Development and Characterization of Murinized Listeria monocytogenes Strains Carrying the Most Common Forms of Internalin A Premature Stop Codons

Introduction: Internalin A (InlA) is used by Listeria monocytogenes (Lm) to invade host epithelial cells. InlA binds certain isoforms of E-cadherin (human and guinea pig) to cross the intestinal barrier; however, it shows limited interaction with murine E-cadherin. 

Purpose: Engineer murinized Lm strains carrying the most common InlA premature stop codons (PMSCs) and characterize the invasion phenotype and virulence of resultant strains. 

Methods: Site directed mutagenesis was used to introduce InlA PMSC3 and PMSC4 into a previously murinized strain (EGD-eInlA^m*). Growth and cell invasion capabilities were investigated using EGD-e, EGD-eInlA^m*, EGD-eInlA^m*:PMSC3, and EGD-eInlA^m*:PMSC4. For growth curves, BHI broth was inoculated with populations of the four strains (6.68 log (CFU/ml)) and was maintained at 37°C for 12 h.  Invasion was assayed in mouse CT-26 and human Caco-2 cells to characterize invasiveness of all strains. BALB/c mice were intragastrically infected with EGD-e, EGD-eInlA^m*, or EGD-eInlA^m*:PMSC3 at 9.3 log CFU and recovery of strains from internal organs was used to define infection. 

Results: All isolates grew similarly at 37°C and exceeded 9.7 log (CFU/ml) by 12 h.  In CT-26 cells, invasion of EGD-eInlA^m* was significantly (P < 0.05) higher compared to all strains. As expected, invasion did not differ (P >0.05) between EGD-e and EGD-eInlA^m* in Caco-2 cells, and both PMSC strains showed attenuated invasion (P < 0.05). After intragastric infection, EGD-eInlA^m* showed higher (P < 0.05) bacterial levels in the liver, spleen, mesenteric lymph nodes, and small intestines as compared to EGD-e and EGD-eInlA^m*:PMSC3.  Importantly, EGD-eInlA^m*:PMSC3 displayed attenuated virulence as determined by lower bacterial loads in livers and spleens. 

Significance: We engineered murinized Lm strains with the most common InlA PMSCs and demonstrated predicted invasion and virulence characteristics of these strains. Because immunological reagents are unavailable for the guinea pig model, it is critical to obtain a murine Lm oral infection model permitting investigation of immunological responses to InlA PMSC strains, which are commonly isolated from ready-to-eat foods (>45%).