Biomedical subjects

Michael J Cromie

Publications and source records attributed to Michael J Cromie.

5 recordsLinked to original sources

PubMed2006

Identification of the lipopolysaccharide modifications controlled by the Salmonella PmrA/PmrB system mediating resistance to Fe(III) and Al(III).

Iron is an essential metal but can be toxic in excess. While several homeostatic mechanisms prevent oxygen-dependent killing promoted by Fe(II), little is known about how cells cope with Fe(III), which kills by oxygen-independent means. Several Gram-negative bacterial species harbour a regulatory system - termed PmrA/PmrB - that is activated by and required for resistance to Fe(III). We now report the identification of the PmrA-regulated determinants mediating resistance to Fe(III) and Al(III) in Salmonella enterica serovar Typhimurium. We establish that these determinants remodel two regions of the lipopolysaccharide, decreasing the negative charge of this major constituent of the outer membrane. Remodelling entails the covalent modification of the two phosphates in the lipid A region with phosphoethanolamine and 4-aminoarabinose, which has been previously implicated in resistance to polymyxin B, as well as dephosphorylation of the Hep(II) phosphate in the core region by the PmrG protein. A mutant lacking the PmrA-regulated Fe(III) resistance genes bound more Fe(III) than the wild-type strain and was defective for survival in soil, suggesting that these PmrA-regulated lipopolysaccharide modifications aid Salmonella's survival and spread in non-host environments.

Aluminum↗

PubMed2006

An RNA sensor for intracellular Mg(2+).

Most RNA molecules require Mg(2+) for their structure and enzymatic properties. Here we report the first example of an RNA serving as sensor for cytoplasmic Mg(2+). We establish that expression of the Mg(2+) transporter MgtA of Salmonella enterica serovar Typhimurium is controlled by its 5' untranslated region (5'UTR). We show that the 5'UTR of the mgtA gene can adopt different stem-loop structures depending on the Mg(2+) levels, which determine whether transcription reads through into the mgtA coding region or stops within the 5'UTR. We could recapitulate the Mg(2+)-regulated transcription using a defined in vitro transcription system with RNA polymerase as the only protein component. The initiation of mgtA transcription responds to extracytoplasmic Mg(2+) and its elongation into the coding region to cytoplasmic Mg(2+), providing a singular example in which the same ligand is sensed in different cellular compartments to regulate disparate steps in gene transcription.

5' Untranslated Regions↗

PubMed2004

Transcriptional control of the antimicrobial peptide resistance ugtL gene by the Salmonella PhoP and SlyA regulatory proteins.

The PhoP/PhoQ two-component system is a master regulator that governs the ability of Salmonella to cause a lethal infection in mice, the adaptation to low Mg(2+) environments, and resistance to a variety of antimicrobial peptides. We have recently established that the PhoP-activated ugtL gene is required for resistance to the antimicrobial peptides magainin 2 and polymyxin B. Here we report that ugtL transcription requires not only the PhoP protein but also the virulence regulatory protein SlyA. The PhoP protein footprinted two regions of the ugtL promoter, mutation of either one of which was sufficient to abolish ugtL transcription. Although the SlyA protein is a transcriptional activator of the ugtL gene, it footprinted the ugtL promoter at a region located downstream of the transcription start site. The PhoP protein footprinted the slyA promoter, indicating that it controls slyA transcription directly. The slyA mutant was hypersensitive to magainin 2 and polymyxin B, suggesting that the virulence attenuation exhibited by slyA mutants may be caused by hypersensitivity to antimicrobial peptides. We propose that the PhoP and SlyA proteins control ugtL transcription using a feed-forward loop design.

Antimicrobial Cationic Peptides↗

PubMed2004

PhoP-regulated Salmonella resistance to the antimicrobial peptides magainin 2 and polymyxin B.

In Salmonella enterica, the PhoP-PhoQ two-component system governs resistance to structurally different antimicrobial peptides including the alpha-helical magainin 2, the beta-sheet defensins and the cyclic lipopeptide polymyxin B. To identify the PhoP-regulated determinants mediating peptide resistance, we prepared a plasmid library from a phoP mutant, introduced it into a phoP mutant and selected for magainin-resistant clones. One of the clones harboured the PhoP-activated ugtL gene, deletion of which rendered Salmonella susceptible to magainin 2 and polymyxin B, but not defensin HNP-1. We established that ugtL encodes an inner membrane protein that promotes the formation of monophosphorylated lipid A in the lipopolysaccharide. Inactivation of both ugtL and the regulatory gene pmrA, which controls lipid A modifications required for resistance to polymxyin B (but not to magainin 2) and is post-transcriptionally activated by the PhoP-PhoQ system, resulted in a strain that was as susceptible to polymyxin B as a phoP mutant. The most frequently recovered clone harboured the yqjA gene, which we show is PhoP regulated and required for resistance to magainin 2 but not to polymyxin B or defensin HNP-1. Our results indicate that different PhoP-mediated modifications in lipid A are necessary for resistance to different antimicrobial peptides.

Anti-Bacterial Agents↗

PubMed2002

Fe(III)-mediated cellular toxicity.

Because it can undergo reversible changes in oxidation state, iron is an excellent biocatalyst but also a potentially deleterious metal. Iron-mediated toxicity has been ascribed to Fe(II), which reacts with oxygen to generate free radicals that damage macromolecules and cause cell death. However, we now report that Fe(III) exhibits microbicidal activity towards strains of Salmonella enterica, Escherichia coli and Klebsiella pneumoniae defective in the Fe(III)-responding PmrA/PmrB signal transduction system. Fe(III) bound to a pmrA Salmonella mutant more effectively than to the isogenic wild-type strain and exerted its microbicidal activity even under anaerobic conditions. Moreover, Fe(III) permeabilized the outer membrane of the pmrA mutant, rendering it susceptible to vancomycin, which is normally non-toxic to Gram-negative species. On the other hand, Fe(III) did not affect the viability of a mutant defective in Fur, the major regulator of cytosolic iron homeostasis, which is hypersensitive to Fe(II)-mediated toxicity. A functional pmrA gene was necessary for bacterial survival in soil. Our results indicate that Fe(III) exerts its microbicidal activity by a mechanism that is oxygen independent and different from that mediated by Fe(II).

Bacterial Proteins↗