Blockade of endoplasmic reticulum stress-induced cell death by Ureaplasma parvum vacuolating factor.

Previously, we found that Ureaplasma parvum internalised into HeLa cells and cytosolic accumulation of galectin-3. U. parvum induced the host cellular membrane damage and survived there. Here, we conducted vesicular trafficking inhibitory screening in yeast to identify U. parvum vacuolating factor (UpVF). U. parvum triggered endoplasmic reticulum (ER) stress and upregulated the unfolded protein response-related factors, including BiP, P-eIF2 and IRE1 in the host cells, but it blocked the induction of the downstream apoptotic factors. MicroRNA library screening of U. parvum-infected cells and UpVF-transfected cells identified miR-211 and miR-214 as the negative regulators of the apoptotic cascade under ER stress. Transient expression of UpVF induced HeLa cell death with intracellular vacuolization; however, some stable UpVF transformant survived. U. parvum-infected cervical cell lines showed resistance to actinomycin D, and UpVF stable transformant cell lines exhibited resistance to X-ray irradiation, as well as cisplatin and paclitaxel. UpVF expressing cervical cancer xenografts in nude mice also acquired resistance to cisplatin and paclitaxel. A mycoplasma expression vector based on Mycoplasma mycoides, Syn-MBA (multiple banded antigen)-UpVF, reduced HeLa cell survival compared with that of Syn-MBA after 72 hr of infection. These findings together suggest novel mechanisms for Ureaplasma infection and the possible implications for cervical cancer malignancy. TAKE AWAYS: • Ureaplasmal novel virulence factor, UpVF, was identified. • UpVF triggered ER stress but suppressed apoptotic cascade via miR-211 and -214. • UpVF conferred resistance to anticancer treatments both in vivo and in vitro. • Dual expression of MBA and UpVF in JCVI-syn3B showed host cell damage.

A unique virulence factor for proliferation and dwarfism in plants identified from a phytopathogenic bacterium.

One of the most important themes in agricultural science is the identification of virulence factors involved in plant disease. Here, we show that a single virulence factor, tengu-su inducer (TENGU), induces witches' broom and dwarfism and is a small secreted protein of the plant-pathogenic bacterium, phytoplasma. When tengu was expressed in Nicotiana benthamiana plants, these plants showed symptoms of witches' broom and dwarfism, which are typical of phytoplasma infection. Transgenic Arabidopsis thaliana lines expressing tengu exhibited similar symptoms, confirming the effects of tengu expression on plants. Although the localization of phytoplasma was restricted to the phloem, TENGU protein was detected in apical buds by immunohistochemical analysis, suggesting that TENGU was transported from the phloem to other cells. Microarray analyses showed that auxin-responsive genes were significantly down-regulated in the tengu-transgenic plants compared with GUS-transgenic control plants. These results suggest that TENGU inhibits auxin-related pathways, thereby affecting plant development.

Phytoplasmas: bacteria that manipulate plants and insects.

TAXONOMY: Superkingdom Prokaryota; Kingdom Monera; Domain Bacteria; Phylum Firmicutes (low-G+C, Gram-positive eubacteria); Class Mollicutes; Candidatus (Ca.) genus Phytoplasma. HOST RANGE: Ca. Phytoplasma comprises approximately 30 distinct clades based on 16S rRNA gene sequence analyses of approximately 200 phytoplasmas. Phytoplasmas are mostly dependent on insect transmission for their spread and survival. The phytoplasma life cycle involves replication in insects and plants. They infect the insect but are phloem-limited in plants. Members of Ca. Phytoplasma asteris (16SrI group phytoplasmas) are found in 80 monocot and dicot plant species in most parts of the world. Experimentally, they can be transmitted by approximately 30, frequently polyphagous insect species, to 200 diverse plant species. DISEASE SYMPTOMS: In plants, phytoplasmas induce symptoms that suggest interference with plant development. Typical symptoms include: witches' broom (clustering of branches) of developing tissues; phyllody (retrograde metamorphosis of the floral organs to the condition of leaves); virescence (green coloration of non-green flower parts); bolting (growth of elongated stalks); formation of bunchy fibrous secondary roots; reddening of leaves and stems; generalized yellowing, decline and stunting of plants; and phloem necrosis. Phytoplasmas can be pathogenic to some insect hosts, but generally do not negatively affect the fitness of their major insect vector(s). In fact, phytoplasmas can increase fecundity and survival of insect vectors, and may influence flight behaviour and plant host preference of their insect hosts. DISEASE CONTROL: The most common practices are the spraying of various insecticides to control insect vectors, and removal of symptomatic plants. Phytoplasma-resistant cultivars are not available for the vast majority of affected crops.

Interaction between the membrane protein of a pathogen and insect microfilament complex determines insect-vector specificity.

Many insect-transmissible pathogens are transmitted by specific insect species and not by others, even if they are closely related. The molecular mechanisms underlying such strict pathogen-insect specificity are poorly understood. Candidatus Phytoplasma asteris, OY strain, line W (OY), is a phytopathogenic bacterium transmitted from plant to plant by sap-feeding insect vectors (leafhoppers). Our study focused on an abundant cell-surface membrane protein of the phytoplasma named antigenic membrane protein (Amp), which is not homologous with any reported functional protein. Immunofluorescence microscopy of the phytoplasma-infected insect showed that OY phytoplasma was localized to the microfilaments of the visceral smooth muscle surrounding the insect's intestinal tract. The affinity column assay showed that Amp forms a complex with three insect proteins: actin, myosin heavy chain, and myosin light chain. Amp-microfilament complexes were detected in all OY-transmitting leafhopper species, but not in the non-OY-transmitting leafhoppers, suggesting that the formation of the Amp-microfilament complex is correlated with the phytoplasma-transmitting capability of leafhoppers. Although several studies have reported interactions between pathogens and mammalian microfilaments, this is an example of host-specific interactions between a bacterial surface protein and a host microfilament in insect cells. Our data also suggest that the utilization of a host microfilament may be a universal system for pathogenic bacteria infecting mammals or insects.

Secretion of immunodominant membrane protein from onion yellows phytoplasma through the Sec protein-translocation system in Escherichia coli.

A gene that encodes a putative SecE protein, which is a component of the Sec protein-translocation system, was cloned from the onion yellows phytoplasma (OY). The identification of this gene and the previously reported genes encoding SecA and SecY provides evidence that the Sec system exists in phytoplasma. In addition, a gene encoding an antigenic membrane protein (Amp) (a type of immunodominant membrane protein) of OY was cloned and sequenced. The OY amp gene consisted of 702 nt encoding a protein of 233 aa which was highly similar to Amp of aster yellows phytoplasma (AY). Part of OY Amp was overexpressed in Escherichia coli, purified, and used to raise an anti-Amp polyclonal antibody. The anti-Amp antibody reacted specifically with an OY-infected plant extract in Western blot analysis and was therefore useful for the detection of OY as well as Amp. Amp has a conserved protein motif that is known to be exported by the Sec system of E. coli. A partial OY Amp protein expressed in E. coli was localized in the periplasm as a shorter, putatively processed form of the protein. It had probably been exported from the cytoplasm to the periplasm through the Sec system. Moreover, OY Amp protein expressed in OY and detected in OY-infected plants was apparently also processed. Because phytoplasmas cannot be cultured or transformed, little information is available regarding their protein secretion systems. This study suggests that the Sec system operates in this phytoplasma to export OY Amp.