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Cloning and sequencing of a part of the heat shock protein 65 gene (hsp65) of "Tropheryma whippelii" and its use for detection of "T. whippelii" in clinical specimens by PCR.

ABSTRACT: Using broad-spectrum primers, we have amplified, cloned, and sequenced a 620-bp fragment of the "Tropheryma whippelii" heat shock protein 65 gene (hsp65) from the heart valve of a patient with Whipple's endocarditis. The deduced amino acid sequence shows high similarity to those from actinobacteria, confirming that "T. whippelii" is indeed a member of this phylum. Based on the nucleotide sequence, we have developed a "T. whippelii"-specific seminested PCR. Seventeen patients shown to be positive by 16S ribosomal DNA (rDNA) PCR and/or internal transcribed spacer (ITS) PCR were also positive by hsp65 PCR. All 33 control specimens from patients without Whipple's disease and negative for "T. whippelii" by both seminested 16S rDNA and ITS PCR remained negative. All amplicons digested with XhoI revealed an identical restriction pattern. Eight of the 17 hsp65 amplicons representing all three previously described ITS types were sequenced. Three of the amplicons showed slight differences, but none of the mutations detected affected the amino acid sequence of the corresponding protein. We conclude that the hsp65 gene is a suitable target for the specific detection of "T. whippelii." Its product represents a putative antigen for a future serodiagnostic assay.

SUBMITTER: Morgenegg S

PROVIDER: S-EPMC86774 | biostudies-literature | 2000 Jun

REPOSITORIES: biostudies-literature

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Cloning and sequencing of a part of the heat shock protein 65 gene (hsp65) of "Tropheryma whippelii" and its use for detection of "T. whippelii" in clinical specimens by PCR.

Morgenegg S S Dutly F F Altwegg M M

Journal of clinical microbiology 20000601 6

Using broad-spectrum primers, we have amplified, cloned, and sequenced a 620-bp fragment of the "Tropheryma whippelii" heat shock protein 65 gene (hsp65) from the heart valve of a patient with Whipple's endocarditis. The deduced amino acid sequence shows high similarity to those from actinobacteria, confirming that "T. whippelii" is indeed a member of this phylum. Based on the nucleotide sequence, we have developed a "T. whippelii"-specific seminested PCR. Seventeen patients shown to be positive ...[more]

PMID: 10834984

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Project description:Whipple's disease is a rare bacterial infection that may involve any organ system in the body. It occurs primarily in Caucasian males older than 40 years. The gastrointestinal tract is the most frequently involved organ, with manifestations such as abdominal pain, malabsorption syndrome with diarrhea, and weight loss. Other signs include low-grade fever, lymphadenopathy, skin hyperpigmentation, endocarditis, pleuritis, seronegative arthritis, uveitis, spondylodiscitis, and neurological manifestations, and these signs may occur in the absence of gastrointestinal manifestations. Due to the wide variability of manifestations, clinical diagnosis is very difficult and is often made only years or even decades after the initial symptoms have appeared. Trimethoprim-sulfamethoxazole for at least 1 year is usually considered adequate to eradicate the infection. The microbiological diagnosis of this insidious disease is rendered difficult by the virtual lack of culture and serodiagnostic methods. It is usually based on the demonstration of periodic acid-Schiff-positive particles in infected tissues and/or the presence of bacteria with an unusual trilaminar cell wall ultrastructure by electron microscopy. Recently, the Whipple bacteria have been characterized at the molecular level by amplification of their 16S rRNA gene(s). Phylogenetic analysis of these sequences revealed a new bacterial species related to the actinomycete branch which was named "Tropheryma whippelli." Based on its unique 16S ribosomal DNA (rDNA) sequence, species-specific primers were selected for the detection of the organism in clinical specimens by PCR. This technique is currently used as one of the standard methods for establishing the diagnosis of Whipple's disease. Specific and broad-spectrum PCR amplifications mainly but not exclusively from extraintestinal specimens have significantly improved diagnosis, being more sensitive than histopathologic analysis. However, "T. whippelii" DNA has also been found in persons without clinical and histological evidence of Whipple's disease. It is unclear whether these patients are true asymptomatic carriers or whether differences in virulence exist among strains of "T. whippelii" that might account for the variable clinical manifestations. So far, six different "T. whippelii" subtypes have been found by analysis of their 16S-23S rDNA spacer region. Further studies of the pathogen "T. whippelii" as well as the host immune response are needed to fully understand this fascinating disease. The recent cultivation of the organisms is a promising major step in this direction.

Project description:We assessed the ability of an in-house database, consisting of 111 hsp65 sequences from putative and valid Mycobacterium species or described groups, to identify 689 mycobacterial clinical isolates from 35 species or groups. A preliminary assessment indicated that hsp65 sequencing confirmed the identification of 79.4% of the isolates from the 32 species examined, including all Mycobacterium tuberculosis complex isolates, all isolates from 13 other species, and 95.6% of all M. avium-M. intracellulare complex isolates. Identification discrepancies were most frequently encountered with isolates submitted as M. chelonae, M. fortuitum, M. gordonae, M. scrofulaceum, and M. terrae. Reexamination of isolates with discrepant identifications confirmed that hsp65 identifications were correct in a further 40 isolates. This brought the overall agreement between hsp65 sequencing and the other identification methods to 85.2%. The remaining 102 isolates had sequence matches below our acceptance criterion, had nondifferential sequence matches between two or more species, were identified by 16S rRNA sequencing as a putative taxonomic group not contained in our database, or were identified by hsp65 and 16S rRNA gene sequencing as a species not in our biochemical test database or had conflicting identifications. Therefore, to incorporate the unconfirmed isolates it was necessary to create 29 additional entries in our hsp65 identification database: 18 associated with valid species, 7 indicating unique sequences not associated with valid or putative species or groups, and 4 associated with unique, but currently described taxonomic groups. Confidence in the hsp65 sequence identification of a clinical isolate is best when sequence matches of 100% occur, but our data indicate that correct identifications can be confidently made when unambiguous matches exceeding 97% occur, but are dependent on the completeness of the database. Our study indicates that for hsp65 sequencing to be an effective means for identifying mycobacteria a comprehensive database must be constructed. hsp65 sequencing has the advantage of being more rapid and less expensive than biochemical test panels, uses a single set of reagents to identify both rapid- and slow-growing mycobacteria, and can provide a more definitive identification.

Dataset Information

Cloning and sequencing of a part of the heat shock protein 65 gene (hsp65) of "Tropheryma whippelii" and its use for detection of "T. whippelii" in clinical specimens by PCR.

Publications

Cloning and sequencing of a part of the heat shock protein 65 gene (hsp65) of "Tropheryma whippelii" and its use for detection of "T. whippelii" in clinical specimens by PCR.

Similar Datasets

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