Gut microbiota dysbiosis in a cohort of patients with psoriasis.

BACKGROUND: There is increasing evidence of the key role that the gut microbiota plays in inflammatory diseases. OBJECTIVES: To identify differences in the faecal microbial composition of patients with psoriasis compared with healthy individuals in order to unravel the microbiota profiling in this autoimmune disease. METHODS: 16S rRNA gene sequencing and bioinformatic analyses were performed with the total DNA extracted from the faecal microbiota of 19 patients with psoriasis and 20 healthy individuals from the same geographic location. RESULTS: Gut microbiota composition of patients with psoriasis displayed a lower diversity and different relative abundance of certain bacterial taxa compared with healthy individuals. CONCLUSIONS: The gut microbiota profile of patients with psoriasis displayed a clear dysbiosis that can be targeted for microbiome-based therapeutic approaches. What's already known about this topic? Psoriasis is a chronic inflammatory immune-mediated skin disease, the aetiology of which remains unclear. The human microbiota is a complex microbial community that inhabits our body and has been related with the maintenance of a healthy status. Several studies have focused on the skin microbiome and its connection with psoriasis although less attention has been focused on the potential role of the gut microbiota in psoriatic disease. What does this study add? This study unravels the gut microbiome dysbiosis present in a cohort of patients with psoriasis, compared with a healthy control group from the same geographical location. This study shows a lower bacterial diversity and different relative abundance of certain bacterial taxa in patients with psoriasis. We gain knowledge and insight into the microbiome alterations in psoriatic disease, opening new avenues for therapeutic approaches to reshape the human microbiome towards a healthy status.

How Diet and Lifestyle Can Fine-Tune Gut Microbiomes for Healthy Aging.

Many physical, social, and psychological changes occur during aging that raise the risk of developing chronic diseases, frailty, and dependency. These changes adversely affect the gut microbiota, a phenomenon known as microbe-aging. Those microbiota alterations are, in turn, associated with the development of age-related diseases. The gut microbiota is highly responsive to lifestyle and dietary changes, displaying a flexibility that also provides anactionable tool by which healthy aging can be promoted. This review covers, firstly, the main lifestyle and socioeconomic factors that modify the gut microbiota composition and function during healthy or unhealthy aging and, secondly, the advances being made in defining and promoting healthy aging, including microbiome-informed artificial intelligence tools, personalized dietary patterns, and food probiotic systems.

Establishment and development of lactic acid bacteria and bifidobacteria microbiota in breast-milk and the infant gut.

The initial establishment of lactic acid bacteria (LAB) and bifidobacteria in the newborn and the role of breast-milk as a source of these microorganisms are not yet well understood. The establishment of these microorganisms during the first 3 months of life in 20 vaginally delivered breast-fed full-term infants, and the presence of viable Bifidobacterium in the corresponding breast-milk samples was evaluated. In 1 day-old newborns Enterococcus and Streptococcus were the microorganisms most frequently isolated, from 10 days of age until 3 months bifidobacteria become the predominant group. In breast-milk, Streptococcus was the genus most frequently isolated and Lactobacillus and Bifidobacterium were also obtained. Breast-milk contains viable lactobacilli and bifidobacteria that might contribute to the initial establishment of the microbiota in the newborn.

Mediterranean diet and faecal microbiota: a transversal study.

Despite the existing evidence on the impact of olive oil and red wine on the intestinal microbiota, the effect of the global Mediterranean Diet (MD) has not been sufficiently studied. We explored the association between the adherence to a Mediterranean dietary pattern, and its components, with faecal microbiota in a cohort of adults with non-declared pathology. This transversal study involved 31 adults without a previous diagnosis of cancer, autoimmune or digestive diseases. Based on the data obtained by means of an annual food frequency questionnaire (FFQ), and the information existing in the literature, a Mediterranean Diet Score (MDS) was calculated. Dietary fibre was obtained from Marlett et al. tables and Phenol-Explorer Database was used for phenolic compounds intake. Quantification of microbial groups was performed by Ion Torrent 16S rRNA gene-based analysis and quantification of short-chain fatty acids (SCFAs) was performed using gas chromatography-mass spectrometry (MS). MDS was associated with a higher abundance of Bacteroidetes (p = 0.001), Prevotellacea (p = 0.002) and Prevotella (p = 0.003) and a lower concentration of Firmicutes (p = 0.003) and Lachnospiraceae (p = 0.045). Also, in subjects with MDS ≥ 4, higher concentrations of faecal propionate (p = 0.034) and butyrate (p = 0.018) were detected. These results confirm the complexity of the diet-microbiota interrelationship.

Structure-function analysis of multidrug transporters in Lactococcus lactis.

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette (ABC) superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in L. lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

Molecular pharmacological characterization of two multidrug transporters in Lactococcus lactis.

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in Lactococcus lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.

Degenerate PCR primers for detecting putative priming glycosyltransferase genes in Bifidobacterium strains.

A new PCR-based method to detect putative exopolysaccharide (EPS) producers from the genus Bifidobacterium was developed based on the detection of two priming glycosyltransferase genes: rfbP (undecaprenyl-phosphate sugar phospho-transferase) and cpsD (galactosyl-transferase). An in silico analysis of the genomes of 28 bifidobacterial strains, belonging to 8 different species, allowed us to detect rfbP, cpsD, or both, in the large majority of the genomes. Based on DNA sequence homology studies, 24 degenerated primers were synthesised in order to select the primer pairs with the broadest capacity to detect the presence of these genes. Four primer pairs targeting internal regions of rfbP and cpsD were selected, allowing the detection of at least one of the two genes in 63 out of 99 bifidobacterial strains analysed, whereas control strains from other genera yielded negative results, suggesting that these genes are widely spread in this genus. The use of these primers is recommended to screen for the potential of Bifidobacterium strains to produce EPS.

Characterization of plasmids from Listeria monocytogenes and Listeria innocua strains isolated from short-ripened cheeses.

The plasmid content of 30 isolates of Listeria monocytogenes and 18 isolates of Listeria innocua obtained from short-ripened cheeses was analysed. The isolates of L. monocytogenes serogroup 1 harboured a single plasmid, pLM33 (33.2 kbp), whereas the serogroup 4 isolates did not contain plasmids. One group of L. innocua strains harboured the plasmid pLI71 (71 kbp) and another one contained two plasmids: pLI59 (59.5 kbp) and pLI56 (56.5 kbp). These plasmid groups were in accordance with clusters previously defined by pulsed-field gel electrophoresis analysis of the chromosomal DNA of Listeria isolates. Plasmids pLM33, pLI71 and pLI59 shared homology regions of at least 20 kbp. Plasmid pLI56 did not encode genes for any known character (such as carbohydrate fermentation, resistance to antibiotics, heavy metals or disinfectants, growth at low pH, NaCl tolerance or thermal inactivation by pasteurisation) and displayed different characteristics to the other three plasmids. It was also the only one cured from the parent strain and the sole plasmid not digested by the restriction enzyme PstI. In addition, its lack of homology with pLM33, pLI71 and pLI59 enhanced the possibility of a different origin for plasmid pLI56.

Duplication of the beta-galactosidase gene in some Lactobacillus plantarum strains.

Curing of a plasmid that encoded a beta-galactosidase gene (beta-gal) from the Lactobacillus plantarum strain of dairy origin LL441 was not accompanied by complete loss of the lactose utilization phenotype. DNA-DNA hybridization, using an internal fragment of the beta-gal gene as a probe, revealed a second determinant located on the chromosome of the cured derivatives. The chromosomal copy was present in all of a series of beta-Gal+ L. plantarum and Lactobacillus pentosus strains from different origins. In addition, four other L. plantarum strains harboured plasmid encoded beta-gal genes as well. Since both sequences cross-hybridized and present a similar genetic organization, it is postulated that the plasmid copy was generated through gene duplication and, probably, selected by growth of the strains in lactose rich environments.

A proteomic approach to cold acclimation of Staphylococcus aureus CECT 976 grown at room and human body temperatures.

Staphylococcus aureus is an important pathogenic microorganism that has been associated with serious infection problems in different fields, from food to clinic. In the present study, we have taken into account that the main reservoirs of this microorganism are the human body and some parts of food processing plants, which have normal temperatures of around 37 and 25°C, respectively. It can be expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food matrices. Since temperature abuse for foods occurs at approximately 12°C, it is expected that S. aureus must acclimate its metabolism to colder temperatures before growing in food. For this reason, we have performed a proteomic comparison between exponential- and stationary-phase cultures of S. aureus CECT 976 acclimated to 12°C after growing at 25°C or 37°C. The analysis led to the identification of two different protein patterns associated with cold acclimation, denominated pattern A and pattern B. The first was characteristic of cultures at stationary phase of growth, grown at 25°C and acclimated to 12°C. The second appeared in the rest of experimental cases. Pattern A was distinguished by the presence of glycolytic proteins, whereas pattern B was differentiated by the presence of general stress and regulatory proteins. Pattern A was related through physiological experiments with a cross-resistance to acid pH, whereas pattern B conferred resistance to nisin. This prompted us to conclude that both molecular strategies could be valid, in vivo, for the process of acclimation of S. aureus to cold temperatures.

Secondary and tertiary structure changes of reconstituted LmrA induced by nucleotide binding or hydrolysis. A fourier transform attenuated total reflection infrared spectroscopy and tryptophan fluorescence quenching analysis.

LmrA, a membrane protein of Lactococcus lactis, extrudes amphiphilic compounds from the inner leaflet of the cytoplasmic membrane, using energy derived from ATP hydrolysis. A combination of total reflection Fourier transform infrared spectroscopy, (2)H/H exchange, and fluorescence quenching experiments was used to investigate the effect of nucleotide binding and/or hydrolysis on the structure of LmrA reconstituted into proteoliposomes. These measurements allowed us to describe secondary structure changes of LmrA during the catalytic cycle. The structure of LmrA is enriched in beta-sheet after ATP binding, and the protein recovers its initial secondary structure after ATP hydrolysis, when P(i) has been released. (2)H/H exchange and fluorescence quenching studies indicate that the protein undergoes two distinct tertiary structure changes during the hydrolysis process. Indeed, the protein alone is poorly accessible to the aqueous medium but adopts a more accessible conformation when ATP hydrolysis takes place. After ATP hydrolysis, but when P(i) is still associated with the protein, the accessibility is intermediate between these two states.

The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism.

The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters.

The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids.

Lactococcus lactis possesses an ATP-binding cassette transporter, LmrA, which is a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein, and is able to transport a broad range of structurally unrelated amphiphilic drugs. A histidine tag was introduced at the N-terminus of LmrA to facilitate purification by nickel affinity chromatography. The histidine-tagged protein was overexpressed in L. lactis using a novel protein expression system for cytotoxic proteins based on the tightly regulated, nisin-inducible nisA promoter. This system allowed us to get functional overexpression of LmrA up to a level of 30% of total membrane protein. For reconstitution, LmrA was solubilized with dodecylmaltoside, purified by nickel-chelate affinity chromatography, and reconstituted in dodecylmaltoside-destabilized, preformed liposomes prepared from L. lactis phospholipids. The detergent was removed by adsorption onto polystyrene beads. The LmrA protein was reconstituted in a functional form, and mediated the ATP-dependent transport of the fluorescent substrate Hoechst-33342 into the proteoliposomes. Interestingly, reconstituted LmrA also catalyzed the ATP-dependent transport of fluorescent phosphatidylethanolamine, but not of fluorescent phosphatidylcholine. These data demonstrate that LmrA activity is independent of accessory proteins and support the notion that LmrA may be involved in the transport of specific lipids or lipid-linked precursors in L. lactis.

Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA.

Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.

Multidrug resistance in lactic acid bacteria: molecular mechanisms and clinical relevance.

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. The secondary multidrug transporter LmrP and the ATP-binding cassette (ABC) type multidrug transporter LmrA in Lactococcus lactis are representatives of the two major classes of multidrug transporters found in pro- and eukaryotic organisms. Therefore, knowledge of the molecular properties of LmrP and LmrA will have a wide significance for multidrug transporters in all living cells, and may enable the development of specific inhibitors and of new drugs which circumvent the action of multidrug transporters. Interestingly, LmrP and LmrA are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA will represent an intriguing new area of research.

Polymorphism of Listeria monocytogenes and Listeria innocua strains isolated from short-ripened cheeses.

Thirty isolates of Listeria monocytogenes and 18 of L. innocua obtained from different short-ripened cheeses manufactured in Asturias (northern Spain), were compared with each other and with reference strains using serotype, phage type and pulsed-field restriction endonuclease digestion profiles analysis of the total DNA. Restriction enzymes ApaI and SmaI defined five clusters in L. monocytogenes (m1 to m5) and two main clusters in L. innocua (i1 and i2). Cluster i2 was further arranged into three subclusters (i2a, i2b and i2c) based on the different Eco52I (XmaIII) and Crf42I (SacII) patterns of its isolates. Clusters of L. innocua were clearly different whereas those of L. monocytogenes were more closely related to each other. In this latter species, serotype 4b isolates (m4 and m5) constituted a more homogeneous group than serogroup I isolates (m1, m2 and m3). Cluster m3 contained two strains of serotype 1/2a whereas m1 and m2 harboured strains of both serotypes, 1/2a and 1/2b. Therefore, the combined use of restriction patterns and serotype may be useful to differentiate L. monocytogenes strains showing identical restriction profiles but differing in serotype. The cheese source of Listeria strains proved that isolates from cluster m1 were repeatedly detected as a contaminant in the same type of cheese. Comparison of L. monocytogenes ApaI profiles showed a genetic proximity of m4 and m5 to the recognized pathogenic strains ATCC 13932 and NCTC 11994, responsible for meningitis cases in other countries. Finally, bacteriophage typing data indicated that m4, the sole phage typable group, had a phage type resembling that of strains causing the Auckland (New Zealand) outbreak of listeriosis in 1969. These data suggest a wide distribution of closely related types which might cause, under several circumstances, sporadic cases of listeriosis.

Structure and dynamics of the membrane-embedded domain of LmrAinvestigated by coupling polarized ATR-FTIR spectroscopy and (1)H/(2)H exchange.

Bacterial LmrA, an integral membrane protein of Lactococcus lactis, confers multidrug resistance by mediating active extrusion of a wide variety of structurally unrelated compounds. Similar to its eucaryotic homologue P-gp, this protein is a member of the ATP-binding cassette (ABC) superfamily. Different predictive models, based on hydropathy profiles, have been proposed to describe the structure of the ABC transporters in general and of LmrA in particular. We used polarized attenuated total reflection infrared spectroscopy, combined with limited proteolysis, to investigate the secondary structure and the orientation of the transmembrane segments of LmrA. We bring the first experimental evidence that the membrane-embedded domain of LmrA is composed of transmembrane-oriented alpha-helices. Furthermore, a new approach was developed in order to provide information about membrane domain dynamics. Monitoring the infrared linear dichroism spectra in the course of (1)H/(2)H exchange allowed to focus the recording of exchange rates on the membrane-embedded region of the protein only. This approach revealed an unusual structural dynamics, indicating high flexibility in this antibiotic binding and transport region.