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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.

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.

Effective cultivation methods, total cost, and biomass preservation are key factors that have a significant impact on the commercialisation and effectiveness of probiotics, such as Lactobacillus. Sugar polymers, milk and whey proteins have been suggested as good additives for industrial preparations. Alternative compounds, such as phytophenols, are a more attractive option, given their potential benefits to human health. The overall goal of this study was to determine if the addition of blueberry phytophenols improves the survival of Lactobacillus johnsonii N6.2 during the freeze-drying process. The addition of blueberry aqueous extract (BAE) stimulated the growth of L. johnsonii under aerobic conditions and improved the stationary phase survival of the bacteria. Furthermore, the addition of BAE to the culture media improved the endurance of L. johnsonii N6.2 to freeze-drying stress, as well as to storage at 4 °C for up to 21 weeks. Moreover, blueberry extract performed more effectively as a lyophilising additive compared to skim milk and microencapsulation with whey protein/sodium alginate. In sum, this study demonstrates that BAE is an effective additive to increase the growth and survival of L. johnsonii N6.2 when added to the culture medium and/or used as a lyophilising preservative. Moreover, BAE or other polyphenols sources might likely enhance growth and increase survival of more probiotic lactic acid bacterial strains.

BackgroundInfants in the neonatal intensive care unit (NICU) often require feeding tubes (FT) for weeks to months. Because FTs are in near constant contact with human milk and/or formula, rapid and extensive bacterial growth is possible. Due to their immature immunologic and gastrointestinal (GI) systems, infants may be at significant health risk due to FT colonization. In adults, length of time FTs remain in place (dwell time) affects the degree of colonization and biofilm formation which is important in infants whose tubes remain in place up to 30 days.ObjectiveThe purpose of this review was to describe and summarize the evidence regarding FT bacterial colonization in infants and identify gaps needing further investigation.MethodsMedline, CINAHL, and Embase databases were searched for clinical and/or laboratory-based observational and randomized controlled studies investigating the presence of bacteria in neonatal FTs.ResultsThis review of 10 studies found evidence that neonatal FTs may contain high quantities of potentially pathogenic and antibiotic resistant bacteria and longer dwell times may increase the bacterial load. Furthermore, evidence suggests FT colonization may be nosocomial in origin and contribute to adverse infant health. Feeding tubes are an unrecognized source of bacterial colonization which may increase morbidity in premature infants and thus the presence of bacteria in FTs is an important area of investigation in the nutritional care of vulnerable infants in the NICU.ImplicationsFurther appropriately powered studies which are clinically based, use appropriate analyses, and control for potential covariates are necessary to make clinical recommendations.

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.

Mother's own milk provides personalized nutrition and immune protection to the developing infant. The presence of healthy microbes plays an important role in the infant's gut by programming the microbiota and excluding potential pathogens. This review describes the important components in mother's own milk that contribute to its superiority for infant nutrition and suggest potential strategies to replicate these factors in alternative feedings when sufficient milk is unavailable. Current strategies to supplement, substitute and replicate mother's own milk including microbial restoration, use of unpasteurized donor human milk, probiotics and fortification are discussed. Critical work remains to be done in understanding the human milk microbiome and metabolome and in improving lactation support for mothers of preterm infants. Increasing delivery of mother's own milk and milk components to infants would likely positively impact infant mortality and health worldwide.

Citrus greening disease, or huanglongbing, may entirely eradicate all varieties of citrus cultivars worldwide in the near future. This disease is caused by non-cultivable bacteria of the genus Liberibacter; among them, the more pathogenic being Liberibacter asiaticus. The complexity of the host–pathogen relationship, associated with the impossibility of performing research using axenic cultures, has severely hindered the basic research on microbiology. Since its genome sequence was published in 2009, most of the scientific publications in the field were dedicated to in silico analysis and selection of targets to design early detection methods. The knowledge gained with these approaches felt short to articulate effective methods to control the disease progression. There is a critical need to understand the basic biology of bacteria to design effective strategies to inactivate central mechanisms of pathogenesis. In this review, we summarize the scientific progress made by studying L. asiaticus’ biology through direct experimentation. The evidence collected thus far is not enough to understand L. ­asiaticus’ fundamental biology. It is imperiously necessary to increase the basic research to identify relevant biological clues to control citrus greening. The gained knowledge may also help to prevent potential catastrophic diseases in other crops of significant importance caused by other unculturable Liberibacter species.

Summary Huanglongbing or citrus greening disease is causing devastation to the citrus industry. Liberibacter asiaticus, an obligate intracellular pathogen of citrus, is one the causative agents of the disease. Most of the knowledge about this bacterium has been deduced from the in silico exploration of its genomic sequence. L. asiaticus differentially expresses genes during its transmission from the psyllid vector, Diaphorina citri, to the plant. However, the regulatory mechanisms for the adaptation of the bacterium into either hosts remain unknown. Here we show that LdtR, a MarR family transcriptional regulator, activates or represses transcription genome-wide. We performed a double approach to identify the components of the LdtR regulon: a transcriptome analysis in both the related bacterium Liberibacter crescens and citrus-infected leaves, strengthened with an in silico prediction of LdtR regulatory sites. Our results demonstrated that LdtR controls the expression of nearly 180 genes in L. asiaticus, distributed in processes such as cell motility, cell wall biogenesis, energy production, and transcription. These results provide new evidence about the regulatory network of L. asiaticus, where the differential expression of genes from these functional categories could be of great importance during the adaptation of the bacterium to either hosts.

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. Fil: Loto, Flavia del Valle. University of Florida; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; Argentina Fil: Coyle, Janelle F.. University of Florida; Estados Unidos Fil: Padgett, Kaylie A.. University of Florida; Estados Unidos Fil: Pagliai, Fernando A.. University of Florida; Estados Unidos Fil: Gardner, Christopher L.. University of Florida; Estados Unidos Fil: Lorca, Graciela L.. University of Florida; Estados Unidos Fil: Gonzalez, Claudio F.. University of Florida; Estados Unidos

Herein, an analytical method was developed for extraction and quantification of benzbromarone and tolfenamic acid in citrus and soil matrices using liquid-liquid extraction followed by liquid chromatography-triple quadrupole-tandem mass spectrometry analysis. The compounds were extracted using 0.1% formic acid in 6:4 ethyl acetate and n-hexane solution, and the analytes were separated using a mixture of 0.1% formic acid in ultrapure water and 0.1% formic acid in acetonitrile as mobile phase. A six-point in-matrix calibration curve was constructed providing good linearity with coefficients of determination R2 ≥ 0.98. The limits of detection and quantification for benzbromarone and tolfenamic acid were 3.0 and 10.0 μg/kg in roots, peel, juice, and soil, and 4.0 and 12.0 μg/kg for leaves samples, respectively. The method yielded excellent recoveries between 81.3 and 101.2%, with relative standard deviation ≤9.5% in the matrices. The developed technique provides a simple and sensitive method for the determination of the chemicals and can be applied to agricultural practices.

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. Fil: Zuo, Ran. University of Florida; Estados Unidos Fil: Oliveira, Aline de. University of Florida; Estados Unidos Fil: Bullita, Enrica. University of Florida; Estados Unidos Fil: Torino, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. University of Florida; Estados Unidos Fil: Padgett Pagliai, Kaylie A.. University of Florida; Estados Unidos Fil: Gardner, Christopher L.. University of Florida; Estados Unidos Fil: Harrison, Natalie A.. University of Florida; Estados Unidos Fil: Silva, Danilo da. University of Florida; Estados Unidos Fil: Merli, Marcelo Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. University of Florida; Estados Unidos Fil: Gonzalez, Claudio Fabricio. University of Florida; Estados Unidos Fil: Lorca, Graciela L.. University of Florida; Estados Unidos

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase β-subunit and a specific sequence on the promoter region. The constitutive expression of prbP observed upon chemical inactivation of PrbP-DNA interactions in vivo indicated that the expression of prbP was not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved. In silico genome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed. In vitro transcription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activity in vitro. The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast to prbP of L. asiaticus (prbPLas), mRNA levels of ferR of L. asiaticus (ferRLas) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbPLas is modulated via interactions with FerRLas. The induction of ferRLas expression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals in L. asiaticus. IMPORTANCE The rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent, Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important for L. asiaticus survival in the plant host. In this study, we investigated the interactions between PrbP in L. asiaticus (PrbPLas) and a ferredoxin-like protein (FerR) in L. asiaticus, FerRLas. We show that the presence of FerR stabilizes and augments the activity of PrbPLas. In addition, we demonstrate that the expression of ferR is induced by increases in osmolarity in Liberibacter crescens. Altogether, these results suggest that FerRLas and PrbPLas may play important roles in the regulation of gene expression in response to changing environmental signals during L. asiaticus infection in the citrus host.

The purpose of this prospective cross-sectional cohort pilot study is to explore the initial microbial community of gastric aspirate fluid as collected immediately after birth and its relationships with mode of delivery and preterm birth. Twenty-nine gastric aspirate samples collected immediately after birth from infants born between 24–40 weeks gestation were analyzed for microbial composition. Total microbial content was low in many samples, with a substantial number sharing taxonomic composition with negative controls. qPCR targeting the 16S rRNA gene showed that infants delivered vaginally had a higher microbial load than infants delivered by C-section. Some pre-term samples showed high relative abundance of genus Ureaplasma, consistent with previous literature that has implicated infections with this taxon as a potential cause of pre-term birth. Vaginally born term infant samples, by contrast, had significantly higher levels of genus Lactobacillus with Lactobacillus crispatus the most dominant species. Microbial evaluation showed that vaginally born term infant gastric aspirate samples had higher levels of lactobacilli than pre-terms. Samples from many infants had low microbial load near the edge of the detection limit.

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase β-subunit and a specific sequence on the promoter region. The constitutive expression of prbP observed upon chemical inactivation of PrbP-DNA interactions in vivo indicated that the expression of prbP was not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved. In silico genome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed. In vitro transcription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activity in vitro. The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast to prbP of L. asiaticus (prbP(Las)), mRNA levels of ferR of L. asiaticus (ferR(Las)) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbP(Las) is modulated via interactions with FerR(Las). The induction of ferR(Las) expression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals in L. asiaticus. IMPORTANCE The rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent, Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important for L. asiaticus survival in the plant host. In this study, we investigated the interactions between PrbP in L. asiaticus (PrbP(Las)) and a ferredoxin-like protein (FerR) in L. asiaticus, FerR(Las). We show that the presence of FerR stabilizes and augments the activity of PrbP(Las). In addition, we demonstrate that the expression of ferR is induced by increases in osmolarity in Liberibacter crescens. Altogether, these results suggest that FerR(Las) and PrbP(Las) may play important roles in the regulation of gene expression in response to changing environmental signals during L. asiaticus infection in the citrus host.

The cells of the gastrointestinal (GI) epithelium are the first to contact the microbiota and food components. As a direct consequence of this, these cells are the first line of defence and key players in priming the immune response. One of the first responses against GI insults is the formation of the inflammasome, a multiprotein complex assembled in response to environmental threats. The formation of the inflammasome regulates caspase-1 by cleaving it into its active form. Once activated, caspase-1 can cleave interleukin-1β (IL-1β), which promotes adaptive and humoral immunity. Some strains, like Lactobacillus johnsonii N6.2, are able to modulate the biosynthesis of important host metabolites mediating inflammation. Of these metabolites are the pro-inflammatory kynurenines. L. johnsonii N6.2 is able to downregulate kynurenines biosynthesis via a redox active mechanism negatively affecting indoleamine 2,3-dioxygenase activity. In this study, we evaluated the effects of L. johnsonii N6.2 combined with the natural antioxidant and anti-inflammatory molecule rosmarinic acid (RA). Inflammasome assembly and the kynurenine pathway were evaluated in GI samples of BioBreeding diabetes-prone (BB-DP) rats. In this work, BB-DP rats were fed daily with RA, L. johnsonii N6.2; or both combined. The transcriptional rate and proteins levels of inflammasome and kynurenine pathway components in ileum tissue were evaluated. Elevated levels of pro-caspase-1 were observed in rats fed with L. johnsonii, while RA had no effect on pro-caspase-1 expression. Western blot assays demonstrated that L. johnsonii fed rats showed lower levels of mature caspase-1, when compared to the other treatments. Furthermore, IL-1β maturation followed a similar pattern across the treatments. Differences were also observed between treatments in expression levels of key enzymes in the kynurenine pathway. These findings support the role of L. johnsonii in modulating the assembly of the inflammasome as well as some steps of the pro-inflammatory kynurenine pathway.

Citrus greening disease, or huanglongbing, may entirely eradicate all varieties of citrus cultivars worldwide in the near future. This disease is caused by non-cultivable bacteria of the genus Liberibacter; among them, the more pathogenic being Liberibacter asiaticus. The complexity of the host-pathogen relationship, associated with the impossibility of performing research using axenic cultures, has severely hindered the basic research on microbiology. Since its genome sequence was published in 2009, most of the scientific publications in the field were dedicated to in silico analysis and selection of targets to design early detection methods. The knowledge gained with these approaches felt short to articulate effective methods to control the disease progression. There is a critical need to understand the basic biology of bacteria to design effective strategies to inactivate central mechanisms of pathogenesis. In this review, we summarize the scientific progress made by studying L. asiaticus' biology through direct experimentation. The evidence collected thus far is not enough to understand L. -asiaticus' fundamental biology. It is imperiously necessary to increase the basic research to identify relevant biological clues to control citrus greening. The gained knowledge may also help to prevent potential catastrophic diseases in other crops of significant importance caused by other unculturable Liberibacter species.

Background: Acetylation and efflux of carbohydrates during cellular metabolism is a well-described phenomenon associated with a detoxification process to prevent metabolic congestion. It is still unclear why cells discard important metabolizable energy sources in the form of acetylated compounds. Methods: We describe the purification and characterization of an approximately 28-kDa intracellular carboxylesterase (YjfP) and the analysis of gene and protein expression by qRT-PCR and Western blot. Results: qRT-PCR and Western blot, respectively, showed that yjfP is upregulated during the diauxic lag in cells growing with a mixture of glucose and lactose. The β-galactosidase activity in the ΔyjfP strain was both delayed and half the magnitude of that of the wild-type strain. YjfP-hyperproducing strains displayed a long lag phase when cultured with glucose and then challenged to grow with lactose or galactose as the sole carbon source. Conclusion: Our results suggest that YjfP controls the intracellular concentration of acetyl sugars by redirecting them to the main metabolic circuits. Instead of detoxification, we propose that sugar acetylation is utilized by the cell for protection and to prevent the metabolism of a necessary minimal intracellular sugar pool. Those sugars can eventually be exported as a side effect of these mechanisms.

Huanglongbing (HLB; “citrus greening” disease) has caused significant damages to the global citrus industry as it has become well established in leading citrus-producing regions and continues to spread worldwide. Insecticidal control has been a critical component of HLB disease management, as there is a direct relationship between vector control and Candidatus Liberibacter spp. (i.e., the HLB pathogen) titer in HLB-infected citrus trees. In recent years, there have been substantial efforts to develop practical strategies for specifically managing Ca. Liberibacter spp.; however, a literature review on the outcomes of such attempts is still lacking. This work summarizes the greenhouse and field studies that have documented the effects and implications of chemical-based treatments (i.e., applications of broad-spectrum antibiotics, small molecule compounds) and nonchemical measures (i.e., applications of plant-beneficial compounds, applications of inorganic fertilizers, biological control, thermotherapy) for phytopathogen control. The ongoing challenges associated with mitigating Ca. Liberibacter spp. populations at the field-scale, such as the seasonality of the phytopathogen and associated HLB disease symptoms, limitations for therapeutics to contact the phytopathogen in planta, adverse impacts of broad-spectrum treatments on plant-beneficial microbiota, and potential implications on public and ecosystem health, are also discussed.

The American Academy of Pediatrics recommends that extremely preterm infants receive mother’s own milk (MOM) when available or pasteurized donor breast milk (DBM) when MOM is unavailable. The goal of this study was to determine whether DBM could be inoculated with MOM from mothers of preterm infants to restore the live microbiota (RM). Culture dependent and culture independent methods were used to analyze the fluctuations in the overall population and microbiome, respectively, of DBM, MOM, and RM samples over time. Using MOM at time = 0 (T0) as the target for the restoration process, this level was reached in the 10% (RM-10) and 30% (RM-30) mixtures after 4 h of incubation at 37°C, whereas, the larger dilutions of 1% (RM-1) and 5% (RM-5) after 8 h. The diversity indexes were similar between MOM and DBM samples, however, different genera were prevalent in each group. Interestingly, 40% of the bacterial families were able to expand in DBM after 4 h of incubation indicating that a large percentage of the bacterial load present in MOM can grow when transferred to DBM, however, no core microbiome was identified. In summary, the microbiome analyses indicated that each mother has a unique microbiota and that live microbial reestablishment of DBM may provide these microbes to individual mothers’ infants. The agreement between the results obtained from the viable bacterial counts and the microbiome analyses indicate that DBM incubated with 10–30% v/v of the MOM for 4 h is a reasonable restoration strategy.

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that was found to regulate gene expression through interactions with the RNA polymerase β-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.

Stable associations between plants and microbes are critical to promoting host health and productivity. The objective of this work was to test the hypothesis that restructuring of the core microbiota may be associated with the progression of huanglongbing (HLB), the devastating citrus disease caused by Liberibacter asiaticus , Liberibacter americanus , and Liberibacter africanus . The microbial communities of leaves ( n = 94) and roots ( n = 79) from citrus trees that varied by HLB symptom severity, cultivar, location, and season/time were characterized with Illumina sequencing of 16S rRNA genes. The taxonomically rich communities contained abundant core members (i.e., detected in at least 95% of the respective leaf or root samples), some overrepresented site-specific members, and a diverse community of low-abundance variable taxa. The composition and diversity of the leaf and root microbiota were strongly associated with HLB symptom severity and location; there was also an association with host cultivar. The relative abundance of Liberibacter spp. among leaf microbiota positively correlated with HLB symptom severity and negatively correlated with alpha diversity, suggesting that community diversity decreases as symptoms progress. Network analysis of the microbial community time series identified a mutually exclusive relationship between Liberibacter spp. and members of the Burkholderiaceae , Micromonosporaceae , and Xanthomonadaceae . This work confirmed several previously described plant disease-associated bacteria, as well as identified new potential implications for biological control. Our findings advance the understanding of (i) plant microbiota selection across multiple variables and (ii) changes in (core) community structure that may be a precondition to disease establishment and/or may be associated with symptom progression. IMPORTANCE This study provides a comprehensive overview of the core microbial community within the microbiomes of plant hosts that vary in extent of disease symptom progression. With 16S Illumina sequencing analyses, we not only confirmed previously described bacterial associations with plant health (e.g., potentially beneficial bacteria) but also identified new associations and potential interactions between certain bacteria and an economically important phytopathogen. The importance of core taxa within broader plant-associated microbial communities is discussed.

OBJECTIVES/SPECIFIC AIMS: Aim 1: To compare frozen MOM to fresh MOM over time as an agent to inoculate DHM and measure the enrichment of commensal microbes and their beneficial bioactive components similar to MOM. Hypothesis: Frozen or fresh MOM inoculated in DHM will produce similar microbial content to MOM over time allowing for the production of beneficial bacterial compounds that may contribute to host immune response. Aim 2: To determine the effect of MOM storage (fresh vs frozen) on the expansion of bioactive components from live microbiota in DHM. Hypothesis: Both fresh and frozen MOM will produce similar results when inoculated into DHM to restore the microbial content (including their bioactive components) similar to each MOM sample. Aim 3: To compare the microbiome found in a mother’s MOM to the microbiome in her infant’s stool. Hypothesis: The mother/infant pair will share a common microbiome between the mother’s MOM and her infant’s stool. METHODS/STUDY POPULATION: Subjects will include 12 pump-dependent mothers of infants born < 34 weeks gestation admitted to the University of Florida Health Shands Hospital, Neonatal Intensive Care Unit (NICU). Inclusion criteria consists of mothers expressing over 100 ml of MOM per day, producing at least 45 ml of MOM at an expression session, at least 18 years of age, and speak English. Mothers are excluded if they have taken antibiotics within 3 days of sample collection, are HIV+, or delivered an infant who has a chromosomal abnormality or is severely ill. An expressed MOM sample will be collected and divided into two fractions: (A) fresh and (B) frozen at -20C for 24 h. The fresh fraction (A) will be processed immediately while the frozen fraction (B) will be processed after 24 h. Each MOM will be inoculated in DHM at dilutions of 10% and 30% and incubated at different time points: 0 h (T0), 2 h (T2), 4 h (T4) at 37°C. At each time point, total viable cell counts as well as microbiome analysis through 16S ribosomal shotgun sequencing will be performed and compared for differences. Bacteria isolated from each MOM will be saved, identified through 16S ribosomal sequencing and grown in culture for future studies. Fecal samples from each corresponding infant will be collected within 48 h after collection of the MOM sample. Stools will be homogenized and subjected to DNA extraction to perform 16S ribosomal shotgun sequencing. Microbiome analysis will be conducted, compared between fecal samples as well as with the microbiome of the MOM. RESULTS/ANTICIPATED RESULTS: Study is ongoing. We anticipate similar results with fresh or frozen MOM to that of a previous pilot study, where enriched microbiota similar to MOM was found when fresh MOM was inoculated and incubated in DHM. The microbiome analysis of the infant fecal samples may illustrate the influence that the microbiome of the MOM may have on the development of the infants’ gastrointestinal microbiota. DISCUSSION/SIGNIFICANCE OF IMPACT: The purpose of this study is to provide evidence on the ability, timing, and efficacy of inoculating DHM with fresh and frozen MOM. Study results will inform future studies to support the implementation of an inoculation procedural protocol to be used in clinical practice and human milk banking. The description of the MOM microbiome, as well as the gastrointestinal microbiome, will expand scientific knowledge on the role breast milk has on the origins of health and disease.

In this study we uncover two genes in Lactobacillus brevis ATCC367, tstT and tstR, encoding for a rhodanese and a transcriptional regulator involved in cyanide detoxification. TstT (LVIS_0852) belongs to a new class of thiosulfate:cyanide sulfurtransferases. We found that TstR (LVIS_0853) modulates both the expression and the activity of the downstream-encoded tstT. The TstR binding site was identified at −1 to +33, from tstR transcriptional start site. EMSA revealed that sulfite, a product of the reaction catalyzed by TstT, improved the interaction between TstR:PtstR, while Fe(III) disrupted this interaction. Site-directed mutagenesis in TstR identified M64 as a key residue in sulfite recognition, while residues H136-H139-C167-M171 formed a pocket for ferric iron coordination. In addition to its role as a transcriptional repressor, TstR is also involved in regulating the thiosulfate:cyanide sulfurtransferase activity of TstT. A 3-fold increase in TstT activity was observed in the presence of TstR, which was enhanced by the addition of Fe(III). Overexpression of the tstRT operon was found to increase the cyanide tolerance of L. brevis and Escherichia coli. The protein-protein interaction between TstR and TstT described herein represents a novel mechanism for regulation of enzymatic activity by a transcriptional regulator.

In our previous work, we found that feeding Lactobacillus johnsonii to BioBreeding diabetes-prone (BBDP) rats decreased the incidence of diabetes development. The aim of this study was to investigate host pathways affected by L. johnsonii, with specific focus on the rate-limiting enzyme of tryptophan catabolism, indoleamine 2,3-dioxygenase (IDO). Suspensions of L. johnsonii or an equal volume of vehicle were orally administered to BBDP rats. Tissue IDO was investigated using quantitative RT-PCR and Western blot, whereas tryptophan, kynurenine, and 5-hydroxytryptamine (5-HT) concentrations were quantified by HPLC and ELISA. IDO activity was also investigated using L. johnsonii culture cell-free supernatant (CFS) with affinity-purified IDO and HT-29 intestinal epithelial cells. L. johnsonii feeding resulted in a 17% reduction in serum kynurenine compared with that in vehicle-fed controls, correlating with a 1.4-fold elevation in 5-HT levels. H₂O₂ produced by L. johnsonii abolished IDO activity in vitro, and L. johnsonii feeding resulted in a 3.9-fold increase in ileum lumen H₂O₂. L. johnsonii CFS significantly reduced IDO activity in HT-29 intestinal epithelial cells (47% reduction) compared with that in vehicle-treated controls, an effect abolished by catalase treatment. These data support the role of H₂O₂ in commensal bacteria-host interactions and highlight the influence of commensal bacteria-derived H₂O₂ on host physiology.

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 β-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. Fil: Gardner, Christopher L.. University of Florida; Estados Unidos Fil: Pagliai, Fernando A.. University of Florida; Estados Unidos Fil: Pan, Lei. University of Florida; Estados Unidos Fil: Bojilova, Lora. University of Florida; Estados Unidos Fil: Torino, Maria Ines. University of Florida; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Lorca, Graciela L.. University of Florida; Estados Unidos Fil: Gonzalez, Claudio F.. University of Florida; Estados Unidos

Phytophenols are found ubiquitously among all plants. They are important in diets rich in fruits and vegetables because these compounds provide health benefits to the host, ultimately decreasing the incidence of chronic diseases. These compounds act as natural antioxidants and provide anti-inflammatory, antiviral, antibiotic, and antineoplastic properties. Reactive oxygen species (ROS) are produced under normal physiological functions, and low/moderate levels are required for cellular turnover and signaling. However, when ROS levels become too high, oxidative stress can occur. Phytophenols quench ROS and ultimately avoid the damaging effects ROS elicit on the cell. The highest source of bioavailable phytophenols comes from our diet as a component usually esterified in plant fiber. For phytophenols to be absorbed by the body, they must be released by esterases, or other related enzymes. The highest amount of esterase activity comes from the gastrointestinal (GI) microbiota; therefore, the host requires the activity of mutualis‐ tic bacteria in the GI tract to release absorbable phytophenols. For this reason, mutualis‐ tic bacteria have been investigated for beneficial properties in the host. Our laboratory has begun studying the interaction of Lactobacillus johnsonii N6.2 with the host since it was found to be negatively correlated with type 1 diabetes (T1D). Analyses of this strain have revealed two important characteristics: (1) It has the ability to release phytophenols from dietary fiber through the secretion of two strong cinnamoyl esterases and (2) L. johnso‐ nii also has the ability to generate significant amounts of H2O2, controlling the activity of indoleamine 2,3-dioxygenase (IDO), an immunomodulatory enzyme. Fil: Kling, Danielle N.. University of Florida; Estados Unidos Fil: Marcial, Guillermo Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. University of Florida; Estados Unidos Fil: Roberson, Dana N.. University of Florida; Estados Unidos Fil: Lorca, Graciela L.. University of Florida; Estados Unidos Fil: Gonzalez, Claudio Fabricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina