Increased Intracellular Cyclic di-AMP Levels Sensitize Streptococcus gallolyticus subsp. gallolyticus to Osmotic Stress and Reduce Biofilm Formation and Adherence on Intestinal Cells

Abstract

Cyclic di-AMP is a recently identified second messenger exploited by a number of Gram-positive bacteria to regulate important biological processes. Here, we studied the phenotypic alterations induced by the increased intracellular c-di-AMP levels in Streptococcus gallolyticus, an opportunistic pathogen responsible for septicemia and endocarditis in the elderly. We report that an S. gallolyticus c-di-AMP phosphodiesterase gdpP knockout mutant, which displays a 1.5-fold higher intracellular c-di-AMP levels than the parental strain UCN34, is more sensitive to osmotic stress and is morphologically smaller than the parental strain. Unexpectedly, we found that a higher level of c-di-AMP reduced biofilm formation of S. gallolyticus on abiotic surfaces and reduced adherence and cell aggregation on human intestinal cells. A genome-wide transcriptomic analysis indicated that c-di-AMP regulates many biological processes in S. gallolyticus, including the expression of various ABC transporters and disease-associated genes encoding bacteriocin and Pil3 pilus. Complementation of the gdpP in-frame deletion mutant with a plasmid carrying gdpP in trans from its native promoter restored bacterial morphology, tolerance to osmotic stress, biofilm formation, adherence to intestinal cells, bacteriocin production, and Pil3 pilus expression. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in S. gallolyticus that may be important for S. gallolyticus pathogenesis. IMPORTANCE Streptococcus gallolyticus is an opportunistic pathogen responsible for septicemia and endocarditis in the elderly and is also strongly associated with colorectal cancer. S. gallolyticus can form biofilms, express specific pili to colonize the host tissues, and produce a specific bacteriocin allowing killing of commensal bacteria in the murine colon. Nevertheless, how the expression of these colonization factors is regulated remains largely unknown. Here, we show that c-di-AMP plays pleiotropic roles in S. gallolyticus, controlling the tolerance to osmotic stress, cell size, biofilm formation on abiotic surfaces, adherence and cell aggregation on human intestinal cells, expression of Pil3 pilus, and production of bacteriocin. This study indicates that c-di-AMP may constitute a key regulatory molecule for S. gallolyticus host colonization and pathogenesis.

Cyclic Di-adenosine Monophosphate Regulates Metabolism and Growth in the Oral Commensal Streptococcus mitis

“…Our success in creating a cdaA -deletion mutant was somewhat unexpected, since it has proven difficult to inactivate the sole diadenylate cyclase CdaA in several streptococci by conventional protocols. Whereas deletion of cdaA was possible in S. pyogenes [ 3 ] and S. mutans [ 45 , 46 ], all attempts to delete cdaA have been unsuccessful in S. pneumoniae [ 15 ] and S. gallolyticus [ 47 ]. In S. agalactiae , it was only possible to delete the chromosomal copy of cdaA in a strain carrying an inducible copy of cdaA on a plasmid [ 44 ].…”

Identification of the main glutamine and glutamate transporters in Staphylococcus aureus and their impact on c‐di‐AMP production

“…Previously, we found that inactivation of the main glycine betaine transporter OpuD (SAUSA300_1245) as well as the predicted amino acid transporter AlsT (SAUSA300_1252) allows an S. aureus dacA mutant to grow in rich medium in the absence of c‐di‐AMP (Zeden et al, ). In several other Firmicutes, including B. subtilis, Lactococcus lactis, Streptococcus pneumoniae, S. agalactiae, Streptococcus pyogenes and L. monocytogenes , inactivating mutations have also been identified in osmolyte and potassium transport systems that allow these bacteria to grow in the absence of c‐di‐AMP (Bai et al, ; Corrigan et al, ; Devaux et al, ; Fahmi et al, ; Gundlach, Commichau, et al, ; Gundlach, Herzberg, Hertel, et al, ; Pham et al, ; Pham & Turner, ; Quintana et al, ; Rocha et al, ; Teh et al, ; Whiteley et al, , ; Witte et al, ; Zarrella et al, ; Zeden et al, ) . This suggests that potassium and osmolyte transporters are more active in the absence of c‐di‐AMP, resulting in the accumulation of toxic levels of potassium and osmolytes in the cell.…”

Unique Roles for Streptococcus pneumoniae Phosphodiesterase 2 in Cyclic di-AMP Catabolism and Macrophage Responses

“…The present results with pneumococcus grown on solid media (a more biofilm-like condition) contrast with prior studies of these bacteria grown in a strictly planktonic liquid broth culture (26), suggesting that c-di-AMP metabolism varies across types of bacterial growth. This is plausible given the many roles c-di-AMP plays in bacterial physiology, including evidence that c-di-AMP itself helps determine planktonic vs. biofilm organization in streptococci (22,(35)(36)(37)(38). Some virulence factors, like pneumolysin which can induce macrophage cytotoxicity as observed here, are more strongly expressed in biofilm-like growth states of pneumococci (32,33).…”