Mycobacterium tuberculosis FadD18 Promotes Proinflammatory Cytokine Secretion to Inhibit the Intracellular Survival of Bacillus Calmette-Guérin

Abstract

Mycobacterium tuberculosis causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a Bacillus Calmette-Guérin (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive fadD18 gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that fadD18 expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.

Keywords: FadD18; Mycobacterium tuberculosis; adhesin; inflammation; intracellular survival; invasion.

Figure 1

Evaluation of invasion and adhesion ability of BCG. An MOI of 10:1 was used for infecting A549 cells with BCG strains at 37 °C for 1 h for the invasion assay or 4 °C for 30 min for the adhesion assay. The CFU assay was used to quantify the number of intracellular BCG mutants. (a) The workflow for BCG transposon mutant library construction and screening. (b) A comparative analysis was undertaken to evaluate the invasive potential of different BCG mutants, with each mutant strain being assessed against the wild-type BCG in all tests. (c) Invasion and (d) adhesion of A549 cells by both the wild-type (BCG) and B2909 mutants. Data are presented as means ± SEMs, with significance denoted by ** for p < 0.01 and *** for p < 0.001. Each sample was assessed in triplicate.

Figure 2

Bioinformatic analysis of FadD18. (a) Type and number of amino acids of FadD18. The ProtParam web server was used to determine the grand average of hydropathicity index (GRAVY) values and the aliphatic index of the FadD18. (b) TMHHM2.0 was used to predict transmembrane domains. (c) Sequence alignment of FadD18 and FadD19. An alignment of protein homologs was constructed with Clustal Omega and ESPript 3.0. Red represents identity. (d) Domains and conserved motifs identified in FadD18 and homologs. The conserved domain in FadD18 and its homologs was analyzed using the NCBI CDD search tool, while the MEME suite motif search tool was utilized to identify conserved motifs in these protein sequences.

Figure 3

Growth rate and morphology of B2909 strain. Morphology and area of single colonies of wild-type BCG (BCG) (a), B2909 (b), and B2909 complement (B2909C) (c) BCG strains. (d) Area of colonies of BCG, B2909, and B2909C BCG strains. *** p < 0.001.

Figure 4

Growth curve and survival ability of BCG strains. (a) The growth curves of the BCG, B2909, and B2909C strains were evaluated in 7H9 liquid medium at 37 °C by monitoring the optical density at 600 nm every three days. (b) THP-1 cells were infected with BCG, B2909, and B2909C strains at an MOI of 10:1 for 4 h. Following infection, cells were washed with PBS three times and then maintained in fresh media. Cell lysis was carried out 0, 24, 48, or 75 h PI, and CFUs of intracellular bacteria were quantified by plating cell lysates on 7H11 agar plates, followed by a 3-week incubation period for colony enumeration. Data are expressed as means ± standard errors of the means. Significance was determined as *** p < 0.001.

Figure 5

Expression of proinflammatory cytokines in THP-1 macrophages. Subsequently, macrophages were harvested at 12 and 24 h. The mRNA expression levels of IL-6 (a), IL-1β (b), and TNF-α (c) were quantified by RT-qPCR. The protein concentrations of IL-6 (d), IL-1β (e), and TNF-α (f) were measured using ELISA. The mRNA levels of cytokines were standardized relative to the levels of β-actin mRNA. Data are presented as means ± SEMs. Significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

FadD18 significantly augmented the activation of NF-κB and MAPK signaling pathways induced by BCG. (a) THP-1 macrophages were treated with BCG and mutant strain B2909 at an MOI of 10:1 for a specific infection time. Cells were then lysed on ice for 30 min and analyzed via Western blotting to measure the levels of total and phosphorylated forms of p65, p38, JNK, and ERK1/2 proteins. (b–e) The gray-scale values of p-p65/p65 (b), p-p38/p38 (c), p-JNK/JNK (d), and p-ERK/ERK (e) were determined. Data are presented as means ± SEMs. Significance is indicated as ns p > 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

Proposed model of FadD18 interaction with host cells. Schematic of the proposed interaction of FadD18 with host cells, showing the enhancement of mycobacteria’s adhesion and invasion capabilities, as well as the promotion of proinflammatory cytokine production through the NF-κB and MAPK signaling pathways.