Pangenome analytics reveal two-component systems as conserved targets in ESKAPEE pathogens

AbstractBacteria sense and respond to environmental stimuli through two-component systems (TCSs), that are composed of histidine kinase sensing and response regulator elements. TCSs are ubiquitous and participate in numerous cellular functions. TCSs across the ESKAPEE pathogens, representing the leading causes of nosocomial infections, were characterized using pangenome analytics, including annotation, mapping, pangenomic status, gene orientation, sequence variation, and structure. Our findings fall into two categories. 1) phylogenetic distribution of TCSs: (i) the number and types of TCSs varies between species of the ESKAPEE pathogens; (ii) TCSs are group-specific, i.e., Gram-positive and Gram-negative, except for KdpDE; (iii) most TCSs are conserved among genomes of an ESKAPEE, except in Pseudomonas aeruginosa. 2) sequence variation: (i) at the operon level, the genomic architecture of a TCS operon stratifies into a few discrete classes; and (ii) at the gene sequence level, histidine kinases, responsible for signal sensing, show sequence and structural variability as compared to response regulators that show a high degree of conservation. Taken together, this first comprehensive pangenomic assessment of TCSs reveals a range of strategies deployed by the ESKAPEE pathogens to manifest pathogenicity and antibiotic resistance. It further suggests that the conserved features of TCSs makes them an attractive group of potential targets with which to address antibiotic resistance.

Download Full-text

Multiple Mechanisms of Action for Inhibitors of Histidine Protein Kinases from Bacterial Two-Component Systems

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.7.1693 ◽

1999 ◽

Vol 43 (7) ◽

pp. 1693-1699 ◽

Cited By ~ 130

Author(s):

Jamese J. Hilliard ◽

Raul M. Goldschmidt ◽

Lisa Licata ◽

Ellen Z. Baum ◽

Karen Bush

Keyword(s):

Pathogenic Bacteria ◽

Response Regulator ◽

Membrane Integrity ◽

Bacterial Killing ◽

Gram Positive Bacteria ◽

Component Systems ◽

Two Component ◽

Two Component Systems ◽

Bacterial Cell Membrane ◽

Cellular Incorporation

ABSTRACT Many pathogenic bacteria utilize two-component systems consisting of a histidine protein kinase (HPK) and a response regulator (RR) for signal transduction. During the search for novel inhibitors, several chemical series, including benzoxazines, benzimidazoles, bis-phenols, cyclohexenes, trityls, and salicylanilides, were identified that inhibited the purified HPK-RR pairs KinA-Spo0F and NRII-NRI, with 50% inhibitory concentrations (IC50s) ranging from 1.9 to >500 μM and MICs ranging from 0.5 to >16 μg/ml for gram-positive bacteria. However, additional observations suggested that mechanisms other than HPK inhibition might contribute to antibacterial activity. In the present work, representative compounds from the six different series of inhibitors were analyzed for their effects on membrane integrity and macromolecular synthesis. At 4× MIC, 17 of 24 compounds compromised the integrity of the bacterial cell membrane within 10 min, as measured by uptake of propidium iodide. In this set, compounds with lower IC50s tended to cause greater membrane disruption. Eleven of 12 compounds inhibited cellular incorporation of radiolabeled thymidine and uridine >97% in 5 min and amino acids >80% in 15 min. The HPK inhibitor that allowed >25% precursor incorporation had no measurable MIC (>16 μg/ml). Fifteen of 24 compounds also caused hemolysis of equine erythrocytes. Thus, the antibacterial HPK inhibitors caused a rapid decrease in cellular incorporation of RNA, DNA, and protein precursors, possibly as a result of the concomitant disruption of the cytoplasmic membrane. Bacterial killing by these HPK inhibitors may therefore be due to multiple mechanisms, independent of HPK inhibition.

Download Full-text

A survey of HK, HPt, and RR domains and their organization in two-component systems and phosphorelays proteins of organisms with fully sequenced genomes

10.7287/peerj.preprints.1267 ◽

2015 ◽

Author(s):

Baldiri Salvado ◽

Ester Vilaprinyo ◽

Albert Sorribas ◽

Rui Alves

Keyword(s):

Response Regulator ◽

Signal Transmission ◽

Pr Proteins ◽

Component Systems ◽

Two Component ◽

Domains Of Life ◽

Two Component Systems ◽

Operon Organization ◽

The Individual ◽

Selection Of

Two Component Systems and Phosphorelays (TCS/PR) are environmental signal transduction cascades in prokaryotes and, less frequently, in eukaryotes. The internal domain organization of proteins and the topology of TCS/PR cascades play an important role in shaping the responses of the circuits. It is thus important to maintain updated censuses of TCS/PR proteins in order to identify the various topologies used by nature and enable a systematic study of the dynamics associated with those topologies. To create such a census, we analyzed the proteomes of 7609 organisms from all domains of life with fully sequenced and annotated genomes. To begin, we survey each proteome searching for proteins containing domains that are associated with internal signal transmission within TCS/PR: Histidine Kinase (HK), Response Regulator (RR) and Histidine Phosphotranfer (HPt) domains, and analyze how these domains are arranged in the individual proteins. Then, we find all types of operon organization and calculate how much more likely are proteins that contain TCS/PR domains to be coded by neighboring genes than one would expect from the genome background of each organism. Finally, we analyze if the fusion of domains into single TCS/PR proteins is more frequently observed than one might expect from the background of each proteome. We find 50 alternative ways in which the HK, HPt, and RR domains are observed to organize into single proteins. In prokaryotes, TCS/PR coding genes tend to be clustered in operons. 90% of all proteins identified in this study contain just one of the three domains, while 8% of the remaining proteins combine one copy of an HK, a RR, and/or an HPt domain. In eukaryotes, 25% of all TCS/PR proteins have more than one domain. These results might have implications for how signals are internally transmitted within TCS/PR cascades. These implications could explain the selection of the various designs in alternative circumstances.

Download Full-text

The two-component system CopRS maintains femtomolar levels of free copper in the periplasm of Pseudomonas aeruginosa using a phosphatase-based mechanism

10.1101/2020.05.13.092544 ◽

2020 ◽

Author(s):

Lorena Novoa-Aponte ◽

Fernando C. Soncini ◽

José M. Argüello

Keyword(s):

Pseudomonas Aeruginosa ◽

Phosphatase Activity ◽

Response Regulator ◽

Redox Enzymes ◽

Two Component System ◽

Component System ◽

Component Systems ◽

Two Component ◽

Two Component Systems ◽

Systems Control

ABSTRACTTwo component systems control periplasmic Cu+ homeostasis in Gram-negative bacteria. In characterized systems such as Escherichia coli CusRS, upon Cu+ binding to the periplasmic sensing domain of CusS, a cytoplasmic phosphotransfer domain phosphorylates the response regulator CusR. This drives the expression of efflux transporters, chaperones, and redox enzymes to ameliorate metal toxic effects. Here, we show that the Pseudomonas aeruginosa two component sensor histidine kinase CopS exhibits a Cu-dependent phosphatase activity that maintains a non-phosphorylated CopR when the periplasmic Cu levels are below its activation threshold. Upon Cu+ binding to the sensor, the phosphatase activity is blocked and the phosphorylated CopR activates transcription of the CopRS regulon. Supporting the model, mutagenesis experiments revealed that the ΔcopS strain showed constitutive high expression of the CopRS regulon, lower intracellular Cu+ levels, and larger Cu tolerance when compared to wild type cells. The invariant phospho-acceptor residue His235 of CopS was not required for the phosphatase activity itself, but necessary for its Cu-dependency. To sense the metal, the periplasmic domain of CopS binds two Cu+ ions at its dimeric interface. Homology modeling of CopS based on CusS structure (four Ag+ binding sites) clearly explains the different binding stoichiometries in both systems. Interestingly, CopS binds Cu+/2+ with 30 × 10−15 M affinities, pointing to the absence of free (hydrated) Cu+/2+ in the periplasm.IMPORTANCECopper is a micronutrient required as cofactor in redox enzymes. When free, copper is toxic, mismetallating proteins, and generating damaging free radicals. Consequently, copper overload is a strategy that eukaryotic cells use to combat pathogens. Bacteria have developed copper sensing transcription factors to control copper homeostasis. The cell envelope is the first compartment that has to cope with copper stress. Dedicated two component systems control the periplasmic response to metal overload. This manuscript shows that the copper sensing two component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of the response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the sensor is activated by copper levels compatible with the absence of free copper in the cell periplasm. This emphasizes the diversity of molecular mechanisms that have evolved in various bacteria to manage the copper cellular distribution.

Download Full-text

Two Component Regulatory Systems and Antibiotic Resistance in Gram-Negative Pathogens

International Journal of Molecular Sciences ◽

10.3390/ijms20071781 ◽

2019 ◽

Vol 20 (7) ◽

pp. 1781 ◽

Cited By ~ 22

Author(s):

Anjali Y. Bhagirath ◽

Yanqi Li ◽

Rakesh Patidar ◽

Katherine Yerex ◽

Xiaoxue Ma ◽

...

Keyword(s):

Antibiotic Resistance ◽

Antimicrobial Resistance ◽

Pathogenic Bacteria ◽

Current Knowledge ◽

Regulation Of Gene Expression ◽

Gram Negative ◽

Environmental Signals ◽

Component Systems ◽

Two Component ◽

Two Component Systems

Gram-negative pathogens such as Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are the leading cause of nosocomial infections throughout the world. One commonality shared among these pathogens is their ubiquitous presence, robust host-colonization and most importantly, resistance to antibiotics. A significant number of two-component systems (TCSs) exist in these pathogens, which are involved in regulation of gene expression in response to environmental signals such as antibiotic exposure. While the development of antimicrobial resistance is a complex phenomenon, it has been shown that TCSs are involved in sensing antibiotics and regulating genes associated with antibiotic resistance. In this review, we aim to interpret current knowledge about the signaling mechanisms of TCSs in these three pathogenic bacteria. We further attempt to answer questions about the role of TCSs in antimicrobial resistance. We will also briefly discuss how specific two-component systems present in K. pneumoniae, A. baumannii, and P. aeruginosa may serve as potential therapeutic targets.

Download Full-text

Molecular Characterization of Two-Component Systems of Helicobacter pylori

Journal of Bacteriology ◽

10.1128/jb.182.8.2068-2076.2000 ◽

2000 ◽

Vol 182 (8) ◽

pp. 2068-2076 ◽

Cited By ~ 126

Author(s):

Dagmar Beier ◽

Rainer Frank

Keyword(s):

Helicobacter Pylori ◽

Histidine Kinase ◽

Response Regulator ◽

Response Regulators ◽

Reading Frame ◽

Component Systems ◽

Two Component ◽

Two Component Systems

ABSTRACT Two-component systems are frequently involved in the adaptation of bacteria to changing environmental conditions at the level of transcriptional regulation. Here we report the characterization of members of the two-component systems of the gastric pathogenHelicobacter pylori deduced from the genome sequence of strain 26695. We demonstrate that the response regulators HP166, HP1043, and HP1021 have essential functions, as disruption of the corresponding genes is lethal for the bacteria, irrespective of the fact that HP1043 and HP1021 have nonconserved substitutions in crucial amino acids of their receiver domains. An analysis of the in vitro phosphorylation properties of the two-component proteins demonstrates that HP244-HP703 and HP165-HP166 are cognate histidine kinase-response regulator pairs. Furthermore, we provide evidence that the variability of the histidine kinase HP165 caused by a poly(C) tract of variable length close to the 3′ end of open reading frame 165/164 does not interfere with the kinase activity of the transmitter domain of HP165.

Download Full-text

Cross-regulation between RpfG and HtsHs to regulate antibiotic biosynthesis in Lysobacter

10.1101/2020.07.13.201541 ◽

2020 ◽

Author(s):

Kaihuai Li ◽

Gaoge Xu ◽

Bo Wang ◽

Guichun Wu ◽

Fengquan Liu

Keyword(s):

Gene Expression ◽

Environmental Changes ◽

Antibiotic Biosynthesis ◽

Two Component System ◽

Independent Manner ◽

Heat Stable ◽

Component Systems ◽

Two Component ◽

Two Component Systems ◽

Sense And Respond

AbstractBacterial two-component systems (TCSs) sense and respond to environmental changes and modulate downstream gene expression. However, the mechanism of cross-talk between multiple TCSs is unclear. In this study, we report a previously uncharacterized mechanism by which the TCS protein RpfG interacts with hybrid two-component system (HyTCS) proteins HtsH1, HtsH2 and HtsH3 to regulate antibiotic biosynthesis in Lysobacter. RpfG, a phosphodiesterase (PDE), can degrade c-di-GMP to 5’-pGpG and can regulate antibiotic heat-stable antifungal factor (HSAF) biosynthesis in a PDE- independent manner. Thus, we wondered whether RpfG regulate HSAF biosynthesis through interactions with other factors. Subsequently, we demonstrated that RpfG interacts with three HyTCS proteins (HtsH1, HtsH2 and HtsH3), that can inhibit the PDE enzymatic activity of RpfG. Importantly, deletion of htsH1, htsH2 and htsH3 resulted in significantly decreased HSAF production, and we showed that HtsH1, HtsH2 and HtsH3 depend on their phosphorylation activity to directly regulate HSAF biosynthesis gene expression. Our results reveal that RpfG does not depend on PDE activity to regulate HSAF biosynthesis, rather it interacts with HtsH1, HtsH2 and HtsH3 to do so, a regulatory mechanism that may be a conserved paradigm in Lysobacter and Xanthomonas.

Download Full-text

Two-Component Systems of S. aureus: Signaling and Sensing Mechanisms

Genes ◽

10.3390/genes13010034 ◽

2021 ◽

Vol 13 (1) ◽

pp. 34

Author(s):

Lisa Bleul ◽

Patrice Francois ◽

Christiane Wolz

Keyword(s):

Transcriptional Activation ◽

Drug Targets ◽

Bacterial Survival ◽

Histidine Kinases ◽

Signal Perception ◽

Signaling Mechanisms ◽

Component Systems ◽

Two Component ◽

Two Component Systems ◽

Sense And Respond

Staphylococcus aureus encodes 16 two-component systems (TCSs) that enable the bacteria to sense and respond to changing environmental conditions. Considering the function of these TCSs in bacterial survival and their potential role as drug targets, it is important to understand the exact mechanisms underlying signal perception. The differences between the sensing of appropriate signals and the transcriptional activation of the TCS system are often not well described, and the signaling mechanisms are only partially understood. Here, we review present insights into which signals are sensed by histidine kinases in S. aureus to promote appropriate gene expression in response to diverse environmental challenges.

Download Full-text

Unique Patterns and Biogeochemical Relevance of Two-Component Sensing in Marine Bacteria

mSystems ◽

10.1128/msystems.00317-18 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 7

Author(s):

Noelle A. Held ◽

Matthew R. McIlvin ◽

Dawn M. Moran ◽

Michael T. Laub ◽

Mak A. Saito

Keyword(s):

Marine Environment ◽

Marine Bacteria ◽

Response Regulator ◽

Bacterial Species ◽

South Pacific ◽

Response Regulators ◽

Orphan Genes ◽

Component Systems ◽

Two Component ◽

Two Component Systems

ABSTRACTTwo-component sensory (TCS) systems link microbial physiology to the environment and thus may play key roles in biogeochemical cycles. In this study, we surveyed the TCS systems of 328 diverse marine bacterial species. We identified lifestyle traits such as copiotrophy and diazotrophy that are associated with larger numbers of TCS system genes within the genome. We compared marine bacterial species with 1,152 reference bacterial species from a variety of habitats and found evidence of extra response regulators in marine genomes. Examining the location of TCS genes along the circular bacterial genome, we also found that marine bacteria have a large number of “orphan” genes, as well as many hybrid histidine kinases. The prevalence of “extra” response regulators, orphan genes, and hybrid TCS systems suggests that marine bacteria break with traditional understanding of how TCS systems operate. These trends suggest prevalent regulatory networking, which may allow coordinated physiological responses to multiple environmental signals and may represent a specific adaptation to the marine environment. We examine phylogenetic and lifestyle traits that influence the number and structure of two-component systems in the genome, finding, for example, that a lack of two-component systems is a hallmark of oligotrophy. Finally, in an effort to demonstrate the importance of TCS systems to marine biogeochemistry, we examined the distribution ofProchlorococcus/Synechococcusresponse regulator PMT9312_0717 in metaproteomes of the tropical South Pacific. We found that this protein’s abundance is related to phosphate concentrations, consistent with a putative role in phosphate regulation.IMPORTANCEMarine microbes must manage variation in their chemical, physical, and biological surroundings. Because they directly link bacterial physiology to environmental changes, TCS systems are crucial to the bacterial cell. This study surveyed TCS systems in a large number of marine bacteria and identified key phylogenetic and lifestyle patterns in environmental sensing. We found evidence that, in comparison with bacteria as a whole, marine organisms have irregular TCS system constructs which might represent an adaptation specific to the marine environment. Additionally, we demonstrate the biogeochemical relevance of TCS systems by correlating the presence of the PMT9312_0717 response regulator protein to phosphate concentrations in the South Pacific. We highlight that despite their potential ecological and biogeochemical relevance, TCS systems have been understudied in the marine ecosystem. This report expands our understanding of the breadth of bacterial TCS systems and how marine bacteria have adapted to survive in their unique environment.

Download Full-text