Small molecule photocatalysis enables drug target identification via energy transfer.

Over half of new therapeutic approaches fail in clinical trials due to a lack of target validation. As such, the development of new methods to improve and accelerate the identification of cellular targets, broadly known as target ID, remains a fundamental goal in drug discovery. While advances in sequencing and mass spectrometry technologies have revolutionized drug target ID in recent decades, the corresponding chemical-based approaches have not changed in over 50 y. Consigned to outdated stoichiometric activation modes, modern target ID campaigns are regularly confounded by poor signal-to-noise resulting from limited receptor occupancy and low crosslinking yields, especially when targeting low abundance membrane proteins or multiple protein target engagement. Here, we describe a broadly general platform for photocatalytic small molecule target ID, which is founded upon the catalytic amplification of target-tag crosslinking through the continuous generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead tag from the small molecule ligand, catalytic signal amplification results in unprecedented levels of target enrichment, enabling the quantitative target and off target ID of several drugs including (+)-JQ1, paclitaxel (Taxol), dasatinib (Sprycel), as well as two G-protein-coupled receptors-ADORA2A and GPR40.

µMap Photoproximity Labeling Enables Small Molecule Binding Site Mapping.

The characterization of ligand binding modes is a crucial step in the drug discovery process and is especially important in campaigns arising from phenotypic screening, where the protein target and binding mode are unknown at the outset. Elucidation of target binding regions is typically achieved by X-ray crystallography or photoaffinity labeling (PAL) approaches; yet, these methods present significant challenges. X-ray crystallography is a mainstay technique that has revolutionized drug discovery, but in many cases structural characterization is challenging or impossible. PAL has also enabled binding site mapping with peptide- and amino-acid-level resolution; however, the stoichiometric activation mode can lead to poor signal and coverage of the resident binding pocket. Additionally, each PAL probe can have its own fragmentation pattern, complicating the analysis by mass spectrometry. Here, we establish a robust and general photocatalytic approach toward the mapping of protein binding sites, which we define as identification of residues proximal to the ligand binding pocket. By utilizing a catalytic mode of activation, we obtain sets of labeled amino acids in the proximity of the target protein binding site. We use this methodology to map, in vitro, the binding sites of six protein targets, including several kinases and molecular glue targets, and furthermore to investigate the binding site of the STAT3 inhibitor MM-206, a ligand with no known crystal structure. Finally, we demonstrate the successful mapping of drug binding sites in live cells. These results establish µMap as a powerful method for the generation of amino-acid- and peptide-level target engagement data.

µMap-Red: Proximity Labeling by Red Light Photocatalysis.

Modern proximity labeling techniques have enabled significant advances in understanding biomolecular interactions. However, current tools primarily utilize activation modes that are incompatible with complex biological environments, limiting our ability to interrogate cell- and tissue-level microenvironments in animal models. Here, we report µMap-Red, a proximity labeling platform that uses a red-light-excited SnIV chlorin e6 catalyst to activate a phenyl azide biotin probe. We validate µMap-Red by demonstrating photonically controlled protein labeling in vitro through several layers of tissue, and we then apply our platform in cellulo to label EGFR microenvironments and validate performance with STED microscopy and quantitative proteomics. Finally, to demonstrate labeling in a complex biological sample, we deploy µMap-Red in whole mouse blood to profile erythrocyte cell-surface proteins. This work represents a significant methodological advance toward light-based proximity labeling in complex tissue environments and animal models.

Identification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae.

Several important human pathogens are represented in the Corynebacterineae suborder, including Mycobacterium tuberculosis and Corynebacterium diphtheriae. These bacteria are surrounded by a multilayered cell envelope composed of a cytoplasmic membrane, a peptidoglycan (PG) cell wall, a second polysaccharide layer called the arabinogalactan (AG), and finally an outer membrane-like layer made of mycolic acids. Several anti-tuberculosis drugs target the biogenesis of this complex envelope, but their efficacy is declining due to resistance. New therapies are therefore needed to treat diseases caused by these organisms, and a better understanding of the mechanisms of envelope assembly should aid in their discovery. To this end, we generated the first high-density library of transposon insertion mutants in the model organism C. glutamicum. Transposon-sequencing was then used to define its essential gene set and identify loci that, when inactivated, confer hypersensitivity to ethambutol (EMB), a drug that targets AG biogenesis. Among the EMBs loci were genes encoding RipC and the FtsEX complex, a PG cleaving enzyme required for proper cell division and its predicted regulator, respectively. Inactivation of the conserved steAB genes (cgp_1603-1604) was also found to confer EMB hypersensitivity and cell division defects. A combination of quantitative microscopy, mutational analysis, and interaction studies indicate that SteA and SteB form a complex that localizes to the cytokinetic ring to promote cell separation by RipC-FtsEX and may coordinate its PG remodeling activity with the biogenesis of other envelope layers during cell division.

A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier.

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the ß-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

Sequential assembly of the septal cell envelope prior to V snapping in Corynebacterium glutamicum.

Members of the Corynebacterineae, including Corynebacterium and Mycobacterium, have an atypical cell envelope characterized by an additional mycomembrane outside of the peptidoglycan layer. How this multilayered cell envelope is assembled remains unclear. Here, we tracked the assembly dynamics of different envelope layers in Corynebacterium glutamicum and Mycobacterium smegmatis by using metabolic labeling and found that the septal cell envelope is assembled sequentially in both species. Additionally, we demonstrate that in C. glutamicum, the peripheral peptidoglycan layer at the septal junction remains contiguous throughout septation, forming a diffusion barrier for the fluid mycomembrane. This diffusion barrier is resolved through perforations in the peripheral peptidoglycan, thus leading to the confluency of the mycomembrane before daughter cell separation (V snapping). Furthermore, the same junctional peptidoglycan also serves as a mechanical link holding the daughter cells together and undergoes mechanical fracture during V snapping. Finally, we show that normal V snapping in C. glutamicum depends on complete assembly of the septal cell envelope.

Click-EM for imaging metabolically tagged nonprotein biomolecules.

EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.

Who were the Nataruk people? Mandibular morphology among late Pleistocene and early Holocene fisher-forager populations of West Turkana (Kenya).

Africa is the birthplace of the species Homo sapiens, and Africans today are genetically more diverse than other populations of the world. However, the processes that underpinned the evolution of African populations remain largely obscure. Only a handful of late Pleistocene African fossils (∼50-12 Ka) are known, while the more numerous sites with human fossils of early Holocene age are patchily distributed. In particular, late Pleistocene and early Holocene human diversity in Eastern Africa remains little studied, precluding any analysis of the potential factors that shaped human diversity in the region, and more broadly throughout the continent. These periods include the Last Glacial Maximum (LGM), a moment of extreme aridity in Africa that caused the fragmentation of population ranges and localised extinctions, as well as the 'African Humid Period', a moment of abrupt climate change and enhanced connectivity throughout Africa. East Africa, with its range of environments, may have acted as a refugium during the LGM, and may have played a critical biogeographic role during the heterogene`ous environmental recovery that followed. This environmental context raises a number of questions about the relationships among early Holocene African populations, and about the role played by East Africa in shaping late hunter-gatherer biological diversity. Here, we describe eight mandibles from Nataruk, an early Holocene site (∼10 Ka) in West Turkana, offering the opportunity of exploring population diversity in Africa at the height of the 'African Humid Period'. We use 3D geometric morphometric techniques to analyze the phenotypic variation of a large mandibular sample. Our results show that (i) the Nataruk mandibles are most similar to other African hunter-fisher-gatherer populations, especially to the fossils from Lothagam, another West Turkana locality, and to other early Holocene fossils from the Central Rift Valley (Kenya); and (ii) a phylogenetic connection may have existed between these Eastern African populations and some Nile Valley and Maghrebian groups, who lived at a time when a Green Sahara may have allowed substantial contact, and potential gene flow, across a vast expanse of Northern and Eastern Africa.

Selective and context-dependent effects of chemical stress across trophic levels at the basis of marine food webs.

Human activities increasingly impact the functioning of marine food webs, but anthropogenic stressors are seldom included in ecological study designs. Diet quality, as distinct from just diet quantity, has moreover rarely been highlighted in food web studies in a stress context. We measured the effects of metal and pesticide stress (copper and atrazine) on the contribution of a benthic intertidal diatom community to two processes that are key to the functioning of intertidal systems: biomass (diet quantity) and lipid (diet quality) production. We then examined if stressors affected diatom functioning by selectively targeting the species contributing most to functioning (selective stress effects) or by changing the species' functional contribution (context-dependent effects). Finally, we tested if stress-induced changes in diet quality altered the energy flow to the diatoms' main grazers (harpacticoid copepods). Diatom diet quantity was reduced by metal stress but not by low pesticide levels due to the presence of an atrazine-tolerant, mixotrophic species. Selective effects of the pesticide reduced diatom diet quality by 60% and 75% at low and high pesticide levels respectively, by shifting diatom community structure from dominance by lipid-rich species toward dominance by an atrazine-tolerant, but lipid-poor, species. Context-dependent effects did not affect individual diatom lipid content at low levels of both stressors, but caused diatoms to lose 40% of their lipids at high copper stress. Stress-induced changes in diet quality predicted the energy flow from the diatoms to their copepod consumers, which lost half of their lipids when feeding on diatoms grown under low and high pesticide and high metal stress. Selective pesticide effects were a more important threat for trophic energy transfer than context-dependent effects of both stressors, with shifts in diatom community structure affecting the energy flow to their copepod grazers at stress levels where no changes in diatom lipid content were detected.

Acute Modulation of Mycobacterial Cell Envelope Biogenesis by Front-Line Tuberculosis Drugs.

Front-line tuberculosis (TB) drugs have been characterized extensively in vitro and in vivo with respect to gene expression and cell viability. However, little work has been devoted to understanding their effects on the physiology of the cell envelope, one of the main targets of this clinical regimen. Herein, we use metabolic labeling methods to visualize the effects of TB drugs on cell envelope dynamics in mycobacterial species. We developed a new fluorophore-trehalose conjugate to visualize trehalose monomycolates of the mycomembrane using super-resolution microscopy. We also probed the relationship between mycomembrane and peptidoglycan dynamics using a dual metabolic labeling strategy. Finally, we found that metabolic labeling of both cell envelope structures reports on drug effects on cell physiology in two hours, far faster than a genetic sensor of cell envelope stress. Our work provides insight into acute drug effects on cell envelope biogenesis in live mycobacteria.

Visualization of mycobacterial membrane dynamics in live cells.

Mycobacteria are endowed with a highly impermeable mycomembrane that confers intrinsic resistance to many antibiotics. Several unique mycomembrane glycolipids have been isolated and structurally characterized, but the underlying organization and dynamics of glycolipids within the cell envelope remain poorly understood. We report here a study of mycomembrane dynamics that was enabled by trehalose-fluorophore conjugates capable of labeling trehalose glycolipids in live actinomycetes. We identified fluorescein-trehalose analogues that are metabolically incorporated into the trehalose mycolates of representative Mycobacterium, Corynebacterium, Nocardia, and Rhodococcus species. Using these probes, we studied the mobilities of labeled glycolipids by time-lapse microscopy and fluorescence recovery after photobleaching experiments and found that mycomembrane fluidity varies widely across species and correlates with mycolic acid structure. Finally, we discovered that treatment of mycobacteria with ethambutol, a front-line tuberculosis (TB) drug, significantly increases mycomembrane fluidity. These findings enhance our understanding of mycobacterial cell envelope structure and dynamics and have implications for development of TB drug cocktails.

New evidence suggesting a dissociated etiology for cribra orbitalia and porotic hyperostosis.

OBJECTIVES: Porotic hyperostosis (PH), characterized by porotic lesions on the cranial vault, and cribra orbitalia (CO), a localized appearance of porotic lesions on the roof of the orbits, are relatively common osteological conditions. Their etiology has been the focus of several studies, and an association with anemia has long been suggested. Anemia often causes bone marrow hypertrophy or hyperplasia, leading to the expansion in trabecular or cranial diploic bone as a result of increased hematopoiesis. Hypertrophy and/or hyperplasia is often coupled with a disruption of the remodeling process of outer cortical bone, cranially and/or postcranially, leading to the externally visible porotic lesions reported in osteological remains. In this article, we investigate whether individuals with CO have increased thickness of the diploë, the common morphological direct effect of increased hematopoiesis, and thus test the relationship between the two conditions, as well as explore the type of anemia that underlie it. METHODS: An analysis of medical CT scans of a worldwide sample of 98 complete, young to middle-aged adult dry skulls from the Duckworth Collection was conducted on male and female cribrotic individuals (n = 23) and noncribrotic individuals (n = 75), all of whom lacked any evidence of porotic lesions on the vault. Measurements of total and partial cranial thickness were obtained by virtual landmark placement, using the Amira 5.4 software; all analyses were conducted in IBM SPSS 21. RESULTS: Cribriotic individuals have significantly thinner diploic bone and thicker outer and inner tables than noncribriotic individuals, contrary to the expected diploic expansion that would result from anemic conditions associated to bone marrow hypertrophy or hyperplasia. Additionally, individuals without CO and those with the condition have distinctive cranial thickness at particular locations across the skull and the severity to which CO is expressed also differentiates between those with mild and those with a moderate to severe form of the condition. CONCLUSIONS: Our results suggest a complex pattern of causality in relation to the pathologies that may lead to the formation of porotic lesions on the vault and the roof of the orbits. A form of anemia may be behind the osteological changes observed in PH and CO, but it is unlikely to be the same type of anemic condition that underlies both types of osteological lesions. We suggest that CO may be associated to anemias that lead to diploic bone hypocellularity and hypoplasia, such as those caused by anemia of chronic disease and, to a lesser extent, of renal failure, aplastic anemia, protein deficiency, and anemia of endocrine disorders, and not those that lead to bone marrow hypercellularity and hyperplasia and potential PH. This leads us to the conclusion that the terms PH and CO should be used to reflect different underlying conditions.

The disruption of prenylation leads to pleiotropic rearrangements in cellular behavior in Staphylococcus aureus.

Prenylation is the addition of prenyl groups to peptide chains or metabolites via the condensation of geranyl- or isopentenyl-diphosphate moieties by geranyltranstransferases. Although this process is extensively studied in eukaryotes, little is known about the influence of prenylation in prokaryotic species. To explore the role of this modification in bacteria, we generated a mutation in the geranyltranstransferase (IspA) of Staphylococcus aureus. Quite strikingly, the ispA mutant completely lacked pigment and exhibited a previously undescribed small colony variant-like phenotype. Further pleiotropic defects in cellular behavior were noted, including impaired growth, decreased ATP production, increased sensitivity to oxidative stress, increased resistance to aminoglycosides and cationic antimicrobial peptides, and decreased resistance to cell wall-targeting antibiotics. These latter effects appear to result from differences in envelope composition as ispA mutants have highly diffuse cell walls (particularly at the septum), marked alterations in fatty acid composition and increased membrane fluidity. Taken together, these data present an important characterization of prokaryotic prenylation and demonstrate that this process is central to a wealth of pathways involved in mediating cellular homeostasis in S. aureus.

Confirming Sterility of an Autoclaved Infected Femoral Component for Use in an Articulated Antibiotic Knee Spacer: A Pilot Study.

Antibiotic spacer designs have proven effective at eradicating infection during a two-stage revision arthroplasty. Temporary reuse of the steam-sterilized femoral component and a new all poly tibia component has been described as an effective articulating antibiotic spacer, but sterility concerns persist. Six explanted cobalt chrome femurs from patients with grossly infected TKA's and six stock femurs inoculated with different bacterial species were confirmed to be bacteria-free after autoclaving under a standard gravity-displacement cycle. The effect of steam sterilization on cobalt chrome fragments contaminated with MRSA biofilm was analyzed microscopically to quantify remaining biofilm. The autoclave significantly reduced the biofilm burden on the cobalt chrome fragments. This study confirmed sterility of the femur after a standard gravity-displacement cycle (132°C, 27 PSIG, 10 minutes).

The membrane protein PrsS mimics σS in protecting Staphylococcus aureus against cell wall-targeting antibiotics and DNA-damaging agents.

Staphylococcus aureus possesses a lone extracytoplasmic function (ECF) sigma factor, σ(S). In Bacillus subtilis, the ECF sigma factor, σ(W), is activated through a proteolytic cascade that begins with cleavage of the RsiW anti-sigma factor by a site-1 protease (S1P), PrsW. We have identified a PrsW homologue in S. aureus (termed PrsS) and explored its role in σ(S) regulation. Herein, we demonstrate that although a cognate σ(S) anti-sigma factor currently remains elusive, prsS phenocopies sigS in a wealth of regards. Specifically, prsS expression mimics the upregulation observed for sigS in response to DNA-damaging agents, cell wall-targeting antibiotics and during ex vivo growth in human serum and murine macrophages. prsS mutants also display the same sensitivities of sigS mutants to the DNA-damaging agents methyl methane sulfonate (MMS) and hydrogen peroxide, and the cell wall-targeting antibiotics ampicillin, bacitracin and penicillin-G. These phenotypes appear to be explained by alterations in abundance of proteins involved in drug resistance (Pbp2a, FemB, HmrA) and the response to DNA damage (BmrA, Hpt, Tag). Our findings seem to be mediated by putative proteolytic activity of PrsS, as site-directed mutagenesis of predicted catalytic residues fails to rescue the sensitivity of the mutant to H2O2 and MMS. Finally, a role for PrsS in S. aureus virulence was identified using human and murine models of infection. Collectively, our data indicate that PrsS and σ(S) function in a similar manner, and perhaps mediate virulence and resistance to DNA damage and cell wall-targeting antibiotics, via a common pathway.

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands.

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.

The lone S41 family C-terminal processing protease in Staphylococcus aureus is localized to the cell wall and contributes to virulence.

Staphylococcus aureus is a versatile pathogen of humans and a continued public health concern due to the rise and spread of multidrug-resistant strains. As part of an ongoing investigation into the pathogenic mechanisms of this organism we previously demonstrated that an intracellular N-terminal processing protease is required for S. aureus virulence. Following on from this, here we examine the role of CtpA, the lone C-terminal processing protease of S. aureus. CtpA, a member of the S41 family, is a serine protease whose homologues in Gram-negative bacteria have been implicated in a range of biological functions, including pathogenesis. We demonstrate that S. aureus CtpA is localized to the bacterial cell wall and expression of the ctpA gene is maximal upon exposure to conditions encountered during infection. Disruption of the ctpA gene leads to decreased heat tolerance and increased sensitivity when exposed to components of the host immune system. Finally we demonstrate that the ctpA(-) mutant strain is attenuated for virulence in a murine model of infection. Our results represent the first characterization of a C-terminal processing protease in a pathogenic Gram-positive bacterium and show that it plays a critical role during infection.

The impact of CodY on virulence determinant production in community-associated methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is a leading human pathogen of both hospital and community-associated diseases worldwide. This organism causes a wealth of infections within the human host as a result of the vast arsenal of toxins encoded within its genome. Previous transcriptomic studies have shown that toxin production in S. aureus can be strongly impacted by the negative regulator CodY. CodY acts by directly, and indirectly (via Agr), repressing toxin production during times of plentiful nutrition. In this study, we use iTRAQ-based proteomics for the first time to study virulence determinant production in S. aureus, so as to correlate transcriptional observations with actual changes in protein synthesis. Using a codY mutant in the epidemic CA-MRSA clone USA300 we demonstrate that deletion of this transcription factor results in a major upregulation of toxin synthesis in both post-exponential and stationary growth. Specifically, we observe hyper-production of secreted proteases, leukocidins and hemolysins in both growth phases in the USA300 codY mutant. Our findings demonstrate the power of mass spectrometry-based quantitative proteomics for studying toxin production in S. aureus, and the importance of CodY to this central process in disease causation and infection.

Chemically Modified Bacterial Sacculi as a Vaccine Microparticle Scaffold.

Vaccine scaffolds and carrier proteins increase the immunogenicity of subunit vaccines. Here, we developed, characterized, and demonstrated the efficacy of a novel microparticle vaccine scaffold comprised of bacterial peptidoglycan (PGN), isolated as an entire sacculi. The PGN microparticles contain bio-orthogonal chemical handles allowing for site-specific attachment of immunogens. We first evaluated the purification, integrity, and immunogenicity of PGN microparticles derived from a variety of bacterial species. We then optimized PGN microparticle modification conditions; Staphylococcus aureus PGN microparticles containing azido-d-alanine yielded robust conjugation to immunogens. We then demonstrated that this vaccine scaffold elicits comparable immunostimulation to the conventional carrier protein, keyhole limpet hemocyanin (KLH). We further modified the S. aureus PGN microparticle to contain the SARS-CoV-2 receptor-binding domain (RBD)─this conjugate vaccine elicited neutralizing antibody titers comparable to those elicited by the KLH-conjugated RBD. Collectively, these findings suggest that chemically modified bacterial PGN microparticles are a conjugatable and biodegradable microparticle scaffold capable of eliciting a robust immune response toward an antigen of interest.

NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus.

Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.