Inhibitor of streptokinase gene expression improves survival after group A streptococcus infection in mice

Abstract

The widespread occurrence of antibiotic resistance among human pathogens is a major public health problem. Conventional antibiotics typically target bacterial killing or growth inhibition, resulting in strong selection for the development of antibiotic resistance. Alternative therapeutic approaches targeting microbial pathogenicity without inhibiting growth might minimize selection for resistant organisms. Compounds inhibiting gene expression of streptokinase (SK), a critical group A streptococcal (GAS) virulence factor, were identified through a high-throughput, growth-based screen on a library of 55,000 small molecules. The lead compound [Center for Chemical Genomics 2979 (CCG-2979)] and an analog (CCG-102487) were confirmed to also inhibit the production of active SK protein. Microarray analysis of GAS grown in the presence of CCG-102487 showed down-regulation of a number of important virulence factors in addition to SK, suggesting disruption of a general virulence gene regulatory network. CCG-2979 and CCG-102487 both enhanced granulocyte phagocytosis and killing of GAS in an in vitro assay, and CCG-2979 also protected mice from GAS-induced mortality in vivo. These data suggest that the class of compounds represented by CCG-2979 may be of therapeutic value for the treatment of GAS and potentially other gram-positive infections in humans.

Fig. 1.

Compound structures. Compounds (A–J) were identified in the original high-throughput screen. CCG-2979 was chosen as the lead compound (details in the text). CCG-102487 was identified as a commercially available analog of CCG-2979.

Fig. 2.

Effect of CCG-2979 and CCG-102487 on SK expression and GAS growth. (A) Effects of CCG-2979 and CCG-102487 on the production of SK activity. Normalized SK activity of GAS treated with CCG-2979 or CCG-102487 at concentrations from 5 to 100 μM (SK activity of culture media was divided by OD₆₀₀ and then normalized to the value for DMSO-treated GAS, which was defined as 100%). The value was presented as mean ± SE for a total of 18 samples (pooled from six independent experiments with triplicates). (B) GAS growth as measured by OD₆₀₀ of an overnight culture in the presence of CCG-2979 at concentrations of 5–100 μM normalized to the OD₆₀₀ of control GAS grown in DMSO vehicle alone. The value was presented as mean ± SE for a total of 18 samples (pooled from six independent experiments with triplicates). (C) Growth curves for UMAA2616 in the presence of 0.1% DMSO, 50 μM CCG-2979, or 50 μM CCG-102487. Mean and SE for a total of six samples (two independent experiments with triplicates) are presented for each time point. (D) Decreased GAS resistance to phagocytosis in the presence of CCG-2979 or CCG-102487. Each point represents an individual experiment.

Fig. 3.

The effect of CCG-102487 on the GAS gene expression program. Growth curves for MGAS2221 in the presence of CCG-102487 (46.7 μM) or DMSO vehicle alone as determined by OD (A) or cfu (B). (C) Graphic summary of gene expression changes. Genes down-regulated on treatment with CCG-102487 are indicated in green, and genes up-regulated on treatment with CCG-102487 are indicated in red. Genes of known homology or function were included. Complete data for gene expression changes are summarized in Table S1.

Fig. 4.

Treatment with CCG-2979 improves survival in an in vivo mouse model for GAS infection. (A) Effect on survival of CCG-2979 [12.7 nmol (5 μg) per mouse] injected daily for 5 d beginning 24 h after infection of PLGTg+ mice with UMAA2616. Data were pooled from four independent experiments (with GAS inoculation of 8.3, 2.8, 3.6, or 4.0 × 10⁵ cfu/mouse, respectively). A total of 27 mice were represented in the control group treated with vehicle alone (solid line), and 27 mice were represented in the CCG-2979–treated group (dashed line). (B) The same model as in A except that CCG-2979 was injected at a dose of 101.4 nmol (40 μg) per mouse per day for 4 d. Data were pooled from three independent experiments (with GAS inoculation of 4.22, 3.16, or 1.24 × 10⁶ cfu/mouse, respectively). A total of 22 mice were represented in the control group (solid line), and 19 mice were represented in the CCG-2979–treated group (dashed line). (C) Pooled data from A and B, including all data for CCG-2979. (D) Pooled data for both compounds. CCG-102487 [93.3 nmol (40 μg) per mouse] was injected daily for 4 d beginning 24 h after infection of PLGTg+ mice with UMAA2616. Data for CCG-102487 (dotted line) are from one experiment (total of 10 mice with GAS inoculation of 4.22 × 10⁶ cfu/mouse). P values were calculated with all pairwise multiple comparison procedures (Bonferroni method).