Increasing efforts are being made to understand the role of intermittent, transient, high-power burst events of neural activity. These events have a temporal, spectral, and spatial domain. Here, we argue that considering all three domains is crucial to fully reveal the functional relevance of these events in health and disease. Neural activity recorded from the scalp [magneto-/electroencephalogram (M/EEG)], cortical surface [electrocorticogram (EcoG)], or inside the brain [local field potential (LFP)] is traditionally analysed by averaging tens to hundreds of trials. The trial-wise average of spectrograms commonly shows periods of sustained high or low spectral power. The underlying single trial activity may manifest as oscillations  with periods of high or low amplitude. However, as recently demonstrated, sustained high spectral power in the averaged spectrogram can also arise from the accumulation of burst-like events across trial , 5., 6..Events have a temporal, spectral, and spatial domain. However, most work so far has focussed on temporal event characteristics, such as event amplitude , event duration, or event interval time A. EventsThe spatial domain is commonly reduced by extracting the time series from a single spatial location, or summarising the time series of several spatial locations through their average or a linear combination . Similarly, the spectral domain is often reduced by selecting a single peak frequency, averaging the signal or amplitude envelope within a specified frequency band, or by fitting a state-wise frequency profile from a time-delay embedded or autoregressive-based Hidden Markov Model (HMM) .Rarely, both the temporal and spectral characteristics of events are analysed together C have noDomain reduction can have several unwanted consequences. First, different domain reduction methods likely result in differences in event characteristics. Focussing on the temporal domain, , the event has a temporospatial gradient. Therefore, reducing the spatial domain can result in an underestimation of the actual event duration or missing the event altogether.Furthermore, domain reduction can obscure interactions between domains, which may lead to ambiguous inferences and conclusions drawn from these analyses. For example, if events change their location in the spatial and/or spectral domain over the course of the event, domain reduction can lead to misestimation of the event duration. The second event in Thus, it becomes apparent that domain reduction can hide important information about mass neural signals. Therefore, even when only one domain is of interest, considering all domains is most likely necessary to accurately describe each individual domain. This increases sensitivity to detect differences between conditions, individuals, or groups, and to minimise spurious inference.Considering all domains simultaneously yields a more detailed description of the underlying signal and allows interactions across domains to be characterised. These interactions can have many forms, and here we illustrate four such interactions between domains C with a As shown in These examples support the idea of interactions across the three domains. They illustrate how multidomain analysis can reveal gradients that would otherwise remain obscure but that are relevant for revealing the mechanistic role of events.Analysis of all three domains  benefits from high signal-to-noise recordings of neural activity, which have become more readily accessible through recent advances in acquisition techniques of mass neural activity, such as large-array, high-density ECoG, high-precision MEG, or optically pumped magnetometers. Parallel development in signal processing, particularly effective spatial leakage correction, precise beamformer, high-resolution time-frequency analysis, and nonstationary approaches for event identification constitute additional advances for analysing events precisely across domains. These advances notwithstanding, challenges, such as varying signal-to-noise across time points, frequencies, and spatial locations, tools for analysing multidimensional event characteristics effectively across events and individuals, and, for M/EEG data, accuracy of spatial localisation (forward and inverse modelling), remain. Furthermore, the resolution of the temporal and spectral domains constitutes a compromise and depends on the choice of ‘epoch’ length in the time–frequency analysis.In summary, intermittent, transient, events have a temporal, spectral, and spatial domain. Considering all three domains will yield a more accurate description of individual event characteristics and their interactions. This is likely to underpin identification of more precise fingerprints of mechanisms underlying differences in population activity measures, such as spectral power. In turn, this can guide the development of rational and mechanistically grounded treatments that target neurological and psychiatric conditions. While we use sensorimotor β activity here as a showcase for discussing the impact of domain reduction and opportunities to consider all three domains, these points likely extend to other brain areas, frequency ranges, and pathologies.


Innate immunity-mediated neuroinflammation actively contributes to the onset and progression of neurodegenerative diseases. Inflammasomes are involved in the progression of the innate immune response and are responsible for the maturation of caspase-1 and inflammatory cytokines during neuroinflammation. The nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome, which is one of the most intensively investigated inflammasomes, has been reported to play a key role in neurodegenerative diseases. Here, we reviewed the mechanisms, role, and latest developments regarding the NLRP3 inflammasome with respect to three neurodegenerative diseases: Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). Patient and animal model studies have found that abnormal protein aggregation of Aβ, synuclein, or copper–zinc superoxide dismutase-1 (SOD1), which are the main proteins expressed in the three diseases, respectively, can activate microglial cells, induce increased interleukin-1β (IL-1β) release, and activate the NLRP3 pathway, leading to neurodegeneration. In contrast, a deficiency of the components of the NLRP3 pathway may inhibit Aβ, synuclein, or SOD1-induced microglial activation. These studies indicate a positive correlation between NLRP3 levels and abnormal protein aggregation. However, in the case of ALS, not only microglia but also astrocytes express increased NLRP3 levels and contribute to activation of the NLRP3 pathway. In addition, in this review article, we also focus on the therapeutic implications of targeting novel inhibitors of the NLRP3 inflammasome or of novel drugs that mediate the NLRP3 pathway, which could play a role  Neurodegenerative diseases are neuronal disorders characterized by the loss of a large number of neurons in the human brain, for example, Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington’s disease. Although the location and etiology of these diseases differ, they share the common characteristic of neurodegeneration. AD and PD mainly occur in middle and old ages, so as the population ages, the incidences of AD and PD are increasing. It is generally believed that the prevalence of dementia in the population over 65 years old is 4% and that the annual incidence rate is 0.6% to 1.2%. The prevalence of PD is second only to that of AD, with PD mainly occurring in those of middle age or older at a prevalence of 2% in the population over 65 years old. However, Huntington’s disease and ALS can occur in patients of different ages, and there is no cure available for these diseases. One of the pathological hallmarks of neurodegenerative diseases is the aggregation of abnormal protein in the central nervous system (CNS). However, innate immunity-mediated neuroinflammation actively contributes to the onset and progression of neurodegenerative diseases  protein, and procaspase-1. The sensors, which can detect PAMPs, DAMPs, and cytosolic double-stranded DNA, are classified into three types: nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs); absent in melanoma-like receptors (ALRs); and pyrin , and a C-terminal leucine-rich repeat domain . The noncanonical inflammasome is activated by lipopolysaccharide (LPS) in the cytosol released from gram-negative bacteria, such as AD is mainly caused by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs) in the brain, involving the increased production, oligomerization, and aggregation of Aβ peptides cleaved from the amyloid β-precursor protein (APP) by the β- and γ-secretase complexes. Aβ is the main component of extracellular senile plaques, which can cause neurotoxicity in AD /presenilin-1 (PS-1) transgenic mice  pathway and the NLRP3 inflammasome  neurons in the substantia nigra (SN) and accumulation of Lewy bodies, which are constituted of fibrillar α-synuclein /NLRP3/caspase-1-mediated pyroptosis  fyn, a nonreceptor Src family tyrosine kinase, contributes to α-syn-induced NLRP3 inflammasome priming via intracerebroventricular infusion reduces movement impairment and attenuates DA neurodegeneration in the midbrain of PD mice , Astragaloside IV , and bushen-yizhi formula  were shown to significantly ameliorate DA neuron degeneration and alleviate motor impairment by suppressing NLRP3 inflammasome activation in the MPTP mouse model  agonists have been shown to suppress the inflammatory reaction and NLRP3 inflammasome activation. Administration of GW501516, a selective PPARβ/δ agonist, ALS is a progressive neurodegenerative disease caused by the deterioration of motor neurons in the brain and spinal cord. Among the cases of ALS, more than 90% are sporadic ALS (sALS) patients, and 5–10% are familial ALS (fALS) patients. Most fALS cases involve autosomal genetic diseases, such as superoxide dismutase-1 (SOD1) and nerve microfilament defects. Neuroinflammation is believed to significantly contribute to ALS disease progression (Byrne et al., The SOD1G93A mutation was used to create the SOD1G93A mouse (Ince et al., However, other results from ALS patients and from SOD1G93A mice indicate that microglia do not express NLRP3. Caspase-1 activation and IL-1β production in microglia have been shown to occur independently of NLRP3 (Meissner et al., In addition, besides microglia, astrocytes are also capable of contributing to innate immune signaling by increasing their release of cytokines, including IL-1β and IL-18 (Dong and Benveniste, In general, these findings suggest that astroglial NLRP3 inflammasome complexes are critically involved in neuroinflammation in ALS. In ALS, microglia and astrocytes switch from a neuroprotective to a proinflammatory phenotype by releasing potentially neurotoxic cytokines.A number of studies have been focused on understanding the role of the inflammasome in neurodegenerative diseases. Increasing evidence indicates a link between NLRP3 and neurodegenerative diseases, although the exact roles and mechanisms by which the NLRP3 inflammasome regulates neurodegenerative diseases remain unclear and require further confirmation. Most importantly, inhibition of NLRP3 in animal models reduces the inflammatory response, decreases abnormal protein deposition, and corrects behavioral abnormalities associated with neurodegenerative diseases, suggesting the possibility of targeting NLRP3 inflammasome to treat such diseases. However, many important topics still need to be explored, such as how the activation or inhibition of the NLRP3 inflammasome influences neurodegenerative diseases. The solution of such questions is important for studying the pathogenesis of neurodegenerative diseases and for providing more evidence and targets for exploring novel treatments. Despite the fact that inhibition of NLRP3 inflammasome activation has been shown exert beneficial effects in animal models of neurodegenerative diseases, the effectiveness and safety of specific NLRP3 inflammasome inhibitors in patients have yet to be verified in clinical trials.FH and YG contributed to the conception and organization and wrote the first draft of the manuscript. FH contributed to manuscript revision. All authors approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since MUSes find applications in different constraint domains and new applications still arise, there have been proposed several domain agnostic algorithms. Such algorithms can be applied in any constraint domain and thus theoretically serve as ready-to-use solutions for all the emerging applications. However, there are almost no domain agnostic tools, i.e. tools that both implement domain agnostic algorithms and can be easily extended to support any constraint domain. In this work, we close this gap by introducing a domain agnostic tool called MUST. Our tool outperforms other existing domain agnostic tools and moreover, it is even competitive to fully domain specific solutions.In many areas of computer science, we are given an unsatisfiable set of constraints with the goal to provide an insight into the unsatisfiability. One of common approaches is to identify minimal unsatisfiable subsets (MUSes) of the constraint set. The more MUSes are identified, the better insight is obtained. However, since there can be up to exponentially many MUSes, their complete enumeration might be intractable. Therefore, we focus on algorithms that enumerate MUSes


Relative orientation estimation between the hand and its fingers is important in many applications, such as virtual reality (VR), augmented reality (AR) and rehabilitation. It is still quite a big challenge to do the estimation by only exploiting inertial measurement units (IMUs) because of the integration drift that occurs in most approaches. When the hand is functionally used, there are many instances in which hand and finger tips move together, experiencing almost the same angular velocities, and in some of these cases, almost the same accelerations are measured in different 3D coordinate systems. Therefore, we hypothesize that relative orientations between the hand and the finger tips can be adequately estimated using 3D IMUs during such designated events (DEs) and in between these events. We fused this extra information from the DEs and IMU data with an extended Kalman filter (EKF). Our results show that errors in relative orientation can be smaller than five degrees if DEs are constantly present and the linear and angular movements of the whole hand are adequately rich. When the DEs are partially available in a functional water-drinking task, the orientation error is smaller than 10 degrees. Hand-finger movement tracing is useful in many areas, such as virtual reality (VR), augmented reality (AR), ergonomic assessment and especially medical applications ,2,3,4,5.Our goal is to estimate relative 3D orientations between finger tips and the dorsal side of the hand with only IMUs, essentially getting rid of magnetic disturbance by not using magnetometers. In order to reduce the integration drift, we exploit information during the daily life rather than using the biomechanical constraint. The information is based on the assumption that there are many instances in which hand and finger tips move together, experiencing almost the same angular velocities and accelerations represented in different 3D coordinate systems. The method was verified with a small sensor configuration: one sensor on the dorsal side of hand, and one on the most distal finger segment of interest.In order to estimate the relative orientation, the information from the gyroscope and accelerometer and extra information during DEs need to be combined in an optimal way. Therefore, an extended Kalman filter (EKF) was introduced to estimate 3D relative orientations between the dorsal side of the hand and finger tips, assuming angular velocities and accelerations are the same, but just represented in a different coordinate system. The process model is based on integrating relative angular velocity, the measurement model is mainly based on the information during the DE. The quality of the DE is considered in the measurement variance. When the DE is available with small variance, we trust the measurement model more; otherwise, we trust the process model more. Thus, the information from process and measurement models is optimally fused to estimate relative 3D orientations during functional hand and finger movements.The gain error and non-orthogonality error are assumed to be time-invariant and can be obtained through sensor calibration; thus, the outputs of calibrated gyroscope can be expressed asFor the calibrated accelerometer, the outputs on the hand and finger tips areThe process model is based on integrating the relative angular velocity between the hand and its fingers in its own frame. We choose the quaternion x expressed in the coordinate frame of object y. h represents the hand and f represents the finger tip. Combining Equations (1) and (5), we find:x relative to frame y. f can be expressed asThe measurement update of EKF is based on the DE. During the DE, the hand and fingers share the same angular velocity in different coordinate framesessed asahh=qhf⊗aIn order to assess the relative confidence in the measurement model (based on our DE assumptions) and the process model, the measurement variance is determined. According to the assumption that a hand and finger share approximately the same angular velocity and acceleration based on Equation , the difEquation .(17)dgyEquation , the errk.k. Based on the Gaussian approximation, as described in Equations (17) and (20), it is essential to know the rotation quaternion The sensor system includes three IMUs fixed on the most distal segments of the thumb and index finger and the dorsal side of the hand, as shown in z-axis of global Vicon system was vertical upward; gravity in this frame was g is the local gravity value). When we have at least two poses, we obtain more than two vectors expressed in the marker frame and IMU frame respectively, which is enough to determine the relative orientation between the IMU and marker frame.For evaluation of the IMU-based 3D relative orientation estimation using the optical system, it is essential to calibrate the relative orientation between the sensor and marker-based reference frame. Here, we used the accelerometer for this marker system’s IMU calibration. Holding the system static, we obtained the gravity in the IMU frame from the accelerometer readings. Meanwhile, we obtained the orientation from the global Vicon frame to marker frame Before the experiment, IMU errors were calibrated according to D Tedaldi et al.’s and WT Fong et al.’s research ,27, inclIn this experiment, the two measurement systems were synchronized by recording the sensed responses of an induced impact at the start and end of each experiment. At the start and end of every experiment, we hit the IMU on a desk, resulting in an acceleration peak measured by the IMU system and a minimum vertical position of the Vicon markers simultaneously, which was used to synchronize the two systems.In order to demonstrate the feasibility of our approach, an experimental part was designed to estimate the accuracy of the algorithm compared with the optical system. Our feasibility experiment involved three participants. The protocol was reviewed, approved and conducted under the auspices of the Ethics Committee EEMCS, Univerisity of Twente. The following tasks were performed:Task1: Movements and rotations of the hand, while not varying relative orientations between hand and fingers: IMUs were fixed on fingers and the dorsal side of the hand. Then, the participant did the pronation and supination movements with the arm while the axis of pronation and supination was continuously changing. The orientation was changed over approximately 160Task2: Simple functional task. The subject was asked to place the hand on the desk; then rise the hand and grasp a cup; subsequently drink some water and place the cup back; and finally place the hand on the original position. The illustration of the movement can be seen in For task 1, the orientation reference was directly derived from the IMUs, because the relative orientation was imposed by the hand, and therefore, known and not varying. For task 2, the reference measurement was performed using the optical VICON system (software version 2.8.2).The error angle used was the arccos of the first component of quaternion error qerr :(24)qerrThe estimation may be influenced by the repetition rate of movements. Equation . For theAccording to rror see c. The reWe proposed and evaluated an IMU-based setup for estimating 3D relative orientation between hand and finger tips. Compared with the IMU-based data glove system described by Salchov-Homer et al.  and KortMost previous IMU-based systems ,28 for fFor the result in task 1, the relative orientation estimation is less sensitive to an increase of repetition rate of the same movement when using gyroscopes or gyroscopes plus an accelerometer than the accelerometer only. That is because as the difference among the accelerometer signals from the hand and finger becomes larger, the non-gravitational acceleration caused by increasing angular velocity or angular acceleration becomes relatively more important compared to the gravity component.Position estimation only based on inertial sensors is quite challenging and limited by integration drift. Our further research will concentrate on relative position estimation based on IMUs combined with sensing the magnetic field of a magnetic source. For this to be feasible, an adequate estimate of relative orientation is required, so the 3D magnetic field measurement can be expressed in the coordinate system of the magnetic source. This is an essential first step in estimating relative positions. In this research, only one healthy participant was involved since we are mainly concentrating on verifying the performance of the algorithm. Subsequently, the proposed relative orientation and position estimation methods for the hand and finger using a small sensing configuration need to be evaluated in healthy subjects and patients during more complex daily tasks, in order to assess the applicability in clinical and daily-life settings. To make the system more friendly to users, the system could be wireless in the future.In conclusion, IMUs can be used to estimate the relative orientation between the hand and fingers without using magnetometers. Compared with previous systems, we only exploit IMUs on finger tips and the dorsal side of the hand rather than having IMUs on every segment. The performance is dependent on how well the hand and fingers move together, which influences the accuracy of the estimate. The median value of estimation error can be smaller than five degrees when IMUs are on our hand and fingers if their relative orientation is not variant over time, while the object or hand is moving. During the water-drinking task, the estimation error can be smaller than 10 degrees during periods when the hand and fingers approximately move together, which may be adequately accurate to provide useful information to clinicians when judging.


First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Stéphanie Cottier is first author on ‘ Stéphanie CottierWhat is your scientific background and the general focus of your lab?I am a molecular and cellular biologist. I earned my PhD from the University of Heidelberg, working in Benedikt Kost's lab on polar growth in pollen tubes. Then I completed a post-doc at the Max Planck Institute for Plant Breeding Research in the laboratory of Erich Kombrink with a focus on small molecules involved in plant-pathogen interactions. Currently, I am working in the laboratory of Roger Schneiter at the University of Fribourg. For this post-doc, I have not only changed country (from Germany to Switzerland), but also the focus of my research. I am now working in a biochemistry lab studying lipid homeostasis in yeast cells. We got interested in the CAP proteins as they were identified to mediate the export of sterols in yeast. Since then we are working on the characterization of their physiological function in different organisms.How would you explain the main findings of your paper to non-scientific family and friends?I am using yeast, the same organism you are using for baking, to study the function of a family of proteins that have a common domain: the CAP domain. CAP domain proteins are not only found in yeast, but also in human, where they have been associated with fertility and cancer. It is therefore important to understand their mode of action.My protein of interest is called Pry3. It has been shown that cells having high amounts of Pry3 have a reduced mating efficiency. We used this reduction in mating efficiency to identify the domains within Pry3, and even the single amino acids (the building blocks of proteins) within the CAP domain that are critical for this inhibition. Interestingly, these amino acids are also found in the CAP domain proteins of other organisms. We were also able to fluorescently tagged Pry3 and analysed its localization by microscopy. When Pry3 is present in high amounts, it loses its polarized localization in the cell wall, and instead spreads all over the cell surface, exerting a deleterious function on the yeast mating reaction.What are the potential implications of these results for your field of research?CAP proteins are present in many different organisms and they have been involved in a wide variety of processes including plant and mammalian immune defence, venom toxicity, sperm maturation, fertilisation, as well as in prostate and brain cancer. However, the mode of action of these conserved proteins remains largely elusive. The identification of residues in the CAP domain of Pry3 that are important for mating inhibition, and shown to be conserved in a number of CAP proteins from different organisms, gives novel cues to decipher the function of the CAP domain.What has surprised you the most while conducting your research?How complex it is to answer a tiny question. The amazing number of methods and paths nowadays science offers to do so, yet without forgetting the very basic constraints that are time and money.What changes do you think could improve the professional lives of early-career scientists?Promoting multidisciplinary research. While collaborations are common, in my opinion they should not be limited to sharing results, but also knowledge and techniques, and give a view of different laboratory organization and philosophy.“In research, there is always ‘the next question’!”What's next for you?We have identified residues within Pry3 that are required for mating inhibition, and we are addressing the possibility that these residues form part of a catalytic active site of the CAP domain.In research, there is always ‘the next question’!


However, the distribution of these TMD-containing effectors remains unknown. Here, we discover prePAAR, a conserved motif found in over 6000 putative TMD-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMDs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions. Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.Type VI secretion systems (T6SSs) deliver antibacterial effector proteins between neighboring bacteria. Many effectors harbor N-terminal  Bacteria secrete proteins to facilitate interactions with their surrounding environment. In Gram-negative bacteria, the transport of proteins across cellular membranes often requires the use of specialized secretion apparatuses found within the cell envelope. One such pathway is the type VI secretion system (T6SS), which in many bacterial species functions to deliver antibacterial effector proteins from the cytoplasm directly into an adjacent bacterial cell via a one-step secretion event . A critihemolysin co-regulated protein (Hcp) capped by a single homotrimer of valine-glycine repeat protein G (VgrG)  domain-containing protein that forms the tip of the VgrG spike (The T6SS is comprised of two main components: a cell envelope-spanning membrane complex and a cytoplasmic bacteriophage tail-like complex. The latter contains a tube structure formed by many stacked copies of hexameric ring-shaped G (VgrG) . TogetheG (VgrG) . AdditiorG spike . AltogetrG spike . Prior trG spike .In addition to its role in effector export, Hcp also possesses chaperone-like properties that facilitate cytoplasmic accumulation of Hcp-interacting effectors prior to their secretion . This cheffector-associated gene (Eag) chaperones, interact with so-called ‘evolved’ PAAR proteins in which the PAAR and toxin domains are found as a single polypeptide chain  for the N-terminal 60 residues of Tse6 using an iterative search procedure that queried the UniProtKB database  and further reduced redundancy by clustering sequences with 95% identity. Remarkably, in all but two of the remaining 1166 prePAAR-containing proteins, we identified a PAAR domain, indicating a probable functional relationship between prePAAR and PAAR. The two prePAAR-containing proteins lacking a PAAR domain were either adjacent to a gene encoding a PAAR domain-containing protein or directly beside T6SS structural genes. Finally, we searched 1166 prePAAR-containing proteins for TMDs and found that all protein sequences contained predicted TMDs with 86% having one region of TMDs and 14% having two regions of TMDs. In sum, our prePAAR-based search procedure identified thousands of candidate effector proteins possessing properties consistent with the requirement for an Eag chaperone for T6SS-dependent export.Examination of our putative effector sequences revealed that prePAAR is substantially enriched in bacterial genera with characterized T6SSs including analyses , List C.analyses . To deteanalyses . The locanalyses . We seareag gene . In apprrearrangement hotspot (Rhs) proteins that function to encapsulate secreted toxins  fusions of RhsA and Tne2 to facilitate the enrichment of these proteins from P. protegens and confirmed that these fusions did not compromise the ability of these effectors to intoxicate recipients  and co-expressed this truncated protein with EagR1 in E. coli. Consistent with our hypothesis, affinity purification of RhsA∆NT showed that this truncated variant does not co-purify with EagR1   to examine the configuration of each subunit within this complex. To facilitate the accurate identification of each component, we obtained class averages of purified VgrG1, RhsA complex . As expeectively . Intriguectively . Lastly,ectively . Interesclasses) . When taP. protegens EagR1 with the prePAAR and TMD-containing N-terminus of RhsA were unsuccessful. However, the EagR1 homologue SciW from Salmonella Typhimurium crystallized in isolation and in the presence of the N-terminus of the class I prePAAR effector Rhs1 (Rhs1NT), allowing us to determine apo and effector-bound structures to resolutions of 1.75 Å and 1.90 Å, respectively   .P. aeruginosa EagT6, although each chaperone differs in its electrostatic surface properties  a high-molecular-weight species corresponding to Tse6-VgrG1a complex and (2) a low-molecular-weight species indicative of free Tse6 . Deletioractions . Using t1A, H15A . We nexth VgrG1a .E. coli, which does not contain additional components such as TMDs or a toxin domain , while the second contains prePAAR fused to PAAR and thus lacks only its TMDs (Tse6∆TMDs). We next assessed the ability of these truncated forms of Tse6 to form a complex with VgrG1a in vivo, as described above. In these experiments, we found that prePAAR and PAAR are together both necessary and sufficient for the formation of Tse6-VgrG1a complexes in vivo , whereas the TMD of Tne2 has an alanine residue (A26) that aligns with A26 of Tse6. Although our structural models and bioinformatic analyses of chaperone-effector pairs suggest this pattern of Eag chaperone specificity is widespread, additional biochemical experimentation will be needed to demonstrate the generalizability of these findings.As demonstrated in Another important difference observed in the class I and class II chaperone-effector interactions is the position of the prePAAR region in each chaperone-effector pair. The asymmetric chaperone-TMD interaction allows SciW to ‘shield’ the hydrophobic regions of Rhs1’s N-terminus from the aqueous milieu while also positioning its prePAAR motif in such a way that would allow it to interact with PAAR. By contrast, the pseudosymmetric binding mode of Tse6 to EagT6 appears to be much more dynamic as interpretable electron density for bound Tse6 was only observed when the effector fragment was held in place by interactions with an adjacent complex in the crystallographic asymmetric unit. Consequently, we speculate that even though the Tse6’s prePAAR motif appears less accessible than that of Rhs1, it is likely highly dynamic in solution and thus may adopt a markedly different conformation when in complex with PAAR.Despite containing a primarily beta-sheet secondary structure, Eag chaperones interact with effector TMDs by mimicking the interactions that occur between the helices of alpha-helical membrane proteins, which, to our knowledge, is a unique mechanism for a chaperone-effector interaction. Upon binding their cognate effector, we hypothesize that Eag chaperones not only shield effector TMDs from solvent but also distort their structure to prevent potential hairpin formation and erroneous insertion into the inner membrane of the effector-producing cell. Because Eag-interacting TMDs have likely evolved to insert into bacterial membranes, a mechanism to prevent self-insertion is probably necessary prior to export. Recent work studying the secretion of TMD-containing effectors of the bacterial type III and type IV secretion systems found that shielding TMDs to prevent inner membrane insertion is a critical step for proper targeting to the secretion apparatus . HoweverCurrent evidence also suggests that Eag chaperones are not secreted by the T6SS . This le+ and NADP+, ADP-ribosylation of FtsZ, pyrophosphorylation of ADP and ATP, and deamination of cytidine bases in double-stranded DNA  at 37°C (P. aeruginosa) or 30°C (P. protegens). Solid media contained 1.5% or 3% agar. Media were supplemented with gentamicin (30 μg mL−1) and IPTG (250 μM) as needed.ens Pf-5 . Both orEscherichia coli strains XL-1 Blue, SM10 and BL21 (DE3) Gold or CodonPlus were used for plasmid maintenance and toxicity experiments, conjugative transfer and protein overexpression, respectively , 0.1% (w/v) rhamnose or 40 μg mL−1 X-gal.ectively . All E. jackhmmer (HmmerWeb v2.41.1) using the first 60 amino acids of Tse6 (PA0093) protein to obtain 2378 sequences. We removed any UniProtKB deprecated sequences entries  and further filter, cluster, and analyze the remaining 975 effector sequences as stated below .For the analysis of all effectors in UniprotKB we used six iterations of Pseudomonas, Burkholderia, Enterobacter, Escherichia, Salmonella, Serratia, Shigella, Vibrio, and Yersinia were downloaded from NCBI using ncbi-genome-download . hmmsearch (v3.1b2) and the effector HMM were used to identify the effectors in all genome assemblies using the ` -Z 45638612 -E 1000` options and we further filtered for a bitscore greater than 40. We further filtered to include effectors that included the prePAAR (AARxxDxxxH) motif, which we searched for using regular expressions, identifying 6129 prePAAR-containing sequences across 5584 genomes. To be included in the analysis of http://pfam.xfam.org/family/PF08786#tabview=tab6) and hmmsearch with the same parameter and bitscore cutoff as the effector search. For hmmsearch (as described above) to capture the known diversity of the PAAR motif. We started broadly by using Pfam’s PAAR motif HMM (http://pfam.xfam.org/family/PF05488#tabview=tab4) to identify 895/1166 PAAR motif containing sequences. For the 271 sequences that were predicted to not have a PAAR motif, we then generated an HMM using three iterations jackhmmer and the PAAR motif of the Tse6 (PA0093) protein (L75 to G162) to identify 219/271 PAAR motifs. We generated a third PAAR motif HMM using 60–160 amino acids of a randomly selected sequence (GCF_001214785.1 in contig NZ_CTBP01000066.1) and two iterations of jackhmmer that was not identified to have a PAAR motif in the previous search but was identified to have a PAAR domain using phmmer (HmmerWeb version 2.41.1). We identified 42/52 sequences had a PAAR domain using the third PAAR motif. For the fourth PAAR domain HMM, we used the 60–160 amino acid sequence of GCF_005396085.1 in the NZ_BGGV01000116 contig and three iterations of jackhammer to identify 8/10 sequences that had a PAAR motif. The remaining two sequences with no PAAR domain were manually analyzed and were determined to either be directly associated with a PAAR domain downstream (GCF_001425105.1) or directly beside T6SS machinery gene (GCF_001034685.1). We predicted the transmembrane (TM) helices in proteins first using TMHMM (v2.0), Phobius web server, and TMbase  .The genome assemblies of ftware/) . Using Tftware/) . We consftware/) . The phyftware/) . For Figftware/) . All scrhttp://pfam.xfam.org/family/PF05488#tabview=tab1) and only obtained the 1923 sequences with one PAAR motif architecture. We then aligned and trimmed the alignment of the 1923 orphan PAAR sequences. We then used the previously mentioned 2054 effector sequences from UniProtKB and filtered to only use 1765 sequences with an AARxxDxxxH motif. To identify Zn2+-binding residues in orphan and prePAAR effector sequence logos, we used logo maker (v0.8) to create sequence logos for the first 200 amino acids (hmmbuild (v3.1b2) and eight PAAR domains annotated using Phyre2 (hmmsearch (v3.1b2) in the first 300 amino acids of the prePAAR effector sequences. For each sequence, we identified an envelope boundary (amino acid coordinates) of where the PAAR domain is predicted to be and we truncated the sequence at these positions. All scripts and intermediate files can be found in: https://github.com/karatsang/effector_chaperone_T6SS/tree/master/ZnBindingResidues .To collect orphan PAAR sequences, we used the Pfam database’s information on the PAAR motif . Phusion polymerase, restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (NEB). Sanger sequencing was performed by Genewiz Incorporated.Plasmids used for heterologous expression were pETDuet-1, pET29b, and pSCrhaB2-CV. Mutant constructs were made using splicing by overlap-extension PCR and standard restriction enzyme-based cloning procedures were subsequently used to ligate PCR products into the plasmid of interest.P. aeruginosa and P. protegens were made using the pEXG2 plasmid as described previously  containing 5% (w/v) sucrose for sacB-based counter-selection. Deletions were confirmed by colony PCR in strains that were resistant to sucrose, but sensitive to gentamicin. Chromosomal point mutations or tags were constructed similarly with the constructs harboring the mutation or tag cloned into pEXG2. Sucrose-resistant and gentamicin-sensitive colonies were confirmed to have the mutations of interest by Sanger sequencing of appropriate PCR amplicons.In-frame chromosomal deletion mutants in eviously . Brieflyeviously . DeletioD1404A or RhsA∆NTD1404A into the rhamnose-inducible pSCrhaB2-CV vector. These plasmids were co-transformed with an IPTG-inducible pPSV39 vector containing or lacking EagR1, respectively (see −6 in 10-fold increments and each dilution was spotted onto LB agar plates containing 0.1% (w/v) L-rhamnose, 250 µM IPTG, trimethoprim 250 µg mL−1 and 15 µg mL−1 gentamicin. Photographs were taken after overnight growth at 37°C.We previously showed that a D1404A mutation was sufficient to attenuate, but not abolish, the toxicity of RhsA and allows for the cloning of this toxin in the absence of its immunity gene . Therefovely see . StationE. coli (DE3) BL21 CodonPlus, P. aeruginosa ∆retS or P. protegens were inoculated into 2 mL or 50 mL LB at a ratio of 1:500, respectively. Cultures were grown at 37°C (E. coli and P. aerugionsa) or 30°C (P. protegens) to OD 0.6–0.8. Upon reaching the desired OD, all samples were centrifuged at 7600 x g for 3 min. The secreted fraction in P. aeruginosa or P. protegens samples was prepared by isolating the supernatant and treating it with TCA ) as described previously , 250 mM NaCl, 10 mM imidazole and purified according to the protocol described below (see Protein expression and purification).Stationary-phase overnight cultures of eviously . The cellacZ-expression cassette was inserted into a neutral phage attachment site (attB) of recipient P. aeruginosa and P. protegens strains to differentiate these strains from unlabeled donors. P. protegens recipient strains also contain a ΔpppA mutation to stimulate T6SS effector secretion to induce a T6SS ‘counterattack’ from P. protegens donor strains  ratio.For intraspecific competitions between −1 X-gal. The remainder of each competition mixture was spotted (10 µL per spot) in triplicate on a 0.45 μm nitrocellulose membrane overlaid on a 3% LB agar plate and incubated face up at 37°C for 20–24 hr. Competitions were then harvested by resuspending cells in LB and counting colony forming units by plating on LB agar containing 40 μg mL−1 X-gal. The final ratio of donor:recipient colony forming units were normalized to the initial ratios of donor and recipient strains.Initial ratios of donors:recipients were counted by plating part of the competition mixtures on LB agar containing 40 μg mLP. protegens proteins and Tse6 prePAAR mutants), RhsA-RhsI-EagR1-VgrG complex for negative-stain EM, Hcp (PFL_6089) and RhsA∆NT used for antibody development or the SciW, EagT6-Tse6NT complex and the SciW-Rhs1NT complex used for crystallization were expressed in E. coli BL21 (DE3) Gold or CodonPlus cells. Important differences in expression strategy used are indicated below.All plasmids used for co-purification experiments , Salmonella Typhimurium , Shigella flexneri , Enterobacter cloacae  and Serratia proteamaculans  were co-expressed using pET29b containing the predicted chaperone and pETDuet-1 harboring the full-length effector and its predicted immunity determinant. A similar co-expression strategy was employed for the RhsA∆NT-RhsI complex, RhsA-RhsI-EagR1-VgrG1 complex, Tse6 and the Tse6 prePAAR variants, Tsi6 and EagT6  from: E. coli) until OD 0.6–0.8. 40 mL overnight cultures were grown for all other of expression strains and were diluted into 2 L of LB broth and grown to OD6000.6–0.8 in a shaking incubator at 37°C. For most samples, protein expression was induced by the addition of 1 mM IPTG and cells were further incubated for 4.5 hr at 37°C. Expression of large protein complexes (>150 kDa) in E. coli, such as the chaperone-effector pairs from Salmonella and Enterobacter, RhsA∆NT-RhsI and RhsA-RhsI-EagR1-VgrG1 complexes were induced at 18°C and incubated at this temperature overnight. One millilitre overnight cultures of P. protegens strains expressing the desired tagged protein was diluted in 50 mL of LB broth and grown at 30°C (P. protegens) unitl OD 0.8. Cells were harvested by centrifugation at 9800 g for 10 min following incubation. For the RhsA-EagR1-VgrG1 complex and the experiments containing Tse6 prePAAR mutants, the pellets for cells expressing the cognate VgrG were combined with the pellets containing effectors, as described above. Pellets from 50 mL culture were resuspended in 3.5 mL lysis buffer , whereas those from 2 L of culture were resuspended in 25 mL of lysis buffer prior to rupture by sonication . Cell lysates were cleared by centrifugation at 39,000 g for 60 min and the soluble fraction was loaded onto a gravity flow Ni-NTA column that had been equilibrated in lysis buffer. Ni-NTA-bound complexes were washed twice with 25 mL of lysis buffer followed by elution in 10 mL of lysis buffer containing 400 mM imidazole. The Ni-NTA purified complex was further purified by gel filtration using a HiLoad 16/600 Superdex 200 column equilibrated in 20 mM Tris-HCl pH 8.0 150 mM NaCl or phosphate buffered saline (for samples used for antibody development only).For the expression of chaperone-effector pairs and the Tse6 prePAAR mutants, a 1 mL overnight culture of expression strains was diluted in 50 mL of LB broth and grown at 37°C (sciW (SL1344_0285) was synthesized with codon optimization for E. coli and cloned into the vector pRSETA with the restriction sites NdeI/HindIII (Life Technologies). This construct includes an N-terminal 6-his tag and an HRV 3C protease cleavage site (MGSSHHHHHHSSDLEVLFQGPLS). SciW-Rhs1NT and EagT6-Tse6NT complexes were co-expressed using pETDUET-1. Note that the EagT6 construct has a C-terminal VSV-G tag . The lysate was cleared by centrifugation at 16,000 rpm for 30 min and the supernatant passed over a nickel NTA gravity column (Goldbio) followed by washing with 50 column volumes of chilled lysis including PMSF, DNase I, and MgCl2. Proteins were eluted with five column volumes elution buffer then purified by gel-filtration using an SD75 16/60 Superdex gel filtration column equilibrated in gel-filtration buffer (GF) with an AKTA pure . For SciW, after affinity purification the protein was dialyzed in GF buffer O/N at 4°C and the His-tag removed during dialysis using HRV 3C protease. The digested SciW was passed over a nickel NTA gravity column and the flow through was collected. SciW was further purified using an SD75 16/60 Superdex gel filtration column equilibrated in GF buffer.Preparation of samples for crystallization  tag see . Cells wNT and EagT6-Tse6NT complexes lysis buffer ; elution buffer ; and GF buffer .The buffers used were as follows: SciW lysis buffer ; SciW elution buffer ; SciW GF buffer ; SciW-Rhs1−1 for initial screening using commercially available screens (Qiagen) by sitting-drop vapor diffusion using a Crystal Gryphon robot (Art Robbins Instruments). The crystallization conditions for SciW were 22 mg mL−1with a 1:1 mixture of 0.1 M Tris HCL pH 8.5, 25% (v/v) PEG 550 MME at 4°C. EagT6-Tse6NT complex was concentrated to 5, 10 and 20 mg mL−1 and screened for crystallization conditions as per SciW. The final crystallization conditions were 20 mg mL−1 with a 1:1 mixture of 0.2M Magnesium chloride, 0.1M Bis-Tris pH 5.5, and 25% (w/v) PEG 3350 at 4°C. SciW-Rhs1NT complex was concentrated to 15, 20, and 25 mg mL−1 and screened for crystallization as per SciW. The crystallization conditions were 25 mg mL−1 protein with a 1:1 mixture of 0.2M Ammonium sulfate, 0.1M Bis-Tris pH 5.5, and 25% (w/v) PEG 3350 at 4°C.SciW was concentrated to 7, 14, and 22 mg mLNT complex were collected in-house at 93K using a MicroMax-007 HF X-ray source and R-axis 4++ detector (Rigaku). Diffraction data from SciW-Rhs1NT crystals were collected at the Canadian Light Source at the Canadian Macromolecular Crystallography Facility Beam line CMCF-ID (08ID-1). SciW crystals were prepared by cryo-protection in mother liquor plus 38% PEG 550 MME and flash freezing in liquid nitrogen. Crystals of EagT6-Tse6NT and SciW-Rhs1NT complexes were prepared in the same manner with increasing the concentration of PEG3350 to 35–38%. All diffraction data were processed using XDS (NT and SciW-Rhs1NT complexes were obtained by molecular replacement using EagT6 (PDB: 1TU1) and SciW as search models, respectively, with the Phenix package. Initial models were built and refined using Coot, Refmac and the CCP4 suite of programs, Phenix, and TLS refinement thiocysteine). The coordinates and structure factors have been deposited in the Protein data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NY (http://www.rcsb.org). Molecular graphics and analysis were performed using Pymol  and UCSF Chimera  uranyl formate solution and grids stored at RT until usage.Four microlitres of each protein sample at a concentration of approximately 0.01 mg mL6 cathode and 4k × 4 k CMOS detector F416 (TVIPS), operating at 120 kV. For VgrG1, RhsA∆NT, the EagR1-RhsA complex and EagR1-RhsA-VgrG1 complex, a total of 99, 140, 100 and 120 micrographs, respectively, were collected with a pixel size of 2.26 Å. Particles for the VgrG1, RhsA∆NT, EagR1-RhsA complex and EagR1-RhsA-VgrG1 complex were selected automatically with crYOLO using individually pre-trained models, resulting in 18,676, 23,907, 32,078, and 31,409 particles, respectively   and detected with anti-rabbit horseradish peroxidase-conjugated secondary antibodies  (P. protegens) was used at a 1:5000 dilution. Western blots were developed using chemiluminescent substrate  and imaged with the ChemiDoc Imaging System (Bio-Rad).Western blot analyses of protein samples were performed as described previously for rabbit anti-Tse1 , rabbit anti-FLAG , rabbit anti-VSV-G , rabbit anti-Hcp1  . Rabbit  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by two peer reviewers, including Rajan Sankaranarayanan as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by John Kuriyan as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Dor Salomon (Reviewer #3).Thank you for submitting your article "Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:The manuscript deals with the elucidation of the mechanism by which bacteria deliver a set of effectors using type VI secretion system to other bacteria. Using bioinformatics approaches, the authors have identified a prePAAR motif in a set of effectors and classified them into two major families. Using structural, biochemical and in vivo approaches, the authors show the importance of the motif in loading the effector molecule on to the type VI assembly using Eag chaperone. The results are also interesting in demonstrating that the chaperone-effector pairing is specific. Overall, the work provides an in-depth molecular understanding on previously open questions in the T6SS field, especially those related to chaperone/adapter protein activity.Essential revisions:1) The identification and the role of prePAAR motif is central to the manuscript. Therefore, mutational data of key residues in the motif showing its role in the direct interaction with the PAAR domain will significantly strengthen the major claim of the manuscript (see reviewer #1 concern b). However, in the present situation, if the authors feel that they will not be able to do this in a short frame of time, you can tone down the claims accordingly.2) The schematic given in Figure 7 can be modified based on the comments of the reviewer.3) Make appropriate revisions by addressing the other concerns of both the reviewers.Reviewer #1:This manuscript significantly contributes to our understanding of the mechanism by which a subset of effectors is delivered by type VI secretion system (T6SS) from one bacteria to another. The authors have identified a prePAAR motif in Eag-dependent TMD containing effectors of T6SS and segregate them into two broad classes. Further, the authors provide in vitro and in vivo evidence to show that the effector and Eag binding is specific. Earlier, the same group has shown that TMD containing effectors require chaperone assistance for loading on to VgrG protein of the type VI assembly and for eventually getting delivered to the recipient bacteria. Here, the authors have also solved the crystal structure of both class I and class II effector molecules in complex with their cognate chaperones which revealed the atomic details of the interaction. Interestingly, the newly identified prePAAR motif is shown to be essential for loading the effector molecule onto VgrG and its delivery. Overall, the work is well done and I would recommend publication after addressing the concerns that are listed below.a) The authors justify the use of "prePAAR" by quoting the earlier work by Zhang et al., 2011. However, it looks like the term prePAAR was not introduced then and the Zhang et al. work refers to the N-terminal region to PAAR domain, which includes the newly identified motif (current study) as well as the TMD. Therefore, the authors have to clearly define what exactly do they mean by prePAAR at the first instance and follow the same throughout. To provide better clarity, since “prePAAR” indicates everything N-terminal to PAAR domain therefore it may be better to use an alternative term for the newly identified motif.b) Based on the data shown in Figure 6F, the authors conclude that prePAAR interacts with PAAR domain. However, this claim needs to be directly proven by including a pull-down of PAAR domain with an N-terminal construct having prePAAR point mutants  or just prePAAR, instead of the N-terminal construct (prePAAR + TMD).c) One interesting finding of the manuscript is the fidelity of the chaperone-effector pairing. However, the authors fail to comment on this fidelity based on the crystal structure information they have obtained. By attempting a simple modelling approach of cross-pairs, they may be able to provide a rationale in terms of the structural basis for this cognate pairing.d) The authors have an interesting proposition that H15 of prePAAR motif participates in Zn+2 binding that eventually helps in PAAR domain folding. However, it is perplexing that the D11A mutant of the effector is compromised in VgrG binding and also inducing toxicity in the target bacteria, while the H15A mutant is similar to wild type . Therefore, this proposition needs a reconsideration. Can the authors provide any evidence for the importance of Zn+2 binding in the folding of the PAAR domain?e) The authors should consider the following suggestions for preparing a more coherent schematic of the entire finding depicted in Figure 7. (1) prePAAR can be depicted at the tip of PAAR domain as the current depiction is misleading where prePAAR is shown at the interface of PAAR domain and VgrG. However, the authors do not seem to have provided any evidence for prePAAR and VgrG interaction. (2) As per the structural knowledge (from this work and previous studies), it is clear that the chaperones are symmetric dimers and therefore the schematics should represent the same throughout the manuscript. (3) From the model , it appears that binding of prePAAR to PAAR domain is dependent on TMD-chaperone binding. However, there is no experimental evidence for such sequential binding. (4) Authors should provide an explanation for their statement that split PAAR is an “evolved” version of PAAR domain and the same needs to be included in the Discussion. Why it cannot be the other way around? Also, it is worth considering the possibility that in split-PAARs the stabilization of PAAR domain with prePAAR may provide structural rigidity to the TMD for effective insertion into the membrane.f) In the Abstract, the authors state "…our findings define the mechanism of chaperone assisted secretion of a widespread family of T6SS membrane protein effectors". It appears that the significant part of this claim is already made in their earlier work  and therefore the authors need to make a clear distinction.g) The authors have shown that mutations in pre-PAAR affect the binding of the effector to VgrG. However, there is no evidence which shows that prePAAR has any direct role in PAAR domain folding and therefore the lines in the Abstract, "…we find that prePAAR functions to facilitate proper folding of the downstream PAAR domain…" needs to be looked at.h) In the Abstract and Introduction, the author should also mention the number of genera which are spanned by these 6000 putative TMD containing effectors as this number does not directly signify the impact of the work.Reviewer #3:eLife, and the authors provide a convincing body of evidence to support their claims. The work is solid, nicely presented and easy to follow. I note that I am not a structural biologist and therefore unable to provide an expert opinion on the structural portions of the work, although they seem reasonable. I have no major comments and no additional experiments are required.The results presented by Ahmad et al. provide an in-depth molecular understanding and novel concepts on previously open questions in the T6SS field, especially those related to chaperone/adapter protein activity. The work is appropriate for publication in  Essential revisions:1) The identification and the role of prePAAR motif is central to the manuscript. Therefore, mutational data of key residues in the motif showing its role in the direct interaction with the PAAR domain will significantly strengthen the major claim of the manuscript (see reviewer #1 concern b). However, in the present situation, if the authors feel that they will not be able to do this in a short frame of time, you can tone down the claims accordingly.P. aeruginosa that show prePAAR is both necessary and sufficient for PAAR-dependent Tse6-VgrG interactions in vivo, even in the absence of effector TMDs and EagT6 . However, in contrast to our in vivo findings, we found that the Tse6D11A and Tse6D11A, H15A prePAAR mutants still interact with PAAR in vitro. These experiments using purified components have revealed the complexities associated with assessing interdomain effector interactions in vitro and a proper dissection of this interaction will require extensive structural and biophysical characterization that is beyond the scope of this work. In light of this, we have removed the data from Figure 6F in the original manuscript, readjusted the figure and added in a new Figure 6D that shows our new in vivo results. As per the suggestion of the reviewer, we have also toned down our claims for the role of prePAAR in catalyzing the folding of PAAR.We agree that a point mutant control is an important addition to Figure 6F that would bolster the claims presented in the manuscript. As we describe in detail in our responses to reviewer #1, we were able to partially address this reviewer’s concern by generating new chromosomal mutations in 2) The schematic given in Figure 7 can be modified based on the comments of the reviewer.We have adjusted the figure according the reviewer’s recommendations. As described below, the only part we did not change was the sequential binding of the chaperone to the TMD because our current data suggest that chaperone-TMD binding should precede prePAAR-PAAR binding.3) Make appropriate revisions by addressing the other concerns of both the reviewers.All other concerns were also addressed in the revised text.Reviewer #1:This manuscript significantly contributes to our understanding of the mechanism by which a subset of effectors is delivered by type VI secretion system (T6SS) from one bacteria to another. The authors have identified a prePAAR motif in Eag-dependent TMD containing effectors of T6SS and segregate them into two broad classes. Further, the authors provide in vitro and in vivo evidence to show that the effector and Eag binding is specific. Earlier, the same group has shown that TMD containing effectors require chaperone assistance for loading on to VgrG protein of the type VI assembly and for eventually getting delivered to the recipient bacteria. Here, the authors have also solved the crystal structure of both class I and class II effector molecules in complex with their cognate chaperones which revealed the atomic details of the interaction. Interestingly, the newly identified prePAAR motif is shown to be essential for loading the effector molecule onto VgrG and its delivery. Overall, the work is well done and I would recommend publication after addressing the concerns that are listed below.a) The authors justify the use of "prePAAR" by quoting the earlier work by Zhang et al., 2011. However, it looks like the term prePAAR was not introduced then and the Zhang et al. work refers to the N-terminal region to PAAR domain, which includes the newly identified motif (current study) as well as the TMD. Therefore, the authors have to clearly define what exactly do they mean by prePAAR at the first instance and follow the same throughout. To provide better clarity, since “prePAAR” indicates everything N-terminal to PAAR domain therefore it may be better to use an alternative term for the newly identified motif.The reviewer is correct, Zhang et al. use the term “PrePAARTM”, which is used to refer to both prePAAR and the first TMD region preceding PAAR. Because our work demonstrates that prePAAR and the TMDs likely have distinct roles in T6S, we decided to refer to prePAAR and the TMD regions separately. We apologize for our lack of clarity with regards to our nomenclature and we have clarified this in our revised manuscript (subsection “prePAAR is a motif found in TMD-containing effectors that interact with Eag chaperones”).b) Based on the data shown in Figure 6F, the authors conclude that prePAAR interacts with PAAR domain. However, this claim needs to be directly proven by including a pull-down of PAAR domain with an N-terminal construct having prePAAR point mutants  or just prePAAR, instead of the N-terminal construct (prePAAR + TMD).P. aeruginosa. Second, we found that the nature of the interaction between the prePAAR motif and the PAAR domain are more nuanced that previously appreciated and challenging to assess in vitro. We have summarized three separate experiments that were attempted to address the reviewer’s point and based on the data described below, we have removed the blot in Figure 6F, readjusted the figure by moving some panels to Figure 6—figure supplement 1 for clarity and adding a new Figure 6D, which contains our new in vivo findings. A comprehensive structural and biophysical characterization of prePAAR-PAAR interactions will be the subject of future work in our lab but we feel that this is beyond the scope of the current study. As is, we believe that the in vivo data shown in Figure 6A and D and the in vitro data shown in 6E is sufficient to highlight the importance of prePAAR in affecting the PAAR-dependent Tse6-VgrG interaction and have removed parts of the text that alluded to prePAAR having a direct role in PAAR folding.The reviewer brings up a good point. Over the past month, we conducted a set of experiments designed to address this concern and these experiments revealed two important insights. First, we found that a prePAAR-PAAR fusion can form stable Tse6-VgrG complexes in vivo in P. aeruginosa chromosomal mutant in tse6 that results in prePAAR being fused directly to PAAR by deleting both effector TMDs and conducted a western blot to assess the formation of a PAAR-dependent Tse6-VgrG complex in vivo. As we have shown in this work and our previous work , the formation of this complex allows us to determine if PAAR is functional in vivo and in the context of this work, can be used to determine if prePAAR influences PAAR function. We found that the SDS-resistant Tse6-VgrG complex forms in a manner that is dependent on the prePAAR motif. This suggests that in conjunction with PAAR, prePAAR alone is sufficient for VgrG interaction with Tse6 and therefore it likely affects PAAR folding.Experiment 1: we generated a D11A, H15A) and conducted a pull-down using the N-terminal Tse6 fragments as bait, as done for the wild-type fragment in Figure 6F. We found that both the wild-type and double mutant pulled-down PAAR. Based on these data, we concluded that studying these two purified protein fragments in vitro does not recapitulate the molecular interactions that we observe in cells .Experiment 2: we co-expressed Eag-VSV-G and PAAR-FLAG with a double mutation-containing N-terminal fragment of Tse6 (NT-Tse6 6-tagged MBP fusions or PAAR (the prey protein in either case was FLAG-tagged) for this experiment. The constructs were tested both when co-expressed or expressed separately and co-purified. We found that in all cases, the presence of the prePAAR motif did not affect the amount of PAAR or MBP-prePAAR recovered. As with experiment 2, these data also indicate that our in vitro system is not recapitulating what we observe in P. aeruginosa.Experiment 3: we fused prePAAR to the C-terminus of maltose-binding protein (MBP). We used prePAAR sequences from Tse6 and Rhs1 and attempted pull-down experiments using the Tse6 or Rhs1 PAAR proteins. We used either HisP. aeruginosa would be redundant with our point mutant data on the full-length effector shown in Figure 6A. Additionally, because the strain expressing the Tse6 prePAAR-PAAR fusion has significantly less detectable Tse6-VgrG complex, we would prefer to test multiple fusions of prePAAR-PAAR and subsequently generate the double mutant in those strains as well to conduct a more comprehensive analysis of prePAAR-PAAR binding both in vivo and in vitro.For experiment 1, it is important to note that we detected less Tse6-VgrG complex in a strain expressing the prePAAR-PAAR fusion compared to the parental background. This may be attributable to a miscalculation in determining the ideal boundaries to truncate the effector and suggests that the PAAR domain is sensitive to misfolding when the effector is truncated. Therefore, for experiments 2 and 3, it is possible that the boundaries we used to study the PAAR domain in isolation inadvertently exposed surfaces on this protein that promote promiscuous binding independent of the prePAAR motif. Our predictions for the PAAR domain boundaries are based on orphan PAAR sequences, which, as shown in the manuscript, differ from the PAAR sequences found in Tse6/Rhs1-like effectors. The cryo-EM density for the Tse6 PAAR domain from our previous work  is also too low resolution to make an accurate prediction for domain boundaries. Further experimentation will be needed to identify the exact boundaries that produce stable variants of these domains that behave similar to the full-length proteins in vivo. We also realize we can further extend experiment 1 to include a D11A/H15A double mutation. However, we felt that the generation of this mutant in Based on these experiments and the conclusions we have drawn from them, we have adjusted our manuscript in order to tone down our assertion that prePAAR plays a role in catalyzing the folding of PAAR domains. These changes were made to the Abstract, Introduction, Results and Discussion.c) One interesting finding of the manuscript is the fidelity of the chaperone-effector pairing. However, the authors fail to comment on this fidelity based on the crystal structure information they have obtained. By attempting a simple modelling approach of cross-pairs, they may be able to provide a rationale in terms of the structural basis for this cognate pairing.We agree with the reviewer and have added additional discussion on how our co-crystal structures may provide a model for Eag-effector cognate pairing. We have added these points in the Discussion section entitled “Role of Eag chaperones in binding effector TMDs and prePAAR” Briefly, there are symmetrical hydrophobic pockets present in chaperones that interact with class I prePAAR effectors that allow for the insertion of large hydrophobic residues present in the effector TMD . These hydrophobic pockets are absent in chaperones that interact with class II effectors and thus we predict that a steric clash would occur that would prevent non-cognate pairing.We have a provided a small figure of the TMD alignments in d) The authors have an interesting proposition that H15 of prePAAR motif participates in Zn+2 binding that eventually helps in PAAR domain folding. However, it is perplexing that the D11A mutant of the effector is compromised in VgrG binding and also inducing toxicity in the target bacteria, while the H15A mutant is similar to wild type . Therefore, this proposition needs a reconsideration. Can the authors provide any evidence for the importance of Zn+2 binding in the folding of the PAAR domain?2+ binding in PAAR domain folding. Our structural model of the Tse6 PAAR domain and its structural alignment with the orphan PAAR c1882 shows that the H15 residue in Tse6 structurally overlaps with the first histidine in c1882, which is known to coordinate Zn2+ . These data, in addition to the informatics analysis showing the conservation of the H residue in prePAAR and orphan PAARs , only suggest that H15 in Tse6 takes part in Zn2+ binding. However, we show that mutation of this residue in the prePAAR motif of Tse6 does not affect the formation of a Tse6H15A-VgrG complex in vivo or in vitro but does sensitize this complex to denaturation by boiling . Thus, the PAAR domain of the Tse6H15A complex may lack some structural integrity but maintains its overall architecture and its ability to bind VgrG.The reviewer brings up an important point about the role of Zn2+-binding residues are conserved across homologs and always appear to be localized to the “tip” of the PAAR protein . Because there are multiple residues that bind Zn2+ and these residues are all located far away from the base of the protein, it could be that a single mutation at this site would not significantly alter the folding of the PAAR domain and thus abrogate its binding to VgrG. The D11 residue, however, is predicted to be localized near the base of the PAAR domain, where it potentially mediates important intramolecular interactions that are necessary to stabilize the folding of this region and mediate its interaction with VgrG. It is possible that the H15 residue tethers the prePAAR motif to the PAAR domain, whereas the D11 residue contributes important electrostatic interactions that are necessary for overall PAAR domain folding.In the initial characterization of PAAR proteins, it was found that Zn2+ affects PAAR folding is out of the scope of this paper because we feel that the high-resolution structural characterization of an Eag chaperone in complex with the prePAAR-TMD-PAAR region of an effector is the most direct way to determine the specific interactions between prePAAR residues and the PAAR domain or its bound zinc ion.At this time, the question of whether Zne) The authors should consider the following suggestions for preparing a more coherent schematic of the entire finding depicted in Figure 7. (1) prePAAR can be depicted at the tip of PAAR domain as the current depiction is misleading where prePAAR is shown at the interface of PAAR domain and VgrG. However, the authors do not seem to have provided any evidence for prePAAR and VgrG interaction. (2) As per the structural knowledge (from this work and previous studies), it is clear that the chaperones are symmetric dimers and therefore the schematics should represent the same throughout the manuscript. (3) From the model , it appears that binding of prePAAR to PAAR domain is dependent on TMD-chaperone binding. However, there is no experimental evidence for such sequential binding. (4) Authors should provide an explanation for their statement that split PAAR is an “evolved” version of PAAR domain and the same needs to be included in the Discussion. Why it cannot be the other way around? Also, it is worth considering the possibility that in split-PAARs the stabilization of PAAR domain with prePAAR may provide structural rigidity to the TMD for effective insertion into the membrane.The reviewer brings up several important points about the figure and we have addressed their concerns in sequence. (1) We have changed the position of prePAAR in the figure to depict an interaction solely with the PAAR domain and not VgrG. (2) We have changed the cartoon of the chaperone to be symmetric in both Figures 1B, 7B and C. (3) While the reviewer is correct in that there is no direct evidence to show that the chaperone must bind the TMD before prePAAR can bind PAAR, we believe that it is important to show this interaction in this way. The chaperone gene and effector gene are found in the same operon and co-transcribed, as shown in Figure 1A. Based on our current and previous work, we know that the TMDs of prePAAR effectors are unstable and must be stabilized by the chaperone. If the chaperone is translated first, it is likely that it will bind the TMDs as they are translated, rather than bind a fully translated prePAAR-TMD-PAAR effector. Binding prePAAR and the TMDs in this way may orient these regions to facilitate proper binding with PAAR . (4) We agree with the reviewer that an explanation of the relationship between orphan PAARs and split PAARs is required in the Results and in the Discussion. To understand the relationship between these PAAR proteins, we constructed a phylogram of orphan and split PAAR (prePAAR-TMD-PAAR) proteins. For this analysis, we used the 564 orphan PAAR and 1,765 split PAAR sequences from Figure 6—figure supplement 1C. The split PAAR sequences are significantly longer than the orphan PAAR sequences due to the presence of the TMD. To more accurately identify boundaries for the PAAR domain in the split PAAR sequences, we built a hidden Markov model using the representative PAAR proteins from our analysis in Figure 1G. We used the hidden Markov model established from this analysis to identify the PAAR domain across the 1,765 split PAAR sequences and constructed a phylogenetic tree using the same methods as done for analysis in Figure 1. We found that all orphan PAAR sequences grouped at the root of the tree suggesting that they are a common ancestor to the PAAR domain of prePAAR-containing effector sequences .f) In the Abstract, the authors state "…our findings define the mechanism of chaperone assisted secretion of a widespread family of T6SS membrane protein effectors". It appears that the significant part of this claim is already made in their earlier work  and therefore the authors need to make a clear distinction.We agree with the reviewer. To address this, we have changed the last sentence of the Abstract to the following: “Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.”g) The authors have shown that mutations in pre-PAAR affect the binding of the effector to VgrG. However, there is no evidence which shows that prePAAR has any direct role in PAAR domain folding and therefore the lines in the Abstract, "…we find that prePAAR functions to facilitate proper folding of the downstream PAAR domain…" needs to be looked at.We have changed the sentence to the following: “In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions.”h) In Abstract and Introduction, the author should also mention the number of genera which are spanned by these 6000 putative TMD containing effectors as this number does not directly signify the impact of the work.We agree that the number of effectors is only significant if we state the number of genera these are found in. Therefore, we have changed the Abstract to include the line “encoded predominantly by 15 genera of Proteobacteria” to better highlight the widespread distribution of these effectors.


Homozygous calsequestrin 2 (CASQ2) point mutations leads to catecholaminergic polymorphic ventricular tachycardia: a common pathogenetic feature appears to be the drastic reduction of mutant CASQ2 in spite of normal transcription. Comparative biochemical analysis of R33Q and D307H knock in mutant mice identifies different pathogenetic mechanisms for CASQ2 degradation and different molecular adaptive mechanisms. In particular, each CASQ2 point mutation evokes specific adaptive cellular and molecular processes in each of the four adaptive pathways investigated. Thus, similar clinical phenotypes and identical cellular mechanism for cardiac arrhythmia might imply different molecular adaptive mechanisms. Homozygous mutations in the cardiac calsequestrin (CASQ2) gene are linked to the recessive form of catecholaminergic polymorphic ventricular tachycardia (CPVT), a life-threatening genetic disease causing dramatic episodes of cardiac arrhythmia and syncope, in the absence of macroscopic structural abnormalities. The genetic background of CPVT is heterogeneous  components and (4) activation of small Heat Shock Proteins (sHSPs).The present work was undertaken to understand whether two different CASQ2 mutants (R33Q versus D307H) trigger either similar or different adaptive processes. We have identified and compared four different, relevant pathways: (1) activation of ER stress markers such as GRP78, GRP94 and calreticulin (CRT) as well as the compensative and protective protein, Bcl-2, (2) protein degradation pathways, (3) change of Store Operated CaLittle is known about the degradative fate of CASQ2; numerous and adaptable degradative pathways might be involved in the heart depending upon age, anatomy, state of health and disease among which are to be recalled the following: the ubiquitin-proteasome system, micro-autophagy, chaperone-mediated autophagy, ER-stress-induced pre-emptive quality control and the calpain system   and CASQ2 knock-out (KO) mice, from birth to adulthood  and by Tel Aviv University Animal Research Ethics Committee (M-14-063).n = 4 for each group; body weight 23–30 g).Experiments were carried out on male gene targeted C57BL/6j mice, homozygous for R33Q CASQ2 . Equal amounts (100 µg) of protein were subjected to SDS-PAGE and transferred to nitrocellulose membranes by western blot transfer system. Nonspecific binding was blocked with 10% nonfat milk in either PBS or TBST and incubated overnight at 4 °C with antibodies diluited in either PBS or TBST. After washing, membranes were incubated with anti-rabbit  or anti-mouse  secondary antibodies conjugated to either alkaline phosphatase or horseradish peroxidase. In the latter case, visualization was achieved using ECL Western Blotting substrate (Pierce) and images were taken through UVTech instrument (Eppendorf).Intensity of each band was determined by Scion Image software. Protein-signal densities were normalized to the corresponding actin-signal densities within a linear relationship of antigen concentration versus signal density , anti-Hsp 20 , anti-S16 Hsp-20 , anti-Hsp27 , anti-S82 Hsp27 , anti-S59 αB-crystallin  and anti-S45 αB-crystallin  from Abcam; anti GRP78 , anti-CASQ2  and anti-CRT  from Thermo; anti Bcl-2  and anti-CNX  from Santa Cruz; anti-Actin , anti-STIM1 , anti-TRPC3 , anti-Orai1 , anti-EDEM1  and anti-Herp  from Sigma; anti-Beclin1  and anti-HRD1 (1:1000) from Novus Biologicals; anti-GRP94  from Stressgen; mono- and polyubiquitinated conjugates  from EnzoLifeScience. Anti Derlin-1  was a generous gift of Doriana Sandonà.Data were expressed as mean ± standard error (SE). Comparisons between means of two groups were performed by unpaired two-tailed Student’s t-test. Comparisons among means of 3 groups were performed by one-way ANOVA Bonferroni’s test. Differences were considered significant at *P < 0.05,**P < 0.01 and ***P < 0.005.p < 0.005). R33Q CASQ2 and D307H CASQ2 are missense mutations of CASQ2 and are likely to be post-translationally down-regulated since they might be recognized as misfolded protein inside the ER/SR, thus activating an ER stress response. The conspicuous decrease of D307H CASQ2 protein was not accompanied by up-regulation of ER stress markers, at variance with findings on R33Q CASQ2 , in accordance with previous observations on both R33Q KI and KO models , no difference in Orai1 expression (109.7 ± 3.7) and drastic reduction of the TRPC3 channel , in comparison to WT control.Alterations in the Store Operated Calcium Entry (SOCE) complex were observed for both R33Q KI and KO models, as judged by expression of STIM1, and Orai1, TRPC3, two SOCE related CaIn conclusion, the D307H KI mouse model shows adaptative mechanisms that markedly differ from the R33Q KI model. In particular, the absence of ER stress activation in D307H KI hearts suggests differences in activation of quality control systems.ER-associated degradation (ERAD) is a quality control system, by which unfolded or misfolded proteins are retrotranslocated from the ER into the cytosol and degraded by the ubiquitin-proteasome system. ERAD occurs in a multistep process including recognition, extraction and ubiquitination of ER proteins. Many ER chaperones are likely to participate in recognition and recruitment of misfolded proteins to the ERAD complex, including GRP78, calnexin (CNX) and EDEM1  in D307H KI hearts but not in R33Q KI hearts (116.3 ± 18.0%). On the other hand, Beclin-1 expression was significantly increased  in R33Q KI hearts but not in D307H KI hearts (87.6 ± 7.7%).As shown in Fig. Content of total mono- and poly-ubiquitinated proteins were studied and we observed a mild increase in D307H KI in comparison to WT and R33Q KI Fig. c.In conclusion, R33Q and D307H KI models show different molecular adaptations, the first involving the autophagy system, the second involving the ERAD pathway, in particular the retrotranslocation machinery towards proteasome.sHsps are proteins with molecular mass ranging from 15 to 30 kDa and a conserved α-crystallin domain. sHsps act as molecular chaperones by preventing either aggregation or misfolding of proteins and allow their correct refolding under stress conditions.p < 0.05), S59 and S45 of αB-crystallin , and S16 of Hsp20 (349.3 ± 84.0% p < 0.05) was observed in R33Q KI hearts, whereas increased phosphorylation of S82 Hsp27  was observed in D307H KI hearts. No alteration whatsoever of phosphorylation state of Hsp27, Hsp20 and αB-crystallin was observed for KO model suggesting that up-regulation of sHsps phosphorylation state was due to the presence of a mutant CASQ2 rather than drastic reduction of CASQ2 content.We monitored the expression of Hsp27, Hsp20 and αB-crystallin. No differences in sHsps were observed between D307H and R33Q KI hearts (Fig. In conclusion, R33Q CASQ2 triggers a complex sHSPs-mediated protective response that appears to be different from that evoked by D307H CASQ2.The goal of this study was to understand how hearts adapt to the presence of mutant CASQ2s comparing two KI mouse models of recessively inherited human CPVT: D307H and R33Q. The present results indicate qualitative differences in induction of ER stress, change of SOCE components, regulation of protein degradation pathways and activation of sHSP protection, and provide new insight in the pathogenesis of CPVT.Characterization of D307H KI heart with regard to ER stress markers and SOCE components highlights no changes in markers of ER stress, such as GRP78, GRP94 and CRT, up-regulation of Bcl-2 and STIM1 and down-regulation of TRPC3.2+ handling during diastolic phase (Valle et al. In D307H KI mice, a different adaptation of SOCE components was observed; in R33Q KI and KO mice, we recently found up-regulation of STIM1 matched to up-regulation of TRPC channels leading to speculation that SOCE did not represent a complementary process to excitation-contraction coupling but it was crucial in Ca2+ concentrations, indicating a defect in its ability to dynamically handle Ca2+ (Kalyanasundaram et al. Previous works (Song et al. 2+ transients and cell shortening in tachypaced HL-1 cardiomyocytes and this protective effect is phosphorylation-dependent (Brundel et al. 2+ handling may involve either direct modulation of ion channel function or modulation of specific kinases, resulting in the conservation of ion currents (Christ et al. 2+ cycling in the sarcoplasmic reticulum and enhances the contractile function of the cardiomyocytes (Qian et al. + channels (Kashlan et al. 2-terminus, respectively and preventing calpain cleaving (Lu et al. 2+ cycling in the sarcoplasmic reticulum and enhances the contractile function of the cardiomyocyte (Qian et al. In the heart some sHSPs are expressed at high levels, especially Hsp27, αB-crystallin and Hsp20 and have various functions. First, sHSPs act as molecular chaperones to facilitate protein folding, localization, degradation, and function, thereby maintaining proteostasis and preventing various forms of cardiomyocyte damage (Westerheide and Morimoto, For Hsp27 and αB-crystallin, phosphorylation increases chaperone activity and reduces oligomeric size whereas the phosphorylation of Hsp20 at serine 16 was found to protect against cardiac hypertrophy (Qian et al. Very high phosphorylated forms for all the three sHSPs were observed in R33Q KI, whereas only for phosphorylated Hsp27 in D307H KI. In KO hearts we did not find variations in sHSPs content and in phosphorylation forms suggesting that adaptive changes are dependent upon the specific point-mutation and not related to the low CASQ2 level.The present results suggest that R33Q KI hearts might be totally protected from potential damages and probably against hypertrophy since Hsp20 is more phosphorylated. On the contrary, D307H KI hearts could be more susceptible to hypertrophy and accordingly enlarged atria and ventricles were previously reported at elderly age (Song et al. 2+ release activating the electrogenic sarcolemmal NCX to extrude the cytosolic Ca2+, causing membrane depolarization and triggering early or delayed after depolarizations (Knollmann et al. The proposed cellular mechanism for CPVT pathogenesis is based on the aberrant SR Ca