How Can Leptospira Interrogans Be Distinguished From Other Spirochetes?

• plos.org
• create business relationship
• sign in

• Loading metrics

Open Admission

Peer-reviewed

Enquiry Article

Leptospira interrogans Enolase Is Secreted Extracellularly and Interacts with Plasminogen

• Sarah Veloso Nogueira,
• Brian T. Backstedt,
• Alexis A. Smith,
• Jin-Hong Qin,
• Elsio A. Wunder Jr,
• Albert Ko,
• Utpal Pal

ten

• Published: October eighteen, 2013
• https://doi.org/10.1371/journal.pone.0078150

Figures

Abstruse

Leptospira interrogans is the amanuensis for leptospirosis, an important zoonosis in humans and animals across the globe. Surface proteins of invading pathogens, such as L. interrogans, are thought to be responsible for successful microbial persistence in vivo via interaction with specific host components. In item, a number of invasive infectious agents exploit host proteolytic pathways, such equally one involving plasminogen (Pg), which aid in efficient pathogen dissemination within the host. Hither we show that L. interrogans serovar Lai binds host Pg and that the leptospiral factor production LA1951, annotated every bit enolase, is involved in this interaction. Interestingly, dissimilar in related pathogenic Spirochetes, such as Borrelia burgdorferi, LA1951 is not readily detectable in the L. interrogans outer membrane. We show that the antigen is indeed secreted extracellularly; however, information technology tin can reassociate with the pathogen surface, where it displays Pg-binding and measurable enzymatic action. Hamsters infected with 50. interrogans as well develop readily detectable antibody responses against enolase. Taken together, our results suggest that the L. interrogans enolase has evolved to play a role in pathogen interaction with host molecules, which may contribute to the pathogenesis of leptospirosis.

Commendation: Nogueira SV, Backstedt BT, Smith AA, Qin J-H, Wunder EA Jr, Ko A, et al. (2013) Leptospira interrogans Enolase Is Secreted Extracellularly and Interacts with Plasminogen . PLoS ONE eight(x): e78150. https://doi.org/ten.1371/journal.pone.0078150

Editor: Janakiram Seshu, The University of Texas at San Antonio, U.s.a. of America

Received: July 31, 2013; Accustomed: September 13, 2013; Published: Oct eighteen, 2013

Copyright: © 2013 Nogueira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original writer and source are credited.

Funding: This study was supported by funding from National Institutes of Health (AI080615, AI088752 and AI052473). The funders had no role in report design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Leptospirosis is a systemic illness of humans and domestic animals. In fact, information technology is regarded as 1 of the nearly widespread zoonotic illnesses caused past pathogenic spirochetes of the genus Leptospira [ane-5]. While these organisms are extremely motile, they are deadening-growing obligate aerobes with an optimal in vitro growth temperature of 30°C and can be distinguished from other spirochetes on the basis of their unique hook-shaped ends [2]. Leptospira interrogans constitutes the major pathogenic leptospiral species that is responsible for human infection. L. interrogans can readily penetrate abraded pare and mucous membrane barriers to found a systemic infection via haematogenous broadcasting and subsequently colonizes multiple organs, specially the kidneys and liver. While wild rodents serve equally natural reservoirs, humans and a few other domesticated animals are accidental hosts in the manual cycle of leptospirosis [three,6]. Equally 50. interrogans are shed in the urine of reservoir hosts and tin can survive in the environment, such as in water or soil for weeks to months, proper sanitation is a key intervention in reducing the manual of leptospirosis [seven,viii]. Moreover, the disease has emerged every bit a global health threat in impoverished populations, specially in developing countries and tropical regions where inadequate sanitation has produced the perfect weather for this rodent-borne disease [ii]. The incidence of human infection is generally higher in the tropics than in temperate regions, but transmission to humans can occur in both industrialized and developing countries [ix]. Over the past decade, a number of factors, including unexpected outbreaks during sporting events, chance tourism, and natural disasters, take underscored the ability of leptospirosis to get a public wellness trouble even in nontraditional settings [4]. Incidence is thought to be significantly underestimated because of the lack of awareness as well equally relatively imprecise diagnosis [9]. Due to a wide diversity of clinical symptoms and manifestations shared with many other diseases, diagnosis of leptospirosis is particularly challenging and depends on a variety of laboratory assays [ten]. Spirochetes tin can exist detected in cultures of infected urine or tissue samples, and diagnostics normally employ methods based on direct detection of spirochetes or their antigens using dark-field microscopy, immunostaining, or PCR, likewise every bit indirect approaches based on host allowed responses [ane,nine-11].

Although antibiotics are effective in treating leptospirosis in humans, preventive strategies such as vaccination remain an important focus of leptospirosis enquiry due to the high case fatality rate (four-twoscore%) [8] and lack of efficient diagnostic tools, which have in turn hampered timely initiation of handling. In particular, efforts take focused on the identification of immunogenic and novel virulence factors [2,6,12-19] and evolution of subunit vaccines. Specifically, inquiry is focused on identifying surface-associated proteins that are conserved among pathogenic isolates and serve equally antigenic targets for bactericidal immune responses [2]. Cell surfaceomes, especially outer membrane (OM) proteins of pathogenic spirochetes are the focus of relatively intensive investigation [14,15,20-24]. Several candidate OM proteins have been evaluated, nonetheless, with a limited caste of success. For example, immunization with LipL32, a lipoprotein constituting more than 50% of the total OM protein content [25] and that plays a disposable function supporting astute or chronic infection with L. interrogans [26], has yielded equivocal results of host protection against Leptospira [1,2,xiv,27].

Man-to-human transmission of L. interrogans is nonexistent [1]; rather, leptospirosis is acquired from an animal source or from contaminated water or soil. Thus, transmission of leptospirosis requires continuous enzootic apportionment of the pathogen among creature reservoirs and long-term persistence within the host [2]. However, the intricate mechanisms by which spirochetes evade immune defenses to persist in the host and cause disease are poorly understood. The plasmin(ogen) (Pg) system is 1 of the near common host defense mechanisms, constituting the central pathway for dissolution of fibrin clots [28]. This organization acts as a host surveillance mechanism that is essential in maintaining tissue homeostasis and facilitates cell migration by assisting the cellular penetration of protein barriers [29]. Pg is the proenzyme of the broad-spectrum serine protease plasmin, the primary fibrinolytic enzyme that is highly abundant in homo tissues and plasma. Conversion of Pg to active plasmin is mediated by proteolytic activation through a number of mammalian plasminogen activators (PA), such as tissue-type plasminogen activator (tPA) and urokinase (uPA). Plasmin is involved in intravascular fibrinolysis and deposition of extracellular matrix (ECM) materials, which is relevant for cell invasion [xxx]. Pg contains kringle domains, which mediate its zipper to cell surfaces by binding proteins with accessible carboxyl-final or internal lysine residues. Together, these data indicated that the Pg organisation displays a unique part in host defense force and maintenance of cellular homeostasis [29].

Certain cellular proteins integral to the glycolytic pathway, such as enolase, although primarily office as metabolic enzymes, are also known to translocate to the cell surface, where they play an of import role in host-pathogen interactions [31]. In many bacterial pathogens, enolase has been plant to play a major part in microbial recruitment of Pg [32]. Past serving as a surface receptor for Pg, enolase could mediate microbial virulence [33,34]. Although L. interrogans serovar Copenhageni [19,23,35] and serovar Pomona [36] accept already been shown to express multiple Pg-binding proteins, the existence of additional, more widely-known Pg-bounden proteins, such as enolase, remains a possibility. Hither we show that Fifty. interrogans serovar Lai indeed binds human Pg via enolase that is secreted extracellularly. We also prove that leptospiral enolase retains weak but measurable enzymatic activity integral to the glycolytic pathway. Identification of cell-surface proteins that are involved in host-pathogen interaction is central to our agreement of microbial pathogenesis and could contribute to the development of novel preventive strategies against infection.

Materials and Methods

Ethics Argument

The hamster serum samples used in the current work originated from Yale Academy from a written report canonical by the Institutional Fauna Care and Use Committee (Yale University IACUC Protocol #2011-11424). The serum samples were obtained through cardiac puncture after euthanasia of the hamsters using CO₂, and animals were handled according to the in a higher place-mentioned Yale University IACUC approved protocol.

Bacteria

A man pathogenic strain, 50. interrogans serovar Lai str. 56601 [37], was used in this study. The bacteria were grown at 30°C in liquid Elinghausen-McCullough-Johnson-Harris (EMJH) media.

Production of recombinant enolase and antibody

The Fifty. interrogans open reading frame (LA1951) encoding enolase was amplified by PCR using specific primers: LA1951 sense: 5'-CGG AAT TCC TCT CAT CAC TCT CAA ATT CA-3' and LA1951 antisense: five'-CCG CTC GAG TAA ATT ATA AAA AGT TTC CC-three'. Recombinant enolase was produced in Escherichia coli using the bacterial expression vector pGEX-6P-1 (GE Healthcare). Purification of the protein, including removal of the glutathione-S-transferase fusion tag, was performed as detailed by the manufacturer (GE Healthcare). Generation of murine polyclonal antibodies against recombinant enolase as well every bit decision of titer and specificity of the antibodies using ELISA and immunoblotting analyses were performed as detailed previously [38,39].

Assessment of 50. interrogans outer membrane proteins

Fifty. interrogans outer membrane (OM) and protoplasmic cylinder (PC) fractions were isolated as described previously [xl] with minor modifications. Five hundred milliliters of L. interrogans was grown to mid to belatedly log phase, centrifuged at 10,000 rpm for xx min, washed with PBS, and finally resuspended in xx mL of water ice cold membrane isolation buffer [twenty mM Tris-HCl, pH 9.0, i Thou NaCl, two mM EDTA] containing 27% sucrose. The solution was stirred with a magnetic bar at room temperature (RT) for ii h, after which, the sucrose concentration was reduced to 13% by the addition of the same buffer and centrifuged at 7650 yard for 30 min. The supernatant was nerveless and further centrifuged at 141,000 chiliad for 2 h. The resulting pellet was resuspended in 6 mL buffer, layered onto a discontinuous sucrose gradient (56%, 42%, 26%), and centrifuged at 100,000 g for 16 h at iv°C. And so, the OM (upper band) and PC (lower ring) were removed past needle aspiration, diluted 5 to 7 fold in cold buffer, and centrifuged at 141,000 g for 4 h at 4°C. The resulting OM pellet was resuspended in one mL buffer, applied to 12 mL of a continuous 10-42% (wt/wt) sucrose gradient, and centrifuged at 100,000 g for xvi h at 4°C. Finally, the OM pellet was removed by needle aspiration, diluted 5 to 7 fold in common cold PBS, centrifuged at 141,000 g for 4 h at 4°C, and resuspended in l-100 µL PBS containing ane mM PMSF. Equivalent amounts of whole-jail cell lysate, OM, and PC were separated by SDS-Page and immunoblotted with antibodies specific for enolase, the known OM poly peptide LipL32, and known inner membrane poly peptide LipL31 [25].

Identification of secreted proteins of L. interrogans

50. interrogans were grown until mid-log phase, and the viability of the spirochete culture was determined by microscopy. The supernatant samples were collected from cultures of intact viable cells by balmy centrifugation and filtered through a 0.2 µm membrane. The bovine serum albumin (BSA) was removed from the nerveless media using a commercial kit (ProteoPrep^TM Blue Albumin Depletion Kit, Sigma). Protein concentration in the supernatant was quantified, and identification of specific secreted proteins was achieved by two-dimensional (2nd) SDS-Page and immunoblotting.

Gel electrophoresis and immunoblotting

Ii-dimensional gel electrophoresis was performed as detailed [41] with the following modifications. Samples were solubilized in rehydration solution composed of seven mM urea, 2 M thiourea, 0.5% (v/v) Triton X-100, 0.v% (5/five) IPG buffer pH iii-10, and 60 mM DTT. Immobiline DryStrips (GE Healthcare) were placed in rehydration solution overnight [41,42]. Later on isoelectric focusing, the strips were incubated in equilibration buffer containing 50 mM Tris-HCl pH 8.eight, six M urea, 29.three% (v/five) glycerol, 2% (w/5) SDS, 0.002% (west/5) bromophenol blue, and 0.1% DTT and resolved using SDS-PAGE. Gels were finally stained with Coomassie Vivid Blue or transferred to a membrane for immunoblotting using specific antibodies confronting recombinant enolase or a command leptospiral protein, LipL31, equally detailed [41].

Detection of enolase-specific antibody response during host infection was accomplished by immunoblot analysis equally detailed [38,39]. Briefly, recombinant enolase was separated by SDS-Page and transferred to a nitrocellulose membrane before immunoblotting using antisera nerveless from hamsters that were infected with L. interrogans serovar Copenhageni strain Fiocruz L1-130.

Plasminogen bounden assay

For cellular assays, microtiter wells were coated with L. interrogans cells in the presence of glutaraldehyde, which facilitates immobilization of cells [31]. Briefly, 10⁷ cells were coated in i% glutaraldehyde in phosphate buffered saline (PBS), incubated for 10 min at 37°C, and washed with PBS to remove unattached cells. Wells were and then blocked with one% BSA in PBS for 1 h and incubated with human being Pg (hPg - 0.5, i.0, and two µg) (Sigma) for an additional hour. Contest experiments were performed by the addition of increasing concentrations (1, 2.5, and v µg) of recombinant enolase for 1 h prior to the addition of a abiding corporeality (i µg) of hPg. Another set of contest experiments was performed past the addition of anti-enolase or LipL32 antibodies or normal mouse serum (NMS) prior to the add-on of hPg. Binding was assessed by incubation with anti-Pg monoclonal antibody (R&D Systems). Plates were done iii times with 0.1% Tween 20 in PBS. Horseradish peroxidase was added to the wells and incubated for 1 h at 37°C. The absorbance was measured at A ₄₅₀ using a microplate reader.

In another prepare of experiments, wells of microtiter plates were coated with 1 μg of recombinant enolase diluted in carbonate buffer (pH 9.half-dozen). Subsequently blocking and washing, as described above, different amounts (0.5, 1.0, and two µg) of hPg were added to the plates. Binding was adamant by incubation with anti-Pg monoclonal antibody (R&D Systems). Alternatively, the plates were also coated with ane μg of hPg diluted in the carbonate buffer and incubated overnight at iv °C. Plates were then blocked with 1% (wt/vol) BSA in PBS for one h followed by three washes with 0.1% Tween 20 in PBS. Competition experiments were performed by the addition of increasing concentrations of the lysine analogue ε-aminocaproic acrid (ε-ACA, Sigma) to L. interrogans or Pg-coated wells. All reactions were carried out at 37°C, after which the wells were washed three times with 1% BSA in PBS. Binding was determined by incubation with anti-enolase antibody. Following iii washes, the wells were developed as described in a higher place.

Plasminogen activation analysis

The Pg activation analysis was performed by measuring the amidolytic activity of generated plasmin as detailed [31]. The wells of microtiter plates were coated with 50. interrogans fixed in the presence of ane% glutaraldehyde and incubated with 1 µg hPg, three µg of a plasmin chromogenic substrate (D-valyl-50-lysyl-p-nitroaniline hydrochloride) (Sigma), and fifteen ng of tissue plasminogen activator (tPA) (Sigma). Control experiments were performed by measuring the generation of plasmin in either the absence of tPA or presence of ε-ACA. Plates were incubated at RT for ii h, and optical densities were read at A ₄₅₀.

Deposition of fibrin in jellified matrices

The fibrinolysis assay was performed equally described [38,43] with small-scale modifications. Briefly, x⁷ L. interrogans cells were preincubated with Pg (l µg) for 3 h in the presence or absence of tPA (l ng) in a final volume of 1 ml. Thereafter, the mixtures were washed three times with PBS to remove gratis Pg molecules. The resulting cell pellets were placed in wells of a fibrin substrate gel matrix that contained one.25% low-melting-temperature agarose, hPg (100 µg), fibrinogen (four mg), and thrombin in a final volume of 2 ml. Controls consisted of untreated cells (without Pg) or no cells. The jellified matrix was incubated in a humidified chamber at 37°C for 8 h. Plasmin activity was detected past the ascertainment of articulate hydrolysis haloes within the opaque jellified-fibrin-containing matrix and recorded by a Catechism Rebel T2 digital photographic camera.

Detection of enolase on the microbial surface and its interaction with outer membrane proteins

For detection of enolase on the surface of intact L. interrogans, microtiter plates were coated with intact or lysed L. interrogans (ten⁹/ml). Later on blocking nonspecific sites with BSA, indistinguishable wells were separately incubated with antibodies confronting recombinant versions of enolase, LipL31, or LipL32. Leap antibody was detected using HRP-labeled secondary antibodies and TMB substrate for color development. For cess of direct enolase interaction with OM proteins, L. interrogans were fractionated into OM vesicle (OMV) and PC fractions and solubilized, and poly peptide preparations were jump to microtiter plates past overnight incubation at four°C in PBS. Afterward blocking in 1% BSA, fixed or increasing concentrations of recombinant enolase were added to the wells. After i hour of incubation at RT, the wells were done in PBS with 0.05% Tween 20 followed by incubation with primary (anti-enolase) and secondary detection antibodies and developed with TMB substrate.

Enzymatic assays

Enolase activity was adamant by measuring the transformation of NADH۰H⁺ to NAD⁺, as described elsewhere [38,44] with the following minor modifications. Briefly, the enzymatic reactions were performed at 25°C in 100 mM HEPES buffer pH 7.4 containing 500 mM MgSO₄ with 2 K KCl, 56 mM 2-phosphoglycerate solution (2-PGE) (Sigma), seven mM β-NADH (Sigma), 20 mM ADP (Sigma), lactate dehydrogenase/pyruvate kinase (PK/LDH Enzyme Solution, Sigma), and ane.six µg of the protein in a final reaction volume of 200 µl. Enolase activity was measured in terms of the charge per unit of reduction in the absorbance at 340 nm (i.e. increment in the production of NAD from NADH). For kinetic analyses, reactions were performed in 100 mM HEPES buffer pH vii.0, x mM MgSO_iv, and 7.7 mM KCl and using varied concentrations (one to 6 mM) of ii-PGE in a terminal volume of 200 µl. Changes in the absorbance per minute were measured at 340 nm at ii min intervals for a period of twenty min.

Surface enolase activeness of intact Fifty. interrogans cells

The enolase activeness of intact L. interrogans or East. coli (negative control) cells was measured by a direct analysis as described previously [38,44]. Briefly, bacteria were centrifuged and washed 3 times in a reaction buffer (100 mM HEPES, pH seven.0, x mM MgSO₄, vii.7 mM KCl) with or without 2-phosphoglycerate (two-PGE) in a final volume of 200 µl. Following 5 min of incubation at RT, bacteria were removed by centrifugation (10,000 rpm for ane min), and enzymatic activeness in the supernatants was measured at A ₃₄₀ nm for the product of phosphoenolpyruvate.

Statistical analysis

Results were presented as means (±) standard mistake mean. Statistical comparisons were performed using Educatee's t examination. Statistical significance was accustomed for P <0.05 or lower values.

Results

L. interrogans serovar Lai binds and activates plasminogen

The interaction betwixt L. interrogans serovar Lai and human plasminogen (hPg) was investigated in order to identify a potential Pg receptor. To accomplish this, intact L. interrogans cells were fixed onto microtiter wells, incubated with increasing concentrations of hPg, and jump proteins were detected using secondary antibodies. The results show that hPg binds L. interrogans in a concentration-dependent style (Figure 1A). Such 50. interrogans-hPg interaction also leads to the activation of plasmin, as in the presence of tissue plasminogen activator (tPA), spirochetes promoted the degradation of a chromogenic substrate (Figure 1B) that was blocked with the addition of a known Pg inhibitor, a lysine analogue (ε-ACA). Similarly, ε-ACA also reduced L. interrogans-hPg interaction (Figure 1C), suggesting an involvement of lysine residues of well-known Pg receptors, such as enolase. Additionally, every bit fibrinogen is ane of the major plasmin substrates in vivo, and jellified matrices containing fibrinogen accept been used to appraise plasmin action [43,45], nosotros adjacent evaluated whether the association of L. interrogans with Pg and tPA promotes fibrinolysis. As shown in Figure 1D, spirochetes readily promoted the lysis of fibrinogen, which was not detectable in the absenteeism of either hPg or tPA. Taken together, the to a higher place serial of assays indicated a specific interaction between hPg and 50. interrogans cells, which leads to the activation of plasmin and subsequent deposition of fibrinogen.

Effigy 1. Plasminogen (Pg) binds 50. interrogans enolase and activates plasmin.

The error bars indicate the standard deviations from three independent experiments performed in triplicate. * P < 0.05. (A) Pg binds to L. interrogans in a concentration-dependent manner. Microtiter plates were coated with fixed cells and incubated with various concentrations of human plasminogen (hPg). (B) Fifty. interrogans converts Pg into plasmin in the presence of tissue plasminogen (tPA). Well-leap L. interrogans cells were incubated with hPg (1µg) and a chromogenic substrate (D-valyl-L-lysyl-p-nitroaniline hydrochloride) in the presence or absence of tPA and a known Pg inhibitor, a lysine analogue, ε-ACA. (C) A Pg inhibitor blocks L. interrogans-hPg interaction. Microtiter plates were coated with stock-still L. interrogans and increasing concentrations (0 to 100 mM) of ε-ACA were incubated with a stock-still amount (1µg) of hPg. (D) Fibrinolytic activeness of Pg-bound Fifty. interrogans. Panels represent L. interrogans cells in the presence or absence of hPg and tPA. Arrow denotes fibrinolytic activity of spirochetes only in the presence of both hPg and tPA.

https://doi.org/ten.1371/journal.pone.0078150.g001

Enolase Specifically Interacts with Plasminogen

50. interrogans gene product LA1951 is annotated as enolase. As our previous experiments (Figure 1B and C) suggested that enolase, a well-known Pg receptor found on the surface of other pathogenic bacteria, is potentially involved in L. interrogans-hPg interaction, we adjacent assessed the ability of recombinant enolase to bind hPg direct. A dose-dependent increase in the amount of bound hPg was observed when increasing amounts of hPg were added to immobilized recombinant enolase (Figure 2A). Inversely, enolase also leap to immobilized hPg and such interaction was significantly inhibited past a known competitor, a lysine analogue ε-ACA (Figure 2B), suggesting that the exposed lysine residue(due south) in enolase are likely responsible for its interaction with hPg. Soluble recombinant enolase (Figure 2C) too as polyclonal enolase antibodies (Figure 2D) both competitively reduced the interaction between hPg and intact L. interrogans cells. Similarly, antibodies against an arable OM protein, LipL32, which has as well been reported to have Pg-binding power [19], were able to reduce Leptospira-hPg interaction. Notably, compared to with LipL32 antibodies, the magnitude of inhibition of hPg-L. interrogans interaction was greater in the instance of anti-enolase antibodies (Figure 2nd), suggesting enolase may be a more predominant Pg receptor in L. interrogans.

Figure 2. Interaction between human being Pg and Fifty. interrogans enolase.

The error bars point the standard deviations from iii independent experiments performed in triplicates, * P <0.05. (A) Pg directly binds to immobilized recombinant enolase. Various concentrations of hPg were incubated with a stock-still corporeality (1 μg) of enolase immobilized on microtiter wells, and detected using Pg antibodies. (B) Recombinant enolase directly binds to immobilized Pg. Various concentrations of enolase were incubated with a stock-still amount (1 μg) of hPg immobilized on microtiter wells in the absence or presence of 50 or 100 mM ε-ACA. (C) Recombinant enolase competitively inhibits bounden of L. interrogans to Pg. Microtiter plates were coated with stock-still 50. interrogans and incubated with increasing amounts of enolase. (D) Enolase antibody significantly inhibits L. interrogans binding to Pg. Microtiter plates were coated with fixed L. interrogans and incubated in the absence or presence of anti-enolase or LipL32 antibodies prior to the improver of hPg (1µg/well). Normal mouse serum (NMS) was used equally a control.

https://doi.org/10.1371/journal.pone.0078150.g002

Enolase is secreted extracellularly past Fifty. interrogans

To function as a Pg receptor, a microbial ligand must exist associated with its jail cell surface. Nonetheless, like to surface-exposed enolases in other pathogenic microorganisms [38,46,47], leptospiral enolase likewise lacks an amino-concluding leader peptide. To determine the localization of enolase in 50. interrogans, nosotros separated OM vesicle (OMV) and PC fractions from intact cells and used them in Western absorb analyses. While a known OM poly peptide, LipL32, was readily detectable in the OM fraction, enolase or an inner membrane poly peptide, LipL31, remained undetectable in the OM fraction (Figure 3A). However, unlike LipL31, enolase was strongly detected in immunoblots of supernatants isolated from intact feasible L. interrogans culture (Figure 3B), suggesting that enolase is secreted extracellularly.

Figure 3. Cellular localization of L. interrogans enolase.

(A) Subcellular localization of enolase. Fifty. interrogans whole cells (WC) were separated into outer membrane (OM) and protoplasmic cylinder (PC) fractions, resolved by SDS-Folio, and immunoblotted with antibodies specific for enolase or proteins known to localize in the OM (LipL32) or in the inner membrane (LipL31). (B) Enolase is secreted extracellularly. Viability of the 50. interrogans civilisation was determined past microscopy, and the supernatant was nerveless from a culture of intact viable cells. The samples were filtered, full-bodied, and analyzed by 2nd gel electrophoresis followed by immunoblotting assays using antibodies against enolase (upper panel) or a subcellular protein LipL31 (lower panel).

https://doi.org/ten.1371/journal.pone.0078150.g003

Detection of enolase on the microbial surface and its interaction with outer membrane proteins

As extracellularly secreted enolase has been shown to reassociate with cell surfaces in other infectious bacteria [46], nosotros next assessed whether enolase also binds to the leptospiral surface by examining the ability of enolase antibodies to direct bind intact, fixed even so nonpermeabilized leptospiral cells. Enolase antibodies readily bind immobilized L. interrogans (Effigy 4A), and the binding is enhanced in a concentration-dependent manner (data not shown), suggesting that enolase is accessible at the L. interrogans surface. To further appraise the specificity of enolase localization, microtiter plates were coated with PBS or intact or lysed L. interrogans and incubated with antibodies against either enolase, a known surface poly peptide (LipL32) or a subsurface poly peptide (LipL31). Results indicated that unlike with LipL31, antibodies against both enolase and LipL32 are significantly bound to the surface of intact bacteria, suggesting potential localization of their corresponding antigens on the pathogen surface (Figure 4A). To farther understand the clan of enolase on the leptospiral surface, we next assessed whether enolase can interact with OM proteins. OMVs were purified from 50. interrogans, and solubilized proteins were coated on microtiter plates. Immobilized OM proteins were probed with excess recombinant enolase, and bound proteins were detected using enolase antibodies. Enolase bound to unidentified OM poly peptide(s) in a saturable mode (Figure 4B).

Effigy 4. Enolase can be detected on L. interrogans surfaceand specifically interacts with outer membrane proteins.

(A) Detection of enolase on the surface of intact 50. interrogans. Microtiter plates were coated in the absence or presence of intact or lysed L. interrogans (10^nine/ml) and incubated with enolase antibody. Leap antibody was detected using HRP-labeled secondary antibodies and TMB substrate for color development, which was recorded at A ₄₅₀. A known surface-exposed and outer membrane protein, LipL32, and a subsurface inner membrane poly peptide, LipL31, were used as controls. (B) Interaction of enolase with OM proteins. A stock-still amount (1µg) of solubilized proteins from isolated OM vesicles were coated on microtiter plates and assessed for binding with increasing amount of recombinant enolase, as described in panel B. The binding of enolase to immobilized OM proteins reached saturation at 5 µg, equally there is a significant increase (P < 0.001) in individual OD values between 0-5 µg, while the difference between 5 and x µg values is nonsignificant (P > 0.05). (C) Recognition of recombinant enolase by infected hamster serum as assessed by immunoblotting. Recombinant enolase was probed with antibodies produced in immunized mice or antiserum collected from hamster infected with L. interrogans. The pointer indicates the position of enolase. Migration of protein standards is shown to the left.

https://doi.org/10.1371/journal.pone.0078150.g004

In agreement with the to a higher place information suggesting extracellular secretion and surface clan of enolase, we also found that an antibody response against the antigen is readily detectable during experimental infection of hamsters with L. interrogans (Figure 4C).

Recombinant and native surface enolase retains enzymatic activities

As enolase possesses specific Pg-binding properties and is detected on the spirochete surface, we finally assessed whether Fifty. interrogans enolase retains the enzymatic activity integral to the glycolytic pathway. The activity of recombinant enolase was assessed by measuring the transformation of NADH۰H⁺ to NAD⁺ as described elsewhere [44]. The results show saturation of enolase activity over time (Figure 5A) and increasing substrate concentration (Figure 5B), suggesting measurable and specific enzymatic activities. To determine whether enolase retains enzymatic activity on the spirochete surface, conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP) was measured in the presence of intact L. interrogans cells. The results indicated measurable enolase activity of Fifty. interrogans cells but not of control Gram-negative bacteria (Effigy 5C).

Figure 5. Enzymatic activities of recombinant and native surface-associated L. interrogans enolase.

(A) Enolase action of the recombinant enolase is highly saturable over time. Enzyme activity was measured by recoding the catalysis of 2- phosphoglycerate to phosphoenolpyruvate for a period of 20 min using 1.6 µg of recombinant enolase. (B) Substrate-dependent saturation of enzymatic activities of recombinant enolase. Increasing concentrations of the substrate (2- phosphoglycerate) were incubated with a fixed amount (4 µg) of enolase. (C) Enolase activeness is detectable on the surface of intact 50. interrogans. Conversion of 2- phosphoglycerate to phosphoenolpyruvate was used to measure enolase activity in the presence of intact Fifty. interrogans or Due east. coli cells. The error bars indicate the standard deviations from iii independent experiments performed in triplicates, * P <0.05.

https://doi.org/10.1371/journal.pone.0078150.g005

Give-and-take

The Pg-binding holding of many pathogenic spirochetes facilitates their invasiveness, thereby supporting bacterial survival in the host [19,35,38,47-51]. Interaction of host Pg with a specific microbial surface ligand can lead to the activation of plasmin, which mediates degradation of intravascular clots and extracellular proteolysis, thus influencing a wide variety of physiological and pathological processes [ix,29,30]. Here we evidence that L. interrogans enolase specifically interacts with recombinant Pg and that the native poly peptide is secreted extracellularly by L. interrogans. The exact mechanism by which enolase is secreted past spirochetes remains enigmatic. As described in studies using other bacteria [46,52], enolase secretion might non exist a consequence of cell lysis or membrane shedding only rather through a procedure in which protein structure, such as a hydrophobic α-helical domain of enolase, is a contributing gene [52]. Involvement of a secretion system besides remains a possibility, equally the L. interrogans genome encodes for type I and II secretion-like genes [2,37,53]. In either case, our information propose that one time secreted by a yet-unknown mechanism, enolase probably localizes on the bacterial surface by reassociation. Although the nature of secreted enolase binding to the L. interrogans surface and the identity of the cellular receptor remain interesting subjects of time to come investigation, a contempo study involving Streptococcus pyogenes raises an intriguing possibility that cell surfaces play a office in enolase-Pg interaction [54]. The interaction of enolase with the prison cell surface is thought to produce a conformation of enolase capable of binding to host plasminogen. Despite its ability to interact with a ligand, L. interrogans enolase, either in recombinant form or as the native surface-associated protein, retains measurable enzymatic activities; this is an expected finding, as ClustalW analyses of enolase sequences from various microorganisms (data not shown) as well show that L. interrogans enolase retains the motif 'SHRSGETED' integral to its catalytic properties [45]. Whether L. interrogans uses the glycolytic pathway as a source of free energy [37,55] or how the enzymatic activity of enolase contributes to leptospiral physiology, even so, remains unknown. Although the biological significance of enolase-Pg interaction in leptospiral virulence remains to be studied, our data showing the generation of enolase-specific antibiotic responses in infected hosts as well as extracellular or microbial surface-associated localization of enolase suggest that the protein may facilitate the pathogen's infection in the host.

Microbial-Pg interaction has been shown to assist pathogens in establishing infection in hosts [31,38,48,49,56-58]. Our initial search for potentially common virulence factors in pathogenic spirochetes infecting humans focused on enolase, which is a relatively conserved antigen among many species. Enolase has been shown to role as a Pg receptor on the jail cell surface of a diversity of other pathogens [38,44,45,58-sixty]. The interactions of pathogenic spirochetes L. interrogans, B. burgdorferi, and T. denticola with Pg has been studied [35,38,47,49,l,61]; especially in the example of Lyme illness spirochetes, enolase-Pg interaction has been suggested to back up B. burgdorferi survival in the vector [38]. However, potential contribution of enolase in Fifty. interrogans-Pg interaction and in infectivity remains unexplored. Our current studies showing a dose-dependent inhibition of the Fifty. interrogans-Pg bounden activity by enolase antibodies or directly past recombinant enolase suggested that this antigen is a predominant Pg-binding protein in L. interrogans and too confirmed the high specificity of such interaction. The binding of Pg to its receptors is mediated by its five kringle domains, which have an affinity for lysine residues [29], and in fact, lysine-dependent bounden is a salient feature of pathogen-Pg interaction [44]. Hither, nosotros show that the lysine analogue εACA and recombinant enolase significantly inhibited L. interrogans-Pg binding, suggesting the importance of kringle domains in such host-pathogen interaction. In add-on, previous work on S. pneumoniae enolase revealed that Pg binding is mediated non only past C-terminal lysine residues only besides by an internal Pg-binding motif with the sequence FYDKERKVY located betwixt the amino acids 248 and 256, although full conservation of this motif is non required for optimal bounden [33,62]. Notably, ClustalW analyses revealed the presence of the internal motif FYDKSKKKY located between the amino acids 251 and 259 in L. interrogans enolase (data not shown). Thus, we hypothesized that L. interrogans binds Pg via surface enolase, which facilitates conversion of bound Pg into plasmin, thereby armoring the pathogen with the potential ability to degrade fibrin and efficiently disseminate within hosts, as reported in other microorganisms [45].

Many microbial virulence factors are cell surface proteins that mediate pathogen interaction with specific host molecules. Appropriately, leptospiral surface proteins are likely to facilitate host jail cell-pathogen interaction [2] and thus contribute to virulence. Although, how enolase potentially contributes to L. interrogans virulence via host-Pg interaction remains a puzzling question. Leptospiral enolase lacks a recognizable leader peptide and could not readily be detectable in isolated OM preparations; yet, nosotros present show that the protein is secreted extracellularly. Notably, using specific antibodies, native enolase tin can exist detected on the Fifty. interrogans surface, and recombinant enolase specifically interacts with OM protein(s). These observations strongly suggest a potential reassociation of the protein with the pathogen surface. In other invasive pathogens, such as in S. pneumoniae, enolase is too secreted and tin reassociate by interacting with receptors on the pneumococci surface [33] via Pg interaction to facilitate infection. Therefore, we speculate that L. interrogans enolase, either as an anchorless protein or via its potential reassociation with the microbial surface, interacts with host Pg, aiding tissue invasion by L. interrogans. However, why pathogenic Fifty. interrogans strains, such every bit serovar Copenhageni, are shown to produce many additional Pg-binding proteins, such every bit LipL32, LIC10494, LIC12730, Lp29, Lp49, LipL40, MPL36, and LIC12238 [19,35], is perplexing. Arguably, such a large cohort of microbial ligands likely results in higher analogousness of the spirochetes towards host Pg. As sure pathogenic bacteria differentially produce surface antigens in specific environments that contribute to their survival [4,63], we speculate that evolution of a various repertoire of Pg receptors in pathogenic Leptospira could be linked to the ability of this remarkable and highly invasive pathogen to infect multiple hosts or a variety of tissues within the same host, facilitating dissemination and colonization in a wide array of environments.

Acknowledgments

Nosotros sincerely give thanks David A. Haake for providing us the L. interrogans serovar Lai strain used in the current report. We also thank Organized religion Kung and Xiuli Yang for their assistance with the study.

Author Contributions

Conceived and designed the experiments: SVN UP. Performed the experiments: SVN BB AS JQ. Analyzed the data: SVN JQ UP. Contributed reagents/materials/assay tools: JQ EW AK. Wrote the manuscript: SVN Up.

References

1. 1. Adler B, de la Peña Moctezuma A (2010) Leptospira and leptospirosis. Vet Microbiol 140: 287-296. doi:https://doi.org/ten.1016/j.vetmic.2009.03.012. PubMed: 19345023.
   • View Article
   • Google Scholar
2. ii. Ko AI, Goarant C, Picardeau M (2009) Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol 7: 736-747. doi:https://doi.org/10.1038/nrmicro2208. PubMed: 19756012.
   • View Article
   • Google Scholar
3. 3. Levett PN (2001) Leptospirosis. Clin Microbiol Rev 14: 296-326. doi:https://doi.org/10.1128/CMR.14.ii.296-326.2001. PubMed: 11292640.
   • View Commodity
   • Google Scholar
4. 4. Haake DA, Dundoo M, Cader R, Kubak BM, Hartskeerl RA et al. (2002) Leptospirosis, h2o sports, and chemoprophylaxis. Clin Infect Dis 34: e40-e43. doi:https://doi.org/10.1086/339942. PubMed: 11941571.
   • View Article
   • Google Scholar
5. 5. Palaniappan RU, Ramanujam S, Chang YF (2007) Leptospirosis: pathogenesis, immunity, and diagnosis. Curr Opin Infect Dis xx: 284-292. doi:https://doi.org/10.1097/QCO.0b013e32814a5729. PubMed: 17471039.
   • View Article
   • Google Scholar
6. half-dozen. Cinco G (2010) New insights into the pathogenicity of leptospires: evasion of host defences. New Microbiol 33: 283-292. PubMed: 21213586.
   • View Article
   • Google Scholar
7. 7. Ko AI, Galvão Reis M, Ribeiro Dourado CM, Johnson WD Jr., Riley LW (1999) Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet 354: 820-825. doi:https://doi.org/x.1016/S0140-6736(99)80012-9. PubMed: 10485724.
   • View Article
   • Google Scholar
8. 8. Reis RB, Ribeiro GS, Felzemburgh RD, Santana FS, Mohr Due south et al. (2008) Touch on of environs and social gradient on Leptospira infection in urban slums. PLoS Negl Trop. Drosophila Inf Serv ii: e228.
   • View Article
   • Google Scholar
9. nine. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM et al. (2003) Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 3: 757-771. doi:https://doi.org/10.1016/S1473-3099(03)00830-2. PubMed: 14652202.
   • View Article
   • Google Scholar
10. 10. Levett PN, Co-operative SL, Whittington CU, Edwards CN, Paxton H (2001) Two methods for rapid serological diagnosis of astute leptospirosis. Clin Diagn Lab Immunol 8: 349-351. PubMed: 11238220.
    • View Article
    • Google Scholar
11. 11. Palaniappan RU, Chang YF, Chang CF, Pan MJ, Yang CW et al. (2005) Evaluation of lig-based conventional and real time PCR for the detection of pathogenic leptospires. Mol Prison cell Probes 19: 111-117. doi:https://doi.org/10.1016/j.mcp.2004.10.002. PubMed: 15680212.
    • View Commodity
    • Google Scholar
12. 12. Adler B, Lo M, Seemann T, Murray GL (2011) Pathogenesis of leptospirosis: the influence of genomics. Vet Microbiol 153: 73-81. doi:https://doi.org/ten.1016/j.vetmic.2011.02.055. PubMed: 21440384.
    • View Commodity
    • Google Scholar
13. 13. Barbosa AS, Abreu PA, Neves FO, Atzingen MV, Watanabe MM et al. (2006) A newly identified leptospiral adhesin mediates attachment to laminin. Infect Immun 74: 6356-6364. doi:https://doi.org/10.1128/IAI.00460-06. PubMed: 16954400.
    • View Article
    • Google Scholar
14. fourteen. Chang YF, Chen CS, Palaniappan RU, He H, McDonough SP et al. (2007) Immunogenicity of the recombinant leptospiral putative outer membrane proteins as vaccine candidates. Vaccine 25: 8190-8197. doi:https://doi.org/10.1016/j.vaccine.2007.09.020. PubMed: 17936448.
    • View Article
    • Google Scholar
15. xv. Cullen PA, Haake DA, Adler B (2004) Outer membrane proteins of pathogenic spirochetes. FEMS Microbiol Rev 28: 291-318. doi:https://doi.org/10.1016/j.femsre.2003.10.004. PubMed: 15449605.
    • View Commodity
    • Google Scholar
16. 16. Lee SH, Kim S, Park SC, Kim MJ (2002) Cytotoxic activities of Leptospira interrogans hemolysin SphH as a pore-forming protein on mammalian cells. Infect Immun 70: 315-322. doi:https://doi.org/10.1128/IAI.seventy.1.315-322.2002. PubMed: 11748197.
    • View Article
    • Google Scholar
17. 17. Merien F, Truccolo J, Baranton G, Perolat P (2000) Identification of a 36-kDa fibronectin-binding protein expressed past a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae. FEMS Microbiol Lett 185: 17-22. doi:https://doi.org/10.1111/j.1574-6968.2000.tb09034.10. PubMed: 10731601.
    • View Article
    • Google Scholar
18. 18. Ristow P, Bourhy P, da Cruz McBride FW, Figueira CP, Huerre One thousand et al. (2007) The OmpA-similar protein Loa22 is essential for leptospiral virulence. PLOS Pathog 3: e97. doi:https://doi.org/x.1371/periodical.ppat.0030097. PubMed: 17630832.
    • View Article
    • Google Scholar
19. 19. Vieira ML, Atzingen MV, Oliveira TR, Oliveira R, Andrade DM et al. (2010) In vitro identification of novel plasminogen-binding receptors of the pathogen Leptospira interrogans. PLOS 1 5: e11259. doi:https://doi.org/10.1371/periodical.pone.0011259. PubMed: 20582320.
    • View Article
    • Google Scholar
20. 20. Cullen PA, Xu 10, Matsunaga J, Sanchez Y, Ko AI et al. (2005) Surfaceome of Leptospira spp. Infect Immun 73: 4853-4863. doi:https://doi.org/10.1128/IAI.73.8.4853-4863.2005. PubMed: 16040999.
    • View Article
    • Google Scholar
21. 21. Haake DA (2000) Spirochaetal lipoproteins and pathogenesis. Microbiology 146 ( vii): 1491-1504. PubMed: 10878114.
    • View Article
    • Google Scholar
22. 22. Nally JE, Whitelegge JP, Bassilian S, Blanco DR, Lovett MA (2007) Characterization of the outer membrane proteome of Leptospira interrogans expressed during acute lethal infection. Infect Immun 75: 766-773. doi:https://doi.org/10.1128/IAI.00741-06. PubMed: 17101664.
    • View Commodity
    • Google Scholar
23. 23. Vieira ML, Pimenta DC, de Morais ZM, Vasconcellos SA, Nascimento AL (2009) Proteome Analysis of Leptospira interrogans Virulent Strain. Open up Microbiol J 3: 69-74. doi:https://doi.org/10.2174/1874285800903010069. PubMed: 19590580.
    • View Article
    • Google Scholar
24. 24. Faisal SM, Yan W, Chen CS, Palaniappan RU, McDonough SP et al. (2008) Evaluation of protective immunity of Leptospira immunoglobulin similar poly peptide A (LigA) DNA vaccine confronting claiming in hamsters. Vaccine 26: 277-287. doi:https://doi.org/x.1016/j.vaccine.2007.x.029. PubMed: 18055070.
    • View Article
    • Google Scholar
25. 25. Cullen PA, Cordwell SJ, Bulach DM, Haake DA, Adler B (2002) Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai. Infect Immun 70: 2311-2318. doi:https://doi.org/x.1128/IAI.70.five.2311-2318.2002. PubMed: 11953365.
    • View Article
    • Google Scholar
26. 26. Murray GL, Srikram A, Hoke DE, Wunder EA Jr., Henry R et al. (2009) Major surface protein LipL32 is not required for either acute or chronic infection with Leptospira interrogans. Infect Immun 77: 952-958. doi:https://doi.org/10.1128/IAI.01370-08. PubMed: 19103763.
    • View Article
    • Google Scholar
27. 27. Grassmann AA, Félix SR, dos Santos CX, Amaral MG, Neto Seixas et al. (2012) Protection against lethal leptospirosis subsequently vaccination with LipL32 coupled or coadministered with the B subunit of Escherichia coli heat-labile enterotoxin. Clin Vaccine Immunol 19: 740-745. doi:https://doi.org/ten.1128/CVI.05720-eleven. PubMed: 22379066.
    • View Article
    • Google Scholar
28. 28. Rijken DC, Lijnen 60 minutes (2009) New insights into the molecular mechanisms of the fibrinolytic arrangement. J Thromb Haemost 7: four-thirteen. doi:https://doi.org/10.1111/j.1538-7836.2008.03220.x. PubMed: 19017261.
    • View Commodity
    • Google Scholar
29. 29. Plow EF, Herren T, Redlitz A, Miles LA, Hoover-Plow JL (1995) The jail cell biology of the plasminogen system. FASEB J 9: 939-945. PubMed: 7615163.
    • View Article
    • Google Scholar
30. 30. Vassalli JD, Sappino AP, Belin D (1991) The plasminogen activator/plasmin system. J Clin Invest 88: 1067-1072. doi:https://doi.org/10.1172/JCI115405. PubMed: 1833420.
    • View Commodity
    • Google Scholar
31. 31. Mundodi V, Kucknoor AS, Alderete JF (2008) Immunogenic and plasminogen-bounden surface-associated alpha-enolase of Trichomonas vaginalis. Infect Immun 76: 523-531. doi:https://doi.org/10.1128/IAI.01352-07. PubMed: 18070902.
    • View Article
    • Google Scholar
32. 32. Miles LA, Dahlberg CM, Plescia J, Felez J, Kato Thou et al. (1991) Office of jail cell-surface lysines in plasminogen binding to cells: identification of blastoff-enolase as a candidate plasminogen receptor. Biochemistry 30: 1682-1691. doi:https://doi.org/10.1021/bi00220a034. PubMed: 1847072.
    • View Article
    • Google Scholar
33. 33. Bergmann S, Rohde M, Chhatwal GS, Hammerschmidt S (2001) alpha-Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface. Mol Microbiol 40: 1273-1287. doi:https://doi.org/10.1046/j.1365-2958.2001.02448.ten. PubMed: 11442827.
    • View Article
    • Google Scholar
34. 34. Crowe JD, Sievwright IK, Auld GC, Moore NR, Gow NA et al. (2003) Candida albicans binds human plasminogen: identification of 8 plasminogen-binding proteins. Mol Microbiol 47: 1637-1651. doi:https://doi.org/10.1046/j.1365-2958.2003.03390.10. PubMed: 12622818.
    • View Article
    • Google Scholar
35. 35. Vieira ML, Vasconcellos SA, Gonçales AP, de Morais ZM, Nascimento AL (2009) Plasminogen acquisition and activation at the surface of Leptospira species lead to fibronectin degradation. Infect Immun 77: 4092-4101. doi:https://doi.org/10.1128/IAI.00353-09. PubMed: 19581392.
    • View Article
    • Google Scholar
36. 36. Verma A, Brissette CA, Bowman AA, Shah ST, Zipfel PF et al. (2010) Leptospiral endostatin-similar protein A is a bacterial cell surface receptor for man plasminogen. Infect Immun 78: 2053-2059. doi:https://doi.org/x.1128/IAI.01282-09. PubMed: 20160016.
    • View Article
    • Google Scholar
37. 37. Ren SX, Fu G, Jiang XG, Zeng R, Miao YG et al. (2003) Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing. Nature 422: 888-893. doi:https://doi.org/x.1038/nature01597. PubMed: 12712204.
    • View Article
    • Google Scholar
38. 38. Nogueira SV, Smith AA, Qin JH, Pal U (2012) A surface enolase participates in Borrelia burgdorferi-plasminogen interaction and contributes to pathogen survival inside feeding ticks. Infect Immun 80: 82-90. doi:https://doi.org/10.1128/IAI.05671-11. PubMed: 22025510.
    • View Article
    • Google Scholar
39. 39. Pal U, Wang P, Bao F, Yang X, Samanta Southward et al. (2008) Borrelia burgdorferi basic membrane proteins A and B participate in the genesis of Lyme arthritis. J Exp Med 205: 133-141. doi:https://doi.org/ten.1084/jem.20070962. PubMed: 18166585.
    • View Article
    • Google Scholar
40. 40. Haake DA, Chao One thousand, Zuerner RL, Barnett JK, Barnett D et al. (2000) The leptospiral major outer membrane poly peptide LipL32 is a lipoprotein expressed during mammalian infection. Infect Immun 68: 2276-2285. doi:https://doi.org/x.1128/IAI.68.4.2276-2285.2000. PubMed: 10722630.
    • View Article
    • Google Scholar
41. 41. Promnares Thou, Kumar 1000, Shroder DY, Zhang X, Anderson JF et al. (2009) Borrelia burgdorferi small lipoprotein Lp6.6 is a member of multiple protein complexes in the outer membrane and facilitates pathogen manual from ticks to mice. Mol Microbiol 74: 112-125. doi:https://doi.org/10.1111/j.1365-2958.2009.06853.10. PubMed: 19703109.
    • View Article
    • Google Scholar
42. 42. Yang Ten, Promnares K, Qin J, He M, Shroder DY et al. (2011) Characterization of multiprotein complexes of the Borrelia burgdorferi outer membrane vesicles. J Proteome Res ten: 4556-4566. doi:https://doi.org/x.1021/pr200395b. PubMed: 21875077.
    • View Article
    • Google Scholar
43. 43. Jong AY, Chen SH, Stins MF, Kim KS, Tuan TL et al. (2003) Bounden of Candida albicans enolase to plasmin(ogen) results in enhanced invasion of human brain microvascular endothelial cells. J Med Microbiol 52: 615-622. doi:https://doi.org/10.1099/jmm.0.05060-0. PubMed: 12867553.
    • View Article
    • Google Scholar
44. 44. Pancholi V, Fischetti VA (1998) alpha-enolase, a novel strong plasmin(ogen) binding poly peptide on the surface of pathogenic streptococci. J Biol Chem 273: 14503-14515. doi:https://doi.org/10.1074/jbc.273.23.14503. PubMed: 9603964.
    • View Article
    • Google Scholar
45. 45. Agarwal Southward, Kulshreshtha P, Bambah Mukku D, Bhatnagar R (2008) alpha-Enolase binds to human plasminogen on the surface of Bacillus anthracis. Biochim Biophys Acta 1784: 986-994. doi:https://doi.org/10.1016/j.bbapap.2008.03.017. PubMed: 18456007.
    • View Article
    • Google Scholar
46. 46. Chhatwal GS (2002) Anchorless adhesins and invasins of Gram-positive leaner: a new class of virulence factors. Trends Microbiol x: 205-208. doi:https://doi.org/10.1016/S0966-842X(02)02351-X. PubMed: 11973142.
    • View Article
    • Google Scholar
47. 47. Floden AM, Watt JA, Brissette CA (2011) Borrelia burgdorferi enolase is a surface-exposed plasminogen binding protein. PLOS ONE half-dozen: e27502. doi:https://doi.org/10.1371/journal.pone.0027502. PubMed: 22087329.
    • View Article
    • Google Scholar
48. 48. Coleman JL, Gebbia JA, Piesman J, Degen JL, Bugge Th et al. (1997) Plasminogen is required for efficient broadcasting of Borrelia burgdorferi in ticks and for enhancement of spirochetemia in mice. Cell 89: 1111-1119. doi:https://doi.org/10.1016/S0092-8674(00)80298-half dozen. PubMed: 9215633.
    • View Article
    • Google Scholar
49. 49. Coleman JL, Sellati TJ, Testa JE, Kew RR, Furie MB et al. (1995) Borrelia burgdorferi binds plasminogen, resulting in enhanced penetration of endothelial monolayers. Infect Immun 63: 2478-2484. PubMed: 7790059.
    • View Article
    • Google Scholar
50. 50. Fenno JC, Tamura M, Hannam PM, Wong GW, Chan RA et al. (2000) Identification of a Treponema denticola OppA homologue that binds host proteins present in the subgingival surround. Infect Immun 68: 1884-1892. doi:https://doi.org/x.1128/IAI.68.four.1884-1892.2000. PubMed: 10722578.
    • View Article
    • Google Scholar
51. 51. Siqueira GH, Atzingen MV, Alves IJ, de Morais ZM, Vasconcellos SA et al. (2013) Characterization of Three Novel Adhesins of Leptospira interrogans. Am J Trop Med Hyg. PubMed: 23958908.
52. 52. Yang CK, Ewis HE, Zhang X, Lu CD, Hu HJ et al. (2011) Nonclassical protein secretion by Bacillus subtilis in the stationary phase is not due to cell lysis. J Bacteriol 193: 5607-5615. doi:https://doi.org/ten.1128/JB.05897-11. PubMed: 21856851.
    • View Article
    • Google Scholar
53. 53. Zeng L, Zhang Y, Zhu Y, Yin H, Zhuang X et al. (2013) Extracellular Proteome Analysis of Leptospira interrogans serovar Lai. Omics. [Epub alee of print]. doi:https://doi.org/10.1089/omi.2013.0043. PubMed: 23895271.
    • View Article
    • Google Scholar
54. 54. Kornblatt MJ, Kornblatt JA, Hancock MA (2011) The interaction of canine plasminogen with Streptococcus pyogenes enolase: they bind to one another merely what is the nature of the structures involved? PLOS ONE 6: e28481. doi:https://doi.org/10.1371/journal.pone.0028481. PubMed: 22174817.
    • View Article
    • Google Scholar
55. 55. Nascimento AL, Ko AI, Martins EA, Monteiro-Vitorello CB, Ho PL et al. (2004) Comparative genomics of 2 Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. J Bacteriol 186: 2164-2172. doi:https://doi.org/x.1128/JB.186.7.2164-2172.2004. PubMed: 15028702.
    • View Commodity
    • Google Scholar
56. 56. Bergmann S, Hammerschmidt Due south (2007) Fibrinolysis and host response in bacterial infections. Thromb Haemost 98: 512-520. PubMed: 17849039.
    • View Commodity
    • Google Scholar
57. 57. Goto H, Kawaoka Y (1998) A novel mechanism for the conquering of virulence by a human flu A virus. Proc Natl Acad Sci U S A 95: 10224-10228. doi:https://doi.org/10.1073/pnas.95.17.10224. PubMed: 9707628.
    • View Article
    • Google Scholar
58. 58. Nogueira SV, Fonseca FL, Rodrigues ML, Mundodi V, Abi-Chacra EA et al. (2010) Paracoccidioides brasiliensis enolase is a surface protein that binds plasminogen and mediates interaction of yeast forms with host cells. Infect Immun 78: 4040-4050. doi:https://doi.org/10.1128/IAI.00221-10. PubMed: 20605975.
    • View Commodity
    • Google Scholar
59. 59. López-Villar E, Monteoliva L, Larsen MR, Sachon Due east, Shabaz Yard et al. (2006) Genetic and proteomic evidences back up the localization of yeast enolase in the jail cell surface. Proteomics 6 Suppl 1: S107-S118. doi:https://doi.org/10.1002/pmic.200500479. PubMed: 16544286.
    • View Article
    • Google Scholar
60. 60. Pancholi V (2001) Multifunctional alpha-enolase: its office in diseases. Cell Mol Life Sci 58: 902-920. doi:https://doi.org/10.1007/PL00000910. PubMed: 11497239.
    • View Article
    • Google Scholar
61. 61. Toledo A, Coleman JL, Kuhlow CJ, Crowley JT, Benach JL (2012) The enolase of Borrelia burgdorferi is a plasminogen receptor released in outer membrane vesicles. Infect Immun eighty: 359-368. doi:https://doi.org/10.1128/IAI.05836-11. PubMed: 22083700.
    • View Article
    • Google Scholar
62. 62. Stie J, Bruni G, Play tricks D (2009) Surface-associated plasminogen binding of Cryptococcus neoformans promotes extracellular matrix invasion. PLOS ONE iv: e5780. doi:https://doi.org/10.1371/journal.pone.0005780. PubMed: 19492051.
    • View Article
    • Google Scholar
63. 63. Velineni S, Asuthkar South, Sritharan K (2006) Fe limitation and expression of immunoreactive outer membrane proteins in Leptospira interrogans serovar icterohaemorrhagiae strain Lai. Indian J Med Microbiol 24: 339-342. doi:https://doi.org/10.4103/0255-0857.29414. PubMed: 17185872.
    • View Commodity
    • Google Scholar

myershandep.blogspot.com

Source: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078150

Share this post