Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins.

Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. SIGNIFICANCE: Enterococcal infections, especially bacteremia and endocarditis, are challenging to treat because E. faecium have acquired resistance to multiple classes of antimicrobials, including ampicillin, aminoglycosides, and glycopeptides. Thus, research on different modes of enterococcal pathogenicity is warranted. This study utilized a proteomic approach to identify MV-associated proteins of different nosocomial E. faecium strains representing four clinically relevant sequence types (STs), namely ST17, ST18, ST78, and ST192. The presented data suggest that E. faecium MVs are involved in virulence and antimicrobial resistance.

3D microfluidic liver cultures as a physiological preclinical tool for hepatitis B virus infection.

With more than 240 million people infected, hepatitis B virus (HBV) is a major health concern. The inability to mimic the complexity of the liver using cell lines and regular primary human hepatocyte (PHH) cultures pose significant limitations for studying host/pathogen interactions. Here, we describe a 3D microfluidic PHH system permissive to HBV infection, which can be maintained for at least 40 days. This system enables the recapitulation of all steps of the HBV life cycle, including the replication of patient-derived HBV and the maintenance of HBV cccDNA. We show that innate immune and cytokine responses following infection with HBV mimic those observed in HBV-infected patients, thus allowing the dissection of pathways important for immune evasion and validation of biomarkers. Additionally, we demonstrate that the co-culture of PHH with other non-parenchymal cells enables the identification of the cellular origin of immune effectors, thus providing a valuable preclinical platform for HBV research.

NOD-like receptor activation by outer membrane vesicles from Vibrio cholerae non-O1 non-O139 strains is modulated by the quorum-sensing regulator HapR.

Vibrio cholerae is an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. It has also emerged as an important cause of different kinds of inflammatory responses, and in particular, V. cholerae strains of the non-O1 non-O139 serogroups (NOVC) have been associated with such infections in human. We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by human epithelial cells and induced inflammatory responses. Small interfering RNA (siRNA)-mediated gene knockdown revealed that the inflammatory potential of NOVC OMVs was partially mediated by the nucleotide-binding domain-, leucine-rich repeat-containing family member NOD1. Physiochemical analysis of the content of these OMVs, in conjunction with NOD1 and NOD2 reporter assays in HEK293T cells, confirmed the presence of both NOD1 and NOD2 active peptidoglycan in the OMVs. Furthermore, we show that deletion of the quorum-sensing regulator HapR, which mimics an infective life style, specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. In conclusion, our study shows that NOVC OMVs elicit immune responses mediated by NOD1 and NOD2 in mammalian host cells. Moreover, we provide evidence that the quorum-sensing machinery plays an important regulatory role in this process by attenuating the inflammatory potential of OMVs under infective conditions. This work thus identifies a new facet of how Vibrio affects host immune responses and defines a role for the quorum-sensing machinery in this process.

Type 1 fimbriation and its phase switching in diarrheagenic Escherichia coli strains.

Type 1 fimbriae can be expressed by most Escherichia coli strains and mediate mannose-sensitive (MS) adherence to mammalian epithelial cells. However, the role of type 1 fimbriae in enteric pathogenesis has been unclear. Expression of type 1 fimbriae in E. coli is phase variable and is associated with the inversion of a short DNA element (fim switch). Forty-six strains of diarrheagenic E. coli were examined for the expression of type 1 fimbriae. Only four of these strains were originally type 1 fimbriated. Seventeen strains, originally nonfimbriated, expressed type 1 fimbriae in association with off-to-on inversion of the fim switch, after serial passages in static culture. The switching frequencies of these strains, from fimbriate to nonfimbriate, were greater than that of the laboratory strain E. coli K-12. None of the 16 strains of serovar O157:H7 or O157:H(-) expressed type 1 fimbriae after serial passages in static culture. The nucleotide sequence analysis of the fim switch region revealed that all of the O157:H7 and O157:H(-) strains had a 16-bp deletion in the invertible element, and the fim switch was locked in the "off" orientation. The results suggest that expression of type 1 fimbriae may be regulated differently in different E. coli pathogens causing enteric infections.

Heteromeric interactions among nucleoid-associated bacterial proteins: localization of StpA-stabilizing regions in H-NS of Escherichia coli.

The nucleoid-associated proteins H-NS and StpA in Escherichia coli bind DNA as oligomers and are implicated in gene regulatory systems. There is evidence for both homomeric and heteromeric H-NS-StpA complexes. The two proteins show differential turnover, and StpA was previously found to be subject to protease-mediated degradation by the Lon protease. We investigated which regions of the H-NS protein are able to prevent degradation of StpA. A set of truncated H-NS derivatives was tested for their ability to mediate StpA stability and to form heteromers in vitro. The data indicate that H-NS interacts with StpA at two regions and that the presence of at least one of the H-NS regions is necessary for StpA stability. Our results also suggest that a proteolytically stable form of StpA, StpA(F21C), forms dimers, whereas wild-type StpA in the absence of H-NS predominantly forms tetramers or oligomers, which are more susceptible to proteolysis.

Cytocidal and apoptotic effects of the ClyA protein from Escherichia coli on primary and cultured monocytes and macrophages.

Cytolysin A (ClyA) is a newly discovered cytolytic protein of Escherichia coli K-12 that mediates a hemolytic phenotype. We show here that highly purified ClyA and ClyA-expressing E. coli were cytotoxic and apoptogenic to fresh as well as cultured human and murine monocytes/macrophages.

A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila.

Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 degrees C reached the nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates. The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.

Resuscitation of viable but nonculturable cells of Vibrio parahaemolyticus induced at low temperature under starvation.

Vibrio parahaemolyticus is known to exist in a viable but nonculturable state when incubated at low temperature under starvation. It has long been debated whether the culturable cells which appear after temperature upshift are the result of true resuscitation or regrowth of a few residual culturable cells. Starved V. parahaemolyticus cells at 4 degrees C reached the nonculturable stage in about 12 days. The true resuscitation of nonculturable cells of V. parahaemolyticus occurred after spreading them onto an agar medium supplemented with H(2)O(2)-degrading compounds such as catalase or sodium pyruvate. The proposed method may be applicable to detecting the enteropathogen from environmental samples.

How Vibrio cholerae survive during starvation.

Vibrio cholerae, a Gram-negative, motile, aquatic bacterium, is the causal agent of the diarrheal disease cholera. Cholera is a serious epidemic disease that has killed millions of people and continues to be a major health problem world-wide. The hypothesis that V. cholerae occupies an ecological niche in the estuarine environment requires that this organism is able to survive the dynamics of physiochemical stresses, including nutrient starvation. As a result of these stresses, bacteria in nature often exist in non-growth or very slow growth states with a low metabolic activity. Because microorganisms have little ability to control their environment, environmental changes have led to changes in cell function and structure. Such cellular responses can originate in one of two ways: by changes in genetic constitution or by phenotypic adaptation. In this review, we will focus on the phenotypic responses of V. cholerae of a given genotype to starvation stress.

Functional and structural diversities of C-reactive proteins present in horseshoe crab hemolymph plasma.

Limulin, a sialic-acid-binding and phosphorylethanolamine-binding hemagglutinin in the hemolymph plasma of the American horseshoe crab (Limulus polyphemus), is a hemolytic C-reactive protein [Armstrong, P.B., Swarnakar, S., Srimal, S., Misquith, S., Hahn, E.A., Aimes, R. T. & Quigley, J.P. (1996) J. Biol. Chem. 271, 14717-14721]. We have now identified three types of C-reactive protein in the plasma of the Japanese horseshoe crab (Tachypleus tridentatus), based on different affinities against fetuin-agarose and phosphorylethanolamine-agarose determined by quantitative precipitin assays using fetuin and an artificial phosphorylethanolamine-protein conjugate. Partial amino acid sequences of the isolated C-reactive proteins identified homologous proteins which were named Tachypleus tridentatus CRP-1 (tCRP-1), tCRP-2 and tCRP-3, each of which possibly constitute isoprotein mixtures. tCRP-2 and tCRP-3, but not tCRP-1, agglutinated mammalian erythrocytes. tCRP-1, the most abundant C-reative protein in the plasma, exhibited the highest affinity to the phosphorylethanolamine-protein conjugate but lacked both sialic-acid-binding and hemolytic activities. tCRP-2 bound to both fetuin-agarose and phosphorylethanolamine-agarose, and exhibited Ca2+-dependent hemolytic and sialic-acid-binding activities, suggestive of limulin-like properties. Furthermore, tCRP-2 exhibited a higher affinity to colominic acid, a bacterial polysialic acid. By contrast, tCRP-3 shows stronger hemolytic, sialic-acid-binding and hemagglutinating activities than tCRP-2. tCRP-3 has no affinity to phosphorylethanolamine-agarose, phosphorylethanolamine-protein conjugate and colominic acid. This suggests tCRP-3 is a novel hemolytic C-reactive protein lacking a common characteristic of phosphorylethanolamine-agarose binding affinity. Twenty-two clones of tCRPs with different deduced amino acid sequences were obtained by PCR using oligonucleotide primers based on the N-terminal and C-terminal sequences of tCRPs and with templates including genomic DNA and cDNA of hemocytes or hepatopancreas derived from one individual. The translation products of the tCRP clones possess high molecular diversity which falls into three related groups, consistent with classification based on their biological activities. Only tCRP-3 contained a unique hydrophobic nonapeptide sequence that appears in the transmembrane domain of a major histocompatibility complex class I heavy chain of rainbow trout, suggesting the importance of the hydrophobic patch to the hemolytic activity of tCRP-3. The structural and functional diversities of tCRPs provide a good model for studying the properties of innate immunity in invertebrates, which survive without the benefit of acquired immunity.

Horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen.

We have characterized and cloned newly isolated lectins from hemolymph plasma of the horseshoe crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a horseshoe crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of "bouquet arrangement" of ficolins and collectins. Electron microscopy revealed that TLs-5 form two- to four-bladed propeller structures. The horseshoe crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the horseshoe crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonself-recognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.

Cloning, sequencing, and functional expression in Escherichia coli of chaperonin (groESL) genes from Vibrio cholerae.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.

Restoration of culturability of starvation-stressed and low-temperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds.

Late-exponential-phase cells of Escherichia coli O157:H- strain E32511/HSC became nonculturable in sterilized distilled water microcosms at 4 degrees C. Plate counts declined from 3 x 10(6) to less than 0.1 CFU/ml in about 21 days. However, when samples of microcosms at 21 days were inoculated onto an agar medium amended with catalase or nonenzyme peroxide-degrading compounds such as sodium pyruvate or alpha-ketoglutaric acid, plate counts increased to 10(4)-10(5) CFU/ml within 48 h. The proposed mode of action of the catalase or pyruvate is via the degradation of the metabolic by-product H2O2, rather than through supplementation of a required nutrient in the recovery of nonculturable cells. Our studies were based on the assumption that E32511/HSC strain responds to starvation and a low temperature by entering a nonculturable state and that the correction of oxidative stress upon the inoculation of bacteria on agar plates promotes recovery of nonculturable cells.

Expression, purification, crystallization and preliminary X-ray diffraction results from Campylobacter jejuni ferritin.

The prokaryotic ferritin gene of Campylobacter jejuni was overexpressed in Escherichia coli under control of the bacteriophage T7 promoter and the protein (Cj-FTN) purified. Preliminary crystallization experiments have been performed using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. Diffraction studies show the crystals belong to the I432 space group (a = 151.52 A). Structure solution by molecular replacement is in progress while crystal quality improvement is carried out.

Isolation and characterization of rugose form of Vibrio cholerae O139 strain MO10.

An extracellular exopolysaccharide (slime) is produced by Vibrio cholerae O139 MO10 in response to nutrient starvation. The presence of this slime layer on the cell surface and its subsequent release have been shown to be associated with biofilm formation and the change from a normal smooth colony morphology to a rugose one. An immunoelectron microscopic examination demonstrated that there is an epitope common to the exopolysaccharide antigen of V. cholerae O1 and that of O139 MO10.

Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide materials that determine colony morphology, stress resistance, and biofilm formation.

Vibrio cholerae O1 strain TSI-4 (El Tor, Ogawa) can shift to a rugose colony morphology from its normal translucent colony morphology in response to nutrient starvation. We have investigated differences between the rugose and translucent forms of V. cholerae O1 strain TSI-4. Electron microscopic examination of the rugose form of TSI-4 (TSI-4/R) revealed thick, electron-dense exopolysaccharide materials surrounding polycationic ferritin-stained cells, while the ferritin-stained material was absent around the translucent form of TSI-4 (TSI-4/T). The exopolysaccharide produced by V. cholerae TSI-4/R was found to have a composition of N-acetyl-D-glucosamine, D-mannose, 6-deoxy-D-galactose, and D-galactose (7.4:10.2:2.4:3.0). The expression of an amorphous exopolysaccharide promotes biofilm development under static culture conditions. Biofilm formation by the rugose strain was determined by scanning electron microscopy, and most of the surface of the film was colonized by actively dividing rod cells. The corresponding rugose and translucent strains were compared for stress resistance. By having exopolysaccharide materials, the rugose strains acquired resistance to osmotic and oxidative stress. Our data indicated that an exopolysaccharide material on the surface of the rugose strain promoted biofilm formation and resistance to the effects of two stressing agents.

Overproduction of Campylobacter ferritin in Escherichia coli and induction of paracrystalline inclusion by ferrous compound.

The ferritin gene (cft) of Campylobacter jejuni was overexpressed in cells of Escherichia coli using a T7 RNA polymerase expression system. Many round particles which were the same size as the ferritin particles purified from C. jejuni were observed in the lysate of the cft-overexpressed E. coli cells. Since most of them were devoid of a central electron dense core consisting of ferric irons, the Campylobacter ferritins over-produced in E. coli seemed to be apoferritin. When large amounts of ferrous iron (supplied as FeSO4) were added to culture medium, the cft-overexpressed cells formed large inclusion bodies of paracrystalline arrays comprised of ferritin particles with central electron dense cores. The addition of ferric irons did not produce paracrystalline inclusion.

A structural analysis of the regularly arranged porin on the outer membrane of Campylobacter jejuni based on correlation averaging.

A negatively stained electron micrograph of regularly arranged porin proteins of Campylobacter jejuni on the isolated outer membrane of bacteria was analyzed in detail by the correlation averaging method using a computer-assisted program. The results showed that the porin of C. jejuni had a trimeric structure separated by about 10.4 +/- 0.15 nm. In addition, the pores in the trimers were also separated by about 4.3 +/- 0.1 nm.

Construction of a ferritin-deficient mutant of Campylobacter jejuni: contribution of ferritin to iron storage and protection against oxidative stress.

The ferritin-encoding gene (cft) of Campylobacter jejuni was cloned and sequenced. The nucleotide sequence of cft had a 501 bp open reading frame for a protein with 167 amino acids and a predicted molecular mass of 19 180 Da, and showed a high similarity to that of Helicobacter pylori and Escherichia coli ferritin genes. To determine the biological function of ferritin in C. jejuni, a ferritin-deficient mutant was constructed. The growth of ferritin-deficient strain SNA 1 was clearly inhibited under iron deprivation. The ferritin-deficient mutant was more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These findings demonstrate that ferritin in C. jejuni makes a significant contribution to both iron storage and protection from intracellular iron overload, and resulting iron-mediated oxidative stress.