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BioRxiv : the Preprint Server For... Mar 2024Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we...
Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.
PubMed: 38464199
DOI: 10.1101/2024.03.01.582947 -
IScience Oct 2023The mechanism by which a bacterial cell senses external nutrients remains largely unknown. In this study, we identified a bacterial cell sensing system for polycyclic...
The mechanism by which a bacterial cell senses external nutrients remains largely unknown. In this study, we identified a bacterial cell sensing system for polycyclic aromatic hydrocarbons (PAHs) in a common marine PAH-using bacterium, . It consists of an outer membrane receptor (PahS) and a periplasmic protein (PahP) in combination with a two-component sensing system (TCS) that ensures a rapid response to PAH occurrence by directly controlling serial reactions including chemotactic sensing and movement, PAH uptake and intracellular PAH metabolism. PahS protrudes from the cell and acts as a PAH sensor, transducing the PAH signal across the outer membrane to its periplasmic partner PahP, which in turn transduces the PAH signal across the periplasm to a specialized TCS. This sensing system plays a critical role in sensing and promoting the metabolism of PAHs, which can be scavenged by various hydrocarbon-degrading bacteria.
PubMed: 37841585
DOI: 10.1016/j.isci.2023.107912 -
The Journal of Biological Chemistry Nov 2023Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas...
Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.
Topics: Acetylation; Alginates; Bacterial Proteins; Biofilms; Periplasm; Protein Processing, Post-Translational; Pseudomonas aeruginosa
PubMed: 37797696
DOI: 10.1016/j.jbc.2023.105314 -
Scientific Reports Nov 2023Periplasmic solute-binding proteins (SBPs) specific for chitooligosaccharides, (GlcNAc) (n = 2, 3, 4, 5 and 6), are involved in the uptake of chitinous nutrients and...
Periplasmic solute-binding proteins (SBPs) specific for chitooligosaccharides, (GlcNAc) (n = 2, 3, 4, 5 and 6), are involved in the uptake of chitinous nutrients and the negative control of chitin signal transduction in Vibrios. Most translocation processes by SBPs across the inner membrane have been explained thus far by two-domain open/closed mechanism. Here we propose three-domain mechanism of the (GlcNAc) translocation based on experiments using a recombinant VcCBP, SBP specific for (GlcNAc) from Vibrio cholerae. X-ray crystal structures of unliganded or (GlcNAc)-liganded VcCBP solved at 1.2-1.6 Å revealed three distinct domains, the Upper1, Upper2 and Lower domains for this protein. Molecular dynamics simulation indicated that the motions of the three domains are independent and that in the (GlcNAc)-liganded state the Upper2/Lower interface fluctuated more intensively, compared to the Upper1/Lower interface. The Upper1/Lower interface bound two GlcNAc residues tightly, while the Upper2/Lower interface appeared to loosen and release the bound sugar molecule. The three-domain mechanism proposed here was fully supported by binding data obtained by thermal unfolding experiments and ITC, and may be applicable to other translocation systems involving SBPs belonging to the same cluster.
Topics: Humans; Periplasmic Binding Proteins; Chitosan; Chitin; Carrier Proteins; Molecular Dynamics Simulation; Ligands; Translocation, Genetic; Crystallography, X-Ray
PubMed: 37996461
DOI: 10.1038/s41598-023-47253-y -
Microbiology Spectrum Aug 2023Outer membrane protein A (OmpA) is the most abundant porin in bacterial outer membranes. KJΔOmpA, an C-terminal in-frame deletion mutant of Stenotrophomonas...
Outer membrane protein A (OmpA) is the most abundant porin in bacterial outer membranes. KJΔOmpA, an C-terminal in-frame deletion mutant of Stenotrophomonas maltophilia KJ, exhibits pleiotropic defects, including decreased tolerance to menadione (MD)-mediated oxidative stress. Here, we elucidated the underlying mechanism of the decreased MD tolerance mediated by Δ. The transcriptomes of wild-type S. maltophilia and the KJΔOmpA mutant strain were compared, focusing on 27 genes known to be associated with oxidative stress alleviation; however, no significant differences were identified. was the most downregulated gene in KJΔOmpA. KJΔOmpA complementation with the chromosomally integrated gene restored MD tolerance to the wild-type level, indicating the role of OmpO in MD tolerance. To further clarify the possible regulatory circuit involved in defects and downregulation, σ factor expression levels were examined based on the transcriptome results. The expression levels of three σ factors were significantly different (downregulated levels of and upregulated levels of and ) in KJΔOmpA. Next, the involvement of the three σ factors in the Δ-mediated decrease in MD tolerance was evaluated using mutant strains and complementation assays. downregulation and upregulation contributed to the Δ-mediated decrease in MD tolerance. OmpA C-terminal domain loss induced an envelope stress response. Activated σ decreased and expression levels, in turn decreasing swimming motility and oxidative stress tolerance. Finally, we revealed both the Δ-- regulatory circuit and - cross regulation. The cell envelope is a morphological hallmark of Gram-negative bacteria. It consists of an inner membrane, a peptidoglycan layer, and an outer membrane. OmpA, an outer membrane protein, is characterized by an N-terminal β-barrel domain that is embedded in the outer membrane and a C-terminal globular domain that is suspended in the periplasmic space and connected to the peptidoglycan layer. OmpA is crucial for the maintenance of envelope integrity. Stress resulting from the destruction of envelope integrity is sensed by extracytoplasmic function (ECF) σ factors, which induce responses to various stressors. In this study, we revealed that loss of the OmpA-peptidoglycan (PG) interaction causes peptidoglycan and envelope stress while simultaneously upregulating σ and σ expression levels. The outcomes of σ and σ activation are different and are linked to β-lactam and oxidative stress tolerance, respectively. These findings establish that outer membrane proteins (OMPs) play a critical role in envelope integrity and stress tolerance.
Topics: Stenotrophomonas maltophilia; Regulon; Peptidoglycan; Sigma Factor; Oxidative Stress; Bacterial Proteins; Gene Expression Regulation, Bacterial
PubMed: 37284772
DOI: 10.1128/spectrum.01080-23 -
Microorganisms Jul 2023Prokaryotic extracellular vesicles (EVs) are vesicles that bud from the cell membrane and are secreted by bacteria and archaea. EV cargo in Gram-negative bacteria...
Prokaryotic extracellular vesicles (EVs) are vesicles that bud from the cell membrane and are secreted by bacteria and archaea. EV cargo in Gram-negative bacteria includes mostly periplasmic and outer membrane proteins. EVs are clinically important as their cargo can include toxins associated with bacterial virulence and toxicity; additionally, they have been proposed as efficient vaccine agents and as the ancestors of the eukaryotic endomembrane system. However, the mechanistic details behind EV cargo selection and release are still poorly understood. In this study, we have performed bioinformatics analysis of published data on EV proteomes from 38 species of bacteria and 4 archaea. Focusing on clusters of orthologous genes (COGs) and using the EggNOG mapper function, we have identified cargo proteins that are commonly found in EVs across species. We discuss the putative role of these prominent proteins in EV biogenesis and function. We also analyzed the published EV proteomes for conserved signal sequences and discuss the potential role of these signal sequences for EV cargo selection.
PubMed: 37630535
DOI: 10.3390/microorganisms11081977 -
Current Opinion in Microbiology Jun 2024Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is... (Review)
Review
Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is multilayered, with two membranes sandwiching the periplasmic space that contains the peptidoglycan cell wall. Understanding how this complicated surface architecture is assembled during cell growth and division is a major fundamental problem in microbiology. Additionally, because the envelope is an important antibiotic target and determinant of intrinsic antibiotic resistance, understanding the mechanisms governing its assembly is relevant to therapeutic development. In the last several decades, most of the factors required to build the Gram-negative envelope have been identified. However, surprisingly, little is known about how the biogenesis of the different cell surface layers is co-ordinated. Here, we provide an overview of recent work that is beginning to uncover the links connecting the different envelope biosynthetic pathways and assembly machines to ensure uniform envelope growth.
Topics: Gram-Negative Bacteria; Cell Wall; Peptidoglycan; Cell Membrane; Bacterial Proteins
PubMed: 38718542
DOI: 10.1016/j.mib.2024.102479 -
BMC Biology Feb 2024Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the...
BACKGROUND
Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39 °C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37 °C, by using RNAseq and physiological assays including in vivo experiments.
RESULTS
In general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes in both species. Altered metabolic features at 39 °C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39 °C, they upregulated ethanol oxidation-related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα, and pmsβ genes at 39 °C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphyloxantin production, and a decrease in biofilm formation at 39 °C. On the side of PA monocultures, we observed an increase in extracellular lipase and protease and biofilm formation at 39 °C along with a decrease in the motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in vitro and in vivo. S. aureus monocultured at 39C showed a decrease in cellular invasion and an increase in IL-8-but not in IL-6-production by A549 cell line. PA also decreased its cellular invasion when monocultured at 39 °C and did not induce any change in IL-8 or IL-6 production. PA strongly increased cellular invasion when co-cultured at 37 and 39 °C. Finally, we observed increased lethality in mice intranasally inoculated with S. aureus monocultures pre-incubated at 39 °C and even higher levels when inoculated with co-cultures. The bacterial burden for P. aeruginosa was higher in liver when the mice were infected with co-cultures previously incubated at 39 °C comparing with 37 °C.
CONCLUSIONS
Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.
Topics: Mice; Animals; Staphylococcus aureus; Virulence; Pseudomonas aeruginosa; Temperature; Interleukin-6; Interleukin-8; Pseudomonas Infections; RNA, Messenger; Biofilms; Staphylococcal Infections
PubMed: 38317219
DOI: 10.1186/s12915-024-01830-3 -
Microbiology Spectrum Feb 2024Bacteria absorb different forms of iron through various channels to meet their needs. Our previous studies have shown that TseF, a type VI secretion system effector for...
Bacteria absorb different forms of iron through various channels to meet their needs. Our previous studies have shown that TseF, a type VI secretion system effector for Fe uptake, facilitates the delivery of outer membrane vesicle-associated quinolone signal (PQS)-Fe to bacterial cells by a process involving the Fe(III) pyochelin receptor FptA and the porin OprF. However, the form in which the PQS-Fe complex enters the periplasm and how it is moved into the cytoplasm remain unclear. Here, we first demonstrate that the PQS-Fe complex enters the cell directly through FptA or OprF. Next, we show that inner membrane transporters such as FptX, PchHI, and FepBCDG are not only necessary for to absorb PQS-Fe and pyochelin (PCH)-Fe but are also necessary for the virulence of toward larvae. Furthermore, we suggest that the function of PQS-Fe (but not PQS)-mediated quorum-sensing regulation is dependent on FptX, PchHI, and FepBCDG. Additionally, the findings indicate that unlike FptX, neither FepBCDG nor PchHI play roles in the autoregulatory loop involving PchR, but further deletion of and can reverse the inactive PchR phenotype caused by deletion and reactivate the expression of the PCH pathway genes under iron-limited conditions. Finally, this work identifies the interaction between FptX, PchHI, and FepBCDG, indicating that a larger complex could be formed to mediate the uptake of PQS-Fe and PCH-Fe. These results pave the way for a better understanding of the PQS and PCH iron absorption pathways and provide future directions for research on tackling infections.IMPORTANCE has evolved a number of strategies to acquire the iron it needs from its host, with the most common being the synthesis, secretion, and uptake of siderophores such as pyoverdine, pyochelin, and the quorum-sensing signaling molecule quinolone signal (PQS). However, despite intensive studies of the siderophore uptake pathways of , our understanding of how siderophores transport iron across the inner membrane into the cytoplasm is still incomplete. Herein, we reveal that PQS and pyochelin in share inner membrane transporters such as FptX, PchHI, and FepBCDG to mediate iron uptake. Meanwhile, PQS and pyochelin-mediated signaling operate to a large extent via these inner membrane transporters. Our study revealed the existence of shared uptake pathways between PQS and pyochelin, which could lead us to reexamine the role of these two molecules in the iron uptake and virulence of .
Topics: Iron; Pseudomonas aeruginosa; Membrane Transport Proteins; Receptors, Cell Surface; Bacterial Outer Membrane Proteins; Siderophores; Bacterial Proteins; Phenols; Thiazoles; Quinolones
PubMed: 38171001
DOI: 10.1128/spectrum.03256-23 -
The Journal of Biological Chemistry May 2024Sphingolipids are produced by nearly all eukaryotes where they play significant roles in cellular processes such as cell growth, division, programmed cell death,...
Sphingolipids are produced by nearly all eukaryotes where they play significant roles in cellular processes such as cell growth, division, programmed cell death, angiogenesis, and inflammation. While it was previously believed that sphingolipids were quite rare among bacteria, bioinformatic analysis of the recently identified bacterial sphingolipid synthesis genes suggests that these lipids are likely to be produced by a wide range of microbial species. The sphingolipid synthesis pathway consists of three critical enzymes. Serine palmitoyltransferase catalyzes the condensation of serine with palmitoyl-CoA (or palmitoyl-acyl carrier protein), ceramide synthase adds the second acyl chain, and a reductase reduces the ketone present on the long-chain base. While there is general agreement regarding the identity of these bacterial enzymes, the precise mechanism and order of chemical reactions for microbial sphingolipid synthesis is more ambiguous. Two mechanisms have been proposed. First, the synthesis pathway may follow the well characterized eukaryotic pathway in which the long-chain base is reduced prior to the addition of the second acyl chain. Alternatively, our previous work suggests that addition of the second acyl chain precedes the reduction of the long-chain base. To distinguish between these two models, we investigated the subcellular localization of these three key enzymes. We found that serine palmitoyltransferase and ceramide synthase are localized to the cytoplasm, whereas the ceramide reductase is in the periplasmic space. This is consistent with our previously proposed model wherein the second acyl chain is added in the cytoplasm prior to export to the periplasm where the lipid molecule is reduced.
Topics: Bacterial Proteins; Serine C-Palmitoyltransferase; Sphingolipids; Oxidoreductases; Protein Transport; Cytoplasm; Caulobacter crescentus; Escherichia coli
PubMed: 38588805
DOI: 10.1016/j.jbc.2024.107276