Studies on bacterial motility as a virulence
factor and stimulation response
著者 Xu Jun
学位授与機関 Tohoku University
学位授与番号 11301甲第18939号
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Studies on bacterial motility as a virulence
factor and stimulation response
(病原因子及び刺激応答としての細菌運動
に関する研究
)
専 攻 生物産業創成科学 指導教員 米山裕 教授 学籍番号 氏 名 許 駿2
Index
Preface ... 3
Chapter 1………... 7
Introduction ... 7
Materials & Methods ... 10
Result... 14
Discussion... 19
Chapter 2……….…………... 22
Introduction ... 22
Materials & Methods ... 25
Results ... ... 28
Discussion ... 31
Chapter 3……….…………... 36
Introduction ... 36
Materials & Methods ... 37
Results ... 40
Discussion ... 42
References ... 44
Acknowledgement ... 61
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Preface
Bacteria occupy every corner on the planet, even where it might be extreme to mankind. Robustness and diversity of the bacterial ecosystem is achieved by their excelled adaptation, modification and resilience capable of responding variation of circumstances. These include the interaction of bacteria with the outer environment and other species around, in some cases, leading to symbiosis of multiple species or the establishment of a successful infection on the prefer animal hosts. Although bacterial infection involves various virulence factors (e.g., toxins, enzymes, adhesivity, etc.) and physiological controls including temporal survival at the viable but nonculturable (VBNC) state, many species rely on motility.
Bacterial motility is mechanically different in different species though, many motile species exhibits flagellum-dependent motility. A thin, helical filament is rotated by a basal motor (flagellar motor) that propels the cell in liquids. The flagellar motor is fueled by an electrochemical gradient called ion motive force, which consists of membrane voltage (Δψ) and ion concentration gradient (e.g. pH difference between the cell exterior and cell interior, ΔpH). The correlation between
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motility and pathogenicity has been reported in many bacterial species such as Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Salmonella and Eecherichia. coli. Spirochetes such as Leptospira interrogans, the causative agent of worldwide zoonosis leptospirosis, and Brachyspira hyodysenteriae, the pathogen of swine dysentery, possess their flagella beneath the outer membrane. The motility of spirochetes is considered as a crucial virulence factor as with other motile bacterial pathogens.
Although the majority of motile bacteria swim using flagella, the way for movement is diverse. For instance, Mycoplasma mobile glides over surfaces via abundant leg-like complexes that reside on the cell surface, and Leptospira spp. show crawling using adhesive outer membrane molecules such as lipopolysaccharides (see Chapter 3). In either type of motility, bacteria can move towards or away from a variety of stimuli, including a concentration gradient of chemicals, light, temperature, gravity, and a magnetic field. Survival and thrive of bacteria are attributed to a reliable response to environmental stimuli.
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and serves as a virulence factor. Moreover, bacteria control motility in response to changes in environmental conditions. For better understanding of their pathogenesis and ecosystem, it is important to know detailed dynamic processes of individual cells by linking with their physiologies behind. To consider significance of the bacterial motility together with physiological insight in terms of various aspects, this thesis describes the following three topics.
1. Bacterium-Bacterium interaction: Pathogen vs
Commensal.
The first topic concerns “the regulation of bacterial motility by the biological process of other microbial species”. This topic will be given by discussing a study on the organic acids produced from the fermentation of Clostridium ramosum, in which showcase the regulatory effect on the motility of E. coli cells (9).
2. Bacterium-Host interaction: Pathogen vs Host
The second topic concerns “the bacterial motility to the host”. This is the continuation of the candidate’s previous studies on the inhibitory effect of mannose-binding lectin to the bacterial motility (10, 11), motility of Salmonella inside the egg (12), whereas the recent studies focus on the gliding
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motility of pathogenic Leptospira cells on the host animal cells in order to reveal complicated host-preference on Leptospira infection.
3. Bacterium response to environmental stimuli
The third topic concerns “the bacterial motility with the environmental factors”. Motility of Leptospira under environmental factors such as light will be discussed. In detail, a recently isolated Leptospira species that showcases the photoresponsivity will be introduced in the topic.
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Chapter 1.
Effects of fermentation products of the
commensal bacterium Clostridium ramosum on
motility, intracellular pH, and flagellar synthesis
of enterohemorrhagic Escherichia coli
Introduction
Although the mechanism of bacterial motility is different in different species, many motile species exhibits flagellum-dependent motility. A thin, helical filament is rotated by a basal motor (flagellar motor) that propels the cell in liquids. The flagellar motor is fueled by an electrochemical gradient called ion motive force, which consists of membrane voltage (Δψ) and ion concentration gradient (e.g. pH difference between the cell exterior and cell interior, ΔpH). In Escherichia coli, proton flux through a proton channel that exists in a stator complex is coupled to the rotation. Although the flagellar motor is bi-directional, a counterclockwise (CCW)-biased rotation bundles the flagella, allowing the cell to travel smoothly. The rotational bias is regulated by environmental stimuli (e.g., chemicals, pH, temperature, and light), and it is responsible for taxis behavior to explore
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preferred regions. Namely, a reversal from CCW to clockwise (CW) unravels the flagellar bundle so that the cell transiently tumbles; by restarting CCW rotation, the flagella are bundled again, and the cell swims in the direction determined randomly during tumbling (Berg 2003).
For pathogenic species, motility and flagella are crucial
virulence factors (Haiko and
Westerlund-Wikström 2013). Virulence of V. cholerae, the pathogen that causes cholera, is attenuated by the inhibition of motility (Guentzel and Berry 1975). In many cases, distinct roles of motility and flagella in infection have been reported, although they are involved in invasion and adhesion in general. H. pylori requires motility to migrate towards the gastric epithelium, but the flagella are not involved in adhesion; instead, other adhesins would be responsible for the adhesion (Clyne et al. 2000). The swimming ability and chemotaxis enable Salmonella species to invade a host, but whether the flagella are associated with their pathogenicity depends on serovars. Flagella are indispensable for the adhesion of Salmonella Enteritidis and Salmonella Dublin but not for Salmonella Typhimurium (Olsen et al. 2013). Motility is also a crucial virulence factor for some spirochete
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species such as L. interrogans, the causative agent of the zoonotic disease leptospirosis, and B. hyodysenteriae, the pathogen that causes swine dysentery. Since the spirochete flagella reside beneath the outer membrane, i.e. within the periplasmic space, their flagella are not direct virulent factors. E. coli also uses motility and flagella for infection, but their roles as virulence factors differ according to the strains and serotypes: The H2 or H6 flagella of enteropathogenic E. coli (EPEC) are used not only for motility to invade the host but also for adhesion (Girón et al. 2002). The Shiga-toxin-producing E. coli (STEC) O113:H21 uses motility for invasion, but the H21 flagella are dispensable for adhesion (Rogers et al. 2012). Quorum sensing by enterohemorrhagic E. coli (EHEC) O157:H7 upregulates the expression of genes related to flagellar synthesis, motility (motA/motB), and chemotaxis (Sperandio et al. 2001), and the H7 flagella play the role of adhesin (Mahajan et al. 2009). Such motilities of pathogens are expected to be a potential target for treatment. Fermentation products from L. lactis subsp. lactis containing organic acids such as acetate and lactate decrease the intracellular pH of various pathogens and inhibit their motility (Nakamura et al. 2015), implying the potential use of
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probiotics as inhibitors of pathogen motility.
In this study, we focused on fermentation products from C. ramosum, a harmless intestinal bacterium that commonly inhabits the human gut. High-performance liquid chromatography has shown that C. ramosum produces various organic acids, and the top three are formic acid (20.1 mM), acetic acid (10.2 mM), and lactic acid (12.7 mM) (Koyanagi et al. submitted for publication). In view of the inhibitory effects of organic acids produced by L. lactis subsp. lactis on bacterial pathogens (Nakamura et al. 2015), we examined the influences of C. ramosum fermentation products on the EHEC physiology and behavior, showing that the fermentation products of C. ramosum decrease the intracellular pH of EHEC and inhibit motility.
Materials & Methods
Bacterial strain, media, and chemicals
In this study, E. coli strain O157:H7 EDL931k (Marques et al. 1986), and commercially available C. ramosum strain JCM1298 were used. E. coli strain O157:H7 EDL931k was obtained by re-cloning of EDL931. For preparation of the C. ramosumculture supernatant (CR sup), the cells were
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anaerobically grown in brain heart infusion (BHI) medium (Merck KGaA, Germany) at 37 °C for 24 h until
1.7 × 109 cfu ml−1 were obtained. The grown cells were killed
by incubation at 65 °C for 1 h and centrifuged at 8,000×g for 10 min. The supernatant was collected, passed through a filter unit (0.22 μm; Merck Millipore, Ireland) twice, and stored at 4 °C. The filtered CR sup was cultured at 37 °C for 24 h to ensure that all bacteria were properly killed and removed. The pH of the original CR was measured with a pH meter (UltraBasic Benchtop Meters; Denver Instrument, US), and the pH of the media was adjusted to 6 by adding HCl or NaOH, as necessary. The EHEC cells were grown in L-broth (DB, NJ, USA) at 37 °C.
Motility analysis
The EHEC cells were harvested in the early stationary phase and diluted 1:20 with fresh BHI medium or CR sup. The cells were infused into a flow chamber composed of a coverslip (upper) and glass slide (bottom) and observed using a dark-field microscope (BH2, Splan 40×, NA 0.70; Olympus, Tokyo Japan). The free-swimming cells were recorded using a computer through a high-speed charge-coupled device
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camera (ICL-B0620M-KC, Imperx; FL, USA) at 30 frames per second. The swimming trajectories and speeds of individual EHEC cells were determined from the movies using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and VBA-based macros in Microsoft Excel (Microsoft, Bellevue, WA, USA), developed previously (Nakamura et al. 2015).
Rotation assay for a single flagellum
The rotation of the EHEC flagellar motor was analyzed with the tethered cell assay (Silverman and Simon 1974; Xu et al. 2016). Five hundred microliters of EHEC culture was centrifuged at 8,000×g for 2 min, and the supernatant was removed. The flagellar filaments were sheared by passing the suspension through a 27-gauge needle and 1-ml syringe to reduce the number of flagellar filaments per cell. The cells were spontaneously tethered on glass via the sheared filaments. Rotations of the tethered cells were recorded at 10-ms intervals, and their speeds and reversals were measured using ImageJ and Microsoft Excel macros developed previously (Nakamura et al. 2015; Xu et al. 2016).
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Measurement of the number and length of flagellar filaments
The EHEC cells were grown overnight in L-broth in the presence or absence of CR sup. Flagellar filaments of more than 100 cells were observed using dark-field microscopy, and the number and length were analyzed with ImageJ and OriginPro 8 (OriginLab Corp., MA USA) software.
Measurement of intracellular pH
Changes in the intracellular pH of the EHEC cells were measured using pH-sensitive enhanced green fluorescent protein (EGFP); an absorption spectrum of EGFP varies in a pH-dependent manner (Haupts et al. 1998). Cells carrying a plasmid encoding egfp (vector pEGFP, GenBank accession no. U76561; Clontech, CA, USA) were grown overnight in
L-broth with 100 μg ml−1ampicillin at 37 °C. One milliliter of the
culture was washed twice with fresh broth by centrifugation (8,000×g, 2 min). The cell suspension was diluted using broth or CR sup, and the fluorescent absorption spectra were measured with a fluorescence spectrophotometer (BSFL-101; Biolab Scientific, Toronto, Canada) with excitation at 490 nm and room temperature. The intensity at 509 nm was
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determined.
Statistical analysis
All experiments were performed in triplicate, and the statistical analysis (Student’s t test) was performed to evaluate the significance in all experiments. The data analysis was performed with Microsoft Excel and OriginPro 8.
Results
Effects of C. ramosum fermentation products on EHEC swimming
To examine the effects of the fermentation products of C. ramosum on EHEC motility, I quantified its swimming ability in the presence and absence of CR sup. Figure 1 shows that exposure to CR sup (pH 5.9–6.0) significantly decreased the
swimming speeds of EHEC cells: 27.4 ± 6.1 μm s−1 (n = 38)
in BHI and 18.1 ± 6.6 μm s−1 (n = 67) in CR sup (Fig. 1).
Swimming speeds measured in BHI adjusted to pH 6.0 by the addition of HCl (BHI + HCl) were comparable to those in
fresh BHI: 26.0 ± 5.1 μm s−1 (n = 33 cells). Addition of
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produced by C. ramosum (Koyanagi et al. submitted), to BHI and pH adjustment to 6 decreased the swimming speeds of EHEC cells to the same level as that in CR sup:
19.3 ± 7.2 μm s−1 (n = 83 cells). When the pH values of CR
sup and BHI + F/A/L were adjusted to 7 by the addition of NaOH, inhibition of motility was not observed. These results suggest that the inhibition of EHEC motility is attributed to the production of organic acids and acidification of media by C. ramosum fermentation.
Effects of C. ramosum fermentation products on the rotation of EHEC flagella
I examined the effect of CR sup on the rotation of the EHEC flagellum using the tethered cell assay (Fig. 2a, inset). In BHI, cells tethered on a glass surface via a single flagellum showed CCW-biased rotation (Fig. 2A, gray thick line). Exposure to CR sup remarkably increased motor reversal, biasing the direction of motor rotation to CW (Fig. 2A, black thick line). A time fraction of CW rotation (CW bias) was about 0.15 and 0.3 in BHI and CR sup, respectively (Fig. 2B). Both CCW and CW rotations were slowed by exposure to CR sup (Fig. 2C). BHI + HCl (pH 6.0) did not affect the tethered cell
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rotation, whereas BHI + F/A/L (pH 6.0) decreased the rotation rate and increased reversal frequency. These results are consistent with those of the swimming assay, suggesting that flagellar rotation is disturbed by CR sup; the existence of organic acids under acidic conditions resulted in anomalies in swimming motility.
Effects of C. ramosum fermentation products on the intracellular pH of EHEC
Organic acids such as acetate and benzoate permeate the cytoplasmic membrane in the protonated form and dissociate protons within the cytoplasm; decreased external pH (i.e., medium pH) facilitates the permeation of protonated acids, resulting in the reduction of intracellular pH (Kihara and Macnab 1981; Minamino et al. 2003; Nakamura et al. 2009; Repaske and Adler 1981). We evaluated the effect of CR sup on the intracellular pH of EHEC using EGFP. Absorption of EGFP around 490 nm is decreased by decreasing the pH (Haupts et al. 1998). We measured the absorption in the presence of benzoate as the positive control and observed a significant reduction in intracellular pH (Fig. 3). Exposure to CR sup decreased the intracellular pH of EHEC to a similar
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level as benzoate, whereas the effect of BHI + HCl (pH 6.0) was slight. BHI + F/A/L decreased the intracellular pH of EHEC as much as CR sup. These results suggest that organic acids produced by C. ramosum would affect the flagellar rotation of EHEC by decreasing the intracellular pH.
Effects of C. ramosum fermentation products on EHEC flagella
The flagellar filament of EHEC consists of flagellin, and monomers of the protein are secreted by the type III secretion system (T3SS). The proton motive force (PMF), sum of Δψ and ΔpH, is the driving force for not only flagellar rotation but also T3SS (Minamino and Namba 2008; Paul et al. 2008). A change in Δψ or ΔpH, even as the total value of PMF is maintained, affects protein secretion through T3SS, suggesting that T3SS discriminates between the two energy components (Minamino et al. 2011). These facts raise the possibility that CR sup affects the synthesis of flagella in EHEC. We cultivated EHEC cells in the presence and absence of CR sup and compared the number and length of the flagella. Table 1 shows that the flagella were significantly shortened when the cells were grown in the presence of CR
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sup. As predicted from the results of the pH measurement, EHEC flagella were also shortened in BHI + F/A/L but not in BHI + HCl. The number of flagella per cell did not depend on the culture conditions
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Discussion
In this study, we showed that the fermentation products of C. ramosum decreased the intracellular pH of EHEC, thereby interfering with flagellar rotation and swimming. Under acidic conditions, the effect of CR sup on EHEC motility was reproduced by the organic acid mixture composed of formate, acetate, and lactate, the top three acids produced by C. ramosum (Koyanagi et al. in press). An organic acid can permeate the cell membrane in a non-charged form and dissociate protons within the cytoplasm. Therefore, acidification of the cell exterior promotes permeation of organic acids, decreasing intracellular pH until equilibrium is achieved (Kihara and Macnab 1981; Repaske and Adler 1981). Thus, inhibition of the EHEC motility would be induced by the existence of organic acids and acidification of media.
CR sup did not change the number of flagella, suggesting that adhesion via flagella would be possible even in the presence of C. ramosum. In contrast, flagellar filaments of EHEC cells cultivated with CR sup were significantly shorter than those grown in the absence of CR sup (Table 1). Shorter flagella may result in slow swimming, but a hydrodynamic
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model based on the resistive force theory (Holwill and Burge 1963; Magariyama et al. 1995) predicts that the shortening of flagella from 5.3 to 3.2 μm does not affect swimming speed. Specifically, motility inhibition of EHEC would not involve shortening of the flagella. Instead, a decrease in intracellular pH inhibits proton flux coupled to rotation, thereby slowing motor rotation (Minamino et al. 2003; Nakamura et al. 2009). Also, reduction of intracellular pH induces ‘pH taxis’ (Kihara and Macnab 1981; Repaske and Adler 1981), increasing CW bias of motor rotation, as shown in Fig. 2. Thus, the observed motility inhibition of EHEC would be attributable to the anomaly in flagellar rotation and not flagellar synthesis.
The fundamental structure and rotation mechanism of the flagellar motor are common across species. Therefore, although EHEC was investigated in this study, we believe that the inhibitory effects by C. ramosum are more general for various motile pathogens. Furthermore, acid production by C. ramosum, a commensal member of gut flora, should be noted because short-chain fatty acids produced by gut microbes are beneficial for the host (Fukuda et al. 2011; Kles and Chang 2006; Saulnier et al. 2009), e.g. acetate
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generated by Bifidobacterium prevents EHEC infection (Fukuda et al. 2011). Acid production by C. ramosum and the resultant reduction of intracellular pH may affect the physiologies of other microbes living in the host gut and intestinal tissues and influence host health.
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Chapter1-Fig. 1
Swimming speed of EHEC. The average and standard deviation values are shown: 38 cells for BHI; 67 cells for CR sup; 83 cells for BHI + F/A/L (pH 6.0); 33 cells for BHI + HCl (pH 6.0); 30 cells for CR sup + NaOH (pH 7.0); 28 cells for BHI + F/A/L + NaOH (pH 7.0). The statistical analysis was performed using Student’s t test (*P < 0.01)
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Chapter1-Fig. 2
Rotation measurements of a single flagellar motor using the tethered cell assay. A, revolution vs. time plot obtained for BHI (gray thick line), CR sup (black thick line), BHI + F/A/L pH 6.0 (gray thin line), and BHI + HCl pH 6.0 (black thin line). Positive and negative revolutions indicate CCW and CW, respectively. The inset shows a schematic of the tethered cell assay. C, the time fraction for CW rotation; the ratio of the total time for CW rotation to observation time was computed for each cell. C, the rotation rate of EHEC tethered cells; CCW and CW rotations were analyzed separately in each cell. Average and standard deviation values for 31 cells (BHI), 26 cells (CR sup), 28 cells (BHI + F/A/L pH 6.0), and 28 cells (BHI + HCl pH 6.0). Asterisks indicate a statistically significant difference from BHI (P < 0.01)
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Chapter1-Fig. 3
Effects of C. ramosum fermentation products and organic acids on the intracellular pH of EHEC. Absorption at 490 nm was measured; absorption is reduced with decreasing pH. The average and standard deviation values of three independent experiments are shown (*P < 0.01)
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Chapter1-Table 1
Flagellar number and length of EHEC grown in the absence and presence of C. ramosumsupernatant or organic acids
*Indicate a statistically significant difference from BHI (P < 0.01)
BHI CR sup BHI + F/L/N BHI + HCl
Flagellar length (μm) 5.3 ± 1.1 3.2 ± 1.1* 3.7 ± 0.9* 5.4 ± 1.2 Flagellar number/cell 6.1 ± 2.4 5.8 ± 1.9 5.9 ± 3.0 6.4 ± 2.0
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Chapter 2.
Involvement of adhesion and crawling behavior
in Leptospira infection and host-preference
Introduction
Leptospirosis is a zoonotic disease caused by the pathogenic spirochete Leptospira (Faine 1982). It has been reported worldwide, affecting lager range of hosts, such as livestock, companion animals, and humans, with mild to fatal symptoms (Bharti et al. 2003). These hosts, including humans are divided into accidental hosts and maintenance hosts. The infection with the accidental hosts usually cause severe and fatal symptoms such as diarrhea, hemorrhage, jaundice and nephritis. The maintenance hosts are in which
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infection is more chronic and the bacteria are maintained by the infection of the renal tubules (Levett 2001). There are more than 300 serovars identified under the 20 species of genus Leptospira, and many of them are the pathogenic strains (Cerqueira 2009). These pathogenic serovars are highly host-preferring during the infection. Although whether the infection of Leptospira results in colonization in the kidney or clinical symptom depends on host animal species, the mechanism by which Leptospira serovars prefer to specific species remains unclear.
Leptospira have periplasmic flagella beneath the outer cell membrane. In the externally flagellated bacteria such as E. coli and Salmonella spp., the flagella are rotating to generate the thrust by interaction with surrounding fluids. In contrast, the role of Leptospira periplasmic flagella is not propelling the cell but transforming the cell body (Bromley and Charon, 1979, Kan and Wolgemuth, 2007, Nakamura et al., 2014). Leptospira cells can smoothly swim when the anterior end is spiral shaped, and the posterior end is hook shaped (Spiral– Hook). During swimming, the morphology of the cell body
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frequently changes. When both ends of the cell body are hook shaped (Hook–Hook) or spiral shaped (Spiral–Spiral), cells move neither forward nor backward and spin at one position (Goldstein and Charon 1988). Although swimming is known as the major method of Leptospira motility, an early study back in 1974 suggested that Leptospira possess the alternative crawling motility once the bacteria move on the top of solid surfaces (Cox, et al., 1976). The mechanism of such ‘swimming-crawling’ motility in Leptospira has been descripted very recently by Tahara et al. as the two-phase motility (Tahara, et al., 2018). Crawling no longer requires the asymmetrical morphology, but only the anchoring molecules which help the bacterial cell to attach to the solid surfaces and the rotation of the cell body. However, the crawling of Leptospira on the real host animal cells has never been observed so far.
Motility is a crucial factor to enhance the pathogenicity in many bacteria. The causative bacteria of gastric ulcer H. pylori requires motility to migrate towards the epithelial tissue of stomach (Clyne et al. 2000). Inhibition of motility in V.
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cholerae attenuates the symptoms of cholera (Guentzel and Berry 1975). Although it might not directly get involved in the invasion, the motility and chemotaxis are the key to guide Salmonella species to invade a host (Olsen et al. 2013, Siitonen et al. 1992). Similar story happened during the Leptospira infection as well. In fact, motility deficient pathogenic Leptospira strains are not capable of persisting inside the host anymore (Wunder et al 2016). During the infection, Leptospira can penetrate the skin and mucous membrane and migrate in host tissue (Bharti et al. 2003). Miyahara et al. observed that Leptospira cells attached to the liver surface and invaded through paracellular routes by scanning electron microscopy (Miyahara, et al., 2014). The observation raises the possibility that the motility, kinetics of Leptospira on the tissue surfaces influences the consequence of leptospiral infection; in other words, adhesivity and crawling on host cells can be involved in the host preference of Leptospira. To verify the hypothesis, we examined the adhesivity and crawling motility of different Leptospira serovars on various animal cells.
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Materials & Methods
Bacterial strains
Pathogenic serovars of L. interrogans (serovar
Icterohaemorrhagiae and Manilae) and saprophytic L. biflexa (serovar Patoc I) were used. The bacterial cells were cultured in enriched Ellinghausen-McCullough-Johnson-Harris liquid medium (BD Difco, NJ, USA) containing 25 μg/mL spectinomycin at 30 °C for 2 to 4 days until the stationary phase. The bacterial cells carry a plasmid harboring the gfp gene in order to visualize the bacterial cell without the background noise caused by the animal cells.
Animal cells and media
Animal kidney epithelial cell lines: MDCK-NBL2 (dog), NRK-52E (rat), Vero (monkey), MDBK-NBL1 (cow) and TCMK-1 (mouse) were used. MDCK, Vero, TCMK and MDBK cells were maintained in Eagle’s minimum essential medium (Sigma-Aldrich, Darmstadt, Germany) containing in 10% fetal bovine serum (Nacalai Tesque, Kyoto, Japan) or 10%
horse serum (Nacalai Tesque) at 37 °C and 5% CO2. NRK
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medium (Thermo Fisher Scientific, MA, USA) with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose and 5% bovine calf serum (Nacalai
Tesque) at 37 °C and 5% CO2. All media for animal cells
contained a 5% antibiotic/antimycotic mixed solution (Nacalai Tesque). The cells were treated with a 0.1% trypsin-EDTA solution (Nacalai Tesque) for dislodging during the passage process.
Microscopy observation and adhesion-crawling assay Animal cells were harvested with 0.1% trypsin and 0.02% EDTA in a balanced salt solution (Nacalai Tesque) and plated in a chamber slide (Iwaki, Tokyo, Japan) in corresponding media without antibiotics as described above (Fig.1a). The slides were incubated for 48 h and washed twice with corresponding media without antibiotics to remove nonadherent cells. Leptospira cells were harvested by centrifugation (1,000xg for 10 min), washed twice with PBS, then resuspended in the corresponding animal cells culture
media without antibiotics at 37 °C to a concentration of 107
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into the corresponding chamber slides containing animal cells, then the chamber slides were incubated at 37 °C for 1 h (Fig.1a). Finally, the moving behaviors such as swimming, adhesion and crawling of Leptospira cells on the animal cells were observed by a dark-field microscope with epi-fluorescent system (BX53, Olympus, Tokyo, Japan) and recorded by a CCD-camera (WAT-910HX, Watec Co., Yamagata, Japan). Further analysis on motility was conducted on a computer with previously developed VBA macro (Microsoft, WA, USA) (Nakamura, et al., 2014), ImageJ (NIH, MD, USA) and OriginPro (OriginLab Corp., MA., USA). Experiments were performed in triplicate.
Results
Swimming, adhesion and crawling equilibrium
To assess the moving behaviors of different serovars of Leptospira cells on the animal cells, we assumed a certain area of observation zone as a closed system. Leptospira cells in such systems appeared to demonstrate three distinct types of motility (Fig.2): “swimming” cells were the Leptospira floating above without physical contact to the animal cells
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below, and performed typical swimming motility; “adhered” cells were the Leptospira cells attached to the animal cells, but showed no substantial displacement in motility; and “crawling” cells were the Leptospira cells not only attached to, but literally performed crawling motility on the animal cells. The dynamic fractions of each type of motility were calculated respectively. The ratio of two fractions is the equilibrium constant between those two types of motility (i.e. K[swimming/adhered] or K[adhered/crawling]).
We calculated the equilibrium constants in different Leptospira serovar-animal kidney cell combinations. The
equilibrium constant between swimming and adhesion (Ks↔a)
and between adhesion and crawling (Ka↔c) were both
showing obvious difference when it came to different combinations. For instance, saprophytic serovar Patoc I was
showing fair values on the Ks↔a constant, which means there
were considerable amount of Patoc cells attached to the surfaces of animal cells (Fig.3a). However, only few of these were eventually preforming crawling and most attached cells
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small (Fig.3b). In contrast, pathogenic serovar
Icterohaemorrhagiae showed a slightly lower Ks↔a constant
to dog kidney cells compared to Patoc I (Fig.3a), whereas its
high Ka↔c constants referred that an extremely large portion
of these attached cells were crawling on the dog cells (Fig.3b). These data suggested that pathogenic Leptospira serovars might possess the better overall affinity to the animal host cells.
Crawling speed
The moving speed of crawling Leptospira cells was analyzed to further understand the behavior difference. The crawling speeds in the results were not as different as the equilibrium constants among each serovar-animal cell combination. Serovar Icterohaemorrhagiae appeared to possess the most decent overall crawling motility on the all types of animal cells, with the crawling speed from 10.3 μm/s to 13.2 μm/s, followed by serovar Manilae, and saprophytic serovar Patoc I showed relatively slow crawling on all cell types (Fig.4).
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To further investigate the potential role of adhesion and crawling motility on Leptospira host-preference. We presented the two equilibrium constants and the crawling speed of each serovar-cell combination in a three-dimensional pairwise plot (Fig.5). pathogenic Serovar Icterohaemorrhagiae showed remarkably high scores of adhesivity and crawling motility on dog, monkey and mouse kidney cells. Pathogenic serovar Manilae on rat and mouse cells, Icterohaemorrhagiae on rat and cow cells lay on the middle of the plot. Combinations such as saprophytic serovar Patoc I to all and Manilae on dog cell showed relatively low scores, lay on the bottom of the plot. The severity of symptoms during the Leptospira infection of each host animal species that the cells represented were indicated by red (usually fatal), yellow (mostly persistent and occasionally fatal) and green (mostly shed in urine or occasionally persistent) colors, based on the scientific studies, clinical reports and other available literatures related to leptospirosis. These data suggested that the adhesivity and crawling motility could contribute to the host-preference and the severity of symptoms during the infection of Leptospira.
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Those host-animal cell combinations with decent adhesivity and crawling motility to the corresponding animal cells are known as the accidental hosts with severe symptoms (red dots), while poorer adhesivity and crawling are mostly known as the maintenance hosts (yellow and green).
Discussion
The crawling motility was first discussed by an early study of Cox and Twigg in 1974 (Cox, et al., 1974). After several decades, the mist of Leptospira crawling motility was finally revealed by Tahara et al as described as the “swimming-crawling” two-phase motility (Tahara, et al., 2018). These findings are the cornerstone and allow us to explore further on the Leptospira infection from the aspect of motility, in a in vivo way. In this study, we constructed such a semi-in vivo system and observed the adhesivity and crawlsemi-ing motility of Leptospira cells to the kidney epithelial cells from several animal hosts. The varied results from each serovar-host combination matched with the common understanding of leptospirosis in a certain degree. The pathogenic L. interrogans serovar Icterohaemorrhagiae are the major
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cause of Weil disease, which not only occurs to human but other mammals as well (Faine 1982). For instance, rats undergo bacteremia after initial infection, while no severe symptoms cause the life of rats appear (Seguin et al., 1986); Leptospira cells settle in the kidneys of rats then being shed in urine to the environment, Accidental hosts such as dog (Ayral, et al., 2014, Fraune, et al., 2013, Klopfleisch, et al., 2010). monkey (Perolat, et al., 1992, Merien, et al., 1997) and human (Faine, 1982) are infected by the contact with contaminated water and aerosols. As mentioned, the clinical signs for these hosts are more severe including jaundice, hemolytic anemia, myalgia, and hepatorenal failure. We found that serovar Icterohaemorrhaegiae demonstrated relatively better adhesivity and crawling motility to cells of these hosts. Interestingly, the kidney cell line of mouse used in this study was the TCMK-1, which was originally from the mouse strain C3H (Black, et al., 1963). Strain C3H mouse develops into highly acute symptoms after being infected by L. interrorgans serovar Icterohaemorrhagiae, (Viriyakosol, et al., 2006, Richer, et al., 2015) and serovar Manilae (Koizumi, et al., 2003), although majority of the mouse strains such as
39
CBA, BALB/c, A/J or C57BL/6 are asymptomatic during the infection (Gomes-Solecki, et al., 2017). However, our results showed that both of serovar Icterohaemorrhaegiae and serovar Manilae were showing the decent adhesivity and crawling to the mouse cells. These outcomes could be explained by the important role of immune response in different species of host during the Leptospira infection, which could be even more crucial in determining the host-preference of Leptospira.
Despite the discrepancy, pathogenic serovars of L. interrogans were superior in both adhesivity and crawling motility to the animal cells compared to the saprophytic serovar L. biflexa Patoc I. According to Tahara et al., the lipopolysaccharides embedded in the bacterial cell membrane are the adhesive molecules in charge of adhesivity and participate in the crawling process as the anchoring molecules (Tahara, et al., 2018). The differences on the adhesivity and crawling between pathogenic and saprophytic Leptospira, and between different serovar-animal cell combinations also raise more questions to the
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detailed study of this LPS-based crawling model. For instance, the amount of effective LPS molecules responsible for adhesion and crawling could be differed by the species or serovars, or the possible counter molecules on the host cells to be paired with the LPS on Leptospira to manifest the crawling process.
Although bacterial infection is a complicated process that must be considered with immune responses of the hosts to pathogens, our study addressed the possibility that adhesivity and crawling motility are involved in the host-preference of Leptospira serovars at early stage of colonization. Further kinetic measurements and molecular study are required to fully understand host-preference during Leptospira infection.
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Chapter2-Fig.1a
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43
44
45
46
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Figure legend
Fig.1. The schematic of the chamber slide used to observe the behavior of Leptospira cells on animal cells (a), and the sample clips recorded by using a dark-field microscope equipped with epi-fluorescent system: L. interrogans serovar Icterohaemorrhaegiae (b), Manilae (c) and L. biflexa serovar Patoc I on NRK cells
Fig.2. The kinetic model assuming three states of behaviors
of Leptospira cells in on the animal cell surface. (Ks↔a) and
(Ka↔c) refer to the two equilibrium constants between
swimming and adhesion, and between adhesion and crawling, respectively.
Fig.3. The equilibrium constants between swimming and adhesion (a), and between adhesion and crawling (b) in different serovar-animal cell combinations. letter M, I and P
refer to the Leptospira serovar Manilae,
Icterohaemorrhaegiae and Patoc I, respectively. (*P<0.05, **P<0.01)
Fig.4. The Crawling speed of crawling Leptospira cells on different animal cells. letter M, I and P refer to the Leptospira serovar Manilae, Icterohaemorrhaegiae and Patoc I, respectively. (*P<0.05, **P<0.01)
Fig.5. The three-dimensional pairwise plot between crawling speed and two equilibrium constants. Combinations in red colored are usually with severe to fatal symptoms, in yellow are mostly the maintenance groups or with mild symptoms, while in green are mostly unharmed or maintenance groups. “Mani vs Monkey” shows in grey due to the unavailability of
48
related data. Mani, Icte and Patoc refer to the Leptospira serovar Manilae, Icterohaemorrhaegiae and Patoc I, respectively. (*P<0.05, **P<0.01)
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Chapter 3.
Photoresponsivity in the spirochete Leptospira
Introduction
Light is a ubiquitous environmental factor that plays an important role in the life of bacteria. Many species of bacteria have light-sensitive proteins to control biological functions that respond to changes in light. Light not only plays a role in regulating processes in photosynthetic prokaryotes, but also in non-photosynthetic bacteria. An early example of light perception in bacteria is the formation of fruiting bodies in the non-photosynthetic myxobacterium Stigmatella aurantica. Starved S. aurantica forms aggregates, which then may develop into fruiting bodies. The formation of stalks that support fruiting bodies only takes place after aggregates are exposed to light (Qualls et al, 1978). How S. aurantica senses light and transforms a light signal into a cellular response is still unknown. With increasing numbers of genomes sequenced, more bacteria have been found to contain genes encoding light receptors, or the cell processes that are affected by light. The importance of light sensing has been shown in a wide range of bacteria. A light-sensing
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membrane protein BlsA found in Acinetobacter baumanni is known to be involved in biofilm formation, motility and the pathogenicity (Mussi et al., 2010). Synechocystis sp. possess multiple proteins in terms of phototaxis and chromatic adaptation (Hirose et al., 2008, Bhaya et al., 2001, Yoshihara et al.,2000). Halobacterium salinarum can sense the light with sensory rhodopsin I (SRI) and II (SRII) (Spudich et al., 1984).
This chapter covers a new light-sensing clade that are found in the recently identified Leptospira species, L. kobayashii, which were isolated from the soil in central Japan (Vincent et al., 2019). Leptospira in this clade are showing photoresponsivity-relevant motility in different wavelength of light. The involvement of light-sensory rhodopsins in the process is highly relevant. Rhodopsins are membrane embedded proteins that were first described as light activated ion channels in Archea. Later rhodopsins were found that did not function as ion channels but instead functioned as light sensors that control motility and phototaxis (Bogomolni et al., 1982). These sensory rhodopsins interact with cytoplasmic effector proteins that in
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turn relay the signal to the cytosol. For example, the well-studied sensory rhodopsins SRI and SRII in H. salinarum regulate phototaxis through a cytoplasmic domain fused to the membrane-embedded rhodospsin (Spudich et al., 2014). Sensory rhodopsins contain a retinal cofactor that is covalently attached to a lysine residue. This retinal cofactor absorbs light in the blue and green light region. Once excited by a photon, the retinal cofactor will undergo isomerization of a carbon-carbon double bond from 13-cis to all-trans. Under dark conditions, the retinal reverts to its 13-cis state. The protein moiety of rhodopsins can have a strong influence on the spectrum and properties of the retinal cofactor.
In this study, we analyzed the motility of the light sensing Leptospira kobayashii and other Leptospira species and showed the evidence of the involvement of sensory rhodopsin in the process.
Materials and Methods
Bacterial strains
L. kobayashii (strains E30, E107, E154, E101), L. idonii strain AB7, L. ryugenii strain YH101, L. inadai strain AY6. L.
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johnsonii strain E8 and L. biflexa strain Patoc I were used. The bacterial cells were cultured in enriched Ellinghausen-McCullough-Johnson-Harris liquid medium (BD Difco, NJ, USA) at 30 °C for 2 to 4 days until the stationary phase. L. kobayashii is the newly identified species, whereas others are previously reported species.
Motility assay with light and phylogenetic analysis
The methods which are used to measure the motile fraction and swimming speed are described previously by Xu et al. (2015). Briefly, 2-4 days cultured Leptospira cells in the
density of 1.0 × 108 cells/ml, were observed under dark-field
microscope (BH2; Olympus, Tokyo, Japan),
the locomotion of bacteria in different intensities and wavelengths of light source was captured by a charge-coupled device camera (WAT-910HX, Watec Co., Yamagata, Japan) at a frame rate of 30 fps (Fig.1). Appropriately captured DVD videos were converted to AVI movies, for further analysis. Swimming trajectory and speed of bacterial cells were analyzed by using software ImageJ (National Institutes of Health, Bethesda, MD) and Macros of Excel (Microsoft, Redmond, WA), which were developed from the
53
previous study. (Nakamura et al. 2006).
To capture every single gyration of a rotating cell, High speed CCD camera (B0620; Imperx, FL, USA) was equipped to the dark-field microscope. The rotation in hook shaped end of rotating cell was intensively focused, appropriate parts of the video were captured on computer for further analysis.
At least four individual trials were carried out in each assay. Statistical analysis was accomplished by using Microsoft Excel and Origin (OriginLab, Northampton, MA).
Phylogenetic analysis was accomplished by using the online NCBI database and the online tools nBlast and BioClould.
Rhodopsin color screening assay
Leptospira cells were inoculated in an enriched EMHJ medium for 3 days until the stationary phase. Cells were harvested by centrifugation in 1,000 xg, 10 min. A solution of all-trans-retinal (Wako Pure Chemical, Osaka, Japan) in EMJH was added to the cells in the final concentration of 50 μm after removing the supernatant. Mixed the cells and solution gently then cultured the cell at 30 °C for 3 h.
54
subsequently, centrifugation was performed after cultivation to remove the unbound retinal molecules before observation.
Results
Light-dependent activation of the L. kobayashii
To assess the light-dependent motility in light sensing L. kobayashii. We analyzed the swimming speed and cell rotation under different light intensities. L. biflexa were swimming in the speed around 10 μm/s under both dark and bright light conditions. L. kobayashii showed the
increased swimming from 10 μm/s to 21 μm/s with the light intensity increased (Fig.2).
We measured the rotation rate of Leptospira cells hook-end. Although both of L. kobayashii and L. biflexa showed almost the same rotation rate around 12 Hz under dark conditions, while intensive light significantly increased the rotation rates of L. kobayashii to 22 Hz, whereas L. biflexa stayed constant (Fig.2).
Photoresponsivity of species related to L. kobayashii In order to give an overview of the photoresponsivity of the
55
relevant Leptospira species, we further analyzed the cell rotation rate of multiple species and strains which are genetically closed to L. kobayashii. Most of the Leptospira species and strains used in this study were found with
photoresponsivity in certain degree (Fig.3). We then plotted the percentage of increase in cell rotation rate as the
intensity of photoresponsivity, to the 16s rRNA similarity of each species and strain with L. kobayashi strain E30 to show the correlation between photoresponsivity and
phylogenetic distance. The results suggested that these two factors are correlated in a certain degree with the R=0.79 (Fig.4)
Confirmation of light sensory rhodopsin
We performed a motility assay with the addition of chromophore retinal to the L. kobayashii cells for the analysis of the presence of rhodopsin. The results showed that cells in dark environment could also response to the light once the retinal was added (Fig.5a). This can be explained by the presence of retinal that enhances the function of rhodopsin ion pumps, due to the fact that retinal is one of the most common chromophore of the rhodopsin in the light cycle.
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Conformational change happens to the rhodopsin molecule and it appears in red-colored. This was consistent with our experiment results showing that cells were colored in red by incubation in the presence of all-trans-retinal. The results suggested the contribution of rhodopsin-like sensors to the observed photoresponsivity (Fig.5b).
Discussion
The new isolated L. kobayashii is a novel saprophytic leptospiral species, which was isolated from soil in Japan. As with other Leptospira sp., L. kobayashii swims by gyration of the spiral-shaped, anterior cell-end and rolling of the coiled protoplasmic cylinder. Here, we report that L. kobayashii shows light-dependent modulation of swimming motility. L. kobayashii recognized red and blue-green lights and their intensities, resulting in distinct changes in motility. Red light accelerated flagellar rotation, but the cell could not translate because of remained symmetric morphology; leptospires swim smoothly when the anterior and posterior ends are spiral- and hook-shaped, respectively; when both cell-ends exhibit hook- or spiral-shape, the cell rotates but moves
57
neither forward nor backward. Blue-green light induced asymmetric cell morphology, allowing the cell to swim smoothly. L. kobayashii cells were colored in red by incubation in the presence of all-trans-retinal (Fig. 5b), suggesting the contribution of rhodopsin-like sensors to the observed photoresponsivity. The genus Leptospira consists of four clades, of which the species in the same clade as L. kobayashii showed light-controlled motility. Identification of genes associating with the photoresponsivity and characterization of the sensor dynamics are going on.
The current results indicate that L. kobayashii strain E30 and its relatives are unprecedented spirochetes possessing the rhodopsin-dependent dichromatism. Thus, we propose “the photo-responsive clade” in the genus Leptospira.
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Chapter3-Fig.2
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Chapter3-Fig.5a
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