Viral Coinfection Replaces Effects of Suilysin onStreptococcus suisAdherence to and Invasion of Respiratory Epithelial Cells Grown under Air-Liquid Interface Conditions

ABSTRACTStreptococcus suisis an important zoonotic pathogen which can infect humans and pigs worldwide, posing a potential risk to global public health. Suilysin, a pore-forming cholesterol-dependent cytolysin, is considered to play an important role in the pathogenesis ofS. suisinfections. It is known that infection with influenza A viruses may favor susceptibility to secondary bacterial infection, resulting in more severe disease and increased mortality. However, the molecular mechanisms underlying these coinfections are incompletely understood. Applying highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface (ALI) conditions, we analyzed the contribution of swine influenza viruses (SIV) to the virulence ofS. suis, with a special focus on its cytolytic toxin, suilysin. We found that during secondary bacterial infection, suilysin ofS. suiscontributed to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas the cytotoxic effects induced by SIV became prominent at later stages of infection. Prior infection by SIV enhanced the adherence to and colonization of porcine airway epithelial cells by a wild-type (wt)S. suisstrain and a suilysin-negativeS. suismutant in a sialic acid-dependent manner. A striking difference was observed with respect to bacterial invasion. After bacterial monoinfection, only the wtS. suisstrain showed an invasive phenotype, whereas the mutant remained adherent. When the epithelial cells were preinfected with SIV, the suilysin-negative mutant also showed an invasion capacity. Therefore, we propose that coinfection with SIV may compensate for the lack of suilysin in the adherence and invasion process of suilysin-negativeS. suis.

Download Full-text

Murine Tracheal and Nasal Septal Epithelium for Air–Liquid Interface Cultures: A Comparative Study

American Journal of Rhinology ◽

10.2500/ajr.2007.21.3068 ◽

2007 ◽

Vol 21 (5) ◽

pp. 533-537 ◽

Cited By ~ 44

Author(s):

Bradford A. Woodworth ◽

Marcelo B. Antunes ◽

Geeta Bhargave ◽

James N. Palmer ◽

Noam A. Cohen

Keyword(s):

Epithelial Cells ◽

Respiratory Epithelium ◽

Liquid Interface ◽

Respiratory Epithelial Cells ◽

Cell Counts ◽

Total Cell Population ◽

Respiratory Epithelial ◽

Air Liquid Interface ◽

Ciliated Epithelial Cells

Background Air–liquid interface cultures using murine tracheal respiratory epithelium have revolutionized the in vitro study of airway diseases. However, these cultures often are impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. The ability to study ciliary physiology in vitro is of utmost importance in the research of chronic rhinosinusitis (CRS). Our hypothesis is that the murine nasal septum is a better source of ciliated respiratory epithelium to develop respiratory epithelial air–liquid interface models. Methods Nasal septa and tracheas were harvested from 10 BALB/c mice. The nasal septa were harvested by using a simple and straightforward novel technique. Scanning electron microscopy was performed on all specimens. Cell counts of ciliated respiratory epithelial cells were performed at one standard magnification (1535×). Comparative analysis of proximal and distal trachea, midanterior and midposterior nasal septal epithelium, was performed. Results Independent cell counts revealed highly significant differences in the proportion of cell populations (p < 0.00001). Ciliated cell counts for the trachea (106.9 ± 28) were an average of 38.7% of the total cell population. Nasal septal ciliated epithelial cells (277.5 ± 16) comprised 90.1% of the total cell population. Conclusion To increase the yield of respiratory epithelial cells harvested from mice, we have found that the nasal septum is a far superior source when compared with the trachea. The greater surface area and increased concentration of ciliated epithelial cells has the potential to provide an eightfold increase in epithelial cells for the development of air–liquid interface cultures.

Download Full-text

Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions

Scientific Reports ◽

10.1038/srep26748 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 17

Author(s):

Fandan Meng ◽

Nai-Huei Wu ◽

Maren Seitz ◽

Georg Herrler ◽

Peter Valentin-Weigand

Keyword(s):

Epithelial Cells ◽

Streptococcal Infection ◽

Liquid Interface ◽

Interface Conditions ◽

Respiratory Epithelial Cells ◽

Respiratory Epithelial ◽

Air Liquid Interface ◽

Porcine Respiratory

Download Full-text

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Journal of Visualized Experiments ◽

10.3791/2513 ◽

2011 ◽

Cited By ~ 10

Author(s):

Hilaire C. Lam ◽

Augustine M.K. Choi ◽

Stefan W. Ryter

Keyword(s):

Epithelial Cells ◽

Cigarette Smoke ◽

Liquid Interface ◽

Respiratory Epithelial Cells ◽

Respiratory Epithelial ◽

Air Liquid Interface

Download Full-text

Real-time measurement of cellular bioenergetics in fully differentiated human nasal epithelial cells grown at air-liquid-interface

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00414.2019 ◽

2020 ◽

Vol 318 (6) ◽

pp. L1158-L1164

Author(s):

Emily Mavin ◽

Bernard Verdon ◽

Sean Carrie ◽

Vinciane Saint-Criq ◽

Jason Powell ◽

...

Keyword(s):

Epithelial Cells ◽

Real Time ◽

Aerobic Glycolysis ◽

Airway Epithelial Cells ◽

Respiratory Epithelium ◽

Liquid Interface ◽

Human Nasal Epithelial Cells ◽

Respiratory Epithelial ◽

Air Liquid Interface

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease, but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus, to date, our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture, or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic, but not mitochondrial, ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium, and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes.

Download Full-text

Primary Swine Respiratory Epithelial Cell Lines for the Efficient Isolation and Propagation of Influenza A Viruses

Journal of Virology ◽

10.1128/jvi.01091-20 ◽

2020 ◽

Vol 94 (24) ◽

Author(s):

Victoria Meliopoulos ◽

Sean Cherry ◽

Nicholas Wohlgemuth ◽

Rebekah Honce ◽

Karen Barnard ◽

...

Keyword(s):

Influenza Virus ◽

Epithelial Cells ◽

Cell Lines ◽

Influenza A ◽

Vaccine Development ◽

Mdck Cells ◽

Influenza Viruses ◽

Clinical Samples ◽

Respiratory Epithelial Cells ◽

Respiratory Epithelial

ABSTRACT Influenza virus isolation from clinical samples is critical for the identification and characterization of circulating and emerging viruses. Yet efficient isolation can be difficult. In these studies, we isolated primary swine nasal and tracheal respiratory epithelial cells and immortalized swine nasal epithelial cells (siNEC) and tracheal epithelial cells (siTEC) that retained the abilities to form tight junctions and cilia and to differentiate at the air-liquid interface like primary cells. Critically, both human and swine influenza viruses replicated in the immortalized cells, which generally yielded higher-titer viral isolates from human and swine nasal swabs, supported the replication of isolates that failed to grow in Madin-Darby canine kidney (MDCK) cells, and resulted in fewer dominating mutations during viral passaging than MDCK cells. IMPORTANCE Robust in vitro culture systems for influenza virus are critically needed. MDCK cells, the most widely used cell line for influenza isolation and propagation, do not adequately model the respiratory tract. Therefore, many clinical isolates, both animal and human, are unable to be isolated and characterized, limiting our understanding of currently circulating influenza viruses. We have developed immortalized swine respiratory epithelial cells that retain the ability to differentiate and can support influenza replication and isolation. These cell lines can be used as additional tools to enhance influenza research and vaccine development.

Download Full-text

Role of the Zinc Uptake ABC Transporter of Moraxella catarrhalis in Persistence in the Respiratory Tract

Infection and Immunity ◽

10.1128/iai.00589-13 ◽

2013 ◽

Vol 81 (9) ◽

pp. 3406-3413 ◽

Cited By ~ 20

Author(s):

Timothy F. Murphy ◽

Aimee L. Brauer ◽

Charmaine Kirkham ◽

Antoinette Johnson ◽

Mary Koszelak-Rosenblum ◽

...

Keyword(s):

Epithelial Cells ◽

Respiratory Tract ◽

Abc Transporter ◽

Divalent Cations ◽

Zinc Uptake ◽

Wild Type ◽

Human Respiratory Tract ◽

Respiratory Epithelial Cells ◽

Content Type ◽

Respiratory Epithelial

ABSTRACTMoraxella catarrhalisis a human respiratory tract pathogen that causes otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. We have identified and characterized a zinc uptake ABC transporter that is present in all strains ofM. catarrhalistested. A mutant in which theznugene cluster is knocked out shows markedly impaired growth compared to the wild type in medium that contains trace zinc; growth is restored to wild-type levels by supplementing medium with zinc but not with other divalent cations. Thermal-shift assays showed that the purified recombinant substrate binding protein ZnuA binds zinc but does not bind other divalent cations. Invasion assays with human respiratory epithelial cells demonstrated that the zinc ABC transporter ofM. catarrhalisis critical for invasion of respiratory epithelial cells, an observation that is especially relevant because an intracellular reservoir ofM. catarrhalisis present in the human respiratory tract and this reservoir is important for persistence. Theznuknockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the zinc uptake ABC transporter mediates uptake of zinc in environments with very low zinc concentrations and is critical for full virulence ofM. catarrhalisin the respiratory tract in facilitating intracellular invasion of epithelial cells and persistence in the respiratory tract.

Download Full-text

Pneumococcal Interactions with Epithelial Cells Are Crucial for Optimal Biofilm Formation and ColonizationIn VitroandIn Vivo

Infection and Immunity ◽

10.1128/iai.00488-12 ◽

2012 ◽

Vol 80 (8) ◽

pp. 2744-2760 ◽

Cited By ~ 100

Author(s):

Laura R. Marks ◽

G. Iyer Parameswaran ◽

Anders P. Hakansson

Keyword(s):

Epithelial Cells ◽

Biofilm Formation ◽

Invasive Disease ◽

Respiratory Epithelial Cells ◽

Symptomatic Infection ◽

Content Type ◽

Abiotic Surfaces ◽

Horizontal Spread ◽

Respiratory Epithelial

ABSTRACTThe human nasopharynx is the main reservoir forStreptococcus pneumoniae(the pneumococcus) and the source for both horizontal spread and transition to infection. Some clinical evidence indicates that nasopharyngeal carriage is harder to eradicate with antibiotics than is pneumococcal invasive disease, which may suggest that colonizing pneumococci exist in biofilm communities that are more resistant to antibiotics. While pneumococcal biofilms have been observed during symptomatic infection, their role in colonization and the role of host factors in this process have been less studied. Here, we show for the first time that pneumococci form highly structured biofilm communities during colonization of the murine nasopharynx that display increased antibiotic resistance. Furthermore, pneumococcal biofilms grown on respiratory epithelial cells exhibited phenotypes similar to those observed during colonizationin vivo, whereas abiotic surfaces produced less ordered and more antibiotic-sensitive biofilms. The importance of bacterial-epithelial cell interactions during biofilm formation was shown using both clinical strains with variable colonization efficacies and pneumococcal mutants with impaired colonization characteristicsin vivo. In both cases, the ability of strains to form biofilms on epithelial cells directly correlated with their ability to colonize the nasopharynxin vivo, with colonization-deficient strains forming less structured and more antibiotic-sensitive biofilms on epithelial cells, an association that was lost when grown on abiotic surfaces. Thus, these studies emphasize the importance of host-bacterial interactions in pneumococcal biofilm formation and provide the first experimental data to explain the high resistance of pneumococcal colonization to eradication by antibiotics.

Download Full-text

Acidosis increases the susceptibility of respiratory epithelial cells toPseudomonas aeruginosa-induced cytotoxicity

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00524.2016 ◽

2017 ◽

Vol 313 (1) ◽

pp. L126-L137 ◽

Cited By ~ 6

Author(s):

Iviana M. Torres ◽

Sally Demirdjian ◽

Jennifer Vargas ◽

Britton C. Goodale ◽

Brent Berwin

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Antimicrobial Peptides ◽

Bacterial Infection ◽

Cell Cytotoxicity ◽

Bacterial Survival ◽

Ph Homeostasis ◽

Respiratory Epithelial Cells ◽

Underlying Mechanisms ◽

Respiratory Epithelial

Bacterial infection can lead to acidosis of the local microenvironment, which is believed to exacerbate disease pathogenesis; however, the mechanisms by which changes in pH alter disease progression are poorly understood. We test the hypothesis that acidosis enhances respiratory epithelial cell death in response to infection with Pseudomonas aeruginosa. Our findings support the idea that acidosis in the context of P. aeruginosa infection results in increased epithelial cell cytotoxicity due to ExoU intoxication. Importantly, enforced maintenance of neutral pH during P. aeruginosa infection demonstrates that cytotoxicity is dependent on the acidosis. Investigation of the underlying mechanisms revealed that host cell cytotoxicity correlated with increased bacterial survival during an acidic infection that was due to reduced bactericidal activity of host-derived antimicrobial peptides. These findings extend previous reports that the activities of antimicrobial peptides are pH-dependent and provide novel insights into the consequences of acidosis on infection-derived pathology. Therefore, this report provides the first evidence that physiological levels of acidosis increase the susceptibility of epithelial cells to acute Pseudomonas infection and demonstrates the benefit of maintaining pH homeostasis during a bacterial infection.

Download Full-text