title: A systematic analysis of the in vitro and in vivo functions of the HD-GYP domain proteins of Vibrio cholerae.
abstract: BACKGROUND
  The second messenger cyclic diguanylate (c-di-GMP) plays a central role in bacterial adaptation to extracellular stimuli, controlling processes such as motility, biofilm development, cell development and, in some pathogens, virulence. The intracellular level of c-di-GMP is controlled by the complementary activities of diguanylate cyclases containing a GGDEF domain and two classes of c-di-GMP phosphodiesterases containing an EAL or HD-GYP hydrolytic domain. Compared to the GGDEF and EAL domains, the functions of HD-GYP domain family proteins are poorly characterized. The human diarrheal pathogen Vibrio cholerae encodes nine putative HD-GYP domain proteins. To determine the contributions of HD-GYP domain proteins to c-di-GMP signaling in V. cholerae, we systematically analyzed the enzymatic functionality of each protein and their involvement in processes known to be regulated by c-di-GMP: motility, biofilm development and virulence.
  
  
  RESULTS
  Complementary in vitro and in vivo experiments showed that four HD-GYP domain proteins are active c-di-GMP phosphodiesterases: VC1295, VC1348, VCA0210 and VCA0681. Mutation of individual HD-GYP domain genes, as well as combinatorial mutations of multiple HD-GYP domain genes, had no effect on motility or biofilm formation of V. cholerae under the conditions tested. Furthermore, no single HD-GYP domain gene affected intestinal colonization by V. cholerae in an infant mouse model. However, inactivation of multiple HD-GYP domain genes, including the four encoding functional phosphodiesterases, significantly attenuated colonization.
  
  
  CONCLUSIONS
  These results indicate that the HD-GYP family of c-di-GMP phosphodiesterases impacts signaling by this second messenger during infection. Altogether, this work greatly furthers the understanding of this important family of c-di-GMP metabolic enzymes and demonstrates a role for HD-GYP domain proteins in the virulence of V. cholerae.
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 125 Mason Farm Rd, CB# 7290, Chapel Hill, NC, USA. robert_mckee@med.unc.edu.
authors.forename: Robert W
authors.initials: RW
authors.lastname: McKee
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 125 Mason Farm Rd, CB# 7290, Chapel Hill, NC, USA. akariisa@gmail.com.
authors.forename: Ankunda
authors.initials: A
authors.lastname: Kariisa
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 125 Mason Farm Rd, CB# 7290, Chapel Hill, NC, USA. benmudrak@gmail.com.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Mudrak
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 125 Mason Farm Rd, CB# 7290, Chapel Hill, NC, USA. cmwhitak@live.unc.edu.
authors.forename: Courtney
authors.initials: C
authors.lastname: Whitaker
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 125 Mason Farm Rd, CB# 7290, Chapel Hill, NC, USA. rtamayo@med.unc.edu.
authors.forename: Rita
authors.initials: R
authors.lastname: Tamayo
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D018441:Biofilms / Q000254:growth & development; D002771:Cholera / Q000382:microbiology / Q000473:pathology; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D004195:Disease Models, Animal; D008124:Locomotion; D051379:Mice; D009154:Mutation; D015398:Signal Transduction; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014774:Virulence
chemical_list: C062025:bis(3',5')-cyclic diguanylic acid; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP