Molecular and chemical dialogues in bacteria-protozoa interactions

Abstract

Protozoan predation of bacteria can significantly affect soil microbial community composition and ecosystem functioning. Bacteria possess diverse defense strategies to resist or evade protozoan predation. For soil-dwelling Pseudomonas species, several secondary metabolites were proposed to provide protection against different protozoan genera. By combining whole-genome transcriptome analyses with (live) imaging mass spectrometry (IMS), we observed multiple changes in the molecular and chemical dialogues between Pseudomonas fluorescens and the protist Naegleria americana. Lipopeptide (LP) biosynthesis was induced in Pseudomonas upon protozoan grazing and LP accumulation transitioned from homogeneous distributions across bacterial colonies to site-specific accumulation at the bacteria-protist interface. Also putrescine biosynthesis was upregulated in P. fluorescens upon predation. We demonstrated that putrescine induces protozoan trophozoite encystment and adversely affects cyst viability. This multifaceted study provides new insights in common and strain-specific responses in bacteria-protozoa interactions, including responses that contribute to bacterial survival in highly competitive soil and rhizosphere environments.

Figure 1

(A) Transcriptomic analysis of P. fluorescens SS101-N. americana interaction. Each point represents one annotated gene in the SS101 genome, with the X-axis showing the gene order, and the Y-axis showing the log2 of gene transcript abundance in the interaction. The identities of highly modulated, well-characterized gene clusters are shown. 1. massA; 2. massB, massC; 3. alkane oxidation gene clusters; 4. agmatinase encoding gene speB. (B) Organization of the lipopeptide (LP) gene cluster in P. fluorescens SS101. The three LP biosynthesis genes are designated massA, massB and massC. In the boxes of the genes are the fold changes in their expression during P. fluorescens-N. americana interaction. (C) Organization of the alkane oxidation gene cluster in SS101. The reference strain used is P. protegens CHA0 (previously described as P. fluorescens). In the boxes of the genes are the fold changes in their expression during P. fluorescens-N. americana interaction. (D) Organization of the putrescine encoding gene speB and its flanking genes. In the boxes of the genes are the fold changes in their expression during P. fluorescens-N. americana interaction.

Figure 2

(A) Time to encystment of amoeboid and flagellate forms of N. americana exposed to increasing concentrations of putrescine. For each putrescine concentration, the average of three replicates is shown. Error bars refer to the standard error of the mean. (B) Representative images of trophozoite viability at 0, 7, 20 and 30 seconds after exposure to 250 mM putrescine. A replicate consisted of an individual sample containing the protist incubated in the indicated putrescine concentration from which a 6 μl sample was extracted and all cysts or trophozoites in that sample were enumerated.

Figure 3

(A) Experimental setup to study Pseudomonas-protozoa interactions by MALDI imaging mass spectrometry (IMS). The green box-line indicates the protozoan predator N. americana alone; the red box-line indicates the interface of P. fluorescens SS101-N. americana; the yellow box-line indicates P. fluorescens SS101 alone. (B) MALDI-IMS analysis of the Pseudomonas-protozoa interaction, including imaging of metabolite classes, spatial segmentation and co-localization of the MALDI IMS data. (C) MS/MS network analysis and annotation of ion clusters from the P. fluorescens SS101-N. americana interaction. Ion clusters in the black squares represent the lipopeptide massetolide A and its derivatives; the black circle represents the 325-477 m/z ion cluster; the grey square represents the 766-796 m/z ion cluster. MS/MS analysis further indicated that the parent ion with 1162.70 m/z detected in the P. fluorescens SS101- N. americana interaction is most likely massetolide A. Complete lists of the ion clusters detected in the network analysis are given in Tables S4, S5 and S6.

Figure 4

(A) MALDI imaging mass spectrometry (IMS) shows production of massetolide A and its derivatives during the P. fluorescens SS101-N. americana interaction. a.u. = arbitrary units. (B) Box plots depicting the production of massetolide A and its derivatives (m/z 1142, 1163, 1165 and 1179) in P. fluorescens SS101 alone (SS101), N. americana alone (Protozoa), P. fluorescens SS101-N. americana interaction (SS101_Protozoa) and massA mutant alone (ΔmassA). (B) The box plots represent the median intensity in arbitrary units after TIC normalization (horizontal line), the upper and lower quartiles (box layout, spectra in which the intensities are within a range of 25%–75% of the data), the upper and lower quantiles (dashed lines, spectra in which the intensities are within a range of 0%–99%) as well as the outliers (spectra with intensities greater than 99% of the data). The green box-line indicates N. americana alone; the red box-line indicates P. fluorescens SS101-N. americana interaction; the yellow box-line indicates P. fluorescens SS101 alone.

Figure 5

(A) MALDI imaging mass spectrometry (IMS) shows production of 249–688 m/z ions in the MS/MS network of the P. fluorescens SS101-N. americana interaction. a.u. = arbitrary units. (B) MALDI imaging mass spectrometry (IMS) shows production of 752–796 m/z ions cluster in the MS/MS network of the P. fluorescens SS101-N. americana interaction. The green box-line indicates N. americana alone; the red box-line indicates P. fluorescens SS101-N. americana interaction; the yellow box-line indicates P. fluorescens SS101 alone.