scholarly journals Inducible Antisense RNA Expression in the Characterization of Gene Functions in Streptococcus mutans

2005 ◽  
Vol 73 (6) ◽  
pp. 3568-3576 ◽  
Author(s):  
Bing Wang ◽  
Howard K. Kuramitsu
Keyword(s):  
Streptococcus Mutans ◽  
Gene Function ◽  
Antisense Rna ◽  
Coenzyme A ◽  
Essential Gene ◽  
Essential Genes ◽  
Expression Vectors ◽  
Rna Expression ◽  
Gene Encoding ◽  
Antisense Dna

ABSTRACT In order to examine gene function in Streptococcus mutans, we have recently initiated an antisense RNA strategy. Toward this end, we have now constructed and evaluated three Escherichia coli-S. mutans shuttle expression vectors with the fruA and scrB promoters from S. mutans, as well as the tetR-controlled tetO promoter from Staphylococcus aureus. Among these, the tetO/tetR system proved to be the most tightly controlled promoter. By using this shuttle plasmid system, modulation of gene function by inducible antisense RNA expression was demonstrated for comC antisense fragments of different sizes as well as for distinct gtfB antisense fragments. It was demonstrated that the size, but not the relative position, of an antisense DNA fragment is important in mediating the antisense phenomenon. Furthermore, by constructing and screening random DNA libraries with the tet expression shuttle system, 78 growth-retarded transformants harboring antisense DNA fragments were also identified. Almost all of them corresponded to homologous essential genes in other bacteria. In addition, a novel essential gene, the coaE gene, encoding dephospho-coenzyme A kinase, which is involved in the final step of coenzyme A catabolism in S. mutans, was identified and characterized. These results suggest that the antisense RNA strategy can be useful for identifying novel essential genes in S. mutans bacteria as well as further characterizing the physiology (including potential virulence factors) of these organisms.

scholarly journals ParA: A Novel Target for Anti-Tubercular Drug Discovery

2021 ◽  
Author(s):  
◽  
Shahista Yasmin Nisa
Keyword(s):  
Cell Division ◽  
Atpase Activity ◽  
Broad Spectrum ◽  
Antisense Rna ◽  
Essential Gene ◽  
Competitive Inhibitor ◽  
Culture Conditions ◽  
Essential Genes ◽  
Rna Expression ◽  
In Silico Docking

<p>Tuberculosis (Tb) continues to be one of the world's greatest challenges in the public health arena. The current treatment for Tb entails a long duration of therapy making adherence to the whole course difficult. This has given rise to drug resistant strains of Mycobacterium tuberculosis which are posing a significant threat to Tb control strategies. To counteract this problem, there is an urgent need to develop alternative anti-tuberculous drugs which target processes that are critical for the growth and/or survival of this microbe. To identify such targets in M. tuberculosis, I used comparative genomics and mutagenesis data to identify conserved essential genes as viable targets for the development of broad-spectrum antibiotics. In addition, I validated the essentiality of three cell division genes in Mycobacterium smegmatis using conditional antisense RNA expression under different culture conditions. Furthermore, I performed high-throughput screens (HTS) using a differential susceptibility assay against one of the validated targets to identify its cognate inhibitor(s). Lastly, I developed a novel biochemical assay of the target to validate the specificity of the inhibitors identified in the HTS and evaluated the potency of the inhibitors against M. tuberculosis. This study identified 261 conserved putative essential genes as broad-spectrum targets. I hypothesized that antisense RNA expression of such genes will lead to its down-regulation and thereby affect the viability of the cells if these genes are essential. I also hypothesized that an essential gene will be required under all culture conditions. One gene, parA, demonstrated that it was essential under various culture conditions. This gene encodes for a protein which contain the conserved Walker A motif thus I theorized that it may posses ATPase activity. The results illustrated that the M. tuberculosis ParA protein possesses ATPase activity. This biochemical activity was used to validate two specific inhibitors of ParA, phenoxybenzamine and octoclothepin, which were identified in the cell-based HTS. Kinetic studies suggest that phenoxybenzamine is a mixed inhibitor while octoclothepin is a competitive inhibitor of ParA. This data is also supported by in silico docking. Both these compounds show low minimum inhibitory concentrations in M. smegmatis under nitrogen starvation conditions. In summary, this thesis illustrates that ParA is a viable target for anti-tubercular drugs. It demonstrates that ParA is an ATPase which has the potential to bind competitive and non-competitive inhibitors that can be exploited to target cell division in M. tuberculosis. Finally, this study presents phenoxybenzamine and octoclothepin as inhibitors of ParA. In conclusion, these compounds can either be developed to increase potency or be used as reference structures to screen for more potent inhibitors of the enzyme.</p>

scholarly journals ParA: A Novel Target for Anti-Tubercular Drug Discovery

2021 ◽  
Author(s):  
◽  
Shahista Yasmin Nisa
Keyword(s):  
Cell Division ◽  
Atpase Activity ◽  
Broad Spectrum ◽  
Antisense Rna ◽  
Essential Gene ◽  
Competitive Inhibitor ◽  
Culture Conditions ◽  
Essential Genes ◽  
Rna Expression ◽  
In Silico Docking

<p>Tuberculosis (Tb) continues to be one of the world's greatest challenges in the public health arena. The current treatment for Tb entails a long duration of therapy making adherence to the whole course difficult. This has given rise to drug resistant strains of Mycobacterium tuberculosis which are posing a significant threat to Tb control strategies. To counteract this problem, there is an urgent need to develop alternative anti-tuberculous drugs which target processes that are critical for the growth and/or survival of this microbe. To identify such targets in M. tuberculosis, I used comparative genomics and mutagenesis data to identify conserved essential genes as viable targets for the development of broad-spectrum antibiotics. In addition, I validated the essentiality of three cell division genes in Mycobacterium smegmatis using conditional antisense RNA expression under different culture conditions. Furthermore, I performed high-throughput screens (HTS) using a differential susceptibility assay against one of the validated targets to identify its cognate inhibitor(s). Lastly, I developed a novel biochemical assay of the target to validate the specificity of the inhibitors identified in the HTS and evaluated the potency of the inhibitors against M. tuberculosis. This study identified 261 conserved putative essential genes as broad-spectrum targets. I hypothesized that antisense RNA expression of such genes will lead to its down-regulation and thereby affect the viability of the cells if these genes are essential. I also hypothesized that an essential gene will be required under all culture conditions. One gene, parA, demonstrated that it was essential under various culture conditions. This gene encodes for a protein which contain the conserved Walker A motif thus I theorized that it may posses ATPase activity. The results illustrated that the M. tuberculosis ParA protein possesses ATPase activity. This biochemical activity was used to validate two specific inhibitors of ParA, phenoxybenzamine and octoclothepin, which were identified in the cell-based HTS. Kinetic studies suggest that phenoxybenzamine is a mixed inhibitor while octoclothepin is a competitive inhibitor of ParA. This data is also supported by in silico docking. Both these compounds show low minimum inhibitory concentrations in M. smegmatis under nitrogen starvation conditions. In summary, this thesis illustrates that ParA is a viable target for anti-tubercular drugs. It demonstrates that ParA is an ATPase which has the potential to bind competitive and non-competitive inhibitors that can be exploited to target cell division in M. tuberculosis. Finally, this study presents phenoxybenzamine and octoclothepin as inhibitors of ParA. In conclusion, these compounds can either be developed to increase potency or be used as reference structures to screen for more potent inhibitors of the enzyme.</p>

scholarly journals Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function

2020 ◽  
Vol 16 (3) ◽  
pp. e1008344 ◽  
Author(s):  
Robert C. Shields ◽  
Alejandro R. Walker ◽  
Natalie Maricic ◽  
Brinta Chakraborty ◽  
Simon A. M. Underhill ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Gene Function ◽  
Essential Gene

scholarly journals A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Hadi H. Choudhary ◽  
Maria G. Nava ◽  
Brina E. Gartlan ◽  
Savannah Rose ◽  
Sumiti Vinayak
Keyword(s):  
Cryptosporidium Parvum ◽  
Gene Function ◽  
Essential Gene ◽  
Critical Role ◽  
Essential Genes ◽  
Parasite Line ◽  
Asexual Parasite ◽  
Content Type ◽  
Genetic Tool ◽  
Transgenic Parasites

ABSTRACT Cryptosporidium spp., protozoan parasites, are a leading cause of global diarrhea-associated morbidity and mortality in young children and immunocompromised individuals. The limited efficacy of the only available drug and lack of vaccines make it challenging to treat and prevent cryptosporidiosis. Therefore, the identification of essential genes and understanding their biological functions are critical for the development of new therapies. Currently, there is no genetic tool available to investigate the function of essential genes in Cryptosporidium spp. Here, we describe the development of the first conditional system in Cryptosporidium parvum. Our system utilizes the Escherichia coli dihydrofolate reductase degradation domain (DDD) and the stabilizing compound trimethoprim (TMP) for conditional regulation of protein levels in the parasite. We tested our system on the calcium-dependent protein kinase-1 (CDPK1), a leading drug target in C. parvum. By direct knockout strategy, we establish that cdpk1 is refractory to gene deletion, indicating its essentiality for parasite survival. Using CRISPR/Cas9, we generated transgenic parasites expressing CDPK1 with an epitope tag, and localization studies indicate its expression during asexual parasite proliferation. We then genetically engineered C. parvum to express CDPK1 tagged with DDD. We demonstrate that TMP can regulate CDPK1 levels in this stable transgenic parasite line, thus revealing the critical role of this kinase in parasite proliferation. Further, these transgenic parasites show TMP-mediated regulation of CDPK1 levels in vitro and an increased sensitivity to kinase inhibitor upon conditional knockdown. Overall, this study reports the development of a powerful conditional system that can be used to study essential genes in Cryptosporidium. IMPORTANCE Cryptosporidium parvum and Cryptosporidium hominis are leading pathogens responsible for diarrheal disease (cryptosporidiosis) and deaths in infants and children below 5 years of age. There are no effective treatment options and no vaccine for cryptosporidiosis. Therefore, there is an urgent need to identify essential gene targets and uncover their biological function to accelerate the development of new and effective anticryptosporidial drugs. Current genetic tool allows targeted disruption of gene function but leads to parasite lethality if the gene is essential for survival. In this study, we have developed a genetic tool for conditional degradation of proteins in Cryptosporidium spp., thus allowing us to study the function of essential genes. Our conditional system expands the molecular toolbox for Cryptosporidium, and it will help us to understand the biology of this important human diarrheal pathogen for the development of new drugs and vaccines.

scholarly journals MCC Regulator of WNT Signaling Pathway (MCC) Is a Podocyte Essential Gene

2021 ◽  
Vol 8 ◽  
Author(s):  
Hui Song ◽  
Lulu Zhuang ◽  
Xiaodong Xu ◽  
Jingsong Shi ◽  
Weixin Hu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Essential Gene ◽  
Wnt Signaling Pathway ◽  
Essential Genes ◽  
Actin Stress Fiber ◽  
Gene Encoding ◽  
Filtration Barrier ◽  
And Function

Podocytes are an integral part of the glomerular filtration barrier. Many genes are already known to be essential for podocyte survival, structure and function, but there are more podocyte essential genes to be identified. By single-cell RNA-seq of mouse podocytes, we detected the expression of gene encoding MCC regulator of WNT signaling pathway (MCC) in majority of the podocytes and speculated that MCC is essential for podocytes. We confirmed MCC expression in mouse podocytes and further showed its expression in human podocytes. To experimentally prove the essentiality of MCC for podocytes, we knocked down MCC in cultured podocytes and found marked morphological change of cell shape, cytoskeletal F-actin stress fiber disruption, increased apoptosis, and downregulation of podocyte essential genes, CD2AP and WT1, demonstrating that MCC is essential for podocytes. Since MCC has been implicated in cell cycle and β-catenin signaling, we examined the expression of cell cycle related genes and activity of β-catenin in the MCC knockdown podocytes, but did not find significant changes. To further explore the mechanism underlying the role of MCC in podocytes, we performed RNA-sequencing and bioinformatics analysis of MCC knockdown podocytes and found a significant enrichment of the regulated genes in lamellipodia formation. Consistently, we found that MCC is present in lamellipodia and MCC knockdown resulted in loss of lamellipodia in the cells. Lastly, we found that MCC was downregulated in podocytes treated with puromycin aminonucleosides and in glomeruli of diabetic mice and FSGS patients, implicating MCC is involved in the development of podocytopathy and proteinuria. In conclusion, MCC is potentially essential for podocytes and its downregulation may be involved in podocytopathy.

Paradoxical production of target protein using antisense RNA expression vectors

Gene ◽  
1994 ◽  
Vol 149 (1) ◽  
pp. 21-24 ◽  
Author(s):  
Renée Williard ◽  
Christopher C. Benz ◽  
John D. Baxter ◽  
Peter Kushner ◽  
C. Anthony Hunt
Keyword(s):  
Antisense Rna ◽  
Target Protein ◽  
Expression Vectors ◽  
Rna Expression
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