Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB  and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB through Western blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A Bothrops genus was responsible for most of the reported cases, followed by Crotalus, Lachesis, and Micrurus genera  (~120 kDa) have been proposed [Monoclonal antibodies , proposed as therapeutic agents ,19,20,21proposed . The lowproposed ,32.In addition to conventional IgG, camelids produce functional antibodies devoid of light chains and CH1 domains, referred to as camelid heavy-chain antibodies (HCAbs) with approximately 90 kDa . The antLama glama VHHs able to inhibit the phospholipase, cytotoxic and myotoxic activities of CTX isolated from C. durissus terrificus venom.In analyzing camelid VHH characteristics, along with the need to develop more effective antivenoms, this study aimed to select and characterize L. glama immunization schedule, using complete and incomplete Freund’s adjuvant, proved to be satisfactory. An antiserum maximum titer (1 × 106) was obtained after the second immunization, as observed by ELISA assays , 76 (34%) and 2 (2%) VHHs showed absorbance values higher than the stipulated cut-off point (2 mean OD from negative samples plus two standard deviations), considered positive for CTX, CB, and CA, respectively (The A assays A. Followectively B.E. coli strain BL21 (DE3), the clones were purified by IMAC. Approximately 2 to 6 mg of recombinant protein per liter of culture were obtained.Ten clones, which showed the best absorbances in ELISA, were sequenced and a multiple sequence alignment between them revealed four sequence profiles for CB and one profile for CA. These sequences were deposited in the GenBank database under the following accession numbers: KF498602 (clone Ctx-17), KF498603 (clone Ctx-21), KF498604 (clone Cb-16), KF498605 (clone Cb-42) and KF498606 (clone Ca-12). All clones presented the established VHH hallmark amino acid in FR2 , with the exception of KF498606. Besides that, the KF498606 showed a shorter CDR3 region with only five amino acid residues (FRGGVW) C. With 9C. durissus terrificus venom, purified KF498602, KF498603, KF498604, and KF498605 clones were able to interact with C. durissus cascavella and C. durissus collilineatus venoms. However, when tested against toxins and venoms from the Bothrops genus, they showed no reactivity in ELISA, demonstrating their specificity for the Crotalus genus , were determined  ratio, whereas KF498605 demonstrated inhibition of above 60% at a ratio of 1:20 B. The KFp < 0.05), when compared to the control carried out with CB alone (p < 0.05) CK level reduction . The results demonstrated a significant reduction in released LDH levels  were revealed through a BLAST search. The structures selected for modeling are shown in Antivenoms able to neutralize toxins in the blood and deep tissues and to present minimal adverse effects associated with the administration of non-human immunoglobulins are challenges the scientific community has faced over the years. Thus, different approaches have been proposed for innovative and cost-effective technologies to develop a new generation of antivenoms, given the global antivenom crisis ,17,18,39C. durissus terrificus snake venom, as well as the biotechnological versatility and therapeutic potential of camelid single domain antibodies, we selected anti-crotoxin VHHs following construction of an immunized L. glama VHH phage library.Considering these challenges, crotalic envenoming’s epidemiology in Brazil, the relevance of CTX in envenoming by L. glama immunization, performed following “the low-dose, low-volume, multi-site immunization protocol” [6), comparable with other immunization schedules [12 clones, the diversity of the immune VHH library was to be expected upon exploring antibody specificity and affinity developed naturally after immunization.rotocol” , proved chedules ,42. WithThree rounds of biopanning, performed in order to select high affinity clones , were enCrotalus, thus demonstrating specificity, an important feature for snakebite diagnosis or serumtherapy [C. durissus terrificus venom [C. durissus cascavella, C. durissus collilineatus and C. durissus cumanensis venoms is about 72.0%, 67.4% and 2.6%, respectively [The four clones responsive to CB presented cross-reactivity only with snake venom from the genus mtherapy ,51. Seleus venom . Moreoveectively . This vaectively , confirm1 [In regards to thermal stability, the two analyzed VHH profiles demonstrated high resistance to heat-induced denaturation. Their stability against temperature changes, especially between 25 and 55 °C, is an important advantage for therapeutic and diagnostic applications, mainly in tropical and subtropical countries. It has been observed that VHH melting temperature values vary between 60 and 80 °C ,54,55, j1  are rend1 . Perform1 ,42. SPR 1  and antiKF498604 and KF498605 demonstrated significant inhibition of CTX and CB phospholipase activity in vitro. It has been observed that polyclonal and monoclonal antibodies, capable of inhibiting CB phospholipase activity in vitro, are potential inhibitors of CTX’s myotoxic and neurotoxic activity in vivo. This demonstrates the participation of CB in CTX’s toxic effects ,62. Becap < 0.05) by the VHH KF498604 by up to 78% at the highest ratio (1:40 w/w), demonstrating this VHH’s potential protective effect in vivo. It was demonstrated that the myotoxic activity of the CB is related to its phospholipase activity, which is reduced by chemically modifying His48, an important residue of the catalytic site along with Asp99 [CK release, related to the myotoxic effect of CTX in mice, was significantly inhibited , which do not block substrate access [N-terminal region, exposed to the solvent, may constitute an important pharmacological site of CTX. This region may allow CTX presynaptic acceptor binding in neuromuscular junctions, with subsequent dissociation of the CA subunit, enabling interaction of CB’s C-terminal with the target, and liberation of CB’s catalytic site, consequently triggering its neurotoxicity. Thus, although they do not inhibit phospholipase activity, these clones may be useful in studies on the neurotoxic mechanisms of CTX and neutralization assays.Molecular docking suggested that KF498604 and KF498605 VHHs interact with the CA–CB interface of CTX, and, despite no contact with the catalytic site , the clones sterically block the substrate’s access F. Most ce access . As was e access , CB’s N-Given the characteristics of the selected VHHs, as well as the involvement of systemic myotoxicity in Acute Kidney Injury (AKI), one of the main causes of death among victims of crotalic envenomation ,69,70,71http://www.mct.gov.br/upd_blob/0228/228451.pdf). The licenses related to access to Brazilian genetic resources for scientific purposes are: Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis–IBAMA, Instituto Chico Mendes de Conservação da Biodiversidade–ICMBio (Number: 27131-1) and Conselho de Gestão do Patrimônio Genético–CGEN/Brazil (Number 010627/2011-1).Procedures involving animals were performed in accordance with the recommendations of the National Council for the Control of Animal Experimentation (CONCEA), and were approved by the Ethics Commission on Animal Use (CEUA) of Fiocruz Rondônia under protocol numbers 2012/11 and 2014/11. The animals’ health status was checked routinely by a veterinarian. All animal groups (mice) were monitored regularly  until the end of the experimental period and there was no unintended death of animals. After topical anesthesia  for retro-orbital bleeding, the mice were euthanized by cervical dislocation, according to CONCEA Normative Resolution No. 13, from 20 September 2013 , CB and CA subunits (50 µg and 100 µg each), plus complete or incomplete Freund’s adjuvant   .L. glama’s immune response was monitored by ELISA immunoassay. Microtiter plate wells  were coated with 1 µg of CTX, CB or CA diluted in PBS  and incubated overnight at 4 °C. After being washed three times with PBST (0.05% Tween-20 in PBS), unspecific sites were blocked with PBSM (1% skimmed milk in PBS) for 24 h. Then, serial dilutions  of the serum in PBSM, collected each week, were added to the wells and the plates were incubated for 24 h at 4 °C. The samples were washed with PBST, and rabbit anti-llama IgG2/IgG3 [gG2/IgG3  at a 1:17 cells using Trizol Reagent , and cDNA synthesis was carried out with the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). A VHH gene repertoire, as well as an immune library, and recombinant phages expressing VHHs fused to VCSM13 helper phage protein III were obtained according to [Three days following the final boost, fifty milliliters of animal blood were collected to isolate lymphocytes using Ficoll-Paque Plus . Total RNA was extracted from 1.5 × 10rding to ,42.E. coli TG1 (A600 0.5) cultures and the samples were incubated for 1 h with no agitation and for 1 h with agitation at 37 °C. Following centrifugation at 4000× g, the supernatants were discarded, the pellets resuspended in 2YT, plated on 2YT/amp/glu, and incubated overnight at 30 °C. Individual clones were collected for two subsequent rounds of biopanning. Selected clones were analyzed by colony PCR and by ELISA in order to verify the presence and reactivity of VHHs against selected targets, respectively.Selection of anti-crotoxin, anti-CB and anti-CA VHHs was performed by biopanning. To this, MaxiSorb immunotubes (Sigma-Aldrich) were adsorbed separately with 1 mg of CTX, CB, and CA in PBS and incubated for 24 h at 4 °C. Excess CTX, CB and CA were removed by washing with PBST and the samples were blocked in PBSM for 2 h. After being washed three times with PBST, recombinant VHH-phages, previously incubated in PBSM, were added to the immunotubes and the samples were incubated at 37 °C under homogenization for 30 min. Subsequently, samples were washed 15 times with PBST and 15 times with PBS. Bound VHH-phage particles were eluted with 100 mM HCl and neutralized with 1 M Tris-HCl, pH 7.5. The eluants were transferred separately to 10 mL of E. coli TG1 (A600 0.5) cultures and the samples were incubated for 1 h with no agitation and for 1 h with agitation at 37 °C. Following centrifugation at 4000× g, the supernatants were discarded, the pellets resuspended in 2YT, plated on 2YT/amp/glu, and incubated overnight at 30 °C. Individual clones were collected for two subsequent rounds of biopanning. Selected clones were analyzed by colony PCR and by ELISA in order to verify the presence and reactivity of VHHs against selected targets, respectively. Clones were cultivated in 1 mL (2YT/amp), and VHH expression was induced with 3 mM isopropyl-d-thiogalactopyranoside (IPTG) (A600 0.9) for 16 h at 30 °C. After centrifugation, 50 µL of supernatants containing soluble VHHs were used to carry out the assay, as described previously. Llama pre- and immune sera were used as negative and positive controls, respectively. Positive clones were sequenced, analyzed and submitted to GenBank.To perform the ELISA, positive mL of CATATGGCCGA(G/C)GT(G/C)-′3) and VHXHOR (5′-CCGCTCGAGTGAGGAGACGG-′3). The restriction endonuclease recognition sites for NdeI and XhoI are underlined.Four positive clones were subcloned into a pET-22b+ vector . Thus, selected recombinant VHHs, cloned into pHEN-1-6xHis plasmid, were amplified by PCR using the following primers: VHNDEF  tag sequence. After transformation into E. coli BL21 (DE3) , the clones were grown in 30 mL of LB medium containing 100 μg/mL of ampicillin (LB amp), under agitation at 37 °C, overnight. Ten milliliters of these cultures were transferred to 1 L of LB amp and when its absorbance (A600) reached about 0.7, protein expression was induced with 1 mM IPTG. Cultures were incubated under agitation at 37 °C for 8 h. Following centrifugation, pellets were resuspended in 20 mL of 50 mM Tris-HCl (pH 8.0). In order to perform bacterial cell disruption, the samples were pretreated with 1 mg/mL of lysozyme at room temperature for 1 h and sonicated for 3.5 min, with 1 min pulses . Inclusion bodies were retrieved after centrifugation at 15,000× g for 15 min, resuspended in wash buffer  and incubated on ice for 10 min. This step was repeated once more. Triton X-100 was removed with Tris-HCl buffer. The samples were centrifuged and resuspended in dissolution buffer , and then incubated at 4 °C for 1 h and centrifuged. VHHs were purified through immobilized metal affinity chromatography (IMAC) using Talon Co2+-precharged resin , under denaturing conditions, according to the manufacturer’s instructions. Renaturation of VHHs was performed by diafiltration in PBS, using 10 kDa Amicon Ultra-2 filter devices . Soluble VHH protein concentration was determined by the Smith method (1985) [VHH fragments, composed of approximately 400 bp, were excised from agarose gel, digested with d (1985)  with a BBothrops and Crotalus genera. Microtiter plate wells were coated with 1 µg of snake venoms obtained from Serpentário de Proteínas Bioativas, Batatais, Brazil , along with 1 µg of eight different toxins . The plates were incubated at 4 °C overnight, washed with PBST and nonspecific sites were blocked with PBSM for 5 h. Subsequently, one microgram of the purified VHHs (pre-incubated in PBSM) was added and the assay was carried out as described previously. Llama immune serum (1:1000) was used as the positive control and an anti-BthTX VHH (KC329718) [In order to verify the specificity of the purified VHHs , cross-immunoreactivity assays were performed by ELISA with different venoms and toxins from the C329718)   at 4 °C overnight. After being washed three times with TBS/0.1% Tween 20 (TBST), the strips were incubated with 0.2 mg/mL of each anti-crotoxin VHH  overnight. The strips were washed again with TBST and incubated with rabbit anti-llama IgG2/IgG3 (1:1000 in 5% TBSM) overnight. After being washed, the samples were incubated with peroxidase conjugated mouse anti-rabbit IgG (1:3000 in 5% TBSM) for 6 h. The strips were washed, and the reactive signals were detected after incubation with hydrogen peroxide in diaminobenzidine (DAB) solution . Llama immune serum (1:1000) was used as the positive control and an anti-BthTX VHH (KC329718-0.2 mg/mL) , as the g, at room temperature for 5 min, and the supernatant concentrations were determined using the Smith method (1985) [Thermal stability studies were performed in order to verify the binding capacity of selected anti-crotoxin VHHs in ELISA assays, after their exposure to different temperatures, according to Tabares-da Rosa et al. , with mod (1985) . Solublev/v) mixture of 0.4 M EDC (1-Ethyl-3-3-dimethylaminopropyl carbodiimide) and 0.1 M NHS (N-hidroxysuccinimide) at a flow rate of 5 µL/min. CTX was diluted in C2H3NaO2 buffer  and injected into a selected flow cell for immobilization. A solution of 1 M H2NCH2CH2OH-HCl was injected in order to block the remaining reactive groups in the flow cell. The control flow cell was prepared with H2NCH2CH2OH–HCl alone. For kinetic measurements, CM5-CTX was subjected to serial dilutions of VHHs (2.5 to 0.002 µM) into the running buffer HBS-p . The kinetic assay was performed at a flow rate of 30 µL/min at 25 °C. The interaction analysis between KF498602 and KF4998604 clones and CTX was performed by SPR using a Biacore T200 system , according to , with mo2H2O4, H3PO4, CH2O2, and C3H4O4, each at 0.15 M, pH 5.0; C-20 mM EDTA; I-KSCN (0.46 M), MgCl2 (1.83 M), urea (0.92 M), guanidine-HCl (1.83 M); W-deionized water) according to Andersson; Hämäläinen and Malmqvist [In order to verify whether or not the CTX interaction epitope is common for both clones, an additional assay was carried out. After saturation of KF498604 (2.5 μM)-CM5-CTX interaction, the sensor chip was washed with HBS-p running buffer, and KF498202 (1:1 VHH/VHH molar ratio) was injected. Following sensogram obtention, chip regeneration was performed with an AIW solution followed by an ICW solution for 30 s each  was reconstituted in chloroform, dried over a low flow of N2, and dissolved in 150 mM NaCl to obtain a 1 mg/mL stock solution. Assays were performed in 150 µL in opaque plates, at wavelengths of 460 nm (excitation) and 534 nm (emission), 37 °C and at intervals of 3 s between each read, over 5 min, using Fluorescence Spectrometer Spectra Max M4  and SoftMax 6.0 Software . The standard reaction medium contained 20 mM Tris–HCl (pH 7.5), 8 mM CaCl2 and 5 µM of phospholipids. In order to verify the VHHs’ ability to inhibit phospholipase activities, 0.08 µg and 0.04 µg of CTX and CB, respectively, were pre-incubated with the VHHs KF498202 and KF498604  for 30 min at 37 °C. The standard reactions with CTX or CB alone were used as positive controls. These reactions produced 186 and 115 fluorescence units, respectively, corresponding to 100% PLA2 activity. The negative control was performed with the standard reaction medium alone.Inhibition of CTX and CB phospholipase activities on fluorescent lipids were performed as described by . The flu2 bottles containing 5 mL of Dulbecco’s Modified Eagle’s Medium , supplemented with 10% fetal bovine serum , HEPES 2 g/L, HCO3− 3.7 g/L, gentamicin (50 µg/mL) and incubated in a 5% CO2 atmosphere at 37 °C. After reaching full confluence, non-adherent cells were removed by washing with DMEM and 2 mL of 500 U/mL trypsin-EDTA solution (Sigma) was added. After incubation at 37 °C for 5 min, 5 mL of supplemented DMEM medium was added and the flasks were centrifuged at 300× g, 24 °C for 10 min. Cell pellets were resuspended in 1 mL of DMEM. An aliquot was diluted by 1:100 (v/v) in 0.4% trypan blue solution (Sigma) in order to count viable cells under an optical microscope (40×) using a Neubauer chamber. Cells were transferred to Costar®-type 96-well microplates (Sigma) at an initial density of 2.0 × 104 cells/well and incubated under the same conditions until reaching 100% confluency. The supernatants were removed and 200 µL of DMEM supplemented with 1% FBS and gentamicin (50 µg/mL) was added for cell differentiation on myotubes [w/w) in PBS. Then, 150 µL of the solution was transferred to wells containing the myotube culture in 50 μL of DMEM supplemented with 1% FBS. After incubation at 37 °C for 3 h, in a 5% CO2 atmosphere, supernatants were collected to determine LDH activity using diagnostic kits . The results were expressed as LDH U/L. 0.1% Triton X-100 and 20 µg/well bothropstoxin II were used as positive controls and PBS as the negative control. All assays were performed in triplicate.Inhibition of CB cytotoxicity by anti-crotoxin VHHs was evaluated by determining the lactic dehydrogenase (LDH) release from damaged murine myoblasts cells, differentiated in myotubes, as was previously described ,75. C2C1myotubes . Four tow/w) in PBS. Then, groups containing 5 male mice (28–30 g) received an i.m. injection of 50 µL of toxin/VHH solutions in the gastrocnemius muscle. Positive control animals were injected with CTX and negative controls received 50 µL of PBS or VHH. After 3 h, blood was collected from the orbital plexus in order to determine the creatine kinase activity using diagnostic kits . Results were expressed as CK U/L.The neutralizing effect on myotoxicity induced by CTX in mice was analyzed by determining the serum creatine kinase (CK) activity. Thus, CTX at its previously determined minimum myotoxic dose (MMD) (1.5 µg)  was incu2CBb (PDB accession code: 3R0L). The antibody mode was selected with the non-CDR regions masked automatically [In order to investigate amino acid residues that participate in the antigen–antibody recognition, we performed homology modeling of five selected VHHs, following the methodology described by . After matically . The VHHatically .


Aspergillus and Trichoderma, have also been used for textile effluents treatment. The main aim of the present study was to requalify a Brazilian Trichoderma culture collection of 51 Trichoderma strains, isolated from different sources in Brazil and preserved in the oldest Latin-American Fungal Service Culture Collection, The Micoteca URM WDCM 804 . Fungal isolates were re-identified through a polyphasic approach including macro- and micro-morphology and molecular biology, and screened for their capability to decolourise real effluents collected directly from storage tanks of a textile manufacture. Trichoderma atroviride URM 4950 presented the best performance on the dye decolourisation in real textile effluent and can be considered in a scale-up process at industrial level. Overall, the potential of Trichoderma strains in decolourising real textile dye present in textile effluent and the production of the oxidative enzymes Lac, LiP and MnP was demonstrated. Fungal strains are available in the collection e-catalogue to be further explored from the biotechnological point of view.Water contamination with large amounts of industrial textile coloured effluents is an environmental concern. For the treatment of textile effluents, white-rot fungi have received extensive attention due to their powerful capability to produce oxidative  enzymes. In addition, other groups of fungi, such as species of  Textile industries waste large water quantities that in some cases return to environment as untreated or incorrectly treated effluents . Due to There are mainly three types of treatment for textile effluents: physical, chemical and biological. In recent years, relevant methods based on biological textile effluents degradation have been developed using bacteria and fungi in aerobic and anaerobic processes ,7,8,9,10Aspergillus and Trichoderma) [For the treatment of textile effluents, white-rot fungi have been extensive assessed due to their powerful capability to produce oxidative, including ligninolytic , lignin peroxidase (LiP) and manganese peroxidase (MnP)) enzymes . Other ghoderma) ,11. In ahoderma) ,5,6,14.Trichoderma is a very genetically diverse genus with interesting features, which make some of its species of agricultural and industrial interest. Species of Trichoderma are widely used in the production of enzymes for both pulp treatment and food production [oduction . In addioduction ,16,17,18Trichoderma an important fungal taxonomic group for biotechnological application. As a matter of consequence, for microbial culture collections that have Trichoderma in their catalogues it is important to have these assets identified and characterised according to the current state-of-the-art. Considering this, the main aim of the present study was to requalify a Brazilian Trichoderma culture collection of 51 Trichoderma strains, isolated from different sources in Brazil and preserved in the oldest Latin-American Fungal Service Culture Collection, The Micoteca URM WDCM 804 . The requalification work was based on the fungal identification through a polyphasic approach including macro- and micro-morphology, molecular biology and screening their capability to decolourise real effluents collected directly from storage tanks of a textile manufacture.All these features make Trichoderma strains previously isolated from different substrates of geographic regions of Brazil (see www.ufpe.br/micoteca). The Micoteca URM is a culture collection registered at the World Federation for Culture Collections (WFCC) under the number WDCM 604 and certified in the ISO 9001:2015 standard under the Certificate Number BR018207-1. Fungal strains were obtained from preserved form under mineral oil, revived according to the instructions issued by the Micoteca URM culture collection, and grown and maintained in Potato Dextrose Agar plates .Fifty-one azil see  were obtTrichoderma strains, macro- and micro-morphological analyses were performed  at 35 °C, in the dark without humidity control and under aerobic conditions.In order to confirm the taxonomical identification of the 51 rmed see . For macFor micro-morphological analysis, strains were inoculated on Cornmeal Agar Medium supplemented with dextrose plates . Fungi were incubated at 25 °C for 3 days in the same conditions described above. Afterwards, 6 mm diameter plugs of each CMD fungal culture were inoculated on PDA plates and grown for 4 days in the same conditions but with a regime of 12:12 h light:dark.Trichoderma [Conidia and phialides, presence of chlamydospores and sterile hyphae were observed using 90% lactic acid slides on a light microscopy. Final morphological identification followed the taxonomic keys and guides available for choderma ,20.2PO4; 6 g (NH4)2SO4, 1 g MgSO4·7H2O, 5 mg FeSO4·7H2O, 1.6 mg MnSO4·H2O, 1.4 mg ZnSO4·7H2O and, 2 mg CaC12·2H2O) at 28 °C under static condition for 96 h. Mycelia were filtered and collected for extraction of genomic DNA according to the method previously described elsewhere [tef1) were performed in 50 µL mixture containing: buffer Taq DNA polymerase 1×; 1.5 mM of MgCl2; 0.4 μM each primer  [For the molecular identification, the fungal strains were cultivated in Minimum Liquid Medium  ,23,24; 0TM under UV light and then photographed and purified using Fermentas® kit . The amplified DNA fragments were sequenced in the Sequencing Platform of the Central Laboratory of the Centre of Biological Sciences, Federal University of Pernambuco, Recife, Brazil.The thermal cycling parameters consisted of initial denaturation at 94 °C for 2 min followed by 30 cycles of denaturation at 94 °C for 30 s, primer annealing at 55 °C for 30 s, primer extension at 72 °C for 1 min and a final extension for 10 min at 72 °C ,21. Ampltef1 gene regions were aligned and edited together with the retrieved from the database using the software MEGA 5.0. The phylogenetic tree was constructed using the Neighbour Joining method and the Maximum Parsimony (MP), with 1000 bootstrap resampling using the software MEGA 5.0 [Electropherograms were analysed and edited in the software Staden Package 2.0. Sequences obtained were used to search the most similar sequences deposited in the GenBank by using the Basic Local Alignment Search Tool (BLAST). The experimental sequences of ITS1-5.8S-ITS2 and MEGA 5.0 .Samples of textile effluent were collected directly from storage tanks in a textile manufacture at the municipality of Toritama, State of Pernambuco, Brazil. The effluent samples were kept at 4 °C and transferred to the Laboratory of Environmental and Quality Engineering  to perform the physical-chemical analyses and decolourisation assay.The physical-chemical analyses involved the Chemical Oxygen Demand (COD) by colorimetric method, Biochemical Oxygen Demand (BOD) by azide modification of a Winkler titration method, Colour , Turbidity , pH using the potentiometric measurement principle, and Sedimentable Solids (SD) by gravimetric through a 0.45 μm filter membrane, using the standard methods for the examination of water and wastewater .Trichoderma strains was assessed by using 6 mm diameter plugs of fungal cultures previously grown at 30 °C for 7 days on PDA plates. Plugs were transferred to 125 mL Erlenmeyer flasks containing 30 mL of Malt Broth  supplemented with 1.9 × 10−2 mM Indigo Carmine dye .The screening of textile dye decolourisation by 650 nm = 3.1 × 103 M−1·cm−1). The Indigo Carmine was used as standard and MB free of inoculum containing dye was used as abiotic control. The decolourisation percentage of Indigo Carmine dye was calculated according to Miranda et al. [Fungal cultures were incubated in the dark at 28 °C for 8 days. Fungal biomass was then removed by filtration through a 0.45 µm Millipore membrane and absorbance of filtrates was assessed in a Hitachi U-5100 spectrophotometer at 650 nm which is the maximum absorption of Indigo Carmine  wheat bran, and (3) textile effluent supplemented with 0.05% (w/v) extract yeast. Cultures were incubated under static conditions, in the dark at 28 °C for 8 days. Afterwards, 2 mL aliquots were transferred into tubes and centrifuged at 1035 g for 15 min at 4 °C. Supernatants were analysed at 650 nm in a Hitachi U-5100 spectrophotometer. Previous to evaluate the decolourisation of the dye present on the real textile effluent, this was sterilised by autoclave at 121 °C during 15 min. Three different experiments were performed. In each case, 3 plugs of 6 mm diameter of fungal cultures grown for 7 days at 30 °C on PDA plates were added to 125 mL Erlenmeyer flasks containing 30 mL of: (1) textile effluent; (2) textile effluent supplemented with 0.5% . Fungal incubation occurred under static condition in the dark, at 28 °C for 8 days. The biomass was then separated by filtration through a 0.45 µm Millipore membrane. In order to determine the activity of each enzyme, absorbance of filtrates was assessed in a spectrophotometer Hitachi U-5100 and one unit (U) of enzyme was defined as the release of 1.0 µmol product formed per min under the assay conditions.In order to determine the activity of Lac, LiP and MnP, 6 mm diameter plugs of fungal cultures previously grown for 7 days at 30 °C on PDA plates were transferred into 125 mL Erlenmeyer flasks containing 50 mL of Basal Culture Medium , 0.1 mL sodium acetate buffer  and 0.1 mL enzymatic extract was used [420 nm = 3.6 × 104 M−1·cm−1. LiP activity was assessed through the oxidation of veratryl alcohol. For this reaction, a mixture containing 1 mL sodium tartrate buffer solution , 500 µL veratryl alcohol (10 mM), 500 µL hydrogen peroxide (2 mM) and 500 µL enzymatic extract was used. The reaction was started by adding hydrogen peroxide to the mixture and the production of veratraldehyde was determined at 310 nm [310 nm = 9.3 × 103 M−1·cm−1.Lac activity was measured from the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). For this reaction, a mixture containing 0.8 mL ABTS (0.03% t 310 nm  with ε31w/v), 50 µL sulphate manganese (2 mM), 50 µL hydrogen peroxide (2 mM) and 1.0 mL sodium succinate buffer . The reactions were performed at 30 °C for 5 minutes and were stopped by adding 2 N NaOH (40 µL). MnP activity were determined at 610 nm with ε610 nm = 2.2 × 103 M−1·cm−1.MnP activity was assessed by using the methodology of phenol red oxidation measurement as previously described elsewhere . BrieflyOverall, the enzymatic results were submitted to analysis of variance and means compared by Tukey test at 5% probability in the ASSISTAT software .Trichoderma species, such as: conidia shape, ornamentation, colour, length and width, and phialide length and base width, and presence of sterile hyphae and chlamydospore, and colony diameter on PDA at 30, 35 and 40 °C and SNA at 35 °C. The analysis was made using the hierarchical clustering with the complete linkage method and was performed with the statistical package jmp 8.0.2 for Macintosh .A cluster analysis was applied and a dendrogram of phenotype relatedness was constructed. The analysis was performed using the phenotype traits that showed at least high association with each Trichoderma were evaluated. Data obtained from the morphological analysis  are normally very good data for species identification when combined with other traits [Fifty-one fungal strains obtained from the Micoteca URM culture collection and belonging to the genus analysis  show a cr traits ,32.Trichoderma is not great. However, related species can often be differentiated by slight but consistent differences in size. The conidial surface appears smooth in most species in light microscope observations, although many species with apparently smooth-appearing conidia are delicately ornamented when examined by SEM. In most species, terminal phialides tend to be more elongate and narrower, and frequently more or less subulate. Chlamydospores are common in many species, although they tend to be uniformly globose or ellipsoidal, terminal and intercalary, smooth-walled, colourless, yellowish or greenish, and 6–15 um diameter in most species. Vegetative hyphae show few characters useful for identification.It is well known that the overall range of variation in conidial dimensions in tef1 gene regions, respectively.This means that the identification based on the classical morphology techniques can be used as presumptive approach. However, to obtain a sound and reliable identification, molecular biology must be performed . ConsequTrichoderma species were unclear , Hamatum ; Longibrachiatum (Trichoderma longibrachiatum and T. ghanense); Lone lineages , Virens (Hypocrea virens/Trichoderma virens), and Harzianum (Hypocrea lixii/Trichoderma harzianum).Through the phylogenetic analysis based on ITS region, inaccurate clades were observed and the groupings observed for some  unclear . For insTrichoderma species, as shown in the Trichoderma asperelloides (8 strains), T. asperellum (4), T. atroviride (6), T. erinaceum (3), T. ghanense (1), T. hamatum (4), T. harzianum (13), T. koningiopsis (4), T. longibrachiatum (1), T. spirale (1) and T. virens (6) (T. hamatum (URM 3492), T. harzianum  and T. virens .Based on the former identification available in the records of the Micoteca URM culture collection, the 51 fungal isolates were distributed into six rens (6) . OverallTrichoderma strains assessed for decolourisation of Indigo Carmine in MB, 18 of them presented over 70% decolourisation, which corresponds to 0.57 × 10−2 mM or less residual dye , followed by T. atroviride URM 4950 and URM 3735, 680.0 U·L−1 and 617.3 U·L−1, respectively.In a previous study performed by Adnan et al. , T. atroT. virens URM 4996 presented a Lac activity of 15.7 U·L−1 which is numerically greater than those observed for the other species assessed  was assessed. After eight days of the experiment, Lac activities assessed were 1.3, 2.0, 8.3 and 2.3 U·L−1, respectively  (The six above mentioned  mg·L−1) .w/v) wheat bran; and (3) textile effluent supplemented with 0.05% (w/v) extract yeast, for each strain, and after statistical analysis of averages, a significant difference between the means was observed . In addition, T. asperellum URM 4926 (84.6%), T. erinaceum URM 3881(66%) and T. harzianum URM 2842 (60.6%) presented a good performance of decolourisation when textile effluent supplemented with extract yeast was used.Based on the data obtained for: (1) textile effluent; (2) textile effluent supplemented with 0.5% (observed . AccordiT. atroviride URM 3735 (43.0%), T. erinaceum URM 3881 (35.6%) and T. virens URM 4996 (23.8%)  .T. harzianum TSK8 (JQ809340) was observed when this strain was grown in supplemented medium with 5.8 mg·L−1 yeast extract. In addition, Adnan et al. [T. atroviride. Authors observed that yeast extract was a good source of nitrogen for growth T. atroviride and to stimulate it to decolourise Reactive Black 5 up to 91.1%. Furthermore, authors evaluated dye decolourisation by using ammonium nitrate and ammonium chloride and, in contrast, observed only a decolourisation rate of 29.1% and 71.7%, respectively.According to Saravanakumar and Kathiresan , a largen et al.  evaluateTrichoderma asset preserved at the Micoteca URM culture collection was re-identified and made available in the culture collection e-catalogue to be further explored from the biotechnologically point of view. In the specific case of dye decolourisation screening using real textile effluent, the strain T. atroviride URM 4950 gave the best results and can be considered in a scale-up process at industrial level. This strain is herein indicated to be used as a major target for future studies related with the treatment of industrial textile effluent and bioremediation of environments contaminated with dyes. Overall, the data obtained suggest that the dye decolourisation in real textile effluent is not a one-step process. More than one oxidative enzyme can be involved in this complex process. The contribution of these enzymes for decolourisation of textile effluent differs from fungus to fungus. The potential of Trichoderma strains in decolourising real textile dye present in textile effluent and the production of the oxidative enzymes Lac, LiP and MnP were successfully demonstrated. Finally, in this study, the strains available through Micoteca URM culture collection are identified according the current state-of-the-art and better characterised to underpinning life sciences and biotechnology.The Brazilian


Anterior cervical discectomy and fusion (ACDF) is the classic surgical treatment for symptomatic cervical degenerative disc disease (CDDD). However, there is controversy over the best surgical management in patients with two noncontiguous symptomatic levels of CDDD.From April 2011 to May 2014, 44 patients with two noncontiguous symptomatic levels of CDDD underwent skip-level ACDFs. In Group NoPlate, 23 cases underwent 2 noncontiguous levels of ACDF using zero-profile anchored spacer; and in Group Plate, 21 cases underwent 2 noncontiguous levels of ACDF using cages and plates. Operation-related paraeters for each group were recorded and compared. Japanese Orthopedic Association (JOA) scores and Neck Disability Index (NDI) scores at preoperation and postoperation were compared with at least a 2-year follow-up. Cervical lordosis was analyzed before surgery, 1 month after surgery, 3 months after surgery, and at final follow-up.p < 0.05), and there were no significant difference in terms of JOA, NDI scores, cervical lordosis and fusion rate between the two groups (P > 0.05). The operation time in Group NoPlate was significantly shorter than in Group Plate (p < 0.05), and the incidence of dysphagia and adjacent segment degeneration in Group NoPlate was significantly lower than in Group Plate (p < 0.05).Mean follow-up was 35.4 ± 6.5 (range 24–48) months. Significant improvement on the JOA, NDI scores and cervical lordosis was noted in each group (ROI-C and cages with plate fixation were both effective in two-level noncontiguous ACDF, and there were no significant difference in clinical outcomes, fusion rate, and cervical lordosis. However, ROI-C was associated with shorter operative time, lower incidence of dysphagia and adjacent segment degeneration. Anterior cervical discectomy and fusion (ACDF) was first reported in the 1950s . Many stIn 2-level noncontiguous ACDF, 2 plates are often used to provide the immediate postoperative stability and improve the fusion rate. However, the procedure of implanting plates is associated with the risk of perforation of esophagus and dysphagia. Besides, it may raise the risk of adjacent-level ossification , 11. PreFrom April 2011 to May 2014, a total of 44 patients underwent skip-level ACDFs.Inclusion criteria were (1) symptomatic CDDD which was unresponsive to non-operative management; and (2) spinal cord or root compression at two noncontiguous intervertebral levels confirmed by magnetic resonance imaging (MRI). Exclusion criteria were: (1) significant instability of cervical spine; (2) developmental cervical spinal stenosis; (3) severe cervical deformity; (4) a previous history of cervical spine surgery; and (5) fracture, tumor, ossification of posterior longitudinal ligament and any serious general illness.P > 0.05) (Table The 44 patients were divided into 2 groups: Group NoPlate 23 patients), who underwent fusion using ROI-C  Table . This stOperations were performed in supine position under general anesthesia using the standard right-sided anterior approach. The basic procedures including exposure, discectomy, and decompression were performed as described previously . To achiZero-profile anchored spacers were used in Group NoPlate. In Group Plate, PEEK cages were inserted, and anterior plates were applied. In both groups, each appropriate-sized cage was packed with 0.25 mg of recombinant human bone morphogenetic protein  and excised local osteophytes.Patients were allowed to sit up on the first day postoperatively and walk on the second day postoperatively with a Philadelphia neck collar, which was applied for 4 weeks.Collected data included age, gender, operated segments, intraoperative blood loss, operative time, complications, and clinical and radiologic outcomes. The complete cervical spine X-ray, computed tomography (CT), and magnetic resonance imaging (MRI) were performed preoperatively. After surgery, the cervical spine X-ray were routinely taken. Clinical and radiological evaluations were performed preoperatively (1 day before operation) and postoperatively at 2 weeks, 1 month, 3 months, and the final follow-up. Radiological outcomes were measured using the Picture Archiving and Communication System (PACS) imaging system.The Japanese Orthopaedic Association  scoring system was used to evaluate the neurological status . Neck DiX-ray and CT scan reconstructions were used to evaluate the bony fusion. Satisfactory fusion include the following conditions : (1) no t test or Chi-square test (Fisher’s exact test).The paired data were compared with a paired sample t test. All the analyses were performed using the Statistical Package for the Social Sciences  and the difference was considered statistically significant at the P < 0.05 level.Data are shown as mean ± standard deviation. Intergroup comparisons were made using n = 13; Group Plate, n = 8), and C4–5 and C6–7 . Group NoPlate had a mean blood loss of 70.0 ± 15.8 ml and the average operative time was 126.0 ± 13.2 min. Group Plate had a mean blood loss of 75.4 ± 23.0 ml and the average operative time was 143.4 ± 17.9 min. No significant difference existed in age, gender, operated levels and blood loss between the 2 groups (Table P < 0.05).The operated noncontiguous levels were C3–4 and C5–6 . The mean JOA RR was (62.6 ± 15.1)% in Group NoPlate and (66.8 ± 14.6)% in Group Plate, which was not statistically different (P > 0.05).Patients were followed up from 24 to 48 mean: 35.4 ± 6.5) months. In both groups, the JOA and NDI scores were significantly improved postoperatively . The fusion rate at 3 months postoperatively was 91.3% (21/23) in Group NoPlate and 95.2% (20/21) in Group Plate, which was not significantly different (P > 0.05). All patients achieved solid fusion at final follow-up.The cervical lordosis was significantly corrected postoperatively and the correction was maintained at last follow-up in both groups  on 2 weeks postoperatively, 3 patients had dysphagia (1 moderate and 2 mild) 1 month postoperatively, and no patients had dysphagia 3 months after the operation. In Group Plate, 12 patients complained of dysphagia  on 2 weeks postoperatively, 9 patients had dysphagia (4 moderate and 5 mild) 1 month postoperatively, 5 patients had dyphagia (1 moderate and 4 mild) 3 months postoperatively, and 4 patients still suffered from mild dyphagia at the last follow-up. There were significant differences in the presence of dysphagia between the two groups at all follow-up time points , 3-level ACDF and 2-level noncontiguous ACDF. Compared with ACDF, ACCF has more bleeding, higher incidences of postoperative complications, and less improvements of cervical lordosis. Three-level ACDF sacrifices an additional motion segment, and has been associated with increased rates of pseudarthrosis and poorer clinical outcomes than shorter constructs , 9. FinnThere is controversy over the use of anterior plate in a single-level ACDF. However, it is beneficial to implant anterior plates in a multilevel ACDF , for theDysphagia is reported to be the most common postoperative complication after ACDF . In mostIt is reported that the presence of anterior plates increased the incidence of adjacent segment degeneration . Park etThis study has several limitations. First, it was retrospective. Second, the number of patients was small. Third, the follow-up period was relatively short. Finally, the final follow-up time was not consistent, which caused variation in adjacent segment degeneration. Thus, the results of this study need to be confirmed by large sample, prospective, randomized studies with long-term follow-up.ROI-C and cages with plate fixation were both effective in two-level noncontiguous ACDF, and there were no significant difference in clinical outcomes, fusion rate, and cervical lordosis. However, ROI-C was associated with shorter operative time, lower incidence of dysphagia and adjacent segment degeneration.


Bacteroidetes phylum, has been studied mainly in Flavobacterium johnsoniae and Porphyromonas gingivalis. Among the 18 genes, essential for T9SS function, a group of four, porK-N  or gldK-N (F. johnsoniae) belongs to a co-transcribed operon that expresses the T9SS core membrane complex. The central component of this complex, PorM (or GldM), is anchored in the inner membrane by a trans-membrane helix and interacts through the outer membrane PorK-N complex. There is a complete lack of available atomic structures for any component of T9SS, including the PorKLMN complex. Here we report the crystal structure of the GldM and PorM periplasmic domains. Dimeric GldM and PorM, each contain four domains of ~180-Å length that span most of the periplasmic space. These and previously reported results allow us to propose a model of the T9SS core membrane complex as well as its functional behavior.Type IX secretion system (T9SS), exclusively present in the  No structural data for the bacterial type IX secretion system (T9SS) are available so far. Here, the authors present the crystal structures of the periplasmic domains from two major T9SS components PorM and GldM, which span most of the periplasmic space, and propose a putative model of the T9SS core membrane complex. In diderm bacteria, protein secretion occurs either as a one-step process, in which substrates are translocated directly from the cytoplasm to the external milieu, or as a two-step process, in which the substrates first cross the inner membrane (IM) into the periplasm using the Sec, Tat, or holins pathways and then cross the outer membrane (OM) through a specialized translocon2. After secretion, the substrates might stay attached to the OM surface, be released into the extracellular milieu, or be injected into a target cell2. The type IX secretion system6 (T9SS) uses a two-step process. Depending on the bacterial strain, the T9SS confers very distinct functions. In F. johnsoniae, the T9SS contributes to gliding motility by secreting SprB, a cell-surface adhesin that is required for movement on solid surfaces7. P. gingivalis, a non-motile bacterium, is a human oral pathogen and a major causative agent of periodontitis, as its T9SS secretes potent proteolytic enzymes called gingipains8 that degrade host cell tissues and interfere with innate host defense mechanisms9. To date, 18 genes have been identified as essential for T9SS function in P. gingivalis10. Among them are a group of five genes, porP–porK–porL–porM–porN, which belong to a co-transcribed operon3. The last four genes have orthologues in the F. johnsoniae genome, gldK–gldL–gldM–gldN, together with other extra orthologues of porM11. PorK, PorL, PorM, and PorN, assemble as a >1.4 MDa trans-membrane complex1. PorK (or GldK) is a lipoprotein anchored to the OM that interacts with the periplasmic protein PorN12. PorL and PorM (GldL and GldM) are IM proteins that interact via their trans-membrane segments. The core of PorL resides in the cytoplasm, whereas PorM, similar to GldM, has a long periplasmic domain3. PorM interacts with both PorK and PorN complex, and therefore spans the entire periplasm by being anchored in the IM and interacting with the OM complex3. PorM (or GldM) is therefore a central structural component of the T9SS and an interesting target for structure and function studies. Here, we present the atomic structures of the periplasmic domains of both PorM and GldM, that exhibit 22% amino-acid identity, and provide information regarding the contribution of each domain for interaction with PorK and PorN.Bacteria, especially Gram-negative species, have assembled and evolved complex and specific cellular machines, known as secretion systems, to secrete proteins or DNA through the cell envelope into the surrounding medium or inside other cellsThe GldM (accession number GI: 58531935) and PorM periplasmic domains (GI: 188595218)  were cloned from residues 36–513 and 36–516, respectively. GldMp crystallized readily, and its structure was solved using the Se edge of a SeMet derivative for phasing  is formed by helices 1–5 in an up and down fold. The D1 domain dimers are packed together through helices α1. The D2 (233–320) and D3 (321–405) domains are exclusively formed of β-strands. Each D2 domain swaps its β-strands 1 and 2 with the other D2 domain, whereas D3 domains swap β-strand 7. The main plane of domain D3 is perpendicular to that of domain D2  was purified and crystallized16  from the resulting library17 that bound to PorMp with high affinity (KD = 4.5 nM)16. We cloned a PorMp fragment between residues 224–516, co-crystallized it with nb130, and determined the structure of the complex . The D4 domain was easily traced in the electron density map because it was stabilized by nb130 binding, and it was introduced into the PorMp224 structure, generating a complete model. The resulting PorMp224 structure exhibits three domains that resemble GldMp domains D2–D4  , which was probably cleaved during crystallization  experiments. We found that the D2–D3 construct oligomerizes, whereas the D1 and D4 isolated domains do not interact with themselves Fig. . On the The most striking difference observed between GldMp and PorMp is their overall topology, which results from the PorMp kink between D2 and D3  is necessary to properly interact with PorK 2 complex1 and from Vincent et al.3, together with the structures reported here, we speculated on the possible architecture of the T9SS core machinery. We used Symmdock software18 to identify which part of PorM/GldM might fit together to form a multimer of dimers. Using the straight GldMp structure, both threefold and fourfold symmetry created associations involving mainly the D1 domain, and the rest of the structures exhibited a topology resembling that of tulip petals , an event that initiates T6SS core complex assembly at the OM15. In turn, PorN binds to the PorK lipoprotein; therefore, the T9SS PorK–PorN complex might represent a functional equivalent of the T6SS TssJ lipoprotein, with equivalent binding affinities for PorM or TssM. In the T6SS core complex, TssJ also binds the C-terminal D4 domain of TssM15.It has been reported by us1 isolated a core membrane complex of T9SS from P. gingivalis that was extracted using DDM. Analysis of this complex by western blot and SDS-PAGE revealed that it contains four components, PorK, PorL, PorM, and PorN, and that its mass is slightly larger than 1.4 MDa1. We recently reported that PorM, PorN, and PorK form homodimers, whereas PorL forms homotrimers3. Hence, the stoichiometry of the assembly is expected to be PorL3/PorM2/PorN2–PorK2, resulting in an overall mass of ~410 kDa. Therefore, three or four copies of the above-described assembly would be necessary to form the ~1.4 MDa isolated by Sato et al.1Sato et al.12 reported data that differ from those reported above. In their report, a P. gingivalis membrane fraction was purified leading to large rings that were analyzed with electron microscopy. These rings, which were attached to the membrane, measure ~50 nm in diameter  and are formed of 32–36 1:1 PorK:PorN complexes. They did not observe the presence of PorM or PorL. These authors proposed that the native complex therefore contains 32–36 copies. The same rings were observed on the surface membrane of P. gingivalis mutants lacking porL, porM, and porP. Strangely enough, despite the strong interaction measured between PorM and PorN, PorM was not observed in the complex. Furthermore, a pore of 350 Å would be very difficult to occlude during non-secretion periods. We therefore suspect that the gigantic pore reported in Gorasia et al. might be due to the absence of PorM/PorL in the preparations, resulting either from purification or from the use of cells encoding a porM− mutation. As often observed with protein-forming rings , ring stoichiometry might vary in the absence of controlling elements.In contrast, Gorasia et al.3/PorM2/PorN2–PorK2 moieties , and PorMpNt (residues 44–217) were cloned into the pET28a+ derivative vector pLIC03  and was cloned into pLIC03 with the same protocol as for PorM constructs , PorMp6–516, Po17. In brief, a llama (Lama glama) was immunized with purified PorMp (Ardèche-lamas France). PorMp was injected subcutaneously four times at 1-week intervals using incomplete Freund’s adjuvant, followed by a fifth injection 2 weeks later. Blood samples were collected aseptically 5 days after the last boost. Lymphocytes were isolated from blood samples, and cDNA was synthesized from the acquired RNA using a reverse PCR protocol. A nanobody phage display library of ~109 independent transformants was generated using the phagemid vector pHEN425. Phage display selection and screening of specific nanobodies were performed as previously published26. After enrichment of antigen-specific clones by rounds of selection on solid-phase-coated antigen, PorM-specific nanobodies were identified, and the inserts of the corresponding pHEN4-derived plasmids were sequenced and cloned into the pHEN6 vector. E. coli WK6 cells carrying the pHEN6 derivatives were grown at 37 °C in terrific broth supplemented with 0.1% glucose and 100 μg/mL ampicillin to an optical density ~0.6–1.0 and the expression of the nanobodies was induced by the addition of 1 mM IPTG for 16 h at 28 °C. The periplasmic fraction containing the nanobodies was prepared using mild osmotic shock and the His-tagged nanobodies were immobilized on a 5-mL Ni-NTA column equilibrated in 50 mM Tris–HCl, pH 8, 300 mM NaCl, and 10 mM imidazole. Nanobodies were eluted in 250 mM imidazole and concentrated using the Amicon-technology (10-kDa cut-off) prior to loading on a HiLoad 16/60 Superdex 75 gel filtration column equilibrated in 20 mM Tris–HCl, pH 8, 50 mM NaCl.The PorM-specific nanonbodies nb01 and nb130 were obtained and purified by standard methodsAll crystallization trials were performed using the sitting-drop vapor-diffusion method at 293K in 96-well Greiner plates. Drops were prepared by mixing different volumes  of protein solution and 100 nL of precipitant solution, and were equilibrated against 150 μL reservoir volume.224 and PorMp224 SeMet derivatives were performed with the PEGs Suite (Qiagen). After optimization, the final crystallization conditions were 0.02 M sodium acetate pH 4.8–5.8, 0.2 M zinc acetate, 15–25% (w/v) PEG3350, with a protein-to-well ratio of 3:1 (v/v). Crystallization trials of PorMp315/nb130 was performed with the 1/1 complex at 10 mg/mL with 0.2 M Ammonium citrate tribasic pH 7.0 20% (w/v) PEG3350 as precipitant. Crystallization trials of PorMNt/nb01 was performed with the 1/1 complex at 10 mg/mL with 0.1 M Hepes pH 7.5, 0.2 M NaCl, and 25% (w/v) PEG3350 as precipitant. Crystals were briefly soaked in crystallization solution supplemented with 20% (v/v) propylene glycol before being flash-frozen in a nitrogen gas stream at 100K.Crystallization trials of PorMp224 diffraction data were collected to 2.85 Å resolution on beamline ID23–1 at the European Synchrotron Research Facilities , using a Pilatus detector. PorMp224 SeMet MAD data were collected to 3.1 Å on the same beamline. The data sets were integrated with XDS27 and were scaled with SCALA from CCP4 Suite v6.3.028.Native PorMp315/nb130 and PorMpNt/nb01 were collected at beamline PROXIMA 1 at SOLEIL, Paris, France. Data were integrated and scaled as for PorMp224. Data collection statistics are reported in Table Complete data sets of PorMp27 and were scaled with SCALA from CCP4 Suite28. More technical details are provided elsewhere17. Data collection statistics are reported in Table Initial crystallization trials of GldMp and SeMet GldMp were performed by the sitting-drop vapor-diffusion method at 293K in 96-well Swissci plates using a Mosquito Crystal robot (TTP Labtech) with the following screens: Stura Footprint Screens (Molecular Dimensions), Structure Screen and Structure Screen 2 (Molecular Dimensions), PEGs Suite and PEGs II Suite (Qiagen), JCSG+ Suite (Qiagen), and Index (Hampton Research). Crystallization hit occurred in condition No. 8 of the Stura Footprint Screen #2  PEG 600). After optimization, the final crystallization conditions were 0.1 M Hepes pH 7–8, 26–46% (w/v) PEG 600. Crystals were briefly soaked in crystallization solution supplemented with 10% (v/v) ethylene glycol and 10% (v/v) glycerol for native and SeMet GldMp, respectively. Native GldMp diffraction data were collected to 2 Å resolution on beamline ID30A-3 at the European Synchrotron Research Facility (ESRF), Grenoble, France. SeMet GldMp single-wavelength anomalous diffraction (SAD) data were collected to 2.4 Å resolution on beamline Proxima-1 at SOLEIL, Paris, France. A fluorescence scan was performed to determine the peak wavelength (0.97908 Å). The data sets were integrated with XDS224 was solved by the multiple-wavelength anomalous diffraction (MAD) method using the SeMet PorMp224 data set at 3.1 Å resolution. Heavy-atom substructure determination, positional refinement, phase calculations, and solvent flattening were performed using autoSHARP29, SHARP30, and SOLOMON31. The partial model of SeMet PorMp224 was built using Turbo-Frodo32, and was subsequently used as model for molecular replacement with MOLREP33 to solve the structure of native PorMp224 at 2.85 Å.The structure of PorMp315/nb130 was solved by molecular replacement with MOLREP33 using the partial model of domains D3 and D4 of PorMp224, and the structure of nb13017 as models. The building of domain D4 of PorMp224 and PorMp315 was then completed manually with COOT34.The structure of the complex PorMpNt/nb01 was solved by combining molecular replacement with MOLREP33 using the structure of nb0117 as starting model, and several cycles of automatic building of PorMpNt in the extra density with BUCCANEER35 followed by refinement with autoBUSTER36. The building of PorMpNt was then completed manually with COOT34.The structure of the complex PorMp29, SHARP30, and SOLOMON31. The partial model of SeMet GldMp was automatically built with BUCANEER35, and was subsequently used as model for molecular replacement with MOLREP33 to solve the structure of native GldMp at 2 Å. The building of GldMp was then completed manually with COOT34.The structure of GldMp was solved by the SAD method using the SeMet GldMp data set collected at 2.4 Å. Heavy-atom substructure determination, positional refinement, phase calculations, and solvent flattening were performed using autoSHARP36, COOT34, and Molprobity37, respectively. More technical details are provided elsewhere17. Refinement statistics are reported in Table Refinement, correction, and validation of the different structures were performed with autoBUSTER18 complex modeling was performed using the straight GldMp structure as input, with both threefold and fourfold symmetry. The two best solutions were found to be close together resulting in a tight-packed N-terminal domain. To note, Symmdock works by maximizing the contact surface between monomers and minimizing the steric clashes.Symmdock softwareBordetella adenylate cyclase have been engineered by restriction-free ligation. BACTH experiments have been performed as previously3. After introduction of the two plasmids producing the fusion proteins into the reporter BTH101 strain, plates were incubated at 30 °C for 48 h. Three independent colonies for each transformation were inoculated into 600 μL of LB medium supplemented with ampicillin, kanamycin, and IPTG (0.5 mM). After overnight growth at 30 °C, 10 μL of each culture were dropped onto LB plates supplemented with ampicillin, kanamycin, IPTG, and X-Gal, and incubated for 16 h at 30 °C. Controls include interaction assays with TolB/Pal, or MalF/MalG, two protein pairs unrelated to the T9SS. The experiments were done at least in triplicate and a representative result is shown.PorM–D1, PorM–D2–D3–D4, PorM–D2–D3, and PorM–D4 domains fused to the T18 and T25 domains of the 7. After gene induction, cells were lysed, and the total membrane fractions obtained after ultracentrifugation were subjected to 10%-acrylamide SDS-PAGE, transfer to nitrocellulose, and immunodetection using monoclonal anti-FLAG antibody. Figure Cysteine codons were introduced by Quick change site-directed mutagenesis into the plasmid encoding the C92S variant of FLAG-tagged PorM224, complexes PorMpNt/nb01 and PorMp315/nb130, and GldMp were deposited in the Protein Data Bank (PDB) under accession numbers 6EY5, 6EY0, 6EY6, and 6EY4, respectively. Other data are available from the corresponding authors upon reasonable request.Structures of PorMpSupplementary Information


CyC) analogs have been investigated against Mycobacterium tuberculosis H37Rv (M. tb) grown either in broth medium or inside macrophages. Our compounds displayed a diversity of action by acting either on extracellular M. tb bacterial growth only, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth with very low toxicity towards host macrophages. Among the eight potential CyCs identified, CyC17 exhibited the best extracellular antitubercular activity (MIC50 = 500 nM). This compound was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 23 potential candidates, most of them being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA and HsaD, have previously been reported as essential for in vitro growth of M. tb and/or survival and persistence in macrophages. Overall, our findings support the assumption that CyC17 may thus represent a novel class of multi-target inhibitor leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes participating in important physiological processes.A new class of Cyclophostin and Cyclipostins ( Mycobacterium tuberculosis (M. tb) the causative agent of tuberculosis (TB) has become the number one global public health emergency worldwide. With 10.4 million new cases and 1.8 million deaths caused by M. tb, as reported by WHO in 20161, TB is now the deadliest infectious disease around the world and remains a great challenge, especially in sub Saharan Africa, Russia and Eastern Europe. The emergence of multiple drug-resistant (MDR), extensively drug-resistant (XDR) and totally drug-resistant (TDR)3 strains over the years and the controversial results of the Gates-backed TB vaccine (MV85A)4 highlight the pressing need for novel therapeutic approaches6.M. tb as a pathogen is its ability to evade host immunity and to establish a chronic and persistent infection7. Several unusual characteristics contribute to this success, the first one being its unique lipid-rich cell wall8. Indeed, the mycobacterial waxy coat, essential for bacterial viability and pathogenicity, possesses unique features. The complex architecture and impermeability of the cell wall are responsible for the inherent resistance of M. tb to many antibiotics9. Most current available drugs including first-line drugs such as isoniazid and ethambutol inhibit cell wall biosynthetic enzymes5. The same comment remains true for new antituberculosis/antibiotics currently evaluated in clinical phase II or III trials, comprising either repurposed drug or new analogues of known anti-mycobacterial drugs10. A posteriori, such target-specificity may not address sufficiently nor efficiently the global spreading of the disease. Following this point of view, in 2013, Zumla et al. stated that “there is growing awareness of the need for drugs that can kill M. tuberculosis in its different physiological states”10.The key feature in the success of i.e., persisting bacilli) from turning into active TB disease5. The inherent difficulty to be compliant to such long treatments is in part responsible for the emergence of resistant strains and represents a new challenge to achieve control of the disease. In this context, continuous efforts for developing innovative chemotherapeutic approaches to treat TB are needed.Another important issue resides in the fact that current treatments consist in a quadritherapy for 2 months, which has to be extended with a 4- to 7-months bitherapy to prevent latent TB infections (CyC1) and Cyclipostins )  than those of rifampicin and penicillin G13. From a chemical point of view, Cyclipostins family members possess a bicyclic enol-organophosphorus core structure similar to that of Cyclophostin, but are phosphate esters of long chain lipophilic alcohols 14 or human hormone-sensitive lipase 15.These natural compounds were also shown to be potent inhibitors of either acetylcholinesterase (CyC1) and Cyclipostins P (CyC18)15 and their respective biological activity against purified lipolytic enzymes. Similar studies were conducted with their phosphate (CyC16-17) and phosphonate (CyC2-15) analogs19 ate analogs of Cyclophostin (and the Cyclipostins) as a new class of selective serine/cysteine enzyme inhibitors in mycobacteria. The selectivity of the CyC derivatives to inhibit the mycobacterial but not the human enzymes, is therefore highly valuable and prompted us to consider these compounds as potential antitubercular agents.Moreover, modulation of the lipophilicity by varying the nature and chain length of the alkyl group either at the C-5 carbon atom (CyC molecule has been tested against M. tb for i) its capacity to inhibit in vitro growth; ii) its antitubercular activity on M. tb-infected macrophages, and iii) its eventual cytotoxicity towards macrophages. Unexpectedly, whereas few analogs were found to inhibit M.tb growth in vitro and in macrophages similarly to isoniazid, they all showed absence of toxicity in mammalian cells. Importantly, potential targets of CyC17, the most potent inhibitor, were identified via an activity-based protein profiling (ABPP) approach, and further validated by the constructions of overexpressing mycobacterial strains.Herein, each CyC compounds 19 and to significantly improve the lipophilicity, CyC13 was synthesized by introducing simultaneously a C16-side alkyl chain  and a C10-side alkyl chain at the C-5 carbon atom , leading to an hybrid compound between CyC7 and CyC11  and the CC50 (concentration leading to 50% host cell toxicity) as reported earlier21. Among the 27 analogs, eight potential candidates exhibited very promising antitubercular activities (Table CyC7(β) and CyC8(α) exhibited moderate (16–40 µM) and good (3–4 µM) activity against extracellular and intramacrophagic M. tb, respectively. In contrast, CyC6(β), CyC7(α) and CyC8(β) appeared to be active only on infected macrophages; whereas CyC17 and Cyclipostins P, i.e. CyC18(α) and CyC18(β), impaired selectively M. tb growth in culture broth medium with MIC50 up to the nanomolar range (MIC50 ≅ 500 nM for CyC17). More particularly, both (α) and (β) isomers of CyC7 as well as CyC8(α) were found to exhibit similar or higher MIC50 values towards intramacrophagic bacilli than the first line antibiotics used as references (Table The set of 27 50) >100 μM, similarly to isoniazid (CC50 > 150 µM) and ethionamide (CC50 ≥ 120 µM), two potent antitubercular agents.Beside antibacterial activity, significantly, all the latter inhibitors displayed very low toxicity towards host macrophages, with cytotoxic concentration  of small molecules selected from whole cell screens. The abovementioned results with the 22 for targets identification. The so-called activity-based probes (ABPs), following labelling and enrichment procedures, allows to isolate selective sets of low-copy-number enzymes in complex proteomic mixtures through the chemical recognition of a specific catalytic mechanism without interference from the more represented proteins22. ABPs typically contain (i) a reactive group which forms a covalent and irreversible adduct with the target; (ii) a linker region that allows to control the specificity of the probe; and (iii) a tag for visualization (fluorescent tag)23 and/or enrichment and isolation24 of the covalently labelled proteins.This prompted us to apply an Activity-based protein profiling (ABPP) approachCyC analogs on catalytic serine or cysteine active residues  or a biotin  reporter tag, were selected  and could also be detected in Coomassie blue staining after capture/enrichment of total lysate by Desthiobiotin-FP . In contrast, pre-treatment with CyC17 . Indeed, the enzymes previously inactivated by CyC17 inhibitor will thus be unable to further react with the FP-ABP. Proteins corresponding to bands 1-9 were then excised from the gel, digested with trypsin and the resulting peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for subsequent protein identification. To overcome the potential overlap of proteins, the proteins that were also present at the same position in the control experiment  have not been taken into account, therefore leading to 23 distinct protein candidates , lipid metabolism (5 proteins), cell wall/cell processes (4 proteins), and virulence/detoxification/adaptation (1 protein) (Table As expected from previous ABPP studies on n) Table .31; the methyltransferase SerA1 (Rv2996c) involved in the L-serine biosynthetic process32; glyA1 (Rv1093) annotated as a serine hydroxymethyltransferase with possible role in serine to glycine conversion33; and UmaA (Rv0469) a S-adenosyl-L-methionine-dependent methyltransferase capable of catalyzing the conversion of phospholipid-linked oleic acid to essential tuberculostearic acid34, a major constituent of mycobacterial membrane phospholipids. Little is known about the catalytic reactions of these enzymes. However, our results, in line with previous findings using fluorophosphonate ABPs30, suggest the presence of at least one nucleophilic  serine or cysteine residue involved in the formation of a covalent adduct with CyC inhibitors.Enzymes involved in metabolic processes included the alcohol dehydrogenase AdhB (Rv0761c) thought to catalyze the reversible oxidation of ethanol to acetaldehyde with the concomitant reduction of NAD; the putative L-lactate dehydrogenase LidD2 (Rv1872c)35 recently proposed as being a functional serine hydrolase30. Five members of the lipase family Lip  were detected; a number significantly lower than the 13 active M. tb Lip enzymes reported using Desthiobiotin-FP29 or the 8 lipases using an alkyne-PEG-FP probe30. Among the five captured Lip proteins, LipH (Rv1399c)37 and LipV (Rv3203)38 had been functionally characterized previously. LipH is known to hydrolyze short-chain ester and may participate in the detoxification pathway of the intracellular lipid metabolism while LipV posseses a broad range substrate specificity and is also active at low pH, suggesting a role in M. tb’s adaptation to acidic conditions into the phagosome. Beside members of the Lip family, six additional enzymes with lipolytic activity were isolated: Rv0183, a monoacylglycerol lipase that degrades host-cell lipids40; Cfp21 (Rv1984c), a cutinase-like protein preferentially active against medium-chain carboxylic esters and monoacylglycerols42; as well as the esterase Rv0045c43 proposed to participate in lipid hydrolysis.The remaining 18 enzymes belong to the serine/cysteine hydrolase family proteins. Among them, a few hydrolases were identified: two putative β-lactamases Rv1730c (currently annotated as a possible penicillin-binding protein) and Rv1367c, both possibly involved in cell wall biosynthesis; two amidases AmiC (Rv2888c) and AmiB2 (Rv1263); and BpoC a possible peroxidase (Rv0554)CyC17: the antigen 85 complex Ag85A (Rv3804c) and Ag85C (Rv0129c), the thioesterase TesA (Rv2928), the carboxylesterase CaeA (Rv2224c) and the hydrolase HsaD (Rv3569c); the latter two proteins being annotated as essential enzymes44.Five additional lipolytic enzymes appeared as highly promising target candidates of 46. Moreover, inhibition of Ag85C was found to block TDM synthesis and to disrupt the integrity of the cell envelope47. Similarly, TesA has been found to be required for the synthesis of both phenolic glycolipids and phthiocerol dimycocerosate (PDIM). Inactivation of TesA in M. marinum was correlated with an important decrease in virulence and increase susceptibility to drugs48. CaeA  is a cell wall-associated carboxylesterase involved in cell wall biosynthesis and/or integrity49. CaeA was also found to play important roles in virulence49, multidrug-resistance50 and innate immunity51. The absence of CaeA enhanced host innate immune responses and compromised the intracellular survival of M. tb in macrophages52. The hydrolase HsaD was first described as participating in cholesterol catabolism53 and then found to be essential for intramacrophage survival of M. tb51. HsaD has recently been proposed as a novel therapeutic target and awaits further developments54.Ag85A and Ag85C express both a mycolyl transferase activity. They catalyze the transfer of mycolic acids from trehalose monomycolate (TMM) to produce trehalose dimycolate (TDM) and are also responsible for the covalent attachment of mycolic acids to arabinogalactanM. tb  (Table CyC17. Compared to WT strain (MIC50 = 0.55 ± 0.023 μM), overexpression of LipH caused a slight increase in the MIC50 value of around 1.3-fold , while overexpression of HsaD led to a significant 2.2-fold increase in MIC50 value .To examine whether these six overexpression strains were affected on their susceptibility to 50 value of the strain overexpressing HsaD prompted us to explore the potential interactions occurring at the enzyme’s active site following CyC17 binding. In silico molecular docking experiments were conducted, as described previously17 using the recently reported crystal structures of HsaD bound to three different inhibitors54: 3,5-dichloro-4-hydroxybenzoic acid (PDB id: 5JZS), 3,5-dichloro-4-hydroxybenzenesulphonic acid (PDB id: 5JZ9) and 3,5-dichloro-benzenesulfonamide (PDB id: 5JZB).The increased MICi.e., lowest energy complex) indicated that the reactive seven-membered monocyclic enolphosphorus ring adopted a productive orientation  and thus the formation of a covalent bond. It is also noteworthy that a high level of concordance was observed between this favorable docked conformation of CyC17 and the structure of the 3,5-dichloro-4-hydroxybenzoic acid found to bind in the vicinity of the HsaD active site  and the active site residues Ser114-Asp241-His269 of the protein during the binding process. The Ligplot + analysis clearly shows that the reactive phosphorous atom is stabilized by H-bonding with Asn244 and His269 residues 54. Specifically, all residues involved in H-bonding and hydrophobic contacts in each of the above mentioned complex structures are also present in stabilizing CyC17, therefore reinforcing the accuracy of our model. Taken together, this network of interactions presumably allows the formation of a stable and productive binding mode, and might provide a clear picture of the inhibition of HsaD by CyC17.From these findings, fight” against TB. Herein, we have evaluated the anti-tubercular activities of a set of 27 CyC analogs  inside infected macrophages, providing the cells a foamy appearance59. In foamy macrophages (FM), bacilli accumulate lipids within intracytoplasmic lipid inclusions (ILI)7, which allow the bacteria to persist in a non-replicating state. To persist inside FM, M. tb hydrolyzes host lipids triacylglycerols (TAG) from LB into fatty acids that are reprocessed as lipid reserves within ILI59. During the reactivation phase, these ILIs are hydrolyzed by M. tb and used to fuel the replication of mycobacteria during their exit from the hypoxic non-replicating state60. Therefore, finding ways to inhibit the activity of such mycobacterial lipolytic enzymes may pave the way for discovering new modalities for TB treatment.Drug discovery developments to generate new lead compounds along with their corresponding targets and mode of action represent a major need in the “ogs Fig. , that wemPPOX compound61, Orlistat62, or more recently the human lysosomal acid lipase inhibitor Lalistat28, have already been described to block M. tb growth with MICs ranging from 25-50 μM. Despite these moderate inhibition activities, a strong synergistic effect on in vitro M. tb growth was reached for the combined application of both latter inhibitors with vancomycin, resulting in a MIC drop of 16-fold for Orlistat (MIC~6 µM)62 and 4-fold for Lalistat (MIC~6–12 µM)28.Some known lipase inhibitors such as the oxadiazolone MCyC analogs, eight showed moderate (16–40 µM), potent (3–4 µM) or very good (0.5 µM) activity as judged by their MIC50 values (Table CyC6(α), CyC7 and CyC8 showed a clear preference against intracellularly-replicating mycobacteria. This supposes that the intracellular mode of action of this class of inhibitors differs from that of those acting exclusively on extracellularly-replicating bacilli. It can therefore be hypothesized that vulnerability of the corresponding target(s) of these inhibitors becomes more apparent and critical during the intracellular lifestyle of M. tb. A specific response of the macrophage induced by the action of these compounds and leading to bacterial death cannot however be excluded. In contrast, CyC17 and CyC18 showed high activity exclusively on extracellular bacteria, a property already observed previously for 1,2,4-Oxadiazole EthR Inhibitors21 and was correlated to limited bioavailability and to the hydrophilicity of these compounds. From a structure-activity relationships (SAR) perspective, some trends have emerged with respect to the effects of the CyC analogs tested. Regarding the natural Cyclophostin (CyC1) and its phosphonate derivatives CyC2-10;15-16, it is noteworthy that identified bioactive compounds CyC7 and CyC8 bearing medium C10- and C12-side alkyl chains, respectively, are corresponding to the most potent and also “less” selective inhibitors of various bacterial enzymes, as compared to the other CyC analogs which were found to exhibit a greater selectivity towards pure recombinant mycobacterial lipase over human counterparts17. With Cyclipostin P, the potent antibacterial activity of CyC18 is in good agreement with the in vitro growth inhibition reported earlier on various mycobacteria13. Moreover, the fact that only the monocyclic enolphosphate CyC17 displays antituberculous activity when compared to the non-active enolphosphonate derivatives CyC11, CyC12 and CyC14, emphasizes the specific need for a phosphate moiety in such heptacyclic analogs to exhibit bactericidal activity against M. tb growth in vitro.In our study, among the 27 tested moderate 6–40 µM, CyC tested  were also captured from M. bovis BCG lysates using an Orlistat-alkyne analog and click chemistry for targets enrichment27; they were not detected, however, when a Lalistat-like probe was directly incubated with M. tb cells prior to lysis and chemical proteomics. Such finding is in agreement with a complementary target profile exerted by each lipase inhibitor given their respective physico-chemical properties. Since the MIC of these two lipase inhibitors towards M. tb growth (around 25-50 μM) was however 50- to 100-times higher than that of CyC17 (0.5 µM), it is thus tempting to speculate that the shared preference for a specific set of enzymes is responsible for the high growth inhibitory potency of our CyC monocyclic enolphosphate.To gain access to the mechanisms of action, an ABPP approach was successfully applied allowing the identification of mycobacterial enzymes impaired by the inhibitors during mycobacterial growth. Selective labelling and enrichment of captured enzymes using appropriate fluorophosphonate probes in combination with CyC17, genes encoding the identified targets  were overexpressed in M. tb mc26230. Whereas overexpression of LipH or HsaD led to slight, but statistically significant increased resistance levels, thereby suggesting that these two lipolytic enzymes could be effective drug targets; overexpression of Rv0183, Ag85A, Ag85C or TesA did not change the susceptibility/resistance profile to CyC17  analogs both in vitro, and ex vivo in infected macrophages. This led to the selection of a set of promising CyC candidates that are devoid to cytotoxic properties towards host cells. By targeting multiple enzymes either involved in lipid metabolism and/or in cell wall biosynthesis, these compounds are emerging as a novel class of multi-target anti-TB candidates which should open up new chemotherapeutic opportunities in the fight against TB. By blocking extracellular and/or intracellular M. tb growth, we anticipate these compounds could prevent the entry of M. tb in the persistence phase and/or reactivation of dormant bacilli residing within the granuloma and the foamy macrophages. To our knowledge, there is no other family of compounds able to target and impair replicating bacteria as well as intracellular bacteria. The dual activity of the CyC inhibitors is of major importance as it may affect the different stages of the infection process. Because lipid storage in bacteria is thought to drive the infection process, CyC inhibitors can also be viewed as attractive candidates to further dissect the fate of the bacteria in the context of infected foamy macrophages.A priority for new drug-development to efficiently treat TB must be focused on the discovery of novel therapeutic targets and approaches. In this work, we evaluated the antitubercular activities of a series of Cyclipostins and Cyclophostin  and CyC2(β)14, the monocyclic enolphosphonates CyC3-416 and the trans-(α) and cis-(β) diastereoisomers CyC5-1017; as well as the trans-(α) and cis-(β) Cyclipostin P CyC1815 and the corresponding monocyclic phosphonate CyC11-1217, difluorophosphonate CyC14-15 and phosphate CyC16-1719 analogs were obtained at 98% purity as described previously. Stock solutions (10 mM) in which the CyC compounds were found to be completely soluble in dimethyl sulfoxide (DMSO), were prepared prior to extracellular and intracellular drug susceptibility testing.The synthesis of natural Cyclophostin CyC13 was prepared via a transesterification reaction from racemic CyC7 using established techniques already reported for CyC16-17 synthesis19, giving desired compound as a mixture of diastereoisomers. Briefly a solution of CyC7  in 1,4-dioxane (360 µL) was added to a flask containing tetrabutylammonium iodide  followed by hexadecyl bromide . The flask was placed in an oil bath preheated to 105 °C. After 4.5 hours, the solution was cooled and concentrated in vacuo. The residue was purified by column chromatography  to give the oily product  as a mixture of trans-(α) and cis-(β) diastereoisomers. The two isomers were further separated by preparative reversed phase HPLC  as follows. Preparative HPLC Specifications and Conditions. Manual preparative injector: Rheodyne 1700 (3725i-119) with 20 mL loop; Solvent A – MeOH; Solvent B – H2O; Varian ProStar Model 210 pumps equipped with 25 mL/min Rainin/Gilson type pump heads. Kromasil 100-10C18-2025 column; 10 µm particle diameter; 250 mm × 20 mm i.d. Spectra-Physics Spectra 100 UV detector with prep cell. LKB 2211 Superac fraction collector. 100% MeOH at a flow rate of 10 mL/min.The new lipophilic enolphosphonate 1H, 13C, and particularly the 31P NMR spectra, and high resolution mass spectrometry  (15.8 mg). HPLC RT 38 min; IR  2293, 2853 1718, 1651 cm−1; 1H NMR  δ 4.19 , 4.06 , 3.75 , 2.90 , 2.17 , 2.15–1.85 , 1.75–1.45 , 1.30 , 0.88 ; 13C NMR  δ 169.3, 155.1 , 123.1 , 66.5 , 51.9, 37.5, 32.1 , 31.3, 30.7 , 29.9–29.7 (multiple overlapping peaks), 29.6 , 29.4, 27.9, 25.7, 25.6 , 23.9, 22.8, 22.1, 21.3 , 14.3; 31P NMR  δ 22.1 ppm; HRMS  calcd for C34H66O5P: 585.4648, found 585.4664. Slow eluting isomer CyC13(α) (17.8 mg). HPLC RT 48 min; IR  2923, 2853, 1718, 1652 cm−1; 1H NMR  δ 4.13 , 3.73 , 2.98 , 2.22 , 2.15–1.85 , 1.75–1.45 , 1.30 , 0.88 ; 13C NMR  δ 169.2 , 156.1 , 123.2 , 66.1 ,, 52.0, 37.4, 32.1 , 30.9, 30.6 , 29.9–29.7 (multiple overlapping peaks), 29.5 , 29.3, 27.8, 25.7, 25.1 , 23.3, 22.9, 21.6, 21.5, 14.3; 31P NMR  δ 24.9 ppm; HRMS  calcd for C34H66O5P: 585.4648, found 585.4634.Fast eluting isomer M. tb H37Rv expressing GFP20 was grown for 14 days in 7H9 medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase , 0.5% glycerol, 0.05% Tween 80 and 50 µg/mL hygromycin B (Euromedex). For target identification, the experiments were conducted using M. tb mc26230 (H37Rv ΔRD1 ΔpanCD) a derivative of H37Rv which contains a deletion of the RD1 region and panCD, resulting in a pan(−) phenotype64. M. tb mc26230 was grown in 7H9 medium supplemented with 10% OADC (BD Difco), 0.5% glycerol, 0.05% Tween 80 and 24 µg/mL D-panthothenate (Sigma-Aldrich). Cultures were kept at 37 °C without shaking.For intra and extracellular assays, M. tb H37Rv-GFP strain in macrophages was monitored by automated fluorescence confocal microscope  as already described20. Briefly, bacteria were washed twice with PBS and resuspended in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum . Murine (Raw264.7) macrophages were infected at a multiplicity of infection (MOI) of 2:1 and incubated 2 hours at 37 °C in RPMI 1640 medium containing 10% FBS. Cells were then washed, treated with 50 µg/mL amikacin (Euromedex) for 1 hour at 37 °C to kill all extra-cellular bacteria, washed again and finally seeded in 384-well plates (5 × 105 cells/mL), containing 2-fold dilutions of compounds in DMSO. The final volume of DMSO was kept under 0.3%. Plates were incubated for 5 days at 37 °C, 5% CO2. Infected cells were stained for 30 min using Syto60 dye (Invitrogen) at a final concentration of 5 µM before reading using fluorescence confocal microscope . Sigmoidal dose-response curves were fitted using Prism software . The concentration required to inhibit 50% of M. tb intracellular growth (MIC50) was determined using ten-point dose-response curves as an average of the MIC50 of all parameters, the ratio of infected cells and the bacterial area per infected cell.The growth of M. tb H37Rv-GFP was washed twice with PBS and resuspended in 7H9 medium containing 10% OADC, 0.5% glycerol, 0.05% Tween 80 and 50 µg/mL hygromycin B. Bacteria were seeded in 384 well plates (7 × 105 bacteria/mL) containing 2-fold dilutions of the compounds in DMSO. The final volume of DMSO was kept under 0.3%. Plates were incubated at 37 °C, 5% CO2 for 5 days. Bacterial fluorescence levels (RFU) were recorded using a fluorescent microplate reader . The MIC50 of all tested compounds were determined using ten-point dose-response curves.A 14 days old culture of 600~1), M. tb mc26230 cells were harvested by centrifugation at 4,000 g for 15 min. Pellets were washed 3 times with PBS containing 0.05% Tween 80. The cell pellets were resuspended in PBS buffer at a 1:1 (w/v) ratio. The bacterial cells were then mixed with the same volume of 0.1 mm diameter glass beads (BioSpec) and disrupted during 4 min of violent shaking using Mini-Beadbeater-96 (BioSpec). The lysate was then centrifuged at 4 °C and at 12,500 g for 15 min to remove the cell debris and unbroken cells. Supernatants were adjusted to a concentration of 2 mg/mL of total proteins, snap frozen in liquid nitrogen and stored at −80 °C until further use.From 1 L of culture at the logarithmically growth stage  were incubated with 2 μM ActivX TAMRA-FP probe (ThermoFisher Scientific) or DMSO (unlabelled control) for 90 min at room temperature and in absence of light. The reaction was stopped by adding 5X Laemmli reducing sample buffer and boiling at 95 °C for 5 min. The labelled proteins were further analyzed by SDS-PAGE electrophoresis (12% Bis-Tris gel) followed by fluorescent gel scanning  and detection using the Cy®3 filter of a ChemiDoc MP Imager (Bio-Rad). Alternatively, the gel was stained with Coomassie blue R250 staining solution and was destained with solution of 10% ethanol and 30% acetic acid.M. tb mc26230 lysates  were incubated with 2 µM ActivX Desthiobiotin-FP probe (ThermoFisher Scientific) or DMSO (unlabelled control) for 90 min at 37 °C. For inhibitor studies, lysates were pre-incubated with 580 µM CyC17 at 37 °C for 90 min prior to Desthiobiotin-FP treatment. The reaction was next stopped by adding 0.3 g of urea  to denature proteins completely. Unreacted probes were removed using Zeba Spin desalting column  and labelled proteins were further captured by 200 µg Nanolink streptavidin magnetic beads 0.8 µm (Solulink), according to the manufacturer’s instructions.M. tb mc26230 treated-lysate sample was enriched for labelled proteins by transfer to the previously washed beads (around 200 µg). The lysate/beads suspensions were incubated for 1 hour at room temperature with mild shaking. The tubes were then placed on the magnetic stand for 2 min to collect the beads, and the supernatant was removed. The beads containing bound, biotinylated proteins were washed three time carefully with the Wash Buffer, as described above, and resuspended in 25 µL PBS buffer pH 7.4 containing 50 mM free biotin. The resulting solution was mixed with 5X Laemmli reducing sample buffer, and heated at 95 °C for 5 min. This step allowed the recovery of the captured labelled proteins by exchanging the initially captured desthiobiotin/streptavidin complex to the greater affinity biotin/streptavidin complex.First, 20 µL of a 10 mg/mL NanoLink streptavidin magnetic beads was transferred into a 1.5 mL Eppendorf tube. The Wash Buffer  was then added to bring the final volume to 250 µL and the resulting mixture was mixed gently to resuspend and wash the beads. The tube was placed on a magnetic stand for 2 min. and the supernatant was discarded. The tube was removed from the magnetic stand and the beads were washed two more times with the Wash Buffer (250 µL). Each The released proteins were resolved by SDS-PAGE at 160 V for 1 hour. The gel was stained with Coomassie blue R250 staining solution and was destained with solution of 10% ethanol; 30% acetic acid. To check for unspecific binding, a DMSO-treated lysate sample was incubated only with the streptavidin-magnetic beads in absence of Desthiobiotin-FP probe treatment, and processed as described above.v/v) were followed by reduction (10 mM dithiothreitol for 1 h. at 56 °C), alkylation (55 mM iodoacetamide for 30 min at room temperature) and digestion by a trypsin solution  containing ProteaseMAX 0.025% (w/v) (Promega) in 50 mM ammonium bicarbonate overnight at 37 °C. Tryptic peptides were extracted by 0.1% TFA in water/acetonitrile (50:50 v/v) and dried into a speed vacuum. Mass spectrometry was performed on a Q Exactive Plus mass spectrometer  equipped with a nanospray ion source and coupled to an Ultimate 3000 nano UPLC . Dried tryptic peptides were dissolved in 2% acetonitrile/0.05% TFA in water and desalted on a C18 µ-precolumn  before elution onto a C18 column . Peptides were eluted with a linear gradient from 6 to 40% of mobile phase B  in A (0.1% formic acid in water) for 52 min. Peptides were detected with a workflow combining full MS (350- 1900 m/z range at 70000 resolution)/data dependent MS/MS Top 10 .The bands of interest were first excised from gels. Classical steps of washes ; enzyme, trypsin; missed cleavages, 2; dynamic modification, Oxidation Met + 15.995 Da; static modification, Carbamidomethyl Cys + 57.021 Da; minimum length of peptides, 6 amino acids; precursor mass tolerance, 10 ppm; fragment mass tolerance, 0.02 Da. Proteins were considered as identified by at least two unique peptides passing the high confidence filter (Relaxed Target FDR:0.05 and Maximum Delta Cn: 0.05). For more details about proteins identification, i.e. sequence coverage and number of identified peptides see Table Mass spectra were processed using Proteome Discoverer software v. 2.1.0.81 (ThermoFisher Scientific) based on SequestHT algorithm. The following parameters were used: organism, UniProt ag85A (Rv3804), ag85C (Rv0129c), rv0183, lipH (Rv1399c), hsaD (Rv3569c) and tesA (Rv2928) were amplified by PCR from M. tb H37Rv genomic DNA. Specific primers  or the MscI (for ag85A and ag85C) restriction site at the 5′ end and BamHI at the 3′ end for all the genes. Amplicons were digested with the corresponding restriction enzymes (ThermoFisher Scientific), gel purified using Nucleospin Gel and PCR Clean-up kit (Macherey-Nagel) and cloned into proper restriction sites of pMV261  or pVV16 in frame with a C-Terminus 6-His tag , both harbouring the hsp60 promoter. The DNA sequences of each insert were confirmed by DNA sequencing (GATC Biotech).M. tb mc26230 electrocompetent cells were prepared as described previously by Goude et al.65. Briefly, 100 mL of M. tb mc26230 cells were cultivated up to mid-log phase  and glycine was added to a final concentration of 0.2 M and incubated during 16 hours. Cells were harvested, washed four times with 10% glycerol solution at room temperature and finally resuspended in 1/100 of the original volume. 200 µL of competent cells were mixed with 1 µg of DNA and transferred to a 2 mm gap electroporation cuvette. A single pulse of 2.5 kV, 25 µF with resistance set at 600 Ω was provided. Culture media was immediately added to the mycobacterial suspension and then incubated during 24 hours at 37 °C. Bacteria were plated on 7H10 Middlebrook agar supplemented with 10% OADC and 50 µg/mL of both kanamycin and hygromycin. Plates were incubated at 37 °C during 3 weeks. Positive transformants were further grown in liquid medium up to OD 1 and the overexpression of the recombinant proteins was checked by Western blot using either the specific monoclonal antibody Mab 32/15 kindly provided by Dr. K. Huygen directed against the M. tb Ag85 complex66, specific rabbit polyclonal antibodies directed against Rv018339, or HisProbe HRP conjugated (ThermoFisher Scientific) for the other proteins.M. tb mc26230 mutant strains overexpressing Ag85A, Ag85C, Rv0183, LipH, HsaD or TesA were determined in 96-well flat-bottom Nunclon Delta Surface microplates with lid  using the resazurin microtiter assay (REMA68). Briefly, log-phase bacteria  were diluted to a cell density of 5 × 106 CFU/mL. Then 100 µL of the above inoculum was added to each well containing 100 µL 7H9 medium, serial two-fold dilutions of CyC17 or controls to a final volume of 200 µL . Growth controls containing no inhibitor , inhibition controls containing 50 µg/mL isoniazid (Euromedex) and sterility controls  without inoculation were also included. Plates were incubated at 37 °C in a humidity chamber69 to prevent evaporation. After 10–14 days of incubation, 20 µL of a 0.020% (w/v) resazurin (Sigma-Aldrich) solution was added to each well, and the plates were incubated at 37 °C for 24 hours for color change from blue to pink or violet and for a reading of fluorescence units (FU). Fluorescence corresponding to the resazurin reduction was quantified using a Tecan Spark 10 M multimode microplate reader  with excitation at 530 nm and emission at 590 nm. For fluorometric MIC determinations, a background subtraction was performed on all wells with a mean of M wells. Relative fluorescence unit was define as: RFU% = (test well FU/mean FU of B wells) × 100. MIC values were determined by fitting the RFU% sigmoidal dose-response curves in Kaleidagraph 4.2 software (Synergy Software). The lowest drug concentrations inhibiting 50% of growth were defined as the MIC50.Susceptibility testing was performed in 7H9 medium supplemented with 10% OADC, 0.5% glycerol, 0.05% Tween 80, 24 µg/mL D-panthothenate and kanamycin (50 µg/mL) when needed. Assays were carried out in triplicate. MICs of each Supporting informationIdentified Proteins