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Engineering of a minimal bacterial therapeutic chassis

Periodic Reporting for period 1 - MYCOCHASSIS (Engineering of a minimal bacterial therapeutic chassis)

Reporting period: 2015-11-01 to 2017-04-30

Biofilm production by infectious diseases is one of the biggest challenges in human health. Infections involving biofilm formation have been found in all parts of the body such as upper respiratory infections (Pseudomonas aeruginosa) urinary tract infections (UTIs) (uropathogenicEscherichia coli [UPEC], Klebsiella pneumoniae) periodontitis (mixed biofilms of Streptococcus mutans and other bacteria) catheter-induced and other device-associated infections (E. coli, Enterococcus faecalis, and others). It is now clear that many chronic infections result from biofilm formation. These chronic infections are resistant to antibiotic therapy and the innate, and adaptive immune and inflammatory response of the host. Inside the host, the matrix protects biofilm bacteria from exposure to innate immune defenses (such as opsonization and phagocytosis)\ and antibiotic treatments ((62)). Antibiotic tolerance in in vitro models have shown that biofilms can withstand treatment with very high dosages of antibiotics that are up to 1000 times the minimal inhibitory concentration. Although this could be in part due to matrix compounds that quench the antibiotic, as well as the existence of bacteria in different growing stages. As a result, biofilm-forming pathogens persist, establishing chronic and recalcitrant infections more especially in immunocompromised patients where infections by opportunistic biofilm-forming pathogens can lead in many instances to death. Also in the biofilm interbacterial interactions can through DNA exchange promote the spread of drug resistance markers and other virulence factors. The matrix of biofilms (eps) is generally composed of DNA, proteins, lipids and polysaccharides. While bacteria are currently in preclinical and clinical trials for treating different human diseases (see above), there is no current public project that contemplates their use to treat bacterial biofilms in lung infections. Current projects using living systems for these diseases are focused on bacteriophages.
Here we propose to rationally engineer the mildly human lung pathogen M. pneumoniae, for animal and human therapy that will deliver therapies to diseased human lungs.

Importance for society:
Current treatments of many diseases use chemical drugs. Those in many cases are effective but they are inert from the point of view that they cannot react to changes in the human body. On the other hand living systems like bacteria if harnessed, they could interact with the human physiology responding to the needs of the patient and delivering locally and continuously an active compound. Evidently, this is easier in human parts of the body open to the exterior, like mouth, nose, skin, digestive system, genital tracts, eyes and lungs where there is a reduced risk of general infection. Getting therapeutic bacteria could be a major step in personalized medicine and could allow targeting complex diseases which now are poorly treated

Overall Objectives:
Aim 1. Whole-cell modeling of the bacterium
Developing a whole-cell computational model based on organism-specific experimental data. This model will be used to minimize the genome and to make reliable predictions about the in vivo behavior of engineered components.
Aim 2. Engineering of the therapeutic chassis
Using the whole-cell model and deletion of non-essential genes we will create a non-pathogenic therapeutic chassis of M. pneumoniae with safety circuits to prevent uncontrolled growth in the host.
Aim3. Displaying proteins for lung and biofilm recognition and engineering of Biofilm dispersion capability
Using the therapeutic chassis we will engineer orthogonal gene circuits to secrete and dissolve in vitro biofilms caused by S. aureus and P. aeruginosa as well as do preliminary tests in mouse models.
During the reporting period we have developed the tools needed for the genetic manipulation of M. pneumoniae. We have developed a system for homologous recombination, a cre-lox system for removing antibiotic resistance, several inducible promoters and the CrispR. Using these tools we have engineered a chassis that is non-pathogenic on which to introduce therapeutic genes. We have also introduce different proteins that dissolve or kill S. aureus and/or P. aeruginosa biofilms and demonstrated that they work In Vitro and in mice models (collaboration with Navarra, Spain). We have also determined with high precision protein copy numbers, RNA copy numbers, protein half lifes, RNA half-lifes and metabolite concentrations and developed an ODE model (collaboration ) of metabolism. We also have done probably one of the most comprehensive analysis done so far about transcription regulation of a bacterium.
We have already a proof of concept engineered M. pneumoniae that can dissolve biofilms of S. aureus in mice. We are currently starting to contact venture capital and pharma companies to explore the possible commercialization of our engineered bacterium. We are also planning to apply for an ERC PoC.