Lorena Rodríguez-Rubio

Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics

Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. In contrast to endolysins, PGHs that mediate lysis of the host bacteria at the end of the lytic cycle to release of phage progeny, the action of VAPGHs generates a small hole through which the phage tail tube crosses the cell envelope to eject the phage genetic material at the beginning to the infection cycle. The antimicrobial activity of VAPGHs was first discovered through the observation of the phenomenon of ‘lysis from without’, in which the disruption of the bacterial cell wall occurs prior to phage production and is caused by a high number of phages adsorbed onto the cell surface. Based on a unique combination of properties of VAPGHs such as high specificity, remarkable thermostability, and a modular organization, these proteins are potential candidates as new antibacterial agents, e.g. against antibiotic-resistant bacteria in human therapy and veterinary as well as biopreservatives in food safety, and as biocontrol agents of harmful bacteria in agriculture. This review provides an overview of the different VAPGHs discovered to date and their potential as novel antimicrobials.

Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

ABSTRACT Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.

Phage lytic proteins: biotechnological applications beyond clinical antimicrobials

Abstract Most bacteriophages encode two types of cell wall lytic proteins: endolysins (lysins) and virion-associated peptidoglycan hydrolases. Both enzymes have the ability to degrade the peptidoglycan of Gram-positive bacteria resulting in cell lysis when they are applied externally. Bacteriophage lytic proteins have a demonstrated potential in treating animal models of infectious diseases. There has also been an increase in the study of these lytic proteins for their application in areas such as food safety, pathogen detection/diagnosis, surfaces disinfection, vaccine development and nanotechnology. This review summarizes the more recent developments, outlines the full potential of these proteins to develop new biotechnological tools and discusses the feasibility of these proposals.

Bacteriophages as Weapons Against Bacterial Biofilms in the Food Industry

Microbiological contamination in the food industry is often attributed to the presence of biofilms in processing plants. Bacterial biofilms are complex communities of bacteria attached to a surface and surrounded by an extracellular polymeric material. Their extreme resistance to cleaning and disinfecting processes is related to a unique organization, which implies a differential bacterial growth and gene expression inside the biofilm. The impact of biofilms on health, and the economic consequences, has promoted the development of different approaches to control or remove biofilm formation. Recently, successful results in phage therapy have boosted new research in bacteriophages and phage lytic proteins for biofilm eradication. In this regard, this review examines the environmental factors that determine biofilm development in food-processing equipment. In addition, future perspectives for the use of bacteriophage-derived tools as disinfectants are discussed.

The Phage Lytic Proteins from the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 Display Multiple Active Catalytic Domains and Do Not Trigger Staphylococcal Resistance

The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we analyzed the specific cleavage sites on the staphylococcal peptidoglycan produced by three phage lytic proteins. The investigated cell wall lytic enzymes were the endolysin LysH5 derived from the S. aureus bacteriophage vB_SauS-phi-IPLA88 (phi-IPLA88) and two fusion proteins between lysostaphin and the virion-associated peptidoglycan hydrolase HydH5 (HydH5SH3b and HydH5Lyso). We determined that all catalytic domains present in these proteins were active. Additionally, we tested for the emergence of resistant Staphylococcus aureus to any of the three phage lytic proteins constructs. Resistant S. aureus could not be identified after 10 cycles of bacterial exposure to phage lytic proteins either in liquid or plate cultures. However, a quick increase in lysostaphin resistance (up to 1000-fold in liquid culture) was observed. The lack of resistant development supports the use of phage lytic proteins as future therapeutics to treat staphylococcal infections.

From endolysins to Artilysin®s: novel enzyme-based approaches to kill drug-resistant bacteria.

One of the last untapped reservoirs in nature for the identification of new anti-microbials is bacteriophages, the natural killers of bacteria. Lytic bacteriophages encode peptidoglycan (PG) lytic enzymes able to degrade the PG layer in different steps of their infection cycle. Endolysins degrade the bacterial cell wall at the end of the infection cycle, causing lysis of the host to release the viral progeny. Recombinant endolysins have been successfully applied as anti-bacterial agent against antibiotic-resistant Gram-positive pathogens. This has boosted the study of these enzymes as new anti-microbials in different fields (e.g. medical, food technology). A key example is the recent development of endolysin-based anti-bacterials against Gram-negative pathogens in which the exogenous application of endolysins is hindered by the outer membrane (OM). These novel anti-microbials, termed Artilysin®s, are able to pass through the OM and reach the PG where they exert their action. In addition, mycobacteria whose cell wall is structurally different from both Gram-positive and Gram-negative bacteria have also been reported to be inhibited by mycobacteriophage-encoded endolysins. Endolysins and endolysin-based anti-microbials can be considered as ideal candidates for an alternative to antibiotics for several reasons: (1) their unique mode of action and activity against bacterial persisters (independent of an active host metabolism), (2) their selective activity against both Gram-positive and Gram-negative pathogens (including antibiotic resistant strains) and mycobacteria, (3) the limited resistance development reported so far. The present review summarizes and discusses the potential applications of endolysins as new anti-microbials.

Potential of the Virion-Associated Peptidoglycan Hydrolase HydH5 and Its Derivative Fusion Proteins in Milk Biopreservation

Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88], and three different fusion proteins created between HydH5 and lysostaphin has been assessed. The lytic activity of the five proteins (HydH5, HydH5Lyso, HydH5SH3b, CHAPSH3b and lysostaphin) was confirmed using commercial whole extended shelf-life milk (ESL) in challenge assays with 104 CFU/mL of the strain S. aureus Sa9. HydH5, HydH5Lyso and HydH5SH3b (3.5 µM) kept the staphylococcal viable counts below the control cultures for 6 h at 37°C. The effect is apparent just 15 minutes after the addition of the lytic enzyme. Of note, lysostaphin and CHAPSH3b showed the highest staphylolytic protection as they were able to eradicate the initial staphylococcal challenge immediately or 15 min after addition, respectively, at lower concentration (1 µM) at 37°C. CHAPSH3b showed the same antistaphyloccal effect at room temperature (1.65 µM). No re-growth was observed for the remainder of the experiment (up to 6 h). CHAPSH3b activity (1.65 µM) was also assayed in raw (whole and skim) and pasteurized (whole and skim) milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skim milk at both temperatures. This effect was most dramatic at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no re-growth detected for the duration of the experiment (360 min). Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4°C for 3 days. These results might facilitate the use of the peptidoglycan hydrolase HydH5 and its derivative fusions, particularly CHAPSH3b, as biocontrol agents for controlling undesirable bacteria in dairy products.

Role of the Pre-neck Appendage Protein (Dpo7) from Phage vB_SepiS-phiIPLA7 as an Anti-biofilm Agent in Staphylococcal Species

Staphylococcus epidermidis and Staphylococcus aureus are important causative agents of hospital-acquired infections and bacteremia, likely due to their ability to form biofilms. The production of a dense exopolysaccharide (EPS) matrix enclosing the cells slows the penetration of antibiotic down, resulting in therapy failure. The EPS depolymerase (Dpo7) derived from bacteriophage vB_SepiS-phiIPLA7, was overexpressed in Escherichia coli and characterized. A dose dependent but time independent response was observed after treatment of staphylococcal 24 h-biofilms with Dpo7. Maximum removal (>90%) of biofilm-attached cells was obtained with 0.15 μM of Dpo7 in all polysaccharide producer strains but Dpo7 failed to eliminate polysaccharide-independent biofilm formed by S. aureus V329. Moreover, the pre-treatment of polystyrene surfaces with Dpo7 reduced the biofilm biomass by 53–85% in the 67% of the tested strains. This study supports the use of phage-encoded EPS depolymerases to prevent and disperse staphylococcal biofilms, thereby making bacteria more susceptible to the action of antimicrobials.

The tape measure protein of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has an active muramidase domain.

ABSTRACT Tailed double-stranded DNA (dsDNA) bacteriophages frequently harbor structural proteins displaying peptidoglycan hydrolytic activities. The tape measure protein from Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has a lysozyme-like and a peptidase_M23 domain. This report shows that the lysozyme-like domain (TG1) has muramidase activity and exhibits in vitro lytic activity against live S. aureus cells, an activity that could eventually find use in the treatment of infections.

‘Artilysation’ of endolysin λSa2lys strongly improves its enzymatic and antibacterial activity against streptococci

Endolysins constitute a promising class of antibacterials against Gram-positive bacteria. Recently, endolysins have been engineered with selected peptides to obtain a new generation of lytic proteins, Artilysins, with specific activity against Gram-negative bacteria. Here, we demonstrate that artilysation can also be used to enhance the antibacterial activity of endolysins against Gram-positive bacteria and to reduce the dependence on external conditions. Art-240, a chimeric protein of the anti-streptococcal endolysin λSa2lys and the polycationic peptide PCNP, shows a similar species specificity as the parental endolysin, but the bactericidal activity against streptococci increases and is less affected by elevated NaCl concentrations and pH variations. Time-kill experiments and time-lapse microscopy demonstrate that the killing rate of Art-240 is approximately two-fold higher compared to wildtype endolysin λSa2lys, with a reduction in viable bacteria of 3 log units after 10 min. In addition, lower doses of Art-240 are required to achieve the same bactericidal effect.