Roles of three carbonic anhydrases in in vitro growth, pH homeostasis, metabolism, and intracellular survival of Neisseria gonorrhoeae

Abstract In many bacterial species, carbonic anhydrases (CAs) have been shown to play crucial roles in metabolism, cellular pH homeostasis, growth, and/or pathogenicity. Thus, these enzymes are considered as vital targets of drug development. We investigated the importance of CAs to growth, cellular metabolism, pH homeostasis, and intracellular survival of Neisseria gonorrhoeae. BLAST searches of N. gonorrhoeae genome sequence revealed the presence of three genes respectively homologous to αCA, βCA, and γCA genes of other bacteria. The putative αCA protein of N. gonorrhoeae was found to localize to the periplasm, whereas the other two proteins were predicted to reside in the cytoplasm. All three CAs of N. gonorrhoeae shared up to 99% identity with CAs of other bacteria. Three isogenic mutants were constructed by disrupting the αCA, βCA, and γCA genes. The mutants were complemented by reincorporating the disrupted CA genes into each of the mutants. All three CA mutants were similar to the parent N. gonorrhoeae in terms of their growth at 5% CO2. Nevertheless, unlike the parent or the other CA mutants, the ΔβCA mutant failed to grow at ambient air or 2% or 3% CO2, highlighting the importance of the βCA for CO2 hydration in decreased CO2 environments. The pH of the culture medium decreased during proliferation of N. gonorrhoeae cells, nevertheless, the least decline of pH was observed in the ΔβCA mutant culture. These findings suggest that N. gonorrhoeae prefers slightly acidic growth environment and βCA is important for pH homeostasis. Acetyl-CoA, which is a substrate for Malonyl-CoA biosynthesis in cell, was recovered in relatively increased amounts from the ΔαCA and ΔβCA mutant cells, underscoring a possible relationship between the αCA and βCA functions and cellular metabolism. Relative to the parent strain, the ΔαCA and ΔβCA mutants were recovered from infected ME-180 cells in decreased numbers, highlighting the importance of the αCA and βCA in intracellular survival and replication. Several other publications have reported αCA as an essential protein for the viability of N. gonorrhoeae. Since our findings on αCA were not in agreement with those published reports, we constructed an additional mutant of αCA gene by deleting the entire ORF of this gene. This second ΔαCA mutant was similar to our first ΔαCA mutant and the parent strain in terms of growth in ambient CO2 concentrations. Thus, we conclude that αCA is dispensable for the viability of N. gonorrhoeae. Unlike our ΔαCA or ΔβCA mutants, our ΔγCA mutant was not different from the parent in growth, metabolism, or intracellular survival. Overall, we conclude that αCA is dispensable for the viability, growth and pH homeostasis but is important for metabolism and intracellular survival of N. gonorrhoeae. The βCA is indispensable for the viability of N. gonorrhoeae, and also plays important roles in growth, pH homeostasis, metabolism, and intracellular survival. Contrary to the other two CAs, the γCA does not play detectable roles in growth, metabolism, pH dynamics, or intracellular survival.