Virulence, cellular masking, and grazing resistance in the struggle to survive under long term protozoan predation; the genetics of survival

ABSTRACT: Purpose: Generalist protozoan predators change the pace of evolution in microbial communities, but investigations into the molecular consequences of predation for the genomic evolution of defence or coadaptation are scarce. We have previously performed a 90-day co-evolution experiment to address these effects using the bacterium Pseudomonas fluorescens SBW25 on solid media in the presence and absence of a wild Acanthamoeba sp. predator. Coevolution led to genomic divergence, altered phenotypes and resistance to predation in coevolved bacterial lineages. Strong parallel phenotypic evolution was observed among the coevolved replicates leading to Wrinkly Spreader, Volcano and Mountain colony phenotypes. Here, we describe the genomic mutations underlying this parallelism with a focus on these morphotypes. Methods: Pseudomonas fluorescens SBW25 and a wild Acanthamoeba sp. isolate as a predator prey pair co-evolved for 90 days yielded novel colony morphotype (Mountain; MNT, and Volcano; VOL) isolates with conferred grazing resistance. These isolates were subjected to RNAseq profiling with and without predation to determine transcriptional changes contributing to grazing resistance. Results: We made a profile of differentially expressed genes (DEG) for the coevolved Vol-L1 (Volcano L1; ∆fadD2-fadD1 & luxR T2675G) while predated by ancestral Acanthamoebae. WT P. fluorescens SBW25 was used as the baseline and significant differential expression was considered for genes with log2 FC ≥ 2, P-value ≤ 0.05 between conditions. We identified a total of 953 DEGs, of which 293 were upregulated and 660 were downregulated. Differentially expressed gene (DEG) profiles were made for the coevolved strains Mnt-L2 (Mountain L2; ∆4974-4975 & PFLU_0924 C790T) while under predation by ancestral Acanthamoebae. In this case we also used WT P. fluorescens SBW25 without predation as a baseline as described previously. In this condition and compared to the WT SBW25, we identified a total of 209 DEGs, of which 118 were upregulated and 91 were downregulated. Conclusions: In this study, from the DEG profile of Mnt-L2 and Vol-L1 while predated by the ancestral Acanthamoebae we found 12 and 21 upregulated genes in O-antigen biosynthesis, respectively. The polysaccharide capsule (LPS) is considered as one of the main virulence factors produced by pathogenic bacteria to protect bacteria against phagocytosis by the host’s immune system as well. We found enrichments in lipopolysaccharide-A production and three viscosin genes in Vol-L1 (a finding unique to the Vol-L1 strain). Vol-L1 had significant downregulation of seven efflux genes, most significantly PFLU_3263 (an efflux transporter), as well as a fatty acid transporter (PFLU4903). These results, taken with the finding of altered survivability only when evolved strains are grown with Acanthamoebae and the abundance of mutations found in FadD genes, led to our hypothesis that the evolved bacteria might be altering transporters to counter increased exposure to LC fatty acids from the Acanthamoeba by increased oxidative stress from LC fatty acid degradation, masking, etc. Furthermore, we found upregulation of an efflux RND transporter downstream of the deletion (PFLU_4976) and a unique enrichment (5 genes) in the cyclophilin-like domain (immunophilin) in Mnt-L2. Among all DEGs, 72 upregulated and 66 downregulated genes were shared between the Vol-L1 and Mnt-L2 conditions.

ORGANISM(S): Acanthamoeba sp. Pseudomonas fluorescens

PROVIDER: GSE206989 | GEO | 2022/06/30

REPOSITORIES: GEO