Evaluating the Contribution of the Predicted Toxin-Antitoxin System HigBA to Persistence, Biofilm Formation, and Virulence in Burkholderia pseudomallei

Melioidosis is an underreported human disease caused by the Gram-negative intracellular pathogen Burkholderia pseudomallei (Bpm). Both the treatment and the clearance of the pathogen are challenging, with high relapse rates leading to latent infections. This has been linked to the bacterial persistence phenomenon, a growth arrest strategy that allows bacteria to survive under stressful conditions, as in the case of antibiotic treatment, within a susceptible clonal population. At a molecular level, this phenomenon has been associated with the presence of toxin-antitoxin (TA) systems. We annotated the Bpm K96243 genome and selected 11 pairs of genes encoding for these TA systems, and their expression was evaluated under different conditions (supralethal antibiotic conditions; intracellular survival bacteria). The predicted HigB toxin (BPSL3343) and its predicted antitoxin HigA (BPS_RS18025) were further studied using mutant construction. The phenotypes of two mutants (Delta higB and Delta higB Delta higA) were evaluated under different conditions compared to the wild-type (WT) strain. The Delta higB toxin mutant showed a defect in intracellular survival on macrophages, a phenotype that was eliminated after levofloxacin treatment. We found that the absence of the toxin provides an advantage over the WT strain, in both in vitro and in vivo models, during persister conditions induced by levofloxacin. The lack of the antitoxin also resulted in differential responses to the conditions evaluated, and under some conditions, it restored the WT phenotype, overall suggesting that both toxin and antitoxin components play a role in the persister-induced phenotype in Bpm.

Automated summary

Melioidosis, caused by Burkholderia pseudomallei, is an underreported human disease with challenging treatment and clearance. This study reveals the association between bacterial persistence and toxin-antitoxin systems, specifically the HigB toxin and its HigA antitoxin, in the context of levofloxacin-induced persister conditions.