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.

NrnA Is a Linear Dinucleotide Phosphodiesterase with Limited Function in Cyclic Dinucleotide Metabolism in Listeria monocytogenes.

Listeria monocytogenes produces both c-di-AMP and c-di-GMP to mediate many important cellular processes, but the levels of both nucleotides must be regulated. c-di-AMP accumulation attenuates virulence and diminishes stress response, and c-di-GMP accumulation impairs bacterial motility. An important regulatory mechanism to maintain c-di-AMP and c-di-GMP homeostasis is to hydrolyze them to the linear dinucleotides pApA and pGpG, respectively, but the fates of these hydrolytic products have not been examined in L. monocytogenes. We found that NrnA, a stand-alone DHH-DHHA1 phosphodiesterase, has a broad substrate range but with a strong preference for linear dinucleotides over cyclic dinucleotides. Although NrnA exhibited detectable cyclic dinucleotide hydrolytic activities in vitro, NrnA had negligible effects on their levels in the bacterial cell, even in the absence of the c-di-AMP phosphodiesterases PdeA and PgpH. The ΔnrnA mutant had a mammalian cell infection defect that was fully restored by Escherichia coli Orn. Together, our data indicate that L. monocytogenes NrnA is functionally orthologous to Orn, and its preferred physiological substrates are most likely linear dinucleotides. Furthermore, our findings revealed that, unlike some other c-di-AMP- and c-di-GMP-producing bacteria, L. monocytogenes does not employ their hydrolytic products to regulate their phosphodiesterases, at least at the pApA and pGpG levels in the ΔnrnA mutant. Finally, the ΔnrnA infection defect was overcome by constitutive activation of PrfA, the master virulence regulator, suggesting that accumulated linear dinucleotides inhibit the expression, stability, or function of PrfA-regulated virulence factors. IMPORTANCE Listeria monocytogenes produces both c-di-AMP and c-di-GMP and encodes specific phosphodiesterases that degrade them into pApA and pGpG, respectively, but the metabolism of these products has not been characterized in this bacterium. We found that L. monocytogenes NrnA degrades a broad range of nucleotides. Among the tested cyclic and linear substrates, it exhibits a strong biochemical and physiological preference for the linear dinucleotides pApA, pGpG, and pApG. Unlike in some other bacteria, these oligoribonucleotides do not appear to interfere with cyclic dinucleotide hydrolysis. The absence of NrnA is well tolerated by L. monocytogenes in broth cultures but impairs its ability to infect mammalian cells. These findings indicate a separation of cyclic dinucleotide signaling and oligoribonucleotide metabolism in L. monocytogenes.

High Temperature Requirement A (HtrA) protease of Listeria monocytogenes and its interaction with extracellular matrix molecules.

High Temperature Requirement A (HtrA) was identified as a secreted virulence factor in many pathogenic bacteria, including Listeria monocytogenes. Recently, it was discovered that Helicobacter pylori and Campylobacter jejuni HtrAs can directly cleave the human cell-adhesion molecule E-cadherin, which facilitates bacterial transmigration. HtrAs also interact with extracellular matrix (ECM) molecules. However, only a limited number of studies have been carried out in this regard. In the present study, the protease and ECM binding properties of L. monocytogenes HtrA (LmHtrA) were studied using native rLmHtrA, catalytically inactive rLmHtrA(S343A) and rLmHtrA lacking the PDZ domain (∆PDZ) to gain more insights into HtrA-ECM molecule interaction. The results show that (1) native rLmHtrA cleaves fibrinogen, fibronectin, plasminogen and casein in a time and temperature dependent manner, (2) interaction of rLmHtrA with various host proteins was found in the micromolar to nanomolar range, (3) in the absence of PDZ domain, rLmHtrA exhibits no drastic change in binding affinity toward the host molecules when compared with native rLmHtrA and (4) the PDZ domain plays an important role in the substrate cleavage as rLmHtrA1-394∆PDZ cleaves the substrates only under certain conditions. The proteolysis of various ECM molecules by rLmHtrA possibly highlights the role of HtrA in L. monocytogenes pathogenesis involving ECM degradation.

Bacterial phospholipases C with dual activity: phosphatidylcholinesterase and sphingomyelinase.

Bacterial phospholipases and sphingomyelinases are lipolytic esterases that are structurally and evolutionarily heterogeneous. These enzymes play crucial roles as virulence factors in several human and animal infectious diseases. Some bacterial phospholipases C (PLCs) have both phosphatidylcholinesterase and sphingomyelinase C activities. Among them, Listeria monocytogenes PlcB, Clostridium perfringens PLC, and Pseudomonas aeruginosa PlcH are the most deeply understood. In silico predictions of substrates docking with these three bacterial enzymes provide evidence that they interact with different substrates at the same active site. This review discusses structural aspects, substrate specificity, and the mechanism of action of those bacterial enzymes on target cells and animal infection models to shed light on their roles in pathogenesis.

Tyrosyl Radical-Mediated Sequential Oxidative Decarboxylation of Coproporphyrinogen III through PCET: Theoretical Insights into the Mechanism of Coproheme Decarboxylase ChdC.

The peroxide-dependent coproheme decarboxylase ChdC from Geobacillus stearothermophilus catalyzes two key steps in the synthesis of heme b, i.e., two sequential oxidative decarboxylations of coproporphyrinogen III (coproheme III) at propionate groups P2 and P4. In the binding site of coproheme III, P2 and P4 are anchored by different residues (Tyr144, Arg217, and Ser222 for P2 and Tyr113, Lys148, and Trp156 for P4); however, strong experimental evidence supports that the generated Tyr144 radical acts as an unique intermediary for hydrogen atom transfer (HAT) from both reactive propionates. So far, the reaction details are still unclear. Herein, we carried out quantum mechanics/molecular mechanics calculations to explore the decarboxylation mechanism of coproheme III. In our calculations, the coproheme Cpd I, Fe(IV) = O coupled to a porphyrin radical cation (por•+) with four propionate groups, was used as a reactant model. Our calculations reveal that Tyr144 is directly involved in the decarboxylation of propionate group P2. First, the proton-coupled electron transfer (PCET) occurs from Tyr144 to P2, generating a Tyr144 radical, which then abstracts a hydrogen atom from the Cß of P2. The ß-H extraction was calculated to be the rate-limiting step of decarboxylation. It is the porphyrin radical cation (por•+) that makes the PCET from Tyr144 to P2 to be quite easy to initiate the decarboxylation. Finally, the electron transfers from the Cß• through the porphyrin to the iron center, leading to the decarboxylation of P2. Importantly, the decarboxylation of P4 mediated by Lys148 was calculated to be very difficult, which suggests that after the P2 decarboxylation, the generated harderoheme III intermediate should rebind or rotate in the active site so that the propionate P4 occupies the binding site of P2, and Tyr144 again mediates the decarboxylation of P4. Thus, our calculations support the fact that Tyr144 is responsible for the decarboxylation of both P2 and P4.

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.

The Crystal Structure of the Ca2+-ATPase 1 from Listeria monocytogenes reveals a Pump Primed for Dephosphorylation.

Many bacteria export intracellular calcium using active transporters homologous to the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Here we present three crystal structures of Ca2+-ATPase 1 from Listeria monocytogenes (LMCA1). Structures with BeF3- mimicking a phosphoenzyme state reveal a closed state, which is intermediate between the outward-open E2P and the proton-occluded E2-P* conformations known for SERCA. It suggests that LMCA1 in the E2P state is pre-organized for dephosphorylation upon Ca2+ release, consistent with the rapid dephosphorylation observed in single-molecule studies. An arginine side-chain occupies the position equivalent to calcium binding site I in SERCA, leaving a single Ca2+ binding site in LMCA1, corresponding to SERCA site II. Observing no putative transport pathways dedicated to protons, we infer a direct proton counter transport through the Ca2+ exchange pathways. The LMCA1 structures provide insight into the evolutionary divergence and conserved features of this important class of ion transporters.

Shape-function insights into bifunctional O-GlcNActransferase of Listeria monocytogenes EGD-e.

O-GlcNAcylation is an important post-translational modification of proteins. O-GlcNAcylated proteins have crucial roles in several cellular contexts both in eukaryotes and bacteria. O-GlcNActransferase (OGT) is the enzyme instrumental in O-GlcNAcylation of proteins. OGT is conserved across eukaryotes. The first bacterial OGT discovered is GmaR in Listeria monocytogenes. GmaR is a GT-2 family bifunctional protein that catalyzes glycosylation of the flagellin protein FlaA and controls transcription of flagellar motility genes in a temperature-dependent manner. Here, we provide methods for heterologous expression and purification of recombinant GmaR and FlaA, in vivo/in vitro glycosylation assays, analysis of the molecular form of recombinant GmaR and detailed enzyme kinetics. We study the structure and functional dynamics of GmaR. Using solution small-angle X-ray scattering and molecular modeling, we show that GmaR adopts an extended shape with two distinctly spaced structural units in the presence of cofactor Mg2+ and with donor UDP-GlcNAc and cofactor combined. Comparisons of restored structures revealed that in-solution binding of Mg2+ ions brings about shape rearrangements and induces structural-rigidity in hyper-variable regions at the N-terminus of GmaR protein. Taking function and shape data together, we describe that Mg2+ binding enables GmaR to adopt a shape that can bind the substrate. The manuscript provides the first 3D solution structure of a bacterial OGT of GT-2 family and detailed biochemical characterization of GmaR to facilitate its future applications.

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.

Morin inhibits Listeria monocytogenes virulence in vivo and in vitro by targeting listeriolysin O and inflammation.

BACKGROUND: Listeria monocytogenes (L. monocytogenes) is a global opportunistic intracellular pathogen that can cause many infections, including meningitis and abortion in humans and animals; thus, L. monocytogenes poses a great threat to public safety and the development of the aquaculture industry. The isolation rate of Listeria monocytogenes in fishery products has always been high. And the pore-forming toxin listeriolysin O (LLO) is one of the most important virulence factors of L. monocytogenes. LLO can promote cytosolic bacterial proliferation and help the pathogen evade attacks from the host immune system. In addition, L. monocytogenes infection can trigger a series of severe inflammatory reactions. RESULTS: Here, we further confirmed that morin lacking anti-Listeria activity could inhibit LLO oligomerization. We also found that morin can effectively alleviate the inflammation induced by Listeria in vivo and in vitro and exerted an obvious protective effect on infected cells and mice. CONCLUSIONS: Morin does not possess anti-Listeria activity, neither does it interfere with secretion of LLO. However, morin inhibits oligomerisation of LLO and morin does reduce the inflammation caused during Listeria infection.

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.

Amino acids other than glutamate affect the expression of the GAD system in Listeria monocytogenes enhancing acid resistance.

The Glutamate Decarboxylase (GAD) system is important for survival of L. monocytogenes and other microorganisms under acidic conditions. Environmental conditions influence the function of the GAD system. Until now, the only conditions known to lead to increased transcription of the GAD system are the stationary phase in rich media and anoxic conditions. Previously, we showed that transcription of the GAD system requires unidentified compounds other than glutamate present in rich media. Following a test looking at various compounds we identified for first time that peptone, tryptone and casamino acids activate the GAD system under oxic conditions suggesting that amino acid(s) other than glutamate and/or peptides are important for the above process. The defined medium, where the GAD system is inactive, once it is supplemented with the above compounds results in an active intracellular and extracellular GAD system and increased acid resistance. Through functional genomics we show that these compounds are required for GadD2 activity and although we previously showed that GadD3 is active part of the intracellular GAD system, the supplementation did not activate this gene. The above is explained by the fact that only gadD2 transcription was upregulated by these compounds while the transcription of gadD1 and gadD3 remained unaffected. Together our results show that the L. monocytogenes GadD2 decarboxylase is activated in the presence of amino acids or peptides other than glutamate, a finding that has important implications for acid tolerance and food safety.

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.

Phosphocholine Antagonizes Listeriolysin O-Induced Host Cell Responses of Listeria monocytogenes.

BACKGROUND: Bacterial toxins disrupt plasma membrane integrity with multitudinous effects on host cells. The secreted pore-forming toxin listeriolysin O (LLO) of the intracellular pathogen Listeria monocytogenes promotes egress of the bacteria from vacuolar compartments into the host cytosol often without overt destruction of the infected cell. Intracellular LLO activity is tightly controlled by host factors including compartmental pH, redox, proteolytic, and proteostatic factors, and inhibited by cholesterol. METHODS: Combining infection studies of L. monocytogenes wild type and isogenic mutants together with biochemical studies with purified phospholipases, we investigate the effect of their enzymatic activities on LLO. RESULTS: Here, we show that phosphocholine (ChoP), a reaction product of the phosphatidylcholine-specific phospholipase C (PC-PLC) of L. monocytogenes, is a potent inhibitor of intra- and extracellular LLO activities. Binding of ChoP to LLO is redox-independent and leads to the inhibition of LLO-dependent induction of calcium flux, mitochondrial damage, and apoptosis. ChoP also inhibits the hemolytic activities of the related cholesterol-dependent cytolysins (CDC), pneumolysin and streptolysin. CONCLUSIONS: Our study uncovers a strategy used by L. monocytogenes to modulate cytotoxic LLO activity through the enzymatic activity of its PC-PLC. This mechanism appears to be widespread and also used by other CDC pore-forming toxin-producing bacteria.

A novel role for the glutamate decarboxylase system in Listeria monocytogenes; protection against oxidative stress.

The GAD system is widely present in several types of organisms and is known to play an important role in bacterial acid tolerance. There is only one account of this system playing a role in oxidative stress in bacteria and one in yeasts. Here we show for first time that it affects the oxidative stress resistance of a Gram-positive bacterium, (L. monocytogenes, tested in three strains; 10403S, EGD-e, and LO28). We found a statistically significant reduction in survival after H2O2 exposure in ΔgadD3 and ΔgadD2 of EGD-e and in ΔgadD1 of LO28. Furthermore, we observed a lag phase prolongation in ΔgadD3 of 10403S and EGD-e and a larger inhibition zone in disk diffusion assay for ΔgadD1 and ΔgadD3 of EGD-e upon H2O2 exposure. All GAD genes playing a role in oxidative stress resistance are part of GADi system and this occurs partly through catalase activity, while the most potent GADe system plays no role. The latter effects could occur through the GABA shunt, but we show here that mutants in succinate semialdehyde dehydrogenase do not show a phenotype suggesting that either effects are through the GABA transaminase or, this pathway is not involved. Our study highlights for first time the role of the GAD system in oxidative stress resistance of a Gram-positive bacterium, which could be used in Food Hurdle Technology to eliminate pathogens such as L. monocytogenes, while it gives an insight on the general mechanism.

Crystal structures and calorimetry reveal catalytically relevant binding mode of coproporphyrin and coproheme in coproporphyrin ferrochelatase.

Coproporphyrin ferrochelatases (CpfCs, EC 4.99.1.9) insert ferrous iron into coproporphyrin III yielding coproheme. CpfCs are utilized by prokaryotic, mainly monoderm (Gram-positive) bacteria within the recently detected coproporphyrin-dependent (CPD) heme biosynthesis pathway. Here, we present a comprehensive study on CpfC from Listeria monocytogenes (LmCpfC) including the first crystal structure of a coproheme-bound CpfC. Comparison of crystal structures of apo-LmCpfC and coproheme-LmCpfC allowed identification of structural rearrangements and of amino acids involved in tetrapyrrole macrocycle and Fe2+ binding. Differential scanning calorimetry of apo-, coproporphyrin III-, and coproheme-LmCpfC underline the pronounced noncovalent interaction of both coproporphyrin and coproheme with the protein (ΔTm  = 11 °C compared to apo-LmCpfC), which includes the propionates (p2, p4, p6, p7) and the amino acids Arg29, Arg45, Tyr46, Ser53, and Tyr124. Furthermore, the thermodynamics and kinetics of coproporphyrin III and coproheme binding to apo-LmCpfC is presented as well as the kinetics of insertion of ferrous iron into coproporphyrin III-LmCpfC that immediately leads to formation of ferric coproheme-LmCpfC (kcat /KM  = 4.7 × 105  m-1 ·s-1 ). We compare the crystal structure of coproheme-LmCpfC with available structures of CpfCs with artificial tetrapyrrole macrocycles and discuss our data on substrate binding, iron insertion and substrate release in the context of the CPD heme biosynthesis pathway. ENZYME: EC 4.99.1.9 DATABASE: pdb-codes of structural data in this work: 6RWV, 6SV3.

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.

Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target.

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (Km = 53 µM; vmax = 180 nmol mg-1 min-1). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Š(Rfree = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D5-symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality.

Cell Shape and Antibiotic Resistance Are Maintained by the Activity of Multiple FtsW and RodA Enzymes in Listeria monocytogenes.

Rod-shaped bacteria have two modes of peptidoglycan synthesis: lateral synthesis and synthesis at the cell division site. These two processes are controlled by two macromolecular protein complexes, the elongasome and divisome. Recently, it has been shown that the Bacillus subtilis RodA protein, which forms part of the elongasome, has peptidoglycan glycosyltransferase activity. The cell division-specific RodA homolog FtsW fulfils a similar role at the divisome. The human pathogen Listeria monocytogenes carries genes that encode up to six FtsW/RodA homologs; however, their functions have not yet been investigated. Analysis of deletion and depletion strains led to the identification of the essential cell division-specific FtsW protein, FtsW1. Interestingly, L. monocytogenes carries a gene that encodes a second FtsW protein, FtsW2, which can compensate for the lack of FtsW1, when expressed from an inducible promoter. L. monocytogenes also possesses three RodA homologs, RodA1, RodA2, and RodA3, and their combined absence is lethal. Cells of a rodA1 rodA3 double mutant are shorter and have increased antibiotic and lysozyme sensitivity, probably due to a weakened cell wall. Results from promoter activity assays revealed that expression of rodA3 and ftsW2 is induced in the presence of antibiotics targeting penicillin binding proteins. Consistent with this, a rodA3 mutant was more susceptible to the ß-lactam antibiotic cefuroxime. Interestingly, overexpression of RodA3 also led to increased cefuroxime sensitivity. Our study highlights that L. monocytogenes genes encode a multitude of functional FtsW and RodA enzymes to produce its rigid cell wall and that their expression needs to be tightly regulated to maintain growth, cell division, and antibiotic resistance.IMPORTANCE The human pathogen Listeria monocytogenes is usually treated with high doses of ß-lactam antibiotics, often combined with gentamicin. However, these antibiotics only act bacteriostatically on L. monocytogenes, and the immune system is needed to clear the infection. Therefore, individuals with a compromised immune system are at risk to develop a severe form of Listeria infection, which can be fatal in up to 30% of cases. The development of new strategies to treat Listeria infections is necessary. Here we show that the expression of some of the FtsW and RodA enzymes of L. monocytogenes is induced by the presence of ß-lactam antibiotics, and the combined absence of these enzymes makes bacteria more susceptible to this class of antibiotics. The development of antimicrobial agents that inhibit the activity or production of FtsW and RodA enzymes might therefore help to improve the treatment of Listeria infections and thereby lead to a reduction in mortality.