Molecular and structural characterization of Listeria bacteriophage-host interactions and the identification of a novel CRISPR-Cas system for the modification of virulent bacteriophage

Abstract

Bacteriophages (phages) are viruses that exclusively infect bacteria and constitute their natural enemies. They are the most abundant biological entity on earth with an estimated 10e31 particles at any given time and outnumber bacteria by at least two orders of magnitude. In the advent of antibiotic resistance, the use of phages as antimicrobials is a re-emerging field of interest. Phages are already successfully employed for biocontrol and detection of foodborne pathogens such as Listeria or Salmonella. However, their application in human medicine is currently limited. One reason is the resistance development of bacteria to phage infection, which demands the design of mixed phage cocktails for efficient treatment. One way for bacteria to acquire resistance to phage are genetic mutations to change cell surface receptors, which the phage needs for host recognition. The understanding of phage receptor binding proteins (RBPs) and the early steps of infection remains incomplete. This knowledge could be of practical use for the rational design of optimized phages as antimicrobials, which is becoming feasible due to advances in genome engineering. I address these research objectives within this study investigating four different, yet related, topics. These include Listeria phage attachment to its host, conformational changes in Listeria phage A511 induced by first binding events and causes of bacterial resistance to avoid phage predation in Listeria. Further, we engineered a biotechnological tool to modify virulent Listeria phage. The first manuscript examines numerous RBPs of previously uncharacterized Listeria phages. Listeria possess a large diversity of cell wall associated carbohydrate polymers called wall teichoic acids. These have been established as receptor molecules in phage infection. In this study, we confirmed the importance of wall teichoic acids for phage infection of two phages infecting Listeria innocua and Listeria ivanovii (B025 and B110) and two phages infecting Listeria monocytogenes serovar type 4b (A500 and PSA). We employed fluorescence microscopy with recombinant Listeria phage receptor binding proteins to observe different binding patterns of RBPs to constructed cell wall mutants in L. monocytogenes. Our data indicates that PSA/A500 RBP cell surface binding depends on terminal galactose residues in wall teichoic acids and is ribitol-C4 specific. In contrast, recombinant receptor binding proteins of phages B025 and B110 only bound to strains featuring terminal sugars in ribitiol-C2 position, however, indiscriminant of galactose or glucose decoration. Despite different binding patterns, we found strong homologies in amino acid sequence of several receptor binding proteins indicating a recent speciation in evolution. The second manuscript depicts a cooperative effort with the groups of Prof. Petr Leiman and Prof. Takashi Ishikawa. We investigated Listeria phage A511, a phage belonging to the Myoviridae. These phages feature a long phage tail that contracts upon attachment to its bacterial host. We examined the structure of the phage A511 baseplate as a model for a contractile infection apparatus in phages infecting Gram-positive bacteria. The structure of the pre- and post-attachment state was described using cryo-electron microscopy (cryo-EM), cryo-electron tomography and X-ray crystallography. We could show that upon attachment drastic conformational changes occur within the baseplate, changing from three to six-fold symmetry. The baseplate double ring structure changed to a more planar state mainly caused by a “flip” of the large structural Gp106 (VrlC-like) protein. Furthermore, we observed phages “in between” both states revealing that the contraction of the tail sheath originates from the baseplate and propagates upwards towards the capsid as a wave. The elucidation of the crystal structure of baseplate protein Gp105 helped to interpret the electron density map, and thus we assigned the putative proteins of the A511 baseplate to its genes. In the third manuscript, we identified and characterized a Listeria phage resistance mechanism. The mechanism is known as CRISPR (clustered regularly interspaced short palindromic repeats). It represents an adaptive immune system in bacteria, which cuts incoming foreign DNA elements by a nuclease-targeting complex in a sequence specific manner. We found a functional CRISPR-Cas type II system in Listeria ivanovii. Functional gene knockouts and phage adsorption experiments confirmed that phage resistance is conferred by the CRISPR-Cas system, although we found that additional defense measures must exist. Further, we engineered the CRISPR-Cas system as biotechnological tool to modify virulent Listeria phages. For that purpose, we constructed a minimal CRISPR-Cas targeting system for introduction into Listeria strains of choice. This contained the gene of the identified nuclease (Cas9). We provided DNA targeting sequences for chosen phages in trans. This conferred resistance to engineered CRISPR strains from targeted phage genomes thereby confirming functionality of our platform. The mechanism enabled us to use CRISPR targeting to increase the efficiency of phage genome engineering in homologous recombination. In homologous recombination, sequences homologous to the target phage genome are introduced into bacteria in trans, containing modifications of choice. During phage infection, DNA may be recombined with the help of the bacterial recombinase system. However, this process is rare producing mixtures of unmodified and recombinant phage with little chance of finding modified phage. We used our constructed minimal CRISPR-Cas system as negative counter selection for unmodified phages. Unmodified phages are targeted within the bacterial host by CRISPR, while only the recombinant phage are able to survive. This allowed us to isolate recombinant phage without the use of any markers. In a proof-of-concept study, we introduced the Staphylococcus peptidoglycan hydrolase gene for lysostaphin into Listeria phage A511. Engineered phage are able to clear co-cultures of S. aureus and Listeria illustrating the potential of engineered phages for applications beyond single pathogen control. In conclusion, this thesis provides new insights into the early infection mechanisms of Listeria phages, identified a phage resistance mechanism for Listeria, which has not been characterized within this Genus before, and resulted in the development of a biotechnological tool to modify virulent Listeria phage. Taken together these results can be of practical use to engineer phages with customized host ranges or antimicrobial properties for further application.