cytochalasin-d and monodansylcadaverine

Peyer's patches constitute both an inductive immune site and an enteropathogen invasion route. Peyer's patch mucosae from porcine jejunum were mounted in Ussing chambers, and either Salmonella choleraesuis vaccine strain SC-54 or non-pathogenic rodent and porcine Escherichia coli strains contacted the Peyer's patch mucosa for 90 min. Internalized bacteria were quantified by a gentamicin resistance assay. Monodansylcadaverine (300 microM, luminal addition), an inhibitor of clathrin-mediated endocytosis, significantly inhibited internalization of both E. coli strains relative to tissues untreated with the inhibitor; internalization of SC-54 was unaffected. The actin-disrupting agent cytochalasin D (10 microM, luminal addition), inhibited internalization of pig-adapted E. coli but not that of rodent-adapted E. coli or SC-54. Internalization of SC-54 and non-pathogenic E. coli in Peyer's patches appears to occur through different cellular routes.

Topics: Actins; Animals; Cadaverine; Clathrin; Cytochalasin D; Electric Conductivity; Endocytosis; Escherichia coli; Female; Jejunum; Male; Peyer's Patches; Salmonella; Swine

The initial interaction between mosquito-borne flavivirus West Nile and mosquito cells is poorly characterized. This study analyzed the endocytic and the associated signaling pathway that mediate the infectious entry of West Nile virus (WNV) into mosquito cell line (C6/36). Pretreatment of C6/36 cells with pharmacological drugs that blocks clathrin-mediated endocytosis significantly inhibited virus entry. Furthermore, the transfection of functional blocking antibody against clathrin molecules and the overexpression of dominant-negative mutants of Eps15 in C6/36 cells caused a marked reduction in WNV internalization. WNV was shown to activate focal adhesion kinase (FAK) to facilitate the endocytosis of virus but not the mitogen-activated protein kinases (ERK1 and ERK2). Subsequent to the internalization of WNV, the virus particles are translocated along the endosomal pathway as revealed by double-immunofluorescence assays with anti-WNV envelope protein and cellular markers for early and late endosomes. Specific inhibitor for protein kinase C (PKC) was shown to be highly effective in blocking WNV entry by inhibiting endosomal sorting event. The disruption of the microtubule network using nocodazole also drastically affects the entry process of WNV but not the disruption of actin filaments by cytochalasin D. Finally, a low-pH-dependent step is required for WNV infection as revealed by the resistance of C6/36 cells to WNV infection in the presence of lysosomotropic agents.

Topics: Actin Cytoskeleton; Aedes; Animals; Antigens, Viral; Cadaverine; Cell Line; Chlorpromazine; Clathrin; Cytochalasin D; Endocytosis; Endosomes; Enzyme Inhibitors; Filipin; Focal Adhesion Protein-Tyrosine Kinases; Intracellular Signaling Peptides and Proteins; Microscopy, Confocal; Microscopy, Fluorescence; Microtubules; Mitogen-Activated Protein Kinases; Nocodazole; Protein Kinase C; Sucrose; West Nile virus

We examined the in vitro ability of Enterococcus faecalis clinical isolates to adhere to and to invade HeLa cells, suggested to be a valuable model system to study bacteria-directed endocytosis. Using a variety of compounds that act on eukaryotic cell structures, both microtubules and microfilaments were found to be involved in enterococcal entry into cells. Two distinct modes of interaction were observed: in one, a close proximity of bacteria with the cell membrane was observed, possibly leading to direct engulfment of the bacterial cell. In the other mode, cellular pseudopodal formation seemed to be stimulated by vicinity of bacterial cells; in some cases, such associations involved formation of clathrin-coated-like vesicles before internalizing enterococci. The above-mentioned experimental data together with the use of monodansylcadaverine, amiloride and NH4Cl, all involved in cytosol acidification and inhibition of receptor-mediated endocytosis (RME), led us to conclude that E. faecalis is internalized within HeLa cells by more than one invasion pathway. One, sensitive to amiloride, is most likely a macropinocytic, actin-dependent uptake mechanism, which determines the production of large smooth-membrane vacuoles engulfing enterococci. The other is RME, in which entry is dependent on both microfilament and microtubule structural integrity.

Topics: Acids; Bacterial Adhesion; Cadaverine; Cells, Cultured; Colchicine; Cytochalasin D; Endocytosis; Enterococcus faecalis; Gram-Positive Bacterial Infections; HeLa Cells; Humans; Microscopy, Electron

Invasion of Edwardsiella ictaluri into cultured mammalian, fish and enzymatically harvested catfish enteric epithelial cells is described. Gentamicin survival assays were used to demonstrate the ability of this catfish pathogen to invade IEC-6 (origin: rat small intestinal epithelium), Henle 407 (origin: human embryonic intestinal epithelium), fathead minnow (FHM, minnow epithelial cells) and trypsin/pepsin-harvested channel catfish enteric epithelial cells. Invasion of all cell types occurred within 2 h of contact at 26 degrees C, in contrast to Escherichia coli DH5 alpha, which did not invade cells tested. Eight Edwardsiella ictaluri isolates from diseased catfish and the ATCC (American Type Culture Collection) strain were evaluated for invasion efficiency using FHM cells. All isolates were invasive, but at differing efficiencies. Invasion blocking assays using chemical blocking agents were performed on a single isolate (LA 89-9) using IEC-6 epithelial cells. Preincubation of IEC-6 cells with cytochalasin D (microfilament depolymerizer) and monodansylcadaverine (blocks receptor-mediated endocytosis) significantly reduced invasion by E. ictaluri, whereas exposure to colchicine (microtubule depolymerizer) had no effect on bacterial internalization. Results indicate that actin polymerization and receptor-mediated endocytosis are involved in uptake of E. ictaluri by IEC-6 epithelial cells. Invasion trials using freshly harvested cells from the intestine of the natural host, Ictalurus punctatus, show that invasion occurs, but at a low efficiency. This is possibly due to loss of outer membrane receptors during enzymatic cell harvest. This study provides the first documentation of the invasion of cultured mammalian and fish cells by E. ictaluri, and identifies possible mechanisms used for intracellular access. Additionally, the study describes several functional in vitro invasion models using commercially available cell lines as well as cells from the natural host (channel catfish, I. punctatus).

Topics: Animals; Bacterial Adhesion; Cadaverine; Catfishes; Cell Line; Colchicine; Cyprinidae; Cytochalasin D; Edwardsiella ictaluri; Endocytosis; Enterobacteriaceae Infections; Enzyme Inhibitors; Epithelial Cells; Fish Diseases; Humans; Intestinal Mucosa; Nucleic Acid Synthesis Inhibitors; Rats

We studied MHC class II (MHC-II)-restricted antigen processing of viable Streptococcus pyogenes by murine macrophages for presentation of two CD4 T cell epitopes of the surface M5 protein. We show that presentation of both epitopes was prevented if actin polymerization was inhibited by cytochalasin D, but not if clathrin-dependent receptor-mediated endocytosis was prevented, suggesting uptake of streptococci by phagocytosis or macropinocytosis was required for presentation of the surface M protein. However, treatment of macrophages with amiloride, which selectively blocks membrane ruffling and subsequent macropinocytosis, inhibited the response to one epitope (M5(308-319)), but had no effect on presentation of the other (M5(17-31)). The effect of the inhibitors on uptake of streptococci was analyzed by electron microscopy. Cytochalasin D completely blocked uptake of streptococci, while dimethyl-amiloride only inhibited uptake into spacious compartments. Neither of the inhibitors altered the cell-surface expression of MHC-II and costimulatory molecules analyzed by flow cytometry. The data suggest that distinct epitopes of a protein associated with viable bacteria may be presented optimally following different uptake mechanisms in the same antigen-presenting cells.

Topics: Amiloride; Amino Acid Sequence; Animals; Antigen Presentation; Antigens, Bacterial; Antigens, CD; B7-1 Antigen; B7-2 Antigen; Cadaverine; CD4-Positive T-Lymphocytes; CD40 Antigens; Cell Line; Cytochalasin D; Epitopes; Histocompatibility Antigens Class II; Macrophages; Membrane Glycoproteins; Mice; Microscopy, Electron; Molecular Sequence Data; Streptococcus pyogenes

Staphylococcus aureus is an important bone pathogen, and evidence shows that this organism is internalized by chick osteoblasts. Here we report that S. aureus is internalized by human osteoblasts. Internalization was inhibited by monodansylcadaverine and cytochalasin D and to a lesser extent by ouabain, monensin, colchicine, and nocodazole. We propose that internalization occurs via a receptor-mediated pathway, requiring the participation of cytoskeletal elements, principally actin.

Topics: Actins; Animals; Cadaverine; Cells, Cultured; Chick Embryo; Cytochalasin D; Humans; Microscopy, Electron; Osteoblasts; Staphylococcus aureus; Virulence

Staphylococcus aureus is a bacterial pathogen causing approximately 80% of all cases of human osteomyelitis. This bacterium can adhere to and become internalized by osteoblasts and previous studies indicate that osteoblasts are active in the internalization process. In the current study, we examined the roles of microfilaments, microtubules and clathrin-dependent receptor-mediated endocytosis in the internalization of S. aureus by MC3T3-E1 mouse osteoblast cells. Microfilament and microtubule polymerization was inhibited with cytochalasin D and colchicine. Clathrin-coated pit formation was examined by using the transaminase inhibitor, monodanslycadaverine. The results of this study indicate that mouse osteoblasts utilize actin microfilaments, microtubules and clathrin-coated pits in the internalization of S. aureus; however, microfilaments seem to play the most significant role in the invasion process.

Topics: Animals; Cadaverine; Cell Line; Cell Survival; Colchicine; Cytochalasin D; Dimethyl Sulfoxide; Gentamicins; Mice; Osteoblasts; Staphylococcus aureus

Organisms of some Legionella species are known to internalize and multiply within epithelial cell lines. During the study on interaction between Legionella spp. and Vero cells, we found that L. dumoffii Tex-KL (ATCC 33343) can enter into Vero cells approximately four to 20 times more often than five other strains of four species of legionella. The mode of entry between L. dumoffii Tex-KL and L. pneumophila Philadelphia-1 was compared and studied by treating Vero cells with reagents which inhibit phagocytosis and endocytosis. Monodansylcadaverine, cytochalasin D and nocodazol were used as inhibitors of receptor-mediated endocytosis, microfilament-dependent phagocytosis and polymerization of microtubules, respectively. The uptake of L. dumoffii Tex-KL required receptor-mediated endocytosis by Vero cells, while the uptake of L. pneumophila Philadelphia-1 used mainly microfilament-dependent phagocytosis. Polymerization of microtubules was necessary for Vero cells for the uptake of both strains of legionella. An electron microscopic examination revealed that some organisms of the L. dumoffii strain Tex-KL escaped from endosomal vacuoles into cytoplasm in the early stage of infection, and proliferated in the cytoplasm. At that period, most of the bacteria were surrounded by rough endoplasmic reticula. In contrast, L. pneumophila Philadelphia-1 proliferated only within ribosome-lined endosome. We suggest that L. dumoffii Tex-KL internalize and proliferate in Vero cells in a different way to L. pneumophila Philadelphia-1, and that there is a variety of the mode of interaction between Legionella spp. and epithelial cells.

Topics: Actin Cytoskeleton; Animals; Cadaverine; Chlorocebus aethiops; Cytochalasin D; Cytoplasm; Endocytosis; Endosomes; Hexoses; Legionella; Legionella pneumophila; Macrophages, Peritoneal; Mice; Microscopy, Electron; Microtubules; Nocodazole; Phagocytosis; Receptors, Cell Surface; Vero Cells

Bovine mammary epithelial cells were pretreated with inhibitors of protein kinase activity, actin polymerization and receptor-mediated endocytosis. In addition, mammary epithelial cells and Streptococcus dysgalactiae were pretreated with inhibitors of protein synthesis. Results showed that activity of tyrosine protein kinases, intact microfilaments and de novo eukaryotic protein synthesis was required for uptake of S. dysgalactiae by bovine mammary epithelial cells; a process that appeared to occur via receptor-mediated endocytosis. In contrast, de novo bacterial protein synthesis was not required for uptake of S. dysgalactiae by MAC-T cells. This study provides insight into bacterial and cellular mechanisms involved in early host-pathogen interactions, putting into perspective the role of mammary epithelial cells in the development and establishment of intramammary infections by S. dysgalactiae.

Topics: Actins; Animals; Cadaverine; Cattle; Cell Line; Chloramphenicol; Cytochalasin B; Cytochalasin D; Endocytosis; Enzyme Inhibitors; Epithelial Cells; Female; Genistein; HeLa Cells; Humans; Mammary Glands, Animal; Protein Kinase Inhibitors; Staurosporine; Streptococcus

Tetanus toxin (TeTX) has been demonstrated to inhibit transmitter release by two mechanisms: Zn(2+)-dependent proteolytic cleavage of synaptobrevin and activation of a neuronal transglutaminase. Herein, attenuation of TeTX-induced blockade of noradrenaline release from synaptosomes was achieved by prior disassembly of microfilaments with cytochalasin D or breakdown of microtubules by colchicine or nocodazole. These drugs and monodansylcadaverine, a transglutaminase inhibitor, displayed some additivity in antagonizing the inhibitory effect of the toxin on synaptosomal transmitter release; as none of them reduced synaptobrevin cleavage, all appear to work independently of the toxin's proteolytic action. Prior stabilization of microtubules with taxol prevented the antagonism seen with colchicine, highlighting that this cytoskeletal component is the locus of the effect of colchicine. Replacement of Ca2+ with Ba2+ caused disappearance of the fraction of evoked secretion whose inhibition by TeTX is reliant on polymerized actin but did not alter the blockade by toxin that is dependent on microtubules. Two temporally distinguished phases of release were reduced by TeTX, and colchicine lessened its effects on both. Blockade of the fast phase (< or = 10 s) of secretion by TeTX was unaffected by cytochalasin D, but it clearly antagonized the toxin-induced inhibition of the slow (10-s to > or = 5-min) component; it is notable that such antagonism was accentuated during a second bout of evoked release. These findings are consistent with sustained release requiring dissociation of synaptic vesicles from the microfilaments, a step that seems to be perturbed by TeTX.

Topics: Actin Cytoskeleton; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Barium; Cadaverine; Calcium; Cerebral Cortex; Colchicine; Cytochalasin D; Cytoskeleton; Endopeptidases; Energy Metabolism; Enzyme Inhibitors; Gout Suppressants; Membrane Proteins; Microtubules; Nerve Tissue Proteins; Nocodazole; Norepinephrine; Nucleic Acid Synthesis Inhibitors; Paclitaxel; Potassium; R-SNARE Proteins; Rats; Synaptic Vesicles; Synaptosomes; Tetanus Toxin; Transglutaminases

The mechanisms which enable entry into cultured human epithelial cells by Klebsiella pneumoniae were compared with those of Salmonella typhi Ty2. K. pneumoniae 3091, isolated from a urine sample of a patient with a urinary tract infection, invaded human epithelial cells from the bladder and ileocecum and persisted for days in vitro. Electron microscopic studies demonstrated that K. pneumoniae was always contained in endosomes. The internalization mechanism(s) triggered by K. pneumoniae was studied by invasion assays conducted with different inhibitors that act on prokaryotic and eukaryotic cell structures and processes. Chloramphenicol inhibition of bacterial uptake revealed that bacterial de novo protein synthesis was essential for efficient invasion by K. pneumoniae and S. typhi. Interference with receptor-mediated endocytosis by g-strophanthin or monodansylcadaverine and inhibition of endosome acidification by monensin reduced the number of viable intracellular K. pneumoniae cells, but not S. typhi cells. The depolymerization of microfilaments by cytochalasin D inhibited the uptake of both bacteria. Microtubule depolymerization caused by colchicine, demecolcine, or nocodazole and the stabilization of microtubules with taxol reduced only the invasion ability of K. pneumoniae. S. typhi invasion was unaffected by microtubule depolymerization or stabilization. These data suggest that the internalization mechanism triggered by K. pneumoniae 3091 is strikingly different from the solely microfilament-dependent invasion mechanism exhibited by many of the well-studied enteric bacteria, such as enteroinvasive Escherichia coli, Salmonella, Shigella, and Yersinia strains.

Topics: Bacterial Proteins; Cadaverine; Cells, Cultured; Chloramphenicol; Colchicine; Cytochalasin D; Demecolcine; Endocytosis; Epithelial Cells; Epithelium; Humans; Ionophores; Klebsiella pneumoniae; Microtubules; Monensin; Nocodazole; Nucleic Acid Synthesis Inhibitors; Ouabain; Paclitaxel; Protein Synthesis Inhibitors; Salmonella typhi; Urinary Tract; Urinary Tract Infections

In this work, we found that rotavirus infection induces an early membrane permeabilization of MA104 cells and promotes the coentry of toxins, such as alpha-sarcin, into the cell. This cell permeability was shown to depend on infectious virus and was also shown to be virus dose dependent, with 10 infectious particles per cell being sufficient to achieve maximum permeability; transient, lasting no more than 15 min after virus entry and probably occurring concomitantly with virus penetration; and specific, since cells that are poorly permissive for rotavirus were not permeabilized. The rotavirus-mediated coentry of toxins was not blocked by the endocytosis inhibitors dansylcadaverine and cytochalasin D or by the vacuolar proton-ATPase inhibitor bafilomycin A1, suggesting that neither endocytocis nor an intraendosomal acidic pH or a proton gradient is required for permeabilization of the cells. Compounds that raise the intracellular concentration of calcium ([Ca2+]i) by different mechanisms, such as the calcium ionophores A23187 and ionomycin and the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, did not block the coentry of alpha-sarcin or affect the onset of viral protein synthesis, suggesting that a low [Ca2+]i is not essential for the initial steps of the virus life cycle. Since the entry of alpha-sarcin correlates with virus penetration in all parameters tested, the assay for permeabilization to toxins might be a useful tool for studying and characterizing the route of entry and the mechanism used by rotaviruses to traverse the cell membrane and initiate a productive replication cycle.

Topics: Abrin; Allergens; Animals; Antibodies, Viral; Antigens, Plant; Cadaverine; Calcium; Cell Line; Cell Membrane Permeability; Cytochalasin D; Cytotoxins; Endocytosis; Endopeptidases; Endoribonucleases; Fungal Proteins; Haplorhini; Humans; Mice; N-Acetylneuraminic Acid; Neutralization Tests; Proton-Translocating ATPases; Ribonucleases; Rotavirus; Tumor Cells, Cultured; Vacuolar Proton-Translocating ATPases; Virus Replication

Although murine L cells bind and internalize rotavirus as well as permissive cell lines, L cells are essentially nonpermissive for rotaviruses. In nonpermissive cell lines such as L cells, internalized rotavirus fails to uncoat and remains as infectious, double-shelled particles. This block in the infectious cycle can be overcome by direct lipofection of viral particles into the L cell cytoplasm. We hypothesized that the internalized rotavirus particles within L cells are sequestered in the endocytic pathway and are unable to initiate infection. L cells were pretreated with a variety of inhibitors of endocytosis prior to infection with rhesus rotavirus. While agents which inhibit acidification of endosomes had no effect on rotavirus infection, two potential direct inhibitors of vesicular transport, dansylcadaverine and cytochalasin D, enhanced rotavirus infection of L cells 5- to 10-fold. All of the drugs, including both inhibitors of endocytosis and lysosomotrophic agents, significantly reduced infection of L cells by serotype 1 reovirus which is known to infect L cells by the endocytic pathway. Time course studies demonstrated that the drugs were effective in promoting rotavirus infection of L cells in only the early phases of infection. Pretreatment of L cells with dansylcadaverine significantly decreased the number of intact, double-shelled rotavirus particles sequestered within the cells. Inhibition of endocytosis may increase the efficiency of infection of L cells by rotavirus by allowing an increased proportion of attached rotavirus virions to enter cells by a productive route which is probably direct membrane penetration.

Topics: Animals; Cadaverine; Coated Pits, Cell-Membrane; Cytochalasin D; Endocytosis; L Cells; Macaca mulatta; Mice; Microscopy, Electron; Orthoreovirus; Rotavirus; Virus Replication

Previous studies with three isolates from diarrhoeal stools suggested that Providencia alcalifaciens is an invasive enteric pathogen that also causes actin condensation in infected cells. These findings were extended in the present study with a further 14 diarrhoeal stool isolates of P. alcalifaciens and HEp-2 cell monolayers for invasion assays. Studies on invasion characteristics with two selected isolates suggested that P. alcalifaciens required prior growth at 37 degrees C for better invasion. Invasion and actin condensation were inhibited by an agent that inhibits microfilament formation, but not by agents that inhibit receptor-mediated endocytosis, microtubule formation, endosome acidification or receptor recycling. In time-course assays with HEp-2 cell monolayers maintained in medium containing gentamicin, P. alcalifaciens showed a small degree of multiplication after invasion of the cells, but viable bacteria could not be recovered over a 24-h period although the integrity of the cell monolayer was preserved during this period.

Topics: Adolescent; Adult; Ammonium Chloride; Cadaverine; Cell Line; Child; Child, Preschool; Chloroquine; Colchicine; Cytochalasin D; Diarrhea; Diarrhea, Infantile; Enterobacteriaceae Infections; Female; Humans; Immunosuppressive Agents; Infant; Male; Middle Aged; Providencia; Temperature; Time Factors

The binding, internalization, and proliferation of Ehrlichia risticii in P388D1 cells and equine polymorphonuclear (PMN) leukocytes were studied by immunofluorescent staining and flow cytometric analysis. The binding of ehrlichiae to P388D1 cells at 4 degrees C was dose dependent, and the antigens of bound organisms were susceptible to pronase treatment. Additionally, the binding of ehrlichiae to P388D1 cells was diminished when either P388D1 cells or ehrlichiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 15 min. These results indicate that the ehrlichial ligand and host cell receptor are likely surface proteins. Following incubation at 37 degrees C, bound E. risticii and/or its antigens were removed with pronase and indirect immunofluorescent staining in the presence of saponin was used to examine intracellular ehrlichiae. Our results indicate that E. risticii was internalized into P388D1 cells within 3 h and proliferated by 48 h of incubation. The microfilament-disrupting agent cytochalasin D and the transglutaminase inhibitor monodansylcadaverine were used to differentiate between phagocytosis (sensitive to cytochalasin) and receptor-mediated endocytosis (sensitive to monodansylcadaverine) of E. risticii by P388D1 cells. In concentrations that produced distinctive morphological changes and inhibited phagocytosis of polystyrene latex beads, cytochalasin D did not suppress the infectivity of E. risticii. Binding, internalization, or proliferation of E. risticii was not affected by cytochalasin D. However, monodansylcadaverine inhibited infection of E. risticii in a dose-dependent manner. The agent did not affect the attachment of ehrlichiae to host cells, but it did suppress internalization and proliferation. These results suggest that E. risticii is internalized by receptor-mediated endocytosis and that productive infection by E. risticii does not depend on phagocytosis by the P388D1 cells. Although E. risticii did not bind to the surface of equine PMN leukocytes at 4 degrees C, organisms were taken up by this cell at 37 degrees C. E. risticii, however, failed to survive in equine PMN leukocytes.

Topics: Animals; Bacterial Adhesion; Cadaverine; Cell Division; Cell Line; Cytochalasin D; Ehrlichia; Endocytosis; Flow Cytometry; Horses; Macrophages; Mice; Mice, Inbred DBA; Neutrophils; Paraldehyde; Trypsin

The characteristics associated with the internalization and intracellular behavior of Campylobacter jejuni during short-term and long-term cultivation with INT 407 cells were examined. The internalization of C. jejuni by INT 407 cells was inhibited by cytochalasin dansylcadaverine, chemicals that disrupt microfilament formation and inhibit receptor cycling, respectively. Ammonium chloride and methylamine, two chemicals that inhibit endosomal acidification, did not affect C. jejuni internalization. Once internalized, C. jejuni were found exclusively with membrane-bound vacuoles. With regard to intracellular survival, a decline in the number of viable intracellular bacteria, as determined by protection from gentamicin, occurred during the initial phase of infection and when a low level of the antibiotic was maintained in the culture medium. However, the number of intracellular C. jejuni increased markedly after the removal of the antibiotic. In the absence of antibiotic, the infection led to the deterioration of the cell monolayers, indicating that C. jejuni is able to survive within epithelial cells and elicit a cytotoxic effect. The ability of C. jejuni to enter and exert deleterious effects on cells may reflect a pathogenic mechanism associated with enteritis caused by this organism.

Topics: Cadaverine; Campylobacter jejuni; Cells, Cultured; Cytochalasin D; Epithelial Cells; Epithelium; Fluorescent Dyes; Gentamicins; Humans; Intestines; Kinetics; Microscopy, Electron; Microscopy, Fluorescence

Recent reports have demonstrated that Bordetella pertussis has invasive behavior in vivo and in vitro. In this study, we investigated the ability of a virulent strain, avirulent mutants, and mutants deficient in specific virulence factors to enter and survive intracellularly in human macrophages in vitro. Uptake of virulent B. pertussis was dose dependent and occurred in the absence of serum or specific antibody, with entry occurring via a microfilament-dependent phagocytic process. The virulent wild-type parental strain was internalized and persisted intracellularly over the 3 days of experiments, as determined by transmission electron microscopy and by recovery of viable plate counts. This is the first report of long-term survival of B. pertussis in human macrophages. Avirulent mutants entered macrophages, but at only an average of 1.5% of virulent parental levels, and did not survive intracellularly. Mutants which did not express adenylate cyclase toxin, filamentous hemagglutinin, or pertussis toxin had decreased abilities to enter and to survive inside macrophages. The results suggest that the internalization process, as well as intracellular survival, is virulence dependent and that mutations which inactivate expression of virulence factors may affect both. The ability of B. pertussis to enter and persist inside macrophages may be important not only for survival of the bacteria but also in the pathogenesis of whooping cough.

Topics: Bordetella pertussis; Cadaverine; Cytochalasin D; Humans; Macrophages; Microscopy, Electron; Phagocytosis; Time Factors