3-6. CBD from bacterial genome

Although there has been an increasing interest in the use of CBDs to detect bacterial pathogens as alternatives to currently used antibodies, identifying a novel CBD from a phage endolysin is time-consuming and labor-intensive due to the following reasons. First, a novel phage should be isolated and its genome should be extracted and sequenced. Second, the full genome annotation requires skilled person and considerable time. Therefore, a novel method to find a CBD is needed for the wide use of CBDs as bioprobes. Here, I present a facile and efficient method for identifying a novel CBD from a sequenced bacterial genome. This method relies on the idea that most bacteria harbor phage genes within their genome as prophages or short phage remnants (Brussow et al., 2004). For example, the genome of Escherichia coli O157:H7 strain Sakai contains 16% of prophage elements (Hayashi et al., 2001), and in Streptococcus pyogenes, phage-related sequences account for more than 10% of the total genome (Beres et al., 2002). These phage genes are often involved in lateral gene transfer and they have a major impact on bacterial genome evolution (Canchaya et al., 2003).

Based on this idea, I hypothesized that I could exploit these sequences as potential reservoir for extracting CBDs of various target pathogens. In this

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thesis, I chose C. perfringens as a model organism because anaerobic nature of this bacterium makes it tricky to grow, which in turn leads to difficulties in C. perfringens phage isolation.

III-3-6-1. Analysis of lytic enzymes in C. perfringens ATCC 13124

C. perfringens ATCC 13124 originally isolated from a gas gangrene patient (Myers et al., 2006) was used as a source for finding a putative CBD within a bacterial genome as its complete genome sequence is available at NCBI database (reference sequence: NC_008261.1). I used amino acid sequences of catalytic domains of the selected endolysins for BLAST search because the catalytic domains are conserved relatively higher than the full-length endolysins, which generally contain host-specific cell wall binding domain(s). The BLAST results showed four Psm homologs and one Ply3626 homolog in the genome of C. perfringens ATCC 13124 (Table III-8).

Among these five putative lytic enzymes, YP_695420 is identical to the well-characterized muramidase, PlyCM (Schmitz et al., 2011), and encoded within prophage region of the genome containing phage terminase, holin, and capsid protein encoding genes. YP_696011 is almost same as Psm (96%

identity) and resides in another prophage region. The absence of phage associated genes nearby both YP_695964 and YP_696189 encoding genes

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suggests that these two lytic enzymes may be bacterial cell wall autolysins.

Especially, YP_696189 (putative amidase) showed a 64% amino acid sequence identity with the phage endolysin Ply3626, indicating close relationship between phage endolysins and bacterial autolysins (Loessner et al., 1997). YP_694826 seemed to reside in small prophage remnant region due to the lack of nearby phage-related genes except a holin (YP_694827).

Table III-8. Homologs of Psm and Ply3626 in C. perfringens ATCC 13124

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III-3-6-2. Identification of CBD from CPF369

Among the five lytic enzymes, I analyzed YP_694826 (Gene symbol:

CPF_0369, I hereafter refer to YP_694826 protein as CPF369) further for two reasons. First, the CPF369 protein contains an terminal catalytic N-acetylmuramidase (glucoside hydrolase 25 family) domain and two C-terminal tandem repeated SH3_3 domains (Fig. III-13A), which are presumed to function in bacterial cell wall binding (Xu et al., 2009). Second, I could predict the protein structure of CPF369 more accurately (Fig. III-13B) because the crystal structure of Psm endolysin, which shares significant amino acid identity (71%) with the CPF369 (Fig. III-13C), is available at the Protein Data Bank (PDB ID: 4KRT) (Tamai et al., 2014). Although both YP_696011 and YP_695420 (PlyCM) also fulfill these criteria, I preferred to give priority to dissimilarity of CPF369 to previously characterized endolysins. As shown in Fig. III-13B, I could identify putative linker region between N-terminal enzymatic active domain (EAD) and C-terminal CBD consisting of two SH3_3 domains. These results made it easier for me to predict CBD of CPF369 (CPF369_CBD) starting from Val 203 to stop codon.

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Fig. III-13. Schematic description of CPF369. (A) Schematic description of CPF369. (B) Deduced three-dimensional model of CPF369. An enzymatic active domain (GH25, red) and tandem-repeated SH3_3 domains (green) are connected by flexible linker (yellow). (C) Amino acid sequence alignment among CPF369-related endolysins. PlyCM, endolyins of C. perfringens phage phiSM101; Psm, endolysin of C. perfringens phage phi3626.

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III-3-6-3. C. perfringens-specific binding activity of CPF369_CBD

The EGFP-CPF369_CBD fusion protein was expressed in E. coli and easily purified to homogeneity by single Ni-NTA affinity chromatography because of the N-terminal decahistidine tag Fig. III-14A.

EGFP-CPF369_CBD could bind all C. perfringens strains tested, whereas other genus or species bacteria could not be labeled by the fusion protein Fig.

III-14B and Table III-9). EGFP alone did not label the C. perfringens cells (Fig. III-14C). These results confirmed that CPF369_CBD specifically binds to C. perfringens cells. The effects of NaCl and pH on the binding activity of EGFP-CPF369_CBD fusion protein were investigated (Fig. III-14D, E). The optimum NaCl concentration was 50 mM, but the CPF369_CBD retained a certain degree (43%) of its binding activity even at 2 M of NaCl. The optimum pH condition for binding activity of CPF369_CBD was between pH 7 and 8. The binding activity decreased rapidly at pH 10, resulting in only 17% of binding compared to the value at pH 7.

In sum, I successfully identified a CBD from a genome (NCBI gene ID: 4201291) of C. perfringens ATCC 13124 and confirmed its specific binding to C. perfringens. This method only requires simple homology analysis; neither phage isolation nor phage genome sequencing is needed. I

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think that it could be a general approach for CBD identification from sequenced bacterial genomes. With the rapid increase of bacterial genome announcements, my method will be useful for discovering novel bioprobes for detection of pathogenic bacteria.

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Fig. III-14. C. perfringens-specific binding activity of EGFP-CPF369_CBD and the effect of NaCl and pH on the binding activity of EGFP-CPF369_CBD. (A) Purified EGFP-CPF369_CBD fusion protein. M, prosi prestained protein marker (GenDepot). (B) C. perfringens-specific binding activity of CPF369_CBD. (C) EGFP alone did not bind to C.

perfringens cells. Scale bars, 10 um. (D-E) Binding of EGFP-tagged CPF369_CBD on C. perfringens cells under different NaCl (D) and pH (E) conditions. All assays were carried out in triplicate.

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Table III-9. Binding spectrum of CPF369_CBD

ATCC, American Type Culture Collection; NCTC, National Collection of Type Cultures.

172 endolysin consisting of three distinct domains: an N-terminal CHAP domain (PF05257), a central amidase_2 domain (PF01510), and a C-terminal SH3_5 domain (PF08460) (Fig. III-15A). Since the SH3_5 domain was presumed to be CBD, I cloned and produced the EGFP tagged SA13_CBD protein in E.

coli. The fusion protein was overexpressed in the soluble form, and purified by Ni-NTA chromatography (Fig. III-15B). Florescence microscopy analysis revealed that SA13_CBD showed broad binding spectrum against S. aureus cells as all of tested S. aureus strains, including methisillin-resistant S.

aureus (MRSA) strains, were decorated by SA13_CBD (Fig. III-15C, Table III-10). Not only S. aureus strains, but also other Staphylococcal strains (except S. cohinii) were labeled by the SA13_CBD. SA13_CBD did not bind to other genera of bacteria. Taken together, SA13_CBD has broad binding properties towards Staphylococcus cells, supposing that conserved epitopes of cell wall of Staphylococcal cells may be the binding target of SA13_CBD.