Chapter 14Biosynthetic Pathways Related to Cell Structure and Function

Krishnamurthy P, Phadnis SH, DeLoney CR, et al.

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In vivo Helicobacter pylori has a tightly spiraled shape, but in vitro it grows as curved rods, which, after prolonged incubation, evolve into metabolically active but nonculturable coccoid forms (2, 6, 8, 57). The spiral morphology of H. pylori appears to confer an advantage to the bacterium in the viscous gastric mucus, since its spiral forms move more effectively in media with high viscosity than more conventional rod-shaped organisms (40). Motility is an essential virulence factor in H. pylori, as nonmotile variants do not colonize the gastric surface in the gnotobiotic piglet animal model (18). Further, the phenomenon of nonculturable but viable coccoid forms of H. pylori may explain the paradox of H. pylori spread via the fecal-oral route in the absence of culturable bacteria from fecal specimens (2, 8). The peptidoglycan of H. pylori, which forms the backbone of the cell wall, probably plays a significant role in maintaining the spiral morphology and in the morphological transformation of H. pylori from spiral to coccoid forms.

Bacterial Peptidoglycans

Peptidoglycans are the stress-bearing structures of bacteria that maintain the integrity of the cell wall and the shape of the bacterium (28, 42). The typical bacterial peptidoglycan (PG) is a heteropolymer of glycan strands made up of the amino sugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) linked by β1 → 4 glycosidic bonds (9, 28, 73). Attached to the carboxyl group of each muramic acid by an amide linkage is the short peptide, l-alanyl-d-glutamyl-l-meso-diaminopimelyl-d-alanyl-d-alanine (12, 66). The MurNAc at the end of each strand is present as a nonreducing 1,6 anhydro sugar. Together the β1 → 4 linked amino sugars and the amide-linked short peptide constitute a monomer and form the basic muropeptide repeating unit of the murein, the specific PG of bacterial cell walls. Between different bacteria the glycan strands exhibit only few structural variations such as O-acetylation or de-N-acetylation of either amino sugar, but the structural diversity of the peptide moiety is substantial, particularly the interpeptide bridges that cross-link peptides from different glycan strands (62, 64, 65). A unique feature of the peptide backbone of all PG is the alternating sequence of optical isomers (l-d-l-d amino acids). Adjacent PG strands are generally cross-linked to each other through the peptide side chains by transpeptidation between the carboxyl group of the d-alanine in position 4 of one peptide and the free amino group of di-aminopimelic acid (DAP) in the adjacent strand. These peptide cross-linking bonds between amino acids located on adjacent glycan chains lead to the formation of cross-linked dimers (peptide cross-linking between two monomers on adjacent glycan strands), trimers, and tetramers. In Escherichia coli, a significant percentage of the peptide cross-links do not involve d-alanine-DAP residues, but involve DAP-DAP residues of neighboring chains; the relative number of both types of cross-links depends on the growth phase of the bacterium (2931).

Analysis of the muropeptide structures in the PG of E. coli by pulse-chase labeling experiments depict changes in the muropeptide composition as the bacteria reach the stationary phase (4, 10, 30, 61). These muropeptide changes include a decrease in pentapeptide side chains, increase in tripeptide side chains, increase in the extent of cross-linking, and decrease in the average chain length of the glycan strands. Not only are the covalent bonds cleaved during the transition to the stationary stage, but there is also release of muropeptides from the PG matrix (36, 37).

H. pylori Peptidoglycan

H. pylori murein has been isolated and characterized by Costa et al. (13) and Krishnamurthy et al. (51). High-performance liquid chromatograms of PG fragments obtained by muramidase digestion of H. pylori murein show a relatively small number of peaks, indicating a simple PG structure. The disaccharide backbone of H. pylori PG consists of GlcNAc β1 → 4 linked to MurNAc acid. Adjacent glycan strands are cross-linked via traditional DAP–d-alanine peptide cross bridges. Unlike many gram-negative bacteria, the murein of H. pylori possessed no detectable DAP-DAP cross-links. Monomers (64.8%) and dimers (35.1%) constitute virtually all of the muropeptides of H. pylori, and its PG lacks detectable muropeptide trimers and tetramers (Table 1). The major muropeptide monomer is pentapeptide. The ratio of nonreducing 1,6-anhydromuramic acid residues at the end of the glycan chains to reducing muramic acid residues is 1:11, suggesting a very low glycan chain length (Table 1). The only amino acids detected by tandem mass spectroscopic analysis are Ala, Glu, Gly, and DAP, linked to the muramic acid moiety in the order l-alanine, d-glutamic acid, DAP, d-alanine, and d-alanine (13, 51).

Table 1. Structure of peptidoglycan isolated from H. pylori strains SP1, 84–183, NCTC 11637 and E. coli MC 4100.

Table 1

Structure of peptidoglycan isolated from H. pylori strains SP1, 84–183, NCTC 11637 and E. coli MC 4100.

H. pylori shows significant variation in murein composition between early log phase (24 h) and late log phase/stationary phase (96 h) cultures (51), suggesting alteration in PG composition as the bacteria change from a helical to a coccoid morphology (13) (Fig. 1). Muropeptides with a pentapeptide side chain are prevalent throughout the growth phase (13). However, the proportion of Gly-containing muropeptides doubles rapidly when cells go into the stationary growth phase (13). There is an overall decrease in monomers as the bacteria age, while the percentage of dimers, "anhydro" residues, and dipeptide monomers increases.

Figure 1

Figure 1

. General composition and age changes in H. pylori PG. As H. pylori cells age from early to late log phase, the composition of their PG changes. The shaded region represents the amino acids that are removed from the PG in late log phase, essentially resulting (more...)

Unique Features of H. pylori Peptidoglycan

The structure of H. pylori PG has several unique features as shown in Table 1. The degree of cross-linking that reflects the percentage of the total number of DAP residues that are engaged in cross-linking peptide bonds is defined as 0.5 × percent dimers + 0.667 × percent trimers + 0.8 × percent tetramers. In H. pylori murein it is 17%, one of the lowest degrees of cross-linking identified to date. For comparison, the percent cross-linking of E. coli PG is 25 to 30% (69, 73); Neisseria spp., 36 to 44% (63); Vibrio spp., 30% (46, 74); Proteus spp., 33 to 37% (47); Moraxella spp., 37% (54); and Pseudomonas spp., 25 to 45% (21, 41, 54). H. pylori PG also has an unusually high proportion of glycan chain terminating "anhydro"-muropeptides, consistent with a very short glycan chain length of 9 to 11 disaccharides, one of the lowest average lengths so far reported. In comparison, E. coli PG has an average glycan chain length of 21 disaccharide units (39). The lack of detectable trimers or tetramers in H. pylori murein indicates that cross-linking does not occur between three or more glycan chains, whereas cross-linking between three or four glycan chains is detectable in E. coli (32). No DAP-DAP cross bridges were identified in H. pylori in contrast to E. coli. The exact function of DAP-DAP cross-links found in E. coli is unclear, but it has been suggested that they perform unique functions since lipoprotein is found attached to muropeptide dimers with these cross-links, and not to DAP-d-Ala linked dimers (23). The absence of l-d–DAP-DAP bond suggests that, unlike E. coli (28), H. pylori has no alternative mechanism to allow the formation of peptide bridges, other than the typical DAP-d-Ala cross-linking. Finally, the presence of pentapeptide as the main fraction of H. pylori muropeptides indicates that it possesses little carboxypeptidase activity. The genome sequence of H. pylori supports this last conclusion (1, 70).

Significance of Unique Characteristics of H. pylori Peptidoglycan

In vivo H. pylori has tight spiral morphology during log growth phase, and a coccoid form in the late log phase of growth (7). In other spiral bacteria including leptospirae, borreliae, treponemas, and campylobacters, the coccoid forms are considered to be degenerate forms (48). However, as the mode of transmission of H. pylori remains unresolved, it has been suggested that coccoid forms are nonculturable but viable organisms that form part of a complex life cycle of the bacterium (53). Further studies are required to understand the pathological significance of low glycan chain length and the low percentage of cross-link seen in H. pylori PG. However, by comparison with the strong and tight murein from organisms such as Staphylococcus spp., which have nearly 90% cross-linking, it is possible to hypothesize that the short glycan chain length and low peptide cross-linking of H. pylori might form a PG that is weakly and loosely held together, enabling the organism to change its morphology from spiral to coccoid to spiral forms much more efficiently than other spiral organisms.

Biosynthetic Pathway of Peptidoglycan Formation

On the basis of protein sequence homologies, the genome of H. pylori appears to code for homologs of all the enzymes involved in the cytoplasmic synthesis of the disaccharide pentapeptide, starting with the synthesis of UDP-N-acetylmuramic acid and finishing with UDP-disaccharide pentapeptide linked to an undecaprenyl lipid carrier (Table 2). The second step in building the murein sacculus is the polymerization reaction, accompanied by the insertion of the newly made material into the existing PG layer. This function is carried out by the penicillin-binding proteins (24, 26, 33). Genes coding for three penicillin-binding proteins have been identified in the genome of H. pylori, based on comparisons of encoded amino acid sequences (Table 3).

Table 2. List of H. pylori 26695 putative genes that are involved in PG biosynthesis.

Table 2

List of H. pylori 26695 putative genes that are involved in PG biosynthesis.

Table 3. H. pylori 26695 ORFs that show sequence motifs of PBPsa.

Table 3

H. pylori 26695 ORFs that show sequence motifs of PBPsa.

Some gram-negative organisms exhibit murein turnover. In this process substantial amounts of PG fragments are released from the murein sacculus, and some of the soluble muropeptides are reutilized for PG assembly (34, 35). In E. coli, lytic transglycosylases and endopeptidases are responsible for releasing 1,6-anhydromuropeptides in the periplasm, and the turnover products are reutilized for PG assembly after uptake into the cytoplasm via active transport systems (35). From the analysis of the H. pylori genome, it is uncertain if murein fragments are recycled by H. pylori. The bacterium has orthologs of the genes slt and amiA, which encode a lytic transglycosylase and an N-acetylmuramyl-l-alanine amidase, respectively (1, 70). However, other genes coding for enzymes of the recycling pathway of murein turnover products have not been identified; for example, ampG, encoding a membrane protein required for uptake of intact muropeptides; ampD, coding for a cytoplasmic amidase with specificity for the 1,6-anhydromuramyl-l-Ala bond; and mpl, a specific tripeptide ligase, which forms UDP MurNAc peptides (16, 42).

Penicillin-Binding Proteins and Other Related Proteins

Penicillin-binding proteins (PBPs) are specialized acyl serine transferases involved in the assembly, maintenance, and regulation of the features of the PG structure. Most of these proteins are anchored in the bacterial inner membrane, with their active sites accessible in the periplasmic space. On the basis of size, the PBPs identified to date have been classified into the broad categories of low-molecular-weight and high-molecular-weight proteins (25). In general, high-molecular-weight PBPs have transglycosylase and/or transpeptidase activities, and low-molecular-weight PBPs have carboxypeptidase and/or endopeptidase activities (24, 27, 33); some of the low-molecular-weight PBPs also display transpeptidase activity (24, 25). Although PBPs fulfill different functions in PG biosynthesis, their catalytic centers have a remarkably well-conserved topology defined by three amino acid groupings, referred to as "motifs" (33). These motifs occur in the same order and with roughly the same spacing along the polypeptide chains, defining common amino acid sequence signatures, and polypeptide folding brings the three motifs close to each other (33, 67). Motif 1 is SXXK, where "S" is the essential serine residue and "X" is a variable amino acid residue; motif 2 is S/YXN/C, and defines one side of the catalytic center; and motif 3 is [K,H][T,S]G, and defines the other side of the catalytic center (25, 33, 67).

H. pylori Penicillin-Binding Proteins

Most experimental studies to date on the PBPs of H. pylori have been conducted using labeled β-lactams, which form a covalent bond with these proteins. PBPs in H. pylori were first reported by Ikeda et al., who identified three proteins using [14C] penicillin G (44). More recently eight additional PBPs have been identified in H. pylori employing penicillin derivatives linked to various reporter groups (14, 17, 38, 52). Altogether, the combined data from the above studies indicate the presence of 11 PBPs in H. pylori ranging in molecular mass from 28 to 100 kDa.

In the genomes of H. pylori strains 26695 and J99, the genes encoding five of these PBPs have been identified and correspond to the open reading frames (ORFs) HP0597/jhp544, HP1565/JHP1473, HP1556/JHP1464, HP0160/JHP0148, and HP0211/JHP0197 given in Table 3 (1, 16, 70). ORFs HP0597/jhp544, HP1565/JHP1473, HP1556/JHP1464 represent high molecular weight proteins and show sequence homology to PBPs from other bacteria. They are designated PBP1, PBP2, and PBP3, respectively (Table 3). ORFs HP0160/JHP0148 and HP0211/JHP0197 represent low-molecular-weight proteins and do not show sequence homology to any known proteins (11, 52). The gene sequences encoding the other six putative PBPs have not been identified (14, 38).

Proposed Functional Roles

Known monofunctional PBPs possess transpeptidase, carboxypeptidase, or endopeptidase activity, and multimodular PBPs have transglycosylase/transpeptidase or carboxypeptidase/endopeptidase activities (22, 27, 56, 67, 68). H. pylori PBP1, PBP2, and PBP3 display sequence similarities with biosynthetic multimodular PBPs from other bacteria, suggesting that they carry out transglycosylation and/or transpeptidation reactions. Hierarchical analysis of the amino acid sequences of 63 multimodular PBPs indicated a close similarity between H. pylori PBP1 and E. coli PBP1b, suggesting that the H. pylori enzyme has transglycosylase/transpeptidase activities (33). This analysis also revealed relationships between H. pylori PBP3 and E. coli PBP2, an enzyme involved in cell wall expansion and maintenance of cell shape, and H. pylori PBP3 and E. coli PBP3, which is involved in septum formation in the latter bacterium (33).

Protein sequences deduced from the H. pylori genome do not show homology to enzymes with carboxypeptidase activity (1, 70). This finding is in agreement with the characteristics of H. pylori murein. The very high percentage of disaccharide pentapeptide is consistent with low carboxypeptidase activity, which would remove the terminal d-Ala from the pentapeptide chain. In the H. pylori genome there are no genes ortholog to those found in other organisms encoding PBP endopeptidases capable of cleaving murein crosslinks (22, 28, 33); however, this activity is essential for the growth of H. pylori PG. This type of endopeptidase need not be a PBP; for example, in E. coli, the MepA protein cleaves PG cross-links. Analyses of the H. pylori genome sequences from two different strains reveal no proteins similar to E. coli MepA. It is possible that any of the multimodular PBPs outlined above could function as an endopeptidase. In addition, H. pylori shows accumulation of glucosaminyl-muramyl-dipeptide, indicating the presence of an enzyme capable of cleaving the l-d bond between DAP and d-Glu.

Unique Features of the Newly Identified HP160 and HP211

Neither HP160 nor HP211 shows sequence homology to known proteins, and both of these proteins are rich in cysteine (5%) (11, 52). HP160 shows regulated expression; the protein is found in both the membrane and soluble fractions and has a unique arrangement of penicillin-binding motifs (52). Mutational analysis of HP160 suggests that the gene product is essential for the growth and survival of H. pylori since it was not possible to obtain viable null mutants (50). HP211 has been shown to bind and hydrolyze penicillin derivatives, suggesting that this protein is a β-lactamase (55). Further studies are needed to understand the role of these PBPs in H. pylori PG biosynthesis.

Amoxicillin and Aztreonam Binding Characteristics of H. pylori Penicillin-Binding Proteins

Amoxicillin resistance was characterized initially by Dore et al. (17) in clinical isolates in which a PBP with a molecular mass of 30 to 32 kDa could no longer be detected in amoxicillin-resistant strains (MIC, 16 to 32 μg/ml). In these studies amoxicillin resistance was found to be unstable upon freezing and subculturing, but the resistance could be "rescued" in some strains by plating the bacteria on amoxicillin gradient plates. More recently DeLoney and Schiller reported preferential binding of amoxicillin to PBP3 (14, 15) whereas Harris et al. (38) reported preferential binding of amoxicillin to a 72-kDa PBP. DeLoney and Schiller also characterized amoxicillin resistance in H. pylori as arising from an alteration in the affinity of PBP1 for β-lactam antibiotics (15). In these studies resistance was found to be stable upon freezing and subculturing. Kusters et al. (unpublished results) also found an alteration in PBP1 from a clinical isolate with a stable amoxicillin MIC of 8 μg/ml. Further studies will serve to establish whether amoxicillin resistance in H. pylori can be transferred between strains.

Aztreonam at sub-MIC induces filamentation in H. pylori (14). DeLoney and Schiller reported aztreonam to bind preferentially to a PBP with a molecular mass of 63 kDa (14). In E. coli aztreonam has been shown to bind to PBP3, a transpeptidase that is specific for septum formation and cell division (45). Since genetic analysis shows 31% homology between H. pylori and E. coli PBP3, it was concluded that the H. pylori enzyme may be involved in septum formation in this bacterium, in agreement with the conclusions of the hierarchical analysis of modular PBP. Genetic studies will further elucidate the role of PBP (molecular mass, 63 kDa) in H. pylori.

Other Proteins with Putative Murein-Related Functions

In the genome of H. pylori ORFs HP0772/JHP0709 and HP0645/JHP0590 code for enzymes homologous to an amidase and a lytic transglycosylase, respectively (1, 70). These are not penicillin-binding proteins but are known to perform murein-related functions. N-acetylmuramyl-l-alanine amidases specifically cleave the amide bond between the lactyl group of muramic acid and the α-amino group of d-alanine, the first amino acid of the stem peptide (71, 72). Lytic transglycosylases catalyze the transfer of the glycosyl bond onto the hydroxyl group of the carbon 6 of the same muramic acid, thereby catalyzing an intramolecular glycosyl transferase reaction and forming 1,6-anhydromuramic acid (3, 19, 43). The functions of these non-PBPs in H. pylori have not been characterized yet. However, preliminary results from mutational analysis suggest that these gene products are essential for the growth and survival of H. pylori (49).


H. pylori colonizes a hostile gastric environment where few other organisms can survive. Its prolonged presence in this milieu is associated with gastric diseases such as gastritis, ulcers, and gastric cancer (5, 20, 5860). One of the basic features that may aid the bacterium to colonize this niche is a unique PG with a low level of cross-linking, short glycan chain length, and lack of trimers and tetramers. These features suggest that the murein of H. pylori is loosely constructed, and thus readily susceptible to alterations enabling the organism to change its morphology according to environmental needs and, in particular, to the process of colonizing new subjects.

Analysis of the H. pylori genome shows the presence of genes coding for all the enzymes of the biosynthetic pathway leading to the disaccharide pentapeptide, which is the basic building block of murein. Also identified are genes encoding the putative murein synthases PBP1, PBP2, and PBP3, which exhibit strong sequence similarities to penicillin-binding proteins encoding transglycosylase and transpeptidase domains from other bacteria, including E. coli, Haemophilus influenzae, and Bacillus subtilis, and which are involved in the construction of the murein sacculus, maintenance of cell shape, and cell division. There is some experimental evidence that indeed these are their functions in H. pylori.

Regarding murein hydrolases required for murein turnover and recycling, as well as for cell proliferation, the data obtained from genome analysis are much more fragmentary. Only a few ortholog genes to those present in other bacteria have been found, and their functions in H. pylori remain to be characterized.

Several other proteins able to bind labeled β-lactams have been detected in H. pylori, but nothing is known yet about their identity and further studies are required to understand the function of these putative PBPs.


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