Bovine serum albumin promotes reactivation of viable but non-culturable Mycobacterium tuberculosis via activation of protein kinase-dependent cell division processes

Abstract

in shade. Excessive dye was removed using 3% hydrochloric acid ethanol for 15 minutes. Next, 164 the smear was covered with 10 µg/mL Nile Red in ethanol and incubated at room temperature 165 for 15 minutes. Finally, the smear was counterstained with 0.1% (w/v) potassium permanganate 166 solution with 1 minute. Stained slides were air-dried and mounted as described previously. 167 Fluorescence microscopy was performed at 100× magnification for green (acid-fastness   The VBNC reactivation assay was performed as described elsewhere (18)  into 24-well plates in 1.5 mL increments and sealed with gas-permeable film. The plate was 215 incubated for 20 days at 37˚C under 5% CO2. At the end of incubation, the number of colonies 216 grown was measured as described previously.

217
Evaluation of the reactivation-promoting effect of fatty acid and globulin-free BSA 218 To determine the effect of BSA Cohn fraction V contaminants, we measured the reactivation-219 promoting activity of fatty acid and globulin-free BSA toward DPI-treated VBNC Mtb cells.      for the untreated control population and 4.64 ± 6.10 × 10 3 CFU/mL for DPI-treated population. culturability. The addition of albumin alone into the medium could also induce reactivation.

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The addition of FBS in BSA-free Dubos medium slightly reduced the regrowth rate; however, BSA promotes reactivation of VBNC M. tuberculosis cells were successfully reactivated at the end of incubation with or without FBS. These 284 phenomena were also observed in H37Ra (Suppl. Fig 2). Thus, we used the H37Rv strain for 285 the further analyses in this study.

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Incubation with sodium pyruvate, which was reported to have a reactivation-promoting 287 effect on VBNC cells elsewhere, led to transient regrowth by day 15 and a slight reduction at 288 day 20. (Suppl. Fig. 3[A] and [B]). 289 We also confirmed that the presence of small population of intact Mtb cells (10 3 CFU/mL) 290 could grow normally in BSA-free Dubos medium, suggesting that the reactivation might not 291 be due to the presence of small number of culturable cells after DPI-treatment (data not shown).

IV.
The antioxidative property or the fatty acid from BSA did not promote 293 reactivation. 294 As shown in Fig. 4(A) and (B), the reactivation-promoting effect of BSA was specific to 295 bovine and human serum albumin. Ovalbumin did not show a reactivation-promoting effect 296 but maintained the number of culturable cells in this system. NAC (antioxidative agent) and 297 D-mannitol (free radical scavenger) did not show any reactivation capacity. 298 We also checked whether the purity of albumin affects the promotion of reactivation using 299 fatty acid and globulin-free BSA and confirmed that there was no significant difference in 300 reactivation-promoting effects (Suppl. Fig 4). These results suggest that commercially 301 available albumin including fatty acids do not affect the reactivation of DPI-treated Mtb. incubation with 10 µM or 30 µM H89 resulted in a CFU/mL value for 3.42 × 10 6 ± 7.45 × 10 5 308 CFU/mL and 3.63 ± 1.70 × 10 2 CFU/mL, while incubation without H89 resulted in 3.00 × 10 8 309 ± 5.07 × 10 7 CFU/mL. We also confirmed that staurosporine, which is known as mycobacterial 310 protein kinase PknB inhibitor (35), suppressed reactivation at 10 µM and resulted in a CFU/mL 311 value for 2.09 × 10 3 ± 7.62 × 10 2 CFU/mL, while incubation without staurosporine resulted in 312 3.58 × 10 8 ± 5.78 × 10 7 CFU/mL (Fig 5[A] and [B]). All inhibitors used in this study did not 313 cause a reduction of the growth of intact MTB cells (Suppl. Fig. 5A and 5B). 314 We also performed molecular docking simulation of these inhibitors toward their 315 considerable targets on Mtb. As shown in Suppl. In this study, we confirmed that DPI could induce a VBNC state in H37Rv as well as H37Ra.

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The mechanism underlying the effect of DPI is considered to involve the inhibitory effect of 320 NADH oxidase, which results in the inhibition of the electron transport system of Mtb. This with Nile Red (Fig. 2[F]). This could also reveal the distribution of the lipid body in the cell as 330 some foci of relatively strong signals of Nile Red, suggesting that the transformation from 331 growing state to VBNC, with drastic alteration of the lipid metabolism.

BSA promotes reactivation of VBNC M. tuberculosis
Secondly, we found that DPI-induced VBNC could facilitate reactivation not only by 333 incubation with FBS but also with OADC supplementation and BSA alone, suggesting albumin 334 might act as a reactivation-promoting agent in both H37Rv and H37Ra (Fig .3 and Suppl. Fig.   335 2). These findings were contrary to the findings of the previous study, which showed that only 336 FBS could facilitate reactivation (18). We considered the reason for the difference may be due further analyses were performed using H37Rv.

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We also tested the reactivation-promoting effect of BSA in Wayne's hypoxic culture, which 343 is widely used for inducing a VBNC of Mtb, and found that there was no significant difference 344 with or without BSA (data not shown).

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Pyruvate, which is known to act as reactivation promoting agent for both gram-negative In this study, we should note that impurities 385 of albumin did not affect reactivation. Although the underlying mechanism is still unclear, both 386 fatty acid and globulin-free BSA and BSA Cohn fraction V showed similar reactivation-387 promoting effect toward DPI-treated Mtb (Suppl. Fig. 4).

388
The reactivation inhibition assay by SQ22536, H89 and staurosporine gave us an important 389 clue for understanding the effect of BSA. In the present study, we could suppress the which was shown in PknB (64) and staurosporine, which act as PknB inhibitor, could also 408 inhibit reactivation of DPI-induced VBNC Mtb (Fig. 5). 409 Our study suggested that the inhibition of mycobacterial protein kinase by H89 and BSA promotes reactivation of VBNC M. tuberculosis staurosporine seems to critically affect several important cellular processes, followed by 411 reactivation (Fig. 6).