Future Directions

Elucidating the role of the Chromosomal Type III SecretionSystem structural protein SsaV

Jackson O. Osaghae-Nosa1*, Miqdad O. Dhariwala1, Gilbert A. Cortes1 and Deborah M. Anderson1

1Department of Veterinary Pathobiology, University of Missouri-Columbia

Abstract

MethodsIntroduction

1. Dieye, Yakhya, Keith Ameiss, Melha Mellata, and Roy Curtiss. "The Salmonella Pathogenicity Island (SPI) 1 contributes more than SPI2 to the colonization of the chicken by Salmonella enterica serovar Typhimurium." BMC Microbiology 9.1 (2009): 3. Web. 12 Apr. 2015. <http://www.biomedcentral.com/1471-2180/9/3>.2. Harding, Robert. How the Plague Is Transmitted. Digital image. Nature. Nature Publishing Group, 2014. Web.3. Plague Worldwide. Digital image. Centers for Disease Control and Prevention. N.p., 2013. Web.

Results

References

Growth CurvesCultures of EO-55 and CO92 pCD1- were grown in 5mL of Heart Infusion Broth (HIB) overnight at 26⁰C. These cultures were then diluted to an OD600 of 0.50 and were monitored over the course of 12 hours at both 26⁰C and 37⁰C. An OD600 was taken at every hour as well as at a 24 hour time point. An OD600 correlates to approximately 1.0X10^9 colony forming units (CFU) of Yersinia pestis. Polymixin B sensitivityCultures of EO-55 and CO92 pCD1- were grown in 5mL of Heart Infusion Broth (HIB) overnight at 26⁰C. 15 ml conical tubes at 8 different concentrations of Polymixin B were then infected with 1x10^6 CFU. An OD600 was taken 16 hours after incubation in 26⁰C for the lowest three concentrations supporting growth.Intracellular SurvivalA phagocytosis assay was performed at 2, 8, and 24 hours post infection. Cultures were grown in 5mL of Heart Infusion Broth (HIB) overnight at 26⁰C. Then, cultures were diluted at 37⁰C for approximately two hours before infection. RAW 264.7 macrophages were then infected with a multiplicity (of infection (MOI) of 20. At each time point, macrophages were lysed by exposing them to a 0.1% solution of Triton for 10min. Viable intracellular bacteria was then plated. Lactose dehydrogenase (LDH) assayA LDH assay was performed at 8 and 24 hours post infection. Cultures were grown in 5ml of HIB overnight at 26⁰C. Then cultures were diluted at 37⁰C for two hours before infection. RAW 264.7 macrophages were then infected with a multiplicity of infection (MOI) of 20.

To determine significance, two biological replicates were pooled and a Two Way Anova with a repeated measures analysis was run. No significance was found at 26⁰C. However, a significant difference was determined at 37⁰C.

Yersinia pestis, a Gram-negative rod shaped bacterium, is the causative agent of plague. The bacterium encodes several virulence factors such as the Type 3 Secretion System (T3SS), Siderophore encoding pigmentation locus, an altered Lipopolysaccharide (LPS) and an anti-phagocytic capsule among others. The T3SS of Y. pestis, located on the CD1 plasmid is the most significant virulence factor associated with the pathogen. It induces apoptosis in mammalian macrophage cells. For this study we used one mutant, EO-55. It merits further research because it harbors a mutation in a Secretion system apparatus protein (SsaV). SsaV is a structural protein that is encoded in the chromosomal T3SS. In order to understand how the chromosomal T3SS interacts with eukaryotic host cells, phagocytic assays were conducted to study bacterial intracellular survival and uptake. RAW 264.7 cell line were infected with EO-55 and its parent strain, CO92pCD1-. Furthermore, Lactate dehydrogenase (LDH) assays were conducted to determine the cytotoxicity of these strains post infection. Preliminary data from our lab suggests that intracellular survival of Y. pestis is not impacted by mutation of SsaV and our goal is to further assess its function. This data enhances the understanding of the role of the chromosomal T3SS within Y. pestis.

Hypothesis

The project described was supported by the IMSD EXPRESS Fellows Program (or simply the IMSD EXPRESS Program) via grant number R25GM056901 from the National Institute of General Medical Science (NIGMS), a component of the National Institutes of Health (NIH).Also I would like to thank the members of the Anderson lab for their valuable input and help in discussing the data and designing experiments.

Acknowledgments

The mutant strain, EO-55, possessing a nonfunctional Chromosomal T3SS has been recognized as a hypocytotoxic strain compared to its parental strain, CO92 pCD1-. Therefore, we expect the mutant to induce lower amounts of cell death. With that in mind, we hypothesize that if more cell death is induced in the presence of the functional Chromosomal T3SS then it must be important for the pathogenesis of the disease plague.

Conclusion

• The disease plague manifests in three main forms: bubonic septicemic, and pneumonic plague.

• The flea vector as well as infected mammalian hosts are responsible for the transmission of this lethal disease.

• Historically, the bacteria Yersinia pestis, is believed to have been the cause of the Black Death which eliminated 30-60% of European population in the 14th Century.

• Cases of plague infection still occur in today’s world with most reported cases occurring in Congo and Madagascar.

•Several instances of the disease plague have been reported in the United States in the last decade.

A mechanism that many gram-negative bacteria possess is the ability to utilize the type three secretion systems. These bacteria include Salmonella enterica, Escherichia coli, Shigella flexneri and Pseudomonas aeruginosa among others. It is a protein appendage that allows the bacteria to inject virulence proteins, called effectors, into the eukaryotic host cell. These effectors ultimately manipulate the cellular functions of the infected host and facilitate the progression of the infection. For this study we used Y. pestis strains that were randomly mutagenized by transposon Tn5-lacZ and a library of 8000 insertion mutants were screened for defects in killing macrophages with the goal to identify bacterial genes required for cytotoxicity during infection. Candidates were further screened for growth defects and for increased sensitivity to Polymixin B. One mutant, EO-55, harbors a mutation in a Secretion system apparatus protein (SsaV). SsaV is a structural protein that is encoded in the

• The role of the Chromosomal T3SS is one that is less understood. However, the data shown here demonstrates that the lack of this T3SS doesn’t impact the survival of Y. pestis.

• Based on the data collected, a mutation in SsaV of the Chromosomal T3SS negatively impacts the mutant EO-55 when compared to Kim6+, Which also doesn’t have the chromosomal T3SS, at two and twelve point four hours post infection.

• This data runs counter current with our original hypothesis because we expected to see lower amounts of intracellular survival when the Chromosomal T3SS was inactive.

• Furthermore, these data may suggest that the chromosomal T3SS contributes to the pathogenesis of the disease plague particularly within mammalian hosts.

• Conduct an intracellular survival assay using CO92 pCD1- and EO-55.• The strain, EO-55, has a transposon gene interruption at both YPO0266. To understand if this gene

will give back virulence, we will make a complementation mutant.• More Cytotoxicity assays will be performed which will measure cell death.• Fine tune the concentration where Polymixin b inhibits both CO92 pCD1- and EO-55.• Infect mice with EO-55 and EO-55 complementing mutant• Determine method of cell death using Electron microscopy

Nine biological replicates were pooled and a comparison T-test was run at each time point. Significant difference was found between Kim6+ and the other two strains at two and twelve point four hours post infection. However no significance was found at seventeen point four hours post infection.

chromosomal T3SS. This protein has been characterized on one of the two T3SS of Salmonella. The T3SS in Salmonella is present on two different loci within the chromosome and uses pH as an inducer for T3SS-2 assembly and secretion of the effector proteins. SsaV, which is present on T3SS-2 is responsible for the secretion of effector proteins through the needle and syringe like machinery. SsaV prevents fusion of the endosome/lysosome with Salmonella-containing vacuoles.

0 5 10 15 20 25 300

0.20.40.60.8

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Growth curve at 26˚C

CO92pCD1- EO-55

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Growth curve at 37˚C

CO92pCD1- EO-55

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Lowest 3 concentrations supporting growth with Polymixin B

Strains and concentrations

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Max lysis uninfected CO92pCD1- EO-550

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One experiment was run and no statistics besides standard error have been conducted. Here, the uninfected are releasing more lactose dehydrogenase than the infected cells.

To determine significance, three biological replicates were pooled and statistical test were run. No significance was found at these concentration.