First insights into host-dependent regulation of biofilm formation and virulence by cNMP second messengers in Photorhabdus luminescens

Photorhabdus luminescens interacts with different eukaryotic hosts - insects, nematodes and plants. While the bacteria form a mutualistic symbiosis with nematodes and plants, they are highly pathogenic towards insects. The bacteria exist in two phenotypic different cell forms designated as primary (1°) and secondary (2°) cells, whereas only the 1° cells interact with the nematodes and 2° cells with plants. Both cell forms are pathogenic against insects. P. luminescens needs to specifically adapt to the different hosts, especially by regulation of sessility and pathogenicity. In other bacteria, second messengers play a central role for those biotic interactions. In P. luminescens we found presence of 2',3'- and 3',5'-cyclic nucleotide monophosphates (cNMPs), while no cyclic di-GMP could be detected. In many bacteria, c-di-GMP is often involved in the regulation of motility and sessility. To get first insight into the role of second messengers in the regulation of P. luminescens-host interactions, we incubated the bacteria in the presence of the three different host signals and analysed the cells for presence of different cNMPs. Thereby, increased levels of 3',5'-cAMP were detected when host signals were present. For that reason, a bioinformatics analysis was conducted, proposing 3',5'-cAMP to regulate virulence in P. luminescens. While adenylate and guanylate cyclases involved in 3',5'-cNMP production are well characterized and could be bioinformatically identified in P. luminescens, the synthesis and regulatory role of the non-canonical 2',3'-cNMPs was vastly unknown. As the prominent second messenger cyclic di-GMP is absent in P. luminescens, we investigated the role of 2',3'-cNMPs for biofilm formation. We identified a RNase T2 (RnsA) that is a homologue to RNase1 of E. coli where it is known to be involved in 2',3'-cNMP production and regulation of sessility. The deletion of rnsA let to a 10-fold decrease of the 2',3'-cNMP levels compared to the wildtype in both P. luminescens 1° and 2° cells. Moreover, we observed an increased biofilm formation in both cell types but especially in 1° cells, suggesting a central role of these second messengers for regulation of sessility and therefore probably also for host colonization. Overall, here we identified RnsA as a central enzyme for 2',3'-cNMP synthesis and raised first insights into the regulation of sessility by 2',3'-cNMPs and virulence by 3',5'-cAMP.