AvrA effector protein of Salmonella enterica serovar Enteritidis is expressed and translocated in mesenteric lymph nodes at late stages of infection in mice

Abstract

Salmonellosis is a major health problem worldwide. Salmonella enterica serovar Enteritidis (S. Enteritidis) has been a primary cause of Salmonella outbreaks in many countries. AvrA is an SPI-1 effector protein involved in the enteritis pathway, with critical roles in inhibiting inflammation and apoptosis. In this work, we constructed an AvrA-FLAG-tagged strain of S. Enteritidis to analyse the expression profile of AvrA in vitro, in cell culture and in vivo. AvrA expression and secretion were observed in vitro under culture conditions that mimicked intestinal and intracellular environments. In agreement, bacteria isolated from infected cell monolayers expressed and translocated AvrA for at least 24 h post-inoculation. For in vivo experiments, BALB/c mice were inoculated by the natural route of infection with the AvrA-FLAG strain. Infecting bacteria and infected cells were recovered from mesenteric lymph nodes (MLN). Our results showed that AvrA continues to be synthesized in vivo up to day 8 post-inoculation. Moreover, AvrA translocation was detected in the cytosol of cells isolated from MLN 8 days after infection. Interestingly, we observed that AvrA is secreted by both type three secretion system (T3SS)-1 and T3SS-2. In summary, these findings indicate that AvrA expression is not constrained to the initial host–bacteria encounter in the intestinal environment as defined previously. The AvrA effector may participate also in systemic S. Enteritidis infection.

Animal Models of Type III Secretion System-Mediated Pathogenesis

“…This section covers selected in vivo techniques to study the T3SS. Mice are the most common rodent used to study T3SS pathogenesis [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50]. They are small, inexpensive, and well described in comparison to other models.…”

“…The T3SS is responsible for the secretion of effector proteins, and their expression can be correlated to T3SS function and inhibition [11,44,45]. Many T3SS inhibitors have been discovered using in vitro secretion assays [33].…”

“…These assays can also be performed in animals, giving a significantly more realistic model of infection, pharmacokinetics, and immune response. Another strategy for measuring T3SS expression is to monitor the transcription of T3SS genes using reverse transcription PCR [45,143]. This allows for quantification of gene expression for specific effectors or regulatory genes.…”

“…The Giacomodonato laboratory has published multiple papers describing strains of S. Typhimurium that produce FLAG-tagged T3SS effector proteins such as SopA, SopB, and AvrA [44,45]. These proteins can be quantified by immunoblotting due to the FLAG tag.…”

Patterns of Expression and Translocation of the Ubiquitin Ligase SlrP in Salmonella enterica Serovar Typhimurium

“…The transition from T3SS1 to T3SS2 expression, observed in certain infection models, is driven by processes of differential regulation and cross talk between regulatory proteins. There are, however, examples of expression of T3SS1 effectors at late stages of infection (11,12) and circumstances of coexpression and cooperation between both T3SSs, depending on the host cell type or the entry mode (13).…”