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Different strategies lead to the emergence of bacterial subpopulations in vivo

A "bet hedging" strategy (see "Insect immune response and impact of the nematobacterial complex") is displayed in Photorhabdus giving a mixture of pre-existing subpopulations that ensure heterogeneity of resistance to antimicrobial peptides (AMPs). Most cells are sensitive to AMPs but some are pre-adapted to resist them, ensuring bacterial survival in the hostile environment of the insect's hemolymph and thus successful infection (Mouammine et al, 2017).

While many publications describe the bacterial factors involved in the early stages of infection in the insect, very little data are available to decipher what happens in the cadaver after the insect dies. Xenorhabdus and Photorhabdus isolated from nematodes are typically described as belonging to a so-called "primary" phenotypic state. However, in all species of symbionts of both genera of Xenorhabdus and Photorhabdus, variants appear when they reach the advanced stationary phase in culture media and in insect cadavers. Thus, a part of the bacterial population converts into forms called "secondary" (or variant 2), or even "tertiary" in Xenorhabdus (Figure 1). In collaboration with J.-B. Ferdy (Univ. Toulouse), we have shown that variants 2 of Xenorhabdus are mutated in a global transcriptional regulator (Lrp). A high survival (called "GASP" for Growth advantage in stationary-phase) was observed in these variants during the advanced stationary phase explaining why they reach a high load during late infections. However, the impact of the variants on the fitness of the overall system (measured by the number of nematodes produced after emergence) is small. Our hypothesis is that the existence of these variants allows Xenorhabdus to develop a "division of labor" strategy (Cambon et al, 2019).

Figure 1. Selection scenario for so-called GASP variants in long-term batch cultures and during the life cycle of X. nematophila. (A) Bacterial growth during infection. (B) Key stages in the life cycle of X. nematophila. From 3 days post-infection (dpi), GASP variants (group 2 and group 3 lrp mutants in red and green) are detected and rapidly increase in frequency as they are more resistant to stationary phase conditions than primary variants (blue). When nematodes begin to disperse around 10-15 days post-infection, the X. nematophila population in the host cadaver may include a high proportion of GASP variants. In principle, therefore, GASP variants could contribute to transmission. However, our data suggest that nematodes carrying GASP variants have a lower probability of successfully infecting new insects (Cambon et al, 2019).

From phenotypic heterogeneity to methylomics

To investigate the mechanistic origin of the emergence of subpopulations, we also studied epigenetic phenomena related to the methylation status of the genome (methylome). DNA methylation can cause differences in the expression of certain genes, including those encoding factors involved in host-bacterial interactions. Due to competition between regulatory proteins and methyltransferases for access to certain sites in promoter regions, gene expression varies depending on the adenine or cytosine methylation status of promoter sequences. A powerful tool (SMRT sequencing for Single Molecule Real-Time) has recently been developed to characterize the methylome. With collaborators from LIPM (INRAE, Toulouse), we have combined powerful tools (Single Molecule Real-Time sequencing and Bisulfite-sequencing) to perform the first description of the methylome of an entomopathogenic bacterium (Payelleville et al, 2018). We have:

• Identified a high level of DNA methylation across the genome that was stable during growth (Figure 2).
• Showed that the emergence of the AMP-resistant subpopulation was not due to DNA methylation.
• Highlighted the existence of unmethylated regions in some promoters.
• Showed that overexpression of Dam methyltransferase in Photorhabdus leads to a significant decrease in motility (underexpression of flagellar genes) and virulence after injection into lepidopteran larvae of Spodoptera littoralis (Payelleville et al, 2017).

Figure 2. First methylome of an entomopathogenic bacterium. We determined that the methylation of the Photorhabdus genome is stable at the different growth phases tested. This work provides key insights into bacterial methylomes by showing that some loci are not methylated by Dam methylase and thus encourages further research to discover transcriptional regulators protecting these loci from DNA methylation (Payelleville et al, 2018).

Finally, in collaboration with the laboratory of D. Clarke (Cork University, Ireland), we evaluated the impact of methylation on the mutualistic relationship between Photorhabdus and the nematode. While no difference in the amount of infective juveniles (IJs) emerging from the cadaver was observed between the two strains, a significant increase in TL50 (time to kill 50% of the insects) appears upon infestation of the insects by IJs associated with the methyltransferase-overexpressing strain (Payelleville et al, 2019). These results confirm that the Photorhabdus Dam methyltransferase plays a role in the pathogenicity of the nematobacterial complex.

Bibliography

Cambon, M.C., Parthuisot, N., Pages, S., Lanois, A., Givaudan, A., Ferdy, J.-B. 2019. Selection of bacterial mutants in late infections: when vector transmission trades off against growth advantage in stationary phase. mBio 10, 1-14. DOI : 10.1128/mBio.01437-19.

Mouammine, A., Pages, S., Lanois Nouri, A., Gaudriault, S., Jubelin, G., Bonabaud, M., et al. 2017. An antimicrobial peptide-resistant minor subpopulation of Photorhabdus luminescens is responsible for virulence. Sci Rep 7, 43670. DOI : 10.1038/srep43670.

Payelleville, A., Lanois, A., Gislard, M., Dubois, E., Roche, D., Cruveiller, S., et al. 2017. DNA adenine methyltransferase (Dam) overexpression impairs Photorhabdus luminescens motility and virulence. Front Microbiol 8, 14 p. DOI : 10.3389/fmicb.2017.01671.

Payelleville, A., Legrand, L., Ogier, J.-C., Roques, C., Roulet, A., Bouchez, O., et al. 2018. The complete methylome of an entomopathogenic bacterium reveals the existence of loci with unmethylated Adenines. Sci Rep 8, 1-14. DOI : 10.1038/s41598-018-30620-5.

Payelleville, A., Blackburn, D., Lanois, A., Pages, S., Cambon, M.C., Ginibre , N., et al. 2019. Role of the Photorhabdus Dam methyltransferase during interactions with its invertebrate hosts. PLoS One 14, 14 p. DOI : 10.1371/journal.pone.0212655.