RESUMO
New mutations leading to structural variation (SV) in genomes-in the form of mobile element insertions, large deletions, gene duplications, and other chromosomal rearrangements-can play a key role in microbial evolution. Yet, SV is considerably more difficult to predict from short-read genome resequencing data than single-nucleotide substitutions and indels (SN), so it is not yet routinely identified in studies that profile population-level genetic diversity over time in evolution experiments. We implemented an algorithm for detecting polymorphic SV as part of the breseq computational pipeline. This procedure examines split-read alignments, in which the two ends of a single sequencing read match disjoint locations in the reference genome, in order to detect structural variants and estimate their frequencies within a sample. We tested our algorithm using simulated Escherichia coli data and then applied it to 500- and 1000-generation population samples from the Lenski E. coli long-term evolution experiment (LTEE). Knowledge of genes that are targets of selection in the LTEE and mutations present in previously analyzed clonal isolates allowed us to evaluate the accuracy of our procedure. Overall, SV accounted for ~25% of the genetic diversity found in these samples. By profiling rare SV, we were able to identify many cases where alternative mutations in key genes transiently competed within a single population. We also found, unexpectedly, that mutations in two genes that rose to prominence at these early time points always went extinct in the long term. Because it is not limited by the base-calling error rate of the sequencing technology, our approach for identifying rare SV in whole-population samples may have a lower detection limit than similar predictions of SNs in these data sets. We anticipate that this functionality of breseq will be useful for providing a more complete picture of genome dynamics during evolution experiments with haploid microorganisms.
RESUMO
Modification of bacterial surface structures, such as the lipid A portion of lipopolysaccharide (LPS), is used by many pathogenic bacteria to help evade the host innate immune response. Helicobacter pylori, a gram-negative bacterium capable of chronic colonization of the human stomach, modifies its lipid A by removal of phosphate groups from the 1- and 4'-positions of the lipid A backbone. In this study, we identify the enzyme responsible for dephosphorylation of the lipid A 4'-phosphate group in H. pylori, Jhp1487 (LpxF). To ascertain the role these modifications play in the pathogenesis of H. pylori, we created mutants in lpxE (1-phosphatase), lpxF (4'-phosphatase) and a double lpxE/F mutant. Analysis of lipid A isolated from lpxE and lpxF mutants revealed lipid A species with a 1 or 4'-phosphate group, respectively while the double lpxE/F mutant revealed a bis-phosphorylated lipid A. Mutants lacking lpxE, lpxF, or lpxE/F show a 16, 360 and 1020 fold increase in sensitivity to the cationic antimicrobial peptide polymyxin B, respectively. Moreover, a similar loss of resistance is seen against a variety of CAMPs found in the human body including LL37, ß-defensin 2, and P-113. Using a fluorescent derivative of polymyxin we demonstrate that, unlike wild type bacteria, polymyxin readily associates with the lpxE/F mutant. Presumably, the increase in the negative charge of H. pylori LPS allows for binding of the peptide to the bacterial surface. Interestingly, the action of LpxE and LpxF was shown to decrease recognition of Helicobacter LPS by the innate immune receptor, Toll-like Receptor 4. Furthermore, lpxE/F mutants were unable to colonize the gastric mucosa of C57BL/6J and C57BL/6J tlr4 -/- mice when compared to wild type H. pylori. Our results demonstrate that dephosphorylation of the lipid A domain of H. pylori LPS by LpxE and LpxF is key to its ability to colonize a mammalian host.
