Biosynthesis of polyhydroxybutyrate by Methylorubrum extorquens DSM13060 is essential for intracellular colonization in plant endosymbiosis.

Methylorubrum extorquens DSM13060 is an endosymbiont that lives in the cells of shoot tip meristems. The bacterium is methylotrophic and consumes plant-derived methanol for the production of polyhydroxybutyrate (PHB). The PHB provides protection against oxidative stress for both host and endosymbiont cells through its fragments, methyl-esterified 3-hydroxybutyrate (ME-3HB) oligomers. We evaluated the role of the genes involved in the production of ME-3HB oligomers in the host colonization by the endosymbiont M. extorquens DSM13060 through targeted genetic mutations. The strains with deletions in PHB synthase (phaC), PHB depolymerase (phaZ1), and a transcription factor (phaR) showed altered PHB granule characteristics, as ΔphaC had a significantly low number of granules, ΔphaR had a significantly increased number of granules, and ΔphaZ1 had significantly large PHB granules in the bacterial cells. When the deletion strains were exposed to oxidative stress, the ΔphaC strain was sensitive to 10 mM HO· and 20 mM H2O2. The colonization of the host, Scots pine (Pinus sylvestris L.), by the deletion strains varied greatly. The deletion strain ΔphaR colonized the host mainly intercellularly, whereas the ΔphaZ1 strain was a slightly poorer colonizer than the control. The deletion strain ΔphaC lacked the colonization potential, living mainly on the surfaces of the epidermis of pine roots and shoots in contrast to the control, which intracellularly colonized all pine tissues within the study period. In earlier studies, deletions within the PHB metabolic pathway have had a minor effect on plant colonization by rhizobia. We have previously shown the association between ME-3HB oligomers, produced by PhaC and PhaZ1, and the ability to alleviate host-generated oxidative stress during plant infection by the endosymbiont M. extorquens DSM13060. Our current results show that the low capacity for PHB synthesis leads to poor tolerance of oxidative stress and loss of colonization potential by the endosymbiont. Altogether, our findings demonstrate that the metabolism of PHB in M. extorquens DSM13060 is an important trait in the non-rhizobial endosymbiosis.

Facile synthesis of multi-faceted, biomimetic and cross-protective nanoparticle-based vaccines for drug-resistant Shigella: a flexible platform technology.

BACKGROUND: No commercial vaccines are available against drug-resistant Shigella due to serotype-specific/narrow-range of protection. Nanoparticle-based biomimetic vaccines involving stable, conserved, immunogenic proteins fabricated using facile chemistries can help formulate a translatable cross-protective Shigella vaccine. Such systems can also negate cold-chain transportation/storage thus overcoming challenges prevalent in various settings. METHODS: We explored facile development of biomimetic poly (lactide-co-glycolide)/PLGA 50:50 based nanovaccines (NVs), encapsulating conserved stabilized antigen(s)/immunostimulant of S. dysenteriae 1 origin surface-modified using simple chemistries. All encapsulants (IpaC/IpaB/LPS) and nanoparticles (NPs)-bare and modified (NV), were thoroughly characterized. Effect of IpaC on cellular uptake of NPs was assessed in-vitro. Immunogenicity of the NVs was assessed in-vivo in BALB/c mice by intranasal immunization. Cross-protective efficacy was assessed by intraperitoneally challenging the immunized groups with a high dose of heterologous S. flexneri 2a and observing for visible diarrhea, weight loss and survival. Passive-protective ability of the simplest NV was assessed in the 5-day old progeny of vaccinated mice. RESULTS: All the antigens and immunostimulant to be encapsulated were successfully purified and found to be stable both before and after encapsulation into NPs. The ~ 300 nm sized NPs with a zeta potential of ~ - 25 mV released ~ 60% antigen by 14th day suggesting an appropriate delivery kinetics. The NPs could be successfully surface-modified with IpaC and/or CpG DNA. In vitro experiments revealed that the presence of IpaC can significantly increase cellular uptake of NPs. All NVs were found to be cytocompatible and highly immunogenic. Antibodies in sera of NV-immunized mice could recognize heterologous Shigella. Immunized sera also showed high antibody and cytokine response. The immunized groups were protected from diarrhea and weight loss with ~ 70-80% survival upon heterologous Shigella challenge. The simplest NV showed ~ 88% survival in neonates. CONCLUSIONS: Facile formulation of biomimetic NVs can result in significant cross-protection. Further, passive protection in neonates suggest that parental immunization could protect infants, the most vulnerable group in context of Shigella infection. Non-invasive route of vaccination can also lead to greater patient compliance making it amenable for mass-immunization. Overall, our work contributes towards a yet to be reported platform technology for facile development of cross-protective Shigella vaccines.

Stable Recombinant Invasion Plasmid Antigen C (IpaC)-Based Single Dose Nanovaccine for Shigellosis.

Shigellosis, caused by the bacteria Shigella, is the leading cause of bacterial diarrhea and the second leading cause of diarrheal death among children under the age of five. Unfortunately, Shigella strains have acquired resistance to antibiotics, and a commercial vaccine is yet to be available. We have previously demonstrated that Shigella dysenteriae serotype 1 (Sd1)-based recombinant, stabilized, "invasion plasmid antigen C" (IpaC; 42 kDa) protein can induce robust immune responses in BALB/c mice against a challenge of a high dose of heterologous Shigella when immunized via three intranasal doses of IpaC without an adjuvant. In this work, in order to reduce the frequency of dosing and increase possible patient compliance, based on our previous screening, the minimum protective dose of stabilized IpaC (20 µg) was encapsulated in biodegradable polymeric poly(lactide-co-glycolide) nanoparticles (∼370 nm) and intranasally administered in BALB/c mice in a single dose. Interestingly, a single intranasal dose of the developed vaccine particles encapsulating only 20 µg of Sd1 IpaC led to a temporal increase in the antibody production with an improved cytokine response compared to free IpaC administered three times as described in our previous report. Upon intraperitoneal challenge with a high dose of heterologous Shigella flexneri 2a (common in circulation), the immunized animals were protected from diarrhea, lethargy, and weight loss with ∼67% survival, while all the control animals died by 36 h of the challenge. Overall, the developed nanovaccine could be explored as a potential noninvasive, cross-protective, single-dose, single-antigen Shigella vaccine amenable for scale-up and eventual mass immunization.

A Comprehensive Computational Investigation into the Conserved Virulent Proteins of Shigella species Unveils Potential Small-Interfering RNA Candidates as a New Therapeutic Strategy against Shigellosis.

Shigella species account for the second-leading cause of deaths due to diarrheal diseases among children of less than 5 years of age. The emergence of multi-drug-resistant Shigella isolates and the lack of availability of Shigella vaccines have led to the pertinence in the efforts made for the development of new therapeutic strategies against shigellosis. Consequently, designing small-interfering RNA (siRNA) candidates against such infectious agents represents a novel approach to propose new therapeutic candidates to curb the rampant rise of anti-microbial resistance in such pathogens. In this study, we analyzed 264 conserved sequences from 15 different conserved virulence genes of Shigella sp., through extensive rational validation using a plethora of first-generation and second-generation computational algorithms for siRNA designing. Fifty-eight siRNA candidates were obtained by using the first-generation algorithms, out of which only 38 siRNA candidates complied with the second-generation rules of siRNA designing. Further computational validation showed that 16 siRNA candidates were found to have a substantial functional efficiency, out of which 11 siRNA candidates were found to be non-immunogenic. Finally, three siRNA candidates exhibited a sterically feasible three-dimensional structure as exhibited by parameters of nucleic acid geometry such as: the probability of wrong sugar puckers, bad backbone confirmations, bad bonds, and bad angles being within the accepted threshold for stable tertiary structure. Although the findings of our study require further wet-lab validation and optimization for therapeutic use in the treatment of shigellosis, the computationally validated siRNA candidates are expected to suppress the expression of the virulence genes, namely: IpgD (siRNA 9) and OspB (siRNA 15 and siRNA 17) and thus act as a prospective tool in the RNA interference (RNAi) pathway. However, the findings of our study require further wet-lab validation and optimization for regular therapeutic use for treatment of shigellosis.

Development of a Self-Adjuvanting, Cross-Protective, Stable Intranasal Recombinant Vaccine for Shigellosis.

With the acquirement of antibiotic resistance, Shigella has resulted in multiple epidemics of shigellosis, an infectious diarrheal disease, causing thousands of deaths per year. Unfortunately, there are no licensed vaccines, primarily due to low or serotype-specific immunogenicity. Thus, conserved subunit vaccines utilizing recombinant invasion plasmid antigens (Ipa) have been explored as cross-protective vaccine candidates. However, achieving cross-protection against Shigella dysenteriae 1, which caused multiple pandemics/epidemics in the recent past, has been difficult. Therefore, a rational approach to improve cross-protection in the preparation for a possible pandemic should involve conserved proteins from S. dysenteriae 1 (Sd1). IpaC is one such conserved immunogenic protein that is less explored as an independent vaccine due to its instability/aggregation. Therefore, to improve cross-protection and potential immunogenicity and to be prepared for a future epidemic/pandemic, herein, we stabilized recombinant Sd1 IpaC, expressed without its chaperone, using a previously reported stabilizing detergent (LDAO) in a modified protocol and assessed its vaccine potential without an adjuvant. The protein assembled into heterogeneous complex spherical structures in the presence of LDAO and showed improved stability at storage temperatures of -80, -20, 4, 25, and 37 °C while providing enhanced yield and concentration. The protein could also be stably lyophilized and reconstituted, increasing the convenience of transportation and storage. Upon intranasal administration in BALB/c mice, the stabilized-IpaC-immunized groups generated significant antibody response and were not only protected against a high intraperitoneal dose of homologous S. dysenteriae 1 but also showed 100% survival against heterologous Shigella flexneri 2a without an adjuvant, while the control animals showed visible diarrhea (bloody-Sd1 challenge), lethargy, and weight loss with 0% survival. Overall, this work demonstrates that stabilized IpaC can be explored as a minimalist, self-adjuvanting, cross-protective, intranasal, single-antigen Shigella vaccine.

A non-invasive nanoparticle-based sustained dual-drug delivery system as an eyedrop for endophthalmitis.

Endophthalmitis is an infectious disease that affects the entire eye spreading to the internal retinal layers and the vitreous and causes severe sight-threatening conditions. Current treatment strategies rely on intraocular injections of antibiotics that are invasive, may lead to procedural complications and, ultimately, blindness. In this study, we developed a non-invasive strategy as an eyedrop containing nanoparticle-based dual-drug delivery system in which the hydrophobic poly-L-lactide core was loaded with azithromycin or triamcinolone acetonide, and the hydrophilic shell was made of chitosan. The developed nanoparticles were ~200-250 nm in size, spherical in shape, moderately hydrophilic, lysozyme tolerant, cytocompatible, and hemocompatible. Application of these chitosan-coated nanoparticles as eye drops to C57BL/6 mice showed higher bioavailability in choroid and retina when compared to the uncoated nanoparticles. The delivery system showed sustained release of drug for 300 h and exhibited antimicrobial effects against Gram-positive and Gram-negative bacteria and anti-inflammatory effects on activated microglial cells. Interestingly, the combination of the nanoparticles loaded with azithromycin and the nanoparticles loaded with triamcinolone acetonide acted synergistically as compared to either of the nanoparticles/drugs alone. Overall, the developed dual-drug delivery system is non-invasive, has antimicrobial and anti-inflammatory effects, and shows potential as an eye drop formulation against endophthalmitis.

Biofertilizers and Biocontrol Agents for Agriculture: How to Identify and Develop New Potent Microbial Strains and Traits.

Microbiological tools, biofertilizers, and biocontrol agents, which are bacteria and fungi capable of providing beneficial outcomes in crop plant growth and health, have been developed for several decades. Currently we have a selection of strains available as products for agriculture, predominantly based on plant-growth-promoting rhizobacteria (PGPR), soil, epiphytic, and mycorrhizal fungi, each having specific challenges in their production and use, with the main one being inconsistency of field performance. With the growing global concern about pollution, greenhouse gas accumulation, and increased need for plant-based foods, the demand for biofertilizers and biocontrol agents is expected to grow. What are the prospects of finding solutions to the challenges on existing tools? The inconsistent field performance could be overcome by using combinations of several different types of microbial strains, consisting various members of the full plant microbiome. However, a thorough understanding of each microbiological tool, microbial communities, and their mechanisms of action must precede the product development. In this review, we offer a brief overview of the available tools and consider various techniques and approaches that can produce information on new beneficial traits in biofertilizer and biocontrol strains. We also discuss innovative ideas on how and where to identify efficient new members for the biofertilizer and biocontrol strain family.