RESUMEN
Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex-like structure. mCD14-EGFP was functional in mediating binding of and responses to LPS. BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface. However, within 5-10 min, the BODIPY-LPS distributed to intracellular vesicles that did not contain mCD14-EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.
Asunto(s)
Endocitosis , Exocitosis , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Astrocitoma , Transporte Biológico , Compuestos de Boro , Compartimento Celular , Glicosilfosfatidilinositoles , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Receptores de Lipopolisacáridos/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Responses of leukocytes to low concentrations of LPS require the expression of membrane-associated CD14 (mCD14) on the cell surface; mCD14 is, however, a glycosyl-phosphatidylinositol-anchored protein and, therefore, a poor candidate for transducing signals across the plasma membrane. The role of other cell surface proteins in responses of leukocytes to LPS was tested by measuring IL-6 secretion of cultured human monocytes and adhesion of PMN in response to LPS after treatment of the cells with trypsin. Trypsin abolished leukocyte integrin-mediated adhesion of PMN in response to LPS, but the trypsinized cells remained responsive to the alternate agonists TNF, formyl peptide, and PMA. Similarly, trypsin treatment of monocytes inhibited IL-6 production in response to LPS, but not to formyl peptide or PMA. No change in cell surface expression of mCD14 was observed by cytofluorometry, and no proteolysis of mCD14 was detected by immunoblot analysis. These results suggest that digestion of a cell surface protein distinct from mCD14 must account for the loss of responsiveness to LPS. The uptake by monocytes of [3H]LPS presented in LPS-soluble CD14 complexes was also inhibited by trypsin treatment. Monomeric LPS was taken up superstoichiometrically to mCD14 with a ratio of up to 15:1, and trypsin treatment decreased this uptake by more than half. This suggests that a cell surface protein may function in accepting LPS from mCD14. Together the results suggest that one or more trypsin-sensitive cell surface proteins distinct from CD14 participate in both the uptake of LPS by leukocytes and the initiation of the signaling process.
Asunto(s)
Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/inmunología , Proteínas de la Membrana/inmunología , Monocitos/inmunología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Adhesión Celular/inmunología , Células Cultivadas , Humanos , Lipopolisacáridos/farmacología , Activación Neutrófila/efectos de los fármacosRESUMEN
Human polymorphonuclear leukocytes (PMN) respond to LPS with strongly increased integrin-mediated adhesion. While the first step of this process has been identified as the interaction of LPS with CD14 on the cell surface, subsequent steps remain to be elucidated. The experiments presented here suggest that monomeric LPS is internalized in vesicles, and uptake may be required for signaling. Fluorescently labeled LPS presented as monomeric complexes with soluble CD14 appeared in the plasma membrane of PMN by 5 min and was concentrated in cytoplasmic vesicles by 20 min. Adhesion in response to LPS/soluble CD14 occurred only after a 15- to 20-min lag period, consistent with endocytosis occurring before signal generation. In contrast, there was no time lag for adhesion in response to the formyl peptide formyl-norleucyl-leucyl-phenylalanine (fNLLP). Adhesion in response to LPS, but not fNLLP, was completely blocked by lowering the temperature to 19 degrees C, a procedure that prevents vesicle fusion. These studies indicated that an event with the time and temperature dependence of endocytosis precedes signaling by LPS. Cytochalasin D, an inhibitor of phagocytosis, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase that blocks vesicle fusion and phagocytosis, both completely blocked adhesion in response to LPS but not in response to fNLLP. These results support the idea that LPS internalization and early endosomal fusion may be required for signal transduction. Parallel studies showed that the adhesion response to TNF had time, temperature, and inhibitor sensitivities nearly identical with those of LPS, suggesting that responses to TNF may also include an obligate vesicle fusion step.
Asunto(s)
Lipopolisacáridos/farmacología , Neutrófilos/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Adhesión Celular/efectos de los fármacos , Membrana Celular/fisiología , Células Cultivadas , Citocalasina D/farmacología , Endocitosis , Activación Enzimática/efectos de los fármacos , Humanos , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , TemperaturaRESUMEN
Sensitive responses of monocytes, macrophages, and neutrophils to bacterial LPS require membrane-bound CD14 (mCD14) and a plasma protein called LPS-binding protein (LBP). Cells lacking mCD14 respond to complexes of LPS and soluble CD14 (sCD14); these responses do not require LBP. To determine whether LBP is necessary for responses of mCD14-bearing cells to LPS, we measured responses of macrophages and neutrophils to complexes of LPS and sCD14 formed in the absence of LBP. We found that the amount of LPS needed to induce adhesive responses of neutrophils or cytokine production by macrophages was the same whether LPS was added with LBP or as LPS-sCD14 complexes, and was >100-fold less than when LPS was added alone. This result supports the view that LBP transfers LPS to CD14, but is not directly involved in responses of CD14-bearing cells to LPS. Responses of neutrophils to LPS-sCD14 complexes could be inhibited partially by blocking mCD14, suggesting that LPS may move rapidly from sCD14 to mCD14. Additionally, we found that responses of neutrophils to LBP and smooth LPS were made 30 to 100 times more sensitive when sCD14 was added. Our findings show that LBP is not necessary for the activation of CD14-bearing cells with LPS, and suggest that LPS-sCD14 complexes are an important intermediate in the inflammatory responses of leukocytes to LPS.
Asunto(s)
Proteínas de Fase Aguda , Receptores de Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/administración & dosificación , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Glicoproteínas de Membrana , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Secuencia de Bases , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Humanos , Técnicas In Vitro , Interleucina-6/biosíntesis , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Datos de Secuencia Molecular , Neutrófilos/metabolismoRESUMEN
Shigella species cause bacillary dysentery in humans by invading colonic epithelial cells. IpaB and IpaC, two major invasins of these pathogens, are secreted into the extracellular milieu. We show here that IpaB and IpaC form a complex in the extracellular medium and that each binds independently to a 17 kDa polypeptide, IpgC, in the bacterial cytoplasm. The IpgC polypeptide was found to be necessary for bacterial entry into epithelial cells, to stabilize the otherwise unstable IpaB protein, and to prevent the proteolytic degradation of IpaC that occurs through its association with unprotected IpaB. We propose that IpgC, which is not secreted and thus acts as a molecular chaperone, serves as a receptor that prevents premature oligomerization of IpaB and IpaC within the cytoplasm of Shigella cells.
Asunto(s)
Adhesinas Bacterianas , Proteínas Bacterianas/metabolismo , Compartimento Celular , Citoplasma/metabolismo , Shigella flexneri/patogenicidad , Secuencia de Bases , Transporte Biológico , Células Cultivadas , Células Epiteliales , Células HeLa , Humanos , Datos de Secuencia Molecular , Operón/genética , Unión Proteica , Shigella flexneri/metabolismo , Virulencia/genética , Virulencia/fisiologíaRESUMEN
Shigella flexneri invades eucaryotic cells and grows in the cytoplasm. Lysis of the phagosomal membrane is a prerequisite for both intracellular multiplication and movement of the bacteria that gain direct access to the host cell actin. In HeLa cells, bacteria generate their own movement essentially by inducing actin polymerization. Polymerization of actin enables them to move rapidly and randomly in the cytoplasm and to spread from one cell to another through protrusions of the host cell membrane. This movement was designated the Ics phenotype. In contrast, in chicken embryo fibroblasts, bacteria move along actin filaments in a very organized manner, following the cytoskeletal architecture; this movement was designated the Olm phenotype. Bacterial movement is a major virulence factor in that it is necessary for efficient colonization of the intestinal epithelium of infected macaque monkeys. Further characterization of the cellular events that lead to colonization of the colonic intestinal epithelium was needed. In order to characterize the movement in vitro in a cell assay system more closely related to the intestinal epithelium, we used human colonic epithelial Caco-2 cells. The movement of bacteria as observed by using immunofluorescence and confocal microscopy appeared to result from the expression of both the Olm and Ics phenotypes. The former allowed colonization of cells along the actin filament ring of the perijunctional area. The latter promoted passage from one cell to adjacent cells. This in vitro pattern of movement and multiplication gives S. flexneri, once it has entered an epithelial cell, the unique capacity to spread through the entire epithelial layer without having further contact with the extracellular compartment.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Colon/microbiología , Shigella flexneri/crecimiento & desarrollo , Línea Celular , Epitelio/microbiología , Humanos , Movimiento , Polímeros/metabolismoRESUMEN
The commonly accepted view that enteroinvasive bacteria enter cells of the intestinal epithelial lining through the apical surface can be challenged in the case of shigellosis. This study is based on in vitro experiments that showed that the invasion of human colonic Caco-2 cells by Shigella flexneri occurred through the basolateral pole of these cells. In these experiments, the few bacteria that interacted with the apical surface either bound to microvilli of the cell dome without causing detectable alteration or bound at the level of intercellular junctions at which they demonstrated a limited capacity for paracellular invasion, which permitted subsequent entry through the lateral domain of the cells. Treatment of Caco-2 cell monolayers with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), which disrupts intercellular junctions, greatly enhanced the rate of cell infection. These observations suggest a physiopathological paradox that may have important consequences for the understanding of the process of colonic invasion in vivo during shigellosis.
Asunto(s)
Colon/microbiología , Shigella flexneri/patogenicidad , Adhesión Bacteriana/inmunología , Calcio/farmacología , Línea Celular , Disentería Bacilar/fisiopatología , Ácido Egtácico/farmacología , Eritrocitos/microbiología , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas In Vitro , Microscopía Electrónica de Rastreo , Receptores de Transferrina/inmunología , Virulencia/efectos de los fármacosRESUMEN
icsA (virG), a gene located on pWR100, the virulence plasmid of Shigella flexneri serotype 5 (M90T), encodes a 120-kDa outer membrane protein. This protein promotes a random intracellular movement of the bacteria and leads to the infection of adjacent cells by the formation of protrusions. This movement, which involves the nucleation, polymerization, and subsequent polarization of actin, is referred to as the Ics phenotype (intra/intercellular spread). Here we present evidence that a second, distinct form of locomotion is also elaborated by S. flexneri in chicken embryo fibroblasts in which the Ics phenotype is not expressed. Using a combination of phase-contrast microcinematography and confocal microscopy, we have demonstrated that bacteria adopt parallel orientations by interacting with stress fibers. This interaction subsequently results in bacterial movement along the stress fibers themselves. This phenomenon occurs independently of the presence of a phagocytic vacuole which is lysed shortly after entry of the bacteria into the cell. It is expressed by M90T and SC560, its icsA mutant. This movement has been termed organelle-like movement (Olm phenotype) and is thought to account for the early accumulation of bacteria seen near the nucleus.
Asunto(s)
Actinas/fisiología , Shigella flexneri/fisiología , Animales , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/genética , Embrión de Pollo , Fibroblastos/microbiología , Microtúbulos/fisiología , Modelos Biológicos , Movimiento , FenotipoRESUMEN
Production of colanic acid in Escherichia coli is regulated by two negative regulators, Lon and RcsC, and by two positive regulators, RcsA and RcsB. Two genes of Klebsiella pneumoniae, rmpA and rmpB, have been shown to positively control colanic acid synthesis in E. coli. While colanic acid production is activated by RmpA only in a lon strain of E. coli, a plasmid carrying both rmpA and rmpB can stimulate colanic acid synthesis in a Lon+ strain. In this work, we present the determination of the nucleotide sequence of rmpB and, on the basis of comparison of the predicted RmpA and RmpB sequences with those of RcsA, B and C and two-component regulatory proteins, we propose that RmpA acts as a transcriptional activator of the structural genes involved in colanic acid biosynthesis.
Asunto(s)
Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Klebsiella pneumoniae/genética , Polisacáridos/biosíntesis , Secuencia de Bases , Klebsiella pneumoniae/patogenicidad , Datos de Secuencia Molecular , Plásmidos , VirulenciaRESUMEN
In Klebsiella pneumoniae, the mucoid phenotype, which is a virulence factor, is distinct from capsule production. It is positively controlled by a plasmid gene, designated rmpA. When introduced into certain Escherichia coli strains, rmpA induces expression of a mucoid phenotype, which results from overproduction of colanic acid at 30 degrees C but not at 37 degrees C. In E. coli, production of colanic acid is regulated by three genes: rcsA and rcsB which act as positive regulators, and rcsC which is a negative effector. In this work we present evidence that the rmpA gene complemented an rcsA, Ion double mutant of E. coli, but not an rcsA, Ion+ isolate. This leads to the suggestion that rmpA expressed an rcsA-like activity and like rcsA, was negatively controlled at post-transcriptional level by the Lon protease. The nucleotide sequence of rmpA is reported. No homology could be found between the 27 kiloDalton RcsA protein and the deduced amino acid sequence of the 15.5 kiloDalton RmpA protein. Another gene, rmpB, which was required in E. coli recA isolates for full expression of rmpA at 30 degrees C, has been identified on the K. pneumoniae virulence plasmid and shown to encode a 37 kiloDalton protein. Although rmpB was closely linked to rmpA, it was not present on the same transcriptional unit. These results suggested that induction of colanic acid synthesis by the K. pneumoniae virulence gene rmpA, was, at least in E. coli, under the control of the RecA network via rmpB, which may act as a positive regulator of rmpA. We conclude that these plasmid genes may function in K. pneumoniae as regulatory genes controlling the mucoid phenotype, which is itself encoded by the chromosome.