scholarly journals Haemophilus ducreyi Requires the flp Gene Cluster for Microcolony Formation In Vitro

2002 ◽  
Vol 70 (6) ◽  
pp. 2965-2975 ◽  
Author(s):  
Joseph R. Nika ◽  
Jo L. Latimer ◽  
Christine K. Ward ◽  
Robert J. Blick ◽  
Nikki J. Wagner ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Rabbit Model ◽  
Etiologic Agent ◽  
Open Reading Frames ◽  
Haemophilus Ducreyi ◽  
Periodontal Pathogen ◽  
Pcr Analysis ◽  
Phenotypic Trait ◽  
Human Foreskin Fibroblasts

ABSTRACT Haemophilus ducreyi, the etiologic agent of chancroid, has been shown to form microcolonies when cultured in the presence of human foreskin fibroblasts. We identified a 15-gene cluster in H. ducreyi that encoded predicted protein products with significant homology to those encoded by the tad (for tight adhesion) locus in Actinobacillus actinomycetemcomitans that is involved in the production of fimbriae by this periodontal pathogen. The first three open reading frames in this H. ducreyi gene cluster encoded predicted proteins with a high degree of identity to the Flp (fimbria-like protein) encoded by the first open reading frame of the tad locus; this 15-gene cluster in H. ducreyi was designated flp. RT-PCR analysis indicated that the H. ducreyi flp gene cluster was likely to be a polycistronic operon. Mutations within the flp gene cluster resulted in an inability to form microcolonies in the presence of human foreskin fibroblasts. In addition, the same mutants were defective in the ability to attach to both plastic and human foreskin fibroblasts in vitro. An H. ducreyi mutant with an inactivated tadA gene exhibited a small decrease in virulence in the temperature-dependent rabbit model for experimental chancroid, whereas another H. ducreyi mutant with inactivated flp-1 and flp-2 genes was as virulent as the wild-type parent strain. These results indicate that the flp gene cluster is essential for microcolony formation by H. ducreyi, whereas this phenotypic trait is not linked to the virulence potential of the pathogen, at least in this animal model of infection.

scholarly journals Haemophilusducreyi Requires an Intact flp Gene Cluster for VirulenceinHumans

2003 ◽  
Vol 71 (12) ◽  
pp. 7178-7182 ◽  
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.

scholarly journals Haemophilus ducreyi Secretes a Filamentous Hemagglutinin-Like Protein

1998 ◽  
Vol 180 (22) ◽  
pp. 6013-6022 ◽  
Author(s):  
Christine K. Ward ◽  
Sheryl R. Lumbley ◽  
Jo L. Latimer ◽  
Leslie D. Cope ◽  
Eric J. Hansen
Keyword(s):  
Monoclonal Antibody ◽  
Culture Supernatant ◽  
Sequence Similarity ◽  
Rabbit Model ◽  
Protein Product ◽  
Haemophilus Ducreyi ◽  
Pcr Analysis ◽  
Amino Acid Sequence Similarity ◽  
Filamentous Hemagglutinin

ABSTRACT We have identified two extremely large open reading frames (ORFs) in Haemophilus ducreyi 35000, lspA1 andlspA2, each of which encodes a predicted protein product whose N-terminal half is approximately 43% similar to the N-terminal half of Bordetella pertussis filamentous hemagglutinin (FhaB). To the best of our knowledge, lspA1 (12,500 nucleotides [nt]) and lspA2 (14,800 nt) are among the largest prokaryotic ORFs identified to date. The predicted proteins, LspA1 and LspA2, are 86% identical overall to each other and also have limited amino acid sequence similarity at their N termini to other secreted bacterial proteins, including certain hemolysins. Southern blot analysis indicated that lspA1 and lspA2sequences were present in 15 other geographically diverse H. ducreyi strains. Reverse transcriptase PCR analysis of total RNA isolated from H. ducreyi 35000 grown in liquid medium, grown on solid agar medium, and isolated from lesions of H. ducreyi-infected rabbits indicated that lspA1 andlspA2 were transcribed both in vitro and in vivo. A 260-kDa protein present in culture supernatant from eight virulentH. ducreyi strains reacted with both polyclonal serum from rabbits infected with H. ducreyi 35000 and a monoclonal antibody predicted to bind both LspA1 and LspA2. This 260-kDa protein in H. ducreyi 35000 culture supernatant was shown to be the protein product of thelspA1 ORF based on its reactivity with a monoclonal antibody specific for LspA1. Four H. ducreyi strains, previously shown to be avirulent in the temperature-dependent rabbit model for chancroid, did not produce either LspA1 or LspA2 in vitro. This finding raised the possibility that LspA1, LspA2, or both may be involved in the ability of H. ducreyi to cause lesions in this animal model.

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

1999 ◽  
Vol 67 (7) ◽  
pp. 3317-3328 ◽  
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.

scholarly journals Identification, Expression, and Functions of the Somatostatin Gene Family in Spotted Scat (Scatophagus argus)

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 194
Author(s):  
Peizhe Feng ◽  
Changxu Tian ◽  
Xinghua Lin ◽  
Dongneng Jiang ◽  
Hongjuan Shi ◽  
...  
Keyword(s):  
Gene Family ◽  
Growth Regulation ◽  
Expression Patterns ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Sequence Alignments ◽  
Scatophagus Argus ◽  
Gene Structure And Expression ◽  
Liver Fragments

Somatostatins (SSTs) are a family of proteins consisting of structurally diverse polypeptides that play important roles in the growth regulation in vertebrates. In the present study, four somatostatin genes (SST1, SST3, SST5, and SST6) were identified and characterized in the spotted scat (Scatophagus argus). The open reading frames (ORFs) of SST1, SST3, SST5, and SST6 cDNA consist of 372, 384, 321, and 333 bp, respectively, and encode proteins of 123, 127, 106, and 110 amino acids, respectively. Amino acid sequence alignments indicated that all SST genes contained conserved somatostatin signature motifs. Real-time PCR analysis showed that the SST genes were expressed in a tissue specific manner. When liver fragments were cultured in vitro with synthetic peptides (SST1, SST2, or SST6 at 1 μM or 10 μM) for 3 h or 6 h, the expression of insulin-like growth factor 1 and 2 (Igf-1 and Igf-2) in the liver decreased significantly. Treatment with SST5 had no significant effect on Igf-1 and Igf-2 gene expression. This study provides an enhanced understanding of the gene structure and expression patterns of the SST gene family in S. argus. Furthermore, this study provides a foundation for future exploration into the role of SST genes in growth and development.

scholarly journals Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase and d-Cycloserine Synthetase Found in the Biosynthetic Pathway

2013 ◽  
Vol 57 (6) ◽  
pp. 2603-2612 ◽  
Author(s):  
Narutoshi Uda ◽  
Yasuyuki Matoba ◽  
Takanori Kumagai ◽  
Kosuke Oda ◽  
Masafumi Noda ◽  
...  
Keyword(s):  
Gene Cluster ◽  
High Performance ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Homocysteine Thiolactone ◽  
Putative Gene ◽  
Content Type ◽  
Dna Fragment ◽  
Layer Chromatography

ABSTRACTWe have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic,d-cycloserine. The gene cluster is composed of 10 open reading frames, designateddcsAtodcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization ofO-ureidoserine. DcsD is similar toO-acetylserine sulfhydrylase, which generatesl-cysteine usingO-acetyl-l-serine with sulfide, and therefore, DcsD may be a synthase to generateO-ureido-l-serine usingO-acetyl-l-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase convertingO-ureido-d-serine intod-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed inEscherichia coliand purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substratesO-acetyl-l-serine and hydroxyurea, synthesis ofd-cycloserine was successfully attained. Thesein vitrostudies yield the conclusion that DcsD and DcsG are necessary for the syntheses ofO-ureido-l-serine andd-cycloserine, respectively. DcsD was also able to catalyze the synthesis ofl-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclicd-amino acid analogs, such asd-homocysteine thiolactone.

scholarly journals The Gene Cluster forpara-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

2014 ◽  
Vol 80 (19) ◽  
pp. 6212-6222 ◽  
Author(s):  
Jun Min ◽  
Jun-Jie Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Gene Knockout ◽  
Gene Clusters ◽  
Sole Source ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Biochemical Analyses ◽  
Nitrophenol Degradation

ABSTRACTBurkholderiasp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) orpara-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of thepnpgenes in thepnpABA1CDEFcluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activityin vitroin the conversion of 2C4NP to CBQ. Genetic analyses indicated thatpnpAplays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

Identification of a gene cluster for cell-surface genes of the SRS superfamily inNeospora caninumand characterization of the novelSRS9gene

Parasitology ◽  
2011 ◽  
Vol 138 (14) ◽  
pp. 1832-1842 ◽  
Author(s):  
V. RISCO-CASTILLO ◽  
V. MARUGÁN-HERNÁNDEZ ◽  
A. FERNÁNDEZ-GARCÍA ◽  
A. AGUADO-MARTÍNEZ ◽  
E. JIMÉNEZ-RUIZ ◽  
...  
Keyword(s):  
Western Blot ◽  
Gene Cluster ◽  
Neospora Caninum ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Specific Expression ◽  
Initial Characterization ◽  
Hydrophilic Region

SUMMARYHere we present the detection of a gene cluster forNeospora caninumsurface genes, similar to theToxoplasma gondiiSRS9 locus, and the cloning and characterization of the NcSRS9gene. PCR genome walking, using NcBSR4gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to theSRS5and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtainedin vitroand RT-PCR analysis showed no significant variations of NcSRS9transcripts duringin vitrotachyzoite-bradyzoite switch, probably due to incomplete maturity ofin vitrobradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding ofN. caninumpersistence.

Selective Ablation of Human T-Cell Lymphotropic Virus Type 1 p12I Reduces Viral Infectivity In Vivo

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4701-4707 ◽  
Author(s):  
Nathaniel D. Collins ◽  
Garret C. Newbound ◽  
Björn Albrecht ◽  
Jennifer L. Beard ◽  
Lee Ratner ◽  
...  
Keyword(s):  
T Cell ◽  
Virus Type ◽  
Rabbit Model ◽  
Etiologic Agent ◽  
Peripheral Blood Mononuclear ◽  
Antigen Production ◽  
Human T Cell

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia and HTLV-1-associated myelopathy. Novel, yet conserved RNA transcripts encoded from open reading frames (ORFs) I and II of the viral pX region are expressed both in vitro and in infected individuals. The ORF I mRNA encodes the protein p12I, which has been shown to localize to cellular endomembranes, cooperate with bovine papillomavirus E5 in transformation, as well as bind to the IL-2 receptor β and γ chains and the H+ vacuolar ATPase. It is unknown what role p12I plays in the viral life cycle. Using an infectious molecular clone of HTLV-1 (ACH) and a derivative clone, ACH.p12I, which fails to produce the p12Imessage, we investigated the importance of p12I in infected primary cells and in a rabbit model of the infection. ACH.p12I was infectious in vitro as shown by viral passage in culture and no qualitative or quantitative differences were noted between ACH and ACH.p12I in posttransfection viral antigen production. However, in contrast to ACH, ACH.p12I failed to establish persistent infection in vivo as indicated by reduced anti-HTLV-1 antibody responses, failure to demonstrate viral p19 antigen production in peripheral blood mononuclear cell (PBMC) cultures, and only transient detection of provirus by polymerase chain reaction in PBMC from ACH.p12I-inoculated rabbits. These results are the first to show the essential role of HTLV-1 p12I in the establishment of persistent viral infection in vivo and suggest potential new targets in antiviral strategies to prevent HTLV-1 infection.

Selective Ablation of Human T-Cell Lymphotropic Virus Type 1 p12I Reduces Viral Infectivity In Vivo

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4701-4707 ◽  
Author(s):  
Nathaniel D. Collins ◽  
Garret C. Newbound ◽  
Björn Albrecht ◽  
Jennifer L. Beard ◽  
Lee Ratner ◽  
...  
Keyword(s):  
T Cell ◽  
Virus Type ◽  
Rabbit Model ◽  
Etiologic Agent ◽  
Peripheral Blood Mononuclear ◽  
Antigen Production ◽  
Human T Cell

Abstract Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia and HTLV-1-associated myelopathy. Novel, yet conserved RNA transcripts encoded from open reading frames (ORFs) I and II of the viral pX region are expressed both in vitro and in infected individuals. The ORF I mRNA encodes the protein p12I, which has been shown to localize to cellular endomembranes, cooperate with bovine papillomavirus E5 in transformation, as well as bind to the IL-2 receptor β and γ chains and the H+ vacuolar ATPase. It is unknown what role p12I plays in the viral life cycle. Using an infectious molecular clone of HTLV-1 (ACH) and a derivative clone, ACH.p12I, which fails to produce the p12Imessage, we investigated the importance of p12I in infected primary cells and in a rabbit model of the infection. ACH.p12I was infectious in vitro as shown by viral passage in culture and no qualitative or quantitative differences were noted between ACH and ACH.p12I in posttransfection viral antigen production. However, in contrast to ACH, ACH.p12I failed to establish persistent infection in vivo as indicated by reduced anti-HTLV-1 antibody responses, failure to demonstrate viral p19 antigen production in peripheral blood mononuclear cell (PBMC) cultures, and only transient detection of provirus by polymerase chain reaction in PBMC from ACH.p12I-inoculated rabbits. These results are the first to show the essential role of HTLV-1 p12I in the establishment of persistent viral infection in vivo and suggest potential new targets in antiviral strategies to prevent HTLV-1 infection.

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

2003 ◽  
Vol 71 (10) ◽  
pp. 5994-6003 ◽  
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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