More Like this

1. Localized cardiolipin synthesis is required for the assembly of MreB during the polarized cell division of Chlamydia trachomatis

   https://doi.org/10.1371/journal.ppat.1010836  

   Ouellette, Scot P.; Fisher-Marvin, Laura A.; Harpring, McKenna; Lee, Junghoon; Rucks, Elizabeth A.; Cox, John V. (September 2022, PLOS Pathogens)

   Derré, Isabelle (Ed.)

   Pathogenic Chlamydia species are coccoid bacteria that use the rod-shape determining protein MreB to direct septal peptidoglycan synthesis during their polarized cell division process. How the site of polarized budding is determined in this bacterium, where contextual features like membrane curvature are seemingly identical, is unclear. We hypothesized that the accumulation of the phospholipid, cardiolipin (CL), in specific regions of the cell membrane induces localized membrane changes that trigger the recruitment of MreB to the site where the bud will arise. To test this, we ectopically expressed cardiolipin synthase (Cls) and observed a polar distribution for this enzyme in Chlamydia trachomatis . In early division intermediates, Cls was restricted to the bud site where MreB is localized and peptidoglycan synthesis is initiated. The localization profile of 6xHis tagged Cls (Cls_6xH) throughout division mimicked the distribution of lipids that stain with NAO, a dye that labels CL. Treatment of Chlamydia with 3’,6-dinonylneamine (diNN), an antibiotic targeting CL-containing membrane domains, resulted in redistribution of Cls_6xH and NAO-staining phospholipids. In addition, 6xHis tagged MreB localization was altered by diNN treatment, suggesting an upstream regulatory role for CL-containing membranes in directing the assembly of MreB. This hypothesis is consistent with the observation that the clustered localization of Cls_6xH is not dependent upon MreB function or peptidoglycan synthesis. Furthermore, expression of a CL-binding protein at the inner membrane of C . trachomatis dramatically inhibited bacterial growth supporting the importance of CL in the division process. Our findings implicate a critical role for localized CL synthesis in driving MreB assembly at the bud site during the polarized cell division of Chlamydia . 

   more » « less
2. Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae

   https://doi.org/10.1073/pnas.1816018116  

   Perez, Amilcar J.; Cesbron, Yann; Shaw, Sidney L.; Bazan Villicana, Jesus; Tsui, Ho-Ching T.; Boersma, Michael J.; Ye, Ziyun A.; Tovpeko, Yanina; Dekker, Cees; Holden, Seamus; et al (February 2019, Proceedings of the National Academy of Sciences)

   Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen,Streptococcus pneumoniae. Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZmutant and anotherStreptococcusspecies. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells andftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling inS. pneumoniaecells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate inS. pneumoniae. 

   more » « less
3. Inhibition of tRNA Synthetases Induces Persistence in Chlamydia

   https://doi.org/10.1128/IAI.00943-19  

   Hatch, Nathan D.; Ouellette, Scot P.; Roy, Craig R. (March 2020, Infection and Immunity)

   ABSTRACT Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and Chlamydia pneumoniae causes community-acquired respiratory infections. In vivo , the host immune system will release gamma interferon (IFN-γ) to combat infection. IFN-γ activates human cells to produce the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Consequently, there is a reduction in cytosolic Trp in IFN-γ-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has significantly reduced its genome size and content, as it relies on the host cell for various nutrients. Importantly, C. trachomatis and C. pneumoniae are Trp auxotrophs and are starved for this essential nutrient when the human host cell is exposed to IFN-γ. To survive this, chlamydiae enter an alternative developmental state referred to as persistence. Chlamydial persistence is characterized by a halt in the division cycle, aberrant morphology, and, in the case of IFN-γ-induced persistence, Trp codon-dependent changes in transcription. We hypothesize that these changes in transcription are dependent on the particular amino acid starvation state. To investigate the chlamydial response mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryote-specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of Trp and leucine, respectively. We show that these drugs block chlamydial growth and induce changes in morphology and transcription consistent with persistence. Importantly, growth inhibition was reversed when the compounds were removed from the medium. With these data, we find that indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently of IFN-γ. 

   more » « less
4. Organization of peptidoglycan synthesis in nodes and separate rings at different stages of cell division of Streptococcus pneumoniae

   https://doi.org/10.1111/mmi.14659  

   Perez, Amilcar J.; Boersma, Michael J.; Bruce, Kevin E.; Lamanna, Melissa M.; Shaw, Sidney L.; Tsui, Ho‐Ching T.; Taguchi, Atsushi; Carlson, Erin E.; VanNieuwenhze, Michael S.; Winkler, Malcolm E. (December 2020, Molecular Microbiology)

   Abstract Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid‐shapedStreptococcus pneumoniae(Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examinedSpncells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D‐SIM (structured‐illumination microscopy). Labeling with fluorescent D‐amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin‐binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading‐edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA‐marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced “nodes” of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division. 

   more » « less
5. Elongasome core proteins and class A PBP1a display zonal, processive movement at the midcell of Streptococcus pneumoniae

   https://doi.org/10.1073/pnas.2401831121  

   Perez, Amilcar J; Lamanna, Melissa M; Bruce, Kevin E; Touraev, Marc A; Page, Julia E; Shaw, Sidney L; Tsui, Ho-Ching Tiffany; Winkler, Malcolm E (June 2024, Proceedings of the National Academy of Sciences)

   Ovoid-shaped bacteria, such asStreptococcus pneumoniae(pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed nonprocessive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body. 

   more » « less