Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air

Abstract

Bacterial b-class carbonic anhydrases (CAs) are zinc metalloenzymes catalysing reversible hydration of CO 2. They maintain the intracellular balance of CO 2 /bicarbonate required for biosynthetic reactions and represent a new group of antimicrobial drug targets. Genome sequence analysis of Pseudomonas aeruginosa PAO1, an opportunistic human pathogen causing life threatening infections, identified three genes, PAO102, PA2053 and PA4676, encoding putative b-CAs that share 28-45 % amino acid sequence identity and belong to clades A and B. The genes are conserved among all sequenced pseudomonads. The CAs were cloned, heterologously expressed and purified. Metal and enzymic analyses confirmed that the proteins contain Zn 2+ and catalyse hydration of CO 2 to bicarbonate. PAO102 (psCA1) was 19-26-fold more active, and together with PA2053 (psCA2) showed CA activity at both pH 7.5 and 8.3, whereas PA4676 (psCA3) was active only at pH 8.3. Circular dichroism spectroscopy suggested that psCA2 and psCA3 undergo pH-dependent structural changes. Taken together, the data suggest that psCA1 may belong to type I and psCA3 to type II b-CAs. Immunoblot analysis showed that all three CAs are expressed in PAO1 cells when grown in ambient air and at 5 % CO 2 ; psCA1 appeared more abundant under both conditions. Growth studies of transposon mutants showed that the disruption of psCA1 impaired PAO1 growth in ambient air and caused a minor defect at high CO 2 . Thus, psCA1 contributes to the adaptation of P. aeruginosa to low CO 2 conditions and will be further studied for its role in virulence and as a potential antimicrobial drug target in this organism.

Structure and inhibition studies of a type II beta-carbonic anhydrase psCA3 from Pseudomonas aeruginosa

“…16 The fundamental dimer and the active site at dimer interface is shown in Figure 2A and B respectively. b-CAs possess a b-sheet core comprised of 4 or 5 strands with four or more a-helices surrounding this core 4,9 (Fig.…”

“…22 Cloning, protein expression and purification were performed as described previously in. 16 A pET15b plasmid construct containing the psCA3 gene PCR-amplified from the PAO1 genomic DNA, was transformed into E. coli Tuner BL21 (DE3) cells for the production of a 6Â His fusion protein. The cells were grown in Luria-Bertani (LB) broth containing 100 mg ml À1 ampicillin and 0.05 mM ZnSO 4 at 310 K. At an optical density (Ab 600 ) of $0.6, protein expression was induced for 3 h by the addition of isopropyl b-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM in the presence of 0.5 mM ZnSO 4 (to prevent Zn 2+ depletion).…”

“…16 Though, the functional roles of the psCAs are not fully characterized, by using transposon mutants with individually disrupted psCAs, psCA1 was shown to play a role in PAO1 growth at ambient CO 2 levels, similar to the role played by the b-CAs PCA in Streptococcus pneumoniae and Can in Escherichia coli. 8,16,17 Furthermore, the most recent studies of the group suggest that psCA2 and psCA3 are involved in the formation of extracellular calcium deposits (Lotlikar et.al., in preparation). This ability of the organism may increase its virulence by contributing to soft tissue calcification or stone formation during bacterial infections which was observed, for example, in late stages of cystic fibrosis or primary ciliary dyskinesia.…”

“…16 A multiple sequence alignment of psCA1, psCA2 and psCA3 shows that these enzymes share a 27-49% amino acid sequence identity amongst themselves. When aligned with the sequences of the b-CA whose crystal structures are available in the Protein Data Bank (PDB), psCA1 shares the highest sequence identity-32% with P. sativum PSCA (PDB ID: 1EKJ) a type I b-CAs, while psCA2 and psCA3 show 32% and 53% sequence identity to Salmonella enterica, SECCA, (PDB ID: 3QY1) and E. coli, ECCA, (PDB ID: 1I6P), respectively, both type II b-CAs ( Supplementary Fig.…”

Carbon dioxide-sensing in organisms and its implications for human disease

“…P. aeruginosa infection is a major clinical challenge in a hospital and immunocompromised setting. Recently three functionally active carbonic anhydrase isoforms were identified in P. aeruginosa PAO1 with the most abundant and active (psCA1) playing an important role in PAO1 survival at high CO 2 [32]. Thus, a better understanding of bacterial adaptation and survival across a range of CO 2 environments may be of importance in the development of future antimicrobial therapies.…”