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Volume 156,
Issue 12,
2010
Volume 156, Issue 12, 2010
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Microbial communication and virulence: lessons from evolutionary theory
More LessAt the heart of tackling the huge challenge posed by infectious micro-organisms is the overwhelming need to understand their nature. A major question is, why do some species of bacteria rapidly kill their host whilst others are relatively benign? For example, Yersinia pestis, the causative organism of plague, is a highly virulent human pathogen whilst the closely related Yersinia pseudotuberculosis causes a much less severe disease. Using molecular techniques such as mutating certain genes, microbiologists have made significant advances over recent decades in elucidating the mechanisms that govern the production of virulence factors involved in causing disease in many bacterial species. There are also evolutionary and ecological factors which will influence virulence. Many of these ideas have arisen through the development of evolutionary theory and yet there is strikingly little empirical evidence testing them. By applying both mechanistic and adaptive approaches to microbial behaviours we can begin to address questions such as, what factors influence cooperation and the evolution of virulence in microbes and can we exploit these factors to develop new antimicrobial strategies?
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- Cell And Molecular Biology Of Microbes
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Transcriptional autoregulation of the RcsCDB phosphorelay system in Salmonella enterica serovar Typhimurium
More LessThe RcsCDB (Rcs) phosphorelay system is involved in the regulation of many envelope genes, such as those responsible for capsule synthesis, flagella production and O-antigen chain length, as well as in other cellular activities of several enteric bacteria. The system is composed of three proteins: the sensor RcsC, the response regulator RcsB, and the phospho-transfer intermediary protein RcsD. Previously, we reported two important aspects of this system: (a) rcsB gene expression is under the control of P rcsDB and P rcsB promoters, and (b) rcsD gene transcription decreases when the bacteria reach high levels of the RcsB regulator. In the present work, we demonstrate that the RcsB protein represses rcsD gene expression by binding directly to the P rcsDB promoter, negatively autoregulating the Rcs system. Furthermore, we report the physiological role of the RcsB regulator, which is able to modify bacterial swarming behaviour when expressed under the control of the P rcsB promoter.
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Telomere position effect is regulated by heterochromatin-associated proteins and NkuA in Aspergillus nidulans
Gene-silencing mechanisms are being shown to be associated with an increasing number of fungal developmental processes. Telomere position effect (TPE) is a eukaryotic phenomenon resulting in gene repression in areas immediately adjacent to telomere caps. Here, TPE is shown to regulate expression of transgenes on the left arm of chromosome III and the right arm of chromosome VI in Aspergillus nidulans. Phenotypes found to be associated with transgene repression included reduction in radial growth and the absence of sexual spores; however, these pleiotropic phenotypes were remedied when cultures were grown on media with appropriate supplementation. Simple radial growth and ascosporogenesis assays provided insights into the mechanism of TPE, including a means to determine its extent. These experiments revealed that the KU70 homologue (NkuA) and the heterochromatin-associated proteins HepA, ClrD and HdaA were partially required for transgene silencing. This study indicates that TPE extends at least 30 kb on chromosome III, suggesting that this phenomenon may be important for gene regulation in subtelomeric regions of A. nidulans.
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Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition
More LessBacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the core α, β and β′ subunits. The ω subunit is conserved between Gram-positive and Gram-negative bacteria, while the δ subunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits via in vitro assays, very little is known about their in vivo roles. In this work we constructed a pair of vectors to investigate the subcellular localization of the δ and ω subunits in Bacillus subtilis with respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealed δ to be present at equimolar levels with RNAP and ω to be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAP β′ and ω subunits in Escherichia coli was also investigated. Similar to B. subtilis, β′ and ω closely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlike ω in B. subtilis, ω levels in E. coli were close to parity with those of β′. These results indicate that δ is likely to be an integral RNAP subunit in Gram-positives, whereas ω levels differ substantially between Gram-positives and -negatives. The ω subunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.
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DevT (Alr4674), resembling a Ser/Thr protein phosphatase, is essential for heterocyst function in the cyanobacterium Anabaena sp. PCC 7120
Heterocyst-forming cyanobacteria are able to perform oxygenic photosynthesis and nitrogen fixation simultaneously in the same filament, by restricting the highly O2-sensitive nitrogenase to specialized cells, the heterocysts. A remarkable change in morphology and metabolism accompanies the differentiation of heterocysts, which only occurs when no source of combined nitrogen is available. In this study, we characterized DevT (Alr4674), a putative protein phosphatase from Anabaena PCC 7120. Mutants defective in devT are able to form morphologically mature heterocysts, which however cannot fix N2, and the mutant cannot survive without a source of combined nitrogen. DevT shows homology to phosphatases of the PPP family and displays a Mn2+-dependent phosphatase activity that can be inhibited by phosphatase inhibitors and oxidizing conditions. DevT is constitutively expressed in both vegetative cells and heterocysts, and is not regulated by NtcA. The heterocyst regulator HetR may exert a certain inhibition on the expression of devT. Under diazotrophic growth conditions, DevT protein accumulates specifically in mature heterocysts. Therefore DevT plays a still unknown role in a late essential step of heterocyst differentiation.
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Peroxynitrite stress is exacerbated by flavohaemoglobin-derived oxidative stress in Salmonella Typhimurium and is relieved by nitric oxide
More LessOxidative and nitrosative stresses including nitric oxide (NO), superoxide (
) and peroxynitrite play key roles in determining the outcome of bacterial infections. In order to survive within the host and allow proliferation within immune cells such as macrophages, Salmonella isolates have a number of inducible proteins that are able to detoxify these highly reactive species, notably the anoxically functioning NO reductase NorVW, and the aerobically functioning flavohaemoglobin, Hmp, which catalyses the reaction between oxygen and NO to produce relatively inert nitrate. However, in the absence of NO but in the presence of reducing substrates and oxygen,
is generated from Hmp-mediated electron transfer to bound oxygen and may form a variety of further oxidative species. Hence, Hmp expression is under tight negative regulation by the transcription factor NsrR, abolition of which causes an increase in the production of Hmp. In a previous study, this increase in Hmp levels conferred resistance to the nitrosating agent S-nitrosoglutathione but, perhaps surprisingly, the organism became more sensitive to killing by macrophages. Here, we report that an nsrR mutant that constitutively overexpresses Hmp is also hypersensitive to peroxynitrite in vitro. This sensitivity is alleviated by deletion of the hmp gene or pre-incubation of growing bacteria with NO-releasing agents. We hypothesize that Hmp-expressing cells, in the absence of NO, generate reactive oxygen species, the toxicity of which is exacerbated by peroxynitrite in vitro and in macrophages. RT-PCR confirmed that peroxynitrite causes oxidative stress and upregulation of katG and ahpC, whilst hmp and norV expression are affected very little. The katG gene upregulated by peroxynitrite encodes a catalase peroxidase enzyme with well-established roles in detoxifying peroxides. Here, we report that KatG is also able to enhance the breakdown of peroxynitrite, suggesting that the protective role of this enzyme may be wider than previously thought. These data suggest that spatial and temporal fluctuations in the levels of NO and reactive oxygen species will have important consequences for bacterial survival in the macrophage.
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Crystal structure and mutagenesis analysis of chitinase CrChi1 from the nematophagous fungus Clonostachys rosea in complex with the inhibitor caffeine
Chitinases are a group of enzymes capable of hydrolysing the β-(1,4)-glycosidic bonds of chitin, an essential component of the fungal cell wall, the shells of nematode eggs, and arthropod exoskeletons. Chitinases from pathogenic fungi have been shown to be putative virulence factors, and can play important roles in infecting hosts. However, very limited information is available on the structure of chitinases from nematophagous fungi. Here, we present the 1.8 Å resolution of the first structure of a Family 18 chitinase from this group of fungi, that of Clonostachys rosea CrChi1, and the 1.6 Å resolution of CrChi1 in complex with a potent inhibitor, caffeine. Like other Family 18 chitinases, CrChi1 has the DXDXE motif at the end of strand β5, with Glu174 as the catalytic residue in the middle of the open end of the (β/α)8 barrel. Two caffeine molecules were shown to bind to CrChi1 in subsites −1 to +1 in the substrate-binding domain. Moreover, site-directed mutagenesis of the amino acid residues forming hydrogen bonds with caffeine molecules suggests that these residues are important for substrate binding and the hydrolytic process. Our results provide a foundation for elucidating the catalytic mechanism of chitinases from nematophagous fungi and for improving the pathogenicity of nematophagous fungi against agricultural pest hosts.
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A quadruple-enterotoxin-deficient mutant of Bacillus thuringiensis remains insecticidal
More LessBacillus thuringiensis is the leading biopesticide used to control insect pests worldwide. Although they have a long record of safe use, under certain conditions commercial strains of B. thuringiensis have the ability to produce numerous putative enterotoxins that have been associated with food poisoning attributed to Bacillus cereus. Therefore, we designed a strategy to delete the genes encoding these toxins. B. thuringiensis strain VBTS 2477 contained genes encoding NHE, CytK-2 and three homologues of haemolysin BL (HBL, HBLa1 and HBLa2). This is the first report, to our knowledge, of a strain of B. cereus or B. thuringiensis containing three sets of hbl operons. The genes encoding HBLa1 and HBLa2 were 96–97 % identical to each other and 76–84 % identical to those encoding HBL. The hbla2 operon was detected by PCR amplification only after hbla1 was deleted. We used sequential gene replacement to replace the wild-type copies of the NHE and three HBL operons with copies that contained internal deletions that span the three genes in each operon. The insecticidal activity of the quadruple-enterotoxin-deficient mutant was similar to that of the wild-type strain against larvae of Trichoplusia ni, Spodoptera exigua and Plutella xylostella. This demonstrates that the genes for enterotoxins can be deleted, eliminating the possibility of enterotoxin production without compromising the insecticidal efficacy of a strain of B. thuringiensis.
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Heterologous expression of the surface-layer-like protein SllB induces the formation of long filaments of Escherichia coli consisting of protein-stabilized outer membrane
Escherichia coli is one of the best studied micro-organisms and is the most widely used host in genetic engineering. The Gram-negative single cells are rod-shaped, and filaments are usually not found. Here, we describe the reproducible formation of elongated E. coli cells. During heterologous expression of the silent surface (S)-layer protein gene sllB from Lysinibacillus sphaericus JG-A12 in E. coli BL21(DE3), the cells were arranged as long chains which were surrounded by highly stable sheaths. These filaments had a length of >100 μm. In the stationary growth phase, microscopic analyses demonstrated the formation of unusually long transparent tube-like structures which were enclosing separate single cells. The tube-like structures were isolated and analysed by SDS-PAGE, infrared-spectroscopy and different microscopic methods in order to identify their unusual composition and structure. The tube-like structures were found to be like outer membranes, containing high levels of proteins and to which the recombinant S-layer proteins were attached. Despite the entire structure being indicative of a disordered cell division, the bacterial cells were highly viable and stable. To our knowledge, this is the first time that the induction of drastic morphological changes in E. coli by the expression of a foreign protein has been reported.
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- Environmental And Evolutionary Microbiology
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Variation in the Neisseria meningitidis FadL-like protein: an evolutionary model for a relatively low-abundance surface antigen
More LessThe molecular diversity of a novel Neisseria meningitidis antigen, encoded by the ORF NMB0088 of MC58 (FadL-like protein), was assessed in a panel of 64 diverse meningococcal strains. The panel consisted of strains belonging to different serogroups, serotypes, serosubtypes and MLST sequence types, of different clinical sources, years and countries of isolation. Based on the sequence variability of the protein, the FadL-like protein has been divided into four variant groups in this species. Antigen variants were associated with specific serogroups and MLST clonal complexes. Maximum-likelihood analyses were used to determine the relationships among sequences and to compare the selection pressures acting on the encoded protein. Furthermore, a model of population genetics and molecular evolution was used to detect natural selection in DNA sequences using the non-synonymous : synonymous substitution (d N : d S) ratio. The meningococcal sequences were also compared with those of the related surface protein in non-pathogenic commensal Neisseria species to investigate potential horizontal gene transfer. The N. meningitidis fadL gene was subject to only weak positive selection pressure and was less diverse than meningococcal major outer-membrane proteins. The majority of the variability in fadL was due to recombination among existing alleles from the same or related species that resulted in a discrete mosaic structure in the meningococcal population. In general, the population structuring observed based on the FadL-like membrane protein indicates that it is under intermediate immune selection. However, the emergence of a new subvariant within the hyperinvasive lineages demonstrates the phenotypic adaptability of N. meningitidis, probably in response to selective pressure.
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- Genes And Genomes
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Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum
More LesspCGR2 and pCG1 belong to different subfamilies of the pCG1 family of Corynebacterium glutamicum plasmids. Nonetheless, they harbour homologous putative antisense RNA genes, crrI and cgrI, respectively. The genes in turn share identical positions complementary to the leader region of their respective repA (encoding plasmid replication initiator) genes. Determination of their precise transcriptional start- and end-points revealed the presence of short antisense RNA molecules (72 bp, CrrI; and 73 bp, CgrI). These short RNAs and their target mRNAs were predicted to form highly structured molecules comprising stem–loops with known U-turn motifs. Abolishing synthesis of CrrI and CgrI by promoter mutagenesis resulted in about sevenfold increase in plasmid copy number on top of an 11-fold (CrrI) and 32-fold (CgrI) increase in repA mRNA, suggesting that CrrI and CgrI negatively control plasmid replication. This control is accentuated by parB, a gene that encodes a small centromere-binding plasmid-partitioning protein, and is located upstream of repA. Simultaneous deactivation of CrrI and parB led to a drastic 87-fold increase in copy number of a pCGR2-derived shuttle vector. Moreover, the fact that changes in the structure of the terminal loops of CrrI and CgrI affected plasmid copy number buttressed the important role of the loop structure in formation of the initial interaction complexes between antisense RNAs and their target mRNAs. Similar antisense RNA control systems are likely to exist not only in the two C. glutamicum pCG1 subfamilies but also in related plasmids across Corynebacterium species.
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ccrAB Ent serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species groups and are expressed in E. faecium
The presence, distribution and expression of cassette chromosome recombinase (ccr) genes, which are homologous to the staphylococcal ccrAB genes and are designated ccrAB Ent genes, were examined in enterococcal isolates (n=421) representing 13 different species. A total of 118 (28 %) isolates were positive for ccrAB Ent genes by PCR, and a number of these were confirmed by Southern hybridization with a ccrA Ent probe (n=76) and partial DNA sequencing of ccrA Ent and ccrB Ent genes (n=38). ccrAB Ent genes were present in Enterococcus faecium (58/216, 27 %), Enterococcus durans (31/38, 82 %), Enterococcus hirae (27/52, 50 %), Enterococcus casseliflavus (1/4, 25 %) and Enterococcus gallinarum (1/2, 50 %). In the eight other species tested, including Enterococcus faecalis (n=94), ccrAB Ent genes were not found. Thirty-eight sequenced ccrAB Ent genes from five different enterococcal species showed 94–100 % nucleotide sequence identity and linkage PCRs showed heterogeneity in the ccrAB Ent flanking chromosomal genes. Expression analysis of ccrAB Ent genes from the E. faecium DO strain showed constitutive expression as a bicistronic mRNA. The ccrAB Ent mRNA levels were lower during log phase than stationary phase in relation to total mRNA. Multilocus sequence typing was performed on 39 isolates. ccrAB Ent genes were detected in both hospital-related (10/29, 34 %) and non-hospital (4/10, 40 %) strains of E. faecium. Various sequence types were represented by both ccrAB Ent positive and negative isolates, suggesting acquisition or loss of ccrAB Ent in E. faecium. In summary, ccrAB Ent genes, potentially involved in genome plasticity, are expressed in E. faecium and are widely distributed in the E. faecium and E. casseliflavus species groups.
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- Microbial Pathogenicity
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Candida albicans forms biofilms on the vaginal mucosa
More LessCurrent understanding of resistance and susceptibility to vulvovaginal candidiasis challenges existing paradigms of host defence against fungal infection. While abiotic biofilm formation has a clearly established role during systemic Candida infections, it is not known whether C. albicans forms biofilms on the vaginal mucosa and the possible role of biofilms in disease. In vivo and ex vivo murine vaginitis models were employed to examine biofilm formation by scanning electron and confocal microscopy. C. albicans strains included 3153A (lab strain), DAY185 (parental control strain), and mutants defective in morphogenesis and/or biofilm formation in vitro (efg1/efg1 and bcr1/bcr1). Both 3153A and DAY815 formed biofilms on the vaginal mucosa in vivo and ex vivo as indicated by high fungal burden and microscopic analysis demonstrating typical biofilm architecture and presence of extracellular matrix (ECM) co-localized with the presence of fungi. In contrast, efg1/efg1 and bcr1/bcr1 mutant strains exhibited weak or no biofilm formation/ECM production in both models compared to wild-type strains and complemented mutants despite comparable colonization levels. These data show for the first time that C. albicans forms biofilms in vivo on vaginal epithelium, and that in vivo biotic biofilm formation requires regulators of biofilm formation (BCR1) and morphogenesis (EFG1).
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Heterogeneous distribution of Candida albicans cell-surface antigens demonstrated with an Als1-specific monoclonal antibody
More LessDespite an abundance of data describing expression of genes in the Candida albicans ALS (agglutinin-like sequence) gene family, little is known about the production of Als proteins on individual cells, their spatial localization or stability. Als proteins are most commonly discussed with respect to function in adhesion of C. albicans to host and abiotic surfaces. Development of a mAb specific for Als1, one of the eight large glycoproteins encoded by the ALS family, provided the opportunity to detect Als1 during growth of yeast and hyphae, both in vitro and in vivo, and to demonstrate the utility of the mAb in blocking C. albicans adhesion to host cells. Although most C. albicans yeast cells in a saturated culture are Als1-negative by indirect immunofluorescence, Als1 is detected on the surface of nearly all cells shortly after transfer into fresh growth medium. Als1 covers the yeast cell surface, with the exception of bud scars. Daughters of the inoculum cells, and sometimes granddaughters, also have detectable Als1, but Als1 is not detectable on cells from subsequent generations. On germ tubes and hyphae, most Als1 is localized proximal to the mother yeast. Once deposited on yeasts or hyphae, Als1 persists long after the culture has reached saturation. Growth stage-dependent production of Als1, coupled with its persistence on the cell surface, results in a heterogeneous population of cells within a C. albicans culture. Anti-Als1 immunolabelling patterns vary depending on the source of the C. albicans cells, with obvious differences between cells recovered from culture and those from a murine model of disseminated candidiasis. Results from this work highlight the temporal parallels for ALS1 expression and Als1 production in yeasts and germ tubes, the specialized spatial localization and persistence of Als1 on the C. albicans cell surface, and the differences in Als1 localization that occur in vitro and in vivo.
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Streptococcal inhibitor of complement-mediated lysis (SIC): an anti-inflammatory virulence determinant
Since the late 1980s, a worldwide increase of severe Streptococcus pyogenes infections has been associated with strains of the M1 serotype, strains which all secrete the streptococcal inhibitor of complement-mediated lysis (SIC). Previous work has shown that SIC blocks complement-mediated haemolysis, inhibits the activity of antibacterial peptides and has affinity for the human plasma proteins clusterin and histidine-rich glycoprotein; the latter is a member of the cystatin protein family. The present work demonstrates that SIC binds to cystatin C, high-molecular-mass kininogen (HK) and low-molecular-mass kininogen, which are additional members of this protein family. The binding sites in HK are located in the cystatin-like domain D3 and the endothelial cell-binding domain D5. Immobilization of HK to cellular structures plays a central role in activation of the human contact system. SIC was found to inhibit the binding of HK to endothelial cells, and to reduce contact activation as measured by prolonged blood clotting time and impaired release of bradykinin. These results suggest that SIC modifies host defence systems, which may contribute to the virulence of S. pyogenes strains of the M1 serotype.
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Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis
More LessPathogenic strains of mycobacteria produce copious amounts of glutamine synthetase (GS) in the culture medium. The enzyme activity is linked to synthesis of poly-α-l-glutamine (PLG) in the cell walls. This study describes a glnA-1 mutant of Mycobacterium bovis that produces reduced levels of GS. The mutant was able to grow in enriched 7H9 medium without glutamine supplementation. The glnA-1 strain contained no detectable PLG in the cell walls and showed marked sensitivity to different chemical and physical stresses such as lysozyme, SDS and sonication. The sensitivity of the mutant to two antitubercular drugs, rifampicin and d-cycloserine, was also increased. The glnA-1 strain infected THP-1 cells with reduced efficiency and was also attenuated for growth in macrophages. A Mycobacterium smegmatis strain containing the M. bovis glnA-1 gene survived longer in THP-1 cells than the wild-type strain and also produced cell wall-associated PLG. The M. bovis mutant was not able to replicate in the organs of BALB/c mice and was cleared within 4–6 weeks of infection. Disruption of the glnA-1 gene adversely affected biofilm formation on polystyrene surfaces. The results of this study demonstrate that the absence of glnA-1 not only attenuates the pathogen but also affects cell surface properties by altering the cell wall chemistry of the organism via the synthesis of PLG; this may be a target for drug development.
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Role of Hcp, a type 6 secretion system effector, of Aeromonas hydrophila in modulating activation of host immune cells
More LessRecently, we reported that the type 6 secretion system (T6SS) of Aeromonas hydrophila SSU plays an important role in bacterial virulence in a mouse model, and immunization of animals with the T6SS effector haemolysin co-regulated protein (Hcp) protected them against lethal infections with wild-type bacteria. Additionally, we showed that the mutant bacteria deleted for the vasH gene within the T6SS gene cluster did not express the hcp gene, while the vasK mutant could express and translocate Hcp, but was unable to secrete it into the extracellular milieu. Both of these A. hydrophila SSU mutants were readily phagocytosed by murine macrophages, pointing to the possible role of the secreted form of Hcp in the evasion of the host innate immunity. By using the ΔvasH mutant of A. hydrophila, our in vitro data showed that the addition of exogenous recombinant Hcp (rHcp) reduced bacterial uptake by macrophages. These results were substantiated by increased bacterial virulence when rHcp was added along with the ΔvasH mutant in a septicaemic mouse model of infection. Analysis of the cytokine profiling in the intraperitoneal lavage as well as activation of host cells after 4 h of infection with the ΔvasH mutant supplemented with rHcp indicated that this T6SS effector inhibited production of pro-inflammatory cytokines and induced immunosuppressive cytokines, such as interleukin-10 and transforming growth factor-β, which could circumvent macrophage activation and maturation. This mechanism of innate immune evasion by Hcp possibly inhibited the recruitment of cellular immune components, which allowed bacterial multiplication and dissemination in animals, thereby leading to their mortality.
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Intracellular survival of Salmonella enterica serovar Typhi in human macrophages is independent of Salmonella pathogenicity island (SPI)-2
More LessFor successful infection, Salmonella enterica secretes and injects effector proteins into host cells by two distinct type three secretion systems (T3SSs) located on Salmonella pathogenicity islands (SPIs)-1 and -2. The SPI-2 T3SS is involved in intracellular survival of S. enterica serovar Typhimurium and systemic disease. As little is known regarding the function of the SPI-2 T3SS from S. enterica serovar Typhi, the aetiological agent of typhoid fever, we investigated its role for survival in human macrophages. Mutations in the translocon (sseB), basal secretion apparatus (ssaR) and regulator (ssrB) did not result in any reduction in survival under many of the conditions tested. Similar results were obtained with another S. Typhi strain or by using human primary cells. Results were corroborated based on complete deletion of the SPI-2 T3SS. Surprisingly, the data suggest that the SPI-2 T3SS of S. Typhi is not required for survival in human macrophages.
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Phenotypic diversification in vivo: Pseudomonas aeruginosa gacS− strains generate small colony variants in vivo that are distinct from in vitro variants
Pseudomonas aeruginosa has long been known to produce phenotypic variants during chronic mucosal surface infections. These variants are thought to be generated to ensure bacterial survival against the diverse challenges in the mucosal environment. Studies have begun to elucidate the mechanisms by which these variants emerge in vitro; however, too little information exists on phenotypic variation in vivo to draw any links between variants generated in vitro and in vivo. Consequently, in this study, the P. aeruginosa gacS gene, which has previously been linked to the generation of small colony variants (SCVs) in vitro, was studied in an in vivo mucosal surface infection model. More specifically, the rat prostate served as a model mucosal surface to test for the appearance of SCVs in vivo following infections with P. aeruginosa gacS− strains. As in in vitro studies, deletion of the gacS gene led to SCV production in vivo. The appearance of these in vivo SCVs was important for the sustainability of a chronic infection. In the subset of rats in which P. aeruginosa gacS− did not convert to SCVs, clearance of the bacteria took place and healing of the tissue ensued. When comparing the SCVs that arose at the mucosal surface (MS-SCVs) with in vitro SCVs (IV-SCVs) from the same gacS− parent, some differences between the phenotypic variants were observed. Whereas both MS-SCVs and IV-SCVs formed dense biofilms, MS-SCVs exhibited a less diverse resistance profile to antimicrobial agents than IV-SCVs. Additionally, MS-SCVs were better suited to initiate an infection in the rat model than IV-SCVs. Together, these observations suggest that phenotypic variation in vivo can be important for maintenance of infection, and that in vivo variants may differ from in vitro variants generated from the same genetic parent.
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Cytokeratin 8 interacts with clumping factor B: a new possible virulence factor target
More LessStaphylococcus aureus is a human pathogen of growing clinical significance, owing to its increasing levels of resistance to most antibiotics. Infections range from mild wound infections to severe infections such as endocarditis, osteomyelitis and septic shock. Adherence of S. aureus to human host cells is an important step, leading to colonization and infection. Adherence is mediated by a multiplicity of proteins expressed on the bacterial surface, including clumping factor B. In this study, we aimed to identify new targets of clumping factor B in human keratinocytes by undertaking a genome-wide yeast two-hybrid screen of a human keratinocyte cDNA library. We show that clumping factor B is capable of binding cytokeratin 8 (CK8), a type II cytokeratin. Using a domain-mapping strategy we identified amino acids 437–464 as necessary for this interaction. Recombinantly expressed fragments of both proteins were used in pull-down experiments and confirmed the yeast two-hybrid studies. Analysis with S. aureus strain Newman deficient in clumping factor B showed the clumping factor B-dependence of the interaction with CK8. We postulate that the clumping factor B–CK8 interaction is a novel factor in S. aureus infections.
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)
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