Borrelia burgdorferi surface‐located Lmp1 protein processed into region‐specific polypeptides that are critical for microbial persistence

One of the Borrelia burgdorferi virulence determinants, annotated as Lmp1, is a surface‐exposed, conserved, and potential multi‐domain protein involved in various functions in spirochete infectivity. Lmp1 contributes to host–pathogen interactions and evasion of host adaptive immunity by spirochetes. Here, we show that in diverse B. burgdorferi species, Lmp1 exists as distinct, region‐specific, and lower molecular mass polypeptides encompassing 1 or more domains, including independent N‐terminal and middle regions and a combined middle and C‐terminal region. These polypeptides originate from complex posttranslational maturation events, partly supported by a periplasmic serine protease termed as BbHtrA. Although spirochete persistence in mice is independently supported by domain‐specific Lmp1 polypeptides, transmission of B. burgdorferi from ticks to mammals requires essential contributions from both N‐terminal and middle regions. Interference with the functions of Lmp1 domains or their complex posttranslational maturation events may aid in development of novel therapeutic strategies to combat infection and transmission of pathogens.     

The apparent non‐host resistance of Ethiopian mustard to a radish‐infecting strain of Turnip mosaic virus is largely determined by the C‐terminal region of the P3 viral protein

Two different isolates of Turnip mosaic virus (TuMV: UK 1 and JPN 1) belonging to different virus strains were tested on three different Brassica species, namely turnip (Brassica rapa L.), Indian mustard (Brassica juncea L.) and Ethiopian mustard (Brassica carinata A. Braun). Although all three hosts were readily infected by isolate UK 1, isolate JPN 1 was able to establish a visible systemic infection only in the first two. Ethiopian mustard plants showed no local or systemic symptoms, and no virus antigens could be detected by enzyme‐linked immunosorbent assay (ELISA). Thus, this species looks like a non‐host for JPN 1, an apparent situation of non‐host resistance (NHR). Through an experimental approach involving chimeric viruses made by gene interchange between two infectious clones of both virus isolates, the genomic region encoding the C‐terminal domain of viral protein P3 was found to bear the resistance determinant, excluding any involvement of the viral fusion proteins P3N‐PIPO and P3N‐ALT in the resistance. A further determinant refinement identified two adjacent positions (1099 and 1100 of the viral polyprotein) as the main determinants of resistance. Green fluorescent protein (GFP)‐tagged viruses showed that the resistance of Ethiopian mustard to isolate JPN 1 is only apparent, as virus‐induced fluorescence could be found in discrete areas of both inoculated and non‐inoculated leaves. In comparison with other plant–virus combinations of extreme resistance, we propose that Ethiopian mustard shows an apparent NHR to TuMV JPN 1, but not complete immunity or extreme resistance.     

Two Clip Domain Family of Serine Proteases and a Serine Protease Homolog from the Fall Webworm, Hyphantria cunea; cDNA Cloning and Characterization

Two types of serine proteases and a serine protease homologue cDNAs were isolated from Hyphantria cunea larvae induced immune response due to an injection of a microorganism through RT‐PCR and cDNA library screening, and their characteristics were examined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, which encoded 388, 390, 580 amino acid residues, and were designated as HcPE‐1, HcPE‐2 and HcPE‐3, respectively. They were revealed as serine proteases or a serine protease homologue with the clip domain through a database search. The deduced amino acid sequence comparison showed high homology of 72‐78% among them. Six Cys residues of the N‐terminal clip domain forming the disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter‐bridge between clip domain and catalytic domain were also well preserved. Three amino acid residues, His, Asp, and Ser, within the active site were perfectly conserved in HcPE‐2 and HcPE‐3, however, His was replaced with Gln¹⁷⁸ in HcPE‐1. The Arg residues (HcPE‐1, Arg¹³²; HcPE‐2, Arg¹³⁴; HcPE‐3, Arg³²⁵) known as the activation sites by proteolytic cleavage were preserved well in all three types of protein. In case of HcPE‐3, three continuous clip‐like domains existed in the N terminal. As the result of phylogenetic analysis, three clip domain family of protein from H. cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showed all three genes were induced through an injection of Escherichia coli, but expression patterns were varied.     

Streptococcus pneumoniae IgA1 protease: A metalloprotease that can catalyze in a split manner in vitro

IgA1 proteases (IgA1P) from diverse pathogenic bacteria specifically cleave human immunoglobulin A1 (IgA1) at the hinge region, thereby thwarting protective host immune responses. Streptococcus pneumoniae (S. pneumoniae) IgA1P shares no sequence conservation with serine or cysteine types of IgA1Ps or other known proteins, other than a conserved HExxH Zn‐binding motif (1604‐1608) found in metalloproteases. We have developed a novel expression system to produce the mature S. pneumoniae IgA1P and we have discovered that this form is both attached to the bacterial cell surface and released in its full form. Our data demonstrate that the S. pneumoniae IgA1P comprises two distinct regions that associate to form an active metalloprotease, the first such example of a metalloprotease that can be split in vitro and recombined to form an active enzyme. By capitalizing on this novel domain architecture, we show that the N‐terminal region of S. pneumoniae IgA1P comprises the primary binding region for IgA1, although the C‐terminal region of S. pneumoniae IgA1P is necessary for cleavage of IgA1. Our findings lend insight into the protein domain architecture of the S. pneumoniae IgA1P and function of this important virulence factor for S. pneumoniae infection.     

The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication‐associated inclusion bodies

Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa‐Pro (nuclear inclusion‐a protease) and VPg (viral protein genome‐linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa‐Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV‐infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2‐associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV.     

The rust transferred proteins—a new family of effector proteins exhibiting protease inhibitor function

Only few fungal effectors have been described to be delivered into the host cell during obligate biotrophic interactions. RTP1p, from the rust fungi Uromyces fabae and U. striatus, was the first fungal protein for which localization within the host cytoplasm could be demonstrated directly. We investigated the occurrence of RTP1 homologues in rust fungi and examined the structural and biochemical characteristics of the corresponding gene products. The analysis of 28 homologues showed that members of the RTP family are most likely to occur ubiquitously in rust fungi and to be specific to the order Pucciniales. Sequence analyses indicated that the structure of the RTPp effectors is bipartite, consisting of a variable N‐terminus and a conserved and structured C‐terminus. The characterization of Uf‐RTP1p mutants showed that four conserved cysteine residues sustain structural stability. Furthermore, the C‐terminal domain exhibits similarities to that of cysteine protease inhibitors, and it was shown that Uf‐RTP1p and Us‐RTP1p are able to inhibit proteolytic activity in Pichia pastoris culture supernatants. We conclude that the RTP1p homologues constitute a rust fungi‐specific family of modular effector proteins comprising an unstructured N‐terminal domain and a structured C‐terminal domain, which exhibit protease inhibitory activity possibly associated with effector function during biotrophic interactions.     

Activation and polar sequestration of PopA,a c‐di‐GMP effector protein involved in Caulobacter crescentus cell cycle control

When Caulobacter crescentus enters S‐phase the replication initiation inhibitor CtrA dynamically positions to the old cell pole to be degraded by the polar ClpXP protease. Polar delivery of CtrA requires PopA and the diguanylate cyclase PleD that positions to the same pole. Here we present evidence that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co‐option as c‐di‐GMP effector protein. While the C‐terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N‐terminal receiver domain, functional adaptation has reversed signal transduction in PopA with the GGDEF domain adopting input function and the receiver domain serving as regulatory output. We show that the N‐terminal receiver domain of PopA specifically interacts with RcdA, a component required for CtrA degradation. In contrast, the GGDEF domain serves to target PopA to the cell pole in response to c‐di‐GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S‐phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit components of the CtrA degradation pathway to the protease specific old cell pole of C. crescentus.     

Structure of the N‐terminal domain of the metalloprotease PrtV from Vibrio cholerae

The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre‐pro‐protein that undergoes several N‐ and C‐terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N‐terminal domain (residues 23–103) that contains two short α‐helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C‐terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.     

The Hypervariable Amino-Terminus of P1 Protease Modulates Potyviral Replication and Host Defense Responses

The replication of many RNA viruses involves the translation of polyproteins, whose processing by endopeptidases is a critical step for the release of functional subunits. P1 is the first protease encoded in plant potyvirus genomes; once activated by an as-yet-unknown host factor, it acts in cis on its own C-terminal end, hydrolyzing the P1-HCPro junction. Earlier research suggests that P1 cooperates with HCPro to inhibit host RNA silencing defenses. Using Plum pox virus as a model, we show that although P1 does not have a major direct role in RNA silencing suppression, it can indeed modulate HCPro function by its self-cleavage activity. To study P1 protease regulation, we used bioinformatic analysis and in vitro activity experiments to map the core C-terminal catalytic domain. We present evidence that the hypervariable region that precedes the protease domain is predicted as intrinsically disordered, and that it behaves as a negative regulator of P1 proteolytic activity in in vitro cleavage assays. In viral infections, removal of the P1 protease antagonistic regulator is associated with greater symptom severity, induction of salicylate-dependent pathogenesis-related proteins, and reduced viral loads. We suggest that fine modulation of a viral protease activity has evolved to keep viral amplification below host-detrimental levels, and thus to maintain higher long-term replicative capacity.     

PopW of Ralstonia solanacearum,a new two‐domain harpin targeting the plant cell wall

Harpins are extracellular glycine‐rich proteins eliciting a hypersensitive response (HR). In this study, we identified a new harpin, PopW, from Ralstonia solanacearum strain ZJ3721. This 380‐amino‐acid protein is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into the leaves of tobacco (non‐host), PopW induced a rapid tissue collapse via a heat‐stable but protease‐sensitive HR‐eliciting activity. PopW has an N‐terminal harpin domain (residues 1–159) and a C‐terminal pectate lyase (PL) domain (residues 160–366); its HR‐eliciting activity depends on its N‐terminal domain. Analyses of subcellular localization and plasmolysis demonstrated that PopW targeted the onion cell wall. This was further confirmed by its ability to specifically bind to calcium pectate, a major component of the plant cell wall. However, PopW had no detectable PL activity. Western blotting revealed that PopW was secreted by the type III secretion system in an hrpB‐dependent manner. Gene sequencing indicated that popW is conserved among 20 diverse strains of R. solanacearum. A popW‐deficient mutant retained the ability of wild‐type strain ZJ3721 to elicit HR in tobacco and to cause wilt disease in tomato (a host). We conclude that PopW is a new cell wall‐associated, hrpB‐dependent, two‐domain harpin that is conserved across the R. solanacearum species complex.     

Expression,purification and molecular modeling of the NIa protease of Cardamom mosaic virus

The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8?M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.     

C‐terminal domains of bacterial proteases: structure,function and the biotechnological applications

C‐terminal domains widely exist in the C‐terminal region of multidomain proteases. As a β‐sandwich domain in multidomain protease, the C‐terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C‐terminal protease domains are described. Furthermore, the application prospects of C‐terminal domains, including polycystic kidney disease, prepeptidase C‐terminal and collagen‐binding domain, in the area of medicine and biological artificial materials are also discussed.     

Structure of a putative ClpS N‐end rule adaptor protein from the malaria pathogen Plasmodium falciparum

The N‐end rule pathway uses an evolutionarily conserved mechanism in bacteria and eukaryotes that marks proteins for degradation by ATP‐dependent chaperones and proteases such as the Clp chaperones and proteases. Specific N‐terminal amino acids (N‐degrons) are sufficient to target substrates for degradation. In bacteria, the ClpS adaptor binds and delivers N‐end rule substrates for their degradation upon association with the ClpA/P chaperone/protease. Here, we report the first crystal structure, solved at 2.7 Å resolution, of a eukaryotic homolog of bacterial ClpS from the malaria apicomplexan parasite Plasmodium falciparum (Pfal). Despite limited sequence identity, Plasmodium ClpS is very similar to bacterial ClpS. Akin to its bacterial orthologs, plasmodial ClpS harbors a preformed hydrophobic pocket whose geometry and chemical properties are compatible with the binding of N‐degrons. However, while the N‐degron binding pocket in bacterial ClpS structures is open and accessible, the corresponding pocket in Plasmodium ClpS is occluded by a conserved surface loop that acts as a latch. Despite the closed conformation observed in the crystal, we show that, in solution, Pfal‐ClpS binds and discriminates peptides mimicking bona fide N‐end rule substrates. The presence of an apicoplast targeting peptide suggests that Pfal‐ClpS localizes to this plastid‐like organelle characteristic of all Apicomplexa and hosting most of its Clp machinery. By analogy with the related ClpS1 from plant chloroplasts and cyanobacteria, Plasmodium ClpS likely functions in association with ClpC in the apicoplast. Our findings open new venues for the design of novel anti‐malarial drugs aimed at disrupting parasite‐specific protein quality control pathways.     

Fungal incompatibility: Evolutionary origin in pathogen defense?

In fungi, cell fusion between genetically unlike individuals triggers a cell death reaction known as the incompatibility reaction. In Podospora anserina, the genes controlling this process belong to a gene family encoding STAND proteins with an N‐terminal cell death effector domain, a central NACHT domain and a C‐terminal WD‐repeat domain. These incompatibility genes are extremely polymorphic, subject to positive Darwinian selection and display a remarkable genetic plasticity allowing for constant diversification of the WD‐repeat domain responsible for recognition of non‐self. Remarkably, the architecture of these proteins is related to pathogen‐recognition receptors ensuring innate immunity in plants and animals. Here, we hypothesize that these P. anserina incompatibility genes could be components of a yet‐unidentified innate immune system of fungi. As already proposed in the case of plant hybrid necrosis or graft rejection in mammals, incompatibility could be a by‐product of pathogen‐driven divergence in host defense genes.     

The acidic domain is a unique structural feature of the splicing factor SYNCRIP

The splicing factor SYNCRIP (hnRNP Q) is involved in viral replication, neural morphogenesis, modulation of circadian oscillation, and the regulation of the cytidine deaminase APOBEC1. It consists of three globular RNA‐recognition motifs (RRM) domains flanked by an N‐terminal acid‐rich acidic sequence segment domain (AcD_12–97) and a C‐terminal domain containing an arginine–glycine‐rich sequence motif (RGG/RXG box), which are located near to the N‐ and C‐terminals, respectively. The acid‐rich sequence segment is unique to SYNCRIP and the closely related protein hnRNP R, and is involved in interactions with APOBEC1. Here, we show that while AcD_12–97 does not form a globular domain, structure‐based annotation identified a self‐folding globular domain with an all α‐helix architecture, AcD_24–107. The NMR structure of AcD_24?107 is fundamentally different from previously reported AcD molecular models. In addition to negatively charged surface areas, it contains a large hydrophobic cavity and a positively charged surface area as potential epitopes for intermolecular interactions.     

Large‐scale gene expression reveals different adaptations of Hyalopterus persikonus to winter and summer host plants

Host alternation, an obligatory seasonal shifting between host plants of distant genetic relationship, has had significant consequences for the diversification and success of the superfamily of aphids. However, the underlying molecular mechanism remains unclear. In this study, the molecular mechanism of host alternation was explored through a large‐scale gene expression analysis of the mealy aphid Hyalopterus persikonus on winter and summer host plants. More than four times as many unigenes of the mealy aphid were significantly upregulated on summer host Phragmites australis than on winter host Rosaceae plants. In order to identify gene candidates related to host alternation, the differentially expressed unigenes of H. persikonus were compared to salivary gland expressed genes and secretome of Acyrthosiphon pisum. Genes involved in ribosome and oxidative phosphorylation and with molecular functions of heme–copper terminal oxidase activity, hydrolase activity and ribosome binding were potentially upregulated in salivary glands of H. persikonus on the summer host. Putative secretory proteins, such as detoxification enzymes (carboxylesterases and cytochrome P450s), antioxidant enzymes (peroxidase and superoxide dismutase), glutathione peroxidase, glucose dehydrogenase, angiotensin‐converting enzyme, cadherin, and calreticulin, were highly expressed in H. persikonus on the summer host, while a SCP GAPR‐1‐like family protein and a salivary sheath protein were highly expressed in the aphids on winter hosts. These results shed light on phenotypic plasticity in host utilization and seasonal adaptation of aphids.     

Molecular dynamic studies on the impact of mutations on the structure,stability, and N‐terminal orientation of annexin A1: Implications for membrane aggregation

Multiple MD simulations were performed for the full‐length wild‐type A1, the full length A1 mutations S27E and S27A, as well as the N‐terminal peptide (AMVSEFLKQAWFIDNEEQEYIKTVKG S ²⁷ KGGPGSAVSPYPTFN) of wild‐type A1 and mutations S27E and S27A. The MD simulation trajectories of about 350 ns were generated and analyzed to examine the changes of core domain calcium binding affinity, core domain and N‐terminal domain structures, and N‐terminal domain orientation. Our results indicated that S27A and S27E mutations caused little changes on the calcium‐binding affinity of the core domain of A1. However, the S27A mutation made the N‐terminal domain of A1 less helical, and made the N‐terminal domain migrate faster toward the core domain; these impacts on A1 are beneficial to the membrane aggregation process. On the contrary, the S27E mutation made the N‐terminal domain of A1 more stable, and hindered the migration to the core domain; these changes on A1 are antagonistic for the membrane aggregation process. Our results using MD simulations provide an atomistic explanation for experimental observations that the S27E mutant showed a higher calcium concentration requirement and lower maximal extent of aggregation, while the wild‐type and two mutants S27E and S27A required identical calcium concentrations for liposome binding. Proteins 2014; 82:3327–3334. © 2014 Wiley Periodicals, Inc.     

Tmprss2 Is Essential for Influenza H1N1 Virus Pathogenesis in Mice

Annual influenza epidemics and occasional pandemics pose a severe threat to human health. Host cell factors required for viral spread but not for cellular survival are attractive targets for novel approaches to antiviral intervention. The cleavage activation of the influenza virus hemagglutinin (HA) by host cell proteases is essential for viral infectivity. However, it is unknown which proteases activate influenza viruses in mammals. Several candidates have been identified in cell culture studies, leading to the concept that influenza viruses can employ multiple enzymes to ensure their cleavage activation in the host. Here, we show that deletion of a single HA-activating protease gene, Tmprss2, in mice inhibits spread of mono-basic H1N1 influenza viruses, including the pandemic 2009 swine influenza virus. Lung pathology was strongly reduced and mutant mice were protected from weight loss, death and impairment of lung function. Also, after infection with mono-basic H3N2 influenza A virus body weight loss and survival was less severe in Tmprss2 mutant compared to wild type mice. As expected, Tmprss2-deficient mice were not protected from viral spread and pathology after infection with multi-basic H7N7 influenza A virus. In conclusion, these results identify TMPRSS2 as a host cell factor essential for viral spread and pathogenesis of mono-basic H1N1 and H3N2 influenza A viruses.