Research On The Motility And Cytoskeleton Of Eriocheir Sinensis

Eriocheir sinensis is the aquaculture species of coastland of China.Recently,because of big breeding scale and high intensive degree,the disease caused by virus,bacteria and parasite become more and more seriously.It is giving a disastrous effects on the aquaculture economy in China.Wang et al.proved that S.eriocheir is is a pathogen of mass mortality in Chinese mitten crab,Eriocheir sinensis,causing tremor disease and infects almost all the artificial breeding crustaceans.S.eriocheiris is a helical bacterium,measuring 2.0 to 10.0 μm long,and can swim up to 5 μm per second in a viscous medium without flagella.Motility is essential for invading,but there is no report about the relationship between pathogenicity and motility of S.eriocheiris.Here,based on the genome analysis of S.eriocheiris,we used chemotactic model,electron microscopy and mass spectrum to study the chemotaxis and components of internal structure.We showed that it has a novel chemotaxis mechanism depending on cell polarity and aerobic condition,and also novel internal structures likely involved in the swimming mechanisms and their component proteins including four actin homologs.This study can help to investigate the pathogenesis of S.eriocheiris in-depth.1.Chemotactic behaviours of S.eriocheirisS.eriocheiris can swim very fast without flagella and have a well-defined shape.In this study,we used capillary assay,microscopic agar-drop assay(MAA)and tethered cell assays to investigate the chemotactic behaviour of S.eriocheiris.In the capillary assay,the cells were attracted to 10 mM methionine,20 mM glucose and 5 mg/ml phosphatidylcholine.This chemotactic is more obvious when the cells cultured under anaerobic condition.A drop of agar containing putative attractant chemicals placed around the center of chamber.We counted cell numbers and analyzed the cell behaviours.The results of MAA showed that the different positions from agar drops have different cell density,and it presented a population peak.We also analyzed the chemotactic behaviors of tethered cells.When the cells exposed to a brief diffusive wave of attractant,they changed their behaviors from free twisting to swimming forward and backward.The results suggest that S.eriocheiris are capable of sensing an attractive chemical and finding a comfortable concentration of the chemoattractants.In our previous studies,we finished the genomes of S.eriocheiris(unpublished)and no two-component-systems including chemotactic genes were found in the genome.This may indicate that the spiroplasma probablely has a novel chemotactic system.2.Cytoskeletal structure and component study of S.eriocheirisS.eriocheiris have a well-defined shape.Its distinct morphology and unique swimming way were defined by the internal cytoskeleton.In this study,we stripped the membrane by Triton X-100.A unique and well-defined internal cytoskeleton of S.eriocheiris was identified.In this structure,a Mycoplasma pneumonia rod-like tip structure and a flat ribbon filament structure were found.The ribbon is connected with the Tip structure and follows the shortest helical line of the polar cell from end to end.Spiroplasma’s cytoskeleton is assembled by seven pairs of fibrils but with opposite polarities,those filaments of a 55 kDa protein that assemble into a helical ribbon,termed the fibril ribbon.The basic structural and functional unit of the motor is some ring-like tetramers with about 10 nm wide and 8.2 nm long and lateral repeats.Differential length changes of the fibrils may generate a wide dynamic spectrum of helical and non-helical geometries allowing for directional motility in low Reynolds number environments.We identified 16 proteins as the components by mass spectrometry and especially,Fib and 4 MreBs homolog proteins were identified.The MreB gene family encodes actin-like proteins that determine cell shape by directing cell wall synthesis and often exists in one to three copies in the genomes of non-spherical bacteria,which are both characterized by cell contractility.The Tip structures appeared frequently attached or close to the plasma membrane.These findings suggest that the Tip structure may be involved in the orientation and attachment of spiroplasma helices in relation to their host cells,and thus may be functionally comparable to the "attachment organelle" of mycoplasmas.Cytoskeletal of Tip structure combined with ribbon filaments may be the internal motor of spiroplasma.3.The observation of S.eriocheiris by QFDR EM and phase contrast microscope and its energetics of swimmingTo understand the unique cell morphology and swimming mechanism,the morphology parameters are very useful.In this study,we used phase contrast microscope and Quick Freeze Deep Etch Replication electronic microscope(QFDE EM)to observe and analyse the eriocheiris and S.melliferum cells pitch parameters.And also,we proposed some physical equations on the energetics of spiroplasma cell body dynamics.The helical pitch of S.eriocheiris and S.melliferum range between 500 nm to 1000 nm.The pitch and height of the pitch are increasing from the Tip pole to another pole,but the cell diameter different showed significant change.The angle of kink has not significant different between S.eriocheiris and S.melliferum.The kink angle of S.eriocheiris is about 127.17±12.86 degree,and the kink angle of S.melliferum is about 132.45±14.59 degree.The swimming speed and kink propagation speed show different when cells were in different concentration of methylcellulose(MC).The ratio of swimming speed and kink propagation speed showed positive relationship.we reproduced several key features of Spiroplasma motility observed experimentally:(1)the cell itself moved in a direction opposite to the kink propagation;(2)the linear swimming velocity was proportional to the kink velocity,with a proportionality constant in fair agreement with experiments;(3)The pitch and height of the pitch were increasing from the tip pole to another pole.4.The structural and proteomic correlation for S.eriocheiris in response to colchicineThis work was designed to study the effects of colchicine on S.eriocheiris characters of growth,cell morphology,and genes expressions.In this study,different concentrations of colchicine(0 g/L,1 g/L,3 g/L,5 g/L)were added into the culture medium of spiroplasma,and then the cells were cultured for 24 hours,until the cells were in exponential phase.We found that,in the presence of colchicine,the spiroplasma lost their helicity,and the length of the cells in the experimental group was longer than that of the control group.Under the different concentrations of colchicine treatment,the total time taken by the spiroplasma to achieve a stationary phase was increased,and the cell population was decreased.We analyzed the differential proteins by iTRAQ quantification.208 differentially expressed proteins were reliably quantified by iTRAQ analysis,including 77 up-regulated proteins and 131 down-regulated proteins subsequent to colchicine stimulation.In the up-regulated proteins,5 were involved in energy metabolism process,17 were DNA replication and translation related proteins,6 were in transport systems proteins and transferase,11 were glycometabolism proteins,11 amino acid and protein metabolism,3 oxidordeuctases and 23 were listed as unknown/hypothetical proteins.Of thedown-regulated proteins,12 were grouped within the ribosomal proteins,7 were involved with energy metabolism processes,9 were related to carbohydrate metabolism and 13 were working in amino acid and protein metabolism process amino acid and protein metabolism,18 were DNA replication and cell division related proteins,2 ionic regulation related proteins,6 oxidordeuctases,13 were in transport systems proteins and transferase,5 lipoprotein and lipid metabolism,45 were in the unknown/hypothetical protein category.The qRT-PCR was conducted to detect the genes expressions during the incubation.7 genes were selected from the iTRAQ results 4 up-regulated:F0F1 ATP synthase subunit delta,Chromosome partitioning protein ParB,DNABs,Putative NAD(FAD)-dependent dehydrogenase and 3 down-regulated:Cell division protein FtsY,Putative spiralin,NADH oxidase.The 7 genes expressions were investigated during the incubation process by 3%colchicine at 1,3,5,7,9,11 hours and different on of colchicine.The qRT-PCR results were consistent with the iTRAQ results.All of our results indicate that colchicine may have a strong impact on the cell morphology and cellular metabolism of spiroplasma.