Characterization of a Bacterial Kinase That Phosphorylates Dihydrosphingosine to Form dhS1P.

Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis synthesizes a variety of sphingolipids, similar to its human host. Studies have shown that synthesis of these lipids (dihydroceramides [DHCs]) is involved in oxidative stress resistance, the survival of P. gingivalis during stationary phase, and immune modulation. Here, we constructed a deletion mutant of P. gingivalis strain W83 with a deletion of the gene encoding DhSphK1, a protein that shows high similarity to a eukaryotic sphingosine kinase, an enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate. Our data show that deletion of the dhSphK1 gene results in a shift in the sphingolipid composition of P. gingivalis cells; specifically, the mutant synthesizes higher levels of phosphoglycerol DHCs (PG-DHCs) than the parent strain W83. Although PG1348 shows high similarity to the eukaryotic sphingosine kinase, we discovered that the PG1348 enzyme is unique, since it preferentially phosphorylates dihydrosphingosine, not sphingosine. Besides changes in lipid composition, the W83 ΔPG1348 mutant displayed a defect in cell division, the biogenesis of outer membrane vesicles (OMVs), and the amount of K antigen capsule. Taken together, we have identified the first bacterial dihydrosphingosine kinase whose activity regulates the lipid profile of P. gingivalis and underlies a regulatory mechanism of immune modulation. IMPORTANCE Sphingoid base phosphates, such as sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (dhS1P), act as ligands for S1P receptors, and this interaction is known to play a central role in mediating angiogenesis, vascular stability and permeability, and immune cell migration to sites of inflammation. Studies suggest that a shift in ratio to higher levels of dhS1P in relation to S1P alters downstream signaling cascades due to differential binding and activation of the various S1P receptor isoforms. Specifically, higher levels of dhS1P are thought to be anti-inflammatory. Here, we report on the characterization of a novel kinase in Porphyromonas gingivalis that phosphorylates dihydrosphingosine to form dhS1P.

A Novel Regulation of K-antigen Capsule Synthesis in Porphyromonas gingivalis Is Driven by the Response Regulator PG0720-Directed Antisense RNA.

The periodontal pathogen Porphyromonas gingivalis strain W83 displays at least three different surface glycans, specifically two types of lipopolysaccharides (O-LPS and A-LPS) and K-antigen capsule. Despite the importance of K-antigen capsule to the virulence of P. gingivalis, little is known as to how expression of genes involved in the synthesis of this surface glycan is regulated. The genes required for K-antigen capsule synthesis are located in a locus that encodes a number of transcripts, including an operon (PG0104 to PG0121, generating ~19.4-kb transcript) which contains a non-coding 77-bp inverted repeat (77 bpIR) region near the 5'-end. Previously, we identified a 550-nucleotide antisense RNA molecule (designated asSuGR for antisense Surface Glycan Regulator) encoded within the 77-bpIR element that influences the synthesis of surface glycans. In this study, we demonstrate that the DNA-binding response regulator PG0720 can bind the promoter region of asSuGR and activate expression of asSuGR, indicating that PG0720 may indirectly influence transcript levels of the K-antigen capsule operon expressed from the sense strand. The data show that deletion of the PG0720 gene confers a defect in the presentation of surface polysaccharides compared with the parent strain and quantitative RT-PCR (qPCR) analysis determined that the overall expression of genes involved in K-antigen capsule synthesis were down-regulated in the PG0720 mutant. Furthermore, the defects of the PG0720 deletion mutant were restored by complementation. Importantly, the PG0720 deletion mutant showed reduced virulence. Altogether, our data show that the response regulator PG0720 regulates expression of asSuGR, a trans-acting antisense RNA molecule involved in modulating the production of surface polysaccharides in P. gingivalis strain W83. The data provide further evidence that surface glycans are key virulence determinants and significantly advances our understanding of the molecular mechanisms controlling the synthesis of P. gingivalis K-antigen capsule, a key virulence determinant.

Chlamydial MreB Directs Cell Division and Peptidoglycan Synthesis in Escherichia coli in the Absence of FtsZ Activity.

Cell division is the ultimate process for the propagation of bacteria, and FtsZ is an essential protein used by nearly all bacteria for this function. Chlamydiae belong to a small group of bacteria that lack the universal cell division protein FtsZ but still divide by binary fission. Chlamydial MreB is a member of the shape-determining MreB/Mbl family of proteins responsible for rod shape morphology in Escherichia coliChlamydia also encodes a homolog of RodZ, an MreB assembly cytoskeletal protein that links MreB to cell wall synthesis proteins. We hypothesized that MreB directs cell division in Chlamydia and that chlamydial MreB could replace FtsZ function for cell division in E. coli Overexpression of chlamydial mreB-rodZ in E. coli induced prominent morphological changes with production of large swollen or oval bacteria, eventually resulting in bacterial lysis. Low-level expression of chlamydial mreB-rodZ restored viability of a lethal ΔmreB mutation in E. coli, although the bacteria lost their typical rod shape and grew as rounded cells. When FtsZ activity was inhibited by overexpression of SulA in the ΔmreB mutant of E. coli complemented with chlamydial mreB-rodZ, spherical E. coli grew and divided. Localization studies using a fluorescent fusion chlamydial MreB protein indicated that chlamydial RodZ directs chlamydial MreB to the E. coli division septum. These results demonstrate that chlamydial MreB, in partnership with chlamydial RodZ, acts as a cell division protein. Our findings suggest that an mreB-rodZ-based mechanism allows Chlamydia to divide without the universal division protein FtsZ.IMPORTANCE The study of Chlamydia growth and cell division is complicated by its obligate intracellular nature and biphasic lifestyle. Chlamydia also lacks the universal division protein FtsZ. We employed the cell division system of Escherichia coli as a surrogate to identify chlamydial cell division proteins. We demonstrate that chlamydial MreB, together with chlamydial RodZ, forms a cell division and growth complex that can replace FtsZ activity and support cell division in E. coli Chlamydial RodZ plays a major role in directing chlamydial MreB localization to the cell division site. It is likely that the evolution of chlamydial MreB and RodZ to form a functional cell division complex allowed Chlamydia to dispense with its FtsZ-based cell division machinery during genome reduction. Thus, MreB-RodZ represents a possible mechanism for cell division in other bacteria lacking FtsZ.

PBP1B Glycosyltransferase and Transpeptidase Activities Play Different Essential Roles during the De Novo Regeneration of Rod Morphology in Escherichia coli.

Peptidoglycan is a vital component of nearly all cell wall-bearing bacteria and is a valuable target for antibacterial therapy. However, despite decades of work, there remain important gaps in understanding how this macromolecule is synthesized and molded into a three-dimensional structure that imparts specific morphologies to individual cells. Here, we investigated the particularly enigmatic area of how peptidoglycan is synthesized and shaped during the first stages of creating cell shape de novo, that is, in the absence of a preexisting template. We found that when lysozyme-induced (LI) spheroplasts of Escherichia coli were allowed to resynthesize peptidoglycan, the cells divided first and then elongated to recreate a normal rod-shaped morphology. Penicillin binding protein 1B (PBP1B) was critical for the first stage of this recovery process. PBP1B synthesized peptidoglycan de novo, and this synthesis required that PBP1B interact with the outer membrane lipoprotein LpoB. Surprisingly, when LpoB was localized improperly to the inner membrane, recovering spheroplasts synthesized peptidoglycan and divided but then propagated as amorphous spheroidal cells, suggesting that the regeneration of a normal rod shape depends on a particular spatial interaction. Similarly, spheroplasts carrying a PBP1B variant lacking transpeptidase activity or those in which PBP1A was overproduced could synthesize new peptidoglycan and divide but then grew as oddly shaped spheroids. We conclude that de novo cell wall synthesis requires the glycosyltransferase activity of PBP1B but that PBP1B transpeptidase activity is needed to assemble cell walls with wild-type morphology.IMPORTANCE Bacterial cell wall peptidoglycan is synthesized and modified by penicillin binding proteins (PBPs), which are targeted by about half of all currently prescribed antibiotics, including penicillin and its derivatives. Because antibiotic resistance is rising, it has become increasingly urgent that we fill the gaps in our knowledge about how PBPs create and assemble this protective wall. We report here that PBP1B plays an essential role in synthesizing peptidoglycan in the absence of a preexisting template: its glycosyltransferase activity is responsible for de novo synthesis, while its transpeptidase activity is required to construct cell walls of a specific shape. These results highlight the importance of this enzyme and distinguish its biological roles from those of other PBPs and peptidoglycan synthases.

Colanic Acid Intermediates Prevent De Novo Shape Recovery of Escherichia coli Spheroplasts, Calling into Question Biological Roles Previously Attributed to Colanic Acid.

UNLABELLED: After losing their protective peptidoglycan, bacterial spheroplasts can resynthesize a cell wall to recreate their normal shape. In Escherichia coli, this process requires the Rcs response. In its absence, spheroplasts do not revert to rod shapes but instead form enlarged spheroids and lyse. Here, we investigated the reason for this Rcs requirement. Rcs-deficient spheroids exhibited breaks and bulges in their periplasmic spaces and failed to synthesize a complete peptidoglycan cell wall, indicating that the bacterial envelope was defective. To determine the Rcs-dependent gene(s) required for shape recovery, we tested spheroplasts lacking selected RcsB-regulated genes and found that colanic acid (CA) biosynthesis appeared to be involved. Surprisingly, though, extracellular CA was not required for recovery. Instead, lysis was caused by mutations that interrupted CA biosynthesis downstream of the initial glycosyl transferase, WcaJ. Deleting wcaJ prevented lysis of spheroplasts lacking ensuing steps in the pathway, and providing WcaJ in trans to a mutant lacking the entire CA operon triggered spheroplast enlargement and lysis. Thus, CA is not required for spheroplast recovery. Instead, CA intermediates accumulate as dead-end products which inhibit recovery of wall-less cells. The results strongly imply that CA may not be required for the survival E. coli L-forms. More broadly, these findings mandate that previous conclusions about the role of colanic acid in biofilm formation or virulence must be reevaluated. IMPORTANCE: Wall-less bacteria can resynthesize their walls and recreate a normal shape, which in Escherichia coli requires the Rcs response. While attempting to identify the Rcs-dependent gene required for shape recovery, we found that colanic acid (CA) biosynthesis appeared to be involved. Surprisingly, though, cell death was caused by mutations that interrupted CA biosynthesis downstream of the initial step in the pathway, creating dead-end compounds that inhibited recovery of wall-less cells. When testing for the biological role of CA, most previous experiments used mutants that would accumulate these deadly intermediates, meaning that all prior conclusions must be reexamined to determine if the results were caused by these lethal side effects instead of accurately reflecting the biological purpose of CA itself.

The Rcs stress response and accessory envelope proteins are required for de novo generation of cell shape in Escherichia coli.

Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown. To address this question, we treated Escherichia coli with lysozyme to remove the peptidoglycan wall while leaving intact the inner and outer membranes and periplasm. The resulting lysozyme-induced (LI) spheroplasts recovered a rod shape after four to six generations. Recovery proceeded via a series of cell divisions that produced misshapen and branched intermediates before later progeny assumed a normal rod shape. Importantly, mutants defective in mounting the Rcs stress response and those lacking penicillin binding protein 1B (PBP1B) or LpoB could not divide or recover their cell shape but instead enlarged until they lysed. LI spheroplasts from mutants lacking the Lpp lipoprotein or PBP6 produced spherical daughter cells that did not recover a normal rod shape or that did so only after a significant delay. Thus, to regenerate normal morphology de novo, E. coli must supplement the classic FtsZ- and MreBCD-directed cell wall systems with activities that are otherwise dispensable for growth under normal laboratory conditions. The existence of these auxiliary mechanisms implies that they may be required for survival in natural environments, where bacterial walls can be damaged extensively or removed altogether.

Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties.

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by ß-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.

Small molecule functional analogs of peptides that inhibit lambda site-specific recombination and bind Holliday junctions.

Our lab has isolated hexameric peptides that are structure-selective ligands of Holliday junctions (HJ), central intermediates of several DNA recombination reactions. One of the most potent of these inhibitors, WRWYCR, has shown antibacterial activity in part due to its inhibition of DNA repair proteins. To increase the therapeutic potential of these inhibitors, we searched for small molecule inhibitors with similar activities. We screened 11 small molecule libraries comprising over nine million individual compounds and identified a potent N-methyl aminocyclic thiourea inhibitor that also traps HJs formed during site-specific recombination reactions in vitro. This inhibitor binds specifically to protein-free HJs and can inhibit HJ resolution by RecG helicase, but only showed modest growth inhibition of bacterial with a hyperpermeable outer membrane; nonetheless, this is an important step in developing a functional analog of the peptide inhibitors.

Novel antibiotics: macrocyclic peptides designed to trap Holliday junctions.

[reaction: see text] Described are the syntheses of eight macrocyclic peptides designed to trap Holliday junctions in bacteria, thereby inhibiting bacterial growth. These macrocycles were designed from linear dimerized hexapeptides that bind to the C-2 symmetrical Holliday junction. They were synthesized from three monomers using a combinatorial-like strategy that permits elucidation of the monomer role in accumulation of Holliday junctions and antibiotic activity. These macrocycles are an important step in designing and synthesizing a new class of antibiotics.