In Silico Analysis of Genetic Vapc Profiles from the Toxin-Antitoxin

microorganisms

Article In Silico Analysis of Genetic VapC Proﬁles from the Toxin-Antitoxin Type II VapBC Modules among Pathogenic, Intermediate, and Non-Pathogenic Leptospira

Alexandre P. Y. Lopes 1,* , Bruna O. P. Azevedo 1,2, Rebeca C. Emídio 1,2, Deborah K. Damiano 1, Ana L. T. O. Nascimento 1 and Giovana C. Barazzone 1

1 Laboratório Especial de Desenvolvimento de Vacinas—Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brazil, 1500, 05503-900 São Paulo, Brazil; [email protected] (B.O.P.A.); [email protected] (R.C.E.); [email protected] (D.K.D.); [email protected] (A.L.T.O.N.); [email protected] (G.C.B.) 2 Programa de Pós-Graduação Interunidades em Biotecnologia, Instituto de Ciências Biomédicas, USP, Avenida Prof. Lineu Prestes, 1730, 05508-900 São Paulo, Brazil * Correspondence: [email protected]; Tel.: +55-11-2627-9475

Received: 29 January 2019; Accepted: 15 February 2019; Published: 20 February 2019

Abstract: Pathogenic Leptospira spp. is the etiological agent of leptospirosis. The high diversity among Leptospira species provides an array to look for important mediators involved in pathogenesis. Toxin-antitoxin (TA) systems represent an important survival mechanism on stress conditions. vapBC modules have been found in nearly one thousand genomes corresponding to about 40% of known TAs. In the present study, we investigated TA profiles of some strains of Leptospira using a TA database and compared them through protein alignment of VapC toxin sequences among Leptospira spp. genomes. Our analysis identified significant differences in the number of putative vapBC modules distributed in pathogenic, saprophytic, and intermediate strains: four in L. interrogans, three in L. borgpetersenii, eight in L. biflexa, and 15 in L. licerasiae. The VapC toxins show low identity among amino acid sequences within the species. Some VapC toxins appear to be exclusively conserved in unique species, others appear to be conserved among pathogenic or saprophytic strains, and some appear to be distributed randomly. The data shown here indicate that these modules evolved in a very complex manner, which highlights the strong need to identify and characterize new TAs as well as to understand their regulation networks and the possible roles of TA systems in pathogenic bacteria.

Keywords: Toxin-antitoxin; VapBC; VapC; Leptospira

1. Introduction Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. It is a zoonotic disease affecting humans and a wide range of animals worldwide with signiﬁcant impact. The genus Leptospira comprises saprophytic and pathogenic species (family Leptospiraceae, order Spirochaetales) and were named based on their spiral shape. They are mobile and measure 6 to 20 µm in length by 0.1 µm in diameter [1,2]. The most serious manifestation of pathogenic leptospirosis results in a syndrome known as Weil’s disease, which is characterized by a devastating kidney and liver failure [2,3]. Based on 16S rRNA phylogeny, DNA-DNA hybridization, pathogenicity, virulence, and in vitro growth characteristics, the genus Leptospira includes at least 21 species arranged in three groups: pathogenic, intermediate pathogenic, and non-pathogenic or saprophytic [4,5], which in turn are divided into over 200 serovars deﬁned by agglutination with homologous antigen [3].

Microorganisms 2019, 7, 56; doi:10.3390/microorganisms7020056 www.mdpi.com/journal/microorganisms Microorganisms 2019, 7, 56 2 of 18

Group I pathogens produce disease in people, mostly severe, caused by bacteria belonging to the evolutionarily-related species L. interrogans, L. kirschneri, and L. noguchii. Group II intermediate pathogens grow better in culture and cause predominantly mild self-resolving illnesses without fatal complications. Group III saprophytic Leptospira are free-living environmental microorganisms [4]. Toxin-antitoxin (TA) systems represent an important mechanism of bacteria survival during stress conditions, such as starvation or antibiotic pressure. TA operons are widely distributed among bacteria and are characterized by a pair of genes encoding for a stable toxin and an unstable antitoxin. The antitoxin acts as an antagonistic regulator that prevents the toxin from exerting its toxicity, except when some environmental conditions determine a decrease in antitoxin concentration, exposing the cell to toxic effects, leading to a reversible cessation of growth [6,7]. TA systems have been involved in potentially harmful aspects of an infection, such as antimicrobial resistance, persistence, and biofilm formation [8]. In some pathogens, including L. interrogans [9], Mycobacterium tuberculosis [10,11], Escherichia coli [12], Haemophilus influenzae [13], and Salmonella enterica [14], they participate in the bacterial physiology during the infection. Experimental data of toxin-antitoxin and in silico analysis on prokaryotic chromosomes have shown the widespread presence of TA modules among bacteria with few exceptions, like the spirochetes Borrelia burgdorferi, Treponema pallidum, and other obligated host associated bacteria [15–18]. TAs are classified into types based on the nature—nucleic acid or protein—of their toxin and antitoxin and on the kind of interaction between them. To date, six types of TA systems have been described [19,20], with the type II system being the most abundant. Type II TA systems are composed of an inhibitory proteic antitoxin that interacts with the toxic protein [21]. Type II TA modules are grouped into different families according to the toxin structure and protein sequence similarity [6,22]. VapBC is the main TA type II family, with about 1900 VapBC modules identified in 960 genomes, corresponding to 30–40% of the known TA modules (URL:http: //bioinfo-mml.sjtu.edu.cn/TADB/)[23]. They are classified based on the presence of a PIN domain (PilT N-terminal) VapC, which is presumed to confer ribonuclease activity to the toxin. Like VapCs, toxins of the RelBE, MazEF, and HicAB families have been described as endoribonucleases, also called interferase RNAs [20,24], which hydrolyze different and specific RNA targets. The RelE toxin cleaves mRNA in the ribosomal A site with high codon specificity [25]. The HicA toxins also cleave mRNA, but independently of the ribosome [26]. Toxins of MazF family have been shown to cleave mRNA [24], rRNA [27], and also tRNA [28]. Most of the few characterized VapC toxins have been shown to exert their activities on the initiator tRNA in a very specific manner. Up until now, the initiator tRNA (tRNAfMet) has been the specific biological target found in the largest number of bacterial species: Leptospira interrogans [29], Salmonella enterica [30], Shigella flexneri [30], and Haemophilus influenzae [31]. Other specific tRNA, such as tRNACys-GCA, tRNALeu-CAG, tRNASer-TGA, CGA, and tRNATrp-CCA, have been identified as substrates of VapCs of Mycobacterium tuberculosis [32]. Additionally, two VapCs of M. tuberculosis cleave 23S rRNA at the sarcin-ricin loop (SRL) [32,33]. The substantial progress of genomic and proteomic studies has allowed for high-throughput studies of leptospiral proteins aimed mainly at the identification of potential antigens for vaccine and diagnostic development [34]. Most of the studies on the genome of leptospiral species have focused mainly on the search for surface-exposed antigens or important proteins to vital metabolic routes [35–38]. More recently, TA modules have achieved more prominence [39,40]. The high diversity among Leptospira species makes their studies very complex and challenging, providing a rich ground to look for specific mediators with importance for bacterial virulence and pathogenesis. In this work, we have investigated the diversity among toxin-antitoxin type II systems among Leptospira species, with a focus on the toxin of the VapBC family. We searched for and compared the putative TA operons of the whole sequenced genomes of pathogenic L. interrogans and L. borgpetersenii, intermediate-pathogenic L. licerasiae, and saprophytic L. biflexa strains within 20 Leptospira ssp., classified according to the pathogenicity phylogenetic tree [5]. This extensive analysis aimed to study the conservation of Microorganisms 2019, 7, 56 3 of 18 these TA modules and to evaluate a possible correlation of their presence in the three phenotypes of pathogenicity.

2. Materials and Methods

2.1. Analysis of Type II TA Modules The analyses of bacterial type II toxin-antitoxin loci were performed using TADB 2.0 database (http://bioinfo-mml.sjtu.edu.cn/TADB2/)[23]. To explore the whole set of putative TA operons of each Leptospira we have used the tool TAfinder (http://202.120.12.133/TAfinder/TAfinder.php) from TADB by entering the Refseq Accession Number of the following bacteria: L. interrogans serovar Copenhageni strain Fiocruz L1-130 (Refseq NC_005823), L. interrogans serovar Lai strain 56601 (Refseq NC_004342), L. borgpetersenii serovar Hardjo-bovis strain JB197 (Refseq NC_008510) and strain L550 (Refseq NC_008508), L. biflexa serovar Patoc strain Patoc 1 (Ames) (Refseq NC_010842) and strain Patoc 1 (Paris) (Refseq NC_010602). The set of TA modules of L. licerasiae serovar Varillal strain VAR010 was obtained from the manuscript of Ricaldi et al. [39].

2.2. Evaluation of the Presence of vapBC among Leptospira spp. To evaluate the presence of putative vapBC operons in the 20 Leptospira spp. comprised in this study, we used the program Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm. nih.gov/Blast.cgi), specifically the protein-protein BLAST tool (BLASTp), to compare the protein sequences of the putative VapC toxins. The VapC query sequences were obtained directly from the TADB database or through locus identification and numbered according to the order of appearance on each genome (base pair numbering) of L. interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC), L. borgpetersenii serovar Hardjo-bovis strain JBL97 (LBJ), L. licerasiae serovar Varillal strain VAR 010 (LEP1GSC185), and L. biflexa serovar Patoc strain Patoc 1 (Ames) (LBF). The subject taxonomic ID (taxid) of the Leptospira used to browse homologous proteins were: Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 (taxid:267671), L. interrogans serovar Lai str. Lai (taxid:1049911), L. borgpetersenii serovar Hardjo-bovis str. JB197 (taxid:355277), L. borgpetersenii serovar Hardjo-bovis str. L550 (taxid:355276), L. biflexa serovar Patoc strain ‘Patoc 1 (Paris)’ (taxid:456481), L. biflexa serovar Patoc strain ‘Patoc 1 (Ames)’ (taxid:355278), Leptospira (taxid:171), L. interrogans (taxid:173), L. kirschneri (taxid:29507), L. noguchii (taxid:28182), L. borgpetersenii (taxid:174), L. weilii (taxid:28184), L. santarosai (taxid:28183), L. alexanderi (taxid:100053), L. alstoni (taxid:28452), L. kmetyi (taxid:408139), L. wolffii (taxid:409998), L. licerasiae (taxid:447106), L. inadai (taxid:29506), L. fainei (taxid:48782), L. broomii (taxid:301541), L. wolbachii (taxid:29511), L. meyeri (taxid:29508), L. biflexa (taxid:172), L. vanthielii (taxid:293085), L. terpstrae (taxid:293075), and L. yanagawae (taxid:293069).

“Conservation Value” Index In order to easily visualize the conservation between two VapCs sequences, we established the “Conservation value”(C-value) formed from values provided by BLASTp analyses, expressed as a frequency between 0 and 1. For each query protein, the C-value was calculated as follows:

(i + p) C value = x c (1) 2 + where “i” is the amino acid identity, “p” refers to a positive or similar amino acid, and “c” is the amino acid coverage sequence, given by the ratio between the length of the highest scoring matching sequence and the query length. Arbitrarily, we considered sequences with the result of “p” and “c” > 0.5 as having a considerable degree of “C-value”. Microorganisms 2019, 7, 56 4 of 18

2.3. Protein Alignment Alignment between two amino acid sequences and identity (%) were performed using the tool Global Align from BLAST, and multiple sequences alignment was computed using COBALT (Constraint-based Multiple Alignment Tool) (https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt. cgi), which considers the proteic conserved domain and sequence similarity information.

3. Results We have been working on the genomics and proteomics of L. interrogans serovar Copenhageni strain Fiocruz L1-130 [5,35,41]. Therefore, this strain was chosen as the basis for the comparative studies presented here. Furthermore, we have studied the TA profile of the sequenced genomes of the pathogenic species L. interrogans serovar Lai [37] and two strains of L. borgpetersenii [42], as well as two strains of the saprophytic L. biflexa [43], available in the TADB database. In the case of the intermediate pathogenic L. licerasiae, a detailed TA profile was described in its genome analysis manuscript [39]. Briefly, 28 TA modules were identified, which code for 15 VapBC, one HigAB, one ChpIK, one MazEF, five ArsR/Aha1, one HTH/Aha1, one XRE/COG2856, one RHH/UNK, one RHH/COG2929, and one RHH/DUF497. We have focused this work on the study of the VapBC family because it is the most abundant type II TA family, comprising about 40% of the identified TA modules in bacterial genomes, allowing us to discuss the variability of TA systems in Leptospira. Moreover, data from the International Leptospira Genome Project [5], made it possible to relate TAs of the genome sequences of 20 Leptospira species in order to build up several comparative tables.

3.1. Analysis and Comparison of TA Proﬁles within Leptospira Species

3.1.1. L. interrogans Serovar Copenhageni Fiocruz L1-130 and L. interrogans Serovar Lai Strain 56601 Investigation of type II TA modules in the genome of L. interrogans serovar Copenhageni strain Fiocruz L1-130 and L. interrogans serovar Lai strain 56601 by the TAfinder tool of TADB resulted in the identification of nine modules in the strain Fiocruz L1-130 (Figure1A) coding for four VapBC, one MazEF (ChpIK), and four TA modules of unclassified families (according TADB), in which the domains are: HEPN/MNT, cd00090/COG3146, COG4118/COG1246, and ArsR/COG3832. The L. interrogans serovar Lai strain 56601 was shown to have all the operons identified in the strain Fiocruz L1-130, except for the TA pair LIC12711/12712. In addition, the serovar Lai strain presented three TA modules, one MazEF, and two extra possible VapBC, inserted in the genome between the modules 4 and 5 of the L. interrogans serovar Copenhageni. The large chromosomal inversion described in the genome of the two L. interrogans strains investigated [44] can be observed by the inverted position of seven TAs of the serovar Copenhageni in relation to serovar Lai, except for the last TA of both chromosomes LIC 13407/13408–LA4258/4259 that are parallelly located (Figure1B).

3.1.2. L. borgpetersenii Serovar Hardjo-bovis Strain JB197 and Strain L550 Search by TAfinder and BLASTp comparison identified nine conserved TA modules in both strains of L. borgpetersenii analyzed here. They code for three VapBC, one MazEF, and five TA modules of unclassified families, in which the domains are: HEPN/MNT, PRK13696/cd09981, cd00090/COG3146, RHH/COG2929, and ArsR/COG3832. TAs of both L. borgpetersenii strains are displayed in completely inverted positions in their genomes, as shown in supplementary results (Table S1).

3.1.3. L. biflexa Serovar Patoc Strain Patoc 1 (Ames) and Strain Patoc 1 (Paris) The same analysis (TAfinder and BLASTp) of TAs of the genomes of L. biflexa resulted in the identification of 23 conserved TA modules. They code for eight VapBC, two MazEF, six RelBE, one phd/doc, and six TA modules of unclassified families, in which the domains are: HEPN/MNT, Microorganisms 2019, 7, 56 5 of 18

RHH-COG2929,Microorganisms 2019 and, 7 four, x FOR ArsR/COG3832. PEER REVIEW The TA positionings in the genomes of both strains 5 of studied 19 here are fully parallel (Table S2).

Figure 1. (A) Location map of toxin-antitoxin (TA) type II systems on the chromosome of L. interrogans

serovar Copenhageni. (B) Comparison of the TAs sets of L. interrogans serovar Copenhageni and L. interrogansFigure 1. serovar(A) Location Lai. map In thisof toxin-antitoxin schema, horizontal (TA) type black II systems lines represent on the chromosome the chromosomes of L. of Copenhageniinterrogans and serovar Lai serovars. Copenhageni. Redand (B) blueComparison lines indicate of the the TAs amino setsacid of L. sequence interrogans correspondence serovar of toxinsCopenhageni and antitoxins, and L. interrogans respectively. serovar Identity Lai. betweenIn this schema, linked ho toxinsrizontal isgreater black lines than represent 90%. Toxins the and chromosomes of Copenhageni and Lai serovars. Red and blue lines indicate the amino acid sequence antitoxins are identified by family or domains, as they appear in TAfinder. * denotes TA modules that correspondence of toxins and antitoxins, respectively. Identity between linked toxins is greater than were experimentally characterized and published. 90%. Toxins and antitoxins are identified by family or domains, as they appear in TAfinder. * denotes 3.2. VariabilityTA modules of Amino that Acidswere experimenta Sequenceslly of characterized VapCs within and Leptospira published. Strains Based3.2. Variability on the of fact Amino that Acids the toxins Sequenc ofes TA of VapCs modules within are Leptospira the elements Strains responsible for the function and substrateBased specificity, on the and fact also that forthe the toxins family of TA classification modules are of the these elements systems, responsible we have for focused the function this study on theand distribution substrate specificity, and conservation and also for of the the family VapC cla toxins,ssification from of the these VapBC system family,s, we have which focused are known this to havestudy poor conservationon the distribution of the and primary conservation sequences of the but VapC high toxins, conservation from the ofVapBC the PIN family, domain which structural are fold [known45]. to have poor conservation of the primary sequences but high conservation of the PIN domain Accordingstructural fold to [45]. the search conducted on TAfinder, the L. interrogans serovar Copenhageni strain Fiocruz L1-130According has fourto the vapBC search loci,conducted all on on chromosome TAfinder, the I, whichL. interrogans encode serovar four toxin-antitoxin Copenhageni strain modules of theFiocruz VapBC L1-130 family. has Based four onvapBC the numericalloci, all on order chromosome of the genes I, which in the encode chromosome, four toxin-antitoxin only VapBC-3 modules of the VapBC family. Based on the numerical order of the genes in the chromosome, only (LIC12660–12659) was experimentally characterized and thus considered a bona fide element [29], VapBC-3 (LIC12660–12659) was experimentally characterized and thus considered a bona fide while the others remain to be elucidated. The alignment of the amino acid sequences of these four VapCs (Figure2) shows the diversity between their primary sequences, but with conservation of the Microorganisms 2019, 7, x FOR PEER REVIEW 6 of 19

element [29], while the others remain to be elucidated. The alignment of the amino acid sequences of these four VapCs (Figure 2) shows the diversity between their primary sequences, but with conservation of the set of three or four acid residues responsible for coordinating Mg2+ or Mn2+ ions at the catalytic site. The cognate VapB antitoxins partners comprise different types of protein domains (RHH, AbrB, and PHD) and therefore cannot have their primary structures compared. In order to investigate whether the amino acid sequences of the toxins from the VapBC modules Microorganismsare divergent2019, 7within, 56 their own bacterial strain, we have evaluated the VapCs’ identities using the 6 of 18 tool “Global Align” for the four VapCs of L. interrogans serovar Copenhageni Fiocruz L1-130, the 15 VapCs L. licerasiae serovar Varillal strain VAR010, and the eight VapCs L. biflexa serovar Patoc 1 2+ 2+ set of(Ames) three (Table or four 1, acidFigure residues S1). The responsible analysis of the for sequence coordinating identities Mg withinor Mn each ionsof the at strains the catalytic were site. Thefound cognate to VapBbe mostly antitoxins very low, partners varying comprise from 13% different to 46%, types with ofmost protein of them domains being around (RHH, AbrB,20%, and PHD)corroborating and therefore the cannotwell-known have low their amino-acid primary identity structures among compared. VapC toxins.

(VapBC-1) MKDKVFLDTNLFIYNFDTENKTKHEKSKEIVLTALAENNYVISYQVIQEFSNVALKKFQI--PLKPKDLAIYL----KRV 74 (VapBC-2) M--KYLLDVNVLISLCDSNHVF-HEKAWKWF-DRKARNGWATCPITQNALVRIMSHSSYPGNPGGVEVVSAILHSLLKVK 76 (VapBC-3) M---YLLDTNICIFLIKKKNAT-LLENLKKK--LNKD--LFVSSLTVAELEFGIQKSEFK----EKNKVALIE-FLTIFN 67 (VapBC-4) M--KYLLDTHVILWIIGSSNLL-SKKAKATI--ENSENKIYVSSVSLWEISLKFRLGKLSLSGMKPSQIPEIL-SKSNIE 74

(VapBC-1) 75 MFPLCSVYYTNENILNAIEIRNRYKLSFYDSVLIGSAIEANCK--TLLSEDLQDGLQIKGLQITNPFNSTIKKKK 147 (VapBC-2) 77 GHQFIPDNISINSSLFYLNIVSVSSKQITDVYLLALSVHHKVK---FATFDSKIPYDSVDRGKEHLELIAA 144 (VapBC-3) 68 ILSFSDKDAESYGIIRADLERKGNVIGSIDMLLAAQAIANNYIFVTNNTKEFKRIKALKIENWTQ 132 (VapBC-4) 75 TINLESADASTYDQLKVIHHR-----DPFDRMLIWQCILRKFT---LISKDSKMKKYRSHGLKTLW 132

FigureFigure 2. Alignment 2. Alignment of theof the VapCs VapCs of ofL. L. interrogans interrogansserovar serovar Copenhageni.Copenhageni. Alignment Alignment was was done done using using the COBALTthe COBALT tool (Constraint-based tool (Constraint-based Multiple Multiple Alignment Alignment Tool), whichTool), which aligns thealigns sequences the sequences via considering via the aminoconsidering acid the sequence amino acid and sequence protein and domains. protein Thedomains. numbers The numbers of VapCs of VapCs in parentheses in parentheses are basedare on the numericalbased on the order numerical of the order genes of the in thegenes chromosome in the chromosome and refer and refer to the to locithe loci described described in in Table Table1 . The highlighted1. The highlighted acidic amino acidic acids amino comprise acids comprise the residues the essentialresidues essential to the catalytic to the catalytic activity activity and classiﬁcation and withinclassification the PIN (PilT within N-terminal) the PIN (PilT domain. N-terminal) domain.

Microorganisms 2019, 7, 56 7 of 18

Table 1. Amino acid sequence identities of VapCs within the same Leptospira strains.

A—L. interrogans Serovar Copenhageni str. Fiocruz B—L. biﬂexa Serovar Patoc Strain Patoc 1 (Ames)—Identity (%) L1-130—Identity (%)

VapC-1 VapC-2 VapC-3 VapC-4 VapC-1 VapC-2 VapC-3 VapC-4 VapC-5 VapC-6 VapC-7 VapC-8

VapC-1 100 19 23 18 VapC-1 100 17 13 24 19 19 27 21 VapC-2 100 19 17 VapC-2 100 18 23 19 22 20 17 VapC-3 100 26 VapC-3 100 27 22 16 16 19 VapC-4 100 VapC-4 100 22 22 22 28 VapC-5 100 36 19 46 VapC-6 100 17 38 VapC-7 100 22 VapC-8 100 C—L. licersiae Serovar Varillal str. VAR 010—Identity (%)

VapC-1 VapC-2 VapC-3 VapC-4 VapC-5 VapC-6 VapC-7 VapC-8 VapC-9 VapC-10 VapC-11 VapC-12 VapC-13 VapC-14 VapC-15

VapC-1 100 26 18 19 28 17 22 14 24 18 17 14 23 21 14 VapC-2 100 13 19 31 21 18 17 19 16 19 17 20 21 17 VapC-3 100 21 14 17 19 17 20 17 24 17 22 23 17 VapC-4 100 18 21 28 25 21 18 22 25 20 31 25 VapC-5 100 23 18 21 24 20 18 21 21 19 21 VapC-6 100 16 18 32 19 16 18 21 21 18 VapC-7 100 32 18 19 18 32 18 22 32 VapC-8 100 23 19 20 100 15 23 100 VapC-9 100 17 20 23 19 25 23 VapC-10 100 20 19 12 21 19 VapC-11 100 20 20 17 20 VapC-12 100 15 23 100 VapC-13 100 20 15 VapC-14 100 23 VapC-15 100 (A) VapCs of L. interrogans serovar Copenhageni Fiocruz L1-130. (B) VapCs of L. biﬂexa serovar Patoc 1 (Ames). (C) VapCs of L. licerasiae serovar Varillal strain VAR010. Microorganisms 2019, 7, 56 8 of 18

In order to investigate whether the amino acid sequences of the toxins from the VapBC modules are divergent within their own bacterial strain, we have evaluated the VapCs’ identities using the tool “Global Align” for the four VapCs of L. interrogans serovar Copenhageni Fiocruz L1-130, the 15 VapCs L. licerasiae serovar Varillal strain VAR010, and the eight VapCs L. biﬂexa serovar Patoc 1 (Ames) (Table1, Figure S1). The analysis of the sequence identities within each of the strains were found to be mostly very low, varying from 13% to 46%, with most of them being around 20%, corroborating the well-known low amino-acid identity among VapC toxins.

3.3. Distribution of VapCs of Pathogenic, Intermediate, and Saprophytic Leptospira Strains In order to study how the amino acid sequences of VapCs are conserved among leptospiral pathogenicity groups (pathogenic, intermediate, and saprophytic) and to infer functional and evolutionary relationships between sequences, we have carried out an extensive study using the BLAST tool, in which we submitted the identified VapCs sequences of two pathogenic strains (L. interrogans and L. borgpetersenii), one intermediate strain (L. licerasiae), and one saprophytic strain (L. biflexa) to compare with sequence database of the Leptospira taxid for each of the species described in the methods section. To enable this comparison, we developed a parameter to indicate “conservation” between sequences named C-value (Cv) (see the methods section), which takes into account three results provided by BLAST analysis: the coverage (“c”), the number of identical amino acids (“i”), and also the number of conserved amino acids designed as positive (“p”), which varies from 0 to 1. We did not use the e-value given by BLAST because it varies according to protein size and it does not consider conservative mutations, which are known to be important to structure and function, as previously indicated for VapC-3 [29]. We present here the results comparing the four strains mentioned above. All tables were colored to indicate, through color intensity, the degree of conservation among the toxins evaluated by C-value. Results for L. interrogans serovar Copenhageni are shown in Table2. Detailed results for the remaining three strains are shown in Tables S3–S5. In addition to the C-values, the results also include the “i”, “p”, and “c” values. It is important to consider that BLAST searches for similar sequences based on a large number of entries from several isolates and that each of the analyzed species have (for the most part) many entries too, and that some of the entries of specific strains do not have the serovar group defined. With that said, it should be noted that when one cell of the Tables2–5 shows that a particular VapC is present in a given species (e.g., L. interrogans or L. noguchii), it is not necessarily present in all members of this species, but in one or some isolates of them. Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 19

Microorganisms 2019, 7, x FOR PEER MicroorganismsREVIEW 2019, 7, x FOR PEER REVIEW 9 of 19 9 of 19

Table 2. Basic Local Alignment Search Tool (BLAST) analysis of VapCs of L. interrogans serovar Copenhageni Fiocruz L1-130.

MicroorganismsMicroorganismsMicroorganisms 2019 ,2019 7, x ,FOR2019 7, Tablex ,FORPEER7, 56 PEER2. REVIEW Basic REVIEW Local AlignmentTable VapCSearch of 2. L. Tool interrogansBasic (BLAST) Local Serovar Alignment analysis Copenhageni of SearchVapCs str. Fiocruz Tool of L. (BLAST) interrogansL1-130 analysis serovar of Copenhageni VapCs of L. interrogans Fiocruz L1-130. serovar Copenhageni9 Fiocruzof 919 of 19 9L1-130. of 18 LIC10866 VapC-1 LIC12116 VapC-2 LIC12660 VapC-3 LIC12713 VapC-4 Leptospira spp Identity Positives Cover IdentityVapC ofPositives L. interrogans Cover Serovar CopenhageniVapCIdentity str. of FiocruzL. interrogansPositives L1-130 Serovar Cover Copenhageni Identitystr. Fiocruz Positives L1-130 Cover C-value C-value C-value C-value (%) (%) LIC10866(%) VapC-1 (%) LIC10866(%) LIC12116 VapC-1(%) VapC-2 (%) LIC12116(%) LIC12660 VapC-2(%) VapC-3 (%) (%) LIC12660 LIC12713(%) VapC-3 VapC-4 LIC12713 VapC-4 Table 2. Basic Local Alignment Search Tool (BLAST) analysis of VapCs of L. interrogans serovar Copenhageni Fiocruz L1-130. L. Leptospirainterrogans spp TableIdentityTable 2. Basic 2. PositivesBasic LocalLeptospira Local AlignmentCover Alignment spp SearchIdentity Search ToolIdentity Tool (BLAST)Positives (BLAST)Positives analysisCover analysisCover of VapCs of VapCs Identityof L.Identity of interrogans L. Positivesinterrogans Positives serovar Cover serovarCover Copenhageni CopenhageniIdentity FiocruzIdentity FiocruzPositives L1-130.Positives L1-130. Cover Cover Identity Positives Cover 100 100 100 1.00 C-value100 100 100 C-value1.00 C-value 100 100 100 C-value1.00 C-value 100 100 100 1.00C-value C-value C-value L. interrogans (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) L. interrogans L. interrogans VapCVapC of VapCL. interrogansof L. of interrogansL. interrogans Serovar Serovar CopenhageniSerovar Copenhageni Copenhageni str. Fiocruz str. Fiocruz L1-130 str. Fiocruz L1-130 L1-130 L. kirschneri 99 100 99 100 100 100 0.99 1.00 97100 100 98100 100 100100 100 0.981.00 1.00 50100 100 66100 100 97100 100 0.561.00 1.00 97100 100 97100 100 100100 100 0.971.00 1.00 100 100 100 1.00 L. interrogans LIC10866LIC10866 VapC-1L. VapC-1interrogans LIC10866 VapC-1 LIC12116 LIC12116 VapC-2 LIC12116VapC-2 VapC-2 LIC12660 LIC12660 LIC12660 VapC-3 VapC-3 VapC-3 LIC12713LIC12713 VapC-4 VapC-4 VapC-4 L. noguchii Leptospira- spp. - - - 97 99 100 0.98 44 64 97 0.52 - - - - LeptospiraLeptospiraL. kirschneri spp spp Identity99Identity Positives Positives99 CoverL. kirschneri100Cover 0.99 99Identity 97Identity Positives99 Positives98 100Cover 100Cover 0.99 0.98 97Identity 50Identity Positives98 Positives66 100Cover 97Cover 0.98 0.56 Identity50 97Identity Positives66 Positives97 97Cover 100Cover 0.56 0.97 97 97 100 0.97 L. borgpetersenii - - Identity- Positives- C-valueC-value Cover- - Identity- Positives- C-valueCoverC-value 48 68Identity 97Positives 0.56CoverC-value C-value - Identity- Positives- Cover- C-valueC-value L. noguchii (%) - (%) (%) - (%) (%)L. noguchii - (%) - - (%)97 C-value (%) - (%)99 (%) - (%)100 (%) - 0.98 97 (%)C-value44 (%) 99 (%)64 (%)100 (%) 97 (%)0.98 0.52 C-value44(%) - (%) 64(%) - (%) 97(%) - (%)0.52 - C-value- - - - L. weilii - - (%)- (%)- (%)87 95 (%)97 (%)0.88 (%) 90 95 (%) 100(%) 0.93(%) 48 (%)66 (%)100 (%)0.57 L.L. interrogans borgpeterseniiL. interrogans - - L. borgpetersenii------48 - 68 - 97 - 0.56 48 - 68 - 97 - 0.56 - - - - - Pathogenic Pathogenic L. santarosai - L.100 interrogans 100 - 100 100- 100 100- 1.00 1.0083 100 10090 100 100100 100 1000.87 1.00 1.0066 100 10083 100 100100 100 1000.75 1.00 1.0064 100 10075 100 100100 100 1000.70 1.00 1.00 L. interrogansL. interrogans 100 100 100 1.00 100 100 100 1.00 100 100 100 1.00 100 100 100 1.00 L. alexanderiL. weilii 93L. interrogans- 95 - 100 L.- weilii 0.94 - 85- 87 94- 95 100- 97 0.90- 0.88 8987 90 9495 95 10097 100 0.920.88 0.93 - 90 48 - 95 66 -100 100 -0.93 0.57 48 66 100 0.57 Pathogenic Pathogenic Pathogenic Pathogenic L. kirschneriL. kirschneri 99 99 99 99 100 100 0.99 0.99 97 97 98 98 100 100 0.98 0.98 50 50 66 66 97 97 0.56 0.56 97 97 97 97 100 100 0.97 0.97 L. alstoniL. santarosai - L. kirschneri- - - 99- L. santarosai- -99 - 10086- 83 0.9995- 90 97100- 10098 0.91- 0.87100 9083 0.9866 9690 5083 100100 66 100 0.930.87 97 0.75 610.5666 64 739783 75 97100 100 1000.650.75 0.70 0.9764 75 100 0.70 L. noguchiiL. noguchii ------97 97 99 99 100 100 0.98 0.98 44 44 64 64 97 97 0.52 0.52 ------L. kmetyiL. alexanderi - L. noguchii93 - 95 - L. alexanderi100 - - 0.94 -93- 85 - -95 94 97 100- 10099 0.94- 0.90100 -85 0.9889 -94 4494 -100 64 100 0.90- 97 0.92 -0.52 89 - -- 94 - -100 - --0.92 ------L. borgpeterseniiL. borgpetersenii ------48 48 68 68 97 97 0.56 0.56 ------L. wolffiiL. alstoni -L. borgpetersenii- - - - L. -alstoni ------86 - - - 95 - - - 100- - - 0.91- 4486 90- 6895 4896 97100 68 100 0.540.91 97 0.93 450.5690 61 62-96 73 100-100 97 0.54-0.93 0.65 -61 73 97 0.65 L. weiliiL. weilii ------87 87 95 95 97 97 0.88 0.88 90 90 95 95 100 100 0.93 0.93 48 48 66 66 100 100 0.57 0.57 L. licerasiaeL. kmetyi - L.- weilii - - - L. -kmetyi ------87 - - -95 - - -97 70- 0.88- 87- 90 - 99- 95 - 0.78-100 - 440.93 - - 6048 - - 10066 - - 1000.52- - 0.57- - - -

Pathogenic Pathogenic L. Pathogenic santarosaiL.L. wolffii santarosai L.- santarosai ------L. -wolffii ------83 - 83- - 90 - 8390 - 100 -90 100 - 0.87100- 0.87 - 660.8744 66 - 836668 83 - 8310097 100 -100 0.750.54 0.75 0.7544 6445 64 6468 7562 7575 97100 100 100 1000.54 0.70 0.54 0.70 0.70 45 62 100 0.54

L. inadai Pathogenic ------63 84 99 0.73 65 78 100 0.72 L. alexanderiL. alexanderi 93 93 95 95 100 100 0.94 0.94 85 85 94 94 100 100 0.90 0.90 89 89 94 94 100 100 0.92 0.92 ------L. faineiL. licerasiae - L. alexanderi- - - 93- L. licerasiae- -95 - 100- - - 0.94- - - 85 - - -94 - - 100- - - 0.9070 - - 8987 - - 94 99 - -100 0.78 -0.92 70 44 -- 87 60 - 99 100 --0.78 0.52 -44 60 100 0.52

Intermediary L. alstoniL. alstoni L.- alstoni------86 86 95 95 100 100 0.91 0.91 90 90 96 96 100 100 0.93 0.93 61 61 73 73 97 97 0.65 0.65 L. broomiiL. inadai - - - - - L. -inadai ------86 - - -95 - - 100- - - 0.9163 - - 9084 - - 96 99 - -100 0.73 630.9363 65 756184 78 1007399 100 0.69970.73 0.72 0.6565 78 100 0.72 L. kmetyiL. kmetyi L.- kmetyi------L. wolbachiiL. fainei - - - - - L.- fainei ------66- - 83- - 99- - 0.74- - 64 - - 75 - - 100- - 0.70------Intermediary Intermediary L. wolffiiL. wolffii L.- wolfﬁi------44 - 44 6844 68 6897 97 970.54 0.54 0.5445 45 45 62 6262 100 100100 0.54 0.540.54 L. meyeriL. broomii 44 - 70 - 95 L. broomii- 0.54 ------66- - 84- - 99- - 0.74- - 62 - 63 74 - 75 100- 100 0.68- 0.69 63 75 100 0.69 L. licerasiaeL. licerasiae L.- licerasiae- - - - L.- wolbachii------70 - 70 - 8770 87 - 8799 99 - 990.78 0.78 0.7866 44 44 4483 60 6060 99100 1001000.74 0.52 0.520.52 64 75 100 0.70 L. biflexaL. wolbachii ------62 66 82 83 99 99 0.71 0.74 28 64 55 75 93 100 0.39 0.70 L. L.inadai meyeriL. inadai L.- 44 inadai - - 70 - - L.- 95 meyeri - - -0.54 - 44- - - - - 70 - - -- 95 - - - -0.54 - - - - - 6366 - 63 - 846384 84 - 849999 99 - 990.730.74 0.73 0.7366 6562 65 6584 7874 7878 99100 100 100 1000.74 0.72 0.68 0.72 0.72 62 74 100 0.68 L. vanthielii 46 L. fainei69 95- 0.55------51 - 69 - 98 - 0.59 - 61 - 75- 100- 0.68- - L.L. fainei biflexaL. fainei ------L.- -biflexa ------62 - - - 82 - - - 99 - - - -0.71 - 62 - 28 - 82 - 55 - 99 - 93 - 0.71- 0.39 - 28 55 93 0.39

Saprophytic L. terpstrae ------Intermediary Intermediary Intermediary L. broomii ------63 75 100 0.69 Conservation valuesL.L. broomii vanthielii(C-valuesL. broomii ) were- 46 expressed- - 69 as- aL. frequency- vanthielii95 - -0.55 between - 46 - 0- and- 69 1. -Th - e -colors 95 - -indicate: -0.55 - - - - - 51 - - - 69 - - - 98 - - -0.59 - 51 6361 63 69 7575 75 98100 100 100 0.59 0.69 0.68 0.69 61 75 100 0.68 L. yanagawae - L. wolbachii------48 - 70 66 98 83 0.58 99 -0.74 64- 75- 100- 0.70

L. wolbachiiL. wolbachii - - - Saprophytic ------66 66 83 83 99 99 0.74 0.74 64 64 75 75 100 100 0.70 0.70 Saprophytic L. terpstrae - - L. terpstrae------Conservation values (C-valuesL. meyeri) wereConservation expressed44 valuesas a frequency70 (C-values95 )between were 0.54expressed 0 and 1.- asTh ae frequencycolors- indicate: -between - 0 and 1.66 The colors84 indicate:99 0.74 62 74 100 0.68 L. meyeriyanagawaeL. meyeri 44 - 44 70 - 70 L.95 yanagawae - 95 0.54 - 0.54 ------very- 6648 highly 66 - conserved8470 84 - 99 98 (0.85 99 ≤0.74 C-value0.58 0.74 48 ≤ 62 1.0); - 62 70 74 highly - 74 98conserved100 - 1000.58 0.68 (0.7- 0.68 ≤ - - - - L. biﬂexa ------62 82 99 0.71 28 55 93 0.39 C-value ≤ 0.84); L. biflexa moderatelyL. biflexa conserved- - - (0.4 -≤ C-value- - ≤ 0.69);- - poorly- - conserved- - (C-value- - ≤- -0.39); - - 62 no62 hits. 82 Positive 82 99and 99cover 0.71 values 0.71 28below 28 50%55 were55 not93 included.93 0.39 0.39 L. vanthielii 46 69 95 0.55 - - - - very highly51 conserved69 (0.8598 ≤ C-value0.59 very highly≤ 1.0);61 conserved 75 highly (0.85conserved100 ≤ C-value0.68 (0.7 ≤ ≤ 1.0); highly conserved (0.7 ≤ L. vanthieliiL. vanthielii 46 46 69 69 95 95 0.55 0.55 ------51 51 69 69 98 98 0.59 0.59 61 61 75 75 100 100 0.68 0.68 L. terpstrae ------

C-value ≤ 0.84); Saprophytic moderately conservedC-value ≤ 0.84); (0.4 ≤ C-value moderately ≤ 0.69); conserved poorly (0.4 conserved ≤ C-value (C-value ≤ 0.69); ≤ 0.39); poorly no conserved hits. Positive (C-value and ≤ cover 0.39); values no below hits. Positive 50% were and not cover included. values below 50% were not included. ConservationConservationSaprophytic Saprophytic L. values terpstraeL. values terpstrae (C-values (C-values L. yanagawae-) were-) were expressed- expressed- - as- a asfrequency- a- frequency- - between- -between - 0- and 0- and1. Th-- 1.e Thcolors- e -colors- indicate:- indicate:- - - - - 48- - 70- - 98- - 0.58------L. yanagawaeL. yanagawae ------48 48 70 70 98 98 0.58 0.58 ------Conservation values (C-values) were expressed as a frequency between 0 and 1. The colors indicate: veryvery very highly highly highly conserved conserved (0.85 (0.85≤ (0.85 C-value≤ C-value ≤ C-value≤ 1.0); ≤ 1.0); ≤ 1.0);highly highlyconserved highly conserved conserved (0.7 ≤ (0.7C-value ≤(0.7 ≤ ≤ C-valueC-value ≤ 0.84); ≤ 0.84);0.84); moderately moderately moderately conserved conserved conserved (0.4 (0.4 ≤(0.4≤ C-valueC-value ≤ C-value ≤≤ 0.69);0.69); ≤ 0.69); poorly poorly poorly conserved conserved conserved (C-value (C-value (C-value≤ 0.39); ≤ 0.39); ≤ 0.39); no hits. no hits. Positive no hits.Positive and Positive cover and values andcover cover belowvalues values 50% below were below 50% not included.50%were were not included.not included. Microorganisms 2019, 7, x FOR PEER REVIEW Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 19 9 of 19 Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 19

Table 2. Basic Local Alignment Search Tool (BLAST) analysis of VapCs of L. interrogans serovar Copenhageni Fiocruz L1-130. Table 2. Basic Local Alignment Search ToolMicroorganisms (BLAST) analysis 2019, 7 ,of x FORVapCs PEER of REVIEWL. interrogans serovar Copenhageni Fiocruz L1-130. 9 of 19 Microorganisms 2019, 7, 56 10 of 18 Table Microorganisms2. Basic Local 2019 Alignment, 7, x FOR SearchPEER REVIEW Tool (BLAST) analysis ofVapC VapCs of L. of interrogans L. interrogans Serovar serovar Copenhageni Copenhageni str. FiocruzVapC L1-130ofFiocruz L. interrogans L1-130. Serovar Copenhageni str. Fiocruz L1-130 9 of 19 LIC10866 VapC-1 LIC12116LIC10866 VapC-2 VapC-1 LIC12660 LIC12116 VapC-3 VapC-2 LIC12713 LIC12660 VapC-4 VapC-3 LIC12713 VapC-4 VapC of L. interrogans Serovar Copenhageni str. Fiocruz L1-130 Leptospira spp Identity Positives CoverLeptospira spp IdentityIdentity Positives Positives Cover Cover IdentityIdentity Positives Positives Cover Cover IdentityIdentity Positives Positives Cover Cover Identity Positives Cover LIC10866 VapC-1 LIC12116 VapC-2C-value LIC12660C-value VapC-3C-value LIC12713C-value VapC-4C-valueTable 2. Basic Local AlignmentC-value SearchC-value Tool (BLAST) analysis of VapCsC-value of L. interrogans serovar Copenhageni Fiocruz L1-130. (%) (%) (%) Table 3. (%)Conservation (%) (%) analysis(%) (%) of(%) VapCs of L.(%) borgpetersenii (%) (%) serovar(%) (%) Hardjo-bovis(%) strain(%) (%) JB197. (%) (%) (%) (%) (%) (%) (%) Leptospira spp Identity Positives Cover Identity Positives Cover Identity Positives Cover Identity Positives Cover L. interrogans TableC-value 2. Basic Local AlignmentL. interrogans SearchC-value Tool (BLAST) analysis of VapCsC-value of L. interrogans serovar CopenhageniC-value Fiocruz L1-130. VapC of L. interrogans Serovar Copenhageni str. Fiocruz L1-130 (%) (%) (%) 100 (%)100 100(%) 1.00(%) 100 100 (%)100 100 (%)100 100 1.00(%) 1.00 100 100 (%)100 100 (%)100 100 1.00(%) 1.00 100 100 100 100 100 100 1.00 1.00 100 100 100 1.00 L. interrogans L. interrogansVapC of L. borgpetersenii Serovar Hardjo-bovis str. JB197—Conservation value (C-value)LIC10866 VapC-1 LIC12116 VapC-2 LIC12660 VapC-3 LIC12713 VapC-4 L. interrogans VapC of L. interrogans Serovar Copenhageni str. Fiocruz L1-130 100 100L. kirschneri100 1.0099 10099 100 L. 0.99kirschneri100 1.0097 99 10098 99 100 100 0.98100 0.99 1.0050 97 10066 98Leptospira 10097 100 spp 0.56100 0.98Identity 1.0097 50Positives 97 66Cover 100 97 0.97 0.56Identity 97Positives 97 Cover 100 0.97Identity Positives Cover Identity Positives Cover L. interrogans LIC10866 VapC-1 Leptospira LIC12116spp. VapC-2 LBJ_0624 VapC-1 LIC12660 LBJ_0764 VapC-3 VapC-2 LBJ_2077 VapC-3LIC12713 VapC-4C-value C-value C-value C-value L. noguchii - - - L. noguchii- 97 - 99 - 100 - 0.98 - 44 97 64 99 97 100 0.52 0.98 (%)- 44 (%)- 64 (%)- 97 - 0.52 (%) - (%) - (%) - - (%) (%) (%) (%) (%) (%) L. kirschneri 99 99Leptospira 100 spp 0.99Identity 97Positives 98Cover 100 0.98Identity 50Positives L. interrogans 66Cover 97 0.56Identity 97Positives 97Cover 100 0.97Identity Positives Cover L. borgpetersenii - - - L. borgpeterseniiC-value------C-value- - 0.9548 - 68 - L.970.90 interrogans - 0.56C-value - - 0.3948 - 68 - 97 C-value- 0.56 - - - - L. noguchii - - - - (%) 97 (%) 99 (%) 100 0.98(%) 44 L.(%) interrogans 64 (%) 97 0.52(%) - (%) - (%) - 100- (%) 100 (%) 100 (%) 1.00 100 100 100 1.00 100 100 100 1.00 100 100 100 1.00 L. weilii - - - L.- weilii 87 - 95 - 97 - 0.88 - 90 87 95 95 100L. interrogans 97 0.93 0.88 48 90 66 95 100 100 0.57 0.93 48 66 100 0.57 L. borgpetersenii - - L. interrogans------48 L. kirschneri68 97 0.560.49 - -0.89 - - 0.38 Pathogenic Pathogenic L. santarosai - 100 - 100 - 100 Pathogenic L. santarosai- 1.00 83 100 - 90 100 - 100100 - 0.871.00 - 66 10083 83 10090 100L. kirschneri100 100 0.75 1.00 0.87 9964 100 66 9975 100 83 100100 100 100 0.99 0.70 1.00 0.75 97 64 98 75 100 100 0.98 0.70 50 66 97 0.56 97 97 100 0.97 L. interrogans L. noguchii 0.93 - 0.39 L. weilii - - L. alexanderi- 93- 8795 10095 0.9497 0.8885 9094 10095 0.90100 0.9389 4894 10066 0.92100 0.57- - - - L. alexanderi 93 L. borgpetersenii95 100 0.94 1.00 85 94 L.1.00 noguchii100 0.90 - 1.0089 - 94 - 100 - 0.92 97 - 99 - 100 - 0.98 - 44 64 97 0.52 - - - - Pathogenic Pathogenic L. santarosai - - L. kirschneri- - 99 83 99 90 100 1000.99 0.87 97 66 98 83 100 1000.98 0.75 50 64 66 75 97 1000.56 0.70 97 97 100 0.97 L. alstoni - - - L. -alstoni 86 - 95 - 100 - 0.91 - 90 86 96 95 L.100 borgpetersenii 100 0.93 0.91 - 61 90 - 73 96 -97 100 0.65- 0.93 - 61 - 73 - 97 - 0.65 48 68 97 0.56 - - - - L. noguchii - - - - 97 99 L. weilii100 0.98 0.9444 64 0.8997 0.52 -0.96 - - - L. alexanderi 93 95 L. kmetyi100 0.94- 85- 94- 100- 0.90- 89- 94- 100- 0.92------L. kmetyi - L. santarosai- - - 0.49 - - 0.87L. weilii- - - 0.40------87 - 95 - 97 - 0.88 - 90 95 100 0.93 48 66 100 0.57 L. alstoni - - L. borgpetersenii- - - 86 - 95 - 100 - Pathogenic 0.91 - 90 - 96 - 100 - 0.93 48 61 68 73 97 970.56 0.65 - - - - L. wolffii - - - L.- wolffii ------44 - 68 Pathogenic - 97L. santarosai- 0.54 - - 45 44 - 62 68 100- 97 0.54- 0.54 83 45 90 62 100 100 0.87 0.54 66 83 100 0.75 64 75 100 0.70 L. kmetyi - - L. weilii------87 - L.95 alexanderi- 97 - 0.88 0.52- 90 - 95 -0.48 100 - 0.93 - 48 - 66 100 0.57 L. licerasiae - - - L. licerasiae------70 - 87 - 99L. alexanderi- 0.78 - 9344 70 9560 87 100100 990.94 0.52 0.78 85 44 94 60 100 100 0.90 0.52 89 94 100 0.92 - - - - L. wolffii - Pathogenic - L. santarosai------83 44 90L. alstoni68 100 970.87 0.540.95 66 45 83 620.88 100 1000.75 0.54 640.44 75 100 0.70 L. inadai - - - L.- inadai ------63 - 84 - 99L. alstoni- 0.73 - - 65 63 - 78 84 100- 99 0.72- 0.73 86 65 95 78 100 100 0.91 0.72 90 96 100 0.93 61 73 97 0.65 L. alexanderi 93 95 100 0.94 85 94L. kmetyi100 0.90 - 89 94 100- 0.92 - - - - - L. licerasiae - - L. fainei ------70- 87- -99 0.78- 44- 60- 100- 0.52- - - - L. fainei - L.- wolfﬁi - - 0.49 - - L.- kmetyi------L. inadai - Intermediary - L. alstoni------86 63 95 84 100 990.91 0.73 90 65 96 78 100 1000.93 0.72 61 73 97 0.65 L. broomii - - - Intermediary L. broomii------L. wolffii- - - - 63 - - 75 - 100- - 0.69- - - 63 - 75 - 100 - 0.69 44 68 97 0.54 45 62 100 0.54 L. fainei - - L. kmetyi------L.- licerasiae- - - - 0.49------0.43 - - - L. wolbachii - - - L. wolbachii------66 - 83 - 99L. licerasiae- 0.74 - - 64 66 - 75 83 100- 99 0.70- 0.74 - 64 - 75 - 100 - 0.70 70 87 99 0.78 44 60 100 0.52 Intermediary L. broomii - - L. wolffii------L. inadai------44 63 68 75 -97 1000.54 0.69 450.38 62 100 0.54 L. meyeri 44 70 95 L.0.54 meyeri - 44 - 70 - 95 - 0.54 66 - 84 - 99L. inadai- 0.74 - - 62 66 - 74 84 100- 99 0.68- 0.74 - 62 - 74 - 100 - 0.68 63 84 99 0.73 65 78 100 0.72 L. wolbachii - - L. licerasiae------66 - L. fainei83 - 99 - 0.74- 70 64 87 75 -99 1000.78 0.70 44 - 60 100 0.52 L. biflexa - - - L. -biflexa ------62 - 82 - 99 L. fainei- 0.71 - - - 28 62 - 55 82 -93 99 0.39- 0.71 - 28 - 55 - 93 - 0.39 ------

L. inadai - - - - Intermediary - -L. broomii- - - 63 84 -99 0.73 65 - 78 100 0.72

L. meyeri 44 70 95 0.54 - - - - 66 84 99 0.74 62 Intermediary 74 100 0.68 L. vanthielii 46 69 95 L.0.55 vanthielii - 46 - 69 - 95 - 0.55 51 - 69 - 98L. broomii- 0.59 - - 61 51 - 75 69 100- 98 0.68- 0.59 - 61 - 75 - 100 - 0.68 - - - - 63 75 100 0.69 L. biflexa - - L. fainei------62 L.- wolbachii82 - 99 - 0.710.51 - 28 - 55 -- 93 - 0.39 -0.38 - - -

Saprophytic L. terpstrae ------

Saprophytic L. terpstrae ------L. wolbachii------66 83 99 0.74 64 75 100 0.70 L. vanthielii 46Conservation 69Intermediary valuesL. broomii 95(C-values 0.55) were - expressed- - Conservation as- a -frequency values- - (betweenC-values- - )0 were and51 1.expressed- L.Th meyerie colors69 -as indicate:a frequency98 - 0.590.83 between - 61 0 and- 1. Th75 -e- colors100 indicate: - 0.68 63 - 75 100 0.69 L. yanagawae - - - L. yanagawae------48 - 70 - 98L. meyeri- 0.58 44 - 48 70 - 70 95- 980.54 - 0.58 ------66 84 99 0.74 62 74 100 0.68 L. wolbachii ------L. biﬂexa - - 0.5366 83 -99 0.74 640.38 75 100 0.70

Saprophytic L. terpstrae ------Conservation values (C-values) were expressed as a frequency between 0 and 1. The colors indicate: very highly conservedL. biflexa (0.85 ≤ C-value- ≤ 1.0); - highlyconserved- (0.7 ≤- - - - - 62 82 99 0.71 28 55 93 0.39 L. yanagawae - - L. meyeri- - 44 - 70 - 95 -0.54 - 48 L.- vanthielii70 - 98 - 0.580.82 66 - 84 -0.47 99 - 0.74 - 620.38 very highly74 conserved100 0.68(0.85 ≤ C-value ≤ 1.0); highly conserved (0.7 ≤ L. vanthielii 46 69 95 0.55 - - - - 51 69 98 0.59 61 75 100 0.68 C-value ≤ 0.84); L. biflexa moderately -conserved - (0.4 ≤ C-value- ≤ 0.69);- - poorly conservedL.- terpstrae (-C-value ≤- - 0.39); - 62 no hits.82 Positive -99 and cover0.71 values28 0.35below 50%55 were not93 included.0.39 C-value ≤ 0.84); moderatelySaprophytic conserved (0.4 ≤ C-value ≤ 0.69); poorly conserved (C-value ≤ 0.39); no hits. Positive and cover values below 50% were not included.

very highly conserved (0.85 ≤ C-value ≤ 1.0);Saprophytic L. terpstrae highly conserved- (0.7 ≤------L. vanthielii 46 69 95 0.55 - L.- yanagawae- - 0.5151 Conservation69 values-98 (C-values0.59 ) were61 - expressed75 as a 100frequency 0.68 between 0 and 1. The colors indicate: C-value ≤ 0.84); moderately conserved (0.4 ≤ C-value ≤ 0.69); poorly conserved (C-value ≤ 0.39); no hits. Positive and cover values belowL. 50% yanagawae were not included.------48 70 98 0.58 - - - - Saprophytic L. terpstrae ------Conservation values (C-values) were expressed as aConservation frequency valuesbetween(C-values 0 and) were 1. Th expressede colors asindicate: a frequency between 0 and 1. The colors indicate: very highly L. yanagawae ------48 70 98 0.58 - - - - very highly conserved (0.85 ≤ C-value ≤ 1.0); highly conserved (0.7 ≤ conserved (0.85 ≤ C-value ≤ 1.0); highly conserved (0.7 very≤C-valueC-value highly ≤ conserved0.84);0.84); moderately moderately(0.85 ≤ C-value conserved conserved ≤ 1.0); (0.4 (0.4≤ highly ≤ C-value conserved ≤ 0.69); (0.7 ≤ poorly conserved (C-value ≤ 0.39); no hits. Positive and cover values below 50% were not included. C-value ≤ 0.84); moderately conserved (0.4 ≤ C-valueC-value ≤ 0.69);0.69); poorly poorly conserved conserved (C-value (C-value≤ 0.39);≤ 0.39); no hits.no hits. Positive Positive and coverand cover values values below below 50% were 50% were not included. not included. Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 19

Microorganisms 2019, 7, x FOR PEER MicroorganismsREVIEW 2019, 7, x FOR PEER REVIEW 9 of 19 9 of 19

Table 2. Basic Local Alignment Search Tool (BLAST) analysis of VapCs of L. interrogans serovar Copenhageni Fiocruz L1-130.

MicroorganismsMicroorganisms 2019, 20197, x FOR, 7, Tablex PEERFOR PEER 2.REVIEW Basic REVIEW Local AlignmentVapCTable Search of 2.L. Toolinterrogans Basic (BLAST) Local Serovar Alignment analysis Copenhageni of SearchVapCs str. Fiocruz Tool of L. L1-130 (BLAST)interrogans analysis serovar of Copenhageni VapCs of L. interrogans Fiocruz L1-130. serovar Copenhageni9 ofFiocruz 199 of 19 L1-130. LIC10866 VapC-1 LIC12116 VapC-2 LIC12660 VapC-3 LIC12713 VapC-4 Leptospira spp Identity Positives Cover IdentityVapC Positives of L. interrogans Cover Serovar CopenhageniIdentityVapC str. of FiocruzL.Positives interrogans L1-130 CoverSerovar CopenhageniIdentity str. Fiocruz Positives L1-130 Cover C-value C-value C-value C-value (%) (%) LIC10866(%) VapC-1 (%) LIC10866(%) LIC12116 VapC-1(%) VapC-2 (%) (%) LIC12116 LIC12660 VapC-2(%) VapC-3 (%) (%) LIC12660LIC12713(%) VapC-3 VapC-4 LIC12713 VapC-4 L. interrogansLeptospira spp TableTableIdentity 2. Basic 2. PositivesBasic LocalLeptospira Local AlignmentCover Alignment spp SearchIdentity Search ToolIdentity Tool(BLAST)Positives (BLAST)Positives analysisCover analysisCover of VapCs of VapCs ofIdentity L. Identityofinterrogans L. Positivesinterrogans Positives serovar Cover serovar CoverCopenhageni CopenhageniIdentity FiocruzIdentity Fiocruz PositivesL1-130. Positives L1-130. Cover Cover Identity Positives Cover 100 100 100 1.00 C-value100 100 100 1.00C-value C-value 100 100 100 1.00C-value C-value 100 100 100 1.00C-value C-value C-value L.Microorganisms interrogans 2019, 7,(%) 56 (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) 11 of(%) 18 (%) L. interrogans L. interrogans VapC VapCof L. interrogans of L. interrogans Serovar Serovar Copenhageni Copenhageni str. Fiocruz str. Fiocruz L1-130 L1-130 L. kirschneri 99 100 99 100 100 1000.99 1.00 97100 100 98100 100 100100 1000.98 1.00 1.00 50100 100 66100 100 97100 1000.56 1.00 1.00 97 100 100 97 100 100100 100 1000.97 1.00 1.00 100 100 100 1.00 L. noguchiiL. interrogans - - LIC10866LIC10866 VapC-1- L. VapC-1interrogans - 97 99 LIC12116 LIC12116100 VapC-2 VapC-2 0.98 44 64 LIC12660 LIC1266097 VapC-3 VapC-30.52 - - LIC12713LIC12713- VapC-4 VapC-4 - LeptospiraLeptospiraL. kirschneri spp spp Identity99Identity Positives Positives99 CoverL. kirschneri100Cover 0.99 Identity99 97Identity Positives99 Positives98 100Cover 100Cover 0.99 0.98 Identity97 50Identity Positives98 Positives66 100Cover 97Cover 0.98 0.56 Identity50 97Identity Positives66 Positives97 Cover97 100Cover 0.56 0.97 97 97 100 0.97 L. borgpetersenii - - - - C-valueTableC-value 4.- Conservation - analysis- of VapCs- C-valueC-value of L.48 licerasiae 68serovar 97 Varillal 0.56 strainC-value VAR010.C-value - - - - C-valueC-value L. weiliiL. noguchii - (%) - (%)- (%) - (%)- (%)L. noguchii - (%)- - 87 - (%) 97 (%)95 - (%) 99 (%)97 - (%)100 0.88(%) - 0.98 90 97(%) 44 (%)95 99(%) 64 (%)100100 (%) 970.93 (%)0.98 0.52 48 44(%) - (%)66 64(%) - (%)100 97(%) - 0.57(%)0.52 - - - - - L.L. interrogans borgpeterseniiL. interrogans - - L. borgpetersenii------48 - 68 - 97 - 0.56 48 - 68 - 97 - 0.56 - - - - - Pathogenic Pathogenic L. santarosai - 100 100- 100 100- 100 100- 1.00 1.0083 100 VapC10090 of L.100 licerasiae 100100 Serovar100 0.87100 Varillal 1.00 str.1.00 VAR66 010—100 Conservation10083 100 value100100 (C-value100 0.75100) 1.00 1.0064 100 10075 100 100 100 0.70100 1.00 1.00 L. interrogansL.L. weiliiinterrogans - - L.- weilii - - 87 - 95 - 97 - 0.88 87 90 95 95 97 100 0.88 0.93 90 48 95 66 100 100 0.93 0.57 48 66 100 0.57 L. alexanderi 93 95LEP1GSC 100 LEP1GSC0.94 LEP1GSC85 LEP1GSC94 100LEP1GSC 0.90 LEP1GSC89 LEP1GSC94 LEP1GSC100 0.92LEP1GSC - LEP1GSC- LEP1GSC- -LEP1GSC LEP1GSC Pathogenic Pathogenic Pathogenic Pathogenic L. kirschneriL. kirschneri 99 99 99 99 100 100 0.99 0.99 97 97 98 98 100 100 0.98 0.98 50 50 66 66 97 97 0.56 0.56 97 97 97 97 100 100 0.97 0.97 L. alstoniL. Leptospirasantarosai spp.- - -185_0307 - - 185_0418L. santarosai- - 185_0630- 86 - 83 185_192295 - 90 100185_2251 - 1000.91 - 185_25800.87 90 83 66185_3193 96 90 83 185_3530100100 1000.93 0.87 185_3550 0.75 61 66 185_355364 73 83 75185_3557 97 100 1000.65 185_35610.75 0.70 64185_3566 75 100 0.70 L. noguchiiL. noguchii ------97 97 99 99 100 100 0.98 0.98 44 44 64 64 97 97 0.52 0.52 ------L. kmetyiL. alexanderi - 93 -—VapC-1 95 - —VapC-2L. alexanderi100 - 0.94—VapC-3 - 93 85 —VapC-4- 95 94 -—VapC-5 100 100 - 0.94—VapC-6 0.90 - 85 89—VapC-7 - 94 94—VapC-8 - 100 *100 - 0.90—VapC-9 0.92 - 89 —VapC-10- - 94 —VapC-11- - 100 - —VapC-13- 0.92 - —VapC-14- - - - L. interrogans L. borgpeterseniiL. borgpeterseniialstoni - - - 0.48- - - -L. 0.36 -alstoni - - - 0.66- - - 86 - -- - 95 - - 0.53- 100 - - - 0.910.80 - 86 48 90 0.5448 9568 96 680.64 10097 100 970.91 0.560.78 0.93 0.56 90 - 61- - 96 - 73 0.87- 100- 97 - 0.930.82- 0.65 - 61 0.92 73 97 0.65 L. wolffii L. interrogans------44 68 97 0.54 45 62 100 0.54 L. L.weilii kmetyiL. weilii ------L. -kmetyi - - - - - 87 - 87 - 95 - 95 - 97 - 97 -0.88 - 0.88 - 90 - 90 - 95 - 95 - 100 - 100 -0.93 - 0.93 - 48 - 48 - 66 - 66 -100 - 100 0.57- - 0.57 - - - - L. licerasiae L. kirschneri------0.66- - - - 0.53 - 0.4170 0.4187 990.64 0.78 0.56 44 - 60 1000.82 0.52 - 0.90

Pathogenic Pathogenic L. Pathogenic santarosaiL. santarosai ------83 83 90 90 100 100 0.87 0.87 66 66 83 83 100 100 0.75 0.75 64 64 75 75 100 100 0.70 0.70 L. inadaiL. wolffiiL. noguchii------L.- -wolffii - - 0.65------0.20 - - - 0.46- 63 - 44 0.4284 - 68 990.63 - 970.73 - -0.54 65 44 45- 78 68 62 1000.54 97 1000.72 0.54 0.85 0.54 45 0.92 62 100 0.54 L. alexanderiL. alexanderiL. borgpetersenii 93 93 95- 95 100 - 100 0.94 0.940.48 85 85 - 94 94 0.20100 100 0.90 0.790.90 89 0.4689 94 940.67 100 100 0.920.55 0.92 ------0.92 L. faineiL. licerasiae - - - - - L. licerasiae------70 - - 87 - - 99 - - 0.78 - 70 44 - 87 60 - 99 100 - 0.78 0.52 44 60 100 0.52 L. weilii - - 0.48 - 0.67 0.80 0.45 0.66 0.93 0.91 0.86 - 0.77

Intermediary L. alstoniL. inadaiL. alstoni ------L. -inadai - - - - - 86 - 86 - 95 - 95 - 100 - 100 -0.91 - 0.91 - 90 63 90 - 96 84 96 - 100 99 100 -0.93 0.73 0.93 6361 65 61 8473 78 73 9997 100 970.73 0.65 0.72 0.65 65 78 100 0.72 L. broomii L. santarosai- - - - 0.37 - 0.46- - - - 0.54 - - - 0.42- - 0.63 - 0.89 63 - 75 1000.54 0.69 0.94 0.72 L. Pathogenic kmetyiL. kmetyi ------L. wolbachiiL. faineiL. alexanderi------L.-- fainei - - 0.65------0.20 - - - 0.80- 66 - - 0.4183 - - 990.64 - - 0.74 - 0.78- 64 - - - 75 - - 1000.86 - - 0.70 ------Intermediary Intermediary L. wolffiiL. wolffii ------44 44 68 68 97 97 0.54 0.54 45 45 62 62 100 100 0.54 0.54 L. meyeriL. broomiiL. 44 alstoni - 70 0.65 - 95 L.0.40 broomii- 0.54 - 0.48------0.50 - - - 0.79- 66 - - 0.7584 - - 990.66 - - 0.74 - 0.78- 62 - 63- 74 - 75 1000.87 - 1000.68 - - 0.69 63 0.77 75 100 0.69 L. licerasiaeL. wolbachiiL. licerasiae L. kmetyi ------L.- wolbachii------70 66 70- - 87 83 87 -- 99 99 99 -0.78 -0.74 0.78 6644 64- 44 8360 75 60- 99100 100 100 0.74 0.52- 0.70 0.52 64 0.92 75 100 0.70 L. biflexa L. wolfﬁi- - 0.38 - 0.78 - -- -0.81 - 0.80 - - 0.4262 0.4182 99 - 0.71 0.53 28 - 55 0.9693 0.39 - 0.97 L. inadaiL. meyeriL. inadai - 44 - - 70 - -L. 95 meyeri - - 0.54 - 44 - - - 70 - - - 95 - - -0.54 - - - - 63 66 63 - 84 84 84 - 99 99 99 -0.73 0.74 0.73 6665 62 65 8478 74 78 99100 100 100 0.74 0.72 0.68 0.72 62 74 100 0.68 L. vanthielii L. licerasiae46 69 1.00 95 1.000.55 1.00- -1.00 - 1.00 - 1.0051 1.0069 981.00 0.59 1.00 61 1.0075 1001.00 0.68 1.00 1.00 L. faineiL. biflexaL. fainei L. inadai- - - 0.77- - - -L. 0.38 -biflexa ------0.55------62 0.39- - - 82 - 0.87- - 99 - - - 0.88- 0.71 - 62 - 28- - 82 - 55 0.94- 99 - 93 - 0.71-- 0.39 - 28 0.91 55 93 0.39

Saprophytic L. terpstrae ------ConservationIntermediary valuesIntermediary L. broomii (C-valuesL. broomii L. fainei) were- expressed- -- as- a frequency- - - - between-- - 0 and- 1.- Th- e colors- -- indicate:------63 - 63 75 0.9375 100 100 0.69- 0.69 - L. yanagawaeL. vanthielii - 46 - 69 - L. vanthielii95 - 0.55 - 46 - - 69 - - 95 - 0.55 - 48 - 51 70 - 69 98 - 980.58 - 0.59 - 51 61 - 69 75 - 98 100 - 0.59 0.68 61 75 100 0.68

Intermediary L. broomii - 0.41 - - 0.56 - - 0.40 - - - - -

L. wolbachiiL. wolbachii - - - Saprophytic ------66 66 83 83 99 99 0.74 0.74 64 64 75 75 100 100 0.70 0.70 Saprophytic L. terpstrae - - L. terpstrae------Conservation values L.(C-values wolbachii ) wereConservation expressed- valuesas0.39 a frequency (C-values- )between were expressed -0 and 1. asTh0.52 ae frequencycolors indicate:0.42 between 00.42 and 1. The 0.64colors indicate:0.85 - 0.81 - 0.37 L. meyeriL. meyeri 44 44 70 70 95 95 0.54 0.54 ------very66 highly66 conserved84 84 99 (0.85 99 ≤0.74 C-value 0.74 ≤ 621.0); 62 highly74 74 conserved100 100 0.68 (0.7 0.68 ≤ L. yanagawaeL. meyeri - - - L. yanagawae0.38- - 0.65 ------0.54 - - 0.78 - 48 0.41 - 70 0.64- 98 0.850.58 48 - - 70 - 0.5598 - 0.58- - - 0.73 - - - C-value ≤ 0.84); L. biflexa moderatelyL. biflexaL. biﬂexa conserved- - --(0.4 ≤- C-value- 0.42 ≤- 0.69);- -- poorly- - conserved- - - (0.37C-value- - ≤ -0.39); - 0.77- - 62 no very 0.4062hits. highly 82Positive 82 conserved0.66 and99 cover99 (0.850.710.80 values 0.71≤ C-value very28below - highly≤28 1.0); 50%55 wereconserved55 - highly not93 included. (0.85conserved93 0.39- ≤ C-value 0.39 (0.7 ≤0.73 ≤ 1.0); highly conserved (0.7 ≤ L. vanthieliiL. vanthielii L. vanthielii 46 46 69- 69 950.39 95 0.55 0.550.61 - - - - - 0.53- - - 0.78- 51 0.4151 69 690.65 98 98 0.590.56 0.59 61 - 61 75 0.5475 100 100 0.750.68 0.68 0.37 C-value ≤ 0.84); moderatelyL. terpstrae conservedC-value- ≤ 0.84); (0.4 ≤ - C-value moderately ≤ 0.69);- conserved poorly- (0.4 conserved ≤ C-value- (C-value ≤ 0.69);- ≤ 0.39); poorly 0.41 no conserved hits. 0.66Positive (C-value and ≤ -cover 0.39); values - no below hits. Positive 50%- were and not cover -included. values -below 50% were not included. Saprophytic

Saprophytic L.Saprophytic terpstraeL. terpstrae ------ConservationConservation values values (C-valuesL. yanagawae (C-values) were) were expressed- expressed as a- frequencyas a frequency -between between 0 and- 0 1.and Th 1.e colorsTh- e colors indicate: indicate:0.42 - - 0.56 - 0.55 0.74 - L. yanagawaeL. yanagawae ------48 48 70 70 98 98 0.58 0.58 ------Conservation values (C-values) were expressed as a frequency between 0 and 1. The colors indicate: veryvery veryhighly highly highly conserved conserved conserved (0.85 (0.85≤ (0.85≤C-value C-value ≤ C-value≤ ≤1.0); 1.0); ≤ 1.0);highly highly conserved highly conserved conserved (0.7 ≤ (0.7C-value ≤ (0.7 ≤ ≤ C-valueC-value ≤ 0.84); ≤ 0.84); 0.84); moderately moderately moderately conserved conserved conserved (0.4 (0.4 ≤ (0.4≤ C-valueC-value ≤ C-value ≤≤ 0.69);0.69); ≤ 0.69); poorly poorly poorly conserved conserved conserved (C-value (C-value (≤C-value0.39); ≤ 0.39); ≤ 0.39); no hits. no Positivehits. no hits.Positive and Positive cover and values coverand belowcover values 50%values below were below not50% included. were50% were not * VapCs included. not included. 8, 12, and 15 (LEP1GSC185:2580, LEP1GSC185:3559, and LEP1GSC185:3880) share the same amino-acid sequence. VapC-12 and VapC-15 were not shown here. Microorganisms 2019, 7, x FOR PEER REVIEW Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 19 9 of 19 Microorganisms 2019, 7, x FOR PEER REVIEW 9 of 19

Table 2. Basic Local Alignment Search Tool (BLAST) analysis of VapCs of L. interrogans serovar Copenhageni Fiocruz L1-130. Microorganisms 2019Table, 7, 56 2. Basic Local Alignment Search ToolMicroorganisms (BLAST) analysis 2019, 7 ,of x FORVapCs PEER of REVIEWL. interrogans 12 serovar of 18 Copenhageni Fiocruz L1-130. 9 of 19 Table Microorganisms2. Basic Local 2019 Alignment, 7, x FOR SearchPEER REVIEW Tool (BLAST) analysis ofVapC VapCs of L. of interrogans L. interrogans Serovar serovar Copenhageni Copenhageni str. FiocruzVapC L1-130ofFiocruz L. interrogans L1-130. Serovar Copenhageni str. Fiocruz L1-130 9 of 19 LIC10866 VapC-1 LIC12116LIC10866 VapC-2 VapC-1 LIC12660 LIC12116 VapC-3 VapC-2 LIC12713 LIC12660 VapC-4 VapC-3 LIC12713 VapC-4 VapC of L. interrogans Serovar Copenhageni str. Fiocruz L1-130 Leptospira spp Identity Positives CoverLeptospira spp IdentityTableIdentity 5.PositivesConservation Positives Cover Cover analysis of VapCsIdentityIdentity of L.Positives biflexaPositives serovarCover Cover Patoc 1 (Ames).IdentityIdentity Positives Positives Cover Cover Identity Positives Cover LIC10866 VapC-1 LIC12116 VapC-2C-value LIC12660C-value VapC-3C-value LIC12713C-value VapC-4C-valueTable 2. Basic Local AlignmentC-value SearchC-value Tool (BLAST) analysis of VapCsC-value of L. interrogans serovar Copenhageni Fiocruz L1-130. (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) Leptospira spp Identity Positives Cover Identity Positives Cover Identity Positives Cover Identity Positives Cover L. interrogans TableC-value 2. Basic Local AlignmentL. interrogans SearchC-value VapC Tool of L.(BLAST) biflexa serovaranalysis Patoc of VapCs strainC-value Patocof L. interrogans 1 (Ames)—Conservation serovar Copenhageni value (C-value)C-value Fiocruz L1-130. VapC of L. interrogans Serovar Copenhageni str. Fiocruz L1-130 (%) (%) (%) 100 (%)100 100(%) 1.00(%) 100 100 (%)100 100 (%)100 100 1.00(%) 1.00 100 100 (%)100 100 (%)100 100 1.00(%) 1.00 100 100 100 100 100 100 1.00 1.00 100 100 100 1.00 L. interrogans L. interrogans LIC10866 VapC-1 LIC12116 VapC-2 LIC12660 VapC-3 LIC12713 VapC-4 L. interrogans VapC of L. interrogansLBF_0418 SerovarLBF_2142 CopenhageniLBF_ str. 2175 FiocruzLBF_2183 L1-130 LBF_2185 LBF_2276 LBF_2813 LBF_3285 100 100L. kirschneri100 1.0099 10099 100 L.Leptospira 0.99kirschneri100 spp.1.0097 99 10098 99 100 100 0.98100 0.99 1.0050 97 10066 98Leptospira 10097 100 spp 0.56100 0.98Identity 1.0097 50Positives 97 66Cover 100 97 0.97 0.56Identity 97Positives 97 Cover 100 0.97Identity Positives Cover Identity Positives Cover L. interrogans LIC10866 VapC-1 LIC12116VapC-1 VapC-2VapC-2 VapC-3 VapC-4 LIC12660VapC-5 VapC-3VapC-6 VapC-7 VapC-8LIC12713 VapC-4C-value C-value C-value C-value L. noguchii - - - L. noguchii- 97 - 99 - 100 - 0.98 - 44 97 64 99 97 100 0.52 0.98 (%)- 44 (%)- 64 (%)- 97 - 0.52 (%) - (%) - (%) - - (%) (%) (%) (%) (%) (%) L. kirschneri 99 99Leptospira 100 spp 0.99Identity 97Positives 98Cover 100 L. interrogans0.98Identity 50Positives 66Cover 97 0.56Identity 97Positives 97Cover 100 0.97Identity Positives Cover L. borgpetersenii - - - L. borgpeterseniiC-value- - - - 0.40- - 0.60- C-value- - - 48 - 0.7868 - 0.72L.97 interrogans - 0.830.56C-value - 0.83- 48 - 0.5968 - 97 C-value- 0.56 - - - - L. noguchii - - - - (%) 97 (%) 99 (%) 100 L. interrogans0.98(%) 44 (%) 64 (%) 97 0.52(%) - (%) - (%) - 100- (%) 100 (%) 100 (%) 1.00 100 100 100 1.00 100 100 100 1.00 100 100 100 1.00 L. weilii - - - L.- weilii 87 - 95 - 97 - 0.88 - 90 87 95 95 100L. interrogans 97 0.93 0.88 48 90 66 95 100 100 0.57 0.93 48 66 100 0.57 L. borgpetersenii - - L. interrogans- - - - - L. kirschneri- 48 0.40 68 - 97 - 0.56 0.78- 0.55- - -0.83 0.73 Pathogenic Pathogenic L. santarosai - 100 - 100 - 100 Pathogenic L. santarosai- 1.00 83 100 - 90 100 - 100100 - 0.871.00 - 66 10083 83 10090 100L. kirschneri100 100 0.75 1.00 0.87 9964 100 66 9975 100 83 100100 100 100 0.99 0.70 1.00 0.75 97 64 98 75 100 100 0.98 0.70 50 66 97 0.56 97 97 100 0.97 L. interrogans L. noguchii - 0.61 - 0.77 0.48 0.47 0.54 0.52 L. weilii - - L. alexanderi- 93- 8795 10095 0.9497 0.8885 9094 10095 0.90100 0.9389 4894 10066 0.92100 0.57- - - - L. alexanderiL. borgpetersenii 93 0.3595 100- 0.940.27 850.78 94 0.55L. noguchii100 0.89 0.90 - - 89 - 0.7394 - 100 - 0.92 97 - 99 - 100 - 0.98 - 44 64 97 0.52 - - - - Pathogenic Pathogenic L. santarosai - - L. kirschneri- - 99 83 99 90 100 1000.99 0.87 97 66 98 83 100 1000.98 0.75 50 64 66 75 97 1000.56 0.70 97 97 100 0.97 L. alstoni - - - L. -alstoni 86 - 95 - 100 - 0.91 - 90 86 96 95 L.100 borgpetersenii 100 0.93 0.91 - 61 90 - 73 96 -97 100 0.65- 0.93 - 61 - 73 - 97 - 0.65 48 68 97 0.56 - - - - L. alexanderi 93 95 L. noguchii100 0.94 - 85 - 94 - 100 - L.0.90 weilii 97 89 990.42 94 1000.59 1000.98 0.600.92 44 0.78- 64 0.83- 97 0.89- 0.52 -0.83 - 0.73- - - L. kmetyi - - - L. -kmetyi ------L. weilii------87 - 95 - 97 - 0.88 - 90 95 100 0.93 48 66 100 0.57 L. borgpetersenii - - - - L. santarosai- 0.41- -0.62 - - 48 0.78 68 0.78 97 0.550.56 0.53- 0.75- - - L. alstoni - - - - 86 95 Pathogenic 100 0.91 90 96 100 0.93 61 73 97 0.65 L. wolffii - - - L.- wolffii ------44 - 68 Pathogenic - 97L. santarosai- 0.54 - - 45 44 - 62 68 100- 97 0.54- 0.54 83 45 90 62 100 100 0.87 0.54 66 83 100 0.75 64 75 100 0.70 L. kmetyi - - L. weilii------L. alexanderi- 87 - 950.35 - 970.59 - 0.88 0.60 - 90 0.77- 95 0.72- 100 0.88- 0.93 -0.84 48 0.7266 100 0.57 L. licerasiae - - - L. licerasiae------70 - 87 - 99L. alexanderi- 0.78 - 9344 70 9560 87 100100 990.94 0.52 0.78 85 44 94 60 100 100 0.90 0.52 89 94 100 0.92 - - - - L. wolffii - Pathogenic - L. santarosai------L. alstoni- 83 44 90- 68 100 - 970.87 0.590.54 66 0.7945 83 0.7262 100 0.851000.75 0.540.83 64 0.7375 100 0.70 L. inadai - - - L.- inadai ------63 - 84 - 99L. alstoni- 0.73 - - 65 63 - 78 84 100- 99 0.72- 0.73 86 65 95 78 100 100 0.91 0.72 90 96 100 0.93 61 73 97 0.65 L. alexanderi 93 95 100 0.94 L. kmetyi85 94- 100 - 0.90 - 89 - 94 - 100 - 0.92 ------L. licerasiae - - L. fainei ------70- 87- -99 0.78- 44- 60- 100- 0.52- - - - L. fainei L. wolffii ------0.52 L. kmetyi- 0.44 - - 0.80 - - 0.75------L. inadai - Intermediary - L. alstoni------86 63 95 84 100 990.91 0.73 90 65 96 78 100 1000.93 0.72 61 73 97 0.65 L. broomii - - - Intermediary L. broomii------L. wolffii- - - - 63 - - 75 - 100- - 0.69- - - 63 - 75 - 100 - 0.69 44 68 97 0.54 45 62 100 0.54 L. fainei - - L. kmetyi------L. licerasiae- - - 0.44------0.56- - 0.80- - 0.79- - -0.82 - 0.55- - - L. wolbachii - - - L. wolbachii------66 - 83 - 99L. licerasiae- 0.74 - - 64 66 - 75 83 100- 99 0.70- 0.74 - 64 - 75 - 100 - 0.70 70 87 99 0.78 44 60 100 0.52 Intermediary L. broomii - - L. wolffii------L. inadai- - - 0.41------44 0.6363 68 0.7975 97 0.331000.54 0.690.80 45 0.5062 100 0.54 L. meyeri 44 70 95 L.0.54 meyeri - 44 - 70 - 95 - 0.54 66 - 84 - 99L. inadai- 0.74 - - 62 66 - 74 84 100- 99 0.68- 0.74 - 62 - 74 - 100 - 0.68 63 84 99 0.73 65 78 100 0.72 L. wolbachii - - L. licerasiae------L. fainei- - 66 - - 83 - - 99 - - 0.74 70 - 64 87 -75 99 100- 0.78 0.700.79 44 60- 100 0.52 L. biflexa - - - L. -biflexa ------62 - 82 - 99 L. fainei- 0.71 - - - 28 62 - 55 82 -93 99 0.39- 0.71 - 28 - 55 - 93 - 0.39 ------L. inadai - - - - L. broomii- 0.42- - - - - 63 - 84 - 99 - 0.73 - 65 78- 100 0.72 Intermediary L. meyeri 44 70 95 0.54 - - - - 66 84 99 0.74 62 Intermediary 74 100 0.68 L. vanthielii 46 69 95 L.0.55 vanthielii - 46 - 69 - 95 - 0.55 51 - 69 - 98L. broomii- 0.59 - - 61 51 - 75 69 100- 98 0.68- 0.59 - 61 - 75 - 100 - 0.68 - - - - 63 75 100 0.69 L. biflexa - - L. fainei------L. wolbachii- - - 62 0.40- 82 -0.94 99 - 0.990.71 - 0.8828 - 0.9255 - 0.4593 - 0.390.87 - 0.92- - -

Saprophytic L. terpstrae ------

Saprophytic L. terpstrae ------L. wolbachii------66 83 99 0.74 64 75 100 0.70 L. vanthielii 46Conservation 69Intermediary valuesL. broomii 95(C-values 0.55) were - expressed- - Conservation as- a -frequency values- - (betweenL.C-values meyeri- - )0 were and51 1.expressed0.43- The colors69 -0.95as indicate:a frequency98 - 0.96 0.59 between - 0.9661 0 and- 1.0.84 Th75 e- colors0.97100 indicate: - 0.680.52 63 0.9175 100 0.69 L. yanagawae - - - L. yanagawae------48 - 70 - 98L. meyeri- 0.58 44 - 48 70 - 70 95- 980.54 - 0.58 ------66 84 99 0.74 62 74 100 0.68 L. wolbachii - - - - L. biﬂexa- 1.00- -1.00 - 1.00 66 1.00 83 1.00 99 1.000.74 1.0064 1.0075 100 0.70

Saprophytic L. terpstrae ------Conservation values (C-values) were expressed as a frequency between 0 and 1. The colors indicate: very highly conservedL. biflexa (0.85 ≤ C-value- ≤ 1.0); - highlyconserved- (0.7 ≤- - - - - 62 82 99 0.71 28 55 93 0.39 L. yanagawae - - L. meyeri- - 44 - 70 - 95 -0.54 L. vanthielii- 48 0.43- 70 -0.95 98 - - 0.58 66 0.91- 84 0.56- 99 0.97- 0.74 -0.53 62 very highly0.9374 conserved100 0.68(0.85 ≤ C-value ≤ 1.0); highly conserved (0.7 ≤ L. vanthielii 46 69 95 0.55 - - - - 51 69 98 0.59 61 75 100 0.68 C-value ≤ 0.84); L. biflexa moderately -conserved - (0.4 ≤ C-value- ≤ 0.69);- L. terpstrae - poorly conserved- - (-C-value - ≤- - 0.39);- 62 no0.91 hits.82 Positive - 99 and cover- 0.71 values- 28 below 50%55- were not93 included.0.39

C-value ≤ 0.84);Saprophytic moderately conserved (0.4 ≤ C-value ≤ 0.69); poorly conserved (C-value ≤ 0.39); no hits. Positive and cover values below 50% were not included.

very highly conserved (0.85 ≤ C-value ≤ 1.0);Saprophytic L. terpstrae highly conserved- (0.7 ≤------L. vanthielii 46 69 95 0.55L. yanagawae- - - -0.95 - 0.97 51 Conservation- 69 0.57 values98 0.44(C-values0.59 ) 0.53were61 expressed0.9575 as a 100frequency 0.68 between 0 and 1. The colors indicate: C-value ≤ 0.84); moderately conserved (0.4 ≤ C-value ≤ 0.69); poorly conserved (C-value ≤ 0.39); no hits. Positive and cover values belowL. 50% yanagawae were not included.------48 70 98 0.58 - - - - Saprophytic L. terpstrae ------Conservation values (C-values) were expressed as aConservation frequency valuesbetween(C-values 0 and) were 1. Th expressede colors asindicate: a frequency between 0 and 1. The colors indicate: very highly L. yanagawae ------48 70 98 0.58 - - - - very highly conserved (0.85 ≤ C-value ≤ 1.0); highly conserved (0.7 ≤ conserved (0.85 ≤ C-value ≤ 1.0); highly conserved (0.7 very≤C-valueC-value highly ≤ conserved0.84);0.84); moderately moderately(0.85 ≤ C-value conserved conserved ≤ 1.0); (0.4 (0.4≤ highly ≤ C-value conserved ≤ 0.69); (0.7 ≤ poorly conserved (C-value ≤ 0.39); no hits. Positive and cover values below 50% were not included. C-value ≤ 0.84); moderately conserved (0.4 ≤ C-valueC-value ≤ 0.69);0.69); poorly poorly conserved conserved (C-value (C-value≤ 0.39);≤ 0.39); no hits.no hits. Positive Positive and coverand cover values values below below 50% were 50% were not included. not included.

3.3.1. L. interrogans Serovar Copenhageni Fiocruz L1-130 BLAST analysis of the four VapC sequences showed that VapC-3, previously described and characterized [29], is the toxin most widely distributed, with high C-value, among the 20 Leptospira species studied, followed by VapC-4 (Table2). In contrast, VapC-1 amino acid sequence shows high C-value only for the pathogenic species L. kirschneri and L. alexanderi. Interestingly, VapC-2 is the only one of the four toxins that is highly conserved exclusively among most of the pathogenic species with the exception of L. borgpetersenii and L. kmetyi species. It should be noted that these two pathogenic species only showed conservation within the VapCs of L. interrogans serovar Copenhageni Fiocruz L1-130. In parallel, we evaluated the similarity of these four VapCs among 12 other important human pathogenic bacteria species, such as Mycobacterium tuberculosis and Shigella sonnei (see Supplementary Material—Table S6), but no conclusions could be taken from it. Interestingly, as for the Leptospira species, VapC-3 is the toxin most distributed among investigated bacteria, followed by VapC-4. VapC-1 and VapC-2 showed medium conservation only with P. aeruginosa and M. tuberculosis.

3.3.2. L. borgpetersenii Serovar Hardjo-Bovis Strain JB197 We also analyzed the conservation of the VapCs of pathogenic L. borgpetersenii (Table3), whose genome was completely sequenced. From the nine TA modules identiﬁed by TAﬁnder, three belong to the VapBC family. VapC1 shows the highest distribution among the species analyzed, concentrating high C-value among the pathogenic species. Similar to VapC-2 of L. interrogans serovar Copenhageni, L. borgpetersenii VapC-2 is highly conserved only among pathogenic species. VapC-3 amino acid sequence is highly conserved only in L. weilii, with diffuse distribution and low C-value among others. Microorganisms 2019, 7, 56 13 of 18

3.3.3. L. licerasiae Serovar Varillal strain VAR010 Among the 28 TAs identiﬁed in the intermediate pathogenic L. licerasiae [39], 15 are VapBC modules (Table4). We observed a diffuse pattern of conservation to the toxins. VapCs 1, 2, 4, and 10 appear with low C-values among most species, while VapCs 3, 5, 7, and 8 exhibit wide distribution and medium conservation C-values. VapCs 6, 9, 11, 13, and 14 present a larger distribution with high C-values indicating higher conservation of sequences. It should be noted that VapCs 8, 12, and 15 (LEP1GSC185:2580, LEP1GSC185:3559, and LEP1GSC185:3880) share the same amino-acid sequence, probably stemming from genetic duplication events.

3.3.4. L. biflexa Serovar Patoc 1 (Ames) When the nonpathogenic L. biflexa strain was analyzed (Table5), we observed a more homogenous pattern of distribution (i.e., we noted that, with the exception of VapC1, all the others displayed high C-values in the other saprophytic strains, and the VapCs 4, 5, 6, 7, and 8 appear with relatively high conservation in the pathogenic species as well). It is interesting to note that among the eight VapCs of L. biflexa, three of them (VapC-4, VapC-5, and VapC-6) resemble VapC-3 of L. interrogans Copenhageni, which might suggest some evolutionary link and explain their strong presence among the pathogenic species. Taken all together, our results describing the distribution of these TA modules among leptospiral strains of varied phenotypes of pathogenicity showed that VapCs are unequally distributed, with some toxins apparently being randomly disseminated, to others present in unique species or specific pathogenicity groups.

4. Discussion Toxin-antitoxin systems have been involved in potentially harmful aspects of an infection, such as antimicrobial resistance, persistence, and biofilm formation, and therefore have been the subject of intensive efforts to elucidate biochemical and functional effects besides their role on the physiology of infection. Nevertheless, due to the high number of types, families, and high divergence among them, all these efforts on the elucidation of biochemical and biological functions are still incipient and remain controversial. Currently, three type II TAs were described and experimentally characterized in the genome of L. interrogans serovar Copenhageni strain Fiocruz L1-130 and serovar Lai strain Lai: one VapBC [29,46], one ChpIK, and one MazEF [9,47], and therefore can be considered bona fide elements, all the others need to be confirmed. It should be pointed out that the ChpKI module has also been called as MazEF [48] since both ChpI and MazF toxins share the structurally and functionally similar domain PemK, which is a sequence-specific endoribonuclease [49]. Even though both chpIK and mazEF, found in L. interrogans serovar Lai strain Lai, have been described as extensively distributed and the sequences conserved in different pathogenic Leptospira [9], the mazEF operon (LA1780/1781) was not identified in the L. interrogans serovar Copenhageni strain Fiocruz L1-130 using BLAST analysis, but it is present only in the L. interrogans serovar Copenhageni strain HAIO156 from the 19 isolates available of this species-serovar. Considering the strict curation of Fiocruz L1-130 and Lai genomes, this fact indicates that the function of MazEF in pathogenesis is not essential or, more likely, redundant, and could be replaced by other TAs in these strains. The analysis of the whole genomes of Leptospira has shown that there is considerable genomic plasticity even within the same species, as in the case of the large inversion in chromosome I and an ~54 kb genomic island that differentiates the genomes of L. interrogans serovars Lai and Copenhageni, which share ~99% similarity at amino acid level of ortholog genes [44]. Our data shows that the disposition of seven TAs along the chromosome of both bacteria is inverted as expected, but more significantly, have the addition of three TA modules. Similarly, we have found that the genomes of both strains of L. borgpetersenii analyzed (JB197 and L550) displayed total inversion of the seven Microorganisms 2019, 7, 56 14 of 18

TA sequences along the chromosome, which share exactly the same amino acid sequences (data not shown). Differently, the analysis of the 23 TA modules of the genomes of the two strains of L. biflexa (Ames and Paris) did not show any variation between them, even in their position on the chromosome, which is in agreement with data in the literature describing the high conservation in the genomes within this species [43]. The low intra-species amino acid sequence identity among VapCs of the L. interrogans, L. licerasiae, and L. biflexa strains evaluated in our study might indicate that they act on different substrates producing distinct effects, however, no link between toxin homologs and a specific physiological function has been established [50,51]. Additionally, it remains controversial whether such TA systems are redundant or not in relation to their physiological functions. It has been postulated that TA systems are responsive to environmental changes, both within their hosts or in free-living conditions. In the case of type II TAs, such changes would trigger the activity of enzymes such as Lon protease [52], which would cause antitoxin degradation, de-repressing transcription, increasing TA expression, and finally releasing the toxins to perform their functions. TAs have been proposed to participate in bacterial pathogenicity based on the idea that adaptation of pathogenic bacteria to the host is intrinsically linked to the expression of virulence genes. VapBC modules have been suggested to be involved with bacterial phenotypes both under intra-host and free-living conditions, which was shown by the loss of adaptability to stressing conditions when knocking out the operons vapBC2ST of Salmonella typhimurium [53], vapBC-1 of Haemophilus influenzae [13], and vapBC of the thermophilic Sulfolobus solfataricus [54]. In L. interrogans Copenhageni, VapBC-3 has been biochemically characterized to act on the lysis of the initiator tRNAfMet and therefore to inhibit translation [29], but information of its role in the physiology of the bacteria is still lacking. TAs are represented by a highly variable number of modules in several bacteria, as highly variable as the extreme environmental or host differences to which these organisms must adapt to live, suggesting it might contribute to their diversification and evolution. Unsurprisingly, the resulting phenotype of each species is affected by a diversity of features including the whole genomic background, among which the diversity of TA modules can be included and may interfere in the response of individual TA systems during adaptation stresses [55]. In the case of Leptospira, the overall set of TA modules characteristic among species living in saprophytic or pathogenic conditions reveals that the L. biflexa has a considerable larger number of TAs then L. interrogans and L. borgpetersenii, which can indicate a relatively large subset of adaptive facilities. However, the intermediate L. licerasiae, whose genomic analysis led to the conclusion of its greater proximity to pathogenic strains than to saprophytic [39], has an even greater set of TA systems, in general, and vapBC specifically. Although the data available to date are very incipient, we could hypothesize that this larger number of modules found in the intermediate pathogenic L. licerasiae, which could also be referred as “intermediate saprophytic” [56], would be related to its ability to survive in both the environment and animal hosts. There are examples in the literature showing that proposed TAs, exhibiting a typical TA genetic organization (e.g., XreA-Ant and Bro-XreB of S. pneumonia [57] and VapBC of M. tuberculosis [58]), did not appear to act as bona fide TAs when experimentally tested. Therefore, it is important to reinforce that the results and hypothesis discussed here are based on in silico analysis and questions related to the possible redundancy and functionality of the VapBC modules analyzed here need to be confirmed by in vivo experiments. It is not clear how acquisition by horizontal gene transfer of the set of TA loci influenced the evolution of each bacterial species in its broad set of genes, and whether TAs may play roles in determining phenotypic aspects such as virulence and persistence [14,51]. It has been suggested that TAs should more likely be considered as just selfish elements that would improve the fitness of bacteria to eventual stressing conditions, driving the evolution of bacterial genomes [21]. By providing an overview of the distribution of vapBC operons among Leptospira species, through the analysis of the conservation of their toxic elements, we could outline a rather complex picture indicating that these modules evolved differently. It is remarkable that, out of all VapC sequences Microorganisms 2019, 7, 56 15 of 18 of pathogenic species submitted to BLAST analysis, VapC-2 of L. interrogans serovar Copenhageni and VapC-2 of L. borgpetersenii serovar Hardjo-bovis are, at the same time, widely and strictly highly conserved only among pathogenic species. Interestingly, proteomic analysis showed an increase of the antitoxin component from VapBC-2 of L. interrogans serovar Copenhageni during stress induced by antibiotic treatment (Ciprofloxacin) [59]. Thus, they might represent potential candidates for participating in the adaptation of the pathogen to the infection host and have a role in pathogenesis. Even though in recent years we have accumulated an increase in the knowledge about TA systems, information on how they have evolved is still missing. One reason that may help to explain this lack of knowledge is the fact that most TA loci are acquired by horizontal gene transfer [39,60] and thus are often not conserved in different isolates belonging to the same bacterial species. According to that explanation and corroborating our results, a study dealing with dissemination type II TAs in Escherichia coli showed that they are unevenly distributed among E. coli phylogroups and are not a universal feature of the species [61]. In parallel studies, our attempts at using molecular phylogenetic analysis to try to identify some branching points and thus infer some correlation of VapCs with pathogenic, intermediate, and saprophytic species of Leptospira has failed. Except in the specific cases mentioned above, our data were inconclusive and unable to infer this kind of correlation, most likely due to the high diversity of their amino acid sequences. Moreover, it is very important to consider that protein primary sequences are the groundings for the three-dimensional structure folding, which ultimately is responsible for toxin and antitoxin activities. The large variability in numbers and the low sequence conservation among VapCs shown here emphasizes the strong need to identify and characterize new TAs as well to understand the regulation nets and roles of TA systems in pathogenic bacteria.

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/7/2/56/s1. Author Contributions: Conception of the work: A.P.Y.L., A.L.T.O.N., and G.C.B.; Data acquisition: A.P.Y.L., B.O.P.A., R.C.E., and D.K.D.; Interpretation: A.P.Y.L., B.O.P.A., R.C.E., D.K.D., A.L.T.O.N., and G.C.B.; Writing and revising: A.P.Y.L., B.O.P.A., R.C.E., D.K.D., A.L.T.O.N., and G.C.B. Funding: This work was financially supported by FAPESP (grant 14/50981-0), CNPq (grant 301229/2017-1), and Fundação Butantan—Brazil. This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)—Finance Code 001. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments: We would like to thank Marcelo A. Ventura for helping with the design of the figures. We also thank Martin Wesley for his help with English revisions. Conflicts of Interest: The authors declare no conflict of interest.

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