The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

Two component system bacterial response regulators are typically DNA-binding proteins which enable the genetic regulation of many adaptive bacterial behaviors. Despite structural similarity across response regulator families, there is a diverse array of DNA-binding mechanisms. Bacteria usually encode several dozen two-component system response regulators, but Francisella tularensis only encodes three. Due to their simplified response regulatory network, Francisella species are a model for studying the role of response regulator proteins in virulence. Here, we show that Francisella response regulators QseB, KdpE, and BfpR all utilize different DNA-binding mechanisms. Our evidence suggests that QseB follows a simple mechanism whereby it binds a single inverted repeat sequence with a higher affinity upon phosphorylation. This behavior is independent of whether QseB is a positive or negative regulator of the gene as demonstrated by qseB and priM promoter sequences, respectively. Similarly, KdpE binds DNA more tightly upon phosphorylation, but also exhibits a cooperative binding isotherm. While we propose a KdpE binding site, it is possible that KdpE has a complex DNA-binding mechanism potentially involving multiple copies of KdpE being recruited to a promoter region. Finally, we show that BfpR appears to bind a region of its own promoter sequence with a lower affinity upon phosphorylation. Further structural and enzymatic work will need to be performed to deconvolute the KdpE and BfpR binding mechanisms.

Since 2014, mass mortalities of mussels Mytilus spp. have occurred in production areas on the Atlantic coast of France. The aetiology of these outbreaks remained unknown until the bacterium Francisella halioticida was detected in some mussel mortality cases. This retrospective study was conducted to assess the association between F. halioticida and these mussel mortalities. Mussel batches (n = 45) from the Atlantic coast and English Channel were selected from archived individual samples (n = 863) collected either during or outside of mortality events between 2014 and 2017. All mussels were analysed by real-time PCR assays targeting F. halioticida; in addition, 185 were analysed using histological analysis and 178 by 16S rRNA metabarcoding. F. halioticida DNA was detected by real-time PCR and 16S rRNA metabarcoding in 282 and 34 mussels, respectively. Among these individuals, 82% (real-time PCR analysis) and 76% (16S rRNA metabarcoding analysis) were sampled during a mortality event. Histological analyses showed that moribund individuals had lesions mainly characterized by necrosis, haemocyte infiltration and granulomas. Risk factor analysis showed that mussel batches with more than 20% of PCR-positive individuals were more likely to have been sampled during a mortality event, and positive 16S rRNA metabarcoding batches increased the strength of the association with mortality by 11.6 times. The role of F. halioticida in mussel mortalities was determined by reviewing the available evidence. To this end, a causation criteria grid, tailored to marine diseases and molecular pathogen detection tools, allowed more evidence to be gathered on the causal role of this bacterium in mussel mortalities.

Tularemia is a vector-borne disease caused by the Gram-negative bacterium Francisella tularensis. Known hosts and vectors in Europe are hare and ticks. F. tularensis is transmitted from ticks and animals, but also from the hydrotelluric environment and the consumption of contaminated water or food. A changing climate expands the range in which ticks can live and consequently might contribute to increasing case numbers of tularemia. Two subspecies of F. tularensis are human pathogenic. Francisella tularensis tularensis (Ftt) is endemic in North America, while Francisella tularensis holarctica (Fth) is the only subspecies causing tularemia in Europe. Ft is classified as a category A bioterrorism agent due to its low infectious dose, multiple modes of transmission, high infectivity and potential for airborne transmission and has become a global public health concern. In line with the European survey and previous phylogenetic studies, Switzerland shows the co-distribution of B.6 and B.12 strains with different geographical distribution and prevalence within the country. To establish itself in different host environments of ticks and mammals, F. tularensis presumably undergoes substantial changes on the transcriptomics and proteomic level. Here we investigate the transcriptomic and proteomic differences of five strains of Fth upon infection of rabbit macrophages and tick cells.

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In Escherichia coli, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins, while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR, which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/VKOR, represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using E. coli cells expressing a periplasmic β-Galactosidase sensor (β-Galdbs), which is only active when disulfide bond formation is inhibited. Here, we report the construction of plasmids that allows fine-tuning of the expression of the β-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when expressed in Pseudomonas aeruginosa UCBPP-PA14, which harbors two DsbB homologs, β-Galdbs behaves similarly as in E. coli, and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these β-Galdbs reporter plasmids provide a basis to identify novel inhibitors of DsbA and DsbB/VKOR in multidrug-resistant gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria.
OBJECTIVE: Disulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria because inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.

In the last 10 years, an increase in tularemia cases has been observed in both humans and animals in Switzerland. In these, infection with Francisella tularensis, the causative agent of the zoonotic disease tularemia, can occur through arthropod vectors or contact to infected animals or exposure to contaminated environmental sources. Currently, we are only able to postulate potential aetiologies: (i) behavioral changes of humans with more exposure to endemic habitats of infected arthropod vectors; (ii) an increased rate of tularemia infected ticks; (iii) increasing number and geographical regions of tick biotopes; (iv) increasing and/or more diverse reservoir populations; (v) increasing presence of bacteria in the environment; (vi) raised awareness and increased testing among physicians; (vii) improved laboratory techniques including molecular testing. To approach these questions, a one-health strategy is necessary. A functioning collaboration between public health, human medicine, and diagnostic and veterinary units for the control of tularemia must be established. Furthermore, the public should be included within citizen-supported-science-projects.

Tularemia is caused by subspecies of Francisella tularensis and can manifest in a variety of disease states, with the pneumonic presentation resulting in the greatest mortality. Despite decades of research, there are no approved vaccines against F. tularensis in the United States. Traditional vaccination strategies, such as live-attenuated or subunit vaccines, are not favorable due to inadequate protection or safety concerns. Because of this, novel vaccination strategies are needed to combat tularemia. Here we discuss the current state of and challenges to the tularemia vaccine field and suggest novel vaccine approaches going forward that might be better suited for protecting against F. tularensis infection.

Two clinically important subspecies, Francisella tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are responsible for most tularaemia cases, but these isolates typically form a weak biofilm under in vitro conditions. Phase variation of the F. tularensis lipopolysaccharide (LPS) has been reported in these subspecies, but the role of variation is unclear as LPS is crucial for virulence. We previously demonstrated that a subpopulation of LPS variants can constitutively form a robust biofilm in vitro, but it is unclear whether virulence was affected. In this study, we show that biofilm-forming variants of both fully virulent F. tularensis subspecies were highly attenuated in the murine tularaemia model by multiple challenge routes. Genomic sequencing was performed on these strains, which revealed that all biofilm-forming variants contained a lesion within the wbtJ gene, a formyltransferase involved in O-antigen synthesis. A ΔwbtJ deletion mutant recapitulated the biofilm, O-antigen and virulence phenotypes observed in natural variants and could be rescued through complementation with a functional wbtJ gene. Since the spontaneously derived biofilm-forming isolates in this study were a subpopulation of natural variants, reversion events to the wbtJ gene were detected that eliminated the phenotypes associated with biofilm variants and restored virulence. These results demonstrate a role for WbtJ in biofilm formation, LPS variation and virulence of F. tularensis.

Low-molecular weight (LMW) thiols, encompassing peptides and small proteins with active cysteine residue(s), are important to bacteria as they are involved in a wide range of redox reactions. They include the tripeptide glutathione (GSH) and the small redox proteins, thioredoxins and glutaredoxins. We review the low MW thiols and related molecules in Francisella species and what role they may play in growth and virulence. Genes for GSH biosynthesis, metabolism and thioredoxins are present in all strains of Francisella, including the fully human-virulent strains. GSH and cysteine (CSH) are the major LMW thiols in Francisella extracts. We explore the potential role of the LMW thiols to overcome the nutritional challenges of intracellular growth (high GSH conditions) as well as the nutritional challenges of planktonic growth (low GSH conditions), and their contribution to Francisella\'s sensing its environmental location. Francisella may also use GSH as a source of CSH, for which it is auxotrophic. \"Glutathione stealing\" from the host may be an important part of Francisella\'s success strategy as a facultative intracellular pathogen both to detect its location and obtain CSH. An understanding of GSH metabolism in Francisella provides insights into the interaction of this pathogen with its host and may reveal additional targets for therapeutic intervention for tularemia infections.

Fluoroquinolones lack approval for treatment of tularemia but have been used extensively for milder illness. Here, we evaluated fluoroquinolones for severe illness.
In an observational study, we identified case-patients with respiratory tularemia from July to November 2010 in Jämtland County, Sweden. We defined severe tularemia by hospitalization for >24 hours and severe bacteremic tularemia by Francisella tularensis subsp. holarctica growth in blood or pleural fluid. Clinical data and drug dosing were retrieved from electronic medical records. Chest images were reexamined. We used Kaplan-Meier curves to evaluate time to defervescence and hospital discharge.
Among 67 case-patients (median age, 66 years; 81% males) 30-day mortality was 1.5% (1 of 67). Among 33 hospitalized persons (median age, 71 years; 82% males), 23 had nonbacteremic and 10 had bacteremic severe tularemia. Subpleural round consolidations, mediastinal lymphadenopathy, and unilateral pleural fluid were common on chest computed tomography. Among 29 hospitalized persons with complete outcome data, ciprofloxacin/levofloxacin (n = 12), ciprofloxacin/levofloxacin combinations with doxycycline and/or gentamicin (n = 11), or doxycycline as the single drug (n = 6) was used for treatment. One disease relapse occurred with doxycycline treatment. Treatment responses were rapid, with median fever duration 41.0 hours in nonbacteremic and 115.0 hours in bacteremic tularemia. Increased age-adjusted Charlson comorbidity index predicted severe bacteremic tularemia (odds ratio, 2.7 per score-point; 95% confidence interval, 1.35-5.41). A 78-year-old male with comorbidities and delayed ciprofloxacin/gentamicin treatment died.
Fluoroquinolone treatment is effective for severe tularemia. Subpleural round consolidations and mediastinal lymphadenopathy were typical findings on computed tomography among case-patients in this study.