A small step towards an important goal: fragment screen of the c-di-AMP-synthesizing enzyme CdaA.

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.

Investigating the Roles of Listeria monocytogenes Peroxidases in Growth and Virulence.

Bacteria have necessarily evolved a protective arsenal of proteins to contend with peroxides and other reactive oxygen species generated in aerobic environments. Listeria monocytogenes encounters an onslaught of peroxide both in the environment and during infection of the mammalian host, where it is the causative agent of the foodborne illness listeriosis. Despite the importance of peroxide for the immune response to bacterial infection, the strategy by which L. monocytogenes protects against peroxide toxicity has yet to be illuminated. Here, we investigated the expression and essentiality of all the peroxidase-encoding genes during L. monocytogenes growth in vitro and during infection of murine cells in tissue culture. We found that chdC and kat were required for aerobic growth in vitro, and fri and ahpA were each required for L. monocytogenes to survive acute peroxide stress. Despite increased expression of fri, ahpA, and kat during infection of macrophages, only fri proved necessary for cytosolic growth. In contrast, the proteins encoded by lmo0367, lmo0983, tpx, lmo1609, and ohrA were dispensable for aerobic growth, acute peroxide detoxification, and infection. Together, our results provide insight into the multifaceted L. monocytogenes peroxide detoxification strategy and demonstrate that L. monocytogenes encodes a functionally diverse set of peroxidase enzymes. IMPORTANCE Listeria monocytogenes is a facultative intracellular pathogen and the causative agent of the foodborne illness listeriosis. L. monocytogenes must contend with reactive oxygen species generated extracellularly during aerobic growth and intracellularly by the host immune system. However, the mechanisms by which L. monocytogenes defends against peroxide toxicity have not yet been defined. Here, we investigated the roles of each of the peroxidase-encoding genes in L. monocytogenes growth, peroxide stress response, and virulence in mammalian cells.

c-di-AMP Accumulation Impairs Muropeptide Synthesis in Listeria monocytogenes.

Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger among bacteria. c-di-AMP regulates many cellular pathways through direct binding to several molecular targets in bacterial cells. c-di-AMP depletion is well known to destabilize the bacterial cell wall, resulting in increased bacteriolysis and enhanced susceptibility to cell wall targeting antibiotics. Using the human pathogen Listeria monocytogenes as a model, we found that c-di-AMP accumulation also impaired cell envelope integrity. An L. monocytogenes mutant deleted for c-di-AMP phosphodiesterases (pdeA pgpH mutant) exhibited a 4-fold increase in c-di-AMP levels and several cell wall defects. For instance, the pdeA pgpH mutant was defective for the synthesis of peptidoglycan muropeptides and was susceptible to cell wall-targeting antimicrobials. Among different muropeptide precursors, we found that the pdeA pgpH strain was particularly impaired in the synthesis of d-Ala-d-Ala, which is required to complete the pentapeptide stem associated with UDP-N-acetylmuramic acid (MurNAc). This was consistent with an increased sensitivity to d-cycloserine, which inhibits the d-alanine branch of peptidoglycan synthesis. Finally, upon examining d-Ala:d-Ala ligase (Ddl), which catalyzes the conversion of d-Ala to d-Ala-d-Ala, we found that its activity was activated by K+ Based on previous reports that c-di-AMP inhibits K+ uptake, we propose that c-di-AMP accumulation impairs peptidoglycan synthesis, partially through the deprivation of cytoplasmic K+ levels, which are required for cell wall-synthetic enzymes.IMPORTANCE The bacterial second messenger c-di-AMP is produced by a large number of bacteria and conditionally essential to many species. Conversely, c-di-AMP accumulation is also toxic to bacterial physiology and pathogenesis, but its mechanisms are largely undefined. We found that in Listeria monocytogenes, elevated c-di-AMP levels diminished muropeptide synthesis and increased susceptibility to cell wall-targeting antimicrobials. Cell wall defects might be an important mechanism for attenuated virulence in bacteria with high c-di-AMP levels.

Ultrasensitive peptide-based multiplexed electrochemical biosensor for the simultaneous detection of Listeria monocytogenes and Staphylococcus aureus.

A novel electrochemical biosensor is reported for simultaneous detection of two of the most common food-borne pathogens: Listeria monocytogenes and Staphylococcus aureus. The biosensor is composed of an array of gold nanoparticles-modified screen-printed carbon electrodes on which magnetic nanoparticles coupled to specific peptides were immobilized via streptavidin-biotin interaction. Taking advantage of the proteolytic activities of the protease enzymes produced from the two bacteria on the specific peptides, the detection was achieved in 1 min. The detection was realized by measuring the percentage increase of the square wave voltammetric peak current at 0.1 V versus a Ag/AgCl reference electrode in ferro/ferricyanide redox couple after incubation with the bacteria protease. The integration of the specificity of the bacterial enzymes towards their peptide substrates with the sensitivity of the electrochemical detection on the sensor array allows the rapid, sensitive and selective quantification of the two bacteria. Outstanding sensitivities were achieved using this biosensor array platform with limit of detection of 9 CFU mL-1 for Listeria monocytogenes and 3 CFU mL-1 for Staphylococcus aureus. The multiplexing capability and selectivity of the array voltammetric biosensor were demonstrated by analysing samples of Staphylococcus aureus, Listeria monocytogenes or E. coli and also containing a mixture of two or three bacteria. Using this biosensor, the two bacteria were successfully quantified simultaneously in one step without the need for DNA extraction or amplification techniques. This platform offers promise for rapid, simple and cost-effective simultaneous detection of various bacteria. Graphical abstract.

An extracytoplasmic protein and a moonlighting enzyme modulate synthesis of c-di-AMP in Listeria monocytogenes.

The second messenger cyclic di-AMP (c-di-AMP) is essential for growth of many bacteria because it controls osmolyte homeostasis. c-di-AMP can regulate the synthesis of potassium uptake systems in some bacteria and it also directly inhibits and activates potassium import and export systems, respectively. Therefore, c-di-AMP production and degradation have to be tightly regulated depending on the environmental osmolarity. The Gram-positive pathogen Listeria monocytogenes relies on the membrane-bound diadenylate cyclase CdaA for c-di-AMP production and degrades the nucleotide with two phosphodiesterases. While the enzymes producing and degrading the dinucleotide have been reasonably well examined, the regulation of c-di-AMP production is not well understood yet. Here we demonstrate that the extracytoplasmic regulator CdaR interacts with CdaA via its transmembrane helix to modulate c-di-AMP production. Moreover, we show that the phosphoglucosamine mutase GlmM forms a complex with CdaA and inhibits the diadenylate cyclase activity in vitro. We also found that GlmM inhibits c-di-AMP production in L. monocytogenes when the bacteria encounter osmotic stress. Thus, GlmM is the major factor controlling the activity of CdaA in vivo. GlmM can be assigned to the class of moonlighting proteins because it is active in metabolism and adjusts the cellular turgor depending on environmental osmolarity.

Mutational and Functional Analyses of Substrate Binding and Catalysis of the Listeria monocytogenes EutT ATP:Co(I)rrinoid Adenosyltransferase.

ATP:Co(I)rrinoid adenosyltransferases (ACATs) catalyze the transfer of the adenosyl moiety from co-substrate ATP to a corrinoid substrate. ACATs are grouped into three families, namely, CobA, PduO, and EutT. The EutT family of enzymes is further divided into two classes, depending on whether they require a divalent metal ion for activity (class I and class II). To date, a structure has not been elucidated for either class of the EutT family of ACATs. In this work, results of bioinformatics analyses revealed several conserved residues between the C-terminus of EutT homologues and the structurally characterized Lactobacillus reuteri PduO (LrPduO) homologue. In LrPduO, these residues are associated with ATP binding and formation of an intersubunit salt bridge. These residues were substituted, and in vivo and in vitro data support the conclusion that the equivalent residues in the metal-free (i.e., class II) Listeria monocytogenes EutT (LmEutT) enzyme affect ATP binding. Results of in vivo and in vitro analyses of LmEutT variants with substitutions at phenylalanine and tryptophan residues revealed that replacement of the phenylalanine residue at position 72 affected access to the substrate-binding site and replacement of a tryptophan residue at position 238 affected binding of the Cbl substrate to the active site. Unlike the PduO family of ACATs, a single phenylalanine residue is not responsible for displacement of the α-ligand. Together, these data suggest that while EutT enzymes share a conserved ATP-binding motif and an intersubunit salt bridge with PduO family ACATs, class II EutT family ACATs utilize an unidentified mechanism for Cbl lower-ligand displacement and reduction that is different from that of PduO and CobA family ACATs.

Molecular and functional characterization of the Listeria monocytogenes RecA protein: Insights into the homologous recombination process.

The recombinases present in the all kingdoms in nature play a crucial role in DNA metabolism processes such as replication, repair, recombination and transcription. However, till date, the role of RecA in the deadly foodborne pathogen Listeria monocytogenes remains unknown. In this study, the authors show that L. monocytogenes expresses recA more than two-fold in vivo upon exposure to the DNA damaging agents, methyl methanesulfonate and ultraviolet radiation. The purified L. monocytogenes RecA protein show robust binding to single stranded DNA. The RecA is capable of forming displacement loop and hydrolyzes ATP, whereas the mutant LmRecAK70A fails to hydrolyze ATP, showing conserved walker A and B motifs. Interestingly, L. monocytogenes RecA and LmRecAK70A perform the DNA strand transfer activity, which is the hallmark feature of RecA protein with an oligonucleotide-based substrate. Notably, L. monocytogenes RecA readily cleaves L. monocytogenes LexA, the SOS regulon and protects the presynaptic filament from the exonuclease I activity. Altogether, this study provides the first detailed characterization of L. monocytogenes RecA and presents important insights into the process of homologous recombination in the gram-positive foodborne bacteria L. monocytogenes.

Crystal structures of the c-di-AMP-synthesizing enzyme CdaA.

Cyclic di-AMP (c-di-AMP) is the only second messenger known to be essential for bacterial growth. It has been found mainly in Gram-positive bacteria, including pathogenic bacteria like Listeria monocytogenes CdaA is the sole diadenylate cyclase in L. monocytogenes, making this enzyme an attractive target for the development of novel antibiotic compounds. Here we report crystal structures of CdaA from L. monocytogenes in the apo state, in the post-catalytic state with bound c-di-AMP and catalytic Co2+ ions, as well as in a complex with AMP. These structures reveal the flexibility of a tyrosine side chain involved in locking the adenine ring after ATP binding. The essential role of this tyrosine was confirmed by mutation to Ala, leading to drastic loss of enzymatic activity.

Copper-Induced Expression of a Transmissible Lipoprotein Intramolecular Transacylase Alters Lipoprotein Acylation and the Toll-Like Receptor 2 Response to Listeria monocytogenes.

Bacterial lipoproteins are globular proteins anchored to the extracytoplasmic surfaces of cell membranes through lipidation at a conserved N-terminal cysteine. Lipoproteins contribute to an array of important cellular functions for bacteria, as well as being a focal point for innate immune system recognition through binding to Toll-like receptor 2 (TLR2) heterodimer complexes. Although lipoproteins are conserved among nearly all classes of bacteria, the presence and type of α-amino-linked acyl chain are highly variable and even strain specific within a given bacterial species. The reason for lyso-lipoprotein formation and N-acylation variability in general is presently not fully understood. In Enterococcus faecalis, lipoproteins are anchored by an N-acyl-S-monoacyl-glyceryl cysteine (lyso form) moiety installed by a chromosomally encoded lipoprotein intramolecular transacylase (Lit). Here, we describe a mobile genetic element common to environmental isolates of Listeria monocytogenes and Enterococcus spp. encoding a functional Lit ortholog (Lit2) that is cotranscribed with several well-established copper resistance determinants. Expression of Lit2 is tightly regulated, and induction by copper converts lipoproteins from the diacylglycerol-modified form characteristic of L. monocytogenes type strains to the α-amino-modified lyso form observed in E. faecalis Conversion to the lyso form through either copper addition to media or constitutive expression of lit2 decreases TLR2 recognition when using an activated NF-κB secreted embryonic alkaline phosphatase reporter assay. While lyso formation significantly diminishes TLR2 recognition, lyso-modified lipoprotein is still predominantly recognized by the TLR2/TLR6 heterodimer.IMPORTANCE The induction of lipoprotein N-terminal remodeling in response to environmental copper in Gram-positive bacteria suggests a more general role in bacterial cell envelope physiology. N-terminal modification by lyso formation, in particular, simultaneously modulates the TLR2 response in direct comparison to their diacylglycerol-modified precursors. Thus, use of copper as a frontline antimicrobial control agent and ensuing selection raises the potential of diminished innate immune sensing and enhanced bacterial virulence.

A Listeria monocytogenes ST2 clone lacking chitinase ChiB from an outbreak of non-invasive gastroenteritis.

An outbreak with a remarkable Listeria monocytogenes clone causing 163 cases of non-invasive listeriosis occurred in Germany in 2015. Core genome multi locus sequence typing grouped non-invasive outbreak isolates and isolates obtained from related food samples into a single cluster, but clearly separated genetically close isolates obtained from invasive listeriosis cases. A comparative genomic approach identified a premature stop codon in the chiB gene, encoding one of the two L. monocytogenes chitinases, which clustered with disease outcome. Correction of this premature stop codon in one representative gastroenteritis outbreak isolate restored chitinase production, but effects in infection experiments were not found. While the exact role of chitinases in virulence of L. monocytogenes is still not fully understood, our results now clearly show that ChiB-derived activity is not required to establish L. monocytogenes gastroenteritis in humans. This limits a possible role of ChiB in human listeriosis to later steps of the infection.

A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity.

ATP:Co(I)rrinoid adenosyltransferases (ACATs) are involved in de novo adenosylcobamide (AdoCba) biosynthesis and in salvaging complete and incomplete corrinoids from the environment. The ACAT enzyme family is comprised of three classes of structurally and evolutionarily distinct proteins (i.e., CobA, PduO, and EutT). The structure of EutT is unknown, and an understanding of its mechanism is incomplete. The Salmonella enterica EutT ( SeEutT) enzyme is the best-characterized member of its class and is known to be a ferroprotein. Here, we report the identification and initial biochemical characterization of an enzyme representative of a new class of EutTs that does not require a metal ion for activity. In vivo and in vitro evidence shows that the metal-free EutT homologue from Listeria monocytogenes ( LmEutT) has ACAT activity and that, unlike other ACATs, the biologically active form of LmEutT is a tetramer. In vitro studies revealed that LmEutT was more efficient than SeEutT and displayed positive cooperativity. LmEutT adenosylated cobalamin, but not cobinamide, showed specificity for ATP and 2'-deoxyATP and released a triphosphate byproduct. Bioinformatics analyses suggest that metal-free EutT ACATs are also present in other Firmicutes.

Spectroscopic Study of the EutT Adenosyltransferase from Listeria monocytogenes: Evidence for the Formation of a Four-Coordinate Cob(II)alamin Intermediate.

The EutT enzyme from Listeria monocytogenes ( LmEutT) is a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes that catalyze the biosynthesis of adenosylcobalamin (AdoCbl) from exogenous Co(II)rrinoids and ATP. Apart from EutT-type ACATs, two evolutionary unrelated types of ACATs have been identified, termed PduO and CobA. Although the three types of ACATs are nonhomologous, they all generate a four-coordinate cob(II)alamin (4C Co(II)Cbl) species to facilitate the formation of a supernucleophilic Co(I)Cbl intermediate capable of attacking the 5'-carbon of cosubstrate ATP. Previous spectroscopic studies of the EutT ACAT from Salmonella enterica ( SeEutT) revealed that this enzyme requires a divalent metal cofactor for the conversion of 5C Co(II)Cbl to a 4C species. Interestingly, LmEutT does not require a divalent metal cofactor for catalytic activity, which exemplifies an interesting phylogenetic divergence among the EutT enzymes. To explore if this disparity in the metal cofactor requirement among EutT enzymes correlates with differences in substrate specificity or the mechanism of Co(II)Cbl reduction, we employed various spectroscopic techniques to probe the interaction of Co(II)Cbl and cob(II)inamide (Co(II)Cbi+) with LmEutT in the absence and presence of cosubstrate ATP. Our data indicate that LmEutT displays a similar substrate specificity as SeEutT and can bind both Co(II)Cbl and Co(II)Cbi+ when complexed with MgATP, though it exclusively converts Co(II)Cbl to a 4C species. Notably, LmEutT is the most effective ACAT studied to date in generating the catalytically relevant 4C Co(II)Cbl species, achieving a >98% 5C → 4C conversion yield on the addition of just over one mol equiv of cosubstrate MgATP.

Antimicrobial activity and acetylcholinesterase inhibition by extracts from chromatin modulated fungi

ABSTRACT Major health challenges as the increasing number of cases of infections by antibiotic multiresistant microorganisms and cases of Alzheimer's disease have led to searching new control drugs. The present study aims to verify a new way of obtaining bioactive extracts from filamentous fungi with potential antimicrobial and acetylcholinesterase inhibitory activities, using epigenetic modulation to promote the expression of genes commonly silenced. For such finality, five filamentous fungal species (Talaromyces funiculosus, Talaromyces islandicus, Talaromyces minioluteus, Talaromyces pinophilus, Penicillium janthinellum) were grown or not with DNA methyltransferases inhibitors (procainamide or hydralazine) and/or a histone deacetylase inhibitor (suberohydroxamic acid). Extracts from T. islandicus cultured or not with hydralazine inhibited Listeria monocytogenes growth in 57.66 ± 5.98% and 15.38 ± 1.99%, respectively. Increment in inhibition of acetylcholinesterase activity was observed for the extract from P. janthinellum grown with procainamide (100%), when compared to the control extract (39.62 ± 3.76%). Similarly, inhibition of acetylcholinesterase activity increased from 20.91 ± 3.90% (control) to 92.20 ± 3.72% when the tested extract was obtained from T. pinophilus under a combination of suberohydroxamic acid and procainamide. Concluding, increases in antimicrobial activity and acetylcholinesterase inhibition were observed when fungal extracts in the presence of DNA methyltransferases and/or histone deacetylase modulators were tested.

Dynamics of P-type ATPase transport revealed by single-molecule FRET.

Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.

Listeria monocytogenes PdeE, a phosphodiesterase that contributes to virulence and has hydrolytic activity against cyclic mononucleotides and cyclic dinucleotides.

We have identified and partially characterized a putative HD domain hydrolase, LMOf2365_2464, which is highly expressed during listerial intracellular replication. LMOf2365_2464 is annotated as a putative HD domain-containing hydrolase. The ability of an isogenic mutant strain, F2365Δ2464, to adhere, invade and replicate in intestinal epithelial cells (Caco-2) was significantly lower than parent strain F2365. Colonization of mouse liver and spleen by L. monocytogenes F2365 was significantly higher than it was for the mutant. The recombinant protein showed phosphodiesterase activity in the presence of divalent metal ions, indicating its role in nucleotide metabolism. It has activity against several cyclic nucleotides and cyclic dinucleotides, but its strongest activity is against cyclic di-AMP and cyclic AMP. Based on this enzymatic activity, we designated LMOf2365_2464 phosphodiesterase E (PdeE).

Listeria monocytogenes behaviour and quality attributes during sausage storage affected by sodium nitrite, sodium lactate and thyme essential oil.

The effects of the addition of nitrite at 200 ppm (N), sodium lactate 1.5% (L) and thyme essential oil at 100 ppm (T1) on Listeria monocytogenes behaviour and ATPase activity inhibition were evaluated, as well as lipid oxidation through the quantification of malonaldehydes, in sausage stored at 8 ℃ for 41 days and at 30 ℃ for 14 days. The changes in the colour profile were performed during storage time at 8 ℃. Quantitative descriptive sensory analyses were performed after two days at 4 ℃. At 8 ℃, the treatments with the highest inhibition on L. monocytogenes were L and N, without significant differences. In turn, at 30 ℃, the bacterium was most inhibited with treatment L, followed by T1 and N, without significant differences. A 44.1% and 19% inhibition of ATPase activity was detected in L and T1 treatments, respectively. At 8 ℃ and 30 ℃, malonaldehydes content was not different between the treatments. N presented the highest values of a* and concentration of metmyoglobin after 41 days at 8 ℃. The panel detected differences between T1 and N for the aroma in the descriptors spices and herbal.

8-Thioalkyl-adenosine derivatives inhibit Listeria monocytogenes NAD kinase through a novel binding mode.

Increased resistance of pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Biosynthetic pathways of several cofactors important for bacterial growth, such as nicotinamide adenine dinucleotide phosphate (NADP), have been proposed as a promising source of antibiotic targets. Nicotinamide adenine dinucleotide kinases (NADK; EC 2.7.1.23) are attractive for inhibitor development, since they catalyze the phosphorylation of NAD to NADP, which is an essential step of NADP metabolism. We previously synthesized diadenosine derivatives that inhibited NADK from two human pathogens, Listeria monocytogenes and Staphylococcus aureus, in the micromolar range. They behave as NAD mimics with the 5',5'-diphosphate group substituted by a 8,5' thioglycolic bridge. In an attempt to improve inhibitory potency, we designed new NAD mimics based on a single adenosine moiety harboring a larger derivatization attached to the C8 position and a small group at the 5' position. Here we report the synthesis of a series of 8-thioalkyl-adenosine derivatives containing various aryl and heteroaryl moieties and their evaluation as inhibitors of L. monocytogenes NADK1, S. aureus NADK and their human counterpart. Novel, sub-micromolar inhibitors of LmNADK1 were identified. Surprisingly, most LmNADK1 inhibitors demonstrated a high selectivity index against the close staphylococcal ortholog and the human NADK. Structural characterization of enzyme-inhibitor complexes revealed the original binding mode of these novel NAD mimics.

Engineering a Prototypic P-type ATPase Listeria monocytogenes Ca(2+)-ATPase 1 for Single-Molecule FRET Studies.

Approximately 30% of the ATP generated in the living cell is utilized by P-type ATPase primary active transporters to generate and maintain electrochemical gradients across biological membranes. P-type ATPases undergo large conformational changes during their functional cycle to couple ATP hydrolysis in the cytoplasmic domains to ion transport across the membrane. The Ca(2+)-ATPase from Listeria monocytogenes, LMCA1, was found to be a suitable model of P-type ATPases and was engineered to facilitate single-molecule FRET studies of transport-related structural changes. Mutational analyses of the endogenous cysteine residues in LMCA1 were performed to reduce background labeling without compromising activity. Pairs of cysteines were introduced into the optimized low-reactivity background, and labeled with maleimide derivatives of Cy3 and Cy5 resulting in site-specifically double-labeled protein with moderate activity. Ensemble and confocal single-molecule FRET studies revealed changes in FRET distribution related to structural changes during the transport cycle, consistent with those observed by X-ray crystallography for the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). Notably, the cytosolic headpiece of LMCA1 was found to be distinctly more compact in the E1 state than in the E2 state. Thus, the established experimental system should allow future real-time FRET studies of the structural dynamics of LMCA1 as a representative P-type ATPase.

Rapid colorimetric sensing platform for the detection of Listeria monocytogenes foodborne pathogen.

Listeria monocytogenes is a serious cause of human foodborne infections worldwide, which needs spending billions of dollars for inspection of bacterial contamination in food every year. Therefore, there is an urgent need for rapid, in-field and cost effective detection techniques. In this study, rapid, low-cost and simple colorimetric assay was developed using magnetic nanoparticles for the detection of listeria bacteria. The protease from the listeria bacteria was detected using D-amino acid substrate. D-amino acid substrate was linked to the carboxylic acid on the magnetic nanoparticles using EDC/NHS chemistry. The cysteine residue at the C-terminal of the substrate was used for the self-assembled monolayer formation on the gold sensor surface, which in turn the black magnetic nanobeads will mask the golden color. The color will change from black to golden color upon the cleavage of the specific peptide sequence by the Listeria protease. The sensor was tested with serial dilutions of Listeria bacteria. It was found that the appearance of the gold surface area is proportional to the bacterial concentrations in CFU/ml. The lowest detection limit of the developed sensor for Listeria was found to be 2.17×10(2) colony forming unit/ml (CFU/ml). The specificity of the biosensor was tested against four different foodborne associated bacteria (Escherichia coli, Salmonella, Shigella flexnerii and Staphylococcus aureus). Finally, the sensor was tested with artificially spiked whole milk and ground meat spiked with listeria.

Rifampin phosphotransferase is an unusual antibiotic resistance kinase.

Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds.