procedures-for-vitro-cultivation-treponema-pallidum-syphilis

Procedures for In Vitro Cultivation of <i>Treponema pallidum</i>, the Syphilis Spirochete

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and ...

University of Texas Health Science Center at Houston - McGovern Medical School

Aspects of the biology of T. pallidum subsp. pallidum, the agent of syphilis, are examined in the context of a century of experimental studies and the recently determined genome sequence. T. pallidum and a group of closely related pathogenic spirochetes have evolved to become highly invasive, persistent pathogens with little toxigenic activity and an inability to survive outside the mammalian host. Analysis of the genome sequence confirms morphologic studies indicating the lack of lipopolysaccharide and lipid biosynthesis mechanisms, as well as a paucity of outer membrane protein candidates. The metabolic capabilities and adaptability of T. pallidum are minimal, and this relative deficiency is reflected by the absence of many pathways, including the tricarboxylic acid cycle, components of oxidative phosphorylation, and most biosynthetic pathways. Although multiplication of T. pallidum has been obtained in a tissue culture system, continuous in vitro culture has not been achieved. The balance of oxygen utilization and toxicity is key to the survival and growth of T. pallidum, and the genome sequence reveals a similarity to lactic acid bacteria that may be useful in understanding this relationship. The identification of relatively few genes potentially involved in pathogenesis reflects our lack of understanding of invasive pathogens relative to toxigenic organisms. The genome sequence will provide useful raw data for additional functional studies on the structure, metabolism, and pathogenesis of this enigmatic organism.

Biology of Treponema pallidum: correlation of functional activities with genome sequence data.

The proteome of the spirochete bacterium Borrelia burgdorferi, the tick-borne agent of Lyme disease, has been characterized by two different approaches using mass spectrometry, providing a launching point for future studies on the dramatic changes in protein expression that occur during transmission of the bacterium between ticks and mammals.

The dynamic proteome of Lyme disease Borrelia.

A common mechanism of immune evasion in pathogenic bacteria and protozoa is antigenic variation, in which genetic or epigenetic changes result in rapid, sequential shifts in a surface-exposed antigen. In this issue of Molecular Microbiology, Dai et al. provide the most complete description to date of the vlp/vsp antigenic variation system of the relapsing fever spirochaete, Borrelia hermsii. This elaborate, plasmid-encoded system involves an expression site that can acquire either variable large protein (vlp) or variable small protein (vsp) surface lipoprotein genes from 59 different archival copies. The archival vlp and vsp genes are arranged in clusters on at least five different plasmids. Gene conversion occurs through recombination events at upstream homology sequences (UHS) found in each gene copy, and at downstream homology sequences (DHS) found periodically among the vlp/vsp archival genes. Previous studies have shown that antigenic variation in relapsing fever Borrelia not only permits the evasion of host antibody responses, but can also result in changes in neurotropism and other pathogenic properties. The vlsE antigenic variation locus of Lyme disease spirochaetes, although similar in sequence to the relapsing fever vlp genes, has evolved a completely different antigenic variation mechanism involving segmental recombination from a contiguous array of vls silent cassettes. These two systems thus appear to represent divergence from a common precursor followed by functional convergence to create two distinct antigenic variation processes.

Antigenic variation with a twist--the Borrelia story.

Out of the woods: the remarkable genomes of the genus Borrelia.

Analysis of the intergenic sequences provided by Feria-Arroyo et al. does not support the claim of high Borrelia burgdorferi tick infection rates in Texas and northeastern Mexico.

Feria-Arroyo et al. had reported previously that, based on PCR analysis, 45% of Ixodes scapularis ticks collected in Texas and Mexico were infected with the Lyme disease spirochete Borrelia burgdorferi (Parasit. Vectors 2014, 7:199). However, our analyses of their initial data (Parasit. Vectors 2014, 7:467) and a recent response by Esteve-Gassent et al. (Parasit. Vectors 2015, 8:129) provide evidence that the positive PCR results obtained from both ribosomal RNA intergenic sequences and the flagellin gene flaB are highly likely due to contamination by the B. burgdorferi B31 positive control strain.

Response to Esteve-Gassent et al.: flaB sequences obtained from Texas PCR products are identical to the positive control strain Borrelia burgdorferi B31.

The spirochetes that cause Lyme disease have an elaborate antigenic variation system that produces millions of variants, thus evading the immune response. Verhey et al. have applied next-generation sequencing and computational analysis to gain new insights into how these bacteria keep 'one step ahead' of elimination by the host.

Catching up with Lyme Disease Antigenic Variation Computationally.

Investigation of  subsp. , the spirochete that causes syphilis, has been hindered by an inability to culture the organism continuously  despite more than a century of effort. In this study, long-term logarithmic multiplication of  was attained through subculture every 6 to 7 days and periodic feeding using a modified medium ( culture medium 2 [TpCM-2]) with a previously described microaerobic, rabbit epithelial cell coincubation system. Currently, cultures have maintained continuous growth for over 6 months with full retention of viability as measured by motility and rabbit infectivity. This system has been applied successfully to the well-studied Nichols strain of , as well as to two recent syphilis isolates, UW231B and UW249B. Light microscopy and cryo-electron microscopy showed that -cultured  retains wild-type morphology. Further refinement of this long-term subculture system is expected to facilitate study of the physiological, genetic, pathological, immunologic, and antimicrobial susceptibility properties of  subsp.  and closely related pathogenic  species and subspecies. Syphilis, a sexually transmitted disease with a global distribution, is caused by a spiral-shaped bacterium called  subspecies  Previously,  was one of the few major bacterial pathogens that had not been cultured long-term  (in a test tube), greatly hindering efforts to better understand this organism and the disease that it causes. In this article, we report the successful long-term cultivation of  in a tissue culture system, a finding that is likely to enhance our ability to obtain new information applicable to the diagnosis, treatment, and prevention of syphilis.

Long-Term  Culture of the Syphilis Spirochete  subsp. .

In 1967, Harland and Lee made a startling discovery: in some humans, the colonic epithelium is covered with a "forest" of spirochetes (W. A. Harlan, and F. D. Lee, Br Med J 3:718-719, 1967, https://doi.org/10.1136/bmj.3.5567.718). In this issue of , Thorell et al. present a systematic analysis of the prevalence and diversity of the spirochetes  and  in the human colon. These and prior studies provide avenues toward resolving important questions: what bacterial and host parameters contribute to this extensive colonization, and what impact does it have on human health?

Article	Year
Biology of Treponema pallidum: correlation of functional activities with genome sequence data. Journal of molecular microbiology and biotechnology\| PubMed ID: 11200228	2001
The dynamic proteome of Lyme disease Borrelia. Genome biology\| PubMed ID: 16563176	2006
Antigenic variation with a twist--the Borrelia story. Molecular microbiology\| PubMed ID: 16796669	2006
Out of the woods: the remarkable genomes of the genus Borrelia. Journal of bacteriology\| PubMed ID: 22001507	2011
Analysis of the intergenic sequences provided by Feria-Arroyo et al. does not support the claim of high Borrelia burgdorferi tick infection rates in Texas and northeastern Mexico. Parasites & vectors\| PubMed ID: 25428816	2014
Response to Esteve-Gassent et al.: flaB sequences obtained from Texas PCR products are identical to the positive control strain Borrelia burgdorferi B31. Parasites & vectors\| PubMed ID: 26050617	2015
Catching up with Lyme Disease Antigenic Variation Computationally. Trends in microbiology\| PubMed ID: 29903419	2018
Long-Term Culture of the Syphilis Spirochete subsp. . mBio\| PubMed ID: 29946052	2018
Hiding in Plain Sight: Colonic Spirochetosis in Humans. Journal of bacteriology\| PubMed ID: 31405918	2019

Steven J. Norris

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