scholarly journals TheAspergillus flavus rtfAGene Regulates Plant and Animal Pathogenesis and Secondary Metabolism

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Jessica M. Lohmar ◽  
Olivier Puel ◽  
Jeffrey W. Cary ◽  
Ana M. Calvo
Keyword(s):  
Oxidative Stress ◽  
Cell Wall ◽  
Secondary Metabolism ◽  
Elongation Factor ◽  
Host Tissue ◽  
Infection Model ◽  
Global Regulator ◽  
Pol Ii ◽  
Content Type ◽  
Transcription Elongation Factor

ABSTRACTAspergillus flavusis an opportunistic fungal plant and human pathogen and a producer of mycotoxins, including aflatoxin B1(AFB1). As part of our ongoing studies to elucidate the biological functions of theA. flavusrtfAgene, we examined its role in the pathogenicity of both plant and animal model systems.rtfAencodes a putative RNA polymerase II (Pol II) transcription elongation factor previously characterized inSaccharomyces cerevisiae,Aspergillus nidulans, andAspergillus fumigatus, where it was shown to regulate several important cellular processes, including morphogenesis and secondary metabolism. In addition, an initial study inA. flavusindicated thatrtfAalso influences development and production of AFB1; however, its effect on virulence is unknown. The current study reveals that thertfAgene is indispensable for normal pathogenicity in plants when using peanut seed as an infection model, as well as in animals, as shown in theGalleria mellonellainfection model. Interestingly,rtfApositively regulates several processes known to be necessary for successful fungal invasion and colonization of host tissue, such as adhesion to surfaces, protease and lipase activity, cell wall composition and integrity, and tolerance to oxidative stress. In addition, metabolomic analysis revealed thatA. flavusrtfAaffects the production of several secondary metabolites, including AFB1, aflatrem, leporins, aspirochlorine, ditryptophenaline, and aflavinines, supporting a role ofrtfAas a global regulator of secondary metabolism. Heterologous complementation of anA. flavusrtfAdeletion strain withrtfAhomologs fromA. nidulansorS. cerevisiaefully rescued the wild-type phenotype, indicating that thesertfAhomologs are functionally conserved among these three species.IMPORTANCEIn this study, the epigenetic global regulatorrtfA, which encodes a putative RNA-Pol II transcription elongation factor-like protein, was characterized in the mycotoxigenic and opportunistic pathogenA. flavus. Specifically, its involvement inA. flavuspathogenesis in plant and animal models was studied. Here, we show thatrtfApositively regulatesA. flavusvirulence in both models. Furthermore,rtfA-dependent effects on factors necessary for successful invasion and colonization of host tissue byA. flavuswere also assessed. Our study indicates thatrtfAplays a role inA. flavusadherence to surfaces, hydrolytic activity, normal cell wall formation, and response to oxidative stress. This study also revealed a profound effect ofrtfAon the metabolome ofA. flavus, including the production of potent mycotoxins.

scholarly journals VelA and LaeA are Key Regulators of Epichloë festucae Transcriptomic Response during Symbiosis with Perennial Ryegrass

2019 ◽  
Vol 8 (1) ◽  
pp. 33 ◽  
Author(s):  
Mostafa Rahnama ◽  
Paul Maclean ◽  
Damien J. Fleetwood ◽  
Richard D. Johnson
Keyword(s):  
Cell Wall ◽  
Secondary Metabolism ◽  
Perennial Ryegrass ◽  
Global Regulator ◽  
Symbiotic Interaction ◽  
Fungal Cell ◽  
Transcriptomic Response ◽  
Epichloë Festucae ◽  
Genes Encoding ◽  
The Impact

VelA (or VeA) is a key global regulator in fungal secondary metabolism and development which we previously showed is required during the symbiotic interaction of Epichloë festucae with perennial ryegrass. In this study, comparative transcriptomic analyses of ∆velA mutant compared to wild-type E. festucae, under three different conditions (in culture, infected seedlings, and infected mature plants), were performed to investigate the impact of VelA on E. festucae transcriptome. These comparative transcriptomic studies showed that VelA regulates the expression of genes encoding proteins involved in membrane transport, fungal cell wall biosynthesis, host cell wall degradation, and secondary metabolism, along with a number of small secreted proteins and a large number of proteins with no predictable functions. In addition, these results were compared with previous transcriptomic experiments that studied the impact of LaeA, another key global regulator of secondary metabolism and development that we have shown is important for E. festucae–perennial ryegrass interaction. The results showed that although VelA and LaeA regulate a subset of E. festucae genes in a similar manner, they also regulated many other genes independently of each other suggesting specialised roles.

scholarly journals Positive Transcription Elongation Factor b Phosphorylates hSPT5 and RNA Polymerase II Carboxyl-terminal Domain Independently of Cyclin-dependent Kinase-activating Kinase

2001 ◽  
Vol 276 (15) ◽  
pp. 12317-12323 ◽  
Author(s):  
Jae B. Kim ◽  
Phillip A. Sharp
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase ◽  
Elongation Factor ◽  
Rna Pol Ii ◽  
Factor B ◽  
Pol Ii ◽  
Positive Transcription Elongation Factor ◽  
Transcription Elongation Factor ◽  
Immunodeficiency Virus ◽  
Carboxyl Terminal

The CDK9-cyclin T kinase complex, positive transcription elongation factor b (P-TEFb), stimulates the process of elongation of RNA polymerase (Pol) II during transcription of human immunodeficiency virus. P-TEFb associates with the human immunodeficiency virus Tat protein and with the transactivation response element to form a specific complex, thereby mediating efficient elongation. Here, we show that P-TEFb preferentially phosphorylates hSPT5 as compared with the carboxyl-terminal domain of RNA Pol IIin vitro. Phosphorylation of hSPT5 by P-TEFb occurred on threonine and serine residues in its carboxyl-terminal repeat domains. In addition, we provide several lines of evidence that P-TEFb is a CDK-activating kinase (CAK)-independent kinase. For example, CDK9 was not phosphorylated by CAK, whereas CDK2-cyclin A kinase activity was dramatically enhanced by CAK. Therefore, it is likely that P-TEFb participates in regulation of elongation by RNA Pol II by phosphorylation of its substrates, hSPT5 and the CTD of RNA Pol II, in a CAK-independent manner.

scholarly journals Postantibiotic and Sub-MIC Effects of Exebacase (Lysin CF-301) Enhance Antimicrobial Activity againstStaphylococcus aureus

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Jun Taek Oh ◽  
Cara Cassino ◽  
Raymond Schuch
Keyword(s):  
Antimicrobial Activity ◽  
Cell Wall ◽  
The United States ◽  
Infection Model ◽  
Postantibiotic Effect ◽  
Content Type ◽  
Wall Ultrastructure ◽  
Bacterial Fitness

ABSTRACTCF-301 (exebacase) is a recombinantly produced bacteriophage-derived lysin (cell wall hydrolase) and is the first agent of this class to enter clinical development in the United States for treating bacteremia including endocarditis due toStaphylococcus aureus. Whereas rapid bactericidal activity is the hallmarkin vitroandin vivoresponse to CF-301 at exposures higher than the MIC, prolonged antimicrobial activity, mediated by cell wall damage, is predicted at concentrations less than the MIC. In the current study, a series ofin vitropharmacodynamic parameters, including the postantibiotic effect (PAE), postantibiotic sub-MIC effect (PA-SME), and sub-MIC effect (SME), were studied to determine how short-duration and sub-MIC CF-301 exposures affect the growth of surviving staphylococci and extend its antimicrobial activity. Mean PAE, PA-SME, and SME values up to 4.8, 9.3, and 9.8 h, respectively, were observed against 14 staphylococcal strains tested in human serum; growth delays were extended by 6 h in the presence of daptomycin. Exposures to CF-301 at sub-MIC levels as low as 0.001× to 0.01× MIC (∼1 to 10 ng/ml) resulted in aberrant cell wall ultrastructure, increased membrane permeability, dissipation of membrane potential, and inhibition of virulence phenotypes, including agglutination and biofilm formation. A mouse thigh infection model designed to study the PAE was used to confirm our findings and demonstratein vivogrowth delays of ≥19.3 h. Our findings suggest that at CF-301 concentrations less than the MIC during therapeutic use, sustained reductions in bacterial fitness and virulence may substantially enhance efficacy.

scholarly journals JMJD6 cleaves MePCE to release positive transcription elongation factor b (P-TEFb) in higher eukaryotes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Schuyler Lee ◽  
Haolin Liu ◽  
Ryan Hill ◽  
Chunjing Chen ◽  
Xia Hong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Factor B ◽  
Pol Ii ◽  
Positive Transcription Elongation Factor ◽  
Transcription Elongation Factor ◽  
Higher Eukaryotes

More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II CTD by positive transcription elongation factor b (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown. In this report, we utilize mouse and human models to reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex.

scholarly journals VelA and LaeA are key regulators of the Epichloë festucae transcriptomic response during symbiosis with perennial ryegrass

2019 ◽  
Author(s):  
M. Rahnama ◽  
P. Maclean ◽  
D.J. Fleetwood ◽  
R.D. Johnson
Keyword(s):  
Cell Wall ◽  
Secondary Metabolism ◽  
Perennial Ryegrass ◽  
Comparative Transcriptomics ◽  
Global Regulator ◽  
Symbiotic Interaction ◽  
Fungal Cell ◽  
Transcriptomic Response ◽  
Epichloë Festucae ◽  
The Impact

AbstractVelA (or VeA) is a key global regulator in fungal secondary metabolism and development which we previously showed is required during the symbiotic interaction of Epichloë festucae with perennial ryegrass. In this study, comparative transcriptomics analyses of ΔvelA mutant compared to wild type E. festucae, under three different conditions (in culture, infected seedlings and infected mature plants) were performed to investigate the impact VelA on the E. festucae transcriptome. These comparative transcriptomics studies showed that VelA regulates the expression of genes encoding proteins involved in membrane transport, fungal cell wall biosynthesis, host cell wall degradation and secondary metabolism, along with a number of small secreted proteins and a large number of proteins with no predictable functions. In addition, these results were compared with previous transcriptomics experiments studying the impact of LaeA, another key global regulator of secondary metabolism and development that we have shown is important for the E. festucae- perennial ryegrass interaction. The results showed that although VelA and LaeA regulate a sub-set of E. festucae genes in a similar manner, they also regulated many other genes independently of each other suggesting specialised roles.

scholarly journals Polo-Like Kinase 1 Inhibits the Activity of Positive Transcription Elongation Factor of RNA Pol II b (P-TEFb)

PLoS ONE ◽  
2013 ◽  
Vol 8 (8) ◽  
pp. e72289 ◽  
Author(s):  
Liangzhen Jiang ◽  
Yan Huang ◽  
Min Deng ◽  
Ting Liu ◽  
Wenbin Lai ◽  
...  
Keyword(s):  
Transcription Elongation ◽  
Elongation Factor ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Positive Transcription Elongation Factor ◽  
Transcription Elongation Factor

scholarly journals RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans

2017 ◽  
Vol 199 (24) ◽  
Author(s):  
C. J. Kovacs ◽  
R. C. Faustoferri ◽  
R. G. Quivey
Keyword(s):  
Cell Wall ◽  
Stress Tolerance ◽  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Galleria Mellonella ◽  
Infection Model ◽  
Critical Function ◽  
Content Type ◽  
The Impact

ABSTRACT Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In Streptococcus mutans, the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in S. mutans biology have not been well investigated. The present study characterizes the impact of RGP disruption as a result of the deletion of rgpF, the gene encoding a rhamnosyltransferase involved in the construction of the core polyrhamnose backbone of RGP. The ΔrgpF mutant strain displayed an overall reduced fitness compared to the wild type, with heightened sensitivities to various stress-inducing culture conditions and an inability to tolerate acid challenge. The loss of rgpF caused a perturbation of membrane-associated functions known to be critical for aciduricity, a hallmark of S. mutans acid tolerance. The proton gradient across the membrane was disrupted, and the ΔrgpF mutant strain was unable to induce activity of the F1Fo ATPase in cultures grown under low-pH conditions. Further, the virulence potential of S. mutans was also drastically reduced following the deletion of rgpF. The ΔrgpF mutant strain produced significantly less robust biofilms, indicating an impairment in its ability to adhere to hydroxyapatite surfaces. Additionally, the ΔrgpF mutant lost competitive fitness against oral peroxigenic streptococci, and it displayed significantly attenuated virulence in an in vivo Galleria mellonella infection model. Collectively, these results highlight a critical function of the RGP in the maintenance of overall stress tolerance and virulence traits in S. mutans. IMPORTANCE The cell wall of Streptococcus mutans, the bacterium most commonly associated with tooth decay, is abundant in rhamnose-glucose polysaccharides (RGP). While these structures are antigenically distinct to S. mutans, the process by which they are formed and the enzymes leading to their construction are well conserved among streptococci. The present study describes the consequences of the loss of RgpF, a rhamnosyltransferase involved in RGP construction. The deletion of rgpF resulted in severe ablation of the organism's overall fitness, culminating in significantly attenuated virulence. Our data demonstrate an important link between the RGP and cell wall physiology of S. mutans, affecting critical features used by the organism to cause disease and providing a potential novel target for inhibiting the pathogenesis of S. mutans.

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