Peptidoglycan maturation controls outer membrane protein assembly.

Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial for the maintenance of cellular integrity and enable survival in challenging environments1-5. The function of the outer membrane is dependent on outer membrane proteins (OMPs), which are inserted into the membrane by the ß-barrel assembly machine6,7 (BAM). Growing Escherichia coli cells segregate old OMPs towards the poles by a process known as binary partitioning, the basis of which is unknown8. Here we demonstrate that peptidoglycan underpins the spatiotemporal organization of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little effect on its activity, leading to preferential insertion of OMPs at division sites. The synchronization of OMP biogenesis with cell wall growth results in the binary partitioning of OMPs as cells divide. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation, a potential vulnerability that could be exploited in future antibiotic design.

An NlpC/P60 protein catalyzes a key step in peptidoglycan recycling at the intersection of energy recovery, cell division and immune evasion in the intracellular pathogen Chlamydia trachomatis.

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs.

Suppression and synthetic-lethal genetic relationships of ΔgpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin-binding protein interactions in Streptococcus pneumoniae D39.

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side-wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division-protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co-IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP-PBP2x.

Prevalence of dental fluorosis in Mexico 2005-2015: a literature review.

OBJECTIVE:: To perform a literature review regarding current dental fluorosis prevalence in Mexico reported from 2005 to 2015. MATERIALS AND METHODS:: A comprehensive scientific literature review, in both English and Spanish, was performed in four databases up to June 2015. Search terms: fluorosis or dental fluorosis (mesh), prevalence (mesh), distribution (mesh), cases (mesh), epidemiology (mesh), Mexico. RESULTS:: 17 publications were included. Reported prevalence of dental fluorosis in Mexico ranged from 15.5 to 100%. Most of the studies were conducted in areas where water fluoride levels are low or optimal (≤1.5ppmF) and in which a prevalence of 15.5 to 81.7% was observed. In areas with higher levels of naturally fluoridated water (>1.5ppmF), prevalence ranged from 92 to 100%. Fluorosis severity ranged from questionable to severe. CONCLUSION:: High prevalence of dental fluorosis was observed even in areas where fluoride concentration in water was low or optimal. In addition to fluoride in groundwater, there are multiple risk factors that should be controlled.

Monitoring the Interaction of the Peptidoglycan with the Bacterial ß-Barrel Assembly Machinery.

Gram-negative bacteria coordinate the biosynthesis of their different cell envelope components. Growth of the outer membrane (OM) requires the essential ß-barrel assembly machine (BAM), which inserts OM proteins (OMPs) into the OM. The underlying peptidoglycan (PG) sacculus grows by the insertion of nascent glycan chains. We have previously identified interactions between BAM and PG in E. coli and showed that these interactions coordinate OM biogenesis with PG growth. BAM responds to the maturation state of the PG, and this mechanism activates preferentially BAM complexes at sites of active PG synthesis. Here we present protocols to purify soluble Bam proteins and full-length BamABCDE, isolate PG and soluble PG fragments, and study BAM-PG interactions with the isolated components. We also describe the protocol to detect interactions between Bam proteins and PG in cells.

Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors.

The development of new antibiotics is particularly problematic in Gram-negative bacteria due to the presence of the outer membrane (OM), which serves as a permeability barrier. Recently, the ß-barrel assembly machine (BAM), located in the OM and responsible for ß-barrel type OM protein (OMP) assembly, has been validated as a novel target for antibiotics. Here, we identified potential BAM complex inhibitors using a screening approach that reports on cell envelope σE and Rcs stress in Escherichia coli. Screening a library consisting of 316 953 compounds yielded five compounds that induced σE and Rcs stress responses, while not inducing the intracellular heat-shock response. Two of the five compounds (compounds 2 and 14) showed the characteristics of known BAM complex inhibitors: synergy with OMP biogenesis mutants, decrease in the abundance of various OMPs, and loss of OM integrity. Importantly, compound 2 also inhibited BAM-dependent OMP folding in an in vitro refolding assay using purified BAM complex reconstituted in proteoliposomes.

The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes.

Bacterial growth and cell division requires precise spatiotemporal regulation of the synthesis and remodelling of the peptidoglycan layer that surrounds the cytoplasmic membrane. GpsB is a cytosolic protein that affects cell wall synthesis by binding cytoplasmic mini-domains of peptidoglycan synthases to ensure their correct subcellular localisation. Here, we describe critical structural features for the interaction of GpsB with peptidoglycan synthases from three bacterial species (Bacillus subtilis, Listeria monocytogenes and Streptococcus pneumoniae) and suggest their importance for cell wall growth and viability in L. monocytogenes and S. pneumoniae. We use these structural motifs to identify novel partners of GpsB in B. subtilis and extend the members of the GpsB interactome in all three bacterial species. Our results support that GpsB functions as an adaptor protein that mediates the interaction between membrane proteins, scaffolding proteins, signalling proteins and enzymes to generate larger protein complexes at specific sites in a bacterial cell cycle-dependent manner.

Prevalence of dental fluorosis in Mexico 2005-2015: a literature review / Prevalencia de fluorosis dental reportada en México 2005-2015: revisión de la literatura

Abstract: Objective: To perform a literature review regarding current dental fluorosis prevalence in Mexico reported from 2005 to 2015. Materials and methods: A comprehensive scientific literature review, in both English and Spanish, was performed in four databases up to June 2015. Search terms: fluorosis or dental fluorosis (mesh), prevalence (mesh), distribution (mesh), cases (mesh), epidemiology (mesh), Mexico. Results: 17 publications were included. Reported prevalence of dental fluorosis in Mexico ranged from 15.5 to 100%. Most of the studies were conducted in areas where water fluoride levels are low or optimal (≤1.5ppmF) and in which a prevalence of 15.5 to 81.7% was observed. In areas with higher levels of naturally fluoridated water (>1.5ppmF), prevalence ranged from 92 to 100%. Fluorosis severity ranged from questionable to severe. Conclusion: High prevalence of dental fluorosis was observed even in areas where fluoride concentration in water was low or optimal. In addition to fluoride in groundwater, there are multiple risk factors that should be controlled.

Resumen: Objetivo: Realizar una revisión de la literatura sobre la prevalencia de fluorosis dental en México reportada durante 2005-2015. Material y métodos: Se realizó una revisión exhaustiva hasta junio de 2015 en cuatro bases de datos de literatura científica en inglés y español. Términos de búsqueda: fluorosis o fluorosis dental (mesh), prevalencia (mesh), distribución (mesh), casos (mesh), epidemiología (mesh), México. Resultados: Se incluyeron 17 publicaciones. La prevalencia reportada en México fue de 15.5 a 100%. La mayoría de los estudios se realizaron en áreas donde el nivel de flúor en agua es bajo u óptimo (≤1.5ppmF), en las cuales se observó una prevalencia de 15.5 a 81.7%. En las zonas con mayor nivel de flúor (>1.5ppmF) en agua natural fue de 92 a 100%. La gravedad de fluorosis varió de dudosa a severa. Conclusión: Existe una alta prevalencia de fluorosis dental incluso en zonas donde la concentración de fluoruro en el agua es baja u óptima. Además de fluoruro en el agua, existen múltiples factores de riesgo que deben ser controlados.