Published Ahead of Print 21 September 2012.
2012, 78(23):8467. DOI:
Appl. Environ. Microbiol.
Ambroise Lambert, Naoko Takahashi, Nyles W. Charon and
Nonpathogenic Leptospira Species
Chemotactic Behavior of Pathogenic and
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Chemotactic Behavior of Pathogenic and Nonpathogenic Leptospira
Ambroise Lambert,a,bNaoko Takahashi,c* Nyles W. Charon,cand Mathieu Picardeaua
Institut Pasteur, Unité de Biologie des Spirochètes, Paris, Francea; Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, Franceb; and Department of
Microbiology, Immunology, and Cell Biology, West Virginia University, Health Sciences Center, Morgantown, West Virginia, USAc
spiracells.WeidentifiedTween80,glucose,sucrose,andpyruvateasattractantsfor Leptospira cells;aminoacidsandvitamin
exceeding 10% (5). Leptospira spp. belong to the spirochete phy-
lum and consist of both saprophytic and pathogenic species, with
Leptospira species are highly motile spirochetes, and their unique
motility likely plays a role in their ability to rapidly disseminate
into the host (9, 10, 16). Spirochete motility is unique as it allows
stop the motility of peritrichous bacteria (3, 12). Leptospira mo-
tility depends on the presence of two periplasmic flagella, each
arising from one of the subpolar ends of the cell without overlap-
ping at the cell center (6). The direction of flagellar rotation is
modulated by chemotaxis, which is defined by the movement of
an organism toward or away from a chemical compound. The
latter is called an attractant if it induces a movement toward itself
regulation of the flagellarly based motility in relation to che-
motaxis is present in many living organisms and has been well
studied in model bacteria such as enterobacteria (22).
Among the spirochetes, chemotaxis has been studied to a lim-
rochaeta aurantia, and Treponema denticola (11, 15, 20, 24). In
Leptospira spp., hemoglobin was found to be an attractant (27),
molecules, including diffusible molecules and small peptides, but
not to intact proteins.
previously used to analyze B. burgdorferi chemotaxis (2, 21) that
Leptospira biflexa serovar Patoc strain Patoc I with the pathogen
Leptospira interrogans serovar Manilae strain L495.
actively motile exponential-phase Leptospira cells in Elling-
hausen-McCullough-Johnson-Harris (EMJH) culture medium
(optical density at 420 nm of 0.5, which corresponds to approxi-
mately 5 ? 108bacteria/ml) were centrifuged at low speed and
gently resuspended in motility buffer consisting of 7 mM
pended cells were then preincubated overnight at 30°C to allow
bacteria to recover motility and to deplete nutrients that were
uman leptospirosis is an emerging disease with more than
1,000,000 cases occurring annually, with a case fatality rate
were translating (less than 5% were nonmotile, with the remain-
ing cells usually showing gyrating ends without translational
in EMJH medium (data not shown).
The chemotaxis chamber used was based on the one described
for B. burgdorferi (2, 21). Briefly, a 96-well plate (1.2-ml square
well storage plate; Thermo Scientific) was filled with 200 ?l of
suspension (2 ? 107L. interrogans cells counted using a Petroff-
Hausser chamber) per well, and the inverted plate was perforated
at the bottom of each well facing the cell suspension to place the
capillary tubes (75-mm, 60-?l, 0.95-mm-diameter hematocrit
capillary tubes; Hirschmann Laborgeräte) containing hypotheti-
eluted from the capillary tube, and the total genomic DNA was
extracted using a cell DNA purification kit (Maxwell; Promega).
Enumeration of Leptospira cells entering in the capillary tubes by
viable plate counts is a low-throughput method, especially for L.
interrogans, as it takes several weeks to form colonies (10).
dorferi cells (2, 21), we developed a SYBR green (SSoFAST
EvaGreen Supermix; Bio-Rad) quantitative PCR (qPCR) assay
targeting lipL32 or rpoB to enumerate L. interrogans and L. biflexa
cells, respectively, in the capillary tubes (18). Standard curves for
the quantification of leptospires were constructed using DNA ex-
tracts from known numbers of leptospires counted in a Petroff-
Hausser chamber. All PCR assays were performed in duplicate,
and control reactions without template were included in each as-
say. For each assay, at least two independent experiments with
three to four capillary tubes were performed.
To begin to identify chemoattractants, we first tested specific
nutrients present in the EMJH culture medium. EMJH is a com-
plex medium composed of Tween 80, glycerol, pyruvate, BSA,
vitamin B12, thiamine, and salts. Pyruvate, which stimulates the
Received 20 July 2012 Accepted 3 September 2012
Published ahead of print 21 September 2012
Address correspondence to Mathieu Picardeau, firstname.lastname@example.org.
*Present address: Naoko Takahashi, Suzuka University of Medical Science, Suzuka
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
December 2012 Volume 78 Number 23Applied and Environmental Microbiology p. 8467–8469aem.asm.org
on June 10, 2014 by guest
tractant of both pathogenic and saprophytic strains. The capillary
tubes were filled with 100 mM pyruvate, and comparisons were
made to tubes filled with motility buffer, which served as a nega-
tive control. The accumulation of L. biflexa and L. interrogans in
the capillary tubes filled with pyruvate reached a plateau in both
strains after 30 min of incubation at 30°C or 37°C. In contrast, no
significant increase in spirochete numbers increased in the buffer
control (data not shown). Based on these findings, an incubation
time of 1 h at 30°C was selected for further experiments.
Identification of chemoattractants. The relative chemotactic
the capillary tube with attractant compared to that in tubes with-
out attractant. These responses were 7 and 8 with pyruvate at 100
mM for L. biflexa and L. interrogans, respectively (Table 1). To
confirm we were assaying for chemotaxis, pyruvate was added in
the bacterial suspension prior to the assay, therefore lowering the
gradient of the attractant. Under these conditions, the number of
bacteria in the capillary tubes decreased, further confirming that
capillary tubes increased with the concentration of pyruvate
We then tested numerous compounds present in EMJH me-
dium for their ability to serve as chemoattractants (Table 1).
Tween 80, which was chosen for its role as a source of long-chain
fatty acids, such as oleate, palmitate, and stearate, in Leptospira
(10, 13), functioned as an attractant for both pathogenic and sap-
rophytic strains. A previous study demonstrated that free fatty
acids are often toxic to Leptospira (25), and we confirmed this
observation when testing chemoattraction toward oleate and in-
stead found it to act as a lysing agent for Leptospira (data not
shown). In contrast, stearate and palmitate were not found to be
toxic under our conditions. Interestingly, pathogenic and sapro-
tate, as it served as attractant for L. interrogans but not L. biflexa
(relative chemotactic response of 1 versus 16 in L. biflexa and L.
which is the most common long-chain fatty acid in Leptospira
olized by ?-oxidation in Leptospira (10).
pathogenic and saprophytic strains (Table 1). These findings sug-
gest that glucose, which stimulates the growth of some Leptospira
strains (4, 8), might be sensed by Leptospira. Sucrose was also
by Leptospira, as both strains lack the genes that code for enzymes
that break down this sugar. However, sucrose could still serve as
an attractant without being metabolized by binding directly or
indirectly to a chemoreceptor. Thus, E. coli shows strong attrac-
tion to the nonmetabolizable galactose analog fucose (1).
Leptospiral attraction toward amino acids was relatively weak.
This includes leucine, which has been shown to be readily trans-
L. biflexa nor L. interrogans displayed strong attraction toward
vitamin B12, which is essential for Leptospira growth (23).
Our experiments indicated positive chemotaxis of L. interro-
gans, but not L. biflexa, toward hemin, which can be used as a
source of both iron and heme (19) (Table 1).
Conclusion. Our capillary tube assay in combination with
real-time PCR is likely to have several applications, such as the
analysis of chemotactic Leptospira mutants or the study of the
FIG 1 Chemotaxis increases with increasing concentration gradient of at-
bacterial suspensions of L. interrogans in wells (W) containing 0, 50, and 100
mM pyruvate. Capillary tubes filled with chemotaxis buffer were used as a
negative control. Fewer cells enter capillaries containing pyruvate as its con-
centration in the bacterial suspensions is increased. Data are representative of
one experiment and are the means of PCR duplicates of three to four capillary
tubes. The error bars represent standard deviations.
TABLE 1 Chemotactic responses of L. biflexa and L. interrogans to
Relative chemotactic responseb
L. biflexa L. interrogans
Hemin 0.3 mM13
aAll compounds were tested in a range of 0.1 mM to 100 mM (1 to 2 % for Tween 80).
Stearic and palmitic acids were first dissolved in chloroform, which by itself had no
chemotactic response. The results shown are the concentrations that gave the
bThe relative chemotactic response is the ratio of cells that migrate into attractant-filled
versus motility-buffer-filled capillary tubes. Compounds eliciting a relative chemotactic
response of ?2 were considered chemoattractants (2). The data shown are
representative of at least two independent experiments.
Lambert et al.
aem.asm.orgApplied and Environmental Microbiology
on June 10, 2014 by guest
tors in the natural host cycle of the bacteria. Our data suggest that
the chemotactic behavior of a pathogenic strain is not identical to
ecology and metabolic requirements of the two species. The an-
notated chemotaxis proteins in Leptospira genomes show that L.
interrogans has 13 chemoreceptor homologs, whereas the sapro-
phyte L. biflexa has twice as many (http://mistdb.com/). Chemo-
receptors in L. biflexa likely play a role in the adaptation of this
aquatic bacterium to a wide range of environmental conditions.
of a larger panel of Leptospira strains from distinct sources. Simi-
larly, other spirochetes, including B. burgdorferi, B. hyodysente-
riae, S. aurantia, and T. denticola, varied significantly in their at-
traction to a panel of chemical compounds (2, 11, 15, 20, 21, 24).
edge of the mechanisms that allow chemotaxis in these unique
We thank Azad Eshghi for critical reading of the manuscript.
This work was supported by the Institut Pasteur, the French Ministry
of Research (ANR-08-MIE-018), and the doctoral program of the Uni-
versity Paris Diderot. In addition, N.W.C. and N.T. were supported by
USPHS grants AI29743 and DE04645.
1. Adler J. 1969. Chemoreceptors in bacteria. Science 166:1588–1597.
2. Bakker RG, Li C, Miller MR, Cunningham C, Charon NW. 2007.
Identification of specific chemoattractants and genetic complementation
of a Borrelia burgdorferi chemotaxis mutant: flow cytometry-based capil-
lary tube chemotaxis assay. Appl. Environ. Microbiol. 73:1180–1188.
3. Berg HC, Turner L. 1979. Movement of microorganisms in viscous en-
vironments. Nature 278:349–351.
4. Bey RF, Johnson RC. 1978. Protein-free and low-protein media for the
cultivation of Leptospira. Infect. Immun. 19:562–569.
5. Bharti AR, et al. 2003. Leptospirosis: a zoonotic disease of global impor-
tance. Lancet Infect. Dis. 3:757–771.
6. Bromley DB, Charon NW. 1979. Axial filament involvement in the mo-
tility of Leptospira interrogans. J. Bacteriol. 137:1406–1412.
7. Charon NW, Russell C, Johnson C, Peterson D. 1974. Amino acid
biosynthesis in the spirochete Leptospira: evidence for a novel pathway of
isoleucine biosynthesis. J. Bacteriol. 117:203–211.
8. Ellinghausen HC. 1968. Stimulation of leptospiral growth by glucose.
Am. J. Vet. Res. 29:191–199.
9. Faine S, Vanderhoeden J. 1964. Virulence-linked colonial and morpho-
logical variation in Leptospira. J. Bacteriol. 88:1493–1496.
10. Faine SB, Adler B, Bolin C, Perolat P. 1999. Leptospira and leptospirosis,
2nd ed. MediSci, Melbourne, Australia.
11. Greenberg EP, Canale-Parola E. 1977. Chemotaxis in Spirochaeta auran-
tia. J. Bacteriol. 130:485–494.
12. Greenberg EP, Canale-Parola E. 1977. Relationship between cell coiling
13. Johnson RC, Harris VG. 1967. Differentiation of pathogenic and sapro-
phytic leptospires. J. Bacteriol. 94:27–31.
14. Johnson RC, Walby J, Henry RA, Auran NE. 1973. Cultivation of
parasitic leptospires: effect of pyruvate. Appl. Microbiol. 26:118–119.
15. Kennedy MJ, Yancey RJ. 1996. Motility and chemotaxis in Serpulina
hyodysenteriae. Vet. Microbiol. 49:21–30.
16. Lambert A, et al. 2012. FlaA proteins in Leptospira interrogans are essen-
tial for motility and virulence but are not required for formation of the
flagellum sheath. Infect. Immun. 80:2019–2025.
17. Livesley MA, Thompson IP, Bailey MJ, Nuttall PA. 1993. Comparison of
the fatty acid profiles of Borrelia, Serpulina and Leptospira species. J. Gen.
18. Lourdault K, Aviat F, Picardeau M. 2009. The use of quantitative real-
time PCR to study the dissemination of Leptospira interrogans in the
guinea pig infection model of leptospirosis. J. Med. Microbiol. 58:648–
19. Louvel H, et al. 2006. Comparative and functional genomic analyses of
iron transport and regulation in Leptospira spp. J. Bacteriol. 188:7893–
20. Lux R, Miller JN, Park NH, Shi W. 2001. Motility and chemotaxis in
tissue penetration of oral epithelial cell layers by Treponema denticola.
Infect. Immun. 69:6276–6283.
21. Motaleb MA, Miller MR, Bakker RG, Li C, Charon NW. 2007. Isolation
and characterization of chemotaxis mutants of the Lyme disease spiro-
etry, and cell tracking. Methods Enzymol. 422:421–437.
22. Porter SL, Wadhams GH, Armitage JP. 2011. Signal processing in com-
plex chemotaxis pathways. Nat. Rev. Microbiol. 9:153–165.
23. Shenberg E. 1967. Growth of pathogenic Leptospira in chemically defined
media. J. Bacteriol. 93:1598–1606.
24. Shi W, Yang Z, Geng Y, Wolinsky LE, Lovett MA. 1998. Chemotaxis in
Borrelia burgdorferi. J. Bacteriol. 180:231–235.
lysis in synthetic medium. J. Bacteriol. 88:55–59.
26. Westfall HN, Charon NW, Peterson DE. 1983. Multiple pathways for
virulence. Infect. Immun. 61:2270–2272.
as a negative control. Data are representative of one experiment and are the means of PCR duplicates of three to four capillaries. The error bars represent the
Chemotaxis in Leptospira spp.
December 2012 Volume 78 Number 23 aem.asm.org 8469
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