Chapter 18

Mechanisms of Penicillin Resistance in Streptococcus pneumoniae: Targets, Gene Transfer and Mutations
Regine Hakenbeck, Dalia Denapaite, and Patrick Maurer

18.1

Evolution of Penicillin Resistance in S. pneumoniae Epidemiological Aspects

S. pneumoniae belongs to the most penicillin-sensitive species. Unlike many other species, b-lactamase producing S. pneumoniae have not been discovered so far. Early attempts to isolate penicillin resistant mutants in the laboratory were extremely cumbersome. Nevertheless, after over 300 passages and during 24 months increasing selection pressure, variants were obtained with an over 1,000-fold increase in resistance to benzylpenicillin [46]. It therefore came as a surprise when the rst reports of b-lactam resistant clinical isolates appeared. Initially, the increase in MIC values was not dramatic and the number of isolates was low (e.g., only 2 out of 200 strains had elevated MIC values in 1965 as reported by Kislak et al. (see Table 18.1)). However, the numbers increased to 12% in Papua, New Guinea in 1971, and it took only a few more years to document high level penicillin and multiple antibiotic resistant isolates in South Africa [64]. Meanwhile, penicillin-resistant S. pneumoniae (PRSP) are reported with increasing frequency worldwide ([32, 60, 96], and references within). The factor mainly responsible for this development is the use of antibiotics [42]. There are some features associated with this epidemiological scenario that are noteworthy (Table 18.1). The MIC ranges over a wide spectrum of antibiotic concentrations, indicating that the mode of resistance development is variable and/or complex. Second, in areas where the use of new generations cephalosporins was encouraged, high-level cephalosporin resistant strains appeared [95]. Last but not least in countries with a high number of resistant isolates, the frequency of resistant

R. Hakenbeck (*) D. Denapaite P. Maurer Department of Microbiology, University of Kaiserslautern, Paul Ehrlich Strasse 23, Kaiserslautern, D-67663, Germany e-mail: hakenb@rhrk.uni-kl.de T.J. Dougherty and M.J. Pucci (eds.), Antibiotic Discovery and Development, DOI 10.1007/978-1-4614-1400-1_18, Springer Science+Business Media, LLC 2012 593

594 Table 18.1 Development of b-lactam resistant Streptococcus spp MIC PenG Streptococcus pneumoniae 1940 Worldwide 0.01 1965 Boston 0.10.2 1967 Papua 0.6 1977 South Africa 48 1990s Hungary >10 1990s USA 0.01 Commensal Streptococcus spp 1990s 1994 CTX cefotaxime Hungary/Spain Germany 20 >50

R. Hakenbeck et al.

MIC CTX 0.02

Reference [69] [73] [58, 59] [64] [90] [95] [112] [74]

2 32 20 >60

strains of closely related commensal oral streptococci was also high, including those with MIC levels far above those reported for S. pneumoniae [24, 74]. In Spain, high level resistant S. pneumoniae in Europe were reported in the early 80s, with clones of the capsular type 9V, 6B, 14, 19F, 23F representing the majority of PRSP (penicillin-resistant Streptococcus pneumoniae), serotypes that are also common among healthy carriers. Clones have been named by an international network The Pneumococcal Molecular Epidemiology Network (PMEN) [97]. These prevalent clones have spread to varying degrees to other countries. The Spain23F-1 clone represents the most widespread one, members of which having since been isolated in South Africa, the US, Europe, and Asia [96, 101]. Macrolide resistant variants of Spain23F-1 contribute to the dissemination of this clone in Europe [114]. In contrast, clone Spain14-5 apparently has spread less to other countries [87]. Type 19A clones with unusually high MIC values were described rst in Hungary in the 1980s [90] and in the Czech Republic and Slovakia [34]. Other resistant 19A clones are increasing in many countries, probably since this serotype is not included in the 7-valent vaccine [18]. A remarkable epidemiological scenario in Iceland documents the increase of penicillin non-susceptible S. pneumoniae (PNSP) mainly due to the spread of a particular type 6B clone Spain6B-2 in the late 1980s/early 1990s which carried resistance determinants against another ve antibiotics [118]. High numbers of PSNP are now reported in Spain (3050%) [108], Asian countries (up to over 90%) [81], and Italy with 69% [86]. No difference in disease potential was found between resistant and sensitive clones [128]. Although PNSP clones of a rare serotype 35B have only been found among patients with invasive disease in the US and only among carriers in Sweden, one cannot deduce a specic disease pattern since it is not known how common this clone is among carriers in the US [4, 61]. Serotype switching within clones has been noted, probably due to immunological pressure in the human population. Examples are 19F variants of the Spain23F-1 clone, and type 14 variants of the Spain9V-3 clone (for review, see [60, 96]). There might be an impact on the clonal structure of resistant S. pneumoniae, due to vaccination

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against the prevalent serotypes. In fact, an increase in the relative proportion of resistant S. pneumoniae causing invasive disease after the introduction of a 7-valent anti-pneumococcal vaccine has been reported [70].

18.2

Penicillin-Binding Proteins Interaction with b-lactams

There are three main players involved in the evolution of resistance in S. pneumoniae: the penicillin target enzymes PBP2x, PBP2b, and PBP1a. Whereas PBPs of sensitive bacteria are inhibited at low concentrations by b-lactams, the altered PBPs of resistant isolates interact with and thus are functionally inhibited by the antibiotic at much higher concentrations. In clinical isolates these PBP genes have a mosaic structure (i.e., regions highly divergent from those of sensitive strains). The mosaicism of the PBP genes is the result of interspecies gene transfer fed by the common gene pool available to commensal and pathogenic streptococci, combined with the selection of point mutations. Apparently, mutations affecting the interaction with the antibiotics also have some impact on enzymatic activity, and thus compensatory mutations may occur. Mutations in non-PBP genes are also involved in the resistance process in clinical isolates and laboratory mutants as well. PBPs are multidomain proteins, which are grouped into three main classes: the high molecular weight (hmw) PBPs of class A and B, and the low molecular weight (lmw) PBPs (for review see [40, 145]). Each species contains a set of PBPs, which are numbered in descending order according to their apparent molecular weight as revealed on SDS-polyacrylamide gels. The hmw PBPs are anchored into the membrane via a short N-terminal hydrophobic region, whereas lmw PBPs contain an amphiphilic helix at the C-terminus that functions as membrane attachment. In all cases, the functional domains are located in the periplasm outside the cytoplasmic membrane. All PBPs and the related b-lactamases contain a penicillin-binding domain which functions during late steps of murein (peptidoglycan) biosynthesis as transpeptidase/carboxypeptidase, representing the penicillin-sensitive steps. It contains three conserved motifs: SXXK with the active site serine which is directly involved in the transpeptidation reaction, and becomes acylated upon binding to b-lactam antibiotics, an (S/Y)XN and a (K/H)(S/T)G box. These sites are located in close proximity and represent crucial parts of the active site cavity in the three dimensional arrangement of the PBP. Mutations relevant for the resistance development resulting in a decreased afnity for b-lactams are located in this domain. The class A hmw PBPs contain an N-terminal transglycosylase domain, the target of the antibiotic moenomycin [93, 138, 141]; the function of the N-terminal domain of class B hmw PBPs is not known. PBPs interact with b-lactams according to the following scheme [37]: K E+I K2 EI K3 EI*

E+P

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where E = active enzyme, I = b-lactam compound, EI = non-covalent complex, EI* = covalent acyl-enzyme complex, and P = biologically inactive product. K is the dissociation constant, k2 and k3 are rst-order rate constants for the acylation respectively the deacylation step, and the second order rate constant k2/K represents the acylation efciency. Due to their ability to covalently bind b-lactams, PBP can easily be visualized after incubation with radioactive or uorescent antibiotic and separation on SDS polyacrylamide gels. Since the hmw PBPs of S. pneumoniae are notoriously difcult to resolve on SDS gels, and PBP variants especially of clinical isolates may have a different electrophoretic mobility, specic antisera and monoclonal antibodies have been produced to label individual PBPs. The different ways to visualize PBPs have been reviewed recently [117]. S. pneumoniae contains six PBPs mysteriously named PBP1a, 1b, 2x, 2a, 2b, and 3. The nomenclature has evolved, due to improved resolution of rst generation SDS gels which resolved PBP1, 2, and 3 to PBP1a + 1b, and 2a + 2b in the late 1970s; PBP2x was discovered later during biochemical characterization of pneumococcal PBPs. PBP2x and PBP2b are class B hmw PBPs, and class A hmw PBPs are represented by PBP1a, 1b, and 2a.

18.3

PBP Function

Due to the lack of true substrates, thiolester compounds have been used to analyze the transpeptidation reaction of PBP2b and PBP2x, and the depsipeptide S2d has been used in many studies [1, 65, 94]. The thiolesters give rise to linear acylenzymes, which are easily hydrolysed thereby mimicking the transpeptidation reaction carried out by PBPs. Although PBPs may react differently with this compound compared to the natural muropeptide substrates, the kinetic parameters help to dene the effect of mutations on peptide hydrolysis of PBP variants. Isolated PBP2a derivatives contain transglycosylase activity in vitro [22]. The glycosyltransferase (GT) domain of class A PBP1b showed moenomycin sensitive binding to lipid II, an indirect evidence that it functions as transglycosylase as well [21]. The lmw PBP3 functions as a D,D-carboxypeptidase in vitro [52]. PBP2x and PBP2b are believed to be essential, since it is not possible to obtain deletion mutants in these two genes [72]. It is curious that in closely related bacteria, S. thermophilus, the PBP2b homologue could be deleted, resulting in altered morphology and defects in exopolysaccharide synthesis [137] and in S. gordonii as well, leading to aberrant septation and early lysis [47], indicating that special functions are associated with PBP2b of S. pneumoniae which are absent in the other species. The class A hmw PBP1a, 1b and 2a are individually dispensable, suggesting that the putative transglycosylase and transpeptidase activities of these PBPs can complement each other. The pbp2a mutant showed a higher susceptibility to moenomycin, and PBP2a was therefore suggested to be the main transglycosylase in

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S. pneumoniae [62, 107]. Double mutants have also been obtained except for the pair pbp1a/pbp2a [62, 107]. Nonetheless, the class A PBP double mutants were severely affected, being unable to synthesize regular division septa, and lysed earlier after reaching the stationary phase [107]. This indicates that PBP1b alone cannot complement for the activities of the other two hmw PBPs. PBP3 deletion mutants can readily be obtained, but they grow poorly and have aberrant shapes: often multiple division septa are found irregularly distributed in the cells, they contain a thickened cell wall and shed wall material into the growth medium [120]. Biochemical alterations of the murein conrmed its D,Dcarboxypeptidase activity [124]. Immunogold-labeling using anti-PBP3 antibodies revealed that PBP3 is evenly distributed over the entire surface [50]. It appears to be absent at the division septum in wild type cells, and the rings formed by hmw PBPs and that of FtsZ are no longer colocalized in PBP3 mutants [98].

18.4

Gene Transfer and the Evolution of Mosaic Genes in Clinical Isolates

The PBP genes in resistant isolates encoding low afnity PBPs are highly variable due to the presence of sequence blocks that differ approximately 20% on the DNA level resulting in up to 10% amino acid changes compared to corresponding sequences in sensitive strains. Mosaic genes have been described in all three key players of the resistance process: pbp2b [26], pbp2x [80], and pbp1a [89]. Despite extensive sequence variations, the number of amino acids is constant with a few exceptions in PBP2b and PBP1a as outlined below. The mosaic structure of PBPs might result in electrophoretic mobility shifts as has been detected already in the rst reports describing PBP pattern in penicillin resistant clinical isolates [55, 109, 148], most prominent detectable in PBP1a variants [48, 80], although their calculated molecular weight is almost identical. Even among sensitive strains different PBP patterns can be distinguished [49]. Since these changes are generally clone specic, the PBP proles on SDS PAGE in combination with antibody reactivity pattern have been used as clonal markers [48]. Restriction fragment length polymorphism (RFLP) of PBP genes has also been used as a DNA based screen to establish clonal relatedness [14, 101]. Although these methods are useful for screening a large number of isolates, small changes in the size of the mosaic blocks and point mutations in pbp genes that are important for the deduction of the evolutionary history might not affect the restriction sites or the epitopes and are thus missed in such analyses. In general, the S. pneumoniae clones as identied by MLST analysis (multilocus sequence typing [85]) are either resistant or sensitive. However, there are a few cases where sensitive isolates were detected that belong to the same clone in agreement with the introduction of resistant genes into a sensitive population. Variation of PBPs has also been noted within resistant clones from Hungary and Poland,

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a
Spain6B-2 (670)

M3 (SA)

S. mitis sensitive

S. pneumoniae resistant

Spain23F-1 (2349) USA23F-4 (CS111) CZ14-10 (29044) 197 (Sp) S. mitis Uo5 (Hu) S. oralis B6 (G)

resistant

S. mitis

S. pneumoniae sensitive

R6

S337TMK

S395SN

K547TG

M3 670 2349 29044 197 Uo5 111 B6 R6

22222223333333333333333444444444455555555555555555555555556666 66788891334445566677888001466889900001111233334566777899990001 58012431893675846918249017425680115670346316786057246657890456 LPQLEVLNTMTSSSYLIAIDTRSMSNNLFTNSVKEDALTNILYIITVTSNVTNYYAAQLSNE IT......A......F..T..GL..KS......NKE.TNH...............GI..ATD ........A......F..T..GL..KS......NKE.TNH...............GI..AT. I...Q...AL.....F.VT..GL.....L............I.............GI..AT. .....L..G......FM.TE.SL..KS......NKE.TNH...............GI..AT. .....L..G......FM.TE.SL..KS......NKE.TNH...............GI..AT. ......S.AFM....F..TE.G.T.KS......NKE.TNH.......A.......GI..AT. M.H.....A......FMVTG.GL..KS.........T.....Q..I...D...SLTPWFA.D I..Q...D..MAAGV....EG...T..I.PDTANK.....VVSTV.L.LDASS..GI..A.D

MIC CTX 0.02 0.5 1 4 6 12 12 60 0.02

Fig. 18.1 Distribution of a family of mosaic PBP2x genes. (a) Mosaic structure. One group of mosaic PBP2x genes contain sequences highly related to the penicillin susceptible S. mitis M3 (red). Related mosaic pbp2x have been identied in S. pneumoniae and oral streptococci isolated in different geographic areas as indicated. White: homology to penicillin susceptible S. pneumoniae; red: homology to PBP2x of S. mitis M3. Grey shading indicates the transpeptidase domain. Arrows point to the active site motifs. (b) Amino acid variation of PBP2x of the strains shown in 1A with different MIC values for cefotaxime. Only changes within the transpeptidase domain are shown that are distinct from PBP2x of the sensitive S. mitis M3. It should be noted that the MIC values reect alterations in other PBPs as well

indicating that PBP genes have been introduced into a clone on several occasions from different sources [63, 113]. Sequences highly related to mosaic regions of mosaic pbp2x [126] and pbp2b [25] of S. pneumoniae were detected in susceptible S. mitis strains, in agreement with the assumption that low afnity PBPs have evolved in commensal streptococci prior to interspecies gene transfer into the pneumococcal population. One major class of mosaic pbp2x can be recognized in different S. pneumoniae clones and in resistant S. mitis and S. oralis strains as well [11, 112] (Fig. 18.1). In addition, a surprisingly large number of distinct pbp2x variants exists with unique mosaic patterns all being approximately 20% divergent from each other [51, 99], indicating multiple intra- and interspecies gene transfer events. The high diversity of the

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mosaic genes is surprising due to the fact that pbp2x is a highly variable gene in sensitive S. mitis and S. oralis ([126]; and own unpublished results). Recombination events resulting in altered pbp genes can occur within the gene or in anking regions. It has been observed in PBP2x from resistant isolates that the border of the mosaic blocks on the DNA level reects the domain structure of the protein in many cases [89, 126]. This suggests a selective pressure on the function of the protein. The mosaic structure might extend into adjacent genes such as ddl upstream of pbp2b [31] and ftsL upstream of pbp2x (own unpublished results). Since the capsular gene locus is anked by the pbp1a and pbp2x genes, intraspecies transformation of resistance can result in capsular switching as well [140] as has been shown to occur in natural populations [12, 15]. PBP genes that are located at a great distance on the chromosome such as pbp2b and pbp2x, or pbp2a and pbp2x, can be introduced in a single transformation step as has been shown with chromosomal DNA from resistant S. mitis, indicating that genes located elsewhere on the chromosome can be altered easily during interspecies transformation events [53, 112].

18.5

PBPs and b-Lactam Resistance: Physiology of Resistant Isolates

Only PBP2x and PBP2b are primary targets for b-lactams (i.e., alterations in PBP2x or PBP2b alone confer a resistance phenotype albeit to only low levels). PBP2x mutants can easily be selected with cefotaxime resulting in MIC values for cefotaxime between 0.030.3 mg/ml depending on the particular mutation (i.e. confer a 1.530-fold increase in resistance compared to sensitive strains (0.02 mg/ml)) [43, 77, 78, 127]. Single mutations in PBP2b result only in a 1.52-fold increase in piperacillin MICs [43, 54]. Since PBP2b does not interact with cefotaxime over a wide concentration range (or other third generation cephalosporins and aztreonam which has a similar side chain as well), it is not a target for this class of compounds [56]. Therefore, pbp2x together with pbp1a of resistant clinical isolates are sufcient for cefotaxime resistance [3, 27, 53, 102]. Indeed, high level cefotaxime resistant clones have been described in the USA and South Africa with altered PBP2x and 1a, but which did not contain alterations in PBP2b [13, 95, 129]. Whereas penicillin are highly lytic antibiotics for S. pneumoniae, cefotaxime leads to much slower lysis and cells are also killed at a much lower rate [56]. This suggests that inhibition of PBP2b is somehow coupled with cell lysis. In agreement with this notion, is the nding that high-level penicillin-resistant strains which usually contain a low afnity PBP2b appear to be tolerant [82]. S. Pneumoniae strains containing a low afnity PBP2b as the only altered PBP have also been shown to display a tolerant response for penicillin antibiotics [43, 112]. The fact that PBP2b mutants are less prone to lysis suggests that cells with a low afnity PBP2b are

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better survivors; thus they might have an advantage over wild type cells even in the absence of antibiotic selection. In this context, it is a curious observation that strains harboring either an altered PBP2b or PBP2x were signicantly less virulent in a murine peritonitis model [115]. The PBP2x mutants remained stable in both resistance phenotype and virulence, and thus the authors suggested that PBP2x plays an essential role during growth, whereas virulent revertants of PBP2b mutants were obtained. The location of the compensatory mutations remains to be claried.

18.6 18.6.1

Mutations in PBPs PBP2x Laboratory Mutants versus Clinical Isolates

Mutations in PBP2x have been extensively studied, due to the fact that they can be selected easily in the laboratory, and their effect can be tested directly via transformation of sensitive S. pneumoniae strains using cefotaxime for selection (Fig. 18.2a). Moreover, it was the rst and for a considerable time the only PBP where soluble, active derivatives were available enabling biochemical studies in vitro. The diversity of mutations observed and physiological characterization of PBP2x mutants suggests a complex evolution of resistance by introduction of mutations that might affect its enzymatic function as well, coupled with complementing mutations in the protein or in other genes as well as outlined below. After all, PBP2x is an essential enzyme, and mutations that affect the interaction of the b-lactam (i.e., affect the overall active site topography) should not severely affect the interaction of the in vivo substrate. Already after one selection step, different mutations in PBP2x occur [104, 127], and six independent laboratory mutants obtained after a multistep selection procedure resulted in six distinct PBP2x mutant proteins with up to four point mutations in the transpeptidase domain [77, 78]. It is remarkable that most of the mutations did not map close to the active site except for the two mutations: T550A and Q552E adjacent to the K547SG box, and H394Y next to the S395SN motif. The mutation T550A confers high level cefotaxime resistance and simultaneously hypersensitivity to penicillins in laboratory mutants [43, 78, 79]; occasionally, it occurs in mosaic PBP2x of high level cephalosporin resistant and penicillin sensitive clinical isolate [13, 119] and as a single PBP2x mutation in low level resistant strains as well [2]. A second substitution in the same codon 550 results in a T550G mutation which increases the cefotaxime resistance even further [43]. Curiously, the reverse substitution, A235T, at the homologous site of TEM b-lactamase resulted in an enzyme with an extended substrate prole that could hydrolyze cefotaxime, an antibiotic which is not a substrate for the original protein [16]. It has therefore been speculated that the T550A substitution is directly related to cefotaxime selection [78]. The T550A mutation results in a 20-fold decrease acylation efciency for cefotaxime [99],

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a
266

S337TMK S395SN

K547SG

616
(750 aa)

PBP2x N-terminal transpeptidase C-terminal

H394Y F388Y

R512W Q552E

laboratory mutants
M289T L403F Q458K T526S S596L L600W

G422D R426C L364F M400T T337A/G/P/S

T550A/G G597D G601E/V

Q552E N605T

clinical isolates
H394Y M339F I371T R384G T550A Y595F

b
L403 T550 S526 G601 G597 G422 Q458 R426 M289 L600

Fig. 18.2 Mutations in PBP2x. (a) Only mutations within the transpeptidase domain of PBP2x (black) have been described. The central black area indicates the transpeptidase domain, and the hatched area at the N-terminus indicates the hydrophobic membrane domain. The three active site boxes are indicated on top. Positions implicated in resistance identied in laboratory mutants and clinical isolates are indicated. (b) Three dimensional arrangement of mutations in two groups of laboratory mutants. Left: mutations occurring in group I; right: mutations in C505 (group II) which result in complete abolishment of b-lactam binding

probably due to the abolition of the hydrogen bond between T550 and the carboxylate moiety which is attached to the six-member ring of second and third-generation cephalosporins [41]. The H394Y change has also been identied in PBP2x of clinical

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isolates [103]; the effect of H394L that occurs occasionally in clinical isolates has not been experimentally investigated. At least two mutational patterns in PBP2x have been observed (Fig. 18.2b) [94]. In all PBP2x of group i: (1) mutations occur at the end of the transpeptidase domain (positions 596601), (2) and mutations in other regions also are similar in some of the mutants. Noteworthy is the group ii PBP2x of one laboratory mutant C505 where no binding to any b-lactam could be detected, even if concentrations up to 10 mg/ml were applied and PBP visualized with anti-benzylpenicilloyl antibodies [79]. The combination of only three mutations in PBP2x-C505T526S-L403F-Q458K abolishes the interaction with cefotaxime as well as penicillin almost completely as measured in a puried soluble protein derivative, and L403F is crucial for this effect [94]. A possible impact of this mutational arrangement on the topology of the active site can be deduced from the three dimensional structure [94]. Mutations located at the end of the penicillin-binding domain in C206G601V-G597D only affect the acylation efciency towards cefotaxime (k2/K = 8,600), possibly affecting indirectly the topology of the active site. Other combinations have an impact on both cefotaxime and penicillin binding [94]. The depsipeptide S2d has been used to determine the rate of hydrolysis for estimating the activity of PBP2x. In all cases studied so far, PBP2x mutants which showed a reduced acylation efciency also reacted considerably poorer with the depsipeptide S2d [65, 94]. Curiously, the amount of PBP2x was also reduced in some mutants, but the molecular basis for this phenomenon is not clear [94]. Mutations in PBPs of resistant clinical isolates cannot easily be deduced from sequence analysis due to the multitude of amino acid alterations and the variability of the mosaic blocks, which is apparent even when comparing related mosaic PBPs (see Fig. 18.1b for examples). Comparison of a large number of diverse mosaic PBP2x revealed only two sites common to almost all highly divergent mosaic designs: T338 adjacent to the active site S337 is altered in one group of mosaic PBP2x (T338(A/G/P/S)), whereas another group contains the mutation Q552E in most cases without the T338 mutation [51, 100]. All other mutations revealed so far occur only in subgroups of mosaic PBP2x or in single rare variants. Kinetic parameters of isolated soluble PBP2x derivatives conrmed that the overall binding efciency of a resistant PBP2x is much slower than that for sensitive PBP2x (k2/ Kd values of 100,000200,000 M1 s1 for sensitive PBP2x compared to 11,00085 M1 s1 and lower for resistant PBP2x containing multiple alterations) [6, 66, 84, 94]. The impact on resistance and b-lactam afnity has been demonstrated by a combination of mutagenesis and biochemical characterization of the protein for T338(A/G/P), M339F, and Q552E [9, 100, 110, 134, 146], which are close to active site residues. The T338(A/G/P) mutations are special since they can be selected primarily with oxacillin [146], probably explaining why they have not been found in the cefotaxime-selected laboratory mutants. The side chain of T338 has been implicated in hydrogen bonding to a buried water molecule [100] and indeed this molecule is absent in a resistant PBP2x containing the T338A substitution [20]. The combination of T338A/M339F reduced the acylation efciency by penicillin over

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1,000-fold, a result of slower acylation (300-fold lower k2) and weaker pre-acylation binding (4-fold higher Kd) [9, 84]. The M339F substitution also contributes to 4070fold faster deacylation kinetic [9, 23]. The structure of PBP2xT338A/M339F has been solved, revealing a distortion of the active site and a reorientation of the hydroxyl group of the catalytic Ser337 [9]. The structure of a mosaic resistant PBP2x carrying the Q552E substitution reveals a distinct mechanism involved in resistance. The b3 strand with the K597TG motif is displaced [110], resulting in narrowing of the active site. The negative charge of the glutamate residue also hinders binding of negatively charged b-lactams [110]. Whereas introduction of the single Q552E mutation into the sensitive R6 PBP2x results in 34-fold reduction of the acylation efciency, mosaic PBP2x with the Q552E substitution have a 7-fold reduction [94] and 15-fold reduction was observed in a mosaic PBP2x which also contained the T338A substitution [110]. Moreover, L364F, I371T, R384G, M400T, Y595F, and N605T [2, 6, 100, 134] are supposedly involved in resistance. The structure of a mosaic resistant PBP2x revealed that the substitutions I371T and R384G result in a slight displacement of the SXN motif, leading to a more accessible open active site, and it has been suggested that thereby branched muropeptide substrates can be better accommodated [20]. Many alterations that have been suggested to contribute to resistance curiously occur also in PBPs of sensitive streptococci ([25, 126]; and own unpublished results). In PBP2x, they include Q447M, S449A and N514H, which have been proposed to contribute to structural alterations of the active site in resistant strains [20, 110]. Also, the R384G change, which has an impact on the susceptibility [6, 134] and altered the acylation efciency of the protein [6], occurs in a sensitive S. mitis (own unpublished results). It is possible that different muropeptide substrates are used in sensitive S. mitis. Thus, the evolution of resistant PBPs in S. pneumoniae includes not only the reduction of b-lactam binding, but also the functional alterations in resistant PBPs as well. Some (but not all) resistant S. pneumoniae indeed contain a different cell wall with altered interpeptide bridges compared to sensitive isolates [38]. The acquisition of altered genes involved in the biosynthesis of such branched muropeptides might be a consequence of an altered PBP function affecting their substrate specicity (see below).

18.7

PBP2b

In PBP2b, G660D at the C-terminal end of the protein, G617A within the K615TG motif [54], and T446A close to the S443SN box [43] have been selected with piperacillin in the laboratory (see Fig. 18.3). A change within the KTG motif has also been observed in clinical isolates (T616S; [136]). The T446(A/S) change occurs in many resistant clinical isolates and E476G as well [25, 33, 119, 133]; alterations at the C-terminal end of PBP2b have been implicated in the resistance process also of clinical isolates [10, 26]. The T446A substitution displays a 60% reduction in penicillin afnity in vitro, and in a PBP2b containing this, up to another 43 amino acids

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PBP2b

(680 aa)

T446A* V338A
S370TMK

G617A* G660D* T616S

K557TG

E476G
S466SN

PBP1a

(719 aa)

T371(A/S)

L539W TSQF574-577NTGY

Fig. 18.3 Mutations in PBP2b and 1a. The central black area indicates the transpeptidase domain, and the hatched area at the N-terminus indicates the hydrophobic membrane domain. The three active site boxes are indicated on top. *: mutations in PBP2b whose impact on resistance has been demonstrated

change the afnity and is reduced up to 99% [106]. Only one case has been reported bearing a change within the SVVK motif (V388A) [71]. Multiple changes in PBP2b between residues 590641 have been observed in high amoxicillin resistant isolates and might contribute to this phenotype [28, 75]. Among resistant PBP2b are rare examples of the presence of additional amino acids in the protein: clinical isolates from Japan were found to contain a duplication of a region encoding three amino acid residues S423WY [143]. Recently, the structure of PBP2b from a wild-type and a high-level penicillin resistant strains has been resolved [17]. Similar to PBP2x and 1a variants, the main structural consequence of alterations concerned the active site, and it has been suggested that active site breathing could be a common mechanism employed by S. pneumoniae PBPs to interfere with b-lactam binding.

18.8

PBP1a

Resistance mediated by PBP1a can only be measured in combination with a low afnity PBP2x and/or PBP2b. In resistant PBP1a of clinical isolates, T371A or T371S close to the active site S370 occur frequently and contribute to resistance [2, 33, 89, 103, 105, 135]. L539W present in PBP1a present in a high level resistant Hungarian isolate [132], and the alteration of four consecutive residues T574SQF to NTGY have

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been associated with resistance [68, 132, 135] as summarized in Fig. 18.3. The crystal structure of a resistant PBP1a derivative shows that the T371A mutation results in loss of a hydrogen bond, causing a shift of the active site S370 [67]. These changes in combination with the other alterations present the mosaic variant results in a narrower, discontinuous active site cavity. PBP1a mutants containing the N574TGY substitutions have a lower acylation efciency in vitro [67]. Again, these positions are also altered in PBP1a genes of sensitive S. mitis (own unpublished results), and thus might also be related to functional properties in respect to the in vivo substrates. Generally, mosaic PBP1a derivatives have a greater effect on the interaction with penicillin compared to a cephalosporin with 8164-fold decreased acylation rates towards penicillin G versus cefotaxime (225-fold) [67]. It has generally been accepted that the introduction of a low afnity PBP1a in a strain carrying a low afnity PBP2x results in elevated resistance. That the situation is much more complex has been shown recently, using low afnity PBP2x variants from laboratory mutants in comparison with mosaic PBP2x from resistant clinical isolates. Introduction of a mosaic pbp1a into the PBP2xT338G mutant, or into a PBP2x carrying three mutations of a laboratory mutant, did not lead to resistance increase [146]. It has been hypothesized that PBP2x and PBP1a interact with each other on some level and that alterations of both PBPs in resistant clinical isolates have evolved to ensure cooperation between both proteins. The data are in agreement with the observation that PBP1a variants can confer different levels of resistance although acylation efciencies were very similar, and it has been postulated that dependent on the mosaic variant the physiological function of PBP1a varies [145].

18.9

PBP2a, 1b, and 3

Alterations in the other three PBPs associated with resistance have been described in rare cases. An altered PBP2a has rst been observed in laboratory mutants, which contain a low afnity PBP2x [79]. Curiously, PBP2a in three such mutants could not be visualized using common labeling procedures, or even when high concentrations of penicillin and anti-penicilloyl antibodies were used for the detection of PBP- b-lactam complexes. In fact, PBP2a is absent in the mutants due to mutations in the genes that lead to premature termination of the transcript (M. van der Linden, J. Rutschmann, and R. Hakenbeck, unpublished results), but such mutations have not been found in clinical isolates. Further evidence that PBP2a is involved in resistance development also of clinical isolates came from experiments where DNA from b-lactam resistant S. mitis or S. oralis was used to transfer the resistance into S. pneumoniae, resulting in transformants which contained a low afnity PBP2a [53, 112]. Some especially high level resistant clinical isolates of S. pneumoniae indeed contained a low afnity PBP2a, diverging from sensitive PBP2a only in up to 3% as changes ([7, 29, 53, 119, 130], and own unpublished results). Whereas in early studies an altered pbp2a of resistant S. pneumoniae could not be transformed using b-lactam selection [29];

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this was possible with pbp2a of another isolate, conrming the potential role as resistance determinant for PBP2a [130]. It is remarkable that these resistant PBP2a mutations anking the active site Ser410 (T411A) occur frequently. PBP2a has a relatively low afnity especially to penicillins, and it has been suggested that therefore PBP1a mutations are selected before PBP2a becomes a player in the resistance development [147]; however, in transformation experiments using DNA of resistant commensal Streptococcus spp., a low afnity PBP2a is transferred into the recipient S. pneumoniae before a low afnity PBP1a is selected [53, 112]. Nevertheless, it is almost impossible to deduce the evolutionary history of high level resistant clinical isolates, and it is quite possible that different routes of gene acquisition occur in the natural environment. In high level resistant S. pneumoniae strains, no changes in PBP1b could be detected [29, 53, 119]; however, PBP1b could not be labeled in particular resistant S. pneumoniae transformants obtained with DNA from a high level resistant S. mitis [53]. The PBP1b gene in the S. mitis strain contains a point mutation resulting in premature stop within the transpeptidase domain, probably resulting in absence of the entire protein (own unpublished results). This is the rst case where a deletion mutation of a PBP has been identied in a resistant isolate. The fact that no growth defects are apparent in the S. mitis strain agrees with the assumption that the putative transglycosylation activities of the three class A hmw PBPs can complement each other. Whether the PBP1b mutation plays a role in resistance, whether it is associated with alterations in all other four hmw PBPs, or a rare coincidence unrelated to resistance remains to be claried. A PBP3 mutation T242I associated with resistance has only been described in one particular laboratory mutant C604, again in the immediate vicinity of the K239TG the mutation [76], and a reduced amount of PBP3, which occurs in some laboratory strains, due to mutations in the promoter region also seems to be related to cefotaxime resistance [122]. Particular clones of clinical isolates contain a PBP3 with altered electrophoretic mobility [76], but these variants do not affect the afnity towards b-lactams and are thus most likely unrelated to resistance.

18.10

Murein Chemistry and Penicillin Resistance

The peptidoglycan of Gram-positive bacteria contains interpeptide bridges which are L-Ser-L-Ala and L-Ala-L-Ala in S. pneumoniae [39]. These amino acids are added to the lipid II substrate by MurM and MurN, also named FibA and FibB [83, 111], encoded by the murMN (bAB) operon. In the cell wall of some high level resistant clinical isolates, such branched muropeptides are present in higher quantity compared to sensitive strains [38]. A mosaic structure of murM is associated with resistance increase in some clones [38, 131], but is not always involved in high level resistance [5, 125]. In vitro studies using lipid II substrates and recombinant MurM and MurN enzymes revealed that a much greater catalytic efciency of MurM from resistant strains compared to the sensitive MurM is mainly responsible

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for the different murein structure [83] whereas MurN from both, resistant and sensitive strains, showed similar enzymatic function [19]. Curiously, disruption of murM/bA results in an almost complete collapse of resistance to a level far below that mediated by the primary resistance determinants PBP2x and PBP2b, and such mutants contain an altered murein with a large reduction of crosslinked muropeptides [36, 142]. This is similar to Staphylococcus aureus where disruption of the fem genes (factor essential for methicillin resistance) in MRSA resulted in a methicillin sensitive phenotype (for review, see [116]); moreover, MurMN mutants are hypersensitive to other cell wall antibiotics, whereas the overexpression of the MurMN genes reduces the lytic response to these compounds [35]. The reason for the resistance-breakdown in MurM mutants remains obscure. PBPs catalyze the crosslinking between two muropeptides, and thus must use the substrates which are the product of MurM/N function. PBP mediated resistance and altered muropeptide composition can be separated in transformation experiments [53, 123], and MurM mutants show no major growth defects [36, 142]. These experiments demonstrate that resistant PBPs can function with either linear or branched precursors in the absence of b-lactams [36, 142]. Thus, MurM (i.e., branched muropeptides) appears to be only crucial during MIC determination (i.e., in the presence of b-lactams), indicating that some of the low afnity PBPs responsible for resistance use branched muropeptides as substrates [53, 123]. It has been suggested that the branched muropeptide precursors are superior competitors against b-lactams for some resistant PBPs or that they might act as signals for some processes during cell wall biosynthesis [36]. Since muropeptides are also the substrate for the sortase enzyme attaching cell surface anchor proteins (LPXTG motif containing proteins) to the peptidoglycan layer, this reaction might also be affected by an altered murein chemistry interfering indirectly with the bacterial response to b-lactams. However, clinical isolates containing identical MurM and PBP alleles differed signicantly in their resistance level [8]. Pneumococcal transformants obtained with chromosomal DNA from high-level resistant oral streptococci also did not reach the resistance level of the donor strains by far, although transfer of PBP genes as well as MurM was achieved ([53, 123] and own unpublished results). These data strongly suggest that other still unknown factors are also involved in b-lactam resistance of clinical isolates.

18.11

Non-PBP Mutations in Laboratory Mutants

In laboratory mutants it was noted for some time that in addition to PBP changes, mutations in non-PBP genes also occur during the selection with b-lactams. Curiously, distinct mutational routes were detected when selection was done with the highly lytic b-lactam piperacillin compared to cefotaxime. In piperacillin resistant mutants, mutations in a putative membrane associated glycosyltransferase CpoA were identied [44]. Its function as a lipid glycosyltrans-

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ferase has recently been veried biochemically in vitro [30]. The cpoA mutants showed a pleiotropic phenotype, including a reduced susceptibility to piperacillin, less PBP1a, and a reduction in growth rate, genetic competence, and stationary phase lysis. CpoA has been veried as being responsible for the addition of the second sugar moiety to the major pneumococcal glycolipid GalGlcDAG, which suggests an indirect compensatory effect against the lytic action of piperacillin (C. Volz, B. Henrich, and R. Hakenbeck, unpublished results). GalGlcDAG represents the lipid anchor for LTA, conrming early suggestions that teichoic acid biosynthesis might be affected in CpoA mutants [44]. Some piperacillin and all cefotaxime-resistant mutants contained mutations in the histidine protein kinase CiaH, with every mutant containing a different ciaH allele [45, 144]. The CiaRH two component system apparently is required during cell wall stress: deletion mutants in ciaR are unusually lysis prone and hypersensitive to a wide variety of early and late cell wall inhibitors, whereas mutants with an activated CiaRH system were highly resistant to many different lysis inducing conditions [91]. Moreover, deletion of the response regulator in mutants containing a low afnity PBP2x showed severe growth defects and lysed rapidly [91]. This defect was especially marked with PBP2x from laboratory mutants containing the T550A change, whereas it was less pronounced in the presence of resistant PBP2x from clinical isolates. CiaR deletion mutants also revealed a complex interactive scenario concerning PBP2x and PBP1a, in that the presence of a mosaic PBP1a can compensate for growth defects apparent in pbp2x/ciaR double mutants [146]. This strongly suggests that PBP2x mutations are functionally not neutral, and that this defect can be balanced by a functional CiaRH system. Mutations in CiaH have not yet been observed in clinical isolates. Since CiaH mutations have a complex phenotype and affect the genetic competence as well [92], it might be required in the in vivo situation in agreement with the nding that CiaRH mutants are attenuated in mouse models [88, 121, 139]. The CiaRH regulon has been described on the basis of target sequences of the CiaR response regulator, present in 15 promoters including ve regulatory RNAs [57], but the signal detected by the sensor kinase CiaH is still unknown. These ndings imply that inhibitors of LTA biosynthesis and histidine protein kinases are important targets for new antimicrobial agents. CiaR mutants containing a low afnity PBP2x could be screened for anti-histidine kinase antibiotics in that they are hypersensitive to such compounds, and gene products involved in LTA biosynthesis might represent useful proteins for in vitro screens. In summary, the evolution of resistance in S. pneumoniae represents a highly complicated scenario, involving target proteins such as PBPs and non-PBP components as well. Laboratory experiments clearly documented that the kind of mutations and genes selected during resistance development varies enormously depending on the selective compound. Moreover, the complex mosaic structures found in resistant clinical isolates suggests that many different ways for the restructuring of PBPs exist, similar to what has been found in laboratory mutants.
Acknowledgment This work was supported by the DFG (Ha 1011/11-1) and the EU (LSHM-CT-2003-503413 and 503335).

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