Global Regulation

The two-component system PhoP/PhoR

Two-component regulatory proteins function in bacteria as sensory and adaptive factors in response to a wide range of environmental stimuli. Some two-component systems, such as PhoP/PhoQ, control the transcription of key virulence genes in several intracellular bacterial pathogens, including Salmonella spp., Shigella spp. and Yersinia spp. In collaboration with Carlos Martín’s laboratory (University of Zaragoza, Spain), we have constructed, by allelic replacement, phoP and phoQ mutants of several M. tuberculosis strains. These mutants are impaired for in vivo multiplication. These mutants are not only attenuated, but also protect against a challenge with M. tuberculosis wild type strains.

The particular morphological and cytochemical properties of phoP and phoP-phoR knock-out mutants of M. tuberculosis then led us to investigate the lipid content of these strains. Our results indicated that PhoP coordinately and positively regulates the synthesis of methyl-branched fatty acid-containing acyltrehaloses known to be restricted to the pathogenic species of the M. tuberculosis complex, namely diacyltrehaloses, polyacyltrehaloses, and sulfolipids. The analysis of transcriptional profiles in phoP mutants has evidenced a complex network of regulation including many genes involved in lipid biosynthesis. More in depth genetic studies will define what are the genes that are directly regulated by PhoP/PhoQ. Similarities between M. tuberculosis phoP-phoR mutants and the attenuated laboratory strain M. tuberculosis H37Ra in terms of morphological and cytochemical properties, lipid content, gene expression and virulence attenuation then prompted us to analyze the functionality of this two-component regulator in the latter strain. Sequence analysis revealed a base substitution resulting in a one amino acid change in the likely DNA-binding region of PhoP in H37Ra relative to H37Rv. Using gel-shift assays, and in collaboration with Deshmuk Gopaul’s team (Institut Pasteur), we then showed that this mutation abrogated the ability of the H37Ra PhoP protein to bind to a 40-bp segment of its own promoter. Complementation of H37Ra with phoP from H37Rv fully restored sulfolipid, diacyltrehalose and polyacyltrehalose synthesis clearly indicating that the lack of production of these lipids in H37Ra is solely due to the point mutation in phoP. Finally, using a pks2-3/4 knock-out mutant of M. tuberculosis H37Rv, evidence was further provided that the above mentioned polyketide-derived acyltrehaloses do not significantly contribute to the virulence of the tubercle bacillus in a mouse model of infection. Reasons for the attenuation of H37Ra thus most likely stand in other virulence factors, many of which are expected to belong to the PhoP regulon and another of which, unrelated to PhoP, appears to be the lack of production of DIM in this strain.


The M. tuberculosis PhoP regulon.
The PhoP regulon was identified by comparing transcriptional profiles of the M. tuberculosis wild type and the phoP mutant using DNA microarrays. Some of the more relevant genes to virulence and intracellular survival are listed and grouped by function. Green and red arrows indicate genes whose expression is positively or negatively regulated by PhoP, respectively. (From Martin et al. PLoS ONE 2008)