Synthesis and biochemical characterization of naphthoquinone derivatives targeting bacterial histidine kinases.

Waldiomycin is an inhibitor of histidine kinases (HKs). Although most HK inhibitors target the ATP-binding region, waldiomycin binds to the intracellular dimerization domain (DHp domain) with its naphthoquinone moiety presumed to interact with the conserved H-box region. To further develop inhibitors targeting the H-box, various 2-aminonaphthoquinones with cyclic, aliphatic, or aromatic amino groups and naphtho [2,3-d] isoxazole-4,9-diones were synthesized. These compounds were tested for their inhibitory activity (IC50) against WalK, an essential HK for Bacillus subtilis growth, and their minimum inhibitory concentrations (MIC) against B. subtilis. As a result, 11 novel HK inhibitors were obtained as naphthoquinone derivatives (IC50: 12.6-305 µM, MIC: 0.5-128 µg ml-1). The effect of representative compounds on the expression of WalK/WalR regulated genes in B. subtilis was investigated. Four naphthoquinone derivatives induced the expression of iseA (formerly yoeB), whose expression is negatively regulated by the WalK/WalR system. This suggests that these compounds inhibit WalK in B. subtilis cells, resulting in antibacterial activity. Affinity selection/mass spectrometry analysis was performed to identify whether these naphthoquinone derivatives interact with WalK in a manner similar to waldiomycin. Three compounds were found to competitively inhibit the binding of waldiomycin to WalK, suggesting that they bind to the H-box region conserved in HKs and inhibit HK activity.

Roles of Two-Component Signal Transduction Systems in Shigella Virulence.

Two-component signal transduction systems (TCSs) are widespread types of protein machinery, typically consisting of a histidine kinase membrane sensor and a cytoplasmic transcriptional regulator that can sense and respond to environmental signals. TCSs are responsible for modulating genes involved in a multitude of bacterial functions, including cell division, motility, differentiation, biofilm formation, antibiotic resistance, and virulence. Pathogenic bacteria exploit the capabilities of TCSs to reprogram gene expression according to the different niches they encounter during host infection. This review focuses on the role of TCSs in regulating the virulence phenotype of Shigella, an intracellular pathogen responsible for severe human enteric syndrome. The pathogenicity of Shigella is the result of the complex action of a wide number of virulence determinants located on the chromosome and on a large virulence plasmid. In particular, we will discuss how five TCSs, EnvZ/OmpR, CpxA/CpxR, ArcB/ArcA, PhoQ/PhoP, and EvgS/EvgA, contribute to linking environmental stimuli to the expression of genes related to virulence and fitness within the host. Considering the relevance of TCSs in the expression of virulence in pathogenic bacteria, the identification of drugs that inhibit TCS function may represent a promising approach to combat bacterial infections.

Host - Bacterial Pathogen Communication: The Wily Role of the Multidrug Efflux Pumps of the MFS Family.

Bacterial pathogens are able to survive within diverse habitats. The dynamic adaptation to the surroundings depends on their ability to sense environmental variations and to respond in an appropriate manner. This involves, among others, the activation of various cell-to-cell communication strategies. The capability of the bacterial cells to rapidly and co-ordinately set up an interplay with the host cells and/or with other bacteria facilitates their survival in the new niche. Efflux pumps are ubiquitous transmembrane transporters, able to extrude a large set of different molecules. They are strongly implicated in antibiotic resistance since they are able to efficiently expel most of the clinically relevant antibiotics from the bacterial cytoplasm. Besides antibiotic resistance, multidrug efflux pumps take part in several important processes of bacterial cell physiology, including cell to cell communication, and contribute to increase the virulence potential of several bacterial pathogens. Here, we focus on the structural and functional role of multidrug efflux pumps belonging to the Major Facilitator Superfamily (MFS), the largest family of transporters, highlighting their involvement in the colonization of host cells, in virulence and in biofilm formation. We will offer an overview on how MFS multidrug transporters contribute to bacterial survival, adaptation and pathogenicity through the export of diverse molecules. This will be done by presenting the functions of several relevant MFS multidrug efflux pumps in human life-threatening bacterial pathogens as Staphylococcus aureus, Listeria monocytogenes, Klebsiella pneumoniae, Shigella/E. coli, Acinetobacter baumannii.

Acid-Sensing Histidine Kinase With a Redox Switch.

The EvgS/EvgA two-component signal transduction system in Escherichia coli is activated under mildly acidic pH conditions. Upon activation, this system induces the expression of a number of genes that confer acid resistance. The EvgS histidine kinase sensor has a large periplasmic domain that is required for perceiving acidic signals. In addition, we have previously proposed that the cytoplasmic linker region of EvgS is also involved in the activation of this sensor. The cytoplasmic linker region resembles a Per-ARNT-Sim (PAS) domain, which is known to act as a molecular sensor that is responsive to chemical and physical stimuli and regulates the activity of diverse effector domains. Our EvgS/EvgA reporter assays revealed that under EvgS-activating mildly acidic pH conditions, EvgS was activated only during aerobic growth conditions, and not during anaerobic growth. Studies using EvgS mutants revealed that C671A and C683A mutations in the cytoplasmic PAS domain activated EvgS even under anaerobic conditions. Furthermore, among the electron carriers of the electron transport chain, ubiquinone was required for EvgS activation. The present study proposes a model of EvgS activation by oxidation and suggests that the cytoplasmic PAS domain serves as an intermediate redox switch for this sensor.

An immuno-dot blot assay for screening histidine kinase inhibitors.

Two-component signal transduction systems (TCSs), consisting of a histidine kinase (HK) and its cognate response regulator, are ubiquitous among bacteria and are associated with the virulence of pathogens. TCSs are potential targets for alternative antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in bacterial TCSs. Here, we describe an immuno-dot blot assay for the inhibition profiling of HKs using the anti-N3-phosphohistidine antibody. This simple method promises reliable detection of HK activity, and it is likely applicable in high-throughput screening of HK inhibitors.

Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos-tag Magenta/Cyan fluorescent dyes.

In the bacterial signaling mechanisms known as two-component systems (TCSs), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos-tag dye technology is suitable for the fluorescent detection of His- and Asp-phosphorylated proteins separated by SDS-PAGE. The dynamics of the His-Asp phosphorelay of recombinant EnvZ-OmpR, a TCS derived from Escherichia coli, were examined by SDS-PAGE followed by simple rapid staining with Phos-tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1 min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag Cyan gel staining. We believe that the Phos-tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.

Identification of an internal cavity in the PhoQ sensor domain for PhoQ activity and SafA-mediated control.

The PhoQ/PhoP two-component signal transduction system is conserved in various Gram-negative bacteria and is often involved in the expression of virulence in pathogens. The small inner membrane protein SafA activates PhoQ in Escherichia coli independently from other known signals that control PhoQ activity. We have previously shown that SafA directly interacts with the sensor domain of the periplasmic region of PhoQ (PhoQ-SD) for activation, and that a D179R mutation in PhoQ-SD attenuates PhoQ activation by SafA. In this study, structural comparison of wild-type PhoQ-SD and D179R revealed a difference in the cavity (SD (sensory domain) pocket) found in the central core of this domain. This was the only structural difference between the two proteins. Site-directed mutagenesis of the residues surrounding the SD pocket has supported the SD pocket as a site involved in PhoQ activity. Furthermore, the SD pocket has also been shown to be involved in SafA-mediated PhoQ control.

Essentiality of WalRK for growth in Bacillus subtilis and its role during heat stress.

WalRK is an essential two-component signal transduction system that plays a central role in coordinating cell wall synthesis and cell growth in Bacillus subtilis. However, the physiological role of WalRK and its essentiality for growth have not been elucidated. We investigated the behaviour of WalRK during heat stress and its essentiality for cell proliferation. We determined that the inactivation of the walHI genes which encode the negative modulator of WalK, resulted in growth defects and eventual cell lysis at high temperatures. Screening of suppressor mutations revealed that the inactivation of LytE, an dl-endopeptidase, restored the growth of the ΔwalHI mutant at high temperatures. Suppressor mutations that reduced heat induction arising from the walRK regulon were also mapped to the walK ORF. Therefore, we hypothesized that overactivation of LytE affects the phenotype of the ΔwalHI mutant. This hypothesis was corroborated by the overexpression of the negative regulator of LytE, IseA and PdaC, which rescued the growth of the ΔwalHI mutant at high temperatures. Elucidating the cause of the temperature sensitivity of the ΔwalHI mutant could explain the essentiality of WalRK. We proved that the constitutive expression of lytE or cwlO using a synthetic promoter uncouples these expressions from WalRK, and renders WalRK nonessential in the pdaC and iseA mutant backgrounds. We propose that the essentiality of WalRK is derived from the coordination of cell wall metabolism with cell growth by regulating dl-endopeptidase activity under various growth conditions.

Characterization of H-box region mutants of WalK inert to the action of waldiomycin in Bacillus subtilis.

The WalK/WalR two-component system is essential for cell wall metabolism and thus for cell growth in Bacillus subtilis. Waldiomycin was previously isolated as an antibiotic that targeted WalK, the cognate histidine kinase (HK) of the response regulator, WalR, in B. subtilis. To gain further insights into the action of waldiomycin on WalK and narrow down its site of action, mutations were introduced in the H-box region, a well-conserved motif of the bacterial HKs of WalK. The half-maximal inhibitory concentrations (IC50s) of waldiomycin against purified WalK protein with triple substitutions in the H-box region, R377M/R378M/S385A and R377M/R378M/R389M, were 26.4 and 55.1 times higher than that of the wild-type protein, respectively, indicating that these residues of WalK are crucial for the inhibitory effect of waldiomycin on its kinase activity. Surprisingly, this antibiotic severely affected cell growth in a minimum inhibitory concentration (MIC) assay, but not transcription of WalR-regulated genes or cell morphology in B. subtilis strains that harbored the H-box triple substitutions on the bacterial chromosome. We hypothesized that waldiomycin targets other HKs as well, which may, in turn, sensitize B. subtilis cells with the H-box triple mutant alleles of the walK gene to waldiomycin. Waldiomycin inhibited other HKs such as PhoR and ResE, and, to a lesser extent, CitS, whose H-box region is less conserved. These results suggest that waldiomycin perturbs multiple cellular processes in B. subtilis by targeting the H-box region of WalK and other HKs.

Bacterial signal transduction networks via connectors and development of the inhibitors as alternative antibiotics.

Bacterial cells possess a signal transduction system that differs from those described in higher organisms, including human cells. These so-called two-component signal transduction systems (TCSs) consist of a sensor (histidine kinase, HK) and a response regulator, and are involved in cellular functions, such as virulence, drug resistance, biofilm formation, cell wall synthesis, cell division. They are conserved in bacteria across all species. Although TCSs are often studied and characterized individually, they are assumed to interact with each other and form signal transduction networks within the cell. In this review, I focus on the formation of TCS networks via connectors. I also explore the possibility of using TCS inhibitors, especially HK inhibitors, as alternative antimicrobial agents.

Angucycline antibiotic waldiomycin recognizes common structural motif conserved in bacterial histidine kinases.

Two-component signal transduction systems (TCSs), composed of a histidine kinase sensor (HK) and its cognate response regulator, sense and respond to environmental changes and are related to the virulence of pathogens. TCSs are potential targets for alternative antibiotics and anti-virulence agents. Here we found that waldiomycin, an angucycline antibiotic that inhibits a growth essential HK, WalK, in Gram-positive bacteria, also inhibits several class I HKs from the Gram-negative Escherichia coli. NMR analyses and site-directed mutagenesis studies using the osmo-sensing EnvZ, a prototypical HK of E. coli, showed that waldiomycin directly binds to both H-box and X-region, which are the two conserved regions in the dimerization-inducing and histidine-containing phosphotransfer (DHp) domain of HKs. Waldiomycin inhibits phosphorylation of the conserved histidine in the H-box. Analysis of waldiomycin derivatives suggests that the angucyclic ring, situated near the H-box in the waldiomycin-EnvZ DHp domain complex model, is responsible for the inhibitory activity. We demonstrate that waldiomycin is an HK inhibitor binding to the H-box region and has the potential of inhibiting a broad spectrum of HKs.

Fe3+-dependent epistasis between the CpxR-activated loci and the PmrA-activated LPS modification loci in Salmonella enterica.

Bacteria utilize varying combinations of two-component regulatory systems, many of which respond and adapt closely to stress conditions, thus expanding their niche steadily. While mechanisms of recognition and avoidance of the specific Fe3+ signal by the PmrA/PmrB system is well understood, those of the CpxR/CpxA system are more complex because they can be induced by various stress conditions, which, in turn, expresses a variety of phenotypes. Here, we highlight another aspect of the CpxR/CpxA system; mutations in degP and yqjA genes, which are under the control of the system, exhibit an iron sensitive phenotype in the mutant background defective in the PmrA-dependent gene products that alter the pyrophosphate status of the lipid A moiety of lipopolysaccharide in Salmonella enterica. Therefore, after the PmrA/PmrB-mediated Fe3+-dependent control of the pyrophosphate status on the cell surface, the CpxR/CpxA system is one of the second layers of envelope stress response that allows adaptation to high Fe3+ conditions in this bacterium.

Identification of the Three Genes Involved in Controlling Production of a Phytotoxin Tropolone in Burkholderia plantarii.

UNLABELLED: Tropolone, a phytotoxin produced by Burkholderia plantarii, causes rice seedling blight. To identify genes involved in tropolone synthesis, we systematically constructed mutations in the genes encoding 55 histidine kinases and 72 response regulators. From the resulting defective strains, we isolated three mutants, KE1, KE2, and KE3, in which tropolone production was repressed. The deleted genes of these mutants were named troR1, troK, and troR2, respectively. The mutant strains did not cause rice seedling blight, and complementation experiments indicated that TroR1, TroK, and TroR2 were involved in the synthesis of tropolone in B. plantarii However, tropolone synthesis was repressed in the TroR1 D52A, TroK H253A, and TroR2 D46A site-directed mutants. These results suggest that the putative sensor kinase (TroK) and two response regulators (TroR1 and TroR2) control the production of tropolone in B. plantarii IMPORTANCE: A two-component system is normally composed of a sensor histidine kinase (HK) and a cognate response regulator (RR) pair. In this study, HK (TroK) and two RRs (TroR1 and TroR2) were found to be involved in controlling tropolone production in B. plantarii These three genes may be part of a bacterial signal transduction network. Such networks are thought to exist in other bacteria to regulate phytotoxin production, as well as environmental adaptation and signal transduction.

Study on in vivo effects of bacterial histidine kinase inhibitor, Waldiomycin, in Bacillus subtilis and Staphylococcus aureus.

Two-component signal transduction systems (TCSs) represent one of the primary means by which bacteria sense and respond to changes in their environment, both intra- and extracellular. The highly conserved WalK (histidine kinase)/WalR (response regulator) TCS is essential for cell wall metabolism of low G+C Gram-positive bacteria and acts as a master regulatory system in controlling and coordinating cell wall metabolism with cell division. Waldiomycin, a WalK inhibitor, has been discovered by screening metabolites from actinomycetes and belongs to the family of angucycline antibiotics. In the present study, we have shown that waldiomycin inhibited autophosphorylation of WalK histidine kinases in vitro from Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans at half-maximal inhibitory concentrations of 10.2, 8.8, 9.2, and 25.8 µM, respectively. Quantitative RT-PCR studies of WalR regulon genes have suggested that waldiomycin repressed the WalK/WalR system in B. subtilis and S. aureus cells. Morphology of waldiomycin-treated S. aureus cells displayed increased aggregation instead of proper cellular dissemination. Furthermore, autolysis profiles of S. aureus cells revealed that waldiomycin-treated cells were highly resistant to Triton X-100- and lysostaphin-induced lysis. These phenotypes are consistent with those of cells starved for the WalK/WalR system, indicating that waldiomycin inhibited the autophosphorylation activity of WalK in cells. We have also confirmed that waldiomycin inhibits WalK autophosphorylation in vivo by actually observing the phosphorylated WalK ratio in cells using Phos-tag SDS-PAGE. The results of our current study strongly suggest that waldiomycin targets WalK histidine kinases and inhibits the WalR regulon genes expression, thereby affecting both cell wall metabolism and cell division.

Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases.

Hybrid sensor kinase, which contains a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, conveys signals to its cognate response regulator by means of a His-Asp-His-Asp phosphorelay. We examined the multistep phosphorelay of a recombinant EvgAS system in Escherichia coli and performed in vitro quantitative analyses of phosphorylation by using Phos-tag SDS-PAGE. Replacement of Asp in the receiver domain of EvgS by Ala markedly promoted phosphorylation at His in the HK domain compared with that in wild-type EvgS. Similar Ala-substituted mutants of other hybrid sensor kinases BarA and ArcB showed similar characteristics. In the presence of sufficient ATP, autophosphorylation of the HK domain in the mutant progressed efficiently with nearly pseudo-first-order kinetics until the phosphorylation ratio reached a plateau value of more than 95% within 60 min, and the value was maintained until 180 min. However, both wild-type EvgS and the Ala-substituted mutant of His in the HPt domain showed a phosphorylation ratio of less than 25%, which gradually decreased after 10 min. These results showed that the phosphorylation level is regulated negatively by the receiver domain. Furthermore, our in vivo assays confirmed the existence of a similar hyperphosphorylation reaction in the HK domain of the EvgS mutant in which the Asp residue was replaced with Ala, confirming the validity of the control mechanism proposed from profiling of phosphorylation in vitro [corrected].

Alkali metals in addition to acidic pH activate the EvgS histidine kinase sensor in Escherichia coli.

Two-component signal transduction systems (TCSs) in bacteria perceive environmental stress and transmit the information via phosphorelay to adjust multiple cellular functions for adaptation. The EvgS/EvgA system is a TCS that confers acid resistance to Escherichia coli cells. Activation of the EvgS sensor initiates a cascade of transcription factors, EvgA, YdeO, and GadE, which induce the expression of a large group of acid resistance genes. We searched for signals activating EvgS and found that a high concentration of alkali metals (Na(+), K(+)) in addition to low pH was essential for the activation. EvgS is a histidine kinase, with a large periplasmic sensor region consisting of two tandem PBPb (bacterial periplasmic solute-binding protein) domains at its N terminus. The periplasmic sensor region of EvgS was necessary for EvgS activation, and Leu152, located within the first PBPb domain, was involved in the activation. Furthermore, chimeras of EvgS and PhoQ histidine kinases suggested that alkali metals were perceived at the periplasmic sensor region, whereas the cytoplasmic linker domain, connecting the transmembrane region and the histidine kinase domain, was required for low-pH perception.

Waldiomycin, a novel WalK-histidine kinase inhibitor from Streptomyces sp. MK844-mF10.

WalK, a histidine kinase, and WalR, a response regulator, make up a two-component signal transduction system that is indispensable for the cell-wall metabolism of low GC Gram-positive bacteria. WalK inhibitors are likely to show bactericidal effects against methicillin-resistant Staphylococcus aureus . We discovered a new WalK inhibitor, designated waldiomycin, by screening metabolites from actinomycetes. Waldiomycin belongs to the family of angucycline antibiotics and is structurally related to dioxamycin. Waldiomycin inhibits WalK from S. aureus and Bacillus subtilis at IC50s 8.8 and 10.2 µM, respectively, and shows antibacterial activity with MICs ranging from 4 to 8 µg ml(-1) against methicillin-resistant S. aureus and B. subtilis.

Mechanism of activation of PhoQ/PhoP two-component signal transduction by SafA, an auxiliary protein of PhoQ histidine kinase in Escherichia coli.

The PhoQ/PhoP two-component signal transduction system in Escherichia coli is activated by SafA, a small membrane protein that modifies the PhoQ histidine kinase. The SafA C-terminal domain (41-65 aa) interacts directly with the sensory domain of PhoQ at the periplasm. We used in vitro and in vivo strategies to elucidate the way SafA modifies the PhoQ/PhoP phosphorelay system. First, the enzymatic activities of membranes from cells overexpressing PhoQ and cells expressing both PhoQ and SafA were compared in vitro. Increased autophosphorylation of PhoQ was observed in the presence of SafA, but it did not increase the dephosphorylation of phospho-PhoP by PhoQ. In addition, SafA increased the phospho-PhoP level on the phosphotransfer assay. We confirmed that induction of SafA results in an accumulation of phospho-PhoP in vivo by the Phos-tag system. Our results suggest that the accumulation of phospho-PhoP is linked to activation of PhoQ autophosphorylation by SafA.

A connecter-like factor, CacA, links RssB/RpoS and the CpxR/CpxA two-component system in Salmonella.

BACKGROUND: Bacteria integrate numerous environmental stimuli when generating cellular responses. Increasing numbers of examples describe how one two-component system (TCS) responds to signals detected by the sensor of another TCS. However, the molecular mechanisms underlying this phenomenon remain poorly defined. RESULTS: Here, we report a connector-like factor that affects the activity of the CpxR/CpxA two-component system in Salmonella enterica serovar Typhimurium. We isolated a clone that induced the expression of a cpxP-lac gene fusion from a high-copy-number plasmid pool of random Salmonella genomic fragments. A 63-amino acid protein, CacA, was responsible for the CpxA/CpxR-dependent activation of the cpxP gene. The CpxR-activated genes cpxP and spy exhibited approximately 30% and 50% reductions in transcription, respectively, in a clean cacA deletion mutant strain in comparison to wild-type. From 33 response regulator (RR) deletion mutants, we identified that the RssB regulator represses cacA transcription. Substitution mutations in a conserved -10 region harboring the RNA polymerase recognition sequence, which is well conserved with a known RpoS -10 region consensus sequence, rendered the cacA promoter RpoS-independent. The CacA-mediated induction of cpxP transcription was affected in a trxA deletion mutant, which encodes thioredoxin 1, suggesting a role for cysteine thiol-disulfide exchange(s) in CacA-dependent Cpx activation. CONCLUSIONS: We identified CacA as an activator of the CpxR/CpxA system in the plasmid clone. We propose that CacA may integrate the regulatory status of RssB/RpoS into the CpxR/CpxA system. Future investigations are necessary to thoroughly elucidate how CacA activates the CpxR/CpxA system.

The connector SafA interacts with the multi-sensing domain of PhoQ in Escherichia coli.

Sensor histidine kinases of two-component signal transduction systems (TCSs) respond to various environmental signals and transduce the external stimuli across the cell membrane to their cognate response regulators. Recently, membrane proteins that modulate sensory systems have been discovered. Among such proteins is SafA, which activates the PhoQ/PhoP TCS by direct interaction with the sensor PhoQ. SafA is directly induced by the EvgS/EvgA TCS, thus connecting the two TCSs, EvgS/EvgA and PhoQ/PhoP. We investigated how SafA interacted with PhoQ. Bacterial two-hybrid and reporter assays revealed that the C-terminal region (41-65 aa) of SafA activated PhoQ at the periplasm. Adding synthetic SafA(41-65) peptide to the cell culture also activated PhoQ/PhoP. Furthermore, direct interaction between SafA(41-65) and the sensor domain of PhoQ was observed by means of surface plasmon resonance. NMR spectroscopy of (15) N-labelled PhoQ sensor domain confirmed that SafA and Mg(2+) provoked a different conformational change of PhoQ. Site-directed mutagenesis studies revealed that R53, within SafA(41-65), was important for the activation of PhoQ, and D179 of the PhoQ sensor domain was required for its activation by SafA. SafA activated PhoQ by a different mechanism from cationic antimicrobial peptides and acidic pH, and independent of divalent cations and MgrB.