The PilA protein of non‐typeable Haemophilus influenzae plays a role in biofilm formation, adherence to epithelial cells and colonization of the mammalian upper respiratory tract

Abstract

SummaryWe recently described the expression of type IV pili (Tfp) by non-typeable Haemophilus influenzae (NTHI), a common respiratory tract pathogen. Prior to that report, Tfp were not thought to be produced by NTHI as they are not observed on NTHI when grown on chocolate agar or other commonly used growth media. To further characterize growth conditions permissive for the expression of NTHI Tfp, as well as determine their role in colonization and virulence, we transformed an NTHI otitis media isolate with a reporter plasmid containing the lux gene cluster driven by the pilA promoter. Transcription from the pilA promoter was demonstrated under a variety of in vitro growth conditions and, importantly, by ex vivo imaging of luciferase-producing NTHI in infected chinchillas. Luciferase-producing NTHI were also identified within a biofilm formed by NTHI in vivo. We further demonstrated a role for NTHI PilA in adherence to human respiratory epithelial cells, in colonization of the chinchilla respiratory tract as well as a requirement for PilA in biofilm development, both in vitro and in vivo. Collectively, our data demonstrate that NTHI express PilA in vivo, and that PilA plays an important role in the pathogenesis of an upper respiratory tract infection induced by NTHI.

Type IV Pilin Proteins: Versatile Molecular Modules

“…All classes of T4P, and even the T2S hyperpseudopili produced upon overexpression of the major pseudopilin, can participate in biofilm development in a wide variety of bacterial and archaeal species by promoting initial (and often highly tenacious) adherence to surfaces, as well as cell-cell interactions (122,128,208,209,328,398,400). O'Toole and Kolter (304) first reported that P. aeruginosa mutants lacking T4P are deficient in biofilm formation.…”

Nontypeable Haemophilus influenzae releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery

“…4C. The NTHI Tfp is an adhesive protein that is extruded through the outer-membrane pore formed by ComE (10,44,45). In addition to its role in adherence and twitching motility, expression of the ComE pore and Tfp is also required for the uptake of DNA from the extracellular environment (44,45).…”

“…NTHI expresses Tfp from a single subpolar location along the long axis of the bacterial cell. These Tfp are essential for twitching motility, adherence to respiratory tract epithelial cells, colonization of the mammalian respiratory tract, and competence (10,44,45,50,51). The Tfp machinery in NTHI is encoded by two operons, pilABCD and comABCDEF.…”

“…Extracellular DNA (eDNA) is a major constituent of the EPS formed by multiple human pathogens (5-7) and serves diverse roles within the bacterial biofilm. eDNA provides structural stability to the matrix, acts as a sink for antimicrobial peptides, protects resident bacteria from the host immune response, provides a source of "common goods" for the resident bacteria, acts as a universal structural material conducive to microbial community architecture, and facilitates the uptake of genetic material between bacterial species via a process known as horizontal gene transfer (5,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Members of the DNABII family of DNA-binding proteins, integration host factor (IHF) and histone-like protein (HU), are known for their strong structural influences on DNA intracellularly (18)(19)(20)(21)(22)(23)(24), and are also critical to the stability of the lattice-like 3D DNA structure found in biofilm matrices extracellularly (13,(25)(26)(27)(28)(29)(30)(31).…”

Bacterial luciferase reporters: The Swiss army knife of molecular biology

“…Other studies have used the V. harveyi luciferase as a reporter for circadian promoter activity in cyanobacteria. 65 73 Acinetobacter baylyi, 74 Bacillus anthracis, 75 Haemophilus influenza 76 and the phytopathogen Agrobacterium tumefaciens.…”