Drug Repurposing: Tolfenamic Acid Inactivates PrbP, a Transcriptional Accessory Protein in Liberibacter asiaticus

Abstract

CLIBASIA_01510, PrbP, is a predicted RNA polymerase binding protein in Liberibacter asiaticus. PrbP was found to regulate expression of a small subset of ribosomal genes through interactions with the β-subunit of the RNA polymerase and a short, specific sequence on the promoter region. Molecular screening assays were performed to identify small molecules that interact with PrbP in vitro. Chemical hits were analyzed for therapeutic efficacy against L. asiaticus via an infected leaf assay, where the transcriptional activity of L. asiaticus was found to decrease significantly after exposure to tolfenamic acid. Similarly, tolfenamic acid was found to inhibit L. asiaticus infection in highly symptomatic citrus seedlings. Our results indicate that PrbP is an important transcriptional regulator for survival of L. asiaticus in planta, and the chemicals identified by molecular screening assays could be used as a therapeutic treatment for huanglongbing disease.

Understanding the Physiology of <b><i>Liberibacter asiaticus</i></b>: An Overview of the Demonstrated Molecular Mechanisms

“…As a result of significant genome reduction in L. asiaticus, only 2% of the genome (11 genes) encodes the transcription factors that regulate all gene expressions [Duan et al, 2009;Gardner et al, 2016;Moran, 2002;Pagliai et al, 2014]. Consequently, the identification of high affinity chemical inhibitors targeting a single transcription factor could result in pleiotropic effects, greatly hindering bacterial adaptation and survival [Pagliai et al, 2014].…”

“…As the bacterium senses environmental changes during the transition from psyllid to citrus host, regulation is altered to activate genes involved in survival and pathogenicity. Among the affected genes, 6 transcription factors are upregulated in citrus, including the 2 transcription factors that have been functionally characterized via biochemical techniques, LdtR and PrbP [Gardner et al, 2016;Pagliai et al, 2014Pagliai et al, , 2015Pagliai et al, , 2017Yan et al, 2013].…”

“…PrbP belongs to the CarD_CdnL_TRCF superfamily; it is a transcriptional activator that has a suggested role in stabilizing the RNA polymerase (RNAP)/open promoter complex formation at some rRNA gene promoters [Gardner et al, 2016;Srivastava et al, 2013;Stallings et al, 2009;Stallings and Glickman, 2011]. This is accomplished through direct interactions between PrbP and the β-subunit of RNAP and a recognition sequence in the promoter region DNA [Gardner et al, 2016].…”

“…This is accomplished through direct interactions between PrbP and the β-subunit of RNAP and a recognition sequence in the promoter region DNA [Gardner et al, 2016]. The transcription of rRNA is a rate-limiting step in bacterial growth, and the formation of the RNAP/open promoter complex is tightly regulated [Bartlett et al, 2000;Häkkinen et al, 2013;Jin et al, 2012;Zhou and Jin, 1998].…”

“…The transcription of rRNA is a rate-limiting step in bacterial growth, and the formation of the RNAP/open promoter complex is tightly regulated [Bartlett et al, 2000;Häkkinen et al, 2013;Jin et al, 2012;Zhou and Jin, 1998]. Small molecules targeting PrbP disrupt the open complex formation, decreasing the transcription and reducing viability of L. asiaticus in the citrus plant [Gardner et al, 2016]. A functional in vitro screening identified tolfenamic acid (TA) as a PrbP ligand that disrupted PrbP-RNAP and PrbP-DNA interactions.…”