Virus-induced fever might change bacteria from commensal to pathogen

Stem-loopNeisseria meningitidis may cause septicemia (bacteria in the blood) and meningitis (infection of the membrane surrounding the brain), but the bacterium colonizes the nasopharynx in 10-20% of the human population without causing disease. Although understanding how the bacterium changes from a commensal to a pathogen has been elusive, an important property is believed to be the ability to resist destruction by the immune response. Fever caused by a viral infection might be the trigger that makes N. meningitidis evade immunity.

A property of N. meningitidis that makes it cause disease is resistance to complement, a collection of proteins in the blood that help clear pathogens. N. meningitidis has evolved several mechanisms to avoid destruction by complement, including the production of a polysaccharide capsule, addition of sialic acid to a component of the bacterial outer membrane, and the production of a protein that binds one of the complement proteins. It is not clear why a commensal organism would have evolved such evasion mechansims – invading the blood and the brain are dead ends, as they do not lead to transmission to a new host.

An answer to this question comes from the finding that N. meningitidis proteins essential for resistance to complement are under the control of an RNA thermosensor. This control element is an RNA stem loop structure (pictured) formed by base pairing of local sequences within the RNA. Buried in the base-paired stem is a short sequence called the ribosome binding site that is essential for translation of the mRNAs into protein. At 30°C, the stem loop structure is intact, preventing binding of ribosomes to the mRNA; protein synthesis is blocked. At elevated temperatures – 37 or 40°C – abundant protein synthesis takes place, because the RNA secondary structure is denatured, allowing ribosomes to more efficiently access the ribosome binding site on the mRNA.

How do these findings explain why N. meningitidis becomes a pathogen? The temperature of the upper respiratory tract, where the bacterium normally colonizes, is low, so the RNA sensor is intact, preventing production of proteins needed for resistance to complement. If the respiratory tract is infected with a virus, a local immune response occurs which is accompanied by high temperatures – a fever. The RNA thermosensors of N. meningitidis have evolved to sense elevated temperature and turn on the synthesis of proteins that help it to avoid destruction by the immune response. Unfortunately, inflammation also damages the mucosal barriers that normally prevent microbes from invading the underlying tissues, where they have access to the bloodstream. N. meningitidis enters the blood stream, and because it is resistant to complement, it is not cleared. The result may be septicemia and infection of the brain. Moving away from its niche in the respiratory tract is probably not part of the microbe’s plan, but rather a consequence of the fact that it has evolved to survive immune responses to other pathogens in the respiratory tract.

There is some epidemiological support for this scenario: peaks of Neisseria meningitidis disease may follow outbreaks of influenza.

The conversion of commensal bacteria into pathogens by a second infection may be more common than we know. Streptococcus pneumoniae is a human nasopharyngeal commensal that colonizes 10 to 40% of healthy individuals. The bacterium is also a leading cause of respiratory disease. There is evidence that infection with influenza virus releases S. pneumoniae bacteria from biofilms; the free-living bacteria are then able to cause respiratory disease. One of the influenza virus-induced host signals responsible for changing S. pneumoniae from a commensal to a pathogen is fever.  For more on this story, listen to This Week in Microbiology #62.

TWiV 112: Creating a killer poxvirus

dickson despommierHosts: Vincent RacanielloAlan Dove, and Rich Condit

On episode #112 of the podcast This Week in Virology, Vincent, Alan, and Rich review the making of a virulent poxvirus by insertion of the gene encoding IL-4, and severe 2009 H1N1 influenza due to pathogenic immune complexes.

Click the arrow above to play, or right-click to download TWiV #112(71 MB .mp3, 98 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 – The Scientist’s Top 10 Innovations 2010
Alan – Avian Vocalizations Center
Vincent – Microbial soap from Cleaner Science (thanks, Nadia!)

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.

Natural antibody protects against viral infection

galactose-a1-3-galactoseAntibodies produced by infection with a virus, or after immunization with viral vaccines, are effective at preventing viral disease. However humans and higher primates contain “natural antibodies” which are present in serum before viral infection. Natural antibodies can activate the classical complement pathway leading to lysis of enveloped virus particles long before the adaptive immune response is activated.

Many natural antibodies are directed against the disaccharide galactose α(1,3)-galactose (α-Gal) (illustration), which is found as a terminal sugar on glycosylated cell surface proteins. Humans, apes, and Old World monkeys lack the gene encoding the enzyme galactosyltransferase, which attaches α-Gal to membrane proteins. Lower primates, most other animals, and bacteria synthesize the disaccharide.

Human serum contains high levels of antibodies specific for α-Gal because the human gut contains bacteria that produce this sugar. Over 2% of serum IgM and IgG antibodies are directed against α-Gal. This antibody binds to the membrane of enveloped viruses that contain α-Gal antigens and triggers the classical complement cascade, leading to lysis of virions and loss of infectivity.

The anti-α-Gal antibody-complement reaction is probably the main reason why humans and higher primates are not infected by enveloped viruses of other animals. Many of these viruses infect human cells efficiently in culture because the complement proteins in serum are inactivated by heating. For example, when vesicular stomatitis virus, human immunodeficiency virus type 2, and human foamy virus are grown in non-human cells which produce α-Gal, the virions can be inactivated by fresh (i.e. not heat-inactivated) human serum. When the same viruses are propagated in human cells, which lack α-Gal, fresh human serum has no effect on viral infectivity. Because of these findings, virus vectors for gene therapy are produced in cells lacking galactosyltransferase to avoid complement-mediated inactivation of the viruses in humans.

When strains of mice that cannot produce any antibodies are infected with vesicular stomatitis virus, titers in the kidney and brain are 10-100 times higher than in normal mice. Furthermore, virus titers in lymph nodes are 10-100 times lower than in antibody-producing mice.

Anti-α-Gal antibodies are an example of how the adaptive immune system cooperates with the innate complement cascade to provide immediate action before specific anti-viral antibodies are developed. Natural antibodies prevent the spread of viruses to vital organs, and improve immunogenicity by enhancing the trapping of antigen in secondary lymphoid organs such as lymph nodes.

Takeuchi Y, Liong SH, Bieniasz PD, Jäger U, Porter CD, Friedman T, McClure MO, & Weiss RA (1997). Sensitization of rhabdo-, lenti-, and spumaviruses to human serum by galactosyl(alpha1-3)galactosylation. Journal of Virology, 71 (8), 6174-8 PMID: 9223512

Ochsenbein AF, Fehr T, Lutz C, Suter M, Brombacher F, Hengartner H, & Zinkernagel RM (1999). Control of early viral and bacterial distribution and disease by natural antibodies. Science (New York, N.Y.), 286 (5447), 2156-9 PMID: 10591647

C1q and the collectins

c1q-bindingThe classical complement pathway begins when the initiator protein C1q binds directly to the surface of a pathogen, or to an antibody that is bound to a microbe.

There is a binding site for C1q on the Fc portion of antibody molecules. C1q can also recognize molecular patterns characteristic of pathogens, much like the Toll-like receptors.

C1q binding to antibody or a pathogen surface initiates an unusual protease cascade with one or more members of a set of seven activating enzymes. This set of cleavages, which occurs on the surface of the microbe, leads to the formation of the membrane attack complex that produces holes in membranes of cells and viruses. Other products of the cascade include mediators of inflammation, which recruit white blood cells to the site of infection. Still more cascade components remain on the surface of the microbe and lead to phagocytosis.

The alternative complement pathway has a distinctive mode of initiation: the abundant C3 protein in plasma is spontaneously hydrolyzed to C3b. The latter protein binds to any membrane surface. Complement regulatory proteins in the membranes of normal cells block further action. When C3b binds to the membrane of a pathogen, there are no complement regulatory proteins present, and therefore the cascade continues and amplifies. Ultimately the membrane attack complex, and mediators of inflammation and opsonization are formed as in the classical pathway.

The mannan-binding pathway is initiated by the mannose-binding collectins that bind to complex carbohydrate residues on the surface of pathogens. Collectins are sugar binding proteins (lectins) that contain collagen and require calcium for binding. When mannose-binding lectins bind to the microbial surface, a protease cascades is initiated that leads to formation of the membrane attack complex or opsonization.

C1q and the collectins are an important and often overlooked component of the first line of defense against pathogens. These proteins can bind to microbes, leading to lysis or phagocytosis, long before the adaptive immune response is activated.

The animal sera used to supplement cell culture media is usually heated for 55°C for 30 minutes before its use in the propagation of enveloped viruses. The purpose of this step is to inactivate the complement proteins which might bind and lyse virions. I don’t bother with heat inactivation as the viruses I work with lack envelopes.

P.S. Wouldn’t C1q and the collectins be a terrific name for a rock band?

The complement system

The complement system is a collection of blood and cell surface proteins that is a major primary defense and a clearance component of innate and adaptive immune responses. At least 30 different complement proteins act sequentially to produce a wide ranges of activities, from cell lysis to augmentation of the adaptive response. The complement system has four major antimicrobial functions.

Lysis – Polymerization of specific activated complement components on a foreign cell or enveloped virus leads to the formation of pores. The lipid bilayer of the cell or virus is disrupted.

Activation of inflammation – Several peptides produced by proteolytic cleavage of complement proteins bind to vascular endothelial cells and lymphocytes. These cells then produce cytokines which stimulate inflammation and enhances responses to foreign antigens.

Opsonization – Certain complement proteins can bind to virions. Phagocytic cells with receptors for these complement proteins can then engulf the virus particles and destroy them. This process is called opsonization.

Solubilization of immune complexes – Some virus infections that are not cytopathic – the virus does not kill cells – lead to the accumulation of antibody-virus complexes. When these immune complexes lodge in blood vessels they can cause damage. An example is glomerulonephritis caused by deposition of antibody-antigen complexes in the kidney. Some complement proteins can disrupt these complexes and facilitate their clearance from the circulatory system.

There are three different complement pathways: classical, alternative, and mannan-binding. Unfortunately the nomenclature of the complement proteins is confusing, because they were named as they were discovered, not according to their function (see illustration below of the classical pathway). We’ll discuss the different pathways in the course of several posts. Don’t be daunted by the apparent complexity; stay with me and you’ll have a good appreciation of an extremely important part of our immune defense system.

And yes, viruses have evolved to modify the complement system.

classical-complement-s(click for a large version)