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, 14 (3), e1006922
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Genetic Mechanisms of Coxiella Burnetii Lipopolysaccharide Phase Variation

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Genetic Mechanisms of Coxiella Burnetii Lipopolysaccharide Phase Variation

Paul A Beare et al. PLoS Pathog.

Abstract

Coxiella burnetii is an intracellular pathogen that causes human Q fever, a disease that normally presents as a severe flu-like illness. Due to high infectivity and disease severity, the pathogen is considered a risk group 3 organism. Full-length lipopolysaccharide (LPS) is required for full virulence and disease by C. burnetii and is the only virulence factor currently defined by infection of an immunocompetent animal. Transition of virulent phase I bacteria with smooth LPS, to avirulent phase II bacteria with rough LPS, occurs during in vitro passage. Semi-rough intermediate forms are also observed. Here, the genetic basis of LPS phase conversion was investigated to obtain a more complete understanding of C. burnetii pathogenesis. Whole genome sequencing of strains producing intermediate and/or phase II LPS identified several common mutations in predicted LPS biosynthesis genes. After passage in broth culture for 30 weeks, phase I strains from different genomic groups exhibited similar phase transition kinetics and elevation of mutations in LPS biosynthesis genes. Targeted mutagenesis and genetic complementation using a new C. burnetii nutritional selection system based on lysine auxotrophy confirmed that six of the mutated genes were necessary for production of phase I LPS. Disruption of two of these genes in a C. burnetii phase I strain resulted in production of phase II LPS, suggesting inhibition of the encoded enzymes could represent a new therapeutic strategy for treatment of Q fever. Additionally, targeted mutagenesis of genes encoding LPS biosynthesis enzymes can now be used to construct new phase II strains from different genomic groups for use in pathogen-host studies at a risk group 2 level.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Antibody detection of C. burnetii LPS forms.
LPS was extracted from C. burnetii NMI (RSA493), NMC (RSA514), and NMII (RSA439) following culture in ACCM-2 for 7 days. (A) LPS was separated by SDS-PAGE and silver stained, (B) LPS was separated by SDS-PAGE, blotted, and probed with antibodies specific to phase I, intermediate, or phase II LPS. NMI, NMC and NMII have unique LPS profiles consisting of multiple bands above 10 kDa, a single band of ~11 kDa and a single band at ~3 kDa, respectively. (C) Model of putative LPS structures of NMI and NMC is based on chemical composition and the previously determined LPS structure of NMII. Bonds between sugars of the outer core and O-antigen are not shown as the structure is unknown. Bold text in the key indicates highly abundant sugars. KDO is defined as 3-deoxy-D-manno-2-octulosonic acid.
Fig 2
Fig 2. Phase transition of strains from different genomic groups following serial passage in axenic media.
NMI (RSA363), S (Q217), G (Q212), and Dugway (7E65-68) were passaged weekly in ACCM-2 for 30 weeks. LPS was extracted at passage 2, 10, 20 and 30, separated by SDS-PAGE, and silver stained. Each strain has a phase I LPS profile at passage 2. Strains subject to additional passage produce increasing amounts of intermediate and phase II LPS.
Fig 3
Fig 3. C. burnetii strains serologically in phase II have intermediate and/or phase II LPS.
LPS was extracted from Australia (RSA297), Australia (RSA425), California 16 (RSA350), California 16 (RSA350) C2, and M44 (RSA461) C1, which are serologically in phase II [58], following culture in ACCM-2 for 7 days. LPS was separated by SDS-PAGE and compared to LPS from NMI, NMC, and NMII by (A) silver stain and (B) immunoblot using LPS-specific antibodies. Australia (RSA297) and Australia (RSA425) show an ~11 kDa intermediate and ~3 kDa phase II LPS. California 16 (RSA350), California 16 (RSA350) C2, and M44 (RSA461) C1 contain phase II LPS. A small amount of phase I LPS is observed only with the California 16 (RSA350) sample, indicating bacteria are producing phase I or phase II LPS. Despite a phase II band by silver stain, only intermediate LPS is detected in Australia (RSA297), suggesting the strain produces an antigenically distinct phase II LPS. Otherwise, banding patterns recapitulate those of silver stain gels. (C) The presence (+) or absence (-) of deleterious mutations within four LPS genes are indicated. Strains with a mixed population of an individual mutation are denoted as +/-.
Fig 4
Fig 4. Domain structure of CBU1655 and CBU0678.
(A) Schematic of the domain structure of the NMI CBU1655, a predicted D-glycero-D-manno-heptose-7-phosphate 1-kinase/D-glycero-D-manno-heptose-1-phosphate adenylyltransferase. Also shown is the domain structure of CBU0678 from phase II strains compared to that of NMI (RSA493). The location of the predicted active site residue, aspartate 455 (D455), is shown. CBU1655 is full-length in all strains. CBU0678 has a reversed domain structure compared to CBU1655. All depicted phase II strains contain a frameshift mutation in cbu0678 that results in a truncated protein. (B) Schematic for the putative ADP-D-glycero-β-D-manno-heptose synthesis pathway in C. burnetii. C. burnetii genes predicted to be involved at each step are printed in bold. The last step is displayed in blue as C. burnetii LPS does not contain ADP-L-glycero-β-D-manno-heptose.
Fig 5
Fig 5. CBU0678 is essential for production of phase I LPS.
LPS was extracted from NMI, NMC, NMI cbu0678tr, or NMI cbu0678trcomp-I following culture in ACCM-2 for 7 days. LPS was separated by SDS-PAGE then analyzed by (A) silver stain or (B) immunoblot probed with LPS-specific antibodies. NMI cbu0678tr produces intermediate and phase II LPS, but not phase I LPS. Expression of wild type cbu0678 in NMI cbu0678tr restores production of phase I LPS, indicating CBU0678 is essential for synthesis of phase I LPS. LPS from NMII, NMII Δcbu1655, and NMII Δcbu1655comp-II was isolated using a modified microextraction protocol following culture in ACCM-2 for 7 days. LPS was separated by SDS-PAGE and visualized by (C) glycoprotein staining or (D) immunoblot probed with anti-phase II LPS antibody. NMII Δcbu1655 produces a deep-rough phase II LPS (< 3 kDa), smaller than that of NMII, which is not recognized by anti-phase II LPS antibody. Expression of wild type cbu1655 in NMII Δcbu1655 restores production of typical phase II LPS and antibody recognition. These data are consistent with a predicted role of CBU1655 in producing the first heptose of the C. burnetii inner core, which is a component of the epitope recognized by anti-phase II LPS antibody. (E) Model of putative LPS structures of NMI cbu0678tr and NMII Δcbu1655 compared to those of NMI, NMC, and NMII. Bold text in the key indicates highly abundant sugars. KDO is defined as 3-deoxy-D-manno-2-octulosonic acid.
Fig 6
Fig 6. Mutation of cbu0533 results in phase II LPS of NMII.
LPS was extracted from NMI Δcbu0533, NMI Δcbu0533comp-I, and NMI Δcbu0533comp-II following culture in ACCM-D for 7 days. LPS was separated by SDS-PAGE and compared to LPS from NMI, NMC, and NMII by (A) silver stain and (B) immunoblot using LPS-specific antibodies. NMI Δcbu0533 produces a phase II LPS similar to that of NMII. Expression of a wild type NMI copy (comp-I) of cbu0533, but not a NMII copy (comp-II) of cbu0533, in NMI Δcbu0533 restores production of phase I LPS. LPS was extracted from NMII cbu0533comp-I and NMI Δcbu0533comp-D156C following culture in ACCM-D for 7 days. Samples were compared to LPS from NMI, NMC, and NMII. LPS was separated by SDS-PAGE and compared to LPS from NMI, NMC, and NMII by (C) silver stain and (D) immunoblot using LPS-specific antibodies. Expression of wild type cbu0533 in NMII results in production of intermediate LPS, consistent with the presence of a large deletion in NMII preventing restoration back to phase I LPS. Expression of the active site mutant cbu0533-D156C in NMI Δcbu0533 does not restore synthesis of phase I LPS. These data demonstrate that mutation of cbu0533 causes production of phase II LPS in NMII. (E) Model of putative LPS structures of NMI Δcbu0533, NMI Δcbu0533comp-I and NMII cbu0533comp-I compared to those of NMI, NMC, and NMII. Bold text in the key indicates highly abundant sugars. KDO is defined as 3-deoxy-D-manno-2-octulosonic acid.
Fig 7
Fig 7. Mutations in cbu0845 result in production of phase II LPS.
LPS was extracted from California 16 (RSA350), California 16 (RSA350) cbu0845comp, California 16 (RSA350) C2, California 16 (RSA350) C2 cbu0845comp, M44 (RSA461) C1, and M44 (RSA461) C1 cbu0845comp following culture in ACCM-2 for 7 days. LPS was separated by SDS-PAGE and compared to LPS from NMI, NMC, and NMII by (A) silver stain and (B) immunoblot using LPS-specific antibodies. Expression of cbu0845 in California 16 (RSA350) C2 and M44 (RSA461) C1 results in production of intermediate LPS due to the presence of a secondary mutation in cbu0678. Expression of cbu0845 in California 16 (RSA350), which has bacteria with or without the cbu0678 mutation, results in increased production of phase I LPS. (C) The presence (+) or absence (-) of deleterious mutations in cbu0678 and cbu0845 are indicated. Strains that have bacteria with or without gene mutations are denoted +/-. These data indicate that mutation of cbu0845 results in production of phase II LPS. (D) Model of putative LPS structures of California 16 (RSA350), California 16 (RSA350) C2, M44 (RSA461) C1, California 16 (RSA350) cbu0845comp, California 16 (RSA350) C2 cbu0845comp, and M44 (RSA461) C1 cbu0845comp compared to those of NMI, NMC, and NMII. Bold text in the key indicates highly abundant sugars. KDO is defined as 3-deoxy-D-manno-2-octulosonic acid.
Fig 8
Fig 8. Mutation of cbu1657 in Australia (RSA297) results in truncation of phase II LPS.
LPS was extracted from NMII Δcbu1657 and NMII Δcbu1657comp-II following culture in ACCM-2 for 7 days. LPS was separated by SDS-PAGE and compared to LPS from NMI and NMII by (A) silver stain and (B) immunoblot probed with anti-phase II LPS antibody. NMII Δcbu1657 produces a smaller phase II LPS than NMII that does not react with anti-phase II LPS antibody. Expression of wild type cbu1657 in NMII Δcbu1657 restores phase II reactivity. LPS was extracted from Australia (RSA297) and Australia (RSA297) cbu1657comp-II following culture in ACCM-2 for 7 days. LPS was separated by SDS-PAGE and compared to LPS from NMI, NMC, and NMII by (C) silver stain or (D) immunoblot probed with anti-phase II LPS antibody. Expression of wild type cbu1657 in Australia (RSA297) restores production of phase II LPS and anti-phase II LPS antibody reactivity. These data confirm that mutation of cbu1657 results in truncation of phase II LPS in Australia (RSA297). (E) Model of putative LPS structures of NMII Δcbu1657, Australia (RSA297), and Australia (RSA297) cbu1675comp-II compared to those of NMI, NMC, and NMII. Bold text in the key indicates highly abundant sugars. KDO is defined as 3-deoxy-D-manno-2-octulosonic acid.
Fig 9
Fig 9. Mutation of cbu0839 explains the LPS profile of NMI (RSA363) after 30 passages.
LPS was extracted from NMI (RSA363) P30, NMI Δcbu0839, and NMI Δcbu0839comp-I following culture in ACCM-D for 7 days. LPS was separated by SDS-PAGE and compared to LPS from NMI, NMC, and NMII by (A) silver stain and (B) immunoblot probed with anti-phase I LPS antibody. Thirty passage NMI (RSA363) contains a mutation in cbu0839 (Table 1). NMI Δcbu0839 and NMI (RSA363) P30 contain phase II LPS by silver stain that does not react with anti-phase I LPS antibody. Expression of a wild type cbu0839 in NMI Δcbu0839 restores production of phase I LPS. These data indicate that mutation of cbu0839 results in the LPS profile of NMI (RSA363) P30. (C) Model of putative LPS structure of NMI Δcbu0839 compared to those of NMI, NMC, and NMII. Bold text in the key indicates highly abundant sugars. KDO is defined as 3-deoxy-D-manno-2-octulosonic acid.
Fig 10
Fig 10. Mutations that accumulate in LPS genes affect protein function.
LPS was extracted from NMI Δcbu0533comp-I and NMI Δcbu0533comp-T138M following culture in ACCM-D for 7 days. LPS was separated by SDS-PAGE and compared to LPS from NMI, NMC, and NMII by (A) silver stain or (B) immunoblot probed with anti-phase I LPS antibody. Expression of cbu0533-T138M in NMI Δcbu0533 fails to fully restore production of phase I LPS, indicating the mutation affects CBU0533 function. LPS was extracted from NMI cbu0678tr and NMI cbu0678tr expressing wild type cbu0678, cbu0678comp-P74A, cbu0678comp-G369R, or cbu0678comp-P74A/G369R following culture in ACCM-2 for 7 days. LPS was separated by SDS-PAGE and compared to LPS from NMI, NMC, and NMII by (C) silver stain or (D) immunoblot probed with anti-phase I LPS antibody. NMI cbu0678tr expressing cbu0678-P74A produces a modified phase I LPS. NMI cbu0678tr expressing cbu0678-G369R or cbu0678comp-P74A/G369R also does not produce phase I LPS. These data confirm that identified point mutations in cbu0678 can affect CBU0678 function. (E) Model of putative LPS structures of NMI Δcbu0533comp-T138M, NMI cbu0678tr cbu0678comp-P74A, and NMI cbu0678tr cbu0678comp-G369R compared to those of NMI, NMC, and NMII. Bold text in the key indicates highly abundant sugars. KDO is defined as 3-deoxy-D-manno-2-octulosonic acid.
Fig 11
Fig 11. Model of LPS phase transition by C. burnetii.
Depicted are mutational steps occurring during phase variation. Changes in phase transition are shown using putative LPS structures associated with mutation of specific genes. A black arrow indicates mutations that cause direct transition from phase I to phase II LPS, as exemplified by NMII. Red arrows depict mutations that result in transition from phase I to phase II via an intermediate LPS stage, as exemplified by M44 (RSA461) C1. Blue arrows depict additional mutations that modify phase II LPS, as exemplified by Australia (RSA297). The grey arrow indicates transition from phase I to an alternative phase II LPS, as exemplified by NMI (RSA363) following 30 passages in axenic medium. Bold text in the key indicates highly abundant sugars. KDO is defined as 3-deoxy-D-manno-2-octulosonic acid.

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