Projects

Chlamydial Cell Division
Chlamydia
is amongst the rare bacteria that lack critical cell division proteins and how these organisms manage to divide in their absence is an intriguing and unanswered question.  The cell division apparatus in bacteria forms a supra-molecular interaction complex of multiple proteins that ultimately separates two equally-sized daughter cells in a process called binary fission.  Canonically, the building of this complex proceeds in a step-wise fashion such that early proteins recruit later ones.  Where Chlamydia is unusual is that it has no homologues of the early proteins, in particular FtsZ, which is considered the central organizer of the division apparatus.

We demonstrated that Chlamydia uses an alternate protein to substitute for FtsZ (Ouellette et al., 2012; Lee et al., 2020).  This protein, MreB, has similar characteristics to FtsZ, can form a ring at the septum, and can interact with other cell division proteins, in part mediated by a unique N-terminal domain.  Given that cell division proteins do interact, we exploited this trait to identify novel components of the system (Ouellette et al., 2014b).  We are implementing recently described techniques for the transformation of Chlamydia and have used these to identify and partially characterize new division proteins that were previously not annotated in the genome (Ouellette et al., 2015).  Work with collaborators from UTHSC in Memphis indicated that chlamydiae divide by a budding mechanism rather than by binary fission (AbdelRahman et al., 2016; Ouellette et al., 2020).  This represents an exciting finding that we are actively pursuing further, including how division is inhibited during different stress states (see below in Chlamydial Persistence).  We hypothesize that Chlamydia uses an MreB-dependent polarized division mechanism.  Current project goals are: 

  1. to determine how the chlamydial septum is localized to a specific site on one side of the mother cell,
  2. to understand the role of phospholipids in constructing the budding daughter cell and factors that regulate phospholipid synthase activity/localization,
  3. to identify novel chlamydial division proteins and regulators that interact with MreB using a system for monitoring protein-protein interactions in bacteria. 

Most recently, we identified and characterized a novel cytoskeletal protein involved in chlamydial cell shape and are investigating its function (Brockett, Lee et al., 2021).  We currently employ a variety of molecular, biochemical, and cell biology techniques to image chlamydial division and investigate the proteins of interest involved in this system.  A better understanding of chlamydial cell division may lead to the identification of novel therapeutic targets that would eliminate Chlamydia specifically without disrupting normal flora.  This project is funded by an R35 MIRA award from the NIGMS/NIH and NSF Collaborative award and is a collaboration with the Cox Lab at the University of Tennessee Health Science Center in Memphis.


Chlamydial Persistence

While productive growth through the normal developmental cycle results in the generation of EBs, non-productive growth leads to the establishment of persistent forms of the organism.  Persistent chlamydiae are most readily identified by their aberrant morphology: they resemble enlarged RBs that fail to undergo cell division.
 
 
Electron micrographs of HEp-2 cells infected with Chlamydia pneumoniae and grown for 48hr in the absence (left) or presence (right) of IFN-gamma. The nucleus has a black border whereas the inclusions have a white background with grey circular shapes in them. The inclusion in the IFN-gamma treated cell is smaller with fewer organisms that are enlarged compared to the untreated infected cell that has dozens of RBs. From Ouellette et al., 2006.

Induction of persistence is a reversible process defined by viable, culture-negative growth that does not result in the production of infectious EBs.  This requires a long-term association with the host cell.  Chronic sequelae associated with chlamydial infection may be caused by a persistent form of the organism.  A better understanding of the mechanisms chlamydiae use to maintain a persistent growth state will lead to improved diagnostic and therapeutic strategies.  Host immune effectors, b-lactam antibiotic treatment, and nutrient deprivation can induce persistence in vitro.  The mechanisms by which each of these elicits persistence differ.  In humans, the immune cytokine IFN-gamma activates target cells to produce indoleamine-2,3-dioxygenase (IDO).  IDO decyclizes tryptophan to N-formyl-kynurenine, which results in an enzymatically controlled tryptophan-limiting environment.  Because chlamydiae are dependent on the host cell for tryptophan, the bacteria are effectively starved for this essential amino acid.  Penicillin and other b-lactam antibiotics have the phenotypic effect of blocking cell division, presumably by interfering with the action of penicillin-binding proteins in this process.  Starving chlamydiae for essential nutrients such as vitamins and iron can also induce a persistent growth state.  A critical parameter of persistence is that it is reversible: the organism remains viable such that it can re-enter the productive growth cycle once the inducing stress has been removed.  There are data to suggest that persistent chlamydiae are refractory to clinical antibiotic treatment, which may also explain treatment failures.
 
Effect of different stressors on chlamydial morphology. C. trachomatis L2 infected cells were treated or not with different stressors including low tryptophan (Low W), bipyridyl to chelate iron (Low Fe), or penicillin (Pen). Chlamydiae were then stained with an outer membrane marker presented in green and imaged by confocal microscopy (60x). Note the large sizes of individual organisms in the treated versus untreated (UTD) conditions. The image in Pen represents a single, aberrantly enlarged bacterium!

Most bacteria respond to amino acid limitation by engaging a stringent response, which is a transcriptional program used to adapt to nutrient-poor conditions.  Interestingly, the stringent response is also necessary for inducing persister cells, which survive stressful conditions (such as antibiotic treatment) without becoming genetically resistant to the stress.  Chlamydia lacks the genes necessary for implementing a stringent response but are capable of persisting.  Additionally, Chlamydia has very few identified regulatory elements thus how it responds to stress is an intriguing question.  We have previously shown (Ouellette et al., 2006) that Chlamydia has an unusual response to IDO-mediated tryptophan limitation: it globally increases transcription while translation is globally decreased.  This disconnect between transcription and translation is unusual in bacteria.  Our goals are to understand how and why Chlamydia responds in this way.  We hypothesize that, having lost the “canonical” response pathways, Chlamydia’s response to amino acid starvation is the consequence of a lack of regulation.  We have observed that Chlamydia increases the transcription of tryptophan codon-containing genes in response to tryptophan limitation (Ouellette et al., 2016), and this is also an unusual response.  Our results suggest that ribosome stalling at tryptophan codons leads to the destabilization of downstream mRNA due to Rho transcript termination (Ouellette et al., 2018).  Current efforts are designed to mechanistically understand this observation as well as the consequences to the bacterium when this occurs.  Having characterized the ability of tRNA synthetase inhibitors to phenocopy the effects of IFN-gamma (Hatch and Ouellette, 2020), we will use these tools to investigate the genetic mechanisms that control this response in bacteria with a variety of transcriptional techniques ranging from traditional (e.g. Northern blots and qPCR) to cutting-edge (e.g. RNAseq, ChIP-seq, and ribosome profiling).  Results will lead to the identification of novel therapeutic and diagnostic targets that have the potential to identify and treat asymptomatic chlamydial infections.  This project is funded by a CAREER award from the National Science Foundation.  Other persistence related projects supported by an R01 from the NIAID/NIH focus on how chlamydial cell division is inhibited during persistence.
 

Differentiation through Degradation
As Chlamydia differentiates from one developmental stage to the other (i.e. RB to EB or vice versa), they must necessarily reorganize their protein content between these morphologically and functionally distinct forms – this requires the action of proteases. We hypothesize that protease activity is essential for chlamydial differentiation.  Chlamydia has a number of conserved protease systems, and we are investigating several of them for their function in chlamydial growth and differentiation.

 
 
 
Of particular interest is the Clp protease system, composed of a proteolytic subunit, ClpP, and a substrate recognition subunit, ClpX or ClpC, that identifies and unfolds proteins into ClpP for degradation.  ClpP is a validated drug target in various bacteria, including Chlamydia (Seleem et al., 2020).  Unusually, Chlamydia possesses two ClpP isoforms, and we have shown that they have differential effects in Chlamydia (Wood et al., 2018).  Interestingly, disrupting the activity of ClpX results in non-functional EBs (Wood et al., 2020).  Current work is focused on (i) identifying substrates of ClpX as well as the critical domains and residues important for its function and (ii) characterizing the function of the related unfoldase ClpC and its substrate preferences.  This work is currently supported by a bridge award from the NIAID/NIH and is a collaboration with the Fisher Lab at Southern Illinois University at Carbondale.
 
Miscellaneous Projects
We have identified a novel transcriptional regulatory circuit and are investigating its effects.  With the recent development of genetic tools for Chlamydia, the Ouellette Lab is also taking advantage of these advances by developing inducible repression systems based on CRISPR interference to knockdown gene expression in Chlamydia (Ouellette et al., 2021).
 

Test of CRISPRi system in Chlamydia.
Cells were infected with a mix of inducible CRISPRi transformants (target IncA) and wild-type Chlamydia in the presence of penicillin (selective antibiotic for the plasmid). Samples were induced with anyhydrotetracycline (aTc) at 12 hr post-infection (hpi) and assessed at 16 and 24 hpi, as indicated. Pulse indicates that aTc was present from 12hpi until fixation, whereas pulse/chase indicates aTc was washed out at 16hpi, then fixed at 24hpi. MOMP=chlamydial major outer membrane protein. IncA is the target of the system. DAPI stains DNA. Aberrant Chlamydia (those without the CRISPRi plasmid) are indicated with ‘A’ and still express IncA, while Chlamydia expressing the CRISPRi construct do not express IncA, unless aTc is removed (4hr pulse/8hr chase).

Wolbachia in Aquatic Insects
In collaboration with Dr. Jeff Wesner (http://jeffwesner.wordpress.com/) in the Biology Department at the University of South Dakota, we are investigating the presence of another obligate intracellular bacteria, Wolbachia, in insects from the Missouri river tributary systems around Vermillion, SD.  Our initial analyses have revealed that Wolbachia infects a number of aquatic insect species (Sazama et al., 2017), and we are currently trying to determine what effect this has on the insect and how Wolbachia is transferred between individuals of a species and between species.