Repeated Social Defeat Stress Induces an Inflammatory Gut Milieu by Altering the Mucosal Barrier Integrity and Gut Microbiota Homeostasis.

Background: Posttraumatic stress disorder (PTSD) is a mental health condition triggered by exposure to traumatic events in an individual's life. Patients with PTSD are also at a higher risk for comorbidities. However, it is not well understood how PTSD affects human health and/or promotes the risk for comorbidities. Nevertheless, patients with PTSD harbor a proinflammatory milieu and dysbiotic gut microbiota. Gut barrier integrity helps to maintain normal gut homeostasis and its dysregulation promotes gut dysbiosis and inflammation. Methods: We used a mouse model of repeated social defeat stress (RSDS), a preclinical model of PTSD. Behavioral studies, metagenomics analysis of the microbiome, gut permeability assay (on mouse colon, using an Ussing chamber), immunoblotting, and immunohistochemical analyses were performed. Polarized intestinal epithelial cells and 3-dimensional crypt cultures were used for mechanistic analysis. Results: The RSDS mice harbor a heightened proinflammatory gut environment and microbiota dysbiosis. The RSDS mice further showed significant dysregulation of gut barrier functions, including transepithelial electrical resistance, mucin homeostasis, and antimicrobial responses. RSDS mice also showed a specific increase in intestinal expression of claudin-2, a tight junction protein, and epinephrine, a stress-induced neurotransmitter. Treating intestinal epithelial cells or 3-dimensional cultured crypts with norepinephrine or intestinal luminal contents (fecal contents) upregulated claudin-2 expression and inhibited transepithelial electrical resistance. Conclusions: Traumatic stress induces dysregulation of gut barrier functions, which may underlie the observed gut microbiota changes and proinflammatory gut milieu, all of which may have an interdependent effect on the health and increased risk of comorbidities in patients with PTSD.

Exosomes derived from differentiated human ADMSC with the Schwann cell phenotype modulate peripheral nerve-related cellular functions.

Peripheral nerve regeneration remains a significant clinical challenge due to the unsatisfactory functional recovery and public health burden. Exosomes, especially those derived from mesenchymal stem cells (MSCs), are promising as potential cell-free therapeutics and gene therapy vehicles for promoting neural regeneration. In this study, we reported the differentiation of human adipose derived MSCs (hADMSCs) towards the Schwann cell (SC) phenotype (hADMSC-SCs) and then isolated exosomes from hADMSCs with and without differentiation (i.e., dExo vs uExo). We assessed and compared the effects of uExo and dExo on antioxidative, angiogenic, anti-inflammatory, and axon growth promoting properties by using various peripheral nerve-related cells. Our results demonstrated that hADMSC-SCs secreted more neurotrophic factors and other growth factors, compared to hADMSCs without differentiation. The dExo isolated from hADMSC-SCs protected rat SCs from oxidative stress and enhanced HUVEC migration and angiogenesis. Compared to uExo, dExo also had improved performances in downregulating pro-inflammatory gene expressions and cytokine secretions and promoting axonal growth of sensory neurons differentiated from human induced pluripotent stem cells. Furthermore, microRNA (miRNA) sequencing analysis revealed that exosomes and their parent cells shared some similarities in their miRNA profiles and exosomes displayed a distinct miRNA signature. Many more miRNAs were identified in dExo than in uExo. Several upregulated miRNAs, like miRNA-132-3p and miRNA-199b-5p, were highly related to neuroprotection, anti-inflammation, and angiogenesis. The dExo can effectively modulate various peripheral nerve-related cellular functions and is promising for cell-free biological therapeutics to enhance neural regeneration.

Whole Genomic analysis of a clinical isolate of Uropathogenic Escherichia coli strain of Sequence Type - 101 carrying the drug resistance NDM-7 in IncX3 plasmid.

The emerging NDM-producing Enterobactereciae is a major threat to public health. The association of NDM-7 with sequence type 101 E.coli is identified in very few numbers. Therefore, it is of interest to analyse the whole genome sequence of NDM-producing uropathogenic E. coli XA31 that was found to carry numerous drug resistance genes of different antibiotic classes. The isolate E. coli belongs to ST-101 carrying blaNDM-7 coexisting with several resistance genes blaOXA-1, blaTEM1-A, blaCTX-M15, aac(6')-Ib-cr, catB3, tetB. Resfinder predicts this and four other plasmid replicons were identified using the Plasfinder in the CGE platform. The high transferable IncX3 plasmid was found to carry the NDM-7 gene. Thus, we the report the combination of NDM-7-ST101-IncX3 in India. The combination of this epidemic clone with NDM-7 is highly required to develop an effective infection control strategy.

Gut Microbiota and Metabolic Specificity in Ulcerative Colitis and Crohn's Disease.

Background: Inflammatory bowel disease (IBD) represents multifactorial chronic inflammatory conditions in the gastrointestinal tract and includes Crohn's disease (CD) and ulcerative colitis (UC). Despite similarities in pathobiology and disease symptoms, UC and CD represent distinct diseases and exhibit diverse therapeutic responses. While studies have now confirmed that IBD is associated with dramatic changes in the gut microbiota, specific changes in the gut microbiome and associated metabolic effects on the host due to CD and UC are less well-understood. Methods: To address this knowledge gap, we performed an extensive unbiased meta-analysis of the gut microbiome data from five different IBD patient cohorts from five different countries using QIIME2, DIAMOND, and STAMP bioinformatics platforms. In-silico profiling of the metabolic pathways and community metabolic modeling were carried out to identify disease-specific association of the metabolic fluxes and signaling pathways. Results: Our results demonstrated a highly conserved gut microbiota community between healthy individuals and IBD patients at higher phylogenetic levels. However, at or below the order level in the taxonomic rank, we found significant disease-specific alterations. Similarly, we identified differential enrichment of the metabolic pathways in CD and UC, which included enriched pathways related to amino acid and glycan biosynthesis and metabolism, in addition to other metabolic pathways. Conclusions: In conclusion, this study highlights the prospects of harnessing the gut microbiota to improve understanding of the etiology of CD and UC and to develop novel prognostic, and therapeutic approaches.

A Holistic Systems Approach to Characterize the Impact of Pre- and Post-natal Oxycodone Exposure on Neurodevelopment and Behavior.

Background: Increased risk of oxycodone (oxy) dependency during pregnancy has been associated with altered behaviors and cognitive deficits in exposed offspring. However, a significant knowledge gap remains regarding the effect of in utero and postnatal exposure on neurodevelopment and subsequent behavioral outcomes. Methods: Using a preclinical rodent model that mimics oxy exposure in utero (IUO) and postnatally (PNO), we employed an integrative holistic systems biology approach encompassing proton magnetic resonance spectroscopy (1H-MRS), electrophysiology, RNA-sequencing, and Von Frey pain testing to elucidate molecular and behavioral changes in the exposed offspring during early neurodevelopment as well as adulthood. Results: 1H-MRS studies revealed significant changes in key brain metabolites in the exposed offspring that were corroborated with changes in synaptic currents. Transcriptomic analysis employing RNA-sequencing identified alterations in the expression of pivotal genes associated with synaptic transmission, neurodevelopment, mood disorders, and addiction in the treatment groups. Furthermore, Von Frey analysis revealed lower pain thresholds in both exposed groups. Conclusions: Given the increased use of opiates, understanding the persistent developmental effects of these drugs on children will delineate potential risks associated with opiate use beyond the direct effects in pregnant women.

Genome Investigation of a Cariogenic Pathogen with Implications in Cardiovascular Diseases.

The proportion of people suffering from cardiovascular diseases has risen by 34% in the last 15 years in India. Cardiomyopathy is among the many forms of CVD s present. Infection of heart muscles is the suspected etiological agent for the same. Oral pathogens gaining entry into the bloodstream are responsible for such infections. Streptococcus mutans is an oral pathogen with implications in cardiovascular diseases. Previous studies have shown certain strains of S. mutans are found predominantly within atherosclerotic plaques and extirpated valves. To decipher the genetic differences responsible for endothelial cell invasion, we have sequenced the genome of Streptococcus mutans B14. Pan-genome analysis, search for adhesion proteins through a special algorithm, and protein-protein interactions search through HPIDB have been done. Pan-genome analysis of 187 whole genomes, assemblies revealed 6965 genes in total and 918 genes forming the core gene cluster. Adhesion to the endothelial cell is a critical virulence factor distinguishing virulent and non-virulent strains. Overall, 4% of the total proteins in S. mutans B14 were categorized as adhesion proteins. Protein-protein interaction between putative adhesion proteins and Human extracellular matrix components was predicted, revealing novel interactions. A conserved gene catalyzing the synthesis of branched-chain amino acids in S. mutans B14 shows possible interaction with isoforms of cathepsin protein of the ECM. This genome sequence analysis indicates towards other proteins in the S. mutans genome, which might have a specific role to play in host cell interaction.

Draft Genome Sequences for Five Photorhabdus Bacterial Symbionts of Entomopathogenic Heterorhabditis Nematodes Isolated from India.

Photorhabdus bacteria exhibit contrasting lifestyles; they are virulent insect pathogens but symbionts of the entomopathogenic Heterorhabditis nematodes. Photorhabdus genomes encode several secondary metabolites and insecticidal protein toxins. Here, we present the draft genome sequences for five Photorhabdus strains isolated from Heterorhabditis nematodes collected from various geographical regions of India.

Development and evaluation of a core genome multilocus sequence typing (cgMLST) scheme for Brucella spp.

Brucellosis is a zoonotic disease caused by Brucella spp. Brucella spp. can be sub-typed by multilocus sequence typing (MLST) method, which targets a set of housekeeping genes. We have developed a core genome MLST (cgMLST) typing scheme to distinguish and differentiate species of Brucella up to biovar level. A total of 407 whole (complete and draft) genome sequences of different Brucella strains were used in this study. Genome sequences were filtered using the BLAST score ratio (BSR)-based allele calling algorithm, and we found that 164 cgMLST target loci are shared in all the 407 genome sequences. These 164 loci were used to develop the cgMLST scheme and further evaluated to sub-type different species of Brucella. Based on our cgMLST scheme, Brucella spp. were classified into 287 sequence types (STs). A phylogenetic tree was constructed based on the STs derived from the cgMLST analysis. The phylogenetic tree differentiated all the 11 Brucella spp. and five biovars of B. suis. B. vulpis formed the outmost clade followed by B. inopinata and B. microti. Among the four subgroups of B. abortus, group A and B were differentiated based on their geographic origins. Similarly, three subgroups of B. melitensis were separated based on their geographical origins with few exceptions. B. neotomae that infect rodents were distinguished from other Brucella spp. B. canis showed the closest relationship with B. suis bv. 4, followed by B. suis bv. 3 and bv. 1. Brucella spp. associated with the marine mammals, such as B. ceti and B. pinnipedialis were closely related. Of these, B. ceti strains isolated from dolphins and porpoise were differentiated into two groups. We incorporated our cgMLST tool in BrucellaBase (http://www.dbtbrucellosis.in/brucella_cgmlst.html), which will be helpful to predict the cgMLST allelic profile and the ST of a newly sequenced genome.

Identification of genetic variants of Brucella spp. through genome-wide association studies.

Brucellosis is an important zoonotic disease caused by Brucella spp. We present a phylogeny of 552 strains based on genome-wide single nucleotide polymorphisms (SNPs) determined by an alignment-free k-mer approach. A total of 138,029 SNPs were identified from 552 Brucella genomes. Of these, 31,152 and 106,877 were core and non-core SNPs, respectively. Based on pan-genome analysis 11,937 and 972 genes were identified as pan and core genome, respectively. The pan-genome-wide analysis studies (Pan-GWAS) could not identify the group-specific variants in Brucella spp. Therefore, we focused on SNP based genome-wide association studies (SNP-GWAS) to identify the species-specific genetic determinants in Brucella spp. Phylogenetic tree representing eleven recognized Brucella spp. showed 16 major lineages. We identified 143 species-specific SNPs in Brucella abortus that are conserved in 311 B. abortus genomes. Of these, 141 species-specific SNPs were confined in the positively significant SNPs of B. abortus using SNP-GWAS. Since conserved in all the B. abortus genomes studied, these SNPs might have originated very early during the evolution of B. abortus and might be responsible for the evolution of B. abortus with cattle as the preferred host. Similarly, we identified 383 species-specific SNPs conserved in 132 Brucella melitensis genomes. Of these 379 species-specific SNPs were identified as positively associated using GWAS. Interestingly, >98% of the SNPs that are significantly, positively associated with the traits showed 100% sensitivity and 100% specificity. These identified species-specific core-SNPs identified in Brucella genomes could be responsible for the speciation and their respective host adaptation.

Identification of potential antigens from non-classically secreted proteins and designing novel multitope peptide vaccine candidate against Brucella melitensis through reverse vaccinology and immunoinformatics approach.

Brucella melitensis is an intracellular pathogen resides in the professional and non-professional phagocytes of the host, causing zoonotic disease brucellosis. The stealthy nature of the Brucella makes it's highly pathogenic, and it is hard to eliminate the bacteria completely from the infected host. Hitherto, no licensed vaccines are available for human brucellosis. In this study, we identified potential antigens for vaccine development from non-classically secreted proteins through reverse vaccinology approach. Based on the systemic screening of non-classically secreted proteins of B. melitensis 16M, we identified nine proteins as potential vaccine candidates. Among these, Omp31 and Omp22 are known immunogens, and its role in the virulence of Brucella is known. Roles of other proteins in the pathogenesis are yet to be studied. From the nine proteins, we identified six novel antigenic epitopes that can elicit both B-cell and T-cell immune responses. Among the nine proteins, the epitopes were predicted from Omp31 immunogenic protein precursor, Omp22 protein precursor, extracellular serine protease, hypothetical membrane-associated protein, iron-regulated outer membrane protein FrpB. Further, we designed a multitope vaccine using Omp31 immunogenic protein precursor, Omp22 protein precursor, extra cellular serine protease, iron-regulated outer membrane protein FrpB, hypothetical membrane-associated protein, and LPS-assembly protein LptD and polysaccharide export protein identified in the previous study. Epitopes were joined using amino acid linkers such as EAAAK and GPGPG. Cholera toxin subunit B, the nontoxic part of cholera toxin, was used as an adjuvant and it was linked to the N-terminal of the multitope vaccine candidate. The designed vaccine candidate was modeled, validated and the physicochemical properties were analyzed. Results revealed that the vaccine candidate is soluble, stable, non-allergenic, antigenic and 87% of residues of the designed vaccine candidate is located in the favored region. In conclusion, the computational analysis showed that the newly designed multitope protein could be used to develop a promising vaccine for human brucellosis.

Identification of OtpR regulated sRNAs in Brucella melitensis expressed under acidic stress and their roles in pathogenesis and metabolism.

Small RNAs (sRNAs) are the small regulatory molecules that regulate various biological processes in bacteria. Though the functions of sRNAs are well documented, very little information is available on the sRNAs of Brucella spp. The otpR is the response regulator of a two-component regulatory system, which plays a significant role in Brucella virulence. In this study, we identified the sRNAs expressed in B. melitensis 16M and its otpR mutant under acidic stress from the RNAseq dataset. We identified 94 trans-encoded and 948 cis-encoded sRNAs in B. melitensis 16M. In B. melitensis 16M ΔotpR under acidic stress 99 trans-encoded and 877 cis-encoded sRNAs were identified. Among these, 12 trans-encoded and 43 cis-encoded sRNAs were upregulated in B. melitensis 16M ΔotpR, with an adjusted P-value≤0.05. The mRNA targets of these sRNAs were predicted. Further, the levels of mRNA targets were examined, and the sRNA-mediated regulatory network was predicted. Functional classification and pathway analysis of mRNA targets provided evidence that sRNAs are involved in different metabolic pathways including carbohydrates, amino acids, lipids, nucleotides transport and metabolism, cell membrane biogenesis and intracellular trafficking of Brucella. We also found that eight transcriptional regulators including a quorum sensing regulator, vjbR are down-regulated by sRNAs. These transcriptional regulators might be responsible for the regulation of several other genes in the otpR mutant. The trans-encoded BsnR88 and cis-encoded BsnR980, BsnR998, BsnR881, BsnR1001, BsnR891, BsnR883, BsnR905 are regulating virB operon genes coding for type IV secretion system (T4SS), which is the major virulence factor of Brucella.

BrucellaBase: Genome information resource.

Brucella sp. causes a major zoonotic disease, brucellosis. Brucella belongs to the family Brucellaceae under the order Rhizobiales of Alphaproteobacteria. We present BrucellaBase, a web-based platform, providing features of a genome database together with unique analysis tools. We have developed a web version of the multilocus sequence typing (MLST) (Whatmore et al., 2007) and phylogenetic analysis of Brucella spp. BrucellaBase currently contains genome data of 510 Brucella strains along with the user interfaces for BLAST, VFDB, CARD, pairwise genome alignment and MLST typing. Availability of these tools will enable the researchers interested in Brucella to get meaningful information from Brucella genome sequences. BrucellaBase will regularly be updated with new genome sequences, new features along with improvements in genome annotations. BrucellaBase is available online at http://www.dbtbrucellosis.in/brucellabase.html or http://59.99.226.203/brucellabase/homepage.html.

Omics of Brucella: Species-Specific sRNA-Mediated Gene Ontology Regulatory Networks Identified by Computational Biology.

Brucella is an intracellular bacterium that causes the zoonotic infectious disease, brucellosis. Brucella species are currently intensively studied with a view to developing novel global health diagnostics and therapeutics. In this context, small RNAs (sRNAs) are one of the emerging topical areas; they play significant roles in regulating gene expression and cellular processes in bacteria. In the present study, we forecast sRNAs in three Brucella species that infect humans, namely Brucella melitensis, Brucella abortus, and Brucella suis, using a computational biology analysis. We combined two bioinformatic algorithms, SIPHT and sRNAscanner. In B. melitensis 16M, 21 sRNA candidates were identified, of which 14 were novel. Similarly, 14 sRNAs were identified in B. abortus, of which four were novel. In B. suis, 16 sRNAs were identified, and five of them were novel. TargetRNA2 software predicted the putative target genes that could be regulated by the identified sRNAs. The identified mRNA targets are involved in carbohydrate, amino acid, lipid, nucleotide, and coenzyme metabolism and transport, energy production and conversion, replication, recombination, repair, and transcription. Additionally, the Gene Ontology (GO) network analysis revealed the species-specific, sRNA-based regulatory networks in B. melitensis, B. abortus, and B. suis. Taken together, although sRNAs are veritable modulators of gene expression in prokaryotes, there are few reports on the significance of sRNAs in Brucella. This report begins to address this literature gap by offering a series of initial observations based on computational biology to pave the way for future experimental analysis of sRNAs and their targets to explain the complex pathogenesis of Brucella.

A genome-wide SNP-based phylogenetic analysis distinguishes different biovars of Brucella suis.

Brucellosis is an important zoonotic disease caused by Brucella spp. Brucella suis is the etiological agent of porcine brucellosis. B. suis is the most genetically diverged species within the genus Brucella. We present the first large-scale B. suis phylogenetic analysis based on an alignment-free k-mer approach of gathering polymorphic sites from whole genome sequences. Genome-wide core-SNP based phylogenetic tree clearly differentiated and discriminated the B. suis biovars and the vaccine strain into different clades. A total of 16,756 SNPs were identified from the genome sequences of 54 B. suis strains. Also, biovar-specific SNPs were identified. The vaccine strain B. suis S2-30 is extensively used in China, which was discriminated from all biovars with the accumulation of the highest number of SNPs. We have also identified the SNPs between B. suis vaccine strain S2-30 and its closest homolog, B. suis biovar 513UK. The highest number of mutations (22) was observed in the phosphomannomutase (pmm) gene essential for the synthesis of O-antigen. Also, mutations were identified in several virulent genes including genes coding for type IV secretion system and the effector proteins, which could be responsible for the attenuated virulence of B. suis S2-30.

Computational prediction of secretion systems and secretomes of Brucella: identification of novel type IV effectors and their interaction with the host.

Brucella spp. are facultative intracellular pathogens that cause brucellosis in various mammals including humans. Brucella survive inside the host cells by forming vacuoles and subverting host defence systems. This study was aimed to predict the secretion systems and the secretomes of Brucella spp. from 39 complete genome sequences available in the databases. Furthermore, an attempt was made to identify the type IV secretion effectors and their interactions with host proteins. We predicted the secretion systems of Brucella by the KEGG pathway and SecReT4. Brucella secretomes and type IV effectors (T4SEs) were predicted through genome-wide screening using JVirGel and S4TE, respectively. Protein-protein interactions of Brucella T4SEs with their hosts were analyzed by HPIDB 2.0. Genes coding for Sec and Tat pathways of secretion and type I (T1SS), type IV (T4SS) and type V (T5SS) secretion systems were identified and they are conserved in all the species of Brucella. In addition to the well-known VirB operon coding for the type IV secretion system (T4SS), we have identified the presence of additional genes showing homology with T4SS of other organisms. On the whole, 10.26 to 14.94% of total proteomes were found to be either secreted (secretome) or membrane associated (membrane proteome). Approximately, 1.7 to 3.0% of total proteomes were identified as type IV secretion effectors (T4SEs). Prediction of protein-protein interactions showed 29 and 36 host-pathogen specific interactions between Bos taurus (cattle)-B. abortus and Ovis aries (sheep)-B. melitensis, respectively. Functional characterization of the predicted T4SEs and their interactions with their respective hosts may reveal the secrets of host specificity of Brucella.

Identification of Recombination and Positively Selected Genes in Brucella.

Brucella is a facultative intracellular bacterium belongs to the class alpha proteobacteria. It causes zoonotic disease brucellosis to wide range of animals. Brucella species are highly conserved in nucleotide level. Here, we employed a comparative genomics approach to examine the role of homologous recombination and positive selection in the evolution of Brucella. For the analysis, we have selected 19 complete genomes from 8 species of Brucella. Among the 1599 core genome predicted, 24 genes were showing signals of recombination but no significant breakpoint was found. The analysis revealed that recombination events are less frequent and the impact of recombination occurred is negligible on the evolution of Brucella. This leads to the view that Brucella is clonally evolved. On other hand, 56 genes (3.5 % of core genome) were showing signals of positive selection. Results suggest that natural selection plays an important role in the evolution of Brucella. Some of the genes that are responsible for the pathogenesis of Brucella were found positively selected, presumably due to their role in avoidance of the host immune system.

Draft Genome Sequence of the Intermediate Rough Vaccine Strain Brucella abortus S19Δper Mutant.

Here, we report the genome sequence of the intermediate rough vaccine strain mutant, Brucella abortus S19Δper. The length of the draft genome was 3,271,238 bp, with 57.2% G+C content. A total of 3,204 protein-coding genes and 56 RNA genes were predicted.

Novel Vaccine Candidates against Brucella melitensis Identified through Reverse Vaccinology Approach.

Global health therapeutics is a rapidly emerging facet of postgenomics medicine. In this connection, Brucella melitensis is an intracellular bacterium that causes the zoonotic infectious disease, brucellosis. Presently, no licensed vaccines are available for human brucellosis. Here, we report the identification of potential vaccine candidates against B. melitensis using a reverse vaccinology approach. Based on a systematic screening of exoproteome and secretome of B. melitensis 16 M, we identified eight proteins as potential vaccine candidates, including LPS-assembly protein LptD, a polysaccharide export protein, a cell surface protein, heme transporter BhuA, flagellin FliC, 7-alpha-hydroxysteroid dehydrogenase, immunoglobulin-binding protein EIBE, and hemagglutinin. Among these, the roles of BhuA and hemagglutinin in the virulence of Brucella are essential to establish infection. Roles of other proteins in the virulence are yet to be studied. Prediction of protein-protein interactions revealed that these proteins can interact with other proteins involved in virulence, secretion system, metabolism, and transport. From these eight potential vaccine candidates, we predicted three surface exposed novel antigenic epitopes that can induce both B-cell and T-cell immune responses. These peptides can be used for the development of either exclusive peptide vaccines or multi-component vaccines against human brucellosis. Reverse vaccinology is an important strategy for discovery of novel global health therapeutics.

Draft Genome Sequence of Brucella abortus Virulent Strain 544.

Here, we present the draft genome sequence and annotation of Brucella abortus virulent strain 544. The genome of this strain is 3,289,405 bp long, with 57.2% G+C content. A total of 3,259 protein-coding genes and 60 RNA genes were predicted.

Draft Genome Sequences of Two Brucella abortus Strains Isolated from Cattle and Pig.

We report the draft genome sequences of two Brucella abortus strains LMN1 and LMN2 isolated from cattle and pig. The LMN1 and LMN2 have the genome size of 3,395,952 bp and 3,334,792 bp, respectively. In addition to the conserved genes of Brucella, few novel regions showing similarity to the phages were identified in both strains.