On Burkholderiales order microorganisms and cystic fibrosis in Russia

Abstract Background Microbes infecting cystic fibrosis patients’ respiratory tract are important in determining patients’ functional status. Representatives of Burkholderiales order are the most dangerous. The goal of our investigation was to reveal the diversity of Burkholderiales, define of their proportion in the microbiome of various parts of respiratory tract and determine the pathogenicity of the main representatives. Results In more than 500 cystic fibrosis patients, representing all Federal Regions of Russia, 34.0% were infected by Burkholderia cepacia complex (Bcc), 21.0% by Achromobacter spp. and 12.0% by Lautropia mirabilis. B. cenocepacia was the most numerous species among the Bcc (93.0%), and A. ruhlandii was the most numerous among Achromobacter spp. (58.0%). The most abundant genotype in Bcc was sequence type (ST) 709, and in Achromobacter spp. it was ST36. These STs constitute Russian epidemic strains. Whole genome sequencing of strains A. ruhlandii SCCH3:Ach33–1365 ST36 and B. cenocepacia GIMC4560:Bcn122 ST709 revealed huge resistomes and many virulence factors, which may explain the difficulties in eradicating these strains. An experience of less dangerous B. cenocepcia ST710 elimination was described. Massively parallel sequencing of 16S rDNA amplicons, including V1-V4 hypervariable regions, was used to definite “healthy” microbiome characteristics. Analysis of maxillary sinus lavage of 7 patients revealed infection with Proteobacteria of the same ST as pathogens from sputum, suggesting that the maxillary sinus is a source of infection in cystic fibrosis patients. Conclusions Characterization of the Russian epidemic bacterial strains in the sputum and sinuses of cystic fibrosis patients have better defined the importance of Burkholderiales bacteria. This information may aid in the development of effective approaches for treatment of this disease

The Variability of the Order Burkholderiales Representatives in the Healthcare Units

Background and Aim. The order Burkholderiales became more abundant in the healthcare units since the late 1970s; it is especially dangerous for intensive care unit patients and patients with chronic lung diseases. The goal of this investigation was to reveal the real variability of the order Burkholderiales representatives and to estimate their phylogenetic relationships. Methods. 16S rDNA and genes of the Burkholderia cenocepacia complex (Bcc) Multi Locus Sequence Typing (MLST) scheme were used for the bacteria detection. Results. A huge diversity of genome size and organization was revealed in the order Burkholderiales that may prove the adaptability of this taxon’s representatives. The following variability of the Burkholderiales in Russian healthcare units has been revealed: Burkholderiaceae (Burkholderia, Pandoraea, and Lautropia), Alcaligenaceae (Achromobacter), and Comamonadaceae (Variovorax). The Burkholderia genus was the most diverse and was represented by 5 species and 16 sequence types (ST). ST709 and 728 were transmissible and often encountered in cystic fibrosis patients and in hospitals. A. xylosoxidans was estimated by 15 genotypes. The strains of first and second ones were the most numerous. Conclusions. Phylogenetic position of the genus Lautropia with smaller genome is ambiguous. The Bcc MLST scheme is applicable for all Burkholderiales representatives for resolving the epidemiological problems

Burkholderia contaminans Biofilm Regulating Operon and Its Distribution in Bacterial Genomes

Biofilm formation by Burkholderia spp. is a principal cause of lung chronic infections in cystic fibrosis patients. A “lacking biofilm production” (LBP) strain B. contaminans GIMC4587:Bct370-19 has been obtained by insertion modification of clinical strain with plasposon mutagenesis. It has an interrupted transcriptional response regulator (RR) gene. The focus of our investigation was a two-component signal transduction system determination, including this RR. B. contaminans clinical and LBP strains were analyzed by whole genome sequencing and bioinformatics resources. A four-component operon (BiofilmReg) has a key role in biofilm formation. The relative location (i.e., by being separated by another gene) of RR and histidine kinase genes is unique in BiofilmReg. Orthologs were found in other members of the Burkholderiales order. Phylogenetic analysis of strains containing BiofilmReg operons demonstrated evidence for earlier inheritance of a three-component operon. During further evolution one lineage acquired a fourth gene, whereas others lost the third component of the operon. Mutations in sensor domains have created biodiversity which is advantageous for adaptation to various ecological niches. Different species Burkholderia and Achromobacter strains all demonstrated similar BiofilmReg operon structure. Therefore, there may be an opportunity to develop a common drug which is effective for treating all these causative agents

Lactaptin induces p53-independent cell death associated with features of apoptosis and autophagy and delays growth of breast cancer cells in mouse xenografts.

Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs

The Characteristics of Ubiquitous and Unique Leptospira Strains from the Collection of Russian Centre for Leptospirosis

Background and Aim. Leptospira, the causal agent of leptospirosis, has been isolated from the environment, patients, and wide spectrum of animals in Russia. However, the genetic diversity of Leptospira in natural and anthropurgic foci was not clearly defined. Methods. The recent MLST scheme was used for the analysis of seven pathogenic species. 454 pyrosequencing technology was the base of the whole genome sequencing (WGS). Results. The most wide spread and prevalent Leptospira species in Russia were L. interrogans, L. kirschneri, and L. borgpetersenii. Five STs, common for Russian strains: 37, 17, 199, 110, and 146, were identified as having a longtime and ubiquitous distribution in various geographic areas. Unexpected properties were revealed for the environmental Leptospira strain Bairam-Ali. WGS of this strain genome suggested that it combined the features of the pathogenic and nonpathogenic strains and may be a reservoir of the natural resistance genes. Results of the comparative analysis of rrs and rpoB genes and MLST loci for different Leptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separate Leptospira species. Conclusions. Thus, the natural and anthropurgic foci supported ubiquitous Leptospira species and the pool of genes important for bacterial adaptivity to various conditions

Identification of RL2-interacting proteins A and B.

<p>Protein identification was based on the SwissProt database using MALDI spectra of protein typsin lysates.</p

RL2 regulates activity of apoptosis-related genes.

<p><b>A.</b> MDA-MB-231 cells were treated with RL2 (0.2 mg/mL) for the indicated time followed by total RNA isolation. Levels of transcripts were analysed by real time RT PCR with specific primers and were presented as relative values normalized to the level of GAPDH mRNA. n = 3; the error bars represent standard deviations. The asterisks indicate significant difference from control (0 h), *p<0.05, or between indicated groups, **p<0.05, ns - not significant difference between groups (p>0.05). <b>B</b>. MDA-MB-231 cells were treated with RL2 (0.2 mg/mL) followed by preparation of whole-cell lysates, which were then subjected to Western blot with the indicated antibodies. Tubulin was used as the internal control. Results shown are representative of five independent experiments.</p

Separation of proteins of RL2-bound fraction.

<p>Coomassie stained gel. RL2-bound fractions of MCF-7 lysates were prepared as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093921#s2" target="_blank">Materials and methods</a>’ section and then were separated by 15% SDS-PAGE gel. Bands indicated (*) were excised followed by in-gel trypsin digestion for mass spectrum analysis. Other bands were not investigated. Shown is representative gel of two independent experiments. Lane 1 – fraction eluted from non-modified Sepharose (control); Lane 2 – fraction eluted from RL2-modified Sepharose with 300 mM NaCl, flow rate was 1 mL/min and detection wavelength was 280 nm. M – molecular weights marker (14.4–116.0 kDa).</p

Enhanced cytotoxic outcome in combination of RL2 with CQ.

<p>MDA-MB-231 cells were used as a model. <b>A</b>. Dose-dependence of CQ cytotoxicity was calculated using MTT data of three independent experiments. Shown are mean values ± SD. <b>B</b>. Cells were treated with different concentrations of RL2 in the presence or absence of CQ, and after 48 h cell viability was measured by MTT assay. <b>C</b>. Cells were incubated with RL2 (0.2 mg/mL), CQ (5 μM) or a combination for 24 h and the percentage of cells with active caspase -3,-7 was analysed using flow cytometry. Shown are mean values ± SD of three independent experiments. The asterisks indicate significant difference from control (*, p<0.05). <b>D</b>. Cells were incubated with RL2 (0.2 mg/mL), CQ (5 μM) or a combination for 24–48 h. Cathepsin D activity was analysed using a fluorescence substrate (labeled with methyl cumaryl amide) by fluorimetry as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093921#s2" target="_blank">Materials and methods</a>’ section. RFU – relative fluorescence units per microgram protein of sample. Shown are mean values ± SD of three independent experiments. The asterisks indicate significant difference between indicated groups (**, p<0.05), ns - not significant difference between groups (p>0.05).</p

RL2 treatment induces autophagic changes in MDA-MB-231 cells.

<p><b>A.</b> Cells were treated with RL2 (0.2 mg/mL) or non-treated (control) for 8 h and fixed with 4% paraformaldehyde followed by immunostaining with anti-LC3A/B primary and FITC-conjugated secondary antibody. Images are representative of two independent experiments. <b>B</b>. MDA-MB-231 cells were treated with RL2 (0.2 mg/mL) for various time points (0, 2, 6 and 24 h). Total lysates were prepared and subjected to Western blot analysis. Shown are representative blots of five independent experiments.</p