Projects

Question: How does Chlamydia do so much with so little?

Overview
The Ouellette lab is interested in the consequences of reductive evolution on bacterial physiology.  As Chlamydia has adapted to an intracellular niche, it has lost many genes that are present in free-living bacteria.  These genes/gene pathways that have been lost through reductive evolution (Muller’s Ratchet) typically encode metabolic pathways to synthesize, for example, amino acids.  Since Chlamydia relies on its host cell for many nutrients, it is not surprising that it would eliminate these types of genes.  However, Chlamydia has also eliminated genes that are considered essential for viability or pathogenesis in many other bacteria.  This raises many interesting questions that are the focus of Dr. Ouellette’s research as described below.  Conversely, when Chlamydia has retained genes that are atypical for Gram-negative bacteria (e.g. genes normally found in Gram-positive bacteria), this is also interesting and suggests a function that is important to chlamydial growth - otherwise these genes would have been deleted.

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.  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 continue to study, 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.  Given that Chlamydia is a coccoid bacterium, we are interested in understanding how its septum is formed at a specific site on one side of the cell. Current project goals are:

1. to determine how the chlamydial septum is localized to a specific site on one side of the mother cell,(Ouellette et al., 2022), 
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. 

                                  

          The localization of MreB is shown in untreated (UTD) chlamydiae at different stages of
           division. Note its localization to one side initially and then to the septum between the 
           daughter and mother cell. Treating with a drug (diNN) to disrupt cardiolipin delocalizes
           MreB from the membrane. (See Ouellette et al, 2022).

Most recently, we identified and characterized a novel cytoskeletal protein involved in chlamydial cell size and are investigating its function (Brockett, Lee et al., 2021).  A follow-up study has characterized the unique N-terminal domain of BacA, and we have performed assays to identify potential interaction partners of this protein, which are under investigation.  Other projects under investigation include how cell division is regulated over the course of the developmental cycle.  We currently employ a variety of molecular, biochemical, and cell biology techniques for these projects and investigate the proteins of interest involved in these systems.  A better understanding of chlamydial cell division and size regulation may lead to the identification of novel therapeutic targets that would eliminate Chlamydia specifically without disrupting normal flora.  This project 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.  Recent work has shown that both Chlamydia and Streptococcus pyogenes (another Trp auxotroph) respond similarly to Trp starvation (Ouellette et al., 2021). We hypothesize that ribosome stalling at Trp codon-rich sequences negatively impacts transcription and translation, resulting in the phenotypes observed. 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 along with CRISPRi 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 a multi-PI R01 from the NIAID/NIH with Drs. Carabeo and Rucks focus on how chlamydial cell division is inhibited during persistence (see Riffaud et al., 2023).