Aggregation of α-synuclein plays a key role in the development of Parkinson’s disease. Soluble aggregates are present not only within human brain but also the CSF and blood. Characterizing the aggregates present in these biofluids may provide insights into disease mechanisms and also have potential for aiding diagnosis.We used two optical single-molecule imaging methods called aptamer DNA-PAINT and single-aggregate confocal fluorescence, together with high-resolution atomic force microscopy for specific detection and characterization of individual aggregates with intermolecular β-sheet structure, present in the CSF and serum of 15 early stage Parkinson’s disease patients compared to 10 healthy age-matched controls.P = 1.7 × 10−5 for each species)]. The ratio of the number of β-sheet α-synuclein aggregates to β-sheet amyloid-β aggregates in serum extracted using our super-resolution method discriminated Parkinson’s disease cases from controls with an accuracy of 98.2% .We found aggregates ranging in size from 20 nm to 200 nm, in both CSF and serum. There was a difference in aggregate size distribution between Parkinson’s disease and control groups with a significantly increased number of larger aggregates (longer than 150 nm) in the serum of patients with Parkinson’s disease. To determine the chemical composition of the aggregates, we performed aptamer DNA-PAINT on serum following α-synuclein and amyloid-β immunodepletion in an independent cohort of 11 patients with early stage Parkinson’s disease and 10 control subjects. β-Sheet aggregates in the serum of Parkinson’s disease patients were found to consist of, on average, 50% α-synuclein and 50% amyloid-β in contrast to 30% α-synuclein and 70% amyloid-β in control serum [the differences in the proportion of these aggregates were statistically significant between diseased and control groups (Our data suggest that studying the protein aggregates present in serum can provide information about the disruption of protein homeostasis occurring in Parkinson’s disease and warrants further investigation as a potential biomarker of disease. et al. reveal changes in the composition and size of individual α-synuclein and amyloid-β aggregates in the serum and CSF of patients with Parkinson's disease compared to controls, with a higher proportion of larger aggregates and an increased α-synuclein/amyloid-β ratio developing in disease.Using a suite of new biophysical methods, Lobanova  In Alzheimer’s disease and Parkinson’s disease, there is accumulation of aberrantly processed and misfolded proteins such as amyloid-β,10 The presence of secreted forms of α-syn has been reported in mouse and human brains showing significant levels of α-syn in the interstitial fluid of both Parkinson’s disease and non-Parkinson’s disease cases.11 In particular, total α-syn concentration in the interstitial fluid of the A53T transgenic mice was 0.5 ng/ml compared to 0.15 ng/ml in control mice. Interstitial fluid in human (non-Parkinson’s disease) brain contained 0.5–8 ng/ml of total α-syn with comparable levels in CSF. In contrast, CSF concentrations of α-syn oligomers were reported at 50–100 pg/ml12 in healthy individuals with an approximate 1.5-fold increase in Parkinson’s disease patients, based on sensitive ELISA measurements. Levels of amyloid-β in CSF are considerably lower: Alzheimer’s disease CSF contains only 1–2 pg/ml of amyloid-β oligomers13 compared to 0.4–0.7 pg/ml in aged control CSF. This is likely to be due to the fact that most of the amyloid-β aggregates are deposited into inert insoluble amyloid-β plaques in the brain. However, 40% of all amyloid-β produced in the brain is removed in the periphery.14 Measurements of the rate of amyloid-β deposition in the brain by Masters and colleagues15 show that in normal brain 2 mg of amyloid-β is deposited in 19 years compared to 6 mg in an Alzheimer’s disease brain, which is a surprisingly small difference in deposition by a factor of 3 over an extended time period. Thus, the existing data suggest that a major route of monomer and aggregate removal is secretion by a variety of mechanisms into the interstitial fluid, where they are either deposited in insoluble aggregates (in the case of amyloid-β predominantly) or enter the CSF for removal.A major route of aggregate removal from neurons is secretion into the interstitial fluid and removal by phagocytosis and endocytosis by glial cells, but aggregates also reach the CSF via the glymphatic system, and efflux into the peripheral blood and lymphatic system, where they are removed by macrophages and tissues such as the liver.16 Using this method, we found that there was no change in the aggregate number in CSF during the development of Alzheimer’s disease but a change in the length distribution. In combination with high-resolution atomic force microscopy (AFM), our methods showed that a higher proportion of longer aggregates were present in the CSF of Alzheimer’s disease patients leading to an increased inflammatory response in vitro. Such an approach of measuring aggregates of multiple protein types may also be of particular relevance in Parkinson’s disease, given that co-pathologies are known to occur in this condition, with a significant proportion of patients having Alzheimer’s type pathology within the brain.17 We have therefore used our quantitative super-resolution imaging methods combined with high-resolution imaging of aggregate morphology and immunodepletion to detect and characterize the aggregates present in the CSF and serum of Parkinson’s disease patients, compared to similarly-aged control subjects.Surprisingly, the characteristics of the soluble oligomeric species that are present in human brain and biofluids have not been clearly established, nor which species are most neurotoxic and contribute to disease onset and progression. We have, to date, lacked the tools to effectively measure and quantify these oligomeric species due to their low concentration down to subpicomolar scale, and heterogeneity in both size and structure. However, characterization of the aggregates present in both CSF and serum provides an opportunity to follow the changes in protein aggregation during the development of neurodegenerative disease. To date, the main method used to detect protein aggregates in serum and CSF has been based on antibody capture, using ELISAs. These measurements provide information about the total mass of soluble aggregates present but cannot distinguish between a small number of large aggregates or a large number of small aggregates. Furthermore, in most studies only aggregates of one protein are measured rather than measuring both the amyloid-β and α-syn aggregates present in the same sample. In contrast, we have recently developed a super resolution-based method to measure the number and size of both amyloid-β and α-syn β-sheet protein aggregates in CSF.http://www.cambridgebioresource.org.uk) or were healthy spouses or carers of patients attending our Research Clinic. Demographic data were collected and all participants completed neuropsychological testing including the Addenbrooke’s Cognitive Examination version 3 (ACE-III). Participants with Parkinson’s disease were also assessed with the Movement Disorder Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) and disease stage was determined using the Hoehn and Yahr scale  included in each experiment together with participant demographics are specified in Venous blood samples were collected in 7.5 ml S-Monovette tubes from 26 Parkinson’s disease patients and 20 healthy control subjects. After collection, the samples were left to clot at room temperature for 15 min and then centrifuged at 2000 rpm for 15 min. The supernatant (serum) was collected and stored at −80°C until use. Lumbar puncture was performed with 1% lignocaine local anaesthetic in 15 Parkinson’s disease patients and 10 control subjects. CSF (5 ml) was collected and centrifuged for 10 min at 30018 In brief, a 488 nm laser beam  was directed to the back aperture of an inverted microscope (Nikon Eclipse TE2000-U). The beam was reflected by a dichroic mirror  and focused to a concentric diffraction-limited spot, 10 μm into the sample in a microfluidic detection channel through a high numerical aperture oil-immersion objective . Fluorescence was collected with the same objective, passing through the dichroic mirror, and imaged onto a 50 μm pinhole (Melles Griot) to remove out-of-focus light. The emission was filtered  and directed to an avalanche photodiode . A custom-programmed field-programmable gate array , was used to count the signals from the APD and combine these into time bins, which were selected according to the expected residence time of molecules passing through the confocal probe volume. At each time point data were collected for 10 min .Aliquots of CSF or serum were diluted by a factor of 2 or 40, respectively, in PBS buffer with pFTAA dye (30 nM). Diluted samples were withdrawn through a single-channel microfluidic device , at a flow velocity of 0.56 cm/s to a syringe  via polyethylene tubing . Flow control was achieved with syringe pumps (Harvard apparatus PhD Ultra), and the device placed on the microscope stage. Single-molecule confocal experiments were performed on the instrument as described previously.The experimental output data were collected using an FPGA card and analysed in Python using custom-written code. To compensate for the variable noise within and between samples the background was first calculated as the rolling average of 3000 time bins. With an approximate event frequency of 1/3000 bins, the presence of even bright events had a negligible effect on the background value calculated. To count the number of events in each sample, the threshold was set at double the background plus 20 AFU. This formula was applied equally to each sample.Samples were diluted 100-fold in PBS buffer and imaged on freshly cleaved mica substrates using AFM. Diluted samples (10 μl) were deposited on the substrate at room temperature. The samples were incubated for 10 min followed by rinsing with 1 ml milli-Q water, then dried using a gentle flow of nitrogen gas.19 This set-up was equipped with a silicon tip with a nominal radius of <10 nm and spring constant of 5 N/m (PPP-NCHR).AFM maps of 3D morphology of all the samples were acquired in a regime of constant phase change, with 2–4 nm/pixel resolution using a NX10 (Park Systems) operating in non-contact mode.n > 150). The average level of noise for each image was measured using SPIP software, which was in the order of 0.1 nm.20 All the measurements were performed at room temperature.Scanning probe image processor (SPIP)  software was used for image flattening and single aggregate statistical analysis  was then attached on the top of the coverslip. The wells were cleaned with the coating buffer (PBS/1% Tween-20) for 45 min followed by an additional incubation with fiducial markers , diluted in the coating buffer for 15 min. The respective biofluid (10 µl) (CSF or neat serum) or supernatant from the immunodepletion experiment (diluted or neat as specified below) were added to each well and incubated for 30 min to allow any aggregates contained in the fluid to stick to the Tween-20-coated glass surface. In the immunodepletion experiments, the serum in Cohort 2  was performed as previously described.Cohort 2  had a siCohort 2  and was n = 11 Parkinson’s disease and 10 control subjects, assayed in duplicate) was performed using the following immunoprecipitation protocol: 5 μg of purified mouse anti-α-syn antibody  or purified mouse anti-amyloid-β antibody  or mouse IgG1 isotype control antibody  (a negative control) were incubated with 1.5 mg magnetic Dynabeads® protein G  in a total volume of 200 μl 0.02% Tween-20 (PBST) in separate 1.5 ml Protein LoBind Tubes (Eppendorf AG) on a rotating wheel at 4°C for 2 h. After spinning down the immunoprecipitated antibody-bead complexes, the Eppendorf tubes were placed on a magnetic rack and the supernatants were aspirated. The beads were then washed twice with 500 μl PBST to remove non-specific binding. The respective serum (200 μl) was added to each Eppendorf tube and rotational incubation was continued overnight at 4°C. For specificity control immunoprecipitation experiments, α-syn or amyloid-β1-40 aggregates at 500 nM or a mixture of α-syn and amyloid-β1-40 aggregates at 500 nM and 2 μM concentrations for both were incubated with the antibody-bead complexes in parallel. After the incubation and centrifugation steps, the supernatants were collected, and 40 µl aliquots were transferred into fresh 0.5 ml Protein LoBind Tubes (Eppendorf AG). The collected aliquots were kept in the freezer at −80°C until further evaluation with AD-PAINT.Immunodepletion of α-syn and amyloid-β proteins from serum samples  to a concentration of 70 µM and incubated at 37°C under constant shaking (200 rpm) for 3 days. Amyloid-β1-40 (AS-24235) was purified according to the suppliers protocol. Monomeric amyloid-β1-40 was diluted in prefiltered PBS (pH 8.0) to a final concentration of 20 µM and incubated at 37°C for 2 days. Afterwards, the aggregates were kept at 4°C prior to use for a maximum of 1 week.Monomeric α-syn was expressed and purified in P < 0.05. Data are shown as mean ± standard deviation (SD) with each dot representing individual participants. The lower and upper boundaries of the box indicate the 25th and 75th percentiles, respectively. The relationship between the number of aggregates as well as the mean length of aggregates in serum versus CSF was assessed using the Pearson’s correlation analysis for the serum-CSF matched samples. The correlation (R) was considered to be statistically significant when P < 0.05. The statistical difference in aggregate numbers between Parkinson’s disease patients and healthy controls in matched CSF and serum was examined by fitting a linear regression model23 The optimal threshold values for relative α-syn content and the β-sheet α-syn/amyloid-β ratio were assessed using logistic regression.All data were analysed using MATLAB (R2020b). Statistical differences for each variable of interest between the Parkinson’s disease and control groups were assessed by the permutation (exact) test, except for the aggregate length and the sample size, for which the Wilcoxon rank sum test for medians and the binomial test were used, respectively. Statistical signiﬁcance was indicated when The data generated in this study are available from the corresponding author upon reasonable request.18 These experiments show that β-sheet structured aggregates are present in both CSF and serum. The serum was diluted 40-fold and the CSF was diluted 2-fold and a comparable number of counts were detected, indicating about 20-fold more PFTAA active aggregates present in the serum than the CSF. We found no significant difference in the number of PFTAA active aggregates between Parkinson’s disease patients and controls for both CSF and serum  and control  subjects  using linear regression analysis . There was a greater difference in aggregate size between Parkinson’s disease and control serum of 5%, with Parkinson’s disease serum containing a significantly higher proportion of aggregates longer than 150 nm (P = 0.03). In the matched samples, this cumulative difference was ∼2.5% and 5% for CSF and serum, respectively  and control  groups  groups .20  leading to the overlap of their levels between Parkinson’s disease and control serum. This finding implies that the absolute levels of protein aggregates may not be a useful biomarker in Parkinson’s disease but, in contrast, the ratio of aggregates of different proteins is a promising candidate biomarker. Furthermore, the utility of the α-syn to amyloid-β ratio as a biomarker was strengthened by our finding that this ratio discriminated Parkinson’s disease from control serum in two independent cohorts, despite the fact that serum storage time and total aggregate concentration differed in the two cohorts. However, since we have observed an effect of storage time on absolute aggregate concentration in our serum samples, it is important that future studies should carefully control for this parameter and take it into consideration when using the α-syn to amyloid-β ratio as a biomarker.The reported increase in levels of oligomeric α-syn in Parkinson’s disease compared to control CSF in previous ELISA experiments29 α-Syn aggregates are also detectable in erythrocytes at higher levels in Parkinson’s disease versus controls.30 It is also possible that the aggregates secreted from the brain via the CSF may accumulate in the blood, due to slow removal, particularly the larger and more mature fibrillar aggregates, which is in line with our experiments. Irrespective of their peripheral or central origin, our data highlight that detection of aggregates in serum may be a useful biomarker in Parkinson’s disease.Our measurements of aggregate size distribution showed that a higher proportion of larger aggregates were present in serum versus CSF. Interestingly, our PFTAA experiments also indicated that aggregates with higher intermolecular β-sheet content were 20-fold more concentrated in serum than CSF. This is a solution-based method and detects all aggregates that flow through the confocal volume and hence provides a reliable estimate of the relative concentrations. Given that one would expect a significant dilution when the aggregates from the CSF enter the blood, this finding suggests that a high proportion of serum aggregates might be of peripheral origin. α-Syn aggregates are found in the gut early in the course of Parkinson’s disease and may propagate via the vagus nerve to the brainstem.Our data show that there are detectable aggregates of both α-syn and amyloid-β present in both the CSF and serum in Parkinson’s disease patients and healthy control subjects, consistent with the concept that secretion is a major route of aggregate removal by cells under normal conditions, and that these aggregates are altered in size and in their relative proportions in the early stages of Parkinson’s disease. The fact that we observed a small fraction of large aggregates over 100 nm in both control and Parkinson’s disease CSF and serum suggests that some aggregates spend sufficient time in the cell to grow to this size before secretion, as the concentration of α-syn and amyloid-β is too low in the CSF or serum for growth to continue after the aggregates are secreted. Any cellular stress due, for instance, to inflammation or mitochondrial dysfunction, might reduce the cell’s secretion capacity resulting in aggregates growing larger before secretion and possibly also leading to accumulation of aggregates in neurons. Disruption of protein homeostasis in Parkinson’s disease appears to preferentially lead to small increases in the number and size of secreted aggregates of α-syn, compared to aggregates of amyloid-β. It is also possible that small increases in the α-syn aggregates over a prolonged time period may play a role in driving the disease. Importantly, we have observed changes in both α-syn and amyloid-β aggregates, reinforcing the idea that co-pathologies are involved in Parkinson’s disease, and highlighting the importance of measuring both these species.We have also observed a negative correlation in total number of aggregates between matched serum and CSF samples in healthy controls, but a lack of correlation between CSF and serum aggregates in Parkinson’s disease. One possible explanation for the observed serum versus CSF correlation in healthy serum is that aggregates in controls are mainly generated in the brain, and are cleared from CSF into the blood at different rates of efficiency . In contrast, in patients with Parkinson’s disease, it is likely that aggregates are generated both in the periphery and in the brain, hence the lack of correlation between CSF and serum aggregates. A larger study would be needed to confirm this.In conclusion, we found that fibrillar serum aggregates range in size from 20 to 200 nm with an increased number and proportion of larger aggregates (longer than ∼150 nm) in the serum in early-stage Parkinson’s disease patients compared to control subjects. This may reflect accumulation of brain-derived aggregates or peripherally-generated aggregates in the blood over time. These serum aggregates consist of both α-syn and amyloid-β species with a significantly higher proportion of α-syn aggregates being found in Parkinson’s disease patients compared to control subjects. The serum α-syn/amyloid-β ratio measured using our single-aggregate method has high diagnostic potential with accuracy of almost 100% (AUC = 98.2%), albeit in a relatively small group of 11 Parkinson’s disease patients and 10 control subjects. Nonetheless, our results identify a new potential biomarker for Parkinson’s disease diagnosis. We now plan to simplify our methodology, which is currently quite time consuming, prior to validation work in a larger cohort of samples to confirm Parkinson’s disease versus control differences, and to investigate longitudinal changes in Parkinson’s disease patients over time to assess whether detection of α-syn and amyloid-β aggregates is a useful strategy for monitoring Parkinson’s disease progression and the effectiveness of therapies.awab306_Supplementary_DataClick here for additional data file.


Although the structure of AimR has been elucidated for phages SPβ and phi3T, there is still controversy regarding the molecular mechanism of AimR function, with two different proposed models for SPβ. In this study, we deepen our understanding of the system by solving the structure of an additional AimR that shows chimerical characteristics with the SPβ receptor. The crystal structures of this AimR  together with in vitro and in vivo analyses confirm a mechanism of action by AimP-induced conformational restriction, shedding light on peptide specificity and cross regulation with relevant biological implications.The arbitrium system is employed by phages of the SPbeta family to communicate with their progeny during infection to decide either to follow the lytic or the lysogenic cycle. The system is controlled by a peptide, AimP, that binds to the regulator AimR, inhibiting its DNA-binding activity and expression of  The arbitrium system is a peptide-based communication system to coordinate the lysis-lysogenic cycle of phages infecting bacteria. Here Gallego del Sol et al. provide the crystal structure of Aim-receptor of Katmira phage infecting B. subtilis in apo, peptide-bound and DNA-bound form. These peptides are sensed in the medium by membrane-bound histidine kinases from two component systems, or in the bacterial cytoplasm by regulatory receptors after active importation. The RRNPP family  is the largest QS family of cytoplasmic regulatory receptors in Firmicutes. RRNPP receptors regulate a wide array of key biological processes including virulence, sporulation, biofilm formation and natural competence2. Despite the low levels of sequence homology, RRNPP members show a similar architecture consisting of a C-terminal peptide-binding domain and an N-terminal effector domain that, with the exception of the Rap subfamily, possess DNA-binding capacity. The peptide-binding domain consists of 5-9 tetratricopeptide repeats (TPR) that adopt a superhelical fold with a concave inner groove where the regulatory peptide is accommodated2. Binding of the specific peptide to the TPR domain induces conformational changes that regulate the activity of the effector domain but strikingly for proteins with similar architecture, the specific allosteric changes vary amongst receptors even within the same subfamily3.Communication between different members of a community is not restricted to highly evolved animals but is also common in microorganisms that use quorum-sensing (QS) mechanisms for cell-cell information exchange. In Gram-positive bacteria, QS is mostly dominated by the synthesis and secretion of small peptides that work as signalling molecules monitoring population densityBacillus subtilis SPbeta group of phages. This system uses a six amino acid (aa) peptide as a communication signal and plays a key role in lysis–lysogeny decisions during infection4. The arbitrium system is composed of three genes; aimP, which encodes the arbitrium peptide, aimR, encoding a transcription factor that recognises and binds to AimP, and aimX, which exerts a negative regulatory effect on lysogeny, thus promoting lysis by a mechanism that is poorly understood5. AimR is a transcription factor that in its apo peptide-free form promotes aimX expression. In the initial stages of infection when the number of active phages is low, the arbitrium peptide is absent and AimR activates aimX expression, which maintains the lytic cycle of the phage. The arbitrium peptide is initially synthesised as a ~40 aa pro-peptide that via a secretion-internalisation process generates the mature AimP. During phage replication AimP will accumulate in the medium, increasing the intracellular concentration of mature AimP peptide until it reaches the threshold level required to bind to its cognate AimR receptor. Since AimR can no longer activate aimX transcription, a switch to the lysogenic cycle occurs thus preventing the killing of the entire bacterial population by the phage4. This simple, direct and elegant communication system allows phages to decide between lytic and lysogenic life cycles after infection. Importantly, it has been recently described that the arbitrium system is also essential for prophage induction7.Recently, it has been found that phages can also exploit QS to communicate and make collective decisions. As a case in point, a novel system termed arbitrium, has been described in the 4. This proposition has a pivotal biological significance, because although homologous arbitrium systems were initially found in genomes of SPbeta-like phages, recent homology searches in the bacterial, archaeal and phages genomes have revealed that this type of communication system is widespread in the microbial world5. Arbitrium-like systems with the prototypical aimR-aimP organisation are found in many different types of phages, other mobile genetic elements (MGEs) and in the host bacteria. All of these systems can be clustered into nine clades according to their preferential peptide communication code plus a tenth clade that groups systems not present in MGEs, being considered as a possible non-arbitrium outgroup5. Although some clades seem to be exclusive to certain types of elements such as phages or MGEs, other clades encompass a variety of phages, MGEs and host bacteria5. Therefore, the arbitrium system could be a mechanism of communication among species.It is assumed that AimR receptors from different SPbeta phages are specifically regulated by their cognate arbitrium peptide, suggesting that phages only communicate with their own progeny2, subsequent structural characterisation of the AimR from B. subtilis SPβ phage, after which the SPbeta family is named, confirmed that it is a bona fide member of this family8. We and others have recently obtained the crystal structures of the AimRs from the B. subtilis phages phi3T and SPβ , both in their apo and AimP-bound forms12. These studies have shown that these receptors have a HTH N-terminal DNA-binding domain (DBD) encompassing three helices followed by a C-terminal peptide-binding domain composed of nine highly degenerated TPR motifs. Moreover, our previous study identified the AimR-binding sites present in the aimX promoter region. Interestingly, the AimR-binding site exhibited a unique architecture composed of two six base-pair (bp) palindromic sequences separated by an unusually long 25 bp spacer8. The AimRPhi and AimRSPβ structures in complex with their specific AimPs showed an identical way of peptide recognition and binding. But noticeably, the mechanism of action proposed for AimP was somewhat different. For phi3T, the structural data obtained from an AimRPhi receptor with a double mutation showed that AimP induces AimRPhi dimer dissociation9, thus preventing binding to the palindromic operator. However, recent in vitro data with the AimRPhi wild-type receptor showed that AimP does not undergo dimer-to-monomer conversion, with AimRPhi being a dynamic dimer with conformational states that are selectively stabilised by the target DNA or the inhibitory AimP peptide13. Similarly, the different structures solved for AimRSPβ confirm that the arbitrium receptor is constitutively a dimer and binding to its cognate peptide does not disrupt the dimeric organisation. Strikingly however, two mechanisms of action for AimP have been proposed for the AimRSPβ receptor. In the initial structures reported by other groups, the conformation exhibited by AimRSPβ, both in its apo or AimP-bound forms, remained invariable12. By contrast, we have recently proposed that AimRSPβ undergoes drastic conformational changes upon peptide-binding8. In our studies, the overall structure of the individual AimRSPβ protomers are quite similar to those presented in previous studies, however huge differences in dimer arrangement have been demonstrated. These discrepancies have led us to suggest an alternative mechanism of AimR inhibition by the AimP peptide. The structures solved by our group support that apo AimR possesses a high degree of plasticity as a receptor. Once it recognises and binds to AimP, this interaction induces a compact and rigid conformation that approaches the AimR-DNA recognition helices, preventing them from reaching the distal operator palindromic sequences8. However, the alternative structures presented by other groups proposed a radically different mechanism based on the fact that AimR had an open conformation both in the apo and AimP-bound states. In the apo form, the authors proposed that binding to the DNA would force a more compact conformation allowing the AimR DBDs to bind to the palindromic sequences present in the aimX promoter region. Contrary to what we proposed, in this alternative mechanism the authors hypothesised that binding to AimP would stabilise the extended AimR conformation, a disposition that prevents the recognition helices to reach the distal boxes present in the aimX operator10.Although the initial phylogenetic analyses did not identify AimR as a member of the RRNPP familyIn this work, using multiple and complementary experimental approaches, we clarify this discrepancy and clearly establish the molecular mechanism by which AimP alters AimR function. More importantly, our results reveal that AimR receptors have a unique domain organisation that can allow cross regulation among different AimRs. We anticipate these results may have crucial ecological and evolutionary implications in the microbial world.SPβ homologues, we identified a very interesting chimeric AimR, present in a resident phage of B. subtilis KATMIRA1933 strain (hereafter Katmira phage), and recently also found in Bacillus phage vB_BsuS-Goe1114. AimRSPβ and the AimR from Katmira phage (AimRKat) have a 66.9% sequence identity overall but this identity is not equally distributed. Instead, it is localised in specific regions of the proteins. In their N-terminal regions, including the DBD and the two initial TPRs (residues 1–125), AimRKat and AimRSPβ presented almost identical sequences (only a single conservative change in TPR2). However, these proteins showed only 50% identity for the rest of the protein (residues 126–386)  Fig. . This va3–9 Fig. , which a5. In this clade, the mature form of AimP has been proposed to be 6 residues long5. Based on the characterisitics defined for AimP8, as well as the predicted localisation of aimP in the phage genome, we proposed that the active Katmira AimP peptide (AimPKat) would correspond to GIVRGA  responsible for AimP recognition are not conserved in these AimR proteins, we hypothesised that the mature AimP peptides should be also different. The arbitrium system of Katmira phage belongs to clade 2 of clustered arbitrium systems identified during previous phylogenetic analysesRGA Fig. . This peaimX localises 3′ of aimP. We therefore scrutinised this region in the Katmira phage genome to identify the DNA-binding region recognised by AimRKat. A putative AimRKat binding box, consisting of 6 bp inverted repeats separated by 25 bp (ATCACT-25-AGTGAT), was identified . Interestingly, and in support of the fact that both AimRSPβ and AimRKat have identical DBDs, the putative aimX-boxKat was almost identical to that found for AimRSPβ downstream of aimPSPβ (aimX-boxSPβ) aside from two substitutions in the spacer region8. Further, both aimX-boxKat and aimX-boxSPβ sites possess high adenine and thymine content, providing flexibility in this region  and biolayer interferometry (BLI) assays. The EMSA analyses using AimRKat and AimRSPβ and DNA fragments encompassing their cognate DNA-binding regions (aimX-boxKat and aimX-boxSPβ) showed almost identical binding profiles  used as negative control . The experiment showed a biphasic thermogram, suggesting an allosteric effect between the two AimP-binding sites, one in each AimRKat monomer, within the dimer. However, the peptide GIVRGA binds to the AimRKat dimer with a similar high affinity at both peptide sites , supporting a weak but existing cooperativity between the two AimP-binding sites on the dimeric AimR receptor  nor SAIRGA   Fig.  affectedbox Fig. . Taken t7. We showed that after treatment of the wild-type (wt) and aimR-mutant prophages with mitomycin C (MC), which activates the bacterial SOS response, the aimR-mutant showed a 50× reduction in the phage titre compared to that observed with the wt phage along with a cloudy-diffuse morphology of the plaques6 6, confirming that the formation of the AimP-AimR complex interferes with prophage induction   HTH DBD followed by a C-terminal regulatory domain composed of 19 helices (α4-α22) arranged as 9 degenerated TPRs  and the TPRC-ter (TPR7-9) connected by a linker region of 30 residues that includes one α helix (α16) , but differs in the dimerisation surface with the AimRSPβ structures reported by other groups12  are virtually identical , almost identical unit cells and similar crystallisation conditions  for further structural comparisons. While the AimRSPβ protomers in the AimRSPβ-II structures are quite similar to those observed in the apo AimRSPβ-I and AimRKat structures (RMSDs < 0.9 Å for protomers superimposition), the conformation of the AimRSPβ-II dimer varies dramatically among these two groups  in the AimRSPβ-II model, which is a consequence of the separation of the two protomers  and TPR9 C-terminal capping helix (α22) and the other one located at the TPRN-ter subdomain generated by residues from TPR3 (α9 and α10) and TPR4 (α11) 9, a distantly related AimR receptor which has 40% identity with AimRSPβ, showing the closed dimer and presenting both the conserved C-terminal leucine zipper and the variable slipping dimerisation surfaces  Figs. , 3a. The6) Figs. , an olig6) Figs. . The dimKat peptide inhibition we attempted to co-crystallise AimRKat with AimPKat. The structure of the complex (AimR-AimPKat) was determined at a resolution of 2.7 Å by molecular replacement (Supplementary Table SPβ complex (PDB 6PH5) as a model. The AimR-AimPKat asymmetric unit contains two AimR molecules arranged as a dimer, with one AimPKat bound to the TPR domain of each AimRKat protomer . This results in a more compact structure with a shorter distance between the TPRN-ter and TPRC-ter subdomains, reducing the TPR superhelix pitch up to 5 Å  in the presence of AimPKat, confirming that the cognate peptide strongly stabilises AimRKat. Conversely, AimPSPβ or AimPPhi, used as control peptides, did not have any effect  the Tm of AimRSPβ, supporting the idea that AimP induces a similar conformational compaction that stabilises this type of AimR receptor8. Indeed, the reporter comparison for AimRSPβ-I in its apo and AimPSPβ-bound structures  showed a similar closing movement for the protomers in the presence of the peptide (reduction of ∼5 Å in the superhelix pitch) resulting in more structurally compact AimR protomers8  have been reported by the same groups that solved the AimRSPβ-II structures12    which includes the aimX-boxKat with the two 6 bp inverted repeats recognised by DBD domain  and a 25 bp spacer. AimRKat-DNA crystals belong to space group P21 and diffracted to a resolution of 2.5 Å  as a model. The AimRKat-DNA asymmetric unit contains two AimR molecules arranged as a dimer and one duplex aimX-boxKat molecule , however, RMSD differences are not constant throughout the entire molecule. RMSD differences are minimal in the N-terminal part, which is bound to the DNA, and increase towards the C-terminal domain  and the identical inverted repeats in their target DNA boxes , N109 (loop connecting α7 and α8), R143 and K145 (loop connecting α9 and α10) mediate hydrogen-bound salt bridges with the DNA phosphates along the entire length of the spacer  extension that is partially visible in several of the structures with the ‘open’ conformation  from the tag enlarges the leucine zipper of the capping C-terminal helix that orchestrates AimR dimerisation. The tag E388 establishes intermolecular contacts with residues from the second protomer and some His present in the tag (up to three are visible in some AimRSPβ-II PDBs) are projected towards the slipping dimerisation surface, interacting with residues from TPRN-ter and TPRC-ter subdomains  and AimR-AimPKat (RMSDs 0.8–1 Å), with the exception of the C-terminal capping helices that are displaced around 4.5 Å   showing both constant and slipping dimerisation surfaces10, which are similar to the AimRKat-DNA reported here  and the second one from the protein including the C-terminal poly-His10(PDB 6JG8). Inspection of the tagged structures showed that only the tag for one monomer was visible and it protrudes outside the AimR dimer , both use an identical peptide recognition mechanism  by a hydrogen bond and a salt bridge, respectively. On the opposite end, the C-terminal carboxylate establishes a salt bridge with the R228 side chain (TPR6). These residues, involved in anchoring the ends of the peptide, are highly conserved among the clade 2 family of arbitrium receptors8, supporting an identical peptide accommodation in the TPR domain. A majority of TPRs possess side chains that provide a mainly hydrophobic cleft where AimPKat is accommodated while the peptide main chain is stabilised by hydrogen bonds with AimRKat N202 and N273 side chains  and, consequently, the residues surrounding these AimP side chains also show differences between AimRKat and AimRSPβ  that anchors AimPKat V3 by interacting with its N and O backbone. Conversely, in AimPSPβ the Pro residue at this peptide position eliminates the hydrogen bridge within the peptide N backbone and also its rigid pyrrolidine side-chain would introduce steric hinderance with the AimRKat N273 side chain  is accommodated by both AimRs in a similar large hydrophobic pocket without major steric constrictions  to emulate AimRSPβ, and its AimP-binding capacity was tested. ThermoFluor assays showed that AimPSPβ stabilises AimRKat-N273A but not AimRKat  with respect to wt AimRKat and has acquired the capacity to bind AimPSPβ, although with low affinity   separated by the prototypical 25 bp spacer  exhibits a much lower (<40%) sequence identity . The fact that these structures presented an unusual DBD separation even in the apo form prompted the authors to propose that in the absence of peptide, the DNA would induce a conformational change that would approach the DBDs to a more canonical separation. Since in the presence of the AimP peptide these authors did not see any change in the AimR structure, they hypothesised that the peptide would block the DNA-induced conformational change in AimR, preventing DNA binding11. In contrast, the structures solved by our group showed high plasticity in the apo form of AimR and that AimP induces, as in other members of the RRNPP family2, major conformational changes that cause a more compact and “closed” conformation in AimR. Further, our study revealed that the AimRSPβ box presents an unusual DNA operator composed of two palindromic 6 bp repeats separated by a long 25 bp spacer8. Therefore, the atypical DBD separation observed in the AimR apo form is necessary to accommodate the non-canonical DNA operator without any modification to the AimR structure. This was confirmed after solving the structure of the AimRSPβ-DNA complex8. In this way, the “closed” conformation observed in the structure of AimRSPβ bound to the regulatory AimP explains how the conformational changes to the DBDs prevent the DNA binding. It should be noted that this plasticity of the apo form and the atypical separation of the DBDs is biologically relevant, as our assays showed that AimRSPβ binds alternative operators to aimX in the phage genome whose boxes are spaced by more than 28 bp8.The recent description of the arbitrium communication system was a game changer in the perception of phage ecologyaimX expression in the SPbeta phages, but also have explained the origin of these discrepancies. While all our structures were obtained after removing the His-tag present in the AimR proteins, this was not the case for the other structures, which were obtained using AimRs that retained the His-tag in their C-terminal regions. Unfortunately, the presence of this extra C-terminal tag altered the AimR conformation obtained in the crystals. In fact, the extra residues L387, E388, H389 and H390 are clearly defined in the experimental map, being embedded in the dimer interface. In this position, the C-terminal tag residues participate in crucial interactions with TPR3 and TPR4, pulling away from this second dimerisation interface, which works as a slipping surface for receptor plasticity. This case is only one example of how the introduction of additional elements (His-tag residues) can distort results and emphasises the need for caution in their use when attempting to understand mechanistic inferences. Several reported examples have shown that the introduction of even a small purification tag may modify the protein behaviour, altering (increasing or decreasing) its intrinsic activity or conferring new capabilities20. In general, it has been observed that tags do not usually have major structural impacts21. However, this is not true in some cases and some structures have shown how the tags establish multiple interactions23, inducing conformational changes that, in the absence of other structural data, may lead to erroneous mechanistical interpretations, exemplified by some of the published AimR structures. Moreover, the structural characterisation of the AimR of phage phi3T was carried out in parallel with that of phage SPβ showing striking differences. For AimRPhi, a dimer-monomer transition induced by the AimP was observed, proposing different mechanisms of action for the inhibitory peptide. However, these structural analyses were performed with a double mutant of AimRPhi that, precisely, involved two residues located in the dimerisation region9. The recent biophysical characterisation of AimRPhi wt has shown that the inhibitory peptide does not induce monomerisation of the receptor, and that the receptor behaves as a dimer in the presence and absence of AimP13, supporting a mechanism of action similar to that observed for AimRSPβ and AimRKat. In parallel, these recent observations with AimRPhi confirm that the use of modified variants (by tags or mutations) can have undesirable parallel functional impact and that it is necessary to be cautious about the biological mechanism deduced.In this manuscript, we have not only confirmed and clarified the mechanism of action by which AimP blocks 8, the conservation of DBDs would suggest that AimR could be controlling similar and essential processes for the phage life cycle beyond aimX. This could lead to a scenario where the presence of two related phages infecting the same host activate similar pathways, even though they do not communicate directly with each other. Therefore, the arbitrium system could allow the development of social behaviours such as altruism or cooperation, which have already been observed for other viruses24. Furthermore, the structures of AimRKat confirm that in the presence of the peptide, the DNA recognition helices remain exposed in a competent DNA-binding disposition, as was also observed for AimRSPβ8. However, the relative distance of the helices in the peptide-stabilised state is different in each receptor, which indicates that in this conformation the AimR could regulate different operators with alternative spacing between the boxes. This attractive proposition, which is currently under analysis, would open the door to a dual regulation role for arbitrium, one general for “relative” phages and one species-specific modulated by the cognate AimP.The confirmation of AimRs as receptors with a high degree of plasticity, whose intrinsic flexibility is restricted by the peptide, has important biological and evolutionary implications. Here, we have confirmed the AimR modularity with DBD effector  and TPR regulatory  portions that, interestingly, seem to show different evolutionary pressures. Identical DNA operators are recognised by AimRs that are regulated by different, but related, peptides. Given that the plasticity of AimR allows for the recognition of alternative operators with different spacingKat and AimRSPβ structures revealed that a limited number of residues provide the peptide specificity in related AimRs. By changing only one of these residues we modified the receptor specificity, making the AimR responsive to a non-cognate AimP. Similarly, a single residue mutation in RapF, another member of the RRNPP family, caused a change in peptide specificity21. These results would support the hypothesis that phages can modify their affinity and selectivity for regulatory peptides with minimal mutational changes in their AimRs. In this way, phages would be rapidly segregated during their evolution so that, as proposed, evolved phages would only communicate with their own progeny4. However, the results also indicate that these minimal changes do not result in complete isolation, as the receptors can still “hear”, albeit with different intensity, peptides from related phages. Therefore, in this evolutionary process, the receptors could exhibit crosstalk capabilities allowing communication between different phages. This possibility has not been evaluated experimentally as studies of arbitrium systems have been reduced to a very limited number of cases and, moreover, belonging to very distinct groups of phages. Our vision, which is entirely speculative, is that related phages may not only present cross regulation but also crosstalk, showing the arbitrium system to be involved in complex phage-phage interactions. The confirmation of this hypothesis would open the interesting possibility that this quorum-sensing mechanism could confer social behaviours to arbitrium-carrying phages.The comparative analyses of AimRB. subtilis strains 168 and Δ6 were obtained from the Bacillus Genetic Stock Centre (BGSC) and B. subtilis subsp. KATMIRA1933 was a gift from professor Wilfried J.J Meijer. B. subtilis strains were grown at 37 °C on LB (Miller) agar plates or in LB (Miller) broth liquid medium shaking at 210 rpm. Escherichia coli DH5α was grown at 37 °C on LB (Lennox) agar plates or in LB (Lennox) broth shaking at 210 rpm. When required, antibiotics were utilised at the following concentrations: kanamycin (10 µg ml−1), ampicillin (100 µg ml−1) or spectinomycin (100 µg ml−1).Bacterial strains used in this study are listed in Supplementary Table SPβ and AimRKat were cloned into the amyE integration vector pDR110 under the control of the IPTG inducible promoter Pspank25. Cloning was performed after PCR amplification of the appropriate template DNA with the same forward primer (AimR-SPβ-1mH) but different reverse primer . Competent cells preparation and transformation was performed as described by Bron et al.26. Briefly B. subtilis cells were grown in minimum medium to early stationary phase to induce natural competence and 1 µg of plasmid was added and incubated at 37 °C for 20 min with shaking at 210 rpm. Then, 50 μl of 10% casamino acids were added to the cells and incubated for a further 90 min with the same conditions. The culture was centrifuged at 8000 × g for 5 min, 800 μl of the supernatant removed, and the pellet re-suspended and plated out onto the relevant antibiotic plates. Plates were incubated at 37 °C for 24 h.Plasmids generated in this study are listed in Supplementary Table Kat protein expression, the Katmira aimR gene was amplified using primers Kat33Plic_FW and Kat33Plic_RV and genomic DNA from Bacillus subtilis subsp. KATMIRA1933 as template. The PCR product was purified and cloned into pLicSGC1 plasmid using Ligation-Independent Cloning (LIC) system as previously described27. The resulting pLIC-AimRKat33 plasmid expressed AimRKat (residues 1–386) with an N-terminal 6xHistag followed by a TEV protease cleaving site. The AimRKat-N273A mutant was generated using Q5 Mutagenesis Kit (NEB), pLIC-AimRKat33 plasmid as template and primers Kat33N273A_FW and Kat33N273A_RV. For production of AimRSPβ with the C-terminal His-tag, the aimR gene from the SPβ phage was amplified using primers SPbCter_FW and SPbC-ter_RV and genomic DNA from B. subtilis strain 168 as template. In parallel, pET21b vector (Novagen) was amplified using primers pet21_FW and pet21_RV. Both PCR products were purified and assembled using NEBuilder kit (NEB). The resulting plasmid expressed AimRSPβ (residues 1–386) plus LEYAHHHHH C-terminal tag (AimRSPβ-II).For AimR−1 was added to the culture. Where experiments were performed to test the effect of peptide on phage titre, 5 μM of the relevant peptide was added before starting the second growth to ensure the bacteria cells were able to incorporate the peptide. The induced cultures were incubated at 30 °C with 80 rpm shaking for 4 h and then left overnight at room temperature. Following lysis, samples were filtered using 0.2 μm filters and lysates were stored at 4 °C until use.For induction, an overnight culture was diluted 1/100 in LB media supplemented with 0.1 mM MnCl2 and 5 mM MgCl2 and then grown at 37 °C with 210 rpm shaking until reaching absorbance 0.2 at 600 nm. This step was repeated twice to ensure the cells were in exponential growth. After the second growth, Mitomycin C (MC) at 0.5 μg mlB. subtilis ∆6 or with the corresponding plasmid) was diluted 1/100 in LB supplemented with 0.1 mM MnCl2 and 5 mM MgCl2 and then grown at 37 °C with 210 rpm shaking until reaching absorbance 0.2 at 600 nm. When required 1 mM IPTG was added. Then, 100 μl of recipient bacteria was infected with 100 μl of serial dilutions of phage lysate in phage buffer  at room temperature for 10 min and 3 ml of phage top agar (LB media supplemented with 0.1 mM MnCl2 and 5 mM MgCl2 and 0.7 % agar) at 55 °C was added to the culture-phage mix and immediately poured over phage base agar plates (LB media supplemented with 0.1 mM MnCl2 and 5 mM MgCl2 and 1.5% agar). Plaques were counted after overnight growth at 37 °C temperature and photographed.The number of phage particles contained in the phage lysate of interest was quantified by a titering assay. An overnight culture of the relevant recipient strain  RIL (Agilent) carrying the corresponding expression plasmid  and disrupted by sonication on ice. The sonicated lysate was clarified by centrifugation at 10,000 × g for 1 h. The supernatant was loaded onto a 5 ml HisTrap FF , washed with lysis buffer and eluted with lysis buffer supplemented with 500 mM imidazole. In order to remove the His-tag, AimRKat was digested with TEV protease (50:1 molar ratio protein:TEV) and dialysed against dialysis buffer . The sample was concentrated in a centrifugal filter (Amicon ultra 30 KDa) and loaded onto a Hi-Load Superdex 200 16/60  gel filtration column previously equilibrated with lysis buffer. Fractions containing the purest protein were pooled, concentrated at 90 mg ml−1 and stored at −80 °C. Typical yields were 25 mg recombinant protein/L of culture medium. AimRKat-N273A was purified following the same protocol. AimRSPβ was produced and purified as previously described8. AimRSPβ -II was purified using the same protocol, but excluding the TEV digestion step.AimRKat crystallised in 0.1 M Lithium sulfate, 0.1 M HEPES 7.0 and 30% w/v Polyvinylpyrrolidone. Crystals of AimRKat in complex with GIVRGA (AimR-AimPkat) peptide were obtained adding 1 mM of the peptide to the protein solution and using 0.2 M Potassium acetate, 0.1 M MES 6.0, 15% v/v Pentaerythritol ethoxylate (15/4 EO/OH) and 3% v/v Jeffamine T-403 as precipitant. Crystals of AimRKat in complex with DNA were obtained using protein at 10 mg/ml mixed with duplex DNA (IDT) at final molar stoichiometry 2:1 protein:DNA and 50% PEG400, 0.1 M Sodium acetate and 0.2 M Lithium sulphate as precipitant. Crystals grew in 1–7 days and were directly flash frozen in liquid nitrogen. Diffraction data was collected from single crystals at 100 °K at XALOC beamline (ALBA Synchrotron), I24 beamline from DLS (Diamond Light Source) and ID23-2 from ESRF (European Synchrotron Radiation Facility). Data sets were processed with XDS28 and reduced using Scala29(CCP4). The data-collection statistics for data sets used in structure determination are shown in Supplementary Table The crystals were grown in hanging drops at 21 °C with a vapour-diffusion approach. Initial crystallisation trials were set up in the Cristalogenesis service of the IBV-CSIC using commercial screens JBS I, JBS II (JENA Biosciences) and MIDAS (Molecular Dimensions) in 96-well plates (Swissci MRC2) using equal volumes of protein at 10 mg/ml in lysis buffer and precipitant. The apo form of AimR30 (CCP4). To solve AimRKat structure the PDB 6HP3 was used as search model in the molecular replacement. Then, the AimRKat refined model was used to AimR-AimPKat structure. Finally, the structure of AimRKat-DNA complex was solved using PDB 6HP7 as search model. To generate the final models, several rounds of manual model building were completed using COOT31 and computational refinement with Refmac32 (CCP4). Refinement statistics are summarised in Supplementary Table https://pymol.org/2/).The structures reported in the manuscript were solved by molecular replacement using Phaser33. Briefly, samples of 20 μl buffer (20 mM Tris pH 8 and 250 mM NaCl) containing 5× Sypro Orange (Sigma-Aldrich) and 20 μM of protein were loaded in 96-well PCR plates. To calculate the Tm in the presence of the peptide, 0.5 μM of GMPRGA or GIVRGA peptide was added to the mixture. Samples were heated from 25 to 85 °C in steps of one degree. Fluorescent intensity was plotted versus temperature and integrated with GraphPad Prism software using a Boltzmann model to calculate melting temperatures.The thermal shift assay was conducted in a 7500 Fast Real time PCR System (Applied Biosystems) as previously described−1) and AimR (from 0.5 μM) protein was mixed in EMSA buffer . The samples were incubated for 10 min at room temperature. For peptide inhibition assay, protein was preincubated with 0.5 μM peptide for 10 min before DNA addition. Electrophoresis was then performed in 8% polyacrylamide gels in Tris-Borate-EDTA (TBE) buffer for about 150 min at 100 V at 4 °C.AimR binding to its operator and the inhibition induced by the arbitrium peptide was analysed by native polyacrylamide and agarose gel electrophoresis. Double strand DNA primer probes were purchased from Macrogen. DNA (10 ng µlKat or AimRKat-N273A) and peptides concentration were 10 and 50 μM, respectively, and both were diluted in buffer 25 mM Tris-HCl pH 8, 250 mM NaCl. The experiments were performed at 25 °C. The data obtained was integrated, corrected and analysed using the NanoAnalyze software (TA Instruments) with a single-site or two-site binding models34.ITC assays were carried out in a Nano ITC Low Volume (TA instruments). In the assays, proteins (AimRD), association (kon) and dissociation (koff) rate constants between AimRs and DNA were measured by biolayer interferometry (BLI) using the BLITz system (FortéBio). Biotinilated DNA probes (IDT)  supplemented with 0.01% tween and 0.1% BSA. Proteins were diluted in PBS. At least four different dilutions of AimR were used in a range between 0.015 and 2.5 μM. A blank using a chip without bounded DNA was used to subtract unspecific binding. Kinetic value calculations and data analysis were performed using BLItz Pro 1.2 software employing a 1:1 model to fit the data.The Binding affinity (K35. 50 μl of protein at 5 mg ml−1 were injected in a KW-803 (Shodex) column equilibrated with 25 mM Tris pH 8, 250 mM NaCl. When testing the peptide in the AimRKat oligomeric state, 1 mM peptide was added to the injected sample and the running buffer was supplemented with peptide at a final concentration of 1 μM. The Astra 7.1.2 software from the manufacturer (Wyatt) was used for acquisition and analysis of the data.SEC-MALS experiments were performed using a Wyatt DAWN HELEOS-II MALS instrument and a Wyatt Optilab rEX differential refractometer (Wyatt) coupled to an AKTA pure system Peptides used in this study were purchased from Proteogenix at 95% purity.Further information on research design is available in the Supplementary informationPeer Review FileReporting summary


FAM111A are causative for Kenny-Caffey syndrome type 2. Patients with Kenny-Caffey syndrome suffer from severe growth retardation, skeletal dysplasia, hypoparathyroidism, hypocalcaemia, hyperphosphataemia and hypomagnesaemia. While recent studies have reported FAM111A to function in antiviral response and DNA replication, its role in regulating electrolyte homeostasis remains unknown. In this study, we assessed the role of FAM111A in the regulation of serum electrolyte balance using a Fam111a knockout (Fam111a/−−) C57BL/6 N mouse model. Fam111a/−− mice displayed normal weight and serum parathyroid hormone (PTH) concentration and exhibited unaltered magnesium, calcium and phosphate levels in serum and 24-hour urine. Expression of calciotropic , magnesiotropic  and phosphotropic  genes in the kidneys, duodenum and colon were not affected by Fam111a depletion. Only Slc34a2 expression was significantly upregulated in the duodenum, but not in the colon. Analysis of femurs showed unaffected bone morphology and density in Fam111a−/− mice. Kidney and parathyroid histology were also normal in Fam111a−/− mice. In conclusion, our study is the first to characterise the function of FAM111A in vivo and we report that mice lacking FAM111A exhibit normal electrolyte homeostasis on a standard diet.Autosomal dominant mutations in  In addition, dental and ocular defects are frequently observed6. While KCS2 patients can reach adulthood, a similar but more severe disorder called gracile bone dysplasia  is perinatally lethal7. Both KCS2 and GCLEB were found to be caused by mutations in the FAM111A gene located on chromosome 11q12.14. While all known mutations are located in and around the serine peptidase domain and the autocleavage site, the specific mutations are different between KCS2 and GCLEB17.Kenny-Caffey syndrome type 2  is a rare hereditary disorder characterised by short stature, cortical thickening and medullary stenosis of the long tubular bones, delayed closure of the anterior fontanelle, hypoparathyroidism and electrolyte imbalances: hypocalcaemia, hyperphosphataemia and hypomagnesaemia19. It plays a role in DNA replication, loading proliferating cell nuclear antigen (PCNA) onto chromatin19 and preventing fork stalling, potentially through proteolytic removal of protein obstacles from the genomic DNA18. While FAM111A is required for DNA replication19, its overexpression also reduces DNA replication20 and leads to caspase-dependent programmed cell death in the osteosarcoma cell line U2OS20. In addition, FAM111A was identified as a host range restriction factor, meaning that it could prevent viral replication by certain viruses, in particular simian vacuolating virus 40 (SV40), rabbitpox virus and vaccinia virus22. This effect is achieved through inhibition of viral replication centre formation and viral DNA replication by disrupting the host cell nuclear barrier function24. Viruses that contain SV40 large T antigen (LT) or serine protease inhibitor 1 (SPI-1), on the other hand, can overcome the host defence through inhibition of FAM111A22. While we are beginning to understand some of the molecular functions of FAM111A, it remains unclear how these relate to the observed phenotype in KCS2 and GCLEB patients.Over the last years, several studies aimed to investigate the function of FAM111A in more detail. FAM111A has been postulated to be a protease expressed in a cell-cycle dependent manner that exhibits autocleavage2+ and Mg2+ homeostasis mainly occurs by absorption from the intestine, storage in bone and reabsorption by the kidneys. The thick ascending limb (TAL) and the distal convoluted tubule (DCT) segments of the nephron are important for the renal handling of these ions by expressing specific channels and transporters, thereby regulating ion excretion into the urine according to the requirements of the body26. Disturbed electrolyte homeostasis is an essential feature of KCS2, but the primary cause of the electrolyte disturbances has not been established. Hypoparathyroidism has been proposed as the main cause, as parathyroid tissue was absent in a KCS2 patient27. Indeed, primary hypoparathyroidism is in line with patients’ increased bone density as well as hypocalcaemia and hypomagnesaemia. However, Isojima and coworkers demonstrated that magnesium supplementation restored parathyroid and calcium levels in a patient, suggesting that hypomagnesaemia is the primary defect3. Interestingly, in a disorder called hypomagnesaemia with secondary hypocalcaemia (HSH), caused by mutations in the magnesium channel TRPM6, patients also present with secondary hypoparathyroidism28. Of note: hypoparathyroidism only occurs as a consequence of hypomagnesaemia if serum magnesium levels are severely low29. A major obstacle to the investigation of FAM111A in electrolyte homeostasis is the absence of a suitable physiological study model, as cell models do not represent the complex interplay between organs or cell types in vivo.Regulation of CaFam111a knockout (Fam111a−/−), heterozygous (Fam111a/−+) and wild type (Fam111a/++) mice. The mice were kept in metabolic cages to collect faeces, blood and urine for biochemical analysis. The parathyroid glands, kidneys and intestines were subjected to immunohistochemistry and expression analyses, and bone structure was analysed by micro-computed tomography (micro-CT).To increase our understanding of the electrolyte disturbances in KCS2 patients, we generated Fam111a/−+ mice, the protein coding region of Fam111a was substituted by the ZEN-Ub1 cassette which contains the E. colilacZ reporter gene and neomycin resistance gene , Fam111a/−+ (20) and Fam111a−/− (8), which was not significantly different from the expected proportion . The male to female ratios of the animals used for the study were 4:4, 5:3 and 3:4, respectively. Genotyping confirmed the presence of the cassette in Fam111a−/− and Fam111a/−+ mice and the Fam111a allele in Fam111a/++ and Fam111a/−+ animals  using RNA isolated from the kidneys, liver, proximal duodenum and distal colon. Fam111a expression was significantly reduced (p < 0.05) by approximately 50% in all tested tissues except the distal part of the colon of Fam111a/−+ mice compared to the Fam111a/++ group. Fam111a expression was absent in the Fam111a−/− mice in all the measured tissues  levels, which are decreased in the serum of KCS2 patients, did not differ between Fam111a/++, Fam111a/−+ and Fam111a−/− mice in serum  and in urine  (Table 2+) concentration is low in KCS2 patients, but was not altered in the Fam111a−/− mice compared to Fam111a/++ and Fam111a/−+ groups . Urine excretion of Mg2+ was similar in Fam111a/++, Fam111a/−+ and Fam111a−/− mice . Serum phosphate (PO43−) levels are high in KCS2 patients, but no differences were found between the Fam111a/++, Fam111a/−+ and Fam111a−/− mice  and urine PO43− levels were unchanged . There were no differences in serum sodium (Na+) levels between the groups  and urine Na+ was similar for all the groups as well . Finally, potassium (K+) levels were not altered in serum  and urine  of Fam111a/++, Fam111a/−+ and Fam111a−/− mice. Albumin serum levels were measured as a control and were the same in each group .Calcium Ca+ levels,Fam111a−/− animals and no fibrosis or infiltration of immune cells was observed  staining of the kidney tissue showed normal glomerular and tubular structures  in ved Fig. A,B. To iFam111a−/− compared with Fam111a/++ mice. The expression of Fam111a and the mitochondrial genes Glycine N-acyltransferase (Glyat) and Glycine N-acyltransferase-like protein (Gm4952) was significantly downregulated in the Fam111a−/− mice compared to the Fam111a/++ group . Solute carrier protein 47 member 1 (Slc47a1) and the E3 ubiquitin-protein ligase Midline-1 (Mid1) showed significantly higher level of transcripts in Fam111a−/− mice than in the Fam111a/++ group .RNA sequencing (RNAseq) was performed to identify differences in the RNA transcriptional profile of kidneys from 2+ and Ca2+ by modulating transcellular reabsorption via TRPM6/7 Mg2+ channels and TRPV5 Ca2+ channels26. To study whether there are differences in the length of the distal convoluted tubule (DCT) between Fam111a/++ and Fam111a−/− mice, immunohistochemical staining was performed on kidney sections  is important in determining the urine excretion of Mgons Fig. A,B. The oup Fig. C. The siice Fig. C. The raals Fig. C.Figure Fam111a/++, Fam111a/−+ and Fam111a−/− mice. The expression levels of Trpm6, Trpm7, Cnnm4, Slc41a1 and Slc34a2 in the distal part of the colon of Fam111a/−+ and Fam111a−/− mice were similar to those in the Fam111a/++ group  , or the structure model index (plate- versus rodlike appearance of trabecular bone).KCS2 patients have a particular bone phenotype including cortical thickening and medullary stenosis of the long bones. To investigate a potential role of FAM111A in skeletal development, micro-CT was used to characterise structural remodeling of femoral bone in Fam111a−/− mice. The polar moment of inertia (a proxy for bone strength) was also the same among the groups. Taken together, in contrast to patient findings where increased cortical thickening was described, there are no observable alterations in the bone microarchitecture upon deletion of Fam111a.The bone cortical indices were also monitored for structural changes Fig. B. CorticFam111a−/− animals was similar to that in the Fam111a/++ group  of mouse kidney tissues identified a cluster of early DCT-like cells with high enrichment of cell cycle and replication factors suggesting a proliferative potential in these DCT cells31. However, total DCT area as well as total early DCT area was similar in the Fam111a/++ and Fam111a−/− mice.Expression analysis demonstrated that duodenal Fam111a−/− mice, including: (1) the nature of FAM111A mutations (loss of function vs gain of function), (2) compensatory mechanisms for Fam111a deletion, (3) differences between mouse and man. We will elaborate on these points in the following sections.Several explanations can contribute to the absence of the patient phenotype in FAM111A patient mutations on the protein function are unclear: specifically, whether it leads to a gain or loss of protein function. Indeed, the presence of mutations in the FAM111A catalytic site in some KCS2 patients are in line with a loss of function effect32. However, recent in vitro studies have pointed towards a gain of function effect on the protease activity of FAM111A20. On the other hand, because the FAM111A protease activity can result in autocleavage, it has been also hypothesized that activating mutations in FAM111A eventually will result in loss of function by enhanced autocleavage18. The present study seems to support the viewpoint that a gain of function rather than a loss of function of FAM111A is responsible for the observed pathology, as we did not observe an electrolyte phenotype in the knockout mice. Mouse models of electrolyte disorders due to hyperactivating mutations such as autosomal dominant hypocalcemia (ADH) and familial hyperkalemic hypertension are generated by knock in of the relevant mutation in mice37. Similarly, transgenic knock in mouse models expressing the FAM111A patient mutations may better reflect the phenotype seen in patients with KCS2.The exact consequences of the nature of the 18. Spartan (SPRTN), the most well characterised DPC protease, possesses some overlapping functions with FAM111A in preventing replication fork obstacles by Top1-cleavage complexes (Top1cc) and formaldehyde38. Loss of function mutations in spartan result in Ruijs-Aalfs syndrome (RJALS) with features such as premature aging, cataracts, stunted growth, skeletal deformities and liver cancer39. However, unlike Fam111a−/− mice, complete deletion of spartan in mouse models is embryonically lethal while hypomorphic mice develop symptoms of RJALS40. It is thus possible that unidentified proteases may compensate for the loss of FAM111A in mice.Based on our study, we cannot exclude that the function of FAM111A is rescued by compensatory mechanisms. FAM111A has been recently shown to function as a DNA–protein crosslinking (DPC) protease44. Also, knockout mouse models have been useful in identifying electrolyte related function of genes that do not encode for transporters (indirect regulation). For example, the roles of Adenylate cyclase 3 (Adcy3) and calcineurin in regulating Mg2+ handling were described using knockout mouse models46. However, not all knockout mouse models recapitulate the phenotype associated with the human disease. For example, Fxyd2 knockout mice have a normal serum Mg2+ concentration, whereas loss-of-function mutations in FXYD2 are responsible for isolated dominant hypomagnesemia in patients48. In addition, Trpm6 knockout mice did not display hypomagnesaemia as is the case in the disease hypomagnesaemia with secondary hypocalcaemia (HSH) caused by mutations in Trpm6. Similar observations have been made in mice deficient in49Cnnm2, suggesting that magnesium homeostasis in mice may be differently regulated compared to man. Mice are also commonly used to study parathyroid function, but Günther et al. found that glial cells missing homolog 2 (Gcm2) deficient mice and parathyroidectomised mice had normal serum PTH levels, as the thymus provided a backup mechanism of PTH secretion50. Indeed, mice harbouring a point mutation in Tbce had no parathyroid glands, but normal PTH levels51. Therefore, mice may not be a suitable model for studying the role of FAM111A in electrolyte handling.Electrolyte imbalance is a prominent feature of Kenny-Caffey syndrome 2 and knockout mouse models have been employed extensively in elucidating the transport function of genes that encode transport proteins23. Deletion of FAM111A in HAP1 cells did not affect cell viability and proliferation unless they were treated with DNA-damage agents18. It would be interesting to see how Fam111a−/− mice would respond to stress conditions where FAM111A is known to play a role such as response to viral stress (LT-virus and orthopoxvirus) or DNA replication stress induced by PARP1 and/or topoisomerase 1 (TOP1) inhibitors. In addition, dietary restriction of electrolytes could be a trigger to develop electrolyte related symptoms. An example was described in serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice which displayed subtle changes in electrolyte regulation compared to control mice in normal conditions and challenging them with dietary NaCl restriction impaired renal sodium handling52. Finally, DCT cells are known to structurally adapt to different stimuli, such as furosemide and low sodium or potassium diets55. Investigation of how these mice will respond to these stimuli may explain a potential role of Fam111a in DCT replication and remodelling.FAM111A has been described as a DNA replication stress response protein in in vitro studies by preventing replication fork stalling caused by poly-ADP-ribose polymerase 1 (PARP1) accumulation as well as by responding to viral stress, preventing viral DNA replicationFam111a−/− mice display normal electrolyte homeostasis, PTH levels, growth, and skeletal development. Expression of relevant electrolyte transporters was not altered in the kidneys and intestines. Hence, knockout of FAM111A does not affect renal electrolyte handling in mice on a standard diet.In conclusion, our data show that Fam111a mouse strains used for this research project were created from embryonic stem (ES) cell clone 19174A-G2, 19174A-A1 generated by Regeneron Pharmaceuticals, Inc. This ES cell clone was subsequently used by the KOMP Repository (https://www.komp.org/) and the Mouse Biology Program (www.mousebiology.org) at the University of California Davis to produce chimera mice. Methods used to create the Velocigene-targeted alleles have been reported previously56. The non-conditional allele Fam111atm1(KOMP)Vlcg was generated by insertion of ZEN-UB1 cassette targeting chromosome 19 in ES cells, which results in the deletion of 4,553 nucleotides  corresponding to the entire protein coding region of Fam111a. ES cell clones were selected based on PCR genotyping and then blastocysts were microinjected into C57BL/6 N background mice. Balb/c donors were used for microinjection to produce the chimera. Sperm rederivation was performed to produce Fam111a/−+ animals. Fam111a−/− and Fam111a/++ mice were generated by breeding Fam111a/−+ mice.The C57BL/6 N Fam111a/++) allele: forward primer (5′-AGCCAAAATGAGCTGTAAGAAGC-3′) and reverse primer (5′-GTGTCATTGTCCCATTCAAC-3′). The following primers were used for genotyping the presence of the ZEN-UB1 cassette: forward primer (5′-TCATTCTCAGTATTGTTTTGCC-3′) and reverse primer (5′-TGAACTTTCTCACATCGTAGC-3′).The following primer pair was used for genotyping the wild type  and during the experiments they were individually housed for 48 h in metabolic cages , after which they were placed back into their own cages. Cage enrichment  was used in the standard cages, but not the metabolic cages. Specific humane endpoints were defined as follows: (1) The animal has visible signs of discomfort, such as weight loss , hunchback, bad fur and/or bad movement pattern, (2) The animal loses more than 20% weight during housing in a metabolic cage, (3) Specific for the low serum magnesium value: the animal has visible muscle cramps, the animal shows lack of movement, the animal shows a severe abnormal walking pattern (wobbling), the animal has severe exorotation of the heel (indication of muscle weakness) or the animal is not able to hold on to the cage (indication of muscle weakness). These endpoints were monitored daily through weighing and visual monitoring.Animal studies were carried out at the Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands. All methods and protocols performed in this study were approved by the local Ethics committee of the Radboud University Nijmegen (RU DEC 2015-0112-006) and the national Ethics committee of the Dutch Central Commission for Animal Experiments (AVD103002016382). This study was performed in compliance with the ARRIVE guidelines. The methods were in accordance with relevant guidelines/regulations, as follows. 3. During the next 24 h, the mice were weighed and urine and faeces were collected. In addition, water and food consumption were measured. The following day, the mice were anaesthetised by 4% (v/v) isoflurane inhalation, serum/blood was collected and the mice were sacrificed. Kidney, thyroid glands, duodenum and colon were collected and snap frozen in liquid nitrogen and stored at -80ºC until further analysis.Mice (8–10 weeks old) were placed in metabolic cages and left to acclimatise for 24 h. Mice were kept on a normal diet, containing 1% calcium (w/w), 0.22% magnesium (w/w), 0.7% phosphate (w/w) and 1000 IU/kg vitamin D43−, Na+ and K+ were determined by the clinical laboratory of the Radboud University Medical Center using an automated system . Mg2+ concentrations were determined by xylidil blue colorimetric assay kit  and read at a wavelength of 600 nm on a BioRad plate reader . Serum and urinary Ca2+ concentrations were measured using a chromogen-based colorimetric assay and read at a wavelength of 570 nm . The values obtained were calibrated using a Precinorm standard solution . Serum albumin was measured using the BCG albumin assay kit  according to the manufacturer’s recommendation and read at a wavelength of 620 nm on a BioRad plate reader . PTH serum concentrations were measured using the mouse PTH 1-84 ELISA kit  according to the manufacturer’s instructions.Serum and urine POTissues were fixed for at least 24 h in 4% (v/v) formalin. Samples were dehydrated in ethanol, embedded in paraffin and cut into sections of 5 μm. The sections were deparaffinised in xylene for 10 min and then rehydrated in graded concentrations of ethanol . The sections were incubated 5 min in haematoxylin  and differentiated in tap water after which the sections were stained with eosin (Merck) and dehydrated in in graded concentrations of ethanol ). Slides were cleared in xylene and mounted using Pertex mounting medium . Tissue slices were imaged using a Zeiss Axiocam 503 color camera mounted onto a Zeiss Axio Imager M2.Gapdh expression. Relative mRNA expression was analysed using the Livak method (2−ΔΔCT) and annotated as fold change of expression compared to the Fam111a/++ group. Primer sequences are provided in the Supplementary Table Total RNA was extracted from kidney and intestinal tissues using TRIzol reagent  according to the manufacturer’s protocol. The isolated RNA was treated with DNase  to prevent contamination from genomic DNA. RNA concentrations and quality were checked using the Nano-Drop 2000c Spectrophotometer . To generate cDNA, 1.5 µg RNA was reverse transcribed using Moloney murine leukemia virus (M-MLV) reverse transcriptase  at 37 °C for 1 h. Gene expression levels were quantified by SYBR-Green  on a CFX96 real-time PCR detection system (BioRad) and normalised for Fam111a/++ (n = 4) and Fam111a−/− (n = 3) male mice were sent for RNA sequencing. Quality control and RNAseq were performed by BaseClear B.V. . Counts of transcripts per sample were generated and quantified using Salmon and expressed as transcripts per million58. The reference transcriptome was downloaded from the BioMart repository at Ensembl . The raw and normalized count data were further analysed using R . Raw and processed data files are available in the Gene Expression Omnibus database (GSE196466).Total RNA was extracted from kidney tissue using TRIzol reagent (Invitrogen) according to manufacturer’s protocol and described earlier. RNA samples from 49. Briefly, X-ray power was set at 40 kV and tube current at 250 mA. Beam hardening (20%) was reduced using a 1 mm aluminum filter, ring-artefacts was set at 5 and an average of three photos (frame averaging) at each angle (0.8°) were taken to generate the final images. Cortical bone parameters were performed in the diaphyseal cortex which included a scan area of 0.45 mm in the femoral center. Trabecular bone measurements were obtained by scanning the distal metaphysis, a total scan area of 1.35 mm from the distal growth plate to the femoral center.Femurs were dissected and fixed in 4% (v/v) formalin for 24 h and subsequently kept in 70% (v/v) ethanol. Femurs were scanned using the Skyscan 1076 in vivo X-ray computed tomographer  with a voxel size of 8.88 μm and an exposure time of 2300 ms as described previously59. Briefly, co-staining for parv and NCC were performed on 4 μm sections of formalin fixed and paraffin embedded kidney samples. Sections were incubated with blocking buffer  to prevent unspecific antibody binding. Sections were incubated overnight at 4 °C in rabbit anti-parvalbumin  and sheep anti-NCC  in blocking buffer. For detection, sections were incubated with Alexa Fluor-conjugated secondary antibodies . Cell nuclei were stained with DAPI  for 10 min at room temperature. Images were taken with an AxioObserver camera and stitched with AxioVision software . NCC or parv positive regions of interest were obtained by thresholding and total positive area per kidney slice was measured using the analyse particle function in Fiji60.Immunohistochemistry was performed as previously describedp value of < 0.05 were considered statistically significant. For the RNAseq, additionally, a false discovery rate (FDR) ≤ 0.05 was used to determine statistical significance.Data are expressed as mean ± SD. Analysis was done using Graphpad Prism v8 . For statistical analysis of three groups, the assumption of equal variances was tested using the Brown-Forsythe test and normality was tested using the Shapiro–Wilk test. For two groups, the assumption of equal variances was tested using an F test. If assumptions were not met, data was first logarithmithically transformed to achieve equal normality. Then, for three groups one-way ANOVA was used to test for differences in the data, followed by a Tukey’s multiple comparison test in case the null hypothesis was rejected. In the case of two groups, an unpaired t-test was used. Differences with a Supplementary Information.