Streptococcus pyogenes SpyCEP Influences Host-Pathogen Interactions during Infection in a Murine Air Pouch Model

Streptococcus pyogenes is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action in vivo, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels in situ. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 in situ were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against S. pyogenes, and we showed that surface-associated SpyCEP was recognized by specific antibodies. In vitro, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, α-SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis.     

Ex vivo rapamycin generates apoptosis-resistant donor Th2 cells that persist in vivo and prevent hemopoietic stem cell graft rejection

Because ex vivo rapamycin generates murine Th2 cells that prevent Graft-versus-host disease more potently than control Th2 cells, we hypothesized that rapamycin would generate Th2/Tc2 cells (Th2/Tc2.R cells) that abrogate fully MHC-disparate hemopoietic stem cell rejection more effectively than control Th2/Tc2 cells. In a B6-into-BALB/c graft rejection model, donor Th2/Tc2.R cells were indeed enriched in their capacity to prevent rejection; importantly, highly purified CD4+ Th2.R cells were also highly efficacious for preventing rejection. Rapamycin-generated Th2/Tc2 cells were less likely to die after adoptive transfer, accumulated in vivo at advanced proliferative cycles, and were present in 10-fold higher numbers than control Th2/Tc2 cells. Th2.R cells had a multifaceted, apoptosis-resistant phenotype, including: 1) reduced apoptosis after staurosporine addition, serum starvation, or CD3/CD28 costimulation; 2) reduced activation of caspases 3 and 9; and 3) increased anti-apoptotic Bcl-xL expression and reduced proapoptotic Bim and Bid expression. Using host-versus-graft reactivity as an immune correlate of graft rejection, we found that the in vivo efficacy of Th2/Tc2.R cells 1) did not require Th2/Tc2.R cell expression of IL-4, IL-10, perforin, or Fas ligand; 2) could not be reversed by IL-2, IL-7, or IL-15 posttransplant therapy; and 3) was intact after therapy with Th2.R cells relatively devoid of Foxp3 expression. We conclude that ex vivo rapamycin generates Th2 cells that are resistant to apoptosis, persist in vivo, and effectively prevent rejection by a mechanism that may be distinct from previously described graft-facilitating T cells.     

Reformulation of BMI and Percent Body Fat to Remove the Height Bias in 8‐year‐olds

BMI and percent body fat (%BF) are both related to height (Ht) in prepubertal children, so may misrepresent childhood adiposity, especially in tall or short children. We sought to construct replacement functions for BMI and %BF that are independent of Ht. Fat mass (FM) was measured using dual‐energy X‐ray absorptiometry, together with Ht and body mass (BM) in 746 healthy boys and girls aged 8 years (0.34 s.d.). Relationships between BM, FM, and Ht were measured and values of p and q derived such that the functions BM. Ht^?p and FM.BM^?q were unrelated to Ht. BM was not directly proportional to Ht², BMI being significantly related to Ht in both boys and girls (P < 0.001). BM was proportional to Ht³, BM. Ht^?3 being independent of Ht. Similarly, FM was not directly proportional to BM and %BF was significantly related to Ht (P < 0.001). While FM was proportional to BM², FM.BM^?1.5 was the function found to be independent of Ht. Using the 85th and 95th percentiles as the cutoffs for overweight and obesity respectively, 6.4% of the boys and 6.8% of the girls were classified differently by BMI and the Ht independent measure BM. Ht^?3. Similarly, 10.1% boys and 13.7% girls were classified differently by %BF and the Ht independent measure FM.BM^?1.5. We propose that improved diagnostic accuracy of body composition in 8‐year‐olds is provided by the BM function (BMF, BM. Ht^?3) and FM function (FMF, FM.BM^?1.5) replacing BMI and %BF, which both overestimate the adiposity of taller children and underestimate it in shorter children.     

Functional Characterization of a Newly Identified Group B Streptococcus Pullulanase Eliciting Antibodies Able to Prevent Alpha-Glucans Degradation

Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade α-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of α-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of α-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections.     

The effect of Staphylococcus aureus carriage in late pregnancy on antibody levels to staphylococcal toxins in cord blood and breast milk

We investigated the effect of carriage of Staphylococcus aureus in the later stages of pregnancy on levels of antibody specific to the S. aureus toxins, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC) and toxic shock syndrome toxin-1 (TSST-1), in cord blood and breast milk and also explored the relationship between levels of antibody in antenatal serum and cord blood. Nasopharyngeal swabs and stool samples were collected on two occasions, from 96 women, during the last 6 weeks of pregnancy. Samples were cultured and S. aureus isolates were identified. Antenatal and cord blood samples from the same women and their infants were analysed for IgG antibody to SEB, SEC and TSST-1 by enzyme-linked immunosorbent assay. Breast milk samples were analysed for IgA antibody to the same toxins. We found that S. aureus carriage in pregnancy is common and exposure to a toxin-producing isolate boosts immunity. Over 89% of women and infants have some protective antibody to the toxins, and antitoxin IgG levels are higher in cord blood samples compared with antenatal samples. Levels of cord blood IgG and breast milk IgA specific for the staphylococcal toxins vary. Some infants lack protection and could be at risk of toxin-induced disease.     

A species of Schellackia (Apicomplexa: Lankesterellidae) parasitising east and southeast Asian lizards

Schellackia orientalis n. sp. parasitises the Japanese lizard Takydromus tachydromoides on Honshu and Takydromus sexlineatus in Thailand. Merogony and gamogony occur in the epithelium of the small intestine, and octonucleate oöcysts form in the lamina propria. In infections induced by inoculation of infected blood, sporozoites appeared in blood cells 17–56 days post-inoculation, initially in any type of white blood cell but most commonly in macrophages and monocytes. Both erythroid and leucocytic cells were parasitised after five days. This haemococcidian is another component of the symbiotic complex found in Takydromus species of eastern and southeastern Asia.     

Two new trypanosome species from East African cordylid lizards

Two species ofTrypanosoma are described from East African cordylid lizards.Trypanosoma zonuri n. sp. was found in a disjunct population ofCordylus cordylus on the western slope of Ngorongoro Crater in Arusha Region, northern Tanzania. It has the appearance of a delicate and fragile trypanosome, slightly over half the size of the robust, leaf-likeT. cordyli n. sp. that parasitisesCordylus t. tropidosternum in the Rondo Forest, Lindi Region, southern Tanzania. These are the first trypanosome species recorded from the saurian family Cordylidae in East Africa.     

SALMFamide2 and serotonin immunoreactivity in the nervous system of some acoels (Xenacoelomorpha)

Acoel worms are simple, often microscopic animals with direct development, a multiciliated epidermis, a statocyst, and a digestive parenchyma instead of a gut epithelium. Morphological characters of acoels have been notoriously difficult to interpret due to their relative scarcity. The nervous system is one of the most accessible and widely used comparative features in acoels, which have a so‐called commissural brain without capsule and several major longitudinal neurite bundles. Here, we use the selective binding properties of a neuropeptide antibody raised in echinoderms (SALMFamide2, or S2), and a commercial antibody against serotonin (5‐HT) to provide additional characters of the acoel nervous system. We have prepared whole‐mount immunofluorescent stainings of three acoel species: Symsagittifera psammophila (Convolutidae), Aphanostoma pisae, and the model acoel Isodiametra pulchra (both Isodiametridae). The commissural brain of all three acoels is delimited anteriorly by the ventral anterior commissure, and posteriorly by the dorsal posterior commissure. The dorsal anterior commissure is situated between the ventral anterior commissure and the dorsal posterior commissure, while the statocyst lies between dorsal anterior and dorsal posterior commissure. S2 and serotonin do not co‐localise, and they follow similar patterns to each other within an animal. In particular, S2, but not 5‐HT, stains a prominent commissure posterior to the main (dorsal) posterior commissure. We have for the first time observed a closed posterior loop of the main neurite bundles in S. psammophila for both the amidergic and the serotonergic nervous system. In I. pulchra, the lateral neurite bundles also form a posterior loop in our serotonergic nervous system stainings.