Phasevarions in
Haemophilus influenzae
biogroup
aegyptius
control expression of multiple proteins
Authors: Tram Greg , Jen Freda E.-C. , Phillips Zachary N. , Lancashire John F. , Timms Jamie , Poole Jessica , Jennings Michael P. , Atack John M. 
Published: December 2023

Haemophilus influenzae
biogroup
aegyptius
is the causative agent of Brazilian purpuric fever (BPF), an invasive disease with high mortality, that sporadically manifests in children previously suffering conjunctivitis. Though strains of
H. influenzae
biogroup
aegyptius
that cause BPF are distinct from conjunctival strains, individual factors that directly correlate with the manifestation of BPF have not been conclusively identified. Phase variation is the rapid and reversible switching of gene expression, and is typically associated with bacterial surface proteins. However, over the last ~15 years, the importance of phase-variable, cytoplasmic DNA methyltransferases in bacterial gene regulation has been reported. Variable expression of a DNA methyltransferase results in genome-wide methylation differences and regulation of multiple genes by epigenetic mechanisms. These systems are called phasevarions (phase-variable regulons). An analysis of the
modA
alleles present in genomes of
H. influenzae
biogroup
aegyptius
demonstrated that all BPF causing isolates encoded the uncharacterized
modA16
allele. The majority of non-BPF strains encoded the ModA13 allele, previously unstudied in
Haemophilus
spp. We demonstrate biphasic ON-OFF switching of both ModA13 and ModA16 and determine the distinct DNA motifs methylated by these enzymes. Differential methylation in isogenic ModA ON-OFF strain pairs results in the control of distinct phasevarions. These phasevarions include several previously characterized virulence factors. Comparison of these isogenic ON-OFF pairs of strains encoding ModA13 or ModA16 determined that switching of these phasevarions did not alter serum resistance, a key characteristic of BPF causing strains of
H. influenzae
biogroup
aegyptius
. This study provides new information about how key virulence determinants are regulated in
H. influenzae
biogroup
aegyptius
.

IMPORTANCE

Haemophilus influenzae
biogroup
aegyptius
is a human-adapted pathogen and the causative agent of Brazilian purpuric fever (BPF), an invasive disease with high mortality, that sporadically manifests in children previously suffering conjunctivitis. Phase variation is a rapid and reversible switching of gene expression found in many bacterial species, and typically associated with outer-membrane proteins. Phase variation of cytoplasmic DNA methyltransferases has been shown to play important roles in bacterial gene regulation and can act as epigenetic switches, regulating the expression of multiple genes as part of systems called phasevarions (phase-variable regulons). This study characterized two alleles of the ModA phasevarion present in
H. influenzae
biogroup
aegyptius
, ModA13, found in non-BPF causing strains and ModA16, unique to BPF causing isolates. Phase variation of ModA13 and ModA16 led to genome-wide changes to DNA methylation resulting in altered protein expression. These changes did not affect serum resistance in
H. influenzae
biogroup
aegyptius
strains.