Analysis of production levels of InlA and InlB invasion factors in Listeria monocytogenes isolates collected in the Russian Federation

Background. Listeria monocytogenes is characterized by the presence of epidemic hypervirulent clones. A key feature of L. monocytogenes is its capacity to invade non-professional phagocytic cells. Hypervirulent clones are strongly associated with the increased production and/or the presence of certain isoforms of invasion factors InlA and InlB. The purpose of the study is to create a test system for InlA and InlB detection and to measure the InlA and InlB production levels in L. monocytogenes isolates belonging to clonal groups with different virulence potential. Materials and methods. The study was performed using 32 L. monocytogenes strains belonging to epidemic clones ECII, ECIV, ECVII (clonal complexes CC1, CC2, CC7) and hypovirulent clonal complex CC9. Sequencing of inlA and inlB genes was performed. The indirect enzyme-linked immunosorbent assay was used to analyze the production levels of InlA and InlB proteins. Results. The variability of InlA was revealed among strains belonging to the same clonal complex: 3 InlA isoforms were identified among strains belonging to CC7; out of 8 strains belonging to CC9, one strain had a stop codon in the inlA gene, leading to the loss of function of the InlA protein. The differences between inlB alleles correlated with the specificity of strains belonging to a certain clonal complex. Differences in production levels of invasion factors were measured. In strains belonging to CC9, the InlA production level was 2.5 times as low compared to strains belonging to CC1, CC2, and CC7. In strains belonging to phylogenetically related CC1 and CC2, the InlB production level was on average 4 times as high compared to strains belonging to CC7 and CC9. Conclusion. The obtained results confirm the variability of major invasion factors both among clonal complexes and strains of the same complex. The increased production of invasion factors InlA and InlB correlates with the potential virulence of strains

OBTAINING THE RECOMBINANT PHOSPHATIDIL-INOSITOL-SPECIFIC PHOSPHOLIPASE C LISTERIA MONOCYTOGENES AND EVALUATION OF ITS DIAGNOSTIC IMPORTANCE

The purpose of the work: studying the expression of the cloned gene phosphatidil-inositol-specifis phospholipase C L. monocytogenes in E.coli and possibilities of its use as the species-specific marker. The processes of transcription and translation of the cloned gene Phospholipase L. monocytogenes in E.coli have been studied, the hybride protein Protein A::phospholipase, and it has been shown, that in its composition the phospholipase preserves its antigenous determinatns, the strain-specific differences in the level of production of phospholipase by the lysterias have been revealed. The two amplification systems have been developed for revealation of L.monocytogenes. The results of the investigations have been used in the Moscow Medical Academy. The field of application: the microbiology and epidemiologyAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

ANTHROPOMETRICAL INDEXES OF PUPILS OF RURAL MUNICIPAL ESTABLISHMENTS IN ULYANOVSK REGION

Physical development is one of objective indicators of a state of health of the population that changes now very sharply, as well as other indicators (diseases, death rate, infantile death rate, average life expectancy, etc.). Methods of the statistical accounting and analysis of the data about physical development of the population have received a deep scientific substantiation and are widely used in practical research activity of establishments of public health services. As a result of research the assessment of anthropometrical indicators of schoolboys of rural municipal unions of the Ulyanovsk region is made. Researches were made in the territory of the Ulyanovsk region from 2008 till 2011. Eight areas with different quality of social and ecological factors of environment have been chosen for the research. Among schoolboys of the senior and average age group schoolboys from only two areas have harmonious development

<it>Listeria monocytogenes </it>virulence factor Listeriolysin O favors bacterial growth in co-culture with the ciliate <it>Tetrahymena pyriformis</it>, causes protozoan encystment and promotes bacterial survival inside cysts

<p>Abstract</p> <p>Background</p> <p>The gram-positive pathogenic bacterium <it>Listeria monocytogenes </it>is widely spread in the nature. <it>L. monocytogenes </it>was reported to be isolated from soil, water, sewage and sludge. Listeriolysin O (LLO) is a <it>L. monocytogenes </it>major virulence factor. In the course of infection in mammals, LLO is required for intracellular survival and apoptosis induction in lymphocytes. In this study, we explored the potential of LLO to promote interactions between <it>L. monocytogenes </it>and the ubiquitous inhabitant of natural ecosystems bacteriovorous free-living ciliate <it>Tetrahymena pyriformis</it>.</p> <p>Results</p> <p>Wild type <it>L. monocytogenes </it>reduced <it>T. pyriformis </it>trophozoite counts and stimulated encystment. The effects were observed starting from 48 h of co-incubation. On the day 14, trophozoites were eliminated from the co-culture while about 5 × 10⁴cells/ml remained in the axenic <it>T. pyriformis </it>culture. The deficient in the LLO-encoding <it>hly </it>gene <it>L. monocytogenes </it>strain failed to cause mortality among protozoa and to trigger protozoan encystment. Replenishment of the <it>hly </it>gene in the mutant strain restored toxicity towards protozoa and induction of protozoan encystment. The saprophytic non-haemolytic species <it>L. innocua </it>transformed with the LLO-expressing plasmid caused extensive mortality and encystment in ciliates. During the first week of co-incubation, LLO-producing <it>L. monocytogenes </it>demonstrated higher growth rates in association with <it>T. pyriformis </it>than the LLO-deficient isogenic strain. At latter stages of co-incubation bacterial counts were similar for both strains. <it>T. pyriformis </it>cysts infected with wild type <it>L. monocytogenes </it>caused listerial infection in guinea pigs upon ocular and oral inoculation. The infection was proved by bacterial plating from the internal organs.</p> <p>Conclusions</p> <p>The <it>L. monocytogenes </it>virulence factor LLO promotes bacterial survival and growth in the presence of bacteriovorous ciliate <it>T. pyriformis</it>. LLO is responsible for <it>L. monocytogenes </it>toxicity for protozoa and induction of protozoan encystment. <it>L. monocytogenes </it>entrapped in cysts remained viable and virulent. In whole, LLO activity seems to support bacterial survival in the natural habitat outside of a host.</p

Experimental Listeria–Tetrahymena–Amoeba food chain functioning depends on bacterial virulence traits

Abstract Background Some pathogenic bacteria have been developing as a part of terrestrial and aquatic microbial ecosystems. Bacteria are consumed by bacteriovorous protists which are readily consumed by larger organisms. Being natural predators, protozoa are also an instrument for selection of virulence traits in bacteria. Moreover, protozoa serve as a “Trojan horse” that deliver pathogens to the human body. Here, we suggested that carnivorous amoebas feeding on smaller bacteriovorous protists might serve as “Troy” themselves when pathogens are delivered to them with their preys. A dual role might be suggested for protozoa in the development of traits required for bacterial passage along the food chain. Results A model food chain was developed. Pathogenic bacteria L. monocytogenes or related saprophytic bacteria L. innocua constituted the base of the food chain, bacteriovorous ciliate Tetrahymena pyriformis was an intermediate consumer, and carnivorous amoeba Amoeba proteus was a consumer of the highest order. The population of A. proteus demonstrated variations in behaviour depending on whether saprophytic or virulent Listeria was used to feed the intermediate consumer, T. pyriformis. Feeding of A. proteus with T. pyriformis that grazed on saprophytic bacteria caused prevalence of pseudopodia-possessing hungry amoebas. Statistically significant prevalence of amoebas with spherical morphology typical for fed amoebas was observed when pathogenic L. monocytogenes were included in the food chain. Moreover, consumption of tetrahymenas fed with saprophytic L. innocua improved growth of A. proteus population while L. monocytogenes-filled tetrahymenas provided negative effect. Both pathogenic and saprophytic bacteria were delivered to A. proteus alive but only L. monocytogenes multiplied within amoebas. Observed differences in A. proteus population behaviour suggested that virulent L. monocytogenes might slow down restoration of A. proteus ability to hunt again and thus restrict the size of A. proteus population. Comparison of isogenic bacterial pairs that did or did not produce the haemolysin listeriolysin O (LLO) suggested a role for LLO in passing L. monocytogenes along the food chain. Conclusions Our results support the idea of protozoa as a means of pathogen delivery to consumers of a higher order and demonstrated a dual role of protozoa as both a “Trojan horse” and “Troy.

Phylogenetically Defined Isoforms of Listeria monocytogenes Invasion Factor InlB Differently Activate Intracellular Signaling Pathways and Interact with the Receptor gC1q-R

The pathogenic Gram-positive bacterium Listeria monocytogenes has been evolving into a few phylogenetic lineages. Phylogenetically defined substitutions were described in the L. monocytogenes virulence factor InlB, which mediates active invasion into mammalian cells via interactions with surface receptors c-Met and gC1q-R. InlB internalin domain (idInlB) is central to interactions with c-Met. Here we compared activity of purified recombinant idInlB isoforms characteristic for L. monocytogenes phylogenetic lineage I and II. Size exclusion chromatography and intrinsic fluorescence were used to characterize idInlBs. Western blotting was used to study activation of c-Met-dependent MAPK- and PI3K/Akt-pathways. Solid-phase microplate binding and competition assay was used to quantify interactions with gCq1-R. Isogenic recombinant L. monocytogenes strains were used to elucidate the input of idInlB isoforms in HEp-2 cell invasion. Physicochemical parameters of idInlB isoforms were similar but not identical. Kinetics of Erk1/2 and Akt phosphorylation in response to purified idInlBs was lineage specific. Lineage I but not lineage II idInlB specifically bound gC1q-R. Antibody against gC1q-R amino acids 221&ndash;249 inhibited invasion of L. monocytogenes carrying lineage I but not lineage II idInlB. Taken together, obtained results suggested that phylogenetically defined substitutions in idInlB provide functional distinctions and might be involved in phylogenetically determined differences in virulence potential

Route of Injection Affects the Impact of InlB Internalin Domain Variants on Severity of Listeria monocytogenes Infection in Mice

The facultative intracellular pathogen Listeria monocytogenes causes a severe food-borne infection in humans and animals. L. monocytogenes invasion factor InlB interacts with the tyrosine kinase c-Met via the N-terminal internalin domain. Previously, distinct variants of the InlB internalin domain (idInlB) have been described in L. monocytogenes field isolates. Three variants were used to restore full-length InlB expression in the L. monocytogenes strain EGDeΔinlB. Obtained isogenic L. monocytogenes strains were tested in the invasion assay and intravenous, intraperitoneal, and intragastric models of infection in mice. All idInlBs were functional, restored InlB activity as an invasion factor, and improved invasion of the parental strain EGDeΔinlB into human kidney HEK23 cells. Meanwhile, distinct idInlBs provided different mortality rates and bacterial loads in internal organs. When recombinant strains were compared, the variant designated idInlB14 decreased severity of disease caused by intravenous and intraperitoneal bacterial administration, whereas this variant improved intestine colonization and stimulated intragastric infection. Obtained results demonstrated that naturally occurring idInlBs differed in their impact on severity of L. monocytogenes infection in mice in dependence on the infection route

Experimentally Created Magnetic Force in Microbiological Space and On-Earth Studies: Perspectives and Restrictions

Magnetic force and gravity are two fundamental forces affecting all living organisms, including bacteria. On Earth, experimentally created magnetic force can be used to counterbalance gravity and place living organisms in conditions of magnetic levitation. Under conditions of microgravity, magnetic force becomes the only force that moves bacteria, providing an acceleration towards areas of the lowest magnetic field and locking cells in this area. In this review, we consider basic principles and experimental systems used to create a magnetic force strong enough to balance gravity. Further, we describe how magnetic levitation is applied in on-Earth microbiological studies. Next, we consider bacterial behavior under combined conditions of microgravity and magnetic force onboard a spacecraft. At last, we discuss restrictions on applications of magnetic force in microbiological studies and the impact of these restrictions on biotechnological applications under space and on-Earth conditions