Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenzae

Abstract

Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe “transformed recombinant enrichment profiling” (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2A Hi375 by hmw1A 86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A 86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A 86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A 86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation.

Inactivation of the Thymidylate Synthase thyA in Non-typeable Haemophilus influenzae Modulates Antibiotic Resistance and Has a Strong Impact on Its Interplay with the Host Airways

“…NTHi self-aggregates, which may promote microcolony formation on host cell surfaces (Meng et al, 2011 ; Mell et al, 2016 ). We asked whether the observed thymidine auxotrophy-driven increased bacterial size differs in its self-aggregation, by using tube-settling assays and monitoring the optical density of bacterial suspensions over time.…”

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

“…This suggests the presence of a regulatory mechanism controlling the fate of the individual cells within the NTHi population, determining the conditions under which NTHi does or does not enter host cells. The role of HMW1/HMW2 extends to entry, with a recent experimental study highlighting how its transfer from a clinical strain to a laboratory strain lacking those genes, alters its phenotype . Transformation of 86‐028NP hmw1A into a laboratory strain lacking it (Rd KW20), or another strain with a different allele (Hi375), indicated a 100–1000‐fold increased invasion of A549 cells .…”

“…The role of HMW1/HMW2 extends to entry, with a recent experimental study highlighting how its transfer from a clinical strain to a laboratory strain lacking those genes, alters its phenotype . Transformation of 86‐028NP hmw1A into a laboratory strain lacking it (Rd KW20), or another strain with a different allele (Hi375), indicated a 100–1000‐fold increased invasion of A549 cells . The increased self‐aggregation and host cell adherence seen was likely to promote group invasion of the host cells rather than individually , implying that a dynamic group population of bacteria can mediate entry as well as colonizing cells.…”

Genome-wide analysis of DNA uptake across the outer membrane of naturally competent Haemophilus influenzae

“…Many bacteria are naturally competent, able to actively bind DNA fragments at the cell surface, and pull them into the cytoplasm, where the incoming fragments may contribute nucleotides to cellular pools or recombine with homologous genomic sequences ( Lorenz and Wackernagel, 1994 ). The genetic exchange associated with this latter process contributes to adaptation and is known to have promoted resistance to antibiotics ( Bae et al., 2014 ) and increased strains' intracellular invasiveness ( Mell et al., 2016 ) and vaccine resistance ( Kress-Bennett et al., 2016 ; Straume et al., 2015 ). Thus, understanding how different genomic regions evolve via natural transformation processes could be used to predict the spread of pathogenic traits.…”