1. IntroductionGram-negative bacteria have a complex cell envelope consisting of an inner membrane (IM), a thin peptidoglycan layer, and an outer membrane (OM)(1Molecular basis of bacterial outer membrane permeability revisited.). The OM exhibits an asymmetric lipid bilayer with lipopolysaccharide (LPS) in the outer leaflet and phospholipids (GP) in the inner leaflet. The specific structure serves as an effective and selective permeability barrier that protects the cells from toxic agents such as antibiotics, bile salts, and detergents, allowing bacterial survival under adverse conditions(2The bacterial outer membrane is an evolving antibiotic barrier.).Klebsiella pneumoniae (K. pneumoniae) is a type of Gram-negative bacteria, which is best known as a pathogen of the human respiratory system that causes pneumonia. The emergence of multidrug resistance especially polymyxin resistance in K. pneumoniae necessitates a critical need for new treatments(3

Centers for Disease Control and Prevention (CDC). (2013) Antibiotic resistance threats in the United States, 2013.

,4Nang S.C. Azad M.A.K. Velkov T. Zhou Q.T. Li J. Rescuing the Last-Line Polymyxins: Achievements and Challenges.). The common mechanisms of polymyxin resistance involve LPS modification, which is mediated by arnBCADTEF operon activated by two-component regulatory systems of PhoPQ, PmrAB, CrrAB in K. pneumoniae(5Trimble M.J. Mlynarcik P. Kolar M. Hancock R.E. Polymyxin: Alternative Mechanisms of Action and Resistance.,6Liu Y.Y. Wang Y. Walsh T.R. Yi L.X. Zhang R. Spencer J. Doi Y. Tian G. Dong B. Huang X. Yu L.F. Gu D. Ren H. Chen X. Lv L. He D. Zhou H. Liang Z. Liu J.H. Shen J. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.). LPS consists of lipid A, core oligosaccharide, and O-specific antigen. Gram-negative bacteria mainly decorate the structure of lipid A, the bioactive component of LPS, in response to polymyxin treatments to promote antimicrobial resistance and interfere with the ability of host recognition. The general lipid A remodeling contains 4-amino-4-deoxy-L-arabinose (L-Ara4N), phosphoethanolamine (PEtN) addition, and palmitoylation, which would lead to perturbation of OM permeability(7Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis.). As major constituents of the OM, GP contents may vary with the lipid A alterations to maintain a healthy balance between membrane integrity and lipid A modification. Given that the maintenance of OM is essential for bacterial survival, understanding the GP regulation of OM may empower the development of new therapeutics.In E. coli, the cell membranes are composed of three major GP species including ∼75% glycerophosphoethanolamines (PE), ∼20% glycerophosphoglycerols (PG), and 5% cardiolipin (CL)(8Biosynthesis and function of phospholipids in Escherichia coli.,9Lopez-Lara I.M. Geiger O. Bacterial lipid diversity.). Like E. coli, the membrane glycerophospholipids of K. pneumoniae mainly consist of PE, PG, and CL. Each phospholipid class comprises a multitude of molecular species defined by acyl chain compositions. GP are synthesized in the cytoplasm or inner membrane, and transported to the OM by specialized transporters. Unlike LPS transport, GP transport between the IM and the OM is bidirectional, which still requires thorough investigation(10Translocation of phospholipids between the outer and inner membranes of Salmonella typhimurium.). It has been demonstrated that the Mla system and the Tol-Pal complex were important for retrograde transport of GP(11Shrivastava R. Jiang X. Chng S.S. Outer membrane lipid homeostasis via retrograde phospholipid transport in Escherichia coli.,12Malinverni J.C. Silhavy T.J. An ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane.). Recently, AsmA-like proteins YhdP, TamB, and YdbH were reported to be necessary for proper anterograde transport of GP(13Douglass M.V. McLean A.B. Trent M.S. Absence of YhdP, TamB, and YdbH leads to defects in glycerophospholipid transport and cell morphology in Gram-negative bacteria.,14Ruiz N. Davis R.M. Kumar S. YhdP, TamB, and YdbH Are Redundant but Essential for Growth and Lipid Homeostasis of the Gram-Negative Outer Membrane.).To prevent uncontrolled breakdown resulting from surrounding mislocalized GP, Gram-negative bacteria regulate the GP contents from the outer leaflet through several mechanisms. PagP is an OM palmitoyltransferase that removes sn-1 palmitate residue (C16:0) from GP and transfers it to lipid A or PG, producing hepta-acylated Lipid A or palmitoylated PG with lysophospholipid (lyso-PL) as byproducts(15Dalebroux Z.D. Miller S.I. Salmonellae PhoPQ regulation of the outer membrane to resist innate immunity., 16Dalebroux Z.D. Matamouros S. Whittington D. Bishop R.E. Miller S.I. PhoPQ regulates acidic glycerophospholipid content of the Salmonella Typhimurium outer membrane., 17Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R. Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria.). PagP is expressed at a low level in unstressed cells, but can be induced by PhoPQ or membrane damage(18Jia W. El Zoeiby A. Petruzziello T.N. Jayabalasingham B. Seyedirashti S. Bishop R.E. Lipid trafficking controls endotoxin acylation in outer membranes of Escherichia coli.). Unlike PagP, another OM phospholipase PldA removes both sn-1 and sn-2 fatty acid side chains from GP and lyso-PL that have miscompartmentalized to the outer leaflet of the OM(19Outer-membrane phospholipase A: known structure, unknown biological function.). Moreover, it is known that accumulated GP in the OM can be translocated back to the IM by retrograde (OM to IM) GP transport to maintain the OM homeostasis. The Mla system consisting of an ABC transporter MlaFEDB, a periplasmic protein MlaC, and an OM lipoprotein MlaA has been shown to be a phospholipid transport system that is important for maintaining OM asymmetry(12Malinverni J.C. Silhavy T.J. An ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane.,20Kamischke C. Fan J. Bergeron J. Kulasekara H.D. Dalebroux Z.D. Burrell A. Kollman J.M. Miller S.I. The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane.).Studies on two-component systems including PhoPQ, PmrAB, and CrrAB, regulators of polymyxin resistance, mainly focused on LPS modification(21Bhagirath A.Y. Li Y. Patidar R. Yerex K. Ma X. Kumar A. Duan K. Two Component Regulatory Systems and Antibiotic Resistance in Gram-Negative Pathogens.). However, our understanding of GP alterations varies with lipid A modification is still lacking. Mutations in crrB have only recently been reported to regulate high-level polymyxin resistance through CrrAB activation(22Sun L. Rasmussen P.K. Bai Y. Chen X. Cai T. Wang J. Guo X. Xie Z. Ding X. Niu L. Zhu N. You X. Kirpekar F. Yang F. Proteomic Changes of Klebsiella pneumoniae in Response to Colistin Treatment and crrB Mutation-Mediated Colistin Resistance.). Here we analyzed the GP alterations regulated by CrrAB using lipidomic analysis and found that CrrAB activated PagP, which appears to act as a bifunctional palmitoyltransferase that transfers palmitate chain from GP to both lipid A and PG within the OM, generating palmitoylated lipid A and acyl-PG in K. pneumoniae. The altered outer membrane may attenuate lipopolysaccharide activation of TLR4 signal transduction pathway and contribute to selective cationic antimicrobial peptide (CAMP) resistance. Our work presented the phospholipid regulation varies with lipopolysaccharide modification, which provides insight into possible antimicrobial strategies that target the OM barrier.4. DiscussionRecent advances have revealed that CrrAB activates LPS modification including L-Ara4N and pEtN, leading to polymyxin resistance(22Sun L. Rasmussen P.K. Bai Y. Chen X. Cai T. Wang J. Guo X. Xie Z. Ding X. Niu L. Zhu N. You X. Kirpekar F. Yang F. Proteomic Changes of Klebsiella pneumoniae in Response to Colistin Treatment and crrB Mutation-Mediated Colistin Resistance.,27McConville T.H. Annavajhala M.K. Giddins M.J. Macesic N. Herrera C.M. Rozenberg F.D. Bhushan G.L. Ahn D. Mancia F. Trent M.S. Uhlemann A.C. CrrB Positively Regulates High-Level Polymyxin Resistance and Virulence in Klebsiella pneumoniae.,30Cheng Y.H. Lin T.L. Lin Y.T. Wang J.T. Amino Acid Substitutions of CrrB Responsible for Resistance to Colistin through CrrC in Klebsiella pneumoniae.). Here we investigated that CrrAB activation not only stimulates LPS remodeling but also regulates the GP components especially acyl-PG species to maintain a balance within the OM.It’s of note that although LPS was modified by L-Ara4N and palmitoylation via elevated expression of arnBCADTEF and pagP, CrrAB activation appeared to have a minor impact on the LPS biosynthesis and degradation pathway since the expression of proteins linked to LPS biosynthesis (LpxACDHBK/WaaA/LpxL), transport (LptACDE), and degradation (YciM/FtsH) remained stable compared to the wild type (data not shown)(31Guest R.L. Rutherford S.T. Silhavy T.J. Border Control: Regulating LPS Biogenesis.,32Fivenson E.M. Bernhardt T.G. An Essential Membrane Protein Modulates the Proteolysis of LpxC to Control Lipopolysaccharide Synthesis in Escherichia coli.). As a consequence, CrrAB activation regulated the ratio of modified LPS to unmodified LPS, but presumably didn’t alter the total LPS level. Since lipid A modified with two L-Ara4N and a palmitoylation was the last-eluted on reversed-phase HPLC, the production of palmitoylated lipid A increased the hydrophobicity of the outer leaflet in the OM. Meanwhile, the ratio of acyl-PG species to PE and PG species in the crrB mutant was significantly elevated with the OM (∼2-fold), which increased the hydrophobicity of the inner leaflet in the OM. Interestingly, GP contents within the IM were almost invariable. Thus, the increased acyl-PG within the OM was thought to cooperate with the palmitoylated lipid A to maintain the balance of OM. The alteration of GP in the inner leaflet and the modified LPS in the outer leaflet increases the hydrophobicity of the OM. Previous studies have reported that elevated hydrophobicity could block surface association and membrane intercalation of amphipathic alpha-helical CAMP, promoting resistance to various CAMPs such as C18G(7Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis.,33Guo L. Lim K.B. Poduje C.M. Daniel M. Gunn J.S. Hackett M. Miller S.I. Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides.). According to this, elevated acyl-PG accompanied with palmitoylated lipid A in the crrB mutant may co-contribute to certain CAMP resistance. It is of note that the palmitoylation of lipid A caused by PagP wasn’t the determinant of polymyxin resistance since the polymyxin MIC was still above 2048 μg/mL after blocking the palmitoylation of lipid A and PG by pagP knockout in the crrB mutant. PagP is required for Bordetella bronchiseptica to persist in the mouse respiratory tract(34Preston A. Maxim E. Toland E. Pishko E.J. Harvill E.T. Caroff M. Maskell D.J. Bordetella bronchiseptica PagP is a Bvg-regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract.) and to resist antibody-mediated complement lysis(35Pilione M.R. Pishko E.J. Preston A. Maskell D.J. Harvill E.T. pagP is required for resistance to antibody-mediated complement lysis during Bordetella bronchiseptica respiratory infection.). Previous studies reported that PagP-mediated lipid A palmitoylation can attenuate LPS activation of the TLR4/MD2/CD14 innate immune receptor, conferring resistance to host immune defenses(36Pushing the envelope: LPS modifications and their consequences., 37Miller S.I. Ernst R.K. Bader M.W. LPS, TLR4 and infectious disease diversity., 38Kawasaki K. Ernst R.K. Miller S.I. Purification and characterization of deacylated and/or palmitoylated lipid A species unique to Salmonella enterica serovar Typhimurium.), which was in accordance with that crrB mutants display an increase in vivo virulence(27McConville T.H. Annavajhala M.K. Giddins M.J. Macesic N. Herrera C.M. Rozenberg F.D. Bhushan G.L. Ahn D. Mancia F. Trent M.S. Uhlemann A.C. CrrB Positively Regulates High-Level Polymyxin Resistance and Virulence in Klebsiella pneumoniae.).Acyl-PG, also known as hemi bis(monoacylglycero) phosphate (HBMP), is a specific lipid species with undefined biosynthesis and function(39Showalter M.R. Berg A.L. Nagourney A. Heil H. Carraway 3rd, K.L. Fiehn O. The Emerging and Diverse Roles of Bis(monoacylglycero) Phosphate Lipids in Cellular Physiology and Disease.). Our results showed that acyl-PG species are enriched in membrane especially OM in K. pneumoniae (Fig. 3C-D). Combined with the untargeted and targeted lipidomics, we presented the acyl-PG profiles of K. pneumoniae, including 22 kinds in bond levels with more fatty acyl compositions by fragmentation patterns in both positive and negative ion modes (Table 1). The fragmentation patterns of acyl-PG in negative ion mode were already described by Hsu FF et al and Coulon D et al, which offered the fragment ion of the fatty acid chains at sn-1, sn-2 or sn-3(40Acylphosphatidylglycerol (acyl-PG) or N-acylphosphatidylethanolamine (NAPE)?.,41Hsu F.F. Turk J. Shi Y. Groisman E.A. Characterization of acylphosphatidylglycerols from Salmonella typhimurium by tandem mass spectrometry with electrospray ionization.). However, the corresponding position of fatty acid chains was difficult to determine. Here we found that the molecular ion [M+Na+] of acyl-PG fragmented at the linkage between phosphate and a carbon atom on both sides, producing ions [R3COOCH2CH(OH)CH2OPO3HNa]+ and [R1COOR2COOCH2CH(OH)CH2OPO3HNa]+, offering the information of fatty acid chain at sn-3 position (Fig. S1). The increased level of acyl-PG in the crrB mutant backing towards the wild type after pagP knockout revealed that PagP is one of the enzymes responsible for acyl-PG generation, which corresponds to a previous study that purified PagP transfers palmitate to PG(16). PagP is a lipid A palmitoyltransferase possessing precise catalyzation activated by PhoPQ or OM damage(17Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R. Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria.). The different alterations of palmitoylated and non-palmitoylated PE before and after the removal of PE are corresponding to the results that PagP can distinguish palmitate from other acyl chains found in GP(29The lipid A palmitoyltransferase PagP: molecular mechanisms and role in bacterial pathogenesis.) (Fig. 4C). Considering that PagP was an OM protein remodeling the distribution of OM lipids, acyl-PG was thought to be synthesized in the OM(42). The decreased acyl-PG ratio and increased PG ratio within the IM after pagP knockout suggested that enriched OM acyl-PG may translocate to IM, and PG synthesized in the IM may transfer to OM as substrates when PagP was activated (Fig. 3D). To date, the pathways for anterograde and retrograde of GP are less understood. The Mla system has been implicated in both retrograde and anterograde GP transport(43Intermembrane transport: Glycerophospholipid homeostasis of the Gram-negative cell envelope.,44Hughes G.W. Hall S.C.L. Laxton C.S. Sridhar P. Mahadi A.H. Hatton C. Piggot T.J. Wotherspoon P.J. Leney A.C. Ward D.G. Jamshad M. Spana V. Cadby I.T. Harding C. Isom G.L. Bryant J.A. Parr R.J. Yakub Y. Jeeves M. Huber D. Henderson I.R. Clifton L.A. Lovering A.L. Knowles T.J. Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system.). Mla proteins including MlaB, MlaD, MlaE, MlaF were over expressed in the crrB mutant(Fig. 2B). However, the GP contents including PE, PG, acyl-PG, Lyso-PE within the OM and IM in the crrB mutant lack significant changes in the defect of the Mla system. Thus, we suspected that Mla pathway targets a relatively minor population of OM GP molecules or there were unknown lipid trafficking systems offsetting the function of Mla system in the process of CrrAB activation. Previous studies have reported that the MsbA-dependent flip of lipids from the IM to the OM is required for lipid A palmitoylation in vivo(18Jia W. El Zoeiby A. Petruzziello T.N. Jayabalasingham B. Seyedirashti S. Bishop R.E. Lipid trafficking controls endotoxin acylation in outer membranes of Escherichia coli.) and it has been demonstrated to possess phospholipid flippase activity in vitro using fluorescent phospholipid derivatives(45The reconstituted Escherichia coli MsbA protein displays lipid flippase activity.). Since a previous study reported that the active site of PagP was located on the outer leaflet of the OM, phospholipids like PG and PE, normally found in the inner leaflet, may translocate to the outer leaflet as substrates, and acyl-PG products were transported to the inner leaflet when CrrAB was activated(42Hwang P.M. Choy W.Y. Lo E.I. Chen L. Forman-Kay J.D. Raetz C.R. Prive G.G. Bishop R.E. Kay L.E. Solution structure and dynamics of the outer membrane enzyme PagP by NMR.).

Except for acyl-PG, phospholipids including PE, PG, lyso-PE altered in the crrB mutant to some extents. Interestingly, the level of acyl-PG (18:1_18:1)_18:0 and acyl-PG (18:1_18:1)_18:1 that shouldn’t be affected by the expression of PagP were elevated in the crrB mutant. Furthermore, the level of PE without palmitate like PE 12:0_14:0, PE 12:0_16:1, PE 14:0_16:1, PE 14:1_16:1, PE 16:1_16:1, PE 16:1_18:1 that shouldn’t affected by PagP was diminished in the crrB mutant. However, the expression of proteins belonging to de novo pathway of PE, PG and CL like CdsA, PssA, Psd, PgsA, PgpA/B/C, ClsA, PldA showed no difference between the crrB mutant and the wild type. Hence, in order to keep a balance between membrane integrity and lipid A modification, CrrAB activation may modulate the GP contents through other pathways.

Our study demonstrates that CrrAB two-component system regulated lipid A palmitoylation and the outer membrane GP through elevated palmitoyltransferase PagP in K. pneumoniae. Although studies about PagP are extensive, most studies focused on lipid A palmitoylation. Our study suggests that PagP acts as a bifunctional palmitoyltransferase to PG and lipid A, contributing to acyl-PG generation. The finding broadens our understanding of PL metabolism and regulation, assisting in new therapeutic strategies targeting on OM barrier.

Article InfoPublication History

Accepted: June 29, 2022

Received in revised form: June 28, 2022

Received: June 10, 2022

Publication stageIn Press Accepted ManuscriptFootnotes

Funding sources: This work was supported by grants from the National Natural Science Foundation of China (32141003, 82104249, and 82104248), and the CAMS Innovation Fund for Medical Science (2021-1-I2M-030).

Author contributions

Lang Sun: Conceptualization, Methodology, Investigation, Writing-Original Draft. Youwen Zhang: Methodology, Resources. Tanxi Cai: Methodology. Xue Li: Supervision. Na Li: Supervision. Zhensheng Xie: Methodology. Fuquan Yang: Conceptualization, Writing-Reviewing and Editing. Xuefu You: Writing-Reviewing and Editing.

Identification

DOI: https://doi.org/10.1016/j.jlr.2022.100251

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© 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.

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