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The penicillin-binding proteins of S. pneumoniae have been subjected to intense investigation with regards to their role in !-lactam resistance. Of the six PBPs from

S. pneumoniae, PBP1a, PBP2b and PBP2x are regarded as the major resistance determinants (as described in Section 1.10.3). In resistant clinical isolates (such as S. pneumoniae 5204), these enzymes are found to be genetically altered; certain mutations are integral to the resistance mechanism and confer a reduced affinity of the PBP for !-lactam antibiotics (Zapun et al., 2008).

3.1.1. S. pneumoniae D39 and 5204 strains

The work in this Chapter focuses on PBPs from two different strains of S. pneumoniae: D39 and 5204. S. pneumoniae D39 is a penicillin-susceptible strain and is the progenitor of S. pneumoniae R6 (Williams et al., 2007), which is a common standard used in comparative experiments of !-lactam sensitive and resistant strains (examples are included in Chesnel et al. (2005), Contreras-Martel et al. (2009) and Job et al. (2008)). There are no deviations at the nucleotide level of the pbp genes between the D39 and R6 strain as identified by sequence comparisons.

S. pneumoniae 5204 was first isolated from sputum in 1999 in Grenoble, France (Chesnel et al., 2003). It is highly resistant to !-lactam antibiotics: penicillin G MIC is 6.0 µg/ml; cefotaxime MIC is 12 µg/ml (Chesnel et al., 2005). Given these high MICs, the 5204 strain can be used as a credible reference for low affinity PBPs.

3.1.2. Generation of PBPs with a low affinity for !-lactam antibiotics

The S. pneumoniae pbps of penicillin-sensitive isolates are genetically uniform (Dowson, et al., 1989). However, the pbps from penicillin-resistant isolates exhibit diverse genetic alterations, which develop either through a series of step-wise spontaneous point mutations (Grebe and Hakenbeck, 1996) or by recombination

events between homologous pbp genes of S. pneumoniae and related species (Dowson et al., 1989; 1990). The pbp1a, pbp2b and pbp2x genes from penicillin- resistant pneumococci contain sequence blocks that are identical in the homologous genes of at least two streptococcal species (Dowson et al., 1989; Laible et al., 1991; Martin et al., 1992). Thus, these processes can generate highly mosaic pbp genes. For example, the sequence of pbp2b from a penicillin-resistant strain of S. pneumoniae (DN87/557) was found to contain blocks of nucleotides that diverged

from the S. pneumoniae R6 sequence by up to 21 % (Dowson et al., 1990). Genes

conferring low affinity for the antibiotic can be disseminated by horizontal gene transfer. Evidence for this process is provided by the >99.6 % nucleotide sequence identity of PBP2b between penicillin-resistant strains of S. pneumoniae and S. oralis, whereas sequences of PBP2b between penicillin-susceptible strains diverge by ~20 % (Coffey et al., 1993). The resulting amino acid substitutions have a higher occurrence in the transpeptidase domain (exemplified in Table 3.1), indicating that the antibiotic pressure promotes genetic rearrangement in the antimicrobial target region (Sanbongi et al., 2004). Only a restricted set of the substitutions are important in the !-lactam resistance mechanism (Chesnel et al., 2003).

Enzyme

Total number of amino acid substitutions

Number of amino acid substitutions specific to the

transpeptidase domain Reference

PBP1a 48 45 Job et al. (2008)

PBP2b 58 44 Contreras-Martel(2009), Pagliero et al. et al.

(2004)

PBP2x 80 41 Chesnel et al. (2003)

Table 3.1: Amino acid substitutions in the major resistance determinants of S. pneumoniae 5204.

3.1.3. Mutations of the S. pneumoniae resistance determinants implicated in !-lactam resistance

The following section discusses important mutations that are relevant in the !-lactam

resistance mechanism of S. pneumoniae PBP1a, PBP2b and PBP2x.

3.1.3.1. PBP2x

PBP2x is normally the first PBP to be modified under the selection pressure of !- lactam antibiotics (Grebe and Hakenbeck, 1996). The sequences of PBP2x from

clinical strains are normally categorised into three groups. The first includes sequences that are comparable to the !-lactam sensitive reference, S. pneumoniae

R6. The other two groups represent PBP2x sequences from !-lactam resistant stains and are classified according to the presence of either T338A or Q552E mutation (Chesnel et al., 2003). The different substitutions confer a reduced susceptibility of PBP2x for !-lactam antibiotics via two independent mechanisms, which are not necessarily mutually exclusive (Carapito et al., 2006). S. pneumoniae 5204 PBP2x is a member of the T338A family of resistant protein sequences (Carapito et al., 2006).

Out of the numerous amino acid substitutions in S. pneumoniae 5204 PBP2x, only

six have been shown to be essential for high levels of resistance (Carapito et al., 2006). The individual roles of the mutations are described in Table 3.2.

Amino acid

substitution Role in the !-lactam resistance mechanism

Reference

Q552E The substitution indroduces a negative charge at the

entrance to the active site, disfavouring the binding of the negatively charged !-lactam.

Mouz et al. (1999)

T338A Located in the first conserved motif, the substitution

reduces the acylation efficiency of the catalytic serine. It has been implemented in the loss of a water molecule, which results in the destabilisation of the active site.

Mouz et al. (1998)

M339F The substitution in conjunction with T338A, causes

the distortion of the active site, causing the reorientation of the active site serine hydroxyl group.

Chesnel et al. (2003)

M400T The substitution potentiates the effect of the M339F

mutation.

Carapito et al. (2006) I371T and

R384G

Co-operation between the two mutations is believed to increase the flexibility of the loop in which they are situated, remodelling the active site and

consequently weakening the non-covalent

interactions with the antibiotic and potentially affecting the reactivity of the catalytic serine.

Carapito et al. (2006)

N605T The substitution directly impacts the acylation

efficiency of the catalytic serine by destabilising a hydrogen bond network. The loss of a hydrogen bond could participate in the disruption of a loop that borders the active site.

Carapito et al. (2006)

Table 3.2: The roles of the essential amino acid substitutions in S. pneumoniae 5204 PBP2x implemented in high levels of !-lactam resistance. Although it is not found in the sequence of S. pneumoniae 5204 PBP2x, the role of the Q552E substitution is described as it is an important mutation in other resistant sequences of PBP2x.

Carapito et al. (2006) found that reversion of the six amino acid substitutions in PBP2x (strain 5204) back to the original amino acids of the !-lactam sensitive sequence caused the acylation efficiency for the !-lactam to increase by 1000-fold. However, if the converse mutations were made to !-lactam sensitive sequence of PBP2x, the acylation efficiency decreases by only 100-fold, an order of magnitude lower. This discrepancy is believed to result from the cooperativity of additional uncharacterised mutations that collectively impact the level of resistance.

3.1.3.2. PBP2b

PBP2b has not been subjected to such a vigorous investigation unlike PBP2x. This is presumably due to the difficulties in obtaining X-ray crystal structural information of truncated forms of PBP2b (Zapun et al., 2008). The T446A mutation is prominent in all identified !-lactam resistant sequences and lies downstream of the SSN conserved motif. It is believed to directly affect the affinity of antibiotics, although its effect is propagated by other uncharacterised substitutions (Pagliero et al., 2004). Applying this mutation to the S. pneumoniae R6 sequence decreases the affinity of PBP2b for penicillin G by 60 %. Other substitutions including E476G and T489S/A are frequently found in the PBP2b sequences of highly resistant strains, although their roles have not been thoroughly investigated (Pagliero et al., 2004).

3.1.3.3. PBP1a

PBP1a is clinically important due to its essential requirement for the development of high levels of !-lactam resistance. A common substitution found in sequences of resistant strains is T371A, which lies adjacent to the catalytic serine (Smith and Klugman, 1998). This mutation results in the loss of a hydrogen bond, which normally forms between T371 and W368, and causes the catalytic serine to change its orientation (Job et al., 2008). Similar blocks of mutations have been identified in the PBP1a sequences from !-lactam resistant clinical isolates; in the majority of drug resistant strains, NTGY replaces the TSQF sequence at positions 574-577 (Job et al., 2008; Smith and Klugman, 1998). This sequence is situated in a loop that borders the active site. The substituted residues are proposed to increase the local

hydrophilicity, which can interfere with the antibiotic recognition. Substituting NTGY into the PBP1a sequence from the R6 strain causes the acylation efficiency for penicillin G to decrease 49-fold (Job et al., 2008).

3.1.4. PBPs to be investigated in this study

The major resistance determinants of S. pneumoniae strains D39 and 5204 are the focal point of this thesis. The key objective is to obtain a greater understanding of these PBPs in terms of their natural enzymology and the complex !-lactam resistance mechanisms they impart. This chapter discusses the cloning, expression and purification of PBP1a, PBP2b and PBP2x, which is a prerequisite for future biochemical and structural characterisations.