Source: CORNELL UNIVERSITY submitted to
MODULATION OF INNATE IMMUNITY BY A BACTERIAL PHOSPHOLIPASE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
0224178
Grant No.
(N/A)
Project No.
NYCV-433864
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Project Director
Marquis, HE.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Microbiology And Immunology
Non Technical Summary
The social and economic impact of infectious diseases is enormous. It is estimated that 300 million people suffer from AIDS, malaria or tuberculosis worldwide, which account for about half of all infectious diseases. There is a critical need for new interventions that modulate the immune response to infection, either to hasten clearance of pathogenic microbes or enhance the efficacy of vaccines. Our long-term goal is to develop a new and safe approach to hasten and enhance the immune response to microbial pathogens. Listeria monocytogenes is a foodborne bacterial pathogen that has the ability to multiply in the cytosol of infected cells and to spread from cell to cell using an actin-based mechanism of motility. Escape from vacuoles, which are formed upon cell-to-cell spread, is mediated in part by a phospholipase C (PC-PLC), whose activation is dependent on a decrease in vacuolar pH. Therefore, active PC-PLC is secreted specifically in acidified vacuoles during the intracellular life cycle of L. monocytogenes. We have generated a mutant strain of L. monocytogenes that constitutively secretes active PC-PLC in the cytosol of infected cells. Loss of regulation of PC-PLC activity during infection does not affect host cell viability or the ability of bacteria to multiply intracellularly. Similarly, loss of regulation of PC-PLC activity does not change the initial course of infection in mice; however, beginning at 2 days post-infection there is an increase in the efficiency of immune clearance. Our hypothesis is that PC-PLC up regulates the immune response to infection when delivered as an active enzyme in the cytosol of host cells, consequently conferring a protective effect against infection. The objectives of this application are: (1) to determine how PC-PLC influences the transcriptional and translational response to infection in macrophages, and (2) to systematically characterize the effects of PC-PLC on the immune response to infection in vivo. If our results confirm that PC-PLC acts as an adjuvant to hasten and enhance the immune response to infection, we will next investigate methods to efficiently and safely deliver PC-PLC as a novel treatment modality. The studies described herein have the capability to significantly impact our ability to manipulate the immune response to other infectious diseases, potentially leading to the development of new prophylactic and curative approaches.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7224010109050%
7224010110025%
7224010116025%
Knowledge Area
722 - Zoonotic Diseases and Parasites Affecting Humans;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1090 - Immunology; 1160 - Pathology; 1100 - Bacteriology;
Goals / Objectives
Goals /Objectives Listeria monocytogenes is a foodborne bacterial pathogen that multiplies in the cytosol of infected cells and spreads from cell to cell using an actin-based mechanism of motility (2). Escape from vacuoles formed upon cell-to-cell spread is mediated in part by a broad-range phospholipase C (PC-PLC), whose activation is dependent on a decrease in vacuolar pH (1). Active PC-PLC is secreted specifically in acidified vacuoles during the intracellular life cycle of L. monocytogenes. We have generated a strain of L. monocytogenes that constitutively secretes active PC-PLC in the cytosol of infected cells (L. monocytogenes plcB∆pro). Loss of regulation of PC-PLC activity during infection does not affect host cell viability or the ability of bacteria to multiply intracellularly in vitro (3). Similarly, loss of regulation of PC-PLC activity does not change the initial course of infection in mice; however, it increases the efficiency of immune clearance starting at 2 days post-infection. Our hypothesis is that PC-PLC up regulates the immune response to infection when delivered as an active enzyme in the cytosol of host cells, consequently conferring a protective effect against infection. 1. Determine how PC-PLC influences the transcriptional and translational response to infection in macrophages We will test the hypothesis that PC-PLC up regulates the immune response to infection when delivered as an active enzyme in the cytosol of host cells. We will mechanistically and systematically determine how the loss of regulation of PC-PLC activity influences the host transcriptional expression profile, and the extent to which the loss of regulation of PC-PLC activity influences the synthesis and secretion of soluble mediators of inflammation in macrophages. 2. Systematically characterize the effects of PC-PLC on the immune response to infection in vivo We will test the hypothesis that bacterial delivery of active PC-PLC in the cytosol of infected cells has a protective effect against infection. This will be tested by infecting mice concomitantly with the L. monocytogenes plcB∆pro mutant strain and the isogenic wild-type strain, and by following the kinetics of bacterial growth and clearance in tissues. We will also evaluate the pathology associated with infection and characterize the immune response in infected tissues. Lastly, we will differentiate PC-PLC-mediated effects that are due to MyD88 vs type 1 IFN immune response to infection, using MyD88 and IFNAR (IFNa receptor) knock out mice. 1. Marquis, H., H. Goldfine, and D. A. Portnoy. 1997. Proteolytic pathways of activation and degradation of a bacterial phospholipase C during intracellular infection by Listeria monocytogenes. J. Cell Biol. 137:1381-1392. 2. Tilney, L. G., and D. A. Portnoy. 1989. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J. Cell Biol. 109:1597-1608. 3. Yeung, P. S., Y. Na, A. J. Kreuder, and H. Marquis. 2007. Compartmentalization of the broad-range phospholipase C activity to the spreading vacuole is critical for Listeria monocytogenes virulence. Infect. Immun. 75:44-51.
Project Methods
AIM 1. Determine how PC-PLC influences the transcriptional and translational response to infection in macrophages 1a. Determine how PC-PLC affects the transcriptional response of macrophages to infection We will use a tissue culture model of infection to determine the extent to which the loss of regulation of PC-PLC activity affects the transcriptional expression profile of genes coding for soluble mediators of inflammation and their receptors. These experiments will be carried out with primary cultures of murine bone marrow-derived macrophages for several major reasons. 1b. Determine how PC-PLC affects the translational response of macrophages to infection The profile of soluble mediators of inflammation synthesized by infected bone marrow-derived macrophages will be determined to evaluate translational activities that are either consistent with or independent of the transcriptional profile. Uninfected cells will be used as controls. We will assess the levels of secreted and intracellular mediators at 8 and 18 hours post-infection, but additional time points will be added if necessary. AIM 2. Systematically characterize the effects of PC-PLC on the immune response to infection in vivo 2a. Determine whether PC-PLC can protect mice against listeriosis This concept will be tested by infecting groups of BALB/c mice with the wild-type strain of L. monocytogenes, or the plcB∆pro mutant, or a combination of both strains. The kinetics of bacteria growth and clearance in tissues will be followed by sacrificing groups of 5 mice at 1, 2, and 4 days post-infection 2b. Define the severity of histopathological lesions associated with infection As a complement to following the kinetics of bacteria growth and clearance in tissues of infected mice, we will assess differences in histopathological lesions associated with infection at various time-points post-infection. 2c. Define the immune cell response to infection Numbers and types of immune cells infiltrating infected tissues will be determined to better define the effect of PC-PLC on the immune response in vivo. The different populations of stained cells will be analyzed on a BD FACSCantoII system (available for a fee in the department of Microbiology and Immunology) and data will be processed using FlowJO software. We will analyze three experimental replicates per time point. Experimental data will be analyzed statistically using a two-tailed t-test.

Progress 10/01/10 to 09/30/12

Outputs
OUTPUTS: This project consisted of two aims. Aim 2 had been completed by the end of the first year. During the second year of the project, we completed aim 1. However, we have not completely finished the analysis of the data. A manuscript is in progress. Results from these studies were presented at the 112th ASM General Meeting. PARTICIPANTS: Bryant Blank is a veterinarian who was doing a residency in Lab Animal Medicine at Cornell University. As part of his residency, he completed a Master degree. He successfully defended his thesis project in June of 2012. He is now working as a Clinical Veterinarian for Cornell University Lab Animal Care. Delbert Abi Abdullah (PhD) is a post-doctoral associate in the Marquis lab. Dr. Abi Abdullah performed the microscopy experiments to analyze mitochondrial fragmentation in infected cells. Alan Pavinski Bitar is a technician in the Marquis lab who has provided technical assistance to Dr. Blank all along this project. Kirk Maurer (DVM PhD) and Helene Marquis (DVM PhD) co-directed this project. Dr. Maurer was an adjunct faculty in the College of Veterinary Medicine and a clinical veterinarian for lab animals. He has recently moved to Darmouth University. Dr. Helene Marquis is an associate professor in the College of Veterinary Medicine. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Listeria monocytogenes is an intracellular bacterial pathogen that causes disease in humans and animals. Escape from vacuoles, which is required for bacteria to multiply in the cytosol of host cells, is mediated in part by a bacterial phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation occurs specifically in acidifying vacuoles. A mutant strain (plcB∆pro) that has lost the ability to control the activity of PC-PLC is attenuated in mice. This attenuation does not appear to be due to a lack of bacterial fitness, as initial colonization of intestines is equal to wild type in the first 28 hrs post-oral infection and significant differences are not seen in the spleen and liver until 24 and 48 hrs post-i.v. infection, respectively. In the second year of this project, we determined how PC-PLC influences the transcriptional and translational response to Listeria monocytogenes infection in macrophages. We tested the hypothesis that PC-PLC up regulates the immune response to infection when delivered as an active enzyme in the cytosol of host cells. We determined how the loss of regulation of PC-PLC activity influences the host transcriptional expression profile, and the extent to which the loss of regulation of PC-PLC activity influences the synthesis and secretion of soluble mediators of inflammation. The transcriptional profile of infected mouse bone marrow derived macrophages was analyzed at 3, 6, and 9 hours post-infection. Contrary to our hypothesis, the results indicated that the mutant did not induce a stronger inflammatory immune response in these cells. However, we detected differences in mitochondrial gene expression. Further investigations revealed that the mutant is less efficient than wild-type strain at inducing mitochondria fragmentation. This last result could, at least in part, explain the attenuation profile of this mutant, as mitochondria fragmentation is a mechanism by which Listeria temporarily impedes the host cell to respond to infection. In previous experiments, we had observed that, the mutant strain compromises the ability of wild-type L. monocytogenes to multiply in the mouse liver. To assess how the mutant modifies the hepatic immune response to infection, we monitored the kinetics of immune cell recruitment in the liver of infected mice and levels of serum inflammatory cytokines up to three days post-infection. We observed a lack of increase in neutrophil counts in the liver of mice infected with the plcB∆pro mutant. In addition, pro-inflammatory cytokines were detected at much lower levels in the serum of mice infected with the mutant strain compared to those infected with wild-type strain. The in vivo data and the transcriptional analysis of infected macrophages together indicate that PC-PLC potentiates the host ability to clear L. monocytogenes in a way that does not involve an increase in the inflammatory response to infection.

Publications

  • Bryant S. Blank, Alan Pavinski Bitar, Lauren A. Griggs, Kirk J. Maurer, and Helene Marquis. 2012. A Phospholipase C of Listeria monocytogenes Modulates the Innate Immune Response to Infection. Published poster abstract for ASM General Meeting.


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis in humans and animals. During infection, the bacterium invades cells and escapes from vacuoles to multiply in the cytosol of infected cells. Escape from vacuoles is mediated in part by a broad-range phospholipase C (PC-PLC), whose activation occurs specifically in vacuoles. We have generated a strain of L. monocytogenes that constitutively secretes active PC-PLC in the cytosol of infected cells (plcBdeltapro). Loss of regulation of PC-PLC activity during infection does not affect host cell viability or the ability of bacteria to multiply intracellularly in vitro. Similarly, loss of regulation of PC-PLC activity does not change the initial course of infection in mice; however, it increases the efficiency of immune clearance starting at 2 days post-infection. Our central hypothesis was that PC-PLC up regulates the immune response to infection when delivered as an active enzyme in the cytosol of host cells, consequently conferring a protective effect against infection. To test this hypothesis, we devised two aims. Aim 1 relates to determining how PC-PLC influences the transcriptional and translational response to infection in macrophages, whereas aim 2 relates to characterizing the effects of PC-PLC on the immune response to infection in vivo. During the first year of funding, we made progress on both aims. Aim 1. The transcriptional response of macrophages to infection. Murine bone marrow-derived macrophages from BALB/c mice were infected with the wild type strain of L. monocytogenes or the plcBdeltapro mutant. An additional group of uninfected cells served as control. Samples of infected cells were lysed at 3, 6, and 9h post-infection to determine intracellular bacterial counts. A parallel set of infected and uninfected cells was used to harvest RNA for transcriptional analysis. Total RNA was purified and the samples were processed by the CU Life Sciences Core Laboratories Center. Cy3-labelled cRNA was hybridized to Agilent Whole Mouse Genome Microarray 4x44K. The slides were scanned, the background subtracted, and non-uniform outliers excluded. We are now beginning to analyze the data. Aim 2. The effect of PC-PLC on wild type infection. We assessed whether the mutant strain (plcBdeltapro) affects the growth of wild type bacteria during a mixed infection. Three groups of mice were infected i.v.: the 1st group received the wild type strain, the 2nd group received twice as many of the mutant strain, and the third group received the same inoculum as groups 1 and 2 together. Bacterial counts in spleen and liver were determined at 72 hours post-infection and the wild type and mutant strains were differentiated by plating isolated colonies on egg yolk agar to detect phospholipase activity. Results from this research were presented internally within the College of Veterinary Medicine at Cornell University during the Biological and Biomedical Sciences Symposium and the Veterinary Investigator Program Symposium, and nationally at the Merial NIH National Veterinary Scholars Symposium. PARTICIPANTS: Bryant Blank is a veterinarian who is doing a residency in Lab Animal Medicine at Cornell University. As part of his residency, Bryant is working on a Master degree. This grant is providing the funds for his research project. Bryant worked in the Marquis lab on all the experiments described in this report. Bryant presented a poster related to this work at the 2011 Biological and Biomedical Sciences Symposium at Cornell University. Lauren Griggs is a veterinary student at Cornell University who participated to the Veterinary Investigator Program (VIP) the summer before entering veterinary school. During that summer she worked with Bryant Blank in the Marquis lab and performed experiments related to aim 2 of this project. She gave an oral presentation about her research results at the end of the summer at the VIP Symposium. She also presented a poster related to her research work at the 2011 Merial NIH National Veterinary Scholar Symposium sponsored by the University of Florida. Alan Bitar is a technician in the Marquis lab who has helped Bryant and Lauren with their experiments and who maintains the lab supplies for this project. Kirk Maurer is a DVM, PhD, and lab animal board certified adjunct faculty in the department of Biomedical Sciences and clinical veterinarian for the Cornell Center for Animal Resources and Education at Cornell University. Kirk co-directed this research project with Helene Marquis. Helene Marquis is a DVM and PhD who is an associate professor in the department of Microbiology and Immunology at Cornell University. Helene co-directed the project with Kirk Maurer. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Aim1: Microarray results are in the process of being evaluated. Aim 2: Results from experiments related to aim 2 indicate that the plcBdeltapro mutant affects the virulence of wild type bacteria during a mixed infection in a tissue specific manner. There was a reproducible and statistically significant difference in the numbers of wild type bacteria recovered from the liver of mice when comparing single strain infections to mixed infections. But, no significant changes in numbers of plcBdeltapro bacteria were seen in the liver of mice when comparing single strain infections to mixed infections. Also, no significant changes in numbers of wild type and plcBdeltapro bacteria were seen in the spleen of mice when comparing single strain infections to mixed infections. Overall, these results show that wild type L. monocytogenes does not affect the growth of the plcB∆pro mutant, but the plcBdeltapro mutant has a negative effect on the growth or survivability of the wild type strain in the liver of co-infected mice. Future experiments will aim at identifying host factors that are affected by the bacterial phospholipase and that contribute to the attenuation of wild type strain during a mixed infection.

Publications

  • No publications reported this period