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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 -
The Journal of Biological Chemistry Mar 2024The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when... (Review)
Review
The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when homeostasis is challenged during host-pathogen interactions. In particular, the presence of free radical groups and excess copper in the periplasm causes noxious reactions, such as sulfhydryl group oxidation leading to enzymatic inactivation and protein denaturation. In response to this, canonical and accessory oxidoreductase systems are induced, performing quality control of thiol groups, and therefore contributing to restoring homeostasis and preserving survival under these conditions. Here, we examine recent advances in the characterization of the Dsb-like, Salmonella-specific Scs system. This system includes the ScsC/ScsB pair of Cu-binding proteins with thiol-oxidoreductase activity, an alternative ScsB-partner, the membrane-linked ScsD, and a likely associated protein, ScsA, with a role in peroxide resistance. We discuss the acquisition of the scsABCD locus and its integration into a global regulatory pathway directing envelope response to Cu stress during the evolution of pathogens that also harbor the canonical Dsb systems. The evidence suggests that the canonical Dsb systems cannot satisfy the extra demands that the host-pathogen interface imposes to preserve functional thiol groups. This resulted in the acquisition of the Scs system by Salmonella. We propose that the ScsABCD complex evolved to connect Cu and redox stress responses in this pathogen as well as in other bacterial pathogens.
Topics: Bacterial Proteins; Copper; Homeostasis; Oxidation-Reduction; Oxidoreductases; Salmonella; Sulfhydryl Compounds; Carrier Proteins
PubMed: 38309504
DOI: 10.1016/j.jbc.2024.105710 -
FEMS Microbiology Ecology Feb 2024Himalayan glaciers are receding at an exceptional rate, perturbing the local biome and ecosystem processes. Understanding the microbial ecology of an exclusively...
Himalayan glaciers are receding at an exceptional rate, perturbing the local biome and ecosystem processes. Understanding the microbial ecology of an exclusively microbe-driven biome provides insights into their contributions to the ecosystem functioning through biogeochemical fluxes. Here, we investigated the bacterial communities and their functional potential in the retreating East Rathong Glacier (ERG) of Sikkim Himalaya. Amplicon-based taxonomic classification revealed the dominance of the phyla Proteobacteria, Bacteroidota, and candidate Patescibacteria in the glacial sites. Further, eight good-quality metagenome-assembled genomes (MAGs) of Proteobacteria, Patescibacteria, Acidobacteriota, and Choloflexota retrieved from the metagenomes elucidated the microbial contributions to nutrient cycling. The ERG MAGs showed aerobic respiration as a primary metabolic feature, accompanied by carbon fixation and complex carbon degradation potentials. Pathways for nitrogen metabolism, chiefly dissimilatory nitrate reduction and denitrification, and a complete sulphur oxidation enzyme complex for sulphur metabolism were identified in the MAGs. We observed that DNA repair and oxidative stress response genes complemented with osmotic and periplasmic stress and protein chaperones were vital for adaptation against the intense radiation and stress conditions of the extreme Himalayan niche. Current findings elucidate the microbiome and associated functional potentials of a vulnerable glacier, emphasizing their significant ecological roles in a changing glacial ecosystem.
Topics: Ecosystem; Ice Cover; Metagenomics; Bacteria; Metagenome; Microbiota; Proteobacteria; Sulfur
PubMed: 38305149
DOI: 10.1093/femsec/fiae012 -
Microbiology Spectrum Mar 2024Tuberculosis, a lung disease caused by (), remains a major global health problem ranking as the second leading cause of death from a single infectious agent. One of the...
UNLABELLED
Tuberculosis, a lung disease caused by (), remains a major global health problem ranking as the second leading cause of death from a single infectious agent. One of the major factors contributing toward success as a pathogen is its unique cell wall and its ability to counteract various arms of the host's immune response. A recent genome-scale study profiled a list of candidate genes that are predicted to be essential for survival of host-mediated responses. One candidate was FtsEX, a protein complex composed of an ATP-binding domain, FtsE, and a transmembrane domain, FtsX. FtsEX functions through interaction with a periplasmic hydrolase, RipC. Homologs of FtsEX exist in other bacteria and have been linked with playing a key role in regulating peptidoglycan hydrolysis during cell elongation and division. Here, we report on FtsE, FtsX, and RipC and their protective roles in stressful conditions. We demonstrate that the individual genes of FtsEX complex and RipC are not essential for survival in normal growth conditions but conditionally essential in low-salt media and antibiotic-treated media. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. Our results suggest that FtsE, FtsX, and RipC are required for both normal cell elongation and division and ultimately for survival in stressful conditions.
IMPORTANCE
Mycobacterial cell growth and division are coordinated with regulated peptidoglycan hydrolysis. Understanding cell wall gene complexes that govern normal cell division and elongation will aid in the development of tools to disarm the ability of mycobacteria to survive immune-like and antibiotic stresses. We combined genetic analyses and scanning electron microscopy to analyze morphological changes of mycobacterial FtsEX and RipC mutants in stressful conditions. We demonstrate that FtsE, FtsX, FtsEX, and RipC are conditionally required for the survival of during rifampicin treatment and in low-salt conditions. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. We also show that the FtsEX-RipC interaction is essential for the survival of in rifampicin. Our results suggest that FtsE, FtsX, and RipC are required for normal cell wall regulation and ultimately for survival in stressful conditions.
Topics: Cell Cycle Proteins; Bacterial Proteins; Rifampin; Peptidoglycan; Mycobacterium smegmatis; Osmolar Concentration; Anti-Bacterial Agents
PubMed: 38289931
DOI: 10.1128/spectrum.02515-23 -
Antibiotics (Basel, Switzerland) Dec 2023Multidrug efflux transporters are major contributors to the antibiotic resistance of in clinical settings. Previous studies showed that these transporters are tightly...
Multidrug efflux transporters are major contributors to the antibiotic resistance of in clinical settings. Previous studies showed that these transporters are tightly integrated into the physiology of and have diverse functions. However, for many of the efflux pumps, such functions remain poorly defined. In this study, we characterized two putative drug efflux pumps, AmfAB and AmfCD (Acinetobacter Major Facilitator), that are homologous to EmrAB-like transporters from and other Gram-negative bacteria. These pumps comprise the Major Facilitator Superfamily (MFS) transporters AmfB and AmfD and the periplasmic membrane fusion proteins AmfA and AmfC, respectively. We inactivated and overproduced these pumps in the wild-type ATCC 17978 strain and its derivative strains lacking the major efflux pumps from the Resistance-Nodulation-Division (RND) superfamily and characterized antibiotic susceptibilities and growth of the strains under stresses typical during human infections. We found that neither AmfAB nor AmfCD contribute to the antibiotic non-susceptibility phenotypes of The two pumps, however, are critical for the adaptation and growth of the bacterium under acidic stress, whereas AmfCD also contributes to growth under conditions of low iron, high temperature, and in the presence of bile salts. These functions are dependent on the presence of the RND pumps, the inactivation of which further diminishes survival and growth. Our results suggest that MFS transporters contribute to stress survival by affecting the permeability properties of the cell envelope.
PubMed: 38275317
DOI: 10.3390/antibiotics13010007 -
Frontiers in Molecular Biosciences 2023is a bacterium that colonizes the gastric epithelium, which affects millions of people worldwide. infection can lead to various gastrointestinal diseases, including...
is a bacterium that colonizes the gastric epithelium, which affects millions of people worldwide. infection can lead to various gastrointestinal diseases, including gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Conventional antibiotic therapies face challenges due to increasing antibiotic resistance and patient non-compliance, necessitating the exploration of alternative treatment approaches. In this study, we focused on Hp0231 (DsbK), an essential component of the Dsb (disulfide bond) oxidative pathway, and investigated peptide-based inhibition as a potential therapeutic strategy. Three inhibitory peptides designed by computational modeling were evaluated for their effectiveness using a time-resolved fluorescence assay. We also examined the binding affinity between Hp0231 and the peptides using microscale thermophoresis. Our findings demonstrate that -designed synthetic peptides can effectively inhibit Hp0231-mediated peptide oxidation. Targeting Hp0231 oxidase activity could attenuate virulence without compromising bacterial viability. Therefore, peptide-based inhibitors of Hp0231 could be candidates for the development of new targeted strategy, which does not influence the composition of the natural human microbiome, but deprive the bacterium of its pathogenic properties.
PubMed: 38274095
DOI: 10.3389/fmolb.2023.1335704 -
Molecular & Cellular Proteomics : MCP Mar 2024We propose a pipeline that combines AlphaFold2 (AF2) and crosslinking mass spectrometry (XL-MS) to model the structure of proteins with multiple conformations. The...
We propose a pipeline that combines AlphaFold2 (AF2) and crosslinking mass spectrometry (XL-MS) to model the structure of proteins with multiple conformations. The pipeline consists of two main steps: ensemble generation using AF2 and conformer selection using XL-MS data. For conformer selection, we developed two scores-the monolink probability score (MP) and the crosslink probability score (XLP)-both of which are based on residue depth from the protein surface. We benchmarked MP and XLP on a large dataset of decoy protein structures and showed that our scores outperform previously developed scores. We then tested our methodology on three proteins having an open and closed conformation in the Protein Data Bank: Complement component 3 (C3), luciferase, and glutamine-binding periplasmic protein, first generating ensembles using AF2, which were then screened for the open and closed conformations using experimental XL-MS data. In five out of six cases, the most accurate model within the AF2 ensembles-or a conformation within 1 Å of this model-was identified using crosslinks, as assessed through the XLP score. In the remaining case, only the monolinks (assessed through the MP score) successfully identified the open conformation of glutamine-binding periplasmic protein, and these results were further improved by including the "occupancy" of the monolinks. This serves as a compelling proof-of-concept for the effectiveness of monolinks. In contrast, the AF2 assessment score was only able to identify the most accurate conformation in two out of six cases. Our results highlight the complementarity of AF2 with experimental methods like XL-MS, with the MP and XLP scores providing reliable metrics to assess the quality of the predicted models. The MP and XLP scoring functions mentioned above are available at https://gitlab.com/topf-lab/xlms-tools.
Topics: Glutamine; Furylfuramide; Periplasmic Proteins; Mass Spectrometry; Protein Conformation; Membrane Proteins
PubMed: 38266916
DOI: 10.1016/j.mcpro.2024.100724 -
Frontiers in Cellular and Infection... 2023is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and...
is a World Health Organization priority pathogen and a significant clinical concern for infections of the respiratory and urinary tracts due to widespread and increasing resistance to antimicrobials. In the absence of a vaccine, there is an urgent need to identify novel targets for therapeutic development. Bacterial pathogens, including , require the -block metal ion zinc as an essential micronutrient, which serves as a cofactor for ~6% of the proteome. During infection, zinc acquisition necessitates the use of high affinity uptake systems to overcome niche-specific zinc limitation and host-mediated nutritional immunity. Here, we report the identification of ZnuCBA and ZniCBA, two ATP-binding cassette permeases that are highly conserved in species and contribute to AJ218 zinc homeostasis, and the high-resolution structure of the zinc-recruiting solute-binding protein ZniA. The Znu and Zni permeases appear functionally redundant with abrogation of both systems required to reduce zinc accumulation. Disruption of both systems also exerted pleiotropic effects on the homeostasis of other -block elements. Zinc limitation perturbed cell morphology and compromised resistance to stressors, such as salt and oxidative stress. The mutant strain lacking both systems showed significantly impaired virulence in acute lung infection models, highlighting the necessity of zinc acquisition in the virulence and pathogenicity of .
Topics: Klebsiella pneumoniae; Zinc; Virulence; Klebsiella; Membrane Transport Proteins
PubMed: 38249299
DOI: 10.3389/fcimb.2023.1322973 -
Antibiotics (Basel, Switzerland) Jan 2024Gram-negative bacteria are intrinsically more resistant to many frontline antibiotics, which is attributed to the permeability barrier of the outer membrane, drug efflux...
Gram-negative bacteria are intrinsically more resistant to many frontline antibiotics, which is attributed to the permeability barrier of the outer membrane, drug efflux pumps and porins. Consequently, discovery of new small molecules antibiotics to kill drug-resistant Gram-negative bacteria presents a significant challenge. Thanatin, a 21-residue insect-derived antimicrobial peptide, is known for its potent activity against Enterobacter Gram-negative bacteria, including drug-resistant strains. Here, we investigated a 15-residue N-terminal truncated analog PM15 (PIIYCNRRTGKCQRM) of thanatin to determine modes of action and antibacterial activity. PM15 and the P to Y and A substituted variants PM15Y and PM15A delineated interactions and permeabilization of the LPS-outer membrane. In antibacterial assays, PM15 and the analogs showed growth inhibition of strains of Gram-negative bacteria that is largely dependent on the composition of the culture media. Atomic-resolution structures of PM15 and PM15Y in free solution and in complex with LPS micelle exhibited persistent β-hairpin structures similar to native thanatin. However, in complex with LPS, the structures of peptides are more compact, with extensive packing interactions among residues across the two anti-parallel strands of the β-hairpin. The docked complex of PM15/LPS revealed a parallel orientation of the peptide that may be sustained by potential ionic and van der Waals interactions with the lipid A moiety of LPS. Further, PM15 and PM15Y bind to LptA, a monomeric functional variant of LptA, the periplasmic component of the seven-protein (A-G) complex involved in LPS transport. Taken together, the structures, target interactions and antibacterial effect of PM15 presented in the current study could be useful in designing thanatin-based peptide analogs.
PubMed: 38247633
DOI: 10.3390/antibiotics13010074 -
BMC Microbiology Jan 2024The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial genomes, including many disease-causing species. Here we have...
BACKGROUND
The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial genomes, including many disease-causing species. Here we have constructed a genomic Lig E knock-out in the obligate human pathogen Neisseria gonorrhoeae and characterised its growth and infection phenotype.
RESULTS
This demonstrates that N. gonorrhoeae Lig E is a non-essential gene and its deletion does not cause defects in replication or survival of DNA-damaging stressors. Knock-out strains were partially defective in biofilm formation on an artificial surface as well as adhesion to epithelial cells. In addition to in vivo characterisation, we have recombinantly expressed and assayed N. gonorrhoeae Lig E and determined the crystal structure of the enzyme-adenylate engaged with DNA substrate in an open non-catalytic conformation.
CONCLUSIONS
These findings, coupled with the predicted extracellular/ periplasmic location of Lig E indicates a role in extracellular DNA joining as well as providing insight into the binding dynamics of these minimal DNA ligases.
Topics: Humans; DNA Ligase ATP; Neisseria gonorrhoeae; DNA Ligases; DNA; Biofilms
PubMed: 38245708
DOI: 10.1186/s12866-024-03193-9