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Chronic pancreatitis (CP) is an etiologically and genetically heterogeneous inflammatory syndrome characterised by progressive damage to the exocrine and endocrine components of the pancreas [ 1]. The multigenic paradigm of CP has sparked research in recent years [ 2]. We aimed to expand the current knowledge of genetic susceptibility of pancreatitis in patients of Indian origin. By employing whole-exome sequencing in an Indian hospital cohort, we dissect the genetic landscape associated with CP or recurrent acute pancreatitis (RAP). Notably, all patients had at least one genetic variant identified in a pancreatitis-risk gene, and most had a co-occurrence of a second variant in an additional risk gene. Based on the presence of both acinar and ductal gene variants in individual patients, we propose a two-hit hypothesis where variants in proteins expressed in both acinar and ductal cells are critical for RAP/CP development., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Enterotoxigenic Escherichia coli (ETEC) strains, which produce the heat-stable enterotoxin (ST) either alone or in combination with the heat-labile enterotoxin, contribute to the bulk of the burden of child diarrheal disease in resource-limited countries and are associated with mortality. Developing an effective vaccine targeting ST presents challenges due to its potent enterotoxicity, non-immunogenicity, and the risk of autoimmune reaction stemming from its structural similarity to the human endogenous ligands, guanylin, and uroguanylin. This study aimed to assess a novel synthetic vaccine carrier platform employing a single chemical coupling step for making human ST (STh) immunogenic. Specifically, the method involved cross-linking STh to an 8-arm N-hydroxysuccinimide (NHS) ester-activated PEG cross-linker. A conjugate of STh with 8-arm structure was prepared, and its formation was confirmed through immunoblotting analysis. The impact of conjugation on STh epitopes was assessed using ELISAs with polyclonal and monoclonal antibodies targeting various epitopes of STh. Immunization of mice with the conjugate induced the production of anti-STh antibodies, exhibiting neutralizing activity against STh., (© 2024 The Author(s). Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

Although suppressed cAMP levels have been linked to cancer for nearly five decades, the molecular basis remains uncertain. Here, we identify endosomal pH as a novel regulator of cytosolic cAMP homeostasis and a promoter of transformed phenotypic traits in colorectal cancer. Combining experiments and computational analysis, we show that the Na+/H+ exchanger NHE9 contributes to proton leak and causes luminal alkalinization, which induces resting [Ca2+], and in consequence, represses cAMP levels, creating a feedback loop that echoes nutrient deprivation or hypoxia. Higher NHE9 expression in cancer epithelia is associated with a hybrid epithelial-mesenchymal (E/M) state, poor prognosis, tumor budding, and invasive growth in vitro and in vivo. These findings point to NHE9-mediated cAMP suppression as a pseudostarvation-induced invasion state and potential therapeutic vulnerability in colorectal cancer. Our observations lay the groundwork for future research into the complexities of endosome-driven metabolic reprogramming and phenotype switching and the biology of cancer progression., Implications: Endosomal pH regulator NHE9 actively controls cytosolic Ca2+ levels to downregulate the adenylate cyclase-cAMP system, enabling colorectal cancer cells to acquire hybrid E/M characteristics and promoting metastatic progression., (©2024 American Association for Cancer Research.)

Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways these bacteria utilize cAMP. We identified universal stress proteins, Rv1636 and MSMEG_3811 in Mycobacterium tuberculosis and Mycobacterium smegmatis, respectively, as abundantly expressed, novel cAMP-binding proteins. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. In slow-growing mycobacteria, intrabacterial concentrations of Rv1636 were equivalent to the concentrations of cAMP present in the cell. In contrast, levels of intrabacterial MSMEG_3811 in M. smegmatis were lower than that of cAMP and therefore, overexpression of Rv1636 increased levels of "bound" cAMP. While msmeg_3811 could be readily deleted from the genome of M. smegmatis, we found that the rv1636 gene is essential for the viability of M. tuberculosis and is dependent on the cAMP-binding ability of Rv1636. Therefore, Rv1636 may function to regulate cAMP signaling by direct sequestration of the second messenger. This is the first evidence of a "sponge" for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intrabacterial "free" pool of cAMP., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in receptor guanylyl cyclase C (GC-C) cause severe gastrointestinal disease, including meconium ileus, early onset acute diarrhea, and pediatric inflammatory bowel disease that continues into adulthood. Agonists of GC-C are US Food and Drug Administration-approved drugs for the treatment of constipation and irritable bowel syndrome. Therapeutic strategies targeting GC-C are tested in preclinical mouse models, assuming that murine GC-C mimics human GC-C in its biochemical properties and downstream signaling events. Here, we reveal important differences in ligand-binding affinity and GC activity between mouse GC-C and human GC-C. We generated a series of chimeric constructs of various domains of human and mouse GC-C to show that the extracellular domain of mouse GC-C contributed to log-orders lower affinity of mouse GC-C for ligands than human GC-C. Further, the Vmax of the murine GC domain was lower than that of human GC-C, and allosteric regulation of the receptor by ATP binding to the intracellular kinase-homology domain also differed. These altered properties are reflected in the high concentrations of ligands required to elicit signaling responses in the mouse gut in preclinical models and the specificity of a GC inhibitor towards human GC-C. Therefore, our studies identify considerations in using the murine model to test molecules for therapeutic purposes that work as either agonists or antagonists of GC-C, and vaccines for the bacterial heat-stable enterotoxin that causes watery diarrhea in humans., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Evolutionarily conserved genes often play critical roles in organismal physiology. Here, we describe multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. We observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2023 Gupta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bacterial infections of the gut are one of the major causes of morbidity and mortality worldwide. The interplay between the pathogen and the host is finely balanced, with the bacteria evolving to proliferate and establish infection. In contrast, the host mounts a response to first restrict and then eliminate the infection. The intestine is a rapidly proliferating tissue, and metabolism is tuned to cater to the demands of proliferation and differentiation along the crypt-villus axis (CVA) in the gut. As bacterial pathogens encounter the intestinal epithelium, they elicit changes in the host cell, and core metabolic pathways such as the tricarboxylic acid (TCA) cycle, lipid metabolism and glycolysis are affected. This review highlights the mechanisms utilized by diverse gut bacterial pathogens to subvert host metabolism and describes host responses to the infection., (© 2022 Federation of European Biochemical Societies.)

Introduction: Hereditary pancreatitis (HP) is a rare debilitating disease with incompletely understood etio-pathophysiology. The reduced penetrance of genes such as PRSS1 associated with hereditary pancreatitis indicates a role for novel inherited factors. Methods: We performed whole-exome sequencing of three affected members of an Indian family (Father, Son, and Daughter) with chronic pancreatitis and compared variants with those seen in the unaffected mother. Results: We identified a novel frameshift mutation in exon 11 of TRPV6 (c.1474_1475delGT; p.V492Tfs*136), a calcium channel, in the patients. Functional characterization of this mutant TRPV6 following heterologous expression revealed that it was defective in calcium uptake. Induction of pancreatitis in mice induced Trpv6 expression, indicating that higher expression levels of the mutant protein and consequent dysregulation of calcium levels in patients with chronic pancreatitis could aggravate the disease. Discussion: We report a novel frameshift mutation in TRPV6 in an Indian family with HP that renders the mutant protein inactive. Our results emphasize the need to expand the list of genes used currently for evaluating patients with hereditary pancreatitis., Competing Interests: The authors declare 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 © 2023 Shah, Prasad, Banerjee, Kurien, Chowdhury and Visweswariah.)

Three-dimensional (3D) printing techniques for scaffold fabrication have shown promising advancements in recent years owing to the ability of the latest high-performance printers to mimic the native tissue down to submicron scales. Nevertheless, host integration and performance of scaffolds in vivo have been severely limited owing to the lack of robust strategies to promote vascularization in 3D printed scaffolds. As a result, researchers over the past decade have been exploring strategies that can promote vascularization in 3D printed scaffolds toward enhancing scaffold functionality and ensuring host integration. Various emerging strategies to enhance vascularization in 3D printed scaffolds are discussed. These approaches include simple strategies such as the enhancement of vascular in-growth from the host upon implantation by scaffold modifications to complex approaches wherein scaffolds are fabricated with their own vasculature that can be directly anastomosed or microsurgically connected to the host vasculature, thereby ensuring optimal integration. The key differences among the techniques, their pros and cons, and the future opportunities for utilizing each technique are highlighted here. The Review concludes with the current limitations and future directions that can help 3D printing emerge as an effective biofabrication technique to realize tissues with physiologically relevant vasculatures to ultimately accelerate clinical translation.

Receptor Guanylyl Cyclase C (GC-C) was initially characterized as an important regulator of intestinal fluid and ion homeostasis. Recent findings demonstrate that GC-C is also causally linked to intestinal inflammation, dysbiosis, and tumorigenesis. These advances have been fueled in part by identifying mutations or changes in gene expression in GC-C or its ligands, that disrupt the delicate balance of intracellular cGMP levels and are associated with a wide range of clinical phenotypes. In this review, we highlight aspects of the current knowledge of the GC-C signaling pathway in homeostasis and disease, emphasizing recent advances in the field. The review summarizes extra gastrointestinal functions for GC-C signaling, such as appetite control, energy expenditure, visceral nociception, and behavioral processes. Recent research has expanded the homeostatic role of GC-C and implicated it in regulating the ion-microbiome-immune axis, which acts as a mechanistic driver in inflammatory bowel disease. The development of transgenic and knockout mouse models allowed for in-depth studies of GC-C and its relationship to whole-animal physiology. A deeper understanding of the various aspects of GC-C biology and their relationships with pathologies such as inflammatory bowel disease, colorectal cancer, and obesity can be leveraged to devise novel therapeutics., Competing Interests: The authors declare 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 Prasad, Mathew and Visweswariah.)

Humans are exposed to numerous synthetic foreign particles in the form of drug delivery systems and diagnostic agents. Specialized immune cells (phagocytes) clear these particles by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particles may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize Escherichia coli (E. coli) faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and Staphylococcus epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable abiotic and biotic material., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cyclic GMP is produced by enzymes called guanylyl cyclases, of which the membrane-associated forms contain an intracellular pseudokinase domain that allosterically regulates the C-terminal guanylyl cyclase domain. Ligand binding to the extracellular domain of these single transmembrane-spanning domain receptors elicits an increase in cGMP levels in the cell. The pseudokinase domain (or kinase-homology domain) in these receptors appears to be critical for ligand-mediated activation. While the pseudokinase domain does not possess kinase activity, biochemical evidence indicates that the domain can bind ATP and thereby allosterically regulate the catalytic activity of these receptors. The pseudokinase domain also appears to be the site of interaction of regulatory proteins, as seen in the retinal guanylyl cyclases that are involved in visual signal transduction. In the absence of structural information on the pseudokinase-guanylyl cyclase domain organization of any member of this family of receptors, biochemical evidence has provided clues to the physical interaction of the pseudokinase and guanylyl cyclase domain. An α-helical linker region between the pseudokinase domain and the guanylyl cyclase domain regulates the basal activity of these receptors in the absence of a stimulatory ligand and is important for stabilizing the structure of the pseudokinase domain that can bind ATP. Here, we present an overview of salient features of ATP-mediated regulation of receptor guanylyl cyclases and describe biochemical approaches that allow a clearer understanding of the intricate interplay between the pseudokinase domain and catalytic domain in these proteins., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Activating mutations in receptor guanylyl cyclase C (GC-C), the target of gastrointestinal peptide hormones guanylin and uroguanylin, and bacterial heat-stable enterotoxins cause early-onset diarrhea and chronic inflammatory bowel disease (IBD). GC-C regulates ion and fluid secretion in the gut via cGMP production and activation of cGMP-dependent protein kinase II. We characterize a novel mouse model harboring an activating mutation in Gucy2c equivalent to that seen in an affected Norwegian family. Mutant mice demonstrated elevated intestinal cGMP levels and enhanced fecal water and sodium content. Basal and linaclotide-mediated small intestinal transit was higher in mutant mice, and they were more susceptible to DSS-induced colitis. Fecal microbiome and gene expression analyses of colonic tissue revealed dysbiosis, up-regulation of IFN-stimulated genes, and misregulation of genes associated with human IBD and animal models of colitis. This novel mouse model thus provides molecular insights into the multiple roles of intestinal epithelial cell cGMP, which culminate in dysbiosis and the induction of inflammation in the gut., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Mishra et al.)

Millions of deaths caused by Mycobacterium tuberculosis ( Mtb ) are reported worldwide every year. Treatment of tuberculosis (TB) involves the use of multiple antibiotics over a prolonged period. However, the emergence of resistance leading to multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) is the most challenging aspect of TB treatment. Therefore, there is a constant need to search for novel therapeutic strategies that could tackle the growing problem of drug resistance. One such strategy could be perturbing the functions of novel targets in Mtb , such as universal stress protein (USP, Rv1636), which binds to cAMP with a higher affinity than ATP. Orthologs of these proteins are conserved in all mycobacteria and act as "sink" for cAMP, facilitating the availability of this second messenger for signaling when required. Here, we have used the cAMP-bound crystal structure of USP from Mycobacterium smegmatis , a closely related homolog of Mtb , to conduct a structure-guided hunt for potential binders of Rv1636, primarily employing molecular docking approach. A library of 1.9 million compounds was subjected to virtual screening to obtain an initial set of ~2,000 hits. An integrative strategy that uses the available experimental data and consensus indications from other computational analyses has been employed to prioritize 22 potential binders of Rv1636 for experimental validations. Binding affinities of a few compounds among the 22 prioritized compounds were tested through microscale thermophoresis assays, and two compounds of natural origin showed promising binding affinities with Rv1636. We believe that this study provides an important initial guidance to medicinal chemists and biochemists to synthesize and test an enriched set of compounds that have the potential to inhibit Mtb USP (Rv1636), thereby aiding the development of novel antitubercular lead candidates., Competing Interests: The authors declare 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 © 2021 Chakraborti, Chakraborty, Bose, Srinivasan and Visweswariah.)

Mycobacteria harbor a unique class of adenylyl cyclases with a complex domain organization consisting of an N-terminal putative adenylyl cyclase domain fused to a nucleotide-binding adaptor shared by apoptotic protease-activating factor-1, plant resistance proteins, and CED-4 (NB-ARC) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal helix-turn-helix (HTH) domain. The products of the rv0891c-rv0890c genes represent a split gene pair, where Rv0891c has sequence similarity to adenylyl cyclases, and Rv0890c harbors the NB-ARC-TPR-HTH domains. Rv0891c had very low adenylyl cyclase activity so it could represent a pseudoenzyme. By analyzing the genomic locus, we could express and purify Rv0890c and find that the NB-ARC domain binds ATP and ADP, but does not hydrolyze these nucleotides. Using systematic evolution of ligands by exponential enrichment (SELEX), we identified DNA sequences that bound to the HTH domain of Rv0890c. Uniquely, the HTH domain could also bind RNA. Atomic force microscopy revealed that binding of Rv0890c to DNA was sequence independent, and binding of adenine nucleotides to the protein induced the formation of higher order structures that may represent biocrystalline nucleoids. This represents the first characterization of this group of proteins and their unusual biochemical properties warrant further studies into their physiological roles in future., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Pore forming toxins (PFTs) are the largest class of bacterial toxins playing a central role in bacterial pathogenesis. They are proteins specifically designed to form nanochannels in the membranes of target cells, ultimately resulting in cell death and establishing infection. PFTs are broadly classified as α- and β-PFTs, depending on secondary structures that form the transmembrane channel. A unique feature about this class of proteins is the drastic conformational changes and complex oligomerization pathways that occur upon exposure to the plasma membrane. A molecular understanding of pore formation has implications in designing novel intervention strategies to combat rising antimicrobial resistance, targeted-cancer therapy, as well as designing nanopores for specialized technologies. Central to unraveling the pore formation pathway is the availability of high resolution crystal structures. In this regard, β-toxins are better understood, when compared with α-toxins whose pore forming mechanisms are complicated by an incomplete knowledge of the driving forces for amphiphatic membrane-inserted helices to organize into functional pores. With the publication of the first crystal structure for an α-toxin, cytolysin A (ClyA), in 2009 we embarked on an extensive multiscale study to unravel its pore forming mechanism. This Account represents the collective mechanistic knowledge gained in our laboratories using a variety of experimental and theoretical techniques which include large scale molecular dynamics (MD) simulations, kinetic modeling studies, single-molecule fluorescence imaging, and super-resolution spectroscopy. We reported MD simulations of the ClyA protomer, oligomeric intermediates, and full pore complex in a lipid bilayer and mapped the conformational transitions that accompany membrane binding. Using single-molecule fluorescence imaging, the conformational transition was experimentally verified by analysis of various diffusion states of membrane bound ClyA. Importantly, we have uncovered a hitherto unknown putative cholesterol binding motif in the membrane-inserted helix of ClyA. Distinct binding pockets for cholesterol formed by adjacent membrane-inserted helices are revealed in MD simulations. Cholesterol appears to play a dual role by stabilizing both the membrane-inserted protomer as well as oligomeric intermediates. Molecular dynamics simulations and kinetic modeling studies suggest that the membrane-inserted arcs oligomerize reversibly to form the predominant transmembrane oligomeric intermediates during pore formation. We posit that this mechanistic understanding of the complex action of α-PFTs has implications in unraveling pore assembly across the wider family of bacterial toxins. With emerging antimicrobial resistance, alternate therapies may rely on disrupting pore functionality or oligomerization of these pathogenic determinants utilized by bacteria, and our study includes assessing the potential for dendrimers as pore blockers.

Although impaired intestinal sodium transport has been described for decades as a ubiquitous feature of inflammatory bowel disease (IBD), whether and how it plays a pivotal role in the ailment has remained uncertain. Our identification of dominant mutations in receptor guanylyl cyclase 2C as a cause of IBD-associated familial diarrhea syndrome brought a shift in the way we envision impaired sodium transport. Is this just a passive collateral effect resulting from intestinal inflammation, or is it a crucial regulator of IBD pathogenesis? This review summarizes the mutational spectrum and underlying mechanisms of monogenic IBD associated with congenital sodium diarrhea. We constructed a model proposing that impaired sodium transport is an upstream pathogenic factor in IBD. The review also synthesized emerging insights from microbiome and animal studies to suggest how sodium malabsorption can serve as a unifying mediator of downstream pathophysiology. Further investigations into the mechanisms underlying salt and water transport in the intestine will provide newer approaches for understanding the ion-microbiome-immune cross-talk that serves as a driver of IBD. Model systems, such as patient-derived enteroids or induced pluripotent stem cell models, are warranted to unravel the role of individual genes regulating sodium transport and to develop more effective epithelial rescue and repair therapies., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tissue injury is one of the most severe environmental perturbations for a living organism. When damage occurs in adult Drosophila, there is a local response of the injured tissue and a coordinated action across different tissues to help the organism overcome the deleterious effect of an injury. We show a change in the transcriptome of hemocytes at the site of tissue injury, with pronounced activation of the Toll signaling pathway. We find that induction of the cytokine upd-3 and Toll receptor activation occur in response to injury alone, in the absence of a pathogen. Intracellular accumulation of hydrogen peroxide in hemocytes is essential for upd-3 induction and is facilitated by the diffusion of hydrogen peroxide through a channel protein Prip. Importantly, hemocyte activation and production of reactive oxygen species (ROS) at the site of a sterile injury provide protection to flies on subsequent infection, demonstrating training of the innate immune system., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Guanylyl cyclase C (GC-C) is the receptor for the heat-stable enterotoxin, which causes diarrhea, and the endogenous ligands, guanylin and uroguanylin. GC-C is predominantly expressed in the intestinal epithelium and regulates fluid and ion secretion in the gut. The receptor has a complex domain organization, and in the absence of structural information, mutational analysis provides clues to mechanisms of regulation of this protein. Here, we review the mutational landscape of this receptor that reveals regulatory features critical for its activity. We also summarize the available information on mutations in GC-C that have been reported in humans and contribute to severe gastrointestinal abnormalities. Since GC-C is also expressed in extra-intestinal tissues, it is likely that mutations thus far reported in humans may also affect other organ systems, warranting a close observation of these patients in future., (© 2020 International Union of Biochemistry and Molecular Biology.)

Misregulation of gut function and homeostasis impinges on the overall well-being of the entire organism. Diarrheal disease is the second leading cause of death in children under 5 years of age, and globally, 1.7 billion cases of childhood diarrhea are reported every year. Accompanying diarrheal episodes are a number of secondary effects in gut physiology and structure, such as erosion of the mucosal barrier that lines the gut, facilitating further inflammation of the gut in response to the normal microbiome. Here, we focus on pathogenic bacteria-mediated diarrhea, emphasizing the role of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in driving signaling outputs that result in the secretion of water and ions from the epithelial cells of the gut. We also speculate on how this aberrant efflux and influx of ions could modulate inflammasome signaling, and therefore cell survival and maintenance of gut architecture and function., (© 2019 Federation of European Biochemical Societies.)

Universal stress proteins (USPs) are present in many bacteria, and their expression is enhanced under various environmental stresses. We have previously identified a USP in Mycobacterium smegmatis that is a product of the msmeg_4207 gene and is a substrate for a cAMP-regulated protein lysine acyltransferase (KATms; MSMEG_5458). Here, we explored the role of this USP (USP 4207 ) in M. smegmatis and found that its gene is present in an operon that also contains genes predicted to encode a putative tripartite tricarboxylate transporter (TTT). Transcription of the TTT-usp 4207 operon was induced in the presence of citrate and tartrate, perhaps by the activity of a divergent histidine kinase-response regulator gene pair. A usp 4207 -deleted strain had rough colony morphology and reduced biofilm formation compared with the WT strain; however, both normal colony morphology and biofilm formation were restored in a Δ usp 4207 Δ katms strain. We identified several proteins whose acetylation was lost in the Δ katms strain, and whose transcript levels increased in M. smegmatis biofilms along with that of USP 4207 , suggesting that USP 4207 insulates KATms from its other substrates in the cell. We propose that USP 4207 sequesters KATms from diverse substrates whose activities are down-regulated by acylation but are required for biofilm formation, thus providing a defined role for this USP in mycobacterial physiology and stress responses., (© 2020 Samanta et al.)

The availability of genome sequence information and a large number of protein structures has allowed the cataloging of genes into various families, based on their function and predicted biochemical activity. Intriguingly, a number of proteins harbor changes in the amino acid sequence at residues, that from structural elucidation, are critical for catalytic activity. Such proteins have been categorized as 'pseudoenzymes'. Here, we review the role of the pseudokinase (or kinase-homology) domain in receptor guanylyl cyclases. These are multidomain single-pass, transmembrane proteins harboring an extracellular ligand-binding domain, and an intracellular domain composed of a kinase-homology domain that regulates the activity of the associated guanylyl cyclase domain. Mutations that lie in the kinase-homology domain of these receptors are associated with human disease, and either abolish or enhance cGMP production by these receptors to alter downstream signaling events. This raises the interesting possibility that one could identify molecules that bind to the pseudokinase domain and regulate the activities of these receptors, in order to alleviate symptoms in patients harboring these mutations., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Pore-forming toxins (PFTs) form nanoscale pores across target membranes causing cell death. Cytolysin A (ClyA) from Escherichia coli is a prototypical α-helical toxin that contributes to cytolytic phenotype of several pathogenic strains. It is produced as a monomer and, upon membrane exposure, undergoes conformational changes and finally oligomerizes to form a dodecameric pore, thereby causing ion imbalance and finally cell death. However, our current understanding of this assembly process is limited to studies in detergents, which do not capture the physicochemical properties of biological membranes. Here, using single-molecule imaging and molecular dynamics simulations, we study the ClyA assembly pathway on phospholipid bilayers. We report that cholesterol stimulates pore formation, not by enhancing initial ClyA binding to the membrane but by selectively stabilizing a protomer-like conformation. This was mediated by specific interactions by cholesterol-interacting residues in the N-terminal helix. Additionally, cholesterol stabilized the oligomeric structure using bridging interactions in the protomer-protomer interfaces, thereby resulting in enhanced ClyA oligomerization. This dual stabilization of distinct intermediates by cholesterol suggests a possible molecular mechanism by which ClyA achieves selective membrane rupture of eukaryotic cell membranes. Topological similarity to eukaryotic membrane proteins suggests evolution of a bacterial α-toxin to adopt eukaryotic motifs for its activation. Broad mechanistic correspondence between pore-forming toxins hints at a wider prevalence of similar protein membrane insertion mechanisms., Competing Interests: The authors declare no conflict of interest.

Nontyphoidal Salmonella disease contributes toward significant morbidity and mortality across the world. Host factors, including gamma interferon, tumor necrosis factor alpha, and gut microbiota, significantly influence the outcome of Salmonella pathogenesis. However, the entire repertoire of host protective mechanisms contributing to Salmonella pathogenicity is not completely appreciated. Here, we investigated the roles of receptor guanylyl cyclase C (GC-C), which is predominantly expressed in the intestine and regulates intestinal cell proliferation and fluid-ion homeostasis. Mice deficient in GC-C ( Gucy2c -/ - ) displayed accelerated mortality compared with that for wild-type mice following infection via the oral route, even though both groups possessed comparable systemic Salmonella infection burdens. Survival following intraperitoneal infection remained similar in both groups, indicating that GC-C offered protection via a gut-mediated response. The serum cortisol level was higher in Gucy2c -/- mice than wild-type ( Gucy2c +/+ ) mice, and an increase in infection-induced thymic atrophy with a loss of immature CD4 + CD8 + double-positive thymocytes was observed. Accelerated and enhanced damage in the ileum, including submucosal edema, epithelial cell damage, focal tufting, and distortion of the villus architecture, was seen in Gucy2c -/- mice concomitantly with a larger number of ileal tissue-associated bacteria. Transcription of key mediators of Salmonella -induced inflammation (interleukin-22/Reg3β) was altered in Gucy2c -/- mice in comparison to that in Gucy2c +/+ mice. A reduction in fecal lactobacilli, which are protective against Salmonella infection, was observed in Gucy2c -/- mice. Gucy2c -/- mice cohoused with wild-type mice continued to show reduced amounts of lactobacilli and increased susceptibility to infection. Our study, therefore, suggests that the receptor GC-C confers a survival advantage during gut-mediated Salmonella enterica serovar Typhimurium pathogenesis, presumably by regulating Salmonella effector mechanisms and maintaining a beneficial microbiome., (Copyright © 2018 American Society for Microbiology.)

The mycobacterial cell envelope is unique in its chemical composition, and has an important role to play in pathogenesis. Phthiocerol dimycocerosates (PDIMs) and glycosylated phenolphthiocerol dimycocerosates, also known as phenolic glycolipids (PGLs), contribute significantly to the virulence of Mycobacterium tuberculosis. FadD22 is essential for PGL biosynthesis. We have recently shown in vitro that FadD22 is a substrate for lysine acylation by a unique cAMP-dependent, protein lysine acyltransferase found only in mycobacteria. The lysine residue that is acylated is at the active site of FadD22. Therefore, acylation is likely to inhibit FadD22 activity and reduce PGL biosynthesis. Here, we show accumulation of PGLs in a strain of M. bovis BCG deleted for the gene encoding the cAMP-dependent acyltransferase, katbcg, with no change seen in PDIM synthesis. Complementation using KATbcg mutants that are deficient in cAMP-binding or acyltransferase activity shows that PGL accumulation is regulated by cAMP-dependent protein acylation in vivo. Expression of FadD22 and KATbcg mutants in Mycobacterium smegmatis confirmed that FadD22 is a substrate for lysine acylation by KATbcg. We have therefore described a mechanism by which cAMP can regulate mycobacterial virulence as a result of the ability of this second messenger to modulate critical cell wall components that affect the host immune response.

Cell membranes are believed to be highly complex dynamical systems having compositional heterogeneity involving several types of lipids and proteins as the major constituents. This dynamical and compositional heterogeneity is suggested to be critical to the maintenance of active functionality and response to chemical, mechanical, electrical and thermal stresses. However, delineating the various factors responsible for the spatio-temporal response of actual cell membranes to stresses can be quite challenging. In this work we show how biomimetic phospholipid bilayer membranes with variable lipid fluidity determine the optimal assembly mechanism of the pore-forming protein, listeriolysin O (LLO), belonging to the class of cholesterol dependent cytolysins (CDCs). By combining atomic force microscopy (AFM) and super-resolution stimulated emission depletion (STED) microscopy imaging on model membranes, we show that pores formed by LLO in supported lipid bilayers can have variable conformation and morphology depending on the fluidity of the bilayer. At a fixed cholesterol concentration, pores formed in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes were larger, flexible and more prone to coalescence when compared with the smaller and more compact pores formed in the lower fluidity 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. In contrast, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes did not show any evidence of pore formation. Fluorescence correlation spectroscopy (FCS) in STED mode revealed the appearance of a length scale, ξ, below which lipid dynamics, under the influence of LLO protein binding and assembly, becomes anomalous. Interestingly, the magnitude of ξ is found to correlate with both lipid fluidity and pore dimensions (and flexibility) in DOPC and POPC bilayers. However this length scale dependent crossover, signalling the onset of anomalous diffusion, was not observed in DMPC bilayers. Our study highlights the subtle interplay of lipid membrane mediated protein assembly and lipid fluidity in determining proteo-lipidic complexes formed in biomembranes and the significant insight that STED microscopy provides in unraveling critical aspects of nanoscale membrane biophysics.

Pore-forming toxins (PFTs) bind to cell membranes and form nanoscale pores that allow leakage of cellular components, resulting in cell death. The water-soluble, monomeric form of these toxins shows a dramatic conformational change during pore formation, as exemplified by crystal structures of the monomer and functional pore of cytolysin A (ClyA). The solvent-exposed, C-terminal residues of the protein are essential for activity, but the mechanism by which this region regulates pore formation remains unknown. We show here that deletion of the C-terminus of ClyA did not alter its ability to bind to the membrane or oligomerize in detergent. However, the truncated toxin lysed erythrocytes poorly, was more susceptible to proteolysis and thermal unfolding, and showed low calcein leakage from small unilamellar vesicles. Using fully atomistic molecular dynamics (MD) simulations, we find that deletion of C-terminal residues from the ClyA monomer significantly altered stability and unfolding trajectories in the transmembrane N-terminal helix, a region that is pivotal in maintaining the structural integrity of the helical bundle. MD simulations of pores with or without the C-terminus showed minor differences, implying that if oligomerization could be induced prior to the addition to vesicles, then an active pore could be generated. Via generation of oligomers in a detergent prior to the addition to vesicles, the truncated toxin could induce calcein leakage from vesicles, albeit to a lower extent. Therefore, regions of pore-forming toxins, not directly involved in the pore structure, are not passive players but have important roles in undergoing the transition through intermediary steps leading to successful pore formation in a membrane environment.

The two second messengers in signalling, cyclic AMP and cyclic GMP, are produced by adenylyl and guanylyl cyclases respectively. Recognition and discrimination of the substrates ATP and GTP by the nucleotidyl cyclases are vital in these reactions. Various apo-, substrate- or inhibitor-bound forms of adenylyl cyclase (AC) structures from transmembrane and soluble ACs have revealed the catalytic mechanism of ATP cyclization reaction. Previously reported structures of guanylyl cyclases represent ligand-free forms and inactive open states of the enzymes and thus do not provide information regarding the exact mode of substrate binding. The structures we present here of the cyclase homology domain of a class III AC from Mycobacterium avium (Ma1120) and its mutant in complex with ATP and GTP in the presence of calcium ion, provide the structural basis for substrate selection by the nucleotidyl cyclases at the atomic level. Precise nature of the enzyme-substrate interactions, novel modes of substrate binding and the ability of the binding pocket to accommodate diverse conformations of the substrates have been revealed by the present crystallographic analysis. This is the first report to provide structures of both the nucleotide substrates bound to a nucleotidyl cyclase., Database: Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 5D15 (Ma1120 CHD +ATP.Ca 2+ ), 5D0E (Ma1120 CHD +GTP.Ca 2+ ), 5D0H (Ma1120 CHD (KDA→EGY)+ATP.Ca 2+ ), 5D0G (Ma1120 CHD (KDA→EGY)+GTP.Ca 2+ )., Enzymes: Adenylyl cyclase (EC number: 4.6.1.1)., (© 2016 Federation of European Biochemical Societies.)

Membrane-protein interactions play a central role in membrane mediated cellular processes ranging from signaling, budding, and fusion, to transport across the cell membrane. Of particular significance is the process of efficient protein olgomerization and transmembrane pore formation on the membrane surface; the primary virulent pathway for the action of antimicrobial peptides and pore forming toxins (PFTs). The suggested nanoscopic length scales and dynamic nature of such membrane lipid-protein interactions makes their detection extremely challenging. Using a combination of super-resolution stimulated emission depletion nanoscopy with fluorescence correlation spectroscopy (STED-FCS) we unravel the emergence of nanoscale lateral heterogeneity in supported bilayer membranes made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol upon interaction with the PFT, listeriolysin O (LLO). A distinct length scale-dependent dynamical crossover (<200 nm) from a Brownian diffusive regime is observed at 33 and 50% cholesterol compositions, indicating the partitioning of lipids into domains with variable cholesterol content. At 25% cholesterol content, this dyamical crossover is observed only in bilayers incubated with LLO providing evidence for the existence of sub ∼100 nm dynamical lipid nanodomains bound to LLO pore assemblies. By introducing asymmetry in cholesterol composition across the bilayer leaflets we infer that this domain formation is driven largely due to active cholesterol sequestration and transient trapping of lipids to the membrane bound motifs present in the toxins, en route to LLO oligomerization and subsequent pore formation. Bilayers prepared with labeled lipids present in either the proximal or distal leaflet allow us to track the dynamical perturbation in a leaflet-dependent manner upon LLO incubation. From the differences in the extent and intensity of the dynamical crossover as observed with STED-FCS, these experiments reveal that the affinity for cholesterol in the membrane binding motifs of the LLO subdomains induce cholesterol and lipid reorganization to a greater extent in the distal (upper) leaflet when compared with the proximal (lower) leaflet. The observed length scale-dependent membrane reorganization that occurs due to invasion by LLO could be generalized to other cholesterol-dependent cytolysins and emphasizes the significant advantage of using super-resolution STED nanoscopy to unravel complex lipid-protein interactions in membrane and cellular biophysics.

Objective: Congenital sodium diarrhoea (CSD) refers to a form of secretory diarrhoea with intrauterine onset and high faecal losses of sodium without congenital malformations. The molecular basis for CSD remains unknown. We clinically characterised a cohort of infants with CSD and set out to identify disease-causing mutations by genome-wide genetic testing., Design: We performed whole-exome sequencing and chromosomal microarray analyses in 4 unrelated patients, followed by confirmatory Sanger sequencing of the likely disease-causing mutations in patients and in their family members, followed by functional studies., Results: We identified novel de novo missense mutations in GUCY2C, the gene encoding receptor guanylate cyclase C (GC-C) in 4 patients with CSD. One patient developed severe, early-onset IBD and chronic arthritis at 4 years of age. GC-C is an intestinal brush border membrane-bound guanylate cyclase, which functions as receptor for guanylin, uroguanylin and Escherichia coli heat-stable enterotoxin. Mutations in GUCY2C were present in different intracellular domains of GC-C, and were activating mutations that enhanced intracellular cyclic guanosine monophosphate accumulation in a ligand-independent and ligand-stimulated manner, following heterologous expression in HEK293T cells., Conclusions: Dominant gain-of-function GUCY2C mutations lead to elevated intracellular cyclic guanosine monophosphate levels and could explain the chronic diarrhoea as a result of decreased intestinal sodium and water absorption and increased chloride secretion. Thus, mutations in GUCY2C indicate a role for this receptor in the pathogenesis of sporadic CSD., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Many bacterial transcription factors do not behave as per the textbook operon model. We draw on whole genome work, as well as reported diversity across different bacteria, to argue that transcription factors may have evolved from nucleoid-associated proteins. This view would explain a large amount of recent data gleaned from high-throughput sequencing and bioinformatic analyses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

An adenylyl cyclase from Mycobacterium avium, Ma1120, is a functional orthologue of a pseudogene Rv1120c from Mycobacterium tuberculosis. We report the crystal structure of Ma1120 in a monomeric form and its truncated construct as a dimer. Ma1120 exists as a monomer in solution and crystallized as a monomer in the absence of substrate or inhibitor. An additional α-helix present at the N-terminus of the monomeric structure blocks the active site by interacting with the substrate binding residues and occupying the dimer interface region. However, the enzyme has been found to be active in solution, indicating the movement of the helix away from the interface to facilitate the formation of active dimers in conditions favourable for catalysis. Thus, the N-terminal helix of Ma1120 keeps the enzyme in an autoinhibited state when it is not active. Deletion of this helix enabled us to crystallize the molecule as an active homodimer in the presence of a P-site inhibitor 2',5'-dideoxy-3'-ATP, or pyrophosphate along with metal ions. The substrate specifying lysine residue plays a dual role of interacting with the substrate and stabilizing the dimer. The dimerization loop region harbouring the second substrate specifying residue, an aspartate, shows significant differences in conformation and position between the monomeric and dimeric structures. Thus, this study has not only revealed that significant structural transitions are required for the interconversion of the inactive and the active forms of the enzyme, but also provided precise nature of these transitions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG&#95;3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem (GAFab domain). In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET) experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.

Calcineurin-like metallophosphoesterases (MPEs) form a large superfamily of binuclear metal-ion-centre-containing enzymes that hydrolyse phosphomono-, phosphodi- or phosphotri-esters in a metal-dependent manner. The MPE domain is found in Mre11/SbcD DNA-repair enzymes, mammalian phosphoprotein phosphatases, acid sphingomyelinases, purple acid phosphatases, nucleotidases and bacterial cyclic nucleotide phosphodiesterases. Despite this functional diversity, MPEs show a remarkably similar structural fold and active-site architecture. In the present review, we summarize the available structural, biochemical and functional information on these proteins. We also describe how diversification and specialization of the core MPE fold in various MPEs is achieved by amino acid substitution in their active sites, metal ions and regulatory effects of accessory domains. Finally, we discuss emerging roles of these proteins as non-catalytic protein-interaction scaffolds. Thus we view the MPE superfamily as a set of proteins with a highly conserved structural core that allows embellishment to result in dramatic and niche-specific diversification of function.

Objective: To investigate the level and location of phosphodiesterase 5 (PDE5) expression in rat prostate., Methods: The ventral, dorsal, and lateral lobes of rat prostate were examined for PDE5 expression by Western blotting. Intact rat urogenital complex, including the urinary bladder and accessory reproductive glands, was examined for PDE5 expression by immunohistochemistry. Individual prostatic lobes were further examined by immunofluorescence for expression of PDE5, α-smooth muscle actin, and rat endothelial cell antigen., Results: Western blot analysis showed that PDE5 was expressed at a significantly lower level in dorsal lobe (DL) than in ventral lobe (VL) or lateral lobe (LL). Immunohistochemistry and immunofluorescence analyses showed that PDE5 was expressed in both acinar epithelium and periacinar smooth muscle. However, although similar levels of smooth muscle PDE5 expression were observed in all 3 prostatic lobes, significantly lower level of epithelial PDE5 expression was found in DL compared with VL or LL. In prostatic blood vessels, PDE5 expression was clearly visible in the endothelium but not as easily detectable in the smooth muscle., Conclusion: PDE5 was expressed in the acinar epithelium and periacinar smooth muscle of rat prostate. However, the epithelial PDE5 expression was significantly less in DL than in VL or LL. Regardless, the acinar wall, not the blood vessel wall, is the predominant PDE5 expression site in rat prostate., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Bacteria can utilize multiple sources of carbon for growth, and for pathogenic bacteria like Mycobacterium tuberculosis, this ability is crucial for survival within the host. In addition, phenotypic changes are seen in mycobacteria grown under different carbon sources. In this study, we use Raman spectroscopy to analyze the biochemical components present in M. smegmatis cells when grown in three differently metabolized carbon sources. Our results show that carotenoid biosynthesis is enhanced when M. smegmatis is grown in glucose compared to glycerol and acetate. We demonstrate that this difference is most likely due to transcriptional upregulation of the carotenoid biosynthesis operon (crt) mediated by higher levels of the stress-responsive sigma factor SigF. Moreover, we find that increased SigF and carotenoid levels correlate with greater resistance of glucose-grown cells to oxidative stress. Thus, we demonstrate the use of Raman spectroscopy in unraveling unknown aspects of mycobacterial physiology and describe a novel effect of carbon source variation on mycobacteria., (© FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

All organisms have the capacity to sense and respond to environmental changes. These signals often involve the use of second messengers such as cyclic adenosine monophosphate (cAMP). This second messenger is widely distributed among organisms and coordinates gene expression related with pathogenesis, virulence, and environmental adaptation. Genomic analysis in Mycobacterium tuberculosis has identified 16 adenylyl cyclases (AC) and one phosphodiesterase, which produce and degrade cAMP, respectively. To date, ten AC have been biochemically characterized and only one (Rv0386) has been found to be important during murine infection with M. tuberculosis. Here, we investigated the impact of hsp60-driven Rv2212 gene expression in Mycobacterium bovis Bacillus Calmette-Guerin (BCG) during growth in vitro, and during macrophage and mice infection. We found that hsp60-driven expression of Rv2212 resulted in an increased capacity of replication in murine macrophages but an attenuated phenotype in lungs and spleen when administered intravenously in mice. Furthermore, this strain displayed an altered proteome mainly affecting proteins associated with stress conditions (bfrB, groEL-2, DnaK) that could contribute to the attenuated phenotype observed in mice.

The cyclic AMP receptor protein (CRP) family of transcription factors consists of global regulators of bacterial gene expression. Here, we identify two paralogous CRPs in the genome of Mycobacterium smegmatis that have 78% identical sequences and characterize them biochemically and functionally. The two proteins (MSMEG&#95;0539 and MSMEG&#95;6189) show differences in cAMP binding affinity, trypsin sensitivity, and binding to a CRP site that we have identified upstream of the msmeg&#95;3781 gene. MSMEG&#95;6189 binds to the CRP site readily in the absence of cAMP, while MSMEG&#95;0539 binds in the presence of cAMP, albeit weakly. msmeg&#95;6189 appears to be an essential gene, while the Δmsmeg&#95;0539 strain was readily obtained. Using promoter-reporter constructs, we show that msmeg&#95;3781 is regulated by CRP binding, and its transcription is repressed by MSMEG&#95;6189. Our results are the first to characterize two paralogous and functional CRPs in a single bacterial genome. This gene duplication event has subsequently led to the evolution of two proteins whose biochemical differences translate to differential gene regulation, thus catering to the specific needs of the organism.

cAMPhor: In the presence of cAMP, cAMPhor folds into a structure that binds DFHBI (green), increasing its fluorescence, while Alexa 647 (red) functions as a normalizing dye. It can thus be used to spatially image cAMP quantitatively in membrane-bound compartments., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Despite highly conserved core catalytic domains, members of the metallophosphoesterase (MPE) superfamily perform diverse and crucial functions ranging from nucleotide and nucleic acid metabolism to phospholipid hydrolysis. Unique structural elements outside of the catalytic core called "cap domains" are thought to provide specialization to these enzymes; however, no directed study has been performed to substantiate this. The cap domain of Rv0805, an MPE from Mycobacterium tuberculosis, is located C-terminal to its catalytic domain and is dispensable for the catalytic activity of this enzyme in vitro. We show here that this C-terminal extension (CTE) mediates in vivo localization of the protein to the cell membrane and cell wall as well as modulates expression levels of Rv0805 in mycobacteria. We also demonstrate that Rv0805 interacts with the cell wall of mycobacteria, possibly with the mycolyl-arabinogalactan-peptidoglycan complex, by virtue of its C terminus, a hitherto unknown property of this MPE. Using a panel of mutant proteins, we identify interactions between active site residues of Rv0805 and the CTE that determine its association with the cell wall. Finally, we show that Rv0805 and a truncated mutant devoid of the CTE produce different phenotypic effects when expressed in mycobacteria. Our study thus provides a detailed dissection of the functions of the cap domain of an MPE and suggests that the repertoire of cellular functions of MPEs cannot be understood without exploring the modulatory effects of these subdomains., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Chromatin immunoprecipitation identified 191 binding sites of Mycobacterium tuberculosis cAMP receptor protein (CRP(Mt)) at endogenous expression levels using a specific α-CRP(Mt) antibody. Under these native conditions an equal distribution between intragenic and intergenic locations was observed. CRP(Mt) binding overlapped a palindromic consensus sequence. Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRP(Mt) during exponential growth, and in response to nutrient starvation. Differential expression of genes with a CRP(Mt)-binding site represented only a minor portion of this transcriptional reprogramming with ∼ 19% of those representing transcriptional regulators potentially controlled by CRP(Mt). The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation. CRP(Mt) can function as a classical transcription factor in M. tuberculosis, though this occurs at only a subset of CRP(Mt)-binding sites., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c(-/-), mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence.

The Rv0805 gene in Mycobacterium tuberculosis encodes a metallophosphoesterase which shows cAMP-hydrolytic activity. Overexpression of Rv0805 has been used as a tool to lower intracellular cAMP levels and thereby elucidate the roles of cAMP in mycobacteria. Here we show that levels of cAMP in M. tuberculosis were lowered by only ∼30% following overexpression of Rv0805, and transcript levels of a number of genes, which include those associated with virulence and the methyl citrate cycle, were altered. The genes that showed altered expression were distinct from those differentially regulated in a strain deleted for the cAMP-receptor protein (CRP(Mt)), consistent with the relatively low dependence on cAMP of CRP(Mt) binding to DNA. Using mutants of Rv0805 we show that the transcriptional signature of Rv0805 overexpression is a combination of catalysis-dependent and independent effects, and that the structurally flexible C-terminus of Rv0805 is crucial for the catalysis-independent effects of the protein. Our study demonstrates the dissociation of Rv0805 and cAMP-regulated gene expression, and reveals alternate functions for this phosphodiesterase from M. tuberculosis., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)