Inactivation of the gene encoding the cationic antimicrobial peptide resistance factor MprF increases biofilm formation but reduces invasiveness of Listeria monocytogenes

Abstract

Aims To understand the genetics involved in surface attachment and biofilm formation of Listeria monocytogenes. Methods and Results An in vitro screen of a Himar1 transposon library of L. monocytogenes strain 15G01 identified three transposants that produced significantly different biofilm levels when compared to the wild‐type strain; two mutants exhibited enhanced biofilm formation and one produced less biofilm biomass than the wild‐type. The mutant 15G01 mprF::Himar1, which had a transposon insertion in the mprF gene, was selected for further analysis. The mutant produced a more densely populated biofilm on solid surfaces such as stainless steel and polystyrene, as determined using scanning electron and light microscopy. The 15G01 mprF::Himar1 mutant remained viable in biofilms, but showed an increase in sensitivity to the cationic antimicrobial gallidermin. The mutant also displayed reduced invasiveness in CaCo‐2 intestinal cells, suggesting virulence properties are compromised by the inactivation of mprF. Conclusions Biofilm formation and gallidermin resistance of L. monocytogenes is influenced by mprF, but this trait is associated with a compromise in invasiveness. Significance and Impact of the Study The presence of pathogenic microorganisms in the food processing environment can cause a significant problem, especially when these microorganisms are established as biofilms. This study shows that the inactivation of the mprF gene results in enhanced biofilm formation and abiotic surface attachment of L. monocytogenes.

LysX2 is a Mycobacterium tuberculosis membrane protein with an extracytoplasmic MprF-like domain

“…Additionally, we observed that the presence of MtLysX2 strongly delayed the onset of biofilm development and maturation at pH 5.5, but not at pH 6.8. The biofilm phenotype is consistent with observation that Listeria monocytogenes and Enterococcus faecalis mprF deletion mutants produced significantly more biofilm than the wild type [41,42]. Given the importance of cell surface contacts in biofilm formation, these phenotypes may be related to a modification of net charge on the bacterial cell surface.…”

“…This suggests that exposure to mild acidic pH could have an effect either on the activity of MtLysX2 or on its function. Then we explored the ability of MtLysX2 to interfere with biofilm formation in M. smegmatis, since MprF from Listeria monocytogenes and Enterococcus faecalis were shown to delay the formation of biofilms [41,42]. MS322 and MS321 were cultivated in Sauton medium at pH 6.8 or 5.5 in 12-well plates.…”

Biofilm Formation by Listeria monocytogenes 15G01, a Persistent Isolate from a Seafood-Processing Plant, Is Influenced by Inactivation of Multiple Genes Belonging to Different Functional Groups

“…L. monocytogenes 15G01 was kept as glycerol stocks in a -80°C freezer and recovered in a three-step process by first growing in Tryptic Soy Broth (TSB) enriched with 0.6% yeast extract (TSBYE) (Difco, BD, USA) overnight at 37°C, then secondly plating on Tryptic Soy Agar enriched with 0.6% yeast extract (TSAYE) (Difco, BD, USA) and, lastly, subculturing and storing on Columbian sheep blood agar (Fort Richard, New Zealand) at 4°C. A library of 6,500 mutants of L. monocytogenes 15G01, created by The New Zealand Plant and Food Research Limited using the Himar1 mariner-based transposition system (22) according to a method described previously (76), was kept on 96-well master plates in glycerol at -80°C, subcultured twice before use and stored on TSAYE plates supplemented with erythromycin (Duchefa, Biochemie, The Netherlands) at a final concentration of 5 ppm. Media and agar plates were supplemented with erythromycin at a final concentration of 5 ppm for the studies with the mutants (the transposon contains the erythromycin resistance gene) and with additional kanamycin (MP Biomedicals, Illkirch, France) at a concentration of 50 ppm for the complemented strains.…”

“…The PCR was performed in two steps with the Mastercycler gradient (Eppendorf, Germany). BioMix Red (Bioline, UK) was used as the mastermix in the first round and the second round was run using AccuPrime Hifi Taq polymerase (Invitrogen, US) as described previously (22). The annealing temperature was adjusted for each mutant if necessary to minimise non-specific annealing or to increase annealing.…”

“…This isolate exhibited low invasion in mammalian cell cultures (20), which is linked to truncation of the primary virulence factor internalin A (InlA) (21). As the capacity to form biofilms is believed to be a major contributing factor in persistence of L. monocytogenes and subsequent contamination of food in foodprocessing premises, a library of mutants of L. monocytogenes 15G01, previously generated using the Himar1 mariner-based transposition system (22), was screened for mutants with altered biofilm formation using the crystal violet assay. This study aimed to reveal additional genes in L. monocytogenes 15G01 that are associated with biofilm formation and to further characterise the role of these genes through visualisation of the structure by using scanning electron and fluorescence microscopy to visualise the structure of the altered biofilms.…”