Genome-wide analysis of ruminant Staphylococcus aureus reveals diversification of the core genome

Staphylococcus aureus causes disease in humans and a wide array of animals. Of note, S. aureus mastitis of ruminants, including cows, sheep, and goats, results in major economic losses worldwide. Extensive variation in genome content exists among S. aureus pathogenic clones. However, the genomic variation among S. aureus strains infecting different animal species has not been well examined. To investigate variation in the genome content of human and ruminant S. aureus, we carried out whole-genome PCR scanning (WGPS), comparative genomic hybridizations (CGH), and the directed DNA sequence analysis of strains of human, bovine, ovine, and caprine origin. Extensive variation in genome content was discovered, including host- and ruminant-specific genetic loci. Ovine and caprine strains were genetically allied, whereas bovine strains were heterogeneous in gene content. As expected, mobile genetic elements such as pathogenicity islands and bacteriophages contributed to the variation in genome content between strains. However, differences specific for ruminant strains were restricted to regions of the conserved core genome, which contained allelic variation in genes encoding proteins of known and unknown function. Many of these proteins are predicted to be exported and could play a role in host-pathogen interactions. The genomic regions of difference identified by the whole-genome approaches adopted in the current study represent excellent targets for studies of the molecular basis of S. aureus host adaptation.     

Major histocompatibility complex class II-peptide complexes internalize using a clathrin- and dynamin-independent endocytosis pathway

Major histocompatibility complex (MHC) class II molecules (MHC-II) function by binding antigenic peptides and displaying these peptides on the surface of antigen presenting cells (APCs) for recognition by peptide-MHC-II (pMHC-II)-specific CD4 T cells. It is known that cell surface MHC-II can internalize, exchange antigenic peptides in endosomes, and rapidly recycle back to the plasma membrane; however, the molecular machinery and trafficking pathways utilized by internalizing/recycling MHC-II have not been identified. We now demonstrate that unlike newly synthesized invariant chain-associated MHC-II, mature cell surface pMHC-II complexes internalize following clathrin-, AP-2-, and dynamin-independent endocytosis pathways. Immunofluorescence microscopy of MHC-II expressing HeLa-CIITA cells, human B cells, and human DCs revealed that pMHC enters Arf6(+)Rab35(+)EHD1(+) tubular endosomes following endocytosis. These data contrast the internalization pathways followed by newly synthesized and peptide-loaded MHC-II molecules and demonstrates that cell surface pMHC-II internalize and rapidly recycle from early endocytic compartments in tubular endosomes.     

How to allow SAR collapse across local and continental scales: a resolution of the controversy between Storch et al. (2012) and Lazarina et al. (2013)

Up‐scaling species richness from local to continental scales is an unsolved problem of macroecology. Macroecologists hope that proper up‐scaling can uncover the hidden rules that underlie spatial patterns in species richness, but a machinery to up‐scale species richness also has a purely practical side at the scales and for the habitats where direct observations cannot be performed. The species–area relationship (SAR) could provide a tool for up‐scaling, but no valid method has yet been put forward. Such a method would have resulted from Storch et al.'s (2012) suggestion that there is a universal curve to which each rescaled SAR collapses, if Lazarina et al. (2013) had not shown that it does not: both arguments were supported by data analyses. Here we present an analytical model for mainland SAR and argue in favour of the latter authors. We identify 1) the variation in mean species‐range size, 2) the variation in forces that drive SAR at various scales, and 3) the finite‐area effect, as the reasons for the absence of collapse. Finally, we suggest a rescaling that might fix the problem. We conclude, however, that ecologists are still far from finding a practical, robust and easy‐to‐use solution for up‐scaling species richness from SARs.     

Prediction of basal metabolism from organ size in the rat: relationship to strain, feeding, age, and obesity

Use of the weight of various organs and tissues together with their specific metabolic activity for prediction of basal metabolism (BM) seems to be promising. In this study we compared the use of this method with those based on simple or multiple regression analyses. We observed that 97.4% of differences in BM in a group of nine adult male Wistar rats weighing 273--517 g could be accounted for by changes in tissue and organ weights. BM measured in lean Zucker and Sprague-Dawley rats did not diverge from the prediction of the model by >1.6%. According to the organ-based model as well as multiple regression analyses, but not simple regression analyses, BM was increased 18--21% in young rats, decreased 6--7% in food restricted/refed rats, and decreased 19--21% in aged rats. Only with obese rats did the predictions of the two methods diverge. The main reason for this discrepancy seems to be the way adipose tissue size and metabolism are taken into account.     

Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity

Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium. As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate. This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio. The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined. Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio. Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.     

Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen

Development of the basement membrane zone (BMZ) occurs postnatally in the rhesus monkey. The purpose of this study was to determine whether house dust mite allergen (HDMA) plus ozone altered this process. Rhesus monkeys were exposed to a regimen of HDMA and/or ozone or filtered air for 6 mo. To detect structural changes in the BMZ, we measured immunoreactivity of collagen I. To detect functional changes in the BMZ, we measured perlecan and fibroblast growth factor-2 (FGF-2). We also measured components of the FGF-2 ternary signaling complex [fibroblast growth factor receptor-1 (FGFR-1) and syndecan-4]. The width of the BMZ was irregular in the ozone groups, suggesting atypical development of the BMZ. Perlecan was also absent from the BMZ. In the absence of perlecan, FGF-2 was not bound to the BMZ. However, FGF-2 immunoreactivity was present in basal cells, the lateral intercellular space (LIS), and attenuated fibroblasts. FGFR-1 immunoreactivity was downregulated, and syndecan-4 immunoreactivity was upregulated in the basal cells. This suggests that FGF-2 in basal cells and LIS may be bound to the syndecan-4. We conclude that ozone and HDMA plus ozone effected incorporation of perlecan into the BMZ, resulting in atypical development of the BMZ. These changes are associated with specific alterations in the regulation of FGF-2, FGFR-1, and syndecan-4 in the airway epithelial-mesenchymal trophic unit, which may be associated with the developmental problems of lungs associated with exposure to ozone.     

Molecular analyses of Salmonella enterica isolates from fish feed factories and fish feed ingredients

Isolates of the most commonly observed salmonella serovars in Norwegian fish feed factories from 1998 to 2000 (Salmonella enterica serovar Agona, S. enterica serovar Montevideo, S. enterica serovar Senftenberg, and S. enterica serovar Kentucky) were studied by pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis and compared to isolates of the same serovars from fish feed ingredients, humans, and other sources (a total of 112 isolates). Within each serovar, a variety of distinct PFGE types (with similarity levels less than 90%) were observed in the feed ingredients and other sources, while only two distinct types of each serovar were identified in the factories. The combined results of PFGE and plasmid analyses showed that each factory harbored only a few S. enterica clones. Some of these clones persisted for at least 3 years in the factories, indicating that there was long-lasting contamination probably due to inadequate decontamination procedures.     

Differences in backbone dynamics of two homologous bacterial albumin-binding modules: implications for binding specificity and bacterial adaptation

Proteins G and PAB are bacterial albumin-binding proteins expressed at the surface of group C and G streptococci and Peptostreptococcus magnus, respectively. Repeated albumin-binding domains, known as GA modules, are found in both proteins. The third GA module of protein G from the group G streptococcal strain G148 (G148-GA3) and the second GA module of protein PAB from P.magnus strain ALB8 (ALB8-GA) exhibit 59% sequence identity and both fold to form three-helix bundle structures that are very stable against thermal denaturation. ALB8-GA binds human serum albumin with higher affinity than G148-GA3, but G148-GA3 shows substantially broader albumin-binding specificity than ALB8-GA. The (15)N nuclear magnetic resonance spin relaxation measurements reported here, show that the two GA modules exhibit mobility on the picosecond-nanosecond time scale in directly corresponding regions (loops and termini). Most residues in G148-GA3 were seen to be involved in conformational exchange processes on the microsecond-millisecond time scale, whereas for ALB8-GA such motions were only identified for the beginning of helix 2 and its preceding loop. Furthermore, and more importantly, hydrogen-deuterium exchange and saturation transfer experiments reveal large differences between the two GA modules with respect to motions on the second-hour time scale. The high degree of similarity between the two GA modules with respect to sequence, structure and stability, and the observed differences in dynamics, binding affinity and binding specificity to different albumins, suggest a distinct correlation between dynamics, binding affinity and binding specificity. Finally, it is noteworthy in this context that the module G148-GA3, which has broad albumin-binding specificity, is expressed by group C and G streptococci known to infect all mammalian species, whereas P.magnus with the ALB8-GA module has been isolated only from humans.     

Fibroblast growth factor-2 during postnatal development of the tracheal basement membrane zone

Thickening of the basement membrane zone (BMZ) is a characteristic of several airway diseases; however, very little is known about how this process occurs. The purpose of this study was to define development of the BMZ in the trachea of growing rhesus monkeys at 1, 2, 3, and 6 mo of age. We measured immunoreactivity of collagen types I, III, and V to detect structural changes in the developing BMZ. To detect more dynamic, functional components of the epithelial-mesenchymal trophic unit, we evaluated the distribution of perlecan, fibroblast growth factor-2 (FGF-2), and fibroblast growth factor receptor-1 (FGFR-1). One-month-old monkeys had a mean collagen BMZ width of 1.5 +/- 0.7 microm that increased to 4.4 +/- 0.4 microm in 6-mo-old monkeys. Perlecan was localized in the BMZ of the epithelium at all ages. FGF-2 was strongly expressed in basal cells at 1-3 mo. At 6 mo, FGF-2 was expressed throughout the BMZ and weakly in basal cells. FGFR-1 immunoreactivity was expressed by basal cells and cilia and weakly in the nuclei of columnar cells at all time points. These data indicate that development of the BMZ is a postnatal event in the rhesus monkey that involves FGF-2.