Protein–protein interactions in the Mla lipid transport system probed by computational structure prediction and deep mutational scanning

MacRae, M.R.; Puvanendran, Dhenesh; Haase, Max A. B.; Coudray, Nicolas; Kolich, Ljuvica; Lam, Cherry; Baek, Minkyung; Bhabha, Gira; Ekiert, Damian

doi:10.1016/j.jbc.2023.104744

Cited by 14 publications

(17 citation statements)

References 45 publications

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“…Due to its strong hydrophobicity, the OM reduces the entry of toxic compounds such as antibiotics, bile salts, and detergents ( 59 ). The Mla (maintenance of lipid asymmetry) pathway is used to transport phospholipids between the inner membrane and OM of gram-negative bacteria ( 60 – 63 ), while the Bam pathway is needed for assembly of OM proteins ( 64 ). The sensitivity of VT101 toward EDTA and SDS resembles similar sensitivity of E. coli and Acinetobacter baumannii Δ mla mutants ( 61 , 62 ) and the E. coli Δ bamD mutant ( 65 ), each of which displays defects in OM lipid asymmetry.…”

Section: Discussionmentioning

confidence: 99%

Klebsiella pneumoniae DedA family proteins have redundant roles in divalent cation homeostasis and resistance to phagocytosis

Tiwari,

Sharma,

Braga

et al. 2024

Microbiol Spectr

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The DedA superfamily is a highly conserved family of membrane proteins. Deletion of Escherichia coli yqjA and yghB , encoding related DedA family proteins, results in sensitivity to elevated temperature, antibiotics, and alkaline pH. The human pathogen Klebsiella pneumoniae possesses genes encoding DedA family proteins with >90% amino acid identity to E. coli YqjA and YghB. We hypothesized that the deletion of K. pneumoniae yqjA and yghB will impact its physiology and may reduce its virulence. The K. pneumoniae Δ yqjA Δ yghB mutant (strain VT101) displayed a growth defect at 42°C and alkaline pH sensitivity, not unlike its E. coli counterpart. However, VT101 retained mostly wild-type resistance to antibiotics. We found VT101 was sensitive to the chelating agent EDTA, the anionic detergent SDS, and agents capable of alkalizing the bacterial cytoplasm such as bicarbonate or chloroquine. We could restore growth at alkaline pH and at elevated temperature by addition of 0.5–2 mM Ca 2+ or Mg 2+ to the culture media. VT101 displayed a slower uptake of calcium, which was dependent upon calcium channel activity. VT201, with similar deletions as VT101 but derived from a virulent K. pneumoniae strain, was highly susceptible to phagocytosis by alveolar macrophages and displayed a defect in the production of capsule. These findings suggest divalent cation homeostasis and virulence are interlinked by common functions of the DedA family. IMPORTANCE Klebsiella pneumoniae is a dangerous human pathogen. The DedA protein family is found in all bacteria and is a membrane transporter often required for virulence and antibiotic resistance. K. pneumoniae possesses homologs of E. coli YqjA and YghB, with 60% amino acid identity and redundant functions, which we have previously shown to be required for tolerance to biocides and alkaline pH. A K. pneumoniae strain lacking yqjA and yghB was found to be sensitive to alkaline pH, elevated temperature, and EDTA/SDS and displayed a defect in calcium uptake. Sensitivity to these conditions was reversed by addition of calcium or magnesium to the growth medium. Introduction of Δ yqjA and Δ yghB mutations into virulent K. pneumoniae resulted in the loss of capsule, increased phagocytosis by macrophages, and a partial loss of virulence. These results show that targeting the Klebsiella DedA family results in impaired divalent cation transport and, in turn, loss of virulence.

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Section: Discussionmentioning

confidence: 99%

Klebsiella pneumoniae DedA family proteins have redundant roles in divalent cation homeostasis and resistance to phagocytosis

Tiwari,

Sharma,

Braga

et al. 2024

Microbiol Spectr

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“…Whole genome sequencing data is available in the National Center for Biotechnology Information (NCBI) sequence read archive (SRA) under BioProject: PRJNA1020123 and PRJNA656143 ( 22 ).…”

Section: Data Availabilitymentioning

confidence: 99%

“…MlaBDEF forms an IM ATP-binding cassette (ABC) transporter with ATPase activity, MlaC is a periplasmic protein, and MlaA is an OM lipoprotein that forms a complex with OmpC/F ( 16 – 19 , 20 ). While the Mla system is not required for growth in lysogeny broth, bacteria lacking a functional Mla system are more sensitive to the membrane stress SDS/EDTA ( 16 , 17 , 21 – 23 ). Inactivating mlaF or mlaC in multiple species results in the loss of function of the Mla system and increased sensitivity to membrane stressors, antibiotics, and the host ( 21 , 24 – 26 ).…”

Section: Introductionmentioning

confidence: 99%

“…However, Mann et al, whose structural work in A. baumannii supports an anterograde lipid transport model, speculate that nucleotide state or solubilization approach of the MlaBDEF studies may explain the differences in conclusions ( 48 ). Recent structures of MlaBDEF from E. coli and A. baumannii and E. coli MlaC in complex with MlaA or MlaD were solved and provide mechanistic insight on lipid binding but do not provide clear evidence for either direction of lipid transport ( 22 , 49 , 50 ).…”

Section: Introductionmentioning

confidence: 99%

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Genetic synergy between Acinetobacter baumannii undecaprenyl phosphate biosynthesis and the Mla system impacts cell envelope and antimicrobial resistance

Noel,

Keerthi,

Ren

et al. 2024

mBio

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Acinetobacter baumannii is a Gram-negative bacterial pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The maintenance of lipid asymmetry (Mla) system is the main homeostatic mechanism by which Gram-negative bacteria maintain outer membrane asymmetry. Loss of the Mla system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978: 17978VU and 17978UN. Here, ∆ mlaF mutants in the two ATCC 17978 strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. Although allele differences in obgE were previously reported to synergize with ∆ mlaF to affect growth and stringent response, obgE alleles do not affect membrane stress resistance. Instead, a single-nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS , results in decreased enzymatic rate and decrease in total Und-P levels in 17978UN compared to 17978VU. The UppS UN variant synergizes with ∆ mlaF to reduce capsule and lipooligosaccharide (LOS) levels, increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier required for the biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii cell envelope and antibiotic resistance. IMPORTANCE Acinetobacter baumannii is a critical threat to global public health due to its multidrug resistance and persistence in hospital settings. Therefore, novel therapeutic approaches are urgently needed. We report that a defective undecaprenyl pyrophosphate synthase (UppS) paired with a perturbed Mla system leads to synthetically sick cells that are more susceptible to clinically relevant antibiotics and show reduced virulence in a lung infection model. These results suggest that targeting UppS or undecaprenyl species and the Mla system may resensitize A. baumannii to antibiotics in combination therapies. This work uncovers a previously unknown synergistic relationship in cellular envelope homeostasis that could be leveraged for use in combination therapy against A. baumannii .

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“…To date, a major unknown in OM biogenesis is the mechanism by which PLs are transported to the OM. The most extensively characterised intermembrane PL transport pathway remains the E. coli Maintenance of Lipid Asymmetry (Mla) pathway (2, 7), which consists of a periplasmic protein that shuttles PLs between an IM ABC transporter and an OM complex (8–19). However, whilst some studies indicate a role in anterograde transport (20, 21), the majority of research suggests the Mla pathway facilitates retrograde transport, removing mislocalised PLs from the outer leaflet of the OM (2, 10, 14, 15, 17, 22, 23).…”

Section: Introductionmentioning

confidence: 99%

Phospholipid transport to the bacterial outer membrane through an envelope-spanning bridge

Cooper,

Clark,

Kudhail

et al. 2023

Preprint

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The outer membrane of Gram-negative bacteria provides a formidable barrier, essential for both pathogenesis and antimicrobial resistance. Biogenesis of the outer membrane requires the transport of phospholipids across the cell envelope. Recently, YhdP was implicated as a major protagonist in the transport of phospholipids from the inner membrane to the outer membrane however the molecular mechanism of YhdP mediated transport remains elusive. Here, utilising AlphaFold, we observe YhdP to form an elongated assembly of 60 β strands that curve to form a continuous hydrophobic groove. This architecture is consistent with our negative stain electron microscopy data which reveals YhdP to be approximately 250 Å in length and thus sufficient to span the bacterial cell envelope. Furthermore, molecular dynamics simulations and in vivo bacterial growth assays indicate essential helical regions at the N- and C-termini of YhdP, that may embed into the inner and outer membranes respectively, reinforcing its envelope spanning nature. Our in vivo crosslinking data reveal phosphate-containing substrates captured along the length of the YhdP groove, providing direct evidence that YhdP transports phospholipids. This finding is congruent with our molecular dynamics simulations which demonstrate the propensity for inner membrane lipids to spontaneously enter the groove of YhdP. Collectively, our results support a model in which YhdP bridges the cell envelope, providing a hydrophobic environment for the transport of phospholipids to the outer membrane.

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Protein–protein interactions in the Mla lipid transport system probed by computational structure prediction and deep mutational scanning

Cited by 14 publications

References 45 publications

Klebsiella pneumoniae DedA family proteins have redundant roles in divalent cation homeostasis and resistance to phagocytosis

Klebsiella pneumoniae DedA family proteins have redundant roles in divalent cation homeostasis and resistance to phagocytosis

Genetic synergy between Acinetobacter baumannii undecaprenyl phosphate biosynthesis and the Mla system impacts cell envelope and antimicrobial resistance

Phospholipid transport to the bacterial outer membrane through an envelope-spanning bridge

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