Clathrin-mediated Endocytosis Facilitates Internalization of Magnaporthe oryzae Effectors into Rice Cells

Many filamentous eukaryotic plant pathogens, such as fungi and oomycetes, deliver effectors into living plant cells to suppress defenses and control plant processes needed for infection. To date, little is known about the mechanism by which these pathogens translocate effector proteins across the plasma membrane into the plant cytoplasm. The blast fungus Magnaporthe oryzae secretes cytoplasmic effectors into a specialized biotrophic interfacial complex (BIC) before translocation. Here we show that cytoplasmic effectors within BICs are packaged into dynamic vesicles that can occasionally be found separated from BICs in the host cytoplasm. Live cell imaging with fluorescently-labelled rice lines, showed that BICs are enriched in plant plasma membrane, actin, and Clathrin Light Chain-1, a marker for clathrin-mediated endocytosis (CME). We report that a novel cytoplasmic effector, Bas83, labels empty membrane vesicles surrounding BICs. Inhibition of CME using VIGS and chemical treatments results in a distinctive swollen BIC phenotype lacking effector vesicles. In contrast, fluorescent marker co-localization, VIGS and chemical inhibitor studies failed to implicate clathrin-independent endocytosis in effector vesicle formation. Taken together, this study provides evidence that cytoplasmic effector translocation is mediated by clathrin-mediated endocytosis in BICs and suggests a role for M. oryzae effectors in co-opting plant endocytosis.

Osmoregulation and the Anthozoan-Dinoflagellate Symbiosis

<p>This study investigated the responses of the temperate anemone Anthopleura aureoradiata, and the tropical coral Acropora aspera to osmotic stress and the role that free amino acids (FAAs) may play in the osmoregulatory mechanism of these anthozoan-dinoflagellate symbioses. Specimens were exposed to a range of hypo- and hyper-saline conditions for durations of 1, 12, 48 and 96 hours, whereupon respiration and photosynthetic rates were measured as physiological indicators of osmotic stress. High performance liquid chromatography was used to quantify 15 FAAs within the anthozoan host tissues to establish the response of FAA pools to osmotic stress and whether FAAs are used in an osmoregulatory capacity. Aposymbiotic specimens of A. aureoradiata were similarly tested to establish if the presence of symbiotic dinoflagellates alters the host’s capacity to respond to osmotic stress given that the symbionts are known to release FAAs into the host cytoplasm. In A. aureoradiata, significant changes in respiration were only observed with exposure to the extreme hypo-osmotic salinity of 12‰, with respiration decreasing by 67% after 1 hour of exposure. No significant changes in respiration were seen at 25, 43 or 50‰, despite a 52% decrease in respiration seen at the hyper-saline treatment of 50‰. The response of the coral A. aspera was markedly different, showing an increase in respiration in response to hypo-salinity (22 and 28‰). Interestingly, the most pronounced respiratory increase of up to 460% occurred in the less extreme hypo-saline treatment of 28‰. The response of photosynthesis also showed differences between the two species. In the symbiotic A. aureoradiata, photosynthesis declined by 61% after the 1 hour exposure to 12‰ and further decreased to 72% below control rates after 96 hours. While in A. aspera, photosynthesis showed no significant deviation from control levels at any of the treatment salinities. FAA pools in both A. aureoradiata and A. aspera showed significant responses to osmotic stress. In symbiotic A. aureoradiata, exposure to 12‰ caused total FAA pools to decline by 50% after 1 hour, after which a seemingly stable state was reached. A hyper-osmotic treatment of 50‰ resulted in a similar trend with a more than 50% decrease after 1 hour of exposure. In A. aspera, the response of the FAA pool was markedly different, with the concentration increasing by up to 200% with exposure to 22‰ and by more than 260% at 28‰. Interestingly, one on the main constituents of FAA pools in A. aureoradiata, Taurine (15% of FAA pools at 35‰), was not present in measurable quantities within A. aspera host tissue. In aposymbiotic individuals of A. aureoradiata exposed to extreme hypo- and hyper-saline treatments of 12 and 50‰ a significant impact on respiration was only observed at 12‰, with a 77% decrease in respiration after 96 hours. Changes in FAA pools of aposymbiotic A. aureoradiata were only seen after 12 hours exposure to 50‰ with a significant 26% decrease. However, the direct comparison between symbiotic and aposymbiotic A. aureoradiata did serve to highlight the contribution of symbiont-derived FAAs to the host pool of FAAs, with FAA pools in aposymbiotic anemones up to 41% lower than those found in symbiotic anemones. The results seen here were not suggestive of FAAs being regulated for the explicit use as compatible organic osmolytes. Rather, changes in FAA pools showed changes consistent with other stress responses. Moreover, the response of anthozoan-dinoflagellate symbioses to osmotic stress appears to be species specific, or at least taxa specific, as the responses of respiration, photosynthesis and FAA pools were very different between the temperate anemone A. aureoradiata and the tropical coral A. aspera. Nevertheless, differences in the respiratory response between symbiotic and apo-symbiotic anemones did indicate some influence of the dinoflagellate symbionts on the ability of the anthozoan host to mediate osmotic stress. It may therefore be that other symbiont-derived compounds are utilised as compatible organic osmolytes (COOs), with a primary candidate being glycerol. This warrants further investigation.</p>

Osmoregulation and the Anthozoan-Dinoflagellate Symbiosis

<p>This study investigated the responses of the temperate anemone Anthopleura aureoradiata, and the tropical coral Acropora aspera to osmotic stress and the role that free amino acids (FAAs) may play in the osmoregulatory mechanism of these anthozoan-dinoflagellate symbioses. Specimens were exposed to a range of hypo- and hyper-saline conditions for durations of 1, 12, 48 and 96 hours, whereupon respiration and photosynthetic rates were measured as physiological indicators of osmotic stress. High performance liquid chromatography was used to quantify 15 FAAs within the anthozoan host tissues to establish the response of FAA pools to osmotic stress and whether FAAs are used in an osmoregulatory capacity. Aposymbiotic specimens of A. aureoradiata were similarly tested to establish if the presence of symbiotic dinoflagellates alters the host’s capacity to respond to osmotic stress given that the symbionts are known to release FAAs into the host cytoplasm. In A. aureoradiata, significant changes in respiration were only observed with exposure to the extreme hypo-osmotic salinity of 12‰, with respiration decreasing by 67% after 1 hour of exposure. No significant changes in respiration were seen at 25, 43 or 50‰, despite a 52% decrease in respiration seen at the hyper-saline treatment of 50‰. The response of the coral A. aspera was markedly different, showing an increase in respiration in response to hypo-salinity (22 and 28‰). Interestingly, the most pronounced respiratory increase of up to 460% occurred in the less extreme hypo-saline treatment of 28‰. The response of photosynthesis also showed differences between the two species. In the symbiotic A. aureoradiata, photosynthesis declined by 61% after the 1 hour exposure to 12‰ and further decreased to 72% below control rates after 96 hours. While in A. aspera, photosynthesis showed no significant deviation from control levels at any of the treatment salinities. FAA pools in both A. aureoradiata and A. aspera showed significant responses to osmotic stress. In symbiotic A. aureoradiata, exposure to 12‰ caused total FAA pools to decline by 50% after 1 hour, after which a seemingly stable state was reached. A hyper-osmotic treatment of 50‰ resulted in a similar trend with a more than 50% decrease after 1 hour of exposure. In A. aspera, the response of the FAA pool was markedly different, with the concentration increasing by up to 200% with exposure to 22‰ and by more than 260% at 28‰. Interestingly, one on the main constituents of FAA pools in A. aureoradiata, Taurine (15% of FAA pools at 35‰), was not present in measurable quantities within A. aspera host tissue. In aposymbiotic individuals of A. aureoradiata exposed to extreme hypo- and hyper-saline treatments of 12 and 50‰ a significant impact on respiration was only observed at 12‰, with a 77% decrease in respiration after 96 hours. Changes in FAA pools of aposymbiotic A. aureoradiata were only seen after 12 hours exposure to 50‰ with a significant 26% decrease. However, the direct comparison between symbiotic and aposymbiotic A. aureoradiata did serve to highlight the contribution of symbiont-derived FAAs to the host pool of FAAs, with FAA pools in aposymbiotic anemones up to 41% lower than those found in symbiotic anemones. The results seen here were not suggestive of FAAs being regulated for the explicit use as compatible organic osmolytes. Rather, changes in FAA pools showed changes consistent with other stress responses. Moreover, the response of anthozoan-dinoflagellate symbioses to osmotic stress appears to be species specific, or at least taxa specific, as the responses of respiration, photosynthesis and FAA pools were very different between the temperate anemone A. aureoradiata and the tropical coral A. aspera. Nevertheless, differences in the respiratory response between symbiotic and apo-symbiotic anemones did indicate some influence of the dinoflagellate symbionts on the ability of the anthozoan host to mediate osmotic stress. It may therefore be that other symbiont-derived compounds are utilised as compatible organic osmolytes (COOs), with a primary candidate being glycerol. This warrants further investigation.</p>

Secreted Effectors Modulating Immune Responses to Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that chronically infects a third of humans. It can cause life-threatening encephalitis in immune-compromised individuals. Congenital infection also results in blindness and intellectual disabilities. In the intracellular milieu, parasites encounter various immunological effectors that have been shaped to limit parasite infection. Parasites not only have to suppress these anti-parasitic inflammatory responses but also ensure the host organism’s survival until their subsequent transmission. Recent advancements in T. gondii research have revealed a plethora of parasite-secreted proteins that suppress as well as activate immune responses. This mini-review will comprehensively examine each secreted immunomodulatory effector based on the location of their actions. The first section is focused on secreted effectors that localize to the parasitophorous vacuole membrane, the interface between the parasites and the host cytoplasm. Murine hosts are equipped with potent IFNγ-induced immune-related GTPases, and various parasite effectors subvert these to prevent parasite elimination. The second section examines several cytoplasmic and ER effectors, including a recently described function for matrix antigen 1 (MAG1) as a secreted effector. The third section covers the repertoire of nuclear effectors that hijack transcription factors and epigenetic repressors that alter gene expression. The last section focuses on the translocation of dense-granule effectors and effectors in the setting of T. gondii tissue cysts (the bradyzoite parasitophorous vacuole).

Capturing Salmonella SspH2 Host Targets in Virus-Like Particles

In the context of host-pathogen interactions, gram-negative bacterial virulence factors, such as effectors, may be transferred from bacterial to eukaryotic host cytoplasm by multicomponent Type III protein secretion systems (T3SSs). Central to Salmonella enterica serovar Typhimurium (S. Typhimurium) pathogenesis is the secretion of over 40 effectors by two T3SSs encoded within pathogenicity islands SPI-1 and SPI-2. These effectors manipulate miscellaneous host cellular processes, such as cytoskeleton organization and immune signaling pathways, thereby permitting host colonization and bacterial dissemination. Recent research on effector biology provided mechanistic insights for some effectors. However, for many effectors, clearly defined roles and host target repertoires—further clarifying effector interconnectivity and virulence networks—are yet to be uncovered. Here we demonstrate the utility of the recently described viral-like particle trapping technology Virotrap as an effective approach to catalog S. Typhimurium effector-host protein complexes (EH-PCs). Mass spectrometry-based Virotrap analysis of the novel E3 ubiquitin ligase SspH2 previously shown to be implicated in modulating actin dynamics and immune signaling, exposed known host interactors PFN1 and−2 besides several putative novel, interconnected host targets. Network analysis revealed an actin (-binding) cluster among the significantly enriched hits for SspH2, consistent with the known localization of the S-palmitoylated effector with actin cytoskeleton components in the host. We show that Virotrap complements the current state-of-the-art toolkit to study protein complexes and represents a valuable means to screen for effector host targets in a high-throughput manner, thereby bridging the knowledge gap between effector-host interplay and pathogenesis.

Temporal compartmentalization of viral infection in bacterial cells

Virus infection causes major rearrangements in the subcellular architecture of eukaryotes, but its impact in prokaryotic cells was much less characterized. Here, we show that infection of the bacterium Bacillus subtilis by bacteriophage SPP1 leads to a hijacking of host replication proteins to assemble hybrid viral–bacterial replisomes for SPP1 genome replication. Their biosynthetic activity doubles the cell total DNA content within 15 min. Replisomes operate at several independent locations within a single viral DNA focus positioned asymmetrically in the cell. This large nucleoprotein complex is a self-contained compartment whose boundaries are delimited neither by a membrane nor by a protein cage. Later during infection, SPP1 procapsids localize at the periphery of the viral DNA compartment for genome packaging. The resulting DNA-filled capsids do not remain associated to the DNA transactions compartment. They bind to phage tails to build infectious particles that are stored in warehouse compartments spatially independent from the viral DNA. Free SPP1 structural proteins are recruited to the dynamic phage-induced compartments following an order that recapitulates the viral particle assembly pathway. These findings show that bacteriophages restructure the crowded host cytoplasm to confine at different cellular locations the sequential processes that are essential for their multiplication.

The bacterial toxin ExoU requires a host trafficking chaperone for transportation and to induce necrosis

Pseudomonas aeruginosa can cause nosocomial infections, especially in ventilated or cystic fibrosis patients. Highly pathogenic isolates express the phospholipase ExoU, an effector of the type III secretion system that acts on plasma membrane lipids, causing membrane rupture and host cell necrosis. Here, we use a genome-wide screen to discover that ExoU requires DNAJC5, a host chaperone, for its necrotic activity. DNAJC5 is known to participate in an unconventional secretory pathway for misfolded proteins involving anterograde vesicular trafficking. We show that DNAJC5-deficient human cells, or Drosophila flies knocked-down for the DNAJC5 orthologue, are largely resistant to ExoU-dependent virulence. ExoU colocalizes with DNAJC5-positive vesicles in the host cytoplasm. DNAJC5 mutations preventing vesicle trafficking (previously identified in adult neuronal ceroid lipofuscinosis, a human congenital disease) inhibit ExoU-dependent cell lysis. Our results suggest that, once injected into the host cytoplasm, ExoU docks to DNAJC5-positive secretory vesicles to reach the plasma membrane, where it can exert its phospholipase activity

The mechanism of artemisinin resistance of Plasmodium falciparum malaria parasites originates in their initial transcriptional response.

The emergence and spread of artemisinin resistant Plasmodium falciparum, first in the Greater Mekong Subregion (GMS), and now in East Africa, is a major threat to global malaria eliminations ambitions. To investigate the artemisinin resistance mechanism, transcriptome analysis was conducted of 577 P. falciparum isolates collected in the GMS between 2016–2018. A specific artemisinin resistance-associated transcriptional profile was identified that involves a broad but discrete set of biological functions related to proteotoxic stress, host cytoplasm remodeling and REDOX metabolism. The artemisinin resistance-associated transcriptional profile evolved from initial transcriptional responses of susceptible parasites to artemisinin. The genetic basis for this adapted response is likely to be complex.

Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion.

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there is little data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites. We discovered that conoid fibre number varied, but there was a fixed spacing between fibres, leading to conoids of different sizes. Associated apical structures varied in size to accommodate a larger or smaller conoid diameter. However, the number of subpellicular microtubules on the apical polar ring surrounding the conoid did not vary, suggesting a control of apical complex size. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins (ROPs) may be trafficked between the parasite and the host cytoplasm

Disruption of the Interfacial Membrane Leads to Magnaporthe oryzae Effector Re-location and Lifestyle Switch During Rice Blast Disease

To cause the devastating rice blast disease, the hemibiotrophic fungus Magnaporthe oryzae produces invasive hyphae (IH) that are enclosed in a plant-derived interfacial membrane, known as the extra-invasive hyphal membrane (EIHM), in living rice cells. Little is known about when the EIHM is disrupted and how the disruption contributes to blast disease. Here we show that the disruption of the EIHM correlates with the hyphal growth stage in first-invaded susceptible rice cells. Our approach utilized GFP that was secreted from IH as an EIHM integrity reporter. Secreted GFP (sec-GFP) accumulated in the EIHM compartment but appeared in the host cytoplasm when the integrity of the EIHM was compromised. Live-cell imaging coupled with sec-GFP and various fluorescent reporters revealed that the loss of EIHM integrity preceded shrinkage and eventual rupture of the rice vacuole. The vacuole rupture coincided with host cell death, which was limited to the invaded cell with presumed closure of plasmodesmata. We report that EIHM disruption and host cell death are landmarks that delineate three distinct infection phases (early biotrophic, late biotrophic, and transient necrotrophic phases) within the first-invaded cell before reestablishment of biotrophy in second-invaded cells. M. oryzae effectors exhibited infection phase-specific localizations, including entry of the apoplastic effector Bas4 into the host cytoplasm through the disrupted EIHM during the late biotrophic phase. Understanding how infection phase-specific cellular dynamics are regulated and linked to host susceptibility will offer potential targets that can be exploited to control blast disease.