A model symbiosis reveals a role for sheathed-flagellum rotation in the release of immunogenic lipopolysaccharide
Bacterial flagella are important virulence factors that facilitate tissue tropism in host–microbe interactions . Besides mediating such migration , flagella serve additional functions , such as adherence to or invasion of host cells ( Young et al . , 2000; Hayashi et al . , 2001; Giron et al . , 2002 ) . However , in most vertebrate models of pathogenesis , it has been difficult to separate the distinct contribution of flagellar motility in bacterial migration from its other roles , such as immune-system activation ( Feuillet et al . , 2006; Andersen-Nissen et al . , 2007 ) . Bacterial flagella are highly conserved on a molecular level , but display diversity in morphology , number and subcellular localization between species . One such variation is the presence of an outer membrane-derived sheath ( Fuerst and Perry , 1988; Geis et al . , 1993; Ferooz and Letesson , 2010 ) . While the flagella of most bacteria are unsheathed , such a structure surrounds the filament of several polarly flagellated bacteria , including several with a host-associated lifestyle . The biological function ( s ) of the sheath remains unknown , but it has been posited to allow these bacteria to evade innate immune recognition of the enclosed flagellins ( Gewirtz et al . , 2004; Yoon and Mekalanos , 2008 ) . This hypothesized role for the flagellar sheath presents a conundrum: what is the effect of coating a rapidly spinning structure with lipopolysaccharide ( LPS ) , another potent immunostimulatory molecule ? As the tools with which to study the flagellar sheath are limited , we sought to use the model symbiosis between Vibrio fischeri and the Hawaiian bobtail squid , in which the events of initial colonization can be observed in real-time , to probe whether the sheathed flagellum plays a role in inducing a symbiont-specific host response . V . fischeri is a gram-negative , bioluminescent bacterium that is found both in seawater and as a beneficial symbiont colonizing the light-emitting organs of certain fishes and squids . In the mutualism with the Hawaiian bobtail squid , Euprymna scolopes , symbiosis begins when a newly hatched squid specifically recruits V . fischeri cells from ambient seawater . The bacteria first aggregate on ciliated appendages of the light organ , and then migrate through pores and down ducts , ultimately reaching internal crypts ( Figure 1A ) where the symbiont population is housed ( Nyholm and McFall-Ngai , 2004 ) . Successful colonization of the crypts ultimately requires flagellar motility , as amotile mutants are unable to colonize the light-organ to luminous levels ( Graf et al . , 1994; Brennan et al . , 2013 ) . However , the symbionts are sensed by host cells even before they reach the crypts . Indeed , as few as 3–5 bacteria interacting with the ciliated cells of the surface appendages signal both organ-wide changes in host transcription and immune-cell ( hemocyte ) trafficking ( Koropatnick et al . , 2007; Kremer et al . , 2013 ) . Colonizing V . fischeri cells also induce an extensive morphogenesis of the light organ , beginning with the programmed cell death ( apoptosis ) of the ciliated appendages that begins within 6 hr of initial exposure to the symbiont , and continues until their complete regression ( Montgomery and McFall-Ngai , 1994; Foster et al . , 2000 ) . 10 . 7554/eLife . 01579 . 003Figure 1 . Motility mutants of V . fischeri do not induce early-stage apoptosis . ( A ) Morphology of the juvenile light organ , highlighting the external ciliated epithelium ( CE; false-colored blue ) in a scanning electron micrograph ( left ) , and the internal features with which the bacteria interact during initiation of symbiotic colonization in a schematic diagram ( right ) . DC , deep crypts; AC , antechamber; D , duct; P , pore; and MF , mucus field . ( B–E ) Representative laser-scanning confocal microscopy ( LSCM ) images of acridine orange ( AO ) -stained juvenile light organs , after exposure to either: no V . fischeri ( B , apo ) ; wild type ( C ) ; flrA ( D ) ; or motB1 ( E ) , as described in the ‘Materials and methods’ . White arrowhead in ( C ) indicates one of the numerous AO-positive nuclei ( yellow ) . Scale bar in ( B ) represents 100 μm for all the images . ( F ) Counts of AO-positive nuclei in the light-organ ciliated epithelium of squid exposed to either the indicated V . fischeri strains , or no V . fischeri ( apo ) . ( *** ) , p<0 . 001 by Kruskal–Wallis Analysis of Variance ( ANOVA ) , followed by Dunn’s Multiple Comparison test . The absence of apoptosis in the aposymbiotic squid confirms that the experimental addition of peptidoglycan ( PGN ) ( ‘Materials and methods’ ) is insufficient to induce AO staining . ( G ) Bacterial localization under conditions used for measurement of early-stage apoptosis ( panel F ) . Localization was determined by examining squid exposed to the indicated strains , genetically labeled to express GFP , using LSCM . Each point represents the bacterial localization in a single light-organ lobe . ( H ) Level of colonization ( colony-forming units , CFU ) under conditions used for measurement of early-stage apoptosis ( panel F ) . Light organs were dissected from anesthetized and pithed squid , and then homogenized and plated to determine symbiont number . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 00310 . 7554/eLife . 01579 . 004Figure 1—figure supplement 1 . Soft-agar motility of wild-type , flrA and motB1 strains . Wild-type , motB1 and flrA strains were grown to mid-log phase and inoculated into SWT medium supplemented with 0 . 3% agar . Plates were visualized after incubating for 11 hr at 28°C . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 00410 . 7554/eLife . 01579 . 005Figure 1—figure supplement 2 . Negative-stain transmission electron micrograph of a representative flagellated motB1 cell . The motB1 mutant was grown to mid-log phase in SWT broth , and visualized by transmission electron microscopy ( TEM ) after negative-staining . Scale bar indicates 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 00510 . 7554/eLife . 01579 . 006Figure 1—figure supplement 3 . Negative-stain transmission electron micrograph of a representative aflagellate flrA cell . The flrA mutant was grown to mid-log phase in SWT broth , and visualized by negative-stain TEM . Scale bar indicates 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 00610 . 7554/eLife . 01579 . 007Figure 1—figure supplement 4 . Representative LSCM image of an AO-stained light-organ lobe isolated from a squid treated with exogenous V . fischeri lipid A . As described in the ‘Materials and methods’ , squid were exposed for 10 hr to purified V . fischeri lipid A , then stained with acridine orange . White arrowheads indicate two of the AO-positive nuclei ( yellow ) . Scale bar represents 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 00710 . 7554/eLife . 01579 . 008Figure 1—figure supplement 5 . Induction of early-stage apoptosis in response to exogenous lipid A . As described in the ‘Materials and methods’ , squid were exposed for 10 hr to either no V . fischeri ( apo ) , wild-type V . fischeri , or purified V . fischeri lipid A alone . Early-stage apoptosis was determined by counting the number of acridine orange ( AO ) -positive nuclei in the ciliated field of one lobe of the light organ . ( *** ) , p<0 . 001 by Kruskal–Wallis ANOVA , followed by Dunn's Multiple Comparison test . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 008
To address whether the sheathed flagellum mediates symbiont recognition , we first compared the appearance of apoptosis within the host’s ciliated epithelium ( Figure 1A ) in response to inoculation with either wild-type V . fischeri , or one of two amotile mutants: motB1 ( Brennan et al . , 2013 ) , which bears normal flagella that do not rotate ( Figure 1—figure supplements 1 and 2 ) ; and flrA , which is both amotile and aflagellate ( Millikan and Ruby , 2003; Brennan et al . , 2013; Figure 1—figure supplements 1 and 3 ) . Such amotile mutants do not successfully colonize the light-organ deep crypts ( Graf et al . , 1994 ) . However , as this apoptosis proceeds over the first few days of symbiotic colonization , we assessed this phenotype an early time-point ( 10 hr post-inoculation ) to optimize apoptopic induction by V . fischeri while selecting conditions under which amotile mutants would not yet exhibit a colonization defect . As previously reported ( Montgomery and McFall-Ngai , 1994 ) , squid that were not exposed to V . fischeri ( i . e . , aposymbiotic , or ‘apo’ ) exhibited much lower levels of apoptosis ( Figure 1B , F ) than squid exposed to wild-type V . fischeri ( Figure 1C , F ) . In contrast , exposure to either the motB1 or the flrA strain did not induce early-stage apoptosis above the background levels ( Figure 1D–F ) . Because the actual location at which amotile mutants are arrested in symbiotic initiation has not been defined , we confirmed that both the location and number ( Figure 1G , H ) of bacteria at this early stage were indistinguishable in squid exposed to wild-type , flrA or motB1 cells . These data show that flagellar motility plays a role in inducing a specific host immune response that is independent of its later role in migration into the light-organ crypts ( Brennan et al . , 2013 ) , suggesting that biosynthesis of the flagellum , and its subsequent functions , may mediate multiple aspects of symbiotic initiation . Reports of flagellar-mediated activation of immunity have thus far been limited to responses to the filament protein , flagellin ( Hayashi et al . , 2001; Franchi et al . , 2006 ) . Despite the flagellar sheath , flagellin monomers are a prevalent component of the secretome of both V . cholerae ( Xicohtencatl-Cortes et al . , 2006 ) and V . fischeri ( unpublished data ) . However , the failure of the fully flagellated motB1 mutant to induce normal apoptosis demonstrated that the expression of immunogenic flagellin is insufficient to activate apoptosis in the light organ and , instead , suggests that apoptosis is driven by flagellar rotation itself . Previous work ( Foster et al . , 2000 ) has shown that apoptosis of the light-organ ciliated epithelium is induced specifically by the lipid-A component of LPS ( Figure 1—figure supplements 4 and 5 ) ; indeed , LPS is a common activator of innate-immune defenses across the animal kingdom ( Choi et al . , 1998; Manna and Aggarwal , 1999 ) . However , no effect of flagellar motility on LPS presentation has been reported previously . We posited that , as sheath production and flagellar biogenesis may not be tightly coordinated ( Richardson et al . , 1990; Josenhans et al . , 1995; Ferooz and Letesson , 2010 ) , a sheathed flagellum might shed LPS due to a rotation-weakened association of the sheath with the spinning flagellar filament . This hypothesis is supported by negative-stain transmission electron microscopy ( TEM ) of sheathed flagella , which has revealed vesicle-like structures ( Figure 2A ) , often at the distal tip of the flagellum , and ∼10–80 nm in diameter ( Geis et al . , 1993; Millikan and Ruby , 2004; Ferooz and Letesson , 2010 ) . Such images are indicative of weaknesses between the flagellar sheath and shaft , which may be analogous to the foci of disassociation between the outer membrane and peptidoglycan required for the formation of cell-derived outer-membrane vesicles ( OMVs ) ( McBroom and Kuehn , 2007 ) . Thus , rotation of the flagellum would further destabilize these interactions , likely releasing LPS and activating an LPS-dependent immune response , rather than simply shielding against a host response . 10 . 7554/eLife . 01579 . 009Figure 2 . Motility mutants of V . fischeri release less LPS into culture supernatants . ( A ) Negative-stain TEM of a wild-type V . fischeri cell . Dashed box , shown larger within the solid box , highlights the distal tip of two flagella , one of which displays dissociation of the sheath from the filament in the form of a membrane vesicle-like structure . Scale bar indicates 1 μm . ( B ) Reactogenic LPS in cell-free supernatants of V . fischeri mid-log ( OD600 ≈ 0 . 5 ) cultures grown in seawater tryptone ( SWT ) medium at 28°C was measured by LAL assay . ( * ) , p<0 . 05; ( ** ) , p<0 . 01; and ( *** ) , p<0 . 001 , as analyzed by one-way repeated measures ANOVA , with a posthoc Bonferroni correction . ( C ) Total LPS levels in cell-free supernatants from mid-log ( OD600 ≈ 0 . 5 ) cultures grown in SWT at 28°C for indicated strains were determined by quantitative SDS-PAGE analysis ( detailed in Figure 2—figure supplement 1 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 00910 . 7554/eLife . 01579 . 010Figure 2—figure supplement 1 . Growth of V . fischeri wild-type , motB1 and flrA strains . Indicated strains were inoculated into SWT and grown at 28°C under microaerobic conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 01010 . 7554/eLife . 01579 . 011Figure 2—figure supplement 2 . Quantification of total LPS in cell-free supernatants by SDS-PAGE analysis . ( A ) Silver-stained gel image of purified V . fischeri LPS separated by SDS-PAGE used to generate a standard curve by densitometry . ( B ) Total dry weight following dialysis of 50 ml of cell-free supernatants of the indicated strains , and band intensity of 125 μg samples of the purified lipid fractions , visualized by silver staining after SDS-PAGE analysis . Image has been cropped to remove irrelevant lanes , but all samples were separated on the same gel . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 011 We determined whether the motB1 and flrA mutants released less of this signal molecule than wild-type V . fischeri using the Limulus amoebocyte lysate ( LAL ) assay to quantify the amount of reactogenic LPS in cell-free supernatants prepared from exponentially growing cultures ( Figure 2B ) . While all strains had similar growth characteristics ( Figure 2—figure supplement 1 ) , we observed a significant reduction in shed LPS by the motB1 mutant , whose flagella do not rotate , and an even greater reduction by the aflagellate flrA mutant . To confirm that these strains actually released less ( and not just a less reactive ) LPS , we directly measured the total LPS shed from these strains by purifying the lipid fraction from the cell-free culture supernatants , and visualizing the LPS by quantitative SDS-PAGE ( Figure 2—figure supplement 2 ) . This analysis revealed a substantial reduction in total LPS released by both the motB1 and flrA strains ( Figure 2C ) , consistent with the data from the LAL assay ( Figure 2B ) . To further test our hypothesis , we next examined whether ( i ) genetic complementation of the motB1 mutant would restore normal levels of LPS shedding; ( ii ) disruption of flagellar rotation by a motB1-independent method would similarly lessen the LPS observed in culture supernatant; and , ( iii ) reduced LPS shedding would be observed when flagellar rotation is disrupted in other sheathed bacteria as well . As anticipated , expression of motB1 in trans restored both soft-agar motility and reactogenic LPS levels in cell-free culture supernatants ( Figure 3 , Figure 3—figure supplement 1 ) . In addition , cell-free culture supernatants from a V . fischeri motX mutant , which lacks a different component of the flagellar motor than motB1 , yet is also flagellated but amotile ( Figure 4—figure supplements 1 and 2 ) , had similarly reduced levels of reactogenic LPS ( Figure 4 ) . Taken together , these data support the notion that flagellar rotation promotes LPS release by V . fischeri cells . 10 . 7554/eLife . 01579 . 012Figure 3 . Genetic complementation restores supernatant LPS levels to the motB1 mutant strain . Levels of reactogenic LPS in mid-log culture supernatants ( OD600 ≈ 0 . 5 ) of indicated strains , as measured using the LAL assay . ( * ) , p<0 . 05 , and ( ** ) , p<0 . 01 , as analyzed by one-way repeated measures ANOVA with a posthoc Bonferroni correction . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 01210 . 7554/eLife . 01579 . 013Figure 3—figure supplement 1 . Soft-agar motility of genetically complemented motB1 strains . Indicated strains were grown to mid-log phase and inoculated into SWT medium containing 0 . 3% agar . Plates were visualized after incubation for 11 hr at 28°C . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 01310 . 7554/eLife . 01579 . 014Figure 4 . Disruption of motX in V . fischeri reduces supernatant LPS levels . Levels of reactogenic LPS , as measured using the LAL assay , in cell-free supernatants of mid-log cultures ( OD600 ≈ 0 . 5 ) of V . fischeri wild-type and motX strains . ( ** ) , p<0 . 01 as determined by paired Student’s t test . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 01410 . 7554/eLife . 01579 . 015Figure 4—figure supplement 1 . Soft-agar motility of V . fischeri motX mutant . Wild-type and motX strains were grown to mid-log phase and inoculated into SWT medium containing 0 . 3% agar . Plates were visualized after incubating for 8 hr at 28°C . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 01510 . 7554/eLife . 01579 . 016Figure 4—figure supplement 2 . Negative-stain transmission electron micrograph of a representative motX cell . The motX mutant was grown to mid-log phase in SWT broth , and visualized by negative-stain TEM , revealing normal sheathed flagella . Scale bar indicates 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 016 Finally , we asked whether flagellar rotation-mediated LPS release is characteristic of other bacteria with sheathed flagella by comparing the amount of LPS shed by wild-type Vibrio cholerae O395N1 and its amotile motAB-deletion derivative . As in V . fischeri , disruption of flagellar rotation in V . cholerae significantly reduced LPS release ( Figure 5A ) , supporting the conclusion that this mechanism of LPS release is not unique to V . fischeri , but is conserved among bacteria with sheathed flagella . In contrast , a similar analysis of an Escherichia coli mutant unable to rotate its ( unsheathed ) flagella revealed no alteration in levels of released LPS ( Figure 5B ) , further indicating that this behavior may be specific to bacteria that sheath their flagella . Nevertheless , it remains possible that rotation-mediated LPS release still occurs in other non-sheathed bacteria , or , perhaps , under other growth conditions . 10 . 7554/eLife . 01579 . 017Figure 5 . Loss of flagellar rotation reduces LPS release by sheathed V . cholerae , but not unsheathed E . coli . ( A ) Reactogenic LPS released into mid-log ( OD600 ≈ 0 . 5 ) culture supernatants by wild-type V . cholerae O395N1 and its motAB ( i . e . , pomAB ) derivative , grown in SWT at 37°C , was measured by the LAL assay . ( * ) , p<0 . 05 by paired Student’s t test . ( B ) Reactogenic LPS in mid-log ( OD600 ≈ 0 . 5 ) culture supernatants of wild-type E . coli and its motAB derivative grown in tryptone medium at 30°C , as measured using the LAL assay . DOI: http://dx . doi . org/10 . 7554/eLife . 01579 . 017
Using the model squid-vibrio symbiosis as a tool to probe the effect of the enigmatic flagellar sheath on host recognition , we uncover a previously unrecognized role for the rotation of sheathed flagella in mediating the release of the immunogenic molecule LPS in both V . fischeri and its pathogenic congener V . cholerae . The flagellar sheath has proved problematic to study; indeed , to our knowledge , there are no mutations that result in the loss of a sheath in otherwise sheathed flagella . Intriguingly , a few Vibrio species , including the human pathogen Vibrio parahaemolyticus , encode two independent flagellar structures: one set producing sheathed , the other unsheathed flagella ( McCarter , 1999 ) . However , pleiotropic effects of mutations in these distinct pathways as well as other technical limitations make a direct comparison of unsheathed and sheathed flagella within this species difficult to interpret ( L McCarter , personal communication ) . Thus , the functional role of the sheath , even as it relates to bacterial motility , remains poorly characterized , and observation of this new phenomenon for the flagellar sheath furthers our understanding of this enigmatic structure and its contribution to bacterial virulence . As this behavior is difficult to probe experimentally , novel approaches , such as the modeling of the biophysical properties underlying lipid-membrane behavior at a sub-micron scale , are needed to inform our understanding of the interactions between the sheath and filament along the length of the flagellum , as well as the physical forces at play between a rotating flagellar sheath and the contiguous , but static , cellular outer membrane . While vesicle-like structures have only been reported at the flagellar tip of V . fischeri ( Millikan and Ruby , 2004 ) , another possible region from which LPS may be shed during rotation is the base of the flagellum where the basal body perturbs the outer membrane . Interestingly , cryotomographic imaging has revealed unusual features in the Vibrio flagellar basal body that may help stabilize this interaction ( Chen et al . , 2011 ) . Additionally , our results show that flagellar rotation in V . fischeri promotes release of the immunogenic molecule LPS that , during initiation of symbiosis , is required for normal induction of apoptotic tissue development ( Foster et al . , 2000 ) . Further , by activating the squid’s developmental program in response to symbiont exposure , the flagellar sheath may serve to promote recognition by the host immune system , suggesting the context-dependent nature ( mutualism or pathogenesis ) of this behavior . Perhaps masking one immunostimulatory molecule ( e . g . , flagellin ) with another ( e . g . , LPS ) can serve to modulate , rather than simply avoid ( Gewirtz et al . , 2004; Yoon and Mekalanos , 2008 ) , detection by the host immune system . Surprisingly , although both the flrA and motB1 mutants still release LPS in culture , albeit at a reduced level , we observed complete attenuation of apoptotic induction , suggesting that ( i ) the threshold of LPS sensed by host cells is exquisitely calibrated to the amount of LPS shed by V . fischeri; ( ii ) because V . fischeri cells are not dividing during symbiotic initiation , flagellar-mediated LPS release is the predominant source of LPS to which host cells are exposed; and/or ( iii ) the LPS released by wild-type V . fischeri is somehow presented in a more immunogenically active form than that released by either the flrA or motB1 mutant . This last point emphasizes the microheterogeneous chemical structures of LPS made by bacteria like V . fischeri ( Phillips et al . , 2011 ) . This heterogeneity , as well as LPS’s insolubility in aqueous solution and the toxicity of spent medium to juvenile squid , makes directly comparing the chemical concentration or specific activity of LPS between culture and symbiotic settings problematic . In this symbiosis , the LPS is produced by a few non-growing bacterial cells at a tissue interface , in high flow-field conditions that further confound quantification . Finally , if LPS is shed from the flagellar tip as vesicles , other molecules such as flagellin might be enclosed , and these components may act synergistically to induce host responses ( although flagellin has not yet been characterized as a morphogen in this association ) . Alternatively , LPS released from either the tip or base of the flagellum might form vesicles with a smaller diameter than the cell-derived OMVs , thereby enabling tighter interaction with the host-cell surfaces . Further work will build upon these discoveries and clarify the mechanisms underlying signaling during initiation of the squid-vibrio symbiosis; such work includes characterizing the composition of LPS released by V . fischeri during flagellar rotation , identifying the squid receptors that recognize V . fischeri LPS ( Goodson et al . , 2005; Krasity et al . , 2011 ) , and determining any role flagellin may have in activating host responses . While thus far demonstrated only in V . fischeri and V . cholerae , the phenomenon of flagellar-mediated LPS release may be conserved across other animal-associated bacteria with sheathed flagella . The human pathogens Brucella melitensis and Helicobacter pylori , whose sheathed flagellum and/or flagellar motility serve as important virulence factors ( Ottemann and Lowenthal , 2002; Petersen et al . , 2011 ) , may similarly release the immunogenic molecule LPS during flagellar rotation and , thereby , regulate immune recognition during infection . However , the complexity of mammalian models of host–microbe interactions can limit efforts to separate the various contributions of flagellar motility , and highlights the role of invertebrate model systems in revealing the underlying principles that govern shared mechanisms of host–microbe interactions .
V . fischeri strains used in this study include: the wild-type E . scolopes light-organ isolate , ES114 ( Boettcher and Ruby , 1990 ) ; flrA::Tnerm , MB21407 ( Brennan et al . , 2013 ) ; motB1::Tnerm , MB06357 ( Brennan et al . , 2013 ) ; and motX::Tnerm , MB12561 ( Brennan et al . , 2013 ) . Strains were grown at 28°C in either Luria–Bertani salt ( LBS ) medium ( per liter , 10 g Bacto-tryptone , 5 g yeast extract , 20 g NaCl , and 50 ml of 1 M Tris–HCl buffer , pH 7 . 5 , in deionized water ) for overnight growth , or seawater tryptone ( SWT ) medium ( per liter , 5 g Bacto-tryptone , 3 g yeast extract , 3 ml glycerol , 700 ml Instant Ocean [Aquarium Systems , Inc . , Mentor , OH] at a salinity of 33–35 ppt , and 300 ml distilled water ) for experimental use . V . cholerae strains O395N1 and its ΔpomAB derivative ( Gosink and Hase , 2000 ) ( referred to as motAB within this work , for consistency with the V . fischeri and E . coli nomenclature ) were grown at 37°C in either SWT for LPS measurements , or Luria-Bertani medium ( per liter , 10 g Bacto-tryptone , 5 g yeast extract , and 10 g NaCl ) for overnight growth . E . coli strains RP437 and its ΔmotAB derivative , RP6894 ( Zhou et al . , 1998 ) , were grown at 30°C in tryptone medium ( per liter , 10 g Bacto-tryptone and 5 g NaCl ) . When appropriate , antibiotics were added to media at the following concentrations: erythromycin , 5 μg/ml; and chloramphenicol , 2 . 5 μg/ml . Growth media were solidified with 1 . 5% agar unless otherwise indicated . For growth curves , overnight cultures were used to inoculate 100 µl volumes of SWT , arrayed in a 96-well plate , and a plate-reader was used to measure optical density ( OD ) at 600 nm over 9 hr of growth at 28°C with shaking . Data are presented as the mean , and standard error of the mean , for three biological replicates . For soft-agar motility assays , cells were grown to OD600 ≈ 0 . 3–0 . 4 . Cultures were then normalized to an OD600 of 0 . 3 , and 2 μl of each strain were inoculated into plates containing SWT supplemented with 0 . 3% agar . Plates were grown at 28°C for 8–11 hr . The diameter of migration by the cells is a measure of their level of flagellar motility . For examination of flagellar structures , cells were grown in SWT broth with shaking at 28°C to an OD600 of ∼0 . 3 , applied to Pioloform-coated copper grids ( Ted Pella Co . , Tustin , CA ) for 1 min , washed with sterile water for 30 s , and negatively stained for 1 min with NanoW ( Nanoprobes , Yaphank , NY ) . Grids were immediately examined using a Philips CM120 transmission electron microscope ( University of Wisconsin Medical School Electron Microscope Facility , Madison , WI ) . The motB1 open reading frame was amplified by PCR using primer pair motB1_compF ( 5′-GCTCTAGACTAACACACAGGAAACAGCTATGGAAGATGAAAACGACTGCA-3′ ) , which introduces an XbaI restriction enzyme site and a ribosomal binding site , and motB1_compR ( 5′ GCGGTACCGACCTAATCTAAGGCGCA-3′ ) , which introduces a KpnI restriction enzyme site . The resulting product was cloned into the XbaI/KpnI-digested fragment of pVSV105 ( Dunn et al . , 2006 ) . Both the complementation construct ( pCAB98 ) and vector control were conjugated into wild-type V . fischeri and into the motB1 mutant using standard techniques ( Stabb and Ruby , 2002 ) . V . fischeri cultures were prepared by growth in SWT broth with shaking to mid-log phase ( OD600 ≈ 0 . 5 ) . Cells were removed by two successive rounds of centrifugation , and the resultant supernatant was passed through a 0 . 22-μm pore-sized filter , yielding a purified , cell-free supernatant . Cell-free supernatants were serially diluted in pyrogen-free water , and reactogenic LPS was detected from these samples using the ToxinSensor Chromogenic LAL Endotoxin Assay Kit ( Genscript , Piscataway , NJ ) , per manufacturer’s instruction , except in half–volume reactions . Reactogenic LPS units for each sample were normalized to the OD600 of the culture . The LPS in uninoculated media , serially diluted in pyrogen-free water , was determined to be fewer than 50 reactogenic LPS units per ml . Data are presented as the mean , and standard error of the mean , for at least three sets of biological replicates . Statistical analyses were performed with a repeated-measures ANOVA , or paired Student’s t test when appropriate , to normalize for variability in the basal levels of LPS released by wild-type cells across different experiments . To remove salts from 50 ml of purified cell-free supernatants of V . fischeri ( described above in ‘Measurement of reactogenic LPS using a chromogenic Limulus amoebocyte lysate ( LAL ) assay’ ) , samples were dialyzed against four liters of distilled , deionized H2O using 3500 MWCO SnakeSkin dialysis tubing ( ThermoScientific , Rockford , IL ) and five water changes . The dialyzed samples were lyophilized and resuspended in 10 ml DNaseI buffer ( 10 mM Tris–HCl , pH 7 . 6 , 2 . 5 mM MgCl2 , 0 . 5 mM CaCl2 ) and treated with 1 mg/ml DNaseI/RNaseA ( Roche , Indianapolis , IN ) overnight at 37°C . Following nuclease treatment , an equal volume of 95% phenol at 65°C was added . Samples were vortexed and incubated at 65°C for 30 min , then cooled on ice and centrifuged at 3000×g for 10 min at 4°C . The aqueous layer was collected and the phenol layer was back extracted with an equal volume of deionized water pre-warmed to 65°C . The aqueous layers were combined and residual phenol was removed by addition of one-tenth volume of 0 . 3 M sodium acetate , and precipitation three times with 3 volumes of absolute ethanol . The resulting pellets were resuspended in HPLC-grade water , frozen , and again lyophilized . Total purified LPS samples from each 50 ml of culture were measured for dry weight , resuspended to a final concentration of 25 μg/μl , and analyzed by SDS-polyacrylamide gel electrophoresis ( PAGE ) using NuPAGE pre-cast 4–12% Bis-Tris polyacrylamide gels ( Novex , Grand Island , NY ) . The gels were loaded with 5 μl from each preparation , as well as known masses of purified V . fischeri LPS to produce a standard curve . Following electrophoresis , gels were analyzed by silver staining and densitometry using ImageJ . A standard curve was generated based on the band intensities of the purified V . fischeri LPS fractions , and the concentrations of LPS in the culture supernatants were estimated from this standard curve . Freshly hatched juvenile squid were exposed to 100 μg of lysozyme-treated peptidoglycan per ml of filter-sterilized Instant Ocean at 32–36 ppt ( PGN-FSIO ) for 2 hr to induce mucus shedding ( Nyholm et al . , 2002 ) . In addition to enhancing the solubility of the peptidoglycan , this lysozyme treatment can result in smaller , more immunogenic breakdown products ( like TCT ) . As indicated in individual experiments , either V . fischeri , at a final concentration of ∼5000 colony-forming units ( CFU ) per ml , or 1 μg/ml V . fischeri lipid A were added to the PGN-FSIO . After 10 hr , squid were exposed to 0 . 001% acridine orange ( AO ) for 5 min , washed in filter-sterilized Instant Ocean ( FSIO ) , and anesthetized in 2% ethanol in FSIO . Squid were then ventrally dissected to expose the light organ , and examined by laser-scanning confocal microscopy ( LSCM ) . AO-positive nuclei in the ciliated epithelial field were counted for one light-organ lobe per squid as a measure of cell-death induction ( Delic et al . , 1991; Foster et al . , 2000 ) . Data are presented as the mean , and standard error of the mean , for 32–38 squid per condition . For experiments in which symbiont number was determined , squid were colonized as above and , at 10 hr post-inoculation , rinsed three times in FSIO , anesthetized in 2% ethanol in FSIO , and ventrally dissected to remove the light organ . Individual light organs were then homogenized , and each homogenate was diluted and spread onto LBS agar for CFU . Data are presented as the mean , and standard error of the mean , for 36–37 squid per condition . To visualize bacterial localization , squid were similarly colonized with bacterial strains carrying pVSV102 , which harbors a green-fluorescent protein ( GFP ) -encoding gene under constitutive expression ( Dunn et al . , 2006 ) , using the conditions described for assaying early-stage apoptosis . After 9 . 5 hr in PGN-FSIO containing the V . fischeri inoculum , animals were exposed to Cell Tracker Orange CMRA ( Invitrogen Molecular Probes , Carlsbad , CA ) for 30 min to label host tissue . Squid were then anesthetized in 2% ethanol in FSIO , pithed , and examined by LSCM as described previously ( Nyholm et al . , 2000; Sycuro et al . , 2006 ) . Bacterial localization was determined by the presence of GFP-fluorescing cells , relative to notable features of the juvenile light organ ( Figure 1A ) . Data indicate the location of the bacterial aggregate that has progressed the furthest in a single light-organ lobe , for 13–15 squid per condition . Thus , the decreased induction of apoptosis by these mutants does not result simply from a differential arrest earlier in migration into the light organ crypts . Scanning electron microscopy was performed on freshly hatched squid , as previously described ( Koch et al . , 2013 ) .
Bacterial flagella mediate host–microbe interactions through tissue tropism during colonization , as well as by activating immune responses . The flagellar shaft of some bacteria , including several human pathogens , is encased in a membranous sheath of unknown function . While it has been hypothesized that the sheath may allow these bacteria to evade host responses to the immunogenic flagellin subunit , this unusual structural feature has remained an enigma . Here we demonstrate that the rotation of the sheathed flagellum in both the mutualist Vibrio fischeri and the pathogen Vibrio cholerae promotes release of a potent bacteria-derived immunogen , lipopolysaccharide , found in the flagellar sheath . We further present a new role for the flagellar sheath in triggering , rather than circumventing , host immune responses in the model squid-vibrio symbiosis . Such an observation not only has implications for the study of bacterial pathogens with sheathed flagella , but also raises important biophysical questions of sheathed-flagellum function .
While a few of the bacteria that live in and on the bodies of humans and other animals are harmful and can cause disease , most others can offer benefits to their hosts . Many bacteria—including some important human pathogens—have tails called flagella that rotate to move the bacteria inside its host . However , the immune system can detect parts of these flagella and eliminate the pathogen . Bacterial flagella are made from filaments of proteins , and some flagella are also enclosed by a sheath that is similar to the outer membrane that encloses certain bacteria . The function of this sheath is unclear , although some researchers have suggested that it might prevent the immune system from detecting the proteins in the flagellum . Now , by studying the interactions between the Hawaiian bobtail squid and a marine bacterium , Brennan et al . show that the sheath can actually alert the host that the bacteria are around . The Hawaiian bobtail squid collects bioluminescent bacteria within a so-called ‘light organ’ . This organ undergoes a number of developmental changes to house the bacteria , and the squid then uses the light from the bacteria to mask its own shadow , which helps it to avoid being detected by predators . Brennan et al . compared how wild-type bacteria and mutant bacteria that either had no flagella , or had flagella that did not rotate , interacted with young squid . Only bacteria with working flagella were able to trigger the normal development of the squid’s light organ , which suggests that the rotating flagella are releasing the signal that tells the squid that the beneficial bacteria are present . Brennan et al . demonstrated that the rotation of sheathed flagella led to the release of a molecule called lipopolysaccharide . This molecule is known to activate the immune system in animals , and it is one of the bacterial signals that the squid responds to . Moreover , when the flagella of other bacteria with sheaths—such as those that cause cholera—are rotating , there is also an increase in the release of lipopolysaccharide . However , rotation of the flagella of bacteria without sheaths has no such effect . The next challenge will be to test the importance of this release of lipopolysaccharide from rotating flagella on the outcome of bacterial diseases of humans and other animals .