Francisella Species Research Articles

Tolfenpyrad Derivatives Exhibit Potent Francisella-Specific Antibacterial Activity without Toxicity to Mammalian Cells In Vitro.

Tularemia is a deadly disease caused by Francisella tularensis, an emerging intracellular bacterial pathogen that can be disseminated rapidly through aerosols and vector-borne transmission. Recent surveillance data demonstrate an increasing incidence in several countries. Although clinical isolates of Francisella strains are sensitive to currently used antibiotics, engineered or horizontal acquisition of antibiotic resistance is a constant threat to public health. Therefore, the identification of antibiotics that target previously undrugged pathways is required to safeguard human health. An environmental pesticide that is registered for use in multiple countries, tolfenpyrad, shows promising activity to block Francisella growth; however, it is not a suitable antimicrobial candidate for use in vivo due to potential toxicity in humans and other animals. In this study, we applied a structure-activity relationship approach to tolfenpyrad to generate compounds with improved antibacterial activity and reduced toxicity. Through screening of a library of derivatives, we identified analogs with improved therapeutic windows compared with tolfenpyrad. Although structural diversity exists among these analogs, they inhibit the growth of Francisella species but not other Gram-negative or Gram-positive species. These compounds block intramacrophage growth of F. novicida and pathogenesis in an in vivo arthropod model of infection. Although the biochemical activity of these drugs is unknown, they appear to target the same pathway as the parent molecule because F. novicida mutants that are resistant to tolfenpyrad are also resistant to its analogs. Taken together, these findings suggest that these tolfenpyrad-derived compounds comprise a new class of Francisella-targeted antimicrobials and merit further evaluation and development.

Francisella sciaenopsi sp. nov. isolated from diseased red drum Sciaenops ocellatus in Florida, USA.

Piscine francisellosis is one of the most important bacterial diseases affecting various fish species worldwide. Francisella orientalis, F. noatunensis, and F. salimarina (F. marina) have been reported as etiological agents of disease in fish. A Francisella sp. was isolated from several diseased red drum Sciaenops ocellatus experiencing morbidity in Florida, USA, in 2008. In this study, molecular and phenotypic characterization of the recovered isolate was conducted. Phenotypically, the isolate showed a biochemical reaction profile distinct from that of F. orientalis and F. salimarina. Although the 16S rRNA sequence of this isolate shared 99.61% identity to the type strain of F. philomiragia O#319LT, whole genome analysis (average nucleotide identity <95%; digital DNA-DNA hybridization <70%) and a multilocus sequence analysis of 8 concatenated housekeeping genes in comparison with other Francisella spp. indicated that this isolate was a novel Francisella species, more closely related to F. orientalis. Immersion, intracoelomic injection, and co-habitation challenges using a Nile tilapia Oreochromis niloticus fingerling model of infection were done to investigate virulence in a piscine model. Variably pigmented granulomas and pigmented macrophage aggregates were observed in the kidneys and spleens of the challenged fish, but no mortality was recorded during the 15 d challenge period, suggesting that this novel Francisella sp. might be an opportunistic pathogen of fish. Based on the phenotypic and genotypic differences from other Francisella spp. observed in this study, we propose the name Francisella sciaenopsi sp. nov. for this novel isolate.

Structural proteomics guided annotation of vaccine targets and designing of multi-epitopes vaccine to instigate adaptive immune response against Francisella tularensis

Francisella tularensis can cause severe disease in humans via the respiratory or cutaneous routes and a case fatality ratio of up to 10 % is reported due to lack of proper antibiotic treatment, while F. novicida causes disease in severely immunocompromised individuals. Efforts are needed to develop effective vaccine candidates against Francisella species. Thus, in this study, a systematic computational work frame was used to deeply investigate the whole proteome of Francisella novicida containing 1728 proteins to develop vaccine against F. tularensis and related species. Whole-proteome analysis revealed that four proteins including (A0Q492) (A0Q7Y4), (A0Q4N4), and (A0Q5D9) are the suitable vaccine targets after the removal of homologous, paralogous and prediction of subcellular localization. These proteins were used to predict the T cell, B cell, and HTL epitopes which were joined together through suitable linkers to construct a multi-epitopes vaccine (MEVC). The MEVC was found to be highly immunogenic and non-allergenic while the physiochemical properties revealed the feasible expression and purification. Moreover, the molecular interaction of MEVC with TLR2, molecular simulation, and binding free energy analyses further validated the immune potential of the construct. According to Jcat analysis, the refined sequence demonstrates GC contents of 41.48 % and a CAI value of 1. The in-silico cloning and optimization process ensured compatibility with host codon usage, thereby facilitating efficient expression. Computational immune simulation studies underscored the capacity of MEVC to induce both primary and secondary immune responses. The conservation analysis further revealed that the selected epitopes exhibit 100 % conservation across different species and thus provides wider protection against Francisella.

Diverse roles of low-molecular weight thiol GSH in Francisella’s virulence, location sensing and GSH-stealing from host

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.

Ex vivo infection model for Francisella using human lung tissue.

Tularemia is mainly caused by Francisella tularensis (Ft) subsp. tularensis (Ftt) and Ft subsp. holarctica (Ftt) in humans and in more than 200 animal species including rabbits and hares. Human clinical manifestations depend on the route of infection and range from flu-like symptoms to severe pneumonia with a mortality rate up to 60% without treatment. So far, only 2D cell culture and animal models are used to study Francisella virulence, but the gained results are transferable to human infections only to a certain extent. In this study, we firstly established an ex vivo human lung tissue infection model using different Francisella strains: Ftt Life Vaccine Strain (LVS), Ftt LVS ΔiglC, Ftt human clinical isolate A-660 and a German environmental Francisella species strain W12-1067 (F-W12). Human lung tissue was used to determine the colony forming units and to detect infected cell types by using spectral immunofluorescence and electron microscopy. Chemokine and cytokine levels were measured in culture supernatants. Only LVS and A-660 were able to grow within the human lung explants, whereas LVS ΔiglC and F-W12 did not replicate. Using human lung tissue, we observed a greater increase of bacterial load per explant for patient isolate A-660 compared to LVS, whereas a similar replication of both strains was observed in cell culture models with human macrophages. Alveolar macrophages were mainly infected in human lung tissue, but Ftt was also sporadically detected within white blood cells. Although Ftt replicated within lung tissue, an overall low induction of pro-inflammatory cytokines and chemokines was observed. A-660-infected lung explants secreted slightly less of IL-1β, MCP-1, IP-10 and IL-6 compared to Ftt LVS-infected explants, suggesting a more repressed immune response for patient isolate A-660. When LVS and A-660 were used for simultaneous co-infections, only the ex vivo model reflected the less virulent phenotype of LVS, as it was outcompeted by A-660. We successfully implemented an ex vivo infection model using human lung tissue for Francisella. The model delivers considerable advantages and is able to discriminate virulent Francisella from less- or non-virulent strains and can be used to investigate the role of specific virulence factors.

Intraspecific Differentiation of &lt;i&gt;Francisella tularensis&lt;/i&gt; Strains Using Multilocus Real-Time Polymerase Chain Reaction

The aim of the study was to develop a method for intraspecific differentiation of the tularemia microbe: subspecies tularensis (subpopulations AI and AII), holarctica (biovars japonica, EryS/R), mediasiatica, and novicida using multilocus real-time PCR. Materials and methods. We used 48 strains of F. tularensis of various subspecies, biovars, and subpopulations. Intraspecific appurtenance of the strains was carried out on the basis of the analysis of the RD-1 region variability applying PCR, the sdhA gene by Sanger fragment sequencing and by the disk diffusion method using disks with erythromycin. The selection of primers and probes was performed using the software available at www.genscript.com and GeneRunner 6.5.52. Sequence homology was assessed using the BLAST algorithm and the GenBank NCBI database. Results and discussion. New data on the structure and occurrence of the differentiation regions RD-8, RD-12, RD-28 of FTT1122c gene and its homologous sequences in strains of tularemia microbe of various subspecies have been obtained. Novel RDhm 346 bp in size, characteristic of strains of the subsp. mediasiatica, holarctica, which is deleted in subsp. tularensis and absent in subsp. novicida has been detected. Based on the detection of the FTT1670, FTT1122с, FTT1067, FTW_2084 loci, a multilocus real-time PCR has been developed – “F. tularensis 4c”, providing for identification of all subspecies of the tularemia microbe, separately for the biovar japonica of the Holarctic subspecies and subpopulations AI, AII of the subspecies tularensis. The PCR specificity was confirmed in the study of strains of tularemia microbe from the fund of the “State Collection of Pathogenic Bacteria” at the premises of the Russian Reserarch Anti-Plague Institute “Microbe”. The results obtained expand the concept of intraspecific genetic heterogeneity of tularemia microbe and possibilities of identifying the causative agent of tularemia using molecular-genetic methods. They are important for understanding the processes of adaptation of the pathogen to circulation in the host organism and environmental objects, the course of evolution and formation of new species of Francisella.

Applying RNA-Seq to investigate the transition into a Viable But Non Culturable State (VBNC) for the intracellular pathogen, Francisella tularensis LVS.

Many species of bacteria, under conditions of stress or nutrient limitation, enter a state of dormancy referred to as viable but non-culturable (VBNC). VBNC bacteria persist in the environment, are difficult to detect and identify by many standard laboratory methods and can be altered in their susceptibility to antibiotics. Entry into the VBNC state is often accompanied by morphological changes; however, the mechanisms underlying this are poorly understood. Francisella transitions rapidly and spontaneously to a VBNC state, and therefore has the potential to be an excellent model organism for the study of this phenomenon. To investigate the transcriptome of VBNC F. tularensis LVS we extracted RNA from culturable and VBNC cells and carried out an RNA-Seq analysis using both Long-read Nanopore and Illumina Sequencing. Differentially expressed genes (DEGs) were identified using a DE-Seq pipeline. Over 300 genes were significantly upregulated and ~100 genes were down regulated in VBNC cells. Amongst the upregulated genes were some involved in the transport of metals or small molecules, and we have also identified putative transcriptional regulators that may be master controllers of the VBNC process. Our data represents the first transcriptomic analysis of F. tularensis LVS as it transitions into the VBNC state. Identifying genes that are involved in this transition is a critical first step in understanding the mechanisms that drive F. tularensis LVS into the VBNC state and will help us identify factors that allow the resuscitation of these bacteria. This will have important implications for understanding both environmental persistence and pathogenicity of Francisella species. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)

Acute Pancreatitis Associated with Atypical Bacterial Pneumonia: Systematic Literature Review

Extra-pulmonary features sometimes occur in association with atypical bacterial pneumonia and include neurologic manifestations, diarrhea, rashes, altered liver enzymes, or kidney injury, among other conditions. Acute pancreatitis has been associated with atypical pneumonias since 1973. We performed a systematic review of the literature in the Excerpta Medica, National Library of Medicine, and Web of Science databases. We retained 27 reports published between 1973 and 2022 describing subjects with an otherwise unexplained pancreatitis temporally associated with an atypical pneumonia. The reports included 33 subjects (19 males, and 14 females; 8 children and 25 adults) with acute pancreatitis temporally associated with atypical pneumonia caused by Mycoplasma pneumoniae (n = 18), Legionella species (n = 14), or Coxiella burnetii (n = 1). Approximately 90% of patients (n = 29) concurrently presented with respiratory and pancreatic diseases. No cases associated with Chlamydophila pneumoniae, Chlamydophila psittaci, or Francisella species were found. Acute pancreatitis has been associated with various infectious agents. The present review documents the association with atypical pneumonia induced by Mycoplasma pneumoniae, Legionella species, and Coxiella burnetii.

Genomic characterization of Francisella tularensis and other diverse Francisella species from complex samples.

Francisella tularensis, the bacterium that causes the zoonosis tularemia, and its genetic near neighbor species, can be difficult or impossible to cultivate from complex samples. Thus, there is a lack of genomic information for these species that has, among other things, limited the development of robust detection assays for F. tularensis that are both specific and sensitive. The objective of this study was to develop and validate approaches to capture, enrich, sequence, and analyze Francisella DNA present in DNA extracts generated from complex samples. RNA capture probes were designed based upon the known pan genome of F. tularensis and other diverse species in the family Francisellaceae. Probes that targeted genomic regions also present in non-Francisellaceae species were excluded, and probes specific to particular Francisella species or phylogenetic clades were identified. The capture-enrichment system was then applied to diverse, complex DNA extracts containing low-level Francisella DNA, including human clinical tularemia samples, environmental samples (i.e., animal tissue and air filters), and whole ticks/tick cell lines, which was followed by sequencing of the enriched samples. Analysis of the resulting data facilitated rigorous and unambiguous confirmation of the detection of F. tularensis or other Francisella species in complex samples, identification of mixtures of different Francisella species in the same sample, analysis of gene content (e.g., known virulence and antimicrobial resistance loci), and high-resolution whole genome-based genotyping. The benefits of this capture-enrichment system include: even very low target DNA can be amplified; it is culture-independent, reducing exposure for research and/or clinical personnel and allowing genomic information to be obtained from samples that do not yield isolates; and the resulting comprehensive data not only provide robust means to confirm the presence of a target species in a sample, but also can provide data useful for source attribution, which is important from a genomic epidemiology perspective.

Ulceroglandular Infection and Bacteremia Caused by Francisella salimarina in Immunocompromised Patient, France.

Although Francisella tularensis is a well-known, highly virulent bacterium that causes tularemia in humans, other Francisella species have been associated with sporadic human infections. We describe a human cutaneous infection with bacteremia caused by F. salimarina, a Francisella species recently identified from seawater and fishes, in an immunocompromised patient in France.

Francisella novicida can utilize Paramecium bursaria as its potential host.

Francisella novicida is a facultative intracellular pathogen and the causative agent of tularemia. Although cases of infection caused by exposure to contaminated water have been reported, its natural host and ecology in the environment remain unclear. In this study, we investigated in vitro the possibility that Paramecium bursaria may be a useful tool as a protist host model of F. novicida. Experimental infection with F. novicida resulted in a stable intracellular relationship within P. bursaria. This symbiotic intracellular relationship was not observed in experimental infections with other Francisella species and Legionella pneumophila. We found that F. novicida showed similar behaviour to that of the eukaryotic endosymbiont of P. bursaria, the green algae Chlorella, in the internalization process. In addition, stable intracellular localization of F. novicida was possible only when Chlorella was not present. Although we investigated the type VI secretion system of F. novicida as a candidate for the bacterial factor, we found that it was not involved in the establishment of an intracellular relationship with P. bursaria. These results suggested that P. bursaria is potentially a protist host model for F. novicida and may be a useful tool for understanding the relationship between protist hosts and their symbionts.

Presence of Francisella tularensis subsp. holarctica DNA in the Aquatic Environment in France.

In 2018, the incidence of tularemia increased twofold in the west of France, with many pneumonic forms, suggesting environmental sources of infection. We investigated the presence of Francisella tularensis subsp. holarctica and other Francisella species DNA in the natural aquatic environment of this geographic area. Two sampling campaigns, in July 2019 and January 2020, allowed the collection of 87 water samples. Using a combination of real-time PCR assays, we tested the presence of either Francisella sp., F. tularensis/F. novicida, and F. tularensis subsp. holarctica, the latter being the only tularemia agent in Europe. Among 57 water samples of the first campaign, 15 (26.3%) were positive for Francisella sp., nine (15.8%) for F. tularensis and/or F. novicida, and four (7.0%) for F. tularensis subsp. holarctica. Ratios were 25/30 (83.3%), 24/30 (80.0%), and 4/30 (13.3%) for the second campaign. Among the thirty sites sampled during the two campaigns, nine were positive both times for Francisella sp., seven for F. tularensis and/or F. novicida, and one for F. tularensis subsp. holarctica. Altogether, our study reveals a high prevalence of Francisella sp. DNA (including the tularemia agent) in the studied aquatic environment. This aquatic environment could therefore participate in the endemicity of tularemia in the west of France.

Novel Genotypes of Nidicolous Argas Ticks and Their Associated Microorganisms From Spain.

The knowledge of the distribution, richness and epidemiological importance of soft ticks of the genus Argas is incomplete. In Spain, five Argas species have been recorded, including three ornitophilic nidicolous ticks, but their associated microorganisms remain unknown. This study aimed to investigate ticks from bird nests and their microorganisms. Ticks were collected extensively from natural cavities and nest-boxes used by European rollers (Coracias garrulus) and little owls (Athene noctua) in Southeastern and Central Spain. Ticks were morphologically and genetically identified and corresponding DNA/RNA tick extracts were analyzed [individually (n = 150) or pooled (n = 43)] using specific PCR assays for bacteria (Anaplasmataceae, Bartonella, Borrelia, Coxiella/Rickettsiella, and Rickettsia spp.), viruses (Flaviviruses, Orthonairoviruses, and Phenuiviruses), and protozoa (Babesia/Theileria spp.). Six Argas genotypes were identified, of which only those of Argas reflexus (n = 8) were identified to the species level. Two other genotypes were closely related to each other and to Argas vulgaris (n = 83) and Argas polonicus (n = 33), respectively. These two species have not been previously reported from Western Europe. Two additional genotypes (n = 4) clustered with Argas persicus, previously reported in Spain. The remaining genotype (n = 22) showed low sequence identity with any Argas species, being most similar to the African Argas africolumbae. The microbiological screening revealed infection with a rickettsial strain belonging to Rickettsia fournieri and Candidatus Rickettsia vini group in 74.7% of ticks, mainly comprising ticks genetically related to A. vulgaris and A. polonicus. Other tick endosymbionts belonging to Coxiella, Francisella and Rickettsiella species were detected in ten, one and one tick pools, respectively. In addition, one Babesia genotype, closely related to avian Babesia species, was found in one tick pool. Lastly, Anaplasmataceae, Bartonella, Borrelia, and viruses were not detected. In conclusion, five novel Argas genotypes and their associated microorganisms with unproven pathogenicity are reported for Spain. The re-use of nests between and within years by different bird species appears to be ideal for the transmission of tick-borne microorganisms in cavity-nesting birds of semiarid areas. Further work should be performed to clarify the taxonomy and the potential role of soft Argas ticks and their microorganisms in the epidemiology of zoonoses.

First Description of a Temperate Bacteriophage (vB_FhiM_KIRK) of Francisella hispaniensis Strain 3523.

Here we present the characterization of a Francisella bacteriophage (vB_FhiM_KIRK) including the morphology, the genome sequence and the induction of the prophage. The prophage sequence (FhaGI-1) has previously been identified in F. hispaniensis strain 3523. UV radiation induced the prophage to assemble phage particles consisting of an icosahedral head (~52 nm in diameter), a tail of up to 97 nm in length and a mean width of 9 nm. The double stranded genome of vB_FhiM_KIRK contains 51 open reading frames and is 34,259 bp in length. The genotypic and phylogenetic analysis indicated that this phage seems to belong to the Myoviridae family of bacteriophages. Under the conditions tested here, host cell (Francisella hispaniensis 3523) lysis activity of KIRK was very low, and the phage particles seem to be defective for infecting new bacterial cells. Nevertheless, recombinant KIRK DNA was able to integrate site-specifically into the genome of different Francisella species after DNA transformation.

Amoebae can promote the survival of Francisella species in the aquatic environment

ABSTRACT Francisella tularensis, a tier 1 select agent, is the causative bacterium of tularemia, a zoonosis with a large animal reservoir. However, F. tularensis, like many other Francisella species, is assumed to have an aquatic reservoir. The mechanisms of Francisella species persistence in surface water remain poorly characterized. In this study, we deeply investigated the long-term interactions of the tularemia agent F. tularensis subsp. holarctica, F. novicida or F. philomiragia with amoebae of the Acanthamoeba species. In amoeba plate screening tests, all the Francisella species tested resisted the attack by amoebae. In in vitro infection models, intra-amoebic growth of Francisella varied according to the involved bacterial species and strains, but also the amoeba culture medium used. In co-culture models, the amoebae favoured Francisella survival over 16 days, which was likely dependent on direct contact between bacteria and amoebae for F. novicida and on amoeba-excreted compounds for F. novicida and for F. tularensis. In a spring water co-culture model, amoebae again enhanced F. novicida survival and preserved bacterial morphology. Overall, our results demonstrate that amoebae likely promote Francisella survival in aquatic environments, including the tularemia agent F. tularensis. However, bacteria-amoebae interactions are complex and depend on the Francisella species considered.

Illuminating the bacterial microbiome of Australian ticks with 16S and Rickettsia-specific next-generation sequencing

Next-generation sequencing (NGS) studies show that mosquito and tick microbiomes influence the transmission of pathogens, opening new avenues for vector-borne pathogen control. Recent microbiological studies of Australian ticks highlight fundamental knowledge gaps of tick-borne agents. This investigation explored the composition, diversity and prevalence of bacteria in Australian ticks (n = 655) from companion animals (dogs, cats and horses). Bacterial 16S NGS was used to identify most bacterial taxa and a Rickettsia-specific NGS assay was developed to identify Rickettsia species that were indistinguishable at the V1-2 regions of 16S. Sanger sequencing of near full-length 16S was used to confirm whether species detected by 16S NGS were novel. The haemotropic bacterial pathogens Anaplasma platys, Bartonella clarridgeiae, “Candidatus Mycoplasma haematoparvum” and Coxiella burnetii were identified in Rhipicephalus sanguineus (s.l.) from Queensland (QLD), Western Australia, the Northern Territory (NT), and South Australia, Ixodes holocyclus from QLD, Rh. sanguineus (s.l.) from the NT, and I. holocyclus from QLD, respectively. Analysis of the control data showed that cross-talk compromises the detection of rare species as filtering thresholds for less abundant sequences had to be applied to mitigate false positives. A comparison of the taxonomic assignments made with 16S sequence databases revealed inconsistencies. The Rickettsia-specific citrate synthase gene NGS assay enabled the identification of Rickettsia co-infections with potentially novel species and genotypes most similar (97.9–99.1%) to Rickettsia raoultii and Rickettsia gravesii. “Candidatus Rickettsia jingxinensis” was identified for the first time in Australia. Phylogenetic analysis of near full-length 16S sequences confirmed a novel Coxiellaceae genus and species, two novel Francisella species, and two novel Francisella genotypes. Cross-talk raises concerns for the MiSeq platform as a diagnostic tool for clinical samples. This study provides recommendations for adjustments to Illuminaʼs 16S metagenomic sequencing protocol that help track and reduce cross-talk from cross-contamination during library preparation. The inconsistencies in taxonomic assignment emphasise the need for curated and quality-checked sequence databases.

Alternative pathways utilize or circumvent putrescine for biosynthesis of putrescine-containing rhizoferrin

The siderophore rhizoferrin (N1,N4-dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure.

Behavioral characteristics and endosymbionts of two potential tularemia and Rocky Mountain spotted fever tick vectors.

Due to climate change-induced alterations of temperature and humidity, the distribution of pathogen-carrying organisms such as ticks may shift. Tick survival is often limited by environmental factors such as dryness, but a predicted hotter and wetter world may allow the expansion of tick ranges. Dermacentor andersoni and D. variabilis ticks are morphologically similar, co-occur throughout the Inland Northwest of Washington State, U.S.A., and both can be injected with pathogenic Rickettsia and Francisella bacteria. Differences in behavior and the potential role of endosymbiotic Rickettsia and Francisella in these ticks are poorly studied. We wanted to measure behavioral and ecological differences between the two species and determine which, if any, Rickettsia and Francisella bacteria - pathogenic or endosymbiotic - they carried. Additionally, we wanted to determine if either tick species may be selected for if the climate in eastern Washington becomes wetter or dryer. We found that D. andersoni is more resistant to desiccation, but both species share similar questing behaviors such as climbing and attraction to bright light. Both also avoid the odor of eucalyptus and DEET but not permethrin. Although both tick species are capable of transmitting pathogenic species of Francisella and Rickettsia, which cause tularemia and Rocky Mountain Spotted Fever, respectively, we found primarily non-pathogenic endosymbiotic strains of Francisella and Rickettsia, and only one tick infected with F. tularensis subspecies holarctica.

Francisella tularensis Subspecies holarctica and Tularemia in Germany.

Tularemia is a zoonotic disease caused by Francisella tularensis a small, pleomorphic, facultative intracellular bacterium. In Europe, infections in animals and humans are caused mainly by Francisella tularensis subspecies holarctica. Humans can be exposed to the pathogen directly and indirectly through contact with sick animals, carcasses, mosquitoes and ticks, environmental sources such as contaminated water or soil, and food. So far, F. tularensis subsp. holarctica is the only Francisella species known to cause tularemia in Germany. On the basis of surveillance data, outbreak investigations, and literature, we review herein the epidemiological situation—noteworthy clinical cases next to genetic diversity of F. tularensis subsp. holarctica strains isolated from patients. In the last 15 years, the yearly number of notified cases of tularemia has increased steadily in Germany, suggesting that the disease is re-emerging. By sequencing F. tularensis subsp. holarctica genomes, knowledge has been added to recent findings, completing the picture of genotypic diversity and geographical segregation of Francisella clades in Germany. Here, we also shortly summarize the current knowledge about a new Francisella species (Francisella sp. strain W12-1067) that has been recently identified in Germany. This species is the second Francisella species discovered in Germany.

Increased Sensitivity of Amoeba-Grown Francisella Species to Disinfectants.

Francisella tularensis is a highly infectious, intracellular bacterium and it is the causative agent of tularemia. The bacterium has been isolated from more than 250 species, including protozoa. Previous studies have shown that the growth of Legionella pneumophila within the amoeba results in a dramatic increase in the resistance to disinfectants. Since Francisella persists in the environment for years, this study investigates whether Acanthamoeba castellanii-grown F. novicida exhibits an alteration in the resistance to disinfectants. The disinfectants used are didecyldimethylammonium chloride (DDAC) combined with isopropyl alcohol (D1), benzalkonium chloride combined with DDAC and formic acid (D2), and polyhexamethylene biguanide (PHMB, D3). The effect of disinfectants on the bacterial viability is determined by a colony-forming unit (CFU), by transmission electron microscopy (TEM), by fluorescence microscopy, and the damage of the bacterial membrane. Our data has shown that only a one-log10 loss in bacterial viability is exhibited upon treatment of agar-grown Francisella, while in amoeba-grown Francisella there was a three-log10 difference with D3. The D1 disinfectant sterilized the bacteria within 10 s. The treatment of agar-grown F. novicida with D2 reduces bacterial viability by seven-log10 within 10 s and 15 min, respectively. Surprisingly, the treatment of amoeba-grown F. novicida with D2 results in a total loss of bacterial viability. In conclusion, A. castellanii-grown F. novicida is more susceptible to many disinfectants.