The Capricious Nature of Bacterial Pathogens: Phasevarions and Vaccine Development

Abstract

Infectious diseases are a leading cause of morbidity and mortality worldwide, and vaccines are one of the most successful and cost-effective tools for disease prevention. One of the key considerations for rational vaccine development is the selection of appropriate antigens. Antigens must induce a protective immune response, and this response should be directed to stably expressed antigens so the target microbe can always be recognized by the immune system. Antigens with variable expression, due to environmental signals or phase variation (i.e., high frequency, random switching of expression), are not ideal vaccine candidates because variable expression could lead to immune evasion. Phase variation is often mediated by the presence of highly mutagenic simple tandem DNA repeats, and genes containing such sequences can be easily identified, and their use as vaccine antigens reconsidered. Recent research has identified phase variably expressed DNA methyltransferases that act as global epigenetic regulators. These phase-variable regulons, known as phasevarions, are associated with altered virulence phenotypes and/or expression of vaccine candidates. As such, genes encoding candidate vaccine antigens that have no obvious mechanism of phase variation may be subject to indirect, epigenetic control as part of a phasevarion. Bioinformatic and experimental studies are required to elucidate the distribution and mechanism of action of these DNA methyltransferases, and most importantly, whether they mediate epigenetic regulation of potential and current vaccine candidates. This process is essential to define the stably expressed antigen target profile of bacterial pathogens and thereby facilitate efficient, rational selection of vaccine antigens.

Keywords: DNA methyltransferase; DNA modification enzyme; epigenetics; gene expression; phase variation; vaccine.

Figure 1

Phase variation and immune evasion. (A) For a phase-variable outer-membrane protein, slipped strand mispairing and changes in DNA sequence repeats in the gene during genome replication lead to ON/OFF expression of the encoded protein (blue). Antibodies to this antigen will not be effective if the protein has phased varied OFF. It is typically easy to predict phase-variable expression of these proteins due to the presence of DNA repeats (simple sequence repeat) in the coding region of the gene. (B) In phasevarions, phase-variable expression of a DNA methyltransferase causes genome-wide changes in DNA methylation, and expression differences in multiple genes due to epigenetic regulation. If these genes encode antigenic proteins/vaccine candidates, then methylation-dependent loss of expression (red protein) or reduced expression (purple protein) can lead to immune evasion as antibodies lose efficacy. However, due to the epigenetic nature of the phase-variable regulation, it is difficult to predict which proteins will have altered expression.

Figure 2

Phase-variable DNA methyltransferases. (A) The three main types of restriction–methylation (R–M) systems: type I consists of separate restriction (R), methyltransferase (M), and specificity (S) components, encoded by hsdR, hsdM, and hsdS genes, respectively. For restriction to occur, a pentameric R₂M₂S complex must form, but methylation can occur independently through a trimeric M₂S complex. The HsdS subunits dictate the DNA sequences that are restricted and methylated. Type II systems are encoded by individual genes, often located separately on the chromosome. The resulting restriction (R) and methyltransferase (M) enzymes recognize and act independently upon the same DNA motif. Type III systems consist of colocalized mod [modification; encoding a methyltransferase, Mod (M)] and res [restriction; encoding a restriction enzyme, Res (R)] genes. Res proteins require Mod to restrict DNA (R₂M₂), but Mod enzymes are active as stand-alone methyltransferases (M₂). (B) Phase variation of type I R–M systems via recombination between expressed (hsdS) and silent (hsdS') specificity genes. Each hsdS gene contains two target recognition domains (TRDs), each contributing half to the sequence recognized by the HsdS protein. Shuffling of each TRD via recombination between homologous inverted repeats (gray at 5′ end, yellow in center) leads to four possible combinations, and therefore, four different methyltransferase specificities in this example. (C) Phase variation of type III R–M systems via slipped strand mispairing (SSM) of simple sequence repeats in the open reading frame of the mod genes. Loss or gain of a repeat unit leads to variation in the open reading frame and either expression of a functional Mod protein (Mod ON), or transcriptional termination through the presence of a premature stop codon (Mod OFF).