A novel approach to develop anti-virulence agents against group A streptococcus

The emergence of antibiotic resistance among human pathogens is a major cause of concern for public health. In recent years, an alternative approach has been proposed to target virulence of pathogens instead of their growth to generate new antimicrobial agents. We discovered a chemical series of novel anti-virulence agents against group A streptococcus (GAS), a common human pathogen. The anti-virulence agents were able to inhibit the gene expression of multiple virulence factors and also protect mice against group A streptococcal infection. This proof of concept study supports the new paradigm of developing novel therapeutic approaches targeting gene expression of bacterial virulence factors.

The discovery and wide use of antibiotics have greatly attenuated the threat of infectious diseases and contributed to the increase in lifespan since the 1940s. However, the alarming rise of antibiotic resistance among almost all major human bacterial pathogens has become a serious public health problem, which is further aggravated by a diminished developmental pipeline for new antibiotics.

The antibiotics currently in clinical use generally target essential bacterial functions, leading to bacterial death or inhibition of growth. As a result, strong selective pressure will favor bacterial strains that have developed resistance. Antibiotic resistance is now a serious problem for nearly all major bacterial pathogens, including resistance to multiple antibiotics, which has developed in large part due to inappropriate and excessive use of antibiotics.

Recently, a novel approach has been proposed aiming to block pathogen virulence without inhibiting bacterial growth, thereby minimizing selection for the resistance. Several proof-of-concept studies have been published. Hung et al. reported discovery of a novel small compound that inhibited the gene expression of two critical cholera virulence factors, cholera toxin and toxin coregulated pilus. This compound demonstrated in vivo efficacy, protecting infant mice from intestinal colonization by Vibrio cholerae (Hung et al., Science 2005). A small compound was also identified that can inhibit the membrane-embedded sensor histidine kinase QseC and protected mice against infection by S. typhimurium and Francisella tularensis (Rasko et al., Science 2008).

In our previous studies, we demonstrated that group A streptococcus utilized the host hemostatic system to facilitate invasion (Sun et al., Science 2004). GAS, also known as Streptococcus pyogenes, is a major human pathogen, causing over 700 million infections each year worldwide. GAS can cause tonsillitis, impetigo and invasive diseases such as streptococcal toxic shock-like syndrome and necrotizing fasciitis. Impetigo is endemic among children of developing countries (Parks et al., Curr Opin Infect Dis 2012). Post-infection complications of impetigo and streptococcal pharyngitis include rheumatic fever and rheumatic heart disease, causing 15.6–19.6 million and 282,000 new cases, respectively, and resulting in 233,000 to 294,000 deaths each year worldwide. Approximately 663,000 cases of invasive GAS disease occur annually with approximately 25% mortality (Carapetis et al., Lancet Infect Dis 2005).

GAS produces streptokinase (SK) that can activate human plasminogen to form plasmin, which is the central enzyme of the fibrinolysis system. We reported that the interaction of SK with human plasminogen was critical for GAS pathogenicity. GAS is a strict human pathogen and the host specificity of GAS was speculated to be due to the species-specific interaction between human plasminogen and SK. We established a murine model in which human plasminogen was expressed by a transgene. The transgenic mice expressing human plasminogen demonstrated significantly increased susceptibility to GAS infection compared with wild-type mice. When the transgenic mice were infected with a GAS mutant in which SK had been inactivated, this increased susceptibility was largely abolished. These data suggested that both human plasminogen and SK were required for susceptibility to GAS infection. SK was thus considered as a potential therapeutic target for anti-GAS agents.

We developed a simple growth-based turbidometric high throughput screening approach to search for low molecular weight compounds that inhibit expression of the SK gene (Sun et al., Proc Natl Acad Sci USA 2012). A modified GAS strain SKKanGAS was generated carrying a kanamycin resistance gene driven by the SK gene promoter in an extrachromosomal plasmid. A control GAS strain UMAA2641 with the same kanamycin resistance gene driven by a different promoter was used for counter screening. A total of 55,000 small molecules were screened to identify compounds capable of inhibiting the growth of the SKKanGAS strain under kanamycin selection without interfering with UMAA2641 growth. Positive hits from this screen were tested in a secondary screen for inhibition of wild-type GAS SK protein expression without growth inhibition.

Compounds that inhibit SK expression with minimal interference of GAS growth were selected. A lead compound (CCG-2979) was selected based on its ability to inhibit SK expression with little inhibitory effect on GAS growth. A commercially available analog of CCG-2979 also demonstrated potency at inhibiting SK expression. The global effect of this SK expression inhibitor on GAS gene expression was characterized by a mRNA microarray analysis.

Surprisingly, a wide range of genes was affected (Fig. 1). As expected, SK was downregulated. A number of other key virulence factors, including multiple adhesins, antiphagocytic factors and cytolytic toxins, were also found to be affected at the mRNA expression level (Fig. 1). In addition to virulence factors, the SK expression inhibitor also changed the expression of genes involved in metabolism and energy production.

Figure 1. Novel small compound affected expression of Streptococcus pyogenes genes involved in virulence, metabolism and energy production

Gene regulation has been extensively studied in GAS. Gene regulatory systems in GAS include “stand-alone” response regulators and two component systems (TCS) (Kreikemeyer et al., Trends Microbiol 2003). “Stand-alone” regulators are transcriptional regulatory proteins that regulate expression of multiple genes in response to the environment. Two component systems are used by bacteria to sense and respond to environmental stimuli. They consist of a membrane-bound sensor and cytoplasmic response regulator and control the up or downregulation of gene expression of multiple virulence factors (Kreikemeyer et al., Trends Microbiol 2003). The gene expression profile changes we observed suggest that these compounds target a major regulation pathway that affect multiple genes involved in virulence and potentially other cellular functions.

Both compounds decreased GAS resistance to phagocytosis. The in vivo efficacy of these two compounds was tested in our previously established human plasminogen transgenic mouse model (Sun et al., Science 2004). Mice were treated with the small compounds one day after infection with GAS and a significant improvement in survival was observed in mice treated with the lead compound CCG-2979. It is worth noting that the streptokinase gene is conserved in some pathogenic streptococci such as Streptococcus dysgalactiae and Streptococcus equi. Streptococcus equi is an important horse pathogen, causing strangles, which is one of the most frequently encountered equine diseases, accounting for almost 30% of infectious cases. Thus, this class of compounds could also inhibit gene expression of virulence factors of these important human and domesticated animal pathogens.

In summary, our current study demonstrates the feasibility of developing agents against GAS infection by targeting regulators of gene expression of SK and other virulence factors. The novel anti-virulence agents function through a different pathway from common antibiotics. As a result, this class of anti-microbial agents may complement the current antibiotics and increase the efficacy of the antibiotic treatment (Waldor, N Engl J Med 2006).

Sun H, Xu Y, Sitkiewicz I, Ma Y, Wang X, Yestrepsky BD, et al. Inhibitor of streptokinase gene expression improves survival after group A streptococcus infection in mice. Proc Natl Acad Sci U S A. 2012;109:3469–74. doi: 10.1073/pnas.1201031109.