Cx43-mediated sorting of miRNAs into extracellular vesicles.

Through the exchange of lipids, proteins, and nucleic acids, extracellular vesicles (EV) allow for cell-cell communication across distant cells and tissues to regulate a wide range of physiological and pathological processes. Although some molecular mediators have been discovered, the mechanisms underlying the selective sorting of miRNAs into EV remain elusive. Previous studies demonstrated that connexin43 (Cx43) forms functional channels at the EV surface, mediating the communication with recipient cells. Here, we show that Cx43 participates in the selective sorting of miRNAs into EV through a process that can also involve RNA-binding proteins. We provide evidence that Cx43 can directly bind to specific miRNAs, namely those containing stable secondary structure elements, including miR-133b. Furthermore, Cx43 facilitates the delivery of EV-miRNAs into recipient cells. Phenotypically, we show that Cx43-mediated EV-miRNAs sorting modulates autophagy. Overall, our study ascribes another biological role to Cx43, that is, the selective incorporation of miRNAs into EV, which potentially modulates multiple biological processes in target cells and may have implications for human health and disease.

Expression in Escherichia coli, Refolding, and Purification of Plant Aspartic Proteases.

Aspartic proteases (APs) are widely distributed in plants. The large majority of genes encoding putative APs exhibit distinct features when compared with the so-called typical APs, and have been grouped as atypical and nucellin-like APs. Remarkably, a diverse pattern of enzymatic properties, subcellular localizations, and biological functions are emerging for these proteases, illustrating the functional complexity among plant pepsin-like proteases. However, many key questions regarding the structure-function relationships of plant APs remain unanswered. Therefore, the expression of these enzymes in heterologous systems is a valuable strategy to unfold the unique features/biochemical properties among members of this family of proteases. Here, we describe our protocol for the production and purification of recombinant plant APs, using a procedure where the protein is refolded from inclusion bodies by dialysis. This method allows the production of untagged versions of the target protease, which has revealed to be critical to disclose differences in processing/activation requirements between plant APs. The protocol includes protein expression, washing and solubilization of inclusion bodies, refolding by dialysis, and a protein purification method. Specific considerations on critical aspects of the refolding process and further suggestions for evaluation of the final recombinant product are also provided.

Moonlighting in Rickettsiales: Expanding Virulence Landscape.

The order Rickettsiales includes species that cause a range of human diseases such as human granulocytic anaplasmosis (Anaplasma phagocytophilum), human monocytic ehrlichiosis (Ehrlichia chaffeensis), scrub typhus (Orientia tsutsugamushi), epidemic typhus (Rickettsia prowazekii), murine typhus (R. typhi), Mediterranean spotted fever (R. conorii), or Rocky Mountain spotted fever (R. rickettsii). These diseases are gaining a new momentum given their resurgence patterns and geographical expansion due to the overall rise in temperature and other human-induced pressure, thereby remaining a major public health concern. As obligate intracellular bacteria, Rickettsiales are characterized by their small genome sizes due to reductive evolution. Many pathogens employ moonlighting/multitasking proteins as virulence factors to interfere with multiple cellular processes, in different compartments, at different times during infection, augmenting their virulence. The utilization of this multitasking phenomenon by Rickettsiales as a strategy to maximize the use of their reduced protein repertoire is an emerging theme. Here, we provide an overview of the role of various moonlighting proteins in the pathogenicity of these species. Despite the challenges that lie ahead to determine the multiple potential faces of every single protein in Rickettsiales, the available examples anticipate this multifunctionality as an essential and intrinsic feature of these obligates and should be integrated into available moonlighting repositories.

Spotted Fever Group Rickettsia Trigger Species-Specific Alterations in Macrophage Proteome Signatures with Different Impacts in Host Innate Inflammatory Responses.

The molecular details underlying differences in pathogenicity between Rickettsia species remain to be fully understood. Evidence points to macrophage permissiveness as a key mechanism in rickettsial virulence. Different studies have shown that several rickettsial species responsible for mild forms of rickettsioses can also escape macrophage-mediated killing mechanisms and establish a replicative niche within these cells. However, their manipulative capacity with respect to host cellular processes is far from being understood. A deeper understanding of the interplay between mildly pathogenic rickettsiae and macrophages and the commonalities and specificities of host responses to infection would illuminate differences in immune evasion mechanisms and pathogenicity. We used quantitative proteomics by sequential windowed data independent acquisition of the total high-resolution mass spectra with tandem mass spectrometry (SWATH-MS/MS) to profile alterations resulting from infection of THP-1 macrophages with three mildly pathogenic rickettsiae: Rickettsia parkeri, Rickettsia africae, and Rickettsia massiliae, all successfully proliferating in these cells. We show that all three species trigger different proteome signatures. Our results reveal a significant impact of infection on proteins categorized as type I interferon responses, which here included several components of the retinoic acid-inducible gene I (RIG-1)-like signaling pathway, mRNA splicing, and protein translation. Moreover, significant differences in protein content between infection conditions provide evidence for species-specific induced alterations. Indeed, we confirm distinct impacts on host inflammatory responses between species during infection, demonstrating that these species trigger different levels of beta interferon (IFN-ß), differences in the bioavailability of the proinflammatory cytokine interleukin 1ß (IL-1ß), and differences in triggering of pyroptotic events. This work reveals novel aspects and exciting nuances of macrophage-Rickettsia interactions, adding additional layers of complexity between Rickettsia and host cells' constant arms race for survival. IMPORTANCE The incidence of diseases caused by Rickettsia has been increasing over the years. It has long been known that rickettsioses comprise diseases with a continuous spectrum of severity. There are highly pathogenic species causing diseases that are life threatening if untreated, others causing mild forms of the disease, and a third group for which no pathogenicity to humans has been described. These marked differences likely reflect distinct capacities for manipulation of host cell processes, with macrophage permissiveness emerging as a key virulence trait. However, what defines pathogenicity attributes among rickettsial species is far from being resolved. We demonstrate that the mildly pathogenic Rickettsia parkeri, Rickettsia africae, and Rickettsia massiliae, all successfully proliferating in macrophages, trigger different proteome signatures in these cells and differentially impact critical components of innate immune responses by inducing different levels of beta interferon (IFN-ß) and interleukin 1ß (IL-1ß) and different timing of pyroptotic events during infection. Our work reveals novel nuances in rickettsia-macrophage interactions, offering new clues to understand Rickettsia pathogenicity.

The Retropepsin-Type Protease APRc as a Novel Ig-Binding Protein and Moonlighting Immune Evasion Factor of Rickettsia.

Rickettsiae are obligate intracellular Gram-negative bacteria transmitted by arthropod vectors. Despite their reduced genomes, the function(s) of the majority of rickettsial proteins remains to be uncovered. APRc is a highly conserved retropepsin-type protease, suggested to act as a modulator of other rickettsial surface proteins with a role in adhesion/invasion. However, APRc's function(s) in bacterial pathogenesis and virulence remains unknown. This study demonstrates that APRc targets host serum components, combining nonimmune immunoglobulin (Ig)-binding activity with resistance to complement-mediated killing. We confirmed nonimmune human IgG binding in extracts of different rickettsial species and intact bacteria. Our results revealed that the soluble domain of APRc is capable of binding to human (h), mouse, and rabbit IgG and different classes of human Ig (IgG, IgM, and IgA) in a concentration-dependent manner. APRc-hIgG interaction was confirmed with total hIgG and normal human serum. APRc-hIgG displayed a binding affinity in the micromolar range. We provided evidence of interaction preferentially through the Fab region and confirmed that binding is independent of catalytic activity. Mapping the APRc region responsible for binding revealed the segment between amino acids 157 and 166 as one of the interacting regions. Furthermore, we demonstrated that expression of the full-length protease in Escherichia coli is sufficient to promote resistance to complement-mediated killing and that interaction with IgG contributes to serum resistance. Our findings position APRc as a novel Ig-binding protein and a novel moonlighting immune evasion factor of Rickettsia, contributing to the arsenal of virulence factors utilized by these intracellular pathogens to aid in host colonization. IMPORTANCE Many Rickettsia organisms are pathogenic to humans, causing severe infections, like Rocky Mountain spotted fever and Mediterranean spotted fever. However, immune evasion mechanisms and pathogenicity determinants in rickettsiae are far from being resolved. We provide evidence that the highly conserved rickettsial retropepsin-type protease APRc displays nonimmune immunoglobulin (Ig)-binding activity and participates in serum resistance. APRc emerges then as a novel Ig-binding protein from Gram-negative bacteria and the first to be identified in Rickettsia. Bacterial surface proteins capable of Ig binding are known to be multifunctional and key players in immune evasion. We demonstrate that APRc is also a novel moonlighting protein, exhibiting different actions on serum components and acting as a novel evasin. This work strengthens APRc as a virulence factor in Rickettsia and its significance as a potential therapeutic target. Our findings significantly contribute to a deeper understanding of the virulence strategies used by intracellular pathogens to subvert host immune responses.

Introduction of a Glycine Linker Connecting the Heavy and Light Chains in Synthetic Cardosin B-Derived Rennet Changes the Specificity of Subpocket S3'.

The development of plant-based synthetic rennets is of high commercial interest, due to the current great consumer demand for animal product alternatives. A previously developed recombinant form of the aspartic protease cardosin B with a three-glycine linker showed great potential due to its good performance in milk coagulation. This enzyme was found to be more specific and less proteolytically active than the native form for milk clotting, but the underlying structural causes for these activity changes were not completely clear. Here, we have performed molecular dynamics simulations with the recombinant enzyme with and without the linker. Our results showed that the introduction of the linker changes the subpocket S3', which is located more than 4 nm away. These results showcase how small modifications in proteins can have significant effects in distant regions in the protein structure that affect their biotechnological applications.

Use of recombinant proteins as a simple and robust normalization method for untargeted proteomics screening: exhaustive performance assessment.

The label-free quantitative mass spectrometry methods, in particular, the SWATH-MS approach, have gained popularity and became a powerful technique for comparison of large datasets. In the present work, it is evaluated the use of recombinant proteins as internal standards for untargeted label-free methods. The proposed internal standard strategy reveals a similar intragroup normalization capacity when compared with the most common normalization methods, with the additional advantage of maintaining the overall proteome changes between groups (which is buffered with the use of other methods). Therefore, the proposed strategy is able to maintain a good performance even when large qualitative and quantitative differences in sample composition are observed, such as the ones induced by biological regulation (as observed in secretome and other biofluids' analyses) or by enrichment approaches (such as immunopurifications). Moreover, this approach corresponds to a cost-effective and simple normalization method altrenative, therefore being an appealing strategy for large quantitative screening, as the analysis of clinical cohorts for biomarker discovery.

Macrophages Infected by a Pathogen and a Non-pathogen Spotted Fever Group Rickettsia Reveal Differential Reprogramming Signatures Early in Infection.

Despite their high degree of genomic similarity, different spotted fever group (SFG) Rickettsia are often associated with very different clinical presentations. For example, Rickettsia conorii causes Mediterranean spotted fever, a life-threatening disease for humans, whereas Rickettsia montanensis is associated with limited or no pathogenicity to humans. However, the molecular basis responsible for the different pathogenicity attributes are still not understood. Although killing microbes is a critical function of macrophages, the ability to survive and/or proliferate within phagocytic cells seems to be a phenotypic feature of several intracellular pathogens. We have previously shown that R. conorii and R. montanensis exhibit different intracellular fates within macrophage-like cells. By evaluating early macrophage responses upon insult with each of these rickettsial species, herein we demonstrate that infection with R. conorii results in a profound reprogramming of host gene expression profiles. Transcriptional programs generated upon infection with this pathogenic bacteria point toward a sophisticated ability to evade innate immune signals, by modulating the expression of several anti-inflammatory molecules. Moreover, R. conorii induce the expression of several pro-survival genes, which may result in the ability to prolong host cell survival, thus protecting its replicative niche. Remarkably, R. conorii-infection promoted a robust modulation of different transcription factors, suggesting that an early manipulation of the host gene expression machinery may be key to R. conorii proliferation in THP-1 macrophages. This work provides new insights into the early molecular processes hijacked by a pathogenic SFG Rickettsia to establish a replicative niche in macrophages, opening several avenues of research in host-rickettsiae interactions.

A Pathogen and a Non-pathogen Spotted Fever Group Rickettsia Trigger Differential Proteome Signatures in Macrophages.

We have previously reported that Rickettsia conorii and Rickettsia montanensis have distinct intracellular fates within THP-1 macrophages, suggesting that the ability to proliferate within macrophages may be a distinguishable factor between pathogenic and non-pathogenic Spotted fever group (SFG) members. To start unraveling the molecular mechanisms underlying the capacity (or not) of SFG Rickettsia to establish their replicative niche in macrophages, we have herein used quantitative proteomics by SWATH-MS to profile the alterations resulted by the challenge of THP-1 macrophages with R. conorii and R. montanensis. We show that the pathogenic, R. conorii, and the non-pathogenic, R. montanensis, member of SFG Rickettsia trigger differential proteomic signatures in macrophage-like cells upon infection. R. conorii specifically induced the accumulation of several enzymes of the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid ß-oxidation, and glutaminolysis, as well as of several inner and outer membrane mitochondrial transporters. These results suggest a profound metabolic rewriting of macrophages by R. conorii toward a metabolic signature of an M2-like, anti-inflammatory activation program. Moreover, several subunits forming the proteasome and immunoproteasome are found in lower abundance upon infection with both rickettsial species, which may help bacteria to escape immune surveillance. R. conorii-infection specifically induced the accumulation of several host proteins implicated in protein processing and quality control in ER, suggesting that this pathogenic Rickettsia may be able to increase the ER protein folding capacity. This work reveals novel aspects of macrophage-Rickettsia interactions, expanding our knowledge of how pathogenic rickettsiae explore host cells to their advantage.

Atypical and nucellin-like aspartic proteases: emerging players in plant developmental processes and stress responses.

Members of the pepsin-like family (A1) of aspartic proteases (APs) are widely distributed in plants. A large number of genes encoding putative A1 APs are found in different plant genomes, the vast majority of which exhibit distinct features when compared with the so-called typical APs (and, therefore, grouped as atypical and nucellin-like APs). These features include the absence of the plant-specific insert; an unusually high number of cysteine residues; the nature of the amino acids preceding the first catalytic aspartate; and unexpected localizations. The over-representation of atypical and nucellin-like APs in plants is suggestive of greater diversification of protein functions and a more regulatory role for these APs, as compared with the housekeeping function generally attributed to typical APs. New functions have been uncovered for non-typical APs, with proposed roles in biotic and abiotic stress responses, chloroplast metabolism, and reproductive development, clearly suggesting functional specialization and tight regulation of activity. Furthermore, unusual enzymatic properties have also been documented for some of these proteases. Here, we give an overview of the current knowledge on the distinctive features and functions of both atypical and nucellin-like APs, and discuss this emerging pattern of functional complexity and specialization among plant pepsin-like proteases.

An atypical aspartic protease modulates lateral root development in Arabidopsis thaliana.

Few atypical aspartic proteases (APs) present in plants have been functionally studied to date despite having been implicated in developmental processes and stress responses. Here we characterize a novel atypical AP that we name Atypical Aspartic Protease in Roots 1 (ASPR1), denoting its expression in Arabidopsis roots. Recombinant ASPR1 produced by transient expression in Nicotiana benthamiana was active and displayed atypical properties, combining optimum acidic pH, partial sensitivity to pepstatin, pronounced sensitivity to redox agents, and unique specificity preferences resembling those of fungal APs. ASPR1 overexpression suppressed primary root growth and lateral root development, implying a previously unknown biological role for an AP. Quantitative comparison of wild-type and aspr1 root proteomes revealed deregulation of proteins associated with both reactive oxygen species and auxin homeostasis in the mutant. Together, our findings on ASPR1 reinforce the diverse pattern of enzymatic properties and biological roles of atypical APs and raise exciting questions on how these distinctive features impact functional specialization among these proteases.

Cardoon-based rennets for cheese production.

The use of crude aqueous extracts of Cynara cardunculus flowers as coagulants in the production of high-quality sheep and goat cheeses-as are the cases of several Portuguese and Spanish cheese varieties with Protected Designation of Origin status-has been maintained since ancient times. The unique rheological attributes and sensory properties characteristic of these cheeses have always suggested that this plant coagulant (and, therefore, its isolated milk-clotting proteases) could be used as alternative rennet in the dairy industry, particularly suited for the production of sheep and goat cheeses. However, the lack of standardization of C. cardunculus crude flower extracts, whose quality and performance depends on numerous factors, has always hampered the application of this plant rennet in industrial production scales. To overcome these limitations, and to aim at developing more effective solutions with potential for scalability of production and commercial application, several strategies have been undertaken in more recent years to establish new cardoon-based rennets. This review provides an overview on these developments and on the currently available solutions, which range from producing standardized formulations of native cardoon enzymes, to the optimization of the heterologous production of cardosins and cyprosins to generate synthetic versions of these milk-clotting enzymes. Challenges and emerging opportunities are also discussed.

Functional and structural characterization of synthetic cardosin B-derived rennet.

The potential of using a synthetic cardosin-based rennet in cheese manufacturing was recently demonstrated with the development and optimization of production of a recombinant form of cardosin B in Kluyveromyces lactis. With the goal of providing a more detailed characterization of this rennet, we herein evaluate the impact of the plant-specific insert (PSI) on cardosin B secretion in this yeast, and provide a thorough analysis of the specificity requirements as well as the biochemical and structural properties of the isolated recombinant protease. We demonstrate that the PSI domain can be substituted by different linker sequences without substantially affecting protein secretion and milk clotting activity. However, the presence of small portions of the PSI results in dramatic reductions of secretion yields in this heterologous system. Kinetic characterization and specificity profiling results clearly suggest that synthetic cardosin B displays lower catalytic efficiency and is more sequence selective than native cardosin B. Elucidation of the structure of synthetic cardosin B confirms the canonical fold of an aspartic protease with the presence of two high mannose-type, N-linked glycan structures; however, there are some differences in the conformation of the flap region when compared to cardosin A. These subtle variations in catalytic properties and the more stringent substrate specificity of synthetic cardosin B help to explain the observed suitability of this rennet for cheese production.

Differences in Intracellular Fate of Two Spotted Fever Group Rickettsia in Macrophage-Like Cells.

Spotted fever group (SFG) rickettsiae are recognized as important agents of human tick-borne diseases worldwide, such as Mediterranean spotted fever (Rickettsia conorii) and Rocky Mountain spotted fever (Rickettsia rickettsii). Recent studies in several animal models have provided evidence of non-endothelial parasitism by pathogenic SFG Rickettsia species, suggesting that the interaction of rickettsiae with cells other than the endothelium may play an important role in pathogenesis of rickettsial diseases. These studies raise the hypothesis that the role of macrophages in rickettsial pathogenesis may have been underappreciated. Herein, we evaluated the ability of two SFG rickettsial species, R. conorii (a recognized human pathogen) and Rickettsia montanensis (a non-virulent member of SFG) to proliferate in THP-1 macrophage-like cells, or within non-phagocytic cell lines. Our results demonstrate that R. conorii was able to survive and proliferate in both phagocytic and epithelial cells in vitro. In contrast, R. montanensis was able to grow in non-phagocytic cells, but was drastically compromised in the ability to proliferate within both undifferentiated and PMA-differentiated THP-1 cells. Interestingly, association assays revealed that R. montanensis was defective in binding to THP-1-derived macrophages; however, the invasion of the bacteria that are able to adhere did not appear to be affected. We have also demonstrated that R. montanensis which entered into THP-1-derived macrophages were rapidly destroyed and partially co-localized with LAMP-2 and cathepsin D, two markers of lysosomal compartments. In contrast, R. conorii was present as intact bacteria and free in the cytoplasm in both cell types. These findings suggest that a phenotypic difference between a non-pathogenic and a pathogenic SFG member lies in their respective ability to proliferate in macrophage-like cells, and may provide an explanation as to why certain SFG rickettsial species are not associated with disease in mammals.

Enzymatic properties, evidence for in vivo expression, and intracellular localization of shewasin D, the pepsin homolog from Shewanella denitrificans.

The widespread presence of pepsin-like enzymes in eukaryotes together with their relevance in the control of multiple biological processes is reflected in the large number of studies published so far for this family of enzymes. By contrast, pepsin homologs from bacteria have only recently started to be characterized. The work with recombinant shewasin A from Shewanella amazonensis provided the first documentation of this activity in prokaryotes. Here we extend our studies to shewasin D, the pepsin homolog from Shewanella denitrificans, to gain further insight into this group of bacterial peptidases that likely represent ancestral versions of modern eukaryotic pepsin-like enzymes. We demonstrate that the enzymatic properties of recombinant shewasin D are strongly reminiscent of eukaryotic pepsin homologues. We determined the specificity preferences of both shewasin D and shewasin A using proteome-derived peptide libraries and observed remarkable similarities between both shewasins and eukaryotic pepsins, in particular with BACE-1, thereby confirming their phylogenetic proximity. Moreover, we provide first evidence of expression of active shewasin D in S. denitrificans cells, confirming its activity at acidic pH and inhibition by pepstatin. Finally, our results revealed an unprecedented localization for a family A1 member by demonstrating that native shewasin D accumulates preferentially in the cytoplasm.

Biophysical characterization of laforin-carbohydrate interaction.

Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood. Recently, the structure of laforin with bound maltohexaose was determined and despite the importance of such breakthrough, some molecular interaction details remained missing. We herein report a thorough biophysical characterization of laforin-carbohydrate interaction using soluble glycans. We demonstrated an increased preference of laforin for the interaction with glycans with higher order of polymerization and confirmed the importance of tryptophan residues for glycan interaction. Moreover, and in line with what has been described for other CBMs and lectins, our results confirmed that laforin-glycan interactions occur with a favourable enthalpic contribution counter-balanced by an unfavourable entropic contribution. The analysis of laforin-glycan interaction through the glycan side by saturation transfer difference (STD)-NMR has shown that the CBM-binding site can accommodate between 5 and 6 sugar units, which is in line with the recently obtained crystal structure of laforin. Overall, the work in the present study complements the structural characterization of laforin and sheds light on the molecular mechanism of laforin-glycan interaction, which is a pivotal requisite to understand the physiological and pathological roles of laforin.

Structure of RC1339/APRc from Rickettsia conorii, a retropepsin-like aspartic protease.

The crystal structures of two constructs of RC1339/APRc from Rickettsia conorii, consisting of either residues 105-231 or 110-231 followed by a His tag, have been determined in three different crystal forms. As predicted, the fold of a monomer of APRc resembles one-half of the mandatory homodimer of retroviral pepsin-like aspartic proteases (retropepsins), but the quaternary structure of the dimer of APRc differs from that of the canonical retropepsins. The observed dimer is most likely an artifact of the expression and/or crystallization conditions since it cannot support the previously reported enzymatic activity of this bacterial aspartic protease. However, the fold of the core of each monomer is very closely related to the fold of retropepsins from a variety of retroviruses and to a single domain of pepsin-like eukaryotic enzymes, and may represent a putative common ancestor of monomeric and dimeric aspartic proteases.

Engineering a cardosin B-derived rennet for sheep and goat cheese manufacture.

Different sheep and goat cheeses with world-renowned excellence are produced using aqueous extracts of Cynara cardunculus flowers as coagulants. However, the use of this vegetable rennet is mostly limited to artisanal scale production, and no effective solutions to large-scale industrial applications have been reported so far. In this sense, the development of a synthetic rennet based on the most abundant cardoon milk-clotting enzymes (cardosins) would emerge as a solution for scalability of production and for application of these proteases as alternative rennets in dairy industry. In this work, we report the development of a new cardosin B-derived rennet produced in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. Using a stepwise optimization strategy-consisting of culture media screening, complemented with a protein engineering approach with removal of the plant-specific domain, and a codon optimization step-we successfully improved cardosin B production yield (35×) in K. lactis. We demonstrated that the secreted enzyme displays similar proteolytic properties, such as casein digestion profiles as well as optimum pH (pH 4.5) and temperature (40 °C), with those of native cardosin B. From this optimization process resulted the rennet preparation Vegetable Rennet (VRen), requiring no downstream protein purification steps. The effectiveness of VRen in cheese production was demonstrated by manufacturing sheep, goat, and cow cheeses. Interestingly, the use of VRen resulted in a higher cheese yield for all three types of cheese when compared with synthetic chymosin. Altogether, these results clearly position VRen as an alternative/innovative coagulant for the cheese-making industry.

RC1339/APRc from Rickettsia conorii is a novel aspartic protease with properties of retropepsin-like enzymes.

Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii) is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a novel bona fide member of the retropepsin family of aspartic proteases, APRc emerges as an intriguing target for therapeutic intervention against fatal rickettsioses.