Abstract
Bacterial motility is crucial for many pathogenic species in the process of invasion and/or dissemination. The spirochete bacteria Leptospira spp. cause symptoms, such as hemorrhage, jaundice, and nephritis, in diverse mammals including humans. Although loss-of-motility attenuate the spirochete’s virulence, the mechanism of the motility-dependent pathogenicity is unknown. Here, focusing on that Leptospira spp. swim in liquid and crawl on solid surfaces, we investigated the spirochetal dynamics on the host tissues by infecting cultured kidney cells from various species with pathogenic and non-pathogenic leptospires. We found that, in the case of the pathogenic leptospires, a larger fraction of bacteria attached to the host cells and persistently traveled long distances using the crawling mechanism. Our results associate the kinetics and kinematic features of the spirochetal pathogens with their virulence.
Introduction
Many bacteria utilize motility to explore environments for survival and prosperity. For some pathogenic species, the motility is a virulence factor (). For example, Helicobacter pylori requires motility to migrate toward the epithelial tissue in the stomach (), and motility and chemotaxis are key factors that guide host invasion in different Salmonella serovars (; ). Movement and adhesion of the Lyme disease spirochete Borrelia burgdorferi in blood vessels are thought to be important during the process of host cell invasion (). In the enteric pathogens, such as the enteropathogenic Escherichia coli (EPEC), the motility machinery flagella are also important for adhesion to the host intestinal epithelium ().
In this study, we address the association of bacterial motility with pathogenicity in the worldwide zoonosis leptospirosis. The causative agent of leptospirosis Leptospira spp. are Gram-negative bacteria belonging to the phylum Spirochaetes. The genus Leptospira is comprised of two major clades, “Saprophytes (S)” and “Pathogens (P)” and is further divided into four subclades called P1, P2, S1, and S2 (). Leptospira spp. are classified into over 300 serovars defined based on the structural diversity of lipopolysaccharide (LPS) (; ). The pathogenic species affect various mammalian hosts such as livestock (cattle, pigs, horses, and others), companion animals (dogs and others), and humans, causing severe symptoms, such as hemorrhage, jaundice, and nephritis in some host-serovar pairs (; ; ). The leptospires can be maintained in the renal tubules of recovered animals or reservoir hosts, and the urinary shedding of leptospires to the environment leads to infection in humans and other animals through contact with contaminated soil or water. Although the pathogenic mechanism of leptospirosis is not well elucidated, their motility is known to be somehow involved in pathogenicity using loss-of-motility mutants in animal models (; ).
Leptospira spp. possess two periplasmic flagella (PFs) beneath the outer cell membrane (Figure 1A). The rotation of the PFs gyrates both ends of the cell body and rotates the coiled cell body (protoplasmic cylinder), allowing the spirochete to swim in fluids (; ). Leptospira cells have high adhesivity to solid surfaces and show diverse behaviors over surfaces: some of the adhered leptospires stay at the same position (so-called adhesion state), whereas the others retain mobility on surfaces (). The surface motility designated as “crawling” is a slip-less movement given by rotation of the protoplasmic cylinder and unidentified adhesins that are freely move on the outer membrane (). Adherence and entry of pathogenic leptospires in the conjunctival epithelium () and in the paracellular routes of hepatocytes () were previously observed using scanning electron microscopy, suggesting adhesion to the host tissue surfaces and subsequent crawling of pathogenic leptospires. To verify this hypothesis, assuming the transition of leptospires between swimming and adhesion states and between adhesion and crawling states in the equilibrium (Figure 1B), we investigated the adhesion and crawling motility of Leptospira on the cultured kidney cells of various mammalian species.
FIGURE 1
Materials and Methods
Leptospira Strains and Growth Conditions
Pathogenic serovars of L. interrogans serovar Icterohaemorrhagiae (strain WFA135), an isolate from Rattus norvegicus in Tokyo, Japan, serovar Manilae (strain UP-MMC-NIID) (; ; ) and a saprophytic L. biflexa serovar Patoc (strain Patoc I) were used in this study. The serovar of WFA135 was determined by multiple loci variable number of tandem repeats analysis (MLVA) and DNA sequencing of the lic12008 gene (; ). Bacteria were cultured in enriched Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid medium (BD Difco, NJ, United States) containing 25 μg/mL spectinomycin at 30°C for 2 (L. biflexa) or 4 (L. interrogans) days until log phase. To track Leptospira cells when in co-culture with mammalian kidney cells, a green fluorescent protein (GFP) was constitutively expressed in each strain (Supplementary Movie S1).
Mammalian Cells and Media
Mammalian kidney epithelial cell lines used included MDCK-NBL2 (dog), NRK-52E (rat), Vero (monkey), MDBK-NBL1 (cow), TCMK-1 (mouse), and HK-2 (human). MDCK, Vero, TCMK, and MDBK cells were maintained in Eagle’s minimum essential medium (MEM) (Sigma-Aldrich, Darmstadt, Germany) containing 10% fetal bovine serum or 10% horse serum (Nacalai Tesque, Kyoto, Japan) at 37°C and 5% CO2. NRK cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, MA, United States) with 4 mM L-glutamine, adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, and with 5% bovine calf serum (Nacalai Tesque) at 37°C and 5% CO2. HK-2 cells were maintained in keratinocyte serum-free (KSF) medium with 0.05 mg/mL bovine pituitary extract and 5 ng/mL human recombinant epidermal growth factor (Gibco – Thermo Fisher Scientific, Waltham, MA, United States). All culture conditions contained a 5% antibiotic/antimycotic mixed solution (Nacalai Tesque). Cells were treated with a 0.1% trypsin – EDTA solution (Nacalai Tesque) to dislodge the cells during each passage process.
GFP Expression in L. interrogans and L. biflexa
For the construction of a replicable plasmid in L. interrogans, the corresponding rep-parB-parA region of the plasmid pGui1 from the L. interrogans serovar Canicola strain Gui44 plasmid pGui1 () was amplified from a L. interrogans serogroup Canicola isolate, and the amplified product was cloned into the PCR-generated pCjSpLe94 () by NEBuilder HiFi DNA Assembly cloning (New England BioLabs), generating pNKLiG1. The flgB promoter region (; ) and gfp were amplified from pCjSpLe94 and pAcGFP1 (Clontech), respectively, and the amplified products were cloned into the SalI-digested pNKLiG1 for L. interrogans or the SalI-digested pCjSpLe94 for L. biflexa. The plasmids were transformed into strains WFA135, UP-MMC-NIID or Patoc I by conjugation with E. coliβ2163 harboring the plasmid (). We used the Leptospira strains stored at –80°C when created the GFP-expressing transformant. After the strains were recovered from –80°C, we passaged them once or twice prior to the conjugation experiment. The colonies of the transformant were cultured in EMJH and passaged twice, and then log phase cultures were stored at −80°C. The GFP-expressing strains were passaged less than four times for the motility assays, that is, less than eight times of passage after recovered from −80°C for the experiments. Primer sequences used in this study are listed in Supplementary Table S1. Expression of GFP did not affect motility in the Leptospira serovars (Supplementary Figure S1).
Preparation of Kidney Cells and Leptospira Cells in a Chamber Slide
Kidney cells were harvested with 0.1% trypsin and 0.02% EDTA in a balanced salt solution (Nacalai Tesque) and plated onto a chamber slide (Iwaki, Tokyo, Japan) using their corresponding media without antibiotics. The slides were incubated for 48 h until a monolayer was formed and washed twice with media to remove non-adherent cells. The cells were incubated for a further 2 h at 37°C and 5% CO2. Approximately 500 μL of stationary phase Leptospira cells were harvested by centrifugation at 1,000 × g for 10 min at room temperature, washed twice in PBS, then resuspended in the corresponding kidney cell culture media without antibiotics at 37°C to a concentration of 107 cells/mL. These suspensions (1 mL) were then added into the corresponding chamber slides containing the kidney cell layer, and the chamber slides were incubated at 37°C for 1 h.
Microscopy Observation and Adhesion-Crawling Assay
The movement behaviors, swimming, adhesion and crawling of the Leptospira cells on the kidney cells were observed using a dark-field microscope (BX53, Splan 40×, NA 0.75, Olympus, Tokyo, Japan) with an epi-fluorescent system (U-FBNA narrow filter, Olympus) and recorded by a CCD-camera (WAT- 910HX, Watec Co., Yamagata, Japan) at 30 frames per second. Leptospira cells were tracked using an ImageJ (NIH, MD, United States)-based tracking system and the motion parameters such as motile fraction, velocity and the mean square displacement (MSD) were analyzed using Excel-based VBA (Microsoft, WA, United States). The two-dimensional MSD of individual leptospiral cells during a period Δt was calculated by the following equation: MSD(Δt) = (xi + Δt−xi)2 + (yi + Δt−yi)2, where (xi, yi) is the bacterial position at I (see also Supplementary Figure S2). Swimming cells were distinguished from adhesion or crawling cells by the difference in a focus (see Figure 2C), and adhesion was discriminated from crawling based on criteria of both crawling speed (<1 μm/s) and MSD slope (<0.5).
FIGURE 2
Results
Steady-State Analysis of Leptospira on the Kidney Cells
We infected cultured kidney cells from six different host species (rat, dog, monkey, mouse, cow, and human) with three Leptospira strains (the pathogenic L. interrogans serovars Icterohaemorrhagiae and Manilae, and the non-pathogenic L. biflexa serovar Patoc) expressing GFP within a chamber slide (Figure 2A). We observed the Leptospira cells by epi-fluorescent microscopy (Figure 2B) and measured the fractions of swimming [S], adhered [A], and crawling [C] bacteria on the kidney cells (Figure 2C). Figures 2D,E show that almost half of the pathogenic population transited from the swimming state to the adhesion and crawling states, whereas >75% non-pathogenic leptospires remained in the swimming state. We calculated the equilibrium constant between swimming and adhesion states (KS–A) and between adhesion and crawling states (KA–C) from the cell fractions using KS–A = [A]/[S] and KA–C = [C]/[A], respectively. The pathogenic leptospires had a significantly larger KA–C in comparison with the non-pathogenic strain (P < 0.05); KS–A did not seem to correlate with virulence (Figure 2F). These thermodynamic parameters suggest that the biased transition from adhesion to crawling would be responsible for the virulence of Leptospira (Figure 2G).
Crawling Motility
Taking the results of the steady-state analysis, we focused on the crawling motility of individual Leptospira cells on the kidney cells. Although the crawling speed varied among the measured host–bacterium pairs, L. interrogans serovar Icterohaemorrhagiae showed significantly faster speed than the others, indicating the species/serovar dependence of the crawling ability (Figure 3A). On the other hand, there is no difference in crawling speed between L. interrogans serovar Manilae and L. biflexa serovar Patoc, suggesting that the crawling speed itself is not related to leptospiral virulence. Meanwhile, we observed that some leptospiral cells attached to the kidney cells and moved smoothly for periods, migrating over long-distances (upper panels of Figures 3B,C and Supplementary Movie S2), whereas others frequently reversed the crawling direction (lower panels of Figures 3B,C and Supplementary Movie S3). Cell movements with reversals are considered to be diffusive and thus can be evaluated by plotting MSD against time, a general methodology for diffusion (Brownian motion) analysis (). The MSD of simple diffusion without directivity is proportional to time, and therefore double-logarithmic MSD plots from such non-directional diffusion represent slopes of ∼1, whereas those from directive movements show MSD slopes of ∼2, representing the relatively long distance traveled by the cells (Supplementary Figure S2). Double-logarithmic MSD plots obtained from each individual leptospires showed a wide range of MSD slopes (example data are shown in Figure 3D) and differed for each host–Leptospira pair (Figure 3E left and Supplementary Figure S3). The non-pathogenic strain showed the slope of ∼1, while the pathogenic strains had significantly larger slopes that denote directive motion (Figure 3E, right). Thus, concerning the crawling motility, directivity and persistency rather than speed could be crucial for virulence.
FIGURE 3
Discussion
Our results suggested the importance of adhesion to and persistent crawling on the host tissue for the pathogenicity of Leptospira. The thermodynamic and kinematic parameters are associated in Figure 4A, showing the tendency that pathogenic species are biased to the crawling state and can migrate longer distance on the host tissue surfaces. The crawling motility of Leptospira is caused by the attachment of the spirochete cell body to surfaces via adhesive cell surface components (; ). The successive alternation in the attachment and detachment of adhesins allows for this progressive movement by the spirochetes, however, an excessively strong adhesion can inhibit crawling (). LPS, the molecular basis for the identification of the different Leptospira serovars (; ), is thought to be a crucial adhesin important for this crawling motion (). Thus, it is possible that compatibility of the serological characteristics of leptospires and the surface properties of the host tissue might affect the crawling behavior over the tissue surfaces and the subsequent clinical consequences. The results of our biophysical experiments outline a plausible framework for the adhesion and crawling-dependent pathogenicity of Leptospira (Figure 4B). The biased transition from the adhesion to the crawling state and the long-distance, persistent crawling allows leptospires to explore the host’s cell surfaces, increasing the probability of encountering routes for invasion through their intracellular tight junctions (Figure 4B, left). In contrast, the swimming or weakly attached leptospires can be swept by external forces, such as intermittent urination. Furthermore, since leptospires cannot be disseminated over host tissues by diffusive crawling, such strains have less chance to discover routes for invasion (Figure 4B, right).
FIGURE 4
Other than LPS, abundant leptospiral proteins that are exposed to the cell exterior are known to possess the binding ability against extracellular matrix (ECM) components, such as fibronectin, laminin, and collagens (; ). For example, Lig (leptospiral immunoglobulin-like) proteins () mediate the Leptospira adhesion via binding to fibronectin, laminin, collagen I, and collagen IV (). LenA (leptospiral endostatin-like protein A) () that was formally called LfhA (leptospiral factor H-binding protein A)/Lsa24 (leptospiral surface adhesin 24) have binding property to the complement regulatory protein factor H () and laminin (). OmpL1 serves as an adhesin through binding to laminin and plasma fibronectin (; ). Some of the adhesins are expressed only in pathogenic strains, suggesting that their combination could be involved in directive and persistent crawling observed in the pathogenic strains. Further study using other saprophytic and pathogenic Leptospira spp. is required for defining the detailed roles of these adhesive molecules for crawling. Also, in vivo experiments demonstrating direct correlation between crawling and virulence of Leptospira should be conducted.
Some bacterial pathogens are specialized to invade a very limited array of hosts, whereas others can infect multiple host species. The host range differs for each pathogen and the clinical symptoms depend on each host–pathogen combination. The same applies for leptospirosis, the outcome of Leptospira infection depends on the host-serovar association, and some animal species can become an asymptomatic reservoir for particular Leptospira serovars. The present experiments also provided data allowing us to discuss the host dependence of the leptospiral dynamics. Among the investigated materials, serovar Manilae vs. rats and serovar Icterohaemorrhagiae vs. rats are typically asymptomatic pairs, and Figure 4A shows that the pairs with reservoirs have lower scores in comparison with those causing severe symptoms, such as Manilae vs. humans, Icterohaemorrhagiae vs. dogs, and others. This implies that the surface dynamics of the spirochete could be related to their host-dependent pathogenicity. Understanding the mechanism of the host preferences by pathogens is important for prevention of the infection spread. There still remain gaps in our knowledge on the host–pathogen interaction in leptospirosis, but crucial insights into the issue have been provided by several research groups. Microarray analyses have revealed that regulation of gene expression in Leptospira is affected by its interaction with host cells (; ). Although leptospiral LPS that is believed to be an adhesin for crawling induces the expression of proinflammatory cytokine genes through the recognition by the host TLR, the host-dependent difference in the TLR recognition was shown between human and murine cells: TLR1 and TLR2 predominantly mediate the activation in human cells, whereas TLR2 and TLR4 act for the recognition in mouse cells (; ). Pathogenic leptospires evade the host immunity through the interaction with the complement system (). Also, the binding of L. interrogans to thrombin inhibits fibrin coagulation, which results in hemorrhage (). In relation to hemorrhage by L. interrogans, the pathogens acquire iron, that is believed to be essential during infection, from host erythrocyte through hemolytic activity of sphingomyelinases-like proteins (Sph) (). Thus, various Leptospira-host interactions such as immune evasion and nutrition acquisition could inclusively determine the host preference of Leptospira. Such abundant factors in both bacteria and hosts should be investigated for a deeper understanding of the host-dependent pathogenicity.
Statements
Data availability statement
All datasets presented in this study are included in the article/Supplementary Material.
Author contributions
JX, NK, and SN planned the project and wrote the manuscript. JX and NK carried out the experiments. SN set up the optical system and programs for data analysis. JX and SN analyzed the data. All authors contributed to the article and approved the submitted version.
Funding
This work was supported by the JSPS KAKENHI: 18K07100 for SN, 19K07571 for NK, and 18J10834 for JX.
Acknowledgments
We thank Dr. H. Nishimura (Sendai Medical Center) and Dr. C. Toma (University of the Ryukyus) for the generous gift of animal cell lines, and Dr. E. Isogai (Tohoku University) and Dr. H. Yoneyama (Tohoku University) for the experiment reagents and the insightful discussion. This manuscript has been released as a pre-print at bioRxiv ().
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.01886/full#supplementary-material
FIGURE S1Effect of GFP expression on the Leptospira motility.
FIGURE S2Explanation of MSD plot.
FIGURE S3Histograms of the MSD slopes.
TABLE S1Primer sequences used in this study.
MOVIE S1Epi-fluorescent images of L. interrogans on the rat kidney cell.
MOVIE S2Progressive, long-distance crawling of L. interrogans on the monkey kidney cells.
MOVIE S3Crawling of L. interrogans with highly frequent reversal on the dog kidney cells.
References
1
AdlerB.de la Peña MoctezumaA. (2010). Leptospira and leptospirosis.Vet. Microbiol.140287–296. 10.1016/j.vetmic.2009.03.012
2
BarbosaA. S.AbreuP. A. E.NevesF. O.AtzingenM. V.WatanabeM. M.VieiraM. L.et al (2006). A newly identified Leptospiral adhesin mediates attachment to laminin.Infect. Immun.746356–6364. 10.1128/IAI.00460-06
3
BarbosaA. S.IsaacL. (2020). Strategies used by Leptospira spirochetes to evade the host complement system.FEBS Lett.10.1002/1873-3468.13768 (in press).
4
BaubyH.Saint GironsI.PicardeauM. (2003). Construction and complementation of the first auxotrophic mutant in the spirochaete Leptospira meyeri.Microbiology149689–693. 10.1099/mic.0.26065-0
5
BhartiA. R.NallyJ. E.RicaldiJ. N.MatthiasM. A.DiazM. M.LovettM. A.et al (2003). Leptospirosis: a zoonotic disease of global importance.Lancet Infect. Dis.3757–771. 10.1016/s1473-3099(03)00830-2
6
BonoJ. L.EliasA. F.KupkoJ. J.StevensonB.TillyK.RosaP. (2000). Efficient targeted mutagenesis in Borrelia burgdorferi.J. Bacteriol.1822445–2452. 10.1128/JB.182.9.2445-2452.2000
7
CerqueiraG. M.PicardeauM. (2009). A century of Leptospira strain typing.Infect. Genet. Evol.9760–768. 10.1016/j.meegid.2009.06.009
8
CharonN. W.LawrenceC. W.O’BrienS. (1981). Movement of antibody-coated latex beads attached to the spirochete Leptospira interrogans.Proc. Natl. Acad. Sci. U.S.A.787166–7170. 10.1073/pnas.78.11.7166
9
CheneyC. P.SchadP. A.FormalS. B.BoedekerE. C. (1980). Species specificity of in vitro Escherichia coli adherence to host intestinal cell membranes and its correlation with in vivo colonization and infectivity.Infect. Immun.281019–1027.
10
ChoyH. A.KelleyM. M.ChenT. L.MøllerA. K.MatsunagaJ.HaakeD. A. (2007). Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen.Infect. Immun.752441–2450. 10.1128/IAI.01635-06
11
ClyneM.OcroininT.SuerbaumS.JosenhansC.DrummB. (2000). Adherence of isogenic flagellum-negative mutants of Helicobacter pylori and Helicobacter mustelae to human and ferret gastric epithelial cells.Infect. Immun.684335–4339. 10.1128/iai.68.7.4335-4339.2000
12
CoxP. J.TwiggG. I. (1974). Leptospiral motility.Nature250260–261. 10.1038/250260a0
13
EbadyR.NiddamA. F.BoczulaA. E.KimY. R.GuptaN.TangT. T.et al (2016). Biomechanics of Borrelia burgdorferi vascular interactions.Cell Rep.162593–2604. 10.1016/j.celrep.2016.08.013
14
FernandesL. G.de MoraisZ. M.VasconcellosS. A.NascimentoA. L. T. O. (2015). Leptospira interrogans reduces fibrin clot formation by modulating human thrombin activity via exosite I.Pathog. Dis.73:ftv001. 10.1093/femspd/ftv001
15
FernandesL. G. V.VieiraM. L.KirchgatterK.AlvesI. J.MoraisZ. M.de VasconcellosS. A. (2012). OmpL1 is an extracellular matrix- and plasminogen-interacting protein of Leptospira spp.Infect. Immun.803679–3692. 10.1128/IAI.00474-12
16
FujitaR.KoizumiN.SugiyamaH.TomizawaR.SatoR.OhnishiM. (2015). Comparison of bacterial burden and cytokine gene expression in golden hamsters in early phase of infection with two different strains of Leptospira interrogans.PLoS One10:e0132694. 10.1371/journal.pone.0132694
17
GoldsteinS. F.CharonN. W. (1990). Multiple-exposure photographic analysis of a motile spirochete.Proc. Natl. Acad. Sci. U.S.A.874895–4899. 10.1073/pnas.87.13.4895
18
HaakeD. A.MatsunagaJ. (2010). Leptospira: a spirochaete with a hybrid outer membrane.Mol. Microbiol.77805–814. 10.1111/j.1365-2958.2010.07262.x
19
JosenhansC.SuerbaumS. (2002). The role of motility as a virulence factor in bacteria.Int. J. Med. Microbiol.291605–614. 10.1078/1438-4221-00173
20
KoizumiN.IzumiyaH.MuJ.-J.ArentZ.OkanoS.NakajimaC.et al (2015). Multiple-locus variable-number tandem repeat analysis of Leptospira interrogans and Leptospira borgpetersenii isolated from small feral and wild mammals in East Asia.Infect. Genet. Evol.36434–440. 10.1016/j.meegid.2015.08.013
21
KoizumiN.WatanabeH. (2004). Leptospiral immunoglobulin-like proteins elicit protective immunity.Vaccine221545–1552. 10.1016/j.vaccine.2003.10.007
22
KusumiA.SakoY.YamamotoM. (1993). Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.Biophys. J.652021–2040. 10.1016/S0006-3495(93)81253-0
23
LambertA.PicardeauM.HaakeD. A.SermswanR. W.SrikramA.AdlerB.et al (2012). FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath.Infect. Immun.802019–2025. 10.1128/IAI.00131-12
24
MatsunagaJ.BarocchiM. A.CrodaJ.YoungT. A.SanchezY.SiqueiraI.et al (2003). Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily.Mol. Microbiol.49929–946. 10.1046/j.1365-2958.2003.03619.x
25
MiyaharaS.SaitoM.KanemaruT.VillanuevaS. Y. A. M.GlorianiN. G.YoshidaS. (2014). Destruction of the hepatocyte junction by intercellular invasion of Leptospira causes jaundice in a hamster model of Weil’s disease.Int. J. Exp. Path.95271–281. 10.1111/iep.12085
26
NahoriM.-A.Fournié-AmazouzE.Que-GewirthN. S.BalloyV.ChignardM.RaetzC. R. H.et al (2005). Differential TLR recognition of leptospiral lipid A and lipopolysaccharide in murine and human cells.J. Immunol.1756022–6031. 10.4049/jimmunol.175.9.6022
27
NakamuraS.LeshanskyA.MagariyamaY.NambaK.KudoS. (2014). Direct measurement of helical cell motion of the spirochete Leptospira.Biophys. J.10647–54. 10.1016/j.bpj.2013.11.1118
28
NarayanavariS. A.LourdaultK.SritharanM.HaakeD. A.MatsunagaJ. (2015). Role of sph2 gene regulation in hemolytic and sphingomyelinase activities produced by Leptospira interrogans.PLoS Negl. Trop. Dis.9:e0003952. 10.1371/journal.pntd.0003952
29
OlsenJ. E.Hoegh-AndersenK. H.CasadesúsJ.RosenkrantzJ. T.ChadfieldM. S.ThomsenL. E. (2013). The role of flagella and chemotaxis genes in host pathogen interaction of the host adapted Salmonella enterica serovar Dublin compared to the broad host range serovar S.Typhimurium.BMC Microbiol.13:67. 10.1186/1471-2180-13-67
30
PicardeauM. (2008). Conjugative transfer between Escherichia coli and Leptospira spp. as a new genetic tool.Appl. Environ. Microbiol.74319–322. 10.1128/AEM.02172-07
31
PicardeauM. (2017). Virulence of the zoonotic agent of leptospirosis: still terra incognita?Nat. Rev. Micro15297–307. 10.1038/nrmicro.2017.5
32
RobbinsG. T.HahnB. L.EvangelistaK. V.PadmoreL.ArandaP. S.CoburnJ. (2015). Evaluation of cell binding activities of Leptospira ECM adhesins.PLoS Negl. Trop. Dis.9:e0003712. 10.1371/journal.pntd.0003712
33
SantosL. A.AdhikarlaH.YanX.WangZ.FoutsD. E.VinetzJ. M.et al (2018). Genomic comparison among global isolates of L. interrogans serovars Copenhageni and Icterohaemorrhagiae identified natural genetic variation caused by an Indel.Front. Cell Infect. Microbiol.8:193. 10.3389/fcimb.2018.00193
34
SatouK.ShimojiM.TamotsuH.JuanA.AshimineN.ShinzatoM.et al (2015). Complete genome sequences of low-passage virulent and high-passage avirulent variants of pathogenic Leptospira interrogans serovar Manilae strain UP-MMC-NIID, originally isolated from a patient with severe leptospirosis, determined using PacBio single-molecule real-time technology.Genome Announc3:e00882-15. 10.1128/genomeA.00882-15
35
SiitonenA.NurminenM. (1992). Bacterial motility is a colonization factor in experimental urinary tract infection.Infect. Immun.603918–3920. 10.1128/iai.60.9.3918-3920.1992
36
SlamtiL.PicardeauM. (2012). Construction of a library of random mutants in the spirochete Leptospira biflexa using a mariner transposon.Methods Mol. Biol.859169–176. 10.1007/978-1-61779-603-6_9
37
StevensonB.ChoyH. A.PinneM.RotondiM. L.MillerM. C.DeMollE.et al (2007). Leptospira interrogans endostatin-like outer membrane proteins bind host fibronectin, laminin and regulators of complement.PLoS One2:e1188. 10.1371/journal.pone.0001188
38
TaharaH.TakabeK.SasakiY.KasugaK.KawamotoA.KoizumiN.et al (2018). The mechanism of two-phase motility in the spirochete Leptospira: swimming and crawling.Sci. Adv.4:eaar7975. 10.1126/sciadv.aar7975
39
TomaC.MurrayG. L.NoharaT.MizuyamaM.KoizumiN.AdlerB.et al (2014). Leptospiral outer membrane protein LMB216 is involved in enhancement of phagocytic uptake by macrophages.Cell. Microbiol.161366–1377. 10.1111/cmi.12296
40
TomizawaR.SugiyamaH.SatoR.OhnishiM.KoizumiN. (2017). Male-specific pulmonary hemorrhage and cytokine gene expression in golden hamster in early-phase Leptospira interrogans serovar Hebdomadis infection.Microb. Pathog.11133–40. 10.1016/j.micpath.2017.08.016
41
VermaA.HellwageJ.ArtiushinS.ZipfelP. F.KraiczyP.TimoneyJ. F.et al (2006). LfhA, a novel factor H-binding protein of Leptospira interrogans.Infect. Immun.742659–2666. 10.1128/IAI.74.5.2659-2666.2006
42
VincentA. T.SchiettekatteO.GoarantC.NeelaV. K.BernetE.ThibeauxR.et al (2019). Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics.PLoS Negl. Trop. Dis.13:e0007270. 10.1371/journal.pntd.0007270
43
WertsC.TappingR. I.MathisonJ. C.ChuangT. H.KravchenkoV.Saint GironsI.et al (2001). Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism.Nat. Immunol.2346–352. 10.1038/86354
44
WunderE. A.FigueiraC. P.BenaroudjN.HuB.TongB. A.TrajtenbergF.et al (2016). A novel flagellar sheath protein, FcpA, determines filament coiling, translational motility and virulence for the Leptospira spirochete.Mol. Microbiol.101457–470. 10.1111/mmi.13403
45
XuJ.KoizumiN.NakamuraS. (2020). Insight into motility-dependent pathogenicity of the zoonotic spirochete Leptospira.bioRxiv [Preprint]. 10.1101/2020.02.11.944587
46
ZhuW.-N.HuangL.-L.ZengL.-B.ZhuangX.-R.ChenC.-Y.WangY.-Z.et al (2014). Isolation and characterization of two novel plasmids from pathogenic Leptospira interrogans serogroup Canicola serovar Canicola strain Gui44.PLoS Negl. Trop. Dis.8:e3103. 10.1371/journal.pntd.0003103
Summary
Keywords
bacterial motility, pathogenicity, spirochete, leptospirosis, zoonosis, host–pathogen association, biophysics, optical microscopy
Citation
Xu J, Koizumi N and Nakamura S (2020) Crawling Motility on the Host Tissue Surfaces Is Associated With the Pathogenicity of the Zoonotic Spirochete Leptospira. Front. Microbiol. 11:1886. doi: 10.3389/fmicb.2020.01886
Received
29 May 2020
Accepted
20 July 2020
Published
05 August 2020
Volume
11 - 2020
Edited by
Axel Cloeckaert, Institut National de la Recherche Agronomique (INRA), France
Reviewed by
Angela Silva Barbosa, Butantan Institute, Brazil; Pablo Tortosa, Université de la Réunion, France; Mônica Larucci Vieira, Federal University of Minas Gerais, Brazil; Felipe Trajtenberg, Institut Pasteur de Montevideo, Uruguay
Updates
Copyright
© 2020 Xu, Koizumi and Nakamura.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Shuichi Nakamura, naka@bp.apph.tohoku.ac.jp
†Present address: Jun Xu, Department of Bacteriology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan
This article was submitted to Infectious Diseases, a section of the journal Frontiers in Microbiology
Disclaimer
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