Proteomic analysis of cell surface proteins from <b><i>Clostridium difficile</i></b>

Wright, Anne; Wait, Robin; Begum, Shajna; Crossett, Ben; Nagy, Judit; Brown, Katherine A.; Fairweather, Neil F.

doi:10.1002/pmic.200401179

Cited by 80 publications

(84 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, it is surprising to recover this mature form tightly associated with the underlying cell wall. It should be noted that this last result is consistent with a previous proteomic study, where Cwp84 was recovered in the extracts collected after enzymatic degradation of the peptidoglycan (42). One hypothesis is that the secreted cleaved protease could be reassociated with the bacterial surface, since it has been shown for some surface proteins in other bacteria (2,9), and previously proposed as an anchoring mechanism of GroEL at the C. difficile surface (18).…”

Section: Discussionsupporting

confidence: 79%

Localization of the Clostridium difficile Cysteine Protease Cwp84 and Insights into Its Maturation Process

et al. 2011

View full text Add to dashboard Cite

Clostridium difficile is a nosocomial pathogen involved in antibiotic-associated diarrhea. C. difficile expresses a cysteine protease, Cwp84, which has been shown to degrade some proteins of the extracellular matrix and play a role in the maturation of the precursor of the S-layer proteins. We sought to analyze the localization and the maturation process of this protease. Two identifiable forms of the protease were found to be associated in the bacteria: a form of ϳ80 kDa and a cleaved one of 47 kDa, identified as the mature protease. They were found mainly in the bacterial cell surface fractions and weakly in the extracellular fraction. The 80-kDa protein was noncovalently associated with the S-layer proteins, while the 47-kDa form was found to be tightly associated with the underlying cell wall. Our data supported that the anchoring of the Cwp84 47-kDa form is presumably due to a reassociation of the secreted protein.Moreover, we showed that the complete maturation of the recombinant protein Cwp84 30-803 is a sequential process beginning at the C-terminal end, followed by one or more cleavages at the N-terminal end. The processing sites of recombinant Cwp84 are likely to be residues Ser-92 and Lys-518. No proteolytic activity was detected with the mature recombinant protease Cwp84 92-518 (47 kDa). In contrast, a fragment including the propeptide (Cwp84 30-518 ) displayed proteolytic activity on azocasein and fibronectin. These results showed that Cwp84 is processed essentially at the bacterial cell surface and that its different forms may display different proteolytic activities.Clostridium difficile, a Gram-positive spore-forming anaerobic bacterium, is the leading bacterial cause of nosocomial intestinal infection worldwide and is responsible for illness ranging from mild diarrhea to life-threatening pseudomembranous colitis (8, 16). The clinical relevance of C. difficile has increased significantly during the past few years, particularly since 2003, when hypervirulent PCR-ribotype 027 strains have been involved in outbreaks and have been associated with severe disease in North America and Europe (39).As in other pathogenic bacteria, C. difficile expresses several virulence factors. The two large clostridial toxins A (TcdA) and B (TcdB) are the most important and the bestcharacterized virulence factors of C. difficile (24,26,30,37), leading to clinical manifestations by disorganizing the cell actin cytoskeleton. At present, the interactions between C. difficile and the host cell surface are not fully understood, even if several cell surface proteins, including adhesins and flagella, have been shown to mediate bacterial attachment (5,17,18,35,38). The S layer is a paracrystalline array on the outer cell surface that completely coats the bacterium; it is composed of two proteins, the high-molecular-weight Slayer protein (HMW-SLP) and the low-molecular-weight S-layer protein (LMW-SLP), derived from a common precursor, SlpA (6). These two proteins are the major surface proteins in C. difficile and play a role in...

show abstract

Section: Discussionsupporting

confidence: 79%

Localization of the Clostridium difficile Cysteine Protease Cwp84 and Insights into Its Maturation Process

et al. 2011

View full text Add to dashboard Cite

show abstract

“…The C. difficile genome contains 28 genes encoding proteins with domains similar to the HMW-SLP (41). Some of them are located on the cell surface (57). CD0514, a gene controlled by phase variation that encodes the cell wall protein CwpV (17), was upregulated in the sigH mutant, as observed for slpA.…”

Section: Fr)mentioning

confidence: 82%

“…Surface-associated proteins and factors, which play a role in cell adherence and could be involved in intestinal colonization, have been identified in C. difficile (12,57). Several genes encoding surface-associated proteins and adhesion factors were differentially expressed between 4 h and 10 h of growth and/or controlled by SigH (see Table S3 in the supplemental material).…”

Section: Fr)mentioning

confidence: 99%

The Key Sigma Factor of Transition Phase, SigH, Controls Sporulation, Metabolism, and Virulence Factor Expression in Clostridium difficile

et al. 2011

View full text Add to dashboard Cite

Toxin synthesis in Clostridium difficile increases as cells enter into stationary phase. We first compared the expression profiles of strain 630E during exponential growth and at the onset of stationary phase and showed that genes involved in sporulation, cellular division, and motility, as well as carbon and amino acid metabolism, were differentially expressed under these conditions. We inactivated the sigH gene, which encodes an alternative sigma factor involved in the transition to post-exponential phase in Bacillus subtilis. Then, we compared the expression profiles of strain 630E and the sigH mutant after 10 h of growth. About 60% of the genes that were differentially expressed between exponential and stationary phases, including genes involved in motility, sporulation, and metabolism, were regulated by SigH, which thus appears to be a key regulator of the transition phase in C. difficile. SigH positively controls several genes required for sporulation. Accordingly, sigH inactivation results in an asporogeneous phenotype. The spo0A and CD2492 genes, encoding the master regulator of sporulation and one of its associated kinases, and the spoIIA operon were transcribed from a SigH-dependent promoter. The expression of tcdA and tcdB, encoding the toxins, and of tcdR, encoding the sigma factor required for toxin production, increased in a sigH mutant. Finally, SigH regulates the expression of genes encoding surface-associated proteins, such as the Cwp66 adhesin, the S-layer precursor, and the flagellum components. Among the 286 genes positively regulated by SigH, about 40 transcriptional units presenting a SigH consensus in their promoter regions are good candidates for direct SigH targets.

show abstract

“…Heat shock protein was observed on the surface of C. pneumoniae (30), S. agalactiae (20,30), Listeria monocytogenes (54), and Clostridium difficile (60). Pyruvate kinase was also reported as a surface-associated protein in S. pyogenes (8), C. difficile (60), and L. monocytogenes (54).…”

Section: Discussionmentioning

confidence: 95%

“…Combining these technologies becomes an important first step in vaccine development. Recently, proteomic analyses of cell surface-associated proteins from Staphylococcus aureus (34), S. pyogenes (8), Staphylococcus epidermidis (53), Streptococcus agalactiae (20), Streptococcus pneumoniae (24), and Clostridium difficile (60) were reported. In these studies, the detection of cell surfaceassociated proteins was based on digestion of the peptidoglycan with lysozyme, mutanolysin M1, and/or lysostaphin in the presence of osmotic protective agents.…”

mentioning

confidence: 99%

Proteomic Analysis and Identification of Streptococcus pyogenes Surface-Associated Proteins

Severin

Nickbarg²,

Wooters³

et al. 2007

J Bacteriol

142

View full text Add to dashboard Cite

Streptococcus pyogenes is a gram-positive human pathogen that causes a wide spectrum of disease, placing a significant burden on public health. Bacterial surface-associated proteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. The identification of these proteins for vaccine development is an important goal of bacterial proteomics. Here we describe a method of proteolytic digestion of surface-exposed proteins to identify surface antigens of S. pyogenes. Peptides generated by trypsin digestion were analyzed by multidimensional tandem mass spectrometry. This approach allowed the identification of 79 proteins on the bacterial surface, including 14 proteins containing cell wall-anchoring motifs, 12 lipoproteins, 9 secreted proteins, 22 membrane-associated proteins, 1 bacteriophage-associated protein, and 21 proteins commonly identified as cytoplasmic. Thirty-three of these proteins have not been previously identified as cell surface associated in S. pyogenes. Several proteins were expressed in Escherichia coli, and the purified proteins were used to generate specific mouse antisera for use in a whole-cell enzymelinked immunosorbent assay. The immunoreactivity of specific antisera to some of these antigens confirmed their surface localization. The data reported here will provide guidance in the development of a novel vaccine to prevent infections caused by S. pyogenes.Streptococcus pyogenes, also known as group A Streptococcus, is a gram-positive bacterium that causes a wide spectrum of diseases ranging from mild localized infections, such as pharyngitis and impetigo, to severe invasive diseases, such as necrotizing fasciitis and streptococcal toxic shock-like syndrome. Invasive streptococcal disease is associated with high morbidity and mortality rates (37). S. pyogenes is also associated with a variety of autoimmune sequelae such as acute rheumatic fever, which after repeated episodes can result in rheumatic valvular heart disease, the most common cause of pediatric heart disease worldwide (11). In spite of the high mortality and substantial economic losses caused by these diseases, there is currently no licensed vaccine to prevent human S. pyogenes infections.For many years, efforts to develop a vaccine to protect against S. pyogenes infections were focused on the surfaceassociated M protein (19, 38), a major virulence factor of S. pyogenes. However, there are at least two significant limitations for using M protein as a vaccine antigen. First, the M protein contains a highly variable amino-terminal region that determines the S. pyogenes serotype. With over 150 different M serotypes identified, it is difficult to envision using the M protein as a broadly efficacious vaccine. Second, M protein elicits antibodies that are cross-reactive with human cardiac myosin and are associated with the development of acute rheumatic fever (10). To circumvent these issues, we have initiated an alternative strategy to identify other proteins localized to the sur...

show abstract

Proteomic analysis of cell surface proteins from Clostridium difficile

Cited by 80 publications

References 26 publications

Localization of the Clostridium difficile Cysteine Protease Cwp84 and Insights into Its Maturation Process

Localization of the Clostridium difficile Cysteine Protease Cwp84 and Insights into Its Maturation Process

The Key Sigma Factor of Transition Phase, SigH, Controls Sporulation, Metabolism, and Virulence Factor Expression in Clostridium difficile

Proteomic Analysis and Identification of Streptococcus pyogenes Surface-Associated Proteins

Contact Info

Product

Resources

About