**Morphological Characterization of**  *Mycobacterium tuberculosis*

#### Ali Akbar Velayati and Parissa Farnia

*Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease (NRITLD), WHO & UNION Collaborating Centre for TB & Lung Diseases, Shahid Beheshti University (Medical Campus), Darabad, Tehran, Iran* 

#### **1. Introduction**

It is more than 100 years since the first Mycobacterium was isolated by Hansen (1874). That was leprosy bacillus, *Mycobacterium leprae,* which even today is still resisting all attempts to cultivate it in the laboratory*.* The tubercle bacillus, *M. tuberculosis was* discovered eight years later by Robert Koch (1882). The Koch discovery was confirmed by more efficient staining models of Ehrlich (1887) and Ziehl- Neelsen (1883). Under Light microscope, the tubercle bacilli typically appear as straight or slightly curved rods. According to growth conditions and age of the culture, bacilli may vary in size and shape from coccobacilli to long rods. The dimensions of the bacilli have been reported to be 1-10 µm in length (usually 3-5 µm), and 0.2 -0.6 µm width. The possibility of morphological variations in tubercle bacilli was suggested by few investigators like Malassez and Vignal (1883), Nocard and Roux (1887), Metschnikoff (1888), Lubarsch(1899), Fischel(1893), and Vera and Rettger (1939). They showed under unfavorable conditions, i.e., a limited food supply, or oxygen deprivation, *Mycobacterium* assumed a swollen appearance without forming the vacuolar or globoid bodies (Vera and Rettger, 1939). These early reports were based on stained preparations and were subjected of severe criticism (Porter and Yegian, 1945). Today with advances in microscopic technique i.e., transmission electron microscope (TEM), scanning electron (SEM) and atomic force microcopy (AFM), almost all of investigators have been agreed that the Koch bacillus does not always manifest itself in the classical rod shape (figure 1). They become shorter in older cultures, filametous within macrophages and ovoid during starvation (Young *et al*., 2005; Farnia *et al*., 2010; Shleeva *et al.*, 2011) and they may produce buds (Chauhan *et a*l., 2006) and branches in extensively drug resistance strains (XDR-TB) (Velayati *et al* 2010; Farnia *et al* 2010). In the following parts the underlying mechanisms that may help the bacilli to change its morphology was highlighted.

#### **2. The role of cell wall in shape maintenance**

The cell wall of mycobacterium is characterized by a unique structure which is caused by partly distinct chemical compositions in comparison with the cell wall of other bacteria

Morphological Characterization of *Mycobacterium tuberculosis* 151

with the release of uridine monophosphate (UMP)(Crick *et al*., 2001; Yuan *et al.,* 2007). Third the bactoprenol molecule translocates the disaccharide pentapeptide precursor to the outside of the cell. The GlcNAc-MurNAc disaccharide is then attached to a peptidoglycan chain using pyrophosphate link between itself and the bactoprenol as energy to drive the reaction. The pyrophosphobactoprenol is converted back to a phosphobactoprenol and recycled. Fourth, outside the cell but near the membrane surface, peptide chains from adjacent glycan chains are cross-linked to each other by a peptide bond exchange (transpeptidation) between the free amine of the amino acid in the third position of the pentapepide (e.g., lysine) or the N-terminus of the attached pentaglycine chain and the D-alanine at the fourth position of the other peptide chain, releasing the terminal D-alanine

Enzymes involved in remodeling PG can be grouped as either biosynthetic or hydrolytic. Biosynthetic enzymes include transglycosylase and transpeptidase domains, often found on a single, bifuntional protein. Hydrolytic enzymes include muramidase, glucosaminidase, lytic transglycosylase, amidase, endopeptidase and carboxypeptidase (Young, 2003; Cabeen and Jacobs-Wagner, 2005). The reaction of these enzymes may be antagonistic, or they may physically interact to form complexes capable of breaking bonds to generate openings for new monomers, while also forming bonds necessary to unit PG strands. The production of these enzymes should be regulated, otherwise the bacterial cell wall would be degraded and the bacteria would be lyses. There are several ways to governorate these enzymes; one method is through formation of complexes with other proteins (Hett and Rubin, 2008).These proteins could suppress the activity of the enzyme, or they could be enzymes themselves with antagonistic reactions that join rather than degrade PG. Another possibility is that the enzymes are sequestered from their substrate until they needed. A third method could be

that the appropriate substrate is not made available until cleavage of it is required.

The PG –synthesizing enzyme organize into complexes that likely contributes to the resulting shape. Various models have been proposed which explain how this organization affects the bacterial shape. The two –competing sites" model (TCS) for peptidoglycan assembly advocates that, in bacterial rods, the shape depends on the activity of two biochemical reactions (sites) which occur in the terminal stages of peptidoglycan synthesis; one site is responsible for lateral wall elongation , and the other is responsible for septum formation (Lleo *et al*., 1990; Alaedini and Day, 1999). The two sites compete with each other in such a way that the lateral wall is not extended during septum formation and *vice versa*  (Lleo *et al*., 1990; Satta *et al* .,1994). The actual shape of the bacteria is thus determined by the balance between the two competing reactions, correct balance leading to normal rods; abnormal prevalence of the site for lateral wall elongation leads to long rods or filaments, whereas prevalence of the site for septum formation leads to formation of coccobacilli or cocci (Lleo *et al*., 1990). The other bacteria carry only one site for peptidoglycan assembly which can form only septa and can grow only as cocci. Another model is "three –for –one " predict the insertion of PG along a track, using an existing strand of PG as a template

of the precursor (Wietzerbin *et al*., 1974; Ghuysen, 1991).

**4. Control of peptidoglycan synthesis** 

**5. The role of PG in cell shape regulation** 

(Koike and Takeya, 1961; Imaeda and Ogura, 1963; Imaeda *et al*., 1969). These variations are thought to be advantageous in stressful conditions of osmotic shock or desiccation as well as contributing to their considerable resistance to many drugs (Jarlier and Nikaido, 1990). The *Mycobacterial* cell wall, in principal, consists of an inner layer and an outer layer that surround the plasma membrane (Hett and Rubin, 2008). The outer compartment consists of both lipids and proteins (Draper, 1971, 1998; Draper *et al*., 1998; Brennan and Nikaido, 1995; Brennan, 2003). The inner compartment consists of peptidoglycan (PG), arabinogalactan (AG), and mycolic acid (MA) covalently linked together to form a complex known as MA-AG-PG complex that extends from the plasma membrane outward in layers, starting with PG and ending with MAs. The Peptidoglycan, which forms the "backbone' of the cell wall skeleton,was first studied by Misaki *et al* (1966). It belongs to a family of structures possessed by almost all bacteria and blue-green algae but by no other type of living organism (Schleifer and Kandler, 1977); its presence in mycobacteria provides conclusive evidence that they are not, as was once believed, some sort of intermediate stage between bacteria and fungi. The peptidoglycon is made of peptides and glycan strands. The long glycan strand typically consists of repeating N-acetylglucosamines (NAGs) linked to N-acetylmuramic acid (NAM). These strands are cross linked by peptides bound to the lactyl group on NAMs from different glycan strands. These peptide chains normally consist of L-alanyl-D-*iso*-glutaminyl-*meso*-diaminopimelic acid (DAP) from one strand linked to the terminal D-alanine residue from L-alanyl-D-*iso*-glutaminyl-*meso*- DAP-D-alanine from a different strand (Kotani *et al*., 1970; Wietzerbin *et al*., 1974). This highly cross-linked glycan meshwork of PG that surrounds bacteria is the primary agent that maintains bacterial shape. The structure of this stratum differs slightly from that of common bacteria, as it presents some particular chemical residues and unusual high number of cross-links. Indeed, the degree of peptidoglycon cross linking in the cell wall of *M. tuberculosis* is 70-80%, whereas that in *E. coli* is 20-30%. (Matsuhashi, 1994; nVollmer and Holtje,2004). PG isolated from *E. Coli* retains its rod –like shape even in the absence of all other material (Weidel *et al*., 1960; Weidel and Pelzer, 1964), confirming its role in shape maintenance. Also, treatment of bacteria with lysozyme which degrades PG, results in rod shaped cells becoming round spheroplasts (Lederberg, 1956). Spheroplasts, or round bacteria lacking PG, can be formed in *M. smegmatis* through degradation of PG. Upon transfer to growth media, the spherical bacteria are able to regenerate wild-type rod -shaped cells (Udou *et al.*, 1982). This occurs through elongation of bacteria that then branch, septate and fragment. These data argue that shape and size are not simply governed by existing PG, but there must be some genetic heritable determinant also.

### **3. Peptidoglycan synthesize**

Little is known about the biosynthesis of the peptidoglycan of *M. tuberculosis.* However, it is generally assumed to be similar to that of *E. coli* (van Heijenoort, 1998). Generally, peptidoglycan synthesis occurs in four sequential steps. First, inside the cytoplasm, soluble substrates are activated and peptidoglycon units are build. Glucosamine is enzymatically converted into MurNAc and then energetically activated by a reaction with uridine triphosphate (UTP) to produce uridine diphosphate –N-acetylmuramic acid (UDP-MurNAc) (De Smet *et al*., 1999). Second, at cytoplasmic membrane, the units UDP-MurNAc pentapeptide is attached to the bactoprenol " conveyor belt", through a pyrophosphate link

(Koike and Takeya, 1961; Imaeda and Ogura, 1963; Imaeda *et al*., 1969). These variations are thought to be advantageous in stressful conditions of osmotic shock or desiccation as well as contributing to their considerable resistance to many drugs (Jarlier and Nikaido, 1990). The *Mycobacterial* cell wall, in principal, consists of an inner layer and an outer layer that surround the plasma membrane (Hett and Rubin, 2008). The outer compartment consists of both lipids and proteins (Draper, 1971, 1998; Draper *et al*., 1998; Brennan and Nikaido, 1995; Brennan, 2003). The inner compartment consists of peptidoglycan (PG), arabinogalactan (AG), and mycolic acid (MA) covalently linked together to form a complex known as MA-AG-PG complex that extends from the plasma membrane outward in layers, starting with PG and ending with MAs. The Peptidoglycan, which forms the "backbone' of the cell wall skeleton,was first studied by Misaki *et al* (1966). It belongs to a family of structures possessed by almost all bacteria and blue-green algae but by no other type of living organism (Schleifer and Kandler, 1977); its presence in mycobacteria provides conclusive evidence that they are not, as was once believed, some sort of intermediate stage between bacteria and fungi. The peptidoglycon is made of peptides and glycan strands. The long glycan strand typically consists of repeating N-acetylglucosamines (NAGs) linked to N-acetylmuramic acid (NAM). These strands are cross linked by peptides bound to the lactyl group on NAMs from different glycan strands. These peptide chains normally consist of L-alanyl-D-*iso*-glutaminyl-*meso*-diaminopimelic acid (DAP) from one strand linked to the terminal D-alanine residue from L-alanyl-D-*iso*-glutaminyl-*meso*- DAP-D-alanine from a different strand (Kotani *et al*., 1970; Wietzerbin *et al*., 1974). This highly cross-linked glycan meshwork of PG that surrounds bacteria is the primary agent that maintains bacterial shape. The structure of this stratum differs slightly from that of common bacteria, as it presents some particular chemical residues and unusual high number of cross-links. Indeed, the degree of peptidoglycon cross linking in the cell wall of *M. tuberculosis* is 70-80%, whereas that in *E. coli* is 20-30%. (Matsuhashi, 1994; nVollmer and Holtje,2004). PG isolated from *E. Coli* retains its rod –like shape even in the absence of all other material (Weidel *et al*., 1960; Weidel and Pelzer, 1964), confirming its role in shape maintenance. Also, treatment of bacteria with lysozyme which degrades PG, results in rod shaped cells becoming round spheroplasts (Lederberg, 1956). Spheroplasts, or round bacteria lacking PG, can be formed in *M. smegmatis* through degradation of PG. Upon transfer to growth media, the spherical bacteria are able to regenerate wild-type rod -shaped cells (Udou *et al.*, 1982). This occurs through elongation of bacteria that then branch, septate and fragment. These data argue that shape and size are not simply governed by existing PG, but there must be some genetic

Little is known about the biosynthesis of the peptidoglycan of *M. tuberculosis.* However, it is generally assumed to be similar to that of *E. coli* (van Heijenoort, 1998). Generally, peptidoglycan synthesis occurs in four sequential steps. First, inside the cytoplasm, soluble substrates are activated and peptidoglycon units are build. Glucosamine is enzymatically converted into MurNAc and then energetically activated by a reaction with uridine triphosphate (UTP) to produce uridine diphosphate –N-acetylmuramic acid (UDP-MurNAc) (De Smet *et al*., 1999). Second, at cytoplasmic membrane, the units UDP-MurNAc pentapeptide is attached to the bactoprenol " conveyor belt", through a pyrophosphate link

heritable determinant also.

**3. Peptidoglycan synthesize** 

with the release of uridine monophosphate (UMP)(Crick *et al*., 2001; Yuan *et al.,* 2007). Third the bactoprenol molecule translocates the disaccharide pentapeptide precursor to the outside of the cell. The GlcNAc-MurNAc disaccharide is then attached to a peptidoglycan chain using pyrophosphate link between itself and the bactoprenol as energy to drive the reaction. The pyrophosphobactoprenol is converted back to a phosphobactoprenol and recycled. Fourth, outside the cell but near the membrane surface, peptide chains from adjacent glycan chains are cross-linked to each other by a peptide bond exchange (transpeptidation) between the free amine of the amino acid in the third position of the pentapepide (e.g., lysine) or the N-terminus of the attached pentaglycine chain and the D-alanine at the fourth position of the other peptide chain, releasing the terminal D-alanine of the precursor (Wietzerbin *et al*., 1974; Ghuysen, 1991).
