Xanthomonas immunity proteins protect against the cis-toxic effects of their cognate T4SS effectors.

Many bacteria kill rival species by translocating toxic effectors into target cells. Effectors are often encoded along with cognate immunity proteins that could (i) protect against "friendly-fire" (trans-intoxication) from neighboring sister cells and/or (ii) protect against internal cis-intoxication (suicide). Here, we distinguish between these two mechanisms in the case of the bactericidal Xanthomonas citri Type IV Secretion System (X-T4SS). We use a set of X. citri mutants lacking multiple effector/immunity protein (X-Tfe/X-Tfi) pairs to show that X-Tfis are not absolutely required to protect against trans-intoxication by wild-type cells. Our investigation then focused on the in vivo function of the lysozyme-like effector X-TfeXAC2609 and its cognate immunity protein X-TfiXAC2610. In the absence of X-TfiXAC2610, we observe X-TfeXAC2609-dependent and X-T4SS-independent accumulation of damage in the X. citri cell envelope, cell death, and inhibition of biofilm formation. While immunity proteins in other systems have been shown to protect against attacks by sister cells (trans-intoxication), this is an example of an antibacterial secretion system in which the immunity proteins are dedicated to protecting cells against cis-intoxication.

Experimental phasing opportunities for macromolecular crystallography at very long wavelengths.

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.

Tamoxifen associated to the conservative CKD treatment promoted additional antifibrotic effects on experimental hypertensive nephrosclerosis.

CKD progression depends on the activation of an intricate set of hemodynamic and inflammatory mechanisms, promoting renal leukocyte infiltration, inflammation and fibrosis, leading to renal function loss. There are currently no specific drugs to detain renal fibrogenesis, which is a common end-point for different nephropathies. Clinical therapy for CKD is mostly based on the management of hypertension and proteinuria, partially achieved with renin-angiotensin-aldosterone system (RAAS) blockers, and the control of inflammation by immunosuppressive drugs. The aim of the present study was to verify if the administration of tamoxifen (TAM), an estrogen receptor modulator, clinically employed in the treatment of breast cancer and predicted to exert antifibrotic effects, would promote additional benefits when associated to a currently used therapeutic scheme for the conservative management of experimental CKD. Wistar rats underwent the NAME model of hypertensive nephrosclerosis, obtained by daily oral administration of a nitric oxide synthesis inhibitor, associated to dietary sodium overload. The therapeutic association of TAM to losartan (LOS), and mofetil mycophenolate (MMF) effectively reduced the severe hypertension, marked albuminuria and glomerular damage exhibited by NAME animals. Moreover, the association also succeeded in limiting renal inflammation in this model, and promoted further reduction of ECM interstitial accumulation and renal fibrosis, compared to the monotherapies. According to our results, the association of TAM to the currently used conservative treatment of CKD added significant antifibrotic effects both in vivo and in vitro, and may represent an alternative to slow the progression of chronic nephropathy.

Photobiomodulation Reduces the Cytokine Storm Syndrome Associated with COVID-19 in the Zebrafish Model.

Although the exact mechanism of the pathogenesis of coronavirus SARS-CoV-2 (COVID-19) is not fully understood, oxidative stress and the release of pro-inflammatory cytokines have been highlighted as playing a vital role in the pathogenesis of the disease. In this sense, alternative treatments are needed to reduce the level of inflammation caused by COVID-19. Therefore, this study aimed to investigate the potential effect of red photobiomodulation (PBM) as an attractive therapy to downregulate the cytokine storm caused by COVID-19 in a zebrafish model. RT-qPCR analyses and protein-protein interaction prediction among SARS-CoV-2 and Danio rerio proteins showed that recombinant Spike protein (rSpike) was responsible for generating systemic inflammatory processes with significantly increased levels of pro-inflammatory (il1b, il6, tnfa, and nfkbiab), oxidative stress (romo1) and energy metabolism (slc2a1a and coa1) mRNA markers, with a pattern similar to those observed in COVID-19 cases in humans. On the other hand, PBM treatment was able to decrease the mRNA levels of these pro-inflammatory and oxidative stress markers compared with rSpike in various tissues, promoting an anti-inflammatory response. Conversely, PBM promotes cellular and tissue repair of injured tissues and significantly increases the survival rate of rSpike-inoculated individuals. Additionally, metabolomics analysis showed that the most-impacted metabolic pathways between PBM and the rSpike treated groups were related to steroid metabolism, immune system, and lipid metabolism. Together, our findings suggest that the inflammatory process is an incisive feature of COVID-19 and red PBM can be used as a novel therapeutic agent for COVID-19 by regulating the inflammatory response. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials can commence.

Immunization with SARS-CoV-2 Nucleocapsid protein triggers a pulmonary immune response in rats.

The SARS-CoV-2 pandemic have been affecting millions of people worldwide, since the beginning of 2020. COVID-19 can cause a wide range of clinical symptoms, which varies from asymptomatic presentation to severe respiratory insufficiency, exacerbation of immune response, disseminated microthrombosis and multiple organ failure, which may lead to dead. Due to the rapid spread of SARS-CoV-2, the development of vaccines to minimize COVID-19 severity in the world population is imperious. One of the employed techniques to produce vaccines against emerging viruses is the synthesis of recombinant proteins, which can be used as immunizing agents. Based on the exposed, the aim of the present study was to verify the systemic and immunological effects of IM administration of recombinant Nucleocapsid protein (NP), derived from SARS-CoV-2 and produced by this research group, in 2 different strains of rats (Rattus norvegicus); Wistar and Lewis. For this purpose, experimental animals received 4 injections of NP, once a week, and were submitted to biochemical and histological analysis. Our results showed that NP inoculations were safe for the animals, which presented no clinical symptoms of worrying side effects, nor laboratorial alterations in the main biochemical and histological parameters, suggesting the absence of toxicity induced by NP. Moreover, NP injections successfully triggered the production of specific anti-SARS-CoV-2 IgG antibodies by both Wistar and Lewis rats, showing the sensitization to have been well sufficient for the immunization of these strains of rats. Additionally, we observed the local lung activation of the Bronchus-Associated Lymphoid Tissue (BALT) of rats in the NP groups, suggesting that NP elicits specific lung immune response. Although pre-clinical and clinical studies are still required, our data support the recombinant NP produced by this research group as a potential immunizing agent for massive vaccination, and may represent advantages upon other recombinant proteins, since it seems to induce specific pulmonary protection.

Structural basis for effector recognition by an antibacterial type IV secretion system.

Many soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a type IV secretion system (T4SS) specialized in translocating effector proteins into other gram-negative species, leading to target cell death. These effectors, known as X-Tfes, carry a carboxyl-terminal domain of ∼120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for T4SS-dependent translocation. However, the structural basis of the XVIPCD-VirD4 interaction is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stability of the N-terminal region is reduced at and below pH 7.0, a property that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.

The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri.

Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.

Secrete or perish: The role of secretion systems in Xanthomonas biology.

Bacteria of the Xanthomonas genus are mainly phytopathogens of a large variety of crops of economic importance worldwide. Xanthomonas spp. rely on an arsenal of protein effectors, toxins and adhesins to adapt to the environment, compete with other microorganisms and colonize plant hosts, often causing disease. These protein effectors are mainly delivered to their targets by the action of bacterial secretion systems, dedicated multiprotein complexes that translocate proteins to the extracellular environment or directly into eukaryotic and prokaryotic cells. Type I to type VI secretion systems have been identified in Xanthomonas genomes. Recent studies have unravelled the diverse roles played by the distinct types of secretion systems in adaptation and virulence in xanthomonads, unveiling new aspects of their biology. In addition, genome sequence information from a wide range of Xanthomonas species and pathovars have become available recently, uncovering a heterogeneous distribution of the distinct families of secretion systems within the genus. In this review, we describe the architecture and mode of action of bacterial type I to type VI secretion systems and the distribution and functions associated with these important nanoweapons within the Xanthomonas genus.

A Family of T6SS Antibacterial Effectors Related to l,d-Transpeptidases Targets the Peptidoglycan.

Type VI secretion systems (T6SSs) are nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella T6SS antibacterial effector called Tlde1 that is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tldi1). Microscopy analysis reveals that cells expressing Tlde1 stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovers similarities between Tlde1 and the catalytic domain of l,d-transpeptidases. Point mutations on conserved catalytic residues abrogate toxicity. Biochemical assays reveal that Tlde1 displays both l,d-carboxypeptidase activity by cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid3 and d-alanine4 and l,d-transpeptidase exchange activity by replacing d-alanine4 by a non-canonical d-amino acid. Phylogenetic analysis shows that Tlde1 homologs constitute a family of T6SS-associated effectors broadly distributed among Proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition and reveals a different mechanism of bacterial antagonism.

A bipartite periplasmic receptor-diguanylate cyclase pair (XAC2383-XAC2382) in the bacterium Xanthomonas citri.

The second messenger cyclic diguanylate monophosphate (c-di-GMP) is a central regulator of bacterial lifestyle, controlling several behaviors, including the switch between sessile and motile states. The c-di-GMP levels are controlled by the interplay between diguanylate cyclases (DGCs) and phosphodiesterases, which synthesize and hydrolyze this second messenger, respectively. These enzymes often contain additional domains that regulate activity via binding of small molecules, covalent modification, or protein-protein interactions. A major challenge remains to understand how DGC activity is regulated by these additional domains or interaction partners in specific signaling pathways. Here, we identified a pair of co-transcribed genes (xac2382 and xac2383) in the phytopathogenic, Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), whose mutations resulted in opposing motility phenotypes. We show that the periplasmic cache domain of XAC2382, a membrane-associated DGC, interacts with XAC2383, a periplasmic binding protein, and we provide evidence that this interaction regulates XAC2382 DGC activity. Moreover, we solved the crystal structure of XAC2383 with different ligands, indicating a preference for negatively charged phosphate-containing compounds. We propose that XAC2383 acts as a periplasmic sensor that, upon binding its ligand, inhibits the DGC activity of XAC2382. Of note, we also found that this previously uncharacterized signal transduction system is present in several other bacterial phyla, including Gram-positive bacteria. Phylogenetic analysis of homologs of the XAC2382-XAC2383 pair supports several independent origins that created new combinations of XAC2382 homologs with a conserved periplasmic cache domain with different cytoplasmic output module architectures.

Stromal Cell-Derived Factor 2: A Novel Protein that Interferes in Endoplasmic Reticulum Stress Pathway in Human Placental Cells.

Endoplasmic reticulum (ER) stress results from changes in ER homeostasis and folding of proteins. ER stress initiates cellular adaptive mechanisms to rescue cell homeostasis or, if that does not work, to elicit apoptosis. We have previously shown that mouse SDF2 is sublocalized in the ER, is ubiquitously expressed, and shows strong similarities with stromal cell-derived factor (SDF) 2L1 and SDF2-like from Arabidopsis, ER proteins involved in chaperone network and protein folding. Thus, we hypothesized that SDF2 plays a role in the ER stress and unfolded protein response. In this study, we investigated the possible role of SDF2 in the human placenta. Expression of SDF2 was present throughout gestation and was expressed by several cell types. Second-trimester cytotrophoblast cells (CTBs) in the differentiation process, monitored through chorionic gonadotropin production, showed upregulation of SDF2 protein. SDF2 expression, however, was significantly diminished in placentas from neonates small for gestational age and in hypoxic in vitro conditions (P ≤ 0.001, 2% O2), suggesting a link with cellular stress. ER stress-induced cells-CTB and BeWo-also showed SDF2 downregulation in different time points, emphasizing this relationship. SDF2 downregulation was also followed by an increase in binding immunoglobulin protein (BiP) expression, an ER protein-associated chaperone acting as a sensor for misfolded proteins and an ER stress cell survival marker. In line with this, SDF2 siRNA resulted in significant anticipation of BiP expression. Downregulation of SDF2 also interfered with C/EBP homologous protein expression, one of the highest inducible genes during ER stress. These findings suggest that SDF2 may be an important regulatory factor by which trophoblast cells can control cell survival under ER stress. In conclusion, this study identifies a novel factor with the ability to interfere with ER stress proteins, which may contribute to the understanding of ER stress associated with placental-related diseases of pregnancy.

The Xanthomonas type IV pilus.

Type IV pili, a special class of bacterial surface filaments, are key behavioral mediators for many important human pathogens. However, we know very little about the role of these structures in the lifestyles of plant-associated bacteria. Over the past few years, several groups studying the extensive genus of Xanthomonas spp. have gained insights into the roles of played by type IV pili in bacteria-host interactions and pathogenesis, motility, biofilm formation, and interactions with bacteriophages. Protein-protein interaction studies have identified T4P regulators and these, along with structural studies, have begun to reveal some of the possible molecular mechanisms that may control the extension/retraction cycles of these dynamic filaments.

Functional characterization of two SOS-regulated genes involved in mitomycin C resistance in Caulobacter crescentus.

The SOS response is a universal bacterial regulon involved in the cellular response to DNA damage and other forms of stress. In Caulobacter crescentus, previous work has identified a plethora of genes that are part of the SOS regulon, but the biological roles of several of them remain to be determined. In this study, we report that two genes, hereafter named mmcA and mmcB, are involved in the defense against DNA damage caused by mitomycin C (MMC), but not against lesions induced by other common DNA damaging agents, such as UVC light, methyl methanesulfonate (MMS) and hydrogen peroxide. mmcA is a conserved gene that encodes a member of the glyoxalases/dioxygenases protein family, and acts independently of known DNA repair pathways. On the other hand, epistasis analysis showed that mmcB acts in the same pathway as imuC (dnaE2), and is required specifically for MMC-induced mutagenesis, but not for that induced by UV light, suggesting a role for MmcB in translesion synthesis-dependent repair of MMC damage. We show that the lack of MMC-induced mutability in the mmcB strain is not caused by lack of proper SOS induction of the imuABC operon, involved in translesion synthesis (TLS) in C. crescentus. Based on this data and on structural analysis of a close homolog, we propose that MmcB is an endonuclease which creates substrates for ImuABC-mediated TLS patches.

Bacterial killing via a type IV secretion system.

Type IV secretion systems (T4SSs) are multiprotein complexes that transport effector proteins and protein-DNA complexes through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells. Many bacteria of the family Xanthomonadaceae, which occupy diverse environmental niches, carry a T4SS with unknown function but with several characteristics that distinguishes it from other T4SSs. Here we show that the Xanthomonas citri T4SS provides these cells the capacity to kill other Gram-negative bacterial species in a contact-dependent manner. The secretion of one type IV bacterial effector protein is shown to require a conserved C-terminal domain and its bacteriolytic activity is neutralized by a cognate immunity protein whose 3D structure is similar to peptidoglycan hydrolase inhibitors. This is the first demonstration of the involvement of a T4SS in bacterial killing and points to this special class of T4SS as a mediator of both antagonistic and cooperative interbacterial interactions.

Xanthomonas citri subsp. citri type IV Pilus is required for twitching motility, biofilm development, and adherence.

Bacterial type IV pili (T4P) are long, flexible surface filaments that consist of helical polymers of mostly pilin subunits. Cycles of polymerization, attachment, and depolymerization mediate several pilus-dependent bacterial behaviors, including twitching motility, surface adhesion, pathogenicity, natural transformation, escape from immune system defense mechanisms, and biofilm formation. The Xanthomonas citri subsp. citri strain 306 genome codes for a large set of genes involved in T4P biogenesis and regulation and includes several pilin homologs. We show that X. citri subsp. citri can exhibit twitching motility in a manner similar to that observed in other bacteria such as Pseudomonas aeruginosa and Xylella fastidiosa and that this motility is abolished in Xanthomonas citri subsp. citri knockout strains in the genes coding for the major pilin subunit PilAXAC3241, the ATPases PilBXAC3239 and PilTXAC2924, and the T4P biogenesis regulators PilZXAC1133 and FimXXAC2398. Microscopy analyses were performed to compare patterns of bacterial migration in the wild-type and knockout strains and we observed that the formation of mushroom-like structures in X. citri subsp. citri biofilm requires a functional T4P. Finally, infection of X. citri subsp. citri cells by the bacteriophage (ΦXacm4-11 is T4P dependent. The results of this study improve our understanding of how T4P influence Xanthomonas motility, biofilm formation, and susceptibility to phage infection.

Stromal cell derived factor-2 (Sdf2): a novel protein expressed in mouse.

The stromal derived factor (SDFs) family comprises a group of molecules generated by stromal cells. SDF1 and SDF4 are chemokines; SDF2 and SDF5 are not yet functionally and structurally defined. In human and mouse, Sdf2 has a paralogous gene, Sdf2l1, whose protein sequences are 78% similar and 68% identical. Human SDF2L1 is an endoplasmic reticulum-stress inducible-gene. In Arabidopsis thaliana, SDF2-like (39% and 37% amino acid sequence identity with Mus musculus Sdf2 and Sdf2l1) has also been implicated in activating the UPR in ER-stress. Here we have cloned, expressed and purified recombinant Sdf2 and raised an anti-Sdf2 antibody. We demonstrated that the protein is expressed in several tissues and is localized in the endoplasmic reticulum. We suggest that Sdf2, initially predicted as a secretory protein because it lacks the canonical ER retention signals in its C-terminal, could be ER-resident through accessory binding proteins or other amino acid sequence motifs, as suggested for the homolog protein SDF2-like. Furthermore, the crystal structure of SDF2-like from Arabidopsis thaliana is a typical ß-trefoil containing three MIR motifs; all hydrophobic residues considered important for maintaining the bottom layer of the ß-trefoil barrel seem to be conserved in the Sdf2 family. Multiple alignment using 43 sequences for SDF2 and 38 for SDF2L1 paralogous families also revealed a very similar residue conservation profile. Comparing the amino acid sequence and predicted 3D structure with other Sdf2-like proteins we suggest a role of mouse Sdf2 in the Unfolded Protein Response and ER-stress, similar to that of Sdf2l1 from human and mouse and SDF2-like from Arabidopsis thaliana. Chronic ER stress has been associated with many human diseases including cancer and diabetes. Identification of new factors associated with the ER stress pathway can help to identify and define key targets of this response.

Structure of the PilZ-FimXEAL-c-di-GMP Complex Responsible for the Regulation of Bacterial Type IV Pilus Biogenesis.

Signal transduction pathways mediated by cyclic-bis(3'→5')-dimeric GMP (c-di-GMP) control many important and complex behaviors in bacteria. C-di-GMP is synthesized through the action of GGDEF domains that possess diguanylate cyclase activity and is degraded by EAL or HD-GYP domains with phosphodiesterase activity. There is mounting evidence that some important c-di-GMP-mediated pathways require protein-protein interactions between members of the GGDEF, EAL, HD-GYP and PilZ protein domain families. For example, interactions have been observed between PilZ and the EAL domain from FimX of Xanthomonas citri (Xac). FimX and PilZ are involved in the regulation of type IV pilus biogenesis via interactions of the latter with the hexameric PilB ATPase associated with the bacterial inner membrane. Here, we present the crystal structure of the ternary complex made up of PilZ, the FimX EAL domain (FimXEAL) and c-di-GMP. PilZ interacts principally with the lobe region and the N-terminal linker helix of the FimXEAL. These interactions involve a hydrophobic surface made up of amino acids conserved in a non-canonical family of PilZ domains that lack intrinsic c-di-GMP binding ability and strand complementation that joins ß-sheets from both proteins. Interestingly, the c-di-GMP binds to isolated FimXEAL and to the PilZ-FimXEAL complex in a novel conformation encountered in c-di-GMP-protein complexes in which one of the two glycosidic bonds is in a rare syn conformation while the other adopts the more common anti conformation. The structure points to a means by which c-di-GMP and PilZ binding could be coupled to FimX and PilB conformational states.

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents.

PILZ protein structure and interactions with PILB and the FIMX EAL domain: implications for control of type IV pilus biogenesis.

The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3'-->5')cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv citri (PilZ(XAC1133), this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ(XAC1133) shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which PilZ proteins regulate specific processes is not clear. In this study, we show that PilZ(XAC1133) binds to PilB, an ATPase required for T4P polymerization, and to the EAL domain of FimX(XAC2398), which regulates T4P biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimX(XAC2398)in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ(XAC1133). Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ(XAC1133) interactions with FimX(XAC2398) and PilB(XAC3239) are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of "degenerate" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery.