Trade-Offs of Escherichia coli Adaptation to an Intracellular Lifestyle in Macrophages.

The bacterium Escherichia coli exhibits remarkable genomic and phenotypic variation, with some pathogenic strains having evolved to survive and even replicate in the harsh intra-macrophage environment. The rate and effects of mutations that can cause pathoadaptation are key determinants of the pace at which E. coli can colonize such niches and become pathogenic. We used experimental evolution to determine the speed and evolutionary paths undertaken by a commensal strain of E. coli when adapting to intracellular life. We estimated the acquisition of pathoadaptive mutations at a rate of 10-6 per genome per generation, resulting in the fixation of more virulent strains in less than a hundred generations. Whole genome sequencing of independently evolved clones showed that the main targets of intracellular adaptation involved loss of function mutations in genes implicated in the assembly of the lipopolysaccharide core, iron metabolism and di- and tri-peptide transport, namely rfaI, fhuA and tppB, respectively. We found a substantial amount of antagonistic pleiotropy in evolved populations, as well as metabolic trade-offs, commonly found in intracellular bacteria with reduced genome sizes. Overall, the low levels of clonal interference detected indicate that the first steps of the transition of a commensal E. coli into intracellular pathogens are dominated by a few pathoadaptive mutations with very strong effects.

Analyses of herpes simplex virus type 1 latency and reactivation at the single cell level using fluorescent reporter mice.

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in sensory neurons from which the virus can periodically reactivate. Whilst latency establishment is thought to result from a failure to express immediate-early genes, we have previously shown that subpopulations of the latent neuronal reservoir have undergone lytic promoter activation prior to latency establishment. In the present study, we have investigated the biological properties of such latently infected neuronal subpopulations using Ai6 fluorescent reporter mice. Using this system we have determined that prior ICP0 or TK promoter activation does not correlate with increased latent virus DNA loads within individual cells and that neurons with evidence of historical lytic cycle promoter activity exhibit a comparable frequency of reactivation to that of the general latent cell population. Comparison of viral DNA content within cells harbouring latent HSV-1 genomes and those undergoing the earliest stages of reactivation has revealed that reactivation can initiate from cells harbouring a wide range of HSV-1 genome copies, but that exiting latency is biased towards cells bearing higher latent virus DNA loads.

Influence of herpes simplex virus 1 latency-associated transcripts on the establishment and maintenance of latency in the ROSA26R reporter mouse model.

Herpes simplex virus 1 (HSV-1) can establish life-long latent infection in sensory neurons, from which periodic reactivation can occur. During latency, viral gene expression is largely restricted to the latency-associated transcripts (LATs). While not essential for any phase of latency, to date the LATs have been shown to increase the efficiency of both establishment and reactivation of latency in small-animal models. We sought to investigate the role of LAT expression in the frequency of latency establishment within the ROSA26R reporter mouse model utilizing Cre recombinase-encoding recombinant viruses harboring deletions of the core LAT promoter (LAP) region. HSV-1 LAT expression was observed to influence the number of latently infected neurons in trigeminal but not dorsal root ganglia. Furthermore, the relative frequencies of latency establishment of LAT-positive and LAT-negative viruses are influenced by the inoculum dose following infection of the mouse whisker pads. Finally, analysis of the infected cell population at two latent time points revealed a relative loss of latently infected cells in the absence of LAT expression. We conclude that the HSV-1 LATs facilitate the long-term stability of the latent cell population within the infected host and that interpretation of LAT establishment phenotypes is influenced by infection methodology.