Phylogenetic Analysis of the Membrane Attack Complex/Perforin Domain-Containing Proteins in Gossypium and the Role of GhMACPF26 in Cotton Under Cold Stress

The membrane attack complex/perforin (MACPF) domain-containing proteins are involved in the various developmental processes and in responding to diverse abiotic stress. The function and regulatory network of the MACPF genes are rarely reported in Gossypium spp. We study the detailed identification and partial functional verification of the members of the MACPF family. Totally, 100 putative MACPF proteins containing complete MACPF domain were identified from the four cotton species. They were classified into three phylogenetic groups and underwent multifold pressure indicating that selection produced new functional differentiation. Cotton MACPF gene family members expanded mainly through the whole-genome duplication (WGD)/segmental followed by the dispersed. Expression and cis-acting elements analysis revealed that MACPFs play a role in resistance to abiotic stresses, and some selected GhMACPFs were able to respond to the PEG and cold stresses. Co-expression analysis showed that GhMACPFs might interact with valine-glutamine (VQ), WRKY, and Apetala 2 (AP2)/ethylene responsive factor (ERF) domain-containing genes under cold stress. In addition, silencing endogenous GhMACPF26 in cotton by the virus-induced gene silencing (VIGS) method indicated that GhMACPF26 negatively regulates cold tolerance. Our data provided a comprehensive phylogenetic evolutionary view of Gossypium MACPFs. The MACPFs may work together with multiple transcriptional factors and play roles in acclimation to abiotic stress, especially cold stress in cotton.

A Continuous Battle for Host-Derived Glycans Between a Mucus Specialist and a Glycan Generalist in vitro and in vivo

The human gastrointestinal tract is colonized by a diverse microbial community, which plays a crucial role in human health. In the gut, a protective mucus layer that consists of glycan structures separates the bacteria from the host epithelial cells. These host-derived glycans are utilized by bacteria that have adapted to this specific compound in the gastrointestinal tract. Our study investigated the close interaction between two distinct gut microbiota members known to use mucus glycans, the generalist Bacteroides thetaiotaomicron and the specialist Akkermansia muciniphila in vitro and in vivo. The in vitro study, in which mucin was the only nutrient source, indicated that B. thetaiotaomicron significantly upregulated genes coding for Glycoside Hydrolases (GHs) and mucin degradation activity when cultured in the presence of A. muciniphila. Furthermore, B. thetaiotaomicron significantly upregulated the expression of a gene encoding for membrane attack complex/perforin (MACPF) domain in co-culture. The transcriptome analysis also indicated that A. muciniphila was less affected by the environmental changes and was able to sustain its abundance in the presence of B. thetaiotaomicron while increasing the expression of LPS core biosynthesis activity encoding genes (O-antigen ligase, Lipid A and Glycosyl transferases) as well as ABC transporters. Using germ-free mice colonized with B. thetaiotaomicron and/or A. muciniphila, we observed a more general glycan degrading profile in B. thetaiotaomicron while the expression profile of A. muciniphila was not significantly affected when colonizing together, indicating that two different nutritional niches were established in mice gut. Thus, our results indicate that a mucin degrading generalist adapts to its changing environment, depending on available carbohydrates while a mucin degrading specialist adapts by coping with competing microorganism through upregulation of defense related genes.

Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails

The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles but MACPF but pore-forming toxic function are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 15 Å resolution determined by negative stain electron microscopy (NS-EM) and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. Tachylectin is a six-bladed β-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin ..B.. delivery subunit would bind to target membranes, and then its MACPF ..A.. toxic subunit disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions give rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.

Supression of Perforin-like Protein pores inhibitPlasmodiummultistage-growth, transmission and erythrocyte senescence

The pore formingPlasmodiumperforin like proteins (PPLP), expressed in all stages of the parasite life cycle are central drivers for host interactions critical for completion of parasite life cycle and high transmission rates. The high sequence similarity in the central membrane attack complex/ perforin (MACPF) domain and consequent functional overlaps defines them as an attractive target for the development of multi-stage antimalarials. Herein we evaluated the mechanism of pan active function of central, highly conserved region of PPLPs, MACPF domain (PMD) and inhibitory potential of specifically designed anti-PMD chemo. TheE. coliexpressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to haemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence at 48 hours which can account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing anemia during malaria infection. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress of merozoites and protecting against erythrocyte senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage and transmission-blocking potential of these inhibitors. Additionally, the erythrocyte senescence protective potential of PMD inhibitors can be used to occlude PPLPs mediated severe malarial anemia. Further these inhibitors can be developed with a potential to protect against severity of the disease.Author SummaryMalaria continues to be a major global health threat despite of several exciting improvements in the treatment and prevention of the disease. One of the major concerns in the development of therapy is the emergence of the drug resistance. But for the efficient treatment regime, targeting multiple stages including host and vector would serve as an ideal therapy. Perforin like proteins (PLPs) are eukaryotic pore forming proteins that are highly conserved in the apicomplexan parasites. These play crucial roles in entry and exit of parasites from the host cells and establish infection at multiple stages ofPlasmodium spp.life cycle. Understanding the mechanism of pore formation by smaller, functional, pan-active scaffold of PLPs can serve as a target for development of cross stage protection. Here, using various biochemical, biophysical and pharmacological evidences, we validate the activity and characterize the pore formation of PLPs on erythrocytes. Further, our specifically designed inhibitors could restrict this pore formation and impede the exit/entry of the parasites. Moreover, these inhibitors could also exert multiple stage inhibition and rescue the uninfected erythrocytes from death. Together, this study highlights the mechanism of pore formation by PPLPs and evaluates their potential for the development of pan-active inhibitors to provide both symptomatic and transmission blocking cure for malaria.

Control of growth factor signalling by MACPF proteins

Members of the membrane attack complex/perforin-like (MACPF) protein superfamily have long captured interest because of their unique ability to assemble into large oligomeric pores on the surfaces of cells. The best characterised of these act in vertebrate immunity where they function to deliver pro-apoptotic factors or induce the cytolysis and death of targeted cells. Less appreciated, however, is that rather than causing cell death, MACPF proteins have also evolved to control cellular signalling pathways and influence developmental programmes such as pattern formation and neurogenesis. Torso-like (Tsl) from the fruit fly Drosophila, for example, functions to localise the activity of a growth factor for patterning its embryonic termini. It remains unclear whether these developmental proteins employ an attenuated form of the classical MACPF lytic pore, or if they have evolved to function via alternative mechanisms of action. In this minireview, we examine the evidence that links pore-forming MACPF proteins to the control of growth factor and cytokine signalling. We will then attempt to reconcile how the MACPF domain may have been repurposed during evolution for developmental events rather than cell killing.

Structure–function characterization of an insecticidal protein GNIP1Aa, a member of an MACPF and β-tripod families

The crystal structure of the Gram-negative insecticidal protein, GNIP1Aa, has been solved at 2.5-Å resolution. The protein consists of two structurally distinct domains, a MACPF (membrane attack complex/PerForin) and a previously uncharacterized type of domain. GNIP1Aa is unique in being a prokaryotic MACPF member to have both its structure and function identified. It was isolated from aChromobacterium piscinaestrain and is specifically toxic toDiabrotica virgifera virgiferalarvae upon feeding. In members of the MACPF family, the MACPF domain has been shown to be important for protein oligomerization and formation of transmembrane pores, while accompanying domains define the specificity of the target of the toxicity. In GNIP1Aa the accompanying C-terminal domain has a unique fold composed of three pseudosymmetric subdomains with shared sequence similarity, a feature not obvious from the initial sequence examination. Our analysis places this domain into a protein family, named here β-tripod. Using mutagenesis, we identified functionally important regions in the β-tripod domain, which may be involved in target recognition.

Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen

The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1,Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogenListeria monocytogenes. Within a few hours of systemic infection, the massive proliferation ofL. monocytogenesinPerforin-2−/−mice leads to a rapid appearance of acute disease symptoms. We go on to show in culturedPerforin-2−/−cells that the vacuole-to-cytosol transitioning ofL. monocytogenesis greatly accelerated. Unexpectedly, we found that inPerforin-2−/−macrophages,Listeria-containing vacuoles quickly (≤15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation ofL. monocytogenesto its replicative niche in the cytosol. This hypothesis was supported by our finding that aL. monocytogenesstrain expressing virulence factors at a constitutively high level replicated equally well inPerforin-2+/+andPerforin-2−/−macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification ofListeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity.