Poster: 24Mycobacterium abscessus-chelonae prophages have the potential to inhibit phage superinfection threatening phage therapy treatments

Abstract

Background

Non-tuberculosis mycobacteria, such as those in the M. abscessus-chelonae (Mab-chel) complex, cause pulmonary and soft tissue infections posing a major global health threat. Mab-chel infections are common in patients that are elderly, immunocompromised or have chronic pulmonary disease such as cystic fibrosis (CF). Most clinical strains are multi- or completely drug resistant making phage therapy an important alternative treatment strategy. Clinical Mab-chel strains typically carry one or more prophages, integrated bacteriophage genomes, that likely increase the bacterium's resistance to lytic infection by superinfecting phage [1]. Secreted toxin systems encoded by the majority of Mab-chel prophages, potentially interfere with treatment by increasing bacterial virulence, drug resistance and resistance to phage infection. To better understand the effect of prophage-encoded toxins on the lytic dynamics of superinfecting phages, a fluorophage will be engineered that allows the monitoring of early and late lytic gene expression in single cells over time. This project investigates how the presence of a Mab-chel prophage affects the lytic dynamics of superinfecting phage to gain deeper insight into phage lytic dynamics necessary to improve future phage therapy treatments.

Non-tuberculosis mycobacteria, such as those in the M. abscessus-chelonae (Mab-chel) complex, cause pulmonary and soft tissue infections posing a major global health threat. Mab-chel infections are common in patients that are elderly, immunocompromised or have chronic pulmonary disease such as cystic fibrosis (CF). Most clinical strains are multi- or completely drug resistant making phage therapy an important alternative treatment strategy. Clinical Mab-chel strains typically carry one or more prophages, integrated bacteriophage genomes, that likely increase the bacterium's resistance to lytic infection by superinfecting phage [1]. Secreted toxin systems encoded by the majority of Mab-chel prophages, potentially interfere with treatment by increasing bacterial virulence, drug resistance and resistance to phage infection. To better understand the effect of prophage-encoded toxins on the lytic dynamics of superinfecting phages, a fluorophage will be engineered that allows the monitoring of early and late lytic gene expression in single cells over time. This project investigates how the presence of a Mab-chel prophage affects the lytic dynamics of superinfecting phage to gain deeper insight into phage lytic dynamics necessary to improve future phage therapy treatments.

Methods

To investigate the effect of Mab-chel prophage, McProf, on superinfection by secondary phage, BPs, CRISPY-BRED will be used to recombineer the genome of BPs to fluorescently report both early and late lytic phage infection. A GFP fluorophage strain of BPs that reports late lytic gene expression will be modified in a recombineering strain of Mycobacterium smegmatis using an oligo encoding a far-red fluorescent protein under the control of the BPs Cro promoter. Recombinant prophage particles will be selected for using CRISPRi technology [2]. Recombineered phage particles will then be screened and amplified prior to performing wide-field fluorescence microscopy necessary to monitor BPs early and late lytic gene expression in the presence and absence of McProf.

To investigate the effect of Mab-chel prophage, McProf, on superinfection by secondary phage, BPs, CRISPY-BRED will be used to recombineer the genome of BPs to fluorescently report both early and late lytic phage infection. A GFP fluorophage strain of BPs that reports late lytic gene expression will be modified in a recombineering strain of Mycobacterium smegmatis using an oligo encoding a far-red fluorescent protein under the control of the BPs Cro promoter. Recombinant prophage particles will be selected for using CRISPRi technology [2]. Recombineered phage particles will then be screened and amplified prior to performing wide-field fluorescence microscopy necessary to monitor BPs early and late lytic gene expression in the presence and absence of McProf.

Results

Preliminary results with a single fluorophage lysogen (BPsGFPEarly) reveals that most cells are undergoing induction and early BPs lytic gene expression regardless of presence/absence of McProf. However, in the presence of McProf the proportion of lysogens (BPsGFPLate) undergoing late lytic expression decreases compared to strains lacking McProf.

Preliminary results with a single fluorophage lysogen (BPsGFPEarly) reveals that most cells are undergoing induction and early BPs lytic gene expression regardless of presence/absence of McProf. However, in the presence of McProf the proportion of lysogens (BPsGFPLate) undergoing late lytic expression decreases compared to strains lacking McProf.

Conclusions

Mab-chel prophages may play an important role in inhibiting lysis necessary for lytic phage therapy treatments. Therefore, it is important to construct a dual reporter lysogen to determine what fraction of cells that undergo BPs induction achieve late lytic gene expression.

Mab-chel prophages may play an important role in inhibiting lysis necessary for lytic phage therapy treatments. Therefore, it is important to construct a dual reporter lysogen to determine what fraction of cells that undergo BPs induction achieve late lytic gene expression.