Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages

Abstract

Legionella pneumophila, the causative agent of Legionnaire's disease, replicates in human alveolar macrophages to establish infection. There is no human-to-human transmission and the main source of infection is L. pneumophila biofilms established in air conditioners, water fountains, and hospital equipments. The biofilm structure provides protection to the organism from disinfectants and antibacterial agents. L. pneumophila infection in humans is characterized by a subtle initial immune response, giving time for the organism to establish infection before the patient succumbs to pneumonia. Planktonic L. pneumophila elicits a strong immune response in murine, but not in human macrophages enabling control of the infection. Interactions between planktonic L. pneumophila and murine or human macrophages have been studied for years, yet the interface between biofilm-derived L. pneumophila and macrophages has not been explored. Here, we demonstrate that biofilm-derived L. pneumophila replicates significantly more in murine macrophages than planktonic bacteria. In contrast to planktonic L. pneumophila, biofilm-derived L. pneumophila lacks flagellin expression, do not activate caspase-1 or -7 and trigger less cell death. In addition, while planktonic L. pneumophila is promptly delivered to lysosomes for degradation, most biofilm-derived bacteria were enclosed in a vacuole that did not fuse with lysosomes in murine macrophages. This study advances our understanding of the innate immune response to biofilm-derived L. pneumophila and closely reproduces the natural mode of infection in human.

Keywords: Legionella pneumophila; biofilm; caspase-1; flagellin; inflammasome; innate immunity.

Figure 1

Dot/Icm type IV secretion promotes robust L. pneumophila biofilm formation. (A) Representative images showing the Live/Dead staining of WT L. pneumophila biofilm (top) or the type IV secretion mutant (dotA). Images were captured using inverted confocal Zeiss LSM 510 META microscope with a 63× water objective. Z-stacks were captured every 1 μm. Red stain indicates dead bacteria while green indicates live bacteria, scale = 10 μm. (B) Representation of biofilm height in μm. (C) Scanning electron microscopy (SEM) of JR32 and dotA mutant. Larger images were captured with the 1000× objective lens while smaller images were magnified 10,000×, scale = 10 μm. (D) Pore-forming activity of L. pneumophila as determined by contact-dependent hemolysis of sheep red blood cells (sRBC) and measured at A₄₁₅ nm. Data are presented as means ± SD of two independent experiments each performed in quadruplicates.

Figure 2

Biofilm-derived L. pneumophila replicates significantly more and induces less murine macrophages death than the planktonic bacteria. (A) BMDMs were infected with planktonic or biofilm-derived L. pneumophila at an MOI of 0.5. CFUs were scored at 1, 24, 48, 72, and 96 h. Data are presented as mean ± SD of two independent experiments each performed in duplicates. Asterisks indicate significant differences (^***P < 0.001). BMDMs were not infected (NT) or infected with L. pneumophila JR32 (planktonic or biofilm) or the dotA mutant at an MOI of 0.5 for (B) 4 or (C) 24 h. The fold change in LDH release was measured from the overall population of macrophages. Data are presented as means ± SD of two independent experiments each performed in quadruplicates. Asterisks indicate significant differences (^**P < 0.01). (D) The hMDMs were infected with L. pneumophila strain JR32 (planktonic or biofilm). CFUs were quantified at 1, 24, and 48 h post-infection. Data are representative as means ± SD of quintuplicate samples. (E) The hMDMs were not infected (NT) or infected with L. pneumophila JR32 (planktonic or biofilm), dotA or the flaA mutant at an MOI of 0.5 for 4 or 24 h. The fold change in LDH release was measured from the overall population of macrophages. Data are representative of mean ± SD of quadruplicate samples.

Figure 3

Biofilm-derived L. pneumophila did not promote caspase-1 or 7 activation in murine macrophages and showed significantly less IL-1β release due to lack of flagellin expression. (A) Pro and active caspase-1 were detected in cell extracts using caspase-1 antibody. WT BMDMs were either not treated (NT) or infected with L. pneumophila JR32 (biofilm or planktonic), the dotA or the flaA mutant for 2 h. (B) The amount of IL-1β was determined in supernatants of WT infected with JR32 (biofilm or planktonic) or the dotA mutant after 24 h. Data are presented as means ± SD of one experiment performed in quadruplicate. Asterisks indicate significant differences (^***P < 0.001). (C) Activation of caspase-7 was detected in cell extracts using caspase-7 antibody. (D) Western blot analysis of planktonic and biofilm-derived L. pneumophila with flagellin antibody.

Figure 4

Vacuoles harboring biofilm-derived L. pneumophila bacteria significantly evade fusion with lysosomes. (A) Representative images of WT BMDMs infected for 1h with JR32 planktonic or biofilm. Nuclei are stained blue with DAPI and L. pneumophila stained green with L. pneumophila-specific antibody. Lyso-tracker red was used to stain acidified lysosomes. White arrows indicate L. pneumophila colocalization with lysotracker. (B) Percent colocalization of L. pneumophila with lysotracker. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm. Data are presented as means ± SD of three independent experiments each performed in triplicates. Asterisks indicate significant difference (^**P < 0.01).