Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.     

Maternal Control of PIN1 Is Required for Female Gametophyte Development in Arabidopsis

Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of PIN1 and PIN3 during ovule development we have used mutant alleles of both genes and also perturbed PIN1 and PIN3 expression using micro-RNAs controlled by the ovule specific DEFH9 (DEFIFICENS Homologue 9) promoter. PIN1 down-regulation and pin1-5 mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or PIN1-silenced plants. We show that sporophytically expressed PIN1 is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.     

The male determinant of self-incompatibility in Brassica oleracea is located in the pollen coating

An in vitro bioassay has been developed to explore the role of the pollen coating in the pollen/stigma interaction in Brassica oleracea . In the assay, coating is removed from pollen grains, supplemented with protein fractions isolated from coatings of different S (self incompatibility) haplotypes, and then—using micromanipulation—interposed between individual pollen grains and the stigmatic surface. Normally, the coating used is of the same haplotype as the pollen in the experiment—thus constituting an 'extension' of its own coat—but carrying the supplemented protein fractions. Initial experiments confirmed preliminary data that the pollen coating contained the male determinant of self incompatibility (SI); not only did the addition of 'self' coating (i.e. that with the same S -haplotype as the stigma) prevent the success of a compatible cross pollination, but a 'cross' coating (i.e. that with a different S -haplotype from the stigma) could induce the germination and growth of self pollen. Protein supplementation experiments demonstrated that the pollen-held determinant is contained within the water soluble component of the pollen coat, while further analysis revealed that the active molecular species possesses an M_r10 kDa. More extensive fractionation by gel filtration and reverse phase HPLC was used to isolate a family of basic, cysteine-rich proteins (PCP-A: P ollen C oat P roteins-class A)—one of which is known to bind to stigmatically-expressed components of the S -locus in Brassica . Introduction of the PCP-A protein fraction into the bioassay confirmed the male determinant of SI as a protein, and probably a member of the PCP-A protein family.     

Expression of the Cameleon calcium biosensor in fungi reveals distinct Ca2+ signatures associated with polarized growth,development, and pathogenesis

Calcium is a universal messenger that translates diverse environmental stimuli and developmental cues into specific cellular and developmental responses. While individual fungal species have evolved complex and often unique biochemical and structural mechanisms to exploit specific ecological niches and to adjust growth and development in response to external stimuli, one universal feature to all is that Ca²⁺-mediated signaling is involved. The lack of a robust method for imaging spatial and temporal dynamics of subcellular Ca²⁺ (i.e., “Ca²⁺ signature”), readily available in the plant and animal systems, has severely limited studies on how this signaling pathway controls fungal growth, development, and pathogenesis. Here, we report the first successful expression of a FRET (Förster Resonance Energy Transfer)-based Ca²⁺ biosensor in fungi. Time-lapse imaging of Magnaporthe oryzae, Fusarium oxysporum, and Fusarium graminearum expressing this sensor showed that instead of a continuous gradient, the cytoplasmic Ca²⁺ ([Ca²⁺]_c) change occurred in a pulsatile manner with no discernable gradient between pulses, and each species exhibited a distinct Ca²⁺ signature. Furthermore, occurrence of pulsatile Ca²⁺ signatures was age and development dependent, and major [Ca²⁺]_c transients were observed during hyphal branching, septum formation, differentiation into specialized plant infection structures, cell–cell contact and in planta growth. In combination with the sequenced genomes and ease of targeted gene manipulation of these and many other fungal species, the data, materials and methods developed here will help understand the mechanism underpinning Ca²⁺-mediated control of cellular and developmental changes, its role in polarized growth forms and the evolution of Ca²⁺ signaling across eukaryotic kingdoms.     

Effects of annealing on gelatinization and microstructures of corn starches with different amylose/amylopectin ratios

Corn starches with different amylose/amylopectin ratios (waxy 0/100, normal corn 23/77, Gelose 50 50/50, Gelose 80 80/20) were annealed at below their gelatinization temperatures in excess water. The effects of annealing on the gelatinization and microstructures of the starches were studied using DSC, XRD and a microscope equipped with both normal and polarized light. In addition, a high-pressure DSC pan was used to study the effects of high-temperature annealing on the multiphase transitions of starches with different water contents. The granular size of the starches increased after the annealing process, but the size variation rates were different, with higher amylopectin contents resulting in a higher diameter growth rates and final accretion ratios. DSC results showed that annealing increased the gelatinization enthalpy of the amylose-rich starches. The increased enthalpy was mainly attributed to endotherm G – there were no significant changes to endotherms M1, M2 or Z – indicating that annealing mainly affected the helical length of shorter or sub-optional amylopectins, in particular the amylopectin in amylose-rich starches. The XRD traces of all starches after annealing remained unchanged.