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Chapter 61 : Eukaryotic-Like Proteins of as Potential Virulence Factors

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Eukaryotic-Like Proteins of as Potential Virulence Factors, Page 1 of 2

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Abstract:

Bioinformatic analysis of the Philadelphia 1 genome revealed four novel open reading frames (ORFs) that have no known prokaryotic homologues but are predicted to encode products with significant domain homology to eukaryotic proteins. The authors postulated that the gene products of these eukaryotic-like ORFs may interact with eukaryotic host cell proteins and thus play a role in the subversion of host cellular trafficking pathways by and the establishment of the replicative niche inside host cells. RNA was extracted from stationary phase broths of 130b using the Epicentre Masterpure RNA Purification Kit (MCR85102). cDNA was synthesized, and specific primers for each gene were used to amplify 200 to 1,100 base pair portions of each ORF. This revealed that all four ORFs were transcribed in stationary phase. To investigate the potential role that each eukaryotic-like gene under investigation may play in interactions with host cells, insertional mutants were constructed for each ORF. The chapter focuses on localization of the protein products of eukaryotic-like ORFs. To characterize the products of 1905 and 2644, a combination of epitope-tagging and specific polyclonal antibodies were used to localize the proteins within the bacteria.

Citation: M. Sansom F, J. Newton H, L. Hartland E. 2006. Eukaryotic-Like Proteins of as Potential Virulence Factors, p 246-250. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch61

Key Concept Ranking

Type II Secretion System
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Type IV Secretion Systems
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Figures

Image of FIGURE 1
FIGURE 1

Reverse transcription (RT)-PCR analysis of putative genes predicted to encode products with homology to eukaryotic proteins. DNA, genomic DNA control; RT +, reverse transcription PCR; RT-, control PCR reaction without reverse transcription step.

Citation: M. Sansom F, J. Newton H, L. Hartland E. 2006. Eukaryotic-Like Proteins of as Potential Virulence Factors, p 246-250. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch61
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Image of FIGURE 2
FIGURE 2

Interaction of 130b and Δ with A549 epithelial cells. () Percentage of the inoculum that is intracellular after a 2-h infection period ( < 0.01, unpaired two tailed t-test) Percentage of the inoculum that associates with cells after a 2-h infection period ( < 0.02, unpaired two tailed t-test). () Percentage of the total cell-associated bacteria that become intracellular after a 2-h infection period ( < 0.008, unpaired two tailed t-test).

Citation: M. Sansom F, J. Newton H, L. Hartland E. 2006. Eukaryotic-Like Proteins of as Potential Virulence Factors, p 246-250. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch61
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Image of FIGURE 3
FIGURE 3

Western blot analysis of derivatives of 130b detected with anti-FLAG monoclonal antibodies. Lane 1,130b (pMIP:FLAG), whole cell lysate (WCL); lane 2, 130b (pM-MB207) negative control (WCL); lane 3, 130b (pMIP:-FLAG) soluble fraction (S); lane 4, 130b (pMIP:-FLAG), insoluble fraction (I); lane 5,130b (pMIP:-FLAG), supernatant fraction (SN). () Derivatives of 130b detected with monospecific antibodies to recombinant Lpg2644. Lane 1, 130b WCL; lane 2; Δ WCL; lane 3, Δ WCL; lane 4 Δ WCL; lane 5, 130b SN; lane 6, Δ SN; lane 7, Δ SN; lane 8, Δ SN. () Derivatives of 130b detected with monospecific antibodies to recombinant Lpg1905. Lane 1, 130b WCL; lane 2, ΔWCL; lane 3,130b SN; lane 4, Δ SN. Derivatives of 130b detected with monospecific antibodies to recombinant Lpg1905. Lane 1, 130b SN; lane 2, Δ SN; lane 3, Δ SN; lane 4, Δ SN. Derivatives of 130b detected with monospecific antibodies to recombinant Lpg1905. Lane 1; 130b SN, lane 2, Δ SN; lane 3, Δ SN.

Citation: M. Sansom F, J. Newton H, L. Hartland E. 2006. Eukaryotic-Like Proteins of as Potential Virulence Factors, p 246-250. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch61
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References

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Tables

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TABLE 1

Characteristics of the eukaryotic-like ORFs.

Citation: M. Sansom F, J. Newton H, L. Hartland E. 2006. Eukaryotic-Like Proteins of as Potential Virulence Factors, p 246-250. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch61

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