Deep-sea microbes as tools to refine the rules of innate immune pattern recognition

Abstract

The assumption of near-universal bacterial detection by pattern recognition receptors is a foundation of immunology. The limits of this pattern recognition concept, however, remain undefined. As a test of this hypothesis, we determined whether mammalian cells can recognize bacteria that they have never had the natural opportunity to encounter. These bacteria were cultivated from the deep Pacific Ocean, where the genus Moritella was identified as a common constituent of the culturable microbiota. Most deep-sea bacteria contained cell wall lipopolysaccharide (LPS) structures that were expected to be immunostimulatory, and some deep-sea bacteria activated inflammatory responses from mammalian LPS receptors. However, LPS receptors were unable to detect 80% of deep-sea bacteria examined, with LPS acyl chain length being identified as a potential determinant of immunosilence. The inability of immune receptors to detect most bacteria from a different ecosystem suggests that pattern recognition strategies may be defined locally, not globally.

Figure 1.

Bacterial sample collection during the R/V Falkor’s 2017 expedition to the Phoenix Islands Protected Area, Kiribati. (A) Site map showing nine sites sampled. (B) Bray-Curtis non-metric multidimensional scaling ordination of total microbial community composition, based on 16S amplicon data from all stations. (C) Overview of the microbial community composition at stations S1-S9 as determined by 16S rRNA amplicon analysis. (D) Streak-purified bacteria strains isolated and sequenced from seawater and substrates collected at stations S2-S5. (E) Moritella relative 16S amplicon abundance in seawater samples collected from different depths at stations S2-S5.

Figure 2.

Grouping of marine bacterial strains into three categories based on engagement with CD14 and TLR4 on mouse macrophages. (A,B) Surface expression of CD14 (A) and TLR4 (B) as measured by mean fluorescence intensity (MFI) on iBMDMs exposed to live deep-sea bacteria strains was compared to live E. coli (MOI=50). The color of columns represents the predicted acyl chain number for the LPS lipid A expressed, as described in Table 1. Dashed lines are used to delineate whether bacterial strains are stimulatory or silent to CD14 or TLR4, as compared to E. coli. (C) Summary of murine CD14 and TLR4 engagement by strains tested.

Figure 3.

Dose-response curves testing ability of purified LPS from Moritella strains to induce PRR loss from the surface of mouse macrophages in comparison to E. coli LPS. (A) Purified LPS from three Moritella strains predicted to be hexa-acylated did not behave similarly to purified E. coli LPS in flow cytometry assays measuring engagement of CD14 and TLR4 on murine iBMDMs. (B) Purified LPS from nine other Moritella strains predicted to be hexa-acylated behaved similarly to E. coli LPS. Red circles (●) indicate Moritella LPS and black squares (■) indicate E. coli LPS. Statistical analysis based on comparison of 100 ng/mL dose from Moritella LPS with 100 ng/mL dose of E. coli LPS. (*p ≤ 0.01, ns = not significant).

Figure 4.

Functional analysis of purified LPS from silent (#28, 36) or stimulatory (#5, 24) Moritella strains in murine and human macrophages. (A,B) Accumulation of TNFα after 3.5 hrs (A) and phosphorylation of STAT1 after 2.5 hrs (B) measured in wild type and Tlr4^−/− iBMDMs incubated with 100 ng/mL LPS from Moritella strains or E. coli. (C) The release of LDH, 3 hours post-electroporation of wild type and Casp11^−/− iBMDMs with 1 μg of LPS from Moritella strains and E. coli. (D) Cleavage of GSDMD, 3 hours post-electroporation of wild type iBMDMs with 1 μg of LPS from Moritella strains and E. coli. (E) Binding of HA-tagged caspase-11 to LPS supplied in excess (5 μg) from Moritella strains or E. coli, as measured by the ability of LPS to compete off biotinylated E. coli LPS (1 μg). (F) The production of pro-IL-1β, 2.5 hours post-treatment of human THP1 cells incubated with 50 ng/mL of LPS from Moritella strains compared to E. coli LPS. (G) Accumulation of TNFα, 4 hours post-treatment of human THP1 cells incubated with 100 ng/mL of LPS from Moritella strains compared to E. coli LPS. (H) Engagement of human TLR4 in human TLR4/NF-κB/SEAP reporter HEK293 cells by LPS from Moritella strains compared to E. coli LPS. (I) The release of LDH, 2.5 hours post-electroporation of human THP1 cells with 1 μg of LPS from Moritella strains compared to E. coli LPS. (*p ≤ 0.01 and ** p ≤ 0.001)

Figure 5.

Cross-species evasion of LPS receptor activity by deep-sea Moritella strains. (A) Engagement of Limulus polyphemus factor C by LPS from Moritella strains compared to E. coli LPS. (B) Inflammatory response to LPS from Moritella strains in vivo. IL-6 and TNFα plasma levels were measured 4 hours post intraperitoneal injection of mice with LPS from stimulatory (#5, 24) or silent (#28, 36) Moritella strains. (C) 10 μg of E. coli LPS was compared to 10 μg of LPS or lipid A (LA) from Moritella strains visualized on a polyacrylamide gel stained with ProQ Emerald LPS staining solution. (D) Accumulation of TNFα after 3.5 hr stimulations of iBMDMs with 100 ng/mL lipid A compared to LPS from Moritella strains. (E) The release of LDH 24 hours post-electroporation of iBMDMs with 1μg of lipid A or LPS from Moritella strains. (F) Engagement of Limulus polyphemus factor C by LPS and lipid A from Moritella strains. (*p ≤ 0.01, ** p ≤ 0.001 and ***<0.0001)

Figure 6.

Further characterization of immuno-silent and immuno-stimulatory Moritella strains. (A) MALDI-TOF MS spectra of Moritella lipid A generated using the FLAT technique. (B) Relative fatty acid content in lipid A derived from silent and stimulatory Moritella as determined by gas chromatography–mass spectrometry (GC-MS) (*p ≤ 0.01). (C) Whole genome phylogeny of Moritella strains. The amino acid phylogeny was inferred using maximum likelihood from a concatenated alignment of 120 single copy genes (56) generated from the four newly-sequenced Moritella genomes (green squares) and other publicly available assemblies. Black dots on branches indicate bootstrap support >75%. (D) Degree of sequence conservation for enzymes in the lipid A biosynthesis pathway. The maximum likelihood phylogeny at left is based on a concatenated amino acid alignment of the 10 indicated lipid A biosynthesis enzymes from each genome. The heatmap depicts the % amino acid identity for each individual enzyme in the pathway, as compared to Moritella oceanus 28. Black dots on tree branches indicate bootstrap support > 75%.