An inexpensive solid medium for obtaining colony‐forming units of oral spirochetes

Chan, E. C. S.; Ciccio, A. De; McLaughlin, Richard William; Klltorlnos, A.; Slboo, R.

doi:10.1111/j.1399-302x.1997.tb00741.x

Cited by 24 publications

(19 citation statements)

References 8 publications

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“…T. denticola ATCC 35405, ATCC 33520, and OTK and isogenic mutants of 35405 (Table 1) were grown in NOS broth or NOS/GN semisolid medium as previously described (7,24), with erythromycin (Em) (40 g ml…”

Section: Methodsmentioning

confidence: 99%

“…Both identify the translation initiation codon as a GUG codon 112 bp downstream of the initial AUG codon in the ORF. However, predicted protein molecular masses are 17 kDa (JCVI; consistent with the GUG start codon, encoding Val 38 ) and 22 kDa (LANL; consistent with either the first or second AUG as the start codon, encoding Met 1 or Met 7 [resulting in a 21.8-kDa or 21.1-kDa protein, respectively]). Analysis of the TDE0760 ORF by the FGENESB algorithm predicted that translation of the TDE0760 ORF initiates at the second AUG codon (Met 7 ) (data not shown).…”

Section: Conservation Of Prcbmentioning

confidence: 99%

“…Taken together with the observation that PrcB localized to the detergent phase of Triton X-114 extracts, while the 15-kDa PrcB fragment localized to the aqueous phase, we propose that PrcB is a lipoprotein, likely localized to the outer membrane. Since it was not possible to definitively identify the N-terminal residue of the PrcB polypeptide by either computational or biochemical methods, we constructed recombinant E. coli strains in which PrcB-6ϫHis translation starts at Met 1 , Met 7 , or Val 38 . When expression of PrcB-6ϫHis was induced, the Met 1 and Met 7 constructs produced a 22-kDa polypeptide whose migration by SDS-PAGE was not distinguishable from that of PrcB-6ϫHis expressed in T. denticola strains CF499 and CF417, and the Val 38 PrcB-6ϫHis construct produced a 17-kDa polypeptide that did not align with PrcB-6ϫHis expressed in T. denticola (data not shown).…”

Section: Fig 2 Protease Locus Inmentioning

confidence: 99%

“…To compare recombinant expression of PrcB initiating at each of the three possible start codons, DNA fragments including the coding region beginning at Met 1 , Met 7 , and Val 38 were amplified using forward primers CX710, CX711, and CX712, respectively, and the reverse primer CX713. Each amplicon, digested with NdeI and HindIII, was cloned into pET30b such that the resulting plasmids (pCF569, pCF570, and pCF571) expressed PrcB-6ϫHis from a different start codon under the inducible control of T7 RNA polymerase.…”

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confidence: 99%

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Treponema denticola PrcB Is Required for Expression and Activity of the PrcA-PrtP (Dentilisin) Complex

et al. 2010

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The Treponema denticola surface protease complex, consisting of PrtP protease (dentilisin) and two auxiliary polypeptides (PrcA1 and PrcA2), is believed to contribute to periodontal disease by degrading extracellular matrix components and disrupting host intercellular signaling. Previously, we showed that transcription of the protease operon initiates upstream of TDE0760 (herein designated prcB), the open reading frame immediately 5 of prcA-prtP. The prcB gene is conserved in T. denticola strains. PrcB localizes to the detergent phase of Triton X-114 cell surface extracts and migrates as a 22-kDa polypeptide, in contrast to the predicted 17-kDa cytoplasmic protein encoded in the annotated T. denticola genome. Consistent with this observation, the PrcB N terminus is unavailable for Edman sequencing, suggesting that it is acylated. Nonpolar deletion of prcB in T. denticola showed that PrcB is required for production of PrtP protease activity, including native PrtP cleavage of PrcA to PrcA1 and PrcA2. A 6؋His-tagged PrcB protein coimmunoprecipitates with native PrtP, using either anti-PrtP or anti-His-tag antibodies, and recombinant PrtP copurifies with PrcB-6؋His in nickel affinity chromatography. Taken together, these data are consistent with identification of PrcB as a PrtPbinding lipoprotein that likely stabilizes the PrtP polypeptide during localization to the outer membrane.Treponema denticola is an oral spirochete strongly associated with periodontal diseases. The predominance of spirochetes in subgingival plaque in severe periodontal diseases and their spatial location within periodontal lesions are highly suggestive of an important role in periodontal pathogenesis (13). Several T. denticola secreted proteinases and peptidases have been identified that likely contribute to periodontal pathogenesis as a consequence of their roles in processing host tissue proteins and peptides to fulfill the nutritional requirements of these highly motile and invasive organisms (reviewed in references 16 and 32). Among these, the outer membrane serine protease complex (variously designated CTLP [40], dentilisin [26], and the PrtP complex [5]) has several activities consistent with its identification as a virulence determinant in periodontal disease. The PrtP protease complex degrades extracellular matrix proteins (3, 23) as well as serum components involved in tissue homeostasis (22, 40), regulation of the complement pathway (33, 41), and other innate immune responses (1,12,38). PrtP protease activity also contributes to T. denticola coaggregation with Porphyromonas gingivalis (25) and penetration of host tissue (8, 23).The prcA-prtP locus is conserved in oral Treponema species, and the encoded products can be divided into two paralogous families based on phylogenetic analysis and substrate specificity of the protease (9). PrtP is one of only two known acylated members of the subtilisin family, the other being SphB1, a surface-anchored subtilisin autotransporter that catalyzes maturation of the virulence factor FhaB (filamen...

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Section: Methodsmentioning

confidence: 99%

Section: Conservation Of Prcbmentioning

confidence: 99%

Section: Fig 2 Protease Locus Inmentioning

confidence: 99%

mentioning

confidence: 99%

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Treponema denticola PrcB Is Required for Expression and Activity of the PrcA-PrtP (Dentilisin) Complex

et al. 2010

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“…T. denticola is one of the more easily cultured species of this genus, but it is not readily grown on the surface of agar plates and has a long generation time in liquid media (Chan et al, 1993(Chan et al, , 1997. Obtaining a model system for biofilm growth of pure cultures of T. denticola has been reported as problematic, requiring the presence of a specific coating such as fibronectin to facilitate attachment of cells (Vesey & Kuramitsu, 2004), and little work has been conducted on T. denticola biofilm growth.…”

Section: Introductionmentioning

confidence: 99%

Treponema denticola biofilm-induced expression of a bacteriophage, toxin–antitoxin systems and transposases

et al. 2010

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Treponema denticola is an oral spirochaete that has been strongly associated with chronic periodontitis. The bacterium exists as part of a dense biofilm (subgingival dental plaque) accreted to the tooth. To determine T. denticola gene products important for persistence as a biofilm we developed a continuous-culture biofilm model and conducted a genome-wide transcriptomic analysis of biofilm and planktonic cells. A total of 126 genes were differentially expressed with a fold change of 1.5 or greater. This analysis identified the upregulation of putative prophage genes in the T. denticola 35405 genome. Intact bacteriophage particles were isolated from T. denticola and circular phage DNA was detected by PCR analysis. This represents the first, to our knowledge, functional bacteriophage isolated from T. denticola, which we have designated Qtd1. In biofilm cells there was also an upregulation of genes encoding several virulence factors, toxinantitoxin systems and a family of putative transposases. Together, these data indicate that there is a higher potential for genetic mobility in T. denticola when growing as a biofilm and that these systems are important for the biofilm persistence and therefore virulence of this bacterium.

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Laboratory Maintenance of Treponema denticola

Fenno

2006

CP Microbiology

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This unit describes methods, media, and equipment required for routine laboratory culture and handling of the oral anaerobic spirochete Treponema denticola. Topics discussed include nutrient requirements, recommended formulations for growth in liquid and solid media, and expected growth kinetics, as well as methods and equipment necessary to maintain anaerobic conditions. Protocols on isolation of T. denticola from clinical samples are included. With the notable exception of specific individual nutrient requirements and media formulations, these protocols are also appropriate for working with other species of cultivable commensal treponemes.Basic protocols included in this chapter cover topics ranging from isolation of T. denticola from clinical samples (including isolation of pure cultures from single colonies) through culturing techniques applicable to biochemical analysis and genetic manipulation of T. denticola. The isolation protocols include a relatively standard method of dispersion and serial dilution on agar plates as well as a method using liquid medium in a 96-well plate format. Both of these methods employ rifampicin as a selective agent to enrich for treponemes in mixed clinical samples. Several commonly used liquid media and related solid media formulations are described, with comments on relative characteristics and performance of each. Methods, equipment, and conditions for inoculation, incubation, passage, and storage of cultures are discussed. Together, this information should provide the technical background required for routine growth and manipulation of T. denticola in a well-equipped microbiology laboratory.CAUTION: Treponema denticola is a Biosafety Level 1 (BSL-1) organism. Such organisms are not known to consistently cause disease in healthy adult humans, and are of minimal potential hazard to laboratory personnel and the environment. Standard microbiological practices should be followed when working with these organisms. See UNIT 1A.1 and other pertinent resources (see APPENDIX 1B) for more information.NOTE: Most strains of oral spirochetes require strictly anaerobic conditions during transfer and growth of cultures. Unless otherwise specified, all incubations and, where feasible, manipulations described in this unit are to be performed under strictly anaerobic conditions (i.e., nonhumidified 85% N 2 /10% H 2 /5% CO 2 atmosphere) in an anaerobic glove box chamber at 37 • C (see Strategic Planning). STRATEGIC PLANNING Media General requirementsSeveral complex broth media formulations are in common use for growth of T. denticola strains (see Reagents and Solutions). These share common features including sources of peptides, amino acids, and trace nutrients (e.g., tryptone, brain heart infusion, yeast extract), as well as reducing agent(s) (L-cysteine and/or thioglycolate), volatile fatty acids, and heat-inactivated serum. All these media provide very low redox potential (Eh = −185 to −220), which is a critical factor in growth of T. denticola (Socransky et al., 1964). These media were orig...

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An inexpensive solid medium for obtaining colony‐forming units of oral spirochetes

Cited by 24 publications

References 8 publications

Treponema denticola PrcB Is Required for Expression and Activity of the PrcA-PrtP (Dentilisin) Complex

Treponema denticola PrcB Is Required for Expression and Activity of the PrcA-PrtP (Dentilisin) Complex

Treponema denticola biofilm-induced expression of a bacteriophage, toxin–antitoxin systems and transposases

Laboratory Maintenance of Treponema denticola

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