Biology subjects

Lazarus, J. E.

Publications and source records attributed to Lazarus, J. E..

3 recordsLinked to original sources

bioRxiv2021

A genome-scale antibiotic screen in Serratia marcescens identifies YdgH as a conserved modifier of cephalosporin and detergent susceptibility

Serratia marcescens, a member of the order Enterobacterales, is adept at colonizing healthcare environments and an important cause of invasive infections. Antibiotic resistance is a daunting problem in S. marcescens because in addition to plasmid-mediated mechanisms, most isolates have considerable intrinsic resistance to multiple antibiotic classes. To discover endogenous modifiers of antibiotic susceptibility in S. marcescens, a high-density transposon insertion library was subjected to sub-minimal inhibitory concentrations of two cephalosporins, cefoxitin and cefepime, as well as the fluoroquinolone ciprofloxacin. Comparisons of transposon insertion abundance before and after antibiotic exposure identified hundreds of potential modifiers of susceptibility to these agents. Using single gene deletions, we validated several candidate modifiers of cefoxitin susceptibility and chose ydgH, a gene of unknown function, for further characterization. In addition to cefoxitin, deletion of ydgH in S. marcescens resulted in decreased susceptibility to multiple 3rd generation cephalosporins, and in contrast, to increased susceptibility to both cationic and anionic detergents. YdgH is highly conserved throughout the Enterobacterales, and we observed similar phenotypes in Escherichia coli O157:H7 and Enterobacter cloacae mutants. YdgH is predicted to localize to the periplasm and we speculate that it may be involved there in cell envelope homeostasis. Collectively, our findings provide insight into chromosomal mediators of antibiotic resistance in S. marcescens and will serve as a resource for further investigations of this important pathogen.

microbiology↗

bioRxiv2019

A New Suite of Allelic Exchange Vectors for the Scarless Modification of Proteobacterial Genomes

Despite the advent of new techniques for genetic engineering of bacteria, allelic exchange through homologous recombination remains an important tool for genetic analysis. Currently, sacB-based vector systems are often used for allelic exchange, but counter-selection escape, which prevents isolation of cells with the desired mutation, limits its utility. To circumvent this limitation, we engineered a series of \"pTOX\" allelic exchange vectors. Each plasmid encodes one of a set of inducible toxins, chosen for their potential utility in a wide range of medically important Proteobacteria. A codon-optimized rhaS transcriptional activator with a strong synthetic ribosome binding site enables tight toxin induction even in organisms lacking an endogenous rhamnose regulon. Expression of the blue amilCP or magenta tsPurple non-fluorescent chromoproteins facilitates monitoring of successful single- and double-crossover events using these vectors. The versatility of these vectors was demonstrated by deleting genes in Serratia marcescens, Escherichia coli O157:H7, Enterobacter cloacae, and Shigella flexneri. Finally, pTOX was used to characterize the impact of disruption of all combinations of the 3 orthologous S. marcescens peptidoglycan amidohydrolases on chromosomal ampC beta-lactamase activity and corresponding beta-lactam antibiotic resistance. Mutation of multiple amidohydrolases was necessary for high level ampC derepression and beta-lactam resistance. These data suggest why beta-lactam resistance may emerge during treatment less frequently in S. marcescens than in other AmpC-producing pathogens like E. cloacae. Collectively, our findings suggest that the pTOX vectors should be broadly useful for genetic engineering of Gram-negative bacteria.\n\nImportanceTargeted modification of bacterial genomes is critical for genetic analyses of microorganisms. Allelic exchange is a technique that relies on homologous recombination to substitute native loci for engineered sequences. However, current allelic exchange vectors often enable only weak selection for successful homologous recombination. We developed a suite of new allelic exchange vectors, pTOX, which were validated in several medically important Proteobacteria. They encode visible non-fluorescent chromoproteins that enable easy identification of colonies bearing integrated vector, and permit stringent selection for the second step of homologous recombination, yielding modified loci. We demonstrate the utility of these vectors by using them to investigate the effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on beta-lactam antibiotic resistance.

microbiology↗

bioRxiv2018

CRISPR screen reveals that EHEC’s T3SS and Shiga toxin rely on shared host factors for infection

Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors - a type III secretion system (T3SS) and Shiga toxins (Stx) - that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection.\n\nImportanceEnterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors - a type III secretion system (T3SS) and Shiga toxins (Stx) - that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3), and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogens key virulence factors.

microbiology↗