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.IMPORTANCEStreptococcus 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.

Keywords: Streptococcus bovis; Streptococcus gallolyticus; biofilm; c-di-AMP; cell adherence.

FIG 1

GALLO_1455 and GALLO_2236 encode c-di-AMP diadenylate cyclase and phosphodiesterase, respectively. (A and B) Gene locations and the domain architecture of GALLO_1455 (A) and GALLO_2236 (B) resemble the typical properties of DacA and GdpP, respectively. PAS, GGDEF, and DHH/DHHA1 domains were deleted to generate S. gallolyticus subsp. gallolyticus UCN34 ΔgdpP. Sixty-nine base pairs of the gene at the 3′ end of gdpP was left undeleted to preserve the ribosomal binding site of rplI. The arrow at the upstream of GALLO_2236 marks the putative transcriptional start site of GALLO_2236 based on in silico algorithm-based promoter prediction. (C) Liquid chromatograph-mass spectrometry (LC-MS) quantification of the intracellular concentration of c-di-AMP in S. gallolyticus subsp. gallolyticus UCN34, the ΔgdpP mutant, and the ΔgdpP/pgdpP complemented strain. Error bars represent the standard deviation of the measurements from three samples. Ordinary one-way analysis of variance (ANOVA test): **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

FIG 2

Phenotypic changes associated with an increased intracellular c-di-AMP levels resulted from the deletion of gdpP. (A) Representative anaerobic growth kinetics of S. gallolyticus subsp. gallolyticus UCN34, the ΔgdpP mutant, and the ΔgdpP/pgdpP complemented strain. Initial inoculum was prepared from log-phase culture adjusted to approximately 3 × 10⁷ CFU/ml. Growth, reflected in optical density, was measured at 600 nm (OD₆₀₀) at the indicated time point. The arrow indicates the sample collection time point for biofilm assay and the RNA-seq experiment. Error bars represent the standard deviation of the measurements from three samples. (B) Representative phase-contrast microscopy images on the stationary-phase culture of the S. gallolyticus subsp. gallolyticus UCN34, the ΔgdpP mutant, and the ΔgdpP/pgdpP complemented strain. Images were acquired with Carl Zeiss Axio Observer.Z1 inverted wide-field microscope fitted with 100×/1.3-numerical-aperture (NA) objective oil lens. Images were processed using Imaris version 8.2. Scale bars = 3 μm. (C) Cell area measurement of 300 imaged cells from three independent experiments using ImageJ software. Error bars represent the standard deviation of the 300 measurements. Kruskal-Wallis test: ****, P ≤ 0.0001; ns, P > 0.05. (D) Representative images of 5 μl of S. gallolyticus subsp. gallolyticus log-phase culture adjusted to approximately 3 × 10⁷ CFU/ml spotted onto BHI agar and BHI agar supplemented with 0.4 M NaCl. (E) MICs of ampicillin and penicillin G against S. gallolyticus subsp. gallolyticus UCN34, the ΔgdpP mutant, and the ΔgdpP/pgdpP complemented strain. MIC was determined based on the optical density reading at 600 nm on a Tecan microplate reader, Infinite M200Pro.

FIG 3

Accumulation of intracellular c-di-AMP levels inhibits S. gallolyticus subsp. gallolyticus biofilm formation. (A) Biofilm quantification using conventional microtiter plate biofilm assay. Error bars represent the standard deviation from 12 samples from 3 independent experiments. Ordinary one-way ANOVA test: ****, P ≤ 0.0001. (B) Representative biofilm images of S. gallolyticus subsp. gallolyticus UCN34, the ΔgdpP mutant, and the ΔgdpP/pgdpP complemented strain acquired using Carl Zeiss confocal laser scanning microscope LSM780 fitted with Plan Apochromat 100×/1.4-NA oil objective lens, with excitation at 488 nm. Scale bars = 10 μm.

FIG 4

Increased intracellular c-di-AMP levels attenuate the ability of S. gallolyticus subsp. gallolyticus to adhere and to form cell aggregates on human colonic epithelial cells. (A) Quantification of the cell number of S. gallolyticus subsp. gallolyticus attached on a monolayer HT-29 human colorectal adenocarcinoma cells. Error bars represent the standard deviation of 9 samples from 3 independent experiments. Kruskal-Wallis test: **, P ≤ 0.01; ***, P ≤ 0.001; ns, P > 0.05. (B) Representative immunofluorescence images of the S. gallolyticus subsp. gallolyticus adhered on a monolayer of HT-29 human colorectal adenocarcinoma cells. Green, S. gallolyticus subsp. gallolyticus labeled with primary antibody rabbit UCN34 and secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488. Blue, Hoechst 33342-stained DNA of the HT-29 cells. Scale bars = 10 μm (top) and 70 μm (bottom).

FIG 5

c-di-AMP regulates the gallocin production and Pil3 biosynthesis in S. gallolyticus subsp. gallolyticus. (A) Gallocin production by S. gallolyticus subsp. gallolyticus UCN34, the ΔgdpP mutant, and the ΔgdpP/pgdpP complemented strain under anaerobic conditions. Five microliters of S. gallolyticus subsp. gallolyticus UCN34, the ΔgdpP mutant, and the ΔgdpP/pgdpP complemented strain log-phase culture adjusted to approximately 3 × 10⁷ CFU/ml was spotted onto BHI agar flooded with Enterococcus faecalis OG1RF or S. gallolyticus subsp. macedonicus. Zone of clearance reflects the growth inhibition of E. faecalis or S. gallolyticus subsp. macedonicus. The strain deficient in producing the gallocin Δblp mutant was used as the negative control. (B) Western blot analysis of the cell wall proteins from S. gallolyticus subsp. gallolyticus UCN34, the ΔgdpP mutant, and the ΔgdpP/pgdpP complemented strain. Equal amounts of the cell wall proteins were loaded (right) and probed with specific polyclonal antibodies against Pil3B (left). Cell wall proteins of the S. gallolyticus subsp. gallolyticus UCN34 Δpil3 mutant was used as a negative control. Theoretical positions of Pil3B monomers, based on the molecular weights, are indicated (m), and high-molecular-weight species corresponding to pilus polymers are labeled (p).