A CcdB toxin-derived peptide acts as a broad-spectrum antibacterial therapeutic in infected mice.

The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for fluoroquinolones. Here, we present a short cell-penetrating 24-mer peptide, CP1-WT, derived from the Gyrase-binding region of CcdB and examine its effect on growth of Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and a carbapenem- and tigecycline-resistant strain of Acinetobacter baumannii in both axenic cultures and mouse models of infection. The CP1-WT peptide shows significant improvement over ciprofloxacin in terms of its in vivo therapeutic efficacy in treating established infections of S. Typhimurium, S. aureus and A. baumannii. The molecular mechanism likely involves inhibition of Gyrase or Topoisomerase IV, depending on the strain used. The study validates the CcdB binding site on bacterial DNA Gyrase as a viable and alternative target to the fluoroquinolone binding site.

Role of lactoyl-glutathione lyase of Salmonella in the colonization of plants under salinity stress.

Salmonella, a foodborne human pathogen, can colonize the members of the kingdom Plantae. However, the basis of the persistence of Salmonella in plants is largely unknown. Plants encounter various biotic and abiotic stress agents in soil. We conjectured that methylglyoxal (MG), one of the common metabolites that accumulate in plants during both biotic and abiotic stress, plays a role in regulating the plant-Salmonella interaction. The interaction of Salmonella Typhimurium with plants under salinity stress was investigated. It was observed that wild-type Salmonella Typhimurium can efficiently colonize the root, but mutant bacteria lacking MG detoxifying enzyme, lactoyl-glutathione lyase (Lgl), showed lower colonization in roots exclusively under salinity stress. This colonization defect is due to the poor viability of the mutated bacterial strains under these conditions. This is the first report to prove the role of MG-detoxification genes in the colonization of stressed plants and highlights the possible involvement of metabolic genes in the evolution of the plant-associated life of Salmonella.

Comparative genomics reveals the diversity of CRISPR-Cas locus in Azotobacter organisms.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are known to provide adaptive immunity to bacteria against invading bacteriophages. In recent years, CRISPR-based technologies have been used for creating improved plant varieties; however, the indigenous CRISPR-Cas elements of plant growth-promoting bacteria are usually neglected. These indigenous genetic cassettes have evolved over millions of years and have shaped the bacterial genome. Therefore, these genetic loci can be used to study the adaptive capability of the bacteria in the environment. This study aims to bioinformatically analyze the genomes of a common free-living nitrogen-fixing Azotobacter spp. to assess their CRISPR-Cas diversity. Strains of Azotobacter vinelandii and Azotobacter chroococcum were found to harbor a large number of spacers. The phylogeny of different Cas and Cse1 proteins revealed a close evolutionary relationship among A. chroococcum B3, A. chroococcum NCIMB 8003 locus II, and A. vinelandii DJ locus I. The secondary structure of the hairpin loop of the repeat was also analyzed, and a correlation was derived between the structural stability of the hairpin loop and the number of spacers acquired by the CRISPR loci. These findings revealed the diversity and evolution of the CRISPR sequences and Cas proteins in Azotobacter species. Although the adaptive immune system of bacteria against bacteriophage, CRISPR-Cas, has been identified in many bacteria, studies of plant growth-promoting bacteria have been neglected. These indigenous CRISPRs have shaped the genome over millions of years and their study can lead to the understanding of the genome composition of these organisms. Our results strengthen the idea of using A. chroococcum and A. vinelandii as biofertilizer strains as they possess more spacers with highly stable repeat sequences, thereby imparting them higher chance of survival against mobile genetic elements like phages and plasmids.

Lysinibacillus macroides-mediated control of cellulose-producing morphotype of Salmonella.

BACKGROUND: Soil-dwelling human pathogens like Salmonella are transmitted by fresh produce such as tomato, spinach, onion and cabbage. With >2600 serovars, it is difficult to classify the good plant colonizers from the non-colonizers. Generally, soil microbiota are classified as autochthonous or zymogenous organisms, based on their ability to survive in soil. However, such information for soil-dwelling human pathogens is not available Thus there is a need to classify these organisms for designing a strategy to prevent their outbreak. Moreover, soil harbours a plethora of microbes, which can be screened for competitive organisms to control such human pathogens. RESULTS: In this study, we examined whether the morphotype based on the attachment factors (e.g., cellulose and curli fimbri) of Salmonella was important for its colonization of roots. Secondly, we tracked the location of the bacteria in the plant cell. Interestingly, most of the epidermal cells occupied by Salmonella showed propidium iodide-positive nuclei. As an extension of the study, a screening of competitive rhizospheric bacteria was performed. One isolate, identified as Lysinibacillus macroides, was able to inhibit the biofilm of Salmonella and subsequently reduced its colonization on roots. CONCLUSION: Based on this study, we classified the Rdar (red, dry and rough) morphotypes as good plant colonists. The ability to colonize and subsequent kill the live plant cell throws light on the zymogenous life cycle of soil-dwelling Salmonella. Additionally, Lysinibacillus macroides served as a biocontrol agent by reducing the burden of Salmonella in various vegetables. Such organisms can further be explored to prevent contamination of the food chain. © 2022 Society of Chemical Industry.

Co-cultivation of Beta vulgaris limits the pre-harvest colonization of foodborne pathogen (Salmonella spp.) on tomato.

Soil-borne Salmonella is associated with a large number of food-related disease outbreaks linked to pre-harvest contamination of plants (like tomato) in agricultural fields. Controlling the spread of Salmonella at field is very important in order to prevent various food-borne illnesses. One such approach involves the utilization of antimicrobial secondary metabolite of plant origin. We screened common salad vegetables for anti-Salmonella activity. Beta vulgaris root (beetroot) had very low colonization of Salmonella under in vitro conditions. We hypothesized that beetroot can be used to reclaim the soil contaminated with Salmonella. Cultivation of B. vulgaris in Salmonella treated soil brings down its CFU significantly. Since these antimicrobial effects are non-specific, a co-cultivation system of beet and tomato (a Salmonella susceptible plant) was used to analyze the effect on soil and its microbiota. The soil physicochemical properties and bacterial diversity were unaffected when tomato and beet co-cultivation was used. However, Salmonella burden on the tomato was reduced and its yield was restored. Thus, the inclusion of these crops in the crop-rotation or as a mixed/intercrop or as a bio-control crop can be a fruitful tool to reclaim the Salmonella contaminated soil.

Immune modulations and survival strategies of evolved hypervirulent Salmonella Typhimurium strains.

BACKGROUND: Evolving multidrug-resistance and hypervirulence in Salmonella is due to multiple host-pathogen, and non-host environmental interactions. Previously we had studied Salmonella adaptation upon repeated exposure in different in-vitro and in-vivo environmental conditions. This study deals with the mechanistic basis of hypervirulence of the passaged hypervirulent Salmonella strains reported previously. METHODS: Real-time PCR, flow cytometry, western blotting, and confocal microscopy were employed to check the alteration of signaling pathways by the hypervirulent strains. The hypervirulence was also looked in-vivo in the Balb/c murine model system. RESULTS: The hypervirulent strains altered cytokine production towards anti-inflammatory response via NF-κB and Akt-NLRC4 signaling in RAW-264.7 and U-937 cells. They also impaired lysosome number, as well as co-localization with the lysosome as compared to unpassaged WT-STM. In Balb/c mice also they caused decreased antimicrobial peptides, reduced nitric oxide level, altered cytokine production, and reduced CD4+ T cell population leading to increased organ burden. CONCLUSIONS: Hypervirulent Salmonella strains infection resulted in an anti-inflammatory environment by upregulating IL-10 and down-regulating IL-1ß expression. They also evaded lysosomal degradation for their survival. With inhibition of NF-κB and Akt signaling, cytokine expression, lysosome number, as well as the bacterial burden was reverted, indicating the infection mediated immune modulation by the hypervirulent Salmonella strains through these pathways. GENERAL SIGNIFICANCE: Understanding the mechanism of adaptation can provide better disease prognosis by either targeting the bacterial gene or by strengthening the host immune system that might ultimately help in controlling salmonellosis.

Nanoceria-Based Phospholipase-Mimetic Cell Membrane Disruptive Antibiofilm Agents.

Continuous mounting of antibiotic resistance due to the narrow range of mechanisms targeted poses tremendous threat to global health. Highly resistant pathogenic bacteria dwelling in the biofilm mode on the surface of medical devices has increased the susceptibility of chronic as well as healthcare-associated infections. Lantipeptides have shown promising membrane disruption of Gram-positive bacteria, leading to programmed cell death, but they are impermeable and hence ineffective to the outer cell membrane of Gram-negative bacteria. Herein, we report for the first time that a polymer-coated nanoceria (PAA-Cnp) having phospholipase-mimetic activity can target the cell membrane of both Gram-negative and Gram-positive bacteria. The nanozyme shows promising membrane disruption-based bactericidal activity against a broad spectrum of pathogenic as well as biofilm-encased bacteria. The unprecedented nanozyme-based strategy described in this paper is useful in preventing biofilm formation on medical devices such as urinary catheters.

Development of mCherry tagged UdgX as a highly sensitive molecular probe for specific detection of uracils in DNA.

Uracil is not always a mistakenly occurring base in DNA. Uracils in DNA genomes are known to be important in the life cycles of Bacillus subtilis phages (PBS1/2) and the malarial parasite, Plasmodium falciparum; and have been implicated in the development of fruit fly and antibody maturation in B-lymphocytes. Availability of a sensitive, specific and robust technique for the detection uracils in genes/genomes is essential to understand its varied biological roles. Mycobacterium smegmatis UdgX (MsmUdgX), identified and characterised in our laboratory, forms covalent complexes with the uracil sites in DNA in a specific manner. MsmUdgX cleaves the glycosidic bond between uracil and the deoxyribose sugar in DNA to produce uracilate and oxocarbenium ions. The oxocarbenium ion is then captured into a covalent complex by the nucleophilic attack of a histidine side chain of MsmUdgX. Here, we describe the use of a fusion protein, mCherry tagged MsmUdgX (mChUdgX), which combines the property of MsmUdgX to covalently and specifically bind the uracil sites in the genome, with the sensitivity of fluorescent detection of mCherry as a reporter. We show that both the purified mChUdgX and the Escherichia coli cell-extracts overexpressing mChUdgX provide high sensitivity and specificity of detecting uracils in DNA.

Rhizospheric life of Salmonella requires flagella-driven motility and EPS-mediated attachment to organic matter and enables cross-kingdom invasion.

Salmonella is an established pathogen of the members of the kingdom Animalia. Reports indicate that the association of Salmonella with fresh, edible plant products occurs at the pre-harvest state, i.e. in the field. In this study, we follow the interaction of Salmonella Typhimurium with the model plant Arabidopsis thaliana to understand the process of migration in soil. Plant factors like root exudates serve as chemo-attractants. Our ex situ experiments allowed us to track Salmonella from its free-living state to the endophytic state. We found that genes encoding two-component systems and proteins producing extracellular polymeric substances are essential for Salmonella to adhere to the soil and roots. To understand the trans-kingdom flow of Salmonella, we fed the contaminated plants to mice and observed that it invades and colonizes liver and spleen. To complete the disease cycle, we re-established the infection in plant by mixing the potting mixture with the fecal matter collected from the diseased animals. Our experiments revealed a cross-kingdom invasion by the pathogen via passage through a murine intermediate, a mechanism for its persistence in the soil and invasion in a non-canonical host. These results form a basis to break the life-cycle of Salmonella before it reaches its animal host and thus reduce Salmonella contamination of food products.

Root mediated uptake of Salmonella is different from phyto-pathogen and associated with the colonization of edible organs.

BACKGROUND: Pre-harvest contamination of fruits and vegetables by Salmonella in fields is one of the causes of food-borne outbreaks. Natural openings like stomata, hydathodes and fruit cracks are known to serve as entry points. While there are reports indicating that Salmonella colonize and enter root through lateral root emerging area, further investigations regarding how the accessibility of Salmonella to lateral root is different from phyto-pathogenic bacteria, the efficacy of lateral root to facilitate entry have remained unexplored. In this study we attempted to investigate the lateral root mediated entry of Salmonella, and to bridge this gap in knowledge. RESULTS: Unlike phytopathogens, Salmonella cannot utilize cellulose as the sole carbon source. This negates the fact of active entry by degrading plant cellulose and pectin. Endophytic Salmonella colonization showed a high correlation with number of lateral roots. When given equal opportunity to colonize the plants with high or low lateral roots, Salmonella internalization was found higher in the plants with more lateral roots. However, the epiphytic colonization in both these plants remained unaltered. To understand the ecological significance, we induced lateral root production by increasing soil salinity which made the plants susceptible to Salmonella invasion and the plants showed higher Salmonella burden in the aerial organs. CONCLUSION: Salmonella, being unable to degrade plant cell wall material relies heavily on natural openings. Therefore, its invasion is highly dependent on the number of lateral roots which provides an entry point because of the epidermis remodeling. Thus, when number of lateral root was enhanced by increasing the soil salinity, plants became susceptible to Salmonella invasion in roots and its transmission to aerial organs.

Cells producing their own nemesis: understanding methylglyoxal metabolism.

Methylglyoxal, which is technically known as 2-oxopropanal or pyruvaldehyde, shows typical reactions of carbonyl compounds as it has both an aldehyde and a ketone functional group. It is an extremely cytotoxic physiological metabolite, which is generated by both enzymatic and nonenzymatic reactions. The deleterious nature of the compound is due to its ability to glycate and crosslink macromolecules like protein and DNA, respectively. However, despite having toxic effects on cellular processes, methylglyoxal retains its efficacy as an anticancer drug. Indeed, methylglyoxal is one of the well-known anticancer therapeutic agents used in the treatment. Several studies on methylglyoxal biology revolve around the manifestations of its inhibitory effects and toxicity in microbial growth and diabetic complications, respectively. Here, we have revisited the chronology of methylglyoxal research with emphasis on metabolism of methylglyoxal and implications of methylglyoxal production or detoxification on bacterial pathogenesis and disease progression.