The effect of growth temperature on the nanoscale biochemical surface properties of Yersinia pestis.

Yersinia pestis, the causative agent of plague, has been responsible for several recurrent, lethal pandemics in history. Currently, it is an important pathogen to study owing to its virulence, adaptation to different environments during transmission, and potential use in bioterrorism. Here, we report on the changes to Y. pestis surfaces in different external microenvironments, specifically culture temperatures (6, 25, and 37 °C). Using nanoscale imaging coupled with functional mapping, we illustrate that changes in the surfaces of the bacterium from a morphological and biochemical standpoint can be analyzed simultaneously using atomic force microscopy. The results from functional mapping, obtained at a single cell level, show that the density of lipopolysaccharide (measured via terminal N-acetylglucosamine) on Y. pestis grown at 37 °C is only slightly higher than cells grown at 25 °C, but nearly three times higher than cells maintained at 6 °C for an extended period of time, thereby demonstrating that adaptations to different environments can be effectively captured using this technique. This nanoscale evaluation provides a new microscopic approach to study nanoscale properties of bacterial pathogens and investigate adaptations to different external environments.

Structure and biophysics of type III secretion in bacteria.

Many plant and animal bacterial pathogens assemble a needle-like nanomachine, the type III secretion system (T3SS), to inject virulence proteins directly into eukaryotic cells to initiate infection. The ability of bacteria to inject effectors into host cells is essential for infection, survival, and pathogenesis for many Gram-negative bacteria, including Salmonella, Escherichia, Shigella, Yersinia, Pseudomonas, and Chlamydia spp. These pathogens are responsible for a wide variety of diseases, such as typhoid fever, large-scale food-borne illnesses, dysentery, bubonic plague, secondary hospital infections, and sexually transmitted diseases. The T3SS consists of structural and nonstructural proteins. The structural proteins assemble the needle apparatus, which consists of a membrane-embedded basal structure, an external needle that protrudes from the bacterial surface, and a tip complex that caps the needle. Upon host cell contact, a translocon is assembled between the needle tip complex and the host cell, serving as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. Following delivery into the host cytoplasm, effectors initiate and maintain infection by manipulating host cell biology, such as cell signaling, secretory trafficking, cytoskeletal dynamics, and the inflammatory response. Finally, chaperones serve as regulators of secretion by sequestering effectors and some structural proteins within the bacterial cytoplasm. This review will focus on the latest developments and future challenges concerning the structure and biophysics of the needle apparatus.

Physiological levels of glucose induce membrane vesicle secretion and affect the lipid and protein composition of Yersinia pestis cell surfaces.

Yersinia pestis grown with physiologic glucose increased cell autoaggregation and deposition of extracellular material, including membrane vesicles. Membranes were characterized, and glucose had significant effects on protein, lipid, and carbohydrate profiles. These effects were independent of temperature and the biofilm-related locus pgm and were not observed in Yersinia pseudotuberculosis.

Uniquely insidious: Yersinia pestis biofilms.

Bubonic plague, one of history's deadliest infections, is transmitted by fleas infected with Yersinia pestis. The bacteria can starve fleas by blocking their digestive tracts, which stimulates the insects to bite repeatedly and thereby infect new hosts. Direct examination of infected fleas, aided by in vitro studies and experiments with the nematode Caenorhabditis elegans, have established that Y. pestis forms a biofilm in the insect. The extracellular matrix of the biofilm seems to contain a homopolymer of N-acetyl-d-glucosamine, which is a constituent of many bacterial biofilms. A regulatory mechanism involved in Y. pestis biofilm formation, cyclic-di-GMP signaling, is also widespread in bacteria; yet only Y. pestis forms biofilms in fleas. Here, the historical background of bubonic plague is briefly described and recent studies investigating the mechanisms by which these unique and deadly biofilms are formed are discussed.

Inactivation of avirulent Yersinia pestis in Butterfield's phosphate buffer and frankfurters by UVC (254 nm) and gamma radiation.

Yersinia pestis is the causative agent of plague. Although rare, pharyngeal plague in humans has been associated with consumption or handling of meat prepared from infected animals. The risks of contracting plague from consumption of deliberately contaminated food are currently unknown. Gamma radiation is a penetrating form of electromagnetic radiation, and UVC radiation is used for decontamination of liquids or food surfaces. Gamma radiation D10-values (the radiation dose needed to inactivate 1 log unit pathogen) were 0.23 (+/-0.01) and 0.31 (+/-0.03) kGy for avirulent Y. pestis inoculated into Butterfield's phosphate buffer and onto frankfurter surfaces, respectively, at 0 degree C. A UVC radiation dose of 0.25 J/cm2 inactivated avirulent Y. pestis suspended in Butterfield's phosphate buffer. UVC radiation doses of 0.5 to 4.0 J/cm2 inactivated 0.97 to 1.20 log units of the Y. pestis surface inoculated onto frankfurters. A low gamma radiation dose of 1.6 kGy could provide a 5-log reduction and a UVC radiation dose of 1 to 4 J/cm2 would provide a 1-log reduction of Y. pestis surface inoculated onto frankfurters. Y. pestis was capable of growth on frankfurters during refrigerated storage (10 degrees C). Gamma radiation of frankfurters inhibited the growth of Y. pestis during refrigerated storage, and UVC radiation delayed the growth of Y. pestis.

Biogenesis of the fraction 1 capsule and analysis of the ultrastructure of Yersinia pestis.

Analysis of a Yersinia pestis Delta caf1A mutant demonstrated that the Caf1A usher is required for the assembly and secretion of the fraction 1 capsule. The capsule assembled into thin fibrils and denser aggregates on the bacterial surface. Pilus-like fibers were also detected on the surface of Y. pestis. The capsule occasionally coated these fibers, suggesting how the capsule may cloak surface features to prevent host recognition.

Structure and assembly of Yersinia pestis F1 antigen.

Most Gram negative pathogens express surface located fibrillar organelles that are used for adhesion to host epithelia and/or for protection. The assembly of many such organelles is managed by a highly conserved periplasmic chaperone/usher assembly pathway. During the last few years, considerable progress has been made in understanding how periplasmic chaperones mediate folding, targeting, and assembly of F1 antigen subunits into the F1 capsular antigen. In particular, structures representing snapshots of several of the steps involved in assembly have allowed us to begin to draw a detailed molecular-level picture of F1 assembly specifically, and of chaperone/usher-mediated assembly in general. Here, a brief summary of these new results will be presented.

Surface plasmon resonance imaging of pathogens: the Yersinia pestis paradigm.

BACKGROUND: Yersinia pestis, causing deadly plague, is classified as a group A bioterrorism bacterium. Some recent DNA-based methods were used for detection of bioterrorism agents. RESULTS: Y. pestis was used as a model organism to develop an immunosensor based on surface plasmon resonance imaging (SPRi) using monoclonal antibody against Y. pestis F1 antigen. The experimental approach included step-by-step detection of Y. pestis membrane proteins, lysed bacteria, intact bacteria, mock-infected powder and mock-infected clinical specimens. SPRi detected on average 10(6) intact Y. pestis organisms in buffer, in mock-infected powder and in a 1:4 mixture with HEL cells. CONCLUSIONS: This study offers the proof-of-concept of the SPRi-based detection of a human pathogen in both environmental and clinical specimens.

Bacterial filamentation of Yersinia pestis by beta-lactam antibiotics in experimentally infected mice.

OBJECTIVE: To identify alternatives to streptomycin for treating pneumonic plague, we evaluated beta-lactam antibiotics to treat experimental pneumonic plague in mice. METHODS: Mice were exposed to a lethal inhaled dose of Yersinia pestis and treated with beta-lactam antibiotics administered every 6 hours, starting 42 hours postexposure. RESULTS: The mice died or were euthanized in extremis 3 days postexposure. We observed marked bacterial filamentation of Y pestis in the tissues of mice treated with ceftazidime (10/10 mice), aztreonam (9/10 mice), or ampicillin (1/10 mice), but not in the tissues of mice treated with cefotetan, cefazolin, ceftriaxone, or saline. There was no evidence of septation of the filamentous bacteria by light or electron microscopy. The filamentous bacteria were confirmed as Y pestis based on their reactivity with rabbit anti-Y pestis F1 serum. CONCLUSIONS: Marked bacterial filamentation of Y pestis can be produced in vivo by certain beta-lactam antibiotics. This antibiotic-induced morphologic change is important because filamentous bacteria in clinical samples could possibly be confused with filamentous actinomycotic organisms.

[Translocation of transposons Tn10 and Tn5 in the Yersinia pestis chromosome]. / Translokatsiia transpozonov Tn10 i Tn5 v khromosomu Yersinia pestis.

The possibility of translocation of the transposons Tn5 and Tn10 into the genome of Yersinia pestis, with the subsequent mutagenic effect was demonstrated. We revealed transposon harbouring clones at frequency 10(-4) to 10(-2). Derivatives of P1cml clr100ts phage served as vectors. Insertion of Tn10 transposon induced mutations in ilv, ser, arg, pur, pro, leu, nic, tyr, gua genes. The number of the insertion sites on the chromosome obtained for Tn5 was the same, these being arg, ade, pyr, leu, gua, trp, his, pan, ilv. The majority of auxotrophs did not revert. Occasionally, revertants were observed at frequencies 10(-8) to 10(-6). Unlike Escherichia coli, reversion was not accompanied by the loss of transposons. The rearrangements induced by transposons, presumably, near the insertion site, as well as duplications of transposons followed by incorporation of copies into novel sites, led to the appearance of additional defective genes, which made it possible to select various types of polyauxotrophs. Based on reiteration of coinciding double and triple mutant markers, we proposed a linkage group of genes within a segment of Y. pestis chromosome: lys ... tyr - ser - arg - ilv - leu - gua - ade(pur) - pro ... his ... pyr ... trp. The reasons for peculiarities of the behaviour of transposons in Y. pestis bacteria are discussed.

[Examining interaction of phages with microorganisms by fluorometry and electro-orientation spectroscopy]. / Izuchenie vzaimodeistvii fagov i mikroorganizmov s ispol'zovaniem metodov fliuorimetrii i élektroorientatsionni spektroskopii.

Bacterial sensitivity to different various phages was examined by electro-orientation spectroscopy, fluorometry, and electron microscopy. The strains of Pseudomonas aeruginosa, Staphylococcus aureus, Yersinia pestis, Mycobacterium smegmatis, and Xanthomonas campestris were used. The fluorescence intensity of a membranotropic agent in the ANS-cell-phage system was shown to depend on the interaction of a bacterial virus and a microorganism. Fluorometric data correlated with electro-orientation spectroscopic findings. An analysis of the low-frequency site makes it possible to determine phage adsorption on the bacterial surface. The changes in electro-orientation effects at high frequencies suggest that there are barrier dysfunctions in the external membranes and that there is cellular phage reproductions. Whether fluorometry and electro-orientation spectroscopy can be further used for rapid identification of microorganisms by using phages is discussed.

[Electron microscopic study of dry live plague vaccine EB by the scanning method]. / Elektronno-mikroskopicheskoe issledovanie chumnoi zhivoi sukhoi vaktsiny EB metodom skanirovaniia

Dry live plague vaccine EB was examined under microscope; it appeared that in the mentioned preparation the bacterial cells were enclosed in the artificial sucrose-gelatin capsule of the stabilizer which apparently maintained the vital activity of the microorganisms in the state of anabiosis by forming a stable bond with the bacterial body.

[Associations of the soil amoeba Hartmannella rhysodes with the bacterial causative agents of plague and pseudotuberculosis in an experiment]. / Izuchenie assotsiatsii pochvennykh ameb Hartmannella rhysodes s bakteriiami--vozbuditeliami chumy i psevdotuberkuleza v éksperimente.

Some aspects of relationships between soil ameba and the causative agents of plague and pseudotuberculosis, capable of forming natural associations, are considered. Ameba can phagocytose bacteria causing plague and pseudotuberculosis. Cases of the preservation of individual bacterial cells at the stationary phase and in precysts of amebae have been registered.

[The variability of Yersinia pestis in the body of the flea]. / Izmenchivost' Yersinia pestis v organizme blokhi.

Changes in the morphology and ultrastructure of Y.pestis cells at different periods of their stay in the body of fleas (Citellophilus tesquorum altaicus) have been studied. The study, carried out by means of optical and electron microscopy, as well as with the use of a culture medium for the isolation of L-forms, has revealed that in the body of fleas Y.pestis cells undergo the effect of processes leading to their L-transformation. As the result of L-transformation, the infective agent may take altered forms, including L-like variants. Such forms are retained in hungry insects and are capable of prolonged survival in the body of the carrier.

[Localization of antigen F-1 of Yersinia pestis EV by an immunoferritin method]. / Izuchenie lokalizatsii antigena F-1 Yersinia pestis EV immunoferritinovym metodom.

The localization of antigen F-1 in Y. pestis strain EV, line (formula; see text) has been studied by means of specific monoreceptor ferritin-labeled antibodies. The study has given direct proofs that antigen F-1 is localized on the surface of the cell wall and in bacterial microcapsules. The substance of the microcapsule is loosely bound with the cell body and easily diffuses into the intercellular space, the antigenic properties of the substance remaining unchanged.

[Ultrastructural changes in plague microbes of vaccinal strain EB and alveolar macrophages of guinea pigs during their interaction in vitro]. / Ul' trastrukturnye izmeneniia v chumnykh mikrobakh vaktsinnogo shtamma EB i al'veoliarnykh makrofagakh morskikh svinok pri vzaimodeistvii ikh in vitro.

Aerosol immunization with plague vaccine EB was found to stimulate an increase in the number of free alveolar macrophages and to enhance their ingesting activity which correlated with an increase in specific antibody titers. Immunological transformation was accompanied by ultrastructural changes in the cytoplasm and the nuclei of alveolar macrophages. The phagocytosis of microbes occurred by their adsorption onto the microphage surface, invagination and enclosing into the endocytic bubble having the function of the digestive vacuole. The growth of the number of macrophages and an increase in their ingesting activity, progressive changes in the cytoplasmic structures may be used as indicators of immunological transformation resulting from the administration of plague vaccine by the inhalation method.

[Ultrastructural changes in plague microbes, strain EV, and splenic cells of guinea pigs in their interaction in vitro]. / Ul'trastrukturnye izmeneniia chumnykh mikrobov shtamma EV i kletok selezenki morskikh svinok pri vzaimodeistvii in vitro.

Dilatation of the endoplasmic reticulum cavities, an increase in the number of ribosomes near bacteria, deformation of mitochondria and coarsening of cristae were revealed in phagocytosis of Past. pestis, strain EV, by reticular cells in the tissue culture of the spleen of intact guinea pigs. Lipophanerous "reticular" inclusions were found in the differentiated reticular cells of the infected cultures. In the reticular cell cytoplasma besides the intact bacteria there were revealed Past. pestis at the initial stages of involution.

[Adhesion pili in Yersinia pestis]. / Pili adgezii u Yersinia pestis.

Y. pestis cells cultivated at 37 degrees C are capable of agglutinating red blood cells of some animals, which is due to the appearance of pili. The adhesion pili consist of protein subunits with a molecular weight of the order of 12000 daltons, their isoionic point being at pH 4.7. The reaction of hemagglutination was inhibited by the mixture of ganglyosides, while the preliminary treatment of red blood cells with neuraminidases increased its effectiveness. The pili are supposed to take part in the expression of virulence.

[The fibronectin-binding capacity of Yersinia pestis]. / Fibronektinsviazyvaiushchaia sposobnost' Yersinia pestis]

Y. pestis cells have been shown capable of binding fibronectin (FN), the presence of adhesion pili considerably enhancing FN binding. The study has established that, along with FN, native adhesive pili, but not subunits, are capable of binding mucin and ganglioside. Structures similar to FN-binding curlings of Escherichia have been found on the surface of Y. pestis cells. The expression of curling-like structures does not depend on the presence of plasmids in Y. pestis cells.

[The plasmid profile of Yersinia pestis strains in different natural foci of plague in the People's Republic of China]. / Plazmidnyi profil' shtammov Yersinia pestis v razlichnykh prirodnykh ochagakh chumy Kitaiskoi Narodnoi Respubliki.

A total of 159 Y. pestis strains isolated in different geographical regions of China have been studied. Y. pestis isolated in the majority of natural foci of plague in China carry plasmids with molecular weights of 6, 13, 15 and 63 MD. One isolated strain has been found to carry plasmid with a molecular weight of 36 MD. Only a few strains have no plasmids with molecular weights of 6 and 45 MD. In certain geographical zones strains carrying large plasmids of 52 and 92 MD have been detected.