The AgI/II family adhesin AspA is required for respiratory infection by Streptococcus pyogenes

Abstract

Streptococcus pyogenes (GAS) is a human pathogen that causes pharyngitis and invasive diseases such as toxic shock syndrome and sepsis. The upper respiratory tract is the primary reservoir from which GAS can infect new hosts and cause disease. The factors involved in colonisation are incompletely known however. Previous evidence in oral streptococci has shown that the AgI/II family proteins are involved. We hypothesized that the AspA member of this family might be involved in GAS colonization. We describe a novel mouse model of GAS colonization of the nasopharynx and lower respiratory tract to elucidate these interactions. We used two clinical M serotypes expressing AspA, and their aspA gene deletant isogenic mutants in experiments using adherence assays to respiratory epithelium, macrophage phagocytosis and neutrophil killing assays and in vivo models of respiratory tract colonisation and infection. We demonstrated the requirement for AspA in colonization of the respiratory tract. AspA mutants were cleared from the respiratory tract and were deficient in adherence to epithelial cells, and susceptible to phagocytosis. Expression of AspA in the surrogate host Lactococcus lactis protected bacteria from phagocytosis. Our results suggest that AspA has an essential role in respiratory infection, and may function as a novel anti-phagocytic factor.

Figure 1. Bacterial numbers (cfu) within nasopharynx or lungs at various times following intranasal challenge of mice (1×10⁸ cells) with S. pyogenes MGAS10270 (•) or MGAS10270 aspA (Δ) (UB2115).

Data are means ± SEM of 3 experiments.

Figure 2. Bacterial numbers (cfu) within nasopharynx or lungs at various times following intranasal challenge of mice (1×10⁸ cells) with S. pyogenes MGAS6180 (•) or MGAS6180 aspA (Δ) (UB2086).

Data are means ± SEM of 3 experiments.

Figure 3. Adherence levels of MGAS10270 or MGAS6180 and corresponding ΔaspA mutants to A549 lung epithelial cells (pneumocytes) or Detroit 562 pharyngeal epithelial cells.

Adherence is expressed as percentage of input cells (10⁶ cfu) attached. Data are means ± SEM of 3 experiments repeated in triplicate. * = P<0.001 using 2 tail student T-test.

Figure 4. Numbers of bacteria (cfu) killed by macrophages (J774.2 cell line) following 1 h co-incubation with S. pyogenes MGAS6180 or MGAS10270 wild type and aspA mutants (input 5×10³ cfu, 5×10⁴ macrophages).

Percentage killing was calculated from cfu remaining compared with control samples without macrophages. Data are means ± SEM of 3 experiments repeated in triplicate. * = P<0.001 using 2 tail student T-test.

Figure 5. Percentage killing of S. pyogenes MGAS6180 or MGAS10270 wild type and aspA mutants by HL60 neutrophils following 1 h co-incubation as compared with controls.

Percentage killing was calculated from cfu remaining compared with control samples without neutrophils (input 10⁴ cfu, 10⁵ neutrophils). Data are means ± SEM of 5 experiments repeated in triplicate. * = P<0.05 using 2 tail student T-test.

Figure 6. Percentage killing of L. lactis MG1363 wild type, and AspA or SspB expressing strains, by (A) J774.2 macrophages or (B) HL60 neutrophils following 1 h co-incubation compared with controls.

Percentage killing was calculated from cfu remaining compared with control samples without macrophages or neutrophils. Data are means ± SEM of 3 experiments repeated in triplicate. * = P<0.05 using 2 tail student T-test.

Figure 7. Model for the establishment of longer-term respiratory tract colonization by GAS.

Following adhesion of bacterial cells to epithelium, and initial transient colonization, there is depletion of bacterial numbers due to host immune responses. These include innate factors, such as anti-microbial peptides and agglutinins, neutrophils and macrophages. A small number of bacterial cells successfully evade these responses, perhaps associated with up-regulation of AspA or transient internalization by epithelial cells. Expression of AspA brings into play the anti-phagocytic properties and biofilm-enhancing activities of AspA, leading to prolonged colonization of the mucosa. Asterisks denote temporal role for AspA.