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 have recently shown that Chlamydia may use an alternate protein to substitute for FtsZ (Ouellette et al., 2012).  This protein, MreB, has similar characteristics to FtsZ, and can interact with other cell division proteins.  Given that cell division proteins do interact, this trait can be exploited to identify novel components of the system (Ouellette et al., 2014b).  Our current project goals are (i) to determine if the Mre system in Chlamydia is a functional substitute for FtsZ, (ii) to characterize its interactions with the known chlamydial cell division proteins, and (iii) to identify novel chlamydial division proteins and regulators that interact with MreB using a system for monitoring protein-protein interactions in bacteria (Ouellette et al., 2014a).  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).  Recent work with collaborators from UTHSC in Memphis suggests that chlamydiae divide by a budding mechanism rather than by binary fission (AbdelRahman et al., 2016).  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 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.

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 IFNg 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 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.  Current efforts are designed to mechanistically understand this observation as well as the consequences to the bacterium when this occurs.  We are currently investigating the genetic mechanisms that control this response in bacteria using a variety of transcriptional techniques ranging from traditional (e.g. Northern blots and qPCR) to cutting-edge (e.g. RNAseq 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.

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.

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 ( 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.