scholarly journals Potassium Uptake Modulates Staphylococcus aureus Metabolism

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Casey M. Gries ◽  
Marat R. Sadykov ◽  
Logan L. Bulock ◽  
Sujata S. Chaudhari ◽  
Vinai C. Thomas ◽  
...  
Keyword(s):  
Carbon Flux ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Normal Growth ◽  
Cytoplasmic Ph ◽  
Carbon Utilization ◽  
Content Type ◽  
Alkaline Conditions ◽  
Green Fluorescent

ABSTRACT Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization. As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K+) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K+ uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K+ deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K+ uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization.

scholarly journals Neurospora crassa NKIN2, a Kinesin-3 Motor, Transports Early Endosomes and Is Required for Polarized Growth

2013 ◽  
Vol 12 (7) ◽  
pp. 1020-1032 ◽  
Author(s):  
Constanze Seidel ◽  
Sergio David Moreno-Velásquez ◽  
Meritxell Riquelme ◽  
Reinhard Fischer
Keyword(s):  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Early Endosome ◽  
Mechanical Forces ◽  
Structural Components ◽  
Content Type ◽  
Early Endosomes ◽  
Polarized Cells ◽  
Green Fluorescent

ABSTRACT Biological motors are molecular nanomachines, which convert chemical energy into mechanical forces. The combination of mechanoenzymes with structural components, such as the cytoskeleton, enables eukaryotic cells to overcome entropy, generate molecular gradients, and establish polarity. Hyphae of filamentous fungi are among the most polarized cells, and polarity defects are most obvious. Here, we studied the role of the kinesin-3 motor, NKIN2, in Neurospora crassa . We found that NKIN2 localizes as fast-moving spots in the cytoplasm of mature hyphae. To test whether the spots represented early endosomes, the Rab5 GTPase YPT52 was used as an endosomal marker. NKIN2 colocalized with YPT52. Deletion of nkin2 caused strongly reduced endosomal movement. Combined, these results confirm the involvement of NKIN2 in early endosome transport. Introduction of a rigor mutation into NKIN2 labeled with green fluorescent protein (GFP) resulted in decoration of microtubules. Interestingly, NKIN2 rigor was associated with a subpopulation of microtubules, as had been shown earlier for the Aspergillus nidulans orthologue UncA. Other kinesins did not show this specificity.

scholarly journals Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1590 ◽  
Author(s):  
Magda Wąchalska ◽  
Małgorzata Graul ◽  
Patrique Praest ◽  
Rutger D. Luteijn ◽  
Aleksandra W. Babnis ◽  
...  
Keyword(s):  
Antigen Processing ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Peptide Transporter ◽  
Virus Species ◽  
Protein Fusion ◽  
Fluorescent Properties ◽  
Bovine Herpesvirus 1 ◽  
Green Fluorescent

Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).

scholarly journals Role of Src Kinases in Mobilization of Glycosylphosphatidylinositol-Anchored Decay-Accelerating Factor by Dr Fimbria-Positive Adhering Bacteria

2011 ◽  
Vol 79 (7) ◽  
pp. 2519-2534 ◽  
Author(s):  
Christophe J. Queval ◽  
Valérie Nicolas ◽  
Isabelle Beau
Keyword(s):  
Fluorescent Protein ◽  
3D Analysis ◽  
Src Kinases ◽  
Decay Accelerating Factor ◽  
Content Type ◽  
Protein Mutants ◽  
Src Homology ◽  
Signaling Receptors ◽  
Green Fluorescent

ABSTRACTAfa/Dr fimbriae constitute the major virulence factor of diffusely adheringEscherichia coli(Afa/Dr DAEC). After recognizing membrane-bound signaling receptors, they trigger cell responses. One of these receptors is the human decay-accelerating factor (hDAF). It has previously been reported that the binding of Afa/Dr fimbriae to hDAF quickly induces recruitment of hDAF around adhering bacteria. The aim of our study is to analyze the role of Src kinases in the Dr fimbria-induced recruitment of hDAF. Using biochemical methods and confocal microscopy followed by 3-dimensional (3D) analysis, we have shown that the activation and cell membrane targeting of Src kinases are necessary for the recruitment and organization of hDAF around adhering bacteria. We identified c-Src to be the specific kinase involved in this process. Using a set of Src-green fluorescent protein mutants, we showed that the catalytic activity and the Src homology 2 (SH2) and SH3 domains of the Src kinases are necessary for Dr fimbria-induced hDAF mobilization to occur. In addition, using mutated Dr fimbriae and a set of mutated hDAFs in which each of the complement control protein (CCP) domains had successively been deleted, we found that the aspartic acids at position 54 in the Dr fimbriae and in CCP domain 4 of hDAF played pivotal roles in the mobilization of the Src kinases and hDAF, respectively.

scholarly journals Mutant Brucella abortus Membrane Fusogenic Protein Induces Protection against Challenge Infection in Mice

2015 ◽  
Vol 83 (4) ◽  
pp. 1458-1464 ◽  
Author(s):  
Job Alves de Souza Filho ◽  
Vicente de Paulo Martins ◽  
Priscila Carneiro Campos ◽  
Juliana Alves-Silva ◽  
Nathalia V. Santos ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Economic Losses ◽  
Macrophage Infection ◽  
Content Type ◽  
Live Vaccines ◽  
Interferon Regulatory Factor 1 ◽  
Potential Vaccine ◽  
Green Fluorescent

Brucellaspecies can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence ofBrucellaspp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of theBrucellamembrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed thatB. abortusΔmfp::kanand Δomp19::kandeletion mutant strains have reduced persistencein vivoin C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kanor Δomp19::kanstrain expressing green fluorescent protein (GFP) approximately 80% or 65% ofBrucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments.B. abortusΔomp19::kanwas attenuatedin vivobut had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kanstrain had a lower virulence in these same mouse models. Furthermore, Δmfp::kanand Δomp19::kanstrains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kanstrain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kanstrain induced protection similar to that of RB51. Thus, these results demonstrate thatBrucellaMfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kanmutant may serve as a potential vaccine candidate in future studies.

scholarly journals Role of the ESCRT Pathway in Laccase Trafficking and Virulence of Cryptococcus neoformans

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Yoon-Dong Park ◽  
Shu Hui Chen ◽  
Emma Camacho ◽  
Arturo Casadevall ◽  
Peter R. Williamson
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fluorescent Protein ◽  
Extracellular Vesicle ◽  
Multivesicular Bodies ◽  
Melanin Production ◽  
Content Type ◽  
Endosomal Sorting ◽  
Green Fluorescent

ABSTRACT The endosomal sorting complex required for transport (ESCRT) plays a crucial role in the transportation and degradation of proteins. We determined that Vps27, a key protein of the ESCRT-0 complex, is required for the transport of the virulence factor laccase to the cell wall in Cryptococcus neoformans. Laccase activity was perturbed, as was melanin production, in vps27Δ strains. In the absence of VPS27, there was an accumulation of multivesicular bodies with vacuolar fragmentation and mistargeting of the vacuolar carboxypeptidase CPY/Prc1, resulting in an extracellular localization. In addition, deletion of VPS27 resulted in a defect in laccase targeting of a Lac1-green fluorescent protein (GFP) fusion to the cell wall with trapping within intracellular puncta; this deletion was accompanied by reduced virulence in a mouse model. However, the actin cytoskeleton remained intact, suggesting that the trafficking defect is not due to defects in actin-related localization. Extracellular vesicle maturation was also defective in the vps27Δ mutant, which had a larger vesicle size as measured by dynamic light scattering. Our data identify cryptococcal VPS27 as a required gene for laccase trafficking and attenuates virulence of C. neoformans in a mouse intravenous (i.v.) meningitis model.

scholarly journals Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants

2015 ◽  
Vol 82 (4) ◽  
pp. 1305-1315 ◽  
Author(s):  
Yue Zhen Li ◽  
Dan Wang ◽  
Xue Ying Feng ◽  
Jian Jiao ◽  
Wen Xin Chen ◽  
...  
Keyword(s):  
Critical Role ◽  
Nodule Occupancy ◽  
Phosphotransferase System ◽  
Carbon Utilization ◽  
Sinorhizobium Fredii ◽  
Content Type ◽  
Symbiotic Interactions ◽  
Mutant Strains ◽  
Negative Role

ABSTRACTThe nitrogen phosphotransferase system (PTSNtr) consists of EINtr, NPr, and EIIANtr. The active phosphate moiety derived from phosphoenolpyruvate is transferred through EINtrand NPr to EIIANtr.Sinorhizobium frediican establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study,S. frediistrains with mutations inptsPandptsO(encoding EINtrand NPr, respectively) formed ineffective nodules on soybeans, while a strain with aptsNmutation (encoding EIIANtr) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-β-hydroxybutyrate granules in bacteroids were observed in nodules infected by theptsPorptsOmutant strains but not in those infected with theptsNmutant strain. However, these defects of theptsPandptsOmutant strains were recovered inptsP ptsNandptsO ptsNdouble-mutant strains, implying a negative role of unphosphorylated EIIANtrin symbiosis. Moreover, the symbiotic defect of theptsPmutant was also recovered by expressing EINtrwith or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTSNtrin symbiosis was also observed when related PTSNtrmutant strains ofS. frediiwere inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTSNtrin addition to the negative role of unphosphorylated EIIANtr.

scholarly journals Mutations in Conserved Regions of the Predicted RAG2 Kelch Repeats Block Initiation of V(D)J Recombination and Result in Primary Immunodeficiencies

2000 ◽  
Vol 20 (15) ◽  
pp. 5653-5664 ◽  
Author(s):  
Carlos A. Gomez ◽  
Leon M. Ptaszek ◽  
Anna Villa ◽  
Fabio Bozzi ◽  
Cristina Sobacchi ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Critical Role ◽  
Recombination Reaction ◽  
Signal Recognition ◽  
Mutant Proteins ◽  
Recombination Signal ◽  
Loop Regions ◽  
Green Fluorescent ◽  
Processing Steps

ABSTRACT The V(D)J recombination reaction is composed of multiple nucleolytic processing steps mediated by the recombination-activating proteins RAG1 and RAG2. Sequence analysis has suggested that RAG2 contains six kelch repeat motifs that are predicted to form a six-bladed β-propeller structure, with the second β-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Here we demonstrate that mutations G95R and ΔI273 within the predicted second β-strand of repeats 2 and 5 of RAG2 lead to immunodeficiency in patients P1 and P2. Green fluorescent protein fusions with the mutant proteins reveal appropriate localization to the nucleus. However, both mutations reduce the capacity of RAG2 to interact with RAG1 and block recombination signal cleavage, therefore implicating a defect in the early steps of the recombination reaction as the basis of the clinical phenotype. The present experiments, performed with an extensive panel of site-directed mutations within each of the six kelch motifs, further support the critical role of both hydrophobic and glycine-rich regions within the second β-strand for RAG1-RAG2 interaction and recombination signal recognition and cleavage. In contrast, multiple mutations within the variable-loop regions of the kelch repeats had either mild or no effects on RAG1-RAG2 interaction and hence on the ability to mediate recombination. In all, the data demonstrate a critical role of the RAG2 kelch repeats for V(D)J recombination and highlight the importance of the conserved elements of the kelch motif.

scholarly journals Critical role of CFTR-dependent lipid rafts in cigarette smoke-induced lung epithelial injury

2011 ◽  
Vol 300 (6) ◽  
pp. L811-L820 ◽  
Author(s):  
Manish Bodas ◽  
Taehong Min ◽  
Neeraj Vij
Keyword(s):  
Cigarette Smoke ◽  
Lipid Raft ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Zonula Occludens ◽  
Lung Epithelial ◽  
Underlying Mechanisms ◽  
Green Fluorescent ◽  
Ceramide Accumulation

Apoptosis of lung epithelial and endothelial cells by exposure to cigarette smoke (CS) severely damages the lung tissue, leading to the pathogenesis of emphysema, but the underlying mechanisms are poorly understood. We have recently established a direct correlation between decreased lipid raft CFTR expression and emphysema progression through increased ceramide accumulation. In the present work, we investigated the role of membrane CFTR in regulating apoptosis and autophagy responses to CS exposure. We report a constitutive and CS-induced increase in the number of TUNEL-positive apoptotic cells in Cftr−/− murine lungs compared with Cftr+/+ murine lungs that also correlated with a concurrent increase in the expression of ceramide, NF-κB, CD95/Fas, lipid raft proteins, and zonula occludens (ZO)-1/2 ( P < 0.001). We also verified that stable wild-type CFTR expression in CFBE41o− cells controls constitutively elevated caspase-3/7 activity (−1.6-fold, P < 0.001). Our data suggest that membrane CFTR regulates ceramide-enriched lipid raft signaling platforms required for the induction of Fas-mediated apoptotic signaling. In addition, lack of membrane CFTR also modulates autophagy, as demonstrated by the significant increase in constitutive ( P < 0.001) and CSE-induced ( P < 0.005) perinuclear accumulation of green fluorescent protein-microtubule-associated protein 1 light chain-3 (LC3) in the absence of membrane CFTR (CFBE41o− cells). The significant constitutive and CS-induced increase ( P < 0.05) in p62 and LC3β expression in CFTR-deficient cells and mice corroborates these findings and suggest a defective autophagy response in the absence of membrane CFTR. Our data demonstrate the critical role of membrane-localized CFTR in regulating apoptotic and autophagic responses in CS-induced lung injury that may be involved in the pathogenesis of severe emphysema.

scholarly journals Role of ATG8 and Autophagy in Programmed Nuclear Degradation in Tetrahymena thermophila

2012 ◽  
Vol 11 (4) ◽  
pp. 494-506 ◽  
Author(s):  
Ming-Liang Liu ◽  
Meng-Chao Yao
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Critical Role ◽  
Nuclear Periphery ◽  
Unicellular Eukaryote ◽  
Knockout Mutant ◽  
Content Type ◽  
Nuclear Degradation

ABSTRACT Autophagy is an evolutionarily conserved mechanism for the degradation of cellular components, but its role in enucleation during differentiation has not been established. Tetrahymena thermophila is a unicellular eukaryote with two functionally distinct nuclei, the somatic (macro-) and the germ line (micro-) nuclei. These nuclei are produced during sexual reproduction (conjugation), which involves differentiation and selective degradation of several specific nuclei. To examine the role of autophagy in nuclear degradation, we studied the function of two ATG8 genes in Tetrahymena . Through fluorescent protein tagging, we found that both proteins are targeted to degrading nuclei at specific stages, with some enrichment on the nuclear periphery, suggesting the formation of autophagosomes surrounding these nuclei. In addition, ATG8 knockout mutant cells showed a pronounced delay in nuclear degradation without apparently preventing the completion of other developmental events. This evidence provided direct support for a critical role for autophagy in programmed nuclear degradation. The results also showed differential roles for two ATG8 genes, with ATG8-65 playing a more significant role in starvation than ATG8-2 , although both are important in nuclear degradation.

scholarly journals Cyclic Di-GMP-Mediated Repression of Swarming Motility by Pseudomonas aeruginosa PA14 Requires the MotAB Stator

2014 ◽  
Vol 197 (3) ◽  
pp. 420-430 ◽  
Author(s):  
S. L. Kuchma ◽  
N. J. Delalez ◽  
L. M. Filkins ◽  
E. A. Snavely ◽  
J. P. Armitage ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Point Mutations ◽  
Second Messenger ◽  
Swarming Motility ◽  
Content Type ◽  
In The Wild ◽  
Green Fluorescent

The second messenger cyclic diguanylate (c-di-GMP) plays a critical role in the regulation of motility. InPseudomonas aeruginosaPA14, c-di-GMP inversely controls biofilm formation and surface swarming motility, with high levels of this dinucleotide signal stimulating biofilm formation and repressing swarming.P. aeruginosaencodes two stator complexes, MotAB and MotCD, that participate in the function of its single polar flagellum. Here we show that the repression of swarming motility requires a functional MotAB stator complex. Mutating themotABgenes restores swarming motility to a strain with artificially elevated levels of c-di-GMP as well as stimulates swarming in the wild-type strain, while overexpression of MotA from a plasmid represses swarming motility. Using point mutations in MotA and the FliG rotor protein of the motor supports the conclusion that MotA-FliG interactions are critical for c-di-GMP-mediated swarming inhibition. Finally, we show that high c-di-GMP levels affect the localization of a green fluorescent protein (GFP)-MotD fusion, indicating a mechanism whereby this second messenger has an impact on MotCD function. We propose that when c-di-GMP level is high, the MotAB stator can displace MotCD from the motor, thereby affecting motor function. Our data suggest a newly identified means of c-di-GMP-mediated control of surface motility, perhaps conserved amongPseudomonas,Xanthomonas, and other organisms that encode two stator systems.

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