No abstract

Intracellular parasites, such as the apicomplexan Toxoplasma gondii, are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection. TgApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in T. gondii. Here, we show that the abundance of TgApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite’s external environment, increasing in response to decreased [Arg]. Using a luciferase-based ‘biosensor’ strain of T. gondii, we demonstrate that the expression of TgApiAT1 varies between different organs within the host, indicating that parasites are able to modulate TgApiAT1-dependent uptake of Arg as they encounter different nutrient environments in vivo. Finally, we show that Arg-dependent regulation of TgApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in the TgApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient.

14 Intracellular parasites, such as the apicomplexan Toxoplasma gondii, are adept at 15 scavenging nutrients from their host. However, there is little understanding of how 16 parasites sense and respond to the changing nutrient environments they encounter during 17 an infection. TgApiAT1, a member of the apicomplexan ApiAT family of amino acid 18 transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in 19 T. gondii. Here, we show that the abundance of TgApiAT1, and hence the rate of uptake 20 of Arg, is regulated by the availability of Arg in the parasite's external environment, 21 increasing in response to decreased [Arg]. Using a luciferase-based 'biosensor' strain of 22 T. gondii, we demonstrate that parasites vary the expression of TgApiAT1 in different 23 organs within their host, indicating that parasites are able to modulate TgApiAT1-24 dependent uptake of Arg as they encounter different nutrient environments in vivo. 25 Finally, we show that Arg-dependent regulation of TgApiAT1 expression is post-26 transcriptional, mediated by an upstream open reading frame (uORF) in the TgApiAT1 27transcript, and we provide evidence that the peptide encoded by this uORF is critical for 28 mediating regulation. Together, our data reveal the mechanism by which an 29 apicomplexan parasite responds to changes in the availability of a key nutrient. 30 31The uptake of nutrients by T. gondii parasites is mediated primarily by plasma membrane 56 transporters [13]. We recently characterised a family of plasma membrane amino acid 57 transporters that are found throughout apicomplexans and have termed these the Apicomplexan 58Amino acid Transporter (ApiAT) family [14]. We have demonstrated that one member of this 59 family, TgApiAT1, is an Arg transporter that is essential for T. gondii virulence [15]. 60In this study, we have investigated the ability of T. gondii parasites to sense and respond to the 61Arg levels that they encounter in their host. We report Arg-dependent regulation of TgApiAT1 62 expression, and demonstrate that this process is mediated by an upstream open reading frame 63 (uORF) in the TgApiAT1 transcript. We also present evidence, obtained using a luciferase-64 based 'biosensor' strain of T. gondii, that parasites vary the expression of TgApiAT1 in 65 different organs within their host. Our data demonstrate how T. gondii parasites are able to 66 sense and respond to changes in the abundance of a key nutrient, as well as illustrating their 67 ability to do so within the course of an infection. 68 69 RESULTS 70 Regulation of TgApiAT1 protein abundance and parasite arginine uptake. 71 To investigate whether the abundance of TgApiAT1 is dependent upon Arg availability, we 72 introduced a haemagglutinin (HA3) epitope tag into the open reading frame of the TgApiAT1 73 genomic locus. The resultant TgApiAT1-HA3-expressing parasites were grown in modified 74 Roswell Park Memorial Institute 1640 (RPMI) medium in which [Arg] ranged from 10 µM to 75 5 mM. Western blotting revealed that the a...

Background: The medical profession has been slow in embracing email as a means of improving communication with patients.Aims: To explore the attitudes, practices and experiences of senior medical specialists towards email communication with their patients.Methods: A cross-sectional qualitative interview of 30 medical staff specialists employed by Canberra Health Services, administered through one-on-one interviews conducted between June and August 2020 (several months into the COVID-19 pandemic). Main outcome measures are the comments extracted from the interviews that were categorised into five domains: (i) integration of email use with patients; (ii) issues addressed through email; (iii) benefits of email communication; (iv) concerns and barriers to email communication; and (v) practice pointers for email use with patients.Results: Regular email correspondence with patients was not widespread. The main benefits identified were improved efficiency and flexibility, especially in the context of managing chronic disease and patient follow up. Participants also identified barriers, including time commitments, privacy and confidentiality, patient expectations and potential for misuse. Most participants were hesitant to endorse email with patients in their practices, citing concerns over the utility and safety of the medium and lack of established protocols and recommendations for email usage.Conclusions: There is a want and need for comprehensive and accessible professional guidance on email use with patients. Our results indicate opportunities to inform good clinical practice in respect of doctor-patient relationships, clinical workloads and risk management. There is also a need for formal guidelines on emailing with patients. The COVID-19 pandemic has heightened the need for such guidelines.