MsaB activates capsule production at the transcription level in Staphylococcus aureus

Abstract

Staphylococcus aureus produces several virulence factors that allow it to cause a variety of infections. One of the major virulence factors is the capsule, which contributes to the survival of the pathogen within the host as a way to escape phagocytosis. The production of the capsular polysaccharide is encoded in a 16 gene operon, which is regulated in response to several environmental stimuli including nutrient availability. For instance, the capsule is produced in the late- and post-exponential growth phases, but not in the early- or mid-exponential growth phase. Several regulators are involved in capsule production, but the regulation of the cap operon is still poorly understood. In this study, we show that MsaB activates the cap operon by binding directly to a 10 bp repeat in the promoter region. We show that despite the fact that MsaB is expressed throughout four growth phases, it only activates capsule production in the late- and post-exponential growth phases. Furthermore, we find that MsaB does not bind to its target site in the early and mid-exponential growth phases. This correlates with decreased nutrient availability and capsule production. These data suggest either that MsaB binding ability changes in response to nutrients or that other cap operon regulators interfere with the binding of MsaB to its target site. This study increases our understanding of the regulation of capsule production and the mechanism of action of MsaB.

Fig. 1.

cap operon promoter activity was measured in the wild-types, msaABCR mutants and msaB mutants of cap5 (a) and cap8 (b) in different growth phases (early, mid-, late- and post-exponential phases). The vector pCN58 containing luxAB without a promoter was used as a negative control. These results represent the means of three independent experiments. Error bars represent se. Student's unpaired t-test was used to compare the results of the wild-types to their respective mutants. ***P ≤ 0.001.

Fig. 2.

Capsule production assays. qRT-PCR was used to measure the relative fold changes in cap5E and cap8E in the mutants and the complemented mutants compared with the wild-type (a). Total capsule production was assessed in the representative CP5 and CP8 strains, including their respective mutants and complements of msaABCR (b) and msaB (c). Samples were serially diluted as indicated on the left and dot-blotted directly onto the membrane. The blots were processed using CP5- and CP8-specific antibodies. These results are representative of three independent experiments for each sample set.

Fig. 3.

Survival assays were performed using two different methods. Bacterial cells were mixed with freshly isolated human neutrophils to measure their survival rates against phagocytosis (a). Bacterial cells were mixed with heparinized human whole blood to measure their survival rates in the presence of complementing proteins (b). Results are presented as percentages of surviving bacterial cells relative to the control, which contained water instead of neutrophils (a) or by comparing the direct count of bacteria added to the total inoculum with the total c.f.u. enumerated, corrected by any dilution factor (b). Results represent the means of three independent experiments. Error bars represent se. Student's unpaired t-test was used to compare the results of wild-types to their respective mutants. The results are displayed with asterisks using the following P value cut-offs: ** P ≤ 0.01; ***, P ≤ 0.001.

Fig. 4.

(a) ChIP using an anti-MsaB antibody was performed to determine whether the promoter region of the cap operon binds to MsaB from whole-cell extracts in different growth phases (early, mid-, late- and post-exponential phases) from two strains that produce the two clinically relevant capsule serotypes (Mu50 for CP5 and UAMS-1 for CP8). Primers specific for the cap promoter region were used to amplify the DNA after immunoprecipitation. Lanes are labelled as follows: WT represents whole-cell extract from the wild-type strain and anti-MsaB; mutant is the negative control, representing the whole-cell extract from the msaABCR deletion mutant and anti-MsaB; (+) control represents the PCR product amplified from the genomic DNA of the tested strain. (b) Map of the cap promoter region showing the 10 bp repeat region that binds MsaB. DNA probes used in the EMSAs are also shown. EMSAs showed that purified MsaB bound to the DNA probe containing the 10 bp repeat region. All of these results are representative of at least three independent experiments.

Fig. 5.

(a) Western blot of Mu50 and UAMS-1 whole-cell lysates probed with an anti-MsaB antibody. Cells were harvested in the early, mid-, late- and post-exponential phases of growth and lysed, and 25 μg protein was loaded in each lane. These results are representative of three independent experiments for each sample set. (b) Absolute quantification of msaB in the four growth phases. Amplicons of msaB were converted to copies μl^− 1, and then serially diluted and used as templates for qRT-PCR. Standard curves were generated by plotting C _t values against the log of the copy numbers (log SQ). SQs of ‘unknown’ samples were calculated by plotting the respective C _t values on the standard curve. Copy numbers were measured by raising 10 to the power of the SQ (10^SQ). These results are representative of triplicate independently treated samples.

Fig. 6.

Total capsule production was assessed in the representative CP5 and CP8 strains, including the wild-types, the respective mutants and the controls, in nutrient-replenished medium (a) and nutrient-depleted medium (b). Samples were serially diluted as indicated on the left and dot-blotted directly onto the membrane. The blots were processed using CP5- and CP8-specific antibodies. (c) ChIP using an anti-MsaB antibody was performed to determine whether the promoter region of the cap operon binds to MsaB from whole-cell extract under nutrient-depleted conditions in the mid-exponential phase of growth. As in previously described ChIP reactions, primers specific for the cap promoter region were used to amplify the DNA after immunoprecipitation. Lanes are labelled as follows: Mu50 (nutrient depleted) and UAMS-1 (nutrient depleted) represent whole-cell extracts from wild-type CP5 and CP8 strains, respectively, and anti-MsaB antibody; ΔmsaABCR mutant represents whole-cell extract from an msaABCR deletion mutant and anti-MsaB antibody; (+) control represents PCR product amplified from the genomic DNA of the tested strain. These results are representative of three independent experiments.