Bacterial Two-component Systems Research Articles

Development of a two component system based biosensor with high sensitivity for the detection of copper ions

Recent advancements in bacterial two-component systems (TCS) have spurred research into TCS-based biosensors, notably for their signal amplification and broad input responsiveness. The CusRS system in Escherichia coli (E. coli), comprising cusS and cusR genes, is a copper-sensing module in E. coli. However, due to insufficient sensing performance, CusRS-based biosensors often cannot meet practical requirements. To address this issue, we made improvements and innovation from several aspects. CusR and CusS expression were adjusted to enhance the Cu(II) biosensor’s performance. A copy-number inducible plasmid was used for signal amplification, while removing copper detox genes cueO and cusCFBA improved sensitivity and lowered detection limits. Ultimately, in the optimized biosensor of Cu26, the fold-change (I/I0) increased from 1.5-fold to 18-fold at 1 μM, rising to 100-fold after optimizing the cell culture procedure. The biosensor’s high fluorescence enabled rapid, instrument-free detection and an improved analysis strategy reduced the detection limit to 0.01 μM, surpassing traditional methods.

Constitutive activation of two-component systems reveals regulatory network interactions in Streptococcus agalactiae

Bacterial two-component systems (TCSs) are signaling modules that control physiology, adaptation, and host interactions. A typical TCS consists of a histidine kinase (HK) that activates a response regulator via phosphorylation in response to environmental signals. Here, we systematically test the effect of inactivating the conserved phosphatase activity of HKs to activate TCS signaling pathways. Transcriptome analyses of 14 HK mutants in Streptococcus agalactiae, the leading cause of neonatal meningitis, validate the conserved HK phosphatase mechanism and its role in the inhibition of TCS activity in vivo. Constitutive TCS activation, independent of environmental signals, enables high-resolution mapping of the regulons for several TCSs (e.g., SaeRS, BceRS, VncRS, DltRS, HK11030, HK02290) and reveals the functional diversity of TCS signaling pathways, ranging from highly specialized to interconnected global regulatory networks. Targeted analysis shows that the SaeRS-regulated PbsP adhesin acts as a signaling molecule to activate CovRS signaling, thereby linking the major regulators of host-pathogen interactions. Furthermore, constitutive BceRS activation reveals drug-independent activity, suggesting a role in cell envelope homeostasis beyond antimicrobial resistance. This study highlights the versatility of constitutive TCS activation, via phosphatase-deficient HKs, to uncover regulatory networks and biological processes.

Targeting New Functions and Applications of Bacterial Two-Component Systems.

Two-component signal transduction systems (TCSs) are regulatory systems widely distributed in eubacteria, archaea, and a few eukaryotic organisms, but not in mammalian cells. A typical TCS consists of a histidine kinase and a response regulator protein. Functional and mechanistic studies on different TCSs have greatly advanced the understanding of cellular phosphotransfer signal transduction mechanisms. In this concept paper, we focus on the His-Asp phosphotransfer mechanism, the ATP synthesis function, antimicrobial drug design, cellular biosensors design, and protein allostery mechanisms based on recent TCS investigations to inspire new applications and future research perspectives.

The succession of microbial community and distribution resistance gene in response to enrichment cultivation derived from a long-term toxic metal(loid)s polluted soil

Microbial communities as the most important and active component of soil play a crucial role in the geochemical cycling of toxic metal(loid)s in the Pb and Zn smelting site soils. However, the relationships between soil microbial communities and the fractions of toxic metal(loid)s and the succession of soil microbial community and functions after enrichment cultivation have rarely been analyzed. In this study, the diversity and composition of microbial communities in soils before and after enrichment cultivation were investigated by high-throughput sequencing. And the co-occurrence relationships between soil microbial community after enrichment cultivation and MRGs genes were also analyzed through the BacMet database. Results showed that the dominant genus in the soils was Lactobacillus and Stenotrophomonas. The soil microbial community exhibited a notable correlation with Cd, Pb, and As, among which Cd exerted the most profound impact. Alishewanella, Pseudomonas, Massilia and Roseibacillus were significantly correlated with the fraction of Cd. After enrichment cultivation, the number of genera decrease to 96. And the dominant genus changed to Acinetobacter, Bacillus, Comamonas, Lysobacter, and Pseudoxanthomonas. High abundance of metal resistance genes (MRGs) including zntA, fpvA, zipB, cadA, czcA, czcB, czcC, zntA, arsR, pstS and pstB was found in the microbial community after enrichment cultivation. The potential host genus for MRGs was Acinetobacter, Comamonas, Lysinibacillus, Azotobacter, Bacillus, Lysobacter, Cupriavidus, Pseudoxanthomonas, and Thermomonas. Additionally, these microbial community after enrichment cultivation possessing pathways of bacterial chemotaxis and two-component systems was enabled them to adapt to the polluted environment. These observations provided potential guidance for microbe isolation and the development of strategies for the bioremediation of toxic metal(loid)s polluted soils.

Photoreception and signaling in bacterial phytochrome revealed by single-particle cryo-EM.

Phytochromes are red-light photoreceptors discovered in plants with homologs in bacteria and fungi that regulate a variety of physiological responses. They display a reversible photocycle between two distinct states: a red-light-absorbing Pr state and a far-red light-absorbing Pfr state. The photoconversion regulates the activity of an enzymatic domain, usually a histidine kinase (HK). The molecular mechanism that explains how light controls the HK activity is not understood because structures of unmodified bacterial phytochromes with HK activity are missing. Here, we report three cryo-electron microscopy structures of a wild-type bacterial phytochrome with HK activity determined as Pr and Pfr homodimers and as a Pr/Pfr heterodimer with individual subunits in distinct states. We propose that the Pr/Pfr heterodimer is a physiologically relevant signal transduction intermediate. Our results offer insight into the molecular mechanism that controls the enzymatic activity of the HK as part of a bacterial two-component system that perceives and transduces light signals.

Unraveling the role of physicochemical differences in predicting protein-protein interactions.

The ability to accurately predict protein-protein interactions is critically important for understanding major cellular processes. However, current experimental and computational approaches for identifying them are technically very challenging and still have limited success. We propose a new computational method for predicting protein-protein interactions using only primary sequence information. It utilizes the concept of physicochemical similarity to determine which interactions will most likely occur. In our approach, the physicochemical features of proteins are extracted using bioinformatics tools for different organisms. Then they are utilized in a machine-learning method to identify successful protein-protein interactions via correlation analysis. It was found that the most important property that correlates most with the protein-protein interactions for all studied organisms is dipeptide amino acid composition (the frequency of specific amino acid pairs in a protein sequence). While current approaches often overlook the specificity of protein-protein interactions with different organisms, our method yields context-specific features that determine protein-protein interactions. The analysis is specifically applied to the bacterial two-component system that includes histidine kinase and transcriptional response regulators, as well as to the barnase-barstar complex, demonstrating the method's versatility across different biological systems. Our approach can be applied to predict protein-protein interactions in any biological system, providing an important tool for investigating complex biological processes' mechanisms.

Repurposing Hsp90 inhibitors as antimicrobials targeting two-component systems identifies compounds leading to loss of bacterial membrane integrity.

The discovery of novel antimicrobials is of paramount importance in tackling the imminent global health crisis of antimicrobial resistance. The discovery of novel antimicrobials with novel mechanisms of actions, e.g., targeting bacterial two-component signaling systems, is crucial to bypass existing resistance mechanisms and stimulate pharmaceutical innovations. Here, we explore the possible repurposing of compounds developed in cancer research as inhibitors of two-component systems and investigate their off-target effects such as bacterial membrane disruption and toxicity. These results highlight compounds that are promising for further development of novel bacterial membrane disruptors and two-component system inhibitors.

Phosphorylation Mechanism Switching in Histidine Kinases Is a Tool for Fast Protein Evolution: Insights From AlphaFold Models.

Histidine kinases (HKs) are a central part of bacterial environmental-sensing two-component systems. They provide their hosts with the ability to respond to a wide range of physical and chemical signals. HKs are multidomain proteins consisting of at least a sensor domain, dimerization and phosphorylation domain (DHp), and a catalytic domain. They work as homodimers and the existence of two different autophosphorylation mechanisms (cis and trans) has been proposed as relevant for pathway specificity. Although several HKs have been intensively studied, a precise sequence-to-structure explanation of why and how either cis or trans phosphorylation occurs is still unavailable nor is there any evolutionary analysis on the subject. In this work, we show that AlphaFold can accurately determine whether an HK dimerizes in a cis or trans structure. By modeling multiple HKs we show that both cis- and trans-acting HKs are common in nature and the switch between mechanisms has happened multiple times in the evolutionary history of the family. We then use AlphaFold modeling to explore the molecular determinants of the phosphorylation mechanism. We conclude that it is the difference in lengths of the helices surrounding the DHp loop that determines the mechanism. We also show that very small changes in these helices can cause a mechanism switch. Despite this, previous evidence shows that for a particular HK the phosphorylation mechanism is conserved. This suggests that the phosphorylation mechanism participates in system specificity and mechanism switching provides these systems with a way to diverge.

Characterization of the transcriptionally active form of dephosphorylated DctD complexed with dephospho-IIAGlc.

Bacterial enhancer-binding proteins (bEBPs) acquire a transcriptionally active state via phosphorylation. However, transcriptional activation by the dephosphorylated form of bEBP has been observed in DctD, which belongs to Group I bEBP. The formation of a complex between dephosphorylated DctD (d-DctD) and dephosphorylated IIAGlc (d-IIAGlc) is a prerequisite for the transcriptional activity of d-DctD. In the present study, characteristics of the transcriptionally active complex composed of d-IIAGlc and phosphorylation-deficient DctD (DctDD57Q) of Vibrio vulnificus were investigated in its multimeric conformation and DNA-binding ability. DctDD57Q formed a homodimer that could not bind to the DNA. In contrast, when DctDD57Q formed a complex with d-IIAGlc in a 1:1 molar ratio, it produced two conformations: dimer and dodecamer of the complex. Only the dodecameric complex exhibited ATP-hydrolyzing activity and DNA-binding affinity. For successful DNA-binding and transcriptional activation by the dodecameric d-IIAGlc/DctDD57Q complex, extended upstream activator sequences were required, which encompass the nucleotide sequences homologous to the known DctD-binding site and additional nucleotides downstream. This is the first report to demonstrate the molecular characteristics of a dephosphorylated bEBP complexed with another protein to form a transcriptionally active dodecameric complex, which has an affinity for a specific DNA-binding sequence.IMPORTANCEResponse regulators belonging to the bacterial two-component regulatory system activate the transcription initiation of their regulons when they are phosphorylated by cognate sensor kinases and oligomerized to the appropriate multimeric states. Recently, it has been shown that a dephosphorylated response regulator, DctD, could activate transcription in a phosphorylation-independent manner in Vibrio vulnificus. The dephosphorylated DctD activated transcription as efficiently as phosphorylated DctD when it formed a complex with dephosphorylated form of IIAGlc, a component of the glucose-phosphotransferase system. Functional mimicry of this complex with the typical form of transcriptionally active phosphorylated DctD led us to study the molecular characteristics of this heterodimeric complex. Through systematic analyses, it was surprisingly determined that a multimer constituted with 12 complexes gained the ability to hydrolyze ATP and recognize specific upstream activator sequences containing a typical inverted-repeat sequence flanked by distinct nucleotides.

Navigating the signaling landscape of Ralstonia solanacearum: a study of bacterial two-component systems.

Ralstonia solanacearum, the bacterium that causes bacterial wilt, is a destructive phytopathogen that can infect over 450 different plant species. Several agriculturally significant crop plants, including eggplant, tomato, pepper, potato, and ginger, are highly susceptible to this plant disease, which has a global impact on crop quality and yield. There is currently no known preventive method that works well for bacterial wilt. Bacteria use two-component systems (TCSs) to sense their environment constantly and react appropriately. This is achieved by an extracellular sensor kinase (SK) capable of sensing a suitable signal and a cytoplasmic response regulator (RR) which gives a downstream response. Moreover, our investigation revealed that R. solanacearum GMI1000 possesses a substantial count of TCSs, specifically comprising 36 RRs and 27 SKs. While TCSs are known targets for various human pathogenic bacteria, such as Salmonella, the role of TCSs in R. solanacearum remains largely unexplored in this context. Notably, numerous inhibitors targeting TCSs have been identified, including GHL (Gyrase, Hsp, and MutL) compounds, Walk inhibitors, and anti-TCS medications like Radicicol. Consequently, the investigation into the involvement of TCSs in virulence and pathogenesis has gained traction; however, further research is imperative to ascertain whether TCSs could potentially supplant conventional anti-wilt therapies. This review delves into the prospective utilization of TCSs as an alternative anti-wilt therapy, focusing on the lethal phytopathogen R. solanacearum.

Antibacterial mechanism of lactobionic acid against Aeromonas salmonicida by transcriptomic analysis and its application in refrigerated grass carp

Aeromonas salmonicida is ubiquitously distributed in aquatic environments and is one of the major pathogens of freshwater aquaculture fish. Recent studies have demonstrated that A. salmonicida plays a crucial role in fish spoilage. In this study, a novel antibacterial agent, lactobionic acid (LBA), was investigated for its antibacterial effect against A. salmonicida. The results showed that the minimum inhibitory concentration (MIC) of LBA was 16 mg/mL. Bacterial cells dropped below the detection limit within 2 h after exposure to 2 MIC of LBA. The cells treated with LBA became rough and sunken, and LBA caused cellular protein leakage and an increase in conductivity. Transcriptomic analysis exhibited that the expression of 1273 genes in A. salmonicida was altered by LBA, including 700 down-regulated and 573 up-regulated genes. The most representative down-regulated genes were enriched in bacterial chemotaxis, two-component system and ATP-binding cassette transporter pathways. LBA showed an effective inhibitory effect on A. salmonicida in refrigerated grass carp and could prolong the storage time of grass carp. Overall, the intervention of LBA resulted in the decrease of cell chemotaxis and disturbance of cellular metabolic processes, which inhibited A. salmonicida. These results revealed the antibacterial mechanism of LBA against A. salmonicida, which provided a strategy for the control of fish spoilage.

GraS signaling in Staphylococcus aureus is regulated by a single D35 residue in the extracellular loop.

Bacterial two-component systems are crucial features of bacterial pathogens such as methicillin-resistant Staphylococcus aureus to overcome environmental and antimicrobial stresses by activating regulons to interfere with the bactericidal mechanisms. GraRS is a unique subset of two-component systems belonging to the intramembrane-sensing histidine kinase family (IM-HK) and is responsible for resistance to cationic host defense peptides. However, the precise manner by which the short 9-residue extracellular loop of the membrane sensor GraS detects the antimicrobial peptides and transduces the signal is not comprehensively understood. Here, we show that a single point mutation (D35A) in the extracellular loop of GraS blocked activation of GraRS, but this effect was also abrogated with graS mutations in the N-terminal transmembrane segments without any accompanying effect on GraS protein expression. Additionally, mutations in H120 and T172 in the dimerization/histidine phosphotransfer (DHp) domain of GraS increased activation without any accompanying enhancement in dimerization, likely due to disruption of the H120-T172 interaction that restricts rotational movements of the DHp helices since swapping H120 and T172 did not alter GraS activation. Notably, the enhancing effects of H120 and T172 mutations were abolished with a D35 mutation, highlighting the pivotal role of D35 in the 9-residue extracellular loop of GraS in GraR phosphorylation. In summary, our study delivers the significance of the D35 in the extracellular loop of GraS and ensuing changes in the N-terminal transmembrane helices as a model to illustrate signaling in the IM-HK subset of two-component regulatory systems. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen capable of infecting skin, blood, internal organs, and artificial medical devices. Generally, personal hygiene and a robust immune system can limit the spread of this pathogen; however, MRSA possesses an assortment of phenotypic tools to survive the hostile host environment including host defense peptides. More specifically, S. aureus utilizes two-component systems to sense noxious environmental cues to respond to harmful environmental elements. Our study focused on a two-component system called GraRS that S. aureus deploys against host defense peptides. We showed that one single residue in the extracellular loop of GraS and the adjacent membrane segment controlled the activation of GraRS, indicating the importance of a well-tuned-charged residue in the extracellular loop of GraS for sensing activity.

Metal Messengers: Communication in the Bacterial World through Transition-Metal-Sensing Two-Component Systems.

Bacteria survive in highly dynamic and complex environments due, in part, to the presence of systems that allow the rapid control of gene expression in the presence of changing environmental stimuli. The crosstalk between intra- and extracellular bacterial environments is often facilitated by two-component signal transduction systems that are typically composed of a transmembrane histidine kinase and a cytosolic response regulator. Sensor histidine kinases and response regulators work in tandem with their modular domains containing highly conserved structural features to control a diverse array of genes that respond to changing environments. Bacterial two-component systems are widespread and play crucial roles in many important processes, such as motility, virulence, chemotaxis, and even transition metal homeostasis. Transition metals are essential for normal prokaryotic physiological processes, and the presence of these metal ions may also influence pathogenic virulence if their levels are appropriately controlled. To do so, bacteria use transition-metal-sensing two-component systems that bind and respond to rapid fluctuations in extracytosolic concentrations of transition metals. This perspective summarizes the structural and metal-binding features of bacterial transition-metal-sensing two-component systems and places a special emphasis on understanding how these systems are used by pathogens to establish infection in host cells and how these systems may be targeted for future therapeutic developments.

Sequestration of histidine kinases by non-cognate response regulators establishes a threshold level of stimulation for bacterial two-component signaling

Bacterial two-component systems (TCSs) consist of a sensor histidine kinase (HK) that perceives a specific signal, and a cognate response regulator (RR) that modulates the expression of target genes. Positive autoregulation improves TCS sensitivity to stimuli, but may trigger disproportionately large responses to weak signals, compromising bacterial fitness. Here, we combine experiments and mathematical modelling to reveal a general design that prevents such disproportionate responses: phosphorylated HKs (HK~Ps) can be sequestered by non-cognate RRs. We study five TCSs of Mycobacterium tuberculosis and find, for all of them, non-cognate RRs that show higher affinity than cognate RRs for HK~Ps. Indeed, in vitro assays show that HK~Ps preferentially bind higher affinity non-cognate RRs and get sequestered. Mathematical modelling indicates that this sequestration would introduce a ‘threshold’ stimulus strength for eliciting responses, thereby preventing responses to weak signals. Finally, we construct tunable expression systems in Mycobacterium bovis BCG to show that higher affinity non-cognate RRs suppress responses in vivo.

Two-Component System Sensor Kinases from Asgardian Archaea May Be Witnesses to Eukaryotic Cell Evolution

The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms.

NH125 Sensitizes Staphylococcus aureus to Cell Wall-Targeting Antibiotics through the Inhibition of the VraS Sensor Histidine Kinase.

Staphylococcus aureus utilizes the two-component regulatory system VraSR to receive and relay environmental stress signals, and it is implicated in the development of bacterial resistance to several antibiotics through the upregulation of cell wall synthesis. VraS inhibition was shown to extend or restore the efficacy of several clinically used antibiotics. In this work, we study the enzymatic activity of the VraS intracellular domain (GST-VraS) to determine the kinetic parameters of the ATPase reaction and characterize the inhibition of NH125 under in vitro and microbiological settings. The rate of the autophosphorylation reaction was determined at different GST-VraS concentrations (0.95 to 9.49 μM) and temperatures (22 to 40°C) as well as in the presence of different divalent cations. The activity and inhibition by NH125, which is a known kinase inhibitor, were assessed in the presence and absence of the binding partner, VraR. The effects of inhibition on the bacterial growth kinetics and gene expression levels were determined. The GST-VraS rate of autophosphorylation increases with temperature and with the addition of VraR, with magnesium being the preferred divalent cation for the metal-ATP substrate complex. The mechanism of inhibition of NH125 was noncompetitive in nature and was attenuated in the presence of VraR. The addition of NH125 in the presence of sublethal doses of the cell wall-targeting antibiotics carbenicillin and vancomycin led to the complete abrogation of Staphylococcus aureus Newman strain growth and significantly decreased the gene expression levels of pbpB, blaZ, and vraSR in the presence of the antibiotics. IMPORTANCE This work characterizes the activity and inhibition of VraS, which is a key histidine kinase in a bacterial two-component system that is involved in Staphylococcus aureus antibiotic resistance. The results show the effect of temperature, divalent ions, and VraR on the activity and the kinetic parameters of ATP binding. The value of the KM of ATP is vital in designing screening assays to discover potent and effective VraS inhibitors with high translational potential. We report the ability of NH125 to inhibit VraS in vitro in a noncompetitive manner and investigate its effect on gene expression and bacterial growth kinetics in the presence and absence of cell wall-targeting antibiotics. NH125 effectively potentiated the effects of the antibiotics on bacterial growth and altered the expression of the genes that are regulated by VraS and are involved in mounting a resistance to antibiotics.

Engineering transmembrane signal transduction in synthetic membranes using two-component systems

Cells use signal transduction across their membranes to sense and respond to a wide array of chemical and physical signals. Creating synthetic systems which can harness cellular signaling modalities promises to provide a powerful platform for biosensing and therapeutic applications. As a first step toward this goal, we investigated how bacterial two-component systems (TCSs) can be leveraged to enable transmembrane-signaling with synthetic membranes. Specifically, we demonstrate that a bacterial two-component nitrate-sensing system (NarX-NarL) can be reproduced outside of a cell using synthetic membranes and cell-free protein expression systems. We find that performance and sensitivity of the TCS can be tuned by altering the biophysical properties of the membrane in which the histidine kinase (NarX) is integrated. Through protein engineering efforts, we modify the sensing domain of NarX to generate sensors capable of detecting an array of ligands. Finally, we demonstrate that these systems can sense ligands in relevant sample environments. By leveraging membrane and protein design, this work helps reveal how transmembrane sensing can be recapitulated outside of the cell, adding to the arsenal of deployable cell-free systems primed for real world biosensing.

Two-Component System Response Regulator ompR Regulates Mussel Settlement through Exopolysaccharides.

The outer membrane protein (OMP) is a kind of biofilm matrix component that widely exists in Gram-negative bacteria. However, the mechanism of OMP involved in the settlement of molluscs is still unclear. In this study, the mussel Mytilus coruscus was selected as a model to explore the function of ompR, a two-component system response regulator, on Pseudoalteromonas marina biofilm-forming capacity and the mussel settlement. The motility of the ΔompR strain was increased, the biofilm-forming capacity was decreased, and the inducing activity of the ΔompR biofilms in plantigrades decreased significantly (p < 0.05). The extracellular α-polysaccharide and β-polysaccharide of the ΔompR strain decreased by 57.27% and 62.63%, respectively. The inactivation of the ompR gene decreased the ompW gene expression and had no impact on envZ expression or c-di-GMP levels. Adding recombinant OmpW protein caused the recovery of biofilm-inducing activities, accompanied by the upregulation of exopolysaccharides. The findings deepen the understanding of the regulatory mechanism of bacterial two-component systems and the settlement of benthic animals.

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.

Molecular insights into Escherichia coli Cpx envelope stress response activation by the sensor lipoprotein NlpE.

Bacterial two-component signal transduction systems provide sensory inputs for appropriately adapting gene expression. These systems rely on a histidine kinase that phosphorylates a response regulator which alters gene expression. Several two-component systems include additional sensory components that can activate the histidine kinase. In Escherichia coli, the lipoprotein NlpE was identified as a sensor for the Cpx cell envelope stress response. It has remained unclear how NlpE signals to Cpx in the periplasm. In this study, we used a combination of genetics, biochemistry, and AlphaFold2 complex modeling to uncover the molecular details of how NlpE triggers the Cpx response through an interaction with the CpxA histidine kinase. Remarkably, only a short loop of NlpE is required to activate the Cpx response. A single substitution in this loop inactivates NlpE signaling to Cpx and abolishes an in vivo biochemical NlpE:CpxA interaction. An independent AlphaFold multimer prediction supported a role for the loop and predicted an interaction interface at CpxA. Mutations in this CpxA region specifically blind the histidine kinase to NlpE activation but preserve the ability to respond to other cell envelope stressors. Hence, our work additionally reveals a previously unrecognized complexity in signal integration by the CpxA periplasmic sensor domain.