The Wiedenheft Lab

Research Areas

A bacterium under attack. Artistry by Gabriel C. Lander

Effective clearance of an infection requires that the immune system rapidly detects and neutralizes invading parasites, while strictly avoiding self-antigens that would result in autoimmunity. The sophisticated cellular machinery and signaling pathways that coordinate an effective immune response have generally been considered to be properties of the eukaryotic immune system. However, a surprisingly sophisticated adaptive immune system that relies on small RNAs for sequence specific targeting of invading parasites has recently been discovered in bacteria and archaea. Central to this immune system is a diverse family of DNA repeats called CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). These repetitive loci serve as molecular vaccination cards that maintain a genetic record of all prior encounters with foreign transgressors (i.e. viruses and plasmids). The acquisition of foreign DNA and sequence-specific interference is mediated by a diverse set of CRISPR-associated (Cas) proteins that are encoded in gene clusters that flank each CRISPR locus. Phylogenetic analyses preformed using a universally conserved cas gene (Cas1) have identified several distinct versions of the CRISPR system that each consist of a unique set of 4-10 cas genes. In each of these systems, the CRISPR locus is transcribed and the long primary transcripts are processed into a library of short CRISPR-derived RNAs (crRNAs) that each contain a unique sequence complementary to a foreign nucleic acid challenger. Each crRNA is packaged into a large, multi-subunit surveillance complex that patrols the intracellular environment and ‘silences’ invading foreign nucleic acid targets (Fig. 1).

Figure 1: The three main steps in adaptive bacterial immunity via CRISPR systems.

The general steps required for adaptive immunity in bacteria and archaea have been identified, however the mechanisms of foreign nucleic acid recognition, new sequence acquisition and RNA-guided interference are not understood. The Wiedenheft laboratory uses molecular biology to engineer model microbes for functional and structural interrogation of the molecular machines that coordinate an effective immune response. Work in our lab focuses broadly on the three areas below.

Cascade

In Escherichia coli, small CRISPR-derived RNAs (crRNAs) are incorporated into a multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense), which is required for protection against bacteriophages. Cascade is a 405-kDa-ribonucleoprotein complex composed of 11 subunits of five functionally essential Cas proteins (one CasA, two CasB, six CasC, one CasD and one CasE) and a 61-nucleotide crRNA. We aim to understand the structural basis of target surveillance, target binding and recruitment of the trans-acting nuclease Cas3.

Csy-complex

In the human pathogen Pseudomonas aeruginosa (PA14), crRNAs are incorporated into a multisubunit surveillance complex called the Csy-complex (CRISPR system yersinia), which is required for protection against bacteriophages. This complex is a 350-kDa-ribonucleoprotein complex composed of 9 subunits of five functionally essential Cas proteins (one Csy1, one Csy2, six Csy3, and one Csy4) and a 60-nt crRNA-guide. Previously we used small angle x-ray scattering (SAXS) and native mass spectrometry to determine the shape and composition of this complex; however, the arrangement of subunits and the mechanism of target recognition remain largely unknown. In collaboration with Drs. Clint Potter, Bridget Carragher and Gabriel Lander at the National Recourse for Automated Molecular Microscopy (NRAMM) we aim to determine the structure of this complex alone, and in association with its binding partners. High-resolution structure determination using x-ray crystallography and biochemical studies of this complex alone and in association with recently discovered anti-CRISPR proteins from Alan Davidson's (University of Toronto) group are also underway.

Microbiome

Bacteriophages are the most abundant biological entities on earth and the selective pressures imposed by these pervasive predators have a profound impact on the composition and the behavior of microbial communities in every ecological setting. Our work is aimed at understanding the role of bacteriophages in the evolution of bacterial pathogenesis in human and non-human ecosystems.