Global Mapping of Small RNA-Target Interactions in Bacteria.

Small RNAs (sRNAs) associated with the RNA chaperon protein Hfq are key posttranscriptional regulators of gene expression in bacteria. Deciphering the sRNA-target interactome is an essential step toward understanding the roles of sRNAs in the cellular networks. We developed a broadly applicable methodology termed RIL-seq (RNA interaction by ligation and sequencing), which integrates experimental and computational tools for in vivo transcriptome-wide identification of interactions involving Hfq-associated sRNAs. By applying this methodology to Escherichia coli we discovered an extensive network of interactions involving RNA pairs showing sequence complementarity. We expand the ensemble of targets for known sRNAs, uncover additional Hfq-bound sRNAs encoded in various genomic regions along with their trans encoded targets, and provide insights into binding and possible cycling of RNAs on Hfq. Comparison of the sRNA interactome under various conditions has revealed changes in the sRNA repertoire as well as substantial re-wiring of the network between conditions.

Common Adaptive Strategies Underlie Within-Host Evolution of Bacterial Pathogens.

Within-host adaptation is a hallmark of chronic bacterial infections, involving substantial genomic changes. Recent large-scale genomic data from prolonged infections allow the examination of adaptive strategies employed by different pathogens and open the door to investigate whether they converge toward similar strategies. Here, we compiled extensive data of whole-genome sequences of bacterial isolates belonging to miscellaneous species sampled at sequential time points during clinical infections. Analysis of these data revealed that different species share some common adaptive strategies, achieved by mutating various genes. Although the same genes were often mutated in several strains within a species, different genes related to the same pathway, structure, or function were changed in other species utilizing the same adaptive strategy (e.g., mutating flagellar genes). Strategies exploited by various bacterial species were often predicted to be driven by the host immune system, a powerful selective pressure that is not species specific. Remarkably, we find adaptive strategies identified previously within single species to be ubiquitous. Two striking examples are shifts from siderophore-based to heme-based iron scavenging (previously shown for Pseudomonas aeruginosa) and changes in glycerol-phosphate metabolism (previously shown to decrease sensitivity to antibiotics in Mycobacterium tuberculosis). Virulence factors were often adaptively affected in different species, indicating shifts from acute to chronic virulence and virulence attenuation during infection. Our study presents a global view on common within-host adaptive strategies employed by different bacterial species and provides a rich resource for further studying these processes.

Convergent Within-Host Adaptation of Pseudomonas aeruginosa through the Transcriptional Regulatory Network.

Bacteria adapt to their host by mutating specific genes and by reprogramming their gene expression. Different strains of a bacterial species often mutate the same genes during infection, demonstrating convergent genetic adaptation. However, there is limited evidence for convergent adaptation at the transcriptional level. To this end, we utilize genomic data of 114 Pseudomonas aeruginosa strains, derived from patients with chronic pulmonary infection, and the P. aeruginosa transcriptional regulatory network. Relying on loss-of-function mutations in genes encoding transcriptional regulators and predicting their effects through the network, we demonstrate predicted expression changes of the same genes in different strains through different paths in the network, implying convergent transcriptional adaptation. Furthermore, through the transcription lens we associate yet-unknown processes, such as ethanol oxidation and glycine betaine catabolism, with P. aeruginosa host adaptation. We also find that known adaptive phenotypes, including antibiotic resistance, which were identified before as achieved by specific mutations, are achieved also through transcriptional changes. Our study has revealed novel interplay between the genetic and transcriptional levels in host adaptation, demonstrating the versatility of the adaptive arsenal of bacterial pathogens and their ability to adapt to the host conditions in a myriad of ways. IMPORTANCE Pseudomonas aeruginosa causes significant morbidity and mortality. The pathogen's remarkable ability to establish chronic infections greatly depends on its adaptation to the host environment. Here, we use the transcriptional regulatory network to predict expression changes during adaptation. We expand the processes and functions known to be involved in host adaptation. We show that the pathogen modulates the activity of genes during adaptation, including genes implicated in antibiotic resistance, both directly via genomic mutations and indirectly via mutations in transcriptional regulators. Furthermore, we detect a subgroup of genes whose predicted changes in expression are associated with mucoid strains, a major adaptive phenotype in chronic infections. We propose that these genes constitute the transcriptional arm of the mucoid adaptive strategy. Identification of different adaptive strategies utilized by pathogens during chronic infection has major promise in the treatment of persistent infections and opens the door to personalized tailored antibiotic treatment in the future.

Prolonged survival of a patient with active MDR-TB HIV co-morbidity: insights from a Mycobacterium tuberculosis strain with a unique genomic deletion.

Coinfection of HIV and multidrug-resistant tuberculosis (MDR-TB) presents significant challenges in terms of the treatment and prognosis of tuberculosis, leading to complexities in managing the disease and impacting the overall outcome for TB patients. This study presents a remarkable case of a patient with MDR-TB and HIV coinfection who survived for over 8 years, despite poor treatment adherence and comorbidities. Whole genome sequencing (WGS) of the infecting Mycobacterium tuberculosis (Mtb) strain revealed a unique genomic deletion, spanning 18 genes, including key genes involved in hypoxia response, intracellular survival, immunodominant antigens, and dormancy. This deletion, that we have called "Del-X," potentially exerts a profound influence on the bacterial physiology and its virulence. Only few similar deletions were detected in other non-related Mtb genomes worldwide. In vivo evolution analysis identified drug resistance and metabolic adaptation mutations and their temporal dynamics during the patient's treatment course.

Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces neutralizing salivary IgA.

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable "sterilizing immunity" at the mucosal level. Our study uncovers a strong temporary neutralizing mucosal component of immunity, emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus-2 spike protein and demonstrate that these IgAs mediate neutralization. RBD-targeting IgAs were found to associate with the secretory component, indicating their bona fide transcytotic origin and their polymeric multivalent nature. The mechanistic understanding of the high neutralizing activity provided by mucosal IgA, acting at the first line of defense, will advance vaccination design and surveillance principles and may point to novel treatment approaches and new routes of vaccine administration and boosting.

Hierarchy in Hfq Chaperon Occupancy of Small RNA Targets Plays a Major Role in Their Regulation.

Bacterial small RNAs (sRNAs) are posttranscriptional regulators of gene expression that base pair with complementary sequences on target mRNAs, often in association with the chaperone Hfq. Here, using experimentally identified sRNA-target pairs, along with gene expression measurements, we assess basic principles of regulation by sRNAs. We show that the sRNA sequence dictates the target repertoire, as point mutations in the sRNA shift the target set correspondingly. We distinguish two subsets of targets: targets showing changes in expression levels under overexpression of their sRNA regulator and unaffected targets that interact more sporadically with the sRNA. These differences among targets are associated with their Hfq occupancy, rather than with the sRNA-target base-pairing potential. Our results suggest that competition among targets over Hfq binding plays a major role in the regulatory outcome, possibly awarding targets with higher Hfq binding efficiency an advantage in the competition over binding to the sRNA.

A Ubiquitous Platform for Bacterial Nanotube Biogenesis.

We have previously described the existence of membranous nanotubes, bridging adjacent bacteria, facilitating intercellular trafficking of nutrients, cytoplasmic proteins, and even plasmids, yet components enabling their biogenesis remain elusive. Here we reveal the identity of a molecular apparatus providing a platform for nanotube biogenesis. Using Bacillus subtilis (Bs), we demonstrate that conserved components of the flagellar export apparatus (FliO, FliP, FliQ, FliR, FlhB, and FlhA), designated CORE, dually serve for flagellum and nanotube assembly. Mutants lacking CORE genes, but not other flagellar components, are deficient in both nanotube production and the associated intercellular molecular trafficking. In accord, CORE components are located at sites of nanotube emergence. Deleting COREs of distinct species established that CORE-mediated nanotube formation is widespread. Furthermore, exogenous COREs from diverse species could restore nanotube generation and functionality in Bs lacking endogenous CORE. Our results demonstrate that the CORE-derived nanotube is a ubiquitous organelle that facilitates intercellular molecular trade across the bacterial kingdom.