Complications arising from antibiotic-resistant bacteria are becoming one of the key issues in modern medicine. Members of drug-resistant Enterobacteriaceae spp. include opportunistic pathogens (e.g., Salmonella spp.) that are among the leading causes of morbidity and mortality worldwide. Overgrowth of these bacteria is considered a hallmark of intestinal dysbiosis. Microcins (small antimicrobial peptides) produced by some gut commensals can potentially cure these conditions by inhibiting these pathogens and have been proposed as a viable alternative to antibiotic treatment. In this proof-of-concept work, we leverage this idea to develop a genetically engineered prototype probiotic to inhibit Salmonella spp. upon exposure to tetrathionate, a molecule produced in the inflamed gut during the course of Salmonella infection. We developed a plasmid-based system capable of conferring the ability to detect and utilize tetrathionate, while at the same time producing microcin H47. We transferred this plasmid-based system to Escherichia coli and demonstrated the ability of the engineered strain to inhibit growth of Salmonella in anaerobic conditions while in the presence of tetrathionate, with no detectable inhibition in the absence of tetrathionate. In direct competition assays between the engineered E. coli and Salmonella, the engineered E. coli had a considerable increase in fitness advantage in the presence of 1 mM tetrathionate as compared to the absence of tetrathionate. In this work, we have demonstrated the ability to engineer a strain of E. coli capable of using an environmental signal indicative of intestinal inflammation as an inducing molecule, resulting in production of a microcin capable of inhibiting the organism responsible for the inflammation.

Microcin H47 (MccH47) is an antimicrobial peptide produced by some strains of Escherichia coli that has demonstrated inhibitory activity against enteric pathogens in vivo and has been heterologously overexpressed in proof-of-concept engineered probiotic applications. While most studies clearly demonstrate inhibitory activity against E. coli isolates, there are conflicting results on the qualitative capacity for MccH47 to inhibit strains of Salmonella. Here, we rectify these inconsistencies via the overexpression and purification of a form of MccH47, termed MccH47-monoglycosylated enterobactin (MccH47−MGE). We then use purified MccH47 to estimate minimum inhibitory concentrations (MICs) against a number of medically relevant Enterobacteriaceae, including Salmonella and numerous multidrug resistant (MDR) strains. While previous reports suggested that the spectrum of activity for MccH47 is quite narrow and restricted to activity against E. coli, our data demonstrate that MccH47 has broad and potent activity within the Enterobacteriaceae family, suggesting it as a candidate for further development toward treating MDR enteric infections.

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile . Some of them are trans -encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile . Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans -encoded RNAs, new categories of Hfq ligands were found including cis -antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile . Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.

The ability of pathogenic bacteria to stably infect the host depends on their capacity to respond and adapt to the host environment and on the efficiency of their defensive mechanisms. Bacterial envelope provides a physical barrier protecting against environmental threats. It also constitutes an important sensory interface where numerous sensing systems are located. Signal transduction systems include Two-Component Systems (TCSs) and alternative sigma factors. These systems are able to sense and respond to the ever-changing environment inside the host, altering the bacterial transcriptome to mitigate the impact of the stress. The regulatory networks associated with signal transduction systems comprise small regulatory RNAs (sRNAs) that can be directly involved in the expression of virulence factors. The aim of this review is to describe the importance of TCS- and alternative sigma factor-associated sRNAs in human pathogens during infection. The currently available genome-wide approaches for studies of TCS-regulated sRNAs will be discussed. The differences in the signal transduction mediated by TCSs between bacteria and higher eukaryotes and the specificity of regulatory RNAs for their targets make them appealing targets for discovery of new strategies to fight against multi-resistant bacteria.

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found including cis-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.