1. A HD superfamily putative hydrolase plays a role in
Mutagenesis
Overlap extension PCR and allelic exchange used to delete lmof2365_2464 in strain F2365.
Assess virulence
In vivo & In vitro
Mouse virulence assay
6- to 8-week-old female A/J Mice used to compare virulence
Cell Culture Assay
Human adenocarcinoma cell lines Caco-2 & HT-29 used to compare the adhesion, invasion, and survival properties
Recombinant Protein Expression
and Characterization
Activity Assays
Recombinant protein expressed using pET28a+ vector. Phosphodiesterase & Phosphatase activity was tested using Bis-pNPP & pNPP
A HD superfamily putative hydrolase plays a role in
Listeria monocytogenes virulence
Swetha Reddy, Gokul Turaga, Michelle M. Banes, Mark L. Lawrence
INTRODUCTION
Listeria monocytogenes is a facultative intracellular parasite that causes listeriosis and is responsible for food borne epidemics. Based on our comparison of genomes between serotype 4b(virulent) strain F2365 , 1/2a(virulent) strain EGDe, and 4a(avirulent) strain HCC23, we selected genes that are unique to pathogenic isolates and created deletion mutants. In our current study we focused on a hydrolysis (HD) domain super family protein encoded by lmof2365_2464. HD domain proteins play important roles in the metabolism of nucleotides and in signaling by nucleotidase and phosphodiesterase activities. In vivo and in vitro experiments conducted show that LMOf2365_2464 plays a role in virulence and intracellular replication in L. monocytogenes. The recombinant f protein shows phosphodiesterase and phosphatase activities indicating its potential role in signaling during infections. M E T H O D
Temperature sensitive shuttle vector pAUL-A used(1)
The recombinant protein shows hydrolase activity. Preliminary data indicates that it’s a Phosphodiesterase though it also shows phosphatase activity(Figure 4).
The mutant strain was attenuated in mice compared to parent strain F At each infection dose, more mice were infected with F2365 than the mutant (Table 1). A B
Figure 2: Bacterial counts of L. monocytogenes F2365 & LMOf2365_2464 mutant in liver (A) and spleen (B). On the appropriate day, sick mice were euthanized and organs retrieved. Tissues were weighed and homogenized. The homogenate was diluted, spread on BHI agar plates and allowed to grow for 48 hours at 37ºC. CFU/gm for each organ was calculated at each dose.
At all doses, colonization in liver was more for mice infected with F2365 compared to the mutant Δlmof2365_2464, and colonization was more at the highest dose for spleen and thymus in mice infected with F2365 (Figure 2) R E S U L T
Bis-p-Nitrophenyl phosphate
(Colourless)
Phosphodiesterase, Buffer, Co2+
p-Nitrophenyl phosphate
F recombinant protein
Phosphatase
,Buffer ,Co2+
p – Nitro Phenol
(Yellow) * A B C D
Table 1. Number of mice infected with the corresponding listerial strain at any time point.
Infection Dose (CFU’s/mouse)
Number of Mice Infected*
F2365
Δlmof2365_2464
1.674x105 5 1
1.674x104 4
1.674x103 3
1.674x102
*Each treatment contained 5 mice. Each mouse was injected with 0.1 ml of bacterial suspension, and observations were made daily.
Figure 4 : Activity of the enzyme was tested using Bis-p-NPP and pNPP. The product p-nitro phenol obtained after hydrolysis was yellow in color.
* Wells 1A,1C,2A & 2C show Phosphatase activity and wells 3A,3C, 4A & 4C show Phosphodiesterase activity. Wells in row 5 & 6 are positive and negative controls with and without substrate and enzyme respectively. Wells in row are controls with synthetic enzymes.
Mutant Δlmof2365_2464 showed decreased adhesion to Caco-2 cells compared to the wild type strain.
Wild type bacteria persisted in Caco-2 cells better compared to mutant strain, and significant differences in CFU/ml at 4hr,12 hr, 14 hr, 16 hr, and 18 hrs post-infection were observed (Figure 3A).
Infection in HT-29 cells showed significant difference in CFU/ml only at early (4 hrs) and late (22 & 24hr)time points (Figure 3B).
Figure 3: Invasion assay with CaCO-2 (A) & HT-29 (B):cells were seeded into 12-well plates 2 day prior to infection. After a wash step with PBS, cells were infected with bacterial suspension to a MOI of 10 bacteria per cell for 2 hours for Cac0-2 and HT29 and 1 hr for J774 cells. The cells were washed 2 times with PBS and incubated in media containing 100 µg/ml of gentamycin. At appropriate time points, cells were washed, mechanically lysed, and spread on BHI agar plates. CFUs/ml data was statistically analyzed. *Statistically significant difference was observed at these time points. A B
SUMMARY AND FUTURE WORK
Mean separation analysis for the mouse virulence assay and cell adhesion and invasion assay show that putative HD domain protein encoded by lmof2365_2464 contributes to virulence.
Furthermore, in vitro assays indicate that the protein is part of the pathogen’s mechanism for intracellular survival.
Preliminary work with the recombinant protein show that the protein has both Phosphodiesterase and Phosphatase activity and requires Cobalt ions.
Protein shows stronger phosphodiesterase activity and cleaves cyclic nucleotides indicating its role in signaling.
We plan on identifying the specific substrates and inhibitors for the protein and elucidating its mechanism of action and its contribution to virulence in L.monocytogenes.
REFERENCES
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Donaldson, J. R., B. Nanduri, J. R. Pittman, S. Givaruangsawat, S. C. Burgess, and M. L. Lawrence. (2011). Proteomic Expression Profiles of Virulent and Avirulent Strains of Listeria monocytogenes Isolated from Macrophages. Journal of Proteomics.
Aravind, L. & Koonin, E. V. (1998). The HD domain defines a new superfamily of metal-dependent phosphohydrolases. Trends Biochem. Sci. 23, 469–472.
Yakunin, A. F., Proudfoot, M., Kuznetsova, E., Savchenko, A., Brown, G., Arrowsmith, C. H. & Edwards, A. M. (2004). The HD domain of the Escherichia coli tRNA nucleotidyltransferase has 2′,3′- cyclic phosphodiesterase, 2′-nucleotidase, and phosphatase activities. J. Biol. Chem. 279, 36819–36827.
Van Coillie, E., H. Werbrouck, M. Heyndrickx, L. Herman, and N. Rijpens. (2004). Prevalence and typing of Listeria monocytogenes in ready-to-eat food products on the Belgian market. J Food Prot 67:
This project was funded by USDA Agricultural Research Service (Agreement No ).