Deep impact of the inactivation of the SecA2-only protein export pathway on the proteosurfaceome of Listeria monocytogenes.

Listeria monocytogenes presents a dimorphism associated to the SecA2 activity with cells having a normal rod shape or a dysmorphic elongated filamentous form. Besides variation of the cell and colony morphotype, this cell differentiation has profound ecophysiological and physiopathological implications with collateral effects on virulence and pathogenicity, biotope colonisation, bacterial adhesion and biofilm formation. This suggests the SecA2-only protein export could influence the listerial cell surface, which was investigated first by characterising its properties in L. monocytogenes wt and ΔsecA2. The degree of hydrophilicity and Lewis acid-base properties appeared significantly affected upon SecA2 inactivation. As modification of electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteosurfaceome was further investigated by shotgun label-free proteomic analysis with a comparative relative quantitative approach. Following secretomic analysis, the protein secretion routes of the identified proteins were mapped considering the cognate transport and post-translocational maturation systems, as well as protein categories and subcellular localisation. Differential protein abundance profiles coupled to network analysis revealed the SecA2 dependence of 48 proteins, including some related to cell envelope biogenesis, translation and protein export, which could account for modifications of adhesion and surface properties of L. monocytogenes upon SecA2 inactivation. This investigation unravelled the profound influence of SecA2 activity on the cell surface properties and proteosurfaceome of L. monocytogenes, which provides advanced insights about its ecophysiopathology. SIGNIFICANCE: L. monocytogenes is a foodborne zoonotic pathogen and etiological agent of human listeriosis. This species presents a cellular dimorphism associated to the SecA2 activity that has profound physiopathological and ecophysiological implications with collateral effects on bacterial virulence and colonisation. To explore the influence of the SecA2-only protein export on the listerial cell, the surface properties of L. monocytogenes expressing or depleted of SecA2 was characterised by microelectrophoresis, microbial affinity to solvents and contact angles analyses. As modifications of hydrophilicity and Lewis acid-base electrostatic properties would owe to modification in the composition of cell-surface proteins, the proteinaceous subset of the surfaceome, i.e. the proteosurfaceome, was investigated further by shotgun label-free proteomic analysis. This subproteome appeared quite impacted upon SecA2 inactivation with the identification of proteins accounting for modifications in the cell surface properties. The profound influence of SecA2 activity on the cell surface of L. monocytogenes was unravelled, which provides advanced insights about its ecophysiopathology.

Molecular Characterization and Comparative Genomics of IncQ-3 Plasmids Conferring Resistance to Various Antibiotics Isolated from a Wastewater Treatment Plant in Warsaw (Poland).

As small, mobilizable replicons with a broad host range, IncQ plasmids are widely distributed among clinical and environmental bacteria. They carry antibiotic resistance genes, and it has been shown that they confer resistance to ß-lactams, fluoroquinolones, aminoglycosides, trimethoprim, sulphonamides, and tetracycline. The previously proposed classification system divides the plasmid group into four subgroups, i.e., IncQ-1, IncQ-2, IncQ-3, and IncQ-4. The last two subgroups have been poorly described so far. The aim of this study was to analyze five newly identified IncQ-3 plasmids isolated from a wastewater treatment plant in Poland and to compare them with all known plasmids belonging to the IncQ-3 subgroup whose sequences were retrieved from the NCBI database. The complete nucleotide sequences of the novel plasmids were annotated and bioinformatic analyses were performed, including identification of core genes and auxiliary genetic load. Furthermore, functional experiments testing plasmid mobility were carried out. Phylogenetic analysis based on three core genes (repA, mobA/repB, and mobC) revealed the presence of three main clusters of IncQ-3 replicons. Apart from having a highly conserved core, the analyzed IncQ-3 plasmids were vectors of antibiotic resistance genes, including (I) the qnrS2 gene that encodes fluoroquinolone resistance and (II) ß-lactam, trimethoprim, and aminoglycoside resistance genes within integron cassettes.

InlL from Listeria monocytogenes Is Involved in Biofilm Formation and Adhesion to Mucin.

The bacterial etiological agent of listeriosis, Listeria monocytogenes, is an opportunistic intracellular foodborne pathogen. The infection cycle of L. monocytogenes is well-characterized and involves several key virulence factors, including internalins A and B. While 35 genes encoding internalins have been identified in L. monocytogenes, less than half of them have been characterized as yet. Focusing on lmo2026, it was shown this gene encodes a class I internalin, InlL, exhibiting domains potentially involved in adhesion. Following a functional genetic approach, InlL was demonstrated to be involved in initial bacterial adhesion as well as sessile development in L. monocytogenes. In addition, InlL enables binding to mucin of type 2, i.e., the main secreted mucin making up the mucus layer, rather than to surface-located mucin of type 1. InlL thus appears as a new molecular determinant contributing to the colonization ability of L. monocytogenes.