Despite the crucial role of bacterial capsules in pathogenesis, it is still unknown if systemic cues such as the cell cycle can control capsule biogenesis. In this study, we show that the capsule of ...the synchronizable model bacterium Caulobacter crescentus is cell cycle regulated and we unearth a bacterial transglutaminase homolog, HvyA, as restriction factor that prevents capsulation in G1-phase cells. This capsule protects cells from infection by a generalized transducing Caulobacter phage (φCr30), and the loss of HvyA confers insensitivity towards φCr30. Control of capsulation during the cell cycle could serve as a simple means to prevent steric hindrance of flagellar motility or to ensure that phage-mediated genetic exchange happens before the onset of DNA replication. Moreover, the multi-layered regulatory circuitry directing HvyA expression to G1-phase is conserved during evolution, and HvyA orthologues from related Sinorhizobia can prevent capsulation in Caulobacter, indicating that alpha-proteobacteria have retained HvyA activity.
Squalamine is a natural aminosterol that has been discovered in the tissues of the dogfish shark (Squalus acanthias). Studies have previously demonstrated that this promoter compound and its ...derivatives exhibit potent bactericidal activity against Gram-negative, Gram-positive bacteria, and multidrug-resistant bacteria. The antibacterial activity of squalamine was found to correlate with that of other antibiotics, such as colistin and polymyxins. Still, in the field of microbiology, evidence has shown that squalamine and its derivatives have antifungal activity, antiprotozoa effect against a limited list of protozoa, and could exhibit antiviral activity against both RNA- and DNA-enveloped viruses. Furthermore, squalamine and its derivatives have been identified as being antiangiogenic compounds in the case of several types of cancers and induce a potential positive effect in the case of other diseases such as experimental retinopathy and Parkinson’s disease. Given the diverse effects of the squalamine and its derivatives, in this review we provide the different advances in our understanding of the various effects of these promising molecules and try to draw up a non-exhaustive list of the different mechanisms of actions of squalamine and its derivatives on the human organism and on different pathogens.
The large adhesin protein LapA mediates adhesion and biofilm formation by Pseudomonas fluorescens. Although adhesion is thought to involve the long multiple repeats of LapA, very little is known ...about the molecular mechanism by which this protein mediates attachment. Here we use atomic force microscopy to unravel the biophysical properties driving LapA-mediated adhesion. Single-cell force spectroscopy shows that expression of LapA on the cell surface via biofilm-inducing conditions (i.e., phosphate-rich medium) or deletion of the gene encoding the LapG protease (LapA+ mutant) increases the adhesion strength of P. fluorescens toward hydrophobic and hydrophilic substrates, consistent with the adherent phenotypes observed in these conditions. Substrate chemistry plays an unexpected role in modulating the mechanical response of LapA, with sequential unfolding of the multiple repeats occurring only on hydrophilic substrates. Biofilm induction also leads to shortening of the protein extensions, reflecting stiffening of their conformational properties. Using single-molecule force spectroscopy, we next demonstrate that the adhesin is randomly distributed on the surface of wild-type cells and can be released into the solution. For LapA+ mutant cells, we found that the adhesin massively accumulates on the cell surface without being released and that individual LapA repeats unfold when subjected to force. The remarkable adhesive and mechanical properties of LapA provide a molecular basis for the “multi-purpose” adhesion function of LapA, thereby making P. fluorescens capable of colonizing diverse environments.
Living cells use cell surface proteins, such as mechanosensors, to constantly sense and respond to their environment. However, the way in which these proteins respond to mechanical stimuli and ...assemble into large complexes remains poorly understood at the molecular level. In the past years, atomic force microscopy (AFM) has revolutionized the way in which biologists analyze cell surface proteins to molecular resolution. In this Commentary, we discuss how the powerful set of advanced AFM techniques (e.g. live-cell imaging and single-molecule manipulation) can be integrated with the modern tools of molecular genetics (i.e. protein design) to study the localization and molecular elasticity of individual mechanosensors on the surface of living cells. Although we emphasize recent studies on cell surface proteins from yeasts, the techniques described are applicable to surface proteins from virtually all organisms, from bacteria to human cells.
Series of force-distance profiles obtained by stretching adhesins from various microbial species (see text for details): single adhesion peaks reflecting specific recognition (A), sawtooth patterns ...with multiple force peaks corresponding to the force-induced unfolding of protein secondary structures (B), constant force plateaus originating from the mechanical unzipping of amyloid interactions formed between multiple adhesins (C), and single large adhesion force peaks with linear shapes obtained by pulling on Gram-positive bacterial pili (D). AFM imaging enables microbiologists to decipher the nanoscale architecture of cell surfaces and its remodelling upon growth or interaction with drugs. ...force spectroscopy allows us to understand how cell surface receptors are spatially organized (e.g. clustering) and respond to force (e.g. single specific bonds, sequential unfolding, zipper-like adhesion, and spring-like properties).
Although belong to the same genus,
is primarily involved in invasive pulmonary infection, whereas
is a common cause of superficial infection. In this study, we compared conidia (the infective ...propagules) of these two
species. In immunocompetent mice, intranasal inoculation with conidia of
resulted in significantly higher inflammatory responses in the lungs compared to mice inoculated with
conidia.
assays revealed that the dormant conidia of
, unlike
dormant conidia, are immunostimulatory. The conidial surface of
was covered by a rodlet-layer, while that of
were presented with exposed polysaccharides.
harbored significantly higher number of proteins in its conidial cell wall compared to
conidia. Notably, β-1,3-glucan in the
conidial cell-wall showed significantly higher percentage of branching compared to that of
. The polysaccharides ensemble of
conidial cell wall stimulated the secretion of proinflammatory cytokines, and conidial cell wall associated proteins specifically stimulated IL-8 secretion from the host immune cells. Furthermore, the two species exhibited different sensitivities to antifungal drugs targeting cell wall polysaccharides, proposing the efficacy of species-specific treatment strategies. Overall, the species-specific organization of the conidial cell wall could be important in establishing infection by the two
species.
Acm2, the major autolysin of Lactobacillus plantarum, is a tripartite protein. Its catalytic domain is surrounded by an O-glycosylated N-terminal region rich in Ala, Ser, and Thr (AST domain), which ...is of low complexity and unknown function, and a C-terminal region composed of five SH3b peptidoglycan (PG) binding domains. Here, we investigate the contribution of these two accessory domains and of O-glycosylation to Acm2 functionality. We demonstrate that Acm2 is an N-acetylglucosaminidase and identify the pattern of O-glycosylation (21 mono-N-acetylglucosamines) of its AST domain. The O-glycosylation process is species-specific as Acm2 purified from Lactococcus lactis is not glycosylated. We therefore explored the functional role of O-glycosylation by purifying different truncated versions of Acm2 that were either glycosylated or non-glycosylated. We show that SH3b domains are able to bind PG and are responsible for Acm2 targeting to the septum of dividing cells, whereas the AST domain and its O-glycosylation are not involved in this process. Notably, our data reveal that the lack of O-glycosylation of the AST domain significantly increases Acm2 enzymatic activity, whereas removal of SH3b PG binding domains dramatically reduces this activity. Based on this antagonistic role, we propose a model in which access of the Acm2 catalytic domain to its substrate may be hindered by the AST domain where O-glycosylation changes its conformation and/or mediates interdomain interactions. To the best of our knowledge, this is the first time that O-glycosylation is shown to control the activity of a bacterial enzyme.
Background: A range of peptidoglycan hydrolases (PGHs) contain low complexity domains of unknown function.
Results:O-Glycosylation of the low complexity domain of the Lactobacillus plantarum autolysin Acm2 is a major negative modulator of enzymatic activity.
Conclusion:O-Glycosylation represents an autoregulatory control mechanism of PGH activity.
Significance: This is the first functional evidence that glycosylation controls the activity of a bacterial enzyme.
In living cells, sophisticated functional interfaces are generated through the self-assembly of bioactive building blocks. Prominent examples of such biofunctional surfaces are bacterial ...nanostructures referred to as pili. Although these proteinaceous filaments exhibit remarkable structure and functions, their potential to design bioinspired self-assembled systems has been overlooked. Here, we used atomic force microscopy (AFM) to explore the supramolecular organization and self-assembly of pili from the Gram-positive probiotic bacterium Lactobacillus rhamnosus GG (LGG). High-resolution AFM imaging of cell preparations adsorbed on mica revealed pili not only all around the cells, but also in the form of remarkable star-like structures assembled on the mica surface. Next, we showed that two-step centrifugation is a simple procedure to separate large amounts of pili, even though through their synthesis they are covalently anchored to the cell wall. We also found that the centrifuged pili assemble as long bundles. We suggest that these bundles originate from a complex interplay of mechanical effects (centrifugal force) and biomolecular interactions involving the SpaC cell adhesion pilin subunit (lectin-glycan bonds, hydrophobic bonds). Supporting this view, we found that pili isolated from an LGG mutant lacking hydrophilic exopolysaccharides show an increased tendency to form tight bundles. These experiments demonstrate that AFM is a powerful platform for visualizing individual pili on bacterial surfaces and for unravelling their two-dimensional assembly on solid surfaces. Our data suggest that bacterial pili may provide a generic approach in nanobiotechnology for elaborating functional supramolecular interfaces assembled from bioactive building blocks.
The rodlet structure present on the Aspergillus fumigatus conidial surface hides conidia from immune recognition. In spite of the essential biological role of the rodlets, the molecular basis for ...their self-assembly and disaggregation is not known. Analysis of the soluble forms of conidia-extracted and recombinant RodA by NMR spectroscopy has indicated the importance of disulfide bonds and identified two dynamic regions as likely candidates for conformational change and intermolecular interactions during conversion of RodA into the amyloid rodlet structure. Point mutations introduced into the RODA sequence confirmed that (1) mutation of a single cysteine was sufficient to block rodlet formation on the conidial surface and (2) both presumed amyloidogenic regions were needed for proper rodlet assembly. Mutations in the two putative amyloidogenic regions retarded and disturbed, but did not completely inhibit, the formation of the rodlets in vitro and on the conidial surface. Even in a disturbed form, the presence of rodlets on the surface of the conidia was sufficient to immunosilence the conidium. However, in contrast to the parental conidia, long exposure of mutant conidia lacking disulfide bridges within RodA or expressing RodA carrying the double (I115S/I146G) mutation activated dendritic cells with the subsequent secretion of proinflammatory cytokines. The immune reactivity of the RodA mutant conidia was not due to a modification in the RodA structure, but to the exposure of different pathogen-associated molecular patterns on the surface as a result of the modification of the rodlet surface layer. The full degradation of the rodlet layer, which occurs during early germination, is due to a complex array of cell wall bound proteases. As reported earlier, this loss of the rodlet layer lead to a strong anti-fumigatus host immune response in mouse lungs.
There is increasing evidence that many amyloids in living cells have physiological functions. On the surfaces of fungal cells, amyloid core sequences in adhesins can aggregate into 100- to ...1,000-nm-wide patches to form high-avidity adhesion nanodomains on the cell surface. The nanodomains form through interactions that have amyloid-like properties: binding of amyloid dyes, perturbation by antiamyloid agents, and interaction with homologous sequences. To test whether these functional interactions are mediated by typical amyloid interactions, we substituted an amyloid core sequence, LVFFA, from human Aβ protein for the native sequence IVIVA in the 1,419-residue Candida albicans adhesin Als5p. The chimeric protein formed cell surface nanodomains and mediated cellular aggregation. The native sequence and chimeric adhesins responded similarly to the amyloid dye thioflavin T and to amyloid perturbants. However, unlike the native protein, the nanodomains formed by the chimeric protein were not force activated and formed less-robust aggregates under flow. These results showed the similarity of amyloid interactions in the amyloid core sequences of native Als5p and Aβ, but they also highlighted emergent properties of the native sequence. Also, a peptide composed of the Aβ amyloid sequence flanked by amino acids from the adhesin formed two-dimensional sheets with sizes similar to the cell surface patches of the adhesins. These results inform an initial model for the structure of fungal cell surface amyloid nanodomains.
Protein amyloid aggregates are markers of neurodegenerative diseases such as Alzheimer's and Parkinsonism. Nevertheless, there are also functional amyloids, including biofilm-associated amyloids in bacteria and fungi. In fungi, glycoprotein adhesins aggregate into cell surface patches through amyloid-like interactions, and the adhesin clustering strengthens cell-cell binding. These fungal surface amyloid nanodomains mediate biofilm persistence under flow, and they also moderate host inflammatory responses in fungal infections. To determine whether the amyloid-like properties of fungal surface nanodomains are sequence specific, we ask whether a disease-associated amyloid core sequence has properties equivalent to those of the native sequence in a fungal adhesin. A chimeric adhesin with an amyloid sequence from the Alzheimer's disease protein Aβ instead of its native sequence effectively clustered the adhesins on the cell surface, but it showed a different response to hydrodynamic shear. These results begin an analysis of the sequence dependence for newly discovered activities for fungal surface amyloid nanodomains.
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