A response regulator protein with antar domain, AvnR, in Acinetobacter baumannii ATCC 17978 impacts its virulence and amino acid metabolism

Abstract

<jats:p> <jats:italic>Acinetobacter baumannii,</jats:italic> a Gram-negative coccobacillus, is notorious for its involvement in opportunistic infections around the world. Its resistance to antibiotics makes treatment of infections challenging. In this study, we describe a novel response regulator protein, AvnR (A1S_2006) that regulates virulence-related traits in <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2767" xlink:type="simple">A. baumannii</jats:ext-link> </jats:named-content> </jats:italic> ATCC17978. Sequence analysis suggests that AvnR is a CheY-like response regulator and contains the RNA-binding ANTAR (<jats:underline>A</jats:underline>miR and <jats:underline>N</jats:underline>asR <jats:underline>t</jats:underline>ranscription <jats:underline>a</jats:underline>nti-termination <jats:underline>r</jats:underline>egulators) domain. We show that AvnR plays a role in regulating biofilm formation (on glass and plastic surfaces), surface motility, adhesion to A549 cells as well as in nitrogen metabolism in <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2767" xlink:type="simple">A. baumannii</jats:ext-link> </jats:named-content> </jats:italic>. RNA-Seq analysis revealed that <jats:italic>avnR</jats:italic> deletion results in altered expression of more than 150 genes (116 upregulated and 42 downregulated). RNA-Seq data suggest that altered biofilm formation and surface motility observed in the <jats:italic>avnR</jats:italic> deletion mutant is likely mediated by previously unknown pathways. Of note, was the altered expression of genes predicted to be involved in amino acid transport and metabolism in <jats:italic>avnR</jats:italic> deletion mutant. Biolog phenotypic array showed that deletion of <jats:italic>avnR</jats:italic> hampered <jats:italic> <jats:named-content content-type="species"> <jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://doi.org/10.1601/nm.2767" xlink:type="simple">A. baumannii</jats:ext-link> </jats:named-content> </jats:italic> ATCC17978’s ability to metabolize various nitrogen sources, particularly that of glutamic acid, serine, histidine, aspartic acid, isoleucine and arginine. Taken together our data show that AvnR, the first ANTAR protein described in <jats:italic>A. baumannii,</jats:italic> affects virulence phenotypes as well as its ability to metabolize nitrogen sources.</jats:p>

Recent Advances in Genetic Tools for Acinetobacter baumannii

“…Variations include those originally designed in P. aeruginosa and Burkholderia spp. The methods of Choi and Schweizer have been adapted for use in A. baumannii (De Silva and Kumar, 2018;De Silva et al, 2020) and includes the initial step of Splicing Overlap Extension (SOE), which introduces an antibiotic resistance gene flanked by Flp Recombinase Target (FRT) sites into the flanking homologous regions of the target gene to allow for selection of recombinants and antibiotic gene removal, respectively (Choi and Schweizer, 2005). Applications in clinical isolates using this method ( Supplementary Table 1) have been successful depending on the type of selection and counter-selection markers used (Trebosc et al, 2016(Trebosc et al, , 2019Xu Q. et al, 2019).…”

“…There are a variety of iterations of this system including various selection markers, and induction parameters (Supplementary Table 3). Versatility of this system has been shown by its successful use in AB5075 (Pérez-Varela et al, 2020;Williams et al, 2020) as well in the clinical isolates AB030, AB031, and LAC-4 with the chromosomal insertion of constitutive fluorescent markers ( Supplementary Table 4) including mTurquoise, RubyRed, mCherry, and sfGFP (Ducas-Mowchun et al, 2019), as well as complementation of deletion mutants in ATCC R 17978 TM using the gentamicin selectable marker (De Silva et al, 2020). The antibiotic selection marker is removed via the Flp recombinase method described above ( Figure 2C) using pFLP2 derivatives (Supplementary Table 2).…”

“…This enzyme performs the transfructorylation reaction of sucrose into levan in high sucrose environments and the buildup of this metabolite is toxic to many Gram-negative organisms including A. baumannii (Reyrat et al, 1998). This counter-selection marker has been used mainly to select for the second recombination event during homologous recombination-type strategies (Amin et al, 2013;Oh et al, 2015;De Silva et al, 2020) or to remove the Flp recombinase expressing vector (Ducas-Mowchun et al, 2019;De Silva et al, 2020) in type strains but also with some success in clinical isolates such as AB030 and AB031. sacB is by far the most commonly used counter-selection marker.…”

Diversity and prevalence of ANTAR RNAs across actinobacteria

“…Previous studies have shown that ANTAR proteins, upon activation (through phosphorylation) bind to their target-RNAs and regulate downstream gene expression in cis (Ramesh et al 2012;Fox et al 2009;Wilson et al 1996a;Chai and Stewart 1998;Mehta et al 2020;Malaka De Silva et al 2020;Weber et al 2019). Hence we analyzed the genomic locations and contexts of the predicted RNAs.…”

“…We next asked what cellular processes are linked to ANTAR in actinobacteria. Studies in Enterococcus, Pseudomonas, Klebsiella, Acinetobacter and Geobacter reveal that ANTAR-target RNAs are linked to nitrogen utilization (Ramesh et al 2012;Drew and Lowe 1989;Chai and Stewart 1998;Malaka De Silva et al 2020). Only few studies in actinobacteria have investigated the role of ANTAR.…”