Biology subjects

Fairweather, S. J.

Publications and source records attributed to Fairweather, S. J..

5 recordsLinked to original sources

bioRxiv2026

Copper transport is required for metabolism and persistence of Toxoplasma gondii

Copper is a conserved cofactor, required for essential processes including aerobic respiration. Pathogens subvert host copper; however the mechanisms of copper uptake are not well understood. Here, we identify and validate two copper transporters of Toxoplasma gondii and determine the role of copper in parasite metabolism and pathogenesis. We show that Ctr1 is required for copper uptake. Deletion leads to undetectable parasite-associated copper, a significant growth defect and a metabolic shift from mitochondrial respiration with to glycolysis. Absence of Crt1 was fully and rapidly rescued by exogenous copper, which we believe is transported through the lower-affinity transporter Ctr2. Ctr2 is dispensable in rapid growth, however, has a role in parasite persistence in chronic infection, both in vitro and in vivo. Together, these findings reveal for the first time a critical role for copper uptake in shaping metabolic plasticity, and highlights the importance of nutrient availability in regulating apicomplexan metabolism.

cell biology↗

bioRxiv2026

A proton transfer mechanism in the malaria parasite lactate/H+ symporter reveals a channel-like transporter without conformational changes

The malaria parasite Plasmodium falciparum relies on anaerobic glycolysis for energy during the intraerythrocytic stage, producing lactate and H+ that must be extruded via its formate-nitrite transporter (PfFNT) to prevent death by cytosolic acidification and swelling. Unlike bacterial homologues classified as channels, PfFNT mediates saturable lactate transport and was classified as a transporter, raising questions about its transport mechanism and what distinguishes channels from transporters. Here, we combine over 720 s of molecular dynamics simulations, quantum chemical calculations, and transport assays in Xenopus laevis oocytes to characterize the transport cycle of PfFNT. We show a transport mechanism in which protonation of a central histidine (H230) enables lactate binding, and proton transfer from H230 to lactate is required to release the substrate as neutral lactic acid. This mechanism is supported by data indicating that neutral compounds such as lactamide do not enter the cavity, while anions like iodide, which are unlikely to protonate under physiological conditions, bind in the cavity but are not released and thus block lactate transport. As neither lactate nor lactic acid can diffuse passively through the protein, it is the requirement for proton transfer defines PfFNT as a lactate/H+ transporter rather than a channel. Notably, the PfFNT transport process occurs without significant protein conformational changes. Furthermore, our data indicates that under acidic conditions, small neutral molecules such as formic acid can passively diffuse through the protein. This suggests PfFNT challenges standard definitions of channels and transporters and differs from existing examples of channel-like transporters as it i) operates as a transporter without conformational changes and ii) can switch between transporter and channel modes within the same transport pathway.

biophysics↗

bioRxiv2023

Cell-specialized chloroplast signaling orchestrates photosynthetic and extracellular reactive oxygen species for stress responses

Cellular responses to abiotic stress involve multiple signals including secondary messengers such as reactive oxygen species (ROS) and Ca2+, phytohormones such as abscisic acid (ABA) and chloroplast-to-nucleus retrograde signals such as 3-phosphoadenosine 5-phosphate (PAP). Mechanism(s) by which these messengers, produced in different subcellular compartments, intersect for cell regulation remain enigmatic. Previously we showed that the chloroplast retrograde signal PAP, similar to ABA, induces an increase in ROS levels in guard cells (Pornsiriwong et al, 2017). Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate both chloroplast and plasma membrane ROS production. In whole leaves, PAP alters various ROS-related processes including plasmodesmal permeability as well as responses to ozone and the bacterial elicitor flg22, but mainly initiates processes that quench ROS during oxidative stress. Conversely, we show in guard cells, both PAP and ABA induce an increase in ROS levels in both chloroplasts via photosynthetic electron transport, and the apoplast via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). Both subcellular ROS sources were necessary for ABA- and PAP-mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD, instead of RBOHF, for apoplastic ROS mediated stomatal closure. We identified three calcium-dependent protein kinases (CPKs) as the post-translational activators of RBOHD-mediated ROS production. CPK13, CPK32, and CPK34 were transcriptionally induced by PAP and concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two Serine (S) residues, including S120 which is also targeted by the core ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP-induced CPKs rescues stomatal closure in ost1. Our data identify stomatal chloroplasts, to be nodes in the multifaceted cellular stress response networks as they are both sources and mediators of ROS and retrograde signals such PAP. Thus, chloroplasts are not just mediators of photosynthesis in response to, for example, excess light, but can serve as critical nodes in the multifaceted cellular stress response networks in specialized cells via retrograde signals, providing support to the concept of sensory plastids. Significance StatementThe chloroplast is an environmental sensor for stresses such as excess light and drought via the activation of photosynthetic-mediated retrograde signals. However, how does it function in specialized cells for which carbon fixation is secondary? Here we show the chloroplast is an important node to coordinate multiple plant signaling pathways in response to stresses such as drought. The chloroplast retrograde signal 3-phosphoadenosine 5-phosphate (PAP) plays multiple roles in reactive oxygen species (ROS) signaling and homeostasis. While PAP suppresses ROS in photosynthetic tissue, PAP instead induces guard cell ROS in chloroplasts and extracellular space to induce stomatal closure. We decipher how PAP-induced proteins activate both extracellular ROS production and anion channels for stomatal closure, thus providing a mechanism by which chloroplasts provide a strategic complement to canonical hormonal pathways in regulating plant physiological responses in specialized cells.

plant biology↗

bioRxiv2021

Coordinated Action of Multiple Transporters in the Acquisition of Essential Cationic Amino Acids by the Intracellular Parasite Toxoplasma gondii

Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is trans-stimulated by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites. Author summaryThe causative agent of toxoplasmosis, Toxoplasma gondii, is a versatile intracellular parasite that can proliferate within nucleated cells of virtually all warm-blooded organisms. In order to survive, T. gondii must scavenge the cationic amino acids lysine and arginine from their hosts. In a previous study, we demonstrated that a plasma membrane-localized protein called TgApiAT1 facilitates the uptake of arginine into the parasite. We found that parasites lacking TgApiAT1 could proliferate when cultured in medium containing high concentrations of arginine, suggesting the existence of an additional uptake pathway for arginine. In the present study, we demonstrate that this second uptake pathway is mediated by TgApiAT6-1, a protein belonging to the same solute transporter family as TgApiAT1. We show that TgApiAT6-1 is the major lysine transporter of the parasite, and that it is critical for parasite proliferation. Furthermore, we demonstrate that TgApiAT6-1 can transport arginine into parasites in conditions where arginine concentrations are high and lysine concentrations are comparatively lower. These data support a model for the finely-tuned acquisition of essential cationic amino acids that involves multiple transporters, and which likely contributes to these parasites being able to survive and proliferate within a wide variety of host cell types.

microbiology↗

bioRxiv2018

Characterization of the apicomplexan amino acid transporter (ApiAT) family in Toxoplasma gondii

Apicomplexan parasites are auxotrophic for a range of amino acids which must be salvaged from their host cells, either through direct uptake or degradation of host proteins. Here, we describe a family of plasma membrane-localized amino acid transporters, termed the Apicomplexan Amino acid Transporters (ApiATs), that are ubiquitous in apicomplexan parasites. Functional characterization of the ApiATs of Toxoplasma gondii indicate that several of these transporters are important for intracellular growth of the tachyzoite stage of the parasite, which is responsible for acute infections. We demonstrate that the ApiAT protein TgApiAT5-3 is an exchanger for aromatic and large neutral amino acids, with particular importance for L-tyrosine scavenging and amino acid homeostasis, and that TgApiAT5-3 is critical for parasite virulence. Our data indicate that T. gondii expresses additional proteins involved in the uptake of aromatic amino acids, and we present a model for the uptake and homeostasis of these amino acids. Our findings identify a family of amino acid transporters in apicomplexans, and highlight the importance of amino acid scavenging for the biology of this important phylum of intracellular parasites.\n\nAuthor SummaryThe Apicomplexa comprise a large number of parasitic protozoa that have obligate intracellular lifestyles and cause significant human and animal diseases, including malaria, cryptosporidiosis, toxoplasmosis, coccidiosis in poultry, and various cattle fevers. Apicomplexans must scavenge essential nutrients from their hosts in order to proliferate and cause disease, including a range of amino acids. The direct uptake of these nutrients is presumed to be mediated by transporter proteins located in the plasma membrane of intracellular stages, although the identities of these proteins are poorly defined. Using a combination of bioinformatic, genetic, cell biological, and physiological approaches, we have characterized a family of plasma membrane-localized transporter proteins that we have called the Apicomplexan Amino acid Transporters (ApiATs). The family is found in apicomplexans and their closest free-living relatives. We show that TgApiAT5-3, a member of the family in the apicomplexan Toxoplasma gondii, is an exchanger for aromatic and large neutral amino acids. In particular, it is critical for uptake of tyrosine, and for parasite virulence in a mouse infection model. We conclude that ApiATs are a family of plasma membrane transporters that play crucial roles in amino acid scavenging by apicomplexan parasites.

microbiology↗