Viruses help form biofilms

Inoviridae virionBacteria frequently grow in communities called biofilms, which are aggregates of cells and polymers. An example of a biofilm is the dental plaque on your teeth. Biofilms are medically important as they can allow bacteria to persist in host tissues and on catheters, and confer increased resistance to antibiotics and dessication. Therefore understanding how biofilms form is crucial for controlling microbial infections. An advance in our understanding of biofilms formation is the observation that filamentous phages help them assemble, and contribute to their fundamental properties.

Pseudomonas aeruginosa is an important human pathogen which is a particular problem in patients with cystic fibrosis. The ability of this bacterium to form biofilms in the lung is linked to its ability to cause chronic infections. Pseudomonas aeruginosa biofilms contain large numbers of filamentous Pf bacteriophages (pictured; image credit). These viruses lyse cells and release DNA, which becomes one component of the biofilm matrix.

Mixing supernatants of P. aeruginosa cultures with hyaluronan, which is present in airways of cystic fibrosis patients, resulted in the formation of a biofilm – in the absence of bacteria. A major component of P. aeruginosa biofilms was found to be Pf bacteriophages. When purifed Pf bacteriophages were mixed with hyaluronan, biofilms formed. Similar biofilms also formed when the filamentous bacteriophage fd of E. coli was mixed with hyaluronan. Mixtures of Pf bacteriophages and various polymers (alginate, DNA, hyaluronan, polyethylene glycol) formed liquid crystals (matter in a state between a liquid and a solid crystal).

Pf phages were detected in sputum from patients with cystic fibrosis, but not in uninfected patients. Addition of Pf phage to sputum from patients infected with P. aeruginosa made the samples more birefringent, a property of liquid crystals. Compared with a strain of P. aeruginosa that does not produce Pf phage, colonies of virus-producing strains formed liquid crystals. These observations indicate that Pf phage help organize the bacteria into a biofilm matrix.

Some features of biofilms include their ability to adhere to surfaces, to protect bacteria from dessication, and to increase resistance to antibiotics. Addition of phage Pf increased biofilm adhesion and tolerance against dessication. Such addition also made the biofilm more resistant to aminoglycoside antibiotics, because these were sequestered in the biofilm. No phage-mediated increased resistance to ciprofloxacin was observed, probably because this antimicrobial does not interact with polyanions of the biofilm as do aminoglycosides.

These results show that presence of bacteriophage in a biofilm of P. aeruginosa helps organize the matrix while contributing to some of its fundamental properties. It seems likely that filamentous phages of other bacteria will play roles in biofilm formation, suggesting that targeting the phages in these matrices could be effectie strategies for treating biofilm infections.

TWiV 346: A double helical career

Episode #346 of the science show This Week in Virology was recorded at the 34th Annual Meeting of the American Society for Virology, where Vincent, Rich, and Kathy spoke with Joan Steitz, a tireless promoter of women in science and one of the greatest scientists of our generation.

You can find TWiV #346 at www.microbe.tv/twiv.

Your viral past

virusesDid you ever wonder what different virus infections you have had in your lifetime? Now you can find out with just a drop of your blood and about $25.

Immune defense systems of many hosts produce antibodies in response to virus infections. These large proteins, which are generally virus specific, can block or inhibit virus infection, and persist at low levels for many years after the initial infection. Hence it is possible to determine whether an individual has had a virus infection by looking for anti-viral antibodies in the blood. Up to now the process of identifying such antibodies has been slow and limited to one or a few viruses. A new assay called VirScan allows unbiased searches for all the virus antibodies in your blood, providing a picture of all your past infections.

To identify the human antivirome, DNAs were synthesized encoding proteins from all viruses known to infect humans – 206 species and over 1000 strains. These DNAs were inserted into the genome of a bacteriophage, so that upon infecting bacteria, the viral peptides are displayed on the phage capsid. These ‘display’ phages were then mixed with human serum, and those that were bound by antibodies were isolated. The DNA sequence of the phage genomes were then determined to identify the human virus bound by the antibodies.

This method was used to assay samples from 569 humans. The results show that each person had been exposed to an average of 10 viruses, with a range from a few to over 20 (two individuals had antibodies to 84 different virus species!). The most frequently identified viruses included herpesviruses, rhinoviruses, adenoviruses, influenza viruses, respiratory syncytial virus, and enteroviruses. The overall winner, found in 88% of samples, is Epstein-Barr virus.

These results are not unexpected: all of us are infected with at least a dozen viruses at any time, and the viruses identified in this study known to infect much of the human population. What was surprising is the absence of some common viruses, such as rotaviruses, and the ubiquitous polyomaviruses. According to serological surveys, the most common human viruses are the small, single-stranded DNA containing anelloviruses. Yet the related torque teno virus was only found in 1.7% of samples. These differences are likely due to a combination of technical and biological issues (e.g., failure of antibodies to certain viruses to persist in serum).

This new assay may one day become a routine diagnostic tool that is used along with complete blood counts and chemistries to know if a patient’s signs and symptoms might be attributable to a past virus infection. VirScan technology is not limited to virus infections – it can be used to provide a history of bouts with bacteria, fungi, and parasites.

VirScan might also allow us to determine which virus infections are beneficial, and which contribute to chronic diseases such as autoimmune or neurodevelopmental disorders or cancer. The assay can be used to conduct unbiased population-based studies of the prevalence of virus infections and their possible association with these diseases. Such connections were not previously possible with antibody assays that search for one virus at a time. This approach was not only inefficient, but required guessing the responsible virus.

Some other findings of this study are noteworthy. As expected, children had fewer virus infections than adults. HIV-positive individuals had antibodies to more viruses than HIV-negative individuals, also expected given the damage done by this virus to the immune system. Frequencies of anti-viral antibodies were higher outside of the United States, possible due to differences in genetics, sanitation, or population density. In most samples, there was a single dominant peptide per virus, although there were occasional differences among populations. This information might be useful for improving vaccines, or tailoring them to specific countries or regions.

Update: It would be very informative to use VirScan to search for antibodies against viruses that are not known to infect humans. Other animal viruses, plant viruses, insect viruses: to which do a significant fraction of humans respond? The information might identify other viruses that replicate in humans and which might constitute future threats (or present benefits).

TWiV 332: Vanderbilt virology

On episode #332 of the science show This Week in Virology, Vincent visits Vanderbilt University and meets up with Seth, Jim, and Mark to talk about their work on a virus of Wolbachia, anti-viral antibodies, and coronaviruses.

You can find TWiV #332 at www.microbe.tv/twiv.

TWiV 323: A skid loader full of viromes

On episode #323 of the science show This Week in Virology, the family TWiVidae discuss changes in the human fecal virome associated with Crohn’s disease and ulcerative colitis.

You can find TWiV #323 at www.microbe.tv/twiv.

TWiV 301: Marine viruses and insect defense

On episode #301 of the science show This Week in Virology, Vincent travels to the International Congress of Virology in Montreal and speaks with Carla Saleh and Curtis Suttle about their work on RNA interference and antiviral defense in fruit flies, and viruses in the sea, the greatest biodiversity on Earth.

You can find TWiV #301 at www.microbe.tv/twiv.

Viral fiber art

dengue virus pillowViruses inspire many different types of art, but I was unaware of the number of people who make viruses out of fiber!

On This Week in Virology #266 we heard from Emily who had knitted a dengue virus pillow (photograph at left).

The next week on TWiV #267 we heard from Carolyn who had knitted a picornavirus (photo below).

knit picornavirus

The following week (TWiV #268) we heard from Jessica who has also knitted two different icosahedral structures.

knit icoshadedron

This made me wonder how many people knit viruses, so I searched Ravelry for ‘virus’. Here are some of the interesting creations I found.

Cold virus by Krista:

Cold virus

Dawn’s cold virus (rhinovirus):

rhinovirus

Melini’s phage hat:

phage hat

Two H1N1 influenza viruses:

h1n1 knit

And Susan’s bacteriophage:

knit bacteriophage

There are also bacteria, such as this collection (with some viruses) from Clare:

knit microbes

You can find more by searching for ‘microbe’ at Ravelry (login required), where you’ll also find the patterns to reproduce these wonderful creations. Microbes are clearly inspiring and fascinating to fiber artists!

Do you make fiber viruses? If so let me know and we can include a photograph here.

TWiV 262: Wrong form, right professor

On episode #262 of the science show This Week in Virology, Vincent returns to the University of Wisconsin – Madison to speak with Ann Palmenberg about her career in virology.

You can find TWiV #262 at www.microbe.tv/twiv.

Viruses might provide mucosal immunity

T4 HocThe mucosal membranes that line our respiratory, alimentary, and urogenital tracts and the outer surface of the eyes are portals of entry for microbes. The cells at these surfaces have functions that require that they are exposed to the environment – for example, gaseous exchange in the lung between inspired air and the blood. Mucus, pH extremes, enzymes, and immune cells are some of the antimicrobial defenses that are present at various mucosal surfaces. It now appears that bacteriophages – viruses that infect bacteria – might also be part of the mucosal antimicrobial defense system.

A sampling of the ratio of bacteriophages to bacteria in a variety of mucosal surfaces (sea anemone, hard and soft coral, polychaete, teleost, human gum, and mouse intestine) revealed higher ratios when compared to non-mucosal samples (e.g. neighboring sea water or saliva). A model bacteriophage of E. coli, T4, was used to show that phage specifically attach to mucus: adherence to cultured cells was reduced when mucus was not produced or removed by chemical treatment.

The principal macromolecules in mucus are mucin glycoproteins, which consist of a polypeptide chain linked to hundreds of variable, branched sugar molecules. Mucins are continuously produced at mucosal surfaces which gives rise to a thick protective layer. Phage T4 was found to attach specifically to mucins and not other components of mucus such as protein or DNA. The attachment of phage T4 to mucus-producing cultured cells reduced the number of bacteria that could attach to and kill the cells. This antimicrobial effect was substantially reduced when a strain of phage T4 was used that cannot lyse its bacterial host. Therefore phages bound in mucus protect cells by infecting and lysing bacterial invaders.

Phage T4 attaches to mucins through immunoglobulin-like (Ig-like) proteins present in the viral capsid. First discovered in antibody molecules, the Ig domain has since been found in hundreds of different proteins with various functions and appear to be encoded in ~25% of dsDNA phages. The Ig domain, typically 80 amino acids in length, is often involved in interactions with other proteins or ligands. For example, the Ig domains of antibodies interact with antigens, and the poliovirus receptor interacts with poliovirus via an Ig domain on the receptor molecule. The capsid of phage T4 contains 155 copies of an Ig-like protein called Hoc (colored yellow in the image). Deletion of the phage T4 hoc gene reduced binding of the virus to mucin, showing that adherence to mucin requires Ig-like protein domains.

These results demonstrate that a model bacteriophage, T4, attaches to mucus via an interaction between viral Ig-like capsid proteins and mucins. The ability of phages to attach to mucin clearly helps protect cultured cells from bacterial attachment and killing. Bacteriophages may be part of a previously unrecognized mucosal immune defense system. This suggests a symbiotic relationship between phages and metazoan hosts: the phages provide protection to mucosal surfaces, and in turn are provided hosts (bacteria) in which to reproduce. However, additional experiments are required to prove the authors’ conclusion of a “key role of the world’s most abundant biological entities in the metazoan immune system”. It will be necessary to directly demonstrate that phages attaching to mucins in mucus can protect an animal (e.g. mice) from bacterial invasion. This will not be an easy experiment because the phage and bacteria composition of mucus is likely to be complex and continuously changing as mucus is sloughed from cells and new mucins are produced.

The finding that phages play roles in mucosal immunity would have far-reaching consequences for human health. Some fascinating questions that come to mind include: do phage populations play roles in human diseases? Are they altered in human diseases and can we correct these diseases by restoring phage populations? Are the phage populations altered by antimicrobial therapy that alters bacterial populations? Do phages contribute to development of the immune system by modulating bacterial populations? Might mucus-bound phages stabilize the microbiome?

Update: Michael Schmidt and I discussed these remarkable findings on episode #59 of the science show This Week in Microbiology.

Live from the Society for General Microbiology Conference in Manchester, UK

MicrobeWorld and the Society for General Microbiology (UK) to live stream two events from their Spring Conference 2013 in Manchester, England, March 25-28.

Peter Wildy Prize for Microbiology Education
Monday, March 25, 2013 17:20 GMT (1:20 PM EST | 10:20 AM PST)  

David Bhella, Ph.D., will be accepting the Peter Wildy Prize for Microbiology Education, awarded annually by the Society for General Microbiology for an outstanding contribution to microbiology education. Bhella’s acceptance speech will be live streamed at 17:20 GMT (1:20 PM EST | 10:20 AM PST). Vincent Racaniello was awarded the Wildy Prize in 2012.

This Week in Microbiology
Wednesday, March 27, 2013 15:30 GMT (11:30 AM EST | 8:30 AM PST) 

Join Vincent Racaniello and co-host Laura Piddock, Ph.D., with guests Paul Williams, Ph.D., Kalin Vetsigian, Ph.D., and David Harper, Ph.D., for a live-streaming episode of This Week in Microbiology. The live stream starts at 15:30 PM GMT (11:30 AM EST | 8:30 AM PST) and you can watch it below. If you have any questions for Vincent or his guests during the broadcast you can tweet your question using the #sgmman hash tag or type it into the chat function of the video player.

If you live elsewhere in the world, please use www.everytimezone.com, to calculate when the live streams will start in your area.

 

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