Peptidoglycan crosslinking relaxation plays an important role in Staphylococcus aureus WalKR-dependent cell viability

The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl-glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.     

Design of a specific colonic mucus marker using a human commensal bacterium cell surface domain

Imaging living cells and organs requires innovative, specific, efficient, and well tolerated fluorescent markers targeting cellular components. Such tools will allow proceeding to the dynamic analysis of cells and the adaptation of tissues to environmental cues. In this study, we have identified and synthesized a novel non-toxic fluorescent marker allowing a specific fluorescent staining of the human colonic mucus. Our strategy to identify a molecule able to specifically bind to the human colonic mucus was on the basis of the mucus adhesion properties of commensal bacteria. We identified and characterized the mucus-binding property of a 70-amino acid domain (MUB(70)) expressed on the surface of Lactobacillus strains. The chemical synthesis of MUB(70) was achieved using the human commensal bacterium Lactobacillus reuteri AF120104 protein as a template. The synthesized Cy5-conjugated MUB(70) marker specifically stained the colonic mucus on fixed human, rabbit, and guinea pig tissues. Interestingly, murine tissue was not stained, suggesting significant differences in the composition of the murine colonic mucus. In addition, this marker stained the mucus of living cultured human colonic cells (HT29-MTX) and human colonic tissue explants. Using a biotinylated derivative of MUB(70), we demonstrated that this peptide binds specifically to Muc2, the most abundant secreted mucin, through its glycosylated moieties. Hence, Cy5-MUB(70) is a novel and specific fluorescent marker for mammalian colonic mucus. It may be used for live imaging analysis but also, as demonstrated in this study, as a marker for the diagnosis and the prognosis of colonic mucinous carcinomas.     

An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death

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Inhibition of NF-kappa B activation by peptides targeting NF-kappa B essential modulator (nemo) oligomerization

NF-kappa B essential modulator/IKK-gamma (NEMO/IKK-gamma) plays a key role in the activation of the NF-kappa B pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861-27869). To search for drugs inhibiting NF-kappa B activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-kappa B activation was quantified using an NF-kappa B-dependent beta-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-kappa B activation with an IC(50) in the mum range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-kappa B activity. Our results provide the "proof of concept" for a new and promising strategy for the inhibition of NF-kappa B pathway activation through targeting the oligomerization state of the NEMO protein.     

Deciphering the role of individual acyl chains in the interaction network between phosphatidylserines and a single-spanning membrane protein

PMP1 is a small single-spanning membrane protein functioning as a regulatory subunit of the yeast plasma membrane H(+)-ATPase. This protein forms a unique helix and exhibits a positively charged cytoplasmic domain that is able to specifically segregate phosphatidylserines (PSs). A marked groove formed at the helix surface is thought to play a major role in the related lipid-protein interaction network. Mutational analysis and (1)H NMR experiments were therefore performed on a synthetic PMP1 fragment using DPC-d(38) micelles as a membrane-like environment, in the presence of small amounts of POPS. A mutation designed for altering the helix groove was shown to disfavor the POPS binding specificity as much as that affecting the electrostatic interaction network. From POPS titration experiments monitored by a full set of one- and two-dimensional NOESY spectra, the association between the phospholipids and the PMP1 peptide has been followed. Our data reveal that the clustering of POPS molecules is promoted from a stabilized framework obtained by coupling the PMP1 helix groove to a POPS sn-2 chain. To our knowledge, the NOE-based titration plots displayed in this report constitute the first NMR data that directly distinguish the role of the sn-1 and sn-2 acyl chains in a lipid-protein interaction. The results are discussed while taking into account our accurate knowledge of the yeast plasma membrane composition and its ability to form functional lipid rafts.     

Dodecylphosphocholine micelles as a membrane-like environment: new results from NMR relaxation and paramagnetic relaxation enhancement analysis

To further examine to what extent a dodecylphosphocholine (DPC) micelle mimics a phosphatidylcholine bilayer environment, we performed ¹³C, ²H, and ³¹P NMR relaxation measurements. Our data show that the dynamic behavior of DPC phosphocholine groups at low temperature (12 °C) corresponds to that of a phosphatidylcholine interface at high temperature (51 °C). In the presence of helical peptides, a PMP1 fragment, or an annexin fragment, the DPC local dynamics are not affected whereas the DPC aggregation number is increased to match an appropriate area/volume ratio for accommodating the bound peptides. We also show that quantitative measurements of paramagnetic relaxation enhancements induced by small amounts of spin-labeled phospholipids on peptide proton signals provide a meaningful insight on the location of both PMP1 and annexin fragments in DPC micelles. The paramagnetic contributions to the relaxation were extracted from intra-residue cross-peaks of NOESY spectra for both peptides. The location of each peptide in the micelles was found consistent with the corresponding relaxation data. As illustrated by the study of the PMP1 fragment, paramagnetic relaxation data also allow us to supply the missing medium-range NOEs and therefore to complete a standard conformational analysis of peptides in micelles. Received: 16 April 1998 / Revised version: 19 June 1998 / Accepted: 30 July 1998     

Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues

Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H⁺-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H⁺-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185–97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (λ_max), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k_q) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We tentatively assigned the two major Trp lifetimes to the Trp (χ₁=60° and 180°) rotamers, based on the recent lifetime–rotamer correlation proposed for model cyclic peptides (Pan and Barkley 2004, Biophys J 86:3828–35). We also analyzed the role of the hydrophobic anchor, by comparing the micelle binding of fragments of various lengths including the synthesized full-length protein and detected peculiar differences for protein interaction with the polar headgroups of DM or DPC.     

Synergistic impacts of global warming on the resilience of coral reefs

Recent epizootics have removed important functional species from Caribbean coral reefs and left communities vulnerable to alternative attractors. Global warming will impact reefs further through two mechanisms. A chronic mechanism reduces coral calcification, which can result in depressed somatic growth. An acute mechanism, coral bleaching, causes extreme mortality when sea temperatures become anomalously high. We ask how these two mechanisms interact in driving future reef state (coral cover) and resilience (the probability of a reef remaining within a coral attractor). We find that acute mechanisms have the greatest impact overall, but the nature of the interaction with chronic stress depends on the metric considered. Chronic and acute stress act additively on reef state but form a strong synergy when influencing resilience by intensifying a regime shift. Chronic stress increases the size of the algal basin of attraction (at the expense of the coral basin), whereas coral bleaching pushes the system closer to the algal attractor. Resilience can change faster—and earlier—than a change in reef state. Therefore, we caution against basing management solely on measures of reef state because a loss of resilience can go unnoticed for many years and then become disproportionately more difficult to restore.     

Contribution of physiologists to the identification of the humoral component of immunity in the 19th century

The history of antimicrobial humoral immunity usually focuses on the works of the German school at the end of the 19th century, born in the tradition of chemistry and disinfection. Starting from an old quarrel of priority about serotherapy between Emil von Behring (1854–1917) and the French physiologists Charles Richet (1850–1935) and Jules Héricourt (1850–1938), we first confirm that the latter stated the principle of serotherapy in 1888 and put it into practice before the seminal Behring's article in 1890, observing several adverse effects of this new immunotherapy. We also find that researchers who can be considered heirs of the French school of Physiology founded by Claude Bernard (1813–1878) also investigated the field of humoral immunity in the 1870–1880s. Maurice Raynaud (1834–1881), Auguste Chauveau (1827–1917), and eventually Charles Richet applied the experimental method of Claude Bernard to the young field of microbiology, illustrating a movement called by Jacques Léonard “physiologization of the pasteurism.” However, the contribution of physiologists in this field started before Louis Pasteur, leading to the conclusion that physiologists and chemists synergistically contributed to the birth of bacteriology and immunology.     

Differential δC and δN signatures among scallop tissues: implications for ecology and physiology

There have been several studies where the isotopic composition of organisms has been determined seasonally, but fewer have examined separate organs. In this context, separate organs (e.g. gonad, digestive gland and muscle) of a suspension-feeder, the scallop Pecten maximus, were used to assess seasonal changes of both stable isotopes and biochemical components. Our study used multiple indicators [stable carbon and nitrogen isotope ratios, biochemical components and seston chlorophyll-a (chl a)] to track nutritive activity and energy allocation in P. maximus from the Bay of Brest (France). In addition to seasonal variation in the isotopic composition of P. maximus tissues, we found strong differences in the mean isotopic signatures of different organs. This has serious implications for interpretation of animal diets and potential use in animal physiology. Furthermore, we present evidence that seasonal variations of metabolism will cause changes in the isotopic composition not related to changes in the diet. Interpretation of isotopic data may require consideration of values from several separate organs. Finally, δ¹⁵N appears powerful to track metabolite fates in the scallop P. maximus.