In order to reach the cell wall, phage lysins have to cross the cell membrane. However, they generally lack a signal peptide that would allow them to do so. In order to solve such a problem, phage viruses synthesize another protein called holin which binds to the cell membrane and makes holes in it (hence its name), allowing lysins to reach the peptidoglycan matrix. The prototypical holin is the lambda phage S protein, which assists the lambda phage R protein (lysin). All holins embed themselves in the cell membrane and contain at least two transmembrane helical domains. The hole making process is thought to be achieved by holin oligomerization at a specific moment when progeny virions are set to be released. Phage lysins are generally species or subspecies specific, which means that they are only effective against bacteria from which they were produced. While some lysins only act upon the cell walls of several bacterial phylotypes, some broad-spectrum lysins have been found. Similarly, some thermostable lysins are known, which makes them easier to use in biotechnology. Regarding their use as antibacterial agents, lysins have been found effective mainly against Gram-positive bacteria, since Gram-negative bacteria possess an outer membrane that prevents extracellular lysin molecules from digesting peptidoglycan. However, lysins with activity against Gram-negative bacteria, such as OBPgp279, have garnered interest as potential therapeutics. One of the most problematic aspects of the use of phage lysins as antimicrobial agents is the potential immunogenicity of these enzymes. Unlike most antibiotics, proteins are prone to antibody recognition and binding, which means that lysins could be ineffective when treating bacterial infections or even dangerous, potentially leading to a systemic immune response or a cytokine storm. Nonetheless, experimental data from immunologically-rich rabbit serum showed that hyperimmune serum slows down but does not block the activity of pneumococcal lysin Cpl-1. Phage lysins have been successfully tested in animal models to control pathogenic antibiotic-resistant bacteria found on mucous membranes and in blood. The main advantage of lysins compared to antibiotics is not only the low bacterial resistance but also the high specificity towards the target pathogen, and low activity towards the host's normal bacterial flora. Lysins were first used therapeutically in animals in 2001, in a publication in which mice orally colonized with "Streptococcus pyogenes" were decolonized with a single dose of PlyC lysin delivered orally.