Nontypeable Haemophilus influenzae Responds to Virus-Infected Cells with a Significant Increase in Type IV Pilus Expression

Abstract

Nontypeable Haemophilus influenzae (NTHI) colonizes the human nasopharynx, but when the host immune response is dysregulated by upper respiratory tract (URT) virus infection, NTHI can gain access to more distal airway sites and cause disease. The NTHI type IV pilus (T4P) facilitates adherence, benign colonization, and infection, and its majority subunit PilA is in clinical trials as a vaccinogen. To further validate the strategy of immunization with PilA against multiple NTHI-induced diseases, it is important to demonstrate T4P expression under microenvironmental conditions that predispose to NTHI infection of the airway. Because URT infection commonly facilitates NTHI-induced diseases, we examined the influence of ongoing virus infection of respiratory tract epithelial cells on NTHI T4P expression in vitro. Polarized primary human airway epithelial cells (HAEs) were sequentially inoculated with one of three common URT viruses, followed by NTHI. Use of a reporter construct revealed that NTHI upregulated pilA promoter activity when cultured with HAEs infected with adenovirus (AV), respiratory syncytial virus (RSV), or rhinovirus (RV) versus that in mock-infected HAEs. Consistent with these results, pilA expression and relative PilA/pilin abundance, as assessed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblot, respectively, were also significantly increased when NTHI was cultured with virus-infected HAEs. Collectively, our data strongly suggest that under conditions of URT virus infection, PilA vaccinogen induction of T4P-directed antibodies is likely to be highly effective against multiple NTHI-induced diseases by interfering with T4P-mediated adherence. We hypothesize that this outcome could thereby limit or prevent the increased load of NTHI in the nasopharynx that characteristically precedes these coinfections. IMPORTANCE Nontypeable Haemophilus influenzae (NTHI) is the predominant bacterial causative agent of many chronic and recurrent diseases of the upper and lower respiratory tracts. NTHI-induced chronic rhinosinusitis, otitis media, and exacerbations of cystic fibrosis and chronic obstructive pulmonary disease often develop during or just after an upper respiratory tract viral infection. We have developed a vaccine candidate immunogen for NTHI-induced diseases that targets the majority subunit (PilA) of the type IV twitching pilus (T4P), which NTHI uses to adhere to respiratory tract epithelial cells and that also plays a role in disease. Here, we showed that NTHI cocultured with virus-infected respiratory tract epithelial cells express significantly more of the vaccine-targeted T4P than NTHI that encounters mock-infected (healthy) cells. These results strongly suggest that a vaccine strategy that targets the NTHI T4P will be effective under the most common predisposing condition: when the human host has a respiratory tract virus infection.

Z-form extracellular DNA is a structural component of the bacterial biofilm matrix

“…Next, we examined the DNA content of biofilms formed on primary, polarized human airway epithelial cells (HAEs), as a bridge to in vivo experiments. We allowed the airway pathogen, NTHI (Mokrzan et al, 2016) to form a 16 h biofilm on polarized HAEs (Figure 1B) (Mokrzan et al, 2020b;Zhang et al, 2002) and then probed for both B-DNA and Z-DNA by IF. In the absence of NTHI, no visible eDNA strands were observed (Figure 1C).…”

Pathogenesis of nontypeable Haemophilus influenzae infections in chronic suppurative lung disease

“…38 Moreover, infection of human AEC with adenovirus, respiratory syncytial virus, or rhinovirus leads to upregulation of the NTHi type IV pilus, which is its major adherence protein, promoting NTHi ability to infect the respiratory cells. 39 CSLD in children is characterized by chronic airway inflammation, which may also adversely impact upon MCC. [40][41][42] Chronic inflammation in asthma is associated with decreased cilial motility, disorientated beating direction, and ultrastructural damage.…”

“…Rhinoviruses and NTHi also share a common cellular receptor 37 and rhinoviruses inhibit macrophage interleukin (IL)‐1 responses to NTHi and diminish IL‐8 responses via TLR‐2 dependent degradation of IRAK‐1 38 . Moreover, infection of human AEC with adenovirus, respiratory syncytial virus, or rhinovirus leads to upregulation of the NTHi type IV pilus, which is its major adherence protein, promoting NTHi ability to infect the respiratory cells 39 …”

Biofilm aggregates and the host airway-microbial interface

“…Otitis media, one of the most frequent illnesses in young children and the most common reason for antibiotic therapy or surgery in children in the US ( Klein, 2000 ), is characterized by mucociliary dysfunction, blocked drainage of Eustachian tubes into the oropharynx, and defective clearance. While this occurs primarily in infants and young children due to immature facial-cranial anatomy, it is multifactorial ( Bakaletz, 2010 ) and growing evidence suggests that preceding viral infection can play an important role ( Brockson et al., 2012 ; Toone et al., 2020 ; Mokrzan et al., 2020 ; Kiedrowski and Bomberger, 2018 ; Hendricks et al., 2021 ). The middle ear epithelium consists of ciliated respiratory epithelial cells ( Figure 1 ) and when mucociliary function is disrupted, bacteria in the normal oropharyngeal and nasopharyngeal microbiota can ascend the Eustachian tube to the middle ear mucosa ( Bakaletz, 2010 ).…”