is an important human opportunistic pathogen that can cause serious health problems, particularly among newborns and older individuals. contains the CAMP factor, a pore-forming toxin first identified in this bacterium. The CAMP reaction is based on the co-hemolytic activity of the CAMP factor and is commonly used to identify in the clinic. Closely related proteins are present also in other Gram-positive pathogens. Although the CAMP toxin was discovered more than a half century ago, no structure from this toxin family has been reported, and the mechanism of action of this toxin remains unclear. Here, we report the first structure of this toxin family, revealing a structural fold composed of 5 + 3-helix bundles. Further analysis by protein truncation and site-directed mutagenesis indicated that the N-terminal 5-helix bundle is responsible for membrane permeabilization, whereas the C-terminal 3-helix bundle is likely responsible for host receptor binding. Interestingly, the C-terminal domain inhibited the activity of both full-length toxin and its N-terminal domain. Moreover, we observed that the linker region is highly conserved and has a conserved DLDAT sequence motif. Structurally, this linker region extensively interacted with both terminal CAMP factor domains, and mutagenesis disclosed that the conserved sequence motif is required for CAMP factor's co-hemolytic activity. In conclusion, our results reveal a unique structure of this bacterial toxin and help clarify the molecular mechanism of its co-hemolytic activity.
CAMP factor is a unique -helical bacterial toxin that is known for its cohemolytic activity in combination with staphylococcal sphingomyelinase. It was first discovered in the human pathogen Streptococcus agalactiae (also known as group B streptococcus), but homologous genes have been found in many other Gram-positive pathogens. In this study, the efforts that led to the determination of the first structure of a CAMP-family toxin are reported. Initially, it was possible to produce crystals of the native protein which diffracted to near 2.45 Å resolution. However, a series of technical obstacles were encountered on the way to structure determination. Over a period of more than five years, many methods, including selenomethionine labeling, mutations, crystallization chaperones and heavy-atom soaking, were attempted, but these attempts resulted in limited progress. The structure was finally solved using a combination of iodine soaking and molecular replacement using the crystallization chaperone maltosebinding protein (MBP) as a search model. Analysis of native and MBP-tagged CAMP-factor structures identified a conserved interaction interface in the C-terminal domain (CTD). The positively charged surface may be critical for binding to acidic ligands. Furthermore, mutations on the interaction interface at the CTD completely abolished its co-hemolytic activities. This study provides novel insights into the mechanism of the membrane-permeabilizing activity of CAMP factor.
NetF-producing type A Clostridium perfringens is an important cause of canine and foal necrotizing enteritis. NetF, related to the β-sheet pore-forming Leukocidin/Hemolysin superfamily, is considered a major virulence factor for this disease. The main purpose of this work is to demonstrate the pore-forming activity of NetF and characterize the chemical nature of its binding site. Electron microscopy using recombinant NetF (rNetF) confirmed that NetF is able to oligomerize and form large pores in equine ovarian (EO) cell membranes and sheep red blood cells. These oligomeric pores appear to be about 4–6 nm in diameter, and the number of oligomer subunits to vary from 6 to 9. Sodium periodate treatment rendered EO cells non-susceptible to NetF, suggesting that NetF binding requires cell surface carbohydrates. NetF cytotoxicity was also inhibited by a lectin that binds sialic acid, by sialidase, and by free sialic acid in excess, all of which clearly implicate sialic acid-containing membrane carbohydrates in NetF binding and/or toxicity for EO cells. Binding of NetF to sheep red blood cells was not inhibited by the gangliosides GM1, GM2 and GM3, nor did the latter promote membrane permeabilization in liposomes, suggesting that they do not constitute the cellular receptors. In contrast, treatment of EO cells with different proteases reduced their susceptibility to NetF, suggesting that the NetF receptor is a sialic acid-containing glycoprotein.
Displaying chemical structures in LATE X documents currently requires either hand-coding of the structures using one of several LATE X packages, or the inclusion of finished graphics files produced with an external drawing program. There is currently no software tool available to render the large number of structures available in molfile or SMILES format to LATE X source code. We here present mol2chemfig, a Python program that provides this capability. Its output is written in the syntax defined by the chemfig TE X package, which allows for the flexible and concise description of chemical structures and reaction mechanisms. The program is freely available both through a web interface and for local installation on the user’s computer. The code and accompanying documentation can be found at http://chimpsky.uwaterloo.ca/mol2chemfig.
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