During an infection, parasites face a succession of challenges, each decisive for disease outcome. The diversity of challenges requires a series of parasite adaptations to successfully multiply and transmit from host to host. Thus, the pathogen genotypes that succeed during one step might be counterselected in later stages of the infection. Using the bacterium Xenorhabdus nematophila and adult Drosophila melanogaster flies as hosts, we showed that such step-specific adaptations, here linked to GASP (i.e., growth advantage in stationary phase) mutations in the X. nematophila master gene regulator lrp, exist and can trade off with each other. We found that nonsense lrp mutations had lowered the ability to resist the host immune response, while all classes of mutations in lrp were associated with a decrease in the ability to proliferate during early infection. We demonstrate that reduced proliferation of X. nematophila best explains diminished virulence in this infection model. Finally, decreased proliferation during the first step of infection is accompanied by improved proliferation during late infection, suggesting a trade-off between the adaptations to each step. Step-specific adaptations could play a crucial role in the chronic phase of infections in any disease organisms that show similar small colony variants (SCVs) to X. nematophila. IMPORTANCE Within-host evolution has been described in many bacterial diseases, and the genetic basis behind the adaptations has stimulated a lot of interest. Yet, the studied adaptations are generally focused on antibiotic resistance and rarely on the adaptation to the environment given by the host, and the potential trade-offs hindering adaptations to each step of the infection are rarely considered. Those trade-offs are key to understanding intrahost evolution and thus the dynamics of the infection. However, understanding these trade-offs supposes a detailed study of host-pathogen interactions at each step of the infection process, with an adapted methodology for each step. Using Drosophila melanogaster as the host and the bacterium Xenorhabdus nematophila, we investigated the bacterial adaptations resulting from GASP mutations known to induce the small colony variant (SCV) phenotype positively selected within the host over the course of an infection, as well as the trade-off between step-specific adaptations.

16During an infection, parasites face a succession of challenges, each decisive for disease outcome. 17The diversity of challenges requires a series of parasite adaptations to successfully multiply and 18 transmit from host to host. Thus, the pathogen genotypes which succeed during one step might be 19 counter-selected in later stages of the infection. Using the bacteria Xenorhabdus nematophila and 20 adult Drosophila melanogaster as hosts, we showed that such step-specific adaptations, here linked 21 to GASP mutations in the X. nematophila master gene regulator lrp, exist and can trade-off with 22 each other. We found that nonsense lrp mutations had lowered ability to resist the host immune 23 response, while all classes of mutations in lrp were associated with a decrease in the ability to 24 proliferate during early infection. We demonstrate that reduced proliferation of X. nematophila best 25 explains diminished virulence in this infection model. Finally, decreased proliferation during the 26 first step of infection is accompanied with improved proliferation during late infection, suggesting a 27 trade-off between the adaptations to each step.Step-specific adaptations could play a crucial role in 28 the chronic phase of infections in any diseases that show similar small colony variants (also known 29 as SCV) to X. nematophila. Importance. Within-host evolution has been described in many 30 bacterial diseases, and the genetic basis behind the adaptations stimulated a lot of interest. Yet, the 31 studied adaptations are generally focused on antibiotic resistance, rarely on the adaptation to the 32 environment given by the host, and the potential trade-off hindering adaptations to each step of the 33 infection are rarely considered. Those trade-offs are key to understand intra-host evolution, and thus 34 the dynamics of the infection. However, the understanding of these trade-offs supposes a detailed 35 study of host-pathogen interactions at each step of the infection process, with for each step an 36 adapted methodology. Using Drosophila melanogaster as host and the bacteria Xenorhabdus 37 nematophila, we investigated the bacterial adaptations resulting from GASP mutations known to 38 induce small colony variant (SCV) phenotype positively selected within-the-host over the course of 39 an infection, and the trade-off between step-specific adaptations. 40