EnvZ/OmpR Controls Protein Expression and Modifications in Cronobacter sakazakii for Virulence and Environmental Resilience.

Cronobacter sakazakii is a notorious foodborne opportunistic pathogen, particularly affecting vulnerable populations such as premature infants, and poses significant public health challenges. This study aimed to elucidate the role of the envZ/ompR genes in environmental tolerance, pathogenicity, and protein regulation of C. sakazakii. An envZ/ompR knockout mutant was constructed and assessed for its impact on bacterial growth, virulence, environmental tolerance, and protein regulation. Results demonstrate that deletion of envZ/ompR genes leads to reduced growth rate and attenuated virulence in animal models. Additionally, the knockout strain exhibited compromised environmental tolerance, particularly in desiccation and oxidative stress conditions, along with impaired adhesion and invasion abilities in epithelial cells. Proteomic analysis revealed significant alterations in protein expression and phosphorylation patterns, highlighting potential compensatory mechanisms triggered by gene deletion. Furthermore, investigation into protein deamidation and glucose metabolism uncovered a link between envZ/ompR deletion and energy metabolism dysregulation. Interestingly, the downregulation of MalK and GrxC proteins was identified as contributing factors to altered desiccation tolerance and disrupted redox homeostasis, respectively, providing mechanistic insights into the phenotypic changes observed. Overall, this study enhances understanding of the multifaceted roles of envZ/ompR in C. sakazakii physiology and pathogenesis, shedding light on potential targets for therapeutic intervention and food safety strategies.

The Interaction between Human Microbes and Advanced Glycation End Products: The Role of Klebsiella X15 on Advanced Glycation End Products' Degradation.

Previous studies have shown that advanced glycation end products (AGEs) are implicated in the occurrence and progression of numerous diseases, with dietary AGEs being particularly associated with intestinal disorders. In this study, methylglyoxal-beta-lactoglobulin AGEs (MGO-ß-LG AGEs) were utilized as the exclusive nitrogen source to investigate the interaction between protein-bound AGEs and human gut microbiota. The high-resolution mass spectrometry analysis of alterations in peptides containing AGEs within metabolites before and after fermentation elucidated the capacity of intestinal microorganisms to enzymatically hydrolyze long-chain AGEs into short-chain counterparts. The 16S rRNA sequencing revealed Klebsiella, Lactobacillus, Escherichia-Shigella, and other genera as dominant microbiota at different fermentation times. A total of 187 potential strains of AGE-metabolizing bacteria were isolated from the fermentation broth at various time points. Notably, one strain of Klebsiella exhibited the most robust growth capacity when AGEs served as the sole nitrogen source. Subsequently, proteomics was employed to compare the changes in protein levels of Klebsiella X15 following cultivation in unmodified proteins and proteins modified with AGEs. This analysis unveiled a remodeled amino acid and energy metabolism pathway in Klebsiella in response to AGEs, indicating that Klebsiella may possess a metabolic pathway specifically tailored to AGEs. This study found that fermenting AGEs in healthy human intestinal microbiota altered the bacterial microbiota structure, especially by increasing Klebsiella proliferation, which could be a key factor in AGEs' role in causing diseases, particularly intestinal inflammation.

2'-Fucosyllactose Promotes Colonization of Akkermansia muciniphila and Prevents Colitis In Vitro and in Mice.

Akkermansia muciniphila is a potential candidate for ulcerative colitis prevention. Considering that it utilizes 2'-fucosyllactose (2'FL) for growth, 2'FL can be used to enrich the abundance of A. muciniphila in feces. However, whether the crosswalk between 2'FL and A. muciniphila can promote the intestinal colonization of A. muciniphila remains unclear. In this study, we explored the effect and the underlying mechanism of 2'FL on the colonization of A. muciniphila in vitro and in vivo as well as its alleviating effect on colitis. Our results revealed that 2'FL can serve as a carbon source of A. muciniphila to support the growth and increase cell-surface hydrophobicity and the expression of the genes coding fibronectin-binding autotransporter adhesin to promote the adhesion to Caco2/HT29 methotrexate (MTX) cells but not of galactooligosaccharides (GOS) and glucose. Moreover, 2'FL could increase the host mucin formation to promote the adhesion of A. muciniphila to Caco2/HT29 MTX cells but not of GOS and glucose. Furthermore, 2'FL could significantly increase the colonization of A. muciniphila in the gut to alleviate colitis in mice. Overall, the interplay between A. muciniphila and 2'FL is expected to provide an advantageous ecological niche for A. muciniphila so as to confer further health benefits against colitis.

The metabolism and transformation of casein-bound lactulosyllysine in vivo: Promoting dicarbonyl stress and the formation of advanced glycation end products accompanied by systemic inflammation.

Lactulosyllysine (LL) widely exists in thermally processed dairy products, while the metabolism and transformation of LL remain poorly understood. We aimed to elucidate the metabolic pathways of LL and its impact on body health by subjecting C57BL/6 mice to a short-term ll-fortified casein diet. Our findings indicated that casein-bound LL might be metabolized and transformed into 3-deoxyglucosone through fructosamine-3-kinase (FN3K) in vivo, which promoted α-dicarbonyl stress, ultimately leading to the formation of advanced glycation end products (AGEs) in various tissues/organs, accompanied by systemic inflammation. The levels of AGEs formation in tissues/organs at various stages of casein-bound LL intake exhibited dynamic changes, correlating with alterations in the expression of FN3K and α-dicarbonyl compounds metabolic detoxification enzymes. The negative effects induced by casein-bound LL cannot be fully reversed by switching to a standard diet for equal periods. Consumption of dairy products rich in LL raises concerns as a potential risk factor for healthy individuals.

Maltodextrin-binding protein as a key factor in Cronobacter sakazakii survival under desiccation stress.

Cronobacter sakazakii (C. sakazakii) is a notorious pathogen responsible for infections in infants and newborns, often transmitted through contaminated infant formula. Despite the use of traditional pasteurization methods, which can reduce microbial contamination, there remains a significant risk of pathogenic C. sakazakii surviving due to its exceptional stress tolerance. In our study, we employed a comparative proteomic approach by comparing wild-type strains with gene knockout strains to identify the essential genes crucial for the successful survival of C. sakazakii during desiccation. Our investigation revealed the significance of envZ-ompR, recA, and flhD gene cassettes in contributing to desiccation tolerance in C. sakazakii. Furthermore, through our comparative proteomic profiling, we identified the maltodextrin-binding protein encoded by ESA_03421 as a potential factor influencing dry tolerance. This protein is regulated by EnvZ-OmpR, RecA, and FlhD. Notably, the knockout of ESA_03421 resulted in a 150% greater reduction in Log CFU compared to the wild-type C. sakazakii. Overall, our findings offer valuable insights into the mechanisms underlying C. sakazakii desiccation tolerance and provide potential targets for the development of new antimicrobial strategies aimed at reducing the risk of infections in infants and newborns.

Survival of Salmonella in Tea Under Different Storage Conditions and Brewing Methods.

Salmonella is one of the leading causes of bacterial gastroenteritis. High prevalence of Salmonella in environment is partially due to its ability to enter the "viable but non-culturable" (VBNC) state when they encounter unfavorable conditions. Dried teas are traditionally believed to have a low risk of causing salmonellosis. This study investigated the survival of Salmonella in four types of dried teas under different storage conditions and brewing methods. A method that coupled propidium monoazide (PMA) and quantitative PCR was optimized to quantify VBNC Salmonella cells to assess the risk of Salmonella contamination in teas after brewing. Each tea sample was inoculated with Salmonella at an 8 log CFU/ml concentration and stored at 4, 10, and 25°C. Under three storage conditions, the number of survived Salmonella was highest in teas stored at 4°C and lowest in teas stored at 25°C. After storage of 120 days, culturable Salmonella was detected from all samples ranging from 6-7 log CFU/g (4°C storage) to 3-4 log CFU/g (25°C storage). The effectiveness of brewing methods in inactivating Salmonella was assessed by brewing inoculated teas at room temperature, 55, 75, and 100°C for 10 min. Brewing teas at 75 and 100°C significantly (P < 0.05) reduced the number of viable Salmonella, but VBNC Salmonella formed when brewed at 75°C. Altogether, Salmonella can persist in dried teas for over 3 months at a temperature ranging from 4 to 25°C, and thermal treatment delivered during home brewing may not eradicate Salmonella in teas.