scholarly journals Phosphoregulation of Nap1 Plays a Role in Septin Ring Dynamics and Morphogenesis in Candida albicans

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Zhen-Xing Huang ◽  
Pan Zhao ◽  
Gui-Sheng Zeng ◽  
Yan-Ming Wang ◽  
Ian Sudbery ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Mouse Model ◽  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Content Type ◽  
Septin Ring ◽  
Wide Range ◽  
Ring Dynamics ◽  
And Function

ABSTRACT Nap1 has long been identified as a potential septin regulator in yeasts. However, its function and regulation remain poorly defined. Here, we report functional characterization of Nap1 in the human-pathogenic fungus Candida albicans. We find that deletion of NAP1 causes constitutive filamentous growth and changes of septin dynamics. We present evidence that Nap1’s cellular localization and function are regulated by phosphorylation. Phos-tag gel electrophoresis revealed that Nap1 phosphorylation is cell cycle dependent, exhibiting the lowest level around the time of bud emergence. Mass spectrometry identified 10 phosphoserine and phosphothreonine residues in a cluster near the N terminus, and mutation of these residues affected Nap1’s localization to the septin ring and cellular function. Nap1 phosphorylation involves two septin ring-associated kinases, Cla4 and Gin4, and its dephosphorylation occurs at the septin ring in a manner dependent on the phosphatases PP2A and Cdc14. Furthermore, the nap1Δ/Δ mutant and alleles carrying mutations of the phosphorylation sites exhibited greatly reduced virulence in a mouse model of systemic candidiasis. Together, our findings not only provide new mechanistic insights into Nap1’s function and regulation but also suggest the potential to target Nap1 in future therapeutic design. IMPORTANCE Septins are conserved filament-forming GTPases involved in a wide range of cellular events, such as cytokinesis, exocytosis, and morphogenesis. In Candida albicans, the most prevalent human fungal pathogen, septin functions are indispensable for its virulence. However, the molecular mechanisms by which septin structures are regulated are poorly understood. In this study, we deleted NAP1, a gene encoding a putative septin regulator, in C. albicans and found that cells lacking NAP1 showed abnormalities in morphology, invasive growth, and septin ring dynamics. We identified a conserved N-terminal phosphorylation cluster on Nap1 and demonstrated that phosphorylation at these sites regulates Nap1 localization and function. Importantly, deletion of NAP1 or mutation in the N-terminal phosphorylation cluster strongly reduced the virulence of C. albicans in a mouse model of systemic infection. Thus, this study not only provides mechanistic insights into septin regulation but also suggests Nap1 as a potential antifungal target.

scholarly journals The Glycine Lipids ofBacteroides thetaiotaomicronAre Important for Fitness during GrowthIn VivoandIn Vitro

2018 ◽  
Vol 85 (10) ◽  
Author(s):  
Alli Lynch ◽  
Seshu R. Tammireddy ◽  
Mary K. Doherty ◽  
Phillip D. Whitfield ◽  
David J. Clarke
Keyword(s):  
Amino Acid ◽  
Gut Microbiome ◽  
Molecular Mechanisms ◽  
Cellular Membrane ◽  
Bacteroides Thetaiotaomicron ◽  
Human Gut ◽  
Acyltransferase Activity ◽  
Content Type ◽  
Health And Disease ◽  
And Function

ABSTRACTAcylated amino acids function as important components of the cellular membrane in some bacteria. Biosynthesis is initiated by theN-acylation of the amino acid, and this is followed by subsequentO-acylation of the acylated molecule, resulting in the production of the mature diacylated amino acid lipid. In this study, we use both genetics and liquid chromatography-mass spectrometry (LC-MS) to characterize the biosynthesis and function of a diacylated glycine lipid (GL) species produced inBacteroides thetaiotaomicron. We, and others, have previously reported the identification of a gene, namedglsBin this study, that encodes anN-acyltransferase activity responsible for the production of a monoacylated glycine calledN-acyl-3-hydroxy-palmitoyl glycine (or commendamide). In all of theBacteroidalesgenomes sequenced so far, theglsBgene is located immediately downstream from a gene, namedglsA, that is also predicted to encode a protein with acyltransferase activity. We use LC-MS to show that the coexpression ofglsBandglsAresults in the production of GL inEscherichia coli. We constructed a deletion mutant of theglsBgene inB. thetaiotaomicron, and we confirm thatglsBis required for the production of GL inB. thetaiotaomicron. Moreover, we show thatglsBis important for the ability ofB. thetaiotaomicronto adapt to stress and colonize the mammalian gut. Therefore, this report describes the genetic requirements for the biosynthesis of GL, a diacylated amino acid species that contributes to fitness in the human gut bacteriumB. thetaiotaomicron.IMPORTANCEThe gut microbiome has an important role in both health and disease of the host. The mammalian gut microbiome is often dominated by bacteria from theBacteroidales, an order that includesBacteroidesandPrevotella. In this study, we have identified an acylated amino acid, called glycine lipid, produced byBacteroides thetaiotaomicron, a beneficial bacterium originally isolated from the human gut. In addition to identifying the genes required for the production of glycine lipids, we show that glycine lipids have an important role during the adaptation ofB. thetaiotaomicronto a number of environmental stresses, including exposure to either bile or air. We also show that glycine lipids are important for the normal colonization of the murine gut byB. thetaiotaomicron. This work identifies glycine lipids as an important fitness determinant inB. thetaiotaomicronand therefore increases our understanding of the molecular mechanisms underpinning colonization of the mammalian gut by beneficial bacteria.

Functional characterization of a gene cluster responsible for inositol catabolism associated with hospital-adapted isolates of Enterococcus faecium

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Janetta Top ◽  
Jery Baan ◽  
Adinda Bisschop ◽  
Sergio Arredondo-Alonso ◽  
Willem van Schaik ◽  
...  
Keyword(s):  
Mouse Model ◽  
Gene Cluster ◽  
Enterococcus Faecium ◽  
Type Species ◽  
Integration Site ◽  
Functional Characterization ◽  
Donor Strain ◽  
Integrase Gene ◽  
Content Type ◽  
Link Type

Enterococcus faecium is a nosocomial, multidrug-resistant pathogen. Whole genome sequence studies revealed that hospital-associated E. faecium isolates are clustered in a separate clade A1. Here, we investigated the distribution, integration site and function of a putative iol gene cluster that encodes for myo-inositol (MI) catabolism. This iol gene cluster was found as part of an ~20 kbp genetic element (iol element), integrated in ICEEfm1 close to its integrase gene in E. faecium isolate E1679. Among 1644 E. faecium isolates, ICEEfm1 was found in 789/1227 (64.3 %) clade A1 and 3/417 (0.7 %) non-clade A1 isolates. The iol element was present at a similar integration site in 180/792 (22.7 %) ICEEfm1-containing isolates. Examination of the phylogenetic tree revealed genetically closely related isolates that differed in presence/absence of ICEEfm1 and/or iol element, suggesting either independent acquisition or loss of both elements. E. faecium iol gene cluster containing isolates E1679 and E1504 were able to grow in minimal medium with only myo-inositol as carbon source, while the iolD-deficient mutant in E1504 (E1504∆iolD) lost this ability and an iol gene cluster negative recipient strain gained this ability after acquisition of ICEEfm1 by conjugation from donor strain E1679. Gene expression profiling revealed that the iol gene cluster is only expressed in the absence of other carbon sources. In an intestinal colonization mouse model the colonization ability of E1504∆iolD mutant was not affected relative to the wild-type E1504 strain. In conclusion, we describe and functionally characterise a gene cluster involved in MI catabolism that is associated with the ICEEfm1 island in hospital-associated E. faecium isolates. We were unable to show that this gene cluster provides a competitive advantage during gut colonisation in a mouse model. Therefore, to what extent this gene cluster contributes to the spread and ecological specialisation of ICEEfm1-carrying hospital-associated isolates remains to be investigated.

scholarly journals Regulation of OmpA Translation and Shigella dysenteriae Virulence by an RNA Thermometer

2019 ◽  
Vol 88 (3) ◽  
Author(s):  
Erin R. Murphy ◽  
Johanna Roßmanith ◽  
Jacob Sieg ◽  
Megan E. Fris ◽  
Hebaallaha Hussein ◽  
...  
Keyword(s):  
In Silico ◽  
Bacterial Species ◽  
Regulation Of Gene Expression ◽  
Structure And Function ◽  
Shigella Dysenteriae ◽  
Content Type ◽  
Bacterial Physiology ◽  
Wide Range ◽  
Rna Thermometer ◽  
And Function

ABSTRACT RNA thermometers are cis-acting riboregulators that mediate the posttranscriptional regulation of gene expression in response to environmental temperature. Such regulation is conferred by temperature-responsive structural changes within the RNA thermometer that directly result in differential ribosomal binding to the regulated transcript. The significance of RNA thermometers in controlling bacterial physiology and pathogenesis is becoming increasingly clear. This study combines in silico, molecular genetics, and biochemical analyses to characterize both the structure and function of a newly identified RNA thermometer within the ompA transcript of Shigella dysenteriae. First identified by in silico structural predictions, genetic analyses have demonstrated that the ompA RNA thermometer is a functional riboregulator sufficient to confer posttranscriptional temperature-dependent regulation, with optimal expression observed at the host-associated temperature of 37°C. Structural studies and ribosomal binding analyses have revealed both increased exposure of the ribosomal binding site and increased ribosomal binding to the ompA transcript at permissive temperatures. The introduction of site-specific mutations predicted to alter the temperature responsiveness of the ompA RNA thermometer has predictable consequences for both the structure and function of the regulatory element. Finally, in vitro tissue culture-based analyses implicate the ompA RNA thermometer as a bona fide S. dysenteriae virulence factor in this bacterial pathogen. Given that ompA is highly conserved among Gram-negative pathogens, these studies not only provide insight into the significance of riboregulation in controlling Shigella virulence, but they also have the potential to facilitate further understanding of the physiology and/or pathogenesis of a wide range of bacterial species.

scholarly journals Long non-coding RNAs in plants: emerging modulators of gene activity in development and stress responses

Planta ◽  
2020 ◽  
Vol 252 (5) ◽  
Author(s):  
Li Chen ◽  
Qian-Hao Zhu ◽  
Kerstin Kaufmann
Keyword(s):  
Stress Responses ◽  
Translational Regulation ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Reproductive Organs ◽  
Gene Activity ◽  
Protein Coding ◽  
Wide Range ◽  
Non Coding Rnas ◽  
Coding Potential

Abstract Main conclusion Long non-coding RNAs modulate gene activity in plant development and stress responses by various molecular mechanisms. Abstract Long non-coding RNAs (lncRNAs) are transcripts larger than 200 nucleotides without protein coding potential. Computational approaches have identified numerous lncRNAs in different plant species. Research in the past decade has unveiled that plant lncRNAs participate in a wide range of biological processes, including regulation of flowering time and morphogenesis of reproductive organs, as well as abiotic and biotic stress responses. LncRNAs execute their functions by interacting with DNA, RNA and protein molecules, and by modulating the expression level of their targets through epigenetic, transcriptional, post-transcriptional or translational regulation. In this review, we summarize characteristics of plant lncRNAs, discuss recent progress on understanding of lncRNA functions, and propose an experimental framework for functional characterization.

scholarly journals A Family of Small Intrinsically Disordered Proteins Involved in Flagellum-Dependent Motility inSalmonella enterica

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Tamiko Oguri ◽  
Youjeong Kwon ◽  
Jerry K. K. Woo ◽  
Gerd Prehna ◽  
Hyun Lee ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Expression Profiles ◽  
Functional Characterization ◽  
Disordered Proteins ◽  
Wild Type ◽  
Content Type ◽  
Cellular Pathway ◽  
Intrinsically Disordered ◽  
Small Proteins ◽  
Wide Range

ABSTRACTBy screening a collection ofSalmonellamutants deleted for genes encoding small proteins of ≤60 amino acids, we identified three paralogous small genes (ymdF,STM14_1829, andyciG) required for wild-type flagellum-dependent swimming and swarming motility. TheymdF,STM14_1829, andyciGgenes encode small proteins of 55, 60, and 60 amino acid residues, respectively. A bioinformatics analysis predicted that these small proteins are intrinsically disordered proteins, and circular dichroism analysis of purified recombinant proteins confirmed that all three proteins are unstructured in solution. A mutant deleted for STM14_1829 showed the most severe motility defect, indicating that among the three paralogs, STM14_1829 is a key protein required for wild-type motility. We determined that relative to the wild type, the expression of the flagellin protein FliC is lower in the ΔSTM14_1829mutant due to the downregulation of theflhDCoperon encoding the FlhDC master regulator. By comparing the gene expression profiles between the wild-type and ΔSTM14_1829strains via RNA sequencing, we found that the gene encoding the response regulator PhoP is upregulated in the ΔSTM14_1829mutant, suggesting the indirect repression of theflhDCoperon by the activated PhoP. Homologs of STM14_1829 are conserved in a wide range of bacteria, includingEscherichia coliandPseudomonas aeruginosa. We showed that the inactivation of STM14_1829 homologs inE. coliandP. aeruginosaalso alters motility, suggesting that this family of small intrinsically disordered proteins may play a role in the cellular pathway(s) that affects motility.IMPORTANCEThis study reports the identification of a novel family of small intrinsically disordered proteins that are conserved in a wide range of flagellated and nonflagellated bacteria. Although this study identifies the role of these small proteins in the scope of flagellum-dependent motility inSalmonella, they likely play larger roles in a more conserved cellular pathway(s) that indirectly affects flagellum expression in the case of motile bacteria. Small intrinsically disordered proteins have not been well characterized in prokaryotes, and the results of our study provide a basis for their detailed functional characterization.

Commentary on “Reflections on culture, structure and function of an intensive support service centred on positive behavioural support”

2016 ◽  
Vol 21 (4) ◽  
pp. 212-219 ◽  
Author(s):  
Sandy Toogood
Keyword(s):  
Support Service ◽  
Structure And Function ◽  
Content Type ◽  
Behavioural Support ◽  
Wide Range ◽  
Service Models ◽  
History Of ◽  
Positive Behaviour Support ◽  
The Uk ◽  
And Function

Purpose The purpose of this paper is to provide a commentary on Patterson and Berry’s paper “Reflections on culture, structure and function of an intensive support service centred on positive behavioural support”. Design/methodology/approach This paper reviews key ideas presented in Patterson and Berry’s article relative to the recent history of service delivery in the UK and the growing interest being shown in positive behaviour support. Findings Patterson and Berry’s article adds to a modest literature on specialist support services and should stimulate further descriptions of service models and the concepts underpinning them. Originality/value The literature on specialist support service models is limited and this addition should be relevant to a wide range of clinicians, consumers and commissioners.

scholarly journals The Three Clades of the Telomere-AssociatedTLOGene Family of Candida albicans Have Different Splicing, Localization, and Expression Features

2012 ◽  
Vol 11 (10) ◽  
pp. 1268-1275 ◽  
Author(s):  
Matthew Z. Anderson ◽  
Joshua A. Baller ◽  
Keely Dulmage ◽  
Lauren Wigen ◽  
Judith Berman
Keyword(s):  
Candida Albicans ◽  
Gene Family ◽  
Transcription Regulation ◽  
Family Members ◽  
Gene Products ◽  
Content Type ◽  
Human Host ◽  
Wide Range ◽  
N Terminus

ABSTRACTCandida albicansgrows within a wide range of host niches, and this adaptability enhances its success as a commensal and as a pathogen. The telomere-associatedTLOgene family underwent a recent expansion from one or two copies in other CUG clade members to 14 expressed copies inC. albicans. This correlates with increased virulence and clinical prevalence relative to those of otherCandidaclade species. The 14 expressedTLOgene family members have a conserved Med2 domain at the N terminus, suggesting a role in general transcription. The C-terminal half is more divergent, distinguishing three clades: clade α and clade β have no introns and encode proteins that localize primarily to the nucleus; clade γ sometimes undergoes splicing, and the gene products localize within the mitochondria as well as the nuclei. Additionally,TLOα genes are generally expressed at much higher levels than areTLOγ genes. We propose that expansion of theTLOgene family and the predicted role of Tlo proteins in transcription regulation provideC. albicanswith the ability to adapt rapidly to the broad range of different environmental niches within the human host.

scholarly journals Competitive Fitness of Fluconazole-Resistant Clinical Candida albicans Strains

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Christina Popp ◽  
Irene A. I. Hampe ◽  
Tobias Hertlein ◽  
Knut Ohlsen ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Candida Albicans ◽  
Transcription Factors ◽  
Mouse Model ◽  
Ergosterol Biosynthesis ◽  
Fitness Costs ◽  
Content Type ◽  
Constitutive Overexpression ◽  
Fluconazole Resistant

ABSTRACT The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is often caused by gain-of-function mutations in the transcription factors Mrr1 and Tac1, which result in constitutive overexpression of multidrug efflux pumps, and Upc2, which result in constitutive overexpression of ergosterol biosynthesis genes. However, the deregulated gene expression that is caused by hyperactive forms of these transcription factors also reduces the fitness of the cells in the absence of the drug. To investigate whether fluconazole-resistant clinical C. albicans isolates have overcome the fitness costs of drug resistance, we assessed the relative fitness of C. albicans isolates containing resistance mutations in these transcription factors in competition with matched drug-susceptible isolates from the same patients. Most of the fluconazole-resistant isolates were outcompeted by the corresponding drug-susceptible isolates when grown in rich medium without fluconazole. On the other hand, some resistant isolates with gain-of-function mutations in MRR1 did not exhibit reduced fitness under these conditions. In a mouse model of disseminated candidiasis, three out of four tested fluconazole-resistant clinical isolates did not exhibit a significant fitness defect. However, all four fluconazole-resistant isolates were outcompeted by the matched susceptible isolates in a mouse model of gastrointestinal colonization, demonstrating that the effects of drug resistance on in vivo fitness depend on the host niche. Collectively, our results indicate that the fitness costs of drug resistance in C. albicans are not easily remediated, especially when proper control of gene expression is required for successful adaptation to life within a mammalian host.

scholarly journals Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

2011 ◽  
Vol 77 (22) ◽  
pp. 8193-8196 ◽  
Author(s):  
Lucja M. Jarosz ◽  
Bastiaan P. Krom
Keyword(s):  
Candida Albicans ◽  
Real Time ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Rapid Screening ◽  
Content Type ◽  
Wide Range ◽  
Hyphal Formation ◽  
Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

scholarly journals Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

2009 ◽  
Vol 9 (3) ◽  
pp. 405-414 ◽  
Author(s):  
Aaron T. Frank ◽  
Caleen B. Ramsook ◽  
Henry N. Otoo ◽  
Cho Tan ◽  
Gregory Soybelman ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Tandem Repeats ◽  
Cell Aggregation ◽  
Structure And Function ◽  
Hydrophobic Surfaces ◽  
Content Type ◽  
Domain Structures ◽  
Aggregation Activity ◽  
Fungal Adhesins ◽  
And Function

ABSTRACT Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664–1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel β-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677–1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like “classic” subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.

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