bluuebunny/crossref_metadata_2025_split huggingface dataset:
Wsp1 Is Downstream of Cin1 and Regulates Vesicle Transport and Actin Cytoskeleton as an Effector of Cdc42 and Rac1 in Cryptococcus neoformans
Authors: Shen Gui , Zhou Erxun , Alspaugh J. Andrew , Wang Ping 
Published: February 2012

ABSTRACT

Human Wiskott-Aldrich syndrome protein (WASP) is a scaffold linking upstream signals to the actin cytoskeleton. In response to intersectin ITSN1 and Rho GTPase Cdc42, WASP activates the Arp2/3 complex to promote actin polymerization. The human pathogen
Cryptococcus neoformans
contains the ITSN1 homolog Cin1 and the WASP homolog Wsp1, which share more homology with human proteins than those of other fungi. Here we demonstrate that Cin1, Cdc42/Rac1, and Wsp1 function in an effector pathway similar to that of mammalian models. In the
cin1
mutant, expression of the autoactivated Wsp1-B-GBD allele partially suppressed the mutant defect in endocytosis, and expression of the constitutively active
CDC42

Q61L

allele restored normal actin cytoskeleton structures. Similar phenotypic suppression can be obtained by the expression of a Cdc42-green fluorescent protein (GFP)-Wsp1 fusion protein. In addition, Rac1, which was found to exhibit a role in early endocytosis, activates Wsp1 to regulate vacuole fusion. Rac1 interacted with Wsp1 and depended on Wsp1 for its vacuolar membrane localization. Expression of the Wsp1-B-GBD allele restored vacuolar membrane fusion in the
rac1
mutant. Collectively, our studies suggest novel ways in which this pathogenic fungus has adapted conserved signaling pathways to control vesicle transport and actin organization, likely benefiting survival within infected hosts.
Morphology of the Trypanosome Bilobe, a Novel Cytoskeletal Structure
Authors: Esson Heather J. , Morriswood Brooke , Yavuz Sevil , Vidilaseris Keni , Dong Gang , Warren Graham 
Published: February 2012

ABSTRACT
The trypanosome bilobe is a cytoskeletal structure of unclear function. To date, four proteins have been shown to localize stably to it: TbMORN1, TbLRRP1, TbCentrin2, and TbCentrin4. In this study, a combination of immunofluorescence microscopy and electron microscopy was used to explore the morphology of the bilobe and its relationship to other nearby cytoskeletal structures in the African trypanosome procyclic trypomastigote. The use of detergent/salt-extracted flagellum preparations was found to be an effective way of discerning features of the cytoskeletal ultrastructure that are normally obscured. TbMORN1 and TbCentrin4 together define a hairpin structure comprising an arm of TbCentrin4 and a fishhook of TbMORN1. The two arms flank a specialized microtubule quartet and the flagellum attachment zone filament, with TbMORN1 running alongside the former and TbCentrin4 alongside the latter. The hooked part of TbMORN1 sits atop the flagellar pocket collar marked by TbBILBO1. The TbMORN1 bilobe occasionally exhibits tendrillar extensions that seem to be connected to the basal and probasal bodies. The TbMORN1 molecules present on these tendrils undergo higher rates of turnover than those for molecules on the main bilobe structure. These observations have been integrated with previous detailed descriptions of the cytoskeletal elements in trypanosome cells.
Role of a SpoVA Protein in Dipicolinic Acid Uptake into Developing Spores of Bacillus subtilis
Authors: Li Yunfeng , Davis Andrew , Korza George , Zhang Pengfei , Li Yong-qing , Setlow Barbara , Setlow Peter , Hao Bing 
Published: February 2012

ABSTRACTThe proteins encoded by thespoVAoperon, including SpoVAD, are essential for the uptake of the 1:1 chelate of pyridine-2,6-dicarboxylic acid (DPA2,6) and Ca2+into developing spores of the bacteriumBacillus subtilis. The crystal structure ofB. subtilisSpoVAD has been determined recently, and a structural homology search revealed that SpoVAD shares significant structural similarity but not sequence homology to a group of enzymes that bind to and/or act on small aromatic molecules. We find that molecular docking placed DPA2,6exclusively in a highly conserved potential substrate-binding pocket in SpoVAD that is similar to that in the structurally homologous enzymes. We further demonstrate that SpoVAD binds both DPA2,6and Ca2+-DPA2,6with a similar affinity, while exhibiting markedly weaker binding to other DPA isomers. Importantly, mutations of conserved amino acid residues in the putative DPA2,6-binding pocket in SpoVAD essentially abolish its DPA2,6-binding capacity. Moreover, replacement of the wild-typespoVADgene inB. subtiliswith any of thesespoVADgene variants effectively eliminated DPA2,6uptake into developing spores in sporulation, although the variant proteins were still located in the spore inner membrane. Our results provide direct evidence that SpoVA proteins, in particular SpoVAD, are directly involved in DPA2,6movement into developingB. subtilisspores.
The Yersinia pestis Rcs Phosphorelay Inhibits Biofilm Formation by Repressing Transcription of the Diguanylate Cyclase Gene
hmsT
Authors: Sun Yi-Cheng , Guo Xiao-Peng , Hinnebusch B. Joseph , Darby Creg 
Published: February 2012

ABSTRACT

Yersinia pestis
, which causes bubonic plague, forms biofilms in fleas, its insect vectors, as a means to enhance transmission. Biofilm development is positively regulated by
hmsT
, encoding a diguanylate cyclase that synthesizes the bacterial second messenger cyclic-di-GMP. Biofilm development is negatively regulated by the Rcs phosphorelay signal transduction system. In this study, we show that Rcs-negative regulation is accomplished by repressing transcription of
hmsT.
Effect of Periodontal Pathogens on the Metatranscriptome of a Healthy Multispecies Biofilm Model
Authors: Frias-Lopez Jorge , Duran-Pinedo Ana 
Published: February 2012

ABSTRACT

Oral bacterial biofilms are highly complex microbial communities with up to 700 different bacterial taxa. We report here the use of metatranscriptomic analysis to study patterns of community gene expression in a multispecies biofilm model composed of species found in healthy oral biofilms (
Actinomyces naeslundii
,
Lactobacillus casei
,
Streptococcus mitis
,
Veillonella parvula
, and
Fusobacterium nucleatum
) and the same biofilm plus the periodontopathogens
Porphyromonas gingivalis
and
Aggregatibacter actinomycetemcomitans
. The presence of the periodontopathogens altered patterns in gene expression, and data indicate that transcription of protein-encoding genes and small noncoding RNAs is stimulated. In the healthy biofilm hypothetical proteins, transporters and transcriptional regulators were upregulated while chaperones and cell division proteins were downregulated. However, when the pathogens were present, chaperones were highly upregulated, probably due to increased levels of stress. We also observed a significant upregulation of ABC transport systems and putative transposases. Changes in Clusters of Orthologous Groups functional categories as well as gene set enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that in the absence of pathogens, only sets of proteins related to transport and secondary metabolism were upregulated, while in the presence of pathogens, proteins related to growth and division as well as a large portion of transcription factors were upregulated. Finally, we identified several small noncoding RNAs whose predicted targets were genes differentially expressed in the open reading frame libraries. These results show the importance of pathogens controlling gene expression of a healthy oral community and the usefulness of metatranscriptomic techniques to study gene expression profiles in complex microbial community models.
Biochemical Characterization of the CDP-
d
-Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F
Authors: Wang Quan , Xu Yanli , Perepelov Andrei V. , Knirel Yuriy A. , Reeves Peter R. , Shashkov Alexander S. , Guo Xi , Ding Peng , Feng Lu 
Published: February 2012

ABSTRACT

Streptococcus pneumoniae
is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of
d
-arabinitol, which is present in the CPSs of several
S. pneumoniae
serotypes, has never been identified. In this study, the genes
abpA
(previously known as
abp1
) and
abpB
(previously known as
abp2
), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-
d
-arabinitol (the nucleotide-activated form of
d
-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result,
abpA
was identified to be a
d
-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to
d
-xylulose-5-phosphate (
d
-Xlu-5-P) to form CDP-
d
-xylulose, and
abpB
was characterized to be a CDP-
d
-xylulose reductase-encoding gene, responsible for the conversion of CDP-
d
-xylulose to CDP-
d
-arabinitol as the final product. The kinetic parameters of AbpA for the substrates
d
-Xlu-5-P and CTP and those of AbpB for the substrate CDP-
d
-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes
abpA
and
abpB
and their products in the biosynthetic pathway of CDP-
d
-arabinitol.
Biological Roles of Nontypeable Haemophilus influenzae Type IV Pilus Proteins Encoded by the
pil
and
com
Operons
Authors: Carruthers Michael D. , Tracy Erin N. , Dickson Amanda C. , Ganser Kara B. , Munson Robert S. , Bakaletz Lauren O. 
Published: February 2012

ABSTRACT

We previously demonstrated that one or more products of the genes in the
pil
and
com
gene clusters of the opportunistic human respiratory pathogen nontypeable
Haemophilus influenzae
(NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the
pilABCD
and
comABCDEF
gene clusters are operons and that the product of each gene is essential for normal pilus function. Mutants with nonpolar deletions in each of the 10
pil
and
com
genes had an adherence defect when primary human airway cells were used as the target. These mutants were also diminished in their ability to form a biofilm
in vitro
and, additionally, were deficient in natural transformation. Collectively, our data demonstrate that the product of each gene within these operons is required for the normal biogenesis and/or function of NTHI Tfp. Based on the similarity of PilA to other type IV pilins, we further predicted that the product of the
pilA
gene would be the major pilin subunit. Toward that end, we also demonstrated by immunogold labeling and mass spectrometry that PilA is indeed the majority type IV pilin protein expressed by NTHI. These new observations set the stage for experiments designed to dissect the function of each of the proteins encoded by genes within the
pil
and
com
gene clusters. The ability to characterize individual proteins with vital roles in NTHI colonization or pathogenesis has the potential to reduce the burden of NTHI-induced diseases through development of a Tfp-derived vaccine or a pilus-directed therapeutic.
Spontaneous Excision of the O-Polysaccharide
wbkA
Glycosyltranferase Gene Is a Cause of Dissociation of Smooth to Rough Brucella Colonies
Authors: Mancilla Marcos , Marín Clara M. , Blasco José M. , Zárraga Ana María , López-Goñi Ignacio , Moriyón Ignacio 
Published: February 2012

ABSTRACT

The brucellae are Gram-negative pathogens that cause brucellosis, a zoonosis of worldwide importance. The genus
Brucella
includes smooth and rough species that differ in that they carry smooth and rough lipopolysaccharides, respectively.
Brucella abortus
,
B. melitensis
, and
B. suis
are typical smooth species. However, these smooth brucellae dissociate into rough mutants devoid of the lipopolysaccharide O-polysaccharide, a major antigen and a virulence determinant encoded in regions
wbo
(included in genomic island-2) and
wbk
. We demonstrate here the occurrence of spontaneous recombination events in those three
Brucella
species leading to the deletion of a 5.5-kb fragment carrying the
wbkA
glycosyltranferase gene and to the appearance of rough mutants. Analysis of the recombination intermediates suggested homologous recombination between the IS
Bm1
insertion sequences flanking
wbkA
as the mechanism generating the deletion. Excision of
wbkA
was reduced but not abrogated in a
recA
-deficient mutant, showing the existence of both RecA-dependent and -independent processes. Although the involvement of the IS
Bm1
copies flanking
wbkA
suggested a transpositional event, the predicted transpositional joint could not be detected. This absence of detectable transposition was consistent with the presence of polymorphism in the inverted repeats of one of the IS
Bm1
copies. The spontaneous excision of
wbkA
represents a novel dissociation mechanism of smooth brucellae that adds to the previously described excision of genomic island-2. This IS
Bm1
-mediated
wbkA
excision and the different %GC levels of the excised fragment and of other
wbk
genes suggest that the
Brucella
wbk
locus is the result of at least two horizontal acquisition events.
Hfq Regulates Biofilm Gut Blockage That Facilitates Flea-Borne Transmission of Yersinia pestis
Authors: Rempe Katherine A. , Hinz Angela K. , Vadyvaloo Viveka 
Published: February 2012

ABSTRACT

The plague bacillus
Yersinia pestis
can achieve transmission by biofilm blockage of the foregut proventriculus of its flea vector. Hfq is revealed to be essential for biofilm blockage formation and acquisition and fitness of
Y. pestis
during flea gut infection, consistent with posttranscriptional regulatory mechanisms in plague transmission.
Second-Site Suppression of RNase E Essentiality by Mutation of thedeaDRNA Helicase in Escherichia coli
Authors: Tamura Masaru , Kers Johan A. , Cohen Stanley N. 
Published: February 2012

ABSTRACTEscherichia colicells normally require RNase E activity to propagate and form colonies. Using random Tn10insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) toE. colicells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of thernemutant phenotype by inactivation ofdeaDwas partial, asrne deaDdoubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration bydeaDinactivation requires normal expression of the endogenousrnggene in doubly mutantrne deaDcells. Second-site suppression bydeaDmutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality.
Anaerobic
p
-Coumarate Degradation by Rhodopseudomonas palustris and Identification of CouR, a MarR Repressor Protein That Binds
p
-Coumaroyl Coenzyme A
Authors: Hirakawa Hidetada , Schaefer Amy L. , Greenberg E. Peter , Harwood Caroline S. 
Published: February 2012

ABSTRACT

The phenylpropanoid
p
-coumarate and structurally related aromatic compounds are produced in large amounts by green plants and are excellent carbon sources for many soil bacteria. Aerobic bacteria remove the acyl side chain from phenylpropanoids to leave an aromatic aldehyde, which then enters one of several possible central pathways of benzene ring degradation. We investigated the pathway for the anaerobic degradation of
p
-coumarate by the phototrophic bacterium
Rhodopseudomonas palustris
and found that it also follows this metabolic logic. We characterized enzymes for the conversion of
p
-coumarate to
p
-hydroxybenzaldehyde and acetyl coenzyme A (acetyl-CoA) encoded by the
couAB
operon. We also identified a MarR family transcriptional regulator that we named CouR. A
couR
mutant had elevated
couAB
expression. In addition, His-tagged CouR bound with high affinity to a DNA fragment encompassing the
couAB
promoter region, and binding was abrogated by the addition of nanomolar quantities of
p
-coumaroyl-CoA but not by
p
-coumarate. Footprinting demonstrated binding of CouR to an inverted repeat sequence that overlaps the −10 region of the
couAB
promoter. Our results provide evidence for binding of a CoA-modified aromatic compound by a MarR family member. Although the MarR family is widely distributed in bacteria and archaea and includes over 12,000 members, ligands have been identified for relatively few family members. Here we provide biochemical evidence for a new category of MarR ligand.
A primary care Symptoms Clinic for patients with medically unexplained symptoms: pilot randomised trial
Authors: Burton Christopher , Weller David , Marsden Wendy , Worth Allison , Sharpe Michael 
Published: February 2012

ObjectivesTo conduct a pilot trial of a primary care Symptoms Clinic for patients with medically unexplained symptoms and evaluate recruitment and retention, and acceptability of the intervention and to estimate potential treatment effects for a full trial.Trial designRandomised parallel group pilot trial.SettingPrimary care in one locality.ParticipantsPrimary care database and postal questionnaire were used to identify patients with multiple specialist referrals and multiple physical symptoms unlikely to be explained by disease.InterventionsGeneral practitioner (GP) with special interest ‘Symptoms Clinic’ + usual care versus usual care alone. The Symptoms Clinic comprised one long (1 h) and three short (20 min) appointments.OutcomesNumber of patients identified and recruited; acceptability of the intervention (items from Client Satisfaction Questionnaire and interview); Medical Outcomes Survey Short Form 12 (SF-12) physical component summary.RandomisationAutomated blocked randomisation accessed by telephone.BlindingNone.Numbers randomized16 to intervention and 16 to usual care alone.Recruitment72 patients, from seven GP practices, had repeated specialist referrals and a Patient Health Questionnaire (PHQ)-15 score of ≥10 indicating a high probability of medically unexplained symptoms. 15 were ineligible and 25 declined to participate.Numbers analysed26 patients; two patients randomised to the intervention group were incorrectly included, three patients in the intervention group and one control did not complete outcome measures.OutcomeMost patients randomised to the Symptoms Clinic found the intervention acceptable: eight out of 11 reported the intervention helped them to deal with their problems. The mean difference between groups in SF-12 physical component summary, adjusted for baseline, was 3.8 points (SD 6).HarmsNo observed harms.ConclusionsPatients with multiple medically unexplained symptoms can be systematically identified in primary care; a randomised trial comparing the Symptoms Clinic with usual care is feasible and has the potential to show clinically meaningful benefit.Trial registrationISRCTN63083469.
The influence of optimal medical treatment on the ‘obesity paradox’, body mass index and long-term mortality in patients treated with percutaneous coronary intervention: a prospective cohort study
Authors: Schenkeveld Lisanne , Magro Michael , Oemrawsingh Rohit M , Lenzen Mattie , de Jaegere Peter , van Geuns Robert-Jan , Serruys Patrick W , van Domburg Ron T 
Published: February 2012

ObjectiveTo assess whether the obesity paradox persists in the long term and to study the effect of optimal medical treatment on this phenomenon.DesignA retrospective cohort study.SettingA tertiary care centre in Rotterdam.ParticipantsFrom January 2000 to December 2005, 6332 patients undergoing percutaneous coronary intervention for coronary artery disease were categorised into underweight (body mass index (BMI)<18.5), normal (18.5–24.9), overweight (25–29.9) and obese (>30).Primary outcome measureMortality.Secondary outcome measuresCardiac death and non-fatal myocardial infarction.ResultsOptimal medical treatment was more common in obese patients as compared with normal weight patients (85% vs 76%; p<0.001). At a mean of 6.1 years, overweight and obese patients had a lower risk of all-cause mortality (HR: 0.75, 95% CI 0.66 to 0.86 and HR: 0.72, 95% CI 0.60 to 0.87, respectively). After adjusting for OMT in the multivariate analysis, BMI did not remain an independent predictor of long-term mortality (HR: 0.90, 95% CI 0.72 to 1.12 and HR: 1.07, 95% CI: 0.80 to 1.43, respectively).ConclusionBMI is inversely related to long-term mortality in patients treated with percutaneous coronary intervention. Patients with a normal BMI are on suboptimal medical treatment when compared with those with a high BMI. A more optimal medical treatment in the obese group may explain the observed improved outcome in these patients.
Surveillance of unplanned return to the operating theatre in neurosurgery combined with a mortality–morbidity conference: results of a pilot survey
Authors: Marini Hélène , Merle Véronique , Derrey Stéphane , Lebaron Christine , Josset Valérie , Langlois Olivier , Gilles Baray Marie , Frébourg Noëlle , Proust François , Czernichow Pierre 
Published: February 2012

BackgroundUnplanned return to the operating theatre (UROT) is a useful trigger tool that could be used to identify surgical adverse events (SAEs). The present study describes the feasibility of SAE surveillance in neurosurgical patients, based on UROT identification, completed with SAE analysis at a morbidity–mortality conference (MMC) meeting.MethodFor consecutive patients who underwent a neurosurgical procedure between 1 November 2008 and 30 April 2009, return to the operating theatre (ROT) was identified based on the hospital information system associated to prospective payment (HISPP). ROT was classified as planned or unplanned and UROT was further classified as related to the natural history of the disease or related to an adverse event (AE-UROT). MMC meetings were organised to discuss results of UROT surveillance and to analyse AE-UROT.Results1006 neurosurgical procedures were included in the surveillance. HISSP identified 152 ROTs, with 73 UROTs related to an SAE (7.3% (5.7% to 9.0%)): infectious SAE (n=24, 2.4% (1.5% to 3.5%)), haemorrhagic SAE (n=23, 2.3% (1.5% to 3.4%)), other cause SAE (n=26, 2.8% (1.9% to 4.0%)), and infectious and other cause SAE (n=2, 0.2% (0.0% to 0.7%)). Identification of AE-UROT through HISSP required a 4 h/month time frame. Eight UROTs related to SAE cases were discussed during MMC meetings, leading to the identification of non-conforming care processes and practical improvement actions.ConclusionUROT related to SAE surveillance in neurosurgical patients was considered feasible. The association of surveillance and MMCs allowed staff to concentrate on the analysis of most frequent or most severe AEs and was a practical and useful tool to stimulate improvement. The impact on healthcare quality of SAE surveillance associated with MMC warrants further research.