Two-component Response Research Articles

Two-Component Response Regulator OmpR Regulates Mucoviscosity through Energy Metabolism in Klebsiella pneumoniae.

Hypermucoviscosity is a hallmark of hypervirulent Klebsiella pneumoniae (hvKP). However, the molecular basis of its regulation is largely unknown. We hypothesize that hypermucoviscosity is modulated via two-component signal transduction systems (TCSs). In-frame deletion mutants of all 33 response regulators of hvKP ATCC43816 were generated using CRISPR/CAS and evaluated for their impacts on hypermucoviscosity. The response regulator OmpR is required for hypermucoviscosity in vitro and virulence in vivo in a mouse pneumonia model. The ΔompR mutant lost its mucoidy but retained its capsule level and comparable rmpADC expression, so transcriptomic analysis by RNA-Seq was performed to identify differentially expressed genes (DEGs) in ΔompR mutant. The top 20 Gene Ontology terms of 273 DEGs belong to purine ribonucleotide triphosphate biosynthetic and metabolic process, transmembrane transport, and amino acid metabolism. Among the overexpressed genes in the ΔompR mutant, the atp operon encoding F-type ATP synthase and the gcvTHP encoding glycine cleavage system were characterized further as overexpression of either operon reduced the mucoviscosity and increased the production of ATP. Furthermore, OmpR directly bound the promoter region of the atp operon, not the gcvTHP, suggesting that OmpR regulates the expression of the atp operon directly and gcvTHP indirectly. Hence, the loss of OmpR led to the overexpression of F-type ATP synthase and glycine cleavage system, which altered the energetic status of ΔompR cells and contributed to the subsequent reduction in the mucoviscosity. Our study has uncovered a previously unknown regulation of bacterial metabolism by OmpR and its influence on hypermucoviscosity. IMPORTANCE Hypermucoviscosity is a critical virulent factor for Klebsiella pneumoniae infections, and its regulation remains poorly understood at the molecular level. This study aims to address this knowledge gap by investigating the role of response regulators in mediating hypermucoviscosity in K. pneumoniae. We screened 33 response regulators and found that OmpR is essential for hypermucoviscosity and virulence of K. pneumoniae in a mouse pneumonia model. Transcriptomic analysis uncovered that genes involved in energy production and metabolism are highly upregulated in the ΔompR mutant, suggesting a potential link between bacterial energy status and hypermucoviscosity. Overexpression of those genes increased production of ATP and reduced mucoviscosity, recapitulating the ΔompR mutant phenotype. Our findings provide new insights into the regulation of K. pneumoniae hypermucoviscosity by a two-component signal transduction system, highlighting the previously unknown role of OmpR in regulating bacterial energy status and its influence on hypermucoviscosity.

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.

Structure of mycobacterial respiratory complex I

Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the treatment of infection by Mycobacterium tuberculosis and other mycobacteria. The mycobacterial ETC is highly branched with multiple dehydrogenases transferring electrons to a membrane-bound pool of menaquinone and multiple oxidases transferring electrons from the pool. The proton-pumping type I nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) is found in low abundance in the plasma membranes of mycobacteria in typical invitro culture conditions and is often considered dispensable. We found that growth of Mycobacterium smegmatis in carbon-limited conditions greatly increased the abundance of Complex I and allowed isolation of a rotenone-sensitive preparation of the enzyme. Determination of the structure of the complex by cryoEM revealed the "orphan" two-component response regulator protein MSMEG_2064 as a subunit of the assembly. MSMEG_2064 in the complex occupies a site similar to the proposed redox-sensing subunit NDUFA9 in eukaryotic Complex I. An apparent purine nucleoside triphosphate within the NuoG subunit resembles the GTP-derived molybdenum cofactor in homologous formate dehydrogenase enzymes. The membrane region of the complex binds acyl phosphatidylinositol dimannoside, a characteristic three-tailed lipid from the mycobacterial membrane. The structure also shows menaquinone, which is preferentially used over ubiquinone by gram-positive bacteria, in two different positions along the quinone channel, comparable to ubiquinone in other structures and suggesting a conserved quinone binding mechanism.

Escherichia coli has robust regulatory mechanisms against elevated peptidoglycan cleavage by lytic transglycosylases

Peptidoglycan (PG) is an essential and conserved exoskeletal component in all bacteria that protects cells from lysis. Gram-negative bacteria such as Escherichia coli encode multiple redundant lytic transglycosylases (LTs) that engage in PG cleavage, a potentially lethal activity requiring proper regulation to prevent autolysis. To elucidate the potential effects and cellular regulatory mechanisms of elevated LT activity, we individually cloned the periplasmic domains of two membrane-bound LTs, MltA and MltB, under the control of the arabinose-inducible system for overexpression in the periplasmic space in E.coli. Interestingly, upon induction, the culture undergoes an initial period of cell lysis followed by robust growth restoration. The LT-overexpressing E.coli exhibits altered morphology with larger spherical cells, which is in line with the weakening of the PG layer due to aberrant LT activity. On the other hand, the restored cells display a similar rod shape and PG profile that is indistinguishable from the uninduced control. Quantitative proteomics analysis of the restored cells identified significant protein enrichment in the regulator of capsule synthesis (Rcs) regulon, a two-component stress response known to be specifically activated by PG damage. We showed that LT-overexpressing E.coli with an inactivated Rcs system partially impairs the growth restoration process, supporting the involvement of the Rcs system in countering aberrant PG cleavage. Furthermore, we demonstrated that the elevated LT activity specifically potentiates β-lactam antibiotics against E.coli with a defective Rcs regulon, suggesting the dual effects of augmented PG cleavage and blocked PG synthesis as a potential antimicrobial strategy.

Functional characterization of transcriptional activator gene SIARRI in tomato reveals its role in fruit growth and ripening.

Auxins regulate several characteristics of plant development and growth. Here, we characterized a new transcriptional activator SIARRI which binds specific DNA sequences and was revealed in Arabidopsis (ARR1). SIARRI acts as a two-component response regulator and its Arabidopsis homologous gene is AT3G16857. It belongs to the subfamily of type-B response regulators in the cytokinin signaling pathway. The study aimed to characterize the transgenic Micro-Tom plants by the overexpression of Solanum lycopersicum two-component response regulator ARR1. Overexpression of SIARRI results in a pleiotropic phenotype during fruit development and ripening. This study indicates that SIARRI is a primary regulator of leaf morphology and fruit development. Moreover, overexpressed plants showed variations in growth related to auxin as well as shorter hypocotyl elongation, enlarged leaf vascularization, and decreased apical dominance. The qRT-PCR investigation revealed that expression was downregulated at the breaker stage and high at Br+6 at various stages of fruit growth and ripening. In contrast to the fruit color, lycopene and β-carotene concentrations in red-yellow overexpression line fruits were reduced significantly, and also slightly reduced in some red fruits. The quantity of β-carotene in the transgenic fruits was lower than that of lycopene. This study showed that this gene might be a new transcriptional activator in fruit development and ripening. Furthermore, this study will provide new insights into tomato fruit ripening.

Fine Mapping and Identification of SmAPRR2 Regulating Rind Color in Eggplant (Solanum melongena L.)

Rind color is an economically important agronomic trait in eggplant that impacts consumer preferences. In this study, bulked segregant analysis and competitive allele-specific PCR were employed to identify the candidate gene for eggplant rind color through constructing a 2794 F2 population generated from a cross between "BL01" (green pericarp) and "B1" (white pericarp). Genetic analysis of rind color revealed that a single dominant gene controls green color of eggplant peel. Pigment content measurement and cytological observations demonstrated that chlorophyll content and chloroplast number in BL01 were higher than in B1. A candidate gene (EGP19168.1) was fine-mapped to a 20.36 Kb interval on chromosome 8, which was predicted to encode the two-component response regulator-like protein Arabidopsis pseudo-response regulator2 (APRR2). Subsequently, allelic sequence analysis revealed that a SNP deletion (ACT→AT) in white-skinned eggplant led to a premature termination codon. Genotypic validation of 113 breeding lines using the Indel marker closely linked to SmAPRR2 could predict the skin color (green/white) trait with an accuracy of 92.9%. This study will be valuable for molecular marker-assisted selection in eggplant breeding and provides theoretical foundation for analyzing the formation mechanism of eggplant peel color.

A Novel Role of the Two-Component System Response Regulator UvrY in Enterohemorrhagic Escherichia coli O157:H7 Pathogenicity Regulation.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen causing severe diseases, such as hemorrhagic colitis and lethal hemolytic uremic syndrome. The signal-sensing capability of EHEC O157:H7 at specific host colonization sites via different two-component systems (TCSs) is closely related to its pathogenicity during infection. However, the types of systems involved and the regulatory mechanisms are not fully understood. Here, we investigated the function of the TCS BarA/UvrY regulator UvrY in the pathogenicity regulation of EHEC O157:H7. Our results showed that UvrY acts as a positive regulator of EHEC O157:H7 for cellular adherence and mouse colonization through the transcriptional activation of the locus for enterocyte effacement (LEE) pathogenic genes. Furthermore, this regulation is mediated by the LEE island master regulator, Ler. Our results highlight the significance of UvrY in EHEC O157:H7 pathogenicity and underline the unknown importance of BarA/UvrY in colonization establishment and intestinal adaptability during infection.

Dual GGDEF/EAL-Domain Protein RmcA Controls the Type III Secretion System of Pseudomonas aeruginosa by Interaction with CbrB.

Cyclic diguanylate (c-di-GMP) is a major bacterial secondary signaling molecule that controls a multitude of cellular processes. More than 40 genes encoding diguanylate cyclases and phosphodiesterases have been identified in Pseudomonas aeruginosa, and many of them have been intensively investigated. However, the mechanism through which they achieve signaling specificity remains unclear. Here, we revealed that the absence of the dual GGDEF/EAL-domain protein RmcA significantly affected biofilm formation of P. aeruginosa PAO1 and led to upregulated expression of the type III secretion system (T3SS) genes; overexpression of RmcA strongly reduced the expression of T3SS. Further investigation showed that the regulatory function of RmcA was independent of the Gac/Rsm pathway. To identify the interaction partners of RmcA involved in this process, bacterial two-hybrid library screening was performed. We found that RmcA directly interacts with a two-component response regulator CbrB, which is involved in the regulation of biofilm formation and T3SS expression by RmcA. These findings reveal that the dual-domain GGDEF/EAL protein RmcA could achieve specificity of action through physical interaction with CbrB, which extends understanding the complex regulatory network of the c-di-GMP signaling.

Research on global calibration technology of pressure sensitive paint based on airtight chamber

Pressure sensitive paint (PSP) technology has been widely applied in wind-tunnel test. Generally, PSP calibration data are obtained using a calibration sample in laboratory priori calibration device, which the sample is sprayed at the same time with the test model.In this study, taking the real test model as the calibration object, a global calibration technology of PSP coating on the surface of the test model is proposed, which is verified in the airtight chamber, and the effective PSP calibration data are obtained. A two-component fast response PSP coating was used in the study. In the validation test, a flying wing model is used, the flying wing model sprayed with PSP coating was placed in the airtight chamber. 11 groups of calibration tests were carried out in the environment of 0.06 MPa ~ 0.14 MPa and 30°C ~50°C, and the calibration curves obtained showed good consistency.

Chromosomal fragment deletion in APRR2-repeated locus modulates the dark stem color in Cucurbita pepo.

Cp4.1LG15g03420 (CpDsc-1), which encodes a two-component response regulator-like protein (APRR2) in the nucleus, influences dark green stem formation in Cucurbita pepo by regulating the chlorophyll content. Stem color is an important agronomic trait in zucchini (Cucurbita pepo) for robust seeding and high yield. However, the gene controlling the stem color has not been characterized. In this study, we identified a single locus accounting for the dark green stem color of C. pepo (CpDsc-1). Genetic analysis of this trait in segregated populations derived from two parental lines (line 296 with dark green stems and line 274 with light green stems) revealed that stem color was controlled by a single dominant gene (dark green vs. light green). In bulked segregant analysis, CpDsc-1 was mapped to a 2.09-Mb interval on chromosome 15. This region was further narrowed to 65.2kb using linkage analysis of the F2 population. Sequencing analysis revealed a 14kb deletion between Cp4.1LG15g03420 and Cp4.1LG15g03360; these two genes both encoded a two-component response regulator-like protein (APRR2). The incomplete structures of the two APRR2 genes and abnormal chloroplasts in line 274 might be the main cause of the light green phenotype. Gene expression pattern analysis showed that only Cp4.1LG15g03420 was upregulated in line 296. Subcellular localization analysis indicated that Cp4.1LG15g03420 was a nuclear gene. Furthermore, a co-dominant marker, G4563 (93% accuracy rate), and a co-segregation marker, Fra3, were established in 111 diverse germplasms; both of these markers were tightly linked with the color trait. This study provided insights into chlorophyll regulation mechanisms and revealed the markers valuable for marker-assisted selection in future zucchini breeding.

The Oral Microbiome Impacts the Link between Sugar Consumption and Caries: A Preliminary Study

Background: The excessive and frequent intake of refined sugar leads to caries. However, the relationship between the amount of sugar intake and the risk of caries is not always consistent. Oral microbial profile and function may impact the link between them. This study aims to identify the plaque microbiota characteristics of caries subjects with low (CL) and high (CH) sugar consumption, and of caries-free subjects with low (FL) and high sugar (FH) consumption. Methods: A total of 40 adolescents were enrolled in the study, and supragingival plaque samples were collected and subjected to metagenomic analyses. The caries status, sugar consumption, and oral-health behaviors of the subjects were recorded. Results: The results indicate that the CL group showed a higher abundance of several cariogenic microorganisms Lactobacillus, A. gerencseriae, A. dentails, S. mutans, C. albicans, S. wiggsiae and P. acidifaciens. C. gingivalis, and P. gingivalis, which were enriched in the FH group. In terms of gene function, the phosphotransferase sugar uptake system, phosphotransferase system, and several two-component responses–regulator pairs were enriched in the CL group. Conclusion: Overall, our data suggest the existence of an increased cariogenic microbial community and sugar catabolism potential in the CL group, and a healthy microbial community in the FH group, which had self-stabilizing functional potential.

Damage-based strength reduction factor spectra of structures subjected to bidirectional ground motions

The current research on the damage-based strength reduction factor has been completely based on the inelastic single-degree-of-freedom systems that are more suitable for two-dimensional analyses. Therefore, it is necessary to construct damage-based strength reduction factor spectra for systems subjected to bidirectional ground motions. In this study, an inelastic single-mass bi-degree-of-freedom (SMBDOF) system subjected to the orthogonal bidirectional ground motions with the hysteretic property of two-dimensional yield-surface plasticity function is presented, and the x-component of the system is used to construct the damage-based strength reduction factor spectra of the system ( R b spectra). The Park-Ang damage model is used to determine the constant damage index of the R b spectra. In addition, 178 ground motion records for site classes C, D, and E are considered as ground excitations of the SMBDOF system to construct the mean R b spectra with the format of R b- D- T for a given ductility capacity and R b- μ u- T for a given damage level. The R b spectra of the SMBDOF system is compared with the corresponding R spectra of the SDOF system. Statistical analysis considering different values of the natural period, ductility capacity, damage index, period ratio, and site condition is conducted. Analysis results show that the coupling of two-component responses of an inelastic system could increase the strength demand of the system in certain cases; the trend of the R b spectra presented in this study is approximately consistent for different site classes, but the spectral values have the characteristic of site conditions. Therefore, the influence of the period ratio should be considered because a large period difference between two components of the SMBDOF system could aggravate seismic damage in certain situations. Based on the statistical analysis, the expression of R b spectra curves with a period ratio γ = 1 is derived using the regression analysis, and the correction expression is obtained considering the influence of the period ratio on the R b spectra. Then, the construction procedure of the R b spectra is introduced. The predicted R b spectra obtained by the proposed procedure is compared with the actual mean spectra, and comparison results indicate a good match.

Complete Genome Analysis of Highly Pathogenic Non-O1/O139 Vibrio cholerae Isolated From Macrobrachium rosenbergii Reveals Pathogenicity and Antibiotic Resistance-Related Genes.

Non-O1/O139 Vibrio cholerae is a highly virulent pathogen that causes mass mortalities of various aquatic animals. In the present study, we sequenced the whole genome of non-O1/O139 V. cholerae GXFL1-4, isolated from Macrobrachium rosenbergii, to reveal the pathogenicity and antibiotic resistance. The result showed its genome contained two circular chromosomes and one plasmid with a total size of 4,282,243 bp, which harbored 3,869 coding genes. Among them, 3,047, 2,659, and 3,661 genes were annotated in the Clusters of Orthologous Genes (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG), respectively. In addition, 372 potential virulence genes were predicted based on the Virulence Factor Database (VFDB) database, such as type II, III, IV, and VI secretion systems related genes, flagella genes, and pilus formation or motility-related genes. Blast results in the Comprehensive Antibiotic Resistance Database (CARD) database showed that the strain contained 148 antibiotic resistance-related genes belonging to 27 categories, such as efflux pump complex antibiotic resistance genes and antibiotic resistance gene cluster genes. The Pathogen-Host Interaction (PHI) database annotated 320 genes related to pathogen-host interaction, such as T3SS, virulence regulatory factors, transcriptional regulators, and two-component response regulator related genes. The whole-genome analysis suggested that the pathogenic non-O1/O139 V. cholerae strain GXFL1-4 might have a complex molecular mechanism of pathogenicity and antibiotic resistance. This study provides a wealth of information about non-O1/O139 V. cholerae genes related to its pathogenicity and drug resistance and will facilitate the understanding of its pathogenesis as well as the development of prevention and treatment strategies for the pathogen.

Mycobacterium tuberculosis PknK Substrate Profiling Reveals Essential Transcription Terminator Protein Rho and Two-Component Response Regulators PrrA and MtrA as Novel Targets for Phosphorylation.

ABSTRACTThe Mycobacterium tuberculosis protein kinase K regulates growth adaptation by facilitating mycobacterial survival in response to a variety of in vitro and in vivo stress conditions. Here, we further add that pknK transcription is responsive to carbon and nitrogen starvation signals. The increased survival of an M. tuberculosis ΔpknK mutant strain under carbon- and nitrogen-limiting growth conditions compared to the wild-type (WT) H37Rv suggests an integral role of PknK in regulating growth during metabolic stress. To identify the downstream targets of PknK-mediated signaling, we compared phosphoproteomic and transcription profiles of mycobacterial strains overexpressing WT and phosphorylation-defective PknK. Results implicate PknK as a signaling protein that can regulate several enzymes involved in central metabolism, transcription regulation, and signal transduction. A key finding of this study was the identification of two essential two-component response regulator (RR) proteins, PrrA and MtrA, and Rho transcription terminator, as unique targets for PknK. We confirm that PknK interacts with and phosphorylates PrrA, MtrA, and Rho in vivo. PknK-mediated phosphorylation of MtrA appears to increase binding of the RR to the cognate probe DNA. However, dual phosphorylation of MtrA and PrrA response regulators by PknK and their respective cognate sensor kinases in vitro showed nominal additive effect on the mobility of the protein-DNA complex, suggesting the presence of a potential fine-tuning of the signal transduction pathway which might respond to multiple cues.IMPORTANCE Networks of gene regulation and signaling cascades are fundamental to the pathogenesis of Mycobacterium tuberculosis in adapting to the continuously changing intracellular environment in the host. M. tuberculosis protein kinase K is a transcription regulator that responds to diverse environmental signals and facilitates stress-induced growth adaptation in culture and during infection. This study identifies multiple signaling interactions of PknK and provides evidence that PknK can change the transcriptional landscape during growth transitions by connecting distinctly different signal transduction and regulatory pathways essential for mycobacterial survival.

The Lon protease negatively regulates pyoluteorin biosynthesis through the Gac/Rsm-RsmE cascade and directly degrades the transcriptional activator PltR in Pseudomonas protegens H78.

Pyoluteorin (Plt) is a broad-spectrum antibiotic with antibacterial and antifungal activities. In Pseudomonas protegens H78, the Plt biosynthetic operon pltLABCDEFG is transcriptionally activated by the LysR-type regulator PltR and is positively regulated by the Gac/Rsm signal transduction cascade (GacS/A-RsmXYZ-RsmE-pltR/pltAB). Additionally, Plt biosynthesis has been shown to be significantly enhanced by mutation of the Lon protease-encoding gene. This study aims to understand the negative regulation pathway and molecular mechanism by which Lon functions in Plt biosynthesis. lon deletion was first found to improve the antimicrobial ability of strain H78 due to its increased Plt production, while partially inhibiting the growth of H78 strain. Lon protease decreases the abundance and stability of the two-component system response regulator GacA and thus participates in the abovementioned Gac/Rsm cascade and negatively regulates Plt biosynthesis. Similarly, Lon protease also decreases the abundance and stability of transcriptional activator PltR. PltR protein can be directly degraded by the Lon protease but not by a mutated form of Lon protease with an amino acid replacement of S674 -A. In summary, Lon protease negatively regulates Plt biosynthesis via both the Gac/Rsm-mediated global regulatory pathway and the direct degradation of the transcriptional activator PltR in P. protegens H78.

Histidine Kinase Two-Component Response Regulators Ssk1, Skn7 and Rim15 Differentially Control Growth, Developmental and Volatile Organic Compounds Emissions as Stress Responses in Trichoderma Atroviride

Histidine Kinase Two-Component Response Regulators Ssk1, Skn7 and Rim15 Differentially Control Growth, Developmental and Volatile Organic Compounds Emissions as Stress Responses in Trichoderma Atroviride

Histidine kinase two-component response regulators Ssk1, Skn7 and Rim15 differentially control growth, developmental and volatile organic compounds emissions as stress responses in Trichoderma atroviride.

The Skn7, Ssk1 and Rim15 proteins are response regulators involved in osmotic, oxidative and nutritional stress in fungi. In order to verify the involvement of these genes in Trichoderma atroviride IMI206040's growth, conidiation, direct antagonism against plant pathogens (Rhizoctonia solani and Sclerotinia sclerotiorum), production of volatile organic compounds (VOCs) with fungistatic effect, and interaction with plants (growth promotion), single mutants were generated, and the phenotypic patterns were analysed in comparison to the wild-type (wt) strain. The mutants were submitted to osmotic, oxidative, membrane and cell wall stress conditions in vitro. The Δskn7 and Δrim15 mutants did not show either significant differences at morphological level, or marked decreases in mycelial growth and conidiation in relation to wt, whereas Δssk1 had altered phenotypes in most conditions tested. The plant-growth promotion of Arabidopsis thaliana seedlings induced by VOCs was not quantitatively modified by any of the mutants in relation to the wt strain, although possible differences in secondary root hairs was noticed for Δrim15. The fungistatic activity was significantly altered for Δssk1 and Δrim15. Overall, the Δssk1 strain showed remarkable morphological differences, with decrease in mycelial growth and conidiation, being also affected in the antagonistic capacity against plant pathogens. The impacts demonstrated by the deletion of ssk1 suggest this gene has a relevant participation in the signalling response to different stresses in T. atroviride and in the interactive metabolism with phytopathogens and plants. On the other hand, unlike other fungal models, Skn7 did not appear to have a critical participation in the above-mentioned processes; Rim15 seemed to confirm its involvement in modulating cellular responses to nutritional status, although with a possible cross-talk with other cellular processes. Our results suggest that Ssk1 likely plays a key regulatory role, not only in basic metabolisms of T. atroviride, but also in biocontrol-related characteristics.

Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens.

SUMMARYPathogenic bacteria can rapidly respond to stresses such as reactive oxygen species (ROS) using reversible redox-sensitive oxidation of cysteine thiol (-SH) groups in regulators. Here, we use proteomics to profile reversible ROS-induced thiol oxidation in Vibrio cholerae, the etiologic agent of cholera, and identify two modified cysteines in ArcA, a regulator of global carbon oxidation that is phosphorylated and activated under low oxygen. ROS abolishes ArcA phosphorylation but induces the formation of an intramolecular disulfide bond that promotes ArcA-ArcA interactions and sustains activity. ArcA cysteines are oxidized in cholera patient stools, and ArcA thiol oxidation drives in vitro ROS resistance, colonization of ROS-rich guts, and environmental survival. In other pathogens, such as Salmonella enterica, oxidation of conserved cysteines of ArcA orthologs also promotes ROS resistance, suggesting a common role for ROS-induced ArcA thiol oxidation in modulating ArcA activity, allowing for a balance of expression of stress- and pathogenesis-related genetic programs.

A Selective Tether Recruits Activated Response Regulator CheB to Its Chemoreceptor Substrate.

ABSTRACTMethylesterase/deamidase CheB is a key component of bacterial chemotaxis systems. It is also a prominent example of a two-component response regulator in which the effector domain is an enzyme. Like other response regulators, CheB is activated by phosphorylation of an aspartyl residue in its regulatory domain, creating an open conformation between its two domains. Studies of CheB in Escherichia coli and related organisms have shown that its enzymatic action is also enhanced by a pentapeptide-binding site for the enzyme at the chemoreceptor carboxyl terminus. Related carboxyl-terminal pentapeptides are found on >25,000 chemoreceptor sequences distributed across 11 bacterial phyla and many bacterial species, in which they presumably play similar roles. Yet, little is known about the interrelationship of CheB phosphorylation, pentapeptide binding, and interactions with its substrate methylesters and amides on the body of the chemoreceptor. We investigated by characterizing the binding kinetics of CheB to Nanodisc-inserted chemoreceptor dimers. The resulting kinetic and thermodynamic constants revealed a synergy between CheB phosphorylation and pentapeptide binding in which a phosphorylation mimic enhanced pentapeptide binding, and the pentapeptide served not only as a high-affinity tether for CheB but also selected the activated conformation of the enzyme. The basis of this selection was revealed by molecular modeling that predicted a pentapeptide-binding site on CheB which existed only in the open, activated enzyme. Recruitment of activated enzyme by selective tethering represents a previously unappreciated strategy for regulating response regulator action, one that may well occur in other two-component systems.

In Silico Core Proteomics and Molecular Docking Approaches for the Identification of Novel Inhibitors against Streptococcus pyogenes.

Streptococcus pyogenes is a significant pathogen that causes skin and upper respiratory tract infections and non-suppurative complications, such as acute rheumatic fever and post-strep glomerulonephritis. Multidrug resistance has emerged in S. pyogenes strains, making them more dangerous and pathogenic. Hence, it is necessary to identify and develop therapeutic methods that would present novel approaches to S. pyogenes infections. In the current study, a subtractive proteomics approach was employed to core proteomes of four strains of S. pyogenes using several bioinformatic software tools and servers. The core proteome consists of 1324 proteins, and 302 essential proteins were predicted from them. These essential proteins were analyzed using BLASTp against human proteome, and the number of potential targets was reduced to 145. Based on subcellular localization prediction, 46 proteins with cytoplasmic localization were chosen for metabolic pathway analysis. Only two cytoplasmic proteins, i.e., chromosomal replication initiator protein DnaA and two-component response regulator (TCR), were discovered to have the potential to be novel drug target candidates. Three-dimensional (3D) structure prediction of target proteins was carried out via the Swiss Model server. Molecular docking approach was employed to screen the library of 1000 phytochemicals against the interacting residues of the target proteins through the MOE software. Further, the docking studies were validated by running molecular dynamics simulation and highly popular binding free energy approaches of MM-GBSA and MM-PBSA. The findings revealed a promising candidate as a novel target against S. pyogenes infections.