Microarray-based identification of htrA, a Streptococcus pneumoniae gene that is regulated by the CiaRH two-component system and contributes to nasopharyngeal colonization

Abstract

Nasopharyngeal carriage is the reservoir from which most disease with Streptococcus pneumoniae arises. Survival as a commensal in this environment is likely to require a set of adaptations distinct from those needed to cause disease, some of which may be mediated by two-component signal transduction systems (TCSTS). We examined the contributions of nine pneumococcal TCSTS to the process of nasopharyngeal colonization by using an infant rat model. Whereas deletions in all but one of these systems have been associated previously with a high degree of attenuation in a murine model of pneumonia, only the CiaRH system was necessary for efficient carriage. Transcriptional analysis by using microarray hybridization identified a locus consisting of two adjacent genes, htrA and spoJ, that was specifically and strongly downregulated in a DeltaciaRH-null mutant. A S. pneumoniae strain lacking the htrA gene encoding a putative serine protease, but not one lacking spoJ, showed decreased fitness in a competitive model of colonization, a finding consistent with this gene mediating a portion of the carriage deficit observed with the DeltaciaRH strain.

FIG. 1.

Recovery of S. pneumoniae strains from infant rat nasal washes. (A) 0100993 parent strain (♦) and 0100993-SR (□); (B) ΔciaRH (these two symbols represent two independent experiments); (C) ΔhtrA; and (D) ΔspoJ. Data shown for 0100993 are geometric mean values from independent inoculations of four litters; data for 0100993-SR are derived from inoculation of two litters; and data for all other strains represent inoculations of single, randomized litters consisting of 10 to 12 individuals each. Error bars indicate ±1 standard error of the mean. Dashed lines indicate the lower limit of detection of the colonization assay.

FIG. 2.

Magnitude and significance of gene expression changes in the ΔciaRH mutant compared to 0100993. The x axis represents the ΔciaRH/0100993 expression ratio on a logarithmic scale such that the dashed lines at 0.3 and −0.3 correspond to the twofold induction and repression criteria used in defining potential ΔciaRH-regulated genes. The dashed lines at 0.2 and −0.2 indicate criteria used in defining the set of genes excluded based on potential regulation in other TCSTS examined. The y axis represents the degree of confidence assigned to differences in gene expression between the ΔciaRH mutant and strain 0100993 given by P values from individual t tests. The horizontal dashed line indicates the position on the logarithmic scale of the P < 0.05 criterion used in defining potentially regulated genes.

FIG. 3.

Expression of htrA (▪) and spoJ (□) in the 0100993 wild-type strain, the ΔciaRH deletion mutant, and the six other TCSTS mutants for which expression data was obtained. Values were determined by microarray hybridization and are given as the geometric mean fluorescence normalized to the median fluorescence level of the entire set of probes on each array. Error bars represent ±1 standard deviation.

FIG. 4.

Competitive nasopharyngeal colonization assays of the 0100993-SR transformed parent strain (A and B, ▴) versus the ΔhtrA (A, ○) or ΔspoJ (B, •) null mutants. The data shown for each pair of strains are derived from competitive inoculations of one litter consisting of 10 to 12 individuals. A second independent competition experiment of ΔhtrA against 0100993-SR gave similar results. Dashed lines indicate the lower limit of detection of the colonization assay.

FIG. 5.

Organization of the region of the pneumococcal chromosomal surrounding the htrA-spoJ locus as determined from the TIGR4 sequence (28). Arrows indicate ORFs as well as a tRNA^arg sequence.