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To compare the transcriptional differences between the T2DM-susceptible and T2DM-tolerant Bama mini-pigs and to determine the utility of Bama mini-pigs as an animal model for T2DM by comparing the transcriptomes of the animals with T2DM patients. The skeletal muscle transcriptomes of healthy Guangxi Bama mini-pigs (CON), T2DM pigs (T2D), and non-T2DM pigs (NT2D) were determined by RNA sequencing. A total of 290 differentially expressed genes (DEGs) were detected in NT2D relative to the CON groups, 572 DEGs in T2D compared to CON, and 300 DEGs in T2D compared to NT2D. The RNA-seq data was verified by qPCR analysis of PGC1α, NR4A3, CSRP3, MYH7, MYH2, MYH1, GLUT4, PPARγ, LEP, ATP5H, UCP2, and MTOR transcripts. The DEGs in T2D Bama mini-pigs were mainly involved in signaling pathways associated with metabolism, cardiovascular diseases, infectious disease, cancer, inflammation and immune responses, cytoskeleton, and signal transduction. Comparative analysis of the transcriptomes of T2DM Bama mini-pigs and T2DM patients (GSE29221 dataset) revealed 17 differentially co-expressed genes that were assigned to 11 GO terms and 6 annotated pathways, including hypertrophic cardiomyopathy, dilated cardiomyopathy, thyroid cancer, cardiac muscle contraction, tight junction, and calcium signaling pathway, suggesting that the T2D Bama mini-pigs could be used on T2DM research in those pathways. This study represents the first RNAseq transcriptome analysis in T2D Bama mini-pigs and provided a new perspective for the study on the human T2DM pathogenesis by using the T2DM model of Bama mini-pigs.

BACKGROUND The results of the heat irrigating effect of radiofrequency ablation (RFA) are uncertain, and the accurate impact of the heat irrigating effect on regional liver tissue is unknown due to a lack of control experiments. AIM The aim of this study was to determine the influence of the heat irrigating effect of RFA on regional liver tissue in Bama miniature pigs. METHODS Eight Bama miniature pigs were randomly divided into the observation group (group A) and the control group (group B), with 4 pigs/group. An RFA electrode needle was implanted near the hepatic segment vasculature (3-5 mm from the hepatic segment portal vein) under ultrasound guidance in group A. Similarly, an RFA electrode needle was implanted far from the hepatic segment vasculature (8-10 mm from the hepatic segment portal vein) in group B. The left internal lobe and right medial lobe were chosen as RFA sites in each pig. RFA was performed at the left internal lobe on day one in each pig, and at the right medial lobe 7 d later. Each RFA lasted 12 min. The general status of the pigs and serious complications were observed during the perioperative period. The pigs were sacrificed and the livers were removed immediately after RFA on the eighth day. The samples were roughly observed. Hematoxylin-eosin and Ki67 staining, as well as TUNEL detection, were performed on the tissue sections. RESULTS All 8 animals successfully underwent ultrasound-guided RFA. No serious complications, such as massive hemorrhage, biliary fistula, severe pleural effusion, pneumothorax, peripheral organ failure, or renal failure occurred in any of the animals during the perioperative period. The RFA coagulative necrosis lesion was spherical and the surrounding liver tissue showed an inflammatory response. The difference in the Suzuki score of the liver tissue surrounding the ablated portal vein, and its distal area between groups A and B, was statistically significant (P < 0.05). More apoptotic cells were seen in liver tissue surrounding the ablated portal vein and its distal area in group A, while fewer apoptotic cells in the same area were seen in group B. The difference in the apoptotic index of the above area between group A and group B was statistically significant (P < 0.05). Cells staining positive for Ki67 were observed in liver tissue at the left internal lobe around the ablated portal vein and its distal area in group A. No Ki67 staining positive cells were observed in other tissue sections. The difference in the Ki67 staining positive index in the above area was statistically significant (P < 0.05) between group A and group B. CONCLUSION Changes as a result of thermal damage occur in liver tissue around the ablated portal vein and its distal area due to the heat irrigating effect when the RFA electrode tip is close to (< 5 mm) the portal vein.

Recent studies have shown that the glucagon receptor (GCGR) plays an important role in the development of type 2 diabetes mellitus. Both pigs and humans exhibit significantly similar behaviors in their glucose and lipid metabolism. In this study, the obtained Bama minipig GCGR coding sequence was 1437 bp encoding 479 amino acids (AA), which demonstrated higher sequence homology with humans than other species. It showed the highest expression profile in the liver, followed by the lung and kidney. In addition, the three-dimensional structure analysis showed that the porcine GCGR protein also had a classic sevenfold transmembrane region and a stalk region at the N-terminus for ligand binding. The stalk region of GCGR possessed five AA variations. The ligand binding pocket of GCGR has one AA variation in the key region, none of which affected the glucagon binding verified by the crystal structure mutagenesis in humans. There was no variation found in the region of membrane anchoring, hydrophobic bond, salt bridge, and hydrogen bond. However, the Gly40Ser mutation in mice resulted in major diseases, meaning that pigs are more suitable for the evaluation of GCGR-related drugs than mice.

BackgroundIn the past 20 years, the number of adults with diabetes has tripled. For most of the researches are often conducted in rodent T2DM models, and effective drugs developed have low clinical conversion efficiency. Therefore, it is urgent to establish a large animal model to explore the pathogenesis of T2DM and formulate disease prevention and control strategies. MethodsThis study was designed to establish and validate a T2DM model in minipigs with notable hyperglycemia using a high-fat diet and low-dose streptozotocin (STZ)，and examined the influence of STZ infusion time, the difference between a high-fat diet and a high-cholesterol and high-fat diet, and the atherosclerotic lesions accelerated by diabetes. Male Bama minipigs (n=24) were randomly divided into 5 groups; the control group was fed with normal diet for 9 months; STZ+HFD group and STZ+HCFD group were infused with 90 mg/kg STZ and then fed with a high-fat diet or high-cholesterol and high-fat diet for 9 months, respectively; HFD + STZ group and HCFD + STZ group were fed with a high-fat diet or high-cholesterol and high-fat diet, respectively, for nine months (after 3 months, pigs were injected with 90 mg/kg STZ intravenously). ResultsThe results showed the serum glucose concentrations were within the normal range in all groups except for HFD + STZ group and HCFD + STZ group. Animals fed with a high-fat diet for 9 months, did not develop apparent atherosclerotic lesions; nevertheless, atherosclerotic lesions were seen in animals fed with high-cholesterol and high-fat diets. Moreover, hyperglycemia accelerated atherosclerosis (lesions on the intimal surface of the abdominal aorta, 0.44±0.29 vs. 0.28±0.26) in minipigs. ConclusionsHigh-fat/high-cholesterol and high-fat diet combined with low-dose streptozotocin successfully established T2DM in minipigs. High-fat diet could not induce apparent atherosclerosis lesions but high-cholesterol and high-fat diet could during the nine months period. Hyperglycemia accelerated atherosclerosis in the minipigs.

The outer membrane (OM) of gram-negative bacteria functions as a selective permeability barrier. Escherichia coli periplasmic Zn-metallopeptidase BepA contributes to the maintenance of OM integrity through its involvement in the biogenesis and degradation of LptD, a β-barrel protein component of the lipopolysaccharide translocon. BepA either promotes the maturation of LptD when it is on the normal assembly pathway (on-pathway) or degrades it when its assembly is compromised (off-pathway). BepA performs these functions probably on the β-barrel assembly machinery (BAM) complex. However, how BepA recognizes and directs an immature LptD to different pathways remains unclear. Here, we explored the interactions among BepA, LptD, and the BAM complex. We found that the interaction of the BepA edge-strand located adjacent to the active site with LptD was crucial not only for proteolysis but also, unexpectedly, for assembly promotion of LptD. Site-directed crosslinking analyses indicated that the unstructured N-terminal half of the β-barrel-forming domain of an immature LptD contacts with the BepA edge-strand. Furthermore, the C-terminal region of the β-barrel-forming domain of the BepA-bound LptD intermediate interacted with a “seam” strand of BamA, suggesting that BepA recognized LptD assembling on the BAM complex. Our findings provide important insights into the functional mechanism of BepA.

Biocontainment is a safeguard strategy for preventing uncontrolled proliferation of genetically engineered microorganisms (GEMs) in the environment. Biocontained GEMs are designed to survive only in the presence of a specific molecule. The design of a pollutant-degrading and pollutant-dependent GEM prevents its proliferation after cleaning the environment. In this study, we present a biocontained toluene-degrading bacterium based on Acinetobacter sp. Tol 5. The bamA gene, which encodes an essential outer membrane protein, was deleted from the chromosome of Tol 5 but complemented with a plasmid carrying a bamA gene regulated by the Pu promoter and the regulatory protein XylR. The resultant strain (PuBamA) degraded toluene, similarly to the wild-type Tol 5. Although the cell growth of the PuBamA strain was remarkably inhibited after toluene depletion, escape mutants emerged at a frequency of 1 per 5.3 × 10−7 cells. Analyses of escape mutants revealed that insertion sequences (ISs) carrying promoters were inserted between the Pu promoter and the bamA gene on the complemented plasmid. MinION deep sequencing of the plasmids extracted from the escape mutants enabled the identification of three types of ISs involved in the emergence of escape mutants, suggesting a strategy for reducing it. IMPORTANCE GEMs are beneficial for various applications, including environmental protection. However, the risks of GEM release into the environment have been debated for a long time. If a pollutant is employed as a specific molecule for a biocontainment system, GEMs capable of degrading pollutants are available for environmental protection. Nevertheless, to our knowledge, biocontained degraders for real pollutants have not been reported in academic journals so far. This is possibly due to the difficulty in the expression of enzymes for degrading pollutants in a tractable bacterium such as Escherichia coli. On the other hand, bacteria with enzymes for degrading pollutants are often intractable as a host of GEMs due to the shortage of tools for genetic manipulation. This study reports the feasibility of a biocontainment strategy for a toluene degrader. Our results provide useful insights into the construction of a GEM biocontainment system for environmental protection.

Betaine is widely used as feed additives in animal husbandry as it can cause many benefits such as improving antioxidant ability, growth performance, and carcass traits. However, there are limited studies about the effects of betaine on the Bama mini-pigs. The present study was conducted to evaluate the effects of dietary betaine on carcass traits, meat quality, and nitrogen metabolism of pigs. Twenty-six pregnant Bama mini-pigs and then 104 weaned piglets were assigned for experimental treatments. The plasma and muscle samples were collected at 65-, 95-, and 125-d-old pigs, respectively. The results showed that betaine addition in the sow-offspring diets increased the lean meat rate in the 65-d-old pigs, whereas carcass weight, carcass yield, and loin-eye area were increased in the 95-d-old pigs, and carcass weight and backfat thickness in the 125-d-old pigs. Dietary betaine addition in the sow-offspring diets increased the contents of plasma Asp of 65-d-old, Met of 95- and 125-d-old, and Sar of 125-d-old pigs. Moreover, betaine addition increased the contents of Met, His, Ile, and Phe in Longissimus thoracis et lumborum, whereas those contents were decreased in biceps femoris and psoas major muscles at different stages. Betaine addition in the sow and piglets' diets regulated the muscle fiber-type and myogenic regulatory gene expressions. In summary, betaine addition in the sow and sow-offspring diets could improve the carcass traits and meat quality by altering the plasma biochemical parameters, amino acid composition, and gene expressions of skeletal muscle.

There is growing concern about the emergence and spread of multidrug-resistant Neisseria gonorrhoeae. To effectively control antibiotic-resistant bacterial pathogens, it is necessary to develop new antimicrobials and to understand the resistance mechanisms to existing antibiotics. In this study, we discovered the unexpected onset of drug resistance in N. gonorrhoeae caused by amino acid substitutions in the periplasmic chaperone SurA and the β-barrel assembly machinery component BamA. Here, we investigated the i19.05 clinical isolate with mutations in corresponding genes along with reduced susceptibility to penicillin, tetracycline, and azithromycin. The mutant strain NG05 (surAmut bamAmut, and penAmut) was obtained using the pan-susceptible n01.08 clinical isolate as a recipient in the transformation procedure. Comparative proteomic analysis of NG05 and n01.08 strains revealed significantly increased levels of other chaperones, Skp and FkpA, and some transport proteins. Efflux pump inhibition experiments demonstrated that the reduction in sensitivity was achieved due to the activity of efflux pumps. We hypothesize that the described mutations in the surA and bamA genes cause the qualitative and quantitative changes of periplasmic chaperones, which in turn alters the function of synthesized cell envelope proteins.

Background The outer membrane (OM) of Treponema pallidum (Tp) serves as the interface between the syphilis spirochete and its obligate human host and also is key to developing a vaccine with worldwide efficacy. We used structural modeling and bioinformatics to delineate the repertoire of OM proteins (OMPs) in the Tp Nichols strain. The Tp ‘OMPeome’ consists of two ‘stand-alone’ proteins (BamA and LptD) involved in OM biogenesis and four paralogous families involved in influx/efflux of small molecules: 8-stranded β-barrels, long-chain fatty acid transporters (FadLs), OM factors (OMFs) for efflux pumps, and Tp repeat protein (Tprs). Design/Methods Three-dimensional structural OMP models were generated with I-TASSER, Phyre2, and trRosetta. SAXS was used to solve solution structures for TprK and T. denticola MOSP, the Tpr family parental ortholog. Results Tp’s BamA (TP0326) is the central component of a hybrid BAM system in which POTRA1–5 interacts with the DUF domain of TamB (TP0325). Although Tp lacks lipopolysaccharide, its genome encodes a nearly complete LPS transporter apparatus. The LptD ortholog (TP0515) contains a large unstructured C-terminal domain, which models LptE-like inside the β-barrel. Tp encodes four 8-stranded β-barrels, each containing positively charged extracellular loops that could contribute to pathogenesis. Surprisingly, Tp encodes five FadL orthologs, all of which display features (i.e., hatch domain and NPA motifs) that are characteristic of Gram-negative FadLs. Three (TP0548, TP0859 and TP0865) have α-helical extensions that could interact with periplasmic components. The Tp genome encodes Mac and RND efflux pumps that presumably achieve combinatorial diversity via co-expression of four paralogous OMFs. Lastly, we confirmed the bipartite membrane topology of Tprs using SAXS to solve solution structures for MOSPN domains of TprK and MOSP. Conclusions The Tp Nichols OMPeome provides a structural framework to (i) elucidate Tp’s enigmatic parasitic strategies, (ii) identify targets of natural immunity, and (iii) interrogate candidate vaccinogens.

Dwarfism, also known as growth hormone deficiency (GHD), is a disease caused by genetic mutations that result in either a lack of growth hormone or insufficient secretion of growth hormone, resulting in a person's inability to grow normally. In the past, many studies focusing on GHD have made use of models of other diseases such as metabolic or infectious diseases. A viable GHD specific model system has not been used previously, thus limiting the interpretation of GHD results. The Bama minipig is unique to Guangxi province and has strong adaptability and disease resistance, and an incredibly short stature, which is especially important for the study of GHD. In addition, studies of GHR knockout Bama minipigs and GHR knockout Bama minipig fibroblast cells generated using CRISPR/Cas9 have not been previously reported. Therefore, the Bama minipig was selected as an animal model and as a tool for the study of GHD in this work. In this study, a Cas9 plasmid with sgRNA targeting the first exon of the GHR gene was transfected into Bama minipig kidney fibroblast cells to generate 22 GHR knockout Bama minipig kidney fibroblast cell lines (12 male monoclonal cells and 10 female monoclonal cells). After culture and identification, 11 of the 12 male clone cell lines showed double allele mutations, and the rate of positive alteration of GHR was 91.67%. Diallelic mutation of the target sequence occurred in 10 female clonal cell lines, with an effective positive mutation rate of 100%. Our experimental results not only showed that CRISPR/Cas9 could efficiently be used for gene editing in Bama minipig cells but also identified a highly efficient target site for the generation of a GHR knockout in other porcine models. Thus, the generation of GHR knockout male and female Bama fibroblast cells could lay a foundation for the birth of a future dwarfism model pig. We anticipate that the "mini" Bama minipig will be of improved use for biomedical and agricultural scientific research and for furthering our understanding of the genetic underpinnings of GHD.

Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed “all-in-one”-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.

The aim of this study was to identify differentially expressed long non-coding RNAs (lncRNAs) molecules and predict their target genes related to muscle development and lipid metabolism in longissimus dorsi (LD) muscles of Bama Xiang pigs under constant heat stress. Ten male Bama Xiang pigs with an average initial body weight of 14 kg were randomly divided into control group (22°C) and heat stress (35 °C) group. The experiment lasted for 28 days. All the pigs were slaughtered at the end of the experiment, and LD muscles were collected for muscle quality analysis and transcriptome sequencing. Heat stress reduced meat quality of Bama Xiang pigs. lncRNAs in LD were identified systematically by deep RNA sequencing between the two groups. The results showed that 365 lncRNAs from the LD were identified, including 128 intergenic lncRNAs, 82 intronic lncRNAs, and 155 anti-sense lncRNAs. The differences lie in transcript of length, number of exons and wider size distribution, and expression level per KB fragment in three subtypes of lncRNAs. The three types of transposable elements coverage, including Line/L1, SINE/tRNA, and LTR/ERVL-MaLR, are the highest in mRNA and the three subtypes of lncRNAs in pigs. lncRNAs and mRNAs were different in comparison of features. The results predicted the target genes of the significant differentially expressed lncRNAs related to muscle development and lipid metabolism. This is the first study to expand the knowledge about muscle-related lncRNAs biology in Bama Xiang pigs under heat stress and will contribute to the development of alleviating the adverse effects of heat stress on pork quality targeting lncRNAs.

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σE) transcriptional response. σE upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σE-upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

The biogenesis of outer membrane proteins requires the function of β-barrel assembly machinery (BAM), whose function is highly conserved while its composition is variable. The Escherichia coli BAM is composed of five subunits, while Thermus thermophilus seems to contain a single BAM protein, named TtOmp85. To search for the primitive form of a functional BAM, we investigated and compared the function of TtOmp85 and E. coli BAM by use of a reconstitution assay that examines the integration of OmpA and BamA from E. coli and TtoA from T. thermophilus, as well as the translocation of the E. coli Ag43. Our results show that a single TtOmp85 protein can substitute for the collective function of the five subunits constituting E. coli BAM.

The BAM complex in Escherichia coli is composed of five proteins, BamA-E. BamA and BamD are essential for cell viability and are required for the assembly of β-barrel outer membrane proteins. Consequently, BamA and BamD are indispensable for secretion via the classical autotransporter pathway (Type 5a secretion). In contrast, BamB, BamC and BamE are not required for the biogenesis of classical autotransporters. Recently, we demonstrated that TamA, a homologue of BamA, and its partner protein TamB, were required for efficient secretion of proteins via the classical autotransporter pathway. The trimeric autotransporters are a subset of the Type 5-secreted proteins. Unlike the classical autotransporters, they are composed of three identical polypeptide chains which must be assembled together to allow secretion of their cognate passenger domains. In contrast to the classical autotransporters, the role of the Bam and Tam complex components in the biogenesis of the trimeric autotransporters has not been investigated fully. Here, using the Salmonella enterica trimeric autotransporter SadA and the structurally similar YadA protein of Yersinia spp., we identify the importance of BamA and BamD in the biogenesis of the trimeric autotransporters and reveal that BamB, BamC, BamE, TamA and TamB are not required for secretion of functional passenger domain on the cell surface.ImportanceThe secretion of trimeric autotransporters (TAA’s) has yet to be fully understood. Here we show that efficient secretion of TAAs requires the BamA and D proteins, but does not require BamB, C or E. In contrast to classical autotransporter secretion, neither trimeric autotransporter tested required TamA or B proteins to be functionally secreted.

The outer membrane (OM) of Gram-negative bacteria is a robust protective barrier that excludes major classes of antibiotics. The assembly, integrity and functioning of the OM is dependent on β-barrel outer membrane proteins (OMPs), the insertion of which is catalyzed by BamA, the core component of the β-barrel assembly machine (BAM) complex. Little is known about BamA in the context of its native OM environment. Here, using high-affinity fluorescently-labelled antibodies in combination with diffraction-limited and super-resolution fluorescence microscopy, we uncover the spatial and temporal organization of BamA in live Escherichia coli K-12 cells. BamA is clustered into ~150 nm diameter islands that contain an average of 10-11 BamA molecules, in addition to other OMPs, and are distributed throughout the OM and which migrate to the poles during growth. In stationary phase cells, BamA is largely confined to the poles. Emergence from stationary phase coincides with new BamA-containing islands appearing on the longitudinal axis of cells, suggesting they are not seeded by pre-existing BamAs but initiate spontaneously. Consistent with this interpretation, BamA-catalyzed OMP biogenesis is biased towards non-polar regions. Cells ensure the capacity for OMP biogenesis is uniformly distributed during exponential growth, even if the growth rate changes, by maintaining an invariant density of BamA-containing OMP islands (~9 islands/μm2) that only diminishes as cells enter stationary phase, the latter change governing what OMPs predominate as cells become quiescent. We conclude that OMP distribution in E. coli is driven by the spatiotemporal organisation of BamA which varies with the different phases of growth.SignificanceThe integrity and functioning of the outer membrane (OM) of Gram-negative bacteria depends on the β-barrel assembly machinery (BAM). Although the structure and the mechanism of the complex have been widely explored, little information exists about the organization of the BAM complex and how it dictates protein distribution in the OM. Here, we utilized highly specific monoclonal antibodies to study the spatiotemporal organization of BamA, the key component of this complex. We reveal that BAM organization is dynamic and tightly linked to the cell’s growth phase. We further discover that the rate of BAM facilitated OMP biogenesis is significantly reduced near the poles. In turn, these features govern the biogenesis patterns and the distribution of OMPs on the cell surface.

The heteropentomeric β-barrel assembly machine (BAM complex) is responsible for folding and inserting a diverse array of β-barrel outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria. The BAM complex contains two essential proteins, the β-barrel OMP BamA and a lipoprotein BamD, whereas the auxiliary lipoproteins BamBCE are individually nonessential. Here, we identify and characterize three bamA mutations, the E-to-K change at position 470 (bamAE470K), the A-to-P change at position 496 (bamAA496P), and the A-to-S change at position 499 (bamAA499S), that suppress the otherwise lethal ΔbamD, ΔbamB ΔbamC ΔbamE, and ΔbamC ΔbamD ΔbamE mutations. The viability of cells lacking different combinations of BAM complex lipoproteins provides the opportunity to examine the role of the individual proteins in OMP assembly. Results show that, in wild-type cells, BamBCE share a redundant function; at least one of these lipoproteins must be present to allow BamD to coordinate productively with BamA. Besides BamA regulation, BamD shares an additional essential function that is redundant with a second function of BamB. Remarkably, bamAE470K suppresses both, allowing the construction of a BAM complex composed solely of BamAE470K that is able to assemble OMPs in the absence of BamBCDE. This work demonstrates that the BAM complex lipoproteins do not participate in the catalytic folding of OMP substrates but rather function to increase the efficiency of the assembly process by coordinating and regulating the assembly of diverse OMP substrates. IMPORTANCE The folding and insertion of β-barrel outer membrane proteins (OMPs) are conserved processes in mitochondria, chloroplasts, and Gram-negative bacteria. In Gram-negative bacteria, OMPs are assembled into the outer membrane (OM) by the heteropentomeric β-barrel assembly machine (BAM complex). In this study, we probe the function of the individual BAM proteins and how they coordinate assembly of a diverse family of OMPs. Furthermore, we identify a gain-of-function bamA mutant capable of assembling OMPs independently of all four other BAM proteins. This work advances our understanding of OMP assembly and sheds light on how this process is distinct in Gram-negative bacteria.

Pigs are the most promising models for the study of wound healing and hypertrophic scarring because they are anatomically and physiologically similar to human beings. The Red Duroc pig and Mini Bama pig are two swine models that have attracted a lot of attention. The aim of the present study was to examine and compare the scarring process in a red Duroc pig and a Mini Bama pig, providing knowledge for researchers and clinicians to enable them to choose the most suitable pig model for studies., Lay Summary Pigs, with an anatomical and physiological skin architecture similar to that of the human, have been studied extensively as models for wound healing and scarring. However, the red Duroc pig, as a frequently used animal model for human wound/scar-related research, has the shortcoming of rapid growth in size and weight, which gives rise to difficulty in its handling and brings about enormous cost. The Mini Bama pig grows much slower and can be a potential replacement. This study compares wound healing and subsequent scarring between the Duroc and Mini Bama pigs with a time span of 35 weeks. Other than clinical appearance at week 35, when the Duroc scars showed an obvious hyperpigmentation while the Mini Bama scars showed slight hypopigmentation, the trends of change in shape, histological appearance (epidermis thickness) and collagen deposition in the tissues are similar in both types of pigs.

The demand for novel antibiotics is imperative for drug-resistant Gram-negative bacteria which causes diverse intractable infection disease in clinic. Here, a comprehensive screening was implemented to identify potential agents that disrupt the assembly of β-barrel outer-membrane proteins (OMPs) in the outer membrane (OM) of Gram-negative bacteria. The assembly of OMPs requires ubiquitous β-barrel assembly machinery (BAM). Among the five protein subunits in BAM, the interaction between BamA and BamD is essential for the function of this complex. We first established a yeast two-hybrid (Y2H) system to confirm the interaction between BamA and BamD, and then screened agents that specifically disrupt this interaction. From this screen, we identified a compound IMB-H4 that specially blocks BamA–BamD interaction and selectively inhibits the growth of Escherichia coli and other Gram-negative bacteria. Moreover, our results suggest that IMB-H4 disrupts BamA–BamD interaction by binding to BamA. Strikingly, E. coli cells having been treated with IMB-H4 showed impaired OM integrity and decreased the abundance of OMPs. Therefore, an antibacterial agent was identified successfully using Y2H system, and this compound likely blocks the assembly of OMPs by targeting BamA–BamD interaction in Gram-negative bacteria.

Background: Since 2014, seasonal malaria chemoprevention (SMC) with amodiaquine-sulfadoxine-pyrimethamine (AQ-SP) has been implemented on a large scale during the high malaria transmission season in Burkina Faso. We report in this paper the prevalence of microscopic and submicroscopic malaria infection at the outset and after the first round of SMC in children under five years old in Bama, Burkina Faso, as well as host and parasite factors involved in mediating the efficacy and tolerability of SMC. Methods: Two sequential cross-sectional surveys were carried out in the first month of SMC in a rural area in southwest Burkina Faso. Blood smears and dried blood spots were collected from 106 and 93 children under five, respectively, at the start of SMC and again three weeks later. Malaria infection was detected by microscopy and by PCR from dried blood spots. For all children, day 7 plasma concentrations of desethyl-amodiaquine (DEAQ) were measured and CYP2C8 genetic variants influencing AQ metabolism were genotyped. Samples were additionally genotyped for pfcrt K76T and pfmdr1 N86Y, molecular markers associated with reduced amodiaquine susceptibility. Results: 2.8% (3/106) of children were positive for Plasmodium falciparum infection by microscopy and 13.2% (14/106) by nested PCR within 2 days of SMC administration. Three weeks after SMC administration, in the same households, 4.3% (4/93) of samples were positive by microscopy and 14.0% (13/93) by PCR (p=0.0007). CYP2C8*2, associated with impaired amodiaquine metabolism, was common with an allelic frequency of 17.1% (95%CI=10.0-24.2). Day 7 concentration of DEAQ ranged from 0.48 to 362.80 ng/mL with a median concentration of 56.34 ng/mL. Pfmdr1 N86 predominated at both time points, whilst a non-significant trend towards a higher prevalence of pfcrt 76T was seen at week 3. Conclusion: This study showed a moderate prevalence of low-level malaria parasitemia in children 3 weeks following SMC during the first month of administration. Day 7 concentrations of the active DEAQ metabolite varied widely, likely reflecting variability in adherence and possibly metabolism. Our findings highlight factors that may contribute to the effectiveness of SMC in children in a high transmission setting.

Insulin-like growth factor 2 (IGF2) is an important growth factor, which promotes growth and development in mammals during fetal and postnatal stages. Using CRISPR-Cas9 system, we generated multiple founder pigs containing 12 different mutant alleles around a regulatory element within the intron 3 of IGF2 gene. Crossing two male founders passed four mutant alleles onto F1 generation, and these mutations abolished repressor ZBED6 binding and rendered this regulatory element nonfunctional. Both founders and F1 animals showed significantly faster growth, without affecting meat quality. These results indicated that editing IGF2 intron 3-3072 site using CRISPR-Cas9 technology improved meat production in Bama pigs. This is the first demonstration that editing non-coding region can improve economic traits in livestock.

The β-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Given its role in the production of OMPs for survival and pathogenesis, BAM represents an attractive target for the development of therapeutic interventions, including drugs and vaccines against multidrug-resistant bacteria such as Neisseria gonorrhoeae. The first structure of BamA, the central component of BAM, was from N. gonorrhoeae, the etiological agent of the sexually transmitted disease gonorrhea. To aid in pharmaceutical targeting of BAM, we expanded our studies to BamD and BamE within BAM of this clinically relevant human pathogen. We found that the presence of BamD, but not BamE, is essential for gonococcal viability. However, BamE, but not BamD, was cell-surface–displayed under native conditions; however, in the absence of BamE, BamD indeed becomes surface-exposed. Loss of BamE altered cell envelope composition, leading to slower growth and an increase in both antibiotic susceptibility and formation of membrane vesicles containing greater amounts of vaccine antigens. Both BamD and BamE are expressed in diverse gonococcal isolates, under host-relevant conditions, and throughout different phases of growth. The solved structures of Neisseria BamD and BamE share overall folds with Escherichia coli proteins but contain differences that may be important for function. Together, these studies highlight that, although BAM is conserved across Gram-negative bacteria, structural and functional differences do exist across species, which may be leveraged in the development of species-specific therapeutics in the effort to combat multidrug resistance.

Even though salbutamol (SAL) had remarkable effects on the enhancement of growth rate and carcass composition in different livestock species such as cattle, pigs, sheep and poultry, it was banned as a growth promoter because of its adverse effects on health. However, the specific mechanism by which salbutamol enhances growth efficiency remains unknown. In this study, Bama pigs were randomly allocated to receive salbutamol (5 mg/kg) for 30 or 60 days and were compared with untreated pigs. Pigs treated with salbutamol demonstrated enhanced growth rates and carcass composition; however, they showed deterioration in blood biochemical indices and organ development. We hypothesized that salbutamol exerts its effects by modulating the composition of the gut microbiota population. The faecal microbiome of pigs was characterized via pyrosequencing of the bacterial 16S rRNA gene. The gut microbiota population analysis showed that salbutamol caused shifts in the microbial composition of less abundant species. Redundancy analysis indicated an increase in abundance of the phylum Bacteroidetes, class Betaproteobacteria, family Christensenellaceae and genus Lactobacillus, and a decreased ratio of the phylum Firmicutes, class Clostridia and genera Ruminococcus, Blautia and Subdoligranulum. In conclusion, our study provided circumstantial evidence that the various effects of salbutamol are caused by gut microbiota modulation, and several potential candidates were identified for SAL detection via the gut microbiota. Our findings provided new insights into the roles of the gut microbiota during salbutamol treatment, and these findings will aid in the screening of alternative strategies for animal health improvement and production enhancement.

Significance The outer membrane of Gram-negative bacteria presents a formidable barrier to the discovery of new antibiotics needed to combat infections by multidrug-resistant bacteria. Targeting essential proteins or processes directly exposed to the environment could bypass this obstacle. Here, we describe a monoclonal antibody that selectively and potently antagonizes BamA, which folds and inserts integral outer membrane β-barrel proteins, by binding to a surface-exposed BamA epitope and, as a result, inhibits bacterial cell growth. Mechanisms of resistance to the antibody reveal that membrane fluidity affects BamA activity. This antibody validates the potential therapeutic strategy of targeting essential, exposed functions and provides a powerful tool for dissecting the fundamental process of folding integral membrane β-barrel proteins in vivo., The folding and insertion of integral β-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect β-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli β-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its β-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying β-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.

The β-barrel assembly machine (Bam) complex in Gram-negative bacteria and its counterparts in mitochondria and chloroplasts fold and insert outer membrane β-barrel proteins. BamA, an essential component of the complex, is itself a β-barrel and is proposed to play a central role in assembling other barrel substrates. Here, we map the path of substrate insertion by the Bam complex using site-specific crosslinking to understand the molecular mechanisms that control β-barrel folding and release. We find that the C-terminal strand of the substrate is stably held by BamA and that the N-terminal strands of the substrate are assembled inside the BamA β-barrel. Importantly, we identify contacts between the assembling β-sheet and the BamA interior surface that determine the rate of substrate folding. Our results support a model in which the interior wall of BamA acts as a chaperone to catalyze β-barrel assembly.

Gram-negative bacteria shield themselves from antibiotics by producing an outer membrane (OM) that forms a formidable permeability barrier. Multidrug resistance among these organisms is a particularly acute problem that is exacerbated by the OM. The poor penetrance of many available antibiotics prevents their clinical use, and efforts to discover novel classes of antibiotics against Gram-negative bacteria have been unsuccessful for almost 50 years. Recent insights into how the OM is built offer new hope. Several essential multiprotein molecular machines (Bam, Lpt, and Lol) work in concert to assemble the barrier and offer a swathe of new targets for novel therapeutic development. Murepavadin has been at the vanguard of these efforts, but its recently reported phase III clinical trial toxicity has tempered the anticipation of imminent new clinical options. Nonetheless, the many concerted efforts aimed at breaking down the OM barrier provide a source of ongoing optimism for what may soon come through the development pipeline. We will review the current state of drug development against the OM assembly targets, highlighting insightful new discovery approaches and strategies.

The transcript variants of Insulin-like growth factor 1 receptor (IGF-1R) and their expression profiles had never been illuminated in pigs until now. Herein, we identified IGF-1R AS02 as a novel splice variant of IGF-1R gene by RT-PCR and analyzed its mRNA expression level by qRT-PCR in liver, cartilage and muscle tissues, while also detecting the single-nucleotide polymorphism (SNP) site near the splice site of the IGF-1R gene (in intron 19) of BaMa and Landrace pigs. Results demonstrated that the IGF-1R AS02 variant showed a significantly (P less than 0.05) higher expression level in cartilage than in muscle and liver across two pig breeds respectively. The expression level of the normal transcript (IGF-1R ISO01) of IGF-1R in cartilage was markedly lower than that in the other two tissues (P less than 0.05). In cartilage, IGF-1R ISO01 expression was higher in BaMa than in Landrace (P less than 0.05), while the expression level of IGF-1R AS02 was lower in BaMa than in Landrace (P less than 0.05). The SNP was detected in intron 19 of the IGF-1R gene of BaMa and Landrace pigs. These results contributed to facilitating a better understanding of IGF-1R gene in pigs.

Background The failure of current prevention strategies to curtail the spread of syphilis, including mother-to-child transmission, underscores the need for a vaccine with worldwide efficacy. Characterization of variation within T. pallidum (TPA) outer membrane proteins (OMPs) is critical for this goal. Methods ClustalW identified polymorphisms within 21 β-barrel forming OMPs (TPA‘s OMPeome) of 15 unique TPA strains. Structural models and conformational B-cell epitopes (BCEs) were predicted. Clade assignments were made using tp0548 and tp0558 sequences. Results TPA OMPeomes contain four paralogous families (Tpr, FadL, OmpW, and efflux pump OMPs) and orthologs for BamA and LptD. The Tprs group into Subfamilies I (C/D/I), II (E/G/J) and III (B/H/L); Tpr β-barrel domains contain 10 β-strands and five extracellular loops (ECLs) harboring BCEs. TprI is invariant, while TprC and TprD have variability located predominantly in ECLs. The closely related TprE and TprJ β-barrels exhibit extremely low variability. TprG β-barrels variants contain a 23 aa insertion derived from TprE or TprJ. The TprB and TprH β-barrels are fully conserved; there are two TprL β-barrels variants. The FadL orthologs (TP0548, TP0856, TP0858, TP0859, TP0865) consist of 14-stranded β-barrels with seven ECLs harboring predicted BCEs. Variability among FadLs range between fully conserved (TP0856) to highly variable (TP0548). Both OmpWs are fully conserved and consist of 8-stranded β-barrels with four ECLs containing BCEs. The highly conserved efflux pump OMPs (OprJ/TP0966, OprN/TP0967, TolC/TP0969) likely form homotrimeric 12-stranded β-barrels. The 18-stranded BamA/TP0326 β-barrel contains a single aa substitution in ECL4. The LptD/TP0515 β-barrel contains 24 β-strands, 12 ECLs and numerous BCEs, which cluster primarily into two variants. Most OMPs do not align with clade designations. Conclusion 1.) TPA OMPs display variable degrees of sequence and surface antigenic variation. 2.) TPA OMPeomes are mosaics likely resulting from recombination among circulating strains. 3.) Our analysis enables selection of variable and non-variable OMPs for assessment of protective capacity. Disclosure No significant relationships.

DegP, a periplasmic dual-functional protease and chaperone in Gram-negative bacteria, is critical for bacterial stress resistance, but the precise underlying mechanisms are not fully understood. Here, we show that the protease function of DegP is critical for Escherichia coli cells to maintain membrane integrity, particularly under heat shock conditions (42°C). Site-directed photo-cross-linking, mass spectrometry and immunoblotting analyses reveal that both periplasmic proteins (e.g. OppA and MalE) and β-barrel outer membrane proteins (OMPs) are DegP-interacting proteins and that OppA is degraded by DegP in vitro and in vivo at 42°C. In addition, OmpA and BamA, chimeric β-barrel OMPs containing a soluble periplasmic domain, are bound to DegP in both unfolded and folded forms, whereas only the unfolded forms are degradable by DegP. The presence of folded OmpA as a substrate of DegP is attributed to its periplasmic domain, which is resistant to DegP degradation and even generally protects pure β-barrel OMPs from degradation in an intra-molecular way. Furthermore, a pair of residues (R262 and V328) in the PDZ domain-1 of DegP play important roles for binding unfolded and folded β-barrel OMPs, with R262 being critical. Our study, together with earlier reports, indicates that DegP plays a critical role in protein quality control in the bacterial periplasm by degrading both periplasmic proteins and β-barrel OMPs under stress conditions and likely also by participating in the folding of chimeric β-barrel OMPs. A working model is proposed to illustrate the finely tuned functions of DegP with respect to different substrate proteins.

Endothelial dysfunction has been considered as pivotal in the pathogenesis of decompression sickness (DCS) and contributes substantively to subsequent inflammatory responses. Escin is well known for its endothelial protection and anti-inflammatory properties, and its protection against DCS has been proved in a rat model. This study aimed to further investigate the protection of escin against DCS in swine. Sixteen swine were subjected to a two-stage experiment with an interval of 7 days. In each stage, 7 days before a simulated air dive, the swine were treated with escin or saline. The first group received a successive administration of escin for 7 days prior to the first dive and saline for 7 days prior to the second; the second group was treated with saline and then escin. After decompression, signs of DCS and circulating bubbles were monitored, and blood was sampled for platelet count and determination of inflammatory and endothelial related indices. The death rate of DCS was markedly decreased in swine treated with escin compared with that in animals treated with saline, though not statistically significant due to the limited number of animals. Escin had no effect on bubble load but significantly ameliorated platelet reduction and endothelial dysfunction, as well as oxidative and inflammatory responses. The results further suggest the beneficial effects of escin on DCS by its endothelia-protective properties, and escin has the potential to be a candidate drug for DCS prevention and treatment.

The beta-barrel assembly machinery (BAM) is an indispensable complex for protein transportation located at the outer membrane of bacteria. BAM is composed of five subunits (BamA-E) in the model bacterium Escherichia coli. DR_0379 is a BamA homolog in Deinococcus radiodurans, but the other subunits have not been detected in this species. In the present study, deletion of bamA resulted in decreased growth rate and altered morphology of D. radiodurans. ΔbamA cells underwent abnormal cell division, leading to aggregated bacteria of diverse size and shape, and the cell envelope was detached from the cell surface, resulting in reduced resistance to high ionic strength. Oxidative stress resistance was significantly enhanced in the mutant, which may be attributed to increased manganese ion concentration and Mn/Fe ratio. Numerous proteins were released into the medium from ΔbamA cells, including surface layer (S-layer) proteins and various transporters located in the periplasm and outer membrane. These results indicate that BamA affects the synthesis and assembly of the outer membrane and S-layer, and thereby influences material transport and cell division. The findings highlight the special functions of BamA in D. radiodurans, and promote our understanding of the multi-layer structure of the D. radiodurans cell envelope.

Made available in DSpace on 2022-04-29T08:30:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-01 Acinetobacter baumannii is an important nosocomial pathogen. BamA is a protein that belongs to a complex responsible for organizing the proteins on the bacterial outer membrane. In this work, we aimed to evaluate murine immune responses to BamA recombinant protein (rAbBamA) from A. baumannii in an animal model of infection, and to assess cross-reactivity of this target for the development of anti-A. baumannii vaccines or diagnostics. Immunization of mice with rAbBamA elicited high antibody titers and antibody recognition of native A. baumannii BamA. Immunofluorescence also detected binding to the bacterial surface. After challenge, immunized mice demonstrated a 40% survival increase and better bacterial clearance in kidneys. Immunoblot of anti-rAbBamA against other medically relevant bacteria showed binding to proteins of approximately 35 kDa in Klebsiella pneumoniae and Escherichia coli lysates, primarily identified as OmpA and OmpC, respectively. Altogether, our data show that anti-rAbBamA antibodies provide a protective response against A. baumannii infection in mice. However, the response elicited by immunization with rAbBamA is not completely specific to A. baumannii. Although a broad-spectrum vaccine that protects against various pathogens is an appealing strategy, antibody reactivity against the human microbiota is undesired. In fact, immunization with rAbBamA produced noticeable effects on the gut microbiota. However, the changes elicited were small and non-specific, given that no significant changes in the abundance of Proteobacteria were observed. Overall, rAbBamA is a promising target, but specificity must be considered in the development of immunological tools against A. baumannii. Bio-Manguinhos Oswaldo Cruz Foundation Brazilian Ministry of Health Oswaldo Cruz Institute Oswaldo Cruz Foundation Brazilian Ministry of Health National Institute of Science and Technology of Innovation on Diseases of Neglected Populations Center for Technological Development in Health Oswaldo Cruz Foundation São Paulo State University Institute of Biosciences Coastal Campus of São Vicente São Paulo State University Institute of Biosciences Coastal Campus of São Vicente

β-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent β-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving β-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for β-barrel OMP biogenesis.

Zinc (Zn) can accumulate in the body of wild animal and human through bio-magnification effects in the food chain to pose chronic toxicity. Male spermatogenesis was sensitive to excessive Zn and elevated temperature. This study aimed to examine whether or not excessive Zn intake caused testicular toxicity and estimate the interaction between Zn and high temperature (HT) in testes of Bama miniature pigs. Six-month-old pigs were pre-fed with or without additional Zn at 1500 mg/kg diet for 30 d and afterward subjected to HT at 40 °C for 5 h daily for 8 consecutive days. Blood samples were collected on d 31 and d 38 and testes were obtained on d 38 immediately after HT exposure. Our data showed both scrotal surface temperature (T) and body surface T increased after 5-h HT exposure (p 0.05). Pigs fed with additional Zn showed germ cell loss, the decreased testes weight (p 0.01) and the elevated testicular H

Bacteria produce a diverse array of antagonistic compounds to restrict growth of microbial rivals. Contributing to this warfare are bacteriocins: secreted antibacterial peptides, proteins and multi-protein complexes. These compounds typically eliminate competitors closely related to the producer. Lectin-like bacteriocins (LlpAs) constitute a distinct class of such proteins, produced by Pseudomonas as well as some other proteobacterial genera. LlpAs share a common architecture consisting of two B-lectin domains, followed by a short carboxy-terminal extension. Two surface-exposed moieties on susceptible Pseudomonas cells are targeted by the respective lectin modules. The carboxy-terminal domain binds D-rhamnose residues present in the lipopolysaccharide layer, whereas the amino-terminal domain interacts with a polymorphic external loop of the outer-membrane protein insertase BamA, hence determining selectivity. The absence of a toxin-immunity module as found in modular bacteriocins and other polymorphic toxin systems, hints toward a novel mode of killing initiated at the cellular surface, not requiring bacteriocin import. Despite significant progress in understanding the function of LlpAs, outstanding questions include the secretion machinery recruited by lectin-like bacteriocins for their release, as well as a better understanding of the environmental signals initiating their expression. ispartof: FRONTIERS IN MICROBIOLOGY vol:9 issue:NOV ispartof: location:Switzerland status: published

Skin is the body&rsquo, s largest organ, and the main function of skin is to protect underlying organs from possible external damage. Melanocytes play an important role in skin pigmentation. The Bama pig has a &ldquo, two-end-black&rdquo, phenotype with different coat colors across skin regions, e.g., white skin (without melanocytes) and black skin (with melanocytes), which could be a model to investigate skin-related disorders, specifically loss of melanocytes. Here, we generated expression profiles of mRNAs and long noncoding RNAs in Bama pig skins with different coat colors. In total, 14,900 mRNAs and 7549 lncRNAs were expressed. Overall, 2338 mRNAs/113 lncRNAs with FDR-adjusted p-value &le, 0.05 were considered to be differentially expressed (DE) mRNAs/lncRNAs, with 1305 down-regulated mRNAs and 1033 up-regulated mRNAs in white skin with|log2(fold change)| &gt, 1. The genes down-regulated in white skin were associated with pigmentation, melanocyte&ndash, keratinocyte interaction, and keratin, while up-regulated ones were mainly associated with cellular energy metabolisms. Furthermore, those DE lncRNAs were predicted to be implicated in pigmentation, keratin synthesis and cellular energy metabolism. In general, this study provides insight into the transcriptional difference involved in melanocyte-loss-induced keratinocyte changes and promotes the Bama pig as a biomedical model in skin research.

Intestinal colonization by the foodborne pathogen Escherichia coli O157:H7 leads to serious disease symptoms, including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Synthesis of one or more Shiga toxins (Stx) is essential for HUS and HC development. The genes encoding Stx, including Stx2a, are found within a lambdoid prophage integrated in the E. coli O157:H7 chromosome. Enhanced Stx2a expression was reported when specific non-pathogenic E. coli strains were co-cultured with E. coli O157:H7, and it was hypothesized that this phenotype required the non-pathogenic E. coli to be sensitive to stx-converting phage infection. We tested this hypothesis by generating phage resistant non-pathogenic E. coli strains where bamA (an essential gene and Stx phage receptor) was replaced with an ortholog from other species. Such heterologous gene replacement abolished the ability of the laboratory strain E. coli C600 to enhance toxin production when co-cultured with E. coli O157:H7 strain PA2, which belongs to the hypervirulent clade 8. The extracellular loops of BamA (loop 4, 6, 7) were further shown to be important for infection by stx2a-converting phages. However, similar gene replacement in another commensal E. coli, designated 1.1954, revealed a bamA-independent mechanism for toxin amplification. Toxin enhancement by 1.1954 was not the result of phage infection through an alternative receptor (LamB or FadL), lysogen formation by stx2a-converting phages, or the production of a secreted molecule. Collectively, these data suggest that non-pathogenic E. coli can enhance toxin production through at least two mechanisms.

Members of the Omp85 protein superfamily have important roles in Gram-negative bacteria, with the archetypal protein BamA being ubiquitous given its essential function in the assembly of outer membrane proteins. In some bacterial lineages, additional members of the family exist and, in most of these cases, the function of the protein is unknown. We detected one of these Omp85 proteins in the pathogen Klebsiella pneumoniae B5055, and refer to the protein as BamK. Here, we show that bamK is a conserved element in the core genome of Klebsiella, and its expression rescues a loss-of-function ∆bamA mutant. We developed an E. coli model system to measure and compare the specific activity of BamA and BamK in the assembly reaction for the critical substrate LptD, and find that BamK is as efficient as BamA in assembling the native LptDE complex. Comparative structural analysis revealed that the major distinction between BamK and BamA is in the external facing surface of the protein, and we discuss how such changes may contribute to a mechanism for resistance against infection by bacteriophage.

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a “targeted boost-and-sort” strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of β-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.

The core component BamA of the β barrel assembly machinery (BAM) adopts several conformations, which are thought to facilitate the insertion and folding of β barrel proteins into the bacterial outer membrane. Which factors alter the stability of these conformations remains to be quantified. Here, we apply single-molecule force spectroscopy to characterize the mechanical properties of BamA from Escherichia coli. In contrast to the N-terminal periplasmic polypeptide-transport-associated (POTRA) domains, the C-terminal transmembrane β barrel domain of BamA is mechanically much more stable. Exposed to mechanical stress this β barrel stepwise unfolds β hairpins until unfolding has been completed. Thereby, the mechanical stabilities of β barrel and β hairpins are modulated by the POTRA domains, the membrane composition and the extracellular lid closing the β barrel. We anticipate that these differences in stability, which are caused by factors contributing to BAM function, promote conformations of the BamA β barrel required to insert and fold outer membrane proteins.

Most studies characterizing the folding, structure, and function of membrane proteins rely on solubilized or reconstituted samples. Whereas solubilized membrane proteins lack the functionally important lipid membrane, reconstitution embeds them into artificial lipid bilayers, which lack characteristic features of cellular membranes including lipid diversity, composition and asymmetry. Here, we utilize outer membrane vesicles (OMVs) released from Escherichia coli to study outer membrane proteins (Omps) in the native membrane environment. Enriched in the native membrane of the OMV we characterize the assembly, folding, and structure of OmpG, FhuA, Tsx, and BamA. Comparing Omps in OMVs to those reconstituted into artificial lipid membranes, we observe different unfolding pathways for some Omps. This observation highlights the importance of the native membrane environment to maintain the native structure and function relationship of Omps. Our fast and easy approach paves the way for functional and structural studies of Omps in the native membrane., Communications Biology, 1 (1), ISSN:2399-3642

Insulin-like growth factor binding protein 6 (IGFBP6) is a key factor in regulating the effects of insulin-like growth factor 2 (IGF2) on the animal growth and development, but the mechanism is far from known. In this study, the 5'-terminal sequence of the IGFBP6 gene (from −920 to −1 bp) which may regulate gene expression was sequenced in the Bama mini-pigs, the Tibetan mini-pigs, the Landrace pigs, the Large White pigs and the Northeast wild boars to screen for the single nucleotide polymorphisms (SNPs) and analyze the relations with the body size traits of swine through a chi-square test analysis. The genotype frequencies of the SNPs in the 5'-terminal sequence have shown that c.-726C > T, c.-722T > C, c.-535C > A, c.-488T > C, c.-403A > G, and c.-378T > C may be related to the dwarf traits of the Bama mini-pig and the Tibetan mini-pigs (P

CdiB/CdiA proteins mediate inter-bacterial competition in a process termed contact-dependent growth inhibition (CDI). Filamentous CdiA exoproteins extend from CDI(+) cells and bind specific receptors to deliver toxins into susceptible target bacteria. CDI has also been implicated in auto-aggregation and biofilm formation in several species, but the contribution of CdiA-receptor interactions to these multi-cellular behaviors has not been examined. Using Escherichia coli isolate EC93 as a model, we show that cdiA and bamA receptor mutants are defective in biofilm formation, suggesting a prominent role for CdiA-BamA mediated cell-cell adhesion. However, CdiA also promotes auto-aggregation in a BamA-independent manner, indicating that the exoprotein possesses an additional adhesin activity. Cells must express CdiA in order to participate in BamA-independent aggregates, suggesting that adhesion could be mediated by homotypic CdiA-CdiA interactions. The BamA-dependent and BamA-independent interaction domains map to distinct regions within the CdiA filament. Thus, CdiA orchestrates a collective behavior that is independent of its growth-inhibition activity. This adhesion should enable 'greenbeard' discrimination, in which genetically unrelated individuals cooperate with one another based on a single shared trait. This kind-selective social behavior could provide immediate fitness benefits to bacteria that acquire the systems through horizontal gene transfer.

We recently demonstrated that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses characteristic BamA bipartite topology. Herein, we used immunofluorescence analysis (IFA) to show that only the β-barrel domain of TP_0326 contains surface-exposed epitopes in intact T. pallidum . Using the solved structure of Neisseria gonorrhoeae BamA, we generated a homology model of full-length TP_0326. Although the model predicts a typical BamA fold, the β-barrel harbors features not described in other BamAs. Structural modeling predicted that a dome comprised of three large extracellular loops, loop 4 (L4), L6, and L7, covers the barrel's extracellular opening. L4, the dome's major surface-accessible loop, contains mainly charged residues, while L7 is largely neutral and contains a polyserine tract in a two-tiered conformation. L6 projects into the β-barrel but lacks the VRGF/Y motif that anchors L6 within other BamAs. IFA and opsonophagocytosis assay revealed that L4 is surface exposed and an opsonic target. Consistent with B cell epitope predictions, immunoblotting and enzyme-linked immunosorbent assay (ELISA) confirmed that L4 is an immunodominant loop in T. pallidum -infected rabbits and humans with secondary syphilis. Antibody capture experiments using Escherichia coli expressing OM-localized TP_0326 as a T. pallidum surrogate further established the surface accessibility of L4. Lastly, we found that a naturally occurring substitution (Leu 593 → Gln 593 ) in the L4 sequences of T. pallidum strains affects antibody binding in sera from syphilitic patients. Ours is the first study to employ a “structure-to-pathogenesis” approach to map the surface topology of a T. pallidum OMP within the context of syphilitic infection. IMPORTANCE Previously, we reported that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses the bipartite topology characteristic of a BamA ortholog. Using a homology model as a guide, we found that TP_0326 displays unique features which presumably relate to its function(s) in the biogenesis of T. pallidum 's unorthodox OM. The model also enabled us to identify an immunodominant epitope in a large extracellular loop that is both an opsonic target and subject to immune pressure in a human population. Ours is the first study to follow a structure-to-pathogenesis approach to map the surface topology of a T. pallidum rare OMP within the context of syphilitic infection.

Assembly of the β-barrel outer membrane proteins (OMPs) is an essential cellular process in Gram negative bacteria and in the mitochondria and chloroplasts of eukaryotes—two organelles of bacterial origin. Central to this process is the conserved β-barrel OMP that belongs to the Omp85 superfamily. In Escherichia coli, BamA is the core β-barrel OMP, and together with four outer membrane lipoproteins, BamBCDE, constitute the β-barrel assembly machine (BAM). In this paper, we investigated the roles of BamD, an essential lipoprotein, and BamB in BamA biogenesis. Depletion of BamD caused impairment in BamA biogenesis and cessation of cell growth. These defects of BamD depletion were partly reversed by single amino acid substitutions mapping within the β-barrel domain of BamA. However, in the absence of BamB, the positive effects of the β-barrel substitutions on BamA biogenesis under BamD depletion conditions were nullified. By employing a BamA protein bearing one such substitution, F494L, it was demonstrated that the mutant BamA protein could not only assemble without BamD, but it could also facilitate the assembly of wild-type BamA expressed in trans. Based on these data, we propose a model in which the Bam lipoproteins, which are localized to the outer membrane by the BAM-independent Lol pathway, aid in the creation of new BAM complexes by serving as outer membrane receptors and folding factors for nascent BamA molecules. The newly assembled BAM holocomplex then catalyzes the assembly of substrate OMPs and BamA. These in vivo findings are corroborated by recently published in vitro data.

Animal growth and development are complex and sophisticated biological metabolic processes, in which genes plays an important role. In this paper, we employed real-time quantitative PCR (RT-qPCR) to analyze the expression levels of hepatic GHR, JAK2 and IGF-I genes in 1, 30, 180 day of Bama minipig and Landrace with attempt to verify the correlation between the expression of these growth-associated genes and the dwarfism phenotype of Bama minipig. The results showed that the expression levels of these 3 genes in Bama minipigs were down-regulated expressed from 1 day to 30 day, and which was up-regulated expressed in Landrace. The expression levels of the 3 genes on 1, 30, 180 day were prominently higher in Landrace than in Bama minipigs. The significant differences of the 3 genes expression levels on 1 day between this two breeds indicate that different expressions of these genes might occur before birth. It is speculated that the down-regulated expression of the 3 genes may have a close correlation with the dwarfism phenotype of Bama minipig. More investigations in depth of this study is under progress with the help of biochip nanotechnology.