Diversity and Standard Nomenclature of Staphylococcus aureus Hyaluronate Lyases HysA and HysB

ABSTRACT Bacterial hyaluronate lyases (Hys) are enzymes that degrade hyaluronic acid in their host and are known to contribute to the pathogenesis of several illnesses. The first two identified Hys genes in Staphylococcus aureus were registered as hysA1 and hysA2. However, their annotations have been mistakenly reversed in some registered assembly data, and different abbreviations (hysA and hysB) in some reports complicates comparative analysis of Hys proteins. We investigated the hys loci of S. aureus genome sequences registered in public databases, analyzed the homology, and defined hysA as hys located in the core genome surrounded by a lactose metabolic operon and a ribosomal protein cluster present in almost all strains and hysB as that located on the genomic island νSaβ of the accessory genome. Homology analysis of the amino acid sequences of HysA and HysB revealed that they are conserved among clonal complex (CC) groups with a few exceptions. Thus, we propose a new nomenclature for S. aureus Hys subtypes: HysACC*** for HysA and HysBCC*** for HysB, with the asterisks representing the clonal complex number of the S. aureus strain producing the Hys subtype. The application of this proposed nomenclature will facilitate the intuitive, straightforward, and unambiguous designation of Hys subtypes and contribute to enhancing comparative studies in this regard. IMPORTANCE Numerous whole-genome sequence data for Staphylococcus aureus harboring two hyaluronate lyase (Hys) genes have been registered. However, the assigned gene names for hysA1 and hysA2 are incorrect in some assembled data, and in some cases, the genes are annotated differently as hysA and hysB. This creates confusion with respect to the nomenclature of Hys subtypes and complicates analysis involving Hys. In this study, we compared the homology of Hys subtypes and observed that to some extent, their amino acid sequences are conserved in each clonal complex group. Hys has been implicated as an important virulence factor, but relative sequence heterogeneity among S. aureus clones raises the question of whether Hys activities are different among these clones. Our proposed Hys nomenclature will facilitate comparison of the virulence of Hys, as well as discussions of the subject.

IMPORTANCE Numerous whole-genome sequence data for Staphylococcus aureus harboring two hyaluronate lyase (Hys) genes have been registered. However, the assigned gene names for hysA1 and hysA2 are incorrect in some assembled data, and in some cases, the genes are annotated differently as hysA and hysB. This creates confusion with respect to the nomenclature of Hys subtypes and complicates analysis involving Hys. In this study, we compared the homology of Hys subtypes and observed that to some extent, their amino acid sequences are conserved in each clonal complex group. Hys has been implicated as an important virulence factor, but relative sequence heterogeneity among S. aureus clones raises the question of whether Hys activities are different among these clones. Our proposed Hys nomenclature will facilitate comparison of the virulence of Hys, as well as discussions of the subject. KEYWORDS Staphylococcus aureus, hyaluronate lyase, HysA, HysB, amino acid sequences S taphylococcus aureus, a commensal bacterial species that can colonize the skin and mucous membranes of the human body, can cause various infections, ranging from mild skin lesions to severe systemic infections (1).
Hyaluronate lyases (or hyaluronidases) are a group of enzymes that degrade hyaluronate, a high-molecular-weight, linear, unsulfated glycosaminoglycan polymer comprising alternating units of b-D-(1!3)-glucuronic acid and b-D-(1!3)-N-acetylglucosamine in the extracellular matrix. Furthermore, bacterial hyaluronate lyases (Hys), which act as endo-N-acetylhexosaminidases by eliminating the b-(1!4) linkage, resulting in the formation of a double bond in D-glucuronic acid (2), have been identified in various Gram-positive and -negative bacteria and have also been implicated in tissue penetration and invasion (2, 3). Jones et al. reported that proteomic analysis of the proteins secreted by S. aureus UAMS-1 led to the identification of two Hys proteins, encoded by hysA1 and hysA2 (4). Southern blot analysis of various strains further indicated that S. aureus possesses at least one hysA hybridizing band. In contrast, hybridizing bands have not been observed for other species of the genus Staphylococcus, suggesting that Hys is a potential determinant of the virulence of S. aureus (5). The strain UAMS-1 whole-genome sequence used in the study by Hart et al. (5) has been registered in a public database (6). The sequence similarity between the hysA1 and hysA2 genes of UAMS-1 (GenBank assembly accession no. GCA_000788115.1) was found to be 80.3%, while that between the HysA1 and HysA2 amino acid sequences of UAMS-1 was found to be 74.9%. Further, numerous whole-genome sequence data corresponding to S. aureus harboring two hys genes have been registered in the NCBI database (7). However, in some of the registered assembly data, the annotations of hysA1 and hysA2 were mistakenly reversed, and in some cases, they were annotated as hysA and hysB, respectively. This complicates the comparative analysis of Hys.
To clarify this issue, we investigated the hys loci of S. aureus genome sequences registered in public databases and analyzed the sequence similarity. As a result, we redefined the two hys genes (hysA1 and hysA2) with low sequence similarity as "hysA" and "hysB." The hysAand hysB-flanking regions of representative strains are shown in Fig. 1. We defined hysA as hys located in the core genome surrounded by a lactose metabolic operon and the ribosomal protein cluster found in almost all strains. In contrast, we defined hysB as that located on the genomic island Sab of the accessory genome in a limited number of isolates. Klaui et al. classified Sab genomic islands into 15 different types and demonstrated their close correlation with clonal complexes (CCs) (8). Among them, eight Sab types (III, IV, V, VI, VII, VIII, XI, and XIV) were found to carry a sequence of hysB in situ, and of these, two Sab types (VII and XI) were found to possess a truncated form (Fig. 1). We compared the amino acid sequences of HysA and HysB among the various CCs registered in public databases. The sequences used in this study are listed in Table S1 in the supplemental material. We aligned the HysA or HysB sequences of various sequence types (STs) using the ClustalW program with Molecular Evolutionary Genetic Analysis (MEGA) v10.1.5 (9). We then constructed a phylogenetic tree of the aligned amino acid sequences using RAxML-NG v1.0.1 (10), with the best model inferred using ModelTest-NG v0.1.7 (11) and 1,000 bootstrap replicates. Thus, comparative analysis revealed that S. aureus strains positive for HysA and those positive for HysB clustered separately, and each of the clusters shared high homology, forming a triangle (Fig. 2, left). The HysA amino acid sequences of CC8 and CC15 (cluster A18), and CC1, CC72, and CC188 (cluster A22) were 100% identical. CC97 and CC834 (cluster A20), CC6 and CC9 (cluster A23), and CC5 and CC88 (cluster A24) were over 98%, 98.7%, and 99.9% identical, respectively (Fig. 2, left). Therefore, we demonstrated that most hysA genes were conserved to some extent in each CC group. However, as an exception, the HysA sequence identity of ST772 belonging to CC1 and other STs in CC1 was 88.7%, and that between ST93 and ST121 belonging to CC121 was 87.3%. Thus, they were classified into individual clusters represented by the ST number. The HysB-positive CC groups were represented as CC15, CC30, CC59, ST93, CC97, CC101, CC779, CC398, and CC3291 (Fig. 2, left). Comparative analysis further revealed that the HysB amino acid sequence similarity between CC15 and CC97 (cluster B8) and between CC398 and CC3291 (cluster B4) was over 99.6% and 99.8%, respectively. In contrast, the HysB sequences of CC59 were truncated, exhibiting 36% sequence similarity among other CC groups (Table S1). Therefore, similarly to HysA, most of the HysB sequence was conserved among the CC groups.
We also compared the coding sequences of the a-hemolysin gene (hla), which is a ubiquitous virulence gene present in the genomic island, Sag , as a control (Fig. 2, right). Thus, we observed that the sequence similarity of Hla was highly conserved among various STs, showing over 98% identity in almost all the STs. The only exception was Hla in ST36, which exhibited a considerably lower similarity (35%), indicating that it was truncated in ST36.
Given that the absence of specificity or consistency in HysA or HysB nomenclature may lead to confusion in characterizing Hys-producing S. aureus, we propose a standard notation for a large variety of HysA and HysB. According to the nomenclature for staphylococcal superantigens (12), we recommend that hys genes be alphabetized in the order in which they are identified; therefore, a gene should be named hysA if it is located on the core genome and hysB if it is located on a mobile element. Further, we recommend naming new, protein-coding hys genes with 90% homology as "hysA CC*** " for HysA subtypes and "hysB CC*** " for HysB subtypes, with the asterisks indicating the clonal complex number of S. aureus. Hys has been implicated as one of the important virulence factors of S. aureus; however, the relative sequence heterogeneity among S. aureus clones raises the question of whether the observed Hys activities are different, as this may affect the virulence of S. aureus clones. Our study results and the proposed nomenclature will facilitate the intuitive, straightforward, and unambiguous designation of HysA or HysB genes and provide a platform for comparing the Hys activity in S. aureus clones in future studies. We may also need to consider the nomenclature of other virulence factors and enzymes of S. aureus with similar nomenclature conflicts in future studies.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, XLSX file, 0.1 MB.