c-di-AMP Accumulation Regulates Growth, Metabolism, and Immunogenicity of Mycobacterium smegmatis

Abstract

Cyclic dimeric adenosine monophosphate (c-di-AMP) is a ubiquitous second messenger of bacteria involved in diverse physiological processes as well as host immune responses. MSMEG_2630 is a c-di-AMP phosphodiesterase (cnpB) of Mycobacterium smegmatis, which is homologous to Mycobacterium tuberculosis Rv2837c. In this study, cnpB-deleted (ΔcnpB), -complemented (ΔcnpB::C), and -overexpressed (ΔcnpB::O) strains of M. smegmatis were constructed to investigate the role of c-di-AMP in regulating mycobacterial physiology and immunogenicity. This study provides more precise evidence that elevated c-di-AMP level resulted in smaller colonies, shorter bacteria length, impaired growth, and inhibition of potassium transporter in M. smegmatis. This is the first study to report that elevated c-di-AMP level could inhibit biofilm formation and induce porphyrin accumulation in M. smegmatis by regulating associated gene expressions, which may have effects on drug resistance and virulence of mycobacterium. Moreover, the cnpB-deleted strain with an elevated c-di-AMP level could induce enhanced Th1 immune responses after M. tuberculosis infection. Further, the pathological changes and the bacteria burden in ΔcnpB group were comparable with the wild-type M. smegmatis group against M. tuberculosis venous infection in the mouse model. Our findings enhanced the understanding of the physiological role of c-di-AMP in mycobacterium, and M. smegmatis cnpB-deleted strain with elevated c-di-AMP level showed the potential for a vaccine against tuberculosis.

Keywords: M. tuberculosis; Mycobacterium smegmatis; c-di-AMP; immunogenicity; physiology.

FIGURE 1

Deletion of cnpB led to elevated bacterial c-di-AMP level. (A) Schematic diagram showing the cnpB knockout strategy in M. smegmatis. (B) PCR verification for the deletion of cnpB in M. smegmatis. disA (MSMEG_6080) was used as a reference gene. The templates were wild-type strain of M. smegmatis (lane 1), cnpB mutant (lane 2), and negative control without template (lane 3), respectively. (C) CnpB expression was determined using western blot with bacterial lysates of wild-type (M. smegmatis, Ms), cnpB mutant (ΔcnpB), cnpB-complemented strain in ΔcnpB (ΔcnpB::C), and cnpB-overexpressed strain in ΔcnpB (ΔcnpB::O), respectively. Pyridoxamine 5′-phosphate oxidase (PdxH, MSMEG_5675) was used as a loading control. (D) The levels of c-di-AMP in each strain were assayed by HPLC. ^**p < 0.01.

FIGURE 2

Determination of bacterial colony morphologies and bacterial lengths. (A) Colony morphologies on 7H10 + OADC plates after 8-day culture at 37°C. The black rectangle around each colony is 10 mm × 10 mm. (B) The measurement of colony diameter in panel (A) (40–50 colonies). (C) Observation of the indicated bacterial strains with a transmission electron microscope after culturing in 7H9 + OADC medium for 3 days. (D) The length of bacterial cells in panel (C) was measured (about 150 bacteria). *p < 0.05, ^***p < 0.001.

FIGURE 3

Detection of bacteria growth in liquid media (180 rpm). Each strain was inoculated at 2.5 × 10⁶CFU/ml in media of 7H9 (A), 7H9 + OADC (C), Sauton’s (E), and Sauton’s + OADC (G). Bacteria were monitored at OD₆₀₀ (A,C,E,G) and corresponding CFUs (B,D,F,H) were numbered at indicated time points, respectively. The growth curves were drawn with the results of three independent experiments with three duplicate cultures. *p < 0.05, ^**p < 0.01, ^***p < 0.001.

FIGURE 4

Biofilm formation and biomass quantification. Biomass was quantified by a crystal violet assay at 48 (A), 72 (B), and 120 h (C). The corresponding biofilm images for each strain are shown below each histogram. *p < 0.05, ^***p < 0.001, ^****p < 0.0001.

FIGURE 5

Pigment produced by ΔcnpB in long-term stationary culture. (A) The appearance of each strain was cultured statically for 7, 14, 21 and 60 weeks. Images were captured after gentle mixing. (B) At 14-week incubation, the bacterial pellets and supernatants were separated by centrifugation. (C) Observation of wild-type and ΔcnpB supernatants at 14 weeks. Next, ΔcnpB supernatant was salted-out by saturated ammonium sulfate [(NH₄)₂SO₄] and protein was collected by centrifugation. Then, protein precipitation by salting-out was redissolved by PBS. (D) Autofluorescence of 3 days, 14 weeks, and 60 weeks of bacterial cells were monitored by the flow cytometer (FL3, excitation 488 nm with a 610LP emission filter). (E) Absorption spectra of 14-week supernatants were recorded by full-wavelength (100–1,000 nm) scanning. (F) Fluorescence measurements of 14-week supernatants were analyzed at the excitation wavelength (λ_excitation) 400 nm.

FIGURE 6

Transcriptional profiles and genes expression of ΔcnpB. Strains were inoculated in 7H9 + OADC medium and cultivated to the late logarithmic stage (3 d) and bacteria pellets were collected for RNA extraction. Differentially expressed genes (DEGs) were screened by RNA-seq with both false discovery rate (FDR) < 0.05 and | log2FC| > 1. KEGG pathway enrichment is associated with DEGs that are repressed (A) or induced (B) in ΔcnpB compared with wild-type M. smegmatis strain. Count means the number of differential gene reads enriched to indicated terms (A,B). (C) The transcription levels of indicated genes were verified by real-time quantitative PCR. *p < 0.05.

FIGURE 7

Immune responses induced by ΔcnpB in mice. (A) The immunization and M. tuberculosis infection strategy scheme. Briefly, 6- to 8-week-old female BALB/c mice were immunized subcutaneously (s.c) with PBS (naïve group) and 10⁷ CFU of wild-type M. smegmatis and ΔcnpB strains, respectively. Mice were boosted two times at 2-week intervals. Then, 4 weeks after the last immunization, mice were infected intravenously (i.v) with 5 × 10⁴ CFU of M. tuberculosis H37Ra. (B) Specific IgG levels against M. smegmatis protein and CnpB in sera were assayed by ELISA, and dilutions were 1:200 and 1:400, respectively. (C) Splenocyte proliferation of immunized mice stimulated by M. smegmatis protein extract (25 μg/ml) or CnpB (5 μg/ml) in vitro. (D–F) Splenocytes of mice were stimulated with M. smegmatis protein extract (25 μg/ml) for 72 h in vitro, and cytokine productions of IFN-γ (D), IL-2 (E), and IL-10 (F) in the supernatant were detected using ELISA. *p < 0.05, ^**p < 0.01, ^***p < 0.001.

FIGURE 8

Immune responses induced by ΔcnpB post-M. tuberculosis infection and protection efficiency. (A) Specific IgG level against M. tuberculosis protein in sera (1:200) of immunized mice after M. tuberculosis H37Ra infection. (B–D) Splenocytes of mice were stimulated with H37Ra protein extracts (25 μg/ml) for 72 h in vitro, and cytokine productions of IFN-γ (B), IL-2 (C), and IL-10 (D) in the supernatant were detected using ELISA. (E) H&E-stained lung sections of each group of mice at 8 weeks post-M. tuberculosis infection. Gross pathology changes were observed by microscope (10 × 40). (F) At 8 weeks post-M. tuberculosis infection, bacterial burdens in the mice lungs and spleens were counted by plating on 7H10 plates. *p < 0.05, ^**p < 0.01, ^***p < 0.001.