Cellular & Molecular Imaging

We designed an experiment to validate our Single Worm Analysis of Movement Pipeline (SWAMP) assay, which will be used in the field to test schistosome worm response to praziquantel (PZQ) treatment. In this validation experiment, we used our SmLE-PZQ-ER (resistant to PZQ) and SmLE-PZQ-ES (sensitive to PZQ) populations and generated pools of 100 adult male worms that contained different proportions of worms from each population: either 1% or 5% of SmLE-PZQ-ER mixed with SmLE-PZQ-ES worms. We cultured the worms for 5 days with daily media changes, exposed the worms on the second day of culture for 24 h before washing out the drug. Worms were cultured and treated in bulk in 6-well plates for 4 days and plated individually on day 4. Worm motility was recorded at the end of day 5 for ~3 minutes using a camera in a black box positioned below the plate, with lighting from above.

We measured the phenotypic response to praziquantel (PZQ) of Schistosoma mansoni from field populations in Western Kenya. Parasite eggs were sampled from patients at different locations and propagated in the laboratory through snails and hamsters. We cultured adult male worms for 5 days with daily media changes, exposing the worms to PZQ on the second day of culture for 24 h before washing out the drug. Worms were cultured and treated in bulk in 6-well plates and plated individually on day 5 for imaging with our Single Worm Analysis of Movement Pipeline (SWAMP) assay. Worm motility was recorded at the end of day 5 for ~3 minutes using a camera in a black box positioned below the plate, with lighting from above. Data can be analyzed using the SWAMP pipeline.

Indocyanine Green (ICG) has seen widespread use in the operating room as a fluorescent vascular imaging agent. However, its rapid vascular clearance often necessitates redosing, resulting in procedural delays, greater background, and potential adverse physiological effects. Here, we present Chromenylium Green (ChromG), a novel fluorescent tracer exhibiting excellent visualization of vasculature in mice with greater contrast and seven times the vascular half-life relative to ICG. ChromG allows for high-resolution imaging of vasculature at greater depths, enabling whole-body 3D vascular reconstruction with up to 74% connectivity. As a proof of concept of ChromG’s utility in disease contexts, we demonstrate the ability to visualize saphenous artery stenoses above the clinically accepted contrast limit for eight times longer than ICG. With its excellent biocompatibility profile and compatibility with current clinical imaging technologies, ChromG is a promising fluorescent probe for prolonged, high-resolution intraoperative vascular imaging.

Indocyanine Green (ICG) has seen widespread use in the operating room as a fluorescent vascular imaging agent. However, its rapid vascular clearance often necessitates redosing, resulting in procedural delays, greater background, and potential adverse physiological effects. Here, we present Chromenylium Green (ChromG), a novel fluorescent tracer exhibiting excellent visualization of vasculature in mice with greater contrast and seven times the vascular half-life relative to ICG. ChromG allows for high-resolution imaging of vasculature at greater depths, enabling whole-body 3D vascular reconstruction with up to 74% connectivity. As a proof of concept of ChromG’s utility in disease contexts, we demonstrate the ability to visualize saphenous artery stenoses above the clinically accepted contrast limit for eight times longer than ICG. With its excellent biocompatibility profile and compatibility with current clinical imaging technologies, ChromG is a promising fluorescent probe for prolonged, high-resolution intraoperative vascular imaging.

Many bacteria use Type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighbouring bacterial or fungal cells as a means of inter-microbial competition. Compared with numerous antibacterial effectors, few antifungal effectors have been described. Furthermore, how T6SS-delivered effectors reach their site of action in different types of target cell remains poorly understood. Here, we combine structural biology with in vivo approaches to show that Rhs2 from Serratia marcescens Db10 is a dual-kingdom T6SS-dependent DNase effector which hijacks distinct, essential target cell functions in order to reach its site of action in bacterial and fungal cells. In bacterial cells, the Rhs2 toxin domain (Rhs2CT) interacts specifically with the elongation factor, EF-Tu, and, in sibling cells, interacts with the cognate immunity protein in an unusual manner. Interaction with EF-Tu is essential for T6SS-mediated intoxication of bacterial cells by Rhs2, but not for DNase activity or intoxication of fungal cells, implying it facilitates entry of Rhs2CT across the inner membrane to the cytoplasm. Alternatively, in fungal cells, Rhs2CT translocates to the nucleus using the nuclear import machinery. Our findings reveal how a single effector domain can act against targets with distinct cellular architectures and suggest that dual-kingdom effectors may occur widely.

Many bacteria use Type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighbouring bacterial or fungal cells as a means of inter-microbial competition. Compared with numerous antibacterial effectors, few antifungal effectors have been described. Furthermore, how T6SS-delivered effectors reach their site of action in different types of target cell remains poorly understood. Here, we combine structural biology with in vivo approaches to show that Rhs2 from Serratia marcescens Db10 is a dual-kingdom T6SS-dependent DNase effector which hijacks distinct, essential target cell functions in order to reach its site of action in bacterial and fungal cells. In bacterial cells, the Rhs2 toxin domain (Rhs2CT) interacts specifically with the elongation factor, EF-Tu, and, in sibling cells, interacts with the cognate immunity protein in an unusual manner. Interaction with EF-Tu is essential for T6SS-mediated intoxication of bacterial cells by Rhs2, but not for DNase activity or intoxication of fungal cells, implying it facilitates entry of Rhs2CT across the inner membrane to the cytoplasm. Alternatively, in fungal cells, Rhs2CT translocates to the nucleus using the nuclear import machinery. Our findings reveal how a single effector domain can act against targets with distinct cellular architectures and suggest that dual-kingdom effectors may occur widely.

Many bacteria use Type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighbouring bacterial or fungal cells as a means of inter-microbial competition. Compared with numerous antibacterial effectors, few antifungal effectors have been described. Furthermore, how T6SS-delivered effectors reach their site of action in different types of target cell remains poorly understood. Here, we combine structural biology with in vivo approaches to show that Rhs2 from Serratia marcescens Db10 is a dual-kingdom T6SS-dependent DNase effector which hijacks distinct, essential target cell functions in order to reach its site of action in bacterial and fungal cells. In bacterial cells, the Rhs2 toxin domain (Rhs2CT) interacts specifically with the elongation factor, EF-Tu, and, in sibling cells, interacts with the cognate immunity protein in an unusual manner. Interaction with EF-Tu is essential for T6SS-mediated intoxication of bacterial cells by Rhs2, but not for DNase activity or intoxication of fungal cells, implying it facilitates entry of Rhs2CT across the inner membrane to the cytoplasm. Alternatively, in fungal cells, Rhs2CT translocates to the nucleus using the nuclear import machinery. Our findings reveal how a single effector domain can act against targets with distinct cellular architectures and suggest that dual-kingdom effectors may occur widely.

We designed an experiment to validate our Single Worm Analysis of Movement Pipeline (SWAMP) assay, which will be used in the field to test schistosome worm response to praziquantel (PZQ) treatment. In this validation experiment, we used our SmLE-PZQ-ER (resistant to PZQ) and SmLE-PZQ-ES (sensitive to PZQ) populations and generated pools of 100 adult male worms that contained different proportions of worms from each population: either 1% or 5% of SmLE-PZQ-ER mixed with SmLE-PZQ-ES worms. We cultured the worms for 5 days with daily media changes, exposed the worms on the second day of culture for 24 h before washing out the drug. Worms were cultured and treated in bulk in 6-well plates for 4 days and plated individually on day 4. Worm motility was recorded at the end of day 5 for ~3 minutes using a camera in a black box positioned below the plate, with lighting from above.

We measured the phenotypic response to praziquantel (PZQ) of Schistosoma mansoni from field populations in Western Kenya. Parasite eggs were sampled from patients at different locations and propagated in the laboratory through snails and hamsters. We cultured adult male worms for 5 days with daily media changes, exposing the worms to PZQ on the second day of culture for 24 h before washing out the drug. Worms were cultured and treated in bulk in 6-well plates and plated individually on day 5 for imaging with our Single Worm Analysis of Movement Pipeline (SWAMP) assay. Worm motility was recorded at the end of day 5 for ~3 minutes using a camera in a black box positioned below the plate, with lighting from above. Data can be analyzed using the SWAMP pipeline.

We designed an experiment to validate our Single Worm Analysis of Movement Pipeline (SWAMP) assay, which will be used in the field to test schistosome worm response to praziquantel (PZQ) treatment. In this validation experiment, we used our SmLE-PZQ-ER (resistant to PZQ) and SmLE-PZQ-ES (sensitive to PZQ) populations and generated pools of 100 adult male worms that contained different proportions of worms from each population: either 1% or 5% of SmLE-PZQ-ER mixed with SmLE-PZQ-ES worms. We cultured the worms for 5 days with daily media changes, exposed the worms on the second day of culture for 24 h before washing out the drug. Worms were cultured and treated in bulk in 6-well plates for 4 days and plated individually on day 4. Worm motility was recorded at the end of day 5 for ~3 minutes using a camera in a black box positioned below the plate, with lighting from above.