**Lipid Surrounding of Mycobacteria: Lethal and Resuscitating Effects**

Alla A. Selishcheva1,2, Galina M. Sorokoumova1

and Evgeniya V. Nazarova1,3 *1Lomonosov Moscow State Academy of Fine Chemical Technology 2Biology Department, Lomonosov Moscow State University 3Bach Institute of Biochemistry, Russian Academy of Sciences Russia* 

#### **1. Introduction**

238 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

Rua, J., De Arriaga, D., Busto, F. and Soler, J. (1989). Effect of glucose on isocitrate lyase in Phycomyces blakesleeanus. *J Bacteriol*, Vol. 171, No. 11, pp. 6391-6393 Sassetti, C. M. and Rubin, E. J. (2003). Genetic requirements for mycobacterial survival during infection. *Proc Natl Acad Sci U S A*, Vol. 100, No. 22, pp. 12989-12994 Shaila, M. S., Gopinathan, K. P. and Ramakrishnan, T. (1973). Protein synthesis in

Sharma, V., Sharma, S., Hoener zu Bentrup, K., McKinney, J. D., Russell, D. G., Jacobs, W. R.,

Singh, K. K., Dong, Y., Belisle, J. T., Harder, J., Arora, V. K. and Laal, S. (2005). Antigens of

Singh, K. K., Dong, Y., Hinds, L., Keen, M. A., Belisle, J. T., Zolla-Pazner, S., Achkar, J. M.,

Mycobacterium tuberculosis. *J Biol Chem*, Vol. 278, No. 3, pp. 1735-1743 Smith, C. V., Sharma, V. and Sacchettini, J. C. (2004). TB drug discovery: addressing issues of persistence and resistance. *Tuberculosis (Edinb)*, Vol. 84, No. 1-2, pp. 45-55 Thadepalli, H., Bach, V. T. and Webb, D. W. (1979). Antimicrobial activity of

Mycobacterium tuberculosis. *Nat Struct Biol*, Vol. 7, No. 8, pp. 663-668 Singh, B., Ghosh, J., Islam, N. M., Dasgupta, S. and Kirsebom, L. A. (2010). Growth, cell

tuberculosis. *Clin Diagn Lab Immunol*, Vol. 12, No. 2, pp. 354-358

research. *Nucleic Acids Res*, Vol. 37, No. Database issue, pp. D499-508 Roberts, M. C. (1994). Epidemiology of tetracycline-resistance determinants. *Trends* 

*Microbiol*, Vol. 2, No. 10, pp. 353-357

205-213

pp. 165-177

A. and Schoolnik, G. K. (2009). TB database: an integrated platform for tuberculosis

Mycobacterium tuberculosis H37Rv and the effect of streptomycin in streptomycinsusceptible and -resistant strains. *Antimicrob Agents Chemother*, Vol. 4, No. 3, pp.

Jr. and Sacchettini, J. C. (2000). Structure of isocitrate lyase, a persistence factor of

division and sporulation in mycobacteria. *Antonie Van Leeuwenhoek*, Vol. 98, No. 2,

Mycobacterium tuberculosis recognized by antibodies during incipient, subclinical

Nadas, A. J., Arora, V. K. and Laal, S. (2003). Combined use of serum and urinary antibody for diagnosis of tuberculosis. *J Infect Dis*, Vol. 188, No. 3, pp. 371-377 Smith, C. V., Huang, C. C., Miczak, A., Russell, D. G., Sacchettini, J. C. and Honer zu

Bentrup, K. (2003). Biochemical and structural studies of malate synthase from

antituberculosis agents against anaerobic bacteria. *Chest*, Vol. 75, No. 5, pp. 569-570

N., Ramakrishnan, L. and Losick, R. (2010). Do mycobacteria produce endospores?

Mycobacterium tuberculosis through two stages of nonreplicating persistence.

oncogene and its high-level expression in the hippocampus and cerebral cortex of

Traag, B. A., Driks, A., Stragier, P., Bitter, W., Broussard, G., Hatfull, G., Chu, F., Adams, K.

Waksman, S. A. and Schatz, A. (1943). Strain Specificity and Production of Antibiotic

Wayne, L. G. (1994). Dormancy of Mycobacterium tuberculosis and latency of disease. *Eur J* 

Wayne, L. G. and Hayes, L. G. (1996). An in vitro model for sequential study of shiftdown of

Young, D., O'Neill, K., Jessell, T. and Wigler, M. (1988). Characterization of the rat mas

Zhang, Y. (2005). The magic bullets and tuberculosis drug targets. *Annu Rev Pharmacol* 

*Proc Natl Acad Sci U S A*, Vol. 107, No. 2, pp. 878-881

*Clin Microbiol Infect Dis*, Vol. 13, No. 11, pp. 908-914

*Infect Immun*, Vol. 64, No. 6, pp. 2062-2069

*Toxicol*, Vol. 45, No. pp. 529-564

Substances. *Proc Natl Acad Sci U S A*, Vol. 29, No. 2, pp. 74-79

rat brain. *Proc Natl Acad Sci U S A*, Vol. 85, No. 14, pp. 5339-5342

Inside of the host macrophages *Mycobacterium tuberculosis* cells are supposed to face various hostile conditions as the result of immune response: action of reactive oxygen and nitrogen intermediates, hydrolases, increased acidity, and antimicrobial peptides activities [Russell, 2010]. However mycobacterial cells have developed certain mechanisms to resist these defenses. Transcriptome analysis of *M. tuberculosis* showed that even negligible changes of environmental factors cause considerable alterations in global gene expression profile [Boshoff, 2004; Cole, 1998]. It's important that such alterations were observed both in the presence of chemical agents (respiration inhibitors, antituberculosis drugs (ATD), ATP synthesis inhibitors), and during incubation of mycobacteria in modified conditions (medium рН, nature of nutrient, nutrient depletion and starvation, hypoxia, exposure to nitric oxide). As the result, altered properties of the whole cell enable mycobacteria to resist these effects. For instance, increase of the incubation temperature activated synthesis of heat shock proteins which led to higher thermal resistance of the cells; exposure of mycobacteria to the acid environment induces expression of *aprABC* locus responsible for restructuration of lipids of the mycobacterial cell wall and storage lipids that are required for intraphagosome survival [Abramovitch, 2011; Sung N, 2004]. Low concentrations of antibiotics in cultivation medium, that don't affect the cell growth, activate genes responsible for protein pump synthesis, which provides a removal of the antibiotics from the bacterial cell. This is one of the main mechanisms of the ATD resistance. Substitution of the nutrient, e.g. substitution of glycerol to FA (free FA or as part of phospholipids (PL)), activates genes responsible for synthesis of enzymes that switch metabolism to a different pathway of nutrient utilization. In this case mycobacterial cell involves two forms of isocitrate lyase, and utilization of the nutrient in the tricarboxylic acid cycle goes through the glyoxylate shunt [Munoz-Elias, 2005]. Upregulation of the genes encoding isocitrate lyase was shown for *М. tuberculosis* cells cultivated in anaerobic conditions [Lu, 2005], for cells isolated from human lung granulomas [Fenhalls, 2002] and from infected macrophages [Schnappinger, 2003]. All these data prove glyoxylate shunt to be an essential mechanism for survival of mycobacterial cells in phagosomes inside of the host macrophage, where they

Lipid Surrounding of Mycobacteria: Lethal and Resuscitating Effects 241

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.* 

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

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

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

*smegmatis*, and its liposomal form was less effective.

(1 µg/ml).

ATD reaches their targets.

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 resuscitate when appropriate conditions appear.

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

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 medium.
