ABSTRACT
Bloodstream infections caused by invasive, non-typhoidal Salmonella (iNTS) are a major global health concern, particularly in Africa where the pathogenic variant of Salmonella Typhimurium sequence type (ST) 313 is dominant. Unlike S. Typhimurium strains that cause gastroenteritis, iNTS strains cause bloodstream infections and are resistant to multiple first-line antibiotics, thus limiting current treatment options. Here, we developed and implemented multiple small molecule screens under physiological, infection-relevant conditions to reveal chemical sensitivities in ST313 and to identify host-directed therapeutics as entry points to drug discovery to combat the clinical burden of iNTS. Screening ST313 iNTS under host-mimicking growth conditions identified 92 compounds with antimicrobial activity despite inherent multidrug resistance. We characterized the antimicrobial activity of the nucleoside analog 3'-azido-3'-deoxythymidine as an exemplary compound from this screen, which depended on bacterial thymidine kinase activity for antimicrobial activity. In a companion macrophage-based screening platform designed to enrich for host-directed therapeutics, we identified three compounds (amodiaquine, berbamine, and indatraline) as actives that required the presence of host cells for antibacterial activity. These three compounds had antimicrobial activity only in the presence of host cells that significantly inhibited intracellular ST313 iNTS replication in macrophages. This work provides evidence that despite high invasiveness and multidrug resistance, ST313 iNTS remains susceptible to unconventional drug discovery approaches.
ABSTRACT
The type three secretion system (TTSS) locus of Aeromonas salmonicida subsp. salmonicida, located on the plasmid pAsa5, is known to be lost when the bacterium is grown at temperatures of 25 °C. The loss of the locus is due to the recombination of the insertion sequences flanking the TTSS region. However, the mechanism involved in this recombination is still elusive. Here, we analyzed 22 A. salmonicida subsp. salmonicida strains that had already lost their TTSS locus, and we systematically explored another 47 strains for their susceptibility to lose the same locus when grown at 25 °C. It appeared that strains from Europe were more prone to lose their TTSS locus compared to Canadian strains. More specifically, it was not possible to induce TTSS loss in Canadian strains that have AsaGEI2a, a genomic island, and prophage 3, or in Canadian strains without a genomic island. A comparative genomic approach revealed an almost perfect correlation between the presence of a cluster of genes, not yet characterized, and the susceptibility of various groups of strains to lose their locus. This cluster of genes encodes putative proteins with DNA binding capacity and phage proteins. This discovery creates new opportunities in the study of pAsa5 thermosensitivity.
ABSTRACT
The Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is an aquatic pathogen which causes furunculosis to salmonids, especially in fish farms. The emergence of strains of this bacterium exhibiting antibiotic resistance is increasing, limiting the effectiveness of antibiotherapy as a treatment against this worldwide disease. In the present study, we discovered an isolate of A. salmonicida subsp. salmonicida that harbors two novel plasmids variants carrying antibiotic resistance genes. The use of long-read sequencing (PacBio) allowed us to fully characterize those variants, named pAsa5-3432 and pRAS3-3432, which both differ from their classic counterpart through their content in mobile genetic elements. The plasmid pAsa5-3432 carries a new multidrug region composed of multiple mobile genetic elements, including a Class 1 integron similar to an integrated element of Salmonella enterica. With this new region, probably acquired through plasmid recombination, pAsa5-3432 is the first reported plasmid of this bacterium that bears both an essential virulence factor (the type three secretion system) and multiple antibiotic resistance genes. As for pRAS3-3432, compared to the classic pRAS3, it carries a new mobile element that has only been identified in Chlamydia suis. Hence, with the identification of those two novel plasmids harboring mobile genetic elements that are normally encountered in other bacterial species, the present study puts emphasis on the important impact of mobile genetic elements in the genomic plasticity of A. salmonicida subsp. salmonicida and suggests that this aquatic bacterium could be an important reservoir of antibiotic resistance genes that can be exchanged with other bacteria, including human and animal pathogens.
Subject(s)
Aeromonas salmonicida/genetics , Drug Resistance, Microbial/genetics , Animals , Genes, Bacterial , Genome, Bacterial , Swine , Virulence Factors/geneticsABSTRACT
BACKGROUND: Amaryllidaceae alkaloids (AAs) are a large group of plant-specialized metabolites displaying an array of biological and pharmacological properties. Previous investigations on AA biosynthesis have revealed that all AAs share a common precursor, norbelladine, presumably synthesized by an enzyme catalyzing a Mannich reaction involving the condensation of tyramine and 3,4-dihydroxybenzaldehyde. Similar reactions have been reported. Specifically, norcoclaurine synthase (NCS) which catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid biosynthesis. RESULTS: With the availability of wild daffodil (Narcissus pseudonarcissus) database, a transcriptome-mining search was performed for NCS orthologs. A candidate gene sequence was identified and named norbelladine synthase (NBS). NpNBS encodes for a small protein of 19 kDa with an anticipated pI of 5.5. Phylogenetic analysis showed that NpNBS belongs to a unique clade of PR10/Bet v1 proteins and shared 41% amino acid identity to opium poppy NCS1. Expression of NpNBS cDNA in Escherichia coli produced a recombinant enzyme able to condense tyramine and 3,4-DHBA into norbelladine as determined by high-resolution tandem mass spectrometry. CONCLUSIONS: Here, we describe a novel enzyme catalyzing the first committed step of AA biosynthesis, which will facilitate the establishment of metabolic engineering and synthetic biology platforms for the production of AAs.
