Virulence Mechanisms of Moraxella osloensis to ... - COST Action 850

Virulence Mechanisms of Moraxellaosloensis to the Slug Deroceras reticulatum:A Bacterium Associated With AMolluscicidal Nematode PhasmarhabditishermaphroditaParwinder GrewalDepartment of EntomologyThe Ohio State UniversityWooster, Ohio 44691, USA

Slug DamageLeft:D. laeve biting a berry (Regents, University of California);Right:D. reticulatum damage to corn (photo by M. Rice).

Slug-Parasitic Nematode:Phasmarhabditis hermaphroditaInfective juvenile of P.hermaphrodita: two layers of cuticle. closed mouth andanus. Size: about 1 mmlong, 40 µm m bodydiameter.

Host Range of P.hermaphroditaSlugsSnailsArion aterCernuella virgataA. distinctusCochlicella acutaA. intermediusHelix aspersaA. lusitanicusLymnaea stagnalisA. silvaticusMonacha cantianaDeroceras caruanaeTheba pisanaD. laeveD. reticulatumLeidyula floridanaTandonnia budapestensisT. sowerbyi-from Glen and Wilson (1997); Speoser et al. (2001); Grewal et al. (2003).

Left: D. reticulatum infected with P. hermaphrodita.Right: Healthy D. reticulatum. . Note the swollen mantleof the infected slug (photo by R. Harvey).

Parasitic Cycle of P. hermaphroditaSlug mantleInfective juvenile (IJ) HermaphroditeSlug dies in 7 –14 days

Bacteria associated with P. hermaphroditaFamilyAeromonadaceaeBacillaceaeEnterobacteriaceaeFlavobacteriaceaeMoraxellaceaePseudomonadaceaeSphingobacteriaceaeSpeciesAeromonas hydrophilaAeromonas sp.Bacillus cereusProvidencia rettgeriSerratia proteamaculansFlavobacterium breveF. odoratumMoraxella osloensisPseudomonas fluorescens (isolate no. 1a)P. fluorescens (isolate no. 140)P. fluorescens (isolate no. 141)P. paucimobilisSphingobacterium spiritovorum-from Wilson et al. (1995).

Moraxella osloensis is a gram-negativeaerobic bacterium. is considered as anopportunistic humanpathogen. is preferred bacteriumfor mass-producingP.hermaphrodita.

Is M. osloensis vectored into the shellcavity pathogenic to the slug? Wilson et al. (1995) reported that a 24-hrculture of M. osloensis injected into D.reticulatum hemocoel was not pathogenic. Shell cavity in the posterior mantle region of D.reticulatum serves as the main portal of entryfor P. hermaphrodita (Tan and Grewal, 2001a). Important organs, including kidney, lung andheart, are located in the slug mantle region.

Pathogenicity of 60-hrM. osloensis culture to D.reticulatum after injection into the shell cavity% slug mortality100806040200 (Control) 6.006.15 × 10 2 6.15 × 10 46.15 × 10 6 6.15 × 10 8R 2 = 0.54*R 2 = 0.73***** ***01 4 8 12Days after injection

% slug motality10080604020Pathogenicity of M. osloensis cultures ofdifferent ages against D. reticulatum afterinjection into the hemocoel0aSaline (Control)24 hr culture40 hr culture60 hr culturebaaaaab4 8 12 16Days after injectionaaabbR 2 = 0.66aababb

Effect of coinjection of penicillin andstreptomycin with 60-hrM. osloensis culture onthe bacterial pathogenicity to D. reticulatum% slug mortality10080604020aSalineAntibioticsBacteriaBacteria+Antibioticscabbababcaababaabab01 4 8 12Days after infection

Numbers of viable M. osloensis in 20 IJs offresh or aged P. hermaphrodita# of bacteria in 20 nematodes140120100806040200aabFresh 3-month old 8-month oldBatch of nematodesb

Percentage of P. hermaphrodita IJs from afresh, 3-month 3old, and 8-month 8old batchcarrying bacteriaPercentage of nematodes100806040200Fresh3-month old8-month old< 1 1 ~ 4 5 ~ 8 9 ~ 12 13 ~ 15Average # of bacteria per nematode

Pathogenicity of fresh, aged and axenic P.hermaphrodita to D. reticulatum after injectioninto the shell cavity10080*% slug mortality6040200Fresh IJs3-month oldIJs8-month oldIJs8-month oldIJs withantibioticsAxenicJ1/J2s withantibioticsAntibioticsSaline

Conclusions Aged culture of M. osloensis (e.g. 60-hr bacterialculture) is pathogenic to D. reticulatum both in theshell cavity and hemocoel of the slug. Axenic J1/J2 nematodes were non-pathogenic afterinjection into the shell cavity and the pathogenicityof the IJs depended on the number of viablebacteria carried by the nematodes. Therefore, P. hermaphrodita only vectors M.osloensis into the shell cavity of D. reticulatum andthe bacterium is the main killing agent.

Questions:DoesDoes M. osloensis produce a toxin(s)to kill D. reticulatum?Is this an exotoxin or endotoxin(s)?What What is this toxin(s)?

% slug mortalityPathogenicity of different components of 3-day 3M. osloensis cultures to D. reticulatum afterinjection into the shell cavity706050403020100SalineCell-free suspensionWashed cells+AntibioticsIntact cellsbbbbaAntibioticsBroken cells+AntibioticsWashed cellscb b b bc cbcb bc bca a aa a a a a a1 4 8 12Days after injectionababcc

Effect of heat treatment (at 60°C C or 100°C C for 15min) on the endotoxin activity of different typesof M. osloensis cells to D. reticulatum% slug mortality5040302010Antibiotics22°C60°C100°Caaababb b b b b bb0a a a a aWashed 2-DcultureBroken 2-DcultureWashed 3-DcultureBroken 3-Dculture

Effect of proteases (trypsinand chymotrypsin)treatment on the endotoxin activity of differenttypes of M. osloensis cells to D. reticulatum% slug motality50403020100AntibioticsAntibiotics+ProteasesBacterial cellsBacterial cells+Proteasesaa aa aaba a a aWashed 2-DculturebBroken 2-DculturebccWashed 3-DcultureabbBroken 3-Dculture

% slug mortalityEndotoxin activity of purified lipopolysaccharide(LPS) from 3-day 3M. osloensis cultures to D.reticulatum after injection into the shell cavity10080604020R 2 = 0.79P < 0.001LD 50 =48microgram/slug00 20 40 60 80 100Concentration of LPS (microgram/slug)

Conclusions M. osloensis produces an endotoxin(s), which istolerant to heat and protease treatments and killthe slug after injection into the shell cavity. Heat and protease treatments increase theendotoxin activity of the young broken cells butnot that of the young washed cells. M. osloensis LPS is an endotoxin that is activeagainst D. reticulatum.

What are the properties of M.osloensis LPS?

Injection, contact and oral toxicity of purifiedLPS (0.1 mg/slug) from 3-day 3M. osloensiscultures against D. reticulatum% slug mortality100bControl80Treated604020a a aaa0Injection Contact Oral

Toxicity of lipid A or polysaccharide moietyfrom M. osloensis LPS to D. reticulatum afterinjection into the shell cavity100c% slug mortality806040bb20aa0Water Polysaccharide Lipid A Triethylamine Lipid A +Triethylamine

Semiquantitation of M. osloensis LPS byLimulus amebocyte lysate (LAL) assay Each LPS concentration and LAL were mixed andincubated. The formation of a firm gel wasconsidered a positive reaction. The minimal LPS concentration required for theLAL gelation was 1pg/ml for M. osloensis LPS or0.06 endotoxin units (EU)/ml for the standardendotoxin (E.coli 055:B5 LPS). The lipid A moiety of M. osloensis LPS caused apositive reaction in the LAL assay, but thepolysaccharide moiety did not.

Detection of LPSs by silver stain. Samples of 1 µg g were analyzedon SDS-PAGE except as noted. Lane 1 and 2: M. osloensis at 1and 5 µg, respectively; lane 3: E. coli EH100; lane 4: E. coli J5.

Effect of galactosamine or galactosamine/uridinecombination on the susceptibility of D.reticulatum to M. osloensis LPS Galanos et al. (1979) reported that sensitivities ofexperimental mammals to LPS were enhancedseveral thousand fold by coinjection of the LPS withgalactosamine, , which was thought to be a specifichepatotoxic agent. The induced sensitization to LPS could be reversedby uridine, , which can inhibit the liver injury elicitedby galactosamine. D. reticulatum is a lower animal that does not have aliver.

Effect of the lethal dose (1 mg/slug) of galactosamine orgalactosamine/uridine(3 mg/slug) combination on thesusceptibility of D. reticulatum to M. osloensis LPS (0.03mg/slug)10080c% slug mortality6040bbb200aEFW G LPS G+LPS G+LPS+U

Effect of the sublethal dose (0.3 mg/slug) of galactosamine orgalactosamine/uridine(1 mg/slug) combination on thesusceptibility of D. reticulatum to M. osloensis LPS (0.01mg/slug)100% slug mortality806040200baa aaEFW G LPS G+LPS G+LPS+U

Discussion M. osloensis LPS possesses a lethal injection toxicitybut no contact or oral toxicity against D. reticulatumthus suggesting that P. hermaphrodita is a necessarynatural vector of M. osloensis for slug control. D. reticulatum may have some primitive liver-likelikeorgan (tissues or cells), which may be the targets ofgalactosamine. It is also possible that galactosamine hurts otherimportant slug organ(s), leading to the inducedsensitization.

Conclusions Toxicity of M. osloensis LPS resides in the lipid Amoiety but not in the polysaccharide moiety. M. osloensis LPS was quantitated as 6 × 10 7 EU/mg. M. osloensis LPS is a rough-type LPS with anestimated molecular weight of 5,300. Coinjection of galactosamine with the LPS increasedits endotoxin activity to D. reticulatum by 2-424 fold. The galactosamine-induced induced sensitization can beblocked completely by uridine.

Why are the aged M. osloensis cellsmore virulent to the slug? Surface exposed antigens such as LPS, OMPs, , and pilihave been considered as virulence factors for M.catarrhalis or M. bovis. It is possible that OMP(s) and pili-like like projectionsmay also be virulence factors for M. osloensis besidesLPS. We hypothesized that the temporal expression of oneor more surface exposed antigens correlates with thetemporal pattern of M. osloensis virulence towards D.reticulatum.

athogenicity of 1 to 5-day5M. osloensis cultures toD. reticulatum after injection into the shell cavity% slug mortality50403020100aababbbbSaline1-dayculture2-dayculture3-dayculture4-dayculture5-dayculture

Detection of LPS during culture. Proteinase K-digestedlysates of 1 ×10 10 M. osloensis cells were analyzed on SDS-PAGE except as noted.Lane 1 to 5: : 1 to 5-day 5culture, respectively; Lane 6 and 7: purifiedM. osloensis LPS from 3-day 3culture at 1 and 2 µg, respectively.

Detection of OMPs during culture. Purified OMPs from 1 ×10 10 M. osloensis cells were analyzed on SDS-PAGE except asnoted. Lane 1: molecular weight markers (unit: KDa); Lane 2to 6: 1 to 5-day 5cultures, respectively.

D E FLight micrographs of Gram-stainedM. osloensis cellsduring culture. A to F represent M. osloensis cells from0 to 5-day 5cultures, respectively.ABC

CDETransmission electron micrographs of sections of M.osloensis cells during culture. A to E represent an M.osloensis cell from 1 to 5-day 5cultures, respectively.AB

Numbers of pili-like like projections per bacterialsection of M. osloensis from 1 to 5-day 5cultures80# of pili-like projections/section6040200aabdcbc1-dayculture2-dayculture3-dayculture4-dayculture5-dayculture

Discussion Pili-like like projections presented on the bacterial surfacemay enable better and more rapid slug colonization. Aged M. osloensis cells appear to enhance theirpathogenicity to the slug during culture not byincreasing the LPS production but by altering theOMP(s) production, and most probably by developingand increasing the density of the pili-like like projectionson their surfaces. Changes of the pathogenicity of M. osloensis against D.reticulatum during culture strongly correlate withstructural changes in the bacterial cell wall.

Conclusions Average yield of M. osloensis LPS per bacteriumdoes not differ during culture. M. osloensis cells from 3-day 3cultures produce moreOMPs than those from the younger or older M.osloensis cultures. Aggregation of M. osloensis cells increased as thedensity of the pili-like like projections presented on thebacterial surfaces increased during culture. Temporal expression of the projections stronglycorrelates with the temporal pattern of M. osloensisvirulence to D. reticulatum.

Overall Conclusions Shell cavity of D. reticulatum served as the main portalof entry for P. hermaphrodita. P. hermaphrodita vectors M. osloensis into the shellcavity and the bacterium is the main killing agent inthe nematode/bacterium complex. M. osloensis produces an active endotoxin (LPS) to killthe slug, which is a first reported biological toxinagainst mollusks. This is also the first report of galactosamine-inducedinducedsensitization to LPS in an animal without a liver.

Overall Conclusions (continued) Temporal expression of the pili-like like projectionsstrongly correlates with the temporal pattern of M.osloensis virulence to D. reticulatum. Changes in M. osloensispathogenicityagainst Dreticulatumduring culture strongly correlate withstructural changes in the bacterial cell wall. Mutualism between P. hermaphrodita and M. osloensisis parallel to the association between entomopathogenicnematodes and their associated bacteria.

Publications Tan, L., and P. S. Grewal. 2002b. Comparison of two silverstaining techniques for detecting lipopolysaccharides inpolyacrylamide gels. J. Clin. Microbiol. 40:4372-4374. Tan, L., and P. S. Grewal. 2002a. Endotoxin activity ofMoraxella osloensis aganist the grey garden slug, Derocerasreticulatum. Appl. . Environ. Microbiol. 68:3943-3947. Tan, L., and P. S. Grewal. 2001b. Pathogenicity ofMoraxella osloensis, a bacterium associated with thenematode Phasmarhabditis hermaphrodita, to the slugDeroceras reticulatum. Appl. . Environ. Microbiol. 67:5010-5016.

Publications (continued)Tan, L., and P. S. Grewal. 2001a. Infection behavior of therhabditid nematode Phasmarhabditis hermaphrodita to the greygarden slug Deroceras reticulatum. J. Parasitol. 87:1349-1354.Grewal, P. S., S. K. Grewal, and L. Tan. 2003. Molluskparasitism by nematodes: types of associations and evolutionarytrends. J. Nematol., in press.Tan, L., and P. S. Grewal. 2003. Characterization of the firstmolluscicidal lipopolysaccharide from Moraxella osloensis. Appl.Environ. Microbiol., in press.Tan, L., P. S. Grewal, and T. Meulia. Temporal expression ofsurface exposed antigens in Moraxella osloensis correlates withvirulence to the slug Deroceras reticulatum. J. Bacteriol., inreview.

AcknowledgmentsPh.D. Student: Li TanTechnical support on TEM: Tea Meulia, Karli J.Fitzelle, , and Dave Fulton.Slug collection: Judy A. Smith and Sukhbir GrewalFinancial support: a competitive matching fund grantfrom OARDC and MicroBio Ltd., an OARDCGraduate Research Competitive Grant, and an OSUpresidential fellowship.