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Frontiers in Cellular and Infection... 2023Bacterial keratitis (bacterial infection of the cornea) is a major cause of vision loss worldwide. Given the rapid and aggressive nature of the disease, immediate... (Review)
Review
Bacterial keratitis (bacterial infection of the cornea) is a major cause of vision loss worldwide. Given the rapid and aggressive nature of the disease, immediate broad-spectrum antibiotics are essential to adequately treat this disease. However, rising antibiotic resistance continues to accelerate, rendering many commonly used therapeutics increasingly ineffective. As such, there is a significant effort to understand the basic pathogenesis of common causative organisms implicated in keratitis in part, to fuel the development of novel therapies to treat this blinding disease. This review explores two common causes of bacterial keratitis, and , with regards to the bacterial mediators of virulence as well as novel therapies on the horizon.
Topics: Humans; Staphylococcus aureus; Pseudomonas aeruginosa; Staphylococcal Infections; Keratitis; Pseudomonas Infections; Eye Infections, Bacterial
PubMed: 37671149
DOI: 10.3389/fcimb.2023.1250257 -
Frontiers in Cellular and Infection... 2023data indicate that mouse corneas exposed to PM showed early perforation and thinning after infection with . To understand the mechanisms underlying this finding, we...
PURPOSE
data indicate that mouse corneas exposed to PM showed early perforation and thinning after infection with . To understand the mechanisms underlying this finding, we tested the effects of PM and the mitochondria targeted anti-oxidant SKQ1 in immortalized human corneal epithelial cells (HCET) that were challenged with strain 19660.
METHODS
Mouse corneas were infected with strain 19660 after a 2 week whole-body exposure to PM or control air and assessed by clinical scores, slit lamp photography and western blot. HCET were exposed to 100μg/ml PM for 24h before challenge with strain 19660 (MOI 20). A subset of cells were pre-treated with 50nM SKQ1 for 1h before PM exposure. Phase contrast microscopy was used to study cell morphology, cell viability was measured by an MTT assay, and ROS by DCFH-DA. Levels of pro-inflammatory markers and anti-oxidant enzymes were evaluated by RT-PCR, western blot and ELISA. Reduced glutathione (GSH) and malondialdehyde (MDA) levels were evaluated by assay kits.
RESULTS
, whole body exposure to PM vs. control air exposed mouse corneas showed early perforation and/or corneal thinning at 3 days post infection, accompanied by increased TNF-α and decreased SOD2 protein levels. , PM induced a dose dependent reduction in cell viability of HCET and significantly increased mRNA levels of pro-inflammatory molecules compared to control. Exposure to PM before bacterial challenge further amplified the reduction in cell viability and GSH levels. Furthermore, PM exposure also exacerbated the increase in MDA and ROS levels and phase contrast microscopy revealed more rounded cells after strain 19660 challenge. PM exposure also further increased the mRNA and protein levels of pro-inflammatory molecules, while anti-inflammatory IL-10 was decreased. SKQ1 reversed the rounded cell morphology observed by phase contrast microscopy, increased levels of MDA, ROS and pro-inflammatory molecules, and restored IL-10.
CONCLUSIONS
PM induces decreased cell viability, oxidative stress and inflammation in HCET and has an additive effect upon bacterial challenge. SKQ1 protects against oxidative stress and inflammation induced by PM after bacterial challenge by reversing these effects. The findings provide insight into mechanisms underlying early perforation and thinning observed in infected corneas of PM exposed mice.
Topics: Humans; Animals; Mice; Epithelium, Corneal; Interleukin-10; Pseudomonas aeruginosa; Antioxidants; Reactive Oxygen Species; Inflammation; RNA, Messenger; Pseudomonas Infections; Mice, Inbred C57BL
PubMed: 37868351
DOI: 10.3389/fcimb.2023.1240903 -
Applied and Environmental Microbiology Jun 2023Pseudomonas aeruginosa possesses three type VI secretion systems (T6SSs) that are involved in interspecies competition, internalization into epithelial cells, and...
Pseudomonas aeruginosa possesses three type VI secretion systems (T6SSs) that are involved in interspecies competition, internalization into epithelial cells, and virulence. Host-derived mucin glycans regulate the T6SSs through RetS, and attacks from other species activate the H1-T6SS. However, other environmental signals that control the T6SSs remain to be explored. Previously, we determined PitA to be a constitutive phosphate transporter, whose mutation reduces the intracellular phosphate concentration. Here, we demonstrate that mutation in the gene increases the expression of the H2- and H3-T6SS genes and enhances bacterial uptake by A549 cells. We further found that mutation of results in activation of the quorum sensing (QS) systems, which contributes to the upregulation of the H2- and H3-T6SS genes. Overexpression of the phosphate transporter complex genes or knockdown of the phosphate starvation response regulator gene in the Δ mutant reduces the expression of the QS genes and subsequently the H2- and H3-T6SS genes and bacterial internalization. Furthermore, growth of wild-type PA14 in a low-phosphate medium results in upregulation of the QS and H2- and H3-T6SS genes and bacterial internalization compared to those in cells grown in a high-phosphate medium. Deletion of the gene abolished the differences in the expression of the QS and T6SS genes as well as bacterial internalization in the low- and high- phosphate media. Overall, our results elucidate the mechanism of PitA-mediated regulation on the QS system and H2- and H3-T6SSs and reveal a novel pathway that regulates the T6SSs in response to phosphate starvation. Pseudomonas aeruginosa is an opportunistic pathogenic bacterium that causes acute and chronic infections in humans. The type VI secretion systems (T6SSs) have been shown to associate with chronic infections. Understanding the mechanism used by the bacteria to sense environmental signals and regulate virulence factors will provide clues for developing novel effective treatment strategies. Here, we demonstrate a relationship between a phosphate transporter and the T6SSs and reveal a novel regulatory pathway that senses phosphate limitation and controls bacterial virulence factors in P. aeruginosa.
Topics: Humans; Type VI Secretion Systems; Pseudomonas aeruginosa; Persistent Infection; Virulence Factors; Quorum Sensing; Phosphates; Bacterial Proteins; Gene Expression Regulation, Bacterial
PubMed: 37184394
DOI: 10.1128/aem.02094-22 -
Advanced Science (Weinheim,... Sep 2023Hyperinflammation elicited by lipopolysaccharide (LPS) that derives from multidrug-resistant Gram-negative pathogens, leads to a sharp increase in mortality globally....
Hyperinflammation elicited by lipopolysaccharide (LPS) that derives from multidrug-resistant Gram-negative pathogens, leads to a sharp increase in mortality globally. However, monotherapies aiming to neutralize LPS often fail to improve the prognosis. Here, an all-in-one drug delivery strategy equipped with bactericidal activity, LPS neutralization, and detoxification is shown to recognize, kill pathogens, and attenuate hyperinflammation by abolishing the activation of LPS-triggered acute inflammatory responses. First, bactericidal colistin results in rapid bacterial killing, and the released LPS is subsequently sequestered. The neutralized LPS is further cleared by acyloxyacyl hydrolase to remove secondary fatty chains and detoxify LPS in situ. Last, such a system shows high efficacy in two mouse infection models challenged with Pseudomonas aeruginosa. This approach integrates direct antibacterial activity with in situ LPS neutralizing and detoxifying properties, shedding light on the development of alternative interventions to treat sepsis-associated infections.
Topics: Animals; Mice; Lipopolysaccharides; Anti-Bacterial Agents; Colistin; Bacteria; Pseudomonas aeruginosa
PubMed: 37428467
DOI: 10.1002/advs.202302950 -
Nature Communications Sep 2023The bacterial Tight adherence Secretion System (TadSS) assembles surface pili that drive cell adherence, biofilm formation and bacterial predation. The structure and...
The bacterial Tight adherence Secretion System (TadSS) assembles surface pili that drive cell adherence, biofilm formation and bacterial predation. The structure and mechanism of the TadSS is mostly unknown. This includes characterisation of the outer membrane secretin through which the pilus is channelled and recruitment of its pilotin. Here we investigate RcpA and TadD lipoprotein from Pseudomonas aeruginosa. Light microscopy reveals RcpA colocalising with TadD in P. aeruginosa and when heterologously expressed in Escherichia coli. We use cryogenic electron microscopy to determine how RcpA and TadD assemble a secretin channel with C13 and C14 symmetries. Despite low sequence homology, we show that TadD shares a similar fold to the type 4 pilus system pilotin PilF. We establish that the C-terminal four residues of RcpA bind TadD - an interaction essential for secretin formation. The binding mechanism between RcpA and TadD appears distinct from known secretin-pilotin pairings in other secretion systems.
Topics: Secretin; Gastrointestinal Hormones; Bacterial Secretion Systems; Cell Aggregation; Escherichia coli; Pseudomonas aeruginosa
PubMed: 37704603
DOI: 10.1038/s41467-023-41200-1 -
Proceedings of the National Academy of... Jul 2023(PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a...
(PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.
Topics: Humans; Mice; Animals; Mixed Function Oxygenases; Pseudomonas aeruginosa; Polysaccharides; Immunization; Pneumonia
PubMed: 37459522
DOI: 10.1073/pnas.2301538120 -
Frontiers in Cellular and Infection... 2023Biofilm is a structured community of bacteria encased within a self-produced extracellular matrix. When bacteria form biofilms, they undergo a phenotypic shift that...
Biofilm is a structured community of bacteria encased within a self-produced extracellular matrix. When bacteria form biofilms, they undergo a phenotypic shift that enhances their resistance to antimicrobial agents. Consequently, inducing the transition of biofilm bacteria to the planktonic state may offer a viable approach for addressing infections associated with biofilms. Our previous study has shown that the mouse antimicrobial peptide CRAMP-34 can disperse () biofilm, and the potential mechanism of CRAMP-34 eradicate biofilms was also investigated by combined omics. However, changes in bacterial extracellular metabolism have not been identified. To further explore the mechanism by which CRAMP-34 disperses biofilm, this study analyzed its effects on the extracellular metabolites of biofilm cells via metabolomics. The results demonstrated that a total of 258 significantly different metabolites were detected in the untargeted metabolomics, of which 73 were downregulated and 185 were upregulated. Pathway enrichment analysis of differential metabolites revealed that metabolic pathways are mainly related to the biosynthesis and metabolism of amino acids, and it also suggested that CRAMP-34 may alter the sensitivity of biofilm bacteria to antibiotics. Subsequently, it was confirmed that the combination of CRAMP-34 with vancomycin and colistin had a synergistic effect on dispersed cells. These results, along with our previous findings, suggest that CRAMP-34 may promote the transition of PAO1 bacteria from the biofilm state to the planktonic state by upregulating the extracellular glutamate and succinate metabolism and eventually leading to the dispersal of biofilm. In addition, increased extracellular metabolites of myoinositol, palmitic acid and oleic acid may enhance the susceptibility of the dispersed bacteria to the antibiotics colistin and vancomycin. CRAMP-34 also delayed the development of bacterial resistance to colistin and ciprofloxacin. These results suggest the promising development of CRAMP-34 in combination with antibiotics as a potential candidate to provide a novel therapeutic approach for the prevention and treatment of biofilm-associated infections.
Topics: Animals; Mice; Pseudomonas aeruginosa; Vancomycin; Colistin; Anti-Bacterial Agents; Biofilms; Pseudomonas Infections; Microbial Sensitivity Tests
PubMed: 38162583
DOI: 10.3389/fcimb.2023.1295311 -
MBio Oct 2023Chemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play...
Chemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play an important role in infection. These multiple functions largely depend on ligand specificities of chemoreceptors, and the number and identities of chemoreceptors show high diversity between organisms. Similar diversity is observed for the spectra of chemoeffectors, which include not only chemicals of high metabolic value but also bacterial, plant, and animal signaling molecules. However, the systematic identification of chemoeffectors and their mapping to specific chemoreceptors remains a challenge. Here, we combined several and approaches to establish a systematic screening strategy for the identification of receptor ligands and we applied it to identify a number of new physiologically relevant chemoeffectors for the important opportunistic human pathogen . This strategy can be equally applicable to map specificities of sensory domains from a wide variety of receptor types and bacteria.
Topics: Animals; Humans; Pseudomonas aeruginosa; Bacterial Proteins; Chemoreceptor Cells; Chemotaxis; Bacteria
PubMed: 37791891
DOI: 10.1128/mbio.02099-23 -
Microbial Genomics Oct 2023Catheter-associated urinary tract infections (CAUTIs) represent one of the major healthcare-associated infections, and is a common Gram-negative bacterium associated...
Catheter-associated urinary tract infections (CAUTIs) represent one of the major healthcare-associated infections, and is a common Gram-negative bacterium associated with catheter infections in Egyptian clinical settings. The present study describes the phenotypic and genotypic characteristics of 31 . isolates recovered from CAUTIs in an Egyptian hospital over a 3 month period. Genomes of isolates were of good quality and were confirmed to be by comparison to the type strain (average nucleotide identity, phylogenetic analysis). Clonal diversity among the isolates was determined; eight different sequence types were found (STs 244, 357, 381, 621, 773, 1430, 1667 and 3765), of which ST357 and ST773 are considered to be high-risk clones. Antimicrobial resistance (AMR) testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines showed that the isolates were highly resistant to quinolones [ciprofloxacin (12/31, 38.7 %) and levofloxacin (9/31, 29 %) followed by tobramycin (10/31, 32.5 %)] and cephalosporins (7/31, 22.5 %). Genotypic analysis of resistance determinants predicted all isolates to encode a range of AMR genes, including those conferring resistance to aminoglycosides, β-lactamases, fluoroquinolones, fosfomycin, sulfonamides, tetracyclines and chloramphenicol. One isolate was found to carry a 422 938 bp pBT2436-like megaplasmid encoding , the first report from Egypt of this emerging family of clinically important mobile genetic elements. All isolates were able to form biofilms and were predicted to encode virulence genes associated with adherence, antimicrobial activity, anti-phagocytosis, phospholipase enzymes, iron uptake, proteases, secretion systems and toxins. The present study shows how phenotypic analysis alongside genomic analysis may help us understand the AMR and virulence profiles of contributing to CAUTIs in Egypt.
Topics: Humans; Pseudomonas aeruginosa; Egypt; Phylogeny; Genomics; Anti-Bacterial Agents; Urinary Tract Infections; Catheters
PubMed: 37902186
DOI: 10.1099/mgen.0.001125 -
Journal of Bacteriology Aug 2023Pseudomonas aeruginosa is an opportunistic pathogen heavily implicated in chronic diseases. Immunocompromised patients that become infected with P. aeruginosa usually... (Review)
Review
Pseudomonas aeruginosa is an opportunistic pathogen heavily implicated in chronic diseases. Immunocompromised patients that become infected with P. aeruginosa usually are afflicted with a lifelong chronic infection, leading to worsened patient outcomes. The complement system is an integral piece of the first line of defense against invading microorganisms. Gram-negative bacteria are thought to be generally susceptible to attack from complement; however, P. aeruginosa can be an exception, with certain strains being serum resistant. Various molecular mechanisms have been described that confer P. aeruginosa unique resistance to numerous aspects of the complement response. In this review, we summarize the current published literature regarding the interactions of P. aeruginosa and complement, as well as the mechanisms used by P. aeruginosa to exploit various complement deficiencies and the strategies used to disrupt or hijack normal complement activities.
Topics: Humans; Pseudomonas aeruginosa; Pseudomonas Infections; Complement System Proteins
PubMed: 37436150
DOI: 10.1128/jb.00018-23