Jennifer Mahony

T-cell activation by transitory neo-antigens derived from distinct microbial pathways

T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-d-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.

Bacteriophages as biocontrol agents of food pathogens

Bacteriophages have long been recognized for their potential as biotherapeutic agents. The recent approval for the use of phages of Listeria monocytogenes for food safety purposes has increased the impetus of phage research to uncover phage-mediated applications with activity against other food pathogens. Areas of emerging and growing significance, such as predictive modelling and genomics, have shown their potential and impact on the development of new technologies to combat food pathogens. This review will highlight recent advances in the research of phages that target food pathogens and that promote their use in biosanitation, while it will also discuss its limitations.

Structure of the phage TP901-1 1.8 MDa baseplate suggests an alternative host adhesion mechanism

Phages of the Caudovirales order possess a tail that recognizes the host and ensures genome delivery upon infection. The X-ray structure of the approximately 1.8 MDa host adsorption device (baseplate) from the lactococcal phage TP901-1 shows that the receptor-binding proteins are pointing in the direction of the host, suggesting that this organelle is in a conformation ready for host adhesion. This result is in marked contrast with the lactococcal phage p2 situation, whose baseplate is known to undergo huge conformational changes in the presence of Ca2+ to reach its active state. In vivo infection experiments confirmed these structural observations by demonstrating that Ca2+ ions are required for host adhesion among p2-like phages (936-species) but have no influence on TP901-1-like phages (P335-species). These data suggest that these two families rely on diverse adhesion strategies which may lead to different signaling for genome release.

Bacteriophage Orphan DNA Methyltransferases: Insights from Their Bacterial Origin, Function, and Occurrence

ABSTRACT Type II DNA methyltransferases (MTases) are enzymes found ubiquitously in the prokaryotic world, where they play important roles in several cellular processes, such as host protection and epigenetic regulation. Three classes of type II MTases have been identified thus far in bacteria which function in transferring a methyl group from S-adenosyl-l-methionine (SAM) to a target nucleotide base, forming N-6-methyladenine (class I), N-4-methylcytosine (class II), or C-5-methylcytosine (class III). Often, these MTases are associated with a cognate restriction endonuclease (REase) to form a restriction-modification (R-M) system protecting bacterial cells from invasion by foreign DNA. When MTases exist alone, which are then termed orphan MTases, they are believed to be mainly involved in regulatory activities in the bacterial cell. Genomes of various lytic and lysogenic phages have been shown to encode multi- and mono-specific orphan MTases that have the ability to confer protection from restriction endonucleases of their bacterial host(s). The ability of a phage to overcome R-M and other phage-targeting resistance systems can be detrimental to particular biotechnological processes such as dairy fermentations. Conversely, as phages may also be beneficial in certain areas such as phage therapy, phages with additional resistance to host defenses may prolong the effectiveness of the therapy. This minireview will focus on bacteriophage-encoded MTases, their prevalence and diversity, as well as their potential origin and function.

The Lactococcal Phages Tuc2009 and TP901-1 Incorporate Two Alternate Forms of Their Tail Fiber into Their Virions for Infection Specialization

Background: Siphoviridae virions often possess lytic domains facilitating host-penetration. Results: Tuc2009 and TP901-1 virions may contain full-length or truncated tail fibers, possessing or lacking a lytic domain, respectively. Conclusion: Phages with a lytic domain infect stationary phase cells better, whereas truncated derivatives have higher adsorption efficiencies. Significance: The heterogeneous phage population serves to most effectively infect bacteria where levels of cell wall cross-linkage differ. Lactococcal phages Tuc2009 and TP901-1 possess a conserved tail fiber called a tail-associated lysin (referred to as Tal2009 for Tuc2009, and Tal901-1 for TP901-1), suspended from their tail tips that projects a peptidoglycan hydrolase domain toward a potential host bacterium. Tal2009 and Tal901-1 can undergo proteolytic processing mid-protein at the glycine-rich sequence GG(S/N)SGGG, removing their C-terminal structural lysin. In this study, we show that the peptidoglycan hydrolase of these Tal proteins is an M23 peptidase that exhibits d-Ala-d-Asp endopeptidase activity and that this activity is required for efficient infection of stationary phase cells. Interestingly, the observed proteolytic processing of Tal2009 and Tal901-1 facilitates increased host adsorption efficiencies of the resulting phages. This represents, to the best of our knowledge, the first example of tail fiber proteolytic processing that results in a heterogeneous population of two phage types. Phages that possess a full-length tail fiber, or a truncated derivative, are better adapted to efficiently infect cells with an extensively cross-linked cell wall or infect with increased host-adsorption efficiencies, respectively.

Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been implicated as a host receptor of (certain) 936-type phages, is specified by a large gene cluster, which, among different lactococcal strains, contains highly conserved regions as well as regions of diversity. The regions of diversity within this cluster on the genomes of lactococcal strains MG1363, SK11, IL1403, KF147, CV56, and UC509.9 were used for the development of a multiplex PCR system to identify the pellicle genotype of lactococcal strains used in this study. The resulting comparative analysis revealed an apparent correlation between the pellicle genotype of a given host strain and the host range of tested 936-type phages. Such a correlation would allow prediction of the intrinsic 936-type phage sensitivity of a particular lactococcal strain and substantiates the notion that the lactococcal pellicle polysaccharide represents the receptor for (certain) 936-type phages while also partially explaining the molecular reasons behind the observed narrow host range of such phages.

Differences in Lactococcal Cell Wall Polysaccharide Structure Are Major Determining Factors in Bacteriophage Sensitivity

ABSTRACT Analysis of the genetic locus encompassing a cell wall polysaccharide (CWPS) biosynthesis operon of eight strains of Lactococcus lactis, identified as belonging to the same CWPS type C genotype, revealed the presence of a variable region among the strains examined. The results allowed the identification of five subgroups of the C type named subtypes C1 to C5. This variable region contains genes encoding glycosyltransferases that display low or no sequence homology between the subgroups. In this study, we purified an acidic polysaccharide from the cell wall of L. lactis 3107 (subtype C2) and confirmed that it is structurally different from the previously established CWPS of subtype C1 L. lactis MG1363. The CWPS of L. lactis 3107 is composed of pentasaccharide repeating units linked by phosphodiester bonds with the structure 6-α-Glc-3-β-Galf-3-β-GlcNAc-2-β-Galf-6-α-GlcNAc-1-P. Combinations of genes from the variable region of subtype C2 were introduced into a mutant of subtype C1 L. lactis NZ9000 deficient in CWPS biosynthesis. The resulting recombinant mutant synthesized a polysaccharide with a composition characteristic of that of subtype C2 L. lactis 3107 and not wild-type C1 L. lactis NZ9000. By challenging the recombinant mutant with various lactococcal phages, we demonstrated that CWPS is the host cell surface receptor of tested bacteriophages of both the P335 and 936 groups and that differences between the CWPS structures play a crucial role in determining phage host range. IMPORTANCE Despite the efforts of nearly 80 years of lactococcal phage research, the precise nature of the cell surface receptors of the P335 and 936 phage group receptors has remained elusive. This work demonstrates the molecular nature of a P335 group receptor while bolstering the evidence of its role in host recognition by phages of the 936 group and at least partially explains why such phages have a very narrow host range. The information generated will be instrumental in understanding the molecular mechanisms of how phages recognize specific saccharidic receptors located on the surface of their bacterial host. Despite the efforts of nearly 80 years of lactococcal phage research, the precise nature of the cell surface receptors of the P335 and 936 phage group receptors has remained elusive. This work demonstrates the molecular nature of a P335 group receptor while bolstering the evidence of its role in host recognition by phages of the 936 group and at least partially explains why such phages have a very narrow host range. The information generated will be instrumental in understanding the molecular mechanisms of how phages recognize specific saccharidic receptors located on the surface of their bacterial host.

Current taxonomy of phages infecting lactic acid bacteria

Phages infecting lactic acid bacteria have been the focus of significant research attention over the past three decades. Through the isolation and characterization of hundreds of phage isolates, it has been possible to classify phages of the dairy starter and adjunct bacteria Lactococus lactis, Streptococcus thermophilus, Leuconostoc spp., and Lactobacillus spp. Among these, phages of L. lactis have been most thoroughly scrutinized and serve as an excellent model system to address issues that arise when attempting taxonomic classification of phages infecting other LAB species. Here, we present an overview of the current taxonomy of phages infecting LAB genera of industrial significance, the methods employed in these taxonomic efforts and how these may be employed for the taxonomy of phages of currently underrepresented and emerging phage species.

Identification and Characterization of Lactococcal-Prophage-Carried Superinfection Exclusion Genes

ABSTRACT Superinfection exclusion (Sie) proteins are prophage-encoded phage resistance systems. In this study, genes encoding Sie systems were identified on the genomes of Lactococcus lactis subsp. cremoris MG1363 and L. lactis subsp. lactis IL1403. These Sie systems are genetically distinct and yet were shown to act specifically against a particular subset of the 936 phage group. Each of the systems allows normal phage adsorption while affecting plasmid transduction and intracellular phage DNA replication, which points to the blocking of phage DNA injection as their common mode of action. Sie-specifying genes found on the MG1363 prophages are also present in various lactococcal strains, whereas the prophage-encoded Sie systems of IL1403 do not appear to be as widely disseminated.

Lactococcal 936-type phages and dairy fermentation problems: from detection to evolution and prevention

The so-called 936-type phages are the most frequently encountered lactococcal phage species in dairy fermentations, where they cause slow or even failed fermentations with concomitant economic losses. Several dairy phage population studies, performed in different geographical locations, have detailed their dominance in dairy phage populations, while various phage-resistance mechanisms have been assessed in a bid to protect against this virulent phage group. The impact of thermal and chemical treatments on 936 phages is an important aspect for dairy technologists and has been assessed in several studies, and has indicated that these phages have adapted to better resist such treatments. The abundance of 936 phage genome sequences has permitted a focused view on genomic content and regions of variation, and the role of such variable regions in the evolution of these phages. Here, we present an overview on detection and global prevalence of the 936 phages, together with their tolerance to industrial treatments and anti-phage strategies. Furthermore, we present a comprehensive review on the comparative genomic analyses of members of this fascinating phage species.