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• W3087636632 abstract "Clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa from patients with cystic fibrosis (CF) frequently contain mutations in the gene encoding an elongation factor, FusA1. Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the efflux pump, MexXY. However, we wondered whether these mutants might also be affected in other virulence-associated phenotypes. Here, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1P443L) in which mexXY expression was increased. Proteomic and transcriptomic analyses revealed that the fusA1 mutant also exhibited discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI (T6) secretion machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6 secretion system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosome synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable with that in the progenitor strain by expression of the WT fusA1 gene in trans, indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (and also additional contributory resistance mechanisms), mutations in fusA1 can lead to highly selective dysregulation of virulence gene expression. Clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa from patients with cystic fibrosis (CF) frequently contain mutations in the gene encoding an elongation factor, FusA1. Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the efflux pump, MexXY. However, we wondered whether these mutants might also be affected in other virulence-associated phenotypes. Here, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1P443L) in which mexXY expression was increased. Proteomic and transcriptomic analyses revealed that the fusA1 mutant also exhibited discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI (T6) secretion machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6 secretion system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosome synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable with that in the progenitor strain by expression of the WT fusA1 gene in trans, indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (and also additional contributory resistance mechanisms), mutations in fusA1 can lead to highly selective dysregulation of virulence gene expression. Due to its high intrinsic resistance to antibiotics and aggressive virulence, Pseudomonas aeruginosa holds the dubious accolade of consistently occupying a “top ten” slot on lists of clinical threats across the globe. Indeed, the World Health Organization recently classified it as a top priority pathogen for which the development of new antimicrobial interventions is critical. This opportunistic, Gram-negative bacterium is ubiquitous and exhibits a particular predilection for the built environment, making encounters with the human populace commonplace. P. aeruginosa is frequently isolated from burn wounds, the respiratory tract, and the urinary tract and is the leading cause of morbidity and mortality in people with cystic fibrosis (CF) (1McCarthy R.R. Mooij M.J. Reen F.J. Lesouhaitier O. O'Gara F. A new regulator of pathogenicity (bvlR) is required for full virulence and tight microcolony formation in Pseudomonas aeruginosa.Microbiology. 2014; 160 (24829363): 1488-150010.1099/mic.0.075291-0Crossref PubMed Scopus (17) Google Scholar, 2Pereira S.G. Rosa A.C. Ferreira A.S. Moreira L.M. Proença D.N. Morais P.V. Cardoso O. Virulence factors and infection ability of Pseudomonas aeruginosa isolates from a hydropathic facility and respiratory infections.J. Appl. Microbiol. 2014; 116 (24484457): 1359-136810.1111/jam.12463Crossref PubMed Scopus (13) Google Scholar, 3Pang Z. Raudonis R. Glick B.R. Lin T.-J. Cheng Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies.Biotechnol. Adv. 2019; 37 (30500353): 177-19210.1016/j.biotechadv.2018.11.013Crossref PubMed Scopus (486) Google Scholar). CF is a genetic disease characterized by defective targeting or activity of the cystic fibrosis transmembrane conductance regulator. Although it is a multisystem disease affecting many organs, the most obvious manifestations of CF are associated with the respiratory tract. Here, defective mucociliary clearance causes an accumulation of thick mucus plugs within the airways. Such oxygen-limited environments provide the perfect niche for P. aeruginosa to thrive (4Ratjen F. Döring G. Cystic fibrosis.Lancet. 2003; 361 (12606185): 681-68910.1016/S0140-6736(03)12567-6Abstract Full Text Full Text PDF PubMed Scopus (872) Google Scholar, 5Trinh N.T.N. Bilodeau C. Maillé É. Ruffin M. Quintal M.-C. Desrosiers M.-Y. Rousseau S. Brochiero E. Deleterious impact of Pseudomonas aeruginosa on cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells.Eur. Respir. J. 2015; 45 (25792634): 1590-160210.1183/09031936.00076214Crossref PubMed Scopus (34) Google Scholar). Chronic P. aeruginosa infection of the CF lung is associated with the transition from an active, motile lifestyle to a sessile, biofilm-like mode of growth. These are bacterial communities embedded within a self-produced extracellular polymeric matrix, composed of mannose-rich polysaccharides, extracellular DNA, and proteins (6Maunders E. Welch M. Matrix exopolysaccharides; the sticky side of biofilm formation.FEMS Microbiol. Lett. 2017; 364 (28605431): 3440-345210.1093/femsle/fnx120Crossref Scopus (70) Google Scholar). This matrix confers a level of protection against antibiotics and the host immune system (7Alhede M. Bjarnsholt T. Givskov M. Alhede M. Pseudomonas aeruginosa Biofilms.Adv. Appl. Microbiol. 2014; 86 (24377853): 1-4010.1016/B978-0-12-800262-9.00001-9Crossref PubMed Scopus (120) Google Scholar). Biofilm formation is also associated with increased expression of the Type VI (T6) secretion machinery. The function of the P. aeruginosa T6 secretion system has become clearer in the last decade; it appears to play a role in killing other bacterial species (or even “non-self” P. aeruginosa strains) (8Hood R.D. Singh P. Hsu F. Güvener T. Carl M.A. Trinidad R.R.S. Silverman J.M. Ohlson B.B. Hicks K.G. Plemel R.L. Li M. Schwarz S. Wang W.Y. Merz A.J. Goodlett D.R. et al.A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria.Cell Host Microbe. 2010; 7 (20114026): 25-3710.1016/j.chom.2009.12.007Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 9Basler M. Ho B.T. Mekalanos J.J. Tit-for-Tat: type VI secretion system counterattack during bacterial cell-cell interactions.Cell. 2013; 152 (23415234): 884-89410.1016/j.cell.2013.01.042Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), especially in tightly packed biofilms where competition for the same resources is rife. Conversely, “free-swimming” planktonic cells predominate in acute infection scenarios. Here, virulence factors and motility are up-regulated, and pathogenicity is enhanced (10Breidenstein E.B.M. de la Fuente-Núñez C. Hancock R.E.W. Pseudomonas aeruginosa: all roads lead to resistance.Trends Microbiol. 2011; 19 (21664819): 419-42610.1016/j.tim.2011.04.005Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Perhaps the most potent P. aeruginosa virulence determinant is the Type III (T3) secretion system, which mediates the translocation of cytotoxic effector proteins directly into the cytoplasm of neighboring host cells. These effectors subvert the function of the recipient cells, typically by disrupting the cytoskeleton, promoting cell rounding and apoptosis, and therefore assist immune evasion through preventing phagocytosis by host innate immune cells (11Galle M. Carpentier I. Beyaert R. Structure and function of the Type III secretion system of Pseudomonas aeruginosa.Curr. Protein Pept. Sci. 2012; 13 (23305368): 831-84210.2174/138920312804871210Crossref PubMed Scopus (75) Google Scholar, 12Berube B.J. Murphy K.R. Torhan M.C. Bowlin N.O. Williams J.D. Bowlin T.L. Moir D.T. Hauser A.R. Impact of type III secretion effectors and of phenoxyacetamide inhibitors of type III secretion on abscess formation in a mouse model of Pseudomonas aeruginosa infection.Antimicrob. Agents Chemother. 2017; 61 (28807906): e01202-e0121710.1128/AAC.01202-17Crossref PubMed Scopus (32) Google Scholar). Both the T3 and T6 secretion systems are contact-triggered injectisomes; however, they are structurally and mechanistically distinct, as are their targets, and the expression of these two systems appears to be inversely correlated (13Moscoso J.A. Mikkelsen H. Heeb S. Williams P. Filloux A. The Pseudomonas aeruginosa sensor RetS switches type III and type VI secretion via c-di-GMP signalling.Environ. Microbiol. 2011; 13 (21955777): 3128-313810.1111/j.1462-2920.2011.02595.xCrossref PubMed Scopus (180) Google Scholar). In previous work, we showed that the rapidly growing planktonic cells associated with increased virulence factor production display markedly up-regulated expression of the machinery required for macromolecular synthesis, especially proteins involved in translation (14Mikkelsen H. Duck Z. Lilley K.S. Welch M. Interrelationships between colonies, biofilms, and planktonic cells of Pseudomonas aeruginosa.J. Bacteriol. 2007; 189 (17220232): 2411-241610.1128/JB.01687-06Crossref PubMed Scopus (88) Google Scholar). Translation comprises four main steps; initiation, elongation, termination, and recycling. The ribosome-associated protein, elongation factor G (EF-G, encoded by fusA1 and fusA2 in P. aeruginosa), is essential for two of these steps: “elongation” and “recycling” (15Savelsbergh A. Rodnina M.V. Wintermeyer W. Distinct functions of elongation factor G in ribosome recycling and translocation.RNA. 2009; 15 (19324963): 772-78010.1261/rna.1592509Crossref PubMed Scopus (91) Google Scholar). During elongation, EF-G catalyzes the translocation of charged tRNA from the A-site to the P-site and from the P-site to the E-site of the large ribosomal subunit. This is coupled with movement of the ribosome along the mRNA being translated. EF-G is comprised of five domains; domains I and II mediate GTP binding and hydrolysis, and domains III, IV, and V dock to the A-site of the ribosome. The tip of domain IV interacts with the mRNA and promotes tRNA translocation (16Salsi E. Farah E. Netter Z. Dann J. Ermolenko D.N. Movement of elongation factor G between compact and extended conformations.J. Mol. Biol. 2015; 427 (25463439): 454-46710.1016/j.jmb.2014.11.010Crossref PubMed Scopus (23) Google Scholar). This involves large structural rearrangements as the EF-G domains swivel relative to one another (17Belardinelli R. Rodnina V, M. Effect of fusidic acid on the kinetics of molecular motions during EF-G-induced translocation on the ribosome.Sci. Rep. 2017; 7 (28874811): 1053610.1038/s41598-017-10916-8Crossref PubMed Scopus (10) Google Scholar, 18Macé K. Giudice E. Chat S. Gillet R. The structure of an elongation factor G-ribosome complex captured in the absence of inhibitors.Nucleic Acids Res. 2018; 46 (29408956): 3211-321710.1093/nar/gky081Crossref PubMed Scopus (9) Google Scholar). The translocation process is repeated until a stop codon is encountered and release factors catalyze hydrolysis of the peptidyl-tRNA bond, thereby liberating the newly synthesized polypeptide. EF-G then coordinates with ribosome-recycling factor (a structural mimic of tRNA) to promote disassembly of the ribosomal subunits (16Salsi E. Farah E. Netter Z. Dann J. Ermolenko D.N. Movement of elongation factor G between compact and extended conformations.J. Mol. Biol. 2015; 427 (25463439): 454-46710.1016/j.jmb.2014.11.010Crossref PubMed Scopus (23) Google Scholar, 19Wilson D.N. Ribosome-targeting antibiotics and mechanisms of bacterial resistance.Nat. Rev. Microbiol. 2014; 12 (24336183): 35-4810.1038/nrmicro3155Crossref PubMed Scopus (534) Google Scholar). Aminoglycoside antibiotics can disrupt both the elongation and recycling steps, thereby leading to a decrease in the overall number of ribosomes available. Whole-genome sequence analyses have revealed that fusA1 is a hotspot for accruing mutations in P. aeruginosa isolates from patients with CF (20Bolard A. Plésiat P. Jeannot K. Mutations in gene fusA1 as a novel mechanism of aminoglycoside resistance in clinical strains of Pseudomonas aeruginosa.Antimicrob. Agents Chemother. 2018; 62 (29133559): e01817-e0183510.1128/aac.01835-17Crossref Google Scholar). These fusA1 mutants often display increased resistance to aminoglycoside antibiotics. However, little more is known about the phenotypic consequences of such fusA1 mutations on the wider physiology of the cell. In this study, we show that a spontaneous SNP in P. aeruginosa fusA1 gives rise to discrete but large-magnitude changes in key pathophysiological processes. For example, the strain containing the mutated fusA1 allele displayed selective up-regulation of genes encoding the T3 secretion system (T3SS) apparatus, the T6 secretion system (T6SS) apparatus, exopolysaccharide biosynthesis genes, and a multidrug efflux system. These findings suggest a hitherto unexpected subtlety in the chain of events linking transcription, translation, and virulence/antibiotic sensitivity in this organism. At the outset of this investigation, we sought to identify potential regulators of biofilm formation in P. aeruginosa. To do this, we made a stable chromosomal reporter construct in which the promoter of the cdrAB operon (PcdrAB) was fused to a promoter-less lacZ ORF. CdrA is a biofilm-associated extracellular matrix adhesion and is known to be primarily expressed in conditions that favor biofilm formation. The construct was integrated at a neutral site in the PAO1 chromosome using the mini-CTX system (21Calvo B. Bilbao J.R. Pérez De Nanclares G. Vázquez J.A. Castaño L. Integration-proficient Pseudomonas aeruginosa vectors for isolation of single-copy chromosomal lacZ lux gene fusions.BioTechniques. 2000; 29 (11084852): 948-95410.2144/00295bm04Crossref PubMed Scopus (189) Google Scholar). The resulting PcdrAB::lacZ reporter strain, hereafter referred to as EMC0, yielded vivid blue colonies when grown on M9 minimal medium agar plates containing X-Gal and glucose, but far paler colonies on medium containing X-Gal and glycerol. This suggested that PcdrAB is activated during colony growth on glucose. To identify genes that might impinge upon transcription from the cdrAB promoter, we mutagenized EMC0 by introducing the pTnMod-OGm plasposon (22Dennis J.J. Zylstra G.J. Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of Gram-negative bacterial genomes.Appl. Environ. Microbiol. 1998; 64 (9647854): 2710-271510.1128/AEM.64.7.2710-2715.1998Crossref PubMed Google Scholar). The resulting mutants were selected on plates containing gentamicin (to select for likely Tn insertion mutants) and X-Gal + glucose (to establish whether any of the mutants were affected in transcription from PcdrAB). Around 10,000 gentamicin-resistant mutants were screened in all. Several of these yielded a paler pigmentation than EMC0 when grown on X-Gal/glucose plates, indicating a reduction in transcription from PcdrAB (Fig. 1A). Further analysis of one of these isolates (hereafter denoted EMC1) in M9-glucose liquid cultures confirmed the diminished β-gal production. This also indicated that EMC1 had a minor growth defect in this medium (Fig. 1A). We therefore measured growth and β-gal production in a rich medium, LB. To our surprise, EMC1 exhibited an even greater growth defect in this medium (Fig. 1B). Attempts to identify the insertion site of the plasposon in EMC1 using conventional approaches (including “random-primed” PCR-based amplification of the regions flanking the plasposon, or “cloning out” of the plasposon as described previously (22Dennis J.J. Zylstra G.J. Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of Gram-negative bacterial genomes.Appl. Environ. Microbiol. 1998; 64 (9647854): 2710-271510.1128/AEM.64.7.2710-2715.1998Crossref PubMed Google Scholar)) failed. We therefore used whole-genome sequencing (WGS) of EMC1 (and, as a control, also of the EMC0 progenitor strain) to identify the plasposon insertion site. Remarkably, and despite the robust GmR phenotype of the strain (Table 1), we found that EMC1 did not contain a plasposon insertion. It did, however, contain a C→T transition at position 4,770,363 in the genome (Fig. S1). This SNP was located in the fusA1 ORF and resulted in a proline-to-leucine substitution at position 443 in the protein. The SNP was confirmed by PCR amplification of the gene followed by Sanger sequencing of the PCR product. WGS also revealed a selection of additional potential SNPs in EMC1, but these were all subsequently found by PCR/Sanger sequencing to be false positive SNP calls arising from the WGS technology. We conclude that the only difference between EMC0 and EMC1 is the SNP in fusA1.Table 1MICs of antibiotics for EMC0 and EMC1AntibioticMode of actionEMC0 MICEMC1 MICμg/mlGentamicinBinds 30S ribosome and disrupts reading of tRNA210KanamycinBinds 30S ribosome and disrupts reading of tRNA1500>2000TetracyclineBinds 30S ribosome and inhibits binding of tRNA2020ChloramphenicolBinds 50S ribosome and inhibits peptide bond formation250250RifampicinInhibits RNA polymerase100100Fusidic acidInhibits elongation factor G17501750 Open table in a new tab The gentamicin resistance of EMC1 was heritable but could be somewhat unstable. In one experiment, progressive subculturing (via 1:100 dilution of the culture into fresh medium every 24 h) of EMC1 in M9-glucose minimal medium lacking gentamicin gave rise to gentamicin-sensitive (GmS) derivatives. After each 24-h round of subculturing, aliquots were removed, serially diluted, and plated onto nonselective M9-glucose agar. A selection of 40 colonies from these plates were then retested for gentamicin resistance by plating onto M9-glucose + Gm. Following the first round of subculturing (24-h growth), none of the 40 tested colonies were GmS. However, after the second round of subculturing (a cumulative 48 h of growth) 1 of 40 colonies tested was gentamicin-sensitive, rising to 5 of 40 colonies after the third 24-h round of subculturing. PCR amplification and sequencing of the fusA1 gene in these GmS derivatives revealed that they all still contained the fusA1P443L mutation. These data suggest that whatever gives rise to the GmR phenotype in EMC1 can be overridden by secondary mutations elsewhere. However, these bypass mutations do not arise with high frequency, and even when they did, they took time to spread through the culture (with only 1 in 8 of the isolates sampled being GmS after 72 h of growth). Indeed, subsequent independent repeats of the experiment yielded no further GmS isolates upon subculturing EMC1 in the absence of gentamicin. FusA1 encodes one of the two paralogous EF-G proteins in P. aeruginosa and plays a pivotal role in protein synthesis and ribosomal recycling (23Palmer S.O. Rangel E.Y. Hu Y. Tran A.T. Bullard J.M. Two homologous EF-G proteins from Pseudomonas aeruginosa exhibit distinct functions.PLoS ONE. 2013; 8 (24260360): e8025210.1371/journal.pone.0080252Crossref PubMed Scopus (13) Google Scholar). The amino acid sequence of the two paralogues (denoted fusA1 (PA4266) and fusA2 (PA2071)) is highly conserved, with a shared identity of 84%. EF-G1B, encoded by fusA2, is thought to have greater involvement in elongation and polypeptide synthesis (23Palmer S.O. Rangel E.Y. Hu Y. Tran A.T. Bullard J.M. Two homologous EF-G proteins from Pseudomonas aeruginosa exhibit distinct functions.PLoS ONE. 2013; 8 (24260360): e8025210.1371/journal.pone.0080252Crossref PubMed Scopus (13) Google Scholar). By contrast, EF-G1A (encoded by fusA1) has a more dominant role in ribosomal recycling and association with ribosomal recycling factors (23Palmer S.O. Rangel E.Y. Hu Y. Tran A.T. Bullard J.M. Two homologous EF-G proteins from Pseudomonas aeruginosa exhibit distinct functions.PLoS ONE. 2013; 8 (24260360): e8025210.1371/journal.pone.0080252Crossref PubMed Scopus (13) Google Scholar). FusA1 is a conformationally flexible multidomain protein (24Lin J. Gagnon M.G. Bulkley D. Steitz T.A. Conformational changes of elongation factor G on the ribosome during tRNA translocation.Cell. 2015; 160 (25594181): 219-22710.1016/j.cell.2014.11.049Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and proline 443 sits in close proximity to the crucial GTPase “switch” regions (Fig. 2A). The switch regions play an important role in directing GDP-GTP exchange, raising the question of whether the P443L substitution might affect the conformation of the protein. To test this, we measured the intrinsic Trp fluorescence profile of purified WT FusA1 and FusA1P443L. FusA1 contains 6 tryptophan residues. Protein Trp fluorescence is exquisitely sensitive to the microenvironment of each Trp residue and, as such, can be a sensitive reporter of protein conformation. These analyses indicated that purified FusA1P443L had a lower quantum yield at the Trp emission λmax (332 nm) compared with the WT protein (Fig. 2B). This indicates that one or more Trp residues in the mutant protein are likely to exhibit altered solvent accessibility compared with the WT protein, possibly due to conformational differences. One of the more widely used web-based algorithms, mCSM (25Pires D.E.V. Ascher D.B. Blundell T.L. mCSM: predicting the effects of mutations in proteins using graph-based signatures.Bioinformatics. 2014; 30 (24281696): 335-34210.1093/bioinformatics/btt691Crossref PubMed Scopus (488) Google Scholar), predicted that the P443L substitution should destabilize FusA1 by 0.267 kcal/mol. Consistent with this, purified FusA1P443L had a lower melting temperature than the WT protein (Fig. 2C). Taken together, these data indicate that the P443L substitution likely alters the conformation, stability, or dynamics of FusA1. The substantial growth defect associated with EMC1 grown in LB was complementable by expression of the WT fusA1 gene in trans on a plasmid (Fig. S2), suggesting that the WT allele is dominant. The complemented EMC1 also displayed the same MIC for gentamicin as EMC0. Compared with EMC0, EMC1 also exhibited defects in twitching motility and swimming motility, and these too could be complemented by introduction of WT fusA1 in trans (Fig. 3A). EMC1 also displayed lower secreted gelatinase activity, and poor growth on the gelatinase plates. However, the lower secreted gelatinase production and slower growth of EMC1 may be linked because complementation of EMC1 in trans with WT fusA1 restored both phenotypes. Expression of fusA1P443L in trans in EMC0 or EMC1 had no apparent effect on the motility/gelatinase phenotypes of the strains compared with the empty vector control. This suggests that the WT fusA1 allele in EMC0 is dominant. By contrast with the impaired motility and secreted protease phenotypes, exopolysaccharide production was increased in EMC1 compared with EMC0, both on plate assays (Fig. 3A) and in liquid culture (Fig. S3). Expression of WT fusA1 in trans in EMC1 led to a decrease in exopolysaccharide production (compared with EMC1 containing the empty vector control), whereas expression of fusA1P443L enhanced exopolysaccharide synthesis. Exopolysaccharides comprise the extracellular matrix, which “glues together” cells in a biofilm. Interestingly in this regard, the increased exopolysaccharide production in EMC1 was not accompanied by an increase in its biofilm-forming ability compared with EMC0 (data not shown). Exopolysaccharide production is often inversely correlated with expression of the T3 secretion machinery. We therefore examined whether the P443L substitution in FusA1 impacted T3 secretion. To our surprise, cultures of EMC1 overexpressed the T3SS protein, PcrV (Fig. 3B). This increased expression was due to increased transcription of the pcrV-encoding operon, because RT-PCR analyses indicated that the amount of mRNA encoding pcrV was also increased in EMC1 (Fig. 3B and Fig. S4). This increased expression of PcrV could be reversed by supplying WT fusA1 in trans. These observations led us to test whether previously reported GmR fusA1 point mutants also displayed increased PcrV expression. A selection of GmR fusA1 mutants (all in a PAO1 genetic background) recently reported by Bolard et al. (20Bolard A. Plésiat P. Jeannot K. Mutations in gene fusA1 as a novel mechanism of aminoglycoside resistance in clinical strains of Pseudomonas aeruginosa.Antimicrob. Agents Chemother. 2018; 62 (29133559): e01817-e0183510.1128/aac.01835-17Crossref Google Scholar) were grown (alongside the progenitor PAO1 strain from that study) in M9-glucose, and the corresponding cell extracts were probed after SDS-PAGE resolution with anti-PcrV antibodies. None of these mutants displayed the robust PcrV expression associated with EMC1 (Fig. S5). This suggests that the GmR phenotype conferred by these other mutated fusA1 proteins is not linked with PcrV expression. Given that multiple phenotypes were affected by the fusA1P443L mutation in EMC1 and that these phenotypes were not all modulated in the manner expected from previous studies (e.g. exopolysaccharide production and T3 secretion both being up-regulated instead of inversely regulated), this suggested that the P443L mutation may lead to global dysregulation in EMC1. To investigate this further, cultures of EMC0, EMC1, and EMC1 complemented with WT fusA1 expressed from pUCP20 in trans (hereafter EMC1*) were grown to late exponential phase in M9 minimal medium + glucose and were prepared for iTRAQ-based proteomic analysis. To establish whether any of the observed changes in protein profile were also underpinned by transcriptional changes, samples were also harvested for RNA sequencing (from cultures grown in the same conditions). The proteomic analysis resolved 3506 proteins (of a total of 5570 predicted ORFs encoded by P. aeruginosa PAO1). Principal components analysis of the data revealed that the proteome of EMC1 was distinct from that of EMC0 and that the proteomic changes giving rise to this segregation could be largely reversed by expression of WT fusA1 in trans in EMC1* (Fig. S6 (A–C) and Tables S1 and S2). Proteins were considered to be significantly modulated if they exhibited a log2-fold change (FC) > 1 (or < −1, if down-regulated) with a false discovery rate (FDR)-adjusted p value of ≤0.01. Based on these criteria, 128 proteins were up-regulated in EMC1 compared with EMC0, and 166 proteins were down-regulated. The 20 most highly up-regulated proteins are shown in Table 2. Remarkably, and consistent with the earlier phenotypic analyses, over half (12 of 20) of these proteins were involved in T3 secretion. Similarly, and also consistent with our earlier observations, PelA, involved in the biosynthesis of exopolysaccharide, was also up-regulated. A list of the top 20 down-regulated proteins is shown in Table 2. The situation here is more ambiguous, because the majority (13 of 20) of these proteins currently have no assigned function.Table 2List of the top 20 proteins (based on log2FC) modulated in EMC1 versus EMC0ProteinLocus tagProtein functionEMC1 versus EMC0log2FCp valueUp-regulated proteins 1PscLPA1725Type III secretion export protein5.8730.000 2PopNPA1698Type III secretion outer membrane protein4.1870.001 3PscPPA1695Type III secretion translocation protein4.1780.000 4PA3661Hypothetical protein4.0040.002 5PopDPA1709Type III secretion outer membrane protein3.8950.000 6PA1325Hypothetical protein3.6670.000 7ExoYPA2191Adenylate cyclase3.2660.000 8SpcSPA3842ExoS chaperone3.2630.000 9PcrHPA1707Regulatory protein3.2620.000 10ExoSPA3841Exoenzyme S3.2610.000 11PcrGPA1705Type III secretion regulator3.2110.000 12PopBPA1708Type III translocator protein3.1510.000 13ExoTPA0044Exoenzyme T2.8360.000 14AprXPA1245Metalloproteinase2.7980.001 15AprAPA1249Alkaline metalloproteinase2.7550.007 16PelAPA3064Exopolysaccharide biosynthesis2.7250.000 17PA0630Hypothetical protein2.6880.001 18PA0620R-type pyocin, related to P2 phage2.6130.000 19SsuDPA3444Hypothetical protein2.5190.002 20PscFPA1719Type III export protein2.5070.000Down-regulated proteins 1ExaBPA1983Ethanol oxidation−3.1540.000 2PA5086Predicted T6SS lipase immunity protein−3.0880.000 3PA2565Hypothetical protein−2.9620.002 4PA3865Probable amino acid–binding protein−2.9550.001 5PA3785Hypothetical protein−2" @default.
• W3087636632 created "2020-09-25" @default.
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• W3087636632 creator A5013149881 @default.
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• W3087636632 creator A5057438504 @default.
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• W3087636632 date "2020-11-01" @default.
• W3087636632 modified "2023-09-26" @default.
• W3087636632 title "Global reprogramming of virulence and antibiotic resistance in Pseudomonas aeruginosa by a single nucleotide polymorphism in elongation factor, fusA1" @default.
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