Hydrolysis of peptides by the protease (also called peptidase) to free amino acids is one of the central activities within a cell. The degradation of proteins is an important mechanism for regulating many pathways, degrading mis-folded proteins, and degradingproteins during starvation to provide amino acids for energy. Usually, proteases are classified into the different families, based on the evolutionary
relationship between each other. Each family is identified by an upper-case letter representing the catalytic type (S for serine-type, T for threonine-type; C for cysteine- type, A for aspartic-type, M for metallo-type, and U for unknown type) (Puente, et al., 2003). As shown in Tbale 3.2.30, analysis of the genomes of the four Leptospira species revealed a slightly different distribution of the three protease families, C, M, and S.
Table 3.2.30 Protease distribution in Leptospira species.
members in each family Protease
Family pomona grippotyphosa lai copenhageni
A 1 1 1 1 C 2 2 3 4 M 34 38 35 36 T 0 0 0 0 S 7 8 7 8 U 1 1 1 1 3.2.7 Cell Wall 3.2.7.1 Peptidoglycan
In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall consisting mainly of the peptidoglycan, a polymer of a repeating disaccharide-peptide unit, where the pentapeptide chains attached to adjacent sugar molecules are cross-linked. The synthesis of peptidoglycans can be divided into three stages (van Heijenoort, 1998; Mirelman, et al., 1976).
The first stage involves synthesis of two amino sugars precursors UDP-NAG and UDP-NAM in the cytoplasm. After D-glutamate and D-alanyl-D-alanine are synthesized respectively, UDP–NAM is linked to pentapeptide, forming the basic subunit of peptidoglycan.
In the second stage, the lipid carrier (lipid P) transfers UDP–NAM- pentapeptide through the inner membrane to the periplasm, where UDP–NAM- pentapeptide is linked to the UDP-NAG sugar to form the disaccharide precursor. Then, lipid PP is hydrolyzed to lipid P that can reenter the cycle.
In the third stage, after the newly synthesized peptidoglycan subunit is transferred to the growing point of the cell wall's peptidoglycan, the sugars are polymerized, and the peptide chains are cross-linked.
As shown in Table 3.2.31, all the enzymes involved in peptidoglycan synthesis except UDP-N-acetylmuramoylpentapeptide lysine N6-alanyltransferase (EC
2.3.2.10) were found in four Leptospira species. The domain search revealed that one possible ORF could encode the missing transferase as it includes the same functional domain (COG2348: uncharacterized protein involved in methicillin resistance) conserved in UDP-N-acetylmuramoylpentapeptide lysine N6-alanyltransferase. Figure 3.2.29 illustrates peptidoglycan synthesis as reconstructed in the four
Leptospira species.
Table 3.2.31 Enzymes involved in peptidoglycan biosynthesis
EC# Description Genes in
pomona Genes in grippotyphosa Genes in lai Genes in copenhageni 2.6.1.16 glucosamine--fructose-6-phosphate
aminotransferase Yes Yes Yes Yes
5.4.2.- Phosphoglucosamine mutase Yes Yes Yes Yes 2.3.1.157 glucosamine-1-phosphate N-
acetyltransferase Yes Yes Yes Yes
2.7.7.23 UDP-N-acetylglucosamine
pyrophosphorylase Yes Yes Yes Yes
2.5.1.7 UDP-N-acetylglucosamine 1- carboxyvinyltransferase
Yes Yes Yes Yes
1.1.1.158 UDP-N-acetylmuramate dehydrogenase Yes Yes Yes Yes 6.3.2.8 UDP-N-acetylmuramate--alanine ligase Yes Yes Yes Yes
5.1.1.3 glutamate racemase Yes Yes Yes Yes
6.3.2.9 UDP-N-acetylmuramoylalanine--D-
glutamate ligase Yes Yes Yes Yes
6.3.2.13 UDP-N-acetylmuramoylalanyl-D-
glutamate--2,6-diaminopimelate ligase Yes Yes Yes Yes
5.1.1.1 alanine racemase Yes Yes Yes Yes
6.3.2.4 D-alanylalanine synthetase Yes Yes Yes Yes 6.3.2.10 UDP-N-acetylmuramoylalanyl-D-
glutamyl-2,6-diaminopimelate--D- alanyl-D-alanine ligase
Yes Yes Yes Yes
2.7.8.13 phospho-N-acetylmuramoyl-
pentapeptide-transferase Yes Yes Yes Yes
2.4.1.227 UDP-N-acetylglucosamine--N- acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N- acetylglucosamine transferase
Yes Yes Yes Yes
2.4.1.129 peptidoglycan glycosyltransferase Yes Yes Yes Yes 3.6.1.27 undecaprenyl-diphosphatase Yes Yes Yes Yes
6.3.1.2 glutamine synthetase Yes Yes Yes Yes
2.3.2.10 UDP-N-acetylmuramoylpentapeptide
lysine N6-alanyltransferase Yes Yes Yes Yes
2.6.1.21 penicillin-binding protein Yes Yes Yes Yes
3.2.7.2 Lipopolysaccharides (LPS)
Lipopolysaccharide (LPS) contributes greatly to the structural integrity of the bacteria and protects them from the host immune defenses. An LPS contain three parts: lipid A, core polysaccharide, and O-specific chain (Figure 3.2.30).
Figure 3.2.30 A schematic diagram of a lipopolysaccharide molecule (Bulach, 2000) The first part, lipid A, consists of six fatty acyl chains linked to two
glucosamine residues. The second part, core polysaccharide, is attached to lipid A through 3-hydroxy-D-manno-octulosonate (KDO). This core is further divided into two regions, an inner core and an outer core. The inner core consists of KDO, heptose, and phosphate, and the outer core consists of hexoses. The third part, O- specific chain, determines the antigenic specificity of the organism. The O-specific chain consists of four to six sugars that may be repeated up to 50 times, making it the most variable region, while lipid A is the most conserved.
The core and O-specific chain are synthesized in a manner similar to
peptidoglycans, where the sugar residues are synthesized and assembled on a lipid P carrier. Figure 3.2.30 described the biosynthesis pathway of lipid A. All the genes in this pathway except KDO 8-P phosphatase (EC 3.1.3.45) were identified from
Leptospira species. A further domain analysis revealed that no any ORF from Leptospira species includes the functional domain (COG1778: low specificity
phosphatase) conserved in KDO 8-P phosphatase. However, since an amino acid sequence analysis indicates that KDO 8-P phosphatase is a member of the haloacid dehalogenase hydrolase superfamily (Wu and Woodard, 2003) and one haloacid dehalogenase-like hydrolase could be identified in all four Leptospira species, this enzyme may function as the missing KDO 8-P phosphatase (Table 3.2.32).
Figure 3.2.31 Lipid A biosynthetic pathway
Table 3.2.32 Enzymes involved in Lipid A and KDO biosynthesis.
EC# Description Genes in
pomona grippotyphosa Genes in Genes in lai copenhageniGenes in
2.3.1.129 UDP-N-acetylglucosamine
acyltransferase (lpxA) Yes Yes Yes Yes
3.5.1.- UDP-3-O-[3-hydroxymyristoyl] N- acetylglucosamine deacetylase (lpxC)
Yes Yes Yes Yes
2.3.1.-(a) UDP-3-O-[3-hydroxymyristoyl]
glucosamine N-acyltransferase (lpxD) Yes Yes Yes Yes 2.4.1.182 Lipid-A-disaccharide synthase (lpxB) Yes Yes Yes Yes
2.7.1.130 Tetraacyldisaccharide 4'-kinase Yes Yes Yes Yes 2.4.99.- 3-deoxy-D-manno-octulosonic-acid
transferase (KDO transferase) (kdtA) Yes Yes Yes Yes 2.3.1.-(b) Apolipoprotein N-acyltransferase (lnt) Yes Yes Yes Yes
2.3.1.-(c) Lipid A biosynthesis lauroyl
acyltransferase;lauroylacyltransferase (htrB)
Yes Yes Yes Yes
2.5.1.55 KDO 8-P synthase Yes Yes Yes Yes
3.1.3.45 3-deoxy-manno-octulosonate-8-
phosphatase Yes Yes Yes Yes
2.7.7.38 3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) (kdsB)
Yes Yes Yes Yes