The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae . Membrane cholesterol is required for the cytolytic activity of this toxin, but it ...is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K d of 1.88 × 10 ⁻⁵ M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto- N -neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.
Significance The pneumococcus accounts for 25% of deaths in children under 5 y of age in developing countries. One of the most important virulence factors expressed by this pathogen is the pore-forming toxin, pneumolysin (Ply), an example of a Gram-positive cholesterol-dependent cytolysin (CDC). We show that Ply interacts with the Lewis histo-blood group antigen sialyl LewisX and that blocking this interaction can protect RBCs from lysis. We also identify glycan receptors on RBCs for the CDC streptolysin O from group A streptococcus. Our study supports the emerging paradigm shift that CDCs have cellular receptors other than cholesterol that define target cell tropism.
Cholesterol-dependent cytolysins (CDCs) form pores in cholesterol-rich membranes, but cholesterol alone is insufficient to explain their cell and host tropism. Here, we show that all eight major CDCs ...have high-affinity lectin activity that identifies glycans as candidate cellular receptors. Streptolysin O, vaginolysin, and perfringolysin O bind multiple glycans, while pneumolysin, lectinolysin, and listeriolysin O recognize a single glycan class. Addition of exogenous carbohydrate receptors for each CDC inhibits toxin activity. We present a structure for suilysin domain 4 in complex with two distinct glycan receptors, P
antigen and αGal/Galili. We report a wide range of binding affinities for cholesterol and for the cholesterol analog pregnenolone sulfate and show that CDCs bind glycans and cholesterol independently. Intermedilysin binds to the sialyl-TF
-glycan on its erythrocyte receptor, CD59. Removing sialyl-TF from CD59 reduces intermedilysin binding. Glycan-lectin interactions underpin the cellular tropism of CDCs and provide molecular targets to block their cytotoxic activity.
In foot-and-mouth disease (FMD)-endemic countries, vaccination is commonly used to control the disease, whilst in FMD-free countries, vaccination is considered as an option, in addition to culling ...the infected and in contact animals. FMD vaccines are mainly comprised of inactivated virions and stimulate protective antibodies to virus structural proteins. In contrast, infection with FMD virus leads to virus replication and additional antibody responses to viral nonstructural proteins (NSP). Therefore, antibodies against NSPs are used to differentiate infection in vaccinated animals (DIVA), in order to estimate the prevalence of infection or its absence. Another advantage of NSP antibody tests is that they detect FMD infection in the field, irrespective of the serotypes of virus in circulation. In cattle, the NSP tests that target the 3ABC polyprotein provides the highest sensitivity, detecting up to 90% of vaccinated animals that become carriers after exposure to infection, with a specificity of around 99%. Due to insufficient diagnostic sensitivity and specificity, detection of a low level of infection is difficult at the population level with a high degree of confidence. The low level of non-specific responses can be overcome by retesting samples scored positive using a second confirmatory test, which should have at least comparable sensitivity to the first test. In this study, six in-house tests were developed incorporating different NSP antigens, and validated using bovine sera from naïve animals, field cases and experimentally vaccinated and/or infected animals. In addition, two (short and long incubation) new commercial NSP tests based on 3ABC competitive blocking ELISAs (ID Screen
FMD NSP Competition, IDvet, France) were validated in this study. The two commercial ELISAs had very similar sensitivities and specificities that were not improved by lengthening the incubation period. Several of the new in-house tests had performance characteristics that were nearly as good as the commercial ELISAs. Finally, the in-house tests were evaluated for use as confirmatory tests following screening with the PrioCHECK
and ID Screen
FMDV NS commercial kits, to assess the diagnostic performance produced by a multiple testing strategy. The in-house tests could be used in series (to confirm) or in parallel (to augment) with the PrioCHECK
and IDvet
FMDV NS commercial kits, in order to improve either the specificity or sensitivity of the overall test system, although this comes at the cost of a reduction in the counterpart (sensitivity/specificity) parameter.
Abstract
Background
Thymic hyperplasia is a recognized complication of Graves' disease that can present radiologically as an anterior mediastinal mass.
Case Description
We present a unique case of ...massive thymic hyperplasia occurring in a 24-year-old female without a known history of thyroid or other systemic disease in whom Graves' disease first manifested intraoperatively during thymectomy for presumed neoplasia.
Conclusion
We suggest that the work-up of all anterior mediastinal masses should include a comprehensive search for medical causes of reversible thymic enlargement.
Serology is used to predict vaccine induced protection against challenge with a heterologous strain of the same serotype of foot-and-mouth disease virus (FMDV). To evaluate the accuracy of such ...predictions, we compared the protection afforded to cattle vaccinated with the O
1 Manisa strain of FMDV against challenge with either a homologous (O
1 Manisa) or a heterologous strain (O
1 Campos). Serology by virus neutralization test (VNT) using O
1 Manisa antiserum predicted an acceptable protection against such a challenge. Two experiments were carried out to compare the results for consistency. A total of 78 naïve cattle were vaccinated with different antigen payloads (60–0.94
μg) of O
1 Manisa. They were challenged by intradermolingual inoculation with live FMDV, either O
1 Manisa or O
1 Campos. Unvaccinated naïve control cattle (
n
=
20) were also challenged with either the O
1 Manisa or O
1 Campos viruses and all developed generalized FMD. The protection results for the vaccinated cattle revealed that higher payloads of O
1 Manisa vaccine were needed to protect against heterologous challenge compared to that for homologous challenge. The 50% protective dose (PD
50) values for the vaccine in experiments 1 and 2 were found to be 28.78 and 9.44 for the homologous challenge and 3.98 and 5.01 for heterologous challenge. Furthermore, protection against O
1 Campos required a higher level of vaccine-induced antibody against this virus compared to the level of O
1 Manisa neutralizing antibody associated with protection against homologous challenge. The 50% protective level of
in vitro neutralizing antibody was found to be log
10
1.827 for O
1 Campos and log
10
0.954 for O
1 Manisa based on O
1 Manisa based virus neutralization test.
Abstract Serology is used to predict vaccine induced protection against challenge with a heterologous strain of the same serotype of foot-and-mouth disease virus (FMDV). To evaluate the accuracy of ...such predictions, we compared the protection afforded to cattle vaccinated with the O1 Manisa strain of FMDV against challenge with either a homologous (O1 Manisa) or a heterologous strain (O1 Campos). Serology by virus neutralization test (VNT) using O1 Manisa antiserum predicted an acceptable protection against such a challenge. Two experiments were carried out to compare the results for consistency. A total of 78 naïve cattle were vaccinated with different antigen payloads (60–0.94 μg) of O1 Manisa. They were challenged by intradermolingual inoculation with live FMDV, either O1 Manisa or O1 Campos. Unvaccinated naïve control cattle ( n = 20) were also challenged with either the O1 Manisa or O1 Campos viruses and all developed generalized FMD. The protection results for the vaccinated cattle revealed that higher payloads of O1 Manisa vaccine were needed to protect against heterologous challenge compared to that for homologous challenge. The 50% protective dose (PD50 ) values for the vaccine in experiments 1 and 2 were found to be 28.78 and 9.44 for the homologous challenge and 3.98 and 5.01 for heterologous challenge. Furthermore, protection against O1 Campos required a higher level of vaccine-induced antibody against this virus compared to the level of O1 Manisa neutralizing antibody associated with protection against homologous challenge. The 50% protective level of in vitro neutralizing antibody was found to be log10 1.827 for O1 Campos and log10 0.954 for O1 Manisa based on O1 Manisa based virus neutralization test.
Abstract This study investigated the transmission of foot-and-mouth disease virus (FMDV) from experimentally infected Indian buffalo to in-contact naïve and vaccinated cattle and buffalo. In each of ...six rooms, two donor buffalo that had been inoculated with FMDV were housed for five days with four recipient animals, comprising one vaccinated buffalo, one vaccinated calf, one unvaccinated buffalo and one unvaccinated calf. Vaccination was carried out with current Indian vaccine strain (O/IND/R2/75) and challenged on 28 days post-vaccination with an antigenically similar strain (O/HAS/34/05). All 12 donor buffalo and the six unvaccinated cattle and six unvaccinated calves developed clinical signs of foot-and-mouth disease (FMD). In contrast, all six vaccinated cattle (100%) and four out of six vaccinated buffalo (66.6%) were protected from disease but all became infected with FMDV. This confirms that buffalo have the potential to spread FMD by direct contact and that vaccination can block this spread. The numbers of animals in the study were too small to determine if the differences in clinical protection afforded by vaccination of cattle and buffalo are significant and warrant a different dose regime.
Strategies to control transboundary diseases have in the past generated unintended negative consequences for both the environment and local human populations. Integrating perspectives from across ...disciplines, including livestock, veterinary and conservation sectors, is necessary for identifying disease control strategies that optimise environmental goods and services at the wildlife-livestock interface. Prompted by the recent development of a global strategy for the control and elimination of foot-and-mouth disease (FMD), this paper seeks insight into the consequences of, and rational options for potential FMD control measures in relation to environmental, conservation and human poverty considerations in Africa. We suggest a more environmentally nuanced process of FMD control that safe-guards the integrity of wild populations and the ecosystem dynamics on which human livelihoods depend while simultaneously improving socio-economic conditions of rural people. In particular, we outline five major issues that need to be considered: 1) improved understanding of the different FMD viral strains and how they circulate between domestic and wildlife populations; 2) an appreciation for the economic value of wildlife for many African countries whose presence might preclude the country from ever achieving an FMD-free status; 3) exploring ways in which livestock production can be improved without compromising wildlife such as implementing commodity-based trading schemes; 4) introducing a participatory approach involving local farmers and the national veterinary services in the control of FMD; and 5) finally the possibility that transfrontier conservation might offer new hope of integrating decision-making at the wildlife-livestock interface.
Abstract Expression of Physalis mottle tymovirus (PhMV) coat protein (CP) in Escherichia coli ( E. coli ) was earlier shown to self-assemble into empty capsids that are nearly identical to the ...capsids formed in vivo . Aminoacid substitutions were made at the N-terminus of wild-type PhMV CP with single or tandem repeats of infection related B-cell epitopes of foot-and-mouth disease virus (FMDV) non-structural proteins (NSPs) 3B1, 3B2, 3AB, 3D and 3ABD of lengths 48, 66, 49, 51 and 55, respectively to produce chimeras pR-Ph-3B1, pR-Ph-3B2, pR-Ph- 3AB, pR-Ph-3D and pR-Ph-3ABD. Expression of these constructs in E. coli resulted in chimeric proteins which self-assembled into chimeric tymovirus-like particles (TVLPs), Ph-3B1, Ph-3B2, Ph-3AB, Ph-3D and Ph-3ABD as determined by ultracentrifugation and electron microscopy. Ph-3B1, Ph-3B2, Ph-3AB and Ph-3ABD reacted with polyclonal anti-3AB antibodies in ELISA and electroblot immunoassay, while wild-type PhMV TVLP and Ph-3D antigens did not react. An indirect ELISA (I-ELISA) was developed using Ph-3AB to detect FMDV-NSP antibodies in sera of animals that showed clinical signs of FMD. Field serum samples from cattle, buffalos, sheep, goats and pigs were examined by using these chimeric TVLPs for the differentiation of FMDV infected animals from vaccinated animals (DIVA). The assay was demonstrated to be highly specific (100%) and reproducible with sensitivity levels (94%) comparable to the Ceditest kit ( P > 0.05).
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