Challenges for Studying Bacterial Host Interactions
Gene expression studies of bacterial pathogens following host cell interaction have been especially challenging (1, 2). Namely purifying adequate amounts of high quality RNA for bacterial transcriptome analysis has proven to be difficult. Harvesting bacteria and purifying bacterial RNA from infected tissues or in vitro cell cultures without altering gene expression is the first problematic step. In addition, bacterial cell numbers in diseased tissues or organs are frequently small in comparison to the numbers of host cells present. Even when host cells are infected in vitro, the numbers of bacteria that adhere to or invade cells is often low. Therefore, purifying total RNA from a mixture of eukaryotic cells and bacteria most often results in a vast excess of eukaryotic cellular RNA and very little bacterial RNA. Frequently too little bacterial RNA is present for generating adequate signals on microrarrays.
Start By Preserving RNA Expression Profiles
For host-pathogen interaction studies, using Ambion's RNAlater™ to "freeze" RNA expression profiles and preserve RNA integrity is an essential first step prior to harvesting cells (Figure 2). Infected tissues or organs should be immersed in RNAlater immediately upon removal from the host organism. RNAlater will penetrate bacterial and host cell membranes and rapidly inactivate endogenous ribonucleases and other cellular enzymes so that the RNA is left intact and its expression pattern unaltered. With cultured cells, it is best to first remove culture medium then add RNAlater to the culture flask or plate so that all cells are completely covered. Allow approximately 10 minutes for RNAlater penetration before harvesting cells for RNA isolation. For non-adherent eukaryotic cells, collect cells by centrifugation, then thoroughly resuspend them in RNAlater. For bacteria that have adhered to host cells but are not internalized, it may be desirable to wash cells prior to the addition of RNAlater to remove bacteria that are not adhering tightly.
|Figure 2: Recommended Procedures for Purifying Bacterial mRNAs for Whole Genome Expression Analysis Following Bacteria-host Cell Interactions.
Isolating Bacterial RNA From Host-Pathogen Mixtures
After freezing expression profiles and harvesting cells, RNA is purified from the host-pathogen cell mixture. One approach is to isolate RNA from the entire host-pathogen mixture using standard lysis buffers. However, bacteria present in the mixture may not be lysed without harsher methods for cell disruption, such as sonication (3). A more effective approach for increasing yields of bacterial RNA from host-pathogen mixtures is to selectively lyse the host cells, remove host RNA and cellular debris, and then isolate RNA from the bacteria using cell disruption methods appropriate for bacteria, such as Ambion's RiboPure™ Bacteria Kit
. One example of such an approach was reported by Grifantini et al. (4) who used saponin-mediated lysis (1% w/v) of epithelial cells prior to purifying RNA from adherent meningococci. Saponin-mediated lysis has also been used to release intraerythrocytic Plasmodium falciparum (5) and for detection of intracellular pathogenic bacteria in cerebrospinal fluid (6). Saponins can also be used in the presence of RNAlater, which preserves RNA expression profiles during host-cell lysis. Because not all eukaryotic cell types are susceptible to saponin-mediated lysis, this procedure should be evaluated with the host cell line of interest prior to implementation (see the Sidebar, Saponin Mediated Lysis of Eukaryotic Cells Harboring Bacterial Pathogens, for a brief protocol).
Following selective lysis of host cells, bacteria can be harvested and RNA purified with Ambion's RiboPure Bacteria Kit. RiboPure Bacteria is the only commercially available kit containing all reagents necessary for phenol-extraction and glass fiber filter purification of bacterial RNA. It is also compatible with RNAlater. RNA purified with RiboPure Bacteria is of extremely high quality and well suited for genome expression analysis with DNA arrays.
Enriching for Bacterial mRNA
For optimal cDNA synthesis and labeling prior to array analysis, it is useful to remove contaminating eukaryotic RNA from any unlysed host cells remaining with the bacterial cells. Ambion's MICROBEnrich™ Kit
(patent pending) was developed specifically to deplete eukaryotic RNA from mixtures of bacterial and host cell RNA. The MICROBEnrich Kit can be used with large quantities of eukaryotic RNA, even when small amounts of bacterial RNA are present in the mixture, such as when saponin-mediated lysis of host cells is not an option.
Kit employs a novel technology to selectively remove >90% of human, mouse, or rat total RNA from a mixed population of mammalian and prokaryotic RNA. The remaining bacterial RNA can then be used in downstream applications such as array analysis. The MICROBEnrich
reaction is scalable (up to 100 µg). The kit can be used to remove a total of 500 µg of mammalian RNA from a mixed population of mammalian/bacterial RNA. Typically, the MICROBEnrich Kit is used for 20 reactions, each consisting of mixed RNA samples containing up to 25 µg of mouse, rat, or human RNA. For further enrichment of bacterial mRNA, 16S and 23S bacterial rRNA can be removed from the bacterial total RNA using Ambion's MICROBExpress™ Kit
. The MICROBEnrich
procedure can be seamlessly integrated with Ambion's MICROBExpress
Kit for elimination of bacterial large subunit rRNAs by further enrichment of bacterial mRNA.
Sassetti C, Rubin EJ. (2002) Genome analyses of microbial virulence. Curr Opin Microbiol 5: 27-32.
Cummings CA, Rehlman DA. (2000) Using DNA microarrays to study host-microbe interactions. Emerg Inf Dis 6: 513-25.
Staudinger BJ, Oberdoerster MA, Lewis PJ, Rosen H. (2002) mRNA expression profiles for Escherichia coli ingested by normal and phagocyte oxidase-deficient human neutrophils. J Clin Invest 110(8): 1151-63.
Grifantini R, Bartolini E, Muzzi A, Draghi M, Frigimelica E, Berger J, Ratti G, Petracca R, Galli G, Agnusdei M, Giuliani MM, Santini L, Brunelli B, Tettelin H, Rappuoli R, Randazzo F, Grandi G. (2002) Previously unrecognized vaccine candidates against groupB meningococcus identified by DNA microarrays. Nature Biotechnol 20: 914-21.
Hsiao LL, Howard RJ, Aikawa M, Taraschi TF. (1991) Modification of host cell membrane lipid composition by the intra-erythrocytic human malaria parasite Plasmodium falciparum. Biochem J 274: 121-32.
Gould FK, Freeman R, Law D, Moriarty T. (1988) Lysis in detection of intracellular organisms. Lancet 2: 461.