**2. An effect of lipid substances (PL and FA) on growth of mycobacterial cells in actively replicating state**

As it was mentioned above, mycobacterial cells are able to use FA as a nutrient source both in vivo, and in vitro by means of glyoxylate shunt [McKinney, 2000; Munoz-Elias, 2005]. It is well known that mycobacterial cells have lipases of various types [Deb, 2006; Mishra, 2008], as well as phospholipase A2 [Raynaud, 2002; Stonehouse, 2002], which was determined due to fully sequenced *M. tuberculosis* H37Rv genome and due to separation of these enzymes. Being equipped with such enzymes mycobacteria is capable of the hydrolysis of PL with the formation of FA. Therefore primarily we investigated the influence of PL of different classes on the growth rate of pathogenic and nonpathogenic mycobacteria.

#### **2.1 An effect of lipids on growth and susceptibility to ATD of** *M. smegmatis* **mc2 155**

At first we used rapidly growing nonpathogenic *M. smegmatis* mc2155 as the model of *M. tuberculosis*. We have cultivated the cells in medium with or without PL (PC, cardiolipin (CL)) to study their susceptibility to two ATD: isoniazide (INH) and rifabutin (RFB). Growth curve measured as optical density (wavelength 600 nm) of the cultivation medium (meatpeptone broth (MPB)) during growth of mycobacteria for 48 hours is represented in fig. 1. When PC and PC/CL (1:4) liposomes (large unilamellar vesicles) applied, a slight stimulation of growth compared with control was observed. Moreover stimulation with pure PC was stronger than with mixture of PC/CL.

Addition of INH (5 µg/ml) inhibit growth of *М. smegmatis* cells at the beginning, which is reflected in longer lag phase but after 30 hours mycobacteria grow almost with the same rate

use lipids as the main nutrient source. And besides that bacillus has designed the way to use its own lipids to control a state of the immune cell due to release of them into the macrophage internal space followed by exocytosis and transfer to nearby macrophages [Russell, 2009]. The other successful strategy for mycobacteria to survive inside of the host cells is believed to be a transition into nonreplicating dormant state so that they could

An effect of PL on growth of mycobacteria has been studied for a long time and the data obtained are insufficient and contradictory. On the one hand, phosphatidylcholine (PC) in the form of liposomes was demonstrated to serve as the nutrient source for a pathogenic strain of *M. tuberculosis* Н37Rv, but not to effect a growth of nonpathogenic strain H37Ra [Kondo, 1976]. On the other hand, the same research group had found that lysophosphatidylcholine, formed as the result of hydrolysis of PL under action of bacterial phospholipases, suppresses mycobacterial growth [Kondo, 1985]. For *Mycobacterium smegmatis* (rapidly growing nonpathogenic species of the genus *Mycobacterium*, commonly used as a model for *M. tuberculosis*) an influence of PL has been poorly investigated, but it

In the present chapter we summarize results obtained by the authors to discuss correlation between bacilli state in vitro (active cell division, inhibition of growth, dormant state, reactivation) and amount of lipid substances (secreted or added externally) in surrounding

**2. An effect of lipid substances (PL and FA) on growth of mycobacterial cells** 

As it was mentioned above, mycobacterial cells are able to use FA as a nutrient source both in vivo, and in vitro by means of glyoxylate shunt [McKinney, 2000; Munoz-Elias, 2005]. It is well known that mycobacterial cells have lipases of various types [Deb, 2006; Mishra, 2008], as well as phospholipase A2 [Raynaud, 2002; Stonehouse, 2002], which was determined due to fully sequenced *M. tuberculosis* H37Rv genome and due to separation of these enzymes. Being equipped with such enzymes mycobacteria is capable of the hydrolysis of PL with the formation of FA. Therefore primarily we investigated the influence of PL of different classes

**2.1 An effect of lipids on growth and susceptibility to ATD of** *M. smegmatis* **mc2**

At first we used rapidly growing nonpathogenic *M. smegmatis* mc2155 as the model of *M. tuberculosis*. We have cultivated the cells in medium with or without PL (PC, cardiolipin (CL)) to study their susceptibility to two ATD: isoniazide (INH) and rifabutin (RFB). Growth curve measured as optical density (wavelength 600 nm) of the cultivation medium (meatpeptone broth (MPB)) during growth of mycobacteria for 48 hours is represented in fig. 1. When PC and PC/CL (1:4) liposomes (large unilamellar vesicles) applied, a slight stimulation of growth compared with control was observed. Moreover stimulation with

Addition of INH (5 µg/ml) inhibit growth of *М. smegmatis* cells at the beginning, which is reflected in longer lag phase but after 30 hours mycobacteria grow almost with the same rate

**155** 

resuscitate when appropriate conditions appear.

medium.

**in actively replicating state** 

was shown that fatty acids inhibit its growth [Kanetsuna, 1985].

on the growth rate of pathogenic and nonpathogenic mycobacteria.

pure PC was stronger than with mixture of PC/CL.

as without INH. Cultivation of *М. smegmatis* cells with liposomal form of INH is similar at the beginning: there is no growth during first 30 hours and cell division during the later cultivation. However growth rate in the presence of liposomal form of INH was shown to be higher than in the presence of free INH. It's noteworthy that capacity of liposomes to reduce the effect of INH depends on the lipid composition of liposomes and was more pronounced for the mixture of PC/CL (1:4) compared to free PC (fig. 1). Analogous results were obtained for RFB (fig. 1). It's obvious, that RFB 1 µg/ml fully inhibited growth of *M. smegmatis*, and its liposomal form was less effective.

Fig. 1. A growth curve of *M. smegmatis*. Optical density at 600 nm (D600) of cultural medium (meet-peptone broth) with in control (0,99% NaCl) (1) and in the presence of 2) – INH, 5 µg/ml; 3) - PC, 200 µg/ml; 4) - INH, 5 µg/ml + PC, 200 µg/ml; 5) – PC/CL 1:4, 200 µg/ml; 6) – PC/CL 1:4, 200 µg/ml + INH, 5 µg/ml; 7) RFB (1 µg/ml) + PC (200 µg/ml); 8) RFB (1 µg/ml).

Thus, our data indicate that PL in culture medium decrease both ATD (INH and RFB) effect of *M. smegmatis* growth inhibition. INH and RFB have different target in mycobacterial cell: INH inhibit enzymes, responsible for elongation of fatty acid part in mycolic acids [Takayama, 1972], while RFB inhibit DNA-dependent RNA polymerase [Wehrli, 1971]. Our results allow us to conclude that there must be a general mechanism of ATD susceptibility reduction in the presence of PL for mycobacteria. This mechanism has to be realized before ATD reaches their targets.

To determinate the cause of the similar influence of PL on effect of different ATD, we investigated the growth rates of *M. smegmatis* in synthetic laboratory medium Sauton, and in modified analogues, in which glycerol was supplemented on other nutrient sources (acetate or PL), in the presence or absence of one of the involved ATD. We selected such concentrations of substrates that the growth rate of the samples differed slightly in the moment of ATD incorporation (24 h of cultivation). The data is represented in fig. 2 as the

Lipid Surrounding of Mycobacteria: Lethal and Resuscitating Effects 243

**2.2 An effect of lipids on growth and viability of** *Mycobacterium tuberculosis* **H37Rv**  Then we have studied a growth and viability of pathogenic *Mycobacterium tuberculosis*  H37Rv *in vitro* during cultivation in liquid Dubo's medium when PL and products of their hydrolysis (lyso-components) in various concentrations were added (table 1). PL were incorporated in the cultivation medium in the form of large unilamellar vesicles. The cells were grown in atomized system BACTEC, in which the growth is detected due to the alterations in oxygen uptake level by means of fluorescent indicator, that is quenched by

Fig. 3. Growth curves of *M. tuberculosis* H37Rv in the presence of PL liposomes obtained by

Viability of the cells was observed using colony counting on solid medium (Dubo's agar) after isolation of the passage that was exposed to the lipids in the liquid medium (fig. 4). It was found that addition of negatively-charged CL into cultivation medium had an effect on the growth rate and viability of *M. tuberculosis* H37Rv, while electro-neutral PC wasn't active. As it follows from the data shown in table 1 the effect of CL was dose-dependent: low concentration (50 µg/ml) caused the 1-2 day delay in growth of mycobacterial cells, and 250-330 µg/ml concentration fully inhibited growth. Applying Murohashi staining method we demonstrated that such concentration of CL cause lysis of *M. tuberculosis* H37Rv cells

Fig. 4. A photography of macrocolonies of *M. tuberculosis* H37Rv on Dubo's agar by the 18th day of cultivation. 1-H37Rv; 2-H37Rv+CL (50 µg/ml); 3-H37Rv+CL (750 µg/ml).

means of BACTEС. 1-H37Rv; 2-H37Rv+CL (50 µg/ml); 3-H37Rv+CL (750 µg/ml).

high oxygen concentrations (fig. 3).

Reproduced from [Andreevskaya, 2010]

(fig. 5) [Sorokoumova, 2009].

Reproduced from [Sorokoumova, 2009]

ratio (%) between optical density of *M. smegmatis* in culture medium, containing different nutrient source in the presence of INH or RFB and control, i.e. optical density of culture medium in the absence of ATD.

Fig. 2. Alteration of optical density of *M. smegmatis* cultivation medium (% of control numbers) in the presence of ATD. 1. INH (40 µg/ml), 2. RFB (7 µg/ml) with different nutrient source: a) glycerol (0,5 g/l) ; b) acetate (0,7 g/l); c) PC (1 g/l). (Reproduced from [Shakina Y.V., 2007])

Fig.2 demonstrates, the during the growth in medium, that contains glycerol the main carbon source, *M. smegmatis* cells possess a strong resistance to INH, but are highly susceptible to RFB. In other words, when hydrocarbon substrates are utilized in full Krebs cycle, mycobacteria retain susceptibility to RFB. But when glyoxylate pathway is activated, i.e. during the growth of *M. smegmatis* in medium with acetate or PL as the main carbon source, mycobacteria become fully resistant to INH and susceptibility to RFB drops considerably.

It is possible, that mechanism of phenotypic resistance of *M. smegmatis* to two ATDs, that have different targets, may be attributed to expression of transport proteins, that are capable of binding the ATD that entered into the cell, and transport it through the cell wall out. Morris et al. [Morris, 2005] has shown that polyketide (tetracycline), macrolide (erythromycin) and aminoglycoside (streptomycine) antibiotics induce *whiB7* gene of *M. tuberculosis*. Similar effect was detected when mycobacteria were cultivated with FA [Morris, 2005]. *whiB7* affects *tap* (Rv 1258c), which in turn is responsible for synthesis of protein pumps that provide efflux of antibiotics out of cell.

ratio (%) between optical density of *M. smegmatis* in culture medium, containing different nutrient source in the presence of INH or RFB and control, i.e. optical density of culture

Fig. 2. Alteration of optical density of *M. smegmatis* cultivation medium (% of control numbers) in the presence of ATD. 1. INH (40 µg/ml), 2. RFB (7 µg/ml) with different

Fig.2 demonstrates, the during the growth in medium, that contains glycerol the main carbon source, *M. smegmatis* cells possess a strong resistance to INH, but are highly susceptible to RFB. In other words, when hydrocarbon substrates are utilized in full Krebs cycle, mycobacteria retain susceptibility to RFB. But when glyoxylate pathway is activated, i.e. during the growth of *M. smegmatis* in medium with acetate or PL as the main carbon source, mycobacteria become fully resistant to INH and susceptibility to RFB drops

It is possible, that mechanism of phenotypic resistance of *M. smegmatis* to two ATDs, that have different targets, may be attributed to expression of transport proteins, that are capable of binding the ATD that entered into the cell, and transport it through the cell wall out. Morris et al. [Morris, 2005] has shown that polyketide (tetracycline), macrolide (erythromycin) and aminoglycoside (streptomycine) antibiotics induce *whiB7* gene of *M. tuberculosis*. Similar effect was detected when mycobacteria were cultivated with FA [Morris, 2005]. *whiB7* affects *tap* (Rv 1258c), which in turn is responsible for synthesis of protein

nutrient source: a) glycerol (0,5 g/l) ; b) acetate (0,7 g/l); c) PC (1 g/l).

(Reproduced from [Shakina Y.V., 2007])

pumps that provide efflux of antibiotics out of cell.

considerably.

medium in the absence of ATD.
