"Chlamydomonas reinhardtii as a model to study cilia-related disease"

by Dr. Karl Lechtreck, Department of Cellular Biology, University of Georgia

Abstract:

Bacteria use extracellular appendages called type IV pili (T4P) for diverse behaviors including DNA uptake, surface sensing, virulence, protein secretion, and biofilm formation. Dynamic extension and retraction of T4P is essential for their function in these behaviors, yet little is known about the molecular mechanisms controlling these dynamics. Furthermore, due to difficulties in visualizing T4P in live cells, their exact function in many of these processes has remained unclear. Through the development of a labeling method to visualize T4P in live cells in real time, our work has addressed multiple outstanding questions related to T4P biology. Using this labeling method, we defined the mechanism by which individual cells use T4P to take up DNA during natural transformation. We furthermore developed the highly naturally transformable species Acinetobacter baylyi as a new model to dissect the molecular mechanisms of T4P dynamics and T4P localization, with implications for how these structures may contribute to multicellular interactions. This work provides insight into the mechanisms that govern diverse microbial behaviors important for bacterial physiology through direct observation of the T4P appendages that mediate them.

Abstract:

When bacterial cells come into direct contact, antagonism mediated by the delivery of a diverse array of potent toxins frequently ensues. The reported outcomes of such assaults include cell death, growth inhibition, or survival in resistant populations. The potential for interbacterial toxins to have long-term consequences in recipient cells has not been investigated. In this work, we examined the physiological effects of intoxication by DddA, a double-strand DNA-specific cytosine deaminase delivered via the type VI secretion system (T6SS) of Burkholderia cenocepacia. Moreover, we harnessed the biological activity of DddA to generate the first generation of precise genome editing tools for the mitochondrial genome. We find that when expressed in E. coli, DddA leads to cell death by chromosome degradation and arrest of DNA replication. Despite the lethal potential of DddA, several species of bacteria resist killing when confronted by DddA delivered by the T6SS of B. cenocepacia cells. Surprisingly, these targeted cells accumulate mutations characteristic of those induced by the toxin, indicating that even in the absence of killing, interbacterial toxins can have profound consequences on target cell populations. Motivated by the diversity of toxin members in the deaminase superfamily, we investigated whether DNA deaminase activity is a common feature in this group. We discovered that highly divergent deaminases act on DNA, including a novel single-stranded DNA deaminase with a markedly divergent structure. Additionally, in this work, we engineered split-DddA halves bound to programmable DNA-binding proteins, resulting in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyze C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. In total, our work reveals that mutagenic activity is a common feature of bacterial toxins in the deaminase superfamily, and it shows that a surprising consequence of antagonistic interactions in microbial communities can be the generation of genetic diversity. Furthermore, we harnessed the DNA modifying potential of DddA to create the first generation of genome editing tools for mtDNA.

"Establishing Causality in Microbiota Research" 

by Dr. Volker Mai, Department of Epidemiology, University of Florida

Jennifer Kurasz of the Karls Lab is the recipient of the 2020 Dr. Juergen Wiegel Award.  The award was announced on February 25, 2021 at the Dr. Juergen Wiegel seminar. The Dr. Juergen Wiegel Graduate Award in Microbiology was established by Dr. Wiegel, his family, and his former students and colleagues.  This award recognizes graduate students for excellent work in non-medical microbial biodiversity and microbial physiology.  Congratulations Jenn!

Abstract: Cilia and eukaryotic flagella are slender cell projections with motile and sensory functions. They lack ribosomes and all ciliary building blocks need to be imported posttranslationally from the cell body. This task involves intraflagellar transport (IFT), a motor-based protein shuttle. Using single particle imaging in the unicellular alga Chlamydomonas reinhardtii, we determined key aspects of ciliary protein transport such as cargoes and unloading sites. The transport frequencies of structural proteins such as tubulin are upregulated when a cilium is too short revealing that cells sense the length of their cilia and adjust the cargo load of IFT, accordingly. In mammals, cilia malfunction leads to a plethora of diseases, named ciliopathies. Many of the disease-related proteins are conserved in protists. Biochemical analyses of isolated Chlamydomonas cilia and live imaging revealed that the BBSome, an octameric protein complex, is an adapter mediating the export of certain signaling proteins from cilia by IFT. This led to the concept that Bardet-Biedl syndrome (BBS, characterized by obesity, kidney anomalies, polydactyly and blindness) results from the abnormal accumulation of (signaling) proteins in cilia. One of the most common inherited single-gene, life-threatening disorders is autosomal dominate polycystic kidney disease (ADPKD), which affects ~1:1,000 adults. ADPKD results from mutations in the TRP cation channel PKD2, but the role of PKD2 in cilia remains unclear. In Chlamydomonas, PKD2 anchors the mastigonemes, large extracellular glycoprotein polymers, to the ciliary membrane. The PKD2-mastigoneme complexes are arranged in two rows along the axoneme positioning them perpendicular to the plane of the ciliary beating. Association with extracellular components, the cytoskeleton or both is characteristic for many mechanically gated channels in eukaryotes. We proposed that pull on these polymers during bending of the axoneme could generate a force to open the PKD2 channel.

The department will be hosting UGA MIBO alum, Dr. Volker Mai (worked in Juergen Wiegel’s lab), to give a seminar this month (Thurs, Feb 25 – seminar at 11:10am) to celebrate announcing the winner of the 2020 Wiegel Award!

Dr. Volker Mai, Department of Epidemiology, University of Florida

Over the last two decades, the role of the commensal microbiota in human health has received enormous research interest. There has been significant progress in detailing the composition and activities of the microbiota at various anatomic sites and correlating distortions in microbiota with various health and disease endpoints. However, to date there appears to be a sparsity of evidence sufficient to support a causal contribution of microbiota to human health and disease. Establishing causality, beyond simple correlations, is crucial for the future development of microbiota targeting prevention regimen. Using examples from our microbiota research I will elaborate on these concepts and discuss potential approaches to advance the field.