**3. Basic characteristics of cell wall deficient L-forms**

## **3.1 L-conversion, morphology and ultrastructure**

Bacterial L-form conversion, i.e. existence without rigid walls, is universal but difficultly recognized phenomenon in nature (Domingue, 1982; Mattman, 2001; Prozorovski et al., 1981). The term "cell wall deficiency" implies alterations in the constitution of bacterial cell wall, resulting from deletion and faulty synthesis of wall components (Mattman, 2001). It is considered that imbalance of cells' ability to degrade and synthesize its classical thick wall results in cell wall deficiency. Since the peptidoglycan is the stress-bearing structure of bacteria, its loss, respectively the loss of rigidity, is a distinctive characteristic of cell wall deficient forms (L-forms). In fact, morphological variability is an indicative and common feature of all L-forms, regardless of what bacterial species they originated from. Although these forms have been observed in patients' specimens for many decades, most are ignored and generally regarded as diagnostically insignificant staining artifacts or debris

The period between 1882 and 1940, after Robert Koch discovered the cause of tuberculosis, was marked by series of papers reporting about the appearance of L-form elements in cultures of mycobacteria, such as filterable forms, branching filaments, syncytial growth, large spheres and "variegated mycelia", all of which characterize mycobacterial growth. Mattman summarized the known data about the ability of *M. tuberculosis* to convert to cell wall deficient forms and suggested a "L-cycle" for mycobacteria (Mattman et al., 1960;

Despite the long history in tuberculosis research, the nature of cell wall deficiency and its association with persistence in life of mycobacteria still remain obscure. Unfortunately, over the last several decades, investigations on these unusual forms of tubercle bacilli have been ignored and neglected. Information about forming of mycobacterial L-forms *in vitro* (in the laboratory), as well *in vivo* (within the body) is based mainly on studies concerning their morphological appearance. Two periods in L-form research of mycobacteria should be distinguished: before introduction of chemotherapy against tuberculosis, and after. Observations made in the beginning of 20th century on mycobacterial pleomorphism and Lform elements provide evidence for existence of L-forms without contact with antimicrobial drugs (Calmette & Valti, 1926; Much, 1931). In the following decades, examinations regarding modification of morphology and L-form transformation by antimicrobials became the starting point of additional information on mycobacterial properties (Dorozhkova & Volk, 1972; Dorozhkova & Volk, 1973; Kochemasova et al., 1968; Mattman et al. 1960; Wang

Bacterial L-form conversion, i.e. existence without rigid walls, is universal but difficultly recognized phenomenon in nature (Domingue, 1982; Mattman, 2001; Prozorovski et al., 1981). The term "cell wall deficiency" implies alterations in the constitution of bacterial cell wall, resulting from deletion and faulty synthesis of wall components (Mattman, 2001). It is considered that imbalance of cells' ability to degrade and synthesize its classical thick wall results in cell wall deficiency. Since the peptidoglycan is the stress-bearing structure of bacteria, its loss, respectively the loss of rigidity, is a distinctive characteristic of cell wall deficient forms (L-forms). In fact, morphological variability is an indicative and common feature of all L-forms, regardless of what bacterial species they originated from. Although these forms have been observed in patients' specimens for many decades, most are ignored and generally regarded as diagnostically insignificant staining artifacts or debris

**3. Basic characteristics of cell wall deficient L-forms** 

**3.1 L-conversion, morphology and ultrastructure** 

Mattman, 1970, 2001).

Fig. 2. Lida Mattman

& Chen, 2001).

(Domingue, 2010). It is assumed that these pleomorphic forms represent various stages in the life cycle of stressed bacteria.

*M. tuberculosis* is known to exhibit extreme pleomorphism in certain circumstances. Various morphological forms of mycobacteria were observed by many authors and were described as "mycococcus form" (Csillag, 1964), large "amoeba-like cells" (Imaeda, 1975), giant noncellular structures or so called "budding yeast-like structures" (Koch, 2003), "elementary bodies and filament structures" (Merkal et al, 1973) "endospores" (Ghosh et al.,2009; Traag et al, 2010) and "ovoid cells"( Shleeva et al., 2011). Mycobacteria are unique among procaryotes with their cell wall structure, containing tightly packed mycolic acids that provide TB bacilli with efficient protection and remarkable capacity to resist to various exogenous stress conditions. The high concentration of lipids in cell wall of mycobacteria is associated with general insusceptibility to chemical/toxic agents and most antibiotics. The mycolic acids and glycolipids in cell wall of mycobacteria also impedes the entry of nutrient substrates, causing the organisms to grow slowly (Draper, 1998). However, mycobacterial cell wall appears to be a dynamic structure that can be remodeled, as the microorganism is either growing, or persisting in different environments (Kremer & Besra, 2005). Under unfavorable conditions, where mycobacteria are exposed to different damaging factors particularly in face of host defense mechanisms, they may produce cell wall deficient forms (L-forms) (Markova et al.2008a; Markova et al. 2008b). A variety of papers reported about production of mycobacterial L-forms experimentally *in vitro,* using different inducing factors. Wide range of substances (cell wall inhibitors) as antibiotics, lytic enzymes and some amino acids affecting cell wall and especially biosynthesis of peptidoglycan have been used as L-inducing factors (Beran et al., 2006; Hammes et al., 1973; Hines and Styer, 2003; Naser et al., 1993; Udou et al., 1983). Indeed, it is important to understand how mycobacteria regulate the cell wall composition in response to changing environment. In some wall deficient cells pieces of cell wall are synthesized and dutifully pulled through the pores of cell membrane but somehow lack structural detail that would permit them to link together. Mitchel & Moyle have added another interesting aspect to consider, which may explain why a cell is unable to resynthesize its cell wall, once losing it. They postulate that perhaps the building blocks are sufficiently soluble to diffuse spontaneously into the culture medium than remain together against the wall where their union is facilitated (Mitchel & Moyle, 1956).

The ability of strains from *M. tuberculosis* complex to produce L-phase variants after nutrient starvation stress was demonstrated in our experiments (n. d.). Morphological transformations of tubercle bacilli from acid fast to polymorphic non-acid-fast and coccoid forms of varying size were observed (Fig. 3). In contrast to classical tubercle bacilli, which typically appear as straight or slightly curved red stained rods in Ziehl-Neelsen stained smears, mycobacterial L-forms showed marked polymorphism and variability in staining reaction. L-form variants of mycobacteria lost acid fastness completely and resembled the morphology of various other bacteria (Fig. 3 b, c).

It is known that acid fastness is dependent on the integrity of the tubercle bacilli. Sometimes, persistent *M. tuberculosis* bacteria bearing cell wall alterations may remain undetected by the classic Ziehl-Neelsen staining (Seiler et al, 2003). Appearance of polymorphic non-acid fast forms and coccoids in cultures of mycobacteria has been observed by other authors

Cell Wall Deficiency in Mycobacteria: Latency and Persistence 197

capability sufficient to initiate reproduction (Domingue, 2010; Klieneberger-Nobel, 1951;

Findings from transmission electron microscopy yielded additional valuable information about the ultrastructure morphology of mycobacterial L-forms. Examinations of *M. tuberculosis* L-forms obtained *in vitro* after starvation stress (n.d.) or during experimental infection in rats (Markova et al., 2008a) revealed typical fine structure of L-form population. L-phase growth consisted of cells of variable shape and size, completely devoid of bacterial cell wall and bound only by a single unit membrane (Fig. 5). Large and elementary bodies of different electron microscopy density, as well as very small granules and vesicular forms were observed (Fig. 5 b, c, d). Some vesicular forms either appeared empty or contained electron-dense granules (Fig. 5 c, f). Of considerable interest was the observation of large bodies of so called "mother" cells, filled with numerous small spherical L-elements (Fig. 5 f). Such "mother" cells are often internally vesiculated and may produce also small, empty bodies, or membrane bound vesicles. Fragmentation of the cytoplasmic mass in numerous granular forms was the mode of L-form reproduction that was noted. Cytoplasmic condensation at the periphery of the large bodies ending in formation of protrusions and buds was often seen*.* Budding, another mode of L-form replication, was observed as well. It should be noted that nucleoid and ribosomal areas within L-bodies were of variable electron densities and intracellular location .The nucleoids were variable, being sometimes compact and sometimes scattered throughout the cytoplasm. Enucleated L-bodies were also seen. Ribosomes were either packed together or diffusely scattered, usually at the periphery of the cells. Electron-dense L-bodies of different size and giant filamentous forms were found in

clinical isolates of *M. tuberculosis* (Fig.5 g, h; Michailova *et al*, 2005).

clinical strains, isolated from patients (**g, h;** Michailova et al., 2005).

**3.2 Modes of reproduction and morphogenesis of L-cycle** 

Fig. 5. Transmission electron microscopy of classical tubercle bacilli (**a**), and L-forms of *M. tuberculosis* obtained *in vitro* after starvation stress (**b ,c ,d**; n. d.), isolated from rats, experimentally infected with *M. tuberculosis* (**e, f**; Markova et al.,2008a) and observed in

The normal existence of bacteria appears to be a dynamic state of morphological and physiological changes, and the reproducibility in response to established conditions for

Prozorovski et al., 1981).

(Chandrasekhar & Ratnam , 1992; Csillag, 1964; Juhasz, 1962; Miller, 1932; Xalabarder, 1958).

Fig. 3. Ziehl-Neelsen stained smears: (a) control TB bacilli; (b, c) non-acid fast polymorphic cells of *M. tuberculosis* L-forms (n.d.)

Morphological forms of different sizes and shapes (short coccobacilli and long rods, oval or round coccoid cells, large spherical bodies and giant filaments) in mycobacterial L-form cultures obtained after starvation stress, were observed by us with scanning electron microscopy (Fig.4, n. d.). Very small granular elements placed on membrane filters with pore size diameter of 0.22µm, evidencing their ability to pass through bacterial filters i.e. filterable L-form cells, were detected (Fig. 4 f). The filterable forms are considered as minimal reproductive cells, which can be formed from large L-bodies in all possible ways. It is believed that such filterable bodies contain a bacterial genome and minimal metabolic

Fig. 4. Scanning electron microscopy of classical tubercle bacilli (a) and mycobacterial Lforms obtained after stress treatments *in vitro* of *M.tuberculosis* (b), *M.bovis* (c, d) and *M.bovis* BCG (e, f), (n.d.).

(Chandrasekhar & Ratnam , 1992; Csillag, 1964; Juhasz, 1962; Miller, 1932; Xalabarder,

Fig. 3. Ziehl-Neelsen stained smears: (a) control TB bacilli; (b, c) non-acid fast polymorphic

Morphological forms of different sizes and shapes (short coccobacilli and long rods, oval or round coccoid cells, large spherical bodies and giant filaments) in mycobacterial L-form cultures obtained after starvation stress, were observed by us with scanning electron microscopy (Fig.4, n. d.). Very small granular elements placed on membrane filters with pore size diameter of 0.22µm, evidencing their ability to pass through bacterial filters i.e. filterable L-form cells, were detected (Fig. 4 f). The filterable forms are considered as minimal reproductive cells, which can be formed from large L-bodies in all possible ways. It is believed that such filterable bodies contain a bacterial genome and minimal metabolic

Fig. 4. Scanning electron microscopy of classical tubercle bacilli (a) and mycobacterial Lforms obtained after stress treatments *in vitro* of *M.tuberculosis* (b), *M.bovis* (c, d) and *M.bovis*

a b c

a b c

**d** e f

cells of *M. tuberculosis* L-forms (n.d.)

BCG (e, f), (n.d.).

1958).

capability sufficient to initiate reproduction (Domingue, 2010; Klieneberger-Nobel, 1951; Prozorovski et al., 1981).

Findings from transmission electron microscopy yielded additional valuable information about the ultrastructure morphology of mycobacterial L-forms. Examinations of *M. tuberculosis* L-forms obtained *in vitro* after starvation stress (n.d.) or during experimental infection in rats (Markova et al., 2008a) revealed typical fine structure of L-form population. L-phase growth consisted of cells of variable shape and size, completely devoid of bacterial cell wall and bound only by a single unit membrane (Fig. 5). Large and elementary bodies of different electron microscopy density, as well as very small granules and vesicular forms were observed (Fig. 5 b, c, d). Some vesicular forms either appeared empty or contained electron-dense granules (Fig. 5 c, f). Of considerable interest was the observation of large bodies of so called "mother" cells, filled with numerous small spherical L-elements (Fig. 5 f). Such "mother" cells are often internally vesiculated and may produce also small, empty bodies, or membrane bound vesicles. Fragmentation of the cytoplasmic mass in numerous granular forms was the mode of L-form reproduction that was noted. Cytoplasmic condensation at the periphery of the large bodies ending in formation of protrusions and buds was often seen*.* Budding, another mode of L-form replication, was observed as well. It should be noted that nucleoid and ribosomal areas within L-bodies were of variable electron densities and intracellular location .The nucleoids were variable, being sometimes compact and sometimes scattered throughout the cytoplasm. Enucleated L-bodies were also seen. Ribosomes were either packed together or diffusely scattered, usually at the periphery of the cells. Electron-dense L-bodies of different size and giant filamentous forms were found in clinical isolates of *M. tuberculosis* (Fig.5 g, h; Michailova *et al*, 2005).

Fig. 5. Transmission electron microscopy of classical tubercle bacilli (**a**), and L-forms of *M. tuberculosis* obtained *in vitro* after starvation stress (**b ,c ,d**; n. d.), isolated from rats, experimentally infected with *M. tuberculosis* (**e, f**; Markova et al.,2008a) and observed in clinical strains, isolated from patients (**g, h;** Michailova et al., 2005).

#### **3.2 Modes of reproduction and morphogenesis of L-cycle**

The normal existence of bacteria appears to be a dynamic state of morphological and physiological changes, and the reproducibility in response to established conditions for

Cell Wall Deficiency in Mycobacteria: Latency and Persistence 199

It is assumed that cell wall deficient bacterial forms survive storage and unfavorable conditions much longer than classical bacteria (Mattman, 2001). Domingue suggests the role of small electron-dense bodies (filterable granules) as notoriously resistant forms of pathogenic bacteria (Domingue, 1997). Xalabander noted that L-forms of mycobacteria were remarkably different from L-forms of other species in their resistance to physical and chemical agents. Similar to prions, mycobacterial L-forms escape destruction by body's immune system, and are seemingly imperishable. Xalabander also noted that these L-forms contain both RNA and DNA proteins, but do not stain well by ordinary mycobacteria dyes (Xalabander, 1958; 1963). On other hand, it is supposed that the smallest and most resistant to environmental stresses filterable L-granules, containing DNA may exert nuclear functions (Klieneberger-Nobel, 1951). Moreover, chromosomal DNA, especially within L-symplasm, should be regarded as a substantial mass of the nucleoid body, which can dynamically interact with other components (Allan et al, 2009). This problematic question is still under discussion and yet, no matter how small and at first glance, enucleated, some of these L-

Shleeva et al. (2010) believe that dormancy in mycobacteria is related to the formation of different cell forms with various characteristics (less differentiated cyst-like forms, weakly differentiated resting cells and highly differentiated spore-like forms) within a population. According to the same authors, passing into a dormant state is associated with drastically decreased metabolic activity of cells, enhanced resistance to harmful factors, and absence of cell division. The resting cells retain their viability but lose capacity for germination and growth, becoming "nonculturable". It is a generally accepted postulate that TB bacilli are in a true dormant state, undergoing no replication. Dormant cells switch on the mechanisms of division arrest and may persist, due to survival of a small number of bacteria (Kaprelyants et al., 1993; Postgate & Hunter, 1962; Shleeva et al., 2010). Recent data, however, cast doubt on the assumption of such 'inactive' latent state, as there is constant metabolic activity within the TB bacilli (Zumla et al., 2011). Evidence about the role of molecular chaperones and intercellular signalling molecules in control of metabolic activity and composition of the

From the view point of the L-cycle theory, a transition of mycobacteria from acid-fast to non-acid fast state, along with appearance of polymorphic cell wall deficient cells, occurs in response to stress. L-forms develop through several stages and result in formation of polymorphic or coccoid fast growing cells. The initial phase of L-conversion probably corresponds to an "invisible" stage, where bacteria cease forming colonies on solid media and growing in liquid media. We suppose that formation and persistence of giant L-forms structures (filaments, syncytia and "mother" cell) sheltering and embodying many individuals inside a common envelope, represents a unique mechanism of survival and may resemble "invisible" or cryptic state of L-form development. However, at some point of L-form development, these giant spherical or filamentous forms start to disintegrate and are no longer visible, giving place to an abundance of granular and coccoid forms, which sometimes become the prevailing elements within L-population. Coccoid forms of mycobacteria, called "mycococcus", were obtained *in vitro* by Csillag in 1964. Mycococcii were grown from *M. tuberculosis* and were similar to the morphology of staphylococci (Csillag, 1964). Genetic analysis of mycobacterial coccoids however, performed by us through amplification of 16SrRNA gene fragment, 16S-23S rRN gene Internal Transcribed

forms will revert back to virulent mycobacteria.

cell wall has been provided by Henderson et al. (2010).

growth is considered as a "life style". Under certain circumstances, bacteria can enter unbalanced growth and undergo complex life cycles, involving different morphological transformations, known as the bacterial L-cycle. Conversion to L-forms is assumed to be a general property of bacteria and as adaptive reaction to unfavourable environmental factors, which interfere with the normal reproduction, as well as permit the growth of cell wall deficient variants (Dienes & Weinberger, 1951). Loss of rigidity due to the lack of murein layer in L-forms, results in uncoordinated propagation and appearance of highly pleomorphic forms. In contrast to classical bacteria, L-forms can reproduce by great variety of unusual modes, such as irregular binary fission, budding, protrusion-extrusion of elementary bodies and granules from large bodies, multiple division with intracellular fragmentation of cytoplasm or combination of all types (Prozorovski et al., 1981). Variations in the development of morphological units of different sizes and shapes, typical for L-forms, appear in accordance with the changing environmental conditions (Markova et al, 2010). The newly reorganized L-form population continues to exist and replicate by unusual modes, displaying various cells and elements such as elementary and large spherical bodies, granular and filterable forms, vesicular and empty bodies, giant filaments and others. Those giant filaments and large bodies may be serving a two-fold purpose, playing a role in Lform reproduction, as well as protecting them from unfavourable environment.

Our observation of giant L-bodies ("mother" cell) within mycobacterial L-form population releasing, through protrusion or budding, numerous previously generated granules, is also perhaps noteworthy of mentioning. Such granular elements, often released from the terminal sides of filaments (Fig 4 d, e; n. d.), were found to develop into bigger coccoid or large L-bodies, although transformation of granules into rod shaped forms was also noticed. The segmentation of L-bodies and breaking up into small elements, which germinate again, as well as the processes of regeneration initiated by the fusion of certain elements (Klieneberger-Nobel, 1951), challenge the conventional vision about bacterial replication. Although the modes of L-form replication were less effective, it should be noted that, at a point of their development and adaptation, L-forms started multiplying with remarkable rapidity, by releasing numerous small granules from collapsing giant L-structures. The small forms grew into large bodies which subsequently either increased in diameter, or disintegrated into even smaller L-form bodies. The observed by us different arrangements of *M. tuberculosis* L-forms coccoid cells of varied size (singly, in pairs or in irregular clusters) suggest either capability of L-forms to divide in different planes by binary fission or the possibility that they arose *en masse* from huge L-form bodies. In our opinion, L-life style is best understood by taking into consideration the unusual modes of replication, exhibited by L-forms. L-forms behave like an entire population, within which the role of individual organisms and organelles is difficult to determine (Markova et al., 2010). Of all structures in the L-cycle, syncytium, designed as "symplasm" and consisting of numerous nuclei embedded in a cytoplasm within one L-body (Mattman, 2001), is the most incredible. As noted by Mattman, fifty mycobacteria can be made within one sac (L-syncytium). Syncytia were observed to be formed from coalescing aggregates of bacteria, when the cell walls disintegrate and the cytoplasm starts to coalesce. The granules emerging from the symplasm grow into young cells, which reproduce further by fission or by other modes. According to Norris, syncytium-like structures may create a favorable environment for development of a complex prebiotic ecology, in which rearranged hyperstructures give rise to even more complex life forms (Norris, 2011).

growth is considered as a "life style". Under certain circumstances, bacteria can enter unbalanced growth and undergo complex life cycles, involving different morphological transformations, known as the bacterial L-cycle. Conversion to L-forms is assumed to be a general property of bacteria and as adaptive reaction to unfavourable environmental factors, which interfere with the normal reproduction, as well as permit the growth of cell wall deficient variants (Dienes & Weinberger, 1951). Loss of rigidity due to the lack of murein layer in L-forms, results in uncoordinated propagation and appearance of highly pleomorphic forms. In contrast to classical bacteria, L-forms can reproduce by great variety of unusual modes, such as irregular binary fission, budding, protrusion-extrusion of elementary bodies and granules from large bodies, multiple division with intracellular fragmentation of cytoplasm or combination of all types (Prozorovski et al., 1981). Variations in the development of morphological units of different sizes and shapes, typical for L-forms, appear in accordance with the changing environmental conditions (Markova et al, 2010). The newly reorganized L-form population continues to exist and replicate by unusual modes, displaying various cells and elements such as elementary and large spherical bodies, granular and filterable forms, vesicular and empty bodies, giant filaments and others. Those giant filaments and large bodies may be serving a two-fold purpose, playing a role in L-

form reproduction, as well as protecting them from unfavourable environment.

complex life forms (Norris, 2011).

Our observation of giant L-bodies ("mother" cell) within mycobacterial L-form population releasing, through protrusion or budding, numerous previously generated granules, is also perhaps noteworthy of mentioning. Such granular elements, often released from the terminal sides of filaments (Fig 4 d, e; n. d.), were found to develop into bigger coccoid or large L-bodies, although transformation of granules into rod shaped forms was also noticed. The segmentation of L-bodies and breaking up into small elements, which germinate again, as well as the processes of regeneration initiated by the fusion of certain elements (Klieneberger-Nobel, 1951), challenge the conventional vision about bacterial replication. Although the modes of L-form replication were less effective, it should be noted that, at a point of their development and adaptation, L-forms started multiplying with remarkable rapidity, by releasing numerous small granules from collapsing giant L-structures. The small forms grew into large bodies which subsequently either increased in diameter, or disintegrated into even smaller L-form bodies. The observed by us different arrangements of *M. tuberculosis* L-forms coccoid cells of varied size (singly, in pairs or in irregular clusters) suggest either capability of L-forms to divide in different planes by binary fission or the possibility that they arose *en masse* from huge L-form bodies. In our opinion, L-life style is best understood by taking into consideration the unusual modes of replication, exhibited by L-forms. L-forms behave like an entire population, within which the role of individual organisms and organelles is difficult to determine (Markova et al., 2010). Of all structures in the L-cycle, syncytium, designed as "symplasm" and consisting of numerous nuclei embedded in a cytoplasm within one L-body (Mattman, 2001), is the most incredible. As noted by Mattman, fifty mycobacteria can be made within one sac (L-syncytium). Syncytia were observed to be formed from coalescing aggregates of bacteria, when the cell walls disintegrate and the cytoplasm starts to coalesce. The granules emerging from the symplasm grow into young cells, which reproduce further by fission or by other modes. According to Norris, syncytium-like structures may create a favorable environment for development of a complex prebiotic ecology, in which rearranged hyperstructures give rise to even more It is assumed that cell wall deficient bacterial forms survive storage and unfavorable conditions much longer than classical bacteria (Mattman, 2001). Domingue suggests the role of small electron-dense bodies (filterable granules) as notoriously resistant forms of pathogenic bacteria (Domingue, 1997). Xalabander noted that L-forms of mycobacteria were remarkably different from L-forms of other species in their resistance to physical and chemical agents. Similar to prions, mycobacterial L-forms escape destruction by body's immune system, and are seemingly imperishable. Xalabander also noted that these L-forms contain both RNA and DNA proteins, but do not stain well by ordinary mycobacteria dyes (Xalabander, 1958; 1963). On other hand, it is supposed that the smallest and most resistant to environmental stresses filterable L-granules, containing DNA may exert nuclear functions (Klieneberger-Nobel, 1951). Moreover, chromosomal DNA, especially within L-symplasm, should be regarded as a substantial mass of the nucleoid body, which can dynamically interact with other components (Allan et al, 2009). This problematic question is still under discussion and yet, no matter how small and at first glance, enucleated, some of these Lforms will revert back to virulent mycobacteria.

Shleeva et al. (2010) believe that dormancy in mycobacteria is related to the formation of different cell forms with various characteristics (less differentiated cyst-like forms, weakly differentiated resting cells and highly differentiated spore-like forms) within a population. According to the same authors, passing into a dormant state is associated with drastically decreased metabolic activity of cells, enhanced resistance to harmful factors, and absence of cell division. The resting cells retain their viability but lose capacity for germination and growth, becoming "nonculturable". It is a generally accepted postulate that TB bacilli are in a true dormant state, undergoing no replication. Dormant cells switch on the mechanisms of division arrest and may persist, due to survival of a small number of bacteria (Kaprelyants et al., 1993; Postgate & Hunter, 1962; Shleeva et al., 2010). Recent data, however, cast doubt on the assumption of such 'inactive' latent state, as there is constant metabolic activity within the TB bacilli (Zumla et al., 2011). Evidence about the role of molecular chaperones and intercellular signalling molecules in control of metabolic activity and composition of the cell wall has been provided by Henderson et al. (2010).

From the view point of the L-cycle theory, a transition of mycobacteria from acid-fast to non-acid fast state, along with appearance of polymorphic cell wall deficient cells, occurs in response to stress. L-forms develop through several stages and result in formation of polymorphic or coccoid fast growing cells. The initial phase of L-conversion probably corresponds to an "invisible" stage, where bacteria cease forming colonies on solid media and growing in liquid media. We suppose that formation and persistence of giant L-forms structures (filaments, syncytia and "mother" cell) sheltering and embodying many individuals inside a common envelope, represents a unique mechanism of survival and may resemble "invisible" or cryptic state of L-form development. However, at some point of L-form development, these giant spherical or filamentous forms start to disintegrate and are no longer visible, giving place to an abundance of granular and coccoid forms, which sometimes become the prevailing elements within L-population. Coccoid forms of mycobacteria, called "mycococcus", were obtained *in vitro* by Csillag in 1964. Mycococcii were grown from *M. tuberculosis* and were similar to the morphology of staphylococci (Csillag, 1964). Genetic analysis of mycobacterial coccoids however, performed by us through amplification of 16SrRNA gene fragment, 16S-23S rRN gene Internal Transcribed

Cell Wall Deficiency in Mycobacteria: Latency and Persistence 201

We suggest that the lack of cell walls and easier permeation of nutrients is the reason for the unique ability of mycobacterial L- forms to grow faster in comparison to classical tubercle bacilli. Pla Y Armengol (1931) found that a large inoculum of tubercle bacilli grows rapidly on all routine media, appearing as large L-body spheres and also vegetated mycelia. In our study, L-form variants were adapted without difficulties to grow on conventional nutrient agar. Light and electron microscopy also provided interesting results about the appearance of non-acid fast coccoid cell morphology of stressed *M. tuberculosis*, that support observations of other authors. The appearance of non-acid fast coccoids in cultures of mycobacteria has been reported by others in the beginning of the last century but the phenomenon was not clearly explained and proven at that time (Csillag, 1964; Juhasz, 1962; Xalabander, 1958;). More surprising was the fact that mycobacterial coccoid L-forms not only mimicked the morphology of staphylococci or other coccus- shaped bacteria, but also exhibited extremely rapid growth and colonial development in contrast to classical TB bacilli (n. d.). Coccoid cells were initially mistaken by us as contaminants, but the specific DNA testing (amplification of 16SrRNA gene fragment, 16S-23S rRNA gene Internal Transcribed Spacer sequences, IS*6110* PCR and DNA sequencing analysis) identified them as *M. tuberculosis* (n. d.). We suppose that non-acid fast coccoid L-form variants of mycobacteria resulted probably from the more regular mode of multiplication, synchronization and stabilization of L-form cells under specific condition of cultivation. Thus, it can be presumed why such coccoid forms of *M. tuberculosis* remain often

unrecognized or are mistaken for contaminants.

stress (n. d.).

a. b. c.

of L-forms from specimens (Michailova et al., 2005; Markova et al., 2008a).

Fig. 7. Light microscopy of (a) control *M. tuberculosis* rough microcolony and (b, c) typical "fried eggs" shaped colonies of *M.tuberculosis* L-forms obtained after nutrient starvation

Standard plating techniques are often inadequate for accurate enumeration of microbial dormant forms, because some of them may be in a "nonculturable" state (Shleeva et al., 2010). When it comes to L-forms**,** they are considered to be both "difficult-to-cultivate" and "difficultto-identify". Because of their altered morphology and fully changed bacterial life cycle, Lforms are difficult to be identified in clinical materials. The isolation of arising *in vivo* L-forms is generally possible only with special procedures ensuring their enrichment and resuscitation to actively growing state i.e. having an ability to form colonies (Michailova et al., 2000a; Zhang et al., 2001; Zhang, 2004). The use of specially supplemented liquid and semisolid media, as well special techniques, like so called "blind" passages, are absolutely necessary for isolation

Spacer sequences and IS *6110* PCR, verified them as *M. tuberculosis* (n. d.). DNA sequencing analysis is currently in progress (n. d.).We consider that the invisible Lconversion phase is followed by a state of active reproduction of non- acid fast and nonrecognizable as mycobacteria L-forms usually with coccoid morphology. Taken together, these data may argue that the curious morphology and growth characteristics of mycobacterial L-forms, their extremely different habit of existence define them as specific type of unrecognizable and hidden persisters. As seen in Fig.6, L-form conversion cycle of mycobacteria is schematically outlined with emphasis on ability of different L-structures to form colonies. In this sense, L-form persistence phenomenon substantially differs from the current understanding for latency as persistence of few ''non-replicating''or ''dormant'' bacteria.
