Saturday, October 17, 2015

BtB#5- An intro to Bacteriophages


Viruses represents the ultimate of pathogens. They represent simplest of chemical replication machinery, totally host dependent. Interestingly, some viruses themselves have their own set of hackers called as Virophages. It is widely acknowledged, there isn't any life form on Earth that doesn't have its own set of viral infections. Among the different category, bacteriophages is the current leading interest. They can be useful as a therapeutic agent. See my earlier post on Phage therapy. However, far less people understand the basics of a bacteriophage or what we commonly call as the phage.

Fig 1: Generalized phage structure.
Bacteriophages, first described by Frederick Twort and Felix d'Herelle are viruses that attack the bacteria. They ubiquitously distributed in environment. The association of phage and bacteria is so profound that if you can find the phage in a system, it associated bacteria should be there, with reverse also being true. Several phages have been well studied in lab and several new has been described in recent years.

A classic textbook figure of structure is shown in Fig 1. The figure shows the structure of T4 phage. The structure consists of a head which holds the genetic material. The head is made up of capsid which is in turn made up of capsomeres and roughly prism shaped. This structure is more or less the same across all of the bacteriophages. The capsid is a tough covering and usually resistant to environmental stress thus being protective to the genetic material. The second component is the tail which is connected to head through a Neck (or sometimes referred to as collar). Tail is not a constant feature, as many members don't have a tail. Tail is often a hollow tunnel through which the genetic material can be transferred. This is surrounded by a covering called as sheath. Some of the complex phages at the end of tail has a specialized structure called as base plate. This holds tail fibres which are responsible for hooking of phages to particular receptors.

Fig 2: Size distribution of sequenced
Bacteriophage genomes. Source
There is great deal of genetic diversity when it comes to the bacteriophages. Phage metagenomics is a reality and a lot of what is called as genetic dark matter is in these phages. Nothing is known about these sequences. Fig 2 captures a summary of genetic size variation considering some of the known phages. Phages also comes in a variety of genetic makeups- DNA or RNA, single or double stranded. Each different type has a different replication strategy. Depending on the structure and organisation of the phage, there are at least 12 distinct group of bacteriophages known. Their properties are summarized in Table 1 (Adapted from source).

Table 1: Characters of various families of Bacteriophages
The basic steps in bacteriophage replication is quite common- Phage adsorption, Genetic entry, Genetic replication, synthesis of virus particle, assembly and finally phage release. The details differ based on the type of phage and host. The phage receptor complex (PRC) are usually located in an accessible region of the outer membrane of bacterial host (exceptions exist where entry mechanism is more complex). Most of the PRC that I have read about in literature are somehow related to porins regulated by operons. For example, in case of E coli lambda phage, J protein in the tail tip interacts with maltose outer membrane porin (part of maltose operon) and then DNA passes through mannose permease complex in the inner membrane.

There are 2 modes of life cycle when it comes to phages- Lytic and Lysogenic life cycle. Lytic life cycle represents a quick paced infection and replication and then burst out of the cell. Lysogeny is a more relaxed life cycle, where the viral genome integrates with the bacterial genome and keeps silent. The bacteria replicates the gene for virus. Someday, if the viral gene decides that its time to leave, the machinery becomes active and turns up into a lytic cycle. Though it appears superficially to be a random process, each type of life cycle is a carefully guarded decision.

Here is a simple explanation. Lytic life cycle is a process which allows the phage to infect new bacteria. Lysogeny is a mode where the phage takes advantage of bacterial replication machinery, without having to do anything by itself. Lytic life cycle would thus be useful only when the bacterial host is possibly in danger of loss such as stress. In other cases, lysogeny may help. It is arguable that this whole process has to do something with quorum sensing, but I'm not aware of any articles that has shown a direct relationship. Indeed, when the phage is in lysogenic state, phages can be awakened to their lytic state by inducing stress such as UV radiation. Interesting enough, quorum sensing mechanisms are demonstrated to induce resistance to phage invasion.

Fig 3: The lambda repressor switch.  Source
At molecular level, the things are a little bit more complex. As an example, let us take the example of E coli phage lambda. Though the exact mechanism differs from phage to phage the overall principle remains nearly the same. The simplest principle can be stated as follows. There exists a competition between two phage repressor molecule: CI and Cro. In the event that CI repressor gains upper hand, lambda DNA becomes a quiescent prophage that integrates into the host chromosome and expresses only one gene, cI. If Cro gets the lead, then the phage turns on the lytic mode. The decision of which repressor wins is based on the expression pattern of other genes, A simplified circuit is shown in Fig 3. Now what determines the underlying gene expression pattern? One well known idea is the multiplicity of infection. In the event that several phages infect the same bacteria CI is made in more numbers. This leads to lysogenic conversion. This is useful since more phages per cell would mean that there are less number of bacteria available to infect and hence lytic cycle wouldn't be useful.

Table 2: Phage encoded toxins in bacteria. Source
Phage forms a very important interest these days. Phages are the next generation antibiotics. You would not like the phages to undergo lysogenic conversion if they are to be used as an antibiotic. This can be achieved by genetically engineering viral strains to not have lot of CI production. Also, Phages are some of the best medium for genetic exchange in bacteria. Further, phages themselves can contribute genes to bacteria that may increase the virulence. Many bacterial toxins are actually phage encoded genes. There also several examples of phage derived genetic product that are involved in antigenic conversion, effector proteins, enzymes, resistance factors etc.

In this post I have talked about bacteriophages in a very short format, just in a introductory format. The genetics and molecular switches involved in decision making process is quite complex, and probably I could put a post on that on some other day. Bacteriophages form a different entity. Phages exist for others such as fungal and parasites. The working mode of fungal phages are still bizarre. For example, some fungal phages never leave the cell. They are transmitted via spores through mating. Perhaps all these are a topic for another future post.
Hendrix RW (2003). Bacteriophage genomics. Current opinion in microbiology, 6 (5), 506-11 PMID: 14572544

Høyland-Kroghsbo NM, Maerkedahl RB, & Svenningsen SL (2013). A quorum-sensing-induced bacteriophage defense mechanism. mBio, 4 (1) PMID: 23422409

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

Boyd EF, & Brüssow H (2002). Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends in microbiology, 10 (11), 521-9 PMID: 1241961

Wednesday, October 14, 2015

Hitting the RNA switch: A new method of antibiotic design


Once again, we returning back to the discussion of antibiotics. The last time I discussed about antibiotics was a post on use of predatory bacteria as a possible alternative for antibiotics and Teixobactin which was isolated from a soil microbe with potential activity. Though there are several lead compounds in pharmaceutical research which will possibly make a good antibiotic, the claim has been that there is no new class of antibiotic. There have been a couple of publications in the past on chemicals of interest that are completely different in their approach compared to traditional antibiotics. However, to date we don't have any of these in the shelf.

The latest exciting news of potential lead compound acting through a totally different mechanism is thus a great research of interest for the microbial community. Howe etal (Representing Merck) has described a new antibiotic lead named as Ribocil. As Thomas Hermann describes “Finding an antibiotic with a new target has always been one of the holy grails of antibiotics discovery. It looks like that’s what the Merck group has now accomplished.”

As early as the 1950's antibiotics were discovered through screening. Several thousand natural compounds were purified and tested against bacterial isolates to look for potent activity. One such example is streptomycin. Merck used a similar approach. Only that instead of the natural compound, they screened their synthetic compounds library via a method of high throughput phenotypic screen. This lead to identification of ~57,000 synthetic small molecules which potentially inhibited E coli. The researchers then argued that riboflavin pathway was specific to bacteria and hence an inhibitor would be of great interest in this category. So they screened the E. coli strain (MB5746) defective in wild-type lipopolysaccharide (LPS) levels and drug efflux was selected. to identify if the riboflavin pathway was involved in action the researchers setup a rescue assay. The principle being that if riboflavin synthesis is effected, by adding riboflavin the bacteria would be able to grow despite the presence of antibiotic.

Fig 1: Structure of Ribocil.
One such chemical identified in the process was Ribocil. The next step is to identify the mechanism. First, in an independent experiment, they created resistant E coli mutants. Most commonly, it is done by serial subculture of strains at sub lethal MIC levels. These strains were labelled as Ribocil. By comparing whole genome sequences between resistant mutants and parental strain, several mutations were identified. What do you expect? A mutated enzyme. However, it was seen that all the mutations were in a gene called RibB which indeed was an enzyme in the synthesis pathway. Just that mutation was not in the coding region. It was in a non coding RNA region- the Riboswitch. Quoting from the paper, "Based on the functional role of the FMN riboswitch and that all ribocilmutations map to this regulatory element, we hypothesized that ribocil inhibits riboflavin biosynthesis directly by mimicking the FMN ligand and binding to the FMN riboswitch to inhibit ribB expression".

Fig 2: Structure of an mRNA showing Riboswitch. Source
Let us digress a little. What is a Riboswitch? There are several mechanism by which protein transcription is controlled. It depends on environmental condition as to what are the proteins that needs to be produced and what concentrations. One of them is regulation of mRNA itself. Ribsowitches are a region most commonly seen in 5'-UTR regions. They have a specific folding which allows its interaction with other proteins. The loops can be opened and mRNA readable for protein synthesis through the control of this region hence acting as a switch. In this case, as Howe puts it, "So instead of regulating the enzyme itself, ribocil is regulating the production of the enzyme". For the first time ever, a riboswitch-binding molecule that is not a close structural analogue of a metabolite ligand is discovered.

The paper of course goes on a long list of experiments done to validate the binding characteristics, structural X crystallography studies and animal experiments to show its ability to be an antibiotic. The team further tweaked the Ribocil structure to enhance activity. This is a start. As Gerry Wright comments, “I’ve no idea if ribocil will end up being a drug candidate, but the work is a proof of principle, which is very important, and it makes us look to new areas of biology as targets for antibiotics.”

This study has brought up some interesting discussion, on how this study is insightful. For example, HIV is a hard to beat virus. HIV encoded non coding RNA's has been studied for a longtime (Link). It has been suggested that a similar approach maybe tried for HIV to identify potential therapeutics. A article published in 2014, has shown that a site in HCV called as  Internal Ribosomal Entry Site (IRES) can be attacked by using small synthetic molecules (Link).

On an important note, the chemical is still a long way though from making it to the shelf and mutations arise easily.
Howe JA, Wang H, Fischmann TO, Balibar CJ, Xiao L, Galgoci AM, Malinverni JC, Mayhood T, Villafania A, Nahvi A, Murgolo N, Barbieri CM, Mann PA, Carr D, Xia E, Zuck P, Riley D, Painter RE, Walker SS, Sherborne B, de Jesus R, Pan W, Plotkin MA, Wu J, Rindgen D, Cummings J, Garlisi CG, Zhang R, Sheth PR, Gill CJ, Tang H, & Roemer T (2015). Selective small-molecule inhibition of an RNA structural element. Nature PMID: 26416753

Hermann T (2015). Non-coding RNA: Antibiotic tricks a switch. Nature PMID: 26416738

Wednesday, October 07, 2015

Nobel Awards- 2015


Nobel Prize is the worlds most prestigious award, news of which is awaited by scientists every year. The recipients of the award is chosen by the Nobel foundation constituted by Nobel committee of Royal Swedish Academy of Sciences, Nobel committee of Karolinska Institutet and Norwegian Nobel Committee. The award consists of a citation, gold medal and money. However, the fame is considered far superior for the award. The 

For the year, 2015 Nobel awards under different categories of science has been announced. Congratulations to the winners.

1. Physiology/Medicine

This years award under the category has been split into two half. First half goes to William Campbell and Satoshi Omura for their contribution to development of avermectin class of drugs to treat river blindness and lymphatic filariasis. The second half of the award is honored to Youyou Tu (Also won Lasker prize in 2011), for contribution to development of the antimalarial drug artemisinin.

2. Physics

Takaaki Kajita and Arthur McDonald have been awarded under the physics category for their work demonstrating that neutrinos, as they travel can change mass at a varying rate, depending on the state thereby convincingly proving that the ghostly particles have mass. The study finally solved a longstanding problem in "standard model" of particle physics.

3. Chemistry

Recognition under this category has been awarded to Tomas Lindahl, Paul Modrich and Aziz Sancar for their work on DNA repair (Actually that sounds so much like physiology). The work described DNA repair mechanisms- base excision repair (by Tomas Lindahl), nucleotide excision repair (by Aziz Sancar) and mismatch repair (by Paul Modrich).
Callaway, E., & Cyranoski, D. (2015). Anti-parasite drugs sweep Nobel prize in medicine 2015 Nature doi: 10.1038/nature.2015.18507

Elizabeth Gibney& Davide Castelvecchi (2015). Morphing neutrinos win physics Nobel Nature, 526 (175) : doi:10.1038/nature.2015.18513

Daniel Cressey (2015). DNA repair sleuths win chemistry Nobel Nature : doi:10.1038/nature.2015.18515

Monday, October 05, 2015

Outbreak Watch: Dengue in India


The current local news on "Dengue" in India is gaining fast pace. With Delhi on spotlight, dengue cases have become one of the most talked about topic, and has left most people searching for information. Sadly, the news and material is quite scattered and there is no clear picture. So let me put the current picture in perspective.

I have earlier written about dengue in my earlier posts (Link). Here is a quick summary. Dengue is a mosquito-borne infection caused by a flavivirus member. The icosahedral structured virus contains a genome of single positive-stranded RNA, about 11 Kbp in length coding for 3 structural (capsid protein C, membrane protein M, envelope protein E) and 7 non structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). The vectors commonly known to be involved in transmission are Aedes aegypti and Aedes albopictus. They are reportedly prevalent in the north-east region of India. The virus has four closely related types DEN-1 to DEN-4.

Fig 1: Dengue Cases reported in India (2009- 2014).
The epidemiological data on number reported cases of dengue in India is documented, updated and published by NVBDCP. It has been stated that the dengue surveillance is not good in the country and the number of cases reported is far less than actually is the case. Yet from the data, I made a graph, to give an idea on the number of cases. It is evident that in recent history, 2013 represents the worst with a total of 75808 cases. However, the number of deaths was proportionately less. Relying on the same data, I have  plotted a second graph to get you the idea of deaths.

Fig 2: Dengue deaths reported in India (2009- 2014)
So, why all this sudden media attention? As per government's Press Information Bureau, “It was noted that at national level, there has been decrease in the number of dengue cases detected with 75 808 cases in 2013, 40 571 cases in 2014, and nearly 21 000 cases in 2015 up to second week of September and the recovery has been in more than 99.9% of cases”. As of on 5th Oct, 2015 Delhi alone has recorded 6500 confirmed cases. In nearly 15 days, the total number of cases has nearly doubled. The number of case was 3791 as on 20th Sept, 2015. That's why it is a news.

Fig 3: Reported dengue cases in India,
top 10 states shown. Data as of on
20, Sept 2015, for year 2015.
This prompted me to look into the statistics, once again. Is Delhi the only affected? I don't have latest data. So I will once again rely on the NVBDCP, statistics, published as of on 20th Sept, 2015. I selected the top 10 states reporting Dengue cases. Of course, by the time I'm posting this the data is almost obsolete, since the numbers have drastically changed. Bu the following graph will still throw you the idea. It has been speculated that the sudden spurge in number of cases is possibly due to monsoons and increased mosquito breeding. According to the report inefficiency in removing stagnant water from premises and in undertaking fumigation to control mosquitoes, and the emergence of a rare strain (type 4) of the dengue virus, everything contributes.Vector control programs have thus been intensified to control the problem.

Diagnosis is usually through commercially available ELISA kits, testing for IgM antibodies. PCR is the best option but expensive. Currently, a lot of reports is based on rapid diagnostic tests, with good probability of false positive reactions. The government is considering banning such tests so as to get a better diagnostic report.

Dengue Facts
A second issue of concern that has been widely circulating in the news is the factor of under-reporting. Let me clarify the issue. Dengue is not clinically evident in a proportion of population and hence that percentage is lost in reporting. A study by INCLEN Study Group had made a claim that annual number of dengue fever cases in India could be 282-times higher than the number. There are some serious limitation in the original study. First, the data was generalized for the country based on data from Madurai, only on tested dengue cases. Second there was a inability to collect a proportion of data due to lack of documentation.

In case you are still curious, the global statistics are not that great. The latest estimates is about 390 million dengue infections per year of which 96 million (about 24.6%) manifest clinically. Also various reports have indicated that the numbers reported are increasing in several places. However, it is not entirely clear if it is because of increased number of cases or there is simply better reporting.

On a final note, as of yet this is not the worst case scenario as reported by most of the media. The cases is still way below what it was in 2013. But I agree that we need better surveillance and preventive measures.
Bagcchi, S. (2015). Dengue surveillance poor in India The Lancet, 386 (10000) DOI: 10.1016/S0140-6736(15)00315-3

Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, George DB, Jaenisch T, Wint GR, Simmons CP, Scott TW, Farrar JJ, & Hay SI (2013). The global distribution and burden of dengue. Nature, 496 (7446), 504-7 PMID: 23563266