Correlation versus causation: Helicobacter pylori population heterogeneity complicates the identification of mutant strain phenotypes

Helicobacter pylori causes cancer in approximately 1% of infected individuals. A proposed mechanism of H. pylori persistence centers on the ability of the pathogen to form biofilms, yet little is known about specific genetic requirements for this process. Our investigation revealed that during lab passage, H. pylori accumulates genetic changes that impact further phenotypic analyses. Specifically, we first sought to characterize the roles of the flagellar genes, pflA and flgS, in biofilm formation; the flgS mutant strain was biofilm deficient, but the pflA mutant strain was a hyper-biofilm former; however, the pflA mutant strain phenotype was unstable. Analysis and screening of six new pflA mutant strains revealed variable biomass phenotypes. This unexpected result led us to explore how genetic heterogeneity within an H. pylori population may complicate standard mutagenesis processes and the interpretation of downstream phenotypes. Analysis of single colony isolates from multiple wild-type strains similarly yielded different biomass phenotypes despite the expected isogenic nature of these isolates. Genomic sequencing of a subset of these isolates revealed various nucleotide changes. Analysis of some of these changes revealed that mutation of futB, babA, or babB did not affect biofilm formation, while mutation of sabA, which encodes the SabA adhesion, resulted in a significant decrease in H. pylori biofilm formation. Overall, these findings reveal that nucleotide changes that occur during a single passage of H. pylori may impact downstream phenotypic analyses. Moreover, these data emphasize the necessity of genetic confirmation redundancy and/or complementation to conclusively move from correlation to causation when analyzing phenotypes of constructed mutant strains.