**4. Bulgecins as Lt inhibitors**

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**, Bulgecin A).

*Pseudomonas aeruginosa - An Armory Within*

Lt knockouts were unsuccessful.

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

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

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.

As previously indicated, *P. aeruginosa* possesses 11Lts: MltA, MltB/Slt35, MltD,

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

*(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* 

MltF, MltF2, MltG, RlpA, Slt, SltB1 (SltB), SltB2 (SltG), and SltB3 (SltH).

membrane, e.g., RlpA (see below) and likely have distinctive roles [29].

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

**74**

**Figure 2.**

*Pseudomonas aeruginosa in complex with Bulgecin A.*

#### **Figure 3.**

*Bulgecin A, the most active of the bulgecins of Paraburkholderia acidophila and Burkholderia mesoacidophila. The pyrrolidine ring (right side of the molecule) and the N-acetylglucosamine potion (GlcNAC) (left side other molecule) are features of Bulgecin A transition state structure.*

Early research by Takeda Pharmaceuticals Japan led to the natural product isolation and purification of the bulgecins [30, 31]. It was discovered that when Bulgecin A was paired with a third generation cephalosporin, cefmenoxime, which targets PBP 3 of Enterobacteriaceae, large bulges were formed in the bacterial cell wall leading to osmotic lysis of the bacteria [30, 31]. Subsequently, investigators discovered the soluble Lt of *E. coli* and solved crystal structures of SltE in complex with Bulgecin A [34]. Through kinetic experiments, it was determined that Bulgecin A was a noncompetitive inhibitor of SltE with an IC50 of 0.5 μM. [35]. While Bulgecin A appeared to be a potent inhibitor of Lts in pathogenic Enterobacteriaceae and led to bacterial killing when paired with β-lactams affecting PBP3 particularly, development of the drug was halted for unknown reasons. Over the next decade, more advanced generation cephalosporins, as well as β-lactam-β-lactamase inhibitor combinations, carbapenems and fluoroquinolones were introduced into the clinic to address the growing problem of Gram negative resistance. Recently a natural product synthesis of the bulgecins was reported for the first time by Tomoshige et al*.* [36] prompting renewed interest in the use of Bulgecin A as an antimicrobial adjuvant, and possible drug optimization via medicinal chemistry.

Since the original discovery of the bulgecins and Slt in *E coli*, Lts have been characterized in many additional bacteria including *P. aeruginosa, Acinetobacter baumannii, Helicobacter pylori, Neisseria meningitides* and *Campylobacter jejuni* [21–25, 28, 34, 37–50]. In general, these organisms have many Lt enzymes with different functions, including endolytic (within strand) and exolytic (end of strand) cleavage of peptidoglycan. Many of these enzymes including those of *P. aeruginosa* have been expressed for biochemical assays of function, and inhibition by Bulgecin A. Crystal structures of many of the Lts of these organisms have also been obtained, some with substrates or Bulgecin A in the active site (**Figure 2**).

A recent publication shows that while *P. aeruginosa* possesses 11 known Lts, three appear to be the main targets of inhibition by Bulgecin A [27]. This work is discussed further below.

### **5. Microbiological effects of Bulgecin A**

Bulgecin A in combination with cefmenoxime and other β-lactams has been studied against Enterobacteriaceae and reported in the original studies by Takeda Pharamceuticals [30, 31]. Later investigators studied Bulgecin A in combination with ampicillin in mouse models of *Helicobacter pylori* infection and found that the combination was effective in eradicating the organism, and Bulgecin A did not

**77**

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

appear to have specific toxicity in mice [51]. These investigators also studied Bulgecin A with *Neisseria gonorrhea* and *N. meningitides* strains that were resistant to penicillin and amoxicillin [48]. For strains with higher penicillin MICs not due to the presence of TEM-1 β-lactamase, Bulgecin A at concentrations of 19 mg/L, reduced the MICs from 0.5 to 0.09 mg/L for penicillin G, and 0.75 to 0.4 mg/L for amoxicillin.

*L1 MBL (left) of Stenotrophomonas maltophilia with Bulgecin A; Bulgecin A sulphonates (yellow moieties,* 

Other investigators examined the effect of Bulgecin A as a metallo-β-lactamase inhibitor using L1 MBL of *Stenotrophomonas maltophila* as a model B1 (di Zn2+) MBL enzyme (**Figure 4**). Simm et al*.* determined that the KI for Bulgecin A was 150uM [52]. Later, our group investigated inhibition of VIM-1 using a Bulgecin A preparation from *B. mesoacidophila* and found that it also acted as an inhibitor of a second B1 MBL enzyme that is commonly found in *P. aeruginosa* in Europe, Asia and

Our group tested the Bulgecin A extracts from *B. mesoacidophila* against a variety of carbapenem resistant *P. aeruginosa* and *Acinetobacter baumannii* isolates with differing resistance mechanisms [53]. Although these were impure preparations, we found that small amounts were able to inhibit growth of these clinical isolates when combined with typical amounts of carbapenems to which the bacteria were otherwise resistant. The Bulgecin A-meropenem combinations proved effective whether carbapenem resistance was due to the presence of MBLs (VIM-1), hyperproduction of PDC (Amp C enzyme of *P. aeruginosa* in combination with OMP loss) or efflux. Tomoshige et al*.* using synthetic Bulgecin A were able to demonstrate bulge formation in *P. aeruginosa* PA01 as well as lysis in the presence of ceftazidime [36].

**6. Slt, MltD and MltG are the main targets of Bulgecin A inhibition and potentiation of β-lactams that inhibit PBP2 and 3 in** *P. aeruginosa*

Previously it was demonstrated that bulgecin A potentiated the bulge formation and lysis of *P. aeruginosa* in the presence of ceftazidime and meropenem [36] in a swarm assay [54]. Recently, Dik et al. [27] used individual transposon knockouts of. Lts in a susceptible *P. aeruginosa* strain, PA01 and further engineered a green fluorescent protein (GFP) gene into the bacteria. The various Lt knockout strains were exposed to ceftazidime, an inhibitor of PBP3 in *P. aeruginosa* and meropenem, an inhibitor of PBP2,3 and 4 [55] on agar medium containing propidium iodide. Bulge formation and bacterial cell lysis were monitored as a function of time by monitoring green fluorescence from viable cells, and red fluorescence during cell lysis, the red fluorescence arising from bacterial DNA interacting with the propidium iodide in the medium. In the presence of ceftazidime, the Slt and MltD knockouts formed bulges and showed lysis. The Slt knockout demonstrated significant bulge formation within 6 hours of exposure to ceftazidime, and lysis within

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

*right) interacting with the ZnII site and with Asp 14 of the L1 protein.*

Canada, and rarely in the US [53].

**Figure 4.**

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

**Figure 4.**

*Pseudomonas aeruginosa - An Armory Within*

*molecule) are features of Bulgecin A transition state structure.*

**Figure 3.**

Early research by Takeda Pharmaceuticals Japan led to the natural product isolation and purification of the bulgecins [30, 31]. It was discovered that when Bulgecin A was paired with a third generation cephalosporin, cefmenoxime, which targets PBP 3 of Enterobacteriaceae, large bulges were formed in the bacterial cell wall leading to osmotic lysis of the bacteria [30, 31]. Subsequently, investigators discovered the soluble Lt of *E. coli* and solved crystal structures of SltE in complex with Bulgecin A [34]. Through kinetic experiments, it was determined that Bulgecin A was a noncompetitive inhibitor of SltE with an IC50 of 0.5 μM. [35]. While Bulgecin A appeared to be a potent inhibitor of Lts in pathogenic Enterobacteriaceae and led to bacterial killing when paired with β-lactams affecting PBP3 particularly, development of the drug was halted for unknown reasons. Over the next decade, more advanced generation cephalosporins, as well as β-lactam-β-lactamase inhibitor combinations, carbapenems and fluoroquinolones were introduced into the clinic to address the growing problem of Gram negative resistance. Recently a natural product synthesis of the bulgecins was reported for the first time by Tomoshige et al*.* [36] prompting renewed interest in the use of Bulgecin A as an antimicrobial

*Bulgecin A, the most active of the bulgecins of Paraburkholderia acidophila and Burkholderia mesoacidophila. The pyrrolidine ring (right side of the molecule) and the N-acetylglucosamine potion (GlcNAC) (left side other* 

adjuvant, and possible drug optimization via medicinal chemistry.

some with substrates or Bulgecin A in the active site (**Figure 2**).

Since the original discovery of the bulgecins and Slt in *E coli*, Lts have been characterized in many additional bacteria including *P. aeruginosa, Acinetobacter baumannii, Helicobacter pylori, Neisseria meningitides* and *Campylobacter jejuni* [21–25, 28, 34, 37–50]. In general, these organisms have many Lt enzymes with different functions, including endolytic (within strand) and exolytic (end of strand) cleavage of peptidoglycan. Many of these enzymes including those of *P. aeruginosa* have been expressed for biochemical assays of function, and inhibition by Bulgecin A. Crystal structures of many of the Lts of these organisms have also been obtained,

A recent publication shows that while *P. aeruginosa* possesses 11 known Lts, three appear to be the main targets of inhibition by Bulgecin A [27]. This work is

Bulgecin A in combination with cefmenoxime and other β-lactams has been studied against Enterobacteriaceae and reported in the original studies by Takeda Pharamceuticals [30, 31]. Later investigators studied Bulgecin A in combination with ampicillin in mouse models of *Helicobacter pylori* infection and found that the combination was effective in eradicating the organism, and Bulgecin A did not

**76**

discussed further below.

**5. Microbiological effects of Bulgecin A**

*L1 MBL (left) of Stenotrophomonas maltophilia with Bulgecin A; Bulgecin A sulphonates (yellow moieties, right) interacting with the ZnII site and with Asp 14 of the L1 protein.*

appear to have specific toxicity in mice [51]. These investigators also studied Bulgecin A with *Neisseria gonorrhea* and *N. meningitides* strains that were resistant to penicillin and amoxicillin [48]. For strains with higher penicillin MICs not due to the presence of TEM-1 β-lactamase, Bulgecin A at concentrations of 19 mg/L, reduced the MICs from 0.5 to 0.09 mg/L for penicillin G, and 0.75 to 0.4 mg/L for amoxicillin.

Other investigators examined the effect of Bulgecin A as a metallo-β-lactamase inhibitor using L1 MBL of *Stenotrophomonas maltophila* as a model B1 (di Zn2+) MBL enzyme (**Figure 4**). Simm et al*.* determined that the KI for Bulgecin A was 150uM [52]. Later, our group investigated inhibition of VIM-1 using a Bulgecin A preparation from *B. mesoacidophila* and found that it also acted as an inhibitor of a second B1 MBL enzyme that is commonly found in *P. aeruginosa* in Europe, Asia and Canada, and rarely in the US [53].

Our group tested the Bulgecin A extracts from *B. mesoacidophila* against a variety of carbapenem resistant *P. aeruginosa* and *Acinetobacter baumannii* isolates with differing resistance mechanisms [53]. Although these were impure preparations, we found that small amounts were able to inhibit growth of these clinical isolates when combined with typical amounts of carbapenems to which the bacteria were otherwise resistant. The Bulgecin A-meropenem combinations proved effective whether carbapenem resistance was due to the presence of MBLs (VIM-1), hyperproduction of PDC (Amp C enzyme of *P. aeruginosa* in combination with OMP loss) or efflux. Tomoshige et al*.* using synthetic Bulgecin A were able to demonstrate bulge formation in *P. aeruginosa* PA01 as well as lysis in the presence of ceftazidime [36].
