Acid resistance system CadBA is implicated in acid tolerance and biofilm formation and is identified as a new virulence factor of Edwardsiella tarda

Edwardsiella tarda is a facultative intracellular pathogen in humans and animals. The Gram-negative bacterium is widely considered a potentially important bacterial pathogen. Adaptation to acid stress is important for the transmission of intestinal microbes, so the acid-resistance (AR) system is essential. However, the AR systems of E. tarda are totally unknown. In this study, a lysine-dependent acid resistance (LDAR) system in E. tarda, CadBA, was characterized and identified. CadB is a membrane protein and shares high homology with the lysine/cadaverine antiporter. CadA contains a PLP-binding core domain and a pyridoxal phosphate-binding motif. It shares high homology with lysine decarboxylase. cadB and cadA are co-transcribed under one operon. To study the function of the cadBA operon, isogenic cadA, cadB and cadBA deletion mutant strains TX01ΔcadA, TX01ΔcadB and TX01ΔcadBA were constructed. When cultured under normal conditions, the wild type strain and three mutants exhibited the same growth performance. However, when cultured under acid conditions, the growth of three mutants, especially TX01ΔcadA, were obviously retarded, compared to the wild strain TX01, which indicates the important involvement of the cadBA operon in acid resistance. The deletion of cadB or cadA, especially cadBA, significantly attenuated bacterial activity of lysine decarboxylase, suggesting the vital participation of cadBA operon in lysine metabolism, which is closely related to acid resistance. The mutations of cadBA operon enhanced bacterial biofilm formation, especially under acid conditions. The deletions of the cadBA operon reduced bacterial adhesion and invasion to Hela cells. Consistently, the deficiency of cadBA operon abated bacterial survival and replication in macrophages, and decreased bacterial dissemination in fish tissues. Our results also show that the expression of cadBA operon and regulator cadC were up-regulated upon acid stress, and CadC rigorously regulated the expression of cadBA operon, especially under acid conditions. These findings demonstrate that the AR CadBA system was a requisite for the resistance of E. tarda against acid stress, and played a critical role in bacterial infection of host cells and in host tissues. This is the first study about the acid resistance system of E. tarda and provides new insights into the acid-resistance mechanism and pathogenesis of E. tarda.

Production of Cadaverine in Recombinant Corynebacterium Glutamicum Overexpressing Lysine Decarboxylase (ldcC) and Response Regulator dr1558

In this study, the response regulator DR1558 from Deinococcus radiodurans was overexpressed in recombinant Corynebacterium glutamicum with lysine decarboxylase (ldcC). The recombinant C. glutamicum strain overexpressing dr1558 and ldcC produced 5.9 g/L of cadaverine by flask cultivation, whereas the control strain overexpressing only ldcC produced 4.5 g/L of cadaverine. To investigate the mechanism underlying the effect of DR1558, the expression levels of genes related to central metabolism and lysine-biosynthesis were analyzed by quantitative-real time polymerase chain reaction. The results showed that phosphoenolpyruvate carboxykinase (pck) was downregulated, and pyruvate kinase (pyk) and other lysine biosynthesis genes were upregulated. Furthermore, in fed-batch fermentation, C. glutamicum coexpressing dr1558 produced 25.14 g/L of cadaverine, a 1.25-fold increase in concentration relative to the control. These results suggested that the heterologous expression of dr1558 may improve the production of biorefinery products by recombinant C. glutamicum.

Resolving the Contradictory Functions of Lysine Decarboxylase and Butyrate in Periodontal and Intestinal Diseases

Periodontal disease is a common, bacterially mediated health problem worldwide. Mastication (chewing) repeatedly traumatizes the gingiva and periodontium, causing traces of inflammatory exudate, gingival crevicular fluid (GCF), to appear in crevices between the teeth and gingiva. Inadequate tooth cleaning causes a dentally adherent microbial biofilm composed of commensal salivary bacteria to appear around these crevices where many bacteria grow better on GCF than in saliva. We reported that lysine decarboxylase (Ldc) from Eikenella corrodens depletes the GCF of lysine by converting it to cadaverine and carbon dioxide. Lysine is an amino acid essential for the integrity and continuous renewal of dentally attached epithelium acting as a barrier to microbial products. Unless removed regularly by oral hygiene, bacterial products invade the lysine-deprived dental attachment where they stimulate inflammation that enhances GCF exudation. Cadaverine increases and supports the development of a butyrate-producing microbiome that utilizes the increased GCF substrates to slowly destroy the periodontium (dysbiosis). A long-standing paradox is that acid-induced Ldc and butyrate production support a commensal (probiotic) microbiome in the intestine. Here, we describe how the different physiologies of the respective tissues explain how the different Ldc and butyrate functions impact the progression and control of these two chronic diseases.

Biochemical Characterization and Structural Insight into Interaction and Conformation Mechanisms of Serratia marcescens Lysine Decarboxylase (SmcadA)

Inducible lysine decarboxylases (LDCs) are essential in various cellular processes of microorganisms and plants, especially under acid stress, which induces the expression of genes encoding LDCs. In this study, a novel Serratia marcesenes LDC (SmcadA) was successfully expressed in E. coli, purified and characterized. The protein had an optimal pH of 6 and a temperature of 40 °C and phylogenetic analysis to determine the evolution of SmcadA, which revealed a close relation to Enterobacteriaceae, Klebsiella sp., among others. The molecular weight of SmcadA was approximately 75 kDa after observation on SDS-PAGE and structural modeling showed the protein as a decamer, comprised of five interlinked dimers. The biocatalytic activity of the purified wild-type SmcadA (WT) was improved through site directed mutations and the results showed that the Arg595Lys mutant had the highest specific activity of 286.55 U/mg, while the Ser512Ala variant and wild-type SmcadA had 215.72 and 179.01 U/mg, respectively. Furthermore, molecular dynamics simulations revealed that interactions through hydrogen bonds between the protein residues and cofactor pyridoxal-5-phosphate (PLP) are vital for biocatalysis. Molecular Dynamics (MD) simulations also indicated that mutations conferred structural changes on protein residues and PLP hence altered the interacting residues with the cofactor, subsequently influencing substrate bioconversion. Moreover, the temperature also induced changes in orientation of cofactor PLP and amino acid residues. This work therefore demonstrates the successful expression and characterization of the purified novel lysine decarboxylase from Serratia marcesenes and provided insight into the mechanism of protein–cofactor interactions, highlighting the role of protein–ligand interactions in altering cofactor and binding site residue conformations, thus contributing to improved biocatalysis.

Structural and functional analysis of the Francisella lysine decarboxylase as a key actor in oxidative stress resistance

Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.