The pathogenic Gram-positive bacterium Listeria monocytogenes has been evolving into a few phylogenetic lineages. Phylogenetically defined substitutions were described in the L. monocytogenes virulence factor InlB, which mediates active invasion into mammalian cells via interactions with surface receptors c-Met and gC1q-R. InlB internalin domain (idInlB) is central to interactions with c-Met. Here we compared activity of purified recombinant idInlB isoforms characteristic for L. monocytogenes phylogenetic lineage I and II. Size exclusion chromatography and intrinsic fluorescence were used to characterize idInlBs. Western blotting was used to study activation of c-Met-dependent MAPK- and PI3K/Akt-pathways. Solid-phase microplate binding and competition assay was used to quantify interactions with gCq1-R. Isogenic recombinant L. monocytogenes strains were used to elucidate the input of idInlB isoforms in HEp-2 cell invasion. Physicochemical parameters of idInlB isoforms were similar but not identical. Kinetics of Erk1/2 and Akt phosphorylation in response to purified idInlBs was lineage specific. Lineage I but not lineage II idInlB specifically bound gC1q-R. Antibody against gC1q-R amino acids 221–249 inhibited invasion of L. monocytogenes carrying lineage I but not lineage II idInlB. Taken together, obtained results suggested that phylogenetically defined substitutions in idInlB provide functional distinctions and might be involved in phylogenetically determined differences in virulence potential.

Copper has a strong bactericidal effect against multi-drug resistant pathogens and polyethers are known for their resistance to biofilm formation. Herein, we combined Cu nanoparticles (NPs) and a polyether plasma polymer in the form of nanocomposite thin films and studied whether both effects can be coupled. Cu NPs were produced by magnetron sputtering via the aggregation in a cool buffer gas whereas polyether layers were synthesized by Plasma-Assisted Vapor Phase Deposition with poly(ethylene oxide) (PEO) used as a precursor. In situ specific heat spectroscopy and XPS analysis revealed the formation of a modified polymer layer around the NPs which propagates on the scale of a few nanometers from the Cu NP/polymer interface and then transforms into a bulk polymer phase. The chemical composition of the modified layer is found to be ether-deficient due to the catalytic influence of copper whereas the bulk polymer phase exhibits the chemical composition close to the original PEO. Two cooperative glass transition phenomena are revealed that belong to the modified polymer layer and the bulk phase. The former is characterized by constrained mobility of polymer segments which manifests itself via a 30 K increase of dynamic glass transition temperature. Furthermore, the modified layer is characterized by the heterogeneous structure which results in higher fragility of this layer as compared to the bulk phase. The Cu NPs/polyether thin films exhibit reduced protein adsorption; however, the constrained segmental dynamics leads to the deterioration of the non-fouling properties for ultra-thin polyether coatings. The films are found to have a bactericidal effect against multi-drug resistant Gram-positive Methicillin-Resistant Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa.

Aim To study the effects exerted by argon microwave nonthermal plasma (NTP) on cell wall‐lacking Mollicutes bacteria. Methods and Results 10⁸ CFU ml⁻¹ agar plated Mycoplasma hominis and Acholeplasma laidlawii were treated with the nonthermal microwave argon plasma for 30–300 s. The maximal 10‐ and 100‐fold drop was observed for A. laidlawii and Myc. hominis, respectively. Similarly treated Escherichia coli and Staphylococcus aureus demonstrated the 10⁵ and 10³ drop, respectively. Removal of cholesterol affected resistance of A. laidlawii. 10 mmol l⁻¹ antioxidant butylated hydroxytoluene decreased mortality by a factor of 25–200. UV radiation alone caused 25–85% mortality in comparison with the whole NTP. Exogenously added hydrogen peroxide H₂O₂ did not cause mortality. NTP treatment of Myc. hominis triggered growth of microcolonies, which were several tenfold smaller than a typical colony. Conclusions Despite the lack of cell wall, A. laidlawii and Myc. hominis were more resistant to argon microwave NTP than other tested bacteria. Mycoplasma hominis formed microcolonies upon NTP treatment. A role of UV and active species was demonstrated. Significance and Impact of the Study The first study of NTP effects on Mollicutes revealed importance of a membrane composition for bacterial resistance to NTP. New specific Myc. hominis morphological forms were observed. The study confirmed importance of the concerted action of reactive oxygen species (ROS) with UV and other plasma bioactive agents for NTP bactericidal action.

Background/Objectives: Fluorothiazinone (FT), a small molecule of the 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-one class, is known to inhibit the type III secretion system (T3SS) in Gram-negative bacteria and has shown therapeutic potential in animal models and clinical trials. Given the evolutionary relationship between the T3SS and the bacterial flagellar apparatus, this study aimed to investigate the effects of FT on bacterial motility and flagellum assembly. Methods: Motility was assessed in Pseudomonas aeruginosa, Proteus mirabilis, pathogenic Escherichia coli, and Listeria monocytogenes using a semisolid agar assay and a microfluidic motility system. The mechanism of FT’s action was further examined through time-course analysis, Western blotting of surface flagella proteins, and transmission electron microscopy (TEM). Results: FT inhibited motility of P. aeruginosa, P. mirabilis, and E. coli in a dose-dependent manner, while L. monocytogenes motility remained unaffected. The inhibitory effect was not immediate but delayed 2–3 h post FT addition. Western blotting revealed the absence of surface flagella in EHEC grown with FT, and TEM confirmed structural disruption of flagella in P. mirabilis. Conclusions: FT selectively inhibits flagellum-based motility in Gram-negative bacteria. Obtained data suggested FT interference with flagellum biosynthesis rather than disruption of rotation. Motility inhibition can contribute to FT therapeutic effects on Gram-negative bacterial infections.

Wild aquatic birds are a major reservoir of the influenza A virus in natural ecosystems, facilitating its entry into the aquatic microbial food web through their feces. Free-living protozoa and particularly bacterivorous ciliates are essential players of the microbial food web. This study investigates the interactions between the Influenza A(H1N1)pdm09 virus and the ciliated protozoan Tetrahymena pyriformis at the population and ultrastructural levels. Co-cultivation of Influenza A(H1N1)pdm09 and T. pyriformis resulted in a decline and eventual complete elimination of the viral population. The inactivation of the virus was not mediated by products excreted by T. pyriformis but required A(H1N1)pdm09 endocytosis. Viruses ingested by protozoa lost their virulence within 48 hours post infection (hpi) and, as determined by hemagglutination assays, were entirely inactivated within 72 hpi. When lysates infected with A(H1N1)pdm09 T. pyriformis were applied to MDCK cells 1.5 and 24 hpi the undamaged part of ingested virions caused a cytopathic effect. Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) of infected T. pyriformis cells revealed large food vacuoles, including multiple undamaged and partly processed virus particles, at 1.5 and 24 hpi. Furthermore, TEM identified coated and half-coated small one-virus endosomes that predominated at 48 hpi. These results demonstrated that A(H1N1)pdm09 inactivation by T. pyriformis includes two types of endosomes that dominated at different periods of interpopulation interactions. The process of A(H1N1)pdm09 inactivation in protozoan cells occurs rapidly, but not instantaneously, that suggesting a dual role of protozoa in the fate of influenza A viruses in natural ecosystems, both as predators and as potential vectors.