Identification of the znuA-Encoded Periplasmic Zinc Transport Protein of Haemophilus ducreyi

ABSTRACT The znuA gene of Haemophilus ducreyiencodes a 32-kDa (mature) protein that has homology to both the ZnuA protein of Escherichia coli and the Pzp1 protein ofH. influenzae; both of these latter proteins are members of a growing family of prokaryotic zinc transporters. Inactivation of theH. ducreyi 35000 znuA gene by insertional mutagenesis resulted in a mutant that grew more slowly than the wild-type parent strain in vitro unless ZnCl2 was provided at a final concentration of 100 μM. Other cations tested did not restore growth of this H. ducreyi mutant to wild-type levels. The H. ducreyi ZnuA protein was localized to the periplasm, where it is believed to function as the binding component of a zinc transport system. Complementation of the znuAmutation with the wild-type H. ducreyi znuA gene provided in trans restored the ability of this H. ducreyi mutant to grow normally in the absence of exogenously added ZnCl2. The wild-type H. ducreyi znuA gene was also able to complement a H. influenzae pzp1 mutation. The H. ducreyi znuA isogenic mutant exhibited significantly decreased virulence (P = 0.0001) when tested in the temperature-dependent rabbit model for experimental chancroid. This decreased virulence was not observed when the znuA mutant was complemented with the wild-type H. ducreyi znuA gene provided in trans.

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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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Expression of the LspA1 and LspA2 Proteins by Haemophilus ducreyi Is Required for Virulence in Human Volunteers

Infection and Immunity ◽

10.1128/iai.72.8.4528-4533.2004 ◽

2004 ◽

Vol 72 (8) ◽

pp. 4528-4533 ◽

Cited By ~ 35

Author(s):

Diane M. Janowicz ◽

Kate R. Fortney ◽

Barry P. Katz ◽

Jo L. Latimer ◽

Kaiping Deng ◽

...

Keyword(s):

Polymorphonuclear Leukocytes ◽

Double Mutant ◽

Human Subjects ◽

Rabbit Model ◽

Haemophilus Ducreyi ◽

Wild Type ◽

Human Volunteers ◽

Mean Size ◽

Wild Type Parent

ABSTRACT Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HPΩ12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm2) than at parent sites (mean size, 39.1 mm2) 24 h after inoculation (P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.

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Haemophilusducreyi Requires an Intact flp Gene Cluster for VirulenceinHumans

Infection and Immunity ◽

10.1128/iai.71.12.7178-7182.2003 ◽

2003 ◽

Vol 71 (12) ◽

pp. 7178-7182 ◽

Cited By ~ 52

Author(s):

Stanley M. Spinola ◽

Kate R. Fortney ◽

Barry P. Katz ◽

Jo L. Latimer ◽

Jason R. Mock ◽

...

Keyword(s):

Animal Model ◽

Gene Cluster ◽

Rabbit Model ◽

Type Iv Secretion ◽

Haemophilus Ducreyi ◽

Secretion Systems ◽

Temperature Dependent ◽

Type Iv ◽

Human Volunteers

ABSTRACT An intact Haemophilus ducreyi flp operon is essential for microcolony formation in vitro. tadA is the 9th of 15 genes in the operon and has homology to NTPases of type IV secretion systems. Fifteen human volunteers were experimentally infected with both H. ducreyi 35000HP and the tadA mutant, 35000HP.400. Papules developed at similar rates at sites inoculated with the mutant and parent, while pustules formed at 36.4% of parent sites and at 0% of mutant sites (P = 0.001). Compared to 35000HP, 35000HP.400 had only a modest but significant reduction in lesion scores in the temperature-dependent rabbit model of chancroid. These data suggest that proteins secreted by the flp locus are required for full expression of virulence by H. ducreyi in humans but have less of a role in virulence in an animal model of infection.

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Neutropenia Restores Virulence to an Attenuated Cu,Zn Superoxide Dismutase-Deficient Haemophilus ducreyi Strain in the Swine Model of Chancroid

Infection and Immunity ◽

10.1128/iai.67.10.5345-5351.1999 ◽

1999 ◽

Vol 67 (10) ◽

pp. 5345-5351 ◽

Cited By ~ 37

Author(s):

Lani R. San Mateo ◽

Kristen L. Toffer ◽

Paul E. Orndorff ◽

Thomas H. Kawula

Keyword(s):

Superoxide Dismutase ◽

Immune Cell ◽

Cell Killing ◽

Haemophilus Ducreyi ◽

Heterosexual Transmission ◽

Swine Model ◽

Wild Type ◽

Wild Type Parent

ABSTRACT Haemophilus ducreyi causes chancroid, a sexually transmitted cutaneous genital ulcer disease associated with increased heterosexual transmission of human immunodeficiency virus. H. ducreyi expresses a periplasmic copper-zinc superoxide dismutase (Cu,Zn SOD) that protects the bacterium from killing by exogenous superoxide in vitro. We hypothesized that the Cu,Zn SOD would protectH. ducreyi from immune cell killing, enhance survival, and affect ulcer development in vivo. In order to test this hypothesis and study the role of the Cu,Zn SOD in H. ducreyi pathogenesis, we compared a Cu,Zn SOD-deficient H. ducreyi strain to its isogenic wild-type parent with respect to survival and ulcer development in immunocompetent and immunosuppressed pigs. The Cu,Zn SOD-deficient strain was recovered from significantly fewer inoculated sites and in significantly lower numbers than the wild-type parent strain or a merodiploid (sodC+ sodC) strain after infection of immunocompetent pigs. In contrast, survival of the wild-type and Cu,Zn SOD-deficient strains was not significantly different in pigs that were rendered neutropenic by treatment with cyclophosphamide. Ulcer severity in pigs was not significantly different between sites inoculated with wild type and sites inoculated with Cu,Zn SOD-deficient H. ducreyi. Our data suggest that the periplasmic Cu,Zn SOD is an important virulence determinant inH. ducreyi, protecting the bacterium from host immune cell killing and contributing to survival and persistence in the host.

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Prevalence of, Antibody Response to, and Immunity Induced by Haemophilus ducreyi Hemolysin

Infection and Immunity ◽

10.1128/iai.67.7.3317-3328.1999 ◽

1999 ◽

Vol 67 (7) ◽

pp. 3317-3328 ◽

Cited By ~ 24

Author(s):

Susan M. Dutro ◽

Gwendolyn E. Wood ◽

Patricia A. Totten

Keyword(s):

Vaccine Development ◽

Rabbit Model ◽

Etiologic Agent ◽

Haemophilus Ducreyi ◽

Structural Protein ◽

Wild Type ◽

Ulcer Disease ◽

A Cell

ABSTRACT Haemophilus ducreyi, the etiologic agent of chancroid, a genital ulcer disease, produces a cell-associated hemolysin whose role in virulence is not well defined. Hemolysin is encoded by two genes, hhdA and hhdB, which, based on their homology to Serratia marcescens shlA and shlBgenes, are believed to encode the hemolysin structural protein and a protein required for secretion and modification of this protein, respectively. In this study, we determined the prevalence and expression of the hemolysin genes in 90 H. ducreyi isolates obtained from diverse geographic locations from 1952 to 1996 and found that all strains contained DNA homologous to the hhdB andhhdA genes. In addition, all strains expressed a hemolytic activity. We also determined that hemolysin is expressed in vivo and is immunogenic, as indicated by the induction of antibodies to hemolysin in both the primate and rabbit disease models as well as in human patients with naturally acquired chancroid. Wild-type strain 35000 and isogenic hemolysin-negative mutants showed no difference in lesion development in the temperature-dependent rabbit model. However, immunization of rabbits with the purified hemolysin protein reduced the recovery of wild-type H. ducreyi, but not hemolysin-negative mutants, from lesions. Our study indicates that hemolysin is a possible candidate for vaccine development due to its immunogenicity, expression in vitro and in vivo by most, if not all, strains, and the effect of immunization on reducing the recovery of viable H. ducreyi in experimental disease in rabbits.

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Putative Surface Proteins Encoded within a Novel Transferable Locus Confer a High-Biofilm Phenotype to Enterococcus faecalis

Journal of Bacteriology ◽

10.1128/jb.188.6.2063-2072.2006 ◽

2006 ◽

Vol 188 (6) ◽

pp. 2063-2072 ◽

Cited By ~ 66

Author(s):

Preeti M. Tendolkar ◽

Arto S. Baghdayan ◽

Nathan Shankar

Keyword(s):

Cell Wall ◽

Enterococcus Faecalis ◽

Insertional Mutagenesis ◽

Surface Proteins ◽

Conjugative Plasmid ◽

Sequence Information ◽

Wild Type ◽

Abiotic Surfaces ◽

Mutant Bank

ABSTRACT Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.

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Recovery of Fusarium oxysporum Fo47 Mutants Affected in Their Biocontrol Activity After Transposition of the Fot1 Element

Phytopathology ◽

10.1094/phyto.2002.92.9.936 ◽

2002 ◽

Vol 92 (9) ◽

pp. 936-945 ◽

Cited By ~ 19

Author(s):

Sophie Trouvelot ◽

Chantal Olivain ◽

Ghislaine Recorbet ◽

Quirico Migheli ◽

Claude Alabouvette

Keyword(s):

Fusarium Oxysporum ◽

Insertional Mutagenesis ◽

Wild Type Strain ◽

Growth Habit ◽

Antagonistic Activity ◽

Phenotypic Characterization ◽

Wild Type ◽

Biocontrol Activity

To investigate the biocontrol mechanisms by which the antagonistic Fusarium oxysporum strain Fo47 is active against Fusarium wilt, a Fot1 transposon-mediated insertional mutagenesis approach was adopted to generate mutants affected in their antagonistic activity. Ninety strains in which an active Fot1 copy had transposed were identified with a phenotypic assay for excision and tested for their biocontrol activity against F. oxysporum f. sp. lini on flax in greenhouse experiments. Sixteen strains were affected in their capacity to protect flax plants, either positively (more antagonistic than Fo47) or negatively (less antagonistic). The molecular characterization of these mutants confirms the excision of Fot1 and its reinsertion in most of the cases. Moreover, we demonstrate that other transposable elements such as Fot2, impala, and Hop have no transposition activity in the mutant genomes. The phenotypic characterization of these mutants shows that they are affected neither in their in vitro growth habit nor in their competitiveness in soil compared with wild-type strain Fo47. These results show that mutants are not impaired in their saprophytic phase and suggest that the altered biocontrol phenotype should likely be expressed during the interaction with the host plant.

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Role of Adhesin Release for Mucosal Colonization by a Bacterial Pathogen

Journal of Experimental Medicine ◽

10.1084/jem.20021153 ◽

2003 ◽

Vol 197 (6) ◽

pp. 735-742 ◽

Cited By ~ 81

Author(s):

Loïc Coutte ◽

Sylvie Alonso ◽

Nathalie Reveneau ◽

Eve Willery ◽

Brigitte Quatannens ◽

...

Keyword(s):

Respiratory Tract ◽

Bacterial Pathogen ◽

Host Cells ◽

Wild Type ◽

Early Step ◽

Bacterial Surface ◽

Extracellular Milieu ◽

Wild Type Parent

Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors called adhesins. To exert these functions, adhesins are typically surface-exposed, although, surprisingly, some are also released into the extracellular milieu, the relevance of which has previously not been studied. To address the role of adhesin release in pathogenesis, we used Bordetella pertussis as a model, since its major adhesin, filamentous hemagglutinin (FHA), partitions between the bacterial surface and the extracellular milieu. FHA release depends on its maturation by the specific B. pertussis protease SphB1. We constructed SphB1-deficient mutants and found that they were strongly affected in their ability to colonize the mouse respiratory tract, although they adhered even better to host cells in vitro than their wild-type parent strain. The defect in colonization could be overcome by prior nasal instillation of purified FHA or by coinfection with FHA-releasing B. pertussis strains, but not with SphB1-producing FHA-deficient strains, ruling out a nonspecific effect of SphB1. These results indicate that the release of FHA is important for colonization, as it may facilitate the dispersal of bacteria from microcolonies and the binding to new sites in the respiratory tract.

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Use of Recombinase-BasedIn VivoExpression Technology To Characterize Enterococcus faecalis Gene Expression during Infection IdentifiesIn Vivo-Expressed Antisense RNAs and Implicates the Protease Eep in Pathogenesis

Infection and Immunity ◽

10.1128/iai.05964-11 ◽

2011 ◽

Vol 80 (2) ◽

pp. 539-549 ◽

Cited By ~ 34

Author(s):

Kristi L. Frank ◽

Aaron M. T. Barnes ◽

Suzanne M. Grindle ◽

Dawn A. Manias ◽

Patrick M. Schlievert ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Rabbit Model ◽

Microscopic Analysis ◽

Mammalian Host ◽

Wild Type ◽

Antisense Transcripts ◽

Content Type ◽

Antisense Rnas

ABSTRACTEnterococcus faecalisis a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades.E. faecalismust be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-basedin vivoexpression technology (RIVET) to identify promoters on theE. faecalisOG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putativein vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected inin vitro- andin vivo-grown cells, providing the first evidence ofin vivo-expressed antisense RNAs inE. faecalis. Deletions in thein vivo-activated genes that encode glutamate 5-kinase (proB[EF0038]), the transcriptional regulator EbrA (ebrA[EF1809]), and the membrane metalloprotease Eep (eep[EF2380]) did not hinder biofilm formation inin vitroassays. In a rabbit model of endocarditis, the ΔebrAstrain was fully virulent, the ΔproBstrain was slightly attenuated, and the Δeepstrain was severely attenuated. The Δeepvirulence defect could be complemented by the expression of the wild-type gene intrans. Microscopic analysis of early Δeepbiofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.

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Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00479.2009 ◽

2010 ◽

Vol 299 (5) ◽

pp. H1525-H1534 ◽

Cited By ~ 18

Author(s):

Xiao-Qin Ren ◽

Gong Xin Liu ◽

Louise E. Organ-Darling ◽

Renjian Zheng ◽

Karim Roder ◽

...

Keyword(s):

Cell Lines ◽

Rabbit Model ◽

Chinese Hamster ◽

Expression Vectors ◽

Delayed Rectifier ◽

Wild Type ◽

Cho Cell ◽

Surface Plasmon Resonance Analysis ◽

Transgenic Rabbit

We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K+ channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K+ current ( IKs and IKr, respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, IKs and IKr, without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated IKr in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in IKr upon KvLQT1 expression. Deletion of the KvLQT1 NH2-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced IKs in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.

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