The Diadenylate Cyclase CdaA Is Critical for Borrelia turicatae Virulence and Physiology

Abstract

Relapsing fever (RF), caused by spirochetes of the genus Borrelia, is a globally distributed, vector-borne disease with high prevalence in developing countries. To date, signaling pathways required for infection and virulence of RF Borrelia spirochetes are unknown. Cyclic di-AMP (c-di-AMP), synthesized by diadenylate cyclases (DACs), is a second messenger predominantly found in Gram-positive organisms that is linked to virulence and essential physiological processes. Although Borrelia is Gram-negative, it encodes one DAC (CdaA), and its importance remains undefined. To investigate the contribution of c-di-AMP signaling in the RF bacterium, Borrelia turicatae, a cdaA mutant was generated. The mutant was significantly attenuated during murine infection, and genetic complementation reversed this phenotype. Because c-di-AMP is essential for viability in many bacteria, whole genome sequencing was performed on cdaA mutants, and single nucleotide polymorphisms identified potential suppressor mutations. Additionally, conditional mutation of cdaA confirmed that CdaA is important for normal growth and physiology. Interestingly, mutation of cdaA did not affect expression of homologs of virulence regulators whose levels are impacted by c-di-AMP signaling in the Lyme disease bacterium, Borrelia burgdorferi. Finally, the cdaA mutant had a significant growth defect when grown with salts, at decreased osmolarity, and without pyruvate. While the salt treatment phenotype was not reversed by genetic complementation, possibly due to suppressor mutations, growth defects at decreased osmolarity and in media lacking pyruvate could be attributed directly to cdaA inactivation. Overall, these results indicate CdaA is critical for B. turicatae pathogenesis and link c-di-AMP to osmoregulation and central metabolism in RF spirochetes.

Structural basis for c-di-AMP–dependent regulation of the bacterial stringent response by receptor protein DarB

“…Loss of the c-di-AMP synthesizing enzymes is lethal to the bacterial cell ( 7 ). Since its discovery in 2008, numerous studies have reported the presence of c-di-AMP in a wide range of different bacterial species, of which several are well-known human pathogens, for example, Listeria monocytogenes , Mycobacterium tuberculosis , Borrelia turicatea , Enterococcus faecium , Staphylococcus aureus , and Streptococcus pneumoniae ( 22 , 23 , 24 ). With the exception of M. tuberculosis , these bacteria use the diadenylate cyclase CdaA as the sole enzyme responsible for c-di-AMP synthesis.…”

InvL, an Invasin-Like Adhesin, Is a Type II Secretion System Substrate Required for Acinetobacter baumannii Uropathogenesis

“…Cells were then lysed using a CF1 cell disrupter (Constant Systems Ltd., Daventry, UK). Following cell lysis, recombinant InvL was purified from the insoluble fraction by Ni-nitrilotriacetic acid affinity chromatography as previously described ( 107 ). Purified protein was then used by Antibody Research Corporation (St. Peters, MO) to generate polyclonal rabbit antibody.…”

“…To generate soluble recombinant InvL for ELISA (described below), InvL lacking the N-terminal signal sequence (as determined by SignalP 5.0) was expressed with a C-terminal His 10 tag from pET-22b(+):: invL-his 10 (-SS) in Rosetta-gami 2(DE3) cells by addition of 1 mM isopropyl-β- d -thiogalactopyranoside for 3 h at 37°C ( 41 ). Cells were then lysed as described above, and recombinant InvL was purified from the soluble fraction using the procedure previously described with the exception that buffers were not supplemented with 0.3% N -lauryl-sarcosine ( 107 ).…”

InvL, an invasin-like adhesin, is a type II secretion system substrate required for Acinetobacter baumannii uropathogenesis

“…To generate soluble recombinant InvL for ELISAs (see below), InvL lacking the N-terminal signal sequence (as determined by SignalP 5.0) was expressed with a C-terminal His10 tag from pET-22b(+)::invL-his10(-SS) in Rosetta-gami 2(DE3) cells by addition of 1 mM isopropyl-β-d-thiogalactopyranoside for 3 hrs at 37°C (41). Cells were then lysed as above, and recombinant InvL was purified from the soluble fraction using the same procedure as previously described with the exception that buffers were not supplemented with 0.3% N-lauryl-sarcosine (104).…”

“…Cells were then lysed using a CF1 cell disrupter (Constant Systems Ltd, Daventry, UK). Following cell lysis, recombinant InvL was purified from the insoluble fraction by Ni-NTA affinity chromatography as previously described (104). Purified protein was then used by Antibody Research Corporation (St. Peters, MO) to generate polyclonal rabbit antibody.…”