Invloed van getemperde fagen P22 en BTP1 op het gedrag van Salmonella Typhimurium

Salmonella enterica significantly contributes to the infectious disease burden in both developed and developing countries. While non-typhoidal S. enterica (NTS) serovars (e.g. serovar Typhimurium and Enteritidis) typically are an important cause of (self-limiting) gastro-enteritis in developed countries, recent reports warn for the dramatic rise and fatality of invasive NTS (iNTS) strains in sub-Saharan Africa. The latter strains form part of a recently described sequence type of S. Typhimurium (i.e. ST313, with D23580 as its representative type strain), and seem to be equipped with a distinctive set of prophages. Since the individual evolution and physiology of Salmonella strains are often shaped by laterally acquired genetic elements such as prophages, this dissertation aimed to focus on the impact of novel prophages present in iNTS.Isolation and preliminary characterization of a number of prophages from iNTS isolates underscored the prevalence of BTP1-like prophages that share an unusually high degree of spontaneous induction. Moreover, these prophages also harbor a genetic region identical to the pid locus of the temperate Salmonella phage P22 that was previously discovered and characterized by our research group. In fact, expression of the Pid protein during P22 infection was previously found to specifically derepress the S. Typhimurium dgoRKDAT operon that encodes proteins required for galactonate metabolism. Surprisingly, however, despite harboring the exact same pid locus, phage BTP1 did not support the Pid/dgo interaction during infection of S. Typhimurium, highlighting regulatory differences between P22 and BTP1 on this front. Subsequently, a parS/ParB-based fluorescent labeling of the incoming phage chromosome in combination with detection by time-lapse fluorescence microscopy was used to study BTP1 infection and transmission dynamics throughout a S. Typhimurium population at single cell resolution, and to compare these dynamics with those of P22. However, while this labelling method could successfully track the cellular whereabouts of the P22 chromosome during all stages of infection, it seemed that efforts to fluorescently track the BTP1 chromosome somehow affected its normal infection behavior. Nevertheless, careful time-lapse microscopic analysis of wild-type BTP1 infection dynamics suggested that this phage (like P22) was also able to commit to a carrier state association in which the phage pseudolysogenizes its S. Typhimurium host cell without integration into the host chromosome or lytic development.To further study how phage BTP1 could potentially affect the phenotypic behavior of its host, a plasmid-borne BTP1 shotgun library was constructed downstream of a conditional promoter, subsequently expressed in S. Typhimurium, and finally screened for BTP1-borne genetic determinants able to affect the growth of this host. As such, several BTP1 loci could be discovered conferring a lethal interference with host physiology upon overexpression, with subsequent efforts aimed at dissecting the mechanism and potential impact of this toxicity. The screening of this library also revealed that plasmid-borne overexpression of the BTP1 pid locus was able to properly trigger the Pid/dgo interaction in S. Typhimurium, which was previously found absent during BTP1 phage infection.In order to better understand the premise of this Pid/dgo interaction, the P22/BTP1 pid promoter was studied in more detail by mutagenesis and fluorescent reporter fusions. This analysis revealed an unusually large 5’ untranslated region to precede the pid open reading frame on the pid transcript, although its potential regulatory impact requires further study. Further scrutiny also revealed that a specific mutation in the S. Typhimurium rpoB gene (which encodes a subunit of the RNA polymerase) could impair the P22 imposed Pid/dgo interaction, although the underlying mechanism so far remains to be further elucidated. In essence, this dissertation has uncovered a number of interesting characteristics of the BTP1 phage and of the pid locus that it shares with phage P22, although the potential contribution of BTP1 to the emergence of iNTS strains still remains to be established. Further study of the interactions between S. enterica and its laterally acquired genetic elements is bound to yield important clues regarding the behavior of this notorious pathogen.status: publishe