Most species of the genus Bifidobacterium contain the gene cluster PFNA, which is presumably involved in the species-specific communication between bacteria and their hosts. The gene cluster PFNA consists of five genes including fn3 , which codes for a protein containing two fibronectin type III domains. Each fibronectin domain contains sites similar to cytokine-binding sites of human receptors. Based on this finding we assumed that this protein would bind specifically to human cytokines in vitro . We cloned a fragment of the fn3 gene (1503 bp; 501 aa) containing two fibronectin domains, from the strain B. longum subsp. longum GT15. After cloning the fragment into the expression vector pET16b and expressing it in E. coli , the protein product was purified to a homogenous state for further analysis. Using the immunoferment method, we tested the purified fragment’s ability to bind the following human cytokines: IL-1β, IL-6, IL-10, TNFα. We developed a sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. We found that the purified protein fragment only binds to TNFα.
Bifidobacteria are commensal microorganisms that inhabit a wide range of hosts, including insects, birds and mammals. The mechanisms responsible for the adaptation of bifidobacteria to various hosts during the evolutionary process remain poorly understood. Previously, we reported that the species-specific PFNA gene cluster is present in the genomes of various species of the Bifidobacterium genus. The cluster contains signal transduction and adhesion genes that are presumably involved in the communication between bifidobacteria and their hosts. The genes in the PFNA cluster show high sequence divergence between bifidobacterial species, which may be indicative of rapid evolution that drives species-specific adaptation to the host organism. We used the maximum likelihood approach to detect positive selection in the PFNA genes. We tested for both pervasive and episodic positive selection to identify codons that experienced adaptive evolution in all and individual branches of the Bifidobacterium phylogenetic tree, respectively. Our results provide evidence that episodic positive selection has played an important role in the divergence process and molecular evolution of sequences of the species-specific PFNA genes in most bifidobacterial species. Moreover, we found the signatures of pervasive positive selection in the molecular evolution of the tgm gene in all branches of the Bifidobacterium phylogenetic tree. These results are consistent with the suggested role of PFNA gene cluster in the process of specific adaptation of bifidobacterial species to various hosts.
To date, transcriptomics have been widely and successfully employed to study gene expression in different cell growth phases of bacteria. Since bifidobacteria represent a major component of the gut microbiota of a healthy human that is associated with numerous health benefits for the host, it is important to study them using transcriptomics. In this study, we applied the RNA-Seq technique to study global gene expression of B. longum at different growth phases in order to better understand the response of bifidobacterial cells to the specific conditions of the human gut. We have shown that in the lag phase, ABC transporters, whose function may be linked to active substrate utilization, are increasingly expressed due to preparation for cell division. In the exponential phase, the functions of activated genes include synthesis of amino acids (alanine and arginine), energy metabolism (glycolysis/gluconeogenesis and nitrogen metabolism), and translation, all of which promote active cell division, leading to exponential growth of the culture. In the stationary phase, we observed a decrease in the expression of genes involved in the control of the rate of cell division and an increase in the expression of genes involved in defense-related metabolic pathways. We surmise that the latter ensures cell survival in the nutrient-deprived conditions of the stationary growth phase.
As permanent residents of the normal gut microbiota, bifidobacteria have evolved to adapt to the host’s immune response whose priority is to eliminate pathogenic agents. The mechanisms that ensure the survival of commensals during inflammation and maintain the stability of the core component of the normal gut microbiota in such conditions remain poorly understood. We propose a new in vitro approach to study the mechanisms of resistance to immune response factors based on high-throughput sequencing followed by transcriptome analysis. This approach allowed us to detect differentially expressed genes associated with inflammation. In this study, we demonstrated that the presence of the pro-inflammatory cytokines IL-6 and TNFα to the growth medium of the B. longum subsp. longum GT15 strain changes the latter’s growth rate insignificantly while affecting the expression of certain genes. We identified these genes and performed a COG and a KEGG pathway enrichment analysis. Using phylogenetic profiling we predicted the operons of genes whose expression was triggered by the cytokines TNFα and IL-6 in vitro . By mapping the transcription start points, we experimentally validated the predicted operons. Thus, in this study, we predicted the genes involved in a putative signaling pathway underlying the mechanisms of resistance to inflammatory factors in bifidobacteria. Since bifidobacteria are a major component of the human intestinal microbiota exhibiting pronounced anti-inflammatory properties, this study is of great practical and scientific relevance.
Caliciviridae is a family of viral pathogens that naturally infects vertebrates, including humans, and causes a range of highly contagious infectious diseases. Caliciviruses are not well studied because of the lack of a universal approach to their cultivation; however, the development of molecular genetics and bioinformatics methods can shed light on their genetic architecture and evolutionary relationships. Here, we present and characterize the complete genome sequence of calicivirus isolated from a sandpiper—Temminck’s stint (Calidris temminckii), preliminarily named Temminck’s stint calicivirus (TsCV). Its genome is a linear, non-segmented, single-stranded (+sense) RNA with genome organization typical of avian caliciviruses. Comparative studies have shown significant divergence of the nucleotide sequence of the TsCV genome, as well as the amino acid sequence of the major capsid protein from all publicly available genomic and protein sequences, with the highest genome sequence similarity to unclassified Ruddy turnstone calicivirus A (43.68%) and the lowest pairwise divergence of the major capsid protein with unclassified goose calicivirus (57.44%). Phylogenetic analysis, as well as a comparative analysis of the homologous proteins, showed evidence of another separate genus within the Caliciviridae family—previously proposed, but not yet accepted by International Committee on Taxonomy of Viruses (ICTV)—the Sanovirus genus, which combines seven previously unclassified genomic sequences of avian caliciviruses, including the newly discovered TsCV, which we propose to consider as a separate species.
Background Klebsiella pneumoniae, which is frequently associated with hospital- and community-acquired infections, contains multidrug-resistant (MDR), hypervirulent (hv), non-MDR/non-hv as well as convergent representatives. It is known that mostly international high-risk clonal lineages including sequence types (ST) 11, 147, 258, and 307 drive their global spread. ST395, which was first reported in the context of a carbapenemase-associated outbreak in France in 2010, is a less well-characterized, yet emerging clonal lineage. Methods We computationally analyzed a large collection of K. pneumoniae ST395 genomes (n = 297) both sequenced in this study and reported previously. By applying multiple bioinformatics tools, we investigated the core-genome phylogeny and evolution of ST395 as well as distribution of accessory genome elements associated with antibiotic resistance and virulence features. Results Clustering of the core-SNP alignment revealed four major clades with eight smaller subclades. The subclades likely evolved through large chromosomal recombination, which involved different K. pneumoniae donors and affected, inter alia, capsule and lipopolysaccharide antigen biosynthesis regions. Most genomes contained acquired resistance genes to extended-spectrum cephalosporins, carbapenems, and other antibiotic classes carried by multiple plasmid types, and many were positive for hypervirulence markers, including the siderophore aerobactin. The detection of “hybrid” resistance and virulence plasmids suggests the occurrence of the convergent ST395 pathotype. Conclusions To the best of our knowledge, this is the first study that investigated a large international collection of K. pneumoniae ST395 genomes and elucidated phylogenetics and detailed genomic characteristics of this emerging high-risk clonal lineage.
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