Salmonella enterica Growth Conditions Influence Lettuce Leaf Internalization

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.

A Simple Assay to Assess Salmonella enterica Persistence in Lettuce Leaves After Low Inoculation Dose

“…Although S. enterica induces plant defense responses ( Meng et al, 2013 ; Garcia and Hirt, 2014 ; Melotto et al, 2014 ; Oblessuc et al, 2020 ), it can still persist for long periods in the leaf apoplast depending on the bacterial strain and the plant genotype ( Wong et al, 2019 ; Jacob and Melotto, 2020 ). Furthermore, recent studies have shown that variations in the S. enterica culturing conditions, such as temperature and nutrients in the medium ( Kroupitski et al, 2019 ), and environmental conditions for the plant cultivation, such as temperature and humidity ( Deblais et al, 2019 ; Jechalke et al, 2019 ; Roy and Melotto, 2019 ), can interfere mainly with the ability of S. enterica to internalize plant tissues. Nevertheless, variations in environmental conditions not only affect the bacterial internalization, which indeed is an important step during bacterial colonization of plants, but also interfere with the outcome of the plant–pathogen interactions and the persistence phenotype.…”

Comparing the effectiveness of Cinnamomum zeylanicum essential oil and two common household sanitizers to reduce lettuce microbiota and prevent Salmonella enterica recontamination

“…An adhesion reduction due to the treatment was observed, with fewer and ungrouped bacterial cells in SH samples (Figure 3b); moreover, in the same samples, Salmonella internalization within stomata was observed. This pathogen can use open stomata as a portal of entry into deeper tissues of the plant host (Kroupitski, Gollop, Belausov, Pinto, & Sela, 2019), and this is believed to be a protection mechanism to evade the hostile environment of the leaf during surface disinfection (Golberg, Kroupitski, Belausov, Pinto, & Sela, 2011). Thus, it is possible that unfavorable conditions created by SH on leaf surface prompted Salmonella internalization.…”

Determination of Salmonella enterica Leaf Internalization Varies Substantially According to the Method and Conditions Used to Assess Bacterial Localization

“…Inoculation of leaves was performed, essentially as described before (Kroupitski et al, 2009(Kroupitski et al, , 2011(Kroupitski et al, , 2019Golberg et al, 2011), except for the incubation temperature. Briefly, a single tomato leaflet, Arabidopsis leaf, or lettuce piece were each submerged in a single 50-ml sterile polypropylene tube (Labcon, Petaluma, CA) containing 30-ml saline.…”

“…10 8 Salmonella CFU/ml saline. While this high inoculum does not represent real-life conditions, such high inocula were previously used to study Salmonella internalization in vivo (Gu et al, 2011(Gu et al, , 2013a and in vitro (Kroupitski et al, 2009(Kroupitski et al, , 2011(Kroupitski et al, , 2019Golberg et al, 2011). The incubation proceeded for 2 h at 40°C, a temperature that increases stomatal openings in multiple species (Kostaki et al, 2020) to facilitate Salmonella internalization.…”