Different carbon sources result in differential activation of sigma B and stress resistance in Listeria monocytogenes

Listeria monocytogenes is an important food-borne pathogen that is ubiquitous in the environment. It is able to utilize a variety of carbon sources, to produce biofilms on food-processing surfaces and to survive food preservation–associated stresses. In this study, we investigated the effect of three common carbon sources, namely glucose, glycerol and lactose, on growth and activation of the general stress response Sigma factor, SigB, and corresponding phenotypes including stress resistance. A fluorescent reporter coupled to the promoter of lmo2230, a highly SigB-dependent gene, was used to determine SigB activation via quantitative fluorescence spectroscopy. This approach, combined with Western blotting and fluorescence microscopy, showed the highest SigB activation in lactose grown cells and lowest in glucose grown cells. In line with this observation, lactose grown cells showed the highest resistance to lethal heat and acid stress, the highest biofilm formation, and had the highest adhesion/invasion capacity in Caco-2-derived C2Bbe1 cell lines. Our data suggest that lactose utilisation triggers a strong SigB dependent stress response and this may have implications for the resistance of L. monocytogenes along the food chain

Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-Like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines

Diagnosis of sheep fasciolosis caused by Fasciola hepatica using cathepsin L enzyme-linked immunosorbent assays (ELISA)

Publication history: Accepted - 3 July 2021; Published online - 6 July 2021.Fasciolosis, a global parasitic disease of agricultural livestock, is caused by the liver fluke Fasciola hepatica. Management and strategic control of fasciolosis on farms depends on early assessment of the extent of disease so that control measures can be implemented quickly. Traditionally, this has relied on the detection of eggs in the faeces of animals, a laborious method that lacks sensitivity, especially for sub-clinical infections, and identifies chronic infections only. Enzyme linked immunosorbent assays (ELISA) offer a quicker and more sensitive serological means of diagnosis that could detect early acute infection before significant liver damage occurs. The performance of three functionally-active recombinant forms of the major F. hepatica secreted cathepsins L, rFhCL1, rFhCL2, rFhCL3, and a cathepsin B, rFhCB3, were evaluated as antigens in an indirect ELISA to serologically diagnose liver fluke infection in experimentally and naturally infected sheep. rFhCL1 and rFhCL3 were the most effective of the four antigens detecting fasciolosis in sheep as early as three weeks after experimental infection, at least five weeks earlier than both coproantigen and faecal egg tests. In addition, the rFhCL1 and rFhCL3 ELISAs had a very low detection limit for liver fluke in lambs exposed to natural infection on pastures and thus could play a major role in the surveillance of farms and a ‘test and treat’ approach to disease management. Finally, antibodies to all three cathepsin L proteases remain high throughout chronic infection but decline rapidly after drug treatment with the flukicide, triclabendazole, implying that the test may be adapted to trace the effectiveness of drug treatment.This work was supported by a European Research Council Advanced Grant (HELIVAC, 322725) and Science Foundation Ireland (SFI) Professorship grant (17/RP/5368) awarded to J.P. Dalton

Improved diagnosis of SARS-CoV-2 by using nucleoprotein and spike protein fragment 2 in quantitative dual ELISA tests

The novel coronavirus, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), is the causative agent of the 2020 worldwide coronavirus pandemic. Antibody testing is useful for diagnosing historic infections of a disease in a population. These tests are also a helpful epidemiological tool for predicting how the virus spreads in a community, relating antibody levels to immunity and for assessing herd immunity. In the present study, SARS-CoV-2 viral proteins were recombinantly produced and used to analyse serum from individuals previously exposed, or not, to SARS-CoV-2. The nucleocapsid (Npro) and spike subunit 2 (S2Frag) proteins were identified as highly immunogenic, although responses to the former were generally greater. These two proteins were used to develop two quantitative enzyme-linked immunosorbent assays (ELISAs) that when used in combination resulted in a highly reliable diagnostic test. Npro and S2Frag-ELISAs could detect at least 10% more true positive coronavirus disease-2019 (COVID-19) cases than the commercially available ARCHITECT test (Abbott). Moreover, our quantitative ELISAs also show that specific antibodies to SARS-CoV-2 proteins tend to wane rapidly even in patients who had developed severe disease. As antibody tests complement COVID-19 diagnosis and determine population-level surveillance during this pandemic, the alternative diagnostic we present in this study could play a role in controlling the spread of the virus

Molecular investigation into the roles of Sigma B and the Stressosome in the response of Listeria monocytogenes to environmental stress

The alternative sigma factor, SigB, has been studied extensively in Listeria monocytogenes, Bacillus subtilis and Staphylococcus aureus, and is known to regulate the general stress response in these bacteria. The activation of SigB by a stress occurs via a signalling cascade, upstream of which is the stressosome complex. The stressosome is a large protein complex composed of RsbS, RsbT and RsbR proteins, with the RsbR proteins hypothesised to function as stress sensors. RsbR has four paralogues, Lmo0161, Lm0799, Lmo1642 and Lmo1842, of which only Lmo0799 has been established to act as a stress sensor. Lmo0799 is a blue light sensor, but somewhat surprisingly inactivation of the corresponding gene does not result in sensitivity to blue light. In the current study, environmental factors including growth phase and temperature, along with exposure to ethanol or alternative carbon sources, were identified to alter the sensitivity of L. monocytogenes to killing by blue light. Whole transcriptomic analysis of the wild-type, SigB deletion mutant and a mutant for which the conserved cysteine residue critical for blue light sensing in Lmo0799, C56, has been changed to an alanine, designated lmo0799 C56A, in the presence of blue light identified a key role for SigB in altering gene transcription in response to blue light exposure. In addition, it confirmed the ability of the C56A mutant to upregulate genes under the control of SigB in response to blue light exposure, although to a lesser extent than the wild-type. These data provide further evidence that L. monocytogenes is able to sense and respond to secondary stresses associated with blue light exposure, such as ROS. Finally, the reconstruction of Dlmo1642 and Dlmo1842 deletion mutants enabled the confirmation that there is a redundancy between the stress sensing abilities of these proteins and the other RsbR paralogues that has previously been associated with Lmo0161

Molecular investigation into the roles of Sigma B and the Stressosome in the response of Listeria monocytogenes to environmental stress

The alternative sigma factor, SigB, has been studied extensively in Listeria monocytogenes, Bacillus subtilis and Staphylococcus aureus, and is known to regulate the general stress response in these bacteria. The activation of SigB by a stress occurs via a signalling cascade, upstream of which is the stressosome complex. The stressosome is a large protein complex composed of RsbS, RsbT and RsbR proteins, with the RsbR proteins hypothesised to function as stress sensors. RsbR has four paralogues, Lmo0161, Lm0799, Lmo1642 and Lmo1842, of which only Lmo0799 has been established to act as a stress sensor. Lmo0799 is a blue light sensor, but somewhat surprisingly inactivation of the corresponding gene does not result in sensitivity to blue light. In the current study, environmental factors including growth phase and temperature, along with exposure to ethanol or alternative carbon sources, were identified to alter the sensitivity of L. monocytogenes to killing by blue light. Whole transcriptomic analysis of the wild-type, SigB deletion mutant and a mutant for which the conserved cysteine residue critical for blue light sensing in Lmo0799, C56, has been changed to an alanine, designated lmo0799 C56A, in the presence of blue light identified a key role for SigB in altering gene transcription in response to blue light exposure. In addition, it confirmed the ability of the C56A mutant to upregulate genes under the control of SigB in response to blue light exposure, although to a lesser extent than the wild-type. These data provide further evidence that L. monocytogenes is able to sense and respond to secondary stresses associated with blue light exposure, such as ROS. Finally, the reconstruction of Dlmo1642 and Dlmo1842 deletion mutants enabled the confirmation that there is a redundancy between the stress sensing abilities of these proteins and the other RsbR paralogues that has previously been associated with Lmo0161

Microbial stress meeting: from systems to molecules and back

International audienceThe 4th Microbial Stress Meeting: from Systems to Moleculesand Back was held in April 2018 in Kinsale, Ireland. The meeting covered five main topics: 1. Stress at the systems and structural level; 2. Responses to osmotic and acid stress; 3. Stress responses in single cells; 4. Stress in host-pathogen interactions; and 5. Biotechnological optimisation of microorganisms through engineering and evolution, over three days. Almost 130 delegates, from 24 countries and both the industrial and academic sectors, attended the meeting, presenting 9 lectures, 28 short talks and 52 posters. The meeting showcased the diverse and rapid advancements in microbial stress research, from the single cell level to mixed populations. In this report, a summary of the highlights from the meeting is presented

Gliotoxin-mediated bacterial growth inhibition is caused by specific metal ion depletion

Abstract Overcoming antimicrobial resistance represents a formidable challenge and investigating bacterial growth inhibition by fungal metabolites may yield new strategies. Although the fungal non-ribosomal peptide gliotoxin (GT) is known to exhibit antibacterial activity, the mechanism(s) of action are unknown, although reduced gliotoxin (dithiol gliotoxin; DTG) is a zinc chelator. Furthermore, it has been demonstrated that GT synergises with vancomycin to inhibit growth of Staphylococcus aureus. Here we demonstrate, without precedent, that GT-mediated growth inhibition of both Gram positive and negative bacterial species is reversed by Zn2+ or Cu2+ addition. Both GT, and the known zinc chelator TPEN, mediate growth inhibition of Enterococcus faecalis which is reversed by zinc addition. Moreover, zinc also reverses the synergistic growth inhibition of E. faecalis observed in the presence of both GT and vancomycin (4 µg/ml). As well as zinc chelation, DTG also appears to chelate Cu2+, but not Mn2+ using a 4-(2-pyridylazo)resorcinol assay system and Zn2+ as a positive control. DTG also specifically reacts in Fe3+-containing Siderotec™ assays, most likely by Fe3+ chelation from test reagents. GSH or DTT show no activity in these assays. Confirmatory high resolution mass spectrometry, in negative ion mode, confirmed, for the first time, the presence of both Cu[DTG] and Fe[DTG]2 chelates. Label free quantitative proteomic analysis further revealed major intracellular proteomic remodelling within E. faecalis in response to GT exposure for 30–180 min. Globally, 4.2–7.2% of detectable proteins exhibited evidence of either unique presence/increased abundance or unique absence/decreased abundance (n = 994–1160 total proteins detected), which is the first demonstration that GT affects the bacterial proteome in general, and E. faecalis, specifically. Unique detection of components of the AdcABC and AdcA-II zinc uptake systems was observed, along with apparent ribosomal reprofiling to zinc-free paralogs in the presence of GT. Overall, we hypothesise that GT-mediated bacterial growth inhibition appears to involve intracellular zinc depletion or reduced bioavailability, and based on in vitro chelate formation, may also involve dysregulation of Cu2+ homeostasis