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Multifunctional Autoprocessing Repeats-in-Toxin (MARTX) toxins are a diverse effector delivery platform of many Gram-negative bacteria that infect mammals, insects, and aquatic animal hosts. The mechanisms by which these toxins recognize host cell receptors for translocation of toxic effectors into the cell have remained elusive. Here, we map the first surface receptor-binding domain of a MARTX toxin from the highly lethal foodborne pathogen Vibrio vulnificus . This domain corresponds to a 273-amino acid sequence with predicted symmetrical immunoglobulin-like folds. We demonstrate that this domain binds internal N -acetylglucosamine on complex biantennary N-glycans with select preference for L1CAM and other N-glycoproteins with multiple N-glycans on host cell surfaces. This receptor binding domain is essential for V. vulnificus pathogenesis during intestinal infection. The identification of a highly conserved motif universally present as part of all N-glycans correlates with the V. vulnificus MARTX toxin boasting broad specificity and targeting nearly all cell types.

Coronavirus non-structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vacuole that houses the replication-transcription complex. Nsp3 in SARS-like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD-N (Macrodomain 2) and SUD-M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x-ray structure of SARS-CoV SUD-N/M as well as a mutant variant of SARS-CoV-2 SUD-N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD-N and SUD-M are not rigidly associated, but rather, have significant rotational flexibility. Phylogenetic analysis supports that the disulfide bond cysteines are also absent in pangolin-SARS and closely related viruses, consistent with pangolins being the presumed intermediate host in the emergence of SARS-CoV-2. The absence of these cysteines does not impact viral replication or protein translation., Competing Interests: CONFLICTS OF INTEREST K.J.F.S. has a significant financial interest in Situ Biosciences, a contract research organization that conducts research unrelated to this work. T.Y.T. and M.O. are listed as inventors on a patent application filed by the Gladstone Institutes that covers the use of pGLUE to generate SARS-CoV-2 infectious clones and replicons. All other authors declare no conflicts of interest.

Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli , indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCE Stenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa . We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB)., Competing Interests: K.J.F.S. declares she has a financial interest in Situ Biosciences, a contract research organization that conducts research unrelated to this research.

Vibrio vulnificus causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The most commonly present MARTX effector domain, the Makes Caterpillars Floppy-like (MCF) toxin, is a cysteine protease stimulated by host adenosine diphosphate (ADP) ribosylation factors (ARFs) to autoprocess. Here, we show processed MCF then binds and cleaves host Ra s-related proteins in b rain (Rab) guanosine triphosphatases within their C-terminal tails resulting in Rab degradation. We demonstrate MCF binds Rabs at the same interface occupied by ARFs. Moreover, we show MCF preferentially binds to ARF1 prior to autoprocessing and is active to cleave Rabs only subsequent to autoprocessing. We then use structure prediction algorithms to demonstrate that structural composition, rather than sequence, determines Rab target specificity. We further determine a crystal structure of aMCF as a swapped dimer, revealing an alternative conformation we suggest represents the open, activated state of MCF with reorganized active site residues. The cleavage of Rabs results in Rab1B dispersal within cells and loss of Rab1B density in the intestinal tissue of infected mice. Collectively, our work describes an extracellular bacterial mechanism whereby MCF is activated by ARFs and subsequently induces the degradation of another small host guanosine triphosphatase (GTPase), Rabs, to drive organelle damage, cell death, and promote pathogenesis of these rapidly fatal infections., Competing Interests: Competing interests statement:The authors declare no competing interest.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages., Competing Interests: Competing interests statement:K.J.F.S. has a significant interest in Situ Biosciences, a contract research organization that conducts research unrelated to this study. K.J.F.S. and her spouse are 100% owners of Situ Biosciences. All other authors declare no conflicts of interest.

About one-third of all human cancers encode abnormal RAS proteins locked in a constitutively activated state to drive malignant transformation and uncontrolled tumor growth. Despite progress in development of small molecules for treatment of mutant KRAS cancers, there is a need for a pan-RAS inhibitor that is effective against all RAS isoforms and variants and that avoids drug resistance. We have previously shown that the naturally occurring bacterial enzyme RAS/RAP1-specific endopeptidase (RRSP) is a potent RAS degrader that can be re-engineered as a biologic therapy to induce regression of colorectal, breast, and pancreatic tumors. Here, we have developed a strategy for in vivo expression of this RAS degrader via mRNA delivery using a synthetic nonviral gene delivery platform composed of the poly(ethylene glycol)- b -poly(propylene sulfide) (PEG- b -PPS) block copolymer conjugated to a dendritic cationic peptide (PPDP2). Using this strategy, PPDP2 is shown to deliver mRNA to both human and mouse pancreatic cells resulting in RRSP gene expression, activity, and loss of cell proliferation. Further, pancreatic tumors are reduced with residual tumors lacking detectable RAS and phosphorylated ERK. These data support that mRNA-loaded synthetic nanocarrier delivery of a RAS degrader can interrupt the RAS signaling system within pancreatic cancer cells while avoiding side effects during therapy., Competing Interests: CONFLICTS OF INTEREST T.E.E., Y.Q., E.A.S. and K.J.F.S. have filed a provisional patent through Northwestern University on PPDP2 delivery of rrsp-mRNA based on these studies. K.J.F.S. further discloses that she holds a patent on use of RRSP as a cancer therapeutic (Patent # US10829752B2) and is named on a patent on use of RRSP-DTB to treat tumors (Patent #US 10597663B2). E.A.S. has a patent application for the PPDP2 delivery platform (US20240158814). E.A.S. is a founder of the company SNC Therapeutics which is developing PEG-b-PPS based therapeutics. K.J.F.S. has a significant interest in Situ Biosciences LLC, a contract research organization that conducts research unrelated to this work.

SARS-CoV-2 nsp13 helicase is an essential enzyme for viral replication and a promising target for antiviral drug development. This study compares the double-stranded RNA (dsRNA) unwinding activity of nsp13 and the Omicron nsp13 R392C variant, which is predominant in currently circulating lineages. Using in vitro gel- and fluorescence-based assays, we found that both nsp13 and nsp13 R392C have dsRNA unwinding activity with equivalent kinetics. Furthermore, the R392C mutation had no effect on the efficiency of the nsp13-specific helicase inhibitor SSYA10-001. We additionally confirmed the activity of several other helicase inhibitors against nsp13, including punicalagin that inhibited dsRNA unwinding at nanomolar concentrations. Overall, this study reveals the utility of using dsRNA unwinding assays to screen small molecules for antiviral activity against nsp13 and the Omicron nsp13 R392C variant. Continual monitoring of newly emergent variants will be essential for considering resistance profiles of lead compounds as they are advanced towards next-generation therapeutic development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Judd F. Hultquist, Karla J. F. Satchell reports financial support was provided by Robert H. Lurie Comprehensice Cancer Center. Karla J. F. Satchell reports financial support was provided by National Institute of Allergy and Infectious Diseases. Judd F. Hultquist reports financial support was provided by Successful Clinical Response to Pneumonia Therapy Center. Karla J. F. Satchell reports financial support was provided by Institute for Quantitative Biosciences Institute Coronavirus Research Group Antiviral Drug Discovery Center. Karla J. F. Satchell reports a relationship with Situ Biosciences that includes: equity or stocks. Judd F. Hultquist reports a relationship with Gilead Sciences Inc that includes: funding grants. Judd F. Hultquist reports a relationship with Merck that includes: consulting or advisory. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

There is an urgent need for new antibiotics given the rise of antibiotic resistance, and succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) has emerged as a promising bacterial enzyme target. DapE from Haemophilus influenzae ( Hi DapE) has been studied and inhibitors identified, but it is essential to explore DapE from different species to assess selective versus broad-spectrum therapeutics. We have determined the structure of DapE from the ESKAPE pathogen Acinetobacter baumannii ( Ab DapE) and studied inhibition by known inhibitors of Hi DapE. Ab DapE is inhibited by captopril and sulfate comparable to Hi DapE, but Ab DapE was not significantly inhibited by a known indoline sulfonamide Hi DapE inhibitor. Captopril and sulfate both stabilize Hi DapE by increasing the thermal melting temperature ( T m ) in thermal shift assays. By contrast, sulfate decreases the stability of the Ab DapE enzyme, whereas captopril increases the stability. Further, we report two crystal structures of selenomethionine-substituted Ab DapE in the closed conformation, one with Ab DapE in complex with succinate derived from enzymatic hydrolysis of N 6 -methyl-l,l-SDAP substrate and acetate (PDB code 7T1Q, 2.25 Å resolution), and a crystal structure of Ab DapE with bound succinate along with l-(S)-lactate, a product of degradation of citric acid from the crystallization buffer during X-ray irradiation (PDB code 8F8O, 2.10 Å resolution)., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Clostridioides difficile causes life-threatening gastrointestinal infections. It is a high-risk pathogen due to a lack of effective treatments, antimicrobial resistance, and a poorly conserved genomic core. Herein, we report 30 X-ray structures from a structure genomics pipeline spanning 13 years, representing 10.2% of the X-ray structures for this important pathogen., Competing Interests: The authors declare no conflict of interest.

A collaborative, open-science team undertook discovery of novel small molecule inhibitors of the SARS-CoV-2 nsp16-nsp10 2'- O -methyltransferase using a high throughput screening approach with the potential to reveal new inhibition strategies. This screen yielded compound 5a , a ligand possessing an electron-deficient double bond, as an inhibitor of SARS-CoV-2 nsp16 activity. Surprisingly, X-ray crystal structures revealed that 5a covalently binds within a previously unrecognized cryptic pocket near the S -adenosylmethionine binding cleft in a manner that prevents occupation by S -adenosylmethionine. Using a multidisciplinary approach, we examined the mechanism of binding of compound 5a to the nsp16 cryptic pocket and developed 5a derivatives that inhibited nsp16 activity and murine hepatitis virus replication in rat lung epithelial cells but proved cytotoxic to cell lines canonically used to examine SARS-CoV-2 infection. Our study reveals the druggability of this newly discovered SARS-CoV-2 nsp16 cryptic pocket, provides novel tool compounds to explore the site, and suggests a new approach for discovery of nsp16 inhibition-based pan-coronavirus therapeutics through structure-guided drug design.

The function and significance of RAS proteins in cancer have been widely studied for decades. In 2013, the National Cancer Institute established the RAS Initiative to explore innovative approaches for attacking the proteins encoded by mutant forms of RAS genes and to create effective therapies for RAS-driven cancers. This initiative spurred researchers to develop novel approaches and to discover small molecules targeting this protein that was at one time termed "undruggable." More recently, advanced efforts in RAS degraders including PROTACs, linker-based degraders, and direct proteolysis degraders have been explored as novel strategies to target RAS for cancer treatment. These RAS degraders present new opportunities for RAS therapies and may prove fruitful in understanding basic cell biology. Novel delivery strategies will further enhance the efficacy of these therapeutics. In this review, we summarize recent efforts to develop RAS degraders, including PROTACs and E3 adaptor and ligase fusions as cancer therapies. This review also details the direct RAS protease degrader, RAS/RAP1-specific endopeptidase that directly and specifically cleaves RAS., Competing Interests: Declaration of interests K.J.F.S. discloses that she holds a patent on use of RRSP as a cancer therapeutic (Patent # US10829752B2). K.J.F.S. has a significant interest in Situ Biosciences, a contract research organization that conducts research unrelated to this work., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The SARS-CoV-2 nucleocapsid (N) protein is highly immunogenic, and anti-N antibodies are commonly used as markers for prior infection. While several studies have examined or predicted the antigenic regions of N, these have lacked consensus and structural context. Using COVID-19 patient sera to probe an overlapping peptide array, we identified six public and four private epitope regions across N, some of which are unique to this study. We further report the first deposited X-ray structure of the stable dimerization domain at 2.05 Å as similar to all other reported structures. Structural mapping revealed that most epitopes are derived from surface-exposed loops on the stable domains or from the unstructured linker regions. An antibody response to an epitope in the stable RNA binding domain was found more frequently in sera from patients requiring intensive care. Since emerging amino acid variations in N map to immunogenic peptides, N protein variation could impact detection of seroconversion for variants of concern. IMPORTANCE As SARS-CoV-2 continues to evolve, a structural and genetic understanding of key viral epitopes will be essential to the development of next-generation diagnostics and vaccines. This study uses structural biology and epitope mapping to define the antigenic regions of the viral nucleocapsid protein in sera from a cohort of COVID-19 patients with diverse clinical outcomes. These results are interpreted in the context of prior structural and epitope mapping studies as well as in the context of emergent viral variants. This report serves as a resource for synthesizing the current state of the field toward improving strategies for future diagnostic and therapeutic design., Competing Interests: The authors declare a conflict of interest. K.J.F.S. has significant financial interest in Situ Biosciences LLC, a contract research organization that conducts coronavirus research unrelated to this work. K.J.F.S. also received payments from Buoy Health during the time of this investigation for consultation on COVID-19 topics unrelated to this work. All other authors declare no conflicts of interest.

Klebsiella pneumoniae is a leading cause of antibiotic-resistant-associated deaths in the world. Here, we report the deposition of 14 structures of enzymes from both the core and accessory genomes of sequence type 23 (ST23) K1 hypervirulent K. pneumoniae.

The marine bacterium Vibrio vulnificus infects humans via food or water contamination, leading to serious manifestations, including gastroenteritis, wound infections, and septic shock. Previous studies suggest phylogenetic Lineage 1 isolates with the v cgC allele of the vcg gene cause human infections, whereas Lineage 2 isolates with the vcgE allele are less pathogenic. Mouse studies suggest that some variants of the primary toxin could drive more serious infections. A collection of 109 V. vulnificus United States human clinical isolates from 2001 to 2019 with paired clinical outcome data were assembled. The isolates underwent whole-genome sequencing, multilocus-sequence phylogenetic analysis, and toxinotype analysis of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin. In contrast to prior reports, clinical isolates were equally distributed between lineages. We found no correlation between phylogenetic lineage or MARTX toxinotype and disease severity. Infections caused by isolates in Lineage 1 demonstrated a borderline statistically significant higher mortality. Lineage 1 isolates had a trend toward a higher proportion of M-type MARTX toxins compared with Lineage 2, although this was not statistically significant. IMPORTANCE Vibrio vulnificus is an aquatic pathogen that is capable of causing severe disease in humans. Previous studies have suggested that pathogenic isolates were restricted to certain phylogenetic lineages and possibly toxinotype. Our study demonstrated that phylogenetic lineage and multifunctional autoprocessing repeats-in-toxin (MARTX) toxinotype do not predict severity of infection. V. vulnificus strains capable of causing severe human disease are not concentrated in Lineage 1 but are genetically diverse. Thus, food surveillance based on lineage type or toxinotype may not be an appropriate intervention measure to control this rare but serious infection.

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic β-lactamase AmpC. However, OXA-10-family β-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA β-lactamases have a propensity for mutation that leads to extended spectrum cephalosporinase and carbapenemase activity. In this study, we identified isolates from a subclade of the multidrug-resistant (MDR) high risk P. aeruginosa clonal complex CC446 with a resistance to ceftazidime. A genomic analysis revealed that these isolates harbored a plasmid containing a novel allele of bla OXA-10 , named bla OXA-935 , which was predicted to produce an OXA-10 variant with two amino acid substitutions: an aspartic acid instead of a glycine at position 157 and a serine instead of a phenylalanine at position 153. The G157D mutation, present in OXA-14, is associated with the resistance of P. aeruginosa to ceftazidime. Compared to OXA-14, OXA-935 showed increased catalytic efficiency for ceftazidime. The deletion of bla OXA-935 restored the sensitivity to ceftazidime, and susceptibility profiling of P. aeruginosa laboratory strains expressing bla OXA-935 revealed that OXA-935 conferred ceftazidime resistance. To better understand the impacts of the variant amino acids, we determined the crystal structures of OXA-14 and OXA-935. Compared to OXA-14, the F153S mutation in OXA-935 conferred increased flexibility in the omega (Ω) loop. Amino acid changes that confer extended spectrum cephalosporinase activity to OXA-10-family β-lactamases are concerning, given the rising reliance on novel β-lactam/β-lactamase inhibitor combinations, such as ceftolozane-tazobactam and ceftazidime-avibactam, to treat MDR P. aeruginosa infections.

The infectious disease human monkeypox is spreading rapidly in 2022, causing a global health crisis. The genomics of Monkeypox virus (MPXV) have been extensively analyzed and reported, although little is known about the virus-encoded proteome. In particular, there are no reported experimental MPXV protein structures other than computational models. Here, a 1.52 Å resolution X-ray structure of the MPXV protein A42R, the first MPXV-encoded protein with a known structure, is reported. A42R shows structural similarity to profilins, which are cellular proteins that are known to function in the regulation of actin cytoskeletal assembly. However, structural comparison of A42R with known members of the profilin family reveals critical differences that support prior biochemical findings that A42R only weakly binds actin and does not bind poly(L-proline). In addition, the analysis suggests that A42R may make distinct interactions with phosphatidylinositol lipids. Overall, the data suggest that the role of A42R in the replication of orthopoxviruses may not be readily determined by comparison to cellular profilins. Furthermore, these findings support the need for increased efforts to determine high-resolution structures of other MPXV proteins to inform physiological studies of the poxvirus infection cycle and to reveal potential new strategies to combat human monkeypox should this emerging infectious disease with pandemic potential become more common in the future., (open access.)

On October 21-22, 2020 the HESI (Health and Environmental Sciences Institute) Protein Allergens, Toxins, and Bioinformatics Committee, and the Society of Toxicology Food Safety Specialty Section co-hosted a virtual workshop titled "From Protein Toxins to Applied Toxicological Testing". The workshop focused on the safety assessment of novel proteins contained in foods and feeds, was globally represented by over 200 stakeholder attendees, and featured contributions from experts in academia, government and non-government organizations, and agricultural biotechnology developers from the private sector. A range of topics relevant to novel protein safety were discussed, including: the state of protein toxin biology, modes and mechanisms of action, structures and activity, use of bioinformatic analyses to assess the safety of a protein, and ways to leverage computational biology with in silico approaches for protein toxin identification/characterization. Key outcomes of the workshop included the appreciation of the complexity of developing a definition for a protein toxin when viewed from the perspective of food and feed safety, confirming the need for a case-by-case hypothesis-driven interpretation of bioinformatic results that leverages additional metadata rather than an alignment threshold-driven interpretation, and agreement that a "toxin protein database" is not necessary, as the bioinformatic needs for toxin detection may be accomplished by existing databases such as Pfam and UniProtKB/Swiss-Prot. In this paper, a path forward is proposed., (Copyright © 2022. Published by Elsevier Inc.)

Objectives: Previously considered solely an opportunistic pathogen, Clostridium innocuum (CI) was recently reported in Taiwan to be an emerging cause of antibiotic-associated diarrhea and clinically indistinguishable from Clostridioides difficile (CD) infection. We previously identified CI culture supernatant being cross-reactive with commercial CD toxin enzyme immunoassays. We aimed to identify and characterize the cross-reacting protein and determine whether it functioned as a human toxin., Methods: We performed western blots using CI culture supernatants and CD anti-toxin antibodies and identified interacting bands. We identified protein(s) using tandem mass spectrometry and evaluated them by cytotoxicity assays., Results: CI, but not CD, was isolated from stool of 12 children and adults with diarrhea. Culture supernatant from 6/12 CI isolates, and an ATCC reference strain, tested positive for CD toxins (total 7/13 isolates) by commercial EIA. Using two of these isolates, we identified two ∼40 kDa hypothetical proteins, CI_01447 and CI_01448, and confirmed cross-reactivity with CD anti-toxin antibodies by enzyme immunoassay and Western blot. Whole-genome sequencing confirmed all 13 isolates contained both genes, which were highly conserved. We observed no cytopathic or cytotoxic effects to HeLa cells when treated with these proteins. We identified amino acid sequence similarity to the NlpC/P60 family of proteins., Conclusions: Our findings do not suggest CI proteins CI_01448 and CI_01447, which cross-react with antibodies against CD toxins A and B, are toxic to HeLa cells. Further studies are needed to determine the function of these cross-reacting proteins and the potential virulence factors that could be responsible for CI diarrheal disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Alpha-pore-forming toxins (α-PFTs) are secreted by many species of bacteria, including Escherichia coli, Aeromonas hydrophila, and Bacillus thuringiensis, as part of their arsenal of virulence factors, and are often cytotoxic. In particular, for α-PFTs, the membrane-spanning channel they form is composed of hydrophobic α-helices. These toxins oligomerize at the surface of target cells and transition from a soluble to a protomer state in which they expose their hydrophobic regions and insert into the membrane to form a pore. The pores may be composed of homooligomers of one component or heterooligomers with two or three components, resulting in bi- or tripartite toxins. The multicomponent α-PFTs are often expressed from a single operon. Recently, motility-associated killing factor A (MakA), an α-PFT, was discovered in Vibrio cholerae. We report that makA is found on the V. cholerae GI-10 genomic island within an operon containing genes for two other potential α-PFTs, MakB and MakE. We determined the X-ray crystal structures for MakA, MakB, and MakE and demonstrated that all three are structurally related to the α-PFT family in the soluble state, and we modeled their protomer state based on the α-PFT AhlB from A. hydrophila. We found that MakA alone is cytotoxic at micromolar concentrations. However, combining MakA with MakB and MakE is cytotoxic at nanomolar concentrations, with specificity for J774 macrophage cells. Our data suggest that MakA, -B, and -E are α-PFTs that potentially act as a tripartite pore-forming toxin with specificity for phagocytic cells. IMPORTANCE The bacterium Vibrio cholerae causes gastrointestinal, wound, and skin infections. The motility-associated killing factor A (MakA) was recently shown to be cytotoxic against colon, prostate, and other cancer cells. However, at the outset of this study, the capacity of MakA to damage cells in combination with other Mak proteins encoded in the same operon had not been elucidated. We determined the structures of three Mak proteins and established that they are structurally related to the α-PFTs. Compared to MakA alone, the combination of all three toxins was more potent specifically in mouse macrophages. This study highlights the idea that the Mak toxins are selectively cytotoxic and thus may function as a tripartite toxin with cell type specificity.

The lack of effective RAS inhibition represents a major unmet medical need in the treatment of pancreatic ductal adenocarcinoma (PDAC). Here, we investigate the anticancer activity of RRSP-DTB, an engineered biologic that cleaves the Switch I of all RAS isoforms, in KRAS-mutant PDAC cell lines and patient-derived xenografts (PDX). We first demonstrate that RRSP-DTB effectively engages RAS and impacts downstream ERK signaling in multiple KRAS-mutant PDAC cell lines inhibiting cell proliferation at picomolar concentrations. We next tested RRSP-DTB in immunodeficient mice bearing KRAS-mutant PDAC PDXs. Treatment with RRSP-DTB led to ≥95% tumor regression after 29 days. Residual tumors exhibited disrupted tissue architecture, increased fibrosis and fewer proliferating cells compared with controls. Intratumoral levels of phospho-ERK were also significantly lower, indicating in vivo target engagement. Importantly, tumors that started to regrow without RRSP-DTB shrank when treatment resumed, demonstrating resistance to RRSP-DTB had not developed. Tracking persistence of the toxin activity following intraperitoneal injection showed that RRSP-DTB is active in sera from immunocompetent mice for at least 1 hour, but absent after 16 hours, justifying use of daily dosing. Overall, we report that RRSP-DTB strongly regresses hard-to-treat KRAS-mutant PDX models of pancreatic cancer, warranting further development of this pan-RAS biologic for the management of RAS-addicted tumors., (©2022 American Association for Cancer Research.)

Vibrio vulnificus is an opportunistic pathogen that causes gastroenteritis and septicemia in humans. The V. vulnificus multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin is a pore-forming toxin that translocates multiple functionally independent effector domains into target cells and an essential virulence factor for fatal disease. The effector repertoire delivered and thus the mechanism of action of the toxin can differ dramatically across V. vulnificus isolates. Here, we utilize a strain of V. vulnificus that carries an F-type MARTX toxin that delivers an actin cross-linking domain (ACD) and four other effector domains. We demonstrate that ACD is the primary driver of virulence following intragastric infection and of bacterial dissemination to distal organs. We additionally show that ACD activates the transcription of intermediate early response genes in cultured intestinal epithelial cells (IECs). However, the genes activated by ACD are suppressed, at least in part, by the codelivered Ras/Rap1-specific endopeptidase (RRSP). The transcriptional response induced by strains translocating only RRSP results in a unique transcriptional profile, demonstrating that the transcriptional response to V. vulnificus is remodeled rather than simply suppressed by the MARTX toxin effector repertoire. Regardless, the transcriptional response in the intestinal tissue of infected mice is dominated by ACD-mediated induction of genes associated with response to tissue damage and is not impacted by RRSP or the three other effectors codelivered with ACD and RRSP. These data demonstrate that while other effectors do remodel early intestinal innate immune responses, ACD is the dominant driver of disease progression by ACD + V. vulnificus during intestinal infection.

The host immune response is highly effective to detect and clear infecting bacterial pathogens. Given the elaborate surveillance systems of the host, it is evident that in order to productively infect a host, the bacteria often coordinate virulence factors to fine-tune the host response during infection. These coordinated events can include either suppressing or activating the signaling pathways that control the immune response and thereby promote bacterial colonization and infection. This review will cover the surveillance and signaling systems for detection of bacteria in the intestine and a sample of the toxins and effectors that have been characterized that cirumvent these signaling pathways. These factors that promote infection and disease progression have also been redirected as tools or therapeutics. Thus, these toxins are enemies deployed to enhance infection, but can also be redeployed as allies to enable research and protect against infection., Competing Interests: KS holds patents related to RRSP and its use to treat cancer. KS holds a significant interest in Situ Biosciences, which conduct work unrelated to this article. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Woida and Satchell.)

Background: While several demographic and clinical correlates of coronavirus disease 2019 (COVID-19) outcome have been identified, their relationship to virological and immunological parameters remains poorly defined., Methods: To address this, we performed longitudinal collection of nasopharyngeal swabs and blood samples from a cohort of 58 hospitalized adults with COVID-19. Samples were assessed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load, viral genotype, viral diversity, and antibody titer. Demographic and clinical information, including patient blood tests and several composite measures of disease severity, was extracted from electronic health records., Results: Several factors, including male sex, higher age, higher body mass index, higher 4C Mortality score, and elevated lactate dehydrogenase levels, were associated with intensive care unit admission. Of all measured parameters, only the retrospectively calculated median Deterioration Index score was significantly associated with death. While quantitative polymerase chain reaction cycle threshold (Ct) values and genotype of SARS-CoV-2 were not significantly associated with outcome, Ct value did correlate positively with C-reactive protein levels and negatively with D-dimer, lymphocyte count, and antibody titer. Intrahost viral genetic diversity remained constant through the disease course and resulted in changes in viral genotype in some participants., Conclusions: Ultimately, these results suggest that worse outcomes are driven by immune dysfunction rather than by viral load and that SARS-CoV-2 evolution in hospital settings is relatively constant over time., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The COVID-19 pandemic has clearly brought the healthcare systems worldwide to a breaking point, along with devastating socioeconomic consequences. The SARS-CoV-2 virus, which causes the disease, uses RNA capping to evade the human immune system. Nonstructural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small-molecule inhibitors of nsp14 methyltransferase (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S -adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC 50 value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC 50 value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure-activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.

Ras-specific proteases to degrade RAS within cancer cells are under active development as an innovative strategy to treat tumorigenesis. The naturally occurring biological toxin effector called RAS/RAP1-specific endopeptidase (RRSP) is known to cleave all RAS within a cell, including HRAS, KRAS, NRAS and mutant KRAS G13D. Yet, our understanding of the mechanisms by which RRSP drives growth inhibition are unknown. Here, we demonstrate, using isogenic mouse fibroblasts expressing a single isoform of RAS or mutant KRAS, that RRSP equally inactivates all isoforms of RAS as well as the major oncogenic KRAS mutants. To investigate how RAS processing might lead to varying outcomes in cell fate within cancer cells, we tested RRSP against four colorectal cancer cell lines with a range of cell fates. While cell lines highly susceptible to RRSP (HCT116 and SW1463) undergo apoptosis, RRSP treatment of GP5d and SW620 cells induces G1 cell cycle arrest. In some cell lines, growth effects were dictated by rescued expression of the tumor suppressor protein p27 (Kip1). The ability of RRSP to irreversibly inhibit cancer cell growth highlights the antitumor potential of RRSP, and further warrants investigation as a potential anti-tumor therapeutic., (© 2021. The Author(s).)

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'- O -methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S -adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn 2+ , and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'- O -methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'- O -methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

VxrA and VxrB are cognate histidine kinase (HK) - response regulator (RR) pairs of a two-component signaling system (TCS) found in Vibrio cholerae , a bacterial pathogen that causes cholera. The VxrAB TCS positively regulates virulence, the Type VI Secretion System, biofilm formation, and cell wall homeostasis in V. cholerae , providing protection from environmental stresses and contributing to the transmission and virulence of the pathogen. The VxrA HK has a unique periplasmic sensor domain (SD) and, remarkably, lacks a cytoplasmic linker domain between the second transmembrane helix and the dimerization and histidine phosphotransfer (DHp) domain, indicating that this system may utilize a potentially unique signal sensing and transmission TCS mechanism. In this study, we have determined several crystal structures of VxrA-SD and its mutants. These structures reveal a novel structural fold forming an unusual β hairpin-swapped dimer. A conformational change caused by relative rotation of the two monomers in a VxrA-SD dimer could potentially change the association of transmembrane helices and, subsequently, the pairing of cytoplasmic DHp domains. Based on the structural observation, we propose a putative scissor-like closing regulation mechanism for the VxrA HK. IMPORTANCE V. cholerae has a dynamic life cycle, which requires rapid adaptation to changing external conditions. Two-component signal transduction (TCS) systems allow V. cholerae to sense and respond to these environmental changes. The VxrAB TCS positively regulates a number of important V. cholerae phenotypes, including virulence, the Type Six Secretion System, biofilm formation, and cell wall homeostasis. Here, we provide the crystal structure of the VxrA sensor histidine kinase sensing domain and propose a mechanism for signal transduction. The cognate signal for VxrAB remains unknown, however, in this work we couple our structural analysis with functional assessments of key residues to further our understanding of this important TCS., (Copyright © 2021 American Society for Microbiology.)

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog ( m7 GpppA m2'-O ). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae , the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a β-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs. IMPORTANCE Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae , the transcription factor ChiS directly regulates gene expression through a cryptic DNA-binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA-binding domain (DBD) and found that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators., (Copyright © 2021 Klancher et al.)

Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Pathogenic Vibrio species use many different approaches to subvert, attack, and undermine the host response. The toxins they produce are often responsible for the devastating effects associated with their diseases. These toxins target a variety of host proteins, which leads to deleterious effects, including dissolution of cell organelle integrity and inhibition of protein secretion. Becoming increasingly prevalent as cofactors for Vibrio toxins are proteins of the small GTPase families. ADP-ribosylation factor small GTPases (ARFs) in particular are emerging as a common host cofactor necessary for full activation of Vibrio toxins. While ARFs are not the direct target of Vibrio cholerae cholera toxin (CT), ARF binding is required for its optimal activity as an ADP-ribosyltransferase. The makes caterpillars floppy (MCF)-like and the domain X (DmX) effectors of the Vibrio vulnificus multifunctional autoprocessing repeats-in-toxin (MARTX) toxin also both require ARFs to initiate autoprocessing and activation as independent effectors. ARFs are ubiquitously expressed in eukaryotes and are key regulators of many cellular processes, and as such they are ideal cofactors for Vibrio pathogens that infect many host species. In this review, we cover in detail the known Vibrio toxins that use ARFs as cross-kingdom activators to both stimulate and optimize their activity. We further discuss how these contrast to toxins and effectors from other bacterial species that coactivate, stimulate, or directly modify host ARFs as their mechanisms of action., (Copyright © 2020 American Society for Microbiology.)

The nontoxic, anthrax protective antigen/lethal factor N-terminal domain (PA/LF N ) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LF N to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single-chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer-relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA-scFv fusions enable cell-specific delivery of diphtheria toxin catalytic domain and Ras/Rap1-specific endopeptidase to pancreatic cancer cells. Importantly, mPA-scFv fusion-based treatments display potent cell-specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'- O -methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S -adenosylmethionine (SAM), the reaction product S -adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m 7 GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m 7 GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers., Competing Interests: Competing interest statement: M.B. and K.J.F.S. are authors of a pending patent on use of RRSP as a therapeutic for cancer (WO2016019379A1). K.J.F.S. is author of a published patent on use of cysteine protease domain for autoprocessing of proteins (US20100137563A1). R.A.M. and G.L.B. are authors of a published patent on the use of diphtheria toxin for protein delivery and of a pending patent on RRSP-DTB as a RAS-directed therapeutic (US20180080033A1 and WO2019104433A1, respectively). K.J.F.S. has a significant financial interest in Situ Biosciences LLC, which conducts contract research unrelated to this work.

Protein degradation by aminopeptidases is involved in bacterial responses to stress. Escherichia coli produces two metal-dependent M17 family leucine aminopeptidases (LAPs), aminopeptidase A (PepA) and aminopeptidase B (PepB). Several structures have been solved for PepA as well as other bacterial M17 peptidases. Herein, we report the first structures of a PepB M17 peptidase. The E. coli PepB protein structure was determined at a resolution of 2.05 and 2.6 Å. One structure has both Zn 2+ and Mn 2+ , while the second structure has two Zn 2+ ions bound to the active site. A 2.75 Å apo structure is also reported for PepB from Yersinia pestis. Both proteins form homohexamers, similar to the overall arrangement of PepA and other M17 peptidases. However, the divergent N-terminal domain in PepB is much larger resulting in a tertiary structure that is more expanded. Modeling of a dipeptide substrate into the C-terminal LAP domain reveals contacts that account for PepB to uniquely cleave after aspartate., (© 2020 The Protein Society.)

Intimate interactions between the armament of pathogens and their host dictate tissue and host susceptibility to infection also forging specific pathophysiological outcomes. Studying these interactions at the molecular level has provided an invaluable source of knowledge on cellular processes, as ambitioned by the Cellular Microbiology discipline when it emerged in early 90s. Bacterial toxins act on key cell regulators or membranes to produce major diseases and therefore constitute a remarkable toolbox for dissecting basic biological processes. Here, we review selected examples of recent studies on bacterial toxins illustrating how fruitful the discipline of cellular microbiology is in shaping our understanding of eukaryote processes. This ever-renewing discipline unveils new virulence factor biochemical activities shared by eukaryotic enzymes and hidden rules of cell proteome homeostasis, a particularly promising field to interrogate the impact of proteostasis breaching in late onset human diseases. It is integrating new concepts from the physics of soft matter to capture biomechanical determinants forging cells and tissues architecture. The success of this discipline is also grounded by the development of therapeutic tools and new strategies to treat both infectious and noncommunicable human diseases., (© 2020 John Wiley & Sons Ltd.)

Emerging evidence suggests the Pseudomonas aeruginosa accessory genome is enriched with uncharacterized virulence genes. Identification and characterization of such genes may reveal novel pathogenic mechanisms used by particularly virulent isolates. Here, we utilized a mouse bacteremia model to quantify the virulence of 100 individual P. aeruginosa bloodstream isolates and performed whole-genome sequencing to identify accessory genomic elements correlated with increased bacterial virulence. From this work, we identified a specific contact-dependent growth inhibition (CDI) system enriched among highly virulent P. aeruginosa isolates. CDI systems contain a large exoprotein (CdiA) with a C-terminal toxin (CT) domain that can vary between different isolates within a species. Prior work has revealed that delivery of a CdiA-CT domain upon direct cell-to-cell contact can inhibit replication of a susceptible target bacterium. Aside from mediating interbacterial competition, we observed our virulence-associated CdiA-CT domain to promote toxicity against mammalian cells in culture and lethality during mouse bacteremia. Structural and functional studies revealed this CdiA-CT domain to have in vitro tRNase activity, and mutations that abrogated this tRNAse activity in vitro also attenuated virulence. Furthermore, CdiA contributed to virulence in mice even in the absence of contact-dependent signaling. Overall, our findings indicate that this P. aeruginosa CDI system functions as both an interbacterial inhibition system and a bacterial virulence factor against a mammalian host. These findings provide an impetus for continued studies into the complex role of CDI systems in P. aeruginosa pathogenesis., Competing Interests: Competing interest statement: A.R.H. serves on an advisory board and as a consultant for Microbiotix, Inc. to develop type III secretion inhibitors. K.J.F.S. has a significant financial interest in Situ Biosciences, which conducts research unrelated to this work.

Galactarate dehydratase (GarD) is the first enzyme in the galactarate/glucarate pathway and catalyzes the dehydration of galactarate to 3-keto-5-dehydroxygalactarate. This protein is known to increase colonization fitness of intestinal pathogens in antibiotic-treated mice and to promote bacterial survival during stress. The galactarate/glucarate pathway is widespread in bacteria, but not in humans, and thus could be a target to develop new inhibitors for use in combination therapy to combat antibiotic resistance. The structure of almost all the enzymes of the galactarate/glucarate pathway were solved previously, except for GarD, for which only the structure of the N-terminal domain was determined previously. Herein, we report the first crystal structure of full-length GarD solved using a seleno-methoionine derivative revealing a new protein fold. The protein consists of three domains, each presenting a novel twist as compared to their distant homologs. GarD in the crystal structure forms dimers and each monomer consists of three domains. The N-terminal domain is comprised of a β-clip fold, connected to the second domain by a long unstructured linker. The second domain serves as a dimerization interface between two monomers. The C-terminal domain forms an unusual variant of a Rossmann fold with a crossover and is built around a seven-stranded parallel β-sheet supported by nine α-helices. A metal binding site in the C-terminal domain is occupied by Ca 2+ . The activity of GarD was corroborated by the production of 5-keto-4-deoxy-D-glucarate under reducing conditions and in the presence of iron. Thus, GarD is an unusual enolase with a novel protein fold never previously seen in this class of enzymes., (© 2019 The Protein Society.)

Studies have successfully elucidated the mechanism of action of several effector domains that comprise the multifunctional-autoprocessing repeats-in-toxins (MARTX) toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy (MCF)-like effector and its cellular effects remains unknown. In this study, we identify the host cell factors that activate in vivo and in vitro MCF autoprocessing as adenosine diphosphate (ADP)-Ribosylation Factor 1 (ARF1) and ADP-Ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active [guanosine triphosphate (GTP)-bound] form compared to the inactive [guanosine diphosphate (GDP)-bound] form. Subsequent to auto-cleavage, MCF is acetylated on its exposed N-terminal glycine residue. Acetylation apparently does not dictate subcellular localization as MCF is found localized throughout the cell. However, the cleaved form of MCF gains the ability to bind to the specialized lipid phosphatidylinositol 5-phosphate enriched in Golgi and other membranes necessary for endocytic trafficking, suggesting that a fraction of MCF may be subcellularly localized. Traditional thin-section electron microscopy, high-resolution cryoAPEX localization, and fluorescent microscopy show that MCF causes Golgi dispersal resulting in extensive vesiculation. In addition, host mitochondria are disrupted and fragmented. Mass spectrometry analysis found no reproducible modifications of ARF1 suggesting that ARF1 is not post-translationally modified by MCF. Further, catalytically active MCF does not stably associate with ARF1. Our data indicate not only that ARF1 is a cross-kingdom activator of MCF, but also that MCF may mediate cytotoxicity by directly targeting another yet to be identified protein. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression., (© 2019 John Wiley & Sons Ltd.)

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are pore-forming bacterial toxins that translocate multiple functionally independent effector domains into a target eukaryotic cell. Vibrio cholerae colonizes intestinal epithelial cells (IECs) and uses a MARTX toxin with three effector domains-an actin cross-linking domain (ACD), a Rho inactivation domain (RID), and an α/β hydrolase domain (ABH)-to suppress innate immunity and enhance colonization. We investigated whether these multiple catalytic enzymes delivered from a single toxin functioned in a coordinated manner to suppress intestinal innate immunity. Using cultured human IECs, we demonstrated that ACD-induced cytoskeletal collapse activated extracellular signal-regulated kinase, p38, and c-Jun amino-terminal kinase mitogen-activated protein kinase (MAPK) signaling to elicit a robust proinflammatory response characterized by the secretion of interleukin-8 (IL-8; also called CXCL8) and the expression of CXCL8 , tumor necrosis factor ( TNF ), and other proinflammatory genes. However, RID and ABH, which are naturally delivered together with ACD, blocked MAPK activation through Rac1 and thus prevented ACD-induced inflammation. RID also abolished IL-8 secretion induced by heat-killed bacteria, TNF, or latrunculin A. Thus, MARTX toxins use enzymatic multifunctionality to silence the host response to bacterial factors and to the damage caused by the toxins. Furthermore, these data show how V. cholerae MARTX toxin suppresses intestinal inflammation and contributes to cholera being classically defined as a noninflammatory diarrheal disease., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Protein purification is the most basic and critical step for protein biophysical and biochemical studies to understand its function and structure. Various fusion tags and proteases have been developed and assembled in expression and purification system. However, it is one of the fields that continues to innovate to develop improved systems that are more efficient, simpler, and less expensive. An efficient self-cleavage C-terminal fusion system was developed using the inositol hexakisphosphate-inducible Vibrio cholerae MARTX Vc toxin cysteine protease domain (CPD). CPD fusion proteins are expressed from the T7 promoter and purified using a 6xHis-tag with immobilized-metal affinity chromatography. The C-terminal CPD-tag is removed by self-cleavage at the final purification stage. Here, we describe an efficient cloning method using Gibson assembly, followed by expression and purification of tagless recombinant proteins of interest using CPD self-cleavage.

Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δ crp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis -infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection. IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease., (Copyright © 2019 Ritzert et al.)

The crystal structure is reported of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida, a possible drug target to combat tetracycline resistance, in complex with flavin adenine dinucleotide (FAD). The structure was refined at 2.2 Å resolution with four polypeptide chains in the asymmetric unit. Based on the results of pairwise structure alignments, PobA from P. putida is structurally very similar to PobA from P. fluorescens and from P. aeruginosa. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. Additionally, the structure was compared with two enzymes from the broader class of oxygenases: 2-hydroxybiphenyl 3-monooxygenase (HbpA) from P. nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) from Mesorhizobium japonicum. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from P. putida with HbpA from P. nitroreducens and MHPCO from M. japonicum revealed local similarities between these structures. Key secondary-structure elements important for catalysis, such as the βαβ fold, β-sheet wall and α12 helix, are conserved across this expanded class of oxygenases.

Manganese superoxide dismutase (MnSOD) functions as a tumor suppressor; however, once tumorigenesis occurs, clinical data suggest MnSOD levels correlate with more aggressive human tumors, implying a potential dual function of MnSOD in the regulation of metabolism. Here we show, using in vitro transformation and xenograft growth assays that the MnSOD-K68 acetylation (Ac) mimic mutant (MnSOD K68Q ) functions as a tumor promoter. Interestingly, in various breast cancer and primary cell types the expression of MnSOD K68Q is accompanied with a change of MnSOD's stoichiometry from a known homotetramer complex to a monomeric form. Biochemical experiments using the MnSOD-K68Q Ac-mimic, or physically K68-Ac (MnSOD-K68-Ac), suggest that these monomers function as a peroxidase, distinct from the established MnSOD superoxide dismutase activity. MnSOD K68Q expressing cells exhibit resistance to tamoxifen (Tam) and cells selected for Tam resistance exhibited increased K68-Ac and monomeric MnSOD. These results suggest a MnSOD-K68-Ac metabolic pathway for Tam resistance, carcinogenesis and tumor progression.