Multifactor Regulation of the MdtJI Polyamine Transporter in Shigella.

Adriano Leuzzi,Paola Pietrangeli,Mariassunta Casalino,Gianni Prosseda,Lucia Marcocci,Maria Letizia Di Martino,Bianca Colonna,Milena Grossi,Gioacchino Micheli,Marialuisa Barbagallo,Maurizio Falconi,Rosaria Campilongo

doi:10.1371/journal.pone.0136744

Abstract

The polyamine profile of Shigella, the etiological agent of bacillary dysentery in humans, differs markedly from that of E. coli, its innocuous commensal ancestor. Pathoadaptive mutations such as the loss of cadaverine and the increase of spermidine favour the full expression of the virulent phenotype of Shigella. Spermidine levels affect the expression of the MdtJI complex, a recently identified efflux pump belonging to the small multi-drug resistance family of transporters. In the present study, we have addressed the regulation of the mdtJI operon in Shigella by asking which factors influence its expression as compared to E. coli. In particular, after identifying the mdtJI promoter by primer extension analysis, in vivo transcription assays and gel-retardation experiments were carried out to get insight on the silencing of mdtJI in E. coli. The results indicate that H-NS, a major nucleoid protein, plays a key role in repressing the mdtJI operon by direct binding to the regulatory region. In the Shigella background mdtJI expression is increased by the high levels of spermidine typically found in this microorganism and by VirF, the plasmid-encoded regulator of the Shigella virulence regulatory cascade. We also show that the expression of mdtJI is stimulated by bile components. Functional analyses reveal that MdtJI is able to promote the excretion of putrescine, the spermidine precursor. This leads us to consider the MdtJI complex as a possible safety valve allowing Shigella to maintain spermidine to a level optimally suited to survival within infected macrophages and, at the same time, prevent toxicity due to spermidine over-accumulation.

Highlights

Polyamines, such as putrescine, spermidine, and cadaverine, are aliphatic polycationic molecules found in all cells
We investigated whether the MdtJI complex is present in Shigella, a bacterial pathogen which has a high intracellular level of spermidine [16] and shares common evolutionary roots and high genome homology with E. coli [9] but undergoes extensive gene decay as compared to its ancestor [41]
By comparing the genome of Shigella with that of E. coli we find that the genes coding for the mdtJ and mdtI subunits are conserved in Shigella spp. (S. flexneri, S. boydi, S. dysenteriae and S. sonnei) and map at the same location, i.e between the ydgD and tqsA genes

Summary

Introduction

Polyamines, such as putrescine, spermidine, and cadaverine, are aliphatic polycationic molecules found in all cells. PotE and CadB, each constituted by a single protein endowed with twelve transmembrane domains, act as antiporters of putrescine/ornithine and, respectively, cadaverine/lysine. Besides excreting putrescine and cadaverine, PotE and CadB act as uptake proteins for the same polyamines at neutral pH. It has been shown that in E. coli spermidine can be excreted by the MdtJI efflux pump [20] but only when the polyamine over-accumulates within the cell and in the extracellular environment for more than 24 hours. The MdtJI complex belongs to the small multidrug resistance (SMR) family of drug exporters [21] It is encoded by the mdtJI operon which, under physiological conditions, is expressed at a very low level. In Shigella we find that the expression of the mdtJI operon is increased by high levels of spermidine and by the presence of the primary regulator VirF. We find that bile promotes mdtJI expression and that, in a polyamine-free medium, MdtJI confers Shigella the ability to stimulate the excretion of putrescine, the precursor of spermidine

Materials and Methods

Results

Discussion