How Listeria monocytogenes organizes its surface for virulence

Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them is located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work "behind the frontline", either supporting virulence effectors or ensuring the survival of the bacterium within its host.We apologize to authors whose relevant work could not be cited owing to space limitations. Research in the group of Molecular Microbiology is funded by the project "NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions" co-funded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referencia Estrategico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER), the Operational Competitiveness Programme (COMPETE) and FCT (Fundacdo para a Ciencia e Tecnologia), and by projects ERANet Pathogenomics LISTRESS ERA-PTG/0003/2010, PTDC/SAU-MIC/111581/2009FCOMP-FEDER, PTDC/BIA-BCM/100088/2008FCOMP-01-0124-FEDER-008860 and PTDC/BIA-BCM/111215/2009FCOMP-01-0124-FEDER-014178. Filipe Carvalho was supported by FCT doctoral grant SFRH1BD16182512009, and Sandra Sousa by the Ciencia 2008 and FCT-Investigator programs (COMPETE, POPH, and FCT)

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

Virulence genes transcription is reduced in the <i>ΔglnPQ</i> mutant and specifically depends on L-glutamine.

<p>(A) Time course measurements of normalized luminescence (Lum/OD) indicative of <i>hly</i> promoter activity in WT <i>L</i>. <i>monocytogenes</i> (circles), Δ<i>glnPQ</i> (squares) or <i>glnQ</i>-E164A (triangles) bacteria grown in MDM with 0.25 mM of L-glutamine. Shown are representative curves of 3 independent experiments, performed in triplicates. (B) Maximal normalized luminescence (Lum/OD) was measured for WT (circles), Δ<i>glnPQ</i> (squares) or <i>glnQ</i>-E164A (triangles) bacteria grown under the indicated concentrations of L-glutamine. Results are mean ±SD of 3 independent experiments performed in triplicates. (C) RT-qPCR analysis of <i>hly</i>, <i>plcA</i>, <i>plcB</i>, and <i>actA</i> transcript levels in WT <i>L</i>. <i>monocytogenes</i> (black bars) or Δ<i>glnPQ</i> (gray bars) bacteria grown in MDM with 4 mM L-glutamine. Transcription levels presented as relative quantity (RQ), relative to their levels in WT bacteria. Results are mean ±SD of 3 independent experiments performed in triplicates. (D) Time course measurements of normalized luminescence (Lum/OD) indicative of <i>hly</i> promoter activity for WT (circles) and <i>ΔglnPQ</i> (squares) bacteria grown on MDM with 0.5 mM L-glutamine (full symbols) or 0.5 mM NH₄Cl (empty symbols) as the sole nitrogen source. (E) Time course measurements of normalized luminescence (Lum/OD) indicative of <i>hly</i> promoter activity for WT (circles) and Δ<i>glnPQ</i> (squares) bacteria grown on MDM with 0.5 mM L-glutamine (full symbols) or 0.5 mM D-glutamine (empty symbols) as the sole nitrogen source. (F) Time course measurements of normalized luminescence (Lum/OD) indicative of <i>hly</i> promoter activity for WT (circles) and Δ<i>glnPQ</i> (squares) bacteria grown on MDM with 0.5 mM L-glutamine (full symbols) or 0.5 mM L-Gly-L-Gln dipeptide (empty symbols) as the sole nitrogen source.</p

The transcription and activity of virulence genes depends on L-glutamine.

<p>(A) RT-qPCR transcription analysis of <i>hly</i>, <i>plcA</i>, <i>plcB</i>, and <i>actA</i> in WT bacteria grown in MDM with either 4 mM L-glutamine (gray bars) or 4 mM NH₄Cl (black bars) as a sole nitrogen source. Transcription levels are presented as relative quantity (RQ), relative to levels in WT. Results are mean ±SD of 3 independent experiments performed in triplicates. (B) Hemolysis activity of LLO in culture supernatants of WT and <i>ΔglnPQ</i> bacteria grown on minimal defined media with 1 mM L-glutamine (grey) or 4 mM NH₄Cl (black) as the sole nitrogen source. (C) Analysis of PI-PLC activity of PlcA in culture supernatants of WT (circles) and <i>ΔglnPQ</i> (squares) bacteria grown on minimal defined media with 1 mM L-glutamine (full symbols) or 4 mM NH₄Cl (empty symbols) as the sole nitrogen source. Representative graphs of 3 biological repeats are shown. Error bars represent standard deviation of the triplicate.</p

The <i>LMRG_02270–1</i> operon is required for activation of the Type I interferon response during macrophage cell infection.

<p>(A) Schematic representation of the <i>LMRG_02270–1</i> operon, which contains fused transmembrane and substrate binding protein (SBP) domains and an ATPase that together form the complete transporter. The transposon insertion site and the E164A and R105A mutations are indicated. RT-qPCR analysis of transcription of IFN-β (B) or IL-6 (C) in BMDMs 6 h post infection with WT <i>L</i>. <i>monocytogenes</i> (WT) or Δ<i>LMRG_02270–1</i> mutant, are indicated. Transcription levels are represented as relative quantity (RQ), relative to uninfected cells. Data represents at least 3 biological repeats. Error bars represent 95% confidence interval. (D) Intracellular growth curves, described in Colony Forming Units (CFU), of WT <i>L</i>. <i>monocytogenes</i> (circles) or Δ<i>LMRG_02270–1</i> mutant (squares) in BMDM cells. Representative growth curves of 3 biological repeats are shown. Error bars represent standard deviation of triplicates.</p

GlnPQ promotes <i>L</i>. <i>monocytogenes</i> virulence.

<p>(A) Intracellular detection of YFP fluorescence indicative of <i>plcA</i> promoter activity (pPL2-<i>plcA-yfp</i>) in <i>WT L</i>.<i>m</i>., Δ<i>glnPQ</i> and <i>glnQ</i>-E164A, at 3.5 hours post infection of BMDMs grown in glutamine-restricted medium (B) Intracellular growth of WT <i>L</i>. <i>monocytogenes</i> (circles), Δ<i>glnPQ</i> mutant (squares) and <i>glnQ</i>-E164A (triangles) in BMDM cells grown in glutamine-restricted medium. Representative growth curves of 3 biological repeats are shown. Error bars represent standard deviation of the triplicate. Bacterial counts in the spleens (C) and livers (D) of infected animals as analyzed at 72 h post infection. Mice were injected intravenously with 4 × 10⁴ WT <i>L</i>. <i>monocytogenes</i> (circles) or Δ<i>glnPQ</i> (squares) cells.</p

GlnPQ is required for L-glutamine utilization by <i>L</i>. <i>monocytogenes</i>.

<p>(A) Growth of WT <i>L</i>. <i>monocytogenes</i> (WT circles) or Δ<i>glnPQ</i> (squares) in MDM supplemented with 1 mM L-glutamine as the sole nitrogen source. Shown are representative growth curves of 3 independent experiments, performed in triplicates. (B) Optical density (600 nm) measurements after 30 h of growth of WT <i>L</i>. <i>monocytogenes</i> (circles), Δ<i>glnPQ</i> (squares), Δ<i>glnPQ</i>-pGlnPQ (diamonds), or <i>glnQ</i>-E164A (triangles) bacteria in MDM supplemented with the indicated concentrations of L-glutamine as the sole nitrogen source. Results are mean ±SD of 3 independent experiments, performed in triplicates.</p

L-glutamine uptake by GlnPQ is rapid and selective.

<p>(A) 30 nM of ³H L-glutamine were added to WT (circles), Δ<i>glnPQ</i> (squares), Δ<i>glnPQ</i>-pGlnPQ (diamonds) or <i>glnQ</i>-E164A (triangles) bacteria. At the indicated time-points total L-glutamine uptake was measured by the rapid filtration method. (B) The intracellular concentration (black, left axis), and the concentration gradient (C_in/C_out) (grey, right axis) of L-glutamine were determined for WT bacteria at the indicated concentration of external L-glutamine. (C) Uptake of ³H-labeled L-glutamine (in counts per minute) by WT bacteria incubated for 5 min with ³H L-glutamine (3 μM) and in the absence or presence of 900 μM of the indicated competing amino acids. In C, shown is only the amount of accumulated label, not total L-glutamine. Results are mean ±SD of 3 independent experiments performed in triplicates.</p

The SBP domain of LMRG_02270 specifically binds L-glutamine.

<p>Isothermal titration calorimetry was used to determine the binding of (A) L-glutamine or (B) D-glutamine to the SBP domain, or (C) of L-glutamine to the R105A mutant of the SBP domain. Shown are the consecutive injections of 2 μL aliquots from a 500 μM solution of the indicated amino acid, into 200 μL of 50 μM SBP. The upper panels show the calorimetric titration and the lower panels display the integrated injection heat derived from the titrations, for which the best-fit curve was used to calculate the <i>K</i>_<i>D</i>. The experiments were conducted five times, and the <i>K</i>_<i>D</i> value is mean ±SD of 5 independent experiments.</p