PBP2a

PBP2A
Penicillin Binding Protein 2a
Introduced to;
Dr. Eman Mahmoud Elawady Dokla
Lecturer of Pharmaceutical Chemistry, Faculty of Pharmacy - Ain
Shams University, Abbasia, Cairo, Egypt
Prepared& presented by;
Muhammad Talaat
level II, ID. 457.
Faculty of Pharmacy - Ain Shams University, Abbasia, Cairo, Egypt

PBP2A
Penicillin Binding Protein 2a

Objectives of the presentation;
I. PBP “Penicillin Binding Protein”
II. Peptidoglycan synthesis & PBP function.
-Cell wall structure.
-Strategy to handle the bacterial infection.
III. β-LactamANTIBIOTICS
“Antibacterials which Inhibit cell wall synthesis”
-Classification.
-Mech. Of Action.
-Preparation& Nomenclature.
-SAR.
-Inhibitory bacterial transpeptidase enzyme be Penicillin.
-Tackling the β-Lactam problems.
-Methicillin.
IV. PBP2a as antimicrobial.

Cell wall Structure
*The bacterial cell wall has functions of providing support
for the maintenance of cell morphology, protecting the integrity
of the cell membrane, and preventing cell breakage
from the osmotic pressure.
*The cell wall is a huge net-like structure that surrounds the cell.
The main structural component is peptidoglycan, which has a
structure consisting of long glycan strands cross-linked by short peptides.
*The peptidoglycan backbone is made up of repeating disaccharide subunits
N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)
connected by a beta 1,4-glycosidic bond.
*Glycan strands are crosslinked by pentapeptides with the sequence
L-Ala-D-Glu-L-meso-diaminopimelic acid-D-Ala-D-Ala in Bacillus subtilis and E. coli.
D-Ala (4 position) from the side chain of a donor NAM links to diaminopimelic acid in
the side chain of the acceptor NAM .

Peptidoglycan synthesis
Peptidoglycan biosynthesis is a two-stage process.
*In the first stage,
-The basic monomer unit is assembled.
-Enzymes catalyze this process in the cytoplasm or at the inner side of the cytoplasmic
membrane (18).
(The initial precursor, UDP-NAG, is synthesized from fructose-6-P in four steps and is then
used to generate UDP-NAM).
L-Ala, D-Glu, meso-diaminopimelic aid and D-Ala-D-Ala are successively added onto
UDP-NAM by a series of enzymes and thus UDP-NAM-pentapeptide, the end product, is
formed. The phospho-NAM-pentapeptide and NAG moieties are successively transferred
onto the undecaprenyl phosphate carrier lipid by two membrane bound enzymes, the
MraY translocase and MurG transferase, and thus the intermediate GlcNAc-MurNAc-
(pentapeptide)- PP-undecaprenol, “lipid II”, is formed (61). Lipid II is then transferred to
the outer side of the cytoplasmic membrane and the disaccharide units are polymerized.

*In Gram-positive bacteria the cell wall is thick (15-80 nanometers), consisting of
several layers of peptidoglycan (11).
*In Gram-negative bacteria, the cell wall is thin (10 nanometers) and composed of a
single layer of peptidoglycan, with an outer membrane (18).
*There is a unique component in the outer membrane, lipopolysaccharide (LPS or
endotoxin), which is toxic to animals.
*In Gram-negative bacteria the outer membrane is usually thought of as part of the cell
wall.

*In the second process,
-Two enzymatic activities, transglycosylase and transpeptidase, play the most important
roles, and they are found in a family of penicillin-binding proteins (PBPs) (21).The PBPs
have been divided into three classes according to their molecular weight (MW) and
conserved amino acid motifs:
the high MW (≥60kD) Class A PBPs, the high MW Class B PBPs and the low molecular
weight (≤60kD) PBPs(18).
The class A high-MW PBPs are bifunctional with transpeptidase activity in the
C-terminal domain and a glycosyl transferase activity in the N-terminal domain. Glycosyl
transferase activity carries out the polymerization of the glycan strands.
The class B high-MW PBPs were once reported to possess a glycosyl transfer activity, even
though their N-terminal domains do not show homology to those of the class A PBPs.
However, glycosyl transfer activity of the class B PBPs has never been proven convincingly
(14).

Strategy to handle the bacterial Infection
1. Bactericidal Treatment
*Bactericidal antibiotics kill bacteria directly.
How do bactericidal antibiotics actually kill bacteria?
-There are many different antibiotics that all have different mechanisms that achieve
the death of bacteria;
-for example:
The antibiotic polymyxin B injures the plasma membrane of bacteria, allowing their
contents to leak out. Under normal circumstances, bacteria and other cells have to
keep a perfect balance of ions on both sides of the plasma membrane because of
osmosis. Polymyxin B disrupts this balance, and also lets other important molecules,
like DNA and RNA, leak out, so the bacterium is a goner.

Strategy to handle the bacterial Infection
2. Bacteriostatic Treatment
*Bacteriostatic antibiotics stop bacteria from growing.
The bacteria don't die, but they can't grow or replicate either.
How do bacteriostatic antibiotics help clear up an infection, if they don't actually kill
bacteria?!!!
-Bacteria normally divide really quickly in our bodies, and their numbers can get totally
out of control. But if an antibiotic stops them from growing and dividing, the host's
immune system will be able to get rid of the bacteria.
-for example:
The sulfa drugs, they prevent the production of important metabolites that the
bacterium needs in order to make new DNA, RNA and proteins. Again, this doesn't kill
the bacteria, but there's no way they can replicate and make new bacteria without new
DNA, RNA and proteins. Okay, now we understand the difference between bactericidal
and bacteriostatic antibiotics.

Β-LACTAM ANTIBIOTICS
(Antibacterials which Inhibit cell wall
synthesis)

Chemical Classification of β-Lactam antibiotics

Mechanism of action of Penicillins “β-Lactam antibiotics”
The uniquely lethal antibacterial action of these agents has been attributed to a selective
inhibition of bacterial cell wall synthesis through inhibition of transpeptidase enzyme.
Penicillin is chemically similar to the modular pieces that form the peptidoglycan, and
when used as a drug, it blocks the enzymes that connect all the pieces together.
As a group, these enzymes are called penicillin-binding proteins.
Some assemble long chains of sugars with little peptides sticking out in all directions.
Others, like the D-alanyl-D-alanine carboxypeptidase/transpeptidase, then cross-link
these little peptides to form a two-dimensional network that surrounds the cell like a
fishing net.

-Bacterial cells change shape and grow into long filaments.
-As the dosage is increased, the cell surface loses its integrity, as it puffs, swells, and ultimately ruptures.
-Penicillin attacks enzymes that build a strong network of carbohydrate and protein chains, called peptidoglycan, that
braces the outside of bacterial cells.
-Bacterial cells are under high osmotic pressure; because they are concentrated with proteins, small molecules and
ions are on the inside and the environment is dilute on the outside.
-Without this bracing corset of peptidoglycan, bacterial cells would rapidly burst under the osmotic pressure.

Cross-linking of bacterial cell wall catalyzed by bacterial Transpeptidase enzyme

Preparations of penicillin
1. Natural penicillins: They obtained naturally from fermentation of the
fungus penicillium chrysogenum. e.g. benzylpenicillin.
2. Biosynthetic penicillins: They obtained by altering the culture media by
addition of certain different carboxylic acids that may be incorporated as
acyl groups e.g. phenoxymethylpenicillin (penicillin V) which is prepared by
addition of phenoxyacetic acid.
3. Semisynthetic penicillins: The fermentation yielded 6-APA which could
then be treated synthetically to give penicillin analogues. This was achieved
by acylating the isolated 6-APA with a wide range of acid chlorides.
Carboxylic acids can be used for the acylation using N,N-
dicyclohexylcarbodiimide (DCC).

Nomenclature of penicillin
Three simplified forms of Penicillin nomenclature have been adopted for
general use:
1. Chemically, they are (2S,5R,6R)-6-acylamino-3,3-dimethylpenam-2-carboxylic acid.
2. They are also considered as derivatives of 6-aminopenicillanic acid.
3. Trivial nomenclature using the name penicillin as suffix and the acyl portion (R) as
prefix e.g. benzyl penicillin (penicillin G).

Structure-activity relationships

Structure-activity relationships
Structure of penicillins compared to that of Acyl D-
alanine-D-alanine moiety

Structure-activity relationships Study
Inhibition of bacterial transpeptidase enzyme by penicillins

Mech. Steps;
1. The amide of the β-Lactam ring is unusually reactive due to ring
strain and a conformational arrangement which does not allow the
lone pair of the nitrogen to interact with the double bond of the
carbonyl.
2. β-Lactams acylate the hydroxyl grp. on the srine residue of PBP
active site in an irreversible manner.
3. This Rx. is further aided by the oxyanion hole which stabilizes
the tetrahedral intermediate and thereby reduces the transition
state energy.

Mech. Steps;
4.The –OH attacks the amide and forms a tetrahedral
intermediate.

Mech. Steps;
5. The tetrahedral intermediate collapse, the amide bond is
broken, and the nitrogen is reduced.

Mech. Steps;
6. The PBP is now covalently bound by the drug and cannot
perform the cross linking action.

Mechanisms of antimicrobial resistance

Of course, bacteria are quick to fight back. Bacteria reproduce very quickly, with dozens of generations
every day, so bacterial evolution is very fast. Bacteria have developed many ways to thwart the action of
penicillin. Some change the penicillin-binding proteins in subtle ways, so that they still perform their
function but do not bind to the drugs. Some develop more effective ways to shield the sensitive enzymes from
the drug or methods to pump drugs quickly away from the cell. But the most common method is to create a
special enzyme, a beta-lactamase (also called penicillinase) that seeks out the drug and destroys it.
Many beta-lactamases use the same machinery as used by the penicillin-binding proteins--so similar, in fact,
than many researchers believe that the beta-lactamases were actually developed by evolutionary
modification of penicillin-binding proteins. The penicillin-binding proteins, use a serine amino acid in their
reaction, colored purple here. The serine forms a covalent bond with a peptidoglycan chain, then releases it
as it forms the crosslink with another part of the peptidoglycan network. Penicillin binds to this serine but
does not release it, thus permanently blocking the active site. Beta-lactamases, have a similar serine in their
active site pocket.
Penicillin also binds to this serine, but is then released in an inactivated form. Other beta-lactamases do the
same thing, but use a zinc ion instead of a serine amino acid to inactivate the β-Lactam.
Strategy done by bacteria to resist the antibiotic action

Penicillin also binds to this serine, but is then released in an inactivated form. Other beta-lactamases
do the same thing, but use a zinc ion instead of a serine amino acid to inactivate the β-Lactam.

Penicillin’s properties; “ advantages & disadvantages”
Penicillin Advantages;
1. Bactericidal against sensitive strains.
2. Has excellent tissue penetration.
3. Efficacious in treatment of infection.
4. Relatively inexpensive in comparison with other antibiotics.
5. Newer Penicillins are resistant to stomach acid , such as penicillin V, or have a broad
spectrum such as Ampicillin and Amoxicillin.
Penicillin Disadvantages;
1. Short duration of action, so the Penicillin must be a short interval, usually every 4 hrs.
2. Acid sensitivity.
3. Penicillinase sensitivity, about 10% of population has allergy.
4. Narrow spectrum of activity.
5. Allergic reactions.
6. Lack of activity of most of gram-negative bacteria organisms

Solution / ApproachesProblem
Two approaches to overcome this problem:
1.To modify the structure of ß-lactam antibiotic so as to
increase its stability toward ß-lactamases e.g. Methicillin,
Nafcilin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin.
2.To use agents (ß-lactamase inhibitors) those were capable of
inhibiting the bacterial enzyme to protect the ß-lactam
antibiotic from destruction.
1. The sensitivity of penicillin G to ß-lactamases.

The Strategy of the approaches;
Modification of the structure of ß-lactam
antibiotic:
1.The strategy is to block the penicillin from reaching
the penicillinase active site.
2. To do this is: place a bulky group on the side chain.
3. This bulky group can then act as a “shield”, and
prevent the binding of penicillins.
*Note;
1. Methicillin& Nafcillin are;
a. Penicillinase resistant.
b. But acid-sensitive.
2. Oxacillin, Cloxacillin, Dicloxacillin or Floxacillin
are;
a. Penicillinase resistant.
b. Acid-sensitivity resistant.

Methicillin
*Acknowledge;
- Methicillin is used for treatment of Staph. Infection.
- Methicillin is penicillinase-resist penicillin; resist the bacterial enzyme
called penicillinase = β-Lactamase.
- It can NOT be used in solid dosage form as tablets bscause of
inactivation happens by gastric acid, as penicillin, so it’s used in IV or
IM form.
- Side effects’
1.Diarrea
2. Allergic Rx.; skin rash& anaphylaxis.
3. Severe hemorrhage cystitis.
*Resistant against Methicillin is believed to have resulted from
bacterial acquisition of gene that encodes a protein capable of
binding the drug and thus preventing the drug from killing the
organism producing Methicillin-Resistant Staph.aureus (MRSA).
- The mecA gene encodes a protein (PBP2A) that reduces the
binding affinity of β-Lactam drugs.
- May also posses mutations for resistance to other
antimicrobials.

PBP2A
*MRSA differ genetically from methicillin-sensitive S. aureus (MSSA) isolates by the presence, in the chromosome, of
a large stretch of foreign DNA (40-60 Kb), referred to as the mec element, and the presence of the mecA gene that
encodes the 76 KDa penicillin-binding protein, PBP2a (also referred to as PBP2′).
*The mecA gene has been proposed to originate from Staphylococcus sciuri. Although the mechanism of gene
acquisition from this specie is not known, two genes, ccrA and ccrB, present on the mec element from one isolate, have
been shown to code for recombinase proteins that are capable of excising and integrating the mec element into the
chromosome.
*Examination of a large number of MRSA isolates has led to the conclusion that the original acquisition of the mecA
gene has occurred once and that MRSA isolates are descendants of a single clone. Although the arrangement and
composition of the mec element may vary between isolates, the mecA gene itself is highly conserved. In common with
other PBPs, PBP2a has the common structural motifs that are associated with penicillin binding yet its affinity for β-
lactam antibiotics is greatly reduced. Consequently, at therapeutic levels of methicillin that would inhibit the
transpeptidational activities of other PBPs, PBP2a remains active ensuring the cross-linking of the glycan chains in
peptidoglycan.
*PBP2a is not able to completely compensate for the other PBPs since cells grown in the presence of methicillin
exhibit a marked reduction in the degree of cross-linking. However, the limited degree of cross-linking is enough to
ensure survival of the cell.

Tackling of PBP2A strategy
*The study of PBPs in S. aureus is of particular importance because of the clinical relevance of
methicillin-resistant S. aureus (MRSA) strains. The acquired resistance to all β-lactam antibiotics of
MRSA strains has had a devastating impact on the treatment of staphylococcal infections, which are a
major cause of hospital-acquired infections.
*The main determinant for methicillin resistance in S. aureus is PBP2A, encoded by a gene imported from
an extraspecies source, which is capable of transpeptidation even in the presence of high concentrations of
antibiotic, because of its low affinity for β-lactam antibiotics.
*Among other factors, one of the native staphylococcal PBPs – PBP2 – is also required for the full
expression of methicillin resistance.
*PBP2 is a HMW class A PBP with both transpeptidase and transglycosylase domains. In the presence of
high concentrations of methicillin, when PBP2A is responsible for the transpeptidation of the
peptidoglycan, the transglycosylase function of PBP2 is still required for the expression of resistance,
implying that PBP2A and PBP2 functionally cooperate during growth in the presence of high
concentrations of β-lactams.

*The in-vitro activity of oxacillin combined with non-beta-lactam antibiotics, bacitracin, vancomycin,
enduracidin, tunicamycin, flavomycin, fosfomycin, and cycloserine, was investigated in 23 methicillin-
resistant and 15 methicillin-susceptible Staphylococcus aureus strains. In the presence of a non-beta-
lactam antibiotic (0.25 MIC), the MICs of oxacillin for methicillin-resistant S. aureus (MRSA) strains and
methicillin-susceptible S. aureus (MSSA) strains were lowered. This effect was most marked with MRSA
strains, and bacitracin, flavomycin, and tunicamycin increased the susceptibility of MRSA to oxacillin by
greater than 200-fold.
*More than 87% and 77% of MRSA and MSSA strains, respectively, were synergically inhibited by a
combination of oxacillin with bacitracin, tunicamycin, flavomycin or cycloserine (fractional inhibitory
concentration < or = 0.5). Polyanetholesulfonic acid prevented the lysis of MRSA cells treated with a
combination of oxacillin and 0.25 MIC of bacitracin, but did not prevent cell death.
*The penicillin binding protein (PBP) profile of MRSA was not affected by incubation with 0.25 MIC of
non-beta-lactam antibiotics. These results suggest that the increased antibacterial activity of oxacillin in
the presence of non-beta-lactam antibiotic is not the consequence of activation of autolysis or of a decrease
in bulk PBP synthesis.
PBP2 is essential for growth of an MSSA strain but not for growth of an MRSA strain.

PBP2A as antimicrobial
* Mech. Of Antimicrobial Infection
1. Inhibition of cell wall synthesis.
2. Inhibition of protein synthesis.
3. Inhibition of nucleic acid synthesis.
4. Inhibition of metabolic pathways.
5. Interference with cell membrane integrity.

PBP2A as antimicrobial
*What are MRSA and MSSA?
1. Resistant bacteria are called methicillin‐resistant Staphylococcus aureus, or MRSA.
2. The bacteria that can be treated by methicillin class antibiotics are MSSA.
*What happens if we find MSSA or MRSA and how is it treated?
If the results are positive, the treatment is simple and consists of a nasal ointment which will begin
the morning of surgery.
*The conclusion;
- PBP2 is essential for growth of an MSSA strain but not for growth of an MRSA strain.
- PBP2 is essential for survival of methicillin-susceptible S. aureus (MSSA) strain.
So, It’s easier to treat MSSA Infection using the agonist action of PBP2A for growth MSSA from
MRSA strand as MSSA stands for Methicillin Sensitive Staphlococcus Aureus, in other words Staph
which responds well to typical antibiotics.
-PBP2A agonists acting as antimicrobial.

Reference;
1. Alaedini, A., and Day, R.A. (1999) Identification of two penicillin-binding multienzyme complexes in Haemophilus
influenzae. Biochem Biophys Res Commun 264: 191–195.
2. Bi, E.F., and Lutkenhaus, J. (1991) FtsZ ring structure associated with division in Escherichia coli. Nature 354: 161–
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3. Daniel, R.A., and Errington, J. (2003) Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped
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4. Giesbrecht, P., Kersten, T., Maidhof, H., and Wecke, J. (1998) Staphylococcal cell wall: morphogenesis and fatal variations
in the presence of penicillin. Microbiol Mol Biol Rev 62: 1371–1414.PubMed | CAS | Web of Science® Times Cited: 96
5. Hartman, B.J., and Tomasz, A. (1984) Low-affinity penicillin-binding protein associated with beta-lactam resistance in
Staphylococcus aureus. J Bacteriol 158: 513–516.PubMed | CAS | Web of Science® Times Cited: 401
6. http://study.com/academy/lesson/types-of-antibiotics-bacteriocidal-vsbacteriostatic-narrow-spectrum-vs-broad-
spectrum.html#lesson
7. http://agscientific.com/blog/index.php/tag/iptg/
8. http://aac.asm.org/content/33/11/1869.short
9. Holtje, J.V. (1996) A hypothetical holoenzyme involved in the replication of the murein sacculus of Escherichia coli.
Microbiology 142: 1911–1918.CrossRef | PubMed | Web of Science® Times Cited: 73
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