Friday, June 13, 2014

Oh you have bacterial infection? Why don’t you try a virus?


   In previous post, I highlighted that we are moving towards a post antibiotic era. I mentioned of a couple of examples on what the future looks like. One of the most talked about everywhere is the concept of phage therapy. After I published the post, I felt that phage therapy is the most talked about concept and needs a separate blog space. Bacteriophages are viruses of bacteria. For reasons of simplicity, the term phage is used to denote the bacteriophages. It should be noted that a variety of phages have been characterized against fungus, Parasites etc. They too are known as phages and often the term is confusing. In this post, the term phages exclusively refers to bacteriophages.

Table 1:  Examples of phage coding for virulence.
  A phage propagates through one of the modes of a life cycle- Lytic or lysogenic. In lytic life cycle, the phage produces progeny, killing the cell. In contrast, the lysogenic life cycle incorporates the phage genome into host genome and lies dormant, replicating with host. Such incorporation maybe an added advantage for bacteria. Well studied examples of viral genome encoding toxin properties to bacteria which subsequently acts as virulence is shown in table 1. Lysogenised phage can be induced back into lytic cycle by external disturbances. In these situations, a virus may package part of host genome and transfer it into other bacteria. An example of interest is transfer of shiga toxin gene to E coli from Shigella. Such E coli called as EHEC (Enterohemorrhagic Escherichia coli ), is one of the leading concern world over.

   This understanding has important implications. Point is you cannot simply isolate some phage and say "Here's your treatment..." Phage undergoing lysogenic conversion may introduce more problems. So the most important hurdle getting the phage to be only in lytic cycle. How? 

      The decision to undergo lytic or lysogenic cycle is not a random effect as was thought to be. Think of it something like this. If the phage is inside the bacteria, but bacteria is not abundant or in a poor growth medium, the chances that the phage will propagate after lysis is bleak. Because when the new phages will pop out there maynot be enough bacteria in vicinity to infect. In such conditions, the phage will undergo lysogenic conversion and keep quiet. On the other hand if bacteria is abundant. it would be preferable to undergo lytic cycle. This decision is taken by a series of switches called as cI and Cro. The actual genetic circuit consists of more players.

       By a series of complex steps, autoregulation of cI synthesis keeps the cell in a stable state of lysogeny. When the conditions are right, cro is transcribed which negatively regulates cI and enhances its own synthesis. This locks the phage into a lytic cycle. For a step by step look into the process click here. The point is that messing up with the cI production can give you a phage that can only lyse. And that is what exactly we want.

Table 2: Phage therapy, advantages and disadvantages.
  Phage therapy is not a new concept. The concept of use of bacteriophages dates to the work by Felix d’Herelle. Subsequently, multiple studies have been reported in French literature. Many publications have appeared in various journals where phages have been probably used in desperate attempts to save the patient, such as in severe septicemia, surgical site infections etc. Since by definition, phages doesn't infect humans, and studies have shown that they provide a very specific agent of therapy. There were industries and organisations that would produce phages, many of which have dropped the project.

 There are a variety of advantages and disadvantages of phage therapy, listed in Table 2. The greatest advantage of phage therapy is that a small dose of initial inoculum is needed on the site. Since phages will produce new progenies, they will take care of a lot of bacteria. And being very specific, they will not attack any bystander, a feature that has not yet been achieved by any antibiotic designed to date. And source of phages is a huge advantage. It is very easy to find phages in environment with a simple screening procedure. Delete for their potential to become lysogenic and they are ready.

The major disadvantage is we developing antibodies. But considering the huge repertoire of possible phages this is not expected to be a great problem. Even greater is the treatment of bacteria that are inside cells, which phages cannot access. Another difficulty is in administering the dose. The phage has to be put right into the site of infection to be maximally effective. This will require trained personnel which is in sharp contrast to oral antibiotics. A yet another problem is to tell the patient.. "You got severe bacterial infection, Multi drug resistant type. how about trying some virus". Such comments no matter how hard you try will be very difficult for the patient to digest. Of course patient education will help.

    In general, phage therapy is not an interest for pharma giants. The reason for this drawback is that phages are naturally occurring entities, which can be easily replicated and not patentable. And just like antibiotics it is a short course therapy (expectedly still shorter and cheaper for phage therapy). The chances of profit for the company is less. However, companies like Mediphage is devoting a lot of R&D, to developing new phages and phage based therapeutics.

     It should be noted that phage are not the ultimate weapons. As, I have discussed in my previous post on CRISPR (Link), bacteria have strategies to combat back. Bacterial resistance will evolve if we use phages the way we have used antibiotics. But, the argument is we may have too many phages in our arsenal, for the bacteria.

So are we ready for the post antibiotic era???
Oppenheim AB, Kobiler O, Stavans J, Court DL, & Adhya S (2005). Switches in bacteriophage lambda development. Annual review of genetics, 39, 409-29 PMID: 16285866

Abedon, S., Kuhl, S., Blasdel, B., & Kutter, E. (2011). Phage treatment of human infections Bacteriophage, 1 (2), 66-85 DOI: 10.4161/bact.1.2.15845

Chan BK, Abedon ST, & Loc-Carrillo C (2013). Phage cocktails and the future of phage therapy. Future microbiology, 8 (6), 769-83 PMID: 23701332

Tuesday, June 10, 2014

Post antibiotic era- Expectations


Again and again, antibiotic gains the centre stage of discussion. In this blog, I have several times written about antibiotics. Most often readers are left with an impression that antibiotics has been something that has come into picture only decades ago, if not centuries ago. You are right, if you think of the tablets and pure chemical forms that is being used in modern medicine. However, there are studies that have shown, for example traces of tetracycline in human skeletal remains from ancient Sudanese Nubia dating back to 350–550 CE. It is likely, that it was not used in its pure form but was somehow through the folk medicine preparations. Oh yes, microbes have been producing antibiotic compounds for millions of years for their own interest.

But the concept of directed use of antibiotics started with penicillin. Of note, Pyocyanase prepared by Emmerich and Low from Pseudomonas aeruginosa is the actual first hospital drug to be used. For all discussion purposes in literature, we refer to as Pre-Antibiotic and Antibiotic era to denote time before penicillin and after penicillin. Antibiotics was such a big hit in science when it was initially introduced, a statement was made "Battle on infectious disease is won". But over the past 60 years we have been proved wrong. Bacteria is battling hard. So much so is the problem, WHO recently released a warning that we are probably stepping into post antibiotic era. I wouldn't say that we have already entered, but we are definitely on the edge. A recent internet page posted a comment noting that we already have entered the era. This is a little bit of hype. But we are on the process for sure.

The natural question that you could come up with is what is the alternative to antibiotics. That seems to be a very difficult question to answer. For the current generation of practitioners, it would be nearly impossible to think of medical intervention without antibiotics. I could see some possibilities though.

For example, a technique called "nanosponge" is in research for use against MRSA. The technique basically uses real red blood cell membranes around biocompatible polymeric nanoparticles, which act as decoy once in the bloodstream, absorbing the damaging proteins and neutralizing their toxicity. No they are not antibiotics but can be considered as anti virulence compound. Theoretically this could be applied to many more pathogens.

The second example that I can think of is in context with treating sepsis or bacteremia. Blood stream infections are often difficult to treat infections. An approach is to filter the microbe out. Maybe I could call it "pathogen dialysis". The concept proposal is to add nanoparticles with bis-Zn-DPA to a patient’s blood when it is already outside the body (something like dialysis). The bis-Zn-DPA (Bis(zinc(II)-dipicolylamine) coordination complex) selectively binds the bacteria. The synthetic molecule attaches strongly to molecules that are ubiquitous in bacterial cell walls but are not present in membranes of human cells. That means only bacterial cells are bound. The attached nanoparticle is a nanomagnet which can be easily pulled out with a magnet-based system. This is not just a concept proposal. A proof of concept study, using a bovine model system the researchers have been able to pull out E coli with as little as 2 cycles.

The third example that is most popular in current literature is the use of phage therapy. Bacteriophages or simply phages are naturally occurring enemies of bacteria. By using right combinations of phages (referred as phage cocktails), it is possible to selectively attack pathogen in question. By definition, phage doesn't infect mamallian cells and hence should be safe. The concept of using phages is not a new phenomenon. FDA has already approved a bacteriophage spray to be used on vegetables that are eaten raw. The lytic cocktail can kill hard strains such as E. coli O157:H7. A clinical trial against drug resistant P. aeruginosa, in chronic otitis patients have shown good clinical correlates. With more and more MDR appearing on the scene, we should expect more trials.

To conclude, I don't expect that antibiotics will be totally useless. As mentioned in my previous posts, we are discovering new antibiotics. Fast track testing and approval have enabled some incentive for fresh chemicals to be pumped in. But the basic stress on requirement of antibiotics is still very high. The post antibiotic era would see totally novel ideas to combat bacteria. That would reduce the burden of antibiotic usage. But, a complete absence of antibiotics... "I doubt". At least not till we have developed and validated new ideas, which will take a few more decades.
Reardon S. (2014). WHO warns against 'post-antibiotic' era, Nature News, DOI: 

Hu C.M.J., Fang R.H., Copp J., Luk B.T. & Zhang L. (2013). A biomimetic nanosponge that absorbs pore-forming toxins., Nature nanotechnology, PMID: 23584215

Lee J.J., Jeong K.J., Hashimoto M., Kwon A.H., Rwei A., Shankarappa S.A., Tsui J.H. & Kohane D.S. (2013). Synthetic ligand-coated magnetic nanoparticles for microfluidic bacterial separation from blood., Nano letters, PMID: 23367876

Wright A., Hawkins C.H., Anggård E.E. & Harper D.R. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a preliminary report of efficacy., Clinical otolaryngology, PMID: 19673983

Tuesday, June 03, 2014

Tripartite motif 5α in news


HIV is one of the most well studied virus in terms of global health. As I have repeated multiple times in this blog, it still haunts the best brains and no absolute cure or preventive measure is available. Except for a handful of cases, where people have demonstrated complete cure, or seem to be never infected. But just to say that as humans never evolved defence strategies against retrovirus is not true. Indeed our cells have restriction factors for multiple steps but virus has its own counter strategy. Indeed HIV has evolutionary advantage in terms of its speed of evolving which we don't have.

The restriction factors so far described include- Cyclophilin A, APOBEC3, TRIM-5α, SAMHD1, Mx-2 and BST-2 (read my previous post here). Of all these TRIM-5α has a special interest. That is because, HIV doesn't produce any factor to counteract its effects yet seems to be not effected by it. In other words Human TRIM-5α seems to be ineffective against HIV. And second, the monkey version of it is too good in defending from HIV. What is the difference?

Fig 1:Structure of the C3HC4 RING
finger domain. Source
    TRIM 5α (Tripartite motif 5α) or RING finger protein 88, was first identified in monkeys. It was demonstrated that expression of RhMTRIM5 ( Rhesus monkey TRIM 5 ) in human cells, which are normally permissive to HIV-1, restricts HIV-1. In human cells the action of TRIM 5α is very bleak. It is known that the TRIM interacts with capsid of the HIV in cytoplasm. TRIM5 spontaneously forms a hexameric protein lattice, which is greatly enhanced in the presence of the capsid lattice structure.

RING stands for "Really Interesting New Gene". Many different proteins are known to have structure referred as RING domains which belongs to a super family of zinc finger proteins. They basically contain a Cys3HisCys4 amino acid motif which binds two zinc cations. Their major function is in ubiquitination pathways. The probable role is to bind capsids and subsequently process the capsid to ubiquitination. There is some proof that the capsid is an essential element in importing the PIC (Pre integration complex). That explains how TRIM5 is supposed to be useful. 

   But just as I mentioned, TRIM5α seems to be not efficient enough. The question as to what is the reason for inefficiency is explained in the latest paper in nature. Based on a series of experiments the researchers concluded that the huTRIM5α (Human TRIM5α), was much less stable than the orthologue monkey version. By introducing mutations in B30.2/SPRY  domain (as little as 5 mutation), the stability was increased with better restriction activity.

    But that begs a question. Evolutionarily why did we change the sequence into a less stable factor? I don't have a clear explanation for this question. I have been told that TRIM restricts MLV (Murine Leukemia Virus), with a similar kind of capsid attack approach. It is my belief that probably TRIM5 is an important restriction factor for other retrovirus and HIV being a more recent phenomenon there is a lag.

    This study has set a small opening to look into pharmacological designs to improve the stability of TRIM5α. But that also requires us to understand the mechanism of action and the chemistry of interaction. Oh yes, you would say that we have a new perspective to look at.
Perron MJ, Stremlau M, Lee M, Javanbakht H, Song B, & Sodroski J (2007). The human TRIM5alpha restriction factor mediates accelerated uncoating of the N-tropic murine leukemia virus capsid. Journal of virology, 81 (5), 2138-48 PMID: 17135314

Richardson, M., Guo, L., Xin, F., Yang, X., & Riley, J. (2014). Stabilized Human TRIM5α Protects Human T Cells From HIV-1 Infection Molecular Therapy, 22 (6), 1084-1095 DOI: 10.1038/mt.2014.52