Chapter 9 : Modeling Microbial Virulence in a Genomic Era: Impact of Shared Genomic Tools and Data Sets

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This chapter discusses the relevance of model host pathogenesis as a third general approach to studying microbial virulence, in which infections are studied in the context of nonvertebrate whole animal hosts. The chapter explains the combination of four key steps: (i) the development of the model host-pathogen system, (ii) the development of genomic tools in both the pathogen and host, (iii) the distribution and use of these tools by the greater research community beyond the laboratories involved in their initial development, and (iv) the collection and ultimate integration of experimental data from a wide variety of research groups made possible by the widespread use of a common resource and its accompanying Web-accessible public database. PA14 was chosen for the construction of a non-redundant mutant library because it is remarkably virulent in the greatest number of model hosts tested, as well as in a number of murine systems of infection. There are four major advantages to construction of a non-redundant mutant library approach as opposed to the traditional approach of screening a random collection of strains for avirulent or attenuated mutants. A microarray experiment in a mutant defective in a newly defined host defense response gene identified candidate downstream genes important in the response to pathogens.

Citation: Lee D, Liberati N, Urbach J, Wu G, Frederick M. 2007. Modeling Microbial Virulence in a Genomic Era: Impact of Shared Genomic Tools and Data Sets, p 213-231. In Pallen M, Nelson K, Preston G (ed), Bacterial Pathogenomics. ASM Press, Washington, DC. doi: 10.1128/9781555815530.ch9
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Image of FIGURE 1

Accumulation of in the intestine. Micrographs were taken of worms fed on lawns of bacteria on brain heart infusion agar for 3 days. Arrows point to the borders of the intestinal lumen. When feed on their normal laboratory food, strain OP50, very few, if any, intact bacterial cells accumulate in the intestinal lumen, which appears as a narrow channel (A and B). In contrast, there is dramatic distension of the intestine when are fed on , and numerous densely packed cells are visible. In the upper right-hand corner of (A) and (C), the round structure depicted is the pharyngeal grinder organ that physically disrupts ingested bacteria. (A) and (C) show the proximal portion of the intestine immediately following the pharyngeal grinder, and (B) and (D) show a middle portion of the intestinal tract.

Citation: Lee D, Liberati N, Urbach J, Wu G, Frederick M. 2007. Modeling Microbial Virulence in a Genomic Era: Impact of Shared Genomic Tools and Data Sets, p 213-231. In Pallen M, Nelson K, Preston G (ed), Bacterial Pathogenomics. ASM Press, Washington, DC. doi: 10.1128/9781555815530.ch9
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Image of FIGURE 2

Immune signaling pathways in mammals and nematodes. In mammals, a family of TLRs mediates the recognition of PAMPs. Immune receptors have not yet been identified in Mammals and share a conserved p38 MAPK signaling module, but does not encode a transcription factor homologous to mammalian NF-κB. Interestingly, has a TIR domain-containing protein, TIR-1, that functions upstream of the p38 MAPK that is homologous to the mammalian SARM protein, but the role of the SARM protein in mammalian immunity is not known.

Citation: Lee D, Liberati N, Urbach J, Wu G, Frederick M. 2007. Modeling Microbial Virulence in a Genomic Era: Impact of Shared Genomic Tools and Data Sets, p 213-231. In Pallen M, Nelson K, Preston G (ed), Bacterial Pathogenomics. ASM Press, Washington, DC. doi: 10.1128/9781555815530.ch9
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Image of FIGURE 3

Schematic of PA14 library and PA14NR construction. (A) PA14 master library production. Transposon mutagenesis of PA14 was performed and individual colonies were picked and grown overnight in 96-well plates. An aliquot of the overnight culture was set aside for arbitrary (ARB) PCR analysis to map each transposon insertion site. Glycerol was added to the remaining culture and mixed, and the sample was split into three aliquots for storage at –80°C. (B) ARB PCR. ARB PCR was used to map the site of insertion for each mutant. In the schematic diagram, the transposon is indicated by a white rectangle within the PA14 genome (wavy lines). The positions of four PCR primers are indicated in this schematic. An aliquot of the original overnight culture was used as a template for the first PCR reaction. The left primer for the first reaction (1L) anneals to the transposon. The right primer (1R) contains a variable sequence in the 3′ end that (randomly) anneals to the PA14 genome at some distance from the transposon; the 5′ end of the 1R primer has a constant region. A subset of PCR products from this reaction will contain the terminal sequence of the transposon and the flanking genomic sequence (varying lengths of genomic sequence for each mutant strain); other PCR products will be the result of pairs of 1R primers annealing to other regions of the PA14 genome. An aliquot of the first completed PCR reaction was used as a template for a second PCR reaction. The left primer for the second reaction (2L) is a nested primer annealing to the transposon sequence, and the right primer (2R) is identical to the constant 5′ end of the 1R primer. In this manner, only fragments that contain the transposon will be amplified as the dominant product in the second completed PCR reaction. The PCR reaction was processed to remove primers and free nucleotides, and the remaining double-stranded product was subjected to sequencing with a transposon-specific sequencing primer. The resulting sequences were batch processed to (i) trim off low-quality sequence, (ii) identify the transposon sequence, and (iii) check the remaining sequence against the PA14 genome via BLAST to map the site of insertion. (C) Selection of PA14NR set. The insertion sites for each mutant identified in step 3B were mapped onto the predicted PA14 ORFs. An automated script was designed to identify the best candidates for inclusion in the PA14 nonredundant set (PA14NR set). For predicted ORFs in which only one insertion was available, that mutant was selected. In cases where more than one insertion was available, all sequences were filtered to retain only those in which the BLAST match of the obtained high-quality sequence to the PA14 genome had a score of 80 or greater. Of the remaining cases, the insertion that was the most 5′ in the ORF was generally chosen. In some cases, two insertions were chosen, such that 5,459 mutants with insertions in 4,596 genes comprise the current version of the PA14NR set. Finally, the script identified the original position of each chosen mutant in the original master library and remapped the location onto a new set of 96-well plates for the NR set. (D) PA14NR set production. By using the list of mutants identified in step 3C, individual strains were manually picked from one of the three frozen copies of the PA14 master library. Each mutant was streaked onto selective media to isolate a pure colony, which was then used to inoculate overnight cultures in 96-well plates (using the remapped NR set locations in each plate). The overnight cultures were grown in deep-well plates to allow for a sufficient volume to freeze 10 copies of the PA14NR set. One of these copies will subsequently be used to create copies that will be distributed to other laboratories.

Citation: Lee D, Liberati N, Urbach J, Wu G, Frederick M. 2007. Modeling Microbial Virulence in a Genomic Era: Impact of Shared Genomic Tools and Data Sets, p 213-231. In Pallen M, Nelson K, Preston G (ed), Bacterial Pathogenomics. ASM Press, Washington, DC. doi: 10.1128/9781555815530.ch9
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Image of FIGURE 4

Interface between model host pathogenesis and genomics. Elements that are the major focus of this chapter are indicated in bold. The basic premise of the model host pathogenesis system is that the bacterial pathogen (in this case, PA14) is used to infect a simple, genetically tractable host (in this case, ) under conditions in which many aspects of the host-pathogen interaction are thought to reflect those that occur during a human infection. Infections are traditionally performed using wild-type or mutant isolates of the host or pathogen. Screens have been carried out in our laboratory for pathogen mutants with reduced virulence and host mutants with enhanced susceptibility and enhanced resistance to pathogens (Esp and Erp phenotypes, respectively). However, the availability of genome-wide mutant libraries in both organisms (RNAi libraries in , and the PA14 nonredundant library of transposon insertions described in this chapter) allows these screens for novel pathogenesis-related genes to be performed to saturation. In the case of the PA14 nonredundant set (PA14NR set), the library is designed such that mutants can be assayed individually or in pools by TraSH techniques. The interaction of host and pathogen can also be examined by using microarrays or proteomic techniques to analyze changes in the transcriptome or proteome of either organism as a consequence of infection. The use of these additional genomic tools can be coupled with the available mutant libraries; transcriptional or proteomic profiling of candidate virulence mutants compared with their wild-type parent has been used to identify putative downstream targets. The collected experimental data will be deposited in a publicly accessible database. Initially, the database will focus on processing phenotypic data for mutants in the PA14NR set library, but it can ultimately be expanded to incorporate additional types of data gathered by other genomic tools.

Citation: Lee D, Liberati N, Urbach J, Wu G, Frederick M. 2007. Modeling Microbial Virulence in a Genomic Era: Impact of Shared Genomic Tools and Data Sets, p 213-231. In Pallen M, Nelson K, Preston G (ed), Bacterial Pathogenomics. ASM Press, Washington, DC. doi: 10.1128/9781555815530.ch9
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