Hafedh Fessi

Philippe Nicoud

Tomas Serrato

Olivia Gilbert

Cécile Courivaud

Salima Daoud

Michel Thomas

Bernard Canaud

Jean-Paul Cristol
email: jp-cristol@chu-montpellier.fr

Two Years' Experience of Intensive Home Hemodialysis with he Physidia S3 System: Results from the RECAP study

Keywords: intensified hemodialysis, slow aily dialysis, dialysis efficiency, adequacy, dialysis quantification

The RECAP study reports results and outcomes (clinical performances, patient acceptance, cardiac outcomes, and technical survival) achieved with the S3 system used as an intensive home hemodialysis (HHD) platform over a three-year French multicenter study. Ninety-four dialysis patients issued from ten dialysis centers and treated more than 6 months (mean follow-up: 24 months) with S3 were included. A two-hour treatment time was maintained in 2/3 of patients to deliver 25 L of dialysis fluid, while 1/3 required up to 3 h to achieve 30 L. The additional convection volume produced by means of the SeCoHD tool (internal filtration backfiltration) was 3 L/session, and the net ultrafiltration produced to achieve dry weight was 1.4 L/session. On a weekly basis, an average 156 L of dialysate corresponding to 94 L of urea clearance when considering 85% dialysate saturation under low flow conditions was delivered. Such urea clearance was equivalent to 9.2 [8.0-13.0] mL/min weekly urea clearance and a standardized Kt/V of 2.5 [1.1-4.5]. The predialysis concentration of selected uremic markers remained remarkably stable over time. Fluid volume status and blood pressure were adequately controlled by means of a relatively low ultrafiltration rate (7.9 mL/h/kg). Technical survival on S3 was 72% and 58% at 1 and 2 years, respectively. The S3 system was easily handled and kept by patients at home, as indicated by technical survival. Patient perception was improved, while treatment burden was reduced. Cardiac features (assessed in a subset of patients) tended to improve over time. Intensive hemodialysis relying on the S3 system offers a very appealing option for home treatment with quite satisfactory results, as shown in the RECAP study throughout a two-year follow-up time, and offers the best bridging solution to kidney transplantation.

Introduction

Intensive home hemodialysis (IHHD) [START_REF] Marshall | Intensive hemodialysis-keeping the faith[END_REF] is recognized as delivering a more physiological and efficient renal replacement therapy in chronic kidney disease patients with better outcomes [START_REF] Ikizler | Intensive hemodialysis: Back to the beginning?[END_REF][START_REF] Nesrallah | Intensive hemodialysis associates with improved survival compared with conventional hemodialysis[END_REF][START_REF] Tennankore | Intensive home haemodialysis: Benefits and barriers[END_REF][START_REF] Nishio-Lucar | Intensive Home Hemodialysis Survival Comparable to Deceased Donor Kidney Transplantation[END_REF]. IHHD remains underutilized as compared to in-center hemodialysis for various reasons, which include technical complexity, fears of home and selfcare treatment, and poor perception about this modality. Over the last decade, technical advances in manufacturing dialysis machines have considerably reduced technical complexity, facilitated home implementation, and improved patient perception toward this modality.

The NxStage system has paved the way and opened a new therapeutic avenue in the US and then abroad by confirming its safety and attractiveness and then by showing clinical benefits [START_REF] Clark | The NxStage System One[END_REF][START_REF] Floramo | Use of the NxStage system one for daily home hemodialysis[END_REF][START_REF] Takahashi | Future home hemodialysis-Advantages of the NxStage System One[END_REF]. Such progresses have been made possible by the development of dedicated home HD machines featuring more friendly user interfaces and web-based connection, facilitating handling, suppressing needs for water treatment systems via simplified dialysate delivery systems, and finally providing a plug and play approach for HD technology [START_REF] Harasemiw | Human factors testing of the Quanta SC+ hemodialysis system: An innovative system for home and clinic use[END_REF][START_REF] Komenda | Hemodialysis With the Quanta SC+: Efficacy and Safety of a Self-care Hemodialysis Machine[END_REF][START_REF] Haroon | Haemodialysis at home: Review of current dialysis machines[END_REF][START_REF] Plumb | Safety and efficacy of the Tablo hemodialysis system for in-center and home hemodialysis[END_REF]. The Physidia S3 system was developed in France few years later with the aim of further reducing the burden of home HD treatment both for patients and care providers by keeping the concept of more intensive dialysis relying on a low flow and more frequent approach [START_REF] Fessi | Safety and Efficacy of Short Daily Hemodialysis with Physidia S(3) System: Clinical Performance Assessment during the Training Period[END_REF].

In this study, we aimed to report results and outcomes (clinical performances, patient acceptance, cardiac outcomes, and technical survival) achieved with the S3 system used as an intensive home HD platform over a three-year French multicentric study.

Materials and Methods

Description of the Physidia S3 System

The Physidia S3 system (named the S 3 system) is a bagged delivery cycler and portable communicating dialysis monitoring device specifically designed for short daily hemodialysis (HHD) and self-administered treatment [START_REF] Fessi | Safety and Efficacy of Short Daily Hemodialysis with Physidia S(3) System: Clinical Performance Assessment during the Training Period[END_REF]. The S3 device has a compact and portable cubic design (dimension 40 x40 x 40 cm), weighing less than 25 kg and being specifically designed for short daily low-dialysate-flow HHD (dialysate flow rates of 150 to 200 mL/min).

The dialysis fluid is stored in 5 L sterile bags. Dialysis fluid bags are stored in a plastic shelf with 5 to 7 racks. The balance chamber technology with an adjustable ultrafiltration rate is able to provide substantial additional convective volume to weight loss by enhancing internal filtration. The S 3 system is not bound to a specific hemodialyzer choice. The S 3 system has a cartridge setup, automated prime function, removable tablet with touchscreen patient interface, and the ability to transmit treatment data to a physician at the end of the dialysis session via a tablet connected by Bluetooth to the monitor. The S 3 system was approved and introduced on the European market (CE mark) in 2013.

Study Design

RECAP is a retrospective longitudinal multicenter study conducted in France. Ten French dialysis facilities, which are experts in home dialysis treatment, collaborated in this observational study to assess outcomes associated with IHHD using the Physidia S 3 system (Physidia SAS, Saint-Barthélémy-d'Anjou, France). IHHD refers in this case to a short (>/=2h) and more frequent weekly treatment schedule (>/=5 sessions/week). The treatment schedule prescription (treatment time, frequency, and dialysate volume per session and week) was defined by the referent nephrologist in each dialysis facility according to patient needs and tolerance. Patient monitoring and treatment efficacy were led by the referent nephrologist based usually on a quarterly outpatient clinic visit capturing patient key parameters including vital signs, laboratory tests, and operating dialysis conditions. Data captured during this visit were sent to the patient's electronic medical record, anonymized, and shared in a central data repository system.

The RECAP study complied with the General Data Protection Regulation (RGPD) and was conducted in agreement with French legislation on non-interventional studies (reference INDS: MR 3309311018).

Patients

Ten French dialysis and training dialysis centers were selected, in which eligible patients older than 18 years were screened. Ninety-four patients having started IHHD between 1 January 2015 and 30 June 2018 and treated more than 6 months and up to 42 months' followup (mean follow-up was 24 months) with the S 3 system were included in the analysis. All patients were provided an information sheet at least one month prior to the start of the study. Patients who expressed their opposition to data collection were not to start the study. Patients who expressed their opposition to data collection were not included in the study. A flow chart of the study population is presented in Figure 1.

Data Collection

Data were collected retrospectively on a quarterly basis from the first day of use at home with the S3 system until 30 September 2018, or until the last day of use of the S 3 system in the case of permanent discontinuation before the end of the study follow-up. Socio-demographic data, comorbidities, disabilities, status before start of IHHD, previous renal replacement therapy modalities, biological data, and dialysis prescriptions were captured from the electronic Case Report Form (eCRF). Blood pressure before, during, and after the dialysis session were extracted directly from the dialysis monitor session reports; anonymized data were recorded and centralized on a regular basis by Physidia company. The identification number of the monitor S 3 and the date of session were used to match the eCRF data with the S 3 monitor data.

Calculation and Statistical Analysis

Continuous data were expressed as mean values +/-standard deviation (SD), and medians with 25th and 75th percentiles or IQR were calculated for non-normally distributed data. Only dialysis session reports lasting 90 min or more were included in the analysis.

Vital parameters, including body weight, systolic and diastolic blood pressure (SBP and DBP), dialysis parameters, and biochemical tests were captured at baseline and for every 3month period until 24 months of follow-up (due to the small number of patients with a follow up of more than 24 months).

The dialysis dose delivered used as a surrogate marker of clinical performances was calculated. The urea reduction rate (URR) was used to assess the efficacy of the dialysis session over time. In addition, the standard weekly urea Kt/V and the weekly urea clearance equivalent were estimated from blood and dialysate flow using the equation of Michaels adapted by Leypoldt [START_REF] Michaels | Operating parameters and performance criteria for hemodialyzers and other membrane-separation devices[END_REF][START_REF] Leypoldt | Volume of urea cleared as a therapy dosing guide for more frequent hemodialysis[END_REF]. According to this equation, urea dialysate saturation (D/P) reaches 90% for dialysate and blood flows of 180 and 250 mL/min, respectively. Based on this assumption, one may estimate the weekly urea clearance as 0.9 of the total dialysate volume delivered per week. The weekly standard Kt/V was estimated as the ratio of weekly urea clearance and total body water. Total body water was estimated at 0.55 of dry weight for male and 0.50 for female patients.

For each parameter, a mixed linear model for repeated measures with a random intercept for each patient was used to assess the statistical significance of the evolution over time. The model was adjusted for time and gender as fixed effects and center as the random effect. Some biomarkers benefited from additional transformation. For iron markers, namely ferritin and transferrin coefficient saturation, the Napierian logarithm value was used in the model. For bone mineral disorder biomarkers, tertile classes were used to assess the evolution of parathyroid hormone (PTH) (low (<130 pg/mL), normal (130-585 pg/mL), and high (>585 pg/mL)) and phosphorus (low (<0.9 mmol/L), normal (0.9-1.4 mmol/L), and high (>1.4 mmol/L)).

An intradialytic hypotension episode was defined as any SBP drop < 90 mmHg measured during the HD session [START_REF] Sars | Intradialytic Hypotension: Mechanisms and Outcome[END_REF].

Technical survival on the S3 system at 1 and 2 years was estimated using the Kaplan-Meier method. The failure of the method was estimated from the cumulative incidence of returning in-center using a competitive risk method with kidney transplantation as the main competitive risk.

No replacement of the missing values was carried out.

All statistical analyses were performed with SAS software® version 9.4 and SAS/STAT 15.1 on Windows (SAS Institute, Cary, NC, USA).

Results

Patient's Characteristics

Ninety-four patients who fulfilled selection criteria were included in the final analysis. Patients' characteristics upon inclusion are displayed in Table 1. The mean age was 49.7 +/-14.9 years old, and 64% of patients were male. The mean predialysis body weight was 75 kg and 64 kg in males and females, respectively. The mean body mass index was 25.3 +/-5.1 kg/m2. While most patients had a normal body mass (54.2%), 31.3% of patients were overweight and 13.3% obese. The mean Charlson comorbidity score was 3.6+/-2.0 [range 1 -12]. Time spent on dialysis treatment before entering S 3 treatment was 12.2 months [3.2-47.0]. As shown, 79.8% of patients were treated in-center, and 11% were incident-naïve dialysis patients. Overall, 40.4% of patients had at least one kidney transplant, and the latest kidney transplantation was 12+/-7.4 years ago. Through the follow-up period, 27,826 dialysis sessions were extracted from the database and analyzed.

Renal Replacement Treatment Prescription and Operating Conditions

The main features of dialysis prescription and operating conditions are displayed in Table 2. The dialysis treatment time was 133 min [120-180] and 129 min [120-150] for female and male patients, respectively. The vascular access used was an arteriovenous fistula in 97.9% of patients. One patient used two vascular access types during the entire study. Access cannulation relied on the buttonhole method in 89.2% of patients. Two patients were treated by means of a tunneled central venous catheter. Training time to home installation was 6.9+/-3.9 weeks including punction training.

All patients received five sessions or more per week, while two-thirds of them received six sessions weekly, then being considered as a daily treatment (Table 3). A two-hour treatment time was delivered in two-thirds of patients, while one-third received up to 3 h. The dialysis fluid volume exchanged was 25 L per session in three-quarters of patients, while one-quarter received up to 30 L per session. The additional convection volume produced by means of the SeCoHD tool (internal filtration back filtration) was 3 L per session, and the net ultrafiltration produced to achieve dry weight was 1.4 L per session, which translated to a normalized ultrafiltration rate of 7.9 mL/h/kg [range 2 -10]. In brief, the RECAP study delivered, on a weekly basis, an average of 156 L of dialysis fluid corresponding to 94.2 L of urea clearance when considering that the dialysate saturation achieved was 85% in this low flow condition. This is equivalent to a weekly urea clearance of 9.17 +/-0.81 mL/min and a standardized weekly Kt/V of 2.52 +/-0.67.

Clinical Performances and Laboratory Data

Fluid volume management and blood pressure control are presented in Figures 2 and3. The dry weight decreased initially (first 6 months) by 0.5 kg, then slowly went back to initial values and remained relatively constant over the observation time (median value 71.5 kg). The pre-dialysis systolic blood pressure (SBP) was significantly higher in men than in women (p = 0.031) at baseline, remaining relatively stable over time with the same difference (p = 0.9134). Pre-dialysis diastolic blood pressure (DBP) did not differ according to gender at baseline (p = 0.3499), but interestingly it decreased during the first 6 months (p = 0.0071) and then stabilized until the end of follow-up (Figures 2 and3).

The urea reduction rate (URR) ranged from 45.8% to 57.4% throughout the follow-up with a P25 that ranged from 5.9% to 50% and P75 that ranged from 52% to 68.2%. The estimated weekly standard urea Kt/V was 2.40 [2.0-3.2]. The delivered urea clearance equivalent per week was 9.0 mL/min [START_REF] Takahashi | Future home hemodialysis-Advantages of the NxStage System One[END_REF][START_REF] Harasemiw | Human factors testing of the Quanta SC+ hemodialysis system: An innovative system for home and clinic use[END_REF][START_REF] Komenda | Hemodialysis With the Quanta SC+: Efficacy and Safety of a Self-care Hemodialysis Machine[END_REF] (Table 3).

Figure 2. Evolution of the predialysis systolic blood pressure (SBP) (mean ± SEM). Figure 3. Evolution of the predialysis diastolic blood pressure (DBP) (mean+/-SEM).

Plasma phosphate (Figure 4) and PTH concentrations remained relatively stable during the 24-month follow-up period, indicating that divalent ions and bone mineral disorders were adequately controlled (Table 4). The total serum calcium increased from baseline to the 9-12month period and then decreased until the end of the 24 months of follow-up, reaching statistical significance (p = 0.0497).

The plasma creatinine levels did not change over time, suggesting that creatinine clearance and muscle mass remained constant. Plasma 2-microglobulin levels were documented in a subset of patients (27 patients at baseline) and remained stable over time with a value </-25 mg/L (Table 4).

Figure 4. Evolution of plasma phosphate concentrations (median +/-IQR).

Anemia and iron status control were satisfactory. As show, the behavior of hemoglobin levels followed a two-phase change: hemoglobin decreased in the initial phase (11.38 +/-1.61 g/dL at baseline to 10.53 +/-1.52 g/dL at 3-6 months period), then hemoglobin increased back to reach 11.33 +/-1.54 g/dL at the end of the 24-month follow-up (Table 4). Ferritin levels followed the same behavior; they decreased initially (9-12 months) and then improved subsequently, reflecting IV iron supplementation. Finally, serum albumin concentrations did not change significantly, while they tended to increase over time (p = 0.037) (Figure 5).

Cardiac Morphologic Features

In a subset of 20 patients, cardiologic features were more precisely monitored via echocardiography during three months of follow-up. As shown in Table 5, the left ventricular mass reduced by 9.6% at 3 months (103 vs. 114 g/m2), while the ejection fraction remained in the normal range and did not change over time (62.0% vs. 61.5%).

Technique Survival

The probability of permanently continuing HHD with the S 3 monitor at 1 and 2 years was 72% and 58%, respectively. The probability of the continued use of HHD with the S 3 monitor during follow-up, estimated using the same method, is displayed in Figure 6.

The main reason for the permanent discontinuation of HHD with the S 3 monitor was kidney transplantation (n = 19) followed by transfer to in-center hemodialysis (n = 11).

The main causes of transfer to in-center HD were the loss of a caregiver (mandatory in France) and the patient's loss of autonomy, requiring treatment. Other more minor causes of discontinuation of treatment with the S 3 system were burnout, lack of patient compliance, or referral to another home dialysis technique.

Discussion

The RECAP is a two-year retrospective cohort study reporting the results and patient experience of 94 kidney disease patients who received IHHD (i.e., short daily, low flow) with the Physidia S 3 system. The RECAP study confirms the clinical benefits of IHHD and enlarges our knowledge by analyzing a relatively large dialysis population presenting with various clinical and metabolic profiles having used the same S 3 device with a similar protocol, reducing care variations and then providing homogeneous findings [START_REF] Nesrallah | Intensive hemodialysis associates with improved survival compared with conventional hemodialysis[END_REF][START_REF] Tennankore | Intensive home haemodialysis: Benefits and barriers[END_REF][START_REF] Nishio-Lucar | Intensive Home Hemodialysis Survival Comparable to Deceased Donor Kidney Transplantation[END_REF][START_REF] Collins | Intensive Hemodialysis: Time to Give the Therapy Greater Consideration[END_REF][START_REF] Weinhandl | Hospitalization, and Technique Failure in Daily Home Hemodialysis and Matched Peritoneal Dialysis Patients: A Matched Cohort Study[END_REF][START_REF] Schachter | Current state of intensive hemodialysis: A comparative review of benefits and barriers[END_REF].

Evolution of the Professional Situation

The evolution of the professional situation is displayed in Figure 7.

Figure 7. Evolution of the professional situation

Among the 11 unemployed patients at baseline, a professional activity had been resumed during the follow-up period by 3 patients. In total, 16 patients (8 men and 8 women) had a period of unemployment during follow-up, and 25% of these patients returned to work at least once during the study Intensive home hemodialysis stands, in this study, for more frequent (>/=5 sessions weekly), short sessions (120 min) and a low dialysis fluid volume (25 L) [START_REF] Schachter | Current state of intensive hemodialysis: A comparative review of benefits and barriers[END_REF]. As indicated, the treatment schedule was well-accepted and able to deliver an adequate renal replacement treatment at home in the vast majority of patients. Technical survival on the same treatment schedule using the S 3 system was 72% and 58%, at 1 and 2 years, respectively. Interestingly, the main reason for discontinuing home treatment with the S3 system was kidney transplantation in 21% of patients [START_REF] Nishio-Lucar | Intensive Home Hemodialysis Survival Comparable to Deceased Donor Kidney Transplantation[END_REF]. In this case, stopping home HD should be considered as a success in the management of a renal patient's trajectory, suggesting that a hemodialysis cycler specifically designed for home treatment on a plug and play concept offers the best bridging solution to kidney transplant [START_REF] Nishio-Lucar | Intensive Home Hemodialysis Survival Comparable to Deceased Donor Kidney Transplantation[END_REF]. Other reasons for stopping home treatment with the S 3 system and returning to in-center hemodialysis represent home treatment failure but not a technical failure.

A renal replacement treatment schedule was maintained and relatively unchanged over time.

All patients received five sessions or more per week, and among them twothirds received six sessions weekly, then being considered as a daily treatment. A two-hour treatment time was maintained in two-thirds of patients to deliver 25 L of dialysis fluid, while one-third required more up to 3 h to achieve a 30 L dialysis fluid exchange. The additional convection volume produced by means of the SeCoHD tool (internal filtration back filtration) was 3 L per session in addition to the net ultrafiltration produced to achieve dry weight, which was 1.4 L per session. In brief, the RECAP study delivered, on a weekly basis, an average 156 L of dialysate corresponding to 94 L of urea clearance when considering 85% dialysate saturation at low flow conditions. Such urea clearance is equivalent to 9. Fluid volume management and blood pressure control were achieved adequately. Dry weight was maintained over time with rare or timely appropriate weight loss adjustment. Blood pressure was maintained in a narrow target range, as shown by the predialysis mean systolic and diastolic blood pressure values of 140 [135-145] mmHg and 80 [75-85] mmHg, respectively. No significant differences were noted between male and female patients. Hemodynamic stability and dialysis tolerance were quite satisfactory, as highlighted by the very low incidence of intradialytic hypotension and almost no serious intradialytic morbidity. These findings are likely due to the low ultrafiltration rate of 7.9 mL/h/kg [range 2-10], resulting in better hemodynamic stability, while a sustained volume and sodium depletion might be achieved over time [START_REF] Kron | Dynamics of vascular refilling in extended nocturnal hemodialysis[END_REF].

Solute removal capacity and uremic control were satisfactorily achieved with intensive hemodialysis low dialysate volume, as highlighted by various indicators of dialysis efficacy used in this study. The urea reduction rate per session was over 50%, corresponding to a weekly standard Kt/V of 2.5 [START_REF] Leypoldt | Effect of Treatment Duration and Frequency on Uremic Solute Kinetics, Clearances and Concentrations[END_REF][START_REF] Leypoldt | Urea standard Kt/V(urea) for assessing dialysis treatment adequacy[END_REF]. The predialysis concentration of selected uremic markers remained remarkably stable over time: urea concentration was lower than 25 mmol/L; 2-microglobulin concentration was lower than 25 mg/L; phosphate concentration was lower than 1.55 mmol/L. Interestingly, circulating levels of selected uremic markers remained in target while dietary protein intake was preserved. Bone mineral disorder markers (plasma calcium and PTH) were also controlled adequately to maintain in target. Anemia correction, as shown by hemoglobin concentrations, was maintained between 11 and 12 g/dL.

Nutritional status was well preserved, as indicated by key parameters such as albumin, which was maintained between 40 and 41 g/dL. Electrolytic disorders were well controlled in the study, as indicated by relatively stable concentrations of plasma bicarbonate. The individualized prescription of dialysate potassium and bicarbonate may have facilitated this endeavor.

Patients' perspectives also appeared to be improved throughout the study follow-up period.

The patient perception and satisfaction survey previously reported by our group confirmed that the S 3 system was easy to use, easily implemented at home, and facilitated patients' mobility [START_REF] Fessi | Safety and Efficacy of Short Daily Hemodialysis with Physidia S(3) System: Clinical Performance Assessment during the Training Period[END_REF]. Despite the short daily treatment schedule, the S 3 system was considered as a less intrusive treatment modality than previously experienced with in center hemodialysis treatments. In addition, a slow flow treatment system tends to reduce the dialysis burden by preventing intradialytic morbidity and shortening post-dialysis recovery time. In line with these positive clinical effects, almost 25% of unemployed patients tended to return to work during the two-year follow-up period.

In agreement with recent daily dialysis studies, cardiac features as assessed by echocardiography were improved with intensive dialysis at home [START_REF] Culleton | Effect of frequent nocturnal hemodialysis vs conventional hemodialysis on left ventricular mass and quality of life: A randomized controlled trial[END_REF][START_REF] Group | In-center hemodialysis six times per week versus three times per week[END_REF][START_REF] Mccullough | Intensive Hemodialysis, Left Ventricular Hypertrophy, and Cardiovascular Disease[END_REF][START_REF] Nehus | Intensive hemodialysis for cardiomyopathy associated with end-stage renal disease[END_REF]. As shown in a subgroup of patients, left ventricular mass was reduced by 9.6% at 3 months, while the ejection fraction remained remarkably stable in the normal range of 61 to 62%. This finding indicates that cardiac remodeling tends to improve with intensive dialysis, while cardiac function is better preserved. This observation is perfectly in line with the frequent hemodialysis study. Several advantages of intensive home dialysis therapy relying on the S 3 system with a bagged cycler system are illustrated in this study: on one hand, the low flow concept permits one to saturate dialysate (up to 75 and 80%) with uremic compounds and then to reduce dialysis volume consumption and save water and power; on the other hand, the friendly interface, fine edge design, and compacity of the S 3 system facilitate handling operations and reduce the intrusiveness of home treatment.

The study also has some limitations that deserve to be explored in further clinical studies. Firstly, RECAP is a retrospective clinical study and not a prospective one. Secondly, the number of patients included is relatively low, although not negligible if we take into account the low proportion of patients treated using frequent hemodialysis at home in France. Thirdly, this study is limited to France. Fourth, the outcome (clinical/technique) failure should have been compared with other HHD programs that use conventional HD machines. Unfortunately, such a comparison was not possible in the reported study, since patients treated with Physidia S3 did not experience a home HD thrice-weekly treatment schedule with conventional incenter HD material. From this perspective, one can only compare our long-term technical survival rate (52%) to previous studies [START_REF] Jayanti | Technique survival in home haemodialysis: A composite success rate and its risk predictors in a prospective longitudinal cohort from a tertiary renal network programme[END_REF][START_REF] Marshall | Survival on home dialysis in New Zealand[END_REF] reporting higher technical acceptance (70 to 88%) based on a thrice-weekly treatment schedule and using conventional in-center HD material. Such a discrepancy likely reflects the burden of daily treatment and not home treatment with the Physidia S 3 device.

Conclusions

Intensive hemodialysis relying on the S 3 system offers a very appealing option for home treatment with quite satisfactory results, as shown in the RECAP study throughout a two-year follow-up time. The S 3 system was easily handled and adopted by patients at home. Overall, the results confirmed that a more frequent, short and low flow treatment schedule may deliver adequate treatment that fits with the majority of dialysis patients' needs. The most prominent findings are as follows: firstly, patient perception was improved, while treatment burden was reduced despite a more frequent treatment schedule; secondly, cardiac features tended to improve over time, confirming that intensive hemodialysis has the capacity to reduce cardiac health in dialysis patients; thirdly, home treatment based on the S 3 system offers the best bridging solution to kidney transplantation.

Figure 1 .

 1 Figure 1. Flowchart of the study population.

Figure 4 .

 4 Figure 4. Evolution of plasma phosphate concentrations (median +/-IQR).

Figure 5 .

 5 Figure 5. Evolution of plasma albumin concentrations (mean +/-SEM).

Figure 6 .

 6 Figure 6. Technical survival probability of continuing HHD with S3 monitor during the follow-up period.

  2 [range 8.0-13.0] mL/min weekly urea clearance and a standardized Kt/V of 2.52 [range 1.1-4.5] [15,20]. An estimate of dialysis dose delivered in the RECAP study addressed most of the patients' metabolic needs and compares favorably with recent studies reporting on short daily and low dialysis fluid volume. This interesting finding may be partially explained by the additional convective dose (+3 L per session corresponding to 15 to 18 L per week) provided through the SeCoHD option.

Funding:

This research received no external funding.

Institutional Review Board Statement:

The study was approved by the Institutional Review Board committee of Montpellier University Hospital (IRB-MTP_2021_02_202100722). The study was performed in accordance with Good Clinical Research Practice guidelines, conducted in accordance with the declaration of Helsinki, and performed on existing data registry after being anonymized, pooled, and analyzed. Blood sampling was part of regular patient monitoring during the training phase, and no extra blood sampling was taken for this study.

Informed Consent Statement:

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to ethical concern.

Conflicts of Interest:

M.T. is an employee of Physidia. B.C. is a part-time consulting employee for Fresenius Medical Care. The other authors state no conflicts of interest.


Meriam Shahin

Devon Sheppard

Claire Raynaud

Jamie-Lee Berry

Ishwori Gurung

Lisete M Silva

Ten Feizi

Yan Liu

Vladimir Pelicic
email: vladimir.pelicic@inserm.fr

Characterization of a glycan-binding complex of minor pilins completes the analysis of Streptococcus sanguinis type 4 pili subunits

Keywords: Biological Sciences. Microbiology type 4 pili, pilin, adhesion

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Type 4 filaments (T4F) -type 4 pili (T4P) and type 2 secretion systems (T2SS) being the best known -are a superfamily of nanomachines ubiquitous in prokaryotes [START_REF] Berry | Exceptionally widespread nano-machines composed of type IV pilins: the prokaryotic Swiss Army knives[END_REF][START_REF] Denise | Diversification of the type IV filament superfamily into machines for adhesion, protein secretion, DNA uptake, and motility[END_REF]. T4F, which mediate a wide variety of functions [START_REF] Berry | Exceptionally widespread nano-machines composed of type IV pilins: the prokaryotic Swiss Army knives[END_REF], are composed of type 4 pilins, assembled by conserved multi-protein machineries spanning the cell envelope (3).

T4F have been studied for decades because they are virulence factors in many human pathogens.

Our current understanding of T4F is as follows. T4F are polymers of type 4 pilins: one predominant (major) and several low abundance (minor) subunits [START_REF] Giltner | Type IV pilin proteins: versatile molecular modules[END_REF].

These subunits are synthesised as prepilins with a distinctive N-terminal sequence motif -the class 3 signal peptide (SP) (5) -consisting of a short hydrophilic leader peptide ending with a Gly or Ala, followed by a hydrophobic tract of 21 residues, which anchors prepilins in the cytoplasmic membrane (CM). Pilins display a characteristic "lollipop" structure where the hydrophobic tract of the SP constitutes the N-terminal portion (α1N) of an α-helix of approx. 50 residues (α1) that supports a globular head [START_REF] Giltner | Type IV pilin proteins: versatile molecular modules[END_REF]. However, larger minor pilins -displaying extra domains "grafted" onto a pilin moiety and hence defined as modular (6) -are not uncommon, which partly explains the extreme functional versatility of T4F. Pilins are assembled in filaments at the CM (3) in two steps. The leader peptide in prepilins is first cleaved by a dedicated prepilin peptidase (PPase) [START_REF] Kaufman | Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by Gram-negative bacteria[END_REF][START_REF] Nunn | Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase[END_REF][START_REF] Lapointe | The type 4 prepilin peptidases comprise a novel family of aspartic acid proteases[END_REF], before the pilins are polymerised by a machinery centred on a platform protein and a hexameric ATPase [START_REF] Mccallum | The molecular mechanism of the type IVa pilus motors[END_REF]. All T4F are helical polymers where pilins are held together by extensive interactions between their α1-helices, which pack within the core of the filament, with often a portion of α1N "melted" [START_REF] Egelman | Cryo-EM of bacterial pili and archaeal flagellar filaments[END_REF]. However, different helical symmetry parameters are seen in different T4F [START_REF] Kolappan | Structure of the Neisseria meningitidis type IV pilus[END_REF][START_REF] Wang | Cryoelectron microscopy reconstructions of the Pseudomonas aeruginosa and Neisseria gonorrhoeae type IV pili at sub-nanometer resolution[END_REF][START_REF] Lopez-Castilla | Structure of the calcium-dependent type 2 secretion pseudopilus[END_REF][START_REF] Luna Rico | Functional reconstitution of the type IVa pilus assembly system from enterohaemorrhagic Escherichia coli[END_REF][START_REF] Gambelli | An archaellum filament composed of two alternating subunits[END_REF].

While the role of major pilins is clear, i.e., they constitute the backbone of T4F, the role and precise localisation of the minor pilins remain poorly defined [START_REF] Giltner | Type IV pilin proteins: versatile molecular modules[END_REF].

Typically, minor pilins are divided into two categories: widely conserved and non-conserved (or system-specific) [START_REF] Giltner | Type IV pilin proteins: versatile molecular modules[END_REF]. Non-conserved minor pilins are highly variable, and their roles cannot be predicted. In some T4F, these pilins have been well studied, such as for example ComP, PilV, PilX in Neisseria meningitidis T4P. These three pilins are dispensable for filament assembly but are key for different T4Pmediated functions. ComP binds DNA to promote its uptake during natural transformation [START_REF] Cehovin | Specific DNA recognition mediated by a type IV pilin[END_REF][START_REF] Berry | A comparative structure/function analysis of two type IV pilin DNA receptors defines a novel mode of DNA binding[END_REF], PilV promotes bacterial adhesion to human cells [START_REF] Barnier | The minor pilin PilV provides a conserved adhesion site throughout the antigenically variable meningococcal type IV pilus[END_REF], and PilX supports the formation of bacterial aggregates [START_REF] Helaine | 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili[END_REF]. In contrast, a group of four minor pilins -often labelled with the letters H, I, J, K -are widely conserved in different T4F and essential for filament assembly [START_REF] Winther-Larsen | Neisseria gonorrhoeae PilV, a type IV pilusassociated protein essential to human epithelial cell adherence[END_REF][START_REF] Carbonnelle | Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occuring after pilus assembly, essential for fiber stability and function[END_REF][START_REF] Giltner | Pseudomonas aeruginosa minor pilins are incorporated into type IV pili[END_REF]. Their role and mechanism of action are likely to be the same since HIJK sets from different T4F are functionally interchangeable in promoting filament assembly [START_REF] Cisneros | Heterologous assembly of type IV pili by a type II secretion system reveals the role of minor pilins in assembly initiation[END_REF]. These pilins interact to form a quasihelical complex [START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF][START_REF] Escobar | Structural interactions define assembly adapter function of a type II secretion system pseudopilin[END_REF], which is capped by the bulky K subunit. This suggested a localisation at the pilus tip [START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF][START_REF] Escobar | Structural interactions define assembly adapter function of a type II secretion system pseudopilin[END_REF], which was recently confirmed by cryo-electron tomography (cryo-ET) of Myxococcus xanthus T4P [START_REF] Treuner-Lange | PilY1 and minor pilins form a complex priming the type IVa pilus in Myxococcus xanthus[END_REF]. Since T4F are assembled from tip to base (3), the HIJK complex is therefore thought to initiate filament assembly [START_REF] Cisneros | Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation[END_REF][START_REF] Douzi | The XcpV/GspI pseudopilin has a central role in the assembly of a quaternary complex within the T2SS pseudopilus[END_REF].

Recently, the study of T4F in monoderm bacteria opened new research avenues [START_REF] Melville | Type IV pili in Gram-positive bacteria[END_REF], with Streptococcus sanguinis becoming a cutting edge model [START_REF] Pelicic | Monoderm bacteria: the new frontier for type IV pilus biology[END_REF]. This commensal of the human oral cavity [START_REF] Kreth | The road less traveleddefining molecular commensalism with Streptococcus sanguinis[END_REF] is an opportunistic pathogen frequently causing infective endocarditis (IE) [START_REF] Cahill | Infective endocarditis[END_REF]. It uses an elementary machinery to assemble T4P composed of two major (PilE1, PilE2) and three minor (PilA, PilB, PilC) pilins [START_REF] Gurung | Functional analysis of an unusual type IV pilus in the Grampositive Streptococcus sanguinis[END_REF][START_REF] Berry | Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis[END_REF]. This makes it possible to characterise the role of all pilin subunits in S. sanguinis T4P in detail. Since we previously characterised PilE1, PilE2 and PilB [START_REF] Raynaud | PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion[END_REF][START_REF] Gurung | Functional analysis of an unusual type IV pilus in the Grampositive Streptococcus sanguinis[END_REF][START_REF] Berry | Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis[END_REF], here we focused on PilA and PilC. We report the detailed structure/function analysis of these two minor pilins, showing how they interact and how they function.

This provides an integrated view of the role of all the pilin subunits in a defined T4F, which has implications for this widespread superfamily of nanomachines.

Results

PilA and PilC minor pilins have remarkably different characteristics

Previously, we showed that all the genes involved in T4P biology in S. sanguinis 2908 strain cluster together in a 22 kb locus named pil [START_REF] Gurung | Functional analysis of an unusual type IV pilus in the Grampositive Streptococcus sanguinis[END_REF]. This locus encodes five pilins -PilE1, PilE2, PilA, PilB, PilC (Fig. 1A) -which are essential for piliation [START_REF] Gurung | Functional analysis of an unusual type IV pilus in the Grampositive Streptococcus sanguinis[END_REF].

These proteins are processed by the PPase PilD and become subunits of T4P [START_REF] Gurung | Functional analysis of an unusual type IV pilus in the Grampositive Streptococcus sanguinis[END_REF][START_REF] Berry | Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis[END_REF]. PilE1 and PilE2 are the major pilins, whereas PilA, PilB and PilC are minor pilins [START_REF] Berry | Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis[END_REF]. We previously characterised PilE1, PilE2 and PilB [START_REF] Raynaud | PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion[END_REF][START_REF] Berry | Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis[END_REF]. Here, we complete the analysis of the role of pilins in S. sanguinis T4P by focusing on PilA and PilC.

Although it has a typical pilin size -17 kDa for the processed protein -PilA is an unusual pilin. Its class 3 SP, which was identified by visual inspection [START_REF] Gurung | Functional analysis of an unusual type IV pilus in the Grampositive Streptococcus sanguinis[END_REF], is degenerate (Fig. 1B) and could not be identified by any of the available bioinformatic tools, including PilFind dedicated to this purpose [START_REF] Imam | Identification of surprisingly diverse type IV pili, across a broad range of Gram-positive bacteria[END_REF]. This is mainly due to the absence of the typical Glu residue in the fifth position of the processed protein (Glu5) [START_REF] Giltner | Pseudomonas aeruginosa minor pilins are incorporated into type IV pili[END_REF] and the presence of an uncommon Pro4 (Fig. 1B). PilA has no sequence homologs outside of S. sanguinis, which makes it impossible to predict its role.

Compared to PilA, PilC has radically different features. It has a canonical class 3 SP (Fig. 1C), with a typical Glu5 in the processed protein. Processed PilC is unusually large for a pilin, with a theoretical molecular mass of 52.8 kDa. As recently reported for PilB [START_REF] Raynaud | PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion[END_REF], the large size of PilC is due to its predicted modular architecture, with a domain grafted onto what appears to be an unusually short pilin moiety (Fig. 1C).In PilC, this extra module, readily identifiable by sequence-based bioinformatics, belongs to the concanavalin A-like lectin/glucanase domain superfamily (SSF49899), which is composed of carbohydrate-binding proteins in the three domains of life with different ligand specificities. These observations raise the possibility that PilC is a modular pilin with a lectin module promoting T4P-mediated adhesion of S. sanguinis to glycans.

PilA is structurally similar to a widely conserved subunit from a complex of minor pilins found at the tip of many T4F

To better understand the role of PilA, we solved its 3D structure by X-ray crystallography. We produced a 15.7 kDa recombinant protein in Escherichia coli, in which the N-terminal 24 residues of the mature pilin (Fig. 1B) were replaced by a hexahistidine tag (6His). This usually promotes pilin solubility without structurally impacting the rest of the protein [START_REF] Craig | Type IV pilin structure and assembly. X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin[END_REF]. We purified soluble 6His-PilA using a combination of affinity and size-exclusion chromatography (SEC), noticing that it purifies as a dimer. The protein crystallised readily. After optimising the best diffracting crystals and using protein produced in the presence of seleno-methionine (SeMet) for phase determination, we solved a 1.77 Å resolution structure (Table S1).

As can be seen in Fig. 2A, PilA -which looks like an "ice axe" -exhibits a typical pilin architecture, with an N-terminal α1-helix packed against a β-sheet composed of five anti-parallel β-strands. Modelling the full-length PilA protein using AlphaFold [START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF] reveals a canonical lollipop shape (4) (Fig. S1). The almost perfect superposition of our structure with the AlphaFold prediction (Fig. S1) -with a mere 0.58 Å root-meansquare deviation (RMSD) -validates the accuracy of both AlphaFold (38) and our structure.

A closer inspection of the crystal packing showed that PilA forms head-to-toe dimers (Fig. 2B), stabilised by a series of hydrogen bonds between residues in α1helix and the last two β-strands of the β-sheet (β4 and β5) (Fig. S2). This explains why 6His-PilA purifies as a dimer. Strikingly, while PilA exhibits no sequence homology to other pilins, our structure reveals extensive structural similarity to the I subunit of HIJK tip-located complex of conserved minor pilins [START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF][START_REF] Escobar | Structural interactions define assembly adapter function of a type II secretion system pseudopilin[END_REF] in a variety of T4F. For example, PilA superposes with an RMSD of 1.52 Å with GspI (PDB 3CI0) from enterotoxigenic E. coli T2SS (Fig. 2C). Critically, since I interacts with J and K [START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF][START_REF] Escobar | Structural interactions define assembly adapter function of a type II secretion system pseudopilin[END_REF], this suggested to us that PilA might be interacting with another minor pilin (PilB and/or PilC) to form a complex capping S. sanguinis T4P.

PilA interacts with PilC

To identify interactions between PilA and other pilins in S. sanguinis T4P, we performed pull-down assays with proteins purified using two different affinity tags: 6His and Strep-tag II. In brief, (i) purified proteins corresponding to the soluble portion of the pilins were mixed in solution, (ii) the 6His-tagged bait was selectively captured using cobalt-based magnetic beads, and (iii) pull-down of the Strep-tagged prey was assessed by immunoblotting using specific anti-Pil antibodies. We verified beforehand that Strep-tagged proteins were not captured using these beads (although PilB exhibited very limited binding). Interestingly, while the bait proteins were always captured, prey proteins were pulled down only for the PilA/PilC pair (Fig. 3A), both by 6His-PilA and 6His-PilC.

To characterise the PilAC complex, we used SEC coupled with multi-angle light scattering (SEC-MALS), which allows for absolute characterisation of molecular mass and stoichiometry of proteins in solution. SEC-MALS was first used with 6His-PilA and 6His-PilC separately. Both proteins eluted as single, monodisperse peaks with estimated molecular masses (in kDa) of 31.7 ± 0.35, and 57.7 ± 0.63, respectively (Fig. 3B). For 6His-PilA, this value is significantly higher than the theoretical molecular mass (15.7 kDa), confirming that the purified protein is a dimer in solution. When SEC-MALS was performed after mixing equimolar amounts of purified 6His-PilA and 6His-PilC, we observed a single, monodisperse peak of 70.8 ± 0.07 kDa (Fig. 3B), indicating that the PilAC complex has a 1:1 stoichiometry. The disappearance of the PilA-PilA homodimer shows that PilC is the preferred binding partner of PilA. Using isothermal titration calorimetry (ITC), we showed that PilA has high affinity for PilC (Fig. 3C), with a dissociation constant (KD) of 23.8 ± 7.9 nM.

We then assessed how PilA and PilC interact. We first performed pull-down assays using a purified 6His-PilCΔpilin protein lacking the pilin moiety (Fig. 1C). In contrast to 6His-PilC (Fig. 3B), 6His-PilCΔpilin was unable to pull down its Strep-PilA partner (Fig. 4A). This suggests that PilA interacts with the pilin moiety of PilC. Next, we used multidimensional nuclear magnetic resonance (NMR) to identify the portion of PilA binding to PilC. First, we performed a partial NMR assignment of the resonances in 6His-PilA (Fig. S3), highlighting the assigned residues on our crystal structure (Fig. 4B). Then, we identified the chemical shift perturbations occurring in PilA when PilC was added (Fig. S3). Nine chemical shifts perturbations concerned PilA residues not involved in the formation of PilA dimers, which are therefore likely to be involved in the PilA-PilC interaction (Fig. 4B). The PilAC interaction interface involves the α1 and α2 helices and the first 2 β-strands (β1 and β2) of PilA (Fig. 4B).

Curiously, although this interface is distinct from the one involved in the formation of PilA homodimers (Fig. S2), the formation of a PilAC complex led to the disappearance of the PilA dimer. We probed this interaction interface by mutagenesis of Ala68, Lys85 and Thr87 in PilA, and quantification of the ability of these mutant proteins to interact with PilC using ITC (Fig. 4C). This showed that PilAT87A displayed a significantly reduced affinity for PilC, corresponding to 38.3 ± 5.9 % of the wild-type (WT) binding.

Taken together, these experiments show that PilA and PilC interact, confirming our hypothesis that PilA is part of a complex of minor pilins in S. sanguinis T4P.

PilA stabilises PilC

We noticed that purified 6His-PilC was unstable, which prompted us to perform longterm stability tests. We kept the purified protein at 4ºC, took samples once a week, which we analysed by SDS-PAGE/Coomassie. This revealed that 6His-PilC was completely degraded by week two (Fig. 5A), yielding two degradation products.

Edman sequencing showed that proteolysis occurred after Ala101 close to the end of the pilin moiety (Fig. 1C), hence the two degradation products were named PilCNter (smaller N-terminal product) and PilCCter (larger C-terminal product). PilCNter is highly unstable as it was no longer detectable by week two. In the same conditions, 6His-PilA remained unchanged over four weeks, confirming that it is a very stable protein (Fig. 5A). Interestingly, when the PilAC complex was analysed in the same way, PilC degradation was significantly delayed. In the presence of PilA, PilC degradation began only at week three, and even at week four the majority of PilC was still intact (Fig. 5A).

The stabilising effect of PilA on PilC was further characterised by performing trypsin sensitivity assays. In brief, we incubated proteins with trypsin on ice for 60 min, took samples at specific timepoints, which we analysed by SDS-PAGE/Coomassie (Fig. 5B). As above, 6His-PilA was intact over the course of the assay, while 6His-PilC the protein was entirely converted into PilCNter and PilCCter by 20 min. As above, the pilin moiety PilCNter was unstable and no longer detectable after 40 min. In contrast, when it was part of a complex with 6His-PilA, 6His-PilC degradation was delayed (Fig. 5B). In addition, PilCNter was also stabilised as it was still detectable after 60 min.

Taken together, these findings show that the functional consequence of the PilAC interaction is that PilA stabilises PilC, especially its pilin moiety.

Crystal structures of PilC reveal a modular pilin

To confirm the prediction that PilC might be a modular pilin, we solved its 3D structure by X-ray crystallography. Due to the instability of its pilin moiety, we produced a 41.4 kDa recombinant 6His-PilCΔpilin protein in E. coli lacking the pilin moiety (Fig. 1C). This protein crystallised readily. After optimising the best diffracting crystals and using SeMet protein for phase determination, we solved a highresolution structure at 1.45 Å (Table S1). Strikingly, while bioinformatics only predicted that the C-terminus of PilC corresponds to a lectin domain, our structure reveals two distinct modules (Fig. 6A). The first module adopts an Ig-like fold (PilCIg), consisting of two β-sheets packed against each other (Fig. 6B). This is a widespread domain found in proteins of different functions [START_REF] Bork | The immunoglobulin fold. Structural classification, sequence patterns and common core[END_REF], which is thought to be involved in protein-protein interactions. PilCIg thus shows significant structural similarity to multiple structures, including the N-acetylglucosamine-binding protein GbpA from Vibrio cholerae (40) (PDB 2XWX). The Ig-like domains in these two proteins, which display no sequence homology, superpose with an RMSD of 2.17 Å (Fig. 6B).

The second module that corresponds to the lectin (PilClectin), is a β-sandwich characterised by two opposing antiparallel β-sheets of six strands (Fig. 6C), with concave and convex sides. PilClectin shows significant structural similarity to multiple carbohydrate-binding proteins, including galectins such as human Gal-7 (PDB 2GAL) [START_REF] Leonidas | Structural basis for the recognition of carbohydrates by human galectin-7[END_REF]. PilClectin and Gal-7, which display no sequence homology, superpose with an RMSD of 1.95 Å (Fig. 6C). In the Gal-7 structure (Fig. 6C), the protein interacts with galactose via a carbohydrate-recognition domain (CRD) forming a patch on the concave side of the protein [START_REF] Leonidas | Structural basis for the recognition of carbohydrates by human galectin-7[END_REF]. Since the two proteins share good structural homology in that region (Fig. 6C), we speculate that the CRD might be on the concave side of PilClectin.

To try to determine a PilC structure encompassing the pilin moiety, we expressed and crystallised PilC from another S. sanguinis strain SK36 (PilC SK36 ), in which the N-terminal 24 residues of the processed proteain were replaced by 6His.

After collecting a complete dataset, we solved a 1.60 Å resolution structure (Table S1). Surprisingly, although the purified protein contained the pilin moiety, this portion was missing in the crystals. It must therefore have been degraded during prolonged incubation in the crystallisation trays due to its inherent instability. When we compared our two PilC structures, we found that they are essentially identical, superposing onto each other with an RMSD of only 0.79 Å (Fig. 6D). This suggests that these proteins -showing only 57 % sequence identity (Fig. S4) -are likely to bind similar glycan ligands. Finally, we used AlphaFold to model the full-length PilC, revealing a lollipop with a bulky domain grafted (Fig. S5A), which superposes nicely onto our crystal structure (1.03 Å RMSD). Interestingly, the globular head of PilC is strikingly different from known pilins. Instead of the usual β-sheet with several antiparallel β-strands, PilC displays a highly distorted β-sheet composed of three βstrands, where β1 and β3 are orthogonal to each other, and linked by β2 with a 90º kink (Fig. S5B).

PilC binds two types of glycans prevalent in the human glycome

The presence of a lectin domain suggests that PilC might be an adhesin recognising host glycans. We therefore investigated the glycan binding ability and specificity of PilC using a glycan microarray designed using the neoglycolipid (NGL) technology [START_REF] Liu | Neoglycolipid-based oligosaccharide microarray system: preparation of NGLs and their noncovalent immobilization on nitrocellulose-coated glass slides for microarray analyses[END_REF]. 6His-PilC bound to a range of glycan probes -results are shown for 672 sequence-defined oligosaccharide probes (Dataset S1) -corroborating its lectin activity (Fig. 7A). Two major types of negatively charged glycans were bound: sialylated glycans and sulphated glycosaminoglycans (GAG). Most of the sialylated ligands display a α2-3-sialyl linkage and terminate with 3′-sialyllactose (3'-SL) or 3′sialyl-N-acetyllactosamine (3'-SLN). Also well recognised (Fig. 7A) were ganglioside GD1a and four probes terminating with the Sd a antigen, which is known to be present in human secretions including saliva [START_REF] Marsh | Anti-Sdx: a "new" auto-agglutinin related to the Sda blood group[END_REF][START_REF] Morton | The Sda blood group antigen in tissues and body fluids[END_REF]. A few probes displaying other sialyl linkages -including α2-9-polysialic acid -were also bound (Fig. 7A). The sulphated GAG ligands are mainly oligosaccharide probes derived from heparin, representative of the highly sulphated domains of heparan sulphate, which is ubiquitously expressed on the cell surface and in the extracellular matrix. Both sialylated glycans and GAGs are prevalent in the human glycome [START_REF] Cummings | The repertoire of glycan determinants in the human glycome[END_REF]. We observed a similar binding profile for PilC SK36 (Fig. S6), showing that glycan-binding ability is conserved in PilC orthologs.

We tested whether the lectin activity of PilC is affected by its pilin moiety, by probing the glycan microarray with 6His-PilCΔpilin. PilCΔpilin bound the same type of glycans as PilC, suggesting that glycan binding is largely due to the lectin module.

However, the pilin moiety affects the binding (Fig. S7). When compared to PilC, PilCΔpilin binds strongly to heparin oligosaccharide probes and weakly to a more restricted range of α2-3-linked sialyl glycans (Fig. S7). Next, using ITC, we quantified the affinity of PilCΔpilin for 3'-SL, 3'-SLN and their constituent subunits (sialic acid and lactose) (Fig. 7B). While no binding was measured with lactose or sialic acid, PilC bound both 3'-SL and 3'-SLN in accord with the microarray findings. There was a slight preference for 3'-SL over 3'-SLN, with KD of 79.1 ± 0.36 µM and 143.73 ± 7.36 µM, respectively (Fig. 7B).

Finally, we probed the glycan-binding activity of PilC by mutagenesis of residues on the concave side of PilClectin where the CRD is predicted to be (Fig. 6C).

We made a series of PilCΔpilin derivatives in which surface-exposed residues in the putative CRD were changed by site-directed mutagenesis (Fig. 8A). We then purified these mutant proteins and quantified their affinity for 3'-SL using ITC, before comparing it to WT PilC (Fig. 8B). This revealed that PilCT347A and PilCK349A displayed significantly reduced affinity for 3'-SL, which was particularly pronounced for PilCK349A, with 39.3 ± 1.4 % of the WT binding. This finding indicates that the concave side of its lectin module is involved in the ability of PilC to bind specific glycans.

Modelling predicts that the PilAC complex is located at the tip of S. sanguinis

T4P, together with PilB

Since the instability of PilC pilin moiety precluded us from determining a crystal structure of the PilAC complex, we modelled this complex using AlphaFold [START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF]. In the PilAC complex (Fig. 9A), the two proteins are staggered, PilA is on the top and the extra modules in PilC are on the side. PilA extends the β-sheet of PilC (Fig. S8), which is likely to explain its stabilising effect on PilC. The complex is stabilised by a series of 14 hydrogen bonds and five salt bridges (Fig. S8). The predicted PilAC interaction interface is in strikingly good agreement with that identified by NMR (Fig. 4B), including the α1 and α2 helices, and the β1 and β2 strands of PilA. This significantly strengthens the validity of our model. S. sanguinis T4P contain another modular pilin, PilB, which was previously predicted to be at the pilus tip [START_REF] Raynaud | PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion[END_REF]. We therefore wondered if PilA, B, C could co-exist in one pilus, which we assessed by modelling using AlphaFold. The PilABC model suggests that the two modular pilins PilB and PilC can be accommodated in the same complex despite their bulky extra domains (Fig. 9B).The three subunits form a quasihelical complex in which PilA interacts with PilC, which interacts with PilB. Some level of movement for the extra modules in PilB and PilC is expected because the short loops that connect them to their respective pilin moieties are likely to be flexible. Although PilA is predicted to be added first, the extra modules in PilB and PilC are capping the complex and look like "open wings" (Fig. 9B). Due to this peculiar architecture, the PilABC complex could only be accommodated at the tip of the T4P. It is possible that a portion of α1N needs to be melted, as in major pilins in many T4F [START_REF] Egelman | Cryo-EM of bacterial pili and archaeal flagellar filaments[END_REF], to allow proper fitting. Tip localisation is likely to optimise the presentation of the adhesive modules in PilB and PilC, promoting S. sanguinis adhesion to host surfaces.

Discussion

T4F are a superfamily of filamentous nanomachines ubiquitous in bacteria and archaea (1, 2), mediating a wide variety of functions ranging from adhesion to protein secretion. Although T4F have been studied for 40 years -notably T4P (46) -many aspects of their complex biology remain poorly understood. This holds especially true for the filament -the "business end" of T4F nanomachines -where the roles of all pilin subunits are yet to be understood. Therefore, in this report we used the inherent simplicity of T4P in monoderm bacteria -which recently opened new research avenues (30) -to complete a structure/function analysis of all the pilins in S. sanguinis [START_REF] Pelicic | Monoderm bacteria: the new frontier for type IV pilus biology[END_REF] T4P. This led to the notable findings discussed below, shining new light on T4F.

Our first important finding -with widespread implications in T4F biology -is the unexpected discovery that the minor pilin PilA from S. sanguinis is a structural homolog of the I minor pilin of the widely conserved HIJK complex. The broader than predicted conservation of that minor pilin is a testament to the central role it plays in T4F biology. There are multiple analogies between the two systems, suggesting that PilABC is perhaps a rudimentary/ancestral version of HIJK. The H,I,J,K pilins -which are found in different T4F and are functionally interchangeable [START_REF] Cisneros | Heterologous assembly of type IV pili by a type II secretion system reveals the role of minor pilins in assembly initiation[END_REF] -are encoded by an operon, and K lacks the usually conserved Glu5 (4). PilA,B,C pilins are also encoded by an operon, and PilA lacks Glu5 [START_REF] Berry | Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis[END_REF]. Structural studies showed that HIJK interact to form a quasihelical complex [START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF][START_REF] Escobar | Structural interactions define assembly adapter function of a type II secretion system pseudopilin[END_REF], capped by the K subunit because no additional subunits could fit above it without significant steric hindrance [START_REF] Korotkov | Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system[END_REF] and the lack of Glu5 in K would not allow neutralisation of the positively charged N-terminus of a pilin above them. Importantly, this localisation was recently confirmed by cryo-ET [START_REF] Treuner-Lange | PilY1 and minor pilins form a complex priming the type IVa pilus in Myxococcus xanthus[END_REF]. Similarly, we found that PilA,B,C are likely to form a complex at the tip of S. sanguinis T4P. Although PilA is added first, because it lacks a Glu5, the bulky extra modules in PilB and PilC cap the PilABC complex like open wings. Due to this peculiar architecture -resembling the Winged Victory of Samothrace, a masterpiece of Greek sculpture -PilABC could only be accommodated at the tip of the T4P.

Critically, both HIJK [START_REF] Winther-Larsen | Neisseria gonorrhoeae PilV, a type IV pilusassociated protein essential to human epithelial cell adherence[END_REF][START_REF] Carbonnelle | Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occuring after pilus assembly, essential for fiber stability and function[END_REF][START_REF] Giltner | Pseudomonas aeruginosa minor pilins are incorporated into type IV pili[END_REF] and PilABC [START_REF] Gurung | Functional analysis of an unusual type IV pilus in the Grampositive Streptococcus sanguinis[END_REF] pilins are essential for filament assembly, which is compatible with PilA being a nucleator initiating filament assembly, a role attributed to the I subunit in HIJK [START_REF] Cisneros | Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation[END_REF][START_REF] Douzi | The XcpV/GspI pseudopilin has a central role in the assembly of a quaternary complex within the T2SS pseudopilus[END_REF]. For the HIJK complex, it was proposed that (i) I interacts with J in the membrane to form a staggered complex, (ii) the IJ complex interacts with K via I, initiating filament assembly by forming a quasihelical complex ( 25), (iii) the IJK complex interacts with H via J [START_REF] Escobar | Structural interactions define assembly adapter function of a type II secretion system pseudopilin[END_REF], and (iv) the HIJK complex interacts with the major pilin via H [START_REF] Escobar | Structural interactions define assembly adapter function of a type II secretion system pseudopilin[END_REF]. The series of events is similar for PilABC, albeit simpler. (i) PilA interacts with PilC and stabilises it (the I subunit in HIJK might play a similar role according to crystallisation studies (47)), (ii) PilC interacts with PilB, and (iii) the PilABC complex most likely interacts with the major pilins via PilB subunit. In both systems, the I/PilA subunit initiates filament assembly and facilitates the presentation of "effectors" at the tip of filaments.

In S. sanguinis T4P, the effectors are the two modular pilins PilB and PilC with adhesive properties. In T4P in diderm model species, the effector is the non-pilin adhesin PilC/PilY1, an extremely large protein that is absent in monoderms.

Interestingly, cryo-ET analysis of M. xanthus T4P revealed that PilC/PilY1 forms a kinked tip structure [START_REF] Treuner-Lange | PilY1 and minor pilins form a complex priming the type IVa pilus in Myxococcus xanthus[END_REF], which looks like one of the open wings in PilABC.

Drawing a picture of the roles of all the pilin subunits in a model T4F system is the second major achievement in this study. S. sanguinis T4P are composed of five pilins: two major (PilE1, PilE2) and three minor (PilA, PilB, PilC) ones. PilE1 and PilE2 -85 % identical in sequence (34) -follow universally shared principles for processing by the PPase and display lollipop structures [START_REF] Berry | Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis[END_REF]. This suggests that S. sanguinis T4P are canonical T4F, i.e., helical polymers where pilins pack within the core of the filament and are held together by interactions between their α1-helices [START_REF] Egelman | Cryo-EM of bacterial pili and archaeal flagellar filaments[END_REF]. Although unusual -T4F are most often homopolymers of only one major pilinthe heteropolymeric composition of S. sanguinis T4P with two major pilins in nearly equal amounts [START_REF] Berry | Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis[END_REF] is not unique. Recently, the structure of a heteropolymeric archaeal T4F used for swimming by Methanocaldococcus villosus has been elucidated by cryo-EM [START_REF] Gambelli | An archaellum filament composed of two alternating subunits[END_REF], showing an architecture with two alternating major pilin subunits. Whether a similar alternating arrangement of PilE1 and PilE2 exists within the S. sanguinis T4P remains to be tested. As for the three minor pilins in S. sanguinis T4P they are divided into non-modular (PilA) and modular (PilB, PilC). We have now defined their respective roles. PilA, which displays a canonical lollipop structure, interacts strongly and exclusively with PilC. Pull-down experiments suggest that PilA and PilC interact via their pilin moieties, which was confirmed by NMR. The interaction interfaces determined by NMR and predicted by modelling are in strikingly good agreement. As a functional consequence, PilA dramatically stabilises the PilC pilin moiety, which is highly unstable on its own. Modelling suggests that this is done by β-strand complementation. PilA thus acts as an intra-pilus chaperone, protecting PilC from proteolysis by perhaps participating in its folding. We previously showed that PilB displays a modular architecture with a bulky functional module grafted onto a pilin moiety [START_REF] Raynaud | PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion[END_REF]. The vWA module in PilB makes it a bona fide adhesin, playing a key role in S. sanguinis adhesion to host cells by binding protein ligands such as fibronectin and fibrinogen [START_REF] Raynaud | PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion[END_REF]. Since S. sanguinis T4P are critical for disease progression [START_REF] Martini | Type IV pili of Streptococcus sanguinis contribute to pathogenesis in experimental infective endocarditis[END_REF], these findings are consistent with PilB-mediated adhesion being involved in IE. Here, we show that PilC is also a modular pilin with two extra modules, an Ig-like fold followed by a ConA-like lectin/glucanase module (SSF49899). While the function of the Ig-like module remains unclear, we showed that the ConA-like module is a bona fide lectin. PilC binds specifically to two major types of glycans -(i) sialylated glycans often terminated with α2-3-sialyl linkage (3'-SL or 3'-SNL), and (ii) heparin oligosaccharide probes representative of sulphated GAGs -both prevalent in the human glycome [START_REF] Cummings | The repertoire of glycan determinants in the human glycome[END_REF]. The measured affinities for 3'-SL and 3'-SLN are consistent with those previously determined for other SSF49899containing proteins [START_REF] Böcker | Binding characteristics of galectin-3 fusion proteins[END_REF]. Affinities could be much higher for larger glycan determinants, or when there is multivalent presentation (abundance) of a glycan ligand at the cell surface [START_REF] Cummings | The repertoire of glycan determinants in the human glycome[END_REF][START_REF] Böcker | Binding characteristics of galectin-3 fusion proteins[END_REF]. The physiological ligands for PilC are yet to be defined -identifying ligands for lectins is often difficult (45, 50) -but it is likely that this modular pilin is involved in S. sanguinis commensalism or IE. Indeed, 3'-SL or 3'-SLN terminated glycans are abundant in the oral cavity [START_REF] Kreth | The road less traveleddefining molecular commensalism with Streptococcus sanguinis[END_REF], especially on heavily glycosylated salivary mucins [START_REF] Cummings | The repertoire of glycan determinants in the human glycome[END_REF]. The full picture of S. sanguinis T4P that has now emerged is the following. S. sanguinis T4P are heteropolymeric filaments composed of PilE1 and PilE2, with helical symmetry parameters that are yet to be determined, capped by a PilABC complex mediating bacterial adhesion to different eukaryotic receptors. The adhesive modules in PilB and PilC are ideally placed to maximise adhesion. However, although this is less likely, we cannot exclude the possibility that S. sanguinis expresses two distinct pili, capped either by PilB or PilAC.

In conclusion, by providing perhaps the most detailed structure/function characterisation of the role of all pilins in a model T4P, this study sheds light on important aspects of T4F biology. The finding that PilABC might be a rudimentary version of the widely conserved HIJK strengthens the notion that complexes of minor pilins capping T4F facilitate the presentation of different effectors at the tip of filaments, thus directly promoting T4F exceptional functional versatility (1). Our findings have general implications for T4F and pave the way for further investigations that will improve our understanding of these fascinating filaments.

Material and methods

Detailed Material and Methods are described in the Supporting information.

Strains and plasmids

The E. coli strains and plasmids used in this study are listed in Table S2. DH5α was used for cloning, BL21(DE3) was used for routine protein expression and purification, and B834(DE3) was used for purification of SeMet-labelled proteins.

DNA manipulations were performed using standard molecular biology techniques [START_REF] Sambrook | Molecular cloning. A laboratory manual[END_REF]. The primers used in this study are listed in Table S3. We amplified pilA directly from the S. sanguinis 2908 genome, while pilB and pilC were synthetic genes codon-optimised for expression in E. coli (GeneArt).

Protein purification

The recombinant proteins were purified in two steps: affinity chromatography and SEC. 6His-tagged proteins were affinity-purified using Gravity Flow Chromatography columns (Bio-Rad) loaded with Ni-NTA agarose resin (Qiagen). Strep-tagged proteins were affinity-purified on an ÄKTA Purifier using StrepTrap HP columns (GE Healthcare). Both the 6His-and Strep-tagged proteins were then further purified by SEC on an ÄKTA Purifier using a Superdex 200 16/600 GL column (GE Healthcare), and simultaneously buffer-exchanged into 50 mM HEPES pH 7.5, 150 mM NaCl.

SeMet-labelled PilA and PilC for phasing were purified as above, but the bacteria were grown in chemically defined medium containing SeMet. 15 N and/or 13 Clabelled PilA for NMR analysis was purified as above, but the bacteria were grown in M9 medium supplemented with 15 NH4Cl and/or 13 C-labelled glucose.

Protein crystallisation and structure determination

Protein crystals for SeMet-labelled PilA were produced using 0.4 µl sitting drops with a 1:1 ratio protein to mother liquor (0.1 M Tris pH 8, 23 % PEG 2K (w/v), 0.3 M secondary was an anti-rabbit antibody conjugated to horseradish peroxidase (GE Healthcare).

SEC-MALS

SEC-MALS was performed using an ÄKTA Prime system with a S200 10/300 GL column (GE Healthcare) and a MALS detector (Wyatt). The UV, light scattering and refractive index signals were analysed with the ASTRA chromatography software.

ITC

ITC was used to study the interaction between the minor pilins PilA and PilC, as well as the binding of PilCΔpilin to sugar ligands. The experiments were performed on a MicroCal ITC200 Malvern machine. Each experiment was repeated three times. Data analysis was performed on the Origin ITC200 software. Statistical analyses were performed with Prism (GraphPad Software). Comparisons were done by one-way ANOVA, followed by Dunnett's multiple comparison tests with 99 % confidence interval. An adjusted P value <0.01 was considered statistically significant (** P<0.01, *** P<0.001, **** P<0.0001).

NMR assignment and chemical shift perturbations

We performed TROSY-based assignment experiments: HNCA, HNCOCA, HNCO, HNCACO, HNCACB, and CBCACONH. All data were collected at 25ºC on a Bruker Avance III HD 800MHz triple resonance spectrometer with a cryoprobe. Chemical shift perturbation experiments were performed with samples in NMR buffer containing 15 N labelled PilA with or without unlabelled PilC. Chemical shift perturbations were determined using the equation:

∆δ = ! (∆δ H ) 2 + $ γ N γ H ∆γ N %

Protein stability assays

To perform protein stability tests, PilA and PilC were purified and mixed at equal concentrations. In long-term assays, the proteins were kept at 4°C for four weeks.

Samples were taken once a week and analysed by SDS-PAGE/Coomassie staining.

In trypsin sensitivity assays, the proteins were incubated with trypsin (Sigma) for 60 min on ice. Samples -taken at 1, 5, 10, 20, 40 and 60 min -were analysed by SDS-PAGE/Coomassie staining.

Glycan microarrays

The binding specificities of 6His-PilC, 6His-PilCΔpilin and 6His-PilC SK36 were analysed using a NGL-based microarray system [START_REF] Liu | Neoglycolipid-based oligosaccharide microarray system: preparation of NGLs and their noncovalent immobilization on nitrocellulose-coated glass slides for microarray analyses[END_REF]. A previously described broad-spectrum microarray of 672 sequence-defined lipid-linked glycan probes was used [START_REF] Mcallister | Chikungunya virus strains from each genetic clade bind sulfated glycosaminoglycans as attachment factors[END_REF]. The list of glycan probes is given in the Dataset S1. Details of the preparation of the glycan probes and the generation of the microarrays are listed in Table S4 in accordance with the MIRAGE guidelines [START_REF] Liu | The minimum information required for a glycomics experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data[END_REF]. The microarray analyses were performed essentially as described [START_REF] Neu | A structure-guided mutation in the major capsid protein retargets BK polyomavirus[END_REF]. Binding was detected with Alexa Fluor-647labelled streptavidin (Molecular Probes). Imaging and data analysis are described in Table S4.

Bioinformatics and modelling

This was done as previously described [START_REF] Raynaud | PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion[END_REF]. Modelling was done using AlphaFold 2.2.0 [START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF] and AlphaFold-Multimer [START_REF] Evans | Protein complex prediction with AlphaFold-Multimer[END_REF]. Five predictions were generated in total, with a final relaxation step. The predictions were ranked according IPTM+PTM and the top predictions were chosen.

Legends to Figures

Fig. 1 .

 1 Fig. 1. PilA and PilC sequence characteristics. A) Protein architecture of the five prepilins in the T4P-encoding pil locus in S. sanguinis 2908 (34). Each protein

Fig. 2 .

 2 Fig. 2. Crystal structure of PilA. A) Cartoon view of the PilA structure rainbowcoloured from blue (N-terminus) to red (C-terminus). B) Cartoon view of the head-to-

Fig. 3 .Fig. 4 .

 34 Fig. 3. PilA interacts with PilC. A) Interactions between PilA and the four other

Fig. 5 .

 5 Fig. 5. PilA stabilises PilC. A) Long-term protein stability experiments. Purified PilA

Fig. 6 .

 6 Fig. 6. Crystal structures of PilC. A) Cartoon views at 180º rotation of the structure

Fig. 7 .

 7 Fig. 7. PilC specifically binds sialylated glycans and GAG. A) Glycan microarrayscreening analyses of 6His-PilC, as a protein-antibody pre-complex. Multiple

Fig. 8 .

 8 Fig. 8. Probing the putative carbohydrate-recognition domain in PilC by

Fig. 9 .

 9 Fig. 9. Modelling the complex of minor pilins in S. sanguinis T4P. A) Model of

Acknowledgements

This work was funded by the Medical Research Council (MR/P022197/1). The glycan microarray studies were performed in the Carbohydrate Microarray Facility at the Glycosciences Laboratory, which is supported by Wellcome Trust biomedical resource grants (099197/Z/12/Z, 108430/Z/15/Z, and 218304/Z/19/Z) and partially by the March of Dimes Prematurity research centre grant (22-FY18-82). The sequencedefined glycan microarrays contain many saccharides provided by collaborators whom we thank. We acknowledge the use of the crystallisation facility at Imperial College London, which is supported by the Biotechnology and Biological Sciences Research Council (BB/D524840/1) and Wellcome Trust (202926/Z/16/Z). We thank Angelika Gründling (Imperial College London), Sophie Helaine (Harvard Medical School) and Romé Voulhoux (CNRS Marseille) for critical reading of the manuscript.

Mg(NO3)2•6H2O). Protein crystals for SeMet-labelled PilC were produced using 0.2 µl sitting drops with of 1:1 ratio protein to mother liquor (0.1 M Pipes pH 7 15 % w/v PEG broad smear, 0.2 M (NH4)2SO4, 0.0 1M cadmium chloride hemi (pentahydrate)).

Data was collected at the Diamond beamlines i04-1 and i03, respectively. Data was processed using Xia2 [START_REF] Winter | Decision making in xia2[END_REF], and phased using autoSHARP [START_REF] Vonrhein | Automated structure solution with autoSHARP[END_REF]. Initial structure was produced using CRANK2 [START_REF] Skubak | A new MR-SAD algorithm for the automatic building of protein models from low-resolution X-ray data and a poor starting model[END_REF] and autobuild, with some manual building in Coot [START_REF] Emsley | Features and development of Coot[END_REF].

Refinement was performed using Coot and phenix.refine [START_REF] Afonine | Towards automated crystallographic structure refinement with phenix.refine[END_REF]. Structure validation was performed using MolProbity [START_REF] Chen | MolProbity: all-atom structure validation for macromolecular crystallography[END_REF]. Protein crystals for PilC SK36 were produced in 0.2 µl sitting drop with a 1:1 protein to mother liquor (0.2 M MgCl2•6H2O, 20 % w/v PEG 3350). Data was collected at the Diamond beamline i04 and structure was produced using the PilC structure from strain 2908 for molecular replacement.

All the 3D structures determined during this study have been deposited in the PDB and are available under accession codes 7O5Y (PilA), 7OA7 (PilC), and 7OA8 (PilC SK36 ). The PilABC structural model is available in ModelArchive at https://modelarchive.org/doi/10.5452/ma-zz3r2.

Pull-down assays

Pull-down assays were carried out using Dynabeads His-tag Isolation and Pulldown (Invitrogen). The 6His-tagged soluble pilin domains were used as bait, while Streptagged soluble pilins were used as prey. The pull-down assays were performed three times for each combination. For each pull-down reaction, input, flow-through, and elution samples were analysed by immunoblotting.

SDS-PAGE and immunoblotting

SDS-PAGE was carried out using standard molecular biology techniques [START_REF] Sambrook | Molecular cloning. A laboratory manual[END_REF]. Gels were either stained with Bio-Safe Coomassie (Bio-Rad) or transferred to a membrane and analysed by immunoblotting. We used specific antibodies generated in rabbits against PilE1, PilE2, PilA, PilB, PilC, which were previously described [START_REF] Gurung | Functional analysis of an unusual type IV pilus in the Grampositive Streptococcus sanguinis[END_REF][START_REF] Berry | Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis[END_REF]. The