Osmotic stress induces long-term biofilm survival in Liberibacter crescens.

Citrus greening, also known as Huanglongbing (HLB), is a devastating citrus plant disease caused predominantly by Liberibacter asiaticus. While nearly all Liberibacter species remain uncultured, here we used the culturable L. crescens BT-1 as a model to examine physiological changes in response to the variable osmotic conditions and nutrient availability encountered within the citrus host. Similarly, physiological responses to changes in growth temperature and dimethyl sulfoxide concentrations were also examined, due to their use in many of the currently employed therapies to control the spread of HLB. Sublethal heat stress was found to induce the expression of genes related to tryptophan biosynthesis, while repressing the expression of ribosomal proteins. Osmotic stress induces expression of transcriptional regulators involved in expression of extracellular structures, while repressing the biosynthesis of fatty acids and aromatic amino acids. The effects of osmotic stress were further evaluated by quantifying biofilm formation of L. crescens in presence of increasing sucrose concentrations at different stages of biofilm formation, where sucrose-induced osmotic stress delayed initial cell attachment while enhancing long-term biofilm viability. Our findings revealed that exposure to osmotic stress is a significant contributing factor to the long term survival of L. crescens and, possibly, to the pathogenicity of other Liberibacter species.

Lactobacillus johnsonii N6.2 and Blueberry Phytophenols Affect Lipidome and Gut Microbiota Composition of Rats Under High-Fat Diet.

Obesity is considered a primary contributing factor in the development of many diseases, including cancer, diabetes, and cardiovascular illnesses. Phytochemical-rich foods, associated to healthy gastrointestinal microbiota, have been shown to reduce obesity and associated comorbidities. In the present article, we describe the effects of the probiotic Lactobacillus johnsonii N6.2 and blueberry extracts (BB) on the gut microbiota and lipid profile of rats under a high-fat (HF) or low-calorie (LC) diet. L. johnsonii was found to increase the levels of long chain fatty acids (LCFA) in the serum of all animals under HF diet, while reduced LCFA concentrations were observed in the adipose tissue of animals under HF diet supplemented with BB extracts. All animals under HF diet also showed lower protein levels of SREBP1 and SCAP when treated with L. johnsonii. The gut microbiota diversity, ß-diversity was significantly changed by L. johnsonii in the presence of BB. A significant reduction in α-diversity was observed in the ileum of animals under HF diet supplemented with L. johnsonii and BB, while increased α-diversity was observed in the ilium of animals under LC diet supplemented with L. johnsonii or BB. In summary, L. johnsonii and BB supplementation induced significant changes in gut microbiota diversity and lipid metabolism. The phospholipids pool was the lipidome component directly affected by the interventions. The ileum and colon microbiota showed clear differences depending on the diet and the treatments examined.

Identification of Biomarkers for Systemic Distribution of Nanovesicles From Lactobacillus johnsonii N6.2.

The ability of bacterial extracellular vesicles (EV) to transport biological molecules has increased the research to determine their potential as therapeutic agents. In this study, Lactobacillus johnsonii N6.2-derived nanovesicles (NV) were characterized to identify components that may serve as biomarkers in host-microbe interactions. Comparative proteomic and lipidomic analyses of L. johnsonii N6.2 NV and cell membrane (CM) were performed. The lipidomic profiles indicated that both fractions contained similar lipids, however, significant differences were observed in several classes. LC-MS/MS proteomic analysis indicated that NV contained several unique and differentially expressed proteins when compared to the CM. Analysis of Gene Ontology (GO) terms, based on cellular component, showed significant enrichment of proteins in the cytoplasm/intracellular space category for the NV fraction. Based on these results, the proteins T285_RS00825 (named Sdp), Eno3 and LexA were selected for studies of localization and as potential biomarkers for host-microbe interactions. Immunogold staining, followed by scanning and transmission electron microscopy (SEM and TEM, respectively), revealed that Sdp was preferentially localized along the cell wall/membrane, and on NV-like structures surrounding the bacteria. These results were confirmed using immunofluorescence staining in Caco-2 cells incubated with NV. Consequently, we evaluated the potential for NV surface-exposed proteins to generate an immune response in the host. Plasma from individuals administered L. johnsonii N6.2 showed that IgA and IgG antibodies were generated against NV and Sdp domains in vivo. Altogether, these results show that L. johnsonii N6.2 NV have the potential to mediate host interactions through immune modulation.

PrbP modulates biofilm formation in Liberibacter crescens.

In Liberibacter asiaticus, PrbP is a transcriptional regulatory protein involved in survival and persistence during host infection. Tolfenamic acid was previously found to inhibit interactions between PrbP and the promotor region of rplK, resulting in reduced survival of L. asiaticus in the citrus host. In this study, we performed transcriptome analyses to elucidate the PrbP regulon in L. crescens, as it is phylogenetically the closest related species to L. asiaticus that can be grown in laboratory conditions. Chemical inhibition of PrbP with tolfenamic acid revealed that PrbP is involved in the regulation of diverse cellular processes, including stress response, cell motility, cell cycle and biofilm formation. In vitro DNA binding and bacterial two-hybrid assays also suggested that PrbP is a global regulator of multiple transcription factors (RpoH, VisN, PleD, MucR, MocR and CtrA) at both transcriptional and/or post-transcriptional levels. Sub-lethal concentrations of tolfenamic acid significantly reduced the attachment of L. crescens during biofilm formation and decreased long-term persistence in biofilm structures. Overall, our findings show the importance of PrbP in regulating diverse biological processes through direct and indirect interactions with other transcriptional regulators in L. crescens.

Assessment of unconventional antimicrobial compounds for the control of 'Candidatus Liberibacter asiaticus', the causative agent of citrus greening disease.

In this study, newly identified small molecules were examined for efficacy against 'Candidatus Liberibacter asiaticus' in commercial groves of sweet orange (Citrus sinensis) and white grapefruit (Citrus paradisi) trees. We used benzbromarone and/or tolfenamic acid delivered by trunk injection. We evaluated safety and efficacy parameters by performing RNAseq of the citrus host responses, 16S rRNA gene sequencing to characterize citrus-associated microbial communities during treatment, and qRT-PCR as an indirect determination of 'Ca. L. asiaticus' viability. Analyses of the C. sinensis transcriptome indicated that each treatment consistently induced genes associated with normal metabolism and growth, without compromising tree viability or negatively affecting the indigenous citrus-associated microbiota. It was found that treatment-associated reduction in 'Ca. L. asiaticus' was positively correlated with the proliferation of several core taxa related with citrus health. No symptoms of phytotoxicity were observed in any of the treated trees. Trials were also performed in commercial groves to examine the effect of each treatment on fruit productivity, juice quality and efficacy against 'Ca. L. asiaticus'. Increased fruit production (15%) was observed in C. paradisi following twelve months of treatment with benzbromarone and tolfenamic acid. These results were positively correlated with decreased 'Ca. L. asiaticus' transcriptional activity in root samples.

Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus.

Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L. asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeYLas was characterized as an endoribonuclease with activity on 3' and 5' termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeYLas protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site-directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeYLas. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeYLas . Luteolin was subsequently examined for efficacy against L. asiaticus in Huanglongbing-infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed.

Identification of the Tolfenamic Acid Binding Pocket in PrbP from Liberibacter asiaticus.

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that was found to regulate gene expression through interactions with the RNA polymerase ß-subunit and a specific sequence on the promoter region. It was found that inactivation of PrbP, using the inhibitor tolfenamic acid, resulted in a significant decrease in the overall transcriptional activity of L. asiaticus, and the suppression of L. asiaticus infection in HLB symptomatic citrus seedlings. The molecular interactions between PrbP and tolfenamic acid, however, were yet to be elucidated. In this study, we modeled the structure of PrbP and identified a ligand binding pocket, TaP, located at the interface of the predicted RNA polymerase interaction domain (N-terminus) and the DNA binding domain (C-terminus). The molecular interactions of PrbP with tolfenamic acid were predicted using in silico docking. Site-directed mutagenesis of specific amino acids was followed by electrophoresis mobility shift assays and in vitro transcription assays, where residues N107, G109, and E148 were identified as the primary amino acids involved in interactions with tolfenamic acid. These results provide insight into the binding mechanism of PrbP to a small inhibitory molecule, and a starting scaffold for the identification and development of therapeutics targeting PrbP and other homologs in the CarD_CdnL_TRCF family.

Functional characterization of LotP from Liberibacter asiaticus.

Liberibacter asiaticus is an unculturable parasitic bacterium of the alphaproteobacteria group hosted by both citrus plants and a psyllid insect vector (Diaphorina citri). In the citrus tree, the bacteria thrive only inside the phloem, causing a systemically incurable and deadly plant disease named citrus greening or Huanglongbing. Currently, all commercial citrus cultivars in production are susceptible to L. asiaticus, representing a serious threat to the citrus industry worldwide. The technical inability to isolate and culture L. asiaticus has hindered progress in understanding the biology of this bacterium directly. Consequently, a deep understanding of the biological pathways involved in the regulation of host-pathogen interactions becomes critical to rationally design future and necessary strategies of control. In this work, we used surrogate strains to evaluate the biochemical characteristics and biological significance of CLIBASIA_03135. This gene, highly induced during early stages of plant infection, encodes a 23 kDa protein and was renamed in this work as LotP. This protein belongs to an uncharacterized family of proteins with an overall structure resembling the LON protease N-terminus. Co-immunoprecipitation assays allowed us to identify the Liberibacter chaperonin GroEL as the main LotP-interacting protein. The specific interaction between LotP and GroEL was reconstructed and confirmed using a two-hybrid system in Escherichia coli. Furthermore, it was demonstrated that LotP has a native molecular weight of 44 kDa, corresponding to a dimer in solution with ATPase activity in vitro. In Liberibacter crescens, LotP is strongly induced in response to conditions with high osmolarity but repressed at high temperatures. Electrophoretic mobility shift assay (EMSA) results suggest that LotP is a member of the LdtR regulon and could play an important role in tolerance to osmotic stress.

Drug Repurposing: Tolfenamic Acid Inactivates PrbP, a Transcriptional Accessory Protein in Liberibacter asiaticus.

CLIBASIA_01510, PrbP, is a predicted RNA polymerase binding protein in Liberibacter asiaticus. PrbP was found to regulate expression of a small subset of ribosomal genes through interactions with the ß-subunit of the RNA polymerase and a short, specific sequence on the promoter region. Molecular screening assays were performed to identify small molecules that interact with PrbP in vitro. Chemical hits were analyzed for therapeutic efficacy against L. asiaticus via an infected leaf assay, where the transcriptional activity of L. asiaticus was found to decrease significantly after exposure to tolfenamic acid. Similarly, tolfenamic acid was found to inhibit L. asiaticus infection in highly symptomatic citrus seedlings. Our results indicate that PrbP is an important transcriptional regulator for survival of L. asiaticus in planta, and the chemicals identified by molecular screening assays could be used as a therapeutic treatment for huanglongbing disease.

H2O2 production rate in Lactobacillus johnsonii is modulated via the interplay of a heterodimeric flavin oxidoreductase with a soluble 28 Kd PAS domain containing protein.

Host and commensals crosstalk, mediated by reactive oxygen species (ROS), has triggered a growing scientific interest to understand the mechanisms governing such interaction. However, the majority of the scientific studies published do not evaluate the ROS production by commensals bacteria. In this context we recently showed that Lactobacillus johnsonii N6.2, a strain of probiotic value, modulates the activity of the critical enzymes 2,3-indoleamine dioxygenase via H2O2 production. L. johnsonii N6.2 by decreasing IDO activity, is able to modify the tryptophan/kynurenine ratio in the host blood with further systemic consequences. Understanding the mechanisms of H2O2 production is critical to predict the probiotic value of these strains and to optimize bacterial biomass production in industrial processes. We performed a transcriptome analysis to identify genes differentially expressed in L. johnsonii N6.2 cells collected from cultures grown under different aeration conditions. Herein we described the biochemical characteristics of a heterodimeric FMN reductase (FRedA/B) whose in vitro activity is controlled by LjPAS protein with a typical Per-Arnst-Sim (PAS) sensor domain. Interestingly, LjPAS is fused to the FMN reductase domains in other lactobacillaceae. In L. johnsonii, LjPAS is encoded by an independent gene which expression is repressed under anaerobic conditions (>3 fold). Purified LjPAS was able to slow down the FRedA/B initial activity rate when the holoenzyme precursors (FredA, FredB, and FMN) were mixed in vitro. Altogether the results obtained suggest that LjPAS module regulates the H2O2 production helping the cells to minimize oxidative stress in response to environmental conditions.

The transcriptional activator LdtR from 'Candidatus Liberibacter asiaticus' mediates osmotic stress tolerance.

The causal agent of Huanglongbing disease, 'Candidatus Liberibacter asiaticus', is a non-culturable, gram negative, phloem-limited α-proteobacterium. Current methods to control the spread of this disease are still limited to the removal and destruction of infected trees. In this study, we identified and characterized a regulon from 'Ca. L. asiaticus' involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR), and a predicted L,D-transpeptidase (ldtP). In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype) and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of 'Ca. Liberibacter asiaticus', using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease.

MglA/SspA complex interactions are modulated by inorganic polyphosphate.

The transcription factors MglA and SspA of Francisella tularensis form a heterodimer complex and interact with the RNA polymerase to regulate the expression of the Francisella pathogenicity island (FPI) genes. These genes are essential for this pathogen's virulence and survival within host cells. Our goal was to determine if an intracellular metabolite modulate these protein/protein interactions. In this study, we identified inorganic polyphosphate (polyP) as a signal molecule that promotes the interaction of MglA and SspA from F. tularensis SCHU S4. Analysis of the Mgla/SspA interaction was carried out using a two-hybrid system. The Escherichia coli reporter strain contained a deletion on the ppK-ppX operon, inhibiting polyP synthesis. The interaction between MglA and SspA was significantly impaired, as was the interaction between the MglA/SspA complex and the regulatory protein, FevR, indicating the stabilizing effect of polyP. In F. tularensis, chromatin immune precipitation studies revealed that in the absence of polyP, binding of the MglA/SspA complex to the promoter region of the pdpD, iglA, fevR and ppK genes is decreased. Isothermal titration calorimetry (ITC) indicated that polyP binds directly to the MglA/SspA complex with high affinity (KD = 0.3 µM). These observations directly correlated with results obtained from calorimetric scans (DSC), where a strong shift in the mid-transition temperature (Tm) of the MglA/SspA complex was observed in the presence of polyP.

Identification of a small molecule that modifies MglA/SspA interaction and impairs intramacrophage survival of Francisella tularensis.

The transcription factors MglA and SspA of Francisella tularensis form a heterodimer complex and interact with the RNA polymerase to regulate the expression of the Francisella pathogenicity island (FPI) genes. These genes are essential for this pathogen's virulence and survival within host cells. In this study, we used a small molecule screening to identify quinacrine as a thermal stabilizing compound for F. tularensis SCHU S4 MglA and SspA. A bacterial two-hybrid system was used to analyze the in vivo effect of quinacrine on the heterodimer complex. The results show that quinacrine affects the interaction between MglA and SspA, indicated by decreased ß-galactosidase activity. Further in vitro analyses, using size exclusion chromatography, indicated that quinacrine does not disrupt the heterodimer formation, however, changes in the alpha helix content were confirmed by circular dichroism. Structure-guided site-directed mutagenesis experiments indicated that quinacrine makes contact with amino acid residues Y63 in MglA, and K97 in SspA, both located in the "cleft" of the interacting surfaces. In F. tularensis subsp. novicida, quinacrine decreased the transcription of the FPI genes, iglA, iglD, pdpD and pdpA. As a consequence, the intramacrophage survival capabilities of the bacteria were affected. These results support use of the MglA/SspA interacting surface, and quinacrine's chemical scaffold, for the design of high affinity molecules that will function as therapeutics for the treatment of Tularemia.

Lactobacillus brevis responds to flavonoids through KaeR, a LysR-type of transcriptional regulator.

The ability of transcription factors to respond to flavonoids as signal molecules was investigated in Lactobacillus brevis. Through in vitro screening of a small library of flavonoids, LVIS1989 (KaeR), a LysR-type transcriptional regulator (LTTR), was identified as responsive to kaempferol. The modulation of KaeR activity by flavonoids was characterized in vivo and in vitro. DNase I footprint assays identified the binding of KaeR at two distinctive sites, one in the intergenic region between LVIS1988 and kaeR (-39 to +2) and another within LVIS1988 (-314 to -353, from kaeR translational start point). EMSA assays revealed that both binding sites are required for KaeR binding in vitro. Furthermore, KaeR-DNA interactions were stabilized by the addition of kaempferol (20 µM). In vivo qRT-PCR experiments performed in L. brevis confirmed that the divergently transcribed genes LVIS1988, LVIS1987 and LVIS1986 and kaeR are upregulated in the presence of kaempferol, indicating the role of KaeR as a transcriptional activator. Transcriptional lacZ fusions using Bacillus subtilis as a surrogate host showed that expression of kaeR and LVIS1988 were induced by the presence of the flavonoid. These results indicate that KaeR belongs to a small and poorly understood group of LTTRs that are positively autoregulated in the presence of a ligand.

LVIS553 transcriptional regulator specifically recognizes novobiocin as an effector molecule.

In this study we aimed to identify small molecules with high affinity involved in the allosteric regulation of LVIS553, a MarR member from Lactobacillus brevis ATCC367. Using high throughput screening, novobiocin was found to specifically bind LVIS553 with a K(D) = 33.8 +/- 2.9 microM consistent with a biologically relevant ligand. Structure guided site-directed mutagenesis identified Lys(9) as a key residue in novobiocin recognition. The results found in vitro were correlated in vivo. An increased tolerance to the antibiotic was observed when LVIS553 and the downstream putative transport protein LVIS552 were either expressed in a low copy plasmid in L. brevis or as a single copy chromosomal insertion in Bacillus subtilis. We provide evidence that LVIS553 is involved in the specific regulation of a new mechanism of tolerance to novobiocin.