Kinetic Characterization of the WalRKSpn (VicRK) Two-Component System of Streptococcus pneumoniae: Dependence of WalKSpn (VicK) Phosphatase Activity on Its PAS Domain

ABSTRACT The WalRK two-component system plays important roles in maintaining cell wall homeostasis and responding to antibiotic stress in low-GC Gram-positive bacteria. In the major human pathogen, Streptococcus pneumoniae, phosphorylated WalR Spn (VicR) response regulator positively controls the transcription of genes encoding the essential PcsB division protein and surface virulence factors. WalR Spn is phosphorylated by the WalK Spn (VicK) histidine kinase. Little is known about the signals sensed by WalK histidine kinases. To gain information about WalK Spn signal transduction, we performed a kinetic characterization of the WalRK Spn autophosphorylation, phosphoryltransferase, and phosphatase reactions. We were unable to purify soluble full-length WalK Spn . Consequently, these analyses were performed using two truncated versions of WalK Spn lacking its single transmembrane domain. The longer version (Δ35 amino acids) contained most of the HAMP domain and the PAS, DHp, and CA domains, whereas the shorter version (Δ195 amino acids) contained only the DHp and CA domains. The autophosphorylation kinetic parameters of Δ35 and Δ195 WalK Spn were similar [Km (ATP) ≈ 37 μM; k cat ≈ 0.10 min−1] and typical of those of other histidine kinases. The catalytic efficiency of the two versions of WalK Spn ∼P were also similar in the phosphoryltransfer reaction to full-length WalR Spn . In contrast, absence of the HAMP-PAS domains significantly diminished the phosphatase activity of WalK Spn for WalR Spn ∼P. Deletion and point mutations confirmed that optimal WalK Spn phosphatase activity depended on the PAS domain as well as residues in the DHp domain. In addition, these WalK Spn DHp domain and ΔPAS mutations led to attenuation of virulence in a murine pneumonia model.

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The pneumococcal two-component system VisRH is linked to enhanced intracellular survival of Streptococcus pneumoniae in influenza-infected pneumocytes

10.1101/767855 ◽

2019 ◽

Author(s):

Nicolás M. Reinoso-Vizcaíno ◽

Melina B. Cian ◽

Paulo R. Cortes ◽

Nadia B. Olivero ◽

Mirelys Hernandez-Morfa ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Stress Response ◽

Influenza A Virus ◽

Pandemic Influenza ◽

Influenza A ◽

Intracellular Survival ◽

Bacterial Pathogenesis ◽

Two Component System ◽

Component System ◽

Two Component

AbstractThe virus-bacterial synergism implicated in secondary bacterial infections caused by Streptococcus pneumoniae following infection with epidemic or pandemic influenza A virus (IAV) is well documented. However, the molecular mechanisms behind such synergism remain largely ill-defined. In pneumocytes infected with influenza A virus, subsequent infection with S. pneumoniae leads to enhanced pneumococcal intracellular survival. The pneumococcal two-component system VisRH appears essential for such enhanced survival. Through comparative transcriptomic analysis between the ΔvisR and wt strains, a list of 179 differentially expressed genes was defined. Among those, the clpL protein chaperone gene and the psaB Mn+2 transporter gene, which are involved in the stress response, are important in enhancing S. pneumoniae survival in influenza-infected cells. The ΔvisR, ΔclpL and ΔpsaB deletion mutants display increased susceptibility to acidic and oxidative stress and no enhancement of intracellular survival in IAV-infected pneumocyte cells. These results suggest that the VisRH two-component system senses IAV-induced stress conditions and controls adaptive responses that allow survival of S. pneumoniae in IAV-infected pneumocytes.Author summaryS. pneumoniae is an inhabitant of the human nasopharynx that is capable of causing a variety of infections contributing to an estimated 1.6 million deaths each year. Many of these deaths occur as result of secondary S. pneumoniae infections following seasonal or pandemic influenza. Although S. pneumoniae is considered a typical extracellular pathogen, an intracellular survival mechanism has been more recently recognized as significant in bacterial pathogenesis. The synergistic effects between influenza A and S. pneumoniae in secondary bacterial infection are well documented; however, the effects of influenza infections on intracellular survival of S. pneumoniae are ill-defined. Here, we provide evidence that influenza infection increases S. pneumoniae intracellular survival in pneumocytes. We demonstrate that the poorly understood VisRH signal transduction system in pneumococcus controls the expression of genes involved in the stress response that S. pneumoniae needs to increase intracellular survival in influenza A-infected pneumocytes. These findings have important implications for understanding secondary bacterial pathogenesis following influenza and for the treatment of such infections in influenza-stricken patients.

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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.

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Phosphatase activity tunes two-component system sensor detection threshold

Nature Communications ◽

10.1038/s41467-018-03929-y ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 28

Author(s):

Brian P. Landry ◽

Rohan Palanki ◽

Nikola Dyulgyarov ◽

Lucas A. Hartsough ◽

Jeffrey J. Tabor

Keyword(s):

Phosphatase Activity ◽

Detection Threshold ◽

Two Component System ◽

Component System ◽

Two Component ◽

Sensor Detection

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Quantitative Kinetic Analyses of Shutting Off a Two-Component System

mBio ◽

10.1128/mbio.00412-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 13

Author(s):

Rong Gao ◽

Ann M. Stock

Keyword(s):

Phosphatase Activity ◽

Response Regulator ◽

Two Component System ◽

Component System ◽

Signaling Systems ◽

Cellular Environment ◽

Two Component ◽

The Impact

ABSTRACT Cells rely on accurate control of signaling systems to adapt to environmental perturbations. System deactivation upon stimulus removal is as important as activation of signaling pathways. The two-component system (TCS) is one of the major bacterial signaling schemes. In many TCSs, phosphatase activity of the histidine kinase (HK) is believed to play an essential role in shutting off the pathway and resetting the system to the prestimulus state. Two basic challenges are to understand the dynamic behavior of system deactivation and to quantitatively evaluate the role of phosphatase activity under natural cellular conditions. Here we report a kinetic analysis of the response to shutting off the archetype Escherichia coli PhoR-PhoB TCS pathway using both transcription reporter assays and in vivo phosphorylation analyses. Upon removal of the stimulus, the pathway is shut off by rapid dephosphorylation of the PhoB response regulator (RR) while PhoB-regulated gene products gradually reset to prestimulus levels through growth dilution. We developed an approach combining experimentation and modeling to assess in vivo kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity in vivo , which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed in vitro . We quantitatively modeled how strong the phosphatase activity needs to be to suppress nonspecific phosphorylation in TCSs and discovered that strong phosphatase activity of PhoR is required for cross-phosphorylation suppression. IMPORTANCE Activation of TCSs has been extensively studied; however, the kinetics of shutting off TCS pathways is not well characterized. We present comprehensive analyses of the shutoff response for the PhoR-PhoB system that reveal the impact of phosphatase activity on shutoff kinetics. This allows development of a quantitative framework not only to characterize the phosphatase activity in the natural cellular environment but also to understand the requirement for specific strengths of phosphatase activity to suppress nonspecific phosphorylation. Our model suggests that the ratio of the phosphatase rate to the nonspecific phosphorylation rate correlates with TCS expression levels and the ratio of the RR to HK, which may contribute to the great diversity of enzyme levels and activities observed in different TCSs.

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Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis

Tubercle and Lung Disease ◽

10.1054/tuld.2000.0240 ◽

2000 ◽

Vol 80 (3) ◽

pp. 141-159 ◽

Cited By ~ 131

Author(s):

N. Dasgupta ◽

V. Kapur ◽

K.K. Singh ◽

T.K. Das ◽

S. Sachdeva ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Two Component System ◽

Component System ◽

Two Component

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Characterization of a Novel Two-Component System in Burkholderia cenocepacia

Current Microbiology ◽

10.1007/s00284-014-0744-z ◽

2014 ◽

Vol 70 (4) ◽

pp. 556-561 ◽

Cited By ~ 7

Author(s):

Callie R. Merry ◽

Michael Perkins ◽

Lin Mu ◽

Bridget K. Peterson ◽

Rebecca W. Knackstedt ◽

...

Keyword(s):

Burkholderia Cenocepacia ◽

Two Component System ◽

Component System ◽

Two Component

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Characterization of a Two-component System inStreptococcus pyogenesWhich Is Involved in Regulation of Hyaluronic Acid Production

Journal of Biological Chemistry ◽

10.1074/jbc.274.8.4786 ◽

1999 ◽

Vol 274 (8) ◽

pp. 4786-4793 ◽

Cited By ~ 80

Author(s):

Brian Bernish ◽

Ivo van de Rijn

Keyword(s):

Hyaluronic Acid ◽

Acid Production ◽

Two Component System ◽

Component System ◽

Two Component ◽

Hyaluronic Acid Production

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Functional Determinants of a Small Protein Controlling a Broadly Conserved Bacterial Sensor Kinase

Journal of Bacteriology ◽

10.1128/jb.00305-20 ◽

2020 ◽

Vol 202 (16) ◽

Cited By ~ 3

Author(s):

Srujana S. Yadavalli ◽

Ted Goh ◽

Jeffrey N. Carey ◽

Gabriele Malengo ◽

Sangeevan Vellappan ◽

...

Keyword(s):

Membrane Protein ◽

Pathogenic Bacteria ◽

Regulatory Function ◽

Two Component System ◽

Alanine Scanning Mutagenesis ◽

Histidine Kinases ◽

Component System ◽

Small Protein ◽

Content Type ◽

Two Component

ABSTRACT The PhoQ/PhoP two-component system plays a vital role in the regulation of Mg2+ homeostasis, resistance to acid and hyperosmotic stress, cationic antimicrobial peptides, and virulence in Escherichia coli, Salmonella, and related bacteria. Previous studies have shown that MgrB, a 47-amino-acid membrane protein that is part of the PhoQ/PhoP regulon, inhibits the histidine kinase PhoQ. MgrB is part of a negative-feedback loop modulating this two-component system that prevents hyperactivation of PhoQ and may also provide an entry point for additional input signals for the PhoQ/PhoP pathway. To explore the mechanism of action of MgrB, we analyzed the effects of point mutations, C-terminal truncations, and transmembrane (TM) region swaps on MgrB activity. In contrast to two other known membrane protein regulators of histidine kinases in E. coli, we found that the MgrB TM region is necessary for PhoQ inhibition. Our results indicate that the TM region mediates interactions with PhoQ and that W20 is a key residue for PhoQ/MgrB complex formation. Additionally, mutations of the MgrB cytosolic region suggest that the two N-terminal lysines play an important role in regulating PhoQ activity. Alanine-scanning mutagenesis of the periplasmic region of MgrB further indicated that, with the exception of a few highly conserved residues, most residues are not essential for MgrB’s function as a PhoQ inhibitor. Our results indicate that the regulatory function of the small protein MgrB depends on distinct contributions from multiple residues spread across the protein. Interestingly, the TM region also appears to interact with other noncognate histidine kinases in a bacterial two-hybrid assay, suggesting a potential route for evolving new small-protein modulators of histidine kinases. IMPORTANCE One of the primary means by which bacteria adapt to their environment is through pairs of proteins consisting of a sensor and a response regulator. A small membrane protein, MgrB, impedes the activity of sensor protein PhoQ, thereby affecting the expression of PhoQ regulated virulence genes in pathogenic bacteria. However, it is unknown how such a small protein modulates the activity of PhoQ. Here, we studied the functional determinants of MgrB and identified specific amino acids critical for the protein's inhibitory function. Notably, we find that the membrane-spanning region is important for MgrB interaction with PhoQ. Additionally, this region appears to physically interact with other sensors, a property that may be important for evolving small protein regulators of sensor kinases.

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