CcrZ is a pneumococcal spatiotemporal cell cycle regulator that interacts with FtsZ and controls DNA replication by modulating the activity of DnaA.

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

Simultaneous purification and depolymerization of Streptococcus pneumoniae serotype 2 capsular polysaccharides by trifluoroacetic acid.

Development of an effective purification process in order to provide low cost and high-quality vaccine is the necessity of glycoconjugate vaccine manufacturing industries. In the present study, we have attempted to develop a method for simultaneous purification and depolymerization process for capsular polysaccharides (CPS) derived from Streptococcus pneumoniae serotype 2. Trifluoroacetic acid (TFA) was used to precipitate impurities which were then removed by centrifugation. It was observed that the TFA treatment could simultaneously depolymerize the CPS and purify it. The purified and depolymerized CPS was analyzed for its purity, structural identity and conformity, molecular size, antigenicity to meet desired quality specifications. The obtained results showed that the purification and depolymerization of S. pneumoniae serotype 2 CPS did not affect the antigenicity of CPS.

Polyvalent Transition-State Analogues of Sialyl Substrates Strongly Inhibit Bacterial Sialidases*.

Bacterial sialidases (SA) are validated drug targets expressed by common human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, or Clostridium perfringens. Noncovalent inhibitors of bacterial SA capable of reaching the submicromolar level are rarely reported. In this work, multi- and polyvalent compounds are developed, based on the transition-state analogue 2-deoxy-2,3-didehydro-N-acetylneuraminic (DANA). Poly-DANA inhibits the catalytic activity of SA from S. pneumoniae (NanA) and the symbiotic microorganism B. thetaiotaomicron (BtSA) at the picomolar and low nanomolar levels (expressed in moles of molecules and of DANA, respectively). Each DANA grafted to the polymer surpasses the inhibitory potential of the monovalent analogue by more than four orders of magnitude, which represents the highest multivalent effect reported so far for an enzyme inhibition. The synergistic interaction is shown to operate exclusively in the catalytic domain, and not in the flanked carbohydrate-binding module (CBM). These results offer interesting perspectives for the multivalent inhibition of other SA families lacking a CBM, such as viral, parasitic, or human SA.

The Bactericidal Fatty Acid Mimetic 2CCA-1 Selectively Targets Pneumococcal Extracellular Polyunsaturated Fatty Acid Metabolism.

Streptococcus pneumoniae, a major cause of pneumonia, sepsis, and meningitis worldwide, has the nasopharynges of small children as its main ecological niche. Depletion of pneumococci from this niche would reduce the disease burden and could be achieved using small molecules with narrow-spectrum antibacterial activity. We identified the alkylated dicyclohexyl carboxylic acid 2CCA-1 as a potent inducer of autolysin-mediated lysis of S. pneumoniae, while having low activity against Staphylococcus aureus 2CCA-1-resistant strains were found to have inactivating mutations in fakB3, known to be required for uptake of host polyunsaturated fatty acids, as well as through inactivation of the transcriptional regulator gene fabT, vital for endogenous, de novo fatty acid synthesis regulation. Structure activity relationship exploration revealed that, besides the central dicyclohexyl group, the fatty acid-like structural features of 2CCA-1 were essential for its activity. The lysis-inducing activity of 2CCA-1 was considerably more potent than that of free fatty acids and required growing bacteria, suggesting that 2CCA-1 needs to be metabolized to exert its antimicrobial activity. Total lipid analysis of 2CCA-1 treated bacteria identified unique masses that were modeled to 2CCA-1 containing lysophosphatidic and phosphatidic acid in wild-type but not in fakB3 mutant bacteria. This suggests that 2CCA-1 is metabolized as a fatty acid via FakB3 and utilized as a phospholipid building block, leading to accumulation of toxic phospholipid species. Analysis of FabT-mediated fakB3 expression elucidates how the pneumococcus could ensure membrane homeostasis and concurrent economic use of host-derived fatty acids.IMPORTANCE Fatty acid biosynthesis is an attractive antibiotic target, as it affects the supply of membrane phospholipid building blocks. In Streptococcus pneumoniae, it is not sufficient to target only the endogenous fatty acid synthesis machinery, as uptake of host fatty acids may bypass this inhibition. Here, we describe a small-molecule compound, 2CCA-1, with potent bactericidal activity that upon interactions with the fatty acid binding protein FakB3, which is present in a limited number of Gram-positive species, becomes metabolized and incorporated as a toxic phospholipid species. Resistance to 2CCA-1 developed specifically in fakB3 and the regulatory gene fabT These mutants reveal a regulatory connection between the extracellular polyunsaturated fatty acid metabolism and endogenous fatty acid synthesis in S. pneumoniae, which could ensure balance between efficient scavenging of host polyunsaturated fatty acids and membrane homeostasis. The data might be useful in the identification of narrow-spectrum treatment strategies to selectively target members of the Lactobacillales such as S. pneumoniae.

The alternative sigma factor σX mediates competence shut-off at the cell pole in Streptococcus pneumoniae.

Competence is a widespread bacterial differentiation program driving antibiotic resistance and virulence in many pathogens. Here, we studied the spatiotemporal localization dynamics of the key regulators that master the two intertwined and transient transcription waves defining competence in Streptococcus pneumoniae. The first wave relies on the stress-inducible phosphorelay between ComD and ComE proteins, and the second on the alternative sigma factor σX, which directs the expression of the DprA protein that turns off competence through interaction with phosphorylated ComE. We found that ComD, σX and DprA stably co-localize at one pole in competent cells, with σX physically conveying DprA next to ComD. Through this polar DprA targeting function, σX mediates the timely shut-off of the pneumococcal competence cycle, preserving cell fitness. Altogether, this study unveils an unprecedented role for a transcription σ factor in spatially coordinating the negative feedback loop of its own genetic circuit.

Extracellular adenosine signaling reverses the age-driven decline in the ability of neutrophils to kill Streptococcus pneumoniae.

The elderly are susceptible to serious infections by Streptococcus pneumoniae (pneumococcus), which calls for a better understanding of the pathways driving the decline in host defense in aging. We previously found that extracellular adenosine (EAD) shaped polymorphonuclear cell (PMN) responses, which are crucial for controlling infection. EAD is produced by CD39 and CD73, and signals via A1, A2A, A2B, and A3 receptors. The objective of this study was to explore the age-driven changes in the EAD pathway and its impact on PMN function. We found in comparison to young mice, PMNs from old mice expressed significantly less CD73, but similar levels of CD39 and adenosine receptors. PMNs from old mice failed to efficiently kill pneumococci ex vivo; however, supplementation with adenosine rescued this defect. Importantly, transfer of PMNs expressing CD73 from young mice reversed the susceptibility of old mice to pneumococcal infection. To identify which adenosine receptor(s) is involved, we used specific agonists and inhibitors. We found that A1 receptor signaling was crucial for PMN function as inhibition or genetic ablation of A1 impaired the ability of PMNs from young mice to kill pneumococci. Importantly, activation of A1 receptors rescued the age-associated defect in PMN function. In exploring mechanisms, we found that PMNs from old mice failed to efficiently kill engulfed pneumococci and that A1 receptor controlled intracellular killing. In summary, targeting the EAD pathway reverses the age-driven decline in PMN antimicrobial function, which has serious implications in combating infections.

Streptococcus pneumoniae metal homeostasis alters cellular metabolism.

Streptococcus pneumoniae colonizes the human nasopharyngeal mucosa and is a leading cause of community-acquired pneumonia, acute otitis media, and bacterial meningitis. Metal ion homeostasis is vital to the survival of this pathogen across diverse biological sites and contributes significantly to colonization and invasive disease. Microarray and qRT-PCR analysis revealed an upregulation of an uncharacterized operon (SP1433-1438) in pneumococci subjected to metal-chelation by N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN). Supplementation of zinc, cobalt, and nickel following TPEN treatment significantly abrogated induction. BLASTP comparisons and protein topology analysis predicted this locus to encode components of ATP binding cassette (ABC) transporters involved in multidrug resistance (SP1434-1435) and energy-coupling factor (ECF) transporters (SP1436-1438). Inductively coupled plasma mass spectrometry (ICP-MS) analysis identified differences in intracellular metal content in a Δ1434-8 mutant strain compared to parental T4R. Further, analysis of the secreted metabolome of WT and Δ1434-8 strains identified significant changes in pneumococcal glycolytic and amino acid metabolic pathways, indicating a shift towards mixed acid fermentation. Additionally, proteomic analysis revealed differentially expressed proteins in the Δ1434-8 mutant strain, with nearly 20% regulated by the global catabolite repressor, CcpA. Based on these findings, we propose that the transporters encoded by SP1433-1438 are involved in regulating the central metabolism of S. pneumoniae and contributing to bacterial survival during metal stress.

Gradient acoustic focusing of sub-micron particles for separation of bacteria from blood lysate.

Handling of submicron-sized objects is important in many biochemical and biomedical applications, but few methods today can precisely manipulate this range of particles. We present gradient acoustic focusing that enables flow-through particle separation of submicron particles and cells and we apply it for separation of bacteria from blood lysate to facilitate their detection in whole blood for improved diagnostics. To control suspended objects below the classical 2µm size limit for acoustic focusing, we introduce a co-flowing acoustic impedance gradient to generate a stabilizing acoustic volume force that supresses acoustic streaming. The method is validated theoretically and experimentally using polystyrene particles, Staphylococcus aureus, Streptococcus pneumoniae and Escherichia coli. The applicability of the method is demonstrated by the separation of bacteria from selectively chemically lysed blood. Combined with downstream operations, this new approach opens up for novel methods for sepsis diagnostics.

Process intensification for production of Streptococcus pneumoniae whole-cell vaccine.

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.

The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae.

HAMLET is a complex of human α-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bisphosphate aldolase (FBPA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid-phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis.

RocS drives chromosome segregation and nucleoid protection in Streptococcus pneumoniae.

Chromosome segregation in bacteria is poorly understood outside some prominent model strains1-5 and even less is known about how it is coordinated with other cellular processes. This is the case for the opportunistic human pathogen Streptococcus pneumoniae (the pneumococcus)6, which lacks the Min and the nucleoid occlusion systems7, and possesses only an incomplete chromosome partitioning Par(A)BS system, in which ParA is absent8. The bacterial tyrosine kinase9 CpsD, which is required for capsule production, was previously found to interfere with chromosome segregation10. Here, we identify a protein of unknown function that interacts with CpsD and drives chromosome segregation. RocS (Regulator of Chromosome Segregation) is a membrane-bound protein that interacts with both DNA and the chromosome partitioning protein ParB to properly segregate the origin of replication region to new daughter cells. In addition, we show that RocS interacts with the cell division protein FtsZ and hinders cell division. Altogether, this work reveals that RocS is the cornerstone of a nucleoid protection system ensuring proper chromosome segregation and cell division in coordination with the biogenesis of the protective capsular layer.

Prevention of EloR/KhpA heterodimerization by introduction of site-specific amino acid substitutions renders the essential elongasome protein PBP2b redundant in Streptococcus pneumoniae.

The RNA binding proteins EloR and KhpA are important components of the regulatory network that controls and coordinates cell elongation and division in S. pneumoniae. Loss of either protein reduces cell length, and makes the essential elongasome proteins PBP2b and RodA dispensable. It has been shown previously in formaldehyde crosslinking experiments that EloR co-precipitates with KhpA, indicating that they form a complex in vivo. In the present study, we used 3D modeling and site directed mutagenesis in combination with protein crosslinking to further study the relationship between EloR and KhpA. Protein-protein interaction studies demonstrated that KhpA forms homodimers and that KhpA in addition binds to the KH-II domain of EloR. Site directed mutagenesis identified isoleucine 61 (I61) as crucial for KhpA homodimerization. When substituting I61 with phenylalanine, KhpA lost the ability to homodimerize, while it still interacted clearly with EloR. In contrast, both homo- and heterodimerization were lost when I61 was substituted with tyrosine. By expressing these KhpA versions in S. pneumoniae, we were able to show that disruption of EloR/KhpA heterodimerization makes the elongasome redundant in S. pneumoniae. Of note, loss of KhpA homodimerization did not give rise to this phenotype, demonstrating that the EloR/KhpA complex is crucial for regulating the activity of the elongasome. In support of this conclusion, we found that localization of KhpA to the pneumococcal mid-cell region depends on its interaction with EloR. Furthermore, we found that the EloR/KhpA complex co-localizes with FtsZ throughout the cell cycle.

Femtoliter droplet confinement of Streptococcus pneumoniae: bacterial genetic transformation by cell-cell interaction in droplets.

Streptococcus pneumoniae (pneumococcus), a deadly bacterial human pathogen, uses genetic transformation to gain antibiotic resistance. Genetic transformation begins when a pneumococcal strain in a transient specialized physiological state called competence, attacks and lyses another strain, releasing DNA, taking up fragments of the liberated DNA, and integrating divergent genes into its genome. While many steps of the process are known and generally understood, the precise mechanism of this natural genetic transformation is not fully understood and the current standard strategies to study it have limitations in specifically controlling and observing the process in detail. To overcome these limitations, we have developed a droplet microfluidic system for isolating individual episodes of bacterial transformation between two confined cells of pneumococcus. By encapsulating the cells in a 10 µm diameter aqueous droplet, we provide an improved experimental model of genetic transformation, as both participating cells can be identified, and the released DNA is spatially restricted near the attacking strain. Specifically, the bacterial cells, one rifampicin (R) resistant, the other novobiocin (N) and spectinomycin (S) resistant were encapsulated in droplets carried by the fluorinated oil FC-40 with 5% surfactant and allowed to carry out competence-specific attack and DNA uptake (and consequently gain antibiotic resistances) within the droplets. The droplets were then broken, and recombinants were recovered by selective plating with antibiotics. The new droplet system encapsulated 2 or more cells in a droplet with a probability up to 71%, supporting gene transfer rates comparable to standard mixtures of unconfined cells. Thus, confinement in droplets allows characterization of natural genetic transformation during a strictly defined interaction between two confined cells.

Insights into the antibacterial mechanism of PEGylated nano-bacitracin A against Streptococcus pneumonia: both penicillin-sensitive and penicillin-resistant strains.

BACKGROUND: Multidrug-resistant (MDR) Streptococcus pneumonia constitute a major worldwide public health concern. MATERIALS AND METHODS: In our preliminary study, PEGylated nano-self-assemblies of bacitracin A (PEGylated Nano-BA12K) showed strong antibacterial potency against reference S. pneumonia strain (ATCC 49619). In this study, the possibility of applying PEGylated Nano-BA12K against penicillin-resistant S. pneumonia was further investigated. In addition, the underlying antibacterial mechanism of PEGylated Nano-BA12K against both sensitive and resistant S. pneumonia was also clarified systematically, since S. pneumonia was naturally resistant to its unassembled counterpart bacitracin A (BA). RESULTS: PEGylated Nano-BA12K showed strong antibacterial potency against 13 clinical isolates of S. pneumonia, including five penicillin-resistant strains. Structural changes, partial collapse, and even lysis of both penicillin-sensitive and penicillin-resistant bacteria were observed after incubation with PEGylated Nano-BA12K via transmission electron microscopy and atomic force microscopy. Thus, the cell wall or/and cell membrane might be the main target of PEGylated Nano-BA12K against S. pneumonia. PEGylated Nano-BA12K exhibited limited effect on the permeabilization and peptidoglycan content of cell wall. Surface pressure measurement suggested that PEGylated Nano-BA12K was much more tensioactive than BA, which was usually translated into a good membranolytic effect, and is helpful to permeabilize the cell membrane and damage membrane integrity, as evidenced by depolarization of the membrane potential, permeabilization of membrane and leakage of calcein from liposomes. CONCLUSION: Collectively, great cell membrane permeability and formidable membrane disruption may work together for the strong antibacterial activity of PEGylated Nano-BA12K against S. pneumonia. Taken together, PEGylated Nano-BA12K has excellent potential against both penicillin-sensitive and penicillin-resistant S. pneumonia and might be suitable for the treatment of S. pneumonia infectious diseases.

Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification.

Streptococcus pneumoniae evades C3-mediated opsonization and effector functions by expressing an immuno-protective polysaccharide capsule and Factor H (FH)-binding proteins. Here we use super-resolution microscopy, mutants and functional analysis to show how these two defense mechanisms are functionally and spatially coordinated on the bacterial cell surface. We show that the pneumococcal capsule is less abundant at the cell wall septum, providing C3/C3b entry to underlying nucleophilic targets. Evasion of C3b deposition at division septa and lateral amplification underneath the capsule requires localization of the FH-binding protein PspC at division sites. Most pneumococcal strains have one PspC protein, but successful lineages in colonization and disease may have two, PspC1 and PspC2, that we show affect virulence differently. We find that spatial localization of these FH-recruiting proteins relative to division septa and capsular layer is instrumental for pneumococci to resist complement-mediated opsonophagocytosis, formation of membrane-attack complexes, and for the function as adhesins.

Phosphorylation-dependent activation of the cell wall synthase PBP2a in Streptococcus pneumoniae by MacP.

Most bacterial cells are surrounded by an essential cell wall composed of the net-like heteropolymer peptidoglycan (PG). Growth and division of bacteria are intimately linked to the expansion of the PG meshwork and the construction of a cell wall septum that separates the nascent daughter cells. Class A penicillin-binding proteins (aPBPs) are a major family of PG synthases that build the wall matrix. Given their central role in cell wall assembly and importance as drug targets, surprisingly little is known about how the activity of aPBPs is controlled to properly coordinate cell growth and division. Here, we report the identification of MacP (SPD_0876) as a membrane-anchored cofactor of PBP2a, an aPBP synthase of the Gram-positive pathogen Streptococcus pneumoniae We show that MacP localizes to the division site of S. pneumoniae, forms a complex with PBP2a, and is required for the in vivo activity of the synthase. Importantly, MacP was also found to be a substrate for the kinase StkP, a global cell cycle regulator. Although StkP has been implicated in controlling the balance between the elongation and septation modes of cell wall synthesis, none of its substrates are known to modulate PG synthetic activity. Here we show that a phosphoablative substitution in MacP that blocks StkP-mediated phosphorylation prevents PBP2a activity without affecting the MacP-PBP2a interaction. Our results thus reveal a direct connection between PG synthase function and the control of cell morphogenesis by the StkP regulatory network.

Separation of pathogenic bacteria by chain length.

Using Deterministic Lateral Displacement devices optimized for sensitivity to particle length, we separate subpopulations of bacteria depending on known properties that affect their capability to cause disease (virulence). For the human bacterial pathogen Streptococcus pneumoniae, bacterial chain length and the presence of a capsule are known virulence factors contributing to its ability to cause severe disease. Separation of cultured pneumococci into subpopulations based on morphological type (single cocci, diplococci and chains) will enable more detailed studies of the role they play in virulence. Moreover, we present separation of mixed populations of almost genetically identical encapsulated and non-encapsulated pneumococcal strains in our device.

PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae.

Eukaryotic-like serine/threonine kinases (eSTKs) with extracellular PASTA repeats are key membrane regulators of bacterial cell division. How PASTA repeats govern eSTK activation and function remains elusive. Using evolution- and structural-guided approaches combined with cell imaging, we disentangle the role of each PASTA repeat of the eSTK StkP from Streptococcus pneumoniae. While the three membrane-proximal PASTA repeats behave as interchangeable modules required for the activation of StkP independently of cell wall binding, they also control the septal cell wall thickness. In contrast, the fourth and membrane-distal PASTA repeat directs StkP localization at the division septum and encompasses a specific motif that is critical for final cell separation through interaction with the cell wall hydrolase LytB. We propose a model in which the extracellular four-PASTA domain of StkP plays a dual function in interconnecting the phosphorylation of StkP endogenous targets along with septal cell wall remodelling to allow cell division of the pneumococcus.

A programmed cell division delay preserves genome integrity during natural genetic transformation in Streptococcus pneumoniae.

Competence for genetic transformation is a differentiation program during which exogenous DNA is imported into the cell and integrated into the chromosome. In Streptococcus pneumoniae, competence develops transiently and synchronously in all cells during exponential phase, and is accompanied by a pause in growth. Here, we reveal that this pause is linked to the cell cycle. At least two parallel pathways impair peptidoglycan synthesis in competent cells. Single-cell analyses demonstrate that ComM, a membrane protein induced during competence, inhibits both initiation of cell division and final constriction of the cytokinetic ring. Competence also interferes with the activity of the serine/threonine kinase StkP, the central regulator of pneumococcal cell division. We further present evidence that the ComM-mediated delay in division preserves genomic integrity during transformation. We propose that cell division arrest is programmed in competent pneumococcal cells to ensure that transformation is complete before resumption of cell division, to provide this pathogen with the maximum potential for genetic diversity and adaptation.

Novel Electrophilic Scaffold for Imaging of Essential Penicillin-Binding Proteins in Streptococcus pneumoniae.

Peptidoglycan (PG) is a mesh-like heteropolymer made up of glycan chains cross-linked by short peptides and is the major scaffold of eubacterial cell walls, determining cell shape, size, and chaining. This structure, which is required for growth and survival, is located outside of the cytoplasmic membrane of bacterial cells, making it highly accessible to antibiotics. Penicillin-binding proteins (PBPs) are essential for construction of PG and perform transglycosylase activities to generate the glycan strands and transpeptidation to cross-link the appended peptides. The ß-lactam antibiotics, which are among the most clinically effective antibiotics for the treatment of bacterial infections, inhibit PBP transpeptidation, ultimately leading to cell lysis. Despite this importance, the discrete functions of individual PBP homologues have been difficult to determine. These major gaps in understanding of PBP activation and macromolecular interactions largely result from a lack of tools to assess the functional state of specific PBPs in bacterial cells. We have identified ß-lactones as a privileged scaffold for the generation of PBP-selective probes and utilized these compounds for imaging of the essential proteins, PBP2x and PBP2b, in Streptococcus pneumoniae. We demonstrated that while PBP2b activity is restricted to a ring surrounding the division sites, PBP2x activity is present both at the septal center and at the surrounding ring. These spatially separate regions of PBP2x activity could not be detected by previous activity-based approaches, which highlights a critical strength of our PBP-selective imaging strategy.