Syphilis – Treponema pallidum, likely uses a single gene to escape the immune system


The bacterium that causes syphilisTreponema pallidum, likely uses a single gene to escape the immune system, research from UW Medicine in Seattle suggests.

The finding may help explain how syphilis can hide in the body for decades, thereby frustrating the immune system’s attempts to eradicate it.

It might also account for the bacterium’s ability to re-infect people who had been previously been infected and should have acquired some immunity to it.

Although syphilis remains easily treated with penicillin, infection rates in the United States have increased steadily over the past two decades. The count rose to more than 115,000 new U.S. cases of the infection in 2018.

Worldwide there are an estimated 6 million new cases of syphilis among adults. The infection is responsible for an estimated 300,000 fetal and neonatal deaths annually.

However, despite its importance as a cause of disease, relatively little is known about the biology of Treponema pallidum.

One reason for this is that until recently it was impossible to grow it in a laboratory dish. As a consequence, many of the laboratory tools used to study other bacteria had not been developed for syphilis specifically.

In a new study, researchers compared the genomes of syphilis bacteria collected from a man who had been infected four times. He was enrolled in a UW Medicine study of spinal fluid abnormalities in individuals with syphilis conducted by Dr. Christina Marra, professor of neurolgy.

The samples were derived from his blood during two infections that occurred six years apart. Between those infections he had been infected and treated two additional times.

The researchers wanted to see if there were differences between the genomes of bacteria from the first and last infection. This differences might reveal how the genes of the bacteria had changed and how those changes might have enabled the bacteria to infect a person whose immune system had already seen and mounted an immune response to several different strains of syphilis.

Surprisingly, the researchers found that there were very few changes between the genomes from the two different samples—except for one gene.

“Across the about 1.1 million bases that make up the bacteria’s genome there were about 20 changes total. That’s very low,” said Dr. Alex Greninger, assistant professor of laboratory medicine at the UW School of Medicine, who led the research project. “But on this one gene, we saw hundreds of changes.”

That gene, called Treponema pallidum repeat gene K (tprK), provides the instructions for the synthesis of a protein found on the surface of the bacterium. Proteins on the surface of a bacterium are typically more easily seen by immune cells and so are often prime targets for immune attack.

The study builds on decades of work from Drs. Sheila Lukehart and Arturo Centurion-Lara in the Department of Medicine at the University of Washington School of Medicine.

They first showed that TprK generated considerable diversity across seven discrete regions in which DNA sequences from elsewhere in the bacterium’s genome could be swapped in and out. This process is called gene conversion.

Work in their lab demonstrated that bacterial cells with new tprK variants can evade the immune response to cause a persistent infection that can lead to the later stages of syphilis.

Amin Addetia, a research scientist in Greninger’s lab and lead author on the study, said it was as though the bacterium has a deck of cards in its genome from which it can draw and deal to these variable regions, essentially changing the protein’s “hand.”

These substitutions change the protein’s appearance on the surface to allow it to elude the immune system.

“I’ve looked at a lot of bacterial genomes,” Addetia said, “and they’re a lot more interesting than the Treponema’s, except for this one gene.

It can generate an astounding number of diverse sequences within these variable regions without impairing the protein’s ability to function.”

Although bacteria, viruses and parasites may have many proteins on their surfaces that the immune system could detect and attack, in many cases only one protein seems to attract most of the attention. Such proteins are called immunodominant.

They may protect the bacterium by catching the immune system’s attention, Greninger said.

“The protein acts like a distraction that draws the immune system away from proteins that might be the bacterium’s Achilles heel. More work will be required to determine if this is the case in TprK.”

Greninger said he hoped the findings might help researchers develop vaccines that allow the immune system either to attack TprK more effectively or to ignore TprK and target other, less variable syphilis proteins.

Treponema pallidum subsp. pallidum (TPA) is the causative agent of syphilis, a globally occurring disease. Although the worldwide number of syphilis cases dramatically decreased after the introduction of penicillin therapy in the 1940s, the estimated number of new syphilis cases per year remains over 5.6 million.

Especially alarming is the number of congenital syphilis cases, which is approaching one million cases per year (Newman et al., 2012; Peeling et al., 2017). In developed countries, syphilis is often transmitted among MSM patients (men who have sex with men). Moreover, MSM patients with syphilis are often co-infected with HIV (42% in Western Europe) (Dubourg et al., 2015).

It is believed that syphilis facilitates the HIV infection, since syphilitic genital ulcers are infiltrated with lymphocytes (the primary target cells for HIV-infection) and provide a portal of entry for HIV acquisition.

The rising prevalence of syphilis among MSM patients has coincided with the introduction of highly active anti-retroviral drugs leading to decreased HIV-associated mortality and the re-emergence of unsafe sexual behavior among MSM (Stolte et al., 2001).

TPA infections are characterized by early and fast dissemination, immune evasion and long persistence in untreated patients. However, the underlying molecular mechanisms remain poorly understood (Radolf et al., 2016).

In spite of recent advances in in vitro cultivation of TPA (Edmondson et al., 2018), routine laboratory cultivation of this pathogen directly from patient samples is not yet possible.

Therefore, most of the information on TPA genetics comes from genome sequencing studies, where DNA was isolated from bacteria propagated in experimentally infected rabbits (Fraser et al., 1998; Matĕjková et al., 2008; Giacani et al., 2010, 2014; Pětrošová et al., 2012, 2013; Zobaníková et al., 2012; Tong et al., 2017).

The research community uses culture-independent enrichment techniques prior to whole genome sequencing of TPA clinical samples due to the overwhelming levels of human DNA and very low amounts of TPA DNA (1000:1 ratio of human to TPA DNA) found in clinical samples.

However, available enrichment techniques demonstrate low efficiency (e.g., Anti-treponemal antibody enrichment, ATAE) (Grillová et al., 2018b) or are based on sequence-specific protocols (e.g., DNA-capture microarray and “in solution” capture techniques) (Arora et al., 2016; Pinto et al., 2016; Knauf et al., 2018; Marks et al., 2018), thus preventing the recovery of unique sequences not present in the reference genomes.

Genetically, TPA can be divided into two separate groups – SS14-like and Nichols-like strains (Nechvátal et al., 2014; Arora et al., 2016). As revealed by molecular typing studies of TPA isolates, most of the examined patients were infected with SS14-like strains (94.1%) (Woznicová et al., 2007; Flasarová et al., 2012; Grillová et al., 2014, 2018c; Arora et al., 2016; Gallo Vaulet et al., 2017; Mikalová et al., 2017a; Pospíšilová et al., 2018). The reason for the predominance of one genetic group is widely discussed, but was not clarified yet (Arora et al., 2016; Šmajs et al., 2016).

In this study, we performed direct whole genome sequencing of 25 TPA clinical samples isolated from different geographical areas using methyl-directed enrichment prior to next generation sequencing (NGS) (Barnes et al., 2014). Using this approach, we obtained 11 complete genome sequences, which represents the vast majority (92%) of complete TPA genomes sequenced directly from clinical samples.

The subsequent detailed comparative genomic analyses revealed unexpected variability among Nichols-like genomes driven by inter-clade and/or intra-strain recombination events, which were accumulated mainly in the genes encoding predicted outer membrane proteins.

This discovery, beyond being relevant to the understanding of basic biology of treponemes, highlights the presence of different repertoires of alleles coding for potential virulence factors, which circulate in the current human population.

More information: Amin Addetia et al, Comparative genomics and full-length Tprk profiling of Treponema pallidum subsp. pallidum reinfection, PLOS Neglected Tropical Diseases (2020). DOI: 10.1371/journal.pntd.0007921


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