bluuebunny/crossref_metadata_2025_split huggingface dataset:
Calcineurin Controls Drug Tolerance, Hyphal Growth, and Virulence in Candida dubliniensis
Authors: Chen Ying-Lien , Brand Alexandra , Morrison Emma L. , Silao Fitz Gerald S. , Bigol Ursela G. , Malbas Fedelino F. , Nett Jeniel E. , Andes David R. , Solis Norma V. , Filler Scott G. , Averette Anna , Heitman Joseph 
Published: April 2011

ABSTRACT

Candida dubliniensis
is an emerging pathogenic yeast species closely related to
Candida albicans
and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during
C. dubliniensis
infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens
C. albicans
,
Cryptococcus neoformans
, and
Aspergillus fumigatus
. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of
C. dubliniensis
to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to
C. albicans
, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in
C. dubliniensis
. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in
C. dubliniensis
. Similar to
C. albicans
,
C. dubliniensis
calcineurin enhances survival in serum.
C. dubliniensis
calcineurin and
crz1/crz1
mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that
C. dubliniensis
calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging
C. dubliniensis
infections.
Differential Chitinase Activity and Production within Francisella Species, Subspecies, and Subpopulations
Authors: Chandler Jeffrey C. , Molins Claudia R. , Petersen Jeannine M. , Belisle John T. 
Published: April 2011

ABSTRACT

Genotyping of
Francisella tularensis
(A1a, A1b, A2, and type B) and
Francisella novicida
has identified multiple differences between species and among
F. tularensis
subspecies and subpopulations. Variations in virulence, geographic distribution, and ecology are also known to exist among this group of bacteria, despite the >95% nucleotide identity in their genomes. This study expands the description of phenotypic differences by evaluating the ability of
F. tularensis
and
F. novicida
to degrade chitin analogs and produce active chitinases. Endochitinase activities were observed to vary among
F. tularensis
and
F. novicida
strains. The activity observed for
F. tularensis
strains was predominantly associated with whole-cell lysates, while the chitinase activity of
F. novicida
localized to the culture supernatant. In addition, the overall level of chitinase activity differed among the subpopulations of
F. tularensis
and between the species. Bioinformatic analyses identified two new putative chitinase genes (
chiC
and
chiD
), as well as the previously described
chiA
and
chiB
. However, the presence of these four open reading frames as intact genes or pseudogenes was found to differ between
Francisella
species and
F. tularensis
subspecies and subpopulations. Recombinant production of the putative chitinases and enzymatic evaluations revealed ChiA, ChiB, ChiC, and ChiD possessed dissimilar chitinase activities. These biochemical studies coupled with bioinformatic analyses and the evaluation of
chiA
and
chiC
knockouts in
F. tularensis
A1 and A2 strains, respectively, provided a molecular basis to explain the differential chitinase activities observed among the species and subpopulations of
Francisella
.
PtrA Is a Periplasmic Protein Involved in Cu Tolerance in Pseudomonas aeruginosa
Authors: Elsen Sylvie , Ragno Michel , Attree Ina 
Published: April 2011

ABSTRACT

In this work, we demonstrate that PtrA (U. H. Ha et al., Mol. Microbiol. 54:307–320, 2004) is a periplasmic protein, specifically synthesized in the presence of copper, that is involved in the tolerance of
Pseudomonas aeruginosa
to copper. Our biochemical and genetic analyses challenge its role in transcriptional inhibition of the type III secretion system.
Complete Genome Sequence and Immunoproteomic Analyses of the Bacterial Fish Pathogen Streptococcus parauberis
Authors: Nho Seong Won , Hikima Jun-ichi , Cha In Seok , Park Seong Bin , Jang Ho Bin , del Castillo Carmelo S. , Kondo Hidehiro , Hirono Ikuo , Aoki Takashi , Jung Tae Sung 
Published: April 2011

ABSTRACT

Although
Streptococcus parauberis
is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (
Paralichthys olivaceus
) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses.
S. parauberis
contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of
S. parauberis
(serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins.
S. parauberis
possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to
Streptococcus uberis. S. parauberis
antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in
S. parauberis
, by reaction with an antiserum obtained from
S. parauberis
-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the
Streptococcaceae
.
Characterization of CrgA, a New Partner of the Mycobacterium tuberculosis Peptidoglycan Polymerization Complexes
Authors: Plocinski P. , Ziolkiewicz M. , Kiran M. , Vadrevu S. I. , Nguyen H. B. , Hugonnet J. , Veckerle C. , Arthur M. , Dziadek J. , Cross T. A. , Madiraju M. , Rajagopalan M. 
Published: April 2011

ABSTRACT

The role(s) in cell division of the
Mycobacterium tuberculosis
Rv0011c gene product, a homolog of the
Streptomyces
CrgA protein that is responsible for coordinating growth and cytokinesis in sporogenic aerial hyphae, is largely unknown. We show that an enhanced cyan fluorescent protein-
M. tuberculosis
CrgA (ECFP-CrgA
MT
) fusion protein is localized to the cell membrane, midcell, and cell pole regions in
Mycobacterium smegmatis
. Furthermore, the ECFP-CrgA
MT
fusion protein colocalized with FtsZ-enhanced yellow fluorescent protein (EYFP) in
M. smegmatis
. Bacterial two-hybrid assays indicated strong interactions of
M. tuberculosis
CrgA with FtsZ, FtsQ, and the class B penicillin-binding proteins, FtsI (PBPB) and PBPA. The midcell localization of CrgA
MT
was severely compromised under conditions of FtsZ depletion, which indicated that CrgA localizes to the midcell region after assembly of the FtsZ ring.
M. tuberculosis
cells with reduced CrgA levels were elongated and grew more slowly than wild-type cells, which indicated defects in cell division, whereas CrgA overproduction did not show growth defects. A
M. smegmatis
Δ
crgA
strain exhibited a bulged cell morphology, elongated cells with a chain-like phenotype, cells with polar bulbous structures, and a modest growth defect. FtsZ and FtsI levels were not affected in cells producing altered levels of CrgA. Septal and membrane localization of GFP-FtsI was enhanced by CrgA overproduction and was diminished in a Δ
crgA
strain, which indicates that one role of CrgA is to promote and/or stabilize FtsI localization. Overall, these data indicate that CrgA is a novel member of the cell division complex in mycobacteria and possibly facilitates septum formation.
Roles of Cysteine Proteases Cwp84 and Cwp13 in Biogenesis of the Cell Wall of Clostridium difficile
Authors: de la Riva Lucía , Willing Stephanie E. , Tate Edward W. , Fairweather Neil F. 
Published: April 2011

ABSTRACT

Clostridium difficile
expresses a number of cell wall proteins, including the abundant high-molecular-weight and low-molecular-weight S-layer proteins (SLPs). These proteins are generated by posttranslational cleavage of the precursor SlpA by the cysteine protease Cwp84. We compared the phenotypes of
C. difficile
strains containing insertional mutations in either
cwp84
or its paralog
cwp13
and complemented with plasmids expressing wild-type or mutant forms of their genes. We show that the presence of uncleaved SlpA in the cell wall of the
cwp84
mutant results in aberrant retention of other cell wall proteins at the cell surface, as demonstrated by secretion of the proteins Cwp66 and Cwp2 into the growth medium. These phenotypes are restored by complementation with a plasmid expressing wild-type Cwp84 enzyme but not with one encoding a Cys116Ala substitution in the active site. The
cwp13
mutant cleaved the SlpA precursor normally and had a wild-type-like colony phenotype. Both Cwp84 and Cwp13 are produced as proenzymes which are processed by cleavage to produce mature enzymes. In the case of Cwp84, this cleavage does not appear to be autocatalytic, whereas in Cwp13 autocatalysis was demonstrated as a Cys109Ala mutant did not undergo processing. Cwp13 appears to have a role in processing of Cwp84 but is not essential for Cwp84 activity. Cwp13 cleaves SlpA in the HMW SLP domain, which we suggest may reflect a role in cleavage and degradation of misfolded proteins at the cell surface.
The Poor Growth of Rhodospirillum rubrum Mutants Lacking RubisCO Is Due to the Accumulation of Ribulose-1,5-Bisphosphate
Authors: Wang Di , Zhang Yaoping , Pohlmann Edward L. , Li Jilun , Roberts Gary P. 
Published: April 2011

ABSTRACT

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first step of CO
2
fixation in the Calvin-Benson-Bassham (CBB) cycle. Besides its function in fixing CO
2
to support photoautotrophic growth, the CBB cycle is also important under photoheterotrophic growth conditions in purple nonsulfur photosynthetic bacteria. It has been assumed that the poor photoheterotrophic growth of RubisCO-deficient strains was due to the accumulation of excess intracellular reductant, which implied that the CBB cycle is important for maintaining the redox balance under these conditions. However, we present analyses of
cbbM
mutants in
Rhodospirillum rubrum
that indicate that toxicity is the result of an elevated intracellular pool of ribulose-1,5-bisphosphate (RuBP). There is a redox effect on growth, but it is apparently an indirect effect on the accumulation of RuBP, perhaps by the regulation of the activities of enzymes involved in RuBP regeneration. Our studies also show that the CBB cycle is not essential for
R. rubrum
to grow under photoheterotrophic conditions and that its role in controlling the redox balance needs to be further elucidated. Finally, we also show that CbbR is a positive transcriptional regulator of the
cbb
operon (
cbbEFPT
) in
R. rubrum
, as seen with related organisms, and define the transcriptional organization of the
cbb
genes.
Characteristic Phenotypes Associated with
ptsN
-Null Mutants in Escherichia coli K-12 Are Absent in Strains with Functional
ilvG
Authors: Reaves Marshall Louis , Rabinowitz Joshua D. 
Published: April 2011

ABSTRACT

The phosphotransferase system (PTS), encompassing EI, HPr, and assorted EII proteins, uses phosphoenolpyruvate to import and phosphorylate sugars. A paralog of EIIA of the sugar PTS system known as
ptsN
has been purported to regulate organic nitrogen source utilization in
Escherichia coli
K-12. Its known biochemical function, however, relates to potassium homeostasis. The evidence for regulation of organic nitrogen source utilization by
ptsN
is based primarily on the defective growth of Δ
ptsN
mutants on amino acid nitrogen sources and other nutrient combinations. These observations were made with
E. coli
strains MG1655 and W3110, which carry a nonfunctional version of
ilvG
. There are three isozymes that effectively catalyze the first committed step of branched-chain amino acid biosynthesis, but
ilvG
is unique for doing so effectively across a range of potassium concentrations. Here we show that all of the nutrient utilization phenotypes attributed to
ptsN
are manifested selectively in strains lacking functional
ilvG
. We conclude that the
ptsN
gene product does not regulate organic nitrogen source utilization as previously proposed.
Inference of the Transcriptional Regulatory Network in Staphylococcus aureus by Integration of Experimental and Genomics-Based Evidence
Authors: Ravcheev Dmitry A. , Best Aaron A. , Tintle Nathan , DeJongh Matthew , Osterman Andrei L. , Novichkov Pavel S. , Rodionov Dmitry A. 
Published: April 2011

ABSTRACT

Transcriptional regulatory networks are fine-tuned systems that help microorganisms respond to changes in the environment and cell physiological state. We applied the comparative genomics approach implemented in the RegPredict Web server combined with SEED subsystem analysis and available information on known regulatory interactions for regulatory network reconstruction for the human pathogen
Staphylococcus aureus
and six related species from the family
Staphylococcaceae
. The resulting reference set of 46 transcription factor regulons contains more than 1,900 binding sites and 2,800 target genes involved in the central metabolism of carbohydrates, amino acids, and fatty acids; respiration; the stress response; metal homeostasis; drug and metal resistance; and virulence. The inferred regulatory network in
S. aureus
includes ∼320 regulatory interactions between 46 transcription factors and ∼550 candidate target genes comprising 20% of its genome. We predicted ∼170 novel interactions and 24 novel regulons for the control of the central metabolic pathways in
S. aureus
. The reconstructed regulons are largely variable in the
Staphylococcaceae
: only 20% of
S. aureus
regulatory interactions are conserved across all studied genomes. We used a large-scale gene expression data set for
S. aureus
to assess relationships between the inferred regulons and gene expression patterns. The predicted reference set of regulons is captured within the
Staphylococcus
collection in the RegPrecise database (
http://regprecise.lbl.gov
).
Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA
Authors: Demas Allison , Oberstaller Jenna , DeBarry Jeremy , Lucchi Naomi W. , Srinivasamoorthy Ganesh , Sumari Deborah , Kabanywanyi Abdunoor M. , Villegas Leopoldo , Escalante Ananias A. , Kachur S. Patrick , Barnwell John W. , Peterson David S. , Udhayakumar Venkatachalam , Kissinger Jessica C. 
Published: April 2011

ABSTRACT

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting
Plasmodium
, differentiating between
Plasmodium
species, and detecting subclinical infections. Despite available whole-genome sequence data for
Plasmodium falciparum
and
P. vivax
, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of
P. falciparum
and
P. vivax
to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7
P. falciparum
strains and 91
P. falciparum
clinical isolates from Tanzania and 10
P. vivax
strains and 96
P. vivax
clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of
P. falciparum
and
P. vivax
in conventional or multiplex PCR platforms.
Natural Substrate Concentrations Can Modulate the Prophylactic Efficacy of Nucleotide HIV Reverse Transcriptase Inhibitors
Authors: García-Lerma J. Gerardo , Aung Wutyi , Cong Mian-er , Zheng Qi , Youngpairoj Ae S. , Mitchell James , Holder Angela , Martin Amy , Kuklenyik Susan , Luo Wei , Lin Carol Yen-Chin , Hanson Debra L. , Kersh Ellen , Pau Chou-Pong , Ray Adrian S. , Rooney James F. , Lee William A. , Heneine Walid 
Published: April 2011

ABSTRACT

Preexposure prophylaxis (PrEP) with antiretroviral drugs is a novel human immunodeficiency virus (HIV) prevention strategy. It is generally thought that high systemic and mucosal drug levels are sufficient for protection. We investigated whether GS7340, a next-generation tenofovir (TFV) prodrug that effectively delivers tenofovir diphosphate (TFV-DP) to lymphoid cells and tissues, could protect macaques against repeated weekly rectal simian-human immunodeficiency virus (SHIV) exposures. Macaques received prophylactic GS7340 treatment 3 days prior to each virus exposure. At 3 days postdosing, TFV-DP concentrations in peripheral blood mononuclear cells (PBMCs) were about 50-fold higher than those seen with TFV disoproxil fumarate (TDF), and they remained above 1,000 fmol/10
6
cells for as long as 7 days. TFV-DP accumulated in lymphoid and rectal tissues, with concentrations at 3 days exceeding 500 fmol/10
6
mononuclear cells. Despite high mucosal and systemic TFV levels, GS7340 was not protective. Since TFV-DP blocks reverse transcription by competing with the natural dATP substrate, we measured dATP contents in peripheral lymphocytes, lymphoid tissue, and rectal mononuclear cells. Compared to those in circulating lymphocytes and lymphoid tissue, rectal lymphocytes had 100-fold higher dATP concentrations and dATP/TFV-DP ratios, likely reflecting the activated status of the cells and suggesting that TFV-DP may be less active at the rectal mucosa. Our results identify dATP/TFV-DP ratios as a possible correlate of protection by TFV and suggest that natural substrate concentrations at the mucosa will likely modulate the prophylactic efficacy of nucleotide reverse transcriptase inhibitors.
Safety and efficacy of intracranial stenting for acute ischemic stroke beyond 8 h of symptom onset
Authors: Xavier Andrew R , Tiwari Ambooj , Purai Natasha , Rayes Mahmoud , Pandey Paritosh , Kansara Amit , Narayanan Sandra , Chaturvedi Seemant 
Published: April 2011

ObjectiveTo report our experience with stent supported intracranial recanalization for acute ischemic stroke beyond 8 h of symptoms onset.BackgroundAcute ischemic stroke (AIS) therapy is often limited to an 8 h window using mechanical means. However, recent reports have shown delayed recanalization beyond 8 h might be a viable option in a subset of patients.MethodsA retrospective review was performed of our AIS database for patients who underwent stent supported intracranial recanalization beyond 8 h of symptom onset. Clinical and angiographic data were reviewed. Outcome was measured using modified Rankin Scale (mRS) scores at 30 and 90 days.Results12 patients (11 men and one woman) underwent delayed stenting for AIS. Mean age was 49 years (range 37–73) and mean National Institutes of Health Stroke Scale was 17 (range 8–29, median 15). Mean time from stroke onset to intervention was 66.1 h (range 10–168 h, median 46 h). 10 patients presented with a Thrombolysis in Myocardial Infarction (TIMI) score of 0 and the remaining two had a TIMI of 1. Recanalized vessels included: left middle cerebral artery (n=6), basilar trunk (n=2), vertebrobasilar junction (n=3) and internal carotid artery (ICA)-T (n=1). Four patients had prior attempts of embolectomy/thrombolysis using mechanical and chemical means. Stents used included: six balloon mounted stents, five Wingspan and one Enterprise self-expanding intracranial stent. Recanalization, defined as a TIMI score of 2 or more, was achieved in 11 patients. Two patients (17%) had intracranial hemorrhage. Thirty day mRS of ≤3 was achieved in six patients (50%). Seven patients (58%) had a 90 day mRS of ≤2.ConclusionStent supported intracranial recanalization is a safe and feasible approach in a selective group of patients presenting with acute ischemic stroke beyond 8 h of symptom onset.
Absence of MMP2 Expression Correlates with Poor Clinical Outcomes in Rectal Cancer, and Is Distinct from MMP1-Related Outcomes in Colon Cancer
Authors: Wong John C.T. , Chan Simon K. , Schaeffer David F. , Sagaert Xavier , Lim Howard J. , Kennecke Hagen , Owen David A. , Suh Kwang W. , Kim Young-Bae , Tai Isabella T. 
Published: April 2011

Abstract
Purpose: Treatments for colorectal cancer (CRC) are primarily disease stage based. However, heterogeneity in outcome within even a single stage highlights its limitations in predicting disease behavior. Recently, the role of gene expression as predictive and prognostic markers has been explored. Our objectives were to identify consistently differentially expressed genes through meta-analysis of high-throughput gene-expression studies, and evaluate their predictive and prognostic significance in colon (CC) and rectal (RC) cancers.
Experimental Design: Publications applying high-throughput gene- expression technologies to specific CRC stages were identified. A vote counting strategy was used to identify the most significant differentially expressed genes. Their predictive and prognostic values were independently assessed in a tissue microarray of 191 cases of stage II-IV CC/RC from two tertiary care centers. Their biological effects were also examined in vitro.
Results: MMP1 and MMP2 were identified as consistently underexpressed in liver metastasis compared with primary CRC. Shorter time to distant metastasis and overall survival occurred in stage III CC lacking MMP1 expression, and in stage III RC lacking MMP2. MMP1 levels in stage II and III CC were associated with increased likelihood of distant metastasis, whereas the risk of local recurrence in stage III RC could be stratified by MMP2. Promotion of cell invasion of CRC cell lines exposed to MMP1/2 inhibitors were confirmed in vitro.
Conclusions: MMP1 and MMP2 may be useful biomarkers that can help stratify patients at higher risk of developing recurrence in colorectal cancer, and guide individualized treatment decisions to achieve better outcomes. Clin Cancer Res; 17(12); 4167–76. ©2011 AACR.