Multiple Gram-negative Pathogens Research Articles

Cross-talk between QseBC and PmrAB two-component systems is crucial for regulation of motility and colistin resistance in Enteropathogenic Escherichia coli.

The quorum sensing two-component system (TCS) QseBC has been linked to virulence, motility and metabolism regulation in multiple Gram-negative pathogens, including Enterohaemorrhagic Escherichia coli (EHEC), Uropathogenic E. coli (UPEC) and Salmonella enterica. In EHEC, the sensor histidine kinase (HK) QseC detects the quorum sensing signalling molecule AI-3 and also acts as an adrenergic sensor binding host epinephrine and norepinephrine. Downstream changes in gene expression are mediated by phosphorylation of its cognate response regulator (RR) QseB, and 'cross-talks' with non-cognate regulators KdpE and QseF to activate motility and virulence. In UPEC, cross-talk between QseBC and TCS PmrAB is crucial in the regulation and phosphorylation of QseB RR that acts as a repressor of multiple pathways, including motility. Here, we investigated QseBC regulation of motility in the atypical Enteropathogenic E. coli (EPEC) strain O125ac:H6, causative agent of persistent diarrhoea in children, and its possible cross-talk with the KdpDE and PmrAB TCS. We showed that in EPEC QseB acts as a repressor of genes involved in motility, virulence and stress response, and in absence of QseC HK, QseB is likely activated by the non-cognate PmrB HK, similarly to UPEC. We show that in absence of QseC, phosphorylated QseB activates its own expression, and is responsible for the low motility phenotypes seen in a QseC deletion mutant. Furthermore, we showed that KdpD HK regulates motility in an independent manner to QseBC and through a third unidentified party different to its own response regulator KdpE. We showed that PmrAB has a role in iron adaptation independent to QseBC. Finally, we showed that QseB is the responsible for activation of colistin and polymyxin B resistance genes while PmrA RR acts by preventing QseB activation of these resistance genes.

Intrinsic Antimicrobial Peptide Facilitates a New Broad-Spectrum Lysin LysP53 to Kill Acinetobacter baumannii In Vitro and in a Mouse Burn Infection Model

Antimicrobial resistance-related infections of Gram-negative pathogens pose a huge threat to global public health. Lysins, peptidoglycan hydrolases from bacteriophages, are expected as an alternative weapon against drug-resistant bacteria. In the present study, we report a new lysin LysP53 from Acinetobacter baumannii phage 53. Bioinformatic analysis revealed that LysP53 contains a positively charged N-terminal region and a putative peptidase catalytic domain. In vitro biochemical experiments showed that LysP53 is active against multiple antibiotic-resistant Gram-negative bacteria, including A. baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli, with a reduction of 5 logs in viable A. baumannii number after exposure to 100 μg/mL LysP53 for 1 h. Further studies showed that LysP53 contains a functional antimicrobial peptide, i.e., N-terminal 33 aa, with a comparable spectrum of activity to LysP53. In an A. baumannii-associated mouse model of burn infection, a single dose of 14 μg/mouse LysP53 (57.6 μM) showed higher decolonization efficacy than 4 μg/mouse minocycline- (874 μM; p < 0.05) and buffer-treated groups (p <0.001), leading to a bacterial reduction of 3 logs. Our findings collectively establish that LysP53 could be a promising candidate in the treatment of topical infections caused by multiple Gram-negative pathogens.

Structure-Activity Studies with Bis-Amidines That Potentiate Gram-Positive Specific Antibiotics against Gram-Negative Pathogens.

Pentamidine, an FDA-approved antiparasitic drug, was recently identified as an outer membrane disrupting synergist that potentiates erythromycin, rifampicin, and novobiocin against Gram-negative bacteria. The same study also described a preliminary structure–activity relationship using commercially available pentamidine analogues. We here report the design, synthesis, and evaluation of a broader panel of bis-amidines inspired by pentamidine. The present study both validates the previously observed synergistic activity reported for pentamidine, while further assessing the capacity for structurally similar bis-amidines to also potentiate Gram-positive specific antibiotics against Gram-negative pathogens. Among the bis-amidines prepared, a number of them were found to exhibit synergistic activity greater than pentamidine. These synergists were shown to effectively potentiate the activity of Gram-positive specific antibiotics against multiple Gram-negative pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli, including polymyxin- and carbapenem-resistant strains.

Identification of Mammalian Proteins That Collaborate with Type III Secretion System Function: Involvement of a Chemokine Receptor in Supporting Translocon Activity

ABSTRACTThe type III secretion system (T3SS) is a highly conserved protein delivery system found in multiple Gram-negative pathogens, including Yersinia pseudotuberculosis. Most studies of Yersinia species type III intoxication of host cells have focused on the bacterial determinants that promote assembly and function of the secretion system. In this study, we performed a pooled RNA interference (RNAi) screen to identify mammalian host proteins required for the cytotoxic effects associated with the Yersinia translocated substrate YopE, a GTPase-activating protein (GAP) that inactivates the small Rho GTPases. Cell populations were positively selected for short hairpin RNAs (shRNAs) that interfere with YopE activity using a combination of fluorescence resonance energy transfer (FRET) and flow cytometry, and the degree of enrichment was determined by deep sequencing. Analysis of the candidates identified by the enrichment process revealed that many were important for the initial step of Y. pseudotuberculosis T3SS function, YopB/D pore formation. These candidates included shRNA that depleted downstream effectors of RhoA signaling, coated pit formation, and receptors involved in cell signaling, including the chemokine receptor CCR5 (chemokine [C-C motif] receptor 5). Depletion of CCR5 in 293T cells yielded a defect in YopB/D pore formation and effector translocation, while both phenotypes could be complemented by overexpression of CCR5 protein. Yop effector translocation was also decreased in isolated primary phagocytic cells from a Ccr5−/− knockout mouse. We postulate that CCR5 acts to promote translocation by modulating cytoskeletal activities necessary for proper assembly of the YopB/D translocation pore. Overall, this study presents a new approach to investigating the contribution of the host cell to T3SS in Y. pseudotuberculosis.

QseC inhibitors as an antivirulence approach for Gram-negative pathogens.

ABSTRACTInvasive pathogens interface with the host and its resident microbiota through interkingdom signaling. The bacterial receptor QseC, which is a membrane-bound histidine sensor kinase, responds to the host stress hormones epinephrine and norepinephrine and the bacterial signal AI-3, integrating interkingdom signaling at the biochemical level. Importantly, the QseC signaling cascade is exploited by many bacterial pathogens to promote virulence. Here, we translated this basic science information into development of a potent small molecule inhibitor of QseC, LED209. Extensive structure activity relationship (SAR) studies revealed that LED209 is a potent prodrug that is highly selective for QseC. Its warhead allosterically modifies lysines in QseC, impairing its function and preventing the activation of the virulence program of several Gram-negative pathogens both in vitro and during murine infection. LED209 does not interfere with pathogen growth, possibly leading to a milder evolutionary pressure toward drug resistance. LED209 has desirable pharmacokinetics and does not present toxicity in vitro and in rodents. This is a unique antivirulence approach, with a proven broad-spectrum activity against multiple Gram-negative pathogens that cause mammalian infections.

A central metabolic circuit controlled by QseC in pathogenic Escherichia coli

The QseC sensor kinase regulates virulence in multiple Gram-negative pathogens, by controlling the activity of the QseB response regulator. We have previously shown that qseC deletion interferes with dephosphorylation of QseB thus unleashing what appears to be an uncontrolled positive feedback loop stimulating increased QseB levels. Deletion of QseC downregulates virulence gene expression and attenuates enterohaemorrhagic and uropathogenic Escherichia coli (EHEC and UPEC), Salmonella typhimurium, and Francisella tularensis. Given that these pathogens employ different infection strategies and virulence factors, we used genome-wide approaches to better understand the role of the QseBC interplay in pathogenesis. We found that deletion of qseC results in misregulation of nucleotide, amino acid, and carbon metabolism. Comparable metabolic changes are seen in EHEC ΔqseC, suggesting that deletion of qseC confers similar pleiotropic effects in these two different pathogens. Disruption of representative metabolic enzymes phenocopied UPEC ΔqseC in vivo and resulted in virulence factor downregulation. We thus propose that in the absence of QseC, the constitutively active QseB leads to pleiotropic effects, impairing bacterial metabolism, and thereby attenuating virulence. These findings provide a basis for the development of antimicrobials targeting the phosphatase activity of QseC, as a means to attenuate a wide range of QseC-bearing pathogens.