Secondary Vacuoles Research Articles

The vampyrellid amoeba Strigomyxa ruptor gen. et sp. nov. and its remarkable strategy to acquire algal cell contents.

The vampire amoebae (Vampyrellida, Rhizaria) inhabit freshwater, marine, and terrestrial ecosystems and consume a wide range of eukaryotic prey. This includes diverse microalgae, fungi, and microscopic animals. One of the most captivating aspects of the vampyrellids is their ability to extract the cell contents of other eukaryotes after local dissolution of the prey cell wall, a feeding strategy that occurs in several vampyrellid families, but is best studied in Vampyrella species that attack zygnematophyte green algae. Here, we report two new vampyrellid strains from temperate moorlands in Germany with a yet-undescribed feeding strategy: internal protoplast extraction and cell wall regurgitation. This feeding strategy involves the phagocytosis of whole desmid cells (genus Closterium, Zygnematophyceae), internal cleavage of the algal cell wall, extraction of the cell contents, and subsequent exocytosis of bundled empty cell walls. The large primary food vacuole formed during the process has exceptional functions, as it forms internal feeding pseudopodia, packages algal cell contents into smaller secondary vacuoles, and transforms into a "waste vacuole" with cell wall remnants. The new feeding strategy, which - in the widest sense - is reminiscent of the pellet casting of owls, reveals a stunningly sophisticated behavior of single protist cells. Based on morphological, phylogenetic, and autecological data, both vampyrellid strains are nearly identical and here assigned to a new and quite unique vampyrellid taxon, Strigomyxa ruptor gen. et sp. nov. (Leptophryidae, Vampyrellida).

Zygospore development of Spirogyra (Charophyta) investigated by serial block-face scanning electron microscopy and 3D reconstructions.

Sexual reproduction of Zygnematophyceae by conjugation is a less investigated topic due to the difficulties of the induction of this process and zygospore ripening under laboratory conditions. For this study, we collected field sampled zygospores of Spirogyra mirabilis and three additional Spirogyra strains in Austria and Greece. Serial block-face scanning electron microscopy was performed on high pressure frozen and freeze substituted zygospores and 3D reconstructions were generated, allowing a comprehensive insight into the process of zygospore maturation, involving storage compound and organelle rearrangements. Chloroplasts are drastically changed, while young stages contain both parental chloroplasts, the male chloroplasts are aborted and reorganised as 'secondary vacuoles' which initially contain plastoglobules and remnants of thylakoid membranes. The originally large pyrenoids and the volume of starch granules is significantly reduced during maturation (young: 8 ± 5 µm³, mature: 0.2 ± 0.2 µm³). In contrast, lipid droplets (LDs) increase significantly in number upon zygospore maturation, while simultaneously getting smaller (young: 21 ± 18 µm³, mature: 0.1 ± 0.2 and 0.5 ± 0.9 µm³). Only in S. mirabilis the LD volume increases (34 ± 29 µm³), occupying ~50% of the zygospore volume. Mature zygospores contain barite crystals as confirmed by Raman spectroscopy with a size of 0.02 - 0.05 µm³. The initially thin zygospore cell wall (~0.5 µm endospore, ~0.8 µm exospore) increases in thickness and develops a distinct, electron dense mesospore, which has a reticulate appearance (~1.4 µm) in Spirogyra sp. from Greece. The exo- and endospore show cellulose microfibrils in a helicoidal pattern. In the denser endospore, pitch angles of the microfibril layers were calculated: ~18 ± 3° in S. mirabilis, ~20 ± 3° in Spirogyra sp. from Austria and ~38 ± 8° in Spirogyra sp. from Greece. Overall this study gives new insights into Spirogyra sp. zygospore development, crucial for survival during dry periods and dispersal of this genus.

Light and electron microscopic studies of the 139A-H strain of scrapie passaged in hamsters.

We report here the light and electron microscopic neuropathology of the 139A-H strain of scrapie passaged in Syrian golden hamsters. The general neuropathological picture consisted of the spongiform change and severe astrocytic gliosis. The topography of prion protein (PrP) was variable, the highest signal was observed in the CA2-molecular layer, CA1-pyramidal and entorhinal cortex. The electron microscopy consisted of: 1. Spongiform vacuoles - these are always membrane bound and contain secondary vacuoles (i.e. membrane-bound compartments or vesicles within vacuoles) and curled membraned fragments. 2. Tubulovesicular structures (TVS) - these are vesicular structures of approximate 27 nm in diameter within neuronal processes - i.e. axonal terminal or dendrites. TVS are smaller and of higher electron density than synaptic vesicles. The significance of TVS remains unknown. 3. Dystrophic neurites. Dendrites or axonal preterminals and terminals filled with electron-dense bodies, including small autophagic vacuoles. 4. Apoptotic cell nuclei. 5. "Whorls", concentric arrays of membranes were visible. Asignificance of those structures is unknown.

The involvement of programmed cell death in inflated leaf petiole morphogenesis in Trapa pseudoincisa.

Trapa plants (Trapaceae) have an inflated leaf petiole called a spongy airbag. The aims of this study were to assess the involvement of programmed cell death (PCD) in the process of inflated leaf petiole morphogenesis. In this paper, light and transmission electron microscopy (TEM) were used to investigate cytological events and the development of inflated leaf petiole. During this process, the inflated leaf petiole of Trapa pseudoincisa L. undergoes a developmental process, changing from solid to hollow phase. Debris from the degraded cells was seldom observed in the transverse sections of leaf petioles, but some degraded cells with an abnormal morphology were observed in longitudinal sections. Cytoplasmic changes, such as disrupted vacuoles, degraded plastids, and the emergence of secondary vacuoles were observed during leaf petiole morphogenesis. In addition, gel electrophoresis and TUNEL assays were used to evaluate DNA cleavage during petiole morphogenesis. DNA internucleosomal cleavage and TUNEL-positive nuclei indicate that the typical PCD features of DNA cleavage occurred early in the process. These results revealed that PCD plays a critical role in inflated leaf petiole morphogenesis. Additionally, a trans-disciplinary systems approach is required that recognises the necessity for integration of cytological and molecular characteristics for identification of aerenchyma type.

The Female Post-Mating Response Requires Genes Expressed in the Secondary Cells of the Male Accessory Gland in Drosophila melanogaster.

Seminal proteins from the Drosophila male accessory gland induce post-mating responses (PMR) in females. The PMR comprise behavioral and physiological changes that include increased egg laying, decreased receptivity to courting males, and changes in the storage and use of sperm. Many of these changes are induced by a "sex peptide" (SP) and are maintained by SP's binding to, and slow release from, sperm. The accessory gland contains two secretory cell types with distinct morphological and developmental characteristics. Products of these "main" and "secondary" cells work interdependently to induce and maintain the PMR. To identify individual genes needed for the morphology and function of secondary cells, we studied iab-6(cocu) males, whose secondary cells have abnormal morphology and fail to provide products to maintain the PMR. By RNA-seq, we identified 77 genes that are downregulated by a factor of >5× in iab-6(cocu) males. By functional assays and microscopy, we tested 20 candidate genes and found that at least 9 are required for normal storage and release of SP in mated females. Knockdown of each of these 9 genes consequently leads to a reduction in egg laying and an increase in receptivity over time, confirming a role for the secondary cells in maintaining the long-term PMR. Interestingly, only 1 of the 9 genes, CG3349, encodes a previously reported seminal fluid protein (Sfp), suggesting that secondary cells may perform essential functions beyond the production and modification of known Sfps. At least 3 of the 9 genes also regulate the size and/or abundance of secondary cell vacuoles, suggesting that the vacuoles' contents may be important for the machinery used to maintain the PMR.

Alterations in the sugar metabolism and in the vacuolar system of mesophyll cells contribute to the desiccation tolerance of Haberlea rhodopensis ecotypes.

Haberlea rhodopensis belongs to the small group of resurrection plants having the unique ability to survive desiccation to air dry state retaining most of its chlorophyll content and then resume normal function upon rehydration. It prefers the shady valleys and northward facing slopes of limestone ridges in mountain zones with high average humidity. Nevertheless, it can be found rarely on rocks directly exposed to the sunlight, without the coverage of the canopy. In the present study, we follow the alterations in the subcellular organization of mesophyll cells and sugar metabolism upon desiccation of shade and sun H. rhodopensis plants. Composition and content of soluble carbohydrates during desiccation and rehydration were different in plants grown below the trees or on the sunny rocks. Sucrose, however, was dominating in both ecotypes. The amount of starch grains in chloroplasts was inversely related to that of sugars. Concomitantly with these changes, the number of vacuoles was multiplied in the cells. This can be explained by the development of small (secondary) vacuoles peripherally in the cytoplasm, rather than by the fragmentation of the single vacuole, proposed earlier in the literature. Accordingly, the centripetal movement of chloroplasts and other organelles may be a result of the dynamic changes in the vacuolar system. Upon rehydration, the inner vacuoles enlarged and the organelles returned to their normal position.

Targeting of organelles into vacuoles and ultrastructure of flower petal epidermis of Petunia hybrida

Autophagy in flower petal epidermal cells is a little studied process. However, it is of interest regarding the physiology of chlorophylless plant tissues. We show that the utilization of organelles in the Petunia hybrida L. flower petal epidermis occurs via the autophagic-like organelle targeting into vacuole during the particular process of vacuole fragmentation and growth of secondary vacuoles (ATVF). It differs from macroautophagy (formation of autophagosome) and microautophagy. Peroxisomes were shown to be subjected to both macroautophagy (as single microbodies and their assemblies) and to ATVF when they are in complexes with mitochondria and chromoplasts. In addition, we have demonstrated that ATVF in flower petals is accompanied with the sequestration of the cytoplasmic sectors located between the neighboring cell wall ingrowths, the ultrastructure of which has been reported in the present work for the first time. It has been shown there are more numerous mitochondria, a more pronounced ATVF, and a thicker cytoplasmic layer in the blue tissues, possibly reflecting more intensive metabolic processes in them compared to white tissues. We assume that these differences may be of interest for the further studies and may be discussed including in terms of anthocyanin-dependent photochemical effects.

The Shigella flexneri type 3 secretion system is required for tyrosine kinase-dependent protrusion resolution, and vacuole escape during bacterial dissemination.

Shigella flexneri is a human pathogen that triggers its own entry into intestinal cells and escapes primary vacuoles to gain access to the cytosolic compartment. As cytosolic and motile bacteria encounter the cell cortex, they spread from cell to cell through formation of membrane protrusions that resolve into secondary vacuoles in adjacent cells. Here, we examined the roles of the Type 3 Secretion System (T3SS) in S. flexneri dissemination in HT-29 intestinal cells infected with the serotype 2a strain 2457T. We generated a 2457T strain defective in the expression of MxiG, a central component of the T3SS needle apparatus. As expected, the ΔmxiG strain was severely affected in its ability to invade HT-29 cells, and expression of mxiG under the control of an arabinose inducible expression system (ΔmxiG/pmxiG) restored full infectivity. In this experimental system, removal of the inducer after the invasion steps (ΔmxiG/pmxiG (Ara withdrawal)) led to normal actin-based motility in the cytosol of HT-29 cells. However, the time spent in protrusions until vacuole formation was significantly increased. Moreover, the number of formed protrusions that failed to resolve into vacuoles was also increased. Accordingly, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to trigger tyrosine phosphorylation in membrane protrusions, a signaling event that is required for the resolution of protrusions into vacuoles. Finally, the ΔmxiG/pmxiG (Ara withdrawal) strain failed to escape from the formed secondary vacuoles, as previously reported in non-intestinal cells. Thus, the T3SS system displays multiple roles in S. flexneri dissemination in intestinal cells, including the tyrosine kinase signaling-dependent resolution of membrane protrusions into secondary vacuoles, and the escape from the formed secondary vacuoles.

Leaf-shape remodeling: programmed cell death in fistular leaves of Allium fistulosum.

Some species of Allium in Liliaceae have fistular leaves. The fistular lamina of Allium fistulosum undergoes a process from solid to hollow during development. The aims were to reveal the process of fistular leaf formation involved in programmed cell death (PCD) and to compare the cytological events in the execution of cell death to those in the unusual leaf perforations or plant aerenchyma formation. In this study, light and transmission electron microscopy were used to characterize the development of fistular leaves and cytological events. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays and gel electrophoresis were used to determine nuclear DNA cleavage during the PCD. The cavity arises in the leaf blade by degradation of specialized cells, the designated pre-cavity cells, in the center of the leaves. Nuclei of cells within the pre-cavity site become TUNEL-positive, indicating that DNA cleavage is an early event. Gel electrophoresis revealed that DNA internucleosomal cleavage occurred resulting in a characteristic DNA ladder. Ultrastructural analysis of cells at the different stages showed disrupted vacuoles, misshapen nuclei with condensed chromatin, degraded cytoplasm and organelles and emergence of secondary vacuoles. The cell walls degraded last, and residue of degraded cell walls aggregated together. These results revealed that PCD plays a critical role in the development of A. fistulosum fistular leaves. The continuous cavity in A. fistulosum leaves resemble the aerenchyma in the pith of some gramineous plants to improve gas exchange.

Symbiotic bacteria in the nudibranch mollusk Dendrodoris nigra: fatty acid composition and ultrastructure analysis

In the present study, the hypothesis that marine nudibranch mollusks harbor symbiotic bacteria was tested using analyses of fatty acids as biochemical markers and transmission electron microscopy of the tissues of Dendrodoris nigra (Gastropoda/Opisthobranchia/Nudibranchia). An aberrant level of the odd-numbered carbon chain and branched fatty acids, iso- and anteiso- that are specific for bacteria, was detected in the nudibranch tissues. Their amounts in the notum exceeded significantly that in the viscera. Rod-shaped gram-negative bacteria were revealed in the epithelial cells of the notum and the mantle edge as well as in the adjoining glycocalix. These bacteria were enclosed in secondary vacuoles in the epithelial cells. The consequent stages of inoculation of the bacteria into the cytoplasm of epithelial cells, from adhesion to the apical surface to invagination of the cell membrane and formation of the vacuole with an enclosed bacterium, were observed. The presence of dividing bacteria suggests that the epithelium includes a renewable, dividing population of symbiotic bacteria. No bacteria were detected in the gonads and the digestive system. Probable functions of these symbiotic bacteria such as involvement in protection or defense from predators and environmental impacts as well as their nutritional role in the nudibranch are discussed.

Listeria monocytogenes: No Spreading without NO

In infectious disease immunology, inducible nitric oxide (NO) synthase-derived NO is believed to function primarily as an antimicrobial and immunoregulatory molecule. In this issue of Immunity, Cole etal. (2012) show that NO helps the intracellular bacteria Listeria monocytogenes to spread.

Nitric Oxide Increases Susceptibility of Toll-like Receptor-Activated Macrophages to Spreading Listeria monocytogenes

Toll-like receptor (TLR) stimulation activates macrophages to resist intracellular pathogens. Yet, the intracellular bacterium Listeria monocytogenes (Lm) causes lethal infections in spite of innate immune cell activation. Lm uses direct cell-cell spread to disseminate within its host. Here, we have shown that TLR-activated macrophages killed cell-free Lm but failed to prevent infection by spreading Lm. Instead, TLR signals increased the efficiency of Lmspread from "donor" to "recipient" macrophages. This enhancement required nitric oxide (NO) production by nitric oxide synthase-2 (NOS2). NO increased Lm escape from secondary vacuoles in recipient cells and delayed maturation of phagosomes containing membrane-like particles that mimic Lm-containing pseudopods. NO also promoted Lm spread during systemic invivo infection, as shown by the fact that inhibition of NOS2 with 1400W reduced spread-dependent Lm burdens in mouse livers. These findings reveal a mechanism by which pathogens capable of cell-cell spread can avoid the consequences of innate immune cell activation by TLR stimuli.

Effects of igneous intrusions on coal petrology, pore-fracture and coalbed methane characteristics in Hongyang, Handan and Huaibei coalfields, North China

The effects of igneous intrusions have proved to be important for the exploration and development of coalbed methane in many coal basins. However, the studies of the influences of localized intrusions on coalbed methane reservoirs are still insufficient. In the context of five typical dike/sill intrusion patterns (i.e., dike cut-through, dike cut-in, floor intrusion by sill, roof intrusion by sill, and dual intrusions of roof and floor by sills), this study investigates the changes of coal organic composition and pores/fractures resulting from igneous intrusions. The influences of igneous intrusions on coal composition mainly include the decrease of vitrinites (especially telocollinite) and the increase of inertinites contents, as well as the formation of secondary devolatilization vacuoles and of exogenous desiccation microfractures. The vacuoles have size of 0.1–10 μm, and the microfractures are commonly developed with a wide space and a significantly short length. The microfractures typically have irregular dendritic, filamentous, and turtleback textures, and densities of hundreds per 9 cm2. The influences of igneous intrusions on coal pores and fractures vary significantly depending on the intrusion patterns, the coal ranks after the intrusion, and the nature of the adjacent formation surrounding the intrusion. For the coals with a VRr > 4.2%, the intrusion significantly reduced the coal pore development. For the coals with a VRr from 2.1%–4.2%, the development of the coal pores improved with decreasing distance to the contact. For the coals with a VRr < 2.1%, the intrusion resulted in a slight improvement of the coal pore development. A possible explanation for the increasing gas content near the intrusion is that the secondary vacuoles and microfractures induced an increase in the gas storage capacity and in the permeability of the coal reservoir. The detailed intrusion patterns of sills and dikes contribute to the formations of the coalbed methane accumulation in coal seams, and to the coal-generated gas accumulation in the reservoir of the highly permeable surrounding-rocks.

Ultrastructural Characteristics of Three Chenopod Halophytes Lacking Salt Excretion Structures

Plants maintained in high soil salinity generally develop particular structures to either tolerate or survive such adverse environments. Excretion of excess ions by special salt glands or other similar structures is a well-known phenomenon for regulating the mineral content of many halophytes. However, the three chenopod halophytes of Suaeda inhabit high saline soils, yet they exhibit no signs of salt excretion structures. The current study has been undertaken to assess the structural attributes of these halophytes to reveal their cellular characteristics during growth in salt tolerance. Transmission and scanning electron microscopy, as well as ion chromatography, have been employed for the study. One of the most noticeable features uncovered was the epidermal cutinization shown to be heavy on the outer epidermis and characterized externally by thick wax plates. Numerous vesicles and membranous invagination in the vacuoles were common features within the mesophyll cytoplasm. Invaginations of the vacuolar and/or plasma membrane frequently formed secondary vacuoles which later became distinct, membrane-bound compartments. Significant accumulation of solid sodium chloride salts was well demonstrated in the vacuoles of air-dried epidermis. Finally, salt tolerance mechanisms in these Suaeda have been discussed with respect to other halophyte modifications that improve salt tolerance in various ways.

Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss

In ruminants, the greatest period of embryonic loss coincides with the period of elongation when the embryonic disc is formed and gastrulation occurs prior to implantation. The impact of early embryonic mortality is not only a major obstacle to the cattle breeding industry but also impedes the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy, immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect to ultrastructural features and differentiation markers.

Activation of Cytosolic Phospholipase A2α in Resident Peritoneal Macrophages by Listeria monocytogenes Involves Listeriolysin O and TLR2

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.

Ultrastructural study of the lateral ventricle choroid plexus in experimental hydrocephalus in Wistar rats

Hydrocephalus is one of the most frequent and complex neurological diseases characterized by the abnormal buildup of cerebrospinal fluid (CSF) in the ventricles of the brain, due to an altered CSF dynamics. To detect possible ultrastructural alterations of the lateral ventricles choroid plexus (responsible for the CSF production), rats seven days after birth were submitted to an intracisternal injection of 20% kaolim (hydrated aluminum silicate) for the hydrocephalus induction. Twenty-eight or 35 days after injection, injected animals and respective controls were processed for observation under a transmission electron microscopy. Alterations found: presence of concentric cell membrane fragments, larger number of primary and secondary lysossomes, vacuoles, and cytoplasmic vesicles, and an enlargement of the intercellular space and between the basolateral interdigitation of the choroid epithelium. The alterations observed are probably associated to an increase of the ventricular pressure, inducing morpho-functional effects on the choroid plexus integrity.

Differential function of Listeria monocytogenes listeriolysin O and phospholipases C in vacuolar dissolution following cell-to-cell spread

We investigated the role of listeriolysin O (LLO) and the bacterial phospholipases PI-PLC and PC-PLC in cell-to-cell spread of Listeria monocytogenes. We showed that LLO is essential for cell-to-cell spread in primary murine macrophages. Electron micrographs revealed that in the absence of continued LLO expression, bacteria remain trapped in secondary spreading vacuoles having either a double or single membrane. In bacteria lacking PI-PLC and PC-PLC, cessation of LLO expression after initiation of infection resulted in a significant increase in the proportion of bacteria trapped in double-membrane compartments. We propose that the bacterial phospholipases are involved in the dissolution of the inner membrane of the spreading vacuole, yet are not sufficient for disruption of the outer membrane. As a consequence, we identified LLO as a key factor in the disruption of the outer membrane. This model is consistent with the observation that LLO is dispensable for cell-to-cell spread from human macrophages into a cell type in which LLO is not required for vacuolar escape. These data suggest that during human infection, spreading of L. monocytogenes to distant organs is likely to occur even in the absence of LLO expression, and that the bacterial phospholipases may be sufficient to mediate continued cell-to-cell spread.

Defective glycosylation of decorin and biglycan, altered collagen structure, and abnormal phenotype of the skin fibroblasts of an Ehlers–Danlos syndrome patient carrying the novel Arg270Cys substitution in galactosyltransferase I (β4GalT-7)

The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders affecting skin and joint function. Molecular defects in extracellular matrix proteins, including collagen (type I, III, and V) and tenascin X are associated with different forms of EDS. Compound heterozygous mutations in the B4GALT7 gene, resulting in aberrant glycosylation of the dermatan sulfate proteoglycan decorin, had been described in a single patient affected with the progeroid form of EDS. We have studied the molecular phenotype of decorin, biglycan, and collagen type I containing fibrils in skin fibroblasts of a patient carrying the novel homozygous C808T point mutation in the B4GALT7 gene, which causes an Arg270Cys substitution in beta4GalT-7. Compared to control fibroblasts, galactosyltransferase activity in beta4GalT-7(Arg270Cys) cells was approximately three times reduced over a temperature range of 25-41 degrees C. Pulse-chase experiments and confocal microscopy demonstrated that synthesis and secretion of decorin were normal in beta4GalT-7(Arg270Cys) cells. However, about 50% of decorin were synthesized as a protein core in addition to its proteoglycan form. Biglycan was found in a monoglycanated form in addition to its mature form. Glycosaminoglycan chains were of the dermatan/chondroitin sulfate type both in beta4GalT-7(Arg270Cys) and control cells, and epimerization was reduced for decorin and biglycan. Compared to control cells, beta4GalT-7(Arg270Cys) cells showed altered, highly spread or stretched phenotypes and decreased proliferation rates. At the ultrastructural level, an intracellular accumulation of multiple secondary lysosomes and degenerative vacuoles was seen in beta4GalT-7(Arg270Cys) cells. Furthermore, the collagen suprastructures were altered in the beta4GalT-7(Arg270Cys) cells. The reduced beta4GalT-7 activity resulting in defective glycosylation of decorin and biglycan may be responsible for the complex molecular pathology in beta4GalT-7 deficient EDS patients, given the role of these proteoglycans in bone formation, collagen fibrillogenesis, and skeletal muscle development.

The Metalloprotease ofListeria monocytogenesControls Cell Wall Translocation of the Broad-Range Phospholipase C

Listeria monocytogenes is a gram-positive bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from secondary vacuoles. PC-PLC requires cleavage of an N-terminal propeptide for activation, and Mpl, a metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. Previously, we showed that cell wall translocation of PC-PLC is inefficient, resulting in accumulation of PC-PLC at the membrane-cell wall interface. In infected cells, rapid cell wall translocation of PC-PLC is triggered by a decrease in pH and correlates with cleavage of the propeptide in an Mpl-dependent manner. To address the role of the propeptide and of Mpl in cell wall translocation of PC-PLC, we generated a cleavage site mutant and a propeptide deletion mutant. The intracellular behavior of these mutants was assessed in pulse-chase experiments. We observed efficient translocation of the proform of the PC-PLC cleavage site mutant in a manner that was pH sensitive and Mpl dependent. However, the propeptide deletion mutant was efficiently translocated into host cells independent of Mpl and pH. Overall, these results suggest that Mpl regulates PC-PLC translocation across the bacterial cell wall in a manner that is dependent on the presence of the propeptide but independent of propeptide cleavage. In addition, similarly to Mpl-mediated cleavage of PC-PLC propeptide, Mpl-mediated translocation of PC-PLC across the bacterial cell wall is pH sensitive.