Staphylococcus aureus controls the progression of infection through the coordinated production of extracellular proteases, which selectively modulate virulence determinant stability. This is evidenced by our previous finding that a protease-null strain has a hypervirulent phenotype in a murine model of sepsis, resulting from the unchecked accumulation of virulence factors. Here, we dissect the individual roles of these proteases by constructing and assessing the pathogenic potential of a combinatorial protease mutant library. When strains were constructed bearing increasing numbers of secreted proteases, we observed a variable impact on infectious capacity, where some exhibited hypervirulence, while others phenocopied the wild-type. The common thread for hypervirulent strains was that each lacked both aureolysin and staphopain A. Upon assessment, we found that the combined loss of these two enzymes alone was necessary and sufficient to engender hypervirulence. Using proteomics, we identified a number of important secreted factors, including SPIN, LukA, Sbi, SEK, and PSMα4, as well as an uncharacterized chitinase-related protein (SAUSA300_0964), to be overrepresented in both the aur scpA and the protease-null mutants. When assessing the virulence of aur scpA SAUSA300_0964 and aur scpA lukA mutants, we found that hypervirulence was completely eliminated, whereas aur scpA spn and aur scpA sek strains elicited aggressive infections akin to the protease double mutant. Collectively, our findings shed light on the influence of extracellular proteases in controlling the infectious process and identifies SAUSA300_0964 as an important new component of the S. aureus virulence factor arsenal. IMPORTANCE A key feature of the pathogenic success of S. aureus is the myriad virulence factors encoded within its genome. These are subject to multifactorial control, ensuring their timely production only within an intended infectious niche. A key node in this network of control is the secreted proteases of S. aureus, who specifically and selectively modulate virulence factor stability. In our previous work we demonstrated that deletion of all 10 secreted proteases results in hypervirulence, since virulence factors exist unchecked, leading to overly aggressive infections. Here, using a combinatorial collection of protease mutants, we reveal that deletion of aureolysin and staphopain A is necessary and sufficient to elicit hypervirulence. Using proteomic techniques, we identify the collection of virulence factors that accumulate in hypervirulent protease mutants, and demonstrate that a well-known toxin (LukA) and an entirely novel secreted element (SAUSA300_0964) are the leading contributors to deadly infections observed in protease-lacking strains.

ABSTRACT Autotrophic microorganisms catalyze the entry of dissolved inorganic carbon (DIC; = CO2 + HCO3− + CO32−) into the biological component of the global carbon cycle, despite dramatic differences in DIC abundance and composition in their sometimes extreme environments. “Cyanobacteria” are known to have CO2 concentrating mechanisms (CCMs) to facilitate growth under low CO2 conditions. These CCMs consist of carboxysomes, containing enzymes ribulose 1,5-bisphosphate oxygenase and carbonic anhydrase, partnered to DIC transporters. CCMs and their DIC transporters have been studied in a handful of other prokaryotes, but it was not known how common CCMs were beyond “Cyanobacteria”. Since it had previously been noted that genes encoding potential transporters were found neighboring carboxysome loci, α-carboxysome loci were gathered from bacterial genomes, and potential transporter genes neighboring these loci are described here. Members of transporter families whose members all transport DIC (CHC, MDT and Sbt) were common in these neighborhoods, as were members of the SulP transporter family, many of which transport DIC. 109 of 115 taxa with carboxysome loci have some form of DIC transporter encoded in their genomes, suggesting that CCMs consisting of carboxysomes and DIC transporters are widespread not only among “Cyanobacteria”, but also among members of “Proteobacteria” and “Actinobacteria”.

Acinetobacter baumannii is a formidable opportunistic pathogen that is notoriously difficult to eradicate from hospital settings. This resilience is often attributed to a proclivity for biofilm formation, which facilitates a higher tolerance toward external stress, desiccation, and antimicrobials.

Characterization of transcriptional networks is one of the main strategies used to understand how bacteria interact with their environment. To reveal novel regulatory elements in the human pathogen Staphylococcus aureus , we adapted a traditional transduction protocol to be used in a high-throughput format in combination with the publicly available S. aureus Nebraska Transposon Mutant Library. Specifically, plasmid transductions are performed in 96-well format, so that a single plasmid can be simultaneously transferred into numerous recipient strains. When used in conjunction with bioluminescent reporter constructs, this strategy enables parallel and continuous monitoring of downstream transcriptional effects of hundreds of defined mutations. Here, we use this workflow in a proof-of-concept study to identify novel regulators of the staphylococcal metalloprotease aureolysin. Importantly, this strategy can be utilized with any other bacterium where plasmid transduction is possible, making it a versatile and efficient tool to probe transcriptional regulatory connections.

Tourette syndrome (TS) and chronic tic disorder (CTD) are neurological disorders of childhood onset characterized by the occurrence of tics; repetitive, purposeless, movements or vocalizations of short duration which can occur many times throughout a day. Currently, effective treatment for tic disorders is an area of considerable unmet clinical need. We aimed to evaluate the efficacy of a home‐administered neuromodulation treatment for tics involving the delivery of rhythmic pulse trains of median nerve stimulation (MNS) delivered via a wearable ‘watch‐like’ device worn at the wrist. We conducted a UK‐wide parallel double‐blind sham‐controlled trial for the reduction of tics in individuals with tic disorder. The device was programmed to deliver rhythmic (10 Hz) trains of low‐intensity (1–19 mA) electrical stimulation to the median nerve for a pre‐determined duration each day, and was intended to be used by each participant in their home once each day, 5 days each week, for a period of 4 weeks. Between 18th March 2022 and 26th September 2022, 135 participants (45 per group) were initially allocated, using stratified randomization, to one of the following groups; active stimulation; sham stimulation or to a waitlist (i.e. treatment as usual) control group. Recruited participants were individuals with confirmed or suspected TS/CTD aged 12 years of age or upward with moderate to severe tics. Researchers involved in the collection or processing of measurement outcomes and assessing the outcomes, as well as participants in the active and sham groups and their legal guardians were all blind to the group allocation. The primary outcome measure used to assess the ‘offline’ or treatment effect of stimulation was the Yale Global Tic Severity Scale–Total Tic Severity Score (YGTSS–TTSS) assessed at the conclusion of 4 weeks of stimulation. The primary outcome measure used to assess the ‘online’ effects of stimulation was tic frequency, measured as the number of tics per minute (TPM) observed, based upon blind analysis of daily video recordings obtained while stimulation was delivered. The results demonstrated that after 4‐week stimulation, tic severity (YGTSS‐TTSS) had reduced by 7.1 points (35 percentile reduction) for the active stimulation group compared to 2.13/2.11 points for the sham stimulation and waitlist control groups. The reduction in YGTSS–TTSS for the active stimulation group was substantially larger, clinically meaningful (effect size = .5) and statistically significant (p = .02) compared to both the sham stimulation and waitlist control groups, which did not differ from one another (effect size = −.03). Furthermore, blind analyses of video recordings demonstrated that tic frequency (tics per minute) reduced substantially (−15.6 TPM) during active stimulation compared to sham stimulation (−7.7 TPM). This difference represents a statistically significant (p < .03) and clinically meaningful reduction in tic frequency (>25 percentile reduction: effect size = .3). These findings indicate that home‐administered rhythmic MNS delivered through a wearable wrist‐worn device has the potential to be an effective community‐based treatment for tic disorders.