Investigation of the influence of prophages on the physiology and virulence of emerging invasive non-typhoid Salmonella Typhimurium (iNTS)

Bacteria are engaged in a never-ending struggle with their viral predators, termed bacteriophages or phages. This resulted in millennia of intimate co-evolution between bacteria and their viruses, thereby fueling the development of sophisticated infection dynamics. One of these peculiar interactions, is the formation of a phage carrier state by temperate phage P22 upon infection of its host, Salmonella Typhimurium. This phenomenon precedes lysogenic conversion and arises due to the delay of the integration event of the phage in the host chromosome, thereby leading to the emergence of a phage free subpopulation that is transiently resistant to superinfection due to cytoplasmic inheritance of superinfection exclusion factors.

Screening for genes involved in the phage carrier state yielded an interesting mutation in P22 that provided further insights into this phage’s infection dynamics. When after infection, the lysogenic subpopulation is eliminated by deleting mnt, a P22 gene required for maintenance of the lysogenic state, the phage free subpopulation flourishes due to the additional available resources. In this scenario, a continuously ongoing infection and formation of phage carrier and phage free cells is observed. On the other hand and more importantly, these experiments also demonstrate that when infecting Salmonella with the wild type P22 phage, the vast majority of the bacterial cells in the culture will be lysogens within 24h.

These observations instigated further investigation of the infection dynamics at population level in bioreactors in order to enable interpretation of the influence of the phage carrier state for phage-host ecology. Also the underlying molecular mechanisms driving the dynamics were further scrutinized using time-lapse fluorescence microscopy. This revealed that the phage carrier state brings forth a massively increased spread of virions via in time deferred lytic consumption of the phage free subpopulation that first protectively proliferates due to their transient resistance to superinfection.

The surprising observation that lytic development (and not lysogeny) occurs upon infection of the phage fee siblings exiting transient resistance contradicts available literature reporting that a high phage-to-host ratio generally leads to lysogeny. This peculiarity, we prove, is due to the gradual dilution of the superinfection exclusion factors that enforces single-phage entrance, consequently resulting in host lysis. These results therefore demonstrate that the phage carrier state is able to increase virion production by molecular mechanisms defying the paradigm that high phage-to-host ratios always favor non-lytic development of temperate phages.

The next step in this research was to examine whether the occurrence of this behavior is unique for phage P22, or if other phages employ the same infection strategy. To this end, the infection dynamics of phage BTP1, a P22-like virus that moreover contributes to the virulence of emerging invasive S. Typhimurium strains, were investigated. This work unveils that upon infection with BTP1 also a phage free, transiently resistant subpopulation is formed. This temporary immunity is linked to the overexpression and subsequent diluting out of a superinfection exclusion factor. However, the infection dynamics at population level in batch differ strongly from these observed for P22 with high titers of phage BTP1 produced shortly after infection.