Tracking Bioluminescent ETEC during In vivo BALB/c Mouse Colonization.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea worldwide. Adhesion to the human intestinal tract is crucial for colonization. ETEC adhesive structures have been extensively studied; however, colonization dynamics remain uncharacterized. The aim of this study was to track bioluminescent ETEC during in vivo infection. The promoter region of dnaK was fused with the luc gene, resulting in the pRMkluc vector. E. coli K-12 and ETEC FMU073332 strains were electroporated with pRMkluc. E. coli K-12 pRMkluc was bioluminescent; in contrast, the E. coli K-12 control strain did not emit bioluminescence. The highest light emission was measured at 1.9 OD600 (9 h) and quantified over time. The signal was detected starting at time 0 and up to 12 h. Streptomycin-treated BALB/c mice were orogastrically inoculated with either ETEC FMU073332 pRMkluc or E. coli K-12 pRMkluc (control), and bacterial colonization was determined by measuring bacterial shedding in the feces. ETEC FMU073332 pRMkluc shedding started and stopped after inoculation of the control strain, indicating that mouse intestinal colonization by ETEC FMU073332 pRMkluc lasted longer than colonization by the control. The bioluminescence signal of ETEC FMU073332 pRMkluc was captured starting at the time of inoculation until 12 h after inoculation. The bioluminescent signal emitted by ETEC FMU073332 pRMkluc in the proximal mouse ileum was located, and the control signal was identified in the cecum. The detection of maximal light emission and bioluminescence duration allowed us to follow ETEC during in vivo infection. ETEC showed an enhanced colonization and tropism in the mouse intestine compared with those in the control strain. Here, we report the first study of ETEC colonization in the mouse intestine accompanied by in vivo imaging.

Control mechanisms of tubulin gene expression in Trypanosoma cruzi.

alpha- and beta-Tubulin mRNAs are three to six-fold more abundant in the epimastigote forms than in trypomastigote and amastigote forms of Trypanosoma cruzi. It has been previously shown that the increased abundance of alpha- and beta-tubulin mRNAs found in epimastigotes is due to an increase in their half-lives. By analysing soluble and cytoskeletal protein fractions of the parasite, we found an inverse correlation between tubulin mRNA and the protein levels of free alpha- and beta-tubulin subunits, which are more abundant in trypomastigotes and amastigotes than in epimastigotes. Here we investigated a possible autoregulatory mechanism responsible for the differential accumulation of tubulin mRNAs in T. cruzi by treating epimastigotes with vinblastine and taxol, drugs that disrupt microtubule dynamics by different mechanisms: vinblastine causes significant depolymerisation of tubulin whereas taxol stabilises microtubules. Vinblastine treatment caused significant morphological alterations in epimastigotes whereas taxol does not alter the parasite morphology. Vinblastine, but not taxol, had a specific effect on the levels of alpha- and beta-tubulin mRNAs, causing a five to nine-fold reduction in the steady-state levels of both mRNA populations, whereas the levels of other mRNAs such as gapdh remained unchanged. The reduction in RNA levels caused by vinblastine treatment is mediated by changes in tubulin mRNA half-lives. In an attempt to identify regulatory elements within tubulin mRNAs, plasmids containing luciferase reporter gene associated with 5'-untranslated (UTR), 3'-UTR and part of coding sequence of the tubulin genes were constructed and used for transient DNA transfections of epimastigotes. Determination of luciferase activity in transfected parasites cultured in the presence and absence of vinblastine indicated that sequences located within the alpha-tubulin 3'-UTR and coding region may be involved in modulating the stability of these transcripts in response to changes in the dynamics of T. cruzi microtubules.