Inactivation of the SecA2 protein export pathway in Listeria monocytogenes promotes cell aggregation, impacts biofilm architecture and induces biofilm formation in environmental condition.

Listeria monocytogenes has a dichotomous lifestyle, existing as an ubiquitous saprophytic species and as an opportunistic intracellular pathogen. Besides its capacity to grow in a wide range of environmental and stressful conditions, L. monocytogenes has the ability to adhere to and colonize surfaces. Morphotype variation to elongated cells forming rough colonies has been reported for different clinical and environmental isolates, including biofilms. This cell differentiation is mainly attributed to the reduced secretion of two SecA2-dependent cell-wall hydrolases, CwhA and MurA. SecA2 is a non-essential SecA paralogue forming an alternative translocase with the primary Sec translocon. Following investigation at temperatures relevant to its ecological niches, i.e. infection (37°C) and environmental (20°C) conditions, inactivation of this SecA2-only protein export pathway led, despite reduced adhesion, to the formation of filamentous biofilm with aerial structures. Compared to the wild type strain, inactivation of the SecA2 pathway promoted extensive cell aggregation and sedimentation. At ambient temperature, this effect was combined with the abrogation of cell motility resulting in elongated sedimented cells, which got knotted and entangled together in the course of filamentous-biofilm development. Such a cell differentiation provides a decisive advantage for listerial surface colonization under environmental condition. As further discussed, this morphotypic conversion has strong implication on listerial physiology and is also of potential significance for asymptomatic human/animal carriage.

Mutations in gltB and gltC reduce oxidative stress tolerance and biofilm formation in Listeria monocytogenes 4b G.

The foodborne pathogen Listeria monocytogenes has the capability to persist on surfaces in food-processing environments, and the organism is resistant to environmental stresses. In this study, a Tn917 insertion mutant of L. monocytogenes 4b G showing reduced biofilm formation and sensitivity to oxidative stress was identified and characterized. The transposon insertion site within the gltB gene was identified by inverse PCR. The gltC gene is located upstream and is reported to be transcribed divergently from gltB. Mutants with deletions in gltB and gltC were constructed and both showed reduced biofilm formation and increased sensitivity to H2O2 compared to the wild-type. In the wild-type strain, gltB and gltC expressions were induced approximately 8-fold and 14-fold by quantitative RT-PCR, respectively, with exposure to H2O2, providing further evidence that their gene products may be involved in the response to oxidative stress. In addition, after the induction by H2O2 and compared with the wild-type, the gltB expression in ΔgltC and the gltC expression in ΔgltB were down-regulated about 4-fold (p<0.05) and 3-fold (p<0.05) respectively. These data demonstrate a possible mutual regulation between gltB and gltC expressions under oxidative stress conditions, partly explaining the similar oxidative stress responses of ΔgltB and ΔgltC. Furthermore, ΔgltB and ΔgltC exhibited decreased adherence to a glass surface compared to the wild-type, while the cell motility of wild-type and mutant strains was similar. It is hypothesized that some cell surface characteristics unrelated with cell motility may be introduced into the mutants by the inactivation of gltB or gltC, which might lead to the reduction in biofilm formation. We conclude that both gltB and gltC are involved in the biofilm formation as well as the oxidative stress tolerance in L. monocytogenes 4b G, by pathways that remain yet unclear.

Initial adhesion of Listeria monocytogenes to solid surfaces under liquid flow.

Some strains of the food borne pathogen Listeria monocytogenes persist in food processing environments. The exact reason behind this phenomenon is not known, but strain differences in the ability to adhere to solid surfaces could offer an explanation. In the present work, initial adhesion of nine strains of L. monocytogenes was investigated under liquid flow at two levels of shear stress on six different surfaces using a flow chamber set-up with microscopy measurements. The surfaces tested were glass and PVC, and glass coated with beef extract, casein, and homogenised and unhomogenised milk. In addition, the effect of prior environmental stress (5% NaCl, low nutrient availability) on initial adhesion was investigated. The hydrophobicity of the investigated surfaces was determined by contact angle measurements and the surface properties of the investigated L. monocytogenes strains were determined using Microbial Adhesion To Solvents (MATS). All surfaces with the exception of PVC were found to be hydrophilic. Strain differences were found to significantly influence the initial adhesion rate (IAR) of all nine strains to all the surfaces (p<0.05) at both low and high shear stress. Furthermore, there was a significant effect of the surfaces tested (p<0.05) in the adhesion ability of almost all strains. The IAR was affected by flow rate (shear stress) as seen by a decrease in adhesion at high shear stress for most strains. A significant effect of interactions between strain-surface and strain-shear stress (p<0.001) was observed but not of interactions between surface-shear stress. No correlation between surface hydrophobicity and IAR was observed. Addition of 5% NaCl during propagation resulted in a decrease in IAR whilst propagation in low nutrient media caused an increase indicating a general change in surface characteristics under these conditions. Known persisting strains did not display general better adherence.