The World’s exponentially growing population is becoming a threat to mankind’s sustainable life on the planet. The United Nations (UN) reports that if fertility remains constant in each country at the level of the 2005-2010 period, the world population could reach nearly 27 billion by 2100, whereby the population of the least developed countries is projected to triple by the end of the century. This would be an outcome unlikely to be sustainable.
To encompass a reduced world population growth several programs were launched by international public and private actors such as the Bill and Melinda Gates foundation, various national governments, the WHO and others. Amongst educational programs about family planning and the distribution of condoms, research for new methods of contraception is ongoing.
State of the art contraception methods are often unaffordable for poor families in lesser developed countries and unintended pregnancy comes along with significant social and financial costs. “Bacterial Contraception” scenario suggests a new method of contraception to mitigate those problems. Edible probiotic bacteria that naturally populate the vagina could be utilized to act as an inperceptible, noninvasive and nonsystemic biological contraceptive for women.
Contraception could be achieved by genetically reprogramming those bacteria to biologically immobilize, disorient and kill spermatozoa before they reach the uterus. Such a method could overcome practical problems like the distribution of contraceptives, as living bacteria can be reproduced on a local basis. It might also help to overcome cultural barriers which often prevent the use of physical contraceptives like condoms, since an edible contraception does not require special attention during sexual intercourse. This scenario should help in the practical and theoretical exploration addressing this idea of contraception.
The development of a bacterial contraceptive method for women requires a detailed understanding of the anatomy and physiology of the female reproductive tract, especially of the vagina and cervix and its secretions. The schematic provides a brief overview of human reproduction physiology in the female reproductive tract which could be targeted by bacterial contraception.
In principal, it seems possible to use bacteria to prevent spermatozoa from progressing into the uterus and / or to render them incapable of fertilization. Native vaginal symbiotic bacteria (such as Lactobacillus lactis) could be genetically engineered to express and secrete proteins to interfere with spermatozoa chemotaxis to disorient, alter chemokinesis and may eventually lead to their death.
The chemoattractant proteins could create a gradient with a high concentration in the vaginal environment, decreasing across the cervical canal towards the uterus. Chemotactically active spermatozoa would be directed away from the uterus, capacitate prematurely or die off and thus decrease the chance of fertilization of the oocyte. Other mechanisms, such as secreted proteins altering the mucus consistency in the cervical canal to block the passage of spermatozoa, as well as immunological functions, e.g. expression of antibodies against diseases are also thinkable.
To address how a genetic construct that provides the desired features could look like, a draft design is shown here. Growth of Lactobacillus lactis was tested in vaginal-fluid simulation experiments and found to be potentially suitable as a model organism. Furthermore, there is a plethora of commercial strains and methods available, mostly developed for dairy industries. This pool of knowledge can be expected to considerably accelerate the development of a prototype for bacterial-based contraception in future studies.
Above: Hypothetical bacterial contraception product: Symbiotic Lactobacilli or Lactococci are genetically modified with a plasmid including a resistance gene for increased fitness to survive in a natural environment and an expression cassette for production and secretion of chemoattractant proteins to intercept spermatozoa in the female reproductive tract. The bacteria could be directly applied in the vaginal environment or, for example enriched in yoghurt and ingested by women. The natural passage through the digestive tract into the vagina could be used as a mode of enriching the modified bacteria in the vaginal environment.
The population of modified bacteria could be expanded in the vagina based on resistance genes of food-grade lantibiotics, which they can produce themselves. This would reduce growth of competing naturally occurring strains and give the genetically altered ones a selective advantage. A commercially available, well known food-grade lantibiotic-resistance construct is the NICE vector. The NICE system was developed in the early 1990ies and the patent will expire soon.
The expression of the lantibiotic nisin could be easily and stably integrated into the genome of the lactic acid bacteria – without the need for a marker – via CRISPR-mediated bacterial genome engineering ( see, e.g., http://aem.asm.org/content/81/13/4423.full), in order to establish a stable concentration of nisin in the culture. A widely used strain is L. lactis NZ9000, a modified MG1363 strain was subject of earlier studies. The strain could be engineered to contain a stably integrated, , designed expression cassette in a plasmid– for example based on the commercially available pNZ8123, which contains the nisin-inducible promotor Pnis. The chemoattractant to distort the sperm chemotaxis and/or chemokinesis could be cloned into a plasmid containing the open reading frame (ORF) under the control of the Pnis promotor as well as homology arms for CRISPR-mediated integration. Plasmids that could be used as basis for this purpose have been developed for L. lactis but has also successfully been used in Lactobacilli. Other lantibiotics such as lactocin or bacteriocin HV219 might be considered as alternatives to nisin.
To get the bacterium to secrete the chemoattractant, an export leader peptide and a short peptide linker needs to be fused to the N-terminus of the protein. For this, the recently published SP310mut2 leader in combination with the LEISS linker may be used.In a further plasmid that could be used for CRISPR-mediated genome integration , an ORF of the nisin resistance gene would be controlled by a constitutive promotor.
The genes are flanked by start and stop codons and have a ribosome binding site (RBS) upstream and a terminator downstream of the gene. The RBS could be used in future studies to adjust the expression strength and thus to stabilize the concentration of the expressed chemoattractant. The Pnis controlled expression of the chemoattractant would only allow engineered bacteria to start stronger protein expression when there is a certain concentration of nisin in the vaginal fluid diminishing competing non-resistant bacteria. This would help establishing a stable population of the strain, making it easier to adjust the concentration of chemoattractants in the vaginal fluid.
Above: hypothetical contraception expression cassettes for Lactococcus lactis or vaginal symbiotic Lactobacilli. Pnis Promoter: a nisin-inducible promotor. RBS: Ribosome Binding Site. Start and Stop → codons defining the open reading frame. SP310: export leader sequence. LEISS: artificial peptide linker. Chemoattractant: the genetic sequence coding for a protein for sperm chemoattractant. Nisin Resistance: Gene coding for a protein which provides resistance against the lantibiotic nisin. Terminator: mRNA sequence forming a secondary structure to terminate translation.
Ideally, the final product could consist of a yoghurt, which women ingest in regular intervals, or a cream or pill to be applied directly in the vagina. In case of ingestion, the bacteria would travel through the intestinal system and eventually reach from the anus into the vagina – a naturally occurring phenomenon. In the vaginal environment they would then start expressing the relevant genes which help preventing pregnancy.
Besides the effect of contraception, a very promising use of transgenic vaginal symbiotic bacteria may be to deliver immunity-promoting mechanisms to fight sexually transmitted diseases (STD). There is already at least one ongoing clinical study in trial in phase I using transgenic Lactobacilli. In future, the various described applications for symbiotic bacteria could be combined in different products. Women could choose from the variety of functions depending on personal needs.
Apart from the practical considerations discussed above, such as how the product could work and how to realize it, there is a need to address further questions: Who is going to use it? How will the idea of transgenic bacteria for contraception be perceived? What is the societal impact of the method? And how can an effective distribution to any woman in need of such contraception be guaranteed? Furthermore, legal questions concerning the use of genetically modified organisms need to be considered. Ideally, the development of such a contraceptive will be conducted in a worldwide, non-profit cooperation between scientists, state and private actors. Thus, the concept could be developed quicker and in greater detail, and accustomed to local needs. Moreover, a safe and fair use of the method might be easier to realize so that any person in demand could benefit from it.