Interleukin-8 production by THP-1 cells stimulated by Salmonella enterica serovar Typhimurium porins is mediated by AP-1, NF-kappaB and MAPK pathways.

Interleukin-8 (IL-8) is released in response to inflammatory stimuli, such as bacterial products. Either porins or lipopolysaccharide (LPS) stimulated THP-1 cells to release IL-8 after 24 h. We have previously reported that stimulation of monocytic cells with Salmonella enterica serovar Typhimurium porins led to the activation of mitogen-activated protein kinase cascades and of protein tyrosine kinases (PTKs). In this report, we demonstrate, using two potent and selective inhibitors of MEK activation by Raf-1 (PD-098059) and p38 (SB-203580), that both ERK1/2 and p38 pathways play a key role in the production of IL-8 by porins and LPS. Porin-stimulated expression of activating protein 1 (AP-1) and correlated IL-8 release is also inhibited by PD-098059 or SB-203580 indicating that the Raf-1/MEK1-MEK2/MAPK cascade is required for their activation. Also PTKs modulate the pathway that control IL-8 gene expression, in fact its expression is abolished by tyrphostin. By using N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN) an inhibitor of nuclear factor-kappaB (NF-kappaB) activity, we also observed IL-8 release modulation. Our results elucidate some of the molecular mechanisms by which AP-1 and NF-kappaB regulate IL-8 release and open new strategies for the design of specific molecules that will modulate IL-8 effects in various infectious diseases.

Distribution of mecA among methicillin-resistant clinical staphylococcal strains isolated at hospitals in Naples, Italy.

Two hundred and twenty strains of Staphylococcus isolated in Naples, Italy, were surveyed for the distribution of the mecA, the structural gene for penicillin-binding protein 2a, which is the genetic determinant for methicillin-resistance in staphylococci. Screening by a cloned mecA, revealed that of 220 strains, 43 were methicillin-resistant (19.5%) and 177 were methicillin-susceptible (80.5%). Among the 43 resistant strains 23 (53.5%) carried mecA in their genome and 20 (46.5%) did not carry mecA, in spite of their resistance to methicillin. Every group was submitted to the AP-PCR profiling. A quantitative analysis of the patterns divided strains into four different clusters for methicillin-resistant mecA-negative and two different clusters for methicillin-resistant mecA-positive with primer 1, while no clusters were noted with primer 7. We conclude that these clinical isolates from our area, were not found to belong to a single clone, although the predominance of four methicillin-resistant mecA-negative genotypes were noted.

Monocytic activation of protein tyrosine kinase, protein kinase A and protein kinase C induced by porins isolated from Salmonella enterica serovar Typhimurium.

OBJECTIVES: In the present study a monocytic cell line, U937, was used to investigate the possible involvement of protein tyrosine kinases (NT-PTKs), protein kinase A (PKA) and protein kinase C (PKC) in cell signaling pathways following Salmonella enterica serovar Typhimurium porin stimulation. METHODS: Different concentrations of porins and lipopolysaccharide (LPS) were analysed to evaluate changes in PTK activity by a non radioactive tyrosine kinase assay and in PKA and PKC phosphorylation by Western blotting analysis. The inhibitors of PTK, PKA and PKC activation used, were: 3,4-dihydroxybenzylidene-malononitrile (tyrphostin 23), inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase activity; dihychloride (H-89), a selective inhibitor of PKA which is useful to discriminate between the effects of PKC and PKA; diacylglycerol kinase inhibitor II (R59949), which is useful for elucidating roles of PKC; calphostin C, a specific inhibitor of PKC. RESULTS: Porins of the outer membrane of the ST were isolated to be used as a stimulus in the performed experiments. Following porin treatment, a dose-dependent increase in PTK, PKA and PKC activation was observed. U937 monocytes pretreated with inhibitors induced an evident decrease in PTK activity and PKA and PKC phosphorylation pattern in porin stimulated monocytes. CONCLUSIONS: Our data support the important role played by NT-PTK, PKA and PKC in transducing the activating signal in macrophages stimulated with porins through the activation of the mitogen-activated protein kinase (MAPK) pathway that participate in the regulation of gene expression.

p53 and c-myc activation by Pasteurella haemolytica leukotoxin is correlated with bovine mononuclear cells apoptosis.

To analyse the role of Pasteurella haemolytica Leukotoxin (LKT) in the mechanism of apoptotic cell death of bovine lymphocytes, we evaluated DNA fragmentation and p53 and c-myc expression. P. haemolytica strain ATCC 14003 was cultivated for LKT production. DNA fragmentation was analysed by electrophoresis on Agarose gel. DNA strand breaks in individual apoptotic cells were also detected by an in situ Terminal deoxy nucleotidyl Transferase (TdT). The Polymerase Chain Reaction (PCR) procedure was used for verified p53 and c-myc activation by P. haemolytica LKT. LKT was able to induce DNA fragmentation in a dose and time-dependent fashion. The greatest apoptotic effect was obtained using LKT at a concentration of 0.25 U. The results show that p53 and c-myc activation by LKT is correlated with apoptosis of bovine lymphocytes and monocytes. Our data suggest that LKT may have an important role in the bacterial virulence of Pasteurella haemolytica.

Induction of tyrosine phosphorylated proteins in THP-1 cells by Salmonella typhimurium, Pasteurella haemolytica and Haemophilus influenzae porins.

The effect of porins purified from Salmonella typhimurium, Pasteurella haemolytica and Haemophilus influenzae on induction of tyrosine phosphorylation in THP-1 cells and C3H/HeJ macrophage was investigated. Incubation of porins at concentration of 1.0-5.0 microg ml(-1) with either THP-1 or macrophage from C3H/HeJ mice resulted in tyrosine phosphorylation of specific host cell proteins. After porin stimulation a pattern of tyrosine phosphorylated proteins appeared in the soluble cytoplasmic fraction, in the membrane fraction and in the insoluble protein fraction. The observed effects were dependent on the porin concentrations; they reached a maximal expression at 3 min and declined at 60 min. Porin and lipopolysaccharide treatments induce a similar phosphorylation pattern in all of the three cellular fractions studied. A difference can be observed in the cytoplasmic fraction bands of 50-60 kDa, which are more evident after treatment with lipopolysaccharide, and in the insoluble fraction band of 80 kDa and the cytoplasmic fraction band of 250 kDa, which are more evident after treatment with porins. The events of tyrosine protein phosphorylation were present in macrophage from lipopolysaccaride-hyporesponsive C3H/HeJ mice stimulated with porins, while they were markedly reduced when the cells were stimulated with lipopolysaccharide. Staurosporine, genistein and cytochalasin D induced in the three cellular fractions a different inhibition pattern of tyrosine protein phosphorylation in porin stimulated cells. Porins extracted from the three bacterial species investigated behave in a similar way as stimuli more or less potent; Hib porin seems to be the most powerful stimulator and, moreover, it specifically induces phosphorylation of a 55 kDa band.

Haemophilus influenzae porin contributes to signaling of the inflammatory cascade in rat brain.

In the present study we observed that the Haemophilus influenzae type b (Hib) porin, among the different surface bacterial components, is involved in the pathophysiology of bacterial meningitis. This study demonstrates that inoculation of Hib porin into the fourth cerebral ventricle causes the simultaneous expression of interleukin-1alpha (IL-1alpha), tumor necrosis factor alpha (TNF-alpha), and macrophage inflammatory protein 2 (MIP-2) at 6 h after inoculation. At 24 h, the expression of MIP-2 decreases while the expression of IL-1alpha and TNF-alpha increases. The mRNA expression of IL-1alpha, TNF-alpha, and MIP-2 is correlated with injury to the blood-brain barrier as demonstrated by the appearance of serum proteins and leukocytes in cerebrospinal fluid and by the increase in brain water content.

Role of Pasteurella multocida, Pasteurella haemolytica and Salmonella typhimurium porins on inducible nitric oxide release by murine macrophages.

The aim of this study was to verify whether Pasteurella haemolytica, P. multocida and Salmonella typhimurium porins could affect the inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release by murine resident peritoneal macrophages in vitro. We also compared their effect with that elicited by P. haemolytica, P. multocida and S. typhimurium lipopolysaccharide (LPS) whose biological activity is well known. Variations in NO release and iNOS mRNA expression due to variable concentrations of porins were recorded and compared. We also investigated the synergism between bacterial products and interferon gamma (IFN-gamma). With this aim cells were incubated with porins together with murine rIFN-gamma prior to assessing the presence of NO in the supernatant and mRNA analysis. Porins in themselves were not able to induce NO release by resident peritoneal macrophages. Incubation of macrophages with IFN-gamma in the presence of porins increased NO release, whereas incubation in the presence of the arginine analog N(G)-monomethyl-L-arginine (NMA) inhibited NO release. The greatest NO release was obtained using porins at a concentration of 5 microg/mL. Porins, together with IFN-gamma, were also able to upregulate the mRNA expression of iNOS. Our findings suggest that gram-negative porins are able to modulate inflammatory and immunological responses by affecting the release of NO and the expression of iNOS gene in activated macrophages.

Role of Pasteurella multocida porin on cytokine expression and release by murine splenocytes.

The aim of this study was to verify whether Pasteurella multocida porin can affect the expression and release of IL-1alpha, IL-6, TNF-alpha, IL-4, IFN-gamma, IL-10 and IL-12 by murine splenocytes in vitro. P. multocida porin and lipopolysaccharide (LPS) were able to induce the release of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 in a dose-dependent fashion. The greatest release of these cytokines was obtained using P. multocida porin at a concentration of 5 microg ml(-1) and LPS at a concentration of 1 microg ml(-1). The time-courses of release showed that P. multocida LPS was able to stimulate the production of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 earlier than porin and at a greater rate. No effect was observed on IL-4 and IL-10 release under the same experimental conditions. P. multocida porin and LPS were also able to up-regulate the mRNA expression of IL-1alpha, IL-6, TNF-alpha, IFN-gamma and IL-12 p40. Our findings suggest that P. multocida porin is able to modulate inflammatory and immunological responses by affecting the release of several cytokines and the expression of their genes.

Effect of saline concentration, pH and growth temperature on the invasive capacity of Listeria monocytogenes.

The invasive ability of Listeria monocytogenes was monitored after treatment at different pH, temperature and salt concentrations. We found a complete loss of invasive ability in bacteria grown at pH < or = 4.5 independently of the incubation temperature (4, 22 and 30 degrees C). Increasing salt concentrations at 22 and 30 degrees C had no effect at pH 7, while drastically affecting invasive ability at pH 5. The expression of two proteins of 30 and 88 kDa, extracted from the culture supernatant and the cell wall, respectively, was detected only in cells grown under normal conditions, but not after low pH and high salt concentration treatment.