Invasion of Endothelial Cells by Tissue-invasive M3 Type Group A Streptococci Requires Src Kinase and Activation of Rac1 by a Phosphatidylinositol 3-Kinase-independent Mechanism

Streptococcus pyogenes can cause invasive diseases in humans, such as sepsis or necrotizing fasciitis. Among the various M serotypes of group A streptococci (GAS), M3 GAS lacks the major epithelial invasins SfbI/PrtF1 and M1 protein but has a high potential to cause invasive disease. We examined the uptake of M3 GAS into human endothelial cells and identified host signaling factors required to initiate streptococcal uptake. Bacterial uptake is accompanied by local F-actin accumulation and formation of membrane protrusions at the entry site. We found that Src kinases and Rac1 but not phos pha tidyl ino si tol 3-kinases (PI3Ks) are essential to mediate S. pyogenes internalization. Pharmacological inhibition of Src activity reduced bacterial uptake and abolished the formation of membrane protrusions and actin accumulation in the vicinity of adherent streptococci. We found that Src kinases are activated in a time-de pend ent manner in response to M3 GAS. We also demonstrated that PI3K is dispensable for internalization of M3 streptococci and the formation of F-actin accumulations at the entry site. Furthermore, Rac1 was activated in infected cells and accumulated with F-actin in a PI3K-independent manner at bacterial entry sites. Genetic interference with Rac1 function inhibited streptococcal internalization, demonstrating an essential role of Rac1 for the uptake process of streptococci into endothelial cells. In addition, we demonstrated for the first time accumulation of the actin nucleation complex Arp2/3 at the entry port of invading M3 streptococci.Streptococcus pyogenes or group A streptococcus (GAS)² is an important human pathogen that causes localized infections of the respiratory tract and the skin but also severe invasive disease, sepsis, and toxic shock-like syndrome. Group A streptococci, although traditionally viewed as extracellular pathogens, are able to adhere to and invade into several eukaryotic cell types (1–5).Localized S. pyogenes infections may lead to dissemination of bacteria through the vascular system, resulting in bacteremia and sepsis. For evasion of the vascular system, S. pyogenes may directly interact with the endothelium, which lines the inner surface of blood vessels. M3 type streptococci are, besides the M1 and M28 strains, most commonly associated with invasive GAS infections (6) and have been shown to be internalized into human umbilical vein endothelial cells (HUVEC) in vitro (7).S. pyogenes can express several invasins, but only the signal transduction pathways of two streptococcal factors, SfbI/prtF1 and M1 protein, respectively, have been studied in more detail. Both invasins trigger bacterial uptake by binding to soluble fibronectin, which acts as a bridging molecule and induces the clustering of host integrins, which in turn activates host signaling pathways. In the case of M1-mediated internalization, activation of PI3K, ILK, paxillin, and focal adhesion kinase has been shown, which promotes actin polymerization-based zipper-like bacterial uptake into epithelial cells (8–10). In contrast to this, caveolae were shown to act as entry port for SfbI-expressing S. pyogenes (11), a mechanism distinct from the zipper-like uptake mechanism employed by strains expressing M1 protein (12). SfbI/protein F1-expressing streptococci form a focal complex-like structure that consists of focal adhesion kinase, Src kinases, paxillin, and Rho GTPases, resulting in uptake of the bacteria (13). However, a requirement for PI3K activation, which in turn induced paxillin phosphorylation, was recently shown for M1-mediated as well as SfbI-mediated invasion (10). In contrast, M3 streptococci do not express these two well characterized invasins (14), the mechanism by which M3 streptococci are able to trigger entry into human endothelial cells is still poorly understood, and no information is currently available concerning host cell signaling factors involved in this process.In this study, we characterized the intracellular signals governing internalization of SfbI/prtF1/M1-negative M3 GAS into primary endothelial cells. We found an essential role for host cell protein-tyrosine kinases (PTKs) and identified Src family PTKs to play an essential role during the uptake process. In contrast to the already characterized receptor-mediated bacterial invasion strategies, which rely on PI3K activation, internalization of M3 GAS is PI3K-independent. In addition to Src family PTKs, the GTPase Rac1 was identified as an important factor for M3 S. pyogenes internalization. Rac1 was found to be activated in response to bacterial internalization, and genetic interference with Rac1 function significantly reduced uptake. Rac1 as well as the actin nucleation complex Arp2/3 was found to accumulate at streptococcal entry ports, strengthening the important role of this GTPase for uptake of M3 type streptococci into human endothelial cells.     

Diversity and evolutionary patterns of immune genes in free-ranging Namibian leopards (Panthera pardus pardus)

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for fitness-related genetic variation in wildlife populations. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, we isolated and characterized genetic variation at the adaptively most important region of MHC class I and MHC class II-DRB genes in 25 free-ranging African leopards from Namibia and investigated the mechanisms that generate and maintain MHC polymorphism in the species. Using single-stranded conformation polymorphism analysis and direct sequencing, we detected 6 MHC class I and 6 MHC class II-DRB sequences, which likely correspond to at least 3 MHC class I and 3 MHC class II-DRB loci. Amino acid sequence variation in both MHC classes was higher or similar in comparison to other reported felids. We found signatures of positive selection shaping the diversity of MHC class I and MHC class II-DRB loci during the evolutionary history of the species. A comparison of MHC class I and MHC class II-DRB sequences of the leopard to those of other felids revealed a trans-species mode of evolution. In addition, the evolutionary relationships of MHC class II-DRB sequences between African and Asian leopard subspecies are discussed.     

Inherent high correlation of individual motility enhances population dispersal in a heterotrophic, planktonic protist

Quantitative linkages between individual organism movements and the resulting population distributions are fundamental to understanding a wide range of ecological processes, including rates of reproduction, consumption, and mortality, as well as the spread of diseases and invasions. Typically, quantitative data are collected on either movement behaviors or population distributions, rarely both. This study combines empirical observations and model simulations to gain a mechanistic understanding and predictive ability of the linkages between both individual movement behaviors and population distributions of a single-celled planktonic herbivore. In the laboratory, microscopic 3D movements and macroscopic population distributions were simultaneously quantified in a 1L tank, using automated video- and image-analysis routines. The vertical velocity component of cell movements was extracted from the empirical data and used to motivate a series of correlated random walk models that predicted population distributions. Validation of the model predictions with empirical data was essential to distinguish amongst a number of theoretically plausible model formulations. All model predictions captured the essence of the population redistribution (mean upward drift) but only models assuming long correlation times (minute), captured the variance in population distribution. Models assuming correlation times of 8 minutes predicted the least deviation from the empirical observations. Autocorrelation analysis of the empirical data failed to identify a de-correlation time in the up to 30-second-long swimming trajectories. These minute-scale estimates are considerably greater than previous estimates of second-scale correlation times. Considerable cell-to-cell variation and behavioral heterogeneity were critical to these results. Strongly correlated random walkers were predicted to have significantly greater dispersal distances and more rapid encounters with remote targets (e.g. resource patches, predators) than weakly correlated random walkers. The tendency to disperse rapidly in the absence of aggregative stimuli has important ramifications for the ecology and biogeography of planktonic organisms that perform this kind of random walk.     

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.

Methods

Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.

Results

In vitro binding experiments of ¹²⁵I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.

Conclusions

These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.     

A large-scale study on subjective perception of discomfort during 7 and 1.5 T MRI examinations

A study on subjective perception has been carried out in order to gain further insight into subjective discomfort and sensations experienced during 7 T magnetic resonance imaging (MRI). This study provides information about subjective acceptance, which is essential if 7 T MRI is to become a clinical diagnostic tool. Of 573 subjects who underwent 7 T MRI, 166 were also examined at 1.5 T, providing a means of discriminating field‐dependent discomfort. All subjects judged sources of discomfort and physiological sensations on an 11‐point scale (0 = no side effects, 10 = intolerable side effects) and scores were analyzed separately for exam phases, with and without table movement at each field strength. Results revealed that 7 T MRI was, in general, judged more uncomfortable than 1.5 T; however, most subjects rated the effects as being non‐critical (mean scores between 0.5 and 3.5). Significant differences were detected regarding vertigo and sweating between subjects positioned “head‐first” and “feet‐first” at 7 T (worse in “head‐first”) and between 7 and 1.5 T (worse at 7 T), with the effects being more pronounced in the moving compared to the stationary table position. The most unpleasant factor at 7 T was the extensive examination duration, while potentially field‐dependent sensations were rated less bothersome. In summary, our study indicates that although certain sensations increase at 7 T compared to 1.5 T, they are unlikely to hinder the use of 7 T MRI as a clinical diagnostic tool. Bioelectromagnetics. Bioelectromagnetics 32:610–619, 2011. © 2011 Wiley Periodicals, Inc.     

NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences ( Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n -propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase-like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free-radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane-bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm-fraction than in the pm-fraction, but this was not the reason for the much lower phenol-stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o -dianisidine and H₂O₂ had about the same specific activities in the pm-and icm-fractions.
Neither the phenol-stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane-bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light-induced cytochrome b -reduction in the pm fraction.     

Assessing the complex sponge microbiota: core,variable and species-specific bacterial communities in marine sponges

Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world''s oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations.