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Memorias Do Instituto Oswaldo Cruz 2024The availability of genes and protein sequences for parasites has provided valuable information for drug target identification and vaccine development. One such parasite...
BACKGROUND
The availability of genes and protein sequences for parasites has provided valuable information for drug target identification and vaccine development. One such parasite is Bartonella quintana, a Gram-negative, intracellular pathogen that causes bartonellosis in mammalian hosts.
OBJECTIVE
Despite progress in understanding its pathogenesis, limited knowledge exists about the virulence factors and regulatory mechanisms specific to B. quintana.
METHODS AND FINDINGS
To explore these aspects, we have adopted a subtractive proteomics approach to analyse the proteome of B. quintana. By subtractive proteins between the host and parasite proteome, a set of proteins that are likely unique to the parasite but absent in the host were identified. This analysis revealed that out of the 1197 protein sequences of the parasite, 660 proteins are non-homologous to the human host. Further analysis using the Database of Essential Genes predicted 159 essential proteins, with 28 of these being unique to the pathogen and predicted as potential putative targets. Subcellular localisation of the predicted targets revealed 13 cytoplasmic, eight membranes, one periplasmic, and multiple location proteins. The three-dimensional structure and B cell epitopes of the six membrane antigenic protein were predicted. Four B cell epitopes in KdtA and mraY proteins, three in lpxB and BQ09550, whereas the ftsl and yidC proteins were located with eleven and six B cell epitopes, respectively.
MAINS CONCLUSIONS
This insight prioritises such proteins as novel putative targets for further investigations on their potential as drug and vaccine candidates.
Topics: Proteomics; Bartonella quintana; Bacterial Vaccines; Bacterial Proteins; Humans; Computer Simulation; Virulence Factors; Proteome
PubMed: 38655925
DOI: 10.1590/0074-02760230040 -
BioRxiv : the Preprint Server For... Apr 2024Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the cellulose...
Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the cellulose synthase complex include the catalytically active BcsA enzyme, an associated periplasmic semicircle of hexameric BcsB, as well as the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a conserved periplasmic cellulase of unknown biological function. While events underlying the synthesis and translocation of cellulose by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three copies of BcsG near the nascent cellulose polymer. Further, the terminal subunit of the BcsB semicircle tethers the N-terminus of a single BcsC protein to establish a trans-envelope secretion system. BcsC's TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of mislocalized cellulose. Lastly, we introduce pEtN modification of cellulose in orthogonal cellulose biosynthetic systems by protein engineering.
PubMed: 38645035
DOI: 10.1101/2024.04.04.588173 -
Frontiers in Microbiology 2024Defenses against oxidative damage to cell components are essential for survival of bacterial pathogens during infection, and here we have uncovered that the DmsABC...
Defenses against oxidative damage to cell components are essential for survival of bacterial pathogens during infection, and here we have uncovered that the DmsABC S-/N-oxide reductase is essential for virulence and in-host survival of the human-adapted pathogen, . In several different infection models, Δ strains showed reduced immunogenicity as well as lower levels of survival in contact with host cells. Expression of DmsABC was induced in the presence of hypochlorite and paraquat, closely linking this enzyme to defense against host-produced antimicrobials. In addition to methionine sulfoxide, DmsABC converted nicotinamide- and pyrimidine-N-oxide, precursors of NAD and pyrimidine for which is an auxotroph, at physiologically relevant concentrations, suggesting that these compounds could be natural substrates for DmsABC. Our data show that DmsABC forms part of a novel, periplasmic system for defense against host-induced S- and N-oxide stress that also comprises the functionally related MtsZ S-oxide reductase and the MsrAB peptide methionine sulfoxide reductase. All three enzymes are induced following exposure of the bacteria to hypochlorite. MsrAB is required for physical resistance to HOCl and protein repair. In contrast, DmsABC was required for intracellular colonization of host cells and, together with MtsZ, contributed to resistance to N-Chlorotaurine. Our work expands and redefines the physiological role of DmsABC and highlights the importance of different types of S-oxide reductases for bacterial virulence.
PubMed: 38638903
DOI: 10.3389/fmicb.2024.1359513 -
Life Science Alliance Jun 2024The Cag type IV secretion system (Cag T4SS) has an important role in the pathogenesis of gastric cancer. The Cag T4SS outer membrane core complex (OMCC) is organized...
The Cag type IV secretion system (Cag T4SS) has an important role in the pathogenesis of gastric cancer. The Cag T4SS outer membrane core complex (OMCC) is organized into three regions: a 14-fold symmetric outer membrane cap (OMC) composed of CagY, CagX, CagT, CagM, and Cag3; a 17-fold symmetric periplasmic ring (PR) composed of CagY and CagX; and a stalk with unknown composition. We investigated how CagT, CagM, and a conserved antenna projection (AP) region of CagY contribute to the structural organization of the OMCC. Single-particle cryo-EM analyses showed that complexes purified from Δ or Δ mutants no longer had organized OMCs, but the PRs remained structured. OMCCs purified from a CagY antenna projection mutant (CagYAP) were structurally similar to WT OMCCs, except for the absence of the α-helical antenna projection. These results indicate that CagY and CagX are sufficient for maintaining a stable PR, but the organization of the OMC requires CagY, CagX, CagM, and CagT. Our results highlight an unexpected structural independence of two major subdomains of the Cag T4SS OMCC.
Topics: Helicobacter pylori; Type IV Secretion Systems; Periplasm
PubMed: 38631913
DOI: 10.26508/lsa.202302560 -
PloS One 2024Lyme disease is the most prevalent vector-borne infectious disease in Europe and the USA. Borrelia burgdorferi, as the causative agent of Lyme disease, is transmitted to...
Lyme disease is the most prevalent vector-borne infectious disease in Europe and the USA. Borrelia burgdorferi, as the causative agent of Lyme disease, is transmitted to the mammalian host during the tick blood meal. To adapt to the different encountered environments, Borrelia has adjusted the expression pattern of various, mostly outer surface proteins. The function of most B. burgdorferi outer surface proteins remains unknown. We determined the crystal structure of a previously uncharacterized B. burgdorferi outer surface protein BBK01, known to belong to the paralogous gene family 12 (PFam12) as one of its five members. PFam12 members are shown to be upregulated as the tick starts its blood meal. Structural analysis of BBK01 revealed similarity to the coiled coil domain of structural maintenance of chromosomes (SMC) protein family members, while functional studies indicated that all PFam12 members are non-specific DNA-binding proteins. The residues involved in DNA binding were identified and probed by site-directed mutagenesis. The combination of SMC-like proteins being attached to the outer membrane and exposed to the environment or located in the periplasm, as observed in the case of PFam12 members, and displaying the ability to bind DNA, represents a unique feature previously not observed in bacteria.
Topics: Animals; Borrelia burgdorferi; DNA-Binding Proteins; Lyme Disease; Ticks; Membrane Proteins; DNA; Bacterial Outer Membrane Proteins; Mammals
PubMed: 38626020
DOI: 10.1371/journal.pone.0296127 -
Frontiers in Cellular and Infection... 2024The hemin acquisition system is composed of an outer membrane TonB-dependent transporter that internalizes hemin into the periplasm, periplasmic hemin-binding proteins...
INTRODUCTION
The hemin acquisition system is composed of an outer membrane TonB-dependent transporter that internalizes hemin into the periplasm, periplasmic hemin-binding proteins to shuttle hemin, an inner membrane transporter that transports hemin into the cytoplasm, and cytoplasmic heme oxygenase to release iron. Fur and HemP are two known regulators involved in the regulation of hemin acquisition. The hemin acquisition system of is poorly understood, with the exception of HemA as a TonB-dependent transporter for hemin uptake.
METHODS
Putative candidates responsible for hemin acquisition were selected via a homolog search and a whole-genome survey of . Operon verification was performed by reverse transcription-polymerase chain reaction. The involvement of candidate genes in hemin acquisition was assessed using an in-frame deletion mutant construct and iron utilization assays. The transcript levels of candidate genes were determined using quantitative polymerase chain reaction.
RESULTS
and operons were selected as candidates for hemin acquisition. Compared with the parental strain, and mutants displayed a defect in their ability to use hemin as the sole iron source for growth. However, hemin utilization by the and mutants was comparable to that of the parental strain. expression was repressed by Fur in iron-replete conditions and derepressed in iron-depleted conditions. HemP negatively regulated expression. Like , was repressed by Fur in iron-replete conditions; however, was moderately derepressed in response to iron-depleted stress and fully derepressed when hemin was present. Unlike and , the operon was constitutively expressed, regardless of the iron level or the presence of hemin, and Fur and HemP had no influence on its expression.
CONCLUSION
HemA, HemU, and TonB1 contribute to hemin acquisition in . Fur represses the expression of and in iron-replete conditions. expression is regulated by low iron levels, and HemP acts as a negative regulator of this regulatory circuit. expression is regulated by low iron and hemin levels in a -dependent manner.
Topics: Hemin; Stenotrophomonas maltophilia; Bacterial Proteins; Membrane Proteins; Iron
PubMed: 38596648
DOI: 10.3389/fcimb.2024.1380976 -
Nature Communications Apr 2024The bacterium Bdellovibrio bacteriovorus is a predator of other Gram-negative bacteria. The predator invades the prey's periplasm and modifies the prey's cell wall,...
The bacterium Bdellovibrio bacteriovorus is a predator of other Gram-negative bacteria. The predator invades the prey's periplasm and modifies the prey's cell wall, forming a rounded killed prey, or bdelloplast, containing a live B. bacteriovorus. Redundancy in adhesive processes makes invasive mutants rare. Here, we identify a MIDAS adhesin family protein, Bd0875, that is expressed at the predator-prey invasive junction and is important for successful invasion of prey. A mutant strain lacking bd0875 is still able to form round, dead bdelloplasts; however, 10% of the bdelloplasts do not contain B. bacteriovorus, indicative of an invasion defect. Bd0875 activity requires the conserved MIDAS motif, which is linked to catch-and-release activity of MIDAS proteins in other organisms. A proteomic analysis shows that the uninvaded bdelloplasts contain B. bacteriovorus proteins, which are likely secreted into the prey by the Δbd0875 predator during an abortive invasion period. Thus, secretion of proteins into the prey seems to be sufficient for prey killing, even in the absence of a live predator inside the prey periplasm.
Topics: Bdellovibrio bacteriovorus; Bdellovibrio; Proteomics; Adhesins, Bacterial
PubMed: 38594280
DOI: 10.1038/s41467-024-47412-3 -
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 -
Frontiers in Immunology 2024In the defense against microorganisms like Candida albicans, macrophages recruit LC3(Microtubule-associated protein 1A/1B-light chain 3) to the periplasm, engaging in...
OBJECTIVE
In the defense against microorganisms like Candida albicans, macrophages recruit LC3(Microtubule-associated protein 1A/1B-light chain 3) to the periplasm, engaging in the elimination process through the formation of a single-membrane phagosome known as LC3-associated phagocytosis (LAP). Building on this, we propose the hypothesis that glucocorticoids may hinder macrophage phagocytosis of Candida glabrata by suppressing LAP, and rapamycin could potentially reverse this inhibitory effect.
METHODS
RAW264.7 cells were employed for investigating the immune response to Candida glabrata infection. Various reagents, including dexamethasone, rapamycin, and specific antibodies, were utilized in experimental setups. Assays, such as fluorescence microscopy, flow cytometry, ELISA (Enzyme-Linked Immunosorbent Assay), Western blot, and confocal microscopy, were conducted to assess phagocytosis, cytokine levels, protein expression, viability, and autophagy dynamics.
RESULTS
Glucocorticoids significantly inhibited macrophage autophagy, impairing the cells' ability to combat Candida glabrata. Conversely, rapamycin exhibited a dual role, initially inhibiting and subsequently promoting phagocytosis of Candida glabrata by macrophages. Glucocorticoids hinder macrophage autophagy in Candida glabrata infection by suppressing the MTOR pathway(mammalian target of rapamycin pathway), while the activation of MTOR pathway by Candida glabrata diminishes over time.
CONCLUSION
Our study elucidates the intricate interplay between glucocorticoids, rapamycin, and macrophage autophagy during Candida glabrata infection. Understanding the implications of these interactions not only sheds light on the host immune response dynamics but also unveils potential therapeutic avenues for managing fungal infections.
Topics: Animals; Mice; Candida glabrata; Glucocorticoids; Sirolimus; Mice, Inbred BALB C; Autophagy; Macrophages; Candidiasis; TOR Serine-Threonine Kinases; Mammals
PubMed: 38585259
DOI: 10.3389/fimmu.2024.1367048 -
ISME Communications Jan 2024Nitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially...
Nitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially stimulate the biological oxidation of iron in anoxic groundwater reservoirs. Nitrate-dependent iron-oxidizing (NDFO) bacteria have been extensively studied in laboratory settings, yet their ecophysiology in natural environments remains largely unknown. To this end, we established a pilot-scale filter on nitrate-rich groundwater to elucidate the structure and metabolism of nitrate-reducing iron-oxidizing microbiomes under oligotrophic conditions mimicking natural groundwaters. The enriched community stoichiometrically removed iron and nitrate consistently with the NDFO metabolism. Genome-resolved metagenomics revealed the underlying metabolic network between the dominant iron-dependent denitrifying autotrophs and the less abundant organoheterotrophs. The most abundant genome belonged to a new order, named Siderophiliales. This new species, " Siderophilus nitratireducens," carries genes central genes to iron oxidation (cytochrome ), carbon fixation (), and for the sole periplasmic nitrate reductase (). Using thermodynamics, we demonstrate that iron oxidation coupled to based dissimilatory reduction of nitrate to nitrite is energetically favorable under realistic Fe/Fe and NO/NO concentration ratios. Ultimately, by bridging the gap between laboratory investigations and nitrate real-world conditions, this study provides insights into the intricate interplay between nitrate and iron in groundwater ecosystems, and expands our understanding of NDFOs taxonomic diversity and ecological role.
PubMed: 38577582
DOI: 10.1093/ismeco/ycae008