Infection of Primary Human Bronchial Epithelial Cells by Haemophilus influenzae : Macropinocytosis as a Mechanism of Airway Epithelial Cell Entry

Abstract

Nontypeable Haemophilus influenzae is an exclusive human pathogen which infects the respiratory epithelium. We have initiated studies to explore the interaction of the nontypeableH. influenzae strain 2019 with primary human airway epithelial cells by electron and confocal microscopy. Primary human airway cell cultures were established as monolayers on glass collagen-coated coverslips or on semipermeable membranes at an air-fluid interface. Scanning electron microscopy indicated that bacteria adhered to nonciliated cells in the population. The surface of infected cells showed evidence of cytoskeletal rearrangements manifested by microvilli and lamellipodia extending toward and engaging bacteria. Confocal microscopic analysis demonstrated that infection induced actin polymerization with an increase in cortical actin as well as evidence of actin strands around the bacteria. Transmission electron microscopic analysis showed lamellipodia and microvilli surrounding organisms, as well as organisms adherent to the cell surface. These studies also demonstrated the presence of bacteria within vacuoles inside of airway cells. Confocal microscopic studies with Texas red-labeled dextran (molecular weight, 70,000) indicated that H. influenzae cells were entering cells by the process of macropinocytosis. These studies indicate that nontypeable H. influenzae can initiate cytoskeletal rearrangement within human airway epithelium, resulting in internalization of the bacteria within nonciliated human airway epithelial cells by the process of macropinocytosis.

Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

“…Thus, BALF is likely to harbor, just as serum, the protein factors that can activate signaling pathways that are crucial for the induction of DYNA-IND entry of IAV. In agreement herewith, macropinocytosis has been described as a functional entry pathway of Haemophilus influenzae into primary human bronchial epithelial cells [71] although the factors involved in signaling the process have not been identified yet.…”

Moraxella catarrhalis is internalized in respiratory epithelial cells by a trigger-like mechanism and initiates a TLR2- and partly NOD1-dependent inflammatory immune response

“…(Clerc and Sansonetti, 1987) and Neisseria gonorrhoeae (Zenni et al, 2000) have been shown to trigger macropinocytosis as the prime mechanism for their entrance into epithelial cells. The findings of TEM and SEM hereby described show that M. catarrhalis infection of pulmonary epithelial cells caused ultrastructural changes resembling those changes following macropinocytosis as demonstrated for H. influenzae (Ketterer et al, 1999). Bacterial uptake by macropinocytosis was confirmed by using HRP to track macropinocytotic vesicles in pulmonary epithelial cells (Oliver et al, 1984).…”

“…Besides M. catarrhalis, Haemophilus influenzae and Streptococcus pneumoniae are also known to colonize and infect the lower respiratory tract in patients with COPD (Murphy et al, 2000;Sethi et al, 2002). Both organisms are capable not only to efficiently adhere to but also to invade the airway epithelium (Cundell et al, 1995;Ketterer et al, 1999). The ability to invade epithelial cells has been discussed as a useful bacterial strategy to colonize the respiratory tract and to avoid extracellular immune recognition by pattern recognition receptors such as the transmembranous Toll-like receptors (TLRs) (Cossart and Sansonetti, 2004;Philpott and Girardin, 2004;Inohara et al, 2005;Akira et al, 2006).…”

Intracellular phenotype of Mycobacterium avium enters macrophages primarily by a macropinocytosis‐like mechanism and survives in a compartment that differs from that with extracellular phenotype

“…This process involves the actin-dependent formation of lamellipodia. It is usually associated with the ingestion of an increased number of bacteria and has been reported to be the mechanism of uptake of Salmonella typhimurium (Francis et al, 1993;Garcia-del Portillo and Finlay, 1994), Shigella flexneri (Clerc and Sansonetti, 1987), Haemophilus influenzae (Ketterer et al, 1999) and Neisseria gonorrhoeae by host cells (Zenni et al, 2000). Invasion of cells by S. typhimurium, as well as S. flexneri, involves activation of the type III secretion system upon contact with the host cell (Galan, 1996;Menard et al, 1994).…”

“…Secretion of virulence proteins into the host cells stimulates extensive membrane ruffling. The macropinocytosis associated with H. influenzae invasion of epithelial cells is less extensive and involves the fusion of several lamellipodia (Ketterer et al, 1999).…”