Molecular characterization of virulence genes cctA , nanA , and fliC in Clostridium

: Clostridium chauvoei toxin A (CctA), neuraminidase (NanA), and flagellin (FliC) proteins contribute to the pathogenicity of Clostridium chauvoei, the causative agent of blackleg in cattle. The aim of this study was to analyze the genetic variability of cctA, nanA, and fliC genes in C. chauvoei isolates from the Rio Grande do Sul and São Paulo state- Brazil, during different sampling periods. The presence of these genes was verified through PCR amplification and partial gene sequencing of 17 strains. Alignment of PCR amplicons combined with bioinformatics analysis was used in an attempt to study the variability across C. chauvoei solates. The similarity among the partial sequences of cctA and nanA genes was 100%. The sequencing of fliC revealed three different paralog alleles of flagellin, and two strains were seen to be polymorphic, with amino acid alterations in the predicted protein. Overall, this study indicates that strains of C. chauvoei isolated in Brazil are highly conserved with respect to the virulence factors evaluated.

The β-barrel protein porin, leukocidine CctA, has been shown to constitute the main virulence factor of C. chauvoei. It is likely that C. Ziech et al. chauvoei is not able to cause disease without the cctA gene, which encodes the CctA protein. However, until now, the generation of a knockout mutant of cctA has not been reported. FREY et al. (2012) demonstrated that vaccination of guinea pigs with CctA, in the form of a fusion protein, protected animals against virulent C. chauvoei spores.
Neuraminidases or sialidases have been reported in viruses, bacteria, protozoa, fungi, and metazoans (VIMR et al., 2004), and they have been implicated in the pathogenesis of blackleg. These enzymes cleave N-acetylneuraminic acid from carbohydrate polymers, such as mucin, glycoproteins, gangliosides, and other sialoglycoconjugates located on many mammalian cell membranes (VILEI et al., 2011). VILEI et al. (2011 showed that antibodies produced against recombinant sialidase NanA of C. chauvoei neutralized the activity of the infectious agent to degrade sialic acid, indicating that NanA is a potential candidate antigen for the development of novel vaccines against blackleg. Flagella of C. chauvoei drew particular attention towards characterization of this pathogen. Flagella are not directly involved in pathogenesis of blackleg (STEVENSON et al., 2015). However, they can contribute to the infectious process since they provide mobility to bacteria and enable them to reach the target tissue where the pathogen causes injury and disease. Moreover, flagellar antigens were studied as potential candidates for vaccines (KOJIMA et al., 2000;TAMURA & TANAKA, 1984;TAMURA & TANAKA, 1987). Flagella have also been reported to be important for immune response against other pathogenic bacteria, such as Salmonella enterica (KITAO & HATA, 2017), Vibrio cholerae (YANCEY et al., 1979), and Pseudomonas aeruginosa (MONTIE et al., 1987). In C. chauvoei, twenty genes involved in flagellar biosynthesis have been characterized (FREY & FALQUET, 2014). Flagellin is an immunodominant surface protein of the C. chauvoei, which may have a protective role as well as a diagnostic potential (USHARANI et al., 2015).
Full genome sequences of twenty strains of C. chauvoei, isolated from four different continents, over a period of sixty-four years, revealed that C. chauvoei genome is highly conserved in contrast to other Clostridium species (RYCHENER et al., 2017). Therefore, the aim of this study was to investigate genetic diversity of CctA (cctA), neuraminidase (nanA), and flagellin (fliC) across strains from different origins and periods of isolation in Brazil.

Bacterial source, growth conditions and characterization
The seventeen C. chauvoei isolates used in this study were obtained from Brazilian blackleg cases from 2002 to 2016 (Table 1). In addition, we included the Brazilian reference strain (MT) used in official tests for control of blackleg vaccines, and the C. chauvoei type strain (ATCC 10092) from the American Type Culture Collection (ATCC). All strains were stored in Reinforced Clostridial Medium (Oxoid Microbiology products, Thermo Scientific, Waltham, MA, USA). Isolates were validated using polymerase chain reaction (PCR) amplification of the fliC gene according to the protocol proposed by SASAKI et al. (2002), and further validation was performed by 16S rRNA gene sequencing.

DNA extraction, PCR amplification, sequencing, and nucleotide analysis
Bacterial DNA was extracted according to the protocol proposed by TAKEUCHI et al. (1997). Pairs of primers were designed in the Primer-BLAST (NCBI, USA), targeting the specific region of the leukocidin toxin gene (cctA) and the neuraminidase gene (nanA) of C. chauvoei. For the amplification of the fliC gene, the primers were adapted from USHARANI et al. (2015) ( Table 2).
DNA amplification using PCR was performed in a reaction volume of 25 μL consisting of 5 μL buffer 10X + mM magnesium chloride, 20 mM of each dNTP (Ludwig Biotec, Brazil), 1.25 U Taq DNA polymerase (Promega, USA), 10 mmol of each primer, ultrapure distilled water qsp (Invitrogen, USA), and 1 μL DNA. PCR was carried out in a Veriti 384-well thermal cycler (Applied Biosystems, USA). The cycling conditions used were as follows: initial denaturation of 94 ºC for 5 minutes; followed by 35 cycles of 95 °C for 50 s; 61 °C for 50 s (cctA), 62 °C for 50 s (nanA) or 68 °C for 50 s (fliC); and 72 ºC for 7 minutes. Final extension was carried out at 72 ºC for 4 min. PCR products were sequenced by Sanger sequencing (ACTGene Molecular Analysis LTDA, Biotechnology Center, UFRGS, Porto Alegre, RS, Brazil). In addition, flagellin reads of eight strains (Table 1) were sequenced by Illumina HiSeq (Fasteris SA, Plan-les-Ouates, Switzerland), according to the

RESULTS AND DISCUSSION
The specific fragments of the genes cctA, nanA, and fliC were amplified from all the strains. The identity of the cctA gene sequences from the 17 Brazilian C. chauvoei strains, the reference strain, MT, and the type strain ATCC 10092, was 100%. Minor differences were reported in the strain JF3703, a strain, which was isolated in 1956 in New Zealand (99.62% identity) (RYCHENER et al., 2017). In addition, the nanA gene sequences were 100% identical among the Brazilian strains, as well as the reference strains, MT and ATCC 10092. Minor differences were reported when the nanA gene sequences were compared to the New Zealand strain, JF3703; the sequence identity was 96.50%.
CctA, the major virulence factor of C. chauvoei, belongs to the leucocidin superfamily of bacterial toxins and is not reported in any other clostridia species (FREY et al., 2012). In the present study, no genetic variability was reported for the 17 Brazilian C. chauvoei strains investigated. FREY et al. (2012) reported that the strain JF3703/1956 showed a slightly different allele of cctA, which does not affect the predicted protein sequence (RYCHENER et al., 2017). This high degree of identity among strains suggested that cctA gene is a potential candidate that can be employed to identify C. chauvoei by PCR in clinical veterinary microbiology laboratories.
Immunoblot analysis of culture supernatants from eight Brazilian C. chauvoei strains revealed that CctA is expressed and secreted by all of the strains (Figure 1) at relatively equal amounts. No reactions with anti-CctA serum were observed on immunoblots containing the culture pellet material (not shown). These results indicated that all strains tested secrete most or all of the expressed CctA toxin.
Sialidases are considered virulence factors for several pathogenic organisms. The sialidase activity of the C. chauvoei, NanA, contains a CBM40 module that specifically binds sialic acid residues that has been previously reported for NanJ of C. perfringens (BORASTON et al., 2007;ROTHE et al., 1991;USEH et al., 2006a;USEH et al., 2003). The region amplified by the nanA primers used in this study consisted primarily of CBM40 domain. All strains showed a conserved DNA sequence revealing an identity of 100%, except for the strain JF3703 for which the observed differences were located in this domain (RYCHENER et al., 2017). Sialidase activity of C. perfringens type A has previously been detected by LLANCO et al. (2014), where the nanH, nanI, and nanJ genes were investigated and different patterns were observed, indicating heterogeneity of the microorganism. However, C. chauvoei, unlike a bacterial enteric pathogen, is highly specialized.
The conserved gene sequences might reflect the restricted environment for survival of this pathogen and its adaptation to limited number of hosts (cattle, sheep, and goats) (FREY & FALQUET, 2014;RYCHENER et al., 2017). The multiplex PCR protocol described by SASAKI et al. (2002) has been widely used for the identification and differentiation of C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. Primers for C. chauvoei contain a conserved initial region of the flagellin gene, fliC. SASAKI et al. (2002) demonstrated that these clostridia have at least two copies of the flagellin gene (fliC) arranged in tandem (fliA and fliB). The deduced N and C terminal amino acid sequences of their FliC proteins are well conserved, but their central amino acid sequence is not. Clostridioides difficile strains also exhibit conservation in the N and C termini of the FliC protein, whereas their central regions are less conserved (TASTEYRE et al., 2000). RYCHENER et al. (2017), using the full genome Illumina HiSeq sequencing, observed three putative genes, fliC1, fliC2, and fliC3, encoding three different paralog genes. THOMAS et al. (2017) through single molecule real-time DNA (SMRT DNA) sequencing, also observed different copy number variations of these genes among the two strains studied.
The primer used for amplification of fliC was selected because USHARANI et al. (2015) reported that central region of the flagellin gene is unique for C. chauvoei. However, sequencing of PCR products for the fliC gene showed triple peaks for some sequences among all the seventeen C. chauvoei strains used in this study, suggesting that this primer anneals in a region of the flagellin that amplifies three alleles not reported by USHARANI et al. (2015). For this reason, eight strains were subjected to Illumina HiSeq sequencing. The sequences deposited on GenBank by USHARANI et al. (2015) are identical to the FliC sequence reported by KOJIMA et al. (2000) and FliA sequence reported by SASAKI et al. (2002).
The Flagellar activity is characterized by motility; it is inherent in the wild-type strain and is not just a peculiarity of the original variants (TAMURA et al., 1995). Polymorphisms in flagellin genes might alter the proteins encoded by them and influence flagellar activity that could be related to bacterial virulence (KOJIMA et al., 1999(KOJIMA et al., , 2000. A poor protective immunity was induced by the recombinant flagellin in mice, suggesting that a conformationdependent epitope may play an important role in the development of immunity against blackleg (KOJIMA et al., 2000). Alternatively, antibodies against flagellin alone might not be sufficient to induce protective immunity as neutralizing antibodies against the main toxin. A study by TAYASTERE et al. (2000) indicated that non-flagellated C. difficile serotypes retain the transcription of fliC genes, but the protein products have remained undetected. In C. difficile, the flagella may be important for colonization and adherence, but not sufficient to induce protective immunity (STEVENSON et al., 2015). As the pathogenesis of blackleg caused by C. chauvoei infection and of colitis caused by C. difficile infections are very different, the true involvement of the flagellum in the pathogenesis of blackleg, as well as how the transcription of the copies of fliC is regulated in C. chauvoei needs to be unraveled to elucidate their role in pathogenesis.

CONCLUSION
Overall, the virulence genes cctA and nanA were highly conserved among all Brazilian strains studied as well as the Brazilian reference strain MT and the type strain ATCC 10092. Due to the conservation of CctA and NanA among these strains, these soluble antigens are good candidates for the design and quality control of C. chauvoei vaccines. Moreover, genetic similarity might indicate that, currently, C. chauvoei is not being challenged to develop mutations. However, three different fliC alleles were detected among the strains studied, and they were related to different serotypes of C. chauvoei. Their role in infection and in inducing protective immunity remains to be elucidated.