T. Pallidum

''Treponema pallidum'', formerly known as ''Spirochaeta pallida'', is a microaerophilic, gram-negative, spirochaete bacterium with subspecies that cause the diseases syphilis, bejel (also known as endemic syphilis), and yaws. It is known to be transmitted only among humans and baboons. ''T. pallidum'' can enter the host through mucosal membranes or open lesions in the skin and is primarily spread through sexual contact. It is a helically coiled microorganism usually 6–15 μm long and 0.1–0.2 μm wide. ''T. pallidum'''s lack of both a tricarboxylic acid cycle and processes for oxidative phosphorylation results in minimal metabolic activity. As a chemoorganoheterotroph, ''Treponema pallidum'' is an obligate parasite that acquires its glucose carbon source from its host. Glucose can be used not only as a primary carbon source but also in glycolytic mechanisms to generate ATP needed to power the bacterium given its minimal genome. The treponemes have cytoplasmic and outer membranes. Using light microscopy, treponemes are visible only by using dark-field illumination. ''T. pallidum'' consists of three subspecies, ''T. p. pallidum, T. p. endemicum,'' and ''T. p. pertenue,'' each of which has a distinct related disorder. The ability of ''T. pallidum'' to avoid host immune defenses has allowed for stealth pathogenicity. The unique outer membrane structure and minimal expression of surface proteins of ''T. pallidum'' has made vaccine development difficult. ''Treponema pallidum'' can be treated with high efficacy by antibiotics that inhibit bacterial cell wall synthesis such as the beta-lactam antimicrobial penicillin-G.

Subspecies

Three subspecies of ''T. pallidum'' are known:
* ''Treponema pallidum pallidum'', which causes syphilis
* ''T. p. endemicum'', which causes bejel or endemic syphilis
* ''T. p. pertenue'', which causes yaws

The three subspecies causing yaws, bejel, and syphilis are morphologically and serologically indistinguishable. The three subspecies can be distinguished by genetics, using restriction fragment length polymorphism (RFLP), which utilizes techniques such as PCR, restriction digest and gel electrophoresis. Genes ''TprC'', ''TprI'', and the 5' flanking region of ''tpp15'' can be used to differentiate between the three subspecies based on DNA fragment lengths and location of bands in gel electrophoresis. These bacteria were originally classified as members of separate species, but DNA hybridization analysis indicates they are members of the same species. ''Treponema carateum'', the cause of pinta, remains a separate species because no isolate is available for DNA analysis. Disease transmittance in subspecies ''T. p. endemicum'' and ''T. p. pertenue'' is considered non-venereal. ''T. p. pallidum'' is the most invasive pathogenic subspecies, while ''T. carateum'' is the least invasive of the species. ''T. p. endemicum'' and ''T. p. pertenue'' are intermediately invasive.

Laboratory identification

''Treponema pallidum'' was first microscopically identified in syphilitic chancres by Fritz Schaudinn and Erich Hoffmann at the Charité in Berlin in 1905. Historically, this bacterium was identified in the clinical laboratory through visualization in dark field microscopy. This bacterium can be detected with special stains, such as the Dieterle stain. ''T. pallidum'' is also detected by serology, including nontreponemal VDRL, rapid plasma reagin, treponemal antibody tests (FTA-ABS), ''T. pallidum'' immobilization reaction, and syphilis TPHA test.

Microbiology

Physiology

''Treponema pallidum'' is a helically shaped bacterium with high motility consisting of an outer membrane, peptidoglycan layer, inner membrane, protoplasmic cylinder, and periplasmic space. It is often described as gram-negative, but its outer membrane lacks lipopolysaccharide, which is found in the outer membrane of other gram-negative bacteria. It has an endoflagellum (periplasmic flagellum) consisting of four main polypeptides, a core structure, and a sheath. The flagellum is located within the periplasmic space and wraps around the protoplasmic cylinder. The peptidoglycan layer interacts with the endoflagellum which may aid in motility. ''T. pallidum'''s outer membrane has the most contact with host cells and contains few transmembrane proteins, limiting antigenicity, while its cytoplasmic membrane is covered in lipoproteins. The outer membrane's treponemal ligands' main function is attachment to host cells, with functional and antigenic relatedness between ligands. The genus ''Treponema'' has ribbons of cytoskeletal cytoplasmic filaments that run the length of the cell just underneath the cytoplasmic membrane.

Outer membrane

The outer membrane (OM) of ''T. pallidum'' has several features that have made it historically difficult to research. These include details such as its low protein content, its fragility, and that it contains fewer gene sequences related to other gram negative outer membranes. Progress has been made using genomic sequencing and advanced computational models. The treponemal outer membrane proteins are key factors for the bacterium's pathogenesis, persistence, and immune evasion strategies. The relatively low protein content prevents antigen recognition by the immune system and the proteins that do exist protrude out of the OM, enabling its interaction with the host. ''Treponema's'' reputation as a "stealth pathogen" is primarily due to this unique OM structure, which serves to evade immune detection.

=TP0126

=

The TP0126 protein has been linked to the outer membrane protein family (OMP). This protein will sit in the outer membrane like a porin, which is supported by circular dichroism recombinant TP0126, and will increase the virulence factor. Researchers have classified the TP0126 protein in this class due to the homology between the protein and the porins of the OMPs. This protein is encoded by the tp0126 gene, which is conserved over all strains of ''T. pallidum.''

= TP0326

=
TP0326 is an ortholog of the β-barrel assembly machine Bam A. BamA apparatus inserts newly synthetized and exported outer membrane proteins into the outer membrane.

= TP0453

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TP0453 is a 287 amino acid protein associated with the inner membrane of the microbe's outer membrane. This protein lacks the extensive beta sheet structure that is characteristic of other membrane proteins, and does not traverse the outer membrane. This protein's function has been hypothesized to be involved with control of nutrient uptake.

= TP0624

=
Outer Membrane Protein A (OmpA) domain-containing proteins are necessary for maintaining structural integrity in gram-negative bacteria. These domains contain peptidoglycan binding sites which creates a "structural bridge between the peptidoglycan layer and the outer memebrane." The protein TP0624 found in ''T.'' ''pallidum'' has been proposed to facilitate this structural link, as well as interactions between outer membrane proteins and corresponding domains on the thin peptidoglycan layer.

=TP0751

=

The TP0751 protein is a protein that is unique to ''T. pallidum'', and it is thought to aid in attachment to the host's extra cellular membrane. Since this protein aids in the attachment to the host, it sits on the surface of the cells, and in 2005, it was discovered that the TP0751 protein will attach to the laminin component in the host's extracellular matrix. With that, it is thought that the TP0751 protein plays a key role in dissemination with the host.

= TP0965

=
TP0965 is a protein that is critical for membrane fusion in ''T. pallidum'', and is located in the periplasm. TP0965 causes endothelial barrier dysfunction, a hallmark of late-stage pathogenesis of syphilis. It does this by reducing the expression of tight junction proteins, which in turn increases the expression of adhesion molecules and endothelial cell permeability, which eventually leads to disruption of the endothelial layer.

Treponema repeat family of proteins

The ''Treponema'' repeat family of proteins (Tpr) are proteins expressed during the infection process. Tprs are formed by a conserved N-terminal domain, an amino-terminal stretch of about 50 amino acids, a central variable region, and a conserved C-terminal domain. The many different types of Tpr include TprA, TprB, TprC, TprD, and TprE, but variability of TprK is the most relevant due to the immune escape characteristics it allows.

Antigen variation in TprK is regulated by gene conversion. In this way, fragments of the seven variable regions (V1–V7), by nonreciprocal recombination, present in TprK and the 53 donor sites of TprD can be combined to produce new structured sequences. TprK antigen variation can help ''T. pallidum'' to evade a strong host immune reaction and can also allow the reinfection of individuals. This is possible because the newly structured proteins can avoid antibody-specific recognition. This is possible because the newly structured proteins can avoid antibody-specific recognition. It is also suspected that the genes that encode for the TprK protein are essential in pathogenesis during the infection of syphilis.

To introduce more phenotypic diversity, ''T. pallidum'' may undergo phase variation. This process mainly happens in TprF, TprI, TprG, TprJ, and TprL, and it consists of a reversible expansion or contraction of polymeric repeats. These size variations can help the bacterium to quickly adapt to its microenvironment, dodge immune response, or even increase affinity to its host.

Culture

In the past century since its initial discovery, culturing the bacteria in vitro has been difficult. Without the ability to grow and maintain the bacteria in a laboratory setting, discoveries regarding its metabolism and antimicrobial sensitivity were greatly impaired. However, successful long-term cultivation of ''T.'' ''pallidum'' in vitro was reported in 2017. This was achieved using Sf1Ep epithelial cells from rabbits, which were a necessary condition for the continued multiplication and survival of the system. The medium TpCM-2 was used, an alteration of more simple media which previously only yielded a few weeks of culture growth. This success was the result of switching out minimal essential medium (MEM) with CMRL 1066, a complex tissue culture medium. With development, new discoveries about ''T.'' ''pallidum'''s requirements for growth and gene expression may occur and in turn, yield research beneficial for the treatment and prevention of syphilis, outside of a host. However, continuous efforts to grow ''T. pallidum'' in axenic culture have been unsuccessful, indicating that it does not satisfy Koch's postulates. The challenge likely stems from the organism's strong adaptation to residing in mammalian tissue, resulting in a reduced genome and significant impairments in metabolic and biosynthetic functions.

Genome

The genome of ''T. pallidum'' was first sequenced in 1998. It is characterized by its helical, corkscrew-like shape. ''T. pallidum'' is not obtainable in a pure culture, meaning that this sequencing played an important role in filling gaps of understanding regarding the microbes' functions. The DNA sequences of ''T. pallidum'' species are more than 99.7% identical, and PCR-based assays are effective at differentiating these species. About 92.9% of DNA was determined to be open reading frames, 55% of which had predicted biological functions. ''T. pallidum'' was found to rely on its host for many molecules typically provided by biosynthetic pathways, and it is missing genes responsible for encoding key enzymes in oxidative phosphorylation and the tricarboxylic acid cycle. The ''T. pallidum'' group and its reduced genome is likely the result of various adaptations, such that it no longer contains the ability to synthesize fatty acids, nucleic acids, and amino acids, instead relying on its mammalian hosts for these materials. The recent sequencing of the genomes of several spirochetes permits a thorough analysis of the similarities and differences within this bacterial phylum and within the species. The chromosomes of the ''T. pallidum'' species are small, about 1.14 Mbp. It has one of the smallest bacterial genomes and has limited metabolic capabilities, reflecting its adaptation through genome reduction to the rich environment of mammalian tissue. It conserves almost 99.8% of its small genome, and uses its constantly mutating protein TprK to avoid immune response from its host. To avoid antibodies attacking it, the cell has few proteins exposed on the outer membrane sheath. Its chromosome is about 1000 kilobase pairs and is circular with a 52.8% G + C average. Sequencing has revealed a bundle of 12 proteins and some putative hemolysins are potential virulence factors of ''T. pallidum.'' These virulence factors are thought to contribute to the bacterium's ability to evade the immune system and cause disease.

Clinical significance

The clinical features of syphilis, yaws, and bejel occur in multiple stages that affect the skin. The skin lesions observed in the early stage last for weeks or months. The skin lesions are highly infectious, and the spirochetes in the lesions are transmitted by direct contact. The lesions regress as the immune response develops against ''T. pallidum''. The latent stage that results can last a lifetime in many cases. In a few cases, the disease exits latency and enters a tertiary phase, in which destructive lesions of skin, bone, and cartilage ensue. Unlike yaws and bejels, syphilis in its tertiary stage often affects the heart, eyes, and nervous system, as well.

Syphilis

''Treponema pallidum pallidum'' is a motile spirochete that is generally acquired by close sexual contact, entering the host via breaches in squamous or columnar epithelium. The organism can also be transmitted to a fetus by transplacental