scholarly journals Genetic Diversity of the Q Fever Agent, Coxiella burnetii, Assessed by Microarray-Based Whole-Genome Comparisons

2006 ◽  
Vol 188 (7) ◽  
pp. 2309-2324 ◽  
Author(s):  
Paul A. Beare ◽  
James E. Samuel ◽  
Dale Howe ◽  
Kimmo Virtaneva ◽  
Stephen F. Porcella ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Point Mutations ◽  
Open Reading Frames ◽  
Cellular Functions ◽  
Genetic Changes ◽  
Large Deletions ◽  
Reference Isolate ◽  
Virulence Potential

ABSTRACT Coxiella burnetii, a gram-negative obligate intracellular bacterium, causes human Q fever and is considered a potential agent of bioterrorism. Distinct genomic groups of C. burnetii are revealed by restriction fragment-length polymorphisms (RFLP). Here we comprehensively define the genetic diversity of C. burnetii by hybridizing the genomes of 20 RFLP-grouped and four ungrouped isolates from disparate sources to a high-density custom Affymetrix GeneChip containing all open reading frames (ORFs) of the Nine Mile phase I (NMI) reference isolate. We confirmed the relatedness of RFLP-grouped isolates and showed that two ungrouped isolates represent distinct genomic groups. Isolates contained up to 20 genomic polymorphisms consisting of 1 to 18 ORFs each. These were mostly complete ORF deletions, although partial deletions, point mutations, and insertions were also identified. A total of 139 chromosomal and plasmid ORFs were polymorphic among all C. burnetii isolates, representing ca. 7% of the NMI coding capacity. Approximately 67% of all deleted ORFs were hypothetical, while 9% were annotated in NMI as nonfunctional (e.g., frameshifted). The remaining deleted ORFs were associated with diverse cellular functions. The only deletions associated with isogenic NMI variants of attenuated virulence were previously described large deletions containing genes involved in lipopolysaccharide (LPS) biosynthesis, suggesting that these polymorphisms alone are responsible for the lower virulence of these variants. Interestingly, a variant of the Australia QD isolate producing truncated LPS had no detectable deletions, indicating LPS truncation can occur via small genetic changes. Our results provide new insight into the genetic diversity and virulence potential of Coxiella species.

The spectrum of genetic changes in the RB1 gene in a group of Russian retinoblastoma patients

2021 ◽  
pp. 11-20
Author(s):  
Е.А. Алексеева ◽  
В.М. Козлова ◽  
О.В. Бабенко ◽  
Т.Л. Ушакова ◽  
Т.П. Казубская ◽  
...  
Keyword(s):  
High Throughput ◽  
Molecular Genetic ◽  
Genetic Diagnosis ◽  
Point Mutations ◽  
Missense Mutations ◽  
Rb1 Gene ◽  
Genetic Changes ◽  
Parallel Sequencing ◽  
Large Deletions ◽  
Efficient Detection

Введение. Ретинобластома - злокачественная опухоль детского возраста, причиной которой является биаллельная инактивация гена RB1. Ранняя молекулярно-генетическая диагностика ретинобластомы необходима как для адекватного выбора алгоритма лечения пациента с такой опухолью, так и для медико-генетического консультирования семьи. Цель: охарактеризовать частоту и спектр мутаций в гене RB1 у российских больных с ретинобластомой. Методы. Исследование проведено на материале ДНК лимфоцитов крови, полученном от 492 больных с ретинобластомой. Скрининг точковых мутаций, малых инсерций/делеций в гене RВ1 осуществляли методом полупроводникового высокопроизводительного параллельного секвенирования. Исключение протяженных делеций в гене RВ1 проводили методом MLPA. Результаты. Исследовано 492 неродственных пациента с ретинобластомой, среди которых 38,2% (188/492) с билатеральной формой заболевания и 61,8% (304/492) - с унилатеральной. В группе больных с билатеральной формой ретинобластомы герминальная мутация обнаружена у 96,8% (182/188) пациентов, в группе больных с унилатеральной формой - у 16,4% (50/304). Суммарно в гене RB1 в исследованной группе пациентов обнаружено 339 мутаций: 232 - герминальных и 107 - соматических. Выявлен практически полный спектр молекулярных изменений, включающий нонсенс-мутации - 37,5% (127/339), миссенс-мутации - 5,3% (18/339), мутации, приводящие к сдвигу рамки считывания - 18,9% (64/339), мутации сайтов сплайсинга - 13,9% (47/339) и протяженные делеции - 24,5% (83/339). Выводы. Применение глубокого высокопроизводительного параллельного секвенирования и метода MLPA позволяет эффективно выявлять молекулярно-генетические изменения в гене RB1. Типы мутаций, обнаруженные в исследованной группе, их частота и распределение совпадают с результатами исследователей из других стран. Background. Retinoblastoma is a childhood malignant tumor caused by biallelic inactivation of the RB1 gene. Early molecular genetic diagnosis of retinoblastoma is necessary both for an adequate choice of an algorithm for treating a patient, and for competent medical genetic counseling of the family Objective. To establish the frequency and spectrum of mutations in the RB1 gene in the group of patients with retinoblastoma. Methods. The study was carried out on the DNA of blood lymphocytes from 492 patients with retinoblastoma. Screening of point mutations, small insertions/deletions in the RB1 gene was performed by semiconductor high-throughput parallel sequencing. Exclusion of gross deletions in the RB1 gene was performed by MLPA. Results. 492 unrelated patients with retinoblastoma were studied, including 38.2% (188/492) with bilateral form and 61.8% (304/492) with unilateral form. In the group of patients with bilateral retinoblastoma, germline mutation was found in 96.8% (182/188) patients, and in the group of unilateral patients, in 16.4% (50/304). In total, the RB1 gene in the studied group of patients 339 mutations were found, 232 germline and 107 somatic. An almost complete spectrum of molecular changes was revealed, including nonsense mutations, 37.5% (127/339); missense mutations, 5.3% (18/339); frame shift mutations, 18.9% (64 / 339); splice site mutations, 13.9% (47/339); and large deletions, 24.5% (83/339). Conclusion. The use of deep high-throughput parallel sequencing and the MLPA method allows efficient detection of molecular genetic changes in the RB1 gene. The types of mutations found in the studied group, their frequency and distribution are the same as the results of researchers in other countries.

scholarly journals The Coxiella burnetii Ankyrin Repeat Domain-Containing Protein Family Is Heterogeneous, with C-Terminal Truncations That Influence Dot/Icm-Mediated Secretion

2009 ◽  
Vol 191 (13) ◽  
pp. 4232-4242 ◽  
Author(s):  
Daniel E. Voth ◽  
Dale Howe ◽  
Paul A. Beare ◽  
Joseph P. Vogel ◽  
Nathan Unsworth ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Coxiella Burnetii ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Protein Family ◽  
Ankyrin Repeat ◽  
Reference Isolate ◽  
Type Iv

ABSTRACT Coxiella burnetii is an obligate intracellular bacterium that directs biogenesis of a parasitophorous vacuole (PV) for replication. Effectors of PV maturation are likely translocated into the host cytosol by a type IV secretion system (T4SS) with homology to the Dot/Icm apparatus of Legionella pneumophila. Since secreted bacterial virulence factors often functionally mimic the activities of host proteins, prokaryotic proteins with eukaryotic features are considered candidate T4SS substrates. Genes encoding proteins with eukaryotic-type ankyrin repeat domains (Anks) were identified upon genome sequencing of the C. burnetii Nine Mile reference isolate, which is associated with a case of human acute Q fever. Interestingly, recent genome sequencing of the G and K isolates, derived from human chronic endocarditis patients, and of the Dugway rodent isolate revealed remarkable heterogeneity in the Ank gene family, with the Dugway isolate harboring the largest number of full-length Ank genes. Using L. pneumophila as a surrogate host, we identified 10 Dugway Anks and 1 Ank specific to the G and K endocarditis isolates translocated into the host cytosol in a Dot/Icm-dependent fashion. A 10-amino-acid C-terminal region appeared to be necessary for translocation, with some Anks also requiring the chaperone IcmS for secretion. Ectopically expressed Anks localized to a variety of subcellular regions in mammalian cells, including microtubules, mitochondria, and the PV membrane. Collectively, these data suggest that C. burnetii isolates translocate distinct subsets of the Ank protein family into the host cytosol, where they modulate diverse functions, some of which may be unique to C. burnetii pathotypes.

scholarly journals Candidate Antigens for Q Fever Serodiagnosis Revealed by Immunoscreening of a Coxiella burnetii Protein Microarray

2008 ◽  
Vol 15 (12) ◽  
pp. 1771-1779 ◽  
Author(s):  
Paul A. Beare ◽  
Chen Chen ◽  
Timo Bouman ◽  
Jozelyn Pablo ◽  
Berkay Unal ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Protein Microarray ◽  
Protein S ◽  
In Vitro Transcription ◽  
Cross Reactivity ◽  
Open Reading Frames ◽  
Enzyme Linked Immunosorbent Assay ◽  
Translation System

ABSTRACT Q fever is a widespread zoonosis caused by Coxiella burnetii. Diagnosis of Q fever is usually based on serological testing of patient serum. The diagnostic antigen of test kits is formalin-fixed phase I and phase II organisms of the Nine Mile reference strain. Deficiencies of this antigen include (i) potential for cross-reactivity with other pathogens; (ii) an inability to distinguish between C. burnetii strains; and (iii) a need to propagate and purify C. burnetii, a difficult and potentially hazardous process. Consequently, there is a need for sensitive and specific serodiagnostic tests utilizing defined antigens, such as recombinant C. burnetii protein(s). Here we describe the use of a C. burnetii protein microarray to comprehensively identify immunodominant antigens recognized by antibody in the context of human C. burnetii infection or vaccination. Transcriptionally active PCR products corresponding to 1,988 C. burnetii open reading frames (ORFs) were generated. Full-length proteins were successfully synthesized from 75% of the ORFs by using an Escherichia coli-based in vitro transcription and translation system (IVTT). Nitrocellulose microarrays were spotted with crude IVTT lysates and probed with sera from acute Q fever patients and individuals vaccinated with Q-Vax. Immune sera strongly reacted with approximately 50 C. burnetii proteins, including previously identified immunogens, an ankyrin repeat-domain containing protein, and multiple hypothetical proteins. Recombinant protein corresponding to selected array-reactive antigens was generated, and the immunoreactivity was confirmed by enzyme-linked immunosorbent assay. This sensitive and high-throughput method for identifying immunoreactive C. burnetii proteins will aid in the development of Q fever serodiagnostic tests based on recombinant antigen.

scholarly journals Correlating Genotyping Data of Coxiella burnetii with Genomic Groups

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 604
Author(s):  
Claudia M. Hemsley ◽  
Angela Essex-Lopresti ◽  
Isobel H. Norville ◽  
Richard W. Titball
Keyword(s):  
Coxiella Burnetii ◽  
Classification Scheme ◽  
Q Fever ◽  
Variable Number Tandem Repeat ◽  
Febrile Illness ◽  
Variable Number ◽  
New Classification ◽  
Genetic Lineages ◽  
Virulence Potential ◽  
Multispacer Sequence Typing

Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. Several distinct genetic lineages or genomic groups have been shown to exist, with evidence for different virulence potential of these lineages. Multispacer Sequence Typing (MST) and Multiple-Locus Variable number tandem repeat Analysis (MLVA) are being used to genotype strains. However, it is unclear how these typing schemes correlate with each other or with the classification into different genomic groups. Here, we created extensive databases for published MLVA and MST genotypes of C. burnetii and analysed the associated metadata, revealing associations between animal host and human disease type. We established a new classification scheme that assigns both MST and MLVA genotypes to a genomic group and which revealed additional sub-lineages in two genomic groups. Finally, we report a novel, rapid genomotyping method for assigning an isolate into a genomic group based on the Cox51 spacer sequence. We conclude that by pooling and streamlining existing datasets, associations between genotype and clinical outcome or host source were identified, which in combination with our novel genomotyping method, should enable an estimation of the disease potential of new C. burnetii isolates.

Induced mutations for generating bananas resistant to Fusarium wilt tropical race 4.

2021 ◽  
pp. 366-378
Author(s):  
Joanna Jankowicz-Cieslak ◽  
Florian Goessnitzer ◽  
Sneha Datta ◽  
Altus Viljoen ◽  
Ivan Ingelbrecht ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Fusarium Wilt ◽  
Point Mutations ◽  
Crop Productivity ◽  
Banana Production ◽  
Biotic Stresses ◽  
Large Deletions ◽  
Forward Genetic Screens ◽  
Race 4

Abstract Bananas are a staple for more than 400 million people. Additionally, more than 16.5 million tonnes are exported, making it both an important food security and a cash crop. Productivity of Cavendish-type bananas is threatened by both abiotic and biotic stresses. The fact that triploid bananas are sterile, parthenocarpic and obligate vegetatively propagated makes them particularly susceptible to diseases, including Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) tropical race 4 (Foc TR4). This is because continual clonal propagation has led to loss of genetic diversity. Additionally, lack of meiosis limits methods for breeding. Foc TR4 has been devastating Cavendish bananas in South-east Asia but has recently also been reported from Queensland in Australia, the Middle East and Mozambique, thus threatening global banana production. To address this, we are performing mutagenesis of in vitro propagated bananas to broaden the genetic diversity in order to find new alleles conferring disease resistance. We have developed methods for efficient induction of mutations in isolated apical meristems from shoot tips using chemical mutagens and ionizing radiation. Mutation discovery methods have been adapted to recover mutations including single point mutations and large deletions spanning millions of base pairs. We have created approximately 5000 mutated lines for forward-genetic screens to identify TR4 resistance in greenhouse- evaluated material. A subset of ca. 500 in vitro plantlets was subjected to glasshouse-based screening using a virulent F. oxysporum isolate. To date, 23 lines showing altered resistance responses to Foc TR4 have been identified.

scholarly journals New Genotypes of Coxiella burnetii Circulating in Brazil and Argentina

Pathogens ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 30 ◽  
Author(s):  
Mateus de Souza Ribeiro Mioni ◽  
Karim Sidi-Boumedine ◽  
Felipe Morales Dalanezi ◽  
Sâmea Fernandes Joaquim ◽  
Renan Denadai ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
South America ◽  
Coxiella Burnetii ◽  
Tandem Repeats ◽  
Q Fever ◽  
Variable Number ◽  
Worldwide Distribution ◽  
Variable Number Tandem Repeats ◽  
Mlva Typing ◽  
Multispacer Sequence Typing

Coxiella burnetii, the zoonotic agent of Q fever, has a worldwide distribution. Despite the vast information about the circulating genotypes in Europe and North America, there is a lack of data regarding C. burnetii strains in South America. Here, we show the presence of novel multispacer sequence typing (MST) genotypes of C. burnetii in two clusters detected in Brazil and Argentina that seem to be distant in parenthood. Argentinian strains isolated from a tick belongs to a new phylogenetic branch of C. burnetii, and the Brazilians strains may be related to MST 20 and 61. Multilocus variable number tandem repeats analysis (MLVA) typing provided a deeper resolution that may be related to host clusters of bovines, caprine, ovine, and ticks. Our results corroborate with the reports of geotypes of C. burnetii. Thus, we highlight the need for more genotyping studies to understand the genetic diversity of C. burnetii in South America and to confirm the hypothesis of host-related genotypes. We also emphasize the importance of virulence studies for a better understanding of Q fever in the region, which may help in surveillance and disease prevention programs.

scholarly journals Genome-Wide Analysis of the Temporal Genetic Changes in Streptococcus pneumoniae Isolates of Genotype ST320 and Serotype 19A from South Korea

2021 ◽  
Vol 9 (4) ◽  
pp. 795
Author(s):  
Jin Yang Baek ◽  
Sun Ju Kim ◽  
Juyoun Shin ◽  
Yeun-Jun Chung ◽  
Cheol-In Kang ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
South Korea ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Genetic Changes ◽  
Genome Wide Analysis ◽  
Reference Isolate ◽  
Genome Wide ◽  
A Genome

Since the introduction of the pneumococcal conjugate vaccine, an increase in the incidence of Streptococcus pneumoniae serotype 19A and sequence type 320 (19A-ST320) isolates have been observed worldwide including in South Korea. We conducted a genome-wide analysis to investigate the temporal genetic changes in 26 penicillin-non-susceptible 19A-ST320 pneumococcal isolates from a hospital in South Korea over a period of 17 years (1999; 2004 to 2015). Although the strains were isolated from a single hospital and showed the same genotype and serotype, a whole-genome sequencing (WGS) analysis revealed that the S. pneumoniae isolates showed more extensive genetic variations compared with a reference isolate obtained in 1999. A phylogenetic analysis based on single nucleotide polymorphisms (SNPs) showed that the pneumococcal isolates from South Korea were not grouped together into limited clusters among the 19A-ST320 isolates from several continents. It was predicted that recombination events occurred in 11 isolates; larger numbers of SNPs were found within recombination blocks compared with point mutations identified in five isolates. WGS data indicated that S. pneumoniae 19A-ST320 isolates might have been introduced into South Korea from various other countries. In addition, it was revealed that recombination may play a great role in the evolution of pneumococci even in very limited places and periods.

scholarly journals Validation of a Novel Commercial ELISA Test for the Detection of Antibodies against Coxiella burnetii

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1075
Author(s):  
Salvatore Ledda ◽  
Cinzia Santucciu ◽  
Valentina Chisu ◽  
Giovanna Masala
Keyword(s):  
Diagnostic Accuracy ◽  
Phase Ii ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Animal Health ◽  
Elisa Test ◽  
Clinical Diagnostics ◽  
Complex Life Cycle ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity

Q fever is a zoonosis caused by Coxiella burnetii, a Gram-negative pathogen with a complex life cycle and a high impact on public and animal health all over the world. The symptoms are indistinguishable from those belonging to other diseases, and the disease could be symptomless. For these reasons, reliable laboratory tests are essential for an accurate diagnosis. The aim of this study was to validate a novel enzyme-linked immunosorbent assay (ELISA) test, named the Chorus Q Fever Phase II IgG and IgM Kit (DIESSE, Diagnostica Senese S.p.A), which is performed by an instrument named Chorus, a new device in medical diagnostics. This diagnostic test is employed for the detection of antibodies against C. burnetii Phase II antigens in acute disease. Our validation protocol was performed according to the Italian Accreditation Body (ACCREDIA) (Regulation UNI CEI EN ISO/IEC 17025:2018 and 17043:2010), OIE (World Organization for Animal Health), and Statement for Reporting Studies of Diagnostic Accuracy (STARD). Operator performance was evaluated along with the analytical specificity and sensitivity (ASp and ASe) and diagnostic accuracy of the kit, with parameters such as diagnostic specificity and sensitivity (DSp and DSe) and positive and negative predictive values (PPV and NPV), in addition to the repeatability. According to the evaluated parameters, the diagnostic ELISA test was shown to be suitable for validation and commercialization as a screening method in human sera and a valid support for clinical diagnostics.

scholarly journals Contributions of lipopolysaccharide and the type IVB secretion system to Coxiella burnetii vaccine efficacy and reactogenicity

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Carrie M. Long ◽  
Paul A. Beare ◽  
Diane C. Cockrell ◽  
Jonathan Fintzi ◽  
Mahelat Tesfamariam ◽  
...  
Keyword(s):  
Phase I ◽  
Coxiella Burnetii ◽  
Vaccine Efficacy ◽  
Q Fever ◽  
Secretion System ◽  
Cell Vaccine ◽  
Post Vaccination ◽  
Whole Cell Vaccine ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

AbstractCoxiella burnetii is the bacterial causative agent of the zoonosis Q fever. The current human Q fever vaccine, Q-VAX®, is a fixed, whole cell vaccine (WCV) licensed solely for use in Australia. C. burnetii WCV administration is associated with a dermal hypersensitivity reaction in people with pre-existing immunity to C. burnetii, limiting wider use. Consequently, a less reactogenic vaccine is needed. Here, we investigated contributions of the C. burnetii Dot/Icm type IVB secretion system (T4BSS) and lipopolysaccharide (LPS) in protection and reactogenicity of fixed WCVs. A 32.5 kb region containing 23 dot/icm genes was deleted in the virulent Nine Mile phase I (NMI) strain and the resulting mutant was evaluated in guinea pig models of C. burnetii infection, vaccination-challenge, and post-vaccination hypersensitivity. The NMI ∆dot/icm strain was avirulent, protective as a WCV against a robust C. burnetii challenge, and displayed potentially altered reactogenicity compared to NMI. Nine Mile phase II (NMII) strains of C. burnetii that produce rough LPS, were similarly tested. NMI was significantly more protective than NMII as a WCV; however, both vaccines exhibited similar reactogenicity. Collectively, our results indicate that, like phase I LPS, the T4BSS is required for full virulence by C. burnetii. Conversely, unlike phase I LPS, the T4BSS is not required for vaccine-induced protection. LPS length does not appear to contribute to reactogenicity while the T4BSS may contribute to this response. NMI ∆dot/icm represents an avirulent phase I strain with full vaccine efficacy, illustrating the potential of genetically modified C. burnetii as improved WCVs.

scholarly journals Human Q Fever on the Guiana Shield and Brazil: Recent Findings and Remaining Questions

2021 ◽  
Author(s):  
Loïc Epelboin ◽  
Carole Eldin ◽  
Pauline Thill ◽  
Vincent Pommier de Santi ◽  
Philippe Abboud ◽  
...  
Keyword(s):  
South America ◽  
Geographical Distribution ◽  
Coxiella Burnetii ◽  
French Guiana ◽  
Q Fever ◽  
Guiana Shield ◽  
Animal Reservoir ◽  
The World ◽  
Unique Strain ◽  
Amazonian Region

Abstract Purpose of Review In this review, we report on the state of knowledge about human Q fever in Brazil and on the Guiana Shield, an Amazonian region located in northeastern South America. There is a contrast between French Guiana, where the incidence of this disease is the highest in the world, and other countries where this disease is practically non-existent. Recent Findings Recent findings are essentially in French Guiana where a unique strain MST17 has been identified; it is probably more virulent than those usually found with a particularly marked pulmonary tropism, a mysterious animal reservoir, a geographical distribution that raises questions. Summary Q fever is a bacterial zoonosis due to Coxiella burnetii that has been reported worldwide. On the Guiana Shield, a region mostly covered by Amazonian forest, which encompasses the Venezuelan State of Bolivar, Guyana, Suriname, French Guiana, and the Brazilian State of Amapá, the situation is very heterogeneous. While French Guiana is the region reporting the highest incidence of this disease in the world, with a single infecting clone (MST 117) and a unique epidemiological cycle, it has hardly ever been reported in other countries in the region. This absence of cases raises many questions and is probably due to massive under-diagnosis. Studies should estimate comprehensively the true burden of this disease in the region.

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