Marianne Halberg Larsen

The RNA-Binding Protein Hfq of Listeria monocytogenes: Role in Stress Tolerance and Virulence

In gram-negative bacteria, the RNA-binding protein Hfq has emerged as an important regulatory factor in a variety of physiological processes, including stress resistance and virulence. In Escherichia coli, Hfq modulates the stability or the translation of mRNAs and interacts with numerous small regulatory RNAs. Here, we studied the role of Hfq in the stress tolerance and virulence of the gram-positive food-borne human pathogen Listeria monocytogenes. We present evidence that Hfq is involved in the ability of L. monocytogenes to tolerate osmotic and ethanol stress and contributes to long-term survival under amino acid-limiting conditions. However, Hfq is not required for resistance to acid and oxidative stress. Transcription of hfq is induced under various stress conditions, including osmotic and ethanol stress and at the entry into the stationary growth phase, thus supporting the view that Hfq is important for the growth and survival of L. monocytogenes in harsh environments. The stress-inducible transcription of hfq depends on the alternative sigma factor sigmaB, which controls the expression of numerous stress- and virulence-associated genes in L. monocytogenes. Infection studies showed that Hfq contributes to pathogenesis in mice, yet plays no role in the infection of cultured cell lines. This study provides, for the first time, information on the role of Hfq in the stress tolerance and virulence of a gram-positive pathogen.

BACTERIAL CHITINASES AND CHITIN-BINDING PROTEINS AS VIRULENCE FACTORS

Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities, regulation, secretion and involvement in cellular and animal infection.

Sodium Chloride Enhances Adherence and Aggregation and Strain Variation Influences Invasiveness of Listeria monocytogenes Strains

Some subtypes of Listeria monocytogenes can persist in the food-processing industry, but the reasons for such persistence are not known. In the present study, 10 strains of L. monocytogenes representing known persistent randomly amplified polymorphic DNA (RAPD) types from fish processing plants were compared to eight strains of different RAPD type and origin (clinical, food, and animal). All 18 strains of L. monocytogenes had similar growth patterns at different temperatures (5 or 37 degrees C) or different salinities (0.5 or 5% NaCl), and all strains formed a thin layer of adhered cells on a plastic surface when cultured in tryptone soya broth (TSB) with a total of 1% glucose. Many ready-to-eat foods, such as cold-smoked fish, contain NaCl at concentrations of 2 to 5%, and NaCl is present in the processing environment. Adding NaCl to TSB changed the adhesion patterns of all strains, and all adhered better when NaCl was added. Also, the addition of NaCl caused a marked aggregation of 13 of the strains; however, 5 of the 18 strains did not aggregate in the presence of up to 5% NaCl. The aggregates stuck to the plastic surface, and this occurred in all but one of the persistent RAPD types. Four strains represented one particular RAPD type that has been isolated as a persistent RAPD type in several fish processing plants for up to 10 years. Because this RAPD type often can contaminate fish products, it is important to address its potential virulence. The 18 strains differed markedly in their ability to invade Caco-2 cells, and the four strains representing the universal persistent RAPD type were the least invasive (10(2) to 10(3) CFU/ml), whereas other strains invaded Caco-2 cells at levels of 10(4) to 10(5) CFU/ml. Five of the 18 strains belonged to the genetic lineage 1 and were the most invasive. Although the most commonly isolated persistent RAPD type was low invasive, it is important to understand why moderate salinity facilitates aggregation and biofilm formation, for this understanding can be beneficial in developing procedures to reduce processing plant contamination.

A Small RNA Controls Expression of the Chitinase ChiA in Listeria monocytogenes

In recent years, more than 60 small RNAs (sRNAs) have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo0850, which encodes a small protein of unknown function. LhrA controls the translation and degradation of the lmo0850 mRNA by an antisense mechanism, and it depends on the RNA chaperone Hfq for efficient binding to its target. In the present study, we sought to gain more insight into the functional role of LhrA in L. monocytogenes. To this end, we determined the effects of LhrA on global-wide gene expression. We observed that nearly 300 genes in L. monocytogenes are either positively or negatively affected by LhrA. Among these genes, we identified lmo0302 and chiA as direct targets of LhrA, thus establishing LhrA as a multiple target regulator. Lmo0302 encodes a hypothetical protein with no known function, whereas chiA encodes one of two chitinases present in L. monocytogenes. We show here that LhrA acts as a post-transcriptional regulator of lmo0302 and chiA by interfering with ribosome recruitment, and we provide evidence that both LhrA and Hfq act to down-regulate the expression of lmo0302 and chiA. Furthermore, in vitro binding experiments show that Hfq stimulates the base pairing of LhrA to chiA mRNA. Finally, we demonstrate that LhrA has a negative effect on the chitinolytic activity of L. monocytogenes. In marked contrast to this, we found that Hfq has a stimulating effect on the chitinolytic activity, suggesting that Hfq plays multiple roles in the complex regulatory pathways controlling the chitinases of L. monocytogenes.

Processing plant persistent strains of Listeria monocytogenes appear to have a lower virulence potential than clinical strains in selected virulence models.

Listeria monocytogenes is an important foodborne bacterial pathogen that can colonize food processing equipment. One group of genetically similar L. monocytogenes strains (RAPD type 9) was recently shown to reside in several independent fish processing plants. Persistent strains are likely to contaminate food products, and it is important to determine their virulence potential to evaluate risk to consumers. We compared the behaviour of food processing persistent and clinical L. monocytogenes strains in four virulence models: Adhesion, invasion and intracellular growth was studied in an epithelial cell line, Caco-2; time to death in a nematode model, Caenorhabditis elegans and in a fruit fly model, Drosophila melanogaster and fecal shedding in a guinea pig model. All strains adhered to and grew in Caco-2 cells in similar levels. When exposed to 10(6) CFU/ml, two strains representing the persistent RAPD type 9 invaded Caco-2 cells in lower numbers (10(2)-10(3) CFU/ml) as compared to the four other strains (10(4)-10(6) CFU/ml), including food and human clinical strains. In the D. melanogaster model, the two RAPD type 9 strains were among the slowest to kill. Similarly, the time to reach 50% killed C. elegans worms was longer (110 h) for the RAPD type 9 strains than for the other four strains (80 h). The Scott A strain and one RAPD type 9 strain were suspended in whipping cream before being fed to guinea pigs and the persistent RAPD type 9 strain was isolated from feces in a lower level (approximately 10(2) CFU/g) than the Scott A strain (approximately 10(5) CFU/g) (P<0.05). The addition of NaCl has been shown to cause autoaggregation and increases adhesion of L. monocytogenes to plastic. However, growth in the presence of NaCl did not alter the behaviour of the tested L. monocytogenes strains in the virulence models. Overall, the two strains representing a very common fish processing plant persistent group (RAPD type 9) appear to have a lower virulence potential in all four virulence models than Scott A and a strain isolated from a clinical case of listeriosis.

The response regulator ResD modulates virulence gene expression in response to carbohydrates in Listeria monocytogenes

Listeria monocytogenes is a versatile bacterial pathogen that is able to accommodate to diverse environmental and host conditions. Presently, we have identified a L. monocytogenes two‐component response regulator, ResD that is required for the repression of virulence gene expression known to occur in the presence of easily fermentable carbohydrates not found inside host organisms. Structurally and functionally, ResD resembles the respiration regulator ResD in Bacillus subtilis as deletion of the L. monocytogenes resD reduces respiration and expression of cydA, encoding a subunit of cytochrome bd. The resD mutation also reduces expression of mptA encoding the EIIABman component of a mannose/glucose‐specific PTS system, indicating that ResD controls sugar uptake. This notion was supported by the poor growth of resD mutant cells that was alleviated by excess of selected carbohydrates. Despite the growth deficient phenotype of the mutant in vitro the mutation did not affect intracellular multiplication in epithelial or macrophage cell lines. When examining virulence gene expression we observed traditional induction by charcoal in both mutant and wild‐type cells whereas the repression observed in wild‐type cells by fermentable carbohydrates did not occur in resD mutant cells. Thus, ResD is a central regulator of L. monocytogenes when present in the external environment.

Influence of Sublethal Concentrations of Common Disinfectants on Expression of Virulence Genes in Listeria monocytogenes

ABSTRACT Listeria monocytogenes is a food-borne human pathogen that causes listeriosis, a relatively rare infection with a high fatality rate. The regulation of virulence gene expression is influenced by several environmental factors, and the aim of the present study was to determine how disinfectants used routinely in the food industry affect the expression of different virulence genes in L. monocytogenes when added at sublethal concentrations. An agar-based assay was developed to screen the effect of disinfectants on virulence gene promoter expression and was validated at the transcriptional level by Northern blot analysis. Eleven disinfectants representing four different groups of active components were evaluated in this study. Disinfectants with the same active ingredients had a similar effect on gene expression. Peroxy and chlorine compounds reduced the expression of the virulence genes, and quaternary ammonium compounds (QAC) induced the expression of the virulence genes. In general, a disinfectant had similar effects on the expression of all four virulence genes examined. Northern blot analyses confirmed the downregulation of prfA and inlA expression by Incimaxx DES (a peroxy compound) and their upregulation by Triquart Super (a QAC) in L. monocytogenes EGD. Hence, sublethal concentrations of disinfectants routinely used in the food industry affect virulence gene expression in the human pathogen L. monocytogenes, and the effect depends on the active components of the disinfectant. From a practical perspective, the study underlines that disinfectants should be used at the lethal concentrations recommended by the manufacturers. Further studies are needed to elucidate whether the changes in virulence gene expression induced by the disinfectants have impact on virulence or other biological properties, such as antibiotic resistance.

Effects of stress and adrenalectomy on activity-regulated cytoskeleton protein (Arc) gene expression.

Activity-regulated cytoskeletal-associated protein (Arc) is an effector immediate early gene induced by novelty and involved in consolidation of long-term memory. Since activation of glucocorticoid receptors is a prerequisite for memory consolidation, we therefore aimed to study the effect of acute restraint stress on Arc gene expression in adrenalectomized rats. Acute stress produced a significant increase in Arc gene expression in the medial prefrontal cortex, but not in the parietal cortex or in the pyramidal cell layer of the hippocampus. The basal level of Arc mRNA in adrenalectomized animals was high in the medial prefrontal cortex and unaffected by acute stress in these animals. These data are consistent with the role of Arc as an integrative modulator of synaptic plasticity by emphasizing the potential role of stress and glucocorticoids in the control of Arc gene expression.

Chitin Hydrolysis by Listeria spp., Including L. monocytogenes

ABSTRACT Listeria spp., including the food-borne pathogen Listeria monocytogenes, are ubiquitous microorganisms in the environment and thus are difficult to exclude from food processing plants. The factors that contribute to their multiplication and survival in nature are not well understood, but the ability to catabolize various carbohydrates is likely to be very important. One major source of carbon and nitrogen in nature is chitin, an insoluble linear β-1,4-linked polymer of N-acetylglucosamine (GlcNAc). Chitin is found in cell walls of fungi and certain algae, in the cuticles of arthropods, and in shells and radulae of molluscs. In the present study, we demonstrated that L. monocytogenes and other Listeria spp. are able to hydrolyze α-chitin. The chitinolytic activity is repressed by the presence of glucose in the medium, suggesting that chitinolytic activity is subjected to catabolite repression. Activity is also regulated by temperature and is higher at 30°C than at 37°C. In L. monocytogenes EGD, chitin hydrolysis depends on genes encoding two chitinases, lmo0105 (chiB) and lmo1883 (chiA), but not on a gene encoding a putative chitin binding protein (lmo2467). The chiB and chiA genes are phylogenetically related to various well-characterized chitinases. The potential biological implications of chitinolytic activity of Listeria are discussed.

Regulation of activity-regulated cytoskeleton protein (Arc) mRNA after acute and chronic electroconvulsive stimulation in the rat.

The temporal profile of Arc gene expression after acute and chronic electroconvulsive stimulations (ECS) was studied using semi-quantitative in situ hybridisation in the rat cortex. A single ECS strongly and temporarily increased Arc mRNA levels in dentate granular cells with maximal induction seen up to 4 h after the stimulus, but returned to baseline at 24 h. A single ECS also increased expression of Arc mRNA in the CA1 and the parietal cortex, but the expression peaked within 1 h and returned to baseline levels within 2 h. Repeated or chronic ECS is a model of electroconvulsive therapy and it would be predicted that gene products involved in antidepressant effects accumulate after repeated ECS. However, repeated ECS reduced Arc gene expression in the CA1 24 h after the last stimulus. These results indicate that Arc is an immediate early gene product regulated by an acute excitatory stimulus, but not accumulated by long term repetitive ECS and therefore not a molecular biomarker for antidepressant properties. More likely, Arc is likely a molecular link to the decline in memory consolidation seen in depressive patients subjected to electroconvulsive therapy.