scholarly journals Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi

2016 ◽  
Vol 113 (26) ◽  
pp. 7231-7236 ◽  
Author(s):  
Robert W. Moon ◽  
Hazem Sharaf ◽  
Claire H. Hastings ◽  
Yung Shwen Ho ◽  
Mridul B. Nair ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Plasmodium Knowlesi ◽  
Binding Protein ◽  
Cynomolgus Macaque ◽  
Genomic Analysis ◽  
Human Infection ◽  
Comparative Genomic ◽  
Gene Encoding ◽  
Human Rbcs

The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

Plasmodium knowlesi: a relevant, versatile experimental malaria model

Parasitology ◽  
2016 ◽  
Vol 145 (1) ◽  
pp. 56-70 ◽  
Author(s):  
ERICA M. PASINI ◽  
ANNE-MARIE ZEEMAN ◽  
ANNEMARIE VOORBERG-VAN DER WEL ◽  
CLEMENS H. M. KOCKEN
Keyword(s):  
Parasite Species ◽  
Vaccine Development ◽  
Plasmodium Knowlesi ◽  
Host Interactions ◽  
Model Studies ◽  
Human Red Blood Cells ◽  
Malaria Model ◽  
Human Primate

SUMMARYThe primate malariaPlasmodium knowlesihas a long-standing history as an experimental malaria model. Studies using this model parasite in combination with its various natural and experimental non-human primate hosts have led to important advances in vaccine development and in our understanding of malaria invasion, immunology and parasite–host interactions. The adaptation to long-termin vitrocontinuous blood stage culture in rhesus monkey,Macaca fascicularisand human red blood cells, as well as the development of various transfection methodologies has resulted in a highly versatile experimental malaria model, further increasing the potential of what was already a very powerful model. The growing evidence thatP. knowlesiis an important human zoonosis in South-East Asia has added relevance to former and future studies of this parasite species.

An essential yeast gene encoding a TTAGGG repeat-binding protein

1993 ◽  
Vol 13 (2) ◽  
pp. 1306-1314
Author(s):  
C Brigati ◽  
S Kurtz ◽  
D Balderes ◽  
G Vidali ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Binding Activity ◽  
Insertion Mutation ◽  
Yeast Gene ◽  
Gene Encoding ◽  
Cloned Gene ◽  
Diploid Cells ◽  
Ttaggg Repeat

A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.

scholarly journals Use of In Vivo-Induced Antigen Technology for Identification of Escherichia coli O157:H7 Proteins Expressed during Human Infection

2005 ◽  
Vol 73 (5) ◽  
pp. 2665-2679 ◽  
Author(s):  
Manohar John ◽  
Indira T. Kudva ◽  
Robert W. Griffin ◽  
Allen W. Dodson ◽  
Bethany McManus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Human Infection ◽  
Escherichia Coli O157 ◽  
Standard Laboratory ◽  
E Coli ◽  
Phase Culture ◽  
Stool Specimens

ABSTRACT Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.

scholarly journals Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Canh Phung ◽  
Timothy B. Wilson ◽  
José A. Quinteros ◽  
Peter C. Scott ◽  
Robert J. Moore ◽  
...  
Keyword(s):  
Liquid Culture ◽  
Vaccine Development ◽  
Genomic Analysis ◽  
Bacterial Biomass ◽  
In Silico Analysis ◽  
Culture Method ◽  
Efficient Production ◽  
Culture Density ◽  
Brucella Broth

AbstractCampylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise l-cysteine, l-lysine and l-arginine. It was found that l-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but l-lysine or l-arginine addition did not enhance growth. Brucella broth supplemented with l-cysteine (0.4 mM), l-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

scholarly journals Virulence characterization and comparative genomics of Listeria monocytogenes sequence type 155 strains

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Eva Wagner ◽  
Andreas Zaiser ◽  
Rebekka Leitner ◽  
Narciso M. Quijada ◽  
Nadja Pracser ◽  
...  
Keyword(s):  
Food Processing ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Specific Sequence ◽  
Equal Distribution ◽  
Genetic Traits ◽  
Virulence Potential ◽  
Genetic Features ◽  
Source Category

Abstract Background Listeria (L.) monocytogenes strains show a high diversity regarding stress tolerance and virulence potential. Genome studies have mainly focused on specific sequence types (STs) predominantly associated with either food or human listeriosis. This study focused on the prevalent ST155, showing equal distribution among clinical and food isolates. We evaluated the virulence potential of 20 ST155 strains and performed comparative genomic analysis of 130 ST155 strains isolated from food, food processing environments and human listeriosis cases in different countries and years. Results The in vitro virulence assays using human intestinal epithelial Caco2 and hepatocytic HEPG2 cells showed an impaired virulence phenotype for six of the 20 selected ST155 strains. Genome analysis revealed no distinct clustering of strains from the same source category (food, food processing environment, and clinical isolates). All strains harbored an intact inlA and inlB locus, except four strains, which had an internal deletion in the inlA gene. All strains harbored LIPI-1, but prfA was present in a longer variant in six strains, all showing impaired virulence. The longer PrfA variant resulted in lower expression of inlA, inlB, and prfA, and no expression of hly and actA. Regarding stress-related gene content, SSI-1 was present, whereas qacH was absent in all strains. 34.6% of the strains harbored a plasmid. All but one ST155 plasmids showed high conservation and harbored cadA2, bcrABC, and a triphenylmethane reductase. Conclusions This study contributes to an enhanced understanding of L. monocytogenes ST155 strains, being equally distributed among isolates from humans, food, and food processing environments. The conservation of the present genetic traits and the absence of unique inherent genetic features makes these types of STs especially interesting since they are apparently equally adapted to the conditions in food processing environments, as well as in food as to the human host environment. However, a ST155-specific mutation resulting in a longer PrfA variant impaired the virulence potential of several ST155 strains.

scholarly journals An essential yeast gene encoding a TTAGGG repeat-binding protein.

1993 ◽  
Vol 13 (2) ◽  
pp. 1306-1314 ◽  
Author(s):  
C Brigati ◽  
S Kurtz ◽  
D Balderes ◽  
G Vidali ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Binding Activity ◽  
Insertion Mutation ◽  
Yeast Gene ◽  
Gene Encoding ◽  
Cloned Gene ◽  
Diploid Cells ◽  
Ttaggg Repeat

A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.

scholarly journals Improvement of Immunogenicity of Meningococcal Lipooligosaccharide by Coformulation with Lipidated Transferrin-Binding Protein B in Liposomes: Implications for Vaccine Development

2012 ◽  
Vol 19 (5) ◽  
pp. 711-722 ◽  
Author(s):  
Noëlle Mistretta ◽  
Bruno Guy ◽  
Yves Bérard ◽  
François Dalençon ◽  
Olivia Fratantonio ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Binding Protein ◽  
Hek293 Cells ◽  
Molar Ratio ◽  
Adjuvant Effect ◽  
Content Type ◽  
Endotoxic Activity ◽  
Protein B

ABSTRACTAmong various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group BNeisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluatedin vitrousing theLimulusAmebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, anin vivostudy demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

scholarly journals A Controlled Human Infection Model of Group AStreptococcusPharyngitis: Which Strain and Why?

mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Joshua Osowicki ◽  
Kristy I. Azzopardi ◽  
Liam McIntyre ◽  
Tania Rivera-Hernandez ◽  
Cheryl-lynn Y. Ong ◽  
...  
Keyword(s):  
Animal Model ◽  
Molecular Epidemiology ◽  
Virulence Factors ◽  
Healthy Adult ◽  
Vaccine Development ◽  
Human Infection ◽  
Infection Model ◽  
Vaccine Antigens ◽  
Adult Volunteers

ABSTRACTGroup AStreptococcus(GAS) is a major cause of global infection-related morbidity and mortality. A modern controlled human infection model (CHIM) of GAS pharyngitis can accelerate vaccine development and pathogenesis research. A robust rationale for strain selection is central to meeting ethical, scientific, and regulatory requirements. Multifaceted characterization studies were done to compare a preferred candidateemm75 (M75) GAS strain to three other strains: an alternative candidateemm12 (M12) strain, an M1 strain used in 1970s pharyngitis CHIM studies (SS-496), and a representative (5448) of the globally disseminated M1T1 clone. A range of approaches were used to explore strain growth, adherence, invasion, delivery characteristics, short- and long-term viability, phylogeny, virulence factors, vaccine antigens, resistance to killing by human neutrophils, and lethality in a murine invasive model. The strains grew reliably in a medium without animal-derived components, were consistently transferred using a swab method simulating the CHIM protocol, remained viable at −80°C, and carried genes for most candidate vaccine antigens. Considering GAS molecular epidemiology, virulence factors,in vitroassays, and results from the murine model, the contemporary strains show a spectrum of virulence, with M75 appearing the least virulent and 5448 the most. The virulence profile of SS-496, used safely in 1970s CHIM studies, was similar to that of 5448 in the animal model and virulence gene carriage. The results of this multifaceted characterization confirm the M75 strain as an appropriate choice for initial deployment in the CHIM, with the aim of safely and successfully causing pharyngitis in healthy adult volunteers.IMPORTANCEGAS (Streptococcus pyogenes) is a leading global cause of infection-related morbidity and mortality. A modern CHIM of GAS pharyngitis could help to accelerate vaccine development and drive pathogenesis research. Challenge strain selection is critical to the safety and success of any CHIM and especially so for an organism such as GAS, with its wide strain diversity and potential to cause severe life-threatening acute infections (e.g., toxic shock syndrome and necrotizing fasciitis) and postinfectious complications (e.g., acute rheumatic fever, rheumatic heart disease, and acute poststreptococcal glomerulonephritis). In this paper, we outline the rationale for selecting anemm75 strain for initial use in a GAS pharyngitis CHIM in healthy adult volunteers, drawing on the findings of a broad characterization effort spanning molecular epidemiology,in vitroassays, whole-genome sequencing, and animal model studies.

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