Phage synergy with the immune system

bacteriophage modelNot long after their discovery, viruses that infect bacteria – bacteriophages – were considered as therapeutic agents for treating infections. Despite many years of research on so-called phage therapy, clinical trials have produced conflicting results. They might be explained in part by the results of a new study which show that the host innate immune system is crucial for the efficacy of phage therapy.

When mice are infected intranasally with Pseudomonas aeruginosa (which causes pneumonia in patients with weak immune systems), the bacterium multiplies in the lungs and kills the animals in less than two days. When a P. aeruginosa lytic phage (i.e. that kills the bacteria) is instilled in the nose of the mice two hours after bacterial infection, all the mice survive and there are no detectable bacteria in the lungs. The phage can even be used prophylactically: it can prevent pneumonia when given up to four days before bacterial challenge.

The ability of phage to clear P. aeruginosa infection in the mouse lungs depends on the innate immune response. When bacteria infect a host, they are rapidly detected by pattern recognition receptors such as toll-like receptors. These receptors detect pathogen-specific molecular patterns and initiate a signaling cascade that leads to the production of cytokines, which may stop the infection. Phage cannot clear P. aeruginosa infection in mice lacking the myd88 gene, which is central to the activity of toll like receptors. This result shows that the innate immune response is crucial for the ability of phages to clear bacterial infections. In contrast, neither T cells, B cells, or innate lymphoid cells such as NK cells are needed for phage therapy to work.

The neutrophil is a cell of the immune system that is important in curtailing bacterial infections. Phage therapy does not work in mice depleted of neutrophils. This result suggests that humans with neutropenia, or low neutrophil counts, might not respond well to phage therapy.

A concern with phage therapy is that bacterial mutants resistant to infection might arise, leading to treatment failure. In silico modeling indicated that phage-resistant bacteria are eliminated by the innate immune response. In contrast, phage resistant bacteria dominate the population in mice lacking the myd88 gene.

These results demonstrate that in mice, successful phage therapy depends on a both the innate immune response of the host, which the authors call ‘immunophage synergy’. Whether such synergy also occurs in humans is not known, but should be studied. Even if observed in humans, immunophage synergy might not be a feature of infections in other anatomical locations, or those caused by other bacteria. Nevertheless, should immunophage synergy occur in people, then clearly only those with appropriate host immunity – which needs to be defined – should be given phage therapy.

TWiV 212: Apocalypse TWiV 122112 212

On episode #212 of the science show This Week in Virology, the TWiVerers answer listener email about genetically modified chickens, a hendra vaccine for horses, online education, curing color blindness, Roosevelt and polio, Th cells, and much more.

You can find TWiV #212 at

TWiV 211: Viruses r us

On episode #211 of the science show This Week in Virology, the TWiV four discuss an mRNA-based influenza vaccine, and a phage tubulin that forms a filamentous array in the host cell that is needed for positioning viral DNA.

You can find TWiV #211 at

Milestones in Microbiology: The video

Cold Spring Harbor was designated a Milestones in Microbiology site in August, an event I witnessed and documented. Now a video of the ceremony has been released, featuring comments by Stanley Maloy, Bruce Stillman, and James D. Watson.

The Hershey-Chase food blender

Hershey-Chase blenderShould you ever visit Cold Spring Harbor Laboratory on Long Island, New York, be sure to go see the food blender that was used to carry out the well known Hershey-Chase experiment. The blender is located in the Szybalski Reading Room of the Carnegie Library. After entering the front door of the library, walk straight back, enter the Szybalski Room, and turn right to find the blender in a display case.

At left is a photograph of the blender, partially hidden behind a letter (click the photo for a larger view). You can see the cap of the blender and the main body into which liquids are poured. I presume the motor is hidden from view. Behind the blender is a diagram of the Hershey-Chase experiment.

The letter is also worth reading because it concerns salary negotiations between Hershey and Milislav Demerec, who was the President of Cold Spring Harbor Laboratory from 1940 to 1960 (at that time it was called the Carnegie Institute of Washington). The letter is dated 15 January 1950 and was written while Hershey was at Washington University in St. Louis, MO:

Dear Dr. Demerec:

I have decided that I would like to come to Cold Spring Harbor, provided we can agree about salary. Carnegie is my first choice, but I do not feel that I can make an appreciable monetary sacrifice in its favor. I still do not know what will be done for me here, of course, but I think we can assume that it will be inferior in one way or another to the prospects at Illinois.

I believe I told you that the offer from Illinois was a professorship at $7000. This is the basic salary for nine months, to which is added $1500 if I teach a course in summer school. One can, of course, also supplement income by teaching elsewhere in the summer, as Luria is doing in Colorado this year. In a telephone conversation this morning, Dr. Spiegelman* told me that Dr. Halvorson is willing to go to the administration with my request for a basic salary of $8000, but wants first to have some assurance that I will accept on those terms. I am therefore writing to you first.

Under these circumstances, I feel that the minimum I can accept is $8000. I hope very much that you will be willing to consider this amount.

The letter is interesting not only because of the $8000 yearly salary that Hershey requests, low by today’s standards, but also his candor in writing to Demerec.

In front of the letter is a photograph of Al Hershey in the laboratory. On This Week in Microbiology #40 Waclaw Szybalski noted that Hershey was not happy when his experiments did not work. In the photo he is pipetting and looking very serious; I wonder what was the outcome of that experiment.

Chase letterAlso in the case is a brief handwritten note from Hershey to Demerec written on 12 June 1950:

Dear Demerec,

I shall be very glad to have Ms. Chase if she decides to accept.

Yours, Hershey

Martha Chase of course did become Hershey’s laboratory assistant.

*Sol Spiegelman was on the faculty of the University of Illinois and presumably was attempting to recruit Hershey. Years later Spiegelman was a Professor at Columbia University. In 1982 he interviewed me for a faculty position in the Department of Microbiology. He was instrumental in obtaining extra start-up funds for my laboratory to match other offers that I had obtained. He recruited me to Columbia but he could not attract Hershey to Illinois.

From a food blender to real-time fluorescent imaging

single phage infectionAlthough Avery, MacLeod, and McCarty showed in 1944 that nucleic acid was both necessary and sufficient for the transfer of bacterial genetic traits, protein was still suspected to be a critical component of viral heredity. Alfred Hershey and Martha Chase showed that this hypothesis was incorrect with a simple experiment involving the use of a food blender. The Hershey-Chase conclusion has since been upheld numerous times*, the most recent by a modern-day experiment using real-time fluorescence.

Hershey and Chase made preparations of the tailed bacteriophage T2 with the viral proteins labeled with radioactive sulfur, and the nucleic acids labeled with radioactive phosphorus. The virions were added to a bacterial host, and after a short period of time were sheared from the cell surface by agitation in a blender. After this treatment, the radioactive phosphorus, but not the radioactive sulfur, remained associated with bacterial cells. These infected cells went on to produce new virus particles, showing that DNA contained all the information needed to produce a bacteriophage.

In a modern validation of the Hershey-Chase experiment, bacteriophages are mixed with a cyanine dye which binds to the viral DNA (illustrated). Upon infection of the bacterial host, the phage DNA is injected into the cell together with the dye. In time the dye leaves the phage DNA and binds to the host genome. This process can be observed in real-time (as it happens) by fluorescence microscopy.

This technique was used to visualize single bacteriophages infecting an E. coli host cell. It takes about 5 minutes on average for 80% of bacteriophage lambda DNA to exit the capsid, with a range of 1-20 minutes.

These experiments do not simply provide a visual counterpart to the Hershey-Chase conclusion, but reveal additional insights into how viral DNA leaves the capsid. One interesting observation is that the amount of DNA that remains in the capsid apparently is not the sole determinant of how quickly ejection occurs. The amount of DNA ejected from the capsid does appear to regulate the dynamics of the process.

The kitchen blender experiment contrasts vividly with the complexity of real-time fluorescent imaging. Hershey and Chase did not have the technology to visualize phage DNA entering the host cell; they used what was available to them at the time. While improved technology is important for pushing research forward, simple experiments will always make important contributions to our understanding of science.

*The infectivity of cloned viral DNA is one validation of the Hershey-Chase experiment.

Hershey, AD, Chase, M. 1952. Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36:39-56. 

Van Valen, D., Wu, D., Chen, Y-J, Tuson, H, Wiggins, P, Phillips, R. 2012. A single-molecule Hershey-Chase experiment. Current Biol 22:1339-1343. 

Milestones in Microbiology at Cold Spring Harbor Laboratory

Milestones at Cold Spring HarborLast week I was at Cold Spring Harbor Laboratory to attend a ceremony designating the well-known laboratory on Long Island as a Milestones in Microbiology site. The purpose of this program, which is administered by the American Society for Microbiology, is to recognize institutions that have substantially advanced the science of microbiology. A plaque is installed which explains the science that was done at the site, and also increases public recognition of  these contributions.

Stan Maloy explained why Cold Spring Harbor Laboratory deserves this honor:

An intensive summer course on bacterial viruses (or phage) begun at Cold Spring Harbor Laboratory in 1945 resulted in advances in bacterial and phage research that led to our understanding of what genes are and how they are expressed, and ultimately germinated the field of molecular biology.

In addition, each summer Cold Spring Harbor Laboratory held meetings that facilitated the enthusiastic exchange of new discoveries and ideas in the rapidly growing field of molecular biology, stimulated largely by microbial geneticists. These discoveries have influenced every aspect of microbiology.

To celebrate the 50th anniversary of the first Phage Course, in 1995 the Laboratory published Coming of Phage (pdf), a brief history of phage and bacterial genetics that examines both the science and the personalities over the years.

Research at Cold Spring Harbor Laboratory has always been associated with significant trends in biology: Darwinian evolution, classical genetics, penicillin production, the use of microbes as model organisms, and the development of the field of molecular biology. The Laboratory is truly a mecca for microbiologists.

Cold Spring Harbor Laboratory is the sixth Milestones in Microbiology site. Others are the Waksman Laboratory at Rutgers University; Hopkins Marine Station in Monterey, California; the site of the University of Pennsylvania Laboratory of Hygiene; Scripps Institution of Oceanography; and the Tulane University School of Public Health and Tropical Medicine.

Stanley Maloy, Bruce Stillman, and James D. Watson spoke at the Cold Spring Harbor Laboratory ceremony at which a plaque commemorating the site was

CSH MIM plaque

unveiled (photo above). A video recording of the ceremony will be posted here soon. At right is a photograph of the plaque that will be installed in the Delbruck Laboratory at Cold Spring Harbor (click for a larger version).

Update. We recorded episode #40 of the science show This Week in Microbiology at Cold Spring Harbor Laboratory on the occasion of its designation as a Milestones in Microbiology site. Vincent and Stanley meet with Waclaw Szybalski and John Kirby to reminisce about how the well known laboratory has advanced the science and teaching of microbiology, and discuss John’s work on the soil dwelling, predatory myxobacteria.

TWiV 87: A PHIREside chat with Professor Graham Hatfull

Hosts: Vincent Racaniello, Alan Dove, Rich Condit, and Graham Hatfull

On episode #87 of the podcast This Week in Virology, Vincent, Alan, and Rich hear from Professor Graham Hatfull how students in the Phage Hunters Integrating Research and Education (PHIRE) program learn about scientific inquiry by doing research on bacteriophages.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $75-$500 off a Drobo.

To enter a drawing to receive 50% off the manufacturers suggested retail price of a Drobo S or FS at, fill out the questionnaire here.

Click the arrow above to play, or right-click to download TWiV #87 (62 MB .mp3, 86 minutes)

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Weekly Science Picks

Rich CDC Public Health Image Library
AlanGreat Microbiologists – A Lego Movie
March of the Microbes by John L. Ingraham
GrahamCoral Reefs in the Microbial Seas by Forest Rohwer

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TWiV 81: Be a virus, see the world

Hosts: Vincent Racaniello and Rich Condit

On episode #81 of the podcast This Week in Virology, Vincent and Rich answer listener questions on viruses and gluten allergy, RNA silencing, influenza virus, herpes simplex virus, HIV/AIDS, chronic fatigue syndrome, manicure salons, and the koala tea of Marseilles.

This episode is sponsored by Data Robotics Inc. Use the promotion code TWIVPOD to receive $75-$500 off a Drobo.

Win a free Drobo S! Contest rules here.

Click the arrow above to play, or right-click to download TWiV #81 (68 MB .mp3, 94 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

Weekly Science Picks

Rich Google Chrome browser ‘speed test‘ (and how it was made)
Vincent Inside the Outbreaks by Mark Pendergrast

Send your virology questions and comments (email or mp3 file) to or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at and tag them with twiv.