Phase Variation in HMW1A Controls a Phenotypic Switch in Haemophilus influenzae Associated with Pathoadaptation during Persistent Infection

Genetic variants arising from within-patient evolution shed light on bacterial adaptation during chronic infection. Contingency loci generate high levels of genetic variation in bacterial genomes, enabling adaptation to the stringent selective pressures exerted by the host. A significant gap in our understanding of phase-variable contingency loci is the extent of their contribution to natural infections. The human-adapted pathogen nontypeable Haemophilus influenzae (NTHi) causes persistent infections, which contribute to underlying disease progression. The phase-variable high-molecular-weight (HMW) adhesins located on the NTHi surface mediate adherence to respiratory epithelial cells and, depending on the allelic variant, can also confer high epithelial invasiveness or hyperinvasion. In this study, we characterize the dynamics of HMW-mediated hyperinvasion in living cells and identify a specific HMW binding domain shared by hyperinvasive NTHi isolates of distinct pathological origins. Moreover, we observed that HMW expression decreased over time by using a longitudinal set of persistent NTHi strains collected from chronic obstructive pulmonary disease (COPD) patients, resulting from increased numbers of simple-sequence repeats (SSRs) downstream of the functional P2hmw1A promoter, which is the one primarily driving HMW expression. Notably, the increased SSR numbers at the hmw1 promoter region also control a phenotypic switch toward lower bacterial intracellular invasion and higher biofilm formation, likely conferring adaptive advantages during chronic airway infection by NTHi. Overall, we reveal novel molecular mechanisms of NTHi pathoadaptation based on within-patient lifestyle switching controlled by phase variation. IMPORTANCE Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly switch their expression. Several NTHi adhesins are phase variable. These adhesins are required for colonization but also immunogenic, in such a way that bacteria with lower adhesin levels are better equipped to survive an immune response, making their contribution to natural infections unclear. We show here that the major NTHi adhesin HMW1A displays allelic variation, which can drive a phase-variable epithelial hyperinvasion phenotype. Over time, hmw1A phase variation lowers adhesin expression, which controls an NTHi lifestyle switch from high epithelial invasiveness to lower invasion and higher biofilm formation. This reversible loss of function aligns with the previously stated notion that epithelial infection is essential for NTHi infection establishment, but once established, persistence favors gene inactivation, in this case facilitating biofilm growth

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

The PLOS Pathogens Staff. Notice of Republication.This article was republished on May 13, 2016, to correct the title which was incorrectly published as “Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza”. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.This work was supported by National Institutes of Health Ruth Kirschstein postdoctoral fellowship F32AI084427 (to JCM); a Canadian Institute of Health Research operating grant (to RJR); Genome British Columbia grant SOF122 (to RJR and JCM); the Faculty of Pharmaceutical Sciences, Canadian Foundation for Innovation (to CN); National Institutes of Heath R01 grant DC002873 (to JWSG); and (to JG) grants from Ministerio Economía y Competitividad-MINECO SAF2012-31166 and SAF2015-66520-R, Dpto. Salud Gobierno Navarra 359/2012 and Ministerio de Educación PRX12/00191.Peer reviewe

Preclinical evaluation of the antimicrobial-immunomodulatory dual action of xenohormetic molecules against haemophilus influenzae respiratory infection

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammation and impaired airway immunity, providing an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. In this context, therapies targeting not only overactive inflammation without significant adverse effects, but also infection are of interest. Increasing evidence suggests that polyphenols, plant secondary metabolites with anti-inflammatory and antimicrobial properties, may be protective. Here, a Cistus salviifolius plant extract containing quercetin, myricetin, and punicalagin was shown to reduce NTHi viability. Analysis of these polyphenols revealed that quercetin has a bactericidal effect on NTHi, does not display synergies, and that bacteria do not seem to develop resistance. Moreover, quercetin lowered NTHi airway epithelial invasion through a mechanism likely involving inhibition of Akt phosphorylation, and reduced the expression of bacterially-induced proinflammatory markers il-8, cxcl-1, il-6, pde4b, and tnfα. We further tested quercetin’s effect on NTHi murine pulmonary infection, showing a moderate reduction in bacterial counts and significantly reduced expression of proinflammatory genes, compared to untreated mice. Quercetin administration during NTHi infection on a zebrafish septicemia infection model system showed a bacterial clearing effect without signs of host toxicity. In conclusion, this study highlights the therapeutic potential of the xenohormetic molecule quercetin against NTHi infection

Serial enrichment for invasive recombinants by gentamicin protection.

<p><b>(A)</b> Boxplots of intracellular invasion frequencies from Pools 0–4 (2 experiments in triplicate). <b>(B)</b> Boxplots of adhesion frequencies (2 experiments in triplicate). The values show the combined ability of clones in Pool <i>n</i> to invade or adhere to airway epithelial cells, while the recovered colonies comprise Pool <i>n+</i>1. Nal^R and Nov^R pools are summarized by the left and right boxplots, with individual data points shown as circles and squares, respectively.</p

Model of enhanced intracellular invasion by HMW1_86-028NP.

<p>Bacteria self-aggregate and adhere to the epithelial cell surface in clumps, and HMW1-mediated attachments are maintained during uptake by cells into subcellular compartments with endosomal features.</p

The <i>hmw1A</i>_{<i>86-028NP</i>} gene confers increased adhesion and intracellular invasion.

<p><b>(A)</b> Western blot detection of HMW adhesins with a guinea pig anti-HMW1A gp85 antibody. The 154 kDa protein corresponds to HMW1A_86-028NP; the 168 kDa protein corresponds to HMW1A_Hi375, and the 150 kDa protein corresponds to HMW2A_Hi375. Expression of HMW2A_86-028NP was not detectable with the antibody used. <b>(B)</b> Intracellular invasion and <b>(C)</b> adhesion by NpNN and HiT derivatives including mutant, recombinant and recombinant mutant strains. Blue indicates NpNN, green indicates the HiT recipient and mutant derivatives, and purple indicates the rHiT recombinant and mutant derivatives. Experiments were run in triplicate on three separate days, as indicated by the three distinct symbols.</p

Intracellular bacteria immunofluorescence microscopy.

<p>Intracellular bacteria immunofluorescence microscopy.</p

Invasion and adhesion in the parent strains.

<p><b>(A)</b> “Invaders/CFU” was calculated as the total gentamicin-protected CFU / total input CFU. <b>(B)</b> “Adherents/CFU” was calculated as above with gentamicin treatment excluded. Triplicate experiments were conducted three times for RdS and HiT recipients and five times for the NpNN donor. Boxplots outline the first and third quartiles, with the thick horizontal line indicating the median, and the whiskers extend 1.5 times the interquartile distance.</p

Self-aggregation is increased by <i>hmw1A</i>_{<i>86-028NP</i>}.

<p>Bacteria scraped from chocolate agar plates were suspended into 35 ml of sBHI normalized to OD₆₀₀ = 0.5 in a 50 ml conical tube and allowed to sit on the lab bench. The OD₆₀₀ at the top of the cultures was followed over time as a proxy for clumping/self-aggregation. Error bars indicate the standard deviation from four replicate assays run on different days.</p

Genotype and phenotype of recombinant clones from Pool 4.

<p>Sequencing grouped 16 clones into six genotypes, two RdS Nal^R (A and B), one RdS Nov^R (C), one HiT Nal^R (D) and two HiT Nov^R (E and F). Genome-wide donor allele frequencies of each clone are shown in <b>(A)</b>. The x-axis indicates the recipient genome coordinate and the y-axis shows donor allele frequency from 0 to 1. Blue arrows mark the antibiotic resistance sites; red arrows mark the invasion locus; and the green arrow marks the <i>hmw2</i> artifact. Clone assignments are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005576#ppat.1005576.s020" target="_blank">S8 Table</a> and exact donor segment breakpoints are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005576#ppat.1005576.s021" target="_blank">S9 Table</a>. All clones were phenotyped in triplicate and were aggregated by genotype for plots depicting their invasion <b>(B)</b> and adhesion <b>(C)</b> phenotypes. Different symbols within each genotype indicate the specific isolated colony tested (labeled 1–4, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005576#ppat.1005576.s020" target="_blank">S8 Table</a>).</p