Phosphate sensing by PhoPR regulates the cytotoxicity of Staphylococcus aureus

Phosphate sensing by PhoPR regulates the cytotoxicity of Staphylococcus aureus | 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 Phosphate sensing by PhoPR regulates the cytotoxicity of Staphylococcus aureus Nathanael Palk , Tarcisio Brignoli , Marcia Boura , Ruth C. Massey doi: https://doi.org/10.1101/2025.06.24.661297 Nathanael Palk 1 School of Cellular and Molecular Medicine, University of Bristol , Bristol, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tarcisio Brignoli 1 School of Cellular and Molecular Medicine, University of Bristol , Bristol, UK 2 Department of Biosciences, Università degli Studi di Milano , Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marcia Boura 1 School of Cellular and Molecular Medicine, University of Bristol , Bristol, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ruth C. Massey 1 School of Cellular and Molecular Medicine, University of Bristol , Bristol, UK 3 School of Microbiology, UCC , Cork, Ireland 4 APC Microbiome Ireland, UCC , Cork, Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: r.massey{at}ucc.ie Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Staphylococcus aureus has evolved a complex regulatory network to coordinate expression of virulence factors, including cytolytic toxins, with host environmental signals. Central to this network are two-component systems, in which a histidine kinase senses an external signal and activates a response regulator via phosphorylation, leading to changes in gene expression. Using a comprehensive screen of transposon mutants in each of the non-essential histidine kinase and response regulatory genes in S. aureus , we demonstrate that 11 of these 16 systems regulate cytotoxicity. Further characterisation of a phoP mutant revealed that its impact on cytotoxicity is mediated through the Agr quorum-sensing system. Notably, we found that unphosphorylated PhoP is an activator of Agr activity, while phosphorylated PhoP also acts as a weak activator of Agr activity in high phosphate environments but as a repressor in low phosphate environments. Overall, we have demonstrated that phosphate sensing through PhoPR is a novel regulator of cytotoxicity in S. aureus . Moreover, our study challenges the canonical model of TCSs as simple on/off systems and highlights the importance of unphosphorylated response regulators in gene regulation. Importance The production of cytolytic toxins is the major means by which bacterial pathogens damage host tissue and cause disease. Understanding the activity and regulation of these toxins is critical for the identification of means to block them and prevent the development of disease. In this study we focused on a specific regulatory mechanism, the two-component systems (TCSs), that enable bacteria to sense their environment and adapt accordingly. In the traditional model of a TCS, a response regulator (RR) is phosphorylated by a histidine kinase (HK), which enables it to activate or repress expression of target genes, which may include toxins or regulators of toxins. We found that 11 of S. aureus’ 16 TCSs affect toxin production, highlighting that S. aureus integrates a broad range of environmental cues to regulate toxicity. We focused on one of these TCSs, the PhoPR system and found that sensing of inorganic phosphate is a novel regulator of cytotoxicity in S. aureus . Furthermore, we found that the RR of this system acts as a strong activator of toxicity in its unphosphorylated form, challenging the traditional model of a TCS as only active upon signal activation. Introduction Staphylococcus aureus is a Gram-positive bacterium and opportunistic human pathogen which was associated with more than one million deaths globally in 2019 ( 1 ). It is a leading cause of a wide range of infections, including skin and soft tissue infections, bacteraemia, endocarditis, pneumonia and osteomyelitis ( 2 ). The ability of S. aureus to cause a diverse spectrum of diseases is generally attributed to an impressive arsenal of virulence factors ( 3 - 6 ). These include several cytolytic toxins, which lyse host cells by forming pores or channels in their membranes, referred to from herein as cytotoxicity. Alpha-haemolysin (Hla), the bicomponent leukotoxins (LukSF-PV, LukAB, and LukED), and gamma haemolysin (HlgAB and HlgCB) use a receptor-dependent mechanism to cause lysis. In contrast, phenol-soluble modulins (PSMs) have detergent-like properties ( 7 - 9 ). These have been linked to several aspects of S. aureus pathogenesis, including immune evasion, nutrient acquisition and inflammation ( 8 , 10 , 11 ). Importantly, cytolytic toxins are expressed in coordination with host environmental signals through a complex regulatory network and understanding this is key to linking their pathogenic role to specific disease states ( 12 - 14 ). In bacteria, two component systems (TCSs) are a widespread mechanism used to sense and respond to environmental signals ( 15 ). In response to a specific signal, a membrane-bound histidine kinase (HK) will undergo ATP-dependent autophosphorylation. Subsequently, the phosphoryl group will be transferred to a cytoplasmic DNA-binding response regulator (RR) ( 16 ). Phosphorylation of the RR induces a conformation change and modulates its affinity for gene promoters, where they can act as transcriptional activators or repressors ( 17 ). Some TCSs also contain auxiliary proteins which can be involved in sensing or signal transduction ( 18 , 19 ). The genome of S. aureus contains 16 TCSs, with several of these linked to regulating expression of cytolytic toxins. For example, the accessory gene regulator (Agr) TCS uses quorum sensing to upregulate most S. aureus toxins, including Hla, leukotoxins, delta-hemolysin (Hld) and PSMs ( 20 - 22 ). The staphylococcal accessory element (Sae) TCS responds to phagocytosis-related signals to upregulate expression of Hla, leukotoxins and HlgAB and HlgCB ( 23 - 26 ). Similarly, the autolysis-related locus (Arl) TCS acts as a metabolic sensor and induces expression of leukotoxins in response to nutritional immunity ( 13 , 27 - 29 ). The remaining TCSs have been linked to cell wall biosynthesis and antibiotic resistance (VraRS, GraRS, NsaRS), autolysis (WalRK, LytRS), respiration (SrrBA, AirRS, and NreCB), and nutrient sensing (HssRS, KdpDE, and PhoPR) ( 17 ). Additionally, there is a TCS with a currently unknown function (DesRK). Current knowledge of the TCSs which regulate cytotoxicity in S. aureus is mainly derived from transcriptional comparisons between wild-type strains and mutant pairs in histidine kinase (HK) and/or response regulator (RR) genes. However, we are currently lacking a comprehensive study which evaluates and compares the contribution of all the TCSs in S. aureus to a single phenotype such as cytotoxicity. To address this, we screened the cytotoxicity of transposon mutants in each non-essential HK and RR gene in the S. aureus NTML reference CA-MRSA strain JE2. Using this approach, we identified several TCS mutants with reduced cytotoxicity, including a mutant of the phoP gene, the RR from the PhoPR TCS. The phoP mutant has a lower abundance of PSMs in the bacterial supernatant due to decreased Agr activity. Interestingly, we found that PhoP regulation of Agr activity is differential depending on the concentration of inorganic phosphate (Pi) in growth media. Furthermore, we show that the phosphorylation state of PhoP is a key factor in mediating this differential regulation. Methods Bacterial Strains and Growth Conditions A list of the S. aureus strains, plasmids and primers used in this study are available in Tables 1 , 2 and 3 . S. aureus strains were grown at 37°C on tryptic soy agar (TSA) or in tryptic soy broth (TSB) with shaking at 180rpm. Mutant strains of all HK and RR genes in the genome of USA300 JE2 were obtained from the Nebraska Transposon Mutant Library (NTML) ( 30 ) and grown in media supplemented with erythromycin (5 μg/ml). S. aureus strains carrying the pRMC2 plasmid were selected with chloramphenicol (10 µg/ml) and expression of the inserted gene induced with anhydrotetracycline (aTC) (200 ng/ml). View this table: View inline View popup Table 1. Strains used in this study. View this table: View inline View popup Download powerpoint Table 2. Plasmids used in this study View this table: View inline View popup Table 3. Primers used in this study. THP-1 Cytotoxicity Assay THP-1 cells were exposed to the supernatant from S. aureus overnight cultures as a measure of cytotoxicity as they are sensitive to lysis by 13 of the 15 toxins present in the supernatant ( 34 ). THP-1 cells were sub-cultured every 2-3 days in RPMI 1640 supplemented with heat-inactivated fetal bovine serum (FBS) (10%), L-glutamine (1 μM), penicillin (200 U/ml), and streptomycin (0.1 mg/ml) at 37°C with 5% CO2. For the toxicity assay, cells were harvested by centrifugation (1250rpm, 20°C, 5 minutes) and resuspended in Hank’s Balanced Salt Solution (HBSS) (Biowhittaker) to a final density of 1 × 10 6 to 1.5 × 10 6 cells/ml. Bacterial supernatants were extracted after 18 hours of growth by centrifugation (4000rpm, 4°C, 5 minutes) and incubated with harvested THP-1 cells for 12 minutes at 37°C. Cell death was quantified via trypan blue exclusion with percentage (%) THP-1 killing defined as the number of dead THP-1 cells out of the total number of THP-1 cells counted for each sample. Each assay was performed with three technical replicates and three biological replicates. For comparisons across multiple assays, this percentage was taken relative to the THP-1 killing of the wild-type strain (JE2) in each assay to account for any variability in the sensitivity of the THP-1 cells. Incubation of THP-1 cells with TSB was used as a negative control to ensure appropriate viability of the THP-1 cells for each assay. Complementation of the phoP::tn mutant To verify the phoP::tn mutation was responsible for the reduced cytotoxicity phenotype, the wild-type phoP gene was reintroduced into the transposon mutant via the pRMC2 vector. This vector was selected as it controls of gene expression through an aTC-inducible promoter. A wild-type copy of the phoP gene was PCR amplified from JE2 genomic DNA with KpnI and SacI restriction sites using phoP F and phoP R primers ( Table 3 ). The PCR product was checked for a product of the correct size by agarose gel electrophoresis and purified using the QIAquick® PCR purification kit (Qiagen). Purified phoP and pRMC2 vector were then digested with KpnI and SacI (NEB) restriction enzymes and the products purified again. Insert and plasmid DNA were ligated in a 3:1 molar ratio using T4 DNA ligase (NEB) to generate p phoP . This was transformed into E. coli Mach 1 competent cells via heat shock and successful transformants confirmed by colony PCR. Plasmids were isolated from Mach 1, passaged through S. aureus RN4220 and then transformed into phoP::tn via electroporation. For electroporation, competent cells were generated by culturing strains to an OD 600nm of 0.5 and washing three times in 0.5M sucrose. For transformations, competent cells were incubated in ice with 100ng of plasmid DNA for 30 mins. Cells were transferred into 0.2mm electroporation curvettes (Bio-Rad) for electroporation (25 μF, 2.5 KV and 100 Ω; pulse time of 2.5 msec). Following electroporation, 700μL of TSB was added and cells were incubated at 37°C for 1 hour. Successful transformants were selected for by plating onto TSA with 10μg/ml chloramphenicol. To test for complementation of cytotoxicity, strains carrying the pRMC2 vector were cultured in TSB with 10μg/ml chloramphenicol and 200ng/ml anhydrotetracycline and supernatants extracted after 18 hours of growth. Cytotoxicity in phoP::tn p phoP was compared to a strain carrying the empty vector ( phoP::tn pRMC2) as these could be grown under the same conditions. PSM Abundance To extract the supernatant, 1ml of S. aureus overnight culture was centrifuged (13000rpm, 5 mins). The supernatant was then diluted 2-fold in TSB and combined with 5X protein loading dye (National Diagnostics). Samples were boiled at 98°C for 5 minutes and loaded on to 4-12% nUView Tris-Glycine Precast Gels. Protein was separated at 100-130V until the dye front had run off the gel. Protein bands were visualised using Quick Coomassie Stain (Protein Ark) overnight and gels destained in deionised water for at least 5 hours. Band intensities were quantified using ImageJ. GFP/YFP reporter assays Reporter plasmids (pRNAIII:: gfp , pAH5E or p pstS::yfp ) were transformed into JE2, phoP::tn or agrA::tn via electroporation. To determine RNAIII expression in TSB, strains with pRNAIII:: gfp were grown overnight in TSB with 10ug/ml chloramphenicol and normalised to an OD 600nm of 0.05 in fresh TSB. GFP fluorescence (485nm excitation/520nm emission/1000 gain) and OD 600nm readings were taken every 30 mins for a 24-hour period with 200rpm shaking in between readings. Measurements were taken in a black 96 well plate (Costar) using a CLARIOstar plate reader. GFP fluorescence (485nm excitation/520nm emission/1500 gain) and these were normalised to OD 600nm readings (F/OD). To determine RNAIII and pstS expression under different phosphate concentrations, overnight cultures of strains carrying either p RNAIII ::gfp or ppstS::yfp were washed and resuspended in phosphate-depleted RPMI media (MP Biomedicals). Strains were then normalised to an OD 600nm of 0.05 in phosphate-depleted RPMI media with phosphate levels adjusted using 0.2M sodium phosphate buffer, pH 7.4 (Thermofisher) and incubated at 37°C overnight with shaking. A 200ul volume of culture was aliquoted into a black 96 well plate (Costar). YFP measurements (485nm excitation/520nm emission/1500 gain) were normalised to OD 600nm and the F/OD of a corresponding strain carrying empty pAH5E was subtracted from this value as described previously ( 33 ). Site-directed Mutagenesis Site-directed mutagenesis was performed to mutate wild-type phoP in p phoP to two variants: D53A and D53E. PCR amplification was performed using p phoP from Mach1 E. coli as template DNA with phoP D53A F and phoP D53A R or phoP D53E F and phoP D53E R primers ( Table 3 ). The PCR product was checked for a band of the correct size by agarose gel electrophoresis. 1ul of DpnI was then added and the PCR product was incubated at 37°C for 1 hour to remove template DNA. 5ul of the product was then transformed into Mach 1, passaged through S. aureus RN4220 and then transformed into phoP::tn via electroporation. mRNA Extraction and qRT-PCR S. aureus strains were grown to an OD 600nm of 4 in 50ml TSB and incubated with 200ng/ml aTC for 1 hour at 37°C with shaking at 180 rpm. 2ml of culture was then collected and RNA stabilised using RNAprotect bacterial reagent (Qiagen). RNA extractions were performed as described previously ( 35 ) using the Quick-RNA fungal/bacterial miniprep kit (Zymo research). Genomic DNA was removed from RNA samples using a TURBO DNA-free kit (Thermo Fisher), and reverse transcription was performed using the qScript cDNA synthesis kit (Quantabio). To verify samples were free from DNA contamination, a reverse transcription negative reaction was performed on all RNA samples and qRT-PCR performed. Threshold values were then compared to reverse transcription positive samples. Real-time PCR was performed using a KAPA SYBR fast qPCR kit (Kapa Biosystems), using RNAIII_F and RNAIII_R, pstS _F and pstS _R or gyrB _F and gyrB _R. gyrB was used as the housekeeping gene. All primer sets were validated using 5-fold dilutions of JE2 genomic DNA with efficiencies of 104.19, 106.19%, and 100.67% for pstS , RNAIII and gyrB , respectively. Statistical Analysis Statistical comparisons between two samples were performed with an unpaired two-tailed t-test using GraphPad Prism. A p value of < 0.05 was considered significant. In our screening of cytotoxicity, multiple t -tests were used and a false discovery rate (FDR) of 1% was applied account for multiple comparisons. Therefore, samples were considered significant if they had an adjusted p -value > 0.01. Data is displayed as the mean ± standard deviation of the mean (SD) and experiments were performed with three biological replicates. Results Cytotoxicity is altered in transposon mutants of many TCSs in S. aureus To identify the TCSs in S. aureus that regulate cytotoxicity, we used transposon insertion mutants in the histidine kinase (HK) and response regulator (RR) genes in the genome of S. aureus JE2 from the Nebraska Transposon Mutant Library (NTML) ( 30 ). As the WalKR TCS is essential for cell viability, there was no mutant strain of the walK and walR genes available. Additionally, there was no mutant strain of the airR gene in the NTML. From a screening of 29 transposon mutants, 17 demonstrated a statistically significant difference in cytotoxicity compared to the wild-type strain ( Fig. 1 ). All unadjusted and adjusted p -values are provided in Supplementary Table 1. Importantly, all transposon mutants in TCSs which have been demonstrated in previous studies to directly regulate expression of cytolytic toxins ( agrA::tn, agrC::tn, saeR::tn, saeS::tn, arlR::tn and arlS::tn ) had a significant reduction in THP-1 killing, providing proof of principle of our cytotoxicity assay ( 3 , 12 , 28 ). In addition to the established cytotoxicity linked TCSs, others involved in cell wall biosynthesis and autolysis ( graR::tn, graS::tn, nsaR::tn, nsaS::tn and lytR::tn ) had reduced cytotoxicity in our assay, indicating that there could be a link between regulation of the cell wall and cytotoxicity. Nutrient sensing also appeared to have an impact on cytotoxicity as several TCS mutants ( kdpE::tn, phoP::tn and hptS::tn ) had reduced cytotoxicity compared to the wild-type strain. In contrast, TCSs involved in respiration had a less of an impact on cytotoxicity, with only the nreC::tn mutant displaying reduced THP-1 killing. Interestingly, the HK and RR mutants of DesRK, a TCS with uncharacterised function, had reduced cytotoxicity compared to the wild-type strain. Overall, this data demonstrates that the majority of the TCSs in S. aureus contribute to regulating cytotoxicity. Download figure Open in new tab Figure 1. Cytotoxicity is altered in transposon mutants of TCSs in S. aureus . To quantify cytotoxicity, the bacterial supernatant from transposon mutants in each non-essential histidine kinase (HK) and response regulator (RR) gene was incubated with THP-1 cells and percentage of dead cells quantified using trypan blue exclusion. THP-1 killing was calculated relative to the THP-1 killing of JE2 (WT) in each assay. Each dot represents one biological replicate (n = 3), error bars the standard deviation, and statistical significance was determined using multiple t -tests with a false discovery rate (FDR) of 1% applied. Significant hits, which had an adjusted p -value ≤ 0.01 are highlighted in purple. The phoP::tn Mutant Produces a Lower Abundance of Phenol-Soluble Modulins PhoPR is an inorganic phosphate (Pi) responsive TCS which is present in many bacterial species ( 36 ). PhoR, the HK of the system, undergoes autophosphorylation in response to Pi limitation and then phosphorylates PhoP, the RR, which can activate or repress target genes ( 36 , 37 ). Whilst most of these genes are involved in phosphate homeostasis, the PhoPR TCS has been linked to regulating virulence factors in several other bacterial species such as Escherichia coli and Mycobacterium tuberculosis ( 38 - 40 ). Therefore, we wanted to establish whether phosphate sensing regulates cytotoxicity in S. aureus . Firstly, we sought to rule out any polar effects of the transposon insertion or spurious effects of mutations elsewhere on the chromosome. Therefore, we complemented the phoP mutation using the pRMC2 vector (pRMC2: phoP ). Expression of phoP in the phoP ::tn mutant restored cytotoxicity to wild-type levels ( Fig. 2a ). As THP-1 cells are particularly sensitive to lysis by PSMs in the S. aureus supernatant, we hypothesised that PSM abundance may be reduced in the supernatant of the phoP::tn mutant. To test this, we extracted the bacterial supernatant and analysed PSM abundance using SDS-PAGE ( Fig. 2b and Supplementary Fig. 1). An agrA::tn mutant, which does not produce any PSMs, was used as a control. We found that the phoP::tn mutant has a lower abundance of PSMs in the supernatant compared to the wild-type strain, explaining the reduced cytotoxicity of this strain. Additionally, this phenotype could also be complemented by expressing phoP from the pRMC2 vector. Download figure Open in new tab Figure 2. The phoP::tn mutant has a lower abundance of PSMs in the supernatant. (a) THP-1 cell lysis upon incubation with bacterial supernatant was quantified using trypan blue exclusion. The phoP::tn mutant exhibits lower cytotoxicity to THP-1 cells, an effect which can be complemented by expressing phoP from the pRMC2 plasmid. (b + c) Bacterial supernatants were extracted, separated via SDS-PAGE and protein bands visualised using Coomassie staining. (b) A representative SDS-PAGE gel showing PSMs in strains, which are visualised just below the 10kDa molecular weight marker, as shown previously ( 41 - 43 ). The band is absent in an agrA::tn mutant, which does not produce PSMs and a lower abundance can be seen in the phoP::tn mutant, which can be complemented. (c) Mean grey area of PSMs band calculated using densitometry. Significance in Fig. 2a + c is denoted as * p < 0.05, ** < 0.01, *** < 0.001. The phoP::tn Mutant has Reduced Agr Activity Given that the Agr TCS is known to directly regulate expression of PSMs ( 22 ), we hypothesised that the activity of the Agr system was reduced in the phoP::tn mutant. To test this, we introduced a RNAIII:: gfp fusion plasmid, which acts as a reporter of Agr activity, into the wild-type strain and phoP::tn mutant and measured fluorescence and growth over a 24h period. An agrA::tn mutant, which has no Agr activity, was used as a control. Despite minor differences in the growth curve, there was significant reduction in fluorescence in the phoP::tn mutant, demonstrating that RNAIII transcription was reduced in comparison to the wild-type strain. This explains the observed reduced cytotoxicity and PSM abundance in the phoP::tn mutant. PhoP Differentially Regulates Agr Activity Depending on Phosphate Availability As PhoPR is activated by Pi limitation, we wanted to determine whether PhoP alters Agr activity in response to Pi availability. Induction of pstS expression, a phosphate-binding protein in the pstSCAB operon, can be used as a marker of PhoPR activation and Pi limitation as pstS expression is induced by phosphorylated PhoP ( 17 , 44 ). To establish a model of Pi limitation, we introduced a pstS :: yfp reporter fusion plasmid into the wild-type strain and phoP::tn mutant. We then compared fluorescence of the wild-type strain and phoP ::tn mutant following an overnight growth in phosphate-depleted RPMI media supplemented with a range of Pi concentrations ( Fig. 4a ). In all Pi concentrations we tested (ranging from 0.125mM – 6mM), the wild-type and phoP::tn mutant grew to a similar OD 600nm (Supplementary Fig 2a + b). We detected pstS induction in the wild-type strain when Pi availability was ≤0.25mM. In contrast, pstS induction was absent in the phoP::tn mutant. Next, we measured Agr activity in the same growth conditions. When Pi availability was relatively high (between 1-6mM Pi), the phoP::tn mutant had significantly reduced Agr activity compared to the wild-type strain. This was similar to what was observed in TSB ( Fig. 3b ), which is a phosphate-rich media (∼14mM Pi). However, when Pi availability was ≤0.25mM we found that the phoP::tn mutant had significantly higher Agr activity compared to the wild-type strain. Furthermore, Agr activity increased in the WT strain between 0.125mM and 6mM ( p = 0.0027), further supporting the notion that Pi availability regulates Agr activity. Overall, our data supports a model whereby PhoP exhibits differential regulation of Agr activity depending on Pi availability and its phosphorylation state. When Pi availability is high, PhoP is unphosphorylated and upregulates Agr activity. In contrast, when Pi availability is low and PhoP is phosphorylated, it represses Agr activity. Download figure Open in new tab Figure 3. Activity of the Agr system is reduced in the phoP::tn mutant. (a) Growth of JE2 and the phoP::tn mutant carrying an RNAIII:: gfp fusion plasmid was measured over 24 hours, demonstrating mutation of phoP has no effect on S. aureus growth. An agrA::tn mutant was used as a control. (b) Fluorescence (F) of JE2 and the phoP::tn mutant was measured to quantify activation of the Agr system. The phoP::tn mutant exhibited reduced fluorescence over 24 hours, indicating that this mutant has impaired Agr activity. Area under the curve (AUC) analysis was performed on OD600nm and F/OD values and significance determined using a t -test. Significance is denoted as * p < 0.01 and ** < 0.0001. Download figure Open in new tab Download figure Open in new tab Figure 4. PhoP differentially regulates Agr Activity depending on phosphate availability. (a) Fluorescence (F) of JE2 WT and phoP::tn mutant carrying a pstS :: yfp reporter plasmid was measured following growth in phosphate-depleted RPMI media supplemented with a range of inorganic phosphate (Pi) concentrations to identify PhoPR activation. Expression of pstS occurs below 0.25mM Pi and this induction does not occur in the phoP::tn mutant. (b) Fluorescence of JE2 and the phoP::tn mutant carrying an RNAIII:: gfp reporter plasmid was measured in the same condition to determine Agr activity. The phoP::tn mutant had reduced fluorescence at high Pi concentrations, but higher fluorescence Pi limitation compared to the wild-type strain. Each dot represents one biological replicate (n = 3), error bars the standard deviation, and statistical significance was determined using a t test. Significance is denoted as * p < 0.05; ** < 0.01; < ***0.001. PhoP represses cytotoxicity in low Pi environments Our data demonstrates that PhoP acts as an activator in high Pi environments as the phoP::tn mutant has lower Agr activity and cytotoxicity and overexpression of phoP increases cytotoxicity in these growth conditions. In contrast, we found that the phoP::tn mutant has higher Agr activity in low Pi environments (≤0.25mM), suggesting it may act as a repressor in this environment. To confirm this, we tested the cytotoxicity of strains following growth in either 0.125mM or 0.25mM Pi ( Fig. 5a + b ). In line with Agr activity under low Pi conditions, the phoP::tn mutant had significantly higher cytotoxicity compared to the wild-type strain in both 0.125mM or 0.25mM Pi. Furthermore, expression of phoP in the phoP ::tn mutant significantly repressed cytotoxicity, confirming that PhoP acts as a cytotoxicity repressor in low Pi environments. Download figure Open in new tab Figure 5. PhoP acts as a repressor of cytotoxicity in low Pi environments. (a + b) THP-1 cell lysis upon incubation with bacterial supernatant was quantified using trypan blue exclusion. Supernatants were extracted following growth of strains in phosphate-depleted RPMI media supplemented with either 0.125mM Pi (a) or 0.25mM Pi (b). In both conditions, the phoP::tn mutant has higher cytotoxicity compared to the wild-type strain and expression of phoP from the pRMC2 significantly represses cytotoxicity compared to the phoP::tn mutant complemented with an empty pRMC2 vector. Significance is denoted as * p < 0.05; ** < 0.01; < ***0.001 Regulation of Agr activity by PhoP is Determined by its Phosphorylation State As we identified PhoP can act as either an activator or repressor of cytotoxicity depending on Pi availability, we wanted to verify the impact of PhoP phosphorylation on Agr activity. To test this, we substituted the phosphorylation site of PhoP (D53) in pRMC2:: phoP with an alanine, which prevents phosphorylation (phosphoablative), or a glutamic acid residue, which mimics the conformational change induced by permanent phosphorylation of the PhoP protein (phosphomimetic) ( 17 ). We then induced expression of either D53A or D53E PhoP and measured expression of pstS and RNAIII. As the pRMC2 and pRNAIII:: gfp vectors use the same resistance marker, we measured the levels of pstS and RNAIII using qRT-PCR. Transcription of pstS was elevated in phoP:tn expressing either D53A or D53E PhoP compared to phoP:tn pRMC2. However, this was 1146-fold higher upon expression of D53E PhoP compared to D53A PhoP, suggesting that induction of pstS expression is mediated by phosphorylated PhoP. Transcription of RNAIII was also elevated in both strains. However, expression of D53A PhoP resulted in approximately 2-fold higher RNAIII transcription compared to D53E PhoP. Combined, this data confirms that phosphorylation state affects PhoP regulation of Agr activity. Download figure Open in new tab Figure 6. Phosphorylation state of PhoP determines Agr regulation. Gene expression of either pstS (a) or RNAIII (b) was quantified relative to expression levels of the gyrB housekeeping gene in strains expressing D53A or D53E PhoP. Fold change was then calculated relative to the expression of each target gene in a strain carrying the empty pRMC2 vector. Expression of pstS is significantly higher when expressing D53E PhoP compared to D53A PhoP. In contrast, expression of D53A PhoP resulted in higher levels of RNAIII expression compared to expression of D53E PhoP. Each dot represents one biological replicate (n = 3), error bars the standard deviation, and statistical significance was determined by a t -test. Significance is denoted as * p < 0.05 and ** p < 0.01. Discussion In this study, we systematically compared the contribution to cytotoxicity of all the non-essential TCSs in the genome of S. aureus strain JE2. Our screening identified a link between TCSs involved in cell wall biosynthesis or autolysis and regulation of cytotoxicity as several mutants ( graR::tn, graS::tn, nsaR::tn and lytR::tn ) had reduced THP-1 toxicity. Disruption of these TCSs has profound effects on cell surface properties and morphology ( 18 , 45 - 48 ). Accordingly, changes to the cell surface structure of S. aureus have been previously reported to affect the regulation and secretion of toxins ( 35 , 49 ). Nutrient sensing was also identified as an important signal for regulating cytotoxicity with kdpE::tn, phoP::tn and hptS::tn mutant exhibiting reduced cytotoxicity. As the biosynthesis of cytolytic toxins is an energetically costly process, many pathogenic bacteria synchronise this with nutrient availability ( 14 , 50 ). Furthermore, nutrient availability can vary between different host tissues, acting as a marker for host environments where cytotoxicity is an advantageous phenotype. Having decided to focus here on the effect of PhoPR on cytotoxicity, we demonstrated that the phoP::tn mutant produces less PSMs in the bacterial supernatant during growth in TSB. PSMs include PSMα1-4 and PSMβ1/2, which are directly regulated by phosphorylated AgrA. Additionally, delta-haemolysin (Hld), is encoded within the main effector molecule of the Agr system, RNAIII ( 51 , 52 ). We identified that the phoP::tn mutant has lower RNAIII expression, suggesting that this mutant has reduced amounts of Hld and lower Agr activity. This finding supports the notion that TCSs in S. aureus are a highly interconnected network which frequently interact with each other to fine-tune gene expression ( 3 , 53 ). For example, expression of a phosphomimetic form of WalR activates the Sae TCS ( 54 ). Similarly, glucose-6-phosphate induces expression of both Agr and Sae and this is dependent on the presence of the HptRS TCS ( 55 ). Induction of pstS by PhoP follows the canonical signalling mechanism by which a two-component system activates a target gene. For example, we detected induction of pstS expression in growth media ≤0.25mM Pi, where PhoPR is activated and levels of phosphorylated PhoP significantly increase ( 17 , 44 ). In contrast, no regulation of pstS was detected when Pi levels were ≥0.5mM, where PhoP is unphosphorylated. This mechanism was confirmed through expression of D53E PhoP, which resulted in 1146-fold higher levels of pstS compared to expression of D53A PhoP. However, PhoP-mediated regulation of Agr activity follows an atypical trend. Firstly, unphosphorylated PhoP acts as a strong activator of RNAIII transcription in high Pi environments. This allows for regulation of Agr activity in the absence of an activation signal and explains why a phoR::tn mutant did not show a reduction in cytotoxicity in our initial screening as the experiment was performed at high Pi. Secondly, we identified that phosphorylated PhoP has differential effects on Agr activity depending on Pi concentration, acting as a weak activator in high Pi and a repressor in low Pi. Our current hypothesis is that additional factors expressed at low Pi regulated by PhoP concur in the regulation of Agr activity. This would explain why Agr activity is elevated in a phoP::tn under Pi limitation and overexpression of PhoP in this condition strongly represses cytotoxicity. This also suggests that regulation of cytotoxicity by Pi is a multifactorial process, with additional factors involved in coordination with PhoPR. Download figure Open in new tab Figure 7. Summary of Agr regulation by PhoP. When phosphate (Pi) availability is high, PhoR is inactive and PhoP mainly exists in its unphosphorylated form. Unphosphorylated PhoP acts as a strong activator of Agr activity. Phosphorylated PhoP also acts as a weaker activator of Agr activity. When Pi availability decreases, activation of PhoR occurs. PhoR undergoes autophosphorylation and mediates phosphorylation of PhoP. Phosphorylated PhoP induces expression of genes involved in phosphate transport, including pstS . Furthermore, phosphorylated PhoP acts as a repressor of Agr activity in low Pi environments. The finding that unphosphorylated PhoP is a stronger activator of RNAIII than phosphorylated PhoP in high Pi environments adds to growing experimental evidence which indicates unphosphorylated RRs have important regulatory functions ( 56 ). For example, unphosphorylated WalR from Streptococcus pneumoniae inhibits fabT , a repressor of fatty acid chain elongation ( 57 ). This supports the idea that TCSs can initiate a more complex response than a simple on-off feed forward mechanism, providing several regulatory options depending on their phosphorylation status. One more potential layer of regulation could be represented by the expression levels of PhoP, which may also change depending on Pi availability and affect how PhoPR regulates Agr activity. To date, studies of PhoPR in S. aureus have focused on its role in regulating phosphate transporters ( pstSCAB, nptA and pitA ) in response to phosphate limitation ( 44 ). Importantly, a S. aureus Δ phoPR mutant was found to be attenuated in the heart during a systemic staphylococcal abscess model of infection whereas a Δ pstSCAB Δ nptA double mutant had no defect, suggesting transporter-independent factors contribute to S. aureus pathogenesis in this environment ( 44 ). Our findings that PhoPR regulates cytotoxicity provides a potential explanation for this virulence defect and suggests that this regulation may be relevant during in vivo infections. In conclusion, our systematic screening approach revealed how the regulatory effects of 11 TCS out of 16 converge on cytotoxicity. 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Share Phosphate sensing by PhoPR regulates the cytotoxicity of Staphylococcus aureus Nathanael Palk , Tarcisio Brignoli , Marcia Boura , Ruth C. Massey bioRxiv 2025.06.24.661297; doi: https://doi.org/10.1101/2025.06.24.661297 Share This Article: Copy Citation Tools Phosphate sensing by PhoPR regulates the cytotoxicity of Staphylococcus aureus Nathanael Palk , Tarcisio Brignoli , Marcia Boura , Ruth C. 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