Discovery of an antivirulence compound that targets the Staphylococcus aureus SaeRS two-component system to inhibit toxic shock syndrome toxin-1 production.

Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting the growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed the production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets the virulence of S. aureus, and it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS.

In Staphylococcus aureus, the acyl-CoA synthetase MbcS supports branched-chain fatty acid synthesis from carboxylic acid and aldehyde precursors.

In the human pathogen Staphylococcus aureus, branched-chain fatty acids (BCFAs) are the most abundant fatty acids in membrane phospholipids. Strains deficient for BCFAs synthesis experience auxotrophy in laboratory culture and attenuated virulence during infection. Furthermore, the membrane of S. aureus is among the main targets for antibiotic therapy. Therefore, determining the mechanisms involved in BCFAs synthesis is critical to manage S. aureus infections. Here, we report that the overexpression of SAUSA300_2542 (annotated to encode an acyl-CoA synthetase) restores BCFAs synthesis in strains lacking the canonical biosynthetic pathway catalyzed by the branched-chain α-keto acid dehydrogenase (BKDH) complex. We demonstrate that the acyl-CoA synthetase activity of MbcS activates branched-chain carboxylic acids (BCCAs), and is required by S. aureus to utilize the isoleucine derivative 2-methylbutyraldehyde to restore BCFAs synthesis in S. aureus. Based on the ability of some staphylococci to convert branched-chain aldehydes into their respective BCCAs and our findings demonstrating that branched-chain aldehydes are in fact BCFAs precursors, we propose that MbcS promotes the scavenging of exogenous BCCAs and mediates BCFA synthesis via a de novo alternative pathway.

CodY controls the SaeR/S two-component system by modulating branched-chain fatty acid synthesis in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive, opportunistic human pathogen that is a leading cause of skin and soft tissue infections and invasive disease worldwide. Virulence in this bacterium is tightly controlled by a network of regulatory factors. One such factor is the global regulatory protein CodY. CodY links branched-chain amino acid sufficiency to the production of surface-associated and secreted factors that facilitate immune evasion and subversion. Our previous work revealed that CodY regulates virulence factor gene expression indirectly in part by controlling the activity of the SaeRS two-component system (TCS). While this is correlated with an increase in membrane anteiso-15:0 and -17:0 branched-chain fatty acids (BCFAs) derived from isoleucine, the true mechanism of control has remained elusive. Herein, we report that CodY-dependent regulation of SaeS sensor kinase activity requires BCFA synthesis. During periods of nutrient sufficiency, BCFA synthesis and Sae TCS activity are kept relatively low by CodY-dependent repression of the ilv-leu operon and the isoleucine-specific permease gene brnQ2. In a codY null mutant, which simulates extreme nutrient limitation, de-repression of ilv-leu and brnQ2 directs the synthesis of enzymes in redundant de novo and import pathways to upregulate production of BCFA precursors. Overexpression of brnQ2, independent of CodY, is sufficient to increase membrane anteiso BCFAs, Sae-dependent promoter activity, and SaeR ~P levels. Our results further clarify the molecular mechanisms by which CodY controls virulence in S. aureus.IMPORTANCEExpression of bacterial virulence genes often correlates with the exhaustion of nutrients, but how the signaling of nutrient availability and the resulting physiological responses are coordinated is unclear. In S. aureus, CodY controls the activity of two major regulators of virulence-the Agr and Sae two-component systems (TCSs)-by unknown mechanisms. This work identifies a mechanism by which CodY controls the activity of the sensor kinase SaeS by modulating the levels of anteiso branched-chain amino acids that are incorporated into the membrane. Understanding the mechanism adds to our understanding of how bacterial physiology and metabolism are linked to virulence and underscores the role virulence in maintaining homeostasis. Understanding the mechanism also opens potential avenues for targeted therapeutic strategies against S. aureus infections.

Regulation of Bacterial Two-Component Systems by Cardiolipin.

The regulation of membrane protein activity for cellular functions is critically dependent on the composition of phospholipid membranes. Cardiolipin, a unique phospholipid found in bacterial membranes and mitochondrial membranes of eukaryotes, plays a crucial role in stabilizing membrane proteins and maintaining their function. In the human pathogen Staphylococcus aureus, the SaeRS two-component system (TCS) controls the expression of key virulence factors essential for the bacterium's virulence. The SaeS sensor kinase activates the SaeR response regulator via phosphoryl transfer to bind its gene target promoters. In this study, we report that cardiolipin is critical for sustaining the full activity of SaeRS and other TCSs in S. aureus. The sensor kinase protein SaeS binds directly to cardiolipin and phosphatidylglycerol, enabling SaeS activity. Elimination of cardiolipin from the membrane reduces SaeS kinase activity, indicating that bacterial cardiolipin is necessary for modulating the kinase activities of SaeS and other sensor kinases during infection. Moreover, the deletion of cardiolipin synthase genes cls1 and cls2 leads to reduced cytotoxicity to human neutrophils and lower virulence in a mouse model of infection. These findings suggest a model where cardiolipin modulates the kinase activity of SaeS and other sensor kinases after infection to adapt to the hostile environment of the host and expand our knowledge of how phospholipids contribute to membrane protein function.

Analysis of the CodY RNome reveals RsaD as a stress-responsive riboregulator of overflow metabolism in Staphylococcus aureus.

In Staphylococcus aureus, the transcription factor CodY modulates the expression of hundreds of genes, including most virulence factors, in response to the availability of key nutrients like GTP and branched-chain amino acids. Despite numerous studies examining how CodY controls gene expression directly or indirectly, virtually nothing is known about the extent to which CodY exerts its effect through small regulatory RNAs (sRNAs). Herein, we report the first set of sRNAs under the control of CodY. We reveal that staphylococcal sRNA RsaD is overexpressed >20-fold in a CodY-deficient strain in three S. aureus clinical isolates and in S. epidermidis. We validated the CodY-dependent regulation of rsaD and demonstrated that CodY directly represses rsaD expression by binding the promoter. Using a combination of molecular techniques, we show that RsaD posttranscriptionally regulates alsS (acetolactate synthase) mRNA and enzyme levels. We further show that RsaD redirects carbon overflow metabolism, contributing to stationary phase cell death during exposure to weak acid stress. Taken together, our data delineate a role for CodY in controlling sRNA expression in a major human pathogen and indicate that RsaD may integrate nutrient depletion and other signals to mount a response to physiological stress experienced by S. aureus in diverse environments.

Repression of branched-chain amino acid synthesis in Staphylococcus aureus is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide.

Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY.

Genetic and Biochemical Analysis of CodY-Mediated Cell Aggregation in Staphylococcus aureus Reveals an Interaction between Extracellular DNA and Polysaccharide in the Extracellular Matrix.

The global regulator CodY links nutrient availability to the regulation of virulence factor gene expression in Staphylococcus aureus, including many genes whose products affect biofilm formation. Antithetical phenotypes of both biofilm deficiency and accumulation have been reported for codY-null mutants; thus, the role of CodY in biofilm development remains unclear. codY mutant cells of a strain producing a robust biofilm elaborate proaggregation surface-associated features not present on codY mutant cells that do not produce a robust biofilm. Biochemical analysis of the clinical isolate SA564, which aggregates when deficient for CodY, revealed that these features are sensitive to nuclease treatment and are resistant to protease exposure. Genetic analyses revealed that disrupting lgt (the diacylglycerol transferase gene) in codY mutant cells severely weakened aggregation, indicating a role for lipoproteins in the attachment of the biofilm matrix to the cell surface. An additional and critical role of IcaB in producing functional poly-N-acetylglucosamine (PIA) polysaccharide in extracellular DNA (eDNA)-dependent biofilm formation was shown. Moreover, overproducing PIA is sufficient to promote aggregation in a DNA-dependent manner regardless of source of nucleic acids. Taken together, our results point to PIA synthesis as the primary determinant of biofilm formation when CodY activity is reduced and suggest a modified electrostatic net model for matrix attachment whereby PIA associates with eDNA, which interacts with the cell surface via covalently attached membrane lipoproteins. This work counters the prevailing view that polysaccharide- and eDNA/protein-based biofilms are mutually exclusive. Rather, we demonstrate that eDNA and PIA can work synergistically to form a biofilm.IMPORTANCEStaphylococcus aureus remains a global health concern and exemplifies the ability of an opportunistic pathogen to adapt and persist within multiple environments, including host tissue. Not only does biofilm contribute to persistence and immune evasion in the host environment, it also may aid in the transition to invasive disease. Thus, understanding how biofilms form is critical for developing strategies for dispersing biofilms and improving biofilm disease-related outcomes. Using biochemical, genetic, and cell biology approaches, we reveal a synergistic interaction between PIA and eDNA that promotes cell aggregation and biofilm formation in a CodY-dependent manner in S. aureus We also reveal that envelope-associated lipoproteins mediate attachment of the biofilm matrix to the cell surface.

Nutritional Regulation of the Sae Two-Component System by CodY in Staphylococcus aureus.

Staphylococcus aureus subverts innate defenses during infection in part by killing host immune cells to exacerbate disease. This human pathogen intercepts host cues and activates a transcriptional response via the S. aureus exoprotein expression (SaeR/SaeS [SaeR/S]) two-component system to secrete virulence factors critical for pathogenesis. We recently showed that the transcriptional repressor CodY adjusts nuclease (nuc) gene expression via SaeR/S, but the mechanism remained unknown. Here, we identified two CodY binding motifs upstream of the sae P1 promoter, which suggested direct regulation by this global regulator. We show that CodY shares a binding site with the positive activator SaeR and that alleviating direct CodY repression at this site is sufficient to abrogate stochastic expression, suggesting that CodY represses sae expression by blocking SaeR binding. Epistasis experiments support a model that CodY also controls sae indirectly through Agr and Rot-mediated repression of the sae P1 promoter. We also demonstrate that CodY repression of sae restrains production of secreted cytotoxins that kill human neutrophils. We conclude that CodY plays a previously unrecognized role in controlling virulence gene expression via SaeR/S and suggest a mechanism by which CodY acts as a master regulator of pathogenesis by tying nutrient availability to virulence gene expression.IMPORTANCE Bacterial mechanisms that mediate the switch from a commensal to pathogenic lifestyle are among the biggest unanswered questions in infectious disease research. Since the expression of most virulence genes is often correlated with nutrient depletion, this implies that virulence is a response to the lack of nourishment in host tissues and that pathogens like S. aureus produce virulence factors in order to gain access to nutrients in the host. Here, we show that specific nutrient depletion signals appear to be funneled to the SaeR/S system through the global regulator CodY. Our findings reveal a strategy by which S. aureus delays the production of immune evasion and immune-cell-killing proteins until key nutrients are depleted.

Guanine Limitation Results in CodY-Dependent and -Independent Alteration of Staphylococcus aureus Physiology and Gene Expression.

In Staphylococcus aureus, the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY-regulated targets. In this work, we investigated the impact of guanine nucleotides (GNs) on S. aureus physiology and CodY activity by constructing a guaA null mutant (ΔguaA strain). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA, activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the ΔguaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, ΔguaA cells failed to form robust biofilms when limited for guanine or guanosine. Transcriptome sequencing (RNA-Seq) analysis of the S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alterations of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus exhibits a more invasive lifestyle when limited for guanosine. Further, gene products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections.IMPORTANCEStaphylococcus aureus infections impose a serious economic burden on health care facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of S. aureus in response to environmental cues is critical for developing efficient diagnostics and treatments. De novo purine biosynthesis is essential for both fitness and virulence in S. aureus since inhibiting production cripples S. aureus's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects S. aureus fitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combat S. aureus infections.

Intermediate Levels of Bacillus subtilis CodY Activity Are Required for Derepression of the Branched-Chain Amino Acid Permease, BraB.

The global transcriptional regulator, CodY, binds strongly to the regulatory region of the braB gene, which encodes a Bacillus subtilis branched-chain amino acid (BCAA) permease. However, under conditions that maximize CodY activity, braB expression was similar in wild-type and codY null mutant cells. Nonetheless, expression from the braB promoter was significantly elevated in cells containing partially active mutant versions of CodY or in wild-type cells under growth conditions leading to intermediate levels of CodY activity. This novel pattern of regulation was shown to be due to two opposing mechanisms, negative and positive, by which CodY affects braB expression. A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY. Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY. ScoC-mediated repression of braB in codY null mutants cells was as efficient as direct, CodY-mediated repression in wild-type cells under conditions of high CodY activity. However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression. We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d'être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter. The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

A spectrum of CodY activities drives metabolic reorganization and virulence gene expression in Staphylococcus aureus.

The global regulator CodY controls the expression of dozens of metabolism and virulence genes in the opportunistic pathogen Staphylococcus aureus in response to the availability of isoleucine, leucine and valine (ILV), and GTP. Using RNA-Seq transcriptional profiling and partial activity variants, we reveal that S. aureus CodY activity grades metabolic and virulence gene expression as a function of ILV availability, mediating metabolic reorganization and controlling virulence factor production in vitro. Strains lacking CodY regulatory activity produce a PIA-dependent biofilm, but development is restricted under conditions that confer partial CodY activity. CodY regulates the expression of thermonuclease (nuc) via the Sae two-component system, revealing cascading virulence regulation and factor production as CodY activity is reduced. Proteins that mediate the host-pathogen interaction and subvert the immune response are shut off at intermediate levels of CodY activity, while genes coding for enzymes and proteins that extract nutrients from tissue, that kill host cells, and that synthesize amino acids are among the last genes to be derepressed. We conclude that S. aureus uses CodY to limit host damage to only the most severe starvation conditions, providing insight into one potential mechanism by which S. aureus transitions from a commensal bacterium to an invasive pathogen.

CodY, a master integrator of metabolism and virulence in Gram-positive bacteria.

A growing body of evidence points to CodY, a global regulator in Gram-positive bacteria, as a critical link between microbial physiology and pathogenesis in diverse environments. Recent studies uncovering graded regulation of CodY gene targets reflect the true nature of this transcription factor controlled by ligands and reveal nutrient availability as a potentially critical factor in modulating pathogenesis. This review will serve to update the status of the field and raise new questions to be answered.

Hierarchical expression of genes controlled by the Bacillus subtilis global regulatory protein CodY.

Global regulators that bind strategic metabolites allow bacteria to adapt rapidly to dynamic environments by coordinating the expression of many genes. We report an approach for determining gene regulation hierarchy using the regulon of the Bacillus subtilis global regulatory protein CodY as proof of principle. In theory, this approach can be used to measure the dynamics of any bacterial transcriptional regulatory network that is affected by interaction with a ligand. In B. subtilis, CodY controls dozens of genes, but the threshold activities of CodY required to regulate each gene are unknown. We hypothesized that targets of CodY are differentially regulated based on varying affinity for the protein's many binding sites. We used RNA sequencing to determine the transcription profiles of B. subtilis strains expressing mutant CodY proteins with different levels of residual activity. In parallel, we quantified intracellular metabolites connected to central metabolism. Strains producing CodY variants F71Y, R61K, and R61H retained varying degrees of partial activity relative to the WT protein, leading to gene-specific, differential alterations in transcript abundance for the 223 identified members of the CodY regulon. Using liquid chromatography coupled to MS, we detected significant increases in branched-chain amino acids and intermediates of arginine, proline, and glutamate metabolism, as well as decreases in pyruvate and glycerate as CodY activity decreased. We conclude that a spectrum of CodY activities leads to programmed regulation of gene expression and an apparent rerouting of carbon and nitrogen metabolism, suggesting that during changes in nutrient availability, CodY prioritizes the expression of specific pathways.

GTP dysregulation in Bacillus subtilis cells lacking (p)ppGpp results in phenotypic amino acid auxotrophy and failure to adapt to nutrient downshift and regulate biosynthesis genes.

The nucleotide (p)ppGpp inhibits GTP biosynthesis in the Gram-positive bacterium Bacillus subtilis. Here we examined how this regulation allows cells to grow in the absence of amino acids. We showed that B. subtilis cells lacking (p)ppGpp, due to either deletions or point mutations in all three (p)ppGpp synthetase genes, yjbM, ywaC, and relA, strongly require supplementation of leucine, isoleucine, valine, methionine, and threonine and modestly require three additional amino acids. This polyauxotrophy is rescued by reducing GTP levels. Reduction of GTP levels activates transcription of genes responsible for the biosynthesis of the five strongly required amino acids by inactivating the transcription factor CodY, which represses the ybgE, ilvD, ilvBHC-leuABCD, ilvA, ywaA, and hom-thrCB operons, and by a CodY-independent activation of transcription of the ilvA, ywaA, hom-thrCB, and metE operons. Interestingly, providing the eight required amino acids does not allow for colony formation of (p)ppGpp(0) cells when transitioning from amino acid-replete medium to amino acid-limiting medium, and we found that this is due to an additional role that (p)ppGpp plays in protecting cells during nutrient downshifts. We conclude that (p)ppGpp allows adaptation to amino acid limitation by a combined effect of preventing death during metabolic transitions and sustaining growth by activating amino acid biosynthesis. This ability of (p)ppGpp to integrate a general stress response with a targeted reprogramming of gene regulation allows appropriate adaptation and is likely conserved among diverse bacteria.

Regulation of Bacterial Two-Component Systems by Cardiolipin.

The composition of phospholipid membranes is critical to regulating the activity of membrane proteins for cellular functions. Cardiolipin is a unique phospholipid present within the bacterial membrane and mitochondria of eukaryotes and plays a role in maintaining the function and stabilization of membrane proteins. Here, we report that, in the human pathogen Staphylococcus aureus, cardiolipin is required for full activity of the SaeRS two-component system (TCS). Deletion of the cardiolipin synthase genes, cls1 , and cls2 , reduces the basal activity of SaeRS and other TCSs. Cardiolipin is an indispensable requisite for Sae activation mediated by human neutrophil peptides (HNPs) in the stationary growth phase but not mandatory for Sae induction in the exponential growth phase. Ectopic expression with cls2 , but not with cls1 , in the cls1 cls2 double mutant fully restores Sae activity. Elimination of cardiolipin from the membranes results in decreased kinase activity of the sensor protein SaeS. Purified SaeS protein directly binds to cardiolipin as well as phosphatidylglycerol. A strain lacking cls2 or cls1cls2 renders S. aureus less cytotoxic to human neutrophils and less virulent in a mouse model of infection. Our findings suggest that cardiolipin enables a pathogen to confer virulence by modulating the kinase activity of SaeS and other sensor kinases upon infection.

Involvement of protein acetylation in glucose-induced transcription of a stress-responsive promoter.

In eukaryotes, lysine acetylation is a well-established post-translational modification that has been implicated in virtually all aspects of eukaryotic physiology. Although homologues of the enzymes that catalyse protein acetylation are widely conserved and distributed among bacterial species, not much is known about the impact of protein acetylation on bacterial physiology. Here, we present evidence that the Gcn5-like acetyltransferase YfiQ and the sirtuin deacetylase CobB play crucial roles in the transcription regulation of the periplasmic stress-responsive promoter cpxP when cells of Escherichia coli grow in the presence of glucose, an environment that induces protein acetylation. Under this growth condition, several acetylation sites were detected on three of the RNA polymerase subunits: ß, ß' and α. We focused on acetylations of the carboxy-terminal domain (CTD) of α because of its relative small size and its limited acetylation. We determined that K298 of α is acetylated in a glucose and YfiQ-dependent manner and that K298 is specifically required for glucose-induced cpxP transcription. Because the αCTD aids in promoter recognition by RNA polymerase, we propose its acetylation may influence bacterial physiology through effects on gene expression.

Regulation of the Sae Two-Component System by Branched-Chain Fatty Acids in Staphylococcus aureus.

Staphylococcus aureus is a ubiquitous Gram-positive bacterium and an opportunistic human pathogen. S. aureus pathogenesis relies on a complex network of regulatory factors that adjust gene expression. Two important factors in this network are CodY, a repressor protein responsive to nutrient availability, and the SaeRS two-component system (TCS), which responds to neutrophil-produced factors. Our previous work revealed that CodY regulates the secretion of many toxins indirectly via Sae through an unknown mechanism. We report that disruption of codY results in increased levels of phosphorylated SaeR (SaeR~P) and that codY mutant cell membranes contain a higher percentage of branched-chain fatty acids (BCFAs) than do wild-type membranes, prompting us to hypothesize that changes to membrane composition modulate the activity of the SaeS sensor kinase. Disrupting the lpdA gene encoding dihydrolipoyl dehydrogenase, which is critical for BCFA synthesis, significantly reduced the abundance of SaeR, phosphorylated SaeR, and BCFAs in the membrane, resulting in reduced toxin production and attenuated virulence. Lower SaeR levels could be explained in part by reduced stability. Sae activity in the lpdA mutant could be complemented genetically and chemically with exogenous short- or full-length BCFAs. Intriguingly, lack of lpdA also alters the activity of other TCSs, suggesting a specific BCFA requirement managing the basal activity of multiple TCSs. These results reveal a novel method of posttranscriptional virulence regulation via BCFA synthesis, potentially linking CodY activity to multiple virulence regulators in S. aureus. IMPORTANCE Two-component systems (TCSs) are an essential way that bacteria sense and respond to their environment. These systems are usually composed of a membrane-bound histidine kinase that phosphorylates a cytoplasmic response regulator. Because most of the histidine kinases are embedded in the membrane, lipids can allosterically regulate the activity of these sensors. In this study, we reveal that branched-chain fatty acids (BCFAs) are required for the activation of multiple TCSs in Staphylococcus aureus. Using both genetic and biochemical data, we show that the activity of the virulence activator SaeS and the phosphorylation of its response regulator SaeR are reduced in a branched-chain keto-acid dehydrogenase complex mutant and that defects in BCFA synthesis have far-reaching consequences for exotoxin secretion and virulence. Finally, we show that mutation of the global nutritional regulator CodY alters BCFA content in the membrane, revealing a potential mechanism of posttranscriptional regulation of the Sae system by CodY.

Dissecting complex metabolic integration provides direct genetic evidence for CodY activation by guanine nucleotides.

The global regulator CodY controls the expression of dozens of metabolic genes and genes mediating adaptation to nutrient availability in many low-G+C Gram-positive bacteria. Branched-chain amino acids L-isoleucine, L-leucine, and L-valine (ILV) activate CodY both in vivo and in vitro, and genes that direct their synthesis (ilv, ybgE, and ywaA) are highly repressed by CodY, creating a potential negative feedback loop. The nucleoside triphosphate GTP also activates CodY in vitro, but the evidence for activation by GTP in vivo is limited and indirect. We constructed a Bacillus subtilis strain (ybgE bcd ywaA) that is unable to convert branched-chain α-keto acids to ILV or to use ILV as a precursor for branched-chain fatty acid synthesis. Unexpectedly, the strain was not viable on rich medium. Supplementing rich medium with short, branched-chain fatty acids or derepressing expression of genes for de novo ILV synthesis bypassed the original lethality, restoring growth and showing that the lack of viability was due to insufficient intracellular production of the precursors of branched-chain fatty acids. Spontaneous extragenic suppressor mutants that arose in the triple mutant population proved to have additional mutations in guaA or guaB or codY. Expression of ILV biosynthetic genes in codY mutants was increased. The gua mutations caused guanine/guanosine auxotrophy and led to partial derepression of direct CodY-repressed targets, including ILV biosynthetic genes, under conditions similar to those that caused the original lethality. We conclude that a guanine derivative, most likely GTP, controls CodY activity in vivo.

Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools.

In several Gram-positive bacterial species, the global transcriptional regulatory protein CodY adjusts the expression of many metabolic genes, apparently in response to changes in the pools of specific metabolites, i.e., the branched-chain amino acids (BCAAs) isoleucine, leucine, and valine (ILV) and the nucleoside triphosphate GTP. CodY not only responds to these metabolites as measured in vitro but also regulates the genes that direct their synthesis. We have constructed a set of strains lacking binding sites for the CodY protein in cis at loci coding for the ILV biosynthetic machinery, effectively overexpressing these genes in an attempt to modulate the ILV input signal to CodY. Metabolite analyses of strains derepressed for genes needed for ILV synthesis revealed more than a 6-fold increase in the valine pool and a 2-fold increase in the isoleucine and leucine pools. Accumulation of the branched-chain amino acids was accompanied by a 24-fold induction of the bkd operon (required for branched-chain fatty acid synthesis) and 6-fold hyperrepression of the CodY-regulated yhdG and yufN genes, demonstrating that CodY perceives intracellular fluctuations in at least one if its input signals. We conclude that changes in the rate of endogenous ILV synthesis serve as an important signal for CodY-mediated gene regulation.

Who's in control? Regulation of metabolism and pathogenesis in space and time.

Bacterial pathogens need to sense and respond to their environments during infection to align cell metabolism and virulence factor production to survive and battle host defenses. Complex regulatory networks including ligand-binding transcription factors, two-component systems, RNA-binding proteins, and small non-coding regulatory RNAs adjust gene expression programs in response to changes in metabolic fluxes, environmental cues, and nutrient availability. Recent studies underlined that these different layers of regulation occur along varying spatial and temporal scales, leading to changes in cell behavior and heterogeneity among the bacterial community. This brief review will highlight current research emphasizing that cell metabolism and pathogenesis are inextricably intertwined in both Gram-positive and Gram-negative bacteria.