Abstract
Human pathogens on plants (HPOP) have evolved complex interactions with their plant host. Stomatal internalization is one such mode of interaction, where bacteria are attracted to stomata and penetrate into the substomatal cavity by a process mediated by chemotaxis. Internalization enables HPOP to evade the hostile environment of the leaf surface and find a protected, nutrient-rich niche within the leaf. Numerous studies have documented attachment and entry of the foodborne pathogens, Salmonella enterica and Escherichia coli into stomata. Internalization, however, varies considerably among different pathogens and in different plants, and both bacterial and plant’s factors were reported to influence HPOP attachment and internalization. Here we have studied the effect of laboratory growth conditions, on the internalization of Salmonella enterica serovar Typhimurium (STm) into lettuce leaf. We have further tested the potential involvement of universal stress-proteins in leaf internalization. We found that STm grown in Luria Bertani broth devoid of NaCl (LBNS), or in diluted LB (0.5×LB) internalized lettuce leaf better (62 ± 5% and 59 ± 7%, respectively) compared to bacteria grown in LB (15 ± 7%). Growth under non-aerated conditions also enhanced STm internalization compared to growth under aerated conditions. Growth temperature of 25 and 37°C did not affect STm internalization, however, growth at 42°C, significantly augmented leaf internalization. Since, the tested growth conditions represent moderate stresses, we further investigated the involvement of five universal-stress genes in STm leaf internalization following growth in LBNS medium. Knockout mutations in ydaA, yecG, ybdQ, and uspAB, but not in ynaF, significantly reduced STm internalization compared to the wild-type (wt) strain, without affecting bacterial attachment and motility. Transduction of the mutations back to the parent strain confirmed the linkage between the mutations and the internalization phenotype. These findings support a specific role of the universal-stress genes in leaf internalization. The present study highlights the complexity of bacterial internalization process and may provide partial explanation for the variable, sometimes-contrasting results reported in the literature regarding stomatal internalization by HPOP. Characterization of the regulatory networks that mediate the involvement of usp genes and the tested growth factors in STm internalization should contribute to our understanding of human pathogens-plant interactions.
Highlights
Consumption of fresh vegetables has consistently increased over the last decades, since they are considered an important part of a healthy and low-calorie diet (Tirpanalan et al, 2011)
Bacteria were streaked on LB agar for overnight and fresh colony were re-suspended in 10 ml LB broth and grown with shaking (150 rpm) for 18–20 h at 37◦C to generate the inoculum for the internalization assay
Similar to Salmonella, pregrowth of E. coli strains in LBNS broth resulted in higher leaf internalization compared to growth in LB broth (28 ± 3% and 1.8 ± 1.2%, respectively) (Figure 3)
Summary
Consumption of fresh vegetables has consistently increased over the last decades, since they are considered an important part of a healthy and low-calorie diet (Tirpanalan et al, 2011). There is an increase in outbreaks of food-borne illness associated with the consumption of fresh produce (Brandl and Sundin, 2013; Mritunjay and Kumar, 2015). Lettuce is one of the vegetables, which often linked to outbreaks of foodborne diseases. Efforts to develop chemical and technological means to disinfect vegetables are ongoing (Meireles et al, 2016), there is currently no treatment that can effectively kill foodborne pathogens on fresh produce (Goodburn and Wallace, 2013). Understanding the factors that affect plant internalization by HPOP is needed toward developing new approaches to enhance the safety of fresh produce
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