Functional analysis of the mrd operon in Salmonella : role in biology and pathogenicity

Abstract

Salmonella are major food-borne pathogens responsible for causing a broad spectrum of human disease, ranging from mild gastroenteritis to life-threatening systemic disease. We are interested in analysing the function of the putative mrd operon in Salmonella, whose gene products include PBP2 and RodA; two proteins which are involved in longitudinal cell wall synthesis and essential for rod shapemaintenance in Salmonella. Although PBP2 is fairly well described, surprisingly little is known about the function of RodA or the other mrd operon proteins. This study aimed to characterise the mrd operon, elucidating its contribution to the biology and pathogenicity of Salmonella. Possible links between the structural integrity of the cell wall and the functionality of wall-spanning virulence organelles were also examined. Precise knockout mutations of the mrd operon genes were constructed in Salmonella enterica serovars Typhi and Typhimurium. A range of subsequent phenotypic analyses have revealed that the round-cell ΔpbpA (PBP2) and ΔrodA mutants, though viable and able to stably propagate, are defective in the expression of flagella and the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS), although the SPI-2 T3SS remains active. These virulence defects were successfully complemented by expressing the major SPI-1 and flagella regulators in trans from inducible promoters. A transposon mutagenesis screen was subsequently developed to search for global regulators of the SPI-1 and motility phenotypes in round-cell mutants. This led to the identification of several candidate regulators, which ongoing work seeks to further analyse. This study has demonstrated that round-cell mrd mutants almost completely down-regulate the expression of key invasion-associated virulence genes, although the cell wall in these mutants remains sufficiently robust to enable the assembly and functioning of wall-spanning multi-protein complexes. Collectively these data provide important insights into both the physiological state of mrd operon mutants, and the function of mrd operon genes.