Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than outer membrane. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane. We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the cytoplasmic membrane of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.
Animals have evolved a wide diversity of aggressive behavior often based upon the careful monitoring of other individuals. Bacteria are also capable of aggression, with many species using toxins to kill or inhibit their competitors. Like animals, bacteria also have systems to monitor others during antagonistic encounters, but how this translates into behavior remains poorly understood. Here, we use colonies of Escherichia coli carrying colicin-encoding plasmids as a model for studying antagonistic behavior. We show that in the absence of threat, dispersed cells with low reproductive value produce colicin toxins spontaneously, generating efficient pre-emptive attacks. Cells can also respond conditionally to toxins released by clonemates via autoinduction or other genotypes via competition sensing. The strength of both pre-emptive and responsive attacks varies widely between strains. We demonstrate that this variability occurs easily through mutation by rationally engineering strains to recapitulate the diversity in naturally occurring strategies. Finally, we discover that strains that can detect both competitors and clonemates are capable of massive coordinated attacks on competing colonies. This collective behavior protects established colonies from competitors, mirroring the evolution of alarm calling in the animal world.
Pseudomonas aeruginosa acute and chronic infections are of great concern to human health, especially in hospital settings. It is currently assumed that P. aeruginosa has two antagonistic pathogenic strategies that parallel two different lifestyles; free-living cells are predominantly cytotoxic and induce an acute inflammatory reaction, while biofilm-forming communities cause refractory chronic infections. Recent findings suggest that the planktonic-to-sessile transition is a complex, reversible and overall dynamic differentiation process. Here, we examine how the Gac/Rsm regulatory cascade, a key player in this lifestyle switch, endows P. aeruginosa with both a permissive lifecycle in nature and flexible virulence strategy during infection.
The type VI secretion system (T6SS) is a supramolecular complex involved in the delivery of potent toxins during bacterial competition. Pseudomonas aeruginosa possesses three T6SS gene clusters and several hcp and vgrG gene islands, the latter encoding the spike at the T6SS tip. The vgrG1b cluster encompasses seven genes whose organization and sequences are highly conserved in P. aeruginosa genomes, except for two genes that we called tse7 and tsi7. We show that Tse7 is a Tox-GHH2 domain nuclease which is distinct from other T6SS nucleases identified thus far. Expression of this toxin induces the SOS response, causes growth arrest and ultimately results in DNA degradation. The cytotoxic domain of Tse7 lies at its C terminus, while the N terminus is a predicted PAAR domain. We find that Tse7 sits on the tip of the VgrG1b spike and that specific residues at the PAAR–VgrG1b interface are essential for VgrG1b-dependent delivery of Tse7 into bacterial prey. We also show that the delivery of Tse7 is dependent on the H1-T6SS cluster, and injection of the nuclease into bacterial competitors is deployed for interbacterial competition. Tsi7, the cognate immunity protein, protects the producer from the deleterious effect of Tse7 through a direct protein–protein interaction so specific that toxin/immunity pairs are effective only if they originate from the same P. aeruginosa isolate. Overall, our study highlights the diversity of T6SS effectors, the exquisite fitting of toxins on the tip of the T6SS, and the specificity in Tsi7-dependent protection, suggesting a role in interstrain competition.
Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile mcr genes, necessitates the development of improved diagnostics for the detection of colistin-resistant organisms in hospital settings.
The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.
SignificanceBacteria live in dense environments where competition for space and resources is fierce. For this reason, they often use diffusible toxins to eliminate closely related strains. Some toxins trigger systematic retaliation, raising the question of the role of provocation in bacterial warfare. We combine mathematical modeling and experiments to study the costs and benefits of provocation. In one-to-one encounters, provocation is costly as it leads to strong counterattacks. However, with three or more strains present, provocation can provide benefits via a “divide-and-conquer” effect, whereby a strain forces its opponents to wipe each other out. This effect could be harnessed as a targeted antibacterial approach; adding low levels of certain antibiotics to communities can promote warfare and cross-elimination between strains.
Bacterial growth and pathogenicity depend on the correct formation of disulfide bonds, a process controlled by the Dsb system in the periplasm of Gram-negative bacteria. Proteins with a thioredoxin fold play a central role in this process. A general feature of thiol-disulfide exchange reactions is the need to avoid a long lived product complex between protein partners. We use a multidisciplinary approach, involving NMR, x-ray crystallography, surface plasmon resonance, mutagenesis, and in vivo experiments, to investigate the interaction between the two soluble domains of the transmembrane reductant conductor DsbD. Our results show oxidation state-dependent affinities between these two domains. These observations have implications for the interactions of the ubiquitous thioredoxin-like proteins with their substrates, provide insight into the key role played by a unique redox partner with an immunoglobulin fold, and are of general importance for oxidative protein-folding pathways in all organisms.
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