Research

Our research focuses on:

  • Discovery of the phage nucleus and spindle
  • Antibiotic discovery and mechanism of action

 

The phage nucleus  of jumbo Pseudomonas phage forms  during lytic growth.  A shell of protein (green) formed by gp105 surrounds replicating phage 201phi201 DNA (blue) inside a Pseudomonas chlororaphis cell (red cell membranes). Top: GFP-gp105 (green), Middle: DNA (blue), Bottom, merged.

Discovery of the Phage Nucleus and Spindle.

Our lab has been working on the cell biology of Pseudomonas jumbo phage for more than a decade.  In collaboration with David Agard at UCSF, we identified and characterized a family of divergent tubulin-related proteins, PhuZ, encoded by large bacteriophage that infect Pseudomonas species.  This led to the discovery of a new paradigm for phage reproduction (Kraemer et.al.Cell 2012; Erb Curr Opin Microbiol 2013).

As we investigated further, we observed that after injection of the phage 201φ2-1 genome into the host cell, the DNA migrates to the center of the cell, where it replicates, creating a centrally localized structure we termed the infection nucleoid.  Migration of phage DNA to midcell depends upon the PhuZ protein which forms a simplified tubulin based bipolar spindle. PhuZ has a conserved C-terminal tail that plays a key role in polymerization by making contacts between subunits within a unique triple stranded filament (Kraemer et.al.Cell 2012;  Zher et.al.Structure 2014). PhuZ filaments display dynamic instability in vivo and in vitro (Erb et.al. eLife 2014). This represents the first identification of a prokaryotic tubulin with the properties of microtubules and the ability to form a simplified bipolar spindle.

We recently discovered that these jumbo phage form a nucleus-like structure during lytic growth (Chaikeeratisak et.al. Science 2017; phagenucleus.org). Many Pseudomonas jumbo phage, including PA3, PhiKZ, and 201φ2-1, build a compartment that physically separates viral DNA from the cytoplasm (Chaikeeratisak et.al. Cell Reports 2017). The “phage nucleus” assembles immediately after DNA injection, grows larger as the DNA replicates, and is positioned at midcell by the bipolar PhuZ spindle. Protein localization data show that DNA replication and transcription occur inside the phage nucleus, while translation and metabolic processes, such as nucleotide synthesis, occur outside of the nucleus. Time-lapse microscopy demonstrates that proteins destined for the nucleus are synthesized in the cytoplasm and then accumulate in the phage nucleus.  This suggests a selective two-way exchange of mRNA, protein, and nucleotides between the cytoplasm and nucleus, as in Eukaryotes. Late in infection, viral capsids assemble on the cell membrane  and traffic through the cell along treadmilling PhuZ filaments.  Capsids dock on the surface of the phage nucleus, where DNA packaging occurs. Ultimately, fully assembled viral particles form and are released when the cell lyses.  These results demonstrate that bacteriophage evolved both a spindle and a nucleus, and provide insight into the evolution of these defining structures of eukaryotic cells.

 

BCP allows the rapid identification of antibiotic mechanism of action. In the example above, the DNA of E.coli cells forms toroidal structures when treated with protein synthesis inhibitors.  Toroids are quantitated with automatic image analysis software.
Antibiotic discovery and mechanism of action.

Antibiotic resistance among bacteria is a steadily growing problem. Once easily treated with penicillin, infections caused by methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE) are now very difficult to combat. Cases of extremely drug resistant gram negative bacteria are becoming increasingly common in and threaten to spread throughout the world.  Without effective antibiotics for these extremely drug resistant organisms, even countries with well-developed health care systems will have few treatment options. Therefore, the need for new antibiotics that are active against multidrug resistant pathogens is more urgent than ever before. Unfortunately, the number of new antibiotics being brought to market is currently insufficient to keep pace with the rate of antibiotic resistance development.

We are focused on three related areas of antibiotic research. First, we are studying some of the latest antibiotics to enter the clinic as well as preclinical molecules to determine their mechanisms of action. Second, we are trying to understand how resistance arises to these new antibiotics and how treatment can be modified to lead to a more successful clinical outcome. Third, we have developed a new method for screening for novel antibiotics and determining their mechanisms of action. We are using this method to screen libraries of natural products for novel compounds active against multidrug resistant bacteria. To accomplish these goals, we have set up collaborations with clinicians at the UCSD School of Medicine, with pharmaceutical companies who have promising new antibiotics, and with chemists who can provide novel compounds for screening for new antibiotics.

Bacterial Cytological Profiling. We have developed a new method for antibiotic discovery called Bacterial Cytological Profiling (BCP) in collaboration with Kit Pogliano (Nonejuie et.al.PNAS 2013; Lamsa et.al.Mol. Micro. 2012). BCP can be used to screen for novel chemical scaffolds that hit new targets in bacteria, to determine antibiotic mechanism of action, and for rapid antibiotic susceptibility testing (Nonejuie et.al.PNAS 2013; Lamsa et.al.ACS Chem Biol.2016; Peters et.al.ACS Chem Biol.2018). We are also working to understand how combinations of antibiotics can be used in the case of antibiotic resistant bacteria to lead to a more successful clinical outcome  (Sakoulas et.al.AAC 2013; Werth et.al.AAC 2013; Sakoulas et.al.AAC 2012; Barber et.al.AAC 2014; Sakoulas et.al.AAC 2015). As part of a collaboration with Victor Nizet, Bernhard Palsson,  George Sakoulas, Pieter Dorrestein and Rob Knight, we are attempting to understand how environmental conditions affect bacterial susceptibility to antibiotics and antimicrobial peptides such as LL-37 (Lin et.al., 2015).