**2. Bacteriophage reproduction**

#### **2.1. Lytic cycle**

Phages replicate inside bacterial host and the process finalizes with lysis of the host and spreading of phage progeny. Phage replication includes the following steps [30]:


#### **2.2. Lysogenic cycle**

they reproduce by a lysogenic cycle [24]. Bacteriophages which possess double-strand DNA express highly specific enzymes called viral-associated peptidoglycan hydrolyses (VAPGH) that bind to the bacterial cell surface and cause disruption of the cell wall to inject their DNA into the host cell [25]. The filamentous phage releases their viral progeny without causing the death of the bacteria [18], while nonfilamentous phages cause bacterial lysis by synthesizing endolysins (enzymes encoded by double-strand DNA phages) that hydrolyze peptidoglycan as part of an holin-endolysin system. The endolysins and holins are synthesized at late stages of phage infection. Endolysins accumulate in the cytoplasm until viral particles are assembled and holins form pores in the membrane allowing cytoplasmic translocation of endolysins through the membrane for peptidoglycan degradation [26]. Furthermore, single-stranded DNA or RNA bacteriophages synthesize "lysines" which interfere or inhibit the synthesis of the bacterial peptidoglycan [27]. The VAPGHs and endolysins are able to degrade the peptidoglycan when applied externally, which is why these enzymes represent an alternative to be used as enzybiotics in Gram-positive bacteria [28]. Bacteriophages and their endolysins are highly specific, infecting or hydrolyzing only a single species of bacteria attaching to specific receptors on the surface of host cell. The specificity of interaction between phage attachment

Phages replicate inside bacterial host and the process finalizes with lysis of the host and

**1. Adsorption**. Phage attachment to a specific host cell in a process involving interaction with receptors on the surface of a susceptible host cell and an infecting virus. There are two major types of receptors: components of a bacterial cell like lipopolysaccharide, peptidoglycan, outer membrane proteins and teichoic acids, and fimbriae-type receptors

**2. Nucleic acid injection**. Through the tail, phage injects its genetic material into the cell after peptidoglycan degradation behind pore formation (by VAPGH). The phage coat protein that includes capping head and tail structure remains attached to the bacterial

**3. Replication**. After injection of its nucleic acid, phage expresses early genes that redirect host synthesis machinery to the reproduction of viral nucleic acid and proteins.

**4. Assembly and packing phage particles**. Once the viral components are synthesized, the genetic material is encapsulated in its protein coat, and complete virus particles are

**5. Phage progeny release**. Phage late proteins like holins and endolysins or murein synthesis inhibitors are produced, and they are responsible for the lysis of the host cell and the

spreading of phage progeny. Phage replication includes the following steps [30]:

structures and host cell surface receptors determinates host range. [29].

**2. Bacteriophage reproduction**

like *pilli* or flagella.

surface.

formed.

release of viral particles to the environment.

**2.1. Lytic cycle**

184 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

The lysogenic cycle comprises the same steps as lytic cycle, but after penetration of the genetic material, the phage nucleic acid is inserted into the chromosome of the bacteria and is replicated as a segment of the own bacterial genome for one or more generations without metabolic consequences for the bacterium. After this cycle, the genetic material of the phage can be excised from the bacterial chromosome and enter into a lytic cycle; usually, this occurs under physiological stress or damage of the genetic material.

### **3. Endolysins**

The term endolysin was coined until 1958 to refer to the phage component responsible for the bacterial lysis. Lytic phages present a genetic cassette encoding a holin-endolysin system. At the end of the reproductive cycle, once mature viral particles have been assembled, holins are synthesized in critical concentrations and inserted into the cell membrane, creating pores for the translocation of endolysins, previously accumulated in the cytoplasm, to reach the peptidoglycan structure [19]. Endolysins are classified according to its enzymatic activity (**Figure 1**) in: (1) N-acetylmuramoyl-alanine amidases, which hydrolyze the amide bond

**Figure 1.** Enzymatic activities of endolysins. (A) N-acetyl-muramidase catalyzes the hydrolysis of N-acetylmuramoilβ-1,4-N-acetylglucosamine. (B) N-acetylglucosaminidase catalyzes the hydrolysis of N-acetylglucosaminil-β-1,4-N-acetylmuramine. (C) Endopeptidase hydrolyzes peptidic bonds on amino acids chains linked to the glycan moiety or in the pentapeptidic bridge. (D) N-acetylmuramoyl-L-alaninamidase hydrolyzes the amide bond that connects the glycan with the amino acids. (E) Transglycosylases attach the glycosidic β-1,4 bonds resulting in the formation of a 1,6 anhydrous ring in N-acetylmuramic acid (modified from Barrera-Rivas et al. [19]).

between the N-acetyl-muramic in the glycan chain and the L-alanil residues; (2) endo-β-Nacetylglucosaminidases, which hydrolyzes the N-acetylglucosamine β-1,4-N-acetylmuramine acid linkage; (3) N-acetyl-muramidases, which catalyze the hydrolysis of N-acetylmuramoilβ-1,4-N-acetilglucosamine bond; (4) transglycosylases, which disrupt β-1-4 glycosidic bonds by forming a 1–6 anhydride ring in the N-acetylmuramic residue; (5) endopeptidases, which may hydrolyze both the tetrapeptide linked to the glycosil moieties and the pentapeptide entrecrossing bridge [31, 32].

Endolysins encoded by double-stranded DNA bacteriophages have a molecular weight between 25 and 40 kDa [33]. Most of endolysins are composed of at least two functional domains: one containing the catalytic activity located generally in the N-terminal domain and one responsible for the recognition of a specific substrate associated with the C-terminal domain. In some cases, more than one catalytic domain or more than one recognition domain are present [19]. The recognition domain usually joins to specific molecules in the bacterial cell envelopes such as monosaccharides, coline or teichoic acids [34]. Endolysin activity is usually species specific, although there have been reports of endolysins with a wider substrate range. Besides, the cell wall recognition domain is not always essential for endolysin activity. The endolysin got a wider substrate range, but it conserved certain specificity, since it was no active against all bacteria. Studies of crystallography and mutation analysis with endolysin *PlyL* against *Bacillus anthracis* led to propose that the C-terminal domain of this endolysins inhibits the activity of the catalytic domain by particular intermolecular interactions. This inhibition is released when the C-terminal domain binds to its particular ligands in the target cell wall, thus acting as a regulatory domain [35]. Most of the reported endolysins from phages against *S. aureus* have two catalytic domains and a cell wall recognition domain being LysK one of the must studied endolysin models. LysK has a cysteine/histidine-dependent aminohydrolase/peptidase (CHAP) catalytic domain that hydrolyzes the peptidic bond between the D-alanine of the oligopeptide chain attached to the sugar backbone and the first glycine of the pentaglycine bridge that is typical of *S. aureus* peptidoglycan and confers resistance to lysozyme. CHAP presents the higher activity of both hydrolytic domains. LysK also has an N-acetylmuramoyl L-alanine amidase or amidase-2 (Ami-2) catalytic domain which catalyzes the hydrolysis of the N-glycosidic bond between the N-acetylmuramic residue and the L-alanine of the oligopeptide attached to the sugar backbone. A third domain called SH3b is responsible for the specific recognition of cell wall components, strain specificity and modulator of hydrolytic activities [36, 37]. Endolysin 2638A has similar triple domain structures: an amino-terminal domain with endopeptidase activity, a central Ami-2 domain (with the highest activity in this phage) and a SH3b cell wall recognition domain [38]. Modular structure of *S. aureus* endolysins has allowed the construction of chimeric endolysins by the combination of catalytic and/or recognition domains. An example is the endolysin Ply187AN-KSH3b, which is a translational fusion of the CHAP domain of phage Ply187 and the cell wall recognition domain SH3b from LysK endolysin. This endolysin was effective in a mouse model of endophthalmitis that also decreased inflammatory response and protected the retina from tisular damage [39].
