The Mla Pathway Plays an Essential Role in the Intrinsic Resistance of Burkholderia cepacia Complex Species to Antimicrobials and Host Innate Components

Abstract

Antibiotic resistance is a threat to our modern society, and new strategies leading to the identification of new molecules or targets to combat multidrug-resistant pathogens are needed. Species of the genus , including the complex (Bcc), , and, can be highly pathogenic and are intrinsically resistant to multiple classes of antibiotics. Bcc species are nonetheless sensitive to extracellular products released by in interspecies competition. We screened for transposon mutants with increased sensitivity to spent medium and identified multiple mutants in genes sharing homology with the Mla pathway. Insertional mutants in representative genes of the Bcc Mla pathway had a compromised cell membrane and were more sensitive to various extracellular stresses, including antibiotics and human serum. More precisely, mutants in the Bcc species and were more susceptible to Gram-positive antibiotics (i.e., macrolides and rifampin), fluoroquinolones, tetracyclines, and chloramphenicol. Genetic complementation of insertional mutants restored cell permeability and resistance to Gram-positive antibiotics. Importantly, Bcc mutants were not universally weaker strains since their susceptibilities to other classes of antibiotics were unaffected. Although cell permeability of homologous mutants in or was also impaired, they were not more sensitive to Gram-positive antibiotics or other antimicrobials as was observed in Bcc mutants. Together, the data suggest that the Mla pathway in may play a different biological role, which could potentially represent a-specific drug target in combination therapy with antibiotic adjuvants. The outer membrane of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore compromising this structure could increase sensitivity to currently available antibiotics. In this study, we show that the Mla pathway, a system involved in maintaining the integrity of the outer membrane, is genetically and functionally different in complex species compared to that in other proteobacteria. Mutants in genes of or were sensitive to Gram-positive antibiotics, while this effect was not observed in or The Mla pathway in species may represent an ideal genus-specific target to address their intrinsic antimicrobial resistances.

Structural insight into outer membrane asymmetry maintenance of Gram-negative bacteria by the phospholipid transporter MlaFEDB

“…On the other hand, the molecular mechanism of how PLs are trafficked between the IM and the OM to maintain the OM asymmetry remains unclear. The maintenance of lipid asymmetry (Mla) pathway is believed to be important for maintaining the asymmetry and integrity of the OM by trafficking PLs between the IM and the OM [35][36][37][38][39][40][41] . Mutants of the Mla pathway in Escherichia coli (E. coli) or other pathogenic species show OM permeability and integrity defects and increased susceptibility to antibiotics [36][37][38][39][40][41][42] .…”

“…The maintenance of lipid asymmetry (Mla) pathway is believed to be important for maintaining the asymmetry and integrity of the OM by trafficking PLs between the IM and the OM [35][36][37][38][39][40][41] . Mutants of the Mla pathway in Escherichia coli (E. coli) or other pathogenic species show OM permeability and integrity defects and increased susceptibility to antibiotics [36][37][38][39][40][41][42] . Six proteins are involved in the Mla pathway, including an ATP-binding cassette (ABC) transporter MlaFE associated with an auxiliary periplasmic membrane protein MlaD and an auxiliary cytoplasmic protein MlaB, a periplasmic protein MlaC and an OM protein MlaA associated with an osmoporin OmpF/C 36,43,44 (Figure 1A).…”

Evolution toward maximum transport capacity of the Ttg2 ABC system inPseudomonas aeruginosa

“…In fact, the Ttg2/Mla pathway has proven to be an important virulence factor in other pathogens, like Burkholderia pseudomallei , that need to spread into neighboring cells to infect eukaryotic tissues 72 . In Burkholderia cepacia complex species mla genes are required for swarming motility and serum resistance 28 . Furthermore, in nontypeable Haemophilus influenzae (NTHi), it is considered a key factor for bacterial survival in the human airway upon exposure to hydrophobic antibiotics 27 .…”

“…Accordingly, components of the P. aeruginosa ABC transporter encoded by the PA4452-PA4456 have been named Ttg2A (MlaF), Ttg2B (MlaE), Ttg2C (MlaD), Ttg2D (MlaC) and Ttg2E (MlaB) 32 . Recent studies of mutant strains with disrupted ttg2 or vacJ genes support the contribution of this ABC transport system to the intrinsic resistance of P. aeruginosa to antimicrobials 24, 28, 32, 35 . Yet, one of these studies has challenged the role of this system in P. aeruginosa as an ABC importer mediating phospholipid intermembrane trafficking 32 .…”

“…MlaC would then transport the phospholipids across the periplasm and deliver them to MlaD for active internalization through the IM 23 . Deletion of the genes of this system is known to destabilize the OM, and bacterial strains lacking any of the Mla components are more susceptible to membrane stress agents 19, 24, 25, 26, 27, 28, 29, 30 . More recently, the retrograde transport hypothesis has been questioned and a new role for this system in anterograde phospholipid transport has been suggested 29, 31 .…”