Xue Geng

Jean-Marie Galano

Camille Oger

Grace Y Sun

Thierry Durand

James C Lee

Neuroprotective effects of DHA-derived peroxidation product 4(RS)-4-F4t-Neuroprostane on microglia

Keywords: 4(RS)-4-F4t-Neuroprostane, microglia, antioxidant, and anti-inflammatory

The abundance of docosahexaenoic acid (DHA) in brain membrane phospholipids has stimulated studies to explore its role in neurological functions. Upon released from phospholipids, DHA undergoes enzymatic reactions resulting in synthesis of bioactive docosanoids and prostanoids. However, these phospholipids are also prone to nonenzymatic reactions leading to more complex pattern of metabolites. A non-enzymatic oxidized product of DHA, 4(RS)-4-F 4t -Neuroprostane (44FNP), has been identified in cardiac and brain tissues. In this study, we examined effects of the 44FNP on oxidative and inflammatory responses in microglial cells treated with lipopolysaccharide (LPS). The 44FNP attenuated LPS-induced production of reactive oxygen species (ROS) in both primary and immortalized microglia (BV2). It also attenuated LPS-induced inflammation through suppressing NFκB-p65 and levels of iNOS and TNFα. In addition, 44FNP also suppressed LPS-induced mitochondrial dysfunction and upregulated the Nrf2/HO-1 antioxidative pathway. In sum, these findings with microglial cells demonstrated neuroprotective effects of this 44FNP and shed light into the potential of nutraceutical therapy for neurodegenerative diseases.

Introduction

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid (PUFA) abundant in the phospholipids in brain, and studies have shown a critical role for it to maintain brain health [START_REF] Sun | Docosahexaenoic acid (DHA): An essential nutrient and a nutraceutical for brain health and diseases[END_REF]. Intake of DHA has been correlated to lower cognitive impairment in Alzheimer's disease [START_REF] Morris | Consumption of fish and n-3 fatty acids and risk of incident Alzheimer disease[END_REF][START_REF] Chang | Docosahexaenoic acid promotes dopaminergic differentiation in induced pluripotent stem cells and inhibits teratoma formation in rats with Parkinson-like pathology[END_REF]. There is growing evidence that some of the neuroprotective effects of DHA is due to its conversion to oxidative metabolites such as neuroprotectin D1, resolvins and maresins [START_REF] Mallick | Docosahexaenoic acid,22:6n-3: Its roles in the structure and function of the brain[END_REF].

Besides enzymatic reactions, DHA is also vulnerable to non-enzymatic reactions to produce peroxidation products, such as neuroprostanoids [START_REF] Milne | The isoprostanes-25 years later[END_REF][START_REF] Leung | Current development in non-enzymatic lipid peroxidation products, isoprostanoids and isofuranoids, in novel biological samples[END_REF][START_REF] Reddy | Amyloid precursor protein-mediated free radicals and oxidative damage: implications for the development and progression of Alzheimer's disease[END_REF][START_REF] Domenichiello | Molecular Pathways Linking Oxylipins to Nociception in Rats[END_REF][START_REF] Galano | Isoprostanes, neuroprostanes and phytoprostanes: An overview of 25years of research in chemistry and biology[END_REF][START_REF] Ahmed | Moving forward with isoprostanes, neuroprostanes and phytoprostanes: where are we now?[END_REF]. Among these, 4(RS)-4-F4t-Neuroprostane (44FNP) is one of the most abundant [START_REF] Galano | Non-enzymatic cyclic oxygenated metabolites of adrenic, docosahexaenoic, eicosapentaenoic and α-linolenic acids; bioactivities and potential use as biomarkers[END_REF][START_REF] Roberts | The biochemistry of the isoprostane, neuroprostane, and isofuran pathways of lipid peroxidation[END_REF][START_REF] Galano | Isoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans[END_REF], and has been proposed to be an oxidative damage biomarker of neurological diseases [START_REF] Peña-Bautista | New screening approach for Alzheimer's disease risk assessment from urine lipid peroxidation compounds[END_REF].

To date, studies have unveiled different health effects of 44FNP, e.g., on arrhythmia [START_REF] Roy | Nonenzymatic lipid mediators, neuroprostanes, exert the antiarrhythmic properties of docosahexaenoic acid[END_REF], decreasing infarct and ventricular tachycardia in ischemia reperfusion [START_REF] Roy | Non-enzymatic oxidized metabolite of DHA, 4(RS)-4-F(4t)neuroprostane protects the heart against reperfusion injury[END_REF], inducing production of heme oxygenase-1 (HO-1) in SH-SY5Y cells [START_REF] Lee | Nonenzymatic oxygenated metabolite of docosahexaenoic acid, 4(RS)-4-F(4t)neuroprostane, acts as a bioactive lipid molecule in neuronal cells[END_REF], and proliferation of human breast cancer cells [START_REF] Roy | Polymeric nanocapsules prevent oxidation of core-loaded molecules: evidence based on the effects of docosahexaenoic acid and neuroprostane on breast cancer cells proliferation[END_REF]. However, whether 44FNP can exert neuroprotective effects in microglia has not been investigated in detail.

In order to provide information to further our understanding of mechanism(s) underlying the neuroprotective effects of 44FNP, this study examined oxidative stress, inflammation, and mitochondrial membrane function in microglial cells stimulated with the bacteria toxin lipopolysaccharide (LPS). In addition, this study also evaluated whether 44FNP may confer antioxidation through the Nrf2/HO-1 pathway.

Materials and Methods

Materials

Dulbecco's modified Eagle's medium (DMEM) and penicillin/streptomycin (P/S, 10,000 units/ml) were purchased from Life Technologies (Grand Island, NY), fetal bovine serum (FBS) and lipopolysaccharide (LPS) from Cayman Chemical (Ann Arbor, MI), dimethyl sulfoxide (DMSO), cOmplete™ protease inhibitor cocktail, PhosSTOP™ phosphatase inhibitor cocktail from Sigma-Aldrich (St. Louis, MO), radioimmunoprecipitation assay (RIPA) buffer, bicinchoninic acid (BCA) protein assay kit, SuperSignal™ West Pico plus chemiluminescent substrate, Restore™ PLUS Western blot stripping buffer, TNF-α mouse uncoated ELISA kit, and 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (DCF) from Thermo Fisher Scientific (Waltham, MA). Primary antibodies against HO-1, phospho-NF-κB p65 (p-p65) was purchased from Cell Signaling (Beverly, MA), monoclonal anti-β-actin peroxidase antibody from Sigma-Aldrich (St. Louis, MO), anti-inducible nitric oxide synthase (iNOS) antibody and TMRM Assay Kit (Mitochondrial Membrane Potential) from Abcam (Cambridge, MA), and anti-Nrf2 antibody from GeneTex (Irvine, CA). Neural Tissue Dissociation kit (P), MACS buffer, and CD11b microbeads were purchased from Miltenyi Biotec (Auburn, California). 44FNP (1 mg/ ml in methanol) was synthesized in Dr. T. Durand's laboratory at IBMM, France [START_REF] Oger | The handy use of Brown's P2-Ni catalyst for a skipped diyne deuteration: application to the synthesis of a [D4]-labeled F4t-neuroprostane[END_REF].

Preparation of Primary Mouse Microglia

Timed pregnant C57BL/6 mice were purchased from Charles River (Wilmington, MA). All animal care and experimental protocols were carried out with permission from the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois at Chicago. Preparation of primary mouse microglia from mouse pups (P1-P4) were accomplished using the Miltenyi Biotec MACS cells separation system. Briefly, cerebral cortices were dissected, and meninges removed. Brain tissue was homogenized using the Neural Tissue Dissociation Kit. Cells were collected and resuspended in ice-cold MACS buffer containing CD11b microbeads and then passed through the magnetized LS columns to harvest the primary microglia according to the manufacturer's protocol. Microglial cells were collected and plated as previously described [START_REF] Chuang | Cytosolic phospholipase A2 plays a crucial role in ROS/NO signaling during microglial activation through the lipoxygenase pathway[END_REF].

BV2 Cell Culture

BV2 cells were originally obtained from Dr. Rosario Donato (University of Perugia, Italy) and subsequently propagated and stored in Dr. Grace Sun's laboratory (University of Missouri, MO, USA). BV2 mouse microglial cells were cultured in DMEM supplied with 5% FBS and 1% P/S. For experiments, cells were subcultured to 80% confluence in multiple well plates and serum starved for 3h prior to treatment with 44FNP for 1h and followed by stimulation with 50 ng/mL LPS. 44FNP was dissolved in DMSO diluted in DMEM to the indicated concentrations.

Measurement of ROS Production

Cells were suspended in 96-well plates, pretreated with 44FNP for 1 h, and followed by LPS treatment for 12h. After treatment, 10 μM DCF was added to the cells for 45 min. The fluorescent intensity of DCF in cells was measured using a Synergy H1 Plate Reader (BioTek, St. Louis, MO) with an excitation wavelength of 490 nm and an emission wavelength of 520 nm.

Western Blot Analysis

After treatment, the culture medium was removed, and cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Cell lysates were collected and centrifuged at 14,000×g for 15 min at 4 °C, and supernatants were collected. Protein concentration in cell lysate was determined using BCA assay. Samples were loaded onto SDS-PAGE for electrophoresis. Afterwards, proteins were transferred to 0.2-μm PVDF membranes at 100 V for 1 h at 4 °C. Membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h, and incubated with antibodies overnight at 4 °C. After washing with TBS-T, blots were incubated with secondary antibody for 1 h. Signals were developed using SuperSignal™ West Pico plus chemiluminescent substrate and captured with a myECL imager (Thermo Scientific). The optical density of bands was measured with the Image Studio Lite 5.2 (LI-COR Biotechnology, Lincoln, NE).

TNF-α ELISA Assay

TNF-α in culture medium was determined by sandwich ELISA. Briefly, cells were treated, and medium was collected and centrifuged at 4000×g for 5 min. The levels of TNF-α were assessed using an ELISA kit following manufacturer's instruction.

Mitochondrial membrane potential measurement

After treatment, cells were incubated with 200 nM TMRM in Hank's balanced salt solution (HBSS) for 30 min at 37 °C. Fluorescence was analyzed use Synergy H1 Plate Reader with an excitation wavelength of 548 nm and an emission wavelength of 575 nm. For positive control, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP), a mitochondrial uncoupler known to cause mitochondria depolarization, was used to validate our technique.

Statistical Analysis

Data are expressed as mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis between multiple groups was carried out using oneway ANOVA followed by Tukey's post hoc HSD test in GraphPad Prism (version 8.10). A p value < 0.05 was considered statistically significant.

Results

Effects of 44FNP on LPS-induced ROS production.

We examined the effects of 44FNP (2.5-20 μM) on ROS production in primary mouse microglia and BV2 cells stimulated with LPS (50 ng/ml). In both types of microglia, 44FNP inhibited LPS-stimulated ROS in a dose-dependent manner with a significant decrease at 5 μM (Fig. 1). To verify ROS production through NADPH oxidase, we used the gp91ds-tat peptide, known to block ROS production through disruption of NADPH subunits [START_REF] He | Prolonged exposure of cortical neurons to oligomeric amyloid-β impairs NMDA receptor function via NADPH oxidase-mediated ROS production: protective effect of green tea (-)-epigallocatechin-3-gallate[END_REF]. Addition of gp91ds-tat abolished LPS-induced ROS in these cells, suggesting that ROS production was mainly through NADPH oxidase. Our treatments in all experimental groups did not impose any ill effects on cell viability (data not shown).

Fig. 1. 44FNP attenuated LPS-induced reactive oxygen species (ROS) production in primary mouse microglia and BV2 cells. Primary microglia (A), BV2 cells (B)

were pretreated with 44FNP for 1 h, followed by stimulation with 50ng/ml LPS for 12 h. Data are represented in mean ± standard deviation (SD) (n = 3). Statistical analysis was carried out with one way ANOVA followed by Bonferroni post-tests. *p<0.05, **p<0.01, ***p<0.001 compared with the LPS group; ###p<0.001, ####p<0.0001 compared with the control group.

Effects of 44FNP on LPS-induced inflammatory responses in BV2 cells.

LPS has been reported to activate microglial inflammation through the NFκB-p65 pathway [START_REF] Chuang | Cytosolic phospholipase A2 plays a crucial role in ROS/NO signaling during microglial activation through the lipoxygenase pathway[END_REF]. In this study, we examined effects of 44FNP on p65 and p-p65 expression in control and LPS-stimulated microglial cells. Results show that LPSinduced phosphorylation of p65 expression was attenuated by 44FNP in a dosedependent manner (Fig. 2A).

To further explore the anti-neuroinflammatory effects of 44FNP, results also showed that 44FNP was able to attenuate LPS-induced increase in iNOS (Fig. 2B) and TNFα secretion (Fig. 2C).

Effects of 44FNP on LPS-induced changes in mitochondrial membrane potential.

A leaky mitochondrial membrane is a key characteristic of mitochondrial dysfunction.

Loss of mitochondrial membrane potential (Δψ m ) is related to mitochondrial depolarization. To demonstrate the effects of 44FNP on LPS-induced mitochondrial dysfunction, we applied TMRM to monitor Δψ m in BV2 cells. As shown in Fig. 3, exposing cells to LPS significantly decreased TMRM intensity, indicating the loss of Δψ m . The loss of Δψ m due to LPS was also attenuated by pretreatment of cells with 44FNP. Consequently, these results demonstrated the ability for 44FNP to suppress LPS-induced mitochondrial potential loss in microglial cells.

Discussion

Free radical-induced peroxidation of arachidonic acid, another PUFA found in membrane phospholipids, results in the formation of isoprostanes [START_REF] Morrow | Characterization of steroid interactions with gamma-aminobutyric acid receptor-gated chloride ion channels: evidence for multiple steroid recognition sites[END_REF]. These isoprostanes are elevated in brain and other body organs due to oxidant injury and are related to inflammatory mechanisms [START_REF] Chen | Mapping of a serine-rich domain essential for the transcriptional, antiapoptotic, and transforming activities of the v-Rel oncoprotein[END_REF]. On the contrary, oxidative derivatives of DHA result in production of neuroprostanes which tend to show protective effects. In agreement with recent studies showing evidence for protective effects of DHA-derived 44FNP on the peripheral organs [START_REF] Roy | Non-enzymatic oxidized metabolite of DHA, 4(RS)-4-F(4t)neuroprostane protects the heart against reperfusion injury[END_REF], results in this study demonstrated similar effects of this neuroprostane on microglial cells in the central nervous system.

In this study, we demonstrate the ability for 44FNP to suppress ROS production and inflammatory responses in both primary mouse microglia and immortalized microglial BV2 cells (Fig. 1A). By using gp91ds tat, results also demonstrated ROS production in microglia is mediated mainly through NADPH oxidase.

LPS is known to stimulate the Toll-like Receptor (TLR4) which is responsible for activation of the NF-κB transcriptional pathway [START_REF] Giridharan | Mechanisms of NF-κB p65 and strategies for therapeutic manipulation[END_REF]. Binding of the NF-κB dimer to the DNA consensus sequence of target genes leads to production of proinflammatory cytokines, such as TNFα [START_REF] Albensi | What Is Nuclear Factor Kappa B (NF-κB) Doing in and to the Mitochondrion?[END_REF]. Consistent with the anti-inflammatory effects of 44FNP demonstrated in human macrophage [START_REF] Bosviel | DHA-derived oxylipins, neuroprostanes and protectins, differentially and dose-dependently modulate the inflammatory response in human macrophages: Putative mechanisms through PPAR activation[END_REF], our results with BV2 microglia also demonstrated the ability for 44FNP to suppress LPS-induced iNOS and TNFα through the NF-κB pathway (Fig. 2).

Mitochondrial membrane potential (Δψ m ) is generated from proton pump across the mitochondrial inner membrane and involves complexes I, III, and IV [START_REF] Zorova | Mitochondrial membrane potential[END_REF].

The stability of the Δψ m is essential for many microglia functions [START_REF] Agrawal | Mitochondrial Dysfunction and Alzheimer's Disease: Role of Microglia[END_REF]. In this study, we provided important new evidence showing that 44FNP was capable of suppressing LPS-induced loss of Δψ m in microglia.

Nrf2 is an important transcription factor involving the maintenance of cellular redox state and alleviates expression of antioxidant genes such as HO-1. Some compounds that suppress NF-kB inflammation could also upregulate Nrf2/HO-1 [START_REF] Giridharan | Mechanisms of NF-κB p65 and strategies for therapeutic manipulation[END_REF][START_REF] Yang | Unveiling anti-oxidative and anti-inflammatory effects of docosahexaenoic acid and its lipid peroxidation product on lipopolysaccharidestimulated BV-2 microglial cells[END_REF]. In a recent study, 44FNP was found to elevate HO-1 mRNA expression in primary neurons [START_REF] Lee | Nonenzymatic oxygenated metabolite of docosahexaenoic acid, 4(RS)-4-F(4t)neuroprostane, acts as a bioactive lipid molecule in neuronal cells[END_REF]. However, study with microglia here showed effects of 44FNP on Nrf2/HO-1 only when cells were stimulated with LPS. These results suggest a role for 44FNP as an antioxidative agent in activated microglia.

In sum, this study demonstrated that 44FNP, a non-enzymatic peroxidation product of DHA, to exert neuroprotective effects on LPS-stimulated microglial cells through its abilities to attenuate oxidative stress, inflammatory responses, and mitochondrial dysfunction. Although future studies should include in vivo testing in animal models, this study has provided a better understanding on the mechanism(s) underlying the neuroprotective effects of 44FNP and better insights into its potential for the development of nutraceutical therapy for neurodegenerative diseases.

Fig. 2 .

 2 Fig. 2. 44FNP attenuated LPS-triggered the p65 pathway (A), iNOS (B), TNF-α (C) in BV2 cells. BV2 cells were pretreated with 44FNP for 1 h followed by stimulation with 50 ng/ml LPS for 1 h (A) or 18 h (B, C). Data are represented in mean ± SD (n = 3). Statistical analysis was carried out with one way ANOVA followed by Bonferroni post-tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the LPS group; ###p<0.001, ####p<0.0001 compared with the control group.

Fig. 3 .

 3 Fig. 3. 44FNP attenuated LPS-induced mitochondrial membrane potential loss in BV2 cells. Cells were pretreated with 44FNP for 1 h followed by stimulation with 50 ng/ml LPS for 24 h. Data are represented in mean ± SD (n = 3). Statistical analysis was carried out with one way ANOVA followed by Bonferroni post-tests. *p<0.05, **p<0.01, ***p<0.001 compared with the LPS group; ##p<0.01, ####p<0.0001 compared with the control group.

3. 4 .

 4 Effects of the 44FNP on Nrf2/HO-1 Our previous study demonstrated ability for exogenous DHA and its oxidative derivatives to upregulate the antioxidant pathway involving Nrf2/HO-1 in microglia. In this study, 44FNP dose-dependently upregulated Nrf2 and HO-1 in cells stimulated with LPS, but not in controls without stimulation with LPS (Fig 4).

Fig. 4 .

 4 Fig. 4. 44FNP enhanced LPS-induced Nrf2/HO-1 pathway in BV2 cells. Cells were pretreated with 44FNP for 1 h followed by stimulation with 50 ng/ml LPS for 6 h. Data are represented in mean ± SD (n = 3). Statistical analysis was carried out with one way ANOVA followed by Bonferroni post-tests. #p<0.05, ##p<0.01, ###p<0.001 compared with the control group.

Acknowledgement:

This work was supported by National Institutes of Health R01AG044404 (JCL).


Husam M A B Alsarraf

Kien Lam Ung

Matt D Johansen

Juliette Dimon

Vincent Olieric

Laurent Kremer

Mickaël Blaise
email: mickael.blaise@irim.cnrs.fr

Biochemical, structural, and functional studies reveal that MAB_4324c from Mycobacterium abscessus is an active tandem repeat N-acetyltransferase

Keywords: Mycobacterium abscessus, N-acetyltransferase, GCN5, X-ray structure, macrophage, infection, intracellular survival Mab, Mycobacterium abscessus, CF, Cystic Fibrosis, MmpS, Mycobacterial membrane protein small, MmpL, Mycobacterial membrane protein large, GNAT, GNC5-related N-acetyltransferase

Mycobacterium abscessus is a pathogenic non-tuberculous mycobacterium that possesses an intrinsic drug-resistance profile. Several N-acetyltransferases mediate drug resistance and/or participate in M. abscessus virulence. Mining the M. abscessus genome has revealed genes encoding additional N-acetyltransferases whose functions remain uncharacterized, of which MAB_4324c is among them. We showed that the MAB_4324c protein is an N-acetyltransferase able to acetylate polyamine substrates. The crystal structure of MAB_4324c was solved at high resolution in complex with its cofactor, revealing the presence of two GCN5-related Nacetyltransferase domains and a cryptic binding site for NADPH. Genetic studies demonstrate that MAB_4324c is not essential for in vitro growth of M. abscessus, however overexpression of the protein enhanced the uptake and survival of M. abscessus in THP-1 macrophages.

Introduction

Mycobacterium abscessus (Mab) is an opportunistic non-tuberculous human pathogen 1 . The Mycobacterium abscessus complex encompasses three subspecies M. abscessus sensu stricto, M. abscessus subsp. massiliense and M. abscessus subsp. bolletii. Several epidemiological studies have emphasized a possible human-human transmission cycle of Mab 2, [START_REF] Bryant | Stepwise pathogenic evolution of Mycobacterium abscessus[END_REF] , and demonstrated that only a few dominant clones are circulating [START_REF] Ruis | Dissemination of Mycobacterium abscessus via global transmission networks[END_REF] , which is in favor of global transmission of Mab in humans.

The prevalence of Mab pulmonary infection was estimated to be below 1 per 100000 inhabitants in the United States, however this number might be underestimated and Mab infections are expected to be on the rise [START_REF] Lee | Mycobacterium abscessus Complex Infections in Humans[END_REF] . Patients with underlying genetic and structural abnormalities, such as those with bronchiectasis or CF are particularly susceptible to Mab infection 1 . The prevalence of Mab infection among CF patients is estimated to range from 5 to 20% [START_REF] Floto | US Cystic Fibrosis Foundation and European Cystic Fibrosis Society consensus recommendations for the management of non-tuberculous mycobacteria in individuals with cystic fibrosis[END_REF] . Treatments of Mab infections are particularly long and are often associated with therapeutic failures. The poor efficacy of drug treatments is correlated with the intrinsic resistance of Mab to most common antibiotics, including first-line anti-tubercular isoniazid and rifampicin 1, [START_REF] Luthra | The Role of Antibiotic-Target-Modifying and Antibiotic-Modifying Enzymes in Mycobacterium abscessus Drug Resistance[END_REF] . In addition to the low permeability of the mycobacterial cell wall which limits the penetration of small molecules, Mab has developed multiple resistance mechanisms that include drug efflux activity mediated by MmpS/MmpL transporters [START_REF] Richard | Mechanistic and Structural Insights Into the Unique TetR-Dependent Regulation of a Drug Efflux Pump in Mycobacterium abscessus[END_REF][START_REF] Richard | Mutations in the MAB_2299c TetR regulator confer cross-resistance to clofazimine and bedaquiline in Mycobacterium abscessus[END_REF] , modifications or hydrolysis of the drug 7,10-15 , and/or alteration of the drug target 16, [START_REF] Richard | Dissecting erm(41)-Mediated Macrolide-Inducible Resistance in Mycobacterium abscessus[END_REF] ultimately incapacitating drug activity.

Mab is thought to be an environmental bacterium that colonizes different hosts and can persist within amoebae [START_REF] Ripoll | Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus[END_REF] . While Mab can replicate within amoeba, genomic analyses highlighted the accumulation of non-mycobacterial genes, which are thought to have been acquired to promote environmental persistence and nutrient acquisition. Furthermore, thorough transcriptomic analyses deciphered the differential gene expression pattern in correlation with the lifestyle of Mab, and identified several genes which were shown to be up-regulated when Mab is inside amoebae or in murine macrophages, thus highlighting the occurrence of an adaptive transcriptional program in response to various stresses encountered within the cell [START_REF] Dubois | Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages[END_REF] . To this end, the MAB_4532c gene, encoding the Eis2 protein, plays a critical role in the intramacrophage survival of Mab [START_REF] Dubois | Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages[END_REF] . Eis proteins that belong to the GNAT family indeed play a key role in the intracellular survival of other pathogenic mycobacteria and notably Mycobacterium tuberculosis 20, [START_REF] Duan | Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3[END_REF] . In addition, due to their Nacetyltransferase activity, Eis proteins can modify aminoglycoside antibiotics through acetylation, thereby impairing their binding to the ribosomes and leading to drug resistance 22, [START_REF] Chen | Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB[END_REF] . Moreover, Eis2 confers high-level resistance to aminoglycosides in Mab 12- [START_REF] Ung | Structural analysis of the N-acetyltransferase Eis1 from Mycobacterium abscessus reveals the molecular determinants of its incapacity to modify aminoglycosides[END_REF] . A very close homologue of Eis2, named Eis1, is neither required for intracellular survival [START_REF] Dubois | Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages[END_REF] nor aminoglycoside resistance, suggesting that it possesses another function that remains to be defined 12, [START_REF] Ung | Structural analysis of the N-acetyltransferase Eis1 from Mycobacterium abscessus reveals the molecular determinants of its incapacity to modify aminoglycosides[END_REF] . Together, these findings exemplify the need to experimentally address the substrate specificity and biological function of GNAT proteins despite their high homology levels with well-described homologues.

Transcriptomic approaches have been largely employed to identify key players of the drug resistance and/or virulence response in mycobacteria. As such, gene regulation studies have revealed that MAB_4324c is more highly expressed under intracellular growth conditions as compared to planktonic cultures [START_REF] Dubois | Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages[END_REF] . In contrast, MAB_4324c expression is down-regulated in a Mab whiB7 deletion mutant [START_REF] Hurst-Hess | Mycobacterium abscessus WhiB7 Regulates a Species-Specific Repertoire of Genes To Confer Extreme Antibiotic Resistance[END_REF] . WhiB7 represents a master transcription regulator controlling gene expression involved in the stress response and confers resistance to several classes of antibiotics, opening up the possibility that MAB_4324c might participate in these processes. Furthermore, RNAseq analyses identified several genes including MAB_4324c that were up-regulated upon exposure of Mab to clinically-relevant antibiotics, such as amikacin, clarithromycin, and tigecycline [START_REF] Schildkraut | RNA-sequencing elucidates drug-specific mechanisms of antibiotic tolerance and resistance in M. abscessus[END_REF] .

Considering the importance of GNAT enzymes in mycobacterial drug-resistance mechanisms and virulence, and because MAB_4324c encodes a predicted GNAT, this study was undertaken to functionally characterize the role of MAB_4324c in Mab. Herein, a combination of biochemical and structural approaches was used to demonstrate that MAB_4324c is an active tandem repeat N-acetyltransferase. Genetic studies showed that while MAB_4324c is dispensable for planktonic growth, its overexpression confers an intracellular growth advantage to Mab in THP-1 macrophages.

Materials and Methods

MAB_4324c cloning into the expression plasmid

The MAB_4324c gene was synthesized by Genscript with codon-optimized for Escherichia coli expression and cloned into the expression vector pET-30a. The sequences encoding for the hexahistidine-and S-tags followed by the TEV protease site were included in the 5'-end of the gene.

MAB_4324c expression and purification

The pET30a::MAB_4324c construct was transformed into E. coli BL21 (DE3) resistant to Phage T1 (New England Biolabs) and then transformed with the pRARE2 plasmid encoding rare tRNAs. An overnight pre-culture was used to inoculate a 12L culture of Luria broth (LB) media supplemented with 50 µg.mL -1 and 30 µg.mL -1 of KAN and chloramphenicol. The culture was grown until the exponential growth phase was reached and subjected to cold shock in an ice bath before induction of the protein expression with 1mM of IPTG (Euromedex) overnight at 18ºC. The

Determination of MAB_4324c oligomeric by size-exclusion chromatography

The Superdex 200 Increase 10/300 GL column (Cytiva) was calibrated with the Gel Filtration Markers Kit for protein molecular weight (MW) ranging from 12 400 to 200 000 Da (Sigma-Aldrich). The MW markers and MAB_4324c were eluted with buffer E as described above and at a flow rate of 0.4 mL.min -1 at 8ºC. The partition coefficient (K av ) of each protein was calculated with the following formula K av =(Ve-V0/Vt-V0) where Ve is the volume of elution of the proteins, Vt the volume of the column (24 mL) and V0 the void volume corresponding to the elution volume of dextran blue. The MW of MAB_4324c was extrapolated from the semilogarithmic plot of the partition coefficients, Kav, of the standard proteins marker versus their respective molecular masses on a logarithmic scale.

Crystallization

MAB_4324c crystals were grown in sitting drops using the MR Crystallization Plates™ (Hampton Research) at 18ºC by mixing 1 µL of 12 mg.mL -1 protein supplemented with 2 mM acetyl-CoA (incubated for 1h on ice), with 1 µL of reservoir solution (2 M ammonium sulfate, 0.1 M HEPES pH 7.5 and 2% w/v PEG400). Cryoprotection was not employed before crystals were cryo-cooled in liquid nitrogen, as all cryoprotective conditions tested led to crystal damage.

Consequently, ice rings were present on diffraction data but did not affect data processing or structure refinement.

Data collection, structure determination and, refinement

X-ray data collection was performed at the X06DA-PXIII beamline at the Swiss Light Source (Table II). A 360° native data set was collected at a wavelength of 1-Å with an oscillation of 0.2° and an exposure time of 0.1 s on PILATUS 2M-F detector [START_REF] Henrich | PILATUS: A single photon counting pixel detector for X-ray applications[END_REF] . A single-wavelength anomalous diffraction experiment from intrinsic sulfur atoms (S-SAD) consisting of 12 × 360° scans at varying values of and of the PRIGo multi-axis goniometer [START_REF] Waltersperger | PRIGo: a new multi-axis goniometer for macromolecular crystallography[END_REF] , was collected on a single crystal at 100 K at 2.075-Å wavelength using 0.2° oscillation 0.1 s exposure as previously described [START_REF] Weinert | Fast native-SAD phasing for routine macromolecular structure determination[END_REF] .

Data processing, scaling and merging were performed using XDS and XSCALE [START_REF] Kabsch | Integration, scaling, space-group assignment and post-refinement[END_REF] . The highresolution data cutoff was based on the statistical indicators CC 1/2 [START_REF] Karplus | Linking crystallographic model and data quality[END_REF] . Substructure determination and phasing were performed with SHELX 30,31 using the CRANK2 program [START_REF] Skubák | Automatic protein structure solution from weak X-ray data[END_REF] . The automatically built model using CRANK2 was rebuilt with Coot 33 and refined with Phenix 34 against the native data set (Table II). The structure factors and atomic coordinates were deposited at the Protein Data Bank under the accession entry 7Q3A.

Kinetics assay

MAB_4324c was concentrated to 7.5 mg.mL -1 in buffer E supplemented with 50% glycerol for use in kinetic assays. The reaction was performed at 25ºC in a quartz cuvette and consisted of monitoring the production of 2-thio-5-nitrobenzoic acid (TNB; extinction coefficient of 14150 M -1

cm -1 at 412 nm) resulting in the reduction of DTNB by the free CoA. Data points were recorded with a time interval of 5 sec for 50 to 60 sec using a Nanodrop2000c spectrophotometer (ThermoFisher Scientific). All compounds and the enzyme were dissolved and diluted in buffer E.

MAB_4324c (0.25 μM) was added to the reaction mixture consisting of DTNB (0.5 mM), ACO (0.5 mM) as well as substrates when requested in a final volume of 100 μL. A reaction mixture lacking the substrate was used to determine the reaction background and to normalize the values.

Standard errors were calculated from triplicates and a nonlinear least-squares regression algorithm was used to determine the Michaelis-Menten equation (GraphPad Prism software). colonies were then grown in 10 mL of Middlebrook 7H9 OADC supplemented with 0.025% tyloxapol and 250 µg.mL -1 KAN at 37ºC for 4 days, harvested and frozen at -80ºC.

Generation of an unmarked

Assessment of protein expression by Western blotting

Bacteria were disrupted by bead-beating, sonication and centrifuged at 20 000 g, at 4 ºC.

The total proteins from the crude extract (about 100 μg) were separated by SDS-PAGE, and were then blotted onto a nitrocellulose membrane (Cytiva) in transfer buffer (20 mM Tris, 150 mM glycine and 10 to 20% (v/v) ethanol). The membrane was treated with blocking buffer: PBS 1X, 5%

(w/v) skimmed milk and 0.2% (v/v) Tween 20. Membranes were first incubated either with a primary anti-Strep-tag monoclonal antibody (dilution 1/10000; Sigma-Aldrich) or with a rabbit antiacetyl lysine primary antibody (dilution 1/500-1/1000; abcam21623) and subsequently incubated with a secondary anti-mouse monoclonal antibody or an anti-rabbit antibody conjugated to the horseradish peroxidase (dilution 1/2000-1/5000; Sigma-Aldrich). Immunoreactive bands were revealed using the luminol-based reaction (SuperSignalTM-Thermo Fisher Scientific) and imaged using a Chemidoc (Biorad).

Macrophage infection with M. abscessus strains.

THP-1 macrophages were grown at 37ºC in a 5% CO 2 incubator in RPMI 1640 supplemented with 10% FBS (RPMI FBS ) and differentiated for 48 hrs with 20 ng.mL-1 phorbol myristate acetate (PMA). Exponentially growing Mab strains were harvested and resuspended in PBS 1X. Single-cell bacteria were produced by passage through a 26.5-gauge needle (20 times) and then further filtered through a 5.0 μm filter (Merck Millipore). THP-1 cells were infected at a 2:1 (bacteria:macrophage) Multiplicity of Infection (M.O.I.) and grown at 37ºC in the presence of 5% CO 2 . After 4 hrs incubation, cells were washed thrice with PBS 1X and incubated with RPMI FBS supplemented with 250 µg.mL -1 AMK for 2 hrs to eliminate extracellular bacilli. Infected THP-1 macrophages were cultured in RPMI FBS supplemented with AMK maintained at 50 µg.mL -1 .

Bacterial extracellular growth was prevented by refreshing the culture medium containing AMK 50 µg.mL -1 every two days. Before plating the bacteria, THP-1 macrophages were washed thrice with PBS 1X. Cell lysis was achieved with 100 µL of 1% Triton X100 in PBS 1X. Then, 900 µl of 1x PBS was added to each well and ten-fold dilutions were plated onto LB agar. This procedure was repeated at 0, 1, 3 and 5 days post-infection. After 5 days of incubation at 37ºC, the number of CFU was assessed. The CFUs counts were plotted on a logarithmic scale and the standard error bars were calculated using the GraphPad Prism software.

Growth curves of M. abscessus strains

Cultures of 20 mL were inoculated with single-cell preparations in 7H9 OADC supplemented with 0.025% tyloxapol (starting OD 600nm of 0.05) and incubated at 37ºC under agitation (100 rpm) for 5 days. The OD was monitored daily. The OD values were plotted and error bars were calculated using the GraphPad Prism software.

Drug susceptibility testing

The minimal inhibitory concentration (MIC) experiments were determined for the following antibiotics: bedaquiline, cefoxitin, ethambutol, vancomycin, ciprofloxacin , imipenem, kanamycin B, hygromycin B, amikacin, tigecycline and clofazimine. MICs were determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [START_REF] Woods | Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes[END_REF] . The broth microdilution method was used in Cation-adjusted Mueller-Hinton broth (CaMHB) with single-cell preparations of Mab wild-type, ΔMAB_4324c and corresponding to 5 × 10 6 CFU.mL -1 . Serial dilutions of the drug (100 μL) were added to 100 μL of bacterial suspension and incubated for 3-5 days at 30ºC.

Results and Discussion

MAB_4324c is an active N-acetyltransferase

Sequence analysis of MAB_4324c using the Pfam server [START_REF] Mistry | Pfam: The protein families database in 2021[END_REF] predicted the presence of a wellconserved GNAT domain in its C-terminus as well as a second GNAT domain displaying lower conservation in its N-terminus. This analysis indicates that MAB_4324c may belong to the tandem repeat N-acetyltransferase family. MAB_4324c shares a low protein sequence identity with the well-characterized Eis proteins and does not possess the same modular organization.

To inquire whether MAB_4324c possesses N-acetyltransferase activity, we performed a biochemical analysis of the protein. First, MAB_4324c was recombinantly expressed in E. coli and the soluble protein was subsequently purified following a three-step purification procedure. Highly pure MAB_4324c preparations were obtained (Fig. 1 A) and assessed for molecular weight determination by size-exclusion chromatography (SEC) (Fig. 1 A). The protein was eluted at a volume of 14.5 mL using a Superdex 200 10/300 GL Increase column. This elution profile corresponds to an apparent molecular weight of 42.6 kDa, which is in line with the theoretical mass of 39.1 kDa. This indicates that the protein is a monomer in solution.

We next assessed the activity of MAB_4324c with a few substrates known as substrates of N-acetyltransferases belonging to different classes 39, [START_REF] Pan | Eis, a novel family of arylalkylamine N-acetyltransferase (EC 2.3.1.87)[END_REF] . N-acetyltransferases substrates are highly diverse and, without any closely related protein whose function is known it was difficult to determine the substrate of MAB_4324c. As the Eis members of the GNAT protein in mycobacteria can modify aminoglycosides through acetylation of their primary amines, we first tested if MAB_4324c could modify this class of antibiotics. The near absence of or very low activity could be noticed using APR, HYG or AMK as substrates and ACO as a cofactor (Fig. 1 B). We next tested substrates suited for numerous GNAT and all possessing primary amines notably histamine, tyramine, spermine and spermidine (Fig. 1 B) [START_REF] Ung | Structural analysis of the N-acetyltransferase Eis1 from Mycobacterium abscessus reveals the molecular determinants of its incapacity to modify aminoglycosides[END_REF][START_REF] Pan | Eis, a novel family of arylalkylamine N-acetyltransferase (EC 2.3.1.87)[END_REF] . This screening identified only spermine and spermidine as substrates of MAB_4324c, with a slightly better apparent activity for spermidine (Fig. 1 B). To ensure that the reaction we observed was enzyme-specific, additional control experiments were performed with the heat-inactivated enzyme or with reactions lacking either the enzyme or the cofactor. As expected, the N-acetyltransferase activity was only detected in the presence of a functional MAB_4324c (Fig. S1A). The kinetic parameters of MAB_4324c for spermidine and acetyl-CoA were determined (Table I) (Fig. 1C, 1D and S1B, S1C). The relatively low catalytic efficiency and very low K M suggest that spermidine is not the cognate substrate of MAB_4324c though these kinetic data attest that MAB_4324c is an active N-acetyltransferase capable of transferring acetyl group to primary amines.

MAB_4324c is a tandem repeat N-acetyltransferase

To support the biochemical data, we engaged in structural studies of MAB_4324c. We succeeded in obtaining exploitable crystals of the protein when it was co-crystallized with acetyl-CoA. Structure phasing was achieved through the sulfur-single-wavelength anomalous dispersion method (Table II).

The partial model built thanks to the anomalous map was further used to solve the structure obtained from a 2Å resolution native dataset.

Two monomers occupy the asymmetric unit (Fig. 2A). Most of the residues could be rebuilt for the two monomers except for the first 11 and 12 residues of our expressed construct (that contains one extra Gly residue coming from the TEV cleavage site on top of the MAB_4324c sequence) in monomers A and B respectively. We could not build some residues of the loop between strands 8 and 9 due to very poor electron density in this area.

Crystal packing analysis did not argue in favor of a functional dimer and, in agreement with the oligomeric state of the protein observed by SEC (Fig. 1A). The MAB_4324c overall structure (Fig. 2B) is composed of 12 β-strands and 11 α-helices forming two GNAT domains interconnected to each other with two β-strands (β6 and β12). The structure can be divided into two major subdomains: domain 1 in the N-terminus is containing residues 1-177 (α1-α6 and β1-β6) and is a GNAT domain although the e-value (8.4x10 -5 ) for this signature motif is quite low when analyzed at the Pfam server. Domain 2 in the C-terminus encompasses residues 178-352 (α7-α11 and β7-β12)

and matches a GNAT domain signature with a much better score (e-value=3.9x10 -11 ) than domain 1.

These two domains share 13% sequence identity and their superposition based on their secondary structure and over the main chain of 137 residues led to a r.m.s.d. value of 2.1Å (not shown), attesting to a certain degree of structural divergence but not as high as it would have been expected

based on the sequence homology.

The two monomers from the asymmetric unit are not equivalent in terms of ligand binding (Fig. 2A). The two C-terminal subdomains bind acetyl-CoA (Fig. 2A and3A). This ligand is in fact in two forms as an acetyl-CoA and as another one where acetyl-CoA is cleaved into CoA and acetate. All ligand occupancies were estimated to be 0.5 (Fig. S2). Unexpectedly, a ligand neither corresponding to acetyl-CoA nor CoA was bound to domain 1 in each of the two monomers of the asymmetric unit. The clear simulated annealed omit maps 41 allowed without ambiguity the identification of NADH in one monomer and NADPH in the second one (Fig. 3B). In one monomer of the asymmetric unit, NADH could be placed however in the other monomer, NADPH was modelled. Since none of these ligands were added exogenously during the crystallization process, it appears obvious that they were co-purified during the purification of MAB_4324c. Of note, NADPH was not modelled in both chains as the phosphate group cannot pack due to steric hindrance with the symmetry mate (not shown) in chain B and electron density does not support the presence of the additional phosphate group (Fig. 3B). To ensure that NADH and NADPH are the bound forms rather than their oxidized forms and to assess to which extent MAB_4324c is bound to nicotinamide after purification, we performed a UV spectrum of the concentrated protein. In addition to the protein absorption peak at 280 nm, a second peak was detected at 340 nm and corresponding to NADH/NADPH (Fig. S3). The concentration of the NADH/NADPH in the sample was estimated to 70 μM, which corresponds to the MAB_4324c concentration, indicating that the protein is saturated with the reduced forms of the nicotinamide groups since the oxidized NAD+/NADP+ forms do not display absorption peak at 340 nm.

Acetyl-CoA (ACO) is recognized by twelve residues (Fig. 3A and Fig. 4), of which the side chain of H222 establishes a H-bond via one water molecule with the S atom of ACO. The sulfur is also contacted by the Y334 side chain. H222 and Y334 interact with the ACO S-group and thus are very likely to be important catalytic residues. This is particularly true for Y334 as it was already proposed that the side chain of the catalytic Y residue of the RimI acetyltransferase might be important as a general acid for the protonation of the ACO sulfhydryl group before the transfer of the acetyl group 42, [START_REF] Christensen | Identification of Novel Protein Lysine Acetyltransferases in Escherichia coli[END_REF] . F219 interacts by hydrophobic interaction with the acetyl moiety that is also stabilized by the main chain of L287. The main chains of T328 and V289 form a H-bond with the NH and CO groups of the pantothenate. The phosphates of ADP are interacting with the main chains of W293 (via one water molecule), G295, Q300 and K297, while the latter also binds the phosphate of the ACO ribose with its side chain. Finally, the side chain of R294 stacks the ACO.

Recognition of NADPH involves a complex network of interactions (Fig. 3B and Fig. 4).

The nicotinamide group of NADPH is contacted by the side chains of Q133 and S154 as well as by the main chain of G114. F167, V129 and L166 mediate van der Waals interactions with the nicotinamide group. The hydroxyl of the ribose is contacted via H-bonds by the side chains of R121, T163 and the main chain of V116. The main chain of G122, E123, G124 and G126 tightly bind to the phosphate group of ADP moiety. The last interaction involves the side chain of R169 that establishes a H-bond with the ribose of the ADP. The identification of a binding site for nicotinamide is particularly intriguing since, to our knowledge, GNAT domains have never been reported to bind to NADH/NADPH. It is well documented however that the type III GNAT enzymes are allosterically regulated by metabolic compounds [START_REF] Lammers | Post-translational Lysine Ac(et)ylation in Bacteria: A Biochemical, Structural, and Synthetic Biological Perspective[END_REF] . This is the case for the GNAT enzyme (Rv0998) from M. tuberculosis which is allosterically regulated by cyclic AMP (cAMP) [START_REF] Nambi | cAMP-regulated Protein Lysine Acetylases in Mycobacteria[END_REF] . Activation of the enzyme's activity occurs upon binding of cAMP that triggers a large conformational switch. However, the GNAT domain is not the domain that binds cAMP [START_REF] Lee | Cyclic-AMP regulation of protein lysine acetylation in Mycobacterium tuberculosis[END_REF] . The N-acetyltransferase MxKat from Myxococcus xanthus can bind to NADP+ thanks to its N-terminal domain consisting of a Rossmann fold and binding to NADP+ which inhibits the enzyme activity 47 . To assess if NADH or NADPH could play a role in regulating or modulating the enzymatic activity of MAB_4324c, we compared the kinetic constants of MAB_4324c for spermidine and in presence of 0.1 mM of the reduced nicotinamide forms. The enzymatic properties were however not significantly affected by the addition of NADH or NADPH (Fig. S4 and Table S1). Interestingly, it was also proposed that the N-terminal GNAT domain of the mycothiol synthase is not catalytically active but its capacity to bind CoA may assist the enzyme to fold [START_REF] Vetting | The substrate-induced conformational change of Mycobacterium tuberculosis mycothiol synthase[END_REF] suggesting that NADH or NADPH may be helpful for the folding of MAB_4324c.

We then attempted to search for MAB_4324c structural homologues whose biological function or enzymatic function was elucidated. This led to the identification of only two related structures (Fig. 4): the mycothiol synthase from Mycobacterium tuberculosis (PDB id: 2C27) [START_REF] Vetting | The substrate-induced conformational change of Mycobacterium tuberculosis mycothiol synthase[END_REF] bound to des-acetylmycothiol and CoA that possesses a r.m.s.d. of 1.9Å and sequence identity of 19%, and the structure of glucosamine N-acetyltransferase bound to chitosan, GlmA, from Clostridium acetobutylicum (PDB id : 5kgp) sharing a r.m.s.d. of 2.6 Å and sequence identity of 12% [START_REF] Dopkins | Structural Studies on a Glucosamine/Glucosaminide N-Acetyltransferase[END_REF] . These two enzymes are also tandem repeat N-acetyltransferases but are involved in different pathways. MshD is an essential enzyme for mycobacteria as it catalyzes the last step of mycothiol synthesis, a key component in protection against oxidative stress. Mycothiol is made of an acetylated Cys residue, a glucosamine residue, and an inositol group. GlmA ensures the transfer of an acetyl group from ACO to the amino group of glucosamine 50 and seems to be involved in cell wall recycling. These three enzymes have in common the ability to bind ACO and indeed residues involved in ACO binding are well conserved in the three proteins (Fig. 4). In the MshD structure, glucosamine is tightly bound by V34, E36, Q37, E79, E179, Q180, E234, Y282 and D305 [START_REF] Vetting | The substrate-induced conformational change of Mycobacterium tuberculosis mycothiol synthase[END_REF] . In GlmA W193, E196, D287, W288 and E290 are key residues for carbohydrate-binding [START_REF] Dopkins | Structural Studies on a Glucosamine/Glucosaminide N-Acetyltransferase[END_REF] . Only a few of the residues involved in carbohydrate binding in these two enzymes are however conserved in MAB_4324c, suggesting a different function for the latter (Fig. 4).

The fact that MAB_4324c could be the mycothiol synthase in Mab cannot be excluded. A BLAST search using MshD (Rv0819) retrieves MAB_0745 as the closest homologue of the mycothiol synthase, sharing 51% sequence identity and 61% similarity. A strong experimental argument for excluding MAB_4324c as the main mycothiol synthase relies on the fact that, while the mycothiol synthase is an essential enzyme in mycobacteria, we were able to generate a MAB_4324c deletion mutant in Mab, as shown below (Fig. 5). While this suggests that MAB_4324c is unlikely to be a mycothiol synthase, we cannot rule out a redundant or closely related function that is not essential for Mab.

Nevertheless, the structural analysis attesting strong structural proximity with proteins involved in carbohydrate metabolism/synthetic pathway underlines the possibility that MAB_4324c may participate in the acetylation of substrates possessing a carbohydrate moiety, which remains to be discovered.

Closely related MAB_4324c homologues are mainly found in actinobacteria

We searched for the presence of MAB_4324c orthologues in other mycobacterial species, but none of them were identified in Mycobacterium leprae, Mycobacterium smegmatis, M.

tuberculosis, or Mycobacterium marinum. However, additional searches using the HMMER server identified very closely related homologues in numerous bacteria, mainly within the actinobacteria phylum. Multiple sequence alignments of the best hits highlight a high conservation level with MAB_4324c (Fig. S5). Highly conserved protein sequences were found within the M. abscessus complex including subspecies M. bolletii, and M. massiliense and one orthologue was found in the phylogenetically related Mycobacterium chelonae. Other occurrences were found in other mycobacteria, such as Mycobacterium immunogenum or Mycobacterium wolinskyi and other actinobacteria of the Streptomycetaceae, Microbacteriaceae groups as well as in a few Proteobacteria from the Burkholderiales group. This analysis indicates that the protein is not conserved necessarily in pathogenic bacteria but rather in environmental bacteria.

MAB_4324c is dispensable for M. abscessus planktonic growth

To investigate the role of MAB_4324c, a deletion mutant was generated by the use of a homologous recombination technique enabling the production of unmarked deletion mutants in Mab (Fig. 5A) [START_REF] Richard | Mutations in the MAB_2299c TetR regulator confer cross-resistance to clofazimine and bedaquiline in Mycobacterium abscessus[END_REF] . Gene deletion was obtained in the Mab smooth background. The gene deletion was confirmed by PCR genotyping and sequencing (Fig. 5B). Complementation of the deletion strain was obtained by cloning MAB_4324c into the episomal pMV261 51 yielding pMV261::MAB_4324c. The same construct was used for overexpressing the protein in the WT background. A Strep-tag coding sequence was added in the 5'-end of the gene to monitor the protein expression by Western blotting. MAB_4324c expression was indeed detected in both WT and mutant strains (Fig. S6). All strains grew similarly to the WT progenitor, suggesting that the inactivation of MAB_4324c does not affect the planktonic growth of Mab (Fig. 5C). The results indicate that MAB_4324c is dispensable for Mab growth in planktonic culture.

MAB_4324c does not trigger resistance to clinically relevant drugs

Transcriptomic data attested that MAB_4324c expression is controlled by the transcriptional WhiB7 regulator [START_REF] Hurst-Hess | Mycobacterium abscessus WhiB7 Regulates a Species-Specific Repertoire of Genes To Confer Extreme Antibiotic Resistance[END_REF] . Upon stress responses, WhiB7 upregulates the expression of numerous genes and particularly those involved in antibiotic resistance. It was notably shown that the expression of the N-acetyltransferase Eis2 Mab that inactivates amikacin [START_REF] Ung | Crystal structure of the aminoglycosides N-acetyltransferase Eis2 from Mycobacterium abscessus[END_REF] , is indeed controlled by WhiB7 [START_REF] Hurst-Hess | Mycobacterium abscessus WhiB7 Regulates a Species-Specific Repertoire of Genes To Confer Extreme Antibiotic Resistance[END_REF] .

Further, RNAseq analysis revealed that MAB_4324c among other genes is upregulated upon exposure of Mab to several classes of antibiotics. MAB_4324c is upregulated upon exposure to clinically relevant antibiotics such as amikacin, clarithromycin and tigecycline [START_REF] Schildkraut | RNA-sequencing elucidates drug-specific mechanisms of antibiotic tolerance and resistance in M. abscessus[END_REF] . We thus, tested whether MAB_4324c contributes to antibiotic resistance in Mab. To do so, MICs of a limited set of antibiotics were determined in ΔMAB_4324c and compared with those of the WT strain. However, no major differences were noticed in the drug susceptibility profile between the two strains (Table S2). Albeit, while we only tested a limited number of antibiotics, this study suggests that, in contrast to Eis2 Mab , MAB_4324c is unlikely to play a role in drug resistance mechanisms in Mab.

MAB_4324c is not involved in protein acetylation

Lysine acetylation is a ubiquitous posttranslational modification in bacteria [START_REF] Lammers | Post-translational Lysine Ac(et)ylation in Bacteria: A Biochemical, Structural, and Synthetic Biological Perspective[END_REF] and this is particularly true in mycobacteria. In M. tuberculosis about 140 proteins involved in numerous metabolic processes were shown to be acetylated [START_REF] Liu | Acetylome Analysis Reveals Diverse Functions of Lysine Acetylation in Mycobacterium tuberculosis[END_REF] . We thus addressed the possibility that MAB_4324c participates in protein acetylation. We compared the acetylome of the WT, ΔMAB_4324c and the corresponding MAB_4324c-overexpressing strains by Western blotting using a pan anti-acetylated lysine primary antibody. No major differences were observed when comparing the acetylome of the four strains, suggesting that MAB_4324c cannot acetylate proteins (Fig. S7).

However, more sensitive assays, such as acetylated peptide enrichment coupled to mass spectrometry, are required to support this hypothesis. Additionally, it would be relevant to explore whether MAB_4324c acetylates host proteins, although this is very unlikely given the fact that MAB_4324c is not predicted to be secreted.

MAB_4324c overexpression confers intracellular survival in human macrophages

A recent RNAseq study identified a set of genes that are specifically upregulated during the intramacrophage growth of M. abscessus subspecies massiliense [START_REF] Dubois | Mycobacterium abscessus virulence traits unraveled by transcriptomic profiling in amoeba and macrophages[END_REF] . MYCMA_RS02565 which is the orthologue of MAB_4324c in M. massiliense, was four-time more expressed under the intracellular conditions as compared to in vitro growth conditions. This prompted us to assess whether MAB_4324c plays a role in the intracellular uptake and/or survival in macrophages. On day 0, the uptake of ΔMAB_4324c was not affected as compared to the WT strain, however the MAB_4324c overexpressing strain showed increased internalization by macrophages, as determined by the CFU counts (Fig. 5D). Similarly, while ΔMAB_4324c failed to show alterations in intracellular survival as compared to the WT strains, infections with either the complemented strain or the WT strain overproducing MAB_4324c were associated with higher bacterial loads (Fig. 5D). The sequences of the MAB_4324c orthologues were retrieved with the HMMER server (http://hmmer.org/). Some of the sequences sharing more than 40% protein sequence identity were selected and realigned using the ClustalX software. WT comp.), Δ4324c and Δ4324c complemented strains (Δ4324c comp.). The lysine acetylome was detected with a pan anti-acetyl lysine primary antibody (abcam 21623) and revealed with an antirabbit secondary antibody produced in goats and coupled to the horseradish peroxidase. On the left is shown the membrane after transfer and colored by ponceau red while on the right is the immunoblot revealed by chemiluminescence. 100 μg of total protein were loaded on the 10% SDS-PAGE for high molecular weight (HMW) proteins and 12% SDS-PAGE for proteins of medium molecular weight (MMW) proteins. The primary antibody was diluted 500 and 1000 times for HMW and MMW proteins respectively while the secondary antibody was diluted 5000 and 10000 times. Only a very weak signal could be detected for proteins with a molecular weight below 25 kDa (not shown).

This suggests that overexpression of

  bacteria were homogenized in lysis buffer A (50 mM Tris-HCl pH 8, 0.4 M NaCl, 20 mM imidazole, 1 mM benzamidine and 5 mM β-ME). Bacteria were disrupted by sonication and centrifugated at 28000 g. The soluble fraction was loaded onto a Ni-NTA Sepharose resin (Cytiva) by gravity. The column was washed with buffer A and buffer B (50 mM Tris-HCl pH 8, 1M NaCl and 5 mM β-ME). Proteins were eluted with buffer C (50 mM Tris-HCl pH 8, 0.2 M NaCl, 5 mM β-ME and 400 mM imidazole). The eluate was dialyzed overnight at 4ºC in dialysis buffer D (50 mM Tris-HCl pH 8, 0.2 M NaCl and 5 mM β-ME) in the presence of His-tagged TEV protease in a 50:1 w/w ratio to remove the tags. The TEV protease and the uncleaved MAB_4324c were separated from the untagged proteins by the passage of the dialyzed eluate through a Ni-NTA sepharose column. MAB_4324c was collected in the flow-through and concentrated by ultra-filtration (centricon 10 kDa CW-Sartorius). Finally, MAB_4324c was concentrated to 5mg.mL -1 and loaded onto a sizeexclusion chromatography (SEC) column (Superdex 200 Increase 10/300 column from GE Cytiva) and eluted with buffer E (20 mM Tris-HCl pH 7.4 and 0.2 M NaCl).

  MAB_4324c knockout mutant in M. abscessus. The ΔMAB_4324c M. abscessus strain was generated by the unmarked deletion strategy involving two recombination steps as described previously[START_REF] Richard | Mutations in the MAB_2299c TetR Regulator Confer Cross-Resistance to Clofazimine and Bedaquiline in Mycobacterium abscessus[END_REF] . The MAB_4324c (1059 bp) flanking regions (each around 1500 bp) were PCR-amplified using the Phusion polymerase (). Amplicons were purified and treated with restriction enzymes (all from NewEngland Biolabs), ligated into pUX1-katG Mtb, and digested with NheI and HpaI restriction sites, where the flanking regions were ligated at the MfeI restriction site.The resulting plasmid was transformed into electro-competent Mab CIP104536T, smooth variant[START_REF] Ripoll | Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus[END_REF] . Transformed bacteria were placed at 37ºC for 3 hrs under agitation before plating on Middlebrook 7H10 (BD Difco) supplemented with oleic acid-albumin-dextrose-catalase (OADC) enrichment (7H10 OADC ) and 250 µg.mL -1 KAN. The selection of colonies was based on resistance to KAN and tdTomato expression (red fluorescent colonies) as an indicator for the first recombination event. Selected red colonies were grown after 4 days in Middlebrook 7H9 broth (BD Difco) supplemented with OADC, 0.025% tyloxapol and 250 µg.mL -1 KAN. Bacteria were pelleted, washed thrice and resuspended in an antibiotic-free medium. Cultures were then incubated overnight at 37ºC under agitation to allow for the second recombination event to occur. Bacteria were then plated on 7H10 OADC plates supplemented with 50 µg.mL -1 isoniazid (INH) and incubated at 37ºC for 4 days. INH-resistant and non-fluorescent colonies were genotyped by PCR to identify the mutants. Proper gene deletion was subsequently confirmed by PCR and sequencing.Overexpression of MAB_4324c in M. abscessus.MAB_4324c was amplified by PCR from genomic DNA using the following primers, Fw: , (restriction sites are underlined) and cloned into the episomal pMV261[START_REF] Stover | New use of BCG for recombinant vaccines[END_REF] and into integrative pMV306 both cut with EcoRI and HpaI. This cloning strategy allows gene expression under the control of the strong hsp60 promoter. The cloning strategy enabled the addition of a strep tag at the C-terminus of MAB_4324c. The pMV261::MAB_4324c and pMV306::MAB_4324c construct were introduced into electro-competent Mab WT and ΔMAB_4324c strains. The transformed bacteria were plated on 7H10 OADC agar containing 250 µg.mL -1 KAN. Individual

Figure 1 .

 1 Figure 1. Biochemical characterization of MAB_4324c (A) Coomassie Blue-stained SDS polyacrylamide gel electrophoresis attesting the purity of MAB_4324c obtained after three steps of purification (10 μg of protein were loaded on the gel). Estimation of the oligomeric state of MAB_4324c, depleted from its tags, by SEC. The elution profile of the proteins used for calibration on the Superdex 200 increase 10/300 GL column is displayed as a black line and the elution profile of MAB_4324c is indicated in red. Calibration was established using β-amylase (200 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome C (12.4 kDa), eluted with estimated volumes of 11.07, 13.2, 15.8 and 17.3 mL, respectively. The void volume indicated by the black arrow was estimated at 8.3 mL with dextran blue. The MAB_4324c elution peak at 14.5 mL corresponds to an apparent molecular weight of 42.6 kDa. (B) Substrate specificity assessment of MAB_4324c. The initial rates were determined in presence of 10 mM of each of the following substrates: AMK, apramycin (APR), hygromycin B (HYG), histamine (HIS), tyramine (TYR), spermine (SPR) or spermidine (SPDN). Acetyl-CoA at 0.5 mM was used as a cofactor. (C) Michaelis-Menten curve used to determine the kinetic constants for spermidine in the presence of 0.5 mM of acetyl-CoA. (D) Michaelis-Menten curve was used to determine the kinetic constants for acetyl-CoA in the presence of 40 mM spermidine.

Figure 2 .

 2 Figure 2. Overall crystal structure of MAB_4324c (A) Composition of the asymmetric unit (A.U). Two non-equivalent monomers of MAB_4324c occupy the A.U. The two monomers are similar with the exception that the N-terminal subdomain of chain A binds NADPH while NADH is present in the N-terminal subdomain of chain B. Nt and Ct stand for N-terminus and C-terminus respectively. (B) Overall structure of MAB_4324c. Alpha (α) and 3 10 (η) helices appear as blue cartoons while beta-strands (β) are colored in pink. Loops or coils are in grey.

Figure 3 .

 3 Figure 3. Acetyl-CoA and NADH/NADPH recognition by MAB_4324c (A) Recognition of acetyl-CoA. The simulated-annealed omit maps contoured at the 3 sigma level are displayed for acetyl-CoA/CoA/acetate as seen in chains A and B. The acetyl-CoA (yellow sticks) and CoA/acetate group (white stick) were refined to an occupancy of about 0.5 each (left panels). On the right panel are displayed the interactions between the cofactor and the MAB_4324c active site residues as seen in chain A. Hydrogen bonds are represented by the dashed lines and water molecules are seen as red spheres. Nitrogen, oxygen and phosphate atoms are in blue, red and orange, respectively, while carbon atoms are in grey for MAB_4324c amino acids or yellow for acetyl-CoA. (B) Recognition of NADH/NADPH. The simulated-annealed omit maps contoured at the 3 sigma level are displayed for NADH/NADPH as seen in chains A and B (left panels). The right panel shows the interactions occurring between NADPH and the residues side-chains of MAB_4324c as seen in chain A. The recognition of NADH is equivalent to the one of NADPH and is, therefore, not displayed.

Figure 4 .

 4 Figure 4. Comparison of MAB_4324c with structural homologues (A) Multiple sequence alignment was performed with ENDscript 55 and adjusted manually with the Inkscape software. The secondary structure (α, α-helix, β, β-strand, η, 3 10 -helix) of MAB_4324c obtained from its crystal structure, is indicated above the alignment. Residues involved in NADPH and ACO binding in MAB_4324c are indicated by the blue and green circles, respectively. The pink and black circles indicate the amino acids involved in carbohydrate binding in the Glucosamine/Glucosaminide N-acetyltransferase, GlmA (PDB id: 5KGP) and the mycothiol synthase MshD (PDB id: 2C27) crystal structures. (B) Superposition of the MshD and GlmA (both as a grey cartoon) onto the MAB_4324c (raspberry cartoon) crystal structure. The substrates bound to MshD and GlmA are shown as yellow sticks. The r.m.s.d. values are reported below each structural alignment.

Figure 5 .

 5 Figure 5. Generation and phenotypic analyses of ΔMAB_4324c (A) Genomic region of MAB_4324c in the parental (WT) and deletion (ΔMAB_4324c) strains is indicated. The size of the PCR amplicons used to genotype the ΔMAB_4324c mutant is represented. (B) PCR genotyping demonstrates the proper deletion of the mutant strain. The PCR product of 1079 pb was amplified from ΔMAB_4324c genomic DNA while a band at 2081 pb was obtained for the WT strain. (C) In vitro growth curves in liquid culture and at 37ºC of the WT smooth (Mab S WT), ΔMAB_4324c (Mab S Δ4324c), complemented strains (Mab S Δ4324c comp.) as well as the WT strain overexpressing MAB_4324c (Mab S WT comp.). Experiments were performed in triplicates. (D) Infection of THP-1 macrophages with Mab. Histograms and error bars represent the means and standard deviations calculated from three independent experiments. For statistical analysis, the unpaired t-test with Welch's correction was applied. *, **, *** and **** stands for p<0.05, p<0.005, p<0.001 and p<0.0001, respectively.

  MAB_4324c provides an intracellular growth advantage with an enhanced intracellular survival capacity. While we established that MAB_4324c is an active N-acetyltransferase, the lack of data regarding its genuine substrate makes it difficult to propose a clear function. Nonetheless, the capacity of MAB_4324c bind NADH/NADPH might explain the phenotype observed when overexpressing MAB_4324c in Mab during macrophage infection. Supporting this hypothesis, several bacterial pathogens have developed strategies to reduce the intracellular pool of NAD+, subsequently reducing the capacity of the cell to fight against the pathogen and therefore, thus favoring escape and intracellular survival of the bacteria 53 . Of particular interest, it has been shown that Sirtuin 3 a mitochondrial deacetylase, whose function is dependent on the NAD+ pool, enhances host defense against Mab infection 54 . Thus, by sequestering nicotinamide and diminishing the concentration of NAD+ inside the host cell, MAB_4324c may reduce the host defense and facilitate the persistence of Mab inside macrophages. Another hypothesis somewhat related to the above one would be that MAB_4324c by binding NADH/NADPH controls the metabolic state of this important cofactor which might be needed to convert its planktonic to an intracellular lifestyle. However, a clear demonstration of these different proposed hypotheses awaits further experimentation.

Figure S5 :

 S5 Figure S5: Multiple sequence alignments of the MAB_4324c orthologues

Figure S6 :

 S6 Figure S6: Western blot confirming the expression of MAB_4324cProtein expression assessment of MAB_4324c in the parental (Mab S WT) and knockout strains (Mab S Δ4324c). The Western blotting revealed with anti-Strep tag primary antibodies attests to the expression of MAB_4324c in both strains transformed with pMV261::MAB_4324c (wells 5 and 10). Expression trials with the integrative pMV306::MAB_4324c were not successful (wells 4 and 8). Strains transformed with the empty plasmids were included as negative controls.

Figure S7 :

 S7 Figure S7: M. abscessus acetylome revealed by an anti-acetyl lysine antibody Comparison of the protein lysine acetylation pattern of the WT, WT overexpressing MAB_4324c

Table I . Kinetic parameters of MAB_4324c.

 I

	Substrates	V max (pmol.s -1 )	K M (µM)	k cat (s -1 )	K M /k cat (M -1 s -1 )
	Acetyl-CoA	99.6 ± 0.02	63 ± 0.07	0.39 x 10 -3 ±0.008	0.16
	Spermidine	186.4 ±5	1.66 x 10 3 ±0.22	0.74 x 10 -3 ±3.8 10 -5	2.24

Table II . X-ray data collection and refinement statistics.

 II

		MAB_4324c native	MAB_4324c Sulfur SAD
	Wavelength (Å)	1	2.08
	Resolution range (Å)	49.09 -2.0 (2.072-2.0)	49.26 -2.33 (2.39 -2.33)
	Space group	P 65 2 2	P 65 2 2
	Unit cell (Å,°)	149.96 149.96 183.38, 90 90 120 150.49 150.49 184.14, 90 90 120
	Total reflections	3143788 (302942)	19074956 (230331)
	Unique reflections	82207 (8058)	99388 (7401)
	Multiplicity	38.2 (37.6)	191.9 (31.1)
	Completeness (%)	96.84 (99.98)	99.00 (99.30)
	Mean I/sigma(I)	39.58 (5.79)	32.18 (0.82)
	Wilson B-factor (Å 2 )	26.2	48.55
	R-meas	0.115 (0.87)	0.278 (4.43)
	CC1/2	1 (0.95)	1 (0.45)
	Reflections used in refinement	79620 (8058)
	Reflections used for R-free	1969 (199)
	R-work	0.186 (0.183)
	R-free	0.225 (0.236)
	Number of non-hydrogen atoms	6620
	macromolecules	5437
	ligands	340
	solvent	860
	Protein residues	679
	RMS (bonds, Å)	0.01
	RMS (angles, °)	1
	Ramachandran favored (%)	98.66
	Ramachandran allowed (%)	1.34
	Ramachandran outliers (%)	0
	Rotamer outliers (%)	0.36
	Clash score	3.6
	Average B-factor (Å 2 )	31.5
	macromolecules	29.9
	ligands	37.9
	solvent	39.3

Values between parentheses are for the last resolution shell.

  The alignment was then generated with ESPript (https://espript.ibcp.fr/ESPript/ESPript/). The red and yellow colors indicate strict protein sequence identity conservation or similarity respectively. These protein sequences correspond to the following Uniprot (https://www.uniprot.org/) entries: B1MJN9 for Mycobacterium abscessus sensu stricto, A0A1N5X9F8 for Mycobacterium bolletii, A0A1U3WRU2 for Mycobacterium massiliense, A0A7V8RYX2 for Mycobacterium immunogenum, A0A1S1M967 for Mycobacterium chelonae, A0A4R8SHM6 for Mycobacterium salmoniphilum, A0A4R5PCD3 for Mycobacterium franklinii, A0A235FHP5 for Rhodococcus sp. OK302, A0A1X2F0T9 for Mycobacterium wolinskyi, A0A4Q9HX52 for Streptomyces kasugaensis, A0A7L5AGZ4 for Marisediminicola antarctica, A0A401YSZ2 for Embleya hyalina, A0A3L7A326 for Mycetocola tolaasinivorans, A0A1X9LGC8

for Cnuibacter physcomitrellae, A0A542Y663 Leucobacter komagatae, A0A3L7ARX6 for Mycetocola lacteus, A0A5C8URY9 Lacisediminihabitans profunda, A0A4Q9GUC3 for Glaciihabitans arcticus, A0A1G9AKS2 for Cryobacterium psychrotolerans, A0A4S1E912 for Alcaligenaceae bacterium 429, A0A2P8GRY7 for Labedella gwakjiensis, A0A4Y9R9U9 for Leifsonia flava and A0A5C8HQH6 for Microbacterium mitrae.

Table S2 . Determination of the MIC 99

 S2

	Antibiotics	MIC (µg.mL -1 )	MIC(µg.mL 1 )
		Mab S wild-type	Mab S ΔMAB_4324c
	Bedaquiline	0.16	0.16
	Kanamycin B	25	25
	Amikacin	50	50
	Tigecycline	5	5
	Clofazimine	3.1	1.5
	Imipenem	32-64	32-64
	Ethambutol	>100	>100
	Cefoxitin	61.2	61.2
	Ciprofloxacin	61.2	61.2
	Vancomycin	>250	>250

MIC of the different antibiotics were determined as described in the Materials and Methods.

Acknowledgments

We thank the staff at SLS beamlines for support during data collection. Funding for this work was supported by the Fondation pour la Recherche Médicale (FRM) [grant number DEQ20150331719 to LK] and, the Lundbeck Foundation, Denmark (R303-2018-2964 to HA). KLU Ph.D. fellowship was supported by the National Research Agency [ANR-17-CE11-0008-01 -MyTraM to MB].

M.D.J. received a postdoctoral fellowship granted by Labex EpiGenMed, an "Investissements d'Avenir" program (ANR-10-LABX-12-01). We would like to thank Dr. W. Daher for his help with macrophage preparations. We thank Dr J. Wong for the critical reading of the manuscript.

Data availability

The data that support findings of this study are available from the corresponding author mickael.blaise@irim.cnrs.fr upon reasonable request. The structural data that support these findings are openly available in the wwPDB at https://doi.org/10.2210/pdb7Q3A/pdb.

Conflict of interest statement

The authors declare no conflict of interest.

Author contributions

HA, KLU, JD and MB performed experiments. MB designed research. VO performed sulfur-SAD phasing. MDJ assisted with macrophage experiments. LK and MB supervised the research. MB wrote the first draft with the input of all the authors. S1.

SUPPLEMENTARY MATERIALS

Table S1. Kinetic parameters of MAB_4324c in presence of NADH and NADPH.

Substrates

V max (pmol.s -1 ) K M (µM) k cat (s -1 ) K M /k cat (M -1 s -


Study and development of new materials for high temperature compact thermal storage applications

Imane Mahroug

To cite this version:

  Imane Mahroug. Study and development of new materials for high temperature compact thermal storage applications. Mechanics [physics]. Université de Bordeaux; CIC EnergiGUNE, 2021. English. NNT : 2021BORD0371. tel-03652920