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.

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.

Microbial stress meeting: From systems to molecules and back.

The 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.

Blue Light Sensing in Listeria monocytogenes Is Temperature-Dependent and the Transcriptional Response to It Is Predominantly SigB-Dependent.

Listeria monocytogenes is an important food-borne pathogen that is tolerant to many of the stresses commonly used during food preservation. Outside the host, the bacterium has a saprophytic lifestyle that includes periodic exposure to solar irradiance. The blue component of this light is known to influence the activity of the stress-inducible sigma factor Sigma B (σB). In this study, the influence of temperature and growth phase on the response of L. monocytogenes to blue light was investigated and the global transcriptional response to blue light was elucidated using an RNAseq-based approach. Stationary phase cells were found to be significantly more resistant to killing by blue light (470 nm) than exponential phase cells. Temperature also had a marked effect on blue light resistance with cells cultured at 37°C being much more sensitive than cells grown at 30°C. The role of σB in light tolerance was confirmed but this effect was observed only at 30°C. σB activation by blue light was assessed by measuring the transcriptional response of known σB-dependent genes (sigB, lmo2230, and opuCA) to light. The transcripts were induced by blue light only at 30°C suggesting that blue light fails to activate σB at 37°C. The light-induced transcription at 30°C was dependent on a functional blue light sensor, Lmo0799 (which we rename herein as RsbL). A transcriptomic analysis of the response to sub-lethal levels of blue light found that the changes in transcription were almost entirely σB-dependent. A mutant where the light sensing mechanism of RsbL was inactivated through an amino acid substitution (Cys56Ala) was found to have an attenuated response to blue light, but residual activation of σB-dependent genes suggested that alternative routes for activation of σB by light are likely to exist. Overall, the study highlights the central role of σB in the response of this pathogen to visible light and further shows that light sensing is absent at temperatures that exist within the mammalian host.