Swarming Behavior of and Hemolysin BL Secretion by Bacillus cereus

An association between swarming and hemolysin BL secretion wasobserved in a collection of 42 Bacillus cereus isolates (P =0.029). The highest levels of toxin were detected in swarmersalong with swarm cell differentiation (P = 0.021), suggestingthat swarming B. cereus strains may have a higher virulencepotential than nonswarming strains.

INTRODUCTION

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INTRODUCTION

Bacterial swarming is a specialized form of surface translocationthat enables flagellate bacteria to coordinately move atop solidsurfaces (11). The ability to swarm depends on a complex differentiationprocess that leads short and oligoflagellate swimmer cells toproduce long, multinucleate, and hyperflagellate swarm cellsactively migrating over surfaces in organized groups of tightlybound cells (reviewed in reference 10). The widespread natureof swarming-proficient species suggests that this type of flagellum-aidedmotility is a successful strategy developed by flagellate bacteriato rapidly colonize environmental surfaces (10). Moreover, swarmingcan be influential in host-pathogen interactions, since it contributesto the virulence potential that certain pathogens may exertby facilitating host colonization (1, 7, 8, 14) and/or leadsto an increase in the production of specific virulence factors(2, 15). We have previously described the swarming behaviorexhibited by laboratory strains of Bacillus cereus and Bacillusthuringiensis (9, 19), two closely related species that producecommon genome-encoded virulence factors (17); among these, thetripartite toxin (B, L₁, and L₂ components) hemolysin BL (HBL)exerts enterotoxic, hemolytic, cytotoxic, and dermonecroticactivity (3-5). In B. thuringiensis 407 Cry^–, a mutationin flhA, a component of the flagellar export apparatus (12,16), was found to coordinately abolish swarming and secretionof HBL (9). In B. cereus NCIB 8122, which produces only theL₂ component of HBL, L₂ secretion was detectable exclusivelyin differentiated swarm cells (19). These findings suggestedthat swarming and HBL secretion could be associated phenomena.

In this study, we assessed the motility behavior of and thesecretion of HBL by B. cereus strains isolated from differentsources to evaluate whether (i) HBL secretion requires intactflagella, (ii) swarming and HBL secretion are prevalent traitsin natural isolates, and (iii) an increase in HBL secretionoccurs along with swarm cell differentiation.

Swimming and swarming motility in B. cereus isolates.

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Swimming and swarming motility...

B. cereus strains were collected from clinical, environmental,or food samples (Table 1) and identified by the API 50 CH assay(Bio-Merieux, France). Identification of B. cereus was confirmed,excluding the presence of parasporal crystals, which are discriminativefor B. thuringiensis, in preparations of sporulating culturesstained with 0.5% basic fuchsin. Assays for swimming (on 1%tryptone-0.5% NaCl plates containing 0.25% agar [TrM]) and chemotaxis(on TrM supplemented with 2.0 mM mannitol or glutamine) wereperformed as described previously (9, 19). Swarming differentiation(on 1% tryptone-0.5% NaCl plates containing 0.7% agar [TrA])was ascertained by visualizing the presence of hyperflagellateand elongated cells (at least 2.5 times longer than cells growingin liquid medium).

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TABLE 1. B. cereus strains used in this study: analysis of motility and HBL secretion

Among the 42 strains analyzed, seven failed to swim (16.7%)and six were able to swim but could not perform chemotaxis (14.3%)(Table 1). Hyperflagellate and elongated swarm cells, mainlylocalized at the colony rim, were evidenced for 45.2% of thestrains. The possibility of predicting swarming proficiencyby measuring colony diameter was proven to be inapplicable.Indeed, although the isolates produced differently sized colonies,the strains truly undergoing swarming, as demonstrated by productionof differentiated swarm cells, did not always develop widercolonies than did strains unable to swarm (Fig. 1). As alreadydemonstrated for B. cereus and B. thuringiensis laboratory strains(9, 19), swimming- and chemotaxis-deficient strains were unableto swarm (Table 1), confirming that the integrity of both theflagellar apparatus and the chemotaxis system is required byundomesticated B. cereus strains for mounting a swarming response.

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FIG. 1. Colonies produced by swarming (ATCC 14579 and F837/76) and nonswarming (1230/88 and S1C) B. cereus strains on tryptone-NaCl containing 0.7% agar. Plates were photographed after 24 h of incubation at 37°C. Insets show examples of swarm cells collected from the colony rim.

The finding that the prevalence of swarming in our B. cereuscollection was lower than that reported for Salmonella spp.,approaching 100% of the strains analyzed (13), suggests thatswarming is a less-relevant environmental behavior for thisspore-forming species. Indeed, production of spores should beregarded as an efficient strategy for contributing to bacterialspreading as well as persistence in different environments.

The percentage of swarming-proficient strains was higher withinclinical (62.5%) than within food (31.6%) isolates (Table 1);however, the limited number of strains analyzed did not allowus to infer that swarming behavior is prevalent in strains foundin a given environment or linked to host adaptation.

HBL production and flagella.

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HBL production and flagella.

HBL is a membrane lytic system composed of the antigenicallydistinct proteins B, L₁, and L₂, encoded by hblA, hblD, andhblC, respectively. Strains secreting complete HBL, as demonstratedby the formation of a discontinuous zone of hemolysis aroundcolonies on sheep blood agar plates (3), were 59.5% (n = 25)of the total number of strains (Table 1). Non-HBL-producingstrains (n = 17) were subjected to PCR amplification to evaluatethe presence of hbl genes (see Table S1 in the supplementalmaterial) (18, 20) and to immunoblot analysis with rabbit antiserato the individual HBL components (9) for detecting HBL proteinsreleased into culture supernatants (1% tryptone-0.5% NaCl [TrB]).Twelve out of the 17 strains lacked one to three hbl genes andthe corresponding encoded proteins (Table 2). Among the remainingHBL-negative isolates, ATCC 33018 and D33 secreted L₂ and Bplus L₁, respectively, whereas the corresponding genes werenot detected with two PCR primer sets. This result was interpretedas a consequence of variations at the primer annealing sites,as already reported for other B. cereus isolates secreting HBLproteins but giving negative results for hbl genes (20). Incontrast, S2-8, R-12, and F3502/73 gave positive PCR resultsfor all hbl genes, while not all proteins were detected (Table2). Cell lysates of strain S2-8 never gave a positive signalfor L₁, thus suggesting that this component was not producedor underwent such rapid and extensive intracellular proteolysisas to be undetectable. When cell lysates of R-12 and F3502/73were subjected to immunoblotting with anti-B antibodies, onereactive band appeared at a molecular mass lower than that ofthe extracellular B component of strain ATCC 14579 (41 kDa)(Fig. 2). Since no internal stop codon was detected in hblAof the two strains (sequencing performed with FHA2 and BR1;see Table S1 in the supplemental material), these results suggestedthat the B component was synthesized but not exported and waspartially proteolyzed inside the cell. Failure to secrete intracellularlyproduced HBL has been reported to occur in a B. thuringiensismutant lacking flagella (9) and explained by the function offlagella as secretion systems in addition to locomotion organelles(16).

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TABLE 2. Characterization of discontinuous non-hemolysis-producing B. cereus strains

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FIG. 2. Immunoblot assay with a polyclonal antibody to the B component of HBL of culture supernatants (S) and cell lysates (L) prepared from the B. cereus strains R12, F3502/73, and ATCC 14579. M, molecular size standards. Arrows indicate estimated molecular masses (in kilodaltons) of B in the different strains.

Interestingly, no flagellum was ever visualized in preparationsof strain R-12 and one or two flagella were seen in no morethan 15% of the F3502/73 cells (Table 1). This finding strengthensthe hypothesis that flagella act as a system for protein exportfor HBL secretion also in B. cereus. In this context, the observationthat nonmotile isolates harboring flagella did secrete HBL (S3-4,1230-88, and F4433/73 [Table 1]) can also suggest that the functionalityof the flagellum as a locomotion organelle is not required forits function as an export apparatus.

Swarm cell differentiation is accompanied by a substantial increase in HBL secretion.

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Swarm cell differentiation is...

Although HBL-producing strains were either swarmers (n = 15)or nonswarmers (n = 10) and HBL-defective strains could swarm(n = 4) or not (n = 13), a weak but statistically significantassociation (P = 0.029, Fisher's exact test) between HBL productionand swarming was observed. This finding, together with the demonstrationthat secretion of the L₂ component of HBL was detectable onlyduring swarming in a reference B. cereus strain (19), led usto hypothesize that an increase in toxin secretion could occurin swarmers along with swarm cell differentiation. To this end,the amount of toxin secreted by randomly selected HBL-producingstrains (six swarming and six nonswarming isolates) was quantifiedduring growth under swarming and nonswarming conditions. Conditionsenabling collection of proteins secreted during swarming differentiationwere realized by spotting late-exponential-phase TrB cultures(0.5 µl, approximately 2 x 10⁸ cells/ml) onto Anoporemembranes (0.2-µm pore size) of 10-mm cell culture inserts(Nalge Nunc International) that were placed into 24-well platescontaining 0.5 ml TrB/well. The inserts allowed us to effectivelyseparate the liquid medium from bacteria growing over membranes,thus mimicking bacterial growth atop solid substrates. Well-definedcolonies were developed by all strains, and swarm cells, mainlylocalized at the colony rim, were detected only for the swarming-proficientisolates. After 48 h of incubation at 30°C, the cultureinserts were removed, the number of CFU on the membranes wascounted, and the culture media were collected to quantify theamount of secreted HBL. Quantification of the B component ofHBL was performed by enzyme immunoassay with specific antibodiesto purified B (6). The B concentration in samples was calculatedby using a calibration curve constructed with purified B proteinat concentrations ranging from 0.5 to 10 ng/ml and expressedas the amount of protein for 10⁶ bacterial cells or the totalamount of proteins in cell lysates.

The amount of B secreted under nonswarming conditions (liquidcultures in TrB) ranged from 3.8 ± 0.64 to 33.6 ±3.15 ng/10⁶ cells for nonswarming strains and from 1.25 ±0.21 to 9.65 ± 1.11 ng/10⁶ cells for swarming strains(Table 3; Table S2 in the supplemental material reports theamount of B as µg/mg of total proteins in cell lysates).When the same strains were propagated over the membranes, significantlyhigher levels of secreted B, ranging from 28.33 ± 2.44to 378.35 ± 21.75 ng/10⁶ cells, were detected for swarming-proficientstrains. The ratio of the amount of B secreted by cells growingon the membrane surface to that secreted in liquid varied from9.38 to 44.2 and from 0.74 to 2.40, with a (21.02 ± 14.36)-and a (1.66 ± 0.62)-fold mean increase for the swarmingand nonswarming strains, respectively (Table 3). Statisticalanalysis of the mean ratios for the two groups of strains revealedthat the ability to swarm was associated with a significantincrease in the secretion of the B component (P = 0.021; two-tailedWelch's t test).

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TABLE 3. Quantification of the B component of HBL secreted by swarming and nonswarming Bacillus cereus strains grown in broth or over a solid surface

Conclusions.

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Conclusions.

The novelties of this report rely on the demonstration that(i) the ability to swarm is a relatively widespread behaviorof B. cereus natural isolates and (ii) swarm cell differentiationin B. cereus is accompanied by a significant increase in HBLsecretion. These data highlight the notion that swarming differentiationby B. cereus may contribute to the virulence potential of thisopportunistic human pathogen. Moreover, interesting data werederived from the observations that (i) aflagellate B. cereusisolates do not secrete the intracellularly produced HBL, (ii)hyperflagellate swarm cells secrete an increased amount of toxin,and (iii) flagellate but nonmotile strains export HBL. Theseresults support the idea that the flagellum is required forHBL secretion and that its functionality as export machineryis not dependent on its functionality as a locomotion organelle.

ACKNOWLEDGMENTS

We thank Jean Schoeni for the design of primers Af, Ar, Df,Dr, Cf, and Cr and for immunoblot analysis of some strains.

This work was supported by National Research Project grant 2005058814from the Ministero dell'Istruzione, dell'Università edella Ricerca.

FOOTNOTES

* Corresponding author. Mailing address: Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Università degli Studi di Pisa, Via S. Zeno 35-39, 56127 Pisa, Italy. Phone: 39 050 2213696. Fax: 39 050 2213711. E-mail: ghelardi{at}biomed.unipi.it

Published ahead of print on 20 April 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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