**2. History**

L-forms were first observed by Emmy Klieneberger-Nobel, in 1945, whose typical "fried eggs"-shaped colonies, duplicating Mycoplasma, were isolated from cultures of *Streptobacillus moniliformis.* The wall-less variants of L-forms she named after the institution she worked in – England's Lister Institute.

Fig. 1. Emmy Klieneberger-Nobel – the founder of bacterial L-forms.

Cell Wall Deficiency in Mycobacteria: Latency and Persistence 195

(Domingue, 2010). It is assumed that these pleomorphic forms represent various stages in

*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,

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

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

morphology of various other bacteria (Fig. 3 b, c).

the life cycle of stressed bacteria.

1956).

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; Mattman, 1970, 2001).

#### Fig. 2. Lida Mattman

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 & Chen, 2001).
