Pseudomonas aeruginosa β-carbonic anhydrase, psCA1, is required for calcium deposition and contributes to virulence

• Published in: Cell calcium (Edinburgh) Vol. 84; p. 102080
• Main Authors: Lotlikar, Shalaka R., Kayastha, Biraj B., Vullo, Daniela, Khanam, Sharmily S., Braga, Reygan E., Murray, Akilah B., McKenna, Robert, Supuran, Claudiu T., Patrauchan, Marianna A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 01.12.2019
• Subjects: Calcium carbonate; Carbonic anhydrases; Gene-deletion mutants; qRT-PCR; Transmission electron microscopy; Gene-deletion mutants; Transmission electron microscopy; Carbonic anhydrases; Calcium carbonate; qRT-PCR
• ISSN: 0143-4160; 1532-1991
• DOI: 10.1016/j.ceca.2019.102080

[Display omitted] •This is the first evidence that a β-class carbonic anhydrase (CA), psCA1, plays a major role in the formation of calcium deposits in a human pathogen Pseudomonas aeruginosa.•The expression of psCA1 is increased in the presence of elevated calcium levels. The deletion of psCA1 impaired virulence of the pathogen.•In-silico modeling of three PAO1 β-class CAs identified four amino acids that differ in psCA1 compared to psCA2, and psCA3, and these differences may play a role in catalytic rate and thus calcium deposition.•Among a series of inhibitors tested against the recombinant psCA1, aminobenzene sulfonamide (ABS) and acetazolamide (AAZ) inhibited the enzyme at nano-molar levels. Their addition significantly reduced calcium deposition.•Overall, this is the first report that inhibiting the CA activity in P. aeruginosa significantly reduces its ability to deposit calcium, which may be used to control virulence of the pathogen. Calcification of soft tissue leads to serious diseases and has been associated with bacterial chronic infections. However, the origin and the molecular mechanisms of calcification remain unclear. Here we hypothesized that a human pathogen Pseudomonas aeruginosa deposits extracellular calcium, a process requiring carbonic anhydrases (CAs). Transmission electron microscopy confirmed the formation of 0.1-0.2 μm deposits by P. aeruginosa PAO1 growing at 5 mM CaCl2, and X-ray elemental analysis confirmed they contain calcium. Quantitative analysis of deposited calcium showed that PAO1 deposits 0.35 and 0.75 mM calcium/mg protein when grown at 5 mM and 10 mM CaCl2, correspondingly. Fluorescent microscopy indicated that deposition initiates at the cell surface. We have previously characterized three PAO1 β-class CAs: psCA1, psCA2, and psCA3 that hydrate CO2 to HCO3−, among which psCA1 showed the highest catalytic activity (Lotlikar et. al. 2013). According to immunoblot and RT-qPCR, growth at elevated calcium levels increases the expression of psCA1. Analyses of the deletion mutants lacking one, two or all three psCA genes, determined that psCA1 plays a major role in calcium deposition and contributes to the pathogen’s virulence. In-silico modeling of the PAO1 β-class CAs identified four amino acids that differ in psCA1 compared to psCA2, and psCA3 (T59, A61A, A101, and A108), and these differences may play a role in catalytic rate and thus calcium deposition. A series of inhibitors were tested against the recombinant psCA1, among which aminobenzene sulfonamide (ABS) and acetazolamide (AAZ), which inhibited psCA1 catalytic activity with KIs of 19 nM and 37 nM, correspondingly. The addition of ABS and AAZ to growing PAO1 reduced calcium deposition by 41 and 78, respectively. Hence, for the first time, we showed that the β-CA psCA1 in P. aeruginosa contributes to virulence likely by enabling calcium salt deposition, which can be partially controlled by inhibiting its catalytic activity.