Bacterial hepatocyte growth factor receptor agonist stimulates hepatocyte proliferation and accelerates liver regeneration in a partial hepatectomy rat model.

Hepatocyte growth factor (HGF) is central to liver regeneration. The Internalin B (InlB) protein is a virulence factor produced by the pathogenic bacterium Listeria monocytogenes. InlB is known to mimic HGF activity by interacting with the HGF receptor (HGFR) and activating HGFR-controlled signaling pathways. We expressed and purified the HGFR-binding InlB domain, InlB321/15, cloned from the fully virulent clinical L. monocytogenes strain. HGFR and Erk1/2 phosphorylation was determined using Western blotting. The capacity of InlB321/15 to bind HGFR was measured using microscale thermophoresis. Liver regeneration was studied in a model of 70% partial hepatectomy (70%PHx) in male Wistar rats. The nuclear grade parameters were quantified using manual (percentage of binuclear hepatocytes), automated (nuclear diameters), or combined (Ki67 proliferation index) scoring methods. Purified InlB321/15 stimulated HGFR and Erk1/2 phosphorylation and accelerated the proliferation of HepG2 cells. InlB321/15 bound HGFR with Kd = 7.4 ± 1.3 nM. InlB321/15 injected intravenously on the second, fourth, and sixth days after surgery recovered the liver mass and improved the nuclear grade parameters. Seven days post 70% PHx, the liver weight indexes were 2.9 and 2.0%, the hepatocyte proliferation indexes were 19.8 and 0.6%, and the percentages of binucleated hepatocytes were 6.7 and 4.0%, in the InlB321/15-treated and control animals, respectively. Obtained data demonstrated that InlB321/15 improved hepatocyte proliferation and stimulated liver regeneration in animals with 70% hepatectomy.

An In Vitro Model of Nonattached Biofilm-Like Bacterial Aggregates Based on Magnetic Levitation.

Chronic infections are associated with the formation of nonattached biofilm-like aggregates. In vitro models of surface-attached biofilms do not always accurately mimic these processes. Here, we tested a new approach to create in vitro nonattached bacterial aggregates using the principle of magnetic levitation of biological objects placed into a magnetic field gradient. Bacteria grown under magnetic levitation conditions formed nonattached aggregates that were studied with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) and characterized quantitatively. Nonattached aggregates consisted of bacteria submerged into an extracellular matrix and demonstrated features characteristic of biofilms, such as a polymeric matrix that binds Ruby Red and Congo red dyes, a prerequisite of bacterial growth, and increased resistance to gentamicin. Three quantitative parameters were explored to characterize strain-specific potential to form nonattached aggregates: geometric sizes, relative quantities of aggregated and free-swimming bacteria, and Congo red binding. Among three tested Escherichia coli strains, one strain formed nonattached aggregates poorly, and for this strain, all three of the considered parameters were different from those of the other two strains (P < 0.05). Further, we characterized biofilm formation on plastic and agar surfaces by these strains and found that good biofilm formation ability does not necessarily indicate good nonattached aggregate formation ability, and vice versa. The model and quantitative methods can be applied for in vitro studies of nonattached aggregates and modeling bacterial behavior in chronic infections, as it is important to increase our understanding of the role that nonattached bacterial aggregates play in the pathogenesis of chronic diseases.IMPORTANCE An increasing amount of evidence indicates that chronic infections are associated with nonattached biofilm-like aggregates formed by pathogenic bacteria. These aggregates differ from biofilms because they form under low-shear conditions within the volume of biological fluids and they do not attach to surfaces. Here, we describe an in vitro model that provides nonattached aggregate formation within the liquid volume due to magnetic levitation. Using this model, we demonstrated that despite morphological and functional similarities of nonattached aggregates and biofilms, strains that exhibit good biofilm formation might exhibit poor nonattached aggregate formation, suggesting that mechanisms underlying the formation of biofilms and nonattached aggregates are not identical. The magnetic levitation approach can be useful for in vitro studies of nonattached aggregate formation and simulation of bacterial behavior in chronic infections.

Cu nanoparticles constrain segmental dynamics of cross-linked polyethers: a trade-off between non-fouling and antibacterial properties.

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.

Phylogenetically Defined Isoforms of Listeria monocytogenes Invasion Factor InlB Differently Activate Intracellular Signaling Pathways and Interact with the Receptor gC1q-R.

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.

(Heteroarylmethyl)benzoic Acids as a New Class of Bacterial Cystathionine γ-Lyase Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling.

Antibiotic resistance is one of the most serious global health threats. Therefore, there is a need to develop antimicrobial agents with new mechanisms of action. Targeting of bacterial cystathionine γ-lyase (bCSE), an enzyme essential for bacterial survival, is a promising approach to overcome antibiotic resistance. Here, we described a series of (heteroarylmethyl)benzoic acid derivatives and evaluated their ability to inhibit bCSE or its human ortholog hCSE using known bCSE inhibitor NL2 as a lead compound. Derivatives bearing the 6-bromoindole group proved to be the most active, with IC50 values in the midmicromolar range, and highly selective for bCSE over hCSE. Furthermore, none of these compounds showed significant toxicity to HEK293T cells. The obtained data were rationalized by ligand-based and structure-based molecular modeling analyses. The most active compounds were also found to be an effective adjunct to several widely used antibacterial agents against clinically relevant antibiotic-resistant strains of such bacteria as Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The most potent compounds, 3h and 3i, also showed a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. Finally, compound 3i manifested potentiating activity in pneumonia, sepsis, and infected-wound in vivo models.

Motility provides specific adhesion patterns and improves Listeria monocytogenes invasion into human HEp-2 cells.

Listeria monocytogenes is motile at 22°C and non-motile at 37°C. In contrast, expression of L. monocytogenes virulence factors is low at 22°C and up-regulated at 37°C. Here, we studied a character of L. monocytogenes near surface swimming (NSS) motility and its effects on adhesion patterns and invasion into epithelial cells. L. monocytogenes and its saprophytic counterpart L. innocua both grown at 22°C showed similar NSS characteristics including individual velocities, trajectory lengths, residence times, and an asymmetric distribution of velocity directions. Similar NSS patterns correlated with similar adhesion patterns. Motile bacteria, including both pathogenic and saprophytic species, showed a preference for adhering to the periphery of epithelial HEp-2 cells. In contrast, non-motile bacteria were evenly distributed across the cell surface, including areas over the nucleus. However, the uneven distribution of motile bacteria did not enhance the invasion into HEp-2 cells unless virulence factor production was up-regulated by the transient shift of the culture to 37°C. Motile L. monocytogenes grown overnight at 22°C and then shifted to 37°C for 2 h expressed invasion factors at the same level and invaded human cells up to five times more efficiently comparatively with non-motile bacteria grown overnight at 37°C. Taken together, obtained results demonstrated that (i) NSS motility and correspondent peripheral location over the cell surface did not depend on L. monocytogenes virulence traits; (ii) motility improved L. monocytogenes invasion into human HEp-2 cells within a few hours after the transition from the ambient temperature to the human body temperature.

Human Short Peptidoglycan Recognition Protein PGLYRP1/Tag-7/PGRP-S Inhibits Listeria monocytogenes Intracellular Survival in Macrophages.

PGLYRP1/Tag-7/PGRP-S is one of mammalian peptidoglycan recognition proteins (PGRPs). Here, we demonstrate that human recombinant PGLYRP1/Tag-7/PGRP-S potentiates the response of murine macrophage-like ANA-1 cells and human macrophages to facultative intracellular pathogen Listeria monocytogenes. PGLYRP1/Tag-7/PGRP-S binds to the surface of L. monocytogenes and other bacterial cells but has no effect on their growth in culture. While PGLYRP1/Tag-7/PGRP-S treatment modestly enhanced phagocytosis of bacteria by ANA-1 cells, the intracellular survival of PGLYRP1/Tag-7/PGRP-S treated L. monocytogenes was strongly inhibited 2 h after internalization. PGLYRP1/Tag-7/PGRP-S treatment of bacteria boosted oxidative burst induction and increased the level of proinflammatory cytokine IL-6 produced by ANA-1, however, these effects happened too late to be responsible for decreased intracellular survival of bacteria. Our results thus suggest that PGLYRP1/Tag-7/PGRP-S acts as a molecular sensor for detection of L. monocytogenes infection of mammalian cells that leads to increased killing through a mechanism(s) that remains to be defined.

Hepatoprotective Activity of InlB321/15, the HGFR Ligand of Bacterial Origin, in CCI4-Induced Acute Liver Injury Mice.

HGF (hepatocyte growth factor)/HGFR (HGF receptor) signaling pathway is a key pathway in liver protection and regeneration after acute toxic damage. Listeria monocytogenes toxin InlB contains a HGFR-interacting domain and is a functional analog of HGF. The aim of this work was to evaluate the hepatoprotective activity of the InlB HGFR-interacting domain. The recombinant HGFR-interacting domain InlB321/15 was purified from E. coli. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was used to measure InlB321/15 mitogenic activity in HepG2 cells. Activation of MAPK- and PI3K/Akt-pathways was tracked with fluorescent microscopy, Western blotting, and ELISA. To evaluate hepatoprotective activity, InlB321/15 and recombinant human HGF (rhHGF) were intravenously injected at the same concentration of 2 ng·g-1 to BALB/c mice 2 h before liver injury with CCl4. InlB321/15 caused dose-dependent activation of MAPK- and PI3K/Akt-pathways and correspondent mitogenic effects. Both InlB321/15 and rhHGF improved macroscopic liver parameters (liver mass was 1.51, 1.27 and 1.15 g for the vehicle, InlB321/15 and rhHGF, respectively, p < 0.05), reduced necrosis (24.0%, 16.18% and 21.66% of the total area for the vehicle, InlB321/15 and rhHGF, respectively, p < 0.05). Obtained data suggest that InlB321/15 is a promising candidate for a tissue repair agent.

Microcolonies: a novel morphological form of pathogenic Mycoplasma spp.

Introduction. The Mollicutes class unites cell wall lacking bacteria many of which are membrane parasites and opportunistic bacteria.Aim. This study describes a novel morphological form found in the five species belonging to the bacterial class Mollicutes, and referred to as microcolonies (MCs).Methodology. MCs were obtained as described below and characterized with bacteriological and immunological methods, and microscopy.Results. In contrast to typical colonies (TCs), MCs are characterized by tiny propeller-shaped colonies formed by rod-like cells tightly packed in parallel rows. These colonies were observed within routinely cultivated cultures of type strains 7-12 days post-plating. Rod-like cells were visualized using a scanning electron microscope within TCs with a 'fried-egg-like' appearance. MCs were not observed to revert to TCs. MCs were resistant to antibiotics and other treatments effective against TCs. Pure MC cultures were generated in vitro by treatment of Mycoplasma cultures with hyperimmune serum, antibiotics or argon non-thermal plasma. MCs of Mycoplasma hominis strain H-34 were characterized in detail to confirm that they belonged to that species. MCs tested positive via PCR with M. hominis-specific primers, direct fluorescence and epifluorescence tests, and Western blotting with the camel-derived nanobody aMh-FcG2a, which is specific to the MH3620 transporter protein. Meanwhile, MCs behaved differently in standard bacteriological tests. Pure MC cultures were also isolated directly from clinical samples of the serum, synovial liquid and urine of patients within flammatory urogenital tract diseases, asthma or arthritis. In total, 79 independent MC cultures were isolated from clinical samples including M. hominis (n=70), Mycoplasma pneumoniae (n=2), Mycoplasma fermentans (n=2) and Mycoplasma spp. (n=5).Conclusion. MCs play an unknown role in infection pathology and display prominent antibiotic resistance, making them a challenge for the future studies on Mollicutes.

Topical treatment with the bacterium-derived c-Met agonist InlB321/15 accelerates healing in the abrasion wound mouse model.

Studies of factors affecting wound-healing rates are encouraged by a critical need for new treatments to manage an increasing burden of non-healing wounds. The InlB protein produced by the Gram-positive bacterium Listeria monocytogenes is an agonist of the tyrosine kinase receptor c-Met and a functional analog of the hepatocyte growth factor (HGF), which is a mammalian ligand of c-Met. The recombinant InlB321 protein, which is the c-Met-binding InlB domain (amino acids 31-321), was cloned from the L. monocytogenes serovar 4b clinical strain VIMHA015 and serovar 1/2a strain EGDe (InlB321/15 and InlB321/EGDe, respectively). Both InlB321 variants stimulated proliferation of endothelial HUVEC cells. InlB321/15 was more active in Erk1/2 phosphorylation assay, and more potent than InlB321/EGDe in the 2D-scratch wound-healing assay. Scratch closure reached 86%, 29% and 72% for InlB321/15, InlB321/EGDe and HGF, respectively, 72 h post-wounding (p < 0.05). Topically applied glycerol-mixed InlB321/15 (300 µg ml- 1) increased abrasion wound-healing rates in mice. The 50% wound closing time (CT50) was reduced by InlB321/15 (4.18 ± 0.91 days; CI: 3.05; 5.31) compared with control animals (5.51 ± 1.21 days; CI: 4.01; 7.01; p < 0.05). Taken together, obtained results suggested a potential of InlB321/15 as a means of accelerating wound healing.

Naturally occurring InlB variants that support intragastric Listeria monocytogenes infection in mice.

Listeria monocytogenes is a causative agent of foodborne infection in humans and animals. The virulence factor InlB interacts with mammalian receptor c-Met via its internalin domain to provide L. monocytogenes invasion in non-professional phagocytes. Naturally occurring InlB internalin domain variants form four subclusters on the maximal likelihood tree. Four variants belonging to distinct subclusters were cloned into the vector carrying 3΄ and 5΄-flanking sequences to restore full length inlB and expressed in the L. monocytogenes strain EGDeΔinlB. The substitutions Val132Ile, Thr117Ala and Ile138Leu, Thr251Met/Ser were specific for variants 13, 14 and 1, respectively, the variant 9 carried Ser73Asn, Ile91Val, Leu164Pro, Met251Ser/Thr substitutions. All InlB variants improved invasion of the parental strain in murine colon carcinoma C26 cells with 4.6-fold difference between the most and least effective variants (variants 14 and 13, respectively, P < 0.05). Bacterial loads in livers of intragastrically infected mice were 258, 149 and 92 times higher for variant 14, 13 and 1 carrying strains, respectively, than for EGDeΔinlB (P < 0.01). In contrast, the variant 9 did not noticeably improve infection comparatively to the parental strain. Overall, obtained results demonstrated that naturally occurred InlB internalin domain variants differed in their ability to support intragastric infection in mice.

Route of Injection Affects the Impact of InlB Internalin Domain Variants on Severity of Listeria monocytogenes Infection in Mice.

The facultative intracellular pathogen Listeria monocytogenes causes a severe food-borne infection in humans and animals. L. monocytogenes invasion factor InlB interacts with the tyrosine kinase c-Met via the N-terminal internalin domain. Previously, distinct variants of the InlB internalin domain (idInlB) have been described in L. monocytogenes field isolates. Three variants were used to restore full-length InlB expression in the L. monocytogenes strain EGDeΔinlB. Obtained isogenic L. monocytogenes strains were tested in the invasion assay and intravenous, intraperitoneal, and intragastric models of infection in mice. All idInlBs were functional, restored InlB activity as an invasion factor, and improved invasion of the parental strain EGDeΔinlB into human kidney HEK23 cells. Meanwhile, distinct idInlBs provided different mortality rates and bacterial loads in internal organs. When recombinant strains were compared, the variant designated idInlB14 decreased severity of disease caused by intravenous and intraperitoneal bacterial administration, whereas this variant improved intestine colonization and stimulated intragastric infection. Obtained results demonstrated that naturally occurring idInlBs differed in their impact on severity of L. monocytogenes infection in mice in dependence on the infection route.

Atmospheric pressure nonthermal plasmas for bacterial biofilm prevention and eradication.

Biofilms are three-dimensional structures formed by surface-attached microorganisms and their extracellular products. Biofilms formed by pathogenic microorganisms play an important role in human diseases. Higher resistance to antimicrobial agents and changes in microbial physiology make treating biofilm infections very complex. Atmospheric pressure nonthermal plasmas (NTPs) are a novel and powerful tool for antimicrobial treatment. The microbicidal activity of NTPs has an unspecific character due to the synergetic actions of bioactive components of the plasma torch, including charged particles, reactive species, and UV radiation. This review focuses on specific traits of biofilms, their role in human diseases, and those effects of NTP that are helpful for treating biofilm infections. The authors discuss NTP-based strategies for biofilm control, such as surface modifications to prevent bacterial adhesion, killing bacteria in biofilms, and biofilm destruction with NTPs. The unspecific character of microbicidal activity, proven polymer modification and destruction abilities, low toxicity for human tissues and absence of long-living toxic compounds make NTPs a very promising tool for biofilm prevention and control.

Non-thermal argon plasma is bactericidal for the intracellular bacterial pathogen Chlamydia trachomatis.

Non-thermal plasma (NTP) is a flow of partially ionized argon gas at an ambient macroscopic temperature and is microbicidal for bacteria, viruses and fungi. Viability of the Gram-negative obligate intracellular bacterial parasite Chlamydia trachomatis and its host cells was investigated after NTP treatment. NTP treatment of C. trachomatis extracellular elementary bodies (EBs) diminished the concentration of infectious bacteria by a factor of 9×10(4), as established by the parallel infection of murine fibroblast McCoy cells with treated and control EBs. NTP treatment of infected McCoy cells caused disruption of membrane-restricted vacuoles (inclusions), where C. trachomatis intracellular reticulate bodies (RBs) multiply, and a 2×10(6)-fold reduction in the concentration of infectious bacteria. When the samples were covered with magnesium fluoride glass to obstruct plasma particles and UV rays alone were applied, the bactericidal effect was reduced 1.4×10(1)-fold and 5×10(4)-fold for EBs and RBs, respectively. NTP treatment caused the viability of host McCoy cells to diminish by 19%. Therefore, the results obtained demonstrated that (i) both extracellular and intracellular forms of C. trachomatis are sensitive to NTP treatment; (ii) the reduction in concentration of infectious bacteria after NTP treatment of infected cells is superior to the reduction in viability of host cells; and (iii) the effect of NTP on intracellular bacteria does not depend on UV rays.

Bactericidal effects of non-thermal argon plasma in vitro, in biofilms and in the animal model of infected wounds.

Non-thermal (low-temperature) physical plasma is under intensive study as an alternative approach to control superficial wound and skin infections when the effectiveness of chemical agents is weak due to natural pathogen or biofilm resistance. The purpose of this study was to test the individual susceptibility of pathogenic bacteria to non-thermal argon plasma and to measure the effectiveness of plasma treatments against bacteria in biofilms and on wound surfaces. Overall, Gram-negative bacteria were more susceptible to plasma treatment than Gram-positive bacteria. For the Gram-negative bacteria Pseudomonas aeruginosa, Burkholderia cenocepacia and Escherichia coli, there were no survivors among the initial 10(5) c.f.u. after a 5 min plasma treatment. The susceptibility of Gram-positive bacteria was species- and strain-specific. Streptococcus pyogenes was the most resistant with 17 % survival of the initial 10(5) c.f.u. after a 5 min plasma treatment. Staphylococcus aureus had a strain-dependent resistance with 0 and 10 % survival from 10(5) c.f.u. of the Sa 78 and ATCC 6538 strains, respectively. Staphylococcus epidermidis and Enterococcus faecium had medium resistance. Non-ionized argon gas was not bactericidal. Biofilms partly protected bacteria, with the efficiency of protection dependent on biofilm thickness. Bacteria in deeper biofilm layers survived better after the plasma treatment. A rat model of a superficial slash wound infected with P. aeruginosa and the plasma-sensitive Staphylococcus aureus strain Sa 78 was used to assess the efficiency of argon plasma treatment. A 10 min treatment significantly reduced bacterial loads on the wound surface. A 5-day course of daily plasma treatments eliminated P. aeruginosa from the plasma-treated animals 2 days earlier than from the control ones. A statistically significant increase in the rate of wound closure was observed in plasma-treated animals after the third day of the course. Wound healing in plasma-treated animals slowed down after the course had been completed. Overall, the results show considerable potential for non-thermal argon plasma in eliminating pathogenic bacteria from biofilms and wound surfaces.