Biology subjects

Cartagena, A. J.

Publications and source records attributed to Cartagena, A. J..

3 recordsLinked to original sources

bioRxiv2023

Discovery, Characterization, and Bioactivity of the Achromonodins: Lasso Peptides Encoded by Achromobacter

Through genome mining efforts, we discovered two lasso peptide biosynthetic gene clusters (BGCs) within two different species of Achromobacter, a genus that contains pathogenic organisms that can infect patients with cystic fibrosis. Using gene-refactored BGCs in E. coli, we heterologously expressed two lasso peptides, which we named achromonodin-1 and achromonodin-2. Achromonodin-1 is naturally encoded by certain isolates from the sputum of patients with cystic fibrosis. We solve the NMR structure of achromonodin-1, demonstrating that it is a threaded lasso peptide with a large loop and short tail structure, reminiscent of previously characterized lasso peptides that inhibit RNA polymerase (RNAP). We then show that achromonodin-1 inhibits RNAP in vitro and has potent but narrow-spectrum activity towards Achromobacter pulmonis, another isolate from the sputum of a cystic fibrosis patient. Our efforts expand the repertoire of antimicrobial lasso peptides and provide insights into how Achromobacter isolates from certain ecological niches may interact with each other.

biochemistry↗

bioRxiv2023

The carbapenem inoculum effect provides insight into the molecular mechanisms underlying carbapenem resistance in Enterobacterales

Carbapenem-resistant Enterobacterales (CRE) are important pathogens that can develop resistance via multiple molecular mechanisms, including hydrolysis or reduced antibiotic influx. Identifying these mechanisms can improve pathogen surveillance, infection control, and patient care. We investigated, both phenomenologically and mechanistically, how resistance mechanisms influence the carbapenem inoculum effect (IE), a phenomenon where inoculum size affects antimicrobial susceptibility testing (AST). We demonstrated that any of seven different carbapenemases were sufficient to impart a meropenem IE when transformed into a laboratory strain of Escherichia coli. Across 106 clinical CRE isolates spanning 6 genera and 12 species, the carbapenem IE strictly depended on resistance mechanism: all 36 carbapenemase-producing CRE (CP-CRE) exhibited a clear IE, whereas 43 porin-deficient CRE displayed none. 27 isolates with both carbapenemase production and porin deficiency exhibited high-level resistance at all inocula, and still displayed an IE, albeit smaller in magnitude than CP-CRE with intact porins. Mechanistically, we found that clinical CP-CRE release carbapenemase activity into culture supernatant to protect other cells. Further, this released activity markedly increased upon exposure to lethal antibiotic doses, functionally consistent with altruism. Concerningly, 50% and 24% of CP-CRE isolates changed susceptibility classification to meropenem and ertapenem, respectively, across the allowable inoculum range in clinical testing guidelines. The meropenem IE, and the ratio of ertapenem to meropenem minimal inhibitory concentration (MIC) at standard inoculum, reliably identified CP-CRE. Understanding how resistance mechanisms affect AST could improve diagnosis and guide therapies for CRE infections. ImportanceInfections caused by carbapenem-resistant Enterobacterales (CRE) pose significant threats to patients and public health worldwide. Carbapenem resistance can occur through several molecular mechanisms, including enzymatic hydrolysis by carbapenemases and reduced influx via porin mutations. Identifying carbapenemase-producing isolates could enable tailored antibiotic selection to improve patient outcomes, and infection control measures to prevent further carbapenemase transmission. In a large collection of CRE isolates, we found that only carbapenemase-producing CRE exhibit an inoculum effect, in which their measured resistance varies markedly with cell density, which risks misdiagnosis. Further, this inoculum effect occurs under conditions where bacteria release carbapenemases to the community upon exposure to antibiotic that results in cell death, functionally consistent with altruism. Measuring this inoculum effect, or integrating other data from routine antimicrobial susceptibility testing, enhances carbapenem resistance detection, paving the way for more effective strategies to combat this growing public health crisis.

microbiology↗

bioRxiv2019

Structural basis for transcription activation by Crl through tethering of σS and RNA polymerase

In bacteria, a primary {sigma} factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative {sigma} factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative {sigma} factors are negatively regulated by anti-{sigma} factors. In Escherichia coli, Salmonella enterica, and many other{gamma} -proteobacteria, the transcription factor Crl positively regulates the alternative {sigma}S regulon by promoting the association of {sigma}S with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-{sigma}S-RNAP in an open promoter complex with a {sigma}S regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of {sigma}S ({sigma}S), the structure, along with p-benzoylphenylalanine crosslinking, reveals that Crl interacts with a structural element of the RNAP {beta} subunit we call the {beta}-clamp-toe ({beta}CT). Deletion of the {beta}CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-{beta}CT interaction. We conclude that Crl activates {sigma}S-dependent transcription in part through stabilizing {sigma}S-RNAP by tethering {sigma}S and the {beta}CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated {sigma}-activators.\n\nSignificance StatementIn bacteria, multiple {sigma} factors can bind to a common core RNA polymerase (RNAP) to alter global transcriptional programs in response to environmental stresses. Many {gamma}-proteobacteria, including the pathogens Yersinia pestis, Vibrio cholera, Escherichia coli, and Salmonella typhimurium, encode Crl, a transcription factor that activates {sigma}S-dependent genes. Many of these genes are involved in processes important for infection, such as biofilm formation. We determined a high-resolution cryo-electron microscopy structure of a Crl-{sigma}S-RNAP transcription initiation complex. The structure, combined with biochemical experiments, shows that Crl stabilizes {sigma}S-RNAP by tethering {sigma}S directly to the RNAP.

biophysics↗