ATP and Formyl Peptides Facilitate Chemoattractant Leukotriene-B4 Synthesis and Drive Calcium Fluxes, Which May Contribute to Neutrophil Swarming at Sites of Cell Damage and Pathogens Invasion.

Here, we demonstrate that human neutrophil interaction with the bacterium Salmonella typhimurium fuels leukotriene B4 synthesis induced by the chemoattractant fMLP. In this work, we found that extracellular ATP (eATP), the amount of which increases sharply during tissue damage, can effectively regulate fMLP-induced leukotriene B4 synthesis. The vector of influence strongly depends on the particular stage of sequential stimulation of neutrophils by bacteria and on the stage at which fMLP purinergic signaling occurs. Activation of 5-lipoxygenase (5-LOX), key enzyme of leukotriene biosynthesis, depends on an increase in the cytosolic concentration of Ca2+. We demonstrate that eATP treatment prior to fMLP, by markedly reducing the amplitude of the fMLP-induced Ca2+ transient jump, inhibits leukotriene synthesis. At the same time, when added with or shortly after fMLP, eATP effectively potentiates arachidonic acid metabolism, including by Ca2+ fluxes stimulation. Flufenamic acid, glibenclamide, and calmodulin antagonist R24571, all of which block calcium signaling in different ways, all suppressed 5-LOX product synthesis in our experimental model, indicating the dominance of calcium-mediated mechanisms in eATP regulatory potential. Investigation into the adhesive properties of neutrophils revealed the formation of cell clusters when adding fMLP to neutrophils exposed to the bacterium Salmonella typhimurium. eATP added simultaneously with fMLP supported neutrophil polarization and clustering. A cell-derived chemoattractant such as leukotriene B4 plays a crucial role in the recruitment of additional neutrophils to the foci of tissue damage or pathogen invasion, and eATP, through the dynamics of changes in [Ca2+]i, plays an important decisive role in fMLP-induced leukotrienes synthesis during neutrophil interactions with the bacterium Salmonella typhimurium.

Redox processes are major regulators of leukotriene synthesis in neutrophils exposed to bacteria Salmonella typhimurium; the way to manipulate neutrophil swarming.

Neutrophils play a primary role in protecting our body from pathogens. When confronted with invading bacteria, neutrophils begin to produce leukotriene B4, a potent chemoattractant that, in cooperation with the primary bacterial chemoattractant fMLP, stimulates the formation of swarms of neutrophils surrounding pathogens. Here we describe a complex redox regulation that either stimulates or inhibits fMLP-induced leukotriene synthesis in an experimental model of neutrophils interacting with Salmonella typhimurium. The scavenging of mitochondrial reactive oxygen species by mitochondria-targeted antioxidants MitoQ and SkQ1, as well as inhibition of their production by mitochondrial inhibitors, inhibit the synthesis of leukotrienes regardless of the cessation of oxidative phosphorylation. On the contrary, antioxidants N-acetylcysteine and sodium hydrosulfide promoting reductive shift in the reversible thiol-disulfide system stimulate the synthesis of leukotrienes. Diamide that oxidizes glutathione at high concentrations inhibits leukotriene synthesis, and the glutathione precursor S-adenosyl-L-methionine prevents this inhibition. Diamide-dependent inhibition is also prevented by diphenyleneiodonium, presumably through inhibition of NADPH oxidase and NADPH accumulation. Thus, during bacterial infection, maintaining the reduced state of glutathione in neutrophils plays a decisive role in the synthesis of leukotriene B4. Suppression of excess leukotriene synthesis is an effective strategy for treating various inflammatory pathologies. Our data suggest that the use of mitochondria-targeted antioxidants may be promising for this purpose, whereas known thiol-based antioxidants, such as N-acetylcysteine, may dangerously stimulate leukotriene synthesis by neutrophils during severe pathogenic infection.

Photodynamic inactivation of bacteria: Why it is not enough to excite a photosensitizer.

BACKGROUND: The development of multidrug resistance (MDR) in infectious agents is one of the most serious global problems facing humanity. Antimicrobial photodynamic therapy (APDT) shows encouraging results in the fight against MDR pathogens, including those in biofilms. METHODS: Photosensitizers (PS), monocationic methylene blue, polycationic and polyanionic derivatives of phthalocyanines, electroneutral and polycationic derivatives of bacteriochlorin were used to study photodynamic inactivation of Gram-positive and Gram-negative planktonic bacteria and biofilms under LED irradiation. Zeta potential measurements, confocal fluorescence imaging, and coarse-grained modeling were used to evaluate the interactions of PS with bacteria. PS aggregation and photobleaching were studied using absorption and fluorescence spectroscopy. RESULTS: The main approaches to ensure high efficiency of bacteria photosensitization are analyzed. CONCLUSIONS: PS must maintain a delicate balance between binding to exocellular and external structures of bacterial cells and penetration through the cell wall so as not to get stuck on the way to photooxidation-sensitive structures of the bacterial cell.

Biofilm matrix proteome of clinical strain of P. aeruginosa isolated from bronchoalveolar lavage of patient in intensive care unit.

Extracellular matrix plays a pivotal role in biofilm biology and proposed as a potential target for therapeutics development. As matrix is responsible for some extracellular functions and influence bacterial cytotoxicity against eukaryotic cells, it must have unique protein composition. P. aeruginosa is one of the most important pathogens with emerging antibiotic resistance, but only a few studies were devoted to matrix proteomes and there are no studies describing matrix proteome for any clinical isolates except reference strains PAO1 and ATCC27853. Here we report the first biofilm matrix proteome of P. aeruginosa isolated from bronchoalveolar lavage of patient in intensive care unit. We have identified the largest number of proteins in the matrix among all published studies devoted to P. aeruginosa biofilms. Comparison of matrix proteome with proteome from embedded cells let us to identify several enriched bioprocess groups. Bioprocess groups with the largest number of overrepresented in matrix proteins were oxidation-reduction processes, proteolysis, and transmembrane transport. The top three represented in matrix bioprocesses concerning the size of the GO annotated database were cell redox homeostasis, nucleoside metabolism, and fatty acid synthesis. Finally, we discuss the obtained data in a prism of antibiofilm therapeutics development.

On the possibility of photodynamic inactivation of tracheobronchial tree pathogenic microbiota using methylene blue (in vitro study).

BACKGROUND: The treatment of patients after mechanical ventilation of lungs suffering from a multi-species infection of the tracheobronchial tree can be complicated.. The situation is aggravated in patients with post-intubation tracheal stenosis, where infection plays a leading pathogenetic role in damage to the tracheal wall. As a result of such a pathological process, cicatricial stenosis of the trachea of purulent-inflammatory infectious genesis or infected tracheal stenosis (ITS) may occur. METHODS: In this work, we studied the possibility of photodynamic inactivation of pathogenic microbiota typical for patients with ITS using methylene blue (MB) as a photosensitizer. RESULTS: 13 clinical isolates of 8 species of bacteria from 9 patients were susceptible to photodynamic inactivation with MB. 30 µM of MB at a light irradiation dose of 25 J/cm2 and incubation with MB for 15 min allows to completely inactivate bacteria found in the tracheobronchial secretions of patients with ITS. CONCLUSIONS: MB retains its optico-physical properties in the range of 3-30 µM and provides effective inactivation of isolated Gram-positive and Gram-negative bacteria, including multi- and pan-resistant to antibiotics.

Magic Peptide: Unique Properties of the LRR11 Peptide in the Activation of Leukotriene Synthesis in Human Neutrophils.

Neutrophil-mediated innate host defense mechanisms include pathogen elimination through bacterial phagocytosis, which activates the 5-lipoxygenase (5-LOX) product synthesis. Here, we studied the effect of synthetic oligodeoxyribonucleotides (ODNs), which mimic the receptor-recognized sites of bacterial (CpG-ODNs) and genomic (G-rich ODNs) DNAs released from the inflammatory area, on the neutrophil functions after cell stimulation with Salmonella typhimurium. A possible mechanism for ODN recognition by Toll-like receptor 9 (TLR9) and RAGE receptor has been proposed. We found for the first time that the combination of the magic peptide LRR11 from the leucine-rich repeat (LRR) of TLR9 with the CpG-ODNs modulates the uptake and signaling from ODNs, in particular, dramatically stimulates 5-LOX pathway. Using thickness shear mode acoustic method, we confirmed the specific binding of CpG-ODNs, but not G-rich ODN, to LRR11. The RAGE receptor has been shown to play an important role in promoting ODN uptake. Thus, FPS-ZM1, a high-affinity RAGE inhibitor, suppresses the synthesis of 5-LOX products and reduces the uptake of ODNs by neutrophils; the inhibitor effect being abolished by the addition of LRR11. The results obtained revealed that the studied peptide-ODN complexes possess high biological activity and can be promising for the development of effective vaccine adjuvants and antimicrobial therapeutics.

Synthetic Hexanucleotides as a Tool to Overcome Excessive Neutrophil Activation Caused by CpG-Containing Oligonucleotides.

Mimicking bacterial DNA, synthetic CpG-containing oligodeoxyribonucleotides (CpG-ODNs) have a powerful immunomodulatory potential. Their practical application is mainly associated with the production of vaccines, where they are used as adjuvants, as well as in local antimicrobial therapy. CpG-ODNs act on a wide variety of immune cells, including neutrophilic granulocytes. On the one hand, the stimulatory effect provides both the direct implementation of their antimicrobial and fungicidal mechanisms, and an avalanche-like strengthening of the immune signal due to interaction with other participants in the immune process. On the other hand, hyperactivation of neutrophilic granulocytes can have negative consequences. In particular, the formation of unreasonably high amounts of reactive oxygen species leads to tissue damages and, as a consequence, a spontaneous aggravation and prolongation of the inflammatory process. Under physiological conditions, a large number of DNA fragments are present in inflammation foci: both of microbial and self-tissue origin. We investigated effects of several short modified hexanucleotides on the main indicators of neutrophil activation, as well as their influence on the immunomodulatory activity of known synthetic CpG-ODNs. The results obtained show that short oligonucleotides partially inhibit the prooxidant effect of synthetic CpG-ODNs without significantly affecting the ability of the latter to overcome bacteria-induced pro-survival effects on neutrophilic granulocytes.

Gram-Negative Bacteria Salmonella typhimurium Boost Leukotriene Synthesis Induced by Chemoattractant fMLP to Stimulate Neutrophil Swarming.

Leukotriene synthesis in neutrophils is critical for host survival during infection. In particular, leukotriene B4 (LTB4) is a powerful neutrophil chemoattractant that plays a crucial role in neutrophil swarming. In this work, we demonstrated that preincubation of human neutrophils with Salmonella typhimurium strongly stimulated LTB4 production induced by the bacterial chemoattractant, peptide N-formyl-L-methionyl-L-leucyl-l-phenylalanine (fMLP), while the reverse sequence of additions was ineffective. Preincubation with bacterial lipopolysaccharide or yeast polysaccharide zymosan particles gives weaker effect on fMLP-induced LTB4 production. Activation of 5-lipoxygenase (5-LOX), a key enzyme in leukotrienes biosynthesis, depends on rise of cytosolic concentration of Ca2+ and on translocation of the enzyme to the nuclear membrane. Both processes were stimulated by S. typhimurium. With an increase in the bacteria:neutrophil ratio, the transformation of LTB4 to ω-OH-LTB4 was suppressed, which further supported increased concentration of LTB4. These data indicate that in neutrophils gathered around bacterial clusters, LTB4 production is stimulated and at the same time its transformation is suppressed, which promotes neutrophil swarming and elimination of pathogens simultaneously.

Integrated into Environmental Biofilm Chromobacterium vaccinii Survives Winter with Support of Bacterial Community.

Chromobacterium species are common in tropical and subtropical zones in environmental samples according to numerous studies. Here, we describe an environmental case of resident Chromobacterium vaccinii in biofilms associated with Carex spp. roots in Moscow region, Russia (warm-summer humid continental climate zone). We performed broad characterization of individual properties as well as surrounding context for better understanding of the premise of C. vaccinii survival during the winter season. Genome properties of isolated strains propose some insights into adaptation to habit and biofilm mode of life, including social cheaters carrying ΔluxR mutation. Isolated C. vaccinii differs from previously described strains in some biochemical properties and some basic characteristics like fatty acid composition as well as unique genome features. Despite potential to modulate membrane fluidity and presence of several genes responsible for cold shock response, isolated C. vaccinii did not survive during exposure to 4 °C, while in the complex biofilm sample, it was safely preserved for at least half a year in vitro at 4 °C. The surrounding bacterial community within the same biofilm with C. vaccinii represented a series of psychrophilic bacterial species, which may share resistance to low temperatures with other species within biofilm and provide C. vaccinii an opportunity to survive during the cold winter season.

Polarization-Sensitive Surface-Enhanced In Situ Photoluminescence Spectroscopy of S. aureus Bacteria on Gold Nanospikes.

We report the possibility of a time-resolved bacterial live/dead dynamics observation with the use of plasmonic nanospikes. Sharp nanospikes, fabricated on a 500-nm thick gold film by laser ablation with the use of 1030-nm femtosecond pulses, were tested as potential elements for antibacterial surfaces and plasmonic luminescence sensors. Staphylococcus aureus bacteria were stained by a live/dead viability kit, with the dead microorganisms acquiring the red colour, caused by the penetration of the luminescent dye propidium iodide through the damaged cell membrane. Photoluminescence was pumped by 515-nm femtosecond laser pulses with linear (Gaussian beam), circular, azimuthal and radial (Laguerre-Gaussian beam) polarizations, exciting the transverse plasmon resonance of the nanospikes and their apex lightning-rod near-field. According to the numerical electrodynamic modeling, the observed strong increase in the photoluminescence yield for radial polarization, while slightly lower for circular and azimuthal polarizations, compared with the low luminescence intensities for the linear laser polarization, was related to their different laser-nanospike coupling efficiencies.

The Potential of Telomeric G-quadruplexes Containing Modified Oligoguanosine Overhangs in Activation of Bacterial Phagocytosis and Leukotriene Synthesis in Human Neutrophils.

Human neutrophils are the first line of defense against bacterial and viral infections. They eliminate pathogens through phagocytosis, which activate the 5-lipoxygenase (5-LOX) pathway resulting in synthesis of leukotrienes. Using HPLC analysis, flow cytometry, and other biochemical methods, we studied the effect of synthetic oligodeoxyribonucleotides (ODNs) able to fold into G-quadruplex structures on the main functions of neutrophils. Designed ODNs contained four human telomere TTAGGG repeats (G4) including those with phosphorothioate oligoguanosines attached to the end(s) of G-quadruplex core. Just modified analogues of G4 was shown to more actively than parent ODN penetrate into cells, improve phagocytosis of Salmonella typhimurium bacteria, affect 5-LOX activation, the cytosol calcium ion level, and the oxidative status of neutrophils. As evident from CD and UV spectroscopy data, the presence of oligoguanosines flanking G4 sequence leads to dramatic changes in G-quadruplex topology. While G4 folds into a single antiparallel structure, two main folded forms have been identified in solutions of modified ODNs: antiparallel and dominant, more stable parallel. Thus, both the secondary structure of ODNs and their ability to penetrate into the cytoplasm of cells are important for the activation of neutrophil cellular effects. Our results offer new clues for understanding the role of G-quadruplex ligands in regulation of integral cellular processes and for creating the antimicrobial agents of a new generation.

Surface-Enhanced IR-Absorption Microscopy of Staphylococcus aureus Bacteria on Bactericidal Nanostructured Si Surfaces.

Surface-enhanced IR absorption (SEIRA) microscopy was used to reveal main chemical and physical interactions between Staphylococcus aureus bacteria and different laser-nanostructured bactericidal Si surfaces via simultaneous chemical enhancement of the corresponding IR-absorption in the intact functional chemical groups. A cleaner, less passivated surface of Si nanoripples, laser-patterned in water, exhibits much stronger enhancement of SEIRA signals compared to the bare Si wafer, the surface coating of oxidized Si nanoparticles and oxidized/carbonized Si (nano) ripples, laser-patterned in air and water. Additional very strong bands emerge in the SEIRA spectra on the clean Si nanoripples, indicating the potential chemical modifications in the bacterial membrane and nucleic acids during the bactericidal effect.

Synthetic CpG oligonucleotides as potential modulators of neutrophil survival in PAMP-associated inhibition of apoptosis.

We studied the effects of a synthetic CpG oligonucleotide (CpG ODN2006) on polymorphonuclear leukocyte (PMNL, neutrophil) survival and oxidant status. CpG ODN2006 showed a dose-dependent effect on the apoptosis of resting neutrophils. Without affecting the viability of resting cells, low concentrations of CpG ODN2006 interfered with Salmonella typhimurium-mediated viability prolongation and increased neutrophil apoptosis to control levels. CpG ODN2006 stimulated neutrophil apoptosis by enhancing ROS generation. Even small doses of ODN could induce the production of intracellular superoxide anions. The high superoxide reactogenicity, including with respect to nitrogen oxide, led to increased levels of intracellular ROS and RNS, which ultimately caused apoptosis. The pro-oxidant effect of low concentrations of CpG ODN2006 was not sufficient to trigger irreversible pro-apoptotic mechanisms. However, the sensitivity of PMNLs to ODN2006, a modulator of apoptosis, increased significantly under conditions of infectious inflammation. Inactivated S. typhimurium proved to be suitable for simulating inflammatory conditions in vitro.

G-quadruplex-forming oligodeoxyribonucleotides activate leukotriene synthesis in human neutrophils.

Human polymorphonuclear leukocytes (PMNLs, neutrophils) play a major role in the immune response to bacterial and fungal infections and eliminate pathogens through phagocytosis. During phagocytosis of microorganisms, the 5-lipoxygenase (5-LOX) pathway is activated resulting in generation of leukotrienes, which mediate host defense. In this study, a library of oligodeoxyribonucleotides (ODNs) with varying numbers of human telomeric repeats (d(TTAGGG)n) and their analogues with phosphorothioate internucleotide linkages and single-nucleotide substitutions was designed. These ODNs with the potential to fold into G-quadruplex structures were studied from structural and functional perspectives. We showed that exogenous G-quadruplex-forming ODNs significantly enhanced 5-LOX metabolite formation in human neutrophils exposed to Salmonella Typhimurium bacteria. However, the activation of leukotriene synthesis was completely lost when G-quadruplex formation was prevented by substitution of guanosine with 7-deazaguanosine or adenosine residues at several positions. To our knowledge, this study is the first to demonstrate that G-quadruplex structures are potent regulators of 5-LOX product synthesis in human neutrophils in the presence of targets of phagocytosis. Communicated by Ramaswamy H. Sarma.

Nitric Oxide in Life and Death of Neutrophils.

BACKGROUND: Nitric Oxide (NO) is a key signalling molecule that has an important role in inflammation. It can be secreted by endothelial cells, neutrophils, and other cells, and once in circulation, NO plays important roles in regulating various neutrophil cellular activities and fate. OBJECTIVE: To describe neutrophil cellular responses influenced by NO and its concomitant compound peroxynitrite and signalling mechanisms for neutrophil apoptosis. METHODS: Literature was reviewed to assess the effects of NO on neutrophils. RESULTS: NO plays an important role in various neutrophil cellular activities and interaction with other cells. The characteristic cellular activities of neutrophils are adhesion and phagocytosis. NO plays a protective role in neutrophil-endothelial interaction by preventing neutrophil adhesion and endothelial cell damage by activated neutrophils. NO suppresses neutrophil phagocytic activity but stimulates longdistance contact interactions through tubulovesicular extensions or cytonemes. Neutrophils are the main source of superoxide, but NO flow results in the formation of peroxynitrite, a compound with high biological activity. Peroxynitrite is involved in the regulation of eicosanoid biosynthesis and inhibits endothelial prostacyclin synthase. NO and peroxynitrite modulate cellular 5-lipoxygenase activity and leukotriene synthesis. Long-term exposure of neutrophils to NO results in the activation of cell death mechanisms and neutrophil apoptosis. CONCLUSION: Nitric oxide and the NO/superoxide interplay fine-tune mechanisms regulating life and death in neutrophils.

Ceruloplasmin-derived peptide is the strongest regulator of oxidative stress and leukotriene synthesis in neutrophils.

Ceruloplasmin, an acute-phase protein, can affect the activity of leukocytes through its various enzymatic activities and protein-protein interactions (with lactoferrin, myeloperoxidase, eosinophil peroxidase, serprocidins, and 5-lipoxygenase (5-LOX), among others). However, the molecular mechanisms of ceruloplasmin activity are not clearly understood. In this study, we tested the ability of two synthetic peptides, RPYLKVFNPR (883-892) (P1) and RRPYLKVFNPRR (882-893) (P2), corresponding to the indicated fragments of the ceruloplasmin sequence, to affect neutrophil activation. Leukotriene (LT) B4 is the primary eicosanoid product of polymorphonuclear leukocytes (PMNLs, neutrophils). We studied leukotriene synthesis in PMNLs upon interaction with Salmonella enterica serovar Typhimurium. Priming of neutrophils with phorbol 12-myristate 13-acetate (PMA) elicited the strong regulatory function of P2 peptide as a superoxide formation inducer and leukotriene synthesis inhibitor. Ceruloplasmin-derived P2 peptide appeared to be a strong inhibitor of 5-LOX product synthesis under conditions of oxidative stress.

Effects of phosphodiester and phosphorothioate ODN2216 on leukotriene synthesis in human neutrophils and neutrophil apoptosis.

Polymorphonuclear leukocytes (PMNLs, neutrophils) play a major role in the initiation and resolution of the inflammatory response, and neutrophil apoptosis is a critical step in resolving inflammation. We examined the effects of oligodeoxynucleotide (ODN) species with different numbers of phosphodiester and phosphorothioate bonds on leukotriene synthesis in PMNLs and on neutrophil apoptosis. Our modifications were based on the well-known ODN2216 molecule (Krug et al., 2001). Treatment of cultured human neutrophils with ODN2216 accelerated apoptosis except in the case of a species with only phosphodiester bonds. The ODNs with poly(g) (phosphorothioate) sequences at both ends and a phosphodiester inner core had maximal effects on leukotriene synthesis in neutrophils and inhibited formation of 5-lipoxygenase metabolites. Addition of phosphodiester and phosphorothioate ODNs to PMNLs produced distinct effects on superoxide and nitric oxide formation: phosphorothioate-containing ODNs concomitantly stimulated production of nitric oxide and superoxide, which may rapidly combine to generate peroxynitrite. Altogether, our results describe strong activation of neutrophil's cellular responses by phosphorothioate ODN2216. We propose that phosphorothioate modification of ODNs represents a potential mechanism of PMNL activation.

Burkholderia contaminans Biofilm Regulating Operon and Its Distribution in Bacterial Genomes.

Biofilm formation by Burkholderia spp. is a principal cause of lung chronic infections in cystic fibrosis patients. A "lacking biofilm production" (LBP) strain B. contaminans GIMC4587:Bct370-19 has been obtained by insertion modification of clinical strain with plasposon mutagenesis. It has an interrupted transcriptional response regulator (RR) gene. The focus of our investigation was a two-component signal transduction system determination, including this RR. B. contaminans clinical and LBP strains were analyzed by whole genome sequencing and bioinformatics resources. A four-component operon (BiofilmReg) has a key role in biofilm formation. The relative location (i.e., by being separated by another gene) of RR and histidine kinase genes is unique in BiofilmReg. Orthologs were found in other members of the Burkholderiales order. Phylogenetic analysis of strains containing BiofilmReg operons demonstrated evidence for earlier inheritance of a three-component operon. During further evolution one lineage acquired a fourth gene, whereas others lost the third component of the operon. Mutations in sensor domains have created biodiversity which is advantageous for adaptation to various ecological niches. Different species Burkholderia and Achromobacter strains all demonstrated similar BiofilmReg operon structure. Therefore, there may be an opportunity to develop a common drug which is effective for treating all these causative agents.

Polymorphonuclear leukocyte apoptosis is accelerated by sulfatides or sulfatides-treated Salmonella Typhimurium bacteria.

Neutrophils die by apoptosis following activation and uptake of microbes or enter apoptosis spontaneously at the end of their lifespan if they do not encounter a pathogen. Here we report that sulfatides or sulfatides-treated Salmonella Typhimurium bacteria accelerated human neutrophil apoptosis. Neutrophil apoptosis was examined by flow cytometry. Sulfatides caused prominent increase in percentage of apoptotic cells after 2.5 hrs of incubation. Salmonella Typhimurium bacteria by themselves did not affect the basal level of apoptosis in neutrophil population. When neutrophils were added to S. Typhimurium "opsonized" by sulfatides, apoptotic index significantly increased, whereas the number of phagocyting cells was not influenced. Sulfatides' proapoptotic effect was strongly dependent on the activity of ß-galactosidase; inhibition of this enzyme impaired its potency to accelerate apoptosis. These data support the mechanism of neutrophil apoptosis triggering based on sulfatides' ability to accumulate in intracellular compartments and mediate successive increase in ceramide content resulting from ß-galactosidase activity.

TNF-alpha and gamma-irradiation induced activation of the Salmonella typhimurium reproduction in the organs of infected animals.

The goal of the current work is to determine the role of the TNF-alpha on the activation of the bacterial growth in an in vivo system. In order to reach this goal we studied the dynamics of the reproduction of vegetative forms of Salmonella and the recultivation of non-cultivating forms of Salmonella in infected animals. Experiments have been conducted both on animals that had been injected with exogenous TNF together with bacterial suspension and on animals that had been exposed to gamma-radiation before infection, since it is known that irradiation increases the secretion of TNF. We demonstrate that in all cases the increase in the level of TNF-alpha in animals leads to the activation of the Salmonella growth. Moreover, in this paper we present the data obtained by the method of molecular display on the identification of genes that are highly expressed in the Salmonella cells cultivated in vitro in the presence of TNF.