**3. Lytic transglycosylases of** *P. aeruginosa*

Recently, lytic transglycosylases of *P. aeruginosa* have been extensively characterized [21–27]. These cell wall proteins are found in many other pathogenic bacteria and are classified according to amino acid sequence and function [28]. To date there are 11 *P. aeruginosa* lytic transglycosylases that have been described. Their functions

**73**

**Figure 1.**

*Bulgecins as β-Lactam Enhancers Against Multidrug Resistant (MDR) Pseudomonas aeruginosa*

range from cell division to aiding in the insertion of secretion systems and two component regulatory systems. They are attractive drug targets to enhance the activity of our most commonly used and safest antibiotics, the β-lactam class (penicillins,

Lts in general catalyze a cleavage reaction that breaks the glycosidic bond between the peptidoglycan building blocks, MurNAc and GlcNAc (**Figure 1**).

*Lt reaction in cell wall remodeling in Pseudomonas aeruginosa. When the transpeptidase (crosslinking function) of a PBP is inhibited by a β-lactam, the tranglycosylase function of the PBP continues to produce strands of uncrosslinked peptidoglycan (PG). The soluble Lt in the periplasm of Gram negative bacteria initiates recycling and cleavage of PG via endolytic (within strand) reaction. Once this first cleavage reaction occurs, the 1,6-anhydroMurNAC-GlcNAC containing fragments are cleaved and released. In P. aeruginosa, these 1,6-anhydromuramylpeptide fragments affect regulation of Amp C β-lactamase production. TP designates tetrapeptide.*

This reaction does not involve a water molecule but rather, an active site Glu or Asp residue functions as a general acid, donating a proton to the oxygen in the β-1,4 glycosidic linkage. Then the deprotonated active site residue acts a general base as a nucleophile to break the glycosidic bond. The result is a 1,6-anhydroMurNAc containing final peptide product. This unique cap on the muramyl peptide is a signal and a way for the cell wall peptidoglycan cleavage products to be trafficked for recycling [26]. The reaction shown in **Figure 1** is within the strand or "endolytic".

*DOI: http://dx.doi.org/10.5772/intechopen.85151*

cephalosporins, carbapenems and monobactams).

Some Lts also catalyze an end of strand or "exolytic" cleavage.

*Bulgecins as β-Lactam Enhancers Against Multidrug Resistant (MDR) Pseudomonas aeruginosa DOI: http://dx.doi.org/10.5772/intechopen.85151*

range from cell division to aiding in the insertion of secretion systems and two component regulatory systems. They are attractive drug targets to enhance the activity of our most commonly used and safest antibiotics, the β-lactam class (penicillins, cephalosporins, carbapenems and monobactams).

Lts in general catalyze a cleavage reaction that breaks the glycosidic bond between the peptidoglycan building blocks, MurNAc and GlcNAc (**Figure 1**).

This reaction does not involve a water molecule but rather, an active site Glu or Asp residue functions as a general acid, donating a proton to the oxygen in the β-1,4 glycosidic linkage. Then the deprotonated active site residue acts a general base as a nucleophile to break the glycosidic bond. The result is a 1,6-anhydroMurNAc containing final peptide product. This unique cap on the muramyl peptide is a signal and a way for the cell wall peptidoglycan cleavage products to be trafficked for recycling [26]. The reaction shown in **Figure 1** is within the strand or "endolytic". Some Lts also catalyze an end of strand or "exolytic" cleavage.

#### **Figure 1.**

*Pseudomonas aeruginosa - An Armory Within*

β-lactamase enzyme [13, 14].

**2.5 Penicillin binding proteins (PBPs) of** *P. aeruginosa*

nance of cell wall will be further discussed below.

CTX-M, GES, PER and VEB types; Class A carbapenemases such as KPC variants; metallo-β-lactamases such as the VIM, IMP, NDM and SPM B1 di-Zn2+ enzymes: and OXA carbapenemases [9, 12]. Weak imipenemases in the so-called Class C AmpCs have already been discussed above. In combination with OprD loss and/or upregulation of MEX efflux pumps, high level carbapenem resistance can be seen in *P. aeruginosa* due to acquired β-lactamases. Traditional class A β-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam have *in vitro* activity versus the Class A ESBLs but not against other β-lactamases, e.g., the anti-pseudomonal combination ceftolozane-tazobactam is not effective against KPC, metallo-blactamases, or OXA enzymes [1]. New β-lactam-β-lactamase inhibitor combinations such as ceftazidime-avibactam and meropenem-vaborbactam will be active against isolates with KPC enzymes, and Class C β-lactamases, as long as they lack other resistance mechanisms that increase the β-lactam MIC beyond what is caused by the

PBPs of *P. aeruginosa* have high affinities for so called anti-pseudomonal β-lactams namely piperacillin, ticarcillin, ceftazidime, cefepime, ceftolozane, meropenem, imipenem, doripenem and aztreonam [1]. PBP 3 is the most important target of inhibition as it is essential for growth of the bacteria [15]. PBP3 is the primary target for ceftazidime whereas PBP2 is the target of carbapenems. Mutations in PBPs have not been described in *P. aeruginosa* leading to β-lactam resistance. The interactions of specific PBPs with specific lytic transglycosylases in the mainte-

**2.6 Current therapeutic strategies to treat infections with resistant** *P. aeruginosa*

Given that 15–33% of *P. aeruginosa* isolates are multidrug resistant (have at least one resistance mechanism) [16, 17] and that resistance is associated with up to fivefold greater mortality [18, 19], choosing the right antibiotic combinations have a tremendous impact on patient outcomes. Advances in the rapid diagnosis of *P. aeruginosa*, and use of both rapid phenotypic tests such as CARBA NP [20] or rapid molecular diagnostics to identify specific ESBL and carbapenemase enzymes, have enhanced the clinician's ability to get patients on the right therapy sooner. Identification of patient risk factors, including prior antibiotic exposure, and knowledge of local trends in resistance patterns are useful in selection of empiric antibiotics, until antimicrobial susceptibilities and genotypic results are available for guidance. Carbapenems (meropenem or imipenem) and anti-pseudomonal cephalosporins in combination with colistin, an aminoglycoside or fosfomycin, versus ceftolozane/tazobactam or meropenem/vaborbactam or ceftazidime/avibactam are all good empiric choices for critically ill patients [16], provided multidrug resistance is not present. However, clearly more therapeutic options are needed for infections with extensively drug resistant and pan-resistant *P. aeruginosa*. Lytic

transglycosylases represent a new target for bacterial inhibition.

Recently, lytic transglycosylases of *P. aeruginosa* have been extensively characterized [21–27]. These cell wall proteins are found in many other pathogenic bacteria and are classified according to amino acid sequence and function [28]. To date there are 11 *P. aeruginosa* lytic transglycosylases that have been described. Their functions

**3. Lytic transglycosylases of** *P. aeruginosa*

**72**

*Lt reaction in cell wall remodeling in Pseudomonas aeruginosa. When the transpeptidase (crosslinking function) of a PBP is inhibited by a β-lactam, the tranglycosylase function of the PBP continues to produce strands of uncrosslinked peptidoglycan (PG). The soluble Lt in the periplasm of Gram negative bacteria initiates recycling and cleavage of PG via endolytic (within strand) reaction. Once this first cleavage reaction occurs, the 1,6-anhydroMurNAC-GlcNAC containing fragments are cleaved and released. In P. aeruginosa, these 1,6-anhydromuramylpeptide fragments affect regulation of Amp C β-lactamase production. TP designates tetrapeptide.*

Lts are classified according to amino acid motifs and function, into 6 distinct families. Even within a family, there is little sequence homology; however, the proteins in families do appear to share distinct folds (**Figure 2**). Lts are also divided into membrane (designated M in their nomenclature) and soluble (S) forms. It is hypothesized that these proteins are associated with numerous other cell wall proteins such as PBPs so that even the soluble Lts might be physically associated with the inner membrane of bacteria. Some Lts are also associated with the outer membrane, e.g., RlpA (see below) and likely have distinctive roles [29].

Lts serve many cellular functions including cell wall recycling, cellular division, insertion into cell wall of important structures like secretion systems and flagellar apparati. Lt redundancy is similar to that of the PBPs, and studies looking at gene knockouts of these proteins show that in *P. aeruginosa*, only loss of the RlpA LT is associated with a change in bacterial morphology [29]. Attempts to prepare multiple Lt knockouts were unsuccessful.

Recently significant research has been conducted on the Lts of *P. aeruginosa*, including structural and kinetic studies defining structure function relations in these varied proteins (reviewed in [26]). These studies are summarized next.

#### **3.1 Kinetic studies of** *P. aeruginosa* **lytic transglycosylases**

As previously indicated, *P. aeruginosa* possesses 11Lts: MltA, MltB/Slt35, MltD, MltF, MltF2, MltG, RlpA, Slt, SltB1 (SltB), SltB2 (SltG), and SltB3 (SltH).

In a tour-de-force of biochemical characterization, including synthesis, purification and characterization of the reaction of soluble forms of all 11 *P. aeruginosa* Lts with 4 synthetic substrates and *P. aeruginosa* sacculus to yield 31 distinct peptidoglycan (PG) products, Lee et al. [25] have thoroughly described the structure

#### **Figure 2.**

*(A) Slt70 of E. coli in complex with Bulgecin A. (B) Lt of Neisseria meningitidis in complex with Bulgecin A. (C) Lt Cj0843 of Campylobacter jejuni in complex with Bulgecin A. (D) Slt inactive mutant E503Q from Pseudomonas aeruginosa in complex with Bulgecin A.*

**75**

Bulgecin A).

*Bulgecins as β-Lactam Enhancers Against Multidrug Resistant (MDR) Pseudomonas aeruginosa*

function relationships for *P. aeruginosa* Lts. Of interest is that each solubilized Lt enzyme could perform both endolytic and exolytic reactions on the PG substrates. Using the simplest synthetic substrate, NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide), Lee et al. found that only MltB and the SltB1–3 Lts could recognize this substrate. A second substrate, a NAG-NAM(tetrapeptide)-NAGanhydroNAM(tetrapeptide), incorporated the anhydroNAM that is likely recognized better by the exolytic Lts. For this substrate, MltA as well as MltB and the SltB1–3 Lts were able to react to convert 100% of the substrate to NAGanhdroNAMtetrapeptide product. The soluble Lts, SltB1–3 of *P. aeruginosa* show the greatest activity in assays designed to study soluble proteins, as compared to solubilized membrane Lts [25]. These Lts were able to cleave NAG-NAM(pentapeptide)- NAG-NAM(pentapeptide) with specific activities of 0.4, 0.4 and 0.3 nanomoles of product/min/mg of protein respectively. Slt, the structural homolog of *E. coli* Slt70, showed no reaction with the synthetic peptidoglycan and slower turnover with the

**3.2 Structural studies of the soluble Lts, Slt, SLtB1 and SltB3 of** *P. aeruginosa*

X-ray crystal structures of Slt in its apo form as well as in complex with various synthetic PG substrates and reaction products demonstrated that this Lt has both exolytic and endolytic activity [23]. It is a donut shaped protein like Slt of *E. coli*. Notably, it is only after the binding of substrates that contain pentapeptide stems that it can exhibit endolytic activity due to a conformational change of the protein on substrate binding. A movie of this rearrangement is available in the supplementary material of reference [23]. Additional studies suggest protein–protein interac-

SltB1 [22] and SltB3 [24] have also been studied using x-ray crystallography. SLtB1 protein structures suggest that the protein forms a so-called "catenane" homodimeric structure in which the active sites face one another and are thus completely occluded. It is speculated that this soluble dimer may represent a form of activity regulation [22]. SltB3 is an exolytic enzyme with four distinct enzymatic domains within the donut shaped annular protein [24]. SltB3 can recognize PG substrates that are 4–20 sugars in

X-ray crystal structures of a solubilized MltF [21] show that this Lt binds a tetrapeptide stem of the substrate in an allosteric domain. Binding causes a large conformational change that leads to enzyme activation. In the kinetic studies, this solubilized membrane had very low activity with any of the 4 synthetic substrates or the *P. aeruginosa* sacculus. This raises some questions regarding the actual role of this Lt and whether the

Bulgecins were first described by Imato et al. in the 1980s [30, 31]. These natural analogs of GlcNAC-MurNAC are produced by various bacterial species including *Burkholderia mesoacidophila* and *Paraburkholderia acidophila* [32, 33], part of the *B. cepacia* complex. Bulgecins are produced together with sulfazecin, a monobactam antibiotic. Three different bulgecins are produced by these bacteria. Bulgecin A is produced in the highest amount and is the most active inhibitor of Lts (**Figure 3**,

length. These PG chains thread through the annular structure during catalysis.

conformational changes are relevant when the protein is membrane bound.

*DOI: http://dx.doi.org/10.5772/intechopen.85151*

tetrapeptide substrate: 0.1 nmoles/min/mg.

tions with inner membrane PBPs are also important [26].

**3.3 Structural studies of the endolytic Lt, MltF**

**4. Bulgecins as Lt inhibitors**

#### *Bulgecins as β-Lactam Enhancers Against Multidrug Resistant (MDR) Pseudomonas aeruginosa DOI: http://dx.doi.org/10.5772/intechopen.85151*

function relationships for *P. aeruginosa* Lts. Of interest is that each solubilized Lt enzyme could perform both endolytic and exolytic reactions on the PG substrates.

Using the simplest synthetic substrate, NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide), Lee et al. found that only MltB and the SltB1–3 Lts could recognize this substrate. A second substrate, a NAG-NAM(tetrapeptide)-NAGanhydroNAM(tetrapeptide), incorporated the anhydroNAM that is likely recognized better by the exolytic Lts. For this substrate, MltA as well as MltB and the SltB1–3 Lts were able to react to convert 100% of the substrate to NAGanhdroNAMtetrapeptide product. The soluble Lts, SltB1–3 of *P. aeruginosa* show the greatest activity in assays designed to study soluble proteins, as compared to solubilized membrane Lts [25]. These Lts were able to cleave NAG-NAM(pentapeptide)- NAG-NAM(pentapeptide) with specific activities of 0.4, 0.4 and 0.3 nanomoles of product/min/mg of protein respectively. Slt, the structural homolog of *E. coli* Slt70, showed no reaction with the synthetic peptidoglycan and slower turnover with the tetrapeptide substrate: 0.1 nmoles/min/mg.
