-
Analytical Chemistry Jun 2024Bacterial viability assessment plays an important role in food-borne pathogen detection and antimicrobial drug development. Here, we first used GelRed as a DNA-binding...
Bacterial viability assessment plays an important role in food-borne pathogen detection and antimicrobial drug development. Here, we first used GelRed as a DNA-binding stain for a bacterial viability assessment. It was found that live bacteria were able to exclude GelRed, which however could easily penetrate dead ones and be absorbed nonspecifically on the bacterial periplasm. Cations were used to reduce the nonspecific adsorption and greatly increase the red fluorescence ratio of dead to live bacteria. Combined with SYTO 9 (a membrane-permeable dye) for double-staining, a ratiometric fluorescent method was established. Using O157:H7 as a bacteria model, the ratiometric fluorescent method can probe dead bacteria as low as 0.1%. A linear correlation between the ratiometric fluorescence and the theoretical ratio of dead bacteria was acquired, with a correlation coefficient of 0.97. Advantages in sensitivity, accuracy, and safety of the GelRed/SYTO9-based ratiometric fluorescent method against traditional methods were demonstrated. The established method was successfully applied to the assessment of germicidal efficacy of different heat treatments. It was found that even 50 °C treatment could lead to the death of minor bacteria. The as-developed method has many potential applications in microbial researches, and we believe it could be expanded to the viability assessment of mammalian cells.
PubMed: 38934709
DOI: 10.1021/acs.analchem.4c01737 -
The Journal of Infectious Diseases Jun 2024Histologic and serologic studies suggest the induction of local and systemic Treponema pallidum-specific CD4+ T-cell responses to T. pallidum infection. We hypothesized...
BACKGROUND
Histologic and serologic studies suggest the induction of local and systemic Treponema pallidum-specific CD4+ T-cell responses to T. pallidum infection. We hypothesized that T. pallidum-specific CD4+ T cells are detectable in blood and in the skin rash of secondary syphilis and persist in both compartments after treatment.
METHODS
Peripheral blood mononuclear cells collected from 67 participants were screened by interferon-γ (IFN-γ) ELISPOT response to T. pallidum sonicate. T. pallidum-reactive T-cell lines from blood and skin were probed for responses to 89 recombinant T. pallidum antigens. Peptide epitopes and HLA class II restriction were defined for selected antigens.
RESULTS
We detected CD4+ T-cell responses to T. pallidum sonicate ex vivo. Using T. pallidum-reactive T-cell lines we observed recognition of 14 discrete proteins, 13 of which localize to bacterial membranes or the periplasmic space. After therapy, T. pallidum-specific T cells persisted for at least 6 months in skin and 10 years in blood.
CONCLUSIONS
T. pallidum infection elicits an antigen-specific CD4+ T-cell response in blood and skin. T. pallidum-specific CD4+ T cells persist as memory in both compartments long after curative therapy. The T. pallidum antigenic targets we identified may be high-priority vaccine candidates.
PubMed: 38932740
DOI: 10.1093/infdis/jiae245 -
Marine Drugs Jun 2024Sortase A (SrtA) is a cysteine transpeptidase that binds to the periplasmic membrane and plays a crucial role in attaching surface proteins, including staphylococcal...
Sortase A (SrtA) is a cysteine transpeptidase that binds to the periplasmic membrane and plays a crucial role in attaching surface proteins, including staphylococcal protein A (SpA), to the peptidoglycan cell wall. Six pentacyclic polyketides (-) were isolated from the marine sponge sp., and their structures were elucidated using spectroscopic techniques and by comparing them to previously reported data. Among them, halenaquinol () was found to be the most potent SrtA inhibitor, with an IC of 13.94 μM (4.66 μg/mL). Semi-quantitative reverse transcription PCR data suggest that halenaquinol does not inhibit the transcription of and , while Western blot analysis and immunofluorescence microscopy images suggest that it blocks the cell wall surface anchoring of SpA by inhibiting the activity of SrtA. The onset and magnitude of the inhibition of SpA anchoring on the cell wall surface in that has been treated with halenaquinol at a value 8× that of the IC of SrtA are comparable to those for an -deletion mutant. These findings contribute to the understanding of the mechanism by which marine-derived pentacyclic polyketides inhibit SrtA, highlighting their potential as anti-infective agents targeting virulence.
Topics: Aminoacyltransferases; Cysteine Endopeptidases; Staphylococcus aureus; Cell Wall; Bacterial Proteins; Animals; Porifera; Anti-Bacterial Agents; Polyketides
PubMed: 38921577
DOI: 10.3390/md22060266 -
Microbiology Spectrum Jun 2024Acetic acid bacteria are used in many industrial processes such as the production of vinegar, vitamin C, the antidiabetic drug miglitol, and various artificial...
Acetic acid bacteria are used in many industrial processes such as the production of vinegar, vitamin C, the antidiabetic drug miglitol, and various artificial flavorings. These industrially important reactions are primarily carried out by an arsenal of periplasmic-facing membrane-bound dehydrogenases that incompletely oxidize their substrates and shuttle electrons directly into the respiratory chain. Among these dehydrogenases, GOX in was predicted to be a pyrroloquinoline quinone-dependent dehydrogenase of unknown function. However, after multiple analysis by a number of labs, no dehydrogenase activity has been detected. Reanalysis of GOX1969 sequence and structure reveals similarities to BamB, which functions as a subunit of the β-barrel assembly machinery complex that is responsible for the assembly of β-barrel outer membrane proteins in Gram-negative bacteria. To test if the physiological function of GOX1969 is similar to BamB in , we introduced the gene into an ∆ mutant. Growth deficiencies in the ∆ mutant were restored when was expressed on the plasmid pGox1969. Furthermore, increased membrane permeability conferred by deletion was restored upon expression, which suggests a direct link between GOX1969 and a role in maintaining outer membrane stability. Together, this evidence strongly suggests that GOX1969 is functionally acting as a BamB in . As such, functional information on uncharacterized genes will provide new insights that will allow for more accurate modeling of acetic acid bacterial metabolism and further efforts to design rational strains for industrial use.IMPORTANCE is an industrially important member of the acetic acid bacteria. Experimental characterization of putative genes is necessary to identify targets for further engineering of rational acetic acid bacteria strains that can be used in the production of vitamin C, antidiabetic compounds, artificial flavorings, or novel compounds. In this study, we have identified an undefined dehydrogenase GOX1969 with no known substrate and defined structural similarities to outer membrane biogenesis protein BamB in K12. Furthermore, we demonstrate that GOX1969 is capable of complementing knockout phenotypes in K12. Taken together, these findings enhance our understanding of physiology and expand the list of potential targets for future industrial strain design.
PubMed: 38916353
DOI: 10.1128/spectrum.01060-24 -
BioRxiv : the Preprint Server For... Jun 2024Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria....
Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg , become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from . In the absence of Mg , AcrA becomes increasingly plastic within acidic conditions, but when Mg is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.
PubMed: 38915626
DOI: 10.1101/2024.06.10.597921 -
ACS Omega Jun 2024Membrane permeability is a natural defense barrier that contributes to increased bacterial drug resistance, particularly for Gram-negative pathogens. As such, accurate...
Membrane permeability is a natural defense barrier that contributes to increased bacterial drug resistance, particularly for Gram-negative pathogens. As such, accurate delivery of the antibacterial agent to the target has become a growing research area in the infectious diseases field as a means of improving drug efficacy. Although the efficient transport of siderophore-antibiotic conjugates into the cytosol still remains challenging, great success has been achieved in the delivery of β-lactam antibiotics into the periplasmic space via bacterial iron uptake pathways. Cefiderocol, the first siderophore-cephalosporin conjugate approved by the US Food and Drug Administration, is a good example. These conjugation strategies have also been applied to the precise delivery of β-lactamase inhibitors, such as penicillin-based sulfone , to restore β-lactam antibiotic efficacy in multidrug-resistant bacteria. Herein, we have explored the impact on the bacterial activity of by modifying its iron chelator moiety. A set of derivatives functionalized with diverse iron chelator groups and linkages to the scaffold (compounds -) were synthesized and assayed in vitro. The results on the ability of derivatives - to recover β-lactam antibiotic efficacy in difficult-to-treat pathogens that produce various β-lactamase enzymes, along with kinetic studies with the isolated enzymes, allowed us to identify compound , a novel β-lactamase inhibitor with an expanded spectrum of activity. Molecular dynamics simulation studies provided us with further information regarding the molecular basis of the relative inhibitory properties of the most relevant compound described herein.
PubMed: 38911797
DOI: 10.1021/acsomega.4c02984 -
The Science of the Total Environment Jun 2024Arsenic (As) cycling in groundwater is commonly coupled to the biogeochemical cycling of iron (Fe) and the associated transformation of Fe minerals present. Numerous...
Arsenic (As) cycling in groundwater is commonly coupled to the biogeochemical cycling of iron (Fe) and the associated transformation of Fe minerals present. Numerous laboratory studies suggested that Fe minerals can act as nucleation sites for further crystal growth and as catalysts for abiotic Fe(II) oxidation. In view of the widespread existence of magnetite in anoxic environments where As is often dissolved, we firstly exploited magnetite to enhance As immobilization during nitrate-reducing Fe(II) oxidation (NRFO) induced by Acidovorax sp. strain BoFeN1, a mixotrophic nitrate-reducing Fe(II)-oxidizing bacterium that can oxidize Fe(II) through both enzymatic and abiotic pathways. Subsequently, we investigated how magnetite affects NRFO and As immobilization. Results demonstrated a significant increase in As(III) removal efficiency from 75.4 % to 97.2 % with magnetite, attributed to the higher amount of NRFO and As(III) oxidation promoted by magnetite. It was found that magnetite stimulated the production of extracellular polymeric substances (EPS), which could decrease the diffusion of nitrate in the periplasm of bacteria and shield them against encrustation, resulting in a more rapid reduction of nitrate in the system with magnetite than that without magnetite. Meanwhile, Fe(II) was almost completely oxidized in the presence of magnetite during the whole 72 h experiment, while in the absence of magnetite, 47.7 % of Fe(II) remained, indicating that magnetite could obviously accelerate the chemical oxidation of Fe(II) with nitrite (the intermediates of nitrate bioreduction). Furthermore, the formation of labile Fe(III), an intermediate product of electron transfer between Fe(II) and magnetite, was reasonably deduced to be vital for anoxic As(III) oxidation. Additionally, the XPS analysis of the solid phase confirmed the oxidation of 43.8 % of As(III) to As(V). This study helps to understand the biogeochemical cycling of Fe and As in the environment, and provides a cost-effective and environmentally friendly option for in situ remediation of As-contaminated groundwater.
PubMed: 38909815
DOI: 10.1016/j.scitotenv.2024.173946 -
Journal of Molecular Biology Jun 2024The ability to adapt to osmotically diverse and fluctuating environments is critical to the survival and resilience of bacteria that colonize the human gut and urinary...
The ability to adapt to osmotically diverse and fluctuating environments is critical to the survival and resilience of bacteria that colonize the human gut and urinary tract. Environmental stress often provides cross-protection against other challenges and increases antibiotic tolerance of bacteria. Thus, it is critical to understand how E. coli and other microbes survive and adapt to stress conditions. The osmotically inducible protein Y (OsmY) is significantly upregulated in response to hypertonicity. Yet its function remains unknown for decades. We determined the solution structure and dynamics of OsmY by nuclear magnetic resonance spectroscopy, which revealed that the two Bacterial OsmY and Nodulation (BON) domains of the protein are flexibly linked under low- and high-salinity conditions. In-cell solid-state NMR further indicates that there are no gross structural changes in OsmY as a function of osmotic stress. Using cryo-electron and super-resolution fluorescence microscopy, we show that OsmY attenuates plasmolysis-induced structural changes in E. coli and improves the time to growth resumption after osmotic upshift. Structure-guided mutational and functional studies demonstrate that exposed hydrophobic residues in the BON1 domain are critical for the function of OsmY. We find no evidence for membrane interaction of the BON domains of OsmY, contrary to current assumptions. Instead, at high ionic strength, we observe an interaction with the water channel, AqpZ. Thus, OsmY does not play a simple structural role in E. coli but may influence a cascade of osmoregulatory functions of the cell.
PubMed: 38908784
DOI: 10.1016/j.jmb.2024.168668 -
PloS One 2024Antibiotic resistance genes (ARGs) transfer rapidly among bacterial species all over the world contributing to the aggravation of antibiotic resistance crisis....
BACKGROUND
Antibiotic resistance genes (ARGs) transfer rapidly among bacterial species all over the world contributing to the aggravation of antibiotic resistance crisis. Antibiotics at sub-inhibitory concentration induce horizontal gene transfer (HRT) between bacteria, especially through conjugation. The role of common non-antibiotic pharmaceuticals in the market in disseminating antibiotic resistance is not well studied.
OBJECTIVES
In this work, we indicated the effect of some commonly used non-antibiotic pharmaceuticals including antiemetic (metoclopramide HCl) and antispasmodics (hyoscine butyl bromide and tiemonium methyl sulfate) on the plasmid-mediated conjugal transfer of antibiotic resistance genes between pathogenic E. coli in the gastric intestinal tract (GIT).
METHODS
Broth microdilution assay was used to test the antibacterial activity of the tested non-antibiotic pharmaceuticals. A conjugation mating system was applied in presence of the studied non-antibiotic pharmaceuticals to test their effect on conjugal transfer frequency. Plasmid extraction and PCR were performed to confirm the conjugation process. Transmission electron microscopy (TEM) was used for imaging the effect of non-antibiotic pharmaceuticals on bacterial cells.
RESULTS
No antibacterial activity was reported for the used non-antibiotic pharmaceuticals. Plasmid-mediated conjugal transfer between isolates was induced by metoclopramide HCl but suppressed by hyoscine butyl bromide. Tiemonium methylsulfate slightly promoted conjugal transfer. Aggregation between cells and periplasmic bridges was clear in the case of metoclopramide HCl while in presence of hyoscine butyl bromide little affinity was observed.
CONCLUSION
This study indicates the contribution of non-antibiotic pharmaceuticals to the dissemination and evolution of antibiotic resistance at the community level. Metoclopramide HCl showed an important role in the spread of antibiotic resistance.
Topics: Escherichia coli; Gene Transfer, Horizontal; Plasmids; Metoclopramide; Microbial Sensitivity Tests; Anti-Bacterial Agents; Drug Resistance, Bacterial; Conjugation, Genetic; Drug Resistance, Microbial
PubMed: 38905247
DOI: 10.1371/journal.pone.0304980 -
Applied and Environmental Microbiology Jun 2024Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a...
UNLABELLED
Purple sulfur bacteria (PSB) are capable of anoxygenic photosynthesis via oxidizing reduced sulfur compounds and are considered key drivers of the sulfur cycle in a range of anoxic environments. In this study, we show that (a PSB species) is capable of autotrophic growth using pyrite as the electron and sulfur source. Comparative growth profile, substrate characterization, and transcriptomic sequencing data provided valuable insight into the molecular mechanisms underlying the bacterial utilization of pyrite and autotrophic growth. Specifically, the pyrite-supported cell cultures ("py"') demonstrated robust but much slower growth rates and distinct patterns from their sodium sulfide-amended positive controls. Up to ~200-fold upregulation of genes encoding various - and -type cytochromes was observed in "py," pointing to the high relevance of these molecules in scavenging and relaying electrons from pyrite to cytoplasmic metabolisms. Conversely, extensive downregulation of genes related to LH and RC complex components indicates that the electron source may have direct control over the bacterial cells' photosynthetic activity. In terms of sulfur metabolism, genes encoding periplasmic or membrane-bound proteins (e.g., FccAB and SoxYZ) were largely upregulated, whereas those encoding cytoplasmic proteins (e.g., Dsr and Apr groups) are extensively suppressed. Other notable differentially expressed genes are related to flagella/fimbriae/pilin(+), metal efflux(+), ferrienterochelin(-), and [NiFe] hydrogenases(+). Characterization of the biologically reacted pyrite indicates the presence of polymeric sulfur. These results have, for the first time, put the interplay of PSB and transition metal sulfide chemistry under the spotlight, with the potential to advance multiple fields, including metal and sulfur biogeochemistry, bacterial extracellular electron transfer, and artificial photosynthesis.
IMPORTANCE
Microbial utilization of solid-phase substrates constitutes a critical area of focus in environmental microbiology, offering valuable insights into microbial metabolic processes and adaptability. Recent advancements in this field have profoundly deepened our knowledge of microbial physiology pertinent to these scenarios and spurred innovations in biosynthesis and energy production. Furthermore, research into interactions between microbes and solid-phase substrates has directly linked microbial activities to the surrounding mineralogical environments, thereby enhancing our understanding of the relevant biogeochemical cycles. Our study represents a significant step forward in this field by demonstrating, for the first time, the autotrophic growth of purple sulfur bacteria using insoluble pyrite (FeS2) as both the electron and sulfur source. The presented comparative growth profiles, substrate characterizations, and transcriptomic sequencing data shed light on the relationships between electron donor types, photosynthetic reaction center activities, and potential extracellular electron transfer in these organisms capable of anoxygenic photosynthesis. Furthermore, the findings of our study may provide new insights into early-Earth biogeochemical evolutions, offering valuable constraints for understanding the environmental conditions and microbial processes that shaped our planet's history.
PubMed: 38899885
DOI: 10.1128/aem.00863-24