Jorge do Fusa. (choro). Transcribed by. Paulo Bellinati. GAROTO. (Annibal Augusto Sardinha) d = HIT.. $2 -. -. -. -. C2 - - - - ¢2 - ¢7 - -. 7. ONTV. Fone: (0xx15) / ciepredengunsee.ml - ciepredengunsee.ml: [email protected] ciepredengunsee.ml Page 2. N. Jorge do Fusa. AN w. 1 o. A. 4. NO -. +. 1 ce obo w. Nível: AVANÇADO Jorge do Fusa Choro Garoto Annibal Augusto Sardinha ( ) q»60 > > %≈ ≈ œ 0 œ. # œ œœ n œ œ œœ bn œœ œ b œ 4 ## 2 œœ.
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Whether mucoid strains evolved to resist to phages also exhibit increased virulence remains to be established. Translocation of commensal E. We also found that this pathoadaptative process was characterized by three main paths. The RcsCDB system controls the production of colanic acid, virulence in diverse pathogens  ,  ,  ,  ,  , modulates responses to environmental changes and is activated upon exposure to antimicrobial peptides  ,  ,  , .
Given the repressive function of IgaA on RcsCDB, which controls many traits likely to be important for bacterial fitness, it is likely that the observed IS insertion upstream of yrfF is an adaptive mutation with pleiotropic effects.
If so the adaptive path may proceed through the occurrence of new mutations, which may compensate for the pleiotropic effects of that first adaptive step.
Interestingly, the same amino-acid substitution in fusA occurred in two independent lines.
FusA is an elongation factor and is part of the str operon of E. One of the adaptive paths taken by E.
While the function of yiaW is unknown, the later genes are involved in spermidine transport, which may affect E. Spermidines are polyamines, polycationic molecules, which interact with nucleic acids and have been described as important in escape from phagolysosomes, biofilm formation and protection from oxidative and acidic stress amongst other traits important in bacterial pathogenesis .
The adaptive process was also marked by the occurrence of an IS insertion into the promoter region of the Lon protease. Such insertion was not only likely adaptive it was observed in two independent lineages and it increases mucoidy , but also likely leads to increased rates of transposition.
The mechanisms via which different mutations underlying E. It is likely however, that such mechanisms would interfere with one or two host defense strategies against infections . This should be revealed by increased bacterial burden in the MUC infected hosts, as compared to hosts infected with non-evolved E.
An alternative, but not mutually exclusive, interpretation would be that pathoadaptation is associated with the induction of a immunopathologic response compromising host survival, irrespectively of pathogen burden. This should be revealed by similar bacterial burdens in the MUC infected host, as compared to hosts infected with non-evolved E.
While critical to further understanding of the mechanism via which E. In conclusion, we demonstrate that E.
This pathoadaptive process and the complex dynamics of the evolved phenotypes can be reasonably described by a model of clonal interference, where distinct haplotypes, carrying new transposon insertions and other mutations, increase in frequency and compete for fixation. Strains and media The RAW Evolution experiment Twelve populations were founded from a single MCCFP clone and were therefore genetically uniform in the beginning of the experiments. Cells were then centrifuged rpm for 5 min , re-suspended in 3 ml of fresh RPMI-Strep medium and seeded in well microtiter plates 0.
The same procedure was applied to control populations, except that bacteria were transferred daily. This adjustment results in similar number of generations in both environments.
Evolution occurred during approximately generations in both environments. We note that in the context of a real infection repeated contact with macrophages will not likely occur with a similar period as the one in this experimental setup.
Fitness measurements To estimate competitive fitness of M1—M6 populations, after and generations of evolution, each evolved population was competed against MCYFP reference strain in the same conditions as used in the evolution experiment.
Both evolved and ancestral strains were grown separately in RPMI-Strep, cells of each type were used to inoculate the competition plate. The initial and final ratios of both strains were determined by Flow cytometry.
The fitness of each population was measured 3 times and the fitness of the ancestral strain 10 times to confirm the neutrality of the marker. A measure of relative fitness increase, expressed as selection coefficient, was estimated as:  where Scoeff is the selective advantage of the evolved strain e over the ancestral strain a, Nfe and Nfa are the numbers of evolved e and ancestral a bacteria after competition and Nia and Nie are the initial numbers, before the competition.
Dynamics of infection at 3 h post infection Bacterial uptake was measured by the gentamicin protection assay as previously described  , with modifications, as follows.
After incubation for an additional hour, the medium was removed, the monolayer of macrophages was washed 3 times with PBS, detached using a cell scraper and centrifuged rpm for 10 min to pellet the cells.
These were further resuspended in PBS and the appropriate dilution was plated on LB agar plates to determine the number of intracellular bacteria. Colanic acid purification and quantification The method used to extract colanic acid was based on a procedure described previously . Then 40 ml of the supernatant was precipitated by addition of three volumes of ethanol. The resulting pellet was dissolved in 5 ml of distilled water, dialyzed for 48 h against distilled water membrane MWCO, Da and dried in SpeedVac.
The supernatant was dialyzed for five days against distilled water and dried. The resulting preparation was resuspended in 1 ml of distilled water.
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