Copper tolerance of Listeria monocytogenes strain DRDC8.

Abstract

Listeria monocytogenes is one of the most important food-borne pathogens due to the severity of the disease it can cause. While the virulence factors required for effective colonisation and infection of mammalian hosts have been well described, other genes may modulate disease persistence. For L. monocytogenes strain DRDC8, the ctpA gene encodes a copper transporting P-type ATPase that apparently maintains intra-cellular copper ion homeostasis (Francis & Thomas, 1997a) and is also required for persistent infection of the liver and spleens of mice (Francis & Thomas, 1997b). However, the distribution of this gene is apparently limited to non-clinically derived environmental L. monocytogenes isolates (Bell, 2002). This may be explained by carriage of ctpA on plasmid DNA (Bell, 2002). Based on predictions of function and proximity to the ctpA gene (pCT0020), ORFs pCT0017, pCT0018, pCT0019 and ctpA were identified as a putative a cop-like operon involved in copper ion transport in L. monocytogenes (Bell, 2002). Southern hybridisation analysis was used to confirm that the ctpA gene is carried on plasmid pCT100 in strain DRDC8. In addition, evidence to suggest that ctpA was encoded by bacteriophage DNA was not obtained. Furthermore, sequence analysis of DNA flanking ctpA identified ORFs that encode polypeptide sequences similar to proteins involved in plasmid replication and other plasmid-associated functions. Mating experiments provided evidence to show that plasmid pCT100 is not conjugative. This suggested that lateral transfer of this plasmid between cohabitating organisms may be limited. Sequence analysis of a 37.279 kbp region of plasmid pCT100 from L. monocytogenes strain DRDC8 (GenBank Accession U15554) showed this plasmid had regions of gene content and organisation similar to that of other characterised Listeria plasmids, particularly plasmid pLI100 from L. innocua CLIP11262 and plasmid pLM80 from L. monocytogenes strain 4b H7858. Gene’s common to these plasmids included those implicated in plasmid DNA replication, DNA transposition/insertion and heavy metal (cadmium) transport. Sequence analysis of plasmid pCT100 also identified regions of DNA absent from other Listeria sequences. For example, a DNA region encoding a series of polypeptide sequences similar to chromosomally-encoded proteins involved in copper transport in other Gram-positive bacteria was identified. The ORFs encoded by this region (pCT0017, pCT0018, pCT0019 and pCT0020 (ctpA), pCT0023, pCT0024, pCT0025, pCT0026, pCT0027) represent a novel cluster of genes implicated in copper homeostasis/tolerance that had not been previously described for other Listeria spp. PCR analysis was used to show that carriage of this copper gene cluster may be restricted to only some Australian ctpA positive L. monocytogenes isolates, typically of dairy and poultry origin. In addition to these plasmid-encoded ORFs, PCR and sequence analysis identified a chromosomal ORF (cutR) also implicated in copper homeostasis/tolerance for strain DRDC8. cutR encodes a polypeptide similar to chromosomally-encoded coppertranslocating P-type ATPases from other Listeria species. The role of ORFs cutR, pCT0017, pCT0018 and pCT0019 in copper tolerance was assessed by comparison of the ability of wild type parent strain DRDC8 and variants containing independent mutations (pCT0017::erm, pCT0018::erm, pCT0019::erm or cutR::erm) to tolerate copper ion stress. The impact of loss of these genes (as a result of curing strain DRDC8 and cutR::erm derivatives of plasmid pCT100) on copper tolerance by DRDC8 was also examined. Minimal inhibitory concentration (MIC) and growth experiments showed that inactivation of cutR, pCT0018 or pCT0019, or removal of plasmid-encoded genes by curing DRDC8 of plasmid DNA, had a significant effect on copper tolerance. In addition, loss of plasmid DNA combined with disruption of cutR was shown to render cells completely incapable of growth in high levels of copper (14 mM CuSO4). This data indicated that pCT0018, pCT0019 and cutR are involved in copper tolerance of L. monocytogenes strain DRDC8. MIC experiments also provided evidence to show that ORFs cutR and pCT0018 may play an additional role in tolerance to cadmium. Interestingly, a L. monocytogenes mutant carrying an erm insertion within pCT0017 could not be constructed. However, evidence that showed that ORF pCT0017 encodes a CopY-like negative repressor protein directly implicated this ORF in copper tolerance. DNA gel shift experiments were used to show that pCT0017 protein binds to two ‘cop box-like’ nucleotide sequences located upstream of the pCT0017 translation start site. Binding occurs in a copper-dependant manner that is consistent with published models of CopY-like protein function. Thus pCT0017 protein may regulate expression of ORFs pCT0017, pCT0018, pCT0019 and ctpA in a copper responsive manner. This is consistent with the view that these ORFs form a cop-like operon involved in copper homeostasis. In conclusion, L. monocytogenes strain DRDC8 displayed an exceptional tolerance to high concentrations of copper ions. The data obtained suggested that both chromosomal and plasmid-encoded genes are involved in copper homeostasis/tolerance of DRDC8. This particular strain may have acquired multiple genes involved in copper tolerance from a cohabitating Gram-positive bacterium in response to exposure to high levels of copper within the environment. Given that strain DRDC8 is an Australian dairy isolate, these genes may provide a selective advantage for survival of other L. monocytogenes strains in associated environments.