The modA10 phasevarion of nontypeable Haemophilus influenzae R2866 regulates multiple virulence-associated traits

Abstract

Non-typeable Haemophilus influenzae (NTHi) is a human restricted commensal and pathogen that elicits inflammation by adhering to and invading airway epithelia cells: transcytosis across these cells can result in systemic infection. NTHi strain R2866 was isolated from the blood of a normal 30-month old infant with meningitis, and is unusual for NTHi in that it is able to cause systemic infection. Strain R2866 is able to replicate in normal human serum due to expression of lgtC which mimics human blood group pk. R2866 contains a phase-variable DNA methyltransferase, modA10 which switches ON and OFF randomly and reversibly due to polymerase slippage over a long tetrameric repeat tract located in its open reading frame. Random gain or loss of repeats during replication can results in expressed (ON), or not expressed (OFF) states, the latter due to a frameshift or transcriptional termination at a premature stop codon. We sought to determine if the unusual virulence of R2866 was modified by modA10 phase-variation. A modA10 knockout mutant was found to have increased adherence to, and invasion of, human ear and airway monolayers in culture, and increased invasion and transcytosis of polarized human bronchial epithelial cells. Intriguingly, the rate of bacteremia was lower in the infant rat model of infection than a wild-type R2866 strain, but the fatality rate was greater. Transcriptional analysis comparing the modA10 knockout to the R2866 wild-type parent strain showed increased expression of genes in the modA10 knockout whose products mediate cellular adherence. We conclude that loss of ModA10 function in strain R2866 enhances colonization and invasion by increasing expression of genes that allow for increased adherence, which can contribute to the increased virulence of this strain.

Comparative Analysis of Streptococcus pneumoniae Type I Restriction-Modification Loci: Variation in hsdS Gene Target Recognition Domains

“…Intra-host bacterial evolution is primarily driven by spontaneous mutations (e.g., slipped-strand mispairing, recombination events, and point mutations) in surface-expressed factors/antigens [ 41 ]. For example, a random on/off slipped-strand mispairing over simple sequence repeats in genes encoding phosphorylcholine and other lipooligosaccharide antigens in Haemophilus influenzae can result in high-frequency phase variation (10 −2 /cell per generation) [ 42 , 43 , 44 , 45 ]. This mechanism has also been reported in Helicobactor pylori [ 46 ], Escherichia coli [ 47 ], and Staphylococcus aureus [ 48 , 49 ].…”

Methylomic and phenotypic analysis of the ModH5 phasevarion of Helicobacter pylori

“…The target genes regulated by these phase variable DNA methyltransferases are collectively referred to as phase variable regulons, or phasevarions. Phasevarion-associated DNA methyltranserases are emerging as important epigenetic regulators of prokaryotic virulence factor and surface antigen expression 12 – 14 .…”

Host–pathogen interactions of nontypeable Haemophilus influenzae: from commensal to pathogen

“…One further aspect of adherence of NTHi to host cells is regulated by a DNA methylase gene, mod10A , which has a long tetrameric repeat tract that is subject to polymerase slippage . This slippage changes the number of repeats (15/16) , causing on and off states of expression, where the loss of expression leads to upregulation of genes potentially promoting adherence to host cells.…”

“…This slippage changes the number of repeats (15/16) , causing on and off states of expression, where the loss of expression leads to upregulation of genes potentially promoting adherence to host cells. This adds another layer of complexity to the regulation of NTHi's adhesive ability, as the mod10A phasevarion also contributes in a second function to host cell internalization .…”