Staphylococcus aureus (S. aureus) is a dangerous pathogen as well as a frequent colonizer, threatening human health worldwide. Protection against S. aureus infection is challenging, as the bacteria have sophisticated strategies to escape the host immune response. To maintain equilibrium with S. aureus, both innate and adaptive immune effector mechanisms are required. Dendritic cells (DCs) are critical players at the interface between the two arms of the immune system, indispensable for inducing specific T cell responses. In this review, we highlight the importance of DCs in mounting innate as well as adaptive immune responses against S. aureus with emphasis on their role in S. aureus-induced respiratory diseases. We also review what is known about mechanisms that S. aureus has adopted to evade DCs or manipulate these cells to its advantage. Staphylococcus aureus (S. aureus) can act as a commensal bacterium in humans, where it frequently colonizes the airways, skin and gut. In most cases, the host can maintain equilibrium with the bacteria over long time periods. However, given the opportunity, S. aureus can cause a broad range of infections, ranging from mild, self-limiting skin and soft tissue infections to life-threatening diseases [S. aureus can cause pneumonia, and colonization with the bacteria is associated with allergic airway inflammation, especially with chronic rhinosinusitis with polyps and asthma [S. aureus is frequently found on inflamed skin of patients with atopic dermatitis (AD) [diseases . In the d asthma . Similartis (AD) . S. aureus mainly has an extracellular lifestyle, the microorganism is also able to invade phagocytes as well as epithelial and endothelial cells and persist intracellularly [S. aureus [Although llularly . To contllularly . Innate . aureus .S. aureus by professional phagocytes, such as monocytes (MOs), macrophages (Mφs) and DCs, induces the release of cytokines and chemokines, and the latter recruit neutrophils to the site of infection. Neutrophils are essential for killing the bacteria, either by phagocytosis or by NETosis. Phagocytosis is a process of engulfing and digestion of bacteria inside the cell, whereas NETosis involves trapping of bacteria in net-like structures, called neutrophil extracellular traps (NETs), which consist of DNA and histones as well as the content of neutrophil granules, such as anti-microbial peptides (AMPs) and elastase [S. aureus secretes many proteins that interfere with both the recognition by phagocytes and their chemotaxis to the infection site [Recognition of elastase . As a coion site . The micion site .S. aureus. Complement factors or their fragments can promote opsonization to facilitate microbial clearance (C3b) and drive inflammation (C3a and C5a). These functions are mediated by specific complement receptors (CRs) on host cells. Moreover, complement can kill many bacterial species directly by forming pores in their membranes, the membrane attack complexes (MACs). In return, S. aureus interferes with complement function at many levels. A capsule and a thick peptidoglycan layer protect its membrane from MAC . By iammation ,9. Thesecteremia . In addi defense .S. aureus infection. Most human adults have a broad spectrum of S. aureus- specific antibodies in their body fluids with large inter-individual variation in terms of antibody titers and the spectrum of S. aureus antigens that are recognized [S. aureus infection and/or a less severe disease course [S. aureus infection. In the majority of cases the disease is caused by heterozygous missense mutations and short deletions in signal transducer and activator of transcription 3 (STAT3) leading to an impairment of T cell development, in particular of Th17 cells. Recently it was shown that HIES patients also have very low anti-S. aureus antibody titers, although total serum IgG levels are in the normal range. Presumably, this is due to their impaired T cell response, which we discuss in detail below. Immunoglobulin (Ig)G replacement therapy significantly ameliorates S. aureus control, with concomitant antibiotic treatment, which makes a strong case of a protective role of antibodies [Considering adaptive immunity, there is compelling evidence that antibodies contribute to clinical protection from cognized ,13. Highe course . Converstibodies ,16.S. aureus toxins and other virulence factors [Binding of IgG antibody to Fc receptor on phagocytes can opsonize the bacteria, whereas binding of IgG and IgM to bacteria triggers the complement cascade. Moreover, antibodies can neutralize  factors .S. aureus clearance. In a mouse model of persistent S. aureus infection, deficiency of T cells increased the susceptibility to S. aureus [S. aureus [Recent studies have highlighted the importance of T cell-mediated immune response in . aureus . In addi. aureus . Further. aureus .S. aureus-specific antibody repertoire indicates the existence of numerous S. aureus-specific T cells. Several recent studies have provided evidence for robust CD4+ and CD8+ T cell memory of staphylococcal antigens in humans [It is well known that most B cells require help by T cells to generate high affinity antibodies, such that the observation of a broad n humans ,21,22.S. aureus activates the T cells and directs their differentiation into effector and memory T cell subpopulations, but DCs are bound to be critically involved. DCs have a central role as antigen-presenting cells (APCs) for T cells, and they have a decisive influence on the quality of the adaptive immune reaction.There is limited knowledge about the mechanisms by which S. aureus during colonization and infection. We also address the countermeasures S. aureus uses to divert and disturb the immune response triggered by recognition of the bacteria by DCs, including the induction of allergic inflammation  and tissue distribution in mice [DCs were first described by Paul Langerhans in 1868  as “bran in mice .DCs are highly specialized phagocytic cells. Their main function is antigen presentation to T cells, and they have the unique ability to initiate and regulate both innate and adaptive immune responses against various antigens . DCs oriS. aureus. Although their ability to directly kill S. aureus is limited, they play a major role in the initiation and regulation of the immune response at the infection site [S. aureus infection [In addition to their crucial role at the interface between innate and adaptive immune system as professional APCs, DCs contribute to the clearance of the opportunistic pathogen ion site ,5,26,30.ion site . In addiion site . Moreovenfection .DCs are generated during hematopoiesis from precursors of lymphoid or myeloid origin, which is an antigen-independent process. The progenitors in the bone marrow, called macrophage- and DC precursors (MDP), give rise to DCs and Mφs . In rececDCs originate from pre-cDCs under the influence of granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4. This type of bone marrow-derived DCs has been extensively studied in in vitro experiments, and they are the most valuable source of knowledge about human and murine DCs. In vivo, the precursors of DCs exit the bone marrow and migrate through the blood into lymphoid tissues, bone marrow, spleen and lymph nodes, where they differentiate into cDCs characterized by the integrin CD11c . Since McDCs are key players in the polarization of the T cell response. cDCs1 secrete Th1 polarizing cytokine IL-12p70, but are also capable of producing immune regulatory cytokines such as transforming growth factor beta (TGF-β) and IL-10, both important in immune tolerance . cDC2 inPlasmacytoid DCs (pDCs) were first reported in 1958 by Lennert et al., and named by their appearance, which resemble plasma cells . PrecursDCs can efficiently recognize a wide range of invading microorganisms. The pattern recognition receptors (PRRs), which are expressed on their surface, can sense pathogen-associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs). Among all PRRs, the Toll-like receptors (TLRs) are particularly well studied and characterized. They play a key role in inducing both direct and indirect DC maturation. To date, 10 human TLRs (TLR1–10) and 12 murine TLRs  have been identified. TLR1, 2, 4–6 and 11 are expressed at the cell surface while TLR3 and 7–9 are located intracellularly in the endosomal compartments .S. aureus recognition, through the detection of lipoproteins, wall teichoic and lipoteichoic acid, as well as peptidoglycan [S. aureus has been shown to evade TLR2 activation by secreting SSL3, indicating that TLR2 inhibition is important for staphylococcal pathogenesis [S. aureus, whereas S. aureus strains producing low amounts of PSMs are less detected by TLR2 [S. aureus activity [S. aureus occurs in 90% of AD patients aggravating the inflammation and sometimes leading to severe invasive infections such as endocarditis or bacteremia [S. aureus RNA, a vita-PAMP enabling the assessment of microbial viability [S. aureus CpG-DNA and induces type I IFN signaling [S. aureus by increasing the production of reactive oxygen species (ROS) [S. aureus carriers have a higher expression of TLR9 that is dependent on the carrier’s TLR9 haplotype, sex and hormone status, which could explain why women are more susceptible to S. aureus septicemia than men [TLR2 is the main receptor involved in doglycan ,57. In adoglycan . Counterdoglycan . In a muogenesis . The pro by TLR2 . In AD, activity . Colonizcteremia . TLR8 iniability ,64. TLR9ignaling . In ostees (ROS) . Persistthan men ,68.S. aureus produces many proteins enabling it to invade different cell types, including non-phagocytic cells, via a zipper-type mechanism [S. aureus to the host cell matrix (S. aureus Fnb A and B bind to the host cell integrin α5β1 via a fibronectin bridge. Other MSCRAMMs such as clumping factor (Clf) A and B are also involved in the attachment of S. aureus to host cells. Protein A (SpA), in contrast, interacts directly with host cell receptors [S. aureus is internalized without triggering TLRs [To escape PRR-triggered phagocytosis and avoid the reaction of host cells to danger signals, echanism ,70. Thisechanism . MSCRAMMl matrix . S. aureeceptors . These ming TLRs ,74.S. aureus and re-attach to the bacterial surface non-covalently. The autolysin (Alt) family of proteins, AltA and Aaa, mediate adherence of S. aureus to host components such as fibronectin, gelatin and heparin, facilitating colonization and infection [S. aureus from phagocytosis and inducing abscesses. In this way, S. aureus can aggregate and shield themselves from phagocytes in a tight network thanks to bacterial and host proteins [Other adhesins, known as secretable expanded repertoire adhesive molecule (SERAMs), are released by nfection . Among tnfection ,77. Otheproteins .S. aureus possesses a broad range of factors that can prevent opsonization, either by hiding the targets of the opsonins or by using decoys. Many S. aureus strains are encapsulated by sugar polymers that cover most of the immunogenic surface-exposed proteins [S. aureus clones. Moreover, all S. aureus clinical isolates produce SpA, a major virulence factor, which binds with high affinity to the Fc-portion of Igs, mostly IgG, rendering them unable to bind to the bacterial surface in the correct orientation and thus preventing opsonization [HIII element, inducing apoptotic cell death. This can result in the complete deletion of the respective B cell clones [Opsonization is an efficient way to help phagocytes, including DCs, to take up bacteria and target them for killing. Igs and complement components are the main opsonins in the body fluids. Phagocytes possess specific receptors for IgG as well as the main complement opsonin C3b. DCs express Fcγ receptors on their surface that bind to IgG, helping the DC to immobilize their target for phagocytosis . Howeverproteins . The twoproteins . Interesnization . SpA canl clones .S. aureus with C3b facilitates phagocytosis [S. aureus is adept in complement evasion. Many secreted proteins such as SpA, aureolysin (Aur), staphylokinase (Sak), extracellular fibrinogen-binding protein (Efb), and staphylococcal complement inhibitor (SCIN) interact with components of the complement pathways, preventing this system from fulfilling its purpose in opsonization and killing of pathogens (Reviewed in [DCs possess CR3 CD11b/CD18) and CR4 (CD11c/CD18), such that coating of ocytosis . However1b/CD18 aiewed in ). For iniewed in . Furtheriewed in ,88,89.S. aureus, which is effective even before the bacteria are taken up by the host cells. S. aureus produces bi-component pore-forming toxins called leukocidins (Luks) which play a pivotal role in killing host immune cells, including DCs. The target structures of these toxins on host cells have been identified over the past few years (reviewed in [S. aureus to either kill its host cell or escape from phagosomes [S. aureus isolates from ruminants and is associated with bovine infections. It targets CCR2 and CCR5 on bovine and CCR1 on both human and bovine neutrophils and induces cell death [To kill before being killed is another strategy of iewed in ). LukED iewed in ,92. LukAagosomes . Since sagosomes ,93,94,95ll death . These Cll death . The spell death ,98,99. Hll death ,101.S. aureus is exposed to bactericidal effector mechanisms in DCs. Phagosomes mature and fuse with lysosomes containing hydrolases. In the phagolysosomes, S. aureus is also subjected to oxidative conditions rich in ROS and reactive nitrogen species (RNS). NADPH oxidase (NOX2) consumes oxygen to produce superoxide radical anions (O2•−) and hydrogen peroxide (H2O2). Oxidation modulates the activities of different groups of proteases and thus reduces proteolysis within phagosomes of DCs [S. aureus is also submitted to acidic pH. Acidification of phagosomes in DCs is reduced compared to Mφs or neutrophils because the responsible enzyme, the V-ATPase, is incompletely assembled in immature DCs [After internalization by phagocytosis, s of DCs . In additure DCs . Proteasture DCs . While rS. aureus manages to survive within eukaryotic host cells, including professional and non-professional phagocytes [S. aureus is also highly resistant to oxidative stress, because staphyloxanthin (Sx), the main pigment of S. aureus, works as an antioxidant and prevents membrane peroxidation [2- into H2O2 and then into H2O + O2 [S. aureus from oxidative stress during phagocytosis [S. aureus survival in neutrophils with a trade-off: promoting the induction of an adaptive immune response by DCs.However, in many cases agocytes . Acidifiagocytes . S. aurexidation . SOK  adds an acetyl group to N-acetylmuramic acid in the peptidoglycan, rendering S. aureus resistant to the lysozyme produced in the phagosome [Modification of the bacterial cell wall is another way to avoid degradation. The hagosome . This mahagosome .S. aureus has means to destroy the phago(lyso)some membrane and escape into the cytoplasm. Upon internalization by professional phagocytes, the bacteria produce phenol-soluble modulins (PSMs), similar to the delta hemolysin, which can form membrane pores. In DCs, PSMs help S. aureus to escape from the phagosome, invade the cytoplasm and possibly kill the host cell [agr system, a global regulator that is active in the phagolysosome environment [S. aureus to interfere with antigen processing and presentation on the MHC class II of the DCs, reducing their T cell-priming ability. Another effect of PSMs is the modulation of cytokine production by the host cells [regs [S. aureus is able to replicate in the cytosol and induce host cell death or survive in a dormant state for extended time periods in the shape of small colony variants (SCV) [S. aureus infection are very difficult to treat, and novel approaches are required. Chloroquine improved clearance of S. aureus from lung epithelial cells in combination with antibiotic therapy [Moreover, ost cell ,116,117.ironment . This mest cells ,119,120.ls [regs ,119. Aftts (SCV) . This ab therapy ,122.Autophagy or, more precisely, xenophagy, is used by DCs to kill pathogens and present antigens, once the bacteria have escaped into the cytoplasm. The cytoplasmic invaders become surrounded by double-membrane vacuoles, named autophagosomes, which present microtubule-associated protein 1 light chain 3 (LC3) associated to phosphatidylethanolamine (LC3-II). These fuse with lysosomes, redirecting their cargo from the cytoplasm back into the lysosomal pathway, followed by digestion and presentation of antigenic proteins. Consequently, peptides derived from cytoplasmic antigens, which are usually presented on the MHC class I, are loaded onto the MHC class II molecules, a process known as cross-presentation of antigens.agr system conditions S. aureus survival during autophagy by preventing the fusion between autophagosomes and lysosomes. S. aureus strains with a highly active agr system are not killed in autophagosomes in vitro and display an extended intracellular survival within phagocytes in vivo [S. aureus can prevent fusion of autophagosomes with lysosomes via a novel mechanism, involving activation of MAPK14 and ATG5 phosphorylation [S. aureus under agr control [The  in vivo . S. aurerylation . Autopha control . Indeed, control ,125. S. aureus is able to modulate the cytokine production of immune cells with several mechanisms. The ESAT-6-like secretion system (Ess) is encoded in the conserved ess gene cluster [ess-encoded virulence factor EsxA dampens the pro-apoptotic response in infected DCs and may allow S. aureus to use these cells as a Trojan horse. EsxB dampens the production of regulatory and pro-inflammatory cytokines by the infected DCs, resulting in a reduced production of IFN-γ and IL-17 by activated CD4+ T cells [As previously mentioned, DCs produce cytokines to control the immune response, recruit immune cells on the infection site and activate T cells.  cluster . The ess T cells .S. aureus can also induce an overstimulation of the immune system by secreting superantigens [egc or on mobile genetic elements [antigens . The speelements ,130. As elements . S. aureus and its host with extremely diverse outcome. Around 25 to 35% of adults persistently carry S. aureus in the nose, while the remainder is able to rapidly clear the bacteria from the upper airways [S. aureus can also cause pneumonia, a life-threatening infection of the lungs. It may be community acquired, often in the context of an influenza infection, in which case the mortality rate can increase to 50% [S. aureus infection and develop so-called ventilation-associated pneumonia. There is increasing evidence that, besides commensal and invasive behavior, S. aureus may also drive allergic airway inflammation [S. aureus is associated with childhood wheezing and asthma [S. aureus, in contrast to 33.3% of healthy adults. In case of additional aspirin hypersensitivity, these were even 87.5% [S. aureus enterotoxins and serine protease-like proteins were found in asthmatic patients [The airways are a site of extensive interaction between  airways . Howevere to 50% . In hospammation ,135. Cold asthma . van Zelen 87.5% . Moreovepatients ,139.S. aureus infection by lung DCs is of particular interest. Over the past years, many subsets of DCs have been described in lung immunity. Under steady state conditions, “immature” DCs in the lungs efficiently recognize and capture inhaled materials. DCs that have encountered antigens or allergens undergo maturation, leave the lung and migrate to draining regional lymphoid tissues, where they present the processed antigenic peptides to naïve T cells. This results in T cell activation and polarization, depending on the nature of the antigen [S. aureus with its host.DCs play a critical role in shaping the adaptive immune response at mucosal sites. The modulation of the T helper cell response to  antigen . It has  antigen . Both prS. aureus-primed cDCs are highly responsive and induce T cell differentiation into IFN-γ-producing CD4+ (Th1) and CD8+ (Tc1) cells [S. aureus-specific memory T cells that respond to S. aureus with the secretion of IFN-γ and/or IL-17 [S. aureus failed to produce IL-2 after an antigen-specific T cell response, suggesting that T cells undergo anergy during persistent infection [S. aureus is therefore a double-edged sword, as has been demonstrated in murine pneumonia models (reviewed in [1) cells . In factor IL-17 ,22. The or IL-17 . Moreovenfection . The luniewed in ).S. aureus colonization of the airways is associated with allergic airway disease, but the mechanisms of allergic sensitization or exacerbation by S. aureus are still poorly understood [derstood ,145. Astderstood . In addiderstood . The invderstood , who shoderstood . Recentlderstood .S. aureus alpha-toxin  may act similarly, because the pore-forming toxin activates the host metalloprotease ADAM10, which destroys epithelial adherens junctions [S. aureus pneumonia [S. aureus alpha toxin is under development as adjunct therapy with standard antibiotic treatment [Some airborne allergens such as Der p1 from house dust mites disrupt the epithelial barrier by cleaving the tight junction proteins, thus gaining access to the DCs at the basolateral side of the epithelium . It is tunctions ,152. Morunctions . On the unctions ,155,156.unctions . While qneumonia ,157. Vianeumonia . In the neumonia . Neutralreatment ,160.S. aureus proteins, SEs and serine protease-like proteins (Spls), readily cause allergic sensitization in the airways [S. aureus factors can elicit adaptive immune responses of different quality in the same individual. Similar observations have recently been reported for fungal antigens by Bacher et al. [S. aureus able to initiate the allergic march in susceptible individuals, or does it merely exacerbate pre-existing allergic inflammation? We hypothesize that S. aureus allergens may sensitize vulnerable persons whose allergic reaction is then potentiated by bacterial toxins and the PAMPs in the airways.It was discovered that two classes of  airways . Affecte airways ,162. Thir et al. . We concS. aureus has evolved means to prevent phagocytosis, to resist killing inside phagosomes, and manipulate DCs to its advantage. The multifaceted interactions between S. aureus and its host take place in the airways. The outcomes range from rapid clearance through symptom-free colonization to asthma or life-threatening pneumonia. Remarkably, single S. aureus proteins can elicit immune responses of distinctive cytokine and antibody profiles in the same individual, demonstrating that bacterial antigens have adjuvant properties. It will be worthwhile exploring more closely how DCs affect these processes and how their response is determined by various S. aureus virulence factors. A better understanding of the behavior of DCs, pivots of the immune system exhibiting great plasticity, will also benefit vaccine research.Coordination of the immune response at the interface between innate and adaptive defense mechanisms is an essential function of DCs. These phagocytic leukocytes sense microorganisms in tissues which border on the external environment. Not surprisingly,


Lactobacillus paracasei subsp. paracasei L. casei W8®; L. casei W8) on gut microbiota composition and metabolic profile in high-fat (HF) diet-fed rats. Four male Wistar rat groups (n = 8/group) were fed 10% low-fat, 45% HF, or HF with non-encapsulated or encapsulated L. casei W8 (4 × 107 CFU/g diet) diet for seven weeks. Microbiota composition, intestinal integrity, inflammatory profiles, and glucose tolerance were assessed. Non-encapsulated and pectin-encapsulated probiotic supplementation positively modulated gut microbiota composition in HF-fed male rats. These changes were associated with improvements in gut barrier functions and local and systemic inflammation by non-encapsulated probiotics and improvement in glucose tolerance by encapsulated probiotic treatment. Thus, these findings suggest the potential of using oral non-encapsulated or encapsulated probiotic supplementation to ameliorate obesity-associated metabolic abnormalities.Development of obesity-associated comorbidities is related to chronic inflammation, which has been linked to gut microbiota dysbiosis. Thus, modulating gut microbiota composition could have positive effects for metabolic disorders, supporting the use of probiotics as potential therapeutics in vivo, which may be enhanced by a microencapsulation technique. Here we investigated the effects of non-encapsulated or pectin-encapsulated probiotic supplementation ( Obesity and its associated comorbidities are major health problems in Western countries, particularly the United States where energy-dense diets are commonly consumed . Obesity14 microbial organisms [Inflammation in obesity has been shown to originate, at least in part, from the gastrointestinal (GI) tract .We have rganisms , and incrganisms ,8. Interrganisms ,9, highlrganisms .Clostridium perfringens and/or promote the proliferation of beneficial populations such as Bifidobacteria [Lactobacillus, Streptococcus and Bifidobacterium, have been shown to positively affect hyperlipidemia, obesity and insulin resistance in HF diet-fed animals in association with changes in gut microbiota composition and maintenance of gut epithelial integrity [Probiotics are defined by the FAO/WHO as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host . Probiotbacteria . Probiotbacteria . Furtherbacteria , promotibacteria . GLP-1 ibacteria . The thebacteria , and prontegrity .In order to exert their functions, high concentration of viable probiotic bacteria have to be able to reach the distal part of the intestine where they can interact with commensal microbiota. Probiotics are exposed to environmental conditions during processing and production of foods that decrease cell number and viability, including exposure to oxygen, heat, humidity, light, and shear that damage the cell wall, cell membrane or result in damage related to oxidation . FurtherLactobaccillus casei encapsulated by block de-esterified pectin showed greater survival during storage and greater stability under simulated GI transit [In this regard, the recent emergence of the microencapsulation technique appears to be a potential solution to enhance the survivability of probiotics . Encapsu transit .Lactobacillus paracasei subsp. Paracasei L. casei W8® (L. casei W8), in the prevention of HF diet-induced metabolic disorders. We hypothesized that probiotic supplementation would improve diet-induced inflammation and epithelial barrier integrity associated with positive alterations in intestinal microbiota composition. We anticipated the microencapsulation would significantly enhance delivery and effects of the probiotic. To test this hypothesis, Wistar rats were fed an HF diet supplemented with a non-encapsulated or pectin-encapsulated probiotic for seven weeks and gut microbiota composition, inflammation and glucose tolerance were determined.The present study investigated the potential beneficial effects of probiotic and microencapsulated probiotic bacteria, L. casei cells was completed as described [L. casei cells, hydrated in peptone water to a target of log cfu/mL of 12, and encapsulation by iontropic gelation was achieved by extrusion into 300 mM calcium chloride. Recovered beads were freeze-dried until use. Viable flora was enumerated by plate counting method on MRS agar at 37 °C [7 CFU/g. The blended diets were stored at −20 °C throughout the study.The lactic acid bacterial strain, L. casei W8, was donated by Chr. Hanson, Hørsholm, Denmark and stored at −80 °C until use. Pectin used for charge modification and encapsulation of cells was GENU pectin type B rapid set-Z from citrus peel, DE 72%, Batch NO: GR41649, and was donated by CP Kelco, Copenhagen, Denmark. Pectinmethylesterase was extracted from Valencia citrus pulp . Charge modification and encapsulation of escribed , similarescribed . Brieflyat 37 °C . Encapsun = 8 per group) and fed low fat , HF , HF supplemented with non-encapsulated L. casei W8 , or HF with supplemented with encapsulated L. casei W8  for seven weeks (n (including food at the bottom of the cages and crumbs) from the amount provided on day (n-1). Energy intake  was then calculated using the caloric density of each diet  were single-housed in a temperature-controlled room with a 12-h light-dark cycle. Animals were divided into four groups . Liver, ileum, cecum, colon, and visceral fat pads  were dissected, weighed, and an adiposity index was determined. Serum and all tissues were snap-frozen and stored at −80 °C until analysis. All animal care procedures were approved by the Institutional Animal Care and Use Committee of the University of Georgia .After seven weeks on their respective diets, animals were fasted for 6 h and euthanized via COAfter six weeks on their respective diets, animals were fasted for 5 h before oral gavage with a glucose solution . Glycemia was measured using a glucometer  before (0 min) and after  glucose challenge. Blood samples (~100 μL) were collected by tail bleeding (tail snip) at each time point to determine circulating insulin levels.DNA was extracted from cecal contents  and the eluted DNA was sent to SeqMatic LLC  for PCR, sequence library preparation, and sequencing. Briefly, polymerase chain reaction (PCR) was used to target the V4 region of the 16S rRNA genes using the 515F/806R primer set based on the protocol used by the Earth Microbiome project . SequencSerum samples were used for SCFAs quantification, which was done at the Mayo Clinic Metabolomics Core using gas chromatography–mass spectrometry as previously described .Insulin  and LPS-binding protein  in serum were measured as a via enzyme-linked immunosorbent assay (ELISA) according to manufacturers’ instructions.−ΔΔCt method [mRNA was extracted from distal gut (ileum) and visceral (mesenteric) fat tissues using the RNeasy Mini Kit or Lipid Tissue Mini Kit  to determine expression for inflammatory genes. Ileum tissues were used to assess gene expression of epithelial barrier function markers. Following extraction, mRNA quantity and purity was determined using a NanoDrop ND-1000 spectrophotometer . and complementary DNAs (cDNAs) were synthetized by reverse transcription . cDNAs were then amplified by qPCR  using SYBR Green PCR master mix (Thermo Fisher Scientific) and primers purchased from Integrated DNA Technologies . Gene ext method .p < 0.05.Data are presented as mean ± SEM. Unless noted otherwise , Prism software  was used for statistical analysis. Bodyweight, energy intake, and OGTT were analyzed by two-way repeated measures ANOVA while one-way ANOVA was used to analyze adiposity levels, gene expression data, SCFAs levels, and biochemical analyses. Differences were considered significant if p < 0.05). The HF and HF/Pca groups’ bodyweight was not statistically different from either the LF or HF/Pro group (p > 0.05).There were significant differences in bodyweight at baseline among all treatment groups A. After p < 0.0001; vs. HF, p < 0.05; p < 0.01; vs. HF, p = 0.08) and epididymal fat pad weights  fat pads. All three HF fed groups showed a significant increase in mesenteric fat depot compared to the LF group (p < 0.01).Despite minimal differences in bodyweight, there were differences in adiposity among groups. Generally, probiotics-treated animals exhibited greater adiposity than the LF control group, particularly the naked probiotic rats. Similarly to bodyweight, the HF/Pro group had a significantly higher total adiposity index than LF and HF rats , but not more than the HF/Pca groups (p = 0.056).There were no significant differences in total energy intake among LF, HF and HF/Pca rats throughout the experiment. The HF/Pro rats consumed significantly more overall than LF and HF rats .During the 1st week, HF-fed groups had a significantly higher energy intake than the LF group  and their overall glycemia area under the curve (AUC) over 120 min was also significantly higher compared to LF rats , but not to the HF group (p < 0.01) and their overall AUC was also significantly lower .There were no differences in baseline glucose and insulin levels among groups A,C. FollHF group B. The HFp < 0.05), but not to HF and HF/Pca rats (p < 0.05), which was reduced by probiotic treatment, albeit not significantly , which was normalized by probiotic supplementation in the HF/Pro group , and partially improved by the HF/Pca treatment. Furthermore, HF feeding alone led to significant increases in abundance of Deltaproteobacteria (p < 0.05) and Actinobacteria (p < 0.05), mediated by increases in Coriobacteriales , Coriobacteriaceae , Adlercreutzia  and Desulfovibrionales , Desulfovibrionaceae , Bilophila , respectively , which were driven by the class 4C0d-2 (p < 0.01) and order YS2 , and this change was restored to the LF level in HF/Pro and HF/Pca groups .In LF-fed rats, Lentisphaerae  abundance, which was primarily driven by increased abundance in Lentisphaeria , Victivallaceae , and Victivallales  abundance and this profile was mainly characterized by increased abundance of TM7-3 , CW040 , and F16 . L. paracasei and L. zeae were only present in probiotic-treated groups with a significant difference between HF/Pro and HF/Pca groups . The presence of Prevotella was detected only in LF and HF/Pca groups without a significant difference between them.At the species level F–I, therp < 0.05; propionate: HF vs. HF/Pro, p < 0.01, vs. HF/Pca, p < 0.05). There was no difference in acetate levels among groups. Correlation analysis showed a significantly positive relationship between serum butyrate and abundance of Cyanobacteria  and Prevotella . Serum propionate was negatively correlated with L. paracasei  and L. zeae .Probiotic supplementation led to changes in serum concentration of SCFAs A. HF feeNo significant differences were found among groups for GLP gene expression, but the HF/Pca group had the highest GLP-1 mRNA expression B.p < 0.05; Gene expression of a mucus protein, mucin 2 (MUC2), in the ileum was elevated in the all HF-fed groups, but only the HF/Pro group showed a significant difference compared to the LF group (p < 0.01) and MCP1 , which was normalized by probiotic supplementation (HF/Pro) and partially normalized in the encapsulated group (HF/Pca) , as well as IL-6 and TNFα, but without significance.In the distal gut (ileum), HF feeding was associated with significant upregulation in gene expression of macrophage infiltration markers, CD68  levels among groups C.p < 0.05; MCP1, p < 0.001). MCP1 gene expression was normalized by probiotic supplementation and partially normalized in the encapsulated group  and systemic  inflammation indices, but had no protective effect on glucose tolerance and adiposity. On the other hand, improvements in glucose tolerance and insulin sensitivity were observed only in the encapsulated probiotic-treated group. Interestingly, Prevotella, which is associated with improved glucose control, was present only in the HF/Pca group among HF-fed rats in line with the glucose tolerance outcome of this study  for more than five weeks significantly increases bodyweight, energy intake, and fat mass . In thisUnexpectedly, probiotic treatment did not prevent HF-diet driven increase in adiposity and bodyweight, regardless of encapsulation. The non-encapsulated probiotic-treated group exhibited the strongest obesity phenotype compared to the LF group. Firmicutes abundance and decreased Bacteroidetes abundance. Elevated Firmicutes to Bacteroidetes ratio is a characteristic of obesity-driven disruptions in microbiota, i.e., dysbiosis [Actinobacteria and Deltaproteobacteria, which were primarily driven by the elevated abundance of genus Adlercreutzia and Bilophila, respectively. Studies have demonstrated that an increase in Adlercreutzia abundance is associated with intestinal permeability [Bilophila blooms with HF diets [To understand the limited effects of supplementation on the obesity phenotype, we characterized the animals’ microbiota composition. HF consumption resulted in modest changes in gut microbiota composition, which were modulated by probiotic supplementation. At the phylum level, HF feeding led to increased ysbiosis ,38. HF feability , while BHF diets . The oveHF diets .Firmicutes to Bacteroidetes ratio was notably normalized by the non-encapsulated supplementation and partially improved by the encapsulated treatment. L. paracasei, the probiotic species used in this study, was only present in probiotic-treated groups although it was significantly lower in the HF/Pca group. Interestingly, Lactobacillus species, notably Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus paracasei, appear to be transient passengers rather than to be GI commensals, promoting the growth of indigenous beneficial gut microbes, such as Bifidobacteria and leading to ecological rearrangement of gut microbiota [L. zeae, whose abundance has been shown to be significantly increased by L. casei BL23 treatment [L. paracasei and L. zeae. whose abundances were elevated in the non-encapsulated treated groups and positively correlated with adiposity, indicating a possibly increased energy efficiency by the probiotic treatment. Unsurprisingly, the probiotic treatments modulated gut microbiota composition. The increased crobiota ,42. In treatment , was onlreatment . Low metreatment , and henreatment . The bloreatment . In addireatment . It is freatment ,48, despreatment . Propionreatment . In thisWe hypothesized that changes in gut microbiota would be associated with improved gut epithelial integrity and decreases in local and systemic inflammation. HF consumption promotes intestinal inflammation, leading to the altered intestinal barrier function and increases in gut permeability and translocation pro-inflammatory bacterial products, such as LPS, into the circulation . PreviouInflammatory cytokines can impair glucose tolerance and insulin sensitivity . In respSurprisingly, the glycemic level was further elevated in the HF/Pro group. The HF/Pro group showed higher adiposity than all other groups. Since excess fat mass, especially visceral, has been linked to glucose metabolism dysregulation , high viIt is, however, important to point out that different metabolic outcomes were observed between HF/Pro and HF/Pca groups for inflammatory profiles, and glucose tolerance. While both probiotic-treated groups showed improvements in local and systemic inflammation and gut barrier functions against the HF challenge, the effects were only significant in rats receiving non-encapsulated probiotics, suggesting the encapsulation process did not enhance the probiotic effects. These results cast doubt on the efficacy of encapsulation employed in this study above the efficacy of probiotic addition as discussed previously.Prevotella abundance, which characterized the microbial profile of the HF/Pca group. Prevotella abundance increases with carbohydrate-rich diets, especially fibers [Prevotella abundance in the HF/Pca group may have been due to the presence of pectin.Conversely, during the OGTT, the HF-induced glucose intolerance was further deteriorated in the HF/Pro group while the impairment was alleviated in the HF/Pca group. This suggests a positive effect of encapsulation on glucose tolerance, potentially independent of the probiotics. As discussed previously, pectin was used for encapsulation. Even allowing for its minimal concentration in diets, it is feasible that pectin itself exerted prebiotic and/or synbiotic effects via hydrolysis and oligosaccharides fermentation Selective fermentation of the branched, neutral sugar rich domain of pectin may have generated favorable metabolites , such asy fibers  and is ay fibers . ConsideBifidobacterium abundance was highest in the HF/Pca group, which was significantly higher than the LF group. Bifidobacterium has been found to be associated with improved glucose tolerance by elevating the level of GLP-1, a known incretin [Bifidobacterium abundance was positively correlated with the gene expression of GLP-1 in the ileum in this study . Moreover, as one of the most commonly used probiotics, incretin , in bothincretin , which cIn conclusion, we demonstrated that probiotic-induced changes in gut microbiota composition were associated with improvements in gut barrier functions and local and systemic inflammation in HF-fed rats. Encapsulation of the probiotics with pectin in this study did not enhance the effect of probiotic supplementation. However, pectin encapsulation had positive effects on glucose tolerance, possibly due to prebiotic and/or synbiotic effect of pectin used as encapsulation material. Thus, our findings support the potential of using an oral non-encapsulated or encapsulated probiotic supplementation to ameliorate obesity-associated metabolic abnormalities.


Trypanosoma Gruby, 1843 is constituted by terrestrial and aquatic phylogenetic lineages both harboring understudied trypanosomes from reptiles including an increasing diversity of crocodilian trypanosomes. Trypanosoma clandestinus Teixeira & Camargo, 2016 of the aquatic lineage is transmitted by leeches to caimans. Trypanosoma grayi Novy, 1906 of the terrestrial lineage is transmitted by tsetse flies to crocodiles in Africa, but the vectors of Neotropical caiman trypanosomes nested in this lineage remain unknown.The genus T. grayi in tsetse flies. All trypanosomes found in tabanids clustered in the crocodilian clade  forming six clades: Grayi (African trypanosomes from crocodiles and tsetse flies); Ralphi ; Terena (caimans); Cay03 (caimans and Brazilian tabanids); and two new clades, Tab01 (Brazilian tabanid and tsetse flies) and Kaiowa. The clade Kaiowa comprises Trypanosoma kaiowa n. sp. and trypanosomes from African and Brazilian tabanids, caimans, tsetse flies and the African dwarf crocodile. Trypanosoma kaiowa n. sp. heavily colonises tabanid guts and differs remarkably in morphology from other caiman trypanosomes. This species multiplied predominantly as promastigotes on log-phase cultures showing scarce epimastigotes and exhibited very long flagellates in old cultures. Analyses of growth behavior revealed that insect cells allow the intracellular development of Trypanosoma kaiowa n. sp.Our phylogenetic analyses uncovered crocodilian trypanosomes in tabanids from South America and Africa, and trypanosomes other than Trypanosoma kaiowa n. sp., no crocodilian trypanosome parasitic in tabanid flies had been cultured, morphologically examined by light, scanning and transmission microscopy, and phylogenetically compared with other crocodilian trypanosomes. Additionally, trypanosomes thought to be restricted to caimans were identified in Brazilian and African tabanids, tsetse flies and the dwarf crocodile. Similar repertoires of trypanosomes found in South American caimans, African crocodiles and tabanids from both continents support the recent diversification of these transcontinental trypanosomes. Our findings are consistent with trypanosome host-switching likely mediated by tabanid flies between caimans and transoceanic migrant crocodiles co-inhabiting South American wetlands at the Miocene.Prior to this description of The online version of this article (10.1186/s13071-019-3463-2) contains supplementary material, which is available to authorized users. Trypanosoma grayi Novy, 1906 [Trypanosoma cecili Lainson, 1977 [T. grayi from tsetse flies and crocodiles [Trypanosoma terena Teixeira & Camargo, 2013, Trypanosoma ralphi Teixeira & Camargo, 2013, and Trypanosoma clandestinus Teixeira & Camargo, 2016.Trypanosomes are ubiquitous parasites of all classes of vertebrates, from fishes to mammals, including a few species parasitic in humans –4. More vy, 1906 , 6 and Tocodiles , 8, 9 anocodiles –12 enablTrypanosoma is composed of two major phylogenetic lineages, in line with the main habitats of their vertebrate hosts, the terrestrial and the aquatic lineage [T. clandestinus of caimans [T. grayi, T. terena and T. ralphi [The genus  lineage , 4. Tryp lineage , 13–19 p caimans  cluster . ralphi , 20–23.Crocodylus niloticus Laurenti, Mecistops cataphractus Cuvier, and Osteolaemus tetraspis Cope [Caiman yacare Daudin, Caiman crocodilus Linnaeus, Melanosuchus niger Spix, and Paleosuchus trigonatus Schneider [Crocodylus Laurenti originated in Australasia during the mid-Miocene (8–13 mya) and through transoceanic dispersal reached South America and Africa, although the exact dispersion route remains uncertain [T. grayi are tsetse flies [T. clandestinus of caimans [In Africa, trypanosomes of the terrestrial lineage occur in species of the family Crocodylidae: pis Cope , 12, 24.chneider , 10–12. chneider –28. The ncertain . Fossil ncertain , 28 indincertain –31. We pncertain . The vecse flies , 6, 32.  caimans –36.Trypanosoma theileri Laveran, 1902 of domestic and wild ruminants, carry other trypanosomes of ungulates, and possibly transmit trypanosomes to marsupials in Australia [Ca. yacare.Tabanids cyclically transmit ustralia , 37–41. ustralia –46. Giveustralia , we searCa. yacare) captured in the Miranda River, Mato Grosso do Sul state, in the Pantanal biome, which is a vast wetland in central Brazil and home to a considerable population of caimans , on the margins of the Ji-Paraná River Fig. . In addiarcoding .Trichoplusia sp.) overlaid with TC100 (= Graceʼs) medium containing 10% FBS, and incubated at 25 °C [4 cells/well) were seeded onto 24-well plates containing glass coverslips, incubated for 24–48 h, and then seeded with 105 flagellates/well. After ~8 h, cultures were washed twice with PBS  and incubated with fresh TC100 medium. After 1, 3, 5, 7 and 10 days, cells were washed three times with PBS, and the cells fixed with methanol and stained with Giemsa.Guts of tabanids caught in the Pantanal biome that were found to be microscopically positive for trypanosomes were seeded in cultures of Hi-5 insect feeder cells  and covered with gold in sputtering equipment SCD 050 (Leica Microsystems). A FEI Quanta 250  was used for SEM. For TEM, trypanosomes were fixed and dehydrated as above for SEM, embedded overnight in epoxy resin, trimmed, and cut in thin sections (80–100 nm). Sections were stained with aqueous 2% uranyl acetate and lead citrate before being examined using a LEO 906E at 80 kV . Images were captured by a CCD camera MegaView III.For SEM, trypanosomes from log-phase Hi-5 cultures were washed three times with PBS, fixed in 2.5% glutaraldehyde plus 1% formaldehyde in 0.1 M cacodylate buffer (pH 7.2) at room temperature overnight, and processed using the protocols usually employed for trypanosomatids , 49, 50.DNA from caiman blood and gut contents of tabanid and tsetse flies preserved in ethanol were prepared as previously described , 47. BriSSU rDNA (~900 bp) and gGAPDH (~680 bp) sequences, as previously described [T. clandestinus and trypanosomes of the aquatic clade; (ii) B, including gGAPDH sequences, determined in the present and previous studies, restricted to trypanosomes of the clade crocodilian , plus sequences of closely related trypanosomes from tapirs (Brazil) [SSU rDNA and gGAPDH sequences from isolates representative of each species/clade of the major crocodilian clade, and trypanosomes representative of other main clades within the terrestrial and aquatic lineages. Phylogenetic analyses were inferred by maximum likelihood (ML) and Bayesian inferences (BI) as described previously [DNA from caiman blood samples and the guts of tabanid and tsetse flies were submitted to nested-PCRs for amplification of V7V8 escribed . Sequenc(Brazil)  and rhinSU rDNA ~00 bp andeviously , 12, 18.eviously . Tree seeviously  with GTRPhaeotabanus fervens Linnaeus (48 flies) and Tabanus occidentalis Linnaeus (3 flies), herein identified by cox1 barcoding, were caught while feeding on Ca. yacare captured in the Miranda River, in the Pantanal biome, Brazil  from Amazonia showed a 40% infection rate, and gGAPDH sequences obtained from these flies revealed that they were infected with T. ralphi  were also obtained from these flies. Of the Ethiopian tabanids  11 out of 33 flies yielded sequences of trypanosomes, five of which were highly similar to those of T. kaiowa n. sp. , we obtained one culture, which was cryopreserved as TCC2918. SSU rRNA and gGAPDH gene sequences from the isolate TCC2918 were identical to those of a previous isolate TCC1611 from Ca. yacare [Trypanosoma kaiowa n. sp.  [Trypanosoma shawi Nunes, 1987 [T. clandestinus from caimans transmitted by aquatic leeches clustered with fish trypanosomes in the aquatic lineage.The comparison of V7V8 ces Fig.  of trypadactyla)  were foues, 1987  from capes, 1987 , T. clangGAPDH sequences or concatenated SSU rDNA and gGAPDH sequences strongly supported the partition of trypanosomes within the clade crocodilian into two main groups  using either ups Fig. . One phyups Fig. . Each cliloticus , and theT. kaiowa n. sp. from the remaining species of crocodilian trypanosomes was confirmed by all analyses of the present study  differs from T. kaiowa n. sp. by only 0.5% in gGAPDH sequences and, hence, should be regarded as a genotype of T. kaiowa n. sp. In contrast, large divergences (~8%) separated T. kaiowa n. sp. from T. grayi, so far restricted to African crocodiles.The separation of dy Figs. , 4. It iwa Figs. , 4. The T. kaiowa n. sp. Therefore, blood trypomastigotes of this species still required morphological characterization. However, SSU rDNA and gGAPDH sequences (15 cloned sequences from each isolate) from the isolates of caiman blood (TCC1611) and tabanid gut (TCC2918) were identical, indicating that we have obtained pure cultures of T. kaiowa n. sp. from both the vertebrate host, and its putative vector.Flagellates were not detected in blood smears of any caiman assuredly infected exclusively with Ca. yacare   Fig. b, c infells Fig. c. The malls Fig. c. Flagellls Fig. c. TrypanT. kaiowa n. sp. co-cultivated with a monolayer of Hi-5 insect cells in TC100 medium at 25 °C. In early cultures (24 h), inoculated promastigotes adhere to the surface of the insect cells by their flagella forming clusters of flagellates, and the flagellates likely invade the cells via the flagellum , and fixed (Giemsa stained) cells. Growth behavior of T. kaiowa n. sp. in the supernatant of Hi-5 cultures was similar to cultures in LIT medium described below. Promastigotes multiply in the supernatant in general forming rosettes  cultures.In early log-phase (2–3 days) of LIT cultures at 25 °C, lla Fig. a. The fllla Fig. b, c. A flla Fig. d–f. Unexize Fig. e that grize Fig. h, i. A nT. kaiowa n. sp. flagellates co-cultivated with monolayers of Hi-5 cultures corroborated previous evidence from light microscopy of flagellates adhering to cell surface by their flagella, and apparently .Other hosts: Caiman crocodilus Linnaeus; Paleosuchus trigonatus Schneider; Osteolaemus tetraspis Cope.Type-locality: Miranda River  in the Pantanal, Mato Grosso do Sul, Brazil.Other localities: Tabajara Reserve, Rondonia, Brazilian Amazonia; Lake Chamo, Ethiopia; Lake Edward, Uganda; Lagoas de Cufada Natural Reserve, Guinea Bissau.Type-material: Hapantotype: the culture of the isolate TCC1611 from Caiman yacare. Paratypes: culture TCC2918 from the tabanid Phaeotabanus fervens, and caiman blood and tissue (liver) samples  and tabanid guts  infected with T. kaiowa n. sp. The cultures of T. kaiowa n. sp are cryopreserved at the Trypanosomatid Culture Collection of the University of São Paulo, TCC-USP, registered in the World Culture Collection, Japan. Blood/liver samples of crocodilians were preserved in ethanol at BSC (blood sample collection) and TSC (tissue sample collection), and insect samples were preserved in ethanol at ISC (Insect Sample Collection). DNA from T. kaiowa n. sp. cultures, caiman blood samples and tabanid guts are all conserved at TCC-USP together with Giemsa-stained smears of cultures and tabanid guts infected with T. kaiowa n. sp.Invertebrate host (putative vector): Phaeotabanus fervens Linnaeus . Additional vectors: Tabanus occidentalis Linnaeus, Ancala africana Griffith & Pidgeon, Tabanus taeniola Palisot de Beauvois, and Glossina pallidipes Austen.Site in host: Blood and liver of crocodilians; the digestive tract of tabanids and tsetse flies.Representative DNA sequences: DNA sequences were deposited in the GenBank database as follows: KF546503 and MG680219 (V7V8 SSU rRNA gene); KF546517 and MG680244 (gGAPDH gene).ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN) [e (ICZN) , detailsEtymology: “Kaiowá” refers to a formerly large Indian Nation occupying a vast area of Mato Grosso do Sul, Paraguay, and Argentina. The Kaiowá and Terena are closely related indigenous people, as closely related to each other are T. terena and T. kaiowa found in caimans.T. kaiowa n. sp. are shown in Figs. 8Tabanid flies exhibited clumps of small oval-shaped flagellates attached to the gut walls, and free-swimming slender flagellates lacking perceptible undulating membranes resembling promastigotes, epimastigotes with inconspicuous undulating membranes, and trypomastigote forms. Early hemocultures exhibited long, wide and pointed trypomastigotes that evolved to rosettes of slender promastigotes. Measurements of log-phase promastigotes (LIT cultures) were 8.3–13 (10.3 ± 1.2) µm in length; 1.5–2.5 (2.0 ± 0.32) µm in width, and 12–25 18.3 ± 3.6) µm of free flagellum. A few epimastigotes and very large and pointed flagellates were observed in old cultures. Light microscopy and ultrastructural features of all stages of  µm of frCa. yacare was placed in the clade Terena. However, this isolate was not considered a paratype of T. terena “because of relevant genetic divergences” (see [T. terena and the isolate TCC1611 (T. kaiowa n. sp.) are indeed distinct species. In addition, long and pointed trypomastigotes of T. kaiowa n. sp. differed from the roll-shaped trypomastigotes of T. terena and T. ralphi present in early hemocultures [T. kaiowa n. sp. from these two species, both exhibiting epimastigotes with noticeable undulating membranes [Trypanosoma sp. Cay03 and Trypanosoma sp. Tab01 as candidate new species. Unfortunately, they cannot yet be designated as new species because there are no valid holotypes for them: no cultures are available, and trypanosomes were not detected either in smears of caiman blood/tissue or tabanids single-infected with these trypanosomes.In our previous phylogenetic study, the isolate TCC1611 from es” (see ). In thecultures –12, and Trypanosoma, and drastically improved our understanding of their evolutionary histories, but are still far from counterbalancing the bias of studies towards pathogenic trypanosomatids in humans and livestock. In addition to many fieldwork difficulties and the permits required for capturing wild animals and collecting blood/tissue samples, additional challenges include the proper identification of vectors, the assessment of both host species and geographical ranges, investigation of life-cycles and developmental stages, and potential pathogenicity of any newly discovered species.The improvement of molecular approaches for the straightforward barcoding of Trypanosomatidae, including next-generation sequencing, has unveiling an increasing number of previously unidentified genera and species of trypanosomes in a wide range of vertebrate and invertebrate hosts , 56–62. T. kaiowa n. sp. Our examination of tabanid guts revealed masses of small flagellates attached to the gut epithelium indicating that T. kaiowa n. sp. heavily colonizes tabanid guts. Similarly, during its development in naturally and experimentally infected tsetse flies, T. grayi also exhibited small flagellates attached by their flagella to the gut wall [T. kaiowa n. sp. in tabanids also appears to be like that reported for Trypanosoma melophagium Flu, 1908 and Trypanosoma lewisi Kent, 1880 in the gut epithelium of their vectors hippoboscid flies and fleas (exhibiting intracellular forms), respectively [T. kaiowa n. sp. invades cells of tabanid guts similarly to what occurred herein within cultured Hi5 insect cells. All evidence indicates that tabanids are the vector of caiman trypanosomes, but confirmation still requires experimental infection of laboratory-raised caimans by tabanids, as has been done for T. grayi with tsetse flies and crocodiles [T. clandestinus with aquatic leeches and caimans [This study provides ecological, biological, behavioral and molecular evidence supporting tabanids as vectors of gut wall . The devocodiles , and T.  caimans .T. kaiowa n. sp. is the predominance of promastigote forms in tabanid guts and cultures, whereas typical epimastigotes are scarce in both cultures and tabanid flies. The predominance of promastigotes differentiates T. kaiowa n. sp. from T. terena and T. ralphi both exhibiting epimastigotes with noticeable undulating membranes [A remarkable peculiarity of embranes . In factembranes , and wheT. kaiowa n. sp. can be co-cultivated at 34 °C with LLCMK2 mammalian cells, proliferates in the supernatants of cultures and, apparently, flagellates could enter the cells (data not shown). However, further investigations are necessary to verify a possible intracellular development of T. kaiowa n. sp. The ability of crocodilian trypanosomes to survive in mammals is an interesting question recently raised by the detection of T. grayi DNA in blood of cattle from Cameroon (Africa), where tsetse flies showed large prevalence rates of both T. grayi and trypanosomes of ungulates [Our preliminary analyses demonstrated that ngulates . Moreovengulates , 62.T. kaiowa n. sp. and T. ralphi, plus the still unnamed Trypanosoma sp. Cay03, and Trypanosoma sp. Tab01. All trypanosomes from caimans clustered with T. grayi forming the crocodilian clade of the terrestrial lineage. The only exception was T. clandestinus that remains the sole trypanosome species of caimans nested in the aquatic lineage of Trypanosoma [Ca. yacare, Ca. crocodilus, M. niger and P. trigonatus, which harbour single or mixed trypanosome infections throughout South American hydrographical basins [P. fervens, Ta. occidentalis and Atylotus sp.) can harbour different trypanosomes of the crocodilian clade: T. ralphi, T. kaiowa n. sp., and the putative new species Trypanosoma sp. Cay03 and Trypanosoma sp. Tab01.Our molecular survey unveiled two species of crocodilian trypanosomes in tabanids, panosoma . So far,l basins –12. HereT. grayi, and exclusively transmitted by tsetse flies [Trypanosoma spp. in the guts of tsetse flies from Cameroon, T. grayi was a highly prevalent species [Findings of ‘South American’ trypanosomes in African tabanid and tsetse flies suggest an underestimated diversity of crocodilian trypanosomes on the African continent, before the present study thought to be restricted to se flies , 6. In a species .T. ralphi in Mozambique, and T. kaiowa n. sp. and Trypanosoma sp. Tab01 in Uganda. Heterogeneous sequences of T. grayi were obtained from tsetse flies from The Gambia, and crocodiles from Cameroon [T. clandestinus), while tabanid and tsetse flies transmit trypanosomes of the terrestrial lineage (crocodilian clade). The evolutionary history of trypanosomes relied on their phylogenetic relationships and eco-geographical and paleontological data of their vertebrate and invertebrate hosts. Although all data suggested an ancient and common origin for the trypanosomes of the crocodilian clade, the very close phylogenetic relationships among Neotropical and Afrotropical trypanosomes of crocodilians underline a recent diversification of all trypanosomes so far identified in this clade. There are no caimans in Africa and no African dwarf crocodiles in South America, but these animals share highly similar genotypes of the same species of trypanosomes. This current disjunct distribution of crocodilian trypanosome species is consistent with the transcontinental dispersal of crocodilians harboring trypanosomes [Here, tsetse flies were found infected with three trypanosomes: Cameroon , 64. Theanosomes , 26–28.T. kaiowa n. sp. in the present study relies on its phylogenetic positioning compared with other crocodilian trypanosomes. The description of T. kaiowa n. sp. is complemented by morphological and behavioural features of the flagellates in different growth conditions, and a predicted life-cycle in tabanid flies and caimans. The diversity and geographical dispersion of vertebrate and invertebrate hosts are also included in T. kaiowa n. sp. description. The close phylogenetic relationships of the crocodilian trypanosomes are in accordance with the transoceanic migration of crocodiles during the Miocene, and with the host-switching of trypanosomes between crocodiles and caimans. Our findings revealed that tabanid and tsetse flies are vectors of trypanosomes to crocodilians. Given that Glossinidae flies are absent from all continents other than Africa, adaptation to tsetse transmission would be secondary in the evolution of crocodilian trypanosomes, and tabanids, which are thought to have emerged during the Mesozoic (~140 mya), would be their ancestor vectors. However, this scenario relies on the contemporary geographical range of tsetse flies while records of Glossina-like fossils in the Nearctic and Palearctic at the Oligocene (30–40 mya) suggest that the ancestors of tsetse flies once flourished outside Africa, and possibly worldwide [To the best of our knowledge, data gathered in the present study provide the first evidence that tabanids can cyclically transmit trypanosomes to caimans and crocodiles in the Neotropic and Afrotropic. The description of orldwide . Data onAdditional file 1: Table S1. Host species, geographical origin and gGAPDH and V7V8 SSU rRNA gene sequences of trypanosomes from crocodilians, tabanids and tsetse flies.


AbstractSEM observations, of the fig cyst nematode Heteroderafici isolated from Ficuscarica roots, collected in home and public gardens of Apulia region, southern Italy, are described and illustrated. Seventy-five localities throughout the Apulia region were sampled and one-quarter of the sampled localities had fig roots infested with H.fici, with population densities ranging from 44 to 180 cysts/100 ml of soil. All attempts to detect H.fici on ornamental Ficus spp. as well as on imported bonsai in Italy were unsuccessful. Morphometric characters of the Italian population conform to those of the type and re-description populations reported for H.fici. Molecular analysis using ITS, D2–D3 expansion domains of the 28S rRNA, and the partial 18S rRNA sequences of H.fici newly obtained in this study matched well with the corresponding sequences of H.fici present in the GenBank database. Phylogenetic trees confirmed and supported the grouping of H.fici in the Humuli group. Heteroderafici completes its embryogenic development in 14–16 days at 25 °C. Post-invasion development and maturity in the roots of F.carica seedlings is completed in 64–68 days at 25–28 °C with juveniles and adults showing different parasitic habits, being endoparasitic and semi-endoparasitic respectively. The establishment of permanent feeding sites that consist of the formation of large syncytia causes anatomical modification of vascular elements and general disorder in the root stelar structures. Syncytia structures associated with mature females showed different degrees of vacuolisation, numbers of syncytial cells, and contained nuclei and nucleoli which were constantly hypertrophied.Morpho-biological notes and histopathology, based on LM and  Heteroderafici Kirjanova, 1954 was described from roots of rubber plants, Ficuselastica Roxb. ex Hornem from China by Ficuscarica Linneaus, 1753 was given by H.fici from F.carica seedlings in pots. However, the impact of the nematode on adult fig trees has never been ascertained. Therefore, H.fici is not considered a major pest and its narrow host range is limited to Ficus species only. H.fici was also found for the first time on F.carica maintained in a nursery in Canada   . Based oii group  or in thii group . SubbotiH.fici was rather widespread in southern Italy and in particular in the Apulia region. The main objectives of the present research were to: (i) obtain additional information on distribution, morphology, and molecular details of H.fici from F.carica, (ii) establish the phylogenetic relationships among Heterodera species closely related to H.fici; (iii) obtain additional biological information (embryogenic and post-invasion development); and (iv) to provide morpho-biological details on the host-parasite relationships of this nematode species in fig-nematode-feeding sites and describe the F.carica host responses to the nematode parasitism.Preliminary investigations indicated that H.fici on edible figs (Ficuscarica), an extensive survey, including more than 75 root and soil samples, was conducted in commercial orchards in several localities of the Apulia region and in private and public gardens of Bari city, southern Italy, in late May 2016. Twenty-five bonsai Ficus spp. from several import/export nurseries working with Asiatic Ficus plants, still in their original pots, were included in the present survey. The nematode population selected and used for the present study was collected at Bari University Campus .In the framework of the project “Urban Phytonematology” and on the basis of occasional records of For diagnosis and identification, cysts and eggs were collected directly from infested roots, whereas second-stage juveniles, mature cysts, and males were extracted from soil by the flotation method , and cysSEM) observations. Specimens fixed in formaldehyde (4% solution) were dehydrated in an ethanol gradient, critical-point dried, sputter-coated with gold, and observed according to procedures described by Terminal cone structures of mature cysts were prepared for scanning electron microscopy (J2) were collected. Genomic DNA was extracted from fifteen J2s as described by Individual cysts were crushed with a sterile micro-spatula under a stereo-microscope and the second stage juveniles . Purified DNA fragments were cloned and sequenced, in both directions, at MWG-Eurofin in Germany.H.fici from southern Italy were digested with the following restriction enzymes: Alu I (Roche), Hae III (Roche), Pst I (Roche) and Rsa I (Roche) (5 U of enzyme for each digestion) at 37 °C overnight. The digested DNA fragments were loaded onto 2.5% agarose gel and visualized by gel red staining gel. All gel images were stored digitally.Ten μl of the ITS containing amplicons of BLAST  search at NCBI  was performed in order to confirm nematode origins and species  method using MEGA version 6 software  model for ITS, 28S and 18 S datasets was carried out. Phylogenetic trees were bootstrapped 1000 times to assess the degree of support for the phylogenetic branching indicated by the optimal tree for each method. The newly obtained sequences were submitted to GenBank with the following accession numbers: LT996913 for the ITS region; LT996915 for the D2–D3 expansion domains; LT996914 for the 18S rRNA gene.A  species . The new species  with defhed data . Phylogesoftware . ML analH.fici was studied in Petri dishes, using single-celled eggs obtained (deposited) from newly formed cysts, washed in distilled water, placed in 2% water agar, and maintained in an incubator at 26 ± 2 °C. Microscopic observations and micrographs were taken at six-hour intervals during the first week and daily for the second week.The embryogenic development of F.carica) seedlings transplanted in pots containing 250 ml of pasteurised sand, and inoculated, five days later, by using 1250 juveniles per pot. Inoculated young plants were maintained in a glasshouse at 26–28 °C. Invasion and nematode development was studied by stereoscope observations in acid fuchsin stained roots at seven-day intervals  and transplanted on naturally infested soil with estimated initial population 1500 juveniles + eggs per pot and exposed to the nematode for a three-month period. Plants were removed from pots, the roots washed free of adhering soil, and the nematode populations were recorded.H.fici were studied in nematode-infected fig roots. Infected and healthy root segments were fixed for 48 hours in formalin-acetic acid-ethanol (FAA) solution, dehydrated in tertiary butyl alcohol, and embedded in 56–58 °C melting-point paraffin. Embedded tissues were sectioned transversely and longitudinally in 10–12 µm thick with a rotary microtome, stained with safranin and fast-green, and mounted in dammar xylene for microscopic examinations  as well as in private and public gardens. High infection rates were observed ranging from 44 to 180 cycts/100 ml of soil, 12–36 cysts per g of roots; 1.2–1.6 eggs – J2 /ml of soil, thus suggesting that the nematode might be causing damage. Furthermore, H.fici was detected in one-quarter of the 75 localities sampled throughout the Apulia region. All sampling attempts to detect H.fici from ornamental Ficus spp. as well as from imported bonsai in Italy were unsuccessful.The fig cyst nematode Measurements. See Table Nematode diagnosis. Detailed morphometric observations of the Italian population based on second-stage juveniles, male body length and characteristic of tail, stylet length, adult female and cyst shape, and vulval cone features , and by the prominent nipples on the male tail tip, which are regularly annulated and obtusely rounded in all other members of the group.Observing the morphology Figs –4, as weFemales. Body basically lemon-shaped. Neck elongate, protruding vulval cone prominent. Cyst cuticle with zigzag pattern. Vulval cone well developed. Egg sac present, but few eggs deposited. Cuticular striae, extending to vulval slit are present at fenestral area . Body vermiform; tapering at both extremities, more marked posteriorly. Cuticular annulation prominent. Lateral field with four incisures. Labial region slightly offset, rounded, with three or four annuli. Labial framework moderately sclerotised. Stylet well developed, basal knobs rounded, directed slightly anteriorly. Median bulb ovoid. Pharyngeal lobe usually distinct, with three large nuclei and overlapping anterior part of intestine. Tail long, tapering, terminus rounded. Anus distinct. Phasmid openings small but distinct, 11–14 μm, posterior to anus and anterior to middle of tail. Hyaline tail region half of tail length  protrudes from the root. Fourth-stage females and males were observed one week later .Host-suitability test. The results presented in this paper as well as in the host-suitability test confirmed that H.fici has a narrow host range limited to Ficus species and for this reason it is not considered a major pest, as are other species of Heterodera.Molecular characterization. The sequenced ITS, D2-D3 expansion domains of the 28S rRNA gene, and the 18S rRNA gene are 1035 bp, 780 bp, and 1627 bp long, respectively. BLAST search at NCBI revealed that the ITS, D2-D3 expansion domains of the 28S rRNA and the partial 18S rRNA sequences of H.fici from Italy, newly obtained in this study, matched well with the corresponding sequences of H.fici present in the database. The RFLP patterns of Italian H.fici were identical to those from Portugal, Greece, and another population from Potenza province, Basilicata, Italy (H.fici showed 99% similarity (1027/1029) with the corresponding region of H.fici deposited in GenBank, differing from 1 to 4 nucleotides; the next closest Heterodera species were H.humuli (coverage: 897/962), H.vallicola (coverage: 899/975), H.ripae (coverage: 894/972), and H.litoralis (coverage: 884/962) with 92–93% similarities. Heterodera species belonging to Schachtii group, instead, showed about 82% similarity with the Italian population of H.fici. The D2–D3 expansion domains of the Italian H.fici, obtained for the first time in this study, showed 95% similarity with the corresponding region of H.latipons Franklin, 1969 and H.avenae Wollenweber, 1924, while with H.glycines Ichinohe, 1952 and H.schachtii the similarity was 94%. The 18S rRNA gene of H.fici showed 99% similarity with the corresponding region of H.avenae, H.hordecalis Andersson, 1975, H.schachtii, and H.glycines, while its similarity with H.elachista Ohshima, 1974 was 98%.a, Italy  Fig. 5)MolecularML method are given in Figures Heterodera into different groups  by having ambifenestrate rather than bifenestrate cysts, longer vulval slit, the shape and measurements of J2 tails, and four prominent nipples on the male tail tip. Furthermore, SEM observations of cysts and males showed greater details of various structures compared to the light microscopy  Skarbilovich, 1959 as outgroups. Figure H.fici grouped with all populations of H.fici and clustered with high bootstrap support (99%) in the Humuli group. Close relationships were revealed among the Humuli group, the species H.salixophila Kirjanova, 1969 and the Schachtii group, as already reported, confirming their coevolution with dicots  Chitwood, 1949 and H.fici and, therefore, it was not possible to partition the observed damage between the two nematodes. However, during our survey H.fici was detected in rather large population densities both in soil and roots and appeared widespread (25% of the orchard infested). Therefore, although the impact of H.fici on fig yield still remains to be assessed, we believe that H.fici deserves attention to avoid its spread and subsequent yield loss.In conclusion, our data confirm the occurrence of


Lactococcus lactis strains revealed the presence of 83 plasmids, including two megaplasmids. The limitations of Pacific Biosciences SMRT sequencing in detecting the total plasmid complement of lactococcal strains is examined, while a combined Illumina/SMRT sequencing approach is proposed to combat these issues. Comparative genome analysis of these plasmid sequences combined with other publicly available plasmid sequence data allowed the definition of the lactococcal plasmidome, and facilitated an investigation into (bio) technologically important plasmid-encoded traits such as conjugation, bacteriocin production, exopolysaccharide (EPS) production, and (bacterio) phage resistance.Plasmids are autonomous, self-replicating, extrachromosomal genetic elements that are typically not essential for growth of their host. They may encode metabolic capabilities, which promote the maintenance of these genetic elements, and may allow adaption to specific ecological niches and consequently enhance survival. Genome sequencing of 16  Lactococcus lactis is globally applied as a starter culture for dairy-based food fermentations, such as those involved in the production of Cheddar, Colby, Gouda and blue cheeses, and from an economic and (food) biotechnological perspective represents one of the most important bacteria   lactococci and is important as it leads to the production of a number of volatile flavor compounds  , 2017, iundancy) , 2014c. . lactis  with dai. lactis . Plasmidsduction , but maysduction  Transduc. lactis . Conjuga. lactis  and is o. lactis . Extensilization , casein lization , citratelization , flavor lization , and phalization , all of acR gene . Citrateompounds . Citrate species . Proteol species . The pla species .L. lactis, and investigate plasmid-encoded  traits.Lactococcal phages are recognized as the main cause of fermentation problems within the dairy industry with concomitant economic problems. Lactococcal strains possess an arsenal of phage defense mechanisms, such as R-M systems and abortive infection (Abi) systems, many of which are plasmid-encoded. In the current study, we assess the genetic content of lactococcal plasmids, define the current pan-plasmidome of De novo assemblies were performed on the Pacific Biosciences SMRTPortal analysis platform (version 2.3.1), utilizing the RS_HGAP_Assembly.2 protocol. Assemblies were then repeated with a reduced minimum coverage threshold adjusted to 15X to ensure all plasmid-associated contigs had been detected.In total, 83 plasmids  were sequenced in the context of this study . SequencIn parallel with SMRT sequencing, an Illumina-based approach was applied to the sixteen lactococcal strains to identify strains where plasmids were potentially absent from the completed assemblies. Re-sequencing of genomes was performed on an Illumina MiSeq platform , to an average coverage of ∼100–125×. Sequences obtained were first quality checked using IlluQC.pl from the NGS QC Toolkit (v2.3)  and asse1 with a general minimum cut-off of >50 bp and confirmed using BLASTX v2.2.26 alignments   .Lactococcus lactis subsp. cremoris strains JM1 and JM2 were cultured in M17 broth (Oxoid) supplemented with 0.5% (w/v) lactose at 30°C without agitation overnight. PFGE plugs were then prepared and restricted with SI nuclease  as previously described (escribed .2O) for 120 min under light-limited conditions and destained in distilled water for 60 min. Gels were visualized by UV transillumination.A 1% (wt/vol) PFGE agarose gel was prepared in 0.5X TBE  buffer and the PFGE plugs were melted in and sealed with molten agarose in 0.5X TBE buffer. A CHEF-DR III pulsed-field system  was used to resolve the DNA fragments at 6 V/cm for 18 h in 0.5X TBE running buffer maintained at 14°C with linear increment (interpolation) of pulse time from 3 to 50 s. DNA ladder (Chef DNA lambda) was included in each gel . The gels were stained in ethidium bromide (10 mg/ml)  supplemented with 0.5% (w/v) lactose or glucose (strain-dependent) at 30°C without agitation overnight. 3 μl of overnight culture was spotted on M17 agar supplemented with 0.5% (w/v) glucose and left at 30°C overnight. Cells that had grown on the spotted areas were inactivated by exposure to UV light for 30 min. Plates were then overlaid with a semi-solid M17 agar (0.4% agarose) containing indicator strain Lactococcus lactis subsp. lactis IL1403: AE005176; L. lactis subsp. lactis IO-1: AP012281; L. lactis subsp. lactis 184: CP015895; L. lactis subsp. lactis 229: CP015896; L. lactis subsp. lactis 275: CP015897; L. lactis subsp. lactis UC06: CP015902; L. lactis subsp. lactis UC08: CP015903; L. lactis subsp. lactis UC11: CP015904; L. lactis subsp. lactis UC063: CP015905; L. lactis subsp. lactis UC77: CP015906; L. lactis subsp. lactis UL8: CP015908; L. lactis subsp. lactis C10: CP015898; L. lactis subsp. cremoris SK11: CP000425; L. lactis subsp. cremoris MG1363: AM406671; L. lactis subsp. cremoris NZ9000: CP002094; L. lactis subsp. cremoris A76: CP003132; L. lactis subsp. cremoris UC509.9: CP003157; L. lactis subsp. cremoris KW2: CP004884; L. lactis subsp. cremoris 158: CP015894; L. lactis subsp. cremoris UC109: CP015907; L. lactis subsp. cremoris JM1: CP015899; L. lactis subsp. cremoris JM2: CP015900; L. lactis subsp. cremoris JM3: CP015901; L. lactis subsp. cremoris JM4: CP015909; L. lactis subsp. cremoris 3107: CP031538; L. lactis subsp. cremoris IBB477: CM007353; L. lactis subsp. lactis A12: LT599049; L. lactis subsp. lactis biovar. diacetylactis FM03: CP020604; L. lactis subsp. lactis 14B4: CP028160; and L. lactis subsp. cremoris HP: JAUH00000000.1.In this study the sequences of 83 plasmids were elucidated utilizing a combined PacBio SMRT sequencing and Illumina MiSeq approach, and represent the detected plasmid complement of 16 lactococcal genomes . InitialL. lactis.The sequenced plasmid dataset was combined with a further one hundred and seven plasmids retrieved from the NCBI database  . In totaL. lactis biovar. diacetylactis FM03P), accounting for up to 355 Kbp of extra-chromosomal DNA in a given strain (as is the case for L. lactis JM1). The size of individual lactococcal plasmids varies widely from the smallest L. lactis KLDS4.0325 plasmid 2, with a size of 0.87 Kbp, to the two megaplasmids, each maintained by L. lactis JM1 and L. lactis JM2, with a size of 193 and 113 Kbp, respectively. The GC content of lactococcal plasmids ranges from ∼30–38%, whilst the average GC content of previously sequenced chromosomes is more constrained (34–36%). Only three lactococcal plasmids deviate from this range; pWC1 29.48, pIL105 29.79, and pHP003 40.05%, where the latter is closer to Streptococcus thermophilus genomic GC-content, which ranges from 39 to 40% . These dairy lactococci carry between one and twelve plasmids  members of the L. lactis taxon. To calculate the pan-plasmidome, a pan-genome analysis approach was applied using the PGAP v1.0 pipeline  was conducted . The plaThe second largest constituent of the lactococcal plasmidome is that represented by transposable elements. Transposable elements encompass 825 CDS, or 15.7% of the plasmidome, with members of the IS6, IS30, IS982, and ISL3 insertion families being among the most dominant genetic elements. These mobile elements are responsible for the transfer and recombination of DNA  and are rep gene, mob gene, and hypothetical gene), while pMPJM1 encodes 188 CDS and shares little homology with other lactococcal plasmids. The remaining thirteen clusters did not display subspecies specificity, each cluster containing plasmids from both subsp. lactis and subsp. cremoris hosts.Following MCL analysis, HCL of the pan-plasmidome was used to cluster plasmids based on their genetic content . The higL. lactis plasmids range in size from 1 to 50 Kbp, and, prior to this study, the largest plasmid identified in L. lactis was the self-conjugative mega-plasmid of 155,960 bp in L. lactis subsp. lactis bv. diacetylactis S50 , comprised of an AT-rich region plus three and a half iterons of 22 bp in length]  via the theta-type replication mechanism [based on the identification of the origin of replication  , which can act as an alternative lactose utilization pathway  plasmids pCV56B, pSK08, pKF147A, and pNCDO2118  . The plated food . Alterna pathway . Another pathway . PlasmidNCDO2118 . Due to L. lactis  locus is believed to be responsible for the donor-to-recipient DNA transfer process of conjugation, though the precise mechanistic details of the conjugation process in L. lactis has not yet been fully elucidated. Plasmids which do not encode the tra operon, may also be co-transferred by conjugation in instances where a plasmid contains an oriT sequence and at least one mobilization gene . Additional genes can also be involved in conjugation in L. lactis; an example of this is cluA, which encodes a cell surface-presented protein, and which is involved in cell aggregation and thought to be essential for high efficiency conjugal transfer  are part of a larger gene cluster  along with five occurrences of traE/F also being present .The  systems . Typicaltra gene cluster have yet to be elucidated, though additional tra-encoded functions have been predicted in a small number of cases, the majority based on homology to the trs operon in Staphylococcus  and traK (P-loop NTPase) on plasmids pUC08B, pIBB477a, pUC11B, pAF22, and pMRC01. Plasmids pAF22, pMRC01, and pNP40 have all previously been demonstrated to be capable of conjugation  plasmids in L. lactis (mob (mobilization) genes. Mobilization genes are responsible for nicking the plasmid’s dsDNA at a particular site and forming a relaxosome, which allows the transfer of a single stranded template to a recipient cell. Variants of four main mob genes are distributed throughout the lactococcal plasmidome; mobA and mobD encode nickases, and mobB and mobC, whose protein products are thought to form a relaxosome with an associated nickase (either mobA or mobD) are typically present in the genetic configuration mobABC or mobDC. Comparative analysis identified 422 occurrences of mob genes (any of the afore mentioned mob genes) distributed across the 190 plasmids assessed in this study, including 15 occurrences of a predicted retron-type reverse transcriptase or maturase (located between mobD and mobC) believed to play a role in DNA recombination. The results indicate that 59.5% of plasmids in the lactococcal plasmidome carry at least one or more genes encoding mobilization proteins.While the . lactis ; principmobC and mobD, encoding a nickase and an associated relaxase near a possible secondary replication origin. However, the presence of five transposase-encoding genes and the lack of predicted tra genes with conserved functions suggest that this plasmid is not capable of autonomous conjugation (though mobilization is possible).The lactococcal megaplasmids pMPJM1 and pMPJM2 harbor two (16 Kbp) regions putatively involved in conjugation and/or mobilization. In the case of pMPJM2 the predicted region was found to contain homologs of virB11, whose deduced product acts as a type IV secretory pathway ATPase (pfam00437). Cellular localization analysis of the operon using PsortB was also indicative of a transmembrane complex composed of cytoplasmic, membrane bound, signal and extracellular proteins , in addition to a homolog of proteins . The divL. lactis may have significant commercial impact for their role in functional foods. Analysis of the plasmids assessed in our study identified a number of strains with predicted novel muco-adhesive elements, similar to those found in pKP1  and one collagen-binding domain protein.Mucin-binding proteins, i.e., those allowing adhesion to the mucin layer of the gastrointestinal tract, are considered essential for stable and extended gut colonization by LAB . While l in pKP1 . Plasmid in pKP1 . While n in pKP1 , while pL. lactis subsp. cremoris IBB477 was found to contain two plasmids, pIBB477a and pIBB477b, which encode cell wall-associated peptidases that have been shown to mediate adhesion to bare mucin and fibronectin coated polystyrene and HT29-MTX cells  contained on pNZ4000 has previously been characterized , while the remainder of the gene cluster in each case consists of variable genes. These eps gene clusters consist of a highly conserved region at the proximal end of the cluster and a variable distal region, which is not unlike other lactococcal polysaccharide biosynthesis clusters  has previously been demonstrated to be essential for EPS subunit biosynthesis , glycosyltransferases (epsGHIJ) and a flippase (epsK), together representing the presumed enzymatic machinery responsible for EPS biosynthesis through the addition and export of sugar moieties.Exopolysaccharide production by ed trait . EPS proproducts . The L. cterized  and consd pMPJM1 . In pNZ4 subunit . Homologclusters . The conynthesis . The vareps region is composed of CDSs predicted to encode products with functions are similar to the chromosomally located cwps gene cluster in strain 229. Plasmid pJM3C contains genes predicted to encode a rhamnosyltransferase, UDP-glucose dehydrogenase, capsular biosynthesis protein and five glycosyltransferases. The p275B variable region is heavily rearranged due to the presence of nine transposase-encoding genes. The megaplasmid pMPJM1 encodes a 9 Kbp predicted EPS region with well conserved functional synteny to that of pNZ4000, although with relatively low homology  identified additional putative bacteriocin-encoding gene clusters , which wcas) genes specify an acquired adaptive immunity system against invading DNA in bacteria   (encoded by integrated prophages) , clusterbacteria . To datespacers) . AnalysihsdS genes were identified in the current analysis). The high frequency of these systems in lactococcal plasmids is indicative of host adaptation as they predominantly act as a host defense mechanism against phage infection.Restriction-modification systems are extremely diverse and widespread and are encoded by approximately 90% of all currently available bacterial and archaeal genomes . R-M sysL. lactis  was lost in a retentostat cultivation, while an RCR plasmid was maintained (parA and parB) on these plasmids should also be considered as it has been shown to contribute to the stability and maintenance of large plasmids without selection . As such, these features account for a large proportion of the plasmidome. However, as efforts to isolate new starter cultures for the dairy industry continue   of these genes is indicative of the self-transmissible and/or mobilizable nature of lactococcal plasmids.Megaplasmids have been found in LAB previously, in particular in members of the us genus . In the 155 Kbp) , and proL. lactis is not commonly associated with the human gut. In this study, the lactococcal plasmidome was shown to contain potential gut adhesion factors, which may allow colonization and/or persistence in the gastrointestinal tract. This trait may offer opportunities for the application of L. lactis as a vector for vaccine and biomolecule delivery (L. lactis were investigated including EPS production. Analysis of a large dataset of newly sequenced plasmids facilitated the identification and comparison of a number of novel EPS gene clusters. The major outcome of this work was the definition of “conserved” and “variable” regions within these EPS clusters. The conserved region encodes the transcriptional regulation, export and biosynthesis initiation machinery, while the variable region contains various genes that are predicted to encode glycosyltransferases, which are believed to be responsible for the production of a diverse set of EPS subunits, and thus a polysaccharide with a distinct composition and perhaps different technological properties.There has been limited research performed to date in the area of lactococcal gut adhesion as delivery . FurtherFinally, phage-resistance mechanisms were assessed with particular emphasis on Abi systems. Abi systems confer defense against phage infection and are commonly found in lactococcal strains where they are frequently plasmid-encoded . AnalysiDiscovery of the first lactococcal megaplasmids along with a host of novel features is evidence that the diversity of the lactococcal plasmidome represents a significant amount of unexploited genetic diversity, and suggests that continued future sequencing efforts and subsequent functional analysis will increase the observed diversity carried by these elements, potentially leading to new avenues of research, and applications. The current plasmidome contributes the equivalent of 22.26% of the CDSs contained in the pan-genome of the L. lactis chromosomes demonstrating its significant value to this taxon. The importance of which has been built on a long history of use in food fermentations, particularly in the dairy industry. The fact that both the opp and lac operons which have led to this adaptation remain largely plasmid encoded only further demonstrates the fundamental importance of the lactococcal plasmidome in terms of the evolution, adaptation, and application of lactococci.The datasets generated for this study can be found in NCBI Genbank, CP034577, CP034578, CP034579, CP034580, CP034581, CP034582, CP034583, CP034584, CP034585, and CP034586.PK carried out the data analysis with FB. PK performed the experiments. DS and JM provided materials and strains. PK, JM, and DS wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.