The 20S proteasome alpha(5) subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the Lettuce mosaic potyvirus HcPro protein

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Experimental and theoretical approach of nanocrystalline TiO2 with antifungal activity

Using a solvothermal method for this research we synthesized nanocrystalline titanium dioxide (nc-TiO2) anatase particles with a mean diameter of 5.4 nm and evaluated their potential antifungal effect against planktonic cells of Candida albicans without UV radiation. To complement experimental data, we analyzed structural and electronic properties of both the bulk and the (1 0 1) surface of anatase by first-principles calculations. Based on experimental and theoretical results, a reactive O2H- and OH- species formation mechanism was proposed to explain the key factor which facilitates the antifungal activity. © 2013 Published by Elsevier B.V.

Monocyte Chemoattractant-1 as a Urinary Biomarker for the Diagnosis of Activity of Lupus Nephritis in Brazilian Patients

Objective. Monocyte chemotactic protein (MCP-1), involved in the pathogenesis of lupus nephritis (LN), has recently been indicated as a new biomarker of kidney activity in systemic lupus erythematosus (SLE). Our aim was to assess urinary MCP-1 (uMCP-1) as a biomarker of renal activity in patients with SLE and to compare it to other disease activity markers, using the ELISA. Methods. Seventy-five female Brazilian patients with SLE and a control group participated in our study. Patients with SLE were distributed among 3 groups according to kidney involvement and classified according to disease activity based on clinical and laboratory measures such as urinary sediment, proteinuria, kidney function, C3, C4, anti-dsDNA, disease activity index, and renal SLE disease activity index. The serum and uMCP-1 concentrations were measured by sandwich ELISA. Results. In the A-LN group (active lupus nephritis: SLE with kidney involvement), the concentration of uMCP-1 was significantly higher than in other groups. A cutoff point was established using the results of the control group to apply this test in the detection of LN. A-LN had a higher frequency of positive results for uMCP-1 in comparison to the other groups (p < 0.001). To detect disease activity in patients with LN, a new cutoff was determined based on the results of patients with SLE with kidney involvement. Setting specificity at 90%, the sensitivity of the test was 50%. Conclusion. The high specificity makes uMCP-1 a useful test as a predictor of kidney activity in SLE, especially when associated to other measures used in clinical practice. (First Release Sept 1 2012; J Rheumatol 2012;39:1948-54; doi :10.3899/jrheum.110201)

Leptospiral Immunoglobulin-like Proteins Interact With Human Complement Regulators Factor H, FHL-1, FHR-1, and C4BP

Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular matrix components, coagulation cascade proteins, and human complement regulators. Here we demonstrate that acquisition of factor H (FH) on the Leptospira surface is crucial for bacterial survival in the serum and that these spirochetes, besides interacting with FH, FH related-1, and C4b binding protein (C4BP), also acquire FH like-1 from human serum. We also demonstrate that binding to these complement regulators is mediated by leptospiral immunoglobulin-like (Lig) proteins, previously shown to interact with fibronectin, laminin, collagen, elastin, tropoelastin, and fibrinogen. Factor H binds to Lig proteins via short consensus repeat domains 5 and 20. Competition assays suggest that FH and C4BP have distinct binding sites on Lig proteins. Moreover, FH and C4BP bound to immobilized Ligs display cofactor activity, mediating C3b and C4b degradation by factor I. In conclusion, Lig proteins are multifunctional molecules, contributing to leptospiral adhesion and immune evasion.

Alternative complement pathway and factor B activities in rats with altered blood levels of thyroid hormone

Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32%) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.

Interaction of leptospira elongation factor tu with plasminogen and complement factor h: a metabolic leptospiral protein with moonlighting activities

The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

A metalloproteinase karilysin present in the majority of Tannerella forsythia isolates inhibits all pathways of the complement system

Tannerella forsythia is a poorly studied pathogen despite being one of the main causes of periodontitis, which is an inflammatory disease of the supporting structures of the teeth. We found that despite being recognized by all complement pathways, T. forsythia is resistant to killing by human complement, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with karilysin, a metalloproteinase of T. forsythia, resulted in a decrease in bactericidal activity of the serum. T. forsythia strains expressing karilysin at higher levels were more resistant than low-expressing strains. Furthermore, the low-expressing strain was significantly more opsonized with activated complement factor 3 and membrane attack complex from serum compared with the other strains. The high-expressing strain was more resistant to killing in human blood. The protective effect of karilysin against serum bactericidal activity was attributable to its ability to inhibit complement at several stages. The classical and lectin complement pathways were inhibited because of the efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4 by karilysin, whereas inhibition of the terminal pathway was caused by degradation of C5. Interestingly, karilysin was able to release biologically active C5a peptide in human plasma and induce migration of neutrophils. Importantly, we detected the karilysin gene in >90% of gingival crevicular fluid samples containing T. forsythia obtained from patients with periodontitis. Taken together, the newly characterized karilysin appears to be an important virulence factor of T. forsythia and might have several important implications for immune evasion.

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.

Increased titers of anti-Saccharomyces cerevisiae antibodies in Crohn's disease patients with reduced H-ficolin levels but normal MASP-2 activity

Mannan-binding lectin (MBL) and ficolins are microbial pattern recognition molecules that activate the lectin pathway of complement. We previously reported the association of MBL deficiency with anti-Saccharomyces cerevisiae antibodies (ASCA) in patients with Crohn's disease (CD). However, ASCA are also frequently found in MBL-proficient CD patients. Here we addressed expression/function of ficolins and MBL-associated serine protease-2 (MASP-2) regarding potential association with ASCA.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Human complement component C3 -binding proteins of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that uses the host mononuclear phagocyte as a niche for survival and replication during infection. Complement component C3 has previously been shown to enhance the binding of M. tuberculosis to mononuclear phagocytes. Using a C3 ligand affinity blot protocol, we identified a 30 kDa C3-binding protein in M. tuberculosis as heparin-binding hemagglutinin (HbhA). HbhA was found to be a hydrophobic protein that localized to the cell membrane/cell wall fraction of M. tuberculosis, and this protein has previously been shown by others to be located on the surface of M. tuberculosis. The C3-binding activity of HbhA was localized to the C-terminus of the protein, which consists of lysine-alanine repeats. Full-length recombinant HbhA coated onto latex beads was shown to mediate the adherence of the beads to murine macrophage-like cells in both a C3-dependent and a C3-independent manner. An in-frame 576 by deletion in the hbhA gene was created in a virulent strain of M. tuberculosis using a PCR technique known as gene splicing by overlap extension (SOEing). Using the ΔhbhA mutant, HbhA was found not to be necessary for growth of M. tuberculosis in laboratory media or in macrophage-like cells, nor is HbhA required for adherence of M. tuberculosis to macrophage-like cells. HbhA is, however, required for infectivity of M. tuberculosis in mice. Mice infected with the ΔhbhA mutant show decreased growth in the lungs, liver, and spleen compared to mice infected with the wild-type strain. Using the ΔhbhA mutant strain, we were able to purify and identify a second 30-kDa C3-binding protein, HupB. These data demonstrate that HbhA is required for the in vivo but not the in vitro survival of M. tuberculosis and that HbhA is not necessary for the adherence of M. tuberculosis to the macrophage-like cells used in these studies. The expression of two proteins that bind human C3 may aid in the efficient binding of M. tuberculosis to complement receptors for uptake into mononuclear cells, or may influence other aspects of the host-parasite interaction. ^

Abrogation of the alternative complement pathway by targeted deletion of murine factor B

To investigate the role of complement protein factor B (Bf) and alternative pathway activity in vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with the neor gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1 generation at a frequency suggesting that Bf deficiency alone has no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P = 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly reflecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functions in vivo.

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR–thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.