Salmonella enterica Serovar Typhimurium 14028s Genomic Regions Required for Colonization of Lettuce Leaves

Abstract

Contamination of edible produce leaves with human bacterial pathogens has been associated with serious disease outbreaks and has become a major public health concern affecting all aspects of the market, from farmers to consumers. While pathogen populations residing on the surface of ready-to-eat produce can be potentially removed through thorough washing, there is no disinfection technology available that effectively eliminates internal bacterial populations. By screening 303 multi-gene deletion (MGD) mutants of Salmonella enterica serovar Typhimurium (STm) 14028s, we were able to identify ten genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the bacterial membrane, and biosynthesis of surface appendages. Interestingly, at 21 days post inoculation, seven of these mutants showed increased population titers inside the leaf, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the Salmonella pathogenicity island 2 (SPI-2) showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not Mut3, was significantly impaired in the leaf apoplastic wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. The genetic basis of STm colonization of leaves investigated in this study provides a foundation from which to develop mitigation tactics to enhance food safety.

Human Pathogen Colonization of Lettuce Dependent Upon Plant Genotype and Defense Response Activation

“…These results strongly suggest that the differences in the ability of E. coli O157:H7 and S. Typhimurium 14028s to survive in the leaf apoplast of the genotypes Lollo Rossa and Red Tide are influenced by the variation in the level of defense responses activated against these bacteria. Although the contribution of the type III secretion systems and type III effectors in the colonization of plants by human pathogenic bacteria remains controversial (Schikora et al, 2011;Shirron and Yaron, 2011;Melotto et al, 2014;Chalupowicz et al, 2018;Montano et al, 2020), it is possible that the lettuce genotypes differ in their ability to recognize type III effectors of S. Typhimurium 14028s and E. coli O157:H7 resulting in variation in effector-triggered immunity against these human pathogenic bacteria. Interestingly, Lollo Rossa has been reported as resistant to the disease bacterial leaf spot of lettuce caused by Xanthomonas campestris pv.…”

Leaf infiltration in plant science: old method, new possibilities

“…crispa cv. Salinas— Salmonella enterica Serovar Typhimurium [ 47 ] Z. mays— Pantoea stewartii subsp stewartii [ 45 ] A. thaliana—Pseudomonas syringae pv. tomato DC3000 , S. lycopersicum var.…”

“…In optimizing the procedure, various methods for detection of cytoplasmic contaminants in the collected AWF are used. For this purpose, measurements of the activity of marker enzymes such as malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI) and glucose-6-phosphate dehydrogenase (G6PDH), determination of the level of metabolites such as glucose-6-phosphate (G-6-P), or immunodetection of cytoplasmic proteins including MDH, RuBisCo or ATPase are commonly used [ 2 , 46 , 47 ].…”

“…Leaf infiltration is widely used in the research of plant disease mechanisms, where it has been developed for the controlled induction of plant infections, for example with the model prokaryotic pathogen Pseudomonas syringae , under standard laboratory conditions [ 4 , 5 , 64 ]. Although various bacteria and not only phytopathogens can actively penetrate the internal leaf tissues via stomata or tissue wounds, applying the inoculum to the leaf surface for spontaneous infiltration of microorganisms is a less efficient method than forced infiltration [ 20 , 47 , 65 , 66 ]. Due to the need for spatially precise application of the inoculum and control of microbial load introduced into the plant tissue, it is preferable to use syringe infiltration for this purpose rather than vacuum infiltration [ 4 , 5 , 12 ].…”

“…Studies of the microbes colonizing leaves are also of great importance in the field of bacteriological safety in the food industry [ 63 , 70 ]. It has been shown, for example, that although the enteric pathogen Salmonella enterica, is not a native plant endophyte, can actively infiltrate the deep leaf structures and multiply there [ 47 ]. Studying the diversity of microorganisms in endophytic communities and explaining the interactions between them can help to develop a strategy of the bacteriological protection of edible plants against their colonization with human pathogens.…”