Avison, Matthew B

Calvopiña, Karina

Dulyayangkul, Punyawee

English

Aminoglycoside resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant mechanism is overproduction of the SmeYZ efflux system. By studying laboratory selected mutants and clinical isolates, we show here that damage to the 50S Ribosomal protein L1 (RplA) activates SmeYZ production. We also show that gentamicin and minocycline, which target the ribosome, induce expression of smeYZ These findings explain the role of SmeYZ in both intrinsic and mutationally acquired aminoglycoside resistance.</p

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                          Calvopiña, K., Dulyayangkul, P., & Avison, M. B. (2020). Mutations inRibosomal Protein RplA or Treatment with Ribosomal ActingAntibiotics Activate Production of Aminoglycoside Efflux Pump SmeYZin Stenotrophomonas maltophilia. Antimicrobial Agents andChemotherapy, 64(2), [e01524-19].https://doi.org/10.1128/AAC.01524-19Peer reviewed versionLink to published version (if available):10.1128/AAC.01524-19Link to publication record in Explore Bristol ResearchPDF-documentThis is the author accepted manuscript (AAM). The final published version (version of record) is available onlinevia the American Society for Microbiology at https://aac.asm.org/content/64/2/e01524-19. Please refer to anyapplicable terms of use of the publisher.University of Bristol - Explore Bristol ResearchGeneral rightsThis document is made available in accordance with publisher policies. Please cite only thepublished version using the reference above. Full terms of use are available:http://www.bristol.ac.uk/red/research-policy/pure/user-guides/ebr-terms/ 1  Mutations in Ribosomal Protein RplA or Treatment with Ribosomal Acting Antibiotics Activate Production of Aminoglycoside Efflux Pump SmeYZ in Stenotrophomonas maltophilia.   Karina Calvopiñaa, Punyawee Dulyayangkula, Matthew B. Avisona*   aSchool of Cellular & Molecular Medicine, University of Bristol, Bristol. UK   * Correspondence to: School of Cellular & Molecular Medicine, University of Bristol, Bristol, United Kingdom. bimba@bris.ac.uk    Running Title: Induction of SmeYZ production in S. maltophilia   2  Abstract  Aminoglycoside resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant mechanism is overproduction of the SmeYZ efflux system. By studying laboratory selected mutants and clinical isolates, we show here that damage to the 50S Ribosomal protein L1 (RplA) activates SmeYZ production. We also show that gentamicin and minocycline, which target the ribosome, induce expression of smeYZ. These findings explain the role of SmeYZ in both intrinsic and mutationally acquired aminoglycoside resistance.      3  Text Aminoglycoside resistance in the important opportunistic pathogen Stenotrophomonas maltophilia is multifactorial. Reduced amikacin and tobramycin susceptibility is conferred by a chromosomal aac(6')-Iz gene, which is present in about 50% of clinical isolates (1,2). The chromosomal aph(3')-IIc gene is more widespread amongst S. maltophilia clinical isolates, and responsible for reduced kanamycin and neomycin susceptibility (3). A wide variety of other genes have been shown to play minor roles in intrinsic aminoglycoside susceptibility (4-9) but by far the most significant inducible resistance mechanisms is the SmeYZ efflux system, where SmeZ is an RND-type efflux pump (10). Following mutation, smeYZ can be over-expressed, which leads to hyper-resistance to all aminoglycosides, and, most importantly, smeYZ over-expressing mutants have been seen in the clinic (10). Disruption of smeYZ also affects S. maltophilia virulence (11), suggesting a more pleotropic role in physiology, as is common for RND-type efflux systems (12).  It has been shown that smeYZ expression is controlled by a two-component regulatory system encoded immediately upstream, named SmeSyRy (13). One S. maltophilia aminoglycoside hyper-resistant mutant shown to over-express smeYZ is K M5 (10, 14). This mutant is a derivative of the clinical S. maltophilia isolate K279a (15, 16) and was selected for its ability to grow of amikacin at 4 times MIC (14). In an attempt to identify the mutation responsible for smeYZ over-expression in this K M5, we resurrected it from the freezer and first confirmed using CLSI disc susceptibility testing (17) that it expresses a ‘resistance profile 3’ phenotype, as previously defined (10, 14): particularly reduced zone diameters for aminoglycosides, quinolones and tetracyclines (Table 1). Both SmeY and SmeZ were found to be approximately 8-fold upregulated in K M5 relative to the parental strain, K279a, according to LC-MS/MS envelope proteomics (Figure 1), which was carried out as described previously (18). Whole genome sequencing (WGS) was performed and analysed as before (19) and showed that smeSy and smeRy are wild-type in K M5. Instead, there is only one difference from K279a, confirmed by PCR sequencing using the primers rplA F 5’ GCGAAGGAACCGGATCTGA-3’ and rplA R 5’- CGCCTGCGGTCTTTGAC-3’. The single point mutation in K M5 is predicted to cause a Gly67Asp change in the largest protein from the 50S ribosomal subunit, L1, encoded by the rplA gene. A previously described clinical isolate 9189, which also has the resistance profile 3 phenotype (Table 1) and overexpresses smeYZ (10) was found by PCR sequencing to also carry differences in rplA relative to K279a: predicted to cause a Phe22Leu change in RplA and a frameshift mutation at codon 212 caused by the insertion of a single adenine base. In order to confirm a role for the observed rplA mutation in reduced aminoglycoside susceptibility in K M5, wild-type rplA was amplified alongside the overlapping rplK by PCR using the primers rplAK F 5’-AAAGCGGCCGCATCCAGCTGTAGAGTCGAGC-3’ and rplAK  4  R 5’AAAGCGGCCGCCTGCGGTCTTTGACGGCTAC-3’ cut with NotI (introduced site underlined) and ligated into the broad host range vector pBBR1MCS (20) and the recombinant or empty vector were used to transform K M5 to chloramphenicol resistance (30 µg.mL- 1). Using MIC and disc susceptibility testing, we confirmed that carriage of wild-type rplA by K M5 increased amikacin and gentamicin susceptibility relative to plasmid only control, though not to wild-type levels (Table 2, Figure 2A). The other markers of “resistance profile 3”: reduced susceptibility to fluoroquinolones and tetracyclines were not reversed by complementation of the rplA mutation in K M5, and we have previously shown that this part of the phenotype is due to over-expression of a different pump operon, smeIJK (10). Surprisingly, in fact, fluoroquinolone and tetracycline susceptibility reduced in KM5 pBBR1MCS::rplA compared with the plasmid only control (Figure 2A). It is known that there is an inverse correlation between SmeYZ production and SmeDEF production in S. maltophilia (21) and indeed, proteomics confirmed that as well as SmeY production (Figure 2B) being reduced in K M5 (pBBR1MCS::rplA) relative to plasmid only control (though not to wild-type levels, as seen for aminoglycoside MICs in Table 2), production of SmeD, SmeE and SmeF increased 17.3-fold, 11.2-fold and 17.3-fold, respectively (p<0.001 n=3 for all). SmeDEF is a known tripartite efflux pump for fluoroquinolones and tetracyclines in S. maltophilia (22), explaining our findings (Figure 2A). In P. aeruginosa, MexXY is considered the most important efflux-pump involved in aminoglycoside resistance (23). It has been stated that at least two P. aeruginosa clinical isolates that hyperexpress mexXY have truncations in rplA, though the data were not presented and reported as ‘unpublished’ (24). Mutations in other ribosomal subunit genes have more conclusively been shown to cause mexXY overexpression (25). Given the similarities between MexXY and SmeYZ, this provides precedence for our experimental observation that rplA disruption is the cause of smeYZ overexpression in S. maltophilia.  Expression of mexXY in P. aeruginosa is inducible in response to ribosomal acting antibiotics (25, 26) and since ribosomal protein damage by mutation is associated with SmeYZ overproduction in S. maltophilia (Figure 1) we next tested the inducibility of smeZ expression by the ribosomal acting antibiotics gentamicin and minocycline. Expression of smeZ was assessed by RT-qPCR using RNA prepared from a nutrient broth (NB) culture of K279a exposed to gentamicin (32 µg.mL- 1) or minocycline (0.5 µg.mL) versus untreated control. The method used was as described previously (18) with the primers smeZ RT F 5’-TGTCCAGCGTCAAGCACC-3’ and smeZ RT R 5’-GCCGACCAGCATCAGGAAG-3’. Abundance of smeZ was normalised to rrnB abundance (as an RNA loading control) using the primers rrnB F 5’-GACCTTGCGCGATTGAATG-3’ and rrnB R 5’-CGGATCGTCGCCTTGGT-3’. This experiment confirmed that, as predicted, the normalised expression of smeZ in the  5  gentamicin- or minocycline-treated K279a cultures was significantly more (approximately 8-fold and 3-fold, respectively, p<0.05, n=3 biological replicates, each with 4 technical replicates) than control Figure 3.  Based on our findings, we therefore conclude that RplA damage in S. maltophilia constitutively mimics the effects of treatment with ribosomal acting antibiotics and constitutively activates SmeYZ production, raising aminoglycoside MICs. Our finding that rplA mutations exist in SmeYZ over-producing clinical isolates confirms that such a mutation does not impair fitness or virulence so much that the mutants cannot survive, colonise and cause infections in humans. Indeed, since reduced SmeYZ production reduces S. maltiphilia virulence, at least in vitro and in a mouse model of infection (11) it may be that the advantage of rplA mutation in S. maltophila is greater than simply raising aminoglycoside MICs. Given that S. maltophilia is frequently a coloniser of the lungs of cystic fibrosis patients, for which the aminoglycoside tobramycin is a regular therapy (28), the potential for selecting mutants with elevated SmeYZ production seems high.  Acknowledgements This work was funded by grants MR/N013646/1 and MR/S004769/1 to M.B.A. from the Antimicrobial Resistance Cross Council Initiative supported by the seven United Kingdom research councils. K.C. received a postgraduate scholarship from SENESCYT, Ecuador. We are grateful to Dr Kate Heesom, School of Biochemistry, University of Bristol for performing the proteomics analysis.  We declare no conflicts of interest.   6  Table 1 Disc susceptibility profile of S. maltophilia derivatives and clinical isolates.   Antibiotic (µg)  CAZ (30) LEV (5) CIP (5) MH (30) CN (10) AK (10) C (30) SXT (25) K279a 32 27 23 32 22 24 25 27 K M5 29 18 14 25 6 9 23 22 9189 31 24 18 31 6 11 6 6  Shaded values represent reduced zone diameters (≥5 mm relative to the parental clinical isolate K279a) in K M5 and in the clinical isolate 9189. Abbreviations: CAZ, ceftazidime; LEV, levofloxacin; CIP, ciprofloxacin; MH, minocycline; CN, gentamicin; AK, amikacin; C, chloramphenicol; SXT, sulphamethoxazole/trimethoprim. Numbers in brackets is the amount (µg) of antimicrobial found in each disc.   7  Table 2 Aminoglycoside MICs against S. maltophilia derivatives and clinical isolates Strain MIC Amikacin Gentamicin K279a 16 8 K M5 >256 >256 9189 256 >256 K M5 (pBBR1MCS) >256 >256 K M5 (pBBR1MCS::rplA) 128 32         8  Figure Legends  Figure 1 Production of SmeYZ in K M5.  Protein abundance data for whole envelope proteomics were normalised to the average 30S and 50S ribosomal protein abundance for each. Abundance of SmeY and SmeZ (Uniprot: B2FQ54 and B2FQ55) are reported as mean +/- Standard Error of the Mean (SEM), n=3. Differences between K279a and K M5 were statistically significant (p<0.05).   Figure 2 Effect of complementing K M5 with rplA  (A) Disc susceptibility testing for antimicrobials against K M5 carrying pBBR1MCS alone (white bar) or pBBR1MCS::rplA (black bar). Data are means +/- SEM, n=3. Abbreviations: LEV, levofloxacin, 5 µg disc; CIP, ciprofloxacin, 5 µg disc; MH, minocycline, 30 µg disc; TE, tetracycline, 30 µg disc; CN, gentamicin, 10 µg disc; AK, amikacin, 10 µg disc; SXT, sulphamethoxazole/trimethoprim, 25 µg disc. (B) Abundance of SmeY normalised to average ribosomal protein abundance based on proteomics analysis is reported as mean +/- SEM, n=3. The differences between K279a and K M5 and between K M5(pBBR1MCS) and K M5(pBBR1MCS::rplA) are statistically significant (p<0.05).     Figure 3 RT-qPCR analysis of the effect of ribosomal acting antibiotics on smeZ expression in K279a. K279a cultures were diluted 1:100 from an overnight culture and each grown in NB for 3 h. The experimental group was grown in the presence of gentamicin (CN) (32 µg.mL-1) or minocycline (MH) (0.5 µg.mL-1). RNA was purified and the abundance of smeZ in each RNA preparation was assayed using RT-qPCR calculated using the 2^∆∆Ct method (18) using the rrnB gene as an internal control for RNA abundance. Values for smeZ/rrnB ratio from treated cells were normalised to untreated control (NB). Stars indicate values that are statistically significantly different from control (p<0.05). There were three biological and, for each, four technical replicates.     9  Figure 1       K279a K M50.00.20.40.60.81.01.21.4Abundance(Relative to Ribosomal Proteins)SmeYSmeZ 10  Figure 2 A         B  LEV CIP MH TE CN AK SXT010203040Zone of inhibition (mm) K M5/pBBR1MCSK M5/pBBR1MCS::rplAK279aK M5K M5/pBBR1MCSK M5/pBBR1MCS::rplA0.00.20.40.60.81.01.21.4SmeYAbundance(Relative to Ribosomal Proteins) 11  Figure 3          12  References 1. Lambert T, Ploy MC, Denis F, Courvalin P. 1999. Characterization of the chromosomal aac(6')-Iz gene of Stenotrophomonas maltophilia. Antimicrob Agents Chemother  43:2366-2371. 2. Li XZ, Zhang L, McKay GA, Poole K. 2003. Role of the acetyltransferase AAC(6')-Iz modifying enzyme in aminoglycoside resistance in Stenotrophomonas maltophilia. J Antimicrob Chemother 51:803-811. 3. Okazaki A, Avison MB. 2007. Aph(3')-IIc, an aminoglycoside resistance determinant from Stenotrophomonas maltophilia. Antimicrob Agents Chemother 51:359-360. 4. 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Mutations in Ribosomal Protein RplA or Treatment with Ribosomal Acting Antibiotics Activate Production of Aminoglycoside Efflux Pump SmeYZ in Stenotrophomonas maltophilia

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Mutations in Ribosomal Protein RplA or Treatment with Ribosomal Acting Antibiotics Activate Production of Aminoglycoside Efflux Pump SmeYZ in Stenotrophomonas maltophilia

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