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Trends in Biochemical Sciences Apr 2024The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with β-barrel OM... (Review)
Review
The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with β-barrel OM proteins (OMPs). Before reaching the OM, precursor unfolded OMPs (uOMPs) must cross the whole cell envelope. A network of periplasmic chaperones and proteases maintains unfolded but folding-competent conformations of these membrane proteins in the aqueous periplasm while simultaneously preventing off-pathway aggregation. These periplasmic proteins utilize different strategies, including conformational heterogeneity, oligomerization, multivalency, and kinetic partitioning, to perform and regulate their functions. Redundant and unique characteristics of the individual periplasmic players synergize to create a protein quality control team capable responding to changing environmental stresses.
PubMed: 38677921
DOI: 10.1016/j.tibs.2024.03.015 -
BioRxiv : the Preprint Server For... Jul 2023and cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic...
BACKGROUND
and 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 USA300 and PAO1 microaerobic mono- and co-cultures compared with 37°C, by using RNAseq and physiological assays including 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 on both species. Altered metabolic features at 39°C included arginine biosynthesis and the periplasmic glucose oxidation in and 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 operon and and genes at 39°C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphylxantin production and a decrease in biofilm formation at 39°C. On the side of PA monocultures, we observed increase in extracellular lipase and protease and biofilm formation at 39°C along with a decrease in motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both and . monocultured at 39°C 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°C and 39°C. Finally, we observed increased lethality in mice intranasally inoculated with monocultures pre-incubated at 39°C and even higher levels when inoculated with co-cultures. The bacterial burden for was higher in liver when the mice were infected with co-cultures previously incubated at 39°C comparing with 37°C.
CONCLUSION
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.
PubMed: 36993402
DOI: 10.1101/2023.03.21.529514 -
Applied and Environmental Microbiology Jul 2023Metal ions are essential nutrients for all life forms, and restriction of metal ion availability is an effective host defense against bacterial infection. Meanwhile,...
Metal ions are essential nutrients for all life forms, and restriction of metal ion availability is an effective host defense against bacterial infection. Meanwhile, bacterial pathogens have developed equally effective means to secure their metal ion supply. The enteric pathogen Yersinia pseudotuberculosis was found to uptake zinc using the T6SS4 effector YezP, which is essential for Zn acquisition and bacterial survival under oxidative stress. However, the mechanism of this zinc uptake pathway has not been fully elucidated. Here, we identified the hemin uptake receptor HmuR for YezP, which can mediate import of Zn into the periplasm by the YezP-Zn complex and demonstrated that YezP functions extracellularly. This study also confirmed that the ZnuCB transporter is the inner membrane transporter for Zn from the periplasm to cytoplasm. Overall, our results reveal the complete T6SS/YezP/HmuR/ZnuABC pathway, wherein multiple systems are coupled to support zinc uptake by Y. pseudotuberculosis under oxidative stress. Identifying the transporters involved in import of metal ions under normal physiological growth conditions in bacterial pathogens will clarify its pathogenic mechanism. Y. pseudotuberculosis YPIII, a common foodborne pathogen that infects animals and humans, uptake zinc via the T6SS4 effector YezP. However, the outer and inner transports involved in Zn acquisition remain unknown. The important outcomes of this study are the identification of the hemin uptake receptor HmuR and inner membrane transporter ZnuCB that import Zn into the cytoplasm via the YezP-Zn complex, and elucidation of the complete Zn acquisition pathway consisting of T6SS, HmuRSTUV, and ZnuABC, thereby providing a comprehensive view of T6SS-mediated ion transport and its functions.
Topics: Humans; Animals; Hemin; Yersinia; Biological Transport; Membrane Transport Proteins; Yersinia pseudotuberculosis Infections; Zinc; Bacterial Proteins
PubMed: 37338394
DOI: 10.1128/aem.00240-23 -
Planta Jul 2023Our findings suggest a reconsideration of pollen wall ontogeny process, entailing examination of physical factors, which enable a new understanding of exine...
Our findings suggest a reconsideration of pollen wall ontogeny process, entailing examination of physical factors, which enable a new understanding of exine developmental processes as self-formation. The pollen wall, the most complex cell wall in plants, is especially interesting as a model of ontogeny in miniature. By a detailed study of each developmental stage of Campanula rapunculoides pollen wall, we aimed to understand the establishment of complex pollen walls and the underlying developmental mechanisms. Other aim was to compare our current observations with studies in other species to reveal the common principles. We also tried to analyse the reasons for commonalities in ontogenies of exines in remote species. TEM, SEM, comparative methods were used in this study. The sequence of events leading to exine emergence from early tetrad stage to maturity is as follows: the appearance of spherical micelles in the periplasmic space and de-mixing of the mixture in periplasm (condensed and depleted layers); appearance of plasma membrane invaginations and columns of spherical micelles inside condensed layer; appearance of rod-like units, pro-tectum and thin foot layer; the appearance of spiral substructure of procolumellae and of dendritic outgrowths on the tops of procolumellae, of vast depleted zone in aperture sites; formation of the endexine lamellae on the base of laminate micelles; gradual twisting of dendritic outgrowths (macromolecule chains) into clubs on the tops of columellae and into spines; final sporopollenin accumulation. Our observations are consistent with the sequence of self-assembling micellar mesophases. Complex organisation of the exine is established through processes of self-assembly operating together with another physical process-phase separation. After genomic determination of the exine building substances, purely physical processes which are not under direct genomic control play an important role after genomic control of constructive substances. The comparison of the underlying mechanisms of exine development in remote species occurred to be general and similar to crystallisation. Our ontogenetic experience has shown the commonality of pollen wall ontogenies in remote species.
Topics: Micelles; Pollen; Cell Wall; Campanulaceae
PubMed: 37410162
DOI: 10.1007/s00425-023-04198-w -
Journal of Advanced Research Mar 2024Growing evidence has shown the correlation between periodontitis and atherosclerosis, while our knowledge on the pathogenesis of periodontitis-promoting atherosclerosis...
INTRODUCTION
Growing evidence has shown the correlation between periodontitis and atherosclerosis, while our knowledge on the pathogenesis of periodontitis-promoting atherosclerosis is far from sufficient.
OBJECTIVES
Illuminate the pathogenic effects of Fusobacterium nucleatum (F. nucleatum) on intracellular lipid deposition in THP-1-derived macrophages and elucidate the underlying pathogenic mechanism of how F. nucleatum promoting atherosclerosis.
METHODS AND RESULTS
F. nucleatum was frequently detected in different kinds of atherosclerotic plaques and its abundance was positively correlated with the proportion of macrophages. In vitro assays showed F. nucleatum could adhere to and invade THP-1 cells, and survive continuously in macrophages for 24 h. F. nucleatum stimulation alone could significantly promote cellular inflammation, lipid uptake and inhibit lipid outflow. The dynamic gene expression of THP-1 cells demonstrated that F. nucleatum could time-serially induce the over-expression of multiple inflammatory related genes and activate NF-κB, MAPK and PI3K-AKT signaling pathways. The exoprotein of F. nucleatum, D-galactose-binding protein (Gbp), acted as one of the main pathogenic proteins to interact with the Cyclophilin A (CypA) of THP-1 cells and induced the activation of the NF- κB, MAPK and PI3K-AKT signaling pathways. Furthermore, use of six candidate drugs targeting to the key proteins in NF- κB, MAPK and PI3K-AKT pathways could dramatically decrease F. nucleatum induced inflammation and lipid deposition in THP-1 cells.
CONCLUSIONS
This study suggests that the periodontal pathogen F. nucleatum can activate macrophage PI3K-AKT/MAPK/NF-κB signal pathways, promotes inflammation, enhances cholesterol uptake, reduces lipid excretion, and promotes lipid deposition, which may be one of its main strategies promoting the development of atherosclerosis.
Topics: Humans; NF-kappa B; Cyclophilin A; Fusobacterium nucleatum; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; THP-1 Cells; Atherosclerosis; Inflammation; Periodontitis; Lipids; Calcium-Binding Proteins; Monosaccharide Transport Proteins; Periplasmic Binding Proteins
PubMed: 37100345
DOI: 10.1016/j.jare.2023.04.007 -
The FEBS Journal Sep 2023Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively...
Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.
Topics: Protein Folding; Carrier Proteins; Kinetics; Lysine; Ligands; Laos; Protein Denaturation; Thermodynamics; Protein Conformation
PubMed: 37178351
DOI: 10.1111/febs.16819 -
PloS One 2023Extreme acidophiles thrive in harsh environments characterized by acidic pH, high concentrations of dissolved metals and high osmolarity. Most of these microorganisms...
Extreme acidophiles thrive in harsh environments characterized by acidic pH, high concentrations of dissolved metals and high osmolarity. Most of these microorganisms are chemolithoautotrophs that obtain energy from low redox potential sources, such as the oxidation of ferrous ions. Under these conditions, the mechanisms that maintain homeostasis of proteins (proteostasis), as the main organic components of the cells, are of utmost importance. Thus, the analysis of protein chaperones is critical for understanding how these organisms deal with proteostasis under such environmental conditions. In this work, using a bioinformatics approach, we performed a comparative genomic analysis of the genes encoding classical, periplasmic and stress chaperones, and the protease systems. The analysis included 35 genomes from iron- or sulfur-oxidizing autotrophic, heterotrophic, and mixotrophic acidophilic bacteria. The results showed that classical ATP-dependent chaperones, mostly folding chaperones, are widely distributed, although they are sub-represented in some groups. Acidophilic bacteria showed redundancy of genes coding for the ATP-independent holdase chaperones RidA and Hsp20. In addition, a systematically high redundancy of genes encoding periplasmic chaperones like HtrA and YidC was also detected. In the same way, the proteolytic ATPase complexes ClpPX and Lon presented redundancy and broad distribution. The presence of genes that encoded protein variants was noticeable. In addition, genes for chaperones and protease systems were clustered within the genomes, suggesting common regulation of these activities. Finally, some genes were differentially distributed between bacteria as a function of the autotrophic or heterotrophic character of their metabolism. These results suggest that acidophiles possess an abundant and flexible proteostasis network that protects proteins in organisms living in energy-limiting and extreme environmental conditions. Therefore, our results provide a means for understanding the diversity and significance of proteostasis mechanisms in extreme acidophilic bacteria.
Topics: Proteostasis; Genomics; Computational Biology; Endopeptidases; Peptide Hydrolases; Iron; Adenosine Triphosphate
PubMed: 37682893
DOI: 10.1371/journal.pone.0291164 -
Environmental Science and Pollution... Dec 2023Currently, there is a noticeable scarcity of applications that harness composite microbial inoculants to stimulate straw decomposition, nitrogen fixation, and crop...
Currently, there is a noticeable scarcity of applications that harness composite microbial inoculants to stimulate straw decomposition, nitrogen fixation, and crop growth. This study addresses this gap by selecting and coculturing three bacterial strains to create a composite microbial inoculant named HY-1. This innovative inoculant exhibits multifunctional capabilities, including nitrogen fixation, straw decomposition, and crop growth promotion. Furthermore, we aimed to explore its impact on soil microbial communities. The results showed that the optimal preparation conditions for the compound microbial inoculant HY-1 were 28.5 ± 0.6 °C, pH = 7.34 ± 0.40, and bacteriophage ratio 1:2:1 (Microbacterium: Streptomyces fasciatus: Bacillus amyloliquefaciens). Compared to single strains, the combination exhibited higher levels of cellulose-degrading and nitrogen-fixing enzyme activity, increased the straw degradation rate by 37.91% within 180 days, and significantly promoted the growth of corn seedlings. Under the condition of straw return, the compound bio-fungicide HY-1 effectively improved the soil microbial diversity. At that time, the soil had the highest number of unique bacterial operational taxonomic units (166), and the abundance of Proteobacteria in the soil increased by 7.24%, while that of Acidobacteriota decreased by 2.27%. The biosynthetic function of the cell wall/membrane/periplasm and the metabolic function of transporting inorganic ions were significantly enhanced. In this study, we discovered that employing coculturing techniques to produce the composite microbial inoculant HY-1 and applying it in the field effectively compensates for the limitations of single-strain inoculants, which often exhibit fewer functions and less pronounced effects. This approach demonstrates significant potential for enhancing the quality of agricultural soils.
Topics: Soil; Agricultural Inoculants; Agriculture; Microbiota; Soil Microbiology
PubMed: 37945959
DOI: 10.1007/s11356-023-30887-w -
Biochemical and Biophysical Research... Oct 2023Iron acquisition is an essential process of cell physiology for biological systems. In Klebsiella pneumoniae, the siderophore and ferric-acquisition ABC...
Iron acquisition is an essential process of cell physiology for biological systems. In Klebsiella pneumoniae, the siderophore and ferric-acquisition ABC (ATP-Binding-Cassette) transporter KfuABC is utilized for iron uptake. Initial recognition of the various ferric sources in periplasm and transportation across the cytoplasmic membrane is performed by the substrate-binding protein (SBP) KfuA. Here we report the 2.0 Å resolution crystal structure of KfuA from K. pneumoniae, which crystallizes in the space group P121 with a single monomer in the asymmetric unit. A bound metal ion reveals the residues required for binding ferric ions. Binding analysis shows that ferric iron and the iron-mimicking gallium bind with high affinity to KfuA. Growth curves show that gallium inhibits growth of K. pneumoniae whereas ferric iron enhances it. This work suggests a mechanism whereby gallium effectively competes with ferric iron, disrupting iron-dependent biological functions via binding to KfuA and leading to heightened antimicrobial efficacy. Significantly, humans lack equivalent ABC transporters like SBP KfuA, underscoring the potential of KfuA as an attractive target for therapeutic intervention.
PubMed: 37669596
DOI: 10.1016/j.bbrc.2023.08.068 -
Genes Aug 2023UshA and CpdB are nucleotidases of the periplasm of several Gram-negative bacteria, while several Gram-positives contain cell wall-bound variants. UshA is a...
UshA and CpdB are nucleotidases of the periplasm of several Gram-negative bacteria, while several Gram-positives contain cell wall-bound variants. UshA is a 5'-nucleotidase, a UDP-sugar hydrolase, and a CDP-alcohol hydrolase. CpdB acts as a 3'-nucleotidase and as a phosphodiesterase of 2',3'-cyclic nucleotides and 3',5'-linear and cyclic dinucleotides. Both proteins are pro-virulent for the pathogens producing them and facilitate escape from the innate immunity of the infected host. Recently, the genomic distribution of -like genes in was found to be non-homogeneous among different taxa, and differences occur within single taxa, even at species level. Similitudes and differences between UshA-like and CpdB-like proteins prompted parallel analysis of their genomic distributions in . The presence of -like and -like genes was tested by TBlastN analysis using seven protein probes to query the NCBI Complete Genomes Database. It is concluded that the distribution of -like genes, like that of -like genes, is non-homogeneous. There is a partial correlation between both gene kinds: in some taxa, both are present or absent, while in others, only one is present. The result is an extensive catalog of the genomic distribution of these genes at different levels, from phylum to species, constituting a starting point for research using other in silico or experimental approaches.
Topics: Genomics; Bacteria; Cell Wall; Ethanol; Hydrolases
PubMed: 37628708
DOI: 10.3390/genes14081657