Gain and loss of function screens coupled to next generation sequencing for antibiotic mode of action and resistance studies in Streptococcus pneumoniae [Mechanisms of Resistance]

Two whole genome screening approaches are described for studying the mode of action and the mechanisms of resistance to trimethoprim (TMP) in the Gram positive Streptococcus pneumoniae. The gain of function approach (Int-Seq) relies on a genomic library of DNA fragments integrated into a fucose-inducible cassette. The second approach leading to both gain and loss of function mutations is based on chemical mutagenesis coupled to next-generation sequencing (Mut-Seq). Both approaches pointed at the drug target dihydrofolate reductase (DHFR) as a major resistance mechanism to TMP. Resistance was achieved by dhfr overexpression either through the addition of fucose (Int-Seq) or by mutations upstream of the gene (Mut-Seq). Three type of mutations increased expression by disrupting a predicted Rho-independent terminator upstream of dhfr. Known and novel DHFR mutations were also detected by Mut-Seq and these were functionally validated for TMP resistance. The two approaches also suggested that an increase in the metabolic flux from purine synthesis to GTP and then to folate can modulate the susceptibility to TMP. We finally provide evidence for a novel role of the ABC transporter PatAB in TMP susceptibility. Our genomic screens highlighted novel aspects on the mode of action and resistance mechanisms to antibiotics.

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