Lipid

4 Lisophosphatidyl-

5. Lisophosphatidyl-

<sup>8</sup>Linoleic acid and lisoPC

BACTEC MGIT960.

900

Fig. 5. Murohashi staining of *M. tuberculosis* cells (14th day of growth). 1-H37Rv; 2-H37Rv+CL (50 µg/ml); 3-H37Rv+CL (750 µg/ml). Reproduced from [Mikulovish, 2010]

These data correlate well with the results of mycobacterial DNA detection by real time PCR, according to which appearance CL in culture medium didn't lead to increase of DNA number [Sorokoumova, 2009] (fig. 6).

Fig. 6. Growth curves of *M. tuberculosis* H37Rv in Dubo's medium in the presence of CL liposomes. Growth was detected with real time PCR. 1-H37Rv; 2-H37Rv+CL (50 µg/ml); 3-H37Rv+CL (250 µg/ml) ; 4-H37Rv+CL (500 µg/ml); 5-H37Rv+CL (750 µg/ml). Reproduced from [Sorokoumova G.M., 2009]

CL is an instable compound that transforms in several substances in water phase. Both in Dubo's medium, containing high concentration of iron ions, and in Tris-buffer, which doesn't have an iron, the products of hydrolysis were mainly formed: lyso-, bislysocomponents and FA (fig. 7). Along with this components such products of

Lipid Surrounding of Mycobacteria: Lethal and Resuscitating Effects 247

**3.1 Mycobacterial lipids released into extracellular space during formation of dormant** 

In vitro model of *Mycobacterium smegmatis* dormant state obtained due to absence of potassium ions in growth medium, created by Kaprelyants, A.S. with colleagues., let us to show that during formation of nonculturable ATD-resistant cells they lose up to 90% of their mass, which reflects in relatively balanced decrease in both lipid and protein amounts [Nazarova, 2011] (fig. 8). Nevertheless at the end of this process nonculturable cells have larger percentage of proteins rather than lipids. Qualitative lipid composition of the cells doesn't noticeably change during transition into nonculturable state with the exception of the drop of the trehalose monomycolate level. Trehalose monomycolate is a precursor of the cord-factor trehalose dimycolate, which is inherent for mycobacterial cells in all of the states.

Fig. 8. *Mycobacterium smegmatis* growth in mH-deB medium (1), lipid (2), and protein (3) content in cells during transition into nonculturable state depending on time based on dry

Cell mass loss during transition into dormant state certainly might lead to appearance of detached cell lipids in surrounding medium. To detect such compounds in growth medium, we conducted a chloroform extraction of culture liquid, separated from the cells through the 200 nm filter and lyophilized. Nonculturable forms of *M. smegmatis* were obtained as the result of cultivation in mH-deB medium. We used the cells, grown in nutrient and potassium ion rich Sauton's medium, for control. *M. smegmatis* cells upon metabolically active growth as well as upon formation of nonculturable cells release into culture liquid a considerable range of neutral lipids: FA, mycolic acids (MA), diacylglycerols (DAG), monoacylglycerols (MAG) (fig. 9). It is noteworthy that mycobacteria in none of the states don't release triacylglycerols (TAG), intracellular energy storage component. Qualitative

biomass. Reproduced from [Nazarova, 2011].

**3. Role of lipid components in formation and reactivation of nonculturable** 

**mycobacterial cells** 

**cells** 

destruction as phosphatidic acid, phosphatidylglycerol, as well as the products of their hydrolysis (lysophosphatidic acid and lysophosphatidylglycerol appear in cultivation medium. Mentioned substances were isolated by preparative TLC from water phase, in which CL was cultivated without *M. tuberculosis* at 370 С for 4 or 6 days, and were identified by MS [Smirnova, 2011]. Summarized results of their effect on *M. tuberculosis* growth is represented in table 1.

Fig. 7. Structural formulas of 1 – cardiolipin, 2 – lysocardiolipin, 3 – bislysocardiolipin, 4 – linoleic acid.

It's interesting, that lysophosphatidylglycerol, the product of phosphatidylglycerol hydrolysis, doesn't have any inhibitory activity in involved concentration interval, as well as a mixture of lysophosphatidylglycerol with linoleic acid with concentration lower than 335 µM, which is inhibitory for phosphatidylglycerol. Thus, conducted experiments have shown that only negatively-charged PL (CL, phosphatidic acid, phosphatidylglycerol), but not their lysocomponents, that have negative charge as well, are able to cause the delay in growth and suppress viability of the mycobacterial cells almost in the same concentration range (250 - 335 µМ). Based on the literature one may speculate the mechanisms of negatively-charged PL activity, that inhibit the bacterial cell growth due to:


To conclude, the same PL seems to have various effects on the growth of different species mycobacteria. Perhaps, it may be explained by a different destiny of PL once they were introduced into 'mycobacteria space': weather they serve only as FA source, or may play a role in metabolism of microorganism by themselves. Therefore we further investigated the alterations in lipid surrounding of mycobacteria in different states: in actively replicating and nonculturable.

destruction as phosphatidic acid, phosphatidylglycerol, as well as the products of their hydrolysis (lysophosphatidic acid and lysophosphatidylglycerol appear in cultivation medium. Mentioned substances were isolated by preparative TLC from water phase, in which CL was cultivated without *M. tuberculosis* at 370 С for 4 or 6 days, and were identified by MS [Smirnova, 2011]. Summarized results of their effect on *M. tuberculosis* growth is

Fig. 7. Structural formulas of 1 – cardiolipin, 2 – lysocardiolipin, 3 – bislysocardiolipin,

negatively-charged PL activity, that inhibit the bacterial cell growth due to:


complex, which prevents cell division [Mizushima, 1992].

It's interesting, that lysophosphatidylglycerol, the product of phosphatidylglycerol hydrolysis, doesn't have any inhibitory activity in involved concentration interval, as well as a mixture of lysophosphatidylglycerol with linoleic acid with concentration lower than 335 µM, which is inhibitory for phosphatidylglycerol. Thus, conducted experiments have shown that only negatively-charged PL (CL, phosphatidic acid, phosphatidylglycerol), but not their lysocomponents, that have negative charge as well, are able to cause the delay in growth and suppress viability of the mycobacterial cells almost in the same concentration range (250 - 335 µМ). Based on the literature one may speculate the mechanisms of



To conclude, the same PL seems to have various effects on the growth of different species mycobacteria. Perhaps, it may be explained by a different destiny of PL once they were introduced into 'mycobacteria space': weather they serve only as FA source, or may play a role in metabolism of microorganism by themselves. Therefore we further investigated the alterations in lipid surrounding of mycobacteria in different states: in actively replicating

represented in table 1.

4 – linoleic acid.

[Yamamoto, 2002];

and nonculturable.

### **3. Role of lipid components in formation and reactivation of nonculturable mycobacterial cells**

#### **3.1 Mycobacterial lipids released into extracellular space during formation of dormant cells**

In vitro model of *Mycobacterium smegmatis* dormant state obtained due to absence of potassium ions in growth medium, created by Kaprelyants, A.S. with colleagues., let us to show that during formation of nonculturable ATD-resistant cells they lose up to 90% of their mass, which reflects in relatively balanced decrease in both lipid and protein amounts [Nazarova, 2011] (fig. 8). Nevertheless at the end of this process nonculturable cells have larger percentage of proteins rather than lipids. Qualitative lipid composition of the cells doesn't noticeably change during transition into nonculturable state with the exception of the drop of the trehalose monomycolate level. Trehalose monomycolate is a precursor of the cord-factor trehalose dimycolate, which is inherent for mycobacterial cells in all of the states.

Fig. 8. *Mycobacterium smegmatis* growth in mH-deB medium (1), lipid (2), and protein (3) content in cells during transition into nonculturable state depending on time based on dry biomass. Reproduced from [Nazarova, 2011].

Cell mass loss during transition into dormant state certainly might lead to appearance of detached cell lipids in surrounding medium. To detect such compounds in growth medium, we conducted a chloroform extraction of culture liquid, separated from the cells through the 200 nm filter and lyophilized. Nonculturable forms of *M. smegmatis* were obtained as the result of cultivation in mH-deB medium. We used the cells, grown in nutrient and potassium ion rich Sauton's medium, for control. *M. smegmatis* cells upon metabolically active growth as well as upon formation of nonculturable cells release into culture liquid a considerable range of neutral lipids: FA, mycolic acids (MA), diacylglycerols (DAG), monoacylglycerols (MAG) (fig. 9). It is noteworthy that mycobacteria in none of the states don't release triacylglycerols (TAG), intracellular energy storage component. Qualitative

Lipid Surrounding of Mycobacteria: Lethal and Resuscitating Effects 249

Level of free FA in growth medium in active state decreases (from 29 μМ to 5 μМ), which indicates an involvement of FA in metabolism and biosynthesis of required lipids for

We consider complex of potassium absence and accumulation of FA in growth medium as a cause of transition of mycobacteria into dormant state in the present model. Bovine serum albumin (BSA) is conventionally required for binding of FA excess in culture liquid in laboratory experiments. When applying BSA A1470 Sigma Cohn Analog™, mentioned FA accumulation and formation of nonculturable cells occur, whereas in case when applying Fatty Acid Free-BSA MP Biomedicals, LLC, secreted FA are entirely bound and dormant

To determine the amount of FA, that were introduced initially into growth medium, we have measured the level of free FA, present in the used for experiments Tween-80, and

The mixture of low concentration Tween 80 and water was titrated with 0,014M NaOH using 0.01% thymol blue solution as an indicator. This method let us to estimate the quantity only of free FA, not exposing an ester group for hydrolysis. FA were shown to be brought into growth medium together with Tween 80 in amount of 15% of detergent mass (table 2). We applied a technique, based on combination of methods described by Chen, R.F., and Dole, V.P., for determination of quantity of FA bound with BSA [Chen, 1967; Dole, 1956]. Briefly: 25 ml of extraction mixture (isopropyl alcohol : heptane : 1N H2SO4 (40:10:1)) were added to 5 ml of water solution of BSA (0,1 g/ml), shaken, let to stand for 30 minutes. Then 10 ml of heptane and 15 ml of 0,1N H2SO4 were added to the mixture and shaken again. The upper phase is transferred to another flask, that contains 25ml of 0,01N H2SO4, shaken again. The upper heptane fraction is separated and titrated as described above. It was shown that BSA A1470 Sigma Cohn Analog™ possesses only 15%-binding capability, while Fatty Acid Free-BSA MP Biomedicals, LLC - 87,5% (of the maximum possible) (table 2).

Thus, addition of BSA (Cohn An.), possessing only 15 % (moles) binding capability of the maximum possible, result in the formation of nonculturable cells. Remaining 15%is necessary for the binding of 57,3 μМ of FA, incorporated together with Tween 80. Therefore we may conclude that 33-59 μМ of free FA found in the medium can't be bound with BSA, and as the result have to be accumulated in culture liquid, which may lead to the formation of nonculturable cells in response to stressful conditions. As it was mentioned earlier, these

In the case of more pure BSA (MP Biomedicals, LLC) it is obvious that despite the presence of free FA in Tween 80 and negligible traces in BSA, it is still capable of binding up to 205,2 μМ of FA (i.e. 68,4% of maximum possible 300 μМ). Therefore FA, that might be secreted during the lifetime of mycobacterial cells are removed from them with BSA, which enables

Thus in conducted experiments we demonstrated secretion of lipid substances including FA into surrounding medium during growth upon stressful conditions which results in transition of bacilli into dormant state. Moreover extracellular FA are accumulated in this

FA have biogenic nature, and might be the product of Tween 80 hydrolysis.

more successful adaptation to stressful conditions, and dormant cells do not form.

metabolically active mycobacterial cells.

content of the FA bound with BSA.

state is not achieved.

process.

lipid composition of culture liquids both in the process of active growth and during transition into nonculturable state doesn't change. However only during development of nonculturable cells level of released fatty acids enlarges twice (from 33 μМ to 59 μМ). These data were obtained after isolation of free FA with preparative TLC and quantification of their amount with titration.

Fig. 9. TLC of lipids in system heptane–diethyl ether–acetic acid (55 : 45 : 1 v/v). Lipid extract of *M. smegmatis* cells cultivated for 24 h in Sauton's medium 1); 2) lipid extract of culture liquid of *M. smegmatis* cells grown for 24 h in Sauton's medium; 3) lipid extract of *M. smegmatis* cells cultivated for 24 h in mH-deB medium; 4) lipid extract of culture liquid after cultivation of *M. smegmatis* cells for 24 h in mH-deB medium. Development by phosphomolybdic acid solution with following plate heating at 100°C. Represented from [Nazarova, 2011].

Analysis of *M. smegmatis* culture liquid free-FA content by GLC-MS showed that oleic acid was a prevalent component. NMR-spectra of FA isolated from a culture liquid and of the oleic acid standard appeared to be identical.

To prove a biogenic nature if the found in culture medium FA we incorporated a radioactive carbon isotope in lipids of mycobacteria as the result of [14C]aspartic acid metabolism. After addition of tagged compound to the *M. smegmatis* cells grown in mH-deB for 46.5 h, a part of the sample was taken for control and the rest was left to cultivate for other day at 37°С. Gained lipid extracts of culture medium were divided on separate substance with reversephase and thin-layer chromatography. Measurement of radioactivity of different zones of TLC-plate demonstrated that in zone, corresponding to oleic acid there is an accumulation of radioactivity (12 490 cpm) as compared to control sample (356 cpm). Incorporation of [14C]aspartic acid followed by accumulation of radioactivity in extracellular FA confirms that at least partially FA detected in growth medium are actually secreted.

lipid composition of culture liquids both in the process of active growth and during transition into nonculturable state doesn't change. However only during development of nonculturable cells level of released fatty acids enlarges twice (from 33 μМ to 59 μМ). These data were obtained after isolation of free FA with preparative TLC and quantification of

Fig. 9. TLC of lipids in system heptane–diethyl ether–acetic acid (55 : 45 : 1 v/v). Lipid extract of *M. smegmatis* cells cultivated for 24 h in Sauton's medium 1); 2) lipid extract of culture liquid of *M. smegmatis* cells grown for 24 h in Sauton's medium; 3) lipid extract of *M. smegmatis* cells cultivated for 24 h in mH-deB medium; 4) lipid extract of culture liquid after

phosphomolybdic acid solution with following plate heating at 100°C. Represented from

Analysis of *M. smegmatis* culture liquid free-FA content by GLC-MS showed that oleic acid was a prevalent component. NMR-spectra of FA isolated from a culture liquid and of the

To prove a biogenic nature if the found in culture medium FA we incorporated a radioactive carbon isotope in lipids of mycobacteria as the result of [14C]aspartic acid metabolism. After addition of tagged compound to the *M. smegmatis* cells grown in mH-deB for 46.5 h, a part of the sample was taken for control and the rest was left to cultivate for other day at 37°С. Gained lipid extracts of culture medium were divided on separate substance with reversephase and thin-layer chromatography. Measurement of radioactivity of different zones of TLC-plate demonstrated that in zone, corresponding to oleic acid there is an accumulation of radioactivity (12 490 cpm) as compared to control sample (356 cpm). Incorporation of [14C]aspartic acid followed by accumulation of radioactivity in extracellular FA confirms

cultivation of *M. smegmatis* cells for 24 h in mH-deB medium. Development by

that at least partially FA detected in growth medium are actually secreted.

their amount with titration.

[Nazarova, 2011].

oleic acid standard appeared to be identical.

Level of free FA in growth medium in active state decreases (from 29 μМ to 5 μМ), which indicates an involvement of FA in metabolism and biosynthesis of required lipids for metabolically active mycobacterial cells.

We consider complex of potassium absence and accumulation of FA in growth medium as a cause of transition of mycobacteria into dormant state in the present model. Bovine serum albumin (BSA) is conventionally required for binding of FA excess in culture liquid in laboratory experiments. When applying BSA A1470 Sigma Cohn Analog™, mentioned FA accumulation and formation of nonculturable cells occur, whereas in case when applying Fatty Acid Free-BSA MP Biomedicals, LLC, secreted FA are entirely bound and dormant state is not achieved.

To determine the amount of FA, that were introduced initially into growth medium, we have measured the level of free FA, present in the used for experiments Tween-80, and content of the FA bound with BSA.

The mixture of low concentration Tween 80 and water was titrated with 0,014M NaOH using 0.01% thymol blue solution as an indicator. This method let us to estimate the quantity only of free FA, not exposing an ester group for hydrolysis. FA were shown to be brought into growth medium together with Tween 80 in amount of 15% of detergent mass (table 2).

We applied a technique, based on combination of methods described by Chen, R.F., and Dole, V.P., for determination of quantity of FA bound with BSA [Chen, 1967; Dole, 1956]. Briefly: 25 ml of extraction mixture (isopropyl alcohol : heptane : 1N H2SO4 (40:10:1)) were added to 5 ml of water solution of BSA (0,1 g/ml), shaken, let to stand for 30 minutes. Then 10 ml of heptane and 15 ml of 0,1N H2SO4 were added to the mixture and shaken again. The upper phase is transferred to another flask, that contains 25ml of 0,01N H2SO4, shaken again. The upper heptane fraction is separated and titrated as described above. It was shown that BSA A1470 Sigma Cohn Analog™ possesses only 15%-binding capability, while Fatty Acid Free-BSA MP Biomedicals, LLC - 87,5% (of the maximum possible) (table 2).

Thus, addition of BSA (Cohn An.), possessing only 15 % (moles) binding capability of the maximum possible, result in the formation of nonculturable cells. Remaining 15%is necessary for the binding of 57,3 μМ of FA, incorporated together with Tween 80. Therefore we may conclude that 33-59 μМ of free FA found in the medium can't be bound with BSA, and as the result have to be accumulated in culture liquid, which may lead to the formation of nonculturable cells in response to stressful conditions. As it was mentioned earlier, these FA have biogenic nature, and might be the product of Tween 80 hydrolysis.

In the case of more pure BSA (MP Biomedicals, LLC) it is obvious that despite the presence of free FA in Tween 80 and negligible traces in BSA, it is still capable of binding up to 205,2 μМ of FA (i.e. 68,4% of maximum possible 300 μМ). Therefore FA, that might be secreted during the lifetime of mycobacterial cells are removed from them with BSA, which enables more successful adaptation to stressful conditions, and dormant cells do not form.

Thus in conducted experiments we demonstrated secretion of lipid substances including FA into surrounding medium during growth upon stressful conditions which results in transition of bacilli into dormant state. Moreover extracellular FA are accumulated in this process.

Lipid Surrounding of Mycobacteria: Lethal and Resuscitating Effects 251

resuscitation were concentration dependent (Fig. 11). It is important that the oleic acid concentrations (0.05-3 µg/ml) were in significantly lower range compared to phospholipids (50-250 µg/ml) (Fig. 11). Such low concentration excludes the use of free FA in this process as a nutrient source, and that they are the substances responsible for reactivation, while phospholipids serve as their sources (due to activity of bacterial phospholipases). FA are known to function as signal substances in a number of important processes in bacterial cells. Thus, arachidonic acid serves as chemoattractant for *Dictyostelium discoideum* [Schaloske, 2007]. Another example of FA signal function is their secretion into extracellular space by the *Xanthomonas campestris*, plant pathogen, and their following absorption resulting in increased expression of genes associated with cell pathogenicity [Barber, 1997]. So, we have revealed a new, not previously described signal function of oleic acid as an agent

stimulating reactivation of nonculturable mycobacterial forms.

Fig. 10. Effect of FA and substances of lipid nature (in concentration of 1 µg/ml) on resuscitation of *M. smegmatis* nonculturable cells. Represented from [Nazarova, 2011].

Fig. 11. Effect of phosphatidylcholine (a) and oleic acid (b) in different concentrations on resuscitation of *M. smegmatis* nonculturable cells. Represented from [Nazarova, E.V., 2011].


\* Maximum binding capability of BSA (FA mole / BSA mole) was shown to be ~ 4 [Spector, 1969].

Table 2. Amounts of FA introduced into growth medium along with Tween 80 and BSA with consideration of experimentally used concentrations. Amounts of free FA detected in growth medium.

It is well known that *M. smegmatis* and *M. tuberculosis* dormant cells can be reactivated with resuscitation promoting factor (Rpf) [Shleeva, 2004; Shleeva, 2011]. An influence of FA on resuscitation appears to be interesting in the context of the shown data on their role in the process of adaptation to stressful conditions.

#### **3.2 An effect of lipid substances on reactivation of mycobacterial dormant cells**

*M. smegmatis* dormant nonculturable cells have been studied to be reactivated by their incorporation into fresh medium containing potassium ions and various lipid substances (triacylglycerols, PL, FA) [Nazarova, 2011]. Oleic acid is observed to have the strongest resuscitation effect, and its active concentrations are 0.18-10.6 μМ. Such low concentration range sufficient for reactivation of dormant cells indicates a signal function of this FA in resuscitation.

The capability of phospholipids to stimulate growth of M. tuberculosis cells in the late stationary phase was known previously [Zhang, 2001]. Since FA are components of PL, we supposed that both phospholipids and FA may be able to stimulate reactivation of dormant cells. To study the role of the lipid compounds in resuscitation process, nonculturable cells were transferred into fresh medium containing potassium ions and a certain amount of lipid component, which effect on cell reactivation was estimated by the method of final dilutions.

As it was found different types of lipids are objectively capable to "trigger" the process of resuscitation of nonculturable cells. Addition of oleic acid compared to different length acids, higher alcohols, and TAG in concentration of 1 µg/ml to 106 nonculturable cells was the most efficient for reactivation (Fig. 10). The phospholipid and oleic acid effects on

Tween 80 382 Admixed FA in Tween 80 57.3 Percentage of admixed FA in Tween 80 15%

Maximum binding capacity of BSA at рН 6,6-7 \* 300 BSA (Cohn An.) 75 FA bound with BSA (Cohn An.) 255 Binding capacity of BSA (Cohn An.) 15%

BSA (МР) 75 FA bound with BSA (МР) 37.5 Binding capacity of BSA (МР) 87.5%

\* Maximum binding capability of BSA (FA mole / BSA mole) was shown to be ~ 4 [Spector, 1969]. Table 2. Amounts of FA introduced into growth medium along with Tween 80 and BSA with consideration of experimentally used concentrations. Amounts of free FA detected in

It is well known that *M. smegmatis* and *M. tuberculosis* dormant cells can be reactivated with resuscitation promoting factor (Rpf) [Shleeva, 2004; Shleeva, 2011]. An influence of FA on resuscitation appears to be interesting in the context of the shown data on their role in the

*M. smegmatis* dormant nonculturable cells have been studied to be reactivated by their incorporation into fresh medium containing potassium ions and various lipid substances (triacylglycerols, PL, FA) [Nazarova, 2011]. Oleic acid is observed to have the strongest resuscitation effect, and its active concentrations are 0.18-10.6 μМ. Such low concentration range sufficient for reactivation of dormant cells indicates a signal function of this FA in

The capability of phospholipids to stimulate growth of M. tuberculosis cells in the late stationary phase was known previously [Zhang, 2001]. Since FA are components of PL, we supposed that both phospholipids and FA may be able to stimulate reactivation of dormant cells. To study the role of the lipid compounds in resuscitation process, nonculturable cells were transferred into fresh medium containing potassium ions and a certain amount of lipid component, which effect on cell reactivation was estimated by the method of final dilutions. As it was found different types of lipids are objectively capable to "trigger" the process of resuscitation of nonculturable cells. Addition of oleic acid compared to different length acids, higher alcohols, and TAG in concentration of 1 µg/ml to 106 nonculturable cells was the most efficient for reactivation (Fig. 10). The phospholipid and oleic acid effects on

**3.2 An effect of lipid substances on reactivation of mycobacterial dormant cells** 

Free FA in growth

growth medium.

resuscitation.

process of adaptation to stressful conditions.

medium

**Component of the medium Concentration in culture liquid, µM** 

24 h. 33.2 48 h. 41.4 72 h. 59

(57.3· 100%/382)

((300-255)· 100%/300)

((300-37.5)· 100%/300)

resuscitation were concentration dependent (Fig. 11). It is important that the oleic acid concentrations (0.05-3 µg/ml) were in significantly lower range compared to phospholipids (50-250 µg/ml) (Fig. 11). Such low concentration excludes the use of free FA in this process as a nutrient source, and that they are the substances responsible for reactivation, while phospholipids serve as their sources (due to activity of bacterial phospholipases). FA are known to function as signal substances in a number of important processes in bacterial cells. Thus, arachidonic acid serves as chemoattractant for *Dictyostelium discoideum* [Schaloske, 2007]. Another example of FA signal function is their secretion into extracellular space by the *Xanthomonas campestris*, plant pathogen, and their following absorption resulting in increased expression of genes associated with cell pathogenicity [Barber, 1997]. So, we have revealed a new, not previously described signal function of oleic acid as an agent stimulating reactivation of nonculturable mycobacterial forms.

Fig. 10. Effect of FA and substances of lipid nature (in concentration of 1 µg/ml) on resuscitation of *M. smegmatis* nonculturable cells. Represented from [Nazarova, 2011].

Fig. 11. Effect of phosphatidylcholine (a) and oleic acid (b) in different concentrations on resuscitation of *M. smegmatis* nonculturable cells. Represented from [Nazarova, E.V., 2011].

Lipid Surrounding of Mycobacteria: Lethal and Resuscitating Effects 253

supported by our results where only 0.18-10.6 μМ of oleic acid is required for resuscitation

The authors acknowledge Dr. Arseny S. Kaprelyants and his laboratory at Bach Institute of Biochemistry (Russian Academy of Sciences), and Dr. Larisa N. Chernousova's research group at Central TB research Institute (Russian Academy of Medical Sciences) for consistent

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of dormant bacilli.

**6. References** 

**5. Acknowledgment** 

and efficient collaboration.

2010), pp. 371-374

37297

1998), pp.537-544

All of the presented results support an idea that amount of FA in surrounding medium play a determinative role in formation and reactivation of *M. smegmatis* nonculturable cells (fig. 12). Thus 0.18-10.6 μМ of oleic acid possess a resuscitation effect. A little larger number of FA (33-59 μМ ) accumulates in culture liquid during transition into nonculturable state. And finally, as it was shown previously, oleic acid in amount of 92 μМ inhibits growth of mycobacterial cells.

Fig. 12. Correlation between a state of *M. smegmatis* and a quantity of extracellular FA.
