Analysis of Staphylococcus aureus Gene Expression During PMN Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 104

the importance of bacterial genes in virulence and host interaction was deter-
mined either on an individual gene basis or for a limited number of genes in
any single study. However, this “single-target” approach is being supplanted
by microarray-based technologies, which have dramatically enhanced
our ability to interrogate host-pathogen interactions on a genome-wide
scale (3,4,6,7,8,9,10,11,12) .
Methods for measuring gene expression in Gram-positive bacteria have
existed for several decades (13,14) . Because of the presence of thick cell walls
not easily susceptible to lysis, early methods to isolate RNA from Gram-
positive organisms were very tedious and time consuming. Procedures included
incubating bacteria with detergent, using cesium chloride step-gradients in
combination with ultracentrifugation, and use of enzymes such as lysostaphin
to disrupt bacteria (15,16) . Because 90% of the S. aureus transcripts produced
during log-phase growth have a half-life less than 5 min (17) , enzymatic
treatment of bacteria is not ideal for isolation of RNA due to the time it
takes for the enzyme to lyse the bacteria. Moreover, designing experiments
to measure changes in gene expression in S. aureus during interaction with
host factors should not be complicated by additional treatments that may
independently cause changes in gene expression. Therefore, complex studies
investigating host-pathogen interactions require non-enzymatic means of lysing
S. aureus . Procedures using rapid physical disruption of the lysis-resilient S.
aureus cell walls (18) are more appropriate for investigating pathogen-host
interactions (3,4,6,7,8,10,11,12) . This is accomplished via high-speed (6 m/s)
reciprocating shaking of the sample with 0.1-m silica spheres in a suitable RNA
lysis buffer.
In this chapter, we describe a method for analyzing S. aureus gene
expression during interaction with human PMNs using Affymetrix oligonu-
cleotide microarrays. Except for human PMNs, the methods described herein
utilize commercially available reagents and can be reproduced by most standard
laboratories.
2. Materials
2.1. Bacterial Culture
1. Tryptic soy broth (BD Biosciences, San Jose, CA).
2. Dulbecco's phosphate-buffered saline (DPBS, Gibco/Invitrogen Corporation,
Carlsbad, CA).
3. Normal human serum.
4. RPMI 1640 medium (Gibco/Invitrogen) containing 10 m M HEPES (RPMI/H):
made by adding 5 mL of sterile 1 M HEPES (Sigma-Aldrich, St. Louis, MO) to
500 mL of sterile RPMI 1640 medium.