Achromobacter xylosoxidans: An Emerging Pathogen Carrying Different Elements Involved in Horizontal Genetic Transfer

In the last few years, numerous cases of multidrug-resistant Achromobacter xylosoxidans infections have been documented in immunocompromised and cystic fibrosis patients. To gain insights into the molecular mechanisms and mobile elements related to multidrug resistance in this bacterium, we studied 24 non-epidemiological A. xylosoxidans clinical isolates from Argentina. Specific primers for plasmids, transposons, insertion sequences, bla _ampC, intI1, and intI2 genes were used in PCR reactions. The obtained results showed the presence of wide host range IncP plasmids in ten isolates and a high dispersion of class 1 integrons (n?=?10) and class 2 integrons (n?=?3). Four arrays in the variable region (vr) of class 1 integrons were identified carrying different gene cassettes as the aminoglycoside resistance aac(6′)-Ib and aadA1, the trimethoprim resistance dfrA1 and dfrA16, and the β-lactamase bla _OXA-2. In only one of the class 2 integrons, a vr was amplified that includes sat2-aadA1. The bla _ampC gene was found in all isolates, confirming its ubiquitous nature. Our results show that A. xylosoxidans clinical isolates contain a rich variety of genetic elements commonly associated with resistance genes and their dissemination. This supports the hypothesis that A. xylosoxidans is becoming a reservoir of horizontal genetic transfer elements commonly involved in spreading antibiotic resistance.     

Melittin modulates keratinocyte function through P2 receptor-dependent ADAM activation

Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecule E-cadherin and release of TGF-α, which was accompanied by transactivation of the EGF receptor and ERK1/2 phosphorylation. This was followed by functional consequences such as increased keratinocyte proliferation and enhanced cell migration. Evidence is provided that ATP release and activation of purinergic P2 receptors are involved in melittin-induced ADAM activation. E-cadherin shedding and EGFR phosphorylation were dose-dependently reduced in the presence of ATPases or P2 receptor antagonists. The involvement of P2 receptors was underscored in experiments with HEK cells, which lack the P2X7 receptor and showed strikingly increased response to melittin stimulation after transfection with this receptor. Our study provides new insight into the mechanism of melittin function which should be of interest particularly in the context of its potential use as an anti-inflammatory or anti-cancer agent.     

Characterization of human phosphodiesterase 12 and identification of a novel 2'-5' oligoadenylate nuclease - The ectonucleotide pyrophosphatase/phosphodiesterase 1

The vertebrate 2-5A system is part of the innate immune response and central to cellular antiviral activities. Upon activation by viral double-stranded RNA, 5′-triphosphorylated, 2′-5′-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2′-5′ oligoadenylate synthetases. The 2-5As bind and activate RNase L, an unspecific endoribonuclease, resulting in viral and cellular RNA decay. Given that most endogenous RNAs are degraded by RNase L, continued enzyme activity will eventually lead to cell growth arrest and cell death. This is averted, when 2-5As and their 5′-dephosphorylated forms, the so-called 2-5A core molecules, are cleaved and thus inactivated by 2′-5′-specific nuclease(s), e.g. phosphodiesterase 12, thereby turning RNase L into its latent form. In this study, we have characterized the human phosphodiesterase 12 in vitro focusing on its ability to degrade 2-5As and 2-5A core molecules. We have found that the enzyme activity is distributive and is influenced by temperature, pH and divalent cations. This allowed us to determine V_max and K_m kinetic parameters for the enzyme. We have also identified a novel 2′-5′-oligoadenylate nuclease; the human plasma membrane-bound ectonucleotide pyrophosphatase/phosphodiesterase 1, suggesting that 2-5A catabolism may be a multienzyme-regulated process.     

Rodent seed predation on tree invader species in grassland habitats of the inland Pampa

Woody plant invasion in grassland ecosystems is a worldwide phenomenon, and biotic interactions as competition and predation have been invoked as a possible barrier to woody encroachment in many ecosystems. We evaluated the role of rodents as seed predators in Pampean grasslands, and we assessed the differences in removal by rodents between one native species, Prosopis caldenia (Caldén) and one exotic species, Gleditisia triacanthos (Honey locust). The experiment was conducted at different phases of the rodent population cycle in two grassland communities, a remnant of a native grassland and a post agriculture grassland (old field). The amount of seed loss caused by predation was estimated by a bait-removal experiment in foraging stations. We estimated the frequency of foraging stations with consumption, the overall amount of seed predation and the individual rate of seed predation. The total amount of seed removal and the individual rate of seed removal were higher for P. caldenia than for G. triacanthos, in the native grassland than in the old field, and in autumn when rodent density was maximum. Overall, the role of rodents on woody seed removal varied according to the plant species and depending on the local conditions that vary through time and space.     

Functional and structural effects of seven-residue deletions on the coiled-coil cytoplasmic domain of a chemoreceptor

Chemoreceptors transmit signals from the environment to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors is strongly conserved and consists of a long alpha-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Changes in this domain during evolution are characterized by the presence of seven-residue insertions/deletions located symmetrically with respect to the hairpin turn, suggesting that specific interactions between the helices that form the hairpin are required for function. We assessed the impact of seven-residue deletions on the signalling ability and higher-order organization of the serine chemoreceptor from Escherichia coli. Our results indicate that symmetry alterations between the two branches of the cytoplasmic hairpin seriously compromise chemoreceptor function. Shorter functional versions of Tsr with symmetrical deletions form mixed trimers of dimers when coexpressed with Tar, the aspartate receptor of E. coli. However, Tar function in those cells is impaired, suggesting that the length difference between receptors introduces non-functional distortions into the chemoreceptor cluster. This observation is reinforced by the analysis of coexpression of Tar with chemoreceptors from Rhodobacter sphaeroides that naturally belong to a shorter-length class.     

Anti-IL-2 treatment impairs the expansion of T(reg) cell population during acute malaria and enhances the Th1 cell response at the chronic disease

Plasmodium chabaudi infection induces a rapid and intense splenic CD4(+) T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (T(reg)) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of T(reg) cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4(+) T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4(+)CD25(+)Foxp3(+) cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4(+) T cells, JES6-1 treatment does not impair effector CD4(+) T cell activation and IFN-γ production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-α and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4(+) T cells from non-treated chronic mice, while it further increased the response of CD4(+) T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of T(reg) cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease.     

Limited effect of anti-rheumatic treatment on 15-prostaglandin dehydrogenase in rheumatoid arthritis synovial tissue

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E₂ (PGE₂) displays an important pathogenic role. The enzymes involved in its synthesis are highly expressed in the inflamed synovium, while little is known about 15- prostaglandin dehydrogenase (15-PGDH) that metabolizes PGE₂. Here we aimed to evaluate the localization of 15-PGDH in the synovial tissue of healthy individuals or patients with inflammatory arthritis and determine the influence of common RA therapy on its expression.

Methods

Synovial tissue specimens from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients were immunohistochemically stained to describe the expression pattern of 15-PGDH. In addition, the degree of enzyme staining was evaluated by computer analysis on stained synovial biopsies from two groups of RA patients, before and after RA specific treatment with either intra-articular glucocorticoids or oral methotrexate therapy. Prostaglandins derived from the cyclooxygenase (COX) pathway were determined by liquid-chromatography mass spectrometry in supernatants from interleukin (IL) 1β-activated fibroblast-like synoviocytes (FLS) treated with methotrexate.

Results

15-PGDH was present in healthy and inflamed synovial tissue, mainly in lining macrophages, fibroblasts and vessels. Intra-articular glucocorticoids showed a trend towards reduced 15-PGDH expression in RA synovium (p = 0.08) while methotrexate treatment left the PGE₂ pathway unaltered both in biopsies ex vivo and in cultured FLS.

Conclusions

Early methotrexate therapy has little influence on the expression of 15-PGDH and on any of the PGE₂ synthesizing enzymes or COX-derived metabolites. Thus therapeutic strategies involving blocking induced PGE₂ synthesis may find a rationale in additionally reducing local inflammatory mediators.