Role of the Vibrio cholerae Matrix Protein Bap1 in Cross-Resistance to Antimicrobial Peptides

Abstract

Outer membrane vesicles (OMVs) that are released from Gram-negative pathogenic bacteria can serve as vehicles for the translocation of effectors involved in infectious processes. In this study we have investigated the role of OMVs of the Vibrio cholerae O1 El Tor A1552 strain in resistance to antimicrobial peptides (AMPs). To assess this potential role, we grew V. cholerae with sub-lethal concentrations of Polymyxin B (PmB) or the AMP LL-37 and analyzed the OMVs produced and their effects on AMP resistance. Our results show that growing V. cholerae in the presence of AMPs modifies the protein content of the OMVs. In the presence of PmB, bacteria release OMVs that are larger in size and contain a biofilm-associated extracellular matrix protein (Bap1). We demonstrated that Bap1 binds to the OmpT porin on the OMVs through the LDV domain of OmpT. In addition, OMVs from cultures incubated in presence of PmB also provide better protection for V. cholerae against LL-37 compared to OMVs from V. cholerae cultures grown without AMPs or in presence of LL-37. Using a bap1 mutant we showed that cross-resistance between PmB and LL-37 involved the Bap1 protein, whereby Bap1 on OMVs traps LL-37 with no subsequent degradation of the AMP. Cholera is an epidemic disease that has killed millions of people and continues to be a major health problem worldwide. The bacterium V. cholerae, the causative agent of cholera, is highly resistant to antimicrobial peptides, which are important effectors of human innate immunity. Moreover, it is well-established that different antimicrobial peptides are able to work in synergy against bacteria. Currently, little is known about the mechanisms underlying the resistance of bacteria toward synergistic effects of antimicrobial peptides. For the first time, we provide a mechanistic explanation for cross-resistance between two antimicrobial peptides: PmB and LL-37. We report that bacteria incubated with PmB produce OMVs containing high levels of the Bap1 protein. We also decipher the mechanism by which Bap1 binds to OMVs isolated from V. cholerae incubated in presence of PmB. Finally, our data demonstrated that Bap1 protein associated with OMVs is able to trap LL-37, thereby increasing the minimum inhibitory concentration of LL-37 against V. cholerae when the bacteria were grown with a sub-lethal concentration of PmB and therefore produced abundant Bap1.