‘Candidatus Liberibacter asiaticus’ Multimeric LotP Mediates Citrus sinensis Defense Response Activation

‘Candidatus Liberibacter asiaticus’ is known as the most pathogenic organism associated with citrus greening disease. Since its publicized emergence in Florida in 2005, ‘Ca. L. asiaticus’ remains unculturable. Currently, a limited number of potential disease effectors have been identified through in silico analysis. Therefore, these potential effectors remain poorly characterized and do not fully explain the complexity of symptoms observed in citrus trees infected with ‘Ca. L. asiaticus.’ LotP has been identified as a potential effector and have been partially characterized. This protein retains structural homology to the substrate binding domain of the Lon protease. LotP interacts with chaperones like GroEL, Hsp40, DnaJ, and ClpX and may exercise its biological role through interactions with different proteins involved in proteostasis networks. Here, we evaluate the interactome of LotP—revealing a new protein–protein interaction target (Lon-serine protease) and its effect on citrus plant tissue integrity. We found that via protein–protein interactions, LotP can enhance Lon protease activity, increasing the degradation rate of its specific targets. Infiltration of purified LotP strained citrus plant tissue causing photoinhibition and chlorosis after several days. Proteomics analysis of LotP tissues recovering after the infiltration revealed a large abundance of plant proteins associated with the stabilization and processing of mRNA transcripts, a subset of important transcription factors; and pathways associated with innate plant defense were highly expressed. Furthermore, interactions and substrate binding module of LotP suggest potential interactions with plant proteins, most likely proteases.Fil: Merli, Marcelo Luciano. University of Florida. Department of Microbiology and Cell Science; Estados Unidos. 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; ArgentinaFil: Padgett Pagliai, Kaylie A.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Cuaycal, Alexandra E.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: García, Lucila. 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; ArgentinaFil: Marano, María Rosa. 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; ArgentinaFil: Lorca, Graciela L.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Gonzalez, Claudio F.. University of Florida. Department of Microbiology and Cell Science; Estados Unido

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.Fil: Pan, Lei. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Gardner, Christopher L.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Beliakoff, Reagan. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Da Silva, Danilo. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Zuo, Ran. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Pagliai, Fernando A.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Padgett Pagliai, Kaylie A.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Merli, Marcelo Luciano. University of Florida. Department of Microbiology and Cell Science; Estados Unidos. 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; ArgentinaFil: Bahadiroglu, Erol. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Gonzalez, Claudio F.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Lorca, Graciela L.. University of Florida. Department of Microbiology and Cell Science; Estados Unido

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.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; ArgentinaFil: Coyle, Janelle F.. University of Florida; Estados UnidosFil: Padgett, Kaylie A.. University of Florida; Estados UnidosFil: Pagliai, Fernando A.. University of Florida; Estados UnidosFil: Gardner, Christopher L.. University of Florida; Estados UnidosFil: Lorca, Graciela L.. University of Florida; Estados UnidosFil: Gonzalez, Claudio F.. University of Florida; Estados Unido

Personalization of the Microbiota of Donor Human Milk with Mother’s Own Milk

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

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.Fil: Zuo, Ran. University of Florida; Estados UnidosFil: Oliveira, Aline de. University of Florida; Estados UnidosFil: Bullita, Enrica. University of Florida; Estados UnidosFil: 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 UnidosFil: Padgett Pagliai, Kaylie A.. University of Florida; Estados UnidosFil: Gardner, Christopher L.. University of Florida; Estados UnidosFil: Harrison, Natalie A.. University of Florida; Estados UnidosFil: Silva, Danilo da. University of Florida; Estados UnidosFil: 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 UnidosFil: Gonzalez, Claudio Fabricio. University of Florida; Estados UnidosFil: Lorca, Graciela L.. University of Florida; Estados Unido