Isolation and Characterization of Mutants with Altered Sensitivity to the Natural Antimicrobial trans-Cinnamaldehyde in Listeria monocytogenes

Listeria monocytogenes is a Gram-positive, rod-shaped, non-sporulating, and facultative aerobic member of the bacterial Firmicutes phylum, that causes relatively rare but severe foodborne infections. Foodborne listeriosis has a high hospitalization rate and case-fatality rate, particularly among susceptible persons such as elderly persons, immunocompromised individuals, pregnant women, and newborns. Furthermore, L. monocytogenes is a versatile and resilient organism that thrives well in many natural and man-made environments because of its capacity to grow over a wide range of adverse conditions including temperatures below 0°C and salt concentrations up to 10%. The organism is frequently found in raw materials of plant and animal origin used for food production, but it can also establish in the resident house microbiota of food production facilities, and as such, it is a common contaminant during food production and distribution. Prevention of foodborne listeriosis therefore aims at either eradicating the pathogen by heat treatment or preventing its outgrowth to high concentrations in unheated foods or foods that are subject to recontamination after heating. To prevent the outgrowth of L. monocytogenes, but also other pathogens and spoilage organisms, artificial preservatives are widely used, especially in ready-to-eat foods. However, artificial preservatives are increasingly perceived by the consumer as foreign and undesirable additions. Some, like nitrites, are even under scrutiny for possible adverse health effects. In response to these concerns, plant essential oils and their constituents have attracted increased attention as possible alternatives because of their remarkable and broad antimicrobial properties. The present work focuses on trans-cinnamaldehyde (t-CIN), the major antimicrobial compound of cinnamon bark essential oil, which is known to inhibit many foodborne pathogens including L. monocytogenes, and that displays synergetic effects in combination with other food preservatives or processes. The antimicrobial capacity of t-CIN is thought to stem at least partially from its thiol-reactive α,β-unsaturated aldehyde moiety, but the antimicrobial mechanisms and cellular targets of t-CIN are not well established. An in-depth elucidation of these mechanisms and targets can provide a basis for developing more rational and effective food preservation strategies utilizing t-CIN. A genome-wide random Himar1 transposon mutant library of L. monocytogenes Scott A was previously constructed and screened in our laboratory, yielding several mutants with increased t-CIN sensitivity. Two of these mutants, yvcK::Himar1 and asnB::Himar1, manifested severe cell shape deformation that was exacerbated by t-CIN. Also, a fraction of the cells in wild-type L. monocytogenes populations showed distortions upon exposure to a sub-lethal concentration of t-CIN. Although the functions of YvcK and AsnB in L. monocytogenes were not yet known, these observations suggested a possible role in the adaptation of L. monocytogenes to t-CIN by maintaining the cell wall integrity and homeostasis, and both mutants were therefore further studied. The vulnerability of the yvcK::Himar1 mutant to sublethal concentrations of t-CIN was demonstrated to be caused by increased bacterial lysis invoked by severely impaired cell wall integrity. Evolution experiments to restore t-CIN tolerance in the yvcK mutant resulted in suppressor mutations in several genes involved in the biosynthesis of the peptidoglycan precursor uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc), including the small RNA rli73 that locates immediately upstream of the glmU-prs operon, and nagR. GlmU catalyzes the last two steps of UDP-GlcNAc biosynthesis and NagR represses the uptake and utilization of GlcNAc. Overexpression of UDP-GlcNAc biosynthetic enzymes or supplying GlcNAc restored the t-CIN tolerance of the yvcK mutant, pointing to a pivotal role of YvcK in UDP-GlcNAc biosynthesis in L. monocytogenes, similar to its homolog in B. subtilis. An insufficient substrate flux into the UDP-GlcNAc biosynthesis pathway leading to a peptidoglycan synthesis defect can probably explain the hypersensitivity of the yvcK mutant towards t-CIN. Loss of AsnB functionality resulted in the absence of amidated meso-Diaminopimelic acid (mDAP) residues in peptidoglycan, confirming that AsnB, like several of its homologs in Gram-positive bacteria, is a Gln-hydrolyzing amidotransferase that mediates the amidation of the ε-carboxyl group of mDAP. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. Moreover, AsnB inactivation also abrogated the proper cell wall anchoring of internalin A (InlA), an LPXTG motif-containing surface-exposed protein that normally cross-links to peptidoglycan via the free ε-amino group of mDAP, and that mediates host cell entry during the infection process. As a result, the asnB mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. As a complementary approach to the screening of the transposon mutant library, evolutionary experiments were performed by repeated passaging wild-type L. monocytogenes in a sublethal concentration of t-CIN to acquire spontaneous mutants with increased t-CIN resistance. All isolated mutants were found to contain amino acid substitutions in YhfK, a predicted oxidoreductase of the short-chain dehydrogenases/reductases superfamily that has homologs in several Gram-positive bacteria but whose function was unknown. Analysis of a yhfK deletion mutant demonstrated that this protein conferred tolerance of L. monocytogenes to several α,β-unsaturated aldehydes besides t-CIN. The mutant forms of YhfK obtained from the evolution experiment, however, elevated the tolerance to t-CIN, but not to other α,β-unsaturated aldehydes. Using GC-MS analysis, YhfK was shown to be required for the conversion of t-CIN to the less toxic 3-phenylpropanal in L. monocytogenes cultures, suggesting that it is an ene reductase. Deletion of the yhfK homolog also sensitized Bacillus subtilis to both t-CIN and trans-2-hexenal, suggesting a conserved function of YhfK in bacteria, possibly to cope with reactive and toxic α,β-unsaturated aldehydes in their environment. Ene reductases like YhfK may also have applications as stereoselective biocatalysts for the synthesis of valuable chemicals. In conclusion, this work has generated novel insights into the effects of t-CIN and other α,β-unsaturated aldehydes on the foodborne pathogen L. monocytogenes. The biosynthesis of peptidoglycan precursors was identified as a target, and peptidoglycan mDAP amidation was demonstrated to be important for tolerance to these toxic compounds. Furthermore, a bacterial degradation pathway was identified that detoxifies the compounds. Finally, the work also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and bacterial virulence, and as such illustrates that studying how bacteria cope with stress can result in unexpected novel fundamental insights.

Jaar van publicatie:2022

Toegankelijkheid:Open