Human Pathogen Listeria Monocytogenes Research Articles

The Novel Internalins InlP1 and InlP4 and the Internalin-Like Protein InlP3 Enhance the Pathogenicity of Listeria monocytogenes.

The pathogenicity of the human foodborne pathogen Listeria monocytogenes relies on virulence factors such as internalins. In 2009/2010 two L. monocytogenes strains were responsible for a serious listeriosis outbreak in Austria, Germany, and the Czech Republic. One of these clones, QOC1, which caused 14 cases including five fatalities, encodes the novel internalins inlP1, inlPq and inlP4, and the novel internalin-like protein inlP3 in the genomic region of hypervariable genetic hotspot 9 in addition to the standard set of virulence genes. The in silico prevalence study revealed that these genes rarely occur in L. monocytogenes, mainly in minor clonal complexes. To obtain first insights of the role of these genes in the pathogenicity of L. monocytogenes, we studied the gene expression under conditions mimicking the ingestion in the host. Expression of inlP1, inlP3, inlPq and inlP4 was increased under gastric stress and in intracellular bacteria grown in intestinal epithelial cells. Furthermore, colonization of the liver and the spleen was slightly, but significantly reduced 72 h post infection in an oral mouse infection model when inlP1 or inlP4 was deleted. Moreover, the impact of InlP1 and InlP3 in virulence was shown in vitro in human intestinal epithelial cells. In this study we conclusively demonstrate a potential contribution of uncommon novel internalins and an internalin-like protein to the pathogenicity of L. monocytogenes.

A likelihood ratio-based approach for improved source attribution in microbiological forensic investigations

A common objective in microbial forensic investigations is to identify the origin of a recovered pathogenic bacterium by DNA sequencing. However, there is currently no consensus about how degrees of belief in such origin hypotheses should be quantified, interpreted, and communicated to wider audiences. To fill this gap, we have developed a concept based on calculating probabilistic evidential values for microbial forensic hypotheses. The likelihood-ratio method underpinning this concept is widely used in other forensic fields, such as human DNA matching, where results are readily interpretable and have been successfully communicated in juridical hearings. The concept was applied to two case scenarios of interest in microbial forensics: (1) identifying source cultures among series of very similar cultures generated by parallel serial passage of the Tier 1 pathogen Francisella tularensis, and (2) finding the production facilities of strains isolated in a real disease outbreak caused by the human pathogen Listeria monocytogenes. Evidence values for the studied hypotheses were computed based on signatures derived from whole genome sequencing data, including deep-sequenced low-frequency variants and structural variants such as duplications and deletions acquired during serial passages. In the F. tularensis case study, we were able to correctly assign fictive evidence samples to the correct culture batches of origin on the basis of structural variant data. By setting up relevant hypotheses and using data on cultivated batch sources to define the reference populations under each hypothesis, evidential values could be calculated. The results show that extremely similar strains can be separated on the basis of amplified mutational patterns identified by high-throughput sequencing. In the L. monocytogenes scenario, analyses of whole genome sequence data conclusively assigned the clinical samples to specific sources of origin, and conclusions were formulated to facilitate communication of the findings. Taken together, these findings demonstrate the potential of using bacterial whole genome sequencing data, including data on both low frequency SNP signatures and structural variants, to calculate evidence values that facilitate interpretation and communication of the results. The concept could be applied in diverse scenarios, including both epidemiological and forensic source tracking of bacterial infectious disease outbreaks.

Comparative Analysis of RNA Structures Reveals an mRNA-mRNA Interaction Controlling Listeria Virulence Factor Expression

To understand physiological processes, it is vital to disclose the conformation of biological molecules inside living cells and correlate their structure with their function. Here, we describe a method, FUSE (5ʹ UTR Structure Elucidation), that we developed to study how the structures of 5ʹ UTRs change in response to different conditions inside the human bacterial pathogen Listeria monocytogenes. We discovered a novel RNA thermoswitch and could calculate the ratio of the two conformations that this thermoswitch assumes at different conditions. FUSE also identified the site of base-pairing between a small RNA and mRNA with a single-nucleotide resolution. Strikingly, FUSE discovered a functional mRNA-mRNA interaction, where the stability of an mRNA encoding the PrsA2 chaperone was increased by the direct binding of an mRNA encoding its substrate, the main virulence factor listeriolysin O. Specifically targeting the 5ʹ UTRs, FUSE can be applied in any living organism or virus.

Strand specific RNA-sequencing and membrane lipid profiling reveals growth phase-dependent cold stress response mechanisms in Listeria monocytogenes.

The human pathogen Listeria monocytogenes continues to pose a challenge in the food industry, where it is known to contaminate ready-to-eat foods and grow during refrigerated storage. Increased knowledge of the cold-stress response of this pathogen will enhance the ability to control it in the food-supply-chain. This study utilized strand-specific RNA sequencing and whole cell fatty acid (FA) profiling to characterize the bacterium’s cold stress response. RNA and FAs were extracted from a cold-tolerant strain at five time points between early lag phase and late stationary-phase, both at 4°C and 20°C. Overall, more genes (1.3×) were suppressed than induced at 4°C. Late stationary-phase cells exhibited the greatest number (n = 1,431) and magnitude (>1,000-fold) of differentially expressed genes (>2-fold, p<0.05) in response to cold. A core set of 22 genes was upregulated at all growth phases, including nine genes required for branched-chain fatty acid (BCFA) synthesis, the osmolyte transporter genes opuCBCD, and the internalin A and D genes. Genes suppressed at 4°C were largely associated with cobalamin (B12) biosynthesis or the production/export of cell wall components. Antisense transcription accounted for up to 1.6% of total mapped reads with higher levels (2.5×) observed at 4°C than 20°C. The greatest number of upregulated antisense transcripts at 4°C occurred in early lag phase, however, at both temperatures, antisense expression levels were highest in late stationary-phase cells. Cold-induced FA membrane changes included a 15% increase in the proportion of BCFAs and a 15% transient increase in unsaturated FAs between lag and exponential phase. These increases probably reduced the membrane phase transition temperature until optimal levels of BCFAs could be produced. Collectively, this research provides new information regarding cold-induced membrane composition changes in L. monocytogenes, the growth-phase dependency of its cold-stress regulon, and the active roles of antisense transcripts in regulating its cold stress response.

Persistent Listeria monocytogenes strains isolated from mussel production facilities form more biofilm but are not linked to specific genetic markers

Contamination of mussels with the human pathogen Listeria monocytogenes occurs during processing in the factory, possibly from bacteria persisting in the factory's indoor and outdoor areas. In this study, a selection of persistent (n=8) and sporadic (n=8) L. monocytogenes isolates associated with mussel-processing premises in New Zealand were investigated for their phenotypic and genomic characteristics. To identify traits that favour or contribute to bacterial persistence, biofilm formation, heat resistance, motility and recovery from dry surfaces were compared between persistent and sporadic isolates. All isolates exhibited low biofilm formation at 20°C, however, at 30°C persistent isolates showed significantly higher biofilm formation after 48h using cell enumeration and near significant difference using the crystal violet assay. All 16 isolates were motile at 20°C and 30°C and motility was fractionally higher for sporadic isolates, but no significant difference was observed. We found persistent isolates tend to exhibit greater recovery after incubation on dry surfaces compared to sporadic isolates. Two of the three most heat-resistant isolates were persistent, while four of five isolates lacking heat resistance were sporadic isolates. Comparison of genome sequences of persistent and sporadic isolates showed that there was no overall clustering of persistent or sporadic isolates, and that differences in prophages and plasmids were not associated with persistence. Our results suggest a link between persistence and biofilm formation, which is most likely multifactorial, combining subtle phenotypic and genotypic differences between isolates.

Genotypes Associated with Listeria monocytogenes Isolates Displaying Impaired or Enhanced Tolerances to Cold, Salt, Acid, or Desiccation Stress.

The human pathogen Listeria monocytogenes is a large concern in the food industry where its continuous detection in food products has caused a string of recalls in North America and Europe. Most recognized for its ability to grow in foods during refrigerated storage, L. monocytogenes can also tolerate several other food-related stresses with some strains possessing higher levels of tolerances than others. The objective of this study was to use a combination of phenotypic analyses and whole genome sequencing to elucidate potential relationships between L. monocytogenes genotypes and food-related stress tolerance phenotypes. To accomplish this, 166 L. monocytogenes isolates were sequenced and evaluated for their ability to grow in cold (4°C), salt (6% NaCl, 25°C), and acid (pH 5, 25°C) stress conditions as well as survive desiccation (33% RH, 20°C). The results revealed that the stress tolerance of L. monocytogenes is associated with serotype, clonal complex (CC), full length inlA profiles, and the presence of a plasmid which was identified in 55% of isolates. Isolates with full length inlA exhibited significantly (p < 0.001) enhanced cold tolerance relative to those harboring a premature stop codon (PMSC) in this gene. Similarly, isolates possessing a plasmid demonstrated significantly (p = 0.013) enhanced acid tolerance. We also identified nine new L. monocytogenes sequence types, a new inlA PMSC, and several connections between CCs and the presence/absence or variations of specific genetic elements. A whole genome single-nucleotide-variants phylogeny revealed sporadic distribution of tolerant isolates and closely related sensitive and tolerant isolates, highlighting that minor genetic differences can influence the stress tolerance of L. monocytogenes. Specifically, a number of cold and desiccation sensitive isolates contained PMSCs in σB regulator genes (rsbS, rsbU, rsbV). Collectively, the results suggest that knowing the sequence type of an isolate in addition to screening for the presence of full-length inlA and a plasmid, could help food processors and food agency investigators determine why certain isolates might be persisting in a food processing environment. Additionally, increased sequencing of L. monocytogenes isolates in combination with stress tolerance profiling, will enhance the ability to identify genetic elements associated with higher risk strains.

L-glutamine Induces Expression of Listeria monocytogenes Virulence Genes.

The high environmental adaptability of bacteria is contingent upon their ability to sense changes in their surroundings. Bacterial pathogen entry into host poses an abrupt and dramatic environmental change, during which successful pathogens gauge multiple parameters that signal host localization. The facultative human pathogen Listeria monocytogenes flourishes in soil, water and food, and in ~50 different animals, and serves as a model for intracellular infection. L. monocytogenes identifies host entry by sensing both physical (e.g., temperature) and chemical (e.g., metabolite concentrations) factors. We report here that L-glutamine, an abundant nitrogen source in host serum and cells, serves as an environmental indicator and inducer of virulence gene expression. In contrast, ammonia, which is the most abundant nitrogen source in soil and water, fully supports growth, but fails to activate virulence gene transcription. We demonstrate that induction of virulence genes only occurs when the Listerial intracellular concentration of L-glutamine crosses a certain threshold, acting as an on/off switch: off when L-glutamine concentrations are below the threshold, and fully on when the threshold is crossed. To turn on the switch, L-glutamine must be present, and the L-glutamine high affinity ABC transporter, GlnPQ, must be active. Inactivation of GlnPQ led to complete arrest of L-glutamine uptake, reduced type I interferon response in infected macrophages, dramatic reduction in expression of virulence genes, and attenuated virulence in a mouse infection model. These results may explain observations made with other pathogens correlating nitrogen metabolism and virulence, and suggest that gauging of L-glutamine as a means of ascertaining host localization may be a general mechanism.

Suppressor Mutations Linking gpsB with the First Committed Step of Peptidoglycan Biosynthesis in Listeria monocytogenes.

The late cell division protein GpsB is important for cell wall biosynthesis in Gram-positive bacteria. GpsB of the human pathogen L. monocytogenes interacts with one of the key enzymes of this pathway, penicillin binding protein A1 (PBP A1), and influences its activity. PBP A1 catalyzes the last two steps of cell wall biosynthesis, but it is unknown how GpsB controls PBP A1. We observed that a L. monocytogenes gpsB mutant forms spontaneous suppressors and have mapped their mutations to genes mediating and influencing the first step of cell wall biosynthesis, likely stimulating the influx of metabolites into this pathway. We assume that GpsB is important to ensure productive incorporation of cell wall precursors into the peptidoglycan sacculus by PBP A1.

Structural basis for glutathione-mediated activation of the virulence regulatory protein PrfA in Listeria

Infection by the human bacterial pathogen Listeria monocytogenes is mainly controlled by the positive regulatory factor A (PrfA), a member of the Crp/Fnr family of transcriptional activators. Published data suggest that PrfA requires the binding of a cofactor for full activity, and it was recently proposed that glutathione (GSH) could fulfill this function. Here we report the crystal structures of PrfA in complex with GSH and in complex with GSH and its cognate DNA, the hly operator PrfA box motif. These structures reveal the structural basis for a GSH-mediated allosteric mode of activation of PrfA in the cytosol of the host cell. The crystal structure of PrfAWT in complex only with DNA confirms that PrfAWT can adopt a DNA binding-compatible structure without binding the GSH activator molecule. By binding to PrfA in the cytosol of the host cell, GSH induces the correct fold of the HTH motifs, thus priming the PrfA protein for DNA interaction.

Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes.

The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteria Streptomyces davawensis and Streptomyces cinnabarinus Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (vitamin B2), RoF, and AF in the human pathogen Listeria monocytogenes, a bacterium which is a riboflavin auxotroph. We show that the L. monocytogenes protein Lmo1945 is responsible for the uptake of riboflavin, RoF, and AF. Following import, these flavins are phosphorylated/adenylylated by the bifunctional flavokinase/flavin adenine dinucleotide (FAD) synthetase Lmo1329 and adenylylated by the unique FAD synthetase Lmo0728, the first monofunctional FAD synthetase to be described in bacteria. Lmo1329 generates the cofactors flavin mononucleotide (FMN) and FAD, whereas Lmo0728 produces FAD only. The combined activities of Lmo1329 and Lmo0728 are responsible for the intracellular formation of the toxic cofactor analogs roseoflavin mononucleotide (RoFMN), roseoflavin adenine dinucleotide (RoFAD), 8-demethyl-8-aminoriboflavin mononucleotide (AFMN), and 8-demethyl-8-aminoriboflavin adenine dinucleotide (AFAD). In vivo reporter gene assays and in vitro transcription/translation experiments show that the L. monocytogenes FMN riboswitch Rli96, which controls expression of the riboflavin transport gene lmo1945, is negatively affected by riboflavin/FMN and RoF/RoFMN but not by AF/AFMN. Treatment of L. monocytogenes with RoF or AF leads to drastically reduced FMN/FAD levels. We suggest that the reduced flavin cofactor levels in combination with concomitant synthesis of inactive cofactor analogs (RoFMN, RoFAD, AFMN, and AFAD) explain why RoF and AF contribute to antibiotic activity in L. monocytogenes IMPORTANCE: The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are small molecules which are produced by Streptomyces davawensis and Streptomyces cinnabarinus RoF and AF were reported to have antibacterial activity, and we studied how these compounds are metabolized by the human bacterial pathogen Listeria monocytogenes We found that the L. monocytogenes protein Lmo1945 mediates uptake of AF and RoF and that the combined activities of the enzymes Lmo1329 and Lmo0728 are responsible for the conversion of AF and RoF to toxic cofactor analogs. Comparative studies with RoF and AF (a weaker antibiotic) suggest that the reduction in FMN/FAD levels and the formation of inactive FMN/FAD analogs explain to a large extent the antibiotic activity of AF and RoF.

Two novel members of the LhrC family of small RNAs in Listeria monocytogenes with overlapping regulatory functions but distinctive expression profiles

ABSTRACTMulticopy small RNAs (sRNAs) have gained recognition as an important feature of bacterial gene regulation. In the human pathogen Listeria monocytogenes, 5 homologous sRNAs, called LhrC1-5, control gene expression by base pairing to target mRNAs though 3 conserved UCCC motifs common to all 5 LhrCs. We show here that the sRNAs Rli22 and Rli33-1 are structurally and functionally related to LhrC1-5, expanding the LhrC family to 7 members, which makes it the largest multicopy sRNA family reported so far. Rli22 and Rli33-1 both contain 2 UCCC motifs important for post-transcriptional repression of 3 LhrC target genes. One such target, oppA, encodes a virulence-associated oligo-peptide binding protein. Like LhrC1-5, Rli22 and Rli33-1 employ their UCCC motifs to recognize the Shine-Dalgarno region of oppA mRNA and prevent formation of the ribosomal complex, demonstrating that the 7 sRNAs act in a functionally redundant manner. However, differential expression profiles of the sRNAs under infection-relevant conditions suggest that they might also possess non-overlapping functions. Collectively, this makes the LhrC family a unique case for studying the purpose of sRNA multiplicity in the context of bacterial virulence.

Attenuating Listeria monocytogenes Virulence by Targeting the Regulatory Protein PrfA

SummaryThe transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes cellular uptake by reducing the expression of virulence genes. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA-binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds at two separate sites on the protein: one within a hydrophobic pocket or tunnel, located between the C- and N-terminal domains of PrfA, and the second in the vicinity of the DNA-binding helix-turn-helix motif. At both sites the compound interacts with residues important for PrfA activation and helix-turn-helix formation. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.

The intrinsic cephalosporin resistome of Listeria monocytogenes in the context of stress response, gene regulation, pathogenesis and therapeutics.

Intrinsic resistance to antibiotics is a serious therapeutic problem in the case of many bacterial species. The Gram-positive human pathogen Listeria monocytogenes is intrinsically resistant to broad spectrum cephalosporin antibiotics, which are commonly used in therapy of bacterial infections. Besides three penicillin-binding proteins the intrinsic cephalosporin resistome of L. monocytogenes includes multidrug resistance transporter transporters, proteins involved in peptidoglycan biosynthesis and modification, cell envelope proteins with structural or general detoxification function, cytoplasmic proteins with unknown function and regulatory proteins. Analysis of the regulation of the expression of genes involved in the intrinsic resistance of L. monocytogenes to cephalosporins highlights the high complexity of control of the intrinsic resistance phenotype. The regulation of the transcription of the intrinsic resistome determinants involves the activity of eight regulators, namely LisR, CesR, LiaR, VirR, σ(B) , σ(H) , σ(L) and PrfA, of which the most prominent role play LisR, CesR and σ(B) . Furthermore, the vast majority of the intrinsic resistome determinants contribute to the tolerance of different stress conditions and virulence. A study indicates that O-acetyltransferase OatA is the most promising candidate for co-drug development since an agent targeting OatA should sensitize L. monocytogenes to certain antibiotics, therefore improving the efficacy of listeriosis treatment as well as food preservation measures.

Structure of the bacterial cell division determinant GpsB and its interaction with penicillin-binding proteins.

Each bacterium has to co-ordinate its growth with division to ensure genetic stability of the population. Consequently, cell division and growth are tightly regulated phenomena, albeit different bacteria utilise one of several alternative regulatory mechanisms to maintain control. Here we consider GpsB, which is linked to cell growth and division in Gram-positive bacteria. ΔgpsB mutants of the human pathogen Listeria monocytogenes show severe lysis, division and growth defects due to distortions of cell wall biosynthesis. Consistent with this premise, GpsB interacts both in vitro and in vivo with the major bi-functional penicillin-binding protein. We solved the crystal structure of GpsB and the interaction interfaces in both proteins are identified and validated. The inactivation of gpsB results in strongly attenuated virulence in animal experiments, comparable in degree to classical listerial virulence factor mutants. Therefore, GpsB is essential for in vitro and in vivo growth of a highly virulent food-borne pathogen, suggesting that GpsB could be a target for the future design of novel antibacterials.

Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes.

Cyclic diadenylate monophosphate (c-di-AMP) is a second messenger utilized by diverse bacteria. In many species, including the Gram-positive human pathogen Listeria monocytogenes, c-di-AMP is essential for growth. Here we show that the single diadenylate cyclase of L. monocytogenes, CdaA, is an integral membrane protein that interacts with its potential regulatory protein, CdaR, via the transmembrane protein domain. The presence of the CdaR protein is not required for the membrane localization and abundance of CdaA. We have also found that CdaR negatively influences CdaA activity in L. monocytogenes and that the role of CdaR is most evident at a high growth temperature. Interestingly, a cdaR mutant strain is less susceptible to lysozyme. Moreover, CdaA contributes to cell division, and cells depleted of CdaA are prone to lysis. The observation that the growth defect of a CdaA depletion strain can be partially restored by increasing the osmolarity of the growth medium suggests that c-di-AMP is important for maintaining the integrity of the protective cell envelope. Overall, this work provides new insights into the relationship between CdaA and CdaR. Cyclic diadenylate monophosphate (c-di-AMP) is a recently identified second messenger that is utilized by the Gram-positive human pathogen Listeria monocytogenes. Here we show that the single diadenylate cyclase of L. monocytogenes, CdaA, is an integral membrane protein that interacts with CdaR, its potential regulatory protein. We show that CdaR is not required for membrane localization or abundance of the diadenylate cyclase, but modulates its activity. Moreover, CdaA seems to contribute to cell division. Overall, this work provides new insights into the relationship between CdaA and CdaR and their involvement in cell growth.

Comparative Genomics of Listeria Sensu Lato: Genus-Wide Differences in Evolutionary Dynamics and the Progressive Gain of Complex, Potentially Pathogenicity-Related Traits through Lateral Gene Transfer.

Historically, genome-wide and molecular characterization of the genus Listeria has concentrated on the important human pathogen Listeria monocytogenes and a small number of closely related species, together termed Listeria sensu strictu. More recently, a number of genome sequences for more basal, and nonpathogenic, members of the Listeria genus have become available, facilitating a wider perspective on the evolution of pathogenicity and genome level evolutionary dynamics within the entire genus (termed Listeria sensu lato). Here, we have sequenced the genomes of additional Listeria fleischmannii and Listeria newyorkensis isolates and explored the dynamics of genome evolution in Listeria sensu lato. Our analyses suggest that acquisition of genetic material through gene duplication and divergence as well as through lateral gene transfer (mostly from outside Listeria) is widespread throughout the genus. Novel genetic material is apparently subject to rapid turnover. Multiple lines of evidence point to significant differences in evolutionary dynamics between the most basal Listeria subclade and all other congeners, including both sensu strictu and other sensu lato isolates. Strikingly, these differences are likely attributable to stochastic, population-level processes and contribute to observed variation in genome size across the genus. Notably, our analyses indicate that the common ancestor of Listeria sensu lato lacked flagella, which were acquired by lateral gene transfer by a common ancestor of Listeria grayi and Listeria sensu strictu, whereas a recently functionally characterized pathogenicity island, responsible for the capacity to produce cobalamin and utilize ethanolamine/propane-2-diol, was acquired in an ancestor of Listeria sensu strictu.

Using the chicken embryo to assess virulence of Listeria monocytogenes and to model other microbial infections.

Microbial infections are a global health problem, particularly as microbes are continually developing resistance to antimicrobial treatments. An effective and reliable method for testing the virulence of different microbial pathogens is therefore a useful research tool. This protocol describes how the chicken embryo can be used as a trustworthy, inexpensive, ethically desirable and quickly accessible model to assess the virulence of the human bacterial pathogen Listeria monocytogenes, which can also be extended to other microbial pathogens. We provide a step-by-step protocol and figures and videos detailing the method, including egg handling, infection strategies, pathogenicity screening and isolation of infected organs. From the start of incubation of the fertilized eggs, the protocol takes <4 weeks to complete, with the infection part taking only 3 d. We discuss the appropriate controls to use and potential adjustments needed for adapting the protocol for other microbial pathogens.

RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP.

Cyclic di-AMP (cdiA) is a second messenger predicted to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intracellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore, a flow cytometry assay based on this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, we have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.

Molecular basis for self-assembly of a human host-defense peptide that entraps bacterial pathogens.

Human α-defensin 6 (HD6) is a 32-aa cysteine-rich peptide of the innate immune system. Although HD6 is a member of an antimicrobial peptide family, it exhibits negligible antibacterial activity in vitro. Rather, HD6 possesses a unique innate immune mechanism whereby it self-assembles into oligomers that capture pathogens to prevent microbial invasion of the intestinal epithelium and subsequent dissemination. Molecular-level understanding for why HD6 functions differently from other human defensins remains unclear. To further elucidate the HD6 self-assembly process and its biological activity, we developed robust protocols for obtaining native and mutant HD6 in high purity from overexpression in Escherichia coli. We combined biophysical characterization with biological assays to probe HD6 structure and function. We report that native HD6 readily self-assembles into elongated fibrils observable by transmission electron microscopy, agglutinates both Gram-negative and -positive bacteria, and prevents the human gastrointestinal pathogen Listeria monocytogenes from invading cultured mammalian cells. Mutation of hydrophobic residues (F2A, I22T, V25T, F29A) perturbs self-assembly and results in attenuated biological activity. In particular, the F2A and F29A mutants do not form fibrils under our experimental conditions and neither agglutinate bacteria nor prevent L. monocytogenes invasion. In total, our results demonstrate that the hydrophobic effect is essential for promoting HD6 self-assembly and innate immune function, and indicate that HD6 may provide host defense against Listeria in the gut. This investigation provides a timely description of how variations in amino acid sequence confer diverse physiological functions to members of the defensin family.