Molecular Mechanisms of HipA-Mediated Multidrug Tolerance and Its Neutralization by HipB

Bacterial multidrug tolerance is largely responsible for the inability of antibiotics to eradicate infections and is caused by a small population of dormant bacteria called persisters. HipA is a critical Escherichia coli persistence factor that is normally neutralized by HipB, a transcription repressor, which also regulates hipBA expression. Here, we report multiple structures of HipA and a HipA-HipB-DNA complex. HipA has a eukaryotic serine/threonine kinase–like fold and can phosphorylate the translation factor EF-Tu, suggesting a persistence mechanism via cell stasis. The HipA-HipB-DNA structure reveals the HipB-operator binding mechanism, ∼70° DNA bending, and unexpected HipA-DNA contacts. Dimeric HipB interacts with two HipA molecules to inhibit its kinase activity through sequestration and conformational inactivation. Combined, these studies suggest mechanisms for HipA-mediated persistence and its neutralization by HipB.

[1]  J. Nyborg,et al.  Protein biosynthesis: structural studies of the elongation cycle , 1998, FEBS letters.

[2]  K. Lewis.  Persister cells, dormancy and infectious disease , 2007, Nature Reviews Microbiology.

[3]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[4]  P. Cossart,et al.  Translation elongation factor EF‐Tu is a target for Stp, a serine‐threonine phosphatase involved in virulence of Listeria monocytogenes , 2005, Molecular microbiology.

[5]  Lode Wyns,et al.  Toxin-antitoxin modules as bacterial metabolic stress managers. , 2005, Trends in biochemical sciences.

[6]  H. Moyed,et al.  Conditional impairment of cell division and altered lethality in hipA mutants of Escherichia coli K-12 , 1988, Journal of bacteriology.

[7]  S. Leibler,et al.  Bacterial Persistence as a Phenotypic Switch , 2004, Science.

[8]  H. Moyed,et al.  Molecular cloning and expression of hipA, a gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis , 1986, Journal of bacteriology.

[9]  T. Hill,et al.  Ectopic Overexpression of Wild-Type and Mutant hipA Genes in Escherichia coli: Effects on Macromolecular Synthesis and Persister Formation , 2006, Journal of bacteriology.

[10]  M. Rooman,et al.  Structural classification of HTH DNA-binding domains and protein-DNA interaction modes. , 1996, Journal of molecular biology.

[11]  S. Harrison,et al.  Structure of a phage 434 Cro/DNA complex , 1988, Nature.

[12]  K. Lewis,et al.  Specialized Persister Cells and the Mechanism of Multidrug Tolerance in Escherichia coli , 2004, Journal of bacteriology.

[13]  J. Kuriyan,et al.  The Conformational Plasticity of Protein Kinases , 2002, Cell.

[14]  P. Cary,et al.  DNA structural deformations in the interaction of the controller protein C.AhdI with its operator sequence , 2007, Nucleic acids research.

[15]  E. Koonin,et al.  Kinase Activity of Overexpressed HipA Is Required for Growth Arrest and Multidrug Tolerance in Escherichia coli , 2006, Journal of bacteriology.

[16]  M. Mardis,et al.  Structure and organization of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis , 1991, Journal of bacteriology.

[17]  H. Moyed,et al.  hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis , 1983, Journal of bacteriology.

[18]  J. Bigger.  TREATMENT OF STAPHYLOCOCCAL INFECTIONS WITH PENICILLIN BY INTERMITTENT STERILISATION , 1944 .

[19]  V. Erdmann,et al.  Phosphorylation of Elongation Factor Tu Prevents Ternary Complex Formation (*) , 1995, The Journal of Biological Chemistry.

[20]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[21]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[22]  K. Lewis,et al.  Persister cells and tolerance to antimicrobials. , 2004, FEMS microbiology letters.

[23]  J. Zheng,et al.  Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[24]  K. Gerdes,et al.  Prokaryotic toxin–antitoxin stress response loci , 2005, Nature Reviews Microbiology.

[25]  L. Johnson,et al.  The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2‐independent roles , 2005, The EMBO journal.

[26]  B. Irwin,et al.  Autoregulation of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis , 1994, Journal of bacteriology.

[27]  K. Lewis.  Multidrug tolerance of biofilms and persister cells. , 2008, Current topics in microbiology and immunology.

[28]  V. Erdmann,et al.  Prokaryotic elongation factor Tu is phosphorylated in vivo. , 1993, The Journal of biological chemistry.