RESUMEN
IMPORTANCE: For many years, measles virus (MeV) was assumed to first enter the host via the apical surface of airway epithelial cells and subsequently spread systemically. We and others reported that MeV has an overwhelming preference for entry at the basolateral surface of airway epithelial cells, which led to a fundamental new understanding of how MeV enters a human host. This unexpected observation using well-differentiated primary cultures of airway epithelia from human donors contradicted previous studies using immortalized cultured cells. Here, we show that appropriate differentiation and cell morphology of primary human airway epithelial cells are critical to recapitulate MeV infection patterns and pathogenesis of the in vivo airways. By simply culturing primary cells in media containing serum or passaging primary cultures, erroneous results quickly emerge. These results have broad implications for data interpretation related to respiratory virus infection, spread, and release from human airway epithelial cells.
Asunto(s)
Células Cultivadas , Células Epiteliales , Virus del Sarampión , Sarampión , Sistema Respiratorio , Humanos , Células Epiteliales/virología , Epitelio , Sarampión/virología , Sistema Respiratorio/citologíaRESUMEN
Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14 C] acetate-labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose-dependent neutrophil apoptosis inhibition via a TLR2-dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function.
Asunto(s)
Apoptosis/fisiología , Proteínas Bacterianas/metabolismo , Francisella tularensis/metabolismo , Lipoproteínas/metabolismo , Neutrófilos/fisiología , Polimorfismo de Nucleótido Simple/genética , Receptor Toll-Like 1/genética , Francisella tularensis/genética , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/fisiología , Neutrófilos/metabolismo , Neutrófilos/microbiología , Tularemia/metabolismo , Tularemia/microbiología , Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Measles virus (MeV) infection of airway surface epithelial cells provides a site for final amplification before being released back into the environment via coughing and sneezing. Multiple cell lines have served as models of polarized epithelia for MeV infection, such as Caco2 cells (intestinal derived human epithelia) or MDCK cells (kidney derived canine epithelia). In this chapter, we describe the materials and air-liquid interface (ALI) culture conditions for maintaining four different cell lines derived from human airway epithelial cells: 16HBE14o-, Calu-3, H358, and NuLi-1. We provide methods for confirming transepithelial electrical resistance (TER) and preparing samples for microscopy as well as expected results from apical or basolateral MeV delivery. Polarized human airway derived cells serve as tissue culture models for investigating targeted questions about how MeV exits a human host. In addition, these methods are generalizable to studies of other respiratory viruses or the biology of ALI airway epithelial cells.
Asunto(s)
Técnicas de Cultivo de Célula , Células Epiteliales , Virus del Sarampión , Humanos , Virus del Sarampión/fisiología , Células Epiteliales/virología , Células Epiteliales/citología , Técnicas de Cultivo de Célula/métodos , Sarampión/virología , Línea Celular , Perros , Animales , Mucosa Respiratoria/virología , Mucosa Respiratoria/citología , Impedancia EléctricaRESUMEN
Francisella tularensis, the Gram-negative bacterium that causes tularemia, produces a high molecular weight capsule that is immunologically distinct from Francisella lipopolysaccharide but contains the same O-antigen tetrasaccharide. To pursue the possibility that the capsule of Francisella live vaccine strain (LVS) has a structurally unique lipid anchor, we have metabolically labeled Francisella with [14C]acetate to facilitate highly sensitive compositional analysis of capsule-associated lipids. Capsule was purified by two independent methods and yielded similar results. Autoradiographic and immunologic analysis confirmed that this purified material was largely devoid of low molecular weight LPS and of the copious amounts of free lipid A that the Francisellae accumulate. Chemical hydrolysis yielded [14C]-labeled free fatty acids characteristic of Francisella lipid A but with a different molar ratio of 3-OH C18:0 to 3-OH C16:0 and different composition of non-hydroxylated fatty acids (mainly C14:0 rather than C16:0) than that of free Francisella lipid A. Mild acid hydrolysis to induce selective cleavage of KDO-lipid A linkage yielded a [14C]-labeled product that partitioned during Bligh/Dyer extraction and migrated during thin-layer chromatography like lipid A. These findings suggest that the O-antigen capsule of Francisella contains a covalently linked and structurally distinct lipid A species. The presence of a discrete lipid A-like molecule associated with capsule raises the possibility that Francisella selectively exploits lipid A structural heterogeneity to regulate synthesis, transport, and stable bacterial surface association of the O-antigen capsular layer.
Asunto(s)
Cápsulas Bacterianas/química , Francisella tularensis/inmunología , Lípido A/química , Antígenos O/química , Ácido Desoxicólico , Electroforesis en Gel de Poliacrilamida , Ácidos Grasos/análisis , Concentración de Iones de Hidrógeno , Immunoblotting , Lipopolisacáridos/química , Modelos Biológicos , Peso Molecular , Antígenos O/aislamiento & purificaciónRESUMEN
A hallmark of Francisella tularensis, a highly virulent Gram-negative bacterium, is an unusual LPS that possesses both structural heterogeneity and characteristics that may contribute to innate immune evasion. However, none of the methods yet employed has been sufficient to determine the overall LPS composition of Francisella. We now demonstrate that metabolic labeling of francisellae with [(14)C]acetate, combined with fractionation of [(14)C]acetate-labeled lipids by ethanol precipitation rather than hot phenol-water extraction, permits a more sensitive and quantitative appraisal of overall compositional heterogeneity in lipid A and LPS. The majority of lipid A of different francisellae strains grown in diverse bacteriologic media and within human phagocytes accumulated as very hydrophobic species, including free lipid A, with <10% of the lipid A molecules substituted with O-Ag polysaccharides. The spectrum of lipid A and LPS species varied in a medium- and strain-dependent fashion, and growth in THP-1 cells yielded lipid A species that were not present in the same bacteria grown in brain heart infusion broth. In summary, metabolic labeling with [(14)C]acetate greatly facilitates assessment of the effect of genotypic and/or environmental variables on the synthesis and accumulation of lipid A and LPS by Francisella, including during growth within the cytosol of infected host cells.