An allosteric inhibitor of the PhoQ histidine kinase with therapeutic potential against Salmonella infection.

BACKGROUND: The upsurge of antimicrobial resistance demands innovative strategies to fight bacterial infections. With traditional antibiotics becoming less effective, anti-virulence agents or pathoblockers, arise as an alternative approach that seeks to disarm pathogens without affecting their viability, thereby reducing selective pressure for the emergence of resistance mechanisms. OBJECTIVES: To elucidate the mechanism of action of compound N'-(thiophen-2-ylmethylene)benzohydrazide (A16B1), a potent synthetic hydrazone inhibitor against the Salmonella PhoP/PhoQ system, essential for virulence. MATERIALS AND METHODS: The measurement of the activity of PhoP/PhoQ-dependent and -independent reporter genes was used to evaluate the specificity of A16B1 to the PhoP regulon. Autokinase activity assays with either the native or truncated versions of PhoQ were used to dissect the A16B1 mechanism of action. The effect of A16B1 on Salmonella intramacrophage replication was assessed using the gentamicin protection assay. The checkerboard assay approach was used to analyse potentiation effects of colistin with the hydrazone. The Galleria mellonella infection model was chosen to evaluate A16B1 as an in vivo therapy against Salmonella. RESULTS: A16B1 repressed the Salmonella PhoP/PhoQ system activity, specifically targeting PhoQ within the second transmembrane region. A16B1 demonstrates synergy with the antimicrobial peptide colistin, reduces the intramacrophage proliferation of Salmonella without being cytotoxic and enhances the survival of G. mellonella larvae systemically infected with Salmonella. CONCLUSIONS: A16B1 selectively inhibits the activity of the Salmonella PhoP/PhoQ system through a novel inhibitory mechanism, representing a promising synthetic hydrazone compound with the potential to function as a Salmonella pathoblocker. This offers innovative prospects for combating Salmonella infections while mitigating the risk of antimicrobial resistance emergence.

Insertion sequence IS6770 modulates potassium symporter kup transcription in Enterococcus faecalis JH2-2 under low pH conditions.

Enterococcus faecalis is a phylogenetically and industrially relevant microorganism associated with Lactic Acid Bacteria. Some strains of this bacterium are employed as probiotics in commercial applications, while others serve as the principal component in starter cultures for artisanal regional cheese production. However, over the last decade, this species has emerged as an opportunistic multiresistant pathogen, raising concerns about its impact on human health. Recently, we identified multiple potassium transporter systems in E. faecalis, including the Ktr systems (KtrAB and KtrAD), Kup, KimA and Kdp complex (KdpFABC). Nevertheless, the physiological significance of these proteins remains not fully understood. In this study, we observed that the kup gene promoter region in the JH2-2 strain was modified due to the insertion of a complete copy of the IS6770 insertion sequence. Consequently, we investigated the influence of IS6770 on the expression of the kup gene. To achieve this, we conducted a mapping of the promoter region of this gene in the E. faecalis JH2-2 strain, employing fluorescence gene reporters. In addition, a transcriptional analysis of the kup gene was executed in a strain derived from E. faecalis V583 that lacks the IS30-related insertion element, facilitating the identification of the transcriptional start site. Next, the expression of the kup gene was evaluated via RT-qPCR under different pH stressful conditions. A strong upregulation of the kup gene was observed at an initial pH of 5.0 in the strain derived from E. faecalis V583. However, the activation of transcription was not observed in the E. faecalis JH2-2 strain due to the hindrance caused by the presence of IS6770. Besides that, our computational analysis of E. faecalis genomes elucidates a plausible association between transposition and the regulation of the kup gene. Remarkably, the ubiquitous presence of IS6770 throughout the phylogenetic tree implies its ancient existence within E. faecalis. Moreover, the recurrent co-occurrence of IS6770 with the kup gene, observed in 30 % of IS6770-positive strains, alludes to the potential involvement of this genomic arrangement in the adaptive strategies of E. faecalis across diverse niches.

Enhancing colistin efficacy against Salmonella infections with a quinazoline-based dual therapeutic strategy.

Colistin remains one of the last-resort therapies for combating infections caused by multidrug-resistant (MDR) Enterobacterales, despite its adverse nephro- and neuro-toxic effects. This study elucidates the mechanism of action of a non-antibiotic 4-anilinoquinazoline-based compound that synergistically enhances the effectiveness of colistin against Salmonella enterica. The quinazoline sensitizes Salmonella by deactivating intrinsic, mutational, and transferable resistance mechanisms that enable Salmonella to counteract the antibiotic impact colistin, together with an induced disruption to the electrochemical balance of the bacterial membrane. The attenuation of colistin resistance via the combined treatment approach also proves efficacious against E. coli, Klebsiella, and Acinetobacter strains. The dual therapy reduces the mortality of Galleria mellonella larvae undergoing a systemic Salmonella infection when compared to individual drug treatments. Overall, our findings unveil the potential of the quinazoline-colistin combined therapy as an innovative strategy against MDR bacteria.

Metagenomic approach to infer rumen microbiome derived traits of cattle.

Ruminants enable the conversion of indigestible plant material into animal consumables, including dairy products, meat, and valuable fibers. Microbiome research is gaining popularity in livestock species because it aids in the knowledge of illnesses and efficiency processes in animals. In this study, we use WGS metagenomic data to thoroughly characterize the ruminal ecosystem of cows to infer positive and negative livestock traits determined by the microbiome. The rumen of cows from Argentina were described by combining different gene biomarkers, pathways composition and taxonomic information. Taxonomic characterization indicated that the two major phyla were Bacteroidetes and Firmicutes; in third place, Proteobacteria was highly represented followed by Actinobacteria; Prevotella, and Bacteroides were the most abundant genera. Functional profiling of carbohydrate-active enzymes indicated that members of the Glycoside Hydrolase (GH) class accounted for 52.2 to 55.6% of the total CAZymes detected, among them the most abundant were the oligosaccharide degrading enzymes. The diversity of GH families found suggested efficient hydrolysis of complex biomass. Genes of multidrug, macrolides, polymyxins, beta-lactams, rifamycins, tetracyclines, and bacitracin resistance were found below 0.12% of relative abundance. Furthermore, the clustering analysis of genera and genes that correlated to methane emissions or feed efficiency, suggested that the cows analysed could be regarded as low methane emitters and clustered with high feed efficiency reference animals. Finally, the combination of bioinformatic analyses used in this study can be applied to assess cattle traits difficult to measure and guide enhanced nutrition and breeding methods.

Study of volatile compounds profiles in milk matrices using Enterococcus faecalis EstA and Rhizomucor miehei lipase.

The use of esterase/lipase enzymes of different origins in food industry is a widely employed strategy to enhance the formation of characteristic aromatic compounds derived from fat and diversify flavour. In the present work, we studied EstA enzyme of Enterococcus faecalis and a high purity Rhizomucor miehei lipase (Palatase). EstA was obtained recombinantly in Escherichia coli BL21 (DE3), and optimum esterase activity was detected at pH 6.75 and 40 °C. We evaluated the effect of the enzymes on milk mixtures prepared with different fat contents (2.8 and 6%) and structure (native or homogenized) on volatile compounds profiles. The milk fat structure before and after the application of low homogenization was characterized by dynamic light dispersion and microscopy. Native milk fat mixtures presented particles of 4.6 µm and 184 nm and homogenized mixtures had particles of 1.4 µm and 258 nm; microscopy images were in concordance with these results. Fifteen volatile compounds were identified, including ketones, esters, alcohols, and acids. We showed the key role of milk fat levels and microstructure in the nature of the volatile compounds produced by the R. miehei enzyme. Both in native or homogenized states, the highest content of fat favored a higher production of acids whereas the lowest fat level favored a higher esters production along with a more balanced volatile profile. For EstA enzyme, results showed a limited action on fat, as biosynthesis of esters only increased with the highest fat level homogenized.

Intranasal trans-sialidase-based vaccine against Trypanosoma cruzi triggers a mixed cytokine profile in the nasopharynx-associated lymphoid tissue and confers local and systemic immunogenicity.

Trypanosoma cruzi, the agent of Chagas disease, can infect through conjunctive or oral mucosas. Therefore, the induction of mucosal immunity by vaccination is relevant not only to trigger local protection but also to stimulate both humoral and cell-mediated responses in systemic sites to control parasite dissemination. In a previous study, we demonstrated that a nasal vaccine based on a Trans-sialidase (TS) fragment plus the mucosal STING agonist c-di-AMP, was highly immunogenic and elicited prophylactic capacity. However, the immune profile induced by TS-based nasal vaccines at the nasopharyngeal-associated lymphoid tissue (NALT), the target site of nasal immunization, remains unknown. Hence, we analyzed the NALT cytokine expression generated by a TS-based vaccine plus c-di-AMP (TSdA+c-di-AMP) and their association with mucosal and systemic immunogenicity. The vaccine was administered intranasally, in 3 doses separated by 15 days each other. Control groups received TSdA, c-di-AMP, or the vehicle in a similar schedule. We demonstrated that female BALB/c mice immunized intranasally with TSdA+c-di-AMP boosted NALT expression of IFN-γ and IL-6, as well as IFN-ß and TGF-ß. TSdA+c-di-AMP increased TSdA-specific IgA secretion in the nasal passages and also in the distal intestinal mucosa. Moreover, T and B-lymphocytes from NALT-draining cervical lymph nodes and spleen showed an intense proliferation after ex-vivo stimulation with TSdA. Intranasal administration of TSdA+c-di-AMP provokes an enhancement of TSdA-specific IgG2a and IgG1 plasma antibodies, accompanied by an increase IgG2a/IgG1 ratio, indicative of a Th1-biased profile. In addition, immune plasma derived from TSdA+c-di-AMP vaccinated mice exhibit in-vivo and ex-vivo protective capacity. Lastly, TSdA+c-di-AMP nasal vaccine also promotes intense footpad swelling after local TSdA challenge. Our data support that TSdA+c-di-AMP nasal vaccine triggers a NALT mixed pattern of cytokines that were clearly associated with an evident mucosal and systemic immunogenicity. These data are useful for further understanding the immune responses elicited by the NALT following intranasal immunization and the rational design of TS-based vaccination strategies for prophylaxis against T. cruzi.

Redundant potassium transporter systems guarantee the survival of Enterococcus faecalis under stress conditions.

Enterococcus is able to grow in media at pH from 5.0 to 9.0 and a high concentration of NaCl (8%). The ability to respond to these extreme conditions requires the rapid movement of three critical ions: proton (H+), sodium (Na+), and potassium (K+). The activity of the proton F0F1 ATPase and the sodium Na+ V0V1 type ATPase under acidic or alkaline conditions, respectively, is well established in these microorganisms. The potassium uptake transporters KtrI and KtrII were described in Enterococcus hirae, which were associated with growth in acidic and alkaline conditions, respectively. In Enterococcus faecalis, the presence of the Kdp (potassium ATPase) system was early established. However, the homeostasis of potassium in this microorganism is not completely explored. In this study, we demonstrate that Kup and KimA are high-affinity potassium transporters, and the inactivation of these genes in E. faecalis JH2-2 (a Kdp laboratory natural deficient strain) had no effect on the growth parameters. However, in KtrA defective strains (ΔktrA, ΔkupΔktrA) an impaired growth was observed under stress conditions, which was restored to wild type levels by external addition of K+ ions. Among the multiplicity of potassium transporters identify in the genus Enterococcus, Ktr channels (KtrAB and KtrAD), and Kup family symporters (Kup and KimA) are present and may contribute to the particular resistance of these microorganisms to different stress conditions. In addition, we found that the presence of the Kdp system in E. faecalis is strain-dependent, and this transporter is enriched in strains of clinical origin as compared to environmental, commensal, or food isolates.

Nasal immunization with a L. lactis-derived trans-sialidase antigen plus c-di-AMP protects against acute oral T. cruzi infection.

The new generation of vaccines for Chagas disease, are focused to induce both humoral and cellular response to effectively control Trypanosoma cruzi parasites. The administration of vaccine formulations intranasally has the advantage over parenteral routes that can induce a specific response at mucosal and systemic levels. This study aimed to evaluate and compare the immunogenicity and prophylactic effectiveness of two Trans-sialidase (TS)-based mucosal vaccines against T. cruzi administered intranasally. Vaccines consisted of a recombinant fragment of TS expressed in Lactococcus lactis formulated in two different adjuvants. The first, was an immunostimulant particle (ISPA, an ISCOMATRIX-like adjuvant), while the second was the dinucleotide c-di-AMP, which have shown immunostimulant properties at the mucosal level. BALB/c mice were immunized intranasally (3 doses, one every two weeks) with each formulation (TS + ISPA or TS + c-di-AMP) and with TS alone or vehicle (saline solution) as controls. Fifteen days after the last immunization, both TS + ISPA or TS + c-di-AMP induced an evident systemic humoral and cellular response, as judged by the increased plasma anti-TS IgG2a titers and IgG2a/IgG1 ratio and enhanced cellular response against TS. Plasma derived antibodies from TS + c-di-AMP also inhibit in vitro the invasion capacity of T. cruzi. Furthermore, specific secretory IgA was more enhanced in TS + c-di-AMP group. Protective efficacy was proved in vaccinated animals by an oral T. cruzi-challenge. Parasitemia control was only achieved by animals vaccinated with TS + c-di-AMP, despite all vaccinates groups showed enhanced CD8+IFN-γ+ T cell numbers. In addition, it was reflected during the acute phase in a significant reduction of tissue parasite load, clinical manifestations and diminished tissue damage. The better prophylactic capacity elicited by TS + c-di-AMP was related to the induction of neutralizing plasma antibodies and augmented levels of mucosal IgA since TS + ISPA and TS + c-di-AMP groups displayed similar immunogenicity and CD8+IFN-γ+ T-cell response. Therefore, TS + c-di-AMP formulation appears as a promising strategy for prophylaxis against T. cruzi.

Genomic analysis revealed conserved acid tolerance mechanisms from native micro-organisms in fermented feed.

AIMS: Fermented feed is an agricultural practice used in many regions of the world to improve the growth performance of farm animals. This study aimed to identify and evaluate the lactic acid bacteria and yeast involved in the production of fermented feed. METHODS AND RESULTS: We isolated and described two micro-organisms from autochthonous microbiota origin present in a regional feed product, Lactobacillus paracasei IBR07 (Lacticaseibacillus paracasei) and Kazachstania unispora IBR014 (Saccharomyces unisporum). Genome sequence analyses were performed to characterize both micro-organisms. Potential pathways involved in the acid response, tolerance and persistence were predicted in both genomes. Although L. paracasei and K. unispora are considered safe for animal feed, we analysed the presence of virulence factors, antibiotic resistance and pathogenicity islands. Furthermore, the Galleria mellonella model was used to support the safety of both isolates. CONCLUSIONS: We conclude that IBR07 and IBR014 strains are good candidates to be used as starter cultures for feed fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here will be helpful to explore other biotechnological aspects and constitute a starting point for further studies to establish the consumption benefit of fermented feed in farm animal production.

The assessment of leading traits in the taxonomy of the Bacillus cereus group.

Bacillus cereus sensu lato strains (B. cereus group) are widely distributed in nature and have received interest for decades due to their importance in insect pest management, food production and their positive and negative repercussions in human health. Consideration of practical uses such as virulence, physiology, morphology, or ill-defined features have been applied to describe and classify species of the group. However, current comparative studies have exposed inconsistencies between evolutionary relatedness and biological significance among genomospecies of the B. cereus group. Here, the combined analyses of core-based phylogeny and all versus all Average Nucleotide Identity values based on 2116 strains were conducted to update the genomospecies circumscriptions within B. cereus group. These analyses suggested the existence of 57 genomospecies, 37 of which are novel, thus indicating that the taxonomic identities of more than 39% of the analyzed strains should be revised or updated. In addition, we found that whole-genome in silico analyses were suitable to differentiate genomospecies such as B. anthracis, B. cereus and B. thuringiensis. The prevalence of toxin and virulence factors coding genes in each of the genomospecies of the B. cereus group was also examined, using phylogeny-aware methods at wide-genome scale. Remarkably, Cry and emetic toxins, commonly assumed to be associated with B. thuringiensis and emetic B. paranthracis, respectively, did not show a positive correlation with those genomospecies. On the other hand, anthrax-like toxin and capsule-biosynthesis coding genes were positively correlated with B. anthracis genomospecies, despite not being present in all strains, and with presumably non-pathogenic genomospecies. Hence, despite these features have been so far considered relevant for industrial or medical classification of related species of the B. cereus group, they were inappropriate for their circumscription. In this study, genomospecies of the group were accurately affiliated and representative strains defined, generating a rational framework that will allow comparative analysis in epidemiological or ecological studies. Based on this classification the role of specific markers such as Type VII secretion system, cytolysin, bacillolysin, and siderophores such as petrobactin were pointed out for further analysis.

Enterococcus faecalis MalR acts as a repressor of the maltose operons and additionally mediates their catabolite repression via direct interaction with seryl-phosphorylated-HPr.

Enterococci are gram-positive pathogens and lead to cause hospital-acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α-polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA-independent carbon catabolite repression. The central PTS protein P-Ser-HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions.

Diversity of volatile organic compound production from leucine and citrate in Enterococcus faecium.

Enterococcus faecium is frequently isolated from fermented food; in particular, they positively contribute to the aroma compound generation in traditional cheese. Citrate fermentation is a desirable property in these bacteria, but this feature is not uniformly distributed among E. faecium strains. In the present study, three selected E. faecium strains, IQ110 (cit-), GM70 (cit+ type I), and Com12 (cit+ type II), were analyzed in their production of aroma compounds in milk. End products and volatile organic compounds (VOCs) were determined by solid-phase micro-extraction combined with gas chromatography mass spectrometry (SPME-GC-MS). Principal component analysis (PCA) of aroma compound profiles revealed a different VOC composition for the three strains. In addition, resting cell experiments of E. faecium performed in the presence of leucine, citrate, or pyruvate as aroma compound precursors allowed us to determine metabolic differences between the studied strains. GM70 (cit+ type I) showed an active citrate metabolism, with increased levels of diacetyl and acetoin generation relative to Com12 or to citrate defective IQ110 strains. In addition, in the experimental conditions tested, a defective citrate-fermenting phenotype for the Com12 strain was found, while its leucine degradation and pyruvate metabolism were conserved. In conclusion, rational selection of E. faecium strains could be performed based on genotypic and phenotypic analyses. This would result in a performing strain, such as GM70, that could positively contribute to flavor, with typical notes of diacetyl, acetoin, 3-methyl butanal, and 3-methyl butanol in an adjuvant culture.

The KupA and KupB Proteins of Lactococcus lactis IL1403 Are Novel c-di-AMP Receptor Proteins Responsible for Potassium Uptake.

Cyclic di-AMP (c-di-AMP) is a second messenger involved in diverse metabolic processes, including osmolyte uptake, cell wall homeostasis, and antibiotic and heat resistance. In Lactococcus lactis, a lactic acid bacterium which is used in the dairy industry and as a cell factory in biotechnological processes, the only reported interaction partners of c-di-AMP are the pyruvate carboxylase and BusR, the transcription regulator of the busAB operon for glycine betaine uptake. However, recent studies uncovered a major role of c-di-AMP in the control of potassium homeostasis, and potassium is the signal that triggers c-di-AMP synthesis. In this study, we have identified KupA and KupB, which belong to the Kup/HAK/KT family, as novel c-di-AMP binding proteins. Both proteins are high-affinity potassium transporters, and their transport activities are inhibited by binding of c-di-AMP. Thus, in addition to the well-studied Ktr/Trk potassium channels, KupA and KupB represent a second class of potassium transporters that are subject to inhibition by c-di-AMP.IMPORTANCE Potassium is an essential ion in every living cell. Even though potassium is the most abundant cation in cells, its accumulation can be toxic. Therefore, the level of potassium has to be tightly controlled. In many Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in the control of potassium homeostasis by binding to potassium transporters and regulatory proteins and RNA molecules. In the lactic acid bacterium Lactococcus lactis, none of these conserved c-di-AMP-responsive molecules are present. In this study, we demonstrate that the KupA and KupB proteins of L. lactis IL1403 are high-affinity potassium transporters and that their transport activity is inhibited by the second messenger c-di-AMP.

GeM-Pro: a tool for genome functional mining and microbial profiling.

Gem-Pro is a new tool for gene mining and functional profiling of bacteria. It initially identifies homologous genes using BLAST and then applies three filtering steps to select orthologous gene pairs. The first one uses BLAST score values to identify trivial paralogs. The second filter uses the shared identity percentages of found trivial paralogs as internal witnesses of non-orthology to set orthology cutoff values. The third filtering step uses conditional probabilities of orthology and non-orthology to define new cutoffs and generate supportive information of orthology assignations. Additionally, a subsidiary tool, called q-GeM, was also developed to mine traits of interest using logistic regression (LR) or linear discriminant analysis (LDA) classifiers. q-GeM is more efficient in the use of computing resources than Gem-Pro but needs an initial classified set of homologous genes in order to train LR and LDA classifiers. Hence, q-GeM could be used to analyze new set of strains with available genome sequences, without the need to rerun a complete Gem-Pro analysis. Finally, Gem-Pro and q-GeM perform a synteny analysis to evaluate the integrity and genomic arrangement of specific pathways of interest to infer their presence. The tools were applied to more than 2 million homologous pairs encoded by Bacillus strains generating statistical supported predictions of trait contents. The different patterns of encoded traits of interest were successfully used to perform a descriptive bacterial profiling.

Implications of the expression of Enterococcus faecalis citrate fermentation genes during infection.

Citrate is an ubiquitous compound in nature. However, citrate fermentation is present only in a few pathogenic or nonpathogenic microorganisms. The citrate fermentation pathway includes a citrate transporter, a citrate lyase complex, an oxaloacetate decarboxylase and a regulatory system. Enterococcus faecalis is commonly present in the gastro-intestinal microbiota of warm-blooded animals and insect guts. These bacteria can also cause infection and disease in immunocompromised individuals. In the present study, we performed whole genome analysis in Enterococcus strains finding that the complete citrate pathway is present in all of the E. faecalis strains isolated from such diverse habitats as animals, hospitals, water, milk, plants, insects, cheese, etc. These results indicate the importance of this metabolic preservation for persistence and growth of E. faecalis in different niches. We also analyzed the role of citrate metabolism in the E. faecalis pathogenicity. We found that an E. faecalis citrate fermentation-deficient strain was less pathogenic for Galleria mellonella larvae than the wild type. Furthermore, strains with deletions in the oxaloacetate decarboxylase subunits or in the α-acetolactate synthase resulted also less virulent than the wild type strain. We also observed that citrate promoters are induced in blood, urine and also in the hemolymph of G. mellonella. In addition, we showed that citrate fermentation allows E. faecalis to grow better in blood, urine and G. mellonella. The results presented here clearly indicate that citrate fermentation plays an important role in E. faecalis opportunistic pathogenic behavior.

Genetic Engineering of Lactococcus lactis Co-producing Antigen and the Mucosal Adjuvant 3' 5'- cyclic di Adenosine Monophosphate (c-di-AMP) as a Design Strategy to Develop a Mucosal Vaccine Prototype.

Lactococcus lactis is a promising candidate for the development of mucosal vaccines. More than 20 years of experimental research supports this immunization approach. In addition, 3' 5'- cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that plays a key role in the regulation of diverse physiological functions (potassium and cellular wall homeostasis, among others). Moreover, recent studies showed that c-di-AMP has a strong mucosal adjuvant activity that promotes both humoral and cellular immune responses. In this study, we report the development of a novel mucosal vaccine prototype based on a genetically engineered L. lactis strain. First, we demonstrate that homologous expression of cdaA gen in L. lactis is able to increase c-di-AMP levels. Thus, we hypothesized that in vivo synthesis of the adjuvant can be combined with production of an antigen of interest in a separate form or jointly in the same strain. Therefore, a specifically designed fragment of the trans-sialidase (TScf) enzyme from the Trypanosoma cruzi parasite, the etiological agent of Chagas disease, was selected to evaluate as proof of concept the immune response triggered by our vaccine prototypes. Consequently, we found that oral administration of a L. lactis strain expressing antigenic TScf combined with another L. lactis strain producing the adjuvant c-di-AMP could elicit a TS-specific immune response. Also, an additional L. lactis strain containing a single plasmid with both cdaA and tscf genes under the Pcit and Pnis promoters, respectively, was also able to elicit a specific immune response. Thus, the current report is the first one to describe an engineered L. lactis strain that simultaneously synthesizes the adjuvant c-di-AMP as well as a heterologous antigen in order to develop a simple and economical system for the formulation of vaccine prototypes using a food grade lactic acid bacterium.

Genetic and phenotypic features defining industrial relevant Lactococcus lactis, L. cremoris and L. lactis biovar. diacetylactis strains.

Lactococcus lactis strains constitute one of the most important starter cultures for cheese production. In this study, a genome-wide analysis was performed including 68 available genomes of L. lactis group strains showing the existence of two species (L. lactis and L. cremoris) and two biovars (L. lactis biovar. diacetylactis and L. cremoris biovar. lactis). The proposed classification scheme revealed coherency among phenotypic (through in silico and in vivo bacterial function profiling), phylogenomic (through maximum likelihood trees) and genomic (using overall genome sequence-based parameters) approaches. Strain biodiversity for the industrial biovar. diacetylactis was also analyzed, finding they are formed by at least three variants with the CC1 clonal complex as the only one distributed worldwide. These findings and methodologies will help improve the selection of L. lactis group strains for industrial use as well as facilitate the interpretation of previous or future research studies on this diverse group of bacteria.

Safety assessment and functional properties of four enterococci strains isolated from regional Argentinean cheese.

The members of the Enterococcus genus are widely distributed in nature. Its strains have been extensively reported to be present in plant surfaces, soil, water and food. In an attempt to assess their potential application in food industry, four Enterococcus faecium group-strains recently isolated from Argentinean regional cheese products were evaluated using a combination of whole genome analyses and in vivo assays. In order to identify these microorganisms at species level, in silico analyses using their newly reported sequences were conducted. The average nucleotide identity (ANI), in silico DNA-DNA hybridization, and phylogenomic trees constructed using core genome data allowed IQ110, GM70 and GM75 strains to be classified as E. faecium while IQ23 strain was identified as E. durans. Besides their common origin, the strains showed differences in their genetic structure and mobile genetic element content. Furthermore, it was possible to determine the absence or presence of specific features related to growth in milk, cheese ripening, probiotic capability and gut adaptation including sugar, amino acid, and peptides utilization, flavor compound production, bile salt tolerance as well as biogenic amine production. Remarkably, all strains encoded for peptide permeases, maltose utilization, bile salt tolerance, diacetyl and tyramine production genes. On the other hand, some variability was observed regarding citrate and lactose utilization, esterase, and cell wall-associated proteinase. In addition, while strains were predicted to be non-human pathogens by the in silico inspection of pathogenicity and virulence factors, only the GM70 strain proved to be non-virulent in Galleria mellonella model. In conclusion, we propose that, in order to improve the rational selection of strains for industrial applications, a holistic approach involving a comparative genomic analysis of positive and negative features as well as in vivo evaluation of virulence behavior should be performed.

Multivariate analysis of organic acids in fermented food from reversed-phase high-performance liquid chromatography data.

Multivariate calibration coupled to RP-HPLC with diode array detection (HPLC-DAD) was applied to the identification and the quantitative evaluation of the short chain organic acids (malic, oxalic, formic, lactic, acetic, citric, pyruvic, succinic, tartaric, propionic and α-cetoglutaric) in fermented food. The goal of the present study was to get the successful resolution of a system in the combined occurrence of strongly coeluting peaks, of distortions in the time sensors among chromatograms, and of the presence of unexpected compounds not included in the calibration step. Second-order HPLC-DAD data matrices were obtained in a short time (10min) on a C18 column with a chromatographic system operating in isocratic mode (mobile phase was 20mmolL-1 phosphate buffer at pH 2.20) and a flow-rate of 1.0mLmin-1 at room temperature. Parallel factor analysis (PARAFAC) and unfolded partial least-squares combined with residual bilinearization (U-PLS/RBL) were the second-order calibration algorithms select for data processing. The performance of the analytical parameters was good with an outstanding limit of detection (LODs) for acids ranging from 0.15 to 10.0mmolL-1 in the validation samples. The improved method was applied to the analysis of many dairy products (yoghurt, cultured milk and cheese) and wine. The method was shown as an effective means for determining and following acid contents in fermented food and was characterized by reducibility with simple, high resolution and rapid procedure without derivatization of analytes.

Enzymes Required for Maltodextrin Catabolism in Enterococcus faecalis Exhibit Novel Activities.

Maltose and maltodextrins are formed during the degradation of starch or glycogen. Maltodextrins are composed of a mixture of maltooligosaccharides formed by α-1,4- but also some α-1,6-linked glucosyl residues. The α-1,6-linked glucosyl residues are derived from branching points in the polysaccharides. In Enterococcus faecalis, maltotriose is mainly transported and phosphorylated by a phosphoenolpyruvate:carbohydrate phosphotransferase system. The formed maltotriose-6″-phosphate is intracellularly dephosphorylated by a specific phosphatase, MapP. In contrast, maltotetraose and longer maltooligosaccharides up to maltoheptaose are taken up without phosphorylation via the ATP binding cassette transporter MdxEFG-MsmX. We show that the maltose-producing maltodextrin hydrolase MmdH (GenBank accession no. EFT41964) in strain JH2-2 catalyzes the first catabolic step of α-1,4-linked maltooligosaccharides. The purified enzyme converts even-numbered α-1,4-linked maltooligosaccharides (maltotetraose, etc.) into maltose and odd-numbered (maltotriose, etc.) into maltose and glucose. Inactivation of mmdH therefore prevents the growth of E. faecalis on maltooligosaccharides ranging from maltotriose to maltoheptaose. Surprisingly, MmdH also functions as a maltogenic α-1,6-glucosidase, because it converts the maltotriose isomer isopanose into maltose and glucose. In addition, E. faecalis contains a glucose-producing α-1,6-specific maltodextrin hydrolase (GenBank accession no. EFT41963, renamed GmdH). This enzyme converts panose, another maltotriose isomer, into glucose and maltose. A gmdH mutant had therefore lost the capacity to grow on panose. The genes mmdH and gmdH are organized in an operon together with GenBank accession no. EFT41962 (renamed mmgT). Purified MmgT transfers glucosyl residues from one α-1,4-linked maltooligosaccharide molecule to another. For example, it catalyzes the disproportionation of maltotriose by transferring a glucosyl residue to another maltotriose molecule, thereby forming maltotetraose and maltose together with a small amount of maltopentaose.IMPORTANCE The utilization of maltodextrins by Enterococcus faecalis has been shown to increase the virulence of this nosocomial pathogen. However, little is known about how this organism catabolizes maltodextrins. We identified two enzymes involved in the metabolism of various α-1,4- and α-1,6-linked maltooligosaccharides. We found that one of them functions as a maltose-producing α-glucosidase with relaxed linkage specificity (α-1,4 and α-1,6) and exo- and endoglucosidase activities. A third enzyme, which resembles amylomaltase, exclusively transfers glucosyl residues from one maltooligosaccharide molecule to another. Similar enzymes are present in numerous other Firmicutes, such as streptococci and lactobacilli, suggesting that these organisms follow the same maltose degradation pathway as E. faecalis.