2012 Vmechanisms of Penicillin Resitance on S Neumoniae Targets, Genen Trasnfer and Mutatiosn

8/2/2019 2012 Vmechanisms of Penicillin Resitance on S Neumoniae Targets, Genen Trasnfer and Mutatiosn

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593T.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

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 increas-

ing selection pressure, variants were obtained with an over 1,000-fold increase inresistance to benzylpenicillin [46]. It therefore came as a surprise when the first

reports ofb-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. pneumo-

niae (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 con-

centrations, 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: [email protected]

Chapter 18

Mechanisms of Penicillin Resistance

in Streptococcus pneumoniae: Targets,Gene Transfer and Mutations

Regine Hakenbeck, Dalia Denapaite, and Patrick Maurer


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594 R. Hakenbeck et al.

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 net-

work The Pneumococcal Molecular Epidemiology Network (PMEN) [97]. These

prevalent clones have spread to varying degrees to other countries. The Spain23F-1clone 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 first 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 thespread of a particular type 6B clone Spain6B-2 in the late 1980s/early 1990s which

carried resistance determinants against another five 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 specific 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

Table 18.1 Development ofb-lactam resistant Streptococcus spp

MIC MIC

PenG CTX Reference

Streptococcus pneumoniae

1940 Worldwide 0.01 0.02 [69]

1965 Boston 0.10.2 [73]

1967 Papua 0.6 [58, 59]

1977 South Africa 48 2 [64]

1990s Hungary >10 [90]

1990s USA 0.01 32 [95]

Commensal Streptococcus spp

1990s Hungary/Spain 20 20 [112]

1994 Germany >50 >60 [74]

CTXcefotaxime


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59518 Mechanisms of Penicillin Resistance in Streptococcus pneumoniae

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 sensi-

tive 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 mosa-icism 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 resis-

tance 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, whichare numbered in descending order according to their apparent molecular weight as

revealed on SDS-polyacrylamide gels. The hmw PBPs are anchored into the mem-

brane 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 con-

tains three conserved motifs: SXXK with the active site serine which is directlyinvolved 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 develop-

ment resulting in a decreased affinity 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]:

2 3

E + I E I EI* E + P

K K K


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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. Kis the

dissociation constant, k2

and k3

are first-order rate constants for the acylation respec-

tively the deacylation step, and the second order rate constant k2

/Krepresents the

acylation efficiency.

Due to their ability to covalently bind b-lactams, PBP can easily be visualized

after incubation with radioactive or fluorescent antibiotic and separation on SDS

polyacrylamide gels. Since the hmw PBPs ofS. pneumoniae are notoriously diffi-

cult to resolve on SDS gels, and PBP variants especially of clinical isolates may

have a different electrophoretic mobility, specific antisera and monoclonal antibod-

ies have been produced to label individual PBPs. The different ways to visualize

PBPs have been reviewed recently [117]. S. pneumoniae contains six PBPs mysteri-

ously named PBP1a, 1b, 2x, 2a, 2b, and 3. The nomenclature has evolved, due to

improved resolution of first generation SDS gels which resolved PBP1, 2, and 3 toPBP1a + 1b, and 2a + 2b in the late 1970s; PBP2x was discovered later during bio-

chemical 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 analyzethe 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 acyl-

enzymes, which are easily hydrolysed thereby mimicking the transpeptidation reac-

tion carried out by PBPs. Although PBPs may react differently with this compound

compared to the natural muropeptide substrates, the kinetic parameters help to

define 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 bacte-

ria, 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 func-

tions 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 thatthe putative transglycosylase and transpeptidase activities of these PBPs can com-

plement each other. Thepbp2a mutant showed a higher susceptibility to moenomy-

cin, 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 ear-

lier after reaching the stationary phase [107]. This indicates that PBP1b alone can-

not 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 confirmed its D,D-

carboxypeptidase 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 affinity 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 keyplayers of the resistance process:pbp2b [26],pbp2x [80], andpbp1a [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

first reports describing PBP pattern in penicillin resistant clinical isolates [55, 109,

148], most prominent detectable in PBP1a variants [48, 80], although their calcu-

lated molecular weight is almost identical. Even among sensitive strains different

PBP patterns can be distinguished [49]. Since these changes are generally clone

specific, the PBP profiles on SDS PAGE in combination with antibody reactivitypattern have been used as clonal markers [48]. Restriction fragment length poly-

morphism (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 screen-

ing a large number of isolates, small changes in the size of the mosaic blocks and

point mutations inpbp 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 identified by MLST analysis (multilocus

sequence typing [85]) are either resistant or sensitive. However, there are a fewcases where sensitive isolates were detected that belong to the same clone in agree-

ment 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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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] ofS. pneumoniae were detected in susceptible S. mitis strains, in agreement

with the assumption that low affinity PBPs have evolved in commensal streptococci

prior to interspecies gene transfer into the pneumococcal population. One major

class of mosaicpbp2x 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 distinctpbp2x variants exists with unique mosaic pat-

terns all being approximately 20% divergent from each other [51, 99], indicating

multiple intra- and interspecies gene transfer events. The high diversity of the

M3 (SA) S. mitissensitive

Spain6B-2 (670)

CZ14-10 (29044)

Spain23F-1 (2349)S. pneumoniae

resistant USA23F-4 (CS111)

B6 (G)

197 (Sp) S. mitis

Uo5 (Hu) resistantS. oralis

S. mitis

S337TMK

S395SN

K547TG

S. pneumoniae

sensitiveR6

a

b

22222223333333333333333444444444455555555555555555555555556666 MIC CTX

66788891334445566677888001466889900001111233334566777899990001

58012431893675846918249017425680115670346316786057246657890456

M3 LPQLEVLNTMTSSSYLIAIDTRSMSNNLFTNSVKEDALTNILYIITVTSNVTNYYAAQLSNEM3 0.02

670 IT......A......F..T..GL..KS......NKE.TNH...............GI..ATD 0.5

2349 ........A......F..T..GL..KS......NKE.TNH...............GI..AT. 1

29044 I...Q...AL.....F.VT..GL.....L............I.............GI..AT. 4

197 .....L..G......FM.TE.SL..KS......NKE.TNH...............GI..AT. 6

Uo5 .....L..G......FM.TE.SL..KS......NKE.TNH...............GI..AT. 12

111 ......S.AFM....F..TE.G.T.KS......NKE.TNH.......A.......GI..AT. 12

B6 M.H.....A......FMVTG.GL..KS.........T.....Q..I...D...SLTPWFA.D 60

R6 I..Q...D..MAAGV....EG...T..I.PDTANK.....VVSTV.L.LDASS..GI..A.D 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 mosaicpbp2x have been identified in S. pneumoniae and oral streptococci isolated

in different geographic areas as indicated. White: homology to penicillin susceptible S. pneumo-

niae; red: homology to PBP2x ofS. 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 reflect alterations in other PBPs as well


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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 alteredpbp genes can occur within the gene

or in flanking regions. It has been observed in PBP2x from resistant isolates that the

border of the mosaic blocks on the DNA level reflects 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 ofpbp2b [31] and ftsL upstream ofpbp2x (own unpublished results).

Since the capsular gene locus is flanked by thepbp1a andpbp2x 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 aspbp2b andpbp2x, orpbp2a andpbp2x,

can be introduced in a single transformation step as has been shown with chromo-

somal DNA from resistant S. mitis, indicating that genes located elsewhere onthe chromosome can be altered easily during interspecies transformation events

[53, 112].

18.5 PBPs and b-Lactam Resistance: Physiologyof Resistant Isolates

Only PBP2x and PBP2b are primary targets for b-lactams (i.e., alterations in PBP2xor PBP2b alone confer a resistance phenotype albeit to only low levels). PBP2x

mutants can easily be selected with cefotaxime resulting in MIC values for cefo-

taxime 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 pip-

eracillin 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 withpbp1a of resistant clinical isolates are sufficient forcefotaxime 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 finding that high-level penicillin-resistant strains which usu-

ally contain a low affinity PBP2b appear to be tolerant [82]. S. Pneumoniae strains

containing a low affinity PBP2b as the only altered PBP have also been shown todisplay a tolerant response for penicillin antibiotics [43, 112]. The fact that PBP2b

mutants are less prone to lysis suggests that cells with a low affinity 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 significantly less virulent in a murine peritonitis model

[115]. The PBP2x mutants remained stable in both resistance phenotype and viru-

lence, 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 clarified.

18.6 Mutations in PBPs

18.6.1 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 transfor-

mation of sensitive S. pneumoniae strains using cefotaxime for selection (Fig. 18.2a).

Moreover, it was the first and for a considerable time the only PBP where soluble,

active derivatives were available enabling biochemical studies in vitro. The diver-

sity of mutations observed and physiological characterization of PBP2x mutants

suggests a complex evolution of resistance by introduction of mutations that mightaffect 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 proce-

dure 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: T550

A and Q552

E

adjacent to the K547

SG box, and H394

Y next to the S395

SN 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 T550

G mutation which

increases the cefotaxime resistance even further [43]. Curiously, the reverse substi-

tution, A235

T, at the homologous site of TEM b-lactamase resulted in an enzyme

with an extended substrate profile that could hydrolyze cefotaxime, an antibiotic

which is not a substrate for the original protein [16]. It has therefore been speculated

that the T550

A substitution is directly related to cefotaxime selection [78]. The T550

A

mutation results in a 20-fold decrease acylation efficiency for cefotaxime [99],


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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 H394

Y change has also been identified in PBP2x of clinical

Fig. 18.2 Mutations in PBP2x. (a) Only mutations within the transpeptidase domain of PBP2x

(black) have been described. The central blackarea indicates the transpeptidase domain, and the

hatchedarea at the N-terminus indicates the hydrophobic membrane domain. The three active site

boxes are indicated on top. Positions implicated in resistance identified 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 ofb-lactam binding

S337TMK S395SN K547SG

266 616

C-terminalN-terminal transpeptidase

PBP2x

a

(750 aa)

Q552EH394Y R512WF388Y

L403

F Q458KM289T L600W

laboratory mutants

T550A/G G601E/VG422D

R426C

M400

TL364

F

clinical isolates

Q552ET337A/G/P/S

H394Y T

N605T

YT550AM339F

I371TR384G

Y595F

T526S S596L

G597D

L403

Q458

S526

R426

G422

G601

M289

G597

L600

T550

b


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isolates [103]; the effect of H394

L 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

abol-

ishes the interaction with cefotaxime as well as penicillin almost completely as

measured in a purified soluble protein derivative, and L403

F 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-G597Donly affect the acylation efficiency towards cefotaxime (k

2/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 effi-

ciency also reacted considerably poorer with the depsipeptide S2d [65, 94].

Curiously, the amount of PBP2x was also reduced in some mutants, but the molecu-

lar basis for this phenomenon is not clear [94].

Mutations in PBPs of resistant clinical isolates cannot easily be deduced fromsequence 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 Q552

E 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 confirmed that theoverall binding efficiency of a resistant PBP2x is much slower than that for sensi-

tive PBP2x (k2/K

dvalues of 100,000200,000 M1 s1 for sensitive PBP2x com-

pared to 11,00085 M1 s1 and lower for resistant PBP2x containing multiple

alterations) [6, 66, 84, 94]. The impact on resistance and b-lactam affinity has been

demonstrated by a combination of mutagenesis and biochemical characterization of

the protein for T338

(A/G/P), M339

F, and Q552

E [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 beenimplicated in hydrogen bonding to a buried water molecule [100] and indeed this

molecule is absent in a resistant PBP2x containing the T338

A substitution [20]. The

combination of T338

A/M339

F reduced the acylation efficiency 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 M

339F substitution also contributes to 4070-

fold 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 Q552

E substitution reveals

a distinct mechanism involved in resistance. The b3 strand with the K597

TG 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 Q552

E mutation into the sensitive R6 PBP2x

results in 34-fold reduction of the acylation efficiency, mosaic PBP2x with the

Q552

E substitution have a 7-fold reduction [94] and 15-fold reduction was observed

in a mosaic PBP2x which also contained the T338

A substitution [110].

Moreover, L364F, I371T, R384G, M400T, Y595F, and N605T [2, 6, 100, 134] are suppos-edly involved in resistance. The structure of a mosaic resistant PBP2x revealed that

the substitutions I371

T and R384

G 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 Q447

M, S449

A and N514

H, 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] andaltered the acylation efficiency 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 ofb-lactam binding, but also the functional altera-

tions 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 specificity (see below).

18.7 PBP2b

In PBP2b, G660

D at the C-terminal end of the protein, G617

A within the K615

TG motif

[54], and T446A close to the S443

SN 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 (T616

S; [136]). The T446

(A/S) change occurs in many

resistant clinical isolates and E476

G 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 peni-

cillin affinity in vitro, and in a PBP2b containing this, up to another 43 amino acids


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change the affinity and is reduced up to 99% [106]. Only one case has been reported

bearing a change within the SVVK motif (V388

A) [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 S423

WY [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 mainstructural consequence of alterations concerned the active site, and it has been sug-

gested 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 lowaffinity PBP2x and/or PBP2b. In resistant PBP1a of clinical isolates, T

371A or T

371S

close to the active site S370

occur frequently and contribute to resistance [2, 33, 89,

103, 105, 135]. L539

W present in PBP1a present in a high level resistant Hungarian

isolate [132], and the alteration of four consecutive residues T574

SQF to NTGY have

S386TMK K615TG

PBP2b (680 aa)

T446A* G660D*G617A*

E476G T616SV338A

S370TMK S466SN K557TG

PBP1a (719 aa)

T371(A/S)

TSQF574-577NTGY

L539W

S443SN

Fig. 18.3 Mutations in PBP2b and 1a. The central blackarea indicates the transpeptidase domain,

and the hatchedarea 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


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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 T371

A muta-

tion 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

N574

TGY substitutions have a lower acylation efficiency 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 affinity PBP1a in a

strain carrying a low affinity PBP2x results in elevated resistance. That the situation

is much more complex has been shown recently, using low affinity PBP2x variantsfrom laboratory mutants in comparison with mosaic PBP2x from resistant clinical

isolates. Introduction of a mosaicpbp1a 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 efficiencies 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 first been observed in laboratory mutants, which

contain a low affinity PBP2x [79]. Curiously, PBP2a in three such mutants could

not be visualized using common labeling procedures, or even when high concentra-tions 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 clini-

cal 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 trans-

formants which contained a low affinity PBP2a [53, 112]. Some especially highlevel resistant clinical isolates of S. pneumoniae indeed contained a low affinity

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 alteredpbp2a

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, confirming the potential role as

resistance determinant for PBP2a [130]. It is remarkable that these resistant PBP2a

mutations flanking the active site Ser410

(T411

A) occur frequently. PBP2a has a rela-

tively low affinity 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 affinity PBP2a is transferred into the recipient

S. pneumoniae before a low affinity 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 first case where a deletion

mutation of a PBP has been identified in a resistant isolate. The fact that no growth

defects are apparent in the S. mitis strain agrees with the assumption that the puta-

tive 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 unre-

lated to resistance remains to be clarified.A PBP3 mutation T

242I associated with resistance has only been described in one

particular laboratory mutant C604, again in the immediate vicinity of the K239

TG

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 cefo-

taxime resistance [122]. Particular clones of clinical isolates contain a PBP3 with

altered electrophoretic mobility [76], but these variants do not affect the affinity

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(fibAB) operon. In the cell wall of some high level

resistant clinical isolates, such branched muropeptides are present in higher quan-

tity compared to sensitive strains [38]. A mosaic structure ofmurM is associatedwith 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 efficiency of

MurM from resistant strains compared to the sensitive MurM is mainly responsible


15/24


for the different murein structure [83] whereas MurN from both, resistant and sensitive

strains, showed similar enzymatic function [19].

Curiously, disruption of murM/fibA 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 reduc-

tion of crosslinked muropeptides [36, 142]. This is similar to Staphylococcus

aureus where disruption of the fem genes (factor essential for methicillin resis-

tance) in MRSA resulted in a methicillin sensitive phenotype (for review, see

[116]); moreover,MurMNmutants 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 andaltered muropeptide composition can be separated in transformation experiments

[53, 123], and MurM mutants show no major growth defects [36, 142]. These exper-

iments demonstrate that resistant PBPs can function with either linear or branched

precursors in the absence ofb-lactams [36, 142]. Thus, MurM (i.e., branched muro-

peptides) appears to be only crucial during MIC determination (i.e., in the presence

ofb-lactams), indicating that some of the low affinity PBPs responsible for resis-

tance 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 cellwall 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

significantly 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 stronglysuggest 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 identified [44]. Its function as a lipid glycosyltrans-


16/24


ferase has recently been verified 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 verified 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, confirming 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 hypersensi-

tive to a wide variety of early and late cell wall inhibitors, whereas mutants with anactivated CiaRH system were highly resistant to many different lysis inducing con-

ditions [91]. Moreover, deletion of the response regulator in mutants containing a

low affinity PBP2x showed severe growth defects and lysed rapidly [91]. This defect

was especially marked with PBP2x from laboratory mutants containing the T550

A

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 compen-

sate for growth defects apparent inpbp2x/ciaR double mutants [146]. This strongly

suggests that PBP2x mutations are functionally not neutral, and that this defect canbe 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 situa-

tion in agreement with the finding 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 five

regulatory RNAs [57], but the signal detected by the sensor kinase CiaH is still

unknown.

These findings imply that inhibitors of LTA biosynthesis and histidine proteinkinases are important targets for new antimicrobial agents. CiaR mutants containing

a low affinity 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 compo-

nents 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 resistantclinical 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).


17/24


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