Proteomics as a tool to understand Leptospira physiology and virulence: Recent advances, challenges and clinical implications

Accepted Manuscript Proteomics as a tool to understand Leptospira physiology and virulence: Recent advances, challenges and clinical implications

Edson G. Nascimento Filho, Monica L. Vieira, Aline F. Teixeira, Jademilson C. Santos, Luis G.V. Fernandes, Felipe J. Passalia, Brenda Daroz, Amanda Rossini, Leandro T. Kochi, Maria F. Cavenague, Daniel C. Pimenta, Ana L.T.O. Nascimento PII: DOI: Reference:

S1874-3919(18)30081-2 doi:10.1016/j.jprot.2018.02.025 JPROT 3056

To appear in:

Journal of Proteomics

Received date: Revised date: Accepted date:

31 October 2017 14 February 2018 22 February 2018

Please cite this article as: Edson G. Nascimento Filho, Monica L. Vieira, Aline F. Teixeira, Jademilson C. Santos, Luis G.V. Fernandes, Felipe J. Passalia, Brenda Daroz, Amanda Rossini, Leandro T. Kochi, Maria F. Cavenague, Daniel C. Pimenta, Ana L.T.O. Nascimento , Proteomics as a tool to understand Leptospira physiology and virulence: Recent advances, challenges and clinical implications. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jprot(2017), doi:10.1016/j.jprot.2018.02.025

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ACCEPTED MANUSCRIPT "Proteomics as a tool to understand Leptospira physiology and virulence: recent advances, challenges and clinical implications"

Edson G. Nascimento Filho1,2; Monica L. Vieira1; Aline F. Teixeira1; Jademilson C.

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Santos1; Luis G.V. Fernandes1; Felipe J. Passalia1,2, Brenda Daroz1,2; Amanda

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Rossini1,2, Leandro T. Kochi1,2, Maria F. Cavenague1,2, Daniel C. Pimenta3 and Ana L.

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T. O. Nascimento1*

Laboratório Especial de Desenvolvimento de Vacinas, Instituto Butantan, Avenida

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Vital Brazil, 1500, 05503-900, São Paulo, SP, Brazil

Programa de Pos-Graduação Interunidades em Biotecniologia, Instituto de Ciencias

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Biomedicas, Universidade de Sao Paulo, Sao Paulo, Brazil Laboratório de Bioquímica e Biofísica, Instituto Butantan, Avenida Vital Brazil, 1500,

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05503-900, São Paulo, SP, Brazil

* Correspondence should be addressed to: Ana L. T. O. Nascimento - Laboratorio Especial de Desenvolvimento de Vacinas, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900, São Paulo, SP, Brazil; Phone: (5511) 26279829; e-mail: [email protected]. 2018

ACCEPTED MANUSCRIPT ABSTRACT

Leptospirosis is a severe disease that represents a significant burden to healthcare costs in the absence of prophylactic measures. There is no effective vaccine for humans. Although Leptospira genome sequencing has broadened our current knowledge of the

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life cycle and evolution of these bacteria, there is still much to be understood regarding

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the pathophysiological features of this organism. In tropical, underdeveloped regions, it is the predominant human disease. Pathogenic bacteria of the genus Leptospira are the

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etiological agents of leptospirosis. This review addresses the contributions made to our

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knowledge of leptospirosis based on proteomic strategies. We present here the advances made on several fronts in understanding the infectious agent Leptospira, from the initial

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2D-gel electrophoresis proteomics approach to more advanced mass spectrometry (MS) technologies. This review provides an update on proteomic studies of Leptospira spp.

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and should be constructive in further investigations of these bacteria, in our fight against

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Impact of our work

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leptospirosis.

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Leptospirosis is a severe disease that represents a significant burden to healthcare costs in the absence of prophylactic measures. There is no efficient vaccine for humans. This review addresses the contribution to Leptospira knowledge based on proteomic strategies. From the initial 2D- gel electrophoreses proteomics approach to more sophisticated mass spectrometry (MS) technologies, we have compiled most recent data of authors that have performed proteomic analyses on various approaches of leptospirosis, for instance: (i) authors that have assessed the infected rat urine, searching for markers; (ii) authors that have compared the proteome profiles of bacteria grown in vivo x in vitro; (iii) authors that have searched for vaccine candidates.

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ACCEPTED MANUSCRIPT This is a critical review on this subject pointing out future directions on Leptospira/leptospirosis research proteomics field, envisaging rational future work to

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increase our knowledge of these bacteria and help fight leptospirosis.

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ACCEPTED MANUSCRIPT INTRODUCTION

Leptospirosis is a zoonosis of global importance, which in recent years has been considered one of the leading emerging infectious diseases [1]. It is caused by a group of bacteria belonging to the order Spirochetales, family Leptospiracea. The genus

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Leptospira consists of pathogenic and intermediate species, which cause severe and

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mild leptospirosis, respectively, and non-pathogenic saprophytic species. There are

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more than 250 serovars identified in 24 different serogroups [2]. The Copenhageni and Icterohaemorrhagie are the most pathogenic and prevalent serovars in humans in urban

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centers [3, 4]. The incidence of human infections is mainly in tropical and subtropical countries, where conditions for transmission are favorable [3]. Infection occurs mainly

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through exposure to soil or water contaminated with urine from infected mammals. In humans, the symptoms associated with the disease range from a subclinical

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infection characterized by fever, chills, headaches, and myalgia to more severe forms,

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such as Weil’s syndrome and severe pulmonary hemorrhagic syndrome [1]. The severe manifestations of leptospirosis reach about 10 to 50% mortality in adults aged 30-40

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years [5]. According to the international agencies WHO (World Health Organization) and GBD (Global Burden of Disease) [4], it has been estimated that there are 1 million

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clinical cases of leptospirosis annually, with death rates reaching about 60,000 individuals worldwide. In Brazil, there were 63,302 confirmed clinical cases of leptospirosis and 6,064 deaths due to the disease between 2000 and 2016 [4, 6]. The clinical phases of leptospirosis are divided into two distinct stages. The early phase, also known as the leptospiremic phase, is when leptospires are found in the bloodstream. The symptoms are characterized by sudden onset of fever, headache, myalgia and vomiting; diarrhea, conjunctival hemorrhage and cough may also occur [3].

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ACCEPTED MANUSCRIPT At this clinical stage, due to similarities to other diseases, leptospirosis is frequently misdiagnosed [7]. Hence, leptospirosis is not detected - and not treated - enabling progression to a more serious clinical situation. The late phase is associated with a more serious and potentially lethal clinical manifestation such as Weil's syndrome, characterized by jaundice, renal failure and bleeding [1, 8]. The development of

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pulmonary hemorrhage during leptospirosis shows extreme severity, resulting in acute

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respiratory distress syndrome leading to the patient’s death within the first 24 hours of

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hospitalization [9].

Although leptospirosis can be treated, an early diagnosis is critical for an

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effective antibiotic therapy. The gold standard reference method for serological

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diagnosis of the disease is the microscopic agglutination test (MAT) in which sera from patients are reacted with live bacterial suspensions of leptospiral serovars [10, 11]. In addition to its complexities in controlling and interpreting the results, MAT serology

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has low sensitivity at the onset of the disease as it relies on antibodies to leptospiral antigens not detected in the first days of post-exposure [12-15].

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Presently, there are veterinarian vaccines derived from inactivated whole cell preparations of pathogenic leptospires. These vaccines provide protection, most likely

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through the induction of antibodies against leptospiral lipopolysaccharide [3, 16, 17]. However, there are drawbacks associated with this type of vaccine: it does not provide protection against leptospiral serovars not included in the preparation, and moreover, polysaccharide antigens do not induce long-term protection. Human vaccines are available in Cuba [18], France [19] and China [20], and they have the same limitations as the veterinarian ones. The large number of pathogenic serovars (>200) represents a major limitation to the production of a multi-serovar vaccine. A cost-effective, broadspectrum vaccine preparation against leptospirosis has long been sought [21]. 5

ACCEPTED MANUSCRIPT Due to their location, the outer membrane proteins (OMPs) of Leptospira facilitate direct interactions with the environment and may have important constituents involved in infection, transmission, survival, and adjustment to environmental conditions. Moreover, they are attractive vaccine candidates, having the potential to promote cross-protective immunity against different serovars of pathogenic Leptospira

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[22, 23]. Many laboratories have used various in silico or experimental tools aimed at

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identifying these proteins.

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Whole genome sequencing of different Leptospira species and serovars has been performed. This strategy has yielded substantial amounts of data, elucidating several

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aspects of leptospiral metabolism and pathogenesis [17]. To date, the complete genome

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sequencing of two pathogenic leptospiral species, including several serovars and one saprophytic strain as well, has been reported [24-28]. Besides, the characterization of 20 leptospiral species, including pathogenic, intermediate and saprophyte strains, has

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recently been published [29]. While genomics and comparative genomic analysis show

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the organism’s plasticity and contribute to a better understanding of Leptospira evolution, the presence of genes does not necessarily imply protein expression, so other

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methods should be employed. Microarrays have been used [30-34], and one study that

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focused on transcriptional and translational analysis of leptospiral OMP expression in response to temperature shift provided interesting results. In that work, a divergence was noted between protein amount and transcript levels for a certain number of proteins, suggesting that many regulatory processes remain unknown [34]. The authors of the last cited study stressed the importance of both data to be determined to draw conclusions on protein expression changes. In recent years, high-throughput RNA sequencing (RNA-Seq) has replaced this technique as the method of choice for genomewide transcriptional drafting in bacteria [35-37]. RNA-Seq has also been applied to

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ACCEPTED MANUSCRIPT pathogenic [38], saprophytic Leptospira [39] and pathogenic bacteria inside a dialysis membrane chamber (DMC) model [40]. These works have clarified some aspects of leptospiral pathogenesis, including mammalian host-adaptation, and some putative virulence targets. Nonetheless, mechanisms of virulence and pathogenesis of

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Leptospira are yet to be elucidated.

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Proteomics applied to Leptospira

Through proteomics, one can identify the proteins expressed by an organism at a

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given moment under specific conditions [41]. Accordingly, it is possible to perform quantitative, functional, structural, and post-translational proteomic characterizations.

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Through these approaches, it is also possible to determine the relationships between proteins in biological processes [42]. With this information, it is possible to identify

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putative targets with a role in the pathogenesis of various infectious diseases, including

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leptospirosis. Furthermore, proteins that are overexpressed by pathogenic strains under conditions mimicking an infection are potential candidates for the development of

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vaccines and also for clinical diagnosis [43, 44]. Many studies have been published analyzing the proteome and the secretome of

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pathogenic and nonpathogenic strains of Leptospira. In a pioneer study, twodimensional gels of extracted leptospiral OMPs were probed with monospecific antiserum and convalescent rat serum, followed by mass spectrometry (MS) analysis of the reactive protein spots (see Fig. 1) [45]. The authors reported that a significant increase in the expression of the OMP Loa22 occurred during acute infection of guinea pigs when compared to other OMPs. Quantitative proteome analysis and MS protein identification showed 42 forms of 33 unique proteins whose levels were considerably

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ACCEPTED MANUSCRIPT higher in the pathogenic serovars compared with a non-pathogenic serovar [43]. In a previous work from our laboratory, whole protein extracts of the virulent strain L. interrogans serovar Pomona cultured from kidney and liver of infected hamsters were fractionated by two-dimensional gel electrophoresis, and 895 spots were analyzed by MALDI-TOF MS. These analyses resulted in the identification of 286 protein spots,

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corresponding to 108 distinct leptospiral proteins. The most prevalent and previously

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described leptospiral OMPs, OmpL1, LipL21, LipL31, LipL32/Hap-1, LipL41, LipL45,

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LipL46, LruA/LipL71, and OmpA-like protein Loa22 were all identified by this approach [46]. In addition, we validated in this study a number of proteins first assigned

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as hypothetical in genome annotation and highlighted some proteins differentially expressed under virulent conditions. The hypothetical OMP encoded by the gene

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LIC10314, which was identified only under virulent conditions, was selected for a follow-up study. This protein, named Lsa63, contains a p83/100 domain that is

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conserved in antigens broadly distributed in spirochetes and was shown to mediate

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leptospiral adhesion to the extracellular matrix components laminin and collagen type IV, possibly mediating host-pathogen interactions during infection [47]. The metabolic

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protein elongation factor Tu (LIC12875), identified in multiple isoforms in our analysis [46], was later characterized as a moonlighting protein with outer surface localization

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and plasminogen- and factor H-binding activities [48]. The various elongation factor Tu isoforms possibly reflect post-translational modifications, which render different localization and function of the protein. Malmström and colleagues (2009) [49] published a landmark work in the proteomics of the virulent L. interrogans serovar Copenhageni. They used a strategy that combines three MS-based proteomic methods: (i) absolute quantification using isotope-labeled reference peptides, (ii) label-free quantification, and (iii) high-

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ACCEPTED MANUSCRIPT throughput proteome sequencing by liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Fig. 1). By this approach, 2,221 proteins out of 3,658 predicted ORFs from genome sequencing were identified [24, 25]. Among these 2,221 proteins, 1,864, representing 51% of the predicted proteome, were provided with estimated copy per cell numbers. Moreover, proteins that are up- or down-regulated after antibiotic treatment

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were also evaluated for copy number per cell. Many hypothetical proteins have been

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validated, including those located at the outer membrane, which may have a role in

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host-pathogen interactions. Indeed, several of these proteins of unknown function have been studied by our research group. Many of these proteins were characterized as

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extracellular matrix (ECM)-binding proteins that may have a role in adhesion/ colonization [50-52]. Proteins that are plasminogen-binding capable of generating

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plasmin, a broad-spectrum protease that degrades laminin and fibronectin, might be important in overcoming tissue barriers and facilitating leptospiral penetration [53-56].

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Furthermore, proteins that interact with components of the complement system could be

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involved in immune evasion, helping the bacteria to reach target sites [57-59]. Functional studies are currently being carried out in our laboratory with L. biflexa

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knockin mutants expressing Lsa23, a complement component-binding protein, and preliminary data show that this protein confers serum-resistance to these mutants

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compared to wild-type L. biflexa (Fernandes et al., unpublished work). Malmström and colleagues (2009) [49] also advanced the study of leptospiral OMPs [60], demonstrating that lipoproteins, like in the spirochete Borrelia, were the predominant OMPs in Leptospira. In another work, Weisser et al. (2013) [61] presented an improvement for highthroughput label-free LC–MS/MS datasets. The pipeline is composed of tools from the OpenMS software and can be applied to a large set of heterogeneous regular data of 58

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ACCEPTED MANUSCRIPT LC–MS/MS runs. Furthermore, Schmidt and colleagues (2011) [62], applying a LCMS/MS approach to detect and quantify tryptic digests of L. interrogans under 25 distinct conditions, including ciprofloxacin, doxycycline, penicillin G and serum, determined proteome changes, allowing for the identification of common and specific proteome patterns for antibiotic response and adaptation. The authors anticipated that

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high resolution LC–MS platforms will become a milestone method for systems biology

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of microorganisms.

Figure 1 shows the workflow of proteome strategies applied to leptospires, and Table 1

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summarizes the proteome approaches utilized to detect/compare expressed leptospiral

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proteins from various sources.

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Proteomics applied to vaccines and diagnostic

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Thongboonkerd (2008) [63] reviewed many studies of proteomics in leptospirosis, focusing on the identification of potential leptospiral immunogens useful

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for diagnostics and vaccine development. The proteins identified using the classical proteomics approach, MALDI-TOF-MS, ESI-Q-TOF-MS/MS, 2DE with SYPRO ruby

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stain and μLC-MS/MS and 2DE with deep purple stain and μLC-MS/MS, were mainly the more abundant, previously identified, including the seven immune-reactive spots detected by MALDI-TOF-MS. According to the author, the number of proteomic studies applied to leptospirosis is very small, compared with other fields. Most of these studies utilized 2DE-based technology, which presents major limitations for the analysis of highly hydrophobic or membrane proteins. After this publication, many studies on

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ACCEPTED MANUSCRIPT Leptospira have been performed using more sophisticated technologies and various samples aimed at representing clinical aspects of the disease. Proteomic analysis was used for the development of a potency test for L. interrogan sserovar Canicola vaccines, normally performed by using a large number of animals [64]. Moreover, these 2D-LC/MS-based techniques could also be developed for

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the quality certification stage of vaccine manufacture. Djelouadji et al. used MALDI-

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TOF-MS for the identification of Leptospira isolates at the species level [65]. These

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authors suggested that MALDI-TOF MS could be used to supplement serological and sequencing-based methods for the identification of Leptospira strains in the clinical

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laboratory, improving leptospirosis diagnosis.

The extracellular proteome of L. interrogans serovar Lai grown in protein-free

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medium was studied [66], and 66 proteins were detected by MS/MS, 33 of which were predicted to be extracellular proteins by bioinformatics analysis. Transcriptional levels

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of the corresponding genes of these 33 proteins showed that 15 were up-regulated and

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two were down-regulated in vivo, compared to in vitro conditions. The authors proposed that the extracellular proteome would complement the previous whole proteome data for

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studying proteins with a possible role in the infection process and for choosing vaccine targets or diagnostic development as well [66]. Humphryes and colleagues (2014) [67]

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characterized the proteome of L. interrogans serovar Canicola by 2D-LC/MS techniques and identified 1653 leptospiral proteins, of which 60 were present in serovars Copenhageni and Pomona, and 16 were recognized for their immunogenic capacity. The authors suggested that comparative proteomics of different serovars could be used for the identification of novel broad-spectrum vaccine candidates against leptospirosis. Zeng and coworkers (2014) [68] employed MS/MS analysis to characterize the subproteome of Triton X-114 OMP extracts aiming to identify surface-

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ACCEPTED MANUSCRIPT exposed proteins of two mildly virulent vaccine strains, JDL03 (serovar Canicola) and JDL10 (serovar Hebdomadis) and to compare to the highly virulent vaccine strain 56601 (serovar Lai). Their findings included the presence of 81 cores, 61 dispensable, 122 unique surface-exposed proteins and 10 highly conserved surface-exposed or subsurface proteins comprising two known cross-reactive antigens, LipL32 and

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peptidase M75 /imelysin, and two novel putative antigens, LipL45-like lipoprotein and

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hypothetical protein LA_0505. The authors asserted that further investigations of these

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potential virulence factors were necessary and should offer new insights into leptospiral pathogenesis. We thoroughly checked these studies and decided to construct chimeric

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proteins with known immunogenic epitopes, with the aim of developing a serovarindependent vaccine. The first construct showed partial immune-protective activity in a

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leptospirosis hamster model, and was able to recognize antibodies present in human serum samples in both phases of leptospirosis and is a potential candidate for disease

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diagnosis [69]. The second one is currently under animal challenge evaluation

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(Fernandes et al., unpublished data)

Nally et al. used leptospires shed in the urine of chronically infected rats

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compared to in vitro cultivated bacteria to understand the dynamics of host-bacteria

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interactions. The results revealed a distinct protein and antigen expression between the two sources, as shown by two-dimensional gel electrophoresis. Apparently, proteins are down-regulated in asymptomatic chronic carriers, avoiding clearance by the immune system, thus facilitating their persistence in the renal tubules of the host. The identification of proteins expressed during persistent infection will shed light on the mechanisms of chronic disease [70]. Further work by this group identified differential protein expression in the host, including membrane metalloendopeptidase, napsin A aspartic peptidase, vacuolar H+-ATPase, kidney aminopeptidase and immunoglobulins

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ACCEPTED MANUSCRIPT G and A, whereas increased protein expression in leptospires shed in urine compared to in vitro cultivated bacteria included Loa22 and GroEL [71]. The authors reinforced the notion that differentially expressed proteins either in the host or the pathogen are indeed interesting markers of infection. More recently, this group analyzed the urine of experimentally infected rats by CE-MS to identify urinary biomarkers of chronic

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infection [72]. The data revealed a selective peptide pattern of 43 polypeptides offering

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a potential diagnostic tool with high sensitivity, specificity and accuracy.

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Proteomics to understand the leptospiral infectious processes

By using iTRAQ labeling and LC-ESI-MS/MS complemented with two-

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dimensional gel electrophoresis and MALDI-TOF MS, proteome analyses of L. interrogans serovar Copenhageni were performed comparing bacteria grown under

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laboratory conditions to the ones exposed to iron deprivation and the presence of serum

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[73]. In this work, the authors identified a total of 563 proteins and showed that proteome modifications occurring under conditions that simulate in vivo infection could

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reveal novel putative leptospiral virulence factors. Proteomic data in combination with computational prediction were used to

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perform a relatively reliable genome re-annotation for the human pathogen L. interrogans serovar Lai [74]. According to the authors, the combination of global protein expression and multiple post-translational modifications will elucidate the special status of L. interrogans during evolution as a mammalian pathogen and offer further interpretations of the physiology and pathogenesis of these bacteria. They identified multiple types of post-translational modifications in leptospiral proteins, such as phosphorylation, acetylation and methylation, indicating the presence of eukaryotic-

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ACCEPTED MANUSCRIPT like modification mechanisms in L. interrogans, which opens prospects for therapeutic candidates. In a similar line of investigation, the combination of genomic and proteomic approaches was used to study the genetic variations between the virulent L. interrogans serovar Lai strain 56601 and the non-virulent culture-attenuated L. interrogans serovar Lai strain IPAV [28]. Comparative proteomic analysis based on quantitative liquid

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chromatography (LC)-MS/MS data revealed that modified expression or mutations in

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important genes, including those encoding Ser/Thr kinase, carbon-starvation protein

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CstA, glutamine synthetase, GTP-binding protein BipA, ribonucleotide diphosphate reductase and phosphate transporter, together with the translational modification of

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lipoproteins or OMPs are expected to contribute to virulence attenuation in strain IPAV. Nally and colleagues (2017) [75] used 2-dimensional differential gel electrophoresis (2-

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D DIGE) to compare the whole cell proteome of in vivo-derived leptospires from the DMC peritoneal implant model with that of in vitro-derived culture attenuated

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leptospires at 30°C and 37°C. Their results supported that differential protein post-

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translational modifications, such as methylation and acetylation, are controlled in response to infection. Their data highlighted the need to investigate the regulatory

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processes used by pathogenic leptospires to adapt to the host during infection. Posttranslational modifications were also analyzed in the saprophytic L. biflexa by a

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proteomic approach [76]. Methylation and acetylation of lysine residues were detected in the proteins of the membrane-associated fraction, whereas phosphorylation was mainly observed among soluble proteins. It seems that the free-living species L. biflexa and pathogenic species L. interrogans have post-translational modification systems conserved between them, suggesting an important functional benefit, despite their different life cycles [76].

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ACCEPTED MANUSCRIPT Proteomics can be a powerful tool to study infectious processes. Srivastava and colleagues (2012) [77] employed proteomic analysis to investigate alterations in human serum promoted by Leptospira. They used classical 2DE and 2D-DIGE in combination with MALDI-TOF/TOF MS, and the data revealed differential expression of several serum proteins in leptospirosis patients compared to healthy individuals. These proteins

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are related to several essential physiological processes and the investigation of their

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functional properties is expected to elucidate the pathophysiology of the disease.

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Moreover, it may help identifying target proteins for the detection of leptospirosis and to discriminate from other febrile infectious diseases. By combining structural,

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physical-chemical, and functional proteomic features, Lessa-Aquino and coworkers (2015) [78] identified 191 immuno-dominant protein antigens, representing the

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complete collection of antibodies generated during leptospiral infections. The authors discussed that mounting an immune response against a reduced number of antigens may

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have the benefit of minimizing energy consumption and avoiding an excessive innate

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inflammatory reaction or cross-reactive autoimmune responses. LPHS, leptospiral pulmonary hemorrhagic syndrome, is a particularly severe

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form of leptospirosis. Pathogenic mechanisms of LPHS remain unknown, hampering

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the development of therapeutic measures [3]. Using 2D-DIGE, Schuller et al. (2015) [79] compared the lung proteome of infected and non-infected guinea pigs. Their data revealed differences in the proteins contained in 130 spots. At the early phase of the disease, there was an increase in proteins of LPHS lung tissue, and may reflect a general host response. On the other hand, the decrease in proteins associated with cytoskeletal and cellular organization in LPHS lung tissue suggests that during leptospiral infection, changes occur in the abundance of host proteins involved in cellular architecture and

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ACCEPTED MANUSCRIPT adhesion, resulting in dramatically increased alveolar wall leakage as observed in LPHS.

FUTURE PERSPECTIVES

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Proteomics tools have highlighted several aspects of leptospiral physiology and

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virulence. The validation of novel hypothetical surface proteins of unknown function

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offered the opportunity to study extracellular matrix, plasminogen, and complementbinding proteins, and to assign putative functions to these proteins. Yet, functional

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studies including knockin and knockout mutants remain to be performed to determine the role of these proteins in leptospiral pathogenesis. Although several proteins have

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been evaluated in leptospirosis animal modeld, including the ones detected by proteomics, a broad-spectrum, serovar-independent vaccine against leptospirosis has not

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been found, and more studies will have to be performed. Immuno-proteomics identified

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only a few, overexpressed proteins, and no advances were made in this direction. It is anticipated that gel-free methods will gain importance in the design of large-scale

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proteomes due to the best results obtained (as seen in Table 1). Proteomics initiatives to further understand other aspects of Leptospira and

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leptospirosis are still needed, such as: (i) complete proteome of L. biflexa, similar to proteome-wide cellular protein concentrations of L. interrogans [49], which will help the identification of unique proteins involved in virulence; (ii) in-depth characterization of the secretome to gain insights into novel host-pathogen interaction strategies and secreted virulence factors, (iii) better characterization of post-translational modifications occurring during different conditions, since they contribute to a broader functional diversity than predicted based on the genome or specific physiological conditions; and

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ACCEPTED MANUSCRIPT (iv) systems biology approaches applying the integration of proteomics data with other large-scale datasets such as transcriptomics, genomics and metabolomics, to predict and characterize the dynamic properties of the Leptospira infectious process.

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CONCLUDING REMARKS

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The last decade witnessed the generation of remarkable amounts of data derived from

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proteomic approaches, including studies on protein profiles of Leptospira spp. Many groups working with leptospires under different strategies contributed in many ways to

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the better understanding of these bacteria. From 2D-gel electrophoresis to sophisticated MS platforms, a great amount of information has been compiled, increasing our

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knowledge of Leptospira protein expression, namely which proteins are actually expressed, under which conditions, and to what kind of regulation they are subjected

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(up- or down-regulation under given circumstances). Bacterial responses to different

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antibiotics provided evidence for possible defense mechanisms, and alternatives for proper treatment and/or prevention of leptospirosis. Host-pathogen interactions in

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chronically infected rats revealed differential protein expression patterns that could constitute markers for leptospirosis. Possible virulence factors involved in leptospiral

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pathogenesis were identified, constituting potential vaccine and/or diagnostic candidates that will have to be evaluated against leptospiral virulent challenge in animal models and a collection of serum samples from confirmed leptospirosis patients to validate respectively their effectiveness.

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ACCEPTED MANUSCRIPT DECLARATIONS Ethics approval and consent to participate. Not applicable. Consent for publication. Not applicable.

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Availability of date and material. Not applicable.

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Competing interests. The authors declare that they have no competing interests. Funding. The following Brazilian agencies: FAPESP (grant 14/50981-0), CNPq (grants

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302758/2013-5 and 441449/2014-0) and Fundacao Butantan, financially supported this

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work; AFT, LGVF and MLV have post-doctoral fellowships from FAPESP (2016/11541-0; 2017/06731-8) and CNPq, respectively. LTK, FJP, MFC and BD have

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MSc and PhD FAPESP fellowships (2016/01384-5, 2017/01102-2, 2016/04295-3 and 2017/01411-5, respectively); AR has MSc CAPES fellowship. The funders had no role

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in study design, data collection and analysis, decision to publish, or preparation of the

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manuscript.

Authors’ contributions. All authors participated in the literature revision, discussion

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and preparation of manuscript, including table and figure.

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Acknowledgments. We are grateful to all the members for support on this project. We are indebted to Albert Leyva for English editing this manuscript. Authors’ information. EGN, the first author. ALTON, the corresponding author.

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Figure 1. Protein profiling workflow, including sample sources, protein preparation,

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mass spectrometry and data analysis tools utilized to study Leptospira spp..

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Table 1. Compilation of the main studies that have contributed to research of leptospirosis proteomics. Method

Species/Serovar

Sample

2DE

L. interrogans serovar Copenhageni L. kirschneri serovar Bim

Serum

2DGE/ ESI-MS MALDI-TOF-MS

L. interrogans serovar Lai

OMP

D E

PT

2DE/ LC-MS ESI-MS

2DE MALDI-TOF-MS

T P

I R

Serum samples from leptospirosis patients X other diseases and healthy controls

Guerreiro et al., 2001[80]

37

Outer membrane isolated from leptospires grown at 20ºC, 30ºC or 37ºC in the presence of 10% fetal calf serum and iron-depleted medium

Cullen et al., 2002 [81]

C S U

N A

Reference

7

M

L. interrogans serovar Copenhageni RJ15958

OMVs

40

Outer membrane vesicles isolated from leptospires grown under in vitro conditions

Nally et al., 2005 [23]

4 Leptospira species

Whole-cell

8

Leptospires grown under in vitro conditions in EMJH medium supplemented with 1% fetal bovine serum intact leptspiral cells X sonicated leptospires

Cullen et al., 2005 [82]

E C

AC

Number of Objective Identified Proteins

ACCEPTED MANUSCRIPT

Kositanont et al., 2007 [44]

2DE MALDI-TOF-MS

L. interrogans serovars Australis, Autumnalis, Bataviae, and Bratislava L. biflexa serovar Patoc

Serum

24

Serum samples from leptospirosis patients MAT+X serum samples from nonleptospirosis patients

2DE MS/MS

L. interrogans serovar Copenhageni RJ16441

OMPs

29

Extracted leptospires from infected guinea pig liver X Leptospires grown under in vitro conditions

Nally et al., 2007 [45]

2DE

L. interrogans serovar Copenhageni RJ16441

Urine

Rat urine-isolated leptospires X in vitro-cultivated leptospires

Monahan et al.,

iTRAQ/HPLC LC-MS/MS ESI-MS/MS

L. interrogans serovar Lai

OMPs

1026

Outer membrane isolated from leptospires grown at 30ºC X Outer membrane isolated from leptospires grown at 37ºC

Lo et al., 2009 [34]

iTRAQ/LC-ESI

L. interrogans serovar

Whole-cell

563

Leptospires grown under

Eshghi et. al.,

2DE/MALDI-TOF

Copenhageni

conventional in vitro conditions X leptospires mimicking in vivo conditions

2009 [73]

Virulent leptospires from kidney and liver of infected Hamsters

Vieira et. al.,

Leptospires grown under in vitro conditions and determination of the

Malmström et al.,

2DE/MALDITOF/MS

LC-MS/MS

D E

T P E

A

C C

I R

C S U

N A

M

T P

5

L. interrogans serovar Kennewick strain Pomona Fromm

Kidney and Liver tissue

108

L. interrogans serovar Copenhageni

Whole-cell

2221

2008 [70]

2009 [46]

2009 [49]

27

ACCEPTED MANUSCRIPT

concentration of proteins 2DE

L. interrogans serovars

Q-TOF MS MS/MS

Australis (str.Ballico), Autumnalis (str. Akiyami A) Icterohaemorrhagiae (str. RGA), and Bratislava (str. Jez Bratislava), L. biflexa serovar Patoc (str. PatocI) L. interrogans serovar Copenhageni strain L1-130

LC-MS/MS

LC-MS/MS

Thongboonkerd et al., 2009 [83]

X pathogenic leptospires grown under in vitro conditions

T P

Whole-cell

SC

2221

U N

A M

I R

Leptospires grown under in vitro conditions and stimulated with antibiotics or heat shock

Beck et al., 2009 [84]

Leptospires grown under in vitro conditions and high-coverage proteome analysis of protein expression and identification of posttranslational modifications

Cao et al., 2010 [74]

L. interrogans serovar Copenhageni strain L1-130

Whole-cell

1680

Leptospires grown under in vitro conditions and stimulated with antibiotic and serum

Schmidt et al.,

L. interrogans serovar Lai strains IPAV and 56601

Whole-cell

Virulence-attenuated Leptospires and pathogenic Leptospires grown under in vitro conditions

Zhong et al.,

T P E

C C

A

Non-pathogenic leptospires

2540

D E

LC-MS/MS

33

Whole-cell

L. interrogans serovar Lai Strain 56601

Ying-yang MDLC-MS/MS*

Whole-cell

2608/2673

2011 [62]

2011 [28]

28

ACCEPTED MANUSCRIPT

2DE/nLC-MSMS

2DE MALDI-TOF/MS LC-ESI-MS/MS

L. interrogans serovar Copenhageni RJ16441

Urine

L. interrogans serovar Copenhageni strain L1-130 L. interrogans serovar Pomona type kennewicki strain RM211

Whole-cell

4

7

D E

L. interrogans serovar Canicola

2DE/2D-DIGE MALDI-TOF/ TOF-MS MALDI-TOF-MS

MS/MS

T P E

A

C C

ND

19 Leptospira species

L. interrogans serovar Lai

T P

Nally et al., 2011 [71]

Leptospires grown under in vitro conditions: in EMJH medium at 37ºC, in EMJH medium depleted of iron, in EMJH medium supplemented with 10% fetal bovine serum or in EMJH medium supplemented with 10% fetal bovine serum and depleted of iron

Eshghi et al., 2012 [85]

Five different vaccines released for commercial sale derived from heat-inactivated bacteria or outer membrane proteins

Humphryes et al.,

Serum samples from leptospirosis patients X febrile and healthy controls

Srivastava et. al.,

40 leptospires isolates grown under in vitro conditions

Djelouadji et al.,

Leptospires grown under in vitro conditions in liquid-

Zeng et. al.,

I R

C S U

N A

2D-LC/MS

Rat urine-isolated leptospires X urine from non-infected rat spiked with in vitro cultivated leptospires

M

Vaccines

Serum

Whole-cell

Culture supernatant

4-111

22

ND

66

2012 [64]

2012 [77]

2012 [65]

2013 [66]

29

ACCEPTED MANUSCRIPT

protein-free medium 2D-LC/MS

L. interrogans serovar Canicola

Whole-cell

1653

MALDI-TOF/MS LTQ Orbitrap XL

63 Leptospira strains

Whole-cell

108

LC-MS/MS

L. interrogans serovar Pomona

OMVs

506

(NVSL11000)

L. interrogans serovar Manilae Strain L495

1073-1293 540-712

L. interrogans serovar Copenhageni RJ16441

Lung tissue

87

L. interrogansserovar Copenhageni RJ16441

Urine

L. interrogans serovars Lai (str. 56601), Canicola (str. JDL03), Hebdomadis (str. JDL10)

OMP/ Culture supernatant

D E

PT

2D-DIGE/MS

CE-MS

MS/MS

U N

A M

E C

AC

T P

43

473-619/ 260-347

Humphryes et al., 2014 [67]

Leptospires grown under conventional in vitro conditions

Xiao et al., 2014 [86]

Outer membrane vesicles

Kunjantarachot et al., 2014 [87]

I R

SC

Whole-cell Culture supernatant

nLC-MS/MS

Leptospires grown under in vitro conditions

isolated from leptospires grown under in vitro conditions

Leptospires grown under in vitro conditions in EMJH medium at 30ºC, 37ºC or in modified EMJH medium with 120 mM NaCl at 30ºC Guinea pig lung-isolated leptospires X non-infected animals

Eshghi et al., 2015 [88]

Rat urine-isolated leptospires in experimental model of chronic leptospirosis X noninfected controls

Nally et. al.,

Surface-exposed protein isolated from three vaccine strains

Zeng et al.,

Schuller et. al., 2015 [79]

2015 [72]

2015 [68]

30

ACCEPTED MANUSCRIPT

-

L. interrogans serovar Copenhageni strain Fiocruz L1-130

ORFs

191

Validation of serum reactive antigens detected by protein microarray

2DE LC-MS/MS

L.biflexa serovar Patoc strain Patoc I

OMP

97

Leptospires grown under in vitro conditions in modified EMJH medium with FeSO4

2-D DIGE

L. interrogans serovar

Whole-cell

MALDI-TOF

Copenhageni strain Fiocruz

* Dai et al., 2007 [89]

D E

I R

SC

43

U N

L1-130

T P

Lessa-Aquino et al., 2015 [78]

Stewart et. al., 2016 [76]

Leptospires grown under

Nally et al.,

in vitro conditions X cultived

2017 [75]

Leptospires in DMCs

A M

T P E

C C

A

31

Graphics Abstract

Figure 1