Characterization Of A Chimeric ChaA/Y Two-component Regulator In Acinetobacter Baumannii ATCC17978

[Background]Acinetobacter baumannii is a non-fermentative Gram-negative bacillus,which has a wide distribution and is a clinical important opportunistic pathogen.Most of the current reports focus on its classification,epidemiology,and antibiotic resistance.However,the research on physiological and stress-responsive mechanism of this pathogen still needs to be strenthened.A.baumannii has ultrastrong abilities to adapt to the environment,such as tolerance to dry,flexible growth needs,the formation of biofilm and quorum sensing activity,which make it an emerging nosocomial pathogen.Two-component regulatory system（TCSs）are important approach for bactieria to sense the external environment signals and respond adequately to changes.TCSs have been proven to play an important role in A.baumannii as a strategy of coupling environment changes and cell physiological changes.Previous researches revealed that the two-component regulatory system of A.baumannii was involved in its drug resistance,motility,biofilm formation,morphology and virulence as well.Through bioinformatics analysis,we found that AUO97_RS03045 in A.baumannii ATCC17978 genome possessed four histidine-containing phosphotransfer（HPt）domains,a Che A-like regulatory domain and a CheY-like receiver domain at the C terminus.AUO97_RS03045 may be a hypothetical hybrid sensor histidine kinase/response regulator involved in bacterial chemotaxis signal transduction.The results of the study on the chemotaxis signal transduction system of Ch A protein in other bacteria（CheA in Escherichia coli and Chp A in Pseudomonas aeruginosa;we denominated AUO97_RS03045 product in ATCC17978 as Cha A/Y）showed that ChA protein was related to the motility of bacteria.The system regulates the motility by controlling the direction of rotation of the flagellar in Escherichia coli,whereas by controlling the biosynthesis of type IV pili and（or）retraction of type IV pili in Pseudomonas aeruginosa.So far,there has been no systematic study about this system in A.baumannii.Although it lacks flagella,A.baumannii is motile.The phenotypes of motility in A.baumannii are diverse.Certain A.baumannii strains exhibit a phenomenon called ‘twitching motility’,which is a kind of jerky movement on wet surface.Certain exhibit movements on the wet surface of semi-solid media which was termed as ‘sliding’ or ‘swarming’.And some strains could move beneath the agar or form ‘ditches’.Previous studies showed that the motility of A.baumannii was associated with type IV pili,quorum sensing,sensing of blue light,iron deficiency and 1,3-diaminopropane.Till now,the underlying molecular and genetic basis of A.baumannii’s motility is still ambiguous,even the the definition of the forms of various movement was still relatively confusing.[Objective]The study on chaA/Y gene of A.baumannii ATCC17978 will not only explore the regulation mechanism of two-component regulatory system in A.baumannii,but also help to understand the bacteria metabolism such as its still ambiguous mechanism of motility.These research works will provide experimental basis for clinical prevention and treatment for infection diseases caused by A.baumannii.[Methods and Results]Firstly,we used PCR method to verify that cha A/Y was co-transcribed with four other upstream genes: AUO97_RS03050（pilJ）、AUO97_RS03055（pilI）、AUO97_RS03060（pilH）and AUO97_RS03065（pilG）,which indicated a five-gene operon with cha A/Y as the last gene.We constructed cha A/Y gene knockout strain ΔchaA/Y::FRT by RecAb system.After eliminating the resistance box,only FRT locus of 91 bp was left in the original position of the chaA/Y gene.At the same time,we constructed cha A/Y complementary strain Δcha A/Y::FRT-c with the pMM67 EH plasmid.Then,the phenotypes of the parental strain ATCC17978 and mutant ΔchaA/Y::FRT were observed and compared,including of the growth curve,biofilm formation,surface motility,AHL content in the culture supernatant,antibiotic resistance,presence of pili and extrapolymeric substances（EPS）under transmission electron microscope.Comparing with ATCC17978,the surface motility,formation of biofilm at the gas–liquid interface and AHL content in the culture supernatant under the same experimental condition decreased significantly in cha A/Y knocking-out strain.The number of pilus-like structures on the cell surface and the amount of extrapolymeric substances（EPS）also decreased in chaA/Y null strain.The complement strain partially restored the phenotype.There was no significant change in growth curve and response to 71 kinds of carbon sources and 23 chemical sensitivity tests.No significant change of antibiotic resistance has been detected,except for the resistance to kanamycin.The kanamycin MIC of ΔchaA/Y::FRT was 8 μg/ml,which was 4 times higher than the wild strain.The transcription of abaI,which encodes an N-acylhomoserine lactone synthase in A.baumannii,decreased significantly in chaA/Y null strain,and supplement with synthetic N-（3-oxodecanoyl）homoserine-L-lactone could rescue the phenotype of surface motility and biofilm formation in the null mutant.In order to clarify the genes regulated by cha A/Y in ATCC17978,RNA-sequencing and real-time fluorescence quantitative PCR were used to investigate the transcriptome differences in ATCC17978 and mutant ΔchaA/Y::FRT cells growing on the motility plates.The results showed that after deletion of cha A/Y,the expression of 113 genes was significantly down-regulated and 80 were up-regulated.These down-regulated genes were enriched in the KEGG pathways of pantothenate and Co A biosynthesis,degradation of aromatic compounds,benzoate degradation,starch and sucrose metabolism,fatty acid biosynthesis,ethylbenzene degradation,fatty acid metabolism,and fluorobenzoate degradation.The most up-regulated genes were AUO97_RS16785（nucleoside-diphosphate-sugar epimerase）,AUO97_RS16780（tartrate symporter MFS superfamily protein）and AUO97_RS16840（major facilitator superfamily transporter）.Among the down regulated genes,these genes may be related to the motility and biofilm phenotype of ATCC17978: CU pili（chaperone-usher pili）related genes（csu A/BABCDE,fim A,fimC,fim D）,AUO97_RS06595-06630 operon,AUO97_RS06645（aba I）.The transcription level of AUO97_RS06595-06630 operon decreased most significantly in ΔchaA/Y::FRT.However,there was no significant change in the genes associated with type IV pili（previous studies had shown that the "twitching" motility of A.baumannii was associated with type IV pili）.Real time fluorescent quantitative PCR results verified the reliability of RNA-sequencing results.Previous studies have found that the AUO97_RS06595-06630 operon（A1S₀112-0119 operon in previous references）and abaI of ATCC17978 have been related to the motility and biofilm formation in ATCC17978.AUO97_RS06595-06630 operon was annotated to be responsible for the non-ribosomal production of a lipopeptide,which may act as a surfactant to aid motility.Previous studies showed that AUO97_RS06595-06630 operon were essential for pellicle formation and motility in A.baumannii.The motility and pellicle phenotypes of A.baumannii might be linked to AUO97_RS06595-06630 operon via the expression of cAMP.The AUO97_RS06595-06630 operon was also reported to be activated by aba I.abaI was predicted to be the only autoinducer synthase encoded in the genome in A.baumannii,The encoded product of abaI are involved in the synthesis of N-（3-hydroxydodecanoyl）-L-HSL（3-hydroxy-C12-HSL）,an acyl serine lactone（AHL）,which is a signal molecule of the bacterial quorum sensing system.Supplement with synthetic N-（3-oxodecanoyl）homoserine-L-lactone could rescue the phenotype of surface motility and biofilm formation in the null mutant,suggesting that AHL play an important role in the regulation process.CU pili related gene clusters were significantly downregulated in Δcha A/Y::FRT.Previous studies have shown that CU pili are associated with biofilm formation of Acinetobacter baumannii,but the relationship between CU pili and motility is not clear.Therefore we constructed csu E gene knockout strain Δcsu E::FRT.Through PCR sequencing,we confirmed that full-length csu E was deleted and replaced by 91 bp FRT loci.Commentary strain ΔcsuE::FRT-c was constructed with pABBR_MCS.Under the same experimental conditions,the motility and biofilm formation of Δcsu E::FRT decreased without no significant change in growth status.Commentary strain restored the biofilm and motility phenotype.These results suggested that CU pili was regulated by cha A/Y and participated in the regulation of motility in A.baumannii.[Conclusions]AUO97_RS03045 is a Cha A/Y hypothetical hybrid sensor histidine kinase/response regulator in A.baumannii strain ATCC17978 involved in bacterial chemotaxis signal transduction and biofilm formation.The surface motility of A.baumannii ATCC17978 might not be associated with type IV pili.We speculate that cha A/Y regulates surface motility and biofilm formation not by regulating the expression of genes associated with type IV pili,but regulating the csu operon associated with CU pili and AUO97_RS06595-06630 operon associated with AbaI-dependent quorum-sensing pathway instead.