Our study highlights the roles of surfactant-like molecules in bacterial inflammation with important implications for the prevention and therapy of inflammatory disorders. It describes a potential pathway for the transfer of hydrophobic bacterial lipoproteins, the major TLR2 agonists, from the cytoplasmic membrane of Gram-positive bacteria to the TLR2 receptor at the surface of host cells. Moreover, our study reveals a molecular mechanism that explains how cytoplasmic and membrane-embedded bacterial proteins can be released by bacterial cells without using any of the typical protein secretion routes, thereby contributing to our understanding of the processes used by bacteria to communicate with host organisms and the environment.
The interior of living cells is a dense and polydisperse suspension of macromolecules. Such a complex system challenges an understanding in terms of colloidal suspensions. As a fundamental test we employ neutron spectroscopy to measure the diffusion of tracer proteins (immunoglobulins) in a cell-like environment (cell lysate) with explicit control over crowding conditions. In combination with Stokesian dynamics simulation, we address protein diffusion on nanosecond time scales where hydrodynamic interactions dominate over negligible protein collisions. We successfully link the experimental results on these complex, flexible molecules with coarse-grained simulations providing a consistent understanding by colloid theories. Both experiments and simulations show that tracers in polydisperse solutions close to the effective particle radius R eff = ⟨R i 3⟩1/3 diffuse approximately as if the suspension was monodisperse. The simulations further show that macromolecules of sizes R > R eff (R < R eff) are slowed more (less) effectively even at nanosecond time scales, which is highly relevant for a quantitative understanding of cellular processes.
Protein diffusion is not only an important process ensuring biological function but can also be used as a probe to obtain information on structural properties of protein assemblies in liquid solutions. Here, we explore the oligomerization state of ovalbumin at high protein concentrations by means of its short-time self-diffusion. We employ high-resolution incoherent quasielastic neutron scattering to access the self-diffusion on nanosecond timescales, on which interparticle contacts are not altered. Our results indicate that ovalbumin in aqueous (DO) solutions occurs in increasingly large assemblies of its monomeric subunits with rising protein concentration. It changes from nearly monomeric toward dimeric and ultimately larger than tetrameric complexes. Simultaneously, we access information on the internal molecular mobility of ovalbumin on the nanometer length scale and compare it with results obtained for bovine serum albumin, immunoglobulin, and β-lactoglobulin.
Temperature variations have a big impact on bacterial metabolism and death, yet an exhaustive molecular picture of these processes is still missing. For instance, whether thermal death is determined by the deterioration of the whole or a specific part of the proteome is hotly debated. Here, by monitoring the proteome dynamics of E. coli, we clearly show that only a minor fraction of the proteome unfolds at the cell death. First, we prove that the dynamical state of the E. coli proteome is an excellent proxy for temperature-dependent bacterial metabolism and death. The proteome diffusive dynamics peaks at about the bacterial optimal growth temperature, then a dramatic dynamical slowdown is observed that starts just below the cell's death temperature. Next, we show that this slowdown is caused by the unfolding of just a small fraction of proteins that establish an entangling interprotein network, dominated by hydrophobic interactions, across the cytoplasm. Finally, the deduced progress of the proteome unfolding and its diffusive dynamics are both key to correctly reproduce the E. coli growth rate.
Antibody therapies are typically based on high-concentration formulations that need to be administered subcutaneously. These conditions induce several challenges, inter alia a viscosity suitable for injection, sufficient solution stability, and preservation of molecular function. To obtain systematic insights into the molecular factors, we study the dynamics on the molecular level under strongly varying solution conditions. In particular, we use solutions of antibodies with poly(ethylene glycol), in which simple cooling from room temperature to freezing temperatures induces a transition from a well-dispersed solution into a phase-separated and macroscopically arrested system. Using quasi-elastic neutron scattering during in situ cooling ramps and in prethermalized measurements, we observe a strong decrease in antibody diffusion, while internal flexibility persists to a significant degree, thus ensuring the movement necessary for the preservation of molecular function. These results are relevant for a more dynamic understanding of antibodies in high-concentration formulations, which affects the formation of transient clusters governing the solution viscosity.
Protein denaturation in concentrated solutions consists of the unfolding of the native structure, and subsequent cross-linking into clusters or gel networks. While the kinetic evolution of structure has been studied...
Antimicrobial peptides (AMps) are an important part of the human innate immune system for protection against bacterial infections, however the AMps display varying degrees of activity against Staphylococcus aureus. previously, we showed that inactivation of the Atp synthase sensitizes S. aureus towards the AMp antibiotic class of polymyxins. Here we wondered if the Atp synthase similarly is needed for tolerance towards various human AMPs, including human β-defensins (hBD1-4), LL-37 and histatin 5. Importantly, we find that the ATP synthase mutant (atpA) is more susceptible to killing by hBD4, hBD2, LL-37 and histatin 5 than wild type cells, while no changes in susceptibility was detected for hBD3 and hBD1. Administration of the ATP synthase inhibitor, resveratrol, sensitizes S. aureus towards hBD4-mediated killing. Neutrophils rely on AMPs and reactive oxygen molecules to eliminate bacteria and the atpA mutant is more susceptible to killing by neutrophils than the Wt, even when the oxidative burst is inhibited.these results show that the staphylococcal Atp synthase enhance tolerance of S. aureus towards some human AMps and this indicates that inhibition of the Atp synthase may be explored as a new therapeutic strategy that sensitizes S. aureus to naturally occurring AMps of the innate immune system. Bacterial pathogens that cause disease in humans remain a serious threat to public health and antibiotics are still our primary weapons in treating many bacterial diseases. The ability to eradicate bacterial infections is however challenged by development of resistance for every type of antibiotic introduced to the clinic 1. The majority of the new small molecule antibiotics in clinical development are however inhibiting the same targets as already marketed antibiotics 2. As an alternative to small molecule antibiotics, antimicrobial peptides (AMPs) are also explored in clinical trials, however most of the AMPs are only tested for topical applications due to toxicity issues and low metabolic stability 3. Here we propose a new strategy to combat bacterial infections, namely to sensitize bacteria to the naturally occurring antimicrobial peptides of the human body and hence boosting the antibacterial capabilities of the innate immune system to eradicate bacterial infections. Humans are continuously exposed to numerous, and potentially pathogenic, microorganisms, where the innate immune system provides the first line of defense. AMPs constitute an important defense mechanism of the innate immune system against invading microorganisms, due to their antimicrobial and immune stimulatory properties 4,5. In humans, several classes of AMPs have been identified, such as α-and β-defensins, the cathelicidin LL-37 and histatins 5. The α-defensins consist of six members, which are divided into human neutrophil peptides (HNP1-4) and human α-defensin 5 and 6 (HD5 and HD6). HNP1-4 are highly concentrated in the granules of neutrophils, but are also expressed in monocytes, lymphocytes and natural killer cells. HD5 and HD6 are primarily ...
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