Genomic trajectories to fluoroquinolone resistance in Francisella tularensis subsp. holarctica live vaccine strain.

OBJECTIVES: Fluoroquinolone (FQ)-resistant mutants were previously selected from the live vaccine strain (LVS) of Francisella tularensis (F. tularensis) subsp. holarctica. This study further characterised all genetic changes that occurred in these mutants during the evolutionary trajectory toward high-level FQ resistance, and their potential impact on F. tularensis antibiotic resistance and intracellular fitness. METHODS: The whole genomes of FQ-resistant mutants were determined and compared with those of their parental strain. All detected mutations were evaluated for their potential impact on FQ resistance and intracellular multiplication of F. tularensis. RESULTS: As compared with the parental LVS genome, 28 mutations were found in the derived FQ-resistant mutants. These mutations involved all genes encoding type II topoisomerases (i.e. gyrA, gyrB, parC, and parE). Interestingly, some of them were not previously associated with FQ resistance, warranting further characterisation. Mutations associated with FQ resistance were also found in other genes, including three encoding proteins involved in transport processes. Most of the detected mutations did not alter multiplication of the corresponding mutants in J774 cells. In contrast, all mutations at locus FTL_0439 encoding FupA/B, a membrane protein involved in iron transport, were associated with FQ resistance and fitness loss. CONCLUSION: FQ resistance in F. tularensis is complex and may involve single or combined mutations in genes encoding type II topoisomerases, transport systems and FupA/B. In vivo studies are now required to assess the potential role of these mutations in FQ treatment failures.

Francisella novicida Two-Component System Response Regulator BfpR Modulates iglC Gene Expression, Antimicrobial Peptide Resistance, and Biofilm Production.

Response regulators are a critical part of the two-component system of gene expression regulation in bacteria, transferring a signal from a sensor kinase into DNA binding activity resulting in alteration of gene expression. In this study, we investigated a previously uncharacterized response regulator in Francisella novicida, FTN_1452 that we have named BfpR (Biofilm-regulating Francisella protein Regulator, FTN_1452). In contrast to another Francisella response regulator, QseB/PmrA, BfpR appears to be a negative regulator of biofilm production, and also a positive regulator of antimicrobial peptide resistance in this bacterium. The protein was crystallized and X-ray crystallography studies produced a 1.8 Å structure of the BfpR N-terminal receiver domain revealing interesting insight into its potential interaction with the sensor kinase. Structural analysis of BfpR places it in the OmpR/PhoP family of bacterial response regulators along with WalR and ResD. Proteomic and transcriptomic analyses suggest that BfpR overexpression affects expression of the critical Francisella virulence factor iglC, as well as other proteins in the bacterium. We demonstrate that mutation of bfpR is associated with an antimicrobial peptide resistance phenotype, a phenotype also associated with other response regulators, for the human cathelicidin peptide LL-37 and a sheep antimicrobial peptide SMAP-29. F. novicida with mutated bfpR replicated better than WT in intracellular infection assays in human-derived macrophages suggesting that the down-regulation of iglC expression in bfpR mutant may enable this intracellular replication to occur. Response regulators have been shown to play important roles in the regulation of bacterial biofilm production. We demonstrate that F. novicida biofilm formation was highly increased in the bfpR mutant, corresponding to altered glycogen synthesis. Waxworm infection experiments suggest a role of BfpR as a negative modulator of iglC expression with de-repression by Mg2+. In this study, we find that the response regulator BfpR may be a negative regulator of biofilm formation, and a positive regulator of antimicrobial peptide resistance in F. novicida.

Gallium as an Antibacterial Agent: A DFT/SMD Study of the Ga3+/Fe3+ Competition for Binding Bacterial Siderophores.

The urgency of finding novel antibacterial drugs (not only antibiotics), exhibiting different mechanisms of therapeutic action, is significant and has served as a premise for recognizing bacteria's siderophores as a plausible drug target. Bacteria secrete siderophores in order to sequester iron(III) from the surrounding medium by binding the essential metal with high affinity. Gallium, on the other hand, is an "abiogenic" ion, known for its anticancer, antibacterial, and anti-inflammatory action. The rationale behind its therapeutic effect lies in its close mimicry of the ferric ion. Since both ions share various physicochemical characteristics, it is of particular interest to understand if gallium could compete with the native ferric ion for binding siderophores and to decipher which molecular characteristics favor Ga3+ binding over Fe3+ binding. It is also well-known that some bacteria are susceptible to gallium-based therapy, while others are not. Therefore, many questions arise such as the following: (1) Which main group/groups building the siderophores promote gallium's attack? (2) Does the combination of the building blocks affect the preference toward a metal? (3) Does the environment play a crucial role? (4) Could the pH of the medium influence the balance between the ions? We try to address these questions by evaluating the free energy of the competition between Ga3+ and Fe3+ ions for siderophore ligands of various structures, denticities, and charge states by employing the tools of the computational chemistry at the DFT/SMD level. Our results not only fall in line with recent experimental data but also complement our knowledge about "Trojan horse" gallium-based therapy.

A Survey of Antimicrobial Resistance Determinants in Category A Select Agents, Exempt Strains, and Near-Neighbor Species.

A dramatic increase in global antimicrobial resistance (AMR) has been well documented. Of particular concern is the dearth of information regarding the spectrum and prevalence of AMR within Category A Select Agents. Here, we performed a survey of horizontally and vertically transferred AMR determinants among Category A agents and their near neighbors. Microarrays provided broad spectrum screening of 127 Francisella spp., Yersinia spp., and Bacillus spp. strains for the presence/absence of 500+ AMR genes (or families of genes). Detecting a broad variety of AMR genes in each genus, microarray analysis also picked up the presence of an engineered plasmid in a Y. pestis strain. High resolution melt analysis (HRMA) was also used to assess the presence of quinolone resistance-associated mutations in 100 of these strains. Though HRMA was able to detect resistance-causing point mutations in B. anthracis strains, it was not capable of discriminating these point mutations from other nucleotide substitutions (e.g., arising from sequence differences in near neighbors). Though these technologies are well-established, to our knowledge, this is the largest survey of Category A agents and their near-neighbor species for genes covering multiple mechanisms of AMR.

Francisella novicida and F. philomiragia biofilm features conditionning fitness in spring water and in presence of antibiotics.

Biofilms are currently considered as a predominant lifestyle of many bacteria in nature. While they promote survival of microbes, biofilms also potentially increase the threats to animal and public health in case of pathogenic species. They not only facilitate bacteria transmission and persistence, but also promote spreading of antibiotic resistance leading to chronic infections. In the case of Francisella tularensis, the causative agent of tularemia, biofilms have remained largely enigmatic. Here, applying live and static confocal microscopy, we report growth and ultrastructural organization of the biofilms formed in vitro by these microorganisms over the early transition from coccobacillary into coccoid shape during biofilm assembly. Using selective dispersing agents, we provided evidence for extracellular DNA (eDNA) being a major and conserved structural component of mature biofilms formed by both F. subsp. novicida and a human clinical isolate of F. philomiragia. We also observed a higher physical robustness of F. novicida biofilm as compared to F. philomiragia one, a feature likely promoted by specific polysaccharides. Further, F. novicida biofilms resisted significantly better to ciprofloxacin than their planktonic counterparts. Importantly, when grown in biofilms, both Francisella species survived longer in cold water as compared to free-living bacteria, a trait possibly associated with a gain in fitness in the natural aquatic environment. Overall, this study provides information on survival of Francisella when embedded with biofilms that should improve both the future management of biofilm-related infections and the design of effective strategies to tackle down the problematic issue of bacteria persistence in aquatic ecosystems.

Larva of greater wax moth Galleria mellonella is a suitable alternative host for the fish pathogen Francisella noatunensis subsp. orientalis.

BACKGROUND: Francisella noatunensis subsp. orientalis (Fno) is the etiological agent of francisellosis in cultured warm water fish, such as tilapia. Antibiotics are administered to treat the disease but a better understanding of Fno infection biology will inform improved treatment and prevention measures. However, studies with native hosts are costly and considerable benefits would derive from access to a practical alternative host. Here, larvae of Galleria mellonella were assessed for suitability to study Fno virulence. RESULTS: Larvae were killed by Fno in a dose-dependent manner but the insects could be rescued from lethal doses of bacteria by antibiotic therapy. Infection progression was assessed by histopathology (haematoxylin and eosin staining, Gram Twort and immunohistochemistry) and enumeration of bacteria recovered from the larval haemolymph on selective agar. Fno was phagocytosed and could survive intracellularly, which is consistent with observations in fish. Virulence of five Fno isolates showed strong agreement between G. mellonella and red Nile tilapia hosts. CONCLUSIONS: This study shows that an alternative host, G. mellonella, can be applied to understand Fno infections, which will assist efforts to identify solutions to piscine francisellosis thus securing the livelihoods of tilapia farmers worldwide and ensuring the production of this important food source.

Influence of pH on the activity of finafloxacin against extracellular and intracellular Burkholderia thailandensis, Yersinia pseudotuberculosis and Francisella philomiragia and on its cellular pharmacokinetics in THP-1 monocytes.

OBJECTIVES: Burkholderia pseudomallei, Yersinia pestis and Francisella tularensis are facultative intracellular bacteria causing life-threatening infections. We have (a) compared the activity of finafloxacin (a fluoroquinolone in development showing improved activity at acidic pH) with that of ciprofloxacin, levofloxacin and imipenem against the extracellular and intracellular (THP-1 monocytes) forms of infection by attenuated surrogates of these species (B. thailandensis, Y. pseudotuberculosis, F. philomiragia) and (b) assessed finafloxacin cellular pharmacokinetics (accumulation, distribution, efflux). METHODS: Bacteria in broth or in infected monocytes were exposed to antibiotics at pH 7.4 or 5.5 for 24 hr. Maximal relative efficacies (Emax) and static concentrations (Cs) were calculated using the Hill equation (concentration-response curves). Finafloxacin pharmacokinetics in cells at pH 7.4 or 5.5 was investigated using 14C-labelled drug. RESULTS: Extracellularly, all drugs sterilized the cultures, with finafloxacin being two to six times more potent at acidic pH. Intracellularly, Emax reached the limit of detection (4-5 log10 cfu decrease) for finafloxacin against all species, but only against B. thailandensis and F. philomiragia for ciprofloxacin and levofloxacin, while imipenem caused less than 2 log10 cfu decrease for all species. At acid pH, Cs shifted to two to five times lower values for finafloxacin and to one to four times higher values for the other drugs. Finafloxacin accumulated in THP-1 cells by approximately fivefold at pH 7.4 but up to 20-fold at pH 5.5, and distributed in the cytosol. CONCLUSIONS: Fluoroquinolones have proven to be effective in reducing the intracellular reservoirs of B. thailandensis, Y. pseudotuberculosis and F. philomiragia, with finafloxacin demonstrating an additional advantage in acidic environments.

Effects of a phytogenic, alone and associated with potassium diformate, on tilapia growth, immunity, gut microbiome and resistance against francisellosis.

This work evaluated the effects of dietary supplementation of A-Live (phytogenic) either individually or in combination with Aquaform (potassium diformate, acidifier) on juvenile Nile tilapia (Oreochromis niloticus) growth performance, innate immune parameters, gut microbiome, and resistance against Francisella noatunensis subsp. orientalis challenge. Each experimental group contained 140 fishes (34.3 ± 0.33) in two 150L tanks. The experimental design consisted of five groups: a negative control; treated groups (G1, G2, G3) supplemented with different concentrations of A-Live and Aquaform in the feed; and a positive control (PC) for pathogen infection. Groups G1, G2, G3, and PC were challenged with Francisella spp. after 15 days. After infection, the mortality was significantly lower in groups G1, G2, and G3 (p < 0.01). Furthermore, these groups showed significant increase (p < 0.05) in daily weight gain, feed conversion rate, and specific growth rate. The PC group presented increase (p < 0.05) in the leukocytes and neutrophils number. Innate immunity parameters showed no difference between treatments after infection. Microbiome analysis revealed an increased number of bacteria belonging to the Vibrionaceae family after pathogen infection suggesting a secondary pathogen function of these bacteria. These results validate the beneficial effects of these products in tilapia farming.

Macrophage-targeted drugamers with enzyme-cleavable linkers deliver high intracellular drug dosing and sustained drug pharmacokinetics against alveolar pulmonary infections.

Intracellular bacterial infections localized to the lung alveolar macrophage (AM) remain one of the most challenging settings for antimicrobial therapy. Current systemic antibiotic treatment fails to deliver sustained doses to intracellular bacterial reservoirs, which necessitates prolonged treatment regimens. Herein, we demonstrate a new intracellular enzyme-cleavable polymeric prodrug with tailored ciprofloxacin release profiles in the lungs and AM. The targeted polymeric prodrug, termed "drugamers", incorporates (1) hydrophilic mannose residues to solubilize the antibiotic cargo and to target and enhance AM uptake and intracellular delivery, and (2) enzyme-cleavable linkage chemistry to provide high and sustained intracellular AM drug dosing. Prodrug monomers, derived from the antibiotic ciprofloxacin, were synthesized with either an intracellular protease cleavable dipeptide linker or a hydrolytic phenyl ester linker. RAFT polymerization was used to copolymerize the prodrug monomers and mannose monomer to synthesize well-defined drugamers without requiring a post-polymerization conjugation step. In addition to favorable in vivo safety profiles following intratracheal administration, a single dose of the drugamers sustained ciprofloxacin dosing in lungs and AMs above the minimum inhibitory concentration (MIC) over at least a 48 h period. The enzyme-cleavable therapeutic achieved a >10-fold increase in sustained ciprofloxacin in AM, and maintained a significantly higher whole lung PK as well. Ciprofloxacin dosed in identical fashion displayed rapid clearance with a half-life of approximately 30 min. Notably, inhalation of the mannose-targeted ciprofloxacin drugamers achieved full survival (100%) in a highly lethal mouse model of pneumonic tularemia, contrasted with 0% survival using free ciprofloxacin. These findings demonstrate the versatility of the drugamer platform for engineering the intracellular pharmacokinetic profiles and its strong therapeutic activity in treating pulmonary intracellular infections.

Nramp1 and NrampB Contribute to Resistance against Francisella in Dictyostelium.

The Francisella genus comprises highly pathogenic bacteria that can cause fatal disease in their vertebrate and invertebrate hosts including humans. In general, Francisella growth depends on iron availability, hence, iron homeostasis must be tightly regulated during Francisella infection. We used the system of the professional phagocyte Dictyostelium and the fish pathogen F. noatunensis subsp. noatunensis (F.n.n.) to investigate the role of the host cell iron transporters Nramp (natural resistance associated macrophage proteins) during Francisella infection. Like its mammalian ortholog, Dictyostelium Nramp1 transports iron from the phagosome into the cytosol, whereas the paralog NrampB is located on the contractile vacuole and controls, together with Nramp1, the cellular iron homeostasis. In Dictyostelium, Nramp1 localized to the F.n.n.-phagosome but disappeared from the compartment dependent on the presence of IglC, an established Francisella virulence factor. In the absence of Nramp transporters the bacteria translocated more efficiently from the phagosome into the host cell cytosol, its replicative niche. Increased escape rates coincided with increased proteolytic activity in bead-containing phagosomes indicating a role of the Nramp transporters for phagosomal maturation. In the nramp mutants, a higher bacterial load was observed in the replicative phase compared to wild-type host cells. Upon bacterial access to the cytosol of wt cells, mRNA levels of bacterial iron uptake factors were transiently upregulated. Decreased iron levels in the nramp mutants were compensated by a prolonged upregulation of the iron scavenging system. These results show that Nramps contribute to host cell immunity against Francisella infection by influencing the translocation efficiency from the phagosome to the cytosol but not by restricting access to nutritional iron in the cytosol.

Immunomodulatory properties of Concholepas concholepas hemocyanin against francisellosis in a zebrafish model.

The development of vaccines for aquaculture has been an important milestone in providing a continuous and sustainable production. Most of the vaccines currently on the market for aquaculture include oil as adjuvant. Nevertheless, several studies reported an occurrence of side effects after their use in farmed fish. As a result, there is a need for new and improved adjuvants that can stimulate the immune system while causing as few side-effects as possible. Hemocyanins are versatile macromolecules with strong immunogenic and immunomodulatory properties. Due to these characteristics, hemocyanin from Concholepas concholepas (CCH) has been biochemically characterized and evaluated as vaccine adjuvant in mice and humans. Francisellosis is a chronic granulomatous disease, which can result in high mortality depending on the host. The disease is caused by the facultative intracellular Gram-negative bacteria Francisella noatunensis, which remains an unsolved problem for the aquaculture, as no efficient vaccines are available. The aim of the present work was to investigate the immunoregulatory properties of CCH against francisellosis in an experimental zebrafish model. When immunized with CCH, zebrafish were protected from subsequent challenge with a lethal dose of Francisella noatunensis subsp. orientalis. Subsequently the mRNA expression levels of several immune-related genes were studied, including mhcii, il12a, tnfα and ifng1-1. Taken together, the data report the immunoregulatory properties of CCH and its potential use as a vaccine adjuvant for aquaculture.

Effects of lipid A acyltransferases on the pathogenesis of F. novicida.

Francisella novicida is a gram-negative pathogen commonly used to study infections by the potential bioterrorism agent, Francisella tularensis. The Francisella lipid A structure has been well characterized and showed to affect the pathogenesis of F. novicida. Previous work characterized two lipid A acyltransferases, LpxD1 and LpxD2, and constructed the lpxD1-null and lpxD2-null mutants. Mutational analysis showed the lpxD1-null mutant was attenuated in mice and subsequently exhibited protection against a lethal WT challenge. However, details as how the virulence has been changed have remained elusive. This study aims to analyze effects of lipid A acyltransferases on the pathogenesis of F. novicida. MS and MSn were conducted to confirm the lipid A structures of lpxD1-null and lpxD2-null mutants. The stress tolerance, Toll-like receptor 4 (TLR4) stimulation level, intracellular survival and replication ability and cytotoxicity of lpxD1-null and lpxD2-null mutants were analyzed. The results suggested the lpxD1-null mutant with shorter acyl chains in lipid A is more sensitive to various environmental stresses than F. novicida and lpxD2-null mutant. In addition, the lpxD1-null mutant fails to survive and replicate in cells and shows lower cytotoxicity to infected cells. This study provides insights into the pathogenesis of F. novicida.

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida.

Fluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in Francisella novicida clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the in vitro FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were compared to the MICs of FQs against bacterial clones harboring the same mutations and were further validated through complementation experiments and structural modeling. Whole-genome sequencing of highly FQ-resistant lineages was also done. Supercoiling and DNA cleavage assays demonstrated that GyrA D87 is a hot spot FQ resistance target in F. novicida and pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition in F. novicida is that the first-step mutation occurs in GyrB, with direct or indirect consequences for FQ sensitivity. Insertion of P466 into GyrB leads to a 50% inhibitory concentration (IC50) comparable to that observed for a mutant gyrase carrying the GyrA D87Y substitution, while the D487E-ΔK488 mutation, while not active on its own, contributes to the high level of resistance that occurs following acquisition of the GyrA D87G substitution in double GyrA/GyrB mutants. The involvement of other putative targets is discussed, including that of a ParE mutation that was found to arise in the very late stage of antibiotic exposure. This study provides the first characterization of the molecular mechanisms responsible for FQ resistance in Francisella.

Francisella noatunensis subsp. noatunensis invades, survives and replicates in Atlantic cod cells.

Systemic infection caused by the facultative intracellular bacterium Francisella noatunensis subsp. noatunensis remains a disease threat to Atlantic cod Gadus morhua L. Future prophylactics could benefit from better knowledge on how the bacterium invades, survives and establishes infection in its host cells. Here, facilitated by the use of a gentamicin protection assay, this was studied in primary monocyte/macrophage cultures and an epithelial-like cell line derived from Atlantic cod larvae (ACL cells). The results showed that F. noatunensis subsp. noatunensis is able to invade primary monocyte/macrophages, and that the actin-polymerisation inhibitor cytochalasin D blocked internalisation, demonstrating that the invasion is mediated through phagocytosis. Interferon gamma (IFNγ) treatment of cod macrophages prior to infection enhanced bacterial invasion, potentially by stimulating macrophage activation in an early step in host defence against F. noatunensis subsp. noatunensis infections. We measured a rapid drop of the initial high levels of internalised bacteria in macrophages, indicating the presence and action of a cellular immune defence mechanism before intracellular bacterial replication took place. Low levels of bacterial internalisation and replication were detected in the epithelial-like ACL cells. The capacity of F. noatunensis subsp. noatunensis to enter, survive and even replicate within an epithelial cell line may play an important role in its ability to infect live fish and transverse epithelial barriers to reach the bacterium's main target cells-the macrophage.

Improved Broth Microdilution Method for Antimicrobial Susceptibility Testing of Francisella Noatunensis Orientalis.

In this project we optimized a minimal inhibitory concentration testing protocol for Francisella noatunensis orientalis. Thirty-three F. noatunensis orientalis isolates recovered from different fish species and locations were tested, and Escherichia coli ATCC 25922 was used as a quality control reference strain. A modified cation-adjusted Mueller Hinton broth supplemented with 2% IsoVitalex and 0.1% glucose (MMH) was tested at a pH of 6.4 ± 0.1, 7.1 ± 0.1, and 7.3 ± 0.1. Growth curves generated for F. noatunensis orientalis indicated that MMH at a pH of 6.4 ± 0.1 provided optimal growth. There were no significant differences in the growth curves obtained from isolates recovered from different fish species or from fresh or marine water. The pH of 6.4 ± 0.1 in the MMH media interfered with the inhibitory properties of the potentiated sulfonamides (ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole) when using the E. coli ATCC reference strain. Minimal inhibitory concentrations of eight antimicrobials (gentamicin, enrofloxacin, ampicillin, oxytetracycline, erythromycin, florfenicol, flumequine, and oxolinic acid) were similar for all F. noatunensis orientalis isolates. The in vitro susceptibility data provided here can provide a baseline for monitoring the development of antimicrobial resistance among F. noatunensis orientalis isolates, as well as provide valuable data in the development of potential therapeutics. Received October 27, 2015; accepted April 13, 2016.

Biofilm formation of Francisella noatunensis subsp. orientalis.

Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC) and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon(®), bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in the iglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums.

Acyl carrier protein is a bacterial cytoplasmic target of cationic antimicrobial peptide LL-37.

In addition to membrane disruption, the cathelicidin antimicrobial peptide (AMP) LL-37 translocates through the bacterial inner membrane to target intracellular molecules. The present study aims to identify an alternate mechanism and a cytoplasmic target of LL-37 in Francisella. LL-37 binding proteins from Francisella novicida U112 bacterial lysates were precipitated by using biotinylated LL-37 (B-LL-37) and NeutrAvidin-agarose beads. Bound proteins were identified by LC-MS/MS, validated and characterized by bead pull-down assays and differential scanning fluorimetry (DSF). The cationic AMP (CAMP) LL-37 was able to interact with Francisella cytoplasmic acyl carrier protein (AcpP; FTN1340/FTT1376). Further study confirmed that LL-37 peptide could bind to AcpP and that the sheep cathelicidin SMAP-29 (Sheep Myeloid Antimicrobial Peptide 29) further increased LL-37 binding to AcpP, suggesting a synergistic effect of SMAP-29 on the binding. LL-37 could also bind to both AcpP of Escherichia coli and Bacillus anthracis, implying a mechanism of broad action of LL-37-AcpP binding. Overexpression of the acpP gene in F. novicida led to an increase in LL-37 susceptibility. LL-37 binding to AcpP changed the fatty acid composition profiles. Taken together, we identified a novel cytoplasmic target of LL-37 in Francisella, suggesting a mechanism of action of this peptide beyond membrane permeabilization. Our findings highlight a novel mechanism of antimicrobial activity of this peptide and document a previously unexplored target of α-helical CAMPs.

Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase dependent activity in Francisella novicida.

Development of new therapeutics against Select Agents such as Francisella is critical preparation in the event of bioterrorism. Testing FDA-approved drugs for this purpose may yield new activities unrelated to their intended purpose and may hasten the discovery of new therapeutics. A library of 420 FDA-approved drugs was screened for antibiofilm activity against a model organism for human tularemia, Francisella (F.) novicida, excluding drugs that significantly inhibited growth. The initial screen was based on the 2-component system (TCS) dependent biofilm effect, thus, the QseC dependence of maprotiline anti-biofilm action was demonstrated. By comparing their FDA-approved uses, chemical structures, and other properties of active drugs, toremifene and polycyclic antidepressants maprotiline and chlorpromazine were identified as being highly active against F. novicida biofilm formation. Further down-selection excluded toremifene for its membrane active activity and chlorpromazine for its high antimicrobial activity. The mode of action of maprotiline against F. novicida was sought. It was demonstrated that maprotiline was able to significantly down-regulate the expression of the virulence factor IglC, encoded on the Francisella Pathogenicity Island (FPI), suggesting that maprotiline is exerting an effect on bacterial virulence. Further studies showed that maprotiline significantly rescued F. novicida infected wax worm larvae. In vivo studies demonstrated that maprotiline treatment could prolong time to disease onset and survival in F. novicida infected mice. These results suggest that an FDA-approved drug such as maprotiline has the potential to combat Francisella infection as an antivirulence agent, and may have utility in combination with antibiotics.