Identication and Comparison of Hyaluronic Acid Biosynthetic Genes from Different Capsular Types of Pasteurella Multocida

Pasteurella multocida produces a capsule composed of different polysaccharides according to the capsular serotype (A, B, D, E, and F). Hyaluronic acid (HA) is a component of certain capsular types of this bacterium, especially capsular type A. Previously, two HA biosynthetic genes from a capsular type A strain were studied for the industrial-scale improvement of HA production. Molecular comparison of these genes across different capsular serotypes of P. multocida has not been reported. This study aimed to compare nine HA biosynthetic genes (glck, pgi, pgm, galU, hyaC, glmS, glmM, glmU, and hyaD) of eleven P. multocida strains (A:B:D:F = 8:1:1:1) with those of other organisms using sequence and structural bioinformatics analyses. These nine genes showed a high level of within-species similarity compared to other Only the last gene of two with capsular and one capsular type F strain differed from of Analysis amino acid patterns together with phylogenetic that the HA of the and D strains were closely related compared However, the genes in capsular similar Protein structural analysis similarities

HA is usually synthesized by two distinct pathways that synthesize HA precursors; these pathways begin with the phosphorylation of glucose by glucokinase to produce two precursors (UDP-glucuronic acid and N-acetylglucosamine). In the rst reaction, phosphoglucomutase (Pgm) converts glucose-6-phosphate to glucose-1-phosphate, after which the phosphate group from UTP is transferred to glucose-1-phosphate via UTP-glucose-1-phosphate uridyltransferase (GalU) to produce UDP-glucose. UDP-glucose is oxidized by UDP-glucose 6-dehydrogenase (HyaC), deriving the rst HA precursor, UDP-glucuronic acid. In the second pathway, glucose-6-phosphate is converted to fructose-6-phosphate by glucose-6-phosphate isomerase (Pgi) and then changed to fructose-6-phosphate by adding an amino group from a glutamine residue via L-glutamine:D-fructose-6-phosphate aminotransferase (GlmS) to produce glucosamine-6phosphate, which is later modi ed by phosphoglucosamine mutase (GlmM) to yield glucosamine-1phosphate. Glucosamine-1-phosphate is acetylated and phosphorylated by bifunctional Nacetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (GlmU) to the second precursor, UDP-N-acetylglucosamine. Finally, hyaluronan synthase (HyaD) combines and polymerizes the two precursors, generating the HA polymer [15,16]. Recent reviews by Peng et al. (2019) showed that the capsule biosynthesis genes in cap loci varied among different capsular types, particularly between types A, D, and F and type B. Comparative analysis of the HA biosynthetic genes from various organisms showed that the hyaluronan synthase from P. multocida was the only class II enzyme, while other organisms produced the class I hyaluronan synthase [17]. Class II hyaluronan synthases are different from class I hyaluronan synthases in terms of structural topology and biosynthesis mechanism [18,19]. In this step, the HA synthesized by class II hyaluronan synthases is connected to the cytoplasmic membrane and transported through the membrane directly to the extracellular matrix during chain elongation and not sulfated or chemically modi ed after biosynthesis [17,20,21].
Previous research improved HA production by genetical modi cation of the has operon in Streptococcus sp. However, production has a risk of pathogenic bacterial contamination [22]. HA synthesis has been studied in many other microorganisms (Bacillus sp., Lactococcus sp., and Escherichia. coli) for the production of high-quality and safe-to-use HAs [16,19]. Industrial-scale HA production was developed with recombinant E. coli and Bacillus sp. strains harbouring the hyaD genes from Streptococcus sp. and P. multocida [23,24]. The recombinant B. subtilis strain produced HasA from Streptococcus equisimilis together with the co-expression of TuaD from B. subtilis. This recombinant bacterium produced a high level of HAs and had been used industrially [14]. Another study optimized conditions for HA production in recombinant E. coli based on the expression of the hyaD gene of P. multocida ATCC 15742 (capsular type A:3) and the hyaC gene of E. coli strain K5 [1]. The level of HA production in the recombinant bacterium increased (2.7 to 3.7 g/L (37%)) after varying the amount of supplemented oxygen and glucosamine. The synthesis also increased by up to 70% when fosfomycin was added to inhibit cell wall synthesis [1]. Coexpression of the hyaC and hyaD genes from P. multocida gave the highest level of HAs (5.4 g/L) and stability compared to the co-expression of the hyaC gene from E. coli and hyaD from S. pyogenes [13]. Although variations in the capsule biosynthesis genes of P. multocida have been studied, HA biosynthetic genes have not been explored and could be useful for further improving HA production. Therefore, this study aimed to compare the HA biosynthesis genes in P. multocida using sequence, pattern and structural analyses. The HA genes were compared across different capsular types of P. multocida and to their orthologues in other bacteria and humans. We proposed that variations observed within these genes could be useful targets for genetic engineering-based improvement of HA production.

Results
Identi cation of the hyaluronic acid biosynthetic genes of Pasteurella multocida Eight HA biosynthetic genes were identi ed in 11 strains of Pasteurella multocida, while hyaluronan synthase (hyaD) was absent in strain HN06 of capsular type D. These nine HA biosynthetic genes shared a high percentage of identity (between 82-99%) and percentage of sequence coverage (between 99-100%) with the query sequences from Mao et al. (2009) (Fig. 1). The lowest percentage of identity (82%) was observed in the hyaC and hyaD genes. Comparison of these genes with those of other organisms showed a lower percentage of identity and coverage: 29-100% and 0-79% in H. sapiens, 25-100% and 0-94% in S. pyogenes strain M1, and 35-100% and 1-100% in S. thermophilus. Among these, the enzymes from E. coli shared the highest percentage of identity (32-93%) and coverage (0-100%) with those of P. multocida, and the hyaD gene was absent in E. coli. The results were used to reconstruct the HA biosynthetic pathway of P. multocida, as depicted in Fig. 1.
Sequence similarities and amino acid patterns of the nine HA biosynthetic genes Comparison of the nucleotide and amino acid sequences of these nine HA biosynthetic genes from 11 strains of P. multocida showed a high level of within-species identity (98-99%) when using the ATCC43137 strain of capsular type A as a reference. Only the hyaD gene of strains with capsular type A:3 (PM70 and CRIMBP-0884) and capsular type F (HN07) differed signi cantly from those of other strains (82%), as shown in Table 1. Pattern analysis of the amino acid sequences of these nine HA biosynthetic genes in P. multocida showed that capsular types A and D had a close relationship compared to capsular types B and F. Particularly, capsular type A strain 3480 had the most different pattern among other type A strains and had ve proteins (Pgi, GalU, GlmS, GlmM, and GlmU) closely related to the capsular type D strain HN06 (Fig. 2). The PMTB2.1 strain of capsular type B had four enzymes that were different from the enzymes of other strains, except GalU and HyaC, which were shared in some type A strains, GlmM, which was shared with type A:3 (FDAARGOS_218) and A:3 (PM70) strains, and HyaD, which was similar to the type A strain (Fig. 2). Seven proteins of the capsular type F strain were shared with the capsular type A:3 strain, and three were shared with the type A. This result showed that the HA biosynthetic enzymes across the examined P. multocida strains, even within the same serotype, had different variation patterns.
Phylogenetic relationship of the nine HA biosynthetic genes Phylogenetic analysis of the HA biosynthetic genes in Fig. 3 shows the separation between these nine enzymes from P. multocida and those from other organisms. The glcK and pgi genes of capsular types A:3 and F were closely clustered, while the A:3 and F strains were only grouped in the trees of the pgm, glmM, and hyaD genes. The galU and glmU genes of the A:3 strains were clustered with those of the capsular type B strains. All these genes from most of the remaining capsular type A strains were grouped, except the pgm, glaU, glmS, glmM, and glmU genes of strain 3480, which were together with those of the capsular types B or D strains and the hyaC gene of strain 3480, which was grouped with the type A:3 strains.
The patterns were changed in the protein tree of these nine enzymes (Fig. 4) GlmM was closely related to capsular type A:3. Phylogenetic trees from the concatenated nucleotide and protein sequences of the HA biosynthetic genes in Fig. 5 showed the same clustering pattern of the capsular type A:3 and F strains and the four type A strains (HB01, HB03, 36950, and ATCC43137). The patterns of the gene and protein trees of the capsular type A (3480), A:3 (FDAARGOS_218), and B:2 strains differed ( Fig. 5A and 5B). These phylogenetic results depicted sequence variation and possibly mutational history within the HA biosynthetic genes of different P. multocida strains.

Structural comparison of the hyaluronic acid biosynthetic enzymes
The structures of nine HA biosynthetic enzymes from different P. multocida strains were successfully predicted and compared. Predicted structures of the Pgi, GalU, HyaC, GlmS, and GlmM proteins were highly similar between capsular types A, B, D, and F (Fig. 6 Comparing the binding site pocket of the predicted structures in Fig. 7 could provide clues into the molecular functions. The binding site pockets were predicted, and different pocket surfaces were observed in the structures of Glck from strain HB01; Pgm from strain FDAARGOS_218; GlmU from strains 3480, FDAARGOS_218, and HN06; and HyaD from strains 3480, 36950, FDAARGOS_218, PM70, CRIMBP-0884, and HN70. Certain positions correlated with the active site of the enzymes determined by a sample score greater than 0.50 (Table S1) A score of less than 0.50 showed no relation to the active site of the enzymes. From all predicted binding pockets, the catalytic pocket of GlmU at the P6 position (Fig. 7) was observed only in the capsular type A strain ATCC43137. The catalytic pocket of HyaD at the P5 position was unique to the capsular type A strains ATCC43137, 3480, and 36950 and capsular type A:3 strain FDAAROGOS_218.
Analysis of the conserved domains of these nine enzymes was performed by comparing the domain size and architecture of these proteins using the domains of strain ATCC43137 as a reference. Differences were found in four enzymes, as shown in Fig. 8.  Fig. 8C) was also absent in the strains PM70, CRIMBP-0884, and HN70. Glycosyl transferase-like family 2 (Glycos_transd_2_2) and glycosyl transferase family 2 (Glycos_transd_2_4) were more abundant in the PM70, CIRMBP-0884, and HN07 strains than in the other strains. Glycos_transd_2_2 also showed differences in sizes and positions between strains (Table S4).

Discussion
The hyaluronic acid biosynthesis pathway of P. multocida involves nine enzyme-encoding genes, including glck, pgi, pgm, galU, hyaC, glmS, glmM, glmU and hyaD, similar to the capsular polysaccharide biosynthesis pathway in S. pyogenes [19,[25][26][27]. HAs are known to be primary capsular components of type A strains of P. multocida. This study found that P. multocida strains of capsular types A, B, D, and F shared eight-core HA biosynthetic enzymes that produce UDP-D-glucuronic acid and UDP-N-acetyl-Dglucosamine, which are modi ed to hyaluronic acid (hyaD), chondroitin sulfate, and heparin, and disaccharide repeats of other components that vary across different strains [28]. The hyaluronan synthase (hyaD) of capsular type A is essential in the joining and polymerization of the two precursors for HA biosynthesis. In the capsular type F strain, N-acetyl-D-galactosamine was used instead of N-acetyl-D-glucosamine to generate chondroitin sulfate (CS) by chondroitin synthase [29,30]. Heparin (HP) was made from three precursors (D-glucuronic acid, D-glucosamine, and L-iduronic acid (IdoA)). Members of the HA biosynthetic pathway synthesize two of these precursors. Only L-iduronic acid could be synthesized by an epimerization reaction of D-glucuronic acid and polymerization of these precursors to generate heparin by heparin synthase [31,32]. This result suggested that the rst eight enzymes of the HA biosynthesis pathway could be the core pathway for synthesizing other types of capsular polysaccharides. Modi cation of these enzymes and the pathway allowed the cells to diversify their capsular polysaccharide components.
Comparative sequence analysis of the HA biosynthetic genes from different capsular types showed a high level of within-species similarity because these genes were a member of the capsule biosynthesis locus (cap locus), a gene cluster for capsular polysaccharide biosynthesis [25,28,33]. The absence of the hyaD gene would not affect heparin and chondroitin sulfate biosynthesis because the enzyme is not required. Only the hyaD gene of the capsular types A:3 and F differed signi cantly from that of the other strains, suggesting a functional difference between HA and chondroitin biosynthesis. The disappearance of hyaD in type D could be due to the requirement of a different enzyme to synthesize the heparin capsular component [34]. The protein pattern and phylogenetic analysis supported the sequence alignment of these genes. These results indicated that the HA biosynthetic genes of types A and D had a closer relationship compared to those of types B and F, except hyaD when compared between capsular type A strain 3480 and capsular type D. Although strains with the same capsular type evidently have highly similar genes in the pathway, this study found that the type A strains had diverse patterns of HA biosynthetic genes. The genes of the capsular type F strain shared more similarity to those of the type A:3 strain (previously used in genetically engineered E. coli) than those of the others of type A strains. However, this study was unable to subtype some of the type A samples, and the deviation of type A:3 could potentially affect their enzymatic activities. The HyaD pattern of the type B strain was similar to that of type A, which might indicate the ability to synthesize HA-containing capsules in this capsular type.
Structures of the Glck, Pgm, GlmU, and HyaD proteins showed individual variations across different strains of P. multocida. Analysis of the binding pockets of Glck and Pgm by comparison with the structures of these enzymes in other organisms found that the sites were irrelevant to the catalytic pockets. In contrast, some of the GlmU and HyaD binding pockets were at the catalytic sites. HyaD also contains two active sites: one site has glucuronic acid-transferase activity, which elongates UDPglucuronic acid to oligosaccharides, and the other site has glucosamine-1-P-transferase activity, which prolongs UDP-N-acetyl-D-glucosamine to oligosaccharides [35]. Surface variation in the binding sites of the HyaD protein in this study was consistent with previous studies that identi ed the enzymatic binding site in the glucuronic acid-transferase domain of HyaD [35]. Our study found that the arrangement of DUF2536 and TPR_2 repeats in the HyaD protein differed in these P. multocida strains, suggesting the reason for the structural variation of the binding site and perhaps different molecular activities. HyaD and HyaC were also reported to be rate-limiting enzymes for HA biosynthesis in bacteria and humans due to the limited substrate (UDP-D-glucuronic acid) produced by HyaC and the speci city of HyaD to the polymerization of UDP-D-glucuronic acid and UDP-N-acetyl-D-glucosamine [36][37][38]. Therefore, it is possible that the HyaD protein of the P. multocida strains in this study could have different substrate speci cities and catalytic rates.
The protein structure of GlmU was previously studied in E. coli, and the catalytic site of this protein was also matched to the variable binding regions predicted from the P. multocida strains in this study. These variable regions correlated with N-terminal pyrophosphorylase and C-terminal acetyltransferase catalytic sites [39,40]. GlmU has two active sites: (i) acetyltransferase, which is responsible for CoA-dependent acetylation of D-glucosamine-1-P to N-acetyl-D-glucosamine-1-P at the C-terminal domain, and (ii) pyrophosphorylase, which catalyses the transfer of uridyl from UTP to D-glucosamine-1-P, forming UDP-N-acetyl-D-glucosamine and pyrophosphate at the N-terminal domain [40]. However, compared to previous studies in E. coli and Salmonella enterica, the binding regions of Glck and Pgm were not related to the active sites. Nevertheless, the binding regions of these proteins might help to regulate enzyme activity [41,42]. Consequently, structural variations of these four enzymes could potentially affect molecular function, substrate speci city, and catalytic rate in HA biosynthesis.
The results from all analyses in this study emphasized differences within the enzymes of capsular types A:3, A:1, and A strains. These three types had HA-containing capsules with different somatic serotypes important to virulence [43][44][45]. These three serotypes may evolve different virulence factor-associated genes as well as different capsule and lipopolysaccharide (LPS) compositions. The somatic serotype was found to be more speci c to the disease than the capsule type and quite diverse between strains [46]. Further investigation of the HyaD proteins in these type A strains might help improve industrial HA production, allowing different alternatives to HyaD enzymes, as well as commercial biosynthesis of heparan and chondroitin, where other enzymes related to the HA biosynthetic pathway can be used.

Conclusion
This study identi ed nine genes involved in the HA biosynthetic pathway of P. multocida strains. Eight genes had a high level of within-species similarity (98-99%), while hyaD of strains with capsular type A:3 and F differed signi cantly those of other strains. Sequence, phylogenetic and pattern analysis of these genes showed that capsular types A and D had a close relationship compared to capsular types B and F. Structural comparison revealed variations in the Glck, Pgm, GlmU, and HyaD proteins. The variations within the active sites and domains of HyaD and GlmU could in uence enzymatic function and substrate speci city. Thus, these enzymes will be potential targets for improving HA production.

Materials And Methods
Identi cation of hyaluronic acid biosynthetic genes  [1] were used to search for orthologous nucleotide sequences in the obtained P. multocida genomes using BLASTn [47]. A similar search was performed against the genomes of Homo sapiens, E. coli strain K-12 MG1655, Streptococcus pyogenes strain M1, and S. thermophilus to identify orthologous genes according to previous reports [48,49], which studied the HA production of these strains, except E. coli, which is often used for the expression of recombinant HA biosynthesis enzymes. Identi cation of orthologous genes was considered at a percentage identity cut-off above 35, an e-value higher than 10e-10, and relevant functional information. Protein sequences encoded from these genes were obtained from the NCBI protein database.

Sequence comparison and amino acid pattern analysis
Nucleotide and amino acid sequences of the HA biosynthesis genes were edited and aligned by using the MAFFT program version 7 [50] to identify conserved regions across different capsular types of P. multocida, and the sequences were compared to those of other organisms using iterative re nement methods (G-INS-i) and the Needleman-Wunsch algorithm, which extracted the conserved sequences and truncate anking sequences [51,52]. Amino acid patterns were analysed from the aligned protein sequences to identify possible variations that could impact the enzymatic function. The pattern was considered different if there was at least one position within the aligned sequence block that differed from other samples at the same position. Numerical patterns were assigned to depict sequence variations of the proteins. This pattern analysis assumed that the orthologous proteins would have a similar molecular function if their amino acid sequences were more similar. A numerical pattern of nine digits was obtained for each sample to represent variations within these nine HA biosynthetic enzymes. These numeric sets were then compared within different P. multocida strains and across different organisms.
Phylogenetic analysis of the hyaluronic acid biosynthetic genes Phylogenetic analysis was employed to analyse the evolutionary relationship of these nine HA biosynthetic genes from different P. multocida strains and other organisms, including H. sapiens, E. coli, S. pyogenes strain M1 GAS, and S. thermophilus as an outgroup. The phylogenetic relationships were constructed from the aligned nucleotide and amino acid sequences based on the Tamura-Nei and Dayhoff models using the maximum likelihood (ML) and neighbour-joining (NJ) methods with 1,000 bootstraps performed with the MEGA program version 7.0 [53]. The phylogenetic relationships were compared to the amino acid sequence patterns previously described to understand the relatedness of the HA biosynthetic enzymes. The nucleotide and amino acid sequences of these nine genes were edited and concatenated using the AliView program version 3.0 (GPLv3) [54] before the sequence alignment and phylogenetic analysis as previously described.

Figure 2
Pattern analysis of the amino acid sequences of nine hyaluronic acid biosynthetic proteins from different P. multocidastrains. The patterns were derived from a single amino acid difference on each enzyme based on the multiple aligned sequences. Pattern numbers and colour shades were compared on the same row, representing variations in the same enzyme.