When students' words hurt: 12 tips for helping faculty receive and respond constructively to student evaluations of teaching.

Student evaluations of curricular experiences and instructors are employed by institutions to obtain feedback and guide improvement. However, to be effective, evaluations must prompt faculty action. Unfortunately, evaluative comments that engender strong reactions may undermine the process by hindering innovation and improvement steps. The literature suggests that faculty interpret evaluation feedback as a judgment not just on their teaching ability but on their personal and professional identity. In this context, critical evaluations, even when constructively worded, can result in disappointment, hurt, and shame. The COVID pandemic has challenged institutions and faculty to repeatedly adapt curricula and educational practices, heightening concerns for faculty burnout. In this context, the risk of 'words that hurt' is higher than ever. This article offers guidance for faculty and institutions to support effective responses to critical feedback and ameliorate counterproductive effects of learner evaluations.

Novel use of an OSCE to assess medical students' responses to a request for a low value diagnostic imaging test: A mixed methods analysis.

OBJECTIVE: Evaluate medical students' communication skills with a standardized patient (SP) requesting a low value test and describe challenges students identify in addressing the request. METHODS: In this mixed-methods study, third-year students from two medical schools obtained a history, performed a physical examination, and counseled an SP presenting with uncomplicated low back pain who requests an MRI which is not indicated. SP raters evaluated student communication skills using a 14-item checklist. Post-encounter, students reported whether they ordered an MRI and challenges faced. RESULTS: Students who discussed practice guidelines and risks of unnecessary testing with the SP were less likely to order an MRI. Students cited several challenges in responding to the SP request including patient characteristics and circumstances, lack of knowledge about MRI indications and alternatives, and lack of communication skills to address the patient request. CONCLUSIONS: Most students did not order an MRI for uncomplicated LBP, but only a small number of students educated the patient about the evidence to avoid unnecessary imaging or the harm of unnecessary testing. PRACTICE IMPLICATIONS: Knowledge about unnecessary imaging in uncomplicated LBP may be insufficient to adhere to best practices and longitudinal training in challenging conversations is needed.

Perspectives on the current state of pre-clerkship clinical reasoning instruction in United States medical schools: a survey of clinical skills course directors.

OBJECTIVES: Clinical reasoning skills are essential for sound medical decision-making. Though many have suggested that clinical reasoning instruction should begin in pre-clerkship curricula, neither pre-clerkship clinical skills director perspectives nor extent of instruction is known. This survey study serves as part of a needs assessment for United States medical school pre-clerkship clinical reasoning curricula. METHODS: United States medical school pre-clerkship clinical skills course directors were surveyed about perceived importance of formal instruction on clinical reasoning concepts, inclusion of these concepts in the curricula, barriers to instruction, and familiarity with clerkship curricula. Results were analyzed using descriptive and analytic statistics. Narrative comments were analyzed qualitatively for themes. RESULTS: Of 148 directors surveyed, 102 (69%) participated and 89 (60%) completed all closed-ended items. Each clinical reasoning concept was identified as somewhat to extremely important to include in pre-clerkship curricula by 90-99% of respondents. Pre-clerkship curricula included variable degrees of formal instruction for concepts, though most respondents rated their inclusion as moderate or extensive. Perceived importance of teaching most concepts moderately correlated with the degree of inclusion in the curriculum (Spearman's rho 0.39-0.44). Curricular time constraints and lack of faculty with skills to teach these concepts were the most frequently cited barriers to instruction. Respondents indicated being somewhat 57% (n=54) to extremely 29% (n=27) familiar with clerkship curricula at their institutions. CONCLUSIONS: This study is the first to examine pre-clerkship clinical skills course director perspectives about clinical reasoning instruction and extent of its inclusion in their curricula.

Implementation of a Hypothesis-Driven Physical Exam Session in a Transition to Clerkship Program.

Introduction: The head-to-toe approach to teaching the physical examination (PE) focuses on technique and performing a comprehensive PE whereas core + clusters and hypothesis-driven PE (HDPE) approaches integrate clinical reasoning into performing a focused PE. These approaches can be implemented in a developmental sequence. We report the implementation and evaluation of an HDPE educational session. Methods: We designed a 3-hour HDPE session as part of a transition to clerkship program. For each of five clinical vignettes, rising third-year students worked in pairs and then in small groups to generate a differential diagnosis and determine relevant PE maneuvers. Students next performed these maneuvers on peers with facilitator observation and feedback. Students completed postsession surveys on their retrospective pre- and postsession knowledge and confidence, as well as their satisfaction with the session. We completed quantitative and qualitative analyses on survey data. Results: One hundred ninety-two students participated, and 140 (73%) completed the survey. Students were significantly more likely to report feeling confident generating a differential diagnosis and using it to select PE maneuvers for common complaints postsession. Over 80% of respondents felt the session improved critical thinking about patient presentations and would help them in clerkships. Discussion: Our session increased student confidence in the progression to performing an HDPE just prior to the start of clerkships. The session is feasible and straightforward to implement. It requires a large number of faculty to facilitate, but the breadth of cases used allows inclusion of faculty from all fields.

Assessing Student Competencies in Antibiotic Stewardship and Patient Counseling.

BACKGROUND AND OBJECTIVES: Antibiotic misuse contributes to antibiotic resistance and is a growing public health threat in the United States and globally. Professional medical societies promote antibiotic stewardship education for medical students, ideally before inappropriate practice habits form. To our knowledge, no tools exist to assess medical student competency in antibiotic stewardship and the communication skills necessary to engage patients in this endeavor. The aim of this study was to develop a novel instrument to measure medical students' communication skills and competency in antibiotic stewardship and patient counseling. METHODS: We created and pilot tested a novel instrument to assess student competencies in contextual knowledge and communication skills about antibiotic stewardship with standardized patients (SP). Students from two institutions (N=178; Albert Einstein College of Medicine and Warren Alpert Medical School of Brown University) participated in an observed, structured clinical encounter during which SPs trained in the use of the instrument assessed student performance using the novel instrument. RESULTS: In ranking examinee instrument scores, Cronbach α was 0.64 (95% CI: 0.53 to 0.74) at Einstein and 0.71 (95% CI: 0.60 to 0.79) at Brown, both within a commonly accepted range for estimating reliability. Global ratings and instrument scores were positively correlated (r=0.52, F [3, 174]=30.71, P<.001), providing evidence of concurrent validity. CONCLUSIONS: Similar results at both schools supported external validity. The instrument performed reliably at both institutions under different examination conditions, providing evidence for the validity and utility of this instrument in assessing medical students' skills related to antibiotic stewardship.

Leadership Observation and Feedback Tool: A Novel Instrument for Assessment of Clinical Leadership Skills.

BACKGROUND: While leadership training is increasingly incorporated into residency education, existing assessment tools to provide feedback on leadership skills are only applicable in limited contexts. OBJECTIVE: We developed an instrument, the Leadership Observation and Feedback Tool (LOFT), for assessing clinical leadership. METHODS: We used an iterative process to develop the tool, beginning with adapting the Leadership Practices Inventory to create an open-ended survey for identification of clinical leadership behaviors. We presented these to leadership experts who defined essential behaviors through a modified Delphi approach. In May 2014 we tested the resulting 29-item tool among residents in the internal medicine and pediatrics departments at 2 academic medical centers. We analyzed instrument performance using Cronbach's alpha, interrater reliability using intraclass correlation coefficients (ICCs), and item performance using linear-by-linear test comparisons of responses by postgraduate year, site, and specialty. RESULTS: A total of 377 (of 526, 72%) team members completed the LOFT for 95 (of 519, 18%) residents. Overall ratings were high-only 14% scored at the novice level. Cronbach's alpha was 0.79, and the ICC ranged from 0.20 to 0.79. Linear-by-linear test comparisons revealed significant differences between postgraduate year groups for some items, but no significant differences by site or specialty. Acceptability and usefulness ratings by respondents were high. CONCLUSIONS: Despite a rigorous approach to instrument design, we were unable to collect convincing validity evidence for our instrument. The tool may still have some usefulness for providing formative feedback to residents on their clinical leadership skills.

Using a Curricular Vision to Define Entrustable Professional Activities for Medical Student Assessment.

BACKGROUND: The new UCSF Bridges Curriculum aims to prepare students to succeed in today's health care system while simultaneously improving it. Curriculum redesign requires assessment strategies that ensure that graduates achieve competence in enduring and emerging skills for clinical practice. AIM: To design entrustable professional activities (EPAs) for assessment in a new curriculum and gather evidence of content validity. SETTING: University of California, San Francisco, School of Medicine. PARTICIPANTS: Nineteen medical educators participated; 14 completed both rounds of a Delphi survey. PROGRAM DESCRIPTION: Authors describe 5 steps for defining EPAs that encompass a curricular vision including refining the vision, defining draft EPAs, developing EPAs and assessment strategies, defining competencies and milestones, and mapping milestones to EPAs. A Q-sort activity and Delphi survey involving local medical educators created consensus and prioritization for milestones for each EPA. PROGRAM EVALUATION: For 4 EPAs, most milestones had content validity indices (CVIs) of at least 78 %. For 2 EPAs, 2 to 4 milestones did not achieve CVIs of 78 %. DISCUSSION: We demonstrate a stepwise procedure for developing EPAs that capture essential physician work activities defined by a curricular vision. Structured procedures for soliciting faculty feedback and mapping milestones to EPAs provide content validity.

How clinical supervisors develop trust in their trainees: a qualitative study.

CONTEXT: Clinical supervisors oversee trainees' performance while granting them increasing opportunities to work independently. Although the factors contributing to supervisors' trust in their trainees to conduct clinical work have been identified, how the development of trust is shaped by these factors remains less clear. OBJECTIVES: This study was designed to determine how supervisors develop and experience trust in resident (postgraduate years 2 and 3) trainees in the clinical workplace. METHODS: Internal medicine in-patient supervisors at two institutions were interviewed about the meaning and experience of developing trust in resident trainees. Transcribed data were coded and analysed using a phenomenographic approach. RESULTS: Forty-three supervisors participated. Supervisors characterised the meaning of trust from the perspectives of trainee competence and leadership or from their own perspective of needing to provide more or less supervision. Supervisors initially considered trust to be usually independent of prior knowledge of the resident, and then used sources of information about trust to develop their judgements of trust. Sources, which incorporated inference, included supervisors' comparisons with a standard, direct observation of the trainee as a team leader or care provider, and stakeholder input from team members, patients and families. Barriers against and accelerators to trust formation related to the resident, supervisor, resident-supervisor relationship, context and task. Trust formation had implications for supervisors' roles, residents' increasingly independent provision of care, and team functioning. CONCLUSIONS: From a general starting point, supervisors develop trust in residents informed by observation, inference and information gathered from the team and patients. Judgements of trust yield outcomes defined by supervisors' changing roles, the increasingly independent provision of care by residents, and team functioning. The implications of these findings for graded resident autonomy aligned with learning needs can inform the design of training environments to enable readiness for unsupervised practice.

Assessing 3rd year medical students' interprofessional collaborative practice behaviors during a standardized patient encounter: A multi-institutional, cross-sectional study.

BACKGROUND: To understand how third-year medical student interprofessional collaborative practice (IPCP) is affected by self-efficacy and interprofessional experiences (extracurricular experiences and formal curricula). METHODS: The authors measured learner IPCP using an objective structured clinical examination (OSCE) with a standardized nurse (SN) and standardized patient (SP) during a statewide clinical performance examination. At four California medical schools from April to August 2012, SPs and SNs rated learner IPCP (10 items, range 0-100) and patient-centered communication (10 items, range 0-100). Post-OSCE, students reported their interprofessional self-efficacy (16 items, 2 factors, range 1-10) and prior extracurricular interprofessional experiences (3 items). School representatives shared their interprofessional curricula during guided interviews. RESULTS: Four hundred sixty-four of 530 eligible medical students (88%) participated. Mean IPCP performance was 79.6 ± 14.1 and mean self-efficacy scores were 7.9 (interprofessional teamwork) and 7.1 (interprofessional feedback and evaluation). Seventy percent of students reported prior extracurricular interprofessional experiences; all schools offered formal interprofessional curricula. IPCP was associated with self-efficacy for interprofessional teamwork (ß = 1.6, 95% CI [0.1, 3.1], p = 0.04) and patient-centered communication (ß = 12.5, 95% CI [2.7, 22.3], p = 0.01). CONCLUSIONS: Medical student IPCP performance was associated with self-efficacy for interprofessional teamwork and patient-centered communication. Increasing interprofessional opportunities that influence medical students' self-efficacy may increase engagement in IPCP.

Cross-resistance to nitro drugs and implications for treatment of human African trypanosomiasis.

The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (approximately 27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

Comparative structural, kinetic and inhibitor studies of Trypanosoma brucei trypanothione reductase with T. cruzi.

As part of a drug discovery programme to discover new treatments for human African trypanosomiasis, recombinant trypanothione reductase from Trypanosoma brucei has been expressed, purified and characterized. The crystal structure was solved by molecular replacement to a resolution of 2.3A and found to be nearly identical to the T. cruzi enzyme (root mean square deviation 0.6A over 482 Calpha atoms). Kinetically, the K(m) for trypanothione disulphide for the T. brucei enzyme was 4.4-fold lower than for T. cruzi measured by either direct (NADPH oxidation) or DTNB-coupled assay. The K(m) for NADPH for the T. brucei enzyme was found to be 0.77microM using an NADPH-regenerating system coupled to reduction of DTNB. Both enzymes were assayed for inhibition at their respective S=K(m) values for trypanothione disulphide using a range of chemotypes, including CNS-active drugs such as clomipramine, trifluoperazine, thioridazine and citalopram. The relative IC(50) values for the two enzymes were found to vary by no more than 3-fold. Thus trypanothione reductases from these species are highly similar in all aspects, indicating that they may be used interchangeably for structure-based inhibitor design and high-throughput screening.

Chemical validation of trypanothione synthetase: a potential drug target for human trypanosomiasis.

In the search for new therapeutics for the treatment of human African trypanosomiasis, many potential drug targets in Trypanosoma brucei have been validated by genetic means, but very few have been chemically validated. Trypanothione synthetase (TryS; EC 6.3.1.9; spermidine/glutathionylspermidine:glutathione ligase (ADP-forming)) is one such target. To identify novel inhibitors of T. brucei TryS, we developed an in vitro enzyme assay, which was amenable to high throughput screening. The subsequent screen of a diverse compound library resulted in the identification of three novel series of TryS inhibitors. Further chemical exploration resulted in leads with nanomolar potency, which displayed mixed, uncompetitive, and allosteric-type inhibition with respect to spermidine, ATP, and glutathione, respectively. Representatives of all three series inhibited growth of bloodstream T. brucei in vitro. Exposure to one of our lead compounds (DDD86243; 2 x EC(50) for 72 h) decreased intracellular trypanothione levels to <10% of wild type. In addition, there was a corresponding 5-fold increase in the precursor metabolite, glutathione, providing strong evidence that DDD86243 was acting on target to inhibit TryS. This was confirmed with wild-type, TryS single knock-out, and TryS-overexpressing cell lines showing expected changes in potency to DDD86243. Taken together, these data provide initial chemical validation of TryS as a drug target in T. brucei.

Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods.

The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine). This unique dithiol plays a pivotal role in thiol-redox homeostasis and in defence against chemical and oxidative stress in trypanosomatids. A tetracycline-dependent conditional double knockout of TRYS (cDKO) was generated in bloodstream Trypanosoma brucei. Culture of cDKO parasites without tetracycline induction resulted in loss of trypanothione and accumulation of glutathione, followed by growth inhibition and cell lysis after 6 days. In the absence of inducer, cDKO cells were unable to infect mice, confirming that this enzyme is essential for virulence in vivo as well as in vitro. To establish whether both enzymatic functions were essential, an amidase-dead mutant cDKO line was generated. In the presence of inducer, this line showed decreased growth in vitro and decreased virulence in vivo, indicating that the amidase function is not absolutely required for viability. The druggability of TRYS was assessed using a potent small molecule inhibitor developed in our laboratory. Growth inhibition correlated in rank order cDKO, single KO, wild-type and overexpressing lines and produced the predicted biochemical phenotype. The synthetase function of TRYS is thus unequivocally validated as a drug target by both chemical and genetic methods.

ATP-dependent ligases in trypanothione biosynthesis--kinetics of catalysis and inhibition by phosphinic acid pseudopeptides.

Glutathionylspermidine is an intermediate formed in the biosynthesis of trypanothione, an essential metabolite in defence against chemical and oxidative stress in the Kinetoplastida. The kinetic mechanism for glutathionylspermidine synthetase (EC 6.3.1.8) from Crithidia fasciculata (CfGspS) obeys a rapid equilibrium random ter-ter model with kinetic constants K(GSH) = 609 microM, K(Spd) = 157 microM and K(ATP) = 215 microM. Phosphonate and phosphinate analogues of glutathionylspermidine, previously shown to be potent inhibitors of GspS from Escherichia coli, are equally potent against CfGspS. The tetrahedral phosphonate acts as a simple ground state analogue of glutathione (GSH) (K(i) approximately 156 microM), whereas the phosphinate behaves as a stable mimic of the postulated unstable tetrahedral intermediate. Kinetic studies showed that the phosphinate behaves as a slow-binding bisubstrate inhibitor [competitive with respect to GSH and spermidine (Spd)] with rate constants k(3) (on rate) = 6.98 x 10(4) M(-1) x s(-1) and k(4) (off rate) = 1.3 x 10(-3) s(-1), providing a dissociation constant K(i) = 18.6 nM. The phosphinate analogue also inhibited recombinant trypanothione synthetase (EC 6.3.1.9) from C. fasciculata, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei with K(i)(app) values 20-40-fold greater than that of CfGspS. This phosphinate analogue remains the most potent enzyme inhibitor identified to date, and represents a good starting point for drug discovery for trypanosomiasis and leishmaniasis.

Leishmania trypanothione synthetase-amidase structure reveals a basis for regulation of conflicting synthetic and hydrolytic activities.

The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.

Phenotypic analysis of trypanothione synthetase knockdown in the African trypanosome.

Trypanothione plays a pivotal role in defence against chemical and oxidant stress, thiol redox homoeostasis, ribonucleotide metabolism and drug resistance in parasitic kinetoplastids. In Trypanosoma brucei, trypanothione is synthesized from glutathione and spermidine by a single enzyme, TryS (trypanothione synthetase), with glutathionylspermidine as an intermediate. To examine the physiological roles of trypanothione, tetracycline-inducible RNA interference was used to reduce expression of TRYS. Following induction, TryS protein was reduced >10-fold and growth rate was reduced 2-fold, with concurrent 5-10-fold decreases in glutathionylspermidine and trypanothione and an up to 14-fold increase in free glutathione content. Polyamine levels were not significantly different from non-induced controls, and neither was the intracellular thiol redox potential, indicating that these factors are not responsible for the growth defect. Compensatory changes in other pathway enzymes were associated with prolonged suppression of TryS: an increase in trypanothione reductase and gamma-glutamylcysteine synthetase, and a transient decrease in ornithine decarboxylase. Depleted trypanothione levels were associated with increases in sensitivity to arsenical, antimonial and nitro drugs, implicating trypanothione metabolism in their mode of action. Escape mutants arose after 2 weeks of induction, with all parameters, including growth, returning to normal. Selective inhibitors of TryS are required to fully validate this novel drug target.

Trypanothione biosynthesis in Leishmania major.

Trypanothione plays a crucial role in regulation of intracellular thiol redox balance and in defence against chemical and oxidant stress. Crithidia fasciculata requires two enzymes for the formation of trypanothione, namely glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and a glutathionylspermidine-dependent trypanothione synthetase (TryS; EC 6.3.1.9), whereas Trypanosoma cruzi and Trypanosoma brucei use a broad-specificity trypanothione synthetase to make trypanothione from glutathione (GSH) and spermidine. Here, we report the identification of two genes in Leishmania major with similarity to previously identified GSPS and TRYS. GSPS is an apparent pseudogene containing two frame shift mutations and two stop codons, whereas TRYS is in a single open-reading frame. The enzyme encoded by TRYS was expressed and found to catalyse formation of trypanothione with GSH and either spermidine or glutathionylspermidine. When GSH is varied as substrate the enzyme displays substrate inhibition (apparent Km=89 microM, Ki(s)=1mM, k(cat)=2s-1). At a fixed GSH concentration, the enzyme obeys simple hyperbolic kinetics with the other substrates with apparent Km values for spermidine, glutathionylspermidine and MgATP of 940, 40 and 63 microM, respectively. Immunofluorescence and sub-cellular fractionation studies indicate that TryS localises to the cytosol of L. major promastigotes. Phylogenetic analysis of the GspS and TryS amino acid sequences suggest that in the trypanosomatids, TryS has evolved to replace the GspS/TryS complex in C. fasciculata. It also appears that the L. major still harbours a redundant GSPS pseudogene that may be currently in the process of being lost from its genome.

Properties of trypanothione synthetase from Trypanosoma brucei.

Trypanothione [N(1),N(8)-bis(glutathionyl)spermidine] plays a central role in defence against oxidant damage, ribonucleotide metabolism and in resistance to certain drugs in trypanosomatids. In Crithidia fasciculata, synthesis of trypanothione involves sequential conjugation of two molecules of glutathione (GSH) to spermidine by two enzymes: glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and trypanothione synthetase (TryS; EC 6.3.1.9), whereas in Trypanosoma cruzi both steps are catalysed by an unusual TryS with broad substrate specificity. To determine which route operates in T. brucei, we have cloned and expressed a single copy gene with similarity to C. fasciculata and T. cruzi TRYS. The purified recombinant protein catalyses formation of trypanothione from either spermidine and GSH, or glutathionylspermidine and GSH. The enzyme displays high substrate inhibition with GSH as variable substrate (apparent K(m)=56 microM, K(i)(s)=37 microM, k(cat)=2.9s(-1)). At a fixed subsaturating GSH concentration (100 microM), the enzyme obeys simple hyperbolic kinetics yielding apparent K(m) values for spermidine, glutathionylspermidine and MgATP of 38, 2.4, and 7.1 microM, respectively. Recombinant TryS can also catalyse conversion of spermine to glutathionylspermine and bis(glutathionyl)spermine, as recently reported for T. cruzi. The enzyme has amidase activity that can be inhibited by iodoacetamide. Studies using GSH and polyamine analogues identified GSH as the critical determinant for recognition by the amidase domain. Thus, the biosynthesis and degradation of trypanothione are similar in African and American trypanosomes, and different from the insect trypanosomatid, C. fasciculata.

Bis(glutathionyl)spermine and other novel trypanothione analogues in Trypanosoma cruzi.

Trypanosomatids differ from other cells in their ability to conjugate glutathione with the polyamine spermidine to form the antioxidant metabolite trypanothione (N1,N8-bis(glutathionyl)spermidine). In Trypanosoma cruzi, trypanothione is synthesized by an unusual trypanothione synthetase/amidase (TcTryS) that forms both glutathionylspermidine and trypanothione. Because T. cruzi is unable to synthesize putrescine and is dependent on uptake of exogenous polyamines by high affinity transporters, synthesis of trypanothione may be circumstantially limited by lack of spermidine. Here, we show that the parasite is able to circumvent the potential shortage of spermidine by conjugating glutathione with other physiological polyamine substrates from exogenous sources (spermine, N8-acetylspermidine, and N-acetylspermine). Novel thiols were purified from epimastigotes, and structures were determined by matrix-assisted laser desorption ionization time-of-flight analysis to be N1,N12-bis(glutathionyl)spermine, N1-glutathionyl-N8-acetylspermidine, and N1-glutathionyl-N12-acetylspermine, respectively. Structures were confirmed by enzymatic synthesis with recombinant TcTryS, which catalyzes formation of these compounds with kinetic parameters equivalent to or better than those of spermidine. Despite containing similar amounts of spermine and spermidine, the epimastigotes, trypomastigotes, and amastigotes of T. cruzi preferentially synthesized trypanothione. Bis(glutathionyl)spermine disulfide is a physiological substrate of recombinant trypanothione reductase, comparable to trypanothione and homotrypanothione disulfides. The broad substrate specificity of TcTryS could be exploited in the design of polyamine-based inhibitors of trypanothione metabolism.