Blocking the ability of the bacterial pathogen that causes gonorrhea to uptake the mineral zinc can stop infection

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Blocking the ability of the bacterial pathogen that causes gonorrhea to uptake the mineral zinc can stop infection by this widespread sexually transmitted infection, according to a study by the Institute for Biomedical Sciences at Georgia State University.

The findings, published in the journal PLoS Pathogens, could be used to move gonorrhea vaccine development forward because they provide insight into how to block growth of this pathogen.

No vaccine has been developed to prevent this serious infection.

There are more than 820,000 new cases of gonorrhea each year in the United States, and more than half of these cases are estimated to be among young people 15 to 24 years old, according to the Centers for Disease Control and Prevention.

Neisseria gonorrhoeae, the pathogen that causes gonorrhea, is considered to be an urgent threat because it has developed resistance to nearly every drug used for treatment, making it difficult to cure.

Untreated gonorrhea can cause severe and permanent health problems in women and men, and most women don’t have symptoms.

In women, untreated gonorrhea can cause pelvic inflammatory disease, which leads to infertility and ectopic pregnancies, life-threatening pregnancies that occur when a fertilized egg implants outside the womb.

“Our results are significant because N. gonorrhoeae will die if it can’t get enough zinc,” said Dr. Cynthia Nau Cornelissen, senior author of the study and director of the Center for Translational Immunology in the Institute for Biomedical Sciences, professor in the Institute for Biomedical Sciences and a Distinguished University Professor at Georgia State.

“The study suggests a way to halt the growth of this sexually transmitted infection in the host’s body.”

N. gonorrhoeae is challenging to prevent and treat because it has an arsenal of conserved outer membrane proteins that allow the pathogen to overcome nutritional immunity, the host’s strategy for sequestering essential nutrients away from invading bacteria and handicapping their infectious ability.

The bacterial pathogen produces eight known outer membrane transporters.

Four are used for the acquisition of iron or iron chelates.

Two of the remaining transporters, TdfH and TdfJ, facilitate zinc uptake.

In previous work, Cornelissen’s group has shown that TdfH binds to Calprotectin (a member of the S100 protein family) and extracts its zinc, which is then internalized by N. gonorrhoeae.

This study sought to evaluate whether other proteins in the S100 family have the ability to support gonococcal growth by zinc acquisition.

Cornelissen’s group found that TdfJ binds directly to S100A7, from which it internalizes zinc.

This interaction is only detected with the human version of S100A7, which is significant because gonorrhea infection only occurs in humans, and thus the ability to use only human S100A7 contributes to the species specificity of the pathogen.

This study shows that N. gonorrhoeae overpowers human nutritional immunity through the interaction between TdfJ and human S100A7, allowing the pathogen to overcome the host’s zinc restriction and continue to grow.

With Cornelissen’s new $9.25 million grant from the National Institutes of Health’s National Institute of Allergy and Infectious Diseases, she will use the findings from this study and previous studies on zinc uptake in N. gonorrhoeae to develop a vaccine to protect against the pathogen.

Using an approach called “starve and kill,” she will attempt to starve N. gonorrhoeae of the necessary nutrients (zinc and iron) to prevent it from growing and potentially even infecting the host.

Cornelissen aims to eventually develop a vaccine that blocks uptake of both iron and zinc by N. gonorrhoeae and fully protects the host against this bacterial pathogen.


What is gonorrhea and how is it transmitted?

Gonorrhea is a disease caused by a bacteria called Neisseria gonorrhea.

It is transmitted via vaginal, anal or oral sex. It is also transmitted from skin-to-skin contact with infected genitals, even if there is no penetration, orgasm or ejaculation.

It is not communicated by casual personal contact, however, or by contact with any object touched or used by an infected person, as the bacteria cannot live for long outside the human body.

It can be passed by a pregnant woman to her baby during a vaginal birth (not a C-section) and most commonly affects the eyes of babies infected with the bacteria. Gonorrhea can cause blindness and other serious health problems for newborns, so testing for and treating an infection during pregnancy is preferable.

How prevalent is gonorrhea? Is there a way to avoid it?

According to the World Health Organization (WHO), 500 million new cases of one of four curable STIs (chlamydia, gonorrhea, syphilis and trichomoniasis) occur each year worldwide. In India, estimates place the prevalence of gonorrhea at 6% of the population (that’s 1 in every 17 people), though it may be more common, as many cases of gonorrhea go undiagnosed.

Gonorrhea, like most STIs, tends to be more prevalent in urban areas, and among young adults, who are more likely to be sexually active with multiple partners without using protection — all risk factors for STIs.

That said, anyone who has sex with someone with a gonorrhea infection can catch gonorrhea. Using condoms or dental dams (a piece of thin, soft plastic or latex) are the best preventative methods, though they are not guarantees against the infection.

What are the symptoms of gonorrhea?

Gonorrhea can infect the cervix, anus, penis, throat or eye, depending on what part of the body was exposed to the bacteria. Infections often don’t develop noticeable symptoms; this is especially true for women. When an infection does manifest symptoms, signs of gonorrhea vary between men and women.

In men, gonorrhea symptoms may include:

  • white, yellow or green pus-like discharge from the penis tip
  • pain or burning during urination
  • a painful or swollen testicle(s)

In women, gonorrhea symptoms may include:

  • more vaginal discharge than usual
  • bleeding outside the menstrual cycle
  • pain or burning during urination
  • pelvic or abdominal pain
  • pain during sex

In both men and women, depending on the location of infection, gonorrhea symptoms may include:

  • a sore throat and swollen lymph nodes
  • an itchy anus, a sore anus, pus-like discharge from the rectum, and straining, pain and/or blood spotting during bowel movements
  • eye sensitivity to light, eye pain, and pus-like discharge from the eye(s)

An untreated gonorrhea infection can spread to your joints, a complication known as gonococcal arthritis; symptoms of this gonorrhea complication include: warm, stiff, swollen and painful joints, especially during movement. Similarly, the infection can also spread to the blood and other body parts and cause rashes or skin sores.

An untreated gonorrhea infection can cause fertility problems for both men and women. In men, epididymitis, that is, an inflammation of a portion of the sperm ducts inside the testicles, might be a symptom of an untreated gonorrhea infection. In women, an untreated gonorrhea infection can spread to the the upper reproductive tract and develop into pelvic inflammatory disease (PID). Both of these painful conditions can damage fertility.

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Worldwide, over 78 million people are estimated to acquire the sexually transmitted infection gonorrhea every year (1). Female reproductive health is disproportionately affected by this disease and half of infected women show no symptoms (24).

Serious health consequences are associated with untreated or insufficiently treated gonorrhea, including pelvic inflammatory disease, inflammation of the fallopian tubes, pre-term delivery, miscarriage, or ectopic pregnancy (57).

Additionally, infants born vaginally to infected mothers are exposed to the disease in the birth canal and are thus at risk of developing a sight-threatening conjunctivitis (8).

Although the health consequences to men are not as severe as for women and predominantly manifest as uncomplicated urethritis accompanied by a neutrophil-rich exudate (69), gonorrhea can ascend to the epididymis or the testes and may require surgical removal of the infected site (1012). Infertility can occur in both females and males without proper treatment (913).

The bacterium responsible for gonorrhea, Neisseria gonorrhoeae (Ng), is a highly adaptable pathogen.

Its natural competence and plastic genome have contributed to the extensive spread of antibiotic resistance.

Through a number of horizontally acquired genes and chromosomal mutations, Ng has become resistant to every antibiotic used for its treatment (1416).

The Centers for Disease Control and Prevention (CDC) in the United States currently recommend a dual therapy of intramuscular ceftriaxone combined with oral azithromycin as a first-line treatment for uncomplicated gonorrhea (1718).

However, the first isolates resistant to this combination therapy have begun to emerge (19).

Three new therapeutics for gonorrhea treatment are being evaluated in clinical trials (20), but considering the speed with which the gonococcus develops antibiotic resistance (15), new drugs will not provide a long-term solution.

The development and introduction of a protective vaccine against gonorrhea should therefore be prioritized to limit its spread.

Thus far, only two gonorrhea vaccines, using either killed whole organisms or purified pilin protein, have progressed to clinical trials.

Despite robust antibody responses in both trials, neither vaccine provided protection against acquiring the disease after immunization (2124).

These failures are likely due to a number of factors.

Pilin proteins undergo extensive antigenic variation through frequent recombination with transcriptionally silent pilS gene cassettes (2528).

Experimental infections have demonstrated that multiple pilin variants are isolated from a single individual, and that these variants are antigenically distinct from the inoculating parent strain (2931).

Further, pilin proteins are subjected to phase variation, where protein expression transitions between “on” and “off” states through slipped-strand repair of upstream repeat regions (32).

Antigenic and phase variation of pilin during infection likely contributed to the failure of both vaccine trials.

Another factor that may have led to the whole cell vaccine’s inability to protect from infection is the presence of the reduction modifiable protein (Rmp; also known as protein III) in the vaccine.

Localized to the outer membrane, Rmp is highly conserved and immunogenic, yet antibodies induced by this antigen actively prevent assembly of the complement membrane attack complex in immune serum (3334).

These challenges illustrated the necessity for new approaches in gonorrhea vaccine development.

In the intervening years, vaccine progress has been slow. One of the difficulties is that Nginfection rarely, if ever, leads to an adaptive immune response (3538).

For this reason, mechanisms of protection against gonorrhea are unknown (24), which makes the evaluation of the potential efficacy of vaccine candidates prior to expensive immunization studies challenging.

The serum bactericidal activity of antibodies generated during an immune response strongly predicts protection for vaccines against N. meningitidis [Nm; (3940)], a frequent causative agent of meningitis, so the ability of gonococcal antigens to elicit bactericidal antibodies is currently used as a surrogate mechanism of protection (41).

Based on this criterion, 14 Ng antigens with functions in colonization and invasion, nutrient acquisition, and immune evasion have been proposed for inclusion in a gonorrhea vaccine [reviewed in (41)].

Immunization with each of the candidate proteins, cyclic loop peptides, or lipooligosaccharide epitope mimics elicited bactericidal antibodies, although studies for seven of the antigens were performed only in Nm(41).

Despite the difficulties in developing a vaccine against gonorrhea, several recent advances suggest that a protective vaccine is now within reach.

The first was the development of a female mouse model of lower genital tract infection, in which mice are treated with 17-β estradiol and a cocktail of antibiotics to increase susceptibility to Ng and to reduce the overgrowth of vaginal commensal bacteria, respectively (42).

This model has enabled the study of the immune response to gonococcal infection in a whole organism for which extensive genetic and immunological tools are available (244344).

A series of elegant studies, combining information gathered from experimental murine infections and tissue culture experiments, demonstrated Ng actively suppresses the generation of a productive adaptive immune response.

Both mouse splenic mononuclear cells and human dendritic cells infected with Ng produced elevated levels of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-1β, and IL-23, a set of cytokines that promote terminal differentiation of T-cells toward T helper 17 (Th17) cells (4546).

Production of IL-17 is a characteristic marker of a Th17 response and promotes neutrophil recruitment through the induction of granulocyte-colony stimulating factor and chemokines (45).

In support of gonorrhea promoting Th17 differentiation during an active infection, elevated levels of IL-17 were discovered in female mice challenged with Ng (46). Gonococci are also able to divert T-cell differentiation away from an adaptive Th1/Th2 response by inducing the production of transforming growth factor (TGF)-β and IL-10 (4749).

Furthermore, Ng stimulates the differentiation of macrophages toward a regulatory phenotype and prevents macrophage antigen display.

Through these immunosuppressive activities, the gonococcus is able to further inhibit the generation of a protective T-cell response (5051).

The knowledge gained through these studies into the sophisticated methods Ng uses to promote its own survival and prevent triggering an adaptive immune response will enable a more informed strategy for vaccine development and help avoid the failures of the past.

In studies making use of the insights gathered from a better knowledge of the immune evasion strategies employed by the gonococcus, mice treated intravaginally with micro-encapsulated IL-12 and either infected with a common laboratory strain, FA1090, or immunized with membrane vesicles (MVs) collected from FA1090 were protected against subsequent infections up to 6 months after the initial treatment, even when challenged with heterologous strains (5253).

IL-12 treatment promoted a Th1 response, as well as enhancing serum immunoglobulin A (IgA) and vaginal IgA and IgG levels (54).

Lessons can also be learned from the successful development of the licensed four-component Nm serogroup B vaccine, 4CMenB (BEXSERO; GlaxoSmithKline).

This bacterium presented a daunting vaccination challenge for a number of years due to the polysaccharide capsule surrounding group B meningococci, which is structurally identical to the polysialic acid carbohydrate found on the surface of many human cells. Because of this similarity, immunization with the group B capsule is minimally immunogenic and/or may result in the generation of autoantibodies (55).

To circumvent this problem, a subunit vaccine was developed by identifying conserved open reading frames in the whole genome sequence of Nm serogroup B, a strategy termed reverse vaccinology (5559).

Out of nearly 600 vaccine candidates identified with this strategy, 350 were successfully expressed and purified from Escherichia coli, 28 elicited bactericidal antibodies, and only three recombinant proteins—two of which are composed of a fusion of two proteins—were combined with MVs to formulate 4CMenB (5960).

Finally, a retrospective study found that immunization with another Nm serogroup B vaccine, MeNZB, containing the same MVs as 4CMenB, was 31% effective at preventing gonorrhea (6162). The MeNZB vaccine is no longer available, but these seminal studies provide strong evidence that a protective gonorrhea vaccine is possible.

A comparison of the number of antigens evaluated for the serogroup B vaccine with the number currently being investigated for a gonorrhea vaccine illustrates how far gonorrhea research lags behind meningitis research and emphasizes that new strategies are necessary to increase the pool of vaccine candidates under consideration.

An innovative way to address this gap was to perform reverse vaccinology antigen mining using subcellular fractionation coupled with high-throughput quantitative proteomics followed by bioinformatics (6364), which identified numerous stably expressed proteins and suggested that formulation of a subunit vaccine against gonorrhea would be successful.

Both genome- and proteome-based reverse vaccinology approaches have become more prevalent since the technique was introduced (56). Candidate vaccine antigens have been identified through whole-genome screens of a number of medically important pathogens (6569).

As the availability of bacterial genome sequences has increased, more detailed analyses have become possible, including comparative genomics.

One weakness of using whole genome sequences to search for vaccine candidate antigens is that the pathogens do not necessarily express the proteins discovered through this approach.

Transcriptome analysis provides a way to circumvent this limitation but a low correlation between transcriptomic and proteomic data has been well established [reviewed in (70)]. For this reason, we have chosen to pursue proteomic-based reverse vaccinology, as proteomic studies reveal the biologically relevant population of proteins expressed during exposure to the conditions under examination (636471).

Proteomic approaches also have the potential to specifically identify surface-exposed proteins without the need for extensive bioinformatic predictions (72).

In this article, we provide an overview of proteomic and bioinformatic approaches that have been utilized for gonorrhea antigen mining.

Our focus will also be on functional and structural characterization of proteome-derived antigens to determine their role in gonococcal pathogenesis and physiology as well as to inform the development of next generation vaccines based on structural vaccinology.


More information: Stavros Maurakis et al, The Novel Interaction Between Neisseria gonorrhoeae TdfJ and Human S100A7 Allows Gonococci to Subvert Host Zinc Restriction, PLOS Pathogens (2019). DOI: 10.1371/journal.ppat.1007937

Journal information: PLoS Pathogens
Provided by Georgia State University

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