Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide ...variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions.
Amyloid transthyretin (ATTR) amyloidosis is characterized by the abnormal accumulation of ATTR fibrils in multiple organs. However, the structure of ATTR fibrils from the eye is poorly understood. ...Here, we used cryo-EM to structurally characterize vitreous body ATTR fibrils. These structures were distinct from previously characterized heart fibrils, even though both have the same mutation and type A pathology. Differences were observed at several structural levels: in both the number and arrangement of protofilaments, and the conformation of the protein fibril in each layer of protofilaments. Thus, our results show that ATTR protein structure and its assembly into protofilaments in the type A fibrils can vary between patients carrying the same mutation. By analyzing and matching the interfaces between the amino acids in the ATTR fibril with those in the natively folded TTR, we are able to propose a mechanism for the structural conversion of TTR into a fibrillar form.
The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins ...(+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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With the growing challenges of bacteria becoming resistant to conventional antibiotics, antimicrobial peptides (AMPs) may offer a potential alternative. One of the most studied AMPs, ...the human cathelicidin derived AMP LL-37 is notable for its antimicrobial activity even though its mechanism of action is not fully understood yet. This work investigates the interaction of LL-37 with 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (POPG) vesicles, which were employed as a bacterial membrane model given the common presence of this phospholipid in the bacterial membrane. Experimental techniques including small angle X-ray scattering, transmission electron microscopy and dynamic light scattering were used to characterize the interactions among LL-37 and POPG. Molecular dynamics simulations complement the experimental studies with molecular-level insights into the process. LL-37 was discovered to actively and critically interact with the POPG vesicles, modifying the membrane curvature that eventually leads to structural transformations from vesicles to mixed micelles. The results shed light on the mechanisms underlying the interactions among LL-37 and bacteria mimetic vesicles and can guide the further development of AMP based antimicrobial materials and therapies.
The outer membrane protein A (OmpA) family contains an evolutionary conserved domain that links the outer membrane in Gram-negative bacteria to the semi-rigid peptidoglycan (PG) layer. The clinically ...significant pathogen
carries several OmpA family proteins (OprF, OprL, PA0833, and PA1048) that share the PG-binding domain. These proteins are important for cell morphology, membrane stability, and biofilm and outer membrane vesicle (OMV) formation. In addition to other OmpAs,
analysis revealed that the putative outer membrane protein (OMP) with gene locus PA1041 is a lipoprotein with an OmpA domain and, hence, is a potential virulence factor. This study aimed to evaluate PA1041 as a PG-binding protein and describe its effect on the phenotype. Clinical strains were confirmed to contain the lipoprotein resulting from PA1041 expression with Western blot, and PG binding was verified in enzyme-linked immunosorbent assay (ELISA). By using a Sepharose bead-based ELISA, we found that the lipoprotein binds to
-diaminopimelic acid (mDAP), an amino acid in the pentapeptide portion of PGs. The reference strain PAO1 and the corresponding transposon mutant PW2884 devoid of the lipoprotein were examined for phenotypic changes. Transmission electron microscopy revealed enlarged periplasm spaces near the cellular poles in the mutant. In addition, we observed an increased release of OMV, which could be confirmed by nanoparticle tracking analysis. Importantly, mutants without the lipoprotein produced a thick, but loose and unorganized, biofilm in flow cells. In conclusion, the lipoprotein from gene locus PA1041 tethers the outer membrane to the PG layer, and mutants are viable, but display severe phenotypic changes including disordered biofilm formation. Based upon the phenotype of the
PW2884 mutant and the function of the protein, we designate the lipoprotein with locus tag PA1041 as "peptidoglycan-binding anchor" (Pba).
Intermediate filament (IF)-like cytoskeleton emerges as a versatile tool for cellular organization in all kingdoms of life, underscoring the importance of mechanistically understanding its diverse ...manifestations. We showed previously that, in Streptomyces (a bacterium with a mycelial lifestyle similar to that of filamentous fungi, including extreme cell and growth polarity), the IF protein FilP confers rigidity to the hyphae by an unknown mechanism. Here, we provide a possible explanation for the IF-like function of FilP by demonstrating its ability to self-assemble into a cis-interconnected regular network in vitro and its localization into structures consistent with a cytoskeletal network in vivo. Furthermore, we reveal that a spatially restricted interaction between FilP and DivIVA, the main component of the Streptomyces polarisome complex, leads to formation of apical gradients of FilP in hyphae undergoing active tip extension. We propose that the coupling between the mechanism driving polar growth and the assembly of an IF cytoskeleton provides each new hypha with an additional stress-bearing structure at its tip, where the nascent cell wall is inevitably more flexible and compliant while it is being assembled and matured. Our data suggest that recruitment of cytoskeleton around a cell polarity landmark is a broadly conserved strategy in tip-growing cells.
End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding ...properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.
Pathogens causing pneumonia utilize the complement regulator vitronectin to evade complement-mediated killing. Although vitronectin is associated with several chronic lung diseases, the role of ...bronchoalveolar vitronectin in pneumonia has not been studied. This study sought to reveal the involvement of vitronectin in the bronchoalveolar space during pneumonia, to assess the effect of outer membrane vesicles and endotoxin on vitronectin release, and to determine whether bacterial pathogens utilize pulmonary vitronectin for evasion. Vitronectin was analyzed in cell-free bronchoalveolar lavage fluid harvested from patients with pneumonia (
= 8) and from healthy volunteers after subsegmental endotoxin instillation (
= 13). Vitronectin binding by
and
was analyzed, and subsequent complement evasion was assessed by serum challenge. The effects of outer membrane vesicles on vitronectin production in mouse lungs and human type II alveolar epithelial cells (A549) were determined. We detected increased vitronectin concentrations in lavage fluid during pneumonia (
0.0063) and after bronchial endotoxin challenge (
0.016). The capture of vitronectin by bacteria significantly reduced complement-mediated lysis. Following challenge with vesicles, vitronectin was detected in mouse bronchoalveolar space, and mouse alveolar epithelial cells
as well as A549 cells
contained increased levels of vitronectin. Taken together, outer membrane vesicles and endotoxin from Gram-negative bacteria induce vitronectin, which is released into the bronchoalveolar space, and used for evasion of complement-mediated clearance.
Non-typeable
(NTHi) is a Gram-negative human pathogen that causes a wide range of airway diseases. NTHi has a plethora of mechanisms to colonize while evading the host immune system for the ...establishment of infection. We previously showed that the outer membrane protein P5 contributes to bacterial serum resistance by the recruitment of complement regulators. Here, we report a novel role of P5 in maintaining bacterial outer membrane (OM) integrity and protein composition important for NTHi-host interactions.
analysis revealed a peptidoglycan-binding motif at the periplasmic C-terminal domain (CTD) of P5. In a peptidoglycan-binding assay, the CTD of P5 (P5
) formed a complex with peptidoglycan. Protein profiling analysis revealed that deletion of CTD or the entire P5 changed the membrane protein composition of the strains NTHi 3655Δ
and NTHi 3655
, respectively. Relative abundance of several membrane-associated virulence factors that are crucial for adherence to the airway mucosa, and serum resistance were altered. This was also supported by similar attenuated pathogenic phenotypes observed in both NTHi 3655Δ
and NTHi 3655Δ
. We found (i) a decreased adherence to airway epithelial cells and fibronectin, (ii) increased complement-mediated killing, and (iii) increased sensitivity to the β-lactam antibiotics in both mutants compared to NTHi 3655 wild-type. These mutants were also more sensitive to lysis at hyperosmotic conditions and hypervesiculated compared to the parent wild-type bacteria. In conclusion, our results suggest that P5 is important for bacterial OM stability, which ultimately affects the membrane proteome and NTHi pathogenesis.
The human pathogen
causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles ...(OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including
. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient
compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in "foci" in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for
peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.
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