Functional characterization of PhoPR two component system and its implication in regulating phosphate homeostasis in Bacillus anthracis.
Recent report on importance of phosphate starvation (PS) in Bacillus anthracis (BA) pathogenesis warrants further investigation of the underlying regulatory mechanism. Potential role of PhoPR two component system (TCS) in phosphate homeostasis and virulence of several pathogens necessitates the study of annotated PhoPR in BA.



Expression of phoP and phoR was analyzed using qRT-PCR. PhoPR was characterized biochemically. DNA-protein interaction was analyzed by EMSA. Docking was done to predict PhoPR interacting residues with further validation by mutational studies. pHCMC05 was used to overexpress PhoP in BA.



In silico analysis revealed Bas4483-4484, as putative PhoR-PhoP. Their expression was decreased with increasing phosphate concentration reflecting some role in PS. Both PhoP (response regulator) and PhoR (histidine kinase) showed characteristic property of TCS i.e., autophosphorylation and phosphotransfer. PhoR showed both kinase and phosphatase activity. PhoP bound with promoter of PS marker genes. In silico and in vitro analysis revealed role of PhoRH370and PhoPD10, PhoPD53, PhoPM55in PhoPR interaction. Challenge studies showed decreased survival of mice infected with BApHCMC05-PhoP.



This study affirms that PhoPR forms functional TCS which is upregulated under PS. PhoP binding with promoter of PS marker genes indicates its possible role in regulating PS response. Low survival of mice infected with BApHCMC05-PhoPsuggests its role in BA virulence.



Considering the significance of PS in BA infection, possible role of PhoPR in its regulation and exclusive presence of TCS in prokaryotes, PhoP can be proposed as potential drug target against anthrax.
Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Somya
S
Aggarwal
Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Vikas Kumar
VK
Somani
Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Vatika
V
Gupta
School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Jaspreet
J
Kaur
Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Damini
D
Singh
School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Abhinav
A
Grover
Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India. Electronic address: rakeshbhatnagar@jnu.ac.in.
Rakesh
R
Bhatnagar
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001408:Bacillus anthracis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D003198:Computer Simulation; D004269:DNA, Bacterial / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006706:Homeostasis / Q000187:drug effects; D051379:Mice; D056004:Molecular Dynamics Simulation; D009876:Operon / Q000235:genetics; D010710:Phosphates / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects; D016019:Survival Analysis; D015854:Up-Regulation / Q000187:drug effects; D014774:Virulence / Q000187:drug effects / Q000235:genetics
D000596:Amino Acids; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010710:Phosphates
Bacillus anthracis; Histidine kinase; PhoPR; Phosphate starvation; Response regulator; Two component system