ORIGINAL RESEARCH published: 17 January 2020 doi: 10.3389/fmicb.2019.03075 COLR Acinetobacter baumannii sRNA Signatures: Computational Comparative Identification and Biological Targets Viviana Cafiso 1* , Stefano Stracquadanio 1 , Flavia Lo Verde 1 , Veronica Dovere 2 , Alessandra Zega 1 , Giuseppe Pigola 3 , Jesús Aranda 4 and Stefania Stefani 1 1 Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy, 2 Department of Translational Research and New Technology in Medicine and Surgery, Azienda Ospedaliero Universitaria Pisana, University of Pisa, Pisa, Italy, 3 Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy, 4 Departament de Genètica i Microbiologia, Facultat de Biociènces, Universitat Autònoma de Barcelona, Barcelona, Spain Multidrug-Resistant (MDR) and Extensively Drug Resistant (XDR) Acinetobacter baumannii (Ab) represent a serious cause of healthcare-associated infections worldwide. Edited by: Currently, the available treatment options are very restricted and colistin-based therapies Benjamin Andrew Evans, are last-line treatments of these infections, even though colistin resistant (COLR ) Ab University of East Anglia, United Kingdom have rarely been isolated yet. In bacteria, small non-coding RNAs (sRNAs) have Reviewed by: been implicated in regulatory pathways of different biological functions, however, no Younes Smani, knowledge exists about the sRNA role on the biological adaptation in COLR Ab. Institute of Biomedicine of Seville (IBIS), Spain Our study investigated two Italian XDR isogenic colistin-susceptible/resistant (COLS/R ) Margarita Poza, Ab strain-pairs to discover new sRNA signatures. Comparative sRNA transcriptome Biomedical Research Institute, Spain (sRNAome) analyses were carried out by Illumina RNA-seq using both a Tru-Seq *Correspondence: and a Short Insert library, whilst Ab ATCC 17978 and ACICU Reference Genome Viviana Cafiso v.cafiso@unict.it assembly, mapping, annotation and statistically significant differential expression (q- value ≤ 0.01) of the raw reads were performed by the Rockhopper tool. A computational Specialty section: This article was submitted to filtering, sorting only similarly statistically significant differentially expressed (DE) sRNAs Antimicrobials, Resistance mapping on the same gene in both COLR Ab isolates was conducted. COLR vs. COLS and Chemotherapy, sRNAome, analyzed integrating the DE sRNAs obtained from the two different libraries, a section of the journal Frontiers in Microbiology revealed some statistically significant DE sRNAs in COLR Ab. In detail, we found: (i) Received: 29 October 2019 two different under-expressed cis-acting sRNAs (AbsRNA1 and AbsRNA2 ) mapping in Accepted: 19 December 2019 antisense orientation the 16S rRNA gene A1S_r01, (ii) one under-expressed cis-acting Published: 17 January 2020 sRNA (AbsRNA3 ) targeting the A1S_2505 gene (hypothetical protein), (iii) one under- Citation: Cafiso V, Stracquadanio S, expressed microRNA-size small RNA fragment (AbsRNA4 ) and its pre-microAbsRNA4 Lo Verde F, Dovere V, Zega A, targeting the A1S_0501 gene (hypothetical protein), (iv) as well as an over-expressed Pigola G, Aranda J and Stefani S microRNA-size small RNA fragment (AbsRNA5 ) and its pre-microAbsRNA5 targeting (2020) COLR Acinetobacter baumannii sRNA Signatures: the A1S_3097 gene (signal peptide). Custom TaqMan probe-based real-time qPCRs R Computational Comparative validated the expression pattern of the selected sRNA candidates shown by RNA-seq. Identification and Biological Targets. Front. Microbiol. 10:3075. Furthermore, analysis on sRNA 1A1S_r01, 1A1S_2505 as well as the over-expressed doi: 10.3389/fmicb.2019.03075 A1S_3097 mutants revealed no effects on colistin resistance. Our study, for the Frontiers in Microbiology | www.frontiersin.org 1 January 2020 | Volume 10 | Article 3075 Cafiso et al. XDR A. baumannii sRNA Signatures first time, found the sRNAome signatures of clinical COLR Ab with a computational prediction of their targets related to protein synthesis, host-microbe interaction and other different biological functions, including biofilm production, cell-cycle control, virulence, and antibiotic-resistance. Keywords: XDR Acinetobacter baumannii, colistin resistance, small RNAs, Illumina RNA-seq, bioinformatics INTRODUCTION 2012; Hood et al., 2013; Parra-Millán et al., 2018; Cafiso et al., 2019), however, no investigations have been conducted so far on The Multidrug-Resistant (MDR) Gram-negative pathogens the role that sRNAs exert on the biological adaptations of colistin included recently in the WHO black list (Tacconelli et al., 2018), resistance acquisition. Few studies have analyzed the sRNA i.e., Acinetobacter, Pseudomonas, and various Enterobacterales contribution to the A. baumannii biology and antimicrobial (including Klebsiella, E. coli, Serratia, and Proteus) represent a resistance. Weiss et al. (2016) offer a detailed view of the sRNA serious health problem worldwide. In particular, focusing on the content of Ab and provide new insights into the evolution and Acinetobacter baumannii infections, the therapeutic options for role of these regulatory molecules. In detail, using RNA-seq, 78 their treatment are very limited and mainly draw on colistin- Ab sRNAs were identified in the AB5075 background, grouped based therapies (Hancock and Chapple, 1999; Zavascki et al., in six classes of similar sRNAs, with one particularly abundant 2007; Vila and Pachón, 2012). Therefore, the increasing use and homologous to regulatory C4 antisense RNAs found in of colistin (COL) and the spread of colistin resistant (COLR ) bacteriophages. Sharma et al. (2014) found three new sRNAs, can consequently determine an increase in polymyxin resistance namely AbsR11, AbsR25, and AbsR28, hypothesizing an sRNA onset (Ko et al., 2007; Gales et al., 2011). involvement in the regulation of antibiotic resistance in bacteria, Small non-coding RNAs (sRNAs) [∼30–300 nucleotides (nt) specifically in cryptic A. baumannii. length] have been recognized as a major class of regulatory Our study aimed to gain new insight on the sRNA signature molecules in bacteria (Barquist and Vogel, 2015). They unroll and biological target of clinical COLR Ab by high-throughput a regulatory function affecting gene expression – by base technology RNA-seq. pairing to their related target mRNA – modulating transcription, Our data define the distinctive signatures of COLR Ab translation, mRNA stability, DNA maintenance, and silencing. sRNAs revealing computational predicted targets involved in Functionally, sRNAs are involved in the regulation of a wide the protein synthesis machinery, host-microbe interactions, range of physiological responses, reacting to environmental pathways involved in biofilm production, cell cycle control, signals, such as pH or temperature shifts (Wassarman, 2002). virulence, and antibiotic-resistance. They can help the modulation of changes in cellular metabolism to optimize use of available nutrients and improve the probability for survival, as well as contributing to virulence (Chabelskaya MATERIALS AND METHODS et al., 2010; Gottesman and Storz, 2011; Sayed et al., 2012; Álvarez-Fraga et al., 2017; Kröger et al., 2018). Bacterial Strains sRNAs act in trans or cis depending on the transcription Two Italian Extensively Drug Resistant (XDR) isogenic start position where the sRNA is transcribed with respect to colistin-susceptible/resistant (COLS/R ) clinical Ab strain- their regulated gene. Trans-encoded sRNAs are transcribed at pairs (1-S/R, 2-S/R) were investigated. Ab strain-pair source, a genetic locus separated from the gene that they regulate antimicrobial susceptibility, molecular characterization, genomic and they often work via an imperfect base pairing with their epidemiology, and phylogeny were previously characterized target mRNAs. Conversely, cis-acting sRNAs are transcribed (Cafiso et al., 2019). from the same genetic locus that they regulate even thought in an antisense orientation to the target gene. Since transcribed RNA-Seq proximally to their target, cis-antisense RNAs share a perfect To optimize data, RNA-seq was carried out using the Illumina match with their targets allowing duplex formation and a Mi-seq Standard pipeline on two biological replicates consisting stringent regulation (Thomason and Storz, 2010; Chang et al., of two different libraries of each strain i.e., a Single-end library 2015; Georg and Hess, 2018). with 50 bp reads (Short-Insert library) and a Paired-end Read A third class of small RNA, the microRNA-size small RNA library with 150 bp reads (Tru-Seq library). fragments (∼15–26 nt) have been identified in a few different For RNA extraction, a single colony of each Ab strain was species of bacteria by next generation sequencing (NGS). These grown in 10 ml of Cation-adjusted Mueller-Hinton broth (Ca- molecules could originate from a cut of a longer precursor (pre- MHB) (Becton Dickinson) and incubated at 37◦ C overnight. The sRNA) in analogy with maturation of the eukaryotic microRNAs overnight cultures were then diluted 1:50 in 50 ml of Ca-MHB (Bloch et al., 2017). in a sterile 250 ml flask and incubated with shaking at 250 rpm Few mechanisms have been proposed to the underlying at 37◦ C. Bacterial pellets were harvested at mid-log growth phase colistin resistance in Ab (Li et al., 2006; Adams et al., 2009; Falagas (OD600 0.6 ∼1.5 × 107 CFU/ml, 18 h) according to the strain et al., 2010; Moffatt et al., 2010; Arroyo et al., 2011; Cai et al., growth-curves (Supplementary Figure S1). Frontiers in Microbiology | www.frontiersin.org 2 January 2020 | Volume 10 | Article 3075 Cafiso et al. XDR A. baumannii sRNA Signatures RNA extraction was performed using the NucleoSpin Comparative sRNA Prediction RNA kit (Macherey-Nagel, Düren, Germany) following the A double computational filtering was carried out on the library manufacturer’s protocol with minor modifications according to analysis data outputs, first for the DE sRNAs in the COLR strains the previously published protocols (Cafiso et al., 2019). versus their COLS counterparts and, thus, to identify only those The total RNA quality was checked by the 2200 TapeStation mapping on the same gene in both COLR Ab isolates with a RNA Screen Tape device (Agilent, Santa Clara, CA, United States) statistically significant q-value ≤ 0.01. and the RNA concentrations were determined using an ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, Determination of Small RNA Functional United States). RNA Integrity Number (RIN) values, ranging from 1 to 10 with 10 being the highest quality, was Categories Functional categories of small RNA target genes were investigated determined by the Agilent TapeStation 2200 system. Only by different bioinformatic tools including BLAST, PANTHER RNAs with preserved 16S and 23S peaks and with RIN (Protein ANalysis THrough Evolutionary Relationships) values >8 were used for library construction. The RIN Classification System, Gene Ontology (GO) Consortium, values >8 indicated intact and high quality RNA samples ExPASy, and KEGG. usable for downstream applications as previously published (Fleige and Pfaffl, 2006). RNA-Seq Data Accession Number RNA-seq data of the two different libraries have been deposited in Library Preparation and Sequencing the NCBI GEO database under study accession no. GSE109951. The Tru-Seq library (TS) was an A Paired-end library with reads of 150 bp and average insert size of 350/400 bp. After I-Tasser ab initio Structure Modeling sequencing, raw reads were processed using FastQC v0.11.2 For the I-Tasser (Iterative Threading asseMBLY Refinement) to check data quality and, then, reads were trimmed by analysis (Zhang, 2009; Roy et al., 2010, 2012; Yang and Zhang, Trimmomatic v.0.33.2 to remove the adapters for Paired-end 2015), the first N-terminal 120 AA of the Ab ATCC 17978 reads. A minimum base quality of 15 over a 4-bases sliding- AbsRNA2 target (A1S_2505) was selected as a target to obtain window was required. Only trimmed reads with a length more significant predictions, as the C-terminal part of the above 36 nucleotides were included in the downstream analysis A1S_2505 protein was excluded because of its low coverage (Cafiso et al., 2019). with threading templates identified from the PDB-library: The Short-Insert library (SI) was processed with an A Single- MTYQYHDESIVTELPEDTVFVFGSNMAGQHGSGAARVASQ end stranded library with reads of 50 bp. After sequencing, HFGAVEGVGRGWAGQSFAIPTLNEHIQQMPLSQIEHYVEDF raw reads were processed using FastQC v0.11.2 to evaluate data KVYAKNHPKMKYFVTALGCGIAGYKVSEIAPLFKGIHHN. quality. Reads were then trimmed using Trimmomatic v.0.33.2 For the analysis of the microAbsRNA4 target (A1S_0501) the to remove sequencing adapters for Single-end reads, requiring a whole FASTA sequence of the protein was used as a template. minimum base quality of 15 (Phred scale) and a minimum read length of 15 nucleotides. Only trimmed reads were included in the downstream analysis (Cafiso et al., 2019). Validation of sRNA-Seq Expression AbsRNA3 and microAbsRNA5 were selected to be validated since they are representative of under-expression and over-expression, Tru-Seq and Short-Insert Library respectively. Particularly, their RNA-seq expression levels were Analysis validated by Custom TaqMan Small RNA Assays as follow: R TS and SI RNA-seq reads were aligned on Ab ATCC 17978 a single colony of each strain was grown in 10 ml of Ca- (CP000521.1) and on Ab ACICU (CP000863.1) Reference MHB (Becton Dickinson) and incubated at 37◦ C overnight. The Genomes, assembled and quantified using Rockhopper v.2.03 overnight cultures were diluted 1:50 in 50 ml of Ca-MHB in a opportunely developed to investigate the bacterial gene structures sterile 250 ml flask and incubated with shaking at 250 rpm under and transcriptomes as well as validated to identify novel sRNAs normal atmospheric conditions at 37◦ C. Bacterial pellets were (McClure et al., 2013; Tjaden, 2015; Cafiso et al., 2019). harvested in mid-log growth phase, lysed by adding lysozyme To obtain the data, analyses were carried out with default (10 mg/ml) and incubated for 1 h at 37◦ C. Small RNA extraction parameters and verbose output. Rockhopper normalizes read was performed by using the mirVanaTM miRNA Isolation counts for each sample using the upper quartile gene expression Kit (Ambion, Austin, TX, United States) according to the level. Starting from the p-values calculated according to the manufacture’s protocol following the enrichment procedure for Anders and Huber approach, differentially expressed genes small RNAs. Extracted sRNAs were quantified using Eppendorf (DEGs) were selected as statistically significant by computing BioPhotometer D30 to assess their quality and to properly dilute q-values ≤ 0.01 based on the Benjamini–Hochberg correction them to the amount suggested by Custom TaqMan Small RNA R with a false discovery rate of <1%. In addition, Rockhopper is Assays protocol (Applied BiosystemsTM ), then a stem-loop qRT- a tool using biological replicates when available and surrogates PCR was performed, one of the most commonly used real-time when biological replicates for two different conditions are PCR approaches to quantify sRNAs. The quantification assay unavailable, considering the two conditions under investigation was divided into two steps: i) RNA was reverse-transcribed into surrogate replicates for each other (McClure et al., 2013). cDNA using a stem-loop specific primer, and ii) the quantified RT Frontiers in Microbiology | www.frontiersin.org 3 January 2020 | Volume 10 | Article 3075 Cafiso et al. XDR A. baumannii sRNA Signatures TABLE 1 | Oligonucleotides used in this work. Name Sequence (50 to 30 ) Application A1S_r01intF GTAGCTTGCTACTGGACC Construction of the 1A1S_r01 mutant A1S_r01intR AGTAAATCCGATTAACGC Construction of the 1A1S_r01 mutant A1S_r01extF CTTAACACATGCAAGTCG Confirmation of the 1A1S_r01 mutant A1S_r01extR ATAAGCCGCCTACGCACG Confirmation of the 1A1S_r01 mutant A1S_2505intF GCACGTGTTGCCAGTCAG Construction of the 1A1S_2505 mutant A1S_2505intR TCACTGCCGTGATTGAAG Construction of the 1A1S_2505 mutant A1S_2505extF ATGGCTGGACAACATGGTAG Confirmation of the 1A1S_2505 mutant A1S_2505extR TTAACGGTTAATAGGGTG Confirmation of the 1A1S_2505 mutant A1S_3097FXbaI ATCGTCTAGAATGGGTGTTGTTGCTGATAG Overexpression of the A1S_3097 gene A1S_3097RNcoI ATCGCCATGGTTATGGTTGAACGTCGGC Overexpression of the A1S_3097 gene pETRAFW TTCTTCGTGAAATAGTGATGATTTTT Sequencing primer for pET-RA plasmid pETRARV CTGTTTCATATGATCTGGGTATC Sequencing primer for pET-RA plasmid M13FpUC GTTTTCCCAGTCACGAC Sequencing primer for the pCR-BluntII-TOPO plasmid M13RpUC CAGGAAACAGCTATGAC Sequencing primer for the pCR-BluntII-TOPO plasmid product was consequently used as a template for real-time qPCR were then introduced by electroporation in A. baumannii with TaqMan Fast Advanced Master Mix (Applied Biosystems). R ATCC 17978 strain and selected on kanamycin-containing The stem-loop specific primer used for RT and the specific plates. Recombinant clones were confirmed by sequencing TaqMan probes and primers used for real-time qPCR were R (Macrogen) of the PCR products obtained by using the provided by Custom TaqMan Small RNA Assay Design Tool R appropriate primers (Table 1). For overexpression, the A1S_3097 on the Applied Biosystems websiteTM . All real-time qPCRs were gene was cloned, using the indicated primers (Table 1), performed in triplicate, using Agilent AriaMx Real-Time PCR into XbaI-NcoI sites of the pET-RA vector (Aranda et al., System, with three different biological replicates using one of 2010). The recombinant plasmid was introduced in E. coli the thermal profiles suggested by the Custom TaqMan Small R DH5α and once correct construction was verified by both RNA Assays protocol, which provided a first enzymatic activation PCR and sequencing (Macrogen), in ATCC 17978. Finally, step of 95◦ C for 10 min, followed by 40 cycles of 95◦ C for A. baumannii transformants overexpressing the A1S_3097 gene 15 s and 60◦ C for 60 s (Salone and Rederstorff, 2015). The were selected on rifampicin- and kanamycin-containing plates expression levels of AbsRNA3 and microAbsRNA5 are shown as and confirmed by PCR with the pETRAFW and pETRARV the increment/decrement fold-change (FC) in COLR (1-R, 2-R) primers (Table 1). vs. COLS strains (1-S, 2-S) in RNA-seq and real-time qPCR. Statistics RESULTS AbsRNA3 and microAbsRNA5 expression levels found by Custom TaqMan probe-based real-time qPCRs were expressed R Comparative Transcriptome Analysis as means ± standard deviations and analyzed by the one- To define the characterizing sRNA traits of the two COLR vs. way analysis of variance (ANOVA) using the on-line Free COLS clinical Ab strains, a comparative analysis of the DE sRNAs Statistics Calculators-DanielSoper (Soper, 2019) considering a was conducted by a computational double cross-filtering. p-value ≤ 0.01 as statistically significant. RNA-sequencing generated 1,307,792 – 1,175,327 – 1,173,332 – 1,270,020 total reads in 1-S, 1-R, 2-S, and 2-R, Ab ATCC 17978 Mutant Construction respectively, with 96, 96, 72, and 93% mapped reads on Ab ATCC Due to the difficult to manipulate the clinical A. baumannii 17978; as well as 97, 97, 76, and 95% reads aligned on Ab ACICU strain-pairs related to the lack of antibiotic markers for the for the TS library, whilst 2,353,045 – 2,041,858 – 1,804,167 – mutants selection, A. baumannii ATCC 17978 strain was used 1,819,349 total reads in 1-S, 1-R, 2-S, and 2-R, respectively, with to generate 1A1S_r01 and 1A1S_2505 mutants as well as to 57, 56, 53, and 56% mapped reads on Ab ATCC 17978 and 59, 58, overexpress the A1S_3097 gene. 54, and 57% on Ab ACICU for the SI library. RNome structures Gene inactivation was carried out as previously described were previously published (Cafiso et al., 2019). (Aranda et al., 2010). Briefly, an internal fragment of the As shown in Tables 2, 3 and Supplementary Table S1, target gene was PCR-amplified from the A. baumannii the comparative statistically significant filtering-analysis of ATCC 17978 genome, using the appropriated primers the sRNAome sorting for DE sRNAs of the COLR vs. (listed in Table 1). The internal fragment was cloned into COLS Ab parental strains returned two different under- the pCR-BluntII-TOPO plasmid (Invitrogen), introduced expressed cis-acting sRNAs (AbsRNA1 and AbsRNA2 ) mapping by electroporation in Escherichia coli DH5α (Clontech) and in antisense orientation the 16S rRNA gene A1S_r01, one selected in kanamycin-containing LB plates. Purified plasmids under-expressed cis-acting sRNA (AbsRNA3 ) targeting the A1S_ Frontiers in Microbiology | www.frontiersin.org 4 January 2020 | Volume 10 | Article 3075 Cafiso et al. XDR A. baumannii sRNA Signatures 2505/ACICU_02783 gene (hypothetical protein), one under- Libraryb TS SI SI SI expressed microRNA-size small RNA fragment (AbsRNA4 ) and its pre-microAbsRNA4 targeting the A1S_0501 gene (hypothetical protein), as well as an over-expressed microRNA- sRNA size (bp) size small RNA fragment (AbsRNA5 ) and its pre-microAbsRNA5 targeting the A1S_3097 gene coding a signal peptide involved 107 228 39 70 in the cytokinin biosynthesis. In detail, the two different cis- acting sRNAs – mapping on the Ab ATCC 17978 Reference Genome – targeted two different positions (Table 2) of the RefGen∗ position (nt) 2904426 3577563 Strain-pair 2 A1S_r01 gene with a size of 35 bp in strain-pair 1 (AbsRNA1 ) 193995 545917 and 39 bp in strain-pair 2 (AbsRNA2 ), no mapped regions were found on the Ab ACICU Reference Genome. The 70–75 nt cis-acting AbsRNA3 mapped in an analog position both on Ab 2904495 3577336 194033 546023 ATCC 17978 and Ab ACICU Reference Genomes targeting the A1S_2505/ACICU_02783 gene in both the Ab strain-pairs. The 107 nt pre-microAbsRNA4 covering the A1S_0501 mapping on pre-microAbsRNA4 pre-microAbsRNA5 Ab ATCC 17978 Reference Genome and its smaller fragment of 21 bp (microAbsRNA4 ) were found in Ab strain-pair 2 and in Ab strain-pair 1, respectively. Similarly, a 228 pre-microAbsRNA5 AbsRNA2 AbsRNA3 targeting the A1S_3097 gene in Ab strain-pair 2 and its inner sRNA fragment of 20 nt in Ab strain-pair 1 were found. Furthermore, both Ab strain-pairs presented the same aforementioned sRNAs with similar expression profiles (over- or under-expression), Libraryb SI SI SI SI though the q-value did not allow, in some cases, to return the same fragments in both strain-pairs, considering the two different genomic annotations. Moreover, the only sRNA with sRNA size (bp) a statistically significant expression in Ab ACICU Reference Genome was ACICU_02783 (AbsRNA3 ) in both strain-pairs 35 75 21 20 (Supplementary Table S1). Acinetobacter baumannii; b SI, Short-Insert Library; TS, Tru-Seq Library; ∗ RefGen, Reference Genome. The sRNA nucleotide positions (transcription start and stop) reported by Rockhopper tool were shown in Table 2. In addition, Strain-pair 1 RefGen∗ position (nt) none of these sRNAs targeted the 50 or 30 untranslated regions 2904426 3577464 194546 545822 (UTR) of their target genes. I-Tasser 2904500 3577445 194580 545842 To predict the putative role of AbsRNA2 , an A1S_2505/ACICU_02783 conserved domain (CD) BLAST TABLE 2 | sRNA nucleotide positions in Ab ATCC 17978 reference genome. search and I-Tasser ab initio protein structure prediction microAbsRNA4 microAbsRNA5 were computationally investigated. The three-dimensional structure of a protein can be very informative and useful to AbsRNA1 AbsRNA3 understand functional characteristics of proteins with unknown sRNA functions. This is because the structure of a protein provides the precise molecular details that often facilitate experimental characterization of an expected function. In a case in which there is no expected function, the structure of a protein Aba ATCC 17978 can be used to facilitate its functional predictions by using the structure as a search template for better-characterized Locus Tag A1S_2505 A1S_0501 A1S_3097 A1S_r01 proteins that share regions of structural similarity (Kemege et al., 2011). The CD-BLAST search provided a match with the PHA00684 super family (cl10259) domain related to a protein of unknown function. On the contrary, analyzing the concordances of the highest significant prediction of the microRNA-size small microRNA-size small sRNA interaction I-Tasser TM-align structural alignment and the COACH RNA fragment RNA fragment mechanisms Predicted biological function, we resolved the structure and biological function as similar to the Orphan Macrodomain cis-acting cis-acting Protein (human C6orf130) with O-Acyl-ADP-ribose deacylase a Ab, activity. In particular, the closest structural similarity of Frontiers in Microbiology | www.frontiersin.org 5 January 2020 | Volume 10 | Article 3075 Cafiso et al. XDR A. baumannii sRNA Signatures Expression profile a Ab, Acinetobacter baumannii; b R, general functional prediction only; 1426, Cytoskeletal protein RodZ, contains Xre-like HTH and DUF4115 domains; c GO numbers refer only to the biological process; d RPKM, Reads the targeted A1S_2505 was the PDB-Hit 2lgrA (TM-score 0.925) matching the human protein C6orf130, previously published as an Orphan Macrodomain Protein (human ↓ ↓ ↓ ↑ C6orf130) with an O-Acyl-ADP-ribose deacylase activity, which catalyzes the deacylation of O-acetyl-ADP-ribose, O-propionyl-ADP-ribose, and O-butyryl-ADP-ribose to produce ADP-ribose (ADP-r) with acetate, propionate, and butyrate, 780 respectively. Due to the structural similarity, we can speculate 352 310 2-S 15 that A1S_2505/ACICU_02783 could have a function similar to RPKMd of RNA-seq data annotated on Ab ATCC 17978a q-value ≤ 0.01• O-Acyl-ADP-ribose deacylase. This structural prediction was also supported by the COACH Predicted biological function, 2-R 472 19 6 5 2l8rA, defining the human protein C6orf130 in complex with ADP-ribose (C-score 0.59), matching the O-acetyl-ADP- ribose deacetylase receptor binding the ADP-ribose in the 523 647 696 1-S 0 AA residues – G19 D20 L21 F22 H32 C33 I34 S35 R39 A42 I44 A45 L47 A87 P118 R119 I120 G121 C122 G123 L124 1-R 105 187 111 173 D125 Y150 L152- representing the binding sites. This ligand binding site (BL0101984) showed GO Molecular Functions of purine nucleoside binding (GO:0001883), hydrolase activity GO numberc GO:0016021 GO:0009691 (GO:0016787) and deacetylase activity (GO:0019213) as – – well as the GO Biological Process of the purine nucleoside per kilo base per million mapped reads; • q-value ≤ 0.01 according to the Rockhopper guidelines were considered statistically significant. metabolic process (GO:0042278). Regarding the computational prediction of the putative role of AbsRNA3 , the A1S_0501 hypothetical protein referred to an integral membrane COGb 1426 component (GO:0016021), whilst the CD BLAST search – – R provided a CD of the cytoskeletal protein RodZ containing Xre-like HTH and DUF4115 domains related to the cell- cycle control, cell division, and chromosome partitioning assembles in complex with cell shape protein RodZ (cl34261 Superfamily). This result was also supported by modeling prediction Bacterial actin MreB the I-Tasser predictions. By LOMETS, the A1S_0501 protein O-Acyl-ADP-ribose I-Tasser Ab Initio showed homology with the highest Norm Z-score (1.71) and 0.33 coverage with 2wus hit referred as a bacterial deacylase structural protein actin MreB that can be complexed with the cell shape protein RodZ. As regards the A1S_0501 GO – – and the consensus prediction of GO term, obtained from I-Tasser, the A1S_0501 protein showed a molecular function of DNA polymerase activity (GO:0034061) (GO-score 0.38) Antisense sRNA: signal and DNA binding (GO:0003677) (GO-score 0.34) and the Antisense sRNA: 16S peptide for cytokinin hypothetical protein hypothetical protein biological process of nucleic acid metabolism (GO:0090304) Antisense sRNA: Antisense sRNA: (GO-score 0.38). ribosomal RNA Description biosynthesis Validation of sRNA Expression Profile TABLE 3 | Comparative sRNAs of the A. baumannii strains. Custom TaqMan probe-based real-time qPCRs, dedicated for R the analysis of bacterial sRNAs, validated and confirmed the RNA-seq expression profiles of two selected sRNA Aba ATCC 17978 candidates: AbsRNA3 and microAbsRNA5 in both Ab Locus Tag A1S_2505 A1S_0501 A1S_3097 strain-pairs, as shown in Figure 1. In detail, AbsRNA3 had A1S_r01 a statistically significant under-expression (p-value ≤ 0.01), whilst microAbsRNA5 showed a statistically significant over- expression (p-value ≤ 0.01) in both COLR Ab strains compared with their COLS parents. pre-microAbsRNA4 pre-microAbsRNA5 sRNA Target Mutants microAbsRNA4 microAbsRNA5 No COL MIC changes with respect to the wild-type Ab ATCC 17978 (COL-MIC 1 mg/L) were observed in the 1A1S_r01 Ab AbsRNA1 AbsRNA2 AbsRNA3 AbsRNA ATCC 17978 and 1A1S_2505 Ab ATCC 17978 mutants as well as in the WT + A1S_3097 (Table 4). Frontiers in Microbiology | www.frontiersin.org 6 January 2020 | Volume 10 | Article 3075 Cafiso et al. XDR A. baumannii sRNA Signatures FIGURE 1 | Custom TaqMan probe-based real-time qPCR validation of RNA-seq expression data on colistin resistant (COLR ) characterizing sRNAs.a value of 5-Fold Changes (FC) was indicated for incalculable FC due to the presence of a 0 value in one of the strains. ∗ p-value ≤ 0.01 obtained by a Student’s T-test and †q-value ≤ 0.01 according to the Rockhopper guidelines were considered statistically significant. TABLE 4 | COL MIC in Ab ATCC 17978 mutants. Regarding the novel AbsRNA1 , AbsRNA2 and AbsRNA3 , we can speculatively assume that via a cis-antisense regulation Phenotype of wild type and derivative strains Colistin MIC (mg/L) mechanism they could post-transcriptionally regulate the target Wild type (WT) Ab ATCC 17978 1 translation. On the contrary, no regulation appears at the 1A1S_r01 1 transcriptional level as demonstrated by the lack of statistically 1A1S_2505 1 significant differential expression in these AbsRNA targets WT + clinical strain derived A1S_3097 1 according to previously published transcriptomic data (Cafiso et al., 2019). On top, in COLR strains, AbsRNA1 and AbsRNA2 under-expression could indicate that they are characterized by DISCUSSION sRNAs modulating the protein synthesis machinery and the amount of active ribosomes in agreement with other previous The comparative (sRNAome) integrated to bioinformatics, findings (Fernández-Reyes et al., 2009). The occurrence of computational cross-double filtering and experimental the under-expressed AbsRNA3 – targeting the O-acetyl-ADP- validations of COLR versus COLS Ab strain-pair revealed ribose – provided evidence that COLR Ab are distinguished by distinctive small RNA signatures in COLR Ab. an AbsRNA3 involved in the RNase III inhibition previously Small non-coding RNAs have been identified so far as associated with different biological functions, including biofilm crucial regulatory elements in bacteria showing high structural production, virulence, and antibiotic resistance in Gram-negative diversity and molecular action mechanisms. The most intensively bacteria (Chen et al., 2011; Kim et al., 2013; Song et al., 2014). studied prokaryotic sRNA regulators are cis-acting sRNAs or In fact, the O-acetyl-ADP-ribose is a substrate for several related trans-encoded sRNAs, however, the more recent discovery of macrodomain proteins such as the human MacroD1, human microRNAs in prokaryotes represents a challenging field of MacroD2, Escherichia coli YmdB. In E. coli, YmdB is an RNase investigation regarding bacterial regulatory mechanisms. III inhibitor that modulates many different functions including Our clinical COLR Ab were distinguished by 3 cis-acting biofilm formation (Kim et al., 2013), and E. coli adaptive sRNAs and 2 microRNA-size small RNA fragments involved in resistance to aminoglycosides via an antibiotic stress-induced different biological networks. These distinctive features span a sequential modulation of the endoribonucleolytic activity of different area of bacterial biology involving protein synthesis RNase III and RNase G (Song et al., 2014). apparatus, host-microbe interactions, biofilm production, cell- Regarding the microRNA-size small RNA fragments cycle control, virulence and antibiotic resistance that emerge (microAbsRNA4 and its pre-microAbsRNA4 as well as the as sRNA targets. Notably, they do not seem apparently related microAbsRNA5 and its pre-microAbsRNA5 ), our data suggest to colistin resistance mechanism as demonstrated by our that these sRNAs exist in a premature form (pre-microAbsRNA) preliminary data on 1A1S_r01, 1A1S_2505 and high expressed that act as precursors of their mature form, microAbsRNAs, A1S_3097 Ab ATCC 17978 mutants showing no COL MIC likely obtained cutting the premature form. Moreover, the under- variations of mutants compared to the WT Ab ATCC 17978 – expressed microAbsRNA4 and its pre-microAbsRNA4 targeting reflecting, however, the wide biological adaptations that the co- a gene coding a structural membrane protein with a CD similar existence of colistin resistance and the XDR profile implies. to cytoskeletal protein RodZ related to the cell-cycle control, Frontiers in Microbiology | www.frontiersin.org 7 January 2020 | Volume 10 | Article 3075 Cafiso et al. XDR A. baumannii sRNA Signatures cell division and chromosome partitioning, could speculatively This work offers a model for the identification of sRNA address its regulation of these functions. In fact, the GO-term signatures and the prediction of their targets in A. baumannii. prediction highlighted two possible molecular functions, a DNA Although we do not have clear information on their functions yet, polymerase activity and a DNA-binding function. Furthermore, our bioinformatic analysis may provide indications regarding the the microAbsRNA4 target (A1S_0501) was previously listed as cellular roles of these new sRNAs. a transcript that decreased significantly upon exposure to NaCl in A. baumannii, but no relationship with the mechanisms of antimicrobial tolerance in response to monovalent cations was DATA AVAILABILITY STATEMENT previously found (Hood et al., 2010). For the over-expressed microAbsRNA5 and its pre- The datasets generated for this study can be found in the NCBI microAbsRNA5 , targeting a signal peptide involved in GEO Database: GSE109951. the cytokinin biosynthetic process, we have to keep in mind that A. baumannii is a versatile pathogen that can adhere and invade numerous cell types displaying varying AUTHOR CONTRIBUTIONS degrees of susceptibility to invasion, stimulating the pro- inflammatory immune response (Choi et al., 2008). The VC and SSte conceived and designed the study. VC, SStr, FL, stimuli and signaling pathways implicated in cell death VD, and AZ performed the genomics, transcriptomics, real time are not yet established; however, they involve imbalanced qPCR, and bioinformatics. GP contributed to the bioinformatics calcium homeostasis, pro-inflammatory cytokines, and oxidative analysis. JA carried out the mutant construction. All authors stress, all traits related to strain virulence (Smani et al., analyzed the data and contributed and approved the manuscript. 2011; Mortensen and Skaar, 2012). MicroAbsRNA5 can regulate host-microbe A. baumannii interactions that shape the pathogenesis of Ab infection mediated by the host FUNDING immune response. This study was supported by a research grant PRIN 2017SFBFER In this study, we experimentally and computationally from MIUR, Italy. This study was also supported by grant discovered five statistically significant DE sRNAs characterizing BIO2016-77011-R from the Ministerio de Economía y COLR Ab strains speculatively implicated, as cis-antisense Competitividad. JA is a Serra Húnter Fellow, Generalitat de sRNAs, in the regulation of protein synthesis machinery via Catalunya, Barcelona, Spain. under-expressed AbsRNA1 and AbsRNA2 and in different biological functions, including biofilm production, virulence and aminoglycoside-resistance via an under-expressed ACKNOWLEDGMENTS AbsRNA3 . Likewise, we found two microAbsRNAs that may be involved in cell-cycle control via an under-expressed We wish to thank the Scientific Bureau of the University of microAbsRNA4 and in the host-microbe interaction via an Catania (Italy) for language support. over-expressed microAbsRNA5 . 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Agents Chemother. 50, 2946–2950. doi: 10.1128/AAC.00103-06 Conflict of Interest: The authors declare that the research was conducted in the McClure, R., Balasubramanian, D., Sun, Y., Bobrovskyy, M., Sumby, P., Genco, absence of any commercial or financial relationships that could be construed as a C. A., et al. (2013). Computational analysis of bacterial RNA-seq data. Nucleic potential conflict of interest. Acids Res. 41:e140. doi: 10.1093/nar/gkt444 Moffatt, J. H., Harper, M., Harrison, P., Hale, J. D., Vinogradov, E., Seemann, T., Copyright © 2020 Cafiso, Stracquadanio, Lo Verde, Dovere, Zega, Pigola, Aranda et al. (2010). Colistin resistance in Acinetobacter baumannii is mediated by and Stefani. This is an open-access article distributed under the terms of the Creative complete loss of lipopolysaccharide production. Antimicrob Agents Chemother. Commons Attribution License (CC BY). The use, distribution or reproduction in 54, 4971–4977. doi: 10.1128/AAC.00834-10 other forums is permitted, provided the original author(s) and the copyright owner(s) Mortensen, B. L., and Skaar, E. P. (2012). Host-microbe interactions that shape are credited and that the original publication in this journal is cited, in accordance the pathogenesis of Acinetobacter baumannii infection. Cell Microbiol. 14, with accepted academic practice. No use, distribution or reproduction is permitted 1336–1344. doi: 10.1111/j.1462-5822.2012.01817.x which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 9 January 2020 | Volume 10 | Article 3075