Dear Franck

in my team we work on the development of new antibacterials, and specially on antibacterials active on MDR Gram negative bacteria that modify the outer membrane permeability but not on bacterial cytoskeleton...

I collaborates with the group of Bill Sullivan in Santa Cruz who developed screen for drugs against Wolbachia and some molecules seem affect the FtsZ protein of the bacteria. See Serbus et al. 2012 in PLOS Pathogens. Your antibiotics would certainly be very interesting to test on such system.

Alain Debec, IJM, Paris

Dear Dr. Franck,

I am working on antibiotic targets from MRSA. The studies based on in vivo and in vitro PTMs (mass spec approach). In this I have identified the proteins modified with antibiotic from cytoskeleton.

Best,

yogesh

Dear Franck, Elongation Factor Thermo unstable (EF-Tu) is an interesting bacterial target, one of the several components for protein expression. If I am not wrong Fusidic acid is an inhibitor of FT-G. I have personally worked on FtsZ inhibitors.

Kind regards,

Daniele

Thank you for your interesting remarks.- One of my points is that, according to our findings, EF-Tu is not only involved in protein expression; it seems to have a double function: EF-Tu may be a substantial component of a kind of primordial bacterial cytoskeleton; in vitro, EF-Tu copies form long chains; very similar ultrastructures can be found in bacteria; the number of EF-Tu copies per cell appears to be a multitude of the number of EF-Tu copies needed for its function in protein expression; EF-Tu is a protein composed of three domains; according to available X-ray data one of these domains fits exaclty in a grove on one of the two other domains of an EF-Tu copy nearby, thus forming a pair that is the beginning of a chain if additional EF-Tu protein copies are involved by the same steric mechanism. The third of the three domains is not involved in chain formation; according to available data, this third domain is involved in the mechanism of protein expression. Hence, an antibacterial agent (a small peptide) can be designed that simulates one of the amino acid motifs at the contact region of the domains that are involved in chain formation. If such a small peptide is transferred into the bacterium, it will block chain formation by competitive hinderance of the contact of an EF-Tu copy with a neighboring EF-Tu copy. Such contacts can be assumed to take place during the entire life of the bacterial cell; without a chain formation, the bacterial cell lacks this kind of primordial cytoskeleton. Our experiments did show that bacterial cells start lysis within a period of one or two hours (1 generation in the fedrmenter) if one of the two Domains involved in chain formation is missing.(experiments with truncated EF-Tu). Question: how can small peptides be trasnferred into bacterial cells?

Instead of small peptides, we started a search for low molecular compounds that simulate the function of these specific small peptides and can easily be transferred into bacteria. This search was not continued several years ago.

I am using antimicrobial peptides against various bacteria.

Are these antibacterial peptides acting against targets inside the cytoplasmic membrane of the bacteria?

Frank Mayer

Hi Frank, from your description EF-Tu proteins are capable of self assembling as in the case of the beta-amyloid plaques (one on the possible Alzheimer's causes).

If you have a small peptide in mind to test you could try it against efflux impaired bacteria to see if the idea works, then search for peptide mimetic with the help of a computational group, or alternatively if you have an in vitro assay to measure the inhibition of the ET-Tu couple formation you can consider converting the assay in a HTS searching for a hit to use as starting point for optimisation. Kind regards, Daniele

dear sir I just started to research and do my Ph.D thesis.

have you done something so far?what would you offer me to go for?

I did give the number of my patent related to the question. The patent describes the details. My first stept was to find out, by recombinant techniques, what happens when the one of the genes coding for the three protein subunits making up the cytoskeleton was deleted in part. It turned out that such a deletion caused the breakdown of the integrity of the cell three to four hours after  it was activated. This appears to be caused by insertion of a considerable amount of copies of the defect kind of protein subunit into the  protein complex making up, in the intact cell, the "building unit" of  the - dynamic - cytoskeleton.  One important point is that the gene coding a defect protein subunit should only be activated at a time when the cells have had sufficient time to built an intact cytoskeleotn, i.e.  intact cells. The gene coding the defect protein subunit was activated by a small shift of the growth temperature of the cell culture.  Respective techniques are well known. - Analysis of the state of the cells in a cell culture was done by electron microscopy and by other standard tests. The next step should be the design of small molecules simulating the sequence and three-dimensional organization of those sequences of the intact subunits of the protein complex that make the contact between the protein complexes that make up the catoskeleton. It should be found a way how these small molecules can penetrate the cell wall and the cytoplasmic membrane of the cell. As soon as they are inside the cell, they will compete, during the assembly of the cytoskeleton, with the intact subunits. Hence, intact cytoskeleton cannot be formed, and the cell will die.

Here is additional information regarding your question: yesterday, I described a more general approach for the search of a target for a new antibacterial agent. In our experiments we had first to establish that the protein in question is, at all, a main component of a so far unknown kind of bacterial cytoskeleton. Our approach seems to have been a detour, but the protein was not known before to have this property., In our case it turned out that the protein was not a complex of several subunits, but a single polypeptide chain folded into a protein with three domains. The deletion mentioned in my answer from yesterday was, in fact, concerning one of the domains that are responsible for the contact with the next protein in the row, making up the cytoskeleton. To make your life much easier when you search for a new antibacterial agent: take, as the target, one of the known and meanwhile well characterized proteins making up a major part of one of the bacterial cytoskeletons discovered more recently, analyze the properties of the contact regions, exposed on the surface of the protein, that are responsible for contact, and design a small molecule that can simulate the properties of one of these contact regions. As soon as you manage to get this small kind of molecule, in high amount, into the bacterial cell, it will compete with intact proteins making up the cytoskeleton in the growing bacterial cell, and there will be no chance for the bacterium to form an intact cytoskeleton.