A Coxiella burnetii phospholipase A homolog pldA is required for optimal growth in macrophages and developmental form lipid remodeling
Many gram-negative bacteria produce an outer membrane phospholipase A (PldA) that plays an important role in outer membrane function and is associated with virulence.
In the current study, we characterized a pldA mutant of Coxiella burnetii, an intracellular gram-negative pathogen and the agent of human Q fever. The C. burnetti pldA open reading frame directs synthesis of a protein with conserved PldA active site residues. A C. burnetii ΔpldA deletion mutant had a significant growth defect in THP-1 macrophages, but not axenic medium, that was rescued by complementation. Thin layer chromatography was employed to assess whether pldA plays a role in remodeling membrane lipids during C. burnetii morphological differentiation. Extracted lipids were analyzed from replicating, logarithmic phase large cell variants (LCVs), non-replicating, stationary phase small cell variants (SCVs), and a mixture of LCVs and SCVs. Similar to Escherichia coli, all three forms contained cardiolipin (CL), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). However, PE and PG were present in lower quantities in the SCV while three additional lipid species were present in higher quantities. Co-migration with standards tentatively identified two of the three SCV-enriched lipids as lyso-phosphatidylethanolamine, a breakdown product of PE, and free fatty acids, which are generally toxic to bacteria. Developmental form lipid modifications required the activity of PldA.
Collectively, these results indicate developmentally-regulated lipid synthesis by C. burnetii contributes to colonization of macrophages and may contribute to the environmental stability and the distinct biological properties of the SCV.
KeywordsCoxiella Lipid Fatty acids Phospholipase A Small cell variant
- 18–1 Lyso-PE
acidified citrate cysteine medium-2
- C. burnetii
- E. coli
free fatty acid
large cell variant
multiplicity of infection
polymerase chain reaction
outer membrane phospholipase A
quantitative polymerase chain reaction
small cell variant
thin layer chromatography
Coxiella burnetii is a gram-negative intracellular pathogen noted for high environmental stability and a low infectious dose via the aerosol route of infection . C. burnetii causes an acute flu-like illness known as Q fever. Following infection, the organism traffics to a vacuole with lysosomal characteristics . Replication of the organism proceeds via a bi-phasic developmental cycle, during which it transitions from a large cell variant (LCV) to a small cell variant (SCV) developmental form [3, 4, 5]. The LCV is considered the replicative form and is present during logarithmic growth. As bacterial growth enters stationary phase, LCVs differentiate into SCVs. As compared to LCVs, SCVs have low metabolic activity and increased resistance to osmotic and physical stressors . These resistance properties are thought to promote environmental stability by the SCV .
Despite the apparent importance of SCVs in C. burnetii disease transmission and pathogenesis, relatively little is known about biochemical changes during transition that confer the unique biological properties of the SCV. Ultrastructural differences between LCVs and SCVs predicted to promote SCV stability include a thicker cell envelope, different peptidoglycan cross linking, condensed chromatin, and synthesis of two highly basic DNA binding proteins [4, 5, 6].
The gram-negative cell envelope is composed of an inner and outer membrane, with peptidoglycan separating the two membranes. Both leaflets of the inner membrane are composed of phospholipids, whereas, the outer membrane has an inner leaflet of phospholipids and an outer leaflet of lipopolysaccharide. Phospholipid modifications are common in bacteria and can provide an increase in resistance properties . For example, cyclopropanation of phospholipid acyl chains in Escherichia coli increases resistance to acid stress .
Considering the importance of phospholipids in cell envelope function, few studies of these molecules have been conducted in C. burnetii. A report published in 2002 demonstrated phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylglycerol (PG) in both virulent phase I and avirulent phase II C. burnetii, which produce full-length and truncated lipopolysaccharide, respectively . Phosphatidylinositol (PI) was also detected in phase II bacteria . A subsequent study showed no PI in C. burnetii, which is consistent with the absence of genes responsible for synthesis of this lipid [10, 11]. Moreover, PI is common in eukaryotes but rare in eubacteria . Both studies used C. burnetii purified from infected hen’s eggs; thus, it is possible that contaminating eukaryotic host cell membrane was in bacterial preparations. The majority of C. burnetii fatty acids are branched, with little difference in the profiles between the cell envelope of LCVs and SCVs [13, 14].
In the current study, we utilized axenic culture to characterize the lipid profile of C. burnetii as it transitions from the LCV to SCV developmental form. We show marked changes in lipid content during differentiation that are attributable to the activity of a predicted outer membrane phospholipase A (PldA).
C. burnetii pldA is required for optimal growth in macrophages
C. burnetii produces a unique lipid profile that changes during the LCV to SCV transition
C. burnetii likely produces lyso-phosphatidylethanolamine and free fatty acids
C. burnetii pldA participates in lipid remodeling associated with LCV to SCV transition
Compositional differences between LCV and SCV developmental forms that contribute to their distinct biological and structural properties are poorly defined. A few proteins have been identified that are differentially synthesized by the LCV and SCV, including the small basic DNA binding proteins ScvA and Hq1 that are associated with the condensed chromatin of SCVs [22, 23, 24]. Warrier et al.  defined 15 developmentally regulated small RNAs that may play roles in differentiation. Sandoz and coworkers  recently demonstrated major changes in peptidoglycan structure during LCV to SCV transition. Here, we demonstrate additional changes to the SCV cell envelope that involve PldA-dependent changes in lipid composition.
PldA-dependent breakdown of PE produces FFA and Lyso-PE. The accumulation of FFA by the C. burnetii SCV is perplexing as these molecules are considered toxic to bacteria . In fact, we are aware of only one organism that stores large amounts of medium-to-long chain saturated fatty acids, an anaerobic bacterium known as G12 that is related to Eubacterium cylidroides . This bacterium appears to accumulate FFA during homeoviscous adaptation to environmental stress. Many bacterial species utilize stores of lipophilic compounds as energy and carbon sources, generally in the form of polyhydroxyalkanoates, such as poly(3-hydroxybutyrate) . How C. burnetii tolerates FFA toxicity and the potential role(s) these compounds play in pathogen physiology are intriguing questions. Given that the greatest amount of FFA is seen in the SCV, these lipid molecules may serve as a nutrient source for outgrowth of the LCV during the initial stages of infection, when the CCV may be limited in nutrients [2, 30].
Lysophopholipids affect membrane stiffness and fluidity, which in turn influences membrane permeability and pore function [20, 31]. PldA-dependent accumulation of lysophopholipids in the Helicobacter pylori membrane promotes release of urease and VacA toxin [32, 33]. Both molecules enhance adherence to epithelial cells and development of ulcer disease [32, 33]. In E. coli, PldA facilitates release of bacteriocins [34, 35]. Shigella flexneri needs PldA for efficient type III secretion and to maintain membrane integrity . We show that PldA-deficient C. burnetii clearly has a growth defect in human macrophages, although the precise mechanism of this attenuation remains to be defined. One possibility is that PldA contributes to a recently described Sec-mediated secretion system of C. burnetii . Another possibility is that mutant SCVs are less resistant to the lysosomal environment.
In this work, developmentally-regulated synthesis of C. burnetii lipids was described. PldA was responsible for enrichment of Lyso-PE and FFA in the SCV. These data, along with major modifications of SCV peptidoglycan , indicate the C. burnetii cell envelope undergoes substantial remodeling during morphologic differentiation. PldA also promotes pathogen growth in the harsh, lysosome-like environment of macrophages. Further characterization of PldA and the unusual lipids generated by the enzyme will provide needed insight into C. burnetii resistance and pathogenesis.
Bacterial and mammalian cell culture
Bacterial strains are described in Additional file 2: Table S1. ACCM-2 or ACCM-2 agarose was employed to grow C. burnetii as previously described . E. coli strains were grown in Luria-Bertani (LB) broth at 37 °C. E. coli W3110 cells were used for lipid isolation while E. coli Stellar cells were used for recombinant DNA procedures. LB agar plates containing 50 μg of kanamycin/ml or 10 μg of chloramphenicol/ml were used to select E. coli transformants. The human acute monocytic leukemia cell line THP-1 (TIB-202; American Type Culture Collection) was grown at 37 °C and 5% CO2 in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone).
Recombinant DNA techniques
Plasmids used in the study are listed in Additional file 2: Table S1. Accuprime Pfx DNA polymerase (Invitrogen) and oligonucleotide primers (Integrated DNA Technologies) were employed in PCR. Primer sequences are listed in Additional file 3: Table S2. PCR products were cloned using the In-Fusion PCR cloning system (BD Clontech). Restriction enzymes were purchased from New England Biolabs.
Construction of pJC-amp and pMini-Tn7T-Kan
For construction of pJC-Amp, the 1169 promoter and amp gene were amplified from pJB-CAT by PCR. The PCR products were cloned by In-Fusion into SalI/NheI-digested pJC-CAT to create pJC-Amp. For construction of pMini-Tn7T-Kan, the 1169 P -kan fragment was amplified from pJB-Kan by PCR. The 1169 P -kan amplicon was cloned by In-Fusion into SalI-digested pMini-Tn7T-CAT to create pMini-Tn7T-Kan.
Construction of pJC-amp::pldA-5′3′-CAT for targeted gene deletion
The pldA 5′ and 3′ flanking regions were amplified from Nine Mile (RSA439) genomic DNA by PCR. The PCR products were cloned by In-Fusion into BamHI/SalI-digested pJC-Amp to create pJC-Amp::pldA-5′3′. The 1169 P -cat fragment was amplified from pJB-CAT by PCR. The 1169 P -cat amplicon was cloned by In-Fusion into PstI-digested pJC-Amp::pldA-5′3′ to create the knock out vector pJC-Amp::pldA-5′3′-CAT.
Construction of pMini-Tn7T-Kan::pldAcomp for complementation
The pldA gene and its upstream promoter region was amplified from Nine Mile (RSA439) genomic DNA by PCR. The PCR product was cloned by In-Fusion into EcoRI-digested pMini-Tn7T-Kan to create pMini-Tn7T-Kan::pldA comp.
C. burnetii gene deletion and complementation
Deletion of pldA was achieved as previously described using pJC-Amp::pldA-5′3′-CAT and 3 μg/ml chloramphenicol for antibiotic selection . The mutant strain was cloned by picking colonies propagated on ACCM-2 agarose. Gene deletion was confirmed by PCR. C. burnetii ΔpldA was complemented with single copy pMini-Tn7T-Kan::pldAcomp as previously described using 350 μg/ml kanamycin for antibiotic selection .
Antibody generation and immunoblotting of C. burnetii lysates
Monospecific rabbit polyclonal antibody directed against C. burnetii PldA was generated using the PldA-specific synthetic peptide CRHIKRYDKKTKHY (Alpha Diagnostic International, San Antonio, TX). Cell lysates were prepared by boiling 2 × 108 genomic equivalents (GE) of each C. burnetii strain in Laemmli sample buffer for 10 min. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a 4–20% gel (Bio-Rad), transferred to nitrocellulose, and probed with the anti-PldA antibody. Reacting proteins were detected using anti-rabbit IgG secondary antibodies conjugated to horseradish peroxidase (Thermo Scientific, Waltham, MA) and chemiluminescence using ECL Pico reagent (Thermo Scientific).
Quantification of C. burnetii replication
Replication was quantified by qPCR (quantitative polymerase chain reaction) of C. burnetii GE using a primer and probe set specific to C. burnetii dotA . ACCM-2 cultures were inoculated with 1 × 106 GE. THP-1 monocytes were differentiated into macrophage-like cells with phorbol-12-myristate-13-acetate (PMA), then infected at a multiplicity of infection (MOI) of 0.2 . Samples were taken immediately and 6 days post-inoculation/infection. Each sample was diluted in 150 μl phosphate-buffered saline (PBS) and boiled for 10 min prior to qPCR. Three independent experiments were performed in triplicate.
Immunofluorescence and CCV analysis
THP-1 cells were seeded on coverslips in 24-well plates at a density of 1 × 105 cells per well, stimulated with PMA for 1 day, then infected at an MOI of 10. At 4 days post-infection, cells were fixed for 30 min with 4% paraformaldehyde, then permeabilized and blocked with 0.1% Triton X-100 containing 1% bovine serum albumin. Cells were fluorescently stained for CD63 (mouse monoclonal antibody H5C6, BD Pharmingen) and C. burnetii (rabbit anti-Nine Mile phase II strain antibody). Alexa Fluor-488 goat anti-mouse and Alexa Fluor-647 goat anti-rabbit antibodies were from Life Technologies. For staining nuclei, Hoescht 33342 (ThermoFisher) was used and for visualization of cell borders, the filamentous actin stain, BODIPY 558/568 labeled phalloidin (Life Technologies) was used. Imaging was performed on a Zeiss LSM-710 confocal fluorescence microscope (Carl Zeiss). Fiji (Image J; National Institutes of Health, USA) was used for measuring areas of CCVs, where CD63 served as a CCV membrane marker. A minimum of 80 cells for each infection from 3 independent experiments were used for analysis.
Isolation and analysis of lipid species
E. coli was grown to an optical density of 1, harvested at 4000×g for 20 min, then the pellet washed once with PBS (1.5 mM KH2PO4, 2.7 mM Na2HPO4-7H2O, 155 mM NaCl, [pH 7.2]). C. burnetii was grown for 4, 7, and 14 days, harvested at 20,000×g for 20 min, then the pellet washed once with PBS. Lipids were extracted by the method of Bligh and Dyer . Dry weights were determined and the lipids spotted onto a Silica Gel 60 TLC plate (E. coli, 150 to 200 μg per lane; C. burnetii, 400 to 550 μg per lane). Lipids were separated using a solvent system containing chloroform, methanol, and glacial acetic acid (65:25:10, v/v). To visualize the lipids, TLC plates were sprayed with 10% sulfuric acid in ethanol and heated on a hot plate at 200 °C. Standards were obtained from the following suppliers and used in the indicated quantities: 18:1 Lyso-PE, 50 μg (Avanti Polar Lipids, Alabaster, AL); palmitoleic acid, 5 μg (Sigma-Aldrich, St. Louis, MO).
Statistical analyses were performed using the unpaired Student t test and GraphPad Prism 6.0 software (La Jolla, CA).
We thank Anita Mora for preparation of figures.
This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
CMS and RAH conceived and supervised the project, and interpreted the data. PAB made pldA constructs for allelic exchange. CMS generated the pldA mutant and performed lipid extractions, TLC, and western blotting. CMS and DCC performed macrophage growth assays. CMS and RAH wrote the manuscript. HEM performed confocal microscopy and vacuole measurements All authors reviewed and approved the manuscript.
Ethics approval and consent to participate
The authors declare that they have no competing interests.
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- 5.McCaul TF, Hackstadt T, Williams JC. Ultrastructural and biological aspects of Coxiella burnetii under physical disruptions. In: Burgdorfer W, Anacker RL, editors. Rickettsiae and rickettsial diseases. New York, NY: academic press, Inc; 1981. p. 267–80.Google Scholar
- 11.Beare PA, Unsworth N, Andoh M, Voth DE, Omsland A, Gilk SD, Williams KP, Sobral BW, Kupko JJ 3rd, Porcella SF, et al. Comparative genomics reveal extensive transposon-mediated genomic plasticity and diversity among potential effector proteins within the genus Coxiella. Infect Immun. 2009;77(2):642–56.CrossRefPubMedGoogle Scholar
- 36.Wang X, Jiang F, Zheng J, Chen L, Dong J, Sun L, Zhu Y, Liu B, Yang J, Yang G, et al. The outer membrane phospholipase a is essential for membrane integrity and type III secretion in Shigella flexneri. Open Biol. 2016;6(9):160073.Google Scholar
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