Antibiotic resistance: a global crisis, problems and solutions.

Healthy state is priority in today's world which can be achieved using effective medicines. But due to overuse and misuse of antibiotics, a menace of resistance has increased in pathogenic microbes. World Health Organization (WHO) has announced ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) as the top priority pathogens as these have developed resistance against certain antibiotics. To combat such a global issue, it is utmost important to identify novel therapeutic strategies/agents as an alternate to such antibiotics. To name certain antibiotic adjuvants including: inhibitors of beta-lactamase, efflux pumps and permeabilizers for outer membrane can potentially solve the antibiotic resistance problems. In this regard, inhibitors of lytic domain of lytic transglycosylases provide a novel way to not only act as an alternate to antibiotics but also capable of restoring the efficiency of previously resistant antibiotics. Further, use of bacteriophages is another promising strategy to deal with antibiotic resistant pathogens. Taking in consideration the alternatives of antibiotics, a green synthesis nanoparticle-based therapy exemplifies a good option to combat microbial resistance. As horizontal gene transfer (HGT) in bacteria facilitates the evolution of new resistance strains, therefore identifying the mechanism of resistance and development of inhibitors against it can be a novel approach to combat such problems. In our perspective, host-directed therapy (HDT) represents another promising strategy in combating antimicrobial resistance (AMR). This approach involves targeting specific factors within host cells that pathogens rely on for their survival, either through replication or persistence. As many new drugs are under clinical trials it is advisable that more clinical data and antimicrobial stewardship programs should be conducted to fully assess the clinical efficacy and safety of new therapeutic agents.

Sulfur transfer from the endophytic fungus Serendipita indica improves maize growth and requires the sulfate transporter SiSulT.

A deficiency of the essential macronutrient sulfur leads to stunted plant growth and yield loss; however, an association with a symbiotic fungus can greatly improve nutrient uptake by the host plant. Here, we identified and functionally characterized a high-affinity sulfate transporter from the endophytic fungus Serendipita indica. SiSulT fulfills all the criteria expected of a functional sulfate transporter responding to sulfur limitation: SiSulT expression was induced when S. indica was grown under low-sulfate conditions, and heterologous expression of SiSulT complemented a yeast mutant lacking sulfate transport. We generated a knockdown strain of SiSulT by RNA interference to investigate the consequences of the partial loss of this transporter for the fungus and the host plant (maize, Zea mays) during colonization. Wild-type (WT) S. indica, but not the knockdown strain (kd-SiSulT), largely compensated for low-sulfate availability and supported plant growth. Colonization by WT S. indica also allowed maize roots to allocate precious resources away from sulfate assimilation under low-sulfur conditions, as evidenced by the reduction in expression of most sulfate assimilation genes. Our study illustrates the utility of the endophyte S. indica in sulfur nutrition research and offers potential avenues for agronomically sound amelioration of plant growth in low-sulfate environments.

Biochemical characterization of a high affinity phosphate transporter (PiPT) from root endophyte fungus Piriformospora indica.

We have functionally characterized the high-affinity phosphate transporter (PiPT) from the root endophyte fungus Piriformospora indica. PiPT belongs to the major facilitator superfamily (MFS). PiPT protein was purified by affinity chromatography (Ni-NTA) and Size Exclusion Chromatography (SEC). The functionality of solubilized PiPT was determined in detergent-solubilized state by fluorescence quenching and in proteoliposomes. In the fluorescence quenching assay, PiPT exhibited a saturation concentration of approximately 2 µM, at a pH of 4.5. Proteoliposomes of size 121.6 nm radius, showed transportation of radioactive phosphate. Vmax was measured to be 232.2 ± 11 pmol/min/mg protein. We have found Km to be 45.8 ± 6.2 µM suggesting high affinity towards phosphate.

Key computational findings reveal proton transfer as driving the functional cycle in the phosphate transporter PiPT.

Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from Piriformospora indica (PiPT) that deprotonation of aspartate 324 (D324) triggers phosphate release. Quantum mechanics/molecular mechanics molecular dynamics simulations combined with free energy sampling have been employed here to identify the proton transport pathways from D324 upon the transition from the occluded structure to the inward open structure and phosphate release. The computational insights so gained are then corroborated by studies of D45N and D45E amino acid substitutions via mutagenesis experiments. Our findings confirm the function of the structurally predicted cytosolic proton exit tunnel and suggest insights into the role of the titratable phosphate substrate.

Antibiotic resistance, biofilm formation, and virulence genes of Streptococcus agalactiae serotypes of Indian origin.

BACKGROUND: Group B Streptococcus (GBS) is a causative agent of various infections in newborns, immunocompromised (especially diabetic) non-pregnant adults, and pregnant women. Antibiotic resistance profiling can provide insights into the use of antibiotic prophylaxis against potential GBS infections. Virulence factors are responsible for host-bacteria interactions, pathogenesis, and biofilm development strategies. The aim of this study was to determine the biofilm formation capacity, presence of virulence genes, and antibiotic susceptibility patterns of clinical GBS isolates. RESULTS: The resistance rate was highest for penicillin (27%; n = 8 strains) among all the tested antibiotics, which indicates the emergence of penicillin resistance among GBS strains. The susceptibility rate was highest for ofloxacin (93%; n = 28), followed by azithromycin (90%; n = 27). Most GBS strains (70%; n = 21) were strong biofilm producers and the rest (30%; n = 9) were moderate biofilm producers. The most common virulence genes were cylE (97%), pavA (97%), cfb (93%), and lmb (90%). There was a negative association between having a strong biofilm formation phenotype and penicillin susceptibility, according to Spearman's rank correlation analysis. CONCLUSION: About a third of GBS strains exhibited penicillin resistance and there was a negative association between having a strong biofilm formation phenotype and penicillin susceptibility. Further, both the strong and moderate biofilm producers carried most of the virulence genes tested for, and the strong biofilm formation phenotype was not associated with the presence of any virulence genes.

Functional characterization of a high-affinity iron transporter (PiFTR) from the endophytic fungus Piriformospora indica and its role in plant growth and development.

Iron (Fe) is a micronutrient required for plant growth and development; however, most Fe forms in soil are not readily available to plants, resulting in low Fe contents in plants and, thereby, causing Fe deficiency in humans. Biofortification through plant-fungal co-cultivation might be a sustainable approach to increase crop Fe contents. Therefore, we aimed to examine the role of a Piriformospora indica Fe transporter on rice Fe uptake under low Fe conditions. A high-affinity Fe transporter (PiFTR) from P. indica was identified and functionally characterized. PiFTR fulfilled all criteria expected of a functional Fe transporter under Fe-limited conditions. Additionally, PiFTR expression was induced when P. indica was grown under low Fe conditions, and PiFTR complemented a yeast mutant lacking Fe transport. A knockdown (KD) P. indica strain was created via RNA interference to understand the physiological role of PiFTR. We observed that the KD-PiFTR-P. indica strain transported a significantly lower amount of Fe to colonized rice (Oryza sativa) than the wild type (WT) P. indica. WT P. indica-colonized rice plants were healthier and performed significantly better than KD-PiFTR-P. indica-colonized rice plants. Our study offers potential avenues for an agronomically sound amelioration of plant growth in low Fe environments.

Advanced strategies for development of vaccines against human bacterial pathogens.

Infectious diseases are one of the main grounds of death and disabilities in human beings globally. Lack of effective treatment and immunization for many deadly infectious diseases and emerging drug resistance in pathogens underlines the need to either develop new vaccines or sufficiently improve the effectiveness of currently available drugs and vaccines. In this review, we discuss the application of advanced tools like bioinformatics, genomics, proteomics and associated techniques for a rational vaccine design.

WRKY6 restricts Piriformospora indica-stimulated and phosphate-induced root development in Arabidopsis.

BACKGROUND: Arabidopsis root growth is stimulated by Piriformospora indica, phosphate limitation and inactivation of the WRKY6 transcription factor. Combinations of these factors induce unexpected alterations in root and shoot growth, root architecture and root gene expression profiles. RESULTS: The results demonstrate that P. indica promotes phosphate uptake and root development under Pi limitation in wrky6 mutant. This is associated with the stimulation of PHOSPHATE1 expression and ethylene production. Expression profiles from the roots of wrky6 seedlings identified genes involved in hormone metabolism, transport, meristem, cell and plastid proliferation, and growth regulation. 25 miRNAs were also up-regulated in these roots. We generated and discuss here a list of common genes which are regulated in growing roots and which are common to all three growth stimuli investigated in this study. CONCLUSION: Since root development of wrky6 plants exposed to P. indica under phosphate limitation is strongly promoted, we propose that common genes which respond to all three growth stimuli are central for the control of root growth and architecture. They can be tested for optimizing root growth in model and agricultural plants.

Isolation of genes conferring salt tolerance from Piriformospora indica by random overexpression in Escherichia coli.

Piriformospora indica, a root endophytic fungus identified in the Indian Thar desert, colonizes the roots of plants and provides resistance towards biotic stress as well as tolerance to abiotic stress in the plants. Despite its positive impact on the host, little is known about the P. indica genes that are involved in salt stress tolerance. Therefore this study was conducted to identify and isolate high salinity-tolerance genes from P. indica. Thirty-six salinity-tolerance genes were obtained by functional screening, based on random over expression of a P. indica cDNA library in Escherichia coli grown on medium supplemented with 0.6 M NaCl. The salinity tolerance conferred by these 36 genes in bacteria was further confirmed by using another strain of E. coli (DH5α) transformants. However when the expression of these 36 genes was analysed in P. indica using quantitative RT-PCR, we found only six genes were up-regulated by salt stress. These six genes are involved in different cellular processes, such as metabolism, energy and biosynthetic processes, DNA repair, regulation of protein turnover, transport and salt stress tolerance. This work presents the basis for further molecular analyses of the mechanisms of salt tolerance in P. indica and for the use of this endophyte to confer salt tolerance to plants.

Role of pilus proteins in adherence and invasion of Streptococcus agalactiae to the lung and cervical epithelial cells.

Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. We have performed comparative genomics of prevalent GBS serotypes of Indian origin (i.e. Ia, III, V, and VII). Pilus-proteins were commonly found up-regulated, and their expression was studied by using antiserum for GBS80 (backbone protein of pilus island-I), GBS67 (ancillary protein of PI-2a), and SAN1518 (backbone protein of PI-2b) by whole cell and Western blot analysis. To check the role of pilus proteins in adherence and invasion, an inhibition assay was performed. Comparative immunoblotting experiments revealed that expression of pili proteins does not differ in geographically different selected serotypes, Ia and V, of India and the United States. In the case of A549 cells, we found that GBS VII invasion and adherence was inhibited by pilus protein-specific antiserum SAN1518 significantly (p < 0.001) by 88.5 and 91%, respectively. We found that mutant strains, deficient in the pilus proteins (Δgbs80 and Δsan1518) exhibit a significant decrease in adherence in the case of type Ia, III, and VII. In the case of type VII, we have found a 95% reduction in invasion when Δsan1518 was used with A549 cells. Because the pilus proteins were identified previously as vaccine candidates against GBS serotypes of developed countries, we also found their role in the attachment and invasion of GBS of Indian origin. Thus, the present work supports the idea of making a more effective pilus protein-based vaccine that can be used universally.

Recombinant YopE and LcrV vaccine candidates protect mice against plague and yersiniosis.

No licensed vaccine exists for the lethal plague and yersiniosis. Therefore, a combination of recombinant YopE and LcrV antigens of Yersinia pestis was evaluated for its vaccine potential in a mouse model. YopE and LcrV in formulation with alum imparted a robust humoral immune response, with isotyping profiles leaning towards the IgG1 and IgG2b subclasses. It was also observed that a significantly enhanced expression of IFN-γ, TNF-α, IL-6, IL-2, and IL-1ß from the splenic cells of vaccinated mice, as well as YopE and LcrV-explicit IFN-γ eliciting T-cells. The cocktail of YopE + LcrV formulation conferred complete protection against 100 LD50Y. pestis infection, while individually, LcrV and YopE provided 80 % and 60 % protection, respectively. Similarly, the YopE + LcrV vaccinated animal group had significantly lower colony forming unit (CFU) counts in the spleen and blood compared to the groups administered with YopE or LcrV alone when challenged with Yersinia pseudotuberculosis and Yersinia enterocolitica. Histopathologic evidence reinforces these results, indicating the YopE + LcrV formulation provided superior protection against acute lung injury as early as day 3 post-challenge. In conclusion, the alum-adjuvanted YopE + LcrV is a promising vaccine formulation, eliciting a robust antibody response including a milieu of pro-inflammatory cytokines and T-cell effector functions that contribute to the protective immunity against Yersinia infections. YopE and LcrV, conserved across all three human-pathogenic Yersinia species, provide cross-protection. Therefore, our current vaccine (YopE + LcrV) targets all three pathogens: Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica. However, the efficacy should be tested in other higher mammalian models.

Identification of potential universal vaccine candidates against group A Streptococcus by using high throughput in silico and proteomics approach.

Streptococcus pyogenes or group A Streptococcus (GAS) causes ~700 million human infections each year, resulting in over 500,000 deaths. The development of a commercial GAS vaccine is hampered due to high strain and serotype diversity in different geographical regions, and the generation of cross-reactive antibodies that may induce autoimmune disease. There is an urgent need to search for alternative vaccine candidates. High throughput multigenome data mining coupled with proteomics seems to be a promising approach to identify the universal vaccine candidates. In the present study, in silico analysis led to prediction of 147 proteins as universal vaccine candidates. Distribution pattern of these predicted candidates was explored in nonsequenced Indian GAS strains (n = 20) by using DNA array hybridization validating in silico analysis. High throughput analyses of surface proteins using 1D-SDS-PAGE coupled with ESI-LC-MS/MS was applied on highly (M49) and less (M1) invasive GAS strains of Indian origin. Comparative proteomics analysis revealed that highly invasive GAS M49 had metabolically more active membrane associated protein machinery than less invasive M1. Further, by overlapping proteomics data with in silico predicted vaccine candidate genes, 52 proteins were identified as probable universal vaccine candidates, which were expressed in these GAS serotypes. These proteins can further be investigated as universal vaccine candidates against GAS. Moreover, this robust approach may serve as a model that can be applied to identify the universal vaccine candidates in case of other pathogenic bacteria with high strain and genetic diversity.

Maize Seedlings Colonization with Serendipita indica and Its Colonization Efficiency Analysis.

Maize is one of the most important crops in the world, and ensuring its successful growth and productivity is crucial for global food security. One way to enhance maize growth and productivity is by improving the colonization of its roots by beneficial microorganisms. In this regard, Serendipita indica, a plant growth-promoting fungus, has gained attention for its ability to enhance plant growth and productivity, especially in cereal crops and medicinal plants. Previous studies have shown that S. indica can colonize various plant species, including maize, but the efficiency of the colonization process in maize seedlings has not been extensively characterized. This protocol outlines a method for efficient colonization of maize seedlings with the beneficial fungus S. indica. The protocol includes the preparation of stock solutions, maintenance and growth of S. indica, surface sterilization and germination of seeds, preparation of S. indica chlamydospores, and colonization of maize plants with S. indica. The advantages of this protocol include the use of surface sterilization techniques that minimize contamination, the production of a large number of viable chlamydospores, and efficient colonization of maize seedlings with S. indica. This protocol may be useful for researchers studying the role of S. indica in promoting plant growth and combating biotic and abiotic stress. Additionally, this protocol may be used in the development of biofertilizers using S. indica as a means of increasing crop yields and reducing dependence on synthetic fertilizers. Overall, this protocol offers a reliable and efficient method for colonizing maize seedlings with S. indica and may have potential applications in the agricultural industry. This study also provides a valuable tool for researchers interested in studying plant-microbe interactions in maize and highlights the potential of S. indica as a biocontrol agent to enhance maize productivity under adverse conditions. Key features • This protocol builds upon the method developed by Narayan et al. (2022), and its application optimized for the root endophytic symbiotic fungus S. indica. • This protocol also allows for histochemical analysis to visualize the colonized fungal spores in the root cells of host plant species. • This protocol helps in mathematical calculation of the percent colonization or efficiency of colonization. • This protocol utilizes readily available laboratory equipment, including a light microscope, autoclave, and laminar flow hood, ensuring ease of reproducibility in other research laboratories.

New-age vaccine adjuvants, their development, and future perspective.

In the present scenario, immunization is of utmost importance as it keeps us safe and protects us from infectious agents. Despite the great success in the field of vaccinology, there is a need to not only develop safe and ideal vaccines to fight deadly infections but also improve the quality of existing vaccines in terms of partial or inconsistent protection. Generally, subunit vaccines are known to be safe in nature, but they are mostly found to be incapable of generating the optimum immune response. Hence, there is a great possibility of improving the potential of a vaccine in formulation with novel adjuvants, which can effectively impart superior immunity. The vaccine(s) in formulation with novel adjuvants may also be helpful in fighting pathogens of high antigenic diversity. However, due to the limitations of safety and toxicity, very few human-compatible adjuvants have been approved. In this review, we mainly focus on the need for new and improved vaccines; the definition of and the need for adjuvants; the characteristics and mechanisms of human-compatible adjuvants; the current status of vaccine adjuvants, mucosal vaccine adjuvants, and adjuvants in clinical development; and future directions.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of a cyclophilin A-like protein from Piriformospora indica.

Cyclophilins are widely distributed both in eukaryotes and prokaryotes and have a primary role as peptidyl-prolyl cis-trans isomerases (PPIases). This study focuses on the cloning, expression, purification and crystallization of a salinity-stress-induced cyclophilin A (CypA) homologue from the symbiotic fungus Piriformospora indica. Crystallization experiments in the presence of 56 mM sodium phosphate monobasic monohydrate, 1.34 M potassium phosphate dibasic pH 8.2 yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 121.15, b = 144.12, c = 110.63 Å. The crystals diffracted to a resolution limit of 2.0 Å. Analysis of the diffraction data indicated the presence of three molecules of the protein per asymmetric unit (V(M) = 4.48 Å(3) Da(-1), 72.6% solvent content).

Proteomic analysis of arsenite - mediated multiple antibiotic resistance in Yersinia enterocolitica biovar 1A.

Arsenic is one of the most important global environmental pollutants. In the present study, fifty one clinical strains of Yersinia enterocolitica biovar 1A showed high resistance to arsenite and arsenate. The minimum inhibitory concentration (MIC) of arsenite (0.625-20 mM) was lower than arsenate (10-80 mM). Growth of Y. enterocolitica in 2 mM arsenite led to 2-8 fold increase in MICs of the five antibiotics (amikacin, ciprofloxacin, gentamycin, kanamycin and tetracycline), suggesting expression of arsenite-induced multiple antibiotic resistance among the strains. Proteomic analysis of Y. enterocolitica revealed differential expression of certain proteins following arsenite exposure, which included a putative outer membrane porin (OmpA) and a putative amino acid transporter protein. In conclusion, modulation of membrane permeability may be involved in the induction of arsenite-mediated expression of multiple antibiotic resistance in Y. enterocolitica.

Sulfur nutrition and its role in plant growth and development.

Sulfur is one of the essential nutrients that is required for the adequate growth and development of plants. Sulfur is a structural component of protein disulfide bonds, amino acids, vitamins, and cofactors. Most of the sulfur in soil is present in organic matter and hence not accessible to the plants. Anionic form of sulfur (SO42-) is the primary source of sulfur for plants that are generally present in minimal amounts in the soil. It is water-soluble, so readily leaches out of the soil. Sulfur and sulfur-containing compounds act as signaling molecules in stress management as well as normal metabolic processes. They also take part in crosstalk of complex signaling network as a mediator molecule. Plants uptake sulfate directly from the soil by using their dedicated sulfate transporters. In addition, plants also use the sulfur transporter of a symbiotically associated organism like bacteria and fungi to uptake sulfur from the soil especially under sulfur depleted conditions. So, sulfur is a very important component of plant metabolism and its analysis with different dimensions is highly required to improve the overall well-being of plants, and dependent animals as well as human beings. The deficiency of sulfur leads to stunted growth of plants and ultimately loss of yield. In this review, we have focused on sulfur nutrition, uptake, transport, and inter-organismic transfer to host plants. Given the strong potential for agricultural use of sulfur sources and their applications, we cover what is known about sulfur impact on the plant health. We identify opportunities to expand our understanding of how the application of soil microbes like AMF or other root endophytic fungi affects plant sulfur uptake and in turn plant growth and development.

PERK Pathway Inhibitors Cure Group A Streptococcal Necrotizing Fasciitis in a Murine Model.

Group A streptococcus (GAS) is a Gram-positive human pathogen that causes invasive infections with mild to life-threatening severity, like toxic shock syndrome, rheumatic heart disease, and necrotizing fasciitis (NF). NF is characterized by a clinical presentation of widespread tissue destruction due to the rapid spread of GAS infection into fascial planes. Despite quick medical interventions, mortality from NF is high. The early onset of the disease is difficult to diagnose because of non-specific clinical symptoms. Moreover, the unavailability of an effective vaccine against GAS warrants a genuine need for alternative treatments against GAS NF. One endoplasmic reticulum stress signaling pathway (PERK pathway) gets triggered in the host upon GAS infection. Bacteria utilize asparagine release as an output of this pathway for its pathogenesis. We reported that the combination of sub-cutaneous (SC) and intraperitoneal (IP) administration of PERK pathway inhibitors (GSK2656157 and ISRIB) cures local as well as systemic GAS infection in a NF murine model, by reducing asparagine release at the infection site. This protocol's methodology is detailed below. This protocol was validated in: Sci Transl Med (2021), DOI: 10.1126/scitranslmed.abd7465.