Characterization of a Haemophilus ducreyi Mutant Deficient in Expression of Cytolethal Distending Toxin

ABSTRACT Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that kills HeLa, HEp-2, and other human epithelial cells in vitro. H. ducreyi CDT activity is encoded by a three-gene cluster (cdtABC), and antibody to the cdtC gene product can neutralize CDT activity in vitro (L. D. Cope, S. R. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S. Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 94:4056–4061, 1997). Culture supernatant fluid from a recombinant Escherichia colistrain containing the H. ducreyi cdtABC gene cluster readily killed both HeLa cells and HaCaT keratinocytes and had a modest inhibitory effect on the growth of human foreskin fibroblasts. Insertional inactivation of the cdtC gene in this recombinant E. coli strain eliminated the ability of this strain to kill HeLa cells and HaCaT keratinocytes. This mutatedH. ducreyi cdtABC gene cluster was used to construct an isogenic H. ducreyi cdtC mutant. Monoclonal antibodies against the H. ducreyi CdtA, CdtB, and CdtC proteins were used to characterize protein expression by this cdtCmutant. Culture supernatant fluid from this H. ducreyi cdtCmutant did not detectably affect any of the human cells used in this study. The presence of the wild-type H. ducreyi cdtC gene in trans in this H. ducreyi mutant restored its ability to express a CDT that killed both HeLa cells and HaCaT keratinocytes. The isogenic H. ducreyi cdtC mutant was shown to be as virulent as its wild-type parent strain in the temperature-dependent rabbit model for experimental chancroid. Lack of expression of the H. ducreyi CdtC protein also did not affect the ability of this H. ducreyi mutant to survive in the skin of rabbits.

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Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems

Infection and Immunity ◽

10.1128/iai.69.9.5626-5634.2001 ◽

2001 ◽

Vol 69 (9) ◽

pp. 5626-5634 ◽

Cited By ~ 34

Author(s):

David A. Lewis ◽

Marla K. Stevens ◽

Jo L. Latimer ◽

Christine K. Ward ◽

Kaiping Deng ◽

...

Keyword(s):

Hela Cells ◽

Culture Supernatant ◽

Cell Envelope ◽

Rabbit Model ◽

Haemophilus Ducreyi ◽

Supernatant Fluid ◽

E Coli

ABSTRACT Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtCgenes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously describedH. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900–3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtAmutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

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Mutations in the lspA1 and lspA2 Genes of Haemophilus ducreyi Affect the Virulence of This Pathogen in an Animal Model System

Infection and Immunity ◽

10.1128/iai.71.5.2478-2486.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2478-2486 ◽

Cited By ~ 18

Author(s):

Christine K. Ward ◽

Jo L. Latimer ◽

Joseph Nika ◽

Merja Vakevainen ◽

Jason R. Mock ◽

...

Keyword(s):

Animal Model ◽

Parent Strain ◽

Type Strain ◽

Double Mutant ◽

Wild Type Strain ◽

Culture Supernatant ◽

Haemophilus Ducreyi ◽

Supernatant Fluid ◽

Wild Type ◽

Wild Type Parent

ABSTRACT Haemophilus ducreyi 35000HP contains two genes, lspA1 and lspA2, whose predicted protein products have molecular weights of 456,000 and 543,000, respectively (C. K. Ward, S. R. Lumbley, J. L. Latimer, L. D. Cope, and E. J. Hansen, J. Bacteriol. 180:6013-6022, 1998). We have constructed three H. ducreyi 35000HP mutants containing antibiotic resistance cartridges in one or both of the lspA1 and lspA2 open reading frames. Western blot analysis using LspA1- and LspA2-specific monoclonal antibodies indicated that the wild-type parent strain 35000HP expressed LspA1 protein that was readily detectable in culture supernatant fluid together with a barely detectable amount of LspA2 protein. The lspA2 mutant 35000HP.2 expressed LspA1 protein that was detectable in culture supernatant fluid and no LspA2 protein. In contrast, the H. ducreyi lspA1 mutant 35000HP.1, which did not express the LspA1 protein, expressed a greater quantity of the LspA2 protein than did the wild-type parent strain. The lspA1 lspA2 double mutant 35000HP.12 expressed neither LspA1 nor LspA2. The three mutant strains adhered to human foreskin fibroblasts and to a human keratinocyte cell line in vitro at a level that was not significantly different from that of the wild-type strain 35000HP. Lack of expression of the LspA1 protein by both the lspA1 mutant and the lspA1 lspA2 double mutant was associated with an increased tendency to autoagglutinate. When evaluated in the temperature-dependent rabbit model for chancroid, the lspA1 lspA2 double mutant was substantially less virulent than the wild-type strain 35000HP. The results of these studies indicated that H. ducreyi requires both the LspA1 and LspA2 proteins to be fully virulent in this animal model for experimental chancroid.

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The lpf Gene Cluster for Long Polar Fimbriae Is Not Involved in Adherence of Enteropathogenic Escherichia coli or Virulence of Citrobacter rodentium

Infection and Immunity ◽

10.1128/iai.74.1.265-272.2006 ◽

2006 ◽

Vol 74 (1) ◽

pp. 265-272 ◽

Cited By ~ 21

Author(s):

Ichiro Tatsuno ◽

Rosanna Mundy ◽

Gad Frankel ◽

Yuwen Chong ◽

Alan D. Phillips ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Hela Cells ◽

Human Infection ◽

Intestinal Biopsy ◽

Citrobacter Rodentium ◽

Wild Type ◽

Human Biopsy ◽

In Vitro Organ Culture

ABSTRACT Using the enteropathogenic Escherichia coli (EPEC) genome sequence, we found that EPEC E2348/69 has an lpfABCDE gene cluster homologous (about 60% identical at the protein level) to the Salmonella long polar fimbria (LPF) operon. To determine whether this operon is essential for adherence, the lpfABCDE2 3 genes were deleted from EPEC strain E2348/69 by allelic exchange. Analysis of the resulting EPECΔlpfABCDE23 strain showed no change in adherence to HeLa cells or to human intestinal biopsy cells in the in vitro organ culture (IVOC) system compared to the wild type. Sera from volunteers experimentally infected with E2348/69 showed no antibody response to the major subunit protein, LpfA. These results suggested that the lpfE23 gene cluster is not necessary for EPEC adherence and attaching/effacing (A/E) lesion formation on human biopsy samples and is not expressed during human infection. We also identified an lpf gene cluster in Citrobacter rodentium strain ICC168 (lpfcr ). A ΔlpfAcr mutant of ICC168 retained wild-type adherence and A/E lesion-forming activity on HeLa cells. C3H/HeJ mice were infected with a wild-type C. rodentium strain and its lpfAcr isogenic mutant. Both strains were recovered at high levels in stools, and there were no significant differences between the groups both in terms of the number of CFU/organ (colon and cecum) and in terms of the amount of hyperplasia, as measured by weight. Similar results were observed in a second mouse strain, C57BL/6. These data suggest that in addition to playing no apparent role in EPEC pathogenesis, lpfcr is not required for C. rodentium virulence in either the C3H/HeJ or C57BL/6 mouse model.

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Multiple Elements Controlling Adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells

Infection and Immunity ◽

10.1128/iai.71.9.4985-4995.2003 ◽

2003 ◽

Vol 71 (9) ◽

pp. 4985-4995 ◽

Cited By ~ 108

Author(s):

Alfredo G. Torres ◽

James B. Kaper

Keyword(s):

Escherichia Coli ◽

Hela Cells ◽

Culture Conditions ◽

Enterohemorrhagic Escherichia Coli ◽

Differentially Expressed ◽

Wild Type ◽

Transposon Insertion ◽

Reduced Adherence ◽

Insertion Mutants

ABSTRACT Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

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In Vitro Attachment of Salmonella typhimurium to Chicken Cecal Mucus: Effect of Cations and Pretreatment with Lactobacillus spp. Isolated from the Intestinal Tracts of Chickens

Journal of Food Protection ◽

10.4315/0362-028x-61.3.265 ◽

1998 ◽

Vol 61 (3) ◽

pp. 265-271 ◽

Cited By ~ 22

Author(s):

S. E. CRAVEN ◽

D. D. WILLIAMS

Keyword(s):

Salmonella Typhimurium ◽

Chelating Agents ◽

Culture Supernatant ◽

Divalent Cations ◽

Supernatant Fluid ◽

Specific Pathogen Free ◽

Intestinal Mucus ◽

Specific Pathogen ◽

Lactobacillus Spp

The attachment of radiolabeled Salmonella typhimurium 3333/O cells to immobilized cecal mucus from specific-pathogen-free leghorn chickens was determined in the presence of d-mannose. The attachment of S. typhimurium was inhibited by the chelating agents EDTA and citrate and by lanthanum but was enhanced in the presence of the calcium, barium, and manganese divalent cations. Summary findings of the effect of lectins are included. Attachment of lactobacilli, previously isolated from the intestines of chickens, to mucus was also enhanced by calcium and inhibited by chelators. The pretreatment of immobilized mucus with portions of cultures of five of eight strains of lactobacilli inhibited subsequent attachment of the S. typhimurium strain. Spent culture supernatant fluid and/or washed cells from these cultures inhibited attachment, and inhibition was enhanced by preheating the cells or supernatant fluid at 80°C. Results indicate that S. typhimurium mucus attachment not involving mannosyl-dependent receptors is influenced by presence of cations. Lactobacillus spp. isolated from the intestinal tracts of chickens produce cellular and cell-free components that inhibit this form of attachment to chicken intestinal mucus.

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Regulation of Expression of the Haemophilus ducreyi LspB and LspA2 Proteins by CpxR

Infection and Immunity ◽

10.1128/iai.00292-09 ◽

2009 ◽

Vol 77 (8) ◽

pp. 3402-3411 ◽

Cited By ~ 21

Author(s):

Maria Labandeira-Rey ◽

Jason R. Mock ◽

Eric J. Hansen

Keyword(s):

Culture Supernatant ◽

Fetal Calf Serum ◽

Calf Serum ◽

Sensory Transduction ◽

Haemophilus Ducreyi ◽

Mobility Shift ◽

Regulation Of Expression ◽

Two Component ◽

Electrophoretic Mobility Shift Assays

ABSTRACT The LspA1, LspA2, and LspB proteins of Haemophilus ducreyi comprise a two-partner secretion system that has been shown to be necessary for H. ducreyi to inhibit phagocytosis by immune cells in vitro. Inactivation of lspA1 resulted in increased levels of LspA2, suggesting that these two proteins are differentially controlled (C. J. Ward et al., Infect. Immun. 71:2478-2486, 2003). Expression of LspA2 but not LspA1 was shown to be both growth phase dependent and affected by the presence of fetal calf serum (FCS) in the growth medium. In addition, neither LspA1 nor LspA2 could be detected in culture supernatant fluid in the absence of FCS. DNA microarray analysis revealed that 324 H. ducreyi genes were differentially regulated after growth in the presence of FCS. Among these, the CpxRA two-component sensory transduction system was downregulated by the presence of FCS. Inactivation of cpxR resulted in increased expression of both LspB and LspA2. Electrophoretic mobility shift assays showed that a recombinant H. ducreyi CpxR protein bound the promoter region of the lspB-lspA2 operon. The cpxR and cpxA genes were shown to be part of an operon containing two additional genes in H. ducreyi 35000HP. This is the first description of a two-component sensory transduction system regulating a proven virulence factor of H. ducreyi.

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Cytolethal Distending Toxin Gene Cluster in Enterohemorrhagic Escherichiacoli O157:H− and O157:H7: Characterization and Evolutionary Considerations

Infection and Immunity ◽

10.1128/iai.71.6.3634-3638.2003 ◽

2003 ◽

Vol 71 (6) ◽

pp. 3634-3638 ◽

Cited By ~ 89

Author(s):

Andreas Janka ◽

Martina Bielaszewska ◽

Ulrich Dobrindt ◽

Lilo Greune ◽

M. Alexander Schmidt ◽

...

Keyword(s):

Gene Cluster ◽

Virulence Factor ◽

Toxin Gene ◽

Cytolethal Distending Toxin ◽

Wild Type ◽

Potential Virulence Factor

ABSTRACT We identified a cytolethal distending toxin (cdt) gene cluster in 87, 6, and 0% of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H−, EHEC O157:H7, and E. coli O55:H7/H− strains, respectively. The toxin was expressed by the wild-type EHEC O157 strains and by a cdt-containing cosmid from a library of SF EHEC O157:H− strain 493/89. The cdt flanks in strain 493/89 were homologous to bacteriophages P2 and lambda. Our data demonstrate that cdt, encoding a potential virulence factor, is present in the EHEC O157 complex and suggest that cdt may have been acquired by phage transduction.

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Attenuation of Zika Virus by Passage in Human HeLa Cells

Vaccines ◽

10.3390/vaccines7030093 ◽

2019 ◽

Vol 7 (3) ◽

pp. 93 ◽

Cited By ~ 1

Author(s):

Li ◽

Collins ◽

Widen ◽

Davis ◽

Kaiser ◽

...

Keyword(s):

Hela Cell ◽

Hela Cells ◽

Zika Virus ◽

Infected Animal ◽

Vaccine Development ◽

Nonstructural Protein ◽

A549 Cells ◽

Wild Type ◽

Nonstructural Protein 1

Zika virus (ZIKV) is a mosquito-borne Flavivirus. Previous studies have shown that mosquito-transmitted flaviviruses, including yellow fever, Japanese encephalitis, and West Nile viruses, could be attenuated by serial passaging in human HeLa cells. Therefore, it was hypothesized that wild-type ZIKV would also be attenuated after HeLa cell passaging. A human isolate from the recent ZIKV epidemic was subjected to serial HeLa cell passaging, resulting in attenuated in vitro replication in both Vero and A549 cells. Additionally, infection of AG129 mice with 10 plaque forming units (pfu) of wild-type ZIKV led to viremia and mortality at 12 days, whereas infection with 103 pfu of HeLa-passage 6 (P6) ZIKV led to lower viremia, significant delay in mortality (median survival: 23 days), and increased cytokine and chemokine responses. Genomic sequencing of HeLa-passaged virus identified two amino acid substitutions as early as HeLa-P3: pre-membrane E87K and nonstructural protein 1 R103K. Furthermore, both substitutions were present in virus harvested from HeLa-P6-infected animal tissue. Together, these data show that, similarly to other mosquito-borne flaviviruses, ZIKV is attenuated following passaging in HeLa cells. This strategy can be used to improve understanding of substitutions that contribute to attenuation of ZIKV and be applied to vaccine development across multiple platforms.

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The N-terminal domain of the human eIF2β subunit and the CK2 phosphorylation sites are required for its function

Biochemical Journal ◽

10.1042/bj20050605 ◽

2006 ◽

Vol 394 (1) ◽

pp. 227-236 ◽

Cited By ~ 17

Author(s):

Franc Llorens ◽

Anna Duarri ◽

Eduard Sarró ◽

Nerea Roher ◽

Maria Plana ◽

...

Keyword(s):

Protein Kinase ◽

Hela Cells ◽

Mutant Cell ◽

Phosphorylation Site ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Mutant Form ◽

Wild Type ◽

Main Site

CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2α evidenced the direct involvement of this protein kinase in eIF2β phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2β or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2β phosphorylation, whereas phosphorylation at Ser67 seems more restricted. In vitro phosphorylation of eIF2β also pointed to Ser2 as a preferred site for CK2 phosphorylation. Overexpression of the eIF2β S2/67A mutant slowed down the rate of protein synthesis stimulated by serum, although less markedly than the overexpression of the Δ2–138 N-terminal-truncated form of eIF2β (eIF2β-CT). Mutation at Ser2 and Ser67 did not affect eIF2β integrating into the eIF2 trimer or being able to complex with eIF5 and CK2α. The eIF2β-CT form was also incorporated into the eIF2 trimer but did not bind to eIF5. Overexpression of eIF2β slightly decreased HeLa cell viability, an effect that was more evident when overexpressing the eIF2β S2/67A mutant. Cell death was particularly marked when overexpressing the eIF2β-CT form, being detectable at doses where eIF2β and eIF2β S2/67A were ineffective. These results suggest that Ser2 and Ser67 contribute to the important role of the N-terminal region of eIF2β for its function in mammals.

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Inhibition of Phagocytosis by Haemophilus ducreyi Requires Expression of the LspA1 and LspA2 Proteins

Infection and Immunity ◽

10.1128/iai.71.10.5994-6003.2003 ◽

2003 ◽

Vol 71 (10) ◽

pp. 5994-6003 ◽

Cited By ~ 40

Author(s):

Merja Vakevainen ◽

Steven Greenberg ◽

Eric J. Hansen

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Rabbit Model ◽

Inhibitory Effect ◽

Haemophilus Ducreyi ◽

Bacterial Cells ◽

Wild Type ◽

Temperature Dependent ◽

Live Bacterial Cells

ABSTRACT Haemophilus ducreyi previously has been shown to inhibit the phagocytosis of both secondary targets and itself by certain cells in vitro. Wild-type H. ducreyi strain 35000HP contains two genes, lspA1 and lspA2, whose encoded protein products are predicted to be 456 and 543 kDa, respectively. An isogenic mutant of H. ducreyi 35000HP with inactivated lspA1 and lspA2 genes has been shown to exhibit substantially decreased virulence in the temperature-dependent rabbit model for chancroid. This lspA1 lspA2 mutant was tested for its ability to inhibit phagocytosis of immunoglobulin G-opsonized particles by differentiated HL-60 and U-937 cells and by J774A.1 cells. The wild-type strain H. ducreyi 35000HP readily inhibited phagocytosis, whereas the lspA1 lspA2 mutant was unable to inhibit phagocytosis. Similarly, the wild-type strain was resistant to phagocytosis, whereas the lspA1 lspA2 mutant was readily engulfed by phagocytes. This inhibitory effect of wild-type H. ducreyi on phagocytic activity was primarily associated with live bacterial cells but could also be found, under certain conditions, in concentrated H. ducreyi culture supernatant fluids that lacked detectable outer membrane fragments. Both the wild-type strain and the lspA1 lspA2 mutant attached to phagocytes at similar levels. These results indicate that the LspA1 and LspA2 proteins of H. ducreyi are involved, directly or indirectly, in the antiphagocytic activity of this pathogen, and they provide a possible explanation for the greatly reduced virulence of the lspA1 lspA2 mutant.

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