Klara Kovarski

Florent Caetta

Martial Mermillod c Carole Peyrin

Céline Perez

Lionel Granjon

Richard Delorme

Ariane Cartigny

Tiziana Zalla

Sylvie Chokron
email: sylvie.chokron@gmail.com

Emotional face recognition in autism and in cerebral visual impairments: In search for specificity

Keywords: Visual perception, Magnocellular pathway, Spatial Frequency, Autism, Cerebral Visual Impairment, Emotion. Emotional faces in autism and CVI 1 9

Autism Spectrum Disorder (ASD) is characterized by difficulties in the social domain, but also by hyper and hypo-reactivity. Atypical visual behaviours and processing have often been observed. Nevertheless, several similar signs are also identified in other clinical conditions including Cerebral Visual Impairments (CVI). In the present study we investigated emotional face categorization in groups of children with ASD and CVI by comparing each group to Typically Developing individuals (TD) in two tasks. Stimuli were either non filtered or filtered by low and high spatial frequencies (LSF and HSF). All participants completed the Autism-Spectrum score (AQ) and a complete neurovisual evaluation. The results show that while both clinical groups presented difficulties in the emotional face recognition tasks and atypical processing of filtered stimuli, they did not differ from one another. Additionally, autistic traits were observed in the CVI group and symmetrically, some visual disturbances were present in the ASD group as measured via the AQ score and a neurovisual evaluation, respectively. The present study suggests the relevance of comparing ASD to CVI by showing that emotional face categorization difficulties should not be solely considered as autism-specific but merit investigation for potential dysfunction of the visual processing neural network. These results are of interest in both clinical and research perspectives, indicating that systematic visual examination is warranted for individuals with ASD.

Introduction

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterised by i) impairments in the social and communication domain and by ii) stereotyped and repetitive behaviours as well as by sensory hypo and hyper-responsiveness [START_REF] Apa | Diagnostic and Statistical Manual of Mental Disorders (DSM-5)[END_REF]. These atypical sensory behaviours in all domains, are now considered important diagnostic features of ASD.

Faces have represented preferred visual stimuli in ASD research to investigate social disturbances [START_REF] Elsabbagh | The development of face orienting mechanisms in infants at-risk for autism[END_REF][START_REF] Falck-Ytter | Eye tracking in early autism research[END_REF][START_REF] Klin | Visual fixation patterns during viewing of naturalistic social situations as predictors of social competence in individuals with autism[END_REF] and atypical processing of facial expressions, gender and identity [START_REF] Behrmann | Configural processing in autism and its relationship to face processing[END_REF][START_REF] Boraston | Impaired sadness recognition is linked to social interaction deficit in autism[END_REF][START_REF] Critchley | The functional neuroanatomy of social behaviour: changes in cerebral blood flow when people with autistic disorder process facial expressions[END_REF][START_REF] Deruelle | Spatial Frequency and Face Processing in Children with Autism and Asperger Syndrome[END_REF][START_REF] Deruelle | Attention to low-and high-spatial frequencies in categorizing facial identities, emotions and gender in children with autism[END_REF][START_REF] Pelphrey | Perception of dynamic changes in facial affect and identity in autism[END_REF]. Atypical brain responses to face stimuli have also been reported in autistic individuals when compared with matched controls [START_REF] Batty | Early processing of emotional faces in children with autism: An event-related potential study[END_REF][START_REF] Hadjikhani | Look me in the eyes: constraining gaze in the eye-region provokes abnormally high subcortical activation in autism[END_REF][START_REF] Hileman | Developmental and individual differences on the P1 and N170 ERP components in children with and without autism[END_REF][START_REF] Pelphrey | Perception of dynamic changes in facial affect and identity in autism[END_REF], suggesting that electrophysiological and neuroimaging responses are affected from the earliest stages of processing, in both visual and face-specific brain regions [START_REF] Critchley | The functional neuroanatomy of social behaviour: changes in cerebral blood flow when people with autistic disorder process facial expressions[END_REF][START_REF] Kovarski | Enhanced early visual responses during implicit emotional faces processing in Autism Spectrum Disorder[END_REF][START_REF] Wong | Abnormal spatiotemporal processing of emotional facial expressions in childhood autism: dipole source analysis of event-related potentials[END_REF]. Visual perception of non-social and lowlevel stimuli has been described in ASD as being impaired, suggesting a broad range of disruption of the processing of visual information [START_REF] Bertone | Enhanced and diminished visuospatial information processing in autism depends on stimulus complexity[END_REF].

Difficulties in processing faces might thus be partly explained by broader disruptions in processing visual information [START_REF] Mottron | Enhanced perceptual functioning in autism: an update, and eight principles of autistic perception[END_REF].

Two major visual pathways from the retinal ganglion cells have been identified: the parvocellular (P) and magnocellular (M) pathways. The M-pathway preferentially processes Emotional faces in autism and CVI low-spatial frequencies (LSF) with high-temporal resolution, while the P-pathway conveys high-spatial frequencies (HSF) with low-temporal resolution [START_REF] Laycock | A role for the 'magnocellular advantage' in visual impairments in neurodevelopmental and psychiatric disorders[END_REF]. Moreover, the M-pathway represents a fast-route with specific sub-cortical projections, thus crucially contributing to emotional face processing [START_REF] Vuilleumier | Distinct spatial frequency sensitivities for processing faces and emotional expressions[END_REF]. The hypothesis of a specific dysfunction of the M-pathway has been proposed in ASD to account for atypicalities in the processing of both social stimuli and low-level stimuli of specific spatial frequencies [START_REF] Greenaway | Marked selective impairment in autism on an index of magnocellular function[END_REF][START_REF] Kovarski | Brief report: Early VEPs to pattern-reversal in adolescents and adults with autism[END_REF][START_REF] Mccleery | Abnormal magnocellular pathway visual processing in infants at risk for autism[END_REF][START_REF] Pei | Electrophysiological measures of low-level vision reveal spatial processing deficits and hemispheric asymmetry in autism spectrum disorder[END_REF]. To investigate this hypothesis, stimuli are usually filtered in LSF and HSF, allowing investigators to artificially and preferentially stimulate the M-or the P-pathways respectively [START_REF] Corradi-Dell'acqua | Neural responses to emotional expression information in high-and low-spatial frequency in autism: evidence for a cortical dysfunction[END_REF][START_REF] Deruelle | Face processing in children with autism: The role of high spatial frequency information[END_REF][START_REF] Deruelle | Spatial Frequency and Face Processing in Children with Autism and Asperger Syndrome[END_REF][START_REF] Deruelle | Attention to low-and high-spatial frequencies in categorizing facial identities, emotions and gender in children with autism[END_REF]. The majority of these studies demonstrated an atypical HSF-based preference for processing faces in ASD relative to controls, in line with the M-pathway impairment hypothesis (but see [START_REF] Corradi-Dell'acqua | Neural responses to emotional expression information in high-and low-spatial frequency in autism: evidence for a cortical dysfunction[END_REF].

Deficits in the M-pathway have often been related to other psychiatric and neurodevelopmental disorders (e.g., schizophrenia, developmental dyslexia, [START_REF] Jahshan | Probing the magnocellular and parvocellular visual pathways in facial emotion perception in schizophrenia[END_REF][START_REF] Pellicano | Investigating the functional integrity of the dorsal visual pathway in autism and dyslexia[END_REF], and also to post-stroke cortical visual disorders (also named Cerebral Visual Impairments or CVI) such as homonymous hemianopia, a visual field defect occurring after unilateral post-chiasmatic damage. In these patients, a unilateral occipital lesion induces a deficit in detecting visual stimuli in the contralesional visual field. In addition, a deficit for processing LSF (i.e. M-pathway) has been specifically found in patients with a right occipital lesion and a left homonymous hemianopia [START_REF] Cavezian | Hemisphere-dependent ipsilesional deficits in hemianopia: Sightblindness in the 'intact' visual field[END_REF][START_REF] Perez | An FMRI investigation of the cortical network underlying detection and categorization abilities in hemianopic patients[END_REF][START_REF] Peyrin | Effect of temporal constraints on hemispheric asymmetries during spatial frequency processing[END_REF]. Thus, M-pathway dysfunction Emotional faces in autism and CVI is not specific to autism, confirming the necessity to compare ASD groups not only to typically developing controls (TD), but also to other atypical conditions such as patients with occipital lesions. Comparing visual brain activity or behavioural responses in individuals with ASD only to TD individuals, reveals group differences but has significant limitations in understanding the specificity of such anomalies. Nevertheless, an increasing number of studies have been performed that compare ASD with comorbid or isolated Attention Deficit Hyperactivity Disorder (ADHD), developmental dyslexia, and schizophrenia [START_REF] Bird | The role of alexithymia in reduced eyefixation in Autism Spectrum Conditions[END_REF][START_REF] Gori | Magnocellular-dorsal pathway and sub-lexical route in developmental dyslexia[END_REF][START_REF] Jahshan | Probing the magnocellular and parvocellular visual pathways in facial emotion perception in schizophrenia[END_REF][START_REF] Jang | Rates of comorbid symptoms in children with ASD, ADHD, and comorbid ASD and ADHD[END_REF], shedding light on the necessity to disentangle what is truly specific to autism from what might be generalized among other conditions. Similarly, children with severe visual deficits, or blind children can present several autistic behaviours as well as difficulties in the communication domain, emphasizing the co-occurrence of visual impairments and autism [START_REF] Chokron | The inter-relationships between cerebral visual impairment, autism and intellectual disability[END_REF][START_REF] Fazzi | Autism in children with cerebral and peripheral visual impairment: fact or artifact[END_REF][START_REF] Hobson | Autism and congenital blindness[END_REF][START_REF] Kiani | The relationship between symptoms of autism spectrum disorder and visual impairment among adults with intellectual disability[END_REF]. In the same vein, if as above-mentioned, adults with CVI may present deficits of the magnocellular pathway [START_REF] Cavezian | Hemisphere-dependent ipsilesional deficits in hemianopia: Sightblindness in the 'intact' visual field[END_REF][START_REF] Perez | An FMRI investigation of the cortical network underlying detection and categorization abilities in hemianopic patients[END_REF][START_REF] Peyrin | Effect of temporal constraints on hemispheric asymmetries during spatial frequency processing[END_REF], children with CVI often exhibit a complex neuropsychological profile associating visual, behavioural and cognitive deficits after lesions between the optic chiasm and cortical visual regions [START_REF] Chokron | Impact of cerebral visual impairments on motor skills: implications for developmental coordination disorders[END_REF][START_REF] Martin | Cerebral versus Ocular Visual Impairment: The Impact on Developmental Neuroplasticity[END_REF]. Indeed, children with CVI often present cognitive and motor impairments [START_REF] Chokron | Impact of cerebral visual impairments on motor skills: implications for developmental coordination disorders[END_REF], but also difficulties in language and memory processing or impaired face and object recognition [START_REF] Fazzi | Autism in children with cerebral and peripheral visual impairment: fact or artifact[END_REF][START_REF] Fazzi | Spectrum of visual disorders in children with cerebral visual impairment[END_REF][START_REF] Good | Cortical visual impairment in children[END_REF]. Moreover, they frequently manifest limitations in visual search, and impaired visual guidance of movement, related to paucity of the tracts between the mapping centres in the posterior parietal lobes (the superior longitudinal fasciculi) [START_REF] Merabet | Disentangling How the Brain is "Wired" in Cortical (Cerebral) Visual Impairment[END_REF]. Accordingly, an overlap between CVI and ASD signs should be acknowledged [START_REF] Chokron | The inter-relationships between cerebral visual impairment, autism and intellectual disability[END_REF][START_REF] Chokron | Troubles du spectre de l'autisme et troubles de la fonction visuelle : revue critique, implications théoriques et cliniques[END_REF][START_REF] Chokron | Troubles de la fonction visuelle, troubles de l'interaction et développement cognitif[END_REF][START_REF] Kiani | The relationship between symptoms of autism spectrum disorder and visual impairment among adults with intellectual disability[END_REF] raising the question of the important role of visual functioning in social development. This opens up key questions on the specificity of atypical face processing in ASD, and whether this might explain in part by broader disturbances of visual function [START_REF] Chokron | The inter-relationships between cerebral visual impairment, autism and intellectual disability[END_REF].

In the present study, we investigated recognition of facial emotion expression and its relation to autistic signs and visual functioning in ASD, CVI and TD. All participants took part in two emotional face categorization tasks of different difficulty: i) a difficult categorization task between a negative and a positive emotion (i.e. angry and happy faces) and ii) an easy categorization task between an emotional and non-emotional facial expression (i.e. fearful and neutral faces). Stimuli were either filtered (LSF and HSF) or unfiltered. All participants completed the Autism-spectrum Quotient (AQ, [START_REF] Baron-Cohen | The autismspectrum quotient (AQ): evidence from Asperger syndrome/high-functioning autism, males and females, scientists and mathematicians[END_REF]) and a neurovisual evaluations.

In line with the primary diagnoses of the participants, we expected the ASD group to have higher AQ scores compared to other groups and, similarly, the CVI group to have more severe visual disturbances than other groups. We hypothesised that both ASD and CVI individuals would present difficulties for categorizing faces exhibiting features of emotion. However, because of a possible specific disruption of the M-pathway in autism, difficulties could be greater in ASD individuals when categorizing LSF stimuli. Two tasks of different complexity were used to investigate how the type of categorization could affect the performance of ASD and CVI groups. To our knowledge, this is the first study comparing, within the same protocol, visual performance as well as autistic traits in individuals with ASD with both age matched TD participants and with individuals with CVI. A one-way ANOVA was performed to compare the groups on their AQ results. Post-hoc comparisons were performed between groups.

Materials and methods

Participants

Ninety

Visual acuity was corrected with spectacles to be within the normal range in all participants.

The local ethics committee approved the protocol. Informed written consent was obtained from all participants (and from caregivers for minor participants).

Tasks and procedure

Stimuli comprised sixteen greyscale male or female emotional face images (256 x 256 pixels) selected from the Karolinska database [START_REF] Goeleven | The Karolinska Directed Emotional Faces: A validation study[END_REF]. In one task, Angry (A) and Happy (H) faces were presented, while in the other task Fearful (F) and Neutral (N) faces were displayed (4 male and 4 female faces for each task). These computer screen presented stimuli were either filtered in LSF, HSF or non-filtered (Broad Spatial Frequency, BFS). The spatial frequency content above 2 cycle/degree of visual angle was removed for LSF stimuli (i.e., low-pass cut-off of 16 cycles per image) and below 3 cycles/degree (i.e., high-pass cut-off of 24 cycles per image) for HSF stimuli. Participants set at about 50 cm from the screen in a room with background lighting. Stimuli of 8° degrees of visual angle were presented centrally on a computer (1280 x 800 pixels) with luminance of 185 cd/m². We controlled that all hemianopic patients could detect the central picture and thus perform the categorization tasks. The experiment was programmed with E-Prime (E-Prime Psychology Software Tools Inc., Pittsburgh, USA). Participants had to decide whether the stimulus was an Angry or Happy face (AH task), while in the other task faces could be either Neutral or Fearful (NF task), facilitating investigation of the influence of task difficulty. The order of tasks was counterbalanced across participants. Each task comprised 48 trials (24 for each emotion; 16 for each SF; 8 for each Emotion without SF). The instructions were explained verbally and two training trials were run initially to familiarize the participants with the stimuli and the tasks. Each trial began with a central fixation cross on the screen for 500 ms, to attract attention, followed by the face stimulus (200 ms), then by a backward mask for 20 ms to avoid retinal persistence and finally by two coloured squares on the screen, each square corresponding to a specific facial expression (see Figure 1). Participants were asked to categorize the face stimuli by pressing the corresponding colour keys on a keyboard (colour correspondence and the button was counterbalanced across participants). Accuracy and response times were recorded for each trial.

- ------------------Figure 1 about here ---------------------

Neurovisual evaluation

A neurovisual evaluation was carried out for all participants [START_REF] Cavezian | Assessment of visuo-attentional abilities in young children with or without visual disorder: toward a systematic screening in the general population[END_REF][START_REF] Cavezian | Ophthalmic disorder may affect visuo-attentional performance in childhood[END_REF]. This included oculomotor assessment (fixation, smooth pursuit, and nystagmus detection), visual acuity, contrast sensitivity, visual field, visual extinction test, visual memory (shapes), neglect tests (bells cancellation test for adults, Bear and A cancellation tests for children), local and global attention (embedded figures test) and the simple visual analysis test (shape matching test). These evaluations were not time limited. To provide a broad index of the visual functioning, a Global Visual Score (GVS) was calculated in all participants by measuring the accuracy in all visual tests. A Kruskal-Wallis test was performed to compare groups on the GVS and post-hoc analyses were performed for single comparisons.

Processing and Analyses

To reduce the effect of extreme values for the emotional categorization tasks, RTs for each participant, task and experimental condition (Frequency) results were removed from analysis below 5 th percentile and above 95 th percentile. After rejection, both the CVI (76±6.3) and the ASD (76.7±6.2) groups presented fewer trials compared to the TD group (80.6±2.4) as revealed by a one-factor ANOVA (F(2, 88)=10.12, P < .001, η 2 p= .187). Because this reflects inter-and intra-participants variability in the clinical populations, data from all participants was kept, as they presented a sufficient number of trials (>78% of trials).

Repeated-measures ANOVA on mean Error Rate for both tasks was performed after an Arcsine square root transformation (Asin-ER) to reduce ceiling effects and to ensure variance homogeneity, with Group (ASD, CVI, TD) as between-group factor and Frequency (LSF, BSF, HSF) and Task (AH, NF) as within-group factors. A similar repeated-measures ANOVA was also performed on Log transformed RTs (Log-RTs) for only accurate responses. For both ANOVAs, Bonferroni post-hoc corrections were applied where needed. Corrected P values and effect sizes (partial eta-squares, η 2 p) are provided. The same analyses were also performed with respect to Age as continuous variables to ensure that no Age by Group significant interactions could affect group effects. Asin-ER and Log-RTs (between different frequencies and tasks) were correlated with AQ scores and with the GVS.

Results

3.1Autistic-spectrum Quotient

A significant Group effect was found (F(2,88) = 43.26, P < .001, η 2 p= .496) due to TD participants presenting a smaller AQ score (12.6 ± 5.4) than both the CVI group (19.8 ± 8.8)

and the ASD group (30.1 ± 8.4), but also due to a smaller AQ score for the CVI than the ASD group (all P < .001), meaning that the ASD group presented more autistic traits compared to CVI and TD groups, but that the CVI also presented a greater AQ score than TD individuals.

Neurovisual evaluation

The Global Visual Score (GVS) of the neurovisual evaluation led to a significant Group effect (H(2) = 17.27, P < .001; TD = 96.2% ± 3.1; CVI = 87.2% ± 10.8; ASD = 92.6% ± 6.9) due to a greater visual performance score in the TD group compared to the CVI group (P < .001), while there was no difference between the CVI and ASD groups (P = .16), nor between ASD and TD (P = .30). Nevertheless, individuals with ASD on average presented more visual disturbances as compared to the TD group (e.g., visual field defect and difficulties in visual fixation, visual memory and attention).

Emotion categorization

Accuracy (Asin-ER)

The analyses revealed a main effect of Group (F(2,88) = 11.02, P < .001, η 2 p = .200) due to the TD group presenting a greater performance score as compared to both CVI (P < .001) and ASD (P = .003) groups, while the clinical groups did not differ (Table 1).

The main effect of Task was significant (F(2,88) = 21.93, P < .001, η 2 p = .199) and interacted with Group (F(2,88) = 4.14, P= .019, η 2 p = .086) due to the ASD and CVI groups presenting a worse Asin-ER in the NF task compared to the AH task (P = .05, P = .005, respectively) and to the TD presenting better accuracy compared to both ASD and CVI groups on the NF task only (P = .002 and P < .001, respectively). The ASD and CVI groups did not differ on either task. The main effect of Frequency (F(1.82,160.01) = 52.09, P < .001, η 2 p = .372) was significant, due to Asin-ER being worse for LSF stimuli compared to BSF and HSF (both P < .001). Task interacted with Frequency (F(1.92,169.4) = 4.47, P = .014, η 2 p = .048) as Asin-ER was worse in the NF task compared to the AH task for only LSF filtering (P < .001). The interaction between Task, Group and Frequency was also significant (F(3.85,169.4) = 3.87, P = .005, η 2 p = .081). Post-hoc analyses revealed that ASD and CVI groups differing from TD individuals on HSF only in the NF task (P = .04 and P = .02, respectively). This interaction was further explained by within-group differences in the NF task as the accuracy for LSF stimuli was lower, compared to BSF stimuli in all groups (all P < .001), but only the CVI and TD groups also presented worse accuracy for LSF compared to HSF (P = .01 and P < .001, respectively). Finally, only the CVI group presented difficulties for LSF compared to BSF stimuli in the AH task (P = .006). To test our initial hypothesis of a specific impairment in processing of the LSF in the clinical groups, and to better understand the significant three-way interaction, we performed two additional two-way ANOVAs for each task. The Group by

Frequency interaction was significant only in the AH task (F(4,176) = 3.72, P = .006, η 2 p =.078) as both ASD and CVI groups differed from TD individuals on LSF only (P = .03 and P = .004, respectively).

-------------------Figure 2 and Table 1 about here ---------------------

Reaction-times (Log-RTs)

The analysis revealed a significant effect of Group (F(2,88) = 13.48, P < .001, η 2 p = .235) (see Table 1). Both ASD and CVI groups were slower than the TD group (both P < .001), but clinical groups did not differ from each other (P > .99). The effect of Task was significant Emotional faces in autism and CVI 1 1

(F(1,88) = 30.16, P < .001, η 2 p = .255), due to Log-RTs being greater for the NF task (6.55 ± 0.36) than for the AH task (6.44 ± 0.32). Critically, there was a significant interaction between Group and Task (F(2,88) = 3.12, P = 0.049, η 2 p = .066). Only ASD and CVI groups presented higher Log-RT in the NF task than in the AH task (P = .019 and P = .002, respectively), while the TD group did not present a Task effect (P = .81). Moreover, the ASD group differed from the TD group on the NF task only (P < .001) although a tendency was present for the AH task (P = .06). The CVI group was significantly different from the TD group on both tasks (AH: P = .019 and NF: P < .001). A significant effect of Frequency (F(2,176) = 32.21, P < .001, η 2 p =

.268) and a significant interaction between Group and Frequency were found (F(4,176) = 4.32, P = .002, η 2 p= .089). This interaction was explained by higher Log-RT for LSF and HSF than BSF in the ASD group (P < .001 and P = .002, respectively) while in the CVI group Log-RT was higher for LSF compared to HSF (P < .001) but not to BSF (P = .24) and in the TD group only LSF led to longer Log-RT compared to BSF and HSF (both P < .001). The TD group differed from ASD and CVI groups on the three filters (all P < .05), but the clinical groups were not statistically different (all n.s.).

Age analyses

To ensure that age did not affect group effects in the preceding analyses, the same ANOVAs on Asin-ER and Log-RT were performed with Age as a continuous variable factor. Both analyses did not reveal any Age by Group interaction, thus age did not affect the Group effects.

Correlations

No significant correlations were found for any group between accuracy (Asin-ER) and the GVS score, nor between accuracy and the AQ score. A significant negative correlation between the Log_RT and the performance of the GVS for the ASD group (r = -0.61, P = .005) and the TD group (r = -0.29, P = .038) emerged, revealing that the more severe the visual disturbance is, the slower the responses are. This correlation was not significant in the CVI group, probably because in this group, all patients presented severe neurovisual disturbance (r = -0.04, P = .85).

No significant correlations were found for any group between the Log RT and the AQ.

Discussion

In the present study, facial emotion categorizations of filtered and non-filtered stimuli (LSF, HSF and BSF) in individuals with ASD were compared to both a non-clinical population and a group with CVI. The present research revealed the presence of some visual impairments in the ASD group [START_REF] Coulter | Understanding the visual symptoms of individuals with autism spectrum disorder (ASD)[END_REF][START_REF] Fazzi | Autism in children with cerebral and peripheral visual impairment: fact or artifact[END_REF][START_REF] Little | Vision in children with autism spectrum disorder: a critical review[END_REF][START_REF] Simmons | Vision in autism spectrum disorders[END_REF]. Far from presenting only the frequently reported visual disorders, additional visual deficits were observed in the ASD group. As a consequence, visual functioning impairments should be systematically evaluated in ASD subjects, not only for clinical purposes but also in experimental studies where visual stimuli are used. Moreover, testing individuals with ASD would prevent any confusion between visual deficits and their deleterious effect on various experimental tasks.

Difficulties in identification of the emotions of others are often described in individuals with autism [START_REF] Harms | Facial emotion recognition in autism spectrum disorders: a review of behavioral and neuroimaging studies[END_REF][START_REF] Lozier | Impairments in facial affect recognition associated with autism spectrum disorders: a meta-analysis[END_REF][START_REF] Shanok | The Nature of Facial Emotion Recognition Impairments in Children on the Autism Spectrum[END_REF][START_REF] Xavier | A Multidimensional Approach to the Study of Emotion Recognition in Autism Spectrum Disorders[END_REF], however, ASD participants were able to complete the two categorization tasks. Similarly, because the targets were always presented in their central visual field, hemianopic patients were able to categorize stimuli despite their visual impairments. The results revealed similar difficulties in categorizing emotional faces in the clinical groups, although they differed regarding the processing of the spatial frequency content and the task difficulty. The present findings confirm that CVI patients are characterized by social interaction

Emotional faces in autism and CVI 1 3 difficulties even in the central visual field [START_REF] Fazzi | Autism in children with cerebral and peripheral visual impairment: fact or artifact[END_REF][START_REF] Fazzi | Spectrum of visual disorders in children with cerebral visual impairment[END_REF], that could at least partly overlap with those observed in autism.

Spatial Frequency processing

Our study shows that LSF stimuli yielded to longer RT and worse accuracy in all groups [START_REF] Jahshan | Probing the magnocellular and parvocellular visual pathways in facial emotion perception in schizophrenia[END_REF], but a significant interaction between Frequency and Group was also found. Worse performance for LSF in both clinical groups compared to controls was observed in the AH task, confirming our predictions. This effect was perhaps induced by a specific magnocellular impairment, or higher level cognitive impairments related to LSF processing.

Moreover, this was not generalizable to both tasks, as the TD group also presented difficulties for LSF stimuli in the NF task (see [START_REF] Jahshan | Probing the magnocellular and parvocellular visual pathways in facial emotion perception in schizophrenia[END_REF].

In line with previous studies [START_REF] Deruelle | Categorizing facial identities, emotions, and genders: attention to high-and low-spatial frequencies by children and adults[END_REF][START_REF] Jahshan | Probing the magnocellular and parvocellular visual pathways in facial emotion perception in schizophrenia[END_REF], the present results confirm that LSF and HSF each play a crucial role at different face processing stages [START_REF] Goffaux | From coarse to fine? Spatial and temporal dynamics of cortical face processing[END_REF]. However, even if CVI and ASD groups seem to present similar LSF impairments, it should be noted that for each group different neural processes could be involved in this behavioural effects. According to [START_REF] Bar | Top-down facilitation of visual recognition[END_REF], LSF information would be projected directly to the orbitofrontal cortex, generating early activity (at 130 ms), then guiding recognition in the temporal areas (at 180 ms) in the fusiform gyrus [START_REF] Bar | Top-down facilitation of visual recognition[END_REF], brain regions in which activity is often found to be atypical in ASD [START_REF] Critchley | The functional neuroanatomy of social behaviour: changes in cerebral blood flow when people with autistic disorder process facial expressions[END_REF][START_REF] Kovarski | Enhanced early visual responses during implicit emotional faces processing in Autism Spectrum Disorder[END_REF].

These projections would be at the origin of predictions and thereby guide perceptual processes, allowing the recognition of stimuli [START_REF] Beffara | Reduction of interference effect by low spatial frequency information priming in an emotional Stroop task[END_REF]. Several authors have suggested predictive coding abnormalities in persons with ASD as they could appear less influenced by contextual information and/or past experience [START_REF] Pellicano | When the world becomes 'too real': a Bayesian explanation of autistic perception[END_REF][START_REF] Sinha | Autism as a disorder of prediction[END_REF] as their perception is less distorted and often reported as enhanced [START_REF] Mottron | Enhanced perceptual functioning in autism: an update, and eight principles of autistic perception[END_REF].

Therefore, LSF impairment observed for ASD may be due to a dysfunction of the predictive coding neural network, before even reaching the temporal lobes to give rise to retention and learning.

In addition, LSF are largely involved in the fast activation of the amygdala in response to emotional faces and threat detection [START_REF] Mermillod | Are coarse scales sufficient for fast detection of visual threat[END_REF][START_REF] Vuilleumier | Distinct spatial frequency sensitivities for processing faces and emotional expressions[END_REF], which is also in line with the concept of an amygdala deficit in individuals with ASD [START_REF] Baron-Cohen | The amygdala theory of autism[END_REF][START_REF] Chevallier | The social motivation theory of autism[END_REF].

CVI individuals presented difficulties in categorizing emotional faces and atypical processing of both LSF and HSF, similar to impaired categorization of emotional faces revealed in other psychiatric conditions such as schizophrenia [START_REF] Jahshan | Probing the magnocellular and parvocellular visual pathways in facial emotion perception in schizophrenia[END_REF][START_REF] Laprevote | Patients with schizophrenia are biased toward low spatial frequency to decode facial expression at a glance[END_REF]. Nevertheless, in line with our hypotheses, both clinical groups presented a difficulty in categorizing LSF stimuli only in the AH task, suggesting that the deficit in processing LSF observed in ASD and CVI could depend on the task.

Hemispheric specialization had been described for spatial frequency processing in patients with CVI (Homonymous Hemianopia), with the left hemisphere being more dedicated to HSF processing and the right hemisphere being more specialized for LSF suggesting that further studies should investigate the effect of lesion side on performance [START_REF] Peyrin | Hemispheric specialization for spatial frequency processing in the analysis of natural scenes[END_REF]Peyrin, Chokron, et al., 2006).

Effect of task complexity

While the ASD group presented longer RTs only in the more difficult task (i.e. NF), the CVI group presented difficulties in both tasks, suggesting a broader difficulty in categorizing emotional faces. This significant interaction between Group and Task, suggests that group Emotional faces in autism and CVI 1 5

differences could merely depend on visual categorization difficulty. CVI may present general visual disturbances that affect more generally low-level visual processing regardless of the visual content, while in the ASD group, some individuals might use low-level facial features to process the emotional content of the stimuli (see [START_REF] Kovarski | Enhanced early visual responses during implicit emotional faces processing in Autism Spectrum Disorder[END_REF][START_REF] Wong | Abnormal spatiotemporal processing of emotional facial expressions in childhood autism: dipole source analysis of event-related potentials[END_REF].

Although no significant difference emerged between the Global Visual Score of the ASD and the CVI groups, the visual deficits in the ASD group could be less severe than in the CVI group, in line with the behavioural results. Interestingly, the Global Visual Scores correlated negatively with the reaction times (i.e. LOG_RTs) in the ASD and TD groups only. The fact that the greater the visual deficit the longer the reaction times in emotion recognition, suggests that visual disturbances are related to facial emotion processing in these populations.

Studies on visual emotion recognition in ASD show some inconsistencies (see [START_REF] Harms | Facial emotion recognition in autism spectrum disorders: a review of behavioral and neuroimaging studies[END_REF][START_REF] Lozier | Impairments in facial affect recognition associated with autism spectrum disorders: a meta-analysis[END_REF], with some reporting a deficit for a specific emotion (e.g. fear, [START_REF] Pelphrey | Visual scanning of faces in autism[END_REF] and others describing a generalized difficulty [START_REF] Rump | The development of emotion recognition in individuals with autism[END_REF]. Nevertheless, in line with our results, persons with ASD presented more difficulties in recognizing faces with negative emotions compared to happy faces [START_REF] Lozier | Impairments in facial affect recognition associated with autism spectrum disorders: a meta-analysis[END_REF][START_REF] Shanok | The Nature of Facial Emotion Recognition Impairments in Children on the Autism Spectrum[END_REF]. This could explain the difficulties of the ASD group in the NF task and could be related to a deficits in categorizing fearful and neutral faces [START_REF] Uono | The specific impairment of fearful expression recognition and its atypical development in pervasive developmental disorder[END_REF]. Previous studies on visual functioning in ASD have suggested that testing responses to different degrees of complexity is crucial. Indeed, adults with ASD might present no difficulties, or even enhanced basic visual responses [START_REF] Mottron | Enhanced perceptual functioning in autism: an update, and eight principles of autistic perception[END_REF]), yet impairments might emerge when more complex stimuli or scenarios are presented [START_REF] Bertone | Enhanced and diminished visuospatial information processing in autism depends on stimulus complexity[END_REF][START_REF] Kovarski | Enhanced early visual responses during implicit emotional faces processing in Autism Spectrum Disorder[END_REF].

Although participants with ASD presented more difficulties in the NF task, analysing the tasks separately demonstrated worse performances for LSF stimuli in the AH task, in line with our initial expectations. Even though broad difficulties might be found in a rather more difficult condition (i.e. NF), while deficit in processing specific frequencies might be revealed in an easier condition (i.e. AH), suggesting that testing under different conditions of difficulty within the same populations is crucial.

AQ in CVI

While autistic individuals predictably presented more autistic traits compared to TD and CVI individuals, the latter group also presented some autistic traits as measured by the AQ score. This is in line with an extensive literature showing that individuals with CVI present atypical behaviours often overlapping with autistic features, including communication difficulties, as well as repetitive behaviours and stereotyped patterns [START_REF] Bathelt | Event-related potential response to auditory social stimuli, parent-reported social communicative deficits and autism risk in school-aged children with congenital visual impairment[END_REF][START_REF] Bathelt | Executive abilities in children with congenital visual impairment in mid-childhood[END_REF][START_REF] Butchart | Autism and visual impairment: a review of the literature[END_REF][START_REF] Ek | Relation between blindness due to retinopathy of prematurity and autistic spectrum disorders: a population-based study[END_REF][START_REF] Hobson | Reversible autism among congenitally blind children? A controlled follow-up study[END_REF][START_REF] Jure | Autism Spectrum Disorders (ASD) in blind children: very high prevalence, potentially better outlook[END_REF][START_REF] Kiani | The relationship between symptoms of autism spectrum disorder and visual impairment among adults with intellectual disability[END_REF][START_REF] Mukaddes | Autism in visually impaired individuals[END_REF][START_REF] Wrzesinska | Visual impairment and traits of autism in children[END_REF]. However, the AQ score in individuals with CVI may not reflect a real autistic behaviour but might be due to some specific questions closely related to visual skills, including visual imagery, attention to detail and patterns. This suggests that visual deficits might impact the AQ questionnaire responses, raising the question of the validity of using such tests in the presence of visual deficits.

Some limitations and important perspectives should be acknowledged in the present study.

Firstly, both the ASD and the CVI groups presented an important heterogeneity as revealed by clinical factors (e.g. neurovisual assessment, AQ), and by methodological factors. This was Emotional faces in autism and CVI 1 7

observed, for example, in the number of trials included in the mean slightly fewer in the two clinical groups compared to the control group. Greater variability is not simply a methodological issue, but it represents a clinical feature in both ASD and CVI profiles, as suggested by an increasing literature in the field (Dinstein, Heeger, & Behrmann, 2015;Kovarski, Malvy, et al., 2019). Secondly, in the present study, while a neurovisual evaluation was included, it could be very interesting to determine if our current results could be extended to more general, non-social stimuli in order to investigate the specificity of this effect to emotional face discrimination. More precisely, given the specific processing of social stimuli in ASD (compared to CVI), futures studies should determine if the different processing of spatial frequencies related to emotional expressions could be extended to more general emotional stimuli, but also to non-emotional stimuli filtered at the same spatial frequencies.

Accordingly, the present results warrant replication and further investigation should elucidate the relationship between autistic signs and non-social visual disturbances, to improve the attainment of correct differential diagnosis between these two conditions (see for discussion [START_REF] Chokron | The inter-relationships between cerebral visual impairment, autism and intellectual disability[END_REF][START_REF] Pawlenko | Considerations in Behavioral Diagnoses of CVI: Issues, Cautions, and Potential Outcomes[END_REF] as well as the medical care in ASD and CVI groups.

Conclusions

Sensory anomalies in autism have only recently gained a critical role in the understanding of autism [START_REF] Apa | Diagnostic and Statistical Manual of Mental Disorders (DSM-5)[END_REF][START_REF] Robertson | Sensory perception in autism[END_REF][START_REF] Thye | The impact of atypical sensory processing on social impairments in autism spectrum disorder[END_REF]. The present paper raises the question of the specificity of visual anomalies in demonstrating important similarities between ASD and CVI groups with respect to both autistic and visual signs, but not with a complete overlap [START_REF] Chokron | The inter-relationships between cerebral visual impairment, autism and intellectual disability[END_REF].

Figures captions

  -nine individuals took part in the experiment [age range: 8-47 years]. Fifty-one were TD individuals, 23 presented an ASD, and 25 manifested CVI. Eight participants (2 TD, 4 ASD and 2 CVI) were excluded from the statistical analyses because of poor performance (CVI and ASD outliers) or for behavioural difficulties during the task (i.e. 2 TD were particularly anxious during the task). Poor performance was assessed via outliers detection and/or if mean RTs exceeded 1900 ms after trial rejection (see methods section). Thus, groups included 49 TD (25 males), 19 ASD (15 males), and 23 CVI (12 males). Groups were matched for chronological age (mean±standard deviation: 22.5±11.2 years; TD: 21.6±9.1 years; ASD: 19.8±12.4 years; CVI: 26.5±13.5 years; F(2, 88)=2.23, P =.11). Participants with ASD were recruited by Créteil and Fondation Rothschild hospitals and had been diagnosed according to the DSM-IV and DSM-5 criteria by an experienced team of clinicians. They presented an educational level in accordance with their chronological ages. Patients with CVI were diagnosed at the Fondation Rothschild Hospital by an experienced clinical team. This group included patients with damage and/or malfunction of the retrochiasmatic visual pathways. Within the 23 CVI participants, 19 presented a visual field defect including left homonymous hemianopia (N=9), right homonymous hemianopia (N=5), left superior quadrantanopia (N=1), left inferior quadrantanopia (N=2) and tunnel vision (N=2). The other 4 young participants, (age < 16 years) presented mixed CVI such as disturbances of gaze control, or visuo-spatial and visual cognition deficits in the absence of visual field defects. Participants in the TD group were recruited via advertisements on the laboratory website and by asking volunteers among friends and colleagues. They did not have any direct relationship neither with the CVI group nor with the ASD group. Autistic traits were measured via the AQ (Baron-Cohen, Hoekstra, Knickmeyer, & Wheelwright, 2006; Baron-Cohen et al., 2001) in all participants. The AQ is a questionnaire developed to quantify autistic-like traits covering the domains that are usually atypical in individuals with ASD such as social skills, attention to details, or intolerance to change.

  Illustration of the emotional face categorization task and example of a male fearful face stimulus filtered in low spatial frequencies (LSF, left), non-filterd (BSF, center) and high spatial frequencies (HSF, right).

Figure 2

 2 Figure 2

Table 1 .

 1 Mean accuracy (%), error rate (Asin-ER), correct reaction times in milliseconds (RTs), and correct Log transformed RTs (Log-RTs), for low (LSF), broad (BSF) and high spatial frequency (HSF), with standard errors reported for the three groups (ASD, CVI and TD). Values are provided for the Angry-Happy task (left) and for the Neutral-Fear task (right).

				Angry-Happy task			Neutral-Fear task
			LSF	BSF	HSF	LSF	BSF	HSF
		Accuracy (%) 88.7 ± 12	95.3 ± 10	96.2 ± 6	82.1 ± 15	93.2 ± 9	89.4 ± 12
	ASD	Asin-ER RTs (ms)	1.3 ± 0.23 865 ± 332	1.5 ± 0.2 752 ± 273	1.5 ± 0.17 832.8 ± 349	1.2 ± 0.22 966 ± 261	1.4 ± 0.21 857 ± 271	1.3 ± 0.23 964 ± 329
		Log-RTs	6.61 ± 0.34	6.51 ± 0.33	6.58 ± 0.33	6.8 ± 0.26	6.66 ± 0.29	6.77 ± 0.3
		Accuracy (%) 88.2 ± 12	96.3 ± 6	93.9 ± 11	79.6 ± 17	91.3 ± 10	89.1 ± 14
	CVI	Asin-ER RTs (ms)	1.3 ± 0.22 831 ± 277	1.5 ± 0.16 800 ± 254	1.4 ± 0.22 804 ± 303	1.2 ± 0.25 1095 ± 691	1.3 ± 0.21 1002 ± 493	1.3 ± 0.25 906 ± 278
		Log-RTs	6.62 ± 0.33	6.57 ± 0.33	6.55 ± 0.38	6.82 ± 0.4	6.76 ± 0.38	6.72 ± 0.28
		Accuracy (%) 96.6 ± 4.8	97.3 ± 4.6	96.7 ± 4.6	89.3 ± 10.5	98.2 ± 4.3	98.5 ± 3.2
	TD	Asin-ER RTs (ms)	1.46 ± 0.15 627 ± 181	1.48 ± 0.14 596.3 ± 165	1.46 ± 0.15 592.2 ± 168	1.3 ± 0.21 688.5 ± 226	1.51 ± 0.12 622.3 ± 192	1.52 ± 0.11 642.6 ± 227
		Log-RTs	6.36 ± 0.28	6.31 ± 0.28	6.3 ± 0.28	6.44 ± 0.32	6.35 ± 0.3	6.37 ± 0.32

Acknowledgement

The authors thank the participants for their involvement in the study and the students involved in data recording and tests assessments.

We show the relevance of comparing an autistic population not only to a TD group, but also to one presenting with CVI. This approach suggests that emotional face categorization difficulties should not be considered as autism-specific also depend on the integrity of visual processing.

Symmetrically, attention should be given to socio-emotional difficulties in those with CVI.

Together with the increasing literature, these results highlight that visual functioning (i.e. peripheral and central) should be systematically investigated with adapted tools in individuals with ASD. From a more theoretical point of view, it seems crucial to study the role and potential consequences of visual deficits in autism to provide an optimal understanding of the condition.

This work was supported by Fondation Orange and Fondation de France.

Funding

Data supporting the findings of this study are available from the corresponding author, upon reasonable request.

Conflict of interst

None to declare.


PhD Mickael Blaise
email: mickael.blaise@irim.cnrs.fr

Jaslyn Mei

PhD Mei Wong
email: jwong@mrc-lmb.cam.ac.uk

PhD Mickael Blaise

Jaslyn E M M Wong

Mickaël Blaise

Mickaël Blaise

Mickaël Blaise

Report of false positives when using zymography to assess peptidoglycan hydrolytic activity of an endopeptidase with multiple LysM domains

Keywords: Bacillus subtilis, LysM, zymography, peptidoglycan, NlpC/P60 Bacillus subtilis, LysM, zymography, peptidoglycan, NlpC/P60 endopeptidase

Report of false positives when using zymography to assess peptidoglycan hydrolytic activity of an endopeptidase with multiple LysM domains.

Introduction

Bacteria are shielded from environmental stresses by a cell wall, which contains a peptidoglycan (PG) layer. PG is a three-dimensional mesh-like structure composed of repeating units of MurNAc and GlcNAc. Long polymers containing MurNAc-GlcNAc repeats are linked to each other by peptide stems, enabling the formation of a complex 3D network [START_REF] Meroueh | Threedimensional structure of the bacterial cell wall peptidoglycan[END_REF]. Although the structure of PG is very rigid, it has also to be highly dynamic during bacterial cell division processes, notably, to facilitate the separation of the two daughter cells and neosynthesis of PG [START_REF] Typas | From the regulation of peptidoglycan synthesis to bacterial growth and morphology[END_REF][START_REF] Vollmer | Bacterial growth does require peptidoglycan hydrolases[END_REF][START_REF] Egan | Regulation of peptidoglycan synthesis and remodelling[END_REF]. During these phases, PG remodeling is mediated by autolysins that are a group of PG-cleaving enzymes with different substrate specificities. These enzymes either cleave the carbohydrate backbone, the peptide stem, or the bond between MurNAc and the peptide stem or the peptide stem at specific positions. The complex and insoluble nature of PG renders the development of activity assays to characterize autolysins very challenging [START_REF] Do | Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes[END_REF]. Zymography, a widely used technique allowing in-gel visualization of substrate hydrolysis, helps to overcome this challenge [START_REF] Vandooren | Zymography methods for visualizing hydrolytic enzymes[END_REF]. The principle of zymography is based on the migration of a protein of interest into a native or non-denaturing electrophoresis gel in which the substrate is incorporated. The substrate hydrolysis can be then detected where the protein endmigrates [START_REF] Vandooren | Zymography methods for visualizing hydrolytic enzymes[END_REF]. It has been extensively used to study matrix metalloproteases in eukaryotes [START_REF] Gross | Collagenolytic activity in amphibian tissues: a tissue culture assay[END_REF] but also to study bacterial PG hydrolytic enzymes.

To study autolysin activities by zymography, bacterial cell wall or bulk PG can be incorporated in the polyacrylamide gels. After gel electrophoresis, in native or denaturing conditions of the purified protein of interest or even crude protein extracts, the PG is stained and any presence of a white zone of clearing corresponding to the end migration of the protein would indicate hydrolysis of PG. When separation is performed under denaturing conditions, the gel is extensively washed to remove sodium dodecyl sulfate (SDS) and the protein is refolded slowly by placing the gel into a renaturation buffer prior to staining.

We studied the PG binding properties of the CwlS protein from Bacillus subtilis [START_REF] Wong | Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein[END_REF]. This enzyme is a NlpC/P60 D,L-endopeptidase capable of cleaving the PG peptide stem between the 2 nd and 3 rd amino acids. CwlS possesses a repeat of four LysM domains that are known binding modules of chitin and MurNAc-GlcNAc polymers [START_REF] Buist | LysM, a widely distributed protein motif for binding to (peptido)glycans[END_REF].

The CwlS encoding gene is essential for daughter cell separation in B. subtilis [START_REF] Fukushima | A new D,L-endopeptidase gene product, YojL (renamed CwlS), plays a role in cell separation with LytE and LytF in Bacillus subtilis[END_REF]. In the course of characterizing CwlS and CwlS mutants by biochemical approaches, we noticed that the apparent hydrolytic activity was not correlated to the catalytic properties observed on the zymograms. In this study, we, therefore, report these observations with the aim of drawing attention to the potential misinterpretation of results of this widely used technique.

Materials and methods

Cloning, Protein expression and Purification

CwlS wild-type (CwlS_FL), 3LysM-Cata, 2LysM-Cata, 1LysM-Cata, Cata, FL_Mut and 4LysM constructs were essentially cloned and purified as previously described [START_REF] Wong | Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein[END_REF].

The

CwlS-C326A mutant was generated with the CCTGCCGGTTTTGATGCCAGCGGCTTCATT forward primer and AATGAAGCCGCTGGCATCAAAACCGGCAGG reverse primer using the QuikChange Lightning Site-Directed Mutagenesis Kits (Agilent Technologies) according to the manufacturer's instructions and using the CwlS-FL gene as a template.

This mutant was expressed and purified as similar as wild-type CwlS.

Zymography

To purify B. subtilis cell wall, B. subtilis cells (ATCC 6056) were grown in 100 ml LB media overnight at 30 °C. The cells were centrifuged at 10,500 g for 15 min, and washed twice with 50 mM Phosphate pH 7.5. The cell lysate was then centrifuged at 30,000 g for 20 min, resuspended in 0.5% SDS, and incubated at 60 °C for 30 min. The cells were centrifuged at 30,000 g for 20 min and washed with water. This step was repeated five times. Extracted B. subtilis cell wall was digested with 100 μg ml-1 trypsin in 1 M Tris-HCl pH 7 overnight at 37 °C. The cell wall was then centrifuged at 30,000 g for 20 min and washed with 1 M Tris-HCl pH 7. This step was repeated once with 1 M Tris-HCl pH 7 and 1 M NaCl, once with 1 M Tris-HCl pH 7, and six times with water.

Zymography gels were prepared by adding 0.4% (w/w) cell wall extract to 15% SDS-PAGE. 20 μg of protein in loading buffer were loaded in each lane. 10 μg BSA and 0.5 μg lysozyme were added as negative and positive control respectively. After SDS-PAGE, the gels were incubated in 50 mM Tris-HCl pH 8 buffer for 48 hours, then stained with 0.1% (w/v) methylene blue diluted in 0.01% KOH.

Assessment of the endopeptidase activity using turbidity activity assay

The endopeptidase activity assay was performed as previously described [START_REF] Wong | Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein[END_REF].

Overall protein net charge determination

The overall net charges of the protein variants at pH 7 were determined using this server: https://pepcalc.com/protein-calculator.php. The protein charges were calculated according to the following formula: where

Ni indicates the number, and pKai the pKa values, of the N-terminus and the side chains of positively charged amino acids: Arginine, Lysine, and Histidine. The j-index relates to the C-terminus and the negatively charged residues: Aspartic Acid, Glutamic Acid as well as Cysteine and, Tyrosine amino acids.

Results and discussion

The CwlS study was undertaken to reveal the role of the N-terminal region of the protein containing multiple LysM domains (Figure 1A). The LysM domain is a known PG binding domain [START_REF] Buist | LysM, a widely distributed protein motif for binding to (peptido)glycans[END_REF], so we initially performed a study to assess if the number of LysM repeats was conferring substrate-binding strength. We showed that the CwlS affinity towards PG and chitin polymers is indeed influenced by the numbers of LysM repeats which cooperated to bind these long polymers [START_REF] Wong | Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein[END_REF]. The dissociation constants (Kd) determined for chitin and MurNAc-GlcNAc polymers using the Microscale Thermophoresis method demonstrated that the Kd increased alongside LysM deletion (Figure 1A) [START_REF] Wong | Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein[END_REF]. A positive correlation between carbohydrate affinity and the number of LysM domains was also confirmed in studies using NlpC/P60 from Thermus thermophilus [START_REF] Wong | An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase[END_REF] and the AtlA autolysin from Enterococcus faecalis [START_REF] Mesnage | Molecular basis for bacterial peptidoglycan recognition by LysM domains[END_REF].

In this study, we assessed if the deletion of LysM modules could influence the PG hydrolysis efficacy of CwlS. We performed zymography experiments using nondenaturing gels containing bulk PG from B. subtilis. Full-length wild-type CwlS protein (CwlS-FL) seemed capable of cleaving PG as a white zone of clearing corresponding to the end-migration position of the protein was observed after methylene blue staining of zymography gels (Figure 1B). We then assessed the PG-cleaving abilities of single to four LysM deletion in CwlS mutants and noticed that the more impaired the CwlS mutants were in their PG binding capacities the lower their abilities were to form white zones of clearing on zymogram (Figure 1A and1B). It appeared that CwlS-FL and 3LysM-Cata proteins that have similar dissociation constants for PG, cleaved PG the most efficiently as indicated by most distinct zones of clearing in zymograms compare with 2LysM-Cata, 1LysM-Cata and Cata mutants that were strongly impaired in PG binding. These mutants presented less pronounced zones of clearing. The Cata mutant that did not possess any PG binding module produced barely detectable zone of clearing.

This observation could have been considered in perfect agreement with our previously published results showing that these mutants were impaired in their capacity to hydrolyze Bacillus subtilis culture which could be explained by their loss of ability to anchor to the cell wall [START_REF] Wong | Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein[END_REF].

Counter-intuitively, a protein mutant (4LysM) that did not contain any catalytic domain but only the four LysM domains also generated zones of clearing on zymograms as pronounced as the wild-type protein, CwlS-FL (Figure 2A). Puzzled by the apparent PG hydrolytic activity of a CwlS variant that does not actually contain a catalytic domain, we further generated a catalytically dead mutant (CwlS-C326A) by site-directed mutagenesis of a catalytic cysteine that is present in the active site of the protein; this cysteine residue is strictly conserved in NlpC/P60 endopeptidases and is essential for PG hydrolysis [START_REF] Xu | Structural basis of murein peptide specificity of a gamma-D-glutamyl-l-diamino acid endopeptidase[END_REF][START_REF] Xu | Structure-Guided Functional Characterization of DUF1460 Reveals a Highly Specific NlpC/P60 Amidase Family[END_REF][START_REF] Ruggiero | Structure and functional regulation of RipA, a mycobacterial enzyme essential for daughter cell separation[END_REF]. Surprisingly, the CwlS-C326A mutant also generated a white zone of clearing in zymograms with equal intensity as the wild-type protein (Figure 2A).

To rule out that neither the 4LysM nor the CwlS-C326A mutant possessed PG hydrolytic properties, we assessed their activities on exponentially growing planktonic B. subtilis cultures. We added the purified proteins to B. subtilis cultures and monitored the cell density over 140 min. If the proteins are active, they should lyse the cells, and this is reflected by a drop in turbidity readings. CwlS-FL and the positive control, lysozyme, hydrolyzed B. subtilis as indicated by a drop in optical density of ∼ 0.3 units (Figure 2B) in about 2 hours. In contrast, an increase in optical cell density was observed for cells treated with the 4LysM and CwlS-C326A mutants, similar to the negative controls, BSA or protein buffer. The inability of the 4LysM and CwlS-C326A mutants to hydrolyze B. subtilis cultures (Figure 2B) strongly indicated that their corresponding zones of clearing on the zymograms do not reflect the enzymatic activity.

One could consider in such a case, that zymography was in fact reflecting PG binding event and not PG hydrolysis.

Interestingly, the ChiZ protein from Mycobacterium tuberculosis that is part of the divisome and possesses a single LysM domain in its C-terminus also gave positive results on a zymogram albeit not being confirmed by other methods to be a PG hydrolase [START_REF] Escobar | False positives in using the zymogram assay for identification of peptidoglycan hydrolases[END_REF]. The authors elegantly proposed that the high net positive charge of ChiZ is in fact impairing toluidine blue coloration. Toluidine blue is indeed a positively charged compound that stains negatively charged PG, thus positively charged protein would repulse toluidine blue. The authors further demonstrated that the highly positively charged CrgA protein with no PG-related function also displayed apparent PG hydrolytic activity on zymogram [START_REF] Escobar | False positives in using the zymogram assay for identification of peptidoglycan hydrolases[END_REF].

We therefore calculated the net charge of all our CwlS variants (Figure 1A). generates indeed a rather weak zone of clearing compare with the CwlS-FL that has a net charge of +33. To completely rule out the possibility that the positive zymogram result observed is not due to the loss of peptidoglycan binding strength but is rather related to the net positive charge of the protein, we included an additional mutant (FL-Mut) in our zymography assays. The FL-Mut (Figure 1A) presents twelve mutations, with three residues in each of the four LysM domains mutated into Ala. The mutagenesis was driven by docking studies which revealed amino acids with side chains that could potentially interact with carbohydrates as described in [START_REF] Wong | Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein[END_REF]. This mutant is highly impaired in its capacity to bind PG but very importantly has a similar net positive charge as the wild-type protein (Figure 1A). As assessed on zymogram (Figure 2C), this FL-Mut also resulted in a zone of clearance that was as pronounced as the CwlS-FL protein and the CwlS-C326A catalytic mutant that both possess similar net positive charges. Furthermore, the FL_Mut apparent activity is more pronounced than the CwlS variant that possessed only a catalytic domain and has a much lower net positive charge (Figure 2C). Altogether our data provides compelling evidence that the apparent activity of all the CwlS variants presented in this study, as assessed by zymography, is not correlated to PG binding strength but more probably to the net positive charge of the protein.

Kohler et al., reported similar zymogram false positive when studying the autolysin AtlA from Neisseria gonorrhoeae [START_REF] Kohler | AtlA functions as a peptidoglycan lytic transglycosylase in the Neisseria gonorrhoeae type IV secretion system[END_REF]. The authors noticed that a catalytic dead mutant had apparent PG hydrolysis activity on zymogram although this mutant was, in fact, inactive in PG degradation assay or cell lysis assays [START_REF] Kohler | AtlA functions as a peptidoglycan lytic transglycosylase in the Neisseria gonorrhoeae type IV secretion system[END_REF]. The authors proposed that the protein was able to bind PG, therefore avoiding the PG coloration by toluidine blue. We calculated the net charge of AtlA, and noticed that it also has a high positive value of 10. Altogether, the studies of ChiZ, AtlA and CwlS, all possessing high net positive charges are therefore in favor of the hypothesis of impairment of coloration due to charges repulsion [START_REF] Escobar | False positives in using the zymogram assay for identification of peptidoglycan hydrolases[END_REF].

We hereby demonstrated that zymography could trigger false-positives when probing for PG hydrolysis activities of a multiple containing LysM protein. This communication is nonetheless not intended to discredit the use of zymography, which has been extensively and successfully used in autolysin hydrolysis assays for decades, but to create awareness that the data should be interpreted with caution. Zymography has in most cases been proven to be trustworthy as hydrolytic activities have successfully been confirmed by additional techniques. To avoid such artifact, Escobar et al.,

proposed to compare the results of gels revealed with and without a refolding step [START_REF] Escobar | False positives in using the zymogram assay for identification of peptidoglycan hydrolases[END_REF].

Alternatively or in complement, we would advise to determine the net charge of the protein of interest, and if possible, to assess the activities of a catalytic dead version alongside a wild-type version of the protein when performing zymography assessments, and most certainly to confirm the PG hydrolase activity by another method [START_REF] Do | Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes[END_REF]. The dotted lines signify that the two gels are independent but were run simultaneously in the exact same conditions. Empty lanes in the gels were cropped.

Figures legends

  With the exception of the Cata mutant with a net charge of 5, the other proteins have all very high net positive charges, from 12 to 33 as compared with ChiZ that has a net charge of 10. The intensity of the zones of clearing of the different CwlS version is indeed correlated with net positive charges. The Cata mutant with a net charge of +5

Figure 1 :

 1 Figure 1: Determination of the peptidoglycan hydrolytic activity of CwlS mutant by zymography A-Schematic representation of the CwlS variants assessed in this study with the PGanchoring and catalytic domains indicated. The CwlS PG-anchoring domain corresponds to the LysM modules present in the protein. N-t and C-t stand for Nterminus and C-terminus respectively. *Indicates the dissociation constants (Kd) previously determined by microscale thermophoresis using Staphylococcus aureus PG tetramers [8], ± represents the standard deviation (with 95% confidence interval). N.d. indicates that binding was not detectable and n.t. indicates that binding was not tested. The isoelectric point (pI) and the overall net charges of the CwlS variants (without its peptide signal of secretion) at a pH of 7 are shown. The pI and the overall net charge at pH 7 of the different protein variants were determined using this server: https://pepcalc.com/protein-calculator.php. B-Determination of the apparent PG hydrolytic activity of the different CwlS variants with one, two, three, or all LysM domains deleted. M indicates the protein molecular weight marker. Bovine serum albumin (BSA) and lysozyme were used as negative and positive controls, respectively.

Figure 2 :

 2 Figure 2: Assessment of the PG hydrolytic properties of CwlS mutants by zymography and upon incubation with planktonic B. subtilis culture A-Zymography assessment of the apparent PG hydrolytic activity of wild-type CwlS and mutated versions devoid of a catalytic domain (4LysM) or containing a mutation in the catalytic domain (C326A) rendering it inactive. B-B. subtilis cell lysis capacities of the different CwlS variants. A drop of OD at 600 nm indicates cell lysis. The data is representative of three independent experiments. C-Comparison of the apparent PG hydrolytic activity of the FL_Mut variant versus CwlS_FL, CwlS_C326A and Cata mutants.

Figure2

  Figure2

Acknowledgements

We would like to thank Pr Jens Stougaard for constant support.

All authors declare no conflict of interest.

Authors contribution

JW and MB designed research. JW performed experiments. JW and MB analyzed data. MB supervised research. MB and JW wrote the manuscript.

Funding

Part of this work was funded by the Danish National Research Foundation grant No.

DNRF79

Declarations of interest