Nα-acetylation of EsxA is required for mycobacterial cytosolic translocation and virulence

Mycobacterium tuberculosis virulence factors, EsxA and EsxB, are secreted as a heterodimer (EsxA:B) and play an essential role in mycobacterial phagosomal escape and cytosolic translocation. Current studies support a model that EsxA must dissociate from its chaperon EsxB at low pH in order for EsxA to interact with host membranes. However, the mechanism of the heterodimer separation is not clear. In the present study, we have obtained evidence that the Nα-acetylation of Thr2 on EsxA, a post-translational modification that is present in mycobacteria, but absent in E. coli, is required for the heterodimer separation. The point mutations at Thr2 without Nα-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the membranes, which resulted in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamic simulation showed that at low pH the Nα-acetylated Thr2 made direct and frequent “bind-and-release” contacts with EsxB, which generates a dragging force to pull EsxB away from EsxA.


Introduction
Mycobacterium tuberculosis (Mtb) is the causative agent for tuberculosis (TB), one of the leading infectious diseases in the world with 10 million people falling ill in 2017 and approximately 1.6 million deaths (1,2). It is believed that after the Mtb-containing aerosolized droplets are inhaled into the lung, Mtb is encountered by alveolar macrophages and internalized into the phagosome, where Mtb manages to survive through arresting phagosome maturation, including inhibition of vATPasemediated acidification (3)(4)(5)(6). Recent compelling evidence support that Mtb penetrates the phagosome and translocate into the cytosol (termed cytosolic translocation), where Mtb replicates and undergoes cell-to-cell spreading (7). The ability of Mtb to arrest phagosome maturation and to translocate from the phagosome to the cytosol has been attributed, at least in part, to the Type VII secretion system, named ESX-1 and the secreted virulence factors EsxA (ESAT-6) and EsxB (CFP-10). The Mtb mutants with either gene deletions or defects in secretion of EsxA and/or EsxB, were not able to translocate into the cytosol and showed significant reduction in host cell lysis and cell-tocell spreading (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19).
In our previous studies, we have found that Mtb EsxA exhibits a unique membranepermeabilizing activity that is not present in the homologous EsxA ortholog from non-pathogenic Mycobacterium smegmatis (Ms) (20). EsxA undergoes pH-dependent conformational changes, inserts into the membrane and forms a membrane-spanning complex (21). The essential role of EsxA membrane-permeabilizing activity in phagosome rupture and cytosolic translocation is further confirmed by a recent study, in which single-residue mutations at Gln5 (Q5) of EsxA up or down regulated the membrane-permeabilizing activity and consequently up or down regulated mycobacterial cytosolic translocation and virulence in cultured cells and in zebra fish (22).
The genes encoding EsxA and EsxB are located in the same operon within the ESX-1 locus. EsxA and EsxB are co-expressed and co-secreted as a heterodimer (23). Our earlier study has demonstrated that EsxA, but not EsxB, has the membrane-permeabilizing activity, and EsxB is believed to function as a chaperon (20). Current studies support a model that the heterodimer is dissociated at low pH to allow EsxA to penetrate the membranes (24). However, the data regarding to the heterodimer dissociation are conflicting. The native heterodimer extracted from Mtb culture filtrate was found to be dissociated at low pH (24). Surprisingly, however, the studies using the recombinant proteins prepared from E. coli suggest that the heterodimer was not dissociated by acidification. This is evidenced by one of our earlier studies that the heterodimer prepared from E. coli (now termed Ec-heterodimer) was inactive in membrane disruption. In the absence of lipid membranes, EsxA formed aggregates in the acidic solution due to increased solvent-exposed hydrophobicity. In contrast, the Ec-heterodimer showed little aggregation at pH 4.0, suggesting that EsxB remains bound to EsxA at low pH and prevents EsxA from forming aggregates, which otherwise would be observed if EsxA was released from EsxB (20). Our data are consistent with an earlier CD analysis showing that the Echeterodimer is not dissociated at low pH (25).
We hypothesized that the mycobacteria-produced proteins contain unique features (e.g. posttranslational modifications, PTMs) that are required for heterodimer dissociation at low pH. In line with this hypothesis, the native EsxA protein isolated from the culture filtrate of Mtb was displayed as multiple spots in 2D SDS-PAGE, and some of the spots contained a N αacetylation at the residue Thr2 (26). Moreover, the heterodimer produced from a Ms strain was found to have a N α -acetylation on the Thr2 residue of EsxA (27). Interestingly, EsxB preferred to bind the non-acetylated EsxA, but not the acetylated form in a 2-D overlay assay (26). Deletion of the N α -acetyltransferase in Mycobacterium marinum (Mm) disrupted the homeostasis of EsxA N α -acetylation and attenuated the virulence (28). Together, these studies suggest that the N α -acetylation of EsxA plays an important role in mycobacterial virulence through facilitating heterodimer dissociation at low pH.
In the present study, we have obtained the evidence showing that the N α -acetylation at Thr2 of EsxA is required for EsxA membrane permeabilization, mycobacterial cytosolic translocation and virulence through facilitating heterodimer dissociation.

Generation of T2X mutations on the esxA gene for expression in E. coli, M. smegmatis and M. marinum
For expression in E. coli: Using the previously reported plasmid pET22b-esxA-His6 as the template (20)(21)(22)29), the mutations T2A, T2Q, T2R, and T2S were introduced into the esxA gene by PCR using the primers listed in Table S1. All of the mutations were confirmed by DNA sequencing. The resultant plasmids were transformed into BL21 (DE3) cells for expression. The cells were grown at 37 °C while shaking at 250 rpm until OD600 reached 0.6-0.8. Protein expression was induced by adding 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 3-8 h at 37 °C. The cells were harvested and the proteins were purified as previously described (20)(21)(22)29).
For expression in Ms: The pMyNT plasmid containing the esxB-esxA operon (27) was used as the template. The mutations T2A, T2Q, T2R and T2S were introduced by overlapping PCR using the primers listed in Table S1. All of the mutations were confirmed by DNA sequencing. The pMyNT plasmids carrying various T2X mutations were electroporated into Ms mc 2 155 strain (voltage: 2,500 V, capacitance: 25 µF, resistance: 1,000 Ω). The Ms cultures were grown at 37 °C overnight or until OD600 reached 2.0. Protein expression was induced by adding 0.2% (w/v) acetamide for 12-16 h. The cells were harvested. The soluble heterodimer proteins were purified with immobilized metal ion affinity chromatography by passing through a Ni 2+column, followed by a sizing exclusion chromatography as previously described (27,(30)(31)(32)(33).
For expression in Mm: The T2X mutations (T2A, T2R, T2Q and T2S) were generated by sitedirected mutagenesis (Agilent Quick Change Kit) using the pMH406 plasmid containing esxB-esxA operon as a template. The mutations were confirmed by DNA sequencing. The mutated plasmids were electroporated into MmDEsxA:B as previously described (32).

Liposome leakage assay
The liposome leakage assay was performed as previously described (20,21,29,34,35). Briefly, 20 mg of DOPC (1,2-dioleoyl-sn-glycero-3phosphocholine) was dried with nitrogen air and left in a vacuum overnight. The samples were rehydrated with 1 ml of the buffer (5 mM HEPES, 50 mM ANTS (8-aminonapthalene-1,3,6 trisulfonic acid) and 50 mM DPX (p-xylene-bispyridinium bromide)). The suspension was subjected to 6x freeze-thaw cycles and extruded via a 0.2 µm membrane filter for 20 times. The liposomes were then desalted to remove excess ANTS and DPX using a Hi-trap desalting column. The desalted liposomes were mixed with 150 mM NaCl, 100 mM NaAc at pH 4.0. The reaction mixture was excited at 350 nm and emissions were recorded at 520 nm in an ISS K2 phase modulation fluorometer. 100 µg of the tested protein was injected into the solution after approximately 30 s of the assay and the fluorescence was recorded.

Isolation of EsxA proteins from the Msproduced EsxA:B heterodimer
The EsxA:B heterodimer purified from Ms was incubated in a solution containing 6 M guanidine at 4 °C overnight. The proteins were then passed through a HisTrap column (GE Healthcare). The His-tagged EsxB protein was bound to the column, and the un-tagged EsxA was collected in flow through. The His-tagged EsxB protein was eluted by an imidazole gradient. Both EsxA and EsxB were subjected to an extensive dialysis using a 3,000 MWCO membrane. The samples were then concentrated and passed through gel filtration for a complete buffer exchange.

Detection of N a -acetylation by NBD-Cl
NBD-Cl (4-chloro-7-nitrobenzof-urazan) only reacts with free N-terminal α-amino group in non-acetylated proteins and emits fluorescence, and it does not react with N α -acetylated proteins due to lack of free N-terminal amino group (36). Proteins (6 µM) were incubated with 0.5 mM NBD-Cl at room temperature. At different hours of post-incubation, the samples were subjected to fluorescence measurement with excitation at 460 nm and emission at 535 nm.

Identification of post-translational modifications by mass spectrometry
The purified proteins were digested by FASPtrypsin and analyzed by LC-MS/MS on a QE-Classic. The results were analyzed by PD2.1 search against a combined database containing E. coli BL21 and Ms mc 2 155.

Western Blotting
The Mm strains were cultured in 7H9 medium and grown to mid-log phase. They were washed with PBS and transferred to Sauton's medium while normalizing all cultures to OD600 = 0.8. The bacteria were cultured for 2 days until harvest. The bacterial cells were collected by centrifugation. The proteins in the culture supernatant were precipitated by trichloroacetic acid (TCA). The bacterial cells were resuspended in 1 ml of PBS containing a cocktail of protease inhibitors (Thermofisher) and sonicated at 30% amplitude for 5 cycles of 30 s pulse and 60 s rest. The culture filtrates and total bacterial lysates were applied to SDS-PAGE and transferred onto PVDF membrane. Western blots were performed to detect EsxA using anti-EsxA antibody (sc-57730, Santa Cruz). As controls, Ag85 (secreted in culture filtrate) and GroEL (only in cell lysate) were also detected by anti-Ag85 (NR-13800, BEI) and anti-GroEl antibodies (NR-13813, BEI), respectively.

Live/Dead Cytotoxicity Assay
RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) with penicillin and streptomycin (100U/mL) at 37 °C and 5% CO2. Raw 264.7 macrophages were plated in a 24-well plate with a density of 5 x10 5 /well for infection on the following day. The Mm strains were prepared with a single cell preparation protocol as previously described (22,37). RAW264.7 cells were infected with various Mm strains at a multiplicity of infection (MOI) of 10 for 1 h. The macrophages were washed 3 times with PBS to remove free mycobacteria and incubated for another 3 h. The macrophages were stained using Calcein-AM and ethidium homodimer (Life Sciences) for 30 min, enabling visualization under a fluorescence microscope for green cells (live) and red cells (dead). The numbers of dead cells were quantified from dozens of random fields from each sample.

CCF-4 FRET assay
Mycobacterial cytosolic translocation was measured by CCF-4 FRET assay as previously described (17,22,38). Briefly, RAW264.7 cells were plated in a 6-well plate at a density of 2.5 x 10 6 cells/well. The macrophages were then incubated with CCF4-AM according to the manufacturer's protocol (Liveblazer B/G loading kit, Life Sciences). Cells were infected at a MOI of 10 for 2 h. Following infection, macrophages were washed 3 times using PBS. DMEM media with 10% FBS was added to the cells and incubated for approximately 2 days. The samples were then excited at 409 nm and the emissions were measured at 450 nm and 535 nm. The blue/green ratio was calculated as I450/I535.

Molecular dynamic simulation
The structure of EsxA:B heterodimer was downloaded from Protein Data Bank with PBD ID 1WA8 (39). DelPhiPka web server (40) was used to obtain the protonation states of ionizable residues at pH 4 and pH 7 and assign the respective states with Visual Molecular Dynamics (VMD) (41). N a -acetylation of Thr2 was performed on VMD after removal of Met1. The four structures i.e. non-acetylated and acetylated of EsxA:B heterodimer at either pH 7 or pH 4 were then solvated in water box with TIP3 (42) water model and ionized with 150 mM NaCl in VMD. The final systems were then simulated with molecular dynamics (MD) simulation program NAMD (43). Each simulation was performed for 20 ns employing force field CHARMM27 (44). The temperature was set as 300 K and the pressure was 1 atm. The snapshots from the simulations were taken to study the behaviors of the N-terminal loop of EsxA with and without N α -acetylation of Thr2 at pH 4 and pH 7.

Results
The Ms-produced heterodimer, but not Ecproduced heterodimer, disrupted liposomal membrane at low pH. We hypothesized that Msheterodimer, but not Ec-heterodimer, dissociates at low pH to allow EsxA permeabilize the liposomal membrane. The membranepermeabilizing activity of Ms-heterodimer and Ec-heterodimer was tested with the ANTS/DPX fluorescence de-quenching assay. As expected, Ms-heterodimer permeabilized the membrane at low pH, while Ec-heterodimer did not (Fig. 1A). NBD-Cl (4-chloro-7-nitrobenzof-urazan) only reacts with free N-terminal α-amino group in non-acetylated proteins and emits fluorescence, but it does not react with N α -acetylated proteins due to lack of free N-terminal amino group. Thus, we used NBD-Cl to test the states of N αacetylation for Ms-heterodimer and Echeterodimer. As expected, the Ms-heterodimer exhibited a significantly lower NBD-Cl fluorescence, compared to Ec-heterodimer, indicating that Ms-heterodimer, but not Echeterodimer, is N α -acetylated (Fig. 1B).

The mutations at Thr2 abolished the membrane-permeabilizing activity of the Ms-heterodimers through blocking separation of EsxA and EsxB.
The Q and A residues have been used to functionally mimic acetylation of amino group of an internal K residue, while R serves as a nonacetylated control (45). Thus, we generated T2A, T2Q and T2R mutations and tested the effects of these mutations on the heterodimer membranepermeabilizing activity.
Unexpectedly, all of the mutations abolished the Ms-heterodimer's membrane-permeabilizing activity ( Fig. 2A and B). The result suggests that either the mutations blocked the heterodimer separation or abolished the EsxA membranepermeabilizing activity. To exclude the possibility that the mutations abolished the membrane-permeabilizing activity, we purified the EsxA proteins containing the same mutations from E. coli and applied them to ANTS/DPX dequenching assay. The result showed that the mutations did not affect EsxA membranepermeabilizing activity (Fig. 2C and D), suggesting that the mutations at Thr2 blocked the heterodimer separation at low pH.

The mutations that block separation of EsxA and EsxB do not have N α -acetylation.
To validate the acetylation state of EsxA wild type and the mutants, we developed a protocol and isolated EsxA and EsxB from the Msheterodimers (Fig. 3A). The presence of N αacetylation on the EsxA proteins was measured by NBD-Cl (Fig. 3B). Consistent with the results in Figure 1B, Ec-EsxA(WT) had a significantly higher fluorescence signal than Ms-EsxA(WT). Similar to Ec-EsxA(WT), the Ms-EsxA mutants (T2A, T2Q, and T2R) emitted significantly higher fluorescence signals than Ms-EsxA(WT), suggesting that the Ms-EsxA mutants were not N α -acetylated. Next, we applied Ms-EsxA(WT), Ms-EsxA(T2A) and Ec-EsxA(WT) to LC-MS/MS to further confirm the N α -acetylation states. In addition, Ms-EsxA(T2S), a mutant equivalent to WT, was included in the LC-MS/MS analysis. The results showed that both Ms-EsxA(WT) and Ms-EsxA(T2S) had the first Met residue removed and the second residue (either Thr2 or Ser2) acetylated (Fig. 3C). While Ms-EsxA(T2A) had the first Met residue removed, the second Ala residue was not acetylated. The Ec-EsxA(WT) still has the first Met residue and no acetylation on Thr2. Interestingly, the LC-MS/MS detected multiple acetylation and oxygenation modifications in the internal sequences of both Ms-EsxA and Ec-EsxA and the roles of these modifications are currently unknown.
EsxB preferred to bind the non-acetylated EsxA to inhibit the membrane-permeabilizing activity. An earlier study has shown that EsxB preferred to bind the non-acetylated EsxA than acetylated EsxA in a 2D overlay assay (26). Thus, we hypothesize that EsxB will prefer to inhibit the membrane-permeabilizing activity of the nonacetylated Ms-EsxA(T2A) than that of the acetylated Ms-EsxA(WT). First, we tested and confirmed that the proteins Ms-EsxA(WT) and Ms-EsxA(T2A) that were isolated from the heterodimers had similar membranepermeabilizing activity to Ec-EsxA(WT), which once again confirms that the states of N αacetylation does not affect membranepermeabilizing activity (Fig. 4A and B). Then, Ms-EsxA(WT) and Ms-EsxA(T2B) were incubated with EsxB at a series of EsxB/EsxA molar ratios. As expected, EsxB preferred to inhibit Ms-EsxA(T2A) than Ms-EsxA(WT) in membrane permeabilization, especially at the lower EsxB/EsxA ratios (Fig. 5A, B and C).

The mutations without N α -acetylation attenuated mycobacterial virulence and inhibited cytosolic translocation.
Here, we investigated the effects of the T2X mutations in mycobacterial pathogenesis. The genes carrying T2X mutations were expressed in the MmDEsxA:B strain, in which the endogenous esxB-esxA operon was deleted. We found that the T2X mutations did not affect the expression and secretion of EsxA and EsxB in the Mm strains (Fig. 6A). As expected, the Mm strains carrying the non-acetylated mutations T2A, T2Q and T2R had a significantly lower cytotoxicity than the strain carrying the acetylated mutation T2S (Fig.  6B). MmWT and MmDEsxA:B were used as the positive control and negative control, respectively. Furthermore, using the previously established CCF4-FRET assay, we found that the non-acetylated mutations T2A, T2Q and T2R abolished mycobacterial cytosolic translocation, while T2S maintained a similar activity as the wild type (Fig. 6C). The data is consistent to the previous report that deletion of a N αacetyltransferase in Mm disrupted the homeostasis of EsxA acetylation and attenuated the virulence (28).

Molecular dynamic simulation detects frequent "bind-and-release" contacts between the acetylated Thr2(Ac) and EsxB.
The reported NMR solution structure of EsxA:B heterodimer does not have the N α -acetylation on Thr2, and the structure shows Thr2 is distal from the contact interface between EsxA and EsxB. It is not clear how the acetylation at Thr2 affects the heterodimer separation at low pH. Thus, we performed molecular dynamic (MD) simulation on the heterodimers containing either nonacetylated or acetylated Thr2 at pH 7 and pH 4, respectively (Figure 7). At pH 7 the nonacetylated Thr2 comes in a close vicinity of EsxB, but it is unable to make a direct contact to EsxB (Figure 7A, C). Compared to non-acetylated Thr2, the acetylated Thr2(Ac) moves further away from EsxB at pH 7 ( Figure 7B, D). Interestingly, at pH 4 the non-acetylated Nterminal loop of EsxA has no direct contact with EsxB ( Figure 7E, G), but the acetylated Nterminal loop is able to make direct contacts with EsxB ( Figure 7F, H). The MD simulation shows that the acetylated loop make direct contacts with EsxB in a frequent "bind-and-release" mode (Movie S1)), which generates a pulling force to trigger the dissociation of the complex.

Discussion
EsxA and EsxB are co-expressed and co-secreted as a heterodimer in mycobacteria. The role of ESX-1, EsxA and EsxB in mycobacterial cytosolic translocation and virulence has been confirmed in a series of studies (7,(17)(18)(19). Earlier biochemical studies have demonstrated that EsxA has a pH-dependent membrane-permeabilizing activity, while EsxB appears to function as a chaperone for EsxA (20). Current studies support a model that the EsxA:B heterodimer is dissociated at low pH, which allows EsxA to permeabilize the membranes (24). However, the mechanism of the heterodimer separation is not known. For the first time, the present study tested the heterodimers with or without N α -acetylation of Thr2 and obtained the evidence that the N αacetylation at Thr2 of EsxA facilitates the heterodimer separation at low pH, which allows EsxA to permeabilize liposomal membrane in vitro as well as mediate mycobacterial phagosome escape and cytosolic translocation in mycobacteria-infected macrophages.
As discussed above, the essentiality of EsxA and EsxB in mycobacterial pathogenesis has been well documented in a series of reports. Genetic manipulations that either deleted the gene of esxA or esxB, or abolished the secretion of EsxA and EsxB, have attenuated mycobacterial virulence and inhibited the phagosome rupture, cytosolic translocation and cell-to-cell spreading (7,10,(17)(18)(19). Moreover, the biochemical characterizations have demonstrated that EsxA possesses a unique membrane-permeabilizing activity that is not present in its ortholog in non-pathogenic M. smegmatis (20). Thus, it is reasonable to believe that during the course of infection, the secreted EsxA exerts its membrane-permeabilizing activity to penetrate the phagosome membranes and facilitate mycobacterial cytosolic translocation. We have reported that the mutations at the Gln 5 residue of EsxA (e.g. Q5V and Q5K) have resulted in up-or downregulation of EsxA membrane-permeabilizing activity in vitro. Moreover, these mutations up-or down-regulated the mycobacterial virulence and cytosolic translocation accordingly, demonstrating the specific and accurate correlation between EsxA membranepermeabilizing activity and mycobacterial virulence as well as the ability to penetrate phagosome membrane (22). Once again, the present study provides new evidence that the N αacetylation at Thr2 of EsxA is required for mycobacterial virulence and cytosolic translocation through facilitating the heterodimer separation.
Since Thr2 has no direct contact with EsxB as shown in the reported solution structure of EsxA:B heterodimer, how N α -acetylation on Thr2 affect heterodimer separation had become a puzzle. Here, the MD simulation result has provided a convincing evidence that the acetylated Thr2(Ac) makes frequent "bind-andrelease" contacts with EsxB only at low pH, generating a dragging force to pull EsxB away from EsxA.
A recent study has shown that the recombinant EsxA does not lyse cell membranes, and the lytic activity previously attributed to EsxA is due to residual ASB-14 detergent in the preparation (46). In fact, we had the similar observations that addition of the recombinant EsxA protein to the surface of lung epithelial cell lines WI-26 and A549 did not lyse the cells (data not shown). Moreover, Conrad et. al. showed that blocking phagosomal acidification by Bafilomycin did not decrease the ESX-1-mediated phagosomal permeabilization, suggesting that acidification is not required for membrane permeabilization (46). It is not clear how the discrepancy arises and what is the broken link between the EsxA pHdependent membrane-permeabilizing activity in model membrane and the ability of mycobacteria to rupture phagosome membrane during infection. Other factors from mycobacteria and host cells, even including properties of target membranes, may be involved in this process, which warrants further investigations.