Virologist Roger W. Hendrix died on 15 August 2017. I only met Roger once, at the 2011 ASM meeting in New Orleans where we recorded an episode of This Week in Virology. The video of that episode is below, starting at my conversation with Roger at 30:34. Harmit Malik and Rachel Katzenellenbogen were my other guests on TWiV 135.
Not 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.
Viruses infect every living organism on the planet, but not every habitat has been explored for their presence. The igneous ocean crust had not yet been examined for viruses, but seek and ye shall find: there are plenty of viruses under the seas.
The oceanic basement is an enormous ecosystem that lies at the bottom of the seas, beneath a thick layer of sediment. It is composed of igneous rock through which percolates 20 million cubic kilometers of water. Previous study of cores from this region has revelealed the presence of prokaryotes, but no one had looked for viruses.
Facilitating the study of the oceanic basement are seafloor observatories that have been placed into existing boreholes. Two have been placed in 3.5 million year old rock in the northeastern Pacific Ocean. They penetrate hundreds of meters through sediment and into the basement (illustrated; image credit) and are fitted with plumbing that allows sampling of uncontaminated fluids from different depths in the basement rock.
Analysis of fluids recovered from these sites revealed both prokaryotes (8,000 per ml) and virus particles (90,000 per ml). Ribosomal RNA sequence analysis showed that bacteria dominated these communities, with some Archaea but virtually no eukaryotes.
Examination of the fluids by electron microscopy showed virus particles of different kinds: tailed and untailed icosahedral particles, untailed globular particles, and rod and spindle shaped. My favorite is the lemon shaped particle, for its form and implied taste.
To provide information on the viral genomes in the oceanic basement, sequences were determined from total cellular DNA (material retained on a 0.2 micron filter) extracted from the samples. Most viral sequences likely had archaeal hosts. Some prophage sequences were identified – viral genomes integrated into host DNA – which allowed more certain identification of the infected cell.
Most of the identified archaeal and bacterial virus sequences came from the families Myoviridae and Siphoviridae (think tailed, icosahedral viruses). One complete circular DNA genome identified is 55,906 nucleotides in length with 81 open reading frames. Twenty of these encode proteins with recognizable functions, such as capsid proteins, a primase and a DNA polymerase. No genes encoding tRNAs, such as those found in giant viruses, were identified.
Some sequences were similar to those of giant viruses like mimiviruses and phycodnaviruses. These viruses are known to only infect eukaryotes. Eukaryotic genomes were rare in the basement metagenome collections (1% of the community in one location).
I am not surprised that viruses have been found in ocean’s basement. Still, I’m amazed when I think about how far down they are, in warm water (65 degrees C) in 3.5 million year old rock.
To paraphrase Samuel E. Wright (Under The Sea):
The viruses are always greenerIn somebody else’s lakeYou dream about going up thereBut that is a big mistakeJust look at the viruses around youRight here under the ocean floorSuch wonderful viruses surround youWhat more is you lookin’ for?
The lovely TWiV team explore evolution of our fecal virome, and the antiviral RNA interference response in the nematode C. elegans.
You can find TWiV #433 at microbe.tv/twiv, or listen below.
Become a patron of TWiV!
The TWiVirions reveal bacteriophage genes that control eukaryotic reproduction, and the biochemical basis for increased Ebolavirus glycoprotein activity during the recent outbreak.
You can find TWiV #431 at microbe.tv/twiv, or listen below.
Become a patron of TWiV!
The obligate intracellular bacteria Wolbachia (pictured), which infects 40% of arthropods, can manipulate its host to ensure its maintenance in the population. An example is cytoplasmic incompatibility, which occurs when infected males mate with uninfected females, and causes embryonic lethality (mating with an infected female produces viable offspring). Two Wolbachia genes responsible for this phenotype have been identified, and they are viral (link to paper).
A comparison of genome sequences of different Wolbachia strains that do or do not cause cytoplasmic incompatibility (CI) revealed two genes that were candidates for this phenotype. Both genes are transcribed in the testes of fruit flies, but at lower levels in older male flies which show decreased CI.
When either gene was expressed in male transgenic fruit flies, there was no effect on hatch rates after mating with uninfected females. When both genes were expressed in male flies, mating with uninfected females led to substantially reduced hatch rates. This transgene-induced lethality was rescued when the flies were mated with Wolbachia-infected females.
The two genes that together cause CI are called cytoplasmic incompatibility factor A and B (cifA, cifB). The cytological defects caused by these genes resemble those observed in Wolbachia-induced CI: most embryos do not divide more than two or three times.
Remarkably (or perhaps not!), cifA and cifB are not Wolbachia genes, but are viral. Wolbachia are infected with a bacteriophage called WO; nearly all sequenced Wolbachia genomes contain integrated WO DNA, and it is within this WO prophage that are found cifA and cifB. In other words, the ability of Wolbachia to control the reproduction of its arthropod host is regulated by two viral genes integrated in the bacterial genome.
Because CI caused by Wolbachia is a means of increasing their proportion in the female line (the bacteria are maternally inherited), cifA and cifB also enable spread of WO bacteriophage.
How cifA and cifB cause CI is unknown – most of the encoded proteins have no recognized protein domains with the exception of weak homology to proteases. Understanding this mechanism might also contribute to controlling the spread of arboviruses: Wolbachia is known to inhibit replication of some mosquito borne viruses such as dengue virus and Zika virus.
The TWiVsters explain how superspreader bacteriophages release intact DNA from infected cells, and the role of astrocytes in protecting the cerebellum from virus infection.
You can find TWiV #428 at microbe.tv/twiv, or listen below.
Become a patron of TWiV!
Bacteriophages are the most abundant biological entities on Earth. There are 1031 of them on the planet, and they infect 1023 to 1025 bacteria every second. That’s a lot of lysis, and it leads to the release of huge quantities of DNA that can be taken up by other organisms, leading to new traits. It seems that some bacteriophages are very, very good at releasing intact DNA, and they have been called superspreaders (link to paper).
In a very simple experiment, E. coli cells carrying a plasmid encoding ampicillin resistance were infected with the well studied phages T4 and T7 and also with a collection of 20 phages isolated from soil, water, and feces in Miami and Washington DC. After the cells lysed, DNA was extracted from the culture medium and introduced into antibiotic sensitive E. coli. Two phages, called SUSP1 and SUSP2, were thousands of times better at releasing plasmid DNA that readily conferred antibiotic resistance. These phages are superspreaders.
Superspreader phages can promote transformation by different plasmids, so their unique talent is not sequence specific. When these phages lyse cells, intact plasmid DNA is released. In contrast, phage T4 infection leads to degradation of plasmid DNA in the host cell. Superspreader phages lack genes encoding known endonucleases – enzymes that degrade DNA, possibly explaining why plasmids are not degraded during infection. Other phages that lack such endonucleases, including mutants of lambda and T4, also promote plasmid mediated transformation.
Phages SUP1 and SUP2 don’t just spread plasmids to laboratory strains like E. coli. When crude mixtures of soil bacteria from Wyoming and Maryland were mixed with SUP1 and SUP2 lysates from E. coli, antibiotic resistance was readily transferred. One of the main recipients of plasmid DNA is a member of the Bacillus genus of soil bacteria, showing that superspreaders can move DNA into hosts of a species other than the one they can infect.
With so many bacteriophages on the planet, it is likely that there are many other superspreaders like SUP1 and SUP2 out there. The implication is that massive amounts of intact plasmid DNAs are being released every second. These DNAs can be readily taken up into other bacteria, leading to new phenotypes such as antibiotic resistance, altered host range, virulence, the ability to colonize new niches, and much more.
You might recall learning in high school biology that bacteriophage infection of a host can lead to either replication and cell lysis, or integration of the viral genome into the host (illustrated). The latter event, called lysogeny, spares the host from virus induced killing. For some phages, the decision between lysis and lysogeny appears to be communicated between cells by a small peptide (link to paper).
Evidence that virus-infected cells produce a substance that can regulate the lysis-lysogeny decision came from the observation that conditioned medium from Bacillus subtilis infected with the bacteriophage phi3T – prepared so that is was virus and cell free – protects cells from lysis. The protective component is destroyed by digestion with a proteinase and hence is a protein. Conditioned medium not only inhibits cell lysis, but increases lysogeny, measured by integration of viral DNA into the bacterial genome.
Examination of the genome sequence of phage phi3T suggested that a six amino acid peptide, Ser-Ala-Ile-Arg-Gly-Ala, was the component in conditioned medium that regulates the lytic-lysogenic decision. Addition of the synthetic peptide to infected cells decreased lysis. The levels of this peptide increase during each cycle of phage infection of the Bacillus host.
The authors call the communication peptide ‘arbitrium’ from the Latin word meaning ‘decision’. The gene encoding the peptide is aimP.
AimP appears to work by entering the bacterium through a transporter protein and binding a protein in the bacterial cell called AimR. The AimR protein in turn binds a sequence in the bacterial genome called aimX. When AimR is bound by the peptide, it cannot bind aimX and lysogeny occurs. In the absence of peptide, AimR binds aimX and lysis proceeds. The product of the aimX gene appears to be a regulatory RNA, but how it promotes lysis is not known.
Different phages of B. subtilis also encode peptides that regulate the lysis-lysogeny decision in a phage-specific manner.
These findings describe a viral communication system that determines whether a bacterial host is lysed or lysogenized. When viruses initially infect a host, the result is lysis because levels of peptide are low. After several cycles of infection the AimP concentrations increase, and upon entry of the peptide into bacteria they lead to lysogeny.
The authors of this work suggest that the arbitrium system is a way for the virus to sense the amount of previous infections to decide whether lysis or lysogeny should occur. If many previous infections have taken place, the host population could be too low to support lytic replication, hence lysogeny occurs. Because lysogens can divide, the bacterial population can be restored to a level that can sustain virus infection.
Of course, the virus particle cannot sense anything – it is a bacterial protein that binds AimP and another bacterial gene that controls lysis. In other words, the virus-infected cell, not the virus, can sense the amount of previous infections.
It should be straightforward to search the genome sequences of phages that infect other bacteria to determine if such a communication system is widespread. More interesting is whether viruses that infect eukaryotes also have communication systems that guide decisions about lytic versus non-lytic or latent infection.
A unique feature of eukaryotic cells, which distinguishes them from bacteria, is the presence of a membrane-bound nucleus that contains the chromosomal DNA (illustrated; image credit). Surprisingly, a nucleus-like structure that forms during viral infection of bacteria is the site of viral DNA replication (link to paper).
During infection of Pseudomonas bacteria with the phage 2O1phi2-1, a separate compartment forms in which viral DNA replication takes place. A phage protein, gp105, makes up the outer layer of this compartment, which initially forms near one end of the cell, and then migrates to the center. The migration of the compartment takes place on a spindle made up of the tubulin-like protein PhuZ.
In addition to viral DNA, certain proteins gain entry into this compartment, including viral proteins involved in DNA and mRNA synthesis, and at least one host cell protein. Other proteins, such as those involved in translation and nucleotide synthesis, are excluded. This compartmentalization very much resembles that of the nucleus of eukaryotic cells.
Packaging of the viral DNA takes place on the surface of the viral nucleus. Empty phage capsids form at the bacterial cytoplasmic membrane, then migrate to the compartment where they attach firmly to the surface. By an unknown mechanism, DNA moves from the compartment into the capsid. Then capsids are released from the surface to further mature in the cytoplasm. The completed phages are released from the cell upon bacterial lysis.
These fascinating observations raise a number of unanswered questions. Does infection with other phages lead to assembly of a viral nucleus? How do molecules selectively move in and out of the structure?
Perhaps the most interesting question relates to the origin of viruses and cells. According to one hypothesis, self-replicating, virus-like nucleic acids might have first appeared on Earth, followed by cells without a nucleus. Was the nucleus a viral invention?