Marcin Bugno

Role of rubrerythrin in the oxidative stress response of Porphyromonas gingivalis

Rubrerythrins are non‐haem iron proteins that have been implicated in oxidative stress protection in anaerobic bacteria and archaea. However, up to now, this role has not been confirmed directly by inactivation of a rubrerythrin gene. Here we report generation of an rbr− mutant of Porphyromonas gingivalis, an obligately anaerobic gingival pathogenic bacterium. Characterization of the rbr− strain clearly showed that P. gingivalis produces a rubrerythrin‐like protein that is absent in the rbr− strain, and that the P. gingivalis rbr− strain is more dioxygen‐ and hydrogen peroxide‐sensitive than the wild type. The latter conclusion is based on two independent results, namely, deeper no‐growth zones upon diffusion of the oxidants through soft agar culture tubes and growth impairment of liquid cultures exposed to the oxidants. A same‐site rbr+ revertant showed increased hydrogen peroxide and dioxygen resistance relative to the rbr− strain. Transcription of the P. gingivalis rubrerythrin gene is induced above its constitutive anaerobic level in response to dioxygen or hydrogen peroxide exposures. Purified rubrerythrins from other organisms have been shown to catalyse reduction of hydrogen peroxide, while showing relatively sluggish reaction with dioxygen and little or no catalase or superoxide dismutase activities. Porphyromonas gingivalis contains a superoxide dismutase but lacks catalase and haem peroxidases. We therefore suggest that rubrerythrin provides oxidative stress protection via catalytic reduction of intracellular hydrogen peroxide.

Reprogramming of TIMP-1 and TIMP-3 expression profiles in brain microvascular endothelial cells and astrocytes in response to proinflammatory cytokines.

Cytokine‐dependent regulation of tissue inhibitors of metalloproteinases (TIMPs) expression provides an important mechanism for controlling the activity of matrix metalloproteinases. We present data indicating that during inflammatory processes TIMP‐1 and TIMP‐3 may be involved in the proteolytic remodeling of subendothelial basement membrane of the brain microvascular system, a key step during leukocyte migration into the brain perivascular tissue. In brain endothelial cells the expression of TIMP‐1 is dramatically up‐regulated by major proinflammatory cytokines, with the combination of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNFα) exhibiting the strongest synergistic stimulation. Simultaneously, IL‐1β/TNFα almost completely blocks TIMP‐3 expression. Both synergistic effects are dose‐dependent within the concentration range 0.05–5 ng/ml of both cytokines and correlate with the expression of inducible nitric oxide synthase, an endothelial cell activation marker. Down‐regulation of TIMP‐3 expression is also detected in astrocytes treated with TNFα or IFN‐γ, whereas oncostatin M as well as TNFα up‐regulate TIMP‐1 mRNA level. We propose that the cytokine‐modified balance between TIMP‐1 and TIMP‐3 expression provides a potential mechanism involved in the regulation of microvascular basement membrane proteolysis.

Emerging Family of Proline-Specific Peptidases of Porphyromonas gingivalis: Purification and Characterization of Serine Dipeptidyl Peptidase, a Structural and Functional Homologue of Mammalian Prolyl Dipeptidyl Peptidase IV

ABSTRACT Porphyromonas gingivalis is an asaccharolytic and anaerobic bacterium that possesses a complex proteolytic system which is essential for its growth and evasion of host defense mechanisms. In this report, we show the purification and characterization of prolyl dipeptidyl peptidase IV (DPPIV) produced by this organism. The enzyme was purified to homogeneity, and its enzymatic activity and biochemical properties were investigated. P. gingivalis DPPIV, like its human counterpart, is able to cleave the N terminus of synthetic oligopeptides with sequences analogous to those of interleukins 1β and 2. Additionally, this protease hydrolyzes biologically active peptides including substance P, fibrin inhibitory peptide, and β-casomorphin. Southern blot analysis of genomic DNA isolated from several P. gingivalis strains reveal that a single copy of the DPPIV gene was present in all strains tested.

A Novel Mechanism of Tissue Inhibitor of Metalloproteinases-1 Activation by Interleukin-1 in Primary Human Astrocytes

Reactive astrogliosis is the gliotic response to brain injury with activated astrocytes and microglia being the major effector cells. These cells secrete inflammatory cytokines, proteinases, and proteinase inhibitors that influence extracellular matrix (ECM) remodeling. In astrocytes, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) is up-regulated by interleukin-1 (IL-1), which is a major neuroinflammatory cytokine. We report that IL-1 activates TIMP-1 expression via both the IKK/NF-κB and MEK3/6/p38/ATF-2 pathways in astrocytes. The activation of the TIMP-1 gene can be blocked by using pharmacological inhibitors, including BAY11-7082 and SB202190, overexpression of the dominant-negative inhibitor of NF-κB (IκBαSR), or by the knock-down of p65 subunit of NF-κB. Binding of activated NF-κB (p50/p65 heterodimer) and ATF-2 (homodimer) to two novel regulatory elements located –2.7 and –2.2 kb upstream of the TIMP-1 transcription start site, respectively, is required for full IL-1-responsiveness. Mutational analysis of these regulatory elements and their weak activity when linked to the minimal tk promoter suggest that cooperative binding is required to activate transcription. In contrast to astrocytes, we observed that TIMP-1 is expressed at lower levels in gliomas and is not regulated by IL-1. We provide evidence that the lack of TIMP-1 activation in gliomas results from either dysfunctional IKK/NF-κB or MEK3/6/p38/ATF-2 activation by IL-1. In summary, we propose a novel mechanism of TIMP-1 regulation, which ensures an increased supply of the inhibitor after brain injury, and limits ECM degradation. This mechanism does not function in gliomas, and may in part explain the increased invasiveness of glioma cells.