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1、RESEARCH ARTICLEPathogenic Chlamydia Lack a ClassicalSacculus but Synthesize a Narrow,Mid-cellPeptidoglycan Ring,Regulated by MreB,forCell DivisionGeorge Liechti1,Erkin Kuru2a,Mathanraj Packiam1,Yen-Pang Hsu2,Srinivas Tekkam2,Edward Hall2b,Jonathan T.Rittichier2,Michael VanNieuwenhze2*,Yves V.Brun3*
2、,Anthony T.Maurelli1c*1 Department of Microbiology and Immunology,F.Edward Hbert School of Medicine,Uniformed ServicesUniversity of the Health Sciences,Bethesda,Maryland,United States of America,2 Department ofChemistry,Indiana University,Bloomington,Indiana,United States of America,3 Department of
3、Biology,Indiana University,Bloomington,Indiana,United States of America These authors contributed equally to this work.a Current address:Department of Genetics,Harvard Medical School,Boston,Massachusetts,UnitedStates of Americab Current address:Department of Chemistry,Hanover College,Hanover,Indiana
4、,United States of Americac Current address:Emerging Pathogens Institute and Department of Environmental and Global Health,College of Public Health and Health Professions,University of Florida,Gainesville,Florida,United States ofAmerica*mvannieuindiana.edu(MV);ybrunindiana.edu(YVB);amaurelliphhp.ufl.
5、edu(ATM)AbstractThe peptidoglycan(PG)cell wall is a peptide cross-linked glycan polymer essential for bacte-rial division and maintenance of cell shape and hydrostatic pressure.Bacteria in the Chlamy-diales were long thought to lack PG until recent advances in PG labeling technologiesrevealed the pr
6、esence of this critical cell wall component in Chlamydia trachomatis.In thisstudy,we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolu-tion microscopy to demonstrate that four pathogenic Chlamydiae species each possess a?140 nm wide PGring limited tothe division plane d
7、uring the replicative phase of their develop-mental cycles.Assembly of this PG ring is rapid,processive,and linked to the bacterial actin-like protein,MreB.Both MreB polymerization and PG biosynthesis occur only in the intracellu-lar form of pathogenic Chlamydia and are required for cell enlargement
8、,division,and transi-tion between the microbes developmental forms.Our kinetic,molecular,and biochemicalanalyses suggest thatthe development of this limited,transient,PG ring structure is the resultof pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.Author SummaryPat
9、hogenic Chlamydia do not assemble their peptidoglycan(PG)cell wall in a classical,mesh-like sacculus,but instead apparently confine it to the mid-cell in the activelyPLOS Pathogens|DOI:10.1371/journal.ppat.1005590May 4,20161/30a11111OPEN ACCESSCitation:Liechti G,Kuru E,Packiam M,Hsu Y-P,Tekkam S,Hal
10、l E,et al.(2016)Pathogenic ChlamydiaLack a Classical Sacculus but Synthesize a Narrow,Mid-cell Peptidoglycan Ring,Regulated by MreB,forCell Division.PLoS Pathog 12(5):e1005590.doi:10.1371/journal.ppat.1005590Editor:Christopher M.Sassetti,University ofMassachusetts Medical School,UNITED STATESReceive
11、d:October 26,2015Accepted:April 1,2016Published:May 4,2016Copyright:This is an open access article,free of allcopyright,and may be freely reproduced,distributed,transmitted,modified,built upon,or otherwise usedby anyone for any lawful purpose.The work is madeavailable under the Creative Commons CC0
12、publicdomain dedication.Data Availability Statement:All relevant data arewithin the paper and its Supporting Information files.Funding:This work was supported by:Grant1R01GM113172-01 from the National Institute ofGeneral Medical Sciences,the National Institutes ofHealth,URL:https:/www.nigms.nih.gov/
13、Pages/default.aspx,to MV;Grant GM113172 from theNational Institutes of Health Institute for GeneralMedical Sciences“Dynamics of bacterialpeptidoglycan synthesis”and Grant GM51986 fromthe National Institutes of Health Institute for GeneralMedical Sciences“Control of bacterial morphologydividing,non-i
14、nfectious form.We characterize the assembly and aging of this PG-ringand link its synthesis to MreB,an actin-like protein associated with lateral cell wall synthe-sis in bacteria.As PG is recognized by the host innate immune system,we hypothesizethat the limited amount of PG synthesized by Chlamydia
15、 is an adaptation to the microbesintracellular lifestyle.IntroductionChlamydia is an obligate intracellular pathogen and the single most prominent cause of bacte-rial sexually transmitted infections and infectious blindness worldwide.Frequently referred toas the silent epidemic,chlamydial infections
16、 are often asymptomatic,which results in alengthy delay between infection and the onset of disease symptoms1.Approximately 1.4 mil-lion Chlamydia infections are reported in the United States annually2,3 and an estimated 90million individuals are believed to be infected globally4.Untreated chlamydial
17、 genital infec-tions can result in cervicitis,pelvic inflammatory disease,and ectopic pregnancy in women andurethritis in men.Chlamydia has undergone a lengthy(700 million year)adaptation to an intracellular envi-ronment in addition to its more recent co-evolution with humans and other vertebrate ho
18、sts5.As a result,pathogenic chlamydial species possess significantly smaller genomes comparedto those of extracellular pathogens,free-living microbes,or environmental chlamydiae5,6.Chlamydia exhibit a distinctive,biphasic life cycle wherein they alternate between an infectiousbut non-replicative ele
19、mentary body(EB)and a non-infectious but replicative reticulate body(RB).Under certain conditions Chlamydia can differentiate into an aberrant,metabolicallyactive but non-replicative form.These aberrant bodies form when RBs are exposed to stress-ors,such as nutrient deprivation and certain antibioti
20、cs that inhibit peptidoglycan(PG)cellwall biosynthesis.Aberrant bodies exhibit a state akin to metabolic stasis that can last for days,enhancing persistence of the microbe in both human and animal hosts.When the stress isreleased,aberrant bodies differentiate back to RBs and normal bacterial replica
21、tion continues.PG is a critical cell wall component of nearly all bacteria.It is comprised of a-(1,4)linkedN-acetylglucosamine(GlcNAc)and N-acetylmuramic acid(MurNAc)disaccharide backboneand a pentapeptide stem,i.e.a muropeptide.In Gram negative and some Gram positive bacte-ria,the peptide stem cons
22、ists of L-alanine,D-glutamate,meso-diaminopimelic acid,and adipeptide of D-alanine-D-alanine(DADA)(Fig 1a).Once synthesis of the major structuralcomponent of PG(lipid II)is completed in the bacterial cytoplasm,it is flipped into the peri-plasm where PG assembly proceeds.Sugar moieties of the PG are
23、initially polymerized,result-ing in assembly of the nascent PG strand(Fig 1a).This step is quickly followed by cross-linkingof the stem peptides from multiple strands into a structure that in the vast majority of bacteriacovers the entire bacterium as a mesh-like sacculus.PG is required for cell gro
24、wth and divisionand provides the bacterium a defined,structurally rigid and species-specific shape 7.Theunique composition of PG makes it an excellent marker for detection of bacteria by the humanimmune system.Indeed,PG is one of the major pathogen-associated molecular patterns(PAMPs)recognized by i
25、nnate immune receptors 8.Despite the long recognized susceptibility of pathogenic chlamydial species to commonanti-PG agents such as penicillin and D-cycloserine(DCS),until recently,Chlamydia wasthought to lack PG9,10.James Moulder summed up the seemingly conflicting physiological1113 and biochemica
26、l 1421 findings as the chlamydial anomaly 22.This paradoxdeepened further after the genome of Chlamydia trachomatis was sequenced and found toPeptidoglycan and Cell Division in Pathogenic ChlamydiaPLOS Pathogens|DOI:10.1371/journal.ppat.1005590May 4,20162/30and differentiation”,URL:https:/www.nigms.
27、nih.gov/Pages/default.aspx,to YVB;Grant F32AI112209-02from the National Institute of Allergy and InfectiousDiseases,National Institutes of Health,URL:http:/www.niaid.nih.gov/Pages/default.aspx,to GL;Grant2R01AI044033-11A1 from the National Institute ofAllergy and Infectious Diseases,National Institu
28、tes ofHealth,URL:http:/www.niaid.nih.gov/Pages/default.aspx,to ATM.The funders had no role in studydesign,data collection and analysis,decision topublish,or preparation of the manuscript.Competing Interests:The authors have declaredthat no competing interests exist.possess almost all of the genes of
29、 the PG biosynthesis pathway 23.Numerous studies havesince shown that the vast majority of the proteins encoded by these genes are functional invitro or when expressed in E.coli2429.The chlamydial genome appears to lack only a fewPG synthesis genes,such as glutamate/alanine racemases and transglycos
30、ylases,which areessential for making PG subunits and polymerizing these subunits into PG chains,respectively23(Fig 1a).In addition the Chlamydia genome lacks a gene encoding FtsZ,a cytoskeletal celldivision initiation protein that organizes numerous PG biosynthetic enzymes around the celldivision pl
31、ane in almost all bacteria7.Recently,a method was developed to label bacterial PG using the inherent promiscuity of PGbiosynthetic enzymes for tagged,fluorescent D-amino acid(FDAA)probes as substrate analogs30,31.Our work with the next generation of bioorthogonally tagged D-amino acid dipeptide(DAAD
32、)probes that mimic DADA during PG synthesis(Fig 1a and 1b)revealed PG in C.trachomatis9,providing the first direct evidence of its existence in these organisms.Anotherstudy confirmed the presence of PG in an environmental strain of Parachlamydiaceae,theamoebae-symbiont,Protochlamydia amoebophila6,10
33、,providing evidence that both relatedFig 1.Relevant steps of PG biosynthesis and D-amino acid dipeptide(DAAD)probes used in this study.(a)DAADs are taken up by bacteria wherethey compete with endogenous D-Ala-D-Ala(DADA)for incorporation into PG.De novo synthesis of DADA is inhibited by D-cyloserine
34、.Thepentapeptide PG subunit is then flipped across the inner membrane into the periplasm where it is transglycosylated to form glycan polymers(nascent PG)and crosslinked by penicillin binding proteins(PBPs).Transpeptidation causes cleavage of the terminal D-Ala at position 5.Because the N-terminally
35、 labeledportion of DAAD becomes the amino acid at position 4 of the pentapeptide,the label is resistant to this processing and remains on the stem peptide.(b)PG-labeling reagents used in this study.Clickable DAADs EDADA and ADADA.Star represents the clickable amino acid.doi:10.1371/journal.ppat.1005
36、590.g001Peptidoglycan and Cell Division in Pathogenic ChlamydiaPLOS Pathogens|DOI:10.1371/journal.ppat.1005590May 4,20163/30phyla synthesize PG.Strikingly,while PG in P.amoebophila forms a typical,cell-encompassingsacculus,PG in C.trachomatis forms only a distinct ring-like band at its mid-cell.Desp
37、ite these advances,many questions remain concerning the function of PG in Chlamyd-iae and the significance of its ring-like structure.Through the use of 3D super resolution struc-tured illumination microscopy(SIM)and clickable DAADs we define the ring-like PGstructure of C.trachomatis as a?140 nm wi
38、de,dynamic ring that forms immediately after theprevious cell division and follows cell constriction at the division septum.We show that thislimited PG ring is also present in other pathogenic chlamydial species;C.muridarum,C.caviae,and C.psittaci.Formation of the ring is non-uniform and directly li
39、nked to the bacterialactin-like cytoskeletal protein MreB.When MreB polymerization is inhibited the PG ring israpidly and non-uniformly turned over,suggesting competition between two coordinated,butseparable processes:MreB-linked PG synthesis and an unknown turnover mechanism.Wepropose a reshaping m
40、odel to explain how this narrow PG ring facilitates both cell enlargementand division.We also propose that Chlamydia limits the timing of PG ring assembly and disso-ciation to the intracellular replicative phase,allowing the pathogen to moderate its detection bythe host immune system.These results s
41、uggest that the absence of a PG sacculus by pathogenicChlamydia is the result of pathoadaptation to its intracellular niche within vertebrate hosts.ResultsPathogenic Chlamydia lack a classical PG sacculusDespite the demonstration of a conventional PG sacculus in P.amoebophila10,similar PGisolation a
42、nd labeling techniques(e.g.using FDAAs)proved unsuccessful in studies of patho-genic Chlamydia9,16,19.We hypothesized that distinct morphological differences in PGconfiguration and structure might exist between pathogenic Chlamydia and the distantlyrelated environmental endosymbionts.In our previous
43、 study we used DAADs(Fig 1b)withdiffraction-limited conventional fluorescence microscopy to show that labeling of PG is con-strained to a thin band in C.trachomatis9.This is in stark contrast to the uniform labeling ofcell periphery observed in all other PG-containing bacteria that produce periphera
44、l PG sacculi9.3D super-resolution microscopy confirmed that PG from C.trachomatis grown in thepresence of DAADs for several generations formed as a narrow ring(Fig 2).This unique PG localization is common to the Chlamydia genus,as the DAAD-labeled PGof three evolutionarily representative,pathogenic,
45、veterinary chlamydial species,C.muri-darum,C.caviae,and C.psittaci,also localized to a single,ring-like structure(Fig 2)and noneFig 2.Structured illumination microscopy of DAAD PG labeling in pathogenic Chlamydia.Structuredillumination microscopy(SIM)was conducted on four pathogenic Chlamydia specie
46、s.EDA-DA was added 2hours post infection(hpi)and coverslips were fixed at 18 hpi.PG labeling(represented by EDADA)isshown in green,the major C.trachomatis outer membrane protein(MOMP)is shown in red,and cell nucleiare in blue(this labeling scheme is maintained in all subsequent figures,unless otherw
47、ise stated).MOMPstaining is not shown for the three other species.Images are representative of 20 inclusions viewed perstrain.Scale bar=1 m.doi:10.1371/journal.ppat.1005590.g002Peptidoglycan and Cell Division in Pathogenic ChlamydiaPLOS Pathogens|DOI:10.1371/journal.ppat.1005590May 4,20164/30of thes
48、e species had peripheral PG labeling.In contrast,DAAD labeling of the coccus-shapedStaphylococcus aureus,which synthesizes PG predominantly at the septum to produce a saccu-lus30,resulted in uniform cell surface labeling(S1 Fig).Thus,we conclude that the restrictionof DAAD labeling to a narrow ring
49、at mid-cell is a unique and defining characteristic of patho-genic chlamydial species.Non-replicative chlamydial EBs do not retain PGEvasion of the immune response is a powerful evolutionary driver for many bacterial patho-gens.Because the human innate immune system recognizes and responds to PG 8,w
50、ehypothesized that Chlamydiaceae may limit PG synthesis to where and when it is absolutelyneeded,i.e.the division site of actively replicating cells.Since the immune system is most likelyto interact with the infectious,extracellular EBs,we reasoned that Chlamydia may exclude PGfrom EBs and restrict