Authors: Bernd Zetsche, Jonathan S Gootenberg, Omar O Abudayyeh...

The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.

Topics: Amino Acid Sequence; CRISPR-Cas Systems; Endonucleases; Francisella; Genetic Engineering; HEK293 Cells; Humans; Molecular Sequence Data; Nucleic Acid Conformation; RNA, Guide, CRISPR-Cas Systems; Sequence Alignment

PubMed: 26422227
DOI: 10.1016/j.cell.2015.09.038

Authors: Daan C Swarts, Martin Jinek

CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single-stranded DNAs (ssDNAs) in trans. To elucidate the molecular basis for both DNase cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that guide RNA-target strand DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded DNase activity. In turn, cis cleavage of double-stranded DNA targets is a result of protospacer adjacent motif (PAM)-dependent DNA duplex unwinding, electrostatic stabilization of the displaced non-target DNA strand, and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanisms of both the cis- and the trans-acting DNase activities of Cas12a enzymes, enabling their further exploitation as genome editing tools.

Topics: Bacterial Proteins; CRISPR-Associated Proteins; CRISPR-Cas Systems; DNA, Single-Stranded; Enzyme Activation; Francisella; Gene Editing; Models, Molecular; Nucleic Acid Conformation; Protein Conformation; RNA, Guide, CRISPR-Cas Systems; Structure-Activity Relationship; Substrate Specificity

PubMed: 30639240
DOI: 10.1016/j.molcel.2018.11.021

Review

Authors: Jill Ellis, Petra C F Oyston, Michael Green...

Francisella tularensis is the etiological agent of tularemia, a serious and occasionally fatal disease of humans and animals. In humans, ulceroglandular tularemia is the most common form of the disease and is usually a consequence of a bite from an arthropod vector which has previously fed on an infected animal. The pneumonic form of the disease occurs rarely but is the likely form of the disease should this bacterium be used as a bioterrorism agent. The diagnosis of disease is not straightforward. F. tularensis is difficult to culture, and the handling of this bacterium poses a significant risk of infection to laboratory personnel. Enzyme-linked immunosorbent assay- and PCR-based methods have been used to detect bacteria in clinical samples, but these methods have not been adequately evaluated for the diagnosis of pneumonic tularemia. Little is known about the virulence mechanisms of F. tularensis, though there is a large body of evidence indicating that it is an intracellular pathogen, surviving mainly in macrophages. An unlicensed live attenuated vaccine is available, which does appear to offer protection against ulceroglandular and pneumonic tularemia. Although an improved vaccine against tularemia is highly desirable, attempts to devise such a vaccine have been limited by the inability to construct defined allelic replacement mutants and by the lack of information on the mechanisms of virulence of F. tularensis. In the absence of a licensed vaccine, aminoglycoside antibiotics play a key role in the prevention and treatment of tularemia.

Topics: Animals; Bacterial Vaccines; Disease Models, Animal; Francisella tularensis; Gene Expression Regulation, Bacterial; Humans; Tularemia; Virulence

PubMed: 12364373
DOI: 10.1128/CMR.15.4.631-646.2002

Review

Authors: Jeannine M Petersen, Martin E Schriefer

Francisella tularensis is a gram-negative coccobacillus and the etiologic agent of the zoonotic disease tularemia. First described in 1911 in Tulare County, California, it has since been reported throughout the Northern Hemisphere, with natural infections reported among an unusually wide range of vertebrates and invertebrates. In recent years, tularemia has emerged in new geographic locations, populations, and settings. This review will serve to highlight mechanisms contributing to the recent emergence of tularemia as well as a repertoire of diagnostic tools useful for detecting and diagnosing disease.

Topics: Animals; Communicable Diseases, Emerging; Disease Outbreaks; Francisella tularensis; Global Health; Humans; Tularemia

PubMed: 15845234
DOI: 10.1051/vetres:2005006

Review

Authors: Monique L van Hoek, Ky V Hoang, John S Gunn...

Bacteria alter gene expression in response to changes in their environment through various mechanisms that include signal transduction systems. These signal transduction systems use membrane histidine kinase with sensing domains to mediate phosphotransfer to DNA-binding proteins that alter the level of gene expression. Such regulators are called two-component systems (TCSs). TCSs integrate external signals and information from stress pathways, central metabolism and other global regulators, thus playing an important role as part of the overall regulatory network. This review will focus on the knowledge of TCSs in the Gram-negative bacterium, , a biothreat agent with a wide range of potential hosts and a significant ability to cause disease. While TCSs have been well-studied in several bacterial pathogens, they have not been well-studied in non-model organisms, such as and its subspecies, whose canonical TCS content surprisingly ranges from few to none. Additionally, of those TCS genes present, many are orphan components, including KdpDE, QseC, QseB/PmrA, and an unnamed two-component system (FTN_1452/FTN_1453). We discuss recent advances in this field related to the role of TCSs in physiology and pathogenesis and compare the TCS genes present in human virulent versus. environmental species and subspecies of .

Topics: Animals; Bacterial Proteins; DNA-Binding Proteins; Francisella tularensis; Gene Expression Regulation, Bacterial; Histidine Kinase; Humans; Protein Domains; Signal Transduction; Tularemia; Virulence

PubMed: 31263682
DOI: 10.3389/fcimb.2019.00198

Review

Authors: Girija Ramakrishnan

, the causative agent of tularemia, is a Gram-negative bacterium that infects a variety of cell types including macrophages, and propagates with great efficiency in the cytoplasm. Iron, essential for key enzymatic and redox reactions, is among the nutrients required to support this pathogenic lifestyle and the bacterium relies on specialized mechanisms to acquire iron within the host environment. Two distinct pathways for iron acquisition are encoded by the genome- a siderophore-dependent ferric iron uptake system and a ferrous iron transport system. Genes of the Fur-regulated operon direct the production and transport of the siderophore rhizoferrin. Siderophore biosynthesis involves enzymes FslA and FslC, while export across the inner membrane is mediated by FslB. Uptake of the rhizoferrin- ferric iron complex is effected by the siderophore receptor FslE in the outer membrane in a TonB-independent process, and FslD is responsible for uptake across the inner membrane. Ferrous iron uptake relies largely on high affinity transport by FupA in the outer membrane, while the Fur-regulated FeoB protein mediates transport across the inner membrane. FslE and FupA are paralogous proteins, sharing sequence similarity and possibly sharing structural features as well. This review summarizes current knowledge of iron acquisition in this organism and the critical role of these uptake systems in bacterial pathogenicity.

Topics: Animals; Biological Transport; Disease Models, Animal; Ferric Compounds; Ferrous Compounds; Francisella tularensis; Humans; Iron; Metabolic Networks and Pathways; Virulence

PubMed: 28421167
DOI: 10.3389/fcimb.2017.00107

Comparative Study Review

Authors: Luke C Kingry, Jeannine M Petersen

Francisella tularensis is the causative agent of the acute disease tularemia. Due to its extreme infectivity and ability to cause disease upon inhalation, F. tularensis has been classified as a biothreat agent. Two subspecies of F. tularensis, tularensis and holarctica, are responsible for tularemia in humans. In comparison, the closely related species F. novicida very rarely causes human illness and cases that do occur are associated with patients who are immune compromised or have other underlying health problems. Virulence between F. tularensis and F. novicida also differs in laboratory animals. Despite this varying capacity to cause disease, the two species share ~97% nucleotide identity, with F. novicida commonly used as a laboratory surrogate for F. tularensis. As the F. novicida U112 strain is exempt from U.S. select agent regulations, research studies can be carried out in non-registered laboratories lacking specialized containment facilities required for work with virulent F. tularensis strains. This review is designed to highlight phenotypic (clinical, ecological, virulence, and pathogenic) and genomic differences between F. tularensis and F. novicida that warrant maintaining F. novicida and F. tularensis as separate species. Standardized nomenclature for F. novicida is critical for accurate interpretation of experimental results, limiting clinical confusion between F. novicida and F. tularensis and ensuring treatment efficacy studies utilize virulent F. tularensis strains.

Topics: Animals; Disease Models, Animal; Francisella tularensis; Humans; Sequence Homology, Nucleic Acid; Tularemia; Virulence

PubMed: 24660164
DOI: 10.3389/fcimb.2014.00035

Review

Authors: Richard Hodosi, Maria Kazimirova, Katarina Soltys...

is an obligate hematophagous parasitic arthropod that is responsible for the transmission of a wide range of zoonotic pathogens including spirochetes of the genus spp., , and , which are part the tick´s microbiome. Most of the studies focus on "pathogens" and only very few elucidate the role of "non-pathogenic" symbiotic microorganisms in . While most of the members of the microbiome are leading an intracellular lifestyle, they are able to complement tick´s nutrition and stress response having a great impact on tick´s survival and transmission of pathogens. The composition of the tick´s microbiome is not consistent and can be tied to the environment, tick species, developmental stage, or specific organ or tissue. Ovarian tissue harbors a stable microbiome consisting mainly but not exclusively of endosymbiotic bacteria, while the microbiome of the digestive system is rather unstable, and together with salivary glands, is mostly comprised of pathogens. The most prevalent endosymbionts found in ticks are spp., spp., -like and -like endosymbionts, spp. and Midichloria spp. Since microorganisms can modify ticks' behavior, such as mobility, feeding or saliva production, which results in increased survival rates, we aimed to elucidate the potential, tight relationship, and interaction between bacteria of the microbiome. Here we show that endosymbionts including -like spp., can provide with different types of vitamin B (B2, B6, B7, B9) essential for eukaryotic organisms. Furthermore, we hypothesize that survival of spp., or the bacterial pathogen can be supported by the tick itself since coinfection with symbiotic provides with complete metabolic pathway of folate biosynthesis necessary for DNA synthesis and cell division. Manipulation of tick´s endosymbiotic microbiome could present a perspective way of control and regulation of spread of emerging bacterial pathogens.

Topics: Animals; Ixodes; Microbiota; Coxiella; Symbiosis; Rickettsia; Francisella tularensis

PubMed: 36467722
DOI: 10.3389/fcimb.2022.990889

Authors: Yaxi Wang, Hannah E Ledvina, Catherine A Tower...

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

Topics: Glutathione; Francisella; Francisella tularensis; DNA Transposable Elements; Bacterial Proteins; Phylogeny; Macrophages; Animals; Mice; Tularemia

PubMed: 37453420
DOI: 10.1016/j.chom.2023.06.010

Review

Authors: Aurélie Hennebique, Sandrine Boisset, Max Maurin...

is a Gram-negative, intracellular bacterium causing the zoonosis tularemia. This highly infectious microorganism is considered a potential biological threat agent. Humans are usually infected through direct contact with the animal reservoir and tick bites. However, tularemia cases also occur after contact with a contaminated hydro-telluric environment. Water-borne tularemia outbreaks and sporadic cases have occurred worldwide in the last decades, with specific clinical and epidemiological traits. These infections represent a major public health and military challenge. Human contaminations have occurred through consumption or use of -contaminated water, and various aquatic activities such as swimming, canyoning and fishing. In addition, in Sweden and Finland, mosquitoes are primary vectors of tularemia due to infection of mosquito larvae in contaminated aquatic environments. The mechanisms of survival in water may include the formation of biofilms, interactions with free-living amoebae, and the transition to a 'viable but nonculturable' state, but the relative contribution of these possible mechanisms remains unknown. Many new aquatic species of have been characterized in recent years. likely shares with these species an ability of long-term survival in the aquatic environment, which has to be considered in terms of tularemia surveillance and control.

Topics: Animals; Culicidae; Francisella tularensis; Humans; Tularemia; Waterborne Diseases; Zoonoses

PubMed: 31287787
DOI: 10.1080/22221751.2019.1638734