Organization of peptidoglycan synthesis in nodes and separate rings at different stages of cell division of Streptococcus pneumoniae.
TL;DR: Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.
Abstract: Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid-shaped Streptococcus 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 examined Spn cells 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.
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TL;DR: In this paper , the authors study the genetic diversity of Streptococcus pneumoniae within 468 infants and 145 of their mothers by deep sequencing whole pneumococcal populations from 3,761 longitudinal nasopharyngeal samples.
Abstract: Characterizing the genetic diversity of pathogens within the host promises to greatly improve surveillance and reconstruction of transmission chains. For bacteria, it also informs our understanding of inter-strain competition and how this shapes the distribution of resistant and sensitive bacteria. Here we study the genetic diversity of Streptococcus pneumoniae within 468 infants and 145 of their mothers by deep sequencing whole pneumococcal populations from 3,761 longitudinal nasopharyngeal samples. We demonstrate that deep sequencing has unsurpassed sensitivity for detecting multiple colonization, doubling the rate at which highly invasive serotype 1 bacteria were detected in carriage compared with gold-standard methods. The greater resolution identified an elevated rate of transmission from mothers to their children in the first year of the child's life. Comprehensive treatment data demonstrated that infants were at an elevated risk of both the acquisition and persistent colonization of a multidrug-resistant bacterium following antimicrobial treatment. Some alleles were enriched after antimicrobial treatment, suggesting that they aided persistence, but generally purifying selection dominated within-host evolution. Rates of co-colonization imply that in the absence of treatment, susceptible lineages outcompeted resistant lineages within the host. These results demonstrate the many benefits of deep sequencing for the genomic surveillance of bacterial pathogens.
17 citations
TL;DR: In this paper, the authors used click chemistry to image peptidoglycan synthesis by single-molecule localization microscopy in the ovoid bacterium Streptococcus pneumoniae.
14 citations
TL;DR: A review of the key protein complexes and how they are involved in cell division in pneumococcus pneumoniae is provided in this paper, where the interaction of proteins in the divisome complex that underpin the control mechanisms for cell division and cell wall synthesis and remodelling are discussed.
Abstract: Cell division in Streptococcus pneumoniae (pneumococcus) is performed and regulated by a protein complex consisting of at least 14 different protein elements; known as the divisome. Recent findings have advanced our understanding of the molecular events surrounding this process and have provided new understanding of the mechanisms that occur during the division of pneumococcus. This review will provide an overview of the key protein complexes and how they are involved in cell division. We will discuss the interaction of proteins in the divisome complex that underpin the control mechanisms for cell division and cell wall synthesis and remodelling that are required in S. pneumoniae, including the involvement of virulence factors and capsular polysaccharides.
13 citations
TL;DR: In this article, it was shown that the newly identified EloR/KhpA complex, regulating cell elongation in S. pneumoniae, forms a complex with the essential peptidoglycan transglycosylase MltG at midcell.
Abstract: The ellipsoid shape of Streptococcus pneumoniae is determined by the synchronized actions of the elongasome and the divisome, which have the task of creating a protective layer of peptidoglycan (PG) enveloping the cell membrane. The elongasome is necessary for expanding PG in the longitudinal direction whereas the divisome synthesizes the PG that divides one cell into two. Although there is still little knowledge about how these two modes of PG synthesis are coordinated, it was recently discovered that two RNA-binding proteins called EloR and KhpA are part of a novel regulatory pathway controlling elongation in S. pneumoniae EloR and KhpA form a complex that work closely with the Ser/Thr kinase StkP to regulate cell elongation. Here, we have further explored how this regulation occur. EloR/KhpA is found at midcell, a localization fully dependent on EloR. Using a bacterial two-hybrid assay we probed EloR against several elongasome proteins and found an interaction with the lytic transglycosylase homolog MltG. By using EloR as bait in immunoprecipitation assays, MltG was pulled down confirming that they are part of the same protein complex. Fluorescent microscopy demonstrated that the Jag domain of EloR is essential for EloR's midcell localization and its interaction with MltG. Since MltG is found at midcell independent of EloR, our results suggest that MltG is responsible for recruitment of the EloR/KhpA complex to the division zone to regulate cell elongation.Importance Bacterial cell division has been a successful target for antimicrobial agents for decades. How different pathogens regulate cell division is, however, poorly understood. To fully exploit the potential for future antibiotics targeting cell division, we need to understand the details of how the bacteria regulate and construct cell wall during this process. Here we have revealed that the newly identified EloR/KhpA complex, regulating cell elongation in S. pneumoniae, forms a complex with the essential peptidoglycan transglycosylase MltG at midcell. EloR, KhpA and MltG are conserved among many bacterial species and the EloR/KhpA/MltG regulatory pathway is most likely a common mechanism employed by many Gram-positive bacteria to coordinate cell elongation and septation.
8 citations
21 Feb 2022
TL;DR: In this article , the authors study the genetic diversity of Streptococcus pneumoniae within individual infants and their mothers by deep sequencing whole pneumococcal populations from longitudinal nasopharyngeal samples.
Abstract: Characterising the genetic diversity of pathogens within the host promises to greatly improve surveillance and reconstruction of transmission chains. For bacteria, it also informs our understanding of inter-strain competition, and how this shapes the distribution of resistant and sensitive bacteria. Here we study the genetic diversity of Streptococcus pneumoniae within individual infants and their mothers by deep sequencing whole pneumococcal populations from longitudinal nasopharyngeal samples. We demonstrate deep sequencing has unsurpassed sensitivity for detecting multiple colonisation, doubling the rate at which highly invasive serotype 1 bacteria were detected in carriage compared to gold-standard methods. The greater resolution identified an elevated rate of transmission from mothers to their children in the first year of the child’s life. Comprehensive treatment data demonstrated infants were at an elevated risk of both the acquisition, and persistent colonisation, of a multidrug resistant bacterium following antimicrobial treatment. Some alleles were enriched after antimicrobial treatment, suggesting they aided persistence, but generally purifying selection dominated within-host evolution. Rates of co-colonisation imply that in the absence of treatment, susceptible lineages outcompeted resistant lineages within the host. These results demonstrate the many benefits of deep sequencing for the genomic surveillance of bacterial pathogens.
6 citations
References
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TL;DR: Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis that facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system.
Abstract: Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
43,540 citations
"Organization of peptidoglycan synth..." refers methods in this paper
...Additional image processing was completed using FIJI (Schindelin et al., 2012)....
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TL;DR: A fluorescent derivative of the antibiotic vancomycin is used as a probe for nascent peptidoglycan synthesis in unfixed cells of various Gram-positive bacteria, providing insights into the diverse molecular strategies used by bacteria to control their cellular morphology, as well as suggesting ways in which these strategies may impact on growth rates and cell envelope structure.
751 citations
"Organization of peptidoglycan synth..." refers methods in this paper
...…through cell division was obtained by staining with fluorescent vancomycin (FL-Vanco), which labels PG pentapeptides in areas of PG synthesis (Daniel & Errington, 2003), with fluorescent D-amino acids (FDAAs), which label PG in regions of active PBP TP activity (Hsu et al., 2017; Kuru et…...
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TL;DR: In this Review, Weiser, Ferreira and Paton summarize the mechanisms that allow pneumococci to transmit and progress from colonizer to pathogen.
Abstract: Streptococcus pneumoniae has a complex relationship with its obligate human host. On the one hand, the pneumococci are highly adapted commensals, and their main reservoir on the mucosal surface of the upper airways of carriers enables transmission. On the other hand, they can cause severe disease when bacterial and host factors allow them to invade essentially sterile sites, such as the middle ear spaces, lungs, bloodstream and meninges. Transmission, colonization and invasion depend on the remarkable ability of S. pneumoniae to evade or take advantage of the host inflammatory and immune responses. The different stages of pneumococcal carriage and disease have been investigated in detail in animal models and, more recently, in experimental human infection. Furthermore, widespread vaccination and the resulting immune pressure have shed light on pneumococcal population dynamics and pathogenesis. Here, we review the mechanistic insights provided by these studies on the multiple and varied interactions of the pneumococcus and its host.
523 citations
"Organization of peptidoglycan synth..." refers background in this paper
...Streptococcus pneumoniae (pneumococcus; Spn) is a Gram-positive, commensal bacterium of humans and a major opportunistic pathogen that causes life-threatening illnesses, including pneumonia, bacteremia, and meningitis (Loughran et al., 2019; Weiser et al., 2018)....
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TL;DR: Tracking a bug's life: Peptidoglycan (PG) of diverse bacteria is labeled by exploiting the tolerance of cells for incorporating different non-natural D-amino acids, thereby enabling fine spatiotemporal tracking of cell-wall dynamics in phylogenetically and morphologically diverse bacteria.
Abstract: Tracking a bug's life: Peptidoglycan (PG) of diverse bacteria is labeled by exploiting the tolerance of cells for incorporating different non-natural D-amino acids. These nontoxic D-amino acids preferably label the sites of active PG synthesis, thereby enabling fine spatiotemporal tracking of cell-wall dynamics in phylogenetically and morphologically diverse bacteria. HCC = 7-hydroxycoumarin, NBD = 7-nitrobenzofurazan, TAMRA = carboxytetramethylrhodamine.
522 citations
"Organization of peptidoglycan synth..." refers background or methods in this paper
...FDAAs were synthesized as reported in (Kuru et al., 2012), with the following change: TADA was synthesized as reported for TDL, except that Boc-D-DAP-OH (N-alpha-t-butyloxycarbonylD-2,3-diaminopropionic acid) was used in place of Boc-D-Lys-OH (N-alpha-t-butyloxycarbonyl-D-lysine)....
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...…synthesis (Daniel & Errington, 2003), with fluorescent D-amino acids (FDAAs), which label PG in regions of active PBP TP activity (Hsu et al., 2017; Kuru et al., 2012), and with an activity-based β-lactone fluorescent probe (7FL) specific for bPBP2x (Sharifzadeh et al., 2017; Sharifzadeh et al.,…...
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TL;DR: To understand how these components coordinate to divide cells, visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis and found that the division septum was built at discrete sites that moved around the division plane.
Abstract: The mechanism by which bacteria divide is not well understood. Cell division is mediated by filaments of FtsZ and FtsA (FtsAZ) that recruit septal peptidoglycan-synthesizing enzymes to the division site. To understand how these components coordinate to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We found that the division septum was built at discrete sites that moved around the division plane. FtsAZ filaments treadmilled circumferentially around the division ring and drove the motions of the peptidoglycan-synthesizing enzymes. The FtsZ treadmilling rate controlled both the rate of peptidoglycan synthesis and cell division. Thus, FtsZ treadmilling guides the progressive insertion of new cell wall by building increasingly smaller concentric rings of peptidoglycan to divide the cell.
467 citations
"Organization of peptidoglycan synth..." refers background in this paper
...Labeling Bsu with short, sequential pulses of two colors of FDAAs or an FDAA followed by Boc-FL (Bisson-Filho et al., 2017) gave a different pattern from the regular nodal pattern in Spn (Figures 7 and S7)....
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...Labeling Bsu with an FDAA followed by Boc-FL again suggests a surprisingly limited number of active PBP complexes labeled by Boc-FL adjacent to newly synthesized PG marked by the FDAA (Bisson-Filho et al., 2017)....
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...In this regard, Spn and Eco simultaneously close division septa while separating daughter cells (Perez et al., 2019; Yang et al., 2017), whereas septa formation by PG synthesis occurs before cell separation in Sau and Bsu (Bisson-Filho et al., 2017, Lund et al., 2018, Monteiro et al., 2018)....
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