Effects of Ionizing Radiation and Long-Term Storage on Hydrated vs. Dried Cell Samples of Extremophilic Microorganisms

A main factor hampering life in space is represented by high atomic number nuclei and energy (HZE) ions that constitute about 1% of the galactic cosmic rays. In the frame of the “STARLIFE” project, we accessed the Heavy Ion Medical Accelerator (HIMAC) facility of the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. By means of this facility, the extremophilic species Haloterrigena hispanica and Parageobacillus thermantarcticus were irradiated with high LET ions (i.e., Fe, Ar, and He ions) at doses corresponding to long permanence in the space environment. The survivability of HZE-treated cells depended upon either the storage time and the hydration state during irradiation; indeed, dry samples were shown to be more resistant than hydrated ones. With particular regard to spores of the species P. thermantarcticus, they were the most resistant to irradiation in a water medium: an analysis of the changes in their biochemical fingerprinting during irradiation showed that, below the survivability threshold, the spores undergo to a germination-like process, while for higher doses, inactivation takes place as a consequence of the concomitant release of the core’s content and a loss of integrity of the main cellular components. Overall, the results reported here suggest that the selected extremophilic microorganisms could serve as biological model for space simulation and/or real space condition exposure, since they showed good resistance to ionizing radiation exposure and were able to resume cellular growth after long-term storage.

Biochemical Properties of Two Plasmodium malariae Cysteine Proteases, Malapain-2 and Malapain-4

Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2–4 (MP2–4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.

Regulation of Tomato Specialised Metabolism after Establishment of Symbiosis with the Endophytic Fungus Serendipita indica

Specialised metabolites produced during plant-fungal associations often define how symbiosis between the plant and the fungus proceeds. They also play a role in the establishment of additional interactions between the symbionts and other organisms present in the niche. However, specialised metabolism and its products are sometimes overlooked when studying plant-microbe interactions. This limits our understanding of the specific symbiotic associations and potentially future perspectives of their application in agriculture. In this study, we used the interaction between the root endophyte Serendipita indica and tomato (Solanum lycopersicum) plants to explore how specialised metabolism of the host plant is regulated upon a mutualistic symbiotic association. To do so, tomato seedlings were inoculated with S. indica chlamydospores and subjected to RNAseq analysis. Gene expression of the main tomato specialised metabolism pathways was compared between roots and leaves of endophyte-colonised plants and tissues of endophyte-free plants. S. indica colonisation resulted in a strong transcriptional response in the leaves of colonised plants. Furthermore, the presence of the fungus in plant roots appears to induce expression of genes involved in the biosynthesis of lignin-derived compounds, polyacetylenes, and specific terpenes in both roots and leaves, whereas pathways producing glycoalkaloids and flavonoids were expressed in lower or basal levels.

Circulating Phylotypes of White Spot Syndrome Virus in Bangladesh and Their Virulence

White Spot Syndrome Virus (WSSV) has emerged as one of the most prevalent and lethal viruses globally and infects both shrimps and crabs in the aquatic environment. This study aimed to investigate the occurrence of WSSV in different ghers of Bangladesh and the virulence of the circulating phylotypes. We collected 360 shrimp (Penaeus monodon) and 120 crab (Scylla sp.) samples from the south-east (Cox’s Bazar) and south-west (Satkhira) coastal regions of Bangladesh. The VP28 gene-specific PCR assays and sequencing revealed statistically significant (p < 0.05, Kruskal–Wallis test) differences in the prevalence of WSSV in shrimps and crabs between the study areas (Cox’s Bazar and Satkhira) and over the study periods (2017–2019). The mean Log load of WSSV varied from 8.40 (Cox’s Bazar) to 10.48 (Satkhira) per gram of tissue. The mean values for salinity, dissolved oxygen, temperature and pH were 14.71 ± 0.76 ppt, 3.7 ± 0.1 ppm, 34.11 ± 0.38 °C and 8.23 ± 0.38, respectively, in the WSSV-positive ghers. The VP28 gene-based phylogenetic analysis showed an amino-acid substitution (E→G) at the 167th position in the isolates from Cox’s Bazar (referred to as phylotype BD2) compared to the globally circulating one (BD1). Shrimp PL artificially challenged with BD1 and BD2 phylotypes with filtrates of tissue containing 0.423 × 109 copies of WSSV per mL resulted in a median LT50 value of 73 h and 75 h, respectively. The in vivo trial showed higher mean Log WSSV copies (6.47 ± 2.07 per mg tissue) in BD1-challenged shrimp PL compared to BD2 (4.75 ± 0.35 per mg tissue). Crabs infected with BD1 and BD2 showed 100% mortality within 48 h and 62 h of challenge, respectively, with mean Log WSSV copies of 12.06 ± 0.48 and 9.95 ± 0.37 per gram tissue, respectively. Moreover, shrimp antimicrobial peptides (AMPs), penaeidin and lysozyme expression were lower in the BD1-challenged group compared to BD2 challenged shrimps. These results collectively demonstrated that relative virulence properties of WSSV based on mortality rate, viral load and expression of host immune genes in artificially infected shrimp PL could be affected by single aa substitution in VP28.

Antiviral Property of the Fungal Metabolite 3-O-Methylfunicone in Bovine Herpesvirus 1 Infection

Bovine herpesvirus type-1 (BoHV-1) is a widespread pathogen that provokes infectious rhinotracheitis and polymicrobial infections in cattle, resulting in serious economic losses to the farm animal industry and trade restrictions. To date, non-toxic active drugs against BoHV-1 are not available. The exploitation of bioactive properties of microbial products is of great pharmaceutical interest. In fact, fungi are a promising source of novel drugs with a broad spectrum of activities and functions, including antiviral properties. Hence, the potential antiviral properties of 3-O-methylfunicone (OMF), a secondary metabolite produced by Talaromyces pinophilus, were evaluated on BoHV-1. In this study, during BoHV-1 infection in bovine cells (MDBK), the non-toxic concentration of 5 µM OMF considerably reduced signs of cell death and increased cell proliferation. Furthermore, OMF significantly decreased the virus titer as well as the cytopathic effect and strongly inhibited the expression of bICP0, the major regulatory protein in the BoHV-1 lytic cycle. These findings were accompanied by a considerable up-regulation in the expression of the aryl hydrocarbon receptor (AhR), a multifunctional transcription factor also linked to the host’s response to a herpesvirus infection. Overall, our results suggest that by involving AhR, OMF shows potential against a BoHV-1 infection.

Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse β-Oxidation Pathway in the Presence of Lanthanide

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse b-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

Bridging the Gap: Type III Secretion Systems in Plant-Beneficial Bacteria

Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines translocating effector proteins into the cytoplasm of eukaryotic cells. They have been intensively studied for their important roles in animal and plant bacterial diseases. Over the past two decades, genome sequencing has unveiled their ubiquitous distribution in many taxa of Gram-negative bacteria, including plant-beneficial ones. Here, we discuss the distribution and functions of the T3SS in two agronomically important bacterial groups: the symbiotic nodule-forming nitrogen-fixing rhizobia and the free-living plant-beneficial Pseudomonas spp. In legume-rhizobia symbiosis, T3SSs and their cognate effectors play important roles, including the modulation of the plant immune response and the initiation of the nodulation process in some cases. In plant-beneficial Pseudomonas spp., the roles of T3SSs are not fully understood, but pertain to plant immunity suppression, biocontrol against eukaryotic plant pathogens, mycorrhization facilitation, and possibly resistance against protist predation. The diversity of T3SSs in plant-beneficial bacteria points to their important roles in multifarious interkingdom interactions in the rhizosphere. We argue that the gap in research on T3SSs in plant-beneficial bacteria must be bridged to better understand bacteria/eukaryotes rhizosphere interactions and to support the development of efficient plant-growth promoting microbial inoculants.

Spatiotemporal Changes in Plasmodium vivax msp142 Haplotypes in Southern Mexico: From the Control to the Pre-Elimination Phase

For 20 years, Plasmodium vivax has been the only prevalent malaria species in Mexico, and cases have declined significantly and continuously. Spatiotemporal genetic studies can be helpful for understanding parasite dynamics and developing strategies to weaken malaria transmission, thus facilitating the elimination of the parasite. The aim of the current contribution was to analyze P. vivax-infected blood samples from patients in southern Mexico during the control (1993–2007) and pre-elimination phases (2008–2011). Nucleotide and haplotype changes in the pvmsp142 fragment were evaluated over time. The majority of multiple genotype infections occurred in the 1990s, when the 198 single nucleotide sequences exhibited 57 segregating sites, 64 mutations, and 17 haplotypes. Nucleotide and genetic diversity parameters showed subtle fluctuations from across time, in contrast to the reduced haplotype diversity and the increase in the R2 index and Tajima’s D value from 2008 to 2011. The haplotype network consisted of four haplogroups, the geographical distribution of which varied slightly over time. Haplogroup-specific B-cell epitopes were predicted. Since only high-frequency and divergent haplotypes persisted, there was a contraction of the parasite population. Given that 84% of haplotypes were exclusive to Mesoamerica, P. vivax flow is likely circumscribed to this region, representing important information for parasite surveillance.

Comparative Genomics of Typical and Atypical Aeromonas salmonicida Complete Genomes Revealed New Insights into Pathogenesis Evolution

Aeromonas salmonicida is a global distributed Gram-negative teleost pathogen, affecting mainly salmonids in fresh and marine environments. A. salmonicida strains are classified as typical or atypical depending on their origin of isolation and phenotype. Five subspecies have been described, where A. salmonicida subsp. salmonicida is the only typical subspecies, and the subsp. achromogenes, masoucida, smithia, and pectinolytica are considered atypical. Genomic differences between A. salmonicida subsp. salmonicida isolates and their relationship with the current classification have not been explored. Here, we sequenced and compared the complete closed genomes of four virulent strains to elucidate their molecular diversity and pathogenic evolution using the more accurate genomic information so far. Phenotypes, biochemical, and enzymatic profiles were determined. PacBio and MiSeq sequencing platforms were utilized for genome sequencing. Comparative genomics showed that atypical strains belong to the subsp. salmonicida, with 99.55 ± 0.25% identity with each other, and are closely related to typical strains. The typical strain A. salmonicida J223 is closely related to typical strains, with 99.17% identity with the A. salmonicida A449. Genomic differences between atypical and typical strains are strictly related to insertion sequences (ISs) activity. The absence and presence of genes encoding for virulence factors, transcriptional regulators, and non-coding RNAs are the most significant differences between typical and atypical strains that affect their phenotypes. Plasmidome plays an important role in A. salmonicida virulence and genome plasticity. Here, we determined that typical strains harbor a larger number of plasmids and virulence-related genes that contribute to its acute virulence. In contrast, atypical strains harbor a single, large plasmid and a smaller number of virulence genes, reflected by their less acute virulence and chronic infection. The relationship between phenotype and A. salmonicida subspecies’ taxonomy is not evident. Comparative genomic analysis based on completed genomes revealed that the subspecies classification is more of a reflection of the ecological niche occupied by bacteria than their divergences at the genomic level except for their accessory genome.

Targeting of Regulators as a Promising Approach in the Search for Novel Antimicrobial Agents

Since the discovery of penicillin in the first half of the last century, antibiotics have become the pillars of modern medicine for fighting bacterial infections. However, pathogens resistant to antibiotic treatment have increased in recent decades, and efforts to discover new antibiotics have decreased. As a result, it is becoming increasingly difficult to treat bacterial infections successfully, and we look forward to more significant efforts from both governments and the scientific community to research new antibacterial drugs. This perspective article highlights the high potential of bacterial transcriptional and posttranscriptional regulators as targets for developing new drugs. We highlight some recent advances in the search for new compounds that inhibit their biological activity and, as such, appear very promising for treating bacterial infections.