Construction Of Mycobacterial Recognition Element And Its Application In Mycobacterial Detection And Antimicrobial Susceptibility Testing

Mycobacteria infection seriously threatens human life and health worldwide.In response to this situation,the World Health Organization has established a series of international medical assistance and scientific guidance for the prevention and treatment of mycobacteria infection.So far,the treatment of mycobacteria infection still relies on the usage of long-term antimicrobial treatment,which can cause the problem of drug-resistance.Unfortunately,different types of mycobacteria show highly similar clinical manifestations,thus misdiagnosis often perplexes the treatment of mycobacteria infections.Worse still,with the increasing abuse of antibacterial agents,the emergence of multi-drug-resistant mycobacteria has made diagnosis and treatment to be more difficult.Rapid and accurate detection of mycobacteria is one of the key prerequisites for shortening the treatment period and reducing drug resistance.Timely identification and antimicrobial susceptibility testing of various mycobacteria is also a key precondition in the successful treatment of mycobacteria infection.The existing protocols for antimicrobial susceptibility testing(AST)still rely on extremely time-consuming mycobacteria culture,which could prolong the diagnostic period and aggravate the further formation of multidrug resistance.Mycobacteriophages are special virus that specifically infect the genus or species of mycobacteria hosts.The infection process of mycobacteriophages to the host mycobacteria generally includes four stages.Firstly,mycobacteriophages anchor to the mycobacteria surface with the aid of their tail fiber.Then the anchored mycobacteriophages invade the cellular membrane and inject DNA into mycobacteria cytoplasm,which triggers replication of progeny mycobacteriophages in the host cells.Lastly,the host mycobacteria are disrupted to allow release of mature progeny mycobacteriophages and intracellular contents.Therefore,mycobacteriophage with lytic gene shows potential usage in rapid detection as a specific recognition element for mycobacteria.Meanwhile,the functional protein of mycobacteriophage,as the important recognizing element for mycobacteriophage to absorb host mycobacteria,can be used as a novel potential specific recognition element for mycobacteria detection and drug susceptibility testing.In this work,Mycobacterium smegmatis(M.smegmatis)was used as a mycobacteria model analyte,the specific recognition elements were constructed and used to establish protocols for mycobacteria detection and rapid AST.The contents are described in detail as follows:1.Construction of ATP bioluminescence assay based on whole mycobacteriophage SWU1 functionalized magnetic particles(MPs)for M.smegmatis detection.With the standard double-layer plate protocol for phage culture and counting,mycobacteriophage SWU1 was cultured in Difco?Middlebrook 7H9 agar media.High-concentration mycobacteriophage SWU1 were prepared by infiltration,separation,PEG8000-mycobacteriophage precipitation and chloroform impurity.Then,the obtained mycobacteriophage SWU1 virions were covalently bond with the tosyl-capped MPs through the amino group of mycobacteriophages surface protein.The mycobacteriophage SWU1-functionalized MPs(SWU1-MPs)can act as highly specific carriers for separating the target mycobacteria,as well as a highly specific disruption agent for releasing ATP from mycobacteria cells.After binding for 60 min,the progeny mycobacteriophage in M.smegmatis cells disrupted the mycobacteria and released intracellular ATP.By gathering the ATP-triggered bioluminescence signal of firefly luciferase,we established a novel bioluminescence protocol for detecting the target mycobacteria with high sensitivity and ideal specificity.The detection range of this protocol is 5.0×10~2 to 5.0×10~5 CFU/m L,and the detection limit is 3.8×10~2 CFU/m L(S/N=3).In addition,this bioluminescence protocol shows high specificity for excluding the interference from Mycobacterium phlei,Mycobacterium terreus,Mycobacterium fortuitum and 6 non-mycobacteria species.This bioluminescence protocol shows acceptable reliability for detection of M.smegmatis in human urine,human saliva and human serum with recoveries of 77%-110%.2.Construction of mycobacteriophage tail protein and mycobacteriophage tail protein fused fluorescent protein probe.Because the mycobacteriophage-based detection still heavily rely on its lytic activity,it is possible to build a more sensitive mycobacteria detection by constructing the functional tail protein of mycobacteriophage.DNA sequence of the putative tail protein GP89 was obtained from the NCBI by homology comparison tool.The primers were designed to contain the terminal sequences of GP89 and reverse vector p ET28a.The GP89 plasmid vector was constructed by fusing the GP89 sequence with the reverse vector p ET28a sequence.In addition,the e GFP sequence was spliced to the C-terminal and N-terminal of GP89 sequence by a flexible linker DNA through total synthesis.Then the two e GFP spliced GP89 sequences were fused to the sequence of vector p ET28a to construct two types of GP89 fusion fluorescent protein probes.Three plasmids were transformed into heterologous E.coli expression system to express proteins.After purification,a water-soluble tail protein GP89 with a molecular weight of 34 k Da and two water-soluble GP89fused fluorescent protein probes with a molecular weight of 61 k Da were obtained.An optimal 3D prediction model of GP89 was obtained by further on-line modeling,model screening and on-line verification.According to the model,it was inferred that the fluorescent protein fused at GP89 N-terminal could partly affect the binding activity of GP89.3.Exploration of the recognition mechanism of mycobacteriophage tail protein towards mycobacteria surface polysaccharide receptor.Lipoarabinomannan(LAM)was obtained by lipid extraction,M.smegmatis cells disruption,Triton X-114precipitation,enzymatic hydrolysis digestion and gel column separation.Then the structure of crude LAM was confirmed by monosaccharide composition analysis,bond structure analysis and anomeric carbon configuration.The main components of the obtained LAM areα-arabinose and theα-mannose with a ratio of 0.9:1.0.The main structure of LAM was composed with three parts,the chain ofα-arabinose branch,the chain ofα-mannose chain and the substitution connection ofα-arabinose andα-mannose at C-2 position.The results are consistent with the theoretical LAM structure.The GP89-labeled red fluorescent microspheres with high excitation wavelength shows a specificity binding with the fluorescently labeled LAM.The result indicates that LAM is an important surface receptor in the process of tail protein GP89 for binding the M.smegmatis.4.Construction of fluorescence assay based on tail protein GP89 functionalized magnetic particles(MPs)for M.smegmatis detection and AST.A fluorescent sandwich method based on tail protein GP89 functionalized magnetic particles(MPs)was established for M.smegmatis detection and AST.GP89 and two GP89 fused e GFP protein probes showed good binding activity to M.smegmatis,GP89 was used to functionalize carboxyl MPs(GP89-MPs),two GP89 fusion fluorescent proteins were used as probes to bind with the complex of GP89-MPs and M.smegmatis specifically.Through the comparison of two GP89 fused fluorescent proteins,the fluorescent signal of the probe,which the fluorescent protein was fused at the N-terminal of GP89,could only detect at a concentration higher than 1.0×10~5 CFU/m L.The probe fused fluorescent protein at the C-terminal GP89 has a much better linear relationship with the concentration of M.smegmatis.The detection range of this method is 1.0×10~2-1.0×10~6 CFU/m L,and the detection limit is 69 CFU/m L(S/N=3).This method shows high specificity for excluding the interferences from Streptococcus mutans,Salmonella typhimurium and other species.This fluorescence method was successfully applied to the detection of M.smegmatis in saline,artificial cerebrospinal fluid and simulated body fluid samples with recoveries of78%-120%.This fluorescence method was successfully applied to the AST of M.smegmatis.The minimum inhibitory concentrations(MIC)of amikacin,levofloxacin,tobramycin,imipenem,clarithromycin and cefoxitin were≤16μg/m L,≤1.0μg/m L,≤2.0μg/m L,≤4.0μg/m L,4.0μg/m L and≥64μg/m L,respectively.These results were consistent with those obtained by the standard broth microdilution method.This GP89-based fluorescence method can achieve AST of M.smegmatis within 4 h.5.Fluorescent lateral flow assay based on tail protein GP89 for rapid detection of M.smegmatis.Due to the specific mycobacterium recognition,GP89 was immobilized onto the test strip nitrocellulose membrane as the capture agent.Meanwhile,an aptamer sequence for binding M.smegmatis was labeled on carboxyl fluorescent microspheres to form the aptamer functionalized fluorescent microspheres(apt-FMs).Then the apt-FMs were pre-incubated with M.smegmatis to form a complex.The fluorescence signal of M.smegmatis-apt-FMs complex was determined at the GP89 capturing band.Under the excitation of portable ultraviolet light,the M.smegmatis can be semi-quantitatively detected.Rapid fluorescence quantitative detection of M.smegmatis can be achieved by fluorometer within 15 min.The detection range of this method is 1.0×10~2 CFU/m L-1.0×10~6 CFU/m L,and the detection limit is 24 CFU/m L(S/N=3).The method has been successfully applied to the detection of M.smegmatis in normal saline,urine and saliva samples with recoveries of 75%-110%.In summary,due to the specific mycobacteria binding capacity of mycobacteriophage,the mycobacteria cells were separated and enriched from different complexes.Utilizing the highly sensitive firefly luciferase-ATP bioluminescent system,a rapid and facile protocol was developed for detecting the target mycobacteria.The mycobacterial tail protein and its fused fluorescent protein were successfully expressed by the heterologous protein expression system.Combined with fluorescence analysis,a rapid,sensitive and specific protocol was developed for detecting the target mycobacteria.Compared with mycobacteriophage,tail proteins have broad-spectrum recognition.Moreover,AST was successfully performed in 4 h by using the mycobacteriophage tail protein and GP89fused fluorescent protein probes.The tail protein-based AST showed the potential to be a supplement method for non-cultural mycobacteria AST.Meanwhile,a sensitive and specific lateral flow assay for mycobacteria was constructed based on the tail protein and aptamer fluorescent probe.Compared with the reported phenotype methods,the lateral flow assay based on GP89 can reduce time cost and boost the detection efficiency.Compared with spectroscopy and mass spectrometry,this method is facile,low-cost,and favorable for field screening of massive mycobacteria.Meanwhile,we successfully verified the specific binding of tail protein towards the extracted LAM,which indicated that the GP89 protein can be used for other mycobacteria detection and AST.