Study On The Biological Function Of LrgAB In Staphylococcus Epidermidis

Objective:CidA-LrgAB system is closely related to biological functions such as programmed cell death,bacterial lysis and pyruvate metabolism,but its function in Staphylococcus species is unclear.The lrgAB deletion mutant strain derived from SE1457 was constructed in this study,and the biological phenotypes of SE1457 and its isogenicΔlrgAB mutants were studied,then the role of LrgAB in the biological functions of S.epidermidis such as cell metabolism,biofilm formation and stress factors was preliminarily explored,so as to lay a foundation for preventing and treating persistent infection caused by S.epidermidis.Methods:1.Homology analysis of SE1457 LrgAB and construction of lrgAB deletion mutant:Using the genomic DNA of SE1457 as a template,the upstream and downstream homologous arm fragment（att B1-DS-US-att B2）of lrgAB was obtained by overlapping PCR amplification.After being ligated into shuttle plasmid p KOR1（including attp site and cdd B site）,p KOR1-ΔlrgAB recombinant plasmid was successfully obtained by BP reaction,which was transformed into E.coli DC10B and then into SE1457 by electroporation,and the suspected lrgAB deletion mutant strain（ΔlrgAB）was obtained by counter selection on TSB containing Atc.Similarly,the lrgAB（including SD sequence）amplified from the genome of SE1457 were cloned into plasmid p CN51,and the yielding recombinant plasmid p CN51-lrgAB was introduced intoΔlrgAB mutant and SE1457,respectively.TheΔlrgAB mutant andΔlrgAB（p CN51-lrgAB）complementary strain were verified using PCR,RT-PCR,q RT-PCR and sequencing.2.Study on the biological phenotype of SE1457ΔlrgAB mutant:The drug sensitivity of SE1457 andΔlrgAB mutant was detected by the tube dilution method;The biofilm formation of S.epidermidis was detected by microplate semi-quantitative method,and the amount of e DNA in biofilm was detected by q RT-PCR.Autolysis assay was performed with 1%Triton X-100,and the cell wall was observed under transmission electron microscope.The tolerance of S.epidermidis to stress factors such as osmotic pressure（0.5M,1.0M,1.5M Na Cl）,acid（p H6.0,p H5.0,p H4.0 HCl）and hydrogen peroxide（0.25m M,0.5m M,0.75m M H₂O₂）were conducted.The activities of SDH（succinate dehydrogenase）and NOX（NADH oxidase）of S.epidermidis were detected.3.Transcriptome sequencing（RNA-Seq）:RNA samples was extracted from SE1457 andΔlrgAB mutant,and RNA seq was performed using Illumina sequencing technology,and the differentially expressed genes（DEGs）were verified by q RT-PCR,and as analyzed using GO and KEGG.Results:1.Homology analysis of SE1457 LrgAB:The loci of lrg A/lrg B are serp2026/serp2027,which consists of 459nt lrg A/702nt lrg B nt,encodes a protein composed of 152aa Lrg A/233aa Lrg B amino acids.The lrg A/lrg B in SE1457 shared about 98%/97%identity with that in S.epidermidis RP62A and SE12228 at the nucleotide level,62%/73%with that in S.aureus,and 47.5%/55.5%in Streptococcus mutans.The Lrg A/Lrg B in SE1457 shared about 99%/97%identity with that in RP62A and SE12228 at the amino acid level,and 54%/72%with that in S.aureus,and 29%/47%in Streptococcus mutans.2.Construction and identification of SE1457ΔlrgAB mutant:The lrgAB fragment was not amplified in theΔlrgAB mutant,and the sequencing results suggested that a 1164-bp fragment（lrgAB genes）was knocked out from the genome of SE1457.The lrg A/lrg B genes were not transcribed inΔlrgAB mutant using q RT-PCR.Compared with SE1457 parent strain,the transcription level of lrg A gene inΔlrgAB（p CN51-lrgAB）complementary strain and SE1457（p CN51-lrgAB）control strain was both about 1.8 folds,while the transcription level of lrg B gene was about0.2 folds and 1.3 folds,respectively.3.Effects of lrgAB deletion on the biological phenotype of S.epidermidis:（1）The results of tube dilution method showed that theΔlrgAB mutant increased the susceptibility to vancomycin,ampicillin,bacitracin,chloramphenicol,and gentamicin compared with SE1457 parent strain.WhileΔlrgAB（p CN51-lrgAB）complementary strain and SE1457（p CN51-lrgAB）control strain restored to the susceptibility level of SE1457.（2）The results of microplate assay showed that the biofilm formation(OD₅₇₀)of SE1457 at 6h,12h,24h and 48h were 0.749±0.05,0.951±0.03,1.959±0.02 and2.631±0.05 respectively.Compared with that of SE1457,ΔlrgAB mutant impaired the biofilm at the above time points（0.483±0.01,0.347±0.01,0.286±0.01 and 0.950±0.02）,significantly（P<0.01）.While biofilm formation was restored in the complementary strainΔlrgAB（p CN51-lrgAB）.The biofilm formation of SE1457（p CN51-lrgAB）was higher than that of SE1457.The biofilm formation ofΔlrgAB（p CN51）at each time points were 0.510±0.02,0.332±0.01,0.242±0.02 and 0.573±0.01,respectively.（3）The amount of e DNA in biofilm was quantified using q RT-PCR and showed that theΔlrgAB mutant produced much e DNA significantly in the biofilm compared with that of SE1457（P<0.01）.The complementary strainΔlrgAB（p CN51-lrgAB）restored to the level of SE1457,while the amount of e DNA of SE1457（p CN51-lrgAB）was lower than that of SE1457.（4）The autolysis rate ofΔlrgAB mutant andΔlrgAB（p CN51）vector control reached to about 70%when staphylococcal cells were inoculated in TSB containing Triton X-100 for 6 hrs with shaking.At the same time point,the autolysis rates of SE1457,ΔlrgAB（p CN51-lrgAB）and SE1457（p CN51-lrgAB）were about 37%,44%and 22%,respectively.（5）TEM showed that the cell wall of SE1457 was smooth and complete,and the cell membrane was clear.ΔlrgAB mutant exhibited rough and discontinuous in cell wall.（6）Compared with that of SE1457,ΔlrgAB mutant enhanced the tolerance（about 10 folds）to osmotic pressure（0.5M,1.0M,1.5M Na Cl）,acid（p H6.0,p H5.0,p H4.0）and H₂O₂（0.25m M,0.5m M,0.75m M）.The tolerance ofΔlrgAB（p CN51）was similar to that ofΔlrgAB mutant.When the lrgAB gene was restored to theΔlrgAB mutant,the tolerance decreased to the level of SE1457.These results were consistent with that of the growth curve(OD₆₀₀)in which S.epidermidis were inoculated in TSB containing the above stressors.（7）The SDH and NOX activities were detected in S.epidermidis.ΔlrgAB mutant decreased the SDH and NOX activities（16.08±4.66 and 433.91±114.84 U/mg prot）significantly compared with that of SE1457（34.03±6.49 and 795.53±115.83 U/mg prot）,whileΔlrgAB（p CN51-lrgAB）complementary strain restored completely in the biological activities of SDX and NOX.（8）RNA seq and q RT-PCR showed that there were 385 genes differentially expressed between theΔlrgAB and the SE1457 parent strain.Among them,299 genes were up-regulated and 86 genes were down-regulated,and these genes were involved in autolysis,glucose metabolism,oxidative phosphorylation,protein metabolism and other pathways.The genes related to autolysis-regulation were down-regulated including membrane integrity maintenance related genes（serp0849,serp2187,etc.）,while genes related to esterification of D-alanine phosphoteic acid（dlt A/C/D,etc.）,cytoplasmic hydrolase synthesis（serp2120,serp0422,serp2136,etc.）,and penicillin-binding proteins（serp1412,serp0227,etc.）were up-regulated.The genes related to oxidative phosphorylation-regulation pyruvate synthesis（pfl A,pfl B,fpg,nrd F1,etc.）were down regulated.The proton pump ABC superfamily transporter related gene（serp2003,serp2004,serp2005,serp0021,serp2017,sit A/sit B,etc.）and stress regulating genes（dlt C/dlt D,etc.）were up-regulated.While the genes related to glucose metabolism（gnt K,gnt P,pyc,etc.）,the amino acid synthesis genes（sdh B,dat,etc.）and the drug resistance regulatory genes（serp2380,serp0844,etc.）were down-regulated.There was no significant change of cid A gene in transcriptome sequencing,but q RT-PCR showed that cid A gene was down-regulated by about 10times.No changes were found in transcription levels of ica R and ica A genes.Conclusions:1.TheΔlrgAB mutant of S.epidermidis was successfully constructed using allelic replacement,and theΔlrgAB（p CN51-lrgAB）complementary strain,the control strain SE1457（p CN51-lrgAB）and the vector controlΔlrgAB（p CN51）were constructed.2.LrgAB modulate the transcription of CidA and control a variety of biological functions in S.epidermidis.3.LrgAB affect the autolysis of S.epidermidis by regulating D-alanine esterification and murein hydrolase activity.4.LrgAB regulate the biofilm formation of S.epidermidis in non ica-dependent pathway.5.LrgAB may be involved in pyruvate utilization,proton pumping,sugar,protein and nucleic acid metabolism and other pathways which affectthe tolerance of S.epidermidis to the environmental stressors.