Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Microbiology & Immunology
Non Technical Summary
The goal of this project is to elucidate the function of proteinases SpeB and IdeE2 in streptococcal infections of upper respiratory tract. S. equi and Group A Streptococcus (GAS) are -hemolytic streptococci that mainly cause upper respiratory infections in horses and human, respectively. Why the infections of upper respiratory tract usually do not spread into lower respiratory tract is not well understood. GAS occasionally causes lower respiratory infections and other severe invasive infections, and invasive GAS isolates frequently cmTy mutations in virulence regulators CovRS and RopB. CovRS and RopB mutants have down-regulation of proteinase SpeB expression. We hypothesize that high levels of SpeB production by GAS play a critical role in upper respiratory infections. S. equi and GAS are phylogenetically related to each other and have simiim· set of virulence factors. To take the advantage of extensive knowledge on GAS pathogenesis, we will first determine whether SpeB facilitates infections of upper respiratory tract by allowing infection of epithelial cells and limits infections of lower respiratory tract by enhancing neutrophil-mediated clearance. We will then apply these findings to S. equi by determining whether IdeE2 plays a critical role in infections of epithelial cells and upper respiratory tract. The project will advance the understanding of S. equi pathogenesis and have the potential to identify new targets for developing a safe and effective strangles vaccine.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The goal of this project is to elucidate the function of proteinases SpeB and IdeE2 in streptococcal infections of upper respiratory tract. S. equi and GAS are -hemolytic streptococci that mainly cause upper respiratory infections in horses and human, respectively. Why the infections of upper respiratory tract usually do not spread into lower respiratory tract is not well understood. GAS occasionally causes lower respiratory infections and other severe invasive infections, and invasive GAS isolates frequently caiTy mutations in virulence regulators CovRS and RopB. CovRS and RopB mutants have down-regulation of proteinase SpeB expression. We hypothesize that high levels of SpeB production by GAS play a critical role in upper respiratory infections. S. equi and GAS are phylogenetically related to each other and have similar set of virulence factors. By taking the advantage of a greater array of molecular tools, mutants, and animal models in our GAS study, we will first determine whether SpeB facilitates infections of upper respiratory tract by allowing infection of epithelial cells and limits infections of lower respiratory tract by enhancing neutrophil-mediated clearance. We will then apply these findings to S. equi by determining whether IdeE2 plays a critical role in infections of epithelial cells and upper respiratory tract. We will pursue two objectives.Objective 1: Determine whether SpeB plays a critical role in GAS infection in up-respiratory tractWe will first define the source-sink dynamics of covRS'vtropB'vt GAS and covRS111111 and ropBmut mutants in infections of upper and lower respiratory tracts. We will address the following questions: 1) Whether the lower respiratory tract is the sink habitat for wild-type M3 and Ml Tl GAS but not their covRS and ropB mutants and 2) whether covRS'v'ropB'vt GAS strains GAS2744 and MGAS2221 have higher ability to colonize the throat than their t'icovRS and t'iropB mutants and to infect epithelial cells. We will then determine whether and how the down-regulation of SpeB expression plays a critical role in alteration in innate immune evasion, GAS clearance, and epithelial cells.Objective 2: Determine whether IdeE2 significantly contributes to S. equi infection of epithelial cells and upper-respiratory tract through evasion of autophagic killingWe will generate an ideE2 deletion mutant of S. equi. We will use the intranasal mouse infection model to determine whether IdeE2 contribute to S. equi throat colonization. We will then determine whether IdeE2 helps S. equi infect epithelial cells in vivo and in vitro. Finally, we will detennine whether IdeE2 evades autophagic killing in epithelial cells.
Project Methods
Objective 1: Determine whether SpeB plays a critical role in GAS infection in up-respiratory tractTo achieve Objective 1, we will address the following questions: 1) Whether the lower respiratory tract is the sink habitat for wild-type M3 and M1T1 GAS but not their covRS and ropB mutants, 2) whether covRSwtropBwt GAS strains GAS2744 and MGAS2221 have higher ability to colonize the throat than their DcovRS and DropB mutants and to infect epithelial cells; and 3) whether and how the down-regulation of SpeB expression plays a critical role in the change in innate immune evasion, GAS clearance, and epithelial cells from wild-type GAS to covRS and ropB mutants.To address question 1, we will determine whether covRSwtropBwt M3 and M1T1 GAS strains are cleared by robust neutrophil response whereas their DcovS and DropB mutants are not in murine pulmonary infection. Then, we will determine whether the clearance of GAS with high speB expression are medicated by neutrophils. Pulmonary infections with GAS2744 and MGAS2221 will be conducted in neutropenic mice and KO mice of IL-1 receptor (IL-1R-/-) and phagocyte NADPH oxidase gp91 gene (gp91phox-/-). The purpose of including the infection of IL-1R-/- is to determine whether the detrimental effect of the RB6-8C5 treatment on GAS2744 and MGAS2221 clearance is from the depletion of neutrophils or inflammatory monocytes. The purpose of using gp91phox-/- mice is to determine whether GAS clearance in pulmonary infection is dependent on NADPH oxidase. We expect to establish that M3 and M1T1 GAS with high speB expression are cleared in murine pulmonary infections by neutrophils in a NADPH oxidase-dependent mechanism whereas DcovRS and DropB mutants inhibit neutrophil infiltration, resist clearance, and establish reactions.To address question 2, we will compare GAS2744 and MGAS2221 with their ropB deletion mutants in throat colonization. These bacteria will be inoculated into the nasal nostril of 5 female and 5 male C57BL/6J mice and neutropenic mice. The throat will be swapped once a day for 5 days after GAS inoculation, and the number of GAS in each swab will be determined by plating. Inclusion of the infection in the neutropenic mice has two purposes. We expect that wt GAS strains have higher ability to colonize the throat in mice than their DcovS and DropB mutants. Then, we will determine whether GAS2744 and MGAS2221, but not their DcovS and DropB mutants, can infect and multiply in epithelial cells in vivo and in vitro. For in vivo test, the strains will be inoculated into the trachea of RB6-8C5-treated female and male C57BL/6J mice, and the lung samples will be collected at 30 h after inoculation, and thin lung section will be stained with Gram and H&E staining. Neutrophil infiltration should be scarce in the lung samples that the H&E stain will tell, and Gram-stain will tell whether GAS infects the bronchial epithelium. For in vitro test, human lung A549 epithelial cells will be infected with GAS, and GAS bacteria inside cells at different time in infection will be quantified by plating and visualized by immunostaining using antibodies specific for the group A antigen. We expect that GAS2744 and MGAS2221 will invade the epithelium at the bronchioles in the neutropenic mice and multiply in A549 cells whereas their DcovS and DropB mutants will not multiply in the bronchial epithelium and have lower ability to invade and multiply in A549 cells.To address question 3, we will correct the speB G-68T mutation in GAS2740 and introduce the mutation into GAS2744. Levels of speB mRNA of these strains at stationary growth phase will be measured, and SpeB activity and protein levels in stationary culture supernatant will be compared. These measurements will tell us whether the speB G-68T mutation downregulates speB transcription or translation. Then, these strains will be compared in neutrophil infiltration and clearance in C57BL/6J and invasion of the bronchial epithelium in the neutropenic mice during pulmonary infection. They will also be compared in GAS multiplication in human A549 epithelial cells. If the speB G-68T mutation causes the phenotype changes that are similar with those caused by covRS and ropB mutations, this result would strongly suggest that the downregulation of the SpeB production is a critical factor for covRSmut and ropBmut GAS to inhibit neutrophil infiltration and reduce GAS clearance and epithelial infection.A role of the down-regulation of speB expression in the innate immune evasion and invasion of epithelial cells by covRS and ropB mutants will be further examined as follows. We will replace the promoter 2 of speB in GAS2744 DcovS with the 70-bp sequence that contains the Mga-binding site and the promoter of the emm gene. We expect that the promoter replacement in the presence of ropB mutation or covS deletion will restore high levels of SpeB production. Reciprocally, the speB gene in GAS2744 will be deleted. We will use these isogenic strains to determine whether high levels of SpeB production through the promoter replacement in covRS and ropB mutants lead to robust neutrophil infiltration and GAS clearance in pulmonary infection of mice, infection of the bronchial epithelium and human A549 epithelial cells.Objective 2: Determine whether IdeE2 significantly contributes to S. equi infection of epithelial cells and upper-respiratory tract through evasion of autophagic killingFirst, we will determine whether IdeE2 contributes to S. equi infection of epithelial cells and upper-respiratory tract. We will generate an ideE2 deletion mutant of S. equi 4047. DideE2 will be compared with the parent strain in throat colonization in intranasal infection of mice, infection of the bronchial epithelium in neutropenic mice, and invasion and multiplication in equine lung cell line extEqFL cells.Second, we will determine whether IdeE2 functions through evasion of autophagic killing in epithelial cells. Heterozygous disruption of the atg6 or beclin 1 autophagy gene reduces autophagy. We would test whether S. equi 4047 has higher throat colonization in Atg6- than in C57BL/6J and whether S. equi 4047 infects the bronchial epithelium of neutropenic mice derived from Atg6- mice. Next, we will compare S. equi 4047 and its DideE2 in association with LC3B, the marker for autophagy, using immunostaining with anti-S. equi and anti-LC3B antibodies at different times of infection of extEqFL cells. If IdeE2 evades the autophagic killing, the association of DideE2 with LC3B will be lower than that of the parent bacteria. To further test the possible role of S. equi evasion of autophagic killing, we will prepare recombinant IdeE2 protein and test whether IdeE2, like SpeB, cleaves Ubiquitin-LC3 Adaptor Proteins NDP52, p62, and NBR1 in extEqFL cell lysates. Third, we will screen S. equi surface proteins for proteins that promote invasion of epithelial cells. We have deletions mutants of genes encoding 8 surface proteins in our previous studies. We will compare these mutants with the parent strain in invasion of extEqFL cells. We will infect cultured cells with the bacteria for 4 h and then quantify numbers of intracellular S. equi by platting. If we identify proteins that are important for invasion of epithelial cells, we can target it to reduce infection of epithelial cells as a new strategy to prevent strangles.