Identification of aaNAT5b as a spermine N-acetyltransferase in the mosquito, Aedes aegypti

Guan, Huai; Wang, Maoying; Liao, Chenghong; Liang, Jing; Mehere, Prajwalini; Tian, Mei; Liu, Hairong; Robinson, Howard; Li, Jianyong; Han, Qian

doi:10.1371/journal.pone.0194499

Cited by 11 publications

(14 citation statements)

References 25 publications

(42 reference statements)

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“…Total RNA was extracted from adult mosquitoes for each of the three independent repeats. Quantitative real-time polymerase chain reaction (qPCR) was performed as described previously [37] in a LightCycler 480 system (Roche Applied Science, Mannheim, Germany) using SYBR Green Master I (Roche) according to the manufacturer's instructions, with the following cycling conditions: initial denaturation at 95 ℃ for 30 s followed by 40 cycles of 95 ℃ for 5 s, and 60 ℃ for 30 s. Primers (forward, 5'-CTG TAA GGT CCT GTG AAT ACG-3'; reverse, 5'-GGT TTA TCA GGG AGT TCA CC-3') were used to amplify the 109-bp DNA fragment of Ae-CLIPB15. Primers (forward, 5'-GAT CCT GTC AAA GGC TTC C-3'; reverse, 5'-GTA CTG TCA GTG CGT ATT GG-3') were used to amplify the 236-bp DNA fragment of Ae-aaCLIP22.…”

Section: Rna Extraction and Quantitative Polymerase Chain Reactionmentioning

confidence: 99%

Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti

Wang

Bhowmick

et al. 2021

Parasites Vectors

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Background Clip domain serine proteases (CLIPs), a very diverse group of proteolytic enzymes, play a crucial role in the innate immunity of insects. Innate immune responses are the first line of defense in mosquitoes against the invasion of pathogenic microorganisms. The Toll pathway, immunodeficiency (IMD) pathway and melanization are the main processes of innate immunity in Aedes aegypti. CLIPS are classified into five subfamilies—CLIPA, CLIPB, CLIPC, CLIPD, and CLIPE—based on their sequence specificity and phylogenetic relationships. We report the functional characterization of the genes that code for two CLIPs in Ae. aegypti (Ae): Ae-CLIPB15 and Ae-CLIPB22. Methods Clustal Omega was used for multiple amino acid sequence alignment of Ae-CLIPB15 and Ae-CLIPB22 with different CLIP genes from other insect species. The spatiotemporal expression profiles of Ae-CLIPB15 and Ae-CLIPB22 were examined. We determined whether Ae-CLIPB15 and Ae-CLIPB22 respond to microbial challenge and tissue injury. RNA interference (RNAi) was used to explore the function of Ae-CLIPB15 and Ae-CLIPB22 in the defense of Ae. aegypti against bacterial and fungal infections. The expression levels of nuclear factor kappa B (NF-κB) transcription factors REL1 and REL2 in the Toll pathway and IMD pathway after bacterial infection were investigated. Finally, the change in phenoloxidase (PO) activity in Ae-CLIPB15 and Ae-CLIPB22 knockdown adults was investigated. Results We performed spatiotemporal gene expression profiling of Ae-CLIPB15 and Ae-CLIPB22 genes in Ae. aegypti using quantitative real-time polymerase chain reaction. These genes were expressed in different stages and tissues. The messenger RNA (mRNA) levels for both genes were also up-regulated by Gram-negative bacteria Escherichia coli, Gram-positive bacteria Staphylococcus aureus and fungal Beauveria bassiana infections, as well as in the tissue injury experiments. RNAi-mediated knockdown of Ae-CLIPB15 led to a significant decrease of PO activity in the hemolymph of Ae. aegypti, while other RNAi experiments revealed that both Ae-CLIPB15 and Ae-CLIPB22 were involved in immune defense against bacterial and fungal infections. The mRNA expression of NF-κB transcription factors REL1 and REL2 in the Toll pathway and IMD pathway differed between Ae-CLIPB15 and Ae-CLIPB22 knockdown mosquitoes infected with bacteria and wild type mosquitoes infected with bacteria. Conclusions Our findings suggest that Ae-CLIPB15 and Ae-CLIPB22 play a critical role in mosquito innate immunity, and that they are involved in immune responses to injury and infection. Their regulation of transcription factors and PO activity indicates that they also play a specific role in the regulation of innate immunity. Graphical Abstract

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Section: Rna Extraction and Quantitative Polymerase Chain Reactionmentioning

confidence: 99%

Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti

Wang

Bhowmick

et al. 2021

Parasites Vectors

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“…Interestingly, the intracellular PAs are in greater quantity in the Vero cells than in the C6 / 36 cells (Fig.5), which opens questions regarding the efficiency of replication, since this virus requires PAs and is replicated more efficiently in C6 / 36 cells. Furthermore it has been described that the enzymes involved in the synthesis and catabolism of PAs and catabolism are conserved throughout the kingdoms; recent studies of polyamines in mosquitoes Aedes aegypti showed that it is probable that the content of PAs probably depends on the enzyme aaNAT5b an acetyltransferase (SAT); so it could act as a viral restriction factor, by decreasing intracellular PAs (Guan et al, 2018); However, there are likely to be other mechanisms in the cells that allow the virus to replicate successfully in the absence of polyamines.…”

Section: Discussionmentioning

confidence: 99%

Antiviral activity of N-ω-Chloroacetyl-L-Ornithine on in vitro replication of Chikungunya virus

Luna-Rojas¹,

Am²,

Alcántara-Farfán³

et al. 2019

Preprint

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14The infections causes by Chikungunya virus (CHIKV), family Togaviridae, genus Alfavirus, have become 15 a health problem around the world, due to its widespread occurrence, the high morbidity rate and the 16 absence of vaccines or antiviral treatments. We analyzed a competitive inhibitor of ornithine 17 decarboxylase, enzyme key in the biosynthesis of Polyamines (PAs), N-ω-chloroacetyl-L-ornithine 18 (NCAO), as a possible inhibitor of CHIKV replication because intracellular polyamines participate in the 19 transcription and in vitro translation of CHIKV. NCAO does not have any cytotoxic effect on C6/36 cells 20 even at a concentration of 1000μM at 72h after post-exposure but in Vero cells the cytotoxic effect was 21 presented above 380μM at 48h post-exposure, which was considered when determining the inhibitory 22 effect on viral replication. We demonstrate that NCAO inhibits the replication of CHIKV in Vero and C6/36 23 cells in a dose-dependent manner causing a decrease in the PFU/mL of at least 4-logarithm (p <0.01) in 24 both cell lines. Viral yields were restored by the addition of exogenous polyamines, mainly putrescine. 25 HPLC analyses it shown that NCAO decrease content of intracellular PAs even though in infected cells 26 mainly decreased spermidine and spermine, so NCAO inhibits CHIKV replication, by depleting the 27 intracellular polyamines in Vero and C6/36 cells, suggesting that this compound is a possible antiviral in 28 CHIKV infections. 29 30 Importance 31 The infections caused by Chikungunya virus (CHIKV), genus Alfavirus, have become a worldwide health 32 problem, due to its high morbidity rate and the absence of vaccines or antiviral treatments. It is known 33 that CHIKV use intracellular poliamines during transcription and traduction process, so the depletion of 34 intracellular putrescine, spermidine and spermine reduce the viral replication. N-ω-chloroacetyl-L-35 ornithine (NCAO) is known as a competitive inhibitor of ornithine decarboxylase, key enzyme in 36 Polyamines biosynthesis, but no studies have proven its activity on the inhibition of polyamine-dependent 37 viral replication. Here we demonstrate NCAO inhibits in vitro CHIKV replication, so we propose NCAO as 38 an antiviral candidate.39

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“…Total RNA was independently isolated for each of the three replications. qPCR was performed as described previously (Guan et al ., 2018) in a LightCycler 480 system (Roche Applied Science, Mannheim, Germany) using SYBR green Master I (Roche) according to the manufacturer's instructions, with the following cycling conditions: initial denaturation at 95 °C for 30 sec followed by 40 cycles of 95 °C for 5 sec, and 60 °C for 30 sec. At the end of the PCR reaction, a melt curve was generated to assess the possibility of undesirable side‐products.…”

Section: Methodsmentioning

confidence: 99%

Arylalkalamine N‐acetyltransferase‐1 functions on cuticle pigmentation in the yellow fever mosquito, Aedes aegypti

Zhang

Chen

et al. 2020

Insect Science

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Arylalkylamine N‐acetyltransferase (aaNAT) catalyzes the acetylation of dopamine, 5‐hydroxy‐tryptamine, tryptamine, octopamine, norepinephrine and other arylalkylamines to form respective N‐acetyl‐arylalkylamines. Depending on the products formed, aaNATs are involved in a variety of physiological functions. In the yellow fever mosquito, Aedes aegypti, a number of aaNATs and aaNAT‐like proteins have been reported. However, the primary function of each individual aaNAT is yet to be identified. In this study we investigated the function of Ae. aegypti aaNAT1 (Ae‐aaNAT1) in cuticle pigmentation and development of morphology. Ae‐aaNAT1 transcripts were detected at all stages of development with highest expressions after pupation and right before adult eclosion. Ae‐aaNAT1 mutant mosquitoes generated using clustered regularly interspaced palindromic repeats (CRISPR) – CRISPR‐associated protein 9 had no obvious effect on larval and pupal development. However, the mutant mosquitoes exhibited a roughened exoskeletal surface, darker cuticles, and color pattern changes suggesting that Ae‐aaNAT1 plays a role in development of the morphology and pigmentation of Ae. aegypti adult cuticles. The mutant also showed less blood feeding efficiency and lower fecundity when compared with the wild‐type. The mutation of Ae‐aaNAT1 influenced expression of genes involved in cuticle formation. In summary, Ae‐aaNAT1 mainly functions on cuticular pigmentation and also affects blood feeding efficiency and fecundity.

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Identification of aaNAT5b as a spermine N-acetyltransferase in the mosquito, Aedes aegypti

Cited by 11 publications

References 25 publications

Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti

Functional characterization of two clip domain serine proteases in innate immune responses of Aedes aegypti

Antiviral activity of N-ω-Chloroacetyl-L-Ornithine on in vitro replication of Chikungunya virus

Arylalkalamine N‐acetyltransferase‐1 functions on cuticle pigmentation in the yellow fever mosquito, Aedes aegypti

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