Serratiopeptidase: a well-known metalloprotease with a new non-proteolytic activity against S. aureus biofilm.

BACKGROUND: The use of indwelling medical devices is associated with a significant risk of infections by Staphylococcus aureus (S. aureus) which possesses a variety of virulence factors including many toxins and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The virulence factors above described are often related to proteins exposed on the bacterial surface. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases. Previously reports evaluated the anti-infective properties of serratiopeptidase (Spep), an extracellular metalloprotease produced by Serratia marcescens ATCC 21074 (E-15), in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. However, to date its mechanism of action is unknown. METHODS: Spep gene was PCR amplified and cloned into expression vector pET28b(+). The mutant EspepA was constructed from plasmid pET28b-Spep applying the one-step overlap extension PCR strategy. There sulting plasmids were costransformed in EcBL21(DE3) cells with the plasmid pRuW4inh1 harboring the Erwinia chrysanthemi secretion system. Bacterial pellets and supernatants were collected and analyzed by SDS-PAGE and zymography. The unambiguous identification and a detailed structure characterization of both the wild type and the mutant Spep were obtained by mass spectrometric analyses. The resultant supernatants sterilized by filtration were separately used to condition biofilm formation of S. aureus. Quantification was based on crystal violet method. RESULTS: In this work we constructed Spep mutant by substituting the glutamic acid in the catalytic site with a residue of alanine. In this manner we were able to evaluate the anti-biofilm activity of Spep mutant in absence of proteolytic activity. As expected, this mutant did not display protease activity but it retained its anti-biofilm properties, suggesting that this action is independent by enzymatic activity. CONCLUSIONS: New knowledge obtained from data reported in this paper calls attention to a novel mechanism of action of Spep. This protein could be developed as a potential "antipathogenic agent" capable to impair the ability of S. aureus to form biofilm on prostheses, catheters and medical devices, exploiting a mechanism different from the proteolytic activity.

Anti-biofilm activity of pseudoalteromonas haloplanktis tac125 against staphylococcus epidermidis biofilm: Evidence of a signal molecule involvement?

Staphylococcus epidermidis is recognized as cause of biofilm-associated infections and interest in the development of new approaches for S. epidermidis biofilm treatment has increased. In a previous paper we reported that the supernatant of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 presents an anti-biofilm activity against S. epidermidis and preliminary physico-chemical characterization of the supernatant suggested that this activity is due to a polysaccharide. In this work we further investigated the chemical nature of the anti-biofilm P. haloplanktis TAC125 molecule. The production of the molecule was evaluated in different conditions, and reported data demonstrated that it is produced in all P. haloplanktis TAC125 biofilm growth stages, also in minimal medium and at different temperatures. By using a surface coating assay, the surfactant nature of the anti-biofilm compound was excluded. Moreover, a purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is not due to a polysaccharide molecule but that it is due to small hydrophobic molecules that likely work as signal. The enriched fraction was also used to evaluate the effect on S. epidermidis biofilm formation in dynamic condition by BioFlux system.

Effect of betamethasone in combination with antibiotics on gram positive and gram negative bacteria.

Betamethasone is an anti-inflammatory steroid drug used in cases of anaphylactic and allergic reactions, of Alzheimer and Addison diseases and in soft tissue injuries. It modulates gene expression for anti-inflammatory activity suppressing the immune system response. This latter effect might decrease the effectiveness of immune system response against microbial infections. Corticosteroids, in fact, mask some symptoms of infection and during their use superimposed infections may occur. Thus, the use of glucocorticoids in patients with sepsis remains extremely controversial. In this study we analyzed the in vitro effect of a commercial formulation of betamethasone (Bentelan) on several Gram positive and Gram negative bacteria of clinical relevance. It was found to be an inhibitor of the growth of most of the strains examined. Also the effect of betamethasone in combination with some classes of antibiotics was evaluated. Antibiotic-steroid combination therapy is, in such cases, superior to antibiotic-alone treatment to impair bacterial growths. Such effect was essentially not at all observable on Staphylococcus aureus or Coagulase Negative Staphylococci (CoNS).

Comparison of the action of different proteases on virulence properties related to the staphylococcal surface.

AIMS: The purpose of this study was to evaluate the antimicrobial efficacy of five different proteases belonging to two different families on Staphylococcus aureus and Staphylococcus epidermidis strains. METHODS AND RESULTS: We used three serine proteases and two metalloproteases in single species biofilm formation assays and in human cell invasion processes. Following each protease incubation with bacterial cells, surface protein patterns were analysed by SDS-PAGE and zymography. Some differently expressed proteins were identified by mass spectrometry. CONCLUSIONS: The effect of tested proteases on biofilm formation was not related to the protease category but was strain-dependent and was related to the biofilm formation capacity of each staphylococcal strain. Some proteases showed a nonspecific and indiscriminate effect on surface proteins, while others induced a discrete and reproducible action on protein profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of the surface-related virulence factors is a promising avenue to overcome persistent infections caused by bacterial biofilms. To this end, we show here that proteases, in particular the metalloprotease serratiopeptidase, can interfere with adhesion and invasion of eukaryotic cells and biofilm formation in staphylococci and their use could represent a viable treatment for the development of novel combination therapies.

Bacterial biofilm formation inhibitory activity revealed for plant derived natural compounds.

Use of herbal plant remedies to treat infectious diseases is a common practice in many countries in traditional and alternative medicine. However to date there are only few antimicrobial agents derived from botanics. Based on microbiological screening tests of crude plant extracts we identified four compounds derived from Krameria, Aesculus hippocastanum and Chelidonium majus that showed a potentially interesting antimicrobial activity. In this work we present an in depth characterization of the inhibition activity of these pure compounds on the formation of biofilm of Staphylococcus aureus as well as of Staphylococcus epidermidis strains. We show that two of these compounds possess interesting potential to become active principles of new drugs.

Holo and apo-transferrins interfere with adherence to abiotic surfaces and with adhesion/invasion to HeLa cells in Staphylococcus spp.

Staphylococcus aureus and Staphylococcus epidermidis are the major cause of infections associated with implanted medical devices. Colonization on abiotic and biotic surfaces is often sustained by biofilm forming strains. Human natural defenses can interfere with this virulence factor. We investigated the effect of human apo-transferrin (apo-Tf, the iron-free form of transferrin, Tf) and holo-transferrin (holo-Tf, the iron-saturated form) on biofilm formation by CA-MRSA S. aureus USA300 type (ST8-IV) and S. epidermidis (a clinical isolate and ATCC 35984 strain). Furthermore S. aureus adhesion and invasion assays were performed in a eukaryotic cell line. A strong reduction in biofilm formation with both Tfs was obtained albeit at very different concentrations. In particular, the reduction in biofilm formation was higher with apo-Tf rather than obtained with holo-Tf. Furthermore, while S. aureus adhesion to eukaryotic cells was not appreciably affected, their invasion was highly inhibited in the presence of holo-Tf, and partially inhibited by the apo form. Our results suggest that Tfs could be used as antibacterial adjuvant therapy in infection sustained by staphylococci to strongly reduce their virulence related to adhesion and cellular invasion.

A new anti-infective strategy to reduce adhesion-mediated virulence in Staphylococcus aureus affecting surface proteins.

Staphylococcus aureus is a flexible microbial pathogen frequently isolated from community-acquired and nosocomial infections. The use of indwelling medical devices is associated with a significant risk of infection by this bacterium which possesses a variety of virulence factors, including many toxins, and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The present study evaluates the anti-infective properties of serratiopeptidase, a secreted protein of Serratia marcescens, in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. SPEP seems to exert its action by modulating specific proteins. Proteomic studies performed on surface proteins extracted from SPEP-treated S. aureus cultures revealed that a number of proteins are affected by the treatment. Among these we found the adhesin/autolysin Atl, FnBP-A, SecA1, Sbi, EF-Tu, EF-G, and alpha-enolase. EF-Tu, EF-G and alpha-enolase are known to perform a variety of functions, depending on their cytoplasmic or surface localization. All these factors can facilitate bacterial colonization, persistence and invasion of host tissues. Our results suggest that SPEP could be developed as a potential anti-infective agent capable to hinder the entry of S. aureus into human tissues, and also impair the ability of this pathogen to form biofilm on prostheses, catheters and medical devices.

Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope.

Mutations that abrogate recognition of a viral epitope by class I-restricted cytotoxic T lymphocyte (CTL) can lead to viral escape if the CTL response against that epitope is crucial for viral clearance. The likelihood of this type of event is low when the CTL response is simultaneously directed against multiple viral epitopes, as has been recently reported for patients with acute self-limited hepatitis B virus (HBV) infection. The CTL response to HBV is usually quite weak, however, during chronic HBV infection, and it is generally acknowledged that this is a major determinant of viral persistence in this disease. If such individuals were to produce a mono- or oligospecific CTL response, however, negative selection of the corresponding mutant viruses might occur. We have recently studied two HLA-A2-positive patients with chronic hepatitis B who, atypically, developed a strong HLA-A2-restricted CTL response against an epitope (FLPSDFFPSV) that contains an HLA-A2-binding motif located between residues 18-27 of the viral nucleocapsid protein, hepatitis B core antigen (HBcAg). These patients failed, however, to respond to any of other HLA-A2-restricted HBV-derived peptides that are generally immunogenic in acutely infected patients who successfully clear the virus. Interestingly, DNA sequence analysis of HBV isolates from these two patients demonstrated alternative residues at position 27 (V --> A and V --> I) and position 21 (S --> N, S --> A, and S --> V) that reduced the HLA and T cell receptor-binding capacities of the variant sequences, respectively. Synthetic peptides containing these alternative sequences were poorly immunogenic compared to the prototype HBc18-27 sequence, and they could not be recognized by CTL clones specific for the prototype peptide. While we do not know if the two patients were originally infected by these variant viruses or if the variants emerged subsequent to infection because of immune selection, the results are most consistent with the latter hypothesis. If this is correct, the data suggest that negative selection of mutant viral genomes might contribute to viral persistence in a subset of patients with chronic HBV infection who express a narrow repertoire of anti-HBV CTL responses.

Comparison of anti-bacterial prophylactic properties of two different vascular grafts: action of anti-bacterial graft coating and systemic antibiotic treatment.

The main problem associated with artificial vascular devices is the risk of bacterial infections, mostly sustained by coagulase negative Staphylococci or Staphylococcus aureus. Many efforts have been made to identify materials refractory to bacterial adhesion. The aim of our study is to verify the antimicrobial properties of two kinds of vascular prosthesis to prevent early onset infections and the efficacy of the concomitant action of a systemic antibiotic treatment. Adult male Wistar rats were used. We subcutaneously implanted in four groups a silver-coated prosthesis fragment, and a rifampicin-soaked prosthesis fragment in the remaining four groups. We inoculated in the site of implant a high bacterial burden of S. aureus in four groups and a low burden in the remaining groups. Systemic levofloxacin was administered for seven days in four groups representing the two kinds of prosthesis; after 21 days the rats were sacrificed, prosthesis fragments were sonicated and the corresponding supernatants were plated for bacterial counts. The rifampicin-soaked prostheses explanted from rats treated with levofloxacin were sterile, regardless of the bacterial inoculum. In other groups some prostheses were colonized. In the experimental rat model used, the action of local and systemic antibiotic treatment was able to reduce colonisation of artificial prostheses.

In vitro detection of herpes simplex virus -1 and -2 infection with immunospecific GD3+-CL6-enhanced magnetic resonance imaging.

Herpes simplex virus infections are prevalent viral infections in humans. HSVs are also the most common cause of sporadic viral encephalitis (HSE). Magnetic resonance is the imaging method of choice for HSE because it provides the most sensitive method for detecting early lesions. The objective of this study is to set-up and in vitro test an experimental contrast agent specific for antigens present on HSV-infected cells, bound with a paramagnetic agent detectable by MR imaging. A selected anti-HSV HrFab was labelled with Alexa Fluor 488, 125I and Gd3+Cl6. In order to assess anti-HSV affinity and specificity, ELISA assays were performed. Vero cells infected with HSV strains were visualized by MRI using anti-HSV HrFab/Gd3+Cl6 complex. Results of the ELISA tests demonstrated that the anti-HSV HrFab labelled with Gd3+Cl6 showed similar affinity for the antigens while the 125I immunoconjugate showed reduced affinity. MRI confirmed high affinity and specificity of antibody for the detection of HSV infections.

Long-lasting memory T cell responses following self-limited acute hepatitis B.

The molecular and cellular basis of long-term T cell memory against viral antigens is still largely undefined. To characterize anti-viral protection by memory T cells against non-cytopathic viruses able to cause acute self-limited and chronic infections, such as the hepatitis B virus (HBV), we studied HLA class II restricted responses against HBV structural antigens in 17 patients with acute hepatitis B, during the acute stage of infection and 2.2 to 13 yr after clinical resolution of disease. Results indicate that: (a) significant T cell proliferative responses to HBV nucleocapsid antigens were detectable in all patients during the acute phase of infection and in 14/17 also 2-13 yr after clinical resolution of disease; b) long-lasting T cell responses were sustained by CD45RO+T cells, predominantly expressing the phenotype of recently activated cells; c) limiting dilution analysis showed that in some patients the frequency of HBV-specific T cells was comparable to that observed in the acute stage of infection and, usually, higher than in patients with chronic HBV infection; d) the same amino acid sequences were recognized by T cells in the acute and recovery phases of infection; and e) HBV-DNA was detectable by nested-PCR in approximately half of the subjects. to conclusion, our results show that vigorous anti-viral T cell responses are detectable in vitro several years after clinical recovery from acute hepatitis B. Detection of minute amounts of virus in some recovered subjects suggests that long-term maintenance of an active anti-viral T cell response could be important not only for protection against reinfection but also for keeping the persisting virus under tight control.

Induction of the DNA-binding activity of c-jun/c-fos heterodimers by the hepatitis B virus transactivator pX.

The hepatitis B virus (HBV) X protein (pX) is capable of activating transcription regulated by viral and cellular promoters containing binding sites for different transcription factors, including AP1. In this study we have analyzed the mechanisms of AP1 induction by pX. The hepatitis B virus transactivator was able to activate TRE (12-O-tetradecanoylphorbol-13-acetate response element)-directed transcription in different cell lines, including HepG2, HeLa, CV1, and PLC/PRF/5 cells. pX-induced AP1 activation in HepG2 cells was associated with an increase in the DNA-binding activity of c-Jun/c-Fos heterodimers, which was not dependent either on an increase in the overall amount of c-Fos and c-Jun proteins in the cells or on formation of dimers between pX and the two proteins, thus suggesting the involvement of posttranslational modifications of the transcription factor. The observation that the overexpression of c-Jun and c-Fos in the cells results in a strong augmentation of the effect of pX on TRE-directed transcription is additional evidence indicating the involvement of posttranscriptional modifications of c-Jun/c-Fos heterodimers. The increased AP1 binding observed in the presence of pX was unaffected by the protein kinase C inhibitors calphostin C and sphingosine and by the protein kinase A inhibitor HA1004, while it was almost completely blocked by staurosporine, a potent and nonspecific protein kinase inhibitor, suggesting that protein kinase C- and A-independent phosphorylation events might play a role in the phenomenon. The ability of pX also to increase TRE-directed transcription in cell lines in which AP1-binding activity is not increased (i.e., HeLa, CV1, and PLC/PRF/5 cells) suggests that pX can activate canonical TRE sites by different mechanisms as well.

The hepatitis B virus (HBV) pX transactivates the c-fos promoter through multiple cis-acting elements.

The hepatitis B virus (HBV) X protein (pX) stimulates transcription regulated by cis-acting elements that control many viral and cellular genes, including the c-myc and the c-fos proto-oncogenes. Using several c-fos promoter deletion mutants, we found the serum-responsive element (SRE) located at -315, the modified TPA-responsive element located at -296 (fos-AP-1 binding site, FAP) and the region spanning from nucleotide -220 to -120, which contains an NF1-like site and several stretches of sequence homologous to the AP-2 consensus binding sites, to be responsive to pX. pX does not modify the pattern of the retarded complexes bound to the SRE/FAP region which, in our system, appears to be occupied by SRE-binding factors. The activation of the SRE does not involve complex formation between SRE-binding factors and pX, it is not associated with an increase in serum response factor binding to the SRE and it does not determine changes in SRE mobility-shift pattern.

Infection of circulating and liver infiltrating T cells by hepatitis C virus of different subtypes.

Hepatitis C virus (HCV) infection display a very high rate of progression to chronicity and, like many other viruses causing persistent infections, it displays a tropism for the cells of the immune system. Peripheral blood mononuclear cells (PBMCs) from 21 HCV chronic carriers and long-term T cell clones derived from circulating or liver infiltrating T lymphocytes were tested by cDNA "nested" PCR for positive and negative strand HCV-RNA. The presence of HCV genomes in PBMCs is a frequent, although not constant, finding and can be accompanied by active viral replication, as suggested by the coexistence of negative strand HCV-RNA. Infected T cells are more represented in livers than in periphery, as indicated by comparing HCV-RNA detection in T cell clones isolated from both the compartments. Sequencing of viral genomes present in PBMCs and liver infiltrating lymphocytes showed that all the three major HCV genotypes present in our population of chronic carriers can infect lymphoid cells. Although each clonal population of T cells is infected by a single strain of HCV, in the same patient lymphoid cells can harbor different viral populations, different from those circulating at that moment in the serum.

Primary effusion Burkitt's lymphoma with t(8;22) in a patient with hepatitis C virus-related cirrhosis.

Hepatitis C virus (HCV) infection may be complicated by non-Hodgkin's lymphoma through yet unknown pathogenetic mechanisms. We describe the case of a patient with HCV-related cirrhosis who developed a primary effusion lymphoma (PEL) of Burkitt's type confined to the peritoneal cavity, in the absence of immunodeficiency or autoimmunity. Paracentesis followed by immunophenotyping, karyotyping, and molecular studies allowed us to diagnose a small noncleaved B-cell lymphoma (CD20+, CD24+, CD10+, CD5-, CD23-, lambda+) with the t(8;22) (q24;q11) translocation and clonal rearrangement of the immunoglobulin heavy chain gene. HCV-RNA, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus were not identified within lymphoma cells. The finding of HCV-RNA in the ascitic fluid suggests a link between HCV and development of lymphoma with HCV playing the role of persistent antigenic stimulation to intraperitoneal B-cell clonal expansion(s).

Chronic cryptogenic hepatitis in childhood is unrelated to hepatitis G virus.

OBJECTIVES: The aim of this study was to define the features of chronic cryptogenic hepatitis (CCH) in childhood and to investigate whether it is related to hepatitis G virus infection. METHODS: Forty-six children (24 males; age range, 1.5 to 17 years) with CCH were studied. CCH was diagnosed when serum alanine aminotransferase concentrations were more than 1.5 times normal for longer than 6 months without any apparent cause of liver disease. RESULTS: No patient had acute symptomatic onset or had received a blood transfusion. Three had undergone minor surgical procedures. All appeared to be healthy during follow-up (median, 4.2 years; range, 1 to 10 years). Hypertransaminasemia was the only aberrant liver function test. Elevated serum alanine aminotransferase values alternated with normal values in 40 children (86.9%). Five children (10.8%) had a spontaneous sustained (>12 months) remission of hypertransaminasemia. Twelve (26%) had laboratory signs of autoimmunity, but none fulfilled the criteria for autoimmune hepatitis. Of 20 children who underwent liver biopsy, 13 (65%) had minimal chronic hepatitis, 4 (20%) had mild chronic hepatitis and 3 (15%) had moderate chronic hepatitis. Serum hepatitis G virus RNA was detected in 2 girls (4%) whose risk factor was a hepatitis G virus-infected mother and a minor surgical procedure, respectively. In 12 families at least 1 other member had chronic liver disease. CONCLUSIONS: Childhood CCH seems to be a symptomless disease characterized by isolated hypertransaminasemia with onset during the first 4 years of life and mild to moderate histologic liver lesions. Although the frequency of spontaneous remissions is low, childhood CCH seems, in the short run, to be a nonprogressive disease. Hepatitis G virus does not play a major role in CCH.

Extranodal lymphomas associated with hepatitis C virus infection.

Hepatitis C virus (HCV) infection may be complicated by non-Hodgkin's lymphoma. We describe eight cases of B-cell extranodal non-Hodgkin's lymphoma occurring during the course of chronic HCV-related hepatic disease (low-grade of mucosa-associated lymphoid tissue [MALT]-type; diffuse large cell; Burkitt; diffuse small cell). Some were localized to the liver (2), liver and spleen (1), spleen (1), peritoneal cavity (1), parotid gland (1); others manifested in the nasopharynx (1) and eyelid (1) but were accompanied by nodal disease. Four lymphomatous specimens available for molecular analysis exhibited clonal immunoglobulin gene rearrangements, lacked bcl-2, bcl-6, c-myc genes and p53 alterations, and did not contain replicative intermediate HCV RNA, as documented by a strand-specific reverse transcriptase-polymerase chain reaction. Low levels of positive-strand HCV RNA were detected in a single hepatic lymphoma, suggesting the presence of the virus in residual hepatocytes. The antigen-driven properties of HCV-associated B-cell malignant neoplasms may be considered for hepatic MALT-type lymphoma, which probably originated from lymphoid tissue acquired during long-standing HCV infection.

Detection of replicative intermediates of viral RNA in peripheral blood mononuclear cells from chronic hepatitis C virus carriers.

Clinical and experimental evidence suggests the possible existence of one or more extrahepatic sites of HCV infection. In order to demonstrate the "in vivo" infection of lymphoid cells by HCV, we applied a nested PCR to total cytoplasmic RNA extracted from fresh or cultured peripheral blood mononuclear cells (PBMCs) of HCV chronically infected patients, using primers derived from the highly conserved 5' untranslated region of the HCV genome. The presence of virions in PBMCs occurs frequently, if not always, and is often accompanied by active viral replication. Moreover, the appearance of replicative intermediates after stimulation of cellular growth with mitogens suggests that latent genomes could undergo replication upon cellular activation and/or proliferation.

Recombinant immunoblot assay for hepatitis C virus antibody in chronic hepatitis.

Testing for hepatitis C virus by ELISA requires confirmation by recombinant immunoblot assay (RIBA). The first-generation RIBA uses the same antigen as used in the ELISA and one further antigen. A second-generation RIBA in which two further antigens are present, detects positivity that is not found by either the ELISA or the original RIBA. Consequently, although it is adequate to test ELISA positive sera with the first-generation RIBA, the second-generation assay is recommended for confirming negativity.

Truncated pre-S/S proteins transactivate multiple target sequences.

In order to investigate the transactivational function of HBV truncated preS/S proteins we have constructed two sets of plasmids and have tested their transactivational potential on the c-myc regulatory sequences and the TPA-responsive element. We found that preS/S proteins only become transactivationally active when truncated at the carboxy terminal end. Furthermore, using immunofluorescence microscopy we determined that the proteins are located exclusively in the cytoplasm, apparently ruling out DNA binding and activation of factors in the nucleus.