Biology subjects

Salcedo-Sora, J. E.

Publications and source records attributed to Salcedo-Sora, J. E..

6 recordsLinked to original sources

bioRxiv2025

Rapid accumulation of fluorophores and fast kill identify drugs with bactericidal effects against Gram-negative bacteria

Antimicrobial resistance is a massive threat, but it can take decades to develop a new antibiotic. That time could be drastically reduced if we were able to anticipate desirable properties of a chemical, such as its potential to target specific bacterial compartments. This would provide the opportunity to prioritise the development of molecules that target, for instance, the cell membrane, as this does not rely on transporters and usually results in a fast-acting bactericidal effect. We used flow cytometry and a set of fluorophores together with a group of antibiotics to discriminate between antimicrobials acting on cell membrane versus intracellularly against two Gram-negative bacteria E. coli and A. baylyi. We then chose Rhodamine 123 to screen a commercial library of chemical compounds. Using flow cytometry, several drugs present in the Prestwick library were observed to have cytotoxic effects towards E. coli. This was confirmed with growth inhibitory assays in both E. coli and A. baylyi for Pantoprazole, Theophylline and Zoledronic acid. This represents an approach to the large-scale screening of small molecules with the potential to deliver fast-acting molecules that target cell membranes in Gram-negative bacteria. 3. Impact statementThe discovery of novel antimicrobials is essential to build resilience against infectious diseases. Understanding the mechanism of action of known as well as novel compounds is equally crucial. Achieving this understanding is a long and complex process that is usually addressed on an individual basis for an antibiotic, or a class of antibiotics. Using high-throughput pipelines such as those possible with flow cytometry can help to shorten this process. We present a flow cytometry-based approach capable of determining if a given molecule has rapid antimicrobial effects that involve increasing the permeability of the cell membrane. The latter is a property that usually makes a chemical a very effective antimicrobial. 4. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2025

Membrane proteins ClcB, PtsI and YcaM mediate the bactericidal effects of colistin in Escherichia coli

Bacteria can be killed very effectively by targeting their first line of protection. The cell membranes (outer and inner membranes) of Gram-negative bacteria are directly targeted by antibiotics, such as polymyxins, via electrostatic interactions with their lipopolysaccharide (LPS) fraction. The downstream effects - preceding the cell death - of the disruptions of the bacterial membranes upon the intercalation of these antibiotics are unknown. By screening a set of E. coli membrane protein knockouts, three membrane transporters were shown to mediate the growth-inhibitory effects of colistin. This was corroborated with growth assays on gain-of-function strains, cytotoxic assays and in vivo infections in an invertebrate animal model. This is first-time evidence that the disruption caused to membrane proteins, such as the chloride channel ClcB, the sugar transport system component PtsI and the hypothetical Glutamate:GABA antiporter YcaM, is part of the cytotoxic pathway that follows or is concomitant to the electrostatic intercalations of polymyxins with the Gram-negative bacteria membrane.

microbiology↗

bioRxiv2024

Automated microscopic measurement of fibrinaloid microclots and their degradation by nattokinase, the main natto protease

Nattokinase, from the Japanese fermented food natto, is a protease with fibrinolytic activity that can thus degrade conventional blood clots. In some cases, however, including in Long COVID, fibrinogen can polymerise into an anomalous amyloid form to create clots that are resistant to normal fibrinolysis and that we refer to as fibrinaloid microclots. These can be detected with the fluorogenic stain thioflavin T. We describe an automated microscopic technique for the quantification of fibrinaloid microclot formation, which also allows the kinetics of their formation and aggregation to be recorded. We also here show that recombinant nattokinase is effective at degrading the fibrinaloid microclots in vitro. This adds to the otherwise largely anecdotal evidence, that we review, that nattokinase might be anticipated to have value as part of therapeutic treatments for individuals with Long COVID and related disorders that involve fibrinaloid microclots.

biochemistry↗

bioRxiv2022

Rapid evaporative ionisation mass spectrometry (REIMS): A potential and rapid tool for the identification of insecticide resistance in mosquito larvae.

Insecticide resistance is a significant challenge facing the successful control of mosquito vectors globally. Bioassays are currently the only method for phenotyping resistance. They require large numbers of mosquitoes for testing, the availability of a susceptible comparator strain and often insectary facilities. This study aimed to trial the novel use of rapid evaporative ionisation mass spectrometry (REIMS) for the identification of insecticide resistance in mosquitoes. No sample preparation is required for REIMS and analysis can be rapidly conducted within hours. Temephos resistant Aedes aegypti (Linnaeus) larvae from Cucuta, Colombia and temephos susceptible larvae from two origins (Bello, Colombia, and the lab reference strain New Orleans) were analysed using REIMS. We tested the ability of REIMS to differentiate three relevant variants: population source, lab versus field origin and response to insecticide. The classification of these data was undertaken using linear discriminant analysis (LDA) and random forest. Classification models built using REIMS data were able to differentiate between Ae. aegypti larvae from different populations with 82% ({+/-} 0.01) accuracy, between mosquitoes of field and lab origin with 89% ({+/-} 0.01) accuracy and between susceptible and resistant larvae with 85% ({+/-} 0.01) accuracy. LDA classifiers had higher efficiency than random forest with this data set. The high accuracy observed here identifies REIMS as a potential new tool for rapid identification of resistance in mosquitoes. We argue that REIMS and similar modern phenotyping alternatives should complement existing insecticide resistance management tools.

systems biology↗

bioRxiv2021

Expansive and diverse phenotypic landscape of field Aedes aegypti larvae with differential susceptibility to temephos: beyond metabolic detoxification

Arboviruses including dengue, Zika and chikungunya are amongst the most significant public health concerns worldwide and their control relies heavily on the use of insecticides to control the vector mosquito Aedes aegypti. The success of controlling these vector-pathogen systems is threatened by widespread insecticide resistance. The work presented here profiled the gene expression of the larvae from two field populations of Ae. aegypti with differential susceptibility to temephos. The contrasting phenotypes originated from two Colombian urban locations, Bello and Cucuta, that we have previously reported to have distinctive disease incidence, socioeconomics, and climate. The closeness of the geographical origin of the study populations was suspected to be highly influential in the profiling of the gene expression of resistance since the mosquitos resistance levels themselves are highly dependent upon environmental variables. We demonstrated that an exclusive field-to-lab (Ae. aegypti reference strain New Orleans) comparison generates an over estimation of differential gene expression (DGE) and that the inclusion of a geographically relevant field control, as used here, yields a more discrete, and likely, more specific set of genes. The composition of the obtained DGE profiles is varied, with commonly reported resistance associated genes such as detoxifying enzymes having only a small representation. We identify cuticle biosynthesis, ion exchange homeostasis, an extensive number of long non-coding RNAs, and chromatin modelling among the specifically and differentially expressed genes in field resistant Ae. aegypti larvae. It was also shown that temephos resistant larvae undertake further gene expression responses when temporarily exposed to this insecticide. The results from the sampling triangulation approach undertaken here contributes a discrete DGE profiling with reduced noise that permitted the observation of a greater gene diversity. This deeper gene granularity significantly increases the number of potential targets for the control of insecticide resistant mosquitoes and widens our knowledge base on the complex phenotypic network of the Ae. aegypti mosquito responses to insecticides. Author SummaryAedes aegypti mosquitoes are vectors for several significant human viruses including dengue, Zika and chikungunya. The lack of widely available vaccines and specific antiviral treatments for these viruses means that the principal method for reducing disease burden is through controlling the vector mosquitoes. Mosquito control relies heavily on the use of insecticides and successful vector control is threatened by widespread insecticide resistance in Ae. aegypti. Here, we examined changes in gene expression that occur in temephos resistant populations of Ae. aegypti from two field populations in Colombia. We compare gene expression in resistant larvae from Cucuta with susceptible larvae from Bello and a susceptible laboratory strain of Ae. aegypti (New Orleans). We also compare mosquitoes from Cucuta with and without temephos exposure. We report several differentially expressed genes beyond those usually reported in resistant mosquitoes. We also demonstrate the over estimation in differential gene expression that can occur when field resistant populations are compared against lab susceptible populations only. The identification of new mechanisms involved in the development of insecticide resistance is crucial to fully understanding how resistance occurs and how best it can be reduced.

genetics↗

bioRxiv2018

Disrupting folate metabolism alters the capacity of bacteria in exponential growth to develop persisters to antibiotics

Bacteria can survive high doses of antibiotics through stochastic phenotypic diversification.We present initial evidence that folate metabolism could be involved with the formation of persisters. The aberrant expression of the folate enzyme gene fau seems to reduce the incidence of persisters to antibiotics. Folate impaired bacteria had a lower generation rate for persisters to both antibiotics ampicillin and ofloxacin. Persister bacteria were detectable from the outset of the exponential growth phase in the complex media. Gene expression analyses showed tentatively distinctive profiles in exponential growth at times when bacteria persisters were observed. Levels of persisters were assessed in bacteria with altered, genetically and pharmacologically, folate metabolism. This work shows that by disrupting folate biosynthesis and usage, bacterial tolerance to antibiotics seems to be diminished. Based on these findings there is a possibility that bacteriostatic antibiotics such as antifolates could have a role to play in clinical settings where the incidence of antibiotic persisters seem to drive recalcitrant infections.

microbiology↗