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
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.
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 14Cacetate 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.
Francisella tularensis is a facultative intracellular bacterium that infects many cell types, including neutrophils. We demonstrated previously that F. tularensis inhibits NADPH oxidase assembly and ...activity and then escapes the phagosome to the cytosol, but effects on other aspects of neutrophil function are unknown. Neutrophils are short-lived cells that undergo constitutive apoptosis, and phagocytosis typically accelerates this process. We now demonstrate that F. tularensis significantly inhibited neutrophil apoptosis as indicated by morphologic analysis as well as annexin V and TUNEL staining. Thus, ∼80% of infected neutrophils remained viable at 48 h compared with ∼50% of control cells, and ∼40% of neutrophils that ingested opsonized zymosan. In keeping with this finding, processing and activation of procaspases-8, -9, and -3 were markedly diminished and delayed. F. tularensis also significantly impaired apoptosis triggered by Fas crosslinking. Of note, these effects were dose dependent and could be conferred by either intracellular or extracellular live bacteria, but not by formalin-killed organisms or isolated LPS and capsule, and were not affected by disruption of wbtA2 or FTT1236/FTL0708-genes required for LPS O-antigen and capsule biosynthesis. In summary, we demonstrate that F. tularensis profoundly impairs constitutive neutrophil apoptosis via effects on the intrinsic and extrinsic pathways, and thereby define a new aspect of innate immune evasion by this organism. As defects in neutrophil turnover prevent resolution of inflammation, our findings also suggest a mechanism that may in part account for the neutrophil accumulation, granuloma formation, and severe tissue damage that characterizes lethal pneumonic tularemia.
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.
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 14C 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.
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 14Cacetate, combined with fractionation of 14Cacetate-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 14Cacetate 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.
A fundamental step in the life cycle of Francisella tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required ...for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum nor the receptors that mediate infection of neutrophils have been defined. In this study, human neutrophil uptake of GFP-expressing F. tularensis strains live vaccine strain and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components, we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis, whereas C5 was not. Second, we used purification and immunodepletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-Ag and capsule as prominent targets of these Abs on the bacterial surface. Finally, we demonstrate using receptor-blocking Abs that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-Ag polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3 opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner.
A fundamental step in the life cycle of
F. tularensis
is bacterial entry into host cells.
F. tularensis
activates complement, and recent data suggest that the classical pathway is required for ...complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by
F. tularensis
in nonimmune human serum, nor the receptors that mediate infection of neutrophils has been defined. Herein human neutrophil uptake of GFP-expressing
F. tularensis
strains LVS and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components we demonstrated first that C1q and C3 were essential for
F. tularensis
phagocytosis whereas C5 was not. Second, we used purification and immuno-depletion approaches to identify a critical role for natural IgM in this process, and then used a
wbtA2
mutant to identify LPS O-antigen and capsule as prominent targets of these antibodies on the bacterial surface. Finally, we demonstrate using receptor-blocking antibodies that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized
F. tularensis
by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of
F. tularensis
phagocytosis and support a model whereby natural IgM binds to surface capsular and O-antigen polysaccharides of
F. tularensis
and initiates the classical complement cascade via C1q to promote C3-opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner.
Francisella tularensis
is a facultative intracellular bacterium that infects many cell types including neutrophils. We demonstrated previously that
F. tularensis
inhibits NADPH oxidase assembly and ...activity and then escapes the phagosome to the cytosol, but effects on other aspects of neutrophil function are unknown. Neutrophils are short-lived cells that undergo constitutive apoptosis, and phagocytosis typically accelerates this process. We now demonstrate that
F. tularensis
significantly inhibited neutrophil apoptosis as indicated by morphological analysis as well as Annexin V and TUNEL staining. Thus, ~80% of infected neutrophils remained viable at 48 h as compared with ~50% of control cells, and ~40% of neutrophils that ingested opsonized zymosan. In keeping with this, processing and activation of procaspases-8, -9 and -3 were markedly diminished and delayed.
F. tularensis
also significantly impaired apoptosis triggered by Fas crosslinking. Of note, these effects were dose-dependent and could be conferred by either intracellular or extracellular live bacteria, but not by formalin-killed organisms or isolated LPS and capsule, and were not affected by disruption of
wbtA2
or
FTT1236/FTL0708
, genes required for LPS O-antigen and capsule biosynthesis. In summary, we demonstrate for the first time that
F. tularensis
profoundly impairs constitutive neutrophil apoptosis via effects on the intrinsic and extrinsic pathways, and thereby define a new aspect of innate immune evasion by this organism. As defects in neutrophil turnover prevent resolution of inflammation, our findings also suggest a mechanism that may in part account for the neutrophil accumulation, granuloma formation and severe tissue damage that characterizes lethal pneumonic tularemia.
A hallmark of
Francisella tularensis
, a highly virulent Gram-negative bacterium, is its unusual lipopolysaccharide (LPS). Detailed analyses of
Francisella
LPS have revealed 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
Cacetate, combined with fractionation of
14
Cacetate-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. We show that 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-antigen polysaccharides. The spectrum of lipid A and LPS species varied in both a medium-dependent and strain-dependent fashion, and intracellular 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
Cacetate greatly facilitates assessment of the effect of genotypic and/or environmental variables on the synthesis and net accumulation of lipid A and LPS by
Francisella
, including during growth within the cytosol of infected host cells.
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