Raman imaging of Micrasterias: new insights into shape formation

The algae Micrasterias with its star-shaped cell pattern is a perfect unicellular model system to study morphogenesis. How the indentations are formed in the primary cell wall at exactly defined areas puzzled scientists for decades, and they searched for chemical differences in the primary wall of the extending tips compared to the resting indents. We now tackled the question by Raman imaging and scanned in situ Micrasterias cells at different stages of development. Thousands of Raman spectra were acquired from the mother cell and the developing semicell to calculate chemical images based on an algorithm finding the most different Raman spectra. Each of those spectra had characteristic Raman bands, which were assigned to molecular vibrations of BaSO4, proteins, lipids, starch, and plant cell wall carbohydrates. Visualizing the cell wall carbohydrates revealed a cell wall thickening at the indentations of the primary cell wall of the growing semicell and uniplanar orientation of the cellulose microfibrils to the cell surface in the secondary cell wall. Crystalline cellulose dominated in the secondary cell wall spectra, while in the primary cell wall spectra, also xyloglucan and pectin were reflected. Spectral differences between the indent and tip region of the primary cell wall were scarce, but a spectral mixing approach pointed to more cellulose fibrils deposited in the indent region. Therefore, we suggest that cell wall thickening together with a denser network of cellulose microfibrils stiffens the cell wall at the indent and induces different cell wall extensibility to shape the lobes.

Impact of Drought on Soluble Sugars and Free Proline Content in Selected Arabidopsis Mutants

Water shortage is an increasing problem affecting crop yield. Accumulation of compatible osmolytes is a typical plant response to overcome water stress. Sucrose synthase 1 (SUS1), and glucan, water dikinase 2 (GWD2) and δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) are members of small protein families whose role in the response of Arabidopsis thaliana plants to mild osmotic stress has been studied in this work. Comparative analysis between wild-type and single loss-of-function T-DNA plants at increasing times following exposure to drought showed no differences in the content of water-insoluble carbohydrate (i.e., transitory starch and cell wall carbohydrates) and in the total amount of amino acids. On the contrary, water-soluble sugars and proline contents were significantly reduced compared to wild-type plants regardless of the metabolic pathway affected by the mutation. The present results contribute to assigning a physiological role to GWD2, the least studied member of the GWD family; strengthening the involvement of SUS1 in the response to osmotic stress; showing a greater contribution of soluble sugars than proline in osmotic adjustment of Arabidopsis in response to drought. Finally, an interaction between proline and soluble sugars emerged, albeit its nature remains speculative and further investigations will be required for complete comprehension.

Streptococci and enterococci

The term streptococcus was first used by Billroth in 1874 to describe chain-forming cocci found in infected wounds. The streptococci are a diverse group of Gram-positive pathogenic cocci that cause clinical disease in humans and domestic animals. They are traditionally classified on the basis of serological reactions, particularly Lancefield grouping based on cell-wall carbohydrates, and haemolytic activity on blood agar. Six groups can be defined by genetic analysis: pyogenic streptococci, milleri or anginosus group, mitis group, salivarius group, mutans group, and bovis group. Since the medically important members of the mitis, salivarius, and mutans groups are all oral streptococci and are of clinical relevance predominantly in endocarditis, they will be considered together in this chapter.

Lysibodies are IgG Fc fusions with lysin binding domains targetingStaphylococcus aureuswall carbohydrates for effective phagocytosis

The cell wall of Gram-positive bacteria contains abundant surface-exposed carbohydrate molecules that are highly conserved within and often across species. The potential therapeutic usefulness of high-affinity antibodies to cell wall carbohydrates is unquestioned, however obtaining such antibodies is challenging due to the poor overall immunogenicity of these bacterial targets. Autolysins and phage lysins are peptidoglycan hydrolases, enzymes that have evolved over a billion years to degrade bacterial cell wall. Such wall hydrolases are modular enzymes, composed of discrete domains for high-affinity binding to cell wall carbohydrates and cleavage activity. In this study, we demonstrate that binding domains from autolysins and lysins can be fused to the Fc region of human IgG, creating a fully functional homodimer (or “lysibody”) with high-affinity binding and specificity for carbohydrate determinants on the bacterial surface. Furthermore, we demonstrate that this process is reproducible with three different binding domains specific to methicillin-resistantStaphylococcus aureus(MRSA). Cell-bound lysibodies induced the fixation of complement on the bacterial surface, promoted phagocytosis by macrophages and neutrophils, and protected mice from MRSA infection in two model systems. The lysibody approach could be used to target a range of difficult-to-treat pathogenic bacteria, given that cell wall hydrolases are ubiquitous in nature.