Study On The Biological Role Of Coxiella Burnetii Lipid A And The Mechanism Of Anti-hyperosmotic Property

Coxiella burnetii?C.burnetii?is a world-wide distributed gram-negative intracellular bacterium,which usually transmitted through the contaminated aerosol and caused the acute or chronic human Q fever disease.Traditionally,owing to the similar biological properties,C.burnetii was also called Q fever Rickettsia.However,the long distance of genetic relationship with other Rickettsia members,C.burnetii was categorized to the order Legionellales in the second edition of Bergey's manual of systematic bacteriology.Notablely,the pathogen is highly resistant to hyperosmosis and dessication,which has been known as a classical bio-warfare and bio-terrorism.Lipopolysaccharide?LPS?,the main structure of outer membrane of most gram-negative bacteria,is an important virulence factor.With a few exceptions like Chlamydia trachomatis,LPS in most gram-negative bacteria consist of lipid A,core oligosaccharide and repeating polysaccharide units?O-antigen?.Lipid A?endotoxin?,a glucosamine-based saccharolipid,is the basal component of LPS.With a few exceptions like Neisseria meningitides,Moraxella catarrhalis,and Acinetobacter baumannii so far,the Kdo-lipid A portion of LPS is essential for most gram-negative bacteria growth.LpxC,a conserved emzyme that catalyzes the first non-reversible step of deacetylation in the biosynthesis of lipid A,is widely used as broad-spectrum antibiotic target.The novel compounds such as LPC-011,CHIR-90 are small-molecule inhibitors that specific inactivating LpxC and inhibiting the biosynthesis of lipid A.Recent years,various immunomodulatory functions of C.burnetii LPS have been reported,but the biological role and function of C.burnetii lipid A remains unclear.Here we characterized the biological role of lipid A in Coxiella burnetii growth in axenic media?ACCM-2?,monkey kidney cells?Vero and BGMK?and macrophage-like THP-1 cells by using the potent LpxC inhibitors-LPC-011 and CHIR-90.Additionally,we identified a few proteins of C.burnetii associated with hyperosmosis-resistance in axenic culture system,by utilizing two-dimensional gel electrophoresis combined with mass spectrometry.Firstly,a preliminary experiment on Coxiella-infected Vero cells with originally added of single high dose of LPC-011 was performed,indicating the C.burnetii lipid A may play a non-essential role in monkey kidney fibroblasts.However,a conclusive role of the essentiality of lipid A in C.burnetii growth cannot be reached attribute to the lack of detecting lipid A biosynthesis,together with the sensitivity of LpxC to LPC-011 remains unknown.Then,a surrogate E.coli model was constructed by introducing C.burnetii lpxC and inactiving the endogenous chromosomal copy of E.coli lpxC,in order to evaluate the minimal inhibitory concentration?MIC?of LPC-011against C.burnetii LpxC separately.In E.coli,the MIC of LPC-011 against C.burnetii LpxC is less than 0.05?g/ml-lower than the MIC against E.coli LpxC.Meanwhile,considering the inhibitor's problematic pharmacokinetic properties in vivo and Coxiella's culturing time up to seven days in host cells and axenic culture systems,the stability of LPC-011 was assessed in cell and cell-free cultures by using the growth symbol,optical density at 550 nm(OD₅₅₀)of E.coli as an indicator.It was found that regular refreshment of inhibitor-containing culture media every 48 hours is required to have a constant inhibition of C.burnetii LpxC in cells,while an E.coli-inhibiting concentration of LPC-011 could be maintained under the acidic conditions of axenic media during seven days post-inoculation in low oxygen at37°C.Secondly,owing to the lack of Coxiella lipid A-detection method,we produced a chlamydial kdtA-contained shuttle vector pMMBGKkdt A,which derived from the previously constructed vector pMMBGK.Both of the two vectors were based on the RSF1010 ori conferred plasmid p MMB207,which was widely used for E.coli cloning system.Utilizing the electroporation of p MMBGKkdt A into Nine Mile phase II C.burnetii,the inhibition of lipid A biosynthesis by the inhibitor LPC-011 was shown in a Nine Mile phase II strain transformant carrying plasmid pMMBGKkdt A.Thus,the exogenous chlamydial Kdt A enzyme modifies Coxiella lipid A with an?-Kdo-?2?8?-?-Kdo epitope that can be detected by anti-chlamydia genus monoclonal antibodies,the endogenous Kdo₂-lipid A was modified into Kdo₃-lipid A structure.Under the established constant inhibition of LpxC,the C.burnetii lipid A was visualized by PE-conjucted lgG using the indirect immunofluorescence assay?IFA?.Besides,the parental transformants have reduced growth yields in axenic media and non-phagocytic cells by LPC-011–treatment.The progenies of lipid A-modified C.burnetii strain prepared from inhibitors-treated BGMK cells retained the capability of normally infecting both BGMK and macrophage-like THP-1 cells without inhibitor treatment but can hardly grow under LPC-011 inhibition in THP-1cells.However,the experiments using lipid A-modified strain cannot completely represent the wild-type C.burnetii phase I or phase II strains.Moreover,in Coxiella-infected BGMK cells,the number of vacuoles was decreased about 60%-70%under LPC-011–treatment compared to the mock–treated cells,but the robust growth of C.burnetii in the reduced vacuoles was not affected by LPC-011.Interestingly,the active brownian movement of evenly distributed bacterial particles of the phase I strain inside the vacuoles was not affected by LPC-011,suggesting that C.burnetii LPS is not the only factor contributing to high hydrophilicity of the phase I strain,besides the full-length LPS,other surface structures like glycosylated proteins might also contribute to the hydrophilicity of phase I strains.In inhibitor-treated THP-1 cells,parental coxiella showed severe growth defects characterized by poor vacuole formation and thus dramatically reduced growth yields.C.burnetii progenies prepared from LPC-011-treated BGMK cells maintained the capability of infection and replication in macrophage-like THP-1cells without inhibitor treatment,which excludes a role of lipid A for parental strain infection and early vacuole development.Then,considering C.burnetii has two morphologically distinct cell types that comprise a biphasic developmental growth cycle,a small cell variant?SCV,0.2⁰.5?m in length?,the extracellular survival form,invades the host and develops into a large cell variant?LCV,>1 in length?for replication.An ultrastructural analysis by transmission electron microscopy?TEM?was performed to assess the developmental transitions.The a quite portion of SCV-size bodies could be found in vacuoles on inhibitor-treated BGMK cells after seven days post-infection,suggesting lipid A may not be required for the morphological transition of LCV to SCV.In addition,culturing a large number of Coxiella organisms in ACCM-2 medium adjusted by gradient ion concentrations of sodium and chloride.Siver-stained gels of Coxiella lysates harvested from hyperosmotic,normal-culturing osmotic pressure,and the hypotonic conditions were resolved by two-dimensional electrophoresis.Total of thirteen proteins had significant difference by comparative analysis based on the abundance of protein spots.Seven proteins that greater than ten-fold more abundant in hyperosmotic condition,were identified as GroES,EF-Tu,RopC,IcmK,RplL and a hypothetical protein?CBU₀632?using mass spectrometry?Maldi-TOF-MS?.Six up-or down-regulated proteins were ten-fold greater in hypotonic medium were identified as GcvH,Bcp,Dot C,RuvB,ornithine decarboxylase and a hypothetical protein?CBU₀658?.In all,we characterized the role of lipid A in C.burnetii growth in different culture systems by using a potent LpxC inhibitor-LPC-011.We evaluated the MIC of LPC-011 against C.burnetii LpxC in a surrogate E.coli model.A reliable condition of effectively inhibiting lipid A biosynthesis in C.burnetii was then established in a IFA-based lipid A-reportor system.We then provide evidence that lipid A is nonessential for C.burnetii survival and growth in vitro,and lipid A has variable roles for bacterial robust growth in different culture systems.In addition,a few proteins and their encoding genes that associated with the anti-hyperosmotic property were identified by by proteome analysis.This study set a basis for better understanding the biological role of C.burnetii lipid A,provide evidence for the possibility of constructing C.burnetii lipid A-null muant to further analyze the lipid A biological functions.It also has important implications for clinical Q fever treatment.Additionally,our study also provided a basis for futher investigating the genetic mechanism of anti-hyperosmotic property on transcriptional level and confirming the biological function of corresponded genes in future experiments.