HEp-2 Tissue Culture Cells Research Articles

Salmonella enterica Serovar Typhimurium Requires the Lpf, Pef, and Tafi Fimbriae for Biofilm Formation on HEp-2 Tissue Culture Cells and Chicken Intestinal Epithelium

Recent work has demonstrated that Salmonella enterica serovar Typhimurium forms biofilms on HEp-2 tissue culture cells in a type 1 fimbria-dependent manner. To investigate how biofilm growth of HEp-2 tissue culture cells affects gene expression in Salmonella, we compared global gene expression during planktonic growth and biofilm growth. Microarray results indicated that the transcription of approximately 100 genes was substantially altered by growth in a biofilm. These genes encode proteins with a wide range of functions, including antibiotic resistance, central metabolism, conjugation, intracellular survival, membrane transport, regulation, and fimbrial biosynthesis. The identification of five fimbrial gene clusters was of particular interest, as we have demonstrated that type 1 fimbriae are required for biofilm formation on HEp-2 cells and murine intestinal epithelium. Mutations in each of these fimbriae were constructed in S. enterica serovar Typhimurium strain BJ2710, and the mutants were found to have various biofilm phenotypes on plastic, HEp-2 cells, and chicken intestinal tissue. The pef and csg mutants were defective for biofilm formation on each of the three surfaces tested, while the lpf mutant exhibited a complete loss of the ability to form a biofilm on chicken intestinal tissue but only an intermediate loss of the ability to form a biofilm on tissue culture cells and plastic surfaces. The bcf mutant displayed increased biofilm formation on both HEp-2 cells and chicken intestinal epithelium, while the sth mutant had no detectable biofilm defects. In all instances, the mutants could be restored to a wild-type phenotype by a plasmid carrying the functional genes. This is the first work to identify the genomic responses of Salmonella to biofilm formation on host cells, and this work highlights the importance of fimbriae in adhering to and adapting to a eukaryotic cell surface. An understanding of these interactions is likely to provide new insights for intervention strategies in Salmonella colonization and infection.

M protein mediated adhesion of M type 24 Streptococcus pyogenes stimulates release of interleukin-6 by HEp-2 tissue culture cells

M protein mediated adhesion of M type 24 Streptococcus pyogenes stimulates release of interleukin-6 by HEp-2 tissue culture cells

The role of intimin in the adherence of enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 to HEp-2 tissue culture cells and to bovine gut explant tissues

AbstractIntimin, an outer membrane protein encoded by eaeA, is a key determinant for the formation of attaching and effacing (AE) lesions by enterohaemorrhagic Escherichia coli (EHEC). To investigate the role of intimin in adherence, the eaeA gene was insertionally inactivated in three EHEC O157 : H7 strains of diverse origin. The absence or presence of intimin did not correlate with the extent of adhesion of mutant or wild-type O157 : H7 in tissue culture and neonatal calf gut tissue explant adherence assays. Adherence of the eaeA mutants to HEp-2 cells was diffuse with no evidence of intimate attachment whereas wild-type bacteria formed microcolonies and AE lesions. Intimin-independent adherence to neonatal calf gut explants was demonstrated by eaeA mutants and wild-type strains which adhered in the greatest numbers to colon but least well to rumen tissue. These results confirm that intimin is necessary for intimate attachment and that additional adherence factors are involved in intimin-independent adherence.

Isolation from a sheep of an attaching and effacing Escherichia coli O115:H- with a novel combination of virulence factors.

Attaching and effacing (AE) lesions were observed in the caecum, proximal colon and rectum of one of four lambs experimentally inoculated at 6 weeks of age with Escherichia coli O157:H7. However, the attached bacteria did not immunostain with O157-specific antiserum. Subsequent bacteriological analysis of samples from this animal yielded two E. coli O115:H(-) strains, one from the colon (CO) and one from the rectum (RC), and those bacteria forming the AE lesions were shown to be of the O115 serogroup by immunostaining. The O115:H(-)isolates formed microcolonies and attaching and effacing lesions, as demonstrated by the fluorescence actin staining test, on HEp-2 tissue culture cells. Both isolates were confirmed by PCR to encode the epsilon (epsilon) subtype of intimin. Supernates of both O115:H(-) isolates induced cytopathic effects on Vero cell monolayers, and PCR analysis verified that both isolates encoded EAST1, CNF1 and CNF2 toxins but not Shiga-like toxins. Both isolates harboured similar sized plasmids but PCR analysis indicated that only one of the O115:H(-) isolates (CO) possessed the plasmid-associated virulence determinants ehxA and etpD. Neither strain possessed the espP, katP or bfpA plasmid-associated virulence determinants. These E. coli O115:H(-) strains exhibited a novel combination of virulence determinants and are the first isolates found to possess both CNF1 and CNF2.

Virulence in the chick model and stress tolerance of Salmonella enterica serovar Orion var. 15+

Three Salmonella enterica serovar Orion var. 15+ isolates of distinct provenance were tested for survival in various stress assays. All were less able to survive desiccation than a virulent S. Enteritidis strain, with levels of survival similar to a rpoS mutant of the S. Enteritidis strain, whereas one isolate (F3720) was significantly more acid tolerant. The S. Orion var. 15+ isolates were motile by flagellae and elaborated type-1 and curli-like fimbriae; surface organelles that are considered virulence determinants in Salmonella pathogenesis. Each adhered and invaded HEp-2 tissue culture cells with similar proficiency to the S. Enteritidis control but were significantly less virulent than S. Enteritidis in the one-day-old and seven-day-old chick model. Given an oral dose of 1 × 10 3 cfu to one-day-old chicken, S. Orion var. 15+ isolates colonised 25% of liver and spleens examined at 24 h whereas S. Enteritidis colonised 100% of organs by the same with the same dose. Given an oral dose of 1 × 10 7 cfu at seven-day old, S. Orion var. 15+ failed to colonise livers and spleens in any bird examined at 24 h whereas S. Enteritidis colonised 50% of organs by the same with the same dose. Based on the number of internal organs colonised, one of the three S. Orion var. 15+ isolates tested (strain F3720) was significantly more invasive than the other two (B1 and B7). Also, strain F3720 was shed less than either B1 or B7 supporting the concept that there may be an inverse relationship between the ability to colonise deep tissues and to persist in the gut. These data are discussed in the light that S. Orion var. 15+ is associated with sporadic outbreaks of human infection rather than epidemics.

Fis, a DNA nucleoid-associated protein, is involved in Salmonella typhimurium SPI-1 invasion gene expression.

The ability of Salmonella enterica serovar Typhimurium to cause disease depends upon the co-ordinated expression of many genes located around the Salmonella chromosome. Specific pathogenicity loci, termed Salmonella pathogenicity islands, have been shown to be crucial for the invasion and survival of Salmonella within host cells. Salmonella pathogenicity island 1 (SPI-1) harbours the genes required for the stimulation of Salmonella uptake across the intestinal epithelia of the infected host. Regulation of SPI-1 genes is complex, as invasion gene expression responds to a number of different signals, presumably signals similar to those found within the environment of the intestinal tract. As a result of our continued studies of SPI-1 gene regulation, we have discovered that the nucleoid-binding protein Fis plays a pivotal role in the expression of HilA and InvF, two activators of SPI-1 genes. A S. typhimurium fis mutant demonstrates a two- to threefold reduction in hilA:Tn5lacZY and a 10-fold reduction in invF:Tn5lacZY expression, as well as a 50-fold decreased ability to invade HEp-2 tissue culture cells. This decreased expression of hilA and invF resulted in an altered secreted invasion protein profile in the fis mutant. Furthermore, the virulence of a S. typhimurium fis mutant is attenuated 100-fold when administered orally, but has wild-type virulence when administered intraperitoneally. Expression of hilA:Tn5lacZY and invF:Tn5lacZY in the fis mutant could be restored by introducing a plasmid containing the S. typhimurium fis gene or a plasmid containing hilD, a gene encoding an AraC-like regulator of Salmonella invasion genes.

CL387626 exhibits marked and unusual antiviral activity against respiratory syncytial virus in tissue culture and in cotton rats

CL387626 (4,4′-Bis[4,6-di[3-aminophenyl- N, N-bis(2-carbamoylethyl)-sulfonilimino]-1,3,5-triazine-2-ylamino-biphenyl-2,2′-disulfonic acid, disodium salt), a compound synthesized by Wyeth-Ayerst Research Laboratories, was tested for its cytotoxicity and antiviral activity against respiratory syncytial virus (RSV) in tissue culture and in cotton rats. The median cell inhibitory (IC 50) and median efficacious (EC 50) concentrations of CL387626 against RSV in proliferating HEp2 or Vero tissue culture cells were determined to be 375 and 0.25 μg/ml, respectively, giving the compound an apparent selective index (S.I.) of 1500. This compound also exhibited uncommon antiviral activity against RSV in cotton rats. In multiple experiments, a single 30 mg/kg dose of CL387626 administered intranasally 4 or 5 days prior to virus challenge, significantly inhibited pulmonary replication of RSV compared to that seen in control animals inoculated similarly with placebo (i.e. water). In contrast to these results, most lots of CL387626 failed to significantly inhibit pulmonary RSV replication when administered utilizing therapeutic administration schedules. Although some cytotoxicity was noted in tissue culture assays, no overt toxic effects were noted in any test animal, including those inoculated with >300 mg CL387626/kg, a dose approximately 150 times the apparent minimal efficacious dose (i.e. 1.9 mg/kg).

M protein mediated adhesion of M type 24 Streptococcus pyogenes stimulates release of interleukin-6 by HEp-2 tissue culture cells

We investigated the contributions of lipoteichoic acid and M protein to reversible and irreversible adhesion of group A streptococci and the effects of such adhesion on release of interleukin-6. Streptococci in which lipoteichoic acid was masked by the hyaluronate capsule were readily washed from HEp-2 cells, indicating no attachment. Unencapsulated, M-negative streptococci in which lipoteichoic acid was exposed were removed more slowly, indicating loose attachment. Only unencapsulated streptococci that expressed both lipoteichoic acid and M protein remained stably adherent to HEp-2 cells throughout multiple washes. Streptococci expressing both M protein and lipoteichoic acid induced release of interleukin-6 from HEp-2 cells, whereas an isogenic, M-negative mutant failed to induce release of interleukin-6. These data suggest that lipoteichoic acid mediates reversible adhesion and that M protein is required for irreversible adhesion and for inducing release of interleukin-6 from HEp-2 cells.

Differential effects of the streptococcal fibronectin-binding protein, FBP54, on adhesion of group A streptococci to human buccal cells and HEp-2 tissue culture cells.

We have previously demonstrated that fibronectin mediates streptococcal adhesion to host cells and that streptococci interact primarily with the N-terminal domain of fibronectin. FBP54 is a 54-kDa protein from group A streptococci that binds fibronectin. In this report, we show that the N-terminal domain of fibronectin reacts with FBP54 and preferentially blocks streptococcal adhesion to buccal epithelial cells. FBP54 blocked adhesion to human buccal epithelial cells by 80% in a dose-related fashion. In contrast, FBP54 had little effect on adhesion of group A streptococci to HEp-2 tissue culture cells. The fibronectin-binding domain of FBP54 has been localized to the first 89 N-terminal residues of the protein. Experiments using affinity-purified antibodies to this region indicated that the N terminus of FBP54 is exposed on the surface of streptococci in a manner that can interact with immobilized receptors. Analysis of sera from patients with post-streptococcal glomerulonephritis and acute rheumatic fever indicated that FBP54 is expressed in vivo and is immunogenic in the human host. These data indicate that FBP54 is a streptococcal adhesin that is expressed in the human host and that preferentially mediates adhesion to certain types of human cells.

Adherence characteristics of coagulase-negative staphylococci isolated from patients with infective endocarditis

Coagulase-negative staphylococci isolated from patients with endocarditis were divided according to whether the infection was of native or of prosthetic valves and was acquired either in the community or in hospital. Comparisons were made with strains from intravenous line-associated bacteraemias. All strains were examined by direct and indirect adherence tests. Line-associated bacteraemia strains were more likely to produce slime and were more hydrophilic but were less likely to attach HEp2 tissue culture cells than were endocarditis strains, and almost equally likely to adhere to plastic and extracellular matrix proteins. Amongst the endocarditis strains, there was little difference in slime production but hospital-acquired or prosthetic-valve strains were more hydrophobic and more likely to adhere to silicone than were the native-valve or community-acquired strains. Exposure of extracellular matrix proteins on native valves due to a pre-existing non-infective heart condition may account for the selection of strains able to adhere to fibronectin or laminin.

Analysis of the role of M24 protein in group A streptococcal adhesion and colonization by use of omega-interposon mutagenesis

We recently concluded that M protein mediates adherence of group A streptococci to HEp-2 tissue culture cells, because the N-terminal half of M protein blocked adherence and M+ strains attached in greater numbers than M- streptococci. To further assess the role of M protein in adhesion, an M-, isogenic mutant of M type M-, isogenic mutant of M type 24 group A streptococci was constructed by insertional inactivation of the emm24 gene with the omega-interposon flanked by emm24 gene sequences. Southern blot analysis confirmed that the omega-element inserted only into emm24. The M- isogenic mutant M24-omega 3 did not react with antiserum to M24 protein, not did it survive in whole human blood. Electron micrographs of M24-omega 3 showed a diminution of surface fibrillae and reduced binding of plasma components compared with the parent strain. The adhesion of the M+ parent to HEp-2 cells and to mouse oral epithelial cells was dramatically greater than the adhesion of the M24-omega 3 mutant, although there was no difference between the two in adhesion to human buccal cells. In addition, the parent strain was dramatically more effective than the M24-omega 3 mutant in colonizing the oral cavity of mice. These results indicate that the M24 protein can serve as an adhesin in streptococcal attachment to human cells in tissue culture and is important in the colonization of mouse mucosal surfaces.

The role of pathogenic Escherichia coli in the etiology of veal calf hemorrhagic enteritis

Veal calf hemorrhagic enteritis, a condition that has no identified specific etiology, is a fatal syndrome of veal calves and has recently become a major concern of the veal calf industry in the midwestern United States. To determine the possible role of common enteric pathogens in this disease, 40 veal calves with hemorrhagic enteritis (cases) and 25 dairy calves diagnosed with enteric infection (control) were investigated. The veal calves were negative for several known enteric pathogens except for pathogenic Escherichia coli isolates that expressed multiple virulence attributes. To determine whether such isolates have a significant association with hemorrhagic enteritis in veal calves, we compared the prevalence of pathogenic E. coli in the 40 veal calves with the prevalence of similar E. coli in the dairy calves that were diagnosed with colibacillosis within the same season of the year. Escherichia coli isolates from the two groups of calves were tested for several properties of E. coli related to pathogenicity, i.e. production of verotoxins, heat-stable enterotoxin (ST a), heat-labile enterotoxin, enterohemolysin, K99 fimbrial antigens, hemagglutination activity, and attachment to Hep-2 tissue culture cells. Escherichia coli that produces ST a were more commonly isolated from veal calves with hemorrhagic enteritis (45%) than from dairy calves with enteritis (12%) ( P<0.05). Various patterns of attachment of E. coli to Hep-2 tissue culture cells were studied. The E. coli that demonstrated aggregative patterns of attachment were more commonly represented in veal calves (32%) than in dairy calves (8%). We observed that there was no correlation between ST a production and K99 pili expression among the enterotoxigenic E. coli (ETEC) isolates that were recovered from veal calves. This may indicate the emergence of K99-negative ETEC, probably as a result of the wide use of K99-based vaccines.

In vitro Invasion of Aeromonas spp. to HEp-2 Tissue Culture Cells

In vitro Invasion of Aeromonas spp. to HEp-2 Tissue Culture Cells

Toxicity and antiviral activity of LY253963 against respiratory syncytial and parainfluenza type 3 viruses in tissue culture and in cotton rats

LY253963, the sodium salt of 1,3,4-thiadiazol-2-ylcyanamide, was evaluated in tissue culture and in cotton rats for toxicity and antiviral efficacy against respiratory syncytial (RSV) and parainfluenza type 3 (PIV3) viruses. The selective index (ratio of the median toxic dose: median efficacious dose) of LY253963 in HEp2 tissue culture cells was >100 against both RSV and PIV3. When given intraperitoneally to cotton rats, the minimum protective dose of LY253963 against both of these viruses was between 1 and 3 mg/kg/day. In contrast, doses of LY253963 as high as 30 mg/kg/day, administered orally after experimental inoculation of virus, did not significantly reduce pulmonary virus titers in treated animals compared to control animals given placebo. No toxic effects were noted in cotton rats, even in those given 20 mg/kg/day for eight consecutive days.

The association of enterotoxigenic and enteropathogenicEscherichia coliand other enteric pathogens with childhood diarrhoea in Yugoslavia

The presence of enterotoxigenic and enteropathogenic Escherichia coli (ETEC and EPEC, respectively) was investigated in stool specimens of 1082 preschool children with diarrhoea and in stools of 335 healthy controls in localities in southern Yugoslavia, as well as in 566 children with diarrhoea and in 231 controls living in northern part of the country, during the seasonal peak (August-November) of enteric diseases in 1986. ETEC were found in 114 (10.5%) children with diarrhoea and in 14 (4.2%) controls (P less than 0.001) in the southern part, and in 26 (4.6%) ill children and one (0.4%) well child (P less than 0.005) in the northern part of Yugoslavia. EPEC were isolated from stools of 85 (7.9%) children with diarrhoea and of 14 (4.2%) well children (P less than 0.05) in localities of southern Yugoslavia, and from 22 (3.9%) ill children and from 10 (4.3%) controls in northern Yugoslavia. Nineteen EPEC strains expressed localized adherence to HEp-2 tissue culture cells; all were isolated from stools of ill children. In southern Yugoslavia, where other enteropathogens were sought, the most commonly found agents in ill children were shigellae (17.5%), rotavirus (11.8%), ETEC, and EPEC. Potential pathogens were detected in 44.5% cases of sporadic diarrhoea and in 15.8% controls. This study revealed that ETEC were associated with acute diarrhoeal disease in Yugoslav preschool children. On the other hand, the diagnosis of EPEC-diarrhoea by routine determination of serogroup established the association of these agents with sporadic diarrhoea only in the 0-2 years age categories in all investigated localities. In the less developed southern part of Yugoslavia bacteria were the predominant causative agents of enteric illness during the seasonal peak of this disease.

Adherence to epithelial cells and ultrastructure of fosfomycin-resistant mutants of group A streptococci.

Adherence of three strains of group A streptococci and their fosfomycin-resistant mutants to HEp-2 tissue culture cells was compared with some cell-surface characteristics, i.e. ultrastructure and hydrophobicity. Among Fosr mutants, both well-adhering and weakly adhering mutants were found. Clonal analysis of the mutants proved their greater stability in the adherence. Well-adhering parent strains of streptococci and Fosr mutants exhibited surface fibrillae in contrast to weakly adhering Fosr mutants which were devoid of fibrillae or contined fibrillae of lower electron density. Decrease of adherence of Fosr mutants of two strains was accompanied by a decrease of their hydrophobicity.

Anti-Ro (SS-A) antibodies from Ro (SS-A)-immunized mice.

Immunization of BALB/c mice with immunoaffinity-purified bovine or human Ro (SS-A) induces the production of antibodies reactive with Ro (SS-A). Fusion of spleen cells from the hyperimmunized mice to SP2/0 cells resulted in hybridoma cell lines that produced anti-Ro (SS-A) antibodies. Anti-Ro (SS-A) binding was established by solid-phase immunosorbent assay, immunoblotting, or RNA immunoprecipitation. Most of the anti-Ro (SS-A) antibodies bound to both human and bovine Ro (SS-A) in the solid phase, but only one of the monoclonal antibodies selectively bound to human Ro (SS-A); this suggests that there are species differences between the bovine and human Ro (SS-A) antigens. Indirect immunofluorescence studies demonstrated that most anti-Ro (SS-A) antibodies bound to cytoplasmic or nuclear HEp-2 cellular antigens, whereas others did not bind to fixed HEp-2 tissue culture cells. Nuclear staining of mouse substrates by one of the sera containing anti-Ro (SS-A) demonstrated that autoantibodies were induced by immunization with human Ro (SS-A).

Standardization of the Immunofluorescence Test for Autoantibody to Nuclear Antigens (ANA): Use of Reference Sera of Defined Antibody Specificity

Standardization of the indirect immunofluorescence antinuclear antibody (IF-ANA) test can be improved for a given substrate with iise of reference ANA sera, uniform assay conditions, and standardization of optical systems. To accomplish this, reference sera from the Arthritis Foundation with defined antibody specificities for nDNA, SS-B, RNP, Sm, nucleoli, and “speckled pattern” were reacted with commonly used IF-ANA substrates, mouse kidney sections, KB and HEp-2 tissue culture cells. Reagents and assay conditions used were those provided with the substrates in commercially available IF-ANA kits. A microscope slide with graded intensities of fluorescent beads was used to standardize microscope fluorescence intensity readings. The authors’ fluorescence pattern and intensity results should be directly comparable to results obtained in other laboratories for the six antibodies for which reference sera are available. Although no defined sera are widely available for SS-A, Scl-70, PM-1, and centromere antibodies, the ability of each substrate to detect these antinuclear antibodies as well as mitochondrial, smooth muscle, ribosomal and microsomal antibodies also was tested. HEp-2 cells and KB cells were found to be superior to mouse kidney sections for detection of SS-A, Scl-70, PM-1 and centromere antinuclear antibodies. Mouse kidney sections were superior for screening of sera for the absence of ANA as well as for detection of smooth muscle and liver-kidney microsomal antibodies. Other antibodies were detected with equal sensitivity with all substrates and each of the three ANA kits used in the study performed satisfactorily. Use of reference sera as well as optical standardization is recommended for IF-ANA testing.

A newly recognized cause of travelers' diarrhea: enteroadherent Escherichia coli.

Adherence to HEp-2 tissue culture cells has been proposed as a virulence characteristic of enteropathogenic Escherichia coli (EPEC). A preliminary study revealed that E. coli that adhered to HEp-2 cells, but did not produce conventional enterotoxins and did not belong to recognized EPEC serogroups, could be isolated from adults from the United States who acquired diarrhea in Mexico. The purpose of this study was to determine the prevalence of these enteroadherent E. coli (EAEC) in 188 travelers with diarrhea and in 92 well travelers. EAEC were found in 14.9% of patients with diarrhea and in 7.6% of well individuals. Compared with well travelers, patients with diarrhea in whom no recognized enteropathogen could be identified had a 30.4% prevalence of EAEC (P less than .0003). These results further support our finding that EAEC are associated with diarrhea in travelers to Mexico and may help to explain the effect of antibiotics in the prevention and therapy for travelers' diarrhea in patients with no recognized bacterial enteropathogens.

Relationships between antinuclear and anti-Ro/SS-A antibodies in subacute cutaneous lupus erythematosus

A certain degree of confusion has arisen regarding the relationship between patients having subacute cutaneous lupus erythematosus (SCLE) and those with "antinuclear antibody-negative" systemic lupus erythematosus (ANA-negative SLE). One of the confusing issues relates to published differences in the autoimmune serologic findings of these two patient groups. In order to clarify this issue, we have screened the sera from thirty-seven patients with SCLE for the presence of fluorescent antinuclear antibodies (FANA) on two different substrates--human Hep-2 tissue culture cells and mouse kidney sections. In addition, these same sera were assayed for anti-Ro/SS-A precipitin antibodies. Seventy-eight percent of the sera were FANA-positive at a titer of 1:10 or greater when tested on human Hep-2 cells, whereas 76% were positive at a titer of 1:80 or greater. Fifty-one percent were positive on mouse kidney sections at a titer of 1:10 or greater, whereas 46% were positive at a titer of 1:20 or greater. Twenty-two percent of these sera were completely FANA-negative on both human and mouse substrates. None of these sera that were negative on both substrates contained anti-Ro/SS-A antibodies. However, 69% of the sera that were FANA-positive on both human and mouse substrates were found to have detectable anti-Ro/SS-A antibodies. Ninety percent of the SCLE sera that were FANA-positive on human Hep-2 cells, but negative on mouse kidney sections, contained anti-Ro/SS-A antibodies. These sera gave a speckle-like, or particulate, nuclear immunofluorescence staining pattern.(ABSTRACT TRUNCATED AT 250 WORDS)