultures within 90 min and determines bacterial phenotypic antimicrobial susceptibility testing (AST) results within ∼7 h. A combination of fresh clinical and seeded blood cultures were tested, and results from the Accelerate Pheno system were compared to Vitek 2 results for identification (ID) and broth microdilution or disk diffusion for AST. The Accelerate Pheno system accurately identified 14 common bacterial pathogens and two
Candida
spp. with sensitivities ranging from 94.6 to 100%. Of fresh positive blood cultures, 89% received a monomicrobial call with a positive predictive value of 97.3%. Six common Gram-positive cocci were evaluated for ID. Five were tested against eight antibiotics, two resistance phenotypes (methicillin-resistant
Staphylococcus aureus
and
Staphylococcus
spp. [MRSA/MRS]), and inducible clindamycin resistance (MLSb). From the 4,142 AST results, the overall essential agreement (EA) and categorical agreement (CA) were 97.6% and 97.9%, respectively. Overall very major error (VME), major error (ME), and minor error (mE) rates were 1.0%, 0.7%, and 1.3%, respectively. Eight species of Gram-negative rods were evaluated against 15 antibiotics. From the 6,331 AST results, overall EA and CA were 95.4% and 94.3%, respectively. Overall VME, ME, and mE rates were 0.5%, 0.9%, and 4.8%, respectively. The Accelerate Pheno system has the unique ability to identify and provide phenotypic MIC and categorical AST results in a few hours directly from positive blood culture bottles and support accurate antimicrobial adjustment.
Comparison of Four Serological Methods and Two Reverse Transcription-PCR Assays for Diagnosis and Surveillance of Zika Virus Infection
Authors: Balmaseda Angel , Zambrana José Victor , Collado Damaris , García Nadezna , Saborío Saira , Elizondo Douglas , Mercado Juan Carlos , Gonzalez Karla , Cerpas Cristhiam , Nuñez Andrea , Corti Davide , Waggoner Jesse J. , Kuan Guillermina , Burger-Calderon Raquel , Harris Eva 
Published: January 2018

ABSTRACT
Zika virus (ZIKV) is a mosquito-borne flavivirus that is responsible for recent explosive epidemics in the Americas. Notably, ZIKV infection during pregnancy has been found to cause congenital birth defects, including microcephaly, and ZIKV has been associated with Guillain-Barré syndrome in adults. Diagnosis and surveillance of Zika in the Americas have been challenging due to similar clinical manifestations and extensive antibody cross-reactivity with endemic flaviviral diseases, such as dengue. We evaluated four serological and two reverse transcription-PCR (RT-PCR) methods in acute-phase (mean day, 1.8), early-convalescent-phase (mean day, 16.7), and late-convalescent-phase (mean, ~7 months) samples from the same individuals in a long-term pediatric cohort study in Nicaragua. Well-characterized samples from 301 cases of Zika, dengue, or non-Zika, nondengue febrile illnesses were tested. Compared to a composite reference, an in-house IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the NIAID-Biodefense and Emerging Infections (BEI) MAC-ELISA measuring IgM yielded sensitivities of 94.5% and 70.1% and specificities of 85.6% and 82.8%, respectively. The NS1 blockade-of-binding ELISA measuring anti-ZIKV NS1 antibody levels yielded sensitivities of 85.0% and 96.5% and specificities of 91.4% and 92.6% at early and late convalescence, respectively. An inhibition ELISA detecting total anti-ZIKV antibodies had sensitivity and specificity values of 68.3% and 58.3% for diagnosis and 94.0% and 98.6% for measuring annual infection incidence. Finally, the ZCD and Trioplex real-time RT-PCR assays detecting Zika, chikungunya, and dengue viruses both yielded a sensitivity of 96.1% and specificity of 100%. Together, these assays resolve the urgent need for diagnostic and surveillance tools for countries affected by Zika virus infections.
HLA Class I Downregulation by HIV-1 Variants from Subtype C Transmission Pairs
Authors: Ende Zachary , Deymier Martin J. , Claiborne Daniel T. , Prince Jessica L. , Mónaco Daniela C. , Kilembe William , Allen Susan A. , Hunter Eric 
Published: January 2018

ABSTRACT

HIV-1 downregulates human leukocyte antigen A (HLA-A) and HLA-B from the surface of infected cells primarily to evade CD8 T cell recognition. HLA-C was thought to remain on the cell surface and bind inhibitory killer immunoglobulin-like receptors, preventing natural killer (NK) cell-mediated suppression. However, a recent study found HIV-1 primary viruses have the capacity to downregulate HLA-C. The goal of this study was to assess the heterogeneity of HLA-A, HLA-B, and HLA-C downregulation among full-length primary viruses from six chronically infected and six newly infected individuals from transmission pairs and to determine whether transmitted/founder variants exhibit common HLA class I downregulation characteristics. We measured HLA-A, HLA-B, HLA-C, and total HLA class I downregulation by flow cytometry of primary CD4 T cells infected with 40 infectious molecular clones. Primary viruses mediated a range of HLA class I downregulation capacities (1.3- to 6.1-fold) which could differ significantly between transmission pairs. Downregulation of HLA-C surface expression on infected cells correlated with susceptibility to
in vitro
NK cell suppression of virus release. Despite this, transmitted/founder variants did not share a downregulation signature and instead were more similar to the quasispecies of matched donor partners. These data indicate that a range of viral abilities to downregulate HLA-A, HLA-B, and HLA-C exist within and between individuals that can have functional consequences on immune recognition.

IMPORTANCE
Subtype C HIV-1 is the predominant subtype involved in heterosexual transmission in sub-Saharan Africa. Authentic subtype C viruses that contain natural sequence variations throughout the genome often are not used in experimental systems due to technical constraints and sample availability. In this study, authentic full-length subtype C viruses, including transmitted/founder viruses, were examined for the ability to disrupt surface expression of HLA class I molecules, which are central to both adaptive and innate immune responses to viral infections. We found that the HLA class I downregulation capacity of primary viruses varied, and HLA-C downregulation capacity impacted viral suppression by natural killer cells. Transmitted viruses were not distinct in the capacity for HLA class I downregulation or natural killer cell evasion. These results enrich our understanding of the phenotypic variation existing among natural HIV-1 viruses and how that might impact the ability of the immune system to recognize infected cells in acute and chronic infection.
The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K (gK) Is Required for gB Binding to Akt, Release of Intracellular Calcium, and Fusion of the Viral Envelope with Plasma Membranes
Authors: Musarrat Farhana , Jambunathan Nithya , Rider Paul J. F. , Chouljenko V. N. , Kousoulas K. G. 
Published: January 2018

ABSTRACT
Previously, we have shown that the amino terminus of glycoprotein K (gK) binds to the amino terminus of gB and that deletion of the amino-terminal 38 amino acids of gK prevents herpes simplex virus 1 (HSV-1) infection of mouse trigeminal ganglia after ocular infection and virus entry into neuronal axons. Recently, it has been shown that gB binds to Akt during virus entry and induces Akt phosphorylation and intracellular calcium release. Proximity ligation and two-way immunoprecipitation assays using monoclonal antibodies against gB and Akt-1 phosphorylated at S473 [Akt-1(S473)] confirmed that HSV-1(McKrae) gB interacted with Akt-1(S473) during virus entry into human neuroblastoma (SK-N-SH) cells and induced the release of intracellular calcium. In contrast, the gB specified by HSV-1(McKrae) gKΔ31-68, lacking the amino-terminal 38 amino acids of gK, failed to interact with Akt-1(S473) and induce intracellular calcium release. The Akt inhibitor miltefosine inhibited the entry of McKrae but not the gKΔ31-68 mutant into SK-N-SH cells. Importantly, the entry of the gKΔ31-68 mutant but not McKrae into SK-N-SH cells treated with the endocytosis inhibitors pitstop-2 and dynasore hydrate was significantly inhibited, indicating that McKrae gKΔ31-68 entered via endocytosis. These results suggest that the amino terminus of gK functions to regulate the fusion of the viral envelope with cellular plasma membranes.

IMPORTANCE
HSV-1 glycoprotein B (gB) functions in the fusion of the viral envelope with cellular membranes during virus entry. Herein, we show that a deletion in the amino terminus of glycoprotein K (gK) inhibits gB binding to Akt-1(S473), the release of intracellular calcium, and virus entry via fusion of the viral envelope with cellular plasma membranes.
Germ Line IgM Is Sufficient, but Not Required, for Antibody-Mediated Alphavirus Clearance from the Central Nervous System
Authors: Nilaratanakul Voraphoj , Chen Jie , Tran Oanh , Baxter Victoria K. , Troisi Elizabeth M. , Yeh Jane X. , Griffin Diane E. 
Published: January 2018

ABSTRACTSindbis virus (SINV) infection of neurons in the brain and spinal cord in mice provides a model system for investigating recovery from encephalomyelitis and antibody-mediated clearance of virus from the central nervous system (CNS). To determine the roles of IgM and IgG in recovery, we compared the responses of immunoglobulin-deficient activation-induced adenosine deaminase-deficient (AID−/−), secretory IgM-deficient (sIgM−/−), and AID−/−sIgM−/−double-knockout (DKO) mice with those of wild-type (WT) C57BL/6 mice for disease, clearance of infectious virus and viral RNA from brain and spinal cord, antibody responses, and B cell infiltration into the CNS. Because AID is essential for immunoglobulin class switch recombination and somatic hypermutation, AID−/−mice produce only germ line IgM, while sIgM−/−mice secrete IgG but no IgM and DKO mice produce no secreted immunoglobulin. After intracerebral infection with the TE strain of SINV, most mice recovered. Development of neurologic disease occurred slightly later in sIgM−/−mice, but disease severity, weight loss, and survival were similar between the groups. AID−/−mice produced high levels of SINV-specific IgM, while sIgM−/−mice produced no IgM and high levels of IgG2a compared to WT mice. All mice cleared infectious virus from the spinal cord, but DKO mice failed to clear infectious virus from brain and had higher levels of viral RNA in the CNS late after infection. The numbers of infected cells and the amount of cell death in brain were comparable. We conclude that antibody is required and that either germ line IgM or IgG is sufficient for clearance of virus from the CNS.IMPORTANCEMosquito-borne alphaviruses that infect neurons can cause fatal encephalomyelitis. Recovery requires a mechanism for the immune system to clear virus from infected neurons without harming the infected cells. Antiviral antibody has previously been shown to be a noncytolytic means for alphavirus clearance. Antibody-secreting cells enter the nervous system after infection and produce antiviral IgM before IgG. Clinical studies of human viral encephalomyelitis suggest that prompt production of IgM is associated with recovery, but it was not known whether IgM is effective for clearance. Our studies used mice deficient in production of IgM, IgG, or both to characterize the antibody necessary for alphavirus clearance. All mice developed similar signs of neurologic disease and recovered from infection. Antibody was necessary for virus clearance from the brain, and either early germ line IgM or IgG was sufficient. These studies support the clinical observation that prompt production of antiviral antibody is a determinant of outcome.