Identification of chitinase as a virulence factor in Listeria monocytogenes

Listeria monocytogenes is a food borne Gram positive pathogen that causes the disease listeriosis in humans and animals. L. monocytogenes has been the responsible for a number of food-borne outbreaks of listeriosis among humans. The disease caused by this bacterium can become very severe in immunocompromised people, often resulting in deaths. L. monocytogenes is ubiquitious in nature, and is found in diverse ecological niches such as soil, water and natural vegetation. However, when this free living bacterium enters a mammalian host it often causes disease, and is therefore regarded as an environmental pathogen. Environmental pathogens are those microorganisms that spend substantial parts of their life cycles outside their hosts, but can cause disease when they are inside the host. An important question about environmental pathogens is whether they are able to use molecules that are required for their survival outside the host, as virulence factors when they are inside their hosts. This hypothesis has come into the focus with the emergence of chitinases and chitin binding proteins as virulence factors in two other environmental pathogens, Legionella pneumophila and Vibrio cholerae. Chitinases are enzymes that hydrolyze chitin, which is a linear polymer of N-acetylglucosamine residues linked by (β1, 4) glycosidic bonds. Chitin is present in cell walls of algae and fungi; and in the exoskeletons of insects, mollusks, crustaceans and is the second most abundant organic compound on the earth. Bacterial chitinases and chitin binding proteins are supposed to be used for nutrient acquisition by utilizing chitin as a readily available source of carbon and nitrogen. However, with the works done on L. pneumophila and V. cholerae, it is evident that these pathogenic bacteria use chitinases and chitin binding proteins to enhance their survival in mammalian hosts as well. The goal of this thesis is to find out whether L. monocytogenes uses its chitinase and chitin binding proteins for its survival inside animal models of infection. The thesis has two main parts. The first part will examine whether mutant strains of L. monocytogenes lacking chitinase and/or chitin binding proteins are attenuated in in vitro and in vivo models of infection. The second part of the thesis will examine the probable mechanisms by which chitinases of L. monocytogenes can be used by the bacterium to enhance its survival in the host

Contribution of Chitinases to Listeria monocytogenes Pathogenesis ▿

Listeria monocytogenes secretes two chitinases and one chitin binding protein. Mutants lacking chiA, chiB, or lmo2467 exhibited normal growth in cultured cells but were defective for growth in the livers and spleens of mice. Mammals lack chitin; thus, L. monocytogenes may have adapted chitinases to recognize alternative substrates to enhance pathogenesis

The Listeria monocytogenes ChiA Chitinase Enhances Virulence through Suppression of Host Innate Immunity

Environmental pathogens survive and replicate within the outside environment while maintaining the capacity to infect mammalian hosts. For some microorganisms, mammalian infection may be a relatively rare event. Understanding how environmental pathogens retain their ability to cause disease may provide insight into environmental reservoirs of disease and emerging infections. Listeria monocytogenes survives as a saprophyte in soil but is capable of causing serious invasive disease in susceptible individuals. The bacterium secretes virulence factors that promote cell invasion, bacterial replication, and cell-to-cell spread. Recently, an L. monocytogenes chitinase (ChiA) was shown to enhance bacterial infection in mice. Given that mammals do not synthesize chitin, the function of ChiA within infected animals was not clear. Here we have demonstrated that ChiA enhances L. monocytogenes survival in vivo through the suppression of host innate immunity. L. monocytogenes Delta chiA mutants were fully capable of establishing bacterial replication within target organs during the first 48 h of infection. By 72 to 96 h postinfection, however, numbers of Delta chiA bacteria diminished, indicative of an effective immune response to contain infection. The Delta chiA-associated virulence defect could be complemented in trans by wild-type L. monocytogenes, suggesting that secreted ChiA altered a target that resulted in a more permissive host environment for bacterial replication. ChiA secretion resulted in a dramatic decrease in inducible nitric oxide synthase (iNOS) expression, and Delta chiA mutant virulence was restored in NOS2(-/-) mice lacking iNOS. This work is the first to demonstrate modulation of a specific host innate immune response by a bacterial chitinase

Genetic analysis of the CDI pathway from Burkholderia pseudomallei 1026b.

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIIIBp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIIIBp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIIIBp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTIIBp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI⁺ inhibitor cells to the same extent as BTH_I0986⁺ cells, suggesting that LPS could function as a receptor for CdiAIIBp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIIIBp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species

Genetic analysis of the CDI pathway from Burkholderia pseudomallei 1026b.

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIIIBp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIIIBp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIIIBp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTIIBp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI⁺ inhibitor cells to the same extent as BTH_I0986⁺ cells, suggesting that LPS could function as a receptor for CdiAIIBp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIIIBp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species

Toxicity of CdiA-CTII^Bp1026b expressed inside <i>B</i>. <i>thailandensis</i> cells.

<p>Plasmids pSCBAD and pSCBAD::<i>cdiA-CT</i>_II^Bp1026b were introduced into the indicated <i>B</i>. <i>thailandensis</i> strains by conjugation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#sec002" target="_blank">Materials and Methods</a>. The mating mixtures were split into equal portions and plated onto LB agar with Polymyxin B and Trimethoprim supplemented with either D-glucose (left panels) or L-arabinose (right panels). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#sec002" target="_blank">Materials and Methods</a>.</p

Complementation of CDI^R mutations.

<p>The indicated <i>B</i>. <i>thailandensis</i> strains were co-cultured with Bt81 inhibitors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#pone.0120265.t001" target="_blank">Table 1</a>) that express the CDI_II^Bp1026b system for 24 h on solid medium, and the competitive index was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#sec002" target="_blank">Materials and Methods</a>. The strain labeled <i>cdiI</i>^1026b expresses the cognate CdiI_II^Bp1026b immunity protein. Plasmid-borne copies of BTH_I0359, BTH_I0986 and BTH_II0599 genes were expressed from an L-arabinose inducible promoter. Data represent the mean ± SEM for three independent experiments. Sample values that were statistically different from one another (p < 0.05) are shown by bars with an asterisk (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#pone.0120265.g001" target="_blank">Fig. 1</a>).</p

Selection of CDI^R mutants of <i>B</i>. <i>thailandensis</i> E264.

<p>A) T23 transposon insertion sites were identified by semi-arbitrary PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#sec002" target="_blank">Methods</a>. Orange arrows indicate T23 insertions in the same transcriptional orientation of the disrupted gene and blue arrows indicate insertions in the opposite orientation. The corresponding CDI^R mutant strain number is given above each arrow. Automated gene annotations are given below each ordered locus designation. GT-1, GT-2 and GT-9 indicate predicted glycosyltransferase families and DUF designations indicate domains of unknown function. B) The indicated <i>B</i>. <i>thailandensis</i> strains were co-cultured with Bt81 inhibitors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#pone.0120265.t001" target="_blank">Table 1</a>) that express the CDI_II^Bp1026b system for 24 h on solid medium, and the competitive index was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#sec002" target="_blank">Materials and Methods</a>. The strain labeled <i>cdiI</i>^1026b expresses the cognate CdiI_II^Bp1026b immunity protein. Data represent the mean ± SEM for three independent experiments. Analysis of the data using Student’s <i>t-</i>test is shown at the top, with bars between samples that were statistically significant (* = p< 0.05).</p

Cell-cell binding.

<p>CDI⁺ (Bt81) and CDI^– (wild-type <i>B</i>. <i>thailandensis</i>) cells were labeled with GFP and mixed with the indicated DsRed-labeled target cells, then analyzed by flow cytometry to detect and quantify cell-cell aggregates. Binding was normalized to 1.0 for the interaction between Bt81 and wild-type <i>B</i>. <i>thailandensis</i> cells. Sample values that were statistically different from one another are shown by bars; ** = p < 0.01, and *** = p < 0.001 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#pone.0120265.g001" target="_blank">Fig. 1</a>). We then tested the three CDI^R target strains and found that ΔBTH_I0986 targets interacted poorly with inhibitor cells, similar to the level observed with CDI^– mock inhibitors (Fig. 6). In contrast, the ΔBTH_II0599 mutant showed wild-type binding levels, and ΔBTH_I0359 targets showed increased binding to inhibitor cells (Fig. 6). Together, these results suggest that mutations in BTH_I0986 confer CDI^R by altering the cell surface to prevent stable associations with CDI_II^Bp1026b inhibitor cells.</p

The CDI^R phenotype is specific for CDIII^Bp1026b.

<p>The indicated <i>B</i>. <i>thailandensis</i> strains were co-cultured with wild-type (<i>cdiAIB</i>⁺) <i>B</i>. <i>thailandensis</i> E264 cells for 24 h on solid medium, and the competitive index was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#sec002" target="_blank">Materials and Methods</a> The strain labeled <i>cdiI</i>^E264 expresses the cognate CdiI^E264 immunity protein. Data represent the mean ± SEM for three independent experiments. Sample values that were statistically different from one another (p < 0.01) are shown by a bar with a double asterisk (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120265#pone.0120265.g001" target="_blank">Fig. 1</a>).</p