Novel Cadmium Resistance Determinant in Listeria monocytogenes

Abstract

Listeria monocytogenes is a foodborne pathogen that can cause severe disease (listeriosis) in susceptible individuals. It is ubiquitous in the environment and often exhibits resistance to heavy metals. One of the determinants that enables Listeria to tolerate exposure to cadmium is the cadAC efflux system, with CadA being a P-type ATPase. Three different cadA genes (designated cadA1 to cadA3) were previously characterized in L. monocytogenes. A novel putative cadmium resistance gene (cadA4) was recently identified through whole-genome sequencing, but experimental confirmation for its involvement in cadmium resistance is lacking. In this study, we characterized cadA4 in L. monocytogenes strain F8027, a cadmium-resistant strain of serotype 4b. By screening a mariner-based transposon library of this strain, we identified a mutant with reduced tolerance to cadmium and that harbored a single transposon insertion in cadA4. The tolerance to cadmium was restored by genetic complementation with the cadmium resistance cassette (cadA4C), and enhanced cadmium tolerance was conferred to two unrelated cadmium-sensitive strains via heterologous complementation with cadA4C. Cadmium exposure induced cadA4 expression, even at noninhibitory levels. Virulence assessments in the Galleria mellonella model suggested that a functional cadA4 suppressed virulence, potentially promoting commensal colonization of the insect larvae. Biofilm assays suggested that cadA4 inactivation reduced biofilm formation. These data not only confirm cadA4 as a novel cadmium resistance determinant in L. monocytogenes but also provide evidence for roles in virulence and biofilm formation.IMPORTANCE Listeria monocytogenes is an intracellular foodborne pathogen causing the disease listeriosis, which is responsible for numerous hospitalizations and deaths every year. Among the adaptations that enable the survival of Listeria in the environment are the abilities to persist in biofilms, grow in the cold, and tolerate toxic compounds, such as heavy metals. Here, we characterized a novel determinant that was recently identified on a larger mobile genetic island through whole-genome sequencing. This gene (cadA4) was found to be responsible for cadmium detoxification and to be a divergent member of the Cad family of cadmium efflux pumps. Virulence assessments in a Galleria mellonella model suggested that cadA4 may suppress virulence. Additionally, cadA4 may be involved in the ability of Listeria to form biofilms. Beyond the role in cadmium detoxification, the involvement of cadA4 in other cellular functions potentially explains its retention and wide distribution in L. monocytogenes.KEYWORDS Listeria monocytogenes, cadmium resistance, cadA, biofilm, virulence L isteria monocytogenes is a Gram-positive rod-shaped bacterium widely distributed in nature (1). As a facultative intracellular foodborne pathogen causing the disease listeriosis, L. monocytogenes organisms are responsible for numerous hospitalizations and deaths in the United States every year (2). T...

Genetic and environmental factors influence Listeria monocytogenes nisin resistance

“…Genes encoding proteins involved in cadmium resistance are also variably present or absent among L. monocytogenes strains (Parsons et al . ), and can be carried on plasmids (Mullapudi et al . ).…”

“…For example, L. monocytogenes strains that possess the LG1 genomic island containing emrE have greater resistance to benzalkonium chloride (Kovacevic et al 2015), a commonly used sanitizer in food processing facilities . Genes encoding proteins involved in cadmium resistance are also variably present or absent among L. monocytogenes strains (Parsons et al 2017), and can be carried on plasmids (Mullapudi et al 2010). Another example is the five-gene stress survival islet (SSI-1), which provides resistance to low pH, high osmolarity and nisin Ryan et al 2010).…”

Dissemination and conservation of cadmium and arsenic resistance determinants in Listeria and other Gram‐positive bacteria

“…However, experimental studies are needed to adequately attribute potential impacts on virulence to the different gene cassettes on these large and complex elements, for example, those mediating cadmium or arsenic resistance. Inactivation of cadA4 resulted in enhanced virulence in the Galleria model, suggesting possible involvement of cadA4 as virulence regulator (Parsons, Lee, Jayeola, & Kathariou, ), but similar targeted studies with the arsenic resistance genes on LGI2 or LGI2‐1 genes are lacking.…”

RNA Helicase Mediates Competitive Fitness of Listeria monocytogenes on the Surface of Cantaloupe

“…Bacterial strains that were used in this study are listed in Table 1. Mutant libraries were constructed as described previously [17,18] in the serotype 1/2b strain 2011L-2858 (hereafter designated as strain 2858), isolated from cantaloupe that were implicated in the 2011 cantaloupe outbreak [1,8].…”

“…Genetic complementation of L1E4 was carried out utilizing Listeria shuttle vector pPL2, as previously described [18,22].…”