Characterisation of bacteriophage-host interactions in Lactococcus lactis

Lactococcus lactis is used extensively world-wide for the production of fermented dairy products. Bacteriophages (phages) infecting L. lactis can result in slow or incomplete fermentations, or may even cause total fermentation failure. Therefore, bacteriophages disrupting L. lactis fermentation are of economic concern. This thesis employed a multifaceted approach to investigate various molecular aspects of phage-host interaction in L. lactis. The genome sequence of an Irish dairy starter strain, the prophage-cured L. lactis subsp. cremoris UC509.9, was studied. The 2,250,427 bp circular chromosome represents the smallest among its sequenced lactococcal equivalents. The genome displays clear genetic adaptation to the dairy niche in the form of extensive reductive evolution. Gene prediction identified 2066 protein-encoding genes, including 104 which showed significant homology to transposase-specifying genes. Over 9 % of the identified genes appear to be inactivated through stop codons or frame shift mutations. Many pseudogenes were found in genes that are assigned to carbohydrate and amino acid transport and metabolism orthologous groups, reflecting L. lactis UC509.9’s adaptation to the lactose and casein-rich dairy environment. Sequence analysis of the eight plasmids of L. lactis revealed extensive adaptation to the dairy environment. Key industrial phenotypes were mapped and novel lactococcal plasmid-associated genes highlighted. In addition to chromosomally-encoded bacteriophage resistance systems, six functional such systems were identified, including two abortive infection systems, AbiB and AbiD1, explaining the observed phage resistance of L. lactis UC509.9 Molecular analysis suggests that the constitutive expression of AbiB is not lethal to cells, suggesting the protein is expressed in an un/inactivated form. Analysis of 936 species phage sk1-escape mutants of AbiB revealed that all such mutants harbour mutations in orf6, which encodes the major capsid protein. Results suggest that the major capsid protein is required for activation of the AbiB system, although this requires furrther investigations. Temporal transcriptomes of L. lactis UC509.9 undergoing lytic infection with either one of two distinct bacteriophages, Tuc2009 and c2, was determined and compared to the transcriptome of uninfected UC509.9 cells. Whole genome microarrays performed at various time-points post-infection demonstrated a rather modest impact on host transcription. Alterations in the UC509.9 transcriptome during lytic infection appear phage-specific, with a relatively small number of differentially transcribed genes shared between infection with either Tuc2009 or c2. Transcriptional profiles of both bacteriophages during lytic infection was shown to generally correlate with previous studies and allowed the confirmation of previously predicted promoter sequences. Bioinformatic analysis of genomic regions encoding the presumed cell wall polysaccharide (CW PS) biosynthesis gene cluster of several strains of L. lactis was performed. Results demonstrate the presence of three dominant genetic types of this gene cluster, termed type A, B and C. These regions were used for the development of a multiplex PCR to identify CW PS genotype of various lactococcal strains. Analysis of 936 species phage receptor binding protein phylogeny (RBP) and CW PS genotype revealed an apparent correlation between RBP phylogeny and CW PS type, thereby providing a partial explanation for the observed narrow host range of 936 phages. Further analysis of the genetic locus encompassing the presumed CW PS biosynthesis operon of eight strains identified as belonging to the CW PS C (geno)type, revealed the presence of a variable region among the examined strains. The obtained comparative analysis allowed for the identification of five subgroups of the C type, named C1 to C5. We purified an acidic polysaccharide from the cell wall of L. lactis 3107 (C2 subtype) and confirmed that it is structurally different from the CW PS of the C1 subtype L. lactis MG1363. Combinations of genes from the variable region of C2 subtype were amplified from L. lactis 3107 and introduced into a mutant of the C1 subtype L. lactis NZ9000 (a direct derivative of MG1363) deficient in CW PS biosynthesis. The resulting recombinant mutant synthesized a CW PS with a composition characteristic for that of the C2 subtype L. lactis 3107 and not the wildtype C1 L. lactis NZ9000. The recombinant mutant exhibited a changed phage resistance/sensitivity profile consistent with that of L. lactis 3107, which unambiguously demonstrated that L. lactis 3107 CW PS is the host cell surface receptor of two bacteriophages belonging to the P335 species as well as phages that are member of the 936 species. The research presented in this thesis has significantly advanced our understanding of L. lactis bacteriophage-host interactions in several ways. Firstly, the examination of plasmidencoded bacteriophage resistance systems has allowed inferences to be made regarding the mode of action of AbiB, thereby providing a platform for further elucidation of the molecular trigger of this system. Secondly, the phage infection transcriptome data presented, in addition to previous work, has made L. lactis a model organism in terms of transcriptomic studies of bacteriophage-host interactions. And finally, the research described in this thesis has for the first time explicitly revealed the nature of a carbohydrate bacteriophage receptor in L. lactis, while also providing a logical explanation for the observed narrow host ranges exhibited by 936 and P335 phages. Future research in discerning the structures of other L. lactis CW PS, combined with the determination of the molecular interplay between receptor binding proteins of these phages and CW PS will allow an in depth understanding of the mechanism by which the most prevalent lactococcal phages identify and adsorb to their specific host.

A novel activation mechanism for Clostridial bacteriophage endolysins

Bacteriophage-encoded endolysins are produced at the end of the phage lytic cycle for the degradation of the host bacterial cell. Endolysins offer the potential as alternatives to antibiotics as biocontrol agents or therapeutics. The lytic mechanisms of three bacteriophage endolysins that target Clostridium species living under different conditions were investigated. For these endolysins a trigger and release mechanism is proposed for their activation. During host lysis, holin lesion formation suddenly permeabilises the membrane which exposes the cytosol-sequestered endolysins to a sudden environmental shock. This shock is suggested to trigger a conformational switch of the endolysins between two distinct dimer states. The switch between dimer states is proposed to activate a novel autocleavage mechanism that cleaves the linker connecting the N-terminal catalytic domain and the C-terminal domain to release the catalytic domain for more efficient digestion of the bacterial cell wall. Crystal structures of cleaved fragments of CD27L and CTP1L were previously obtained. In these structures cleavage occurs at the stem of the linker connected to the C-terminal domain. Despite a sequence identity of only 22% between 81 residues of the C-terminal domains of CD27L and CTP1L, they represent a novel fold that is identified in a number of different lysins. Within the crystal structures the two distinct dimerization modes are represented: the elongated head‐on dimer and the side-by‐side dimer. Introducing mutations that inhibit either of the dimerization states caused a decrease in the efficiency of both the autocleavage mechanism and the lytic activity of the endolysins. The two dimer states were validated for the full-length endolysins in solution by using right angle light scattering, small angle X‐ray scattering and cross-linking experiments. Overall, the data represents a new type of regulation governed by the C-terminal domains that is used to activate these endolysins once they enter the bacterial cell wall.

Characterisation of virus transport and attenuation in epikarst using short pulse and prolonged injection multi-tracer testing

Attenuation processes controlling virus fate and transport in the vadose zone of karstified systems can strongly influence groundwater quality. This research compares the breakthrough of two bacteriophage tracers (H40/1 and T7), with contrasting properties, at subsurface monitoring points following application onto an overlying composite sequence of thin organic soil and weathered limestone (epikarst). Short pulse multi-tracer test results revealed that T7 (Source concentration, Co=1.8x106pfu/mL) and H40/1(Co=5.9x106pfu/mL) could reach sampling points 10m below ground less than 30 minutes after tracer application. Contrasting deposition rates, determined from simulated tracer responses, reflected the potential of the ground to differentially attenuate viruses. Prolonged application of both T7 (Co=2.3x104pfu/mL) and H40/1 (Co=1.3x105pfu/mL) over a five hour period during a subsequent test, in which ionic strength levels observed at monitoring points rose consistently, corresponded to a rapid rise in T7 levels, followed by a gradual decline before the end of tracer injection; this reflected reaction-limited deposition in the system. T7’s response contrasted with that of H40/1, whose concentration remained constant over a three hour period before declining dramatically prior to the end of tracer injection. Subsequent application of lower ionic strength tracer-free flush water generated a rapid rise in H40/1 levels and a more gradual release of T7. Results highlight the benefits of employing prolonged injection multi-tracer tests for identifying processes not apparent from conventional short pulse tests. Study findings demonstrate that despite rapid transport rates, the epikarst is capable of physicochemical filtration of viruses and their remobilization, depending on virus type and hydrochemical conditions.

Phenotypic and genotypic variations within a single bacteriophage species.

Background: Although horizontal gene transfer plays a pivotal role in bacteriophage evolution, many lytic phage genomes are clearly shaped by vertical evolution. We investigated the influence of minor genomic deletions and insertions on various phage-related phenotypic and serological properties. Findings. We collected ten different isolates of Pseudomonas aeruginosa bacteriophage KMV. All sequenced genomes (42-43 kb, long direct terminal repeats) are nearly identical, which intuitively implied strongly similar infections cycles. However, their latent periods vary between 21 and 28 minutes and they are able to lyse between 5 and 58% of a collection of 107 clinical P. aeruginosa strains. We also noted that phages with identical tail structures displayed profound differences in host spectra. Moreover, point mutations in tail and spike proteins were sufficient to evade neutralization by two phage-specific antisera, isolated from rabbits. Conclusion: Although all analyzed phages are 83-97% identical at the genome level, they display a surprisingly large variation in various phenotypic properties. The small overlap in host spectrum and their ability to readily escape immune defences against a nearly identical phage are promising elements for the application of these phages in phage therapy. © 2011 Ceyssens et al; licensee BioMed Central Ltd.


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Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis

Marine sponges have never been directly examined with respect to the presence of viruses or their potential involvement in horizontal gene transfer. Here we demonstrate for the first time, the presence of viruses in the marine sponge Hymeniacidon perlevis. Moreover, bacterial 16s rDNA was detected in DNA isolated from these viruses, indicating that phage-derived transduction appears to occur in H. perlevis. Phylogenetic analysis revealed that bacterial 16s rDNA isolated from sponge-derived viral and total DNA differed significantly, indicating that not all species are equally involved in transduction.

Microneedle-mediated transdermal bacteriophage delivery

Interest in bacteriophages as therapeutic agents has recently been reawakened. Parenteral delivery is the most routinely-employed method of administration. However, injection of phages has numerous disadvantages, such as the requirement of a health professional for administration and the possibility of cross-contamination. Transdermal delivery offers one potential means of overcoming many of these problems. The present study utilized a novel poly (carbonate) (PC) hollow microneedle (MN) device for the transdermal delivery of Escherichia coli-specific 14 bacteriophages both in vitro and in vivo. MN successfully achieved bacteriophage delivery in vitro across dermatomed and full thickness skin. A concentration of 2.67 x 10(6) PFU/ml (plaque forming units per ml) was detected in the receiver compartment when delivered across dermatomed skin and 4.0 x 10(3) PFU/ml was detected in the receiver compartment when delivered across full thickness skin. An in vivo study resulted in 4.13 x 10(3) PFU/ml being detected in blood 30 min following initial MN-mediated phage administration. Clearance occurred rapidly, with phages being completely cleared from the systemic circulation within 24 h, which was expected in the absence of infection. We have shown here that MN-mediated delivery allows successful systemic phage absorption. Accordingly, bacteriophage-based therapeutics may now have an alternative route for systemic delivery. Once fully-investigated, this could lead to more widespread investigation of these interesting therapeutic viruses. (c) 2012 Elsevier B.V. All rights reserved.

Development of M13 bacteriophage-based SPR detection using Salmonella detection as a case study

Surface plasmon resonance (SPR)-based biosensor is a popular platform for real-time monitoring and sensitive detection for a myriad of targets. However, only a few studies have reported the use of bacteriophages as specific binders for SPR-based detection. This study aimed to demonstrate how filamentous M13 bacteriophages expressing 12-mer peptides can be employed in an SPR-based assay, using a Salmonella-specific bacteriophage as a model binder to detect the foodborne bacterium Salmonella. Several important factors (immobilization buffers and methods, and interaction buffers) for a successful bacteriophage-based SPR assay were optimized. As a result, a Salmonella-specific bacteriophage-based SPR assay was achieved, with very low cross reactivity with other non-target foodborne pathogens and detection limits of 8.0 × 10⁷ and 1.3 × 10⁷ CFU/mL for one-time and five-time immobilized sensors, respectively. This proof-of-concept study demonstrates the feasibility of using M13 bacteriophages expressing target-specific peptides as a binder in a rapid and label-free SPR assay for pathogen detection.

Atmospheric pressure, nonthermal plasma inactivation of MS2 bacteriophage: effect of oxygen concentration on virucidal activity

AimsThe main aim of this study was to determine the virucidal inactivation efficacy of an in-house-designed atmospheric pressure, nonthermal plasma jet operated at varying helium/oxygen feed gas concentrations against MS2 bacteriophage, widely employed as a convenient surrogate for human norovirus.

Methods and ResultsThe effect of variation of percentage oxygen concentration in the helium (He) carrier gas was studied and found to positively correlate with MS2 inactivation rate, indicating a role for reactive oxygen species (ROS) in viral inactivation. The inactivation rate constant increased with increasing oxygen concentrations up to 075% O-2. 3 log(10) (999%) reductions in MS2 viability were achieved after 3min of exposure to the plasma source operated in a helium/oxygen (9925%:075%) gas mixture, with >7 log(10) reduction after 9min exposure.

ConclusionsAtmospheric pressure, nonthermal plasmas may have utility in the rapid disinfection of virally contaminated surfaces for infection control applications.

Significance and Impact of StudyThe atmospheric pressure, nonthermal plasma jet employed in this study exhibits rapid virucidal activity against a norovirus surrogate virus, the MS2 bacteriophage, which is superior to previously published inactivation rates for chemical disinfectants.