Does prebiotic feeding affect equine gastric health? A study on the effects of prebiotic-induced gastric butyric acid production on mucosal integrity of the equine stomach.

Fructo-oligosaccharides are commonly administered as prebiotics to horses in order to reduce the risk of disruption of microbial populations in the hindgut. Their microbial degradation to SCFA already begins in the stomach potentially resulting in increased gastric concentrations of SCFA such as butyric acid. The impact of butyric acid on the squamous mucosa is postulated to be detrimental, its effects on the glandular mucosa are yet unknown. Thus, the aim of this study was to determine the effects of butyric acid exposure on the functional integrity and morphology of the equine nonglandular and glandular gastric mucosa using butyric acid concentrations equivalent to the ones found in horses subjected to prebiotic fructo-oligosaccharides feeding. Gastric mucosal samples of healthy horses were exposed to butyric acid using the in vitro Ussing chamber technique. Electrophysiological parameters were continuously monitored, mucosal samples were blinded and histomorphological analysis was performed using a scoring system for assessment of histopathologic changes. Exposure to butyric acid resulted in pathohistomorphological changes in the glandular mucosa and in impairment of functional mucosal integrity in the squamous and the glandular mucosa as documented by significant changes in tissue conductances (Gt). Administration of fructo-oligosaccharides as a preventive prebiotic measure to horses should therefore be carefully considered, particularly in horses known to be at risk of developing EGUS.

Immunolocalization of DMRTB1 in human testis with normal and impaired spermatogenesis.

BACKGROUND: The transcription factor DMRTB1 plays a pivotal role in coordinating the transition between mitosis and meiosis in murine germ cells. No reliable data are available for human testis. OBJECTIVES: The present study aims to examine the testicular expression pattern of DMRTB1 in men showing normal and impaired spermatogenesis. MATERIALS AND METHODS: Immunohistochemistry was performed using 54 human testicular biopsy specimens and a commercial rabbit polyclonal anti-DMRTB1 primary antibody. RT-PCR complemented immunohistochemistry. To further characterize immunopositive cells and possible co-localization, the proliferation marker Ki-67, the tumor marker PLAP, and an anti-DMRT1 antibody were used. RESULTS: In men with normal spermatogenesis, a strong immunoreactivity was detectable in a subset of spermatogonia (38.34 ± 2.14%). Some spermatocytes showed a weak immunostaining. Adjacent Sertoli cells were immunonegative. Compared with a hematoxylin and eosin overview staining, these immunopositive cells were almost exclusively identified as Apale and B spermatogonia and primary spermatocytes in (pre-)leptotene, zygotene, and pachytene stages. In patients with spermatogenic arrest at spermatogonial level, an altered staining pattern was found. No immunoreactivity was detected in Sertoli cells in Sertoli cell-only syndrome. In germ cell neoplasia in situ (GCNIS) tubules, except for a few (0.4 ± 0.03%), pre-invasive tumor cells were immunonegative. Seminoma cells showed no immunostaining. DISCUSSION: According to previous findings in mice, it seems reasonable that DMRTB1 is expressed in these normal germ cell populations. Moreover, altered staining pattern in spermatogenic arrest at spermatogonial stage suggests a correlation with mitosis and transformation into B spermatogonia. The absence of DMRTB1 in GCNIS cells and tumor cells might be associated with uncontrolled neoplastic cell proliferation and progression into invasive germ cell tumors. Further research is required to elucidate, for example, the role of DMRTB1 in the malignant transformation of human germ cells. CONCLUSION: Our data indicate a relevant role for DMRTB1 regarding the entry of spermatogonia into meiosis in men.

Testicular morphology and spermatogenesis in harbour porpoises (Phocoena phocoena).

Knowledge about reproductive parameters in male harbour porpoises such as testicular histology and germ cell maturation as well as seasonal changes in spermatogenesis is scarce. Thus, the aim of the present study was to report changes in the histological appearance of the testicular morphology of neonatal and juvenile harbour porpoises during maturation, to identify stages of spermatogenesis in adult males and to detect seasonal modifications. The identification of these stages can be used to assess the developmental profile of gene expression during spermatogenesis and to identify defects in spermatogenesis arising in pathological conditions. Testes of adult male harbour porpoises from the North and Baltic Sea that became stranded or by-caught in the years 1998-2016 were histologically examined using Haematoxylin and Eosin - staining. The Periodic Acid Schiff (PAS) staining was used for spermatogenic staging and the evaluation of the development of the acrosomic cap. For the identification of changes in testes morphology and morphometry during the course of the year, histological characteristics like germ cell associations and diameter of the convoluted seminiferous tubules were noted for each month. The analysis showed that in adult males more than one stage of spermatogenesis could be found per cross section of the convoluted seminiferous tubules similar to findings in men and some ape species. This rare phenomenon is called multi-stage-arrangement. In sexually active males from the peak breeding season (June and July) eight stages of spermatogenesis were identified and all stages occurred simultaneously, while during the low breeding season (August to May) only residual spermatogenesis or constituent germ cell populations were found. Missing germ cell generations were recorded in specimens from July to September. Our investigations provide a detailed staging of spermatogenesis and give new insight into the reproductive biology of male harbour porpoises. With these new basic parameters, indicators for endocrine disruptors can be developed in the future, aiming to detect how environmental factors could affect male fertility in wildlife.

Influence of a specific amino acid pattern in the diet on the course of an experimental Campylobacter jejuni infection in broilers.

Campylobacter jejuni (C. jejuni) is one of the most important zoonotic pathogens worldwide. In Europe, the majority of the cases are caused by consuming contaminated poultry meat. The objective of the present study was to investigate potential effects of different crude protein levels in complete diets for broilers on infection dynamics of C. jejuni after experimental infection. In total, 300 commercial broilers line Ross 308 were divided into 4 different groups, including 5 replications of 15 chickens each. The chickens were fed a conventional diet (212 g CP/kg DM) and a protein-reduced test diet (190 g CP/kg DM) supplemented with essential amino acids. This resulted simultaneously in lower amino-acid concentrations preferentially utilized by C. jejuni, such as aspartate, glutamate, proline, and serine. One group of each feeding concept was infected artificially with C. jejuni at day 21 by applying an oral C. jejuni inoculum containing 4.17 ± 0.09 log10 cfu of C. jejuni to 3 of 15 chickens, called "seeders." Feeding the test diet resulted in a significant reduction (P < 0.001) in CP intake (31.5 ± 1.20 g CP/broiler/day and 27.7 ± 0.71 g CP/broiler/day, respectively), a significant decrease (P < 0.05) in crude mucin in excreta (55.7 ± 8.23 g/kg DM and 51.9 ± 7.62 g/kg DM, respectively), and in goblet cell number in cecal crypts (P < 0.05; 15.1 ± 5.71 vs. 13.6 ± 5.91 goblet cells/crypt). In groups receiving the test diet, the excretion of C. jejuni was significantly reduced in seeders by 1.9 log10 cfu/g excreta at day 23 (3.38a ± 2.55 vs. 1.47b ± 2.20; P = 0.033). At day 25, prevalence of C. jejuni in cloacal swabs amounted to 53.3% in the group fed the test diet and 75.7% in the control group, respectively (P < 0.05). In summary, a definite amino acid pattern in the broiler diets could contribute to a development of an effective feeding strategy to reduce the prevalence of C. jejuni infection in chickens (Patent No 17187659.2-1106).

Characterization of the equine blood-testis barrier during tubular development in normal and cryptorchid stallions.

The formation of the blood-testis barrier (BTB) is defined as occurring with the first appearance of spermatocytes at around puberty and is vital for normal spermatogenesis. This barrier between two adjacent Sertoli cells (SCs) consists of a cell junctional protein complex, which includes tight junctions (TJs), adherens junctions, and gap junctions. In many mammalian species, BTB composition has already been investigated, whereas little is known about the equine BTB. In the present study, immunohistochemistry and qualitative Western Blot analysis were used to assess the expression and distribution patterns of the junctional proteins claudin-11 (TJ), zonula occludens-1 (TJ associated), N-cadherin (adherens junctions), and connexin 43 (gap junctions) in equine testes during tubular development and in testes of stallions exhibiting unilateral cryptorchidism. Therefore, testes of 21 warmblood stallions (aged 12 months-11 years) were obtained during routine surgical castration. In the normal adult equine testis, the junctional proteins are localized at the basolateral region of the seminiferous tubules forming a circumferential seal corresponding to the known BTB localization. N-cadherin is additionally expressed along the lateral SC surface. In immature seminiferous cords still lacking a lumen, a diffuse distribution pattern of the junctional proteins throughout the SC cytoplasm is visible. As lumen formation advances, the immunolocalization shifts progressively toward the basolateral SC membranes. Additionally, apoptotic germ cells were detected and quantified in prepubertal stallions using terminal deoxynucleotidyl transferase dUTP nick end labeling assay and correlated with junctional protein localization. In the retained testis of cryptorchid stallions, which exhibit an aberrant testicular morphology, a deviating expression of the junctional proteins is visible. The present data show for the first time that (1) the equine SC junctional complex contains claudin-11, zonula occludens-1, N-Cadherin, and connexin 43, as already described for men or mice, and that (2) different distribution patterns of these proteins exist during testicular development in the context of lumen formation (lumen scores: 1-7) and in retained testes of unilateral cryptorchid stallions.

The Sda/GM2-glycan is a carbohydrate marker of porcine primordial germ cells and of a subpopulation of spermatogonia in cattle, pigs, horses and llama.

Spermatogonia are a potential source of adult pluripotent stem cells and can be used for testis germ cell transplantation. Markers for the isolation of these cells are of great importance for biomedical applications. Primordial germ cells and prepubertal spermatogonia in many species can be identified by their binding of Dolichos biflorus agglutinin (DBA). This lectin binds to two different types of glycans, which are α-linked N-acetylgalactosamine (GalNac) and ß-linked GalNac, if this is part of the Sda or GM2 glycotopes. We used the MAB CT1, which is specific for the trisaccharides motif NeuAcα2-3(GalNAcß1-4)Galß1-, which is common to both Sda and GM2 glycotopes, to further define the glycosylation of DBA binding germ cells. In porcine embryos, CT1 bound to migratory germ cells and gonocytes. CT1/DBA double staining showed that the mesonephros was CT1 negative but contained DBA-positive cells. Gonocytes in the female gonad became CT1 negative, while male gonocytes remained CT1 positive. In immunohistological double staining of cattle, pig, horse and llama testis, DBA and CT1 staining was generally colocalised in a subpopulation of spermatogonia. These spermatogonia were mainly single, sometimes paired or formed chains of up to four cells. Our data show that the Sda/GM2 glycotope is present in developing germ cells and spermatogonia in several species. Owing to the narrower specificity of the CT1 antibody, compared with DBA, the former is likely to be a useful tool for labelling and isolation of these cells.

Connexin 43: its regulatory role in testicular junction dynamics and spermatogenesis.

Spermatogenesis is an intensely regulated process of germ cell development which takes place in the seminiferous tubules of the testis. In addition to known endocrine and autocrine/paracrine signaling pathways, there is now strong evidence that direct intercellular communication via gap junction channels and their specific connexins represents an important mechanism in the regulation of spermatogenesis. Another possibility is that connexins may indirectly regulate the spermatogenic process through modulation of tight and adherens junction proteins, further main structural components of the Sertoli-Sertoli junctional complexes at the blood-testis barrier site. The present review is focused on connexin 43 and updates its possible roles and functions in testicular junction dynamics and in the initiation and maintenance of spermatogenesis. In addition, testicular phenotypes of recently generated (1) conventional connexin 43 knockout mice, (2) connexin 43 knockin mice and (3) transgenic mice exhibiting a cell-specific (conditional) connexin 43 knockout will be discussed.

Recognising the Sertoli-cell-only (SCO) syndrome: a case study.

Total Sertoli-cell-only (SCO) syndrome is often confused with a focal SCO picture, in which testicular illness caused damage to seminiferous tubules and compromised the Sertoli cell range of maturation and functions, but from which still some spermatozoa can be retrieved for assisted reproductive techniques. Here, a possibly new SCO syndrome phenotype is reported exhibiting complete lack of germ cells despite normal architecture of the seminiferous tubules with presence of mature Sertoli cells and normal Leydig cells in the intertubular tissue. Sertoli cells are immunonegative for the prepubertal differentiation markers cytokeratin-18, anti-Muellerian hormone and M2A antigen, but reveal a positive signal for the gap junctional protein connexin 43 known to be expressed in Sertoli cells with an adult type of differentiation. The complete lack of germ cells in combination with fully differentiated adult-type Sertoli cells in this case is in contradiction with known SCO subtypes and with the current hypothesis of reciprocal regulation of Sertoli and germ cell differentiation.

Prenatal development of murine gonads with special reference to germ cell differentiation: a morphological and immunohistochemical study.

The prenatal differentiation of male and female gonads of the mouse was investigated both morphologically and immunohistochemically. Sexual dimorphism could be detected as early as 12 days post-coitum (dpc) by the appearance of the primary elements of the tunica albuginea and positive immunoreactivity for anti-Muellerian hormone in the Sertoli cells of the male gonad. Male germ cells passed two waves of mitotic activity, a first wave between 12 and 14 dpc, which is followed by apoptosis of the old germ cell generation, and a second wave between 17 and 20 dpc. Oct-4 was expressed as a juxtanuclear ring in the cytoplasm of germ cells up to 17 dpc. Subsequently, it was down-regulated and completely disappeared in 20 dpc full-term fetuses. By contrast, M2A antigen revealed only a weak immunoreaction in some germ cells of 14 dpc gonads, but exhibited strong signals in all germ cells of 20 dpc full-term fetuses. Therefore, we postulate that, in the mouse, prenatal germ cells represent two populations: the first is immunopositive for Oct-4 and disappeared in full-term fetuses, whereas the second appeared in 14 dpc and is immunopositive for M2A antigen.

[Embryonal germ cells and germ cell tumors]. / Embryonale Keimzellen und Keimzelltumoren.

Testicular germ cell tumors comprise of group of pluripotent tumors including seminomas and nonseminomas, arise from intratubular germ cell neoplasia and originate from the primordial germ cells/ gonocytes. Many well characterized markers of embryonic stem cells including CD9, PODXL and centromere-specific histone-H3-like protein CENPA are consistently expressed in TGCTs. In embryonic stem cells, pluripotency and self renewal capacities are provided by a network of OCT3/4, NANOG and SOX2. In testicular germ cell tumors, pluripotency genes OCT3/4 und NANOG are upregulated both, in seminomas and non-seminomas, while SOX2 is differentially upregulated in embryonal carcinomas only. Similar to embryonic stem cells, most histological elements of type II GCTs are sensitive to chemotherapy and irradiation. Furthermore, all invasive TGCTs show a consistent gain of the short arm of chromosome 12, as found in ES cells upon extensive in vitro culturing. Moreover, the genetic constitution of testicular germ cell tumors can also be linked to characteristics of embryonic stem cells, likely related to their specific inability to repair DNA damage and their high sensitivity to apoptotic cell death. In conclusion, testicular germ cell tumors represent embryonic cancers found in adults. Both the seminomas and nonseminomas have their specific population of stem cells representative of the primordial germ cells/gonocytes and for embryonic stem cells, respectively.

Effects of human pituitary FSH isoforms on mouse follicles in vitro.

The effects of human FSH glycoforms on mouse follicle development and function in vitro were analysed, and an attempt was made to relate markers of follicular maturation to the expression of immunolocalized connexin (Cx) 43 and Cx26-based gap junctions. Three FSH fractions comprising discrete pI ranges [7.10-5.99 (pool I), pI 5.62-4.95 (pool II) and <3.75 (pool III)] were studied. Pool I produced the strongest effect on preantral granulosa cell proliferation and oestradiol production, and was highly effective for stimulating antral formation; this isoform also evoked a peripheral distribution of Cx43-containing gap junctions. Pool II was effective in promoting preantral granulosa cell proliferation but required higher FSH doses. This particular isoform provoked a more central distribution of Cx43-containing gap junctions, which was associated with a lower oestradiol production and less effective antral formation. Pool III was the least active for all markers of follicle development, and this was associated with minimal induction of Cx43-based gap junctions. The effects of the three FSH isoform pools on Cx26 expression were similar. The pattern of differences strongly suggests that FSH isoforms have complementary and specific actions on developing follicles, and that a shifting stage specific balance of isoforms is required for optimal follicle development.

Cellular localization of estrogen receptor-alpha (ERalpha) and -beta (ERbeta) mRNA in the boar testis.

Boar testes synthesize high amounts of estrogens which are known to stimulate several male sexual functions in a variety of extragonadal target tissues. Possible effects within the testis depend on the existence of the estrogen receptor subtypes alpha and beta (ERalpha, ERbeta). The precise cellular localization of these subtypes within the testis was, so far, based mainly on protein expression studies using different antibodies in several species including boars shows contradictory results. Therefore, we investigated the ERalpha and ERbeta gene expression using RT-PCR of testis homogenates and RT-PCR after UV-single cell microdissection combined with in-situ hybridization of four fertile boars with an average age of 32 weeks. Both ERalpha and ERbeta mRNA were found in testis homogenates. Using in-situ hybridization and UV-single cell microdissection ERalpha mRNA was present in type A and type B spermatogonia up to mid-pachytene primary spermatocytes in stage V-VIII and stage I of the seminiferous epithelial cycle, but not in other cells. ERbeta mRNA was found only in Sertoli cells. Interstitial Leydig cells revealed neither ERalpha nor ERbeta mRNA. The data suggest a direct impact of estrogen in the boar on Sertoli cell function via ERbeta and germ cell formation via ERalpha.

Regulation of Sertoli cell and germ cell differentation.

Unwanted childlessness affects approximately one in six couples worldwide. According to the World Health Organization, in nearly 40% of cases the cause can be attributed to the female, in 20% to the male, in 25% to both, and in 15% the cause remains unknown. The incidence of male factor infertility in the general population is approximately 7%. The majority of these men experience irreversible idiopathic infertility and cannot father children without some form of medical intervention. Male factor infertility, in addition, may be caused by testicular germ cell cancer, which is known to represent the most common cancer among young men in Western industrialized countries. There is growing evidence that this cancer originates from fetal germ cells exhibiting an aberrant programme of gene expression and that tumour progression may be favoured by an aberrant Sertoli cell-germ cell communication. The present monograph aims to shed more light on the regulation of Sertoli and germ cell differentiation. Involving knockout and transgenic mouse models, the authors focus on (a) male factor infertility that might be related to altered maturation of Sertoli cells, (b) male factor infertility that might be due to incorrect histone-to-protamine exchange in haploid spermatids, and (c) progression of testicular germ cell cancer that might be favoured by an aberrant Sertoli cell-germ cell communication.

Gene signatures of testicular seminoma with emphasis on expression of ets variant gene 4.

Gene expression patterns of testicular seminoma were analysed applying oligonucleotide microarrays in 40 specimens of different tumour stages (pT1, pT2, pT3) and in normal testes. Transcripts of maternally expressed 3 transcripts were expressed in seminoma without correlation with delta-like 1 homologue expression indicating an impaired imprinting status in seminoma. Interestingly, the transcripts of bromodomain-containing 2 and nuclear autoantigenic sperm protein associated with spermatogenesis were significantly upregulated in progressing tumour stages. Transcription factors TEA domain family member 4 and ETS variant gene 4 (ETV4), weakly expressed in normal testis, were strongly augmented during tumourigenesis. For ETV4 expression, a significant correlation with the increased expression of matrix metalloproteinase 2 and a disintegrin and metalloproteinase domain 15 was determined. The ETV4 protein was localised to nuclei of spermatogonia and revealed an intense staining in seminoma cells. Taken together, we characterised additional transcription factors and spermatogenesis-associated genes involved in the progression of seminoma.

Identification of antigenic sites on staphylococcal enterotoxin B and toxoid.

The staphylococcal enterotoxins (SEs) are capable of causing both food poisoning and a toxic shock-like illness in man. In addition, SEs are known to act as superantigens, stimulating T-cells according to their T-cell receptor Vbeta type. Relatively little is known of their antigenic determinants and how these may relate to the structure and function of the toxins. As a step in the study of these relationships, the entire molecule of SEB was synthesized in duplicate as a series of octapeptides overlapping by seven residues. This series thus represented all the potential linear epitopes of eight residues or less. The reactivity of the octapeptide series with antisera raised to purified SEB and to formaldehyde-inactivated SEB has been used to locate several antigenic sites on native SEB and to identify antigenic differences in the toxoid. Three antigenic peptides identified from the antigenic profile were synthesized and characterized. These represented amino acids 21-32, 93-107 and 202-217 of SEB. None of these peptides affected SEB-induced T-cell proliferation. However, the occurrence or absence of cross-reactivity of these peptides with antibodies to native SEB corresponds to the degree of exposure and/or the rigidity of these regions within SEB.

Crystal structure of a biologically inactive mutant of toxic shock syndrome toxin-1 at 2.5 A resolution.

Toxic shock syndrome toxin-1 (TSST-1) is one of a family of staphylococcal exotoxins recognized as microbial superantigens. The toxin plays a dominant role in the genesis of toxic shock in humans through a massive activation of the immune system. This potentially lethal illness occurs as a result of the interaction of TSST-1 with a significant proportion of the T-cell repertoire. TSST-1, like other superantigens, can bind directly to class II major histocompatibility (MHC class II) molecules prior to its interaction with entire families of V beta chains of the T-cell receptor (TCR). The three-dimensional structure of a mutant (His-135-Ala) TSST-1 was compared with the structure of the native (wild-type) TSST-1 at 2.5 A resolution. The replacement of His 135 of TSST-1 with an Ala residue results in the loss of T-cell mitogenicity and toxicity in experimental animals. This residue, postulated to be directly involved in the toxin-TCR interactions, is located on the major helix alpha 2, which forms the backbone of the molecule and is exposed to the solvent. In the molecular structure of the mutant toxin, the helix alpha 2 remains unaltered, but the His to Ala modification causes perturbations on the neighboring helix alpha 1 by disrupting helix-helix interactions. Thus, the effects on TCR binding of the His 135 residue could actually be mediated, wholly or in part, by the alpha 1 helix.

The refined crystal structure of toxic shock syndrome toxin-1 at 2.07 A resolution.

The pyrogenic toxin toxic shock syndrome toxin-1 from Staphylococcus aureus is a causative agent of the toxic shock syndrome disease. It belongs to a family of proteins known as superantigens that cross-link major histocompatibility class II molecules and T-cell receptors leading to the activation of a substantial number of T cells. The crystal structure of this protein has been refined to 2.07 A with an Rcryst value of 20.4% for 51,240 reflections. The final model contains three molecules in the asymmetric unit with good stereochemistry and a root-mean-square deviation of 0.009 A and 1.63 from ideality for bond lengths and bond angles, respectively. The overall fold is considerably similar to that of other known microbial superantigens (staphylococcal enterotoxins). However, a detailed structural analysis shows that toxic shock syndrome toxin-1 lacks several structural features that affect its specificity for V beta elements of the T-cell receptor and also its recognition by major histocompatibility class II molecules.

Definition of sites on HLA-DR1 involved in the T cell response to staphylococcal enterotoxins E and C2.

We have exploited the relative inefficiency of interaction between staphylococcal enterotoxins, SEE or SEC2, and H-2Ek compared to HLA-DR1 molecules to deduce which regions of the major histocompatibility complex (MHC) class II molecule are involved in the T cell response to these superantigens. Transfectants expressing hybrid DR/H-2E MHC class II molecules were used to present SEE to the T cell receptor V beta 8.1-expressing Jurkat cell line, and SEC2 to human peripheral blood T cells. For SEE, the critical region of the class II molecule for T cell reactivity and for binding was the beta 1 domain alpha-helix. The functional data were corroborated by measurements of direct binding. Sequence comparison between DR and H-2E raised the possibility that the glutamic acid at position 84 in the beta chain of H-2Ek, in place of glycine was responsible for the observed functional effects. This suggestion was supported by the finding that DQw2 (glutamine at 84) transfectants supported the SEE response much more efficiently than DQw6 that has glutamic acid at this position. In addition, amino acid substitutions at either position 36 or 39 in the DR alpha 1 domain abolished T cell reactivity without any obvious alteration in binding. For SEC2, use of transfectants expressing exon-shuffled alpha and beta chain genes showed that replacement of the alpha 1, alpha 2 and beta 1 domains with H-2E sequence inhibited the presentation of SEC2. Similarly, the substitutions at positions 36 and 39 in the alpha 1 domain abolished the T cell response to SEC2. Taken together, these data may be best explained by a model in which these two toxins have primary binding sites on the beta 1 domain (SEE) and the alpha 1 and alpha 2 domains (SEC2), but by virtue of a secondary binding site on the opposite surface of the class II molecule, cross-link two adjacent DR molecules. Such cross-linking may be important in the induction of T cell reactivity.

Crystal structure of the superantigen enterotoxin C2 from Staphylococcus aureus reveals a zinc-binding site.

BACKGROUND: Staphylococcus aureus enterotoxin C2 (SEC2) belongs to a family of proteins, termed 'superantigens', that form complexes with class II MHC molecules enabling them to activate a substantial number of T cells. Although superantigens seem to act by a common mechanism, they vary in many of their specific interactions and biological properties. Comparison of the structure of SEC2 with those of two other superantigens--staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1)--may provide insight into their mode of action. RESULTS: The crystal structure of SEC2 has been determined at 2.0 A resolution. The overall topology of the molecule resembles that of SEB and TSST-1, and the regions corresponding to the MHC class II and T-cell receptor binding sites on SEB are quite similar in SEC2. A unique feature of SEC2 is the presence of a zinc ion located in a solvent-exposed region at the interface between the two domains of the molecule. The zinc ion is coordinated to Asp83, His118, His122 and Asp9* (from the neighbouring molecule in the crystal lattice). Atomic absorption spectrometry demonstrates that zinc is also bound to SEC2 in solution. CONCLUSIONS: SEC2 appears to be capable of binding to MHC class II molecules in much the same manner as SEB. However, structure-function studies have suggested an alternative binding mode that involves a different site on the toxin. The zinc ion of SEC2 lies within this region and thus may be important for complex formation, for example by acting as a bridge between the two molecules. Other possible roles for the metal cation, including a catalytic one, are also considered.

Structural basis of superantigen action inferred from crystal structure of toxic-shock syndrome toxin-1.

Superantigens stimulate T cells bearing particular T-cell receptor V beta sequences, so they are extremely potent polyclonal T-cell mitogens. T-cell activation is preceded by binding of superantigens to class II major histocompatibility complex (MHC) molecules. To further the structural characterization of these interactions, the crystal structure of a toxin associated with toxic-shock syndrome, TSST-1, which is a microbial superantigen, has been determined at 2.5 A resolution. The N- and C-terminal domains of the structure both contain regions involved in MHC class II association; the C-terminal domain is also implicated in binding the T-cell receptor. Despite low sequence conservation, the TSST-1 topology is similar to the structure reported for the superantigen staphylococcal enterotoxin B4. But TSST-1 lacks several of the structural features highlighted as central to superantigen activity in the staphylococcal enterotoxin B and we therefore reappraise the structural basis of superantigen action.