An intramolecular cross-talk in D29 mycobacteriophage endolysin governs the lytic cycle and phage-host population dynamics.

D29 mycobacteriophage encodes LysA endolysin, which mediates mycobacterial host cell lysis by targeting its peptidoglycan layer, thus projecting itself as a potential therapeutic. However, the regulatory mechanism of LysA during the phage lytic cycle remains ill defined. Here, we show that during D29 lytic cycle, structural and functional regulation of LysA not only orchestrates host cell lysis but also is critical for maintaining phage-host population dynamics by governing various phases of lytic cycle. We report that LysA exists in two conformations, of which only one is active, and the protein undergoes a host peptidoglycan-dependent conformational switch to become active for carrying out endogenous host cell lysis. D29 maintains a pool of inactive LysA, allowing complete assembly of phage progeny, thus helping avoid premature host lysis. In addition, we show that the switch reverses after lysis, thus preventing exogenous targeting of bystanders, which otherwise negatively affects phage propagation in the environment.

Pseudomonas Phage ZCPS1 Endolysin as a Potential Therapeutic Agent.

The challenge of antibiotic resistance has gained much attention in recent years due to the rapid emergence of resistant bacteria infecting humans and risking industries. Thus, alternatives to antibiotics are being actively searched for. In this regard, bacteriophages and their enzymes, such as endolysins, are a very attractive alternative. Endolysins are the lytic enzymes, which are produced during the late phase of the lytic bacteriophage replication cycle to target the bacterial cell walls for progeny release. Here, we cloned, expressed, and purified LysZC1 endolysin from Pseudomonas phage ZCPS1. The structural alignment, molecular dynamic simulation, and CD studies suggested LysZC1 to be majorly helical, which is highly similar to various phage-encoded lysozymes with glycoside hydrolase activity. Our endpoint turbidity reduction assay displayed the lytic activity against various Gram-positive and Gram-negative pathogens. Although in synergism with EDTA, LysZC1 demonstrated significant activity against Gram-negative pathogens, it demonstrated the highest activity against Bacillus cereus. Moreover, LysZC1 was able to reduce the numbers of logarithmic-phase B. cereus by more than 2 log10 CFU/mL in 1 h and also acted on the stationary-phase culture. Remarkably, LysZC1 presented exceptional thermal stability, pH tolerance, and storage conditions, as it maintained the antibacterial activity against its host after nearly one year of storage at 4 °C and after being heated at temperatures as high as 100 °C for 10 min. Our data suggest that LysZC1 is a potential candidate as a therapeutic agent against bacterial infection and an antibacterial bio-control tool in food preservation technology.

Corrigendum: Separation of Mycobacterium smegmatis from a mixed culture using the cell wall binding domain of D29 mycobacteriophage endolysin.

[This corrects the article DOI: 10.3389/fmicb.2020.01119.].

Separation of Mycobacterium smegmatis From a Mixed Culture Using the Cell Wall Binding Domain of D29 Mycobacteriophage Endolysin.

Pathological infection caused by Mycobacterium tuberculosis is still a major global health concern. Traditional diagnostic methods are time-consuming, less sensitive, and lack high specificity. Due to an increase in the pathogenic graph of mycobacterial infections especially in developing countries, there is an urgent requirement for a rapid, low cost, and highly sensitive diagnostic method. D29 mycobacteriophage, which is capable of infecting and killing M. tuberculosis, projects itself as a potential candidate for the development of novel diagnostic methods and phage therapy of mycobacterial infections. In our previous study, we showed that the cell wall binding domain [C-terminal domain (CTD)] located at the C-terminal end of the D29 mycobacteriophage LysA endolysin very selectively binds to the peptidoglycan (PG) of Mycobacterium smegmatis and M. tuberculosis. Here, by using M. smegmatis as model organism and by exploiting the PG binding ability of CTD, we have developed a method to isolate M. smegmatis cells from a mixed culture via magnetic separation. We show that green fluorescent protein (GFP)-tagged CTD (CTD-GFP) can bind to M. smegmatis cells in vitro after treatment with non-ionic detergent Triton X-100. Fluorescence-based assays show that CTD-GFP binding to M. smegmatis cells is highly specific and stable, and is not disrupted by an excess of either GFP or BSA. We further fused CTD with glutathione-S-transferase (GST) to generate CTD-GST protein and carried out an anti-GST antibody-mediated coating of CTD-GST on Dynabeads. This allowed us to perform successful magnetic separation of M. smegmatis from a mixed culture of bacteria having both Gram-negative and Gram-positive bacteria. Furthermore, the separated cells could be confirmed by a simple PCR. Thus our assay allows us to separate and identify M. smegmatis from a mixed culture.

Understanding the role of the lysozyme-like domain of D29 mycobacteriophage-encoded endolysin in host cell lysis and phage propagation.

Mycobacteriophages are viruses that infect and kill mycobacteria. The peptidoglycan hydrolase, lysin A (LysA), coded by one of the most potent mycobacteriophages, D29, carries two catalytic domains at its N-terminus and a cell wall-binding domain at its C-terminus. Here, we have explored the importance of the centrally located lysozyme-like catalytic domain (LD) of LysA in phage physiology. We had previously identified an R198A substitution that causes inactivation of the LD when it is present alone on a polypeptide. Here, we show that upon incorporation of the same mutation (i.e. R350A) in full-length LysA, the protein demonstrates substantially reduced activity in vitro, even in the presence of the N-terminal catalytic domain, and has less efficient mycobacterial cell lysis ability when it is expressed in Mycobacterium smegmatis. These data suggest that an active LD is required for the full-length protein to function optimally. Moreover, a mutant D29 phage harbouring this substitution (D29R350A) in its LysA protein shows significantly delayed host M. smegmatis lysis. However, the mutant phage demonstrates an increase in burst size and plaque diameter. Taken together, our data show the importance of an intact LD region in D29 LysA PG hydrolase, and indicate an evolutionary advantage over other phages that lack such a domain in their endolysins.

Fission-fusion dynamics and group-size-dependent composition in heterogeneous populations.

Many animal groups are heterogeneous and may even consist of individuals of different species, called mixed-species flocks. Mathematical and computational models of collective animal movement behavior, however, typically assume that groups and populations consist of identical individuals. In this paper, using the mathematical framework of the coagulation-fragmentation process, we develop and analyze a model of merge and split group dynamics, also called fission-fusion dynamics, for heterogeneous populations that contain two types (or species) of individuals. We assume that more heterogeneous groups experience higher split rates than homogeneous groups, forming two daughter groups whose compositions are drawn uniformly from all possible partitions. We analytically derive a master equation for group size and compositions and find mean-field steady-state solutions. We predict that there is a critical group size below which groups are more likely to be homogeneous and contain the abundant type or species. Despite the propensity of heterogeneous groups to split at higher rates, we find that groups are more likely to be heterogeneous but only above the critical group size. Monte Carlo simulation of the model show excellent agreement with these analytical model results. Thus, our model makes a testable prediction that composition of flocks are group-size-dependent and do not merely reflect the population level heterogeneity. We discuss the implications of our results to empirical studies on flocking systems.

Complexity of Intercalation in MXenes: Destabilization of Urea by Two-Dimensional Titanium Carbide.

MXenes are a new class of two-dimensional materials with properties that make them important for applications that include batteries, capacitive energy storage, and electrocatalysis. These materials can be exfoliated and delaminated to create high surface areas with interlayers accessibility. Intercalation is known to be possible, and it is critical for many applications including electrochemical energy storage, water purification, and sensing. However, little is known about the nature of the intercalant and bonding interactions between the intercalant within the MXene. We have investigated urea interaction within a titanium carbide based MXene using inelastic neutron scattering (INS) to probe the state of intercalated species. By comparison with reference materials, we find that under intercalation conditions urea decomposes readily, leading to intercalation of ammonium cations observable by INS and evolving carbon dioxide detected by infrared spectroscopy. Reactive molecular dynamics calculations were conducted to provide atomistic insights about reaction pathways and their energetics. These results have implications for understanding intercalation in active layered materials.

Impact of Parthenium hysterophorus leaf extracts on the fecundity, fertility and behavioural response of Aedes aegypti L.

Different extracts of 1,000 ppm were prepared from the leaves of Parthenium hysterophorus using acetone, benzene, petroleum ether, diethyl ether and hexane as the solvents. The efficacy of each extract was assessed against dengue fever vector, Aedes aegypti by evaluating the variations in fecundity, fertility and behavioural response of the female adults. The leaf extracts could cause 70-100% repellency in the oviposition behaviour of the adults. The diethyl ether extract was found to be the most effective extract resulting in maximum effective repellency (99.7%) leading to the highest levels of reduced fecundity and 100% egg mortality followed by benzene extracts causing 93.8% reduced oviposition and 100% ovicidal effect. Hexane and acetone extracts with the least oviposition deterrence of 70-74% and negligible egg mortality (8-9%) proved to be the least effective extracts. The petroleum ether extract had a moderate impact resulting in 93.2% diminished fecundity and 41% ovicidal effect. The behavioural response of female adults of A. aegypti was evaluated by performing spatial repellency and contact irritancy assays. The most significant spatial repellency behaviour was elicited by acetone extracts leading to escape of 80% mosquitoes. Hexane and diethyl ether extracts could cause moderate response with 50-60% escape, while a slight and no reaction was observed on exposure to petroleum ether and benzene extracts, respectively. An interesting observation was the knocked-down activity caused by the hexane extracts with no recovery even after 24 h. A significant contact irritancy response was noticed in the mosquitoes on exposure to acetone leaf extracts resulting in first flight only after 4 s and a total of 12 flights during exposure. No irritancy behaviour was observed on exposure to diethyl ether and benzene leaf extracts. However, as against controls, a slight irritability response was noticed on exposure to hexane leaf extracts resulting in relative irritability of 1.2. Our results suggest the selective efficiency of Parthenium leaf extracts against A. aegypti, as the most effective oviposition deterrent and ovicidal agent was least effective as irritant extract and vice-versa. Further detailed research is needed to identify the active ingredient in the extracts and implement the effective mosquito management programme.