A small regulatory RNA controls antibiotic tolerance inStaphylococcus aureusby modulating efflux pump expression

A small regulatory RNA controls antibiotic tolerance in Staphylococcus aureus by modulating efflux pump expression | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A small regulatory RNA controls antibiotic tolerance in Staphylococcus aureus by modulating efflux pump expression Kam Pou Ha , View ORCID Profile Philippe Bouloc doi: https://doi.org/10.1101/2024.08.05.606701 Kam Pou Ha 1 Université Paris-Saclay , CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC) , 91198, Gif-sur-Yvette, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Philippe Bouloc 1 Université Paris-Saclay , CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC) , 91198, Gif-sur-Yvette, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Philippe Bouloc For correspondence: philippe.bouloc{at}i2bc.paris-saclay.fr Abstract Full Text Info/History Metrics Preview PDF Abstract Staphylococcus aureus is an opportunistic pathogen that poses a considerable burden to healthcare settings worldwide, aided by its ability to thrive in different environmental growth conditions and survive exposure to antibiotics. Small regulatory RNAs (sRNAs) are crucial in enhancing bacterial fitness by modulating gene expression in response to changing environmental conditions. We investigated the role of sRNAs in S. aureus antibiotic resistance and tolerance. By assessing the fitness of a library of sRNA mutants, we identified that RsaA sRNA is required for bacterial growth when exposed to low concentrations of fluoroquinolone, a class of antibiotics targeting DNA replication. We also found that in the absence of RsaA, S. aureus is less susceptible to β-lactam antibiotics, which act on the cell wall. RsaA has been reported to prevent the expression of MgrA, a master regulatory protein controlling the expression of efflux pumps. Here, we show that RsaA affects the sensitivity of S. aureus to fluoroquinolone and β-lactam antibiotics through MgrA. RsaA has two forms, a short one that is commonly referred to in RsaA studies, and a long form about twice the length, of which not much is known. Interestingly, our phenotype was restored only when complemented with the long form of the gene. This work demonstrates the role of regulatory RNAs in the adaptation of S. aureus to antibiotic resistance and highlights their value as potential therapeutic targets for manipulating individual sRNA responses to promote the efficacy of existing antibiotics. Introduction Antibiotic resistance refers to the ability of bacteria to grow in antibiotic concentrations that are considered inhibitory, while antibiotic tolerance is the capacity of bacteria to survive exposure to bactericidal antibiotics for extended periods without necessarily acquiring genetic changes ( 1 ). Resistance involves the acquisition of resistance genes from other bacteria or by genetic mutation ( 1 – 3 ), leading to mechanisms such as enzymatic inactivation of antibiotics, alteration of antibiotic targets, or efflux pumps that expel antibiotics from bacterial cells ( 2 , 3 ). However, the molecular basis for tolerance is wider in range. Antibiotic tolerance can be mediated by the induction of stress responses, such as the SOS DNA damage response that detects and repairs damage caused by fluoroquinolone antibiotics ( 4 , 5 ), or by the reversible depletion of peptidoglycan in the cell wall, which enables survival in the presence of β-lactam antibiotics ( 6 ). In an extreme subset of tolerance, also called persistence, bacterial cells may enter a dormant or near-dormant state to protect cellular processes against the action of antibiotics ( 7 ). Tolerance has also been demonstrated as a precursor to the development of antibiotic resistance ( 8 – 10 ). Although antibiotic resistance mechanisms have been well studied, less is known about antibiotic tolerance. In addition, while global efforts have primarily focussed on methicillin-resistant S. aureus (MRSA), these strains represent only a subset of the overall S. aureus burden ( 11 ). Indeed, infections from both susceptible and resistant S. aureus strains lead to prolonged illness and increased mortality rates ( 11 , 12 ). Therefore, understanding how S. aureus modulates its sensitivity to antibiotics in the absence of resistance could aid in the development of novel treatment options to prevent resistance from developing. Small non-coding RNAs (sRNAs) are key players in the post-transcriptional regulation of target genes ( 13 ). They enable rapid environmental adaptation by base-pairing with their target mRNAs to modulate mRNA translation and stability ( 13 ). sRNAs act on almost all cellular functions and as a result, have emerged as important regulators of virulence ( 14 ), metabolic adaptation ( 15 ) and antibiotic resistance ( 16 , 17 ). Although many sRNAs have been identified in S. aureus, their functions and targets are still mostly unknown ( 18 , 19 ). The best-described sRNA in S. aureus is RNAIII, a large 514-nucleotide sRNA that is involved in virulence ( 20 ). Other described sRNAs include RsaE, which is associated with the TCA cycle ( 21 , 22 ) and arginine catabolism ( 15 ), and IsrR, which plays a key role in the iron-sparing response ( 23 , 24 ). Although sRNAs play important roles in regulating antibiotic-resistance processes such as cell envelope modifications and drug efflux pumps in Gram-negative species ( 16 , 25 – 27 ), their impact on antibiotic resistance in Gram- positive species has received less attention ( 16 , 17 ). In addition, the role of sRNAs in antibiotic tolerance is poorly documented. To find sRNAs required for S. aureus to adapt to sublethal antibiotic concentrations, we used a fitness assay with sRNA mutants grown in the presence of the fluoroquinolone antibiotic norfloxacin. We identified a single sRNA, RsaA, the loss of which increased sensitivity of the bacterium to fluoroquinolone antibiotics. Further tests revealed that loss of RsaA also decreased the sensitivity of S. aureus to β-lactam antibiotics, and that both effects were mediated by RsaA via the master regulatory protein MgrA. Together, our results demonstrate that sRNAs contribute towards antibiotic tolerance in S. aureus . Results RsaA is required for growth in the antibiotic norfloxacin To determine whether sRNAs are required for S. aureus to adapt to the presence of the fluoroquinolone antibiotic norfloxacin, we employed a well-established technique that enables the competitive fitness of a sRNA mutant library to be assessed ( 18 , 23 , 28 , 29 ). This technique, referred to as the fitness competition assay, uses a library of DNA-tagged deletion mutants in the HG003 strain (Supplementary Table S1) and enables the subtle phenotypes of sRNA mutants to be screened more easily. Three mutants were independently constructed for each sRNA gene, which were then used to establish three independent libraries (Supplementary Table S4). Each library was restricted to 48 mutants of “ bona fide ” sRNAs, defined as those that are genetically independent with their own promoter and terminator, to prevent any interference from nearby coding sequences ( 18 ). sRNA gene sequences were replaced with specific DNA tags to enable each mutant to be identified and the proportion of each mutant within the total population to be calculated. By using indexed PCR primer pairs, up to 40 samples could be tested in one DNA- seq run ( 18 ). Mutants that disappeared or accumulated under a given stress condition indicated a potential role for the corresponding sRNAs under this growth condition. The sRNA libraries were exposed to a sub-lethal concentration of norfloxacin and grown over three days with two serial dilutions. Samples were taken at different time points and growth stages, and the proportion of each mutant within the population was determined ( Figure 1A ). Results were normalised to the same inoculum grown in the same medium without antibiotic and sampled at the same growth phases. Among the 48 tested mutants, the strain containing Tag075 displayed the greatest fitness disadvantage, decreasing by 28-fold [log2(FC)=- 4.8] in the presence of norfloxacin after 72 hours, when compared to the control condition ( Figure 1B ). This strain, in which the rsaA gene has been replaced by Tag075 ( ΔrsaA ), progressively disappeared from the library under the selective pressure of norfloxacin under sub-lethal concentrations. Neither rnaIII , rsaC , rsaD , rsaE , rsaG, isrR nor rsaOG mutants ( 18 ), present in the libraries tested, were impacted by the presence of norfloxacin, indicating that their corresponding sRNAs are unlikely to contribute to adaptation to norfloxacin under the tested conditions. Download figure Open in new tab Figure 1. S. aureus RsaA is required for optimal growth in the presence of norfloxacin. (A) Experimental protocol scheme to identify mutants with altered fitness in media containing the antibiotic norfloxacin. (B) Evolution of mutant proportions in libraries (for composition, see Supplementary Table S4) grown in the presence of 0.25 µg/ml norfloxacin normalised to the same libraries grown in the absence of antibiotic. Error bars indicate the standard deviation from three independent libraries. Left: data with the complete 48 mutant libraries. Tag075 corresponding to the rsaA mutant is highlighted by a red box. For Tag-mutant association, see Supplementary Table S1. Right: selected data for ΔrsaA is shown. Colour code corresponds to the sampling time colour from Figure 1A . (C) Growth of WT and ΔrsaA strains serially diluted 10-fold and spotted onto BHI agar +/- 0.6 µg/ml norfloxacin (n = 3). To assess whether the sensitivity of the rsaA mutant to norfloxacin also occurred in the absence of the 47 other sRNA strains, monocultures of ΔrsaA were grown on agar plates containing norfloxacin. Results from these spot tests revealed that ΔrsaA is 100-fold more sensitive to norfloxacin, when compared to its parental strain HG003, referred to here as the wild-type strain (WT) ( Figure 1C ). In contrast, no difference in growth was observed between either strain when grown in the absence of the antibiotic. Therefore, loss of RsaA leads to greater sensitivity of S. aureus to norfloxacin and this is independent of any effects on growth in a mixed culture. Taken together with the fitness competition results, these findings suggested that the RsaA sRNA is required for optimal growth of S. aureus in the presence of norfloxacin. S. aureus rsaA norfloxacin-dependent phenotype is complemented only by the long form of the rsaA gene To confirm that the increased sensitivity of the rsaA mutant to norfloxacin was solely due to the loss of RsaA, the rsaA mutant was complemented with a wild-type copy of the gene. Since there are two versions of the RsaA transcript, a short form of 144 base pairs in length and a long version of 282 base pairs ( 30 , 31 ) ( Figure 2A ; Supplementary Figure S1), individual plasmids were constructed to reflect complementation with each form of the gene. Download figure Open in new tab Figure 2. The long form of RsaA restores the wild-type phenotype, but not the short form. (A) Schematic diagram of long and short forms of RsaA in S. aureus . Blue and white arrows represent genes, dark blue sticks represent transcription terminators. Gene annotations refer to NCTC8325 nomenclature (CP00025.1) retrieved from Genbank ( 33 ). The predicted secondary structure of RsaA L, along with the experimentally confirmed structure of RsaA S ( 31 ), can be found in Fig. S1. (B) Growth of WT, rsaA mutant, empty vector (pCN38) and mutants complemented with RsaA short form (p rsaA S ) or long form (p rsaA L ), serially diluted 10-fold and spotted onto BHI agar + 0.6 µg/ml norfloxacin (n = 3). Spot tests in BHI broth alone are shown in Fig. S2. (C) Growth curves of WT and rsaA mutants grown in BHI broth containing 0.5 µg/ml norfloxacin (n = 3). OD 600 measurements in BHI broth alone are shown in Fig. S2. Error bars represent the standard deviation of the mean. (D) Growth of WT, rsaA mutant, empty vector (pCN38) and mutants complemented with RsaA short form (p rsaA S ) or long form (p rsaA L ), serially diluted 10-fold and spotted onto BHI agar + 0.15 µg/ml ciprofloxacin (n = 3). When the strains were spotted onto agar containing norfloxacin, the loss of RsaA reduced the growth of S. aureus by 100-fold when compared to the wild-type strain, but no improvement in growth occurred when the rsaA mutant was complemented with the short form of the gene (RsaA S ) ( Figure 2B ), indicating that RsaA S is not enough to restore the wild-type phenotype. This phenotype was also observed when the same strains were grown in liquid media containing norfloxacin ( Figure 2C ), with the difference that growth of ΔrsaA was 3.12-fold slower than the wild-type strain after 20 h rather than 100-fold, which reflects the higher sensitivity of spot tests when compared to growth curves. However, in both solid and liquid cultures, complementation of ΔrsaA with the long form of RsaA (RsaA L ) restored the wild-type phenotype, demonstrating that the long form of the gene is essential for optimal growth of S. aureus in this antibiotic. Loss of RsaA sensitises S. aureus to ciprofloxacin Norfloxacin belongs to the fluoroquinone class of antibiotics that cause DNA damage by blocking DNA replication ( 32 ), which leads to cell death. To determine whether the phenotype was specific to norfloxacin or could also be observed in another antibiotic of the same functional class, the spot test experiment was repeated with ciprofloxacin. As observed in Figure 2D , the rsaA mutant was more sensitive to ciprofloxacin than the WT strain, and this phenotype was restored when complemented with the long form of the gene. Therefore, RsaA L , provided here in multicopy form through a plasmid, leads to a phenotype that affects fluoroquinlones in general. Combined, these data indicate that RsaA is involved in protecting S. aureus against the action of fluoroquinolone antibiotics, and that this function requires the long form of the gene to be present. Loss of RsaA reduces sensitivity of S. aureus to β-lactam antibiotics Having demonstrated that RsaA is required for optimal growth of S. aureus in the presence of fluoroquinolone antibiotics, we tested whether a similar effect could occur for other antibiotic classes. For this, the β-bactam and glycopeptide antibiotic classes, represented by cefazolin and vancomycin respectively, were selected for testing due to their clinical relevance. When the strains were grown in the presence of these two antibiotics, it was revealed that vancomycin had no effect on the growth of the rsaA mutant when compared to the wild-type strain (Figure S3). By contrast, in the presence of cefazolin, S. aureus growth was enhanced in the absence of RsaA by 10-fold in spot tests ( Figure 3A ) and by 1.2-fold in liquid culture after 20 h ( Figure 3B ), when compared to the wild-type strain. Furthermore, the wild-type phenotype was restored only when ΔrsaA was complemented by the long form of the gene. Download figure Open in new tab Figure 3. The rsaA mutant is less sensitive to β-lactam antibiotics when compared to the WT (A) Growth of WT, rsaA mutant, empty vector (pCN38) and mutants complemented with RsaA short form (p rsaA S ) or long form (p rsaA L ), serially diluted 10-fold and spotted onto BHI agar + 0.3 µg/ml cefazolin (n = 3). Spot tests in BHI broth alone are shown in Fig. S2. (B) Growth curves of WT and rsaA mutants grown in BHI broth containing 0.125 µg/ml cefazolin (n = 3). OD 600 measurements in BHI broth alone are shown in Fig. S2. Error bars represent the standard deviation of the mean. (C) Growth of WT, rsaA mutant, empty vector (pCN38) and mutants complemented with RsaA short form (p rsaA S ) or long form (p rsaA L ), serially diluted 10-fold and spotted onto BHI agar + 0.6 µg/ml cefalexin, 0.15 µg/ml cloxacillin or 0.03125 µg/ml penicillin G (n = 3). Spot tests in BHI broth alone are shown in Fig. S2. As a follow up, we repeated the spot tests on a selection of other β-bactam antibiotics: cefalexin, cloxacillin and penicillin G. Results demonstrated that growth of the rsaA mutant is increased in the presence of β-bactams when compared to the wild-type strain ( Figure 3C ). Taken together, these data demonstrate that the loss of RsaA reduces the sensitivity of S. aureus to β-bactam antibiotics. This is opposite to the phenotype found for fluoroquinolone antibiotics, in which the loss of RsaA made S. aureus more sensitive to these antibiotics instead. Nevertheless, in both cases, the long form of RsaA is required for this process. RsaA acts on MgrA to fully mediate its effect on norfloxacin and partly for cefazolin RsaA is known to repress translation of the master regulatory protein, MgrA ( 31 ). The latter regulates S. aureus virulence by promoting genes involved in capsule synthesis and inhibiting genes related to biofilm formation ( 31 ). Separately, MgrA has also been shown to act on several efflux pumps, including the fluoroquinolone efflux pumps NorA ( 34 , 35 ), NorB ( 35 – 37 ) and NorC ( 38 ), and a β-bactam efflux pump, AbcA ( 37 ). Since MgrA is reported to inhibit these Nor efflux pumps and promote AbcA ( 34 – 38 ), this would align with our observed phenotypes in the rsaA mutant, in which MgrA is no longer being repressed. To test this hypothesis, we performed spot tests on single and double mutants of rsaA and mgrA in the presence of norfloxacin or cefazolin. As seen in Figure 4A , loss of RsaA and MgrA led to opposite effects on S. aureus growth when compared to the wild-type strain, supporting previous reports of RsaA as a repressor of MgrA. While ΔrsaA was more sensitive to norfloxacin than wild-type S. aureus , the mgrA mutant showed reduced sensitivity, and the reverse was true of these two strains concerning cefazolin. When the double mutant was tested, its growth in norfloxacin was restored to wild-type levels, indicating that the increased sensitivity of ΔrsaA was most likely caused by derepression of MgrA. In the presence of cefazolin, growth of ΔrsaA ΔmgrA was very slightly increased compared to ΔmgrA but does not reach ΔrsaA levels, again indicating that the phenotype is likely due to the derepression of MgrA. However, in this case, growth also did not reach wild-type levels, suggesting a minor role for another RsaA-mediated mechanism. Download figure Open in new tab Figure 4. The increased sensitivity of the rsaA mutant to norfloxacin is mediated by MgrA Growth of WT, ΔrsaA , ΔmgrA and ΔrsaA ΔmgrA strains, serially diluted 10-fold and spotted onto BHI agar containing (A) 0.6 µg/ml norfloxacin or (B) 0.3 µg/ml cefazolin (n = 3). (B) Growth of WT, ΔmgrA and ΔmgrA complemented with empty vector (pCN38) or long form (p rsaA L ), serially diluted 10-fold and spotted onto BHI agar containing (C) 0.6 µg/ml norfloxacin or (D) 0.3 µg/ml cefazolin (n = 3). Spot tests in BHI alone are shown in Fig. S4. To further investigate the link between RsaA, the derepression of MgrA and its subsequent effect on S. aureus sensitivity to norfloxacin and cefazolin, we generated an mgrA mutant that overproduced RsaA L ( ΔmgrA pRsaA L ) and repeated the spot tests ( Figure 4B ). These results revealed that the overproduction of RsaA L has no additional effect on the growth of ΔmgrA in the presence of norfloxacin, demonstrating that RsaA acts solely through MgrA to mediate its effect on norfloxacin. By contrast, the overproduction of RsaA L led to a slightly increased sensitivity of ΔmgrA to cefazolin, indicating that while RsaA mostly acts through MgrA to mediate its effect on cefazolin, another mechanism may be involved. The full-length rsaA gene is required for expression of the short RsaA form Having confirmed that the long form of RsaA was essential for mediating the sensitivity of S. aureus to growth in norfloxacin and cefazolin, we wanted to determine why this long form was required. To investigate this, we first searched for possible open-reading frames in the RsaA L transcript that could translate to a small protein with a regulatory function. This identified one candidate that spanned most of the long-form region; however, mutation of the predicted ORF to block expression had no effect on antibiotic phenotype in spot tests (Supplementary Figure S5). Next, we examined whether additional interactions are present between mgrA mRNA and RsaA L , as compared to what is already known with mgrA mRNA and RsaA S ( 31 ). However, RNA- RNA interaction prediction software did not find any new interactions present in RsaA L that was not also detected with the short form only (Supplementary Figure S6), supporting that for RsaA to pair with mgrA mRNA, only its short form transcript is required. Finally, we examined the levels of expression between RsaA S and RsaA L . Results showed that there are far higher levels of the short form transcript in the wild-type strain when compared to the long form transcript ( Figure 5A ). This pattern is also reflected in the rsaA mutant when complemented with the long form of the gene ( Figure 5A ), corresponding with our previous observation that only RsaA L restores the wild-type phenotype. However, when the rsaA mutant was complemented with RsaA S , minimal expression of RsaA S was detected ( Figure 5A ), suggesting that while only the short form is required for sRNA function (Supplementary Figure S6), the short form cannot be expressed successfully on its own and expression of the long-form transcript is still needed. When we probed the same Northern blot membranes to detect only RsaA L and the long-form region, it was revealed that for the complemented rsaA mutant that overexpressed RsaA L , we could also see a clear band corresponding to the long-form region only ( Figure 5B ), suggesting a cleavage of the long-form region from the RsaA L transcript. Download figure Open in new tab Figure 5. The long RsaA transcript needs to be expressed for the sRNA to function. (A) Northern blot of WT and ΔrsaA complemented with empty vector (pCN38), RsaA short form (p rsaA S or long form (p rsaA L ), sampled at an OD 600 of 6 and probed for RsaA using a 5’-end probe to detect both short and long forms, with tmRNA probed as a loading control (n = 4). (B) Northern blot using the same membrane as for Fig. 5A , probed for RsaA L and RsaA L-region only using a RsaA L 3’-end probe, and tmRNA (loading control) (n = 4). (C) Growth of WT and ΔrsaA complemented with short form (p rsaA S ), long-region only (p rsaA Lreg ), or both short form and long region (p rsaA S + p rsaA Lreg ), serially diluted 10-fold and spotted onto BHI agar +/- 0.6 µg/ml norfloxacin or 0.3 µg/ml cefazolin (n = 3). To investigate the possibility of a cleavage of the long-form region further, the rsaA mutant was complemented with RsaA S and only the long-form region (RsaA L-region ) on separate plasmids. Spot tests revealed that although neither RsaA S nor RsaA L-region on their own can restore the wild-type phenotype, restoration of this phenotype occurs when both are present, even when they are not present on the same transcript ( Figure 5C ). This supports the hypothesis that the RsaA L transcript is cleaved and that the two cleavage products are required for RsaA to function as a sRNA. Combined, these data demonstrate that expression of RsaA L is required for the short form to be generated, likely through a cleavage event in which both cleavage products are required for RsaA function. Discussion Small regulatory RNAs play a crucial role in regulating key processes related to antibiotic resistance in Gram-negative species ( 16 , 25 ). These processes include cell envelope modifications ( 26 ) and drug efflux pumps ( 27 ). However, their impact on antibiotic resistance in Gram-positive species has received less attention ( 16 ). In S. aureus, the sRNA SprX, also known as RsaOR ( 21 ), influences vancomycin and teicoplanin glycopeptide resistance by repressing SpoVG ( 39 ). Notably, 6S RNA, which targets RNA polymerase, was shown to contribute to increased tolerance to low levels of rifampicin and related antibiotics in S. aureus , as well as in other distantly related bacteria ( 29 ). Antibiotics also influence sRNA gene expression, though connections to antibiotic resistance and/or tolerance are less clear. S. aureus sRNAs are differentially expressed after exposure to vancomycin, linezolid, ceftobiprole, and tigecycline ( 40 ), and exposure of S. aureus to sub-inhibitory concentrations of the β-lactam antibiotic oxacillin enhances bacterial virulence via the sRNA Ssr42 ( 41 ). The data presented here connects the RsaA sRNA to antibiotic resistance mechanisms regarding to two antibiotic classes: fluoroquinolones (norfloxacin and ciprofloxacin) and β-lactams (cefazolin, cefalexin, cloxacillin and penicillin G). Although fluoroquinolones act on DNA replication and β-lactams on the cell wall, mechanisms of resistance to both classes can involve the action of efflux pumps. Our results show that RsaA acts through the master regulator MgrA to mediate the sensitivity of S. aureus to antibiotics, linking RsaA with the efflux pumps NorA, NorB, NorC and AbcA. To our knowledge, RsaA is the first example of sRNA linked to fluoroquinolone resistance mechanisms in S. aureus . Of note, in E. coli , the sRNA SdsR represses the TolC efflux pump and increases sensitivity to fluoroquinolones ( 27 ). SdsR activity is dependent on the RNA chaperon Hfq ( 27 ), which is likely not the case for RsaA ( 42 ). We found that the effect of RsaA with fluoroquinolones was fully regulated via MgrA, but this regulation was only partial for β-lactams. Since sRNAs often control the expression of multiple mRNA targets to enable a coordinated regulation of cellular processes ( 13 ), it is likely that the RsaA-mediated effect on β-lactam sensitivity is mediated by other mRNA targets. A study investigating the RNA targetome of RsaA identified ssaA_2 and ssaA2_3 mRNAs as putative RsaA targets and found that these mRNAs form high-affinity complexes with RsaA in vitro ( 43 ). Since these two genes belong to the family of staphylococcal secretory antigen A (SsaA) proteins involved in peptidoglycan degradation ( 44 ), regulation of these substrates by RsaA may affect changes in sensitivity to β-lactam antibiotics, which function by disrupting peptidoglycan cross- linking during cell wall synthesis ( 45 ). Loss of RsaA was complemented only by the long form of the gene, which demonstrates that the short form of the gene is not sufficient for regulating sensitivity to our tested antibiotics. RsaA L was first detected by Northern blot and believed to originate from read-through at the RsaA S transcriptional terminator ( 30 ). Both RsaA L and RsaA S share the same 5’ end, as determined by RACE experiments ( 22 ). Our study reveals the importance of the long-form rsaA L gene being present for the sRNA to function. As no additional interaction sites with mgrA mRNA were predicted in RsaA L when compared to RsaA S , this correlates with studies on RsaA that have focussed only on its short form ( 30 , 31 , 43 ), which is believed to be the functional region. It must be noted that even if RsaA L was not mentioned in these studies, the rsaA mutant was complemented using a long stretch of the S. aureus genome comprising of both rsaA L and rsaA S genes and the surrounding genomic region ( 30 , 31 , 43 ). Our data demonstrate that the long form of the rsaA gene is needed for regulating staphylococcal sensitivity to norfloxacin and cefazolin, and that for RsaA to function, the long form transcript requires processing, most likely through cleavage into two halves. This finding aligns with measurements of the half-lives of RsaA short and long form transcripts ( 30 ), where RsaA S was found to be > 2-fold more stable than RsaA L after treatment with rifampicin to inhibit RNA polymerase ( 30 ). Based on experimental and homology data, there are believed to be 15 ribonucleases (RNases) in S. aureus that are involved in RNA processing and/or degradation ( 46 ). Of these, the main staphylococcal RNases are the exonucleases RNase J1/J2 and PNPase, and the endonucleases RNase III and RNase Y, the latter two of which have been reported to affect the stability and processing of RsaA L ( 30 , 47 ). In S. aureus, the only other sRNA that is believed to be processed from a longer transcript is RsaE, which enables it to interact more efficiently with a second target, thereby expanding the range of targets recognised by this sRNA ( 48 ). In the case of RsaA, since we were able to restore the wild-type phenotype in a rsaA mutant by expressing both RsaA S and the long region of RsaA L from different plasmids, the RsaA L long region is most likely interacting with the short form of RsaA to enable its function, possibly by stabilising the sRNA and/or by promoting interactions with mgrA mRNA. Small regulatory RNAs control many cellular processes and enable rapid adaptation to stress. Here, we show that staphylococcal RsaA acts via the master regulator MgrA to mediate sensitivity to two different antibiotic classes, fluoroquinolones and β-bactams. In addition, we revealed that this regulation requires the full-length version of the rsaA gene, the transcript of which is likely processed by the cell to produce two halves that interact to produce functional RsaA. Therefore, exposure to sub-inhibitory fluoroquinolone or β-lactam concentrations can alter the expression of their corresponding efflux pumps, leading to changes in antibiotic tolerance. Methods Bacterial strains, plasmids and growth conditions The bacterial strains used in this study are listed in Supplementary Table S1. Experiments were performed with HG003, a Staphylococcus aureus model strain widely used for regulation studies ( 49 ). Plasmids were engineered by Gibson assembly ( 50 ) in E. coli IM08B ( 51 ) as described (Supplementary Table S2), using the indicated appropriate primers (Supplementary Table S3) for PCR amplifications. Plasmids were verified by DNA sequencing and transformed into HG003 or its derivatives, as appropriate. HG003 mgrA and rsaA mgrA double mutants were constructed by transferring mgrA ::tetM ( 52 ) into HG003 and HG003 ΔrsaA ::tag075 using phage-mediated transduction. S. aureus was routinely cultured in Brain Heart Infusion (BHI) broth at 37 °C, with shaking (180 rpm). Escherichia coli was grown in Lysogeny Broth (LB) at 37 °C with shaking (180 rpm). Media were supplemented with antibiotics as required: ampicillin 100 μg/ml for E. coli ; chloramphenicol 5 μg/ml, tetracycline 1 μg/ml, and kanamycin 90 μg/ml for S. aureus . Fitness competition assay Mutants with altered fitness in the presence of norfloxacin were identified and analysed using a reported strategy ( 28 ) with three independent libraries containing 48 mutants (Supplementary Table S4). The libraries were grown at 37°C in BHI or BHI + 0.25 µg/ml norfloxacin for 3 days. Overnight cultures were diluted 1000 times into fresh medium for each day. Samples were withdrawn at an OD 600 of 1 and after overnight growth as indicated ( Figure 1A ). Spot tests To assess mutant phenotypes, bacterial growth in the presence of antibiotics was visualised by spot tests, which enable subtle growth phenotypes to be detected, such as with sRNA mutants. 5 µL of ten-fold serial dilutions of overnight cultures (from neat to 10 -7 dilution) were spotted on agar plates containing either BHI or BHI + antibiotic at the indicated concentrations. Agar plates were incubated for 24 h at 37 °C. Measurement of bacterial growth in liquid culture To measure growth of S. aureus in liquid culture, bacteria were first grown to stationary phase in BHI at 37 °C (180 rpm), then inoculated 1/50 into a flat-bottomed 96-well plate (200 µl total volume) and placed into a CLARIOstar Omega plate reader (BMG Labtech). Antibiotics were added to the media as needed, at the indicated concentrations. Bacteria were grown for 20 h at 37 °C (300 rpm), and absorbance at 600 nm was measured every 30 min. Northern blots Total RNA preparations and Northern blots were performed as previously described ( 53 ). 20 µg of total RNA samples were separated either by agarose (1.3%) gel electrophoresis. Membranes were probed with primers (Table S3) 32 P-labelled using Terminal Deoxynucleotidyl Transferase (ThermoScientific) and scanned using an Amersham Typhoon imager. Acknowledgements USA300 LAC mgrA ::tetM strain used to transduce into HG003 to generate our mgrA mutants was a gift from Alexander Horswill. The research was supported by the “Agence National pour la Recherche” [ANR-19-CE12-0006-01 (sRNA-RRARE)]. References 1. ↵ Westblade LF , Errington J , Dörr T . 2020 . Antibiotic tolerance . PLoS Pathog 16 : e1008892 . OpenUrl 2. ↵ Foster TJ . 2017 . Antibiotic resistance in Staphylococcus aureus . Current status and future prospects . FEMS Microbiol Rev 41 : 430 – 449 . OpenUrl CrossRef PubMed 3. ↵ Darby EM , Trampari E , Siasat P , Gaya MS , Alav I , Webber MA , Blair JMA . 2022 . Molecular mechanisms of antibiotic resistance revisited . Nature Reviews Microbiology 2022 21 : 5 21:280– 295. OpenUrl 4. ↵ Dörr T , Lewis K , Vulić M . 2009 . SOS Response Induces Persistence to Fluoroquinolones in Escherichia coli . PLoS Genet 5 : e1000760 . OpenUrl CrossRef PubMed 5. ↵ Clarke RS , Ha KP , Edwards AM . 2021 . RexAB promotes the survival of Staphylococcus aureus exposed to multiple classes of antibiotics . Antimicrob Agents Chemother 65 . 6. ↵ Shin J , Choe D , Ransegnola B , Hong H , Onyekwere I , Cross T , Shi Q , Cho B , Westblade LF , Brito IL , Dörr T . 2021 . A multifaceted cellular damage repair and prevention pathway promotes high-level tolerance to β-lactam antibiotics . EMBO Rep 22 . 7. ↵ Keren I , Kaldalu N , Spoering A , Wang Y , Lewis K . 2004 . Persister cells and tolerance to antimicrobials . FEMS Microbiol Lett 230 : 13 – 18 . OpenUrl CrossRef PubMed Web of Science 8. ↵ Windels EM , Michiels JE , Fauvart M , Wenseleers T , Van den Bergh B , Michiels J. 2019 . Bacterial persistence promotes the evolution of antibiotic resistance by increasing survival and mutation rates . ISME J 13 : 1239 – 1251 . OpenUrl CrossRef 9. Levin-Reisman I , Ronin I , Gefen O , Braniss I , Shoresh N , Balaban NQ . 2017 . Antibiotic tolerance facilitates the evolution of resistance . Science (1979) 355:826–830. 10. ↵ Liu J , Gefen O , Ronin I , Bar-Meir M , Balaban NQ . 2020 . Effect of tolerance on the evolution of antibiotic resistance under drug combinations . Science (1979) 367:200–204. 11. ↵ Ikuta KS , Swetschinski LR , Aguilar GR , Sharara F , Mestrovic T , Gray AP , Weaver ND , Wool EE , Han C , Hayoon AG , Aali A , Abate SM , Abbasi-Kangevari M , Abbasi-Kangevari Z , Abd-Elsalam S , Abebe G , Abedi A , Abhari AP , Abidi H , Aboagye RG , Absalan A , Ali HA , Acuna JM , Adane TD , Addo IY , Adegboye OA , Adnan M , Adnani QES , Afzal MS , Afzal S , Aghdam ZB , Ahinkorah BO , Ahmad A , Ahmad AR , Ahmad R , Ahmad S , Ahmad S , Ahmadi S , Ahmed A , Ahmed H , Ahmed JQ , Rashid TA , Ajami M , Aji B , Akbarzadeh-Khiavi M , Akunna CJ , Hamad H Al , Alahdab F , Al-Aly Z , Aldeyab MA , Aleman A V. , Alhalaiqa FAN , Alhassan RK , Ali BA , Ali L , Ali SS , Alimohamadi Y , Alipour V , Alizadeh A , Aljunid SM , Allel K , Almustanyir S , Ameyaw EK , Amit AML , Anandavelane N , Ancuceanu R , Andrei CL , Andrei T , Anggraini D , Ansar A , Anyasodor AE , Arabloo J , Aravkin AY , Areda D , Aripov T , Artamonov AA , Arulappan J , Aruleba RT , Asaduzzaman M , Ashraf T , Athari SS , Atlaw D , Attia S , Ausloos M , Awoke T , Quintanilla BPA , Ayana TM , Azadnajafabad S , Jafari AA , Darshan B , Badar M , Badiye AD , Baghcheghi N , Bagherieh S , Baig AA , Banerjee I , Barac A , Bardhan M , Barone-Adesi F , Barqawi HJ , Barrow A , Baskaran P , Basu S , Batiha AMM , Bedi N , Belete MA , Belgaumi UI , Bender RG , Bhandari B , Bhandari D , Bhardwaj P , Bhaskar S , Bhattacharyya K , Bhattarai S , Bitaraf S , Buonsenso D , Butt ZA , Dos Santos FLC , Cai J , Calina D , Camargos P , Cámera LA , Cárdenas R , Cevik M , Chadwick J , Charan J , Chaurasia A , Ching PR , Choudhari SG , Chowdhury EK , Chowdhury FR , Chu DT , Chukwu IS , Dadras O , Dagnaw FT , Dai X , Das S , Dastiridou A , Debela SA , Demisse FW , Demissie S , Dereje D , Derese M , Desai HD , Dessalegn FN , Dessalegni SAA , Desye B , Dhaduk K , Dhimal M , Dhingra S , Diao N , Diaz D , Djalalinia S , Dodangeh M , Dongarwar D , Dora BT , Dorostkar F , Dsouza HL , Dubljanin E , Dunachie SJ , Durojaiye OC , Edinur HA , Ejigu HB , Ekholuenetale M , Ekundayo TC , El-Abid H , Elhadi M , Elmonem MA , Emami A , Bain LE , Enyew DB , Erkhembayar R , Eshrati B , Etaee F , Fagbamigbe AF , Falahi S , Fallahzadeh A , Faraon EJA , Fatehizadeh A , Fekadu G , Fernandes JC , Ferrari A , Fetensa G , Filip I , Fischer F , Foroutan M , Gaal PA , Gadanya MA , Gaidhane AM , Ganesan B , Gebrehiwot M , Ghanbari R , Nour MG , Ghashghaee A , Gholamrezanezhad A , Gholizadeh A , Golechha M , Goleij P , Golinelli D , Goodridge A , Gunawardane DA , Guo Y , Gupta R Das , Gupta S , Gupta VB , Gupta VK , Guta A , Habibzadeh P , Avval AH , Halwani R , Hanif A , Hannan MA , Harapan H , Hassan S , Hassankhani H , Hayat K , Heibati B , Heidari G , Heidari M , Heidari-Soureshjani R , Herteliu C , Heyi DZ , Hezam K , Hoogar P , Horita N , Hossain MM , Hosseinzadeh M , Hostiuc M , Hostiuc S , Hoveidamanesh S , Huang J , Hussain S , Hussein NR , Ibitoye SE , Ilesanmi OS , Ilic IM , Ilic MD , Imam MT , Immurana M , Inbaraj LR , Iradukunda A , Ismail NE , Iwu CCD , Iwu CJ , Linda MJ , Jakovljevic M , Jamshidi E , Javaheri T , Javanmardi F , Javidnia J , Jayapal SK , Jayarajah U , Jebai R , Jha RP , Joo T , Joseph N , Joukar F , Jozwiak JJ , Kacimi SEO , Kadashetti V , Kalankesh LR , Kalhor R , Kamal VK , Kandel H , Kapoor N , Karkhah S , Kassa BG , Kassebaum NJ , Katoto PDMC , Keykhaei M , Khajuria H , Khan A , Khan IA , Khan M , Khan MN , Khan MAB , Khatatbeh MM , Khater MM , Kashani HRK , Khubchandani J , Kim H , Kim MS , Kimokoti RW , Kissoon N , Kochhar S , Kompani F , Kosen S , Koul PA , Laxminarayana SLK , Lopez FK , Krishan K , Krishnamoorthy V , Kulkarni V , Kumar N , Kurmi OP , Kuttikkattu A , Kyu HH , Lal DK , Lám J , Landires I , Lasrado S , Lee SW , Lenzi J , Lewycka S , Li S , Lim SS , Liu W , Lodha R , Loftus MJ , Lohiya A , Lorenzovici L , Lotfi M , Mahmoodpoor A , Mahmoud MA , Mahmoudi R , Majeed A , Majidpoor J , Makki A , Mamo GA , Manla Y , Martorell M , Matei CN , Mcmanigal B , Nasab EM , Mehrotra R , Melese A , Mendoza-Cano O , Menezes RG , Mentis AFA , Micha G , Michalek IM , Des ACMGN , Kostova NM , Mir SA , Mirghafourvand M , Mirmoeeni S , Mirrakhimov EM , Mirza-Aghazadeh-attari M , Misganaw AS , Misganaw A , Misra S , Mohammadi E , Mohammadi M , Mohammadian-Hafshejani A , Mohammed S , Mohan S , Mohseni M , Mokdad AH , Momtazmanesh S , Monasta L , Moore CE , Moradi M , Sarabi MM , Morrison SD , Motaghinejad M , Isfahani HM , Khaneghah AM , Mousavi-Aghdas SA , Mubarik S , Mulita F , Mulu GBB , Munro SB , Muthupandian S , Nair TS , Naqvi AA , Narang H , Natto ZS , Naveed M , Nayak BP , Naz S , Negoi I , Nejadghaderi SA , Kandel SN , Ngwa CH , Niazi RK , Des ATN , Noroozi N , Nouraei H , Nowroozi A , Nuñez-Samudio V , Nutor JJ , Nzoputam CI , Nzoputam OJ , Oancea B , Obaidur RM , Ojha VA , Okekunle AP , Okonji OC , Olagunju AT , Olusanya BO , Bali AO , Omer E , Otstavnov N , Oumer B , Padubidri JR , Pakshir K , Palicz T , Pana A , Pardhan S , Paredes JL , Parekh U , Park EC , Park S , Pathak A , Paudel R , Paudel U , Pawar S , Toroudi HP , Peng M , Pensato U , Pepito VCF , Pereira M , Peres MFP , Perico N , Petcu IR , Piracha ZZ , Podder I , Pokhrel N , Poluru R , Postma MJ , Pourtaheri N , Prashant A , Qattea I , Rabiee M , Rabiee N , Radfar A , Raeghi S , Rafiei S , Raghav PR , Rahbarnia L , Rahimi-Movaghar V , Rahman M , Rahman MA , Rahmani AM , Rahmanian V , Ram P , Ranjha MMAN , Rao SJ , Rashidi MM , Rasul A , Ratan ZA , Rawaf S , Rawassizadeh R , Razeghinia MS , Redwan EMM , Regasa MT , Remuzzi G , Reta MA , Rezaei N , Rezapour A , Riad A , Ripon RK , Rudd KE , Saddik B , Sadeghian S , Saeed U , Safaei M , Safary A , Safi SZ , Sahebazzamani M , Sahebkar A , Sahoo H , Salahi S , Salahi S , Salari H , Salehi S , Kafil HS , Samy AM , Sanadgol N , Sankararaman S , Sanmarchi F , Sathian B , Sawhney M , Saya GK , Senthilkumaran S , Seylani A , Shah PA , Shaikh MA , Shaker E , Shakhmardanov MZ , Sharew MM , Sharifi-Razavi A , Sharma P , Sheikhi RA , Sheikhy A , Shetty PH , Shigematsu M , Shin J Il , Shirzad-Aski H , Shivakumar KM , Shobeiri P , Shorofi SA , Shrestha S , Sibhat MM , Sidemo NB , Sikder MK , Silva LMLR , Singh JA , Singh P , Singh S , Siraj MS , Siwal SS , Skryabin VY , Skryabina AA , Socea B , Solomon DD , Song Y , Sreeramareddy CT , Suleman M , Abdulkader RS , Sultana S , Szócska M , Tabatabaeizadeh SA , Tabish M , Taheri M , Taki E , Tan KK , Tandukar S , Tat NY , Tat VY , Tefera BN , Tefera YM , Temesgen G , Temsah MH , Tharwat S , Thiyagarajan A , Tleyjeh II , Troeger CE , Umapathi KK , Upadhyay E , Tahbaz SV , Valdez PR , Van Den Eynde J , Van Doorn HR , Vaziri S , Verras GI , Viswanathan H , Vo B , Waris A , Wassie GT , Wickramasinghe ND , Yaghoubi S , Yahya GATY , Jabbari SHY , Yigit A , Yigit V , Yon DK , Yonemoto N , Zahir M , Zaman BA , Zaman S Bin , Zangiabadian M , Zare I , Zastrozhin MS , Zhang ZJ , Zheng P , Zhong C , Zoladl M , Zumla A , Hay SI , Dolecek C , Sartorius B , Murray CJL , Naghavi M. 2022 . Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019 . The Lancet 400:2221–2248. 12. ↵ Murray CJ , Ikuta KS , Sharara F , Swetschinski L , Robles Aguilar G , Gray A , Han C , Bisignano C , Rao P , Wool E , Johnson SC , Browne AJ , Chipeta MG , Fell F , Hackett S , Haines-Woodhouse G , Kashef Hamadani BH , Kumaran EAP , McManigal B , Agarwal R , Akech S , Albertson S , Amuasi J , Andrews J , Aravkin A , Ashley E , Bailey F , Baker S , Basnyat B , Bekker A , Bender R , Bethou A , Bielicki J , Boonkasidecha S , Bukosia J , Carvalheiro C , Castañeda-Orjuela C , Chansamouth V , Chaurasia S , Chiurchiù S , Chowdhury F , Cook AJ , Cooper B , Cressey TR , Criollo-Mora E , Cunningham M , Darboe S , Day NPJ , De Luca M , Dokova K , Dramowski A , Dunachie SJ , Eckmanns T , Eibach D , Emami A , Feasey N , Fisher-Pearson N , Forrest K , Garrett D , Gastmeier P , Giref AZ , Greer RC , Gupta V , Haller S , Haselbeck A , Hay SI , Holm M , Hopkins S , Iregbu KC , Jacobs J , Jarovsky D , Javanmardi F , Khorana M , Kissoon N , Kobeissi E , Kostyanev T , Krapp F , Krumkamp R , Kumar A , Kyu HH , Lim C , Limmathurotsakul D , Loftus MJ , Lunn M , Ma J , Mturi N , Munera-Huertas T , Musicha P , Mussi-Pinhata MM , Nakamura T , Nanavati R , Nangia S , Newton P , Ngoun C , Novotney A , Nwakanma D , Obiero CW , Olivas-Martinez A , Olliaro P , Ooko E , Ortiz-Brizuela E , Peleg AY , Perrone C , Plakkal N , Ponce-de-Leon A , Raad M , Ramdin T , Riddell A , Roberts T , Robotham JV , Roca A , Rudd KE , Russell N , Schnall J , Scott JAG , Shivamallappa M , Sifuentes-Osornio J , Steenkeste N , Stewardson AJ , Stoeva T , Tasak N , Thaiprakong A , Thwaites G , Turner C , Turner P , van Doorn HR , Velaphi S , Vongpradith A , Vu H , Walsh T , Waner S , Wangrangsimakul T , Wozniak T , Zheng P , Sartorius B , Lopez AD , Stergachis A , Moore C , Dolecek C , Naghavi M. 2022 . Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis . The Lancet 399:629–655. 13. ↵ Waters LS , Storz G . 2009 . Regulatory RNAs in Bacteria . Cell 136 : 615 – 628 . OpenUrl CrossRef PubMed Web of Science 14. ↵ Chakravarty S , Massé E . 2019 . RNA-Dependent Regulation of Virulence in Pathogenic Bacteria . Front Cell Infect Microbiol 9 : 476676 . OpenUrl 15. ↵ Rochat T , Bohn C , Bohn C , Morvan C , Le Lam TN , Razvi F , Pain A , Toffano-Nioche C , Toffano-Nioche C , Ponien P , Jacq A , Annick Jacq , Jacquet E , Fey PD , Gautheret D , Philippe Bouloc , Bouloc P. 2018 . The conserved regulatory RNA RsaE down-regulates the arginine degradation pathway in Staphylococcus aureus . Nucleic Acids Res 46 : 8803 – 8816 . OpenUrl CrossRef 16. ↵ Dersch P , Khan MA , Mühlen S , Görke B . 2017 . Roles of Regulatory RNAs for Antibiotic Resistance in Bacteria and Their Potential Value as Novel Drug Targets . Front Microbiol 8 : 803 . OpenUrl CrossRef 17. ↵ Mediati DG , Wu S , Wu W , Tree JJ . 2021 . Networks of Resistance: Small RNA Control of Antibiotic Resistance . Trends in Genetics 37 : 35 – 45 . OpenUrl 18. ↵ Liu W , Rochat T , Toffano-Nioche C , Le Lam TN , Bouloc P , Morvan C. 2018 . Assessment of Bona Fide sRNAs in Staphylococcus aureus . Front Microbiol 9 : 228 . OpenUrl CrossRef 19. ↵ Menard G , Silard C , Suriray M , Rouillon A , Augagneur Y . 2022 . Thirty Years of sRNA-Mediated Regulation in Staphylococcus aureus : From Initial Discoveries to In Vivo Biological Implications . International Journal of Molecular Sciences 2022 , Vol 23, Page 7346 23:7346. 20. ↵ Bronesky D , Wu Z , Marzi S , Walter P , Geissmann T , Moreau K , Vandenesch F , Caldelari I , Romby P . 2016 . Staphylococcus aureus RNAIII and Its Regulon Link Quorum Sensing, Stress Responses, Metabolic Adaptation, and Regulation of Virulence Gene Expression . Annu Rev Microbiol 70 : 299 – 316 . OpenUrl CrossRef 21. ↵ Bohn C , Rigoulay C , Chabelskaya S , Sharma CM , Marchais A , Skorski P , Borezée-Durant E , Barbet R , Jacquet E , Jacq A , Gautheret D , Felden B , Vogel J , Bouloc P . 2010 . Experimental discovery of small RNAs in Staphylococcus aureus reveals a riboregulator of central metabolism . Nucleic Acids Res 38 : 6620 – 6636 . OpenUrl CrossRef PubMed Web of Science 22. ↵ Geissmann T , Chevalier C , Cros M-J , Boisset S , Fechter P , Noirot C , Schrenzel J , Francois P , Vandenesch F , Gaspin C , Romby P . 2009 . A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation . Nucleic Acids Res 37 : 7239 – 7257 . OpenUrl CrossRef PubMed Web of Science 23. ↵ Coronel-Tellez RH , Pospiech M , Barrault M , Liu W , Bordeau V , Vasnier C , Felden B , Sargueil B , Bouloc P . 2022 . sRNA-controlled iron sparing response in Staphylococci . Nucleic Acids Res 50 : 8529 – 8546 . OpenUrl 24. ↵ Barrault M , Chabelskaya S , Coronel-Tellez RH , Toffano-Nioche C , Jacquet E , Bouloc P . 2024 . Staphylococcal aconitase expression during iron deficiency is controlled by an sRNA-driven feedforward loop and moonlighting activity . Nucleic Acids Res 1 : 13 – 14 . OpenUrl 25. ↵ Kim T , Bak G , Lee J , Kim K sun . 2015 . Systematic analysis of the role of bacterial Hfq-interacting sRNAs in the response to antibiotics . Journal of Antimicrobial Chemotherapy 70 : 1659 – 1668 . OpenUrl CrossRef PubMed 26. ↵ Acuña LG , Barros MJ , Peñaloza D , Rodas PI , Paredes-Sabja D , Fuentes JA , Gil F , Calderón IL . 2016 . A feed-forward loop between SroC and MgrR small RNAs modulates the expression of eptB and the susceptibility to polymyxin B in Salmonella Typhimurium . Microbiology (United Kingdom ) 162 : 1996 – 2004 . OpenUrl CrossRef 27. ↵ Parker A , Gottesman S . 2016 . Small RNA regulation of TolC, the outer membrane component of bacterial multidrug transporters . J Bacteriol 198 : 1101 – 1113 . OpenUrl Abstract / FREE Full Text 28. ↵ Le Lam TN , Morvan C , Wenfeng Liu , Wenfeng Liu , Liu W , Liu W , Bohn C , Bohn C , Jaszczyszyn Y , Jaszczyszyn Y , Philippe Bouloc , Bouloc P. 2017 . Finding sRNA-associated phenotypes by competition assays: An example with Staphylococcus aureus . Methods 117 : 21 – 27 . OpenUrl 29. ↵ Esberard M , Hallier M , Liu W , Morvan C , Bossi L , Figueroa-Bossi N , Felden B , Bouloc P . 2022 . 6S RNA-Dependent Susceptibility to RNA Polymerase Inhibitors . Antimicrob Agents Chemother 66 . 30. ↵ Efthimia Lioliou , Lioliou E , Sharma CM , Caldelari I , Helfer A-C , Fechter P , Vandenesch F , Vogel J , Romby P , Romby P . 2012 . Global regulatory functions of the Staphylococcus aureus endoribonuclease III in gene expression . PLoS Genet 8 . 31. ↵ Romilly C , Lays C , Tomasini A , Caldelari I , Benito Y , Hammann P , Geissmann T , Boisset S , Romby P , Vandenesch F . 2014 . A Non-Coding RNA Promotes Bacterial Persistence and Decreases Virulence by Regulating a Regulator in Staphylococcus aureus . PLoS Pathog 10 : e1003979 . OpenUrl CrossRef PubMed 32. ↵ Drlica K . 1999 . Mechanism of fluoroquinolone action . Curr Opin Microbiol 2 : 504 – 508 . OpenUrl CrossRef PubMed Web of Science 33. ↵ Benson DA , Cavanaugh M , Clark K , Karsch-Mizrachi I , Lipman DJ , Ostell J , Sayers EW . 2013 . GenBank . Nucleic Acids Res 41 : D36 – D42 . OpenUrl CrossRef PubMed Web of Science 34. ↵ Truong-Bolduc QC , Ding Y , Hooper DC . 2008 . Posttranslational Modification Influences the Effects of MgrA on norA Expression in Staphylococcus aureus . J Bacteriol 190 : 7375 – 7381 . OpenUrl Abstract / FREE Full Text 35. ↵ Truong-Bolduc QC , Hooper DC . 2010 . Phosphorylation of MgrA and Its Effect on Expression of the NorA and NorB Efflux Pumps of Staphylococcus aureus . J Bacteriol 192 : 2525 – 2534 . OpenUrl Abstract / FREE Full Text 36. Truong-Bolduc QC , Dunman PM , Strahilevitz J , Projan SJ , Hooper DC . 2005 . MgrA Is a Multiple Regulator of Two New Efflux Pumps in Staphylococcus aureus . J Bacteriol 187 : 2395 – 2405 . OpenUrl Abstract / FREE Full Text 37. ↵ Truong-Bolduc QC , Hooper DC . 2007 . The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and β-Lactams in Staphylococcus aureus . J Bacteriol 189 : 2996 – 3005 . OpenUrl Abstract / FREE Full Text 38. ↵ Truong-Bolduc QC , Strahilevitz J , Hooper DC . 2006 . NorC, a new efflux pump regulated by MgrA of Staphylococcus aureus . Antimicrob Agents Chemother 50 : 1104 – 1107 . OpenUrl Abstract / FREE Full Text 39. ↵ Eyraud A , Tattevin P , Pierre Tattevin , Chabelskaya S , Felden B . 2014 . A small RNA controls a protein regulator involved in antibiotic resistance in Staphylococcus aureus . Nucleic Acids Res 42 : 4892 – 4905 . OpenUrl CrossRef PubMed 40. ↵ Howden BP , Beaume M , Harrison PF , Hernandez D , Schrenzel J , Seemann T , Francois P , Stinear TP . 2013 . Analysis of the Small RNA Transcriptional Response in Multidrug-Resistant Staphylococcus aureus after Antimicrobial Exposure . Antimicrob Agents Chemother 57 : 3864 – 3874 . OpenUrl Abstract / FREE Full Text 41. ↵ Horn J , Klepsch M , Manger M , Wolz C , Rudel T , Fraunholz M . 2018 . Long Noncoding RNA SSR42 Controls Staphylococcus aureus Alpha-Toxin Transcription in Response to Environmental Stimuli . J Bacteriol 200 . 42. ↵ Bohn C , Rigoulay C , Bouloc P . 2007 . No detectable effect of RNA-binding protein Hfq absence in Staphylococcus aureus . BMC Microbiol 7 : 1 – 9 . OpenUrl CrossRef PubMed 43. ↵ Tomasini A , Moreau K , Chicher J , Johana Chicher , Geissmann T , Vandenesch F , Romby P , Romby P , Marzi S , Caldelari I . 2017 . The RNA targetome of Staphylococcus aureus non-coding RNA RsaA: impact on cell surface properties and defense mechanisms . Nucleic Acids Res 45 : 6746 – 6760 . OpenUrl CrossRef 44. ↵ Dubrac S , Boneca IG , Poupel O , Msadek T . 2007 . New insights into the WalK/WalR (YycG/YycF) essential signal transduction pathway reveal a major role in controlling cell wall metabolism and biofilm formation in Staphylococcus aureus . J Bacteriol 189 : 8257 – 8269 . OpenUrl Abstract / FREE Full Text 45. ↵ Bush K , Bradford PA . 2016 . β-Lactams and β-Lactamase Inhibitors: An Overview . Cold Spring Harb Perspect Med 6 : a025247 . OpenUrl Abstract / FREE Full Text 46. ↵ Bonnin RA , Bouloc P . 2015 . RNA degradation in Staphylococcus aureus : Diversity of ribonucleases and their impact . Int J Genomics 2015 . 47. ↵ Marincola G , Schäfer T , Behler J , Bernhardt J , Ohlsen K , Goerke C , Wolz C . 2012 . RNase Y of Staphylococcus aureus and its role in the activation of virulence genes . Mol Microbiol 85 : 817 – 832 . OpenUrl CrossRef PubMed 48. ↵ Durand S , Braun F , Lioliou E , Romilly C , Helfer AC , Kuhn L , Quittot N , Nicolas P , Romby P , Condon C . 2015 . A Nitric Oxide Regulated Small RNA Controls Expression of Genes Involved in Redox Homeostasis in Bacillus subtilis . PLoS Genet 11 : e1004957 . OpenUrl CrossRef PubMed 49. ↵ Herbert S , Ziebandt AK , Ohlsen K , Schäfer T , Hecker M , Albrecht D , Novick R , Götz F . 2010 . Repair of global regulators in Staphylococcus aureus 8325 and comparative analysis with other clinical isolates . Infect Immun 78 : 2877 – 2889 . OpenUrl Abstract / FREE Full Text 50. ↵ Gibson DG , Young L , Chuang RY , Venter JC , Hutchison CA , Smith HO . 2009 . Enzymatic assembly of DNA molecules up to several hundred kilobases . Nature Methods 2009 6 : 5 6:343–345. OpenUrl 51. ↵ Monk IR , Tree JJ , Howden BP , Stinear TP , Foster TJ . 2015 . Complete bypass of restriction systems for major Staphylococcus aureus lineages . mBio 6 : 1 – 12 . OpenUrl CrossRef 52. ↵ Crosby HA , Schlievert PM , Merriman JA , King JM , Salgado-Pabón W , Horswill AR . 2016 . The Staphylococcus aureus Global Regulator MgrA Modulates Clumping and Virulence by Controlling Surface Protein Expression . PLoS Pathog 12 : e1005604 . OpenUrl CrossRef 53. ↵ Marchais A , Naville M , Bohn C , Bouloc P , Gautheret D . 2009 . Single-pass classification of all noncoding sequences in a bacterial genome using phylogenetic profiles . Genome Res 19 : 1084 – 1092 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted August 06, 2024. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following A small regulatory RNA controls antibiotic tolerance in Staphylococcus aureus by modulating efflux pump expression Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share A small regulatory RNA controls antibiotic tolerance in Staphylococcus aureus by modulating efflux pump expression Kam Pou Ha , Philippe Bouloc bioRxiv 2024.08.05.606701; doi: https://doi.org/10.1101/2024.08.05.606701 Share This Article: Copy Citation Tools A small regulatory RNA controls antibiotic tolerance in Staphylococcus aureus by modulating efflux pump expression Kam Pou Ha , Philippe Bouloc bioRxiv 2024.08.05.606701; doi: https://doi.org/10.1101/2024.08.05.606701 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7644) Biochemistry (17728) Bioengineering (13917) Bioinformatics (42038) Biophysics (21489) Cancer Biology (18637) Cell Biology (25553) Clinical Trials (138) Developmental Biology (13401) Ecology (19941) Epidemiology (2067) Evolutionary Biology (24367) Genetics (15622) Genomics (22547) Immunology (17764) Microbiology (40475) Molecular Biology (17208) Neuroscience (88749) Paleontology (667) Pathology (2842) Pharmacology and Toxicology (4834) Physiology (7659) Plant Biology (15175) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9835) Zoology (2272)