Moraxella catarrhalis phasevarions

Abstract

Moraxella catarrhalis is a human-restricted, Gram-negative, opportunistic bacterial pathogen of the respiratory mucosa. Although frequently found to colonize the nasopharynx asymptomatically, M. catarrhalis is also an important cause of otitis media (OM) in infants and children, and exacerbations of chronic obstructive pulmonary disease (COPD) in the elderly and heavy smokers. Despite the significant burden of M. catarrhalis induced disease, no vaccine is currently available for M. catarrhalis and no vaccine candidates have progressed to clinical trials. While a number of immunogenic proteins have been identified, several of the most promising have been discounted from further development due to the discovery of their phase-variable expression. Phase variation is the random, reversible, ON/OFF or graded switching of gene expression that is typically associated with genes encoding cell surface structures and allows bacteria to adapt to different host microenvironments and evade host defences. Phase variation of genes encoding cytoplasmically located restriction-modification (R-M) systems have also been observed in numerous bacterial pathogens, commonly mediated by simple DNA sequence repeats (SSRs). Phase variation of DNA methyltransferases causes differential methylation of the genome between clonally derived cells, which has been shown to epigenetically alter the expression of multiple genes in a system known as a phasevarion (phase-variable regulon). Six genes with SSR mediated phase-variable expression have been previously described in M. catarrhalis, including four outer membrane proteins (mid/hag, uspA1, uspA2, and uspA2H) and two Type III DNA methyltransferases (modM and modN). However, the complete repertoire of genes epigenetically regulated within M. catarrhalis phasevarions had not been described. Through our bioinformatic analysis we identified two novel phase-variable genes in M. catarrhalis, encoding a Type III DNA methyltransferase (modO) and a well conserved hypothetical permease (MC25239_RS00020), and confirmed their phase-variable expression experimentally. Expression of modO in M. catarrhalis ATCC 23246 was correlated with the number of 5′-CAACG-3′ repeat units in the SSR tract located upstream of the modO open reading frame, with >10 fold higher modO mRNA levels detected when ten repeats were present versus nine or eleven repeats. A modest but statistically significant difference in MC25239_RS00020 mRNA (1.45 fold) was observed between M. catarrhalis CCRI-195ME variants containing eight or nine 5′-GTTC-3′ SSRs in the repeat tract located upstream of MC25239_RS00020, and larger differences in expression are expected to be observed upon isolation of a broader range of SSR variants. The majority of phase-variable Type III DNA methyltransferase genes sequenced to date have multiple alleles that differ in their central target recognition domain (TRD) and it is hypothesized that every allelic variant methylates a different target sequence and regulates the switching of expression of a distinct phasevarion. Three TRD alleles of modM (modM1-3) were previously identified in M. catarrhalis, while modN has only been identified in a single strain. In analysing 51 publicly available M. catarrhalis genomes of diverse origin we have discovered three novel modM alleles (modM4-6), one novel modN allele (modN2), and six novel modO alleles (modO1-6). In all cases the Nand C-terminal regions are well conserved (>90% nucleotide identity), while the central TRD is highly variable (35-55% nucleotide identity) between alleles. In addition, we demonstrate that the modM, modN, and modO containing R-M systems and their respective allelic variants are differentially distributed between the virulent ‘RB1’, avirulent ‘RB2/3’, and distantly related ‘divergent’ M. catarrhalis phylogenetic lineages, potentially contributing to their independent evolution by limiting genetic flux. Further analysis of 648 OM, COPD, and nasopharyngeal carriage isolates with multi-plex PCR revealed that while modM2 is the most frequently occurring allele across all sites of infection, modM3 is overrepresented in child middle ear isolates, suggesting a potential role in OM. We previously demonstrated that phase variation of ModM2, regulates the expression of a phasevarion comprised of 34 genes in via differential methylation of the target sequence 5′-GARm6AC-3′. Expanding on our initial analysis, we show that ModM3 methylates a unique target sequence, 5′-ACm6ATC-3′, and regulates the expression of a distinct phasevarion comprised of 29 genes, including genes involved in growth under biofilm forming conditions and the response to oxidative and nitrosative stress. We also show in an in vivo Chinchilla lanigera model of OM that challenge with the modM3 OFF variant resulted in an increased middle ear bacterial load compared to the modM3 ON variant, highlighting the biological relevance of an important epigenetic regulator. Through investigation of phase variation, R-M systems, and phasevarions in M. catarrhalis, this thesis has increased understanding of M. catarrhalis pathogenesis and will aid in future selection of stably expressed M. catarrhalis vaccine candidates.