The Mla pathway is critical for Pseudomonas aeruginosa resistance to outer membrane permeabilization and host innate immune clearance

Munguia, Jason; LaRock, Doris L.; Tsunemoto, Hannah; Olson, Joshua; Cornax, Ingrid; Pogliano, Joseph; Nizet, Victor

doi:10.1007/s00109-017-1579-4

Cited by 34 publications

(34 citation statements)

References 47 publications

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“…Genes BCAL0302 and BCAL0305 are part of an eight-gene locus coding for ABC transporter systems that share high sequence identities at both gene and protein levels with the Mla pathway ( Fig. 2 and Table 2) found in various Gram-negative bacteria, including Escherichia coli, Shigella flexneri, and P. aeruginosa (17)(18)(19). Reciprocal BLASTP analyses showed that only MlaB from E. coli K-12 or PA4452 from P. aeruginosa strain PAO1 had no homologue in B. cenocepacia (Table 2).…”

Section: Resultsmentioning

confidence: 99%

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

2018

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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.

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Section: Resultsmentioning

confidence: 99%

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

2018

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“…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] .…”

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confidence: 99%

“…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).…”

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confidence: 99%

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

Tang

Chang

Wen

et al. 2020

Preprint

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The asymmetric phospholipid outer membrane (OM) of Gram-negative bacteria serves as the first line of defense against cytotoxic substances such as antibiotics. The Mla pathway is known to maintain the lipid asymmetry of the OM by transporting phospholipids between the inner and outer membranes. Six Mla proteins MlaFEDBCA are involved, with the ABC transporter MlaFEDB acts through a mechanism yet to be elucidated. Here we determine cryo-EM structures of MlaFEDB in apo, phospholipid-, ADP-or AMP-PNP-bound state to 3.3-3.75 Å resolution and establish a proteoliposome-based transport system containing MlaFEDB, MlaC and MlaA/OmpF to reveal the transport direction of phospholipids. Mutagenetic in vitro transport assays and in vivo sensitivity assays reveal functional residues which recognize and transport phospholipids as well as regulate the activity and structural stability of the MlaFEDB complex. Our work provides molecular basis for understanding the mechanism of the Mla pathway which could be targeted for antimicrobial drug development.

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“…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 .…”

Section: Introductionmentioning

confidence: 96%

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

Yero

Costenaro

Conchillo-Solé

et al. 2019

Preprint

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22In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding 23 component of an ABC transport system thought to be involved in maintaining the 24 asymmetry of the outer membrane. The crystallographic structure of Ttg2D at 2.5Å 25 resolution reveals that this protein can bind two diacyl phospholipids. Native and 26 denaturing mass spectrometry experiments confirm that Ttg2D binds two 27 phospholipid molecules, which may have different head groups. Analysis of the 28 available structures of Ttg2D orthologs allowed us to classify this protein family as a 29 novel substrate-binding protein fold and to venture the evolutionary events that 30 differentiated the orthologs binding one or two phospholipids. In addition, gene 31 knockout experiments in P. aeruginosa PAO1 and multidrug-resistant strains show 32 that disruption of this system leads to outer membrane permeabilization. This 33 demonstrates the role of this system in low-level intrinsic resistance against certain 34 antibiotics that use a lipid-mediated pathway to permeate through membranes. 35 36 Introduction 37Pseudomonas aeruginosa are amongst the most important multidrug-resistant (MDR) 38 human pathogens 1 , showing inherent resistance to an important number of the 39 presently available antibiotics 2 . P. aeruginosa are responsible for chronic lung 40 infections in individuals with chronic obstructive pulmonary disease or cystic fibrosis 41 (CF) 3 and account for over a tenth of all nosocomial infections 4 . A number of effective 42 drugs and formulations can treat P. aeruginosa infections, even in CF patients 5 . 43These include frontline antibiotics such as piperazillin-tazobactam, ceftazidime, 44 aztreonam, imipenem, meropenem, ciprofloxacin, levofloxacin, tobramycin, amikacin, 45 and colistin 6 . Yet, resistance to most of these antimicrobials is being increasingly 46 reported 7 . The basis for the inherently high resistance of these microorganisms is 47primarily their low outer-membrane (OM) permeability 8, 9 , complemented by the 48 production of antibiotic-inactivating enzymes (e.g. β-lactamases), the constitutive 49 expression of efflux pumps 10, 11 and the capacity to form biofilms 1, 12 , among other 50 mechanisms. The susceptibility of P. aeruginosa to antimicrobials can be additionally 51 reduced by the acquisition of inheritable traits, including horizontal gene transfers 52 and mutations that decrease uptake and efflux pump overexpression 13, 14, 15 . 53Although a number of genes and mechanisms of resistance to antibiotics are already 54 known in P. aeruginosa, the complex mechanisms controlling the basal, low-level 55 resistance to these compounds are still poorly understood 16, 17 . 56 57The OM of P. aeruginosa is known to be central to its antibiotic-resistance 58 phenotype. Its intrinsically low permeability is partly determined by inefficient OM 59 porin proteins that provide innate resistance to several antimicrobial compounds, 60 mainly hydrophilic 1, 8, 10 . On the other hand, the loss of specific efflux pump ...

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The Mla pathway is critical for Pseudomonas aeruginosa resistance to outer membrane permeabilization and host innate immune clearance

Cited by 34 publications

References 47 publications

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

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

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

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

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