Dynamics of Shigella interaction with the epithelium in the infection process

The study of the adhesion of Shigella flexneri to intestinal mucosal explants from human postabortion fetuses, used as an experimental model, has revealed that the process of interaction between the infective agent and the epithelium develops in accordance with Langmuir's equation of the adsorption isotherm. The specific biological feature of the adhesive interaction between bacteria and the mucous membrane is the fact that the effective adhesion of microbes is possible only in case of their high concentration on the surface of the mucous membrane. In case of their low concentration in the parietal layer no microbial adhesion is observed, whereas epithelial villi infected with shigellae and fixed to the mucous membrane of the explant produce a high parietal concentration of the infective agents, which leads to the increase of adhesion by more than two orders.     

Role of apoptosis in the regulation of the infectious process

Review on the influence of pathogenic bacteria on the apoptosis program of host cells. Many pathogenic bacteria posess mechanisms to control cell death, which makes it possible to maintain the optimum conditions for the development of infection. The mechanisms of the induction and suppression of apoptosis, worked out by bacteria, are very variable and realized as the result of complex interaction of biologically active bacterial molecules with concrete targets of signal paths leading to apoptosis. In case of intracellular parasitism the antiapoptosis activity of bacteria may be regarded as one of the mechanisms of the persistence of infective agents, ensuring the development of chronic infections.     

The action of ribosomal preparations on nonspecific resistance to bacterial infection and on early tolerance to endotoxic shock

Ribosomal preparations from Shigella flexneri and Shigella sonnei, introduced parenterally into mice, enhance their resistance to infection with the causative agents of typhoid fever and staphylococci. This effect is considerably less pronounced than that produced by the preparation of homologous lipopolysaccharide isolated by Boivin's method. After the administration of ribosomes nonspecific resistance to bacterial infective agents lasts for a short time. Ribosomal preparations do not enhance the resistance of mice to the lethal action of endotoxin.     

Bioluminescent signal system: bioluminescence immunoassay of pathogenic organisms.

The Ca(2+)-regulated photoprotein obelin has been examined as a label for bioluminescence immunoassay of infective agents. The hepatitis B virus (HbsAg) and the bacteria Escherichia coli and Shigella sonnei lipopolysaccharide (LPS) were chosen as model antigens. Chemically synthesized obelin-corresponding antibody conjugates were used in a solid-phase microplate immunoassay. The sensitivities achieved by the assay were 0.25 ng/mL for S. sonnei LPS and 0.375 ng/mL for HbsAg. A novel, filter-based immunoassay to determine bacterial admixtures in the environment was proposed. The NanoCeram filters were effectively applied to 'trap' and pre-concentrate pathogens from samples under study for the purposes of further detection and measurement of the absorbed material by bioluminescence immunoassay.     

Development of the controlled model of persisting infection , caused by Pseudomonas aeruginosa and bacteria of the complex Burkholderia cepacia

As the result of testing three different variants, the experimental models of persisting infection for P. aeruginosa and B. cepacia have been developed. These doses differ in the time of administration, doses of antibiotics and the infective doses of the microorganisms. The administration of the sub-inhibiting concentration of antibiotics for 5 days and the subsequent infection of laboratory animals (non-inbred mice) B. cepacia strains in a dose of LD50 leads to a considerable increase in the survival rate of mice and to a longer period (up to 20 days) of obtaining inoculative material from the spleen. The isolated cultures are characterized by a sharply slower growth on artificial culture media (up to 5-7 days as compared with 24-48 hours for the initial culture). The newly developed models have made it possible to control different stages of the infectious process in the induced increase or decrease of the virulent properties of the infective agent and in changes in the immune status of the host. As the result of these studies, in some mice (10%) infected with B. cepacia after the injection of gamma-hydroxybutyric acid lactone the infection has taken the acute form, while in the mice infected with P. aeruginosa no such effect has been observed. On the contrary, in the mice infected with P. aeruginosa and then receiving cyclophosphamide the transition of the infection into the acute form has been observed in 30% of the animals. In the mice infected with B. cepacia no such effect has been noted after the injection of this preparation. Different effects produced by cyclophosphamide and lactone are discussed from the positions of "quorum sensing" in pathogenic bacteria.     

A Virus from Epidemic Vomiting Disease

An illness consisting of vomiting, fever, and mild diarrhoea after a short incubation period was observed in a boarding school and reproduced in volunteers who received filtered extracts of faeces from a typical case. The main discriminatory diagnostic feature of the illness in volunteers was vomiting. The faeces contained no pathogenic bacteria nor any virus that could be detected in tissue cultures and there was no evidence that an infective agent could be grown in organ cultures of human intestine. The agent was shown to be ether stable and passed a 50-nm filter. Laboratory studies on another agent of uncertain significance and obtained in other epidemics are briefly described.     

Natural-focus infections: the key questions and new vantage points

A short history of the concept of natural focal infections is presented: the idea put forward by D. K. Zabolotny?, E. I. Pavlovski?'s teaching, 3 stages of its development. A number of fundamental questions and the modern content of the concept are considered. The natural foci of infections are a combination of surface, soil and/or water ecosystems, including the population of the causative agent of infection. In contrast to surface ecosystems, in soil and water ecosystems the hosts of the causative agents of sapronotic infections ae soil invertebrates and hydrobios, in which these agents may circulate in biocenotic trophic chains. The circulation of the causative agents in natural foci is a discrete process; the mechanisms and forms of the existence of pathogenic bacteria during seasonal and prolonged periods between epidemics is considered. Special attention is given to latent (nonculturable) forms of bacteria. The complex character of the status of the causative agents of natural focal infections is discussed.     

Effect of the influenza A virus on the sensitivity of mice to infection caused by Streptococcus group B

Study of the capacity of group B streptococci for causing the development of infection in mice has revealed the virulence of the cultures for mice to be determined by the serovar of the streptococcus, the infective dose, and the amount of type-specific polysaccharide. Under the conditions of mixed viral-bacterial infection, influenza A virus was shown to influence the development of bacterial infection in the animals in two ways: to increase the virulence of an avirulent strain and to decrease the pathogenicity of a virulent one in streptococcal monoinfections. Simultaneously with viral infection, the stimulation of the multiplication of an avirulent strain in the lungs of mice was observed, while in the control groups of the animals the elimination of bacteria from the lungs was registered. No additional accumulation of the infective virus in the lungs of mice in the presence of streptococci was found.     

A novel caspase-1/toll-like receptor 4-independent pathway of cell death induced by cytosolic Shigella in infected macrophages

Shigella-induced macrophage cell death is an important step in the induction of acute inflammatory responses that ultimately lead to bacillary dysentery. Cell death was previously reported to be dependent upon the activation of caspase-1 via interaction with IpaB secreted by intracellular Shigella, but in this study, we show that Shigella infection of macrophages can also induce cell death independent of caspase-1 or IpaB activity. Time-lapse imaging and electron microscopic analyses indicated that caspase-1-dependent and -independent cell death is morphologically indistinguishable and that both resemble necrosis. Analyses of Shigella mutants or Escherichia coli using co-infection with Listeria suggested that a component common to Gram-negative bacteria is involved in inducing caspase-1-independent cell death. Further studies revealed that translocation of bacterial lipid A into the cytosol of macrophages potentially mediates cell death. Notably, cell death induced by cytosolic bacteria was TLR4-independent. These results identify a novel cell death pathway induced by intracellular Gram-negative bacteria that may play a role in microbial-host interactions and inflammatory responses.     

Experimental study of the preventive properties of immune milk containing Shigella antibodies

The protective properties of the mammary gland secretions of cows immunized with Shigella sonnei into the udder were studied. As a model for this study the intranasal and intraperitoneal infection of white mice was used. Immune milk was found to have pronounced protective properties against S. sonnei. When introduced intraperitoneally, this milk protected the animals infected with S. sonnei from death. When introduced intranasally, it not only protected the animals from death, but perceptibly inhibited the development of the pathological process in the pulmonary tissue, preventing the multiplication of shigellae and accelerating the elimination of the infective agents from the lungs of the infected animals. As a rule, the degree of protective action was determined by the level of antibodies to shigellae in the substrate under test.     

Shigella protein IpaH(9.8) is secreted from bacteria within mammalian cells and transported to the nucleus.

Various pathogenic bacteria such as Shigella deliver effector proteins into mammalian cells via the type III secretion system. The delivered Shigella effectors have been shown to variously affect host functions required for efficient bacterial internalization into the cells. In the present study, we investigated the IpaH proteins for their ability to be secreted via the type III secretion system and their fate in mammalian cells. Upon incubation in a medium containing Congo red, the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD. Immunofluorescence microscopy indicated that IpaH(9.8) is secreted from intracellular bacteria and transported into the nucleus. On microinjection of the protein, intracellular IpaH(9.8) is accumulated at one place around the nucleus and transported into the nucleus. This movement seems to be dependent on the microtubule network, since nuclear accumulation of IpaH(9.8) is inhibited in cells treated with microtubule-destabilizing agents. In nuclear import assay, IpaH(9.8) was efficiently transported into the nucleus, which was completely blocked by treatment with wheat germ agglutinin. The nuclear transport of IpaH(9.8) does not depend on host cytosolic factors but is partially dependent on ATP/GTP, suggesting that, like beta-catenin, IpaH(9.8) secreted from intracellular Shigella can be transported into the nucleus.     

Epizootic characteristic of rabies today

During the 23 year period rabies was registered in the Moscow region in 163 cases among foxes, 22 cases among racoon-like dogs, 92 cases among dogs, 54 cases among cats. In 1991-2000 ten cases of rabies were registered among other wild animal other than foxes and raccoon-like dogs (hares, hedge-hogs, polecats, badgers, hamsters, martens, rats). Under today conditions the generalized epizootological pattern of rabies is characterized by the vector "natural foci-->anthropurgic foci" with wild and domestic animals playing an alternative role in the epizootic process and the circulation of the infective agent. Wild carnivorous animals maintain natural focal infection in time and space, while all domestic animals are a direct or indirect ecological impasse and took no part in the maintenance of the infection. Foxes are the main source of infection for the animals of the anthropurgic cycle: they play a special role in the development of the epizootic situation in the region as the main reservoir and source of the causative agent of rabies as a natural focal infection. Among other wild animals, raccoon-like dogs are involved into epizootic and epidemic chains. Dogs are the main objects to be infected in the anthropurgic cycles, while cats--a progressing group of risk. During the period of 25 years a decrease in the probability of natural rabies was noted.     

Shigellosis

Shigellosis is a global human health problem. Four species of Shigella i.e. S. dysenteriae, S. flexneri, S. boydii and S. sonnei are able to cause the disease. These species are subdivided into serotypes on the basis of O-specific polysaccharide of the LPS. Shigella dysenteriae type 1 produces severe disease and may be associated with life-threatening complications. The symptoms of shigellosis include diarrhoea and/or dysentery with frequent mucoid bloody stools, abdominal cramps and tenesmus. Shigella spp. cause dysentery by invading the colonic mucosa. Shigella bacteria multiply within colonic epithelial cells, cause cell death and spread laterally to infect and kill adjacent epithelial cells, causing mucosal ulceration, inflammation and bleeding. Transmission usually occurs via contaminated food and water or through person-to-person contact. Laboratory diagnosis is made by culturing the stool samples using selective/differential agar media. Shigella spp. are highly fragile organism and considerable care must be exercised in collecting faecal specimens, transporting them to the laboratories and in using appropriate media for isolation. Antimicrobial agents are the mainstay of therapy of all cases of shigellosis. Due to the global emergence of drug resistance, the choice of antimicrobial agents for treating shigellosis is limited. Although single dose of norfloxacin and ciprofloxacin has been shown to be effective, they are currently less effective against S. dysenteriae type 1 infection. Newer quinolones, cephalosporin derivatives, and azithromycin are the drug of choice. However, fluoroquinolone-resistant S. dysenteriae type 1 infection have been reported. Currently, no vaccines against Shigella infection exist. Both live and subunit parenteral vaccine candidates are under development. Because immunity to Shigella is serotype-specific, the priority is to develop vaccine against S. dysenteriae type 1 and S. flexneri type 2a. Shigella species are important pathogens responsible for diarrhoeal diseases and dysentery occurring all over the world. The morbidity and mortality due to shigellosis are especially high among children in developing countries. A recent review of literature (Kotloff et al.,1999) concluded that, of the estimated 165 million cases of Shigella diarrhoea that occur annually, 99% occur in developing countries, and in developing countries 69% of episodes occur in children under five years of age. Moreover, of the ca.1.1 million deaths attributed to Shigella infections in developing countries, 60% of deaths occur in the under-five age group. Travellers from developed to developing regions and soldiers serving under field conditions are also at an increased risk to develop shigellosis.     

Bacterial systems for the delivery of eukaryotic antigen expression vectors

Attenuated bacterial strains allow the administration of recombinant vaccines via the mucosal surfaces. Whereas attenuated bacteria are generally engineered to express heterologous antigens, a novel approach employs intracellular bacteria for the delivery of eukaryotic antigen expression vectors (so-called DNA vaccines). This strategy allows a direct delivery of DNA to professional antigen-presenting cells (APC), such as macrophages and dendritic cells (DC), through bacterial infection. The bacteria used for DNA vaccine delivery either enter the host cell cytosol after phagocytosis by the APC, for example, Shigella and Listeria, or they remain in the phagosomal compartment, such as Salmonella. Both intracellular localizations of the bacterial carriers seem to be suitable for successful delivery of DNA vaccine vectors.     

Bacterial autophagy

Autophagy is a vital process through which cellular material and dysfunctional organelles are degraded and recycled, and it is inhibited by the metabolic checkpoint kinase MTOR. Autophagy also targets intracellular bacteria (a process termed xenophagy) for lysosomal degradation, thereby playing a key role in innate immunity. In the past few years, the identification of molecules, such as CALCOCO2/NDP52, SQSTM1/p62 and ubiquitin, implicated in the specific targeting of intracellular bacteria, received considerable attention. However, it remains unclear how xenophagy is initiated, since this process commonly occurs in metabolically replete cells. In a recent study, we demonstrated that infection with Shigella and Salmonella triggered an early state of intracellular amino acid (AA) starvation causing MTOR dissociation from endomembranes, downregulation of MTOR activity and activation of the EIF2AK4/GCN2-EIF2S1/eIF2α/ATF3 signaling axis. We also observed that AA starvation was caused by host membrane damage, which appeared to be transient in the case of Salmonella and sustained in Shigella-infected cells, thus highlighting the existence of key timing disparities in xenophagy triggering, depending on the bacterial pathogen. Together, our findings demonstrate that xenophagy is only one arm of a more general metabolic switch geared toward AA starvation in bacteria-infected cells.     

Streptococcus pneumoniae multiplication in the allantoic cavity of developing chick embryos

The possibility of the active multiplication of S. pneumoniae (serotypes 1, 3, 6 and 19) in the allantoic cavity of chick embryos has been demonstrated. This multiplication is accompanied by the development of characteristic changes whose intensity and time of manifestation have been found to depend on the infective dose and the age of the embryo. The accumulation of S. pneumoniae in the allantoic cavity of chick embryos in the absence of visible changes in the biological properties of the infective agent after 5 successive subcultures makes it possible to recommend chick embryos as a model for the study of experimental pneumococcal infection.     

A marine bacterial adhesion microplate test using the DAPI fluorescent dye: a new method to screen antifouling agents

AIMS: To develop a method to screen antifouling agents against marine bacterial adhesion as a sensitive, rapid and quantitative microplate fluorescent test. METHODS AND RESULTS: Our experimental method is based on a natural biofilm formed by mono-incubation of the marine bacterium Pseudoalteromonas sp. D41 in sterile natural sea water in a 96-well polystyrene microplate. The 4'6-diamidino-2-phenylindole dye was used to quantify adhered bacteria in each well. The total measured fluorescence in the wells was correlated with the amount of bacteria showing a detection limit of one bacterium per 5 microm(2) and quantifying 2 x 10(7) to 2 x 10(8) bacteria adhered per cm(2). The antifouling properties of three commercial surface-active agents and chlorine were tested by this method in the prevention of adhesion and also in the detachment of already adhered bacteria. The marine bacterial adhesion inhibition rate depending on the agent concentration showed a sigmoid shaped dose-response curve. CONCLUSIONS: This test is well adapted for a rapid and quantitative first screening of antifouling agents directly in seawater in the early steps of marine biofilm formation. Significance AND IMPACT OF THE STUDY: In contrast to the usual screenings of antifouling products which detect a bactericidal activity, this test is more appropriate to screen antifouling agents for bacterial adhesion removal or bacterial adhesion inhibition activities. This screening test focuses on the antifouling properties of the products, especially the initial steps of marine biofilm formation.     

Delivery of biologically active anti-inflammatory cytokines IL-10 and IL-1ra in vivo by the Shigella type III secretion apparatus

Pathogenicity of many Gram-negative bacteria relies on a type III secretion (T3S) apparatus, which is used for delivery of bacterial effectors into the host cell cytoplasm allowing the bacteria to manipulate host cell cytoskeleton network as well as to interfere with intracellular signaling pathways. In this study, we investigated the potential of the Shigella flexneri T3SA as an in vivo delivery system for biologically active molecules such as cytokines. The anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist (IL-1ra) were genetically fused to the first 30 or 60 residues of the Shigella T3S effector IpaH9.8 or to the first 50 residues of the Yersinia enterocolitica effector YopE and the recombinant fusion proteins were expressed in S. flexneri. YopE(50)-IL-10, IpaH(60)-IL-10, and IpaH(60)-IL-1ra were efficiently secreted via the T3S apparatus of Shigella. Moreover, these recombinant proteins did not impair the invasive ability of the bacteria in vitro. In a murine model, Shigella strains expressing YopE(50)-IL-10, IpaH(60)-IL-10, and IpaH(60)-IL-1ra induced a lower mortality in mice that was associated with reduced inflammation and a restricted localization of bacteria within the lung tissues as compared with wild-type Shigella. Moreover, the level of TNF-alpha and IL-1beta mRNA were reduced in the lungs following infection by IL-10- and IL-1ra-secreting Shigella, respectively. These findings demonstrate that the Shigella T3S apparatus can deliver biologically active cytokines in vivo, thus opening new avenues for the use of attenuated bacteria to deliver proteins for immunomodulation or gene therapy purposes.     

Chemoprophylaxis of Shigella flexneri Keratoconjunctivitis in Rabbits

Chloramphenicol, chlortetracycline, streptomycin, and sulfisoxazole prevented keratoconjunctivitis in rabbit eyes infected with Shigella flexneri. The route of administration of the agent and the dose were important variables in controlling the infectious process. This assay system appears to be a useful technique for the in vivo evaluation of chemotherapeutic agents.     

Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.