Team:Hamburg/Biochemistry
Biochemistry
Nor-Pyochelin
Nor-Pyochelin is a chemical derivate of pyochelin, which lacks a single methyl group on its second cyclized cysteine residue. Based on the work of Abdallah et. al., which included a detailed analysis of pyochelin and a couple of similar compounds. We figured that Nor-Pyochelin would outcompete native pyochelin as a potential antibiotic carrier molecule since Abdallah et. Al. measured a 30% higher receptor affinity and iron transport capability for Nor-Pyochelin. An effect that we wanted to replicate in our antibiotic efficiency study.
Pyochelin
At the beginning of our project we decided that we wanted to do something about multiresistant bacteria that cause lung infections. Therefore, pyochelin, one of two native siderophores of Pseudomonas aeruginosa, became one of our prime targets for bio- and chemical synthesis, as P. aeruginosa is known for its manifold antibiotic resistances and oppurtunistic lung infections. Therefore P. aeruginosa poses a big problem in hospitals as one of the main reasons for opportunistic infections in weakened or immunosuppressed patients. Being extremely resilient and undemanding, pseudomonads are known to survive in hostile environments, like the tubes of breathing devices and even disinfectants, it is hard to prevent pseudomonade infections. Since Pyochelin is relatively small and easy to synthesize, and pseudomonas is one of the most resilient bacteria known, we chose pyochelin as our main target for our trojan horse strategy.
However, fighting pseudomonas with a gallium complex with its native siderophore wasn’t enough. Additionally, during the biobricks design for the pyochelin synthesis we found out that pseudomonas gene sequences consist of a very high GC-content which proved to be very difficult for synthesis and other molecular manipulation. Therefore, we had to design our project towards a similar but different approach. The new target became Nor-Pyochelin.
Siderophores
Siderophores are a class of small peptides originating in bacteria. In the next couple of years siderophores will reenter the spotlight of medical research again after a short period in the 90’s where they were a molecule of interest to find a solution for multiresistant bacteria.
However, till today most siderophores are still poorly researched and more and more basic functions of these very versatile peptides become revealed as more and more work groups around the world jump onto the siderophore train. The main aspect why we and most other groups are interested in siderophores is their key ability: Metal-ion-complexation.
Bacteria mostly utilize siderophores to scavenge iron3+-ions from their surroundings, an important trace element direly needed for proliferation. Many studies show that iron is not the only 3+ metal-ion that siderophores are able to form chelate complexes with, aluminum, vanadium, copper and gallium are known to form complexes with siderophores as well.
For our experiments gallium3+ is the ion of interest studies suggesting the cytotoxic abilities of gallium and the biochemical similarities, with the key difference that gallium inhibits enzymes irreversibly, while iron activates them. Other known properties of siderophores are: antimicrobial effects against bacteria that utilize other iron acquisition methods (or other siderophore types), the inactivation of specific toxins, biofilm formation, and of course to outcompete other cells in iron deprived environments.
Yersiniabactin
Now one might think, with Pseudomonas as the bacterial target, what role does Yersiniabactin, a siderophore once originating in Yersinia pestis, which later became a common siderophore in many types of enterobacteria, play in the project design. Well there are two good reasons.
First Pseudomonas mostly causes pneumonia in hospital infections due to its resilience and ability to proliferate in sparse environments, which he has in common with some enterobacteria. Enterobacteria like Klebsiella pneumonia, and Escherichia coli often cause lung infections leading to pneumonia when they carry the genetic sequences for Yersiniabactin synthesis, and tend to be multiresistant as well. Yersiniabactin is an important factor in pneumonia caused by enterobacteria since it has a high efficiency at acquiring iron, and possesses the ability to cleanse ions from human storage proteins like transferrin and lactoferrin. Another important factor for the prevalence of Yersiniabactin in lung infection is the fact, that yersiniabactin, as many siderophores, is bound by Lipocalin 2 in human serum, which significantly reduces the efficiency.
Second, and of utmost importance, was the fact that we had problems acquiring the genes from pseudomonas since a synthesis is extremely difficult due to the high GC-content. During our research we stumbled upon an image showing several pathways of siderophore synthesis, including similarities and the involved enzymes for the non-ribosomal synthesis. A short inquiry made sure that our initial thought was correct. Yersiniabactin is synthesized by two different enzymes, HMWP1 and HMWP2 synthases, of which HMWP2 became the synthase of interest for all our following research. Hence, HMWP2 synthesizes a compound that is known as Nor-Pyochelin. Therefore, we figured that with different induction methods we could create bacterial strains that could be induced to either produce yersiniabactin (utilizing the full casette), or Nor-Pyochelin with HMWP1 turned off. Essentially the gene cassette for Yersiniabactin allows us to synthesize a siderophore from a completely different organism but its own as well. Which leads to the elegant design of our project, not only are we able to target multiple different bacteria specifically by introducing toxic ions with their specific siderophores, but we are able to produce two different siderophores targeting pneumonia mediating multiresistant bacteria strains.
