Surface-associated Proteins Research Articles

Surface anchoring of a bacterial adhesin secreted by the two‐partner secretion pathway

In Gram-negative bacteria, most surface-associated proteins are present as integral outer-membrane proteins. Exceptions include the Haemophilus influenzae HMW1 and HMW2 adhesins and a subset of other proteins secreted by the two-partner secretion system. In the present study we sought to determine the mechanism by which HMW1 is anchored to the bacterial surface. In initial experiments we found that HMW1 forms hair-like fibres on the bacterial surface and is usually present as pairs that appear to be joined together at one end. Further analysis established that HMW1 is anchored to the multimeric HMW1B outer membrane translocator, resulting in a direct correlation between the level of surface-associated HMW1 and the quantity of HMW1B in the outer membrane. Mutagenesis and polyethylene glycol maleimide labelling revealed that anchoring of HMW1 requires the C-terminal 20 amino acids of the protein and is dependent upon disulphide bond formation between two conserved cysteine residues in this region. Immunolabelling studies demonstrated that the immediate C-terminus of HMW1 is inaccessible to surface labelling, suggesting that it remains in the periplasm or is buried in HMW1B. Coexpression of HMW1 lacking the C-terminal 20 amino acids and wild-type HMW1 supported the conclusion that the C-terminus of HMW1 occupies the HMW1B pore. These observations may have broad relevance to proteins secreted by the two-partner secretion system, especially given the conservation of C-terminal cysteine residues among surface-associated proteins in this family.

Staphylococcus aureus capsular material promotes osteoclast formation

Osteomyelitis, which is most frequently due to infection by Staphylococcus aureus, commonly causes bone destruction. S. aureus is known to secrete a number of surface-associated proteins that are potent stimulators of bone resorption. The precise cellular and humoral mechanisms that mediate this stimulatory effect are uncertain. In this study, we have determined whether osteoclast formation and resorption is directly promoted by surface-associated proteins. Surface-associated material (SAM) obtained from a 24-hour culture of S. aureus was added to cultures of mouse and human monocytes. Human monocyte cultures were incubated in the presence and absence of a soluble receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). In cultures where M-CSF, RANKL, and SAM were added together, osteoclast formation did not exceed that seen in cultures with M-CSF and RANKL. In keeping with this finding, SAM did not increase osteoclast formation and resorption when mouse monocytes were cocultured with RANKL-expressing osteoblasts. In the absence of RANKL, however, SAM was capable of inducing osteoclast formation in cultures of human monocytes. This finding was evidenced by the generation of vitronectin receptor and tartrate-resistant acid phosphatasepositive multinucleated cells that were capable of lacunar resorption. Inhibitors of RANKL-dependent (RANK:Fc, OPG) and RANKL-independent (anti-TNF-alpha, gp130, IL-8, TGF-beta) osteoclast formation did not inhibit SAM-induced osteoclast formation. SAM did not stimulate mature osteoclast resorption activity. These findings indicate that RANKL, which is present in the circulation as a soluble factor, does not play a role in osteoclast formation in the presence of S. aureus SAM and that S. aureus SAM contains a soluble factor that promotes osteoclast formation by a RANKL-independent mechanism.

Influence of BrpA on critical virulence attributes of Streptococcus mutans.

Streptococcus mutans, the primary etiological agent of human dental caries, has developed multiple mechanisms to colonize and form biofilms on the tooth surface. The brpA gene codes for a predicted surface-associated protein with apparent roles in biofilm formation, autolysis, and cell division. In this study, we used two models to further characterize the biofilm-forming characteristics of a BrpA-deficient mutant, strain TW14. Compared to those of the parent strain, UA159, TW14 formed long chains and sparse microcolonies on hydroxylapatite disks but failed to accumulate and form three-dimensional biofilms when grown on glucose as the carbohydrate source. The biofilm formation defect was also readily apparent by confocal laser scanning microscopy when flow cells were used to grow biofilms. When subjected to acid killing at pH 2.8 for 45 min, the survival rate of strain TW14 was more than 1 log lower than that of the wild-type strain. TW14 was at least 3 logs more susceptible to killing by 0.2% hydrogen peroxide than was UA159. The expression of more than 200 genes was found by microarray analysis to be altered in cells lacking BrpA (P < 0.01). These results suggest that the loss of BrpA can dramatically influence the transcriptome and significantly affects the regulation of acid and oxidative stress tolerance and biofilm formation in S. mutans, which are key virulence attributes of the organism.

Down-regulation of MT1-MMP expression suppresses tumor cell invasion in metastatic human SW626 ovarian cancer cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) is a key enzyme involved in degradation of extracellular matrix (ECM) and various surface-associated proteins that control cell growth, differentiation and survival, plays crucial roles in molecular carcinogenesis, tumor cell growth, invasion, and angiogenesis. We tested the inhibitory effect of antisense MT1-MMP on the ability of metastatic human ovarian carcinoma cell line SW626 in proliferation and invasion. RT-PCR was used to amplify MT1-MMP cDNA fragments with two different restriction sites at its 5'-end. Antisense MT1-MMP cloned in eukaryotic expression vector pMMP14as was transfected into SW626 cells. MT1-MMP protein expression, activities of MMP-2 and MMP-9, changes of cell proliferation, and cell invasion ability were detected by Western blot, optimized gelatin zymography, MTT assay and matrigel in vitro invasion assay, respectively. After 48 h transfection, decreased expression of endogenous MT1-MMP protein was detected in pMMP14as-transfected SW626 cells and showed significantly lower proliferation level when compared with control cells. The activation of proMMP-2 was inhibited markedly, and the mean percentage of invasive cells was 63.30+/-5.80% in pMMP14as-transfected cells, which was less than that (97.60+/-7.50%) in control cells (P<0.05). Both cell proliferation and invasion in SW626 cells were inhibited effectively by antisense MT1-MMP transfection, suggesting that MT1-MMP may be a proper target molecule for anti-invasion therapy for human ovarian cancers.

A role for Efg1p inCandida albicansinteractions with extracellular matrices

Candida albicans interactions with extracellular matrices extracellular matrices represent hallmarks of invasive candidiasis. We have assessed the role of Efg1p, a key transcriptional regulator, in the interactions between C. albicans and extracellular matrices. Wild-type C. albicans cells were able to bind and subsequently penetrate the extracellular matrices layer, whereas the Deltaefg1/Deltaefg1 mutant strain showed a drastically reduced ability to interact with extracellular matrices. Results indicate that, besides its control of morphogenesis, Efg1p also regulates C. albicans interactions with extracellular matrices. Proteomic analyses indicated that expression of 30 surface-associated proteins is affected in the Deltaefg1/Deltaefg1 mutant, some of which may be involved in adherence to extracellular matrices.

Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells

Surface-associated protein expression by Streptococcus uberis was influenced by the presence of collagen, laminin and bovine mammary epithelial cells in the culture medium. After electrophoresis and silver staining, four proteins stained more intensively in samples from S. uberis cultivated with epithelial cells and extracellular matrix components than in samples from S. uberis cultivated alone. Induction of these proteins was more obvious after multiple bacterial passages. The correlation between the phenotype of S. uberis and its potential virulence status as illustrated by an immunoblotting study with sera obtained from infected cows revealed that these proteins are probably expressed in vivo during infection.

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.

The ErbB2 Signaling Network as a Target for Breast Cancer Therapy

Overexpression of the ErbB2/Her2 receptor tyrosine kinase in breast cancers is associated with the most aggressive tumors. Experimental studies have revealed that ErbB2 shows many features of a therapeutic target: ErbB2 is able to confer many of the characteristics of a cancerous cell, including uncontrolled proliferation, resistance to apoptosis and increased motility; ErbB2 overexpression is specific to tumor cells; as a cell surface-associated protein, it is easily accessible to drugs and as a kinase it is amenable to targeted inhibition by small molecules. Recent clinical results demonstrate the efficacy of ErbB2-targeting therapy and promise an expanding use of ErbB2-targeting drugs for breast cancer treatment. However, as only a fraction of patients responds successfully to therapy and risks of recurrence are still high, further investigation is required for an improved understanding of the complex network of signaling pathways underlying ErbB2-driven cancer progression.

Tumor suppressive maspin and epithelial homeostasis

Maspin is a 42-kDa novel serine protease inhibitor (serpin) with multifaceted tumor suppressive activities. To date, the consensus that maspin expression predicts a better prognosis still largely holds for breast, prostate, colon, and oral squamous cancers. Interestingly, however, more detailed analyses revealed a biphasic expression pattern of maspin in early steps of tumorigenicity and re-expression of maspin in dormant cancer metastatic revertants. These data suggest a sensitivity of maspin expression to changes of epithelial microenvironments, and a role of maspin in epithelial homeostasis. Experimental evidence consistently showed that maspin suppresses tumor growth, invasion and metastasis, induces tumor redifferentiation, and enhances tumor cell sensitivity to apoptosis. Maspin protein isolated from biological sources is a monomer, which is present as a secreted, a cytoplasmic, a nuclear, as well as a cell surface-associated protein. Nuclear maspin is associated with better prognoses of cancer. It is further noted that extracellular maspin is sufficient to block tumor induced extracellular matrix degradation, tumor cell motility and invasion, whereas intracellular maspin is responsible for the increased cellular sensitivity to apoptosis. Despite these exciting developments, the mechanistic studies of maspin have proven challenging primarily due to the lack of a prototype molecular model. Although the maspin sequence has overall homologies with other members in the serpin superfamily, it does not behave like a typical serpin, that is, non-inhibitory toward active serine proteases in solution. This novel feature is in line with the X-ray crystallographic evidence. Several recent studies dedicated to finding the maspin partners support a paradigm shift. The current review is intended to summarize these recent findings and discuss a new perspective of maspin in epithelial homeostasis.

Identification of genes induced in Listeria monocytogenes during growth and attachment to cut cabbage, using differential display.

The food-borne pathogen Listeria monocytogenes is a ubiquitous soil bacterium with the potential to contaminate fresh produce during cultivation and postharvest processing. In order to identify potential mechanisms by which L. monocytogenes may successfully attach to and colonize fresh produce, gene expression in L. monocytogenes cells inoculated onto fresh-cut cabbage was compared to gene expression in cells grown under control conditions. Differential display of reverse transcriptase PCR fragments amplified with a set of 81 arbitrary primers allowed the isolation and identification of 32 L. monocytogenes gene fragments that were observed to be more highly expressed under cabbage-associated conditions. Genes involved in carbohydrate, amino acid, and nucleic acid metabolism, motility and cell division, and transport were identified, as were a number of open reading frames (ORFs) encoding putative proteins with no known functions. Site-directed mutations in two ORFs encoding potential cell surface-associated proteins and a third ORF encoding a putative regulatory protein had no effect on the mutants' capacity to attach to fresh-cut cabbage. Although this study did not show clearly the impact of the differentially expressed genes on growth on cabbage, it is a first step in identifying some of the genetic factors that are potentially involved.

The N-Terminal Domain of Enterococcal Surface Protein, Esp, Is Sufficient for Esp-Mediated Biofilm Enhancement inEnterococcus faecalis

Enterococci have emerged as one of the leading causes of nosocomial bloodstream, surgical site, and urinary tract infections. More recently, enterococci have been associated with biofilms, which are bacterial communities attached to a surface and encased in an extracellular polymeric matrix. The enterococcal cell surface-associated protein, Esp, enhances biofilm formation by Enterococcus faecalis in a glucose-dependent manner. Mature Esp consists of a nonrepeat N-terminal domain and a central region made up of two types of tandem repeats followed by a C-terminal membrane-spanning and anchor domain. This study was undertaken to localize the specific domain(s) of Esp that plays a role in Esp-mediated biofilm enhancement. To achieve this objective, we constructed in-frame deletion mutants expressing truncated forms of Esp in an isogenic background. By comparing strains expressing the mutant forms of Esp to those expressing wild-type Esp, we found that the strain expressing Esp lacking the N-terminal domain formed biofilms that were quantitatively less in biovolume than the strain expressing wild-type Esp. Furthermore, an E. faecalis strain expressing only the N-terminal domain of Esp fused to a heterologous protein anchor formed biofilms that were quantitatively similar to those formed by a strain expressing full-length Esp. This suggested that the minimal region contributing to Esp-mediated biofilm enhancement in E. faecalis was confined to the nonrepeat N-terminal domain. Expression of full-length E. faecalis Esp in heterologous host systems of esp-deficient Lactococcus lactis and Enterococcus faecium did not enhance biofilm formation as was observed for E. faecalis. These results suggest that Esp may require interaction with an additional E. faecalis-specific factor(s) to result in biofilm enhancement.

Ligation of Cell Surface-Associated Glucose-Regulated Protein 78 by Receptor-Recognized Forms of α2-Macroglobulin: Activation of p21-Activated Protein Kinase-2-Dependent Signaling in Murine Peritoneal Macrophages

Previous studies of the plasma proteinase inhibitor alpha2-macroglobulin (alpha2M) demonstrated that alpha2M-proteinase complexes (alpha2M*) modulate immune responses and promotes macrophage locomotion and chemotaxis. Alpha2M* binds to cell surface-associated glucose-regulated protein 78 (GRP78), which activates downstream signaling events. The role of p21-activated protein kinase-1 and -2 (PAK-1 and -2) in promoting cellular motility is well documented. In the current study, we examined the ability of alpha2M* to activate PAK-1 and PAK-2. Upon macrophage stimulation with alpha2M*, PAK-2 is autophosphorylated, resulting in increased kinase activity; however, PAK-1 is negligibly affected. Alpha2M*-stimulated macrophages showed a marked elevation in the levels of Rac x GTP. Receptor tyrosine phosphorylation upon binding of alpha2M* to GRP78, recruits PAK-2 to the plasma membrane via the adaptor protein NCK. Consistent with this hypothesis, silencing of GRP78 gene expression greatly attenuated the levels of membrane-associated PAK-2 and NCK. PAK-2 activity was markedly decreased by inhibition of tyrosine kinases and PI3K before alpha2M* stimulation. We further demonstrate that phosphorylation of Lin-11, Isl-1, Mec-3 (LIM) kinase and cofilin is promoted by treating macrophages with alpha2M*. Thus, alpha2M* regulates activation of the PAK-2-dependent motility mechanism in these cells.

Differential expression of non-cytoplasmic Actinobacillus pleuropneumoniae proteins induced by addition of bronchoalveolar lavage fluid

Actinobacillus (A.) pleuropneumoniae is the causative agent of a porcine pleuropneumonia occurring worldwide. In order to identify novel non-cytoplasmic putative virulence-associated proteins, we prepared fractions enriched in surface-associated proteins for differential proteome analysis by two-dimensional (2D) gel electrophoresis and quadrupole time-of-flight mass spectrometry (Q-Tof MS). Bacteria grown under standard culture conditions were compared to an ex vivo model based on the addition of bronchoalveolar lavage fluid (BALF) to the culture media. Twelve proteins were found to be upregulated upon induction with BALF, among them a superoxide dismutase, a parvulin-like peptidy-prolyl isomerase, a polynucleotide phosphorylase and the highly immunogenic lipoprotein OmlA. Four of the proteins upregulated by BALF were additionally constitutively expressed by an isogenic A. pleuropneumoniae fur deletion mutant and could be identified by Q-Tof MS as the heat shock protein GroES, a putative dipeptide transporter, a putative metal ion transporter and a conserved protein of unknown function. In silico analysis of the putative promoter regions of the encoding genes revealed putative Fur boxes upstream of two genes, one of which encodes part of a putative metal ion transporter. An isogenic mutant with a deletion in this protein was constructed and designated as A. pleuropneumoniae Deltafui. Analysis of the mutant in an aerosol infection model revealed symptoms indistinguishable from those seen upon infection with wild type A. pleuropneumoniae. This result implies that not all proteins upregulated by BALF are directly involved in A. pleuropneumoniae virulence.

Identification and characterization of human surfactant protein A binding protein of Mycoplasma pneumoniae.

Mycoplasma pneumoniae infections represent a major primary cause of human respiratory diseases, exacerbate other respiratory disorders, and are associated with extrapulmonary pathologies. Cytadherence is a critical step in mycoplasma colonization, aided by a network of mycoplasma adhesins and cytadherence accessory proteins which mediate binding to host cell receptors. Furthermore, the respiratory mucosa is enriched with extracellular matrix components, including surfactant proteins, fibronectin, and mucin, which provide additional in vivo targets for mycoplasma parasitism. In this study we describe interactions between M. pneumoniae and human surfactant protein-A (hSP-A). Initially, we found that viable M. pneumoniae cells bound to immobilized hSP-A in a dose- and calcium (Ca(2+))-dependent manner. Mild trypsin treatment of intact mycoplasmas reduced binding markedly (80 to 90%) implicating a surface-associated mycoplasma protein(s). Using hSP-A-coupled Sepharose affinity chromatography and polyacrylamide gel electrophoresis, we identified a 65-kDa hSP-A binding protein of M. pneumoniae. The presence of Ca(2+) enhanced binding of the 65-kDa protein to hSP-A, which was reduced by the divalent cation-chelating agent, EDTA. The 65-kDa hSP-A binding protein of M. pneumoniae was identified by sequence analysis as a novel protein (MPN372) possessing a putative S1-like subunit of pertussis toxin at the amino terminus (amino acids 1 to 226), with the remaining amino acids (227 to 591) exhibiting no homology with other subunits of pertussis toxin, other known toxins, or any reported proteins. Recombinant MPN372 (MPN372) bound to hSP-A in a dose-dependent manner, which was markedly reduced by preincubation with mouse recombinant MPN372 antisera. Also, adherence of viable M. pneumoniae cells to hSP-A was inhibited by recombinant MPN372 antisera, demonstrating that MPN372, a previously designated hypothetical protein, is surface exposed and mediates mycoplasma attachment to hSP-A.

Involvement of theEscherichia coliO157:H7(pO157)ecfOperon and Lipid A Myristoyl Transferase Activity in Bacterial Survival in the Bovine Gastrointestinal Tract and Bacterial Persistence in Farm Water Troughs

Escherichia coli O157:H7 is an important food-borne pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome in humans. Recently, we reported that the pO157 ecf (E. coli attaching and effacing gene-positive conserved fragments) operon is thermoregulated by an intrinsically curved DNA and contains the genes for bacterial surface-associated proteins, including a second copy of lipid A myristoyl transferase, whose chromosomal copy is the lpxM gene product. E. coli O157:H7 survives and persists well in diverse environments from the human and bovine gastrointestinal tracts (GIT) to nutrient-dilute farm water troughs. Transcriptional regulation of the ecf operon by intrinsic DNA curvature and the genetic redundancy of lpxM that is associated with lipid A modification led us to hypothesize that the pO157 ecf operon and lpxM are associated with bacterial survival and persistence in various in vivo and ex vivo environments by optimizing bacterial membrane structure and/or integrity. To test this hypothesis, three isogenic ecf operon and/or lpxM deletion mutants of E. coli O157:H7 ATCC 43894 were constructed and analyzed in vitro and in vivo. The results showed that a double mutant carrying deletions in the ecf and lpxM genes had an altered lipid A structure and membrane fatty acid composition, did not survive passage through the bovine GIT, did not persist well in farm water troughs, had increased susceptibility to a broad spectrum of antibiotics and detergents, and had impaired motility. Electron microscopic analyses showed gross changes in bacterial membrane structure.

Solubilization of cellular membrane proteins fromStreptococcus mutans for two-dimensional gel electrophoresis

Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.

Biofilm formation of Pseudomonas putida IsoF: the role of quorum sensing as assessed by proteomics

Pseudomonas putida strains are frequently isolated from the rhizosphere of plants and many strains promote plant-growth, exhibit antagonistic activities against plant pathogens and have the capacity to degrade pollutants. Factors that appear to contribute to the rhizosphere fitness are the ability of the organism to form biofilms and the utilization of cell-to-cell-communication systems (quorum sensing, QS) to co-ordinate the expression of certain phenotypes in a cell density dependent manner. Recently, the ppu QS locus of the tomato rhizosphere isolate P. putida Iso F was characterized and an isogenic QS-negative ppuI mutant P. putida F117 was generated. In the present study we investigated the impact of QS and biofilm formation on the protein profile of surface-associated proteins of P. putida IsoF. This was accomplished by comparative proteome analyses of the P. putida wild type IsoF and the QS-deficient mutant F117 grown either in planktonic cultures or in 60 h old mature biofilms. Differentially expressed proteins were identified by peptide mass fingerprinting and database search in the completed P. putida KT2440 genome sequence. The sessile life style affected 129 out of 496 surface proteins, suggesting that a significant fraction of the bacterial genome is involved in biofilm physiology. In surface-attached cells 53 out of 484 protein spots were controlled by the QS system, emphasizing its importance as global regulator of gene expression in P. putida IsoF. Most interestingly, the impact of QS was dependent on whether cells were grown on a surface or in suspension; about 50% of the QS-controlled proteins identified in planktonic cultures were found to be oppositely regulated when the cells were grown as biofilms. Fifty-seven percent of all identified surface-controlled proteins were also regulated by the ppu QS system. In conclusion, our data provide strong evidence that the set of QS-regulated proteins overlaps substantially with the set of proteins differentially expressed in sessile cells.

CovS/CovR of group B streptococcus: a two‐component global regulatory system involved in virulence

In this study, we carried out a detailed structural and functional analysis of a Streptococcus agalactiae (GBS) two-component system which is orthologous to the CovS/CovR (CsrS/CsrR) regulatory system of Streptococcus pyogenes. In GBS, covR and covS are part of a seven gene operon transcribed from two promoters that are not regulated by CovR. A DeltacovSR mutant was found to display dramatic phenotypic changes such as increased haemolytic activity and reduced CAMP activity on blood agar. Adherence of the DeltacovSR mutant to epithelial cells was greatly increased and analysis by transmission electron microscopy revealed the presence at its surface of a fibrous extracellular matrix that might be involved in these intercellular interactions. However, the DeltacovSR mutant was unable to initiate growth in RPMI and its viability in human normal serum was greatly impaired. A major finding of this phenotypic analysis was that the CovS/CovR system is important for GBS virulence, as a 3 log increase of the LD(50) of the mutant strain was observed in the neonate rat sepsis model. The pleiotropic phenotype of the DeltacovSR mutant is in full agreement with the large number of genes controlled by CovS/CovR as seen by expression profiling analysis, many of which encode potentially secreted or cell surface-associated proteins: 76 genes are repressed whereas 63 were positively regulated. CovR was shown to bind directly to the regulatory regions of several of these genes and a consensus CovR recognition sequence was proposed using both DNase I footprinting and computational analyses.

Caractérisation d’une protéase extracellulaire de Clostridium difficile

Clostridium difficile is an intestinal pathogen, which produces two main virulence factors, the exotoxins A and B. Other bacterial structures have been implicated in the colonization of the gastrointestinal tract, which is the first step of the pathogenic process. C. difficile expresses adherence factors and also, displays some surface-associated proteolytic activity, which could play a role in the physiopathology of this bacterium. The aim of this work was to study the protein named Cwp84 which displays significant homologies with many cysteine proteases. The coding catalytic domain of this protein has been cloned in the expression system pGEX-6P-1, as an in-frame fusion with the gluthatione S-transferase, and subsequently purified. The purified fraction showed proteolytic activity on gelatine and BAPNA, but not on azocoll, suggesting a highly selective substrate specificity. The results obtained from inhibition experiments confirmed that Cwp84 belongs to the cysteine protease family. Cwp84 could play a role in degrading some specific host proteins or in the maturation of surface-associated bacterial proteins.

CD81 binding regions of hepatitis C virus remain conserved after liver transplantation.

CD81 is a surface-associated protein expressed in the membranes of mammalian cells. It has been suggested that CD81 interacts with hepatitis C virus E2 protein, and thus might facilitate the entry of HCV into hepatocytes. The envelope-binding site appears to involve amino acids (aa) 480-493 and 544-551 within the E2 glycoprotein. Little is known about the quasispecies genetic diversity of these two regions. We studied four patients who underwent transplantation for HCV-related cirrhosis and who developed recurrent hepatitis C. We evaluated HCV quasispecies diversity in serum samples obtained at the time of transplantation and at several time points thereafter. Quasispecies diversity was assessed by cloning and sequencing of viral isolates, with computer analysis of evolution models. The genetic distance in the region that spans aa 480 to 493 was 0.019 +/- 0.004 before the transplant, and 0.039 +/- 0.014 after the transplant (p=0.324). In the aa 544 to 551 region, the pre-transplant genetic distance was 0.012 +/- 0.008 and the post-transplant distance, 0.010 +/- 0.007 (p=0.890). There was also no significant difference between the number of nonsynonymous substitutions per nonsynonymous site before and after transplantation. In conclusion, the HCV genetic sequences of putative CD81 binding regions aa 480-493 and aa 544-551 did not diversify significantly after liver transplantation. This may favor HCV re-infection of the allograft after liver transplantation.