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Amidochelocardin overcomes resistance mechanisms exerted on tetracyclines and natural chelocardin
(2020)
The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.
A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens.
IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.
A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually delaying actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.
Comparative genomics provides structural and functional insights into Bacteroides RNA biology
(2022)
Bacteria employ noncoding RNA molecules for a wide range of biological processes, including scaffolding large molecular complexes, catalyzing chemical reactions, defending against phages, and controlling gene expression. Secondary structures, binding partners, and molecular mechanisms have been determined for numerous small noncoding RNAs (sRNAs) in model aerobic bacteria. However, technical hurdles have largely prevented analogous analyses in the anaerobic gut microbiota. While experimental techniques are being developed to investigate the sRNAs of gut commensals, computational tools and comparative genomics can provide immediate functional insight. Here, using Bacteroides thetaiotaomicron as a representative microbiota member, we illustrate how comparative genomics improves our understanding of RNA biology in an understudied gut bacterium. We investigate putative RNA-binding proteins and predict a Bacteroides cold-shock protein homolog to have an RNA-related function. We apply an in silico protocol incorporating both sequence and structural analysis to determine the consensus structures and conservation of nine Bacteroides noncoding RNA families. Using structure probing, we validate and refine these predictions and deposit them in the Rfam database. Through synteny analyses, we illustrate how genomic coconservation can serve as a predictor of sRNA function. Altogether, this work showcases the power of RNA informatics for investigating the RNA biology of anaerobic microbiota members.
Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.