Pseudomonas aeruginosa LptE is crucial for LptD assembly, cell envelope integrity, antibiotic resistance and virulence

Lipopolysaccharide (LPS) is an essential structural component of the outer membrane (OM) of most Gram-negative bacteria. In the model organism Escherichia coli, LPS transport to the OM requires seven essential proteins (LptABCDEFG) that form a continuous bridge across the cell envelope. In Pseudomonas aeruginosa the recently-demonstrated essentiality of LptD and LptH, the P. aeruginosa LptA homologue, confirmed the crucial role of the Lpt system and, thus, of LPS in OM biogenesis in this species. Surprisingly, independent high-throughput transposon mutagenesis studies identified viable P. aeruginosa insertion mutants in the lptE gene, suggesting that it might be dispensable for bacterial growth. To test this hypothesis, we generated an lptE conditional mutant in P. aeruginosa PAO1. LptE depletion only slightly impairs P. aeruginosa growth in vitro. Conversely, LptE is important for cell envelope stability, antibiotic resistance and virulence in an insect model. Interestingly, the maturation and OM localization of LPS is only marginally affected in LptE-depleted cells, while the levels of the OM component LptD are strongly reduced. This suggests that P. aeruginosa LptE might not be directly involved in LPS transport, although it is clearly essential for the maturation and/or stability of LptD. While poor functionality of LptD caused by LptE depletion is somehow tolerated by P. aeruginosa, this has a high cost in terms of cell integrity, drug resistance and virulence, highlighting LptE function(s) as an interesting target to weaken P. aeruginosa defenses and reduce its infectivity

Study on the antibacterial properties of leathers tanned with natural tannins and their interactions with shoes inhabiting bacteria

Content: Tannins are high molecular weight polyphenols, naturally synthesized by plants to defend themselves against biotic and abiotic stress factors. Their role as antioxidant, antibiotic and antibacterial agent has been known for many years among agriculture, food, pharma and cosmetics industry. If tannins would perform an antibacterial activity in a vegetable tanned leather, the leather itself could be certified as an antibacterial material. This effect could be very interesting for all the applications in which the leather, being in contact with sweat and bacteria, becomes a solution to reduce more or less severe hyperhidrosis and bromhidrosis. The goal of the study was the assessment of the antibacterial activity of vegetable tanned leathers with natural tannins to produce articles in direct contact with human skin and, therefore, their effect on sweat, bacterial growth and metabolite production. Firstly, the antibacterial activity has been evaluated and compared between leathers tanned with Chestnut, Quebracho and Tara extracts, chrome tanned leathers and synthetic materials. The trial was performed in vitro by inoculating gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacterial strains. A later step defined the most suitable blend of tannins to obtain, after tanning and/or retanning, an antibacterial natural leather. Furthermore, the vegetable tanned leathers, made with this tannins blend, have been the target of an in vivo trial during which 15 panelists have worn two differently made shoes. The lining and insole inside the right shoe have been made with vegetable tanned leathers with tannins, while the ones inside the left shoe contained only synthetic material. The shoes have been worn for 28 consecutive days, followed by a molecular and bioinformatic analysis of microbiota samples taken from the inner surface of the shoes by using a sterile swab. Lastly, a biochemical analysis of volatile short chain fatty acids has been carried out to investigate the byproducts of the bacteria responsible for the unpleasant odor of shoes. Take-Away: 1. Vegetable tanned leather is a wonderful antibacterial material thanks to the presence of natural tannins, such as chestnut, quebracho and tara. This property is appreciated in the production of insole leather, lining, leather goods and automotive interiors. 2. The problem of bromhidrosis (bad feet odor) can be avoided by using vegetable tanned leather. 3. In particular, vegetable leathers tanned with tannins used to make inside part of the shoes permit to avoid the formation of cheesy and acidic odours thanks to their antibacterial properties and their capacity to absorb sweat

ActS activates peptidoglycan amidases during outer membrane stress in <i>Escherichia coli</i>

The integrity of the cell envelope of E. coli relies on the concerted activity of multi-protein machineries that synthesize the peptidoglycan (PG) and the outer membrane (OM). Our previous work found that the depletion of lipopolysaccharide (LPS) export to the OM induces an essential PG remodeling process involving LD-transpeptidases (LDTs), the glycosyltransferase function of PBP1B and the carboxypeptidase PBP6a. Consequently, cells with defective OM biogenesis lyse if they lack any of these PG enzymes. Here we report that the morphological defects, and lysis associated with a ldtF mutant with impaired LPS transport, are alleviated by the loss of the predicted OM-anchored lipoprotein ActS (formerly YgeR). We show that ActS is an inactive member of LytM-type peptidoglycan endopeptidases due to a degenerated catalytic domain. ActS is capable of activating all three main periplasmic peptidoglycan amidases, AmiA, AmiB, and AmiC, which were previously reported to be activated only by EnvC and/or NlpD. Our data also suggest that in vivo ActS preferentially activates AmiC and that its function is linked to cell envelope stress

The Lipopolysaccharide Export Pathway in Escherichia coli: Structure, Organization and Regulated Assembly of the Lpt Machinery

The bacterial outer membrane (OM) is a peculiar biological structure with a unique composition that contributes significantly to the fitness of Gram-negative bacteria in hostile environments. OM components are all synthesized in the cytosol and must, then, be transported efficiently across three compartments to the cell surface. Lipopolysaccharide (LPS) is a unique glycolipid that paves the outer leaflet of the OM. Transport of this complex molecule poses several problems to the cells due to its amphipatic nature. In this review, the multiprotein machinery devoted to LPS transport to the OM is discussed together with the challenges associated with this process and the solutions that cells have evolved to address the problem of LPS biogenesis

Study on the antibacterial properties of leathers tanned with natural tannins and their interactions with shoes inhabiting bacteria

Content: Tannins are high molecular weight polyphenols, naturally synthesized by plants to defend themselves against biotic and abiotic stress factors. Their role as antioxidant, antibiotic and antibacterial agent has been known for many years among agriculture, food, pharma and cosmetics industry. If tannins would perform an antibacterial activity in a vegetable tanned leather, the leather itself could be certified as an antibacterial material. This effect could be very interesting for all the applications in which the leather, being in contact with sweat and bacteria, becomes a solution to reduce more or less severe hyperhidrosis and bromhidrosis. The goal of the study was the assessment of the antibacterial activity of vegetable tanned leathers with natural tannins to produce articles in direct contact with human skin and, therefore, their effect on sweat, bacterial growth and metabolite production. Firstly, the antibacterial activity has been evaluated and compared between leathers tanned with Chestnut, Quebracho and Tara extracts, chrome tanned leathers and synthetic materials. The trial was performed in vitro by inoculating gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacterial strains. A later step defined the most suitable blend of tannins to obtain, after tanning and/or retanning, an antibacterial natural leather. Furthermore, the vegetable tanned leathers, made with this tannins blend, have been the target of an in vivo trial during which 15 panelists have worn two differently made shoes. The lining and insole inside the right shoe have been made with vegetable tanned leathers with tannins, while the ones inside the left shoe contained only synthetic material. The shoes have been worn for 28 consecutive days, followed by a molecular and bioinformatic analysis of microbiota samples taken from the inner surface of the shoes by using a sterile swab. Lastly, a biochemical analysis of volatile short chain fatty acids has been carried out to investigate the byproducts of the bacteria responsible for the unpleasant odor of shoes. Take-Away: 1. Vegetable tanned leather is a wonderful antibacterial material thanks to the presence of natural tannins, such as chestnut, quebracho and tara. This property is appreciated in the production of insole leather, lining, leather goods and automotive interiors. 2. The problem of bromhidrosis (bad feet odor) can be avoided by using vegetable tanned leather. 3. In particular, vegetable leathers tanned with tannins used to make inside part of the shoes permit to avoid the formation of cheesy and acidic odours thanks to their antibacterial properties and their capacity to absorb sweat

New Insights into the Lpt Machinery for Lipopolysaccharide Transport to the Cell Surface: LptA-LptC Interaction and LptA Stability as Sensors of a Properly Assembled Transenvelope Complex▿

Lipopolysaccharide (LPS) is a major glycolipid present in the outer membrane (OM) of Gram-negative bacteria. The peculiar permeability barrier of the OM is due to the presence of LPS at the outer leaflet of this membrane that prevents many toxic compounds from entering the cell. In Escherichia coli LPS synthesized inside the cell is first translocated over the inner membrane (IM) by the essential MsbA flippase; then, seven essential Lpt proteins located in the IM (LptBCDF), in the periplasm (LptA), and in the OM (LptDE) are responsible for LPS transport across the periplasmic space and its assembly at the cell surface. The Lpt proteins constitute a transenvelope complex spanning IM and OM that appears to operate as a single device. We show here that in vivo LptA and LptC physically interact, forming a stable complex and, based on the analysis of loss-of-function mutations in LptC, we suggest that the C-terminal region of LptC is implicated in LptA binding. Moreover, we show that defects in Lpt components of either IM or OM result in LptA degradation; thus, LptA abundance in the cell appears to be a marker of properly bridged IM and OM. Collectively, our data support the recently proposed transenvelope model for LPS transport

Study on the antibacterial properties of leathers tanned with natural tannins and their interactions with shoes inhabiting bacteria

Content: Tannins are high molecular weight polyphenols, naturally synthesized by plants to defend themselves against biotic and abiotic stress factors. Their role as antioxidant, antibiotic and antibacterial agent has been known for many years among agriculture, food, pharma and cosmetics industry. If tannins would perform an antibacterial activity in a vegetable tanned leather, the leather itself could be certified as an antibacterial material. This effect could be very interesting for all the applications in which the leather, being in contact with sweat and bacteria, becomes a solution to reduce more or less severe hyperhidrosis and bromhidrosis. The goal of the study was the assessment of the antibacterial activity of vegetable tanned leathers with natural tannins to produce articles in direct contact with human skin and, therefore, their effect on sweat, bacterial growth and metabolite production. Firstly, the antibacterial activity has been evaluated and compared between leathers tanned with Chestnut, Quebracho and Tara extracts, chrome tanned leathers and synthetic materials. The trial was performed in vitro by inoculating gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacterial strains. A later step defined the most suitable blend of tannins to obtain, after tanning and/or retanning, an antibacterial natural leather. Furthermore, the vegetable tanned leathers, made with this tannins blend, have been the target of an in vivo trial during which 15 panelists have worn two differently made shoes. The lining and insole inside the right shoe have been made with vegetable tanned leathers with tannins, while the ones inside the left shoe contained only synthetic material. The shoes have been worn for 28 consecutive days, followed by a molecular and bioinformatic analysis of microbiota samples taken from the inner surface of the shoes by using a sterile swab. Lastly, a biochemical analysis of volatile short chain fatty acids has been carried out to investigate the byproducts of the bacteria responsible for the unpleasant odor of shoes. Take-Away: 1. Vegetable tanned leather is a wonderful antibacterial material thanks to the presence of natural tannins, such as chestnut, quebracho and tara. This property is appreciated in the production of insole leather, lining, leather goods and automotive interiors. 2. The problem of bromhidrosis (bad feet odor) can be avoided by using vegetable tanned leather. 3. In particular, vegetable leathers tanned with tannins used to make inside part of the shoes permit to avoid the formation of cheesy and acidic odours thanks to their antibacterial properties and their capacity to absorb sweat

Linking dual mode of action of host defense antimicrobial peptide thanatin: structures, lipopolysaccharide and LptAm binding of designed analogs

At present, antibiotics options to cure infections caused by drug resistant Gram-negative pathogens are highly inadequate. LPS outer membrane, proteins involved in LPS transport and biosynthesis pathways are vital targets. Thanatin, an insect derived 21-residue long antimicrobial peptide may be exploited for the development of effective antibiotics against Gram-negative bacteria. As a mode of bacterial cell killing, thanatin disrupts LPS outer membrane and inhibits LPS transport by binding to the periplasmic protein LptAm. Here, we report structure-activity correlation of thanatin and analogs for the purpose of rational design. These analogs of thanatin are investigated, by NMR, ITC and fluorescence, to correlate structure, antibacterial activity and binding with LPS and LptAm, a truncated monomeric variant. Our results demonstrate that an analog thanatin M21F exhibits superior antibacterial activity. In LPS interaction analyses, thanatin M21F demonstrate high affinity binding to outer membrane LPS. The atomic resolution structure of thanatin M21F in LPS micelle reveals four stranded -sheet structure in a dimeric topology whereby the sidechain of aromatic residues Y10, F21 sustained mutual packing at the interface. Strikingly, LptAm binding affinity of thanatin M21F has been significantly increased with an estimated Kd~0.73 nM vs 13 nM for thanatin. Further, atomic resolution structures and interactions of Ala based thanatin analogs define plausible correlations with antibacterial activity and LPS, LptAm interactions. Taken together, the current work provides a frame-work for the designing of thanatin based potent antimicrobial peptides for the treatment of drug resistance Gram-negative bacteria.Ministry of Education (MOE)Submitted/Accepted versionThis work was supported by the grant from Ministry of Education (MOE), Singapore

Thanatin Impairs Lipopolysaccharide Transport Complex Assembly by Targeting LptC–LptA Interaction and Decreasing LptA Stability

International audienceThe outer membrane (OM) of Gram-negative bacteria is a highly selective permeability barrier due to its asymmetric structure with lipopolysaccharide (LPS) in the outer leaflet. In Escherichia coli, LPS is transported to the cell surface by the LPS transport (Lpt) system composed of seven essential proteins forming a transenvelope bridge. Transport is powered by the ABC transporter LptB2FGC, which extracts LPS from the inner membrane (IM) and transfers it, through LptC protein, to the periplasmic protein LptA. Then, LptA delivers LPS to the OM LptDE translocon for final assembly at the cell surface. The Lpt protein machinery operates as a single device, since depletion of any component leads to the accumulation of a modified LPS decorated with repeating units of colanic acid at the IM outer leaflet. Moreover, correct machine assembly is essential for LPS transit and disruption of the Lpt complex results in LptA degradation. Due to its vital role in cell physiology, the Lpt system represents a good target for antimicrobial drugs. Thanatin is a naturally occurring antimicrobial peptide reported to cause defects in membrane assembly and demonstrated in vitro to bind to the N-terminal β-strand of LptA. Since this region is involved in both LptA dimerization and interaction with LptC, we wanted to elucidate the mechanism of inhibition of thanatin and discriminate whether its antibacterial effect is exerted by the disruption of the interaction of LptA with itself or with LptC. For this purpose, we here implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to probe in vivo the Lpt interactome in the periplasm. With this system, we found that thanatin targets both LptC-LptA and LptA-LptA interactions, with a greater inhibitory effect on the former. We confirmed in vitro the disruption of LptC-LptA interaction using two different biophysical techniques. Finally, we observed that in cells treated with thanatin, LptA undergoes degradation and LPS decorated with colanic acid accumulates. These data further support inhibition or disruption of Lpt complex assembly as the main killing mechanism of thanatin against Gram-negative bacteria