EnzyPha, an Engineered Helper Phage Developed to Overcome Most of the Limitations Regarding Filamentous Phage Titration and ELISA Tests

Background: Phage display method is a technology which enables expression of exogenous polypeptides on the surface of bacteriophage particles. Phage titration and ELISA are applied for measuring helper phage particles or polypeptide bearing phages and also evaluation of the interaction between polypeptide bearing phages and coated antigens, respectively. Although, several procedures have been introduced to perform phage titration and ELISA but they faced to some limitations such as being time consuming and expensive and also low reproducibility. Objective: We developed a new system called EnzyPha by engineering the M13KO7 expressing Secreted Acid Phosphatase of Mycobacterium tuberculosis (SapM enzyme) on its pIX protein for applying in colorimetric phage titration and ELISA methods. Methods: For this purpose, to evaluate the idea, colorimetric phage titration and ELISA were performed and compared to the traditional methods. Results: SapM enzyme was expressed on pIX protein of M13KO7 properly. The colorimetric phage titration and phage ELISA showed better and comparable results against the traditional approaches. Conclusion: The results showed that the proposed model would titrate phages more sensitive than plating titration method through a shorter timeframe. Moreover, it could be a better alternative to the routine phage ELISA due to time saving, cost effective and higher sensitivity.

How pirate phage interferes with helper phage: Comparison of the two distinct strategies

Pirate phages use the structural proteins encoded by unrelated helper phages to propagate. The best-studied example is the pirate P4 and helper P2 of coliphages, and it has been known that theStaphylococcus aureuspathogenicity islands (SaPIs) that can encode virulence factors act as pirate phages, too. When alone in the host, the pirate phages act as a prophage, but when the helper phage gene is also in the same host cell, the pirate phage has ability to exploit the helper phages structural proteins to produce pirate phage particles and spread, interfering with the helper phage production. The known helper phages in these systems are temperate phages. Interestingly, the interference of the pirate phage to the helper phage occurs in a different manner between the SaPI-helper system and the P4-P2 system. SaPIs cannot lyse a helper lysogen upon infection, while when a helper phage lyse a SaPI lysogen, most of the phage particles produced are the SaPI particles. On the contrary, in the P4-P2 system, a pirate phage P4 can lyse a helper P2 lysogen to produce mostly the P4 particles, while when P2 phage lyses a P4 lysogen, most of the produced phages are the P2 particles. Here, the consequences of these different strategies in the pirate and helper phage spreading among uninfected host is analyzed by using mathematical models. It is found that SaPI’s strategy interferes with the helper phage spreading significantly more than the P4’s strategy, because SaPI interferes with the helper phage’s main reproduction step, while P4 interferes only by forcing the helper lysogens to lyse. However, the interference is found to be weaker in the spatially structured environment than in the well-mixed environment. This is because, in the spatial setting, the system tends to self-organize so that the helper phages take over the front of propagation due to the need of helper phage for the pirate phage spreading.Competing interestsThe author declares no competing interest.

Convergent evolution of pathogenicity islands in helper cos phage interference

Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful ( pac ) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpm A and cpm B genes. Another SaPI subfamily is induced and packaged by cos -type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm , which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution. This article is part of the themed issue ‘The new bacteriology’.