In vitro culture system to determine MICs and MBCs of antimicrobial agents against Treponema pallidum subsp. pallidum (Nichols strain)

Norris, Steven J.; Edmondson, Diane G.

doi:10.1128/aac.32.1.68

Cited by 36 publications

(26 citation statements)

References 29 publications

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“…One key factor that has severely hampered studies of the pathogenic mechanisms used by this important human pathogen is that it cannot be continuously cultured in vitro. Although limited multiplication has been achieved in an in vitro tissue culture system (12,17,32,(34)(35)(36)(37), T. pallidum is a fastidious, obligate human pathogen for which intratesticular inoculation of rabbits is the only reliable method of bacterial propagation. This organism remains refractory to genetic manipulations, thus preventing direct investigation of the functions of individual gene products.…”

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confidence: 99%

Heterologous Expression of theTreponema pallidumLaminin-Binding Adhesin Tp0751 in the Culturable SpirocheteTreponema phagedenis

et al. 2008

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Treponema pallidum subsp. pallidum, the causative agent of syphilis, is an unculturable, genetically intractable bacterium. Here we report the use of the shuttle vector pKMR4PEMCS for the expression of a previously identified T. pallidum laminin-binding adhesin, Tp0751, in the nonadherent, culturable spirochete Treponema phagedenis. Heterologous expression of Tp0751 in T. phagedenis was confirmed via reverse transcriptase PCR analysis with tp0751 gene-specific primers and immunofluorescence analysis with Tp0751-specific antibodies; the latter assay verified the expression of the laminin-binding adhesin on the treponemal surface. Expression of Tp0751 within T. phagedenis was functionally confirmed via laminin attachment assays, in which heterologous Tp0751 expression conferred upon T. phagedenis the capacity to attach to laminin. Further, specific inhibition of the attachment of T. phagedenis heterologously expressing Tp0751 to laminin was achieved by using purified antibodies raised against recombinant T. pallidum Tp0751. This is the first report of heterologous expression of a gene from an unculturable treponeme in T. phagedenis. This novel methodology will significantly advance the field of syphilis research by allowing targeted investigations of T. pallidum proteins purported to play a role in pathogenesis, and specifically host cell attachment, in the nonadherent spirochete T. phagedenis.

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Heterologous Expression of theTreponema pallidumLaminin-Binding Adhesin Tp0751 in the Culturable SpirocheteTreponema phagedenis

et al. 2008

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“…Taken together, these results indicate that heterologous expression of TP_0144 in the ⌬TDE_0143 strain restores its growth and TPP uptake when exogenous TPP is Conclusion. Although tremendous efforts have been undertaken during the last several decades, T. pallidum still cannot be continuously cultivated in vitro (5,42,43). This is primarily due to a lack of understanding of its nutrient requirements and metabolism.…”

Section: Resultsmentioning

confidence: 99%

Evidence that TP_0144 of Treponema pallidum Is a Thiamine-Binding Protein

Bian

Wang

et al. 2015

J. Bacteriol.

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e Thiamine pyrophosphate (TPP), the biologically active form of thiamine (also known as vitamin B 1 ), is an essential cofactor for several important enzymes involved in carbohydrate metabolism, and therefore, it is required for all living organisms. We recently found that a thiamine-binding protein (TDE_0143) is essential for the survival of Treponema denticola, an important bacterial pathogen that is associated with human periodontitis. In this report, we provide experimental evidence showing that TP_0144, a homolog of TDE_0143 from the syphilis spirochete Treponema pallidum, is a thiamine-binding protein that has biochemical features and functions that are similar to those of TDE_0143. First, structural modeling analysis reveal that both TDE_0143 and TP_0144 contain a conserved TPP-binding site and share similar structures to the thiamine-binding protein of Escherichia coli. Second, biochemical analysis shows that these two proteins bind to TPP with similar dissociation constant (K d ) values (TDE_0143, K d of 36.50 nM; TP_0144, K d of 32.62 nM). Finally, heterologous expression of TP_0144 in a ⌬TDE_0143 strain, a previously constructed TDE_0143 mutant of T. denticola, fully restores its growth and TPP uptake when exogenous thiamine is limited. Collectively, these results indicate that TP_0144 is a thiamine-binding protein that is indispensable for T. pallidum to acquire exogenous thiamine, a key nutrient for bacterial survival. In addition, the studies shown in this report further underscore the feasibility of using T. denticola as a platform to study the biology and pathogenicity of T. pallidum and probably other uncultivable treponemal species as well. Spirochetes comprise a monophyletic phylum that exhibits distinct long and coiled cell shapes and a characteristic corkscrew-like motility (1). The phylum comprises nine genera. One of these genera is Treponema, which contains several important human pathogens such as Treponema pallidum, the causative agent of syphilis (2, 3). The genus of Treponema consists of more than 60 different species, and many of these species cannot be cultivated in vitro (e.g., T. pallidum can be grown experimentally only in rabbit testes) (4-7). The lack of reliable in vitro cultivation systems has substantially hindered our understanding of the biology of T. pallidum and syphilis pathogenesis (3). Identifying virulence determinants of T. pallidum still depends on expressing potential virulence genes in heterologous systems such as Escherichia coli (8-11). However, the physiological differences between the spirochetes and E. coli have limited the use of E. coli systems for functional investigations.Among the treponemal species, Treponema denticola, an oral spirochete associated with periodontitis, can be easily cultivated and is genetically manipulable (12-14). Due to the high physiological similarities between T. denticola and T. pallidum, the oral spirochete has been explored to study the physiology, biosynthetic pathways, and virulence determinants of T. pallidum (e.g., heterol...

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“…The minimum inhibitory concentration and the minimum bactericidal concentrations of tetracycline using an in vitro tissue culture system were 0.2 mg/L and 0.5 mg/L, respectively. 12 Doxycycline, a longacting derivative of tetracycline, demonstrates similar treponemicidal activity, but its longer half life allows less frequent dosing and it is better tolerated. 13 Animal data demonstrated high CSF levels of doxycycline, five times the concentration of tetracycline in the brain due to its lipophilic property.…”

Section: Discussionmentioning

confidence: 99%

Oral Doxycycline for Treatment of Neurosyphilis in Two Patients Infected with Human Immunodeficiency Virus

Castel¹,

Prichard²

2010

Pharmacotherapy

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The frequency of syphilis has been increasing during the past 5 years primarily among men who have sex with men, many of whom are infected with the human immunodeficiency virus (HIV). Data on treatment options other than intravenous or intramuscular penicillin for syphilis are very limited. We describe two HIV-infected patients with asymptomatic neurosyphilis who were successfully treated with oral doxycycline. The first patient was a 45-year-old Hispanic man with well-suppressed HIV RNA who had a positive Venereal Disease Research Laboratory (VDRL) titer of 1:128. His cerebral spinal fluid (CSF) revealed a positive VDRL titer of 1:16, and an elevated white blood cell count of 96 cells/mm(3) and protein level of 89 mg/dl. He received high-dose doxycycline 200 mg twice/day for 28 days. Two months later, his CSF VDRL titer, white blood cell count, and protein level decreased to 1:4, 5 cells/mm(3), and 60 mg/dl, respectively. The second patient was a 37-year-old Caucasian man with complications from acquired immunodeficiency disease. A routine VDRL titer was found to be 1:64. Although the CSF VDRL was nonreactive, both his white blood cell count and protein level were elevated at 29 cells/mm(3) and 46 mg/dl, respectively. High-dose doxycycline 200 mg twice/day was prescribed for 28 days. Three months later, the patient's VDRL titer decreased to 1:2; his CSF white blood cell count decreased significantly to 1 cell/mm(3), and his protein level was within normal limits. Clinicians should be aware that an extended course of high-dose, oral doxycycline may be an effective and safe alternative regimen to intravenous or intramuscular penicillin, without requiring hospitalization or home health care, for the treatment of neurosyphilis in HIV-infected patients. Prospective trials are needed to assess the long-term efficacy oral doxycycline for neurosyphilis.

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In vitro culture system to determine MICs and MBCs of antimicrobial agents against Treponema pallidum subsp. pallidum (Nichols strain)

Cited by 36 publications

References 29 publications

Heterologous Expression of theTreponema pallidumLaminin-Binding Adhesin Tp0751 in the Culturable SpirocheteTreponema phagedenis

Heterologous Expression of theTreponema pallidumLaminin-Binding Adhesin Tp0751 in the Culturable SpirocheteTreponema phagedenis

Evidence that TP_0144 of Treponema pallidum Is a Thiamine-Binding Protein

Oral Doxycycline for Treatment of Neurosyphilis in Two Patients Infected with Human Immunodeficiency Virus

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