Identification of protective protein antigens for vaccination against Streptococcus dysgalactiae in cobia (Rachycentron canadum).

Streptococcus dysgalactiae is considered a causative agent of severe infection and economic loss for the cobia industry in Taiwan. In this study, protective antigens of this pathogenic bacterium were identified and screened in cobia (Rachycentron canadum). Outer surface proteins (OMPs) of this pathogen were extracted using mutanolysin digestion. Immunogenic targets were detected by western blot and then subjected to peptide sequencing using NanoLC-MS/MS. Two surface proteins, namely phosphoenolpyruvate protein phosphotransferase (PtsA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), showed strong reactions with cobia antisera against S. dysgalactiae. Recombinant proteins were produced in Escherichia coli cells and their protective efficacies were investigated in cobia. Fish immunised with recombinant proteins, rPtsA + ISA (ISA 763 AVG) and rGAPDH + ISA, elicited higher levels of specific antibody responses against the recombinant proteins and had high levels of lysozyme activity. Notably, vaccinated fish were protected from lethal challenge with relative percentage of survival (RPS) values for rPtsA + ISA and rGAPDH + ISA groups being 91.67% and 83.33%, while 0% RPS value was found in both ISA injected and control groups. The results presented in the study demonstrate that the GAPDH and PtsA are promising vaccine candidates for preventing S. dysgalactiae disease in cobia.

Structural and functional analysis of BB0689 from Borrelia burgdorferi, a member of the bacterial CAP superfamily.

Spirochete Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted from infected Ixodes ticks to a mammalian host after a tick bite. The outer surface protein BB0689 from B. burgdorferi is up-regulated when the tick feeds, which indicates a potential role for BB0689 in Lyme disease pathogenesis. We have determined the crystal structure of BB0689, which revealed that the protein belongs to the CAP superfamily. Though the CAP domain is widespread in all three cellular domains of life, thus far the CAP domain has been studied only in eukaryotes, in which it is usually linked to certain other domains to form a multi-domain protein and is associated with the mammalian reproductive tract, the plant response to pathogens, venom allergens from insects and reptiles, and the growth of human brain tumors. Though the exact function of the isolated CAP domain remains ambiguous, several functions, including the binding of cholesterol, lipids and heparan sulfate, have been recently attributed to different CAP domain proteins. In this study, the bacterial CAP domain structure was analyzed and compared with the previously solved crystal structures of representative CAPs, and the function of BB0689 was examined. To determine the potential function of BB0689 and ascertain whether the functions that have been attributed to the CAP domain proteins are conserved, the binding of previously reported CAP domain interaction partners was analyzed, and the results suggested that BB0689 has a unique function that is yet to be discovered.

Crystal structures of the Erp protein family members ErpP and ErpC from Borrelia burgdorferi reveal the reason for different affinities for complement regulator factor H.

Borrelia burgdorferi is the causative agent of Lyme disease, which can be acquired after the bite of an infected Ixodes tick. As a strategy to resist the innate immunity and to successfully spread and proliferate, B. burgdorferi expresses a set of outer membrane proteins that are capable of binding complement regulator factor H (CFH), factor H-like protein 1 (CFHL-1) and factor H-related proteins (CFHR) to avoid complement-mediated killing. B. burgdorferi B31 contains three proteins that belong to the Erp (OspE/F-related) protein family and are capable of binding CFH and some CFHRs, namely ErpA, ErpC and ErpP. We have determined the crystal structure of ErpP at 2.53Å resolution and the crystal structure of ErpC at 2.15Å resolution. Recently, the crystal structure of the Erp family member OspE from B. burgdorferi N40 was determined in complex with CFH domains 19-20, revealing the residues involved in the complex formation. Despite the high sequence conservation between ErpA, ErpC, ErpP and the homologous protein OspE (78-80%), the affinity for CFH and CFHRs differs markedly among the Erp family members, suggesting that ErpC may bind only CFHRs but not CFH. A comparison of the binding site in OspE with those of ErpC and ErpP revealed that the extended loop region, which is only observed in the potential binding site of ErpC, plays an important role by preventing the binding of CFH. These results can explain the inability of ErpC to bind CFH, whereas ErpP and ErpA still possess the ability to bind CFH.

Structural characterization of CspZ, a complement regulator factor H and FHL-1 binding protein from Borrelia burgdorferi.

Borrelia burgdorferi is the causative agent of Lyme disease and is found in two different types of hosts in nature - Ixodes ticks and various mammalian organisms. To initiate disease and survive in mammalian host organisms, B. burgdorferi must be able to transfer to a new host, proliferate, attach to different tissue and resist the immune response. To resist the host's immune response, B. burgdorferi produces at least five different outer surface proteins that can bind complement regulator factor H (CFH) and/or factor H-like protein 1 (CFHL-1). The crystal structures of two uniquely folded complement binding proteins, which belong to two distinct gene families and are not found in other bacteria, have been previously described. The crystal structure of the CFH and CFHL-1 binding protein CspZ (also known as BbCRASP-2 or BBH06) from B. burgdorferi, which belongs to a third gene family, is reported in this study. The structure reveals that the overall fold is different from the known structures of the other complement binding proteins in B. burgdorferi or other bacteria; this structure does not resemble the fold of any known protein deposited in the Protein Data Bank. The N-terminal part of the CspZ protein forms a four-helix bundle and has features similar to the FAT domain (focal adhesion targeting domain) and a related domain found in the vinculin/α-catenin family. By combining our findings from the crystal structure of CspZ with previous mutagenesis studies, we have identified a likely binding surface on CspZ for CFH and CFHL-1.

Crystal structure of the infectious phenotype-associated outer surface protein BBA66 from the Lyme disease agent Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease is transmitted to the mammalian host organisms by infected Ixodes ticks. Transfer of the spirochaetal bacteria from Ixodes ticks to the warm-blooded mammalian organism provides a challenge for the bacteria to adapt and survive in the different environmental conditions. B. burgdorferi has managed to differentially express genes in response to the encountered changes such as temperature and pH variance or metabolic rate to survive in both environments. In recent years, much interest has been turned on genes that are upregulated during the borrelial transfer to mammalian organisms as this could reveal the proteins important in the pathogenesis of Lyme disease. BBA66 is one of the upregulated outer surface proteins thought to be important in the pathogenesis of B. burgdorferi as it has been found out that BBA66 is necessary during the transmission and propagation phase to initiate Lyme disease. As there is still little known about the pathogenesis of B. burgdorferi, we have solved the crystal structure of the outer surface protein BBA66 at 2.25Å resolution. A monomer of BBA66 consists of 6 α-helices packed in a globular domain, and the overall folding is similar to the homologous proteins BBA64, BBA73, and CspA. Structure-based sequence alignment with the homologous protein BBA64 revealed that the conserved residues are mainly located inwards the core region of the protein and thus may be required to maintain the overall fold of the protein. Unlike the other homologous proteins, BBA66 has an atypically long disordered region at the N terminus thought to act as a "tether" between the structural domain and the cell surface.

Structure of an outer surface lipoprotein BBA64 from the Lyme disease agent Borrelia burgdorferi which is critical to ensure infection after a tick bite.

Lyme disease is a tick-borne infection caused by the transmission of Borrelia burgdorferi from infected Ixodes ticks to a mammalian host during the blood meal. Previous studies have shown that the expression of B. burgdorferi surface-localized lipoproteins, which include BBA64, is up-regulated during the process of tick feeding. Although the exact function of BBA64 is not known, this lipoprotein is critical for the transmission of the spirochete from the tick salivary glands to the mammalian organism after a tick bite. Since the mechanism of development of the disease and the functions of the surface lipoproteins associated with borreliosis are still poorly understood, the crystal structure of the B. burgdorferi outer surface lipoprotein BBA64 was solved at 2.4 Å resolution in order to obtain a better insight into the pathogenesis of B. burgdorferi and to promote the discovery of novel potential preventive drugs against Lyme disease. In this study, the crystal structure of BBA64 was also compared with that of the paralogous protein CspA (also referred to as BbCRASP-1, CRASP-1 or BBA68). CspA is the complement regulator-acquiring surface protein-1 of B. burgdorferi; its structure is known, but its function apparently differs from that of BBA64. It is demonstrated that unlike the homologous CspA, BBA64 does not form a homodimer. Their differences in function could be explained by divergence in their amino-acid sequences, electrostatic surface potentials and overall tertiary structures. The C-terminal part of BBA64 has a different conformation to that of CspA; the conformation of this region is essential for the proper function of CspA.