Proteomic methods reveal cyclophilin a function as a host restriction factor against rotavirus infection

Rotavirus (RV) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. RV infection causes a global shutoff of host proteins as many other viruses do. However, previous studies revealed that RV could selectively upregulated the expression of some host proteins that then played important roles in RV infection. To globally explor such host proteins that were upregulated in early human rotavirus (HRV) infection, proteomic methods were used and a total of ten upregulated host proteins were unambiguously identified. Cyclophilin A (CYPA), a peptidyl‐prolyl cis‐trans isomerase, was among these upregulated host proteins. Following infection, CYPA was recruited to the viroplasm and interacted with HRV structural protein VP2; CYPA reduced host susceptibility to HRV infection and inhibited replication of HRV by repressing the expression of viral proteins. Furthermore, we found that the increased expression of CYPA in enterocytes of small intestine correlated to the period when BALB/c mice became resistant to RV diarrhea. Together, we identified CYPA as a novel host restriction factor that confered protection against RV infection and might contribute to host susceptibility to RV diarrhea.     

Antagonistic effect of two isolates of Trichoderma harzianum against postharvest pathogens of tomato (Lycopersicon esculentum)

Pathogenicity test of all fungi (14 different isolates) isolated from both infected tomato fruits and the surface wash of other healthy fruits had different pathogenicity rates. The genus Rhizopus sp. was the most pathogenic one followed by Fusarium sp. Trichoderma harzianum isolates (T3 and T4) had a different pattern of antagonism against the tested pathogens. In dual plate test of the antagonistic action of T3 and T24 against the postharvest pathogens, clear zone size ranged between 1 and 4 and 3 and 6 mm by T3 and T24, respectively. The antagonists (T3 and T24) didn't show inhibition zone against Rhizopus sp., but they could overgrow it by 100% after 9 days of incubation. Mostly, all the other postharvest isolates showed high degree of overgrowth by T3 than T24. The two antagonists failed to overgrow Aspergillus species except for A. niger (3) which was overgrown by T3. Volatile and non-volatile metabolite tests indicated that mycelial growth of Penicillium stekii was significantly inhibited by T3 and T24 more than the other tested pathogens. The inhibition of A. niger (1) was 12% by non-volatile metabolites of T24 produced after 1 day incubation, and reached to 97% inhibition by the metabolites of 3 days. Interestingly, inhibition of Aspergillus sp. by volatile compounds of T3 and T24 was 2% and 20%, respectively, whereas the inhibition of the same pathogen by non-volatile compounds reached 75% and 87%, respectively. The results of slice assay clearly indicate that T. harzianum (T3 and T24) could provide a complete protection to tomato slices from the infection of the tested pathogens. After 3 days of incubation, Trichoderma suppressed the linear growth of these pathogens on tomato slices and the percentage of suppression was significant and ranged between 80 and 100%, except with Rhizopus sp. the suppression reached 33% only.     

Bacterial cooperation in the wild and in the clinic: Are pathogen social behaviours relevant outside the laboratory?

Individual bacterial cells can communicate via quorum sensing, cooperate to harvest nutrients from their environment, form multicellular biofilms, compete over resources and even kill one another. When the environment that bacteria inhabit is an animal host, these social behaviours mediate virulence. Over the last decade, much attention has focussed on the ecology, evolution and pathology of bacterial cooperation, and the possibility that it could be exploited or destabilised to treat infections. But how far can we really extrapolate from theoretical predictions and laboratory experiments to make inferences about ‘cooperative’ behaviours in hosts and reservoirs? To determine the likely importance and evolution of cooperation ‘in the wild’, several questions must be addressed. A recent paper that reports the dynamics of bacterial cooperation and virulence in a field experiment provides an excellent nucleus for bringing together key empirical and theoretical results which help us to frame – if not completely to answer – these questions.     

The use of fluorescent reporter protein tagging to study the interaction between Root‐Knot Nematodes and Soft Rot Enterobacteriaceae

The study of plant parasitic nematodes such as Meloidogyne spp. and their interactions with phytopathogenic bacteria remains underexplored. One of the challenges towards establishing such interactions is the dependence on symptom development as a measure of interaction. In this study, mCherry was employed as a reporter protein to investigate the interaction between the soft rot Enterobacteriaceae (SRE) Pectobacterium carotovorum subsp. brasiliensis (Pcb) and root‐knot nematode (M. incognita). Pectobacterium carotovorum subsp. brasiliensis was transformed with pMP7604 generating Pcb_mCherry strain. This strain was shown to attach to the surface coat of M.incognita J2 at the optimum temperature of 28°C. This suggests that RKN juveniles may play a role in disseminating Pcb in soils that are heavily infested with Pcb. The presence of RKN juveniles was shown to play a role in introducing Pcb_mCherry into potato tubers potentially acting as a source of latent tuber infections.

Significance and Impact of the Study

This study uses fluorescent reporter protein tagging as a tool to demonstrate the interaction between root‐knot nematode (Meloidogyne incognita) and the soft rot Enterobacteriacea (Pectobacterium carotovorum subsp. brasiliensis). Introduction of Pectobacterium through wounds generated by second‐stage juveniles (J2) into potato tubers was demonstrated. These results suggest that RKN juveniles can facilitate latent infection of potato tubers in the soil. These findings have important implications in the management of RKN and SRE in seed potato production. Furthermore, this tool can be used to study other nematode–bacteria interactions that have not been previously studied.     

Biotechnological approaches to develop bacterial chitinases as a bioshield against fungal diseases of plants

Fungal diseases of plants continue to contribute to heavy crop losses in spite of the best control efforts of plant pathologists. Breeding for disease-resistant varieties and the application of synthetic chemical fungicides are the most widely accepted approaches in plant disease management. An alternative approach to avoid the undesired effects of chemical control could be biological control using antifungal bacteria that exhibit a direct action against fungal pathogens. Several biocontrol agents, with specific fungal targets, have been registered and released in the commercial market with different fungal pathogens as targets. However, these have not yet achieved their full commercial potential due to the inherent limitations in the use of living organisms, such as relatively short shelf life of the products and inconsistent performance in the field. Different mechanisms of action have been identified in microbial biocontrol of fungal plant diseases including competition for space or nutrients, production of antifungal metabolites, and secretion of hydrolytic enzymes such as chitinases and glucanases. This review focuses on the bacterial chitinases that hydrolyze the chitinous fungal cell wall, which is the most important targeted structural component of fungal pathogens. The application of the hydrolytic enzyme preparations, devoid of live bacteria, could be more efficacious in fungal control strategies. This approach, however, is still in its infancy, due to prohibitive production costs. Here, we critically examine available sources of bacterial chitinases and the approaches to improve enzymatic properties using biotechnological tools. We project that the combination of microbial and recombinant DNA technologies will yield more effective environment-friendly products of bacterial chitinases to control fungal diseases of crops.     

Exploring insights for virulent gene inhibition of multidrug resistant Salmonella typhi,Vibrio cholerae,and Staphylococcus areus by potential phytoligands via in silico screening

In our recent studies on prevalence of multidrug resistant pathogens in Byramangala reservoir, Karnataka, India, we identified Salmonella typhi, Staphylococcus aureus, and Vibrio cholerae which had acquired multiple drug resistance (MDR) and emerged as superbugs. Hence, there is a pressing demand to identify alternative therapeutic remedies. Our study focused on the screening of herbal leads by structure-based virtual screening. The virulent gene products of these pathogens towards Kanamycin(aph), Trimethoprim(dfrA1), Methicillin (mecI), and Vancomycin (vanH) were identified as the probable drug targets and their 3D structures were predicted by homology modeling. The predicted models showed good stereochemical validity. By extensive literature survey, we selected 58 phytoligands and their drug likeliness and pharmacokinetic properties were computationally predicted. The inhibitory properties of these ligands against drug targets were studied by molecular docking. Our studies revealed that Baicalein from S. baicalensis (baikal skullcap) and Luteolin from Taraxacum officinale (dandelion) were identified as potential inhibitors against aph of S. typhi. Resveratrol from Vitis vinifera (grape vine) and Wogonin from S. baicalensis were identified as potential inhibitors against dfrA1 of S. typhi. Herniarin from Herniaria glabra (rupture worts) and Pyrocide from Daucus carota (Carrot) were identified as the best leads against dfrA1 of V. cholerae. Taraxacin of T. officinale (weber) and Luteolin were identified as potential inhibitors against Mec1. Apigenin from Coffee arabica (coffee) and Luteolin were identified as the best leads against vanH of S. aureus. Our findings pave crucial insights for exploring alternative therapeutics against MDR pathogens.     

The Measles Virus Nucleocapsid Protein Tail Domain Is Dispensable for Viral Polymerase Recruitment and Activity

Paramyxovirus genomes are ribonucleoprotein (RNP) complexes consisting of nucleoprotein (N)-encapsidated viral RNA. Measles virus (MeV) N features an amino-terminal RNA-binding core and a 125-residue tail domain, of which only the last 75 residues are considered fully mobile on the nucleocapsid surface. A molecular recognition element (MoRE) domain mediates binding of the viral phosphoprotein (P). This P N-tail interaction is considered instrumental for recruiting the polymerase complex to the template. We have engineered MeV N variants with tail truncations progressively eliminating the MoRE domain and upstream tail sections. Confirming previous reports, RNPs with N truncations lacking the carboxyl-terminal 43-residues harboring the MoRE domain cannot serve as polymerase template. Remarkably, further removal of all tail residues predicted to be surface-exposed significantly restores RNP bioactivity. Insertion of structurally dominant tags into the central N-tail section reduces bioactivity, but the negative regulatory effect of exposed N-tail stems is sequence-independent. Bioactive nucleocapsids lacking exposed N-tail sections are unable to sustain virus replication, because of weakened interaction of the advancing polymerase complex with the template. Deletion of the N-MoRE-binding domain in P abrogates polymerase recruitment to standard nucleocapsids, but polymerase activity is partially restored when N-tail truncated RNPs serve as template. Revising central elements of the current replication model, these data reveal that MeV polymerase is capable of productively docking directly to the nucleocapsid core. Dispensable for polymerase recruitment, N-MoRE binding to P-tail stabilizes the advancing polymerase-RNP complex and may rearrange unstructured central tail sections to facilitate polymerase access to the template.     

Induction of Anti-influenza Immunity by Modified Green Fluorescent Protein (GFP) Carrying Hemagglutinin-derived Epitope Structure

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure. Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.     

Highly Divergent T-cell Receptor Binding Modes Underlie Specific Recognition of a Bulged Viral Peptide bound to a Human Leukocyte Antigen Class I Molecule

Human leukocyte antigen (HLA)-I molecules can present long peptides, yet the mechanisms by which T-cell receptors (TCRs) recognize featured pHLA-I landscapes are unclear. We compared the binding modes of three distinct human TCRs, CA5, SB27, and SB47, complexed with a “super-bulged” viral peptide (LPEPLPQGQLTAY) restricted by HLA-B*35:08. The CA5 and SB27 TCRs engaged HLA-B*35:08^LPEP similarly, straddling the central region of the peptide but making limited contacts with HLA-B*35:08. Remarkably, the CA5 TCR did not contact the α1-helix of HLA-B*35:08. Differences in the CDR3β loop between the CA5 and SB27 TCRs caused altered fine specificities. Surprisingly, the SB47 TCR engaged HLA-B*35:08^LPEP using a completely distinct binding mechanism, namely “bypassing” the bulged peptide and making extensive contacts with the extreme N-terminal end of HLA-B*35:08. This docking footprint included HLA-I residues not observed previously as TCR contact sites. The three TCRs exhibited differing patterns of alloreactivity toward closely related or distinct HLA-I allotypes. Thus, the human T-cell repertoire comprises a range of TCRs that can interact with “bulged” pHLA-I epitopes using unpredictable strategies, including the adoption of atypical footprints on the MHC-I.