Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk
Authors: Baddal Buket , Muzzi Alessandro , Censini Stefano , Calogero Raffaele A. , Torricelli Giulia , Guidotti Silvia , Taddei Anna R. , Covacci Antonello , Pizza Mariagrazia , Rappuoli Rino , Soriani Marco , Pezzicoli Alfredo 
Published: November 2015

The ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeable
Haemophilus influenzae
(NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epithelium
in vitro
, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection in
Haemophilus
species. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies.

IMPORTANCE
Simultaneous monitoring of infection-linked transcriptome alterations in an invading pathogen and its target host cells represents a key strategy for identifying regulatory responses that drive pathogenesis. In this study, we report the progressive events of NTHi colonization in a highly differentiated model of ciliated bronchial epithelium. Genome-wide transcriptome maps of NTHi during infection provided mechanistic insights into bacterial adaptive responses to the host niche, with modulation of the central metabolism as an important signature of the evolving milieu. Our data indicate that infected epithelia respond by substantial alteration of the cytoskeletal network and cytokine repertoire, revealing a dynamic cross talk that is responsible for the onset of inflammation. This work significantly enhances our understanding of the means by which NTHi promotes infection on human mucosae and reveals novel strategies exploited by this important pathogen to cause invasive disease.