Microorganisms: Infectious Disease and Ecology

Blog entries about bacteria, viruses, fungi and other microorganisms.

Screening for Antiviral Compounds under Level 4 Containment Conditions

Posted on by Isobel Maciver

Working with bacteria and viruses that cause life-threatening diseases with no currently available treatment options takes guts. Most scientists are familiar with the routine requirements of good aseptic technique, are highly aware of laboratory safety requirements, and are more than familiar with autoclaves and sterilization issues, but if we make a mistake the consequences are usually only lost time or a spoiled experiment—not a lost life.

Scientists working with highly virulent organisms deal with a whole other level of risk that requires adherence to the strictest of safety regulations, and these containment regulations can sometimes place constraints on the type of experiment that can be performed with dangerous pathogens. A paper published in the April 2014 issue of Assay and Drug Development Technologies brought this to my attention and reminded me of the serious issues some scientists face on a daily basis as they research ways to combat infectious diseases.

Continue reading “Screening for Antiviral Compounds under Level 4 Containment Conditions”

His-Tagged Fusion Proteins: Application Update

Posted on by Gary Kobs

Crystal structure of Polyhistidine tagged recombinant catalytic subunit of cAMP-dependent protein kinase. Credit: StructureID=1fmo; DOI=http://dx.doi.org/10.2210/pdb1fmo/pdb;
Crystal structure of Polyhistidine tagged recombinant catalytic subunit of cAMP-dependent protein kinase. Credit: StructureID=1fmo; DOI=http://dx.doi.org/10.2210/pdb1fmo/pdb;

Researchers often need to purify a single protein for further study. One method for isolating a specific protein is the use of affinity tags. Affinity purification tags can be fused to any recombinant protein of interest, allowing fast and easy purification following a procedure that is based on the affinity properties of the tag.

The most commonly used tag to purify and detect recombinant expressed proteins is the polyhistidine tag. Protein purification using polyhistidine tags relies on the affinity of histidine residues for immobilized metal such as nickel, which allows selective protein purification. The metal is immobilized to a support through complex formation with a chelate that is covalently attached to the support.

Polyhistidine tags offer several advantages for protein purification. The small size of the polyhistidine tag renders it less immunogenic than other larger tags. Therefore, the tag usually does not need to be removed for downstream applications following purification.

A large number of commercial expression vectors that contain polyhistidine are available. The polyhistidine tag may be placed on either the N- or C-terminus of the protein of interest.

And finally, the interaction of the polyhistidine tag with the metal does not depend on the tertiary structure of the tag, making it possible to purify otherwise insoluble proteins using denaturing conditions. The resulting purified protein can be used for a variety of applications.

The following references illustrate examples of some of the most common post purification applications with fusion proteins containing a polyhistidine tag:

Enzymatic assays

  1. Negi, V-S. et al. (2014) A carbon nitrogen Lyase from Leucaena leucocephala catalyzes the first Step of mimosine degradation. Plant Physiol. 164,  922–34.
  2. Yu, S. et al. (2013) Syk Inhibits the activity of protein kinase A by phosphorylation tyrosine of the catalytic subunit. J. Biol. Chem. 288, 10870-81.
  3. Rusconi, B. et al. (2013) Discovery of catalases in members of the Chiamydiales order. J. Bact. 195, 3543–51.

Structural analysis

  1. Araiso, Y. et al. (2014) Crystal structure of Saccharomyces cerevisiae mitochondrial GatFAB a novel subunit assembly in tRNA –dependent amidotransferases. Nucl.Acids. Res.(available only online).
  2. Someya, T. et al. (2012) Crystal Structure of Hfq from Bacillus subtilis in complex with SELEX-dervived RNA aptamer: insight into RNA-binding properties of bacterial Hfq. Nucl. Acid Res. 40, 1856-67.

Protein pulldowns

  1. Yun, S-C. et al. (2010) Pmr a Histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function. J.Bact. 192, 4720–31.
  2. Haim, H. et al. (2010)  Cytokeratin 8 interacts with clumping factor B: a new possible virulence factor target. Microbiology 156, 3710-21.

Additional Resources
His-tagged Protein Purification Systems

Elegant Experiments that Changed the World

Posted on by Isobel Maciver

Crystallographic structure of HIV reversed transcriptase. Wikimedia Commons
Crystallographic structure of HIV reverse transcriptase. Wikimedia Commons

Today, reverse transcriptases are commonplace molecular biology tools, easy to obtain and routinely used in labs for everyday cloning and gene expression analysis experiments. Reverse transcriptase inhibitors have also found widespread use as antiviral drugs in the treatment of retroviral infections.

It’s easy to forget that the existence of reverse transcriptase activity—the ability to convert an RNA template into DNA—was once a revolutionary notion not easily accepted by the scientific community. The idea that RNA could be the template for DNA synthesis challenged the “DNA–>RNA–> Protein” central dogma of molecular biology.

The foundational studies that proved the existence of a reverse transcriptase activity in RNA tumor viruses were described in two papers published back-to-back in Nature in June, 1970. Two of the authors of these studies, Howard Temin of the University of Wisconsin and David Baltimore of the Massachusetts Institute of Technology, were awarded a Nobel Prize for their work in 1975.

In appreciation of the significance of these papers, the editorial introduction published in Nature at the time states:

This discovery, if upheld, will have important implications not only for carcinogenesis by RNA viruses but also for the general understanding of genetic transcription: apparently the classical process of information transfer from DNA to RNA can be inverted.

Before these papers were published, it was known that successful infection of cells by RNA tumor viruses required DNA synthesis. Formation of virions could be inhibited by Actinomycin D—an inhibitor of DNA-dependent RNA polymerase—so it was known that synthesis of viral RNA from a DNA template was part of the viral life cycle. The existence of an intracellular DNA viral genome was therefore indicated, and had been postulated by Temin in the mid 1960’s. However, proof of the mechanism whereby this DNA template was generated from the RNA genome of the infecting virus remained elusive. Continue reading “Elegant Experiments that Changed the World”

For Alphavirus Reporters, Location Matters

Posted on by Isobel Maciver
Computer-generated model of an alphavirus.
Computer-generated model of an alphavirus.

Luminescent reporters offer virologists a convenient way to measure replication of viruses and are also used to image the spread of viruses in vivo in experimental systems. These reporter viruses are useful for evaluating the effects of antiviral drug treatments, testing the efficacy of potential vaccines, and studying the ways in which viruses replicate in the body and cause disease. One challenge in the construction of such reporters is the need to ensure that the reporter molecule itself does not alter the virus in ways that affect its ability to cause disease. Another challenge is maintaining the reporter gene throughout several cycles of viral replication. In smaller viruses, it can be particularly difficult to introduce a reporter gene without compromising the ability of the virus to replicate and cause disease.

A 2014 paper was published in J. Virology comparing the effectiveness of various NanoLuc® luciferase alphavirus reporter constructs. The authors of the study, Chengqun Sun et al. from the University of Pittsburgh, placed these reporter genes in three different locations in the genome of several alphaviruses and compared the effect on their ability to replicate in vitro and in vivo. They also assessed the ability of the luciferase genes to persist during infection of cultured cells and in a mouse model. They showed that the size and location of the reporter had a significant effect on successful replication and persistence. They also showed that the reporters could potentially be integrated at different positions within the genome to study different aspects of viral pathogenesis.

Continue reading “For Alphavirus Reporters, Location Matters”

NanoLuc® Luciferase: A Good Thing for Small Packages

Posted on by Isobel Maciver
influenza viruses

A paper published on October 2 in the Journal of Virology describes an exciting development in the world of influenza research—the construction of a luciferase reporter virus that does not affect virulence and can be used to track development and spread of infection in mice.

Insertion of luciferase reporter genes into viruses has been accomplished before for several viruses, but has not been successful for influenza. Construction of influenza reporter viruses is complicated because the viral genome is small and all the viral genes are critical for infection. Therefore, replacement of an existing gene with a reporter gene or insertion of additional reporter sequences without affecting the virus’s ability to replicate and cause infection has proven difficult. To be successful, a reporter gene needs to be small enough to insert into the viral genome without eliminating any other vital functionality.

Continue reading “NanoLuc® Luciferase: A Good Thing for Small Packages”

Testing for Neonatal Sepsis: The Next Generation of Biomarkers

Posted on by Radhika Ganeshan

newbornNeonatal sepsis is a systemic infection prevalent in preterm and very low birth weight infants and causes high morbidity. Most cases of neonatal sepsis are caused by pathogenic bacteria that invade the bloodstream, triggering an abrupt and overwhelming infection in the target organs accompanied by a systemic inflammatory response. Testing for neonatal sepsis is challenging because it does not affect a specific organ and presents multiple symptoms that are often confused with other related conditions (1). Current diagnostic tests for sepsis include those that identify markers of the host response to infection (e.g., procalcitonin, C reactive protein, cytokines, etc.) and those that detect bacterial infection in blood (bacteremia) (2). The lack of specific diagnostic biomarkers for early and accurate detection of neonatal sepsis has spurred the quest for next-generation biomarkers using powerful mass screening technologies such as proteomics. Continue reading “Testing for Neonatal Sepsis: The Next Generation of Biomarkers”

TB Vaccine News

Posted on by Isobel Maciver

Mycobacterium tuberculosis (Ziehl Neelsen stain). Photo credit: Centers for Disease Control and Prevention.
Mycobacterium tuberculosis (Ziehl Neelsen stain). Photo credit: Centers for Disease Control and Prevention.
A paper published last week in Science Translational Medicine describes promising results from a phase 1 clinical trial of a new anti-tuberculosis vaccine. The vaccine, composed of a human Adenoviral vector expressing a Mycobacterium tuberculosis antigen, generated an immune response in people with and without previous exposure to the current anti-tuberculosis (BCG) vaccine.

Mycobacterium tuberculosis, discovered by Robert Koch in 1882, is the organism that causes tuberculosis—commonly known as TB. After introduction of the BCG (Bacille Calmette-Guérin ) vaccine in 1919 and antibiotic treatment in the 1950s, the hope was that TB would be finally consigned to history—that Mycobacteruim tuberculosis would be a name only associated with the pre-antibiotic era and would not be a part of the 21st century world. However, over the last 30 years the emergence of multi-drug resistance and the worldwide HIV epidemic have led to the re-emergence of TB to the point where the following statements are true: Continue reading “TB Vaccine News”

Hepatitis C: A Promising Animal Model, and Reasons to Get Tested

Posted on by Kari Kenefick

Hepatitis C virus infection by source, in the U.S. From Wikipedia.
Hepatitis C virus infection by source, in the U.S. From Wikipedia.

During graduate studies in Medical Microbiology and Immunology at the University of WI-Madison, a favorite class was an infectious disease course that included an exercise in designing the perfect pathogen. This was a thought experiment, a writing exercise. No laboratory experimentation was involved.

You might initially think of a perfect pathogen as one that produces the most spores, allowing the pathogen to spread or seed itself in many locations. Copious slime and mucus production and projectile vomiting and diarrhea were frequently suggested during discussions of the perfect pathogen. And it’s true that these features really get the attention of the infected person and her/his caregiver. There are some pretty scary microbial buggers out there, for instance those that cause hemolytic anemia and/or raging fevers; these are the attention getters of the infectious disease world. Continue reading “Hepatitis C: A Promising Animal Model, and Reasons to Get Tested”

The Power of One: Revealing Microbial Dark Matter Using Single-Cell Sequencing

Posted on by Kelly Grooms

abstract digital backgroundMicroorganisms; they are the most abundant form of life. They are all around us, silent, unseen and undetected. The number of ‘species’ of archaea and bacteria climbs every year and is predicted to rise well past one million (1). Despite their abundance, we know very little about all but a small fraction of these diverse cellular life forms because we are unable to cultivate most in a laboratory setting. In fact, 88% of all our microbial isolates belong to just four bacterial phyla (Proteobacteria, Firmicutes, Actinobacteria and Bacterioidetes; 2). The remaining branches of the microbial phylogenetic tree range from underrepresented to virtually unknown and are collectively referred to as “microbial dark matter”.

If you want to target those shadowy, ill-defined branches where exotic and underrepresented organisms belong, you go to environments that might harbor them. Towards this end, Christian Rinke and a large coalition of co-authors collected samples from a wide and varied choice of habitats including the South Atlantic tropical gyre, the Homestake Mine in South Dakota, the Great Boiling Spring in Nevada, the sediment at the bottom of the Etoliko Lagoon in Greece and even a bioreactor. Continue reading “The Power of One: Revealing Microbial Dark Matter Using Single-Cell Sequencing”