Histidine catabolism and virulence of Brucella: the virB operon coopted regulatory functions of the histidine utilization pathway

RODRIGO SIEIRA; DIEGO J. COMERCI; RODOLFO A. UGALDE

Baeza, España

Workshop; Bacterial Type-IV Secretion Systems in Human Disease; 2008

International University of Andalusia

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:Arial; mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman"; mso-ansi-language:EN-US; mso-fareast-language:EN-US;} h3 {mso-margin-top-alt:auto; margin-right:0cm; mso-margin-bottom-alt:auto; margin-left:0cm; mso-pagination:widow-orphan; mso-outline-level:3; font-size:13.5pt; font-family:Arial; mso-ansi-language:EN-GB; mso-fareast-language:EN-US;} @page Section1 {size:595.45pt 841.7pt; margin:72.0pt 49.7pt 72.0pt 63.8pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Abstract: Histidine catabolism and virulence of Brucella: the virB operon coopted regulatory functions of the histidine utilization pathway   Rodrigo Sieira, Diego J. Comerci, and Rodolfo A. Ugalde. Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín (IIB-INTECH-CONICET), San Martín 1650, Buenos Aires, Argentina.E-mail: rsieira@iib.unsam.edu.ar     The Type-IV Secretion System of Brucella plays an essential role in the pathogenesis of this intracellular pathogen. It is coded by the virB genes, which are arranged as an operon that is tightly regulated in the intracellular environment. After internalization of the bacterium into the host cell, the virB promoter (PvirB) is rapidly activated at 5 hours post infection. Subsequently, the virB expression is repressed before the bacterial multiplication starts in the intracellular replication niche. In order to identify the environmental signals and regulatory pathways that control the intracellular virB expression, we isolated DNA-binding proteins of Brucella that bind specifically to the PvirB. Using ion-exchange columns and electrophoresis mobility shift assays (EMSA) we isolated a 27-kDa protein that binds to PvirB with a high affinity (apparent dissociation constant = 50 nM). Surprisingly, this protein was identified by mass spectrometry as HutC, a transcriptional regulator known to be involved in the regulation of the hut (for histidine utilization) genes, which code for enzymes that catabolize histidine to glutamate in five steps. Competition experiments showed that HutC binds to a region that includes the binding site of IHF, a global regulator which was previously shown to control transcription of the virB operon. EMSA experiments showed that HutC competes with IHF for the binding to PvirB. To study the role of HutC in the transcriptional regulation of the virB operon we constructed in-frame deletion mutants and choromosomal transcriptional fusions between PvirB and lacZ. Analyses of b-Gal activity during intracellular infection of J774 macrophages revealed that HutC exerts a role of co-activator on PvirB. Experiments carried out with bacteria grown in minimal media showed that the expression of the virB operon is HutC-dependent only under defined conditions that resemble the intracellular environment encountered by Brucella within the host cell (pH 4.5 and a nutrient-deprived medium). Our results strongly suggest that during evolution Brucella coopted the regulatory function of HutC to regulate its virulence genes, probably as a way to sense its own metabolic state as an environmental stress signal.