Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer

Many intracellular parasites are capable of penetrating host epithelial barriers. To study this process in more detail we examined the interactions between the pathogenic bacteria Salmonella choleraesuis and polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells grown on membrane filters. Association of bacteria with the MDCK cell apical surface was an active event, requiring bacterial RNA and protein synthesis, and was blocked by low temperatures. Salmonella were internalized within a membrane-bound vacuole and exhibited penetration through, but not between MDCK cells. A maximum of 14 Salmonella per MDCK cell crossed the monolayer per hour to the basolateral surface yet the monolayer remained viable and impermeable to Escherichia coli. Apical S. choleraesuis infection resulted in an increase in paracellular permeability but the MDCK intercellular contacts were not significantly disrupted. Basolateral S. choleraesuis infection was inefficient, and only small numbers of S. choleraesuis penetrated to the apical medium.     

Characterization of a Salmonella choleraesuis mutant that cannot multiply within epithelial cells

A mutant of Salmonella choleraesuis was identified that could invade (enter) and penetrate through polarized monolayers of Caco-2 and MDCK epithelial cells at normal levels but was defective for intracellular multiplication within these cells. It was also able to survive inside cultured J774 macrophage cells. These bacteria remained inside membrane-bound vacuoles, which coalesced at later times in the perinuclear region of the epithelial cell. This mutant exhibited slightly slower growth rates in rich or minimal media than the parental strain but was normal for iron usage, phosphate usage, and anaerobic growth and was a prototroph. The mutant was completely avirulent when administered orally or intravenously to susceptible mice. These results suggest that the ability to multiply within eukaryotic cells may contribute to S. choleraesuis virulence.     

Identification of Salmonella typhimurium invasiveness loci.

Salmonella typhimurium is capable of entering into (invading) nonphagocytic host cells. To systematically identify the bacterial genes necessary for this process, 15,000 Tn10dCm random transposon mutants of S. typhimurium were individually screened for invasiveness, using the human colonic epithelial Caco-2 cell line. Four hundred and eighty-eight mutants had decreased levels of invasiveness; most were nonmotile. However, five mutants, representing four loci, were completely motile. Further characterization of these five mutants showed that they were also unable to enter the dog kidney epithelial cell line MDCK and the mouse macrophage line J774.A1. In contrast to the parental strain, they were unable to disrupt the transepithelial resistance of polarized epithelial monolayers, nor were they able to penetrate across these epithelial barriers. Three of the four classes of mutants remained virulent in mice. The results confirm several aspects of S. typhimurium invasiveness: (i) intact motility enhances invasiveness of cultured cells; (ii) S. typhimurium invasiveness is multifactorial, and at least six distinct genetic loci are involved; and (iii) invasion loci involved in uptake into epithelial cells are also needed for uptake into cultured phagocytic cells. The results also emphasize that decreased levels of invasiveness eliminate bacterial penetration of polarized epithelial barriers and invasiveness loci mutants are not necessarily avirulent.     

猪霍乱沙门菌载体介导猪瘟病毒DNA免疫研究

构建了猪瘟病毒(CSFV)主要保护性抗原E2基因的真核表达质粒pVAXE2。将其电转化猪霍乱沙门氏菌C500疫苗株,得到了携带pVAXE2质粒的猪霍乱沙门氏菌工程菌株S.C500/pVAXE2,对该菌株的特征、培养特性和生化特性进行了鉴定。分别用1×10⁸CFU、2×10⁹CFU S.C500/pVAXE2经口服或肌肉注射免疫小鼠和家兔,间接ELISA检测免疫动物的特异性抗体,在第三次免疫后2周用20ID50猪瘟兔化弱毒和致死量猪霍乱沙门氏菌强毒株对免疫兔进行攻击。结果表明,S.C500/pVAXE2保持了猪霍乱沙门氏菌原有形态特征、培养特性和生化特性,免疫鼠和兔都产生了抗CSFV和猪霍乱沙门菌的ELISA抗体,免疫家兔能抵抗猪瘟兔化弱毒株和猪霍乱沙门氏菌强毒株的攻击。显示了以S.C500为DNA运输载体构建二联或多联猪用疫苗的可行性。     

Epithelial cell polarization is a determinant in the infectious outcome of immunoglobulin A-mediated entry by Epstein-Barr virus.

Diseases of the nasopharyngeal epithelium due to Epstein-Barr virus (EBV) infection typically occur in chronic virus carriers with preexisting virus-specific antibodies. In vitro studies have shown that EBV-specific immunoglobulin A (IgA) promotes infection of human epithelial cells, otherwise refractory to EBV, via the polymeric immunoglobulin receptor (pIgR). To determine if EBV similarly exploits IgA transport mechanisms in vivo, we examined the fate of IgA-EBV complexes in the blood of mice, where pIgR-mediated transcytosis of IgA immune complexes through hepatocytes eliminates exogenous antigens from the circulation. By PCR analysis we showed hepatobiliary transport of IgA-EBV in viremic mice, but without detectable hepatocellular infection by immunostaining. Because efficient transport of EBV immune complexes might avert an infectious outcome, we modulated the transcytotic pathway in polarized Madin-Darby canine kidney (MDCK) cells transfected with pIgR to determine the effect on viral antigen expression. Like hepatocytes in vivo, MDCK cells in polarized monolayers translocated IgA-EBV from the basal cell face into apical medium without evidence for infection. However, when exposed to IgA-EBV as unpolarized single-cell suspensions, MDCK cells expressed EBV immediate-early and early antigens. These results suggest that pIgR-mediated transcytosis of pIgA-EBV through epithelium facilitates endogenous spread of EBV in long-term virus carriers, with infection being confined to cells with altered polarity from prior cytopathology.     

Role of lipopolysaccharide in the receptor function for bacteriophage Ox2

Abstract The sensitivity of Salmonella typhimurium as well as of Escherichia coli to the bacteriophage Ox2 was found to require, in addition to the OmpA protein, a certain type of rough LPS. Heptoseless mutants were resistant and unable to adsorb the phage. Mutants with less defective LPS chemotype were sensitive and could, except the second most defective chemotypes, adsorb Ox2. However, isolated LPS-free OmpA protein could bind the phage, and this binding could not be increased by adding LPS.     

Role of the RecBCD Recombination Pathway in Salmonella Virulence

Mutants of Salmonella enterica lacking the RecBC function are avirulent in mice and unable to grow inside macrophages (N. A. Buchmeier, C. J. Lipps, M. Y. H. So, and F. Heffron, Mol. Microbiol. 7:933-936, 1993). The virulence-related defects of RecBC(-) mutants are not suppressed by sbcB and sbcCD mutations, indicating that activation of the RecF recombination pathway cannot replace the virulence-related function(s) of RecBCD. Functions of the RecF pathway such as RecJ and RecF are not required for virulence. Since the RecBCD pathway, but not the RecF pathway, is known to participate in the repair of double-strand breaks produced during DNA replication, we propose that systemic infection by S. enterica may require RecBCD-mediated recombinational repair to prime DNA replication inside phagocytes. Mutants lacking both RecD and RecJ are also attenuated in mice and are unable to proliferate in macrophages, suggesting that exonucleases V and IX provide alternative functions for RecBCD-mediated recombinational repair during Salmonella infection.     

Trichomonas vaginalis: Ultrastructural Bases of the Cytopathic Effect

ABSTRACT. The in vitro cytopathic effect of Trichomonas vaginalis on epithelial cells was explored through the interaction of trophozoites of the virulent strain GT-10 with MDCK monolayers. The interaction was analyzed through electrophysiology, video microscopy, and transmission and scanning electron microscopy. Electrical measurements revealed that living parasites produced severe damage to the cell monolayers within 30 min, manifested as a rapid decrease in transepithelial resistance. Microscopic observations demonstrated that when placed in contact with epithelial cells, trichomonas formed clumps through interdigitations and transient plasma membrane junctions between adjacent parasites. Also, attached trophozoites adopted an ameboid shape. The in vitro cytopathic action of T. vaginalis on MDCK cells was initially evident by modifications of the plasma membrane, resulting in opening of tight junctions, membrane blebbing, and monolayer disruption. After 15 min of interaction the damage was focal, concentrating at sites where parasite clumps adhered to the monolayer. At 30 min practically all MDCK cells were dead, whether or not trichomonas were attached to them. These events were followed by detachment of lysed cells and complete disruption of the monolayer at 60 min. Electron microscopy demonstrated a peculiar form of adhesion that appears to be specific for trichomonas, in which the basal surface of T. vaginalis formed slender channels through which microvilli and cytoplasmic fragments of epithelial cells were internalized. The same sequence of lytic events was found with the less virulent GT-3 strain. However, the time course of cytolysis with GT-3 parasites was much slower, and lysis was limited to areas of attachment of T. vaginalis.     

Salmonella enterica serovar Typhimurium effectors SopB, SopE, SopE2 and SipA disrupt tight junction structure and function

Salmonella enterica serovar Typhimurium is a major cause of human gastroenteritis. Infection of epithelial monolayers by S. Typhimurium disrupts tight junctions that normally maintain the intestinal barrier and regulate cell polarity. Tight junction disruption is dependent upon the Salmonella pathogenicity island-1 (SPI-1) type 3 secretion system but the specific effectors involved have not been identified. In this study we demonstrate that SopB, SopE, SopE2 and SipA are the SPI-1-secreted effectors responsible for disruption of tight junction structure and function. Tight junction disruption by S. Typhimurium was prevented by inhibiting host protein geranylgeranylation but was not dependent on host protein synthesis or secretion of host-derived products. Unlike wild-type S. Typhimurium, DeltasopB, DeltasopE/E2, DeltasipA, or DeltasipA/sopB mutants, DeltasopB/E/E2 and DeltasipA/sopE/E2 mutants were unable to increase the permeability of polarized epithelial monolayers, did not disrupt the distribution or levels of ZO-1 and occludin, and did not alter cell polarity. These data suggest that SPI-1-secreted effectors utilize their ability to stimulate Rho family GTPases to disrupt tight junction structure and function.     

Differential contribution of sodC1 and sodC2 to intracellular survival and pathogenicity of Salmonella enterica serovar Choleraesuis

Several of the most virulent Salmonella enterica strains possess two genes encoding periplasmic Cu,Zn superoxide dismutase, sodC1 and sodC2, located on a lambdoid prophage and on the chromosome, respectively. These genes contribute to Salmonella virulence by protecting bacteria from superoxide generated by the host's phagocytes. To investigate the respective contributions of sodC1 and sodC2 to the virulence of a clinical isolate of Salmonella enterica serovar Choleraesuis (S. choleraesuis), we have analyzed both the intracellular survival of wild type and sodC mutant strains within J774 macrophages and Caco-2 cells, and their ability to proliferate in intraperitoneally-infected mice in competition assays. In agreement with previous studies, mutant strains lacking one or both sodC genes were equally impaired in their ability to survive within activated macrophages. However, when macrophage killing experiments were carried out with non-opsonized bacteria, sodC2 contributed to intracellular survival more than sodC1, indicating that changes in the pathways of bacterial uptake can modify the relative role of the two sodC genes. More unexpectedly, we have found that the ability of S. choleraesuis to survive within Caco-2 cells was severely affected by inactivation of sodC genes, sodC2 being more important than sodC1. As Caco-2 cells actively produce superoxide, this suggests that oxygen radical production by colonic cells has a role in controlling proliferation of facultative intracellular bacteria. Mouse infection studies confirmed that, in the S. choleraesuis strain under investigation, both sodC genes are required to confer full virulence, sodC2 contributing slightly more than sodC1 to Salmonella pathogenesis. Our findings contrast with the results of other studies carried out in S. enterica serovar Typhimurium and suggest that the relative contributions of sodC1 and sodC2 to host-pathogen interactive biology may vary depending on the Salmonella serovar or strain.     

A Salmonella typhimurium rfaE mutant recovers invasiveness for human epithelial cells when complemented by wild type rfaE (controlling biosynthesis of ADP-L-glycero-D-mannoheptose-containing lipopolysaccharide)

Invasion of host cells is essential for the pathogenicity of Salmonella. The author's group has recently reported the cloning of the rfaE gene of Salmonella typhimurium, previously implicated in biosynthesis of the lipopolysaccharide (LPS)-inner core [Jin et al. (2001); Kim (2002)]. The product of the rfaE gene is involved in ADP-L-glycero-D-manno-heptose biosynthesis. rfaE mutants synthesize heptose-deficient LPS (Re-LPS) consisting only of lipid A and 3-deoxy-D-manno-octulosonic acid (KDO). Mutants that make incomplete LPS are rough mutants and "deep-rough" mutants affected in the heptose region of the inner core have reduced growth rate and increased sensitivity to high temperature. Complementation of S. typhimurium rfaE mutant strain SL1102 (rfaE543) with rfaE demonstrated conclusively that this gene restored the smooth phenotype, and the LPS produced by the complemented strain was indistinguishable from that of wild type smooth strains. In vitro infection experiments showed that complementation with rfaE permitted invasion of human Chang epithelial cells, larynx epidermal carcinoma HEp-2 cells and intestinal epithelial Henle-407 cells. These data imply that the structure of the LPS that is synthesized is critical for Salmonella invasiveness.     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.     

Effects of brefeldin A on endocytosis, transcytosis and transport to the Golgi complex in polarized MDCK cells

We have studied the effects of brefeldin A (BFA) on endocytosis and intracellular traffic in polarized MDCK cells by using the galactose-binding protein toxin ricin as a membrane marker and HRP as a marker of fluid phase transport. We found that BFA treatment rapidly increased apical endocytosis of both ricin and HRP, whereas basolateral endocytosis was unaffected, as was endocytosis of HRP in the poorly polarized carcinoma cell lines HEp-2 and T47D. Tubular endosomes were induced by BFA both apically and basolaterally in some MDCK cells, comparable with those seen in HEp-2 and T47D cells. In addition, in MDCK cells, BFA induced formation of small (< 300 nm) vesicles, labeled both after apical and basolateral uptake of HRP, as well as some very large (> 700 nm) vacuoles, which were only labeled when HRP was present in the apical medium. In contrast, neither in MDCK nor in HEp-2 or T47D cells, did BFA have any effect on lysosomal morphology. Moreover, transcytosis in the basolateral-apical direction was stimulated both for HRP and ricin. Other vesicular transport routes were less affected or unaffected by BFA treatment. Two closely related structural analogues of BFA (B16 and B21), unable to produce the changes in Golgi and endosomal morphology seen after BFA treatment in a number of different cell lines, were also unable to mimic the effects of BFA on MDCK cells.     

Isolation and characterization ofSerratia marcescens mutants defective in prodigiosin biosynthesis

Mutants deficient in the biosynthesis of prodigiosin have been obtained by treatingSerratia marcescens with high doses of ultraviolet radiation. Mutants were selected on the basis of the color characteristics of their colonies when grown on peptone glycerol medium. New types of mutants, with unusual blocks in the biosynthetic pathway of prodigiosin, were obtained. All the mutants were classified under a new scheme on the basis of the syntrophic pigmentation characteristic and infrared spectroscopic analysis of their pigment. By these criteria mutants could be distinguished into eight distinct classes. Classes I to III include mutants of the three classes (M1, B3, and B1) reported previously [Morrison, DA (1966) J Bacteriol 91:1599–1604] and several new ones. Mutants blocked in the methylamylpyrrole (MAP) arm of the bifurcated pathway were assigned to class I. A class II mutant was distinguished by its inability to synthesize methoxybipyrrolecarboxyaldehyde (MBC), but was able to produce norprodigiosin. Class III mutants were deficient in the synthesis of hydroxybipyrrolecarboxaldehyde (HBC). Double mutants were obtained with defects in the expression of both MBC and MAP and were assigned to class IV. Mutants of class V were unable to synthesize HBC and MAP, but could form MBC when furnished with exogenous HBC. Class VI and VII mutants were defective in the synthesis of all three precursors, but differed in their ability to perform the coupling step. Finally, a mutant of class VIII was found to produce the three intermediates, but was deficient in prodigiosin or norprodigiosin biosynthesis, indicative of a defect in the enzymatic condensation of MAP with the bipyrroles MBC and HBC. The anomalous pattern of syntrophism among certain interclass mutants suggests that the physiology of pigment formation inS. marcescens is quite complex.     

Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids

Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect nonpolarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene luciferase (LUC) into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, which were most successful in initial LUC assays, were also tested for transfer of vectors bearing the reporter gene green fluorescent protein (GFP) and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function.     

The characteristics of the O-specific humoral immune response of mice vaccinated with Salmonella choleraesuis antigens]

The work deals with the results of the comparative enzyme immunoassay (EIA) of serum samples taken from (CBA X C57BL/6) F1 mice immunized with O-specific polysaccharides, O-antigens (O-Ag) obtained by Boivin's method and antigenic preparations isolated with hydroxylamine (HA) from S. choleraesuis and S. typhimurium. O-Ag and lipopolysaccharide (LPS) of the corresponding bacterial species were used as antigens for the sensitization of polystyrene plates. The primary and secondary humoral immune response was studied by means of EIA. As revealed in this investigation, the immunization of mice with HA-isolated antigenic preparations and O-Ag, obtained from S. typhimurium, in a single injection (in doses of 1-100 micrograms) led to the development of weak specific immune response to O-Ag. Response to LPS was absent. After the second immunization of the animals pronounced immune response to O-Ag and LPS was observed. It developed as a response of both IgM and IgG type. The immunization of mice, made in a single injection, with HA-isolated antigenic preparations and O-Ag, obtained from S. choleraesuis, did not lead to the development of O-specific immune response. After the immunization of mice with these antigens in two injections sharply pronounced nonspecific activity of IgM and IgG serum antibodies with respect to O-Ag and LPS of homologous and heterologous bacterial species was noted in EIA. Neither S. typhimurium O-polysaccharide, nor S. choleraesuis O-polysaccharide did not induce O-specific immune response even after the second immunization.     

IgG transcytosis and recycling by FcRn expressed in MDCK cells reveals ligand-induced redistribution

The neonatal Fc receptor (FcRn) transports IgG across epithelial cells and recycles serum IgG. FcRn binds IgG at the acidic pH of endosomes and releases IgG at the basic pH of blood. We expressed rat FcRn in polarized MDCK cells and demonstrated that it functions in transcytosis and recycling of IgG. In the absence of IgG, FcRn is distributed predominantly apically, but redistributes to basolateral locations upon IgG addition, indicating that ligand binding induces a signal that stimulates transcytosis. FcRn transcytoses IgG more efficiently in the apical to basolateral than the reverse direction when IgG is internalized by receptor-mediated endocytosis at acidic pH or by fluid phase endocytosis at basic pH. The PI 3-kinase inhibitor wortmannin disrupts basolateral recycling and transcytosis in both directions, but only minimally reduces apical recycling. Confocal imaging and quantitative IgG transport studies demonstrate that apically-internalized IgG recycles to the apical surface mainly from wortmannin-insensitive apical early endosomes, whereas FcRn-IgG complexes that transcytose to the basolateral surface pass through downstream Rab11-positive apical recycling endosomes and transferrin-positive common endosomal compartments.     

Epithelial cell polarity alters Rho-GTPase responses to Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues. Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface. We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen. Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P. aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA. In contrast, P. aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation. There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly. Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases. Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P. aeruginosa infection. Nevertheless, polarized epithelial cells still sense the presence of apically infecting P. aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.     

Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line.

The development of surface polarity has been studied in the epithelial Madin-Darby canine kidney (MDCK) cell line by examining two basolateral markers: a monoclonal antibody against a 58-kd protein and [35S]methionine uptake. The surface distribution of these markers was followed after plating the cells on coverslips or nitrocellulose filters. In subconfluent monolayers the apical surface of many cells was stained with the anti-58-kd antibody. Clearing of the apical surface occurred first after confluency had been reached in cells grown on coverslips. Similarly, in cells grown on filters the basolateral 58-kd protein disappeared from the apical surface concomitantly with the development of a measurable electrical resistance over the cell monolayer. The uptake of [35S]methionine was measured from both sides of filter-grown cells and began to polarize early after seeding, reaching a value of greater than 98% basolateral in the fully polarized monolayer. These results emphasize that the development of surface polarity in MDCK cells is a gradual process, and that extensive cell-cell contacts seem to be required for complete surface polarization.