Many eukaryotic microbes have complex life cycles that include both sexual and asexual phases with strict species specificity. Whereas the asexual cycle of the protistan parasite Toxoplasma gondii can occur in any warm-blooded mammal, the sexual cycle is restricted to the feline intestine. The molecular determinants that identify cats as the definitive host for T. gondii are unknown. Here, we defined the mechanism of species specificity for T. gondii sexual development and break the species barrier to allow the sexual cycle to occur in mice. We determined that T. gondii sexual development occurs when cultured feline intestinal epithelial cells are supplemented with linoleic acid. Felines are the only mammals that lack delta-6-desaturase activity in their intestines, which is required for linoleic acid metabolism, resulting in systemic excess of linoleic acid. We found that inhibition of murine delta-6-desaturase and supplementation of their diet with linoleic acid allowed T. gondii sexual development in mice. This mechanism of species specificity is the first defined for a parasite sexual cycle. This work highlights how host diet and metabolism shape coevolution with microbes. The key to unlocking the species boundaries for other eukaryotic microbes may also rely on the lipid composition of their environments as we see increasing evidence for the importance of host lipid metabolism during parasitic lifecycles. Pregnant women are advised against handling cat litter, as maternal infection with T. gondii can be transmitted to the fetus with potentially lethal outcomes. Knowing the molecular components that create a conducive environment for T. gondii sexual reproduction will allow for development of therapeutics that prevent shedding of T. gondii parasites. Finally, given the current reliance on companion animals to study T. gondii sexual development, this work will allow the T. gondii field to use of alternative models in future studies.

The bacterial pathogen Vibrio cholerae can occupy both the human gut and aquatic reservoirs, where it may colonize chitinous surfaces that induce the expression of factors for three phenotypes: chitin utilization, DNA uptake by natural transformation, and contact-dependent bacterial killing via a type VI secretion system (T6SS). In this study, we surveyed a diverse set of 53 isolates from different geographic locales collected over the past century from human clinical and environmental specimens for each phenotype outlined above. The set included pandemic isolates of serogroup O1, as well as several serogroup O139 and non-O1/non-O139 strains. We found that while chitin utilization was common, only 22.6% of the isolates tested were proficient at chitin-induced natural transformation, suggesting that transformation is expendable. Constitutive contact-dependent killing of Escherichia coli prey, which is indicative of a functional T6SS, was rare among clinical isolates (only 4 of 29) but common among environmental isolates (22 of 24). These results bolster the pathoadaptive model in which tight regulation of T6SS-mediated bacterial killing is beneficial in a human host, whereas constitutive killing by environmental isolates may give a competitive advantage in natural settings. Future sequence analysis of this set of diverse isolates may identify previously unknown regulators and structural components for both natural transformation and T6SS.V ibrio cholerae, the bacterium responsible for the diarrheal disease cholera, can occupy a range of freshwater and marine environments, where it commonly associates with abiotic chitinous material and the biotic surfaces of algae, invertebrates, plants, and fish (1). When water carrying V. cholerae is ingested, cells that survive passage through the acidic stomach may gain access to the small intestine and bind to its mucus layer. Isolates carrying the CTX prophage can secrete cholera toxin (CT), which is responsible for the potentially fatal diarrhea that also aids in transmission from the host.Over 200 O serogroups have been described, with each defined as a group of bacteria that share a surface antigen. Although only the O1 and O139 serogroups carrying the CTX prophage are responsible for major cholera epidemics, other serogroups may be associated with isolated cases of gastroenteritis but so far have not been shown to spread globally (2). The pandemic O1 CTX ϩ isolates are further divided into two biotypes, Classical and El Tor, on the basis of several biochemical and phage susceptibility tests (3-6). Seven cholera pandemics have been described. The O1 Classical biotype was responsible for the sixth and likely prior pandemics but was displaced by the O1 El Tor biotype in the seventh pandemic, which began in Southeast Asia in 1961 (7). In 1992, an El Tor mutant, serotype O139, became responsible for some regional cholera outbreaks and continues to coexist with O1 El Tor, although in a minor capacity (8-10).When inhabiting aquatic environments, V. cholerae can degrade the chi...

Summary Emerging lipidomic technologies have enabled researchers to dissect the complex roles of phospholipases in lipid metabolism, cellular signaling and immune regulation. Host phospholipase products are involved in stimulating and resolving the inflammatory response to pathogens. While many pathogen‐derived phospholipases also manipulate the immune response, they have recently been shown to be involved in lipid remodeling and scavenging during replication. Animal and plant hosts as well as many pathogens contain a family of patatin‐like phospholipases, which have been shown to have phospholipase A2 activity. Proteins containing patatin‐like phospholipase domains have been identified in protozoan parasites within the Apicomplexa phylum. These parasites are the causative agents of some of the most widespread human diseases. Malaria, caused by Plasmodium spp., kills nearly half a million people worldwide each year. Toxoplasma and Cryptosporidium infect millions of people each year with lethal consequences in immunocompromised populations. Parasite‐derived patatin‐like phospholipases are likely effective drug targets and progress in the tools available to the Apicomplexan field will allow for a closer look at the interplay of lipid metabolism and immune regulation during host infection.

Many eukaryotic microbes have complex lifecycles that include both sexual and asexual phases with strict species-specificity. While the asexual cycle of the protistan parasite Toxoplasma gondii can occur in any warm-blooded mammal, the sexual cycle is restricted to the feline intestine 1 . The molecular determinants that identify cats as the definitive 5 host for T. gondii are unknown. Here, we defined the mechanism of species specificity for T.gondii sexual development and break the species barrier to allow the sexual cycle to occur in mice. We determined that T. gondii sexual development occurs when cultured feline intestinal epithelial cells are supplemented with linoleic acid. Felines are the only mammals that lack delta-6-desaturase activity in their intestines, which is required for linoleic acid metabolism, resulting 10 in systemic excess of linoleic acid 2, 3 . We found that inhibition of murine delta-6-desaturase and supplementation of their diet with linoleic acid allowed T. gondii sexual development in mice.This mechanism of species specificity is the first defined for a parasite sexual cycle. This work highlights how host diet and metabolism shape coevolution with microbes. The key to unlocking the species boundaries for other eukaryotic microbes may also rely on the lipid composition of 15 their environments as we see increasing evidence for the importance of host lipid metabolism during parasitic lifecycles 4, 5 . Pregnant women are advised against handling cat litter as maternal infection with T. gondii can be transmitted to the fetus with potentially lethal outcomes. Knowing the molecular components that create a conducive environment for T.gondii sexual reproduction will allow for development of therapeutics that prevent shedding of T. 20 gondii parasites. Finally, given the current reliance on companion animals to study T. gondii sexual development, this work will allow the T. gondii field to use of alternative models in future studies.3 Main: The apicomplexan parasite Toxoplasma gondii causes a chronic infection in nearly one third of the human population and is well-known for causing congenital infections leading to blindness, mental retardation, and hydrocephaly of the developing fetus. T. gondii has a complex lifecycle containing both sexual and asexual phases. The T. gondii asexual cycle can occur in any 5 warm-blooded animal when contaminated food or water is consumed and T. gondii disseminates throughout the host, converting to an encysted form in muscle and brain tissue. In contrast, the T.gondii sexual cycle is restricted to the feline intestinal epithelium, culminating in the excretion of environmentally resistant oocysts 1 . The molecular basis for this species specificity is unknown. 10To determine the molecular mechanisms that define the species specificity of T. gondii sexual development, we generated cat intestinal organoids ( Fig. 1a), then seeded these epithelial cells onto glass coverslips. These monolayers displayed intestinal epithelial properties, including polarization and ...

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of any warm-blooded animal. In a previous screen to identify virulence determinants, disruption of gene TgME49_305140 generated a T. gondii mutant that could not establish a chronic infection in mice. The protein product of TgME49_305140, here named TgPL3, is a 277 kDa protein with a patatin-like phospholipase (PLP) domain and a microtubule binding domain. Antibodies generated against TgPL3 show that it is localized to the apical cap. Using a rapid selection FACS-based CRISPR/Cas-9 method, a TgPL3 deletion strain (ΔTgPL3) was generated. ΔTgPL3 parasites have defects in host cell invasion, which may be caused by reduced rhoptry secretion. We generated complementation clones with either wild type TgPL3 or an active site mutation in the PLP domain by converting the catalytic serine to an alanine, ΔTgPL3::TgPL3 S1409A (S1409A). Complementation of ΔTgPL3 with wild type TgPL3 restored all phenotypes, while S1409A did not, suggesting that phospholipase activity is necessary for these phenotypes. ΔTgPL3 and S1409A parasites are also virtually avirulent in vivo but induce a robust antibody response. Vaccination with ΔTgPL3 and S1409A parasites protected mice against subsequent challenge with a lethal dose of Type I T. gondii parasites, making ΔTgPL3 a compelling vaccine candidate. These results demonstrate that TgPL3 has a role in rhoptry secretion, host cell invasion and survival of T. gondii during acute mouse infection.